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Endocrinology Vol. 144, No. 4 1175-1185
Copyright © 2003 by The Endocrine Society

ARTICLE

Inhibitory and Stimulatory Regulation of Testicular Inhibin B Secretion by Luteinizing Hormone and Follicle-Stimulating Hormone, Respectively, in the Rhesus Monkey (Macaca mulatta)

Suresh Ramaswamy, Gary R. Marshall, Clifford R. Pohl, Robert L. Friedman and Tony M. Plant

Departments of Cell Biology and Physiology (S.R., G.R.M., R.L.F., T.M.P.) and Medicine (G.R.M.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; and Department of Physical Therapy (C.R.P.), School of Health Sciences, Duquesne University, Pittsburgh, Pennsylvania 15282

Address all correspondence and requests for reprints to: Dr. Tony M. Plant, Department of Cell Biology and Physiology, University of Pittsburgh, S 828A Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261. E-mail: plant1{at}pitt.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study examined the relative role of FSH and LH in governing testicular inhibin B secretion in the rhesus monkey. Adult male monkeys, rendered hypogonadotropic and hypogonadal by administration of a GnRH receptor antagonist (acyline), were implanted with testosterone (T)-filled or empty capsules. Following T-induced restoration of spermatogenesis, both groups received recombinant human FSH and vehicle for 12 d. Juvenile male monkeys received an 11-d infusion of single-chain recombinant human LH and recombinant human FSH, either alone or in combination. In adults, chronic hypogonadotropism resulted in a modest reduction of circulating inhibin B levels, which was more than fully reversed by FSH. In the presence of T, which exerted a marked suppression in inhibin B secretion, FSH restored inhibin B levels only to those observed before acyline treatment. In juveniles, treatment with single-chain recombinant human LH led to a suppression of inhibin B secretion and curtailed the FSH-induced stimulation of this testicular hormone. The T-induced decrease in inhibin B secretion was associated with suppression in inhibin-ßB mRNA levels, but FSH stimulation of inhibin B secretion occurred in the absence of clear changes in expression of this subunit gene. These findings indicate that inhibin B secretion by the monkey testis is governed by the inhibitory and stimulatory action of LH and FSH, respectively. The action of LH is presumably indirect and likely mediated by T inhibition of inhibin-ßB gene expression. The molecular basis of the stimulatory action of FSH on inhibin B secretion requires further study.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE RHESUS MONKEY and man, the testicular regulation of the secretion of FSH is achieved primarily by the negative feedback action of inhibin B acting at the level of the pituitary (1, 2). That the secretion of inhibin B is regulated, in turn, by the gonadotropins is indicated by the finding that, in both monkey and man, the pattern of secretion of this testicular hormone from birth to adulthood closely parallels the time course of circulating FSH and LH concentrations during this phase of development (3, 4). Although FSH is generally recognized to stimulate testicular inhibin secretion (5, 6, 7, 8), the action of LH, if any, in this regard is less clear.

We had the opportunity to outline the relative role of FSH and LH in regulating the testicular secretion of inhibin B and expression of the gene encoding the inhibin-ßB subunit in two studies examining the hormonal control of spermatogenesis in the monkey. In one study, adult monkeys rendered hypogonadotropic by treatment with a GnRH receptor (GnRH-R) antagonist were subsequently treated with testosterone (T) and FSH, either alone or in combination. In the other study, juvenile monkeys, in which gonadotropin secretion was arrested by an endogenous control system that delays the onset of puberty in higher primates (9), were stimulated with FSH and LH, either alone or in combination. In both cases, the secretion of inhibin B was monitored by measuring the circulating concentrations of this hormone and testicular expression of the inhibin-ßB gene was quantitated by ribonuclease (RNase) protection assay of RNA derived from whole testes harvested at the time of castration.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Twenty adult [4.5–8 yr of age, 5.5–11 kg body weight, 10–24 ml combined testicular volume (TV)] and 16 juvenile (16–19 months of age, 2.2–3.7 kg body weight) male rhesus monkeys (Macaca mulatta) were used. It should be noted that the results for the juvenile monkeys were obtained during a previously published experiment examining the role of FSH and LH in initiating the pubertal proliferation of Sertoli cells (10). The animals were maintained in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals, and the experimental procedures were approved by the University of Pittsburgh Institutional Animal Care and Use Committee.

Surgical procedures
For the implantation of iv catheters, testicular biopsy, and castration, the monkeys were first sedated with ketamine hydrochloride (50–100 mg im, Ketaject, Phoenix Scientific Inc., St. Joseph, MO). Anesthesia was achieved with sodium pentobarbital (25 mg/kg body weight, iv, plus 5-mg supplements as required, Nembutal sodium solution, Abbott Laboratories, North Chicago, IL). All surgical procedures were performed under aseptic conditions. Postsurgically all animals received a single im injection of penicillin (300,000 U, Bicillin L-A, Wyeth Laboratories, Philadelphia, PA) and iv injections of a broad-spectrum antibiotic (100 mg cefazolin sodium, Kefzol, Apothecon, Princeton, NJ) and an analgesic (1 mg/kg body weight meperidine hydrochloride, Demerol, Elkins-Sinn, Cherry Hill, NJ) twice daily for 4 d. Implantation of SILASTIC capsules was performed under ketamine sedation (100 mg, im, and 50-mg supplements, as required).

Access to venous circulation
SILASTIC (Dow Corning Corp., Midland, MI) venous catheters [inner diameter, 0.040 in. and outer diameter, 0.085 in., Stuart’s Bio-Sil, Sil-med Corp., Taunton, MA] were implanted in an internal jugular and femoral vein. The catheters were tunneled sc from the site of venous insertion to the midscapular region at which they were exteriorized via a small cutaneous fistula. Animals with catheters were fitted with a nylon jacket attached via a flexible stainless steel tether to a swivel device mounted on top of the cage, permitting continuous access to the venous circulation without tranquilization and with minimal restraint. One of the iv catheters was dedicated to hormone infusion, and the other was used for the withdrawal of blood samples. The routine care of monkeys housed in the remote sampling laboratory has been described previously (11).

Collection of blood
Before catheterization, blood samples were collected by femoral venipuncture following sedation with ketamine (100 mg, im). After catheterization, blood samples were drawn via the catheter into heparinized syringes. Serum and plasma were separated and stored at -20 C until required for assay.

Hormones and GnRH-R antagonist
Recombinant human (rh) FSH and single-chain human (sch) LH were kindly provided by the National Hormone and Peptide Program (NIDDK, Harbor-UCLA Medical Center, Torrance, CA). The preparation of stock (rhFSH, 200 IU/ml; schLH, 300 IU/ml) and working solutions (rhFSH, 2.2–2.9 IU/ml; schLH, 3.3–4.8 IU/ml) of the gonadotropin infusates have been described previously (10). T-filled or empty SILASTIC capsules (2 in. long; inner diameter, 0.134 in. and outer diameter, 0.186 in., Dow Corning Corp.) were prepared as described previously (12). The GnRH-R antagonist, acyline (Bioqual, Rockville, MD), was kindly provided by the Contraception and Reproductive Health Branch, Center for Population Research, National Institute of Child Health and Human Development (NICHD). A stock solution of acyline (300 µg/ml) was prepared in 5% aqueous mannitol (AMVET Scientific Products, Yaphank, NY) and stored at 4 C.

Experiments with adults
A schematic of the overall experimental design employing the adult monkeys is presented in Fig. 1Go. Within the framework of this design, four interrelated, albeit independent, experiments were conducted. Each of these is described below.



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  		Figure 1. Schematic of the overall design employed in experiments with adult male monkeys. All monkeys were treated with the GnRH-R antagonist, acyline, for the duration of the experiment (stippled bar). After 15–26 wk of acyline treatment, a testicular biopsy was taken and 0–7 wk later the animals were divided into two groups of 10 monkeys each. One group was implanted with T-filled SILASTIC capsules and the other received empty capsules. After 11–44 wk of T treatment, each group was further subdivided into a FSH- or vehicle-treated subgroup. The experiment was terminated by bilateral castration after injection of 5-bromo-2'-deoxyuridine (BrdU).

 
Establishing a chronic hypogonadotropic state and its impact on circulating concentrations of inhibin B.
Baseline values for circulating hormone concentrations were established for each of the 20 adult males before the hypogonadotropic state was induced. For this purpose, one or more blood samples were collected at approximately 1000 h. Testicular dimensions (length and breadth of each testis) were also measured at this time to determine the combined TV for each animal. A hypogonadotropic state was then induced and maintained throughout the entire study by daily sc injections of the GnRH-R antagonist (30–60 µg acyline/kg body weight; personal communication, Blye, R. P., Contraception and Reproductive Health Branch, Center for Population Research, NICHD). Following the initiation of GnRH-R antagonist treatment, the animals were sedated weekly to collect a blood sample and measure TV. The weekly sedation of monkeys also provided an opportunity to examine injection sites, the fistulae, and other dermal areas under the jacket.

Although all 20 monkeys were rendered hypogonadotropic by GnRH-R antagonist treatment, the changes in circulating inhibin B concentrations associated with this perturbation were determined in only 10 monkeys.

After 15–26 wk of GnRH-R antagonist treatment, a biopsy was taken from one of the testes chosen randomly (Fig. 1Go). The biopsy was fixed in Bouin’s fluid, embedded in paraffin, and 4-µm sections cut and stained with periodic acid-Schiff-hematoxylin for immediate histological examination, as described previously (10). Following confirmation of a full regression in spermatogenesis, the three experiments described below were conducted as illustrated in Fig. 1Go.

Effect of T replacement on the circulating concentrations of inhibin B in the hypogonadotropic hypogonadal monkey.
This experiment was initiated after 20–33 wk of treatment with the GnRH-R antagonist and within 7 wk from the time of biopsy. At this time, the reduction in TV had stabilized at approximately 10% (2.2 ± 0.2 ml, n = 20) of the pre-GnRH-R antagonist treatment value. The monkeys were divided into two groups of 10 each (Fig. 1Go). In an attempt to restore intratesticular content of this steroid, one group was implanted with T-filled capsules (20/monkey; one monkey received an additional set of T-filled capsules 14 wk after the first set was implanted), and the other group was implanted with empty capsules (20/monkey). The collection of blood samples and measurement of TV were continued on a weekly basis. It should be noted here that changes in circulating concentrations of inhibin B in response to T treatment were examined in only 50% of the monkeys.

Effect of stimulation with FSH, in the presence and absence of T, on the secretion of inhibin B and expression of inhibin-ßB in the testis of the hypogonadotropic adult monkey.
Following the restoration and stabilization of TV after at least 8 wk of T treatment at approximately 35% of the pre-GnRH-R antagonist treatment value (6.4 ± 0.5 ml, n = 10), the monkeys from both T- and non-T-treated groups were each divided further into two subgroups of five monkeys each (Fig. 1Go). After 11–44 wk of T treatment or empty capsule implantation, all monkeys were fitted with indwelling iv catheters and housed in remote sampling cages. A subgroup of five monkeys from both the T- and non-T-treated groups was then treated for 12 d with an intermittent iv infusion of FSH (2 IU/kg body weight per pulse, 2 ml as a 3-min bolus infusion every 3 h). The remaining subgroup from the T- and the non-T-treated groups received an infusion of vehicle.

A blood sample was collected immediately before initiation, on d 0, of the intermittent FSH infusion. Frequent blood samples were collected during a 3-h interpulse interval (at 5, 20, 40, 60, and 170 min after a pulse of FSH) on d 0 and d 12 of FSH treatment to describe the time course of hFSH in the circulation. In addition, blood samples were also collected on a daily basis, at 5 and 60 min after a bolus infusion of FSH or vehicle, to determine peak levels of the infused gonadotropin and the response in the secretion of inhibin B, respectively.

On the last day of FSH or vehicle treatment, each monkey received a bolus iv injection of 5-bromo-2'-deoxyuridine (33 mg/kg body weight, Sigma, St. Louis, MO) and was then bilaterally castrated. Each testis was weighed and cut into several portions, and for the purpose of this study, tissue from both testes was frozen in liquid nitrogen and stored at -80 C until the extraction of testicular RNA.

Effect of T replacement on expression of inhibin-ßB in the testis of the hypogonadotropic adult monkey.
The subgroup from the T- and non-T-treated groups that received 12 d of vehicle infusion (n = 5 each) provided the opportunity to examine the impact of T treatment on the testicular expression of the gene encoding inhibin-ßB in the absence of gonadotropin.

Experiment with juveniles
Effect of FSH and LH stimulation, either alone or in combination, on the secretion of inhibin B and expression of inhibin-ßB by the prepubertal testis.
Groups of four monkeys each received intermittent iv infusions (2 ml as a 1-min bolus infusion every 3 h) of schLH (3 IU/kg body weight per pulse) and rhFSH (2 IU/kg body weight per pulse), either alone or in combination, or vehicle for 11 d, as described previously (10). As reported earlier, intermittent infusions of recombinant gonadotropins produced episodic patterns in the circulating concentrations of hFSH and hLH with peak values of 12.1 ± 0.35 and 3.9 ± 0.18 IU/liter, respectively (10). The relationship between the intermittent infusion of LH and the resulting discharge of T, which was also reported earlier (10), was reminiscent of that seen between endogenous LH discharges and episodes of T secretion in adult monkeys (13).

For the purpose of this study, blood samples were collected before initiation on d 0 of gonadotropin or vehicle infusion and, subsequently, at 60 min after a bolus infusion of the hormone or vehicle on d 0, 1, 2, 4, 6, 8, 10, and 11.

On d 11, the monkeys were castrated and one testis or half a testis was frozen in liquid nitrogen and stored at -80 C until required for extraction of testicular RNA.

Assays
Gonadotropins.
Circulating concentrations of endogenous FSH and LH were determined using homologous (cynomolgus) RIA reagents supplied by the National Hormone and Peptide Program, as described previously (14, 15). The sensitivity of the FSH assay ranged from 0.04 to 0.06 ng/ml, and the intra- and interassay coefficients of variation were less than 11.9% and 2.3%, respectively. The sensitivity of the LH assay ranged from 0.05 to 0.16 ng/ml, and the intra- and interassay coefficients of variation were less than 6.6% and 9.8%, respectively. Circulating concentrations of rhFSH were measured using an hFSH assay kit (Technicon Immuno-1 system, Bayer Corp. Diagnostic Division, Tarrytown, NY), as described previously (10).

Inhibin B.
Circulating concentrations of inhibin B were determined using a specific two-site ELISA previously validated for the monkey (16, 17). The sensitivity of the inhibin B assay was less than 20 pg/ml, and the intra- and interassay coefficients of variation were 5% or less and 7% or less, respectively.

Steroids.
Circulating T concentrations were measured using a previously described RIA employing antiserum T3–125 (Endocrine Sciences, Inc., Tarzana, CA) (13). The mean sensitivity of the assay was approximately 0.05 ng/ml, and the intra- and interassay coefficients of variation were 9.1% and 12.6%, respectively. Circulating concentrations of estradiol-17ß (E2) were determined using a RIA kit (Diagnostic Products Corp., Los Angeles, CA). The mean sensitivity of the assay was approximately 2.2 pg/ml, and the intraassay coefficient of variation was 4.6%.

RNA preparation
Total RNA was extracted from testes by the single-step guanidinium thiocyaniate-phenol-chloroform method (18). The integrity of extracted RNA was determined by visualizing ethidium bromide-stained 28S and 18S rRNA bands after migration on agarose gel, and concentration was determined by measuring absorbance at 260 nm.

Complementary DNAs and synthesis of riboprobes
Human inhibin-ßB (920 bases) cDNA was kindly provided by Dr. A. J. Mason (Genentech, Inc., South San Francisco, CA). A 627-base fragment of inhibin-ßB was subcloned into pGEM-3Z vector (Promega Corp., Madison, WI). The subcloned fragment was then linearized with BamH1 and in vitro transcribed with T7 RNA polymerase to synthesize 32P-labeled antisense RNA probe. Sense probe was generated from the same cDNA fragment by linearizing with EcoR1 followed by in vitro transcription with SP6 RNA polymerase. A 117-base fragment of rat cyclophilin (cyclo) cDNA was kindly provided by Dr. J. L. Roberts (Mt. Sinai School of Medicine, New York, NY).

RNase protection assay
RNase protection assay was performed as described previously (15). Increasing amounts of in vitro transcribed sense inhibin-ßB (1, 2, 4, 8, 16, and 32 pg) and cyclo (5, 10, 20, and 40 pg) mRNAs were hybridized with the corresponding 32P-labeled antisense RNA probes and yielded, in each case, a linear relationship between mRNA mass and integrated OD. Increasing amounts of testicular RNA (2.5, 5, 10, 20, and 40 µg) yielded a dose-dependent linear increase in integrated OD for both transcripts, and 20 µg RNA were used routinely.

Testicular RNA was hybridized with the 32P-labeled antisense RNA probes (inhibin-ßB, 300,000 cpm; and cyclo, 15,000 cpm) in solution at 45 C overnight. This was followed by combined RNase A and T1 (40 and 2 µg/ml, respectively) digestion of nonhybridized RNA at 32 C for 1 h. Stable hybrids were extracted with phenol-chloroform, precipitated with ethanol, and denatured and separated on 6% polyacrylamide/8 M urea gels. The dried gel was exposed on a CS molecular imaging screen (Bio-Rad Laboratories, Inc., Hercules, CA) for approximately 48 h, and an image of each gel was acquired using a molecular imager (model GS-525, Bio-Rad Laboratories, Inc.), and the OD of each mRNA hybrid was quantified using Molecular Analyst software (Bio-Rad Laboratories, Inc.). Antisense probes with 20 µg transfer RNA were run as negative controls.

The OD of the inhibin-ßB mRNA hybrid was normalized to that of cyclo and expressed as relative OD. Because heterologous antisense probes were used, in the case of inhibin-ßB, a major hybrid of approximately 200 bases and some minor hybrids of 150 bases or less were noted, and in the case of cyclo, there were two hybrids of approximately 70 and 40 bases. The relative OD values for the major hybrid for inhibin-ßB was quantitated using the cyclo hybrids.

Numerical analysis
The significance of differences between mean values of hormone concentrations was determined by multifactor ANOVA with repeated measures followed by Student-Newman-Keuls multiple range test. Undetectable hormone concentrations were assigned a value equivalent to the sensitivity of the respective assay. The significance of differences in mean testicular weight and mean relative OD of the inhibin-ßB mRNA hybrid were assessed by one-factor ANOVA followed by Student-Newman-Keuls multiple range test. Statistical significance was accepted at P < 0.05. All data are expressed mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Experiments with adults
Establishing a chronic hypogonadotropic state and its impact on circulating concentrations of inhibin B.
Before initiation of GnRH-R antagonist administration, the circulating concentrations of FSH and LH were 0.22 ± 0.04 and 0.44 ± 0.13 ng/ml (mean ± SEM, n = 10), respectively, and within 1 wk of GnRH-R antagonist treatment, the levels of these gonadotropins had reached a nadir (0.07 ± 0.01 and 0.09 ± 0.03, respectively). The corresponding values for circulating T concentrations were 2.6 ± 0.5 and 0.6 ± 0.1 ng/ml, respectively. These immediate and dramatic suppressions in gonadotropin and T levels were sustained for the duration of the study. The hypogonadotropic state was associated with a profound and progressive reduction in mean TV from 18.0 ± 1.3 ml before GnRH-R antagonist administration to reach, by wk 16, a stable plateau that was approximately 10% (2.0 ± 0.2 ml) of the pre-GnRH-R antagonist treatment value.

Circulating concentrations of inhibin B were only modestly reduced in the face of the severe and sustained hypogonadotropic state (Fig. 2Go). Before GnRH-R antagonist treatment, the mean circulating concentration of inhibin B was 1271 ± 136 pg/ml, and this was reduced by 15% to 1086 ± 146 pg/ml after 1 wk of GnRH-R antagonist treatment (Fig. 2Go). After 16 wk of sustained gonadotropin deprivation, circulating concentrations of this testicular hormone had declined significantly to 887 ± 76 pg/ml (Fig. 2Go).



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  		Figure 2. Mean concentrations of circulating LH (top panel), FSH (middle panel), and inhibin B (bottom panel) in 10 adult monkeys before and after the initiation of acyline treatment on wk 0. Error bars indicate SEM. a, Different from all other data points; b, different from wk 0; P < 0.05.

 
Effect of T replacement on the circulating concentrations of inhibin B in the hypogonadotropic hypogonadal monkey.
In the 10 monkeys used for this experiment, circulating concentrations of gonadotropins immediately before the implantation of T-filled or empty capsules were at or below the limit of detection, and those of T were at castrate levels. At this time, the mean inhibin B concentration and mean TV were 635 ± 83 pg/ml and 1.9 ± 0.1 ml, respectively. These values were similar to those observed after 16 wk of GnRH-R antagonist treatment when regression of spermatogenesis was complete.

Implantation of T-filled capsules resulted, within 1 wk, in a significant increase in the circulating concentrations of this steroid to 16.3 ± 4.5 ng/ml, which was sustained for the duration of the experiment (Fig. 3Go). Circulating concentrations of FSH and LH, which were already at or below the limit of detection before implantation, did not appear to be influenced by T treatment (data not shown).



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  		Figure 3. Mean concentrations of circulating T (open symbol) and inhibin B (closed symbol) before (wk 0) and after sc implantation of T-filled (top panel) or empty (bottom panel) capsules in hypogonadotropic hypogonadal adult monkeys (n = 5 each). Note that data for wk 16 are comprised by results from wk 12 or 16. Error bars indicate SEM. a, Different from wk 0; P < 0.05.

 
The sustained elevation in plasma T concentrations was associated with a marked and progressive decline in circulating concentrations of inhibin B which, by wk 4 of T treatment, had fallen significantly to approximately 55% of the pre-T implantation value (Fig. 3Go). By wk 16, inhibin B levels had been further reduced to approximately 40% of the pre-T implantation value (Fig. 3Go).

Circulating concentrations of E2 in the T-treated group were 12.5 ± 0.4 pg/ml before treatment and 61.9 ± 26.4 and 77.0 ± 28.0 pg/ml on wk 2 and 4 of treatment, respectively.

Sixteen weeks of T treatment resulted in a marked and significant restoration in TV from 2.0 ± 0.1 to 6.2 ± 0.7 ml.

Implantation of empty capsules did not influence inhibin B concentrations (Fig. 3Go), and circulating concentrations of gonadotropins and T in this group were maintained at undetectable and castrate levels, respectively. Similarly, changes in TV were not observed.

Effect of stimulation with FSH, in the presence and absence of T, on the secretion of inhibin B and expression of inhibin-ßB in the testis of the hypogonadotropic adult monkey.
At the time of this experiment, circulating levels of T in the steroid-treated group were sustained at a mean of 15.3 ± 2.1 ng/ml, whereas those in the non-T-treated group were maintained at castrate levels. Initiation of the intermittent infusion of rhFSH immediately increased circulating concentrations of hFSH in both T- and non-T-treated monkeys from undetectable levels (<=0.10 IU/liter) to reach, by 24 h, peak concentrations in the range of 19–30 IU/liter. These values were sustained for the duration of the 12-d FSH infusion. In the group receiving vehicle infusion, hFSH levels remained undetectable. Infusion of FSH did not affect circulating T concentration in either the T- or non-T-treated group.

In both the T- and non-T-treated groups, stimulation with FSH elicited a robust, significant increase in the circulating concentrations of inhibin B by d 1 of treatment (Fig. 4Go). It is to be noted here that the concentrations of circulating inhibin B during FSH stimulation in the non-T-treated group were markedly higher than those seen before initiation of GnRH-R antagonist treatment (Fig. 4Go). In contrast, FSH stimulation in the T-treated group restored inhibin B concentrations to those observed before induction of the hypogonadotropic state (Fig. 4Go). The fold increase in inhibin B concentrations, however, was greater (x5) in the T-treated group with concentrations of this testicular hormone increasing from 321 ± 19 to 1684 ± 131 pg/ml at the termination of the FSH infusion. Although the baseline inhibin B concentration in the non-T-treated group (1138 ± 221 pg/ml) was greater than that in the T-treated monkeys, the FSH-induced increase in this testicular hormone was only 2-fold (Fig. 4Go).



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  		Figure 4. Mean concentrations of circulating inhibin B following 12 d of intermittent infusion of rhFSH (closed circle) or vehicle (open circle) initiated on d 0 in hypogonadotropic adult monkeys implanted with empty (left panel) or T-filled (right panel) capsules (n = 5 each). Solid bar at left in both panels represents the mean inhibin B concentration in the eugonadotropic state (n = 10; redrawn from Fig. 1Go, wk 0 value). Error bars indicate SEM a, significantly different from d 0. Note, in both panels, d 1–12 of FSH infusion different from corresponding values in vehicle-treated group; P < 0.05.

 
Interestingly, the robust increase in inhibin B secretion induced by FSH stimulation in the presence and absence of T was not associated with a corresponding change in inhibin-ßB mRNA level (Fig. 5Go).



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  		Figure 5. Inhibin-ßB mRNA levels in the testes of hypogonadotropic adult monkeys at the end of 12 d of rhFSH or vehicle infusion in the absence (A) and presence (B) of T. The upper boxes in both panels show, for two monkeys in each subgroup, autoradiograms of inhibin-ßB and cyclo mRNAs hybridized with respective 32P-labeled antisense probes. The solid bars in both panels show, for all animals per subgroup, the corresponding relative OD (±SEM) of the inhibin-ßB mRNA hybrid.

 
In the absence of T, the mean testicular weight for the vehicle and FSH-infused animals were 9.6 ± 0.6 and 11.7 ± 1.6 g (P > 0.05), respectively. Corresponding values for the T-treated monkeys were 15.2 ± 2.4 and 26.1 ± 4.3 g (P < 0.05).

Effect of T replacement on expression of inhibin-ßB in the testis of hypogonadotropic adult monkeys.
The dramatic, T-induced decrease in inhibin B secretion noted in the hypogonadotropic adult males was associated with a significant suppression in inhibin-ßB mRNA level (Fig. 6Go).



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  		Figure 6. Inhibin-ßB mRNA levels in the testis of hypogonadotropic adult monkeys implanted with empty (left bar) or T-filled capsules (right bar). The upper box shows, for two monkeys in each group, autoradiograms of inhibin-ßB and cyclo mRNAs hybridized with respective 32P-labeled antisense probes. The lower box shows, for all animals per subgroup, the corresponding relative OD (±SEM) of the inhibin-ßB mRNA hybrid. Note the mean inhibin-ßB level in the empty capsule group is reproduced from Fig. 5Go. *, P = <0.05.

 
Experiment with juveniles
The effect of FSH and LH stimulation, alone or in combination, on the secretion of inhibin B and expression of inhibin-ßB by the prepubertal testis.
Stimulation with FSH, alone, resulted within 24 h in a significant, approximately 4-fold increase in the circulating concentrations of inhibin B (Fig. 7Go). Following this initial robust increase, circulating inhibin B levels decreased between d 2 and 4 of the FSH infusion to reach a plateau that was significantly greater than corresponding levels in the vehicle-treated group (Fig. 7Go).



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  		Figure 7. Time course of changes in mean concentrations of circulating inhibin B (closed symbols, top and middle panels) before and after stimulation, initiated on d 0, with rhFSH alone (top panel), schLH alone (middle panel), or vehicle (open symbol, repeated in top and middle panels) in juvenile monkeys. The bottom panel shows the response of circulating T concentrations to stimulation with either schLH (closed symbol) or rhFSH (open symbol). Error bars indicate SEM. a, Different from d 0 within the group and from corresponding values during vehicle treatment and b, different from d 0 within the treatment group; P < 0.05.

 
Treatment with LH alone resulted, by d 1, in a marked increase in circulating T concentrations from 0.4 ± 0.1 to 3.7 ± 0.5 ng/ml. Although circulating inhibin B concentrations during the LH infusion were not different from those during vehicle infusion (Fig. 7Go), when comparisons were made within the LH-treated group, a progressive and significant reduction in inhibin B levels was observed (323 ± 45 pg/ml to 149 ± 36 pg/ml; d 0 and 11, respectively), and this decrease was inversely related to the LH-stimulated increase in circulating T concentrations (Fig. 7Go).

Although addition of LH to the FSH infusion did not modify the acute phase (d 1–4) of FSH-stimulated inhibin B secretion (Fig. 8Go), circulating concentrations of inhibin B during the remainder of the combined gonadotropin infusion (d 6–11) were significantly lower than those during infusion with FSH alone (Fig. 8Go) and, moreover, were indistinguishable from the corresponding levels in the vehicle-treated group. The dramatic blunting in the inhibin B response to FSH stimulation in the combined gonadotropin-treated group was also inversely related to the increase in circulating T concentrations (Fig. 8Go).



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  		Figure 8. Time course of changes in mean concentrations of circulating inhibin B (closed symbol, top panel) and T (closed symbol, bottom panel) before and after combined stimulation, initiated on d 0, with rhFSH and schLH in juvenile monkeys. For comparison, the response of inhibin B (shaded area, top panel) and T (open symbol, bottom panel) to FSH stimulation, alone, is reproduced from Fig. 7Go. Error bars indicate SEM. a, Different from d 0; b, different from corresponding time in the group treated with rhFSH alone; P < 0.05.

 
The inhibin-ßB mRNA levels at the end of the 11 d of gonadotropin treatment are shown in Fig. 9Go. Although the FSH-induced increase in inhibin B secretion was not associated with a corresponding change in the level of testicular inhibin-ßB mRNA, the inhibitory action of LH on inhibin B secretion, observed in the presence and absence of FSH, was correlated with significant decreases in the levels of inhibin-ßB mRNA.



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  		Figure 9. Inhibin-ßB mRNA levels in testis of juvenile monkeys following treatment with vehicle, rhFSH, schLH, and a combination of rhFSH and schLH. The upper box shows, for two monkeys in each treatment group, autoradiograms of inhibin-ßB and cyclo mRNAs hybridized with corresponding 32P-labeled antisense probes. Solid bars represent the corresponding relative OD (±SEM) of inhibin-ßB mRNA hybrid for all animals (n = 4 each). *, Different from vehicle- and FSH-treated groups; P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The findings that treatment of adult male rhesus monkeys with daily injections of the GnRH-R antagonist, acyline, resulted in a rapid decline in circulating gonadotropins to concentrations at or below the sensitivities of the assays, and in those of T to castrate levels together with a dramatic 90% reduction in TV and an arrest of spermatogenesis, confirm that a chronic hypogonadotropic state was successfully achieved. Because the profound and protracted hypogonadotropism induced by GnRH-R antagonist was associated with only a 30% reduction in circulating concentrations of inhibin B, it is reasonable to conclude that, whereas the secretion of this hormone by the postpubertal testis is dependent on gonadotropin stimulation, substantial inhibin B secretion may occur without, or with only minimal, pituitary support. This view is consistent with the results of a study of the cynomolgus macaque, in which substantial amounts of circulating immunoactive inhibin, as measured using the Monash assay, were reported to persist during short-term treatment with a GnRH-R antagonist (19, 20).

Although the effects of elevating the gonadotropin drive on inhibin B production by the normal testis of adult primates have not been studied, a modest elevation in circulating inhibin B concentrations has been noted following stimulation with FSH alone. In men a single injection of a relatively large dose of rhFSH (3000 IU) resulted in less than a 2-fold increase in inhibin B levels (5, 21). Administration of a lower dose of FSH (225 IU) to normal men, however, was ineffective in this regard (22). In two normal adult monkeys, a continuous infusion of recombinant monkey FSH for 5 d that increased the circulating levels of this gonadotropin by 10-fold also elicited only a modest increase in circulating inhibin B concentrations (Marshall, G. R., S. Ramaswamy, and T. M. Plant, unpublished observations).

The stimulatory action of FSH on inhibin B secretion by the monkey testis appears to be amplified in the hypogonadotropic condition. In the present study, 11–12 d of stimulation with FSH in both juveniles, in which gonadotropin secretion is arrested (9), and hypogonadotropic adult males led to a rapid and robust increase in circulating concentrations of inhibin B. This finding is consistent with the 1- to 4-fold increase in inhibin B levels previously reported in response to FSH administration to hypogonadal men (23, 24, 25, 26, 27, 28).

In striking contrast to FSH, LH stimulation of the testis appears to suppress secretion of inhibin B. In the present experiment with juvenile monkeys, an intermittent LH infusion for 11 d reduced circulating inhibin B levels and curtailed the stimulatory action of FSH on inhibin B secretion. The most parsimonious explanation for the inhibitory action of LH on testicular inhibin B secretion is that it is indirect and mediated by a paracrine action of increased T production from the Leydig cells. This view is supported by the findings that the time course of the LH-induced suppression of inhibin B secretion is inversely related to that of T and the implantation of T-filled capsules in hypogonadotropic adult monkeys resulted in an approximately 60% suppression in inhibin B secretion by the regressed testis. In this regard, it is interesting to note that human chorionic gonadotropin treatment of a hypogonadal patient with Leydig cell hypoplasia because of a mutation of the LH receptor resulted in an approximately 70% decline in the circulating concentrations of inhibin B (29). In this patient, circulating T concentrations increased in response to human chorionic gonadotropin stimulation from less than 0.1 to approximately 25 nmol/liter. This may be compared with the range of 15–30 nmol/liter reported for normal men (30). In men with acquired hypogonadotropic hypogonadism, however, LH treatment failed to suppress circulating inhibin B levels (25). The most likely explanation for the negative finding in the latter study is that intratesticular T levels were probably subphysiologic as reflected by the low circulating T levels (4–6 nmol/liter) produced in response to daily injections of LH (25). It is to be noted here that an inhibitory action of T on inhibin B production by rat Sertoli cells in culture has been reported (31).

Parenthetically, the foregoing inhibitory action of T on inhibin B secretion by the primate testis may explain the varied testicular inhibin B responses that have been reported in studies designed to examine the contraceptive efficacy of T-containing steroid regimens. A consistent, marked suppression (approximately 60%) in the circulating concentrations of inhibin B has been noted in studies of cynomolgus monkeys treated with up to 200 mg T per week (32, 33). Similarly, a marked decline in inhibin B secretion was observed in analogous studies of men treated with relatively high doses of T [100–200 mg/wk (5, 34, 35)] but not with a single or lower dose of T [30–80 mg/wk (36, 37, 38, 39, 40)].

The possibility that the actions of T on testicular inhibin B production may be mediated via E2 needs to be considered because T treatment of hypogonadotropic adult monkeys in the present study resulted in an increase in circulating, and presumably testicular, levels of E2 and estrogen receptor-{alpha} and -ß have been localized in Sertoli cells of the primate, as in other species (41). In vitro studies using primary cultures of rat Sertoli cells, however, have not noted an inhibitory action of E2 on inhibin B production (31, 42). Therefore, it seems reasonable to conclude that the action of T to suppress inhibin B secretion by the monkey testis is not mediated by E2.

The opposing action of FSH and LH on testicular inhibin B secretion may mask the range over which this testicular hormone is regulated by gonadotropin drive. A selective reduction in LH release would attenuate the T "brake" on testicular inhibin B secretion, allowing the stimulatory action of FSH to be amplified. This possibility is supported by the finding that FSH treatment of hypogonadotropic adult monkeys led to circulating inhibin B levels that were 2-fold higher than those in normal monkeys. A selective reduction of FSH tone, on the other hand, would reduce the stimulatory gonadotropin input to inhibin B secretion and allow the inhibitory action of LH induced T secretion to dominate, a possibility supported by the dramatic inhibition in inhibin B secretion following T administration to hypogonadotropic adults. The compensatory increase in T secretion by the remaining testis following unilateral orchidectomy may therefore underlie the small increase in inhibin B secretion that occurs in the face of elevated FSH secretion elicited by this experimental perturbation in the adult monkey (17).

That Sertoli cells are the principal source of testicular inhibin B secretion in man and monkeys is supported by several lines of evidences. In the primate testis, inhibin-{alpha}- and ßB-subunit peptides and their corresponding mRNAs have been localized in Sertoli cells (43, 44, 45, 46, 47). Primary cultures of prepubertal monkey and human Sertoli cells secrete substantial amounts of inhibin B (48, 49). Sertoli cells are the only testicular cells that express both androgen receptor (AR) and the FSH receptor (50, 51) and therefore have the necessary molecular machinery to transduce the observed actions of FSH and T on inhibin B production. A positive correlation exists between the circulating levels of inhibin B and the number of Sertoli cells in normal adult monkeys (11). Finally, several situations exist in which substantial amounts of inhibin B are secreted by testes exhibiting only Sertoli cells and undifferentiated spermatogonia in the seminiferous tubules. In this regard, infantile, juvenile (4, 16), and hypogonadotropic adult primates (present study) produce a substantial quantity of inhibin B. In a boy with McCune-Albright syndrome and macroorchidism resulting from a Gs{alpha} gene mutation, adult levels of inhibin B were noted in the absence of Leydig cell activation (52). Because inhibin-ßB mRNA has not been localized in undifferentiated spermatogonia, it would be reasonable to conclude that, in these situations, the Sertoli cell must be the principal cellular source of inhibin B secretion.

The possibility that Leydig cells contribute to the synthesis and secretion of inhibin B must also be considered. In this regard, the mRNAs encoding inhibin-{alpha} and -ßB and the subunits themselves have been localized in Leydig cells of monkey and man (43, 44, 45, 46, 47). In the present study, however, LH stimulation of the juvenile testis did not increase testicular inhibin B secretion, suggesting that the Leydig cell is unlikely to be a major source of testicular inhibin B. Similarly, LH or human chorionic gonadotropin administration to normal men or patients with acquired hypogonadotropic hypogonadism failed to stimulate inhibin B secretion (22, 25, 29). Additionally, Leydig cells in the testis of the juvenile and hypogonadotropic adult are typically hypoplastic (53, 54, 55), again suggesting that sustained testicular inhibin B secretion by this interstitial cell type is unlikely.

It is frequently argued that circulating concentrations of inhibin B reflect the spermatogenic state of the testes and the presence or absence of advanced germ cell types determines, in part, the testicular secretion of inhibin B (56, 57). Moreover, if the function of the FSH-inhibin B feedback loop is to regulate sperm output in primates, as we have recently argued (58), then the germinal epithelium must contribute to the control of inhibin B secretion to satisfy the theoretical requirement that the magnitude of the testicular feedback signal (inhibin B) inhibiting FSH secretion be directly related to sperm output. The foregoing view is supported by an apparent correlation between the circulating concentrations of inhibin B and sperm concentration in normal men (59, 60, 61, 62, 63, 64) and by the observations that the inhibin-ßB subunit mRNA and peptide are localized in advanced germ cell types (43, 47). Furthermore, the dramatic decline in the circulating concentrations of inhibin B that is observed in association with germ cell depletion following chemotherapy or testicular irradiation (65, 66, 67) suggests that advanced germ cells provide a stimulus to inhibin B secretion.

The results of the present experiments, however, fail to provide additional support for the foregoing view. First, in the hypogonadotropic adult, a substantial quantity of inhibin B continued to be secreted despite the complete disappearance of germ cell types more mature than undifferentiated spermatogonia. Second, although T replacement of the hypogonadotropic adults resulted in a qualitative recovery of spermatogenesis, as reflected by histological examination of testicular tissue at the conclusion of the study (Marshall, G. R., S. Ramaswamy, and T. M. Plant, unpublished results), the immediate suppression in circulating inhibin B levels induced by T treatment was sustained for up to 44 wk of T treatment. If advanced germ cells facilitate inhibin B production, it is to be predicted that, in this experiment, inhibin B production would have rebounded in association with the T-mediated restoration of spermatogenesis. Similarly, in the hypogonadal patient with the homozygous mutation of the LH-R, human chorionic gonadotropin treatment for approximately 4 months initiated spermatogenic activity (29), but circulating concentrations of inhibin B remained suppressed at this time. Thus, it would seem reasonable to conclude that the role of putative germ cell factors in determining the magnitude of inhibin B feedback signal requires additional study.

At the molecular level, the suppressive action of T on the testicular secretion of inhibin B, which was presumably mediated by increasing AR activity in Sertoli cells, was associated with a marked reduction in inhibin-ßB mRNA levels in extracts of whole testes in both hypogonadotropic adult and juvenile monkeys. It is recognized that T treatment of hypogonadotropic adults also resulted in a restoration in spermatogenic activity (Ref. 12 ; and Marshall, G. R., S. Ramaswamy, and T. M. Plant, unpublished observations); therefore, the possibility that the increase in germ cell mRNA diluted the level of inhibin-ßB transcripts of Sertoli cells exists. The latter possibility, however, cannot account for the LH-induced decrease in inhibin-ßB gene expression in the testis of the juvenile monkey, which occurred with only a minimal expansion of germ cells and in the face of a marked increase in Sertoli cell numbers (10). Thus, it seems reasonable to conclude that inhibition of inhibin-ßB gene expression is a major factor underlying the suppression of inhibin B secretion by T. Here it is interesting to note that, in the adult rat, an increase in testicular inhibin-ßB mRNA level was observed following the destruction of Leydig cells with ethylene dimethane sulfonate (68). Concomitant T replacement of the ethylene dimethane sulfonate-treated rat, however, did not reverse the change in inhibin-ßB gene expression. Because an AR response element has not been reported for the inhibin-ßB promoter, it appears likely that indirect pathways are involved in regulation of inhibin-ßB gene expression by T.

The present findings that, in the juvenile and hypogonadotropic adult monkey, FSH stimulated the robust secretion of inhibin B without an apparent increase in inhibin ßB mRNA levels is consistent with those in hypophysectomized male rats (69) and suggest that the action of FSH in these situations probably does not involve regulation of inhibin-ßB gene expression. In the FSH-treated monkeys, it is unlikely that Sertoli cell mRNA was diluted by transcripts from an expanding population of germ gels for the following reasons. In the case of the juveniles, FSH treatment resulted in a decrease in the number of undifferentiated type A spermatogonia and the appearance of only an occasional B spermatogonia in a very small proportion of seminiferous cords (10). In the adult, although morphometric analysis of the seminiferous tubule remains to be completed, testicular weights in the FSH and vehicle-treated animals were not different.

In the T-treated, hypogonadotropic adults, on the other hand, testicular weight in the group that received FSH was greater than that in the vehicle-treated animals; therefore, in this paradigm, the possibility that an FSH induced increase in inhibin-ßB gene transcription may have been masked by dilution from increased germ cell mRNA cannot be excluded. Although it is recognized that a classical cAMP response element is not found in the inhibin-ßB promoter (70, 71), cAMP has been reported to stimulate inhibin-ßB mRNA levels by transcriptional mechanisms (72, 73) and expression of many FSH-regulated genes occurs via FSH-signaling pathways that do not include a cAMP response element (74, 75).

Lastly, it is recognized that FSH regulation of inhibin-{alpha} synthesis (76) may contribute to the control of inhibin B secretion.

In conclusion, the present study provides compelling evidence for the view that the gonadotropin control of testicular inhibin B secretion by the primate testes involves opposing stimulatory and inhibitory actions of FSH and LH, respectively. FSH is posited to act directly on the Sertoli cell, although the molecular bases of this action require further study. The action of LH, on the other hand, appears to be mediated by a paracrine action of T from the Leydig cell to regulate inhibin-ßB gene expression by the Sertoli cell. The precise manner by which these opposing endocrine inputs to the Sertoli cell are integrated with putative factors from the germinal epithelium to set the magnitude of the feedback signal (inhibin B) governing the secretion of FSH, which serves as a feed-forward arm of the control system governing the quantitative aspects of spermatogenesis, remains to be established.


   Acknowledgments
 
The authors acknowledge the expert technical assistance of Deborah A. Bollette and Michael A. Cicco and the support of the Primate and Assay Cores of the Center for Research in Reproductive Physiology. We are grateful to Dr. A. S. McNeilly, MRC Reproductive Biology Unit, University of Edinburgh, United Kingdom, for performing some of the inhibin B assays and Dr. A. F. Parlow and the National Hormone and Peptide Program, NIDDK, for the recombinant human gonadotropins and RIA kits used to measure monkey FSH and LH. We also thank Drs. Nancy Alexander and Richard P. Blye and the NICHD for the GnRH-R antagonist (acyline).


   Footnotes
 
Preliminary reports of this work were presented at the Serono Foundation International Workshop on Inhibins, Activins, and Follistatins, Melbourne, Australia, 2000 (Abstract P-23), and the 83rd Annual Meeting of The Endocrine Society, Denver, CO, 2001 (Abstract OR17-6).

This work was supported by NICHD/NIH through cooperative agreement (U54-HD-08610) as part of the Specialized Cooperative Centers Program in Reproduction Research and by Grant HD-32473.

Abbreviations: AR, Androgen receptor; cyclo, cyclophilin; E2, estradiol-17ß; GnRH-R, GnRH receptor; rh, recombinant human; RNase, ribonuclease; sch, single-chain human; T, testosterone; TV, testicular volume.

Received October 17, 2002.

Accepted for publication December 16, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Plant TM 1994 FSH secretion in the adult male rhesus monkey is controlled in a manner consistent with that proposed by the inhibin hypothesis. In: Burger HG, de Kretser DM, Findlay J, Petraglia F, Robertson DM, eds. Frontiers in endocrinology. Rome: Ares-Serono Symposia; 3:135–143
  2. Hayes FJ, Hall JE, Boepple PA, Crowley Jr WF 1998 Differential control of gonadotropin secretion in the human: endocrine role of inhibin. J Clin Endocrinol Metab 83:1835–1841[Free Full Text]
  3. Ramaswamy S, Plant TM 2001 Operation of the follicle-stimulating hormone (FSH)-inhibin B feedback loop in the control of primate spermatogenesis. Mol Cell Endocrinol 180:93–101[CrossRef][Medline]
  4. Andersson AM, Skakkaebaek NE 2001 Serum inhibin B levels during male childhood and puberty. Mol Cell Endocrinol 180:103–107[CrossRef][Medline]
  5. Anawalt BD, Bebb RA, Matsumato AM, Groome NP, Illingworth PJ, McNeilly AS, Bremner WJ 1996 Serum inhibin B levels reflect Sertoli cell function in normal men and men with testicular dysfunction. J Clin Endocrinol Metab 81:3341–3345[Abstract]
  6. Illingworth PJ, Groome NP, Byrd W, Rainey WE, McNeilly AS, Mather JP, Bremner WJ 1996 Inhibin B: a likely candidate for the physiologically important form of inhibin in men. J Clin Endocrinol Metab 81:1321–1325[Abstract]
  7. Majumdar SS, Winters SJ, Plant TM 1997 A study of the relative roles of follicle-stimulating hormone and luteinizing hormone in the regulation of testicular inhibin secretion in the rhesus monkey (Macaca mulatta). Endocrinology 138:1363–1373[Abstract/Free Full Text]
  8. Raivio T, Dunkel L 1997 Inhibins in childhood and puberty. Best Pract Res Clin Endocrinol Metab 16:43–52[CrossRef]
  9. Plant TM 1994 Puberty in primates. In: Knobil E, Neil JD, eds. The physiology of reproduction. 2nd ed. New York: Raven Press Ltd.; 453–485
  10. Ramaswamy S, Plant TM, Marshall GR 2000 Pulsatile stimulation with recombinant single chain human luteinizing hormone elicits precocious Sertoli cell proliferation in the juvenile male rhesus monkey (Macaca mulatta). Biol Reprod 63:82–88[Abstract/Free Full Text]
  11. Ramaswamy S, Marshall GR, McNeilly AS, Plant TM 1999 Evidence that in a physiological setting Sertoli cell number is the major determinant of circulating concentrations of inhibin B in the adult male rhesus monkey. J Androl 20:430–434[Abstract/Free Full Text]
  12. Marshall GR, Zorub DS, Plant TM 1995 Follicle-stimulating hormone amplifies the population of differentiated spermatogonia in the hypophysectomized testosterone-replaced adult rhesus monkey (Macaca mulatta). Endocrinology 136:3504–3511[Abstract]
  13. Plant TM 1981 Time course of concentrations of circulating gonadotropin, prolactin, testosterone, and cortisol in adult male rhesus monkeys (Macaca mulatta) throughout the 24 h light-dark cycle. Biol Reprod 25:244–252[Abstract]
  14. Ramaswamy S, Pohl CR, McNeilly AS, Winters SJ, Plant TM 1998 The time course of follicle-stimulating hormone suppression by recombinant human inhibin A in the adult male rhesus monkey (Macaca mulatta). Endocrinology 139:3409–3415[Abstract/Free Full Text]
  15. El Majdoubi M, Ramaswamy S, Sahu A, Plant TM 2000 Effects of orchidectomy on levels of the mRNAs encoding gonadotropin-releasing hormone and other hypothalamic peptides in the adult male rhesus monkey (Macaca mulatta). J Neuroendocrinol 12:167–176[CrossRef][Medline]
  16. Plant TM, Padmanabhan V, Ramaswamy S, McConnel DS, Winters SJ, Groome NP, Midgley Jr AR, McNeilly AS 1997 Circulating concentrations of dimeric inhibin A and B in the male rhesus monkey (Macaca mulatta). J Clin Endocrinol Metab 82:2617–2621[Abstract/Free Full Text]
  17. Ramaswamy S, Marshall GR, McNeilly AS, Plant TM 2000 Dynamics of the follicle-stimulating hormone (FSH)-inhibin B feedback loop and its role in regulating spermatogenesis in the adult male rhesus monkey (Macaca mulatta) as revealed by unilateral orchidectomy. Endocrinology 141:18–27[Abstract/Free Full Text]
  18. Chomcyznski P, Sacchi N 1987 Single-step method off RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  19. Weinbauer GF, Schubert J, Yeung C-H, Rosiepen G, Nieschlag E 1998 Gonadotropin-releasing hormone antagonist arrests premeiotic germ cell proliferation but does not inhibit meiosis in the male monkey: a quantitative analysis using 5-bromodeoxyuridine and dual parameter flow cytometry. J Endocrinol 156:23–34[Abstract]
  20. Zhengwei Y, Wreford NG, Schlatt S, Weinbauer GF, Nieschlag E, McLachlan RI 1998 Acute and specific impairment of spermatogonial development by GnRH antagonist-induced gonadotropin withdrawal in the adult macaque (Macaca fascicularis). J Reprod Fertil 112:139–147[Abstract/Free Full Text]
  21. Kamischke A, Simoni M, Schrameyer K, Lerchl A, Nieschlag E 2001 Is inhibin B a pharmacodynamic parameter for FSH in normal men? Eur J Endocrinol 144:629–637[Abstract]
  22. Kinniburgh D, Anderson RA 2001 Differential patterns of inhibin secretion in response to gonadotropin stimulation in normal men. Int J Androl 24:95–101[CrossRef][Medline]
  23. Kamischke A, Behre HM, Bergmann M, Simoni M, Schafer T, Nieschlag E 1998 Recombinant human follicle-stimulating hormone for treatment of male idiopathic infertility: a randomized, double-blind, placebo-controlled, clinical trial. Human Reprod 13:596–603[Abstract/Free Full Text]
  24. Liu PY, Turner L, Rushford D, McDonald J, Baker HWG, Conway AJ, Handelsman DJ 1999 Efficacy and safety of recombinant human follicle-stimulating hormone (Gonal-F) with urinary human chorionic gonadotropin for induction of spermatogenesis and fertility in gonadotropin-deficient men. Human Reprod 14:1540–1545[Abstract/Free Full Text]
  25. Young J, Couzinet B, Chanson P, Brailly S, Loumaye E, Schaison G 2000 Effects of human recombinant luteinizing hormone and follicle-stimulating hormone in patients with acquired hypogonadotropic hypogonadism: study of Sertoli and Leydig cell secretions and interactions. J Clin Endocrinol Metab 85:3239–3244[Abstract/Free Full Text]
  26. Foresta C, Bettella A, Merico M, Garolla A, Plebani M, Ferlin A, Rossato M 2000 FSH in the treatment of oligozoospermia. Mol Cell Endocrinol 161:89–97[CrossRef][Medline]
  27. Foresta C, Bettella A, Merico M, Garolla A, Ferlin A, Rossato M 2002 Use of recombinant human follicle-stimulating hormone in the treatment of male factor infertility. Fertil Steril 77:238–244[CrossRef][Medline]
  28. Bouloux PMG, Handelsman DJ, Jockenjoevel F, Nieschlag E, Rabinovici J, Frasa WLH, de Bie JJ, Voortman G, Itskovitz-Eldor J 2001 First human exposure to FSH-CTP in hypogonadotropic hypogonadal males. Human Reprod 16:1592–1597[Abstract/Free Full Text]
  29. Gromoll J, Eiholzer U, Nieschlag E, Simoni M 2000 Male hypogonadism caused by homozygous deletion of exon 10 of luteinizing hormone (LH) receptor: differential action of human chorionic gonadotropin and LH. J Clin Endocrinol Metab 85:2281–2286[Abstract/Free Full Text]
  30. Carlsen E, Olsson C, Petersen JH, Andersson A, Skakkaebaek NE 1999 Diurnal rhythm in serum levels of inhibin B in normal men: relation to testicular steroids and gonadotropins. J Clin Endocrinol Metab 84:1664–1669[Abstract/Free Full Text]
  31. Deputy CE, Mahmoud AM, Dhooge WS, Schoonjans FA, Comhaire FH 1999 Hormonal regulation of inhibin B secretion by immature rat Sertoli cells in vitro: possible use as a bioassay for estrogen detection. J Androl 20:54–62[Abstract/Free Full Text]
  32. Weinbauer GF, Schlatt S, Walter V, Nieschlag E 2001 Testosterone-induced inhibition of spermatogenesis is more closely related to suppression of FSH than to testicular androgen levels in the cynomolgus monkey model (Macaca fascicularis). J Endocrinol 168:25–38[Abstract]
  33. Narula A, Gu Y-Q, O’Donnell L, Stanton PG, Robertson DM, McLachlan RI, Bremner WJ 2002 Variability in sperm suppression during testosterone administration to adult monkeys is related to follicle-stimulating hormone suppression and not to intratesticular androgens. J Clin Endocrinol Metab 87:3399–3406[Abstract/Free Full Text]
  34. Anderson RA, Wallace EM, Groome NP, Bellis AJ, Wu FCW 1997 Physiological relationship between inhibin B, follicle-stimulating hormone secretion and spermatogenesis in normal men and response to gonadotropin suppression by exogenous testosterone. Hum Reprod 12:746–751[Abstract/Free Full Text]
  35. McLachlan RI, O’Donnell L, Stanton PG, Balourdos G, Frydenberg M, de Kretser DM, Robertson DM 2002 Effects of testosterone and medroxyprogesterone acetate on semen quality, reproductive hormones, and germ cell populations in normal young men. J Clin Endocrinol Metab 87:546–556[Abstract/Free Full Text]
  36. Buchter D, von Eckardstein S, von Eckardstein A, Kamischke A, Simoni M, Behre HM, Nieschlag E 1999 Clinical trial of transdermal testosterone and oral levonorgestrel for male contraception. J Clin Endocrinol Metab 84:1244–1249[Abstract/Free Full Text]
  37. Martin CW, Riley SC, Everington D, Groome NP, Riemersma RA, Baird DT, Anderson RA 2000 Dose-finding study of oral desogestrel with testosterone pellets for suppression of the pituitary-testicular axis in normal men. Hum Reprod 15:1515–1524[Abstract/Free Full Text]
  38. Behre HM, Kliesch S, Lemcke B, von Eckardstein S, Nieschlag E 2001 Suppression of spermatogenesis to azoospermia by combined administration of GnRH antagonist and 19-nortestosterone cannot be maintained by this non-aromatizable androgen alone. Hum Reprod 12:2570–2577
  39. Kinniburgh D, Zhu H, Cheng L, Kicman AT, Baird DT, Anderson RA 2002 Oral desogestrel with testosterone pellets induces consistent suppression of spermatogenesis to azoospermia in both Caucasian and Chinese men. Hum Reprod 17:1490–1501[Abstract/Free Full Text]
  40. Anderson RA, Kinniburgh D, Baird DT 2002 Suppression of spermatogenesis by etonogestrel implants with depot testosterone: potential for long-acting male contraception. J Clin Endocrinol Metab 87:3640–3649[Abstract/Free Full Text]
  41. O’Donnell L, Robertson KM, Jones ME, Simpson ER 2001 Estrogen and spermatogenesis. Endocr Rev 22:289–318[Abstract/Free Full Text]
  42. Berlau J, Roemer W, Oettel M, Hipler UC, Patchev V, Schreiber G 1999 17{alpha}-Estradiol and 17ß-estradiol do not affect basal and follicle-stimulating hormone-stimulated inhibin B secretion by highly purified rat Sertoli cells. Andrologia 32:345–349[CrossRef]
  43. Vannelli GB, Barni T, Forti G, Negro-Vilar A, Vale W, Serio M, Balboni GC 1992 Immunolocalization of inhibin {alpha}-subunit in the human testis. A light- and electron-microscopy study. Cell Tissue Res 269:221–227[CrossRef][Medline]
  44. Vliegen MK, Schlatt S, Weinbauer GF, Bergmann M, Groome NP, Nieschlag E 1993 Localization of inhibin/activin subunits in the testis of adult nonhuman primates and men. Cell Tissue Res 273:261–268[CrossRef][Medline]
  45. Zhang T, Zhou HM, Liu YX 1997 Expression of plasminogen activator and inhibitor, urokinase receptor and inhibin subunits in rhesus monkey testes. Mol Hum Reprod 3:223–231[Abstract/Free Full Text]
  46. Anderson RA, Irvine DS, Balfour C, Groome NP, Riley SC 1998 Inhibin B in seminal plasma: testicular origin and relationship to spermatogenesis. Hum Reprod 13:920–926[Abstract/Free Full Text]
  47. Andersson A-M, Mueller J, Skakkebaek NE 1998 Different roles of prepubertal and postpubertal germ cells and Sertoli cells in the regulation of serum inhibin B levels. J Clin Endocrinol Metab 83:4451–4458[Abstract/Free Full Text]
  48. Berensztein E, Saraco N, Belgorosky A, Rivarola MA 2000 Secretion of inhibin B by human prepubertal testicular cells in culture. Eur J Endocrinol 142: 481–485
  49. Majumdar SS, Ramaswamy S, Walker WH, Onate SA, Friedman RL, Plant TM, In vitro studies of the cellular origin of testicular inhibin B and the control of its secretion by FSH and LH in the rhesus monkey. Program of the 84th Annual Meeting of The Endocrine Society, San Francisco, CA, 2002, p 228 (Abstract P1-316)
  50. Tsai MJ, O’Malley BW 1994 Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Ann Rev Biochem 63:451–484[CrossRef][Medline]
  51. Simoni M, Gromoll J, Nieschlag E 1997 The follicle-stimulating hormone receptor: biochemistry, molecular biology, physiology, and pathophysiology. Endocr Rev 18:739–773[Abstract/Free Full Text]
  52. Coutant R, Lumbroso S, Fey R, Lahlou N, Venara M, Rouleau S, Sultan C, Limal J-M 2001 Macroorchidism due to autonomous hyperfunction of Sertoli cells and Gs{alpha} gene mutation: an unusual expression of McCune-Albright syndrome in a prepubertal boy. J Clin Endocrinol Metab 86:1778–1781[Abstract/Free Full Text]
  53. Prince FP 1990 Ultrastructural evidence of mature Leydig cells and Leydig cell regression in the neonatal human testis. Anat Rec 228:405–417[CrossRef][Medline]
  54. Prince FP, Mann DR, Fraser HM 1998 Blockade of the hypothalamic-pituitary-testicular axis with a GnRH receptor antagonist in the neonatal marmoset monkey: changes in Leydig cell ultrastructure. Tissue Cell 30:651–661[CrossRef][Medline]
  55. Saez JM 1994 Leydig cells: endocrine, paracrine, and autocrine regulation. Endocr Rev 15:574–626[Abstract/Free Full Text]
  56. Anderson RA, Sharpe RM 2000 Regulation of inhibin production in the human male and its clinical applications. Int J Androl 23:136–144[CrossRef][Medline]
  57. Meachem SJ, Nieschlag E, Simoni M 2001 Inhibin B in male reproduction: pathophysiology and clinical relevance. Eur J Endocrinol 145:561–571[Abstract]
  58. Plant TM, Marshall GR 2001 The functional significance of FSH in spermatogenesis and the control of its secretion in male primates. Endocr Rev 22:764–786[Abstract/Free Full Text]
  59. Jensen TK, Andersson A-M, Hjollund NHI, Scheike T, Kolstad H, Giwerman A, Henriksen TB, Ernst E, Bonde JP, Olsen J, McNeilly AS, Groome NP, Skakkaebaek NE 1997 Inhibin B as a serum marker of spermatogenesis: correlation to differences in sperm concentration and follicle-stimulating hormone levels—a study of 349 Danish men. J Clin Endocrinol Metab 82:4059–4063[Abstract/Free Full Text]
  60. Klingmueller D, Haidl G 1997 Inhibin B in men with normal and disturbed spermatogenesis. Hum Reprod 12:2376–2378[Abstract/Free Full Text]
  61. Pierik FH, Vreeburgh JT, Stijnen T, De Jong FH, Weber RF 1998 Serum inhibin B as a marker of spermatogenesis. J Clin Endocrinol Metab 83:3110–3114[Abstract/Free Full Text]
  62. Kolb BA, Stanczyk FZ, Sokol RZ 2000 Serum inhibin B levels in males with gonadal dysfunction. Fertil Steril 74:234–238[CrossRef][Medline]
  63. Andersen AG, Jensen TK, Carlsen E, Jorgensen N, Andersson AM, Krarup T, Keiding N, Skakkaebaek NE 2000 High frequency of sub-optimal semen quality in an unselected population of young men. Hum Reprod 15:366–372[Abstract/Free Full Text]
  64. Brugo-Olmedo S, De Vincentiis S, Calamera JC, Urrutia F, Nodar F, Acosta AA 2001 Serum inhibin B may be a reliable marker of the presence of testicular spermatozoa in patients with non-obstructive azoospermia. Fertil Steril 76:1124–1129[CrossRef][Medline]
  65. Wallace EM, Groome NP, Riley SC, Parker AC, Wu FCW 1997 Effects of chemotherapy-induced testicular damage on inhibin, gonadotropin, and testosterone secretion: a prospective longitudinal study. J Clin Endocrinol Metab 82:3111–3115[Abstract/Free Full Text]
  66. Petersen PM, Andersson A-M, Rorth M, Daugaard G, Skakkaebaek NE 1999 Undetectable inhibin B serum levels in men after testicular irradiation. J Clin Endocrinol Metab 84:213–215[Abstract/Free Full Text]
  67. Foppiani L, Schlatt S, Simoni M, Weinbauer GF, Hacker-Kolm UU, Nieschlag E 1999 Inhibin B is a more sensitive marker of spermatogenic damage than FSH in the irradiated non-human primate model. J Endocrinol 162:393–400[Abstract]
  68. Tena-Sempere M, Kero J, Rannikko A, Yan W, Huhtaniemi I 1999 The pattern of inhibin/activin {alpha}- and ßB-subunit messenger ribonucleic acid expression in rat testis after selective Leydig cell destruction by ethylene dimethane sulfonate. Endocrinology 140:5761–5770[Abstract/Free Full Text]
  69. Feng ZM, Bardin CW, Chen CL 1989 Characterization and regulation of testicular inhibin ß-subunit mRNA. Mol Endocrinol 3:939–948[Abstract/Free Full Text]
  70. Feng Z-M, Li Y-P, Chen CL 1989 Analysis of the 5'-flanking regions of rat inhibin {alpha}- and ß-B-subunit genes suggests two different regulatory mechanisms. Mol Endocrinol 3:1914–1925[Abstract/Free Full Text]
  71. Dykema JC, Mayo KE 1994 Two messenger ribonucleic acids encoding the common ßB-chain of inhibin and activin have distinct 5'-initiation sites and are differentially regulated in rat granulosa cells. Endocrinology 135:702–711[Abstract]
  72. Klaij IA, Timmermen MA, Blok LJ, Grootegoed JA, de Jong FH 1992 regulation of inhibin ß-subunit mRNA expression in rat Sertoli cells: consequences for the production of bioactive and immunoactive inhibin. Mol Cell Endocrinol 85:237–246[CrossRef][Medline]
  73. Najmabadi H, Rosenberg LA, Yuan QX, Reyaz G, Bhasin S 1993 Transcriptional regulation of inhibin ßB messenger ribonucleic acid levels in TM.4 or primary rat Sertoli cells by 8-bromo-cyclic adenosine monophosphate. Mol Endocrinol 7:561–569[Abstract/Free Full Text]
  74. Chaudhary J, Whaley PD, Cupp A, Skinner MK 1996 Transcriptional regulation of Sertoli cell differentiation by follicle stimulating hormone at the level the c-fos and transferring promoters. Biol Reprod 54:692–699[Abstract]
  75. Conti M 2002 Specificity of the cyclic adenosine 3', 5'-monophosphate signal in granulosa cell function. Biol Reprod 67:1653–1661[Abstract/Free Full Text]
  76. Keinan D, Madigan MB, Bardin CW, Chen, C-LC 1989 Expression and regulation of testicular inhibin {alpha}-subunit gene in vivo and in vitro. Mol Endocrinol 3:29–35[Abstract/Free Full Text]



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