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Endocrinology Vol. 138, No. 4 1363-1373
Copyright © 1997 by The Endocrine Society

ARTICLES

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)1

S. S. Majumdar2, S. J. Winters and T. M. Plant

Departments of Cell Biology and Physiology (S.S.M., T.M.P.), and Medicine (S.J.W.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Address all correspondence and requests for reprints to: Dr. Tony M. Plant, Department of Cell Biology and Physiology, University of Pittsburgh, S330 Biomedical Science Tower, Pittsburgh, Pennsylvania 15261.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The purpose of this study was to examine the relative roles of FSH and LH in stimulating testicular inhibin secretion in the male rhesus monkey. Recombinant human (rh) FSH and rhCG were used as the gonadotropic stimuli, and juvenile rhesus monkeys, in which the endocrine activity of the pituitary-testicular axis was being driven in an adult manner with an intermittent iv GnRH infusion, were studied. Immunoactive inhibin levels were measured by the Monash RIA. Initiation of an intermittent iv infusion of rhFSH (10 IU every 3 h) resulted, after a delay of 5–6 h, in a progressive increase in the concentrations of immunoactive inhibin, which achieved, after 48 h of stimulation, a value twice that observed during vehicle treatment. Gel filtration chromatography revealed that the FSH-induced elevation in immunoactive inhibin was the result of an increase in three distinct mol wt fractions: peak I (100 kDa), peak II (50–60 kDa), and peak III (31 kDa). Although peak III accounted for most of the inhibin immunoactivity in vehicle-treated animals, peaks I and II were most responsive to FSH stimulation. Application of recently developed enzyme-linked immunosorbent assays for inhibin B and pro-{alpha}-C-related peptides provided additional insights into the nature of the FSH-sensitive forms of circulating immunoactive inhibin. Most notably, the 31-kDa fraction (peak III) was comprised of inhibin B and pro-{alpha}-C. In contrast to FSH stimulation, an intermittent infusion of rhCG (40 IU every 3 h), which markedly elevated testicular testosterone secretion, failed to increase immunoactive inhibin concentrations. These findings indicate that various forms of immunoactive inhibin are present in the circulation of the rhesus monkey, and that in this species, FSH is the principal stimulus of the secretion of testicular inhibins, including inhibin B. Additionally, they further underline the importance of the FSH-inhibin feedback loop in governing testicular function in primates.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PREVIOUS studies by our laboratories provided substantial evidence in support of the view that testicular inhibin is the major regulator of FSH secretion in the rhesus monkey (1, 2, 3). To fully understand the negative feedback control of testicular function in this species, it was important to examine further the role of FSH in the control of inhibin secretion by the macaque testis. In the male rhesus monkey, a single im injection of purified human (h) FSH at a dose of either 10 or 25 IU/kg BW results in a dose-dependent rise in serum immunoactive inhibin that achieves maximal concentrations 24–108 h after administration of the gonadotropin (4), and in juvenile male rhesus monkeys daily injections of this gonadotropin (3 IU/kg·day) lead to an increase in circulating immunoactive levels of the testicular hormone that plateau after 4 weeks of FSH stimulation (5). Similarly, in GnRH antagonist-treated adult male cynomolgus monkeys, im administration of highly purified hFSH (15 IU/monkey, twice a day) is able to maintain serum immunoactive inhibin concentrations at pre-GnRH antagonist control levels (6). In normal men treated with hCG or a long acting testosterone (T) ester to chronically suppress endogenous gonadotropin secretion, administration of hFSH results in a variable restoration of serum immunoactive inhibin (7, 8).

Although FSH is considered to be the major stimulus for the secretion of inhibin (9), in vivo studies in men and rats using purified gonadotropins indicate that LH may also regulate the secretion of this testicular hormone. In normal men, im administration of hCG results in a significant rise in the circulating levels of immunoactive inhibin (10), and in men in whom endogenous gonadotropin secretion and testicular hormone production have been suppressed by treatment with a long acting T ester, chronic administration of either hLH or hCG partially restores serum immunoactive inhibin concentrations (8). Single sc injections of hCG into adult male rats increase the concentration of immunoactive inhibin in peripheral plasma and testicular interstitial fluid within 8 h (11, 12).

Additional evidence for the view that LH may provide a stimulus for testicular inhibin production is provided by in vitro studies. Immature and adult rat Leydig cells in primary culture secrete immunoactive inhibin in a manner that may be augmented by the addition of LH (13, 14, 15). The presence of inhibin subunit polypeptides and their corresponding messenger RNAs (mRNAs) in Leydig cells has been indicated using the techniques of immunohistochemistry, Northern blotting, and in situ hybridization (13, 15, 16, 17).

To begin to examine the relative importance of FSH and LH in stimulating the secretion of inhibin by the primate testes, the present study was undertaken in the GnRH-driven juvenile male monkey. In this macaque, the endocrine activity of the normally quiescent pituitary-testicular axis of the prepubertal animal may be driven in a manner resembling that in the adult by the chronic intermittent iv infusion of GnRH (18). Recombinant (r) hFSH or rhCG was used to amplify either the endogenous FSH or LH signal, respectively. The recombinant gonadotropins were administered individually as an intermittent iv infusion for 48 h in such a manner that each infusion of exogenous FSH or CG was superimposed on a discharge of endogenous gonadotropin elicited by the concomitant pulsatile infusion of GnRH. In addition to assessing quantitative changes in inhibin secretion, as reflected by changes in circulating concentrations of immunoactive inhibin, the qualitative nature of this circulating testicular hormone was also evaluated by gel filtration chromatography, and recently developed enzyme-linked immunosorbent assays (ELISAs) for dimeric inhibin and inhibin pro-{alpha}-C. The GnRH-driven juvenile male monkey was chosen for this experiment because at this stage of development the animal weighs only 2.5–3.5 kg, approximately one third the weight of an adult monkey. Thus, this experimental model enabled us to complete the study with relatively limited stocks of the recombinant hormones.

The present study also provided an opportunity to examine the role, if any, of FSH in the regulation of testicular T secretion in the rhesus monkey.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Eight juvenile male rhesus monkeys (Macaca mulatta) were used. In this species, the onset of puberty, as reflected by the initiation of nocturnal testicular T secretion, occurs at approximately 30 months of age (19). In the present study, monkeys were 17–23.5 months of age and weighed between 2.5–3.4 kg at the time of catheterization. Four monkeys were used to examine the effects of exaggerated FSH stimulation on inhibin secretion. Treatment with FSH or the FSH vehicle was initiated between 22–26.4 months of age and was completed before the age of 27.1 months. Each monkey received both FSH and FSH vehicle (see Experimental design below). After a minimum period of 4 weeks following completion of the FSH experiment, the same four monkeys were treated with rhCG. Treatment with rhCG was completed by 32 months of age. The remaining four animals were studied to examine the effects of the vehicle used to administer CG. Their ages ranged from 19–23 months. All animals were maintained under a controlled photoperiod (lights on, 0600–1800 h), as previously described (20), in accordance with NIH Guidelines for the Care and Use of Experimental Animals.

Hormonal preparations
rhFSH, produced by a transfected Chinese hamster ovary cell line (21), and rhCG, produced by transfected GH3 cells (22, 23), were used. Concentrations of rhFSH and rhCG in media were determined by Dr. Hsueh. A granulosa aromatase bioassay (24) with LER 907 as standard and a commercially available ELISA with an NIH hCG preparation (CR121) as standard were used for FSH and CG, respectively. The rhFSH in Chinese hamster ovary cell culture medium (125 IU/ml) was diluted in Ca2+- and Mg2+-free PBS (without CaCl2 and MgSO4; Life Technologies, Grand Island, NY) containing 200 µg/ml cephazolin sodium (Kefzol, Eli Lilly Co., Indianapolis, IN) to achieve a final concentration of 10 IU rhFSH/ml. The rhFSH infusates were prepared individually for each animal and contained 1% serum from the respective monkey. PBS containing antibiotic and 1% of the appropriate monkey serum was used for vehicle.

The rhCG infusates (2.5 or 5 IU/ml medium) were prepared individually for each animal in a manner similar to that described for rhFSH. Nontransfected GH3 cell line medium containing antibiotic and the appropriate monkey serum was used as vehicle.

Synthetic GnRH (LHRH-2, biological grade) was provided by the National Hormone and Pituitary Program. The stock and working dilutions used in the present study were prepared as previously described (25).

Catheterization procedure
To withdraw blood samples and to administer GnRH and recombinant gonadotropins without restraint or tranquilization, the monkeys were implanted with one or two central venous catheters and housed in remote sampling cages, as described previously (18, 20). Animals were anesthetized before surgery with pentobarbital sodium (~30 mg/kg BW, iv, plus 5-mg supplements as required; Nembutal sodium solution, Abbott Laboratories, North Chicago, IL). Postoperatively, each monkey received approximately 100 mg cephazolin sodium and meperidine hydrochloride (1 mg/kg BW; Demerol, Winthrop Pharmaceuticals, New York, NY) iv twice daily for 4 days.

In animals with only one catheter, each individual infusion of GnRH (2 ml of 0.15 µg GnRH/ml) was automatically introduced into the catheter every 3 h and immediately chased into the animal with a saline bolus (0.8 ml/min for 3 min). Heparinized saline [4 U heparin (Elkins-Sinn, Cherryhill, NJ)/ml isoosmolar NaCl (Abbott Laboratories)] was continuously infused (3.0 ml/h) through the catheter to maintain patency. The GnRH and saline solutions were introduced into the catheter using a set of three three-way stopcocks arranged in series and in combination with three individually programmed peristaltic pumps. In these animals, the single catheter was also used for withdrawing blood and administering rhFSH.

In animals bearing two catheters, one catheter was used for the pulsatile infusion of GnRH, and the other was employed for sampling of blood and intermittent administration of the recombinant gonadotropin. GnRH in these monkeys was administered either as described for animals with one catheter or as a continuous intermittent infusion (0.1 µg/min for 3 min every 3 h), with the GnRH infusate (0.3 µg/ml) remaining in the catheter during the 3-h intervals between infusions.

All animals were allowed to adapt to the remote infusion and withdrawal system for a minimum of 5 weeks before the effects of FSH or CG administration were examined.

Activation of the pituitary-testicular axis in juvenile monkeys
To elicit prematurely an adult pattern of hormonal activity in the pituitary-testicular axis of the juvenile monkey, each animal received a chronic intermittent iv infusion of GnRH (~0.1 µg/min for 3 min every 3 h). The GnRH infusion was initiated 1–7 days after catheterization. This exogenous hypophysiotropic signal to the gonadotrophs of the juvenile monkey elicits a pattern of circulating T and immunoactive inhibin concentrations similar to those produced by spontaneous testicular secretion in adults (18). The progressive activation of the pituitary-testicular axis by pulsatile GnRH treatment was monitored at approximately weekly intervals by tracking plasma T concentrations.

Immunoassays
Immunoactive inhibin concentrations were measured as described previously (26) by a double antibody RIA, using recombinant human inhibin A as the standard (0.03–0.3 ng/tube), purified bovine inhibin as the iodinated tracer, and an antiserum to bovine 31-kDa inhibin (no. 1989) obtained from Dr. David Robertson through the Contraceptive Development Branch, NICHHD. The minimum detectable dose was 0.03 ng. Sample volumes in all assay tubes were maintained constant by adding serum from postmenopausal women because of the nonspecific effects of serum or plasma on tracer binding. Serum from adult male rhesus monkeys produced dose-response curves, which paralleled those of the standard. Samples were assayed in duplicate or triplicate using 25–150 µl plasma. Inhibin was undetectable in plasma from castrated adult monkeys (<0.03 ng/ml). The intraassay coefficient of variation in the midportion of the standard curve was 3.5%. The interassay coefficients of variation of samples of various potencies ranged from 7.5–12.2%.

Inhibin B and inhibin pro-{alpha}-C were assayed using ELISA kits obtained from Serotec (Washington DC). These assays have been described in detail by Groome et al. (27, 28). Column eluates were analyzed singly, and standards were analyzed in duplicate. The mean coefficient of variation of the duplicate standards for the inhibin B and pro-{alpha}-C ELISAs were 4.7% and 14.4%, respectively. Inhibin A, activin A and B, follistatin, and purified human pro-{alpha}-C all had less than 0.5% cross-reactivity in the inhibin B ELISA. Inhibin A and B, activin A and B, and follistatin were reported to cross-react less than 0.02% in the pro-{alpha}-C assay. The alkaline phosphatase ELISA amplification kit was obtained from Life Technologies (Gaithersburg, MD). Absorbencies were read with an E Max precision microplate reader (Molecular Devices Corp., Sunnyvale, CA) at 495 nm. Eluates obtained after gel filtration chromatography of plasma were lyophilized and reconstituted, as described for assay standards, in 0.1 ml FCS for the assay of inhibin B or diluted 1:20 in the manufacturer’s assay diluent for assay of pro-{alpha}-C. FCS produced a strongly positive reaction in the pro-{alpha}-C ELISA.

Plasma concentrations of monkey FSH were measured by a hFSH (NIDDK hFSH I-3):anti-hFSH (NIAMDD 5, National Hormone and Pituitary Program) RIA system that employs a purified rhesus pituitary FSH preparation (WP-XIII-21–42) as standard (25). The minimum detectable concentration in the FSH assay was 2.0 ng/ml, and the intraassay and mean interassay coefficients of variation were 5.5% and 12.5%, respectively. Plasma concentrations of monkey LH were estimated using a RIA kit supplied by the National Hormone and Pituitary Program. It consists of a cynomolgus LH:anti-hCG (rabbit 13, pool D) RIA system that uses a rhesus pituitary preparation (NICHHD rhLH RP-1) as standard (29). The minimum detectable concentration in the LH assay was 10 ng/ml, and the intraassay and mean interassay coefficients of variation were 2.5% and 4.2%, respectively.

Concentrations of rhFSH in monkey serum were measured by a previously described double antibody RIA using reagents anti-hFSH-5, hFSH-I3 (AFP-4822B), and the Second International Reference Preparation of human menopausal gonadotropin as the reference standard (30) from the National Hormone and Pituitary Program. The minimum detectable concentration in the hFSH assay was 2 mIU/ml, and the intra- and interassay coefficients of variation were 2.8–13.0% and 16.0%, respectively. Concentrations of rhCG in monkey serum were measured by a double antibody RIA using an antisera to hLH (RD21, Wellcome Reagents, Beckenham, UK) that cross-reacts 100% with hCG. The tracer was highly purified hLH (Kabi Diagnostics, Stockholm, Sweden), and the RIA standard was the Second International Reference Preparation of human menopausal gonadotropin. The minimum detectable concentration in the hCG assay was 0.45 mIU/ml, and the intra- and interassay coefficients of variation across the standard curve approximated 10%.

Plasma T concentrations were assayed in duplicate by a previously described RIA (31), using antiserum T3–125 (Endocrine Sciences, Torrance, CA). The minimum detectable concentration in the assay was 0.13 ng/ml. The intraassay and mean interassay coefficients of variations were 12.4% and 18.1%, respectively. Plasma levels of estradiol (E2) were measured in duplicate by a double antibody RIA kit (Diagnostic Products Corp., Los Angeles, CA). The minimum detectable concentration of E2 was 3.2 pg/ml. All samples were run in a single assay with an intraassay coefficient of variation of 4.8%.

Gel filtration chromatography
To examine the apparent molecular size of inhibin in the circulation, 2–3 ml plasma were chromatographed on sequential columns of Sephadex G-75 (1.6 x 70 cm; Pharmacia, Piscataway, NJ) and Sephadex G-100 (1.6 x 86 cm) at 4 C with 0.1 M ammonium carbonate buffer. The flow rate was 4 ml/h. Tracer quantities of [125I]rat FSH (Mr, 33 kDa) were cochromatographed with each sample for internal calibration. One-milliliter fractions of elute were collected, counted to locate the radioactive FSH peak, and lyophilized using a Speed-Vac concentrator (Savant Instruments, Farmingdale, NY).

Experimental design
Experiments were initiated after establishing that a robust pattern of episodic T secretion had been induced in the juvenile monkeys by the uninterrupted pulsatile infusion of GnRH for 28–75 days.

Exp 1: effect of exaggerated FSH stimulation on circulating inhibin concentrations. A selective increase in FSH stimulation of the testis was imposed in four monkeys by the intermittent iv administration of rhFSH. For this purpose, rhFSH (10 IU in 1 ml infusate) was manually introduced into a catheter and delivered to the monkey by a 5-ml saline flush over 2–3 min. A FSH injection was administered every 3 h for 48 h, and all FSH infusions were administered within 5 min of the initiation of GnRH infusion. Time courses of circulating concentrations of immunoactive inhibin and T as well as those for rhFSH and macaque LH were characterized during 3-h windows (corresponding to an inter-GnRH pulse interval) before, during, and after rhFSH treatment. Specifically, on day 1 of the experiment, samples were taken immediately before the GnRH infusion at 0900 h and thereafter at 10- to 60-min intervals until the next GnRH infusion at 1200 h. At this time, treatment with rhFSH was initiated and maintained for 48 h; the last injection of rhFSH was given at 0900 h on day 3 of the experiment. Sequential blood samples were collected between the inter-GnRH pulse intervals corresponding to the 1st (0–3 h), 2nd (3–6 h), 4th (9–12 h), 8th (21–24 h), and 16th (45–48 h) injections of FSH. Similar samples were collected during the 1st (0–3 h), 8th (21–24 h), and 32nd (93–96 h) GnRH pulse intervals after termination of FSH treatment. A 10-ml blood sample was also taken immediately after the last FSH injection (45–48 h) for the qualitative analysis of circulating inhibin.

Control experiments with vehicle were conducted in an identical manner. In three monkeys, these were completed 6–12 days before treatment with rhFSH. In the remaining animal, the vehicle infusion was initiated 20 days after termination of FSH treatment.

Exp 2: effect of exaggerated LH stimulation on circulating inhibin concentrations. An increase in the LH stimulus to the testis was obtained in the four monkeys employed in the first experiment by superimposing a brief infusion of rhCG on the discharge of endogenous gonadotropin elicited by the intermittent infusion of GnRH. For this purpose, rhCG (40 IU in 8 or 16 ml infusate) was delivered to the monkey over a 3-min period via the infusion catheter. The CG infusates were administered automatically by a peristaltic pump every 3 h for 48 h, and the CG infusions coincided with those of GnRH. The time courses of circulating concentrations of inhibin, T, and macaque FSH were characterized during 3-h windows (corresponding to an inter-GnRH pulse interval) before, during, and after rhCG treatment using a blood-sampling protocol identical to that employed in Exp 1. Levels of circulating rhCG and E2 were also measured in selected samples.

Control experiments with the CG vehicle (8 ml/pulse) were conducted in four additional animals.

All samples were processed at 4 C after collection; red blood cells were returned to the animal, and plasma was frozen at -20 C until analyzed.

Data analysis
The amplitude of the abrupt increments in either circulating rhFSH or rhCG concentrations produced by the intermittent infusion of the respective gonadotropin was defined as the difference between the basal and peak concentrations that were observed before and after iv infusion of exogenous hormone.

To analyze numerically the effects of rhFSH or rhCG on testicular inhibin secretion, average values were first obtained for inhibin concentrations in the sequential samples collected from each animal during each 3-h inter-GnRH pulse interval examined. Mean inhibin concentrations for the four animals for each treatment were then calculated for each 3-h window studied, and the significance of differences in this parameter were determined using ANOVA for repeated measures followed by Fisher’s test (StatView II, Abacus Concepts, Berkeley, CA). Circulating levels of T and macaque FSH during stimulation with rhCG were also analyzed as described above.

E2 concentrations, which were measured in individual monkeys in pooled samples obtained by combining individual samples collected during a given 3-h inter-GnRH pulse interval, were also analyzed by ANOVA.

The half-lives of rhFSH and rhCG in the circulation of the monkeys were calculated from semilogarithmic plots of the composite profile of plasma concentrations of each hormone vs. time after the first brief infusion of the recombinant gonadotropin.

Elution profiles of immunoactive inhibin after gel filtration were analyzed by calculating the area under the curve described by the immunoactive inhibin peaks using a computer program (32). The mean areas under the inhibin peaks observed during FSH and vehicle treatment were compared by Student’s t test for paired data.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Exp 1
The intermittent iv administration of rhFSH to GnRH driven juvenile male rhesus monkeys produced a pulsatile profile in the circulating concentrations of the heterologous gonadotropin (Fig. 1Go). The mean amplitude of the increments in circulating concentrations of rhFSH was approximately 35 mIU/ml. Nadir levels of the pulsatile rhFSH profile increased from a mean of 9 mIU/ml during the first 3 h of treatment to approximately 45 mIU/ml after the last injection of the gonadotropin; the time when the mean peak concentration of rhFSH (77 mIU/ml) was maximal. Immediately after termination of rhFSH treatment, plasma levels of this gonadotropin began to decline, and within 24 h, concentrations of this hormone were markedly lower than those during treatment. Plasma concentrations of rhFSH returned to pretreatment control values by 96 h after withdrawal of FSH treatment.



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  		Figure 1. Time courses of circulating concentrations of rhFSH (top panel) and endogenous immunoactive inhibin (lower panel) in four GnRH-driven juvenile male rhesus monkeys before, during (stippled area), and after the intermittent iv infusion of rhFSH (10 IU every 3 h) from 0–48 h. Note, although a pulse of FSH was administered every 3 h, circulating hormone profiles were monitored only after the 1st (0–3 h), 2nd (3–6 h), 4th (9–12 h), 8th (21–24 h), and 16th (45–48 h) injections of the gonadotropin. Similarly, samples were collected only during the 1st (0–3 h), 8th (21–24 h), and 32nd (93–96 h) 3-h inter-GnRH pulse intervals after termination of FSH treatment. The nonlinear scale on the abscissa reflects these sampling windows. Vertical bars represent the SEM.

 
The semilogarithmic plot of mean rhFSH concentrations vs. time after the first iv bolus of the recombinant hormone was rectilinear (r2 = 0.94) and provided an estimate of 100 min for the half-life of rhFSH in the circulation of the monkey.

The effect of the pulsatile FSH infusion on circulating immunoactive inhibin concentrations is also shown in Fig. 1Go. After a delay of 5–6 h following initiation of treatment with rhFSH, plasma concentrations of immunoactive inhibin increased progressively to achieve, after 48 h of stimulation with the gonadotropin, a value twice that before the administration of rhFSH. Inhibin levels after 9 h (3.01 ± 0.58 ng/ml; mean ± SE), 21 h (3.84 ± 0.44 ng/ml), and 45 h (4.48 ± 0.39 ng/ml) of stimulation with rhFSH were significantly (P < 0.01) greater than pretreatment control levels (1.97 ± 0.29 ng/ml). During the first 3 h after termination of pulsatile FSH treatment, plasma inhibin remained at concentrations (4.58 ± 0.38 ng/ml) indistinguishable from those observed during the terminal phase of FSH stimulation. Subsequently, plasma concentrations of this testicular hormone declined, reaching, 21 h later, a value intermediate to those observed before initiation and at the end of FSH stimulation. Plasma concentrations of immunoactive inhibin returned to pretreatment control values within 96 h of terminating the FSH infusion.

There was no indication of pulsatile inhibin release during either exogenous FSH stimulation or control conditions.

The intermittent iv infusion of vehicle failed to influence circulating inhibin concentrations. The mean concentration (±SE) of this testicular hormone before and 9, 21, and 45 h after initiation of vehicle infusion were 1.8 ± 0.2, 1.4 ± 0.09, 1.8 ± 0.17, and 1.9 ± 0.23 ng/ml, respectively.

Gel filtration chromatography of plasma from the four vehicle-treated monkeys revealed three peaks of immunoactive inhibin (Fig. 2Go). The largest peak (peak III) accounted for 86.7 ± 3.80% of the total immunoactivity and eluted immediately after rat FSH, but before hCG{alpha}, which served as 33- and 23-kDa mol wt markers, respectively. The peak with the highest mol wt (peak I) eluted immediately after the void volume (100 kDa) and accounted for only a small proportion of total immunoactivity (1.5 ± 0.95%). Peak II, which eluted after BSA but before rat FSH, and was approximately 50–60 kDa and accounted for 11.8 ± 3.27% of the inhibin immunoactivity.



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  		Figure 2. Inhibin immunoactivity (closed circles) in 1-ml fractions after gel filtration chromatography of 2 ml plasma from one GnRH-driven juvenile male rhesus monkey during infusion of vehicle (upper panel) and after treatment with rhFSH (lower panel). [125I]Rat FSH (continuous line), a mol wt (33 kDa) marker, was cochromatographed with each plasma sample. BSA (66 kDa; closed arrow) and [125I]hCG{alpha} (23 kDa; open arrow) were used as additional mol wt markers, but were chromatographed separately. Three peaks of immunoactive inhibin were observed, and these were designated peaks I, II, and III according to decreasing mol wt.

 
rhFSH treatment significantly increased (P < 0.05) each of the three peaks of immunoactive inhibin (Fig. 2Go, lower panel, and Fig. 3Go). The relative increase in inhibin immunoactivity observed upon FSH treatment, however, was exaggerated in peaks I and II. During FSH stimulation, these two peaks accounted for 3.5 ± 0.50% and 33.0 ± 5.27%, respectively, of the total immunoactive inhibin activity; values that compared to 1.5 ± 0.95%, and 11.8 ± 3.17%, respectively, under control conditions.



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  		Figure 3. Comparison of areas of immunoactive inhibin peaks I, II, and III (see Fig. 2Go) eluted after gel filtration chromatography of monkey plasma (n = 4) during vehicle (open bar) and rhFSH infusion (closed bar). Asterisks indicate means significantly (P < 0.05) different from control values.

 
Gel filtration chromatography in July 1992 of a blood sample collected from one animal during vehicle treatment was repeated in July 1996 for examination with the inhibin B ELISA and the pro-{alpha}-C ELISA. Figure 4Go shows that the 31-kDa peak (peak III) of inhibin immunoactivity was again identified by RIA, but that the minor peaks (I and II) were no longer present. Rechromatography of a second plasma sample stored at -20 C for 3.5 yr produced similar results. Although the inhibin B ELISA detected peak III, this assay also identified a leading shoulder of immunoactivity in the 33–36 kDa range as well as immunoactivity in the 90–100 kDa range. The latter overlapped the void volume. The pro-{alpha}-C assay also detected the 31-kDa peak, but, in addition, identified a peak of immunoactivity in the 56–60 kDa range.



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  		Figure 4. Immunoactive inhibin (B), inhibin B (C), and pro-{alpha}-C (D) in 1-ml fractions after gel filtration chromatography of a 4-ml plasma sample from a GnRH-driven juvenile male monkey infused with vehicle and stored at -20 C for approximately 4 yr. The top panel (A) shows the elution profile of [125I]rat FSH (closed circle), a 33-kDa mol wt marker, that was cochromatographed with the plasma sample, and [125I]macaque LH{alpha} (open circle) that was used as an additional mol wt (23-kDa) marker, but was chromatographed separately. Arrows 1, 2, and 3 in A indicate the positions of elution of blue dextran (Vo), BSA (66 kDa), and recombinant human inhibin A (31 kDa), respectively. These additional mol wt markers and NaI125, which eluted in fraction 365 (Vi), were chromatographed separately.

 
The pulsatile discharge of LH and the corresponding episodes of testicular T secretion induced in the juvenile male rhesus monkeys by the chronic intermittent iv infusion of GnRH, which was continued without interruption during the course of these experiments, did not appear to be influenced by the infusion of either rhFSH (Fig. 5Go) or vehicle (data not shown).



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  		Figure 5. Time courses of circulating concentrations of endogenous monkey LH (top panel) and T (lower panel) in four GnRH-driven juvenile male rhesus monkeys before, during (stippled area), and after intermittent infusion of rhFSH (10 IU every 3 h) from 0–48 h. Note, although a pulse of FSH was administered every 3 h, hormone profiles were monitored only after the 1st (0–3 h), 2nd (3–6 h), 4th (9–12 h), 8th (21–24 h), and 16th (45–48 h) injections of the gonadotropin. Similarly, samples were collected only during the 1st (0–3 h), 8th (21–24 h), and 32nd (93–96 h) 3-h inter-GnRH pulse intervals after termination of FSH treatment. The nonlinear scale on the abscissa reflects these sampling windows. Vertical bars represent the SEM.

 
Exp 2
The intermittent iv administration of rhCG to GnRH-driven juvenile male rhesus monkeys produced a pulsatile profile in the circulating concentrations of the heterologous gonadotropin (Fig. 6Go). The mean amplitude of the increments in circulating concentrations of rhCG was approximately 450 mIU/ml. The marked elevations in circulating rhCG concentration that were produced every 3 h after each brief infusion of the recombinant hormone declined to very low levels (<15 mIU) before the animal received the next infusion. The semilogarithmic plot of mean rhCG concentrations vs. time following the first iv bolus of the recombinant hormone was rectilinear (r2 = 0.92) and provided an estimate of 30 min for the t1/2 of rhCG in the circulation of the monkey.



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  		Figure 6. Time courses of circulating concentrations of rhCG (top panel), endogenous immunoactive inhibin (middle panel), and T (lower panel) in four GnRH-driven juvenile male rhesus monkeys before, during (stippled area), and after the intermittent iv infusion of rhCG (13.3 IU/min for 3 min, every 3 h) from 0–48 h. Although a brief infusion of rhCG was administered every 3 h, circulating hormone profiles were monitored only after the 1st (0–3 h), 2nd (3–6 h), 4th (9–12 h), 8th (21–24 h), and 16th (45–48 h) infusions of the gonadotropin. Similarly, samples were collected during the 1st (0–3 h), 8th (21–24 h), and 32nd (93–96 h) 3 h inter-GnRH pulse intervals after termination of rhCG treatment. Note the nonlinear scale on the ordinate after termination of the intermittent rhCG infusion at 51 h. Horizontal bars represent the SEM.

 
The response of circulating inhibin concentrations to the pulsatile rhCG infusion is also shown in Fig. 6Go. Mean plasma inhibin levels during and after treatment with CG were not significantly different (P > 0.05) from the pretreatment control level. The intermittent iv infusion of vehicle failed to influence circulating inhibin concentrations (not shown). There was no indication of pulsatile inhibin release during CG stimulation or control conditions.

Before initiation of the intermittent infusion of rhCG, the time course of circulating T levels in response to endogenous LH discharges was episodic. The mean T concentration rose from a nadir of approximately 2.5 ng/ml to a peak of approximately 8 ng/ml 40 min after the GnRH bolus (Fig. 6Go). The first brief infusion of rhCG, which was superimposed upon the endogenous gonadotropin discharge, resulted in an elevation of the peak T concentration to 11 ng/ml. In contrast to the striking episodic nature of T secretion before treatment, circulating concentrations of this steroid after the first rhCG infusion were sustained with little decrement at concentrations very close to that of the peak. By the second infusion of rhCG, the episodic profile in circulating T was completely obliterated. Mean concentrations of circulating T, however, continued to increase throughout the duration of the rhCG infusion, and at the conclusion of the 2 days of CG stimulation the mean concentration of this steroid was 25.9 ng/ml. Mean T levels after 3, 9, 24, and 48 h of stimulation with CG were significantly (P < 0.05) greater than pretreatment control levels.

During the first 3 h after termination of the pulsatile rhCG infusion, plasma T remained at concentrations indistinguishable from those observed during the terminal phase of CG stimulation. Subsequently, the episodic pattern of circulating T concentrations reappeared, and mean plasma concentrations of this testicular steroid declined (Fig. 6Go). Although nadir concentrations of T returned to pretreatment control values within 96 h of terminating the CG infusion, the peak value of T was higher than the corresponding pretreatment value. The episodic pattern of T secretion remained unchanged as a result of vehicle infusion (not shown).

Circulating concentrations of macaque FSH declined significantly (P < 0.05) after 21 h of rhCG treatment, but infusion of vehicle to four different animals did not influence the plasma profile of this gonadotropin (Fig. 7Go).



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  		Figure 7. Mean plasma concentrations of endogenous FSH before, during (stippled area), and after treatment with rhCG (top panel) or vehicle (lower panel) in GnRH-driven juvenile male rhesus monkeys (n = 4 for both groups). rhCG was administered as an intermittent iv infusion of (13.3 IU/min for 3 min every 3 h) from 0–48 h. Note that although a pulse of CG was administered every 3 h, circulating hormone profiles were only monitored after the 1st (0–3 h), 2nd (3–6 h), 4th (9–12 h), 8th (21–24 h), and 16th (45–48 h) injections of CG. Similarly, samples were collected during the 1st (0–3 h), 8th (21–24 h), and 32nd (93–96 h) 3-h inter-GnRH pulse intervals after termination of CG treatment. The nonlinear scale on the ordinate reflects these sampling windows. Horizontal bars represent the SEM. The asterisk indicates mean levels significantly different from preinfusion control values by ANOVA.

 
Plasma levels of E2 before the CG infusion were generally undetectable (3.5 ± 0.3 pg/ml), but after 24 and 48 h of CG stimulation, circulating concentrations of this steroid were 13.3 ± 5.3 and 19.1 ± 5.2 pg/ml, respectively. Ninety-six hours after CG withdrawal, plasma E2 had returned to pretreatment concentrations. This rise in circulating E2 concentration during the CG infusion was significant (P < 0.01).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The finding that an iv infusion of rhFSH stimulated testicular secretion of immunoactive inhibin in the rhesus monkey demonstrates that in nonhuman primates, as in rodents and man (33, 34, 35), this recombinant gonadotropin is biologically active. Although purified preparations of hFSH have been previously reported to stimulate testicular secretion of inhibin in nonhuman primates (4, 5, 6), the present study, which employed a recombinant preparation of FSH, adds to the earlier findings because the possibility of contamination with LH was eliminated. Moreover, FSH stimulation was provided in an intermittent mode at a frequency of one injection every 3 h, the approximate pulse frequency of endogenous gonadotropin secretion in adult males in which the pituitary-testicular axis is driven by endogenous GnRH (36). Although the temporal pattern of FSH stimulation in the present study may be viewed as physiological, the peak concentrations of FSH achieved (80 mIU/ml) were in all probability supraphysiological. The latter view is based on the observation that during the infusion of rhFSH, plasma levels of this gonadotropin exceeded those reported in adult men by several-fold (37).

Inhibin is known to exist in a variety of molecular configurations in various biological fluids (38, 39, 40, 41). In the adult male rhesus monkey, the major form of circulating immunoactive inhibin, as revealed by gel filtration chromatography, has an approximate Mr of 31 kDa (26). The present results establish that 31-kDa inhibin (peak III) also predominates in the circulation of the juvenile male monkey, in which the pituitary-testicular axis is prematurely activated with an intermittent infusion of GnRH, the experimental model we selected to study the gonadotropin drive to testicular inhibin secretion. The GnRH-driven juvenile monkey also exhibits higher mol wt forms of immunoactive plasma inhibin of 50–60 and 100 kDa (peaks II and I, respectively). These higher mol wt forms of immunoactive inhibin, which are also observed, albeit at lower levels, in the adult male (26), represent approximately 15% of the total activity in the GnRH-driven juvenile male monkey. Treatment with rhFSH significantly increased all three forms of immunoactive inhibin in plasma of GnRH-driven juvenile male monkeys, but the greatest rise (6-fold) was observed for 50- to 60-kDa inhibin (peak II). As a result, the relative contribution of 50- to 60-kDa inhibin rose from 12% to 33% of the total inhibin immunoactivity in plasma. Although there are no previous studies in males of the effects of FSH on the spectrum of circulating forms of immunoactive inhibin, high mol wt forms of inhibin were recently reported in plasma of women during treatment with FSH for ovulation induction (38).

Application of the recently developed ELISAs for dimeric inhibin B and pro-{alpha}-C-related peptides to a limited number of samples provides additional insight into the nature of the three FSH-sensitive circulating forms of immunoactive inhibin. The identities of the immunoactive components of the 31-kDa material (peak III) is of particular interest because there is compelling physiological evidence establishing that inhibin accounts for the testicular component of the feedback loop governing the secretion of FSH in the monkey (1, 2, 3). The present ELISA finding that the 31-kDa peak in the GnRH-driven juvenile male monkey is comprised in part of inhibin B was to be expected because the principal circulating form of dimeric inhibin in men and the adult male monkey is inhibin B (28, 42). Thus, it would seem reasonable to conclude that part of the FSH-induced increase in inhibin immunoactivity in peak III may be accounted for by an elevation in the circulating concentration of inhibin B, an inference that is entirely consistent with the recent finding that in normal men inhibin B levels increase in response to the administration of rhFSH (35).

The relatively broad 31- to 36-kDa peak identified by the inhibin B ELISA indicates that in the monkey, circulating inhibin B is heterogeneous, possibly reflecting variable glycosylation of the combined inhibin {alpha}-subunit, as described previously for recombinant human inhibin A from a mammalian cell line (43), and inhibin {alpha}-C subunit in follicular fluid (27). The inability of the polyclonal antisera, 1989 (RIA), to detect the leading edge of the 33- to 36-kDa peak containing inhibin B may be related to loss of immunoactivity due to changes in tertiary structure resulting from glycosylation of the {alpha}-subunit.

The remainder of the immunoactivity in peak III is presumably comprised, either partly or entirely, of a moiety recognized by the pro-{alpha}-C assay and probably represents a form of uncombined inhibin {alpha}-subunit. Plasma from men has also been reported to contain inhibin {alpha}-subunit (27) in the 31–36 kDa range (38).

The molecular compositions of the 50- to 60- and 100-kDa fractions of immunoactive inhibin (peaks II and I, respectively) identified in the circulation of the GnRH-driven juvenile monkey are less clear. With prolonged storage of plasma at -20 C, these peaks were not detected by the RIA. This may reflect glycoprotein degradation with a change in tertiary structure. A 50- to 60-kDa moiety, however, was detected after storage by the pro-{alpha}-C assay, but not by the {alpha}-ßB ELISA, and probably represents precursors of the inhibin {alpha}-subunit, as suggested previously in human male serum by Western analysis (41). On the other hand, immunoactivity in the 90–100+ kDa range (peak I) was detected by the dimeric inhibin B assay, suggesting that it represents variably processed dimeric inhibin B (38) and/or 31 kDa inhibin bound to plasma proteins (44).

The results of the present study also provide considerable insight into the dynamics of testicular inhibin secretion in response to FSH stimulation. Although circulating concentrations of rhFSH were elevated immediately after the first intermittent infusion of this gonadotropin, secretion of immunoactive inhibin was not stimulated until 5–6 h later. Similarly, after the withdrawal of the FSH infusion and a prompt decline in circulating levels of this gonadotropin, plasma inhibin was sustained for 24 h at concentrations significantly above pretreatment control levels. Recently, a similar latency between an increase and a decrease in the FSH stimulus and the respective response of testicular inhibin secretion, as reflected by circulating inhibin B concentrations, has been observed in normal men (35). Studies of cultured Sertoli cells also provide evidence for the view that the inhibin response to a change in FSH stimulation is heavily damped. Namely, an increase in inhibin secretion from rat Sertoli cells in primary culture was not observed until 8–12 h after initiation of FSH stimulation (45), and after the withdrawal of FSH stimulation, enhanced inhibin secretion was sustained for at least 8 h (46). Although the mechanisms underlying the lag between FSH stimulation and inhibin secretion are unknown, steady state levels of inhibin subunit mRNAs are increased in cultured rat Sertoli cells within 1.5 h after exposure to FSH (47), suggesting that the delay between stimulation and secretion may reflect post-transcriptional processing of the inhibin subunits. On the other hand, the maintenance of plasma concentrations of immunoactive inhibin after the withdrawal of the FSH infusion in the monkey together with a half-life of circulating inhibin of about 30 min (26, 48) may reflect persistent transcription, prolonged half-life of the mRNAs for the inhibin subunits, or a combination of the two.

Presumably, the hysteresis exhibited by the Sertoli cell in response to a change in FSH stimulation is responsible in part for the apulsatile mode of testicular inhibin secretion observed in response to robust intermittent stimulation by either exogenous or endogenous FSH (18, 26). Additionally, the topography of the Sertoli cell within the seminiferous tubule and the spatial characteristics of secretion by this cell type may contribute to the lack of fidelity between acute FSH stimulation and the concomitant circulating inhibin profile. In this regard, in vitro studies of rat and baboon Sertoli cells demonstrated the vectorial secretion of inhibin, with maximal secretion across the apical surface of the cell (49, 50). Therefore, as discussed previously (26), a large proportion of testicular inhibin may first be transported via the rete testis to the epididymis before entering the general circulation. Whatever, the case may be, the absence of moment to moment coupling between pulsatile FSH stimulation and the circulating profile of plasma inhibin in the monkey is to be contrasted with the high fidelity of the relationship between LH discharges and episodic testicular T secretion in this species (36).

To begin to examine the role, if any, of LH in the control of testicular inhibin secretion in the monkey, Exp 2 was designed with the aim of achieving a selective increase in the CG stimulation of the testis. While an increased LH drive to the testis was undoubtedly produced, as demonstrated by the marked elevation of circulating T concentrations during the CG infusion, endogenous FSH secretion declined. Although we had not anticipated a suppression of FSH secretion during the administration of rhCG, the latter result, on reflection, is of little surprise, because the enhanced secretion of T induced by infusion of the recombinant gonadotropin produced a striking increase in circulating E2 concentrations. Although testicular inhibin appears to be the principal gonadal component of the negative feedback loop governing FSH secretion in the male monkey (1, 2, 3), supraphysiological levels of circulating E2, similar to those observed in the present study, provide a potent inhibition of FSH secretion in this species (51). Regardless of the mechanism underlying the suppression of endogenous FSH secretion, the failure to maintain circulating concentrations of this gonadotropin at preinfusion control levels raises a caveat that must be taken into account when interpreting the results of the second experiment. Specifically, it is conceivable that in the face of waning FSH concentrations, a stimulatory action of CG on testicular inhibin secretion may have escaped detection.

The failure of CG to stimulate inhibin secretion in the monkey may, on the other hand, be unrelated to the decrease in FSH secretion. In adult male rats, increased testicular inhibin production and secretion have been achieved after a single sc injection of 100 IU hCG (12). Although circulating levels of hCG were not measured in that study, it is likely that the acute CG stimulus to the rat testis was greater than that achieved after the repetitive iv administration of 40 IU rhCG to 3-kg monkeys. Thus, the relatively low dose of CG used in the present study may not have been of sufficient magnitude to mimic the CG stimulus achieved in rats receiving a single injection of 100 IU hCG. The earlier findings in man that acute and chronic hCG administrations (5000 IU/injection) elicit an increase in circulating immunoactive inhibin levels (8, 10) are more difficult to reconcile with the failure of the monkey testis to respond to hCG administration with an increase in inhibin production. Whatever the case may be, the mechanism by which high doses of CG induce inhibin release is unclear, as in rats, the production of this testicular glycoprotein is not compromised after the destruction of Leydig cells with ethane dimethane sulphonate (12). As proposed by Sharpe et al. (11), the action of hCG to increase inhibin secretion in the male rat may be attributable to the inflammatory-like action of hCG on the testis (52).

The present study also provided an opportunity to examine the idea that FSH enhances the responsiveness of the Leydig cell to LH. Although earlier studies in support of this idea were conducted with purified pituitary FSH preparations (53, 54), Vihko et al. (33) reported that treatment of mature hypophysectomized rats with recombinant FSH for 7 days resulted in a greater than 2-fold increase in testicular androgen production and an associated increase in LH receptor number. Whether the failure of exaggerated FSH stimulation for 48 h to influence the pattern of episodic testicular T secretion in the monkey reflects differences in experimental model or differences between species remains to be determined.

Parenthetically, the half-life of rhFSH in the circulation of the monkey by a one-compartmental model was 100 min. This compares to a value of 240 min for the disappearance of circulating FSH after hypophysectomy in postmenopausal women (55), a value of 274 min for the disappearance of purified urinary rhFSH injected into hypogonadotropic men (56), and a value of 144 min for a preparation of rhFSH injected into adult women (57). The half-life of rhCG in the circulation of the monkey was approximately 30 min. By contrast, the half-life of a highly purified preparation of placental rhCG after rapid iv injection into normal adult men and women was substantially slower and curvilinear. The rapid component of the half-life was 6 h, and the slow component was 36 h (58). Similarly, the postpartum disappearance of rhCG has rapid and slow components of 11 and 23 h, respectively (59). These differences in clearance properties between placental and pituitary gonadotropins, on the one hand, and recombinant preparations, on the other, presumably reflect in part differences in carbohydrate and sialic acid contents leading to differences in binding to hepatocyte receptors and/or renal clearance (60).


   Acknowledgments
 
We thank Drs. Irving Boime and Aaron Hsueh for generously providing the recombinant gonadotropin preparations. We also acknowledge the contributions of Drs. Naohito Mikuma, Ayesh D. Perera, and Toshihiko Tsujii to some of the earlier phases of this work. We are most grateful to the Contraceptive Development Branch, NICHHD, and the NIDDK, through the National Hormone and Pituitary Program, University of Maryland School of Medicine, for the gifts of the assay reagents and GnRH used in this study. The authors acknowledge the expert technical assistance provided by Deborah Berger, Deborah Bolette, Amy Sartori, Joyce Sczcepanski, and the staff of the Primate and Assay Cores of the Center for Research in Reproductive Physiology, University of Pittsburgh School of Medicine.


   Footnotes
 
1 This work was supported by NIH Grants HD-08610, HD-16851 (to T.M.P.), and HD-19546 (to S.J.W.); NICHHD Contract (CD92–9); and the Andrew Mellon Foundation. Preliminary reports of this work were presented at the 74th and 75th Annual Meetings of The Endocrine Society, 1992 and 1993 (Abstracts 579 and 1832, respectively). It should be noted that in Abstract 1832, recombinant hCG is erroneously referred to as recombinant human LH. Back

2 Present address: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India. Back

Received October 7, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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