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KMID : 0350519960490020633
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Journal of Catholic Medical College
1996 Volume.49 No. 2 p.633 ~ p.647
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The Effects of Mullerian Inhibiting Substance on Steroidogenesis and Proliferation of Human Granulosa Cells
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Abstract
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Ovarian follicular growth is a consequence of granulosa cell proliferation, and steroid production by these cells appears to be a major determinant of the endocrine microenvironment of ovum maturation. The gonadotropins, PSH and LH, regulated
directly
the growth and differentiation of the granulosa cells in the ovary, but there is evidence to suggest that the gonadotropins act partly through locally produced growth factors and that this interaction is complex. A number of growth factors, such
as
epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and transforming growth factor-β(TGF-β) are produced in the ovarian follicle and might act in an autocrine and/or paracrine manner to directly control granulosa cell
proliferation
and differentiation. While it has been demonstrated that steroid hormones, gonadotropins and growth factors regulate proliferation and differentiation of ovarian follicles, little is known concerning the factors involved in the inhibition of
ovarian
function.
Recently, Mullerian inhibiting substance (MIS), a non-steroidal testicular Sertoli cell product responsible for the regression of Mullerian duct in male embryo, has been shown to be produced by ovarian granulosa cells in adolescent and adult
females.
Although the function of MIS in the ovary has not been fully delineated, MIS appears to be a regulator of oocyte maturation and follicular development in the rat.
In this study, in order to elucidate the precise timing and locale of MIS immunoreactivity in ovaries from adult normal cycling women and in order to investigate the influence of MIS on steroidogenesis and proliferation of human granulosa cells,
we
performed immunohistochemical staining using the polyclonal anti-human MIS antibody and culture of human granulosa cells. The cells were cultured for 2 to 12 days under two conditions, with and with and without MIS (20 ng/ml). Each condition was
additionally defined by the presence and absence of EGF (20 ng/ml), FSH (10 ng/ml), or LH (10 ng/ml). Progesterone, and testosterone were measured from the spent media by radioimmunoassay and the cell number was determined by trypsinizing the
cells
and
counting them with a Coulter counter.
@ES The results were as follows:
@EN 1. MIS was detected specifically and exclusively in the cytoplasm of granulosa cells in growing follicles by immunohistochemical staining. MIS staining waned in the mature follicle just before ovulation and could not be found in atretic
follicle,
corpus luteum and corpus albicans.
2. There was about 6-fold increase in the final granulosa cell number when the culture were maintained for 12 days in Ham's F-10 supplemented with 10% MIS-free female fetal calf serum (control). FSH and EGF caused a significant increase in
granulosa
cell number compared with the control but LH significantly suppressed cell number after 8 days in culture.
3. MIS caused a significant decrease in granulosa cell number compared with the control after 8 days in culture in the 20ng/ml dose, and on day 12 in the 2ng/ml dose (P<0.05). MIS(20ng/ml) also significantly blocked induced proliferation of
granulosa
cells after 6 days in culture and the maximum effect was observed on day 12 of culture (P<0.01).
4. Estradiol production from granulosa cells was significantly stimulated by both gonadotropins, with LH having the greater effect than FSH after 6 days in culture (P<0.05). However, EGF significantly suppressed estradiol production of granulosa
cells
during 4 days in culture. MIS had no effect on basal estradiol production of granulosa cells, but significantly decreased FSH induced estradiol production of granulosa cells after 4 days in culture (O<0.05).
5. Progesterone production from granulosa cells was significantly stimulated by both gonadotropins and EGF, with LH having the greatest effect. However, MIS had no effect on basal progesterone production of granulosa cells, but significantly
decreased
EGF induced progesterone production on granulosa cells after 4 days in culture (P<0.05) with having the greatest effect on day 10 of culture (P<0.01).
6. Testosterone production from granulosa cells was not detected in all conditions.
In conclusion, these experiments demonstrate that the human granulosa cells produce MIS and suggest that MIS may have relevant function in the ovary as a regulator of follicular development and oocyte maturation during the adult reproduction
cycle.
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KEYWORD
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