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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.

SUBMITTER: Wang X

PROVIDER: S-EPMC6826996 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Ruminal microbiota and metabolites play crucial roles in animal health and productivity. Exploring the dynamic changes and interactions between microbial community composition and metabolites is important for understanding ruminal nutrition and metabolism. Tibetan sheep (Ovis aries) are an important livestock resource on the Qinghai-Tibetan Plateau (QTP), and the effects of various dietary protein levels on ruminal microbiota and metabolites are still unknown. The aim of this study was to investigate the response of ruminal microbiota and metabolites to different levels of dietary protein in Tibetan sheep. Three diets with different protein levels (low protein 10.1%, medium protein 12.1%, and high protein 14.1%) were fed to Tibetan sheep. 16S rRNA gene sequencing and gas chromatography coupled with time-of-flight mass spectrometry (GC-TOF-MS) were used to study the profile changes in each group of ruminal microbes and metabolites, as well as the potential interaction between them. The rumen microbiota in all groups was dominated by the phyla Bacteroidetes and Firmicutes regardless of the dietary protein level. At the genus level, Prevotella_1, Rikenellaceae_RC9_gut_group and Prevotellaceae_UCG-001 were dominant. Under the same forage-to-concentrate ratio condition, the difference in the dietary protein levels had no significant impact on the bacterial alpha diversity index and relative abundance of the major phyla and genera in Tibetan sheep. Rumen metabolomics analysis revealed that dietary protein levels altered the concentrations of ruminal amino acids, carbohydrates and organic acids, and significantly affected tryptophan metabolism (p < 0.05). Correlation analysis of the microbiota and metabolites revealed positive and negative regulatory mechanisms. Overall, this study provides detailed information on rumen microorganisms and ruminal metabolites under different levels of dietary protein, which could be helpful in subsequent research for regulating animal nutrition and metabolism through nutritional interventions.

Project description:The gut microbiota is closely associated with the health and production performance of livestock. Partial studies on ruminant microbiota are already in progress in the Qinghai-Tibetan Plateau Area (QTPA) in China, but large-scale and representative profiles for the QTPA are still lacking. Here, 16S rRNA sequencing was used to analyze 340 samples from yak, cattle, yak-cattle hybrids, and Tibetan sheep, which lived in a shared environment from 4 eco-regions of the QTPA during the same season, and aimed to investigate the fecal microbiota community composition, diversity, and potential function. All samples were clustered into 2 enterotypes, which were derived from the genera Ruminococcaceae UCG-005 and Acinetobacter, respectively. Environment, human activity, species, and parasitization all affected the fecal microbiota. By assessing the relationship between the fecal microbiota and the above variables, we identified a scattered pattern of fecal microbiota dissimilarity based more significantly on diet over other factors. Additionally, gastrointestinal nematode infection could reduce the capacity of the bacterial community for biosynthesis of other secondary metabolites, carbohydrate metabolism, and nucleotide metabolism. Ultimately, this study provided a fecal microbiota profile for ruminants living in 4 eco-regions of the QTPA and its potential future applications in developing animal husbandry regimes. IMPORTANCE Cattle, yak, and sheep reside as the main ruminants distributed throughout most regions of Qinghai-Tibetan Plateau Area (QTPA) in China. However, there is a lack of large-scale research in the QTPA on their fecal microbiota, which can regulate and reflect host health as an internalized "microbial organ." Our study depicted the fecal microbiota community composition and diversity of yak, cattle, yak-cattle hybrids, and Tibetan sheep from 4 eco-regions of the QTPA. Additionally, our results demonstrated here that the ruminant samples could be clustered into 2 enterotypes and that diet outweighed other factors in shaping fecal microbiota in the QTPA. This study provided a basis for understanding the microbiota characteristic of ruminants and its possible applications for livestock production in the QTPA.

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

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