Carcinoembryonic antigen-related cell adhesion molecule 10 expressed specifically early in pregnancy in the decidua is dispensable for normal murine development

Abstract

The carcinoembryonic antigen (CEA) family consists of a large group of evolutionarily and structurally divergent glycoproteins. The murine CEACAM9 and CEACAM11-related proteins as well as the pregnancy-specific glycoproteins (PSG) are secreted members of the CEA family which are differentially expressed in fetal trophoblast cell populations during placental development. PSG are essential for a successful pregnancy, possibly by protecting the semiallotypic fetus from the maternal immune system. In contrast, Ceacam10 mRNA, coding for a protein identical in structure with CEACAM11-related proteins, is expressed in the maternal decidua surrounding the implantation site of the conceptus only during early stages of gestation between day 6.5 and day 10.5 postcoitum. To determine its role during murine development, we inactivated Ceacam10. Ceacam10(-/-) mice developed, like the previously established Ceacam9(-/-) mice, indistinguishably from wild-type littermates with respect to sex ratio, weight gain, and fertility. However, a small but significant reduction of the litter size by 23% was observed in Ceacam10(-/-) matings. Furthermore, combining the Ceacam9 and Ceacam10 null alleles, both located on chromosome 7, by meiotic recombination and subsequent mating of heterozygotes carrying both knockout alleles on one chromosome yielded wild-type and double knockout offspring at the expected Mendelian ratio. Taken together, both Ceacam10 and Ceacam9, alone or in combination, are not essential for either murine placental and embryonic development or for adult life.

FIG. 1.

Analysis of Ceacam10 expression by in situ hybridization during placental development. Unfixed uteri from pregnant BALB/c mice at 6.5 dpc (a, d, g, j, m, and p), 8.5 dpc (b, e, h, k, n, and q) or 10.5 dpc (c, f, i, l, o, and r) were cryosectioned. In situ hybridization was performed with digoxigenin-labeled antisense Ceacam10 (a to c), Ceacam9 (d to f), Psg18 (g to i), Pl-1 (j to l), 4311 (m to o), and Mash-2 (p to r) RNA probes. The hybridized RNA was visualized by reaction with an antidigoxigenin alkaline phosphatase-conjugated antibody and subsequent incubation with the substrate nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate. The mesometrial side of the conceptus is to the right. Arrows point to individually labeled cells or cell cluster. bv, blood vessel; e, embryo; epc, ectoplacental cone; db, decidua basalis; dc, decidua capsularis; gc, giant (trophoblast) cells; ivr, intermediate vascular region; la, labyrinth zone; pgc, primary giant (trophoblast) cells; sgc, secondary giant (trophoblast) cells; sp, spongiotrophoblast; ue, uterine epithelium. Arrows in panels m and n indicate individual cells stained by the 4311 probe. Scale bars (identical within each column, except for panel b), 0.5 mm for 6.5 and 8.5 dpc placentae (1 mm in panel b); 1 mm for 10.5 dpc placentae.

FIG. 2.

Targeted disruption of the murine Ceacam10 gene. (a) Structure of the wild-type allele (upper line), targeting construct (middle), and recombinant locus (bottom line). Gray boxes represent the four exons of Ceacam10. The neo and tk expression cassettes used for the selection of homologous recombinants are shown as open boxes. Arrows indicate the transcriptional direction of neo. The vector sequence within the targeting plasmid is shown as a thin line. The expected sizes of the DNA fragments obtained after digestion with HindIII are 6.1 kb for the wild-type allele and 2.7 kb for the correctly targeted allele, using an SspI/BamHI DNA fragment located directly 5′ of the targeting construct as a probe. B, BamHI; C, ClaI; E, EcoRI; H, HindIII; K, KpnI; S, SstI; Ss, SspI. (b) Southern blot analyses of DNA isolated from G418-FIAU-resistant ES cell clones. DNA was digested with HindIII and hybridized with the ca. 250-bp SspI/BamHI ³²P-labeled genomic 5′ probe. The sizes of marker DNA fragments are indicated in the right margin. The asterisk indicates cross-hybridizing DNA fragments. (c) RT-PCR analysis of Ceacam9 and Ceacam10 mRNA in total RNA isolated from 8.5-dpc placentae and embryos of Ceacam9^−/− and Ceacam10^−/− homozygous matings using gene-specific primers. As a control, β-actin-specific primers were used. The mobility and sizes of marker DNA fragments are shown in the left margin.

FIG. 3.

Hypothetical model of immune regulatory functions of CEACAM9 and CEACAM10 during early murine pregnancy. Based on the spatiotemporal expression pattern of CEACAM1, CEACAM9, and CEACAM10 and the homo- and heterophilic properties of CEA family members, the following hypothetical functional scenario can be sketched: a dimeric, embryo-derived CEACAM9 binds to CEACAM1 on decidual NK cells of the mother. This cross-links CEACAM1, which transmits a negative signal through the immunoreceptor tyrosine-based inhibition motifs (ITIM; black dots) in its cytoplasmic tail. This reaction is counterbalanced by maternally produced CEA CAM10 dimers which function as soluble decoy receptors, thus regulating the invasively growing fetal trophoblast cells.