Isolation And Culturation Of Dairy Goat Male Germline Stem Cells And Induced Differentiation To Sperm Cells

Male germline stem cells are settled in the basement membrane of testis seminiferous tubules. It's the foundation of steady spermatogenesis throughout the whole male life. It can be cultured in vitro for long-term stabily proliferation, induced to differentiate into sperm cells and to dedifferentiate into pluripotent stem cells. Male germline stem cells bring hopes for many problems, such as male infertility and obtaining of pluritent stem cells. The mechanism of dairy goat male germline stem cells'proliferation and differentiation is still largely unclear. Systematic articles on separation and culture of dairy goat germline stem cells are few, which also caused a considerable obstacle for the study of male germline stem cells in the dairy goat.This study compared the conditions to isolate and culture dairy goat male germline stem cells; Test the biological characteristics and biological functions of dairy goat germline stem cells; Induced cells to differentiate into sperm cells by retinoic acid stimulation and transfection of Stra8 gene, verified the possibility of the cells to differentiate into sperm cells in vitro.1. Optimization of the isolation and culture conditions of dairy goat male germline stem cells.In this study, we found digesting the dairy goat testis tissues with 2 mg/mL collagenase +20μg/mL DNase +2 mg/mL Disperse cocktail enzyme (Dispase) (CDD) combined 1 mg/mL trypsin (Trypsin) +10μg/mL DNase (TD) can get sufficient amount and high survival rate of cells; Purify the germline stem cells with 1μg/mL fibronectin (Fibronectin, FN) for 2h differential culture can get dairy goat male germline stem cells with hihg purity (49%); Culture dairy goat male germ stem cells with DM/F12 +20% KSR base fluid added with 2 mmol/L Glu + 0.2 mmol/Lβ-ME + 1% NEAA + 1000 IU/mL LIF + 4 ng/mL bFGF at 37℃, 5% CO2 and saturated humidity conditions found the cells with excellent capacity in colony formation, proliferation and tissue non-specific alkaline phosphatase staining; The age of dairy goat influence the culture efficiency of male germline stem cells, dairy goat male germline stem cells would obtained easier if the dairy goat is younger; cultured with bFGF combined with BIO, the cells may have better capacity in proliferation and colony formation.2. Identification of the biological characteristics of dairy goat male germline stem cells.In this study, we obtained dairy goat male germline stem cells with better proliferative ability. Immunofluorescence staining and RT-PCR determination found that the cells express pluripotency, proliferation and germ cells'markers. The cells can also differentiate into three germ layers cells.3. Attemptation of inducing dairy goat male germline stem cells to sperm cells in vitro.In this study, we found dairy goat germline stem cells have the stimulatory effects of RA. RA stimulated gene Stra8 and meiosis asscioated gene expression was obviously increased. Meiosis genes and genes after meiosis expression increased obviously after Stra8 gene transfected. This shows that dairy goat male germline stem cells can differentiate into sperm cells in vitro.