DOI: 10.5061/dryad.6djh9w112
title: Thy1-positive spermatogonia suppress the proliferation of spermatogonial stem cells by Extracellular vesicles in vitro

description: The self-renewal of mammalian spermatogonial stem cells (SSCs) supports
 spermatogenesis to produce spermatozoa, and this is precisely controlled
 in a stem niche microenvironment in the seminiferous tubules. Although
 studies have revealed the role of the surrounding factors in SSCs, little
 is known about whether the division of SSCs is controlled by extracellular
 vesicles. Here, extracellular vesicles were found in the basal compartment
 of seminiferous tubules in mouse, rat, rabbit and human testes. In the
 mice, the testicular extracellular vesicles are secreted by spermatogonia
 and are taken up by SSCs. Further, the extracellular vesicles from
 thy1-positive spermatogonia were purified by anti-Thy1-coupled magnetic
 beads, and which suppress their proliferation of SSCs but not lead to the
 apoptosis in vitro.
description: <b>Animals</b> C57BL/6 mice, rabbit, and adult Sprague Dawley rats were purchased from SLAC Laboratory Animal Co., Ltd. (Shanghai, P. R. China), All animal care and experiments of this study were performed in accordance with the guidelines and were approved by the Ethics Committee of International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University (Permit number: GKLW 2017-31).   <b>2.2. </b><b>Human testicular biopsies</b> The collection of human testicular tissues was in accordance with institutional guidelines, and <b>all patients provided written informed consent,</b> the study design was approved by the Ethics Committee of the International Peace Maternity and Child Health Hospital. Informed consent was obtained from the participants. Two testicular tissue biopsies were obtained by puncture from men with obstructive azoospermia (age 30 and 37 years) with normal spermatogenesis, cases of obstructive azoospermia were caused by vasoligation. Clinical examinations included the evaluation of secondary sexual characteristics, testicular size and consistency, epididymal distension, presence of the vasa deferentia and varicocele. Both patients had their serum follicle stimulating hormone concentrations measured, with values in the normal range.   <b>2.3. </b><b>Cell culture</b> Spermatogonial stem cells were established from 6-day-old male F1 progeny of DBA/2 ´ C57BL/6 or C57BL/6/Tg14 (act-EGFP-OsbY01) mice as described (17,18). There is no difference when it comes to the percentage and behaviour of the SSCs within the testes of C57BL/6 and DBA/2 mouse (19). They were seeded on mitomycin C-treated mouse embryonic fibroblast (MEF) feeder cells and cultured in SSC medium consisting of StemPro-34 SFM medium supplemented with stemPro supplement (Thermo Fisher Scientific, Waltham, MA, USA), 25 mg/ml insulin, 100 mg/ml transferrin, 60 mM putrescine, 30 nM sodium selenite, 6 mg/ml d–(+)-glucose, 30 mg/ml pyruvic acid, 1 ml/ml d-l-lactic acid (Sigma-Aldrich, St Louis, USA), 5 mg/ml bovine serum albumin (Sigma-Aldrich, St Louis, USA), 2 mM L-glutamine, 10 mM 2-mercaptoethanol (Sigma-Aldrich, St Louis, USA), 1 ´ MEM vitamins solution (Invitrogen, USA), 1 ´ non-essential amino acid solution (Invitrogen, USA), 2 mM l-glutamine (Invitrogen, USA), 1 ´ penicillin/streptomycin solution (Invitrogen, USA), 0.1 mM ascorbic acid, 10 mg/ml d-biotin (Sigma-Aldrich, St Louis, USA), 20 ng/ml recombinant human epidermal growth factor (Invitrogen, USA), 10 ng/ml human basic FGF (Invitrogen, USA), 10 ng/ml recombinant human GDNF (Invitrogen, USA) and 1% fetal bovine serum (FBS) (Gibco/Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA). The medium was replaced every 2–3 days. For MEF preparation, C57BL/6J mouse embryos were minced, digested with trypsin-EDTA (Invitrogen, USA), and then cultured in Dulbecco’s (D) MEM containing 10% FBS supplemented with 2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin (MEF culture medium).