RESUMEN
OBJECTIVE: To investigate the location of ß-1,4-N-acetylgalactosaminyltransferase II (B4GALNT2) and the involvement of this protein and Sd(a) antigen in embryonic implantation. DESIGN: Cell and animal study. SETTING: University. ANIMAL(S): Adult outbred Institute for Cancer Research mice. INTERVENTION(S): B4GALNT2 antibody injected into the uteri of mice in early pregnancy; E3.5 blastocysts and pregnant uterine tissues were collected. MAIN OUTCOME MEASURE(S): Protein expression was detected by immunofluorescence staining and Western blotting. Embryo attachment was assayed via in vitro and in vivo embryo implantation models. RESULT(S): The b4galnt2 gene expression in the 293T cell line showed the protein localized in the plasma membrane. We confirmed that B4GALNT2 was localized on the surface of E3.5 blastocysts but was an intracellular component in uterine epithelia. Finally, anti-B4GALNT2 and lectins inhibition assays demonstrated the involvement of B4GALNT2 and Sd(a) antigen in embryonic attachment in vitro and in vivo via the mouse system and human endometrial cell line (Ishikawa). CONCLUSION(S): B4GALNT2 expressed in the blastocyst may interact with a ligand on the endometrial surface, perhaps via Sd(a) also, to permit embryo implantation. Our data suggest that B4GALNT2 and Sd(a) antigen are essential for embryo implantation.
Asunto(s)
Blastocisto/enzimología , Implantación del Embrión , N-Acetilgalactosaminiltransferasas/metabolismo , Útero/enzimología , Animales , Anticuerpos/administración & dosificación , Blastocisto/efectos de los fármacos , Blastocisto/inmunología , Western Blotting , Membrana Celular/enzimología , Membrana Celular/inmunología , Técnicas de Cultivo de Embriones , Endometrio/enzimología , Endometrio/inmunología , Femenino , Técnica del Anticuerpo Fluorescente , Edad Gestacional , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Antígenos de Histocompatibilidad/metabolismo , Humanos , Inyecciones , Ligandos , Ratones , N-Acetilgalactosaminiltransferasas/antagonistas & inhibidores , N-Acetilgalactosaminiltransferasas/genética , N-Acetilgalactosaminiltransferasas/inmunología , Embarazo , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Útero/efectos de los fármacos , Útero/inmunologíaRESUMEN
OBJECTIVE: To investigate B4galnt2 gene regulation in the female mouse reproductive system (B4galnt2 encodes an enzyme, ß1,4-N-acetylgalactosylaminyltransferase II, that catalyzes the addition of GalNAc to glycoproteins via a ß1,4 linkage). DESIGN: Experimental prospective study. SETTING: Research institute and university. ANIMAL(S): Outbred Institute for Cancer Research (ICR) mice. INTERVENTION(S): Subcutaneous injection of P/E2; uterine tissues were collected after a 3-day injection period and were collected at different times during pregnancy. MAIN OUTCOME MEASURE(S): Gene expression was measured by quantitative real-time polymerase chain reaction after hormonal treatment of ovariectomized mice or pregnant mice. Primary endometrial cell cultivation and a gene promoter assay were used for P regulation analysis. The small interfering RNA (siRNA) technique was used to assess the gene function in embryo implantation in vivo. RESULT(S): Animal experiments, a primary endometrial cell cultivation assay, and a gene promoter assay indicated that B4galnt2 is regulated positively by P and negatively by estrogen. B4galnt2 was expressed in uterine tissue at peri-implantation (embryonic day 3.5) along with a sharp increase in placental P production at embryonic day 10.5, and declined as estrogen increased during pregnancy. Using the siRNA in vivo implantation assay, we have proved that B4galnt2 participated in embryonic implantation during pregnancy in mice. CONCLUSION(S): This study shows for the first time the expression of B4galnt2 in pregnant mice and its regulation by P. We conclude that the naturally occurring up-regulation of B4galnt2 during pregnancy contributes to normal embryo implantation but not to embryo development.