Vansandt Lindsey M, Livesay Janelle L, Dickson Melissa Joy, Li Lei, Pukazhenthi Budhan S, Keefer Carol L
Department of Animal and Avian Sciences, University of Maryland, College Park, Maryland, USA; Center for Species Survival, Smithsonian Conservation Biology Institute, Front Royal, Virginia, USA.
Department of Animal and Avian Sciences, University of Maryland, College Park, Maryland, USA.
Theriogenology. 2016 Sep 1;86(4):1022-1035.e3. doi: 10.1016/j.theriogenology.2016.03.031. Epub 2016 Apr 1.
Spermatogonial stem cells (SSCs) are distinct in their ability to self-renew, transmit genetic information, and persist throughout the life of an individual. These characteristics make SSCs a useful tool for addressing diverse challenges such as efficient transgenic production in nonrodent, biomedical animal models, or preservation of the male genome for species in which survival of frozen-thawed sperm is low. A requisite first step to access this technology in felids is the establishment of molecular markers. This study was designed to evaluate, in the domestic cat (Felis catus), the expression both in situ and following enrichment in vitro of six genes (GFRA1, GPR125, ZBTB16, POU5F1, THY1, and UCHL1) that had been previously identified as SSC markers in other species. Antibodies for surface markers glial cell line-derived neurotrophic factor family receptor alpha 1, G protein-coupled receptor 125, and thymus cell antigen 1 could not be validated, whereas Western blot analysis of prepubertal, peripubertal, and adult cat testis confirmed protein expression for the intracellular markers ubiquitin carboxy-terminal hydrolase 1, zinc finger and BTB domain-containing protein 16, and POU domain, class 5, transcription factor 1. Colocalization of the markers by immunohistochemistry revealed that several cells within the subpopulation adjacent to the basement membrane of the seminiferous tubules and identified morphologically as spermatogonia, expressed all three intracellular markers. Studies performed on cheetah (Acinonyx jubatus) and Amur leopard (Panthera pardus orientalis) testis exhibited a conserved expression pattern in protein molecular weights, relative abundance, and localization of positive cells within the testis. The expression of the three intracellular SSC marker proteins in domestic and wild cat testes confirms conservation of these markers in felids. Enrichment of marker transcripts after differential plating was also observed. These markers will facilitate further studies in cell enrichment and IVC of felid SSCs enabling both production of transgenic domestic cats and preservation of the male genome from rare and endangered felids.
精原干细胞（SSCs）在自我更新、传递遗传信息以及在个体整个生命周期中持续存在的能力方面具有独特性。这些特性使SSCs成为应对各种挑战的有用工具，例如在非啮齿类生物医学动物模型中高效生产转基因动物，或为冻融精子存活率低的物种保存雄性基因组。在猫科动物中应用这项技术的一个必要的第一步是建立分子标记。本研究旨在评估家猫（Felis catus）中先前在其他物种中被鉴定为SSC标记的六个基因（GFRA1、GPR125、ZBTB16、POU5F1、THY1和UCHL1）的原位表达以及体外富集后的表达情况。针对表面标记物胶质细胞系源性神经营养因子家族受体α1、G蛋白偶联受体125和胸腺细胞抗原1的抗体无法得到验证，而对青春期前、青春期周围和成年猫睾丸的蛋白质印迹分析证实了细胞内标记物泛素羧基末端水解酶1、锌指和BTB结构域包含蛋白16以及POU结构域、第5类、转录因子1的蛋白质表达。通过免疫组织化学对标记物进行共定位显示，在生精小管基底膜附近的亚群中，形态学上被鉴定为精原细胞的几个细胞表达了所有三种细胞内标记物。对猎豹（Acinonyx jubatus）和东北豹（Panthera pardus orientalis）睾丸进行的研究显示，在蛋白质分子量、相对丰度以及睾丸内阳性细胞的定位方面存在保守的表达模式。家猫和野猫睾丸中三种细胞内SSC标记蛋白的表达证实了这些标记在猫科动物中的保守性。在差异铺板后也观察到了标记转录本的富集。这些标记将有助于对猫科动物SSCs进行细胞富集和体外培养的进一步研究，从而实现转基因家猫的生产以及珍稀濒危猫科动物雄性基因组的保存。
