Abstract

Plasma and ovarian levels of inhibin were determined by a radioimmunoassay (RIA) at 3-h intervals throughout the 4-day oestrous cycle of hamsters. Plasma concentrations of FSH, LH, progesterone, testosterone and oestradiol-17β were also determined by RIAs. In addition, hamsters were injected at various times with human chorionic gonadotrophin (hCG) to determine the follicular development. The changes in plasma concentrations of FSH after injection of antisera to oestradiol-17β (oestradiol-AS) and inhibin (inhibin-AS) on the morning of day 2 (day 1=day of ovulation) were also determined.

Plasma concentrations of inhibin showed a marked increase on the afternoon of day 1, remained at plateau levels until the morning of day 4, then increased abruptly on the afternoon of day 4 when preovulatory LH and FSH surges were initiated. A marked decrease in plasma concentrations of inhibin occurred during the process of ovulation after the preovulatory gonadotrophin surges. An inverse relationship between plasma levels of FSH and inhibin was observed when the secondary surge of FSH was in progress during the periovulatory period. Plasma concentrations of oestradiol-17β showed three increase phases and these changes differed from those of inhibin. Changes in plasma concentrations of oestradiol-17β correlated well with the maturation and regression of large antral follicles. Follicles capable of ovulating following hCG administration were first noted at 2300 h on day 1. The number of follicles capable of ovulating reached a maximum on the morning of day 3 (24·8± 0·6), and decreased by 0500 h on day 4 (15·0 ± 1·1), corresponding to the number of normal spontaneous ovulations. Plasma concentrations of FSH were dramatically increased within 6 h after inhibin-AS, though no increase in FSH levels was observed after oestradiol-AS. These findings suggest that changes in the plasma levels of inhibin during the oestrous cycle provide a precise indicator of follicular recruitment, and that the changes in plasma concentrations of oestradiol-17β are associated with follicular maturation. These findings also suggest that inhibin may play a major role in the inhibition of FSH secretion during the oestrous cycle of the hamster.

Journal of Endocrinology (1995) 146, 169–176

ABSTRACT

Concentrations of inhibin in peripheral blood were determined by radioimmunoassay in pseudopregnant, pregnant and lactating rats and correlated with the status of follicular development, luteal function in the ovaries and placental function. In pseudopregnant rats, plasma concentrations of inhibin remained relatively high from days 2 to 12 of pseudopregnancy and no significant changes occurred throughout pseudopregnancy. In pregnant rats, a marked decline in plasma concentrations of inhibin occurred between days 6 and 14 followed by an abrupt increase on days 20 and 21. In lactating rats, plasma concentrations of inhibin were consistently lower in dams nursing eight pups than in dams nursing two pups. Plasma concentrations of inhibin remained low during the early stages of lactation followed by a significant increase during the late stages in dams nursing eight pups. These changes in inhibin during the period of lactation correlated with those of plasma concentrations of LH, but not with those of oestradiol-17β, testosterone and progesterone. Throughout pseudopregnancy, pregnancy and lactation, on the other hand, plasma concentrations of FSH were within the range of dioestrous levels.

These findings indicate that concentrations of inhibin in peripheral plasma vary with the number of healthy antral follicles and no positive correlation was observed with luteal function during the luteal phase in rats. The results also suggest that inhibin is a more direct index of follicular development than oestradiol-17β in rats.

Journal of Endocrinology (1989) 121, 545–552

ABSTRACT

While prostaglandin F_2α (PGF_2α) has been thought to be a natural luteolysin in non-primates, a luteolytic effect in the human corpus luteum is less evident. We therefore investigated the action of PGF_2α on monolayer cultures of human luteal cells obtained from mid-luteal phase corpora lutea.

PGF_2α increased basal and human chorionic gonadotrophin (hCG)-stimulated progesterone production by human cultured luteal cells. A potent tumour-promoting phorbol ester, phorbol 12-myristate-13-acetate (PMA), also stimulated progesterone production by cultured human luteal cells.

Although human luteal cells were incubated for 24 h with PMA, hCG was still able to stimulate the production of progesterone by PMA-pretreated cells. However, PMA pretreatment blocked the ability of PGF_2α to stimulate progesterone production. It is possible that the luteotrophic effect of PGF_2α may be mediated, in part, by the activation of protein kinase C.

Addition of PGF_2α to suspensions of human luteal cells preincubated with myo-[2-³H]inositol promoted an increase in labelled inositol phosphates. PGF_2α also rapidly increased intracellular free Ca²⁺ in human luteal cells loaded with the fluorescent Ca²⁺ probe, fura-2.

We conclude that PGF_2α and PMA stimulate progesterone production and that PGF_2α increases the intracellular free calcium and inositol phosphates of human cultured luteal cells in the mid-luteal phase.

Journal of Endocrinology (1992) 133, 451–458

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 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

We have characterized α₁-adrenergic receptor subtypes in functional rat thyroid cells, FRTL, with relation to iodide efflux, and have also examined the effect of TSH on α₁ receptor subtypes. FRTL cells grown in a medium containing 5 mU TSH/ml (6H cells) had five times the number of α₁ receptors of those maintained in TSH-free medium (5H cells) (11·2 fmol/10⁶ cells compared with 2·0 fmol/10⁶ cells). Pretreatment with chlorethylclonidine (CEC; 10 μmol/l), which inactivates only α_1b receptors, caused 98·8% and 97·0% decreases in the density of specific [³H]prazosin-binding sites in 5H and 6H cells respectively. LIGAND computer program analysis of the displacement curves for 2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4 benzodioxane (WB4101) showed that FRTL cells contained mostly low-affinity WB4101 sites. Using the phenoxybenzamine inactivation method, we found a linear relationship between α₁ receptor density and the cytosolic free Ca²⁺ concentration response in FRTL cells. Pre-exposure of intact FRTL cells to CEC caused a 98·7% decrease in noradrenaline-stimulated maximal increase in cytosolic free Ca²⁺. Also, CEC and 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8), but not nicardipine, inhibited noradrenaline-stimulated iodine efflux. The results suggest that FRTL cells contain mostly the α_1b-adrenergic receptor subtype; that the α_1b receptors mediate cytosolic free Ca²⁺ and iodide efflux responses, and that TSH enhances these responses by increasing the α_1b receptor density without affecting the post-receptor mechanism.

Journal of Endocrinology (1990) 124, 433–441

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

The present study was conducted to study the effect of immunoneutralization against endogenous inhibin on FSH, LH, oestradiol-17β 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-17β 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 μl 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 μl inhibin-AS tended to decrease as compared with those in control animals during the second cycle. Plasma concentrations of oestradiol-17β in the animals treated with 200 μl 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 μl 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.

Journal of Endocrinology (1996) 151, 65–75

Plasma concentrations of inhibin pro-alphaC, inhibin A and inhibin B were determined by enzyme-linked immunosorbent assay at 6 h intervals throughout the 4-day oestrous cycle of the golden hamster. Plasma concentrations of follicle-stimulating hormone (FSH) and oestradiol-17beta were also measured by radioimmunoassay during the oestrous cycle. Plasma concentrations of inhibin A increased from the early morning of day 1 (day 1=day of ovulation) and reached plateau levels at 0500 h on day 2. An abrupt increase in plasma concentrations of inhibin A was found at 1700 h on day 4, when the preovulatory FSH surge was observed. An increase in plasma concentrations of inhibin B occurred on day 1 and reached plateau levels at 1700 h on day 1. The levels remained elevated until 0500 h on day 4 and declined gradually by 2300 h on day 4. Plasma concentrations of inhibin pro-alphaC gradually increased with some fluctuation from day 1 to 1700 h on day 4 and then declined. Significant negative relationships were noted between plasma FSH and both dimeric forms of inhibin from day 1 to day 3. Significant positive relationships were found between plasma oestradiol-17beta and inhibin A or inhibin pro-alphaC throughout the oestrous cycle. In contrast, no significant relationship was found between plasma oestradiol-17beta and inhibin B. These findings suggest that both dimeric forms of inhibin play a role in the regulation of FSH secretion during follicular development. These findings also suggest that inhibin pro-alphaC could be secreted primarily by large follicles, and early atretic follicles could also be responsible for inhibin pro-alphaC secretion. On the other hand, the secretory pattern of dimeric inhibins might shift from inhibin B to inhibin A with follicular development.

Macrophage inflammatory protein-1alpha (MIP-1alpha) is a member of the CC chemokines. We have previously reported the use of a whole bone marrow culture system to show that MIP-1alpha stimulates the formation of osteoclast-like multinucleated cells. Here we use rat bone marrow cells deprived of stromal cells, and clones obtained from murine macrophage-like cell line RAW264 to show that MIP-1alpha acts directly on cells in osteoclast lineage. We obtained several types of RAW264 cell clones, one of these clones, designated as RAW264 cell D clone (D clone), showed an extremely high response to receptor activator of NFkappaB ligand (RANKL) and tumor necrosis factor-alpha (TNF-alpha), while the other clone, RAW264 cell N clone (N clone), demonstrated no response to RANKL or TNF-alpha. Although both clones expressed receptor activator NFkappaB (RANK) before being stimulated for differentiation, only the D clone expressed cathepsin K when cells were stimulated to differentiate to osteoclasts. MIP-1alpha stimulated the formation of mononuclear preosteoclast-like cells from rat bone marrow cells deprived of stromal cells. MIP-1alpha also stimulated formation of osteoclast-like multinucleated cells from the D clone, when these cells were stimulated with RANKL and TNF-alpha. These findings provide strong evidence to show that MIP-1alpha acts directly on cells in the osteoclast lineage to stimulate osteoclastogenesis. Furthermore, pretreatment of RAW264 cell D clone with MIP-1alpha significantly induced adhesion properties of these cells to primary osteoblasts, suggesting a crucial role for MIP-1alpha in the regulation of the interaction between osteoclast precursors and osteoblasts in osteoclastogenesis.