Skip to main content
ARS Home » Midwest Area » Columbia, Missouri » Plant Genetics Research » Research » Publications at this Location » Publication #397746
Research Project: Development of Gene-editing Technologies in Livestock to Address Agriculturally Relevant Problems

Location: Plant Genetics Research

Title: Inactivation of growth differentiation factor 9 blocks folliculogenesis in pigs

Author
item Chen, Paula
item UH, KYUNGJUN - University Of Missouri
item MONARCH, KAYLYNN - University Of Missouri
item SPATE, LEE - University Of Missouri
item REESE, EMILY - University Of Missouri
item PRATHER, RANDALL - University Of Missouri
item LEE, KIHO - University Of Missouri

Submitted to: Biology of Reproduction
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/11/2023
Publication Date: 1/16/2023
Citation: Chen, P.R., Uh, K., Monarch, K., Spate, L., Reese, E., Prather, R., Lee, K. 2023. Inactivation of growth differentiation factor 9 blocks folliculogenesis in pigs. Biology of Reproduction. 108(4):611-618. https://doi.org/10.1093/biolre/ioad005.
DOI: https://doi.org/10.1093/biolre/ioad005

Interpretive Summary: Pigs are a highly important agricultural species and have emerged as a leading biomedical model for genetic diseases in humans. Understanding mechanisms of ovarian function is necessary to improve reproductive potential and produce these models. The use of gene editing systems has become extremely useful for determining the function of genes; therefore, we used a bacterial-derived gene editing system to create mutations in a gene, called growth differentiation factor 9, that was predicted to be important for ovarian function in pigs. Disruption of this gene resulted in a pig that did not have estrous cycles nor proper development of the reproductive tract, including the ovaries. Thus, this gene is absolutely essential for female fertility in pigs.

Technical Abstract: Growth differentiation factor 9 (GDF9) is a secreted protein belonging to the transforming growth factor 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.