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Open AccessArticle

Characterization of GLOD4 in Leydig Cells of Tibetan Sheep During Different Stages of Maturity

1
College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070, China
2
Sheep Breeding Biotechnology Engineering Laboratory of Gansu Province, Minqin, 733300, China
3
College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Genes 2019, 10(10), 796; https://doi.org/10.3390/genes10100796
Received: 17 September 2019 / Revised: 10 October 2019 / Accepted: 10 October 2019 / Published: 12 October 2019
(This article belongs to the Special Issue Genomics of Sexual Development and Reproduction in Mammals)
We have previously reported that glyoxalase domain-containing protein 4 (GLOD4) is expressed in sheep testes by proteome analysis, but its roles during testicular development remain unclear. The aim of this study was to understand the expression characteristics and biological functions of the GLOD4 gene in developmental Tibetan sheep testes. The cDNA sequence of the Tibetan sheep GLOD4 gene was cloned by the RT-PCR method, and the structural characteristics of the GLOD4 protein were analyzed using relevant bioinformatics software, including ProtParam, TMHMM, Signal P 4.1, SOPMA, and phyre2. The expression patterns and immunolocalization of GLOD4 were examined in developmental testes derived from three-month-old (3M), one-year-old (1Y), and three-year-old (3Y) Tibetan sheep using quantitative real-time PCR (qRT-PCR), Western blot, immunohistochemistry, and immunofluorescence staining. The sequence analysis showed that the coding sequence (CDS) region of the GLOD4 gene was 729 bp in length and encoded 242 amino acids. Bioinformatics analysis found that the nucleotide and amino acid sequence of Tibetan sheep GLOD4 exhibited the highest sequence similarity with goat and chiru, and the least with zig-zag eel, of the species compared. GLOD4 expressions at both the mRNA and protein levels were significantly higher in the testes of the 1Y and 3Y groups than those in the 3M group (p < 0.01). Immunohistochemistry and immunofluorescence results indicated that the GLOD4 protein was mainly localized in the cytoplasm of Leydig cells from Tibetan sheep testes throughout the development stages. These results taken together suggest that the GLOD4 gene may be implicated in the development of the Leydig cells of Tibetan sheep during different stages of maturity.
Keywords: cloning; GLOD4; Tibetan sheep; testis; Leydig cells cloning; GLOD4; Tibetan sheep; testis; Leydig cells
MDPI and ACS Style

Wang, X.; Li, T.; Liu, N.; Zhang, H.; Zhao, X.; Ma, Y. Characterization of GLOD4 in Leydig Cells of Tibetan Sheep During Different Stages of Maturity. Genes 2019, 10, 796.

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