Gonadotropin-releasing hormone (GnRH) and gonadal steroids regulate synthesis and release of luteinizing hormone (LH). GnRH is secreted intermittently by the hypothalamus, producing pulsatile LH release, and a pulsatile GnRH stimulus is required to maintain LH secretion. We report the regulatory effects of GnRH Recently, cDNAs coding for LH a and /3 subunits were isolated (16,17), and we have used these cDNAs as hybridization probes to develop a sensitive assay for mRNAs using dot-blot hybridization of extracted cytoplasmic RNA from rat pituitaries.We employed this mRNA quantitation assay to study the effects of GnRH pulse injections on expression of the genes for LH a and p subunits in gonadectomized male rats that were given testosterone implants. In this animal model endogenous GnRH secretion is markedly reduced, as judged by infrequent serum LH pulses (18). Thus assessment of the effects of exogenous GnRH pulse injections, given to mimic the intermittent physiologic stimulus, is not unduly complicated by endogenous GnRH secretion. In addition, experiments are performed in the presence of stable physiologic concentrations of testosterone, which avoids the potential effects of fluctuating serum testosterone levels in intact animals (19). This model has been previously used to study the effects of testosterone in regulating GfiRH receptor and gonadotropin responses to a pulsatile GnRH stimulus (19). In the present study we examined the effects of different doses of GnRH per pulse injection on GnRH membrane receptors and LH subunit gene expression to assess their role in the regulation of gonadotropin secretion. MATERIALS AND METHODSMale Sprague-Dawley rats (250-300 g) were castrated and implanted with two 20-mm Silastic implants containing testosterone, to produce a serum testosterone concentration of 2.3 ± 0.12 ng/ml (mean ± SEM). Beginning immediately after recovery from anesthesia, GnRH pulse (10-250 ng per pulse) or control saline injections were given every 30 min for 48 For mRNA quantification pituitaries were individually homogenized in 10 mM Tris/0.5% Nonidet P-40/1 mM EI)TA, pH 7.4, and the homogenate was centrifuged (13,000x g for 5 mmin). DNA was measured (20) in the nuclear pellet, and total RNA was extracted from the cytosol supernatant by using a phenol/chloroform/isoamyl alcohol mixture (100:100:1, Abbreviations: GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; FSH, follicle-stimulating hormone. §To whom reprint requests should be addressed

In this study we examined the changes in alpha and LH beta mRNAs in anterior pituitaries of male and female rats after castration. mRNA concentrations were measured by an optimized RNA dot blot hybridization assay. Rat alpha and LH beta cDNAs were nick-translated to specific activities of 2-5 X 10(8) cpm/micrograms and were used as hybridization probes. The total RNA per assay, RNA per dot, and saturating amounts of probe were optimized. The intra- and interassay coefficients of variation were 5% and 28%, respectively. Both alpha and LH beta mRNA concentrations increased after castration, but marked differences were observed in the kinetics of responses in male and female rats. In males, alpha and LH beta mRNAs were increased by 24 h postcastration (by 25% and 38%, respectively), and 4- to 5-fold increases over intact controls were evident by 18 days. Alpha mRNA rose rapidly and had doubled by 2 days, whereas LH beta mRNA concentrations showed a similar increase by 6-7 days postcastration. The slower rise in LH beta mRNA was associated with a transient decline in serum and pituitary LH concentrations between 2 and 6 days after castration. In female rats, alpha mRNA increased more slowly. Alpha concentrations had doubled by 10 days, while a similar increase in LH beta mRNA occurred 7 days after castration. Thereafter, both subunit mRNAs continued to rise, and by day 20 alpha mRNA was increased 5-fold and LH beta mRNA 16-fold over values in intact females. Serum and pituitary LH concentrations rose gradually, and both were increased by 7-10 days after castration. The increase in serum and pituitary LH followed a time course similar to that of the progressive rise in LH beta mRNA concentrations. These data show that an increase in steady state LH subunit mRNA concentrations is one of the mechanisms involved in increased gonadotropin biosynthesis and secretion after castration. The kinetics of LH subunit mRNA and LH secretory responses are different in male and female rats and suggest that the concentration of LH beta mRNA may be a limiting factor in LH secretion.

Alpha and LH beta subunit mRNAs were measured in pituitaries of 4-day cycling rats during the estrous cycle. A two-fold increase in alpha mRNA occurred between 0800-2000 h on diestrus, but alpha mRNA concentrations were stable during other days of the cycle. LH beta mRNA concentrations were low during estrus and metestrus (11-16 pg cDNA bound/100 micrograms pituitary DNA), but were elevated (27-30 pg) between 0800-2000 h on diestrus. A second increase in LH beta mRNA was observed on the afternoon of proestrus, prior to the onset of the LH surge with maximum values (45 pg) coincident with peak LH secretion. LH beta mRNA concentrations declined rapidly and had fallen to basal values by midnight on proestrus. These data show that alpha and LH beta mRNAs change in a similar manner during metestrus, diestrus and estrus, suggesting coordinate regulation of alpha and LH beta gene expression at these times. During the LH surge, however, LH beta mRNA alone is increased, suggesting that the LH beta gene can be differentially expressed at times when maximum LH secretion is occurring.

Changes in the frequency of GnRH and LH pulses have been shown to occur between the luteal and preovulatory periods in the ovine estrous cycle. We examined the effect of these different frequencies of GnRH pulses on pituitary concentrations of LH and FSH subunit mRNAs. Eighteen ovariectomized ewes were implanted with progesterone to eliminate endogenous GnRH release during the nonbreeding season. These animals then received 3 ng/kg body weight GnRH in frequencies of once every 4, 1, or 0.5 h for 4 days. These frequencies represent those observed during the luteal and follicular phases, and the preovulatory LH and FSH surge of the ovine estrous cycle, respectively. On day 4, the ewes were killed and their anterior pituitary glands were removed for measurements of pituitary LH, FSH, and their subunit mRNAs. Pituitary content of LH and FSH, as assessed by RIA, did not change (P greater than 0.10) in response to the three different GnRH pulse frequencies. However, subunit mRNA concentrations, assessed by solution hybridization assays and expressed as femtomoles per mg total RNA, did change as a result of different GnRH frequencies. alpha mRNA concentrations were higher (P less than 0.05) when the GnRH pulse frequency was 1/0.5 h and 1 h, whereas LH beta and FSH beta mRNA concentrations were maximal (P less than 0.05) only at a pulse frequency of 1/h. Additionally, pituitary LH and FSH secretory response to GnRH on day 4 was maximal (P = 0.05) when the pulse infusion was 1/h.(ABSTRACT TRUNCATED AT 250 WORDS)