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Enrichment and in vitro features of the putative gonocytes from cryopreserved testicular tissue of neonatal bulls
Author(s) -
Cai H.,
Tang B.,
Wu J. Y.,
Zhao X. X.,
Wang Z. Z.,
An X. L.,
Lai L. X.,
Li Z. Y.,
Zhang X. M.
Publication year - 2016
Publication title -
andrology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.947
H-Index - 43
eISSN - 2047-2927
pISSN - 2047-2919
DOI - 10.1111/andr.12229
Subject(s) - gonocyte , biology , cryopreservation , andrology , reproductive technology , somatic cell , microbiology and biotechnology , homeobox protein nanog , spermatogenesis , endocrinology , embryo , embryonic stem cell , sertoli cell , biochemistry , gene , induced pluripotent stem cell , medicine
Summary Enrichment and propagation of gonocytes or spermatogonial stem cells ( SSC s) from cryopreserved testicular tissue is essential to apply SSC s‐related techniques in large domestic animals. We previously reported the cryopreservation of adult bovine testicular tissue. Here, we conducted the enrichment and culture of putative gonocytes from cryopreserved testicular tissues of post‐natal 1‐day‐old bulls. The testicular structure was well maintained after freezing and thawing. Higher mRNA levels of gonocyte/ SSC s markers ( PLZF , GFR α 1 , and UCHL ‐1 ) than those of pluripotency genes ( Oct4 , Sox2 , and Nanog ) were detected in the frozen–thawed sex cords. GFR α1 was specifically detected in the membrane and cytoplasm of gonocytes by immunostaining. Differential plating provided 40–50% enrichment of putative gonocytes. They were single, paired‐, aligned‐cells, or grape cluster‐like colonies in minimum essential medium ( MEM ) containing 2.5% FBS + 2 mM glutamine + 100 IU / mL penicillin‐streptomycin + 40 μg/ mL gentamycin + 15 mM HEPES + 10 mM β‐mercaptoethanol + 0.1 mM non‐essential amino acids + 1 mM sodium pyruvate. On day 3, gonocyte progeny increased and the contaminated somatic cells spread and concurrently divided slowly. On day 5, gonocyte progeny proliferated continuously and typical intercellular bridges formed by incomplete cytokinesis in paired‐cells or aligned‐cysts were observed. Immunochemically, they were still GFR α1 and PLZF positive. These cells expressed significantly higher gonocyte/ SSC s marker mRNA s than pluripotency gene mRNA s, concomitant with a higher level of differentiated spermatogonia marker c‐kit . With time, gonocyte progeny colonies appeared in varied sizes and expanded dramatically on day 7. After cultured for 9–10 days, however, large colonies collapsed and dispersed as some single cells and small syncytial cysts. Together, MEM containing 10% dimethyl sulfoxide + 2.5% newborn calf serum provides efficient cryoprotection for the testicular tissue from 1‐day‐old neonatal bulls. Putative gonocytes enriched from these nascent tissues present robust proliferation capacity, conserved gonocyte/ SSC s markers, and SSC s‐like in vitro features.