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Summary. Aim: The ERBB2 receptors tyrosine kinase, also known as HER2/Neu, play an essential role in early organism development and modulation of cell behavior in varieties of tissue in an adult organism. Our aim was the generation of transgenic rat spermatogonial line capable of inducible expression of a dominant negative form of the ERBB2 protein in vitro and the transgenic rat as an animal model for dissection of the ERBB2 mediated signaling in vitro and in vivo. Materials and Methods: Donor derived rat spermatogonial stem cells that express green fluorescence protein and inducible ERT2CreERT2 recombinase were modified with Sleeping Beauty transposon-based vector that express truncated kinase deficient form of the ERBB2 receptor upon Cre mediated recombination. Clonally selected spermatogonial cell lines were extensively tested in vitro. Animals were generated via spermatogonia mediated transgenesis by transplantation of clonal cell line into testes of chemically sterilized recipients. Obtained progeny were tested for inducibility in vivo and served as donors of spermatogonia for downstream analysis. Results: We obtained animals and clonally derived spermatogonial stem cell lines that express an inducible dominant negative form of the ERBB2 protein. Isogenic nature of induced and uninduced cells allows most accurate morphological and molecular comparison of cells affected by the interruption of normal function of the ERBB2 receptor and cellular dynamics in vitro and in vivo. Conclusions: Clonally derived spermatogonial stem cell lines that express an inducible dominant negative form of ErbB2 demonstrated an obvious difference in morphological appearance and growth kinetics of induced cells comparing to uninduced ones. Western blot analysis of induced and uninduced cells revealed significant differences in presence and phosphorylation state of several tested important proteins involved in the ERBB2 mediated signal transduction, such as S6 ribosomal protein; AKT (the serine/threonine kinase also known as protein kinase B); and three protein kinases that participate in the RAS-RAF-MEK-ERK signal transduction cascade PRAS40, MEK, ERK1/2.

Different cell types demonstrate an individual response to the doses of DMSO in freezing media that cannot be calculated empirically. Such situation requires experimental adjustment of the dose of the main cryoprotectant for each cell type. The purpose of the work was to optimize the basic composition of the cryopreservation medium for the rat spermatogonial cell lines, to investigate the time period of the initial storage of the cells at –80° C, as well as the effect of the ratio of volume of the freezing medium to the number of cells on their subsequent survival after recovery. Our data demonstrate that the 8% DMSO is the most effective concentration of the cryoprotectant. Furthermore, we tested different time of initial storage of cryovials in a commonly used “Mr. Frosty” Freezing Container at –80ºC. Our results suggest that 12 hours period (overnight) provides enough time for primary freezing step at –80ºC. Optimization of the volume of freezing medium revealed, that 0.5 ml, compared with 1.0 ml of medium for rat spermatogonial stem cells allows more effective preservation and long-time cryostorage of low numbers of cells (3×104) with successful recovery of up to 50% of the frozen cell population. Additionally, we attempted to improve the freezing medium composition by supplementing its base formulation with sucrose and/or trehalose. The use of optimal concentration of DMSO (8%) in the medium in combination with a 200mM of trehalose resulted in an increase of spermatogonia viability after recovery by more than 12% compared to the original composition of SG (Spermatogonia Growth) medium containing 10% of DMSO.