RESUMEN
The blood-epididymis barrier (BEB) forms a unique microenvironment that is crucial for the maturation, protection, transport, and storage of spermatozoa in the epididymis. To characterize the function of tight junctions (TJs), which are constitutive components of the BEB, we determined the expression and localization of TJ proteins such as zonula occludens (ZO)-1, 2, and 3, occludin, and claudin3 (Cldn3) during postnatal development in the goat epididymis. To assess the expression patterns of TJ proteins in immature (3 months of age) and mature (14 months of age) goat epididymides, two different experimental methods were used including immunofluorescence labeling and western blotting. We show that, ZO-1, 2, and 3, and occludin, were strictly expressed and localized to the TJs of the goat epididymis, whereas Cldn3 was present in basolateral membranes as well as TJs. All TJ proteins examined were more highly expressed in the immature epididymis compared to levels in mature tissue. In conclusion, our study indicates that at least five TJ proteins, namely ZO-1, ZO-2, ZO-3, occludin, and Cldn3, are present in TJs, and the expression strength and pattern of TJ proteins tend to be age dependent in the goat epididymis. Together, these data suggest that the distinct expression patterns of TJ proteins are essential for regulating components of the luminal contents in the epididymal epithelium and for forming adequate luminal conditions that are necessary for the maturation, protection, transport, and storage of spermatozoa in the goat epididymis.
RESUMEN
Successful in vitro development of embryos is dependent upon maintenance of cellular function in the embryonic microenvironment. However, the molecular aspects involved in the thermoprotection of embryos, against heat and cold stress it is not clear. The aim of this study was to determine the effects of heat and cold shock on the viability and development of porcine diploid parthenotes developing in vitro. Exposure of two-cell stage embryos to 41 degrees C did not affect further cleavage. However, prolonged heat shock, greater than 12h, reduced the percentage of blastocysts that developed from two-cell stage parthenotes, as well as the total number of nuclei in the blastocysts that formed. Furthermore, the degree of apoptosis was increased (P<0.05) in these blastocyst stage parthenotes. In contrast, exposure of two-cell parthenotes to cold (30 degrees C) for 24h did not affect the cleavage rates, development to blastocyst, nor the total cell numbers per blastocyst. Real time PCR revealed that quantitative expression of the Bcl-xL gene was not different, but amounts of HSP 70.2, Bak, and Caspase 3mRNA were significantly increased in the heat shocked embryos, as compared with untreated controls. These results suggest that porcine embryos are more tolerant to cold shock than to heat shock. Heat stress seems to induce apoptosis related gene expression in porcine parthenotes developing in vitro, which results in diminished parthenote viability.