Expression of placenta-specific 8 in human oocytes, embryos, and models of in vitro implantation  Min Li, M.Sc., Dong Liu, M.Sc., Li Wang, Ph.D., Weizhou Wang, B.Sc., Aiming Wang, M.D., Yuanqing Yao, M.D., Ph.D.  Fertility and Sterility  Volume 106, Issue 3, Pages 781-789.e2 (September 2016) DOI: 10.1016/j.fertnstert.2016.05.018 Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Detection of PLAC8 protein expression in human oocytes and different developmental stages of embryos and LoVo cells by immunofluorescence and Western blotting. (A) LoVo cells were used as a positive control. Metaphase 2 (MII) oocytes (n = 12), two-cell embryos (n = 4), four-cell embryos (n = 4), eight-cell embryos (n = 8), morulae (n = 7) and blastocysts (n = 18). Oocytes, embryos, and LoVo cells were stained using a rabbit anti-human PLAC8-specific antibody and secondary anti-rabbit IgG conjugated to FITC (R555) (red). Negative control experiments were conducted by replacing the primary antibody with phosphate-buffered saline and applying the same secondary antibody (R555). The nuclei were labeled with 4,6-diamidino-2-phenylindole (DAPI) (blue). In the oocytes and embryos, PLAC8 was labeled in the cytoplasm of all cells investigated. In the majority of the oocytes and embryos, a distinct, stronger immunoreactivity was localized to the cell cortex, and it was reduced toward the cell center. In the LoVo cells, PLAC8 was labeled in the cytoplasm and asymmetrically located beside the nucleus. Representative images of the experiments are shown. Scale bar = 50 μm. (B) In the positive control LoVo cells, PLAC8 protein could be detected by Western blotting and could be efficiently knocked down by two separate siRNAs. Fertility and Sterility 2016 106, 781-789.e2DOI: (10.1016/j.fertnstert.2016.05.018) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Model of embryo-endometrial stromal cell coculture and different statuses of human embryo invasion. (A–D) Images captured from four typical day 6–7 artificial hatched blastocysts cultured on a layer of artificially induced decidualization of stromal cells. (A, B) The process of implantation and expansion in two hatched blastocysts: adhered to the stromal cells, contracted, expanded, and flattened progressively with outgrowth into the stromal cell layer. (C) The process of implantation without expansion in one hatched blastocyst: adhered to the stromal cells and contracted but did not expand. (D) The process of one hatched blastocyst without implantation: adhered to the stromal cells but ultimately rolled onto the stromal cells. Scale bar = 50 μm. (E) Correlating invasion (area of outgrowth) with embryo quality (morphologic grade). Dotted lines indicated approximate area of invasion for blastocysts of different grades (right panel). The average invasion area of blastocysts of grade 2 (n = 6) was statistically significantly larger than that of blastocysts of grade 3 (n = 15) (left panel; **P<.01). Results shown are mean areas ± standard error of the mean. Fertility and Sterility 2016 106, 781-789.e2DOI: (10.1016/j.fertnstert.2016.05.018) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Detection of PLAC8 protein expression during the implantation process in the embryo-endometrial stromal cell coculture model assessed by immunofluorescence. The coculture models were stained with human PLAC8-specific antibody (red). Vimentin (green) was stained to outline endometrial stromal cells. Nuclei were stained with DAPI (blue). (A) In an implanted and expanded embryo, PLAC8 was labeled in the cytoplasm and nucleolus. The lower channel was enlarged for display. (B) A contracted embryo in the coculture model: no clear immunoreactivity was identified in the cytoplasm or nucleolus. Scale bar = 50 μm. Fertility and Sterility 2016 106, 781-789.e2DOI: (10.1016/j.fertnstert.2016.05.018) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Detection of PLAC8 protein localization in the embryo-endometrial stromal cell coculture model using colabeling with a characterized nucleolus marker B23 by immunofluorescence. The coculture models and LoVo cells were stained with human PLAC8-specific antibody (red). B23 (green) was stained to outline nucleolus. Nuclei were stained with DAPI (blue). (A) An expanded embryo in the coculture model: PLAC8 (red) was labeled in the cytoplasm and nucleolus. Colabeling with B23 in the nucleolus could be detected (indicated by arrows). (B) In the LoVo cells, PLAC8 was labeled in the cytoplasm and asymmetrically beside the nucleus, which also localizing beside B23 positive region. Scale bar = 50 μm. Fertility and Sterility 2016 106, 781-789.e2DOI: (10.1016/j.fertnstert.2016.05.018) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions