Follicular fluid modulation of functional LH receptor induction in pig granulosa cells

Follicular fluid was collected from small (1-2 mm), medium (3-5 mm) and large (6-12 mm) follicles of pigs, treated with charcoal to remove steroids, and tested for effects on the induction of functional LH/hCG receptors in cultures of granulosa cells from small antral pig follicles. Granulosa cells were cultured for 2, 4 or 6 days in Medium 199 + 10% pig serum. Granulosa cells cultured in the presence of purified human FSH (0.1 microgram/ml, LER 8/117), insulin (1 mU/ml), cortisol (0.01 microgram/ml) and thyroxine (10(-7) M) accumulated a 4- to 8-fold increase in LH/hCG receptors compared to control cultures. The amounts of cyclic AMP and progesterone secreted after exposure to ovine LH (1 microgram/ml: NIH-S19) were also increased 2-3-fold and 80-100-fold, respectively. Exposure to FSH alone resulted in lower amounts of LH/hCG receptors with a concomitant decrease in optimum LH responses. Addition of 12.5-50% follicular fluid obtained from small (1-2 mm) follicles led to a dose-dependent inhibition of the FSH plus insulin, cortisol and thyroxine induction of LH/hCG receptors after 4 days of culture. Fluid from medium follicles showed reduced ability to inhibit LH/hCG receptor induction, and fluid from large follicles exerted only a slight inhibition or no inhibition of receptor induction. Fluid from medium-sized and large follicles exerted a progressive dose-dependent stimulation of progesterone secretion by the granulosa cell cultures. The inhibitory activity was precipitated primarily with 70% ethanol and to a lesser degree by 36 and 90% ethanol. These studies demonstrate that induction of functional LH/hCG receptors in cultures of pig granulosa cells from immature follicles is enhanced by including insulin, cortisol and thyroxine, in addition to FSH, in the culture medium, and that follicular fluid modulates both receptor induction and progesterone secretion as a function of follicular maturation.     

Expression and regulation of mRNAs for insulin-like growth factor (IGF-I), IGF-binding protein-2, and LH receptor in the process of follicular atresia

Expression of mRNAs for IGF-I, IGF-binding protein-2 (IGFBP-2), and LH receptor (LHR) as well as their regulations during induced follicular atresia was determined. 26-day-old female rats received 15 IU pregnant mare serum gonadotropins (PMSG). Through detection, it was demonstrated that apoptosis occurred in some small antral follicles after 48 h of PMSG treatment. At 96 h, apoptosis occurred in preovulatory follicles. At 120 h, numerous apoptotic cells appeared in preovulatory follicles. IGF-I was mainly expressed in preantral and small antral follicles from 48 to 120 h. At 48 and 96 h, the theca cells of preantral and antral follicles expressed high level of IGFBP-2 mRNA. At 48 h, there were strong signals of LHR mRNA in granulosa cells, but the LHR signals in granulosa cells significantly decreased at 96 and 120 h (p<0.001). Both epidermal growth factor (EGF) and IGF-I inhibited apoptosis in preantral and antral follicles. Meanwhile, it was observed that EGF promoted IGF-I mRNA expression, and in preovulatory follicles, IGF-I stimulated LHR mRNA expression. These results show that the interaction between ECF and IGF-I may be involved in the regulation of atresia of follicles at different stages of development.     

Expression and regulation of mRNAs for insulin-like growth factor (IGF-I), IGF-binding protein-2, and LH receptor in the process of follicular atresia

Expression of mRNAs for IGF-I, IGF-binding protein-2 (IGFBP-2), and LH receptor (LHR) as well as their regulations during induced follicular atresia was determined. 26-day-old female rats received 15 IU pregnant mare serum gonadotropins (PMSG). Through detection, it was demonstrated that apoptosis occurred in some small antral follicles after 48 h of PMSG treatment. At 96 h, apoptosis occurred in preovulatory follicles. At 120 h, numerous apoptotic cells appeared in preovulatory follicles. IGF-I was mainly expressed in preantral and small antral follicles from 48 to 120 h. At 48 and 96 h, the theca cells of preantral and antral follicles expressed high level of IGFBP-2 mRNA. At 48 h, there were strong signals of LHR mRNA in granulosa cells, but the LHR signals in granulosa cells significantly decreased at 96 and 120 h (p<0.001). Both epidermal growth factor (EGF) and IGF-I inhibited apoptosis in preantral and antral follicles. Meanwhile, it was observed that EGF promoted IGF-I mRNA expression, and in pr     

Autocrine role of estrogens in the augmentation of luteinizing hormone receptor formation in cultured rat granulosa cells

The effects of estrogens on gonadotropin-stimulated luteinizing hormone (LH) receptor formation were examined in primary cultures of rat granulosa cells. Granulosa cells were cultured for 3 days with increasing concentrations of follicle-stimulating hormone (FSH) in the presence or absence of native and synthetic estrogens. Follicle-stimulating hormone stimulated LH receptor formation in a dose-dependent fashion, and estrogens enhanced the FSH-stimulated LH receptor content by decreasing the apparent ED50 of FSH. At 6.25 ng/ml FSH, the enhancement in LH receptor was estrogen dose dependent, with an ED50 value of about 3 X 10(-9) M for 17 beta-estradiol. The increased LH receptor content seen in cells treated with FSH and estrogen was correlated with increased cAMP production by these cells in response to LH stimulation. Time course studies revealed enhancement of FSH-stimulated LH receptor induction at 48 and 72 h of culture. Granulosa cells were also cultured with FSH for 2 days to induce functional LH receptors, then further cultured for 3 days with LH in the presence or absence of estrogens. At 30 ng/ml LH, increasing concentrations of estrogens maintained LH receptor content in a dose-dependent fashion, with their relative estrogenic potencies in keeping with reported binding affinities to estrogen receptors. An autocrine role of estrogens on LH receptor formation was further tested in granulosa cells treated with FSH and an aromatase substrate (androstenedione) to increase estrogen biosynthesis. Cotreatment with semipurified estrogen antibodies partially blocked the FSH stimulation of LH receptors, whereas nonimmune serum was ineffective. Also, inclusion of diethylstilbestrol prevented the inhibitory effect of the estrogen antibodies. Thus, local estrogens in ovarian follicles may play an autocrine role in granulosa cells to enhance LH receptor formation and to increase granulosa cell responsiveness to the LH surge, with subsequent ovulation and adequate corpus luteum formation.     

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.     

Development of preovulatory follicles expected to form short-lived corpora lutea in beef cows

Oestrus, expected to be followed by a short luteal phase, was induced in post-partum cows by weaning their calves at 35 days after parturition. Ovaries containing the first preovulatory follicles (Type F) formed after parturition were collected 3 h after the onset of oestrus. For comparison, preovulatory follicles (Type C) were collected 3 h after the onset of oestrus in normally cycling cows. The number of granulosa cells was determined and the concentrations of receptors for follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in granulosa cells and for LH in theca cells were measured. Concentrations of oestradiol-17 beta, testosterone, androstenedione and progesterone in follicular fluid were also measured. Type F follicles contained about twice the number of granulosa cells (based on DNA) as did Type C follicles (45.8 +/- 11.3 and 24.5 +/- 3.9 micrograms DNA/follicle, respectively; P less than 0.05) but these cells had fewer receptors for LH (0.13 +/- 0.02 vs 0.29 +/- 0.03 fmol/micrograms DNA; P less than 0.01) and FSH (0.61 +/- 0.08 vs 1.3 +/- 0.29 fmol/micrograms DNA; P less than 0.08) than did those from Type C follicles. Additionally, there were fewer receptors for LH in theca tissue from Type F than from Type C follicles (28.3 +/- 5.2 vs 51.3 +/- 6.1 fmol/follicle; P less than 0.01). Concentrations of oestradiol-17 beta (475.8 +/- 85.6 vs 112.9 +/- 40.0 ng/ml; P less than 0.01) and androstenedione (214.1 +/- 48.7 vs 24.7 +/- 7.7 ng/ml; P less than 0.01) in follicular fluid were higher in Type C than in Type F follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epidermal growth factor and gonadotropin-releasing hormone inhibit cyclic AMP–dependent luteinizing hormone receptor formation in ovarian granulosa cells

The induction of luteinizing hormone (LH) receptors was studied in granulosa cells prepared from the ovaries of hypophysectomized diethylstilbestrol-treated immature rats. Incubation of granulosa cells for 48 h with increasing concentrations of follicle-stimulating hormone (FSH) or choleragen caused parallel rises in cAMP levels and LH receptors. These observations, with the finding that 8-Bromo-cAMP also induced LH receptor formation, indicate that hormonal stimulation of LH binding sites is mediated by cAMP. Peptide hormones that inhibited FSH-stimulated cAMP production, such as epidermal growth factor (EGF) and a gonadotropin-releasing hormone agonist (GnRHa), also prevented LH receptor formation. GnRHa and EGF had negligible effects on FSH-stimulated cAMP production from 0 to 24 h of culture, but reduced cAMP accumulation by 80% and 90%, respectively, from 24 to 48 h when the majority of LH receptors appeared. FSH-sensitive adenylate cyclase activity, as measured by the conversion of (³H)-ATP to (³H)-cAMP, was inhibited by GnRHa and EGF at 48 h of culture. EGF and GnRHa also reversed the inhibition of ectophosphodiesterase activity caused by FSH in granulosa cells between 48 and 72 h of culture. Both EGF and GnRHa inhibited induction of LH receptors by 8-Bromo-cAMP, suggesting that their effects are also on cAMP action. Addition of GnRHa, but not EGF, between 36 and 48 h of culture completely prevented further increases in LH receptors induced by 8-Bromo-cAMP, indicating that the inhibitory action of GnRHa can be initiated at later times during granulosa cell differentiation, whereas full expression of EGF action requires a longer period. These results demonstrate that EGF and GnRH inhibit FSH-induced LH receptor formation in the granulosa cell by reducing hormone-dependent cAMP production and also by impairing the ability of cAMP to stimulate LH receptor formation.     

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.     

Gonadotropin surge induces two separate increases in messenger RNA for progesterone receptor in bovine preovulatory follicles

In mice deficient in progesterone receptor (PR), follicles of ovulatory size develop but fail to ovulate, providing evidence for an essential role for progesterone and PR in ovulation in mice. However, little is known about the expression and regulation of PR mRNA in preovulatory follicles of ruminant species. One objective of this study was to determine whether and when PR mRNA is expressed in bovine follicular cells during the periovulatory period. Luteolysis and the LH/FSH surge were induced with prostaglandin F(2alpha) and a GnRH analogue, respectively, and the preovulatory follicle was obtained at 0, 3.5, 6, 12, 18, or 24 h after GnRH treatment. RNase protection assays revealed a transient increase in levels of PR mRNA, which peaked at 6 h after GnRH and declined to the time 0 value by 12 h and a second increase at 24 h. The second objective was to investigate the mechanisms that regulate PR mRNA expression through in vitro studies on follicular cells of preovulatory follicles obtained before the LH/FSH surge. Theca and granulosa cells were isolated and cultured with or without a luteinizing dose of LH or FSH, progesterone, LH + progesterone, or LH + antiprogestin (RU486). Levels of PR mRNA increased in a time-dependent manner in granulosa cells cultured with LH or FSH and in theca cells cultured with LH, peaking at 10 h of culture. In contrast, progesterone (200 ng/ml) did not upregulate mRNA for its own receptor, and neither progesterone nor RU486 affected LH-stimulated PR mRNA accumulation. Furthermore, RU486 completely blocked LH-stimulated expression of oxytocin mRNA, indicating that PR induced by LH in vitro is functional. These results show that the gonadotropin surge induces a rapid and transient increase in expression of PR mRNA in both theca and granulosa cells of bovine periovulatory follicles followed by a second rise close to the time of ovulation and that the first increase in PR mRNA can be mimicked in vitro by gonadotropins but not by progesterone. These results suggest multiple and time-dependent roles for progesterone and PR in the regulation of periovulatory events in cattle.     

Determination of onset of apoptosis in granulosa cells of the preovulatory follicles in the bonnet monkey (Macaca radiata): correlation with mitogen-activated protein kinase activities

During reproductive life, only a selected few ovarian follicles mature and ovulate, while the vast majority of follicles undergo a degenerative process called atresia. Recent studies have indicated that follicular atresia is mediated through apoptosis of follicular granulosa cells. The objectives of the present study were to determine the time of onset of apoptosis in granulosa cells of preovulatory follicles and to evaluate the consequences of gonadotropin withdrawal on mitogen-activated protein (MAP) kinase activities. Bonnet monkeys (Macaca radiata) undergoing controlled ovarian stimulation cycles were utilized for stimulation of multiple follicles, and granulosa cells were retrieved from preovulatory follicles at 24, 48, 72, and 96 h after stopping gonadotropin treatment. Serum and follicular fluid estradiol concentrations were highest at 24 h but declined precipitously (P < 0.05) to reach the lowest concentrations at 96 h; however, progesterone concentrations during this period did not increase, indicating the absence of luteinization. Quantitative analysis of genomic DNA by 3'-end labeling revealed the presence of low-molecular-weight fragments from 48 h onward, but by agarose gel electrophoresis, DNA laddering could be visualized only after 72 h. Messenger RNA expression for Bax, caspase-2, and caspase-3 increased with the onset of apoptosis. Immunoblot analysis of MAP kinases in lysates of granulosa cells (48-72 h) indicated increased (P < 0.05) levels of phosphorylated extracellular response kinase-1 and -2, Jun N-terminal kinase (JNK)-1 and -2, and p38. However, in vitro kinase assay data indicated that only phospho-JNK and -p38 activities were higher at 72 h compared to 24 h. These results demonstrate that granulosa cells of preovulatory follicles undergo apoptosis and that increased activities of phospho-JNK and -p38 are correlated with apoptosis in the primate.     

Expression of mRNA of lipoprotein receptor related protein 8, low density lipoprotein receptor, and very low density lipoprotein receptor in bovine ovarian cells during follicular development and corpus luteum formation and regression

Lipoproteins in the plasma are the major source of cholesterol obtained by the ovarian theca and granulosa cells for steroidogenesis. In this study, we have identified mRNA expression in bovine theca and granulosa cells of two lipoprotein receptors, low density lipoprotein receptor (LDLr) and very low density lipoprotein receptor (VLDLr) in granulosa cells from small antral follicles through preovulatory follicles and in theca cells from large and medium sized antral follicles. In the corpus luteum (CL) both these receptors were found in the developing and differentiating stages whereas only mRNA for VLDLr was detected in the regression stage. This study also described for the first time, the presence of lipoprotein receptor related protein (LRP8) in granulosa cells from small antral follicles through preovulatory follicles and in theca cells from large and medium sized antral follicles. This may indicate a role of LRP8 in cholesterol delivery to steriodogenic cells. LRP8 was not detected in any of the CL stages. The roles of the LDLr superfamily in lipid transport to ovarian cells and its participation in follicular and CL development and regression is discussed.     

Intrafollicular content of luteinizing hormone receptor, alpha-inhibin, and aromatase in relation to follicular growth, estrous cycle stage, and oocyte competence for in vitro maturation in the mare

The intrafollicular content of LH receptor, alpha-inhibin, and aromatase are known good indicators of follicular status. We investigated the amounts of these proteins in granulosa and cumulus cells in relation to oocyte competence for in vitro maturation, follicular growth, and estrous cycle stage in the mare. Follicular punctures were performed 34 h after an injection of crude equine gonadotropins, either during the follicular phase, at the end of the follicular phase, or during the luteal phase. The cumulus-oocyte complex, granulosa cells, and follicular fluid of follicles larger than 5 mm were collected. The nuclear stage of the oocytes after in vitro culture was determined microscopically. Granulosa and cumulus cell amounts of LH receptor, alpha-inhibin, and aromatase were assessed by the semiquantitative Western blot method and image analysis. Follicular fluids were assayed for progesterone (P4) and estradiol-17beta (E2). The three factors were expressed in mural granulosa and cumulus cells from all follicles from the gonadotropin-independent growth period until the preovulatory stage. Considering all the follicles punctured, the amounts of LH receptor and alpha-inhibin in granulosa cells were not different for the three physiological stages studied. The amounts of aromatase in granulosa cells, as well as the E2:P4 ratios, were higher for follicles punctured during the follicular phase than for the two other groups (p < 0.05). Considering the data from the three groups, the E2:P4 ratio and the LH receptor and aromatase contents, but not alpha-inhibin, in granulosa cells increased with an increase in follicular diameter (p < 0.01). The E2:P4 ratios and the amounts of LH receptor, alpha-inhibin, and aromatase in granulosa cells were lower in follicles 5-9 mm in diameter than in larger ones (p < 0.05). In cumulus cells, the amounts of the three factors were different neither between the three groups nor between the follicular diameters. Although we could not establish any obvious relationship to oocyte competence for in vitro maturation, the influence of the follicle diameter on the content of LH receptors, alpha-inhibin, and aromatase in granulosa cells was similar to the influence of follicle diameter on oocyte competence. Therefore, one can hypothesize that, in the mare, there is a link between the acquisition of oocyte competence and the expression of these factors in the follicular cells.     

Preovulatory biosynthesis and granulosa cell secretion of immunoreactive oxytocin by goat ovaries

Oestrous cycles of goats were synchronized hormonally. Immunoreactive oxytocin was undetectable (less than 0.1 ng/mg protein) in media from granulosa cells isolated before the LH surge for small (1-2 mm), medium (3-5 mm) and large (greater than 5 mm diameter) follicles when cultured for 24 h without or with added hormones. Granulosa cells from large and medium, but not small, follicles isolated 6-12 h after spontaneous preovulatory LH surges secreted high concentrations of oxytocin (4-12 ng/mg protein). Addition of PGE-2 (1 microgram/ml) caused a further significant (P less than 0.05) increase in oxytocin secretion by cultured granulosa cells, whereas PGF-2 alpha, FSH and LH were ineffective when added to culture media. Ovarian venous blood and granulosa cells were collected at 0, 6, 12 or 18 h after GnRH injection in hormonally synchronized goats. Peripheral serum LH values were increased significantly in all but 2 of 22 goats within 2 h of GnRH injection. At the earliest sampling time after GnRH (6 h), ovarian venous levels of oxytocin were increased significantly from basal levels of 0.4 pg/ml to 2.4 pg/ml. Oxytocin concentrations in follicular fluid increased from a basal value of 67 pg/ml to 155 pg/ml by 6 h and to 372 pg/ml by 18 h after GnRH injection. Oxytocin secretion by cultured granulosa cells was not increased significantly by 6 h (0.1 ng/mg protein) but rose to 1.4 and 3.5 ng/mg protein at 12 and 18 h, respectively. Approximately parallel increases occurred in progesterone in ovarian venous blood and granulosa cell culture media over the same time period. (ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Oncogene-transformed granulosa cells as a model system for the study of steroidogenic processes

Highly steroidogenic granulosa cell lines were established by transfection of primary granulosa cells from preovulatory follicles with SV40 DNA and Ha-ras oncogene. Progesterone production in these cells was enhanced to levels comparable to normal steroidogenic cells, by prolonged (> 12 h) stimulation with 8-Br-cAMP, forskolin and cholera toxin, which elevate intracellular cAMP. The steroidogenic capacity of individual lines correlated with the expression of the ras oncogene product (p21) and the morphology of the cells. Formation of the steroid hormones was associated with de novo synthesis of the mitochondrial cytochrome P450scc system proteins. Since cholesterol import into mitochondria is essential for steroidogenesis, the expression of the peripheral benzodiazepine receptor (PBR) and the sterol carrier protein 2 was characterized in these cells. The induction of the expression of the genes coding for both proteins appeared to be mediated, at least in part, by cAMP. Stimulation of the PBR by specific agonists enhanced progesterone production in these cells. The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) dramatically suppressed the cAMP-induced steroidogenesis, in spite of enhanced intracellular cAMP levels, suggesting that TPA can modify the effects of cAMP. cAMP stimulation suppressed growth of transformed cells concomitantly with induction of steroidogenesis. The transformed cells lacked receptors for the native stimulants, the gonadotropic hormones. After transfection of the cells with a lutropin (LH) receptor expression plasmid, the LH and hCG response was reconstituted. In these newly established cell lines gonadotropins were able to stimulate the formation of cAMP and progesterone in a dose-dependent manner with an ED₅₀ characteristic of the native receptor. High doses caused desensitization to gonadotropins as observed in normal cells. These newly established oncogene-transformed granulosa cell lines can serve as a useful model to study inducible steroidogenesis and the effect of oncogene expression on this process.     

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.