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Homeobox Genes and the Male Reproductive System

Article · January 2002


DOI: 10.1007/978-1-4615-0679-9_15

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HOMEOBOX GENES AND THE MALE REPRODUCTIVE SYSTEM

Manjeet Rao and Miles F. Wilkinson

Department of Immunology
The University of Texas
M. D. Anderson Cancer Center
Houston, TX 77030

INTRODUCTION

Ever since the pioneering work of Jacob and Monad in the 1960s, it has been clear
that there is a class of genes that dictates biological events by regulating the transcription of
subordinate downstream genes. Drosophila geneticists hypothesized that mutations in
these regulatory genes were responsible for a bizarre set of phenotypes in a class of mutant
flies, in which there were striking partial or complete transformations of body segments.
For example, the bithorax homeotic mutation converts the identity of the haltere-carrying
thoracic segment into a wing-carrying segment, thereby generating four-winged flies. The
antennapedia homeotic mutation transforms the antennae on the head into legs.
The advent of advanced molecular biology techniques in the early 1980s permitted the
isolation of the first genes responsible for these homeotic mutations. By chromosome
walking and hybridization techniques, Gehring, Scott, Weiner, and colleagues isolated the
anntennapedia. foshi tarazu, and Ultrabirthorax genes (Garber et aI., 1983; Scott et al.,
1983; Scott and Weiner, 1984). Each of these homeotic genes contains a similar -180-nt
sequence named the homeobox that encodes a 60 amino-acid domain composed of three
alpha helices. The third helix of this homeodomain makes base-specific contacts with
DNA, consistent with the notion that homeobox proteins are transcriptional regulatory
proteins.
The next breakthrough was the discovery that most homeobox genes are remarkably
highly conserved. Using Southern-blot analysis with D. melanogaster homeobox probes,
cross-hybridizing bands were found in vertebrate DNA. This led to the cloning of
homeobox genes from a variety of species, including Xenopus laevis, chicken, and mouse
(reviewed by Duboule, 1994). The function of the homeobox proteins encoded by many of
these mammalian homeobox genes was determined by generating and analyzing knockout
mice, in which both (null mice) or one allele of a gene is rendered nonfunctional. These
studies showed that homeobox proteins regulate a variety of embryonic events, including
establishment of the anterior-posterior body axis and organogenesis. In contrast, less is
known concerning the role of homeobox genes in postnatal and adult stages. Homeobox
proteins regulate these diverse biological events by controlling the transcription rates of
specific target genes (Duboule, 1994). Thus, the original hypothesis that homeobox genes
encode regulatory proteins was shown to be correct.
More than 30 mouse homeobox genes are expressed in the male reproductive tract. At
least ten of these are expressed in the epididymis (Table 1). In this chapter, we will review

The Epididymis: From Molecules to Clinical Practice


Edited by Robaire and Hinton. Kluwer Academic/Plenum Publishers. 2002 269
what is known about the expression and function of homeobox genes in the epididymis as
well as in other male reproductive organs.

Table 1. Homeobox genes expressed in the epididymis

Gene Expression pattern Epididymal function

Hoxa-lO Embryo: cauda epididymidis Anterior-posterior identity (knockout mice


to prostatic anlagen of the urogenital sinus exhibit homeotic transformation of corpus to
caput epididymidis and proximal ductus
deferens to cauda epididymidis)
Hoxa-ll Embryo: stromal cells ofWolffian Anterior-posterior identity (knockout mice
ducts exhibit partial homeotic transformation
of vas deferens to epididymis)
Hoxa-13 Postnatal: most accessory sex organs, Not known
probably epididymis
Hoxd-IO Late embryo/newborn: epididymis Not known
and testis
Hoxc-8 Postnatal: downregulated Not known
in epididymis after birth
Hoxd-4 Postnatal: epididymis Not known

Pax-2 Embryo: Wolffian duct; Specification of epididymis and the entire


Adult: all regions of epididymis urogenital tract (knockout mice lack the
epididymis)
Pem Androgen regulated; upregulated after birth Not known
until sexual maturity; caput-specific in mice

Pem2 Epididymis specific; induced on Not known


day 23-30 p. p.; androgen independent
Testesl Adult: somatic cells Not known
(Oct-6)

HOXGENES

Homeobox genes are classified on the basis of the specific sequences in their
homeodomains. The largest class of hom eobox genes in mammals is called Hox (Dubuole,
1994). The mammalian Hox genes are organized into four clusters (a-d) on separate
chromosomes. Within each cluster, the Hox genes are numbered 3' to 5' (e.g., the most 3'
Hoxa gene is a-I and the most 5' is a-J3). Remarkably, the linear order ofthe mouse Hox
genes within each cluster is the same as that of their counterpart genes (Hom) in D.
melanogaster. The expression pattern of the Hox genes is also conserved both spatially
and temporally. 3' Hox genes are expressed more anteriorly and earlier in development,
while 5' Hox genes are expressed more posteriorly and later.
Male reproductive tissues in vertebrate embryos express 5' Hox genes (Table 2)
probably because many reproductive tissues arise late in gestation and in the posterior
portion of the developing vertebrate embryo (Dolle et al., 1991 and references therein).
Several 5' Hox genes are required for the normal development of the urogenital tract, as
disruption of them has profound effects, as discussed below.

5' Hoxa Genes

The expression of a-JO is temporally regulated in the Wolffian duct and urogenital
sinus. In situ hybridization analysis demonstrated that a-IO mRNA is present in a region
extending from the caudal epididymis to the prostatic anlagen of the urogenital sinus
(podlasek et al., I 999b). The anterior boundary of this region is defined by a sharp margin
between the cauda epididymis and the ductus deferens. a-IO mRNA levels are maximal

270
during late gestation (embryonic day (e) 18-19) and are then downregulated postnatally
before sex-organ differentiation is complete. a-10 is expressed in cultured e15 or e18
urogenital sinuses in the absence of androgen (dihydrotestosterone). Thus, even though
postnatal sexual differentiation requires androgens, a-10 is expressed in an androgen-
independent manner, at least in vitro.

Table 2. 5' Hox genes expressed in the male reproductive system

Gene Male reproductive pattern Reproductive phenotype in knockout References


expression mice

Hoxa-1O Wolffin duct and urogenital Cryptorchidism Riyli et ai, 1995


sinus Homeotic transformations Benson et aI., 1996
Podlasek et aI., 1999b
Hoxa-ll Urogenital sinus Cryptorchidism Hsieh-Li et aI., 1995
Homeotic transformations
Hoxa-13 Urogenital sinus and Abnormal seminal vesicles in Hd'" Yokouchi et aI., 1995
seminal vesicle heterozygote (HdlHd is embryonic Warot et aI. , 1997
lethal) Podlasek et aI., 1999b
Hoxb-13 Urogenital sinus Being characterized Zelster et aI., 1996
Hoxc-8 Postnatal epididymis Not known Lindsey and Wilkinson,
1996b
Hoxc-ll Genital tubercle No knockout Hostikka & Capecchi,
1998
Hoxd-IO Genital tubercle Sterile as a result of hindlimb defects, Dolle et aI., 1991
no known male reproductive system
defects
Hoxd-ll Urogenital tract Hypofertile for unknown reasons Davis & Capecchi., 1994
Hoxd-12 Genital tubercle Fertile and viable Dolle et aI. , 1991
Hoxd-13 Lower genital tract Hypofertile, probably as a result of Podlasek et aI., 1997
epithelium and several sex-organ defects Warot et ai, 1997
mesenchyme

Evaluation of a-1O'/' knockout mice revealed that a-1O is required for the normal
development of four accessory sex organs: the seminal vesicles, the anterior prostate, the
ductus deferens, and the epididymis. a-IO's role in seminal vesicle formation comes from
the fmding that a-l0 knockout mice have abnormal seminal vesicles with diminished
stromal clefting (Rijli et aI., 1995; Podlasek et aI., I 999b ). a-lOis also important for the
normal branching of the anterior prostate (also called the coagulating gland), which
exhibits decreased branching and is smaller in a-1O'/' mice (podlasek et aI., I999b).
The role of a-lOin specifying the anterior-posterior identity of the epididymis and
other accessory sex organs came to light as a result of three apparent homeotic
transformation events that occur in a-10'/' mice. First, the corpus region of the epididymis
in a-1O'/' mice acquires the morphological characteristics of the more anterior region (the
caput), such that it is wider and the coils of the tubule are more densely packed (Benson et
aI., 1996). Second, the proximal end of the ductus deferens gains the characteristics of the
cauda epididymidis in mutant mice (Benson et aI., 1996). This apparent anteriorization
event was confirmed by the finding that in a-I 0'/' mice, endogenous p-galactosidase
activity, which is normally restricted to the caput and corpus epididymidis, is also present
in the cauda epididymidis and the proximal ductus deferens. Third, about -30% of null
mutants have alterations in the ductal architecture of the anterior prostate that suggest
partial posterior morphological transformation with incomplete penetrance (Podlasek et aI.,
1999b).
These three partial transformation events in a-10'/' mutant mice suggest that a-l0
controls t he identity of the epididymis, ductus deferens, and anterior prostate along the
anterior-posterior axis. To determine the extent of these transformation events, it will be
essential to examine the expression of genes that are normally expressed in these organs.

271
For example, several caput-specific epididymal genes have been identified (Cornwall and
Hahn, 1995; Orgebin-Crist, 1996; see Cornwall et al. and Kirchhoff et aI., this volume) that
could be used as markers to determine how completely the corpus epididymidis has
converted to a caput-like character. Likewise, the expression of cauda epididymidis-
specific genes could be used to assess the extent of proximal ductus deferens
transformation into cauda epididymidis. Such analyses could also provide clues about the
molecules downstream of a-l0 that are responsible for controlling the identity of the
accessory sex organs.
It is interesting that while a- 10.1- male mice exhibit anterior transformation of the
epididymis and ductus deferens, a- 1(fl- females undergo anterior transformation of the
proximal uterus to oviduct (Bensen et al., 1996). This suggests that common molecules
controlled by a-lO are involved in both male and female reproductive organogenesis.
The next gene in the Hoxa cluster, a-ll, is also expressed in embryonic reproductive
tissue. a-l 1 transcripts are not detectable in the gonads themselves but are expressed in the
cells surrounding the Wolffian and Mullerian ducts as well as in the developing kidney
(Hsieh-Li et aI., 1995). Likewise, in adult mice, a-ll transcripts are present in stromal
cells surrounding the vas deferens. No expression is detectable in the cells of the epithelial
lining.
The a-Il locus gives rise not only to sense transcripts encoding a-II protein but also
to antisense transcripts. These antisense RNAs are processed, polyadenylated, and
expressed in various organs of the developing embryo, including the male reproductive
tract. Because sense and antiseqse a-ll transcripts exhibit an inverse pattern of expression
in developing limbs, the antisense transcripts may be negative regulatory molecules that
decrease the levels of the sense-coding transcripts by a ribonuclease H-mediated
mechanism.
a-l rl - adult mice ha ve several de fects in t he m ale reproductive tract. One of the
defects is abnormal spermatogenesis and sterility as a result of unilateral or bilateral
cryptorchidism (Hsieh-Li et al., 1995). Although prior to sexual maturity, the a-l r l - testis
is indistinguishable from the wild-type testis, in adults the testis is smaller and consistently
does not completely descend into the scrotal sac. Spermatogenesis is abnormal, such that
the most severe cases have a "Sertoli-only" phenotype in which many seminiferous tubules
in mutant testes contain few and/or apoptotic germ cells. In addition, mutant epididymides
have few sperm and instead contain mainly cellular debris and fluid. These spermatogenic
defects probably result from the cryptorchidism but it is possible that a-II also has direct
effects on spermatogenesis.
The other major defect in a-l r l - mutant mice is a malformation of the vas deferens
that resembles a partial homeotic transformation of the epididymis (Hsieh-Li et aI., 1995).
The transformed portion of the vas deferens is smaller, more tightly coiled, and has smaller
lumens, particularly at the proximal end near the junction of the vas deferens and cauda
epididymidis. The epithelial lining is also abnormal, as it exhibits less complex mucosal
folding. Mutant epithelial lining cells are narrower and more columnar and the nuclei are
more basal. In contrast to wild-type epithelial lining, which generally has only a single
layer of cells, the mutant has a more stratified organization of epithelial cells lining the vas
deferens.
It is not clear whether such defects in the vas deferens result from the absence of a-II
during embryonic gestation, postnatal development, or both. Because a-ll mRNA is
expressed both by precursors of the vas deferens during embryonic development and in the
vas deferens proper in adult animals, a-II has the potential to act both before and after
birth. Studies examining mutant late-gestation embryos and newborns may clarify the role
of a-II in the specification of the vas deferens.
The most 5' gene in the Hoxa complex, a-13, is also expressed both embryonically and
postnatally in male reproductive tissues. During mouse midgestation (eI2.5-14.5), a-13
mRNA is expressed in the genital tubercle, which first appears as a bud of proliferating
mesenchyme a round e 11 and ul timately gives rise tot he penis or clitoris (Dolle et al.,
1991; Yokouchi et al., 1995; Warot et al., 1997). Expression of a-13 in genital tubercle
conforms to the "temporal colinearity" rule ofHox gene expression, as a-13 is the most 5'
Hoxa gene and the genital tubercle is the last structure to develop along an independent
axis during mammalian embryogenesis. In situ hybridization and reverse transcription-

272
polymerase chain reaction (RT-PCR) analyses demonstrated that a-13 mRNA is also
expressed by both mesenchymal and epithelial cells in the urogenital sinus between e12.5
and e16.5 (Warot et aI., 1997; Podlasek et aI., 1999a). At eI4.5, a-13 mRNA is also
expressed in the caudal portion of the Wolffian ducts and in the walls of the urinary bladder
(Warot et al., 1997).
a-13 mRNA is present in most male reproductive structures in newborn mice (Warot
et al., 1997; Podlasek et aI., 1999a). a-13 mRNA levels in seminal vesicles are low at
birth, peak during the mid-postnatal period (between days 5 and 10 p.p.), when the lateral
branches of the central lumen arise and undergo secondary and tertiary branching; and
expression progressively decreases until adulthood. In the urogenital sinus and prostate,
expression is highest before birth. Although it diminishes slightly after birth, a-I3 mRNA
expression remains relatively high during the postnatal period, when most of the prostatic
main ducts form and prostate ductal morphogenesis is most intense.
a-13-I - mice die in utero between ell.5 and e15.5 and have severe genitourinary
malformations (Warot et aI., 1997). This embryonic-lethal phenotype is unique to a-I3, as
disruption of other Hox genes permits survival into adulthood or causes postnatal lethality
(Chisaka and Capecchi, 1991; Rijli et al., 1995). To examine the reproductive function of
a-l3, hypopdactyly (He!) mutant mice have been studied. These mutant mice possess digit
abnormalities as a result of a deletion in exon 1 of the a-13 gene, which results in a
semidominant mutation with full penetrance. Homozygous HdJHd mutant mice generally
die late in gestation and hence are not useful for examining postnatal male reproductive
function, however, HdJ+ heterozygotes are viable. Although they have normal-sized testes
and are fertile, close analysis of HdJ+ mice revealed that they have abnormal accessory sex
organs (podlasek et aI., 1999a). In particular, the seminal vesicles of most HdJ+ animals
are small and have morphological abnormalities. This defect is specific to the seminal
vesicle, as other derivatives of the Wolffian duct, inc luding the epididymis and duc tus
deferens, are normal. However, derivatives of the urogenital sinus also show abnormalities.
The dorsal, lateral, and ventral prostate lobes of HdJ+ mutant mice are smaller and have
fewer duct tips than do wild-type mice. In addition, HdJ+ mice occasionally exhibit
abnormalities in lobe development, such as having a single coagulating gland main duct (as
opposed to the usual two) and agenesis of the dorsal prostate lobe.
HOXA-13 is the only human HOX gene that has been linked with a genitourinary
disorder. Hand-foot-genital syndrome, which is characterized by abnormalities of the
digits and uterus, is caused by a premature termination mutation in the human A-13 gene
(Mortlock et al., 1997). The encoded protein is shortened by 20 amino acids and probably
has reduced DNA-binding ability. Curiously, this mutation apparently does not affect male
reproductive function. It will be interesting to determine whether other 5' HOX gene
mutations are responsible for male reproductive dysfunction, including cryptorchidism and
hypospadias, which are amongst the most common congenital abnormalities of the human
male reproductive tract.

5' HoxdGenes

Like the 5' Hoxa genes, the 5' Hoxd cluster genes are expressed in several different
male reproductive tissues. Much of what we know about their expression comes from a
comparative in situ hybridization analysis of the five most 5' Hoxd genes from early
rnidgestation (elO.5) to the postnatal period (day 7 p.c.) (Dolle et aI., 1991). This analysis
showed that 5' Hoxd genes are expressed by the genital tubercle as soon as it arises at
elO.5. The expression of each 5' Hoxd gene correlates with its position in the cluster such
that the most 5' gene, d-13, is the most highly expressed, d-ll and d-I2 are expressed at
moderate levels, d-IO is expressed at only trace levels, and d-9 transcripts are not
detectable. These levels are maintained through late gestation but decrease somewhat at
birth and postnatally. The spatial pattern of the expression of the individual 5' genes in late
gestation and newborns corresponds to their position in the locus in the classic Russian-doll
pattern characteristic of Hox genes. The most 5' gene, d-13, is expressed in urinary bladder
and vas deferens but not in epididymis, whereas its neighbor, d-I2, has an overlapping
pattern of expression that extends more anteriorally within the vas deferens. d-IO

273
transcripts are present even more anteriorally, in the epididymis and testis, whereas more
posterior organs have only low dolO mRNA levels.
Paralogs are genes that occupy the same relative position within Hox gene clusters.
Hox gene paralogs often exhibit similar patterns of expression (Duboule, 1994). In
agreement with this generality, d-13 is expressed in a similar pattern in the urogenital tract
as its Hoxa cluster paralog, a-13. During midgestation (eI2.S), both a-13 and d-13
mRNAs are expressed in the mouse genital tubercle and in both mesenchyme and
epithelium of the urogenital sinus (Dolle et al., 1991; Oefelein et al., 1996; Warot et al.,
1997). By eI4.S, both a-13 and d-13 are expressed in the caudal portion of the Wolffian
ducts and in the walls of the urinary bladder (Warot et al., 1997). After birth, both genes
are expressed by all lower genitourinary tract tissues, including the prostate, seminal
vesicles, and epididymis (Podlasek et al., 1997; Warot et al., 1997). Although expression
of d- 13 mRNA persists in the prostate and seminal vesicles of adult mice, the level in
adults is lower than in newborns as a result of a progressive decline in mRNA levels
postnatally (Podlasek et al., 1997).
Both d-ll and d-13 are critical for normal male reproductive function, as both dol r l -
and d-13-I - mice are either infertile or hypo fertile (Davis and Cappacchi, 1994; Dolle et al.,
1993). The basis for the sterility of d-ll null mice is not yet known, as these mice have no
obvious male reproductive tract abnormalities (Davis and Cappacchi, 1994). Sperm are
present in normal amounts in d-l r l - vas deferens, have a normal appearance, and are
motile.
In contrast, d-13-I - mice exhibit obvious sex-organ defects, which may reflect d-13's
broad expression pattern in the male reproductive tract. The most striking abnormalities
observed in d-13-1- mice are in organs generating branching ducts during postnatal
development, including the seminal vesicles, the bulbourethral gland, and several regions
of the prostate (Podlasek et al., 1997). Other organs displaying defects are the ampullary
gland, the bladder neck, and the urethral gland. Interestingly, the levels of d-13 expression
in different tissues do not reflect the necessity of d-13 for normal tissue development. For
example, the epididymis, ductus deferens, and bladder express d-13 mRNA but there are no
obvious defects in these tissues in null mice.
The diverse morphological abnormalities observed in d-13-I - mice suggest that d-13
regulates not only embryonic development but also three postnatal events: remodelling of
mesenchyme in the seminal vesicle, the normal budding process that occurs in the
urogenital sinus, and duc t growth in the prostate (Podlasek et al., 1997). The latter is
consistent with the fact that many homeobox proteins promote cell growth and
oncogenesis. However, Podlasek and colleagues (1997) were unable to demonstrate a
correlation between d-13 expression and the known mitotic activity of cells in particular
segments of male reproductive organs.

S' Hoxa / Hoxd Double Mutants

The expression do mains 0 fa -13 and d- 13 0 vedap spatially and temporarily in the
embryonic genitourinary tract and during postnatal development of the male accessory sex
organs. This allows one to examine the individual contributions of these two highly similar
transcription factors. Mutations in a-13 have a somewhat more dramatic effect on
morphogenesis than do d-l3 mutations, which suggests that a-13 is the more important of
the two for some events (Podlasek et al., 1997; Warot et al. 1997). To elucidate more
clearly the specific relative contributions of a-13 and d-13, mice defective in both genes
have been analyzed. One double mutant mouse line examined has the semidominant Hd
allele of a-13 and is null for d-13. The HdlHd / d-13-I - progeny of this line die in utero
before e16 (as do HdlHd / d-13+ I + mice). These double-homozygous mutant embryos have
defects in the formation of the genital bud and completely lack external genitalia, the lower
urinary tract (bladder and urethra), and derivatives of the urogenital sinus (Kondo et al.,
1997). Another study examined mutant embryos containing a laboratory-generated a-13
null (presumably recessive) allele. These a-13-I - / d-13-I - embryos completely lack the
genital tubercle and do not complete the normal separation of the urogenital sinus and the
terminal hindgut at e12.S (Warot et al., 1997).

274
To examine the relative roles of a-l3 and d-l3 in later developmental events, mice
heterozygous at the a-13 locus were examined, as many of these mice survive into the
postnatal period. These a-13+1• / d-I]"I. double mutants have more abnormalities than do d-
13-1- single mutants (Warot et al., 1997). Double mutants typically lack the preputial gland,
the anterior prostate, and the bulbourethral gland, whereas all of these accessory sex organ
structures are usually present in a-13 or d-13 single mutants (except that the d-13-I- mice
exhibit agenesis of the bulbourethral gland approximately half the time). This clearly
indicates that a-13 and d-l3 have the same function in the development of these organs. In
contrast, a-l3 and d-l3 exert additive effects on the development of the seminal vesicle, as
a-13+I- / d- 13-1- double mutants have severe seminal vesicle hypoplasia, whereas mice
mutant for only one of these genes have only subtle defects of the seminal vesicle
(diminished size and clefting). The function of a-l3 and d-l3 is quite specific to the
accessory s ex 0 rgans, as t he do uble mutants ha ve no obvious morphological defects in
other male reproductive organs, including the epididymis, testis, and ductus deferens.
Double mutants in which the d-13 locus is heterozygous (a-13· I - / d-13+I - mutants)
have normal genital tubercles and a proper separation between the urogenital sinus and
hindgut, but the entire ur ogenital sinus is rudimentary a nd I acks a presumptive ur inary
bladder primordium (Warot et aI., 1997). It remains to be determined why the subtle
reduction in Hox gene dosage in heterozygotes has such a profound effect on the
development of specific structures in the male reproductive tract.

S' Hoxb and Hoxc Genes

Because Hox paralogs are often functionally redundant, the 5' genes in the Hoxb "
Hoxc clusters may also participate in male reproductive tract development or functiol.
Unfortunately, little is known about the expression of most of these genes, and mice null
for Hoxb and Hoxc cluster genes are only just being produced (Table 2). c-ll is one of the
better candidates to regulate male-reproductive events, as it is expressed in the genital
tubercle and the undifferentiated mesenchyme surrounding the urogential sinus at
midgestation (Hostikka and Capecchi, 1998). Preliminary data from c-l r l - mice suggest
that c-11 is important in multiple facets of urogenital development (Mario Capecchi,
personal communication). In contrast, neither b-13 nor c-13 appears to be involved in male
reproductive tract development (Peterson et al., 1994). b-13 rnRNA is expressed in the
urogenital sinus at e12.5, but not in the genital tubercle (Zeltser et aI., 1996), and b-13·I.
mice have no obvious male reproductive tract dysfunction (Mario Cappechhi, personal
communication). Clearly, studies examining more null mice will be required to determine
the precise role of each 5' Hoxb and Hoxc genes in the embryonic and postnatal
development of the male reproductive system.

3' Hox Genes

At least eight 3' Hox genes are expressed in the mouse testis and other urogenital tract
organs (Table 3). The expression patterns of most of these genes have been discussed in
detail elsewhere (Wolgemuth et al., 1991; Lindsey & Wilkinson, 1996). The stage-specific
expression of many of these 3' Hox genes in the testis suggests that the transcription factors
they encode regulate downstream genes critical in particular steps in spermatogenesis.
Surprisingly, however, little or no evidence has surfaced implicating any 3' Hox gene in
testis function (Horan et al., 1994, 1995, Lucie Jeannotte, personal communication) In
some cases, this is because mice with mutations in many 3' Hox genes die soon after birth,
and therefore it has not been possible to assess their function in postnatal and adult testes.
In other cases, mutant mice do survive until adulthood but there is no obvious phenotypic
effect on the testis. This may be because 3' Hox genes have redundant or context-
dependent functions. However, it is also possible that some 3' Hox genes, particularly
those expressed in male germ cells (Table 3), have no function in the testis. It has been
hypothesized that male germ cells express many "irrelevant genes" because these cells
uniquely undergo major alterations in chromatin structure as a result of the replacement of
histones with transition proteins and protamines. These alterations in chromatin structure
may nonspecifically activate the transcription of genes that provide no function in germ

275
cells. This "graveyard of gene expression" hypothesis is supported by the observation that
postmitotic germ cells express many genes, including proliferation-inducing proto-
oncogenes, that probably do not function in nondividing cells (Propost et ai., 1988). Thus,
while 3' Hox genes play important roles in various embryonic events, including limb
development (Duboule, 1994), some may not have a function in male germ cells.

Table 3. 3' Hox genes expressed in the male reproductive system

Gene Male reproductive expression pattern Reproductive phenotype in


knockout mice
Hoxa-3 Pachytene spermatocytes Viable and fertile
Hoxa-4 Spermatocytes (meoitic prophase), spermatids V iable and fertile
Hoxa-5 Testicular germ cells Postnatal lethality
Hoxa-7 Testicular somatic cells Viable and fertile
Hoxb-4 Testicular germ and somatic cells Viable and fertile
Hoxb-7 Testicular germ cells Viable and fertile
Hoxc-8 Testicular germ cells, epididymal somatic cells Postnatal lethality
Hoxd-4 Epididymal epithelium, Interstitial cells (Leydig?) Viable and fertile

OTHER HOMEOBOX GENES

Prd-Class Homeobox Genes

Several Prd-class homeobox genes are expressed in the male reproductive tract (Table
4). Of these, only Pax-2 is known to be expressed in the epididymis (Fickenscher et ai.,
1993). Its expression is apparently restricted to the epididymis and the ductus deferens;
Pax-2 transcripts have not been detected in testis, prostate, kidney, or seminal vesicle
(Oefelein et ai., 1996). Pax-2'/' mice lack most components of the genital tract, including
the epididymis. They also lack ureters and kidneys and hence die soon after birth. These
multiple defects most likely result from a failure to form the ductal and mesenchymal
components of the developing urogenital system (Torres et al., 1995). The role of Pax-2
appears to be conserved among mammals, as mutations in the human PAX2 gene are also
associated with a variety of urogenital abnormalities (Cunliffe et ai., 1998).

Table 4. Prd-Class Genes Expressed in the Male Reproductive System

Gene Male reproductive Reproductive phenotype in References


expression pattern knockout mice
Pax-2 Epididymal epithelium and No ureters and genital tracts; Fickenscher et aI., 1993,
ductus deferens degenerated Wolffian duct Torres et aI., 1995
Pax-5 Adult testis -5% survive and are fertile Adams et aI., 1992
Urbanek et aI., 1994
Alx Adult testis Viable and fertile Rudnick et aI., 1994
Qu et aI., 1999
Cartl Adult testis Homozygous mutant; Zhao et aI., 1996
die soon after birth

Pem and the PEPP Class of Homeobox Genes

Recently, a new class of homeobox genes has been identified that share the distinctive
characteristic of being expressed almost exclusively in reproductive organs. We have
named this class of genes the PEPP homeobox subfamily after the members that have so
far been identified: Pern, Esx-l (Spx-l), Psx-l, and Psx-2 (Gpbox) (Maiti et ai, 2001). The
PEPP family members are probably derived from a common precursor homeobox gene, as
they all encode related homeodomains and they are all on the mouse X chromosome (Lin et

276
al., 1994; Maiti et al., 1996a; Branford et ai, 1997; Li et al., 1997; Sutton and Wilkinson,
1997a; Takasaki et al., 2000). It is likely that the PEPP genes were derived from a gene
related to the D. melanogaster aristaless homeobox gene, as the two introns in the
homeodomain region are located at a signature position unique to aristaless and PEPP
family members (Maiti et al., 1996a, I 996b; Takasaki et al., 2000). Esx-l and Pem will be
discussed in more detail below because they are expressed in male reproductive organs
(Table 4). Psx-l and Psx-2 will not be discussed here, as they are expressed only in
placenta and other female reproductive organs (Chun et ai, 1999; Takasaki et al., 2000).
Esx-l has the potential to be important in testis, as it appears to be exclusively
expressed in testicular germ cells in male mice. Esx-l transcripts are localized to the
spermatogenic cell epithelium of the seminiferous tubule, with abundant expression in
stage IV-VII spermatogoniaipreleptotene spermatocytes and in round spermatids (Branford
et al, 1997; Li et al., 1997). However, the precise function of Esx-l in germ cells in the
testis is unclear, as Esx-l'/' mutant males are viable and fertile (Li & Behringer, 1998).

Table 5. PEPP Family Member Genes Expressed in Male Reproductive Tissue

Gene Male Reproductive Expression Reproductive phenotype References


Pattern in knockout mice
Pem Primordial germ cells; Sertoli Viable and fertile Lindsey & Wilkinson, 1996a,b;
cells and somatic epididymal Maiti et aI., 1996a,b;
cells in post- natal and adults Pitman et al.,1998;
Sutton et aI., 1998
Pem2 Adult rat epididymis Not known Nhim et aI., 1997
Esxl Spermatogonia, pre- leptotene Viable and fertile Branford et aI., 1997;
{Spx} spermatocytes round spermatids Li et ai, 1997;
Li and Behringer, 1998

Pem, the other PEPP family member expressed in male reproductive organs, is first
expressed in primoridal germ cells (PGCs) during embryogenesis (pitman et al., 1998).
Pem protein is first detected between e7.5 and e.8.S in premigratory mouse PGCs. Pem
expression persists in migrating PGCs until just after they arrive in the male and female
genital ridges (between eI4 and eIS).
In neonatal and adult mice, Pem is expressed in somatic cells of the epididymis, testis,
ovary, and placenta. Pem mRNA is first detectable in both the mouse and rat epididymis
on days 10-12 p.p., and then its levels progressively increase until sexual maturity (days
30-37 p.p.) (Lindsey and Wilkinson, I996a; I 996b). In both the rat and mouse epididymis,
Pem is expressed from a "male-specific" proximal promoter (Pp) (Maiti et al., 1996a;
Sutton et al., 1998). Expression from the Pp is androgen dependent, as hypophysectomized
rats express much lower levels of Pem mRNA in the epididymis, and addition of
testosterone increases Pem mRNA to normal levels (Lindsey and Wilkinson, 1996b; Sutton
et al., 1998). This androgen dependence may explain the postnatal time-course of Pem
mRNA expression, which roughly parallels that of androgen levels in the epididymis.
Although the kinetics and androgen dependence of Pem expression are conserved
between mice and rats, there are differences between these two species in Pem's spatial
expression in the epididymis. In the mouse, Pem mRNA and protein are specifically
localized in a subregion of the caput epididymidis (Pitman et al., 1998; Rao, Wayne, and
Wilkinson, unpublished observations; note that the original localization of Pem mRNA in
the corpus and cauda [Lindsey and Wilkinson, 1996a] was incorrect). A mouse Pem
transgene containing -0.6 kb sequences upstream of the Pp transcription-start site was
expressed at high levels exclusively in the same region of the caput epididymidis as the
endogenous Pem gene in all three independent transgenic mouse lines tested (Rao, Wayne,
Sutton, and Wilkinson, unpublished observations). Thus, this transgene contains all of the
sequences necessary to direct gene expression to a subregion of the caput epididymidis.
Unlike the mouse epididymis, the rat epididymis contains a high percentage of Pem-
expressing cells covering a large region of the epididymis extending from the caput to the

277
cauda (Lindsey and Wilkinson, 1996b; Rao and Wilkinson, unpublished observations).
This broad expression pattern in the rat epididymis probably explains why Pern mRNA
levels are much higher in the rat than the mouse epididymis (Sutton et aI., 1998).
The functional relevance of this difference in Pem expression in the rat and mouse
epididymis is not known. One possibility is that Pem acts only in the caput epididymidis
and that its broader expression pattern in the rat serves no purpose. Alternatively, Pem
may exert additional functions in the rat epididymis (requiring expression by more cells in
a broader region) than in the mouse epididymis. Mice and rats have many differences in
male reproductive physiology (Russell et al., 1990) and hence it would not be surprising
that a transcription factor would be differentially expressed in these two rodents.
In the mouse and rat testis, Pem is expressed only in Sertoli cells (Lindsey and
Wilkinson, 1996a; Pitman et aI., 1998; Sutton et aI., 1998; Maiti et aI., 2001). Pern mRNA
and protein in the mouse testis are first detectable on days 8 p.p., peak one or two days
later, and then remain expressed into adulthood (Lindsey and Wilkinson, 1996a; Pitman et
aI., 1998). Pern transcripts in the mouse testis are derived primarily from the Pp, the same
promoter active in the epididymis (Sutton et aI., 1998). Pem expression in mouse testis in
vivo is dependent on androgen, as shown by using hypophysectomized mice, hypo gonadal
gonadotropin-deficient mice, and androgen receptor-deficient mice (Lindsey and
Wilkinson, 1996a). Similarly, in rat testes, the Pem Pp is dependent on androgen for
expression, as demonstrated in EDS-treated rats (Sutton et aI., 1998). Rat testes also
transcribe Pem's other promoter, the distal promoter (Pd), which is also active in ovary,
placenta, and tumor cells of diverse lineages (Maiti et al., 1996b). Expression from the Pd
is androgen independent (Maiti et al., 1996b; Sutton et al., 1998).
The function of Pem in PGCs, epididymal somatic cells, and Sertoli cells is not
known; Pern-I- mice do not display any obvious defects in any of these cell types (Pitman et
aI., 1998). One possibility is that Pem's function in reproductive tissues is context
dependent and hence it is only evident under particular environmental situations. Pem
contains a conserved redox domain (CPAC) at its carboxy terminus, which may act as a
redox sensor or mediate redox catalysis in response to oxidative stress (Maiti et aI., 1996a,
1996b; Sutton and Wilkinson, 1997b).
That Pem has a function in the epididymis and testis is supported by the finding that
the promoter driving Pem expression in these tissues (the Pp) is expressed in a highly
regulated manner exclusively in these two tissues. As described above, the Pp directs
region-specific expression in the epididymis and it is strongly regulated by androgen in
both epididymal somatic cells and Sertoli cells. In addition, the Pp is expressed in a stage-
specific manner in Sertoli cells, suggesting that Pem may regulate stage-specific events in
seminiferous tubules. In particular, Pern mRNA is selectively expressed during the
androgen-dependent stages 0 f the mouse seminiferous epithelium cycle (stages VI-VIII)
(Lindsey and Wilkinson, 1996a). Likewise, Pem protein is present in mouse Sertoli nuclei
only during these stages (Pitman et aI., 1998; Sutton et al., 1998). It seems unlikely that
the Pern Pp would have evolved to be expressed in an androgen-regulated, and stage-,
region-, and cell type-specific manner in male reproductive tissue unless there was a
selective value for Pem expression.
A clue as to Pem's function came from a recent study demonstrating that Pem is a
potent inhibitor of embryonic stem (ES) cell differentiation into endoderm (Fan et aI.,
1999). This regulation may be physiologically relevant, as Pem is constitutively expressed
at low levels in undifferentiated ES and F9 embryonal carcinoma stem cells but is
dramatically upregulated when these cells are induced to differentiate into visceral or
parietal endoderm (Sasaki et al., 1991; Lin et aI., 1994). Physiological relevance is further
suggested by the fmding that this in vitro regulation is recapitulated in the intact mouse.
Pem is expressed at low levels by early mouse embryonic cells (as early as the morula
stage) and then upregulated in visceral and parietal endoderm during midgestation (Sasaki
et aI., 1991; Lin et aI., 1994). Although it is intriguing that Pem regulates endodermal
differentiation, it remains to be determined whether Pem also regulates the differentiation
of PGCs or somatic cells in the testis and epididymis.
Pern is unique among mammalian homebox genes in that its homeodomain has
undergone rapid evolution. Sequence analysis of the Pem homeodomain from 12 different
rodent species revealed that its DNA-binding region (the third helix) is highly conserved

278
but that the regions involved in putative protein-protein interactions have undergone rapid
directional selection (Sutton and Wilkinson, 1997b). This suggests that while Pem's DNA-
binding specificity is the same in different species, other functions, including its ability to
bind other proteins, are altered. Pem homeodomain-binding proteins may have coevolved
with Pem to maintain their ability to bind to Pem. Alternatively, Pem's homeodomain may
have rapidly evolved to allow Pem to interact with different proteins in different species.
Such a switch in protein-protein interactions could be critical for directing subtle
differences in reproductive functions in different species. In this regard, it is interesting
that a Drosophila homeobox gene, OdsH, was recently shown to also contain a
homeodornain undergoing rapid directional selection (Ting et aI., 1998). Importantly,
variant forms of OdsH in different Drosophila species were shown to cause hybrid male
sterility (Ting et al., 1998). Future studies may show that Pem is important for reproductive
isolation in mammals.
We have also isolated a second Pem-like gene, Pem2, from the rat (Nhim et aI., 1997).
Pem2 is a processed gene (i.e., it has no introns) present on chromosome 4 that probably
arose by reverse transcription of an mRNA from the X-linked rat Pem gene. Although
many processed genes are nonfunctional pseudo genes, several lines of evidence suggest
that Pem2 may be functional. First, unlike most pseudogenes, Pem2 is transcribed.
Second, Pem2 transcripts are tissue specific, having been detected only in epididymis, not
in testis or several other reproductive and nonreproductive tissues. Third, Pem2 transcripts
are regulated in a stage- and region-specific manner in the epididymis (Table 5; Nhim et
al., 1997). Fourth, the Pem2 gene has evolved a specific set of splice acceptor and donor
sites that permit it to generate a protein very similar to the carboxy-terminal portion of
Perno
There are several other processed genes that encode functional proteins, including
Pgk-2, Zja, and Pdha-2, which like Pem2, are autosomal processed genes derived from the
X chromosome (Salehi-Ashtiani and Goldberg, 1996). Interestingly, although Pem2 and
these three other processed genes are all expressed specifically in the male reproductive
tract, Pgk-2, Zja, and Pdha-2 are expressed only in the testis, whereas Pem2 is expressed
specifically in the epididymis (Nhim et ai., 1997).

POU-Class Homeobox Genes

The only homeobox gene known to be expressed in PGCs besides Pem is the POU-
class gene Oct-3 (also called Oct-4 and Pou5j1) (Sch51er et al., 1990.). Oct3 is also
expressed in the testes of adult mice (Table 6; Tomolin et al., 1996). Both Oct-3 and Pem
may be involved in early differentiation events, as both are also expressed by ES cells
(Sasaki et aI., 1991). However, they probably differ in their activity in stem cells, as Pem
expression is dramatically upregulated and Oct-3 expression is downregulated in
differenttiating ES cells (Sasaki et ai., 1991). The role of Oct-3 in PGCs and adult testes is
not known, as mouse Oct-T1• mice do not progress beyond the blastocyst stage (Nichols et
al., 1998).
Two other POU-class homeobox genes expressed in the reproductive tract are Sperm-
1 (Sprm-l) and Testes-l (Tst-l or Oct-6). Sprm-l is expressed in male germ cells and Tst-l
is expressed in Sertoli and epididymal cells (Table 6). Sprm-rl . mice are subfertile,
suggesting that Sprm-l directs activities critical for male germ cells (Pearse et al., 1997).
However, as these knockout mice have normal testicular morphology and produce normal
numbers of mobile spermatozoa, the cellular and molecular basis for this subfertile
phenotype is not clear.

Emx2 and Nkx3.1

Among the several other homeobox genes expressed in the male reproductive tract
(Table 6), two have been shown to playa functional role: Emx2 and the androgen-regulated
Nkx3.1 gene. Ernx2 is essential for an early step in male reproductive-tract development as
EmxT1- mice lack gonads as well as other organs of the genital tract (Miyamoto et aI.,
1997). These null mice also exhibit greatly reduced Pax-2 mRNA levels, suggesting that
the Ernx2 transcription factor regulates Pax-2 expression. However, it is not clear whether

279
Emx2 directly activates Pax-2 transcription or whether intermediate factors are involved. It
is also possible that Ernx2 is not a regulatory factor upstream of Pax-2 but rather that its
absence causes Pax-2 levels to decrease because the types of cells that express Pax-2 do not
develop or die by apoptosis.
Nkx3.rl- mice have defects in prostate ductal morphogenesis and secretory protein
production and in the bulbourethral gland (Bhatia-Gaur et aI., 1999). Both homozygous
and heterozygous mutant mice suffer from prostatic epithelial hyperplasia and dysplasia,
suggesting that even small decreases in the levels of this homeobox transcription factor
have severe consequences. Interestingly, the human NXK3.1 gene has been mapped to a
chromosome region frequently deleted in prostate cancer (He et aI., 1997). Thus,
Nkx3.11NKX3.l may not only be necessary for normal prostate development, but its
mutation or aberrant expression may also contribute to prostate cancer.

Table 6. Other Homeobox Genes Expressed in the Male Reproductive System

Gene Male Reproductive Expression Reproductive phenotype References


Pattern in knockout mice
Oct310ct41Pou PGCs and adult testis Emryonic lethal Scholer et aI., 1990
5f1 Nichols et ai, 1998
Sprm-l Spermatogonia (before Subfertile Anderson et aI., I 993·
meiosis), and round spermatids Pearse et aI., 1997
Tst-1IOct-6 Sertoli cells Lethal (within 2 days after Pearse et aI., 1997
birth)
Emx-2 Epithelial components of Absence of gonads and Miyamoto et aI., 1997
urogenital sinus genital tract
Lbx-2 Urogenital system Not yet characterized Chen et aI., 1999

Nkx3.1 Developing testis and prostatic Defects in prostate B ieberich et aI., 1996
buds development Bhatia-G et ai, 1999
Cux-l Round spermatids No defects in mutant Vanden H et al.,1996
Tufarelli et aI., 1998
Gtx Testicular germ cells Not known Komuro et aI., 1993

FUTURE STUDIES

Several approaches will be necessary to precisely defme the function of homeobox


genes in the male reproductive system. First, some knockout mice may have subtle defects
in reproductive function that can be identified by using high-throughput assays such as
gene-array analysis. Second, the functions of context-dependent homeobox genes may
only be revealed when environmental conditions are altered for null mice (e.g., stress is
applied). Third, in cases in which one mutant gene engenders no obvious male
reproductive dysfunction, mice deficient in two homeobox genes suspected of providing
the same redundant function can be examined.
To study homeobox genes that cause embryonic or neonatal lethality when disrupted,
mosaic null or conditional knockout mice can be generated to target the disruption
specifically to the male-reproductive system. Another powerful approach to selectively
knockout homeobox genes in the male reproductive tract is to generate transgenic mice that
express tissue-targeted inhibitors of homeobox gene function (e.g., dominant-negative
mutants). A complementary approach is to generate gain-of-function transgenic mice that
ectopically express or overexpress homebox genes in the urogenital tract. This gain-of-
function approach has been useful in other systems. For example, the Pax-6 homeobox
gene induces the generation of eye-like structures at sites where it is ectopically expressed
(e.g., in limbs), indicating that Pax-6 is a master regulator of eye development (Hanson and
Van Heyningen, 1995). By selecting an appropriate combination of several different in
vivo approaches, the role of homeobox transcription factors in the male reproductive
system should be revealed.

280
Lastly, there is a need for further progress in the applications of homeobox genes to
the clinic. Importantly, mutations in some homeobox genes have been associated with
human urogenital diseases (Mortlock et aI., 1997). Mouse models need to be developed for
studying these human urogenital diseases. Modulators of homeobox gene expression and
function could be identified using such mouse models. Positive modulators may be useful
for treating male infertility disorders, while negative modulators could serve as a reversible
form of male birth control. An understanding of the factors that regulate the region-specific
expression of homeobox genes in the epididymis may be useful for designing such
modulators. Another future therapeutic approach is gene therapy. For example, gene
therapy directed towards PGCs would permit correction of germline mutations. Progress in
this area is currently hindered by our ignorance of PGC biology and gene regulation. An
understanding of how homeobox proteins and other transcription factors regulate the PGC
genetic program will be critical for such gene therapy. Another potential gene-therapy
target is the epididymis. Not only would such an approach be useful for correcting
epididymal disorders, but also it could generate efficient birth-control methods.

ACKNOWLEDGMENTS

We would like to thank Drs. Steven Potter and Wade Bushman for helpful comments
on this chapter and the authors indicated in the text who provided unpublished information.

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