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

Abstract

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.

Unraveling Stage-Dependent Expression Patterns of Circular RNAs and Their Related ceRNA Modulation in Ovine Postnatal Testis Development

“…Tibetan sheep ( Ovis aries ), a well-known Chinese domestic sheep breed, is mainly distributed in Qinghai-Tibet Plateau (QTP) over 3,000 m above sea level, which provides food and livelihoods to the local farmers and herdsmen ( Wang et al, 2019 ), and serves a pivotal role in the QTP ecosystem functions ( Jing et al, 2020 ). Due to the long-term excessive dependence on grazing that lacks supplementary feeding, the main reproductive characteristics of Tibetan sheep are late sexual maturity and low reproductive rates.…”

E2 Site Mutations in S Protein Strongly Affect Hepatitis B Surface Antigen Detection in the Occult Hepatitis B Virus

“…The structural characteristics (signal peptides, physicochemical properties, transmembrane domains, and secondary structures) of S proteins with or without E2 mutations were analyzed as previously reported ( Wang et al, 2019 ; Santos Junior et al, 2020 ). Protein signal peptide prediction was carried out on the SignalP v5.0 server 1 .…”

The Human Glyoxalase Gene Family in Health and Disease

“…It is possible that GLOD4 executes a similar side-reorganization to overcome the absence of metal-binding. Although little is known about the molecular function and role in disease, GLOD4 was also reported to be heavily expressed in the Leydig cells of young versus older sheep, suggesting that GLOD4 may be involved in testicular development . Future studies are clearly needed both in vitro and in vivo to fully understand the functional role of GLOD4 .…”