1 Department of Animal Reproduction, National Institute of Animal Industry, Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki 305-0991; 2 Primate Research Institute, Kyoto University, Inuyama, Aichi 484-8506; and 3 Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
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ABSTRACT |
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We investigated the importance of inhibin and
testosterone in the regulation of gonadotropin secretion in adult male
golden hamsters (Mesocricetus auratus). After castration,
plasma concentrations of inhibin and testosterone were reduced to
undetectable, whereas plasma follicle-stimulating hormone (FSH) and
luteinizing hormone (LH) were increased. After hemicastration, plasma
FSH and LH increased moderately and plasma inhibin decreased to
one-half its initial level. Plasma testosterone levels in hemicastrated
animals decreased 3 h after hemicastration but returned to those in
sham-operated animals at 6 h. Plasma LH in the castrated hamster
declined comparably to intact animals with testosterone treatment;
plasma FSH also decreased but still remained at levels higher than
those in intact animals. After treatment with inhibin in
long-term-castrated animals, plasma FSH decreased, whereas plasma LH
was not altered. Intact males treated with flutamide, an anti-androgen,
showed a significant increase in plasma LH but not in FSH. On the other
hand, treatment with anti-inhibin serum induced a significant elevation
in plasma FSH, but not in LH. Using immunohistochemistry, we showed
that the inhibin -subunit was localized to both Sertoli and Leydig cells. The present study in adult male hamsters indicates that FSH
secretion is regulated mainly by inhibin, presumably from Sertoli and
Leydig cells, and that LH secretion is controlled primarily by
androgens produced from the Leydig cells. This situation is more
similar to that of primates than of rats.
gonadotropin; flutamide; testosterone; immunohistochemistry; testis; follicle-stimulating hormone
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INTRODUCTION |
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INHIBIN IS NOW WELL KNOWN as a major factor in the regulation of follicle-stimulating hormone (FSH) secretion in female rodents (1, 19, 20, 34), ruminants (17, 18, 23, 24), and primates (6). Regarding males, however, there is a controversy concerning the physiological importance of circulating inhibin in the regulation of FSH secretion, especially in rodents such as rats (9, 33). Unilateral orchidectomy results in compensatory hypertrophy of the remaining testis, accompanied by an elevation in circulating FSH in immature male rats (14, 22, 30), but not in mature males (16). Culler and Negro-Vilar (10), using rats with destroyed Leydig cells, demonstrated that the negative feedback effect of inhibin on the secretion of FSH in the adult male rat is masked by the presence of high amounts of testosterone produced by the Leydig cell and that testosterone is a primary factor in the regulation of FSH secretion. These data suggest that a testicular factor, maybe inhibin, is important for the regulation of FSH secretion in immature, but not in adult, male rats.
In the present study, we studied the involvement of testicular inhibin
and androgen in the regulation of gonadotropin secretion in the adult
male golden hamster by determining changes in plasma gonadotropins,
immunoreactive (ir)-inhibin, and testosterone 1) after
bilateral or unilateral orchidectomy, 2) after treatment with
inhibin or testosterone, and 3) after immunoneutralization against inhibin or treatment with anti-androgen. We also investigated the localization of the inhibin -subunit in the testis immunohistochemically.
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MATERIALS AND METHODS |
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Animals. Adult male golden hamsters were used in this study. The animals were housed in groups of 6-8 animals per cage in a room with controlled conditions of temperature, humidity, and lighting (14:10-h light-dark cycle; lights on at 0500). Food and water were available ad libitum.
Inhibin preparation.
Inhibin administered in the present experiment was obtained as follows.
Bovine inhibin was partially purified from bovine follicular fluid by
use of immunoaffinity chromatography, the standard preparation for
radioimmunoassay of inhibin (15). After immunoaffinity chromatography,
the inhibin-rich fraction was concentrated by an ultrafilter (molecular
weight cutoff 10,000; ULTRA FILTER UK-10, 43 mm diameter, ADVANTEC
TOYO, Tokyo, Japan) as described by Miyamoto et al. (29). The final
concentrations of inhibin were adjusted by adding saline. The dose of
inhibin preparation was represented as the value obtained from
radioimmunoassay using antiserum against the -subunit of inhibin.
Inhibin bioactivity at the highest dose (10 µg) of the inhibin
preparation used in the present study corresponded to ~1.3 µg of 32 kDa purified bovine inhibin by use of the rat pituitary cell culture system.
Antiserum against inhibin for injection.
Antiserum against inhibin (inhibin-AS) used in the present study was
the same antiserum as described previously in studies with female
hamsters (19, 20). Briefly, a castrated goat was immunized against
[Tyr30]inhibin--(1
30) conjugated to rabbit
serum albumin (kindly provided by Dr. N. Ling, Neurocrine Bioscience,
San Diego, CA). After immunization, serum samples were collected and
pooled. One lot (TNDK-4) of these was used in the present study.
Control serum was obtained from a castrated goat.
Primary antibodies against inhibin- and
3-hydroxysteroid dehydrogenase for immunohistochemistry.
A rabbit was immunized several times against
[Tyr30]inhibin-
-(1
30) conjugated to rabbit
serum albumin, as described above. After immunization, serum samples
were collected and pooled. One lot (TNDK-1) was used for immunohistochemistry.
Experiment: unilateral or bilateral orchidectomy.
Unilateral or bilateral orchidectomy was performed at 1100 with
hamsters under ether anesthesia, and animals were killed by decapitation at various times up to 48 h after the operation. Sham
operation was performed for control animals. Trunk blood was collected
into heparinized centrifuge tubes, and plasma samples were obtained by
immediate centrifugation at 1,700 g for 15 min at 4°C.
Plasma samples were stored at 20°C until assayed for FSH,
luteinizing hormone (LH), inhibin, and testosterone.
Administration of inhibin.
Partially purified bovine inhibin (0.1-10 µg/ml saline ip) was
injected at 1100 into individual animals that had been orchidectomized >3 wk before. Saline was injected as control. Animals were
decapitated at various times up to 48 h after the injections. Plasma
samples were obtained as described above and stored at 20°C
until assayed for FSH, luteinizing hormone (LH), and inhibin.
Administration of testosterone.
Male animals had been castrated 4 wk before the experiment. These
animals were implanted with a 20-mm-long Silastic tube (1.57 mm ID and
3.18 mm OD; Dow Corning, Midland, MI) containing crystalline
testosterone. Empty tubes were implanted in control animals.
Testosterone-containing tubes were incubated in saline at 37°C for
24 h before implantation to avoid a surge-like release after
implantation. Groups of animals were killed by decapitation at 0, 3, 6, 12, 24, 36, and 48 h after implantation, and plasma samples were
obtained as described above. Plasma samples were stored at
20°C until assayed for FSH, LH, and testosterone.
Treatment with inhibin-AS and/or flutamide. Four groups were assigned to the following four treatments at 1100: 1) a combination of 300 µl of vehicle (ethanol-sesame oil, 1:1) and 200 µl of normal goat serum (control serum) for controls, 2) 50 mg/kg 2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]propanamide (flutamide; Sigma Chemical, St. Louis, MO) in 300 µl of vehicle and 200 µl of control serum like the flutamide-alone treatment group, 3) 300 µl of vehicle and 200 µl of inhibin-AS, like the inhibin-AS-alone treatment group, and 4) 50 mg/kg flutamide in 300 µl of vehicle and 200 µl of inhibin-AS as the combined-treatment group. Vehicle and flutamide were given subcutaneously in the animals' lumber region, and control serum and inhibin-AS were administered via jugular vein under light ether anesthesia.
Groups of animals were killed by decapitation every 6 h until 24 h after treatments. Plasma samples were obtained as described above and were stored atImmunohistochemistry.
Testes obtained in the castration experiments were examined
immunohistochemically. The testes were embedded in paraffin after fixation with 4% paraformaldehyde and were sectioned at 6 µm. The
immunostaining was performed as described previously (31). Briefly,
after castration, the sections were soaked in 0.01 M sodium citrate
buffer (pH. 6.0) and were autoclaved at 121°C for 15 min to expose
the epitope for each antiserum (37). Thereafter, sections were
incubated for 30 min with 0.05 M Tris · HCl (pH 7.6)
containing 0.15 M NaCl (TBS) and 3% H2O2 to
quench endogenous peroxidase activity. Then the sections were incubated
with Block Ace (Dainippon Pharmaceutical, Osaka, Japan) at 37°C for
30 min in a humidified chamber to control for nonspecific reactions. Subsequently, the sections were incubated with the primary antiserum against the inhibin- subunit (TNDK-1) diluted 1:4,000 or 3
-HSD-AS diluted to 5 ng/ml in TBS containing 10% Block Ace overnight at 37°C. After a wash with TBS, the sections were incubated with 0.5%
biotinylated goat anti-rabbit second antibody (Vector Laboratory ABC-peroxidase-staining kit, Elite, CA) diluted in TBS containing 10%
Block Ace at 37°C for 1 h. After three washes with TBS for 5 min
each, the sections were treated with 2% avidin-biotin complex (Vector)
at 37°C for 30 min. The sections were then reacted with 0.5%
3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical, St
Louis, MO) and 0.01% H2O2 to visualize the
bound antibody.
Determination of plasma concentrations of FSH, LH, inhibin, and testosterone. Concentrations of each hormone in the plasma were determined by specific RIAs as follows. Plasma concentrations of FSH and LH were measured using National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) RIA kits for rat FSH and LH, as described previously (4). Iodinated preparations were rat FSH-I-8 and LH-I-9. The antisera used were anti-rat FSH-S-11 and LH-S-10. Results were expressed in terms of NIDDK rat FSH-RP-2 and LH-RP-2. The intra- and interassay coefficients of variation were 4.4 and 14.6% for FSH and 8.9 and 6.7% for LH, respectively.
Plasma concentrations of inhibin were measured as described previously (15, 20). The iodinated preparation was 32-kDa bovine inhibin purified in this laboratory, and the antiserum used was rabbit antiserum against bovine inhibin (TNDH-1). Results were expressed in terms of 32 kDa bovine inhibin. The intra- and interassay coefficients of variation were 8.8 and 14.4%, respectively. Plasma concentrations of testosterone were determined by an double-antibody RIA system with 125I-labeled radioligands as described previously (39). The antiserum against testosterone (GDN 250) (12) was kindly provided by Dr. G. D. Niswender, Colorado State University (Fort Collins, CO). The intra- and interassay coefficients of variation were 6.3 and 7.2%, respectively.Statistics. All data were expressed as means ± SE. One-way ANOVA was performed, and the significance between two means was determined by Student's t-test or the Cochran-Cox test, and the significance among more than two means was determined by Duncan's multiple range test (38). A value of P < 0.05 was considered statistically significant.
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RESULTS |
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Effects of unilateral or bilateral orchidectomy on plasma levels of
FSH, LH, ir-inhibin, and testosterone.
In the bilaterally castrated animals, plasma concentrations of FSH
increased significantly after the operation, and then the levels
continued to increase during the experiment (Fig.
1A). A significant increase (P
< 0.05) in plasma levels of FSH in the animals with unilateral
orchidectomy was also found 12 h after the operation. However, the
increment of FSH was slight, and then the levels remained static during
the rest of the experiment. Plasma levels of LH in the bilaterally
orchidectomized group increased significantly (P < 0.05)
within 12 h of the operation, and the elevated levels were maintained
for 48 h (Fig. 1B). Although plasma levels of LH in the
unilaterally orchidectomized group were also significantly increased,
the increment in plasma LH was slight compared with that in animals
orchidectomized bilaterally.
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Changes in plasma concentrations of FSH, LH, and ir-inhibin after
the treatment with inhibin in long-term-castrated males.
Plasma concentrations of FSH decreased significantly (P < 0.05) compared with initial values within 3 h after treatment with 10 µg inhibin (Fig. 2A). Plasma FSH
reached a nadir at 12 h after treatment. However, this lowest value was
still higher (P < 0.05) than that in intact animals.
Thereafter, plasma concentration of FSH tended to increase after 24 h
of treatment, although the value was still lower than that noted at the
initial time. On the other hand, plasma concentrations of LH at any
time after the treatment with inhibin were not different from the
initial values (Fig. 2B). Both plasma FSH and LH were not
significantly altered by treatment with the lower doses (1 µg or 0.1 µg) of inhibin.
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Changes in plasma levels of FSH, LH, and testosterone after
treatment with testosterone to long-term-castrated males.
Plasma concentrations of FSH and LH in long-term-castrated males were
reduced by the treatment with testosterone (Fig.
3, A and B). Within 12 h
after testosterone treatment, plasma LH was completely suppressed to
levels observed in intact males (see the value at 0 h postcastration,
Fig. 1B); plasma concentration of FSH was still higher than
that observed for intact animals.
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Changes in plasma concentrations of FSH, LH, ir-inhibin, and
testosterone after treatment with inhibin-AS, flutamide, or a
combination of inhibin-AS and flutamide.
Plasma concentrations of FSH were elevated significantly (P
< 0.05) after treatment with inhibin-AS, alone or in combination with flutamide (Fig. 4A). There was
no significant difference in plasma concentrations of FSH between the
groups treated with either flutamide alone or vehicle.
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Localization of ir-inhibin -subunit or
3
-HSD in the testis.
Immunopositive reaction of the inhibin
-subunit was found in the
seminiferous tubules and also in the interstitial cells (Fig.
5, C and D). The reaction
intensity in the seminiferous tubules varied in a pattern consistent
with localization in Sertoli cells. A highly positive reaction against
the inhibin
-subunit was found in Sertoli cells at stages
VIII-IX of seminiferous tubules as classified by
Clermont (8), which then gradually decreased from stage X, and
few positive reactions were found at stages II-VII. The
positive reaction of inhibin
-subunit was also observed in the
interstitial cells. These cells also reacted positively to 3
-HSD
(Fig. 5E).
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DISCUSSION |
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This is the first report to show the physiological importance of circulating inhibin in the regulation of tonic FSH secretion in mature male rodents by use of specific antiserum against inhibin. Mature intact male hamsters treated with inhibin-AS showed a significant increase in plasma concentrations of FSH, suggesting that circulating inhibin in the animals plays a physiological role in the suppression of FSH secretion. Treatment with inhibin to long-term-castrated hamsters caused a reduction in plasma concentrations of FSH. This observation also supports the suggestion that inhibin regulates FSH secretion in the mature male, as well as female, hamster (19, 20). However, Culler and Negro-Vilar (9) and Rivier et al. (33) demonstrated that immunoneutralization against inhibin does not alter plasma concentrations of FSH in adult male rats. Furthermore, Culler and Negro-Vilar concluded that the negative effect of inhibin on FSH secretion is masked by the existence of high amounts of testosterone, which would play the primary role for the regulation of FSH secretion in mature male rats (10). As far as we know, in the fully adult male, only primates, such as monkeys (27), and ruminants, rams (41), and bulls (25) are taxa that show an increase in plasma FSH after passive or active immunization against inhibin. The importance of circulating inhibin in the regulation of FSH secretion in male animals may vary depending on species and age. Therefore, golden hamsters would be a good model animal for studying gonadal regulation of gonadotropin secretion in primates, including humans.
In the present study, treatment with flutamide to intact male hamsters induced an increase in plasma concentrations of LH. Testosterone implants into long-term-castrated hamsters caused the plasma concentrations of LH to be reduced to those of normal intact males. These findings also suggest that androgens in the male hamster act as regulators of LH secretion. Based on the present results after immunoneutralization to inhibin or inhibin administration, circulating inhibin may not play an important role in regulating LH secretion in this model. These findings suggest that circulating androgens are the primary negative regulators of LH secretion in mature male hamsters.
Replacement of testosterone in the long-term-castrated hamster reduced plasma concentrations of FSH in the present study. This finding indicates that testosterone in male hamsters regulates not only LH but also FSH secretion. Contrary to the suggestion obtained from the testosterone replacement study, treatment with flutamide to intact hamsters did not alter plasma concentrations of FSH. In addition, no additive increase in plasma FSH was observed in animals cotreated with flutamide and inhibin-AS. These findings suggest that circulating androgens are important in regulating only LH secretion. The discrepant effects of androgens on FSH secretion obtained from anti-androgen treatment and testosterone replacement experiments might be due to the differences in status of the animals used in each experiment (normal vs. long term castrated). We believe that acutely changing endogenous androgens are not sole regulators of FSH secretion, although testosterone itself exerts a suppressive action on FSH secretion. There is a report that flutamide treatment in normal hamsters causes the elevation of FSH secretion (2). However, in that study, because hamsters were injected with 50 or 100 mg/kg flutamide daily, the response might have been due to a chronic, not acute, effect of anti-androgens on FSH secretion. Therefore, that report does not negate the present results.
In the present study, plasma concentrations of inhibin rapidly
decreased and became only a trace within 6 h after bilateral orchidectomy. This finding indicates that the main source of plasma inhibin is the testis, in the male hamster as well as in other mammals.
Immunohistochemical study indicates that the inhibin- subunit is
localized in the Sertoli cell of the male hamster, as well as the rat
(3, 28, 33). In addition, the present study clearly demonstrated that
the inhibin-
subunit was also localized to interstitial cells.
Because these interstitial cells were positive to 3
-HSD, presumably
they were Leydig cells. It was suggested in some previous reports that
Leydig cells can produce the inhibin-
subunit in rats and sheep (3,
35, 40). In addition, Risbridger et al. (32) reported that the
Leydig cells of adult rats expressed mRNA for the inhibin-
subunit
and produced immunoreactive and bioactive inhibin in an in vitro cell
culture system (32).
In the immunohistochemical study, the intensity of immunostaining for
the inhibin- subunit varied in seminiferous tubules and was less in
the seminiferous tubules containing nearly mature spermatocytes. These
findings suggest that inhibin production may depend on the stage of the
seminiferous epithelium. The same finding was shown in the mRNA levels
of the rat testis (5, 21), and the changes in inhibin production along
with spermatogenesis in seminiferous tubules were also demonstrated in
the previous study using rats (13). Inhibin might, therefore, play a
paracrine role in the differentiation of spermatogenic cells.
In bilaterally orchidectomized animals, two components were observed in the decay of ir-inhibin. The t1/2 of the first phase was ~15 min, and the second t1/2 was ~20 h. These values agreed with a previous study in which the male rat was used (36).
In hemicastrated animals, plasma concentrations of testosterone recovered to intact levels rapidly, after an initial decline. This rapid recovery of steroid hormones suggests a rapid compensatory testosterone synthesis by the remaining testis. This rapid compensation is also demonstrated in the previous study using rats (11). In the hemicastrated animals, ir-inhibin concentrations were one-half to two-thirds of the level of intact animals during the experiment, suggesting that production of inhibin, unlike testosterone, does not recover quickly. A moderate but significant increase in plasma concentrations of FSH was observed after the hemicastration. This finding, together with our inhibin-AS treatment, suggests that low levels of circulating inhibin in the hemicastrated hamster are mainly responsible for the high levels of plasma FSH. On the other hand, plasma concentrations of LH after hemicastration were lower than those after bilateral castration. These lower levels of LH in hemicastrated animals compared with bilaterally castrated animals may be mainly responsible for the quick recovery of plasma testosterone. Although the levels of testosterone would be enough to suppress plasma levels of LH to those of intact animals, slightly higher levels of LH in the plasma were observed in the group undergoing unilateral orchidectomy. It is not clear why this occurs. One possibility is that a longer time would be needed than in intact hamsters to suppress LH levels after recovery of testosterone levels. Another possibility is that some factors that suppress LH secretion would be secreted in the testis and would not be recovered within the experimental period.
In conclusion, the present study suggests that circulating inhibin is the primary factor in the regulation of FSH secretion in the mature male hamster, as well as in females. The present results also suggest that the source of ir-inhibin is the Sertoli cells in the hamster, as in other species, and that the Leydig cells are also a possible source of ir-inhibin. Our results also suggest that testosterone exerts a limited function in the regulation of FSH secretion but plays the major role in the regulation of LH secretion in male hamsters.
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ACKNOWLEDGEMENTS |
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We express our gratitude to Dr. R. J. Hutz, Department of
Biological Sciences, University of Wisconsin-Milwaukee (Milwaukee, WI),
for reading the original manuscript and for valuable suggestions. We
are also grateful to Dr. A. F. Parlow and the Rat Pituitary Hormone
Distribution Program, National Institute of Diabetes and Digestive and
Kidney Diseases (Bethesda, MD), for providing RIA materials; Dr. G. D. Niswender, Animal Reproduction and Biotechnology Laboratory, Colorado
State University (Fort Collins, CO) for providing antiserum to
testosterone (GDN 250); Dr. N. Ling, Neuroendocrine Inc. (San Diego,
CA) for providing [Tyr30]inhibin--(1
30);
Dr. J. I. Mason, Cecil H. and Ida Green Center for Reproductive
Science, University of Texas Southern Medical Center (Dallas, TX) for
providing antiserum to 3
-hydroxysteroid dehydrogenase, and Teikoku
Hormone, MGF Co. Ltd. (Tokyo, Japan) for providing testosterone.
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FOOTNOTES |
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This work was supported by a grant-in-aid for the Liberal Harmonious Research Promotion System from the Science and Technology Agency, Japan, and a grant-in-aid for Scientific Research from the Ministry of Education of Japan (11839003).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: K. Taya, Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, 3-5-8, Saiwai-cho, Fuchu, Tokyo 183-8509, Japan (E-mail: taya{at}cc.tuat.ac.jp).
Received 26 January 1999; accepted in final form 2 November 1999.
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