Intrafollicular action of estrogen in regulating pituitary-induced ovarian progesterone synthesis and oocyte maturation in Rana pipiens: temporal relationship and locus of action

Previous studies demonstrated that estrogen inhibited frog pituitary homogenate (FPH) and progesterone-induced oocyte maturation. In order to determine whether estrogen interfered with intrafollicular progesterone synthesis, experiments were designed to study the effect of exogenous estrogen (estradiol-17 beta) on FPH-induced progesterone production in amphibian (Rana pipiens) ovarian follicles cultured in vitro. Intrafollicular progesterone concentrations were monitored directly using radioimmunoassay and the occurrence of germinal vesicle breakdown (GVBD) was used as a biological indicator of the action of steroids on oocyte maturation. FPH elicited a rapid and dramatic increase in follicular progesterone concentration (1000-3000 pg per follicle) which preceded germinal vesicle breakdown. Addition of estrogen to the culture medium inhibited FPH-induced progesterone production and the accompanying GVBD in a dose-dependent fashion. The presence of estrogen did not enhance the degradation of preloaded progesterone, suggesting estrogen impeded progesterone synthesis rather than enhanced progesterone metabolism. The temporal relationship between estrogen and FPH interaction was assessed by varying the relative time of hormone addition, after which intrafollicular progesterone concentration and GVBD were monitored. Progesterone production and GVBD were drastically inhibited when estrogen was added before or simultaneously with FPH. However, when addition of estrogen was delayed until after FPH simulation, a progressive loss of the inhibitory effect of the steroid on progesterone accumulation and GVBD was observed. Thus, the estrogen-sensitive phase was confined to the early portion of FPH stimulation. Continuous presence of estrogen in the culture system was not required to inhibit FPH-induced events. A short exposure (15 min) of follicles to estrogen was sufficient to inhibit oocyte maturation, whereas progesterone synthesis was not significantly affected. With longer exposure, however, FPH-induced progesterone production was impeded. Washing estrogen-treated follicles did not reverse the inhibitory effect of estrogen, however the follicles remained responsive to exogenous progesterone stimulation and exhibited GVBD. Results suggest that the inhibitory effects of estrogen on FPH action and progesterone production were not reversible under the in vitro culture conditions. To determine whether specific follicular components were involved in estrogen inhibition, progesterone production was assessed following selective removal of different follicle components by microdissection prior to being treated with FPH and estrogen.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro study of the direct ovarian effects of gonadotropin-releasing hormone (GnRH) in the frogs, Rana pipiens and Rana catesbeiana

Ovaries from the leopard frog, Rana pipiens, and bullfrog, R. catesbeiana, were used to study potential direct extrapituitary effects of gonadotropin-releasing hormone (GnRH). GnRH alone did not alter steroid secretion from ovaries in either species. Ovarian fragments containing mature follicles from R. pipiens were incubated in several submaximal doses of homologous pituitary homogenate or purified bullfrog luteinizing hormone (LH) together with 1000 ng/ml GnRH. The addition of GnRH failed to alter testosterone (T) or progesterone secretion or germinal vesicle breakdown over a wide dose range of gonadotropin. The effects of supramaximal doses of pituitary homogenate on R. pipiens and the effects of homologous LH on T secretion by fragments of R. catesbeiana ovaries were also unaffected by the presence of GnRH. The lack of a GnRH effect on the dynamics of T secretion in the bullfrog was further confirmed in a superfusion system with homologous pituitary homogenate. This study fails to demonstrate an action of GnRH at the level of the ovary in two ranid species.     

Role of calcium in the control of steroidogenesis in preovulatory ovarian follicles of the goldfish

The possible involvement of calcium in the regulation of steroidogenesis in the goldfish was investigated using preovulatory ovarian follicles incubated in vitro. Incubation of follicles in media deficient in calcium impaired testosterone production in response to human chorionic gonadotropin (hCG) in both the presence and the absence of the phosphodiesterase inhibitor IBMX. Similarly, addition of calcium channel antagonists (verapamil, nifedipine, nicardipine, and CoCl2) caused a dose-dependent inhibition of hCG-stimulated testosterone production. TMB-8, an inhibitor of intracellular calcium mobilization, also suppressed hCG-stimulated testosterone production. Basal testosterone production was not affected by incubation in calcium-deficient media or with drugs which reduce intracellular calcium availability. In other studies, nifedipine blocked forskolin and dibutyryl cyclic AMP-stimulated testosterone production suggesting that one of the major sites of calcium action is distal to cyclic AMP generation. Two inhibitors of calmodulin, W5 and W7, significantly inhibited hCG-stimulated testosterone production. These findings suggest that calcium derived from intracellular and extracellular pools participate in the expression of gonadotropin effects on steroid production in goldfish ovarian follicles and that these effects are mediated intracellularly by interaction with calmodulin.     

In vitro ovarian responses to pulsatile and continuous gonadotrophin administration on steroid secretion and oocyte maturation in the frogs, Rana pipiens and Rana catesbeiana

An in vitro superfusion system was used to study the relative effects of pulsatile and continuous gonadotrophin administration on steroid secretion and oocyte maturation in Rana pipiens ovaries. Pulsatile (10 min pulse/hr) delivery of pituitary extract (PE) resulted in a slightly (insignificantly) lower level of testosterone (T) secretion over a 12-hr period. Administration of a fivefold lower, subliminal amount of PE instead of hormone-free media between pulses did not change the pattern of T secretion. When the interpulse frequency was increased to 2 or 3 hr, there were notable oscillations in T secretion which corresponded to the peaks in luteinizing hormone associated with the PE. In four experiments ovarian fragments underwent oocyte maturation, but this occurred only in fragments that received continuous PE stimulation. Progesterone (P) secretion was measurable only when oocyte maturation was observed in the ovarian fragment. Rana catesbeiana ovarian fragments exposed to continuous superfusion with homologous PE produced more T than those receiving hourly pulses of PE over a 6-hr period. Oocyte maturation accompanied by P secretion was observed in one experiment under continuous, but not pulsatile administration of PE. These results suggest that the frog ovary may be more resistant than the mammalian ovary to "down-regulation" under continuous gonadotrophic stimulation. The implications of these results on the frog pituitary--gonadal axis are discussed.     

LH effect on the pattern of steroidogenesis in cultured Graafian follicles of the rat: dependence on macromolecular synthesis.

Graafian follicles explanted from proestrous rats before the preovulatory gonadotropin surge secreted predominantly 17beta-estradiol, and only small amounts of progestins and androgens, during 12 h of culture in hormone-free medium. Addition of ovine luteinizing hormone (NIH-LH-S18; 0.1-1.0 mug/ml) to the medium stimulated within 1-2 h the rate of accumulation of these steroids. However, accumulation of androstenedione and estradiol ceased after 4-6 h in the LH-treated cultures, whereas progesterone continued to accumulate and became the major secretory product at 6-12 h. Incubation of the follicles with LH for only 5 min resulted in significant stimulation of the accumulation of progesterone, androstenedione and estradiol during a subsequent 6-h culture period in hormone-free medium containing antibodies to LH; 30 min exposure to the hormone sufficed to elicit a maximal steroidogenic response and to induce ovum maturation in 95% of the follicles. Addition of actinomycin D (act D; 8 mug/ml) within the first hour after exposure of follicles to LH suppressed the LH-effect on progesterone but augmented the LH effect on estradiol accumulation; when addition of this inhibitor was delayed until 2 h, progesterone accumulation continued unabated for a further 10 h. By contrast, puromycin (80 mug/ml) inhibited progesterone accumulation when added to the medium at any time (1, 2 or 3 h) after the hormone. It is suggested that (i) a short-lived protein is essential for the stimulatory effect of LH on follicular steroidogenesis; (ii) the act D-sensitive product (mRNA?) required for the production of this protein is synthesized in adequate amount during the first 2 h of LH action, and is stable for at least 10 h; and (iii) LH may stimulate the production of an additional act D-sensitive regulatory protein that inhibits enzymes engaged in the cleavage of the 17-side-chain of progesterone, or cells equipped with these enzymes.     

Hyperprolactinemia-induced precocious puberty: studies on the mechanism(s) by which prolactin enhances ovarian progesterone responsiveness to gonadotropins in prepubertal rats

Hyperprolactinemia induced in immature female rats by treatment with sulpiride, a dopaminergic receptor blocker, increased the in vitro release of ovarian progesterone (P) in response to different doses of both highly purified hCG and human FSH. The increased P response to gonadotropins was also observed in ovaries of animals injected with ovine PRL or in rats in which the hyperprolactinemic condition was induced by pimozide, a more typical dopaminergic receptor blocker. In addition, the pimozide treatment advanced the time of puberty in a manner similar to that previously observed with sulpiride. In other experiments in which only the ovarian response to hCG, but not that to FSH, was evaluated, it was found that the in vivo treatment of hypophysectomized immature rats with sulpiride did not modify the almost undetectable serum PRL levels of these animals and failed to increase the in vitro ovarian P response to hCG. By contrast, sc injection of PRL to hypophysectomized rats clearly enhanced the in vitro release of P in response to the gonadotropin. Adrenalectomy of otherwise intact rats significantly decreased the in vitro ovarian P response to hCG and blunted the increase in the P response induced by hyperprolactinemia. These effects, however, were almost completely reversed by concomitant corticosterone replacement therapy. The ovarian content of hCG receptor in normal rats was found to increase during juvenile development (days 22-31). Hyperprolactinemic animals showed a greater ovarian hCG receptor content than age-matched 31-day-old controls. In contrast, the FSH receptor contents were similar in both groups. The increase in hCG receptor content induced by hyperprolactinemia was even more clearly manifested in isolated granulosa cells, but, as in the case of the whole ovaries, the FSH receptor content in these cells remained essentially the same in hyperprolactinemic and control rats. The results indicate that the enhanced ovarian P response to hCG induced by PRL in prepubertal rats is, at least in part, mediated by an increase in the LH receptor content of the granulosa cells of the developing follicle. It also appears that the sensitizing effect of PRL on the prepubertal ovarian P response to gonadotropins is modulated by the adrenal gland through an effect exerted by corticosterone. The mechanisms by which PRL enhances the ovarian P response to FSH do not appear to involve changes in FSH receptors. However, the occurrence of enlarged uteri in hyperprolactinemic rats suggest that the PRL-induced increase in the P response to FSH may be related to the presence of more mature, estrogen-secreting follicles resulting from the hyperprolactinemic condition.     

Purification of gonadotropins in the leopard frog (Rana pipiens).

Two gonadotropic preparations having FSH-like and LH-like activity have been separated from pituitaries of the leopard frog Rana pipiens by the use of methods similar to those used for separating gonadotropins from the bullfrog (Rana catesbeiana) and other tetrapod species. The R. pipiens hormones behaved much like those of the bullfrog in several chromatographic systems and showed a high degree of biological and immunochemical similarity to the bullfrog FSH and LH, respectively.     

Inhibitory action of follicular fluid on progesterone and prostaglandin synthesis in bovine follicles.

The present studies examined the inhibitory effect of mid-cycle and preovulatory bovine follicular fluid (bFF) on the secretion of progesterone and prostaglandin F 2 alpha (PGF 2 alpha) by bovine theca and granulosa cells cultured in tissue culture Medium 199. The inhibitory effect of bFF on the secretion of PGF 2 alpha by incubated and cultured hemipituitary glands of male rats was also studied. Addition of 5, 10 or 20% charcoal-extracted, mid-cycle bFF to the culture medium caused a twofold decrease in the accumulation of both progesterone and PGF 2 alpha by the cultured granulosa cells (P less than 0.01). In contrast, when preovulatory bFF was used, there was no inhibitory effect on secretion of progesterone and PGF 2 alpha. The addition of 10% mid-cycle bFF to the culture medium caused a two- to fourfold decrease in the accumulation of both PGF 2 alpha and progesterone by the cultured theca (p less than 0.008). However, in the presence of 1 microgram LH/ml, the inhibitory effect of mid-cycle bFF on progesterone and PGF 2 alpha secretion was abolished. The secretion of PGF 2 alpha was significantly (p less than 0.03) decreased in male rat hemipituitary glands after 5 h of incubation or 18 h of culture. These findings suggest that bFF from mid-cycle follicles inhibits prostaglandin synthetase as well as luteinization. The inhibition disappeared with bFF from preovulatory follicles.     

Effect of sodium on progesterone production in granulosa cells of rapidly growing chicken ovarian follicles

The importance of extracellular sodium (Nao+) in the progesterone production in hen granulosa cells from the first (F1), second (F2), and third (F3) largest preovulatory follicles was investigated in short term incubations. Progesterone synthesis in the absence or presence of the gonadotropin increased with increasing Nao+ concentration. Luteinizing hormone (LH) caused an additional and significant increase in steroidogenesis at every Nao+ concentration tested. However, no significant difference in the ED50 of Nao+ among F1, F2, and F3 cells was observed whether in the absence or the presence of the gonadotropin. Furthermore, although LH provoked steroidogenesis dose dependently in F1, F2, and F3 granulosa cells, no significant change in the ED50 of LH was observed among F1, F2, and F3 cells. The Na+/H+ exchange inhibitor, amiloride, attenuated both basal and LH-stimulated steroidogenesis in a concentration-dependent manner in the presence of Nao+. Amiloride was ineffective in the absence of Nao+. The present studies indicate the importance of Nao+ in the modulation of steroidogenesis in hen granulosa cells. Since steroidogenesis was suppressed by amiloride only in the presence of sodium, it is suggested that Nao+ is important for the maintenance/regulation of intracellular pH by an exchange with intracellular H+. Our studies also suggest that the sodium modulatory mechanism may not be a determinant in the differential progesterone production observed among the three largest preovulatory follicles.     

Effect of prostaglandins on collagen synthesis in rabbit ovarian follicles during the ovulatory process

Collagen fibers in the ovarian follicle undergo drastic changes at ovulation due to the preovulatory increase of collagenolytic activities. The collagen synthesis in ovaries, however, has not been elucidated yet. To clarify the regulatory role of prostaglandins (PGs) in collagen synthesis of the follicular wall in relation to the ovulatory process, we measured prolyl hydroxylase (PH), as well as lysyl oxidase (LO) activity and the content of hydroxyproline (Hyp) in ovarian follicles of the rabbits treated by hCG, hCG/indomethacin (IM) and hCG/IM/various PGs. The experimental groups consisted of; 1) untreat control group 2) ovulatory group receiving hCG 3) non-ovulatory group given PGs 4) ovulatory group given hCG and PGs 5) group in which hCG-induced ovulation was inhibited by IM (4 mg/kg) 6) group in which IM-inhibited ovulation was recovered by PGF2 alpha (1.5 mg/kg) 7) group in which IM-inhibited ovulation was not restored by PGE1 (0.1 mg/kg) and PGE2 (0.7 mg/kg). The peak activities of PH and LO in ovarian follicles were observed at 12-13 hr after hCG injection, namely, immediately after ovulation. Significant changes of these activities after hCG administration were specific to the ovaries. PH activity in the ovaries was suppressed by the administration of IM, but LO activity was not significantly suppressed. In the hCG/IM/PGF2 alpha-treated ovulatory rabbits (Group 6), PH activity recovered to nearly the level of the hCG-treated rabbits (Group 2). By addition of PGE2, ovulation did not recover but PH activity was restored to about 70% of the hCG-treated rabbits. PGE1 did not have any effect on the reversal of ovulation-blockage or restoration of PH activity. The amount of Hyp after hCG administration tended to decrease from 6 hr to 10 hr but was significantly increased from 10 hr to 13 hr. This increase of Hyp after ovulation significantly correlated with the increase of PH and LO activities. In the hCG/IM/PGF2 alpha-treated rabbits (Group 6), the changes of Hyp were similar to those the hCG-treated rabbits (Group 2). In conclusion, collagen synthetic activity, found to be regulated by PH and LO activities in the ovarian follicles, was activated after follicle rupture, resulting in reconstruction of collagen fibers, and PGs play an important role in the ovulatory process by modifying collagen synthesis.     

Control of ovarian steroidogenesis by insulin-like peptides in the blowfly (Phormia regina)

This study investigated the ability of insulin and of insect insulin-like peptides (ILPs) to stimulate ovarian steroidogenesis in the blowfly Phormia regina. Bovine insulin was active on ovaries isolated in vitro, which showed an age-dependent sensitivity; this peptide progressively stimulated steroidogenesis in ovaries isolated from the third day after adult molt, but not in younger ones, and had maximal activity after the fifth day. This stimulatory effect was observed equally from females reared in the presence or in the absence of males, excluding a regulatory effect of mating. The mode of action of insulin in blowflies did not involve cAMP, but triggered a specific and well-conserved transduction cascade. In particular, a peroxovanadium compound, known to activate specifically the insulin receptor in mammals, also stimulated blowfly ovarian steroidogenesis in vitro. Conversely, chemicals known to inhibit the mammalian insulin receptor or downstream elements of its signaling pathway, such as LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), were able to prevent the steroidogenic action of bovine insulin on fly ovaries. Extracts from the median neurosecretory cells (MNCs) of blowfly brains, which are known to contain endogenous ILPs, stimulated ovarian steroidogenesis very efficiently and were also sensitive to inhibition by LY294002. These experiments indicated the involvement of PI3K in the mode of action of MNC extracts and substantiated that their endogenous ILPs are involved in the regulation of ovarian steroidogenesis. This conclusion was corroborated by the effects of synthetic bombyxin II, an ILP originating from silkworm MNCs, which also stimulated steroidogenesis in isolated blowfly ovaries. Altogether, these data suggest that insulinlike neurohormones from MNCs play a crucial role as steroidogenic gonadotropins in female flies.     

Comparison of blood glucose and liver glycogen of larval and adult frogs (Rana pipiens)

Blood glucose levels are lower in tadpoles than adults. Transition to adult levels occurs during the later stages of metamorphosis, but the rise is interrupted by a brief decline in blood glucose at the beginning of metamorphic climax. Liver glycogen rises from 4.6% to 8% during larval life, falls to 1.7% at the beginning of metamorphic climax then returns to 5–7% during late climax and in the frog. Possible mechanisms responsible for the differences in carbohydrate regulation in larval and post metamorphic amphibians, and the biological significance of the differences are briefly discussed.