Cloning and Molecular Characterization of HSL and Its Expression Pattern in HPG Axis and Testis during Different Stages in Bactrian Camel

Abstract

Hormone-sensitive lipase (HSL) is a key enzyme in animal fat metabolism and is involved in the rate-limiting step of catalyzing the decomposition of fat and cholesterol. It also plays an important regulatory role in maintaining seminiferous epithelial structure, androgen synthesis and primordial germ cell differentiation. We previously reported that HSL is involved the synthesis of steroids in Bactrian camels, although it is unclear what role it plays in testicular development. The present study was conducted to characterize the biological function and expression pattern of the HSL gene in the hypothalamic pituitary gonadal (HPG) axis and the development of testis in Bactrian camels. We analyzed cloning of the cDNA sequence of the HSL gene of Bactrian camels by RT-PCR, as well as the structural features of HSL proteins, using bioinformatics software, such as ProtParam, TMHMM, Signal P 4.1, SOPMA and MEGA 7.0. We used qRT-PCR, Western blotting and immunofluorescence staining to clarify the expression pattern of HSL in the HPG axis and testis of two-week-old (2W), two-year-old (2Y), four-year-old (4Y) and six-year-old (6Y) Bactrian camels. According to sequence analysis, the coding sequence (CDS) region of the HSL gene is 648 bp in length and encodes 204 amino acids. According to bioinformatics analysis, the nucleotide and amino acid sequence of Bactrian camel HSL are most similar to those of Camelus pacos and Camelusdromedarius, with the lowest sequence similarity with Mus musculus. In adult Bactrian camel HPG axis tissues, both HSL mRNA and protein expression were significantly higher in the testis than in other tissues (hypothalamus, pituitary and pineal tissues) (p < 0.05). The expression of mRNA in the testis increased with age and was the highest in six-year-old testis (p < 0.01). The protein expression levels of HSL in 2Y and 6Y testis were clearly higher than in 2W and 4Y testis tissues (p < 0.01). Immunofluorescence results indicate that the HSL protein was mainly localized in the germ cells, Sertoli cells and Leydig cells from Bactrian camel testis, and strong positive signals were detected in epididymal epithelial cells, basal cells, spermatocytes and smooth muscle cells, with partially expression in hypothalamic glial cells, pituitary suspensory cells and pineal cells. According to the results of gene ontology (GO) analysis enrichment, HSL indirectly regulates the anabolism of steroid hormones through interactions with various targets. Therefore, we conclude that the HSL gene may be associated with the development and reproduction of Bactrian camels in different stages of maturity, and these results will contribute to further understanding of the regulatory mechanisms of HSL in Bactrian camel reproduction.