Characterization and In Vitro Culture of Male Germ Cells from Developing Bovine Testis
Characterization and In Vitro Culture of Male Germ Cells from Developing Bovine Testis
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DOI：
10.1262/jrd.10-185m
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发表时间：
2011-06-01
影响因子：
1.8
通讯作者：
Imai, Hiroshi
中科院分区：
文献类型：
--
作者：
Fujihara, Mayako;Kim, Sung-Min;Imai, Hiroshi
在新生儿睾丸中，男性原始生殖细胞(性细胞)向A型精原细胞的转变是精子发生的初始过程，也是一个关键过程。然而，在大型家畜中，生殖细胞在发育过程中的生理和生化特征仍然很大程度上是未知的。在这项研究中，我们描述了从新生期到成年期发育的睾丸中的牛生殖细胞。DBA凝集素和UCHL1泛素羧基末端水解酶1的表达仅限于新生儿睾丸的性腺细胞和成人睾丸的精原细胞。性细胞也表达生殖细胞标志物(VASA)和斯特细胞标志物(NANOG和Oct3/4)，而这些标志物在成人睾丸中的表达仅限于分化的精浆细胞，在精原细胞中很少表达。我们随后利用这些标记来鉴定体外培养后的性细胞和精原细胞。从成年睾丸采集的精原细胞在体外仅形成集落一周。而新生睾丸生殖细胞经过1.5个月的培养，每次传代后均能增殖并形成集落。生殖细胞和干细胞标记物的表达证实了这些克隆保留了性腺细胞的未分化状态。此外，使用免疫缺陷小鼠睾丸进行的移植试验表明，来自性腺细胞的长期培养细胞能够在受体睾丸中定植。这些结果表明，牛的性腺细胞在体外可以维持生殖细胞和斯特恩细胞的潜能。
The transition from male primitive germ cells (gonocytes) to type A spermatogonia in the neonatal testis is the initial process and a crucial process in spermatogenesis. However, in large domestic animals, the physiological and biochemical characteristics of germ cells during the developmental processes remain largely unknown. in this study, we characterized bovine germ cells in the developing testis from the neonatal stage to the adult stage. The binding of the lectin Dolichos biflorus agglutinin (DBA) and the expression of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) were restricted to gonocytes in the neonatal testis and spermatogonia in the adult testis. Gonocytes also expressed a germ cell marker (VASA) and stern cell markers (NANOG and OCT3/4), while the expressions of these markers in the adult testis were restricted to differentiated spermatic cells and were rarely expressed in spermatogonia. We subsequently utilized these markers to characterize gonocytes and spermatogonia after culture in vitro. Spermatogonia that were collected from the adult testis formed colonies in vitro only for one week. On the other hand, gonocytes from the neonatal testis could proliferate and form colonies after every passage for 1.5 months in culture. These colonies retained undifferentiated states of gonocytes as confirmed by the expression of both germ cell and stem cell markers. Moreover, a transplantation assay using immunodeficient mice testes showed that long-term cultured cells derived from gonocytes were able to colonize in the recipient testis. These results indicated that bovine gonocytes could maintain germ cell and stern cell potential in vitro.