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Experimental Studies |
Departments of Cell Biology and Physiology (T.M.P., S.R.) and Medicine (S.J.W.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; Reproductive Sciences Program (D.S.McC., A.R.M.), University of Michigan, Ann Arbor, Michigan 48109; Oxford Brooks University (N.G.), Oxford, OX3 0BP, United Kingdom; and the Medical Research Council Reproductive Biology Unit (A.S.McN.), University of Edinburgh, 37 Chalmer Street, Edinburgh EH3 9EW, United Kingdom
Address all correspondence and requests for reprints to: Tony M. Plant, Departments of Cell Biology and Physiology, University of Pittsburg School of Medicine, S330 Biomedical Science Tower, 3500 Terrace Street, Pittsburg, Pennsylvania, 15261.
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Introduction |
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Although immunoactive inhibin concentrations, equivalent in potency to approximately 2 ng/mL of rh-inhibin A, may be measured in peripheral serum of adult male rhesus monkeys (5, 6), the precise form of this circulating testicular hormone in the macaque remains to be determined. The purpose of the present study was to address this issue. To this end, we capitalized on recently developed immunoradiometric assays for the measurement of inhibin A and B (7, 8, 9). These were applied to the monkey to determine the concentrations of these two forms of the glycoprotein hormone in the circulation of intact and castrated adult males. Circulating concentrations of dimeric inhibin were also described in neonatal and juvenile monkeys. In addition, inhibin B levels were determined in testicular venous blood from adults. To more fully place into physiological perspective our previous finding that replacement with rh-inhibin A maintains FSH secretion at intact control levels following castration in the hypophysiotropically clamped monkey (2), circulating concentrations of this form of dimeric inhibin achieved by the iv infusion of the recombinant hormone were measured.
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Materials and Methods |
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Ten neonatal (5–40 days of age; 0.3–0.6 kg BW), 12 prepubertal
(12–27 months of age; 1.2–3.1 kg BW) and 19 adult (>5 yr of age;
8.2–13.2 kg BW) intact and 14 previously castrated male rhesus monkeys
(Macaca mulatta) were used in this study. Eleven of the
gonadectomized males were >4 yr of age (5.1–11.5 kg BW), whereas the
remaining 3 were orchidectomized at 1 week of age and were studied as
neonates (
4 weeks of age, 0.5–0.7 kg BW) and as juveniles (7–13
months, 1.7–2.3 kg BW). Animals were maintained in accordance with the
NIH Guides for the Care and Use of Laboratory Animals. Adult monkeys
and some juveniles were housed in individual cages, and neonates were
maintained with their mothers under a controlled photoperiod (lights
on, 0600–1800 h). The remaining juvenile monkeys were housed in a
group setting in which the artificial photoperiod was supplemented with
natural daylight.
Collection of blood
Peripheral serum samples were generally collected by femoral
venepuncture. For this purpose, juvenile and adult animals were first
sedated with ketamine hydrochloride (50 mg/mL im; Ketaject, Ketamine
Hydrochloride, Phoenix Scientific, St. Joseph, MO). Blood was taken
from infant monkeys without sedation. On occasion, samples were
collected from animals bearing chronic indwelling venous catheters and
housed in remote sampling cages that allow continuous access to the
venous circulation. Testicular venous blood was collected from adult
monkeys under anesthesia with sodium pentobarbital (25 mg/kg BW iv;
Nembutal sodium solution, Abbott Laboratories, North Chicago, IL). All
samples were collected during the day and sera were stored at -20
C.
rh-inhibin A infusions
In a previous study (2), male rhesus monkeys were infused with rh-inhibin A (832 ng/h/kg BW; Biotech Australia Pty. Limited, Roseville, NSW, Australia) for 4 days following castration. At the time, blood was collected to monitor circulating FSH and immunoactive inhibin levels. In the present study, we measured inhibin A concentrations in some of these samples, which had been collected from six animals on day 2 or 3 of the infusion and had been stored at -20 C for approximately 3 yr.
Assays
Inhibin A.
Dimeric inhibin A was measured by a two-site
enzyme linked immunosorbent assay (ELISA) described in detail
previously (7) or by a two-site solid phase immunochemiluminiscent
assay (SPICA) (8). Both assays use rh-inhibin A (Genentech, South San
Francisco, CA) as standard and the same two monoclonal antibodies;
clone R1, directed towards the inhibin 
subunit (10), and clone E4,
directed towards the inhibin ßA subunit (11). The SPICA assay format
consists of the anti--inhibin monoclonal detection antibody
conjugated to an acridinium ester (anti--INH-DMAE) and the
anti-ßA-inhibin monoclonal capture antibody conjugated to
superparamagnetic particles (anti-ßA-INH-PMP) to effect separation.
Peptides related to inhibin such as activin-A and transforming growth
factor-ß and its binding proteins, -2 macroglobulin and
follistatin, showed very little cross-reactivity or interference in
this assay. The sensitivity and the intra- and interassay coefficients
of variation of the inhibin A SPICA assay were, on average, 10 pg/mL
and 6% and 12%, respectively. A pool of serum from castrated male
monkeys was spiked with rh-inhibin A (Biotech Australia Pty. Limited)
at three concentrations (250, 500, and 1000 pg/mL). The mean recovery
of inhibin A from this pool was 91 ± 11%. The ELISA assay had a
sensitivity of 7 pg/mL, and the coefficients of variation were <5%
within and <7% between plates. In the ELISA, serum samples ran
parallel to the rh-inhibin A standard (Genentech), and the standard was
completely recovered when spiked into castrate male serum
(recovery = 97 ± 3%, n = 12). Values reported for the
SPICA are means of triplicate determinations, whereas those for the
ELISA are from duplicate determinations.
Inhibin B. Inhibin B was measured in a two-site ELISA described in detail previously (9). A monoclonal antibody specific to the ßB subunit (C5) was used for capture, and the same F(ab) fraction of a mouse monoclonal antibody (R1) conjugated to alkaline phosphatase used in the inhibin A ELISA was used for detection. Serum samples from male monkeys diluted in parallel to the rh-inhibin B standard (Genentech) and the standard was completely recovered when spiked into castrate male serum (recovery = 98 ± 2%, n = 10). The assay had a sensitivity of 8 pg/mL, and the coefficients of variation were <5% within and <7% between plates.
Immunoactive inhibin.
Immunoactive inhibin concentrations
were measured as described previously (6) by a double antibody RIA,
using rh-inhibin A (Genentech) for standard (0.03–0.3 ng/tube),
purified bovine inhibin as the iodinated tracer, and an antiserum to
bovine 31-kilodalton inhibin (no. 1989) obtained from Dr. David
Robertson through the Contraceptive Development Branch, National
Institute of Child Health and Human Development, National Institutes of
Health. This antiserum recognizes the inhibin subunit in addition
to dimeric hormone. The minimum detectable dose was 0.03 ng. Inhibin
was undetectable in samples from castrated adult monkeys. The
intraassay coefficient of variation in the midpoint of the standard
curve was 7%. The interassay coefficients of variation of samples of
various potencies ranged from 7.5–12%.
Testosterone. Circulating testosterone concentrations were assayed in ether extracts of sera without chromatography by a previously described RIA (12) that employs antiserum T3-125 (Endocrine Science, Tarzana, CA). The sensitivity was 0.1 ng/mL and the intra- and interassay coefficients of variation were <13% and 13%, respectively.
Numerical analysis
Hormone concentrations below the limit of assay detectability were assigned a concentration equivalent to the minimum detectable value in the respective assay. Significance of differences between mean concentrations were determined by one-way ANOVA followed by Fisher PLSD test using StatView II program.
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Results |
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. In intact adults, serum concentrations of inhibin B
ranged from 304-3330 pg/mL, with a mean (± SE) of
1008 ± 184 pg/mL. In contrast, circulating inhibin A
concentrations, as determined by both ELISA and SPICA, were very low
(46 pg/mL or less). Both forms of dimeric inhibin were generally
undetectable in sera from castrated animals.
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). Interestingly, inhibin B
concentrations in juveniles were approximately 40% greater than those
in infants, although ANOVA failed to reveal significance in this
difference when all three developmental stages were analyzed (Table 2).
As in adult castrate animals, inhibin B was generally undetectable in
serum from agonadal neonates (range, <8–16.3 pg/mL) and juveniles
(range, <8–13.3 pg/mL). Circulating inhibin A concentrations in
neonatal and juvenile monkeys were undetectable (<7 pg/mL) by
ELISA.
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).
The mean concentration of inhibin B in testicular venous blood was
1057 ± 223 pg/mL (range 524-1861 pg/mL, n = 5). In three of
the five animals in which testicular venous blood was obtained, inhibin
B concentrations were also determined in peripheral blood collected at
the time of testicular venous sampling. In these three monkeys, the
ratio of inhibin B concentration in spermatic vein to that in
peripheral vein was 1.1:1, 1.5:1, and 1.8:1, respectively. Analogous
ratios for immunoactive inhibin in these three animals were 5.2:1,
4.6:1, and 1.6:1, respectively (Table 3). Corresponding values for
testosterone were 18.7:1, 36.9:1, and 7.0:1, respectively (Table 3).
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Discussion |
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1000 pg/mL) are severalfold greater than those in man. This
quantitative species difference is paralleled by immunoactive inhibin
concentrations (6) and may reflect, in part, the relative size of the
testis in the rhesus monkey (2.5 g/kg BW) vs. humans (0.3
g/kg BW).
That the testes are the source of inhibin B in the monkey is evident
from the finding that circulating concentrations of this dimeric
inhibin are undetectable in castrated males, as previously described in
orchidectomized men (14). Inhibin - and ßB-mRNAs are expressed in
the primate testis (16, 17), and in the rodent testis these mRNAs, as
well as their corresponding peptides, have been demonstrated (18, 19, 20).
The finding that serum inhibin B levels are low in men with impaired
Sertoli cell function (14) suggests that this cell is probably the
major site of testicular inhibin B production. In this regard, inhibin
B has been isolated from conditioned media of cultured rat Sertoli
cells (21), and in the monkey, inhibin bioactivity has been detected in
such media (22). Moreover, an immunocytochemical investigation
utilizing antisera to the inhibin and ß subunits provided results
consistent with the notion that the primate Sertoli cell produces
inhibin B (23). In addition, stimulation of Sertoli cell activity by
administration of rh-FSH to normal men results in a significant
increase in circulating inhibin B levels (14). The relatively low
levels of circulating inhibin A observed in the present study are
probably also of testicular origin, because concentrations of this
dimer are reduced in blood collected from castrates. The mRNA that
encodes the ßA subunit is expressed in the testis of the closely
related cynomolgus monkey (16).
The foregoing considerations may now be incorporated into the concept that the testicular regulation of FSH secretion in primates is governed by a control system consistent with that described by the classical inhibin hypothesis (24). A pivotal line of evidence for this view is the finding that, in the hypophysiotropically clamped monkey, initiation of an iv infusion of rh-inhibin A immediately following orchidectomy maintains FSH synthesis and secretion at precastration control levels (2). In addition to the difference in the molecular form of native monkey inhibin on the one hand, and that of the recombinant hormone used for replacement on the other, the infusion of rh-inhibin A produced circulating immunoactive inhibin concentrations equivalent in potency to approximately 2000 pg/mL of rh-inhibin A (2). Interestingly, in the present study, the inhibin A ELISA revealed that the concentration of this inhibin dimer in a selection of some of these same samples was 4- to 5-fold greater than that determined earlier by RIA. Thus, it appears that the concentrations of circulating inhibin A achieved in castrated animals by the replacement infusion in our earlier study were probably greater than those of endogenous inhibin B in intact adult males. Therefore, in contrast to our original position (2), the rh-inhibin A infusion used in our earlier replacement study may not have been entirely physiological. In addition, the explanation for the different estimates of potency of circulating rh-inhibin A in the ELISA and RIA remains to be established.
In the present study, inhibin B concentrations in testicular venous blood were similar to those observed in the peripheral circulation. Because the testicular/peripheral vein ratio of immunoactive inhibin concentrations in the same samples ranged from 2:1–5:1, different forms of inhibin may not utilize the same route to gain access to the peripheral circulation. Certainly, the very high testicular/peripheral vein ratio for testosterone (7:1–37:1) observed in the present study would suggest that the lymphatic route is important for inhibin access to the peripheral circulation; a notion that we have proposed previously (6). Although information on the testicular/peripheral vein ratio of inhibin B is not available for other species, analogous ratios for immunoactive inhibin concentrations of 2.5:1 and 10:1 have been reported for rat and human, respectively (25, 26, 27).
The present finding that circulating inhibin B concentrations were approximately 40% greater in juveniles than those in infants, which has been recently confirmed in additional animals (28), was unexpected, because the juvenile phase of development in the monkey and other higher primates is characterized by marked hypogonadotropism (29). It had been anticipated that developmental changes in circulating inhibin B levels, like those previously established for immunoactive inhibin (30) and testosterone (31), would parallel the changes in gonadotropin drive to the testis, namely elevations in infancy and adulthood and a reduction in the juvenile (29). This prediction proved to be incorrect. Therefore, although FSH stimulates inhibin B production in normal men (14), the present results indicate that secretion of this form of inhibin during the juvenile phase of development in the monkey occurs in the presence of little or no gonadotropin stimulation. A relative gonadotropin-independent mode of inhibin B secretion has also recently been reported in men with gonadotropin-releasing hormone deficiency (15). It is therefore of interest to note that substantial production of immunoactive 31-kilodalton inhibin by cultures of juvenile monkey Sertoli cells is observed under basal conditions (32). Although the cell biology of inhibin B production during the hypogonadotropic phase of prepubertal development is unclear, the sustained circulating levels of this dimer in the juvenile monkey are probably related to the 6-fold increase in Sertoli cell number that occurs during this developmental stage (33).
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Acknowledgments |
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Footnotes |
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Received December 16, 1996.
Revised March 25, 1997.
Revised April 18, 1997.
Accepted April 28, 1997.
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and ßB subunit messenger RNAs. J Urol. 150:1007–1009.[Medline]
, ßA, and ßB subunits in
various tissues predicts diverse functions. Proc Natl Acad Sci USA. 85:247–251.-subunit mRNA levels by specific germ cell types. Mol Cell
Endocrinol. 72:13–22.[CrossRef][Medline]
ß-B dimer. Mol Cell Endocrinol. 70:109–116.[CrossRef][Medline]
-C in the plasma of neonatal, juvenile and adult
male rhesus monkeys. Presented at the 79th Annual Meeting of The
Endocrine Society, Minneapolis, MN, June, 1997 (abstract P2-337).
concentrations in infant, prepubertal, and
adult male rhesus monkeys (Macaca mulatta) and in juvenile
males during premature initiation of puberty with pulsatile
gonadotropin releasing hormone treatment. Endocrinology. 125:250–256.This article has been cited by other articles:
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  D. R. Simorangkir, G. R. Marshall, J. Ehmcke, S. Schlatt, and T. M. Plant Prepubertal Expansion of Dark and Pale Type A Spermatogonia in the Rhesus Monkey (Macaca mulatta) Results from Proliferation During Infantile and Juvenile Development in a Relatively Gonadotropin Independent Manner Biol Reprod, December 1, 2005; 73(6): 1109 - 1115. [Abstract] [Full Text] [PDF]
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S. J. Winters, S. Kawakami, A. Sahu, and T. M. Plant Pituitary Follistatin and Activin Gene Expression, and the Testicular Regulation of FSH in the Adult Rhesus Monkey (Macaca mulatta) Endocrinology, July 1, 2001; 142(7): 2874 - 2878. [Abstract] [Full Text] [PDF]
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S. C. Chapman and T. K. Woodruff Modulation of Activin Signal Transduction by Inhibin B and Inhibin-Binding Protein (InhBP) Mol. Endocrinol., April 1, 2001; 15(4): 668 - 679. [Abstract] [Full Text]
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A.J. Tilbrook and I.J. Clarke Negative Feedback Regulation of the Secretion and Actions of Gonadotropin-Releasing Hormone in Males Biol Reprod, March 1, 2001; 64(3): 735 - 742. [Abstract] [Full Text]
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F. J. Hayes, S. DeCruz, S. B. Seminara, P. A. Boepple, and W. F. Crowley Jr. Differential Regulation of Gonadotropin Secretion by Testosterone in the Human Male: Absence of a Negative Feedback Effect of Testosterone on Follicle-Stimulating Hormone Secretion J. Clin. Endocrinol. Metab., January 1, 2001; 86(1): 53 - 58. [Abstract] [Full Text]
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F. J. Hayes, S. B. Seminara, S. DeCruz, P. A. Boepple, and W. F. Crowley Jr. Aromatase Inhibition in the Human Male Reveals a Hypothalamic Site of Estrogen Feedback J. Clin. Endocrinol. Metab., September 1, 2000; 85(9): 3027 - 3035. [Abstract] [Full Text]
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S. J. Winters and T. M. Plant Partial Characterization of Circulating Inhibin-B and Pro-{alpha}C During Development in the Male Rhesus Monkey Endocrinology, December 1, 1999; 140(12): 5497 - 5504. [Abstract] [Full Text]
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S. Ramaswamy, C. R. Pohl, A. S. McNeilly, S. J. Winters, and T. M. Plant The Time Course of Follicle-Stimulating Hormone Suppression by Recombinant Human Inhibin A in the Adult Male Rhesus Monkey (Macaca mulatta) Endocrinology, August 1, 1998; 139(8): 3409 - 3415. [Abstract] [Full Text] [PDF]
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F. J. Hayes, J. E. Hall, P. A. Boepple, and W. F. Crowley Jr. Differential Control of Gonadotropin Secretion in the Human: Endocrine Role of Inhibin J. Clin. Endocrinol. Metab., June 1, 1998; 83(6): 1835 - 1841. [Full Text]
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A.-M. Andersson, J. Toppari, A.-M. Haavisto, J. H. Petersen, T. Simell, O. Simell, and N. E. Skakkebæk Longitudinal Reproductive Hormone Profiles in Infants: Peak of Inhibin B Levels in Infant Boys Exceeds Levels in Adult Men J. Clin. Endocrinol. Metab., February 1, 1998; 83(2): 675 - 681. [Abstract] [Full Text]
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