In mammals, adiponectin and its receptors (AdipoR1 and AdipoR2) mRNAs are expressed in various tissues. However, the cellular expression and the role of adiponectin system have never been investigated in rat ovary. Here, we report the presence of adiponectin, AdipoR1 and AdipoR2 in rat ovaries, and we have investigated its role in granulosa cells. Using RT-PCR and western blot, we show that the mRNAs and proteins for adiponectin, AdipoR1 and AdipoR2 are found in the ovaries. Immunohistochemistry localized adiponectin, AdipoR1 and AdipoR2 in theca-interstitial T-I cells, corpus luteum, oocyte and less abundantly in granulosa cells. In the KGN human granulosa cell line, adiponectin mRNA and protein were undetectable; AdipoR2 was weakly expressed, whereas AdipoR1 was clearly present. Human chorionic gonadotrophin (hCG) injection (48 h) after pregnant mare serum gonadotrophin (PMSG) injection (24 h) in immature rats increased the level of adiponectin (protein) by about threefold (P!0.05) and those of AdipoR1 by threefold (mRNA, P!0.05) and 1.5-fold (protein, P!0.05) in ovary, whereas the mRNA and protein levels of AdipoR2 were unchanged. Interestingly, hCG injection (48 h) after the PMSG treatment (24 h) decreased plasma adiponectin levels and increased insulin plasma levels. In vitro in primary rat granulosa cells, human adiponectin recombinant (5 mg/ml) in the presence or absence of follicle-stimulating hormone (10 K8 M, 48 h) had no effect on the steroidogenesis. However, it increased progesterone secretion (P!0.05) by about twofold and oestradiol production (P! 0.05) by about 1.6-fold in response to insulin-like growth factor-I (IGF-I) (10 K8 M). Furthermore, it improved IGF-I-induced IGF-I receptor-b subunit tyrosine phosphorylation and ERK1/2 phosphorylation. In basal state, human adiponectin recombinant also increased rapidly but transiently the ERK1/2, p38 and Akt phosphorylations, whereas it increased more lately the adenosine 5 0 -monophosphate-activated protein kinase (AMPK) phosphorylation. Thus, AdipoR1 and AdipoR2 are regulated by hCG treatment in rat ovary and adiponectin enhances IGF-I-induced steroidogenesis in granulosa cells.

In mammals, IGFs are important for the proliferation and steroidogenesis of ovarian cells. Metformin is an insulin sensitizer molecule used for the treatment of the infertility of women with polycystic ovary syndrome. It is, however, unclear whether metformin acts on ovarian cells. Adenosine 5' monophosphate-activated protein kinase (AMPK) is involved in metformin action in various cell types. We investigated the effects of metformin on bovine granulosa cell steroidogenesis in response to IGF1 and FSH, and studied AMPK in bovine ovaries. In granulosa cells from small follicles, metformin (10 mM) reduced production of both progesterone and estradiol and decreased the abundance of HSD3B, CYP11A1, and STAR proteins in presence or absence of FSH (10(-8) M) and IGF1 (10(-8) M). In cows, the different subunits of AMPK are expressed in various ovarian cells including granulosa and theca cells, corpus luteum, and oocytes. In bovine granulosa cells from small follicles, metformin, like AICAR (1 mM) a pharmaceutical activator of AMPK, increased phosphorylation of both Thr172 of AMPK alpha and Ser 79 of ACACA (Acetyl-CoA Carboxylase). Both metformin and AICAR treatment reduced progesterone and estradiol secretion in presence or absence of FSH and IGF1. Metformin decreased phosphorylation levels of MAPK3/MAPK1 and MAPK14 in a dose- and time-dependent manner. The adenovirus-mediated production of dominant negative AMPK abolished the effects of metformin on secretion of progesterone and estradiol and on MAPK3/MAPK1 phosphorylation but not on MAPK14 phosphorylation. Thus, in bovine granulosa cells, metformin decreases steroidogenesis and MAPK3/MAPK1 phosphorylation through AMPK activation.

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