Inhibin and the regulation of testicular function. Historical and clinical aspects.
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Animals↗
Biomedical subjects
D M Robertson
Publications and source records attributed to D M Robertson.
The effects of exogenously administered testosterone on spermatogenesis in intact and hypophysectomized rats.
The effects of exogenously administered testosterone on the maintenance of spermatogenesis in intact and hypophysectomized rats were examined. Adult male rats were given Silastic implants containing testosterone in lengths ranging from 0.5-20 cm, and their effects on daily sperm production (DSP); serum FSH, LH, and testosterone; and interstitial fluid testosterone were determined in intact and hypophysectomized rats over a 13-week period. In intact rats, DSP levels were suppressed to 4-30% of control values at lower testosterone doses (2- to 6-cm implants), while DSP levels were partially maintained (65-93%) at higher doses (greater than or equal to 8 cm implants). Under these conditions LH levels were suppressed while FSH levels were reduced to 30-60% of control values. A steep testosterone-induced change in DSP levels was observed in both intact (2- to 3-fold) and hypophysectomized (18-fold) animals over a narrow testosterone dose range (implant lengths, 6-8 cm). Interstitial fluid testosterone levels associated with this change in DSP levels range from 6-8% of control values, while serum testosterone levels were elevated 1- to 2-fold above control values. A comparison of the testosterone implant lengths that caused a 50% change in DSP levels after 7 weeks of treatment was similar in intact and hypophysectomized rats. At high testosterone doses (greater than or equal to 10-cm implants), maximal DSP levels decreased 10% by 7 weeks and 35% by 13 weeks. DSP and serum testosterone levels were highly correlated (r = 0.54-0.83), while DSP and interstitial fluid testosterone showed a less correlation (r = 0.36-0.70) under the various experimental conditions examined. In conclusion, the testosterone-induced maintenance of DSP in rats is associated with several dose-related responses or events that appear to be differentially regulated. At low testosterone concentrations, DSP levels are partially maintained in intact rats but are totally suppressed in hypophysectomised rats, suggesting that a pituitary factor, probably FSH, has a potentiating effect at these testosterone concentrations. At intermediate testosterone doses resulting in the testosterone-induced maintenance of DSP, the similarity of dose-response curves (ED50 and maximum response) in intact and hypophysectomised rats would suggest that pituitary hormones are not required for this aspect of the process. The observation that the maintenance of DSP by testosterone under some experimental conditions is poorly correlated with interstitial fluid testosterone levels raises questions as to the mechanisms by which testosterone acts to stimulate spermatogenesis.
Animals↗
Highly polarized secretion of inhibin by Sertoli cells in vitro.
We have studied the regulation of inhibin secretion by rat Sertoli cells grown on extracellular matrix-impregnated porous filters in a twin chamber assembly. Previous studies have established that rat Sertoli cells cultured under these conditions reproduce the morphological and functional polarization observed in the Sertoli cell in situ. Sertoli cells isolated from 18- to 22-day-old Wistar rats were cultured for up to 8 days with daily changes of fully defined supplemented Eagle's Minimum Essential Medium (MEM). Rat inhibin was measured by RIA and pituitary cell bioassay, and transferrin by RIA. Inhibin measured by immunoassay or bioassay was always readily detectable in the upper, but not the lower, chamber. Inhibin secretion into the upper chamber exhibited a dose-dependent stimulation of up to 3.7-fold by ovine FSH, with a medium effective dose of 2.2 micrograms/liter and a constant bio- to immunoreactive ratio (3.6 +/- 0.4). Apically directed secretion accounted for over 80% of inhibit output under basal conditions and over 94% with FSH stimulation. Insulin also stimulated upper chamber inhibin secretion at a high dose (5 mg/liter) but not at lower doses or in conjunction with FSH exposure of Sertoli cells. Testosterone augmented FSH-induced stimulation of inhibin secretion, but was ineffective without FSH exposure. In contrast to inhibin secretion, for which FSH is the principal regulator, transferrin secretion by Sertoli cells is more evenly bidirectional (overall mean upper to lower chamber ratio of 1.5) and requires exposure to other stimuli (insulin, retinoic acid, and testosterone) in addition to FSH to achieve maximal secretion. Both submaximal and maximal FSH stimulation of inhibin output were augmented by a phosphodiesterase inhibitor, isobutylmethylxanthine, and these effects were fully reproduced by forskolin, which suggests the involvement of cAMP in the vectorial secretion of inhibin. The marked polarization of Sertoli cell inhibin secretion in vitro could not be explained by restricted transmembrane passage of inhibin. It is, therefore, suggested that the bulk of inhibin secretion by the immature rat Sertoli cell in vivo may be directed primarily into the seminiferous tubular lumen. Thus, in addition to its role in endocrine negative feedback signaling to the pituitary, inhibin may also have important functions in seminiferous tubular function and the support of spermatogenesis.
Animals↗
Isolation of inhibin alpha-subunit precursor proteins from bovine follicular fluid.
Two proteins with structural characteristics similar to peptide sequences identified in the inhibin alpha-subunit precursor sequence have been isolated from bovine follicular fluid. A side-fraction from the purification of bovine follicular fluid inhibin with high levels of inhibin immunoactivity relative to its inhibin bioactivity was fractionated through a sequence of procedures which included triazine dye affinity and phenyl-Sepharose chromatography, gel permeation chromatography on Sephadex G-100, reverse phase HPLC, and preparative polyacrylamide gel electrophoresis. The first of the two proteins identified had a molecular mass of 25-26K under reducing and nonreducing conditions and a NH2-terminal sequence identical to that of 43K inhibin alpha-subunit and showed minimal activity (less than 2% activity) compared with bovine 31K inhibin in either the inhibin in vitro bioassay or the RIA. These data suggest that this protein is the alpha 1-166 sequence of the bovine inhibin alpha-subunit (designated alpha N-subunit), most likely released after processing of either the inhibin alpha-subunit precursor or the 43K alpha-subunit involved in the conversion of 58K to 31K inhibin. The other protein identified (designated pro-alpha C-subunit) has a molecular mass of 27K under nonreducing conditions and 20K and 6K under reducing conditions. It is inactive in the in vitro bioassay, although highly reactive in the inhibin RIA, and has NH2-termini identical to the pro sequence of the inhibin alpha-subunit precursor and the 20K alpha-subunit sequence. These results suggest that pro-alpha C is a disulfide-linked structure and may represent an intermediate in the dimerisation of alpha- and beta-subunits to form inhibin while the alpha N-subunit is probably a proteolytic product of either the alpha-subunit precursor or 58K inhibin.
Animals↗
Effect of testosterone on serum immunoactive inhibin concentrations in intact and hypophysectomized male rats.
Intact and hypophysectomized rats were treated with graded doses of testosterone via subcutaneous Silastic implants over a 13-week period. Serum inhibin concentrations fell 50% (P less than 0.001) after 2 weeks of hypophysectomy, remaining suppressed at this level for 13 weeks. The administration of testosterone to hypophysectomized rats (serum testosterone values 2-12 ng/ml; control values 5.5 ng/ml) was without effect on serum inhibin values. In contrast, administration of testosterone to intact animals for 7 weeks resulted in an initial fall (P less than 0.05) in inhibin levels to 50-70% of controls then increasing to reach control levels at higher doses. Serum FSH concentrations were similarly biphasic with increasing dose of testosterone and values for these two hormones were significantly correlated (r = 0.44, P less than 0.01). Segments of seminiferous tubules in culture from rats after various times of hypophysectomy showed a partly suppressed secretion of inhibin. The administration of testosterone did not modify inhibin production although inhibin production was sensitive to FSH. It is concluded that (1) serum inhibin concentrations are partly suppressed after hypophysectomy and testosterone has no effect on serum inhibin values; and (2) the suppression of serum inhibin in intact rats treated with increasing doses of testosterone is attributable to the concomitant fall in serum FSH concentrations.
Animals↗
Effects of immunization against recombinant bovine inhibin alpha subunit on circulating concentrations of gonadotrophins in ewes.
Immunization of ewes against a pure recombinant preparation of the alpha subunit of bovine inhibin (alpha-bI) resulted in a three- to fourfold increase in ovulation rate, associated with antibodies in plasma recognizing pure native 31 kDa inhibin. The aim of this study was to examine the effects of this immunization on basal and GnRH-stimulated plasma concentrations of FSH and LH in ewes during the anoestrous and breeding seasons. The groups were untreated control ewes (n = 5), control ewes treated with keyhole limpet haemocyanin (KLH alone, n = 4), ewes treated with alpha-bI alone (n = 4) and alpha-bI-KLH conjugate-treated ewes (n = 3). There were no effects of immunization on basal FSH or LH in anoestrous ewes, despite the presence of antibodies recognizing 31 kDa inhibin. In the breeding season, immunization against alpha-bI resulted in increased basal (follicular phase, P less than 0.1; luteal phase P less than 0.05) and GnRH-stimulated (follicular phase only, P less than 0.001) release of FSH, but not LH. The data are compatible with the hypotheses that the increase in ovulation rate in immunized ewes is due to an increase in circulating FSH concentrations and that inhibin may only have a major peripheral influence on FSH in sheep during the breeding season.
Anestrus↗
Serum inhibin levels in normal men and men with testicular disorders.
Serum concentrations of inhibin, FSH and LH were measured in 39 normal men and 127 men with testicular disorders resulting in infertility. The infertile men were divided into groups on the basis of their mean sperm count, FSH levels and karyotype. The mean (+/- S.D) serum concentrations of inhibin in the normal men was 554 +/- 156 U/l and did not differ significantly from those groups with oligospermia, azoospermia or Klinefelter's syndrome. Combined analyses of all groups did not reveal any significant correlation between serum concentrations of inhibin and FSH or with any other parameter measured. Serum concentrations of FSH and LH were positively correlated, and Leydig cell dysfunction, as evidenced by increased serum LH levels, low testosterone levels or a declining testosterone/LH ratio were found with severe spermatogenic damage. The failure of serum concentrations of inhibin to correlate with those of FSH levels or the degree of testicular damage raise questions as to the clinical value of this parameter alone.
Adult↗
Immunoreactive inhibin concentrations in serum throughout the menstrual cycle of the macaque: suppression of inhibin during the luteal phase after treatment with an LHRH antagonist.
Concentrations of immunoreactive inhibin in serum samples collected daily from six adult stumptailed female macaques during normal menstrual cycles were measured with a heterologous radioimmunoassay. Serum inhibin concentrations were low during the follicular phase of the cycle. After ovulation they began to rise, reaching a plateau between 8 and 11 days, before falling in parallel with the decline in luteal progesterone secretion. The dependence of the inhibin secretion by the corpus lutem on pituitary gonadotrophins was investigated by the administration of an LHRH antagonist [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Trp3,D-hArg(Et2)6,D-Ala10 ]LHRH once daily for 3 days beginning on day 8 of the luteal phase in six macaques. LHRH antagonist treatment markedly suppressed serum levels of inhibin and progesterone and these remained at the level found in the follicular phase for the remainder of the luteal phase. These results show that inhibin in the macaque is secreted into the peripheral blood almost exclusively during the luteal phase, being highest when FSH is at its nadir. Suppression of serum inhibin concentrations during the luteal phase by LHRH antagonist suggests that its secretion is integrated with the LH control of the corpus luteum.
Animals↗
Radioimmunoassay of inhibin in the serum of male monkeys.
A heterologous inhibin radioimmunoassay method to measure inhibin in serum of male cynomolgus (Macaca fascicularis) and rhesus (Macaca mulatta) monkeys has been validated using a specific antibody against bovine 31 kDa inhibin and 125I-labelled 31 kDa inhibin as tracer. A serum pool from male monkeys was used as standard. Serial dilutions of normal monkey serum showed parallel logit-log dose-response curves to purified porcine and bovine inhibin as well as to a female human serum pool. The intra-assay coefficient of variation was 4.2% (n = 10) and the interassay coefficient of variation 5.1% (n = 10). No loss of inhibin immunoactivity was noted after storage at 23 degrees C for 5 days or repeated thawing and freezing of the serum samples. Serum from castrated monkeys showed undetectable levels of inhibin. Treatment with a gonadotrophin-releasing hormone agonist for 15 weeks led to a marked suppression of peripheral serum inhibin to concentrations similar to those after hypophysectomy or pituitary stalk section.
Animals↗
Effects of FSH and testosterone on highly purified rat Sertoli cells: inhibin alpha-subunit mRNA expression and inhibin secretion are enhanced by FSH but not by testosterone.
The effects of FSH and testosterone on inhibin mRNA expression and inhibin production by highly purified Sertoli cell preparations were examined. Sertoli cells were isolated from testes of 22-day-old rats by sequential trypsin, collagenase and hyaluronidase treatments, with subsequent osmotic shock treatment on day 3 of culture. Contamination by peritubular and germ cells was less than 0.5 and 1-3% respectively. Intracellular and secreted inhibin levels were measured by radioimmunoassay, using Sertoli cells which were incubated for 24 h in the absence or presence of FSH and testosterone from days 4 to 5 of culture. FSH stimulated the cellular inhibin content and the secreted inhibin level by four- and sevenfold respectively, with a half-maximal effective dose of 5-50 ng/ml. Under the present incubation conditions, testosterone (1 mumol/l) had no effect on immunoreactive inhibin levels in either the presence or absence of FSH. Similarly, the expression of inhibin alpha-subunit mRNA was increased following FSH stimulation, whereas testosterone had no effect. The expression of inhibin beta B-subunit mRNAs was not influenced by FSH or testosterone. It is concluded that highly purified Sertoli cell preparations, with a very low number of peritubular or germ cells, are fully responsive to FSH with respect to inhibin mRNA expression and inhibin production.
Animals↗
Threat to the New York City water supply--plutonium.
The mayor of the City of New York received an anonymous letter on April 1st 1985 threatening to contaminate the water supply with plutonium unless all criminal charges against Mr Bernhard Goetz, the suspect in a dramatic subway shooting incident, were dismissed by April 11th 1985. Local and Federal authorities were called upon to evaluate the credibility of the threat and to institute a "round the clock" monitoring program by New York City personnel. The Environmental Measurements Laboratory, EML, was requested by the City to analyse a composite, large volume (approximately 175 litres) drinking water sample collected by City personnel on April 16th 1985. The concentration measured was 21 fCi/l which was a factor of 100 greater than previously observed results in our data base, and the mass isotopic content of the plutonium was very unusual. Additional samples were collected one to three months later at various distribution points in the water supply system. The plutonium concentrations were much lower and comparable to EML's earlier data. Mass isotopic analysis of these samples provided more reasonable compositions but with high uncertainties due to very low plutonium concentration. Recent measurements of large volume samples, approximately 1000 litres, collected in the Fall of 1985 from the New York City and New Jersey water supplies showed identical plutonium concentrations of 0.05 fCi/l. Mass isotopic analyses indicated similar 240Pu/239Pu ratios which were slightly lower than global fallout estimates. Due to our inability to confirm the elevated plutonium concentration value for the composite sample of April 16th 1985, it is impossible to conclude whether the threat to contaminate the New York City water supply was actually carried out or whether the sample was contaminated prior to receipt at EML.
Crime↗
Radioimmunoassay of rat serum inhibin: changes after PMSG stimulation and gonadectomy.
A heterologous inhibin radioimmunoassay (RIA) method has been developed which is highly specific and of sufficient sensitivity to detect inhibin in female and male rat serum. Purified bovine 31 kDa inhibin was used in the generation of the antiserum and following iodination as tracer in the assay. Parallel logit-log dose-response lines were observed between a rat ovarian extract used as standard and serial dilutions of female and male serum and testicular interstitial fluid. The within-assay variation based on index of precision was 0.049 (n = 5) and the between-assay variation (n = 4) was 9.8%. The specificity of the assay was assessed from: (a) the failure of a number of structurally related proteins (activin-A, transforming growth factor-beta (TGF-beta), Müllerian inhibitory substance) as well as inhibin subunits to crossreact (less than 0.5%) in the assay relative to bovine 31 kDa inhibin; (b) nondetectable levels of immunoactivity in the serum of gonadectomised rats; and (c) a close correlation (r = 0.96) between serum levels of in vitro biological and immunological activities from rats following pregnant mare serum gonadotropin (PMSG) stimulation. Similar initial t 1/2 values (14-15 min) of serum inhibin following gonadectomy were obtained in both sexes. This RIA method will be useful in the study of the physiology of inhibin in the female and male rat.
Animals↗
Effects of FSH and IGF-I on immature rat Sertoli cells: inhibin alpha- and beta-subunit mRNA levels and inhibin secretion.
Effects of follicle-stimulating hormone (FSH) and insulin-like growth factor-I (IGF-I) on inhibin production by cultured Sertoli cells from 21- to 23-day-old rats were studied. The expression of inhibin alpha- and beta-subunit mRNAs, and inhibin immunoreactivity and in vitro bioactivity were estimated. Using a cDNA probe corresponding to the alpha-subunit of bovine inhibin, specific hybridization with a 1.5-1.7 kilobase (kb)mRNA species was observed. Addition of FSH to the cultured Sertoli cells for 24 h markedly increased the level of this mRNA in a dose-dependent way. IGF-I had no effect on the intensity of the hybridization. Using a cDNA probe corresponding to the beta B-subunit of human inhibin, 3.5 and 4.2 kb mRNA species were detected. FSH and IGF-I had no effect on the hybridization signal. No hybridization was observed with a cDNA probe corresponding to the beta A bovine inhibin subunit. Inhibin activity was detected in cells and medium by immunoassay, and in the medium by in vitro bioassay. FSH stimulated both immunoreactivity and in vitro bioactivity, whereas IGF-I had no effect at all. The present effect of FSH on inhibin alpha-subunit mRNA expression in cultured Sertoli cells indicates that regulation of inhibin production by FSH includes an effect at the transcriptional level. However, this does not exclude additional translational and posttranslational effects.
Animals↗
Advances in the physiology of inhibin and inhibin-related peptides.
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Animals↗
Inhibin: circulating levels in women during ovulation induction and detection in human placenta by specific radioimmunoassay.
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Animals↗
Effects of 31 kilodalton bovine inhibin on follicle-stimulating hormone and luteinizing hormone in rat pituitary cells in vitro: actions under basal conditions.
The effects of purified 31 kilodalton (kDa) bovine inhibin on the basal release and cell contents of FSH and LH in pituitary cells from adult male Sprague-Dawley rats have been investigated for periods up to 28 days in tissue culture. A constant rate of basal FSH release (1.90 +/- 0.24 ng/10(6) cells.h) was observed during the first 10 days of culture and this release was blocked by inhibin in a concentration-dependent manner (IC50 = 0.16 U/ml, IC100 greater than 2.5 U/ml). The constant basal LH release between days 2 and 12 of culture (0.15 +/- 0.02 ng/10(6) cells.h) was also blocked by inhibin in a concentration-dependent manner to a minimum of 25% of control (IC50 = 0.66 U/ml, ICmax greater than 6 U/ml). FSH and LH cell contents decreased exponentially in the presence and absence of inhibin over the first 20 days of culture permitting the calculation of decay half-times (t1/2). Inhibin reduced the FSH cell content t1/2 from 5.0 to 1.8 days and the LH cell content t1/2 from 5.8 to 3.6 days (IC50 = 0.81-0.83 U/ml, ICmax greater than 6 U/ml for both FSH and LH). The sum of the FSH released and FSH cell content (i.e. total FSH) increased with time in culture and this increase was blocked by inhibin. In contrast, total LH decreased with time in all cultures. The data suggest that in pituitary cells separated from hypothalamic and gonadal inputs 1) inhibin primarily suppresses tonic FSH synthesis coupled to its basal release; 2) at higher concentrations inhibin partially suppresses basal LH release and promotes intracellular degradation of FSH and LH. Thus, based on differences in sensitivity, inhibin acts at two or more sites, one associated with FSH tonic synthesis-basal release and the other(s) with FSH/LH mobilization and/or degradation.
Animals↗
Pregnant mare's serum gonadotropin stimulates inhibin subunit gene expression in the immature rat ovary: dose response characteristics and relationships to serum gonadotropins, inhibin, and ovarian steroid content.
We have investigated the temporal changes and dose responses of ovarian inhibin gene expression, circulating inhibin, and ovarian estradiol content in PMSG-primed immature female rats. The relative levels of ovarian inhibin alpha- and beta A-subunit messenger RNA (mRNA), serum inhibin, and ovarian steroid content were measured after stimulation of immature female rats with 20 IU PMSG. Each of these variables rose in parallel and peaked at 48 h (relative ovarian inhibin alpha- and beta A-subunit mRNA increased 3.8-fold and 2.8-fold, respectively, serum inhibin 269 U/ml, vs. control 31, P less than 0.01, ovarian estradiol content 114 pmol/pair of ovaries vs. controls 4.7 P less than 0.001), corresponding to the time of maximal follicular development in this animal model. Subsequently each fell to reach a nadir at 96 h. The data are consistent with the hypothesis that PMSG stimulation of inhibin secretion occurs by a mechanism involving inhibin gene transcription. The increase in ovarian inhibin and estradiol synthesis during folliculogenesis and their simultaneous decline before ovulation suggest that 1) the circulating levels of inhibin and estradiol reflect follicular maturation and 2) these two hormones are regulated via a common mechanism during follicular development in the rat.
Animals↗
Comparison of inhibin immunological and in vitro biological activities in human serum.
A comparison of serum inhibin levels in men and women was undertaken using a sensitive sheep pituitary cell in vitro bioassay and a newly developed heterologous RIA. The RIA was based on an antiserum raised to bovine 31K inhibin using [125I]31K inhibin as tracer. Bovine inhibin alpha- and beta-subunits, bovine activin-A, transforming growth factor-beta, and Mullerian inhibitory substance did not cross-react in the RIA. In both assays, dilutions of serum gave response lines parallel to that of the partially purified human follicular fluid inhibin preparation used as standard. Negligible levels of both bio (B)- and immuno (I) activities were found in serum from women with premature ovarian failure or castrated men. In ovulation-induced cycles, serum B inhibin levels increased progressively from the early to the late follicular phase and remained at the late follicular phase level during the early and midluteal phases. Serum I inhibin levels also rose during the follicular phase, but declined during the early luteal phase before increasing again in the midluteal phase. As a consequence, inhibin B:I ratios varied during the treatment cycle, with high ratios in early follicular (2.86) and early luteal (2.25) phases and a low ratio in the midluteal phase (1.09). Similar changes in serum B:I ratios also occurred during the midcycle and midluteal phases of normal cycles. The B:I ratio was lower (0.35) in normal men. We conclude that the largely similar pattern of inhibin biological and immunological activities in serum obtained during a variety of physiological conditions support the validity of the RIA procedure, and the B:I ratio of serum inhibin varies during the follicular and luteal phases of the cycle and is low in men. Potential reasons for these changes in B:I ratio include the presence of interfering substances in either the bioassay or the RIA, the presence of inhibin isoforms, and/or modulation of secreted forms by sex steroids.
Adult↗