Steroidogenic factor-1 expression is transiently repressed and c-myc expression and deoxyribonucleic acid synthesis are induced in rat granulosa cells during the periovulatory period

The expression of steroidogenic factor 1 (SF-1), cytochrome P450 aromatase (P450arom), and cytochrome P450 cholesterol side-chain cleavage (P450scc) was examined during the periovulatory period. Immature rats were injected with eCG to induce development of ovarian follicles to the preovulatory stage. At 48 h after the eCG injection, the LH surge was simulated by an injection of an ovulatory dose of hCG, and RNA was isolated at 0, 2, 4, 6, 8, and 24 h after hCG injection. The mRNA levels for SF-1, P450arom, and P450scc were relatively high in total ovarian RNA samples from eCG-treated rats. Levels of SF-1 and P450arom mRNA decreased within 2 h after injection of hCG. The SF-1 mRNA levels gradually increased from 4 to 24 h. Aromatase levels remained undetectable until 24 h after hCG. P450scc mRNA levels increased throughout the 24-h period after hCG. Levels of SF-1 and P450arom, but not P450scc, mRNA were also reduced in RNA samples from isolated granulosa cells at 4 h after hCG treatment relative to those from eCG-treated rats. In situ hybridization analysis also revealed that hCG uniformly suppressed SF-1 mRNA levels an all granulosa cells compared to those of eCG-treated controls. The relationship of SF-1 expression to immediate/early gene expression and cell cycle traverse was also examined. C-myc mRNA levels were induced by up to 10-fold at 4 h after hCG injection. Similarly, DNA synthesis, as measured by the percentage of granulosa cells that incorporated 5'-bromodeoxyuridine, was increased from 16 +/- 4% in eCG-treated rats to 61 +/- 7% at 4 h after hCG treatment (p < 0.05). This study provides the novel finding that SF-1 expression is transiently repressed to very low levels in response to the LH surge. Further, these studies suggest that granulosa cells traverse the cell cycle before becoming terminally differentiated luteal cells.     

Expression of androgen receptors and steroidogenic enzymes in relation to follicular growth and atresia following ovulation in pigs

Among the new antral follicles that develop after ovulation in pigs, the incidence of atresia, based on granulosa cell apoptosis, increases between Days 5 and 7 of the estrous cycle. The purpose of this study was to determine how follicular growth and atresia affected the expression of some key enzymes regulating follicular steroidogenesis and androgen receptor on Days 3, 5, and 7 after the onset of estrus. Ovaries were frozen in liquid propane for subsequent sectioning and immunohistochemical analysis. Ninety-six follicles were classified according to size as small (< 3 mm), medium (3-5 mm), or large (> 5 mm). Follicles in the active stages of the cell cycle were identified by the presence of the cell proliferation-associated nuclear antigen Ki-67 in granulosa cells. Follicles with apoptotic cells were identified by in situ 3'-end labeling of DNA. Staining intensity of antigens on sections was assigned a numeric value (0-3). Follicles assigned a value > 1 for 3'-end labeling in their granulosa cells were classified atretic. The percentage of atretic follicles increased (p < or = 0.05) from 5% on Days 3 and 5 to 41% on Day 7. Expression of Ki-67 in granulosa cells was more strongly (p < or = 0.05) associated with nonatretic follicles (98% expressing) than with atretic follicles (41% expressing). Aromatase cytochrome P450 (P450arom) was localized predominantly in granulosa cells of nonatretic follicles and was undetectable in atretic follicles. Androgen receptor in granulosa cells and expression of P450 17 alpha-hydroxylase/C17-20 lyase (P450c17) in theca interna were lower (p < or = 0.001) in atretic follicles than in nonatretic follicles. The expression of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) was localized to the theca interna and was unaffected by follicle atresia. In nonatretic small follicles, the expression of P450arom and P450c17 decreased (p < 0.01) between Days 3 and 7 while expression of Ki-67 was unchanged. In nonatretic follicles, increased follicle size was associated with a decrease (p < 0.01) in androgen receptor expression and increases (p < 0.01) in P450arom, P450c17, and 3 beta HSD expression. In conclusion, increased expression of steroidogenic enzymes was associated with follicular growth. Loss of P450arom expression in vivo is an early event in atresia and is followed by decreased cell proliferation, and decreased expression of androgen receptor and P450c17.     

Induction of Ad4BP/SF-1, steroidogenic acute regulatory protein, and cytochrome P450scc enzyme system expression in newly established human granulosa cell lines

We have established immortalized human granulosa cells by triple transfection of primary cells obtained from in vitro fertilization patients with SV40 DNA, Ha-ras oncogene, and a temperature sensitive (ts) mutant of the tumor suppressor gene p53 (p53val135). Forty-one clones were isolated, and their steroidogenic responses were analyzed. While all the cell lines proliferate rapidly and show only traces of progesterone production, upon stimulation with 50 microM of forskolin (FK), which elevates intracellular cAMP, they become steroidogenic as evidenced by progesterone production. The steroidogenic response of the cell lines was stable even after 20 generations and several cycles of freezing and thawing. A highly responsive cell line (HO-23) was further examined for characteristics of the steroidogenic response. Cells stimulated with FK and 8-Br-cAMP produced high levels of pregnenolone, progesterone, and 20alpha-hydroxy-4-pregnen-3-one (20alpha-OH-progesterone) comparable with amounts produced by highly differentiated primary human granulosa-luteal cells. Hydrocortisone and dexamethasone highly augment the cAMP-stimulated progesterone production, whereas testosterone and PRL enhanced cAMP-induced progesterone synthesis only moderately. Estradiol, insulin-like growth factor I, and insulin showed no significant effect on cAMP-induced steroidogenesis. The phorbol ester TPA, and basic fibroblast growth factor, dramatically suppress cAMP-induced production of progesterone, whereas bovine corneal endothelial cell ECM (BCE/ECM) enhanced cAMP-induced progesterone and antagonized basic fibroblast growth factor suppression of cAMP-induced steroidogenesis. Steroidogenic factor 1 (Ad4BP/SF-1) was expressed in control cells, and its expression was augmented by FK, whereas the steroidogenic acute regulatory protein showed low expression in the nonstimulated cells but was clearly elevated upon cAMP stimulation and was slightly decreased by TPA in cAMP-stimulated cells. Expression of the electron carrier adrenodoxin (ADX), which is a part of the cytochrome P450scc enzyme system, was very low in nonstimulated cells but was dramatically elevated in FK- and 8-Br-cAMP-stimulated cells, whereas no reduction of ADX was evident in cells costimulated with FK and TPA. Immunocytochemical studies revealed a weak staining of ADX in mitochondria of nonstimulated cells and intensive staining in highly clustered mitochondria of FK- or 8-Br-cAMP-stimulated cells. Only moderate reduction in ADX staining was evident in cells costimulated with FK and TPA. These unique cell lines can provide a useful model for the investigation of induced steroidogenesis in human granulosa cells.     

Lamin-binding fragment of LAP2 inhibits increase in nuclear volume during the cell cycle and progression into S phase

It is hypothesized that the two-cell model for estrogen production by the ovarian follicle is preserved in the primate corpus luteum, but there is little direct evidence to support this theory. To determine the sites of androgen and estrogen synthesis within the primate corpus luteum and to ascertain whether changes in steroid hormone levels are related to steroidogenic enzyme expression, the enzymes converting progesterone to androgen (cytochrome P450 17alpha-hydroxylase/17,20 lyase; P450(c17)) and then to estrogen (aromatase; P450(arom)), as well as P450 side-chain cleavage (P450(scc)) and 3beta-hydroxysteroid dehydrogenase (3beta HSD), were detected by immunohistochemistry in macaque luteal tissue throughout the menstrual cycle and simulated early pregnancy. Corpora lutea were collected from rhesus monkeys in the early (Days 2-4 post-LH surge), mid (Days 6-8), mid-late (Days 10-12), and late (Days 14-15) luteal phase and after 1, 3, 6, or 9 days of hCG treatment that began on Day 9 of the luteal phase. Specific cytoplasmic staining for P450(c17), P450(arom), P450(scc), and 3beta HSD was present in luteal cells, but not in the microvasculature, within all luteal tissues examined. P450(c17)-stained luteal cells were located along the vascular tracts and around the periphery of the corpus luteum. Intensely stained luteal cells were associated with blood vessels entering from the outer surface of the corpus luteum, but not with blood vessels returning from the connective tissue centrum. In contrast, P450(arom)-stained luteal cells were distributed throughout the luteal parenchyma. P450(c17) staining intensity was similar at all stages of the luteal phase; however, the number and intensity of P450(arom)-stained cells decreased by late luteal phase. In simulated early pregnancy, cells stained for P450(c17) were present near blood vessels, with some positive cells scattered throughout the corpus luteum. P450(arom) immunostaining was heterogeneous within the corpus luteum; many intensely stained cells were interspersed among others that were only lightly stained. Overall, cellular staining for P450(c17) and P450(arom) remained intense through 9 days of simulated early pregnancy. In contrast, P450(scc) and 3beta HSD immunoreactivity were not located in distinct luteal compartments. These results are consistent with a two-cell model for steroid hormone production in the primate corpus luteum, whereby paraluteal (theca-luteal) cells produce androgen substrate that is converted to estrogens by true (granulosa-) luteal cells. The divergence in enzyme detection as the luteal phase progresses, with P450(c17) labeling high and P450(arom) staining having decreased, suggests a shift in the function of the corpus luteum as it ages. Enzyme localization during chorionic gonadotropin exposure simulating early pregnancy demonstrates the continued capacity of the primate corpus luteum to produce steroid hormones.