G. P. van Rees

LH-RH-dependent synthesis of protein necessary for LH release from rat pituitary glands in vitro

Anterior pituitary glands of intact diestrous and gonadectomized female rats were incubated with a supramaximally active dose of LH-RH. Puromycin (P) and cycloheximide (C) did not consistently affect the LH release from glands of ovariectomized rats. In intact rats, LH release induced by LH-RH occurred in two phases, an initial one (relatively slow rate of release) and a secondary one (high rate of release). P and C had no effect on the initial phase, but prevented the secondary one. Pre-incubation with LH-RH prevented the effect of P and C added after 4 hours. Since P and C did not affect the total amount of LH present in media and hypophyses, the LH-released during the P- and C-sensitive phase is not newly formed LH. It is concluded that LH-RH induces the synthesis of protein which is necessary for LH-RH-induced LH release. The initial phase of LH release is made possible by protein already present in the glands at the beginning of the experiment.

Induction of LH Surges by Continuous Infusion of LH-RH

Preovulatory LH surges were measured in cannulated proestrous rats. Such LH peaks were also obtained by infusing pentobarbital-blocked animals with LH-RH for periods of up to 20 h; this suggested that the pituitary LH stores were exhausted. However, the total amount of LH secreted appeared more or less determined by the LH-RH dose, indicating that the size of the releasable LH pool is not fixed.

The Role Played by the Preoptic Region and the Hypothalamus in Spontaneous Ovulation and Ovulation Induced by Progesterone

In female rats, cuts were made with the use of the Halasz technique around the preoptic area, leaving the connections between the preoptic area and hypothalamus intact. Such cuts do not interfere with spontaneous ovulation, as opposed to cuts made at the level of the optic chiasm. In addition, the effects of cuts around the preoptic area on progesterone-induced secretion of gonadotrophins were studied. In acute experiments, these cuts blocked the activity of progesterone injected on the third day of dioestrus of the 5-day cycle or at pro-oestrus of the 4-day cycle. However, in chronic experiments, progesterone injected at pro-oestrus, followed by pentobarbital, did induce ovulation.

Inhibitory and augmentative effects of oestradiol on LH-RH-induced release of LH by anterior pituitary glands from intact female rats in vitro

Abstract Anterior pituitary glands from intact dioestrous female rats were incubated in vitro and the LH released into the medium was measured at hourly intervals. The effect of a supra-maximally active dose of LH-RH was first inhibited and later augmented by oestradiol added to the medium. The augmented response also occurred when LH-RH was absent during the preceding phase of the incubation. Both effects of oestradiol could be blocked by cyclo-heximide, which agent, however, also partly inhibited the response to LH-RH.

Testicular weight, tubular diameter and number of sertoli cells in rats are decreased after early prepubertal administration of an LHRH-antagonist ; the quality of spermatozoa is not impaired

To suppress gonadotropin secretion during the sensitive period in development of the testes, immature male rats were treated with an antagonist of luteinizing hormone-releasing hormone (LHRH; ORG. 30276) from postnatal days 6-15. Previously, it has been demonstrated that this treatment results in delayed pubertal development, decreased testicular weight, impaired fertility and adult sexual behavior. In the present experiments it was investigated whether the decreased testicular weight was correlated with morphological changes in the testis. Also, by using an artificial insemination technique, the biological activity of spermatozoa of adult male rats, treated during early prepuberty with the LHRH antagonist (LHRH-A), was tested. The present results demonstrated a decrease in the diameter of the testicular tubuli of LHRH-A-treated rats. The number of Sertoli cells per tubular cross-section was also smaller. But qualitatively no differences could be observed in the testis. All stages of maturation of the seminiferous epithelium were equally frequently represented in LHRH-A-treated males compared with controls. Artificial insemination using spermatozoa obtained from the epididymis of LHRH-A-treated rats, resulted in a pregnancy rate of 100%, similar to the control rate. From the present data, we conclude that the infertility in adult male rats, treated with an antagonist to LHRH during prepubertal life, does not result from malfunction in the maturational processes in the germinal cells and the testes as a whole, despite the observation of changes in the testicular morphology. The infertility of LHRH-A-treated male rats can be explained by the observed impairment of sexual behavior. We suggest, that a central action of the antagonist of LHRH when administered to immature male rats may lead to permanent changes in the development of sexual behavior.

Induction and inhibition of ovulation in the rat by intracerebral progesterone implants.

Depending on the moment of implantation, intra-cerebrally-placed progesterone pellets either induce or inhibit ovulation specifically if they are placed into the ventromedial nuclei of the hypothalamus. Similar implants placed in the pre-optic area, the amygdala, or the hippocampus are ineffective. Implants in the ventromedial nuclei can lengthen the ovulatory cycle by only one day. A hypothesis is presented to explain the effect of progesterone on the secretion of gonadotropins in different experimental circumstances.

Effects of LHRH antagonist administration to immature male rats on sexual development

Gonadotropin secretion in immature male rats was inhibited by administration of a potent LHRH antagonist (LHRH-A): from 6 to 15 days of age (early onset/short-term treatment), from 6 to 48 days of age (early onset/long-term treatment) or from 22 to 31 days of age (late onset/short-term treatment). Balano-preputial separation was retarded by 9 or 13 days (short-term treatments) or by about 40 days (long-term treatment). Adult testicular weight was lowered and plasma FSH was increased after early, but not after late onset of LHRH-A treatment. Plasma LH and testosterone levels were not affected by any of the LHRH-A treatments. Fertility was diminished after early onset LHRH-A administration only. Adult precopulatory and copulatory behavior were severely affected after early onset of LHRH-A treatment. Intensity of precopulatory anogenital inspection was increased. The copulatory pattern was incomplete with absence of ejaculatory behavior during sexual behavior tests. Sexual behavior was not affected after late onset of LHRH-A treatment. Thus, administration of LHRH-A to immature male rats delays balano-preputial separation irrespective of the age of onset of LHRH-A treatment. In contrast, effects on adult FSH levels, testicular weight, fertility and sexual behavior depend on age and duration of LHRH-A administration.

Effect of pre-incubation with different concentrations of LH-RH on subsequent LH release caused by supramaximally active amounts of LH-RH; Role of LH-RH-induced protein synthesis

Abstract Anterior pituitary glands from intact diestrous female rats were incubated for two consecutive periods of 3 hours. During the first period various submaximally active amounts of luteinizing hormone-releasing hormone (LH-RH) were added to the media, whereas during the second period a supramaximally active concentration of LH-RH was present. When during the second incubation period protein synthesis was inhibited by cycloheximide, the amount of luteinizing hormone (LH) released during that period was positively correlated to the concentration of LH present during the first incubation period. This relationship was not seen when cycloheximide was absent, or when cycloheximide was present throughout both periods. Total LH was not affected by LH-RH; thus no effect of LH-RH on LH synthesis was observed. It is concluded that the amounts of protein synthesized by the pituitary glands in response to the different amounts of LH-RH during the first incubation period can constitute a limiting factor for the response to the supramaximally active amount of LH-RH added during the second incubation period.

Effect of inhibin-like activity on LH-RH-stimulated release of FSH by pituitary glands from female rats in vitro

During long-term incubation of pituitary glands from intact female rats in the presence of inhibin-like activity, LH-RH-stimulated release of FSH becomes inhibited after 4 h of incubation. However, at the same time inhibition of basal FSH release is included. Therefore, glands were at first incubated for 4 h in the presence of inhibin-like activity to block basal release completely and thereafter LH-RH was added to the medium. It was found that LH-RH still could stimulate FSH release, despite the continuous presence of inhibin-like activity. This means that LH-RH-stimulated release of FSH could be investigated separately from basal release. Using this way of incubation, it was found that part of the action of LH-RH on FSH release was independent of protein synthesis. Also part of LH-RH-stimulated FSH release was independent of the presence of extracellular Ca2+. Furthermore it was found that LH-RH, when added after 4 h of incubation did stimulate FSH synthesis, in the presence as well as absence of inhibin-like activity. The present results indicate that LH-RH-stimulated release of FSH is not affected by inhibin-like activity. Complete inhibition of basal release and synthesis of FSH does not prevent LH-RH from stimulating FSH release and synthesis. It is suggested that two separate releasing mechanisms for FSH could exist in the pituitary gland.

Absence of an inhibitory effect of oestradiol on LH-RH induced release of LH in vitro caused by inhibition of protein synthesis.

Abstract Pituitary glands of ovariectomized rats were incubated for four hours and the LH released into the medium was measured by radioimmunoassay at one-hour intervals. The effect of a maximally active dose of LH-RH (1000 ng/ml) on pituitary LH release was inhibited by oestradiol (15 ng/ml) added to the media. Although cycloheximide (25 μ/ml) and puromycin (54 μ/ml) in the medium did not affect LH-RH-induced LH release consistently, the inhibitory effect of oestradiol was prevented. It is concluded that this effect of oestradiol is dependent on de novo synthesis of protein.