Phospholipase D Family Member 6 Is a Surface Marker for Enrichment of Undifferentiated Spermatogonia in Prepubertal Boars

Abstract

Spermatogonial stem cells (SSCs) have a functional ability to maintain self-renewal and sustain production of spermatozoa throughout the reproductive lifespan of a male. Studies on SSCs can thus better the understandings of spermatogenesis and unravel the mechanisms for self-renewal and differentiation of male germline stem cells. However, the rarity of SSCs in the testis and the lack of reliable surface markers obstruct the related study and further application of SSCs. This is especially the case in livestock animals. In this study, we identified that phospholipase D family member 6 (PLD6) is a surface marker for undifferentiated spermatogonia in boar testes. By magnetic-activated cell sorting, PLD6⁺ cell fraction comprises 84.45% ± 0.35% of undifferentiated spermatogonia (marked by PLZF). Xenotransplantation of PLD6⁺ cells into the recipient mouse testis revealed a ninefold increase of donor cell-derived colony formation compared with that in the unselected cell group, indicating the significant enrichment of SSCs. Furthermore, based on the sorted PLD6⁺ cells with a high SSC content, we established a feeder-free culture system that could maintain porcine undifferentiated spermatogonia for 4 weeks in vitro with the expression of typical markers throughout the culture period. In conclusion, this study demonstrates that PLD6 is a surface marker of undifferentiated spermatogonia in testes of prepubertal boars and could be utilized to unprecedentedly enrich porcine undifferentiated spermatogonia. These data provide the basis for future studies on the refinement of germ cell culture and manipulation of porcine undifferentiated spermatogonia.