Age-related changes in the secretory pattern of FSH and LH in response to LH-RH in prepubertal female rats

Developmental changes in the pituitary responsiveness and the secretory pattern of FSH and LH in response to a single injection of LH-RH (100 ng/rat, s.c.) as estimated by increases in plasma concentrations of FSH and LH 10, 30 and 60 min after the injection were studied in female rats at 5, 10, 15, 20, 25 and 30 days of age. The pituitary responsiveness to LH-RH for both FSH and LH release increased from 5 to 15 days of age, reached a maximum on 15 days of age and declined thereafter, whereas a marked increase in the amount of these hormones in the pituitary occurred between 15 and 20 days of age. An apparent change in the secretory pattern of both FSH and LH was observed from 20 days of age onward. In groups up to 15 days of age, plasma concentrations of FSH and LH remained elevated 60 min after the injection of LH-RH, though the plasma concentration of these hormones returned to preinjection concentrations in groups at 20 days of age or later. These results indicate that the age-related changes in the secretory pattern of LH and FSH in response to LH-RH as well as changes in the pituitary responsiveness were apparent during the prepubertal period.     

Inhibitory control of the pituitary LH secretion by LH-RH in male rats.

Luteinizing hormone-releasing hormone (LH-RH) was administered to prepubertal male rats (intact, castrate or castrate-adrenalectomized, 60 g body weight) for 28 days (1 microgram LH-RH/day, s.c.), at a 10-fold physiological dose, as compared to the minimal FSH-releasing dose of 100 ng/rat s.c. In intact rats, serum LH and weight of androgen-dependent organs (vented prostate, seminal vesicles) were reduced after 14 days of treatment. In castrate rats, the postcastration rise in serum LH was abolished by treatment. Pituitary LH content, FSH secretion and prolactin secretion were not suppressed. Hypothalamic LH-RH was increased at 14 and 21 days. In castrate adrenalectomized male rats, LH secretion was also suppressed by 1 microgram LH-RH s.c. x 28 days. The hypothalamic LH-RH content did not increase. The pituitary LH-RH receptor level was not down-regulated after 14 days treatment either in intact or castrate male rats. Pituitary inhibition (LH release) in rats by a supraphysiological dose of LH-RH given for 28 days indicates that the optimal regime for chronic treatment has to be determined by monitoring LH release at regular intervals. Direct pituitary inhibition by LH-RH may explain some of the unexpected antifertility effects observed with high doses of LH-RH.     

Changes in hypothalamic LH-RH content and blood levels of LH-RH, gonadotropin and estradiol during the preovulatory stage of rat estrous cycle.

In order to investigate the sequence of events concerning gonadotropin surge, serum LH, FSH and estradiol concentrations were measured during the rat estrous cycle as well as hypothalamic and blood levels of LH-RH in the preovulatory stage. Normally cyclic female Wistar rats kept on 12 hr light (from 22.00 hr to 10.00 hr) and 12 hr dark were killed at different times of day during each stage of the cycle. The hypothalamus was quickly dissected out, divided into 3 portions (the anterior, middle and posterior) and extracted in 90% methanol. Blood LH-RH was extracted by affinity chromatography prior to radioimmunoassay. The content of LH-RH in the anterior and middle hypothalamus started to decrease between 1.00 hr-3.00 hr, reached its nadir at 6.00 hr on proestrus and recovered to its previous values on estrus. Almost simultaneously blood LH-RH concentration showed an increase of 18.3 pg/ml-8.8 pg/ml between 1.00 hr-3.00 hr, and then fell to less than 1.0 pg/ml at 6.00 hr. On the other hand, serum estradiol level began to elevate on diestrus II followed by its peak at 6.00 hr on proestrus, while the peaks of serum LH and FSH were observed at 8.00 hr and 10.00 hr, respectively. These studies indicate that the elevation of serum estradiol was followed by the release of LH-RH from the hypothalamus and the LH-RH may be responsible for the preovulatory discharge of gonadotropin.     

Effect of steroids in combination with LH-RH on the release of LH and FSH in LH-RH-primed immature male rat.

LH and FSH release of immature male rats was remarkedably enhanced by LH-RH if primed with LH-RH one hour before. The effect of exogenous steroids on the action of the second LH-RH was investigated. C-18, C-19 and C-21 steroids in different doses were tested. Blood samples were collected from the jugular vein immediately before and 30 min after the second LH-RH injection. Serum LH and FSH were determined by respective radioimmunoassays. The concomitant increase of LH and FSH was not induced by all the steroids administered iv. Estrone or 17alpha-hydroxyprogesterone suppressed LH and FSH release. Estradiol-17beta preferentially suppressed LH release. Cortisone, progesterone or dehydroepiandrosterone significantly facilitated FSH release, whiel 20alpha-dihydroprogesterone or 20beta-dihydroprogesterone selectively promoted LH release. The sc injection of most steroids dissolved in oil tended to augment the acute release of LH but not FSH. 20alpha-Dihydroprogesterone was particularly potent in this concern. Dehydroepiandrosterone or androstenedione was effective in maintaining FSH release for a longer period. These data revealed that potentiated LH and FSH release induced by the second LH-RH was readily modified by steroids administered simultaneously.     

Self-priming effect of LH-RH on pituitary gonadotropins in hyperprolactinemic women

To investigate how various concentrations of serum prolactin (PRL) influence the priming effect of luteinizing hormone releasing hormone (LH-RH) on the pituitary gland, 24 women with various blood PRL concentrations received intravenous injections of 100 micrograms of synthetic LH-RH twice at an interval of 60 minutes and their serum LH and follicle-stimulating hormone (FSH) were measured and analysed. In the follicular phase with a normal PRL concentration (PRL less than 20 ng/ml, n = 6), marked first peaks of the two hormones following the first LH-RH stimulation and enhanced second peaks after the second LH-RH administration were observed, indicating a typical priming effect of LH-RH on gonadotropins, though the second response of FSH was more moderate than that of LH. In hyperprolactinemia, in which the serum PRL concentration was higher than 70 ng/ml (n = 13), the basal concentration of gonadotropins was not significantly changed but the priming effect of LH-RH on LH and FSH was significantly decreased (p less than 0.01). No marked second peaks of LH and FSH were observed, suggesting an inhibitory effect of hyperprolactinemia on the second release of LH and FSH. In contrast, this effect was restored in a group of women whose serum PRL concentration was between 30 and 50 ng/ml (n = 5). Furthermore, enhanced second peaks of both LH and FSH were noted after successful bromocriptine therapy reduced hyperprolactinemia (PRL greater than 70 ng/ml) to less than 25 ng/ml (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)     

Feedback control of FSH secretion in the male rat

Site of feedback control of FSH secretion in the male rat was studied by measuring changes in serum LH, FSH and hypothalamic LH-RH by radioimmunoassay in rats after castration and after 500 rad X-irradiation to the testis. The rise in serum LH and FSH in castrated animals was associated with a significant fall in hypothalamic LH-RH 16 and 24 days after castration. Serum FSH rose significantly after X-irradiation without a significant change in serum LH or hypothalamic LH-RH content up to 30 days after irradiation. When pituitary halves from X-irradiated animals were incubated in vitro in the presence or absence of synthetic LH-RH, there was a significant rise in FSH (but not LH) released in the incubation medium in the absence of added LH-RH. The response of the pituitaries to LH-RH was, however, not different between control and irradiated rats. It is concluded that the testicular FSH-inhibitory substance acts predominantly at the pituitary gland on the LH-RH independent release of FSH.     

Effect of hypothalamic extracts and synthetic LH-RH on proteic synthesis by rat hypothalamus in vitro (author's transl)]

The incorporation of 14C-Leucine in pituitary proteins in rats, in vitro, has been studied. In absence of stimulation, the pituitaries of adult female rats have shown approximately twice the capacity of protein biosynthesis in vitro than the pituitaries of prepuberal female rats (21 days old). For the stimulation in vitro of the pituitaries, synthetic LH-RH or hypothalamic extracts from adult or prepuberal female rats were used. The pituitaries of adult female rats did not respond to any of the stimulation tests employed. The pituitaries of prepuberal female rats increased their biosynthetic activity significantly, when synthetic LH-RH or adult female rat hypothalamic extract was added to the culture medium. The addition of prepuberal female rat hypothalamic extract did not alter the basic response. The female prepuberal rats injected during 5 consecutive days with FSH and LH, have shown a greater sensibility to LH-RH in vitro than the ones injected with estradiol and progesterone, or with synthetic LH-RH.     

Pituitary responsiveness to synthetic LH-RH and pituitary LH content at various reproductive stages in the sheep.

In sheep the basal concentration of LH in jugular vein plasma was significantly higher during the first 50 days of gestation in late pregnancy or at parturition. The pituitary response to a single i.v. injection of 200 microng synthetic LH-RH was determined at different stages of gestation and compared with that of anoestrous and cyclic sheep. Pituitary response to LH-RH decreased progressively with advancing gestation: by 56 days after mating the response had declined to 35% and by parturition to 14% of the value in anoestrous sheep. The pituitary response to LH-RH increased after parturition and the pattern of recovery differed in non-lactating and lactating sheep. By 63 days postpartum the response to LH-RH in non-lactating and lactating animals had returned to values similar to those in sheep during anoestrus and sheep during the luteal phase of the oestrous cycle. A decrease in pituitary responsiveness during pregnancy was associated with a decrease in pituitary content of LH. The quantity of LH released in response to a standard injection of LH-RH was linearly related to pituitary LH content.     

A paradoxical castration effect on LH-RH levels in male rat hypothalamus and serum

Serum and hypothalamic luteinizing hormone releasing hormone (LH-RH) was lowered in young mature male rats after castration. Testosterone injections raised the hypothalamic LH-RH content significantly. The mean value of serum LH level was elevated by testosterone, but not significantly. Hypothalamic LH-RH content was also lowered by hypophysectomy. In this circumstance, testosterone injections significantly increased LH-RH content. These results suggest that there may be a positive feedback of testosterone upon the hypothalamic LH-RH release and synthesis mechanisms.     

Norepinephrine stimulates LH-RH secretion into the hypophysial portal blood of the rat

The present study was designed to investigate the effect of intracerebroventricular infusion of norepinephrine (NE) on the secretion of luteinizing hormone-releasing hormone (LH-RH) into the hypophysial portal blood of steroid-primed ovariectomized rats. Saline infusion into the third ventricle caused no significant change in LH-RH levels. NE infusion (20 micrograms) resulted in a significant release of LH-RH (p less than 0.05) into the portal blood 10-30 min later. This endogenous LH-RH was similar to synthetic LH-RH when characterized by thin-layer chromatography. LH secretion in similarly treated rats but with intact portal vessels, also was significantly elevated (p less than 0.05) at 20 and 40 min after the start of NE infusion. These results show that NE stimulated the secretion of LH-RH into the hypophysial portal blood and this correlated with an enhanced release of LH.     

Alcohol decreases the alpha subunit, LH and testosterone secretion in response to LH-RH]

In our previous study we showed that alcohol disturbed the circadian rhythms of LH, testosterone and its conversion to DHT. To determine the effect of LH-RH on pituitary-gonadal function before and after alcohol, 11 male volunteers aged 24-29 years (mean 25.5) were investigated. Blood for hormonal estimations was withdrawn before and 20, 30, 60, and 120 min after LH-RH. In every case, the LH-RH test was performed twice: 6 hours after placebo and, a week later, 6 hours after alcohol administered orally, in dose of 1.0 g/kg bw. The LH, FSH, alpha-subunit and testosterone concentrations were measured with radioimmunological methods. Results: It was shown that alcohol significantly inhibited LH (p < 0.05), alpha-subunit (p < 0.02) and testosterone (p < 0.001) response to LH-RH stimulation, but not that of FSH.     

Effects of LH-RH on isolated pituitary gonadotropic cells.

Rat anterior pituitary glands were dissociated with Pronase and the cells were separated by velocity sedimentation at unit gravity. After 30 min of incubation of the enriched gonadotropic cells with LH-RH, there was a significant increase in LH and FSH in the incubation medium. LH-RH (100 ng/ml) and 10(-3) M cAMP both caused significant increases in LH in the incubation medium after 24 hr of incubation.     

Effect of LH-RH treatment on hypothalamo-pituitary-gonadal axis and Leydig cell ultrastructure in cryptorchid boys

62 cryptorchid boys aged 2-6 years were selected at random either for surgical or for hormonal LH-RH treatment. As all biopsies from boys operated show typical histological and ultrastructural signs of cryptorchidism, it can be concluded that only true cryptorchid patients are included in our study. LH-RH treatment was successful in 16 (55%) of 31 boys. Median 30-min response values to LH-RH test of LH were initially normal in all boys. Those treated successfully remain normal after treatment whereas unsuccessfully treated patients have significantly lower LH 30-min response values at the end of the 4 weeks of LH-RH treatment. FSH response was not statistically different before or after treatment. This might indicate that a normal LH response is necessary for testicular descent. In the 15 boys operated after 4 weeks of unsuccessful LH-RH treatment, testes biopsies show recruitment of the Leydig cell precursors and development of juvenile Leydig cells with an increase in their content of lipoid droplets and smooth endoplasmic reticulum. No adverse effect on the number of spermatogonia could be observed after 4 weeks of LH-RH treatment.     

Parellel inhibition of LH-RH-induced cyclic AMP accumulation and LH and FSH release by LH-RH antagonists in vitro.

The relative potencies of seven antagonists of LH-RH to inhibit LH-RH-induced cyclic AMP accumulation and LH and FSH release were measured using rat hemipituitaries in vitro. At appropriate concentrations, [Des-His2, D-Ala6] LH-RH, [Des-His2, D-Ala6, des-Gly-NH210] LH-RH ethylamide, [Des-His2, D-Leu6] LH-RH, [D-Phe2] LH-RH, [Des-His2, Des-Gly-NH210] LH-RH propylamide, [D-Phe2, D-Leu6] LH-RH and [D-Phe2, D-Phe6] LH-RH led to parallel inhibition of cyclic AMP accumulation and LH and FSH release. [D-Phe2, D-Leu6] LH-RH and [D-Phe2, D-Phe6] LH-RH can inhibit 50% of LH-RH action at molar ratios of 100 and 30, respectively. These findings of parallel changes of cyclic AMP levels and LH and FSH release add strong support to the already obtained evidence for a mediator role of the adenylate cyclase system in the action of LH-RH in the anterior pituitary gland.     

Hypersensitivity of the human gonadotrophs to the repeated administration of small doses of LH-RH.

The functional capacity of the gonadotrophs was assessed by repeated stimulation with small doses of LH-RH (5 microgram intravenously at 2-hour intervals for 3 injections) in normal women during the early and late follicular phases of the menstrual cycle. The results were compared to those obtained when a single dose (100 microgram) of the neurohormone was administered. During the early follicular phase, the release of LH and FSH remained about equal after the 3 successive injections of the small and after the large dose of LH-RH. During the late follicular phase, the release of LH and fsh increased progressively after the repeated administration of the 5 microgram of the neurohormone while the large dose induced a more pronounced and a more sustained pituitary response. This hypersensitivity of the gonadotrophs is observed when the E2 concentrations are higher than in the early follicular phase of the menstrual cycle.     

Differential effects of LH-RH immunoneutralization on LH and FSH secretion in the ewe

Neutralization of LH-RH by injection of an ovine antiserum to LH-RH in ewes during the late follicular phase of the oestrous cycle resulted in an immediate blockade of pulsatile secretion of LH. Plasma concentrations of FSH gradually rose in the antiserum-treated ewes during the 36-h study period but levels declined in control ewes. These results show that, in the ewe, pulsatile LH secretion is dependent on LH-RH from the hypothalamus, while FSH is largely unresponsive to short-term reduction of LH-RH stimulation. Since reduction in LH secretion is likely to reduce ovarian function, the changes in FSH secretion may be attributed to the removal of a negative feedback influence of an ovarian factor, perhaps oestradiol, on FSH secretion.     

The temporal relationship between plasma levels of FSH and LH in the ram.

Blood samples were collected from adult Soay rams at frequent intervals during the regressed, developing and active phases of the sexual cycle, or after i.v. injections of synthetic LH-RH or standard preparations of ovine FSH and LH, and were assayed for FSH, LH and testosterone. The highest FSH and LH levels occurred during the developing stage of the cycle, and the highest testosterone levels during the active stage. At each phase there were conspicuous short-term fluctuations in the concentrations of all three hormones, and episodic peaks of plasma LH were associated with increases in both FSH and testosterone. Synthetic LH-RH (5 microgram) induced a rise in the plasma values of FSH and LH at all stages and the magnitude and duration of the response could be predicted from a knowledge of the endogenous short-term fluctuations. When 50--250 ng LH-RH were given i.v. plasma LH increased in a way similar to that occurring spontaneously. The ratio of FSH:LH released after LH-RH changed with the stage of the sexual cycle, but the clearance rate of the two hormones was not affected. These findings are consistent with the control of FSH and LH by a single releasing hormone which is secreted in pulses. The different temporal patterns in the circulating FSH and LH result from differences in secretion and clearance.     

Effects of an LH-RH analogue in male rats pretreated with oestradiol benzoate.

Treatment of adult male rats with oestradiol benzoate (OB) for 21 days significantly decreased the body, testicular and accessory sex organ weights but increased anterior pituitary weight. OB treatment also significantly suppressed circulating FSH and LH levels as well as plasma and testicular concentrations of testosterone. The seminiferous tubules and interstitial cells were partly atrophied, and there was some effect on spermatogenesis, with step 14 to 19 spermatids being fewer than normal. Rats treated with OB for 21 days were then treated daily with LH-RH analogue ((D-Leu6, des-Gly-NH2(10))-LH-RH-ethylamide), to see if testicular function could be recovered. Circulating gonadotrophins were significantly elevated, testicular histology was normal and testicular and plasma testosterone concentrations and the accessory sex organ weights remained suppressed. These results suggest possible extra-pituitary effects of the LH-RH analogue, including a direct action on the testes and/or accessory sex organs.     

A comparison of the LH-releasing activities of LH-RH and its agonistic analogue buserelin in the ovariectomized rat

LH-RH and the potent agonistic analogue (D-Ser(But)6-des-Gly10)-LH-RH(1-9)-ethylamide (HOE-766 or buserelin) were at several doses either infused or injected intravenously in 5-weeks-ovariectomized rats, which had been treated with either 3 micrograms estradiol-benzoate (EB) or with oil, 24 h previously. Blood samples for assay of LH were taken during the subsequent 24 h. Pituitary glands were removed at the end of the experiments. Buserelin, when infused, was slightly more effective than LH-RH on releasing LH. When injected, however, buserelin was at the higher dose ranges increasingly more effective as an LH-releasing agent than LH-RH. EB-treatment increased the LH response of the pituitary gland to both peptides in an identical way. It was concluded that buserelin derives its high potency not from its intrinsic LH-releasing activity, which is only slightly greater than that of LH-RH, but from a longer duration of action.     

Effect of bromocriptine on the hypothalamo-pituitary function in adult male rats.

Daily oral administration of bromocriptine (50 μg/kg) to adult male rats, suppressed serum prolactin levels. The pituitary prolactin levels remained unaltered. Serum FSH levels as well as pituitary FSH levels showed no significant change as compared to the controls. Serum LH levels were significantly decreased in spite of the high pituitary LH levels, in bromocriptine treated rats. In the drug treated rats, $\text{in vitro}$ sensitivity of the pituitary to the exogenous LH-RH was not altered; whereas hypothalamic LH-RH content was considerably lowered. These observations suggest the possible effect of bromocriptine on the synthesis of LH-RH in the hypothalamus which leads to the accumulation of LH in the pituitary and decline of serum LH.