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School of Biomedical Sciences (F.J.P.E.), University of Nottingham Medical School, Queen’s 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, Queen’s Medical Centre, Nottingham NG7 2UH, United Kingdom. E-mail: fran.ebling{at}nottingham.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-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 2–3 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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 0.03–0.05 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 Dunnett’s 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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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 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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, 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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, 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 25–54 pg/ml,
below those (100–200 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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, 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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; 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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), 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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 (2–3 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.
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Acknowledgments |
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Footnotes |
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Received December 15, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) within the testis and excurrent ducts of the rat and marmoset
monkey from perinatal life to adulthood. J Endocrinol 153:485–495[Abstract]
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