MICROSCOPY RESEARCH AND TECHNIQUE 32~459-497 (1995)

Spermatogenesis in Nonmammalian Vertebrates

JEFFREY PUDNEY
Fearing Research Laboratory, Brigham and Women's Hospital, Haruard Medical School, Boston, Massachusetts 02115

KEY WORDS Anamniotes, Spermatocysts, Amniotes

ABSTRACT Spermatogenesis appears to be a fairly conserved process throughout the verte-

brate series. Thus, spermatogonia develop into spermatocytes that undergo meiosis to produce
spermatids which enter spermiogenesis where they undergo a morphological transformation into
spermatozoa. There is, however, variation amongst the vertebrates in how germ cell development
and maturation is accomplished. This difference can be broadly divided into two distinct patterns,
one present in anamniotes (fish, amphibia) and the other in amniotes (reptiles, birds, mammals).
For anamniotes, spermatogenesis occurs in spermatocysts (cysts) which for most species develop
within seminiferous lobules. Cysts are produced when a Sertoli cell becomes associated with a
primary spermatogonium. Mitotic divisions of the primary spermatogonium produce a cohort of
secondary spermatogonia that are enclosed by the Sertoli cell which forms the wall of the cyst. With
spermatogenic progression a clone of isogeneic spermatozoa is produced which are released, by
rupture of the cyst, into the lumen of the seminiferous lobule. Following spermiation, the Sertoli
cell degenerates. For anamniotes, therefore, there is no permanent germinal epithelium since
spermatocysts have to be replaced during successive breeding seasons. By contrast, spermatogen-
esis in amniotes does not occur in cysts but in seminiferous tubules that possess a permanent
population of Sertoli cells and spermatogonia which act as a germ cell reservoir for succeeding bouts
of spermatogenic activity. There is, in general, a greater variation in the organization of the testis
and pattern of spermatogenesis in the anamniotes compared to amniotes. This is primarily due to
the fact there is more reproductive diversity in anamniotes ranging from a relatively unspecialized
condition where gametes are simply released into the aqueous environment to highly specialized
strategies involving internal fertilization. These differences are obviously reflected in the mode of
spermatogenesis and this is particularly true of the stage of spermiogenesis where the morphology
of the species-specific spermatozoon is determined. Moreover, unlike amniotes, many anamniotes
display a spermatogenic wave manifest, depending upon the species, either at the level of the cyst
or seminiferous lobule.
This variation in the organization of the testis makes certain anamniotes perfect models for
investigating germ cell development and maturation. For instance, the presence of a spermatogenic
wave provides an opportunity to manually isolate discrete germ cell stages for analysis of specific
Sertoli/germ cell interactions. Furthermore, for many anamniotes, germ cells mature in association
with a morphologically poorly developed Sertoli cell. This seeming independence of Sertoli cell
regulation allows the in vitro culture of isolated germ cells of some species of anamniotes through
several developmental stages. Thus, due either to the anatomical organization of the testis, or
structural simplicity of the germinal units, nonmammalian vertebrates can provide excellent ex-
perimental animal models for investigating many basic problems of male reproduction.
0 1995 Wiley-Liss, Inc.

INTRODUCTION spermatogenesis depend primarily on several parame-

Throughout the vertebrate series it would appear
that germ cell development and maturation proceeds in
a similar fashion. Thus, spermatogonial stem cells di-
vide to produce generations of spermatogonia which
enter the spermatogenic cycle. Spermatogonia develop
into spermatocytes which undergo meiosis to produce
spermatids. These spermatids then proceed through a
morphological metamorphosis during spermiogenesis
to form spermatozoa. When, however, spermatogenesis
is compared between the different classes of verte-
brates it is obvious this process can be accomplished in
a variety of ways. These differences in the pattern of
ters which are not necessarily mutually exclusive; (1)
the organization of the testis, i.e., the anatomical dis-
position of the primary germinal compartment; (2) the
relationship between germ cells and Sertoli cells dur-
ing the spermatogenic cycle; (3) whether fertilization is
external or internal; and (4) whether the species is
Received May 31, 1994; accepted in revised form August 12, 1994.
Address reprint requests to Dr. Jeffrey Pudney, Fearing Research Laboratory,
Brigham and Women's Hospital, 250 Longwood Avenue, Boston, MA 02115.

0 1995 WILEY-LISS, INC.

460 J. PUDNEY

cold-blooded (poikilotherm) or warm-blooded (homeo-

therm).
Since the structural association between germ cells
and Sertoli cells during spermatogenesis largely dic-
tates the morphological appearance of the germinal
compartment, these two factors will be discussed to-
gether.

THE ORGANIZATION OF THE TESTIS AND

SERTOLUGERM CELL RELATIONSHIPS
It is now well established that Sertoli cells play a
pivotal role during spermatogenesis. A recent review is
available dedicated to the Sertoli cell in nonmamma-
lian vertebrates (Pudney, 1993). For this present re-
port, therefore, information on the Sertoli cell will be
restricted to aspects of this cell which affect or impinge
on the pattern of spermatogenesis in different classes
of nonmammalian vertebrates.

THE VERTEBRATE TESTIS Fig. 1. Amphiorus testes develop from coelomic pouches. This tes-
tis is filled with mature spermatozoa.
In the phylum, chordata, nonmammalian verte-
brates include the jawless fishes, lampreys and hagfish
(Agnatha); cartilaginous fishes, sharks, rays, and chi-
meras (Chondrichthys); the bony fishes (Osteichthys);
newts, salamanders, frogs, toads, and caecilians (Am-
phibia); turtles, snakes, lizards, crocodiles, alligators
(Reptilia), and the birds (Aves).
In the protochordate Amphioxus the testes are
formed from metamerically arranged coelomic pouches
(Fig. 1).As vertebrates evolved, however, these rows of
testes tended to form localized compact organs. In most
vertebrates the testes are paired except for the Ag-
natha in which fusion of the testes has occurred and
there are several species of bony fishes which possess a
single testis. The shape of the vertebrate testis varies
from extremely elongated, as in most cartilaginous and
bony fishes, to round or oval as in many amphibia,
reptiles, and birds. For some urodelean amphibia and
bony fishes, however, the testes are composed of dis-
tinct lobes. In nonmammalian vertebrates the testes lie
within the body attached by a mesorchium to the body
wall. Usually the testes are isolated structures but can
be associated with other tissue elements such as he-
mopoietic tissue, the epigonal organ which invests the
testes in selachians or the fat body attached to the tes-
tes of anuran amphibia. An interesting feature in bony Fig. 2. Ambisexual teleost containing an ovary (0)and testis (T).
fishes is the occurrence of ambisexual species which
possess both an ovary and a testis (Fig. 2). These go-
nads may be present simultaneously or develop se- ary conserved feature of the vertebrate testis and even
quentially. Those species in which the testis develops occurs in the testis of Amphioxus (Pudney, 1993).
first are termed protandrous, while those where the
ovary develops first are protogynous. ORGANIZATION OF THE TESTIS
The vertebrate testis is composed of two distinct com- Within the vertebrate series the general structural
partments. One consists of the interstitial tissue, con- appearance of the testis can be divided into two distinct
taining blood vessels, lymphatics and Leydig cells patterns, one present in the anamniotes (fish, am-
which secrete the male hormones or androgens. The phibia) the other in amniotes (reptiles, birds, mam-
second is the seminiferous compartment which con- mals).
tains the germinal epithelium defined as a tripartite
structure consisting of an acellular basement mem- Anamniotes
brane, germ cells and Sertoli cells (Grier, 1993). The In these nonmammalian vertebrates, the unit of
association of germ cells with a somatic element, i.e., spermatogenesis is a spermatocyst, usually referred to
Sertoli cell of the germinal epithelium, is an evolution- as a cyst (von La Valette, St. George, 1876). Incipient
 SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES
B Studies on the morphology of the testis of chondrich-
dG $.
+@ sG--@ @ the production of a basement membrane by the germi-
+ interstitial tissue. These cells are not enclosed by a
Fig. 3. Schematic diagram showing general features of cyst for-
mation in anamniotes. A: Initially a Sertoli cell (SC) becomes associ-
ated with a primary spermatogonium (PG). B: The Sertoli cell com-
pletely surrounds the germ cell to produce a nascent cyst. C: The
primary spermatogonium undergoes repeated mitotic divisions to pro-
duce secondary spermatogonia (SG) enclosed by Sertoli cytoplasm
which forms the wall of the cyst. D Germ cell maturation then pro-
ceeds with the eventual production of a n isogeneic clone of spermato-
zoa which are often orientated with their heads towards the Sertoli
cell nucleus. E: Spermiation is accomplished by rupture of the cyst. F:
The Sertoli cell then undergoes degeneration. (Reproduced from Pud-
ney, 1993, with permission of Cache River Press.)
cysts are formed when a primary spermatogonium be-
comes engulfed by a Sertoli cell (Fig. 3). The primary
spermatogonium then undergoes numerous mitotic di-
visions to produce secondary spermatogonia. The cyst
is now composed of a mass of germ cells surrounded by
Sertoli cytoplasm which forms the wall of the cyst.
Germ cell maturation then occurs with formation of an
isogeneic clone of spermatozoa which are released by
rupture of the cyst wall. Following spermiation, in
most anamniotes, Sertoli cells usually degenerate.
In bony fishes and amphibia, cysts develop within
larger structures that develop from sperm collecting
ducts. In the literature, these blind-ending pouches
have been referred to as either lobules, tubules, ampul-
lae, or follicles. Recently, a consistent and unifying ter-
minology has been proposed for describing the anam-
niote testis (Grier, 1993). In this study, the lobule was
defined as the anatomically correct term for applying
to the germinal compartment of the testis in most bony
fishes and all amphibia.
46 1
thyan fishes have also described the germinal compart-
ment as consisting of cysts contained within lobules (or
follicles or ampullae). This organization of the testes in
these species, however, has been recently shown to be
anatomically incorrect (Grier, 1992). This study was
based on the observation that the germinal comDart-
ment in the vertebrate testis is composed of a i e p i -
thelium (germinal) which by definition rests on a
basement membrane that sequesters it from the sur-
rounding interstitial tissue. Called the extracellular
matrix hypothesis, it was shown that, chronologically,
nal compartment differed in the testes of vertebrate
groups.
Utilizing this hypothesis, it was reported that a ma-
jor factor in how the anamniote testis is organized was
when the germinal elements became isolated from the
interstitial tissue, by the secretion of a basement mem-
brane, to form a separate germinal compartment.
Thus, in the cartilaginous fishes, when germ cells (pri-
mary spermatogonia) and precursor somatic (Sertoli)
cells initially come together they lie free within the
basement membrane and so do not represent a germi-
nal compartment. Then the basement membrane is se-
creted t o surround and isolate the Sertoli cell and germ
cells (now secondary spermatogonia) from the intersti-
tial tissue to produce a distinct germinal compartment,
the spermatocyst. In the chondrichthyan testis, there-
fore. cvsts are not Dresent within lobules. but occur as
isolatid structure; Since the chondrichthyan testis is
composed of a mass of cysts, it is called polyspermato-
cystic (Grier, 1992).
Similarly, the agnathan testis is also made up of nu-
merous individual cysts, i.e., polyspermatocystic. The
composition of cysts, however, differs between the two
vertebrate classes. Cysts in the agnathan testis all con-
tain a single generation of isogeneic germ cells. By
contrast, the chondrichthyan cysts contain many (al-
though fixed in number) Sertoli cells, each of which is
associated with a clone of germ cells. These individual
Sertoli cells plus their cohort of germ cells present
within a cyst have been called spermatoblasts (Callard,
1991). The term spermatoblast was initially used to
define a functional unit of spermatogenesis consisting
of a Sertoli cell with its associated germ cells within
the mammalian seminiferous epithelium (Von Ebner,
1871). It should be noted that in the chondrichthyan
testis, each cyst is composed of spermatoblasts which
all contain germ cells at roughly the same stage of
development.
For the agnatha, most species of bony fishes, and
anuran amphibia, the cysts are stationary within the
testis. By contrast, in the chondrichthyes, several spe-
cies of bony fishes and urodelean amphibia, cysts move
through the testis as they mature during spermatogen-
esis. The direction of this cystic migration, however,
differs amongst these nonmammalian vertebrates. In
urodeles, cysts form in the region of the lobule proxi-
mal to the central sperm collecting duct. During sper-
matogenesis, as cysts mature they move distally to-
wards the blind end of the lobule. Spermiation occurs
462 J. PUDNEY
when these cysts rupture and the released spermatozoa
must then pass down the lobule, through the immature
region, to enter the sperm duct. For certain species of
bony fishes, however, the reverse occurs with nascent
cysts located at the blind end of the lobule which then
migrate, as they mature, down towards the sperm duct.
On reaching the sperm duct these cysts break down to
release spermatozoa either as free entities or as naked
(spermatozeugmata) or encapsulated (spermatophores)
bundles. In the chondrichthyes it is the cyst which mi-
grates through the testis. Developing cysts originate in
a germinal zone, the location of which varies in the
testes of different chondrichthyan species (Pratt, 1988).
For example, in the spiny dogfish Squalus acanthias,
the germinal zone is found on the dorsolateral surface
of the gonad. During spermatogenesis, as cysts mature,
they migrate radially through the testis t o the opposite
side by which time they are filled with spermatozoa.
Spermiation then occurs and the cysts degenerate. This
pattern of cyst development has been called diametri-
cal since it occurs across the diameter of the testis
(Pratt, 1988).
It should be mentioned that the migration of cysts in
lobules of bony fishes and urodeles or testes of chon-
drichthyans does not occur by movement of the cysts
themselves. Cysts are passive structures and it is pre-
sumably the continual formation of cysts and/or growth
and elongation of sperm ducts which cause their mi-
gration either along the lobule or through the testis.
Also, during spermatogenesis, in some anamniotes,
e.g., urodeles, although all cysts contain synchro-
nously developing germ cells, the lobule itself could
consist of different regions composed of cysts at
different stages of maturation, i.e., an immature zone
with spermatogonial cysts and a mature zone with
spermatogenically active cysts. Furthermore, the
stage of maturation reached by cysts in the mature
zone may vary from lobule to lobule within the same
testis, resulting in production of a spermatogenic
wave. A spermatogenic wave also occurs in the
chondrichthyes that involves the progressive matura-
tion of cysts as they migrate through the testis. This
results in a distinct zonation of spermatogenesis in the
testis of these species.
Amniotes
In amniotes there is no cystic form of spermatogen-
esis and therefore the testis has been defined as
postspermatocystic (Grier, 1993). Instead, the seminif-
erous epithelium is composed of a stable population of
Sertoli cells associated with successive stages of germ
cell maturation. For all amniotes the germinal com-
partment is composed of seminiferous tubules which
are single structures except for birds in which exten-
sive branching may occur. The number and length of
these tubules vary greatly among anamniotes. The im-
mature germ cells, the spermatogonia, are located a t
the base of the seminiferous epithelium while sper-
matocytes and spermatids occur at successively higher
levels. Mature, elongate spermatids border the lumen
of the tubules into which they are released during sper-
miation. Therefore, unlike the anamniote testis where
cysts contain germ cells a t the same stage of develop-
ment, in the amniote testis there is a stratification of
germ cells at different phases of maturation within the
seminiferous epithelium. Furthermore, in anamniotes,
following spermiation, Sertoli cells undergo degenera-
tion, whereas in amniotes, Sertoli cells represent a per-
manent feature of the seminiferous epithelium. There
is, in general, less variation in the organization of the
testis and pattern of spermatogenesis in the amniotes
compared to the anamniotes.
SEASONAL CYCLES
In vertebrates the pattern of spermatogenesis varies
according to whether: (1) species are cold-blooded
(poikilotherm), e.g., fish, amphibia, reptiles, or warm-
blooded (homeotherm), e.g., birds, mammals; and (2)
reproduction is seasonal. Poikilotherm vertebrates un-
dergoing reproductive seasonal cycles exhibit postnup-
tial spermatogenesis. This is because spermatogenesis
begins soon after the conclusion of the breeding season.
In contrast, spermatogenesis essentially stops in ho-
meothermic vertebrates following the breeding season.
For these species, therefore, spermatogenesis is pren-
uptial because recrudescence does not occur until the
start of the next breeding season.
In general, all feral vertebrates exhibit a seasonal
cycle of reproductive activity. This is more pronounced
for species that inhabit temperate zones than those liv-
ing in more stable habitats, e.g., tropical, or benthic
environments. For temperate poikilotherms which
practice postnuptial spermatogenesis, germ cell devel-
opment begins in the summer and then proceeds either
slowly throughout winter, or ceases completely during
winter to be reinitiated in the spring. In homeotherms,
following the breeding season, there is complete testic-
ular involution and regression of the germinal epithe-
lium with spermatogonia (usually) as the remaining
germ cells. It should be pointed out that few descriptive
studies have been carried out on the changes the testes
of nonmammalian vertebrates undergo throughout
their annual reproductive cycles.
All amniotes possess a permanent seminiferous epi-
thelium which contains a resident population of Sertoli
cells and a constant population of germ cells, i.e., pri-
mary spermatogonia which act as stem cells and are
responsible for annual reinitiation of spermatogenesis.
By contrast, the only anamniotes reported to possess a
permanent germinal epithelium are anurans (Burgos
and Vitale-Calpe, 196713; Lofts, 1984). This is because,
although the apical Sertoli cytoplasm is shed at sper-
miation, the basal portion remains intact and regener-
ates from one breeding season to the next. For other
anamniotes there is degeneration of cysts following
spermiation and so these germinal units have to be
constantly replenished. Recent observations, however,
suggest that a permanent germinal epithelium occurs
in some teleosts with persistence of a few Sertoli cells
and germ cells (spermatogonia) from one breeding sea-
son to the next (Grier, 1993).
Several reviews are available on the organization of
the testis in nonmammalian vertebrates (Callard,
1991; Grier, 1993; Pilsworth and Setchell, 1981; Pud-
ney, 1987; Roosen-Runge, 1977).
 SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES
Fig. 4. Amphioxus testis. A Germinal epithelium with a layer of
immature germ cells at the periphery (arrowheads) and mature sper-
matozoa in the lumen. B: Spermatogonia are poorly differentiated
with few organelles except for several large mitochondria. C: Sper-
EXTERNAL VERSUS
INTERNAL FERTILIZATION
In general, nonmammalian vertebrates that practice
external fertilization produce a larger number of
poorly differentiated spermatozoa compared t o those
which engage in internal fertilization. The spermato-
zoa of external fertilizing species may be considered
primitive, but a number also produce gametes with
highly specialized features such as the acrosomal fila-
ment of petromyzonids (Nicander and Sjoden, 1971).
Also, spermatozoa of teleosts could be considered prim-
itive since they lack an acrosome. The absence of this
structure, however, is due to the method of fertilization
in which spermatozoa gain access to the ovum via a
micropyle, thus making the presence of an acrosome
obsolete.
PROTOCHORDATES
In Amphioxus, the testes occur as a metamerically
arranged series along the length of the body (Fig. 1).
Each testis contains a typical germinal epithelium
with layers of developing germ cells present a t the pe-
riphery and mature spermatozoa occupying the central
cavity of the testis (Fig. 4A). Germ cells develop in
association with Sertoli cells (see Pudney, 1993). Sper-
matogonia appear poorly differentiated with nuclei
containing finely dispersed chromatin, apparently
lacking a distinct nucleolus and few cytoplasmic or-
ganelles except for several large mitochondria with
lamelliform cristae (Fig. 4B). The spermatozoa posses a
rounded nucleus with clumps of condensed chromatin
and a small, cap-shaped acrosome located on the ante-
rior pole (Fig. 4C). This acrosome appears to be sepa-
rated from the nucleus by a clear zone containing a
flocculent, homogenous substance. The posterior pole
of the nucleus contains a fossa which houses two cen-
463
matozoa appear primitive and possess a small acrosome (arrowhead),
and a single large mitochondrion (MI at the base of the nucleus and
surrounding the flagellum.
trioles orientated roughly a t 90" to each other, one of
which (presumably the distal centriole) gives rise to
the long flagellum. The flagellum contains a typical
axoneme consisting of a 9 + 2 arrangement of doublets.
A single large mitochondrion is located a t the base of
the nucleus and appears to partially surround the cen-
triole region (Fig. 4). It would appear that spermato-
genesis proceeds until the testis is filled with mature
spermatozoa (see Fig. 1)at which time the gonad (pre-
sumably) ruptures to release the gametes to the exter-
nal environment.
AGNATHA
The agnatha consist of the lampreys (Petromyo-
nidae) and hagfish (Myxinoidae). Presumably due to
the difficulty in obtaining specimens and lack of com-
mercial interest in these fish, few studies are available
on spermatogenesis in this group of vertebrates.
Lampreys spend a number of years as larvae, called
ammocetes (which is a nonparasitic feeding stage) be-
fore they metamorphose into mature animals. Al-
though gender is established in the larval stage, go-
nadal growth is postmetamorphosis and in some
species is accomplished in a short period of time, i.e.,
6-9 months. Mitotic activity of germ cells begins fol-
lowing metamorphosis, and cysts develop (Larsen,
1965; Weisbart et al., 1978). As spermatogenesis pro-
ceeds, the testis becomes a mass of cysts containing
spermatozoa. The mature testis eventually fills the
body cavity since, as lampreys spawn once and then
die, other body organs atrophy.
Most myxinoid species do not possess a discrete
breeding season since they live in a constant environ-
ment in the sea at 50-100 meters or more. Hagfish
juvenile males are progynous because during sexual
differentiation the anterior portion of the gonad under-
464 J. PUDNEY
Fig. 5. Polyspermatocystic testis of Myxine sp.
goes early stages of oogenesis. The posterior region of
the gonad remains sexually indifferent until, a t a late
stage in growth, cysts develop containing spermato-
gonia. At this phase the anterior ovarian structures
generally undergo degeneration.
In both myxinoids and petromyzonids the testis is
composed of a mass of cysts, i.e., polyspermatocystic
(Grier, 1992; Fig. 5). Sperm ducts are lacking in ag-
nathans and spermatozoa are shed into the body cavity
where they exit to the outside via the abdominal pore.
In myxinoids, germ cell development is synchronous
within individual cysts although different stages of
spermatogenesis can be found between cysts (Dodd and
Sumpter, 1984). An early light microscope study on
spermatogenesis in Myxine glutinosa is available (Cun-
ningham, 1891). It has been reported that the most
primitive of polyspermatocystic testis occurs in the
hagfish Eptatretus stouti (Grier, 1993). This is a me-
senteric type of testis with regions containing sperma-
tocysts which are interconnected by bridges of tissue
identical to the mesorchium. Nascent cysts are derived
from isolated spermatogonia and Sertoli cells lying free
within the interstitial tissue (Tsuneki and Gorbman,
1977). Prior to meiosis a basement membrane is pro-
duced to isolate Sertoli cells and spermatogonia from
the interstitial tissue, resulting in the formation of
cysts.
Few electron microscopic studies have been carried
out on the agnathan testis and so information on the
ultrastructure of spermatogenesis is lacking. Several
reports, however, are available on the fine structure of
spermatozoa in a few lamprey species, Lampetra fluui-
atilis (Nicander and Sjoden, 1971) and Lampetra plan-
eri (Follenius, 1965; Stanley, 1967). These descriptions
are extremely similar for both species. The long nu-
cleus is rod-shaped and possesses an acrosomal com-
plex. This complex consists of a simple acrosomal ves-
icle positioned on the tip of the nucleus, a dense
subacrosomal ring and an axial nuclear canal lined by
invaginated nuclear membrane that runs the length of
the nucleus and into the flagellum. This endonuclear
canal contains an undulating central fiber that extends
into the flagellum where it is bound by apposing inner
and outer nuclear membranes. Apparently, during fer-
tilization, an acrosome reaction occurs, resulting in the
extrusion of the long, preformed central fiber. This fi-
ber, enclosed by the plasma membrane of the sperma-
tozoon, has been referred to as the acrosomal tubule or
filament (Nicander and Sjoden, 1971).The extension of
the central fiber is thought to be involved in sperma-
tozoal penetration of the egg. Lamprey spermatozoa do
not possess a discernible midpiece. The nuclear im-
plantation fossa contains 2 centrioles both aligned
nearly parallel with the longitudinal axis of the sper-
matozoon. One centriole forms the basal body, from
which the axoneme of the flagellum develops, while
the other remains as an accessory centriole. The fla-
gellum contains a typical axoneme consisting of a 9 -t2
arrangement of doublets. The doublets possess coarse
fibers apposed to their outer surfaces. A number of
elongate mitochondria are located along the proximal
region of the flagellum.
CHONDRICHTHYES
The cartilaginous fishes are composed of two
unequal subclasses, the Elasmobranchii (sharks, dog-
fish, skates, rays) with approximately 600 species and
the Holocephali (ratfish) with only 28 known species.
As for agnathans this group of vertebrates has
received little attention from reproductive biologists.
The detailed process of spermatogenesis is, therefore,
only known for a few species of elasmobranchs and
even less for holocephalans.
It is apparent from the literature that cyst develop-
ment and spermatogenesis, at least as reported at the
light microscope level, is fairly similar for several dif-
ferent species of chondrichthyans: Cetorhinus maximus
(Matthews, 1950); it should be mentioned that in this
account an error in observation was made by Matthews
in that cells described as “spermatogonia” represent
Sertoli cells while the “primary spermatocytes” corre-
spond to spermatogonia: Hydrolagus colliei (Stanley,
1963);Scyliorhinus caniculus (Dobson and Dodd, 1977;
Mellinger, 1965; Stanley, 1966); Torpedo marmorata
(Stanley, 1966); Squalus acanthias (Callard et al.,
1985; Holstein, 1969) and Heterodontus portusjacksoni
(Jones and Jones, 1982). The nuclear changes associ-
ated with germ cell development and maturation have
been described for several species of elasmobranchs
(Moore, 1895) In S. acanthias, gonocytes and undiffer-
entiated Sertoli cell precursors are located in the ger-
minal zone on the rostra1 aspect of the testis. Nascent
cysts form when these Sertoli cells engulf primary
spermatogonia within cytoplasmic processes (Fig. 6A).
According to the extracellular matrix hypothesis
(Grier, 19921,spermatocysts are not produced until the
basement membrane is formed which isolates the Ser-
toli/germ cell unit from the interstitial tissue. By the
time this happens both cell types have undergone sev-
eral mitotic divisions resulting in the formation of a
spermatocyst consisting of a single layer of Sertoli cells
and secondary spermatogonia (Fig. 6B). Cysts then be-
 SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES
Fig. 6. Germinal zone of S . acunthias testis showing cysts a t var-
ious stages of development. A: Cysts form when a Sertoli cell (arrow)
engulfs a gonocyte (arrowhead). Nascent cysts are composed of pri-
mary spermatogonia (P) surrounded by Sertoli cells (S).B: Fully
come attached to the system of sperm ducts in the testis
(Fig. 6B).
Sertoli and germ cells undergo repeated mitotic di-
visions to form a mass of cells within the cyst, which
now contains a lumen. These cells eventually become
organized into a peripheral layer of secondary sper-
matogonia surrounded by Sertoli cytoplasm with Ser-
toli nuclei bordering the lumen of the cyst (Fig. 7A).
For T. marmorata, however, Sertoli and germ cells
form less conspicuous concentric layers (Stanley, 1966).
At this stage the number of secondary spermatogonia
approximates the number of Sertoli cells in the cyst.
Sertoli cells now cease dividing, which heralds the ap-
pearance of spermatoblasts within the cyst. This occurs
when each Sertoli cell encompasses a single sper-
matogonium with cytoplasmic processes (or possibly a
clone of spermatogonia connected by intercellular
bridges) which effectively isolates germ cells from each
other. The spermatogonia continue to divide, causing
the cyst to increase in size. Identifiable spermatoblasts,
consisting of a Sertoli cell plus its cohort of isogeneic
germ cells, can now be identified in cysts (Fig. 7B).
During this period, Sertoli nuclei enlarge and move
from their adlumenal position towards the periphery of
the cyst wall. By the time this migration of Sertoli
nuclei has been accomplished meiosis has occurred and
spermiogenesis proceeds.
It is interesting to note that during the initial devel-
opment of cysts, mitotic divisions appear to occur
within groups of spermatogonia (Fig. 7A). These
groups could possibly represent clones of germ cells
connected by cytoplasmic bridges. During meiosis,
however, division appears to be initiated by a group of
germ cells and then proceeds as a wave to involve other
germ cells present in the cyst (Fig. 7C).
465
formed cyst with a lumen and basal layer of secondary spermatogonia
and Sertoli cells. One cyst (C) has become attached to the system of
sperm excurrent ducts (arrowhead).
During spermiogenesis there is a great deal of trans-
location of germ cells within the spermatoblast. This
eventually results in a single layer of spermatids lining
the inner face of the spermatoblast with their tails pro-
jecting into the spermatoblast cavity (Fig. 7D,E). How
this movement of germ cells within the spermatoblast
is accomplished is unknown. The cyst still possesses a
patent lumen which is bound by the apical margins of
the Sertoli cells. During spermiogenesis, spermatids
elongate and begin to form loose bundles within the
spermatoblast with their tails now projecting into the
lumen of the cyst (Fig. 7F). Spermatids progressively
form tighter bundles as they mature, until a t the end of
spermiogenesis they have aggregated into a closely
packed mass located in a pocket of apical Sertoli cyto-
plasm (Fig. 7G-I). Spermiation involves release of the
mature gametes along with the apical portion of Sertoli
cytoplasm. Spermatozoa exit the cyst via the sperm
duct which has now acquired a patent opening. Follow-
ing spermiation the Sertoli cells forming the cyst un-
dergo fatty degeneration. There are species-specific
variations in chondrichthyan testicular organization
(Hoar, 1969). This includes major differences in the
location of the germinal zone and consequently migra-
tion of spermatocysts through the testis for different
species (Pratt, 1988).
Quantitative estimates of spermatogenesis are facil-
itated in chondrichthyans since germ cells can be eas-
ily identified and hence counted in cysts as they ma-
ture. The number of divisions spermatogonia undergo
are species-specific. Thus, S. acanthias has 13 sper-
matogonial divisions, two of them type A and eleven
involving type B spermatogonia (Holstein, 1969). The
number of mitotic divisions carried out by both Sertoli
cells and spermatogonia are fixed, resulting in cysts
466 J. PUDNEY

Fig. 7.
 SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES 467
containing a specific number of germ cells and Sertoli through the testis. Since the zone represents an ab-
cells. Initially, mitotic divisions are synchronized be- sence of germ cells, when the next breeding season oc-
tween spermatogonia and Sertoli cells through the first curs there is a short arrest in spermatogenic activity.
9 divisions (Stanley, 1966). Sertoli cells then cease di- In contrast, a zone of degeneration is absent from S.
viding, at which time the cyst contains approximately tiburo (Parson and Grier, 1992). For this shark there is
500 of these cells. Spermatogonia continue to undergo widespread complete regression of the testis which only
four more cycles of mitoses before they enter meiosis. contains spermatogonial cysts during the period of tes-
The end product of all these divisions is 64 spermatids ticular involution.
associated with each Sertoli cell (Stanley, 1966). This Few electron microscopic studies have been carried
results roughly in 32,000 spermatozoa per mature cyst. out on the chondrichthyan testis. Ultrastructurally,
For Sphyrna tiburo, it has been estimated that a ma- the primary spermatogonia of S. acanthias are
ture spermatocyst contains approximately 460 sperma- characterized by large, spherical, pale-staining nuclei
toblasts, each possessing 64 spermatozoa resulting in with few chromatin clumps and a paucity of organelles
29,000 spermatozoa per spermatocyst (Parsons and in the cytoplasm. Secondary spermatogonia appear
Grier, 1992). smaller in size with irregularly shaped, dark staining
A rare case of germ line chromatin diminution has nuclei containing many clumps of chromatin and
been reported to occur during spermatogenesis in H . possess many mitochondria, plus profiles of both
colliei (Stanley et al., 1984). At metaphase I in meiosis smooth (SER) and rough (RER) endoplasmic reticulum
of primary spermatocytes, a mass of heterochromatin (Fig. 8). Cytoplasmic bridges were observed connecting
accumulates at the side of the metaphase plate. This groups of secondary spermatogonia (Fig. 8). These
clump of heterochromatin is eventually passed into one connections are maintained by clones of germ cells
of the secondary spermatocytes during cytokinesis at throughout spermatogenesis (Fig. 9). Small desmo-
anaphase I. As the nuclear membrane is restored, a some-like junctions were often detected between
double membrane (possibly of nuclear origin) forms spermatogonia and Sertoli cells. At later stages of
around the mass of heterochromatin which is then spermatogenesis, however, no junctional specializa-
termed the chromatin diminution body. By the end of tions were observed between Sertoli cells and germ
the second meiotic division this body ends up in the cells. Primary spermatocytes appear cytologically
cytoplasm of one of the four spermatids where it re- more differentiated than spermatogonia. They possess
mains unchanged until midspermiogenesis. At this large spherical nuclei with finely dispersed chromatin
time the chromatin body is discarded by means of apo- and the cytoplasm contains numerous mitochondria,
crine exocytosis and is engulfed by the Sertoli cell. cisterns of SER, a well-developed Golgi apparatus as
Seasonal changes in spermatogenesis in elasmo- well as an assortment of vacuoles and dense bodies
branchs have been reviewed (Dodd and Sumpter, 1984; (Fig. 9). An occasional lipid droplet and centriole were
Parsons and Grier, 1992; Wourms, 1977). Due to the observed in some spermatocytes. An excellent detailed
difficulty in obtaining sufficient numbers of specimens study of the fine structure of spermiogenesis has been
throughout the reproductive cycle, few species, mainly carried out for S. suckleyi (Stanley, 1971a,b).
restricted to sharks, e.g., S. acanthias (Simpson and Round spermatids contain spherical nuclei with fine
Wardle, 1967)S. tiburo (Parsons and Grier, 1992),have granular chromatin. A number of mitochondria and a
been completely and adequately studied. For many spe- well-developedGolgi apparatus as well as an extensive
cies a seasonal cycle of spermatogenesis manifests it- system of SER is present in the cytoplasm (Fig. 10A).
self by the development of a zone of breakdown. This There appears to be a distinct polarization of some or-
zone, consisting of degenerating cysts, was first de- ganelles such as the Golgi apparatus which appear to
scribed in S. acanthias (Simpson and Wardle, 1967). be located adjacent to the developing flagellum (Fig.
This zone develops annually, in the spring, due to sper- 10B). A bundle of juxtanuclear filaments is commonly
matogonial degeneration and proceeds to migrate detected in round spermatids (Fig. lOC). Interestingly,
the nuclear envelope next to the Golgi apparatus
exhibits structural modifications, related to adhesion
of the acrosome to the nucleus, before the appearance of
Fig. 7. Cyst development in the testis of S. acanthias. A: Mitotic
the acrosomal vesicle (Stanley, 1971a). In this region
activity of both germ cells and Sertoli cells results in growth of the the nuclear membranes become closely apposed and
cyst which is now composed of several basal layers of secondary sper- hence appear dense in appearance under the electron
matogonia surrounded by Sertoli cytoplasm. Nuclei of the Sertoli cells microscope (Fig. 10D). This membrane modification,
(arrowheads) form a distinct layer bordering the lumen. Note: mitotic therefore, is the initial event that gives nuclear polar-
divisions appear t o occur amongst distinct groups of germ cells. B:
Spermatoblast formation occurs when the cytoplasm of a single Ser- ity and is unusual since normally this results from the
toli cell (arrowheads) surrounds and isolates a cohort of secondary formation of the acrosome. The acrosomal vesicle de-
spermatogonia. C Meiosis occurs as a wave that progresses around velops from the Golgi apparatus located close to the
the cyst. D: and E: Spermatoblasts sectioned longitudinally (D) and developing flagellum and becomes attached to the
transversely (E) containing a cohort of isogeneic spermatids in the
lumen. Arrowheads indicate the Sertoli cytoplasm forming the walls dense nuclear membrane (Fig. 11A). As the acrosome
of the spermatoblast. F-I: These micrographs illustrate the progres- vesicle enlarges to form the acrosome it actually in-
sive condensation of elongating spermatids (F)into loosely organized dents upon the nucleus which accommodates by form-
bundles with their heads orientated toward the basement membrane ing a shallow depression (Fig. 11B). An acrosomal
of the cyst (G) that coalesce into closer packed bundles (HI to even-
tually form a tight mass of mature spermatids embedded in Sertoli granule does not appear to develop (Fig. 11B). The nu-
cytoplasm (I). clear envelope continues to condense over the surface of
468 J. PUDNEY

Fig. 8. Secondary spermatogonia possess irregularly shaped nuclei with clumps of chromatin and
contain numerous organelles and are connected by cytoplasmic bridges (arrowhead) S . acunthias.

the nucleus while at the same time fibrous material
becomes deposited along the outer nuclear membrane
to form the fibrous nuclear sheath (Fig. 1lC). Eventu-
ally this sheath will cover most of the nucleus except
for the posterior pole. There also appears to be a dis-
tinctive pattern of chromatin condensation as sperma-
tids mature. This begins with aggregation of the chro-
matin into coarse granules (see Fig. 10A) which
possibly is initiated in the anterior pole of the nucleus
beneath the acrosome. This process continues but
seems to be limited to the central region of the nucleus,
resulting in a peripheral zone of nucleoplasm virtually
devoid of chromatin granules (see Figs. 10A and 1 1 0 .
At a later stage of nuclear condensation, the chromatin
granules become transformed into a mass of randomly
orientated short chromatin fibers (see Fig. 14A).
Initially the centrioles lie close to the Golgi appara-
tus and near the periphery of the cell (Fig. 11A). For S.
suckleyi it was reported that as the acrosome develops,
the nucleus undergoes a rotation of approximately 180"
that results in the acrosome becoming located at
roughly the opposite pole within the cell in respect to
the developing flagellum (Stanley, 1971a). This now
defines the anterior pole of the cell, containing the ac-
rosome, and posterior pole containing the flagellum. At
this stage two filamentous bundles, one striated and
one nonstriated, become associated with both distal
and proximal centrioles (Fig. 12A). These fibrous bun-
dles are precursors of the axial midpiece rod (a charac-
teristic feature of chondrichthyan spermatozoa) that
will eventually connect the spermatid nucleus to the
developing flagellum. A dense material is also depos-
ited around the flagellum where it projects from the
spermatid that presumably represents the ring centri-
ole or annulus (Fig. 12A). For S. acanthias, however, it
appears that nuclear rotation occurs at a later stage of
spermatid differentiation since the fibrous bundles,
plus a developing flagellum, could still be detected in a
lateral position (with respect to the nucleus) within the
cell (Fig. 12A). Following nuclear rotation, these fi-
brous bundles increase in length to eventually insert in
a deep fossa located at the posterior pole of the nucleus
(Fig. 12B). With further elongation of the spermatid
the bundles of filaments forming the axial component
of the midpiece become twisted upon each other. This
spiraling, however, appears to only affect the nonstri-
ated fibrous bundle which wraps itself around the stri-
ated bundle of filaments (Fig. 12C). A characteristic
feature of round spermatids is the formation of a long
cytoplasmic projection (Fig. 13A).This process contains
 SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES 469

Fig. 9. Primary spermatocytes have round nuclei with evenly dispersed chromatin, abundant or-
ganelles and are connected by cytoplasmic bridges (arrow). Also present in this micrograph is a portion
of the Sertoli cytoplasm (arrowheads) forming the walls of the spermatoblast S. acunthius.

a system of fine filaments with an accumulation of
dense material a t the tip. Often these projections can
be seen to indent the Sertoli cytoplasm of the sperma-
toblast (Fig. 13B). These structures, therefore, would
seem to act as some kind of anchoring device.
As the spermatid develops the nucleus becomes elon-
gated. The acrosome now forms an elongated cap over
the pointed tip of the nucleus (Fig. 14A). Interestingly,
the outer acrosomal membrane is closely associated
with what appears to be a number of electron-dense
bodies that are evenly spaced along this membrane. A
short, subacrosomal rod (perforatorium) develops to oc-
cupy the space between the nucleus and acrosome (Fig.
14A). The subacrosomal rod is attached at the base of
the acrosome and extend towards, and slightly beyond
the tip of the nucleus. The nuclear chromatin has now
condensed to form a mass of coarse strands orientated
along the longitudinal axis of the nucleus (Fig. 14A).
During nuclear condensation the nuclear volume de-
creases from about 180 p3 to about 10 p3 and the sur-
face area from around 154 p.' to 58 p2 (Stanley, 1971b).
It was proposed that the excess nuclear envelope was
discarded by the release of vesicles from the posterior
margin of the nuclear membrane. For S. acanthias the
posterior region of the nucleus is dilated and devoid of
chromatin and encloses the proximal portion of the fi-
brous bundles (see Fig. 12B). Moreover, the inner nu-
clear membrane facing the fibrous bundle is rich in
nuclear pores (see Fig. 12B). This development is rem-
iniscent of the redundant nuclear envelope present in
mammalian spermatozoa.
With further maturation of the spermatid, the elon-
gate nucleus assumes a helical shape and a t this time
the fibrous nuclear sheath disappears. This spiralling
of the nucleus also includes the chromatin which be-
comes orientated into a helical configuration (Fig.
14B). This helical pattern begins posteriorly to ad-
vance anterially and is accompanied by a transient pro-
gressive loosening of the nuclear membrane adjacent to
the chromatin just acquiring a spiral pattern (Fig. 14B;
Stanely, 1971b).
As nuclear elongation and chromatin condensation is
occurring, the two fibrous elements of the axial mid-
piece rod fuse. At the same time elongate mitochondria
aggregate around the axial midpiece rod to form a mi-
tochondrial sheath that now delineates the midpiece.
These mitochondria are associated with glycogen gran-
ules and are enclosed by a fibrous midpiece sheath. The
posterior end of the midpiece rod inserts on the basal
body (distal centriole) of the flagellum. At doublets 3
and 8 the fibrous material surrounding the centrioles
extends down as two longitudinal columns and accom-
panies the axoneme the length of the flagellum (Fig.
15). As spermatids elongate, the cytoplasm is displaced
posteriorly and is reflected to form a sleeve of cyto-
plasm surrounding the proximal portion of the tail. For
470 J. PUDNEY

Fig. 10. Spermiogenesis in testis of S . acanthias. A Round sper-
matids with spherical nuclei and abundant organelles present in lu-
men of the spermatoblast. Note the pattern of chromatin condensa-
tion which occurs centrally within the nucleus leaving a peripheral
ring of nucleoplasm virtually devoid of chromatin granules. B: Golgi
apparatus associated with the developing flagellum. C : A long bundle
of filaments present in the cytoplasm of round spermatids. D Nuclear
membrane adjacent the Golgi apparatus condenses (arrowheads) and
becomes modified for attachment of the acrosome. Arrows point to
what appear to be acrosomal vesicles budding from the Golgi appa-
ratus S. acanthias.
 SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES 471

Fig. 11. Spermiogenesis in S. acanthias. A: Acrosome initially develops in the vicinity of the devel-
oping flagellum. B Acrosomal vesicle attached to nucleus lacks an acrosomal granule. C:Fibrous sheath
(arrowheads) deposited along outer surface of nucleus. Note the pattern of chromatin condensation into
a mass of coarse granules that occurs centrally producing a clear peripheral zone of nucleoplasm.

some species this remnant cytoplasm is discarded dur-
ing spermiation, e.g., S. suckleyi (Stanley, 1971b)
whereas for others it is retained, e.g., H . portusjacksoni
(Jones et al., 1984). During the process of spermiogen-
esis the early round spermatids are located in the lu-
men of the spermatoblast. As spermatids undergo elon-
gation, however, they become embedded within
individual recesses formed by Sertoli cytoplasm. At
this stage the 64 spermatids are loosely arranged but
as the germ cells mature they move closer and closer
together to eventually form a compact bundle that ap-
parently resides in a single pocket formed by Sertoli
cytoplasm (Fig. 15).
The mechanisms involved in bundle formation of
chondrichthyan spermatozoa has been investigated in
the ratfish H . colliei (Stanley and Lambert, 1985). It
was shown that when elongate spermatids indent the
Sertoli cell to occupy a cytoplasmic recess, filaments
present in Sertoli cytoplasm become concentrated
around the developing germ cell. As spermatids elon-
gate further, these filaments condense into bundles
which become aligned parallel to the long axis of each
spermatid and extend down to the base of the Sertoli
cell. Eventually a filamentous sheath is formed imme-
diately surrounding the acrosome and anterior nuclear
region of the spermatid. It was suggested that a close
transmembrane adherence existed between the Sertoli
cell filaments and tip of the spermatid acrosome.
Through the use of NBD-Phallacidin and specific anti-
bodies to actin and myosin it was shown the filamen-
tous sheath contained both contractile proteins. This
suggested that during the formation of spermatid bun-
dles the system of acto-myosin represented a contrac-
tile structure involved in the orientation of spermatid
nuclei towards the periphery of the spermatoblast. It
was postulated these filaments were analogous to those
developed between Sertoli cells and spermatids in the
mammalian testis. Initially described by Brokelmann
(1961) and called junctional specializations (Flickinger
and Fawcett, 19671,they are now commonly referred to
as ectoplasmic specializations (Russell, 1977). Compac-
tion of the spermatids into tight bundles was thought
to occur by a mechanism of endocytosis of Sertoli
plasma membrane located between the germ cells. This
uptake of Sertoli plasma membrane would cause the
spermatids to move closer together, resulting in their
coalescence. Stability of the individual spermatids, as
this process occurred, was maintained by an extracel-
lular dense material present between the tip of the
acrosome and Sertoli plasma membrane which was
thought to act as an adhesive substance.
A recent study has reported on the fate of mitochon-
dria during spermiogenesis in H . colliei (Stanley and
Lambert, 1990). Apparently there is fragmentation of
elongate mitochondria into smaller mitochondria.
These mitochondria then either migrate to surround
the midpiece of the developing spermatid or become
enclosed in large vacuoles. These vacuoles are limited
by a double membrane (with ribosomes along the inner
membrane). The vacuoles containing excess mitochon-
472 J. PUDNEY

Fig. 12. Development of filamentous bundles in spermatids of S.
acanthias. A Fibrous bundles associated with developing flagellum.
The dense material (arrowheads) deposited at the cell membrane in
the vicinity of the flagellum represents the ring centriole or annulus.
Note that in this spermatid, nuclear rotation has not yet occurred
since although the acrosome is well-developed the flagellum projects
laterally from the cell and has not yet moved to the posterior pole of
the nucleus. B The striated (arrowheads) and nonstriated bundles of
filaments insert in a deep fossa at the posterior pole of the nucleus.
Note that the posterior region of the nucleus that surrounds the fila-
mentous bundles is dilated, devoid of chromatin with numerous nu-
clear pores in the inner membrane that faces the bundles of filaments.
C: This micrograph shows the nonstriated bundle of filaments (arrow-
heads) is twisted around the striated bundle of filaments.
 SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES 473

Fig. 13. A characteristic feature of spermatids is the formation of a long cytoplasmic process. A: This
process contains an array of fine filaments. B: Often these processes indented the Sertoli cytoplasm
forming the wall of the spermatoblast and may represent an anchoring device S. ucanthius.

dria are eliminated and phagocytosed by the Sertoli
cell.
The morphology of chondrichthyan spermatozoa is
conservative and appears similar for those species
which have been studied, e.g., the sharks S. suckleyi
(Stanley, 1965a, 1964,1971b);H . portusjacksoni (Jones
et al., 1984); Prionace glauca, Carchurhinus falci-
formis, Centroscymnus owstoni and Chlamydoselachus
anguineus (Hara and Tanaka, 1990); rays Dasyatis ku-
hlii and Dasyatis garouaensis (Hara and Tanaka, 1990)
and the chimaera H . colliei (Stanley, 1965a, 1983) and
Chimaera phantasma (Hara and Tanaka, 1990). The
acrosome tends to be small and elongated with a deep
posterior indentation that fits loosely over the pointed
tip of the nucleus. The long nucleus is attached to the
flagellum via the midpiece which contains the mid-
piece rod surrounded by a small number of mitochon-
dria. The anterior of the midpiece rod inserts into the
nuclear implantation fossa while the centriole complex
is positioned at the posterior end of the midpiece rod.
Unlike many other vertebrates, therefore, flagellar el-
ements are not present in the midpiece of chondrich-
thyan spermatozoa. The flagellum is composed of a typ-
ical axoneme consisting of a 9 + 2 array of doublets plus
a characteristic feature of chondrichthyan spermato-
zoa, the longitudinal columns (Fig. 15).
Species differences in spermatozoon morphology pri-
marily involve flagellar structures. Thus, in H . colliei
it was reported that the proximal centriole is absent
from the basal body (Stanley, 1965a, 1983).Major vari-
ations, however, involve the number and appearance of
the longitudinal columns (Hara and Tanaka, 1990). In
sharks the flagellum is symmetrical with a central ax-
oneme and two equal-sized, oval longitudinal columns
a t doublets 3 and 8. Spermatozoa of rays also have two
longitudinal columns but these are round in cross-sec-
tion. For chimeric species, however, the development of
the longitudinal column adjacent doublet 8 is sup-
pressed, appearing only as a short rod, whereas the one
associated with doublet 3 extends the length of the fla-
gellum (Stanley, 1983; Hara and Tanaka, 1990). This
arrangement results in an asymmetric axoneme in
spermatozoa of the chimera. Glycogen granules appar-
ently are commonly present in chondrichthyan sper-
matozoa. For elasmobranchs this occurs as a small
number of granules dispersed between the mitochon-
dria (Stanley, 1971b). In H. colliei, however, abundant
glycogen granules were detected along the length of
the flagellum (Stanley, 1983).
A characteristic feature of chondrichthyan sperma-
tozoa is their helical shape. The long, pointed head ap-
pears spirally twisted giving it a corkscrew appear-
ance. This helical twisting also involves the acrosome,
and elements of both the midpiece, and flagellum. In S.
acanthias the axoneme is straight but the microtubules
of the doublets follow a helical course (Stanley, 1971b).
By contrast, in H . colliei both axoneme and microtu-
bules exhibit a spiral pattern along the length of the
flagellum (Stanley, 1983). How spiralization of these
components of the spermatozoa is accomplished is un-
known. The helical configuration of the head and
flagellar elements in chondrichthyan spermatozoa are
related to motility. Forward progression in these sper-
matozoa is not achieved by lateral bending of the fla-
gellum but by rotating around their long axis. The
function of the longitudinal columns in chondrichthyan
474 J. PUDNEY

Fig. 14. Spermiogenesis in S. acanthias. A Acrosome forms a loose cap over the elongated tip of the
nucleus and a perfortorium (arrowhead) develops in the subacrosomal space. Note chromatin is organized
as a mass of fibers orientated parallel to the long axis of the nucleus. B Elongate spermatid showing
helical pattern of chromatin fibers and loosening of the nuclear membrane adjacent regions of chromatin
acquiring a spiral configuration.

spermatozoa is unknown although it has been sug-
gested they may restrict lateral movement of the fla-
gellum allowing the spermatozoa to progress by revolv-
ing about its long axis (Stanley, 1971b).
OSTEICHTHYES
The osteichthyes are composed of two classes of bony
fishes, the Crossopterygii and Actinopterygii. The
Crossopterygii include the Dipnoi (lung fishes) and Co-
elacanthini, represented by a living fossil, the coela-
canth (Latimeria). The Actinopterygii consist of the
Chondrostei (sturgeons), Holostei (bowfin, garpike)
and the Teleostei. Of all these groups, the Teleostei are
by far the most important and extensive with more
than 20,000 extant species. Despite the economic im-
portance of teleosts and their ready availability as re-
search subjects, the reproductive biology of relatively
few species has been adequately studied.
The bony fishes are a very diverse group of verte-
brates with extremes of reproductive strategies rang-
ing from external to internal fertilization. This results
in great variation in the organization of the testis. Due
to this, the nomenclature describing the different
structural arrangements of the teleost testis has been
somewhat confusing and inconsistent (Grier, 1981). To
rectify this condition a more uniform nomenclature has
been proposed, based on the distribution of spermato-
gonia within the seminiferous compartment (Grier et
al., 1980; Grier, 1981, 1993). The teleost testis is orga-
nized as a system of either anastomosing tubules, pos-
sibly restricted to lower fish or lobules present in other
species (Grier, 1993). Furthermore, both types of testes
can be further defined by the distribution of spermato-
gonia within the tubules or lobules (Grier et al., 1980;
Grier, 1981). First, there is the unrestricted sper-
matogonial testis type characterized by the presence of
spermatogonia along the entire length of the tubule or
lobule (Fig. 16A). During the nonbreeding season, pri-
mary spermatogonia and Sertoli cells occur as solid
cords within the tubule or lobule which contains a ves-
tigial lumen. With the onset of spermatogenesis, cysts
develop and line the tubules or lobules which now have
a patent lumen. Spermiation results in the breakdown
of the Sertoli cells which form the walls of the cyst to
release spermatozoa into the lumen. This unrestricted
spermatogonial testis type is found in most species of
teleost.
The second pattern of organization found in teleosts
is the restricted spermatogonial testis type. This de-
scribes the situation where primary spermatogonia are
confined to the distal region of the lobule only (Fig.
16B). During spermatogenesis the primary spermato-
gonia associate with Sertoli cells to form cysts. As germ
cells mature, cysts migrate down the lobule toward the
efferent sperm ducts. By the time spermatozoa have
formed, the cyst is located near the sperm duct. Sper-
 SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES
Fig. 15. Cross-section through a bundle of mature spermatids lo-
cated in a pocket formed by the Sertoli cytoplasm. This section passes
through the axoneme illustrating the presence of the longitudinal
columns (arrowheads) located at doublet 3 and 8. S. acunthias.
miation ensues with release of spermatozoa into the
sperm duct. Sertoli cells, depending on the species, ei-
ther degenerate or become incorporated into the epi-
thelium of the efferent ducts. The restricted sper-
matogonial testis type is only found in one group of
teleosts, the Atheriniformes. Although Atheriniforme
species all possess a common testicular structure, there
is great diversity in their reproductive biology depend-
ing upon whether fertilization is external or internal.
Atherinimorph teleosts practicing external fertiliza-
tion include Fundulus heteroclitus (Selman and Wal-
lace, 1986) and Oryzias latipes (Gresik et al., 1973). For
these species, spermatogenesis is a fairly simple pro-
cess. Primary spermatogonia occur at the blind end of
the lobule and undergo several mitotic divisions before
becoming associated with Sertoli cells to form cysts. As
cysts mature they move down the lobule towards the
efferent sperm ducts. Unlike other teleost species, dur-
ing maturation of these cysts, there is little differenti-
ation of Sertoli cells which remain remarkably unde-
veloped through the process of spermatogenesis. At the
completion of spermatogenesis, cysts are located in the
lobular region proximal to the efferent ducts. The Ser-
toli cells, forming the cyst, then fuse with epithelial
cells lining the efferent ducts which results in release
of the contained spermatozoa.
Those atherinimorph species which engage in inter-
nal fertilization demonstrate a more complex process of
spermatogenesis. An adaptation for this reproductive
475
Fig. 16. Schematic diagrams illustrating spermatogenesis in the
Osteichthyes. A: Unrestricted spermatogonial testis type character-
ized by the presence of spermatogonia (PG) along the length of the
lobule. Cysts form when a Sertoli cell (SC) surrounds a primary sper-
matogonium. With the onset of spermatogenesis, cysts (C) mature and
line the lobule. Spermiation occurs with rupture of the cyst and lib-
eration of spermatozoa (SP)into the lobule lumen. B: Restricted sper-
matogonial testis type where spermatogonia (PG) are confined to the
distal end of the lobule. Cysts form when a Sertoli cell (SC) surrounds
a primary spermatogonium. As cysts (C) mature they migrate down
the lobule towards the efferent ducts (ED). By the time cysts reach the
efferent duct they contain spermatozoa. Spermiation results when the
cyst fuses with the efferent duct. (Adapted from Grier, 1993.)
strategy is the production of spermatozoa packaged ei-
ther in naked (spermatozeugmata) or encapsulated
(spermatophores) bundles which are introduced into
the female at insemination. This results in complex
morphological relationships developing between sper-
matids and Sertoli cells not seen in atherinimorph spe-
cies with external fertilization, e.g., Lebistes reticula-
tus (Vaupel, 1929) Poecilia reticulata (Billard, 1970a)
Poecilia latipinna (Grier, 1975a) Mollienisia latipinna
(Van den Hurk et al., 1975). Only one atherinimorph
species, Horaichthys setnai, produces true spermato-
phores, whereas all others develop spermatozeugmata
(Grier et al., 1981). Spermatophores develop within
cysts and the walls of these structures, which encapsu-
late the spermatozoa, are formed by a Sertoli cell se-
cretion. The process of spermatophore formation has
been described for H . setnai (Grier, 1984).Briefly, sper-
matogenesis is initiated and proceeds in a similar fash-
ion to that occurring in species with external fertiliza-
tion until the stage of spermiogenesis is reached. At
this time the Sertoli cell hypertrophies, progressively
acquiring a columnar shape, and becomes highly secre-
tory. The released secretion then self-assemblesto form
the capsule of the spermatophore which surrounds the
spermatozoa within the cyst. Rupture of the cyst occurs
476 J. PUDNEY

with transfer of spermatophores to the efferent ducts.

The remaining Sertoli cells either become incorporated
into the efferent ducts or are sloughed and degenerate.
Spermatozeugonata formation has been reported for
Lebistes reticulatus (Winge, 1922; Vaupel, 1929) Po-
ecilia latipinna (Grier, 1975a) and Mollienisia latipinna
(Van den Hurk et al., 1975). As for other atherini-
morphs, spermatogenesis proceeds normally up to sper-
miogenesis. During this phase the Sertoli cells hyper-
trophy, acquire a columnar shape and in these poeciliid
species spermatids align themselves with their heads
abutting the apical Sertoli cytoplasm to form the sper-
matozeugmata. For goodeide, however, i t is the devel-
oping flagellum that becomes associated with Sertoli
cells, e.g., Ameca splendens, Ataenobius toweri, Chara-
codon lateralis, Xenotoca eiseni (Grier et al., 1978).
Spermatozoa are not held together by junctional spe-
cializations with Sertoli cells. Instead, Sertoli cell pro-
jections anchor spermatozoa at the lumenal margin of
the cyst. When the mature cyst contacts the efferent
duct, spermiation occurs and the contained spermato-
zeugmata are released.
Seasonal reproduction is common for teleosts and the
testis may exhibit large variations in size and histolog-
ical appearance. The length of time following comple-
tion of spermatogenesis and spawning may vary from
species to species (Borg, 1982; Ruby and McMillan
1970) or may occur directly following production of
spermatozoa (Henderson, 1962). For some tropical spe-
cies of teleosts there is no apparent seasonal fluctua-
tion in spermatogenesis. There is diversity in the germ
cells composing the regressed teleost testis. For many
species the quiescent testis contains mainly spermato-
gonia (Shrestha and Khanna, 1978); for others either
primary spermatocytes (Ahsan, 19661, spermatids
(Rosenblum et al., 1987) or spermatozoa (Borg 1982; Fig. 17. Unrestricted spermatogonial testis type seminiferous lob-
ule during the breeding season with a patent lumen and lined by cysts
Ruby and McMillan, 1970). containing maturing germ cells in the testis of the teleost Pugellus sp.
At the light microscope level the process of spermato-
genesis has been described for a number of teleost spe-
cies, e.g., Ictalurus nebulosus (Rosenblum et al., 1987);
Couesius plumbeus (Ahsan, 1966); Gasterosteus aculea- lobule to describe lobules containing spermatozoa with
tus (Borg, 1982); Eucalia inconstans (Ruby and Mc- few scattered cysts; and storage lobule to describe lob-
Millan, 1970); Tandanus tandanus (Davis, 1977); Sci- ules packed with spermatozoa and containing no cysts
aenops ocellatus (Grier et al., 1987). (Grier et al., 1987).
In general, spermatogenesis is very similar for all Germ cell renewal in the lobules has been investi-
these species of teleosts. For most teleosts spermato- gated in several species. It is generally accepted that a
gonia are the prevalent germ cell present in the re- permanent population of primordial stem germ cells
gressed testis. As recrudescence ensues, spermatogonia exist in the lobule wall. During each annual cycle of
undergo repeated mitotic divisions and eventually spermatogenesis these cells undergo mitosis to produce
become associated with Sertoli cells to form cysts. Sper- spermatogonia. The remaining stem cells provide a res-
matogenesis then proceeds and cysts containing matur- ervoir for future spermatogenic cycles. Other studies,
ing germ cells are distributed along the length of the however, have suggested there is a n annual migration
lobule (Fig. 17). of stem cells into the lobules from a n interstitial loca-
Eventually the cysts become filled with spermatozoa tion. Migratory germ cells have been described in the
which are released into the lumen at spermiation re- interstitial tissues of C. plumbeus (Ahsan, 1966); E .
sulting from rupture of the cyst. Few cysts can now be inconstans (Ruby and McMillan, 1970); and Salmo
detected and the lobules contain scattered spermato- gairdneri (Hurk et al., 1978). None of these studies
gonia which will serve as stem cells for the next cycle were able to identify the original source of these germ
of spermatogenesis. To reflect these changes in sper- cells and the morphological evidence (light micros-
matogenesis, the following terms have been proposed copy), illustrating the entry of germ cells into the lob-
to describe the lobules: production lobules to describe ules is not convincing.
the lobule or part that contains maturing cysts, with or A number of reports are available describing differ-
without mature spermatozoa in the lumen; transitional ent aspects of spermatogenesis in various teleost spe-
 SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES
cies a t the electron microscope level, e.g., Lebistes re-
ticulatus (Asai, 1971; Gronberg and Telkka, 1968;
Gronberg and Wartiovaara, 1972; Mattei and Boisson,
1966; Mattei et al., 1967);Oligocottus maculosus (Stan-
ley, 1969); Upeneus prayensis (Boisson et al., 1969);
Pimephales notatus (Schjeide et al., 1972); Albula
vulpes (Mattei and Mattei, 1973); Gambusia affinis
(Grier, 1975b);Poecilia latipinna (Grier, 1973, 1975a);
Trichurus lepturus (Mattei and Mattei, 1976a); An-
guilla australis schmidtii and A. dieffenbachii (Todd,
1976); Oryzias latipes (Grier, 1976; Hamaguchi, 1987);
Lepodogaster lepadogaster (Mattei and Mattei, 1978a);
Liza aurata (Brusle, 1981); Lepomis macrochirus (Sp-
rando et al., 1988); Salmo gairdneri (Billard, 1972,
1983);Poecilia reticulatus (Billard and Flechon, 1969);
Platichthys flesus (Jones and Butler, 1988a,b); Thy-
mallus thymallus (Lahnsteiner et al., 1991); Rhodeus
sericeus sinensis (Guan and Afzelius, 1991); Blennius
pholis (Silveira et al., 1990);Ophidion sp (Mattei et al.,
1989);8 species of blennidae (Lahnsteiner and Patzner,
1990);Paroncheilus sp (Mattei and Mattei, 1984);Zcta-
lurus punctatus (Poirier and Nicholson, 1982);Brachy-
danio rerio (Kessel et al., 1983); a large number of
cyprinids (Clerot, 1976; Thiaw et al., 1986; Thiaw and
Mattei, 1989); several species of gobies (Fishelson et
al., 1990).
In general, the fine structure of germ cell develop-
ment is very similar for all species studied. The sper-
matogonia are characterized by large regular nuclei
usually containing a distinct nucleolus. In P. latipinna
and P. notatus, spermatogonia contain annulate lamel-
lae and the mitochondria are associated with a dense
material of nuclear origin (Grier, 1975a; Schjeide et al.,
1972). In several species of cyprinids, groups of mito-
chondria were located at specific sites in the cytoplasm
of spermatogonia (Clerot, 1976). These mitochondria
were related with a dense material (cement). Annulate
lamellae were also often detected associated with this
intermitochondrial cement. Nuage has been reported
to occur in spermatogonia of B . pholis (Silveira et al.,
1990)and 0. latipes (Hamaguchi, 1987).Spermatocytes
possess a large oval nucleus with coarse chromatin and
a distinct Golgi apparatus (Fig. 18A). Abundant micro-
tubules occur in spermatocytes of some cyprinodonts
(Thiaw and Mattei, 1989) Centriolar division occurs in
spermatocytes. Round spermatids contain a number of
mitochondria dispersed throughout the cytoplasm with
two centrioles located a t the periphery of the cell (Fig.
18B). Annulate lamellae have been described in sper-
matids of B. pholis (Silveira et al., 1990), and glycogen
granules and mitochondria1 cement in spermatids of
blennidae (Lahnsteiner and Patzner, 1990). The distal
centriole lies closer to the plasma membrane than the
proximal centriole which is usually aligned at roughly
90” with respect to the distal centriole. It has been re-
ported that the distal centriole appears to be anchored
to the plasma membrane by a number of fibers (Mattei
and Mattei, 1976a, 1978a). Usually a well-developed
Golgi apparatus is associated with the centrioles. The
flagellum then begins to develop from the distal cen-
triole. Flagella, however, have been detected in sper-
matogonia and spermatocytes (Billard and Flechon,
1969). In several teleost species it has been shown that
477
nuclear pores are distinct in young spermatids but dis-
appear when the flagellum begins to develop (Grier,
1975b; Gronberg and Wartiovaara, 1972).
As round spermatids mature there occurs a distinct
spatial arrangement of organelles that will eventually
define the structural appearance of the resultant sper-
matozoon. Thus, the centrally located nucleus moves to
occupy a position close to the cell surface that (usually)
is adjacent to the Sertoli cell cytoplasm. Then the two
centrioles plus the developing flagellum migrate from
their position at the periphery of the cell to become
located close to the nucleus. During this transloction
the distal centriole carries the flagellum through the
cytoplasm, resulting in the formation of a cytoplasmic
canal (Fig. 18C). The flagellum is covered by reflected
plasma membrane and is therefore separated from the
rest of the cell body (Fig. 18C). In some teleost species
the nucleus undergoes approximately a 90” rotation
and centrioles become located within a nuclear depres-
sion. For other species, this nuclear rotation does not
occur and centrioles do not become associated with a
nuclear depression. At this time the previously dis-
persed mitochondria also move to become preferen-
tially associated with the developing flagellum. Due to
the presence of the cytoplasmic canal these mitochon-
dria do not, as in many other vertebrates, come into
close physical contact with the flagellum. For some
species a submitochondrial net, consisting of filaments
enclosed between two membranes, develops between
the mitochondria and plasma membrane of the cyto-
plasmic canal (Dadone and Narbaitz, 1967; Grier,
1975b). A different location of mitochondria has been
described where these organelles surround the base of
the nucleus in P. flesus (Jones and Butler, 1988a) and
a number of species of blennidae (Lahnsteiner and
Patzner, 1990). The mitochondria occupied recesses de-
veloped by the nucleus. In several species of gobies it
has been reported that small mitochondria present in
round spermatids disappear to be replaced by 2 large
mitochondria (Fishelson et al., 1990). Also in these te-
leosts a peculiar system of alternating RER and SER
was described that formed rings in the cytoplasm. As
the spermatids matured, however, these rings of ER
disappeared. A stranger location of mitochondria oc-
curs in B . pholis with several of these organelles lo-
cated at the anterior pole of the nucleus, resulting in
the absence of a midpiece (Silveira et al., 1990). In T .
thymallus the mitochondria of the midpiece becomes
fused to form a chondriosome which is wound around
the proximal region of the axoneme (Lahnsteiner et al.,
1991).
In L . lepadogaster it has been reported that a re-
gional differentiation occurs at the anterior surface of
spermatid nuclei (Mattei and Mattei, 1978a). In this
area a number of lamellae occur dispersed between the
thickened inner and outer nuclear membranes which
were termed “lamelles perinucleaires intermem-
branaires.” It was suggested this represented an ata-
vistic development of the acrosome which has been lost
in the evolution of the teleosts. Also in S. gairdneri, an
abortive acrosomal vesicle seems to develop that dis-
appears later in spermiogenesis (Billard, 1983).
The association of the developing flagellum with the
478 J. PUDNEY

Fig. 18. Various stages of spermatogenesis in L.macrochrius. A (arrow) can be seen in this spermatid. C: Flagellum located in the
Cyst containing primary spermatocytes. B: Round spermatids possess cytoplasmic canal. (Micrographs courtesy of R.L. Sprando and L.D.
bullet-shaped nuclei with a deep implantation fossa (arrowhead) at Russell.)
the posterior pole. A cross-sectional profile of the proximal centriole

nucleus signals chromatin condensation as well as ini- cent condensed chromatin remains intact. Eventually
tiating morphological changes, e.g., elongation, that the nuclear membrane reforms to preferentially en-
eventually result in the species-specific shape of the close only the condensed chromatin, thus eliminating
head of the mature spermatozoon. It has been shown, in excess membrane.
several species, that the condensation of chromatin in Concomitant with changes in nuclear volume, as
the nuclei of spermatids, as they mature, occurs in a spermatids mature, is also a reduction in cell volume.
specific pattern that often begins in the region adjacent For many teleosts this excess cytoplasm is sloughed,
to the developing flagellum (Boisson et al., 1968b;Rus- towards the end of spermiogenesis, as a residual body
sell, 1988; Sprando et al., 1988; Stanley, 1969). A fine which is usually engulfed by the Sertoli cell. For L.
structural study of nuclear differentiation during sper- macrochirus, however, the process of elimination of ex-
miogenesis has been undertaken for Oncorhynchus tra cytoplasm is accomplished by the formation of
tshawytscha (Zirkin, 1975).The nuclei of early sperma- membrane-bound cytoplasmic packets which migrate
tids contain randomly distributed chromatin fibers to the cell surface and are extruded (Sprando et al.,
similar to other (somatic) cells. As spermatids mature 1988). It was estimated that during spermiogenesis in
the chromatin progresses to highly orientated thick fi- L. macrochirus, both nuclear and cytoplasmic volumes
bers and unusual, irregular ribbon-like elements. Nu- of spermatids were reduced some 80% and 92%, respec-
clei of mature spermatozoa were extremely dense and tively, resulting in a total reduction of 87% in cell vol-
showed little substructure. It was suggested these ume.
structural changes were related to the process of re- Following division of the centriole and initiation of
placement of nuclear histones by protamines. flagellum development, a transitory intercentriolor
As the chromatin condenses there is obviously a lamellated body appears in many species, e.g., L. retic-
great reduction in the volume of the nucleus. This re- ulatus (Asai, 1971; Gronberg and Telkka, 1968; Mattei
sults in a large area of redundant nuclear membrane. and Mattei, 1966); U. prayensis (Boisson et al., 1969);
The removal of this excess membrane has been de- P. Zatipinna (Grier, 1973, 1975a); 0. latipes (Grier,
scribed in several teleost species. This can involve the 1976). This structure has been suggested to be derived
release of small vesicles from either the entire surface, from satellites associated with the proximal centriole
e.g., 0. maculosus (Stanley, 1969)or base, e.g.,P. flesus (Grier, 1973, 1976). The intercentriolar lamellated
(Jones and Butler, 1988a) of the nucleus as the chro- body accompanies the centrioles as they migrate from
matin condenses. A more interesting process, however, the cell periphery to the nucleus. The implantation
has been described for L. macrochirus (Sprando et al., fossa, therefore, houses a centriolar complex consisting
1988). Apparently the nuclear membrane associated of: (1)a proximal centriole from which, for various spe-
with clear, chromatin-free nucleoplasm undergoes in- cies, microtubules radiate towards the nucleus to at-
termittent breakdown. The nuclear membrane adja- tach to the nuclear membrane of the implantation fossa
 SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES
and in some cases to surround the periphery of the
nucleus; (2) an intercentriolar lamellated body dis-
posed between the proximal and distal centriole; and
(3) a distal centriole (basal body) which forms the ax-
oneme of the flagellum. It has been reported that the
microtubles emanating from the proximal centriole
serve to stabilize the nucleus prior to chromatin con-
densation as well as the relationship between the cen-
triolar complex and nucleus during nuclear morpho-
genesis and development of the implantation fossa
(Grier, 1973).The microtubles are transient and disap-
pear as soon as the implantation fossa has formed but
before chromatin condensation has ceased.
For most species the intercentriolar lamellated body
appears during spermiogenesis. In a few other species,
it has been reported to arise in spermatocytes (Grier,
1975b; Gronberg and Telkka, 1968). The proximal cen-
triole and intercentriolar lamellated body are, for
many species, temporary structures and degenerate a t
a later stage of spermiogenesis. A few species, however,
retain either the lamellated body, e.g., Pantodon
buchholzi (VanDeurs and Lastein, 1973) or proximal
centriole, e.g., 0. latipes (Grier, 1976). The exact
function of the intercentriolar lamellated body is
unknown.
As yet, ectoplasmic specializations have not been ob-
served between spermatids and Sertoli cells in the te-
leost testis (Sprando and Russell, 1987a). It was sug-
gested that since spermatids did not enter into close
physical contact with Sertoli cells, ectoplasmic special-
izations are not developed. Occasional “desmosome-
like” structures have been reported between spermato-
cytes and Sertoli cells in L. macrochirus (Sprando and
Russell, 198713).
A rather remarkable observation has been reported
for L . lepadogaster during spermatogenesis (Mattei and
Mattei, 1978a). It was reported that mature spermato-
zoa were commonly seen penetrating spermatocytes
and spermatids. Usually the spermatozoa penetrated
the cytoplasm but occasionally also passed through the
nucleus. A total of 6 spermatozoa were found penetrat-
ing a single spermatid. Apparently the embedded sper-
matozoa did not interfere with maturation of the germ
cells and were released when the residual cytoplasm
was shed. Another unusual report concerns a number of
species of blennidae where immature spermatids were
released from the cyst to mature in the testicular gland
and/or spermatic ducts (Lahnsteiner and Patzner,
1990).
The fine structure of spermatozoa of many species of
Osteichthyes has been reported. For the Crossoptery-
gii, information is available on the ultrastructure of
several species of lungfish, e.g., Protopterus annectans
(Boisson and Mattei, 1965a) and Neoceratodus forsteri
(Jespersen, 1971). The head of the spermatozoon con-
sists of a very elongated nucleus with a small anterior
acrosome. Two rod-shaped structures extend from the
tip of the acrosome, through the nucleus, to terminate
in the cytoplasm. Each rod is enveloped by nuclear
membrane. The midpiece contains two perpendicularly
aligned centrioles. The proximal centriole is attached
to the posterior margin of the nucleus, while the distal
centriole gives rise to the flagellum. The axoneme is
479
composed of a typical 9 + 2 arrangement of doublets.
Along the entire length of the flagellum two broad ex-
tensions or “wings” are present which contain a dense
material.
For Actinopterygii, the fine structure of spermatozoa
has been studied for the Holostein Lepisosteus osseus
(Afzelius, 1978).In this species the spermatozoa are not
highly differentiated and may be considered primative,
since they lack an acrosome, have a short midpiece
with a single ring-shaped mitochondrion and lateral
ridges or “wings” on the flagellum that are roughly in
the plane of the two central doublets of the axoneme.
The two centrioles are aligned perpendicular to each
other and the proximal centriole is joined to a striated
fibrous body which projects into the posterior fossa of
the nucleus. A short cytoplasmic sleeve surrounds the
intital portion of the flagellum which contains a 9 + 2
arrangement of doublets.
The spermatozoa of the Teleostei, however, have
been best studied at the electron microscope level, e.g.,
Cnesterodon decemmaculatus (Sotelo and Trujillo-
Cenoz, 1958);L . reticulatus (Mattei and Boisson, 1966);
J . lineata (Dadane and Narbaitz, 1967); Oligocottus
maculosus (Stanley, 1969); Dactylopterus uolitans
(Boisson et al., 1968a);P. reticulata (Billard, 1970b);F .
heteroclitus, Cyprinodon variegatus (Yasuzumi, 1971);
0. latipes (Grier, 1976).
Spermatozoa of species which practice external fer-
tilization, e.g., 0. latipes are not highly developed mor-
phologically and consist of either a round, bullet-shaped
or slighly elongated head; a simple centriolar complex;
few mitochondria; a poorly differentiated midpiece; and
usually a lateral head to tail articulation. By contrast,
species with internal fertilization produce more mor-
phologically complex spermatozoa, e.g., P. reticulatus
which have elongate heads, a highly modified centriolar
complex, a well-developed midpiece associated with a
large number of mitochondria, and caudal articulation
of the head with the tail.
The number of mitochondria associated with the mid-
piece of teleost spermatozoa varies from species to spe-
cies, e.g., 4 in L. aurata (Brusle, 1981), and 6-10 for L .
lepodogaster (Mattei and Mattei, 1978a). In some spe-
cies a submitochondrial net is present, e.g., J . lineata
(Dadone and Narbaitz, 1967). Species variation also
exists in the morphology of the midpiece. For instance,
in L. lepadogaster a portion of the midpiece is devoid of
mitochondria (Mattei and Mattei, 197813). The flagel-
lum of most species contains a typical 9 + 2 axoneme of
doublets and often develops lateral ridges (“wings”)
along the entire length. The axoneme of some other
species, however, is composed of atypical 9 + 0 organi-
zation of doublets, e.g., Lampanyctus sp. (Mattei and
Mattei, 1976b); Anguilla anguilla (Billard and Gins-
burg, 1973; Ginsburg and Billard, 1972);Albula vulpes
(Mattei and Mattei, 1973);A. australis schmidtii and A.
dieffenbachii (Todd, 1976); Lycodontis afer (Boisson et
al., 1967). Species variation also exists in the number of
flagella developed by spermatozoa. Biflagellated sper-
matozoa are produced by Porichthys notatus (Stanley,
1965b);I . punctatus (Yasuzumi, 1971);Lampanyctus sp.
(Mattei and Mattei, 1976b);L . lepadogaster (Mattei and
Mattei, 1974,1978b);Paroncheilus sp. (Mattei and Mat-
480 J. PUDNEY
tei, 1984) and I . punctatus (Poirier and Nicholson,
1982). The centrioles lie parallel to one another and
each participates in the development of an individual
flagellum. Although a large number of teleosts, repre-
senting different families, produce biflagellated sper-
matozoa, the evolutionary significance (if any) of this
feature is unknown. A more bizarre condition exists in
Gymnarchus niloticus (Mattei et al., 1967b)and a num-
ber of species of mormyres (Mattei et al., 1972b) which
produce aflagellated spermatozoa. Spermiogenesis ap-
pears normal but neither of the two centrioles, which lie
between the nucleus and mitochondria, develops a fla-
gellum. It was suggested these gametes carry out
ameoboid movement.
In a large number of different cyprinodonts it was
reported there was great variation in the presence and
absence of the dynein arms of axonemal doublets
(Thiaw et al., 1986). Despite these differences all sper-
matozoa were found to be motile. A number of teleost
species produces morpholgoically bizzare spermatozoa.
In L. afer (Boisson et al., 1967) the proximal centriole
develops to produce an intracellular flagellum. Fur-
thermore, one of the proximal centriole microtubles be-
comes closely associated with the nuclear membrane to
extend along the periphery of the nucleus. A similar
condition occurs in A. uulpes (Mattei and Mattei, 1973)
where the nuclear membrane penetrates the proximal
centriole, causing disruption and dislocation of the mi-
crotubular triplets which become associated with the
nuclear membrane. These then extend through the cy-
toplasm, associated with a projection of the nuclear
membrane, to form what has been termed a pseudofla-
gellum. Furthermore, a large mitochondrion is not as-
sociated with the centrioles but is located at the ante-
rior of the spermatozoon towards the lateral aspect of
the nucleus. The spermatozoa of P. buchholzi are ex-
tremely aberrant (Van Deurs and Lastein, 1973). The
centriolar complex consists of a single centriole and a
lamellated cap-shaped body situated between the cen-
triole and the nucleus. The midpiece is composed of 9
helical mitochondria1 threads, formed by end-to-end fu-
sion of small, single mitochondria that alternate with 9
helical dense fibers. Posterior to the midpiece is a
structure called the fenestrated sheath composed of in-
ner and outer membranes penetrated by pores. This
sheath surrounds the initial portion of the axoneme.
Many species of anguillidae also produce extremely
morphologically exotic spermatozoa, e.g., A. anguilla
(Billard and Ginsburg, 1973; Ginsburg and Billard,
1972); A. australis schmidtii and A. diefenbachii,
(Todd, 1976). The spermatozoa have a sickle- or cres-
cent-shaped head tapering to a narrow neck region and
a long flagellum. A short striated rod or appendage
projects from the posterior region of the head near the
origin of the flagellum. The proximal centriole devel-
ops and divides into two groups of 4 and 5 microtubular
triplets which then course anteriorly along either the
inner concave or outer concave aspects of the nucleus.
A midpiece is not present because the single mitochon-
drion becomes displaced (in these species) to the ante-
rior pole of the nucleus. The nucleus may partly sur-
round the mitochondrion that lies in an indentation of
its concave side. The presence of this mitochondrion, at
the tip of the spermatozoon, gives a bulbous protubur-
ance to the gamete.
Patches of regular particles have been detected by
freeze-fracture in the cell membrane covering the an-
terior head of spermatozoa in B . rerio (Kessel et al.,
1983) and R. sericeus sinensis (Guan and Afzelius,
1991). It was suggested these regions may act as rec-
ognition andlor adhesion sites allowing spermatozoa t o
fuse with the ova.
AMPHIBIA
The amphibia are composed of three extant orders:
the Anura (frogs, toads) is the largest and more than
two-thirds of amphibians belong to this group; Urode-
lia (newts, salamanders) and Apoda (caecilians) a
small group of legless amphibians. Amphibians are the
most primitive of terrestrial vertebrates and most spe-
cies need to return to an aqueous environment to
breed.
Morphologically, the amphibian testis is similar to
that of teleosts. Spermatogenesis occurs in cysts
present in a seminiferous compartment called lobules.
Based on spermatogonial distribution, three types of
testes have been described for amphibia (Grier, 1993):
one, in urodeles, in which the testis is divided into lobes
and new lobules form from primary spermatogonia and
their associated Sertoli cells within connections of the
testicular lobes, e.g., Taricha granulosa. The other oc-
curs in urodeles that do not have lobed testes and here
primary spermatogonia and Sertoli cells are located in
the lobular region proximal to the sperm duct, e.g.,
Necturus maculosus (Fig. 19A). The third is found in
anuran species in which the primary spermatogonia
are present along the length of the lobule (Fig. 24).
Most amphibian species undergo a postnuptial sper-
matogenic cycle. Initiation of spermatogenesis occurs
soon after spawning and is completed quickly, result-
ing in spermatozoa being retained within the testis
prior to the next breeding season. Temperate amphib-
ians undergo an annual spermatogenic cycle with mat-
uration of spermatozoa occurring in the winter fol-
lowed by spermiation in the spring breeding season.
Urodeles
Many urodeles exhibit a spermatogenic wave in the
testis during the breeding season. This results in a spa-
tial and temporal segregation of germ cells. Spermato-
genesis begins at the caudal end of the testis and
progresses towards the cephalic end. Lobules in se-
quential stages of maturation, therefore, can be found
along the length of the testis with the least mature
located in the cephalic region. Furthermore, spermato-
genesis is confined to regions of the seminiferous lobule
distal t o the main sperm duct. Lobular regions proxi-
mal to the sperm duct contain primary spermatogonia
which act as a reservoir for future waves of spermato-
genesis (Fig. 19B). In many urodeles, therefore, there is
a maturational wave of spermatogenesis occurring lon-
gitudinally in a caudacephalic direction, plus a proxi-
mal-to-distal cycle of differentiating germ cells which
eventually form the maturing segments of the seminif-
erous lobules which initiate seasonal testicular recru-
descence. Spermatogenesis and cyst development has
 SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES
Fig. 19. Organization of N . maculosus testis. A Seminiferous lob-
ules contain an immature proximal segment composed of cysts filled
with spermatogonia. Arrowheads point to sperm duct. B: Primary
spermatogonia are large cells with irregularly shaped nuclei and un-
dergo mitotic activity t o produce secondary spermatogonia. C: Mitotic
divisions of spermatogonia result in growth of the cysts. D: Cross-
been described a t the light microscope level €or several
species of urodeles, e.g., Desmognathus fuscus (Kings-
bury, 1901); Taricha torosa (Miller and Robbins, 1954);
Cynops pyrrohogaster pyrrohogaster (Tanaka and Iwa-
sawa, 1979); N . maculosus (Pudney et al., 1983).
In general, spermatogenesis proceeds in a similar
fashion for most urodeles and resembles that occurring
in teleosts. For instance, in N . maculosus, nascent cysts
481
section of seminiferous lobule containing cysts filled with secondary
spermatogonia. Arrowheads point to Sertoli cytoplasm that forms the
walls of the cysts. Note absence of a lumen in the lobule. E: As sper-
matogenesis proceeds, cysts migrate towards the distal end of the
lobule where spermiation occurs by rupture of the cysts resulting in
the lumen of the lobule containing bundles of spermatozoa.
develop when a primary spermatogonium becomes as-
sociated with a Sertoli cell (Fig. 19B). Subsequent mat-
urational divisions of spermatogonia results in an
increase in size of the cysts (Fig. 19C). During sper-
matogenesis, cysts migrate toward the distal end of
the seminiferous lobule. By the time this occurs, the
cysts contain mature spermatozoa. Spermiation then
occurs, exposing Sertoli cells with isogeneic bundles of
482 J. PUDNEY
spermatozoa embedded in their apical cytoplasm (Fig.
19E). Sertoli cells are either sloughed with the sper-
matozoa or undergo degeneration.
A number of studies have reported on the fine struc-
ture of different aspects of spermatogenesis in several
species of urodels, e.g., Pleurodeles wnltlii (Picheral,
1967, 1972a,b,c, 1979); D. fuscus (Lommen, 1970);
Salamandra salamandra (Bergmann et al., 1982;
Schindelmeiser et al., 1985); Ambystoma mexicanum
(Keyhani and Lemanski, 1981; Miltner and Arm-
strong, 1983).
Primary spermatogonia are large cells characterized
by highly irregularly shaped nuclei and with the cyto-
plasm appearing relatively devoid of organelles except
for a number of mitochondria, several small Golgi re-
gions and an occasional lipid droplet (Fig. 20A). The
secondary spermatogonia appear smaller with large
spherical nuclei, numerous mitochondria, long cister-
nae of SER, several Golgi areas (Fig. 20B) and profiles
of annulate lamellae (Fig. 21B). An interesting feature
of these spermatogonia is that during the early stage of
cyst formation, both develop abundant blunt projec-
tions a t their periphery that invaginate the surround-
ing Sertoli cytoplasm (Fig. 21A). It is assumed these
represent anchoring devices that keep individual sper-
matogonia associated with their corresponding Sertoli
cell during development of the cysts. At a later stage of
cyst development the germ cells have a smooth surface
(see Fig. 20B).
It has been reported that as cysts mature the germ
cells lose close physical contact with Sertoli cells (Pud-
ney, 1993). Furthermore, at this time, germ cells de-
velop filopodia and the intercellular space between Ser-
toli cells and germ cells widens (Fig. 22). It was
suggested this occurred to allow movement and/or
translocation of cells as the cysts become organized
within the seminiferous lobule. Spermatocytes of S.
salamandra have been reported to contain annulate
lamellae and an occassional lipid droplet (Schindel-
meiser et al., 1985). Furthermore, in this species, mi-
tochondria were located at the periphery of spermato-
cytes and this disposition of these organelles was also
observed in early spermatids. Proacrosomal granules
were also reportedly detected in secondary spermato-
cytes. As yet, it would appear that junctional special-
izations have not been described between germ cells
and Sertoli cells at any stage of spermatogenesis.
The fine structure of urodelean spermatozoa has
been best studied in P. waltlii (Picheral, 1967,
1972a,b,c, 1979). The spermatozoa possess an acroso-
ma1 cap that ends in either a sharp point or rounded
knob and in some species develops a hooked barb, e.g.,
P. waltlii, Euproctus aspes. An elongated, conical sub-
acrosomal space contains the perforatorium which con-
sists of an axial rod that extends deep into the nucleus
within an endonuclear canal. The nucleus has promi-
nent ridges composed of nonchromatin material con-
sisting of minute tubules packed together (Fig. 23A).
Many species of urodeles display this ridge on the sper-
matozoal nuclei but which differs in composition rang-
ing from a single bundle, e.g., P. waltlii to several, e.g.,
S. salamandra. The neck, or connecting piece appears
as a long cylinder that fits into a deep fossa on the
posterior pole of the nucleus, resulting in this region
being mostly enclosed by a shell of chromatin. At the
caudal end of the neck lie the two centiroles. The pres-
ence of a ring centriole (annulus) has been reported for
P. waltlii (Picheral, 1972b) and D. fuscus (Lommen,
1970). The spermatozoa of urodeles are unusual since
they develop an undulating membrane that extends
almost the length of the flagellum (Fig. 23B). The fla-
gellum consists of an axial fiber or rod (also called SUP-
porting filament) that extends from the neck region
and the attached thin undulating membrane with the
axoneme located a t the free edge along with a dense
structure called the marginal filament (Fig. 2 3 0 . The
midpiece contains the mitochondria which are associ-
ated only with the axial fiber and so do not surround
the axoneme (Fig. 23D). In A. mexicanurn the mito-
chondria are smaller than in other species and so
closely packed as to exhibit an almost crystalline ar-
rangement (Keyhani and Lemanski, 1981). The flagel-
lum of Hynobius nebulosus lacks mitochondria which
are present in a protoplasmic bead located around the
nucleus (Picheral, 1979).
Anura
Like urodeles, anuran spermatogenesis occurs in
cysts located in seminiferous lobules (Fig. 24A). Ini-
tially cyst development progresses similarly to that oc-
curring in urodeles and teleosts (Fig. 24B), e.g., Rana
temporaria (Witschi, 1915); Rana esculenta (Lofts,
1964);Rana pipiens (Burgos, 1955; Rugh, 1939). There
is a distinct difference, however, in the process of sper-
matogenesis that distinguishes these amphibia from
urodeles. During spermiogenesis, as elongate sperma-
tids become embedded in a single Sertoli cell, the for-
mation of the spermatid flagellum signals rupture of
the cyst. These open cysts now form the wall of the
seminiferous lobule which is lined by highly differen-
tiated Sertoli cells with bundles of spermatids deeply
embedded in their apical cytoplasm (Fig. 24C). At this
stage the seminiferous lobule resembles morphologi-
cally the germinal epithelium present in amniotes.
During this period, at the periphery of the lobule, pri-
mary spermatogonia can be detected surrounded by
Sertoli cells that are preparing to form cysts for the
next round of spermatogenesis (Fig. 24A). Spermiation
then ensues and in some species, e.g., R . esculenta only
the apical portion of the Sertoli cell is shed along with
the spermatozoa. The basal portion of the Sertoli cell,
containing the nucleus, remains to develop the next
generation of cysts. In these amphibian species, there-
fore, a permanent germinal epithelium can be said to
be present.
An unusual mode of spermatogenesis has been re-
ported for Bombina uariegata (Obert, 1976).In this spe-
cies, apparently germ cells develop in the absence of
Sertoli cells and spermatozoa do not aggregate into
bundles but appear as a mass of single cells. It was
unknown whether this represents a primitive condition
or is a peculiarity restricted to discoglossid amphibia.
Electron microscope studies are available on sper-
matogenesis in several species of anuran amphibians,
e.g., Bufo arenarum (Burgos and Fawcett, 1956;
Cavicchia and Moviglia, 1982); Rana clamitans (Poir-
 Fig. 20. Low power electron micrographs of N. maculosus testis merous filopodia at periphery of the germ cell. B Secondary sper-
showing (A) Primary spermatogonia (SP)with irregularly shaped nu- matogonia possess large spherical nuclei. Note that at this stage of
clei which are poorly differentiated and possess a number of mito- development the cells exhibit a smooth outline with no formation of
chondria and a n occasionally lipid droplet. Note development of nu- filopodia.
484 J. PUDNEY

Fig. 21. A: Primary spermatogonia (PG) produce numerous filopodia that invaginate the surrounding
Sertoli cytoplasm. B: Annulate lamellae (arrowheads) present in secondary spermatogonia. N . maculo-
sus.

ier and Spink, 1971); R. pipiens (Poirier and Spink,

1971; Zirkin, 1971); Xenopus laevis (Bernardini et al.,
1986; Reed and Stanley, 1972); Discoglossus pictus
(Sandoz, 1973, 1974); B. variegata (Folliot, 1979);
Megophyrs montana (Asa and Phillips, 1988); Rana
catesbeiana (Sprando and Russell, 1988a); Rhacopho-
rus arboreus and R. schlegelii (Mizuhira et al., 1986).
Morphologically the early stages of spermatogenesis
resemble those occurring in urodeles. Primary sper-
matogonia have large, irregular nuclei with a promi-
nent nucleolus. In X . Zaeuis, spermatocytes possess
abundant SER (Reed and Stanley, 1972). At the
beginning of spermiogenesis the two centrioles are
located close to the Golgi apparatus. Later the
centrioles migrate towards the plasma membrane and
a flagellum develops from the distal entriole. The
centrioles then move towards the nucleus and in doing
so cause retraction of the plasma membrane, resulting
in the formation of a cytoplasmic canal containing the
developing flagellum. The centrioles initially reside in
a small indentation on the anterior pole of the nucleus
adjacent to the acrosome. Then the centrioles become
relocated toward the posterior pole of the nucleus
either by active movement or nuclear rotation. During
spermiogenesis the Golgi apparatus appears poorly
developed and this has been related to the fact that an
acrosomal granule is not produced during formation of
the acrosome, e.g., B. arenarum (Burgos and Fawcett,
1956).X . leavis (Reed and Stanley, 1972);B. uariegata Fig. 22. During cyst formation, secondary spermatogonia lose
(Fo&t, 1979). It has been that distinct close physical contact with Sertoli cells and develop filopodia (arrow-
heads). Furthermore, the intercellular space between germ cells and
modifications of the nuclear envelope occur during Sertoli cells widens. It is assumed this occurs to allow movement of
spermiogenesis of D. pictus (Sandoz, 1974). In early germ cells as cysts mature. N . maculosus.
 SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES
Fig. 23. N. mucolosus spermatozoa. A: Bundle of tightly packed
tubules (arrowheads) associated with nucleus. B Scanning electron
micrograph showing undulating membrane (arrowheads) along
length of flagellum. C: Cross-section through axoneme (arrowhead)
spermatids, nuclear pores are not evenly distributed
over the nuclear surface but are preferentially grouped
in front of the Golgi apparatus. As the acrosomal
vesicle becomes associated with the nucleus, however,
and spreads over its surface, these pores gradually
become displaced towards the posterior pole of the
nucleus. A manchette has been described during
spermiogenesis in D. pictus (Sandoz, 1974). For X .
laevis, as chromatin condenses and nuclear elongation
occurs in maturing spermatids, excess nuclear and
plasma membrane appears to be pinched off into the
intercellular space between these germ cells and
Sertoli cells (Reed and Stanley, 1972). At later stages
485
located next to the marginal filament (small arrow) attached do the
axial rod (large arrow) by the undulating membrane (large thick ar-
row). D: Cross-section through midpiece showing mitochondria asso-
ciated with axial rod (arrowheads).
of spermiogenesis, mitochondria aggregate in the
cytoplasmic canal surrounding the flagellum. A
single, large mitochondrion, however, has been re-
ported to surround the centrioles at the base of the
nucleus in X . laevis (Reed and Stanley, 1972). A
perforatorium, which develops in the space between
the acrosome and tip of the nucleus and consists of
coarse strands, has been described in spermatids of B .
arenurum (Burgos and Fawcett, 1956). During spermi-
ogenesis of R. catesbeiana, the round spermatids are
penetrated by processes of Sertoli cytoplasm which
disappear later in development (Sprando and Russell,
1988a). Studies carried out to specifically identify
486
Fig. 24. Spermatogenesis in R a m sp. A: Primary spermatogonia (arrowheads) occur along the length
of the seminiferous lobule. B Maturing cysts present in seminiferous lobule. C: Open cysts containing
bundles of spermatids embedded in Sertoli cell cytoplasm line the length of the lobule.
cellular junctions in the anuran germinal epithelium
have reported desmosome-likejunctions between sper-
matocytes and Sertoli cells in R. catesbeiana (Sprando
and Russell, 1987b).Furthermore, ectoplasmic special-
izations also have been described between elongate
spermatids and Sertoli cells in this species. (Sprando
and Russell, 1987a). These structures, however, ap-
peared morphologically primitive compared to mam-
malian ectoplasmic specializations. The layer of
filaments (5-7 nm in diameter) was unorganized,
poorly developed and often not associated with
cisternae of ER. Microtubules were often detected
related to the filaments.
Spermatozoa of unusual morphology have been de-
scribed for B . variegatu (Folliot, 1979). Helical-shaped
spermatozoa are produced that cannot be divided into
distinct head and tail regions. This is due to the fact
that the motile apparatus, including the undulating
membrane, effectively extends along the entire length
of the head. Thus, the origin of the flagellum, i.e., the
centriolar apparatus, is located at the anterior region
of the spermatozoa close to the acrosome. Furthermore,
the nuclear chromatin does not fully condense and ma-
ture spermatozoa still retain a portion of residual cy-
toplasm. Even stranger spermatozoa have been de-
scribed for R . arboreus and R. schlegelii (Mizuhira et
al., 1986). These species produce spiral spermatozoa in
the shape of a counter clockwise corkscrew. The poste-
rior region of the nucleus is surrounded by mitochon-
dria and the flagellum has two axonemes enclosed by a
mass of microtubules.
Normally, nuclear condensation and elongation oc-
curs concurrently with cytoplasmic changes during
spermiogenesis (Phillips, 1974). This does not, how-
ever, appear to occur in M. montana (Asa and Phillips,
1988). In this species, development of the flagellum
and shedding of residual cytoplasm is almost complete
J. PUDNEY
before condensation of nuclear chromatin begins.
Moreover, a unique pattern of nuclear morphogenesis
takes place. Initially, the chromatin condenses into a
long, cylindrical coil within the spherical nucleus.
Later the nuclear membrane conforms to the contours
of the cylinder as it uncoils to form the long, tapering,
helical nucleus of the mature spermatozoon. Also, the
flagellum has a double axoneme which is connected by
cross-bridges extending from the region between sub-
tubules a and b of doublets 3 and 8. During spermio-
genesis a manchette does not develop in this species. It
was suggested, therefore, that nuclear shaping may be
controlled by the pattern of DNA and protein aggrega-
tion, indicating that microtubles are not necessarily
universally required to achieved nuclear morphogene-
sis.
The mechanism of spermation has been described in
anurans (Burgos and Vitale-Calpe, 1967a,b; DeRober-
tis et al., 1946; Van Oordt et al., 1954) This is accom-
plished by distension of the apical Sertoli cell cyto-
plasm containing the bundles of spermatozoa. The
swelling of the Sertoli cytoplasm is due to an influx of
fluid and results in displacement of spermatozoa to-
wards the lumen. Continual expansion of the apical
cytoplasm causes the Sertoli cell recesses containing
the spermatozoa to unfold and subsequently flush the
gametes into the lumen of the seminiferous lobule. Fi-
nally, the swollen apical region of the Sertoli cell is
sloughed into the lumen. Further loss of Sertoli cyto-
plasm is prevented by apposition of residual, overlap-
ping processes which preserve the integrity of the re-
maining nucleated portion of the Seroli cell attached to
the basement membrane.
REPTILIA
With the reptiles, the amniote organization of the
seminiferous compartment is attained with a perma-
 SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES
nent germinal epithelium contained in tubules. The
reptilia consists of the Squamata (snakes, lizards),
Chelonia (tortoises, turtles) and Crocodilia (crocodiles,
alligators). For temperate zone reptiles reproduction is
cyclical. The breeding season starts in spring/early
summer followed by a period of testicular regression
with the germinal epithelium reduced to a layer of Ser-
toli cells and spermatogonia. Spermatogenic recrudes-
cence is initiated in the late summedfall. For some
species spermatogenesis is completed before winter
with spermatozoa, for the next breeding season, stored
in the epididymides. This is the typical postnuptial pat-
tern of spermatogenesis. In other species, spermato-
genesis undergoes arrest in the winter (or proceeds at a
very slow rate) to resume again in the spring, and is
completed just before the breeding season. This pattern
of spermatogenesis, therefore, has been referred to as
prenuptial (see Licht, 1984). Spermatogenesis has been
described a t the light microscope level for several rep-
tilian species, e.g., turtles Terrapene carolina (Altland,
1951)Sternotherus odoratus (Risley, 1938); lizards An-
olis carolinensis (Fox, 1958); Cnemidophorus tigris
(Goldberg and Lowe, 1966); snakes Naja naja (Lofts et
al., 1966); Thamnophis elegens terrestris (Fox, 1952).
Electron microscopic analysis has been carried out
on several aspects of spermatogeneis in a variety of
reptilian species, e.g., for lizards Liolaemus weigmani
and Amphisbaena darwinii (Sotelo and “rujillo-Cenoz,
1958);Agama agama (Charnier et al., 1967); Tropidu-
rus torquatus (da Cruz-Hofling and da Cruz-Landim,
1978;da Cruz Landim and da Cruz-Hofling, 1977);An-
olis carolinensis (Clark, 1967); Cnemidophorus lemnis-
catus (Del Conte, 1976)Lacerta uiuipara (Courtens and
Depeiges, 1985)Podarcis (-Lacerta) taurica (Butler and
Gabri, 1984). Turtles, e.g., Clemmys japonica (Yasu-
zumi and Yasuda, 1968); Chelydra serpentina (Mah-
moud et al., 1985) Chrysemys picta (Dubois et al., 1988;
Hess et al., 1991); Pseudemys scripta (Kaplan et al.,
1966;Sprando and Russell, 1988b);snakes, e.g., Python
sebae (Boisson and Mattei, 1965b); Lampropeltis getu-
lus (Austin, 1965; Hamilton and Fawcett, 1968); Con-
strictus constrictus (Hamilton and Fawcett, 1968).
In general, the main features of spermatogenesis are
similar for most reptilian species. At the beginning of
the breeding season the seminiferous tubules contain
spermatogonia which are large cells characterized by
irregularly shaped nuclei. The cytoplasm contains a
large number of organelles including mitochondria,
SER and vacuoles as well as a number of lipid droplets
(Fig. 25). Further maturation and development of the
germ cell is, in general, morphologically unremarkable
for most reptilian species. Several salient points of
spermiogenesis are the formation of a manchette, the
appearance of a tubular body, unusual morphology of
the mitochondria, and formation of the mantle. Ini-
tially the manchette surrounds the elongating nucleus
as a loose helix of microtubules. As the nucleus contin-
ues to elongate and the chromatin condenses, however,
the helix of microtubles unwinds to become orientated
parallel to the long axis of the nucleus (Fig. 26A). In A.
carolinensis (Clark, 1967) it has been reported that mi-
crotubules of the manchette (caudal sheath) insert cau-
dally in a ring of granular material associated with the
487
distal centriole and anteriorly in a ring of granular
material around the base of the acrosome. These mi-
crotubules were apparently linked to each other by
short arms. The manchette disappears at a late stage of
spermiogenesis. A distinctive tubular body of as yet
unknown origin and formation has been described in
spermatids of several reptilian species (Fig. 26B). This
structure has also been called a membranous body
(Clark, 1967).
During spermiogenesis of turtles, the mitochondria
become highly modified with the disappearance of
lamelliform cristae which are replaced by a series of
concentric membranes within the organelles (Fig.
26B). This transformation of mitochondria produce
structures that have been termed mitochondria1 lamel-
lar bodies (Yasuzumi and Yasuda, 1968). In C . picta,
spermatids possess numerous lipid droplets and a well
developed system of SER (see Fig. 26B). As spermatids
mature the acrosome develops to spread down over the
nucleus; as this occurs it is accompanied by a thin cy-
lindrical projection of Sertoli cytoplasm. This Sertoli
cytoplasm which envelops the acrosome is in turn sur-
rounded by spermatid cytoplasm (see Fig. 27) and has
been variously referred to as the spermatid mantle,
hood, or accessory cap (Butler and Gabri, 1984; Del
Conte, 1976; Sprando and Russell, 1988b).
In some species, several intranuclear tubules have
been described in developing spermatids, e.g., C .japon-
ica (Yasuzumi and Yasuda, 1968; Fig. 2 6 0 . Specialized
cellular junctions have been described between germ
cells and reptilian Sertoli cells. Desmosome-like junc-
tions occur between spermatocytes and Sertoli cells in
P. scripta (Sprando and Russell, 1987b). Ectoplasmic
specializations are produced by Sertoli cells associated
with elongate spermatids for several reptilian species
(Baccetti et al., 1980; Sprando and Russell, 1987a).
These structures are characterized by a prominent
layer of filaments plus deeper elements of ER. These
ectoplasmic specializations project for some distance
beyond the head of the spermatid in C. picta (Fig.
26D).
A perforatorium has been reported to develop during
spermiogenesis of several reptilian species (Fig. 26D;
Boisson and Mattei, 196513; Butler and Gabri, 1984;
Clark, 1967; Courtens and Depeiges, 1985; da Cruz-
Landim and da Cruz-Hofling, 1977; Hess et al., 1991;
Yasuzumi, 1974; Yasuzumi and Yasuda, 1968). The
perforatorium in reptiles has been shown to contain
actin (Baccetti, 1979). Changes in nuclear chromatin
have been reported during reptilian spermiogenesis
(Butler and Gabri, 1984; Clark, 1967; da Cruz-Hofling
and da Cruz-Landim, 1978). Nuclei of round sperma-
tids contain dispersed chromatin. As the nucleus elon-
gates this chromatin becomes organized into a system
of fibers that course parallel to the long axis of the
nucleus. For L . uiuipara, however, it has been sug-
gested the chromatin fibers initially follow a helical
pattern before assuming a longitudinal orientation
with respect to the elongating nucleus (Courtens and
Depeiges, 1985).With further condensation of the chro-
matin, the fibers become thicker and tightly packed to
produce the fully condensed nuclei of mature sperma-
tozoa. It is assumed that chromatin condensation re-
488
Fig. 25. Primary spermatogonia (PG) present in germinal epithelium of C . pictu at beginning of
testicular recrudescence.
sults from dehydration of nucleoprotein and nucleo-
plasm elimination (Yasuzumi, 1974).
A study of cell volume changes and cytoplasmic elim-
ination as spermatids mature has been carried out for
P. scripta (Sprando and Russell, 1988b). The elimina-
tion of excess cytoplasm during spermiogenesis is ac-
complished by several mechanisms. There is a n inva-
sion of spermatids by Sertoli processes which sequester
small portions of cytoplasm that are then removed from
the germ cell. Budding of small mitochondrial-rich cy-
toplasmic fragments also occurs from the midpiece re-
gion of elongating spermatids. Finally, residual bodies
of varying sizes also function to eliminate excess cyto-
plasm.
Some unusual morphological findings have been ob-
served during spermiogenesis of L. vivipara (Courtens
and Depeiges, 1985). Nuclear pouches develop which
are formed by invaginations of the nuclear envelope.
These pouches initially appear close to the developing
acrosome and proceed to migrate down towards the pos-
terior pole of the nucleus. This movement could be ac-
complished by a system of microtubules that were seen
J. PUDNEY
to be associated with each pouch. It was proposed these
pouches transport perinuclear material from the ac-
rosomal region to deposit it in the region close to the
centrioles. Structures termed lamellar plates composed
of two sheets of double membranes containing dense
material were also formed. These lamellar plates ap-
pear at the anterior region of the nucleus and eventu-
ally fuse with the nuclear membrane. Both the nuclear
pouches and lamellar plates disappear towards later
stages of spermiogenesis. As spermatids mature follow-
ing regression of the manchette, numerous lipid drop-
lets have been described surrounding the nucleus of
some species (Hamilton and Fawcett, 1968). In L. vzui-
para these have been called tergosomes which have
been reported to fuse with the nuclear membrane to
discharge their contents into the intramembranous
space (Courtens and Depeiges, 1985). For several spe-
cies of snakes, however, it was reported these lipid
droplets are discarded with the residual body (Hamil-
ton and Fawcett, 1968).
Although reptiles are amniotes, several structural
features distinguish their spermatozoa from that of
 SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES 489

Fig. 26. Spermiogenesis in C. pictu. A Manchette (arrowheads)
composed of microtubules orientated parallel to long axis of spermatid
nucleus. B: Spermatids contain a distinctive tubular body (arrow-
heads), many lipid droplets (small arrows) and highly modified mito-
chondria with concentric cristae (large arrows). C: Developing sper-
matids contain intranuclear tubules (arrowhead). D Elongate sper-
matid illustrating presence of the perforatorium (arrowhead) and ec-
toplasmic specialization produced by the Sertoli cell that surrounds
acrosomal region.
490
Fig. 27. Development of mantle of Sertoli cytoplasm (arrowhead)
that envelopes acrosomal region of spermatid. G . domesticus. (Micro-
graph courtesy of R.L. Sprando and L.D. Russell.)
mammalian species. Reptilian spermatozoa (1)retain
both centrioles with the proximal centriole orientated
at roughly 90" to the distal centriole; (2) develop an
additional structure, the neck cylinder, which encom-
passes the centriolar region of the flagellum, and for
snakes display an unusual concentric arrangement of
the mitochondrial and fibrous sheaths, and further-
more do not possess a striated connecting piece. The
neck cylinder is a dense structure which is attached to
the posterior pole of the nucleus and surrounds the
connecting piece and centrioles. The mitochondrial
sheath consists of a number of seperate mitochondria
that spiral around the flagellum. For snakes the mito-
chondrial sheath is extensive and extends a consider-
able distance along the length of the flagellum. A sys-
tem of 9 dense fibers is also present which apparently
develops from the proximal centriole and courses down
to surround the axoneme. The outer fibrous sheath cov-
ers most of the flagellum. For some reptilian sperma-
tozoa (particularly turtles) the mitochondria extend
from the region containing the proximal centriole to
the annulus, thus making the identification of a dis-
crete neck region difficult. Unusual morphological fea-
J. PUDNEY
tures of spermatozoa have been reported for several
reptilian species. Thus, for C . picta spermatozoa have a
perforatorium consisting of 2-3 rods which are contig-
uous with nuclear canals, a connecting collar of dense
material that surrounds the base of the nucleus and a
distal centriole that contains central microtubules ex-
tending the entire length of this structure. In L. getulus
and C . constrictus a distinctive nonmitochondrial com-
ponent of the midpiece has been described (Hamilton
and Fawcett, 1968) This region consists of conspicuous
plaques of dense material, interspersed between the
mitochondria, spaced at irregular intervals along the
length of the midpiece. These plaques were not sur-
rounded by a membrane and were thought to be de-
rived from granules that did not participate in the for-
mation of the neck cylinder.
AVES
Birds are amniotes and the organization of the testis
is essentially similar to that of other amniotes. Those
avian species inhabiting temperate climates exhibit
well-defined seasonal cycles of testicular activity. Dur-
ing testicular regression the seminiferous tubules are
composed only of Sertoli cells and spermatogonia. Since
birds are homeotherms they carry out prenuptial sper-
matogenesis. Due to the high metabolic rate of birds,
however, full testicular recrudescence can be accom-
plished in a matter of weeks.
Although nearly 9,000 extant avian species exist,
spermatogenesis has been studied in only a few. Fur-
thermore, these studies have been essentially re-
stricted to domesticated birds, e.g., fowl, quail, or
commercially available song birds, e.g., canary, bud-
gerigar. At the light microscopic level, several reports
have described spermatogenesis in various avian spe-
cies, e.g., Cygnus olor (Breuker, 1982); Gallus domes-
ticus (DeReviers, 1971; Zlotnik, 1947); Branta cana-
densis interior (Mori and George, 1978).
Although a number of electron microscope studies
have been carried out on avian spermatogenesis they
have been primarily restricted to the stage of spermi-
ogenesis with few investigations on the complete
breeding cycle, e.g., Passer domesticus (Sotelo and Tru-
jillo-Cenoz, 1958); P . montanus saturatus (Yasuzumi,
1956);Gallus domesticus (Cooksey and Rothwell, 1973;
Gunawardana and Scott, 1977; Maretta, 1977; McIn-
tosh and Porter, 1967; Nagano, 1962; Nicander, 1967;
Sprando and Russell, 1988b; Tingari, 1973; Xia et al.,
1986); Streptopelia roseogrisea (Mattei et al., 1972a);
Melopsittacus undulatus (Humphreys, 1975a,b);Gallus
gallus (Okamura and Nishiyama, 1976); Cairina mos-
chata (Marchand, 1977); Cygnus olor (Breuker, 1982);
Lonchura striatu var domestica (Kondo et al., 1988);
Rhea umricans (Phillips and Asa, 1989);and Coturnix
coturnixjaponica (Lin and Jones, 1992).In general, the
process of spermatogenesis in birds is very similar to
that previously described for the reptiles.
An excellent study has been reported describing the
fine structure of spermatogenesis throughout the
breeding season in C . olor (Breuker, 1982). At the be-
ginning of the breeding season spermatogonia are the
primary germ cells present in the seminiferous epithe-
lium. Nuclei of the spermatogonia are round or slightly
 SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES
irregular in shape with a prominent nucleolus. The
cytoplasm contains abundant ribosomes, many mito-
chondria, a well-developed Golgi apparatus, two cen-
trioles orientated at right angles to each other, and an
unusual feature comprised of a single long microtubule
associated with a bundle of fine filaments. The primary
spermatocytes possess many mitochondria closely re-
lated with long cisternae of SER and a chromatoid
body. A species difference has been described for M.
undulatus in which numerous oval mitochondria occur
located at the periphery of spermatocytes (Humphreys,
1975a). In C . olor, secondary spermatocytes are char-
acterized by the presence of annulate lamellae. With
the onset of spermiogenesis the long cisternae of SER
present in spermatocytes break down into vesicles in
spermatids, and the distal centriole initiates the devel-
opment of a flagellum a t the periphery of the cell. The
annulus develops around the flagellum where it ex-
tends from the cell membrane. The proximal centriole
is orientated roughly 90" with respect to the distal cen-
triole. A Golgi apparatus is located in the vicinity of
the centriole and produces an acrosomal vesicle that
apparently lacks an acrosomal granule. Then both cen-
trioles and acrosomal vesicle migrate close to the nu-
cleus. In the flagellar region, five layers of annulate
lamellae develop which often appear attached to the
nucleus. Subsequently, the acrosome and centrioles
move to their respective anterior and posterior posi-
tions on the nucleus.
For S. roseogrisea, however, this alignment of acroso-
ma1 vesicle and flagellum has been reported to occur by
rotation of the nucleus (Mattei et al., 1972a). As the
spermatid nucleus undergoes elongation, a rod (perfo-
ratorium or acrosomal spine) develops beneath the ac-
rosomal vesicle. With further nuclear elongation this
rod lengthens to penetrate the nucleus in an endonu-
clear canal or for other species in an apical nuclear
pocket. The perforatorium is not bound by a mem-
brane. Also in the cytoplasm a tubular body appears
that is similar to that produced by reptilian sperma-
tids.
With further maturation of the spermatid, the annu-
late lamellae separate from the nucleus to lie free in
the cytoplasm, and can often be found in contact with
the tubular body. Also, annulate lamellae occasionally
appear to roll up and so form rings which become de-
tached from the tubular body. During spermiogenesis,
as the spermatid elongates the manchette develops.
Initially the microtubules are spirally organized
around the nucleus. With further elongation and con-
densation of the nucleus, however, the microtubules
become orientated parallel to the long axis of the nu-
cleus. This raises the question, therefore, as t o whether
two different sets of microtubules occur or only one that
becomes reorientated from a spiral to a longitudinal
pattern. At a later stage of spermiogenesis the micro-
tubules of the manchette disappear. Late in spermio-
genesis, a t the level of the annulus, a fibrous sheath is
formed which surrounds the axoneme along the length
of the principal piece. The final step in the formation of
the midpiece is the development of a mitochondria1
sheath which extends from the base of the nucleus to
the annulus. In G . domesticus, about 30 mitochondria
49 1
surround the distal centriole and proximal portion of
the axoneme in the form of a helix (Bakst and
Howarth, 1975).
The onset of testicular regression occurs when the
last generation of spermatids is released with no fur-
ther maturation of the remaining germ cells. This re-
sults in a loss of the synchronized stages of spermato-
genesis, degeneration of residual germ cells and
obliteration of the lumen of the seminiferous tubule.
Eventually the germinal epithelium is reduced to a
layer of spermatogonia and Sertoli cells. An interesting
feature of testicular involution was a massive invasion
of seminiferous tubules by macrophages which appar-
ently assisted the Sertoli cells in the removal of cellu-
lar debris. These macrophages were prevalent both in
the lumen as well as the germinal epithelium. During
spermiogenesis of several avian species a mantle or
hood of Sertoli cytoplasm associated with the acroso-
ma1 region of elongating spermatids, analagous to that
previously described during spermiogenesis of reptiles,
has been described (Fig. 27), e.g., G. domesticus, (Cook-
sey and Rothwell, 1973; Sprando and Russell, 1988b);
5'. roseogrisea (Mattei et al., 1972a); Lonchura striata
(Kondo et al., 1988).
As in P. scripta, the spermatid cytoplasm is pene-
trated by Sertoli cell processes in G . domesticus (Sp-
rando and Russell, 1988b). Although these processes
also sequester portions of spermatid cytoplasm, how-
ever, unlike in reptiles there was no evidence to indicate
these packets of spermatid cytoplasm were removed by
the Sertoli cell. Instead, it appeared that excess cyto-
plasm was primarily eliminated by means of a lobe,
developed by spermatid cytoplasm forming the mantle,
that was pinched off at spermiation to produce a resid-
ual body (Sprando and Russell, 198813).
Several reports on avian spermatogenesis have re-
marked that germ cell stages do not appear to be as
closely synchronized as in mammals, e.g., Breuker
(1982). This is apparently due to the fact that in birds
fewer spermatogonial mitotic divisions are synchro-
nized with the spermatogenic cycle (Lin and Jones,
1992). This explains therefore, why only small portions
of the avian germinal epithelium are occupied by dis-
tinct germ cell stages and is reminiscent of the primate
mosaic pattern of spermatogenesis.
An excellent study has been carried out on the evo-
lution of the RER during spermiogenesis of G. domes-
ticus (Xia et al., 1986). In early round spermatids the
RER is organized into several different systems con-
sisting of either a loose network of tubular cisternae
scattered throughout the cytoplasm, or long flattened
cisternae occurring singly or in layers of 2-3 running
parallel to the nucleus, or a collection of tubular cis-
ternae opposite thickened plaques of the plasma mem-
brane, or as a tight network of anastomosing tubular
cisternae (6-8) associated with spherical vesicles
which was called an alveolar body. All these elements
of the RER appeared contiguous with each other. A
stack of annulate lamellae was usually found in close
proximity to the alveolar body. With elongation of the
spermatid nucleus the flattened cisternae of RER dis-
appear but the tubular cisternae now become ex-
panded. As the spermatid matured further with con-
492 J. PUDNEY
densation of the nuclear chromatin the network of
tubular cisternae disintegrated into vesicles that be-
came highly distended to give the cytoplasm a vacuo-
lated appearance. The alveolar body is apparently
maintained just prior to spermiation when it undergoes
dissolution. All RER remnants are subsequently shed
with the residual body. Although spermatid RER in
this avian species undergoes distinct structural change
during spermiogenesis the exact physiological rele-
vance of these interesting transformations is as yet un-
known.
A great deal of interest has centered on the develop-
ment of the manchette during avian spermiogenesis,
and a detailed study of this structure has been carried
out by McIntosh and Porter (1967) in G. domesticus.
Initially the highly ordered array of microtubules
which surround the elongating nucleus was found t o be
a left-handed double helix with cross-bridges connect-
ing each helix. With further maturation the helical set
of microtubules is replaced by a system of straight mi-
crotubules running parallel to the long axis of the nu-
cleus. It is uncertain from the literature whether two
different transitory sets of microtubules appear during
spermiogenesis, e.g., McIntosh and Porter (1967) and
Nicander (1967), or whether only one system of micro-
tubules occurs which simply changes orientation, e.g.,
Okamura and Nishiyama (1976); Breuker (1982);
Mattei et al, (1972a). This development of two sets of
differently orientated microtubules is characteristic
of nonpasserine spermiogenesis, e.g., G. domesticus
(McIntosh and Porter, 1967; Nicander, 1967); C . olor
(Breuker, 1982).R. americans (Phillips and Asa, 1989).
By contrast, in passerine species only one set of micro-
tubules appears which forms a twisted bundle that
winds around the short nucleus and proximal portion of
the flagellum during formation of the corkscrew-
shaped spermatozoa that is characteristic of these birds
(Kondo et al., 1988; Nicander, 1967).
At a later stage of spermiogenesis, the manchette
disappears. There is some controversy as to the func-
tion of the manchette. Several studies have produced
evidence to suggest microtubules act as a scaffold to
mold the shape of the nucleus as it elongates in the
developing spermatid, e.g. Kondo et al. (1988); McIn-
tosh and Porter (1967). Other reports indicate that the
manchette is not involved in nuclear shaping that is
controlled by intranuclear mechanisms such as conden-
sation of the chromatin (Fawcett et al., 1971).It may be
that species vary in the involvement of the manchette
microtubules in the molding of the nucleus. Certainly
in passerine species there appears good evidence that
the helical microtubules play a role in the formation of
the spiral nucleus characteristic of these birds.
During spermatogenesis a variety of Sertoli cell/
germ cell junctions has been described in the avian
testis. Contact between germ cells (spermatocytes
and/or spermatids) and Sertoli cells via desmosomes,
desmosome-like or presumed gap junctions has been
commonly detected (Breuker, 1982; Cooksey and Roth-
well, 1973; Osman, et al., 1980; Scheib, 1972; Sprando
and Russell, 198713). Ectoplasmic specializations are
developed by Sertoli cytoplasm associated with the ac-
rosomal region of elongate spermatids in G. domesticus
(Cooksey and Rothwell, 1973; Osman et al., 1980; sp-
rando and Russell, 1987a). The structural appearance
of the avian ectoplasmic specializations differ from
those produced by mammalian Sertoli cells. Filaments
(6 nm) do not form an organized linear array but ap-
pear as a tangled mass and were often associated with
microtubules. Furthermore, these studies also reported
the absence of subsurface cisternae of ER which form
part of the mammalian ectoplasmic specializations. Ec-
toplasmic specializations between elongate spermatids
and Sertoli cells were reported not to be present in the
testis of C . olor (Breuker, 1982).
There are several distinctive features of spermato-
genesis that distinguish passerines from nonpasserine
species. As noted previously, passerines develop only
one set of spiral microtubules during spermiogenesis,
whereas nonpasserines produce two sets, one spiral fol-
lowed by a longitudinal array. Furthermore, in contrast
to nonpasserines, passerine spermatozoa do not possess
a perforatorium. Also during spermiogenesis, nonpas-
serine spermatids are arranged as individual cells em-
bedded in Sertoli cytoplasm similar to other amniotes,
whereas in passerines the spermatids are collected into
bundles reminiscent of spermatogenesis in the anam-
niotes (Denehy, 1976; Humphreys, 1972).
Several studies have reported on the fine structure of
the avian spermatozoon, e.g., G. domesticus (Bakst and
Howarth, 1975; Thurston and Hess, 1987; Tingari,
1973);Eudromia elegens elegens (Asa et al., 1986);Grus
vipio (Phillips et al., 1987);Numida meleagrir and Me-
leagris gallopavo (Thurston and Hew, 1987); Rhea
americans (Phillips and Asa, 1989).In general, nonpas-
serines produce what has been termed sauropsid sper-
matozoa due to their resembling reptilian spermatozoa.
These sauropsid spermatozoa are characterized by a
small acrosome, a perforatorium, a well-defined mid-
piece containing two centrioles, and they move by a
lashing motion of the flagellum; by contrast passerines
produce spiral spermatozoa that do not have a perfora-
torium and progress by helical forward movement (Bac-
cetti et al., 1980; Humphreys, 1972; Yasuzumi, 1974).
Species differences obviously occur in the morphology of
avian spermatozoa. Thus, Serinus canaria and P. do-
mesticus produce spermatozoa which possess an undu-
lating membrane that runs from the tip of the acrosome
to the end of the flagellum (Humphreys, 1972; Nagano,
1962). In G. vipio it has been reported that chromatin
condensation is not complete in mature spermatozoa
which was assumed to be responsible for the large vari-
ation in the shape of the sperm head in this species
(Phillips et al., 1987).Spermatozoa of the primitive bird
E. elegens elegens develop a sheath of cytoplasm con-
taining glycogen that surrounds the flagellum and a
long axial canal that extends through the length of the
nucleus (Asa et al., 1986).
CONCLUSION
As can be appreciated from this review, a large body
of literature exists describing spermatogenesis in non-
mammalian vertebrates. This is, however, limited com-
pared to the mass of information that is available on
spermatogenesis in mammals. Furthermore, several
caveats hold concerning the quality and quantity of
 SPERMATOGENESIS I N NONMAMMALIAN VERTEBRATES
reports which can be obtained on mammalian versus
nonmammalian spermatogenesis.
First, relatively few comprehensive studies have
been carried out describing spermatogenesis through-
out the reproductive cycle for nonmammalian species.
Moreover, in many cases cytological analysis of germ
cell development and maturation has been limited to
only a few representative species of a particular non-
mammalian class or order. Due to the extreme diver-
sity in reproductive strategies which can be displayed
by nonmammalian species, it is often difficult, there-
fore, to generalize on the cytology of spermatogenesis
in many classes or even orders of these vertebrates.
Second, this problem is compounded by the fact that
spermatogenesis in few nonmammalian vertebrates
has been analyzed using modern microscopic tech-
niques such as scanning electron microscopy, freeze-
fracture analysis, immunocytochemistry, morphome-
try. This has resulted, unfortunately, in placing
reliance on older reports for cytological descriptions of
spermatogenesis in many nonmammalian vertebrates.
These studies were often carried out using, by contem-
porary standards, suboptimal conditions for preserva-
tion of tissue and microscopic analysis. This is espe-
cially true for many seminal papers on the fine
structure of germ cell maturation of various nonmam-
malian species.
Due to the great variety in the organization of the
testis and pattern of spermatogenesis that can be found
in nonmammalian vertebrates, certain species provide
excellent models for analyzing mechanisms that regu-
late germ cell development and maturation.
For instance, in amniotes it is difficult to investigate
specific Sertoli/germ cell interactions due to the com-
plexity of the germinal epithelium which may contain,
at any one time, several successive generations of ma-
turing germ cells. This problem can be circumvented,
however, by utilizing anamniote species that exhibit a
spermatogenic wave. Thus, in elasmobranchs a sper-
matogenic wave exists at the level of the cysts, result-
ing in a topographical segregation of germ cell devel-
opmental stages in the testis. In S. acanthias this
favorable anatomical arrangement can be visualized
with the naked eye, thus allowing dissection of the tes-
tis into regions containing discrete germ cell stages.
Advantage has been taken of this convenient organi-
zation of the testis to analyze germ cell stage-specific
variations in steroidogenesis in the testis of S. acanth-
zas (Callard et al., 1985). This is just one example that
emphasizes the usefulness of species outside the nor-
mal range of common laboratory animals which, due to
the organization of the testis, provide excellent models
for investigating basic reproductive processes.
There is another interesting feature of spermatogen-
esis in anamniotes that makes them excellent experi-
mental models for analyzing germ cell development
and maturation. Attempts to maintain spermatogene-
sis in vitro using the amniote testis (particularly mam-
mals) has received little success. By contrast, it has
been shown that it is possible to culture in vitro iso-
lated amphibia spermatogonia and maintain them
through several developmental stages even up to elon-
gate spermatids (Abe, 1981; Risley, 1983). Thus, in the
493
amphibia, at least, the advantages of in vitro experi-
mental techniques can be used to further understand
processes involved in maturation of germ cells. The
ability to culture isolated amphibian germ cells, and
possibly germ cells from other anamniote species, is
probably related to the fact that during spermatogen-
esis in vivo, the early germ cells mature in association
with a morphologically poorly differentiated Sertoli
cell. Since usually structure reflects function it is pos-
sible that the Sertoli cells, therefore, are not highly
active and so play a minor role in maintaining and
regulating germ cell development. This would suggest
that in many species of anamniotes the early stages of
spermatogenesis progress mostly independent of the
Sertoli cell.
In conclusion, as previously discussed (Pudney et al.,
19851, to approach and elucidate basic problems in re-
productive biology more attention should be paid to
choosing the correct and appropriate animal model. A
wide range of species displaying different reproductive
strategies is available to investigators of male repro-
ductive biology. No one species per se is capable of pro-
viding all the answers. By judicious selection, however,
a particular species can be chosen because, due to some
novel morphological parameter, it is more suited for
studying one particular aspect of male reproduction.
This illustrates and defines the importance of studying
so-called unconventional animal models to understand
more completely phenomena associated with male re-
production.
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