ABSTRACT
The potential health impact of pharmaceutical waste is now a growing concern. Contraceptive steroids are prominent environmental contaminants and thus may act as endocrine disruptors. Numerous xenobiotics hamper Sertoli cells junctional communication which is known to participate in spermatogenesis control. This has been associated with male subfertility and testicular cancer. We investigated three contraceptive molecules found in the environment for their potential impact on Sertoli cells gap junction functionality: 17a-ethynylestradiol, medroxyprogesterone acetate and levonorgestrel. Four other non-steroid drugs also found in the environment were included in the study. Communication disruption was analyzed in vitro in murine seminiferous tubules and the 42GPA9 Sertoli cell line. Steroids modulated connexin43 trafficking and impaired junctional communication through rapid effects apparently acting on the cell membrane but not on Cx43 expression. The 4 non-steroid compounds showed no effect. Longer exposure to steroids increased gap junction impairment, which was associated in part with Na/K ATPase internalization. Estrogen receptors (ER) did not appear to be involved in gap junction disruption: Sertoli cells are devoid of ERalpha and only express the cytoplasmic beta isoform. ERbeta localization was not modified by either steroid. The threshold level was surprisingly low, around 10(-16) M. We conclude that steroidal pollutants disrupt Sertoli cells junctional communication in vitro at concentrations that can be found in the environment.
Subject(s)
Gap Junctions/drug effects , Signal Transduction/drug effects , Spermatogenesis/drug effects , Steroids/pharmacology , Animals , Cell Line/drug effects , Connexin 43 , Cytoplasm/drug effects , Cytoplasm/physiology , Gap Junctions/physiology , In Vitro Techniques , Male , Mice , Mice, Transgenic , Sertoli Cells/drug effects , Sertoli Cells/metabolism , Signal Transduction/physiology , Spermatogenesis/physiologyABSTRACT
3T3-F442A and BFC-1 cells are widely used for studying adipocyte differentiation and metabolism. Macrophage markers were previously reported in these cell lines. We examined whether 3T3-F442A and BFC-1 would produce interferon-gamma (IFN-gamma), the expression of which is a matter of debate in cells other than T-lymphocytes and natural killer cells, like macrophages or dendritic. IFN-gamma was absent from preadipocytes. However 3T3-F442A, but not BFC-1, presented a differentiation-dependent induction of IFN-gamma mRNA and protein. Immunofluorescence studies showed that IFN-gamma was located in mature adipocytes. IFN-gamma was retrieved in the culture medium. Then, we examined the expression of other markers of T-lymphocytes or macrophages, like the CD3/T-cell receptor complex or Toll-like receptors (TLR) -2 and -9, in these cells. Transcripts for the three subunits of CD3 were undetectable whatever the differentiation stage. In contrast, TLR-2 and -9 genes were expressed differentially during the differentiation process. TLR-2 mRNA was induced early then decreased while TLR9 transcript appeared at later days and increased in parallel to IFN-gamma. In contrast to what was expected from 3T3-F442A cells, IFN-gamma was absent from adipocytes isolated either from subcutaneous or periepidydimal mouse adipose tissue. However, TLR-2 and -9 mRNAs were present in both adipose depots although at various levels. Hence, we detect the presence of two markers of innate immunity, TLR-2 and -9, in in vivo-derived adipocytes and we demonstrate that differentiated 3T3-F442A cells selectively express IFN-gamma and TLR-9 in a manner that resembles what is occurring for natural killer dendritic cells.