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

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.

Keywords: DMRTC2; Tibetan sheep; cloning; spermatogenesis; testis.

Figure 1

Full-length coding sequence (CDS) cloning and sequence analysis of Tibetan sheep DMRTC2 gene. (A) Results for PCR amplification of DMRTC2 cDNA. M, DL2000 marker; 1-2, DMRTC2 RT-PCR product. (B) The nucleotide and deduced amino acid sequences for the cloned DMRTC2 CDS region. (C) Comparisons of sheep DMRTC2 CDS region between the predicted and cloned sequences. (D) Alignment of the deduced amino acid sequences of Tibetan sheep DMRTC2 with that of chiru (accession no: XP_005971442.1), cattle (accession no: AAI09621.1), goat (accession no: XP_005692632.1), white tail deer (accession no: XP_020726841.1), and pig (accession no: XP_020950121.1).

Figure 2

Sequence composition of Tibetan sheep DMRTC2 CDS region at the nucleotide and amino acid levels. (A) Base composition. (B) Amino acid composition.

Figure 3

The secondary and tertiary structures of Tibetan sheep DMRTC2 protein. (A) Secondary molecular structure. Different lines with different colors denote different secondary structures: blue, alpha helix; red, extended strand; green, beta turn; purple, random coil. (B) Tertiary molecular structure.

Figure 4

Phylogenetic tree and domain analysis of DMRTC2. (A) Neighbor-joining phylogenetic tree based on nucleotide sequences of DMRTC2 gene among different mammals. The bootstrap values (greater than 50) and branch lengths (less than 1) were showed above and below each branch, respectively. The closest homology with ovine DMRTC2 is indicated by orange circle. (B) DMRTC2 protein domain prediction among different mammals. Weblogo tool was used to show the amino acid (C) and nucleotide (D) composition of the DM domain.

Figure 5

Temporal expression patterns of DMRTC2 mRNA in developmental testes and epididymides. Data show means ± SD from eight independent experiments. **: p < 0.01, *: p < 0.05, and ns (no significance): p > 0.05. 3 M: three months old, 1 Y: one year old, and 3 Y: three years old.

Figure 6

Temporal expression patterns of DMRTC2 protein during testicular development. (A) Western blot analysis for DMRTC2 protein. (B) Relative expression level of DMRTC2 protein. Data show means ± SD from eight independent experiments. **: p < 0.01, *: p < 0.05, and ns: nonstatistical significance. 3 M: three months old, 1 Y: one year old, and 3 Y: three years old.

Figure 7

Hematoxylin and eosin (H&E) staining and immunofluorescence staining of DMRTC2 protein within Tibetan sheep testes at different development stages. Nuclei were counterstained with DAPI (blue). 3 M: three months old, 1 Y: one year old, and 3 Y: three years old.