Localization and quantification of binding sites for follicle-stimulating hormone, luteinizing hormone, growth hormone, and insulin-like growth factor I in sheep ovarian follicles

Abstract

In sheep, growth and development of ovarian follicles beyond 2 mm in diameter is acutely dependent on gonadotropin support. As a consequence, following hypophysectomy (HPX) or hypothalamic-pituitary stalk disconnection (HPD), growth of follicles beyond 2 mm is arrested and all follicles > 2 mm undergo atresia. Although administration of exogenous gonadotropins stimulates follicular growth and ovulation in HPD ewes, follicles in HPX ewes remain unresponsive unless growth hormone (GH) is also given. To determine whether the difference in follicular sensitivity to gonadotropins after HPD (gonadotropin sensitive) or HPX (gonadotropin insensitive) is related to the distribution and quantity of binding sites for FSH, LH, and/or insulin-like growth factor I (IGF-I), binding sites for these hormones were localized and quantified using topical autoradiography in healthy follicles from control (pituitary-intact), HPD, and HPX ewes. In addition, in situ hybridization was performed to localize mRNA for GH and FSH receptors. Irrespective of treatment, binding of FSH and mRNA for FSH receptor were greatest (p < 0.05) in the membrana granulosa; LH binding was greatest (p < 0.05) in the theca interna; and IGF-I binding was greatest (p < 0.05) in the theca externa. Although the relative number of binding sites for LH did not differ among treatments, those for FSH and IGF-I were lower (p < 0.05) in HPD and HPX ewes compared to controls. Attempts to quantify binding sites for GH were unsuccessful due to high nonspecific binding. However, mRNA for GH receptor was most abundant (p < 0.05) in the membrana granulosa and oocytes of small antral and preantral follicles. Compared to levels in controls and HPD ewes, the level of GH receptor mRNA was lower (p < 0.05) in follicles obtained from HPX ewes. On the basis of these data, failure of small antral follicles in HPX ewes to respond to exogenous gonadotropins is not due to a reduction in receptors for FSH, LH, or IGF-I. The observed reduction of mRNA for GH receptor in the membrana granulosa of follicles from HPX ewes provides evidence that GH may play an important role in early stages of folliculogenesis and that it is involved in the maintenance of sensitivity to gonadotropins.