CLONING AND EXPRESSION ANALYSIS OF HISTONE H2A VARIANT IN OOCYTES OF CHINESE SOFT-SHELLED TURTLE (PELODISCUS SINENSIS)

Histone H2A and its variants play an important role in many cellular processes. However, their role in the germ cells differentiation of turtle has not been well characterized yet. Here, we cloned the Chinese soft-shell turtle (Pelodiscus sinensis) PsH2A and analyzed its expression pattern and cellular location during oogenesis. The 575 bp PsH2A cDNA was isolated from P. sinensis, which contains an open reading frame (ORF) of 399 bp encoding a protein of 133 amino acid residues, a partial 5′ untranslated region (5′-UTR) of 68 bp and a partial 3′ untranslated region (3′-UTR) of 108 bp. Real-time quantitative PCR (RT-qPCR) result showed that PsH2A mRNA expression level was higher in ovary than other tissues (P<0.01), such as heart, liver, spleen, kidney, brain, muscle, and testis. Reverse transcription PCR (RT-PCR) result showed that PsH2A mRNA only detected in ovary. Moreover, in the male and female gonads of 1-year, 2-year and 3-year-old P. sinensis, the PsH2A mRNA was only significantly expressed in ovary (P<0.01). The results of chemical in situ hybridization (CISH) showed that PsH2A mRNA was specifically expressed in oocytes. When primary oocytes developed to stage Ⅲ, strong signal could be detected and evenly distributed in the cytoplasm. From growing follicular stage (stage Ⅶ oocytes) to mature follicular stage (stage Ⅸ oocytes), the PsH2A mRNA signal gradually weakened and spread in the cytoplasm around the nucleus. The signal was distributed in the perinuclear cytoplasm, but was undetected in the peripheral cytoplasm. Furthermore, the relative expression level of PsH2A mRNA also responded to seasonal change in ovarian development in P. sinensis. In 1-year-old and 3-year-old ovaries, the relative level of PsH2A mRNA increased as ovary developing. However, in 2-year-old ovary, the relative level of PsH2A mRNA increased first and then decreased, reaching a peak level in April. In addition, in different season, the relative expression of PsH2A mRNA showed a downward trend with age increasing. In summary, these findings suggest that PsH2A may play an important role in oogenesis of P. sinensis.