In vitro culture and characterization of spermatogonial stem cells on Sertoli cell feeder layer in goat (Capra hircus)

Abstract

Purpose To develop an efficient protocol for isolation, purification and long-term culture of spermatogonial stem cell (SSC) in goat. Methods The isolation of SSC was performed by testicular disaggregation by enzymatic digestion using collagenase IV, trypsin and DNase I. Further SSCs were enriched using Percoll density gradient centrifugation. The purity of SSCs was assessed by immunocytochemistry (ICC) using α6 integrin. The SSCs were co-cultured on Sertoli cell feeder layer. The SSC colonies were characterized by studying the expression of SSC specific markers (viz., α6 integrin and PLZF) using ICC. The abundance of mRNAs encoding the markers of SSC (viz., β1 integrin and Oct-4) and Sertoli cells (viz., vimentin) was also assayed using quantitative real-time PCR (qPCR). Results The viability of isolated testicular cells was>90 % and the Percoll density gradient method resulted in 3.65 folds enrichment with a purity of 82.5 %. Co-culturing of SSCs with Sertoli cell feeder layer allowed the maintenance of stable SSC colonies even after one and half months of culture. The results of ICC analysis showed the expression of α6 integrin and PLZF in almost all the SSC colonies. qPCR analysis revealed a differential expression of mRNAs encoding β1 integrin, Oct-4 and vimentin markers. Conclusion Results of this study demonstrate a simple enzymatic digestion and Percoll density gradient method for isolation and enrichment of SSCs, and suitability of Sertoli cell feeder layer for long term in vitro culture of SSC in goats.Results also suggest the possible application of non-caprine antibodies against SSC specific markers for the identification and subsequent assessment of SSCs in goats.

Derivation, enrichment and characterization of goat (Capra hircus) spermatogonial stem cells from pre-pubertal testes

“…1). This duration was similar to that reported for mice (Kanat et al (Pramod and Mitra 2014). The colonies probably start to develop when spermatogonia and sertoli cells make contact, apparently creating a microenvironment that favours their development.…”

“…Among the transcription factors, PLZF expression has been reported in gonocytes and undifferentiated spermatogonia and has been shown to be essential for spermatogonial stem cell maintenance and self-renewal in mouse (Luo et al 2006), goat (Pramod and Mitra 2014) cattle (Reding et al 2010) and buffalo (Kadam et al 2012). THY1 has been shown to be a unique surface marker of SSCs in mice (Kubota et al 2004b), cattle (Reding et al 2010), buffalo (Kadam et al 2012) and goat (Abbasi et al 2013), while UCHL1 is expressed in pre-meiotic male germ cells.…”

In vitro culture of spermatogonial stem cells isolated from adult alpaca (Vicugna pacos) testes analysed withDolichos biflorusby flow cytometry

“…Koruji, Movahedin, Mowla, Gourabi, and Arfaee () performed a co‐culture of SSC with Sertoli cells in mice, and considered that the contact of these cells and the secretion of growth factors allowed a satisfactory environment that promotes the SSC proliferation. Likewise, Pramod and Mitra () demonstrated the improvement in the proliferation of goat SSC, by co‐cultivating these cells with Sertoli cells. In our case of alpaca spermatogonial cells, they were co‐cultured with Sertoli cells, allowing a suitable microenvironment to be produced for the proliferation of SSCs.…”

“…Furthermore, some studies highlight the importance of others testicular cells, such as Sertoli cells, that compound the cellular niche of SSCs in maintaining their self‐renewal potential, due to either the factors produced by them or cell–cell interactions. In this sense, the co‐culture of SSCs with cells that produce these factors or these niche cells has been already used to increase the proliferative potential of SSCs in species such as mice (Hamidabadi & Bojnordi, ), porcine (Lee et al, ) or goat (Pramod & Mitra, ).…”

Long‐term culture of undifferentiated spermatogonia isolated from immature and adult bovine testes

“…Undifferentiated spermatogonia including SSCs from neonatal and pre-pubertal stage of domesticated animal such as pig (Zhang et al, 2017;Zheng et al, 2013) and goat (Pramod & Mitra, 2014) can also be cultured in vitro. In cattle, although gonocytes and SSCs isolated from neonatal and immature bovine testes can be maintained in culture (Aponte, Soda, van de Kant, & de Rooij, 2006;Fujihara et al, 2011;Izadyar et al, 2002;Oatley, Kaucher, Yang, Waqas, & Oatley, 2016;Sahare et al, 2016), long-term culture systems for SSCs from adult testes have been established only in mice.…”