ABSTRACT

Serum and ovarian concentrations of inhibin during the oestrous cycle of rats were determined by a radioimmunoassay (RIA) based on a porcine inhibin RIA. Serum concentrations of LH, FSH, oestradiol-17β, progesterone and testosterone were also determined during the cycle. Serum concentrations of inhibin were high (8·47 ± 0·58 μg/l) during the morning of pro-oestrus and then dropped sharply to the lowest level (3·21 ± 0·38 μg/l) at 24.00 h on the day of pro-oestrus after the preovulatory LH and FSH surge. Inhibin concentrations then recovered rapidly, with a peak (7·94 ± 0·97 μg/l) at 15.00 h on the day of oestrus. Inhibin concentrations in serum dropped again to their second lowest value (4·00 ± 0·12 μg/l) at 24.00 h on the day of oestrus. The largest FSH surge observed from midnight of pro-oestrus to the morning of oestrus (the so-called second FSH surge) was shown to be inversely related to the changes in serum concentrations of inhibin, suggesting a close relationship between the serum FSH and inhibin concentrations in cyclic rats.

Journal of Endocrinology (1989) 121, 91–100

ABSTRACT

A sensitive and specific radioimmunoassay (RIA) system for porcine ovarian inhibin has been developed. Antisera to porcine inhibin of molecular weight 32 000 (32 kDa inhibin) were raised in male chickens. The recognition site of the antiserum used in the present study was the N-terminal region of the α-subunit of 32 kDa inhibin. The antiserum could recognize higher molecular weight forms of inhibin present in porcine follicular fluid as well as the 32 kDa form. The average effective dose and the least detectable amount of inhibin in this RIA were 643 and 30·7 pg/tube respectively. Non-specific effects of serum on the RIA could be overcome by including 100 μl serum from a castrated pig in the standards and by incubating at 30 °C.

Serum concentrations of inhibin fluctuated between 0·6 and 2·5 μg/l during the oestrous cycles of the pigs. The amount of serum inhibin gradually increased from the late luteal phase to the early follicular phase and reached a maximum of 2·48 μg/l at day −4 (day 0 = day of ovulation). Concentrations then decreased rapidly to reach a minimum of 0·6 μg/l. Two small peaks were also observed during the luteal phase, although the concentration was relatively low during this phase. Changes in serum concentrations of oestradiol-17β did not parallel those of inhibin, especially during the luteal phase when serum concentrations of oestradiol-17β remained quite low. Serum concentrations of FSH were inversely related to those of inhibin rather than to those of oestradiol-17β, suggesting that the secretion of FSH during the oestrous cycles of pigs is mainly controlled by ovarian inhibin.

J. Endocr. (1988) 118, 211–219

ABSTRACT

It is believed that the renin-angiotensin system evolved initially in primitive bony fishes and is absent from elasmobranchs. We have isolated angiotensin I from the incubates of plasma and kidney extracts of an elasmobranch fish, Triakis scyllia, using eel vasopressor activity as an assay system. Its sequence was determined to be H-Asn-Arg-Pro-Tyr-Ile-His-ProPhe-Gln-Leu-OH. Dogfish angiotensin I is teleost-like because of an asparagine residue at position 1 but it is mammalian-like because of an isoleucine residue at position 5. The unique and most important substitution in dogfish angiotensin I is a proline residue at position 3 which may cause significant changes in its tertiary structure. A glutamine residue at position 9 is also unique among all angiotensin Is sequenced to date. Dogfish angiotensin I is more potent than rat angiotensin I in its vasopressor activity in the dogfish but the relationship is reversed in the rat. Thus angiotensin receptors as well as the hormone molecules appear to have evolved during vertebrate phylogeny. Our findings establish the elasmobranch renin-angiotensin system and support the hypothesis that the renin-angiotensin system is a phylogenetically old hormonal system which plays important roles in cardiovascular and fluid homeostasis.

Journal of Endocrinology (1993) 139, 281–285

ABSTRACT

To investigate the physiological importance of inhibin in the regulation of FSH secretion in prepubertal bulls, animals (6-month-old) were passively immunized against inhibin. Five animals were given an i.v. bolus injection of 50 ml inhibin antiserum raised against bovine 32 kDa inhibin in a castrated male goat, and four bulls were given the same amount of castrated male goat serum (control serum) as controls. Treatment with the inhibin antiserum resulted in a marked increase (P < 0·01) in plasma concentrations of FSH within 12 h compared with control animals, and FSH levels in immunized animals remained high until 168 h after the injection. Concentrations of plasma LH and testosterone in the immunized animals were not different from those in the control animals. The present findings provide strong evidence that inhibin plays an important role in the inhibitory regulation of FSH secretion in prepubertal bulls.

Journal of Endocrinology (1993) 137, 15–19

ABSTRACT

To investigate the physiological importance of inhibin in the regulation of FSH secretion in cows, seven cyclic cows were treated with an inhibin antiserum raised against bovine 32 kDa inhibin in a castrated goat. The same animals treated with a castrated goat serum (control serum) served as controls. On day 12 of the oestrous cycle (day 0 = day of oestrus), four of seven cows were injected with 100 ml inhibin antiserum first, and the remaining three cows with 100 ml control serum first. Twelve days after the second oestrus following the first serum injection (42–46 days after the first serum injection), the former four cows were injected with control serum and the latter three with inhibin antiserum. Follicular development after the injections of control serum or inhibin antiserum was assessed by daily ultrasonographic examination.

Treatment with inhibin antiserum resulted in a marked increase (P < 0·01) in plasma concentrations of FSH and oestradiol-17β but not LH or progesterone, compared with those after treatment with control serum. Plasma concentrations of FSH increased significantly (P < 0·01) at 8 h after injection of antiinhibin serum when compared with the control value. Concentrations of FSH in the plasma remained high for 72 h, then declined to the control level by 84 h, concomitant with an abrupt decrease in the titre of free inhibin antibody in the plasma. High concentrations of oestradiol-17β were observed between 36 and 96 h after treatment. Treatment with inhibin antiserum markedly increased the number of small (≥ 4 < 7 mm in diameter), medium (≥ 7 < 10 mm) and large (≥ 10 mm) follicles by 48, 72 and 96 h after treatment when compared with the value before treatment. The number of large follicles returned to the pretreatment value at 168 h, whereas the number of small and medium follicles remained increased.

The present results provide strong evidence that inhibin is an important factor in the inhibitory regulation of FSH secretion during the mid-luteal phase of cows, and demonstrate that an increase in endogenous FSH secretion after immunoneutralization of circulating inhibin stimulates the rapid growth of a large number of follicles.

Journal of Endocrinology (1993) 136, 35–41

The purpose of this study was to investigate the effects of physiologic levels of ghrelin on insulin secretion and insulin sensitivity (glucose disposal) in scheduled fed-sheep, using the hyperglycemic clamp and hyperinsulinemic euglycemic clamp respectively. Twelve castrated Suffolk rams (69.8 ± 0.6 kg) were conditioned to be fed alfalfa hay cubes (2% of body weight) once a day. Three hours after the feeding, synthetic ovine ghrelin was intravenously administered to the animals at a rate of 0.025 and 0.05 μg/kg body weight (BW) per min for 3 h. Concomitantly, the hyperglycemic clamp or the hyperinsulinemic euglycemic clamp was carried out. In the hyperglycemic clamp, a target glucose concentration was clamped at 100 mg/100 ml above the initial level. In the hyperinsulinemic euglycemic clamp, insulin was intravenously administered to the animals for 3 h at a rate of 2 mU/kg BW per min. Basal glucose concentrations (44± 1 mg/dl) were maintained by variably infusing 100 mg/dl glucose solution. In both clamps, plasma ghrelin concentrations were dose-dependently elevated and maintained at a constant level within the physiologic range. Ghrelin infusions induced a significant (ANOVA; P < 0.01) increase in plasma GH concentrations. In the hyperglycemic clamp, plasma insulin levels were increased by glucose infusion and were significantly (P < 0.05) greater in ghrelin-infused animals. In the hyperinsulinemic euglycemic clamp, glucose infusion rate, an index of insulin sensitivity, was not affected by ghrelin infusion. In conclusion, the present study has demonstrated for the first time that ghrelin enhances glucose-induced insulin secretion in the ruminant animal.

The ontogeny of inhibin secretion in the testis of rats was investigated. Testicular localization, content of immunoactive and bioactive inhibin and its molecular size in fetal and neonatal rats (from 16 days of gestation to 5 days of age) were determined. Strong immunostaining with an antiserum against a polypeptide of porcine inhibin alpha-subunit was noted in testicular interstitial cells from 16 days of gestation. Co-localization of inhibin alpha-subunit and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) was observed in the interstitial cells until 2 days of age. Immunoreactive inhibin alpha-subunit in the interstitial tissue had disappeared by 5 days of age, although 3 beta HSD-positive cells were still detected. Weak immunostaining for the inhibin alpha-subunit was detected in the seminiferous tubules, probably in the cytoplasm of Sertoli cells, from 20 days of gestation onward. No inhibin alpha-subunit immunostaining was observed in germ cells throughout the experimental period. Testicular inhibin was detected at 16 days of gestation (49.5 +/- 6.7 pg per testis) by RIA. Testicular immunoreactive inhibin showed a tendency to increase during fetal life and levels were maintained at a similar value after birth (697.0 +/- 46.9 pg per testis at 5 days of age). Inhibin bioactivity and its molecular size in testicular homogenate was examined at 17 days of gestation and 0 and 5 days of age. Although no bioactivity was detected at 17 days of gestation, bioactivity was noted at 0 and 5 days of age (177.7 and 1303.9 pg per testis respectively). Immunoblot analysis with antiserum against inhibin alpha-subunit revealed only approximately 40 kDa molecular masses in the testis at 17 days of gestation, probably inhibin-related proteins, but not inhibin. At 0 and 5 days of age, a protein of 30 kDa molecular mass, possibly inhibin, was detected as well as material of approximately 40 kDa molecular mass. FSH in the plasma was first detected at 19 days of gestation (1197.0 ng/l), increased towards birth, and thereafter decreased (4588.5 +/- 572.3 ng/l at 21 days of gestation and 2400.0 +/- 179.6 ng/l at 5 days of age). These results indicate that Leydig cells in fetal and neonatal rats produce inhibin-related substances with no inhibin bioactivity, whereas Sertoli cells begin to produce inhibin during the perinatal period as a possible regulator of FSH secretion.

ABSTRACT

Brain natriuretic peptide (BNP) is a novel peptide that has actions similar to atrial natriuretic peptide (ANP). The present study investigated BNP localization in the heart, ANP and BNP contents in several organs, and ANP and BNP clearance through these organs. In the morphological study, it was shown that porcine BNP-like immunoreactivity was mainly distributed in the granules of the atrium. The content of porcine BNP-like immunoreactivity in the atrium was extremely high, about 100-fold greater than in the ventricle. From the determination of porcine BNP and ANP contents of such organs as the heart, kidney and liver and also plasma, it was shown that porcine BNP concentration was approximately one order of magnitude lower than that of ANP, and clearance rates of ANP and porcine BNP from these organs were similar and not significantly different between the organs. These results suggest that the modes of secretion and degradation of porcine BNP are not the same as those of ANP.

Journal of Endocrinology (1992) 132, 101–106

ABSTRACT

A sensitive radioimmunoassay (RIA) for the determination of inhibin in peripheral plasma and tissue homogenates of different species has been developed using antisera to partially purified bovine follicular fluid (bFF) inhibin and ¹²⁵I-labelled bFF 32 kDa inhibin. Antisera were produced by immunization of rabbits with partially purified bFF inhibin prepared by immunoaffinity chromatography. Increasing doses of a high titre antiserum could neutralize the suppressing effect of bFF, porcine follicular fluid and rat ovarian homogenate on FSH secretion from rat anterior pituitary cells in culture. Sensitivity of the assay was 3·1 ng International Research Standard of porcine inhibin per tube. Parallel inhibition curves were obtained for inhibin preparations from female and male animals of ten species, i.e. cattle, goats, sheep, cats, dogs, monkeys, pigs, horses, rats and man. Inhibin subunits and related proteins cross-reacted minimally with the antiserum used in the study. Plasma concentrations of inhibin in adult male and female rats were measured by the RIA before and at various times after gonadectomy. Inhibin levels in peripheral plasma before gonadectomy were significantly higher in adult female than in adult male rats. Inhibin levels decreased abruptly after gonadectomy in both sexes and they correlated negatively with plasma concentrations of FSH. This inhibin RIA will facilitate studies of the physiology of inhibin in various species of animals.

Journal of Endocrinology (1989) 122, 697–704

Abstract

We examined vitamin A-deficient chicks to determine whether vitamin A affects the estrogen-induced development of the chick oviduct. When oviduct development was stimulated for 5 days with the synthetic estrogen, diethylstilbestrol, the wet weight of the oviduct in vitamin A-deficient chicks was only half that in control chicks. The DNA content in this tissue showed that the decreased oviduct weight in the vitamin A-deficient chicks was caused by the decreased proliferation of oviduct cells. However, the estrogen-induced expression of the ovalbumin gene was not affected by the vitamin A deficiency, suggesting that estrogen-induced cytodifferentiation is not affected by vitamin A. To clarify the vitamin A action on estrogen-induced development in the oviduct, transcripts of nuclear estrogen receptor (ER) and all-trans-retinoic acid (RARα, β and γ) receptors, which exert the effects of estrogen and vitamin A, were measured. The ER, RARα and RARβ genes, but not that of RARγ, were expressed during oviduct development, indicating that estrogen and vitamin A may control the expression of target genes through their cognate receptors. Thus, we have shown that vitamin A is involved in estrogen-induced cell proliferation but not in cytodifferentiation of the chicken oviduct.

Journal of Endocrinology (1996) 148, 257–265