Murine submucosal glands are clonally derived and show a cystic fibrosis gene-dependent distribution pattern.

Submucosal glands (SMGs) are the major site of expression of the cystic fibrosis (CF) transmembrane conductance regulator gene (CFTR) in the human lung. As such, SMGs may be a critical component of CF lung disease pathogenesis and an important target for gene therapy. Gene-targeted mouse models exist for CF and these are used to validate gene therapy or other interventions and to dissect CF phenotypes. It is important, therefore, to compare human and mouse SMGs. We show that SMGs in the mouse are similar in structure, cell types, and Cftr expression to those in the human. Murine SMGs were found to be present in the proximal regions of the trachea at the same density as in humans but, unlike in humans, did not extend below the trachea. Upon investigation of homozygous Cftr tm1HGU and Cftr tm1G551D mutant mice, SMGs were found to extend more distally than those in wild-type control mice (P < 0.05). To investigate the development of SMGs we generated aggregation chimeric mice. Chimeric offspring contained a contribution of transgenic cells that were detectable either by DNA in situ hybridization (reiterated beta-globin transgene TgN[Hbb-bl]83Clo) or beta-galactosidase histochemistry (Lac Z reporter gene TgR[ROSA26]- 26Sor). Analysis of the distribution of transgenic cells in chimeric SMGs suggests that SMGs are clonally derived.

A quantitative test for developmental neutrality of a transgenic lineage marker in mouse chimaeras.

The mouse transgene, provisionally designated TgN(Hbb-b1)83Clo, was produced by Dr C. Lo by pronuclear injection of the cloned beta-major globin gene and comprises a highly reiterated sequence that is readily detected by DNA in situ hybridization on histological sections. This fulfils many of the requirements of an ideal genetic cell marker and has been widely used for lineage studies with mouse chimaeras. However, it is not known whether it causes cell selection or influences developmental processes, such as cell mixing, in chimaeric tissues. In the present study, non-transgenic genetic markers (electrophoretic polymorphisms of glucose phosphate isomerase and differences in eye pigmentation) revealed no significant effect of the presence of hemizygous transgenic cells on the overall composition, size or gross morphology of 12 1/2 d chimaeric foetuses, placentas or extraembryonic membranes. Also, a previously described maternal genetic effect on the composition of chimaeric tissues occurred in the presence or absence of the transgene. These tests have demonstrated that hemizygous cells are not at a significant selective disadvantage, when incorporated into mouse aggregation chimaeras with non-transgenic cells. Further studies are needed to test whether homozygous transgenic cells are also selectively neutral and to test whether hemizygous or homozygous transgenic cells influence developmental processes, such as cell mixing, that were not tested.

Biochemical evidence for cell fusion in placentas of mouse aggregation chimeras.

Eight series of mouse chimeras were produced by aggregating 8-cell embryos that differed at the Gpi-1s locus, encoding glucose phosphate isomerase (GPI-1). Chimeric blastocysts (Gpi-1sa/Gpi-1sa <--> Gpi-1sb/Gpi-1sb) were transferred to pseudopregnant females, which produced only GPI-1C enzyme. Quantitative electrophoresis of GPI-1 was used to estimate the contribution of each embryo (GPI-1A and GPI-1B enzyme activity) to the fetus and placentas of 12 1/2 day chimeric conceptuses. Chimeric fetuses and placentas were identified by the presence of both GPI-1AA and GPI-1BB homodimers. The overall distribution of the percentage GPI-1A in the placentas was bimodal or U-shaped. It was positively correlated with the %GPI-1A in the fetus in most of the eight series of chimeras analyzed. In the first chimera experiment, involving seven series of chimeras, GPI-1AB heteropolymer was detected in 78/211 (37%) of the placentas. Heteropolymer was not detected in chimeric placentas with an unbalanced composition of GPI-1A and GPI-1B. The production of heteropolymer implies that GPI-1A and GPI-1B monomers are produced in the same cell and that fusion must have occurred between the two genetically distinct cell populations in the placenta. In the second experiment, samples of different regions were dissected from another series of 27 chimeric placentas and analyzed; 12 contained heteropolymer. Although GPI-1AB heteropolymer was widely distributed throughout the placenta it was detected less frequently in the outer part of the placenta. In another experiment, analysis of 34 homozygous (nonchimeric) Gpi-1sb/Gpi-1sb conceptuses transferred to homozygous Gpi-1sa/Gpi-1sa reproductive tracts revealed no evidence for fusion between maternal cells and cells of zygotic origin in the placenta. The chimera studies provide biochemical evidence for fusion between zygotic cells in the murine placenta. This presumably occurs during the formation of the syncytial trophoblast.

A maternal genetic effect on the composition of mouse aggregation chimaeras.

Two series of 12 1/2 day mouse chimaeric conceptuses were produced by aggregating (C57BL x CBA)F2 strain preimplantation embryos with embryos that differed at the Gpi-1s locus that encodes glucose phosphate isomerase, GPI-1. The composition of individual issues was evaluated by quantitative electrophoresis to estimate the % GPI-1A in the chimaeric tissue containing GPI-1A and GPI-1B. In one series of chimaeras, the GPI-1A cells were derived from a backcross between inbred BALB/c strain females and (BC x BALB/c)F1 males, where BC is the partly congenic strain C57BL/Ola.AKR-Gpi-lsa,c/Ws. In the other series of chimaeras, the GPI-1A cells were derived from the reciprocal backcross between (BC x BALB/c)F1 females and inbred BALB/c strain males. The [(BC x BALB/c)F1 female x BALB/c male]<==>(C57BL x CBA)F2 series of chimaeras was reasonably balanced so that GPI-1A and GPI-1B cells were fairly equally represented in the foetuses, placentas and extraembryonic membranes (tissue means: 37-51% GPI-1A). This series did not differ significantly in composition from an earlier series of (BC x BALB/c)F2<==>(C57BL x CBA)F2 chimaeras. However, the [BALB/c female x (BC x BALB/c)F1 male]<==>(C57BL x CBA)F2 series of chimaeras was unbalanced, with mean tissue compositions (28-33% GPI-1A) that were intermediate between the above two balanced series and the unbalanced (BALB/c x BALB/c)<==>(C57BL x CBA)F2 series (tissue means: 14-22% GPI-1A), that was studied previously. Thus, both (BALB/c x BALB/c) and [BALB/c x (BC x BALB/c)F1] embryos contributed less to the tissues of chimaeric conceptuses than either (BC x BALB/c)F2 or [BC x BALB/c)F1 x BALB/c] embryos. This implies that embryos from BALB/c mothers contributed less to the tissues of chimaeric conceptuses than embryos from (BC x BALB/c)F1 mothers. We, therefore, conclude that a maternal genetic effect is responsible for some of the differences in composition among the four groups of chimaeras. This maternal effect must act before the 8-cell stage but it is not yet known whether it is mediated via cytoplasmic inheritance, genomic imprinting or by the reproductive tract. Evidence that a maternal effect retards preimplantation development of embryos from BALB/c females is reviewed and the possibility that this might cause them to contribute poorly to chimaeric conceptuses when aggregated with more precociously developing embryos is discussed.

Restricted distribution of tetraploid cells in mouse tetraploid<==>diploid chimaeras.

Tetraploid mouse embryos were produced by electrofusion at the 2-cell stage, cultured overnight, and aggregated with normal diploid embryos to produce tetraploid<==>diploid (4n<==>2n) chimaeric conceptuses. At 7 1/2 days the 4n<==>2n chimaeras were usually smaller and developmentally retarded compared to control diploid<==>diploid chimaeras. At 12 1/2 days the 4n<==>2n chimaeras had heavier placentas but there was no significant difference in fetal size. Tetraploid cells showed a restricted tissue distribution at both developmental stages studied: 4n cells were commonly present in both the primitive endoderm and the trophectoderm lineages but they rarely contributed to the primitive ectoderm lineage. The overall similarity in the distribution of tetraploid cells at 7 1/2 and 12 1/2 days implies that whatever causes the restricted tissue distribution operates largely before 7 1/2 days. There was no evidence for excessive embryonic losses of 4n<==>2n chimaeras. So, if the restricted distribution of 4n cells was a result of cell selection, the mechanism is more likely to involve loss of 4n cells from the primitive ectoderm early in development rather than selective death of conceptuses with tetraploid cells in this lineage. Alternatively, 4n cells may be preferentially allocated to the trophectoderm and primitive endoderm rather than the primitive ectoderm layer at the blastocyst stage.

Genotypically unbalanced diploid<==>diploid foetal mouse chimaeras: possible relevance to human confined mosaicism.

Two series of mouse chimaeras were produced by aggregating pairs of eight-cell embryos that differed at the Gpi-1s locus, encoding glucose phosphate isomerase (GPI-1); the paired embryos were respectively homozygous Gpi-1sa/Gpi-1sa and Gpi-1sb/Gpi-1sb. Chimaeric blastocysts were transferred to pseudopregnant females, that were homozygous Gpi-1sc/Gpi-1sc and produced only GPI-1C enzyme. Quantitative electrophoresis of GPI-1 was used to estimate the contribution of each embryo (GPI-1A and GPI-1B enzyme activity) to the foetus, placenta and other extraembryonic tissues of 12 1/2 day chimaeric conceptuses. For both series of chimaeras, the distributions of %GPI-1A in different tissues were classified as (1) balanced and typical, (2) balanced but atypical or (3) unbalanced. One series of chimaeras was clearly unbalanced, so that the cells derived from the (C57BL x CBA/Ca)F2 embryo (Gpi-1sb/Gpi-1sb) predominated over those derived from the BALB/c inbred strain (Gpi-1sa/Gpi-1sa) in most foetuses. Two significant observations were made concerning this unbalanced series. Firstly, the mean composition of the placenta and other extraembryonic tissues was similar to that in the foetus, i.e. also unbalanced with (C57BL x CBA/Ca)F2 (abbreviated to BF2) cells predominating. Secondly, despite this generalized deficiency of BALB/c cells, there were differences in the frequency of non-chimaeric tissues between different developmental lineages. In 20/38 [corrected] chimaeric conceptuses in the unbalanced series only BF2 cells were detected in the foetus, whereas both BF2 and BALB/c cells were present in at least one of the extraembryonic tissues. This group of chimaeras, therefore, shows some similarities to human confined mosaicism. Although chimaerism occurred more often in the primitive endoderm (hypoblast) lineage (yolk sac endoderm and parietal endoderm) than in the placenta, this may also be the case in human mosaics. The mosaic status of the human yolk sac endoderm is usually unknown so it is possible that mosaicism often occurs in the yolk sac endoderm as well as the trophectoderm in human 'confined placental mosaicism'. The uniformly unbalanced phenotype seen in the mouse chimaeras may be a result of generalized cell selection against BALB/c cells in all tissues. As an alternative explanation, we propose that most of the BALB/c cells in the blastocyst are allocated to the mural trophectoderm, which has a limited mitotic potential and so contributes little to the mid-gestation conceptus. Further work is required to test these possibilities.

Expression of glucose phosphate isomerase in interspecific hybrid (Mus musculus x Mus caroli) mouse embryos.

Hybrid Mus musculus x Mus caroli embryos were produced by inseminating M. musculus (C57BL/OlaWs) females with M. caroli sperm. Control M. caroli embryos developed more rapidly than did control M. musculus embryos and implanted approximately 1 day earlier. At 1 1/2 days, both the hybrid embryos and those of the maternal species (M. musculus) had cleaved to the 2-cell stage. By 2 1/2 days some of the hybrids were retarded compared to M. musculus, and by 3 1/2 days most were lagging behind. This is consistent with the idea that the rate of development of hybrid embryos declines once it becomes dependent on embryo-coded gene products. We have used this difference in rate of preimplantation development, between hybrid and M. musculus embryos, to try to determine whether the activation of embryonic Gpi-1s genes, that encode glucose phosphate isomerase (GPI-1), is age-related or stage-related. In control M. musculus embryos (both mated and Al groups), the GPI-1AB and GPI-1A allozyme, indicative of paternal gene expression, were detected in 7 of 9 samples of 3 1/2-day compacted morula stage embryos and were seen in all 19 samples of 3 1/2-day blastocysts. In hybrid embryos, these allozymes were detected 1 day later. They were not detected in any 3 1/2-day samples (12 samples of compacted morulae) but were consistently detected at 4 1/2 days (4 samples of blastocysts and 2 samples of uncompacted morulae). Our interpretation of the results is that gene activation in hybrid embryos is stage-specific, rather than age-specific, and probably begins around the 8-cell stage, with detectable levels of enzyme accumulating later. Analysis of GPI-1 electrophoresis indicated that both the paternal (M. caroli) and maternal (M. musculus) Gpi-1s alleles were equally expressed in hybrid embryos and that the paternally derived allele was not activated before the maternally derived allele.

Death of mouse embryos that lack a functional gene for glucose phosphate isomerase.

A null allele of the Gpi-1s structural gene, that encodes glucose phosphate isomerase (GPI-1; E.C. 5.3.1.9), arose in a mutation experiment and was designated Gpi-1sa-m1H. The viability of homozygotes has been investigated. No offspring homozygous for the null allele were produced by intercrossing two heterozygotes, so the homozygous condition was presumed to be embryonic lethal. Embryos were produced by crossing Gpi-1sa/null heterozygous females and Gpi-1sb/null heterozygous males. Homozygous null embryos were identified at different stages of development by electrophoresis and staining either for GPI-1 alone or GPI-1 plus phosphoglycerate kinase (PGK) activity. At 6 1/2 and 7 1/2 days post coitum homozygous null embryos were present at approximately the expected 25% frequency (37/165; 22.4% overall) although at 7 1/2 days the homozygous null embryos tended to be small. By 8 1/2 days most homozygous null embryos were developmentally retarded and had not developed significantly further than at 7 1/2 days; some were dead or dying. By 9 1/2 days the homozygous null conceptus was characterised by a small implantation site that contained trophoblast and often a small amount of extraembryonic membrane. Surviving trophoblast tissue was also detectable at 10 1/2 days. Previous studies have shown that oocyte-coded GPI-1 persists only until 5 1/2 or 6 1/2 days. Survival of homozygous null embryos to 7 1/2 or 8 1/2 days and survival of certain extraembryonic tissue to 10 1/2 days suggests that the homozygous null condition may not be cell-lethal although it is certainly embryo-lethal. Mutant cells that are deficient in glycolysis may use the pentose phosphate shunt to bypass the block in glycolysis created by the deficiency of glucose phosphate isomerase, and/or might be rescued by the transport, from the maternal blood, of energy sources other than glucose (such as glutamine). Either strategy may only permit slow cell growth that would not be adequate to support normal embryogenesis. Transport of maternal nutrients would be more efficient to the trophoblast and extraembryonic membranes and this may help to explain why these tissues survive for longer than the embryo itself. The morphological similarity between homozygous nulls and androgenetic conceptuses, where the trophoblast also survives better than the embryo, is discussed.

Genetic differences in glucose phosphate isomerase activity among mouse embryos.

We have compared mouse embryos of three heterozygous, congenic genotypes (with high, medium and low levels of oocyte-coded glucose phosphate isomerase (GPI-1) activity respectively) to test whether 1) the survival time of oocyte-coded GPI-1 activity in the early embryo is affected by its activity level in the oocyte and 2) whether embryo-coded GPI-1 is detected earlier in embryos that inherit low levels of oocyte-coded GPI-1. The oocyte-coded GPI-1 was entirely GPI-1A allozyme in the high and medium groups but was the less stable GPI-1C allozyme in the low group. We determined total GPI-1 activity and the ratio of different GPI-1 allozymes in early embryos and calculated the activity of oocyte-coded and embryo-coded GPI-1. In all three groups, the oocyte-coded enzyme activity remained at a more or less constant level for the first 21 1/2 days. Some oocyte-coded GPI-1 remained in 4 1/2 day embryos from the high and medium groups but was gone by 5 1/2 days. Very little remained in 4 1/2 day embryos that inherited low levels of a less stable form of the enzyme (GPI-1C allozyme). Despite a 4- to 5-fold difference in initial oocyte-coded GPI-1 activity, no differences were seen among the three genotypically distinct groups of embryos in the time of activation of the embryonic Gpi-1s genes. The embryo-coded GPI-1 was first detectable in 3 1/2 day compacted morulae in all three groups. The level of oocyte-coded GPI-1, in the high group, when embryo-coded GPI-1 was first detected was higher than the level in the low group at any stage prior to detection of embryo-coded GPI-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Non-additive inheritance of glucose phosphate isomerase activity in mice heterozygous at the Gpi-1s structural locus.

The activity of blood glucose phosphate isomerase (GPI-1) in mice heterozygous for various alleles at the Gpi-1s structural locus (heterozygotes a/b, a/c and b/c) was significantly higher than expected, on the basis of additive inheritance, from the levels in parental homozygotes. Moreover, the GPI-1 activity was higher in a/b heterozygotes than in either parent (heterosis). Studies of heat stability with kidney homogenates revealed that the relative stabilities of GPI-1 dimers was AA greater than AB greater than BB greater than AC greater than or equal to BC greater than CC. Differences in dimer stabilities in vivo would affect the total GPI-1 levels in heterozygotes and could account for non-additive inheritance but would be insufficient to explain heterosis for GPI-1 activity. Other possible contributing factors include unequal production or stability of monomers, or higher catalytic activity of heterodimers. Monomers could also associate non-randomly but this would not be sufficient to explain heterosis. It is clear that non-additive inheritance patterns may be produced by variants of either structural or regulatory genes.

Glucose phosphate isomerase activity in mouse and human eggs and pre-embryos.

We have measured the activity of glucose phosphate isomerase (GPI-1) in 12 unfertilized human eggs and five human pre-embryos relative to the GPI-1 activity in C57BL/OlaWs mouse eggs. The GPI-1 activity in the human eggs was approximately 6 times that in the C57BL mouse eggs. This implies that human eggs have approximately twice the activity per unit volume of the C57BL mouse eggs but no more than certain other strains of mice. The activity in five human pre-embryos, the most advanced of which was an early blastocyst, was similar to that seen in the human eggs. No change in GPI-1 activity was seen in mouse pre-embryos up to 2 1/2 days (8- to 12-cell stage) but the activity had declined by 3 1/2 days (compacted morula and early blastocyst stages). It seems that high levels of GPI-1 activity are maintained during the early preimplantation development of both species.

Prediction of ovulation using a dipstick sensitive to urinary luteinising hormone.

Accurate prediction of ovulation is essential for patients receiving donor insemination (AID). In 9 patients, detection of the urinary luteinising hormone (LH) surge by monoclonal antibody immunospecific dipstick tests (Ovustick) for both home and laboratory use was assessed with simultaneous quantitative estimations of plasma and urinary LH. In all patients the Ovustick accurately detected within 24 hours the beginning of the plasma LH surge. This test may prove to be a useful method for ovulation prediction but further evaluation is required.

High activity of an unstable form of glucose phosphate isomerase in the mouse.

Quantitative electrophoretic studies of the three allozymes of glucose phosphate isomerase (GPI-1) produced by Gpi-1sa/Gpi-1sc heterozygous mice revealed two opposing influences on GPI-1 activity. First, the GPI-1AC heterodimer is less stable than GPI-1AA but more stable than the GPI-1CC homodimer. Second, a genetic determinant that maps close to or within the Gpi-1s structural gene causes elevated activity of GPI-1AC and probably also GPI-1CC dimers. The relative lability of these allozymes masks this elevated activity in some tissues but the effect is probably ubiquitous. The significance of these observations is discussed.

Urinary excretion of biologically active chorionic gonadotrophin by the pregnant marmoset (Callithrix jacchus jacchus).

No significant correlation exists between the amount of biologically active marmoset chorionic gonadotrophin (mCG) in urine and results obtained with an immunological pregnancy test. The pregnant marmoset excretes large amounts of oestrogenic steroids, which must be removed, to prevent the enhancement of the response of the bioassay for mCG. More than 99% of these unconjugated and conjugated urinary oestrogens can be removed by extraction with acetone and ether. mCG is excreted throughout pregnancy, maximum levels occurring between the 8th and 9th week of gestation. There is a considerable within- and between-animal variation in the amount of mCG excreted. However, the pattern of gonadotrophin excretion by the pregnant marmoset is similar to that of man and the apes but unlike that of baboons and macaques.