Early Ovary Development, sox3 Gene cDNA Cloning and Expression Analysis of Pseudaspius leptocephalus

Abstract

Pseudaspius leptocephalus is the famous indigenous fish in Northeast China region, with very important economic and research value. In recent years, environmental pollution and overfishing has caused it to become increasingly scarce. We performed P. leptocephalus conservation research, including clarifying the characteristics of ovarian development and the role of the sox3 gene (SRY-related high-mobility-group-box 3) in the ovarian development of P. leptocephalus. The histological characteristics of P. leptocephalus ovarian development were investigated using the paraffin section technique. The full length of the cDNA of the sox3 gene was cloned and analyzed using Rapid Amplification of cDNA End (RACE) technology. The expression pattern of the sox3 gene in different tissues and at different gonad development stages were analyzed using real-time PCR.The results revealed the ovarian cavity was clearly visible in the elliptical gonads, indicating ovary formation occurs around 45 d. At 160 d, the nucleoli of the oocytes in the ovaries were distributed along the nucleus and the testis was preparing to form seminiferous lobules and began to enter early stage Ⅱ development. At 260 d of ovarian development, the yolk nucleus appeared in the cytoplasm of some ovary oocytes, and the ovaries entered the middle stage Ⅱ phase. Also at 260 d, the number of secondary spermatocytes in the testis increased, and remained at stage Ⅱ until 360 d (when the study ceased). The differentiation of the testis began later than that of the ovaries. This result is consistent with the gonadal development in most fish. The results indicate the cDNA of the sox3 gene in P. leptocephalus is 1 800 bp (GenBank: MT952206), and encodes 299 amino acids. Among them, the protein contains an HMG conserved domain. The alignment of the amino acid sequence and the phylogenetic tree analysis indicates the SOX3 sequence in P. leptocephalus contains highly conserved regions. These regions are highly consistent with the SOX3 sequences in Danio rerio and Carassius auratus. Furthermore, the RT-PCR demonstrated that sox3 was highly expressed in the ovary, followed by the brain and eyes. Between the sexes, the expression level of the sox3 gene only varied significantly in the gonads, with the sox3 gene expression in the ovary being significantly higher than that in the testis. In the early stage of gonadal differentiation, the expression of sox3 in the ovaries is obviously higher than that in undifferentiated gonads. The lowest expression levels were detected in the testis. In the developmental stage after gonadal differentiation, sox3 was continuously expressed throughout ovarian development, while the expression in the testis remained at a low level. Therefore, the sox3 gene might play an important role in ovary differentiation and development when compared to the testis, but its specific function and mechanism require further research.

Keywords

• pseudaspius leptocephalus
• ovarian development
• sox3
• gene cloning
• gene expression