Immunoneutralization Of Endogenous Inhibin Research Articles

Induction of Superovulation by Immunoneutralization of Endogenous Inhibin in Immature Rats

The effects of passive immunoneutralization of endogenous inhibin on ovulation rate in immature rats were investigated. Efficiency of superovulation on production of fertilized oocytes was compared between the inhibin antiserum (inhibin-AS) and equine chorionic gonadotropin (eCG) protocols. Immature female Wistar strain rats were superovulated with a single injection of 100-200 microl inhibin-AS, with and without an injection of human chorionic gonadotropin (hCG). A total of 77.8% of the 26-30-day-old rats treated with a single injection of 100-200 microl inhibin-AS ovulated 72 h after treatment, while rats given normal goat serum (NGS; 200 microl) did not ovulate. At 28 days of age, all of the inhibin-AS treated rats ovulated when additional hCG treatment was given, whereas the number of ovulated oocytes was not affected. The number of ovulated oocytes in the inhibin-AS-hCG treated groups was significantly higher than that of the NGS-hCG treated group. In addition, plasma concentrations of FSH in the inhibin-AS-hCG treated group significantly increased compared with the NGS treated group. While the percentage of mated rats in the 200 microl inhibin-AS-hCG treated group was significantly lower than that of the 15 IU eCG-hCG treated group, the fertilization rate was comparable between the two groups. The number of fertilized oocytes in the 200 microl inhibin-AS-hCG treated group was significantly higher in comparison with the 15 IU eCG-hCG treated group. These results suggest that immunoneutralization of endogenous inhibin could be a reliable method for induction of superovulation to collect a large number of normally fertilized oocytes in immature rats.

Immunization against endogenous inhibin increases normal oocyte/embryo production in adult mice.

The objectives of the present study were to induce super-ovulation in adult mice by immunoneutralization of endogenous inhibin and to investigate embryo development in vivo and in vitro. Adult female mice of the ddY strain at 3 mo of age were superovulated with a single ip injection of inhibin antiserum (inhibin-AS; 50, 100, or 200 micro L) at 12:00 h on metestrus of the 4-d estrous cycle. The control group was treated with 100 micro L normal goat serum (control serum). Five mice were sacrificed every 6 h and blood samples were collected for hormonal assay. Individual female mice were bred in the afternoon of proestrus with individual fertile males. Embryos were collected at 08.00 h on d 2 post-coitus and cultured in KSOM solution. Treatment with inhibin-AS significantly increased the concentrations of plasma FSH. Furthermore, there was a significant elevation in plasma concentrations of estradiol-17beta and progesterone in the inhibin-immunized groups in comparison with controls. The superovulated oocytes that were fertilized normally in vivo were able to develop normally to blastocysts in vitro. The number of oocytes and blastocysts developed in animals treated with inhibin-AS was significantly higher than that for mice treated with control serum. Moreover, the rate of fertilization and the rate of blastocyst development were similar in inhibin-AS treated and control groups. These results indicate that immunoneutralization of endogenous inhibin induces superovulation in adult mice without additional treatment with human chorionic gonadotropin (hCG). In addition, the superovulated oocytes obtained by administration of inhibin-AS have normal embryonic developmental competence.

Evidence for a negative intrafollicular role for inhibin in regulation of estradiol production by granulosa cells.

Intrafollicular concentrations of inhibin A and estradiol vary inversely during development of dominant follicles in cattle. Thus, we hypothesized that inhibin has a negative autocrine or paracrine effect on estradiol production by granulosa cells. To examine this hypothesis, a homologous model system was used to test the effects of bovine antibovine inhibin antibodies, bovine inhibin, and a peptide fragment of bovine inhibin (bINH) on capacity of granulosa cells isolated from individual estrogen-active or -inactive dominant or subordinate follicles to produce estradiol during short-term (18 h) serum-free culture. Immunoblot analysis of media demonstrated that granulosa cells basally produce different molecular weight forms of inhibin, similar to those in bovine follicular fluid. Immunoneutralization of endogenous inhibin in culture with different doses (12.5-1000 microg) of highly purified bovine antibovine inhibin antibodies increased estradiol production 2- to 15-fold, compared with controls. Preadsorption of the anti-inhibin antibodies with bINH precursors or bovine pro-alpha(C) suppressed the capacity of anti-inhibin antibodies to enhance estradiol production by granulosa cells, compared with controls. Treatment of granulosa cells with an immunoaffinity-purified preparation of bINH suppressed basal estradiol production 60%, compared with controls. In contrast, treatment of granulosa cells with the bINH peptide increased estradiol production 14-fold, compared with controls. Based on these results, we concluded that both anti-inhibin antibodies and bINH blocked the suppressive local effects of basally produced inhibin on estradiol production during culture of granulosa cells and that inhibin has a negative autocrine or paracrine effect on the in vitro capacity of granulosa cells isolated from dominant or subordinate follicles to produce estradiol.

Production and endocrine role of inhibin during the early development of bull calves.

This study investigated the ontogeny of control of FSH secretion by inhibin during early prepubertal development of bulls by 1) measurements of circulating levels of inhibin and FSH from 1 to 13 wk of age, and 2) immunoneutralization of endogenous inhibin at 7, 21, 60, and 120 days of age. In addition, production and localization of inhibin in testes were examined by immunohistochemistry and Western blots at 7, 21, 60, and 120 days of age. Plasma immunoreactive inhibin levels were relatively low between 1 and 3 wk of age and then showed a tendency to rise (P < 0.1) from 4 wk of age. Circulating concentrations of FSH were low during 3 wk after birth and increased at 5 wk, remained high (P < 0.05) until 16 wk of age. Treatment with inhibin antiserum resulted in a significant (P < 0.05) increase in plasma FSH at 7, 21, 60, and 120 days of age compared to those following injection of control serum; however, the magnitude of the FSH rise after inhibin immunization was greater as bulls aged. There were no significant changes in plasma LH after inhibin immunization. An intense staining of inhibin alpha subunits was found in Sertoli cells within the solid seminiferous cords from 7 to 120 days of age, while no specific immune reaction was found in interstitial cells. Western blot analysis of testicular homogenates isolated from bulls 7-120 days of age revealed presence of a 28.5-kDa molecule that cross-reacted with inhibin alpha subunit and beta(B) subunit-specific antibodies. In this study, before 13 wk of age in bull calves, there was no inverse relationship between plasma concentrations of immunoreactive inhibin and FSH. However, the present immunization study clearly indicates that inhibin participates in the regulation of FSH secretion from infancy to early prepubertal stage, although the endocrine significance of inhibin becomes greater in older bulls. The results also indicate that the major production site of inhibin in the testis is Sertoli cells and that these cells produce inhibin that exerts a negative feedback effect on FSH secretion from early stages of development.

Induction of superovulation by immunoneutralization of endogenous inhibin through the increase in the secretion of follicle-stimulating hormone in the cyclic golden hamster.

The present study was conducted to study the effect of immunoneutralization against endogenous inhibin on FSH, LH, oestradiol-17 beta and progesterone secretion and to investigate the effect of removal of endogenous inhibin on subsequent follicular development in the hamster. After treatment with anti-inhibin serum (inhibin-AS) at 1100 h on day 2 of the oestrous cycle (day 1 = day of ovulation), a marked increase in plasma levels of FSH and a slight increase in plasma levels of LH were noted and pituitary contents of FSH, but not LH, were also increased. In the group treated with inhibin-AS, superovulation occurred on day 1 of the following cycle. Plasma levels of oestradiol-17 beta markedly increased with the increase in the number of ovulations induced by human chorionic gonadotrophin (hCG) as compared with those in control animals. In the second cycle, plasma concentrations and pituitary contents of FSH in the animals given 200 microliters inhibin-AS still showed high values as compared with those in the animals treated with control serum, although superovulation did not occur on day 1 of the third cycle. Plasma concentrations and pituitary contents of LH in the hamster given 200 microliters inhibin-AS tended to decrease as compared with those in control animals during the second cycle. Plasma concentrations of oestradiol-17 beta in the animals treated with 200 microliters inhibin-AS changed in a similar way to controls. A marked increase in plasma concentrations of progesterone was noted on days 1 and 2 of the second cycle in the group receiving inhibin-AS. The twice daily injection of 1 IU hCG during the second cycle to the animals given 200 microliters inhibin-AS induced superovulation on day 1 of the third cycle. These results indicate that circulating inhibin may be an important indicator of the number of developing follicles and may maintain the species-specific number of developing follicles through suppression of FSH secretion in the cyclic hamster. They also suggest that high levels of inhibin slightly suppress plasma levels of LH, indicating that plasma LH may also regulate follicular development in the cyclic hamster.

Regulation of FSH beta messenger ribonucleic acid levels in the rat by endogenous inhibin [corrected and republished article originally printed in Endocrinology 1991 Nov;129(5):2802-4

This study investigated the role of endogenous inhibin in regulating FSH beta mRNA levels subsequent to the gonadotropin surge in the immature, estradiol (E2)-treated female rat. Rats which undergo FSH surges on day 29 have low to undetectable levels of FSH beta mRNA at 0900 h on day 30, whereas those treated simultaneously with E2 and progesterone (P) implants to block these surges have considerably higher levels of FSH beta mRNA. In view of the profound inhibitory effect of inhibin on FSH beta mRNA, we examined the possibility that increased inhibin secretion is responsible for the decline in FSH beta mRNA levels on the morning after the FSH surge by immunoneutralization of endogenous inhibin. Twenty-eight day-old rats which received E2 and blank (B1) or P implants were injected iv with 0.4 ml of a potent anti-rat inhibin serum (anti I alpha, prepared in sheep against rat inhibin alpha (1-26)-Tyr27 coupled to human alpha-globulins) or normal sheep serum at 1700 to 1830 h on day 29 and were killed at 0900 h on day 30. Animals which received the inhibin antiserum showed significantly (P less than 0.001) elevated serum FSH levels (22.9 +/- 1.9 ng/ml [E2 + B1] and 17.1 +/- 0.6 ng/ml [E2 + P]) compared to those which received normal serum (4.4 +/- 0.1 [E2 + B1] and 4.2 +/- 0.1 [E2 + P]). Serum LH was undetectable (less than 0.6 ng/ml) in all groups. Free glycoprotein alpha-subunit was also increased (P less than 0.001) by antiserum to inhibin in E2 + B1-treated rats but was significantly suppressed by P after injection of either normal serum or anti I alpha. Total pituitary RNA was extracted and hybridized to cDNA probes for rat FSH beta, LH beta, and the common alpha-subunit by Northern blot analysis; RNA levels were normalized with beta-actin or cyclophilin probes. As expected, in rats which received normal serum, FSH beta mRNA levels were about 4-fold higher after treatment with E2 + P implants than after treatment with E2 + B1 implants. However, injection with anti-inhibin serum resulted in a striking elevation of FSH beta mRNA levels: 13-fold in animals treated with E2 + B1 implants and 5-fold in animals treated with E2 + P implants. There were no significant differences in levels of LH beta or alpha-subunit mRNAs between rats which received anti-inhibin or normal serum although there was a 30-40% decrease in alpha mRNA after P treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of FSH beta messenger ribonucleic acid levels in the rat by endogenous inhibin.

This study investigated the role of endogenous inhibin in regulating FSH beta mRNA levels subsequent to the gonadotropin surge in the immature, estradiol (E2)-treated female rat. Rats which undergo FSH surges on day 29 have low to undetectable levels of FSH beta mRNA at 0900 h on day 30, whereas those treated simultaneously with E2 and progesterone (P) implants to block these surges have considerably higher levels of FSH beta mRNA. In view of the profound inhibitory effect of inhibin on FSH beta mRNA, we examined the possibility that increased inhibin secretion is responsible for the decline in FSH beta mRNA levels on the morning after the FSH surge by immunoneutralization of endogenous inhibin. Twenty-eight day-old rats which received E2 and blank (B1) or P implants were injected iv with 0.4 ml of a potent anti-rat inhibin serum (anti I alpha, prepared in sheep against rat inhibin alpha (1-26)-Tyr27 coupled to human alpha-globulins) or normal sheep serum at 1700 to 1830 h on day 29 and were killed at 0900 h on day 30. Animals which received the inhibin antiserum showed significantly (P less than 0.001) elevated serum FSH levels (22.9 +/- 1.9 ng/ml [E2 + B1] and 17.1 +/- 0.6 ng/ml [E2 + P]) compared to those which received normal serum (4.4 +/- 0.1 [E2 + B1] and 4.2 +/- 0.1 [E2 + P]). Serum LH was undetectable (less than 0.6 ng/ml) in all groups. Free glycoprotein alpha-subunit was also increased (P less than 0.001) by antiserum to inhibin in E2 + B1-treated rats but was significantly suppressed by P after injection of either normal serum or anti I alpha. Total pituitary RNA was extracted and hybridized to cDNA probes for rat FSH beta, LH beta, and the common alpha-subunit by Northern blot analysis; RNA levels were normalized with beta-actin or cyclophilin probes. As expected, in rats which received normal serum, FSH beta mRNA levels were about 4-fold higher after treatment with E2 + P implants than after treatment with E2 + B1 implants. However, injection with anti-inhibin serum resulted in a striking elevation of FSH beta mRNA levels: 13-fold in animals treated with E2 + B1 implants and 5-fold in animals treated with E2 + P implants. There were no significant differences in levels of LH beta or alpha-subunit mRNAs between rats which received anti-inhibin or normal serum although there was a 30-40% decrease in alpha mRNA after P treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

REGULATION OF FSHβ MESSENGER RIBONUCLEIC ACID LEVELS IN THE RAT BY ENDOGENOUS INHIBIN

This study investigated the role of endogenous inhibin in regulating FSHβ mRNA levels subsequent to the gonadotropin surge in the immature, estradiol (E2)-treated female rat. Rats which undergo FSH surges on day 29 have low to undetectable levels of FSH3 mRNA at 0900 h on day 30, whereas those treated simultaneously with E2 and progesterone (P) implants to block these surges have considerably higher levels of FSHg mRNA. In view of the profound inhibitory effect of inhibin on FSHβ mRNA, we examined the possibility that increased inhibin secretion is responsible for the decline in FSHβ mRNA levels on the morning after the FSH surge by immunoneutralization of endogenous inhibin. Twenty-eight day-old rats which received E2 and blank (Bl) or P implants were injected iv with 0.4 ml of a potent anti-rat inhibin serum (anti lα, prepared in sheep against rat inhibin α(l–26)-Tyr27 coupled to human a-globulins) or normal sheep serum at 1700 to 1830 h on dav 29 and were killed at 0900 h on day 30. Animals which received the inhibin antiserum showed significantly (P< 0.001) elevated serum FSH levels (22.9 ± 1.9 ng/ml [E2 + Bl] and 17.1 + 0.6 ng/ml [E2 + P]) compared to those which received normal serum (4.4 + 0.1 [E2 + Blj and 4.2 ± 0.1 [E2 + PJ). Serum LH was undetectable (<0.6 ng/ml) in all groups. Free glycoprotein α-subunit was also increased (P<0.001) by antiserum to inhibin in E2 + Bl-treated rats but was significantly suppressed by P after injection of either normal serum or anti la. Total pituitary RNA was extracted and hybridized to cDNA probes for rat FSHβ, LHβ, and the common a-subunit by Northern blot analysis; RNA levels were normalized with β-actin or cyclophilin probes. As expected, in rats which received normal serum, FSHβ mRNA levels were about 4-fold higher after treatment with E2 + P implants than after treatment with E2 + Bl implants. However, injection with anti-inhibin serum resulted in a striking elevation of FSHβ mRNA levels: 13-fold in animals treated with E2 + Bl implants and 5-fold in animals treated with E2 + P implants. There were no significant differences in levels of LHβ or α-subunit mRNAs between rats which received anti-inhibin or normal serum although there was a 30-40% decrease in α mRNA after P treatment. These results, taken together with our previous results showing higher serum inhibin concentrations in rats undergoing FSH surges than in those in which the surge is blocked by P, indicate that endogenous inhibin regulates FSHβ mRNA levels in the rat. In addition, progesterone suppresses both secretion of free α-subunit and α mRNA levels. At present these conclusions are limited to the immature female rat; further studies will be necessary to determine if they generalize to the adult female and/or male rat.

Initial ovulation rate and follicle population after injection of inhibin-neutralizing antiserum in the late-prepubertal rat

In late-prepubertal female rats passive immunoneutralization of endogenous inhibin was achieved by injection of inhibin antiserum. Effects on follicle population, timing of sexual maturation, ovulation rate at first and second oestrus and serum FSH levels were studied. Rats were injected with antiserum, (non-immune) control serum from castrated sheep (castrated serum) or their IgG fractions, or with saline on day 33 or 3 or 2 days (days -3/-2) before the expected day of first ovulation, day 38.5 +/- 0.2 (n = 70). Blood was collected from different subgroups at 8, 24 and 48 h, and at first and second oestrus after injection. At necropsy, ovaries were histologically prepared for differential counting of follicles (48 h and first oestrus) and counting of corpora lutea (CL; first and second oestrus) as an index of ovulation rate. Results from rats injected with either serum or its IgG fraction were not different, as was the case when rats were injected with either castrated serum or saline. Thus, results from groups treated with antiserum and antiserum IgG were combined and labelled 'antiserum', and the castrated serum, castrated serum IgG and saline-treated groups were combined and labelled 'control'. The activity of inhibin-neutralizing antibodies in the circulation of antiserum-treated rats was reduced by 43% between 8 h and second oestrus after injection, as determined by the binding of purified bioactive radioiodinated 31 kDa bovine inhibin.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of Leydig Cells and Endogenous Inhibin in Regulating Pulsatile Gonadotropin Secretion in the Adult Male Rat

The purpose of the present study was to determine the parameters of pulsatile gonadotropin secretion in the adult male rat that are regulated by the suppressive feedback influences provided by factors originating in the Leydig cells or by endogenous inhibin, or both. This was achieved by examining the changes in the secretion parameters of FSH and LH that result from selectively destroying the Leydig cells using the toxicant ethane dimethane sulfonate (EDS) or passively immunoneutralizing endogenous inhibin using high-titer anti alpha-inhibin subunit serum, or both. Both FSH and LH were secreted in a pulsatile manner in intact male rats as determined using two different pulse-detection methods. Destruction of the Leydig cells with EDS 6 days before sampling significantly increased basal FSH secretion without affecting pulsatile FSH secretion. Injection of anti-inhibin serum into intact (vehicle treated) males 18 h before sampling produced no observable alteration in any parameter of FSH secretion. Either administration of anti-inhibin serum or castration of rats previously treated with EDS induced further significant, selective increases in the basal parameters of FSH secretion, raising mean FSH to levels comparable to those observed in 6-day castrate rats. When examined individually, however, the parameters of mean trough and peak FSH level and mean pulse amplitude remained significantly higher in the 6-day castrate males. In sharp contrast to the selective effects on basal FSH, destruction of the Leydig cells with EDS dramatically elevated all parameters of LH secretion to levels comparable to those observed in similarly timed castrate rats. Neither immunoneutralization of endogenous inhibin nor castration of EDS-treated rats 18 h before sampling caused any further alteration in any parameter of LH secretion. The results from these studies demonstrate that the Leydig cell provides all of the suppressive influence of the testes on LH secretion and a major portion of the suppressive influence over FSH secretion. This influence is exerted through a suppression of all parameters of LH secretion, but through a selective suppression of basal FSH secretion parameters. Collectively, the results suggest that the Leydig cell-derived influences suppress those parameters of gonadotropin secretion that are mediated by LHRH, acting, at least in part, through a suppression of pituitary sensitivity to LHRH. In the absence of the Leydig cells, endogenous inhibin can be demonstrated to also suppress basal FSH secretion in the adult male rat.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthetic peptide segments of inhibin alpha- and beta-subunits: preparation and characterization of polyclonal antibodies.

The amino acid sequences of human inhibin alpha-, beta A- and beta B-subunits were analyzed for hydrophilicity and chain flexibility to predict regions that are on the surface of the subunits and, therefore, are potential antigenic sites. Based on these analyses, a total of nine peptides were synthesized, and rabbit antisera against the peptides were prepared. Peptides of the N-terminus (residues 1-16 and 13-24) and region 109-123 of the alpha-subunit produced high titer antibodies. Regions 69-79 and 93-105 of the beta A-subunit and region 93-104 of the beta B-subunit were also immunogenic. Immunoblotting of an inhibin preparation with anti-alpha-peptide antiserum revealed that a 32K band (inhibin) and an 18K band (alpha-subunit) were stained. Immunoblotting with anti-beta-peptide antiserum detected a 32K band (inhibin), a 24K band (activin), and a 14K band (beta-subunit). Injection (iv) of these antisera into rats induced dramatic elevation of serum FSH in 6-12 h and suggested immunoneutralization of endogenous inhibin. RIAs for each subunit were developed using radioiodinated peptides as tracers. Competition binding assays indicated crossreactivity with human follicular fluid, semen, serum, plasma, and crude inhibin preparations. Parallel dilution curves were obtained. Antisera against beta A- and beta B-subunit peptide cross-reacted with each other. In immunocytochemical studies, these antisera were used in conjunction with gold-labeled goat antirabbit immunoglobulin G to localize inhibin in cells of the rat testis. Specific staining of inhibin was localized within the Sertoli cells of some tubules in adult rat testis. Positive staining could be blocked by preadsorbing the sera with the appropriate synthetic peptide. These results suggest that antibodies against synthetic inhibin peptides are useful in elucidating the roles of inhibin and activin.

Immunoneutralization of Endogenous Inhibin Modifies Hormone Secretion and Ovulation Rate in the Rat*

Studies were conducted to determine the effects of inhibin immunoneutralization on gonadotropins and sex steroids secretion, as well as ovulation rate and the outcome of pregnancy in the rat. These changes were compared to the normal pattern of hormone release during the estrous cycle, as well as to the ovulatory and gestational history of control animals. Measurement of plasma inhibin levels with antibodies raised against the N-terminus part of the alpha-chain of the molecule indicated an increase in immunoreactive inhibin-alpha release during the afternoon of proestrus, followed by a marked decline after the primary gonadotropin surge. Removal of endogenous inhibin by the iv injection of anti-inhibin serum on diestrus-1 caused the expected rise in plasma FSH, but not LH values. FSH secretion remained elevated for approximately 28 h, then returned to baseline. While not altering vaginal cytology, the injection of the antiserum during diestrus-1 or -2, and to a lesser degree, during estrus, caused a marked increase in the number of tubal ova shed during the subsequent estrus. However, plasma estrogen and progesterone remained comparable at all times between control and treated animals. In female rats mated after inhibin immunoneutralization at diestrus-1, the increase in ovulation rate resulted in a significant increase in developing fetuses, but only ca. 40% of the pups survived. In order to assess whether elevated FSH secretion represented the primary mechanism through which removal of endogenous inhibin increased the number of maturing follicles, ovine FSH was infused for 35 h starting in diestrus-1 or diestrus-2. Similar to the treatment with the anti-inhibin serum, FSH infusion caused an increase the number of eggs present in the oviducts on the next day of estrus. These results support the existence of a close functional relationship between the circulating levels of FSH and inhibin in the female rat. They also indicate that immunization against inhibin increases ovulation rate, a phenomenon probably mediated through elevated levels of circulating FSH.