Functional significance of the signal transduction pathways Akt and Erk in ovarian follicles: in vitro and in vivo studies in cattle and sheep
ABSTRACT The intracellular signalling mechanisms that regulate ovarian follicle development are unclear; however, we have recently shown differences in the Akt and Erk signalling pathways in dominant compared to subordinate follicles. The aim of this study was to investigate the effects of inhibiting Akt and Erk phosphorylation on IGF- and gonadotropin- stimulated granulosa and theca cell function in vitro, and on follicle development in vivo.
Bovine granulosa and theca cells were cultured for six days and stimulated with FSH and/or IGF, or LH in combination with PD98059 (Erk inhibitor) and/or LY294002 (Akt inhibitor) and their effect on cell number and hormone secretion (estradiol, activin-A, inhibin-A, follistatin, progesterone and androstenedione) determined. In addition, ovarian follicles were treated in vivo with PD98059 and/or LY294002 in ewes on Day 3 of the cycle and follicles were recovered 48 hours later.
We have shown that gonadotropin- and IGF-stimulated hormone production by granulosa and theca cells is reduced by treatment with PD98059 and LY294002 in vitro. Furthermore, treatment with PD98059 and LY294002 reduced follicle growth and oestradiol production in vivo.
These results demonstrate an important functional role for the Akt and Erk signalling pathways in follicle function, growth and development.
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ABSTRACT: Follicle development is coordinated by gonadotropins, steroids, and growth factors, which activate multiple signaling pathways. Phosphorylated-MAPK (pMAPK) level was indicated as an early marker of follicle dominance, whereas phosphorylated STAT3 (pSTAT3) was increased in granulosa cells of hypophysectomized rats. We hypothesized that MAPK3/1 and STAT3 pathways are regulated in granulosa cells during follicle deviation in cattle. Cyclic beef cows were synchronized and ovariectomized to recover the two largest follicles. Follicular diameter did not differ on Day 2 but was significantly greater in dominant follicles (DFs) than that in subordinate follicles (SFs) on Days 3 and 4 of the follicular wave. The elevated abundance of CYP19A1 mRNA expression in granulosa cells of DFs and cleaved caspase 3 in Day-4 SFs further validated our in vivo model. Before deviation, pMAPK3/1 levels were significantly higher in granulosa cells of the future DF. STAT3 mRNA and total protein (tSTAT3) were higher in granulosa cells of SFs collected on Day 4. Furthermore, levels of pSTAT3 were dramatically increased in granulosa cells of Day-4 SFs. In conclusion, pMAPK3/1 was increased in the future DF, but such differential abundance between the DF and SF was not evident after deviation. The higher abundance of pSTAT3 in granulosa cells of SFs after deviation suggests that this pathway may be involved in granulosa cell death and follicular atresia. Copyright © 2014 Elsevier Inc. All rights reserved.Theriogenology 09/2014; DOI:10.1016/j.theriogenology.2014.09.026 · 1.85 Impact Factor
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ABSTRACT: A short-term increase in food intake and specifically dietary energy can stimulate folliculogenesis and increase ovulation rate in ewes. The mechanism appears to involve the insulin-glucose metabolic system and its interaction with FSH signalling pathways in the granulosa cells of ovarian follicles. This experiment was designed to investigate the interaction between these two systems in the granulosa cells of ovarian follicles. Thirty six Ile-de-France ewes were used in this controlled experiment to study the effects of intravenous glucose on folliculogenesis. Eighteen ewes were infused with glucose (10 mM/h for 72 h) from day 8 of the oestrous cycle, while the others (controls) received saline. Ovaries were collected when the infusions ended (luteal phase) or 30 h later and after a luteolytic dose of a PGF2alpha analogue (follicular phase). Follicles were dissected and granulosa cells and follicular fluid harvested. The blood concentrations of glucose, insulin, oestradiol and FSH were monitored over the experiment. The levels of Aromatase P450 and of the phosphorylated and non-phosphorylated forms of Akt, AMPK and ERK in granulosa cells and the concentration of oestradiol in follicular fluid, were determined. Glucose increased the circulating concentration of glucose (P < 0.05) and insulin (P < 0.05). It also increased the total number of follicles >1.0 mm in diameter (P < 0.05) and small (P < 0.05) follicles (>1.0 to 2.0 mm in diameter) but not medium (>2.0 to 3.5 mm in diameter) or large (>3.5 mm in diameter) follicles. Glucose decreased circulating oestradiol (P < 0.05) but not that of FSH or progesterone. Glucose reduced Aromatase P450 in granulosa cell (P < 0.05) and it decreased the phosphorylation of Akt (P < 0.05), ERK (P < 0.05) and AMPK (P < 0.05) in granulosa cells. The level of Aromatase P450 was greatest in large oestrogenic follicles and the phosphorylation of Akt (P < 0.05), ERK (P < 0.05) and AMPK (P < 0.05) was lower in small follicles compared to medium and large follicles. These data suggest that the effect of glucose in small follicles is a direct action of glucose that increases the number of small follicles while the effect of glucose in oestrogenic follicles is an indirect insulin-mediated action.Reproductive Biology and Endocrinology 01/2015; 13(1):6. DOI:10.1186/1477-7827-13-6 · 2.41 Impact Factor
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ABSTRACT: Abnormalities in ovarian function including defective oogenesis and folliculogenesis represent a key female reproductive deficiency. Accumulating evidences in literature have shown that PI3K/PTEN/Akt and TSC/mTOR signaling pathways are critical regulators of ovarian function including quiescence, activation and survival of primordial follicles (PFs), granulosa cell (GC) proliferation and differentiation, and meiotic maturation of oocytes. Deregulation of these signaling pathways may contribute to infertility caused by impaired follicular development, intrafollicular oocyte development and ovulation. This article reviews the current state of knowledge on the functional role of PI3K/PTEN/Akt and TSC/mTOR pathways during mammalian oogenesis and folliculogenesis and their association with female infertility.Journal of Molecular Endocrinology 10/2014; 53(3). DOI:10.1530/JME-14-0220 · 3.62 Impact Factor