Fetal-maternal immune interaction: blocking antibody and survival of the fetus.

In the late 1940s it became clear that the homograft reaction was essentially the result of an immune response. Subsequently, Medawar commented on the apparent paradox of the survival of the mammalian fetus in the face of such a potential (cell-mediated) immune response. In an outbred population the fetal-placental unit will be antigenically different to the mother by virtue of its complement of paternal genes and additionally there may be developmental or stage-specific gene products that are immunogenic. Many mechanisms have been proposed to account for the survival of the fetus in the face of a potential immune attack and, while many of these have been investigated in considerable detail, there has been no clear-cut indication that any one plays a predominant role. Either control of immune rejection of the fetus is exercised by an as yet undiscovered mechanism or, more probably, by a combination of some or all of the mechanisms that have been proposed by many workers over the last three decades. Potential controlling processes, which will be reviewed briefly, include: systemic and local modification of maternal responsiveness; altered expression of MHC antigens on extra-embryonic tissues; the placenta as a barrier; and blocking antibody responses. We discuss some of our recent studies in which we have started to look for potential blocking antibodies in a mouse model system. Cells secreting immunoglobulins M and G, characterized in hemolytic plaque assays, have been mapped to areas close to the midgestation mouse embryo, using an immunocryohistological technique. A scaled-down version of hybridoma technology has been used as an analytical probe of the specificity and isotype of immunoglobulin secreted by cells originating either from close to the embryo/fetus or from the para-aortic lymph nodes (PALN). So far monoclonal (IgG1) antibodies with specificity for embryonic cells have been derived together with some monoclonal immunoglobulins with as yet uncharacterized antibody specificity.

Cellular and nuclear volume of primitive red blood cells in digynic and diandric triploid and control diploid mouse embryos.

It has long been known that a relationship exists between ploidy and cellular and nuclear volume. This has been observed in amphibia and plants, and more recently in postimplantation tetraploid mouse embryos. We wished to establish whether a similar relationship exists in digynic and diandric triploid mouse embryos, as well as establishing whether the cellular or nuclear volume of primitive nucleated red blood cells of these two classes of triploids were the same. Spontaneously occurring digynic triploid embryos isolated from LT/Sv strain female mice result from the fertilisation of primary oocytes. These embryos develop to the forelimb-bud stage but invariably possess neural tube and cardiac abnormalities. Diandric triploid embryos were also analysed, and were produced experimentally by standard micromanipulatory techniques. They have a relatively normal morphology, and can survive up to the forelimb--bud stage. Primitive red blood cellular and nuclear volume was analysed in serially sectioned digynic and diandric triploid and in developmentally matched diploid control embryos isolated both as littermates of triploid embryos from LT/Sv strain mice, and from other genetically dissimilar diploid controls. The triploid and diploid embryos were analysed between 8-8.5 and 10-10.5 days of gestation, respectively. The cellular and nuclear volumes of the primitive red blood cells in the digynic and diandric triploid embryos were not significantly different, though they were significantly greater than comparable measurements for diploid embryos. We have therefore confirmed that a predictable relationship exists between cellular and nuclear volume and ploidy in the material analysed. The red blood cellular volume in triploid and diploid embryos increased in a predictable way over time, while their nuclear volume decreased in a predictable way over the same time period. A similar relationship has previously been observed when the nucleated red blood cells of diploid and tetraploid embryos were analysed morphometrically.

Time-dependent differences in the development of somites of four different mouse strains.

In studies on reproductive toxicity and especially teratogenicity, animals are often treated at defined stages of pregnancy. As a result the exposure to a certain teratogen can lead to striking differences in the formation of abnormalities in different strains of the same species. As a contribution to the discussion about the reasons for these differences, we examined the somite development of four different strains of mice during organogenesis. The embryos of pregnant females of the inbred strains DBA/2J, BALB/cJ, and C57BL/6J and of the outbred strain Han:NMRI were studied on days 9, 10, 11, or 12 of gestation. As a criterion for development the somite pairs were evaluated on the respective days. There were remarkable differences in the somite number, even within one litter. The largest variation (minimum vs. maximum) was 14 pairs of somites. The regression curves did not exhibit major differences in the speed of somite development from day 9 to day 12 between the four strains. We have to conclude from our results that the individual embryonic stages within one litter may vary by nearly 1 day, and that there may be a delay of half a day in the embryonic development between different strains of mice.

Segregation analysis of the testis-determining autosomal trait, Tda, that differs between the C57Bl/6J and DBA/2J mouse strains suggests a multigenic threshold model.

The testis-determining autosomal trait (Tda) of the mouse was uncovered when the Y chromosome of the poschiavinus variety of Mus musculus domesticus was introduced into the C57BL/6J laboratory strain background. Testis development is normal in the F1 generation but, in the backcross and subsequent crosses to C57BL/6J females, XY individuals with the poschiavinus Y chromosome expressed bilateral ovaries or various combinations of an ovotestis with a contralateral ovary or testis or bilateral ovotestes and few had testes bilaterally. In other strain backgrounds, such as DBA/2J, XY individuals with the poschiavinus Y chromosome always expressed normal testes bilaterally. The first breeding analysis of this difference in the interaction of strain background with the poschiavinus Y chromosome suggested that the Tda trait was due to a single gene, but attempts to map it failed. We constructed two strains of C57BL/6J and DBA/2J that are consomic for the poschiavinus Y chromosome in order to conduct a segregation analysis of the Tda trait. In the C57BL/6J.Y-POS consomic strain, liability to express incomplete testis development is normally distributed and thresholds in development specify the probability of different classes of ovary, ovotestis, and testis combinations. Testis development is complete in the DBA/2J.Y-POS consomic strain. We demonstrated previously that the Tda trait of C57BL/6J is recessive to that of DBA/2J and the segregating first backcross generation of embryos rejected the single-gene model. We have extended our analysis to a F2 generation of embryos that also rejects a single-gene model. We also report a test mating analysis of the first backcross generation. It was initiated to provide an independent assessment of the single-gene model, but the analysis of the distribution of test mating results suggests that the difference in the Tda trait between C57BL/6J and DBA/2J may be due to a small number of loci, possibly four or five, and that the phenotypic effect between loci may be additive.

Ontogeny of murine I-Ak antigens in tissue of nonlymphoid origin.

The ontogeny of I-Ak antigens in murine tissues of nonlymphoid origin has been evaluated by reacting acetone-fixed cryostat sections of embryonic, neonate, and adult tissues with the MOAb 10-2.16 in indirect immunofluorescence. Thymus dendritic cells appear to be the first to express I-Ak antigens which become detectable only after birth in skin Langerhans cells, gastrointestinal epithelium, septal cells in lung alveoli, and some endothelia. The full expression of I-Ak antigens as detected in adult mice is reached only at 1 month of age.

A new embryonic stem cell line from DBA/1lacJ mice allows genetic modification in a murine model of human inflammation.

The development of embryonic stem (ES) cells and their capacity to generate mice with mutations at specific loci has provided a powerful resource for functional analysis of genes in pathological processes. However, the ability to combine this technology with the large number of existing murine models of human genetic disease has been complicated by the inability to routinely generate ES cell lines from strains other than 129. Here, we report the production of a novel ES cell line derived from an inbred mouse, DBA/1lacJ. This new ES cell line undergoes homologous recombination and efficient colonization of the germline of male chimeric offspring with ES cell microinjection into C57B1/6 embryos. The DBA/1lacJ mouse is a murine model of human inflammation, therefore genetic modifications in the DBA ES cells will allow evaluation of the target gene's role in the inflammatory process.

Ganglioside GM1 elevation in DBA/2 mouse embryos.

The composition of whole embryo gangliosides at embryonic day E-12 was compared among the C57BL/6 (B6) DBA/2 (D2), and C3H mouse strains. N-acetylneuraminic acid was the predominant sialic acid species in the E-12 embryos. N-glycolyneuraminic acid was either undetectable or present in only trace amounts. Whole embryo ganglioside sialic concentration was significantly lower in D2 embryos than in B6 or C3H embryos. GM3 and GD3 were the most abundant ganglioside species in each strain and comprised approximately 75% of the total distribution. The D2 embryos expressed an elevation of GD1a and a reduction of GQ1b relative to B6 and C3H. Also, the level of GM1 was significantly higher in the D2 embryos than in the B6 or C3H embryos. Since a reduction of beta-galactosidase activity and an elevation of GM1 concentration in brain were previously reported in postnatal DBA mice, our results suggest that the elevated GM1 in D2 embryos may result from a reduced activity of GM1 beta-galactosidase.

Gene expression, ontogeny and transplacental induction of hepatic UDP-glucuronosyl transferase activity in mice.

The ontogeny and transplacental inducibility of UDP-glucuronosyl transferase (UDPGT) activities potentially relevant to detoxification of polycyclic aromatic hydrocarbons were studied in (C57BL/6 x DBA/2) F1 or (DBA/2 x C57BL/6) F1 fetal mouse liver, with p-nitrophenol (PNP) and 3-hydroxybenzo[a]pyrene (3-OH-BP) as substrates. Both UDPGT activities developed during the late fetal period and reached almost 60% of the adult activity at term; PNP, but not 3-OH-BP UDPGT decreased significantly on postnatal day 1 before rising to adult levels. A single exposure to beta-naphthoflavone (beta NF; 150 mg/kg) on day 17 of gestation induced the PNP-UDPGT activity significantly (1.5-fold) by day 19 in the B6D2 F1s but not D2B6 F1s. A single dose of 3-methylcholanthrene (MC; 100 mg/kg) or 2,3,7,8,-tetrachlorodibenzo-p-dioxin (10 micrograms/kg) did not induce, but three injections of MC also resulted in significant induction in the fetuses of C57BL/6 mothers. 3-OH-BP-UDPGT was not significantly induced by any of the chemicals in either genetic cross. In a parallel study, a gene for an inducible mouse UDPGT, designated UDPGTm-1, was shown by Northern blotting to be expressed in fetal liver by day 18 of gestation at low levels relative to adults, but was not induced transplacentally by MC, beta NF or phenobarbital (PB). These results show that (1) at least two functionally defined UDPGT activities toward phenolic substrates are present in the late fetal mouse liver; (2) one of these is transplacentally inducible by beta NF and MC, but only in fetuses of C57BL/6 mothers, (3) induction where achieved was relatively small in magnitude, and (4) a gene of a PB-inducible UDPGT was expressed at low levels in the fetuses but was not induced transplacentally.

Clastogenic effects of benzo[a]pyrene in postimplantation embryos with different genetic background.

Certain strains of mice vary in their enzyme inducibility by polycyclic hydrocarbons, i.e., the strain C57 shows high and the strain DBA shows low inducibility of aryl hydrocarbon hydroxylase (AHH) by benzo[a]pyrene (BaP). The effect of this genetically determined difference on the clastogenic response to BaP was studied in 11 day old embryos after transplacental treatment. The four possible crosses, C57 and DBA inter se, C57 X DBA and DBA X C57, were used to determine the influence of the genetic background on the aberration yields in the embryos. Constitutive and induced AHH levels were measured in liver, bone marrow, and placenta of the pregnant females and in their embryos. Enzyme inducibility was high in tissues of C57 dams and in their homozygous or heterozygous embryos. In contrast, induction of AHH activity was low in tissues of DBA females and their homozygous embryos. The high BaP-induced AHH activity found in heterozygous embryos of DBA dams is in agreement with the dominant mode of inheritance for high AHH inducibility. The cytogenetic results showed that the clastogenic response was lowest in homozygous C57 embryos and highest in hybrid embryos independent of the genetic constitution of the dams. Homozygous DBA embryos showed an intermediate aberration yield. The AHH inducibility by BaP did not correlate quantitatively with the induced aberration rates. However, the data suggest that BaP activation in embryonic tissue on day 11 of pregnancy is sufficient to account for the clastogenicity in the fetuses. It is concluded that the genetic endpoint chromosomal breakage is not only determined by the formation of active BaP metabolites but also by genetically controlled detoxification of BaP, repair process, and the rate of transformation of primary DNA lesions into true DNA discontinuities.

Visualization of second polar body chromosomes in fertilized and artificially activated mouse oocytes treated with okadaic acid.

OBJECTIVE: Our objective was to develop a new reliable method for cytogenetic analysis of the chromosome set in second polar bodies (PBs) from one-cell-stage mouse embryos. SETTING: The study took place at the Reproductive Biology and Experimental Cytogenetics Laboratories. METHODS: Oocytes from F1 hybrid and T6/T6 mice were fertilized in vitro and artificially activated with ethanol. Zygotes, parthenogenetic embryos, and isolated second PBs were treated with 10 microM okadaic acid (OA) for 1-2 hr, further cultured in plain medium, and fixed. Chromosomal preparations were made and C-banded, and the number of chromosomes in second PBs and embryos was counted. RESULTS: OA-induced nuclear envelope breakdown in pronuclei as well as in second PB nuclei. Countable chromosome plates were obtained in 92-93% of second PBs treated 4-4.5 hr after activation. The T6 marker chromosome could easily be recognized in second PBs from T6/T6 mice. A haploid set of chromosomes was obtained in 18 of 19 isolated second PBs treated with OA 4-5 hr after activation. CONCLUSION: Treatment of second PBs with OA allows visualization of the PB chromosomes. Cytogenetic analysis of the second PB and the corresponding oocyte constitutes a new approach for the study of meiotic nondisjunction in experimental cytogenetics. The chromosomal study of isolated second PBs seems to be promising for clinical preimplantation cytogenetics.