Progesterone directly inhibits pituitary luteinizing hormone secretion in an estradiol-dependent manner.

We recently demonstrated that progesterone and estradiol inhibit pituitary LH secretion in a synergistic fashion. This study examines the direct feedback of progesterone on the estradiol-primed pituitary. Nine ovariectomized (OVX) ewes underwent hypothalamic-pituitary disconnection (HPD) and were infused with 400 ng GnRH every 2 h throughout the experiment. After 7 days of infusion, estradiol was implanted s.c. Four days later, estradiol implants were exchanged for blank implants in 4 ewes and for progesterone implants in 5 ewes. These implants remained in place for another 4 days. Blood samples were collected around exogenous GnRH pulses before and 0.5 to 96 h after implant insertion and exchange. Serum LH and progesterone concentrations were determined through RIA. One month later, 4 of the HPD-OVX ewes previously implanted with steroids were reinfused with GnRH and the implantation protocol was repeated using blank implants only. In estradiol-primed ewes, progesterone significantly lowered LH secretion after 12 h of implantation and LH secretion remained inhibited while progesterone implants were in place (p less than 0.05). Removing estradiol transiently lowered LH secretion, and this effect was significant only 24 h after estradiol withdrawal (p less than 0.05). These data suggest that progesterone has a direct, estradiol-dependent inhibitory effect on pituitary LH release and that estradiol may sustain pituitary gonadotrope response to GnRH.     

Biphasic stimulatory effects of estrogen on gonadotropin surges induced by continuous administration of gonadotropin-releasing hormone in women

The present experiments were performed to study the effects of preovulatory levels of estrogen on GnRH-induced gonadotropin release. Twelve female volunteers in various phases of the menstrual cycle received estradiol infusion for 66 h at a constant rate of 500 micrograms/24 h which is grossly equivalent to its production rate during the preovulatory follicular phase. In 8 of the women, GnRH was administered concomitantly from 6 h after the initiation of estradiol infusion. The administered doses of GnRH were 2.5 and 5 micrograms/h. Blood samples obtained throughout the infusion were analysed for LH, FSH, estradiol and progesterone. The sole administration of estradiol failed to induce the positive feedback effect on gonadotropin release within the experimental period in the early follicular phase (days 3-7) in 4 women. In 5 women treated during the follicular phase, remarkable LH releases were induced after a lag period by the infusion of both GnRH and estradiol. The induced LH surge formed a prolonged biphasic pattern. Although a similar pattern of FSH was observed in some cases, its response was minimal compared with that of LH. In 3 women during the luteal phase, however, a combined administration of estradiol and GnRH induced only a short term release of LH which was terminated in only 12 h. The present data indicate that 1) Preovulatory levels of estrogen affect the late part of the LH surge which is induced by constant administration of low doses of GnRH resulting in a prolonged biphasic release of LH, and 2) These effects of both hormones are not manifest in the presence of high levels of progesterone. These results indicate the possibility of a role of GnRH and estrogen in the mechanism of the prolonged elevation of a gonadotropin surge at mid-cycle.     

Site of action for endotoxin-induced cortisol release in the suppression of preovulatory luteinizing hormone surges

The study was conducted to identify the mechanisms of endotoxin/cortisol action in the suppression of preovulatory LH surges in heifers infused with Escherichia coli (E. coli ) endotoxin. The hypotheses tested were that 1) endotoxin stimulates the release of progesterone, possibly from the adrenal leading to the LH blockade; 2) cortisol released in response to endotoxin infusion blocks the synthesis of estradiol at the ovarian level, culminating in a failure of the LH surge. Eight Holstein heifers were given two injections of prostaglandin F(2alpha) (PG), 11 d apart, to synchronize estrus. Starting from 25 h after the second injection of PG (PG-2), the uterus of each heifer was infused either with 5 ml of pyrogen-free water (control, n = 3) or with E. coli endotoxin (5 mug/kg of body weight) in 5 ml of pyrogen-free water (treated, n = 5), once every 6 h for 10 treatments. Blood samples were obtained every 15 min for 1 h before infusion and again 2 h after each infusion, then hourly until 1 h before the next infusion. After the tenth infusion, blood was collected daily until estrus. Serum progesterone concentrations remained at baseline values (< 1 ng/ml) in control and treated heifers. The total amount of progesterone measured starting 24 to 84 h after PG-2 injection was not different between control and treated heifers (P 0.05). In the control heifers, serum estradiol concentrations remained basal (< 10 pg/ml) until 4 h before the LH surge. Serum estradiol concentrations increased to 20 +/- 5.6 pg/ml, 4 h before the LH surge in control heifers (LH surge occurred 60 to 66 h after the PG-2 injection). There were no changes in serum estradiol concentrations in treated heifers during the sampling period, and the concentrations remained < 10 pg/ml. The total amount of estradiol measured in control heifers was higher (P < 0.05) than in treated heifers. The results if this study suggest that increases in cortisol concentrations after the infusion of endotoxin might block the synthesis of estradiol at the ovarian level, resulting in the failure of a preovulatory LH surge to occur.     

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.     

Relationship between serum gonadotropins and pituitary immunoreactive gonadotropins and steroid receptors during the first FSH increase of the estrous cycle and following steroid treatment in heifers

The objectives were to determine the effects of (i) time during the first FSH increase of the estrous cycle (time-course study) and (ii) exogenous steroid treatment (steroid feedback study) on the relationship between circulating serum gonadotropins, and the proportions of pituitary cells immunoreactive for gonadotropins and steroid receptors during the estrous cycle in heifers. Pituitaries were collected from heifers (n=40) slaughtered at 13h (n=8), 30h (n=24) and 66h (n=8) after estrous onset, corresponding to before, during and after the first FSH increase of the estrous cycle. Heifers slaughtered during the FSH increase (at 30h) either received no treatment (n=8), or were treated (n=16) with estradiol benzoate and/or progesterone before slaughter. During the time-course study, the proportion of pituitary cells immunoreactive for FSH increased (P<0.05) during the first transient FSH increase reflecting serum concentrations. The proportion of pituitary cells immunoreactive for LH was unaltered, a reflection of serum LH concentrations. The proportion of estrogen receptors (ER)-alpha, but not ER-beta, was decreased (P<0.05) at 30h compared with at either 13 or 66h. During the steroid feedback study, exogenous progesterone with or without estradiol suppressed (P<0.05) the proportions of pituitary cells immunoreactive for gonadotropins, serum FSH concentrations and LH pulse frequency. Steroid treatment did not alter the proportion of pituitary cells positive for estrogen receptors (alpha and beta). While progesterone receptors (PR) were not detected in the anterior pituitary by immunohistochemistry during the early estrous cycle or in response to steroid treatment, quantitative real-time PCR revealed that mRNA for progesterone receptors was expressed at very low levels. The expression of pituitary PR mRNA was decreased (P<0.05) at 30 and 66h compared with 13h, and was suppressed (P<0.05) following steroid treatments. Alterations in pituitary steroid receptors are implicated in the differential regulation of gonadotropin secretion during the first transient FSH rise, but not in response to exogenous steroids. The time-course study and steroid feedback responses support the hypothesis that LH pulse frequency is tightly linked to regulation of GnRH pulse frequency. Serum FSH is regulated by its own synthesis, as reflected by pituitary FSH content and perhaps by alterations in pituitary sensitivity to circulating steroids by changes in steroid receptor content.     

Administration of GnRH in vivo stimulates progesterone and inhibits androgen accumulation by ovarian follicles isolated from pubertal rabbits

A single i.v. injection of gonadotropin releasing hormone (GnRH) to pubertal female rabbits led to an ovulatory pulse of LH but no ovulations resulted. By contrast, 5 i.v. injections over 6 h led to 1-3 ovulations in 5 of 8 animals treated. Twenty-four hours after the initial injection animals were killed and follicles isolated. Large follicles greater than 1 mm dia, from both GnRH treated groups released more progesterone during the control incubation period than those from saline treated. Small follicles less than 1 mm dia, from the same GnRH groups accumulated 3-6 times more progesterone than those from saline treated when stimulated with luteinizing hormone (LH). Testosterone accumulation by small and large follicles was not affected by one injection of GnRH but was depressed in follicles from rabbits treated with 5 injections of GnRH. A single injection of GnRH enhanced the ability of small and large follicles to release estradiol which was depressed 30% in the presence of LH. Multiple GnRH injections depressed estradiol accumulation by small and large follicles. These data suggest the administration of GnRH in vivo can have stimulatory as well as inhibitory effects on subsequent follicular steroid release and accumulation in vitro.     

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.     

Direct pituitary effects of estradiol and progesterone on luteinizing hormone release, stores, and subunit messenger ribonucleic acids

To determine the direct, chronic actions of progesterone (P4) and estrogen (estradiol, E2) on anterior pituitary synthesis and release of LH, 24 western range ewes underwent hypothalamic-pituitary disconnection (HPD) and ovariectomy (OVX) during the breeding season and were pulsed with exogenous GnRH with or without steroid replacement. Sequential blood samples were collected before infusion of GnRH and on Days 7 and 14 of GnRH infusion. Silastic capsules of P4 and/or E2 were implanted s.c. on Day 7 and remained in place throughout the experiment. Control ewes received only GnRH infusion. Blood sampling was centered around three exogenous GnRH pulses. After the final blood sampling, pituitaries were collected and stored at -70 degrees C. Concentrations of LH in serum and pituitaries were determined by RIA. Relative concentrations of LH subunit mRNAs were determined by Fast Blot analysis. Simultaneous implantation of P4 and E2 lowered LH pulse amplitude 70% and mean serum levels 30% compared with controls. Neither steroid alone affected LH release. E2 alone or in combination with P4 lowered LH-beta subunit mRNA concentrations 40% compared with controls while alpha-subunit levels were unchanged. Only E2 alone altered the pituitary content of LH, causing a 60% decrease. We conclude that the combination of P4 and E2 is necessary for inhibition of GnRH-stimulated LH secretion. E2 inhibits GnRH-stimulated LH-beta subunit mRNA concentrations but does not affect alpha-subunit mRNA concentrations. The control of pituitary LH content by P4 and E2 is the result of changes in both LH-beta subunit mRNA concentrations and LH secretion.     

Role of estradiol in inducing an ovulatory-like surge of luteinizing hormone in sheep

Two experiments were performed to examine the effect of estradiol on secretion of luteinizing hormone (LH) and on the number of receptors for gonadotropin-releasing hormone (GnRH) after down regulation of GnRH receptors in ovariectomized ewes. In the first experiment, ovariectomized ewes were administered one of four treatments: Group 1) infusion of GnRH i.v. for 40 h; Group 2) injection of 100 micrograms estradiol i.m.; Group 3) infusion of GnRH i.v. for 16 h followed immediately by an injection of 100 micrograms estradiol i.m.; and Group 4) infusion of GnRH i.v. for 40 h plus injection of 100 micrograms estradiol i.m. after the 16th h of infusion. Ewes in Groups 1, 3 and 4 responded to the infusion of GnRH with an immediate increase in serum concentrations of LH, with maximum values occurring between 2 and 4 h after the start of infusion; serum concentrations of LH then began to decline and were approaching the pretreatment baseline within 16 h. Administration of estradiol resulted in a surge of LH regardless of whether the pituitary had been desensitized by infusion of GnRH or not. In all cases the magnitude of the surge was similar to that induced by the initial infusion of GnRH. In Groups 2 and 3 the surge of LH began at 12.3 +/- 0.1 and 11.9 +/- 0.1 h after administration of estradiol. In contrast, the ewes in Group 4 had a surge of LH beginning 3.7 +/- 0.1 h after administration of estradiol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antagonism of estrogen-induced prolactin release by progesterone

Previous work from our laboratory has shown that during the process of nuclear occupancy of the progesterone receptor complex (1-2 h), nuclear estradiol receptors of the anterior pituitary are depleted. The purpose of this study was to determine whether the depletion of nuclear estradiol receptors by progesterone had functional biological significance. The ovariectomized (26 days of age) immature rat was used as the model for analysis of this question. The ability of estradiol to release prolactin from the anterior pituitary was the function chosen to determine the biological significance of the progesterone and estradiol interactions. In response to estradiol exposure (2 micrograms/rat), prolactin release reached peak values from 8 h to 12 h and returned to control levels by 24 h. A second injection of estradiol 13 h after the initial injection stimulated a second increase in serum prolactin at 25 h. This model of two injections of estradiol 13 h apart served to provide adequate levels of anterior pituitary progesterone receptors and elevated serum prolactin levels upon which superimposed progestin modulation could be examined. A single injection of progesterone (0.8 mg/kg BW) 1 h before the second estradiol injection blocked the increase in serum prolactin. This action was a receptor-mediated event because progesterone had no effect without estrogen priming or when the progesterone antagonist RU486 was used. Finally, when the interval between the progesterone and second estradiol injection was extended to 4 h, a time period when progesterone does not deplete pituitary nuclear estrogen receptors, the estrogen-induced increase in serum prolactin was not blocked.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulatory actions of estradiol and progesterone on phorbol ester-stimulated LH secretion from cultured rat pituitary cells.

We compared the ability of estradiol and progesterone to modulate gonadotropin-releasing hormone (GnRH) and protein kinase C (PKC)-mediated luteinizing hormone (LH) secretion. Long-term (48 h) treatment of rat pituitary cells with 1 nM estradiol enhanced GnRH and phorbol ester (TPA)-stimulated LH secretion. This positive effect was facilitated by additional short-term (4 h) treatment with progesterone (100 nM). However, long-term progesterone treatment, which inhibited GnRH-stimulated LH secretion, did not influence TPA-stimulated gonadotropin release. These steroid actions occurred without an effect on the total amount of LH in the cell cultures (total LH = LH secreted + LH remaining in the cell) and neither the secretagogues nor the steroids altered total LH. Since GnRH or TPA-induced LH secretion depends on Ca2+ influx into the gonadotroph, we also analyzed the effects of estradiol and progesterone under physiological extracellular Ca2+ concentrations and in the absence of extracellular Ca2+. The steroids were able to influence GnRH or TPA-induced LH secretion under both conditions. However, when TPA was used as stimulus in Ca(2+)-deficient medium the relative changes induced by estradiol and progesterone were more pronounced, possibly indicating that the extracellular Ca(2+)-independent component of PKC-mediated LH secretion is more important for the regulation of the steroid effects. It is concluded that estradiol and progesterone might mediate their modulatory actions on GnRH-stimulated LH secretion via an influence on PKC. This effect can occur independently from de novo synthesis of LH and Ca2+ influx into gonadotrophs.     

Regulatory roles of leptin at the hypothalamic-hypophyseal axis before and after sexual maturation in cattle

Studies assessed, either directly or indirectly, the role of GnRH in leptin-mediated stimulation of LH release in cattle before and after sexual maturation. In experiment 1, the objectives were to determine whether leptin could acutely accelerate the frequency of LH pulses, and putatively GnRH pulses, in prepubertal heifers at different stages of development. In experiment 2, we determined directly whether acute, leptin-mediated increases in LH secretion in the fasted, mature female are accompanied by an increase in GnRH secretion. Ten-month-old prepubertal heifers (experiment 1) fed normal- (n = 5) and restricted-growth (n = 5) diets received three injections of saline or recombinant ovine leptin (oleptin; 0.2 microg/kg body weight, i.v.) at hourly intervals during 5-h experiments conducted every 5 wk until all normal-growth heifers were pubertal. Leptin increased mean concentrations of circulating LH regardless of diet, but pulse characteristics were not altered at any age. In experiment 2, ovariectomized, estradiol-implanted cows (n = 5) were fasted twice for 72 h and treated with either saline or oleptin i.v. (as in experiment 1) on Day 3 of each fast. Leptin increased plasma concentrations of LH and third ventricle cerebrospinal fluid concentrations of GnRH, and increased the amplitude of LH and the size of GnRH pulses, respectively, on Day 3 of fasting compared to saline. Overall, results indicate that leptin is unable to accelerate the pulse generator in heifers at any developmental stage. However, leptin-mediated augmentation of LH concentrations and pulse amplitude in the nutritionally stressed, mature female are associated with modifications in GnRH secretory dynamics.     

Estrogen and progesterone receptor content in the pituitary gland and uterus of progesterone-primed and gonadotropin releasing hormone-treated anestrous ewes

The objective of this work was to investigate the effect of progesterone (P) and gonadotropin-releasing hormone (GnRH) treatment on estrogen receptor (ER) and P receptor (PR) concentrations in the pituitary gland and uterus of anestrous ewes. Ewes were either not treated (group C, n = 4); were treated with 0.33 g P-controlled internal drug release (P-CIDR) for 10 days (group P, n = 4), with GnRH, 6.7 ng i.v. injections every 2 h for 18 h followed by a 4 microg bolus administration of Receptal at 20 h (group GnRH, n = 4), or with a combination of the P and GnRH treatment (group P + GnRH, n = 3). Ewes were humanely killed either at the beginning of the experiment (group C), when the CIDR was removed (group P), or 24 h after the GnRH bolus treatment (groups GnRH and P + GnRH). Progesterone treatment increased serum P concentrations, indicating that the treatment was effective. All GnRH treated ewes had similar luteinizing hormone (LH) surges, which lasted 8 h. At slaughter, estradiol (E2) concentrations in the GnRH group were higher than in groups C, P, and P + GnRH. Treatment with GnRH increased more than 10-fold the content of ER and PR in the pituitary gland without altering steroid receptor concentrations in the uterus. When GnRH was combined with P the uterine receptor contents were higher than with P treatment alone. The treatment with P decreased ER and PR content in the uterus, but had no effect on the pituitary gland. The results show that regulation by P and GnRH of ER and PR content in anestrous ewes is tissue-specific.     

Stimulatory and inhibitory effects of progesterone on FSH secretion by the anterior pituitary.

The purpose of this study was to investigate whether progesterone exerted progesterone receptor mediated direct effects on the anterior pituitary in the secretion of FSH and whether such effects were mediated through the 5 alpha-reduction of progesterone. Treatment of anterior pituitary dispersed cells for 48 h with 0.5 nM estradiol reduced the ED50 for gonadotropin releasing hormone (GnRH)-stimulated FSH release from 0.58 to 0.36 ng/ml and the ED50 for GnRH-induced LH release from 0.54 to 0.19 ng/ml. When dispersed pituitary cells were treated with 0.5 nM estradiol and exposed to various doses of progesterone for 1 to 6 h, the most consistent rise in basal and GnRH-stimulated FSH release was observed with the 50 nM dose of progesterone with a 3-h exposure period. All three doses of progesterone elevated basal LH and GnRH-stimulated LH was increased by the 50 and 100 nM doses of progesterone during the 3-h period of treatment. Using the 50 nM dose of progesterone, basal and GnRH-stimulated LH was increased after 2, 3 and 6 h of progesterone treatment. When the period of exposure of progesterone was extended to 12, 36 or 48 h, there was a significant inhibition of GnRH-stimulated FSH release. GnRH-stimulated LH release was inhibited at 36 and 48 but not 12 h after progesterone treatment. These studies showed that the effect of progesterone administered for periods of 1 to 6 h enhanced the secretion of LH and FSH whereas progesterone administered for periods beyond 12 h inhibited FSH and LH release by dispersed pituitary cells in culture. These results are similar to those observed in vivo after progesterone treatment. Furthermore estrogen priming of the dispersed pituitary cells was necessary to observe the effects of progesterone. The progesterone antagonist RU486 prevented the progesterone-induced rise in GnRH-stimulated FSH release. Furthermore the 5 alpha-reductase inhibitor N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane- 17 beta-carboxamide also prevented the progesterone-induced rise in GnRH-stimulated FSH release in estrogen-treated dispersed pituitary cells. These results indicate that the anterior pituitary is a major site of action of progesterone in the release of FSH and that 5 alpha-reduction of progesterone plays an important role in FSH release.     

Pulsatile administration of gonadotropin-releasing hormone advances ovulation in cycling mares

Cycling standardbred mares were infused with saline or 20 micrograms gonadotropin-releasing hormone (GnRH) in a pulsatile pattern (one 5-sec pulse/h, 2 h or 4 h) beginning on Day 16 of the estrous cycle. Although serum concentrations of luteinizing hormone (LH) increased significantly earlier in all three GnRH-treated groups (within one day of the initiation of infusion) compared to saline-infused controls, there were no differences in peak periovulatory LH concentrations among treatments (overall mean +/- SEM, 8.98 +/- 0.55 ng/ml). The number of days from the start of treatment to ovulation was significantly less in mares infused with 20 micrograms GnRH/h (mean +/- SEM, 2.9 +/- 0.6 days after the initiation of treatment, or 18.9 days from the previous ovulation; N = 7) compared to mares treated with saline (5.9 +/- 0.3 days, or 21.9 days from previous ovulation; N = 7) or 20 micrograms GnRH per 4 h (5.4 +/- 0.9 days or 21.4 days from previous ovulation; N = 5). Although mares infused with 20 micrograms GnRH/2 h ovulated after 4.3 +/- 0.7 days of treatment (Day 20.3; N = 7), this was not significantly different from either the control or 20 micrograms GnRH/h treatment groups. Neither the duration of the resulting luteal phase nor the length of the estrous cycle was different between any of the treatment groups (combined means, 14.7 +/- 0.2 days and 21.3 +/- 0.4 days, respectively). We conclude that pulsatile infusion of GnRH is effective in advancing the time of ovulation in cycling mares, but that the frequency of pulse infusion is a critical variable.     

Roles of estradiol and gonadotropin-releasing hormone in controlling negative and positive feedback associated with the luteinizing hormone surge in ovariectomized pigs.

Ovariectomized gilts (n = 63) were given estradiol benzoate (estradiol), antiserum to neutralize endogenous GnRH, and pulses of a GnRH agonist (GnRH-A) to stimulate release of LH. GnRH-A was given as 200-ng pulses hourly from 0 to 54 h and as 100- or 200-ng pulses every 30 or 60 min from 54 to 96 h after estradiol. Estradiol alone suppressed LH from 6 to 54 h and elicited an LH surge that peaked at 72 h. When GnRH-A was given every 30-60 min from 0 to 96 h, estradiol suppressed LH for 6-12 h, but then LH returned to pre-estradiol concentrations. When pulses of GnRH-A were given only between 54 and 96 h after estradiol, the surge of LH was related positively to dose and frequency of GnRH-A. We conclude that 1) estrogen acts at the hypothalamus to inhibit release of GnRH for 54 h and then causes a synchronous release of GnRH; 2) estrogen acts at the pituitary to block its response to GnRH for 6-12 h and enhances the accumulation of releasable LH; and 3) magnitude of the LH surge is dependent on the amount of GnRH stimulation.     

Pituitary receptors for gonadotropin-releasing hormone in relation to changes in pituitary and plasma gonadotropins in ovariectomized hypothalamo/pituitary-disconnected ewes. II. A marked rise in receptor number during the acute feedback effects of estradiol

Ovariectomized (OVX), hypothalamo/pituitary-disconnected (HPD) ewes were used to ascertain the short-term effects of estradiol on the number of gonadotropin-releasing hormone (GnRH) receptors in the pituitary gland. The time course of the study was such that measurements were made during the period of short-term negative feedback and positive feedback. Groups of 4 OVX-HPD ewes were given 250-ng pulses of GnRH each hour and an i.m. injection of oil (Group 1) or 50 micrograms estradiol benzoate in oil (Groups 2-4). Blood samples were collected from each ewe prior to treatment with estradiol or oil and again immediately before slaughter. Groups 2, 3, and 4 were killed 6, 16, and 20 h, respectively, after administration of estradiol. Amplitudes of luteinizing hormone (LH) pulses and average plasma concentrations of LH were reduced 6 h after estradiol treatment. Sixteen and 20 h after injection, the average plasma LH levels were elevated, but pulse amplitudes were similar to preinjection values. The number of GnRH receptors was significantly (p less than 0.01) increased within 6 h of estrogen treatment and further increased 16 and 20 h after treatment. Pituitary content of LH was similar in all groups. These data indicate that the number of GnRH receptors in the pituitary gland of ewes can be acutely influenced by a direct effect of estradiol. However, the magnitude and direction of the change in receptors number does not account for the changes in pituitary responsiveness to GnRH, suggesting estradiol also modifies post-receptor mechanisms that influence secretion of LH.     

In vivo and in vitro studies on the effect of adrenocorticotropic hormone or cortisol on the pituitary response to gonadotropin releasing hormone

Experiment I: Hyperadrenal states were induced in intact heifers (N = 3) or adrenalectomized (ADRX) heifers (N = 3) by constant infusion of ACTH (20.8 micrograms, 1-24 ACTH/h) or hydrocortisone succinate (HS) (30 mg/h), respectively. Control infusions consisted of the saline vehicle. All infusions began on Day 2 of a normal estrous cycle. Exogenous gonadotropin releasing hormone (GnRH) was given as a 100-micrograms bolus i.v. on Days 7, 9, and 11 (intact) or 5, 7, and 9 (ADRX) of the cycle. In intact heifers, the cumulative luteinizing hormone (LH) response was reduced (P less than 0.05) by the ACTH treatment. In ADRX heifers, the HS treatment did not alter the cumulative response but did alter the qualitative response with a time X treatment interaction (P less than 0.01). The LH response in the HS-ADRX animals had a slower onset and lower peak concentrations with a more prolonged response. Experiment II: Dispersed bovine pituitary cells were prepared and incubated at concentrations of 2 X 10(6) viable cells in 2.0 ml per dish. Cells were exposed to cortisol at concentrations of 0.01, 0.10, 0.21 and 1.03 X 10(-6) M for time periods of 8, 14, 20 or 26 h for basal LH secretion studies and 10, 16, 22 and 28 h for GnRH-stimulated LH secretion. Both dosage of cortisol and length of exposure had a depressing effect on basal LH release. The cortisol pretreatment also decreased (P less than 0.001) the LH release following addition of GnRH (8.5 X 10(-8) M) in cultures at all dosages and exposure times of cortisol. However, there was no decrease in LH or protein content of cells. These experiments indicate a direct action of cortisol on the pituitary gland to depress both basal and stimulated LH release.     

Independence of progesterone blockade of the luteinizing hormone surge in ewes from opioid activity at naloxone-sensitive receptors.

Fifteen ovariectomized ewes were treated with implants (s.c.) creating circulating luteal progesterone concentrations of 1.6 +/- 0.1 ng ml-1 serum. Ten days later, progesterone implants were removed from five ewes which were then infused with saline for 64 h (0.154 mol NaCl l-1, 20 ml h-1, i.v.). Ewes with progesterone implants remaining were infused with saline (n = 5) or naloxone (0.5 mg kg-1 h-1, n = 5) in saline for 64 h. At 36 h of infusion, all ewes were injected with oestradiol (20 micrograms in 1 ml groundnut oil, i.m.). During the first 36 h of infusion, serum luteinizing hormone (LH) concentrations were similar in ewes infused with saline after progesterone withdrawal and ewes infused with naloxone, but with progesterone implants remaining (1.23 +/- 0.11 and 1.28 +/- 0.23 ng ml-1 serum, respectively, mean +/- SEM, P greater than 0.05). These values exceeded circulating LH concentrations during the first 36 h of saline infusion of ewes with progesterone implants remaining (0.59 +/- 0.09 ng ml-1 serum, P less than 0.05). The data suggested that progesterone suppression of tonic LH secretion, before oestradiol injection, was completely antagonized by naloxone. After oestradiol injection, circulating LH concentrations decreased for about 10 h in ewes of all groups. A surge in circulating LH concentrations peaked 24 h after oestradiol injection in ewes infused with saline after progesterone withdrawal (8.16 +/- 3.18 ng LH ml-1 serum).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a direct negative effect of estradiol at the level of the pituitary gland in sheep

The purpose of this experiment was to determine if pituitary stores of LH could be replenished by administration of GnRH when circulating concentrations of both progesterone and estradiol-17 beta (estradiol) were present at levels observed during late gestation. Ten ovariectomized (OVX) ewes were administered estradiol and progesterone via Silastic implants for 69 days. One group of 5 steroid-treated OVX ewes was given GnRH for an additional 42 days (250 ng once every 4 h). Steroid treatment alone reduced (p less than 0.01) the amount of LH in the anterior pituitary gland by 77%. Pulsatile administration of GnRH to steroid-treated ewes resulted in a further decrease (p less than 0.01) in pituitary content of LH. Compared to the OVX ewes, concentrations of mRNAs for alpha- and LH beta-subunits were depressed (p less than 0.01) in all steroid-treated ewes, whether or not they received GnRH. The ability of the dosage of GnRH used to induce release of LH was examined by collecting blood samples for analysis of LH at 15 days and 42 days after GnRH treatment was initiated. Two of 5 and 3 of 5 steroid-treated ewes that received pulses of GnRH responded with increased serum concentrations of LH after GnRH administration during the first and second bleedings, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)