Biomedical subjects

G M Kidder

Publications and source records attributed to G M Kidder.

At least 19 recordsLinked to original sources

PubMed1998

Contributions of Na+/H+ exchanger isoforms to preimplantation development of the mouse.

Previous work provided evidence of Na+/H+ exchanger activity in the apical domain of mouse trophectodermal plasma membranes that provides a route for entry of extracellular Na+ (Manejwala et al., 1989). This activity was hypothesized to contribute to the trans-trophectodermal Na+ flux that is required for blastocoel expansion. In the present work, we have used reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry to identify members of the Na+/H+ exchanger (NHE) family that are likely to participate in this process. When cDNA preparations from ovulated oocytes and several stages of preimplantation development were tested with PCR primers specific for the NHE-1, -2, -3, and -4 isoforms of the exchanger, only amplicons representing the NHE-1 and NHE-3 isoforms were detected. The identity of these amplicons was confirmed by direct sequencing. NHE-1 mRNA is present in oocytes and in all preimplantation stages, increasing threefold on a per embryo basis between the 4-cell and blastocyst stages. NHE-3 mRNA, on the other hand, was only detected in oocytes. Immunocytochemical analysis of blastocysts revealed that NHE-1 is localized in the basolateral domain of the trophectoderm, whereas NHE-3 is localized in the apical domain, a situation like that in epithelia of adult organs. We conclude that NHE-3, an oogenetic product that persists into the blastocyst stage, is the Na+/H+ exchanger isoform most likely to be involved in blastocoel expansion.

Animals↗

PubMed1997

Embryonic expression of the putative gamma subunit of the sodium pump is required for acquisition of fluid transport capacity during mouse blastocyst development.

The sodium/potassium pump, Na+,K+-ATPase, is generally understood to function as a heterodimer of two subunits, a catalytic alpha subunit and a noncatalytic, glycosylated beta subunit. Recently, a putative third subunit, the gamma subunit, was cloned. This small protein (6.5 kD) coimmunoprecipitates with the alpha and beta subunits and is closely associated with the ouabain binding site on the holoenzyme, but its function is unknown. We have investigated the expression of the gamma subunit in preimplantation mouse development, where Na+, K+-ATPase plays a critical role as the driving force for blastocoel formation (cavitation). Using reverse transcriptase-polymerase chain reaction, we demonstrated that the gamma subunit mRNA accumulates continuously from the eight-cell stage onward and that it cosediments with polyribosomes from its time of first appearance. Confocal immunofluorescence microscopy revealed that the gamma subunit itself accumulates and is localized at the blastomere surfaces up to the blastocyst stage. In contrast with the alpha and beta subunits, the gamma subunit is not concentrated in the basolateral surface of the polarized trophectoderm layer, but is strongly expressed at the apical surface as well. When embryos were treated with antisense oligodeoxynucleotide complementary to the gamma subunit mRNA, ouabain-sensitive K+ transport (as indicated by 86Rb+ uptake) was reduced and cavitation delayed. However, Na+, K+-ATPase enzymatic activity was unaffected as determined by a direct phosphorylation assay ("back door" phosphorylation) applied to plasma membrane preparations. These results indicate that the gamma subunit, although not an integral component of Na+,K+-ATPase, is an important determinant of active cation transport and that, as such, its embryonic expression is essential for blastocoel formation in the mouse.

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PubMed1997

Altered gap junctional communication, intercellular signaling, and growth in cultured astrocytes deficient in connexin43.

Astrocytes are characterized by extensive intercellular communication mediated primarily by gap junction channels composed of connexin43. To examine this junctional protein in astrocytic functions, astrocytes were cultured from embryonic mice with a null mutation in the connexin43 gene (Reaume et al.: Science 267:1831-1834, 1995). Using anti-Cx43 antibodies, immunoblotting and immunostaining indicated that homozygous null astrocytes were devoid of Cx43. They are also deficient in intercellular dye transfer. Astrocytes cultured from heterozygous embryos express significantly lower Cx43 compared to wild type, and their dye coupling is reduced. Markers of glial differentiation, such as glial fibrillary acidic protein and S100, appeared similar in all genotypes. Measurement of intercellular calcium concentration following mechanical stimulation of confluent astrocytes revealed that the number of cells affected by a rise in intracellular calcium was reduced in homozygous cultures compared to wild type. In fact, the calcium response in homozygous astrocytes was similar to that observed in wild-type astrocytes in the presence of a gap junction blocker. The growth rate of astrocytes lacking Cx43 was reduced compared to wild-type astrocytes. These results suggest that gap junctional intercellular communication mediated by Cx43 is not critical for astrocyte differentiation but is likely involved in the regulation of intercellular calcium signaling and cell growth.

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PubMed1997

Gap junctions promote the bystander effect of herpes simplex virus thymidine kinase in vivo.

Transfer of the herpes simplex thymidine kinase gene (HSVtk) into tumor cells followed by the administration of ganciclovir (GCV) provides a potential strategy for the treatment of some malignancies. During GCV treatment, not only the cells that express the HSVtk gene are killed but also frequently neighboring tumor cells that are not genetically altered. This has been called the "bystander effect." Although the mechanism of the bystander effect in vivo remains elusive, our results suggest that gap junction formation between neighboring cells is an important contributing factor. The C6 rat glioma cell line, which exhibits a low level of intercellular communication by gap junctions and connexin43 (Cx43)-transfected clones of this cell line forming gap junctions from a moderate level (Cx43-12 and Cx43-14) to a high level (Cx43-13), were transduced with HSVtk. Transduced and nontransduced cells were mixed in various concentrations and then cultured in vitro or injected s.c. into C.B-17/SCID-beige mice followed by i.p. injections of GCV. Cx43-transfected clones showed a significant increase of the bystander effect compared with the less coupled C6 parental cell line. In 11 of 12 mice injected with cells of Cx43-transfected clones, no tumors were seen at the inoculation site when a mixture of 50% HSVtk-negative and HSVtk-positive cells was used. Moreover, in mice injected with cells of clone Cx43-13, which exhibits the highest intercellular communication, tumors were frequently undetectable at the inoculation site when using mixtures of 75% HSVtk-negative and 25% HSVtk-positive cells, and even mixtures containing 5% HSVtk-positive cells of Cx43-transfected clones showed tumor size reduction. All animals in control groups (n = 26) developed large tumors at every injection site. These results demonstrate that gap junctions are an important component in mediating the bystander effect in vivo.

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PubMed1997

Ouabain sensitivity and expression of Na/K-ATPase alpha- and beta-subunit isoform genes during bovine early development.

The fluid movements that arise during blastocyst formation (cavitation) are, at least in part, driven by the Na/K-ATPase. In this study, the reverse transcriptase-polymerase chain reaction (RT-PCR) was used to survey bovine pre-attachment embryos for transcripts encoding known isoforms of the Na/K-ATPase alpha- and beta-subunits, including isoforms not previously detected during the first week of mammalian development. Transcripts encoding the Na-K-ATPase alpha 1, alpha 2, alpha 3 and beta 2 isoforms were detected throughout bovine preattachment development. This is the first indication that alpha 2, alpha 3 and beta 2 mRNAs are expressed during this early developmental interval. As in the mouse, beta 1-subunit transcripts were not detected until the morula stage and were also present in blastocysts. Thus, in two mammalian species an increase in abundance of beta 1 isoform transcripts in the morula stage is coincident with the onset of cavitation. Transcripts encoding the recently characterized alpha 4 isoform were not detected. The sensitivity of bovine blastocysts to ouabain (a potent inhibitor of Na/K-ATPase) was determined by assessing the ability of bovine blastocysts to recover in ouabain supplemental culture medium following cytochalasin-induced blastocyst collapse. Re-expansion of bovine blastocysts was inhibited in all ouabain concentrations down to 10(-9) M. Mouse blastocysts, in contrast, were sensitive to ouabain at or above 10(-3)M. These results have established that transcripts encoding multiple isoforms of both the alpha and beta subunits of the Na/K-ATPase are expressed throughout early bovine development and that bovine blastocysts display a greater sensitivity to ouabain than murine blastocysts. Future analysis will determine the possible individual and collective roles of these isoforms during blastocyst formation.

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PubMed1997

Evidence for the presence of sodium- and potassium-dependent adenosine triphosphatase alpha1 and beta1 subunit isoforms and their probable role in blastocyst expansion in the preattachment horse conceptus.

The unusual hypotonicity of equine blastocyst fluid has prompted us to investigate the role of sodium- and potassium-dependent adenosine triphosphatase (Na+,K+-ATPase) in the process of fluid accumulation in the horse conceptus. Nine mares were used for the experiments. Reverse transcriptase polymerase chain reaction was conducted on two sets of five conceptuses recovered between 12 and 28 days (+/- 1 day) after ovulation. Messenger RNAs encoding the alpha1 and beta1 subunit isoforms of Na+,K+-ATPase were detected in all embryonic tissues examined. Western blot analysis showed that alpha1 and beta1 subunits are both present in Day 15 conceptuses. Trophoblast tissues from 19 conceptuses between 8 and 31 days after ovulation were stained immunohistochemically using primary antibodies against the alpha1 and beta1 subunit isoforms of the Na+,K+-ATPase. Both isoforms were detected in all sections. Trophoblastic vesicles, prepared from 6 conceptuses between 12 and 14 days after ovulation, were used to investigate the inhibition of blastocyst expansion with ouabain after collapse induced with cytochalasin D. In normal medium there was a mean 3-fold increase, and in ouabain (10(-6) M) a mean 3-fold decrease, in the volume of vesicles that had been partially collapsed with cytochalasin D. We therefore conclude that, despite the hypotonicity of the blastocyst fluid in the early horse conceptus, the Na+,K+-ATPase plays a role in its accumulation, as in other species.

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PubMed1997

Normal development of preimplantation mouse embryos deficient in gap junctional coupling.

The connexin multigene family (13 characterized members in rodents) encodes the subunits of gap junction channels. Gap junctional intercellular coupling, established during compaction of the preimplantation mouse embryo, is assumed to be necessary for development of the blastocyst. One member of the connexin family, connexin43, has been shown to contribute to the gap junctions that form during compaction, yet embryos homozygous for a connexin43 null mutation develop normally, at least until implantation. We show that this can be explained by contributions from one or more additional connexin genes that are normally expressed along with connexin43 in preimplantation development. Immunogold electron microscopy confirmed that roughly 30% of gap junctions in compacted morulae contain little or no connexin43 and therefore are likely to be composed of another connexin(s). Confocal immunofluorescence microscopy was then used to demonstrate that connexin45 is also assembled into membrane plaques, beginning at the time of compaction. Correspondingly, embryos homozygous for the connexin43 null mutation were found to retain the capacity for cell-to-cell transfer of fluorescent dye (dye coupling), but at a severely reduced level and with altered permeability characteristics. Whereas mutant morulae showed no evidence of dye coupling when tested with 6-carboxyfluorescein, dye coupling could be demonstrated using 2',7'-dichlorofluorescein, revealing permeability characteristics previously established for connexin45 channels. We conclude that preimplantation development in the mouse can proceed normally even though both the extent and nature of gap junctional coupling have been perturbed. Despite the distinctive properties of connexin43 channels, their role in preimplantation development can be fulfilled by one or more other types of gap junction channels.

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PubMed1996

Development of astrocytes and neurons in cultured brain slices from mice lacking connexin43.

Astrocyte and neuronal development was investigated in organotypic brain slice cultures from mouse fetuses with a null mutation in the connexin43 gene. Astrocyte morphology and electrical properties were indistinguishable in null mutant slices and control slices but at 18 days in vitro astrocyte density in the central regions of the null mutant slices was significantly higher than in control slices. Neuronal development assessed morphologically and electrophysiologically appeared normal in the mutant slices. These results suggest that intercellular communication mediated through connexin43 is not essential for the development of astrocytes and neurons but may play a role in regulating astrocytic migration.

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PubMed1996

Role of germinal vesicle on protein synthesis in rat oocyte during in vitro maturation.

To investigate the role of the germinal vesicle (GV) on in vitro maturation (IVM) of rat oocytes, we examined protein synthesis during IVM by comparing polypeptide patterns in control and enucleated oocytes using one and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separation of polypeptides extracted from the cytoplasm of GV by one-dimensional SDS-PAGE revealed that a 55 kDa polypeptide was present only in the GVs of rat oocytes. At 0, 12, 24, 36, and 44 hr after PMSG injection, prior to the initiation of maturation, enucleated oocytes synthesized the same major polypeptides as cumulus intact (CI) oocytes. During meiotic maturation, no major changes were detected in protein synthesis from prophase (GV stage) to prometaphase I (0-6 hr IVM). However, after entry into prometaphase I (7 hr IVM), striking changes were seen; a 24 kDa polypeptide disappeared and expression of a 34 kDa polypeptide became stronger. This pattern lasted until metaphase II. We detected no major differences in the pattern of protein synthesis between CI and enucleated oocytes using two-dimensional PAGE. These results indicate that protein synthesis in the maturing rat oocyte is controlled by cytoplasmic regulators rather than intrinsic nuclear components.

Animals↗

PubMed1996

Multiple members of the connexin gene family participate in preimplantation development of the mouse.

The connexin gene family, of which there are at least 12 members in rodents, encodes the protein subunits intercellular membrane channels (gap junction channels). Because of the diverse structural and biophysical properties exhibited by the different connexins, it has been proposed that each may play a unique role in development or homeostasis. We have begun to test this hypothesis in the preimplantation mouse embryo in which de novo gap junction assembly is a developmentally regulated event. As a first step, we have used reverse transcription-polymerase chain reaction (RT-PCR) to determine the connexin mRNA phenotype of mouse blastocysts, and have identified transcripts of connexins 30.3, 31, 31.1, 40, 43, and 45. Quantitative measurements indicated that all six of these connexin genes are transcribed after fertilization. They can be divided into two groups with respect to the timing of mRNA accumulation: Cx31, Cx43, and Cx45 mRNAs accumulate continuously from the two- or four-cell stage, whereas Cx30.3, Cx31.1, and Cx40 mRNAs accumulate beginning in the eight-cell stage. All six mRNAs were found to co-sediment with polyribosomes from their time of first appearance, indicating that all six are translated. The expression of Cx31.1 and Cx40 was examined by confocal immunofluorescence microscopy; whereas both could be detected in compacting embryos, only Cx31.1 could be seen in punctate membrane foci indicative of gap junctions. Taken together with other results (published or submitted), our findings indicate that at least four connexins (Cx31, 31.1, 43 and 45) contribute to gap junctions in preimplantation development. The expression of multiple connexin genes during this early period of embryogenesis (when there are only two distinct cell types) raises questions about the functional significance of connexin diversity in this context.

Animals↗

PubMed1996

Rat endometrial stromal cells express the gap junction genes connexins 26 and 43 and form functional gap junctions during in vitro decidualization.

Gap junctions form between rat endometrial stromal cells as they undergo decidualization. We have examined the steady-state levels of the gap junction transcripts, connexins 26 and 43 (cx26 and cx43), during artificially induced decidualization in vivo and found that they have a temporal pattern similar to that observed in pregnancy. An in vitro model of decidualization was then used. Endometrial stromal cells from rat uteri sensitized for decidualization were cultured for 24, 48, or 72 h before total RNA was extracted and subjected to Northern blot analyses to determine the steady-state levels of cx26 and cx43 transcripts. The analyses revealed that cx26 transcript steady-state levels decreased, whereas those for cx43 increased, from 24 to 72 h. Using an anti-cx43 antibody, punctate immunofluorescent signals were observed around the periphery of the cells, suggesting that cx43 had been assembled into membrane plaques. The presence of functional gap junctions between the cells was determined in vitro by two dye-coupling methods: preloading and scrape-loading. Calcein (995 Da) and a membrane-bound dye, dioctadecyl-3,3,3',3-tetramethylindocarbocyanine perchlorate (933 Da), were preloaded into 5% of the endometrial stromal cells before plating. The percentage of preloaded cells that transferred calcein to adjacent cells increased from 10% at 3 h after plating to 40% at 6 h. To determine whether or not cells maintain the ability to dye-couple throughout the culture period, carboxyfluorescein (CF; 376 Da) and rhodamine dextran (9.3 kDa) were introduced into cells by scraping the cells with a scalpel, and the distribution of dyes was determined 20 min later. In cells cultured for 24, 48, or 72 h, only CF was transferred to cells distal to the scrape line. The results from these experiments suggest that stromal cells can dye-couple throughout the culture period (3-72 h) and indicate that functional gap junctions form between endometrial stromal cells as they undergo decidualization in vitro.

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PubMed1995

Cardiac malformation in neonatal mice lacking connexin43.

Gap junctions are made up of connexin proteins, which comprise a multigene family in mammals. Targeted mutagenesis of connexin43 (Cx43), one of the most prevalent connexin proteins, showed that its absence was compatible with survival of mouse embryos to term, even though mutant cell lines showed reduced dye coupling in vitro. However, mutant embryos died at birth, as a result of a failure in pulmonary gas exchange caused by a swelling and blockage of the right ventricular outflow tract from the heart. This finding suggests that Cx43 plays an essential role in heart development but that there is functional compensation among connexins in other parts of the developing fetus.

Animals↗

PubMed1995

Temporal control of gap junction assembly in preimplantation mouse embryos.

The de novo assembly of gap junctions during compaction in the 8-cell stage of mouse development is a temporally regulated event. We have performed experiments designed to explore the relationship between this event and DNA replication in the second, third, and fourth cell cycles after fertilization. Inhibition of DNA synthesis by continuous treatment with the DNA synthesis inhibitor, aphidicolin, during the third and fourth cell cycles had no effect on the establishment of gap junctional coupling during compaction. However, a delay of 10 hours in DNA synthesis during the second cell cycle caused by a transient aphidicolin treatment resulted in the failure of gap junctional coupling at the time of compaction. Thus the timing of establishment of gap junctional coupling, like the timing of compaction itself, is linked to DNA replication in the 2-cell stage. Immunofluorescence analysis showed that the failure of gap junctional coupling after aphidicolin treatment in the 2-cell stage is correlated with the failure of nascent connexin43 to be inserted into plasma membranes. We propose that the developmental 'clock' that controls gap junction assembly is set in motion by events surrounding the second cycle of DNA replication, and that this 'clock' ultimately controls the post-translational processing of connexin43.

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PubMed1994

Regulation of Na+,K(+)-ATPase alpha subunit gene expression during mouse preimplantation development.

Several lines of evidence support the hypothesis that a sodium flux, driven by Na+,K(+)-ATPase in the basolateral plasma membranes of mural trophectoderm, drives fluid transport during blastocoel formation in eutherians. In light of the importance of this enzyme for preimplantation development, attention has been focused on the regulation of expression of its alpha and beta subunits. Here we report on the spatial distribution and translation of the alpha subunit mRNA. Although this mRNA accumulates from the 2-cell stage onward the alpha subunit itself could not be detected by immunofluorescence prior to the late morula stage, after which it becomes concentrated in the mural trophectoderm. In the present study we have used a wholemount, fluorescent in situ hybridization technique that takes advantage of the optical sectioning capability of the confocal microscope to show that alpha subunit mRNA, in contrast to the alpha subunit itself, accumulates in all cells of the early blastocyst. This finding demonstrates that the spatial distribution of the alpha subunit is regulated post-transcriptionally. We have also examined the translational regulation of alpha subunit mRNA by preparing polyribosomal and subribosomal ribonucleoprotein fractions for mRNA assay by reverse transcription-polymerase chain reaction. We found that alpha subunit mRNA is in polyribosomes continuously from at least the 4-cell stage. Thus, the abrupt appearance of the alpha subunit in the late morula stage as revealed by immunofluorescence must be determined by post-translational events. In the Discussion, we consider the hypothesis that synthesis of the beta subunit of the enzyme is the rate limiting step in functional expression of the alpha subunit.

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PubMed1994

Possible roles of insulin and insulin-like growth factors in rat preimplantation development: investigation of gene expression by reverse transcription-polymerase chain reaction.

The sensitive mRNA phenotyping technique of reverse transcription-polymerase chain reaction was used to demonstrate that insulin receptor mRNA is present in rat embryos during the preimplantation period. In addition, mRNA encoding insulin-like growth factor (IGF) type I and type II receptors have also been detected in rat preimplantation embryos. IGF-I mRNA was not detected in preimplantation embryos but was found in oviducts and uteri of prepubertal and early pregnant rats. IGF-II mRNA was present in both embryos and in oviducts and uteri during the preimplantation period. These findings suggest that insulin and IGF-I could influence early embryo development in endocrine or in paracrine fashions, whereas IGF-II may have an additional autocrine mode of action in affecting preimplantation embryos in rats.

Animals↗

PubMed1994

Expression of plasminogen activator genes and enzymatic activities in rat preimplantation embryos.

Plasminogen activator has been implicated in tissue invasion and remodelling because of its role in the degradation of the extracellular matrix. Its activity can be detected in mouse embryos as early as day 6 of pregnancy, suggesting that plasminogen activator is involved in the process of implantation. The present study determined the time course of expression of the genes encoding tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) during the preimplantation period in rats by the sensitive mRNA phenotyping procedure of reverse transcription-PCR. The tPA mRNA was present in rat oocytes and two-cell embryos, but was not detected between the four-cell and blastocyst stages. The uPA mRNA was first detected in two-cell rat embryos, and was present through to the blastocyst stage. In chromogenic assays, plasminogen activator activity was detected in oocytes and embryos between two-cell and blastocyst stages. Most plasminogen activator activity present in preimplantation embryos appeared to be uPA, as it could be inhibited by anti-uPA antibody and a specific uPA inhibitor, amiloride, but not by anti-tPA antibody. The present data demonstrate the expression of uPA gene and uPA activity in preimplantation rat embryos, suggesting that embryonic uPA may be involved in early embryo development and implantation.

Amino Acid Sequence↗

PubMed1993

Alterations in the synthesis of insulin-like growth factor binding proteins and insulin-like growth factors in rat C6 glioma cells transfected with a gap junction connexin43 cDNA.

When C6 glioma cells are stably transfected with a connexin43 cDNA and gap junctions are increased, the rate of cellular proliferation is decreased. To determine if this phenomenon is related to alterations in IGFBP and IGF synthesis, we have compared IGFBPs and IGFs in the conditioned media from primary rat astroglia, C6, and transfected C6 clones Cx43-13 (high expresser), and Cx43-12 and Cx43-14 (intermediate expressers). Primary astroglia produced IGFBP-2 (34 kDa) and IGFBP-3 (40-45 kDa). C6 cells synthesized high levels of IGFBP-3 and low levels of IGFBP-2, and a 24 kDa IGFBP (IGFBP-4). Cx43-13 cells did not synthesize IGFBP-3, but produced low levels of IGFBP-2 and high levels of IGFBP-4. Cx43-12 and Cx43-14 secreted IGFBP profiles similar to the parent C6 line, but with reduced levels of IGFBP-2. The lack of IGFBP-3 in Cx43-13 cells was not due to the presence of proteases. Northern analysis showed IGFBP-2 mRNA to be readily detectable only in the primary astroglia. IGFBP-3 mRNA was detected in the primary astroglia, C6, Cx43-12 and Cx43-14, but not in Cx43-13. In contrast, IGFBP-4 mRNA was readily detected only in the Cx43-13. IGF-II concentrations in the media were low to undetectable for both C6 and transfected cells. IGF-I concentrations were significantly lower in the media from transfected cells compared to the C6 cells. Stable mRNA levels for IGF-I were lower in transfected cells, with the lowest levels observed in the Cx43-13 cells. Although C6 cells did not respond mitogenically to exogenous IGF-I or IGF-II, Cx43-13 cells responded to IGF-I or IGF-II in a dose dependent manner. Conditioned media from Cx43-13 cells decreased the DNA synthesis of C6 cells, and this effect could be reversed by the addition of IGF-II. The decreased synthesis of the autocrine/paracrine growth factor IGF-I together with decreased levels of a positive modulator IGFBP-3, and the increased levels of a negative modulator IGFBP-4 in the extracellular milieu, may be responsible for the reduced proliferative capacity in cells expressing abundant connexin43.

Animals↗

PubMed1993

Coexpression of gap junction proteins in the cumulus-oocyte complex.

The connexins constitute a family of proteins that make up the intercellular membrane channels of gap junctions. We had previously reported the presence of two members of this protein family, connexins 32 and 43, in mouse one-cell zygotes (Barron et al., Dev Genet 10:318-323, 1989; Valdimarsson et al., Mol Reprod Dev 30:18-26, 1991), implying that both must be present in the mature oocyte and could be involved in mediating the intercellular coupling that occurs between the oocyte and cumulus granulosa during oogenesis. In the present report we provide evidence for this, based on an analysis of the cumulus-oocyte complex (COC) using reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry with a confocal microscope. Transcripts of both connexin32 (Cx32) and connexin43 (Cx43) were detected by RT-PCR in both components of the COC. Cx32 mRNA in the oocyte declined precipitously following human chorionic gonadotropin (hCG) stimulation of pregnant mare serum gonadotropin (PMSG)-primed ovaries, whereas there was no obvious change in Cx43 mRNA. Peptide-specific antibodies against both connexins provided diffuse cytoplasmic staining of oocytes as well as some punctate staining near the oocyte surface, which could not be unequivocally resolved as cumulus-oocyte gap junctions. However, the two antibodies did provide clear evidence of Cx32 and Cx43 in gap junction-like structures between cumulus cells. We could find no evidence of the incorporation of the oocyte's store of Cx32 into gap junctions during postfertilization development.(ABSTRACT TRUNCATED AT 250 WORDS)

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