Granulosa Cells Of Large Follicles Research Articles

Developmental and hormonal regulation of keratinocyte growth factor expression and action in the ovarian follicle.

The developing ovarian follicle is one of the most rapidly proliferating normal tissues in vivo. Mesenchymal-epithelial cell interactions between theca cells and granulosa cells are essential for this follicular expansion. Ovarian hormones (i.e. estrogen and LH) may promote follicular development by regulating the local production of mesenchymal inducer proteins that mediate theca cell-granulosa cell interactions. Recently, theca cells were shown to produce keratinocyte growth factor (KGF) that can act in a paracrine manner to stimulate granulosa cell growth. In this study, the developmental and hormonal regulation of KGF was examined during follicular development in the bovine ovary. Expression of KGF in theca cells and the KGF receptor (KGFR, or splice variant of the fibroblast growth factor family receptor family, FGFR-2) in granulosa cells was examined using RT-PCR. Both KGF and KGFR were detected throughout follicular development in small (<5 mm), medium (5-10 mm), and large (>10 mm) follicles. Quantitative RT-PCR assays were used to determine steady-state levels of KGF and KGFR messenger RNAs. Developmental regulation of KGF and KGFR was analyzed in freshly isolated theca cells and granulosa cells from small, medium, and large follicles. Observations demonstrated that expression of KGF (in theca cells) and KGFR (in granulosa cells) was highest in large follicles. These results suggest that KGF actions are important for the rapid proliferation of granulosa cells in large follicles. Estrogen and LH are the primary endocrine hormones that regulate theca cell function in vivo. Therefore, hormonal regulation of KGF was analyzed by treating serum-free theca cell cultures with estrogen and human CG (hCG, an LH agonist). Results showed that both estrogen and hCG stimulated KGF gene expression in theca cells. These results suggest that estrogen and LH may promote follicular growth (i.e. granulosa cell proliferation), in part, by stimulating the local production of KGF. Effects of KGF on granulosa cell differentiated functions were examined. Treatment with KGF reduced basal levels and FSH-stimulated levels of aromatase activity in bovine and rat granulosa cells. In addition, KGF inhibited the ability of hCG to stimulate progesterone production by granulosa cells. The inhibition of granulosa cell steroid production by KGF was likely the indirect effect of promoting cellular proliferation. Therefore, KGF directly stimulates granulosa cell proliferation and indirectly inhibits granulosa cell differentiated functions. Combined results suggest that theca cell production of KGF may be important for ovarian folliculogenesis. This is the first report of the regulation of KGF expression in the ovary. The developmental and hormonal regulation of KGF and KGFR during folliculogenesis provides evidence that KGF may be important for hormone-induced granulosa cell proliferation. As a result, KGF may be essential for establishing the microenvironment required for oocyte maturation in the ovary.

Developmental and Hormonal Regulation of Keratinocyte Growth Factor Expression and Action in the Ovarian Follicle

The developing ovarian follicle is one of the most rapidly proliferating normal tissues in vivo. Mesenchymal-epithelial cell interactions between theca cells and granulosa cells are essential for this follicular expansion. Ovarian hormones (i.e. estrogen and LH) may promote follicular development by regulating the local production of mesenchymal inducer proteins that mediate theca cell-granulosa cell interactions. Recently, theca cells were shown to produce keratinocyte growth factor (KGF) that can act in a paracrine manner to stimulate granulosa cell growth. In this study, the developmental and hormonal regulation of KGF was examined during follicular development in the bovine ovary. Expression of KGF in theca cells and the KGF receptor (KGFR, or splice variant of the fibroblast growth factor family receptor family, FGFR-2) in granulosa cells was examined using RT-PCR. Both KGF and KGFR were detected throughout follicular development in small (<5 mm), medium (5–10 mm), and large (>10 mm) follicles. Quantitative RT-PCR assays were used to determine steady-state levels of KGF and KGFR messenger RNAs. Developmental regulation of KGF and KGFR was analyzed in freshly isolated theca cells and granulosa cells from small, medium, and large follicles. Observations demonstrated that expression of KGF (in theca cells) and KGFR (in granulosa cells) was highest in large follicles. These results suggest that KGF actions are important for the rapid proliferation of granulosa cells in large follicles. Estrogen and LH are the primary endocrine hormones that regulate theca cell function in vivo. Therefore, hormonal regulation of KGF was analyzed by treating serum-free theca cell cultures with estrogen and human CG (hCG, an LH agonist). Results showed that both estrogen and hCG stimulated KGF gene expression in theca cells. These results suggest that estrogen and LH may promote follicular growth (i.e. granulosa cell proliferation), in part, by stimulating the local production of KGF. Effects of KGF on granulosa cell differentiated functions were examined. Treatment with KGF reduced basal levels and FSH-stimulated levels of aromatase activity in bovine and rat granulosa cells. In addition, KGF inhibited the ability of hCG to stimulate progesterone production by granulosa cells. The inhibition of granulosa cell steroid production by KGF was likely the indirect effect of promoting cellular proliferation. Therefore, KGF directly stimulates granulosa cell proliferation and indirectly inhibits granulosa cell differentiated functions. Combined results suggest that theca cell production of KGF may be important for ovarian folliculogenesis. This is the first report of the regulation of KGF expression in the ovary. The developmental and hormonal regulation of KGF and KGFR during folliculogenesis provides evidence that KGF may be important for hormone-induced granulosa cell proliferation. As a result, KGF may be essential for establishing the microenvironment required for oocyte maturation in the ovary.

Tumour necrosis factor-alpha inhibits follicle-stimulating hormone-induced granulosa cell oestradiol secretion in the human: dependence on size of follicle.

This study investigated the effects of tumour necrosis factor-alpha (TNF) on human granulosa cells taken from ovaries of 11 premenopausal women undergoing oophorectomy during the luteal phase of the cycle for reasons unrelated to ovarian pathology. Granulosa cells from follicles ranging from 5-10 mm diameter (small) and from >10-25 mm (large) were subjected to culture for 48 and 96 h. Granulosa cells were cultured with human FSH (1 ng/ml), testosterone (1 microM) and human TNF (10 ng/ml), each alone, and in various combinations. In granulosa cells of small follicles, FSH alone increased progesterone and cAMP accumulation and the conversion of testosterone to oestradiol. In granulosa cells of large follicles, FSH increased progesterone and cAMP accumulation but not the conversion of testosterone to oestradiol. Only in granulosa cells of small follicles did TNF significantly inhibit FSH-induced conversion of testosterone to oestradiol but it was not apparent until the second 48 h of culture and concomitantly TNF did not alter the ability of FSH to stimulate progesterone and cAMP accumulation. In granulosa cells of large follicles, TNF did not alter FSH-stimulated oestradiol, progesterone or cAMP accumulation. Interestingly, progesterone accumulation in the presence of TNF and FSH was significantly greater in granulosa cells of large follicles than in granulosa cells of small follicles. The results indicate that TNF suppresses FSH-induced oestradiol secretion in granulosa cells from small follicles and this modulatory effect of TNF appears to be independent of decreases in progesterone and cAMP. The potential physiological significances of these findings is that TNF may be a relevant cytokine in suppressing FSH-induced oestradiol secretion and follicular growth during the luteal phase of the cycle.

Intraovarian immunolocalization of steroidogenic enzymes in a Hokkaido brown bear, Ursus arctos yesoensis during the mating season.

Immunolocalization for four steroidogenic enzymes was performed on an ovary taken from a Hokkaido brown bear during the mating season. This specimen is considered to be in the follicular phase because of the presence of large follicles. In large follicles, cholesterol side-chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) were immunolocalized in theca interna cells and granulosa cells. 17 alpha-hydroxylase/C17-C20 lyase cytochrome P450 (P450c 17) was immunolocalized in theca interna cells but not in granulosa cells. Aromatase cytochrome P450 (P450arom) was immunolocalized only in granulosa cells. In medium follicles, however, P450scc and 3 beta HSD were immunolocalized only in theca interna cells, and the immunoreactivity of P450arom was detected in neither theca interna cells nor granulosa cells. Immunoreactivities of P450scc, 3 beta HSD and P450c 17 but not P450arom were detected in interstitial cells. This study suggests that estrogen biosynthesis takes place through interrelation between theca cells and granulosa cells and is explained by the so-called two-cell mechanism. Furthermore, the granulosa cells in large follicles have the capability for pregnenolone and progesterone biosynthesis, and the interstitial cell in the bear ovary is also a steroidogenic site.

Control of peptides regulating mitosis of granulosa cells in immature rat ovary by oestrogen and gonadotrophin.

Previous studies in this laboratory have demonstrated the presence of a peptide that inhibits granulosa cell proliferation in medium sized follicles. This peptide was produced after 72-96 h of exposure to diethylstilboestrol (DES). This study analyses oestrogen and gonadotrophin modulation of this and another stimulatory peptide found in large follicles. Intact, immature, female rats were assigned to the following study groups: (i) one to four injections of DES (2 mg per rat) given at intervals of 24 h, animals killed 24 h after the last injection; (ii) DES at 0 and 24 h, animals killed 24 or 48 h after the last injection; and (iii) DES plus pentobarbital (37 mg kg-1 body weight) at 30 h, animals killed 48 h after the last injection. Small, medium and large follicles (diameters of < 200, 200-400 and > 400 microns, respectively) were isolated from ovaries, granulosa cells were harvested and follicular fluid supernatant (FFS) was collected. FFS proteins were tested for their effects on incorporation of [3H]thymidine into granulosa cell DNA. Results showed that unfractionated FFS protein (150 micrograms) from medium follicles in groups (i) (three and four injections only), (ii) and (iii) inhibited [3H]thymidine incorporation. Pentobarbital blockage of gonadotrophin secretion had no effect on the inhibitory peptide activity and two injections of diethylstilboestrol were enough to stimulate synthesis of the inhibitory peptide, provided sufficient time was allowed; FFS protein (150 micrograms) from large follicles was stimulatory, but only when it was collected 24 h after the second injection, and the effect was abolished with pentobarbital treatment. Fractionation of pooled FFS into proteins with three molecular mass ranges (< 10 kDa, 10-30 kDa and > 30 kDa) showed that the inhibitory activity was in the < 10 kDa fraction while the > 30 kDa fraction stimulated thymidine incorporation. The number of medium and large follicles increased 24 h after the second DES injection, but the number of granulosa cells in the large follicles was significantly reduced after pentobarbital blockage of gonadotrophins. Taken together, these findings show that (i) the inhibitory peptide is induced in effective amounts in the medium follicles 48 h after the second DES injection and induction is not modulated by gonadotrophins; and (ii) stimulatory activity seen in the large follicles is transient and under gonadotrophic control, since blockage of follicle-stimulating hormone (FSH) secretion led to loss of stimulatory activity and a resulting reduction in number of granulosa cells in large follicles.(ABSTRACT TRUNCATED AT 400 WORDS)

Increase in the expression of thyroid hormone receptors in porcine granulosa cells early in follicular maturation.

Thyroid hormone has been demonstrated to synergize with FSH to exert stimulatory effects on the differentiation of porcine granulosa cells. In order to further characterize the nature of thyroid hormone action on granulosa cells, the presence of triiodothyronine (T3) receptors in the nuclei of porcine granulosa cells was examined, and qualitatively and quantitatively compared during follicular maturation. Then, comparative abilities of granulosa cells from varying follicle stages to respond to T3 were assessed in terms of FSH-induced LH/hCG receptor formation and progesterone secretion. Furthermore, the expression of erb-A was analyzed using Northern blot hybridization of porcine granulosa cell RNA with a v-erb-A probe. Binding experiments with [125I]T3 showed that granulosa cell nuclei obtained from small follicles had a greater ability to bind [125I]T3 compared to those from large follicles. Scatchard analysis revealed the presence of nuclear T3 receptors with a single class of binding sites. There was little difference in the affinity of the T3 receptors during follicular maturation. By contrast, the number of the T3 receptors was higher in small follicle granulosa cells compared to that in large follicle granulosa cells. Thus, the increased T3 binding to small follicle granulosa cells relative to large follicle granulosa cells appears to be attributable to the increased number of the nuclear T3 receptors rather than to a change in the affinity. The magnitude of the stimulatory effects of T3 on granulosa cell functions was maximal in small follicle granulosa cells, but negligible in large follicle granulosa cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Transient Expression of Progesterone Receptor Messenger RNA in Ovarian Granuiosa Cells after the Preovulatory Luteinizing Hormone Surge

The ovarian steroid progesterone affects reproductive physiology by regulating the expression of specific genes in target tissues. In an attempt to address the question of whether the ovary itself is a target tissue for progesterone action, we have examined the localization and regulation of progesterone receptor (PR) mRNA in the rat ovary. We used the polymerase chain reaction to clone the steroid-binding domain of the rat PR from uterine cDNA and used this as a probe to isolate a larger cDNA from a rat placental cDNA library. We used RNA filter hybridization, a quantitative reverse transcription-polymerase chain reaction amplification assay, and in situ hybridization to detect PR mRNA in the rat ovary. Expression of the PR gene was initially studied in an immature animal model; 23-day-old rats were treated with either PMSG or PMSG followed by hCG. We found little or no PR mRNA in the ovaries of control or PMSG-treated animals; however, the mRNA was highly expressed in the granulosa cells of large follicles in the ovaries of animals treated with PMSG followed by hCG. Other cell types, including thecal and interstitial cells, did not express detectable levels of PR mRNA. The PR mRNA was induced more than 20-fold in the immature ovary 5 h after hCG administration and was down-regulated to near-basal levels by 12 h after hCG administration. In a subsequent series of experiments, we examined PR gene expression in adult rats during the estrous cycle. The expression of PR mRNA was transient and was tightly coupled to the preovulatory LH surge on proestrous evening. PR mRNA was localized to the granulosa cells of mature ovarian follicles during the estrous cycle. In cycling animals treated with pentobarbital to block the preovulatory LH surge, no induction of PR mRNA on proestrous evening was observed. This transient, hormonally regulated, and cell-specific expression of the PR gene in the rat ovary strongly suggests an important intraovarian function for progesterone during the rat reproductive cycle.

Developmental and hormonal regulation of bovine granulosa cell function in the preovulatory follicle.

Bovine granulosa cells were isolated from small antral, medium antral, and large Graffian follicles (i.e. small, medium, and large preovulatory follicles). Serum-free cultures of granulosa cells were established and found to be viable for 3-6 days of cell culture. Radiolabeled granulosa cell-secreted proteins were obtained and analyzed electrophoretically. No major changes were detected in the protein profiles of small, medium, and large follicle granulosa cells. FSH and insulin, however, had a dramatic effect on granulosa cell-secreted proteins and increased the apparent production of 200K, 65K, 25K, and 15K proteins. The effects of these hormones on the radiolabeled secreted proteins were similar for small, medium, and large follicle granulosa cells. Aromatase activity was high for the first day of serum-free granulosa cell culture and subsequently declined to low levels. Both FSH and insulin alone stimulated aromatase activity, while a combination of hormones resulted in an additive response similar to the stimulation observed with 10% calf serum. Although the level of aromatase activity increased slightly with the size of the follicle, the effects of hormones were independent of follicle size. Progesterone production was low on days 1 and 2 of serum-free granulosa cell culture and high on days 3 and 6 of cell culture. Interestingly, FSH and insulin suppressed progesterone production on day 1 of cell culture for small and medium follicle granulosa cells, but not for large follicle cells. In contrast, hormones stimulated progesterone production on days 3 and 6 of granulosa cell culture, and the level of progesterone production increased with the size of the follicle. The stimulatory effects of hormones on days 3 and 6 of the culture were similar for medium and large follicle granulosa cells, but were altered for small follicle cells. Results indicate that when aromatase activity is high and stimulated by hormones, progesterone levels are low and generally suppressed by the same regulatory agents. Conversely when progesterone levels are high and hormone responsive, aromatase activity is low. The inverse relationship between aromatase activity and progesterone production implies that bovine granulosa cells alter their differentiated state in culture from an estrogen-producing cell to a progesterone-producing cell. Combined observations indicate that the results obtained on day 1 of culture probably reflect the developmental and hormonal regulation of granulosa cell function in the preovulatory follicle, while data obtained at later times in culture reflect the ability of the cell to synthesize progesterone and develop a luteinization-like activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Internalization of receptor-bound human chorionic gonadotrophin in preovulatory rat granulosa cells in vivo.

The subcellular distribution of 125I-labelled human chorionic gonadotrophin (hCG) in preovulatory rat granulosa cells was studied in vivo. Pregnant mare serum gonadotrophin-pretreated immature female rats received an iv injection of [125I]hCG a few hours before the endogenous preovulatory gonadotrophin surge. The animals were killed at 2 or 6 h after the [125I]hCG injections. Light microscope autoradiographs showed that the mural granulosa cells of large follicles were the most highly labelled cells in the ovaries. Electron microscope autoradiography was used to study the subcellular distribution of radioactivity in the mural granulosa cells. At 2 h 45% of the counted silver grains were associated with the plasma membrane and 10% with the lysosomes, at 6 h the values were 51% and 9%, respectively. The distribution of the observed silver grains was compared with the generated expected source to grain pairs by computerized linear multiple regression analysis. The magnitudes of the regression coefficients revealed that the plasma membrane and the lysosomes were the only specifically 125I-labelled organelles, that a few radioactive molecules were located diffusely over the cytoplasm at 2 h and that the 125I-radioactivity of the nuclei was negligible. The present results suggest that preovulatory rat granulosa cells are in vivo able to internalize into lysosomes [125I]hCG initially bound to LH/hCG receptors of the plasma membrane.

MATURATIONAL CHANGES IN SHEEP OVARIAN FOLLICLES: GONADOTROPHIC STIMULATION OF CYCLIC AMP PRODUCTION BY ISOLATED THECA AND GRANULOSA CELLS

Theca and granulosa tissues isolated from sheep ovarian follicles of different sizes were incubated in the presence of human chorionic gonadotrophin (HCG; 5 IU/ml) or follicle stimulating hormone (FSH; 5 microgram NIH-FSH-S11/ml) for 40 min. Changes in the total amounts of cyclic 3',5'-adenosine monophosphate (cAMP) were used as an index of the responsiveness of these preparations to the hormones. Thecal tissue of both large (4-6 mm in diameter) and small (1-3 mm) follicles responded similarly to gonadotrophins. Granulosa cells from small follicles failed to respond to stimulation by HCG. FSH, however, consistently increased cAMP production in comparison with controls or cells treated with HCG. Granulosa cells of large follicles responded to both HCG and FSH.

The Porcine Ovarian Follicle. II. Electron Microscopic Study of Surface Features of Granulosa Cells at Different Stages of Development1

Surface features of granulosa cells from porcine ovarian follicles were studied at different stages of development by electron microscopy. The presence ofnumerous microvilli on the luminal surface granulosa cells of large follicles and their absence on granulosa cells from small and medium follicles were observed. Microfilaments within the microvilli were also observed. A possible relationship between these structural features and the appearance of trophic hormone receptors is discussed.

Characteristics of gonadotropin receptors of porcine granulosa cells during follicle maturation.

In order to determine why granulosa cells obtained from large preovulatory porcine follicles luteinize spontaneously in culture, whereas cells obtained from adjacent less mature small follicles fail to luteinize spontaneously, or in the presence of exogenous gonadotropin, gonadotropin receptors in granulosa cells obtained from increasingly mature small, medium and large porcine follicles have been studied. Granulosa cells obtained from small, medium and large follicles did not differ significantly in size. Granulosa cells obtained from small (1–2 mm), medium (3– 5 mm) and large porcine follicles were incubated with 125I—hCG. Granulosa cells obtained from large follicles bound 10–lOOO–fold more 125I—hCG (623,300 ± 44,890 cpm/5 X 106 cells; n = 12) compared to small follicles of the same ovary or from other ovaries (7,554 ± 1,983 cpm/5 X 106 cells; n = 16). Medium follicle cells bound only a slightly greater amount of hCG than small follicle cells. Qualitatively the binding of small, medium and large follicle granulosa cells to 125I—hCG was roughly similar. Binding reached a plateau between 30 and 90 minutes. Furthermore, if the concentration of 125I—hCG was raised (from 0.01 to 0.5 μg/ml) there was exhibition of saturation. When unlabeled hCG or LH was added to cells from any follicle size immediately prior to addition of 125I—hCG it could inhibit the binding 90–95%, demonstrating similarity of LH and hCG receptors. Evidence that the binding was strong and that there was slow turnover of hCG at its receptor site has been found. (Endocrinology92: 531, 1973)