Summary. The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase: EC 126.96.36.199) was measured in a microsomal preparation of the granulosa of rapidly growing ovarian follicles of laying hens in the late preovulatory period (2–3 h before expected ovulation). The specific activity of the enzyme was measured in the five largest (F1–F5) preovulatory follicles, F1 being the follicle destined to ovulate first. Enzyme activity increased concomitantly with follicle size. The apparent K m of the enzyme decreased 60–80% from the smallest to the largest preovulatory follicle. There was no significant change in the V max during follicle development. Although our results have demonstrated the presence of HMG/CoA reductase in chicken granulosa cells and the progressive increase of its activity with follicular maturation, the quantitative significance of de-novo synthesized cholesterol as steroid hormone precursor remains to be ascertained.
E. K. Asem and F. Hertelendy
E. K. Asem and F. Hertelendy
Summary. A radiochemical assay was utilized to study the inhibitory effects of clomiphene and tamoxifen on the cholesterol side-chain cleavage enzyme activity in a mitochondrial preparation of granulosa cells isolated from mature ovarian follicles of laying hens. At saturating substrate concentrations, both clomiphene and tamoxifen were able to suppress enzyme activity in a dose-related manner IC50 1·8 × 10−5 m). Double reciprocal plots of kinetic data show that the inhibition is mixed, exhibiting competitive kinetics at low concentrations, whereas at high concentrations, the inhibition is of a non-competitive nature. The competitive inhibition constants as determined from Dixon plots are 2 × 10−55 m for clomiphene and 2·3 × 10−55 m for tamoxifen. It is concluded that, in granulosa cells, clomiphene and tamoxifen directly inhibit the mitochondrial cholesterol side-chain cleavage activity. This inhibition may represent an important aspect of the mode of action of clomiphene and tamoxifen.
E. K. Asem and R. P. Novero
Experiments were conducted in vitro to examine the effect of epidermal growth factor (EGF) and transforming growth factor α (TGF-α) on the production of fibronectin (deposited, secreted into medium and cell-associated) by hen granulosa cells isolated from the largest (F1; about 35 mm in diameter, mature) and third largest (F3; 15–20 mm in diameter) preovulatory follicles, as well as from a pool of immature small yellow follicles (6–8 mm in diameter). The cells were incubated in culture wells coated with type IV collagen or in wells without collagen coating, and the amounts of fibronectin produced were measured using a specific ELISA. The total amount of fibronectin produced by unstimulated cells was greatest in wells containing F1 cells and increased with time. The amount of fibronectin deposited by unstimulated cells was greatest in wells containing F1 cells and was much higher in collagen-coated wells than in uncoated wells. Both EGF and TGF-α increased the quantity of fibronectin deposited by granulosa cells in collagen-coated and uncoated wells. Fibronectin secreted into the medium by unstimulated cells also increased with the stage of follicular maturation and was enhanced by EGF and TGF-α. The quantity of cell-associated fibronectin in granulosa cells in collagen-coated and uncoated wells was also increased by these growth factors. Type IV collagen did not have any appreciable effect on the amount of fibronectin present in the incubation medium or on cell-associated fibronectin. Because of its marked effect on deposited fibronectin, there were greater total quantities of fibronectin in culture wells coated with type IV collagen. These results demonstrate that EGF and TGF-α stimulate the production and deposition of fibronectin by chicken granulosa cells. The results also suggest that in combination with collagen IV, these growth factors can regulate the formation of the extracellular matrix (for example, basal lamina) of the hen ovarian follicle.
B. L. Marrone and E. K. Asem
Summary. LH was used to stimulate cAMP production in theca cells from the 5 largest preovulatory follicles of hens and this was related to LH-stimulated androstenedione production in the same cells. cAMP production was stimulated by LH to the same extent in theca cells from each follicle. However, LH was not effective in stimulating androstenedione production in theca cells from the largest follicle (T1), although androstenedione production was greatly increased by LH in the smaller follicles (T2–T5). Effects similar to those of LH on cAMP production were observed in response to forskolin, indicating that the intrinsic adenylate cyclase activity was similar in theca cells from each follicle. In addition, forskolin was unable to stimulate androstenedione production by T1 cells. Our results provide evidence that the levels of receptor-mediated and non-receptor-mediated cAMP production are similar in theca cells from the 5 largest follicles. We conclude that the step that restricts the ability of T1 cells to produce androgen is distal to cAMP generation.
Keywords: cAMP; theca; LH; follicular maturation; domestic hen
B. K. Tsang, D. F. Mattice, M. Li and E. K. Asem
Summary. The gonadotrophic regulation of progesterone production by rat granulosa cells was examined in a chemically-defined medium containing FSH, dibutyryl cyclic AMP ((Bu)2cAMP) and the calcium ionophore, A23187. FSH and A23187 alone significantly enhanced the production of pregnenolone, progesterone and its metabolite, 20α-hydroxypregn-4-en-3-one (20α-OH-P) from endogenous substrate(s). Stimulation of progesterone production by A23187 was accompanied by an increase in 3β-hydroxysteroid dehydrogenase (3β-HSD) but not 20α-hydroxysteroid dehydrogenase (20α-HSD) activity, as attested by enhancement of the metabolism of exogenous pregnenolone to progesterone but not of progesterone to 20α-OH-P. In contrast, although (Bu)2cAMP increased pregnenolone and progesterone production and the metabolism of exogenous progesterone to 20α-OH-P, it failed to stimulate the conversion of exogenous pregnenolone to progesterone. The increase in progesterone production and in the conversion of exogenous pregnenolone to progesterone by FSH and A23187 was concentration- and time-dependent. Whereas maximal stimulation of de-novo progesterone synthesis by FSH was evident by 6 h (earliest time examined), a significant increase in the conversion of exogenous pregnenolone to progesterone in the presence of FSH or the ionophore was not noted until 12 h of incubation. Although a small but significant increase in progesterone production was also noted as early as 6 h of incubation in the presence of the calcium ionophore, this was markedly smaller than that elicited by FSH.
We conclude that the calcium ionophore A23187 and (Bu)2cAMP have similar as well as distinct effects on progesterone production in rat granulosa cells in vitro. We suggest that, while cAMP may be involved in the more rapid control by gonadotrophin of the production of the steroid via increased synthesis of pregnenolone and/or its metabolism to 20α-OH-P, calcium may be important in the synthesis and metabolism of pregnenolone for the maintenance of steroidogenic capacity of the granulosa cells.
Keywords: calcium; cAMP; 3β-hydroxysteroid dehydrogenase; 20α-hydroxysteroid dehydrogenase; steroidogenesis; granulosa cell; gonadotrophin action; rat