Effects of hyperadrenal states on luteinizing hormone in cattle

The effect of an induced hyperadrenal state on luteinizing hormone (LH) secretion and subsequent ovarian function was examined in both intact and adrenalectomized (ADRX) heifers. Treatments were begun on Day 2 or Day 16 of an estrous cycle in order to examine their effect on corpus luteum development or ovulation, respectively. In Experiment I, continuous intravenous infusion of ACTH (1.0 mg/24 h) to intact heifers decreased LH concentrations during the early phase of the cycle (Days 3-5). Treatment of ADRX heifers with hydrocortisone succinate (HS) (100 mg/24 h) did not appear to change mean LH concentrations, although da Rosa and Wagner (1981) have reported reduced plasma concentrations of progesterone at mid-cycle in these ACTH-treated intact heifers and HS-treated ADRX heifers. ACTH treatment of ADRX heifers had no effect on LH or progesterone. In the second study, there were similar frequencies of LH surges at the anticipated time of ovulation in all treatment groups. HS (100 mg/24 h) in ADRX heifers and ACTH (0.5 mg/24 h) in intact heifers was given continuously beginning on Day 16 of an estrous cycle. Although some animals in all groups exhibited LH surges, the ACTH-treated intact and HS-treated ADRX heifers failed to show a consistent subsequent increase in progesterone concentrations in plasma, suggesting a failure of luteal development. Although no difference was seen in baseline concentrations of LH, there was a greater difference between basal and overall mean LH concentrations in control groups than was observed in ACTH- or HS-treated animals. These induced hyperadrenal states resulted in depression of ovarian function as shown by decreased plasma progesterone during the luteal phase of the cycle. It is not known if other noncorticoid steroids from the adrenal cortex are necessary for a full expression of this effect.     

Effect of cortisol or adrenocorticotropic hormone on luteinizing hormone secretion by pig pituitary cells in vitro

The effect of cortisol or adrenocorticotropic hormone (ACTH) on basal and gonadotropin-releasing hormone (GnRH)-induced secretion of luteinizing hormone (LH) was studied in vitro using dispersed pig pituitary cells. Pig pituitary cells were dispersed with collagenase and DNAase and then grown in McCoy's 5a medium containing 10% dextran charcoal-pretreated horse serum and 2.5% fetal calf serum for 3 days. Cells were preincubated with cortisol or ACTH before GnRH was added. When pituitary cells were incubated with 400 micrograms cortisol/ml medium for 6 h or longer, increase basal secretion of LH was observed. However, GnRH-induced LH release was reduced by cortisol. The degree of this reduction was dependent on cortisol, and a concentration of cortisol higher than 100 micrograms/ml was needed. Cortisol also inhibited the 17 beta-estradiol-induced increase in GnRH response. ACTH-(1-24), ACTH-(1-39), or porcine ACTH had no influence on GnRH-induced LH secretion. Our results show that cortisol can act directly on pig pituitary to inhibit both normal and estradiol-sensitized LH responsiveness to GnRH.     

The inhibitory action of corticotropin-releasing hormone on gonadotropin secretion in the ovariectomized rhesus monkey is not mediated by adrenocorticotropic hormone

Earlier observations in our laboratory indicated that i.v. infusion of human/rat corticotropin-releasing hormone (hCRH) suppresses pulsatile luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release in ovariectomized rhesus monkeys. Since cortisol secretion increased significantly as well, it was not possible to exclude the possibility that this inhibitory effect of hCRH on gonadotropins was related to the activation of the pituitary/adrenal axis. The purpose of the present study was to determine the role of pituitary/adrenal activation in the effect of hCRH on LH and FSH secretion. We compared the effects of 5-h i.v. infusions of hCRH (100 micrograms/h, n = 7) and of human adrenocorticotropic hormone (ACTH) (1-24) (5 micrograms/h, n = 3; 10 micrograms/h, n = 3, 20 micrograms/h, n = 3) to ovariectomized monkeys on LH, FSH, and cortisol secretion. As expected, during the 5-h ACTH infusions, cortisol levels increased by 176-215% of baseline control, an increase similar to that observed after CRH infusion (184%). However, in contrast to the inhibitory effect observed during the CRH infusion, LH and FSH continued to be released in a pulsatile fashion during the ACTH infusions, and no decreases in gonadotropin secretion were observed. The results indicated that increases in ACTH and cortisol did not affect LH and FSH secretion and allowed us to conclude that the rapid inhibitory effect of CRH on LH and FSH pulsatile release was not mediated by activation of the pituitary/adrenal axis.     

Steroid hormone feedback on LH in prepubertal Holstein heifers

A significant dose-response relationship between gonadotropin-releasing hormone (GnRH) and time to luteinizing hormone (LH) peak, peak serum LH and total serum LH was obtained in prepubertal Holstein heifers (28 weeks of age) (Experiment 1). For the second experiment, the effect of steroid feedback on the anterior pituitary was determined. A steady infusion of saline, estradiol-17 beta or progesterone was maintained for 24 h while GnRH, in various schemes, was administered 8 h after the beginning of steroid infusion. Estradiol-17 beta infusion (2.08 micrograms/h), although it did not affect peripheral concentrations of estrogen, caused an LH release 24 to 30 h later in 37.5% of the heifers. This amount of exogenous estrogen did not affect the LH response to a single GnRH (4 micrograms) challenge. When the same GnRH dosage (4 micrograms) was administered 6 times at hourly intervals, the heifers infused with estradiol had a lower response after the first 2 injections of GnRH and a greater response after the last 4 injections than heifers infused with saline. When GnRH was infused (4 micrograms/h) for 6 h, beginning 8 h after steroid infusion, estradiol infusion caused a significantly higher peak LH and total LH release than an infusion of either saline or progesterone (7.3 micrograms/h). The progesterone infusion had no effect on the GnRH-stimulated LH release. We conclude that prepubertal dairy heifers have an anterior pituitary capable of responding to the feedback effect of estrogen in a positive manner.     

Effects of hyperprolactinemia on the control of luteinizing hormone and follicle-stimulating hormone secretion in the male rat

Experiments were conducted to determine the effects of acute hyperprolactinemia (hyperPRL) on the control of luteinizing hormone and follicle-stimulating hormone secretion in male rats. Exposure to elevated levels of prolactin from the time of castration (1 mg ovine prolactin 2 X daily) greatly attenuated the post-castration rise in LH observed 3 days after castration. By 7 days after castration, LH concentrations in the prolactin-treated animals approached the levels observed in control animals. HyperPRL had no effect on the postcastration rise in FSH. Pituitary responsiveness to gonadotropin hormone-releasing hormone (GnRH), as assessed by LH responses to an i.v. bolus of 25 ng GnRH, was only minimally effected by hperPRL at 3 and 7 days postcastration. LH responses were similar at all time points after GnRH in control and prolactin-treated animals, except for the peak LH responses, which were significantly smaller in the prolactin-treated animals. The effects of hyperPRL were examined further by exposing hemipituitaries in vitro from male rats to 6-min pulses of GnRH (5 ng/ml) every 30 min for 4 h. HyperPRL had no effect on basal LH release in vitro, on GnRH-stimulated LH release, or on pituitary LH concentrations in hemipituitaries from animals that were intact, 3 days postcastration, or 7 days postcastration. However, net GnRH-stimulated release of FSH was significantly higher by pituitaries from hyperprolactinemic, castrated males. To assess indirectly the effects of hyperPRL on GnRH release, males were subjected to electrical stimulation of the arcuate nucleus/median eminence (ARC/ME) 3 days postcastration. The presence of elevated levels of prolactin not only suppressed basal LH secretion but reduced the LH responses to electrical stimulation by 50% when compared to the LH responses in control castrated males. These results suggest that acute hyperPRL suppresses LH secretion but not FSH secretion. Although pituitary responsiveness is somewhat attenuated in hyperprolactinemic males, as assessed in vivo, it is normal when pituitaries are exposed to adequate amounts of GnRH in vitro. Thus, the effects of hyperPRL on pituitary responsiveness appear to be minimal, especially if the pituitary is exposed to an adequate GnRH stimulus. The suppression of basal LH secretion in vivo most likely reflects inadequate endogenous GnRH secretion. The greatly reduced LH responses after electrical stimulation in hyperprolactinemic males exposed to prolactin suggest further that hyperPRL suppresses GnRH secretion.     

Effect of serotonin on the basal and gonadotrophin-releasing hormone-induced release of luteinizing hormone from rat pituitary glands in vitro

The effect of serotonin (5-HT) on the basal and gonadotrophin-releasing hormone (GnRH)-stimulated release of luteinizing hormone (LH) was studied in rat adenohypophysis in vitro. Anterior pituitary glands from ovariectomized rats were incubated for 1h in the presence of different doses of 5-HT (0.01 to 3 mumol/l). Serotonin added to the culture medium slightly dimished the basal release of LH and markedly inhibited the release of LH induced by GnRH. Responsiveness to GnRH (3 nmol/l) was significantly reduced, in a dose-dependent manner, by the simultaneous treatment of glands with 5-HT. Maximal inhibition to 65% of the response obtained with GnRH alone, was attained with 1 mumol/l 5-HT. The EC50 value was estimated to be about 1.9 X 10(-7) M. The inhibitory effect of 5-HT was evident within 30 min of incubation. Furthermore, 5-HT appear to exert a short-lasting action, since the rate of basal and GnRH-induced release of LH was reduced during the first hour of incubation, but after 2h the suppressive effects of 5-HT were no longer apparent. Methysergide, a serotonin receptor blocking agent, partially antagonized the inhibitory effect of 5-HT on LH release, either basal or GnRH-stimulated. This suggests that a receptor-mediated component may be involved in the mechanism of 5-HT action. The present results indicate that 5-HT can affect the release of LH by acting directly at the pituitary gland level.     

Direct opioid regulation of pituitary release of bovine luteinizing hormone

Although endogenous opioid peptides (EOP) are thought to alter pituitary release of luteinizing hormone (LH) by modifying the release of gonadotropin-releasing hormone (GnRH) from the brain, EOP may also directly affect the release of LH from pituitary cells. This hypothesis was tested using dispersed cells from the bovine anterior pituitary gland. Pituitaries were enzymatically dissociated, preincubated for 18 h and then cultured for either 2 or 24 h with GnRH, naloxone, methionine-enkephalin (Met-enk) or their combinations. Basal release of LH into media was 18.2 and 38.4 ng/100,000 cells after culture for 2 or 24 h, respectively. When cultured for 2 or 24 h with 10 nM GnRH, LH release was 296% and 131% of the basal release for each culture period. Cellular viability (75% vs 68%) and total (cells + medium) LH (128 vs 134 ng/100,000 cells) did not differ (P greater than .05) between cells cultured for 2 or 24 h. Naloxone (1 microM) increased (P less than .01) basal release of LH by 57% after 2 h of culture but not after 24 h of culture. Naloxone did not augment the amount of LH released in response to 10 nM GnRH. Addition of Met-enk (1 nM to 1 microM) suppressed (P less than .05) basal release of LH (23% to 62%) after 2 h of culture. Similar suppressive effects (8% to 49%) occurred in a dose-dependent manner (0.1 nM to 1 microM) after 24 h of culture. Met-enk (1 and 100 nM) antagonized (P less than .05) the stimulatory effect of naloxone and reduced (P less than .05) the amount of LH released in response to GnRH after 2 h of culture. In summary, the stimulatory effect of naloxone on the basal release of LH suggests that EOP may directly regulate pituitary cell function; the inhibitory effect of physiological concentrations of Met-enk on the basal in vitro release of LH suggests that EOP may directly affect the release of LH in vivo; the antagonism between the stimulatory effect of naloxone and the inhibitory effect of Met-enk is consistent with effects exerted through opioid receptors; and the stimulatory effect of GnRH may be partially reduced by Met-enk. These results are consistent with the hypothesis that opioids may directly modulate the release of LH at the pituitary level.     

Influence of acute stress and the adrenal axis on regulation of LH and testosterone in the male rhesus monkey (Macaca mulatta)

In order to determine the mechanism by which stress may affect the secretion and function of luteinizing hormone (LH) in primates, the response of the adrenal and gonadal axes was followed in male rhesus monkeys during brief restraint in primate chairs and during various hormone treatments. To further assess the responsiveness of the gonadal axis, gonadotropin releasing hormone (GnRH) was administered during the experiments. Corticosteroid levels were elevated throughout the first restraint trial as compared to those in subsequent trials. LH was elevated in the first sample of the first trial as compared to that in the following trials. The responses of LH to GnRH were equivalent in all trials, while the testosterone response to GnRH was attenuated in the first trial. A single injection of adrenocorti-cotropin (ACTH, 40 IU), while increasing circulating corticosteroids similarly to that observed during the first restraint trial, failed to cause an acute initial release of LH. However, ACTH did lower the testosterone response to GnRH. Following 5 days of ACTH treatment (40 IU twice daily), basal LH was suppressed, and the testosterone response to GnRH was decreased. Following 5 days of cortisol injections (100 mg twice daily), basal LH and testosterone were suppressed, but again only the testosterone response to GnRH was attenuated. Acute restraint stress, acting by some mechanism other than the activation of adrenal axis, stimulates a transient release of LH. While the stress-stimulated release of corticosteroids failed to affect the LH response following GnRH administration, it did act directly on the testes to prevent the normal release of testosterone. Finally, chronic elevation of corticosteroids, produced by ACTH or cortisol administration, suppressed basal serum LH and attenuated the response of testosterone to GnRH.     

Effect of neuropeptide Y on GnRH-induced LH release from bovine anterior pituitary cell cultures derived from heifers in a follicular,luteal or ovariectomized state

Objectives were to determine if neuropeptide Y (NPY) had direct effects GnRH induced secretion of LH from the anterior pituitary gland, and if endogenous steroids modulated the effect of NPY. To accomplish these objectives, 15 Hereford heifers were assigned to one of three ovarian status groups: follicular, luteal, or ovariectomized. One animal from each of the three ovarian status groups was slaughtered on each of 5 days and anterior pituitary gland harvested. Anterior pituitary gland cells within ovarian status were equally distributed and randomly assigned to one of three cell culture treatments: no NPY or GnRH (control), 10 nM GnRH, or 100 nM NPY+10 nM GnRH. Anterior pituitary cell cultures were incubated with or without NPY for 4 h and further incubated for an additional 2 h with or without GnRH and supernatant collected for quantification of LH. Treatment of anterior pituitary cell cultures with GnRH or GnRH+NPY did not affect LH release in cultures obtained from follicular (S.E.=5%; P=0.58) or ovariectomized (S.E.=7%; P=0.22) heifers. Both GnRH and GnRH+NPY increased LH release from anterior pituitary cell cultures from heifers in the luteal phase (S.E.=14%; P < or = 0.05) compared to control cultures. Cultures from luteal phase heifers treated with GnRH did not differ from those treated with GnRH+NPY (P=0.34). These data provide evidence to suggest that effects of NPY on LH release may occur primarily at the level of the hypothalamus.     

Differential actions of corticosterone on luteinizing hormone and follicle-stimulating hormone biosynthesis and release in cultured rat anterior pituitary cells: interactions with estradiol

Previous in vivo studies from our laboratory suggested that glucocorticoids antagonize estrogen-dependent actions on LH secretion. This study investigated whether corticosterone (B) may have similar actions on gonadotropin biosynthesis and secretion in vitro. Enzymatically dispersed anterior pituitary cells from adult female rats were cultured for 48 h in alpha-modified Eagle's medium containing 10% steroid-free horse serum with or without 0.5 nM estradiol (E2). The cells were then cultured for 24 h with or without B in the presence or absence of E2. To evaluate hormone release, 5 x 10(5) cells were incubated with varying doses of GnRH (0, 10(-11)-10(-7) M) or pulsatile GnRH (10(-9) M; 20 min/h) for 4 h. Cell and medium LH and FSH were measured by RIA. To evaluate LH biosynthesis, 5 x 10(6) cells were incubated for an additional 24 h with 10(-10) M GnRH, 60 microCi 3H-glucosamine (3H-Gln), 20 microCi 35S-methionine (35S-Met), and the appropriate steroid hormones. Radiolabeled precursor incorporation into LH subunits was determined by immunoprecipitation, followed by SDS-PAGE. Continuous exposure to GnRH stimulated LH release in a dose-dependent manner, and this response was enhanced by E2. B by itself had no effect on LH release, but inhibited LH secretion in E2-primed cells at low concentrations of GnRH (10(-10) M or less). Total LH content was not altered by GnRH or steroid treatment. Similar effects of B were observed in cells that were given a pulsatile GnRH stimulus. In contrast to LH, E2 or B enhanced GnRH-stimulated FSH release at the higher doses of GnRH, while the combination of E2 and B increased basal and further augmented GnRH-stimulated release. Total FSH content was also increased in the presence of B, but not E2 alone, and was further augmented in cells treated with both steroids. There were no effects of the steroids on the magnitude of FSH release in response to GnRH pulses, but the cumulative release of FSH was greater in the E2 + B group compared to controls, indicating an increased basal release. Independent of E2, B suppressed the incorporation of 3H-Gln into LH by more than 50% of control, with only subtle effects on the incorporation of 35S-Met.(ABSTRACT TRUNCATED AT 400 WORDS)     

Independent actions of gonadotropin releasing hormone upon cyclic GMP production and luteinizing hormone release

Gonadotropin releasing hormone (GnRH) and its potent analog [D-Ser(tBu)6]des-Gly10-GnRH N-ethylamide elevate pituitary cyclic GMP levels while stimulating gonadotropin release in cultured pituitary cells. Addition of mycophenolic acid to pituitary cell cultures decreased basal and GnRH-induced cGMP production to undetectable levels, but did not reduce basal or GnRH-stimulated luteinizing hormone (LH) release. Elevation of endogenous cGMP levels by sodium nitroprusside, or addition of cGMP or its potent derivatives, was also without effect on basal or GnRH-stimulated LH release. These findings demonstrate that the elevation of intracellular cGMP during GnRH action does not mediate the release of LH by pituitary cells.     

Long-term superfusion of rat pituitary cells: interaction of 17 beta-estradiol with pulses of gonadotropin-releasing hormone on luteinizing hormone release

In a series of four experiments, the temporal development of acute inhibitory and delayed stimulatory effects of 17 beta-estradiol (E) on luteinizing hormone (LH) release by superfused rat anterior pituitary cells pulsed with gonadotropin-releasing hormone (GnRH) was studied. Dispersed anterior pituitary cells from ovariectomized rats were cultured on Bio-Beads for 3 days and then placed in columns and superfused for up to 24 hr. During superfusion, the cells were exposed to GnRH pulses (3 X 10(-9) M, one 6-min pulse/hr). Cells treated with E (3 X 10(-10) M) either before (only 24 hr prior to superfusion) or before and during superfusion released significantly (P less than 0.05) more LH in response to the first few pulses of GnRH than cells treated with diluent. In contrast, cells treated with E only during superfusion initially released less GnRH-induced LH than cells treated with diluent. In a subsequent experiment, the inhibitory effect of E reached a maximum by 1.5 hr (P less than 0.01), and then gradually disappeared after 4.5 hr. Cells superfused simultaneously with E and fixed "low"-dose GnRH (5 X 10(-10) M) pulses did not exhibit enhanced LH responses with time to that dose of GnRH. However, E-superfused cells responded more than diluent-superfused cells to subsequent stimulation with a higher-dose GnRH pulse. Superfusion of cells with E for 16.5 hr in the absence of GnRH pulses also did not increase release of LH to low-dose (5 X 10(-10) M) pulses of GnRH, yet did cause a transitory increase to subsequent high-dose (10(-8) M) GnRH pulses. In conclusion, these results demonstrate the direct biphasic inhibitory then stimulatory effects of E on GnRH-induced LH release by superfused rat anterior pituitary cells. Expression of the stimulatory effect of E is related to the dose of GnRH.     

Interrelationship between estrogen and thyroxine on the release of luteinizing hormone and gonadotropin-releasing hormone in vitro

The present experiments were designed to study the interaction between estradiol benzoate (EB) and thyroxine (T4) given in vivo on the responsiveness of pituitary luteinizing hormone (LH) to gonadotropin-releasing hormone (GnRH) and the release of GnRH in vitro. Ovariectomized-thyroidectomized (Ovx-Tx) rats were injected s.c. with saline or T4 (2 micrograms/100 g b.wt), and oil or EB (0.1 microgram) once daily for 40 days following a 2 x 2 factorial design. All animals were then decapitated and blood samples were collected. Anterior pituitaries (APs) were incubated in vitro with and without 0.1 ng GnRH at 37 degrees C for 4 h. Mediobasal hypothalami (MBHs) were excised and then incubated with and without APs from Ovx donor rats. Concentrations of LH and GnRH in the medium and that of LH in the serum were measured by radioimmunoassay. The LH level in media containing MBHs and donor APs was used as the index of bioactive GnRH release. In Ovx-Tx rats, T4 injections reduced the serum LH concentration, the pituitary LH response to GnRH, and the bioactive as well as the immunoreactive GnRH release. The serum LH levels and the spontaneous as well as the GnRH-stimulated release of LH in vitro were suppressed in Ovx-Tx rats following administration of EB. By contrast, the serum LH concentration, as well as pituitary LH response to GnRH and GnRH release in vitro, were higher in the group treated with both T4 and EB than in that treated with saline and EB. These results suggest that the differential changes in the LH secretion after thyroidectomy of Ovx versus non-Ovx rats are due to an antagonistic effect between T4 and estrogen on the response of pituitary LH to GnRH, and the release of GnRH.     

Effects of exogenous melatonin on human pituitary and adrenal secretions. Hormonal responses to specific stimuli after acute administration of different doses at two opposite circadian stages in men

We evaluated the effect of an acute oral administration of 2 dosages (100 and 1 mg) of melatonin (MT) vs placebo (PL) on pituitary release of LH, FSH, TSH and PRL after GnRH + TRH and on the adrenocortical release of cortisol, aldosterone and progesterone after ACTH in healthy adult males. We carried out a double blind study on 6 volunteers in winter-early spring, at 2 opposite phases of the circadian cycle: 08(00) and 20(00). Injection of GnRH (100 micrograms), TRH (200 micrograms) and ACTH (10 micrograms of the synthetic analogue ACTH 1-17, alsactide) was performed 1 h after MT or PL ingestion. The measurement of plasma MT levels confirmed its effective gastrointestinal absorption after both doses. The hormonal patterns were superimposable after MT and PL. A higher response of FSH, PRL, cortisol and aldosterone was observed in the evening vs morning protocols independently of previous treatment (MT or PL). Our data demonstrate that the acute oral administration of 2 different doses of MT at 2 opposite circadian stages is ineffective as to the modification of a variety of pituitary and adrenocortical responses in human male subjects. The circadian chronosusceptibility of pituitary and adrenocortical cells to specific stimuli deserves interest to future investigation.     

Serum luteinizing hormone, 13,14-dihydro-15-keto-prostaglandin F2alpha and cortisol profiles during postpartum anestrus in Brahman and Angus cows

Pluriparous suckled Brahman and Angus cows were utilized to evaluate the effect of breed, day after calving and endogenous opioid peptides (EOP) on hormonal profiles during postpartum anestrus. On Days 17 and 34 after calving, blood samples with and without heparin were collected at 15- and 30-min intervals, respectively, for a 7-h period via jugular cannula. Two hours after the start of blood sampling, cows of each breed were administered either 1 mg/kg iv naloxone or saline. Three hours later, all animals received 10 ng/kg iv GnRH. On Day 34 after calving cows received 0.2 IU/kg iv ACTH. Mean LH, basal LH and area under the LH curve increased (P < 0.01) from Day 17 to Day 34 after calving. Height of LH pulses increased (P < 0.05) by Day 34 after calving. Brahman cows had higher (P < 0.05) mean LH, basal LH, LH pulse frequency and area under the LH curve than Angus cows. Naloxone increased postchallenge area under the LH curve in treated cows above that of control cows (P < 0.06). Naloxone also increased the postchallenge area under the LH curve above that of the prechallenge level (P < 0.01). No breed differences in the response to the naloxone challenge were observed. The LH response to naloxone challenge occurred earlier on Day 34 than on Day 17 after calving but the amount of LH released was similar between days. The GnRH-induced LH release was greater in Brahman than in Angus cows (P < 0.04). Mean cortisol concentrations and area under the cortisol curve decreased (P < 0.05) between Day 17 and Day 34 after calving. Mean cortisol concentrations and area under the cortisol curve were lower (P < 0.01) in Brahman than in Angus cows. Cortisol secretion after ACTH treatment was similar between Brahman and Angus cows. The cortisol response after ACTH challenge was positively correlated (r=0.68; P < 0.001) to the prechallenge area under the cortisol curve. Under optimal environmental conditions Brahman cows have a greater LH release and their anterior hypophysis is more sensitive to GnRH challenge than the Angus cows.     

Early pituitary follicle stimulating hormone response to gonadotrophin releasing hormone in ewes

The object of our experiments was to characterize the response of plasma follicle stimulating hormone (FSH) within minutes of an i.v. injection of high or low doses of gonadotrophin releasing hormone (GnRH), especially in relation to contemporary changes in luteinizing hormone (LH) concentrations. In the deep anoestrous period (June), three intact ewes and two ovariectomized ewes were injected with 1 mug synthetic GnRH followed 2 h later by a second identical injection. A week later, the same regimen was repeated with the same sheep but with 50 mug GnRH after an interval of 5 h 20 min. Blood samples were collected every 15 sec for 15 min after each injection (early release), then at longer intervals (main release) till the next treatment, followed by sampling for a further 6-h period after the second treatment. FSH was released as soon as the second minute after GnRH injection in all ewes. The mean pituitary FSH response, during this early release, in intact and ovariectomized ewes was similar after either 1 or 50 mug GnRH. However, the main release was less pronounced in the ovariectomized sheep and was not stimulated after the second treatment in all sheep. Three other ewes were injected with 40 mug GnRH and sampled every 15 sec for seven, 6-min periods during the period of release to compare FSH and LH secretion. The profiles reflected a similarity in sensitivity and responsiveness to GnRH, especially soon after GnRH injection. Increases in both hormones were formed by several grouped associated spikes. It is suggested that a readily releasable pool of FSH exists in the ewe. There are probably differences in the mechanisms of synthesis and/or release between pituitary FSH and LH.     

Plasma LH and FSH responses and ovarian activity in prepubertal heifers treated with repeated injections of low doses of GnRH for 72 h

Twelve 5-month-old Hereford X Friesian heifers were injected i.v. with 2.0 micrograms GnRH at 2-h intervals for 72 h. Blood samples were collected at 15-min intervals from 24 h before the start until 8 h after the end of the GnRH treatment period. Over the 24-h pretreatment period, mean LH concentrations ranged from 0.4 to 2.2 ng/ml and FSH concentrations from 14.1 to 157.4 ng/ml; LH episodes (2-6 episodes/24 h) were evident in all animals. Each injection of GnRH resulted in a distinct episode-like response in LH, but not FSH. Mean LH, but not FSH, concentrations were significantly increased by GnRH treatment. The GnRH-induced LH episodes were of greater magnitude than naturally-occurring episodes (mean maximum concentration 6.7 +/- 0.5 and 4.9 +/- 0.6 ng/ml respectively). Preovulatory LH surges occurred between 17.0 and 58.8 h after the start of treatment in 9/12 heifers, with a coincident FSH surge in 8 of these animals. This was not followed by normal luteal function. There were no apparent correlations between pretreatment hormone concentrations, and either the pituitary response to GnRH or the occurrence of preovulatory gonadotrophin release.     

Effect of adrenocorticotrophic hormone (ACTH1-24) on ovine pituitary gland responsiveness to exogenous pulsatile GnRH and oestradiol-induced LH release in vivo.

The present experiment was designed to determine if and how exogenous ACTH replicates the effects of stressors to delay the preovulatory LH surge in sheep. Twenty-four hours after oestrous synchronisation with prostaglandin in the breeding season, groups of 8-9 intact ewes were injected with 50 microg oestradiol benzoate (0 h) followed 8 h later by 3 injections of saline or GnRH (500 ng each, i.v.) at 2 h intervals (controls). Two further groups received an additional 'late' injection of ACTH (0.8 mg i.m.) 7.5 h after oestradiol, i.e., 0.5 h before the first saline or GnRH challenge. To examine if the duration of prior exposure to ACTH was important, another group of ewes was given ACTH 'early', i.e. 2.5 h before the first GnRH injection. The first GnRH injection produced a maximum LH response of 1.9+/-0.4 ng/ml which was significantly (p < 0.01) enhanced after the second and third GnRH challenge (7.1+/-1.5 ng/ml and 7.0+/-1.7 ng/ml, respectively; 'self-priming'). Late ACTH did not affect the LH response after the first GnRH challenge (1.9+/-0.4 vs. 1.8+/-0.3 ng/ml; p > 0.05) but decreased maximum LH concentrations after the second GnRH to 35% (7.1+/-1.5 vs. 4.6+/-1.1 ng/ml; p = 0.07) and to 40% after the third GnRH (7.0+/-1.7 vs. 4.0+/-0.8 ng/ml; p = 0.05). When ACTH was given early, 4.5 h before the second GnRH, there was no effect on this LH response suggesting that the effect decreases with time after ACTH administration. Concerning the oestradiol-induced LH surge, exogenous GnRH alone delayed the onset time (20.5+/-2.0 vs. 27.8+/-2.1 h; p > 0.05) and reduced the duration of the surge (8.5+/-0.9 vs. 6.7+/-0.6 h; p > 0.05). The onset of the LH surge was observed within 40 h after oestradiol on 29 out of 34 occasions in the saline +/- GnRH treated ewes compared to 11 out of 34 occasions (p < 0.05) when ACTH was also given, either late or early. In those ewes that did not have an LH surge by the end of sampling, plasma progesterone concentrations during the following oestrous cycle increased 2 days later suggesting a delay, not a complete blockade of the LH surge. In conclusion, we have revealed for the first time that ACTH reduces the GnRH self-priming effect in vivo and delays the LH surge, at least partially by direct effects at the pituitary gland.     

Pituitary gonadotropin-releasing hormone receptor content in rats with polycystic ovaries

A single injection of estradiol valerate (EV) induces, after a lag period of 4-6 wk, a chronic anovulatory polycystic ovarian (PCO) condition in adult rats. This condition is associated with a selective compromise of luteinizing hormone (LH) release and/or synthesis reflected in low basal serum LH concentrations, decreased pituitary content of LH, and decreased gonadotropin-releasing hormone (GnRH)-stimulated LH secretion. The present study was undertaken to determine to what extent the aberrant LH release in rats with PCO could be related to alterations in pituitary content of GnRH receptors. Pituitary GnRH-receptor content was assessed by the evaluation of saturation binding of a GnRH analog, [125I]-D-Ala6-des-Gly10-GnRH, to pituitary membrane preparations. The receptor content of pituitaries from rats with PCO was compared to that obtained from intact animals at estrus and diestrus. Receptor levels in ovariectomized normal rats and rats with PCO were also assessed. The pituitary GnRH receptor content in PCO rats was similar to that observed in normal controls at estrus and was significantly lower than that for rats at diestrus. Although a twofold increase in pituitary GnRH receptor content was observed at 28 days following the castration of control rats, GnRH receptor content in the pituitaries of PCO rats, at 28 days following ovariectomy, remained unchanged. Although, castration-induced elevations in mean serum LH and follicle-stimulating hormone (FSH) concentrations were observed in both the PCO and control animals, the rise in both gonadotropins was significantly attenuated in the PCO-castrates when compared to the ovariectomized controls. Since GnRH is a major factor in the regulation of pituitary GnRH receptor content, these findings suggest that hypothalamic GnRH release is impaired in rats with PCO and that this impairment is independent of any influences from the polycystic ovaries.     

Inhibitory effects of the antiprogestin, RU 486, on progesterone actions and luteinizing hormone secretion in pituitary gonadotrophs

The effects of RU 486 on the modulation of LH release by progesterone were investigated in cultured anterior pituitary cells from ovariectomized adult female rats. The inhibitory effect of progesterone on LH secretion was demonstrable in estrogen-treated pituitary cells, in which addition of 10(-6) M progesterone to cells cultured in the presence of 10(-9) M estradiol for 52 h reduced the LH response to GnRH (10(-11) to 10(-7) M). When RU 486 was superimposed upon such combined treatment with estradiol and progesterone, the suppressive effect of progesterone on GnRH-induced LH release was completely abolished. The converse (facilitatory) effect of progesterone on LH secretion was observed in pituitary cells pretreated with 10(-9) M estradiol for 48 h and then with 10(-6) M progesterone for 4 h. When RU 486 was added together with progesterone during the 4 h treatment period, the facilitatory effect of progesterone was blocked and LH release fell to below the corresponding control value. The direct effect of RU 486 on LH secretion in the absence of exogenous progesterone was evaluated in cells cultured in the absence or presence of 10(-9) M estradiol and then treated for 4 to 24 h with increasing concentrations of RU 486 (10(-12) to 10(-5) M) and stimulated with GnRH (10(-9) M) during the last 3 h of incubation. In estrogen-deficient cultures, 4 h exposure to RU 486 concentrations of 10(-6) M and above decreased the LH response to GnRH by up to 50%. In cultures pretreated with 10(-9) M estradiol, GnRH-stimulated LH responses was inhibited by much lower RU 486 concentrations, of 10(-9) M and above. After 24 h of incubation the effects of RU 486 were similar in control and estradiol-pretreated pituitary cell cultures. Thus, RU 486 alone has a significant inhibitory effect on LH secretion that is enhanced in the presence of estrogen. The antiprogestin is also a potent antagonist of both the inhibitory and the facilitatory actions of progesterone upon pituitary gonadotropin release in vitro.