Luo Jinping, Megee Susan, Rathi Rahul, Dobrinski Ina
Department of Clinical Studies, Center for Animal Transgenesis and Germ Cell Research, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, 19348, USA.
Mol Reprod Dev. 2006 Dec;73(12):1531-40. doi: 10.1002/mrd.20529.
Identification and isolation of spermatogonial stem cells (SSCs) are a prerequisite for culture, genetic manipulation, and/or transplantation research. In this study, we established that expression of PGP 9.5 is a spermatogonia-specific marker in porcine testes. The expression pattern of PGP 9.5 in spermatogonia was compared to cell type-specific protein (GATA-4 or PLZF) expression in seminiferous tubules at different ages, and expression levels of PGP 9.5, Vasa, and Oct-4 were compared in different cell fractions. Enrichment of spermatogonia from 2-week-old (2wo) and 10-week-old (10wo) boars by adhesion to laminin, differential plating, or velocity sedimentation followed by differential plating was assessed by identification of spermatogonia using expression of PGP 9.5 as a marker. Compared to the initial samples, spermatogonia were enriched twofold in laminin-selected cells (P < 0.05), and fivefold either in cells remaining in suspension (fraction I) or in cells slightly attached to the culture dish (fraction II) (P < 0.05) after differential plating. Cells in fraction II appeared to be superior for future experiments due to higher viability (>90%) than in fraction I ( approximately 50%). Velocity sedimentation plus differential plating achieved cell populations containing up to 70% spermatogonia with good viability (>80%). Enriched spermatogonia from 2wo and 10wo testes could be maintained in a simple culture medium without additional growth factors for at least 2 weeks and continued to express PGP 9.5. These data provide the basis for future studies aimed at refining conditions of germ cell culture and manipulation prior to germ cell transplantation in pigs.
精原干细胞（SSCs）的鉴定与分离是其培养、基因操作和/或移植研究的前提条件。在本研究中，我们确定了PGP 9.5的表达是猪睾丸中精原细胞特异性标志物。将精原细胞中PGP 9.5的表达模式与不同年龄生精小管中细胞类型特异性蛋白（GATA - 4或PLZF）的表达进行比较，并比较了不同细胞组分中PGP 9.5、Vasa和Oct - 4的表达水平。通过以PGP 9.5的表达作为标志物鉴定精原细胞，评估了通过与层粘连蛋白粘附、差异铺板或速率沉降后再差异铺板从2周龄（2wo）和10周龄（10wo）公猪中富集精原细胞的情况。与初始样本相比，层粘连蛋白选择的细胞中精原细胞富集了两倍（P < 0.05），差异铺板后，悬浮细胞（组分I）或轻微附着于培养皿的细胞（组分II）中的精原细胞富集了五倍（P < 0.05）。由于组分II中的细胞活力（>90%）高于组分I（约50%），因此组分II中的细胞似乎更适合未来的实验。速率沉降加差异铺板获得了含有高达70%精原细胞且活力良好（>80%）的细胞群体。从2wo和10wo睾丸中富集的精原细胞可以在无额外生长因子的简单培养基中维持至少2周，并持续表达PGP 9.5。这些数据为未来旨在优化猪生殖细胞移植前生殖细胞培养和操作条件的研究提供了基础。
