Endocrinology Vol. 141, No. 8 2861-2869
Copyright © 2000 by The Endocrine Society
Estrogenic Induction of Spermatogenesis in the Hypogonadal Mouse1
Francis J. P. Ebling,
A. Nigel Brooks,
Anna S. Cronin,
Hazel Ford and
Jeffrey B. Kerr
School of Biomedical Sciences (F.J.P.E.), University of
Nottingham Medical School, Queens Medical Centre, Nottingham NG7 2UH,
United Kingdom; Zeneca Pharmaceuticals Central
Toxicology
Laboratory (A.N.B.), Alderley Park, Macclesfield, SK10 4TJ, United
Kingdom; Department Anatomy (J.B.K.), Monash University, Clayton,
Victoria 3168, Australia; and Department Anatomy (A.S.C., H.F.),
University of Cambridge, Cambridge CB2 3DY, United Kingdom
Address all correspondence and requests for reprints to: Dr. Francis J. P. Ebling, School of Biomedical Sciences, University of Nottingham Medical School, Queens Medical Centre, Nottingham NG7 2UH, United Kingdom. E-mail: fran.ebling{at}nottingham.ac.uk
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Abstract
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Abnormal sperm production and reduced fertility have been reported in
transgenic male mice lacking the
-subtype of the estrogen receptor
(ER)
or aromatase. The aim of this study was to investigate the role
of estrogen in male reproductive function, by determining the effect of
estradiol on testicular function in hypogonadal (hpg)
mice congenitally lacking gonadotropin; and thus, sex steroid
production. hpg mice were treated, at 23 months of
age, with slow-release estradiol implants, which achieved circulating
estradiol concentrations of approximately 40 pg/ml. Treatment for 35
days reliably induced a 4- to 6-fold increase in testicular weight,
compared with the vestigial testes in the untreated or
cholesterol-treated controls. The degree of testicular growth after 35
days was similar to that in hpg mice receiving an
intrahypothalamic graft of preoptic area tissue taken from neonatal
mice on the day of birth, a procedure known to induce testicular
development in hpg mice by activation of the pituitary
gland. Histological analysis revealed that the testes contained
elongated spermatids after 35 days of estradiol treatment, whereas germ
cell development never progressed beyond the pachytene stage in control
hpg mice. Treatment for 70 days induced full
qualitatively normal spermatogenesis in hpg mice. Testis
weight increased 5-fold, reflecting a 5-fold increase in total
seminiferous tubule volume and a 4- to 5-fold increase in the total
volume of the seminiferous epithelium. In all experiments,
spermatogenesis proceeded in the absence of measurable androgen
concentrations, but circulating FSH concentrations were slightly (but
significantly) elevated, relative to cholesterol-treated control
hpg mice. This stimulatory action of estradiol on FSH
secretion was unexpected, particularly because identical estradiol
treatments significantly decreased serum FSH levels in wild-type
littermates. These results indicate that estrogens may play a role in
spermatogenesis, via stimulatory effects on FSH secretion. An
alternative or complementary explanation, given the recent
identification of estrogen receptors (ER
and ERß) and aromatase
within various cell types in the testis, is that estrogens exert
paracrine actions within the testis to promote spermatogenesis. The
identification of effects of estradiol on testicular function provides
a conceptual basis to reexamine the speculative link between increased
exposure to environmental estrogens and reduced fertility in man.
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Introduction
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CRITICAL ANALYSIS OF the suggested link,
between exposure to xenoestrogens during early development and reduced
fertility in later life, has been limited by the lack of understanding
of the role of estrogens in male reproductive function (see Refs. 1, 2 for review). Estrogens, derived either from local aromatization of
androgens or produced by the testis, can exert feedback effects on the
neuroendocrine components of the male reproductive axis, but recent
evidence suggests that estrogen may also exert paracrine actions within
the testis itself (see Ref. 3 for review). Transgenic male mice lacking
the estrogen receptor (ER)
have low sperm numbers and defective
sperm function resulting in greatly decreased fertility (4, 5). This
reduced sperm production is thought to be a consequence of impaired
fluid resorption within the efferent ducts of the testis (6, 7),
because ER
immunoreactivity has been detected in the efferent ducts
of the rat and developing marmoset (8). The identification of ER
immunoreactivity within Leydig cells and of a second estrogen receptor
(ERß) within several cell types in the rat testis, including germ
cells and Sertoli cells (9, 10), raises the likelihood that estrogens
have additional functions within the testis. Unraveling these
physiological actions is particularly important, given the widespread
interest in the association between environmental estrogens and
fertility in the male (2, 11, 12) and recent demonstrations that
various xenoestrogens can disrupt testicular development (13, 14).
The hypogonadal (hpg) mouse has been widely used to
investigate the hormonal regulation of testicular function because it
lacks the ability to produce mature GnRH decapeptide (15, 16). As a
result, these mice are hypogonadotrophic, with vestigial testes, and
have provided a unique model to investigate the effects of
gonadotropins and androgens in the testis (17, 18, 19), though their
unusual developmental history (for example, the lack of androgenization
in the neonatal period) may need to be considered when extrapolating
findings from this strain to other species. The approach of the current
study was to treat hpg mice with estradiol and to assess the
effects on endocrine function and development of the testes,
epididymides, and seminal vesicles. The effects of chronic estradiol
treatment were compared with the effects of a qualitatively normal
gonadotropic drive induced by grafts of wild-type preoptic area brain
tissue, containing GnRH neurons, into the hypothalamus of
hpg mice, a procedure previously demonstrated to induce
testicular development and spermatogenesis by activation of the
pituitary gland (20, 21).
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Materials and Methods
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Animals
All procedures were licensed under the Animals (Scientific
Procedures) Act of 1986 and were conducted in accordance with the
University of Cambridge Scientific Procedures on Animals Code of
Practice. Studies were carried out in C3H hpg mice
(Mus musculus), 23 months old at the start of the
experiment, supplied from a breeding colony at the Department of
Anatomy, University of Cambridge, originally derived from stock
purchased from Jackson ImmunoResearch Laboratories, Inc. (Bar Harbor, ME). After steroid or graft treatments,
mice were individually housed with food and lab chow available ad
libitum and were maintained on a 12-h light, 12-h dark
photoperiod. Genotype of all individuals was ascertained using a PCR
analysis of DNA extracted from ear punches, as described by Lang
(22).
Steroid treatments and hormone assays
Implants were made by packing 10 mm crystalline cholesterol
(Sigma Chemical Co., Poole, UK), or 17ß-estradiol
(Sigma) mixed 1:50 (2%) or 1:10 (10%), by weight with
cholesterol, or testosterone (Sigma) into Silastic tubing
(1.59-mm id, 3.18-mm od; Osteotec, Christchurch, Dorset, UK) plugged at
either end with silicone medical adhesive (Osteotec). After the
adhesive had been allowed to cure for at least 24 h at room
temperature, the implants were washed three times in 70% ethanol. The
implants were incubated for 24 h in 0.01M PBS, pH7.4,
before implantation, so that release rates would be stabilized by the
time the implants were placed under the skin (23). Mice were
anesthetized with halothane, and the Silastic implants were placed into
a sc pocket on their flank. At the end of treatment periods, blood
samples were collected by cardiac puncture, under terminal anesthesia
(sodium pentobarbitone, ip), and allowed to clot at room temperature.
After centrifugation, serum was harvested and stored at -20 C until
required for assay. All estradiol concentrations were measured in a
single assay after extraction of 150 µl serum in hexane:ether using a
commercially available ELISA kit (Fenzia: Orion Diagnostica, Finland).
The limit of detection was 15 pg/ml, and the intraassay coefficient of
variation (cv), based on all duplicates, was 8.7%. Testosterone
concentrations were measured using an ELISA, after extraction of 150
µl serum in hexane:ether, exactly as previously described (24). The
limits of detection were 0.030.05 ng/ml, the mean intraassay cv was
8.1%, and the interassay cv for an adult male wild-type pool run in
all assays (n = 3) was 7.0%. FSH concentrations were measured in
a single assay using a mouse FSH kit from Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire, UK); the limit of
detection was 1.6 ng/ml, and the intraassay cv was 12.5%.
Morphometric procedures
After blood collection, 3 or 4 mice from each group were
perfused, via the left ventricle of the heart, with 0.01 M
phosphate-buffered 0.9% saline, pH 7.2, followed by 4%
paraformaldehyde, for a minimum of 5 min or until the fixed testes
became hardened. The reproductive tracts were excised, trimmed of fat
and connective tissue, and weighed. Epididymides and seminal vesicles
were processed for paraffin histology, and 7-µm sections through
their long axes were stained with hematoxylin-eosin for light
microscope analysis. Each fixed testis was cut in half in the
midtransverse plane, and each piece was processed for paraffin
histology, with orientation in the block, to yield cross-sections of
the testis. Sections were cut at 7 µm and stained with
hematoxylin-eosin. Point-counting methods were used to measure volume
density (Vv) of seminiferous tubules (or cords),
seminiferous epithelium, and tubular lumen, using a x25 objective and
a x10 eyepiece containing a square lattice with 121 (11 x 11)
intersections as test points. The number of so-called hits, on the
above components, was counted with no overlap or gap in each area
sampled. The whole stained section from each specimen was analyzed with
5 to 10 areas sampled randomly, depending on the size of the testis.
Vv of testicular components were obtained by
dividing the number of hits on each structure by the total number of
points superimposed over the tissue. Volume percentage was calculated
by multiplying Vv by 100. Volumes of components per testis
were determined by multiplying Vv by whole
testicular fixed volume, the latter being numerically equal to fixed
testicular weight because the specific gravity of testicular tissue is
very close to 1.0. The volume density and total volume of Sertoli cell
nuclei per testis were determined by point counting, as above, using a
x40 oil-immersion objective.
The diameters of seminiferous tubules (or cords) were measured across
the minor axis (n = 20 per testis) using an eyepiece micrometer
calibrated with a stage micrometer using a x25 objective and x10
eyepiece. Total length (L) of the seminiferous tubules per testis was
calculated by the equation L =
VST/
r2, where
VST is the total volume of seminiferous tubules
per testis (measured above) and r is the average radius of the
tubule.
Bioassay
Female C3H mice were genotyped using a PCR analysis of DNA
extracted from ear punches, as described by Lang (22); and homozygote
hpg individuals received 2% estradiol implants, as
described above for males, or received implants containing 10%
crystalline estradiol-17ß in cholesterol, or cholesterol alone as a
control. Blood samples were collected for estradiol assay, as described
above, and the reproductive tract was dissected and weighed after 12
days of treatment.
Hypothalamic grafts
Postnatal day 0 pups from wild-type C3H stock were killed
by decapitation, and the brains were rapidly removed and placed on
their dorsal surface in a sterile Petri dish. A block was excised with
an iridectomy knife, with lateral cuts approximately 0.5 mm either side
of midline, a caudal cut through the rostral part of the tuber
cinareum, a rostral cut approximately 1 mm in front of this caudal cut,
and a cut approximately 0.5-mm deep to the ventral surface. The tissue
was gently minced with the iridectomy knife and drawn into the needle
of a 5-µl Hamilton syringe. Male hpg mice were
anesthetized with xylazine/rompun and placed in a Kopf stereotaxic
frame with incisor bar level with the ear bars. A small burr hole was
drilled through the skull at bregma, and the needle containing the
graft was lowered 0.60 cm below the dural surface on midline. The graft
was gently ejected into the third ventricle, and hemorrhage from the
midsaggital sinus was stemmed with gelfoam and compression.
Statistical analysis
Organ weight data from each experiment were analyzed by
factorial ANOVA followed by post hoc Dunnetts t tests.
Hormonal data were analyzed by Kruskal-Wallis tests when concentrations
for control groups fell below the limit of detection of the assay.
Tests were carried out on a Macintosh G3 computer using Statview
software (Abacus Concepts, Berkeley, CA). For all tests,
significance was set at P < 0.05.
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Results
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Exp I
The aim of the initial experiment was to determine whether
estradiol treatment affected testicular development in hpg
mice. Hpg mice were treated, for 35 days, with slow-release
sc implants containing 2% 17ß-estradiol in cholesterol. The main
control group received implants containing cholesterol only, for 35
days. Additional groups of age-matched wild-type or hpg mice
with no implants, and of grafted hpg mice (see above), were
also included as interpretative controls. Testicular weight was
uniformly low in cholesterol-treated hpg males (Fig. 1
, Table 1
)
and in untreated hpg males (Fig. 1
). The estradiol-treated
group showed a 4-fold increase (P < 0.001) in
testicular weight, compared with untreated or cholesterol-treated
control hpg mice (Fig. 1
, Table 1
). The degree of testicular
growth was similar to that in hpg mice receiving an
intrahypothalamic graft of preoptic area tissue taken from neonatal
mice on the day of birth (Fig. 1
), but testis weight in
estradiol-treated or grafted hpg mice was significantly less
than in wild-type littermates of a similar age (Fig. 1
). Despite the
increase in testicular weight, circulating androgen concentrations were
undetectable in the hpg mice treated with estradiol (Fig. 1
, lower panel), whereas a significant increase
(P < 0.01) in circulating androgen occurred when
testicular growth was driven by the hypothalamic graft (Fig. 1
, lower). Estradiol treatment also increased by 4-fold the wet
weight of the epididymides (P < 0.05; Table 1
), but it
decreased body weight (P < 0.05; Table 1
) and
decreased the weight of the epididymal fat pad (P <
0.005; Table 1
), a marker of abdominal fat reserves. Thus, the growth
of the testis and epididymis in estrogen-treated hpg mice
was not a reflection of increased growth overall; the estradiol
treatment was clearly catabolic.

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Figure 1. Exp I: (top left) Paired testes
weights and (bottom left) serum testosterone
concentrations in 2- to 3-month-old hpg mice treated for
35 days with cholesterol implants (CHOL) or 2% 17ß-estradiol
implants (E), no treatment (-), hypothalamic grafts (graft) or in
age-matched wild-type littermates (+/+). Values are group mean ±
SE, n = 4 per group. *, P < 0.05;
**, P < 0.01; ***, P < 0.001;
****, P < 0.0001 vs.
cholesterol-treated control group. Exp II: (top right)
paired testes weights and (bottom right) serum
testosterone concentrations in a second experiment: hpg
mice were treated for 35 days with CHOL or 2% E; values are group
mean ± SE, n = 4 per group. Note that, in both
experiments, estradiol treatment increased testicular weight, but serum
testosterone concentrations remained undetectable, whereas growth
driven by hypothalamic grafts was accompanied by an increase in
circulating testosterone.
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Exp II
A second study was conducted with the aims of examining the
histology of the testis and epididymis and to determine whether the
estradiol treatments were producing hormone concentrations within the
normal physiological range. Estradiol treatment, for 35 days, increased
testes weight by 6-fold, compared with cholesterol-treated
hpg mice (Fig. 1
, top right), and as observed
previously, serum androgen concentrations remained below the limit of
detection (Fig. 1
, bottom right). As before, estradiol
induced a reduction in body weight (cholesterol, 34.5 ± 3.6
g vs. 2% estradiol, 27.7 ± 1.3 g). Testes from
cholesterol-treated hpg mice had a very immature histology,
consisting of seminiferous cords or narrow tubules with small lumina
(e.g. Exp III, see Fig. 4a
). Sertoli cells often
were clustered in the center of the cords. Spermatids were absent, and
primary spermatocyte development was arrested at midpachytene. The
cholesterol-treated hpg mice also had shrunken epididymal
tubules with very small lumina (e.g. Exp III, see
Fig. 4g
). Spermatogenesis was clearly more advanced in the
estradiol-treated hpg mice, all animals exhibiting variable
(but low) numbers of round and elongating spermatids; although the
abundance of the latter seemed much reduced, compared with wild-type
testes. Estradiol caused a marked expansion of the epididymal lumen,
suggestive of increased fluid content, though the gross size of the
epididymis was still less than that found in wild-type males.

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Figure 4. Exp III: a, Seminiferous cords in a testis from a
3-month-old hpg mouse treated with cholesterol only;
note that pachytene primary spermatocytes (P) are the most advanced
germ cell type. Some Sertoli cell nuclei (S) are located centrally. b,
Cross-section of tubules from a wild-type control, shown for
comparison; note the well-developed Leydig cells (L) and elongating
spermatids (E) and mature spermatids (M). c and d, Qualitatively normal
spermatogenesis in hpg mice treated for 70 days with 2%
or 10% 17ß-estradiol, respectively; note the elongating spermatids
(E) at various stages of the spermatogenic cycle and mature spermatids
(M). e, Seminiferous tubules from hpg mice treated with
cholesterol for 35 days and then with testosterone for 35 days; note
the elongating spermatids (E) and patchy spermiogenesis in some tubules
(*). f, Testis from a hpg mouse treated with 2%
17ß-estradiol for 70 days, combined with testosterone for the final
35 days, showing elongating (E) and mature spermatids (M). g,
Epididymal histology in a hpg mouse treated with
cholesterol only. h, Epididymis from a wild-type control, shown for
comparison, with abundant spermatozoa in the lumen. i, Epididymal
tubules from an hpg mouse treated for 70 days with 2%
17ß-estradiol; note the similar density of spermatozoa, compared with
the wild-type mouse. Scale bars in a and g, 100 µm;
applied to all figures, magnification x275.
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Serum estradiol concentrations in male and female hpg mice
treated with cholesterol (Fig. 2
, upper), were undetectable, whereas the 2% and 10%
estradiol implants dose-dependently increased circulating estradiol
concentrations (Fig. 2
, upper). The range of values in
hpg mice treated with the 2% implants was 2554 pg/ml,
below those (100200 pg/ml) reported for proestrous female mice with a
more sensitive RIA (25). In the female hpg mice, 12 days of
treatment with the sc implants produced increases in uterine weight
comparable with nonpregnant age-matched wild-type littermates (Fig. 2
, lower).

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Figure 2. Top, Serum estradiol concentrations
in 2-month-old female hpg mice treated for 12 days with
CHOL, 2% 17ß-estradiol implants (2%E), 10% 17ß-estradiol
implants (10% E) or in males receiving implants for 35 days in Exp II.
Values are group mean ± SE, n = 34 per group.
Bottom, Uterus weights in the hpg females
receiving cholesterol or estradiol implants for 12 days (see
top) or in untreated age-matched wild-type littermates
(+/+).
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Serum FSH concentrations in the male hpg mice treated with
cholesterol (Fig. 3
, left) were
undetectable or close to the limit of detection, whereas FSH levels
were detectable in all hpg mice treated with 2% estradiol
implants. Levels were significantly elevated (P <
0.05) in the estradiol-treated hpg mice, compared with the
cholesterol-treated hpg mice, but were 4-fold lower than in
age-matched male wild-type littermates (Fig. 3
) sampled as a positive
control for the assay. Serum FSH concentrations were also measured in
blood samples collected from a pilot experiment in which adult
wild-type C3H mice were treated for 12 days with sc implants containing
cholesterol, 2% estradiol, or 10% estradiol (Fig. 3
, right). Both estradiol treatments significantly
(P < 0.005) reduced serum FSH levels, relative to the
cholesterol-treated mice (Fig. 3
, right).

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Figure 3. Left, Exp II, Serum FSH
concentrations in male hpg mice treated for 35 days with
cholesterol CHOL or 2%E or in wild-type littermates (+/+); *,
P < 0.05; ***, P < 0.001
vs. cholesterol-treated hpg control
group. Right, Serum FSH concentrations in wild-type
(+/+) adult male C3H mice treated for 12 days with CHOL, 2%E, or 10%
17ß-estradiol implants (10%E); ***, P < 0.005
vs. cholesterol-treated wild-type control group;
arrowhead, assay limit of detection.
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Exp III
Because spermatogenesis in the mice treated with estradiol
for 35 days was qualitatively and quantitatively incomplete, compared
with normal mice, a third study was conducted to investigate whether
complete spermatogenesis could be induced in hpg mice by a
longer period of treatment, or a greater dose of estradiol, or by
additional exposure to testosterone. Three groups of hpg
mice received implants of cholesterol, or 2% or 10% estradiol for 70
days; and two subgroups, treated with cholesterol or 2% estradiol,
also received Silastic implants containing 100% testosterone for the
final 35 days of the experiment. Estradiol treatments for 70 days were
sufficient without additional androgen supplementation to induce full
qualitatively normal spermatogenesis (Fig. 4
; c, d, and i), comparable with that
seen in wild-type controls (Fig. 4
, b and h). The histological
appearance of the testes (Fig. 4
, c and d) and wet weights of the
testes (Fig. 5
, top), epididymides (Fig. 5
, second from bottom
panel), and seminal vesicles (Fig. 5
, bottom)
were similar in the groups treated with either 2 or 10% estradiol.
Serum testosterone concentrations remained below the limit of detection
in the hpg mice treated with 2 or 10% estradiol (Fig. 5
, second from top), but circulating FSH concentrations were
significantly elevated by this treatment (Fig. 5
, middle),
albeit not to the levels seen in male wild-type mice (Fig. 5
, middle).

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Figure 5. Exp III. From top
to bottom, Paired testes weights, serum testosterone
concentrations, serum FSH concentrations, paired epididymides weights,
and paired seminal vesicle weights in hpg mice treated
for 70 days with cholesterol implants (chol), 2% E or 10%
17ß-estradiol implants (2% E or 10% E) and in untreated age-matched
wild-type littermates (+/+). chol+T and 2%E+T groups also received
testosterone implants for the latter 35 days of the experiment. Values
are group mean ± SE. Group size is indicated at the
bottom; except for serum FSH values, where group sizes are n = 3,
or n = 2 where error bars have been replaced with
the values for individual mice. *, P < 0.05; **,
P < 0.01; ***, P < 0.001;
****, P < 0.0001 vs.
cholesterol-treated control group. Note that the additional
testosterone treatment produced physiological concentrations of
testosterone in the circulation and increased growth of the
epididymides and seminal vesicles, but did not induce further testis
growth, compared with the hpg mice only receiving
estradiol treatments.
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Morphometric analysis of paraffin sections stained with hematoxylin and
eosin revealed that estradiol treatment for 70 days induced a 5-fold
increase in total seminiferous tubule volume (Table 2
), which reflected a 4- to 5-fold
increase in the total volume of the seminiferous epithelium (Table 2
).
The total tubule length per testis did not differ significantly between
any of the experimental groups (Table 2
). Testosterone supplementation
produced no further increase in the total volume of seminiferous
tubules or of the seminiferous epithelium (Table 2
), confirming that
estradiol treatment alone increased the spermatogenic activity of the
seminiferous tubules. Both estradiol doses resulted in an abundance of
mature spermatozoa in the epididymis (Fig. 4i
). The estradiol
treatments significantly increased the total volume of Sertoli cell
nuclei in the testis (Table 2
) and, by inference, the number of Sertoli
cells present (though, after estradiol treatment, the total volume of
Sertoli cell nuclei still only reached about one third of that for
wild-type mice (Table 2
). The testosterone treatments produced
circulating androgen concentrations within the normal range for adult
male mice (Fig. 5
) and induced major growth of the seminal vesicles
(Fig. 5
, bottom), additional growth of the epididymides
(Fig. 5
), and partial induction of spermatogenesis (Fig. 4e
),
confirming the bioactivity. However, the supplementary testosterone
treatment did not increase testis weight (Fig. 5
, top), or
stimulate spermatogenesis beyond that produced by estradiol alone (Fig. 4f
). Testosterone treatment alone did not increase the total volume of
Sertoli cell nuclei (Table 2
), and the supplementary testosterone
blocked the stimulatory effect of estradiol treatment on total Sertoli
cell nuclear volume (Table 2
).
 |
Discussion
|
|---|
Although induction of spermatogenesis in hpg mice by
androgens has been previously reported (19), our studies demonstrate
that administration of estradiol within the normal physiological range
(for females) can exert similar effects. The current assay was not
sufficiently sensitive to determine normal circulating and
intratesticular estradiol concentrations in the male mouse, so we
cannot be sure how the levels generated by the 2% implants relate to
true physiological values in the male. Nevertheless, the estradiol
doses we used are certainly less than those previously reported to
alter Sertoli cell replication in the testis when administered to
pregnant rodents (26). The current doses are also far lower than the 15
µg per day estradiol benzoate treatment that had been shown to
increase the proliferation of type A spermatogonia when delivered to
infant rats from 511 days of age (27). Our studies greatly extend
these original observations by Kula (27), who first postulated a
stimulatory role for estradiol in spermatogenesis in the rat, because
we have demonstrated that: 1) qualitatively, full germ cell development
can be induced by estradiol; 2) this can be achieved by concentrations
of estradiol that are likely to be in the physiological range; and 3)
stimulatory actions of estradiol occur in the mouse, as well as in the
rat.
Quantitatively, spermatogenesis was reduced, compared with wild-type
controls; for example, the total mean seminiferous epithelial volume
per testis in estradiol-treated hpg mice was 4-fold less.
This may reflect the greatly reduced proliferation, and thus supply, of
Sertoli cells in the hpg mouse that have not been exposed to
gonadotropin at any stage of development, in contrast to several other
models used to study spermatogenesis, in which endocrine support is
withdrawn during postnatal life; for example, by hypophysectomy (28),
by destruction of Leydig cells using ethane dimethylsulphonate (29), or
by GnRH immunoneutralization (30). If hpg mice are treated
in the neonatal period with FSH, there is marked proliferation of the
Sertoli cell population (31), and subsequent FSH plus androgen
treatments are quantitatively more effective in inducing
spermatogenesis than in hpg mice that are not treated with
FSH during the neonatal period (31). In fact, the induction of
spermatogenesis observed in the estradiol-treated hpg mice
was accompanied by significant increase in the total volume of Sertoli
cell nuclei and, by inference, in the number of Sertoli cells, though
the testes were still markedly deficient in Sertoli cells, when
compared with wild-type controls.
Although the estradiol treatments decreased serum FSH concentrations in
wild-type mice (as expected), they induced a small (but significant)
rise in circulating FSH levels in the male hpg mice. A
previous study, in which female hpg mice were treated with
sc implants of estradiol benzoate for 5 days, failed to detect an
increase in circulating FSH levels, despite the observation of
follicular development in the ovary that suggested stimulation by FSH
(32). The increase in FSH in male hpg mice in the current
study must reflect a direct pituitary action of estradiol, and it is
possible that this paradoxical stimulatory effect of estradiol on the
pituitary reflects incomplete masculinization during the neonatal
period in male hpg mice, which by their very nature, lack
androgen production. This unexpected increase in FSH concentrations in
hpg mice treated with estradiol provides one possible
explanation for the induction of spermatogenesis. A large body of
evidence indicates that FSH and testosterone support the completion of
meiosis and spermiogenesis via actions on Sertoli cells (see Ref. 33
for review), but it is controversial as to whether FSH alone can
initiate spermatogenesis in the absence of androgens. Could the low
levels of FSH induced by the estradiol treatments in the current study
be sufficient to drive spermatogenesis? One study of the hpg
mouse (34) concluded that FSH alone was not sufficient to complete
spermatogenesis, in that FSH treatment given to weanling (day-21) mice
increased proliferation of spermatogonia and primary spermatocytes, but
condensed (i.e. fully elongated) spermatids were not formed
by 70 days of age. However, important differences between that study
and the present observations should be noted. That study (34) used
recombinant human FSH rather than murine FSH, the treatment was given
once daily rather than a continual release that would be predicted in
the current estradiol treatment, and the treatment was only continued
for 49 days; FSH may have been increased for all 70 days of the
estradiol treatment in the current study. Moreover, inspection of the
photomicrographs in the previous study (34) does indicate the presence
of round and early elongating spermatids after FSH treatment alone.
Furthermore, a quantitative assessment of germ cell development was
reported, revealing that approximately one million round spermatids per
testis were formed after FSH treatment, whereas these germ cells were
totally absent in age-matched, untreated hpg mice (34). The
issue of the role of FSH in initiation of spermatogenesis is confounded
by the possibility that FSH might indirectly promote androgen
production by Leydig cells. Treatment of hpg mice, at an
adult age, with purified ovine FSH stimulated the development of round
spermatids and increased the androgenic capacity of the testes, as
determined in vitro (18), though no effects of 10 days of
FSH treatment on serum testosterone or testicular content of androgen
occurred (18). Likewise, in the current study, circulating testosterone
concentrations remained below the limit of detection in the
estradiol-treated hpg mice with increased FSH levels. The
slight (but significant) increase in the weight of the seminal vesicles
in the estradiol-treated hpg mice could indicate small rises
in circulating androgen that our assay was unable to detect. However,
two previous studies of the effects of ovine FSH (18) or human
recombinant FSH (34) in hpg mice failed to detect any
increase in intratesticular testosterone levels; thus, there is little
reason to believe that the small increases in FSH concentrations
induced by estradiol in the current study would have raised
intratesticular testosterone to a level detectable by immunoassay or
ELISA. An unequivocal test of any role for FSH and androgens in
estradiol-induced spermatogenesis may best be obtained by introducing
mutations of the FSH ß-subunit gene or receptor (35, 36) or androgen
receptor (should such mice be produced) into the hpg line,
and then examining whether the stimulatory effects of estradiol on
spermatogenesis are abolished.
Even if FSH alone is sufficient to initiate spermatogenesis, it
is certainly not absolutely necessary, as revealed by the partial
fertility in male mice genetically modified to be deficient in FSH
ß-subunit production (35) or lacking the FSH receptor (36). An
alternative (or perhaps additional) explanation of the current
observations is that there are effects of estrogen directly on the germ
cell line. Whereas ER
is only expressed in Leydig cells, an
immunocytochemical study in the rat has revealed ERß in Sertoli cells
and in germ cells (9), though the abundance of ERß immunoreactivity
in spermatocytes decreased after the pachytene phase of meiotic
maturation (9). Consistent with these findings in the rat, a recent
in situ hybridization study has demonstrated ERß messenger
RNA in both Sertoli cells and germ cells in adult Cynomolgus monkeys
(37). Given the identification of aromatase in germ cells of the mouse
testis (38), it is possible that some of the actions previously
ascribed to androgens acting on Sertoli cells are, in fact, mediated
via aromatization to estradiol acting on germ cells directly, and
that this is what the current experimental treatment is mimicking.
Estrogen synthesis has been demonstrated in the fetal rat testis (39, 40); so, it is possible that because the testis in the hpg
mouse is essentially neonatal throughout life, then such testes remain
sensitive to the effects of estradiol. It was recently reported (41)
that estradiol could induce proliferation of isolated rat fetal
gonocytes in vitro, so there is a precedent for estrogenic
actions on the germ cell line. Although that study (41) used doses of
estradiol in the micromolar range to induce proliferation of gonocytes,
this may not necessarily be supraphysiological, because fetal rat
Sertoli cells would seem to secrete high levels of this steroid (39, 40). There is also in vitro evidence for estrogenic actions
on Sertoli cells themselves, in that physiological doses of estradiol
increase inhibin B production by Sertoli cell cultures derived from
18-day-old rats (42).
The slight growth of the seminal vesicles in the estradiol-treated
hpg mice could also reflect actions of estradiol, in the
male reproductive tract, mediated via androgen receptors. Although we
cannot unequivocally rule out the possibility that the effects of
estradiol on spermatogenesis are mediated via such a pathway, we
consider it unlikely. To date, the evidence for such cross-talk between
estrogen and androgen receptors is based entirely on in
vitro studies employing a prostate cancer cell line (43), and
using supraphysiological concentrations of estradiol (43). Support for
a physiological role of estrogen in spermatogenesis comes from the
recent observations that there is a progressive long-term deterioration
in spermatogenesis in genetically modified mice lacking aromatase (44, 45). Although young (23 month) male ERß knockout mice are reported
to be fertile (46), it is not known whether there is a subsequent
impairment of spermatogenesis with increasing age, as reported for
1-yr-old male mice lacking aromatase (45). The widespread expression of
aromatase, ER
, and ERß in different compartments of the testis
points to a physiological role for estrogen in many aspects of
testicular function. The development of infertility in ER
knockout
mice may be partly attributed to an impairment of fluid reabsorption
from the testicular efferent ducts; but other actions of estrogen in
the testis are beginning to emerge [for example, on the proliferation
of Sertoli cells early in life (26) and on the expression of
steroidogenic factor 1 (13)].
In conclusion, the current observations of induction of spermatogenesis
in hpg mice may reflect indirect actions via stimulation of
FSH secretion, or direct effects within the testis on the development
of the germ cell line, or a combination of these two processes. Further
studies are justified to resolve this and to determine whether the
unusual reproductive development (possible lack of neonatal
androgenization) in this mutant line contribute to the observed effects
of estrogen. Notwithstanding these caveats, these studies identify
potentially important actions of estrogen on reproductive function in
the male that might be mimicked or antagonized by environmental
chemicals with estrogenic properties.
 |
Acknowledgments
|
|---|
We thank staff at the Department of Anatomy for
assistance with animal care; staff in the Pathology Section,
Zeneca Pharmaceuticals, Central Toxicology
Laboratory, for carrying out histological processing; Drs. H.
Charlton FRS and J. Lang for reagents and advice in establishing the
PCR procedure for identification of hpg genotype; and Mr. I.
Swanson (Medical Research Council Reproductive Biology Unit,
Edinburgh) and Dr. J. Herbert (Department of Anatomy, University
of Cambridge) for the gift of reagents for the testosterone ELISA.
 |
Footnotes
|
|---|
1 This work was supported by a Zeneca Pharmaceuticals
Strategic Research Award (SRF 297) and by The Royal Society (University
Research Fellowship, to F.J.P.E). 
Received December 15, 1999.
 |
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