Background: Despite its anticancer activity, cisplatin exhibits severe testicular toxicity when used in chemotherapy. Owing to its wide application in cancer therapy, the reduction of damage to normal tissue is of imminent clinical need. In this study, we evaluated the effects of catechin hydrate, a natural flavon-3-ol phytochemical, on cisplatin-induced testicular injury. Methods: Type 2 mouse spermatogonia (GC-1 spg cells) were treated with 0-100 μM catechin and cisplatin. Cell survival was estimated using a cell proliferation assay and Ki-67 immunostaining. Apoptosis was assessed via flow cytometry with the Dead Cell Apoptosis assay. To determine the antioxidant effects of catechin hydrate, Nrf2 expression was measured using qPCR and CellROX staining. The anti-inflammatory effects were evaluated by analyzing the gene and protein expression levels of iNOS and COX2 using qPCR and immunoblotting. Results: The 100 μM catechin hydrate treatment did not affect healthy GC-1 spg cells but, prevented cisplatin-induced GC-1 spg cell death via the regulation of anti-oxidants and inflammation-related molecules. In addition, the number of apoptotic cells, cleaved-caspase 3 level, and BAX gene expression levels were significantly reduced by catechin hydrate treatment in a cisplatin-induced GC-1 spg cell death model. In addition, antioxidant and anti-inflammatory marker genes, including Nrf2 , iNOS, and COX2 were significantly downregulated by catechin hydrate treatment in cisplatintreated GC-1 cells. Conclusions: Our study contributes to the opportunity to reintroduce cisplatin into systemic anticancer treatment, with reduced testicular toxicity and restored fertility.

In the study for a differentiation and development of spermatogonial cells, the researchers should commonly require a simple, fast and reasonable method that could evaluate the developmental stage of male germ cells without any damage and also relentlessly culture them so far as a cell stage aiming at experimental applications. For developing the efficient method to identify the stage of sperm cells, the morphological characteristics of sperm cells were investigated by staining the cells with blue fluorescent dye Hoechst 33258, and a criterion for male germ cell classification was elicited from results of the previous investigation, then the efficiency of the criterion was verified by applying it to assort the germ cells recovered from male mice in age from 6 to 35 days. As morphological characteristics, spermatogonia significantly differed from spermatocytes in size, appearance and fluorescent patches of nucleus, and spermatids could also be distinguished from spermatozoa by making a difference in the volume and shape of nucleus and the shape and fluorescence of tail. Aforesaid criterion was applicable for classifying in vitro cultured sperm cells by verifying its efficiency and propriety for assorting the stages of testicular germ cells. However, the fluorescent staining showed that germ cells in mouse testis should be dramatically differentiated and developed at 21 days and 35 days of age, which were known as times of sexual puberty and maturity in male mice, respectively. In conclusion, the results indicated that this simple criterion for sperm cell classification using fluorescence staining with Hoechst 33258 may be highly efficient and reasonable for spermatogenesis study.

본 연구는 가축유전자원의 효율적 보존을 위해 정조세포를 줄기세포 형태로 장기보관하면서 필요에 따라 증식, 분화를 통해 가축의 복원에 활용하기 위한 연구의 일부로 진행되었다. 정조세포를 분리하여 배양한 결과 배양온도는 다른 세포들과는 달리 에 세포분열이 활발하였으며, TCM199에 FCS를 첨가한 배양액과 세르톨리세포 공배양으로 정조세포의 배양을 지지하였다. 40일령이 지나면서 정조세포 콜로니 즉 germline stem cells를 형성하였으며, 일부에