Androgen synthesis during follicular development: evidence that rat granulosa cell 17-ketosteroid reductase is independent of hormonal regulation

Although androgens have been implicated in follicular atresia, ovarian follicular androgen synthesis is required for preovulatory follicular growth. To localize the site(s) of androgen biosynthesis and to obtain a better understanding of the regulation of the androgenic pathway(s) in rat ovarian follicles we examined the relative abilities of developing follicles to accumulate specific androgens [testosterone (T) and dihydrotestosterone (DHT)] using both radioimmunoassay (RIA) and 3H-substrate metabolism techniques. Small antral and preovulatory follicles were obtained from control or human chorionic gonadotropin (hCG)-primed immature rats, respectively (Richards and Bogovich, 1982). Small antral follicles, theca and granulosa cells produced little immunoassayable androgen (T + DHT) when incubated with or without 8-bromo-cAMP. In contrast, preovulatory follicles and theca produced more androgen than small antral tissues and in a manner acutely stimulable by cAMP. Granulosa cells produced little androgen under these conditions. Inclusion of [3H] androstenedione in the incubates yielded increased accumulation of [3H] T and [3H] DHT for all small antral and preovulatory tissues. Indeed, granulosa cells from both small antral and preovulatory follicles possessed a remarkable ability to accumulate [3H] T. This ability was not altered by hypophysectomy or subsequent treatment with estradiol and/or follicle-stimulating hormone (FSH). These results suggest that 17-ketosteroid reductase may be a constitutive enzyme in granulosa cells.     

Secretion of 17 alpha-hydroxyprogesterone, androstenedione, and estrogens by porcine granulosa and theca interna cells in culture

The steroid secreting activities of dispersed granulosa and theca interna cells from preovulatory follicles of prepubertal gilts 72 h after pregnant mare's serum gonadotropin treatment (750 IU) were compared. The cells were cultured for 24 h with or without steroid substrate (10(-8) to 10(-5) M progesterone, 17 alpha-hydroxyprogesterone, or androstenedione), FSH (100 ng/mL), LH (100 ng/mL), and cyanoketone (0.25 microM, an inhibitor of 3 beta-hydroxysteroid dehydrogenase). Granulosa cells cultured alone secreted mainly progesterone. Theca interna cells secreted mainly 17 alpha-hydroxyprogesterone and androstenedione, with secretion being markedly enhanced by LH. In the presence of cyanoketone, which inhibited endogenous progesterone production, theca interna but not granulosa cells were able to convert exogenous progesterone to 17 alpha-hydroxyprogesterone and androstenedione, and exogenous 17 alpha-hydroxyprogesterone to androstenedione and estradiol-17 beta in high yield. The secretion of the latter steroids from exogenous substrates was unaffected by LH. Theca interna cells secreted more estradiol-17 beta than did granulosa cells in the absence of aromatizable substrate, but estradiol-17 beta secretion by the latter was markedly increased after the addition of androstenedione. These apparent differences in steroid secreting activity between the cell types suggest that the enzymes responsible for conversion of C21 to C19 steroids, i.e., 17 alpha-hydroxylase and C17,20-lyase, reside principally in the theca interna cells. However, aromatase activity appears to be much higher in granulosa cells.     

Expression and function of Fas antigen vary in bovine granulosa and theca cells during ovarian follicular development and atresia

Fas antigen is a receptor that triggers apoptosis when bound by Fas ligand (FasL). A role for Fas antigen in follicular atresia was studied in follicles obtained during the first wave of follicular development during the bovine estrous cycle (estrus is Day 0). Granulosa and theca cells were isolated from healthy dominant follicles and the two largest atretic subordinate follicles on Day 5, atretic dominant follicles on Days 10-12, and preovulatory follicles on Day 1. Fas antigen mRNA levels were highest in granulosa cells from subordinate as compared to other follicles, and lowest in theca cells from healthy Day 5 dominant as compared to other follicles. FasL alone had no effect on viability of granulosa or theca cells but became cytotoxic in the presence of interferon-gamma (IFN). IFN has been shown to induce responsiveness to Fas antigen-mediated apoptosis in other cell types. In the presence of IFN, killing of granulosa cells by FasL was greater in subordinate compared to healthy dominant follicles on Day 5, did not differ between healthy and atretic dominant follicles, and was similar in theca among all follicles. Granulosa cells from preovulatory follicles, which had been exposed to the LH surge in vivo, were completely resistant to FasL-induced killing. In summary, Fas antigen expression, and responsiveness to Fas antigen-mediated apoptosis, vary during follicular development.     

Steroid metabolism by avian ovarian cells during follicular maturation

The profiles of steroid hormones produced by ovarian cells from the domestic hen were examined. Theca cells from the immature, small white follicles (SWFT), the third largest (T3), and largest (T1) preovulatory follicles, and the ruptured, postovulatory follicle (POFT) were incubated for 3 h at 37 degrees with [3H] progesterone (Prog) or [3H] pregnenolone (Preg). Granulosa cells from the largest preovulatory follicle were incubated with [3H] Preg or were coincubated with theca cells and [3H] Preg. The production of specific steroid metabolites was determined on the basis of coelution of radioactivity with known standard compounds, using an isocratic high-pressure liquid chromatography (HPLC) technique. Granulosa cells converted 93% of [3H] Preg substrate to Prog. More Prog was utilized by T3 cells than by T1 and SWFT cells, either when [3H] Prog was the substrate or when coincubated with granulosa cells and [3H] Preg. The major metabolites of Prog were androstenedione, 17-hydroxyprogesterone, and an unidentified compound with an elution time of 53 min. The POFT cells metabolized [3H] Prog to the same extent as T3 cells did, but their profile of steroidogenesis favored production of the unidentified 53 min metabolite. SWFT cells utilized the least amount of [3H] Preg substrate. The results point to marked changes in enzyme activities in theca cells during maturation and following ovulation.     

Regional pattern of cell maturation and progesterone biosynthesis in the avian granulosa cell layer

The objective of the present study was to compare the structural and functional features of cells derived from histologically different regions of the granulosa cell layer of hen preovulatory follicles. Granulosa cells were isolated from a 0.8-1.5-cm diameter region of the granulosa layer overlying the germinal disc (GD) or from the remainder of the granulosa layer peripheral to the disc region (GP). In the first study, the isolated cells were prepared from each region of the five largest preovulatory follicles; fixed; stained with fluorescent dyes for DNA, total protein, and RNA; and analyzed by use of multiparameter flow cytometry. A greater percentage of cells from the GD region than from the GP region were in proliferative (S and G2/M) stages of the cell cycle in the four largest follicles. In addition, GD cells had lower relative protein content than GP cells in the two largest follicles. In the second study, progesterone biosynthesis in response to treatment with luteinizing hormone (LH) or forskolin was examined in granulosa cells from the GD and the GP regions of the largest preovulatory follicles. GP cells had greater responsiveness to the treatments than GD cells. In addition, conversion of 25-hydroxy-cholesterol to progesterone was greater in GP cells than in GD cells. There were no differences in cyclic adenosine 3',5'-monophosphate (cAMP) production by GD and GP cells in response to LH or forskolin or in the ability of cells from each region to convert pregnenolone substrate to progesterone via 3 beta-hydroxysteroid dehydrogenase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of genistein on steroidogenic response of granulosa cell populations from porcine preovulatory follicles

Genistein affects reproductive processes in animals. However, the mechanism of its action is not fully elucidated and differs among species. The objectives of the current study were: 1/ to establish an in vitro model of granulosa cell culture for studying the intracellular mechanism of phytoestrogen action in porcine ovary; 2/ to determine an in vitro effect of genistein on basal and FSH-stimulated P(4) and E(2) production by porcine granulosa cell populations (antral, mural, total) isolated from large, preovulatory follicles. Granulosa cells were isolated from large (> or =8 mm), preovulatory follicles and separated into antral and mural cell subpopulations. Cells were allowed to attach for 72 h (37 degrees Celsius, 10% serum, 95% air/5% CO2) and than cultured for next 48 hours with or without serum (0, 5 and 10%), FSH (0, 10 or 100 ng/ml) and genistein (0, 0.5, 5 or 50 microM). Basal P(4) and E(2) production did not differ among antral, mural and unseparated granulosa cells isolated form porcine preovulatory follicles. Only mural cells tended to secrete less P(4) and E(2) than other cell populations. FSH stimulated P(4) production in a dose dependent manner in all cell populations and culture systems. Genistein inhibited in a dose dependent manner basal and FSH-stimulated P(4) production by antral, mural and unseparated granulosa cells. However, genistein did not affect E(2) production by granulosa cells. In addition, viability of porcine granulosa cells was not affected by the pyhytoestrogen except the highest dose of genistein. It appears that genistein may be involved in the regulation of follicular function in pigs. Moreover, unseparated porcine granulosa cells may provide a suitable in vitro model for studying the intracellular mechanism of phytoestrogen action in porcine ovary.     

Biphasic effect of gonadotropin releasing hormone on progestin secretion by rat granulosa cells

The effect of an agonistic gonadotropin releasing hormone (GnRH)-analog (D-Ala6, des-Gly10-NH2-GnRH-ethylamide, GnRHa) on granulosa cell steroidogenesis in the presence or absence of follicle-stimulating hormone (FSH) or luteinizing hormone (LH) was studied. Granulosa cells, isolated from preovulatory follicles of pregnant mare's serum gonadotropin (PMSG)-treated immature rats or from the less mature follicles of untreated immature rats, were cultured for a period of 72 h with daily changes of medium, and progesterone and its metabolite, 20 alpha-dihydro-progesterone (20 alpha-OHP), were assayed in the medium. In granulosa cells from preovulatory follicles, LH and FSH caused a much greater stimulation of steroidogenesis than did GnRHa. There appeared to be no interaction between GnRHa and FSH during the first 10 h, but at 24 h and later the presence of GnRHa clearly inhibited the steroidogenic response to LH and FSH. Steroidogenesis in granulosa cells from immature rats was considerably lower and the effects of GnRHa and FSH alone less pronounced. In these cells, FSH-stimulated progesterone secretion was inhibited by GnRHa only at 72 h. In contrast, 20 alpha-OHP secretion in the same cultures was potentiated by the combined presence of FSH and GnRHa. In conclusion, it seems as though the effects of GnRHa on granulosa cell steroidogenesis varies with exposure time, the initial response being stimulatory and the later inhibitory. Furthermore, the response is also to some extent determined by the maturational stage of the granulosa cells.     

Plasminogen activator activity and thymidine incorporation in avian granulosa cells during follicular development and the periovulatory period.

The hormonal and second messenger regulation of plasminogen activator (PA) activities in avian granulosa and theca cells has been documented. However, the physiological role(s) of PAs in the avian ovary remains poorly understood. The present studies were designed to evaluate PA activity in hen granulosa cells collected from the most mature (F1) preovulatory follicle at three discrete time points relative to a spontaneous ovulation and from follicles collected at various stages of follicular development. Levels of PA activity in the granulosa layer of the F1 follicle declined by greater than 90% as follicles were collected closer to their anticipated time of ovulation (e.g., from 17-16 h to 0.75-0.15 h; p less than 0.05). Timing of tissue collection was confirmed by evaluation of serum progesterone levels, which peaked as expected at the 6-5-h time point. During follicular development, PA activity was several times greater in rapidly growing follicles (6-12 mm, 1-3 wk prior to ovulation) than in slowly growing (1-5 mm) or preovulatory (F3 and F1) follicles (p less than 0.05). Granulosa cells of these rapidly growing follicles also incorporated significantly higher levels of 3H-thymidine than did granulosa cells of mature follicles (p less than 0.05), suggesting a higher level of DNA synthesis. Similarly, granulosa cells of the mitotically active germinal disc region of the F1 granulosa layer were found to possess at least 3-fold higher (p less than 0.05) levels of PA activity and a 2-fold greater level of 3H-thymidine incorporation than the more mature granulosa cells isolated from the remaining F1 granulosa layer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative analysis of in-vitro incorporation of [3H]thymidine into hamster follicles during the oestrous cycle

Follicles were isolated from hamster ovaries at 09:00 h and 15:00 h on each of the 4 days of the oestrous cycle (Day 1 = oestrus; Day 4 = pro-oestrus) by microdissection and by a mixture of enzymes and classified into 10 stages with pre-calibrated pipettes (stage 1 = preantral follicles with 1 layer of granulosa cells; stage 10 = preovulatory antral follicles). The follicles at each stage were incubated for 4 h with [3H]thymidine with incorporation expressed per microgram follicular DNA or per follicle. A significant increase in thymidine per follicle occurred at 15:00 h on Days 1 and 3 of the cycle from stage 2 (bilaminar follicle) to stage 6 (7-8 layers granulosa cells plus theca). When expressed as thymidine per follicle or microgram DNA, there was a significant increase in incorporation for stages 1-4 (4 layers granulosa cells) on Day 4 at 15:00 h compared to 09:00 h, presumably as a consequence of the preovulatory increase in gonadotrophins. Follicles in stages 5 to 8 (preantral follicles with 5 or more layers of granulosa cells to small antral follicles), from which the next set of ovulatory follicles will be selected, did not show a significant peak in incorporation per microgram DNA until Day 1 at 09:00 and 15:00 h when the second increase in FSH is in progress. DNA synthesis was similarly sustained throughout Day 1 for stage 1-4 follicles. These results suggest that periovulatory changes in FSH and LH, directly or indirectly, are not only responsible for ovulation and the recruitment of the next set of follicles destined to ovulate but also stimulate DNA replication in smaller follicles which develop over the course of several cycles before they ovulate or become atretic.     

Influence of 5 alpha-reduced androgens on oestradiol secretion by granulosa cells of pregnant mare serum gonadotrophin treated immature rats

The effect of aromatizable androgens (testosterone and androstenedione) and naturally occurring 5 alpha-androstane, 3 alpha 17 beta-diol and 5 alpha-androstane, -3 beta, 17 beta-diol on oestradiol secretion by granulosa cells isolated from preovulatory follicles of PMSG-primed immature rats was investigated. The amount of oestradiol secreted by granulosa cells in the absence of exogenous aromatizable androgen in a 4h incubation was negligible. However, the addition of testosterone or androstenedione resulted in concentration dependent increases in oestradiol secretion. The 5 alpha-reduced androgens inhibit oestradiol and oFSH-stimulated oestradiol secretion by the granulosa cells in the presence of exogenous testosterone. The least potent of the androgens tested in causing this effect being the 5 alpha-androstane-3 alpha, 17 beta-diol. This result suggests that the naturally occurring 5 alpha-reduced androgens have a direct effect on androgen-aromatizing enzymes. The effect of these androgens may have an important connotation with respect to the control of the onset of puberty and regulation of ovarian oestradiol secretion within the microenvironment of an ovarian follicle.     

Determination of steroidogenic potential of ovarian cells of the brushtail possum (Trichosurus vulpecula)

The ovary of the brushtail possum (Trichosurus vulpecula) secretes steroids; however, little is known about the identity of the steroidogenic cells in the ovary. The aim of the present study was to determine the identity of the ovarian cell types expressing mRNAs encoding proteins important for steroidogenesis and determine at what stage of follicular development they are expressed. The genes examined were those for steroidogenic factor-1 (SF-1), steroidogenic acute regulatory protein (StAR), cytochrome p450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase/Delta5,Delta4 isomerase (3betaHSD), cytochrome p45017alphahydroxylase (p45017alphaOH), and p450 aromatase (p450arom). None of the genes examined were expressed in oocytes at any stage of follicular development. SF-1 was expressed in granulosa cells from the type 2 or the primary stage of development and thereafter to the preovulatory stage. In addition, the theca interna of small and medium-size antral but not preovulatory follicles and the interstitial glands and corpora lutea expressed SF-1 mRNA. Granulosa cells of preantral and small to medium-size antral follicles were not capable of synthesizing steroids from cholesterol because they did not contain p450scc mRNA. However, granulosa cells of many of the small to medium-size antral follicles expressed p450arom and 3betaHSD mRNA. The interstitial glands, theca interna, and corpus luteum expressed StAR, p450scc, 3betaHSD, and p45017alphaOH mRNA, suggesting that these tissues are capable of synthesizing progestins and androgens. The corpus luteum expressed p450arom, indicating that this tissue also has the potential to secrete estrogens in this species.     

Secretion of cumulus expansion-enabling factor (CEEF) in porcine follicles

The objective of this study was to find out whether porcine cumulus and mural granulosa cells can secrete cumulus expansion-enabling factor (CEEF). Culture drops of M-199 medium were conditioned with denuded porcine oocytes (1 oocyte/μl), cumulus cells from oocytectomized complexes (1 OOX/μl), pieces of mural granulosa isolated from preantral to preovulatory follicles (1000 cells/μl), or oviductal cells (1000 cells/μl) for 24 hr. The production of CEEF was assessed by the addition of mouse OOX and follicle-stimulating hormone (FSH) (1 μg/ml) to microdrops of the conditioned medium. After 16–18 hr, expansion of the mouse OOX was scored on a scale of 0 to 4 by morphologic criteria. Mouse OOX did not expand in nonconditioned FSH-supplemented medium. Immature porcine oocytes produced +3 to +4 expansion of the mouse OOX. Granulosa cells isolated from preantral and early antral follicles and cumulus cells isolated from all stages of follicle development constitutively secreted CEEF under in vitro conditions. Mural granulosa cells of small, medium, and preovulatory (PMSG) follicles also secreted CEEF in vitro; however, FSH or leutenizing hormone (LH) stimulation was essential for this secretion. Hormonally induced secretion of CEEF was accompanied by expansion of the mural granulosa itself. Granulosa cells isolated from follicles of gilts 20 hr after PMSG and human chorionic gonadotropin (hCG) administration did not produce CEEF and did not expand in response to FSH and LH in vitro. CEEF activity also was found in the follicular fluid of small antral follicles, was reduced in medium follicles, and was not detectable in PMSG-stimulated follicles. However, CEEF activity was reestablished in the follicular fluid of preovulatory follicles by hCG injection, conceivably due to increased production of CEEF by cumulus cells. We conclude that (1) porcine cumulus and mural granulosa cells are capable of CEEF production in vitro and (2) autocrine secretion of CEEF by cumulus cells is involved in regulation of porcine cumulus expansion both in vitro and in vivo. Mol. Reprod. Dev. 49:141–149, 1998. © 1998 Wiley-Liss, Inc.     

Comparison of the effect of 4-hydroxy-4-androstene-3,17-dione on aromatase activity in granulosa cells from preovulatory follicles of rats, rabbits, and humans

The effect of the aromatase inhibitor 4-hydroxy-4-androstene-3,17-dione (4-OH-A) on the synthesis of estradiol (1,3,5 (10)-estratriene-3,17 beta-diol) by granulosa cells from preovulatory follicles of rats, rabbits and humans was examined. Granulosa cells from all three species were incubated for 4 h without treatment (control) or in the presence of androstenedione (4-androstene-3,17-dione, 0.5 microM), 4-OH-A (5 microM), or both compounds together. Estradiol levels were determined in the medium and cells by radioimmunoassay. In all three species, estradiol synthesis was markedly increased by androstenedione and this increase was blocked by 4-OH-A. In the rabbit, however, 4-OH-A alone caused a small but significant increase in radioimmunoassayable estradiol. The apparent increase seen with 4-OH-A alone may be due to a metabolite of 4-OH-A that cross-reacts in the estradiol radioimmunoassay. With granulosa cells from humans, in which 4-OH-A is of potential therapeutic importance, no similar effect of 4-OH-A alone was observed.     

Granulosa cell steroidogenesis and follicular fluid steroid concentrations after the onset of oestrus in cows

Granulosa cell responsiveness at an early (1-2 h) or late (14-16 h) stage of differentiation following the onset of oestrus [and presumably the LH surge] was studied in 16 cows. Follicular fluid collected at the early stage (8 preovulatory follicles) had a higher concentration of testosterone (P less than 0.05), oestradiol (P less than 0.01) and oestrone (P less than 0.01) than did follicular fluid collected at the late stage of oestrus (8 preovulatory follicles). No difference in follicular fluid progesterone was noted between follicles collected at the early and late stages of oestrus. Granulosa cells collected at the early stage of oestrus had a higher in-vitro response (progesterone production) to LH (P less than 0.05), forskolin (P less than 0.08) and diacylglycerol (P less than 0.05) than did granulosa cells collected at the late stage of oestrus. However, later stage granulosa cells produced more (P less than 0.01) progesterone after culture with prostaglandin E-2 than did earlier stage granulosa cells. These results show that follicular fluid oestrogen decreases, which suggests a loss of aromatase activity as oestrus progresses, and that granulosa cells become refractory (low progesterone production) to in-vitro LH, forskolin, and diacylglycerol challenge, yet acquire responsiveness to prostaglandin E-2 as oestrus progresses.     

Stage of ovarian follicular development associated with the initiation of steroidogenic competence in avian granulosa cells.

Previously described models for avian ovarian steroidogenesis, using mature, 25-40-mm preovulatory follicles as the source of tissues, were based on the assumption that interaction of the granulosa layer, as the predominant source of progesterone, with adjacent theca cells is required for maximal production of C21, C19, and C18 steroids. In the present study, we evaluated the steroidogenic capacity of ovarian cells isolated from less mature, 6-8-mm and 9-12-mm follicles in the chicken ovary (representative of a stage of development 2-3 wk prior to ovulation) to determine at which stage of follicular development granulosa and/or theca cells become steroidogenically competent. Granulosa cells collected from 6-8-mm follicles were found to be virtually incompetent to produce steroids, containing extremely low basal levels of progesterone (12 pg/5 x 10(5) cells) and failing to respond with increased steroid output following a 3-h exposure to ovine LH (oLH; 0.1 and 100 ng/0.5 ml), ovine FSH (oFSH; 100, 500, and 1,000 ng/0.5 ml), 8-bromo-cyclic adenosine monophosphate (8-bromo-cAMP; 0.33 and 3.33 mM) or 25-hydroxycholesterol (250 and 2,500 ng/0.5 ml). However, addition of pregnenolone (20 and 200 ng/0.5 ml) to granulosa incubations resulted in significantly increased progesterone levels. Granulosa cells of 6-8-mm follicles also failed to increase cAMP formation in the presence of oLH (10, 100, and 1,000 ng/0.5 ml) and 3-isobutyl-1-methylxanthine (IBMX; 10 microM), but responded to stimulation with 1,000 ng oFSH (4.4-fold increase over basal) or 10 microM forskolin (32-fold increase over basal) in the presence of IBMX. In contrast, granulosa cells isolated from 9-12-mm follicles and incubated for 3 h in vitro were found to contain basal progesterone levels 200-fold higher than those found in granulosa cells of 6-8-mm follicles. Furthermore, granulosa cells of 9-12-mm follicles markedly increased progesterone production following incubation in the presence of oFSH (100-1,000 ng/0.5 ml), 8-bromo-cAMP (0.33 and 3.33 mM), or 25-hydroxycholesterol (250 and 2,500 ng/0.5 ml). However, these granulosa cells remained unresponsive to oLH (0.1, 10, and 100 ng/0.5 ml), failing to increase cAMP accumulation (in the presence of IBMX) and progesterone output. Theca cells of small yellow follicles were found to produce measurable basal levels of progesterone, androstenedione, and estradiol, and levels of each steroid were significantly increased following a 3-h challenge with oLH, 8-bromo-cAMP, 25-hydroxycholesterol, and pregnenolone.(ABSTRACT TRUNCATED AT 400 WORDS)     

A granulosa cell differentiation-stage-specific cytotoxic activity in bovine and rat sera

Heat-inactivated serum is cytotoxic to granulosa cells from preantral follicles but not to cells from preovulatory follicles. A dominant feature of the granulosa cells of preovulatory follicles is the presence of luteinizing hormone (LH) receptors on the surface of the cells. In the present study, we have examined the relationship between the process of LH receptor induction and the acquisition of serum tolerance in granulosa cells in vitro. Granulosa cells from the ovaries of immature rats primed with diethylstilbestrol (DES) were cultured in a 1:1 mixture of Ham's F-12 and Dulbecco's modified Eagle's medium containing 30 ng of ovine follicle-stimulating hormone (oFSH; NIH-15). At either 0, 24, or 48 h of culture, heat-inactivated fetal bovine serum (FBS) was added (10% by volume) to separate groups of culture tubes. All cells were cultured for a total of 72 h, at which time the cultures were assessed for LH receptor (specific 125I-human chorionic gonadotropin [hCG] binding) and DNA content. LH receptors were induced in all FSH-containing serum-free cultures by 48 h. Receptors were not induced, however, when serum was added after either 0 or 24 h of culture. Furthermore, serum addition at these times resulted in a cell loss (assessed by DNA) of 40-60%. Serum addition at 48 h to FSH-containing cultures resulted in an inability to detect LH receptors at 72 h and with no significant effect on the culture DNA content. Addition of a protein extract of FBS at the initiation of cell culture prevented FSH-stimulated LH receptor induction and was cytotoxic. A lipid extract of FSH did not interfere with receptor induction and was not cytotoxic.(ABSTRACT TRUNCATED AT 250 WORDS)     

Androgen-induced changes in rat ovarian granulosa cells in vitro

Ovarian granulosa cells collected from small antral follicles from immature rats were cultured in McCoy's 5A medium, for 1-6 days in the presence of delta 4-androstenedione, testosterone, dihydrotestosterone, and dehydroepiandrosterone (10(-5) M and 10(-7) M). Granulosa cells examined by electron microscopy demonstrated many lipid droplets, mitochondria with tubular cristae and profiles of smooth endoplasmic reticulum, all suggestive of active metabolism in the cell. Cells cultured in androstenedione, testosterone, dihydrotestosterone and dehydroepiandrosterone produced estrogen and progesterone as measured by radioimmunoassay. By day 4, cells cultured in androgen had almost completely degenerated. The control cells acquired none of the aforementioned characteristics and survived up to beyond 6 days, at which time the experiments were terminated. This study supports the hypothesis that high concentrations of androgens in cultured granulosa cells contribute to their degeneration through altered structure, which is associated with functional change.     

Epidermal growth factor and basic fibroblast growth factor suppress the spontaneous onset of apoptosis in cultured rat ovarian granulosa cells and follicles by a tyrosine kinase-dependent mechanism.

Recent biochemical studies have suggested that apoptotic cell death is the molecular mechanism underlying the degeneration of ovarian follicles during atresia. Using a sensitive autoradiographic method for the detection of DNA fragmentation, we studied apoptosis in ovarian granulosa cells or intact follicles placed in serum-free culture as model systems to elucidate the hormonal regulation of atresia. Immature rats (25 days old) were primed for 2 days with 10 IU equine CG to induce a homogeneous population of mature preovulatory follicles. Granulosa cells isolated from these follicles contained predominantly intact high mol wt DNA. However, a time-dependent, spontaneous onset of internucleosomal DNA fragmentation characteristic of apoptotic cell death occurred in granulosa cells during culture. Treatment of granulosa cells with epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), or basic fibroblast growth factor (bFGF) inhibited the spontaneous onset of apoptotic DNA cleavage found during culture by 40-60%. In contrast, insulin-like growth factor I, insulin, TGF beta and tumor necrosis factor-alpha were ineffective. Likewise, activation of the protein kinase A or C pathways with forskolin or phorbol 12-myristate 13-acetate, respectively, did not prevent the onset of DNA fragmentation, although inclusion of a tyrosine kinase inhibitor (genistein) completely blocked the ability of EGF, TGF alpha, and bFGF to suppress apoptosis in granulosa cells. Similar to cultured granulosa cells, a spontaneous onset of apoptosis was also observed to occur in isolated preovulatory follicles during culture. Furthermore, treatment of follicles with EGF or bFGF inhibited the spontaneous initiation of apoptosis, and the suppressive effects of these growth factors were also attenuated by co-treatment with genistein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenylyl cyclase system of the small preovulatory follicles of the domestic hen: responsiveness to follicle-stimulating hormone and luteinizing hormone

The purpose of this study was to determine if the granulosa cells of the small preovulatory follicles of the domestic hen are a target tissue for follicle-stimulating hormone (FSH). The third largest (F3), fourth largest (F4), and fifth largest (F5) follicles were removed from hens at 20, 12, 6 and 2 h before ovulation of the F1 follicle. Basal, FSH- and luteinizing hormone (LH)-stimulable adenylyl cyclase (AC) activities were measured in the granulosa cells. Isolated granulosa cells of the F5 follicle, obtained 20 h before ovulation of the F1 follicle, were incubated with ovine (o) or turkey (t) FSH and progesterone (P4) was assayed in the medium. Basal AC activity was similar for F5, F4 and F3 granulosa cells except for an increase (P less than 0.01) in F3 follicles removed 2 h before ovulation of the F1 follicle. The FSH-stimulable AC activity of F5, F4 and F3 granulosa cells was elevated over basal (P less than 0.01). The greatest responsiveness was seen in the F5 follicle and the least in the F3 follicle. LH-stimulable AC activity was absent in the F5 follicle but present in the F4 and F3 follicles with the greater responsiveness in the F3 follicle. Isolated F5 granulosa cells secreted significant amounts of P4 in response to oFSH and tFSH. The data indicate that: 1) FSH stimulates the AC system of granulosa cells of the smaller preovulatory follicles (F5 greater than F4 greater than F3) while LH stimulates the AC system of granulosa cells of the larger follicles (F3 greater than F4), and 2) FSH promotes P4 production by granulosa cells of F5 follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Progesterone receptor-mediated inhibition of apoptosis in granulosa cells isolated from rats treated with human chorionic gonadotropin

Almost all ovarian follicles undergo atresia during follicular development. However, the number of corpora lutea roughly equals the number of preovulatory follicles in the ovary. Because apoptosis is the cellular mechanism behind follicle and luteal cell demise, this suggests a change in apoptosis susceptibility during the periovulatory period. Sex steroids are important regulators of follicular cell survival and apoptosis. The aim of the present work was to study the role of progesterone receptor-mediated effects in the regulation of granulosa cell apoptosis. The levels of internucleosomal DNA fragmentation were evaluated in rat granulosa cells before and after induction of the nuclear progesterone receptor, using hCG treatment to eCG-primed rats to mimic the naturally occurring LH surge. Granulosa cells isolated from hCG-treated rats showed a several-fold increase in the expression of progesterone receptor mRNA and a 47% decrease (P < 0.01) in DNA fragmentation after 24 h incubation in serum-free medium compared to granulosa cells isolated from rats treated with eCG only. The effect of hCG treatment in vivo was dose-dependently reversed in vitro by addition of antiprogestins (Org 31710 or RU 486) to the culture medium, demonstrated by increased DNA fragmentation as well as increased caspase-3 activity. Addition of antiprogestins to granulosa cells isolated from immature or eCG-treated rats did not result in increased DNA fragmentation. The results suggest that progesterone receptor-mediated effects are involved in regulating the susceptibility to apoptosis in LH receptor-stimulated preovulatory rat granulosa cells.