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Original Studies |
Medical Research Council Reproductive Biology Unit (H.M.F., A.S.M.), Centre for Reproductive Biology, Edinburgh EH3 9EW; and School of Biological and Molecular Sciences, Oxford Brookes University (N.P.G.), Oxford OX3 OBP, United Kingdom
Address all correspondence and requests for reprints to: Dr. H. M. Fraser, Medical Research Council Reproductive Biology Unit, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9EW, United Kingdom. E-mail: h.fraser{at}ed-rbu.mrc.ac.uk
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Abstract |
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Treatment with GnRH antagonist, starting on day 25 of the cycle, abolished the early follicular phase rise in FSH and the associated increase in inhibin B. The same treatment, followed by exogenous FSH, restored the secretion of inhibin B. Treatment with antiestrogen, commencing during the midfollicular phase, induced a supraphysiological rise in FSH, followed by a marked stimulation of inhibin B and estradiol secretion. Despite continued antiestrogen treatment, FSH secretion declined before peak values of inhibin B and estradiol were attained, implying a potential endocrine role for inhibin B, in addition to estradiol, in the negative feedback regulation of FSH. These results show that follicular phase FSH is the major stimulus for inhibin B secretion.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit precursors,
indicated that, in women (2) and nonhuman primates (3), immunoreactive
inhibin was highest during the luteal phase. This contradicted the
classical concept that inhibin was a product of the developing follicle
and played a negative feedback role, together with estradiol-17ß, in
suppressing FSH secretion during the mid- to late follicular phase.
However, with the development of enzyme-linked immunosorbent assays,
specific for dimeric inhibin A and inhibin B, it has been demonstrated
that, whereas the pattern of inhibin A secretion is in accord with the
previous, less specific assay, inhibin B peaks during the follicular
phase of the human menstrual cycle, giving support to a negative
feedback function during the follicular phase (4). A better understanding of the physiological relationships between FSH, inhibin B, and estradiol can be achieved by hormone manipulations using specific antagonists. Macaques seem to be suitable models, because the expression of inhibin and activin subunits in their ovaries is similar to that in the human (5, 6, 7, 8). The aim of the present report was to determine the pattern of inhibin A and inhibin B during the cycle of the stump-tailed macaque, a species we have used previously for studies on the endocrine role of inhibin (8, 9). Because this showed that inhibin B was highest during the early follicular phase, as in women, we went on to explore the effects of manipulating the intercycle rise and the midfollicular phase decline in FSH on inhibin B secretion. This was achieved by: 1) blocking the rise with GnRH antagonist treatment; 2) administering exogenous FSH in GnRH antagonist-treated animals; and 3) preventing the midfollicular phase decline in FSH by administering a specific antiestrogen, Faslodex. The latter experiment also addresses the extent of the negative feedback role of estradiol, in relation to inhibin B, at this time.
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Materials and Methods |
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Treatments
To investigate the effects of blocking the intercycle rise in FSH, GnRH antagonist treatment was initiated during the late luteal phase (day 25 of the cycle). Immediately before injection, the GnRH antagonist [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Pal(3)3,D-(Hci)6,Lys(iPr)8,D-Ala10]GnRH (Antarelix, Europeptides, Argenteuil, France) was dissolved in water containing 5% mannitol, to a concentration of 10 mg/mL. Four macaques were given three daily injections of the GnRH antagonist, administered sc in the thigh, at a dose of 1 mg/kg, as described previously (10). Blood samples were collected daily for 21 days from the start of treatment and 3 times per week thereafter, until the first posttreatment cycle.
To investigate the effects of adding back FSH during GnRH antagonist treatment, four animals commenced treatment with three daily injections of Antarelix on day 25 of the cycle, as described above. Recombinant FSH (Gonal-F, Serono, UK), 7.5 1U per dose, dissolved in sterile water, was administered by sc injection starting at 0900 h, 1 day after initiation of GnRH antagonist treatment and repeated at 1700 h for 5 days. The dose approximates 80 IU in women, equating to a low-dose protocol, and was chosen in an attempt to recreate the physiological profile of endogenous FSH at this time. Blood samples were collected as above.
To examine the effects of blocking estradiol feedback on FSH and inhibin secretion, four macaques in the midfollicular phase of the cycle (day 8) were treated with 0.2 mg/kg Faslodex (ZM182780) (Zeneca Pharmaceuticals, Macclesfield, UK), a steroidal pure antiestrogen (11), dissolved in propylene glycol, once daily for 3 days. Blood samples were collected at 0, 2, 4, 6, 8, and 12 h after the first injection, and daily for the next 10 days.
Assays
Serum inhibin A and inhibin B were measured using the two-site enzyme-linked immunoassays developed for human serum and validated in our laboratory for the macaque, as described previously (12). The assays used 50 µL serum in duplicate and had sensitivities of 4 ng/L for inhibin A and 16 ng/L for inhibin B. Estradiol-17ß and progesterone were measured by RIAs (13), detection limits being 30 pmol/L and 0.7 nmol/L, respectively. Macaque FSH was measured as described (14) using a heterologous RIA with a detection limit of 2 µg/L NICHHD cyn-FSH-RP1, and inter- and intraassay coefficients of variance of 15 and 11%, respectively. LH was measured by RIA, based upon recombinant cynomolgus monkey LH and supplied by the National Hormone and Pituitary Program, NIDDKD. A rabbit antiserum (AFP342994) was used as a final dilution of 1:750,000. Rec-mo LH-RP-1 (AFP-6936H) was used for radioiodination, as instructed, and results were expressed as µg/L of the same preparation. Assay sensitivity was 0.3 µg/L, and inter- and intraassay coefficients of variance were 12 and 7%, respectively. FSH concentrations, after administration of Gonal-F, were measured by a routine human FSH immunoradiometric assay (4), in which macaque FSH is not detected.
To obtain the profile of inhibin A and inhibin B throughout the normal cycle, daily serum samples from eight macaques with normal ovulatory cycles were measured; and the results were plotted, relative to the day of the midcycle LH surge.
Data for hormone concentrations were subjected to one-factor ANOVA for repeated measures. The individual means were further examined using the Newman-Keuls test for pairwise comparisons. Differences were considered significant at a level of P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In
contrast, serum concentrations of inhibin B were highest during the
early-to-midfollicular phase of the cycle, when estradiol was still low
(Fig. 1). Peak values were associated with the early follicular phase
FSH peak (Fig. 2), correlation
coefficient (r = 0.549) rising (to R = 0.82)
(P < 0.01), when the FSH values were shifted forward
by 1 day, implying that FSH was driving the rise in inhibin B.
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). This
was associated with a total absence of the normal follicular phase rise
in inhibin B and a suppression of serum estradiol concentrations. A
high dose of the GnRH antagonist was employed to help ensure that FSH
secretion would be suppressed, because FSH is generally less
susceptible to GnRH antagonist treatment than LH. This probably results
in depot formation and continued slow release of the compound.
Subsequently, the normal late follicular phase rise in estradiol, the
midcycle LH surge, and ovulation all failed to occur; and inhibin A and
inhibin B were maintained at basal levels for 27–36 days. Thereafter,
recovery was evident; and entering the data around the time of
posttreatment ovulation (which occurred 45 ± 4 days, mean ±
SEM, after start of treatment) also provides confirmation
of the association between the reinitiation of pituitary gonadotropin
secretion and the rise in inhibin B followed by the recovery of the
cyclic pattern of estradiol, progesterone, and inhibin A secretion
(Fig. 3).
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). Calculation of the cumulative serum
concentrations of human FSH (achieved during the treatment period in
each macaque), plotted against the cumulative inhibin B values obtained
over the same period, failed to show a significant correlation.
However, when the ages of the animals were taken into account, it was
found that the animal showing the least response was the oldest.
Removal of the data concerning this animal revealed a significant
correlation between the serum FSH concentrations attained and the
inhibin B produced. Inhibin A declined to basal levels and was
uninfluenced by the exogenous FSH. Estradiol secretion did not change
consistently over the treatment period. However, whereas GnRH
antagonist treatment alone resulted in estradiol levels falling to less
than 100 pmol/L (Fig. 3
), with exogenous FSH, values associated with
the late luteal/early follicular phase range (approximately 200 pmol/L)
were observed (Fig. 4).
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). The rise in serum concentrations of
FSH was rapid, being significantly above (P < 0.01)
pretreatment values at 8 h after the first injection. LH followed
a similar pattern, although the magnitude of the rise was different
from that of FSH; whereas the increase in FSH was twice that observed
for the early follicular or preovulatory peaks, that of LH was less
than half that observed during the preovulatory LH surge (compare Figs. 3 and 5). This gonadotropin rise was followed by a supraphysiological
increase in estradiol-17ß and inhibin B, values being significantly
elevated by 12 h and reaching a peak on the day after the third
injection and, like FSH, they were twice the magnitude of the peak
values seen in the normal cycle (Fig. 5). It was of interest that the
subsequent decline in FSH and LH took place as the treatment with
antiestrogen was continuing and the serum concentrations of inhibin B
and estradiol were continuing to rise, so that by treatment day 3, FSH
and LH concentrations had returned to baseline, whereas the rise in
inhibin B and estradiol was maximal. Despite the stimulation of
estradiol secretion, an LH surge and progesterone rise indicative of
ovulation did not follow. Instead, estradiol and inhibin B secretion
declined before rising again to normal preovulatory levels, so that
ovulation was delayed by an estimated 5–7 days. Inhibin A secretion
was not stimulated by antiestrogen treatment; all samples remained
below 20 ng/L (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Further evidence for the relationship between an elevation in follicular phase FSH secretion and a stimulation of inhibin B was provided by the demonstration that exogenous FSH, administered during GnRH antagonist treatment, resulted in a stimulation of inhibin B secretion, albeit at a variable magnitude between animals (17). The animal with the lowest response was the oldest of the group, and the follicles may have required a higher level of stimulation. Finally, the treatment with the antiestrogen showed that high concentrations of FSH can stimulate a marked increase in inhibin B in the mid- to late-follicular phase ovary, a time when inhibin B concentrations are normally beginning to decrease.
With respect to the source of the changes in inhibin B, we know that
the inhibin/activin ß-subunit is highly expressed in the granulosa
cells of developing follicles in the stump-tailed macaque, as in all
primate species reported (5, 6, 7, 8, 17). However, for synthesis of the
inhibin B dimer, the production of the -subunit within the follicle
is essential, and it is therefore -subunit production that is likely
to be the rate-limiting step for dimeric inhibin production (5, 6, 7).
GnRH antagonist treatment does not have acute suppressive effects upon
inhibin/activin ß-subunit messenger RNA (mRNA) in the primate ovary
(18). Thus, the varying patterns of inhibin B secretion, observed after
the hormone manipulations described, are likely to reflect the numbers
of follicles expressing the -subunit.
Although it is concluded from the results that the follicular phase rise in serum concentrations of inhibin B is dependent upon FSH secretion and that increasing the output of FSH leads to supraphysiological rises in inhibin B, the extent of the endocrine role of inhibin B during the follicular phase of the primate menstrual cycle is not yet clear (1). Administration of recombinant inhibin A, during the early follicular phase in the stump-tailed macaque, failed to suppress serum FSH concentrations (9). Treatment of rhesus monkeys, with a dose of inhibin that far exceeded physiological concentrations, did induce a reduction in FSH (18), giving support to the concept of a potential role in the negative feedback regulation of FSH. The potential of inhibin B to exert a negative feedback on FSH secretion may be implied from our observation that FSH subsequently declined during the antiestrogen treatment period, at a time when inhibin B and estradiol concentrations were at their peak. Assuming complete blockade of the estrogen receptor, it may be suggested that the decline in FSH is a result of negative feedback of inhibin B, although other explanations are possible. The negative feedback role of estradiol during the mid- to late follicular phase of the menstrual cycle is well established and is exemplified by the marked increase in FSH after competitive blockade of the estradiol receptor observed in the present study. On the basis of the temporal relationships between FSH, inhibin B, and estradiol during the early to midfollicular phase, it has been implied that inhibin B exerts a negative feedback effect on the rise in FSH before estradiol (4). Further studies, using hormone manipulations, should be useful in resolving this question.
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Acknowledgments |
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Received July 31, 1998.
Revised December 22, 1998.
Accepted January 4, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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