Gene function & annotation

Development of a Sheep Knockout Model to Study the Role of Follicle-Stimulating Hormone in Gametogenesis

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Abstract

Follicle-stimulating hormone (FSH) is a key regulator of ovarian follicle recruitment, growth, and development, and plays an essential role in spermatogenesis during reproductive life. However, its specific role during gamete development remains poorly described in large mammals. The objective of this study was to generate a large animal model of FSH deficiency by knocking out the FSHβ gene in sheep using CRISPR/Cas9, providing a physiologically relevant system to investigate gamete development and gonadal function. We hypothesize that FSHβ knockout females will exhibit diminished ovarian reserve, impaired antral follicle development, and will not ovulate, whereas males will show compromised spermatogenesis. Six single-guide RNAs (sgRNAs) targeting exons 2 and 3 of the ovine FSHβ gene were designed and validated in vitro. For in vivo testing, groups of 100 zygotes were mixed with ribonucleoprotein complexes (sgRNA at 100 ng/µL and Cas9 protein at 200 ng/µL, pre-incubated on ice for 10 min) and electroporated (poring conditions: 40 V, 3.5 ms pulse length, 50 ms interval, two bipolar pulses, 0 decay rate) 6h post-in vitro fertilization. A dual-guide approach (i.e., co-incubating two sgRNAs targeting different locations) was used, resulting in a 90.3% knockout rate (n = 28/31). Following optimization, 33 edited blastocysts were transferred into 11 synchronized ewes (3 embryos each). Pregnancy was confirmed in three ewes (27.3% pregnancy rate), carrying a total of four fetuses detected via ultrasound. Contemporary non-transferred embryos showed an 81.4% (n=48/59) knockout rate. One female and three male lambs were born in April 2025. Genotyping by Sanger sequencing, vector cloning, and whole genome sequencing revealed biallelic loss-of-function mutations in all males, whereas the single female was mosaic, with 12.5% edited alleles consisting of a 1bp deletion mixed with wild-type. While the mosaic genotype of the single female limits definitive conclusions regarding complete FSHβ loss-of-function in females, her inclusion demonstrates the feasibility of generating edited females and provides preliminary insight into early reproductive development under partial FSHβ disruption. One male was euthanized within 24h of birth due to medical complications. The remaining animals reached nine months of age with normal growth and without health issues. Reproductive examination revealed subfertility in males, characterized by reduced testicular volume, oligospermia, decreased sperm motility, and morphologically abnormal sperm. These findings provide evidence that FSHβ disruption compromises male reproductive function in large mammals, supporting and extending observations from rodent studies. Blood was collected periodically to evaluate the reproductive hormonal profile between birth and puberty in knockout and contemporary control lambs and is currently under evaluation. Additional confirmed-female pregnancies have been established, and future work will focus on performing detailed ovarian and follicle analysis. This study successfully produced FSHβ knockout sheep to help elucidate the role of FSH in gametogenesis.

Keywords: 2026

How to Cite:

Casale, M., Werry, N., Trott, J., Fletes, A., O’Grady, E., Wayne, H., Smith, F., McNabb, B., Van Eenennaam, A. & Denicol, A., (2026) “Development of a Sheep Knockout Model to Study the Role of Follicle-Stimulating Hormone in Gametogenesis”, World Congress on Genetics Applied to Livestock Production Digital Archive 2026(1): 2281824. doi: https://doi.org/10.31274/wcgalp.23457

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