Follistatin suppresses steroid-enhanced follicle-stimulating hormone release in vitro in rats.

Previous in vitro and in vivo studies from our laboratory showed that progesterone (P(4)), corticosterone (B), and testosterone (T) increase intracellular content and release of FSH in the anterior pituitary. Activin (Act) and inhibin (Inh) are structurally related proteins with antagonistic actions, as Act stimulates and Inh inhibits FSH secretion from the anterior pituitary. Together with follistatin (FS), a protein that bioneutralizes Act, they form an autocrine-paracrine loop in the anterior pituitary that tightly regulates FSH secretion. The objective of the present study was to test the hypothesis that P(4), B, and T modulate this autocrine-paracrine loop to favor increased FSH secretion. If Act were to mediate steroid-induced FSH release, FS would be expected to block these effects. To test this interaction, cell cultures were prepared from anterior pituitaries of male and female rats, and treated with Act, B, P(4), or T in the absence or presence of FS. Act, B, P(4), and T increased FSH release; FS suppressed both basal and Act- and steroid-stimulated FSH release to approximately 50% below basal levels. Cell cultures from anterior pituitary of female rats were used to compare the interaction of incremental concentrations of FS on dose-related Act- and P(4)-stimulated FSH release. With increasing concentrations of Act, the FS-induced suppression of FSH release was attenuated and eventually abolished; in contrast, maximally stimulatory concentrations of P(4) did not fully overcome the FS-induced suppression of FSH release. The effects of P(4), B, and Act in the presence and absence of estradiol on steady-state mRNA levels of FSHbeta, Actbeta(B), and FS were determined in primary pituitary cell cultures from metestrous female rats by reverse transcription-polymerase chain reaction. Whereas Act, P(4), B increased FSHbeta mRNA levels, only Act raised the level of FS mRNA, and neither steroid increased Actbeta(B) mRNA. The results support the hypothesis that endogenous Act is a common mediator of the action of P(4), B, and T in the rat primary anterior pituitary cell culture. We conclude that the stimulation of FSH release and intracellular content in the gonadotroph by P(4), B, and T is mediated, in part, by Act and involves modulation of a tightly regulated Act/FS autocrine-paracrine loop.

Progesterone receptor A and B messenger ribonucleic acid levels in the anterior pituitary of rats are regulated by estrogen.

In target tissues of most mammalian and avian species, progesterone receptors (PR) are expressed as structurally related, but functionally distinct, isoforms A and B, and they are regulated by estrogen (E) as well as by their cognate ligand, progesterone (P(4)). The objectives of the present work were to identify mRNA expression for the A and B isoforms of PR in the anterior pituitary of the rat, to examine its regulation by gonadal steroids, and to compare this regulation with that in the primary target organ, the uterus. Messenger RNAs for the PR isoforms, determined by two separate reverse transcription-polymerase chain reaction protocols, one that detects PR A and PR B equally and the other specific for PR B, were identified in anterior pituitary of female and male rats. In anterior pituitary of cycling female rats, steady-state mRNA levels for both PR A+B and PR B were highest at 0900 h on proestrus, declined rapidly to nadir values at 0900 h on metestrus (PR A+B) or 0900 h on estrus (PR B), and remained below proestrous values through 2100 h on diestrus. Administration of E to intact proestrous female rats caused significant increases in mRNA for both PR A+B and PR B on estrus and metestrus. Blockade of P(4) action by administration of the antiprogestins RU-486 and ZK-98299 on proestrus had no effect on PR mRNA levels on the morning of estrus. Ovariectomy two and ten days after surgery markedly reduced mRNA levels for both PR A+B and PR B. Whereas treatment of 10-day-ovariectomized rats with E led to marked induction of mRNA for PR A+B and PR B two days later, treatment with P(4) one day after treatment had no effect on basal or E-stimulated PR mRNA. Regulation of PR mRNA expression in the pituitary differed from that in the uterus, in which P(4) treatment of ovariectomized rats antagonized the E-induced rise in mRNA for PR B, and antiprogestins increased mRNA for both isoforms. In addition to induction of PR mRNA in the pituitary of female rats by E in vivo, we also demonstrated induction by E in primary culture of anterior pituitary cells in vitro. We conclude that in the anterior pituitary of female rats, both the A and B isoforms of PR are expressed and regulated by E.

Antiprogestins suppress basal and activin-stimulated follicle-stimulating hormone secretion in an estrogen-dependent manner.

Previous studies from our laboratory, demonstrating that suppression of serum FSH by RU486 requires a high estrogen (E) background, suggested that E-inducible progesterone receptors play a role in the regulation of FSH secretion. We demonstrated further that the type II antiprogestin RU486 and the type I antiprogestin ZK98299 both suppressed the elevated serum FSH and FSHbeta messenger RNA levels similarly on the evening of proestrus, but had divergent effects on the morning of estrus, when only RU486, but not ZK98299, lowered the elevated serum FSH level (secondary FSH surge). In the present work we used primary anterior pituitary cell culture to examine whether RU486 caused direct, E-dependent suppression of basal and recombinant human activin A (activin)-induced FSH secretion in the gonadotrope and to compare this direct effect, if any, with that of ZK98299. Primary cell cultures were prepared from anterior pituitaries collected from cycling female rats either on metestrous or proestrous morning and cultured in DMEM, supplemented with charcoal-stripped serum without or with 10 nM estradiol (E2) for 96 h; exposure to test agents occurred during the last 48 h of culture. FSH released into the medium and intracellular FSH content were determined by RIA. In cells from the anterior pituitary of metestrous rats cultured in E2-free medium, neither antiprogestin (10 nM) affected FSH release; in contrast, when cells were cultured in medium to which E2 had been added, both antiprogestins caused profound suppression of both basal and activin (10 ng/ml)-stimulated FSH release. In cell cultures from proestrous rats, both antiprogestins caused a slight, but significant, suppression of basal FSH release even in the absence of added E2; activin-stimulated FSH release, however, was not affected. Upon exposure of the cells from proestrous rats to E2, the antiprogestins potently suppressed both basal and activin-stimulated FSH secretion. Because the foregoing incubations were performed in culture medium devoid of progesterone (P4), the actions of the antiprogestins on FSH secretion were independent of the natural ligand. Addition of P4 (10 nM) to the cell cultures stimulated basal and activin-induced FSH release more in the presence than in the absence of E2. The FSH response to P4 was completely blocked by both antiprogestins in both the absence and presence of E2. Finally, both RU486 and ZK98299 blocked the stimulatory effect of corticosterone (1 microM) on FSH secretion. The observed effects of P4 and antiprogestins were specific for FSH secretion; LH secretion was not similarly suppressed by either antiprogestin, but was, in fact, stimulated by ZK98299 in E2-treated cells. We conclude that 1) E2-inducible progesterone receptors interact with activin-mediated signal transduction to regulate FSH secretion, and 2) unlike on the morning of estrus in vivo, RU486 and ZK98299 affect FSH secretion similarly in the gonadotrope in vitro.

The antiprogestins RU486 and ZK98299 affect follicle-stimulating hormone secretion differentially on estrus, but not on proestrus.

Previous in vivo studies from our laboratory indicated that administration of the antiprogestin RU486 on proestrus suppresses both the preovulatory gonadotropin surges and the secondary FSH surge, suggesting a role for the progesterone receptor (PR) in the generation of these surges. The present study was designed to test the effects of another antiprogestin, ZK98299, which has been reported to block the PR through a mechanism different from that of RU486, on gonadotropin secretion in vivo. RU486 and ZK98299 (2 and 6 mg/kg) were administered s.c. at 1230 h on proestrus; uterine intraluminal fluid content, serum gonadotropins, and gonadotropin subunit messenger RNAs (mRNAs) were determined at 1830 h on proestrus and at 0900 h on estrus. At 1830 h on proestrus, both RU486 and ZK98299 at both doses caused equal suppression of the preovulatory FSH surge and FSHbeta mRNA. Both antiprogestins also equally attenuated the preovulatory LH surge at this time, with the higher doses causing greater suppression. In contrast, at 0900 h on estrus, the antiprogestins affected serum FSH differentially; only RU486 suppressed the secondary FSH surge despite the fact that both drugs prevented the release of uterine intraluminal fluid, confirming blockade of progesterone action at the level of the uterus. Neither drug had a significant effect on FSHbeta mRNA at 0900 h on estrus. ZK98299 at the higher dose caused a small, but significant, increase in serum LH. In a subsequent experiment, we compared the effects of RU486 and ZK98299 (6 mg/kg, s.c.), administered at 1230 h on proestrus, on serum FSH raised above the natural secondary FSH surge on the morning of estrus by passive immunization with an antiserum to inhibin-alpha (anti-I) at 1700 h on proestrus. Consistent with the results of the first experiment, both antiprogestins blocked the release of uterine intraluminal fluid, but only RU486 lowered serum FSH in both the normal sheep serum-treated controls and anti-I-treated rats; in contrast, ZK98299 actually increased serum FSH in the normal sheep serum-treated control animals. ZK98299 also increased FSHbeta mRNA in the control group; RU486, on the other hand, reduced FSHbeta mRNA only in the anti-I group. The results demonstrate unequivocally that whereas the effects of the two antiprogestins on serum FSH and FSHbeta mRNA are similar on proestrus, they are divergent on estrus. The data suggest that the functional state of the PR/transcriptional activation complex in the gonadotrope on the morning of estrus is different from that on the evening of proestrus.

Corticosterone selectively increases follicle-stimulating hormone beta-subunit messenger ribonucleic acid in primary anterior pituitary cell culture without affecting its half-life.

We demonstrated previously that glucocorticoids differentially affect the levels of the two pituitary gonadotropins, LH and FSH, both in vivo and in vitro. In vivo, the effect of glucocorticoids is GnRH independent, indicating a direct action on the gonadotrope, and it leads to selective up-regulation of the pituitary content of FSH and FSH beta-subunit messenger RNA (mRNA). The objective of the present study was to confirm the direct action of corticosterone (B) on FSH beta-subunit mRNA in primary anterior pituitary cell culture and to assess whether the selective B-induced rise in FSH beta mRNA is mediated through altered stability of the FSH beta transcript. Anterior pituitary glands collected from randomly cycling female rats were dissociated with trypsin. Cells were incubated at 37 C for 48 h and subsequently exposed to vehicle or B (1.7 microM) for an additional 42 h. At the end of the incubation, media were sampled for FSH and LH, cells were lysed, and total RNA was isolated for Northern blot analysis. Exposure to B for 42 h caused direct and selective upregulation of FSH release, FSH content, and FSH beta mRNA; decreased alpha-subunit mRNA; and had no significant effect on LH release, LH content, or LH beta mRNA. To evaluate the mRNA stability of the three subunits, cells were exposed to the transcription blocker actinomycin D (act D; 5 micrograms/ml) for an additional 6 h. The combined 6-h treatment with B and act D slightly, but significantly, suppressed alpha-subunit mRNA and did not change LH beta mRNA, confirming a long half-life of the two gonadotropin subunit mRNAs. In contrast, FSH beta mRNA was significantly suppressed by act D to the same level in vehicle- and B-treated cells. The posttranscriptional decay rate was examined by sampling at 0, 1, 2, 3, and 6 h during the 6-h act D treatment period. Decay curves for FSH beta mRNA were parallel in vehicle- and B-treated cells, indicating that B did not alter FSH beta mRNA stability. We conclude that the selective B-induced rise in FSH beta mRNA is mediated at the level of transcription rather than mRNA stabilization.

Effects of hypoxia and Freon 12 on mechanics of cardiac contraction.

Data are presented which indicate that the mechanism of tension depression and subsequent recovery from dichlorodifluoromethane (Freon 12), an aerosol gas recently described as a potent cardiac depressant agent, differs from that of hypoxia. To analyze these differences, 22 rat papillary muscles, contracting isometrically in a myograph, were studied during and subsequent to 15-min interventions of of hypoxia. Freon 12 with adequate oxygenation, or Freon 12 combined with hypoxia. During each of the three interventions the developed force (F) was markedly depressed, while peak shortening velocity (Vpm) was selectively more depressed by Freon and Freon combined with hypoxia than by hypoxia alone. While hypoxia shortened the time to peak force (TTP) and one-half relaxation time (RT1/2) markedly, Freon 12 with adequate oxygenation slightly shortened RT1/2 (P is less than 0.001) but failed to shorten TTP significantly. In contrast, Freon 12 administered during hypoxia shortened TTP and RT1/2 significantly (P is less than 0.001), more than did hypoxia or Freon 12 alone. Posthypoxic prolongation of TTP and RT1/2 was not seen during recovery from Freon 12. This prolongation was depressed during recovery from Freon 12 given either during hypoxia or during recovery from hypoxia. The results indicate that Freon 12 and hypoxia act synergically, although the mechanisms through which they mediate their actions on myocardial tissue are not identical.