Abstract
Spermatozoa released from testes undergo a maturation process and acquire the capacity to fertilize ova through epididymal transit. The epididymis is divided into four regions, each with unique morphology, gene profile, luminal microenvironment and distinct function. To study the functions of relevant genes in the epididymal initial segment (IS), a novel IS-specific mouse model, Lcn9-Cre knock-in (KI) mouse line was generated via CRISPR/Cas9 technology. The TAG stop codon was replaced by a 2A-NLS-Cre cassette, resulting in the co-expression of Lcn9 and Cre recombinase. IS-specific Cre expression was first observed from postnatal day 17. Using the Rosa26tdTomato reporter mice, the Cre-mediated DNA recombination was detected exclusively in principal cells. The epididymal IS-specific Cre activity in vivo was further confirmed using Lcn9-Cre mice crossed with a mouse strain carrying Tsc1 floxed alleles (Tsc1flox/+). Cre expression did not affect either normal development or male fecundity. Different from any epididymis-specific Cre mice reported previously, the novel Lcn9-Cre mouse line can be used to introduce entire IS-specific conditional gene editing for gene functional study.
Highlights
Infertility affects about 15% of the global population, and approximately half of the cases are attributed to a “male factor” [1, 2]
A novel Cre KI mouse line with epididymal initial segment (IS)-specific Cre activity was generated via the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system
In this Cre mouse line, a 2A-NLS-Cre element was introduced into the endogenous Lcn9 gene locus, ensuring that the Cre expression driven by the Lcn9 promoter is in tandem with Lcn9 expression
Summary
Infertility affects about 15% of the global population, and approximately half of the cases are attributed to a “male factor” [1, 2]. Sperm dysfunction is considered as the most principal cause [3]. Impaired epididymal spermatozoa maturation is an important cause of sperm dysfunction [4]. Immature and nonfunctional spermatozoa pass through the efferent ducts into the epididymis and undergo a complex maturation process through epididymal transit [3, 5]. The epididymis is a conserved part of the male reproductive tract in all vertebrate species that practice internal fertilization [6,7,8].
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