In vitro effects of inhibin on apoptosis and apoptosis related proteins in human ovarian granulosa cells.
OBJECTIVE
To clarify the in vitro effects of inhibin A (I) on apoptotic cell death and its mechanisms in ovarian granulosa cells the immunoexpression patterns of the apoptosis markers caspase-3 and pro- and anti-apoptotic proteins (Bcl-2, Bcl-xl, Bak) were evaluated in ovarian granulosa cells collected from women with different hormonal status.


METHODS
Granulosa cells were isolated from follicles of women participating in an in vitro fertilization (IVF) program, normally cyclic (NC) and premenopausal women (PrM). The obtained cells were cultured for 72 h with inhibin A (Sigma, USA)--10 ng/ml. The concentration of estradiol in the culture medium was determined by radioimmunoassay using the Coat-A-Count kit (Nippon, Japan), whose intra- and interassay coefficients of variations were 6,8% and 6,2% respectively. The expression of caspase-3, Bak, Bcl-2, Bcl-xl was investigated immunohistochemically. The percentages of immunopositive cells were calculated and Student's t-test was used for statistical analysis.


RESULTS
Addition of inhibin A (10 ng/ml) to granulosa cells cultures resulted in increased estradiol production. Maximal stimulation was observed in granulosa cells collected from women participating in IVF whereas minimal effect of inhibin treatment on estradiol production was demonstrated in premenopausal women. Inhibin A exposition enhanced the immunoexpression of prooncogenes (Bcl-2, Bcl-xl) and reduced the expression of caspase-3 and pro-apoptotic protein Bak in ovarian granulosa cells from the three experimental groups.


CONCLUSIONS
Our findings suggest that inhibin A in vitro stimulates the estradiol secretion by granulosa cells dependently of the woman hormonal status, while it inhibits apoptotic process in granulosa cells independently of the hormonal status.
Denkova
R
R
Institute of Experimental Morphology and Anthropology, BAS 1113 Sofia, Bulgaria. rdenkova@hotmail.com
Bourneva
V
V
Staneva-Dobrovski
L
L
Zvetkova
E
E
Baleva
K
K
Yaneva
E
E
Nikolov
B
B
Ivanov
I
I
Simeonov
K
K
Timeva
T
T
Yankov
M
M
D000328:Adult; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002478:Cells, Cultured; D004958:Estradiol / Q000378:metabolism; D005260:Female; D006107:Granulosa Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007265:Inhibins / Q000494:pharmacology*; D008565:Membrane Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D011863:Radioimmunoassay; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051020:bcl-X Protein
C494772:BAK1 protein, human; C494811:BCL2L1 protein, human; D008565:Membrane Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051020:bcl-X Protein; C422362:inhibin A; D004958:Estradiol; D007265:Inhibins; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases