Abstract
The sex-determining region on the Y chromosome (SRY) is thought to be the central genetic element of male sex development in mammals. Pathogenic modifications within the SRY gene are associated with a male-to-female sex reversal syndrome in humans and other mammalian species, including rabbits and mice. However, the underlying mechanisms are largely unknown. To understand the biological function of the SRY gene, a site-directed mutational analysis is required to investigate associated phenotypic changes at the molecular, cellular, and morphological level. Here, we successfully generated a knockout of the porcine SRY gene by microinjection of two CRISPR-Cas ribonucleoproteins, targeting the centrally located "high mobility group" (HMG), followed by a frameshift mutation of the downstream SRY sequence. This resulted in the development of genetically male (XY) pigs with complete external and internal female genitalia, which, however, were significantly smaller than in 9-mo-old age-matched control females. Quantitative digital PCR analysis revealed a duplication of the SRY locus in Landrace pigs similar to the known palindromic duplication in Duroc breeds. Our study demonstrates the central role of the HMG domain in the SRY gene in male porcine sex determination. This proof-of-principle study could assist in solving the problem of sex preference in agriculture to improve animal welfare. Moreover, it establishes a large animal model that is more comparable to humans with regard to genetics, physiology, and anatomy, which is pivotal for longitudinal studies to unravel mammalian sex determination and relevant for the development of new interventions for human sex development disorders.
Highlights
IntroductionThe male and female sex are determined by the presence or absence of the Y chromosome [1]
In mammalian species, the male and female sex are determined by the presence or absence of the Y chromosome [1]
Three targets, including the sex-determining region on the Y chromosome (SRY) and the monoallelic KDM6A genes, both located on the Y chromosome, and the biallelic GGTA1 gene located on chromosome 1 were selected for direct comparison of their copy numbers
Summary
The male and female sex are determined by the presence or absence of the Y chromosome [1]. The SRY gene presumably serves as master regulator for the formation of primary precursor cells of the tubuli seminiferi, leading to the development of testicles from undifferentiated gonads [9]. It is still unknown whether the SRY gene is the only sex-determining gene on the Y chromosome or if other genes such as SOX9 [10,11,12] and SOX3 [13] are involved in sex determination. We provide evidence that genetically male pigs with a knockout of the SRY gene undergo sex reversal of the external and internal genitalia. As the pig shares many genetic, physiological, and anatomical similarities with humans, it provides a suitable large animal model for human sex reversal syndromes, allowing for the development of new interventions for human sex disorders
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