Study On The Character Of In Vitro Separaction And Culturation And Transplantation Of Bovine Male Germ Stem Cells

Male germ stem cells(mGSCs)settle in testicular seminiferous tubules after the differentiation of genital gland and is the sole kind of adult stem cells which can transfer genetic matter to offspring. Some investigators had researched the cytology characters, separation and purifying, the in vitro cultural system, in vitro induction into haploid cells or spermatozoa, gene modification and transplantation of the mGSCs into homogeneous and heterogeneous testis and reached new achievements. But few results of investigation about neonatal bovine mGSCs were reported.According to previous investigations, the following contents were studied in the thesis: development of neonatal bovine testes and mGSCs, different methods and effects of separation and purifying on mGSCs, establishment of cultural systems of mGSCs and observation of in vitro behaviors of mGSCs, directional induction and differentiation of mGSCs into spermatozoa-like cells, analysis of formation and in vitro differentiation potential of ES-like cells from neonatal bovine mGSCs,and finally,the assay of tissues difference between testis tissues of model rabbits deficient in endogenic germ cells and those of model rabbits transplanted with neonatal bovine mGSCs. The conclusion showed as the follows:Bovine testis tissues were observed from 6-week fetuses to neonatal calf. The results demonstrated that male germ cells began to form clone at 6~10 w; and seminiferous tubules appeared at 15 w; and more germ cells located at center of seminiferous tubules at 20 w; and sertoli cells and germ cells located at sub-center of seminiferous tubules and leydig cells was less among seminiferous tubules in neonatal testis.Neonatal testis tissues were procedurally treated through 0.1% collagenase 30 min and 0.25% trypsin +0.04% EDTA+5 ng/mL DNaseI 5 min, and single cells and small cells masses were acquired. Activity and purification degree of mGSCs is better through 3 times isolation with adhesion than through percoll density gradient centrifugation,reaching an activity and purifying of 76.7% and 98.2% respectively. Through 3 times adhering isolation, 2 kinds of mGSCs were acquired,including cells diameter less than 15μm and over 25μm. The remarkable character of the former was cytoplasm fluidity were about 85% of total cells and...