PARENT SESSION

(M310) EFFECTS OF ACROGRANIN ON BLASTOCYST HATCHING AND SUBSEQUENT ADHESION AND OUTGROWTH IN THE MOUSE.

Qin, Junwen¹, Diaz-Cueto, Laura^{2, 3}, Schwarze, Juan-Enrique², Gerton, George², Imakawa, Kazuhiko ¹, ¹ The University of Tokyo, Tokyo, Japan² University of Pennsylvania Medical Center, Philadelphia, PA³ Hospital de Ginecobstetricia, Mexico D. F., Mexico

ABSTRACT- Using cDNA microarray methodology, we had previously shown that transcripts of acrogranin, a recently identified autocrine growth factor, were up-regulated in mouse blastocysts adhered to the filter membrane in an in vitro culture system. In this study, we investigated the expression and effects of acrogranin on blastocyst hatching, adhesion and embryo outgrowth during the peri-implantation period in the mouse. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that substantial amounts of acrogranin mRNA were present in blastocysts during this time period. In situ hybridization demonstrated the presence of acrogranin transcripts in both inner cell mass (ICM) and trophectoderm. Immunohistochemistry analysis revealed that acrogranin was exclusively localized to the surface of the trophectoderm at the stages of early and pre-/post-adhesion blastocysts as well as in both trophoblast cells and ICM of outgrowth embryos. Immunoblotting analysis confirmed that peri-implantation mouse blastocysts secrete a single form of 88 kDa acrogranin into the surrounding medium. NIH3T3 cells that had been transfected with an acrogranin expression construct secreted the 88 kDa form of recombinant acrogranin, from which a 68 kDa form could be generated by deglycosylation. In vitro treatment of blastocysts with recombinant acrogranin promoted blastocyst hatching, adhesion and outgrowth, whereas rabbit anti-mouse acrogranin IgG reduced the incidence of blastocyst hatching, adhesion and outgrowth. Bromodeoxyuridine (BrdU) incorporation and immunodisection of ICM studies revealed that acrogranin was effective on the trophectoderm, not ICM. These results indicate that acrogranin is an important factor for the processes of blastocyst hatching, adhesion and outgrowth, and suggest that the effects of acrogranin on blastocyst adhesion and outgrowth may have been triggered by the prior action of acrogranin to induce hatching of the blastocysts.

KEY WORDS: acrogranin, blastocyst hatching, adhesion, outgrowth