C. F. Wang

The Operating Characteristics of the Hypothalamic–Pituitary System during the Menstrual Cycle and Observations of Biological Action of Somatostatin

Publisher Summary This chapter focuses on the investigations conducted over the past few years on the interaction between the hypothalamic hormone(s) and gonadal steroids in the regulation of cyclic gonadotropin output by the adenohypophysis in humans. It describes hypothalamus as a neuroendocrine regulator. It also reviews the observations on the biological actions of hypothalamic somatostatin in humans. The description of the hypothalamic-hypophysial portal system and the subsequent presentation of a number of anatomical and physiological observations led to the proposal that the hypothalamus is critically involved in the control of anterior pituitary function. The chapter presents the three elements of the hypothalamic-hypophysial portal system: (1) the CNS-hypothalamus complex that may be regarded as a signal generator, (2) the pituitary as a signal transmitter, and (3) the cyclic ovarian steroid output as a signal modulator. It reviews the regulation of the hypothalamic-hypophysial- gonadal system in humans within this general context.

Prolactin inhibition of estrogen production by cultured rat granulosa cells

The effect of prolactin (PRL) treatment on estrogen production by rat granulosa cells was investigated in vitro. Immature, hypophysectomized, DES-treated rats were injected for 2 days with FSH to induce aromatase enzymes and receptors for PRL and LH. After FSH priming, the granulosa cells were cultured for 4 days in serum-free medium containing 10(-7) M androstenedione and purified FSH, LH and/or PRL. A dose-related inhibition of estrogen production from control cells was observed following PRL treatment in which 1 micrograms/ml of PRL inhibited estrogen formation by > 90%. In these same cultures, PRL caused a dose-related increase in progesterone and 20 alpha-dihydroprogesterone secretion. Treatment with purified FSH or LH stimulated estrogen synthesis by 3-10-fold. Concomitant treatment with PRL suppressed the FSH- and LH-induced increases in estrogen production in a dose-dependent manner; 1 micrograms/ml PRL suppressed estrogen production by > 80% during days 2-4 of culture. In these same cultures, PRL did not alter the stimulatory effects of FSH and LH on progesterone and 20 alpha-dihydroprogesterone production. These experiments demonstrate that PRL acts directly on rat granulosa cells in vitro to suppress basal and gonadotropin-induced increases in estrogen production.

Studies on the mechanism of LH receptor control by FSH

Abstract The mechanism of the control of LH/hCG receptor by FSH was investigated using granulosa cells from the immature hypophysectomized estrogen primed rat as a model. The specific binding of [ 125 I]hCG to freshly collected granulosa cells was low (0.18 ± 0.01 fmoles/10 6 cells). By contrast, a marked increase in [ 125 I]hCG binding was observed when granulosa cells were cultured in serum free medium with FSH (10 ng/ml); time course studies revealed that the binding capacity was low at day 1 (1.3 ± 0.4 fmoles/10 6 cells), increased sharply at day 2 (11.7 ± 1.7 fmoles/10 6 cells), then reached a maximum at day 3 (19.2 ± 1.3 fmoles/10 6 cells). Comparable results were obtained when cells were primed with FSH in vivo, thus indicating that the defined culture system is both efficient and physiologic for LH/hCG receptor induction. Cholera toxin (CT), PGE 2 , and cyclic nucleotide analogues mimicked in part the FSH effect; Scatchard analysis showed that the 125 I-high binding sites induced by all agents were of high affinity ( K d = ~1 × 10 −11 ), but the number of binding sites induced by CT, PGE 2 , and the cyclic nucleotides was 70, 45 and 25%, respectively, of that induced by FSH. The effect of the agents on the in vitro induction of hCG binding sites was dose dependent; the ED 50 s of CT, FSH and PGE 2 for receptor induction were 40 pg/ml, 4 ng/ml, and 30 ng/ml, respectively. The maximum induction of hCG-binding sites by FSH, CT and PGE 2 closely paralleled their maximum stimulatory effects on cyclic AMP formation. Autoradiography revealed that the increase in [ 125 I]hCG-binding sites was a heterogenous process: in the FSH, CT and PGE 2 treated cultures, only 47 ± 4, 38 ± 4 and 28 ± 2%, respectively, of the viable cells were labeled. By contrast, all compounds induced 3β-hydroxysteroid dehydrogenase activity in 70–80% of the viable cells. These results demonstrate a role of cyclic AMP in the mechanism of LH/hCG receptor regulation by FSH but reveal that the mechanism is complex and suggest that some non-cyclic AMP actions are also involved.

The role of cyclic AMP in the induction of estrogen and progestin synthesis in cultured granulosa cells

The role of cyclic AMP in the induction of enzymes involved in estrogen and progestin biosynthesis in undifferentiated granulosa cells was investigated. When granulosa cells from immature hypophysectomized, DES-treated rats were cultured for 2 days in serum-free medium with aromatase substrate (10(-7) M androstenedione) together with graded doses of FSH, prostaglandin E2 (PGE2), cholera toxin (CT), or dibutyryl cyclic AMP (Bu2cAmP), there was a dose-related increase in estrogen (E) production. The induction of E production by saturating doses of FSH, PGE2, CT, and Bu2cAmP required a lag phase of approximately 24 h, after which the E response increased sharply to maximum levels at day 3, and then declined gradually to day 5. Treatment for 24 h ((day 0-1) with FSH, together with 1 microgram/ml of either actinomycin D or cycloheximide, completely abolished the stimulatory action of FSH on E production. When the inhibitors were removed, the FSH-induced increases in E returned to near normal levels after a 24-h lag period. Similar effects of the inhibitors upon E production by CT, PGE2 and Bu2cAMP were observed. As with E, the production of progesterone and 20 alpha-dihydroprogesterone was markedly stimulated by FSH, PGE2, CT and Bu2cAmP, and the results of the time course, dose response, and inhibitor experiments were similar to those for E production. These results indicate that FSH induces the de novo synthesis of enzymes required for both estrogen and progestin biosynthesis by undifferentiated granulosa cells and suggest that this action is mediated by cyclic AMP.

LH stimulation of estrogen secretion by cultured rat granulosa cells

The direct effect of LH on estrogen secretion by rat granulosa cells was investigated. Ovarian granulosa cells from immature hypophysectomized diethylstilbestrol-treated rats were primed with FSH for 2 days in vitro to induce LH receptors. After the FSH priming, the granulosa cells were washed, and recultured for 4 additional days in media containing aromatase substrate (10(-7) M androstenedione) and purified FSH or LH. After the incubations, estrogen (E), progesterone (P) and 20 alpha-dihydroprogesterone (20 alpha-OH-P) in the media were measured by RIA. When granulosa cells from hypophysectomized DES-treated rats were cultured for 6 days with FSH and androstenedione, the production of E, P and 20 alpha-OH-P was stimulated to a maximum of 100-, 200- and 270-fold, respectively, above that of control levels. In contrast, LH did not increase steroidogenesis in these cells. Following 2 days of FSH priming in vitro, however, the cultured granulosa cells exhibited marked increases (400-600%) in E, P and 20 alpha-OH-P production in response to LH treatment over a 4-day incubation period. This stimulatory effect of LH on estrogen and progestin production was dose-related; the minimum and maximum effective doses of LH for steroid production were 3 and 30 ng/ml, respectively, and the ED50 was calculated to be 6 ng/ml of LH. As with LH, FSH also stimulated steroidogenesis in a dose-related manner and the apparent ED50 of FSH on steroidogenesis was 45 ng/ml. To investigate whether LH can also stimulate aromatase activity in granulosa cells primed with FSH in vivo, immature hypophysectomized DES-treated rats were injected for 2 days with FSH after which the granulosa cells were isolated and cultured for 4 days in medium containing 10(-7) M androstenedione and LH or FSH. Both LH and FSH stimulated E, P and 20 alpha-OH-P production, and the maximum steroidogenic responses of LH and FSH were similar to those observed in cultured granulosa cells primed with FSH in vitro. THese results have demonstrated that LH is effective in stimulating both estrogen and progestin secretion in rat granulosa cells pretreated with FSH. This suggests an important role of LH in the direct control of both aromatization and luteinization in the granulosa cell.

STEROID MODULATION OF THE HYPOTHALAMIC-PITUITARY SYSTEM IN THE SECRETION OF REPRODUCTIVE HORMONES

The results of a study of the inhibition and amplification of pituitary responses to the synthetic hypothalamic hormones LRF and TRF during estrogen and progesterone experiments in normal and hypogonadal women are reported. Serum luteinizing hormone (LH) follicle stimulating hormone (FSH) prolactin (PRL) and estradiol (E2) and progesterone were measured by radioimmunoassay. In 10 hypogonadal subjects there was a significant decrease in PRL levels during infusion of pharmacological doses of E2 followed by a significant increase in PRL release lasting several hours. Small doses of ethinyl estradiol (EE) induced a significant elevation of serum PRL levels with 1 week of treatment and a continued rise within 3-4 weeks to more than 3 times the initial concentration. The LH and FSH levels fell progressively to premenopausal levels at Week 4. With large doses of EE a more rapid increase of PRL levels was found and a higher elevation. Data indicate that pituitary sensitivity and reserve are modulated by estrogen and progesterone. The sensitivity appeared to be blunted. Results of experiments indicate that the pituitary gland represents a major feedback site for the regulation of optimal delivery of tropic hormones of reproduction via steroid signals. Estrogen provides a time and dose-related positive feedback loop in the augmentation of pituitary PRL secretion. Progesterone in low concentration has a facilitatory action on the pituitary response to LRF depending on prior exposure to estrogen in optimal amount and duration. Progesterone in high concentration in the midluteal phase lessens the estrogen-augmented pituitary response to LRF.

STEROID MODULATION OF THE HYPOTHALAMIC-PITUITARY SYSTEM IN THE SECRETION OF REPRODUCTIVE HORMONES*

Publisher Summary This chapter discusses the steroid modulation of the hypothalamic–pituitary system in the secretion of reproductive hormones. The brain-pituitary system that controls the reproductive hormones, gonadotropins and prolactin (PRL), appears to be distinct from that of metabolic hormones TSH, ACTH, and GH. The pituitary gland represents a major feedback site for the regulation of optimal delivery of tropic hormones of reproduction through steroid signals, and estrogen provides a time and dose related positive feedback loop in the preferential augmentation of pituitary PRL secretion. Based on changes in circulating levels, the overlapping negative and positive feedback effect of estrogen on gonadotropin output can be meaningfully correlated with the estrogen directed changes in pituitary dynamics, a time, and dose related increase in pituitary sensitivity and reserve. Progesterone in low concentration has a facilitatory action on the pituitary response to LRF operative only with prior exposure to an optimal amount, and duration of estrogen.