Trophoblast cell-specific carcinoembryonic antigen cell adhesion molecule 9 is not required for placental development or a positive outcome of allotypic pregnancies

Abstract

The carcinoembryonic antigen (CEA) family consists of a large group of evolutionarily divergent glycoproteins. The secreted pregnancy-specific glycoproteins constitute a subgroup within the CEA family. They are predominantly expressed in trophoblast cells throughout placental development and are essential for a positive outcome of pregnancy, possibly by protecting the semiallotypic fetus from the maternal immune system. The murine CEA gene family member CEA cell adhesion molecule 9 (Ceacam9) also exhibits a trophoblast-specific expression pattern. However, its mRNA is found only in certain populations of trophoblast giant cells during early stages of placental development. It is exceptionally well conserved in the rat (over 90% identity on the amino acid level) but is absent from humans. To determine its role during murine development, Ceacam9 was inactivated by homologous recombination. Ceacam9(-/-) mice on both BALB/c and 129/Sv backgrounds developed indistinguishably from heterozygous or wild-type littermates with respect to sex ratio, weight gain, and fertility. Furthermore, the placental morphology and the expression pattern of trophoblast marker genes in the placentae of Ceacam9(-/-) females exhibited no differences. Both backcross analyses and transfer of BALB/c Ceacam9(-/-) blastocysts into pseudopregnant C57BL/6 foster mothers indicated that Ceacam9 is not needed for the protection of the embryo in a semiallogeneic or allogeneic situation. Taken together, Ceacam9 is dispensable for murine placental and embryonic development despite being highly conserved within rodents.

FIG. 1

Targeted disruption of the murine Ceacam9 gene. (a) Structure of the wild-type allele, targeting construct, and recombinant locus. Gray boxes represent the three exons of Ceacam9. The neo and tk expression cassettes used for the selection of homologous recombinants are shown as open boxes. Arrows indicate their transcriptional directions. The vector sequence within the targeting plasmid is shown as a thin line at the 3′ end of the tk gene. The expected sizes of the DNA fragments obtained were 23 kb after digestion with HindIII for the wild-type allele and 13.5 and 10 kb after digestion with both probes, which are located 5′ and 3′ of the targeting construct, respectively, for the correctly targeted allele. B, BamHI; E, EcoRI; H, HindIII; K, KpnI. (b) Southern blot analyses of DNA isolated from tail biopsies obtained from the progeny of a mating of Ceacam9^+/− parents. The DNAs were digested with HindIII and hybridized with a genomic probe 5′ of the Ceacam9 sequence present in the targeting vector. The blot was rehybridized with a probe from the 3′-flanking region. To exclude additional nonhomologous integration events of the targeting construct, the blot was hybridized with the whole HindIII-linearized ³²P-labeled pGK-neotk vector as a probe, which did not reveal additional hybridizing fragments apart from the 10-kb HindIII DNA fragment. The sizes of the hybridizing DNA fragments are in the right margin. (c) Northern blot analysis of total RNA isolated from days 9.5, 10.5, and 12.5 p.c. placentae of wild-type (+/+) and day 10.5 p.c. placentae of Ceacam9^−/− mice (−/−). The same filter was sequentially hybridized with ³²P-labeled Ceacam9 and β-actin cDNA probes. The mobilities of the 28S and 18S ribosomal RNAs are shown.

FIG. 2

Analysis of trophoblast marker gene expression in placentae from wild-type and Ceacam9^−/− mice by in situ hybridization. Placentae from wild- type (+/+) and homozygous mutant (−/−) mice were isolated day 8.5 p.c., cryosectioned, and hybridized with digoxigenin-labeled antisense RNA probes. Specifically binding RNA was visualized after incubation with anti-digoxigenin antibodies coupled with alkaline phosphatase using NBT-BCIP (nitroblue tetrazolium–5-bromo-4-chloro-3-indolylphosphate) as a chromogenic substrate. No labeling was observed with sense probes (data not shown). The probes used are indicated above and below the panels. Bars, 0.5 mm. De, decidua; ep, embryo proper; epc, ectoplacental cone; pg, primary giant trophoblast cells; sg, secondary giant trophoblast cells.