山羊精原干细胞体外培养分化

Culture and Differentiation of Goat Spermatogonial Stem Cells

探索山羊精原干细胞体外培养体系。收集2月龄关中奶山羊睾丸,一步酶法消化分离曲细精管细胞,台盼兰检测平均存活率82.7%,以1×106个/ml接种含15%胎牛血清DMEM/F12培养瓶,37℃、5%CO2和饱和湿度条件下培养,4周后FBS逐渐降至10%。原代培养以多突起和片状的睾丸体细胞铺壁生长为主,10天左右精原干细胞数量增加,可见二联体和四联体,3周左右有鸟巢状和山脉状集落形成,碱性磷酸酶染色阳性,培养30天集落数不断增加,散在分布有贴壁和漂浮精子,换液后精子丢失。挑取单集落重新接种铺壁的曲细精管体细胞饲养层后陆续有精子细胞及精子形成,主要分布于集落周围。

The objective of this study was to develop a practical in vitro culture system of goat spermatogo- nial stem cells (SSCs) to facilitate further studies on transgenesis. 12 testis from 2-month-old bucks (GuanZhong milk goat) were decapsulated and dissociated enzymatically by one step to recover seminiferous tubule cells. 1×106 cells/ml with a viability 82.7% was cultured in 25 cm2 flasks containing DMEM/F12 supplemented with 15% fetal bovine serum (FBS) for first 4 week, less FBS after each medium exchange thereafter with 10% FBS as final concentration at 37 ℃ in a humidified atmosphere with 5% CO2. During the first two week of culture, the number of SSCs declined then increased, pairs and chains of spermatogonia appeared ,in the third and fourth week, bird- nest-like and mountain-like colonies were observed with positive alkaline phosphatase (AKP) staining. During the second month of culture, more and larger colonies were found, there were anchored and free elongate-spermatid- like cells around colonies, selected the colonies and cultured them confluent monolayer of Sertoli cells, spermatid were formed around. For the first time to our knowledge, goat spermatogonial stem cells have been cultured and differentiated into spermatids in vitro, this culture system would provide targeted cells for tansgenesis, greatly accelerate the research on goat spermatogenesis and utilization of excellent male gene resources.