Search PubMed⌕ Search

Biomedical subjects

G M Kidder

Publications and source records attributed to G M Kidder.

At least 55 records · Page 3Linked to original sources
PubMed1991

Antibodies to a renal Na+/glucose cotransport system localize to the apical plasma membrane domain of polar mouse embryo blastomeres.

Mouse preimplantation embryos were examined for the cell surface expression of epitopes that cross-react with antibodies to a 75-kDa subunit of a purified porcine renal brush border Na+/glucose cotransport system. A Na+ cotransport system is hypothesized to reside in the apical plasma membrane domain of mouse polar blastomeres and to be associated with the induction of their apical-basal polarity. Western blot analysis showed that unfertilized oocytes as well as preimplantation embryos contain a cross-reacting antigen with an apparent molecular weight of about 75,000. Embryos and their isolated blastomeres were double-labeled and assayed by indirect immunofluorescence (IIF) for the expression of epitopes (visualized by labeling with rabbit antiserum or mouse monoclonal IgG to cotransporter followed by the appropriate rhodamine-conjugated second antibodies) and for the development of cell surface polarity (visualized by the apical restriction of fluoresceinated succinylated concanavalin A binding; FS Con A). IIF did not detect these epitopes until after the second cleavage when 4-cell embryos expressed low-to-moderate levels. Although epitopes were expressed on all surfaces of 4-cell blastomeres, some blastomeres expressed more epitopes on their apical surfaces than on their basolateral ones. All precompaction 8-cell embryos expressed epitopes, with expression being greater apically on some blastomeres. The level of expression appeared to reach a maximum on morulae and to decline on cavitating embryos. Assays performed on isolated blastomeres from postcompaction embryos showed that by the 16-cell stage epitope expression appeared to become restricted to FS Con A-labeled apical plasma membrane domains and was no longer evident on basolateral domains. This apparent apical restriction of epitope expression was confirmed by electron microscopic examination of immunogold-labeled isolated polar 16-cell blastomeres. These results demonstrate that preimplantation mouse embryos contain an antigen(s) that is immunologically and structurally similar to a 75-kDa renal Na+/glucose cotransporter. The onset of cell surface expression of this antigen precedes development of the stable polar phenotype.

Animals↗
PubMed1990

Cell polarity and development of the first epithelium.

In the 4 1/2 to 5 days between fertilization and implantation, the mouse conceptus must gain the abilities to implant and produce an embryo. Each of these is the sole developmental responsibility of one of two cell types forming the blastocyst, trophectoderm and inner cell mass (ICM), respectively. Trophectoderm is a polarized transporting epithelium while the ICM is an aggregate of non-epithelial pluripotent stem cells. These two cell types originate from the division of polar blastomeres when their cleavage furrows parallel their apical surfaces. Blastomeres polarize in response to asymmetric cell--cell contact, and understanding the mechanism of this induction is regarded as the key to understanding the origin of trophectoderm and ICM. Here we propose a model based on transcellular ion current loops for the induction of cell polarity during the development of the first epithelium, trophectoderm.

Animals↗
PubMed1990

Expression of Na,K-ATPase alpha and beta subunit genes during preimplantation development of the mouse.

Na,K-ATPase is a plasma membrane enzyme that plays a critical role in eutherian blastocoel formation (cavitation) by pumping Na+ into the extracellular space enclosed by the trophectoderm. Previous experiments with the mouse had shown that the alpha (catalytic) subunit of the enzyme becomes detectable by immunocytochemistry in the late morula, just prior to the onset of cavitation. In the present study we have used cDNAs corresponding to three mRNA isoforms of the alpha subunit and a beta subunit to determine which genes are expressed during preimplantation development and to explore the timing of their expression. Of the three alpha subunit cDNAs tested by Northern blot hybridization with blastocyst RNA, only alpha 1 produced a hybridization signal, recognizing a single mRNA about 4 kb in length. This mRNA is relatively abundant in zygotes but barely detectable by the 2-cell stage and then accumulates steadily thereafter to reach its preimplantation maximum in blastocysts. The beta 1 cDNA detected mRNA of about 2.6-2.8 kb. This mRNA is present in zygotes but could not be detected in 2-, 4-, or 8-cell stages; it is present at a low level in late morulae and is abundant in blastocysts. The temporal profile of accumulation of beta 1 mRNA thus matches more closely than does alpha 1 the timing of appearance of the catalytic subunit. This suggests that the beta subunit may regulate production of the holoenzyme and hence the timing of cavitation.

Animals↗
PubMed1990

Differentiation of an epithelium: factors affecting the polarized distribution of Na+,K(+)-ATPase in mouse trophectoderm.

Na+,K(+)-ATPase is a marker of the basolateral plasma membrane domain of polarized epithelial cells, including the mural trophectoderm of the mammalian blastocyst (Watson and Kidder (1988). Dev. Biol. 126, 80-90). We have used this marker to explore the factors governing the establishment and maintenance of apical/basolateral polarity during differentiation of trophectoderm. A polyclonal antiserum (anti-GP80) against human cell-CAM 120/80, a homolog of the mouse cell-cell adhesion protein, uvomorulin, was used to prevent cell flattening (compaction) and formation of the epithelial junctional complex. The majority of treated embryos failed to develop a blastocoel; instead their blastomeres developed fluid-filled cavities that expanded while untreated control embryos were cavitating. Immunocytochemistry revealed that the catalytic subunit of Na+,K(+)-ATPase was contained within the membranes lining these cavities, as well as within numerous punctate foci in the cytoplasm. The down-regulation of expression of the enzyme that normally occurs in the ICM and polar trophectoderm did not take place, since the immunoreactivity remained equally strong in all blastomeres. The enzyme could not be detected in plasma membranes. We conclude that uvomorulin-mediated cell adhesion is involved in spatially restricting the expression of the catalytic subunit and is a prerequisite for the insertion of enzyme-laden vesicles into plasma membranes, but not for expression of the catalytic subunit gene. When fully developed blastocysts were treated with cytochalasins to disrupt the epithelial junctional complex, the catalytic subunit shifted from the basolateral to the apical plasma membrane. This finding suggests a primary role for the apical plasma membrane in the process of polarization, and implies that tight junctions are a manifestation of polarity that serve to maintain the separation between apical and basolateral markers.

Animals↗
PubMed1989

Connexin32, a gap junction protein, is a persistent oogenetic product through preimplantation development of the mouse.

Gap junctions appear de novo during compaction in the eight-cell stage of mouse development. This is a critical event in the life of the embryo, because gap junctional intercellular communication is an essential requirement for maintaining compaction and, hence, for development of the blastocyst. Recently, a family of genes encoding gap junction proteins (connexins) has been identified and cloned, and we have taken advantage of the availability of antibodies and cDNA probes to investigate the expression of these genes in early development. We found that a protein with antigenic and size similarity to the "liver" gap junction protein, connexin32, is present throughout preimplantation development from the zygote through the late morula. Connexin32 mRNA, however, could not be detected in any preimplantation stage. This, and the presence of connexin32 in zygotes before activation of embryonic transcription, leads us to conclude that this protein is inherited as an oogenetic product that persists well beyond the transition from the oogenetic to embryonic program of gene expression. Furthermore, we found that mRNA for another gap junction protein, connexin43, is fairly abundant in preimplantation embryos. We conclude that it is more likely connexin43, and not connexin32, that is used to assemble new connexons as the level of intercellular coupling increases after compaction.

Animals↗
PubMed1988

Immunofluorescence assessment of the timing of appearance and cellular distribution of Na/K-ATPase during mouse embryogenesis.

We have employed immunofluorescence with a rat kidney Na+/K+-ATPase polyclonal antibody to investigate the cellular distribution and timing of appearance of this enzyme during preimplantation development. The enzyme is first detected in the late morula within the cytoplasm of each blastomere. When cavitation begins this distribution changes dramatically to a ring encircling the blastocoel, restricted to the basolateral cell margins. Using this enzyme as a marker for cavitation, we examined its expression in embryos that had been treated with wheat germ agglutinin (WGA), which causes cleavage arrest and was reported to trigger premature compaction- and cavitation-like events in early cleavage stages (L. V. Johnson, 1986, Dev. Biol. 113, 1-9). Although WGA-treated 2-,4-, and 8-cell embryos quickly underwent compaction- and cavitation-like events, no Na+/K+-ATPase expression was observed. Thus the WGA effect does not likely involve acceleration of the developmental program for cavitation. Embryos arrested at the 8-cell stage but cultured overnight to Day 4, however, expressed the enzyme in the typical blastocyst pattern (around each fluid-filled cavity). We conclude that Na+/K+-ATPase expression is initiated or increases dramatically in the late morula and is independent of cytokinesis. The enzyme assumes a distribution during cavitation consistent with its presumed role in transtrophectodermal fluid transport.

Animals↗
PubMed1987

Gap junction assembly in the preimplantation mouse conceptus is independent of microtubules, microfilaments, cell flattening, and cytokinesis.

Gap junctions first appear during compaction in the eight-cell stage of mouse development. Their assembly can be initiated in the near absence of transcription and protein synthesis from the four-cell stage, indicating the existence of preformed precursors. We have investigated the temporal control of this event, focusing on the possible involvement of the cytoskeleton, cell flattening, and cytokinesis. Embryos in various cleavage stages were treated with cytochalasins, to disrupt microfilaments and block cell flattening, cytokinesis, or both, or nocodazole, to promote microtubule depolymerization. To assess their capacity to initiate gap junction assembly after such treatments, the embryos were then aggregated with communication-competent, compacted embryos that had been labeled with carboxyfluorescein diacetate. Passage of the fluorescent dye, carboxyfluorescein, from labeled to unlabeled embryo was taken as evidence that interembryonic junction formation had occurred. The capacity to assemble gap junctions was acquired at the normal time by embryos prevented by cytochalasin treatment from undergoing cell flattening or any cytokinesis from fertilization onward. Likewise, treatment with nocodazole beginning in the four-cell or early eight-cell stage did not interfere with gap junction assembly. Neither drug affected the inability of four-cell embryos to assemble gap junctions prematurely. We conclude that intact microfilament or microtubule networks are not required for gap junction assembly in this system, nor do they restrain junctional precursors from assembling prematurely. Furthermore, the timing of gap junction assembly is not linked to cell flattening, cytokinesis, or cell number.

Actin Cytoskeleton↗
PubMed1986

Intercellular junctional coupling in preimplantation mouse embryos: effect of blocking transcription or translation.

Gap junction formation in cleavage stage mouse embryos was examined by testing for ionic coupling or by observing the intercellular movement of the fluorescent dye, Lucifer yellow CH. Our results confirm that embryo-wide cell coupling, mediated by cell-to-cell membrane channels (gap junctions), is acquired in the 8-cell stage after compaction has begun. However, not all partially compacted embryos were found to be ionically or dye coupled, suggesting that the initiation of gap junction assembly is not necessarily triggered by the onset of cell flattening. The rate of fluorescent dye movement throughout the embryo was found to increase as embryos proceed through compaction and beyond, indicating that the number of gap junctional channels between blastomeres increases as development progresses. The inhibitors alpha-amanitin and cycloheximide were used to assess the requirement of new transcription and protein synthesis, respectively, for the onset of intercellular coupling and its progressive increase during compaction. Treatment conditions were chosen to bring about suppression of mRNA and protein synthesis within 2 hr. Ionic coupling was detected in almost all compacted 8-cell embryos treated with either inhibitor from the 4-cell stage. On the other hand, dye coupling was weak or undetectable in such embryos. We propose that a limited supply of junctional components is present by the 4-cell stage to serve as a pool of precursors for the first gap junctions to be assembled in the 8-cell stage. However, it is apparent that continued embryonic gene expression is required for the full extent of junctional coupling to be established.

Amanitins↗
PubMed1985

On the use of alpha-amanitin as a transcriptional blocking agent in mouse embryos: a cautionary note.

We have tested the effect of alpha-amanitin at 10, 50 and 100 micrograms/ml, on precursor uptake and incorporation into poly(A)+ RNA and poly(A)- RNA of mouse embryos on days 2, 3 and 4 of gestation. Embryos were pretreated with the inhibitor for 2 hr, then labeled for 2 hr in its continued presence. RNA fractions were separated by affinity chromatography on oligo(dT)-cellulose. alpha-Amanitin did not suppress uptake of RNA precursors at any of the concentrations tested in any stage. At 10 micrograms/ml, we could not detect any effect on incorporation into either RNA fraction in any stage. Only the highest concentration tested, 100 micrograms/ml, was effective in all stages in substantially suppressing incorporation into poly(A)+ RNA within 2 hr. Longer treatments increased the level of suppression to a maximum of about 80%. Incorporation into poly(A)- RNA was suppressed to roughly the same extent. Despite previously reported data, it cannot be assumed that alpha-amanitin at concentrations less than 100 micrograms/ml brings about a quick interruption of mRNA synthesis in preimplantation mouse embryos.

Adenosine↗
PubMed1985

Timing of transcription and protein synthesis underlying morphogenesis in preimplantation mouse embryos.

During preimplantation development of the mouse, embryos pass through a series of morphogenetic events: compaction, fluid accumulation to form the blastocoele (cavitation), and escape from the zona pellucida (hatching). We have used the inhibitors alpha-amanitin and cycloheximide to investigate the timing of transcriptional and translational events underlying these morphogenetic stages. Groups of embryos were transferred from a common pool into medium containing one or the other inhibitor at regular time intervals, and then were scored over the ensuing 24 or more hours for their ability to reach a particular morphogenetic end point. By comparing the time when the control population reached an end point with the time at which embryos had to be transferred into the inhibitor in order to prevent them from reaching that end point, we could determine when in advance of each event the necessary transcription or protein synthesis has been completed. Our results suggest that compaction (as well as cleavage to the eight-cell stage) is an embryonically, rather than maternally, programmed event, although the necessary transcription is completed well in advance, at least by the early four-cell stage. The transcriptional and translational events underlying fluid accumulation, on the other hand, appear to be completed within a few hours of the start of this process. For hatching, there is once again a long delay between the apparent time of completion of the necessary transcriptional events and the process itself, with protein synthesis being completed just a few hours in advance. Our results raise the possibility that post-transcriptional regulatory mechanisms play an important role in the timing of morphogenetic events in early mouse embryos.

Amanitins↗
PubMed1985

Utilization of cytoplasmic poly(A)+RNA for protein synthesis in preimplantation mouse embryos.

The distribution of cytoplasmic poly(A)+RNA between subribosomal (less than 80S) and ribosomal/polysomal (greater than or equal to 80S) ribonucleoprotein particles has been investigated in mouse morulae and blastocysts. After labelling for 24h with [5,6-3H]uridine, late morulae (96h post-hCG), early blastocysts (100h post-hCG), or late blastocysts (120h post-hCG) were homogenized in detergent buffer, and 11 000g supernatants were prepared and centrifuged through 15-40% sucrose gradients. Poly(A)+RNA was isolated from the appropriate gradient fractions by affinity chromatography. In late morulae beginning to undergo cavitation, poly(A)+RNA was evenly distributed between the two types of RNP particles whereas it was almost entirely in the ribosomal/polysomal fraction in early and late blastocysts. The sedimentation profile (in 5-20% sucrose gradients) of poly(A)+RNA from the ribosomal/polysomal fraction of late morulae and blastocysts was the same, with a range of 4S to greater than 28S and a modal peak slightly smaller than 18S. Roughly 75% of this RNA was judged to be functional mRNA based on the EDTA sensitivity of the RNP particles containing it. Poly(A)+RNA from the subribosomal fraction of late morulae displayed a similar range of sedimentation values, but was enriched for a component sedimenting at 6-7S. These results demonstrate that the subcellular distribution of poly(A)+RNA shifts as cavitation begins, with the decline of the non-translating, subribosomal fraction. Although most of this fraction in late morulae is probably too small to constitute translatable mRNA, the remainder may represent a reserve available for recruitment into polyribosomes to support blastocyst expansion. Since little non-translating mRNA exists in blastocysts, the increasing rate of protein synthesis during blastocyst expansion must be driven by mRNA accumulation and/or stabilization.

Animals↗
PubMed1984

Synthesis of putative microtubule-associated proteins by mouse blastocysts during early outgrowth in vitro.

The outgrowth of mouse trophoblast in culture provides a simplified model system analogous in certain ways to blastocyst implantation in vivo. Day-four blastocysts cultured for 3 days in vitro undergo extensive changes in cell shape and motility which are likely to involve the complex cytoskeletal system of the trophoblast cells. To explore the biochemical basis of these changes, one set of cytoskeletal proteins, the microtubule-associated proteins (MAPs), was studied. Day 4 blastocysts were labeled with [35S]methionine and blastocyst outgrowths, after 3 days in culture from the blastocyst stage, were labeled with [3H]methionine. Labeled embryos were disrupted and the soluble supernatants were pooled, and newly synthesized proteins from the two stages were coassembled with taxol-stabilized brain microtubule polymer enriched for MAP-binding sites. Double-labeled coassembly proteins (putative MAPS) were then released from the microtubule polymer by treatment with 0.35 M NaCl and analyzed by one-dimensional polyacrylamide gel electrophoresis. 3H/35S dpm ratios were determined for individual protein bands to compare the relative synthesis rates for day 4 blastocyst and day 3 outgrowth MAPs. In spite of the extensive changes in cell shape and motility associated with blastocyst outgrowth, a common set of putative MAPs characterizes the two stages investigated, including several in the size range of tau factors. No synthesis of high molecular weight MAPs comparable with MAP 1 or MAP 2 from brain was detected. The synthesis rates of individual MAPs relative to each other remain constant over this period and are likely coordinated with total protein and tubulin synthesis.

Alkaloids↗
PubMed1983

Glucose-6-phosphate dehydrogenase isozymes in fish--a comparative study.

The electrophoretic distribution and substrate specificities of isozymes of glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) were studied in seven species of teleost fish. The fish examined included two species of bonefish, Albula neoguinaica and A. glossodonta (Albulidae, Anquilliformes) (Shaklee and Tamaru, '81), and five representatives of the order Perciformes: two species of butterflyfish, Chaetodon miliaris and C. aurega (Chaetodontidae); a goatfish, Upeneus arge (Mullidae); a goby, Bathygobius fuscus (Gobiidae); and a snapper, Pristipomoides filamentosus (Lutjanidae). After horizontal starch gel electrophoresis, gel slices were stained using a variety of substrates and cofactors. In all species except the goby, two groups of isozymes were distinguished, corresponding to the mammalian G6PD (specific for glucose-6-phosphate (G6P) and NADP+) and H6PD (capable of utilizing galactose-6-phosphate and in certain cases other monosaccharide phosphates in addition to G6P). None of the five visible isozymes in the goby was specific for G6P. In each of the other species a single G6P- and NADP+-specific isozyme was noted, having the most rapid mobility toward the anode. In addition, it was found that all of the isozymes in all of the fish examined could catalyze the oxidation of fructose-6-phosphate at a rate comparable to that for G6P, suggesting that glucose-6-phosphate dehydrogenase can obviate the role of glucosephosphate isomerase in monosaccharide metabolism.

Animals↗
PubMed1983

Control of gap junction formation in early mouse embryos.

Intercellular communication via gap junctions begins in the eight-cell stage in early mouse embryos. We have studied the timing of this event in relation to compaction, and have begun to explore some of the possible control mechanisms underlying it. Gap junction formation was inferred by measuring ionic coupling as well as by observing the intercellular transfer of fluorescent dye. Embryos were obtained early on Day 3 of pregnancy by flushing the oviducts of HA/ICR mice that had been mated with CB6F1/J males. Gap junctions were detected only in those embryos which had achieved the fully compacted state. Inhibition of protein synthesis by cycloheximide treatment beginning as early as the late four-cell stage failed to block compaction or the acquisition of gap junctions, demonstrating that the necessary proteinaceous components are present in advance of these events. In order to test the possibility that gap junctions could be induced to form prematurely, fully compacted, communication-competent eight-cell embryos were aggregated with two- or four-cell embryos. Even after 10 hr of aggregation, no interembryonic gap junctions could be detected. Fully compacted eight-cell embryos when aggregated with each other, however, became ionically coupled within 3-5 hr. The number of interembryonic junctional channels was judged to be effectively small, since the aggregated embryos exhibited obvious ionic coupling but very weak dye coupling. In contrast to gap junction formation within embryos, junction formation between embryos was blocked by cycloheximide. These results demonstrate that gap junction formation in early mouse embryos is under precise temporal control, involving the assembly or mobilisation of preexisting components. This stockpile of components is either unavailable or insufficient to allow the formation of additional gap junctions between aggregated communication-competent embryos without new protein synthesis.

Animals↗
PubMed1982

Turnover of embryonic messenger RNA in preimplantation mouse embryos.

We have estimated the average half-life of embryonic messenger RNA in mouse embryos at two stages of preimplantation development. Embryos were collected at 48 and 75 h post-hCG and cultured overnight in the presence of [3H]uridine. Beginning at 65--68 h (morulae) or 92--94 h (early blastocysts), the label was withdrawn and replaced with unlabelled uridine, and samples were taken at intervals thereafter for RNA isolation. Label in cytoplasmic, poly(A)-containing RNA was measured after binding to oligo(dT)-cellulose, and was normalized to label in 28S and 18S ribosomal RNA, separated on sucrose gradients. The stability of rRNA in both stages was verified directly, as was the integrity and purity of the isolated mRNA. With morulae, the mRNA decay curve was monophasic, with an average half-life of 9.5 +/- 0.9 h. In three experiments with early blastocysts the decay curve appeared to be biphasic, consisting of short-lived (less than 6 h) and long-lived (30--50 h) components; in two other experiments a short-lived component was not evident. In all cases, however, the overall average half-life of mRNA in early blastocysts, determined by linear regression assuming monophasic kinetics, was greater than that in morulae. Our data indicate that the stability of embryonic mRNA increases by at least twofold during the morula-to-blastocyst transition. The results are considered in terms of the transcriptional dependency of early mouse embryos and the regulation of maternal and embryonic mRNA.

Animals↗