Progesterone Secretion by Granulosa Cells from Different Sized Follicles of Human Ovaries after Short Term Incubation*

Abstract

Studies on the ability of granulosa to secrete progesterone after hormonal treatment have not addressed the possibility of variations in response associated with the stage of follicular development. Accordingly, granulosa were isolated from follicular fluid obtained from apparently healthy ovaries of patients undergoing tubal bypass. Follicle size was estimated by laparoscopic and ultrasonic measurements, and granulosa cells were prepared by centrifugation and treatment with EGTA and hypertonic sucrose solution to minimize aggregation. Cell viability was determined and 10(6) cells/ml medium 199D were incubated for 24 h without or with ovine LH (1 microgram/ml) or 17 beta-estradiol (5 micrograms/ml). Progesterone secretion by granulosa increased from 6 to 30 ng/24 h/10(6) cells and correlated with follicle sizes of 5--25 mm. Compared to untreated cells obtained from the same follicle, granulosa from follicles ranging from 5--15 mm secreted 2.5 times more progesterone when treated with LH. This effect of LH diminished with further increases in follicle size, with LH having no effect on cells from follicles larger than 20 mm. Compared to untreated cells from the same follicles, 17 beta-estradiol caused a progressive inhibition of progesterone secretion by granulosa that correlated with follicle sizes between 5--18 mm, with almost 60% inhibition occurring in cells from 18-mm follicles. Thereafter, the inhibitory effect of 17 beta-estradiol diminished, and no effect was found in granulosa from follicles larger than 22 mm. These results indicate that LH and 17 beta-estradiol have maximal in vitro effects on progesterone secretion by granulosa from follicles with diameters of 15--18 mm. The change in response to LH and 17 beta-estradiol observed in samples from larger follicles may be due to the fact that these cells already have maximal binding of LH and 17 beta-estradiol to their receptors. Alternatively, it is possible that cells from larger follicles are desensitized to LH and 17 beta-estradiol.

Characterization of long-term monolayer cultures of human granulosa cells from follicles of different size and exposed in vivo to clomiphene citrate and hCG

“…The apparent discrepancy between our present findings and those of Bieszczad et al (1982) concerning the progesterone responsiveness of cells from small compared to large preovulatory follicles may actually be a function of the length of stimulation in vitro. In the 24-h incubation studies (Bieszczad et al, 1982), only granulosa cells from follicles <20 mm responded to ovine LH (1 µg/ml) with enhanced progesterone production, and the authors suggested that the inability of LH to affect progesterone secretion by cells from larger follicles might be due to a maximal binding of LH to the receptors at the time of culture or to desensitization. Our studies of 15-25-mm follicles tend to support the former hypothesis during the first 2 days of culture, but are not consistent with a continued down regulation.…”

Effects of oestrogen on progesterone synthesis and arachidonic acid metabolism in human luteal cells

“…Our results indicate that oestradiol affects luteal progesterone biosynthesis directly and acutely by inhibiting the activities of 3B-HSD/AS-4-isomerase, the enzyme that catalyses oxidation of pregnenolone to progesterone. This is in accord with evidence that oestrogen inhibits the 3/3-HSD/ isomcrase system in microsomes of human placenta and CL of pregnancy (Depp et al, 1973) and in cultured human granulosa cells (Bieszczad et al, 1982;Veldhuis et al, 1983). Oestradiol action at this site is also consistent with earlier work suggesting that oestradiol exerts its inhibitory effect at a step(s) beyond the accumulation of CAMP (Williams et al, 1979).…”

“…The possibility that oestradiol is a locally produced luteolysin is supported by the finding that oestradiol inhibits progesterone accumulation by granulosa-lutein cells in uitro (Bieszczad et al, 1982). It has been suggested that the direct inhibitory effect of oestradiol on luteal cell steroidogenesis could involve inhibition of the 3/3-hydroxysteroid dehydrogenase (3P-HSD) enzyme system (Depp et al, 1973) or a reduction in the LH binding capacity of luteal cells (Sotrel et al, 1981).…”

Effects of androgen on 17.BETA.-estradiol production by cultured human granulosa cells.

“…Also, granulosa cells morphologically differentiate from autosynthetic cells into steroid-secreting cells around the time of the LH surge (Wada et al 1986), and the amount of gonadotropin receptors increases markedly along with this change (Richards and Midgley 1976). Variations in the reaction of granulosa cells to steroid according to the maturity of the sampled cells have also been reported by Jarrell and Belbeck (1985) and Bieszczad et al (1982).…”