Li Zhendong, Li Mingyou, Hong Ni, Yi Meisheng, Hong Yunhan
Department of Biological Sciences, National University of Singapore, Science Drive 4, Singapore, 117543, Singapore.
Cell Tissue Res. 2014 Jul;357(1):71-81. doi: 10.1007/s00441-014-1867-z. Epub 2014 Apr 29.
Primordial germ cell (PGC) formation is pivotal for fertility. Mammalian PGCs are epigenetically induced without the need for maternal factors and can also be derived in culture from pluripotent stem cells. In egg-laying animals such as Drosophila and zebrafish, PGCs are specified by maternal germ plasm factors without the need for inducing factors. In these organisms, PGC formation and cultivation in vitro from indeterminate embryonic cells have not been possible. Here, we report PGC formation and cultivation in vitro from blastomeres dissociated from midblastula embryos (MBEs) of the fish medaka (Oryzias latipes). PGCs were identified by using germ-cell-specific green fluorescent protein (GFP) expression from a transgene under the control of the vasa promoter. Embryo perturbation was exploited to study PGC formation in vivo, and dissociated MBE cells were cultivated under various conditions to study PGC formation in vitro. Perturbation of somatic development did not prevent PGC formation in live embryos. Dissociated MBE blastomeres formed PGCs in the absence of normal somatic structures and of known inducing factors. Most importantly, under culture conditions conducive to stem cell derivation, some dissociated MBE blastomeres produced GFP-positive PGC-like cells. These GFP-positive cells contained genuine PGCs, as they expressed PGC markers and migrated into the embryonic gonad to generate germline chimeras. Our data thus provide evidence for PGC preformation in medaka and demonstrate, for the first time, that PGC formation and derivation can be obtained in culture from early embryos of medaka as a lower vertebrate model.
原始生殖细胞（PGC）的形成对于生育能力至关重要。哺乳动物的PGC是通过表观遗传诱导产生的，无需母体因子，并且也可以从多能干细胞在体外获得。在果蝇和斑马鱼等产卵动物中，PGC由母体生殖质因子指定，无需诱导因子。在这些生物体中，无法从不确定的胚胎细胞中进行PGC的体外形成和培养。在此，我们报告了从青鳉（Oryzias latipes）囊胚中期胚胎（MBE）解离的卵裂球中进行PGC的体外形成和培养。通过使用在vasa启动子控制下的转基因表达生殖细胞特异性绿色荧光蛋白（GFP）来鉴定PGC。利用胚胎扰动来研究体内PGC的形成，并在各种条件下培养解离的MBE细胞以研究体外PGC的形成。体细胞发育的扰动并未阻止活胚胎中PGC的形成。解离的MBE卵裂球在没有正常体细胞结构和已知诱导因子的情况下形成了PGC。最重要的是，在有利于干细胞衍生的培养条件下，一些解离的MBE卵裂球产生了GFP阳性的PGC样细胞。这些GFP阳性细胞含有真正的PGC，因为它们表达PGC标记并迁移到胚胎性腺中以产生种系嵌合体。因此，我们的数据为青鳉中PGC的预形成提供了证据，并首次证明，作为一种低等脊椎动物模型，可以从青鳉的早期胚胎在培养中获得PGC形成和衍生。
