In VitroCulture and Differentiation of Buffalo (Bubalus bubalis) Spermatogonia

Abstract

The objective of this study was to develop a culture system which could support buffalo spermatogonia differentiation into spermatids in vitro. Testes from 3- to 5-month-old buffaloes were decapsulated and seminiferous tubules were enzymatically dissociated to recover spermatogonia and sertoli cells. The cells were cultured in modified Dulbecco modified Eagle medium supplemented with different concentrations of foetal bovine serum, retinol, testosterone for 2 months at 37 degrees C. Spermatogonia and sertoli cells were identified with an antibody against c-kit or GATA4, respectively. The viability of spermatogonia in the media supplemented with different concentrations of serum was all significantly higher (p < 0.05) compared with that in the medium without serum. A-paired or A-aligned spermatogonia and spermatogonial colonies (AP-positive) were observed after 7-10 days of culture and spermatid-like cells with a flagellum (6-8 microm) appeared after 30 days of culture. For cultured conditions, retinol could not significantly promote the formation of spermatid-like cells (p > 0.05), whereas supplementation of testosterone could significantly promote (p < 0.05) the formation of spermatid-like cells after 41 days of culture. The expression of the spermatid-specific marker gene (PRM2) was identified after 30 days of culture by RT-PCR. Yet, the transition protein 1 (TP1, a haploid makers) was not detected. Meanwhile, spermatids developed in vitro were also confirmed by Raman spectroscopy. These results suggest that buffalo spermatogonia could differentiate into spermatids in vitro based on the analysis of their morphology, PRM2 expression and Raman spectroscopy. Yet, the normality of the spermatid-like cells was not supported by TP1 expression.

Fact or fiction

“…Both FSH and testosterone support the differentiation of germ cells most likely via Sertoli cells, pointing out the importance of both, the presence of somatic cells supporting germ cells in culture and the need for endocrine signaling. This demonstrated for the first time that early meiotic mammalian germ cells can complete meiosis into haploid cells in vitro 26 , 28 - 30 . In rats, FSH and testosterone enhance the number of round spermatids in the culture of middle and late pachytene spermatocytes when cocultured with Sertoli cells.…”

Isolation and Identification of Prepubertal Buffalo (<i>Bubalus bubalis</i>) Spermatogonial Stem Cells

“…Cell isolation was performed as described previously (Xie et al, 2010). Briefly, testes from 3 to 7 age of months buffalo were immediately washed several times by PBS containing penicillin (100 U/mL), and streptomycin (100 μg/mL).…”

“…Oct-4 (also as knows Pou5fl), THY-1, c-kit, PGP9.5 (also as knows UCHL-1) and Dolichos biflorus agglutinin (DBA) are used to identify bovine (Izadyar et al, 2002; Oatley et al, 2004; Reding et al, 2010), goat (Heidari et al, 2012; Abbasi et al, 2013), sheep (Rodriguez-Sosa et al, 2006), pig (Lee et al, 2013; Lee et al, 2014), dog (Harkey et al, 2013), mouse and human (von Kopylow et al, 2010) and chicken SSCs (Momeni-Moghaddam et al, 2014; Sisakhtnezhad et al, 2015). Recently, Oct-4 and c-kit were reported to be experessed in buffalo testis and spermatogonail stem cells (Xie et al, 2010; Yu et al, 2014). In the further, it is essential for determination the morphological, physiological and stem cell potential through various markers and different biological methods.…”

Spermatogonial stem cells from domestic animals: progress and prospects

“…By contrast, even though researchers have made strenuous efforts to culture SSCs from domestic animals, the propagation of SSCs in vitro could only be maintained for a short time (no longer than 2 months; Aponte et al 2006, Luo et al 2006, Goel et al 2007, 2009, Kuijk et al 2009, Xie et al 2010, Fujihara et al 2011, Heidari et al 2012, Kala et al 2012, Nasiri et al 2012, Zhu et al 2012, Kadam et al 2013, Zheng et al 2013b. As shown in relevant reports, putative SSCs in domestic testes can be isolated and primary cultured with ease, but the proliferation of putative SSCs experiences a gradual decrease during sub-culture, and over time, differentiation and apoptosis dominate the cellular events, with the final consequence that the propagation of SSCs comes to a standstill.…”

“…At present DMEM (high glucose) and DMEM/F12 are the most widely used media in cultures of SSCs from domestic animals (Luo et al 2006, Goel et al 2007, 2009, Xie et al 2010, Fujihara et al 2011, Heidari et al 2012, Kala et al 2012, Nasiri et al 2012, Zhu et al 2012, Kadam et al 2013, Zheng et al 2013b, probably due to the availability and affordability of the media. The corresponding studies, on the other hand, show that DMEM, in all likelihood, promotes both selfrenewal and differentiation of SSCs.…”