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Experimental Studies |
Department of Physiology, Morehouse School of Medicine (D.R.M., M.A.A.); Department of Psychology, Emory University (K.W.); and Yerkes Regional Primate Research Center (D.R.M., M.A.A., K.G.G., K.W.), Atlanta, Georgia 30310; the Medical Research Council Reproductive Biology Unit (I.S., A.S.M., H.M.F.), Edinburgh, Scotland; and Oxford Brookes University (N.P.G.), Oxford, United Kingdom
Address all correspondence and requests for reprints to: Dr. David R. Mann, Department of Physiology, Morehouse School of Medicine, 720 Westview Drive SW, Atlanta, Georgia 30310.
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Abstract |
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Introduction |
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There is no information available on changes in inhibin-B during development or after manipulation of pituitary function. The neonatal period is an active period of gonadotropin and testosterone secretion in male primates (5, 6, 7, 8, 9, 10). Blockade of neonatal activation of the pituitary-testicular axis with a GnRH agonist in the rhesus monkey (8, 11) or antagonist in the marmoset (9) delayed the onset of puberty in the former and reduced peripubertal changes in LH and testosterone secretion in both species. The neonatal period may also be an important interval in the determination of adult Sertoli cell number in the testis (12, 13). Sertoli cell number increases 5- to 6-fold during the neonatal period, although the overall increase in number of Sertoli cells during the neonatal-juvenile transition is substantially less than that between the juvenile period and adulthood in monkeys (14). No data are available on the impact of disrupting gonadotropin secretion during this period on Sertoli cell function and number in the primate. As ultimately the number of sperm produced is dependent on the number of Sertoli cells and their ability to function normally, it is important to establish the factors that may alter the ability of these cells to perform their critical role in the process of spermatogenesis.
The objectives of this study were to examine the effect of reversibly suppressing gonadotropin and testicular function during the neonatal period with a GnRH antagonist on circulating inhibin-B levels in the neonatal and pubertal male rhesus monkey. In addition, the effect of androgen replacement therapy on circulating inhibin-B levels was determined in these animals.
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Materials and Methods |
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Assays
Serum inhibin-B levels were measured as recently described (1, 2). The inhibin-B assay is a two-site enzyme-linked immunoassay that
uses a capture antibody directed against the C-terminal portion of the
human ß-subunit. The F(ab) fraction of a mouse monoclonal antibody
(Rl) to the N-terminal portion of the inhibin 
-subunit was
conjugated to alkaline phosphatase for detection purposes. The unknowns
were treated with SDS (Sigma Chemical Co., Poole, UK) as previously
described (1), diluted 1:5 with FCS, pretreated for 3 min at 100 C to
reduce nonspecific binding and catalase activity, and then treated for
30 min with hydrogen peroxide. Serial dilution (in FCS) of serum pools
from neonatal (2-month-old), juvenile (1-yr-old), and adult male rhesus
monkeys exhibited linearity with the human standard (dimeric inhibin
derived from human follicular fluid). Inhibin-B levels in the serum of
orchidectomized animals were undetectable; these levels were low, but
detectable (43 pg/mL), in the serum of a stalk-sectioned male monkey.
Random samples from each developmental group were also assayed for
inhibin A, which was undetectable (2). When 400 pg human inhibin-B
standard were spiked into castrate plasma, serially diluted, and
assayed, recovery ranged from 94–110%. The intra- and interassay
coefficients of variation were 4.1% and 10.5%, respectively.
Serum FSH levels were measured using a heterologous RIA as we have previously described (16, 17). Ovine FSH-I-I-NIH was used as tracer, and results were expressed in terms of the NICHHD cyn-FSH-RPI standard. All samples from the study were run in one assay. Levels of detection were 1.6 ng/mL, and the intraassay coefficient of variation was 15%.
Serum LH concentrations were measured by the mouse interstitial cell testosterone bioassay as we have previously described (8). Data are expressed in terms of the monkey pituitary WP-XV-20 standard. The minimum level of detection was 4 ng/mL. Intra- and interassay coefficients of variation were 4% and 13.7%, respectively.
Serum testosterone levels were measured by RIA using a commercial kit (Diagnostic Products Corp., Los Angeles, CA). The minimum level of detection in the testosterone assay was 0.2 ng/mL. The intra- and interassay coefficients of variation for the testosterone assay were 8.7% and 5.0%, respectively.
Statistics
Inhibin-B data for neonates (1 yr old or less) were assessed by ANOVA (vehicle x Ant x Ant/And) followed by the Newman-Keuls test for multiple comparisons. Inhibin-B data from peripubertal animals were partitioned into late prepubertal (27–36 months of age; corresponding to July through April of year 3), pubertal (39–48 months of age; July through April of year 4), and late pubertal periods (49–53 months of age; May through August of year 5) and assessed by repeated measure ANOVA (treatment x age) followed by Tukey’s test for multiple comparisons. The late prepubertal period was defined as the 12-month interval immediately preceding rapid pubertal testicular enlargement, the pubertal period as the period of rapid testicular enlargement (corresponding to the breeding season of year 4), and the late pubertal period corresponding to seasonal decline in testicular size and activity associated with the nonbreeding season. The impact of two covariates (matriline rank and whether animals became sexually mature during the period of observation) on the peripubertal inhibin-B data was also assessed using repeated measure analysis of covariance. Matriline rank is the rank of each subject in the social group based on the rank of the subject’s family. Animals were considered mature if they exhibited a pubertal rise in testicular size or serum testosterone and LH and/or if sperm was recoverable upon electroejaculation. Correlation coefficients between inhibin-B, testicular volume, and serum testosterone and LH were also determined (Pearson’s correlation coefficient) in the group of control animals (n = 5) that reached puberty during the breeding season of their fourth year of life.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Serum concentrations of inhibin-B were higher in controls than in
Ant- or Ant/And-treated animals at 2 (P = 0.029) and 6
months (P = 0.002) of age (Fig. 1
). At 6
months, but not at 2 months, of age, inhibin-B levels were lower in
Ant/And-treated than in Ant-treated animals. Testosterone
concentrations in Ant/And-treated animals remained elevated through 6
months of age, but by this time were less than levels of detection in
control and Ant-treated animals (15). There was no effect of treatment
on inhibin-B at 12 months of age. Serum FSH levels were higher in serum
pools from controls at 2 months of age (8 ± 2 ng/mL) than in Ant-
and Ant/And-treated animals (<1.6 ng/mL). At 6 and 12 months of age
(with the exception of one control pool), serum FSH was less than the
limit of detection.
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A major increase (P < 0.0001) in serum inhibin-B
levels occurred in all treatment groups between 27–36 months of age
(July through April of year 3; Fig. 2). Inhibin-B rose in
controls from 765 ± 103 to 1510 ± 204 pg/mL. At 33 and 36
months of age, inhibin-B levels were lower (P < 0.01)
in Ant/And-treated animals than in controls. Whether the animals were
destined to become sexually mature during the breeding season of their
fourth year had a significant effect (P = 0.04) on
inhibin-B values during this late prepubertal period (see Fig. 3), but matriline rank had no effect on inhibin-B.
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). During this period, five of
six controls compared to four of six Ant-treated and three of six
Ant/And-treated animals became sexually mature. There was a significant
effect of age (P = 0.038) and a significant interaction
(P = 0.013) of treatment and age on inhibin-B
concentrations over this period. Inhibin-B levels were higher in
controls than in Ant/And-treated animals at 45 months and in
Ant-treated animals at 48 months. Neither covariate (matriline rank or
whether the animals reached puberty during the period) had an effect on
inhibin-B. Between 49 and 52 months of age (nonbreeding season of year 5), inhibin-B in controls showed a small decline. During this period, treatment had no effect on inhibin-B. At 52 months of age, levels of inhibin-B in controls were still elevated above levels at 27 months of age (966 ± 79 vs. 765 ± 103 pg/mL).
Inhibin-B data were also partitioned across treatment groups according
to whether animals reached puberty during the sampling period. Of those
that became sexually mature, there was no effect of treatment on
inhibin-B levels. Figure 3 compares inhibin-B levels for all pubertal
and nonpubertal animals regardless of treatment. Inhibin-B levels rose
rapidly between 27 and 39 months of age in animals that were to reach
puberty, but then declined over the next 8 months in association with
the achievement of sexual maturation. Levels of inhibin-B in animals
that were not destined to reach puberty were lower than those in
animals that did reach puberty between 27 and 39 months of age, but
thereafter no differences were observed.
In 27- to 36-month-old controls destined to reach puberty during year
4, inhibin-B levels were positively correlated with age (r =
0.599; P = 0.005) and testicular volume (r =
0.5980; P = 0.005), but not with serum LH (r =
0.1701; P = 0.473) or testosterone (r = 0.235;
P = 0.318; Fig. 4, A–C). When these same
animals were between 33 and 52 months of age, inhibin-B was no longer
correlated with any of these parameters.
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2) than from treated
animals (n = 39; 19 from Ant-treated and 20 from Ant/And-treated
animals). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-inhibin). It
is likely that this apparent conflict between the current and the
earlier report resulted from assay differences, as in a recent
preliminary report (20) in which the same inhibin-B assay was used as
that in the current study, inhibin-B levels in juveniles (12–20 months
of age) did not differ from those in neonates. The elevated levels of inhibin-B in infants may not represent testosterone-induced Sertoli cell activity as has been proposed (19), but, possibly, FSH stimulation of Sertoli cell activity. Control infants had high levels of serum FSH relative to peripubertal animals, and Ant or Ant/And administration suppressed FSH to undetectable levels in 2-month-old animals. Associated with this decrease, inhibin-B levels in Ant- and Ant/And-treated animals at 2 and 6 months of age were lower than those in controls. Testosterone levels were 5- to 6-fold higher in Ant/And-treated animals than in controls (15), but they failed to normalize inhibin-B concentrations, suggesting that FSH is more likely than testosterone to be the driving force for the secretion of this hormone in the neonate. In fact, at 6 months of age, inhibin-B levels were actually lower in Ant/And-treated animals than in animals treated with Ant alone. Alternatively, while circulating levels of testosterone were high in Ant/And-treated infants, intratesticular levels of the hormone (that were not measured in the current study) may have been subnormal. Thus, androgen could conceivably be the driving force for Sertoli cell activity in the neonate.
Sexual maturation in male rhesus monkeys, raised and maintained in a large social group and exposed to external environmental conditions as in the current study, is influenced by matriline rank and season (11, 21, 22). Animals from lower ranking families tend to achieve sexual maturity later than those from higher ranking families, and the achievement of reproductive competence is limited to the breeding season of each calendar year. Most (80–90%) of control males reach puberty during the breeding season of their fourth year, but if for any reason this process is delayed, animals will not mature until the subsequent breeding season (11).
It was, therefore, necessary to consider both matriline rank and whether puberty was achieved as potential covariables in assessing the inhibin-B data. In the animals chosen for this study, matriline rank did not have a significant effect on inhibin-B levels in animals 27–53 months of age, but levels of the hormone were affected by whether puberty was reached during the sampling interval. During the late prepubertal period (27–36 months of age, corresponded to July through April of the animals’ third year of life) inhibin-B levels rose rapidly in all three treatment groups (including all animals sampled), but the rate and magnitude of the rise was lower in the Ant- and Ant/And-treated animals than in the controls. Most of this difference between the control group and the two treated groups probably resulted from the fact that more control animals were destined to become sexually mature during the subsequent breeding season. When the data were partitioned for only those animals that were destined to reach puberty their fourth year, there was no effect of treatment on inhibin-B concentrations during the late prepubertal period. In those animals that would achieve puberty, levels of inhibin-B rose more rapidly than those that would not between 27–39 months of age regardless of treatment group.
In animals 39–48 months of age, inhibin-B concentrations changed significantly with age, and the way they changed was affected by treatment. During the late prepubertal period, inhibin-B concentrations were generally higher in controls than in either Ant- or Ant/And-treated animals. During this period (encompassing the breeding season of the animals’ fourth year), the majority of the animals became sexually mature. As the breeding season continued, inhibin-B values declined gradually in all treatment groups in those animals that became sexually mature. Over this interval, inhibin-B was neither affected by matriline rank or pubertal status. There was a further decline in inhibin-B between 49–52 months of age in association with the nonbreeding season of the animals fifth year. However, levels of inhibin-B remained above levels that were observed at the beginning of the late prepubertal period.
During the late prepubertal period (27–36 months of age), inhibin-B
levels in the circulation were positively correlated with age and
testicular volume, but not with serum LH or testosterone. After this
period (39–52 months of age), inhibin-B concentrations were no longer
correlated with these other parameters. The temporal changes in
inhibin-B during peripubertal development in the present study are
similar to those reported earlier for -inhibin in the male rhesus
monkey (19), cynomolgus monkey (23), chimpanzee (24), and human (25).
Inhibin-A levels in the men and the male monkey have been reported to
be undetectable (2, 3, 20). If dimeric inhibin-A does not exist in
appreciable amounts in the male, then one might expect that an assay
specific for the -subunit of inhibin would show parallel changes
with an assay specific for the dimeric form of inhibin-B.
The rise of inhibin-B during the late prepubertal period appeared to occur earlier than the rise in LH, testosterone, and testicular volume. However, it is possible the changes in inhibin-B are associated with and being driven by the pubertal reactivation of the hypothalamic-pituitary-testicular axis. Only morning, not evening levels of serum LH, FSH, and testosterone were assessed in the present study. It is possible that increased nocturnal secretion of gonadotropin and testosterone may have occurred in concert with the rise of inhibin-B. This speculation is further supported by the observation that in male juvenile monkeys in which the pituitary-testicular axis is prematurely reawakened by chronic pulsatile administration of GnRH, inhibin levels in the circulation rose in association with increased secretion of FSH, LH, and testosterone (19). Alternatively, the rise in inhibin-B during the pubertal period may not be driven by gonadotropins, but, instead, by an additional, as yet unidentified, regulatory mechanism. The current data do not allow us to resolve this matter..
It was not possible to correlate FSH and inhibin-B levels in peripubertal animals in the current study, because macaque FSH RIAs are often insufficiently sensitive to reliably measure FSH in pubertal and adult animals. Only 19% of peripubertal samples had detectable levels of FSH in the current study, making it impossible to fully define the relationship between FSH and inhibin-B during this period.
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Acknowledgments |
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Footnotes |
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Received August 22, 1996.
Revised December 11, 1996.
Revised February 20, 1997.
Accepted March 6, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 125:250–257.[Abstract]
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