Growth differentiation factor-9

In many mammalian species GDF9 is essential for early follicular development through its direct action on the granulosa cells allowing proliferation and differentiation [7] The deletion of ‘’Gdf9’’ results in decreased ovary size, halted follicular development at the stage of the primary follicle and the absence of any corpus lutea.[11] The proliferative ability of granulosa cells is significantly reduced whereby no more than a single layer of granulosa cells is able to surround and thus support the developing oocyte.[7] Any somatic cell formation after the primary layer is atypical and asymmetrical.[11] Normally the follicle becomes atretic and degenerates although this does not occur emphasizing the abnormality of these supporting cells.[11] GDF9 deficiency is further linked with the up regulation of inhibin.[7] The normal expression of GDF9 allows the downregulation of inhibin a and thus promotes the ability of the follicle to progress past the primary stage of development.[12]

In vitro exposure of mammalian ovarian tissue to GDF9 promotes primary follicle progression.[13][14] GDF9 stimulates growth of preantral follicles by preventing granulosa cell apoptosis.[15] This may occur through increased follicle stimulating hormone (FSH) receptor expression or be a result of post-receptor signaling.[7]

Some sheep breeds show a range of fertility phenotypes due to eight single nucleotide polymorphisms (SNP) across the coding region of GDF9.[16] A SNP in the Gdf9 gene resulting in a non conservative amino acid change was identified, whereby ewes homozygous for the SNP were infertile and completely lacked any follicle growth.[17]

Typical of later stages of follicle development is the appearance of cumulus cells.[18] GDF9 causes the expansion of cumulus cells, a characteristic process in normal follicular development.[8] GDF9 induces hyaluronanic synthase 2 (Has2) and suppresses urokinase plasminogen activator (uPA) mRNA synthesis in granulosa cells.[18] This allows an extracellular matrix rich in hyaluronic acid, allowing the expansion of cumulus cells.[19] Silencing of GDF9 expression results in the absence of cumulus cell expansion, this highlights the integral role of GDF9 signaling in altering granulosa cell enzymes and therefore allowing cumulus cell expansion in late stages of folliculogenesis.[18][20]

A lack of GDF9 causes pathophysiological alterations in the oocyte itself in addition to severe follicular abnormality. Oocytes reach normal size and form a zona pellucida although organelles become clustered and cortical granules do not form.[11] In GDF9 deficient oocytes the meiotic ability is significantly altered, where less than half will proceed metaphase 1 or 2 and a large percentage of oocytes have abnormal germinal vesicle breakdown.[11] As cumulus cells surround the oocyte during development and remain with the oocyte once it is ovulated, GDF9 expression in cumulus cells is important in allowing an ideal oocyte microenvironment.[18] The altered phenotype observed in GDF9 deficient oocytes likely results from the lack off somatic cell input in later stages of folliculogenesis.[11]

GDF9 is required just prior to the surge of luteinizing hormone (LH), a key event responsible for ovulation.[7] Prior to the LH surge, GDF9 supports the metabolic function of cumulus cells, allowing glycolysis and cholesterol biosynthesis.[21] Cholesterol is a precursor of many essential steroid hormones such as progesterone. Progesterone levels rise significantly post ovulation to support the early stages of embryogenesis.[7] In preovulatory follicles, GDF9 promotes the production of progesterone via the stimulation of the prostaglandin- EP2 receptor signaling pathway.[22]

GDF9 mutations are present in women with premature ovarian failure, in addition to mothers of dizygotic twins.[7][23] Three particular missense mutations GDF9 ^P103S, GDF9 ^P374L and GDF9 ^R454C have been found, although GDF9 ^P103S is present in women with dizygotic twins as well as women with premature ovarian failure.[7] Given the same mutation is linked with a poly ovulatory phenotype and the failure of ovulation, these mutations are thought to alter the rate of ovulation, rather than specifically increasing or decreasing the rate.[7] Most of these mutations are located in the pro-region of the gene that encodes GDF9, an area essential for the dimerization and hence activation of the encoded protein.[24][25]

PCOS accounts for approximately 90% of anovulation infertility, affecting 5-10% of woman of reproductive age.[26] In women with PCOS, GDF9 mRNA is decreased in all stages of follicular development compared to women without PCOS.[7] In particular, levels of GDF9 increase as the follicle develops from primordial stages to more mature stages.[27] Women with PCOS have considerably lower expression of GDF9 in primordial, primary and secondary stages of folliculogenesis.[27] GDF9 expression is not only reduced in women with PCOS but also delayed.[27] Despite these facts the exact link of GDF9 with PCOS is not well established.[7]