THY1 is a surface marker of porcine gonocytes

Abstract

Gonocytes are important for the study of spermatogenesis. Identification and isolation of gonocytes has been reported in rodents but not in pigs due to a lack of molecular markers for gonocytes. The objective of this study was to identify THY1 expression in porcine testicular tissue and subsequently utilise THY1 as a marker to isolate and enrich porcine gonocytes from testes of newborn piglets. Immunohistochemical analysis showed that THY1 was expressed in gonocytes. Double-immunofluorescent analysis of THY1 and ZBTB16 indicated that THY1 and ZBTB16 were partially co-localised in gonocytes. Double-immunofluorescent analysis of both THY1 and GATA4 suggested that THY1(+) cells were not Sertoli cells. Magnetic-activated cell sorting of THY1(+) cells yielded a cell population with an enrichment of UCHL1(+) gonocytes 3.4-fold of that of the unsorted testicular cell population. Western blot and quantitative reverse transcription-polymerase chain reaction analyses confirmed that the selected THY1(+) fraction had a higher expression of UCHL1 than the unsorted cells. In conclusion, the study demonstrated that THY1 is a surface marker of gonocytes in testes of pre-pubertal boars and could be utilised to identify and isolate porcine gonocytes. The findings will also facilitate culture and manipulation of male germline stem cells.

The histone methyltransferase ESET is required for the survival of spermatogonial stem/progenitor cells in mice

“…MACS with anti-Thy1 IgG has been successfully enriched SSCs in our laboratory and the other groups. 5, 48 The Thy1-positive (as a marker of SSCs) cells were as high as 92% in the MACS-sorted cells (Figures 2a and b). Short hairpin RNAs can be expressed from lentiviruses, allowing for high transfection efficiency of a variety of cell types, including non-dividing cells and stem cells.…”

Effect of sucrose on cryopreservation of pig spermatogonial stem cells

“…The single cell suspensions were then pelleted by centrifugation at 500×g for 5 min at 25°C, and resuspended in DMEM/F12 supplemented with 2% (v/v) fetal bovine serum (FBS, Gibco, UK). Then, the isolated testicular cells were subjected to differential plating three times to purify pSSCs (Zheng et al 2014a). Briefly, the process of differential plating was as follows: the freshly isolated cells were cultured at 37°C in a humidified atmosphere of 5% CO 2 in air for 2 h in complete culture medium containing DMEM/ F12 with 2% FBS, and then unattached cells were collected and reseeded in new dishes for another 6 h. The unattached cells were collected once more and replated in new dishes for another 12 h. The target cells were collected by gently triturating the dishes with a pipette after rinsing with DPBS.…”

Spermatogonial stem cells: Current biotechnological advances in reproduction and regenerative medicine

“…That is the case of the widely accepted marker GFRα1, the receptor for GDNF in SSCs [43] . In domestic animals, besides GFRα1, another important marker for both gonocytes (perinatal germline stem cells) and SSCs is THY1 [44][45][46] . are able to generate very diverse tissues while in cell culture [1,3] .…”