Conservation of spermatogonial stem cell marker expression in undifferentiated felid spermatogonia

Abstract

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.

Molecular markers of putative spermatogonial stem cells in the domestic cat

“…Furthermore, in the mouse and several domestic species, PLZF was expressed in both gonocytes and undifferentiated spermatogonia (Costoya et al., ; Zheng et al., ) and was essential for self‐renewal and maintenance of the stem cell pool (Costoya et al., ). In our study, immunostaining for PLZF in cat sections revealed a similar distribution pattern of positive cells as reported in human (Wu et al., ), mouse (Gassei & Orwig, ; Wu et al., ), bull (Reding et al., ) or cat (Vansandt et al., ) testis. In fact, results for foetal and prepuberal testes were strikingly similar to those observed in other species (Costoya et al., ; Gassei & Orwig, ; Wu et al., ).…”

“…In fact, results for foetal and prepuberal testes were strikingly similar to those observed in other species (Costoya et al., ; Gassei & Orwig, ; Wu et al., ). However, in adult testis, a higher proportion of cells might have been marked by PLZF herein than in a recent study (Vansandt et al., ). The inability to combine PLZF and DDX4 analysis on the same sections precluded definitive identification of potential stem germ cells.…”

“…Additionally, the speckled appearance of PLZF labelling and specific nuclear localization of the protein further strengthened our assumptions (Suliman, Xu, & Williams, ; Wang et al., ). Thus, differences in cell proportions labelled between this and previous studies may relate to different antibodies used possibly recognizing different protein isoforms (Vansandt et al., ). More studies are required to solve such discrepancies in the feline model.…”

“…Indeed, EPCAM (‐) cells along with prepuberal and foetal testis homogenates showed higher relative gene expression levels of purported SSC markers such as POUF51 , CD9 , PLZF and GFRA1 . Notably, a recent report also supports the expression of POUF51 and PLZF by feline spermatogonia, both at the gene and protein levels (Vansandt et al., ). There are reported species differences regarding EPCAM expression profiles in testis tissue.…”

Systematic identification and comparison of expressed profiles of lncRNAs and circRNAs with associated co-expression and ceRNA networks in mouse germline stem cells

“…Germline stem cells belong to adult stem cells, and they possess the ability that transmit genetic information from generation to generation [1–5]. Mammalian male germline stem cells, also know as spermatogonial stem cells (SSCs), were identified in the 1950s [2, 6, 7].…”