바다송사리(Oryzias dancena)의 정소조직 배양 시스템에서 정자형성과정의 특성분석

Abstract

Establishing testis organ culture system is considerably worthwhile in terms of the study of spermatogenic regulation at molecular levels and the application of germline cells to tissue engineering. Based on the previous studies that shown the effectiveness of agarose gel stand in organ culture system, this study evaluated the effectiveness of a whole testis organ culture system using agarose gel stand in marine medaka (Oryzias dancena) in terms of germ cell proliferation and differentiation. Testis organ culture was performed on a basal medium only containing fetal bovine serum and antibiotics. The proliferation and differentiation of spermatogonia were confirmed by 5-bromo-2‘-deoxyuridine (BrdU) incorporation assay and qRT-PCR with synaptonemal complex protein 3 (scp3) as a meiosis marker. In addition, it was evaluated if the organ culture system is able to support the differentiation of foreign germ cells which were artificially introduced into cultured tissues. As the results, testis organ culture was maintained up to day 8 of culture. Proliferation of spermatogonial cells was also observed until day 8 of culture, but the number of cysts with dividing cells was significantly decreased over time. During culture of BrdU-incorporated testes, BrdU-positive spermatids and spermatozoa were observed on day 2 and 8 of culture, respectively, indicating that spermatogenesis happened. However, it was not observed when the testes were stained with BrdU at day 2 of culture and cultured for an additional 6 days indicating that spermatogenesis was not triggered on day 2 of culture. This was supported by the analysis of scp3 expression by culture day that showed a significant decrease of its expression on day 2 of culture. When the germ cell population containing enriched spermatogonia from japanese medaka (Oryzias latipes) were isolated, labeled with fluorescent dye, and introduced into O. dancena testis organ culture system, the differentiated spermatids with short tail structure were identified after 6 days of culture indicating that this system could partially support differentiation of the foreign early-stage germ cells. Collectively, I confirmed that medaka whole testis organ culture system using agarose gel stand could support germ cell proliferation and differentiation during the early phase of culture. This study can be used as the basic data for the study of spermatogenesis in vitro. Further study is necessary to find the optimal conditions to be able to improve spermatogenic activity in this organ culture system.