Cell Tissue Res (1985) 242:145-156
a n d 1"tssue
Selective destruction and regeneration
of rat Leydig cells in vivo
A new method for the study of seminiferous tubular-interstitial tissue interaction
J.B. Kerr, K. Donachie*, and F.F.G. Rommerts**
MRC Reproductive Biology Unit, and *Department of Obstetrics and Gynaecology, Centre for Reproductive Biology, Edinburgh, Scotland;
**Department of Biochemistry, Erasmus University, Rotterdam, The Netherlands
Summary. The effect of a single i.p. administration of eth-
ane dimethanesulphonate (EDS) upon rat testicular histolo-
gy was studied by light microscopy and morphometry up
to 4 weeks after treatment. One day after injection the inter-
stitial tissue exhibited degenerating Leydig cells, abundant
pyknotic interstitial cells, deposition of cellular debris and
extensive networks of fibrillar material. Macrophages con-
tained greatly increased numbers of cytoplasmic inclusion
bodies. From 3 to 7 days morphometric analysis showed
that Leydig cells and cellular debris had disappeared from
the interstitial tissue, leaving only macrophages, fibroblasts
and lymphatic endothelial tissue. A very small number of
new Leydig cells were seen on day 14, often located in
peritubular or perivascular positions. Regeneration of foe-
tal-like Leydig cells occurred by 4 weeks, their cytoplasm
containing large lipid inclusions and, numerous Leydig cells
were often observed closely applied to the walls of the semi-
niferous tubules. The observations suggest that, after exper-
imental destruction and depletion of Leydig cells, an inter-
stitial precursor cell, as yet unidentified, gives rise to a new
Leydig cell population. EDS thus offers a valuable opportu-
nity to study further the interactions between the seminifer-
ous tubules and the interstitial tissue following the destruc-
tion and subsequent regeneration of the Leydig cells.
Key words: Antifertility c o m p o u n d - Ethane dimethanesul-
phonate - Leydig cells - Destruction - Regeneration - Rat
Ethane-l,2-dimethanesulphonate (EDS) has long been re-
cognised to exert an unusual pharmacological action upon
the rat testis resulting in a temporary period of sterility
2-8 weeks after a single treatment (Cooper and Jackson
1970; Jackson 1964; Jackson and Jackson 1984). In con-
trast to the antifertility actions of other simple diesters of
methane sulphonic acid, the anti-spermatogenic effect of
EDS is not attributable to the arrest of germ cell prolifera-
tion, but rather it appears to selectively impair the function
of the Leydig cells (Jackson and Jackson 1984). In the rat
a single dose of EDS inhibits Leydig cell testosterone pro-
duction and suppresses serum androgen levels 24 h after
administration (Bu'Lock and Jones 1976; Morris and
McCluckie 1979), both these markers of Leydig cell func-
Send offprint requests to. Dr. J.B. Kerr, Dept. of Anatomy, Mon-
ash University, Clayton, Victoria 3168, Australia
ten
Research
9 Springer-Verlag1985
tion returning to the normal range during weeks 6-9 (Bu-
'Lock and Jackson 1975). These changes are accompanied
by a temporary decline in testis, seminal vesicle and prostate
weights and impairment but subsequent restoration of sper-
matogenesis and fertility (Cooper and Jackson 1970; Jack-
son and Morris 1977; Jackson et al. 1973). Serum concen-
trations of follicle-stimulating hormone (FSH) and luteiniz-
ing hormone (LH) are initially elevated following EDS
treatment but return to within normal limits with the resto-
ration of spermatogenic function (Jackson and Morris
1977), suggesting re-establishment of the feedback signals
linking the testis and the anterior pituitary. Although these
numerous studies point to a direct cytotoxic effect of EDS
upon Leydig cell function, correlation with the morphologi-
cal response of the Leydig cells has not been available.
In view of much recent interest in the dynamic interactions
between spermatogenesis and the interstitial tissue, which
suggests a paracrine relationship between the Sertoli cells
and Leydig cells (Kerr and Sharpe 1985a; Sharpe 1984),
the possibility that EDS might serve as a valuable tool to
study experimental alterations to this intercellular axis
prompted the present study.
Materials and methods
Treatments and tissue fixation
Adult male Sprague-Dawley rats, approximately 350g
body weight were used from our own laboratory colony.
Twenty five rats (5 per group) received a single intraperiton-
eal injection of EDS (7.5 mg/100 g body weight) in di-
methyl-sulphoxide-water (1:3 V/V). A control group of 5
rats received a single i.p. injection of the vehicle. EDS-
injected rats were killed on days 1, 3, 7, 14 and 28 after
treatment, and controls were killed on day 28. The testes
were fixed by vascular perfusion using methods described
by Kerr et al. (1984). Briefly, under deep ether anaesthesia
the thoracic aorta was cannulated, the right atrium severed
and the testes briefly perfused with physiological saline,
then with a mixture of 5% glutaraldehyde, 3% formalde-
hyde and 0.01% trinitrophenol buffered in 0.1 M sodium
cacodylate, pH 7.4, for 45 rain. The hardened testes were
removed, decapsulated and, after measurement of their vol-
umes by water displacement (Elias and Hyde 1980), cut
into cubes approximately 2 m m on edge and fixed for 3 h
in the same fixative. Blocks were post-fixed for 2 h in os-
mium tetroxide, stained en bloc with uranyl acetate, dehy-
146
drated in ethanol and embedded in a 1 : 1 mixture of Epon-
Araldite. Blocks from all animals were cut at 1 ~tm using
a Reichert OmU3 ultramicrotome and then stained with
Toluidine blue. Sections were examined and photographed
with a Zeiss photomicroscope II using Plan Apo 40/1.0
and 63/1.4 oil-immersion objectives.
Morphometric analysis
Blocks from all testes were cut at 1 lam and examined by
light microscopy to exclude from quantitative analysis any
tissues which exhibited unsatisfactory preservation using
criteria similar to those previously described (Kerr and
Sharpe 1985a). Tissues displaying artifactual spaces be-
tween the seminiferous tubules and the interstitial tissue
which may arise from poorly fixed localised sites within
the tissue or from mechanical damage during the cutting
of glutaraldehyde-fixed blocks were not assigned for mor-
phometry. Testes of 3 of the 5 animals in each group were
thus selected in which the preservation of the tissue was
judged satisfactory for quantitative analysis. A minimum
of 10 one-micron sections (all from different blocks) of
transversely-sectioned seminiferous tubules were selected
from each rat which on average provided an area containing
15 20 seminiferous tubules in each section. Sections were
examined using 10 x eyepieces and a 25 x objective lens.
The volumetric densities of the total interstitial tissue (all
cells, extracellular spaces and blood vessels), the Leydig
cells and macrophages (identified as described below) were
measured by a point-counting technique using a graticule
of 121 points (11 x 11 squares) inserted into one eyepiece.
Volumetric densities of these components were determined
on 6-10 random areas of each section and the scores were
expressed as a percentage of the total number of points
superimposed upon each section. Average volumetric densi-
ties were calculated for each testis and the means +s.d.
for the 3 animals belonging to each group were determined.
The volume of the testis occupied by each component was
obtained by multiplying the volumetric density of the com-
ponent by the volume of the whole testis from which the
sections were derived. Mean absolute volumes of the com-
ponents were calculated for each treatment group. The pos-
sible alteration of volumetric density and absolute volume
of the measured components due to changes in the volume
of tissue blocks during their processing and embedding were
not taken into account since the principal interest of the
study was to compare the morphology of the normal testis
to testes from EDS-treated animals.
Statistics
Morphometric data are expressed as the mean _+S.D. Sta-
tistical significance of the results was determined by Dun-
can's new multiple range test.
Results
Histology
In vehicle-treated control rats, the testicular interstitial tis-
sue contained prominent clusters of Leydig cells often sur-
rounding blood vessels or occupying positions more cen-
trally placed within the interstitial tissue (Fig. 1). Leydig
cells exhibited a wide variety of shapes including ovoid,
polygonal or irregularly elongated profiles. Their nuclei of-
ten showed a prominent nucleolus and dense patches of
heterochromatin were associated with the nuclear mem-
brane. Leydig cell cytoplasm was basophilic after Tolui-
dine-blue staining, and many dense inclusion bodies were
representative of mitochondria. Cytoplasmic lipid inclu-
sions were not commonly noted, a feature characteristic
of adult rat Leydig cells. Macrophages, present in fewer
numbers than Leydig cells, were readily identified by their
pale-staining irregular nuclei together with their character-
istically abundant supplies of cytoplasmic granules and vesi-
cles indicative of their well developed lysosomal compo-
nents fulfilling the phagocytic function of macrophages.
Thin fusiform endothelial cells forming the walls of lym-
phatic spaces together with fibroblasts associated with the
loose interstitial connective tissue were also observed. The
pale-staining interstitial tissue matrix represented the inter-
stitial fluid of the extensive lymphatic sinusoids characteris-
tic of the testis in rats and other rodent species. Twenty-four
hours after EDS treatment, most of the Leydig cells exhib-
ited various degrees of disintegration, few appeared mor-
phologically unaltered and deposition of fibrillar-like mate-
rial was also observed (Figs. 2, 3). By light microscopy the
structural damage to Leydig cells was often so marked that
it became difficult to identify individual cells since their
cytoplasm was fragmented and dispersed throughout large
areas of the intersitital spaces (Fig. 3). Clumps of frag-
mented Leydig cells commonly exhibited dense granular
material and numerous cytoplasmic vesicles. Individual
pyknotic structures were often scattered amongst the exten-
sive cellular debris. Macrophages were always present and
contained increased numbers of dense granules and cyto-
plasmic vesicles. Extensive networks of randomly oriented
fibre-like material was noted (Fig. 4), a feature not seen
in the normal interstitial tissue. Since the endothelial lining
of lymphatic spaces was not seen to partly surround the
degenerating Leydig cells, the fibrillar material was possibly
derived from disorganisation and dispersal of these endo-
thelial cells. Elongated or irregularly-shaped nuclei of endo-
thelial and connective tissue cells were also observed, but
their morphology did not appear different from control
tissues. The seminiferous epithelium appeared qualitatively
normal. On day 3 after EDS treatment, Leydig cells had
disappeared from the testis (Fig. 5) although spermatogene-
sis appeared unaffected. Despite extensive analysis of the
interstitial tissue, none of the interstitial cells could be classi-
fied as adult, immature or atrophied Leydig cells. The cellu-
lar debris and fibrillar-like material present on day 1 after
treatment was not seen on day 3. The interstitial spaces
were filled with interstitial fluid, within which were numer-
ous macrophages although in contrast to their appearance
on day 1, their cytoplasm did not usually contain especially
large granules and vesicles. Interstitial connective tissues
and elongated cytoplasmic profiles of endothelial cells were
a common feature of the interstitial tissue. Occasional lym-
phocytes and plasma cells were noted within the interstitial
lymphatic sinusoids. Seven days after EDS treatment, the
morphology of the seminiferous epithelium was similar to
day 3 although degenerating germ cells were occasionally
present in the basal aspects of seminiferous tubules at stage
VII and VIII of the spermatogenic cycle. Leydig cells were
not observed within the interstitial tissue (Fig. 6). Occasion-
ally, focal areas of the interstitial tissue were filled with
great numbers of mononucleated cells (Fig. 7) commonly
surrounding the interstitial capillaries and venules. In fa-
 147

Fig. 1. Vehicle-treated control testis illustrating the typical morphology of the interstitial tissue containing numerous Leydig cells. Macro-
phages (M) and a monocyte (asterisk) are shown within the fluid-filled interstitial lymphatic sinusoid (LS). Nuclei of lymphatic endothelial
cells are shown (arrowheads). x 470

Fig. 2. 1 day after EDS treatment, showing early disruption of interstitial tissue organisation. Some Leydig cells appear normal while
others (arrowheads) show cytoplasmic damage. Fragments of pyknotic material are seen and macrophages (arrows) often exhibit dense
cytoplasmic inclusions, x 470
148

Fig. 3. 1 day after EDS treatment, illustrating more advanced cellular disintegration resulting in many scattered pyknotic fragments
(arrows). Some Leydig cells (L) appear intact but contain unusual cytoplasmic vesicles. Macrophages (arrowheads') are numerous and
contain many cytoplasmic granules and vesicles. • 470

Fig. 4. 1 day after EDS treatment, in which Leydig cells are absent but cellular debris (arrowhead) and pyknotic material possibly
represent degraded remnants of Leydig cells. Many mononucleated cells are present, including macrophages (asterisk). Fibrillar networks
(arrows) occupy the lymphatic sinusoidal spaces (LS). • 470
 149

Fig. 5. 3 days after EDS treatment, illustrating disappearance of Leydig cells and cellular debris. The lymphatic sinusoid (LS) contains
cells with cytoplasmic filopodia, at times identified as macrophages (arrows). Note perivascular and peritubular nuclei (arrowheads)
of unknown identity. The nucleus of a lymphatic endothelial cell is shown (asterisk). x 470

Fig. 6. 7 days after EDS treatment, illustrating the lymphatic sinusoid (LS) containing macrophages and a monocyte (arrows), the
latter having a characteristic nucleus and granule-free cytoplasm. Nuclei of endothelial cells are indicated (arrowheads). x 470
150

Fig. 7. 7 days alter EDS treatment, illustrating focal infiltration of monocytes and tymphocytes into the interstitial tissue. A cell within
the venule is closely applied to the vessel endothelium (arrow). x 470 Inset." Possible migration of this cell through the endothelial
tissues, x 850

Fig. 8. 14 days after EDS treatment, showing Leydig cells (asterisks) with oval or spherical nuclei. Other interstitial cells (arrowheads)
possibly represent stages in the development of new Leydig cells. Macrophages and mononucleated cells are illustrated (arrows). x 470

Fig. 9. 28 days after EDS treatment, illustrating abundant foetal-like Leydig cells with round nuclei and cytoplasmic lipid inclusions
(L). Macrophages are also shown (arrows). x 470
vourable sections, these cells were also observed within the
vessel lumina and at times appeared to be migrating
through the endothelium of the vessels. These observations
together with their morphology suggested their classifica-
tion as a mixture of monocytes and lymphocytes. Two
weeks following treatment, the majority of the interstitial
tissue spaces presented a similar morphology and cell popu-
lation to that previously seen on days 3 and 7, but occasion-
ally, small numbers of Leydig cells were observed (Fig. 8).
However, the structure of these Leydig cells was not identi-
cal to Leydig cells seen in the adult testis. Their nuclei
were larger and showed a spherical or oval shape, often
containing a single large nucleolus. Although the cytoplas-
mic area was well developed, granules representing mito-
chondria were not especially abundant and small lipid inclu-
sions were often present. Interstitial cells with elongated
or polygonal nuclei were occasionally seen close to the peri-
tubular tissue, and their position and morphology was simi-
lar to the interstitial mesenchymal cells seen in foetal or
immature rat testes. The seminiferous tubules exhibited
patchy disruption of spermatogenesis. The most remarkable
feature of the interstitial tissue 28 days after EDS adminis-
tration was the striking abundance of large Leydig cells
(Fig. 9). Leydig cells often surrounded blood vessels or
formed large clusters of cells, and of particular interest was
their frequent association with the peritubular tissues of
the seminiferous tubules, thereby forming uninterrupted
rows of cells partly surrounding the tubules (Fig. 10). By
comparison with adult Leydig cells from the vehicle-injected
control group, the Leydig cells of the 28 day group exhib-
ited a very different morphology and were similar to the
151
morphology of Leydig cells in the foetal testis. These Leydig
cells contained less irregularly-shaped nuclei which were
usually spherical or slightly ovoid in shape. In addition,
many of the Leydig cells exhibited large cytoplasmic lipid
inclusions, a feature rarely seen in normal adult rat Leydig
cells. The location and morphology of macrophages, con-
nective tissue cells, endothelial cells and lymphatic sinusoids
of this treatment group was similar to their appearance
within control testes. Although some seminiferous tubules
were severely depleted of germ cells most exhibited more
advanced spermatogenesis compared to the 2 week treat-
ment group.
Morphometric analysis
Testis volume was significantly (P<0.01) reduced 2 and
4 weeks after EDS treatment (Table l). The volumetric den-
sities of the measured interstitial tissue components showed
significant increases and decreases in EDS-treated rats com-
pared to vehicle-injected control rats. A better appreciation
of these changes was demonstrated by the conversion of
volumetric density data into absolute volumes per testis
in the various treatment groups. The total volume of inter-
stitial tissue (all interstitial cells, extracellular spaces and
blood vessels) was significantly ( P < 0.05) increased I day
post-EDS treatment compared to vehicle-treated testes, but
at 7 and 14 day post-EDS treatment, it was significantly
decreased (P<0.05 to 0.001). The significant increase in
total interstitial tissue volume 1 day after EDS reflected
a major increase in the total interstitial fluid volume (mea-
sured as extracellular space). The total volume of Leydig
cells within the testes of all EDS-treated groups was signifi-
152

Fig. 10. 28 days after EDS treatment, showing a seminiferous tubule partly surrounded by peritubular foetal-likc Leydig cells. The
lymphatic sinusoidal space (LS) is indicated, x 470

Table 1. Morphometric analysis of interstitial tissue (Means + S.D.; n =3 animals)
Vehicle Days after EDS treatment
control
1 3
Testis volume (cm3) 1.24 + 0.04 1.37+0.11 1.12_+0.12
Volumetric density (%)
Total interstitial tissue l t.4 _ 0.7 13.7 _+0.5 9.6 +1.0 6.8 _+0.3* 11.5 _+2.0 19.6 _+2.0*
Leydig cells 3.0 +0.2 0.8 +0.3** n.d.** n.d.**
Macrophages 0.3 +0.1 0.5 ___0.1 0.8 +0.1"* 0.4 •
Volume per testis (~1)
Total interstitial tissue 141 +6 188 +12" 109 _+20
Leydig cells 37 +2 11 _+5'* n.d.**
Macrophages 4 +l 7 +1" 9 _+2*
n.d., not detected
*P < 0.05 or 0.0t, **P < 0.001, EDS treatments vs. vehicle control
cantly smaller ( P < 0.001) than the total volume within con-
trol testes, but showed a marked restoration towards nor-
mal values between weeks 1 and 4 following EDS adminis-
tration. Total volume of macrophages was significantly
( P < 0.05, 0.01) increased 1 and 3 days after EDS treatment,
but was unchanged at other times compared to the normal
testis.
Discussion
In a series of studies over many years (Jackson 1964; Jack-
son and Jackson 1984) the antispermatogenic and antiferti-
lity action of EDS in rats was thought to be mediated via
a direct cytotoxic inhibition of the function of the Leydig
cells. However, the subsequent recovery of Leydig cell func-
tion accompanying the restoration of spermatogenic activi-
ty and fertility raised the possibility that a proportion of
the Leydig cell population was resistant to EDS action or
alternatively normal androgenic function was restored via
proliferation of Leydig cells from other interstitial cells.
However these views have not been resolved, since the ef-
fects of EDS upon the morphology of the Leydig cells and
other elements of the interstitial tissue have not been stud-
ied. In the present study the use of semi-thin plastic sections
of perfusion-fixed testes together with the application of
contemporary morphometric analysis has provided a signif-
icant step forward in elucidating the morphological re-
sponse of the interstitial tissue following EDS treatment.
The results show that within 24 h of EDS treatment, Leydig
cells have undergone advanced disintegration which results
in their complete elimination from the testis on day 3. Ley-
dig cells are absent from the testis for at least 5 days (days
3-7 after treatment) but remarkably, during weeks 2 4, re-
generation of the Leydig cells occurs resulting in a substan-
tial restoration of their total volume normally seen in the
adult rat testis. We believe these findings to be unique for
two reasons. First, the selective destruction of Leydig cells
in the mammalian testis has heretofore not been described
and secondly, their rapid re-appearance following complete
elimination from the testis has no previous precedent.
A number of fundamental aspects of the cell biology
of the testis are raised by the present study: i) the mecha-
nism by which EDS selectively destroys the Leydig cells
and the involvement of macrophages and lymphocytes
153
7 14 28
1.04___0.05 0.89_+0.04* 0.75+0.07*
O
0.t +0.0"* 2.6 +0.7
0.5 _+0.1 0.4 _+0.t
71 _+1"* 99 _+23* 145 +28
n.d.** t +0"* 19 +4**
3 _+1 4 +_1 3 -+-0
within the interstitial tissue; ii) the question of the origin
of new "foetal-like" Leydig cells and the factors stimulating
their differentiation; and iii) the response of the seminifer-
ous epithelium to a period of androgen deprivation due
to temporary depletion of the Leydig cell population.
In earlier studies, it has been suggested that EDS exerts
a deleterious effect upon rat Leydig cells via an interaction
with the plasma membrane receptors for hCG, since a brief
( 4 1 2 h) prior exposure of the testis to EDS essentially abol-
ishes the capacity of the Leydig cells to produce cyclic AMP
in response to in vitro hCG stimulation (Bu'Lock and Jones
1976). Whether this action involves alkylation of cellular
components similar to the alkylating actions on the rat
seminiferous epithelium observed with other diesters of
methane sulphonic acid is not yet resolved (Morris and
McCluckie 1979). The rapid disintegration of Leydig cells
after EDS treatment is consistent with the acute impairment
of steroidogenic metabolism and inhibition of cyclic AMP
and testosterone production (Bu'Lock and Jackson 1971,
1975). Although the precise mode of action of EDS upon
Leydig cells requires additional study, the present observa-
tions indicate that destruction of Leydig cells elicits an im-
mune response involving macrophage-lymphocyte interac-
tion. At the time of Leydig cell disintegration, the total
volume of macrophages within the testis had increased sig-
nificantly compared to the control testis tissue and their
greatly increased quantities of cytoplasmic granules and va-
cuoles suggested a highly activated state of phagocytosis
of Leydig cell debris. During the induction of immunity,
macrophages are believed to facilitate the engagement of
antigen-sensitized lymphocytes via secretion of lymphokin-
etic factors (Drutz 1976; Roitt 1980). Upon presentation
of antigen to the lymphocytes via macrophages, additional
lymphocytes are attracted to the site of antigen-responding
cells and this response was reflected by the infiltration of
many lymphocytes into focal areas of the interstitial tissue
seen 7 days after EDS treatment. Activated lymphocytes
are known to produce lymphokines which selectively attract
monocytes and their derivatives, the macrophages, to the
sites of antigenic challenge (Rocklin 1976), and may also
stimulate proliferation of fixed macrophages such as those
within the rat testis (Miller et al. 1983). These mechanisms
probably account for the significant increase in total macro-
phage volume during and soon after the destruction of the
154
Leydig cells on days 1-3. Augmentation of increased mac-
rophage phagocytic activity also occurs via lymphocyte-
macrophage interaction mediated by other lymphokine sub-
stances. This phenomenon raises the question of the prima-
ry site of action of EDS. EDS perhaps specifically disrupts
the Leydig cell and in some way presents an antigenic chal-
lenge to the nearby macrophages or alternatively, EDS may
be an allergen specific to the macrophages which precipi-
tates their phagocytic activities and interaction with lym-
phocytes. Although either or both of these suggested modes
of action may conceivably operate, recent in vitro studies
(Rommerts, unpublished observations) have shown that the
addition of EDS to isolated adult rat Leydig cells severely
impairs steroid production within 3 h and induces morpho-
logical alterations by 24 h. It therefore seems likely that
the former mechanism of EDS action upon Leydig cells
may occur in vivo.
The regeneration of abundant numbers of new Leydig
cells 4 weeks after EDS treatment and within 3 weeks fol-
lowing their total depletion raises the question of their ori-
gin from within the interstitial tissue. Since Leydig cells
were absent from the testis during days 3-7, the new genera-
tion of Leydig cells cannot have arisen by mitosis or hyper-
trophy of pre-existing Leydig cells although these mecha-
nisms are thought to explain Leydig cell hyperplasia in the
normal testis following chronic stimulation with hCG
(Chemes et al. 1976; Christensen and Peacock 1980). The
present observations favour an alternative mechanism in
which a more primitive interstitial stem-cell population dif-
ferentiates into histologically recognisable Leydig cells. As
the newly developed Leydig cells exhibited a very similar
morphology to foetal rat Leydig cells (Lording and
deKretser 1972; Tapanainen et al. 1984), we suggest that
their differentiation followed the pattern of development
of Leydig cells in the foetal testis in which precursor mesen-
chymal cells differentiate into histologically recognisable
Leydig cells (Wartenberg 1981). The interstitial tissue in
2- and 4-week EDS-treated rats contained many fusiform
mesenchymal cells in addition to numerous interstitial cells
with a morphology intermediate between the former cell
type and the larger Leydig cells. Occcasionally, mitotic fig-
ures were seen amongst the interstitial cells. These observa-
tions suggest that the regeneration of Leydig cells occurs
initially via mesenchymal cell maturation and possibly to
a lesser extent, by Leydig cell division, in agreement with
the pattern of Leydig cell development observed during foe-
tal and postnatal life (Christensen 1975; Christensen and
Gillim 1969; Gondos et al. 1976, 1977; Prince 1984). Addi-
tional ultrastructural and autoradiographic studies using
tritiated thymidine will be necessary to determine the loca-
tion and identity of Leydig cell precursors and their pattern
of maturation into functional Leydig cells. It is widely be-
lieved that LH (or CG) is the hormone which during post-
natal or foetal life exerts a major stimulatory influence upon
the development of Leydig cells (Faiman et al. 1981;
Swerdloff and Heber 1981). Since serum LH levels are ele-
vated up to five-fold above the normal range for 4 weeks
after EDS treatment (Jackson and Morris 1977), LH is per-
haps responsible for the regeneration of Leydig cells. How-
ever, we have recently shown (Kerr and Sharpe 1985a, b)
that, in the immature rat testis, FSH provokes rapid hyper-
plasia and hypertrophy of Leydig cells and an accompany-
ing reduction in the numbers of mesenchymal cells. These
studies showed that many of the Leydig cells appeared to
develop from the peritubular tissues surrounding the semi-
niferous tubules, an observation also noted in the interstitial
tissue 4 weeks after EDS treatment. Serum FSH levels are
significantly elevated above the normal range within 3 days
after EDS treatment, attaining maximal elevation (3 fold)
at 4 weeks and thereafter decline towards normal values
(Jackson and Morris 1977). Since the target tissue for FSH
is the Sertoli cell (Means et al. 1976), the present observa-
tions together with those mentioned above introduces the
possibility that in addition to the trophic action of LH,
the seminiferous tubules also in some way exert a local
stimulatory effect upon the development and function of
the interstitial tissue, a concept which is attracting increas-
ing support (Aoki and Fawcett 1978; deKretser 1982;
deKretser and Kerr 1983; Parvinen 1982; Parvinen et al.
1984; Sharpe 1983, 1984). Although not studied in detail
here, preliminary observations indicate that occasional de-
generating germ cells were noted 1 week after EDS treat-
ment although maximum impairment of spermatogenesis
occurred approximately 2 weeks after EDS treatment. A
qualitative improvement of the seminiferous epithelium in
most seminiferous tubules was observed in association with
new Leydig cell growth at 4 weeks. A detailed analysis of
impairment and restoration of spermatogenesis is underway
in our laboratory. The seminiferous tubules are thought
to be the site of production of factors modifying both the
size (Bergh 1983) and secretory properties of the Leydig
cells (Parvinen et al. 1984; Sharpe and Cooper 1984; Sharpe
and Bartlett 1985). Taken with the histological evidence
that during pubertal maturation, new Leydig cells may dif-
ferentiate from peritubular interstitial cells (Christensen
1975; deKretser 1967; Fawcett 1973; van Straaten and
Wensing 1978), the development of numerous peritubular
Leydig cells 4 weeks post-EDS treatment suggests a func-
tional relationship between spermatogenesis and Leydig cell
growth. Interstitial cells suggested to be Leydig cell precur-
sors have been noted in association with the peritubular
tissue of the canine testis (Connell and Christensen 1975),
and fully differentiated Leydig ceils are commonly noted
within the peritubular tissue of the human testis in cases
of cryptorchidism or Sertoli cell only syndrome (Mori et al.
1978; Schulze and Holstein 1978). These observations sug-
gest that newly differentiated Leydig cells may arise from
peritubular ceils and subsequent leave the lamina propria
to gain access to the surrounding interstitial tissue.
At present it is not known why extensive degeneration
of spermatogenesis does not occur even in the chronic ab-
sence of the Leydig cells, a condition in which intratesticular
levels of testosterone would presumably be undetectable.
Since quantitative spermatogenesis is dependent upon high
intratesticular testosterone concentrations (Stevens and
Steinberger 1983) failure to observe any significant germ
cell degeneration before 2 weeks post-EDS treatment sug-
gests that the seminiferous tubules conserve their endoge-
nous supply of testosterone possibly via the intratubular
presence of androgen-binding protein (Hansson et al. 1976)
and a recently reported high capacity androgen-binding fac-
tor (Parvinen 1984). Alternatively, disruption of Sertoli cell
- Leydig cell interaction by EDS may activate local control
mechanisms within the seminiferous tubule in an attempt
to preserve intratubular testosterone at levels sufficient to
support qualitative spermatogenesis. We are investigating
the acute and chronic effects of EDS upon the distribution
of intratesticular testosterone levels to define more clearly

the d y n a m i c relationships b e t w e e n the two c o m p a r t m e n t s
o f the testis w h i c h are o f f u n d a m e n t a l i m p o r t a n c e in the
r e g u l a t i o n o f fertility.
Acknowledgements. We thank Professor Sir Alistair Currie (Dept.
of Pathology, University of Edinburgh) for access to ultramicro-
tome equipment. This work was performed while J.B.K. was sup-
ported by a C.J. Martin Research Fellowship from the National
Health and Medical Research Council of Australia.
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Accepted April 22, 1985