Corrigendum to: 198 AGE EFFECT ON RELATIVE GENE EXPRESSION OF BOVINE SPERMATOGONIA

Abstract

Spermatogonial stem cells (SSCs) have important applications in treatments for infertility and animal transgenesis. However, the isolation of bovine SSCs is less efficient than for other species and it is lower for adult bulls. The goal of this study was to verify whether the relative gene expression of spermatogonia is affected by age after the laminin differential plating. Ten grams of parenchyma of testicles from pre-pubertal animals (5 months of age, n = 5) and bulls (3–4 years of age, n = 5) were minced and digested: colagenase (1 mg mL–1) for 30 min at 37°C and trypsin (2.5 mg mL–1) for 5 min at 37°C. Cells were plated (3 × 106 viable cells) in 60-mm culture dish covered with laminin (20 ng mL–1) and they were cultured for 15 min in high-humidity atmosphere with 5% of CO2 at 37°C. The adherent cells were recovered by enzymatic digestion with 0.1% trypsin for 1 min at 37°C. Viable cells were selected by the Trypan exclusion method, RNA was extracted from ~200.000 viable cells (Ilustra RNAspin Mini RNA, GE Healthcare, Waukesha, WI, USA) and cDNA synthesis was performed (SuperScript® VILO™, Invitrogen, Carlsbad, CA, USA). ITGA6 (integrin, α 6), SELP (Selectin P) and ICAM (Intercellular Adhesion Molecule 1) relative gene expression were determined by real-time RT-PCR (Mastercycler ep Realplex, Eppendorf International, Hamburg, Germany); GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) and ACTB (actin β) were used as housekeeping genes. The statistical analysis was performed by QPCR_MIXED (SAS®, SAS Institute Inc., Cary, NC, USA). An α level at 0.05 was always adopted. ITGA6 and ICAM1 relative expression weren't effected by age; respectively, P = 0.2367 and P = 0.3583. However, SELP was highly expressed in adult bulls (P = 0.0022). Previously, ICAM1 and SELP have also been shown to mark aging haematopoietic stem cells and were more highly expressed in spermatogonia from adult mice than younger. However, SELP is expressed in more differentiated spermatogenic cells such as human sperm. The expression levels of ITGA6 did not seem to be significantly altered by age, indicating that age affects only certain SSC properties. Financial support was provided by FAPESP (CEUA/FMVZ/USP 2509/2011).

Bovine male germline stem-like cells cultured in serum- and feeder-free medium

“…Spermatogonial stem cells (SSCs) have important applications in treatments for infertility and production of animal transgenesis (Giassetti et al 2015). Studies have shown that the establishment of multipotent male germline stem cell (mGSC) lines derived from neonate and adult testes has been achieved (Mardanpour et al 2008).…”

“…The gene targeting based on GSC is a promising technique for the production of transgenic offspring, acting as a valuable tool in bovine research. However, the isolation of bovine's SSCs is less efficient than that of other species (Giassetti et al 2015). In this study, we isolated and established pluripotent mGSCs lines from Chinese Qinchuan bovine testes with the usage of serum-and feeder-free medium, their multipotency and differentiation potential in vitro were also tested.…”