Supplementary Material for: Carbonic anhydrase IV deficiency causes intrauterine embryonic loss in mice

Members of the carbonic anhydrase gene family, responsible for the reversible hydration of carbon dioxide, participate in several important biological processes including processes involved in fertilization. CAIV has been shown to play a role in sperm cell capacitation and regulation of sperm motility and is present in mature murine placentae. The present study specifically analyses the distribution of CAIV in female reproductive organs and during early placenta development. Immunostaining for CAIV was performed on female reproductive organs (ovary, fallopian tube, uterus, vagina) of non-pregnant mice and on implantation sites of early pregnancy between 4.5 and 9.5 days post coitum (dpc). Sex typing of embryos was performed by PCR using three separated gene combinations for X- and Y-chromosomes, respectively. Additionally, reproductive outcome of CAIV-deficient mice was determined. CAIV is largely absent in the female reproductive organs of non-pregnant mice. Immunostaining for CAIV was present in the blastocyst and in consecutive stages of the developing embryo. In the endometrial epithelium distant from the implantation chamber, CAIV is induced from 8.5 dpc onwards. Moreover, the yolk sac epithelium, the trophoblast giant cells and the labyrinthine compartment of the developing hemochorial placenta shows a strong immunostaining for CAIV. In heterozygous mating, the number of CAIV knockout pups is significantly reduced than was to be expected according to the mendelian rules, while homozygous mating of CAIV knockout mice results in a significant reduction of litter size which is mainly due to a reduced number of female mice born. Since at 9.5 dpc the number of female embryos is rather higher than that of males, the observed reduction of female offspring appears to be due to a defect in placentation after 9.5 dpc. Thus, CAIV seems to be involved in the signaling network of embryo development, implantation, and placentation.