Abstract：Sex determining region Y-box 9 (SOX9) plays a particularly important role in the process of the individual development of mammals, and widely exists in various mammalian tissues. SOX9 gene mainly expresses on mammalian testicular tissues and is involved in the regulation of proliferation and differentiation of testicular cells. In this study, SOX9 expression was detected by qRT-PCR in various 6-month-old the Small Tail Han sheep (Ovis aries) tissues such as heart, liver, spleen, lung, hypothalamus, pituitary, testis and so on. Furthermore, mRNA expression and cellular localization of SOX9 were checked by qRT-PCR, Western blot and immunohistochemistry in testis during different development stages such as 0, 2, 6, 12, and 24-month-old. The qRT-PCR results showed that SOX9 was expressed in all the tissues detected, which expressed in pituitary with highest quantity, normal quantity in hypothalamus and testis, weak expression in muscle tissues (longissimus dorsi and biceps femoris), and mRNA expression from high to low in turn was: pituitary, hypothalamus, testis, liver, lung, heart, kidney, pineal gland, skin, biceps femoris, longissimus dorsi, spleen. At different developmental stages of testis, the mRNA expression of SOX9 were firstly decreased and then increased with month increasing, in which SOX9 expression in 6-month-old sheep testis was significantly higher than 0, 2-month-old (P＜0.05) while that was remarkably lower than 12, 24-month-old (P＜0.05). Expression trend of SOX9 protein was in accordance with SOX9 mRNA. The Western blot showed that SOX9 protein was lowly expressed in 0, 2, 6-month-old sheep testis, but its expression was higher and had increasing trend in 12, 24-month-old sheep testis. In addition, the immunohistochemical staining revealed that immunoreactivity was located in a small numbers of sustentacular cells and interstitial cells in 0, 2-month-old testis, while positive reaction was located in sustentacular cells, partial interstitial cells, even slight numbers of primary spermatocytes and secondary spermatocytes in 6, 12, 24-month-old testis. This study demonstrated that the SOX9 gene was widely expressed in sheep various tissues, and it plays an important role in the hypothalamic-pituitary-testicular axis, and it has a close relationship with the process of proliferation and differentiation of testicular cells.
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