水牛精原细胞的体外培养及分化

Buffalo spermatogonial culture and differentiation in vitro

以探讨水牛精原细胞体外培养的发育潜能为目的,对3～5月龄水牛睾丸组织进行精原细胞与支持细胞的体外共培养。两步酶法消化睾丸组织来制备水牛生殖细胞悬液,接种于含10%FBS的DMEM培养液中,在37℃、5%CO2、饱和湿度下培养30d,观察细胞的生长和形态变化,并对培养4周的细胞进行RT-PCR分析,以检测PRM-2和TP-1基因的表达情况。结果显示,水牛精原细胞体外培养24h后,精原细胞(10.0～12.5μm)呈圆形并紧贴支持细胞上;培养7～8d后,精原细胞出现聚集状态;培养10d后,有细胞克隆形成;培养30d后,出现似长形精子细胞(8.0～10.0μm)。对培养4周后的细胞进行RT-PCR分析,精子细胞特异表达基因PRM-2被检测出来。结果表明,采用本试验体外培养条件对水牛精原细胞进行培养,能够满足精原细胞体外长期培养的需要,并可以发生增殖与分化而形成似精子细胞。

To explore a developmental potential of buffalo spermatogonial culture in vitro, 3-5-month-old buffaloes were used to spermatogonial and sertoli cells co-culture in vitro. The germ cells were obtained by two-step enzymatic digestion and were cultured in DMEM medium supplemented with 10% fetal bovine serum （FBS） for 30 d at 37℃ in a humidified atmosphere with 5% CO2. Growth and morphologic changes of ceils were observed during cul- ture and cultured cells at 4 weeks were examined by reverse transeription-polymerase chain reaction （RT-PCR） for protamine-2 （PRM-2） and transition protein-1 （TP-1） mRNA. The result showed that buffalo spermatogonial（10.0- 12.5μm） were round and adhered to sertoli cells at 24 h later. After 1 weeks of culture, spermatogonial were congregating growth. Clones were formed after 10 days and elongated spermatid-like cells（8.0-10.0 μm） were observed after 30 days of culture. The expression of the spermatid-specifie marker gene,PRM-2,was confirmed after 4 weeks of culture by RT-PCR. In conclusion, the culture conditions could sustain the survival and allow the proliferation and differentiation of buffalo spermatogonial during long-term culture in vitro, and ultimately spermatid-like cells were formed.