Robert F. Casper

Induction of prolactin release by LRF and LRF-agonist

Abstract Infusion of LRF (0.2 μg/min × 3.5 hrs) and injection of LRF-agonist (50 μg, SC) elicited a prompt and sustained PRL release by the pituitary. This effect is independent of ovarian steroid environment as observed in both normal cycling women and hypogonadal subjects with similar time course and magnitude of PRL release.

Studies on the effect of gonadotropin-releasing hormone and its agonist on human luteal steroidogenesis in vitro

The possible direct effect of gonadotropin-releasing hormone (GnRH) and a potent GnRH agonist [( imBzl )-D- His6 -Pro9-NEt]-GnRH on basal and human chorionic gonadotropin (hCG)-stimulated progesterone, androstenedione, and estradiol production by cultured human luteal cells was examined. Luteal cells from the early or midluteal phase of the menstrual cycle responded to hCG stimulation with two to fivefold increases in steroid production in both short-term (4 hours) and long-term (up to 144 hours) culture in chemically defined medium without serum. After 48 hours in this system, levels of androstenedione and estradiol were very low, and progesterone was the predominant steroid produced. The addition of GnRH or a potent GnRH agonist to the medium had no effect on either basal or hCG-stimulated steroid secretion. When luteal cells were cultured longer (for up to 10 days) in the presence of serum, GnRH agonist caused no significant alteration of either basal or hCG-stimulated progesterone production. Collectively, these results support the conclusion that GnRH and its potent agonist do not act directly on human corpora luteal cells to modulate steroidogenesis.

Intravaginal administration of progesterone: enhanced absorption after estrogen treatment *

A progesterone solution was administered by intravaginal instillation, intramuscular injection, and sublingually to estrogen-deficient women, with or without estradiol (E2) replacement, and serum progesterone (P) concentrations were measured by radioimmunoassay. Intravaginal application to postmenopausal subjects receiving E2 gave the highest values of serum P: 10 times baseline at 15 minutes and 30 to 40 times at 1 to 2 hours, with sustained levels, for 7 hours and decline to 10 times baseline at 24 hours. Intravaginal application to hypoestrogenic women gave similar results, but of much lower magnitude (highest value, 20 times baseline). Intramuscular injection, in contradistinction, showed gradually increasing levels over the study period, up to 30 times basal values at 24 hours. It contrast, sublingual application produced very modest serum increases, to approximately 10 times baseline within 2 hours and return to basal value at 24 hours. Since the most rapid and highest levels were observed by vaginal application to postmenopausal women receiving estrogen, and considering that the vagina has been similarly shown to be very effective for estrogen absorption, it is conceived that full hormone replacement could be accomplished in the deficient states by cyclic vaginal application of both steroids.

Chorionic gonadotropin prevents LRF-agonist-induced luteolysis in the human

The effects of exogenous and endogenous hCG on the luteolytic action of LRF-agonist, [D-Trp6, Pro9NEt]-LRF (LRF-Ag), were evaluated. In sequential studies, 4 normal cycling women treated with LRF-Ag (50 microgram S.C.) on two consecutive days had premature decline of circulating levels of progesterone (P) and estradiol (E2) with a shortened (p < 0.05) luteal phase (11.5 +/- 1.2 days) when compared to the control cycle (15 +/- 0.7 days). When intramuscular hCG was added to the LRF-Ag treatment in doses of 100 IU or 5000 IU daily for 7 days, the luteal function was prolonged (19 +/- 1.2 days, p < 0.05) with significant (p < 0.001) elevation of P and E2 levels compared with the control cycle. Four women with early pregnancy, requesting therapeutic abortion, were given LRF-Ag (50 microgram or 500 microgram S.C.) on 2 consecutive days. None of the 4 women aborted and there was no change in the levels of beta hCG, P or E2 over the course of a week. These results indicate that both exogenous or endogenous hCG can overcome the luteolytic effect of LRF-Ag within the dose and duration used. The possibility that a more prolonged administration of a larger dose of LRF-Ag may negate the luteotropic effect of hCG remains to be explored.

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.

In vitro heteroregulation of LH receptors by prolactin and FSH in rat granulosa cells

The purpose of the present study was to further characterize the regulation of LH/hCG receptors by FSH in granulosa cells and test the hypothesis that the LH/hCG receptor levels are heteroregulated by PRL. Granulosa cells from immature hypophysectomized, DES-treated rats were cultured for 2-4 days in defined medium containing androstenedione (10(-7) M) and/or FSH and PRL, after which [125I]iodo-hCG binding to the granulosa cells was measured. When granulosa cells were cultured for 2 days (days 0-2) with increasing concentrations of FSH (0.1-100 ng/ml), there was a dose related increase in [125I]iode-hCG binding from a control value of 1.05 +/- 0.2 fmoles/10(6) cells to a maximum of 20 +/- 1.8 fmoles/10(6) cells. The miminum, half-maximum (ED50) and maximum doses of FSH were 0.3, 0.5 and 3 ng/ml, respectively. At concentrations of FSH greater than 3 ng/ml there was a progressive decrease in [125I]-iodo-hCG binding to a low value of 6.1 +/- 1 fmoles/10(6) cells at 100 ng/ml of FSH. No changes in [125I]iodo-hCG binding were observed in response to PRL (1 microgram/ml) during the day 0-2 incubation. When granulosa cells were stimulated for 2 days with 20 ng/ml of FSH, washed, and then recultured for another 2 days (days 2-4) with FSH, the LH/hCG receptor content remained high (F leads to F = 17.4 +/- 2.8 fmoles/10(6) cells). In contrast, when FSH-primed cells were recultured for 2 days without FSH, the [125I]iodo-hCG binding decreased sharply to near control levels (F leads to C = 2.5 +/- 0.2 fmoles/10(6) cells). This marked loss of LH/hCG receptors was largely prevented when FSH primed cells were recultured with PRL (F leads to P = 10.3 +/- 1.5 fmoles/10(6) cells). This stimulatory effect of PRL on [125I]iodo-hCG binding was dose-dependent: minimum, ED50, and maximum doses of PRL were 0.2, 0.5 and 1 microgram/ml, respectively. Scatchard-plot analysis revealed that although the dissociation constant (Kd) of the LH/hCG receptors stimulated by FSH and PRL were of similar high affinity (approximately 8 x 10(-11) M), the maximum binding (Bmax) values in the PRL-treated cells were less. Addition of 10(-7) estradiol together with the PRL did not cause a further increase in Bmax values above that observed with PRL alone.

Changes in pituitary hormones during and following transsphenoidal removal of prolactinomas

Abstract Prolactin (PRL) and other pituitary hormones (luteinizing hormone, follicle-stimulating hormone, growth hormone [GH], thyrotropin stimulating hormone) were measured before, during, and after transphenoidal pituitary adenomectomy in 16 patients with hyperprolactinemia. The diagnosis of prolactinoma was made in three of the 16 patients by the absence of PRL response to thyrotropin-releasing factor (TRF) and a dopamine receptor antagonist, metoclopramide, without radiologic evidence of an adenoma. Contrary to findings in subjects with normal PRL values, the PRL rise in response to anesthesia and operation was absent. Other pituitary hormones, with the exception of GH, which increased during anesthesia and operation, exhibited no acute changes. In 11 of 16 cases, complete tumor removal was achieved as determined by the rapid decline of PRL levels to normal values within 24 to 48 hours after operation and by subsequent clinical follow-up. This finding documents that the adenoma is the main source of excessive PRL secretion. The circulating half time of immunoreactive PRL determined by frequent sampling in these patients was variable, ranging from 74 to 190 minutes, significantly longer than the previously reported value of 15 minutes determined by bioassay. Although a transient decline was evident, serum PRL levels remained elevated in those patients with incomplete tumor removal. These findings suggest that a single measurement of serum PRL within 24 to 48 hours following transphenoidal adenomectomy is a reliable indicator of the success or failure of the procedure.

Role of serum-free defined medium in regulation of LH receptor in cultured rat granulosa cells.

The induction of luteinizing hormone (LH) receptor by follicle-stimulating hormone (FSH) in granulosa cells was compared following culture in serum-free or serum-containing medium. Incubation of primary cultures of granulosa cells in serum-free defined medium with purified FSH resulted in dramatic increases in the level of functional LH receptors. This striking enhancement of LH receptor by FSH was completely abolished by concomitant incubation with serum (rat, horse, porcine, human or calf). The serum inhibition of FSH was not readily reversible and could be evoked throughout the culture period. The synthesis of cAMP by FSH was markedly suppressed by serum, suggesting that serum component(s) are inhibiting FSH action at the level of adenylate cyclase. Such an action, however, cannot be the sole mechanism because serum also blocked LH receptor induction by cyclic AMP analogs. In defined medium, addition of insulin, transferrin, dexamethasone or fibronectin alone had no effect on basal levels of LH receptor. However, following incubation with either insulin or dexamethasone, the FSH-induced increases in LH receptor were markedly suppressed. Insulin was found to markedly inhibit FSH-stimulated cyclic AMP formation; this was not the case with dexamethasone. The present results demonstrated the complete inhibition of FSH action by serum in cultured granulosa cells and suggest that the effect is caused by a combination of direct actions of common metabolic hormones which inhibit FSH action at multiple sites. These experiments clearly indicate the obligatory role of defined medium in the hormone-dependent differentiation of the granulosa cell in culture.

Effects of a superactive luteinizing hormone-releasing factor agonist on gonadotropin and ovarian function during the menstrual cycle

The effect of a potent luteinizing hormone-releasing factor (LRF) agonist (D-Trp6, Pro9 NEt)-LRF on pituitary gonadotropin release and its concomitant ovarian response was examined in normal women during the early follicular (EFP), late follicular (LFP), and midluteal (MLP) phases. A single subcutaneous injection of 50 micrograms of LRF agonist in subjects during the EFP caused prompt release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) to levels comparable to those found during spontaneous midcycle gonadotropin surges, while in LFP subjects gonadotropin levels rose 1 1/2 to 2 times above the levels of midcycle surges. The LH/FSH release in the MLP was almost identical to that found in the EFP. The ovarian response as measured by increasing estradiol levels followed a similar pattern during the 48 hours after injection in all three phases of the cycle. The inappropriate gonadotropin surge induced by LRF agonist in EFP subjects resulted in prolonged follicular phases and anovulation. Three of four subjects in the LFP showed evidence of ovulation in response to the same dose of LRF agonist. The pharmacodynamics of gonadotropin-ovarian responses to this potent LRF agonist reported here should provide an important reference for systemic investigation and rational clinical application.