Isolation, proliferation, and induction of Bama mini-pig spermatogonial stem cells in vitro

Abstract

ABSTRACT. Spermatogonial stem cells (SSCs), the unique seed cells of testes, can undergo meiosis and form spermatozoa, thus transmitting genetic information to offspring. Research concerning these cells explores the mechanism underlying spermatogenesis, making possible the induction of their differentiation into spermatozoa in vitro. SSCs have therefore attracted much interest among scientists. Although the proliferation of such cells in vitro has been demonstrated, we are unaware of any long-term laboratory culture of porcine SSCs. The objective of this study was to isolate, characterize, culture, and induce the differentiation of Bama mini-pig SSCs. SSCs were isolated using differential plating and cultured for over 100 days on an STO feeder cell layer without serum. Cell clusters appeared after three passages and continuously formed during subsequent cultivation. Staining showed that these clusters were positive for UCHL1 and CDH1, could be bound by Dolichos biflorus agglutinin, and that some cells expressed OCT4. Ultrastructure observations revealed SSCs in testis tissue to be round in shape, while those cultured in vitro were flat and bound together. Our attempts at inducing differentiation showed that SSCs cultured in vitro could undergo meiosis. In this study, we describe an effective culture system for Bama mini-pig SSCs capable of producing enough cells to establish a platform for further SSC research, such as genetic manipulation or exploration of the mechanism underlying spermatogenesis.

Localization and characterization of SSCs from pre-pubertal bovine testes

“…1). In 3 months old testes, type A spermatogonia (Zhao et al 2016). In 5 months old testis, DBA positive cells were located both in basement membrane and just next to the basement membrane.…”

“…2). UCHL1 expression is restricted to spermatogonial stem cells of prepubertal bull (Herrid et al 2007, Reding et al 2010, cat (Vansandt 2014), prepubertal buffalo (Goel et al 2010) and porcine (Zhao et al 2016). However, in neonatal and adult bovine testis, UCHL1 is expressed in gonocytes and spermatogonia (Fujihara et al 2011).…”

In vitro differentiation of spermatogonial stem cells using testicular cells from Guangxi Bama mini-pig

“…Porcine testicular cells were cultured in vitro and porcine SSC-like colonies could be maintained for 9 passages [ 17 ]. Previously, we reported that SSCs of Bama mini-pig could be maintained for up to 3 months on SIM mouse embryo derived thioguanine- and ouabain-resistant (STO) feeder layer [ 28 ]. However, the cultured SSCs were influenced by unknown factors from serum and feeder layer cells, and no previous report has provided evidence that long-term-cultured SSCs from pig can maintain spermatogenesis potential.…”

The effects of growth factors on proliferation of spermatogonial stem cells from Guangxi Bama mini‐pig

“…Isolation of the SSCs was performed as our previous study (Zhao et al, ). Generally, the testes were obtained from 1 month Bama mini‐pigs at the farm and transported to the laboratory on ice.…”

“…The testes from 1 and 2 months pigs were subjected to immunohistochemical analysis. The staining process was performed as our previous report (Zhao et al, ). Briefly, the testes were cut into small pieces, fixed with Bouin's fixative for 12 hr at room temperature, dehydrated, embedded in paraffin and sectioned into slices (4 µm thick).…”