Freja Kamel

A Detailed Examination of the in vivo and in vitro Effects of ACTH on Gonadotropin Secretion in the Adult Rat

These studies were designed to: (1) determine the effects of continuous infusion of synthetic ACTH(1-24) on postcastration changes in serum and pituitary LH, FSH and prolactin in the male rat; (2) assess the effects of adrenalectomy on the gonadotropin and prolactin response to ACTH, and (3) test the hypothesis that ACTH may directly (not via adrenal factors) alter gonadotropin secretion at the brain and/or pituitary level. Adult male rats were either orchidectomized (ORX) or orchidectomized-adrenalectomized (ORX-ADX), and were treated continuously for 6 days with ACTH(1-24) (10 micrograms/day) or saline using an osmotic minipump. Animals were killed on day 6 following castration. ACTH treatment reduced serum LH and prolactin levels in ORX rats to mean values +/- SE of 204 +/- 25 and 37 +/- 3 ng/ml respectively, compared to 366 +/- 72 and 62 +/- 7 ng/ml in saline-treated ORX animals. Serum FSH concentrations were not altered by ACTH administration. Pituitary concentrations of LH and FSH, but not prolactin were enhanced by ACTH treatment. Adrenalectomy had no effect on serum and pituitary gonadotropin and prolactin levels, but abolished the effects of ACTH on these parameters. Central (intracerebroventricular) infusion of ACTH(1-24) (6 micrograms/day X 4 days) failed to alter the rise in serum LH in male rats following orchidectomy. Acute treatment with large doses of ACTH of perifused anterior pituitary glands from male rats and chronic treatment with ACTH of enzymatically dispersed anterior pituitary cells from female rats did not influence basal or GnRH-stimulated LH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Gonadal steroid modulation of LHRH-stimulated LH secretion by pituitary cell cultures☆

Enzymatically dispersed rat pituitary cells were grown in primary culture, and LHRH-stimulated LH secretion was measured. Testosterone (T) decreased and 17 beta-estradiol (E) increased pituitary responsiveness to LHRH. The effect of E on LH secretion was partly due to an increase in LH content. There was a latent period of 12 h for E and 18 h for T between the onset of steroid treatment and the manifestation of steroid action. Neither steroid was required to be continuously present in order to exert its effects. After steroid withdrawal, the effect of T persisted for 72 h and that of E for more than 96 h. The actions of both steroids were blocked by protein-synthesis inhibitors. These results are consistent with the hypothesis that steroid effects rely on a mechanism involving alterations in protein synthesis; the affected proteins may be involved in the process of LHRH action.

Progesterone modulation of gonadotropin secretion by dispersed rat pituitary cells in culture. I. Basal and gonadotropin-releasing hormone-stimulated luteinizing hormone release.

Dispersed, estradiol-treated, rat pituitary cells were cultured to characterize the influences of a physiologic concentration of progesterone (P, 10(-7) M) on gonadotroph responsiveness to gonadotropin-releasing hormone (GnRH). Acute (less than 6 h) P treatment enhanced and chronic (greater than 12 h) treatment suppressed both basal and GnRH-stimulated luteinizing hormone (LH) release. This modulation took place without any change in intracellular LH stores, indicating that the secretory changes are not attributable to changes in LH synthesis, and were not accompanied by similar alterations in basal or thyrotropin-releasing hormone-stimulated prolactin secretion. Moreover, the timing of these responses was fixed since a 10-fold lower P concentration produced only smaller and briefer alterations in LH release. Analyses of the temporal characteristics of effective P stimuli indicated that a brief 6 h exposure to P inhibited GnRH-stimulated LH secretion 18 h later. In contrast, Ps acute actions rapidly dissipated following removal of the steroid from the culture medium. Finally, P-induced enhancement and suppression of GnRH-stimulated LH release could be blocked by appropriately timed treatments with protein synthesis inhibitors. Our findings are consistent with the hypothesis that P influences gonadotroph secretory function via the production of specific proteins.

Intracellular receptors mediate gonadal steroid modulation of LHRH-induced LH release

Primary cultures of enzymatically dispersed rat pituitary cells were used to study the role of intracellular receptors in gonadal steroid modulation of LHRH-induced LH release. Nuclear receptors for both testosterone (T) and 17 beta-estradiol (E) were observed, with KD values of 6.3 and 0.1 nM respectively. Occupation of these receptors was correlated with modulation of LH secretion. The relationship between these two parameters was nonlinear, so that steroid effects on LH secretion were maximal when fewer than 50% of the receptors were occupied. The androgen antagonist cyproterone blocked both T binding to nuclear receptors and T inhibition of LH secretion. Similarly, the estrogen antagonist CI-628 blocked both E binding and E stimulation of LH secretion. In cultures derived from pseudohermaphrodite rats. T did not bind to nuclear receptors, nor did it inhibit LH secretion. These results, showing a relationship between occupation of nuclear receptors and modulation of LH secretion, suggest that steroid effects on LH secretion are mediated by these receptors.

Progesterone modulation of gonadotropin secretion by dispersed rat pituitary cells in culture. II. Intracellular metabolism and progestin receptors

Dispersed, estradiol (E2)-treated, rat pituitary cell cultures were used to examine the intracellular processing of progesterone (P) associated with its modulation of gonadotropin-releasing hormone (GnRH)-stimulated luteinizing hormone (LH) secretion. Enhancement and suppression of LH release was only observed with acute and chronic exposures to P or other naturally occurring and synthetic progestins avidly bound by pituitary progestin receptors; such responses were inhibited by cotreatment with the antiprogestin RU486 but not with the antiandrogen flutamide, illustrating the importance of the P + receptor interactions. However, cotreatment with a 100-fold molar excess of the 5 alpha-reductase inhibitor 17 beta-N,N-diethyl-carbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one (4-MA) had no effect on the expression of Ps modulatory actions. Additional studies using different E2 pretreatments revealed that P enhanced LH release when progestin receptor levels were elevated. Moreover, the magnitude and duration of Ps influences on LH release increased in cells with higher receptor levels. However, there were several instances in which progestin receptor level and P modulation of LH release did not correlate. In several instances E2-induced progestin receptor levels stabilized at a maximal level whereas P enhancement of LH secretion continued to increase in size and duration. These findings underscore the importance of progestin receptors for P-induced modulation of LH secretion and illustrate that 5 alpha-reduction and further metabolism of P is not obligatory for the expression of these responses. In addition, our data demonstrate that the important cellular mechanisms underlying E2 priming of gonadotroph responsiveness to P entail the induction of progestin receptor levels and other as yet unidentified cellular processes.

Gonadal steroid modulation of LH secretion stimulated by LHRH, Ca2+ and cAMP

Primary cultures of enzymatically dispersed rat pituitary cells were used to examine steroid effects on LH secretion stimulated by LHRH, Ca2+ and cAMP. Cultures were pretreated for 48 h with testosterone (T) or 17 beta-estradiol (E) and then challenged for 4 h with various secretogogues. T did not affect basal LH secretion; it inhibited the responses to Ca2+ and LHRH; and it potentiated the response to cAMP. E stimulated both basal LH secretion and the responses to all secretogogues, without affecting cell LH content. Thus, T affects stimulus-secretion coupling, while E affects secretion per se. All steroid effects were blocked by steroid antagonists, indicating that steroid action is receptor-mediated regardless of the secretogogue involved. The similarity of steroid effects on the responses to LHRH and Ca2+ but not cAMP suggests that Ca2+ rather than cAMP is a second messenger for LHRH, and that steroid action occurs at some step subsequent to LHRH-stimulated Ca2+ mobilization.

Testosterone processing by pituitary cells in culture: An examination of the role of 5α-reduction in androgen action on the gonadotroph

Dispersed rat pituitary cells were exposed to [1,2,6,7-3H]testosterone ([3H]T, 10(-8) M) to assess the role of 5 alpha-reduction in T regulation of gonadotroph secretion. After 4 to 48 hours of exposure, [3H]T metabolites isolated by thin-layer chromatography were characterized in medium and cell homogenates as well as bound to androgen receptors salt-extracted from purified nuclear pellets. Receptor-bound 5 alpha-[3H]dihydrotestosterone ([3H]DHT)/total [3H]androgens rose progressively from 16% at 4 hours to more than 50% at 48 hours. Coincubation with 4-MA (10- to 1,000-fold molar excess) or testosterone-17 beta-carboxylic acid (TCA; 1,000-fold excess) reduced receptor-bound [3H]DHT/[3H]androgen to less than 10% and 20%, respectively, but elevated [3H]T-receptor levels. Despite inhibiting 5 alpha-reductase activity, TCA and 4-MA had no effect on T suppression of gonadotropin-releasing hormone-stimulated luteinizing hormone secretion or T enhancement of total (cell + secreted) follicle-stimulating hormone levels. The results suggest that 5 alpha-reduction to DHT is not essential for the expression of the direct influences of T on gonadotropin synthesis and secretion in rat gonadotrophs.

Behavioral and Neuroendocrine Effects of Long-Term Progesterone Treatment in the Rat

Progesterone and estradiol, alone or in combination, were administered to ovariectomized rats for 2 weeks. Progesterone alone had no effect on body weight, luteinizing hormone (LH) secretion, receptivity or brain cytoplasmic progestin receptors (CPR). Progesterone in combination with estradiol significantly attenuated estrogen suppression of weight gain and estrogen stimulation of receptivity, the LH afternoon surge and induction of CPR, but did not affect the negative feedback of estrogen on morning LH levels. The decrease in CPR after 2 weeks of progesterone is very similar in magnitude to the decrease observed following acute treatment, suggesting that, unlike neurotransmitter agonists and glucocorticoids, progesterone does not cause down regulation of its receptors in the brain following chronic treatment.

Estradiol processing by pituitary cells in culture: An examination of the influences of various exposures to progesterone

Dispersed, estradiol (E2)-treated pituitary cells were used to examine the cellular mechanisms underlying progesterone (P) suppression of GnRH-stimulated LH and FSH secretion. When cells were exposed to 10(-9) M E2 for 48 h prior to GnRH challenge, P (10(-7) M) treatment for the last 24 h suppressed gonadotroph responsiveness to GnRH for both LH and FSH secretion (gonadotropin released/intracellular stores of gonadotropin available for release). To determine if P acts by blocking E2 processing and/or uptake, we exposed cells to 2,4,6,7-3H-E2 +/- P and monitored the level and distribution of 3H-estrogens bound to estrogen receptors salt-extracted from nuclear pellets purified by sucrose density centrifugation. At 1, 4 and 24 h, P had no effect on the level of 3H-estrogen+receptor complexes or on the distribution of receptor-bound 3H-E2, 3H-estrone and 3H-estriol. The results indicate that chronic influences of P to suppress the responsiveness of E2-treated gonadotrophs to GnRH cannot be explained by alterations in estrogen receptor occupation as is the case in reproductive tract tissues.

Aromatization: important for testosterone's developmental influences on the neurocircuits mediating cervically stimulated prolactin secretion in the female rat

Adult female rats treated neonatally with testosterone or estrogen fail to respond to cervical stimulation with episodic prolactin release. The developmental action of testosterone is inhibited by co-administration of 1,4,6-androstatriene-3,17-dione at a dose sufficient to inhibit the testosterone-induced rise in estrogen receptors in limbic brain cell nuclei. These data suggest the importance of aromatization for testosterone action.