Control of plasminogen activator activity in the thecal layer of the largest preovulatory follicle in the hen ovary.

Although the existence of plasminogen activator (PA) activity and the factors that regulate it in ovarian granulosa cells of both mammalian and avian species have been extensively documented, very little information has been generated concerning the control of PA activity in the adjacent thecal layer. This study was conducted to evaluate the effects of several physiological and pharmacological agents on PA activity in dispersed cells from the thecal layer of the largest preovulatory follicle in the hen ovary 17-16 h before ovulation. LH (50 and 100 ng) in the presence of 3-isobutyl-1-methylxanthine (0.01 mM) stimulated an approximate 25% increase in cell-associated PA activity, possibly via elevated levels of cAMP. Prostaglandin E1 and E2 (PGE1 and PGE2; 0.1 and 1 microM), but not PGI2 or PGF2 alpha (1 microM), enhanced PA activity and cAMP formation, effects that were potentiated by 0.01 mM 3-isobutyl-1-methylxanthine. Activation of Gs with cholera toxin (0.01-10 ng/tube) or adenylyl cyclase with forskolin (0.01-10 microM) stimulated cAMP formation and PA activity in a dose-dependent manner. Exposure of cells to the cAMP analog 8-bromo-cAMP (0.1-5 mM) caused similar increases in thecal cell PA activity. Incubation of cells with phorbol 12-myristate 13-acetate (PMA; 3.2-162 nM), an agonist known to activate protein kinase-C, resulted in a dose-dependent increase in PA activity. However, an equimolar concentration of phorbol 13-monoacetate (162 nM), an inactive analog of PMA that does not activate protein kinase-C, was without effect. Coincubation of cells with forskolin (1 microM) and PMA (32 nM) resulted in a synergistic stimulation of secreted PA activity, apparently via an enhancement of adenylyl cyclase activity. Treatment of cells with the calcium ionophore A23187 (0.01-1 microM) suppressed basal PA activity. However, PA activity stimulated by PMA (32 nM) was synergistically increased after coincubation with a 0.05-microM concentration of A23187, but was inhibited at doses of 0.5 and 1 microM. Taken collectively, the data indicate that PA activity is present in the thecal layer of the largest preovulatory follicle in the ovary of the domestic hen. Furthermore, several endocrine factors (i.e. LH and PGs) were found to stimulate PA activity, possibly via both the adenylyl cyclase-cAMP-protein kinase-A and phosphoinositide-protein kinase-C pathways. In light of these findings, we propose that the preovulatory increase in PGs and LH activates PA in the thecal layer of the largest preovulatory follicle, resulting in proteolytic degradation of the follicular connective tissue and, ultimately, ovulation.     

Identification of steroidogenic cell subpopulations in the ovary of the newly hatched chicken

The aim of the present study is the isolation of subpopulations of steroid-producing cells in the ovary of the newly hatched chicken. Cells were obtained by fractional trypsin dissociation of the ovary and isopycnic separation in a continuous metrizamide gradient (0-30%). Testosterone and 17 beta-estradiol secretion was measured by radioimmunoassay in the incubation medium of the isolated cells. Six fractions of ovarian cells were studied. Fraction II (density 1.080) contained typical steroidogenic cells with a positive reaction for 3 beta-hydroxysteroid dehydrogenase, delta 5-isomerase. This fraction secreted testosterone (3.7 ng/10(6) cells/2 hr) but no 17 beta-estradiol secretion was detectable. The majority (85%) of cells obtained in fraction VI (density 1.150) were relatively undifferentiated and contained polyribosomes, mitochondria with lamellar cristae, and few rough endoplasmic reticulum; only 3-5% of the cells of this fraction were similar to those of fraction II. In fraction VI the highest level of 17 beta-estradiol secretion was found (2.9 ng/10(6) cells/2 hr) whereas testosterone was at a minimum level (0.06 ng/10(6) cells/2 hr). Results strongly suggest the existence of two cell subpopulations in the inmature chicken ovary: typical steroidogenic and poorly differentiated cells which secrete testosterone and 17 beta-estradiol, respectively.     

Development and steroidogenic activity of preantral follicles in the neonatal rat ovary

The neonatal rat ovary is completely devoid of antral follicles until the twelfth day of age. During this period the ovary becomes steroidogenically active and responsive to gonadotrophins. The aim of this study was to correlate the onset of ovarian androgen and oestrogen production in vitro with the first appearance of distinct granulosa and theca cells. Although ovarian aromatase activity increased significantly on day 7 of age, ovarian oestrogen production was limited by low progesterone and testosterone production until day 12 of age. Increased aromatase activity on day 7 and androgen production on day 12 were coincident with the first appearance of granulosa and theca cells respectively. These functional and morphological changes were not associated with significant alterations in ovarian weight or concentrations of LH or FSH in serum.     

Prolactin inhibits plasminogen activator activity in the preovulatory follicles

The present study was designed to determine the effects of PRL on changes in morphology and plasminogen activator (PA) activity in the preovulatory follicles. Rabbit ovaries were perfused with hCG alone or with hCG plus at 10, 10(2), or 10(3) ng/ml. PRL at 10(3) ng/ml directly inhibited the degeneration and decomposition of surface epithelial cells induced by hCG exposure. The subsurface connective tissue was visualized by treatment with sodium dodecyl sulfate, which removed surface epithelial cells from the ovary, thereby exposing collagen fibrils and the basal lamina. Sodium dodecyl sulfate treatment revealed inhibition of connective tissue disruption at the apex of the follicle wall in PRL-treated ovaries. PA activity in mature follicles in perfused rabbit ovaries exposed to hCG increased from 1.40 +/- 0.08 to 28.4 +/- 4.25 IU/g tissue after 4 h of perfusion. The addition of PRL to the perfusate inhibited the hCG-stimulated increase in intrafollicular PA activity in a dose-dependent fashion. Although at 7 h mature follicles treated by hCG alone showed greater intrafollicular PA activity than those treated with hCG plus PRL, this difference was not significant. These results suggest that PRL may act directly by interfering with mechanical events within the ovary that are required for the rupture of mature Graafian follicles, probably via the inhibition of intrafollicular tissue PA activity.     

Characterization of granulosa cell subpopulations from avian preovulatory follicles by multiparameter flow cytometry

The objective of the present study was to characterize the granulosa cell populations from individual hen (Gallus domesticus) preovulatory follicles at defined stages of follicular maturation using multiparameter flow cytometry. Granulosa cells were fixed and stained with three fluorochromes that selectively bind to DNA (Hoechst 33342, blue), RNA (pyronin Y, red), or protein (fluorescein isothiocyanate, green). A flow cytometer equipped with a three-laser excitation system was used to analyze three colors of fluorescence from stained cells. Forward angle light scatter and axial light loss measurements were made on each cell to determine relative cell size. In addition, the ratios of RNA to protein and DNA to protein were measured. The major findings obtained from correlated measurements of cell cycle (DNA), protein, RNA, cell size, and ratios were: 1) the percentage of proliferating cells decreased while cell size increased during follicular maturation; 2) two subpopulations of granulosa cells were identified within each follicle based on relatively high and low protein contents; the fraction of cells in the high protein subpopulation increased, and the fraction of cells in the low protein subpopulation decreased during follicular maturation; 3) the high and low protein subpopulations also differed in cell cycle distribution, RNA content, and cell size; and 4) the distribution of cells into the two subpopulations and the degree of proliferation were influenced by stage of the ovulatory cycle, primarily in the most mature follicles. The results demonstrate the dynamic heterogeneity of the granulosa cell populations from individual ovarian follicles and show the influences of follicular maturation and stage of the ovulatory cycle on cell growth and metabolism.     

Arachidonic acid stimulates steroidogenesis in goldfish preovulatory ovarian follicles

The possibility that arachidonic acid (AA) plays a role in the regulation of steroidogenesis in goldfish was investigated using preovulatory ovarian follicles incubated in vitro. AA was shown to act in a time- and dose-dependent manner to stimulate testosterone production. AA in the range of 10(-5) to 10(-4) M increased testosterone production within 2 hr and had a maximal effect by 9 hr. The magnitude of the testosterone response to AA was similar to that observed when ovarian follicles were incubated with human chorionic gonadotropin (hCG). Ovarian follicles incubated with AA and either hCG or forskolin (adenylate cyclase activator) produced more testosterone than follicles incubated with either of these compounds alone. The actions of AA on testosterone production were completely blocked by cyclooxygenase inhibitors (indomethacin or ibuprofen) and were reduced by 50% by the lipoxygenase inhibitor nordihydroguaiaretic acid. Phospholipase C was far more effective than phospholipase A2 in the stimulation of testosterone production. Taken together, these results suggest that AA formed subsequent to the action of phospholipase C on membrane phospholipids has a role in the regulation of steroidogenesis in preovulatory goldfish ovarian follicles.     

Cytochrome p450-dependent lipid metabolism in preovulatory follicles

Estrogen biosynthesis and proteolysis are both important processes involved in ovarian follicular development, which may be influenced by cytochrome P450 (CYP)-dependent fatty acid metabolites. However, CYP-dependent lipid metabolism has not been characterized with respect to follicular maturation in vivo. Therefore, follicular fluid was collected in the hours before and after the LH surge in pigs, and concentrations of epoxy, hydroxy, and dihydroxy lipids were measured by liquid chromatography tandem mass spectrometry. Arachidonate oxidation and epoxyeicosatrienoic acid hydrolysis to dihydroxyeicosatrienoic acids (DHETs) were also assessed in thecal and granulosa tissue fractions, and the expression of CYP epoxygenases was evaluated by immunoblots using available antisera. To evaluate soluble epoxide hydrolase (sEH) expression, the porcine sEH was cloned from ovarian tissue, expressed and purified for antibody generation. The follicular fluid oxylipin concentrations ranged from 1-150 nm depending on the compound and estrous stage. The follicular fluid concentrations of CYP-dependent oxylipins increased at estrus, as did sEH expression; however, significant changes in epoxides were not observed, and the 11,12-DHET peak was delayed. The ratio of 14,15-DHET:11,12-DHET across all samples correlated with the log of follicular fluid estradiol concentrations (P < 0.01). Epoxygenase activities were similar in theca and granulosa, varying little with follicular development, whereas the decline of a single CYP2J isoform at ovulation was observed by immunoblots. The sEH activity was higher in granulosa than in theca. Finally, the dynamic changes in follicular CYP-dependent arachidonic acid metabolites and their modulatory function in vascular models suggest roles for these metabolites in follicular maturation, which may include regulation of estradiol biosynthesis and preovulatory remodeling of the follicular wall that should be fully explored in future studies.     

Estrogen regulation of thecal cell steroidogenesis and differentiation: thecal cell-granulosa cell interactions

Estrogen regulation of thecal cell steroidogenesis and differentiation was investigated with cells from ovarian antral follicles. Bovine theca interna cells were isolated and cultured in serum-free conditions to evaluate the effects of estradiol on thecal cell production of androstenedione, testosterone, and progesterone. Estradiol increased thecal cell androgen production throughout a 6-day culture period; however, the basal and stimulated levels of androgen production diminished after day 3 of culture. Androstenedione accumulation was approximately 10-fold greater than that of testosterone. In contrast to the stimulatory effects that estradiol had on androgen production, estradiol suppressed progesterone production throughout the 6-day culture period. Comparison of the effects of estradiol and hCG on thecal cells from small (less than 5 mm), medium (5-10 mm), and large (greater than 10 mm) antral follicles demonstrated that estradiol stimulated androgen production to a greater extent than hCG with cells from all of these stages of follicle development. Estradiol stimulation of androstenedione was greater in theca from small follicles than in theca from medium or large follicles. In contrast, suppressive effects of estradiol on progesterone were most apparent on thecal cells from medium and large follicles and less apparent on theca from small follicles. Estradiol stimulated androstenedione production in a dose-dependent fashion, with a minimum effective concentration of 10(-9) M and a maximum effective concentration of 10(-7)-10(-6) M. Concentrations greater than 10(-6) M estradiol resulted in a decline in the stimulatory response and may be important in the preovulatory follicle to suppress thecal cell androgen production and initiate the process of luteinization. Progesterone production was slightly stimulated by 10(-9) M estradiol, whereas higher concentrations (10(-7)-5 x 10(-6) M) resulted in a dose-dependent suppression of progesterone production. Interestingly, combined treatment of thecal cells with estradiol and hCG resulted in a greater than additive stimulation of androstenedione production, and estradiol decreased the ability of hCG to stimulate progesterone production. Observations demonstrate that estradiol can dramatically alter thecal cell production of steroids and support a hypothesis that steroid-mediated interactions between granulosa and thecal cells play an important role in regulating cellular function within follicles. The data provide evidence that a local feedback loop may exist in ovarian follicles, where androgens produced by thecal cells are used as a substrate for granulosa cell aromatization into estrogens, which, in turn, may feed back to stimulate thecal cell production of androgens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of messenger ribonucleic acid for the antiapoptosis gene P11 in the rat ovary: gonadotropin stimulation in granulosa cells of preovulatory follicles

P11, a member of the S100 family of calcium-binding proteins, has been shown to interact with BAD (Bcl-xL/Bcl-2-associated death promoter) in the yeast two-hybrid protein-protein interaction assay. Because overexpression of P11 dampens the proapoptotic activity of BAD in transfected cells, we tested the possibility that the expression of this antiapoptotic protein may be regulated by gonadotropins and other survival factors in the ovary. Northern blot analysis of ovaries obtained from prepubertal rats revealed an increased expression of P11 messenger RNA (mRNA) during prepubertal development in the theca cells of preantral and early antral follicles. Treatment of immature rats with PMSG did not affect P11 expression, whereas treatment of PMSG-primed rats with an ovulatory dose of human (h)CG stimulated ovarian P11 mRNA within 6-9 h in the granulosa cells of preovulatory follicles. Treatment of cultured preovulatory follicles in vitro with LH further confirmed the time-dependent stimulation of P11 by gonadotropins. In addition, treatment of cultured preovulatory follicles with MDL-12,330A, an inhibitor of adenylate cyclase, inhibited LH-stimulated P11 mRNA, whereas treatment with forskolin, an adenylate cyclase activator, but not the protein kinase C activator, 2-O-tetradecanol-phorbal-13-acetate, mimicked the LH action, suggesting the role of adenylate cyclase activation in P11 expression. Treatment with other follicle survival factors, including the epidermal growth factor, the basic fibroblast growth factor, and interleukin-1beta, could also stimulate P11 expression in cultured preovulatory follicles. These results demonstrate the expression of P11 mRNA in theca cells of different-sized follicles and in granulosa cells of preovulatory follicles following gonadotropin stimulation, and suggest that P11 may mediate, at least partially, the survival action of gonadotropins during the ovulatory process.     

Macroscopic identification and steroidogenic function of atretic follicles in sheep

The degree of translucency, vascularization of the follicle wall and the integrity of the membrana granulosa, as seen under a stereoscopic microscope, were used to distinguish between non-atretic and atretic isolated follicles. Subsequent histological evaluation showed that both non-atretic and severely atretic follicles could be correctly identified with over 95% accuracy. The concentrations of steroids were measured in both follicular tissue and fluid. The total steroid content of large (3.1-6.0 mm diameter) non-atretic follicles in vivo (115 pmol/follicle) exceeded that of large atretic follicles (54 pmol) and also that of small (2.0-3.0 mm diameter) non-atretic and atretic follicles (6 pmol). Oestrogen accounted for 80%, testosterone 13% and progesterone 7% of the total steroid content in large non-atretic follicles, whereas testosterone accounted for 75% of the steroid content in the other three categories of follicles. A similar pattern is observed when the concentrations of steroids are expressed on the basis of follicular weight. Total oestrogen secretion, as measured by the output of steroid into the culture medium in 24 h, was 937 pmol for large non-atretic, 31 pmol for large atretic, 65 pmol for small non-atretic and 4 pmol for small atretic follicles. In terms of steroid production (pmol/mg tissue), the four follicular categories were equally efficient. However, the type of steroid produced depended on the category of follicle and corresponded to its steroid content.     

Melatonin directly suppresses steroid production by preovulatory follicles in the cyclic hamster

Abstract: The purpose of these studies was to investigate the effects of melatonin on the production of steroids (progesterone, testosterone, and estradiol) and cAMP by preovulatory follicles and to examine changes in melatonin concentrations in the ovary during the estrous cycle. Adult cyclic hamsters were used in this study. Melatonin concentrations in the ovary, pineal gland, and serum were measured at mid-light and mid-dark during the estrous cycle. Effects of melatonin on steroidogenesis by preovulatory follicles, thecae, and granulosa cells were examined, and its effect on cAMP production by preovulatory follicles was also investigated. Melatonin (0.1–10 ng/ml) had no effect on steroid production in the absence of hCG, but melatonin decreased progesterone and estradiol production by preovulatory follicles in a dose-dependent and time-dependent manner in the presence of hCG (100 mlU/ml). The target of melatonin was thecae but not granulosa cells, and melatonin significantly reduced cAMP production by preovulatory follicles. Melatonin concentrations in the ovary showed a similar phasic variation with high levels during mid-dark and low during mid-light, as in the pineal gland and serum. These results show that the ovarian melatonin levels also exhibit a circadian rhythm and suggest that the high melatonin milieu in the ovary may induce gonadal regression in the cyclic hamster.     

Steroid binding to feather follicles in the chicken

Steroid binding profiles in chicken feather follicles were studied in vitro. Progesterone and promegestone were bound specifically with high affinity (dissociation constant = 7.79 nmol/l in females and 2.0 nmol/l in males) and with low capacity to the high-speed supernatant fraction (cytosol) of homogenized feather follicles. The number of progestin-binding sites was significantly (P less than 0.01) higher in females than in males (1.35 +/- 0.07 (mean +/- S.D.) vs 0.605 +/- 0.15 pmol/mg protein). Significant cross-competition was observed between progestins and glucocorticoids for the binding sites. The heat-activated progestin-receptor complexes bound specifically to DNA-cellulose in vitro. The DNA-cellulose binding was 36.6% of the total specific binding. The presence of progestin-binding sites in the feather follicles implies a direct action of progesterone in this structure, which may be an important factor in the regulation of moulting. The lack of oestrogen binding to the cytosol prepared from the feather follicles suggests different regulation of progestin receptors in different target organs.     

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.     

Decreased inhibin gene expression in preovulatory follicles requires primary gonadotropin surges

We have examined the role of the primary gonadotropin surges in regulating inhibin alpha- and beta A-subunit mRNA levels in rat ovarian follicles. Inhibin subunit mRNA levels decline dramatically on the evening of proestrus in follicles of the ovulatory pool. Because this decline is temporally associated with primary gonadotropin surges, we investigated the contribution of LH and FSH to this process. The primary gonadotropin surges were blocked by injection of a GnRH antagonist (WY45760) at 1200 h on proestrus. This resulted in sustained elevation of inhibin mRNA levels through 0700 h of the subsequent day, a time when inhibin mRNA levels would normally be very low. Replacement of either exogenous LH or FSH in ovulatory doses to an antagonist-treated animal at 1530 h on proestrus resulted in a decrease in inhibin mRNA levels by 4-5 h postreplacement. We conclude that LH and FSH act via a common mechanism to repress inhibin mRNA levels in stimulated preovulatory follicles.