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GnRH acts via a single cell surface receptor (GnRH-R), and the number of pituitary GnRH-R increases on proestrus, after gonadectomy, or in response to pulsatile GnRH in the rat. Estradiol (E2) is known to exert a transient positive action to increase GnRH-R number, and the rise in plasma E2 contributes to initiation of the midcycle LH surge. The present study was designed to determine the effect of GnRH pulse amplitude and frequency on GnRH-R messenger RNA (mRNA) levels and to assess the relative contributions of GnRH and gonadal steroids to increasing GnRH-R gene expression. These studies were conducted in vivo using previously characterized GnRH-deficient male (castrate testosterone-replaced) and ovariectomized phenoxybenzamine-treated female models. To investigate the effect of GnRH pulse amplitude, adult male and female rats received GnRH iv (5-250 ng/pulse at 30-min intervals; saline pulses to controls) for 12 or 24 h. In males, GnRH-R mRNA was increased by all pulse doses, with maximal effects (3-fold) at 5-25 ng/pulse. In contrast, only lower doses (5-10 ng/pulse) were effective in females (2-fold increase). In a subsequent study, GnRH pulses (25 ng for males; 10 ng for females) were given at 8-, 30-, or 240-min intervals for 12 or 24 h. Some animals received a continuous GnRH infusion (200 ng/h). In males, GnRH-R mRNA levels were stimulated by all GnRH pulse intervals (maximal after 30-min pulses), whereas continuous GnRH was ineffective. In females, only 30- and 240-min pulse intervals increased GnRH-R mRNA levels, with faster (8-min) pulses or continuous GnRH being ineffective. To determine the relative roles of ovarian steroids and GnRH, ovariectomized phenoxybenzamine-treated female animals received GnRH (10 ng/pulse, 30-min interval), E2 (via sc implants; plasma E2 levels, approximately 50 pg/ml), or their combination for 12-24 h (saline pulses to controls). In the absence of E2, GnRH-R concentrations fell by 70% between 12-24 h. E2 alone tended to increase GnRH-R mRNA at 12 h, with a 2-fold rise observed after 24 h. Pulsatile GnRH alone increased GnRH-R mRNA by 50% at 12 h (compared to saline-pulsed controls; P < 0.05) and by 6-fold after 24 h. When GnRH and E2 were combined, the magnitude of the increase (vs. saline controls) was greater than that seen for either GnRH or E2 alone.(ABSTRACT TRUNCATED AT 400 WORDS)

Gonadotropin subunit gene transcription and messenger RNA (mRNA) levels are differentially regulated by GnRH pulse frequency and amplitude in the male rat. The rapid changes of subunit mRNA levels and LH and FSH secretion during the estrous cycle, particularly the rapid rise in LH-beta subunit mRNA on proestrus afternoon, suggest that physiological changes in the pattern of GnRH action may also be important in female rats. However, in the absence of a GnRH-deficient female model the role of varying GnRH stimulation remains to be determined. We have characterized a GnRH-deficient model by administering the alpha-adrenergic antagonist phenoxybenzamine (PBZ) to ovariectomized (OVX) rats. Initial experiments showed that PBZ given 24 h earlier abolished the afternoon LH surge in OVX estradiol (E2) replaced rats whereas LH responses to exogenous GnRH were preserved. A PBZ regimen of 15 mg/kg ip at OVX followed by 10 mg/kg at 24 h and 5 mg/kg at 48 h prevented the increase in alpha, LH-beta, and FSH-beta mRNAs and LH and FSH secretion for 72 h post-OVX. LH and FSH responses to GnRH pulses were preserved suggesting that PBZ blocked the post-OVX increase in hypothalamic GnRH secretion. The suppressive effect of PBZ appeared to be specific to the hypothalamic-pituitary-ovarian axis as plasma PRL, TSH, and corticosterone were not decreased compared to controls. We have used this GnRH-deficient OVX female model to investigate the effects of exogenous GnRH pulses on subunit mRNA expression. GnRH pulses (5-250 ng/30 min for 12-24 h) were administered via an intraatrial catheter beginning 24 h after OVX and the first PBZ injection (OVX+PBZ+saline pulses to controls). Expression of alpha and FSH-beta mRNAs and LH and FSH secretion were increased by GnRH pulse doses of 5-25 ng to values similar to or greater than those in OVX controls though the higher doses of GnRH/pulse did not increase FSH-beta mRNA or plasma FSH. However, LH-beta mRNA levels were not increased by GnRH pulses. GnRH pulses were also given to rats replaced with proestrus concentrations of estradiol alone or in combination with progesterone (P). Again, no demonstrable increases in LH-beta mRNA expression were observed. alpha-mRNA concentrations were further increased in the presence of E2 alone, and P in combination with E2, produced an augmented response of FSH-beta subunit mRNA. These data suggest that ovarian steroid hormones act directly on the gonadotrope to augment alpha and FSH-beta mRNA responses to GnRH.(ABSTRACT TRUNCATED AT 400 WORDS)

To investigate the mechanism(s) during pubertal development by which androgens alter hypothalamic proopiomelanocortin (POMC) gene expression and beta-endorphin content, we used the technique of in situ hybridization histochemistry and the androgen-insensitive testicular feminized (Tfm) rat. We evaluated POMC mRNA levels in the arcuate nuclei and periarcuate regions of 12 coronal brain slices from prepubertal (age, 30 days) and adult (age, 60 days) normal male and Tfm rats (n = 4 for each group). Hybridizations were performed using an 35S-radiolabeled oligonucleotide probe complementary to a 30-base sequence within POMC mRNA. The tissue sections were sequentially exposed to x-ray film and photographic emulsion with subsequent analysis by both densitometry and computer-assisted grain counting. beta-Endorphin was measured in hypothalamic tissue blocks from similar animals in each of the four experimental groups. The results of densitometry and grain counting were consistent and revealed an increase in POMC mRNA with pubertal development in both the male and Tfm animals. The concentration of hypothalamic beta-endorphin was greater for the adult Tfm animals than for all other groups, which did not differ from each other. These results suggest that androgens may stimulate POMC gene transcription by their action through estrogen receptors after conversion by aromatase. Alternatively, additional pubertal factors may be responsible for act directly through their respective receptors to alter translation, posttranslational processing, or secretion of beta-endorphin, resulting in diminished intracellular hypothalamic peptide concentration.

To investigate the role of somatostatin (SRIF) in regulating sexually dimorphic GH secretion, we used a reverse hemolytic plaque assay and acutely dispersed somatotropes from age-matched normal male, normal female, and androgen receptor-deficient, testicular feminized (Tfm) rats. Hemolytic plaques were developed after a 90-min incubation in the presence of GH antiserum, 10 nM GH-releasing hormone (GHRH), and the following concentrations of SRIF: 0, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 nM. Additional studies were performed with 0 or 100 nM SRIF in the absence of GHRH. The absolute number of somatotropes (x10(6); mean +/- SEM) recovered from the pituitaries of Tfm rats (1.73 +/- 0.18) was significantly greater than that from the males (1.11 +/- 0.13; P = 0.01); the number from female rats (1.30 +/- 0.15) was not different from that of either male or Tfm animals. GHRH-stimulated GH secretion, as estimated by the mean GH plaque area (micron2 x 10(4); mean +/- SEM) in the absence of SRIF, was greater for somatotropes from male rats (3.36 +/- 0.41) than that for either Tfm (2.27 +/- 0.32; P = 0.02) or female (1.78 +/- 0.24; P = 0.001) rats; values for the latter two groups did not differ. However, mean GH plaque areas for each group during maximal SRIF inhibition in either the presence or absence of GHRH were indistinguishable from each other and from mean plaque areas obtained under basal conditions. As demonstrated by a lesser EC50 value (0.04 +/- 0.02 nM; mean +/- SEM), somatotropes from female rats were more sensitive to the inhibitory effect of SRIF than were those from either male (EC50 = 1.82 +/- 0.45; P = 0.0001) or Tfm (EC50 = 0.74 +/- 0.22, P = 0.0001) rats; values for the latter two groups were indistinguishable. These observed differences suggest that gender and/or the gonadal hormone environment may be important determinants of the inhibitory effects of SRIF on GH secretion by the somatotrope. While these gender-associated differences may represent effects of the gonadal hormones directly on the somatotrope, they could reflect modulation of the secretion of hypothalamic SRIF and/or GHRH by the prevailing gonadal hormone environment. Such gender-related differences may contribute to the overall sex-dependent patterns of GH secretion in the intact animal.