Identification, isolation, and in vitro culture of porcine gonocytes
Identification, isolation, and in vitro culture of porcine gonocytes
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DOI：
10.1095/biolreprod.106.056879
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发表时间：
2007-07-01
影响因子：
3.6
通讯作者：
Imai, Hiroshi
中科院分区：
文献类型：
--
作者：
Goel, Sandeep;Sugimoto, Miki;Imai, Hiroshi
生殖母细胞是位于新生儿睾丸生精小管中的原始生殖细胞，产生精原细胞，从而启动精子发生。由于缺乏特异性标记物，这些细胞的分离和培养已被证明在猪中是困难的。在本研究中，我们表明，凝集素，双花扁豆凝集素（DBA），它具有特定的亲和力，生殖嵴中的原始生殖细胞（PCGs），结合特异性的生殖细胞在新生猪睾丸。DBA对生殖细胞的特异性亲和力随着年龄的增长而逐渐丧失。这表明DBA与原始生殖细胞（如生殖母细胞）结合强烈，与原始精原细胞结合较弱，而与精原细胞完全不结合。新生猪睾丸生殖细胞中碱性磷酸酶（AP）活性的存在证实了原始生殖细胞的存在。纯化来自新生猪睾丸的生殖细胞，并获得由约70%的生殖细胞组成的细胞群，如DBA结合测定所示。在不存在任何特异性生长因子的情况下，将纯化的生殖细胞在补充有10%FBS的DMEM/F12中培养7天。细胞在培养中保持活力并活跃地增殖。最初，生殖细胞生长为病灶集落，培养7天后转化为三维集落。培养的生殖细胞表达SSEA-1，胚胎干细胞（ES）的标志物，并为阴性的体细胞标志物的表达。这些结果将有助于建立一个雄性生殖细胞系，可用于研究精子发生在体外和遗传修饰的猪。
Gonocytes are primitive germ cells that reside in the seminiferous tubules of neonatal testes and give rise to spermatogonia, thereby initiating spermatogenesis. Due to a lack of specific markers, the isolation and culture of these cells has proven to be difficult in the pig. In the present study, we show that a lectin, Dolichos biflorus agglutinin (DBA), which has specific affinity for primordial germ cells (PCGs) in the genital ridge, binds specifically to gonocytes in neonatal pig testes. The specific affinity of DBA for germ cells was progressively lost with age. This suggests that DBA binds strongly to primitive germ cells, such as gonocytes, weakly to primitive spermatogonia, and not at all to spermatogonia. The presence of alkaline phosphatase (AP) activity in the germ cells of neonatal pig testis confirmed the existence of primitive germ cells. Gonocytes from neonatal pig testis were purified, and a cell population that consisted of approximately 70% gonocytes was obtained, as indicated by the DBA binding assay. Purified gonocytes were cultured in DMEM/F12 supplemented with 10% FBS in the absence of any specific growth factors for 7 days. The cells remained viable and proliferated actively in culture. Initially, the gonocytes grew as focal colonies that transformed to three-dimensional colonies by 7 days of culture. Cultured germ cells expressed SSEA-1, a marker for embryonic stem (ES) cells, and were negative for the expression of somatic cell markers. These results should help to establish a male germ cell line that could be used for studying spermatogenesis in vitro and for genetic modification of pigs.