Homologous transplantation of fluorescently labelled enriched buffalo (Bubalus bubalis) spermatogonial stem cells to prepubertal recipients

Abstract

Spermatogonial stem cell transplantation provides a unique opportunity to study the biology of spermatogenesis and also offers an alternative approach for genetic modification in large animals. The present study aimed to extend this technique to the water buffalo. Spermatogonial stem cells (SSCs) were isolated from prepubertal buffalo testes (3-6 months of age) using two-step enzymatic digestion method and enriched by differential plating and Percoll density gradient centrifugation. The enriched SSCs expressed numerous spermatogonial transcriptional markers, viz. ID4, THY1, BCL6B, UCHL1, ETV5 and REX1 which confirmed their bonafide SSC identity. Subsequently, the enriched SSCs were labelled with a fluorescent dye PKH26 and transplanted into buffalo calves under ultrasound guidance. The recipient testes were recovered after 7-8 weeks by castration and their fluorescence microscopebased examination exhibited the persistence and localization of the fluorescent donor cells within the recipient seminiferous tubules. Further validation was done by the flow cytometric evaluation of PKH26 labeled donor cells among those isolated by two-step enzymatic digestion of recipient testicular parenchyma. In conclusion, we demonstrated the feasibility of SSC transplantation technique in the water buffalo.