Exploring the Molecular Characteristics and Role of PDGFB in Testis and Epididymis Development of Tibetan Sheep

Abstract

Platelet-derived growth factor B (PDGFB), as an important cellular growth factor, is widely involved in the regulation of cellular events such as cell growth, proliferation, and differentiation. Although important, the expression characteristics and biological functions in the mammalian reproductive system remain poorly understood. In this study, the PDGFB gene of Tibetan sheep was cloned by RT-PCR, and its molecular characteristics were analyzed. Subsequently, the expression of the PDGFB gene in the testes and epididymides (caput, corpus, and cauda) of Tibetan sheep at different developmental stages (3 months, 1 year, and 3 years) was examined by qRT-PCR and immunofluorescence staining. A bioinformatic analysis of the cloned sequences revealed that the CDS region of the Tibetan sheep PDGFB gene is 726 bp in length and encodes 241 amino acids with high homology to other mammals, particularly goats and antelopes. With the increase in age, PDGFB expression showed an overall trend of first decreasing and then increasing in the testis and epididymis tissues of Tibetan sheep, and the PDGFB mRNA expression at 3 months of age was extremely significantly higher than that at 1 and 3 years of age (p < 0.05). The PDGFB protein is mainly distributed in testicular red blood cells and Leydig cells in Tibetan sheep at all stages of development, as well as red blood cells in the blood vessel, principal cells, and the pseudostratified columnar ciliated epithelial cells of each epididymal duct epithelium. In addition, PDGFB protein expression was also detected in the spermatocytes of the 3-month-old group, spermatids of the 1-year-old group, spermatozoa and interstitial cells of the 3-year-old group, and loose connective tissue in the epididymal duct space in each developmental period. The above results suggest that the PDGFB gene, as an evolutionarily conserved gene, may play multiple roles in the development and functional maintenance of testicular cells (such as red blood cells, Leydig cells, and germ cells) and epididymal cells (such as red blood cells, principal cells, and ciliated epithelial cells) during testicular and epididymal development, which lays a foundation for the further exploration of the mechanisms by which the PDGFB gene influences spermatogenesis in Tibetan sheep.

Keywords: PDGFB; development; epididymis; sheep; testis.

Figure 1

A sequencing result of PDGFB cDNA amplification product from Tibetan sheep. Note: (A) PDGFB cDNA amplification product. M (Lane 1): DL3000 marker; 1 (Lane 2), 2 (Lane 3), and 3 (Lane 4): samples harvested from 3−month−old, 1−year−old, and 3−year−old, respectively. (B) Sequence alignment between cloned and predicted PDGFB CDS region.

Figure 2

Amino acid sequence encoded by PDGFB gene in Tibetan sheep.

Figure 3

Physicochemical properties of the protein encoded by PDGFB gene in Tibetan sheep. Note: (A) Hydrophilic analysis; (B) Transmembrane structure analysis; and (C) Signal peptide analysis.

Figure 4

Sequence alignment of PDGFB amino acid sequences and phylogenetic tree among various species. Note: (A) Multiple alignment of PDGFB amino acid sequences from nine different mammalian species; (B) Phylogenetic tree for amino acid sequences of PDGFB protein. The closest homology with ovine DMRTC2 is indicated by four-pointed star.

Figure 5

Spatial structure and interacting protein network analysis of PDGFB protein in Tibetan sheep. Note: (A) Secondary structure composition of Tibetan sheep PDGFB protein; (B) Tertiary structure prediction of Tibetan sheep PDGFB protein; (C) A network analysis of Tibetan sheep PDGFB protein-interacting protein.

Figure 6

PDGFB mRNA expression in testes and epididymides of developed Tibetan sheep. Note: 3M, 3 months old; 1Y, 1 year old; 3Y, 3 years old. **, Extremely significant difference (p < 0.01); *, Significant difference (p < 0.05); ns, Non-significant difference (p > 0.05). Each experiment was independently repeated at least 3 times.

Figure 7

Immunofluorescence staining of PDGFB protein in testicular tissues of Tibetan sheep at different developmental stages. Note: 3M, 3 months old; 1Y, 1 year old; 3Y, 3 years old. LC, Leydig cell; Rc, red blood cell; Sp, spermatid. Scale bar, 50 um. Each experiment was independently repeated at least 3 times.

Figure 8

Immunofluorescence staining of PDGFB protein in each region of the epididymis of Tibetan sheep at different developmental stages. Note: 3M, 3 months old; 1Y, 1 year old; 3Y, 3 years old. PC, principal cell; PCCE, pseudostratified columnar epithelium; Sz, spermatozoon; ED, epididymal duct; RC, red blood cell. Scale bar, 50 um. Each experiment was independently repeated at least 3 times.