Abstract
BackgroundReproductive dysfunction in the diabetic female rat is associated with altered folliculogenesis and steroidogenesis. However, the molecular mechanisms involved in the reduction of steroid production have not been described. Adiponectin is an adipocytokine that has insulin-sensitizing actions including stimulation of glucose uptake in muscle and suppression of glucose production in liver. Adiponectin acts via two receptor isoforms – AdipoR1 and AdipoR2 – that are regulated by hyperglycaemia and hyperinsulinaemia in liver and muscle. We have recently identified AdipoR1 and AdipoR2 in rat ovary. However, their regulation in ovaries of diabetic female rat remains to be elucidated.MethodsWe incubated rat primary granulosa cells in vitro with high concentrations of glucose (5 or 10 g/l) + or - FSH (10-8 M) or IGF-1 (10-8 M), and we studied the ovaries of streptozotocin-induced diabetic rats (STZ) in vivo. The levels of oestradiol and progesterone in culture medium and serum were measured by RIA. We used immunoblotting to assay key steroidogenesis factors (3beta HSD, p450scc, p450 aromatase, StAR), and adiponectin receptors and various elements of signalling pathways (MAPK ERK1/2 and AMPK) in vivo and in vitro. We also determined cell proliferation by [3H] thymidine incorporation.ResultsGlucose (5 or 10 g/l) impaired the in vitro production in rat granulosa cells of both progesterone and oestradiol in the basal state and in response to FSH and IGF-1 without affecting cell proliferation and viability. This was associated with substantial reductions in the amounts of 3beta HSD, p450scc, p450 aromatase and StAR proteins and MAPK ERK1/2 phosphorylation. In contrast, glucose did not affect the abundance of AdipoR1 or AdipoR2 proteins. In vivo, as expected, STZ treatment of rats caused hyperglycaemia and insulin, adiponectin and resistin deficiencies. Plasma progesterone and oestradiol levels were also reduced in STZ rats. However, the amounts of 3beta HSD and p450 aromatase were the same in STZ rat ovary and controls, and the amounts of StAR and p450scc were higher. Streptozotocin treatment did not affect adiponectin receptors in rat ovary but it increased AMPK phosphorylation without affecting MAPK ERK1/2 phosphorylation.ConclusionHigh levels of glucose decrease progesterone and oestradiol production in primary rat granulosa cells and in STZ-treated rats. However, the mechanism that leads to reduced ovarian steroid production seems to be different. Furthermore, adiponectin receptors in ovarian cells are not regulated by glucose.
Highlights
Reproductive dysfunction in the diabetic female rat is associated with altered folliculogenesis and steroidogenesis
We report an investigation of ovarian steroid production and the protein levels of key factors involved in steroidogenesis in two conditions: in primary rat granulosa cells in the presence of high glucose concentrations; and in streptozotocin-treated female rats
A high glucose concentration (5 or 10 g/l) decreased both basal and FSH-or IGF-1-stimulated progesterone and oestradiol production in rat granulosa cells. We investigated whether this inhibitory effect of glucose on the production of both progesterone and oestradiol resulted from the production of smaller amounts of the three key enzymes in steroidogenesis (3βHSD, p450scc and p450 aromatase) and/or of steroidogenic acute regulatory protein (StAR), a major cholesterol carrier
Summary
Reproductive dysfunction in the diabetic female rat is associated with altered folliculogenesis and steroidogenesis. Several studies have shown altered ovarian function in association with diabetes, the molecular alterations in ovarian steroid metabolism that could explain these reproductive dysfunctions remain to be elucidated. We have recently shown that the activation of AMPK, a key regulator of cellular energy homeostasis, reduces progesterone secretion through inhibition of the MAPK ERK1/2 signalling pathway in rat and bovine granulosa cells [13,14]. It remains to be determined whether diabetes is associated with abnormalities in the abundance of ovarian p450scc, 3βHSD, StAR, p450 aromatase or in MAPK ERK1/2 and AMPK phosphorylations
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