ISL1 Expression In The Developing Mouse Gonads And The Establishment Of Isl1 Gene Targeting Mouse Model

Gonadal development and gametogenesis is the foundation of mammalian reproduction.Primordial germ cells(PGCs)migrate into the genital ridge before sex differentiation.In female mice,the primordial follicle pool is established through PGCs proliferation and meiosis and it will become the source of folliclegenesis after sexual maturity.Spermatogenesis occurs in the testes,where spermatogonia develop into mature spermatozoa after mitosis,differentiation,meiosis and spermiogenesis.Gonadal development and gametogenesis are strictly regulated by a variety of factors,including expression and interaction of specific or critical genes.LIM homeobox transcription factor ISL1 acts as an important regulatory factor during embryonic development,but its roles in gonadal development and gametogenesis have not been reported yet.In this study,we detected ISL1 expression in gonadal development and gametogenesis,and then established the inducible and conditional Isll gene targeting mouse models.Firstly,we detected Isll mRNA expression in embryonic gonads and adult tissues using real-time quantitative PCR(qRT-PCR)and the results showed that Isll mRNA was highly expressed in adult ovary and testis.The whole-mount in situ hybridization(WISH)results showed that Isll mRNA located in the seminiferous cords of male testis.Further,we located ISL1 expression by using immunohistochemistry(IHC)and in situ hybridization,and the results showed that ISLl expressed in germ cells.In female,ISLI expressed in the oogonia,oocytes of primordial follicles,growing follicles and mature oocyte.In male,ISL1 expressed in spermatogonia and spermatocytes.However,ISLI expression was not detected in the germ cells undergoing the Lepotene/zygotene stage of prophase I in MI either in the males or females.These results suggest that ISLI regulates mouse gonadal development and gametogenesis.In order to study the functions of Isll in the developing gonads,we established the inducible and conditional Isll gene knockout mouse models.The transgenic mice with LoxP flanked Isll lucs(IsllLoxP/LoxP)are generated through CRISPR/Cas9 method.IsllLoxP/LoxP mice are mated with IsllMCM/+ mice,in which DNA recombinase(Cre)MCM cassettes is under Isll promoter,to produce lsllMCM/LoxP offspring.Tamoxifen administration time and dosage are determined for inducible Cre-mediated gene disruption in lsllMCM/LoxP mice.Moreover,appropriate mouse strain is selected between several mice that specifically express Cre in germ cell at different developmental stages.VasaCre/+;IsllLoxP/? mice(CKO)model,in which Isll is truncated after Cre expression in spermatogonia,is used to investigate effects of ISLl ablation on testicular development and spermatogenesis,compared with VasaCre/+;lsll?/+ controls.the CKO mice exhibit a high incidence of multinucleated giant cells(MGCs)in seminiferous tubules which indicate that ISL1 deficiency affect spermatogenesis.