Mutation of the Sp1 binding site in the 5' flanking region of SRY causes sex reversal in rabbits.

Yuning Song,Zhanjun Li,Yuxin Xu,Mao Chen,Lin Yuan,Haobin Yao,Liangxue Lai,Yi Lu,Jichao Deng,Tingjun Liu,Yong Wang

doi:10.18632/oncotarget.16979

Yuning Song, Zhanjun Li + Show 9 more

Open Access

https://doi.org/10.18632/oncotarget.16979

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Abstract

Sex-determining region Y is a crucial gene that initiates male sex determination in mammals. Mutations of the Sp1-binding site in the 5′ flanking region of SRY are associated with clinical male-to-female sex reversal syndrome, although such occurrences are rare and, until now, have not been reported in animal models. In this study, we mutated Sp1-binding sites in the 5′ flanking region of the rabbit SRY gene using the CRISPR/Cas9 system. As expected, the SRY-Sp1 knockout rabbits had female external and internal genitalia and exhibited normal female copulatory behaviors, but they were infertile, and the adults displayed reduced follicles. Interestingly, we successfully obtained offspring from sex-reversed SRY-Sp1 knockout rabbits using embryo transfer. In summary, our study demonstrates that Sp1 is a major regulator in SRY gene transcription, and mutations of the Sp1 binding sites (Sp1-B and Sp1-C) in the 5′ flanking region of SRY induce sex reversal in rabbits, which can be used as targets for clinical research of male-to-female sex reversal syndrome. Additionally, we provide the first evidence that sex reversal syndrome patients have the potential to become pregnant with the use of embryo transfer.

Highlights

In mammals, the sex-determining gene, SRY, which is expressed in the genital ridges, facilitates gender determination [1, 2]
Co-transfection experiments suggest that two Sp1 binding sites located in the 5’ flanking region are associated with transcriptional activation of SRY in humans [9]
To determine whether the mutations in the Sp1 binding sites induced sex reversal, we examined the external genitalia of the Sp1 KO, XY rabbits when the pups were two months old

Summary

INTRODUCTION

The sex-determining gene, SRY, which is expressed in the genital ridges, facilitates gender determination [1, 2]. The Sp1 transcription factor binds to G-C rich motifs that are 21-22 bp long. It plays a role as a transcriptional activator in most mammalian genes and is essential for the differentiation of spermatids [7, 8]. Co-transfection experiments suggest that two Sp1 binding sites located in the 5’ flanking region are associated with transcriptional activation of SRY in humans [9]. There is a need to further characterize the association between mutations in Sp1 binding sites and female sex reversal and to provide proper animal models for clinical research of male-tofemale sex reversal syndromes. We mutated Sp1-binding sites in the 5’ flanking region of rabbit SRY using CRISPR/Cas, and observed the typical phenotype of sex reversal syndromes in SRY-Sp1 knockout (KO) rabbits. We identified the potential ability of sex-reversed females as surrogates for embryo transfer in the rabbit model

RESULTS

DISCUSSION

MATERIALS AND METHODS

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