Cloning, Molecular Characterization and Expression Patterns of DMRTC2 Implicated in Germ Cell Development of Male Tibetan Sheep.

Taotao Li,Xingxu Zhao,Nana Chen,De′En Yin,Youji Ma,Lingyun Kang,Hongyu Zhang,Xia Wang

doi:10.3390/ijms21072448

Abstract

The double sex and mab-3-related transcription factors like family C2 (DMRTC2) gene is indispensable for mammalian testicular function and spermatogenesis. Despite its importance, what expression and roles of DMRTC2 possesses and how it regulates the testicular development and spermatogenesis in sheep, especially in Tibetan sheep, remains largely unknown. In this study, DMRTC2 cDNA from testes of Tibetan sheep was firstly cloned by the RT-PCR method, and its molecular characterization was identified. Subsequently, the expression and localization patterns of DMRTC2 were evaluated by quantitative real-time PCR (qPCR), Western blot, and immunofluorescence. The cloning and sequence analysis showed that the Tibetan sheep DMRTC2 cDNA fragment contained 1113 bp open reading frame (ORF) capable of encoding 370 amino acids, and displayed high identities with some other mammals, which shared an identical DM domain sequence of 47 amino acids ranged from residues 38 to 84. qPCR and Western blot results showed that DMRTC2 was expressed in testes throughout the development stages while not in epididymides (caput, corpus, and cauda), with higher mRNA and protein abundance in Tibetan sheep testes of one- and three-year-old (post-puberty) compared with that of three-month-old (pre-puberty). Immunofluorescence results revealed that immune staining for DMRTC2 protein was observed in spermatids and spermatogonia from post-puberty Tibetan sheep testes, and gonocytes from pre-puberty Tibetan sheep testes. Together, these results demonstrated, for the first time, in sheep, that DMRTC2, as a highly conserved gene in mammals, is essential for sheep spermatogenesis by regulating the proliferation or differentiation of gonocytes and development of spermatids in ram testes at different stages of maturity.

Highlights

Spermatogenesis occurring in the seminiferous tubule boundaries of mammalian testis is a sophisticated, multistep, and continuous biological event where spermatogonial stem cells undergo mitosis, meiosis, and cell differentiation to generate mature spermatozoa that is needed for the continuation of species [1]
Sequence analysis showed that the cloned cDNA sequence included a 1113 bp open reading frame (ORF) translatable to 370 amino acid, with an ATG (M) start codon and a TAG stop codon (Figure 1B)
The resulting full-length coding sequence (CDS) sequence of Tibetan sheep DMRTC2 has been deposited in GenBank

Summary

Introduction

Spermatogenesis occurring in the seminiferous tubule boundaries of mammalian testis is a sophisticated, multistep, and continuous biological event where spermatogonial stem cells undergo mitosis, meiosis, and cell differentiation to generate mature spermatozoa that is needed for the continuation of species [1]. Meiosis is one of the most critical processes during spermatogenesis [2,3], which is required for perpetuation of species and generation of new variation. This process is finely regulated by numerous genes, which express at the transcriptional and translational levels [4,5]. Double sex and mab-3-related transcription factors (DMRT) like family C2 (DMRTC2, termed as DMRT7), a member of the DMRT family genes, is involved in the regulation of mammalian sex differentiation [6] and the process of spermatogenesis, meiosis [7,8,9]. Previous studies indicated that targeted deletion of DMRTC2 gene for males give rise to abnormal spermatogenesis such as meiotic arrest, while females exhibit normal fecundity [9,11,12]

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Results

Conclusion