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.
Highlights
Water buffalo (Bubalus bubalis) is an inhabitant of the Asian continent and about half of its total world population exists in India
POU5F1 expression in the prepubertal buffalo testes was present in the germ cells, which were distinguishable by their large size, topological position and morphology (Fig. 3A)
To investigate the stem cell characteristics of germ cells in buffalo, we examined the expression of Pou5f1, both at transcript and protein level in prepubertal and adult testes
Summary
Water buffalo (Bubalus bubalis) is an inhabitant of the Asian continent and about half of its total world population exists in India. Several reproductive technologies from bovine have been adapted for improvement of buffalo production, albeit with various limitations. There is a need to explore newer areas that can find application in genetic improvement of buffalo more efficiently. One such area is male germline stem cell technology that still remains unexplored in this species. Water 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; not much information is available in buffalo
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