Spermatogonial Stem Cells (SSCs) in Buffalo (Bubalus bubalis) Testis

Abstract

BackgroundWater buffalo is an economically important livestock species and about half of its total world population exists in India. Development of stem cell technology in buffalo can find application in targeted genetic modification of this species. Testis has emerged as a source of pluripotent stem cells in mice and human; however, not much information is available in buffalo.Objectives and MethodsPou5f1 (Oct 3/4) is a transcription factor expressed by pluripotent stem cells. Therefore, in the present study, expression of POU5F1 transcript and protein was examined in testes of both young and adult buffaloes by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical analysis. Further, using the testis transplantation assay, a functional assay for spermatogonial stem cells (SSCs), stem cell potential of gonocytes/spermatogonia isolated from prepubertal buffalo testis was also determined.ResultsExpression of POU5F1transcript and protein was detected in prepubertal and adult buffalo testes. Western blot analysis revealed that the POU5F1 protein in the buffalo testis exists in two isoforms; large (∼47 kDa) and small (∼21 kDa). Immunohistochemical analysis revealed that POU5F1 expression in prepubertal buffalo testis was present in gonocytes/spermatogonia and absent from somatic cells. In the adult testis, POU5F1 expression was present primarily in post-meiotic germ cells such as round spermatids, weakly in spermatogonia and spermatocytes, and absent from elongated spermatids. POU5F1 protein expression was seen both in cytoplasm and nuclei of the stained germ cells. Stem cell potential of prepubertal buffalo gonocytes/spermatogonia was confirmed by the presence of colonized DBA-stained cells in the basal membrane of seminiferous tubules of xenotransplanted mice testis.Conclusion/SignificanceThese findings strongly indicate that gonocytes/spermatogonia, isolated for prepubertal buffalo testis can be a potential target for establishing a germ stem cell line that would enable genetic modification of buffaloes.

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

“…Further, the use of more numbers of enzymes might cause cell injury while less numbers increase the possibility of cell contamination [13,42]. Previously, in farm animals, attempts were made to isolate the SSCs using either four [10,11], three [14] or two [43] enzymes with variable success. In the present study, we performed testicular disaggregation by enzymatic digestion using three enzymes without any reduction in the total cell count.…”

“…The SSCs have been isolated using two enzymes in human [12,13], three enzymes in buffalo [14] and four enzymes in cattle [11] as well as goat [10]. Use of more enzymes ensures higher purity but it takes more time and may affect the viability of the cells.…”

Maintenance of Potential Spermatogonial Stem Cells In Vitro by GDNF Treatment in a Chondrichthyan Model (Scyliorhinus canicula L.)1

“…For example, the GFRα1 (GDNF Family Receptor α1) has been described as a marker of undifferentiated spermatogonia in mouse [7], equids [8], buffalo [9] or collared peccary [10] and more recently in fishes such as the Nile tilapia [5]. Other undifferentiated spermatogonial markers were identified in various species such as the cell surface marker Thy1 (Thymus cell antigen 1) in mouse [11], bull [12] and carp [13] or the following transcriptional factors: PLZF (Promyelocytic Leukaemia Zinc Finger protein) in mouse [7], equids [8] and zebrafish [14], POU2/POU5F1 in mouse [15], buffalo [9] and carp [13] and ID4 (Inhibitor of DNA binding 4), a more selective marker of SSCs than Thy1, in the mouse [16]. Some of these defined markers were used to improve in vitro culture systems by selecting undifferentiated spermatogonia from the testicular cell population, including potential SSCs, using the MACS technology (Magnetic-Activated Cell Sorting) in mammals as well as in Teleosts.…”

Spermatogonial stem cells from domestic animals: progress and prospects

“…Stage-specific embryonic antigen-1 (SSEA1) specifically marks a subset of undifferentiated spermatogonia in prepubertal boars , and the positive staining of Claudin-8 is discernible in putative SSCs and a handful of Sertoli cells in bovine testes (McMillan et al 2013). Nevertheless, transcription factors, NANOG and OCT3/4, which are related to the pluripotency of stem cells, seem to be conserved but are less dependable markers for undifferentiated spermatogonia from domestic species, due to the dynamic changes in their expression during germ cell development (Goel et al 2008, Fujihara et al 2011, Mahla et al 2012; Table 1). …”