Isolation and Identification of Prepubertal Buffalo (Bubalus bubalis) Spermatogonial Stem Cells.

Wanyou Feng,Dagiang Do,Ben Huang,Yanfei Deng,Chan Luo,Deshun Shi,Qinyou Liu,Shibei Chen,Xiaocan Lei

doi:10.5713/ajas.15.0592

Wanyou Feng, Dagiang Do + Show 7 more

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https://doi.org/10.5713/ajas.15.0592

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Abstract

Isolation and culture of spermatogonial stem cells (SSCs) are attractive for production of genetic modified offspring. In the present study, buffalo spermatogonial stem-like cells were isolated, cultured and expression pattern of different germ cell marker genes were determined. To recover spermatogonia, testes from age 3 to 7 months of buffalo were decapsulated, and seminiferous tubules were enzymatically dissociated. Two types of cells, immature sertoli cell and type A spermatogonia were observed in buffalo testes in this stage. Germ cell marker genes, OCT3/4 (Pou5f1), THY-1, c-kit, PGP9.5 (UCHL-1) and Dolichos biflorus agglutinin, were determined to be expressed both in mRNA and protein level by reverse transcription polymerase chain reaction and immunostaining in buffalo testes and buffalo spermatogonial stem-like cells, respectively. In the following, when the isolated buffalo buffalo spermatogonial stem-like cells were cultured in the medium supplemented 2.5% fetal bovine serum and 40 ng/mL glial cell-derived neurotrophic factor medium, SSCs proliferation efficiency and colony number were significantly improved than those of other groups (p<0.05). These findings may help in isolation and establishing long term in vitro culture system for buffalo spermatogonial stem-like cells, and accelerating the generation of genetic modified buffaloes.

Highlights

Water buffalo is an economically important livestock, which partially distributes in south China, providing high quality of milk, meat and work power (Shi et al, 2012)
Analysis of germ cell marker genes expression in prepubertal buffalo testis The reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that Oct-4, PGP9.5, THY-1 and c-kit were all expressed in testes of prepubertal buffalo, with PCR product fragments of 312 bp for Oct-4, 195 bp for c-kit, 502 bp for THY1, 358 bp for PGP9.5 and 199 bp for β-actin (Figure 1A), respectively
No bands were visible in the case of negative controls, where the cDNA was replaced with purified water

Summary

Introduction

Water buffalo is an economically important livestock, which partially distributes in south China, providing high quality of milk, meat and work power (Shi et al, 2012). Developing of stem cell technology in buffalo has significance in the genetic improvement of this species (Shi et al, 2007; Mahla et al, 2012). Spermatogonial stem cells (SSCs) have received a great deal of attention in recent years, as they have unique capacity for self-renewal and produce large number of differentiating haploid germ cells which could transmit paternal genetic information to generations (Kanatsu-Shinohara and Shinohara, 2013). Sci. 29:1407-1415 successful germ cell transplantation into bull testis had resulted in production of donor-derived sperm cells (Stockwell et al, 2009). In case of establishment livestock species male germ cells line, researchers can facilely create targeted gene editing animals using TALEN or CRISPR/Cas technology (Gaj et al, 2013; Chapman et al, 2015). The most likely reason is lack of sufficient understanding of expression of related markers in the male stem cell, especially water buffalo

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