Secretory Patterns of the Luteinizing Hormone-Releasing Hormone in Female Rats

Abstract

To get more insight into the hypothalamic control of gonadotropin secretion, we attempted to define the secretory patterns of the luteinizing hormone-releasing hormone (LH-RH), by measuring hypothalamic LH-RH contents as well as by observing the effects of immunoneutralization of endogenous LH-RH on serum gonadotropin levels.
Female rats of Wister and Fischer strain with regular 4 or 5 day estrous cycles were used. They were raised in a light controlled (from 5 : 00 to 19 : 00), air conditioned room. Concentrations of serum and pituitary gonadotropins were measured by double antibody radioimmunoassay with NIAMDD kits and anti-LH serum from Dr. G.D. Niswender. Hypothalamic LH-RH was assayed by radioimmunoassay using anti-LH-RH serum from Drs. G. D. Niswender and T. Nett and synthetic LH-RH for iodinated and standard hormones. LH, FSH and LH-RH values were expressed in terms of NIH-LH-Sl, NIH-FSH-Sl and synthetic LH-RH respectively. Blood samples were taken either by decapitation or through the indwelling intra-atrial cannula. The hypothalamus was excised with scissors and stored in 0.1N HCl at-20°C. Before assay for LH-RH, acidic extract of the hypothalmus was neutralized with 0.1N NaOH.
In rats with 4 day cycles, serum LH levels increased with a peak at 16 : 00 to 18 : 00 on the day of proestrus, and serum FSH concentrations also increased with a longer duration than that during the LH surge. Hypothalamic LH-RH contents decreased in the late afternoon of the proestrus. In rats with 5 day cycles, serum gonadotropin and hypothalamic LH-RH changes similar to those in rats with 4 day cycles were observed on the day of proestrus. In long-term ovariectomized animals, an injection of estradiol benzoate (EB 20μg) decreased serum LH and FSH concentrations, and an additional injection of EB (20μg) or progesterone (P 2mg) 72 hrs after the first EB injection evoked an LH surge as observed in rats with normal cycles. However, hypothalamic LH-RH contents failed to change significantly during the steroid induced LH surges. Furthermore, no appreciable changes in the hypothalamic LH-RH contents were induced in the case of LH release evoked by electrochemical stimulation of the medial preoptic area (MPO) in the proestrous rats in which spontaneous LH surge had been blocked with pentobarbital (30mg/kg B.W.) injected at 13 : 00.
Anti-LH-RH serum obtained from male rabbits immunized with LH-RH-BSA conjugate bound I¹²⁵-LH-RH. Synthetic LH-RH and rat hypothalamic extract produced parallel inhibition of the binding. This anti-LH-RH serum showed cross reactivity to neither hypothalamic nor pituitary hormones examined other than LH-RH. From the cross reactivity to the LH-RH fragments, the antibody seemed to have stronger affinity to the C-terminus than to the N-terminus of the LH-RH molecule. This antiserum inhibited spontaneous gonadotropin surges and ovulation when injected at 12 : 00 on the day of proestrus in the volume of 0.5 to 1.0 ml. The cyclicity of vaginal smears was temporarily interrupted with successive leucocytic smears for 1 to 3 cycles after the antiserum injection and thereafter returned to the regular cycle. This anti-LH-RH serum inhibited the rise of serum LH and FSH concentrations induced by EB or EB and P injections. Moreover, it prevented the elevation of serum gonadotropins induced by electrochemical stimulation of the MPO or arcuate nucleus in pentobarbital-blocked proestrous rats.
In order to elucidate the roles of LH-RH in controlling the basal gonadotropin secretion, anti-LH-RH serum was injected into long-term ovariectomized rats. Both serum LH and FSH levels lowered after the antiserum injection with short latency, but FSH levels decreased less than LH levels. In short-term ovariectomized rats, the prompt increase of FSH levels during the 6 hrs after ovariectomy was never inhibited by the anti-LH-RH injection, whereas LH concentrations were below the lowest detectable limits of the assay.