Study On Survival And Proliferation Of Mouse Spermatogonia In Vitro

The maintenance and propagation of any mammalian species depend upon the production of male and female gametes and their union to form an embryo. The male gametes derive from spermatogonial stem cells in adult animals. A type A-single spermatogonium (A5) is spermatogonial stem cell in mammalian testes. Spermatogonial stem cells can self-renew and produce differentiated cells during the whole life span in a adult animal. Spermatogonial stem cells can be cultured, cryopreserved, transplanted and genetically manipulated. Studies on spermatogonial stem cells are of very significant importance for long-term preservation and utilization of animal's genomic resources, saving endangered animals, producing transgenic animals, producing farm animals with superior characters, therapy of infertility and elucidating the process and mechanisms of spermatogenesis. Up to now, spermatogonial stem cells and haemotopoietic stem cells are the only two types of adult stem cells which can be detected by their function, and moreover the former is the only type of stem cells which can be morphologically identified by intercellular bridges. The spermatogonial stem cell, therefore, represents a useful model for studies of biology of the adult stem cells. In the present study, the Kunming white mouse were used to investigate: (1) methods of in vitro detection; (2) biology of in vitro culture, proliferation in vitro; (3) methods of cryopreservation, of spermatogonial stem cells; and (4) obtaining of nutritional factors needed for these cells to proliferate by molecular biological methods. By establishing a long-term culture system, combining with the techniques of cryopreservation, we intended to obtain spermatogonial stem cells that can survive and proliferate in vitro for a long period.Fresh seminiferous epithelial cells dissociated from 6 days of postpartum(dpp), 25 dpp, and adult experimental cryptorchid mice and cryopreserved seminiferous epithelial cells from 6 dpp mice were seeded on STO or BRL feeders in a medium composed of DMEM + 10%FCS + 4 mmol/L glutamine. Sertoli cells and spermatogonia co-culture was conducted at 32 or 37 . During the first week after seeding, differentiated spermatogonia and other more advanced germ cells degenerated gradually, while only some spermatogonia with extremely resistance maintained. With no apparent dividing activities, these cells could survive for more than two and a half months in the co-culture system and could be passaged one or two times or continually cultured after cryopreservation. They showed no obvious clones and differentiation present, remained no distinctive characteristics. They were single cells or paired or aligned or clustered syncytia and adhered on the surface of Sertoli cells or in the concavity formed by Sertoli cell cytoplasm. They took the shape of sphere or oval with high nuclear-cytoplasmic ratio and had a spheral or oval nuclear. Alkaline phosphatase(AP) histochemical staining showed that the cytoplasm and membrane of these cells were positive. These cells were most likely spermatogonial stem cells and the first few differentiated progeny of stem cells according to As spermatogonial stem cell theory and stem cell biological characteristics. When 100 ng/mL human glial cell line-derived neurotrophic factor(hGDNF) was added into the culture medium, the number of spermatogonial stem cell and their clones in established culture system from 25-day-old mouse described above increased 8 times after co-cultured 25-30 days. Conclusions: (1) Freshly dissociated or cryopreserved Sertoli cells and germ cells, co-cultured at 32 ~37 with STO feeders or BRL feeders, all have no significant difference in establishment of a long-term culture system of spermatogonal stem cell. (2)AP histochemical staining can be used to detect spermatogonial stem cells combined with cell morphology in a lone-term in vitro culture system. (3)Both the feeders and adult sertoli cells are needed for establishment of a long-term culture system of spermatogonial stem cell. (4) Proliferation of spermato...