Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 Are Essential for Ovarian Follicular Development in Sheep1

Abstract

The aim of this study was to test the hypothesis that both growth differential factor 9 (GDF9) and bone morphogenetic protein (BMP15; also known as GDF9B) are essential for normal ovarian follicular development in mammals with a low ovulation rate phenotype. Sheep (9-10 per group) were immunized with keyhole limpet hemocyanin (KLH; control), a GDF9-specific peptide conjugated to KLH (GDF9 peptide), a BMP15-specific peptide conjugated to KLH (BMP15 peptide), or the mature region of oBMP15 conjugated to KLH (oBMP15 mature protein) for a period of 7 mo and the effects of these treatments on various ovarian parameters such as ovarian follicular development, ovulation rate, and plasma progesterone concentrations evaluated. Also in the present study, we examined, by immunohistochemistry, the cellular localizations of GDF9 and BMP15 proteins in the ovaries of lambs. Both GDF9 and BMP15 proteins were localized specifically within ovarian follicles to the oocyte, thereby establishing for the sheep that the oocyte is the only intraovarian source of these growth factors. Immunization with either GDF9 peptide or BMP15 peptide caused anovulation in 7 of 10 and 9 of 10 ewes, respectively, when assessed at ovarian collection. Most ewes (7 of 10) immunized with oBMP15 mature protein had a least one observable estrus during the experimental period, and ovulation rate at this estrus was higher in these ewes compared with those immunized with KLH alone. In both the KLH-GDF9 peptide- and KLH-BMP15 peptide-treated ewes, histological examination of the ovaries at recovery (i.e., approximately 7 mo after the primary immunization) showed that most animals had few, if any, normal follicles beyond the primary (i.e., type 2) stage of development. In addition, abnormalities such as enlarged oocytes surrounded by a single layer of flattened and/or cuboidal granulosa cells or oocyte-free nodules of granulosa cells were often observed, especially in the anovulatory ewes. Passive immunization of ewes, each given 100 ml of a pool of plasma from the GDF9 peptide- or BMP15 peptide-immunized ewes at 4 days before induction of luteal regression also disrupted ovarian function. The ewes given the plasma against the GDF9 peptide formed 1-2 corpora lutea but 3 of 5 animals did not display normal luteal phase patterns of progesterone concentrations. The effect of plasma against the BMP15 peptide was more dramatic, with 4 of 5 animals failing to ovulate and 3 of 5 ewes lacking surface-visible antral follicles at laparoscopy. By contrast, administration of plasma against KLH did not affect ovulation rate or luteal function in any animal. In conclusion, these findings support the hypothesis that, in mammals with a low ovulation rate phenotype, both oocyte-derived GDF9 and BMP15 proteins are essential for normal follicular development, including both the early and later stages of growth.

Physiological effects of major genes affecting ovulation rate in sheep

“…The major effects on ovarian phenotype are a significant reduction in ovarian volume, no normal follicular development beyond the type 2 stage, few, if any type 5 (i.e. antral) follicles and significantly larger oocytes in the type 2-4 follicles in both the BMP15 and GDF9 groups [12]. This phenotype is similar to that observed in sheep with the BMP15 and GDF9 mutations.…”

“…Both GDF9 mRNA and protein are present in normal sheep oocytes in non-growing type 1 follicles (i.e. primordial follicles) as well as in growing follicles [3,12] (Fig. 1).…”

“…It was proposed that the mutations in the BMP15 gene in sheep may actually affect the level of GDF9 secretion and that abnormal concentrations of GDF9 are the cause of the Inverdale phenotype. However, when sheep are immunised against peptides specific for BMP15 or GDF9, the evidence is that secreted forms of BMP15 as well as GDF9 are essential for both normal follicular development and ovulation rate [12,18].…”

“…S31 preantral) stage of growth. It is worth noting that GDF9 mRNA and protein are present before follicular formation in sheep as well as in non-growing type 1 follicles in several species including sheep, cows and possum [3,8,12]. Thus, either the form of the protein is biologically inactive, the appropriate receptors are not present on the adjacent granulosa cells in type 1 follicles or that GDF9 is a relatively inactive growth factor when other companion factors are absent.…”

“…The aforementioned gene discoveries have led to a new paradigm in reproductive biology, namely that the oocyte plays a key role in regulating the ovulation rate [10][11][12]. Thus, sheep are proving to be remarkably informative in offering new insights into why some mammals have a predisposition to produce two or three offspring rather than one [18].…”