Growth differentiation factor 9 reverses activin A suppression of steroidogenic acute regulatory protein expression and progesterone production in human granulosa-lutein cells.
BACKGROUND
We have reported that growth differentiation factor 9 (GDF9) can enhance activin A (β(A)β(A))-induced inhibin B (αβ(B)) secretion in human granulosa-lutein (hGL) cells, but its effects on steroidogenic acute regulatory protein (StAR), ovarian steroidogenic enzymes, and progesterone production are unknown. We undertook this study to further evaluate GDF9 in this regard.


METHODS
hGL cells from women undergoing in vitro fertilization treatment were cultured with and without small interfering RNA (siRNA) transfection targeted at inhibin α-subunit or GDF9 before treatment with GDF9, activin A, FSH, or combinations. We compared StAR, P450 side-chain cleavage enzyme, and 3β-hydroxysteroid dehydrogenase expression in hGL cells and progesterone levels in culture media after these treatments. mRNA, protein, and hormone levels were assessed with real-time RT-PCR, immunoblotting, and ELISA, respectively. Data were analyzed by ANOVA followed by Tukey's test.


RESULTS
Activin A alone reduced basal and FSH-induced progesterone production by decreasing the expression of StAR protein, which regulates the rate-limiting step in steroidogenesis but not P450 side-chain cleavage enzyme and 3β-hydroxysteroid dehydrogenase. GDF9 attenuated these activin A effects on StAR and progesterone. After transfection of α-subunit siRNA, activin A level increased (P < 0.001), whereas basal and activin A-induced inhibin B levels (with and without GDF9) decreased. Furthermore, the effects of GDF9 in reversing activin A suppression of progesterone production were attenuated (P < 0.001). Transfection of GDF9 siRNA decreased GDF9 as expected and led to lower StAR expression and progesterone secretion than those observed with activin A treatment alone.


CONCLUSIONS
GDF9 attenuates the suppressive effects of activin A on StAR expression and progesterone production by increasing the expression of inhibin B, which acts as an activin A competitor.
Shi
Feng-Tao
FT
Department of Obstetrics and Gynaecology, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada.
Cheung
Anthony P
AP
Klausen
Christian
C
Huang
He-Feng
HF
Leung
Peter C K
PC
D028341:Activins / Q000037:antagonists & inhibitors*; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D004336:Drug Antagonism; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D055429:Growth Differentiation Factor 9 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000494:pharmacology*; D006801:Humans; D007265:Inhibins / Q000037:antagonists & inhibitors / Q000235:genetics; D008184:Luteal Cells / Q000187:drug effects* / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology; D014162:Transfection; D000098682:Steroidogenic Acute Regulatory Protein
D055429:Growth Differentiation Factor 9; D010750:Phosphoproteins; D034741:RNA, Small Interfering; C422405:activin A; C094377:inhibin-alpha subunit; D000098682:Steroidogenic Acute Regulatory Protein; D028341:Activins; D011374:Progesterone; D007265:Inhibins