Roles of Inhibin and Estradiol in the Regulation of Follicle-Stimulating Hormone and Luteinizing Hormone Secretion during the Estrous Cycle of the Rat

BIOLOGY OF REPRODUCTION 55, 127-133 (1996) Roles of Inhibin and Estradiol in the Regulation of Follicle-Stimulating Hormone and Luteinizing Hormone Secretion during the Estrous Cycle of the Rat' K. Arai, G. Watanabe, K. Taya,2 and S. Sasamoto Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183, Japan The relative contributions of inhibin and estradiol inthe regulation of FSH and LH secretion were examined at various stages of the estrous cycle in the rat. At 1100 h on metestrus, diestrus, or estrus or at 0500 h on proestrus, rats were ovariectomized or given an injection of normal goat serum, antiserum to inhibin (inhibin-AS), antiserum to estradiol (estradiol-AS), or both antisera to examine the role of gonadal hormones in the regulation of tonic gonadotropin secretion. Plasma samples were collected before and at 6, 12, and 24 h after the treatments. Further, to examine the effects of the treatments on preovulatory gonadotropin surges, the five treatments described were carried out at 0500 h on proestrus and blood samples were collected from 1100 h to 2000 h on the same day at 1.5-h intervals. There was a significant rise in the concentration of plasma FSH after injection of inhibin-AS as well as after ovariectomy on each day of the estrous cycle. These treatments, however, had less effect on estrous FSH secretion. The rise in FSH was greater with immunoneutralization against both inhibin and estradiol than with immunoneutralization against inhibin alone on diestrus and proestrus. Basal levels of LH were increased at all stages of the cycle through immunoneutralization against inhibin and were also increased through immunoneutralization against estradiol except at estrus. Especially on diestrus, a remarkable increase in LH secretion was induced at 6 h after immunoneutralization against both inhibin and estradiol (1449.3 ± 100.3% vs. control). The magnitude of the LH surge increased in inhibin-immunized rats, decreased in estradiol-immunized or ovariectomized rats, and remained at normal levels after injections of both antisera. The magnitude of the primary FSH surge increased very markedly in the inhibinimmunized group and decreased in the estradiol-immunized group. These results suggest that both estradiol and inhibin play a role in the regulation of LH secretion and that inhibin is a major regulator of FSH secretion during the estrous cycle of the rat. Furthermore, it is suggested that one or more extragonadal factors suppress estrous FSH secretion. MATERIALS AND METHODS Animals Adult cyclic female rats of the Wistar strain weighing 270-330 g were used. They were kept under conditions of controlled temperature (25 + 2C) and lighting (lights-on from 0500 to 1900 h). Food and water were available ad libitum. Vaginal smears were checked daily, and only rats with at least two consecutive 4-day estrous cycles were used. Inhibin Antiserum (Inhibin-AS) [Tyr 3 0]-Porcine inhibin a(l-30) conjugated to rabbit serum albumin was used as the antigen. This conjugate was provided by Dr. N. Ling (Neurocrine Bioscience Inc., San Diego, CA). The conjugate (3.6 mg) was dissolved in 1 ml saline and mixed with an equal volume of Freund's complete adjuvant. A castrated goat was given 2 ml of the suspension (including 3.6 mg of the conjugate) s.c. at each immunization. First, second, and third immunizations were performed at 2-wk intervals and thereafter, monthly. Blood samples were obtained 2 wk after each injection. The sera were collected and examined for inhibin-AS titer, and titers of the antisera were checked in the following manner. The antisera were diluted with 0.05 M PBS (pH 7.4) containing 1% BSA, and the diluted samples were incubated with 5000 cpm of 125 -I-labeled bovine 32-kDa inhibin (325 Ci/mmol) at 4C for 24 h in a total volume of 200 pIl. To separate bound radioligands, 100 pl1 of 1% bovine gamma globulin in PBS and 500 pl of 25% polyethylene glycol in PBS were added, and the mixture was agitated for 3 min. After centrifugation at 4°C and 1700 X g, radioactivity of the precipitate was counted in a gamma counter. The serum used in the present experiment had a titer of 1:1 000 000 as defined by final dilution of the antiserum required to bind 50% of added 125 -I-labeled bovine 32-kDa inhibin. In vivo efficiency of the antiserum was ensured by an increase in plasma concentrations of FSH after an i.v. injection of six doses (6.25-200 1l) of the antiserum at 1100 h on metes- INTRODUCTION In 4-day-cyclic rats, striking changes in gonadotropin secretion occur from the evening of proestrus to the early morning of estrus [1, 2]. Except during this periovulatory period, plasma concentrations of both FSH and LH remain at low basal levels. Hypothalamic GnRH, gonadal steroids, and inhibin play important roles in the regulation of FSH and LH secretion. Inhibin suppresses both FSH synthesis and release and plays a major role as a regulator of FSH secretion [3]. Passive immunization against inhibin causes an increase in the concentration of FSH in plasma [4, 5] Accepted March 4, 1996. Received September 14, 1995. 'Supported in part by a grant-in-aid (Bio Media Program 96-V-2-2-9) from the Ministry of Agriculture, Forestry and Fisheries, Japan, and CIBAGEIGY Foundation (Japan) for the Promotion of Science. 2Correspondence: Kazuyoshi Taya, Laboratory of Veterinary Physiology, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183, Japan. FAX: 423-67-5767. 127 and in the ovulation rate [6] in the female rat. Furthermore, large changes in inhibin secretion during the periovulatory period are involved in the secondary surge of FSH and result in the difference between the patterns of FSH and LH secretion during the periovulatory period [7-10]. On the other hand, gonadal steroids modulate gonadotropin secretion through GnRH in hypothalamus and gonadotropin gene expression at the level of the pituitary gland [11-18]. However, the relative contributions of estrogen and inhibin to regulation of gonadotropin secretion during the estrous cycle still have not been clarified. The purpose of the present experiments was to examine these problems using cyclic rats through passive immunization against inhibin and/or estradiol as well as ovariectomy at various stages of the estrous cycle. ABSTRACT 128 ARAI ET AL. trus and diestrus. A dose-related increase in the basal secretion of FSH was observed at 24 h after the injection, and the maximal response was noted when 100 1 of the antiserum was injected. The capacity of the antiserum to neutralize the inhibin bioactivity of rat inhibin (ovarian homogenate) was also examined in vitro with use of a dispersed anterior pituitary cell bioassay system. The secretion of FSH from cultured rat anterior pituitary cells was suppressed in a dose-dependent manner by rat ovarian homogenate, and the maximal suppression of the ovarian homogenate could be reve (...truncated)