159 LOCALIZATION OF CLAUDIN FAMILY PROTEINS IN PIG EMBRYOS DURING PRE-IMPLANTATION DEVELOPMENT

Abstract

Tight junctions (TJ) are critical for blastocoel formation in mammalian pre-implantation embryos. Claudin family proteins, which are TJ proteins, are generally important for the barrier function of TJ. However, the expression and localization of claudin proteins are not clarified in mammalian pre-implantation embryos. The present study was designed to examine changes in the localization of claudin isoforms, claudin-1, -2, and -4, in pig parthenogenetic diploids through pre-implantation development. Oocyte–cumulus–granulosa cell complexes were collected from the follicles 4–6 mm in diameter, and then maturation-cultured. Only oocytes with a prominent first polar body were subjected to electro-stimulation after maturation, and they were treated with cytochalasin B to produce parthenogenetic diploids. Presumptive diploids were then cultured for 168 h and observed every 24 h. Embryos of each developmental stage from 2-cell to blastocyst were subjected to immunofluorescence staining of the anti-TJ proteins. Zona-free embryos were fixed and treated with rabbit anti-claudin-1 and -2 polyclonal antibodies or mouse anti-claudin-4 monoclonal antibody, followed by treatment with Alexa fluor 488-labeled goat anti-rabbit IgG antibody or goat anti-mouse IgG antibody. Some embryos were treated with rabbit anti-occludin antibody or anti-ZO-1 antibody for the detection of the TJ-net work. All embryos were counterstained with Hoechst 33342 and observed under an epifluorescence microscope after whole-mounting. The specific fluorescence for all TJ proteins examined was observed in embryos at all pre-implantation stages. Occludin and ZO-1 were detected in the cytoplasm before the morula stage (96 h), and localized toward the boundary region among cells from the morula and early blastocyst stages, reflecting the distribution of the tight junction in embryos. Claudin-1 and -2 localized in the cytoplasm at early-cleaving stages. They were detected in the nucleus after compaction, and the distribution of these proteins in the nucleus was dominant at the blastocyst stage. The distribution of the 2 claudin proteins in the nucleus was quite different; claudin-1 distributed rather homogeneously, but claudin-2 formed several bright spots in the nucleus. Claudin-4 also showed a unique distribution pattern in embryos; it was detected in the cytoplasm with strong fluorescence at the periphery of the nucleus of the 2- and 4-cell embryos. Claudin-4 changed its localization toward the boundary region of cells around the early blastocyst stage (120 h), and then the distribution of claudin-4 was restricted to the boundary region later in the blastocyst stage (144 h). These results indicate that claudin-4, but not claudin-1 and -2, is responsible for the formation of TJ in the pre-implantation embryos, although the function of these claudin proteins in the nucleus is unknown.