Growth differentiation factor-9

Growth/differentiation factor 9 is a protein that in humans is encoded by the ''GDF9'' gene.

Growth factors synthesized by ovarian somatic cells directly affect oocyte growth and function. Growth differentiation factor-9 (GDF9) is expressed in oocytes and is thought to be required for ovarian folliculogenesis. GDF9 is a member of the transforming growth factor-beta (TGFβ) superfamily.

Growth Differentiation Factor 9 (GDF9)

Growth differentiation factor 9 (GDF9) is an oocyte derived growth factor in the transforming growth factor β (TGF-β) superfamily.Otsuka, F., McTavish, K. and Shimasaki, S. (2011). Integral role of GDF-9 and BMP-15 in ovarian function. Mol. Reprod. Dev., 78(1), pp.9-21 It is highly expressed in the oocyte and has a pivotal influence on the surrounding somatic cells, particularly granulosa, cumulus and theca cells. Paracrine interactions between the developing oocyte and its surrounding follicular cells is essential for the correct progression of both the follicle and the oocyte.Castro, F., Cruz, M. and Leal, C. (2015). Role of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 in Ovarian Function and Their Importance in Mammalian Female Fertility — A Review. Asian Australas. J. Anim. Sci, 29(8), pp.1065-1074 GDF9 is essential for the overall process of folliculogenesis, oogenesis and ovulation and thus plays a major role in female fertility.

Signaling Pathway

GDF9 acts through two receptors on the cells surrounding the oocyte, it binds to bone morphogenic protein receptor 2 (BMPRII) and downstream to this utilizes the TGF-β receptor type 1 (ALK5).Gilchrist, R., Lane, M. and Thompson, J. (2008). Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality. Human Reproduction Update, 14(2), pp.159-177 Ligand receptor activation allows the downstream phosphorylation and activation of SMAD proteins. SMAD proteins are transcription factors found in vertebrates, insects and nematodes, and are the intercellular substrates of all TGF-β molecules. Huang, Q., Cheung, A., Zhang, Y., Huang, H., Auersperg, N. and Leung, P. (2009). Effects of growth differentiation factor 9 on cell cycle regulators and ERK42/44 in human granulosa cell proliferation. AJP: Endocrinology and Metabolism, 296(6), pp.E1344-E1353 GDF9 specifically activates SMAD2 and SMAD3 which form a complex with SMAD4, a common partner of all SMAD proteins, that is then able to translocate to the nucleus to regulate gene expression.

Role in Folliculogenesis

Early Follicle Development

In many mammalian species GDF9 is essential for early follicular development through its direct action on the granulosa cells allowing proliferation and differentiation 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.Dong, J., Albertini, D., Nishimori, K., Kumar, T., Lu, N. and Matzuk, M. (1996). Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature, 383(6600), pp.531-535 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. Any somatic cell formation after the primary layer is atypical and asymmetrical. Normally the follicle becomes atretic and degenerates although this does not occur emphasizing the abnormality of these supporting cells. GDF9 deficiency is further linked with the up regulation of inhibin. 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.Elvin, J., Yan, C., Wang, P., Nishimori, K. and Matzuk, M. (1999). Molecular Characterization of the Follicle Defects in the Growth Differentiation Factor 9-Deficient Ovary. Molecular Endocrinology, 13(6), pp.1018-1034

''In vitro'' exposure of mammalian ovarian tissue to GDF9 promotes primary follicle progression.Hreinsson, J., Scott, J., Rasmussen, C., Swahn, M., Hsueh, A. and Hovatta, O. (2002). Growth Differentiation Factor-9 Promotes the Growth, Development, and Survival of Human Ovarian Follicles in Organ Culture. The Journal of Clinical Endocrinology & Metabolism, 87(1), pp.316-321Nilsson, E. (2002). Growth and Differentiation Factor-9 Stimulates Progression of Early Primary but Not Primordial Rat Ovarian Follicle Development. Biology of Reproduction, 67(3), pp.1018-1024 GDF9 stimulates growth of preantral follicles by preventing granulosa cell apoptosis.Orisaka, M., Orisaka, S., Jiang, J., Craig, J., Wang, Y., Kotsuji, F. and Tsang, B. (2006). Growth Differentiation Factor 9 Is Antiapoptotic during Follicular Development from Preantral to Early Antral Stage. Molecular Endocrinology, 20(10), pp.2456-2468 This may occur through increased follicle stimulating hormone (FSH) receptor expression or be a result of post-receptor signaling.

Some sheep breeds show a range of fertility phenotypes due to eight single nucleotide polymorphisms (SNP) across the coding region of GDF9.Hanrahan, J. (2003). Mutations in the Genes for Oocyte-Derived Growth Factors GDF9 and BMP15 Are Associated with Both Increased Ovulation Rate and Sterility in Cambridge and Belclare Sheep (Ovis aries). Biology of Reproduction, 70(4), pp.900-909 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.Nicol, L., Bishop, S., Pong-Wong, R., Bendixen, C., Holm, L., Rhind, S. and McNeilly, A. (2009). Homozygosity for a single base-pair mutation in the oocyte-specific GDF9 gene results in sterility in Thoka sheep. Reproduction, 138(6), pp.921-933

Late Follicle Development

Typical of later stages of follicle development is the appearance of cumulus cells.Elvin, J., Clark, A., Wang, P., Wolfman, N. and Matzuk, M. (1999). Paracrine Actions Of Growth Differentiation Factor-9 in the Mammalian Ovary. Molecular Endocrinology, 13(6), pp.1035-1048 GDF9 causes the expansion of cumulus cells, a characteristic process in normal follicular development. GDF9 induces hyaluronanic synthase 2 (Has2) and suppresses urokinase plasminogen activator (uPA) mRNA synthesis in granulosa cells. This allows an extracellular matrix rich in hyaluronic acid, allowing the expansion of cumulus cells.Zhao, H., Qin, Y., Kovanci, E., Simpson, J., Chen, Z. and Rajkovic, A. (2007). Analyses of GDF9 mutation in 100 Chinese women with premature ovarian failure. Fertility and Sterility, 88(5), pp.1474-1476 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.Gui, L. (2005). RNA Interference Evidence That Growth Differentiation Factor-9 Mediates Oocyte Regulation of Cumulus Expansion in Mice. Biology of Reproduction, 72(1), pp.195-199

Role in Oogenesis and Ovulation

Role in Oogenesis

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. 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. 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. The altered phenotype observed in GDF9 deficient oocytes likely results from the lack off somatic cell input in later stages of folliculogenesis.

Role in Ovulation

GDF9 is required just prior to the surge of luteinizing hormone (LH), a key event responsible for ovulation. Prior to the LH surge, GDF9 supports the metabolic function of cumulus cells, allowing glycolysis and cholesterol biosynthesis.Sugiura, K., Pendola, F. and Eppig, J. (2005). Oocyte control of metabolic cooperativity between oocytes and companion granulosa cells: energy metabolism. Developmental Biology, 279(1), pp.20-30 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. In preovulatory follicles, GDF9 promotes the production of progesterone via the stimulation of the prostaglandin- EP2 receptor signaling pathway. Elvin, J., Yan, C. and Matzuk, M. (2000). Growth differentiation factor-9 stimulates progesterone synthesis in granulosa cells via a prostaglandin E2/EP2 receptor pathway. Proceedings of the National Academy of Sciences, 97(18), pp.10288-10293

Altered GDF9 Expression in Humans

Mutations in GDF9

GDF9 mutations are present in women with premature ovarian failure, in addition to mothers of dizygotic twins.Yan, C., Wang, P., DeMayo, J., DeMayo, F., Elvin, J., Carino, C., Prasad, S., Skinner, S., Dunbar, B., Dube, J., Celeste, A. and Matzuk, M. (2001). Synergistic Roles of Bone Morphogenetic Protein 15 and Growth Differentiation Factor 9 in Ovarian Function. Molecular Endocrinology, 15(6), pp.854-866 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. 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. 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.Laissue, P. (2006). Mutations and sequence variants in GDF9 and BMP15 in patients with premature ovarian failure. European Journal of Endocrinology, 154(5), pp.739-744Shimasaki, S., Moore, R., Otsuka, F. and Erickson, G. (2004). The Bone Morphogenetic Protein System In Mammalian Reproduction. Endocrine Reviews, 25(1), pp.72-101

Link with Polycystic Ovarian Syndrome (PCOS)

PCOS accounts for approximately 90% of anovulation infertility, affecting 5-10% of woman of reproductive age.de Resende, L., Vireque, A., Santana, L., Moreno, D., de Sá Rosa e Silva, A., Ferriani, R., Scrideli, C. and Reis, R. (2012). Single-cell expression analysis of BMP15 and GDF9 in mature oocytes and BMPR2 in cumulus cells of women with polycystic ovary syndrome undergoing controlled ovarian hyperstimulation. Journal of Assisted Reproduction and Genetics, 29(10), pp.1057-1065 In women with PCOS, GDF9 mRNA is decreased in all stages of follicular development compared to women without PCOS. In particular, levels of GDF9 increase as the follicle develops from primordial stages to more mature stages.Wei, L., Huang, R., Li, L., Fang, C., Li, Y. and Liang, X. (2014). Reduced and delayed expression of GDF9 and BMP15 in ovarian tissues from women with polycystic ovary syndrome. Journal of Assisted Reproduction and Genetics, 31(11), pp.1483-1490 Women with PCOS have considerably lower expression of GDF9 in primordial, primary and secondary stages of folliculogenesis. GDF9 expression is not only reduced in women with PCOS but also delayed. Despite these facts the exact link of GDF9 with PCOS is not well established.

Synergistic Interaction

Bone morphogenic protein 15 (BMP15) is highly expressed in the oocyte and the surrounding follicular cells contributing greatly to folliculogenesis and oogenesis. Like GDF9, BMP15 belongs to the TGF-β superfamily. Differences in the synergistic action of BMP15 and GDF9 appear to be species dependent. BMP15 and GDF9 act in an additive manner to increase mitotic proliferation in sheep granulosa cells, although the same effect is not observed in bovine granulosa cells.McNatty, K., Juengel, J., Reader, K., Lun, S., Myllymaa, S., Lawrence, S., Western, A., Meerasahib, M., Mottershead, D., Groome, N., Ritvos, O. and Laitinen, M. (2005). Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function in ruminants. Reproduction, 129(4), pp.481-487 The silencing of ‘’Bmp15’’ in mice results in partial fertility but normal histological appearance of the ovary. Although, when this is combined with the silencing of one allele of ‘’Gdf9’’, mice are completely infertile due to insufficient folliculogenesis and altered cumulus cell morphology. Mice with this genome also fail to release oocytes resulting in trapped oocytes in the corpus lutea. This phenotype is absent in ‘’Gdf9’’ silenced mice and only present a small population of ‘’Bmp15’’ silenced mice. This reveals the synergistic relationship of GDF9 and BMP15 whereby the silencing of both genes results in more severe outcome then either of the genes alone. It is thought that any co operative effects of GDF9 and BMP15 are modulated through the BMPRII receptor.Edwards, S., Reader, K., Lun, S., Western, A., Lawrence, S., McNatty, K. and Juengel, J. (2008). The Cooperative Effect of Growth and Differentiation Factor-9 and Bone Morphogenetic Protein (BMP)-15 on Granulosa Cell Function Is Modulated Primarily through BMP Receptor II. Endocrinology, 149(3), pp.1026-1030

GDF9 plays an important role in the development of primary follicles in the ovary. It has a critical role in granulosa cell and theca cell growth, as well as in differentiation and maturation of the oocyte.

GDF9 has been connected to differences in ovulation rate and in premature cessation of ovary function, therefore has a significant role in fertility.

The cell surface receptor through which GDF9 generates a signal is the bone morphogenetic protein type II receptor ( BMPR2).

References

Further reading

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{{TGFβ receptor superfamily modulators

Growth factors