Milk fat globule epidermal growth factor 8 (MFG-E8): a novel protein in the mammalian endometrium with putative roles in implantation and placentation.

OBJECTIVES: MFG-E8 is a novel endometrial protein with conserved functions in tissue remodeling and angiogenesis in non-uterine tissues. Our aims were: 1. To examine the presence of MFG-E8 protein in the human endometrium during the window of implantation, in human endometrial cell lines, in human placental tissue at different gestational ages, and in murine implantation sites during early gestation; and 2. To study the regulation of MFG-E8 mRNA expression in mice implantation sites. STUDY DESIGN: MFG-E8 protein and its receptor integrin αvß3 were detected by immunostaining in human endometrial biopsies obtained from normal volunteers, in human endometrial cell lines (epithelial: Ishikawa and HEC-1A, stromal: HESC, and endothelial: HEEC), in human products of conception from all trimesters of gestation, and in murine implantation and inter-implantation sites dissected on days 5 and 8 post-coitus. MFG-E8 gene expression was assessed by RT-PCR. MAIN OUTCOME MEASURES: Immunohistochemical determination of MFG-E8 in endometrium and products of conception as well as relative MFG-E8 mRNA expression in mice implantation sites. RESULTS: MFG-E8 protein was present almost exclusively in the epithelial compartment of human endometrium. It was also expressed in the cytotrophoblasts and syncytiotrophoblasts outlining chorionic villi of the human placenta at all trimesters of gestation, and in murine implantation sites. MFG-E8 mRNA was significantly up-regulated in murine implantation sites and with increased gestational age. CONCLUSIONS: MFG-E8 expression in the endometrial epithelium as well as in chorionic villi suggests its possible role in endometrial reorganization during the receptive phase and in events related to normal pregnancy in mammals.

Validation of the hemizona assay (HZA) in a monkey model. II. Kinetics of binding and influence of cryopreserved-thawed spermatozoa.

We compared fresh and frozen-thawed cynomolgus monkey spermatozoa tight binding to the zona pellucida under hemizona assay (HZA) conditions. Monkey oocytes were recovered after superovulation and stored in salt solution. Matching hemizonae were obtained by micromanipulation. Semen, obtained by electroejaculation, was used fresh or was cyropreserved, thawed, and washed by swim-up separation. At the standard initial dilution of 500,000 motile sperm/ml (or 5 x 10(4) motile sperm/hemizona), binding was significantly higher for fresh sperm (P = 0.00004). For frozen-thawed samples, there was a linear increase in the number of tightly bound sperm with increasing sperm concentration (r = 0.95). At 1.5 x 10(6) motile sperm/hemizona, binding of frozen-thawed spermatozoa was similar to that of fresh at a standard concentration. Kinetic studies showed peak binding at 1 hr of gametes coincubation. We conclude that, in this monkey model, the HZA is a valuable bioassay for evaluation of sperm binding to the zona pellucida, the initial requisite for fertilization and embryo development.