Regulation of ovulation rate in mammals: contribution of sheep genetic models

Abstract

Ovarian folliculogenesis in mammals from the constitution of primordial follicles up to ovulation is a reasonably well understood mechanism. Nevertheless, underlying mechanisms that determine the number of ovulating follicles were enigmatic until the identification of the fecundity genes affecting ovulation rate in sheep, bone morphogenetic protein-15 (BMP-15), growth and differentiation factor-9 (GDF-9) and BMP receptor-1B (BMPR-1B). In this review, we focus on the use of these sheep genetic models for understanding the role of the BMP system as an intra-ovarian regulator of follicular growth and maturation, and finally, ovulation rate.

Figure 1

Immunostaining for BMPR-1B in sheep ovary. Photomicrographs of primordial (a, e), primary (b, f), small antral (c, g) and large antral follicles (d, h). The photomicrographs (a-d) correspond to immunostaining with anti-BMPR-1B rabbit polyclonal antibody (1/1000; R&D Systems). The photomicrographs (e-h) correspond to immunostaining with control rabbit IgG.

Figure 2

Progesterone secretion by sheep granulosa cells. Granulosa cells from small antral follicles of 1–3 mm in diameter were cultured for 96 h in serum free media in the presence or absence of FSH (10ng/ml), in combination with varying concentrations of recombinant human BMP-2, BMP-4, BMP-6 (each from R&D Systems), BMP-15 [46] or rat GDF-9 [76]. Each combination of treatments was tested in triplicate in each of 4 independent experiments. Results represent progesterone secretion by 50,000 granulosa cells between 48 h and 96 h of culture. Data are expressed as percentages (mean ± SEM) of the amount of progesterone secreted by cells cultured in control condition. Different letters denote significant difference (p < 0.05).

Figure 3

³H-thymidine incorporation into sheep granulosa cells. Granulosa cells from small antral follicles of 1–3 mm in diameter were incubated with ³H-thymidine for 2 h after 48 h of culture in the presence of 3% fetal ovine serum with or without recombinant human BMP-2 (100 ng/ml), BMP-4 (50 ng/ml), BMP-6 (100ng/ml), BMP-15 (200 ng/ml) or rat GDF-9 (1000 ng/ml). Results represent the labeling index (percentage of ³H-thymidine-labelled cells). Data are expressed as percentages (mean ± SEM) of the labeling index of cells cultured in control condition. Asterisk indicates significant difference (p < 0.05) compared with control.

Figure 4

Schematic representation of the effects of a mutation in fecundity (Fec) gene on folliculogenesis and ovulation rate in sheep. The reduced activity of the BMP signaling system in the ovary of mutated Fec gene carrier (right) compared to non-carrier (left) ewes leads to decrease both the positive action of BMP on granulosa cell (GC) mitosis and its inhibiting action on FSH sensitivity. The consequence is the presence of smaller antral follicles with a reduced number of granulosa cells exhibiting a higher FSH sensitivity, leading to an advance in follicular maturation as attested by precocious LH receptor expression. The smaller matured follicles present in mutated Fec gene carrier each produce reduced amounts of oestradiol (E2) and inhibin, but altogether they produce the same amounts than one larger wild-type follicle. Consequently, the same endocrine dialog can establish between the ovaries and the central nervous system of both genotypes that leads to the selection and ovulation of numerous smaller follicles in mutated Fec gene carrier.

Figure 5

Relationship between BMP system activity and ovulation rate in sheep: working hypothesis. Increased ovulation rate is associated with loss of function mutations in the BMP system with additive or multiplicative effects of the mutated FecX, FecG or FecB genes. Thus, the relationship is based on the concept that ''the more the BMP system activity is reduced, the more the ovulation rate increases''. However, when the level of BMP activity is too low, folliculogenesis is blocked at early stages, leading to sterility as observed in homozygous FecX^mut (mut for alleles I, H, B, G or L) or homozygous FecG^H ewes. At the opposite, over-activity of the BMP system may lead to anovulation, then sterility, due to intensive inhibition of gonadotropin action. It is also hypothesized that crossbreeding between the 3 known mutated Fec genes might lead either to over-increased ovulation rate or to sterility.