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Spermatocytes and round spermatids of rat testis: The difference between in vivo and in vitro protein patterns
Author(s) -
Cossio Gabriela,
Sanchez JeanCharles,
Wettstein Rodolfo,
Hochstrasser Denis F.
Publication year - 1997
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150180335
Subject(s) - in vitro , in vivo , biology , andrology , microbiology and biotechnology , spermatogenesis , significant difference , spermatid , medicine , genetics , endocrinology
During mammalian spermatogenesis meiotic cell division and spermiogenesis occurs. Gene expression during this process is temporally regulated at the transcriptional and translational levels but the mechanisms are not well understood. In this publication we have investigated the synthesis of proteins in vitro to detect the proteins with a high metabolic turnover and to compare them with the in vivo protein map. RNA of spermatocytes and round spermatid cell populations, purified by centrifugal elutriation, and total testis was isolated. The poly A+ mRNA fraction was translated using a rabbit reticulocyte lysate. The translation products were separated by two‐dimensional (2‐D) gel electrophoresis using nonlinear 3.5–10 immobilized pH gradients for the first‐dimensional separation. The gels with 35 S‐translated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes and scanned using a phosphorimager. A highly reproducible and complex protein pattern was obtained using this methodology. Only rat testis messages were translated. Using Melanie 2 software we could compare and detect more than 1000 proteins on 2‐D radioactive images. Some changes could be observed in protein expression between the different cell types but they were not statistically significant. The comparison between the 2‐D rat testis map and the in vitro translated patterns show no matching between any spots. This result suggests that the post‐transcriptional modifications occurring in the reticulocyte system are not the same as those that occur in vivo in the testis. Rabbit reticulocyte proteins were detected by staining PVDF membranes with colloidal gold. Rat testis and reticulocyte patterns were completely different.