Inactivation of growth differentiation factor 9 blocks folliculogenesis in pigs

Abstract

Growth differentiation factor 9 (GDF9) is a secreted protein belonging to the transforming growth factor (TGF)-beta superfamily and has been well characterized for its role during folliculogenesis in the ovary. Although previous studies in mice and sheep have shown that mutations in GDF9 disrupt follicular progression, the exact role of GDF9 in pigs has yet to be elucidated. The objective of this study was to understand the role of GDF9 in ovarian function by rapidly generating GDF9 knockout (GDF9−/−) pigs by using the CRISPR/Cas9 system. Three single guide RNAs designed to disrupt porcine GDF9 were injected with Cas9 mRNA into zygotes, and blastocyst-stage embryos were transferred into surrogates. One pregnancy was sacrificed on day 100 of gestation to investigate the role of GDF9 during oogenesis. Four female fetuses were recovered with one predicted to be GDF9−/− and the others with in-frame mutations. All four had fully formed oocytes within primordial follicles, confirming that knockout of GDF9 does not disrupt oogenesis. Four GDF9 mutant gilts were generated and were grown past puberty. One gilt was predicted to completely lack functional GDF9 (GDF9−/−), and the gilt never demonstrated standing estrus and had a severely underdeveloped reproductive tract with large ovarian cysts. Further examination revealed that the follicles from the GDF9−/− gilt did not progress past preantral stages, and the uterine vasculature was less extensive than the control pigs. By using the CRISPR/Cas9 system, we demonstrated that GDF9 is a critical growth factor for proper ovarian development and function in pigs.

Dietary methionine supplementation during the estrous cycle improves follicular development and estrogen synthesis in rats

“…Knockout of GDF9 and BMP15 impairs mammalian follicle and oocyte development and reduces ovulation and fertilization rates. 57,58 In contrast, increased GDF9 and BMP15 expression appears to contribute to follicular development and granulosa cell proliferation, increasing the ovulation number and the oocyte fertilization rate. 59–63 Furthermore, our in vitro data demonstrated that the addition of 1 mM and 2 mM methionine enhanced follicle diameter increase.…”

The paralogs’ enigma of germ-cell specific genes dispensable for fertility: the case of 19 oogenesin genes

“…In females, several ubiquitously expressed genes were targeted in the oocyte using the conditional deletion approach, leading to dramatic phenotypes, including: a significant alteration of the reserve of primordial follicles (Pten [25]) or the loss of oocytes with an early arrest of follicular growth (Omcg1 [26]; Mdm2 [27]). Several other genes highly or exclusively expressed in the oocyte were invalidated by a global knock-out approach, leading to different phenotypes, like: sterility with a blockage of follicular growth at the primary stage (Gdf9 in the mouse [28] and in the pig [29]), sterility with a significant defect of oocyte maturation (Patl2 [30]), or sterility with an arrest of early embryonic development (e.g., maternal genes Nlrp5, Stella, Nmp2, and Zar1 [31]).…”