Exogenous versus endogenous pulses of luteinizing hormone-releasing hormone and secretory patterns of gonadotropins.

OBJECTIVE: Does an exogenously administered regimen of luteinizing hormone-releasing hormone (LH-RH) pulses override endogenous LH-RH? DESIGN: Pulses of LH-RH were given intravenously during 1 week with intervals of 90 (n = 5) or 120 minutes (n = 5). Before, during, and after treatment serial plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) patterns and pituitary responsiveness to LH-RH were estimated. PATIENTS: Women with virtual absence of gonadal function (postmenopause, gonadal dysgenesis, and premature menopause). RESULTS: During treatment with the 90-minute interval, no LH pulses that were not related to injections of LH-RH were observed. Two spontaneous LH pulses were observed during treatment with the 120-minute interval. Immediately after treatment, a lowered incidence of spontaneous LH pulses was seen of 3 pulses/6 h if LH-RH had been given every 90 minutes and to 1.5 pulses/6 h after the 120-minute interval treatment. Gonadotropin responses to 100 micrograms of LH-RH were attenuated during treatment but recovered within 48 hours after discontinuation of treatment. CONCLUSIONS: (1) Exogenously administered LH-RH can override endogenous LH-RH or its effects on the release of LH in women with hypergonadotropic hypogonadism; (2) during pulsatile LH-RH treatment desensitization of the pituitary occurs to some degree; and (3) immediately after cessation of treatment with pulsatile LH-RH, spontaneous LH pulses are present but with a significantly lower incidence.

Autonomous FSH synthesis in vitro in anterior pituitary glands and grafts from female rats.

When pituitary glands from intact female, but not from ovariectomized rats, are incubated for 8 h in medium TC199 without further additives, FSH is synthesized. This LHRH-independent (or autonomous) FSH synthesis is prevented when bovine follicular fluid (bFF) is added to the incubation medium. Results from preliminary experiments, however, indicate no clear autonomous FSH synthesis after long-term absence of LHRH. To investigate the regulatory mechanisms involved in autonomous FSH synthesis and release, pituitary glands (exposed to endogenous LHRH) and pituitary grafts (not exposed to endogenous LHRH) from intact and ovariectomized rats were incubated for 8 h in medium TC199. Total FSH content (FSH released plus FSH remaining in the tissue) was compared with that in non-incubated glands or grafts, giving an indication of FSH synthesis. In addition, some of the animals were given LHRH pulses for 40 h before incubation. When pituitary tissue was taken from intact female rats, FSH synthesis occurred in the animals' own glands and in grafts from LHRH-pretreated rats. No FSH synthesis was seen in ovariectomized rats with or without pretreatment with bFF and/or LHRH. However, when ovariectomized rats had been pretreated with oestrogen, FSH synthesis was measured in vitro after pulsatile LHRH treatment in vivo. The results indicate that autonomous FSH synthesis in vitro is dependent upon previous (in vivo) exposure of the glands to both oestrogen and LHRH.

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.

Acute desensitization of pituitary FSH response to LHRH in ovariectomized rats: further evidence that in the presence of ovarian proteins the LHRH-dependent, LH-like component of FSH release becomes apparent.

Pulsatile release of LHRH and short-term pituitary desensitization to LHRH in the rat are believed to be necessary for the maintenance of LH pulsatility. In contrast, FSH release is partly induced by LHRH release and is partly LHRH-independent. This LHRH-independent release of FSH is subject to inhibitory feedback control by ovarian proteins (probably inhibin), and may obscure an LHRH-induced short-term loss of pituitary FSH responsiveness to LHRH. The object of this study was to establish whether short-term pituitary desensitization to single doses of LHRH results not only in a loss of LH response, but also of FSH response. Ovariectomized rats were used to eliminate the influence of steroid feedback. A group of ovariectomized rats was pretreated with steroid-free bovine follicular fluid (bFF) to suppress LHRH-independent FSH release, and phenobarbital to suppress LHRH-dependent FSH release respectively, 7 and 1 h before administration of LHRH. Another group received phenobarbital only. The animals were injected sequentially with either low or high doses of LHRH (1.25 or 10 ng/100 g body weight at times 0 and at 80, 120 or 180 min, and 6.25 or 50 ng/100 g at 60 min). Blood was taken for FSH measurements before and 5 and 10 min after each injection. Rats pretreated with bFF and phenobarbital showed an acute FSH response related to the dose of injected LHRH. No dose-response curve was seen in animals which had only been pretreated with phenobarbital.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Regulation by ovarian factors of the LHRH-induced LH response in pituitary glands in situ or grafted under the kidney capsule in intact and ovariectomized rats.

When pituitary glands from intact female rats are incubated with LHRH, the resulting LH release shows a biphasic pattern: an initial low rate of LH release (lag phase) is followed by a high rate. When pituitary glands from long-term ovariectomized rats are incubated, the rate of LH release is high throughout stimulation with LHRH. The disappearance of the lag phase might be due to increased LHRH release after ovariectomy and/or the disappearance of ovarian factors. To distinguish between these possibilities, pituitary glands which had been exposed to endogenous LHRH (pituitary glands in situ) or which had been unexposed to endogenous LHRH (pituitary glands transplanted under the kidney capsule) were incubated in the presence or absence of LHRH. Biphasic LH secretion patterns were observed during incubation with LHRH with the animal's own pituitary gland and with the transplanted pituitary gland from intact, but not from ovariectomized rats. Thus the disappearance of the lag phase after ovariectomy results from the absence of ovarian secretory products, rather than from increased release of LHRH.

Administration of a GnRH-antagonist to immature rats affects subsequent female and male pubertal development differently.

Gonadotropin secretion was inhibited in immature male and female rats by sc administration of the GnRH-antagonist ORG30276 (GnRH-A) on days 6, 9, 12 and 15. In GnRH-A-treated females this resulted in suppression of the temporarily increased plasma LH and FSH levels, which normally occur in prepubertal female rats. Ovarian weight was decreased. Although vaginal opening in GnRH-A-treated rats occurred earlier, the age of 1st estrus and the number of ova shed at first ovulation were not affected. Fertility at 4 months of age was normal. After initial suppression of gonadotropin levels, the FSH levels in GnRH-A-treated males, however, sharply increased to about twice the control levels. Plasma FSH remained elevated at least until 4 months of age. The LH levels at adult age were not affected by antagonist treatment and neither were testosterone levels. Testicular weight was decreased by GnRH-A from about 50% on day 15 to about 30% at 4 months of age. Pubertal development as measured by balano-preputial separation was delayed by about 7-10 days. At 4 months of age fertility was decreased. Thus, suppression of early gonadotropin secretion by GnRH-A treatment had dramatic effects on subsequent pubertal development in the male, but not in the female rat.

The frequency of pulsatile luteinizing hormone-releasing hormone treatment and luteinizing hormone and follicle-stimulating hormone secretion in women with amenorrhea of suprapituitary origin.

The influence of luteinizing hormone-releasing hormone (LH-RH) pulse frequency on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) was studied in hypogonadotropic hypogonadal women. They received three regimens of 5 days of pulsatile LH-RH (5 micrograms/pulse) given at 30-, 90-, or 180-minute intervals, with at least 6 weeks between treatments. On day 1, LH and FSH increased in proportion to the LH-RH pulse frequency. After 5 days of treatment with the 30- and 90-minute intervals, LH was still elevated, but FSH had returned to pretreatment levels together with a decline of the FSH response. The LH response only declined during treatment with the 30-minute pulse interval. During each treatment, estradiol (E2) increased. Explanations for dissociation between LH and FSH secretion during treatment with higher LH-RH pulse frequencies could be: (1) desensitization of FSH rather than LH secretion on LH-RH; (2) a differential effect of E2 on LH and FSH; (3) nonsteroidal ovarian factors selectively regulating LH and/or FSH release.

The self-priming action of LHRH increases the low pituitary LH and FSH response caused by ovarian factors: observations in vitro.

Pituitary glands taken from intact rats on day 2 of dioestrus and incubated with LHRH show a biphasic pattern of LH and FSH release. Initially the release of the gonadotrophins is low (first-phase or lag-phase response), but increases during further incubation with LHRH (second-phase or primed-state response). Removal of the influence of an unidentified ovarian factor either by ovariectomy or prolonged incubation in medium only leads to an increased (lag-phase) response to LHRH. The development of the increased response after prolonged incubation was prevented by the addition of cycloheximide to the media, implicating that this process is dependent upon the synthesis of protein. Steroid-free material (bovine follicular fluid or rat ovarian extracts) prevented the development of this process. In addition, it was shown that steroid-free rat ovarian extracts were also able to induce the development of a lag phase in pituitary glands from ovariectomized rats. Finally, it was found that steroid-free ovarian extracts reversed the self-priming effect of LHRH. The biological activity which reduced the responsiveness of the pituitary gland towards stimulation by LHRH was eliminated after the use of protein-denaturating techniques such as increased temperature or addition of methanol. The presence of this activity in ovaries, did not vary during the oestrous cycle, contrary to inhibin-like activity. Hence the ovarian factor responsible for the low lag-phase response is a protein which is probably not identical to inhibin. It is concluded that a non-steroidal ovarian factor reduces the responsiveness of the anterior pituitary gland to LHRH.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulsatile LH-RH treatment induces a relatively low response of LH and FSH, while discontinuation enhances the response in women with amenorrhea of suprapituitary origin.

Five women with amenorrhea of suprapituitary origin were given intravenous injections of 10 micrograms LH-RH every 90 minutes for 4 days by means of a portable infusion pump. Immediately before and after this, the LH and FSH responses to a test dose of 100 micrograms LH-RH were measured. Four days after discontinuation of the treatment, so that LH and FSH could be measured, blood was sampled every 10 minutes for a period of 6 hours, during which 20 micrograms LH-RH was injected intravenously every hour. Finally, a test dose of 100 micrograms LH-RH was given. The whole procedure was repeated at least 6 weeks later, but this time hourly injections of 100 micrograms LH-RH were given 4 days after discontinuation of the pulsatile LH-RH treatment. Four days after the pulsatile LH-RH treatment was stopped, increased LH and FSH responses to LH-RH were observed. These could be reduced by 6 injections, given hourly, of either 20 or 100 micrograms LH-RH. Although the totally released amount of both LH and FSH did not differ between the two treatment regimens irrespective of the LH-RH dose used, the response of both gonadotropins to the LH-RH test dose after the hourly 100 micrograms LH-RH injections was significantly lower. This indicated that desensitization can be attributed, at least in part, to a lower responsiveness of LH and FSH to LH-RH when pulsatile LH-RH is given. Low responses during treatment with pulsatile LH-RH could not be related to higher concentrations of plasma estradiol. We conclude that women with amenorrhea of suprapituitary origin who are treated with pulsatile LH-RH have a low state of responsiveness to LH-RH, which can be caused by the presence of the LH-RH and might be attributed in part to desensitization by LH-RH. Removal of the LH-RH results in an enhancement of the responsiveness, as the pituitary gland might have recovered from this desensitization.

Pulsatile GnRH treatment of the ovariectomized rat and release of LH and FSH.

The effect of pulsatile GnRH administration on the levels of LH and FSH was investigated in rats that had been ovariectomized 2 weeks earlier. Also the asynchronous occurrence of endogenous and GnRH-induced LH and FSH pulses was analysed. A small pulse dose of GnRH (1.25 ng/100 g) was given iv at a frequency of once every 60 min or once every 120 min during 24 h. A larger dose of 5 ng/100 g was given once every 60 or 120 min during either 24 h or 96 h. Blood was sampled arterially every 5 min around the two first and last GnRH injections and LH and FSH were measured. Only the treatment with the larger GnRH pulse dose resulted in a change of LH and FSH plasma levels. LH levels declined under all circumstances, whereas FSH was found to be increased temporarily after 24 h of treatment. The pituitary LH response to pulses of GnRH (5 ng/100 g) decreased irrespective of the frequency or duration with which GnRH was administered. There was a marked asynchronicity between LH and FSH pulses and almost every injection of GnRH (5 ng/100 g) resulted in clear LH pulses but not in FSH pulses.

The influence of pulsatile GnRH administration to the ovariectomized rat on the pituitary response to GnRH and the occurrence of spontaneous LH pulses.

Otherwise untreated adult ovariectomized rats were given pulses of GnRH (5 ng/100 g body weight iv) once every 60 or 120 min for 24 or 96 h. On the first and last day of the experiment plasma LH was estimated during the administration of GnRH pulses. Endogenous LH pulses between exogenously generated LH pulses were observed in nearly all animals on both the first and the last day, without any change in nadir and amplitude values. Shortly after an injection of GnRH, the spontaneous LH pulses were fewer than expected. The number of these pulses, however, increased again with time after the injections. The response to exogenous GnRH was reduced on the last day of the experiment. However, not all GnRH injections led to LH pulses. Most injections which did not result in an LH pulse appeared to be given within 15 min after a preceding endogenous LH pulse. The results obtained are in agreement with the hypothesis of an acute short-lasting desensitization of the pituitary gland caused by exogenous as well as endogenous pulses of GnRH.

Patterns of LH and FSH in men during high-frequency blood sampling.

The aim of the study was to test the hypothesis that in serial determinations of concentrations of LH and FSH involving blood samples taken every minute, the observed pulses of LH and FSH which last less than 3-4 min might not be a physiological phenomenon but part of the 'noise' of the radioimmunoassay or blood-sampling technique. Blood was sampled every minute for a period of 90 min in six men. During the first 45 min, blood was sampled by means of vacuum tubes only. During the second 45 min, sampling took place with a syringe via a rubber stopper, either using a tourniquet (n = 3) or flushing the cannula with heparinized saline. Three criteria were used to identify variations in the patterns of LH and FSH as true hormonal changes. First, a threshold was used which had to be exceeded by the difference between nadir and maximum values before a pulse could be identified. An average of approximately six pulses per 90 min was found in both the LH and FSH series. The majority of these pulses lasted less than 3-4 min. In two subjects, larger LH pulses of longer duration were measured. Secondly, differences between duplicate measurements of nadir and/or maximum values of more than one-third of the amplitude of a pulse were considered unacceptable. This involved about 75% of the pulses. Thirdly, the reproducibility of the hormone variations was estimated. In one subject, concentrations of LH were measured four times in four separate assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Short-term pituitary desensitization to luteinizing hormone-releasing hormone (LH-RH) after pulsatile LH-RH administration in women with amenorrhea of suprapituitary origin.

The existence of a short-term pituitary desensitization in luteinizing hormone (LH) release to single doses of luteinizing hormone-releasing hormone (LH-RH) in the ovariectomized rat was recently disclosed. The purpose of the present study was to investigate whether this refractoriness is also present in humans. Blood from six women with amenorrhea of suprapituitary origin was sampled every 10 minutes for 300 minutes for determination of LH and follicle-stimulating hormone (FSH). A pulse of 20 micrograms LH-RH was given intravenously 90 and 210 minutes after the first blood sample, and 2 micrograms LH-RH was given 30, 150, 240, and 270 minutes after t0. The mean maximal increments of LH and FSH were compared. The LH response to a 2-micrograms LH-RH bolus given 30 (t240) or 60 (t150) minutes after a 20-micrograms LH-RH pulse was significantly decreased, compared with the initial response to this dose at t30. For both LH and FSH, the response to 2 micrograms LH-RH given 30 minutes after the 20-micrograms pulse (t240) was almost absent, compared with 60 (t150) minutes after the 20-micrograms dose. We conclude that a short-term pituitary refractoriness to LH-RH is present after administration of single pulses of LH-RH in women with amenorrhea of suprapituitary origin and pulses of LH-RH in the physiologic range (2 micrograms) given to these women do not always generate LH and FSH increments that are identifiable as significant hormone pulses.

The involvement of ovarian factors in maintaining the pituitary glands of female rats in a state of low LH responsiveness to LHRH.

The effects of discontinuation and restoration of ovarian influences on the pituitary LH response to LHRH in vitro were investigated. When female rat pituitary glands taken on day 2 of dioestrus were incubated with LHRH the release of LH was low during the first hour (lag phase response) and afterwards a progressive, protein synthesis-dependent increase took place (second phase response), this being the self-priming action of LHRH. Short-term discontinuation (less than 1 day) of ovarian influences on the rat pituitary gland in vivo (ovariectomy) or in vitro (incubation in medium only) resulted in an increased LHRH-induced LH response during the lag phase. The biphasic LH response or the self-priming action of LHRH disappeared completely after long-term discontinuation of ovarian influences on the pituitary gland, LH release being at its maximum from the start of the incubation. The biphasic response was reinstated when ovaries were implanted under the kidney capsules of ovariectomized rats. Auto-implantation of an ovary into the spleen immediately after bilateral ovariectomy did not, however, prevent the disappearance of the LHRH self-priming action. Ovarian activity responsible for the presence of the low LH response during the lag phase was thus effectively removed by the liver, but inhibin-like activity suppressing serum FSH levels remained present. Silicone elastomer implants (s.c.) containing oestradiol-17 beta, implanted for 4 weeks, did not reverse the loss of the biphasic LH response to LHRH. It is concluded that liver-labile factors released by the ovaries keep the pituitary gland in a state of low responsiveness to LHRH.(ABSTRACT TRUNCATED AT 250 WORDS)

Phenobarbital enhances pituitary LH release induced by LRH pulses in the ovariectomized rat.

In the ovariectomized (OVX) rat, the plasma LH response was measured to a pulse of LRH (1.25 or 5 ng/100 g body weight, ia) given before and 1 h after ip administration of phenobarbital (80 mg/kg body weight). The LH response to the LRH pulses was increased 1 h after phenobarbital. In a second experiment, the pituitary LH content of OVX rats was measured 1 h after administration of phenobarbital or saline. No difference in pituitary LH content was found. It is concluded that in the OVX rat, phenobarbital increases the response to a pulse of LRH, presumably by suppressing endogenous pulsatile LRH. This, together with results of earlier experiments, further supports the hypothesis that under conditions where endogenous pulsatile LRH is present, there is always a certain degree of pituitary desensitization or refractoriness and that the removal of this endogenous LRH leads to recovery of pituitary sensitivity to LRH.

Comparison of the time courses of luteinizing hormone (LH) secretion rates during continuous stimulation by LH-releasing hormone (LH-RH) in vivo and in vitro.

The patterns of LH secretion during constant stimulation of the pituitary glands of estradiol-treated ovariectomized rats with a maximally stimulating amount of LH-RH in vivo and in vitro correspond with each other qualitatively and quantitatively. In vitro the changes with time of the LH secretion rate are somewhat retarded, especially the occurrence of desensitization.

Short-term pituitary desensitization to LH-RH after pulsatile LH-RH in the ovariectomized rat: an in vivo experiment.

The development of acute insensitivity of pituitary LH secretion to LH-RH after a short exposure to LH-RH is described. In the first experiment, ovariectomized (OVX), phenobarbital-pretreated rats were given pulses of LH-RH (1.25 or 6.25 ng/100 g body weight (b.w.), intravenously). In rats given 1.25 ng at time 0, 6.25 ng at 60 min, 1.25 ng at 80 min and 1.25 ng at 120 min, there was a substantial increase in plasma LH after the first two injections, no increase after the third injection and a relatively small increase after the fourth one. In other rats treated identically but not given a 1.25-ng dose at 80 min, the plasma LH rise in response to the 1.25-ng dose at 120 min was comparable to that seen after the 1.25-ng dose given at time 0. If the 1.25-ng LH-RH pulses given at times 0 and 80 min were replaced by a rat pituitary extract, the plasma LH rise in response to the 1.25-ng dose at 120 min was comparable to that seen after administration of pituitary extract. In the second experiment, OVX phenobarbital-pretreated rats were given 1.25 ng LH-RH/100 g b.w. at t = 0. They were then divided into three groups, each receiving 1.25, 3.75 or 6.25 ng LH-RH/100 g b.w. at t = 60 min. Each of these three groups was again divided into three groups which received 1.25 ng LH-RH/100 g b.w. at 80, 100 or 120 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of pretreatment of long-term ovariectomized (OVX) rats with a competitive LRH antagonist on FSH release in vitro.

The effect of a single sc injection of an LRH antagonist ((Ac-D-p-Cl-Phe1,2,D-Trp3,D-Phe6,D-Ala10)-LRH, Org 30093) into OVX rats on FSH release 24 h later was studied. Plasma FSH was decreased but pituitary FSH content was not changed. Incubation of the pituitary glands during 4 h resulted in a decreased basal release. FSH release induced by a low concentration of LRH (1 ng/ml) was depressed but that of a high concentration (10 000 ng/ml) was augmented in comparison to FSH release induced in control glands. However, pretreatment with the antagonist had no specific effect on FSH release in vitro induced by high K+ or high K+ plus mbcAMP and theophylline, indicating that the changes of pituitary responsiveness to LRH are not caused by those parts of the secretory mechanism which are stimulated by these secretagogues. Moreover, it is concluded that the changes of pituitary LH release induced by administration of an LRH antagonist also concern FSH.

Divergent effects on LH and FSH synthesis and release from intact female rat pituitary glands in vitro by methylxanthines, cyclic AMP derivatives and sodium fluoride.

FSH release from the female rat pituitary gland consists of an LH-like, LRH-dependent component and an autonomous, inhibin-sensitive component. It was investigated whether cyclic AMP mediated FSH release. BrcAMP, theophylline, MIX or NaF stimulated LH release but inhibited FSH release and synthesis. Although dbcAMP had no inhibitory effect on FSH release, it partly reversed the inhibitory action of theophylline. In view of previous and the present results it is concluded that cyclic AMP may mediate the LRH-dependent LH and FSH release and, through a separate pathway, may mediate the inhibition of autonomous FSH release by the ovarian protein inhibin.