GnRH pulses regulate gonadotropin subunit gene transcription in a frequency-dependent, subunit-specific manner. The alpha-subunit gene is stimulated by constant GnRH and by rapid to intermediate pulse frequencies, while stimulation of LHbeta subunit gene transcription requires intermediate frequency pulses. We have defined the GnRH-responsive elements of the rat LH subunit gene promoters by deletion/mutation analysis and transfection studies in rat pituitary cells and two clonal gonadotrope cell lines. The alpha-subunit gene GnRH-responsive region lies between -411 and -375 bp. The region contains two Ets-domain protein binding sites, and mutating either site obliterates the response. DNA protein binding studies demonstrate the two sites are not equivalent, and that Ets-1 does not mediate this response. Studies of the LHbeta promoter reveal a major GnRH-responsive region between -456 and -342 bp. Within this region, two Sp1 binding sites contribute to the GnRH response, and the 3'Sp1 site is also critical for basal expression. The 5'Sp1 site partially overlaps a CArG box, and mutating the CArG element specifically eliminates the response to pulsatile GnRH. DNA containing this mutation cannot form intermediate mobility complexes with nuclear proteins, but retains Sp1 binding. Mutation of the 3'Sp1 site and either the 5'Sp1 or CArG element partially restores GnRH stimulation, suggesting a downstream element contributes to the full GnRH response. These studies demonstrate that unique composite elements and transcription factors are responsible for GnRH stimulation of the LH subunit genes and may contribute to their differential responses to GnRH pulses.

Gonadotropin secretion and gene expression are differentially regulated by hypothalamic GnRH pulses by unknown mechanisms. GnRH stimulates calcium influx through L-type voltage-gated channels and activates phospholipase C, leading to increased protein kinase C (PKC) and mitogen-activated protein kinase activity. We found differential contributions of these pathways to GnRH-stimulated rat LH subunit transcription in pituitary gonadotropes and cell lines. Endogenous transcription of the alpha- and LHbeta-subunits in rat pituitary cells was stimulated by GnRH. Independent PKC activation by phorbol myristate acid stimulated only the alpha-subunit gene. In contrast, an L-channel antagonist (nimodipine) inhibited only LHbeta stimulation by GnRH, and an L-channel agonist (BayK 8644) stimulated only basal LHbeta transcription. GnRH induction of a rat alpha-subunit promoter construct in alphaT3 cells was unaffected by nimodipine or elimination of external calcium, while both treatments eliminated the LHbeta response. Application of a mitogen-activated kinase kinase (MEK) inhibitor (PD098059) decreased basal and GnRH-stimulated alpha-subunit promoter activity and had no effect on LHbeta promoter activity. In pituitary cells from mice bearing an LHbeta promoter-luciferase reporter transgene, GnRH stimulation was inhibited by nimodipine but not by PD098059. Thus, GnRH induction and basal control of the alpha-subunit gene seem to occur through the PKC/mitogen-activated protein kinase pathway, while induction of the LHbeta gene is dependent on calcium influx. Differential signaling from the same receptor may be a mechanism for preferential regulation of transcription.

Transcriptional regulation of the rat LH beta (rLH beta) gene was studied through the use of transgenic mice bearing a region of the rLH beta gene (from -2 kb to +41 bp) linked to a luciferase (LUC) reporter gene. All 7 founder mice were successfully bred with B6SJLF1 mates and exhibited germ line transmission of the LH beta LUC transgene. Levels of rLH beta LUC activity were highest in the pituitary, but activity was also detected in ovary, testis, and hypothalamus. Pituitary rLH beta LUC activity was found to be regulated by gender, gonadectomy, gonadal steroid replacement, and GnRH antagonist administration. Females had higher basal pituitary rLH beta LUC activity than males. This activity was increased 2- to 4-fold seven days postovariectomy, and stimulated activity was suppressed to intact levels by daily injections of 17 beta estradiol (E2; 300 ng). In males, castration increased pituitary LUC activity 2- to 4-fold, and this suppressed to intact levels by daily injections of 25 micrograms dihydrotestosterone (DHT). The postgonadectomy rise in pituitary rLH beta LUC activity in females and males was blocked by daily administration of the GnRH antagonist Antide (60 micrograms), which also suppressed serum LH and LH beta mRNA levels to a similar extent. Rat LH beta LUC activity measured in the hypothalamus was not altered by gonadectomy or gonadal steroid or Antide treatment, demonstrating that this regulation is pituitary-specific. These results indicate that feedback regulation of pituitary rLH beta LUC gene expression is operational in this transgenic mouse model.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated the requirement for gonadotropin-releasing hormone (GnRH) release in vivo and the pattern of GnRH administration in vitro on the expression of the gonadotropin subunit genes in female rats. Injection of the GnRH antagonist ([Nal-Lys] GnRH; 30 micrograms/100g bw) to ovariectomized rats rapidly suppressed transcription of the alpha-subunit and LH beta genes to 10-25% of control after 24 h, as measured by nuclear run-off assays. The rate of FSH beta gene transcription was also suppressed, but to a lesser extent (to 60-75% of control). Administration of 17 beta-estradiol (20 micrograms/100 g bw) in addition to antagonist did not suppress transcription of the genes beyond that seen with the antagonist alone. Administration of constant levels of GnRH (0.1, 1, or 10 nM) to pituitary fragments in static culture stimulated alpha-subunit mRNA synthesis 2- to 3-fold, but had no significant sustained effects on LH beta or FSH beta transcription. In contrast, pulsatile GnRH administered once per hour (25 ng over 10 min) to pituitary fragments mounted on perifusion columns stimulated both alpha-subunit and LH beta gene transcription 3-fold after 3 h, with inconsistent stimulation of FSH beta. Pulsatile GnRH appears to be crucial for LH beta gene stimulation, as continuous GnRH on the columns stimulated only alpha-subunit mRNA synthesis. Thus, pulsatile GnRH in vivo is required to maintain transcription of the alpha-subunit and LH beta genes, with lesser effects on FSH beta. While continuous GnRH can stimulate alpha-subunit mRNA synthesis, a pulsatile GnRH is required to stimulate the LH beta gene.

Studies were conducted in vitro and in vivo to determine whether or not inhibin affects the transcription rate of the gene for the beta subunit of follicle stimulating hormone (FSHbeta). Pituitary cells in primary culture were incubated with 0-3000 milli-units/ml inhibin; a dose-related decrement in mRNA was obtained but a parallel effect was not observed for the transcription rate of the FSHbeta gene in a nuclear run-on experiment. To determine effects in vivo, ovariectomized ewes were treated with saline (group 1), 75 mug inhibin 6 h before slaughter (group 2), inhibin 6 h and 12 h before slaughter (group 3) or inhibin 12 h before slaughter (group 4). In samples taken each 2 h, plasma FSH levels were seen to be maximally reduced 6 h after a single injection of inhibin; at this time mRNA levels were reduced up to 100% whereas FSHbeta gene transcription rate was reduced by 50%. A second injection at 6 h (group 3) caused a further reduction in plasma FSH levels with no additional effect on transcription rate. In those sheep killed 12 h after a single inhibin injection, transcription rate for the FSHbeta gene, cytoplasmic mRNA levels and plasma FSH concentrations had recovered.These studies show that the rapid effect of inhibin on FSHbeta mRNA levels may be due, in part, to an effect on transcription rate of the FSHbeta gene. An additional mechanism is required, however, to fully explain the inhibin effect on FSHbeta mRNA levels. These effects of inhibin have a time course which allows the maximal effect and full recovery within 12 h.

In order to examine pituitary gonadotropin secretion and responsiveness to GnRH after photic-induced changes in reproductive condition, an in vitro pituitary perifusion system was established for male golden hamster tissue. Anterior pituitaries from adult males which had been maintained on 14 h light:10 h dark (long days) or 6 h light:18 h dark (short days) for 10 weeks were perifused using an Acusyst perifusion system. Perfusates from unstimulated tissue (basal secretion) and from tissue stimulated with hourly pulses of GnRH (25, 50, or 100 ng/ml) were assayed for LH and FSH by RIA. Tissue from short-day animals had lower basal LH secretion than tissue from long day animals, but there were no significant photoperiodic differences for GnRH-stimulated LH secretion. In contrast, there were no photoperiodic differences in basal FSH secretion, but tissue from short-day animals secreted more FSH than tissue from long-day animals when stimulated with GnRH. Bioactivity of a small number of perfusate samples was assessed using in vitro rat granulosa cell and mouse Leydig cell assays for FSH and LH, respectively, and did not show any photoperiodic differences in LH or FSH bioactivity for GnRH-stimulated tissue. These studies indicate that the pituitaries of gonadally regressed hamsters are capable in vitro of responding to GnRH with similar or greater levels of gonadotropin release compared to pituitaries from animals with functional gonads. Therefore, it appears that the lowered serum gonadotropin levels seen in vivo in gonadally regressed animals are not due to a reduction in intrinsic pituitary sensitivity to GnRH.

The in vivo suppression of LH by 17 beta-estradiol (E2) has been documented frequently. However, the demonstration of a direct inhibitory action of E2, in contrast to a stimulatory action, on the secretion of LH from the anterior pituitary has been inconsistent. The aim of this study was to determine if E2 can suppress either basal (unstimulated) or GnRH-stimulated gonadotropin secretion directly at the level of the anterior pituitary gland. Anterior pituitaries were obtained from metestrous and proestrous females rats at 0900 h, and trunk blood was collected for serum measurements of LH, FSH, E2, and progesterone (P). Each anterior pituitary was cut into eighths and placed into a microchamber for perifusion. Pituitary fragments were perifused at a rate of 10 ml/h using medium 199 (without phenol red) that contained E2 (1 nM) or ethanol as a control. Six pulses of GnRH (peak amplitude, 50 ng/ml; duration, 2 min) were administered one per h starting at 60 min. Fractions of perfusate were collected every 5 min for measurement of LH and FSH. The total amounts of LH and FSH secreted during the 1-h interval after each GnRH pulse or corresponding basal hour were calculated. Both basal and LH and FSH responses to GnRH were significantly greater from pituitaries of proestrous compared to metestrous rats. The selective suppression of LH secretion by in vitro treatment with E2 was demonstrated using pituitaries from metestrous rats receiving GnRH pulses, but not using pituitaries from proestrous rats. Thus, a negative feedback effect of E2 on LH secretion was observed only in pituitaries from donors with low serum levels of E2 and high P, but not from donors with high serum levels of E2 and low P. We believe that the in vivo steroid environment determined the subsequent responses to in vitro treatment with E2 on GnRH-stimulated gonadotropin secretion from the isolated pituitary gland.

The hormonal interactions required for the generation of a secondary surge of FSH on the evening of proestrus have not been clearly defined. The role of GnRH in driving a surge of FSH has been questioned by findings in previous studies. In the current study, gonadotropin secretion was measured from pituitary fragments obtained from rats at 0900 and 2400 h on each day of the estrous cycle. Pituitary fragments were perifused in basal (unstimulated) conditions or in the presence of GnRH pulses to determine whether a selective increase in basal release of FSH and/or an increase in the responsiveness to GnRH occurs during the secondary FSH surge. Each anterior pituitary was cut into eighths and placed into a microchamber for perifusion. Seven pulses of GnRH (peak amplitude = 50 ng/ml; duration = approximately 2 min) were administered at a rate of one per hour starting at 30 min. Fractions of perfusate were collected every 5 min and frozen until RIA for LH and FSH. The mean total amount of LH or FSH secreted during the hour interval following each of the last six pulses of GnRH (or the corresponding basal hour) was calculated. Analysis of variance with repeated measures indicated that the evening secretion of LH on proestrus (2400 h) dropped significantly (p less than 0.05) from a maximum on the morning of proestrus (0900 h), whereas the FSH secretion remained elevated at this time. Therefore, the ratio of FSH to LH secreted in response to GnRH pulses was highest during the secondary FSH surge and lowest on the morning of proestrus.(ABSTRACT TRUNCATED AT 250 WORDS)

Pituitary gonadotropin responses to GnRH were measured using both in vitro and in vivo methods to investigate the contribution of increased pituitary responsiveness to GnRH in generating the rise in serum gonadotropin levels after gonadectomy. We compared in vitro GnRH-stimulated secretion rates of LH and FSH of perifused pituitaries obtained from intact female (metestrous) and male rats, and rats gonadectomized 2 or 6 days earlier. GnRH pulses (peak amplitude, 50, 500, or 5000 ng/ml; frequency, one per h) caused significant dose-dependent increases in gonadotropin secretion rates. However, gonadectomy resulted in decreased secretion rates of LH and FSH. Similar findings were observed for in vivo serum gonadotropin responses to a single iv injection of GnRH (males received 250 or 1000 ng; females received 1000 or 4000 ng). These results indicate that increases in serum LH and FSH levels 2 or 6 days after gonadectomy are not mediated by increased responses of the rat anterior pituitary to GnRH. We have also shown that perifused pituitaries from proestrous and diestrous rats exhibit significantly higher GnRH-stimulated gonadotropin secretion rates than pituitaries from metestrous and estrous rats. Therefore, we tested the effect of in vivo pretreatment with 17 beta-estradiol (E2) or testosterone (T) in both female and male rats on the in vitro secretion of LH and FSH. Rats were gonadectomized and received a sc Silastic implant containing E2, T, or no steroid as a control 6 days before perifusion. Perifused pituitaries received pulses of GnRH (peak amplitude, 50 ng/ml; frequency, one per h). In vivo pretreatment with E2, but not T, caused significant increases of in vitro LH and FSH secretion rates for pituitaries of both sexes. Overall, our data demonstrate that gonadectomy does not cause increases in LH and FSH secretory responses to GnRH, and that prior exposure to E2 in vivo has a major stimulatory influence on the in vitro secretion of both gonadotropins regardless of sex.