Tyrosine phosphorylation of the gap junction protein connexin43 is required for the pp60v-src-induced inhibition of communication.

Gap junction communication in some cells has been shown to be inhibited by pp60v-src, a protein tyrosine kinase encoded by the viral oncogene v-src. The gap junction protein connexin43 (Cx43) has been shown to be phosphorylated on serine in the absence of pp60v-src and on both serine and tyrosine in cells expressing pp60v-src. However, it is not known if the effect of v-src expression on communication results directly from tyrosine phosphorylation of the Cx43 or indirectly, for example, by activation of other second-messenger systems. In addition, the effect of v-src expression on communication based on other connexins has not been examined. We have used a functional expression system consisting of paired Xenopus oocytes to examine the effect of v-src expression on the regulation of communication by gap junctions comprised of different connexins. Expression of pp60v-src completely blocked the communication induced by Cx43 but had only a modest effect on communication induced by connexin32 (Cx32). Phosphoamino acid analysis showed that pp60v-src induced tyrosine phosphorylation of Cx43, but not Cx32. A mutation replacing tyrosine 265 of Cx43 with phenylalanine abolished both the inhibition of communication and the tyrosine phosphorylation induced by pp60v-src without affecting the ability of this protein to form gap junctions. These data show that the effect of pp60v-src on gap junctional communication is connexin specific and that the inhibition of Cx43-mediated junctional communication by pp60v-src requires tyrosine phosphorylation of Cx43.     

OVARIAN NUTRITIONAL RESOURCES DURING THE REPRODUCTIVE CYCLE OF THE HEMATOPHAGOUS DIPETALOGASTER MAXIMA (HEMIPTERA: REDUVIIDAE): FOCUS ON LIPID METABOLISM

In this study, we have analyzed the changes of the ovarian nutritional resources in Dipetalogaster maxima at representative days of the reproductive cycle: previtellogenesis, vitellogenesis, as well as fasting‐induced early and late atresia. As expected, the amounts of ovarian lipids, proteins, and glycogen increased significantly from previtellogenesis to vitellogenesis and then, diminished during atresia. However, lipids and protein stores found at the atretic stages were higher in comparison to those registered at previtellogenesis. Specific lipid staining of ovarian tissue sections evidenced remarkable changes in the shape, size, and distribution of lipid droplets throughout the reproductive cycle. The role of lipophorin (Lp) as a yolk protein precursor was analyzed by co‐injecting Lp‐OG (where OG is Oregon Green) and Lp‐DiI (where DiI is 1,10‐dioctadecyl‐3,3,30,30‐tetramethylindocarbocyanine) to follow the entire particle, demonstrating that both probes colocalized mainly in the yolk bodies of vitellogenic oocytes. Immunofluorescence assays also showed that Lp was associated to yolk bodies, supporting its endocytic pathway during vitellogenesis. The involvement of Lp in lipid delivery to oocytes was investigated in vivo by co‐injecting fluorescent probes to follow the fate of the entire particle (Lp‐DiI) and its lipid cargo (Lp‐Bodipy‐FA). Lp‐DiI was readily incorporated by vitellogenic oocytes and no lipoprotein uptake was observed in terminal follicles of ovaries at atretic stages. Bodipy‐FA was promptly transferred to vitellogenic oocytes and, to a much lesser extent, to previtellogenic follicles and to oocytes of ovarian tissue at atretic stages. Colocalization of Lp‐DiI and Lp‐Bodipy‐FA inside yolk bodies indicated the relevance of Lp in the buildup of lipid and protein oocyte stores during vitellogenesis.     

Structural modulation of the surface and cytoplasm of oocytes during vitellogenesis in the lobster,Homarus americanus. An electron microscope-protein tracer study

The present investigation describes the ultrastructural changes which occur at the surface and in the cytoplasm of developing oocytes of the lobster, Homarus americanus, during vitellogenesis. The immature oocytes showed no surface specializations of the oolemma and no pinocytotic activity was observed. Horseradish peroxidase (HRP) tracer studies showed penetration of the tracer into the perivitelline space, but no uptake by the oocytes. The surfaces of oocytes examined during vitellogenesis, when yolk protein accumulation was maximal, exhibited numerous microvilli that projected into the perivitelline space, often appearing to be embedded in the follicular cell mass. In addition, the plasma membrane of vitellogenic oocytes contained many pinocytotic pits frequently situated at the bases of microvilli. The perivitelline space was engorged with electrondense material which appeared similar to that contained in pinocytotic structures of the oocytes. Vitellogenic oocytes incubated in HRP showed uptake of tracer reaction product by the coated pits and vesicles of the oolemma. Aggregation and subsequent fusion of these vesicles into large multivesicular bodies of ingested material were also observed in vitellogenic oocytes. Animals artificially induced to undergo vitellogenesis exhibited modulations of oocyte ultrastructure similar to those of normal vitellogenesis, notably, pinocytotic incorporation of extra-oocytic material and hypertrophy of oocyte surface microvilli. This study supports the hypothesis for a dual source of yolk protein in the American lobster.     

Effect of gap junction uncoupling in full-grown Bufo arenarum ovarian follicles: participation of cAMP in meiotic arrest

The aim of the present study was to determine the presence of the connexins Cx43, Cx32 and Cx26 in Bufo arenarum ovarian follicles during the breeding season as well as to analyse the possible alterations in the meiotic process when connexins are blocked by specific antibodies. Western blot analysis revealed that the Cx43 and Cx32 proteins were present but not Cx26. We demonstrated that the anti-Cx43 and anti-Cx32 antibodies produced the uncoupling of the gap junctions. When these junctions are blocked the maturation process is triggered in the oocytes. We determined that dbcAMP exerts an inhibitory effect on the maturation induced by the uncoupling of the gap junctions when the oocytes are injected or pretreated with this metabolite. We propose the idea that cAMP is the regulatory molecule in meiotic arrest in this amphibian species.     

Comparative study of liver vitellogenin gene expression and oocyte yolk accumulation in wild and captive Atlantic bluefin tuna (Thunnus thynnus L.)

The sequence of vitellogenin A (VgA) and vitellogenin B (VgB) cDNAs in Atlantic bluefin tuna (Thunnus thynnus L.) were determined, and vitellogenin expression levels in the liver and oocyte yolk accumulation were compared in wild and captive-reared individuals. Liver and ovary samples were taken from 31 individuals reared experimentally in three commercial Atlantic bluefin tuna fattening sites in the Mediterranean Sea and from 33 wild individuals caught by commercial traps during the fish's migration towards their Mediterranean spawning grounds. The total length of VgA cDNA was 5585 nucleotides and that of VgB was 5267 nucleotides. The identity and similarity between deduced amino acid sequences of VgA and VgB were 60% and 78%, respectively. The Atlantic bluefin tuna VgA and VgB amino acid sequences have high similarities with those of other teleost fishes. Relative levels of VgA and VgB mRNAs were low in April, increased significantly during the reproductive period in May and June, and declined in July. There was a trend towards higher relative levels of VgA and VgB mRNAs in captive fish compared to wild individuals during the reproductive period. The surface occupied by eosinophilic yolk granules in fully vitellogenic oocytes, as well as the frequency of oocytes in late vitellogenesis, was significantly higher in captive compared to wild individuals. The study suggests that the experimental conditions under which Atlantic bluefin tuna individuals were reared allowed the occurrence of normal vitellogenesis, based on gene expression of VgA and VgB in the liver and yolk accumulation in the oocytes. The higher yolk accumulation and frequency of vitellogenic oocytes observed in the ovaries of captive fish suggest that improvements in feeding practices may result in an improved vitellogenic process.     

Calmodulin transmitted through gap junctions stimulates endocytic incorporation of yolk precursors in insect oocytes

In ovarian follicles of Oncopeltus fasciatus, and of Xylocopa virginica, calmodulin (CaM) of epithelial cell origin is required by oocytes for endocytic uptake of yolk precursor molecules. Furthermore, this 17-19 kDa protein is normally transported to the oocytes via gap junctions. Downregulation of gap junctions by treatment with 1 mM octanol or separation of the epithelial cells from their oocytes terminated precursor uptake, and this activity could be rescued by microinjection of 60 microM CaM, but not by injections of incubation medium, nor solutions of other molecular species tested. That endogenous CaM is required was confirmed by incubating otherwise untreated follicles in physiological salt solution (PSS) containing either calmidazolium or W-7, both known antagonists of CaM. By radioimmunoprecipitation, we show that the epithelial cells surrounding an oocyte synthesized 15 times as much calmodulin as did the oocytes they encircled. Neither octanol-treated follicles nor denuded oocytes incubated in medium containing calmodulin were able to resume endocytosis, arguing against an extracellular route. However, fluorescently labeled calmodulin microinjected into oocytes is shown to have crossed through gap junctions, making epithelial cells distinctly fluorescent.     

Regulation of the vitellogenin receptor during Drosophila melanogaster oogenesis

In many insects, development of the oocyte arrests temporarily just before vitellogenesis, the period when vitellogenins (yolk proteins) accumulate in the oocyte. Following hormonal and environmental cues, development of the oocyte resumes, and endocytosis of vitellogenins begins. An essential component of yolk uptake is the vitellogenin receptor. In this report, we describe the ovarian expression pattern and subcellular localization of the mRNA and protein encoded by the Drosophila melanogaster vitellogenin receptor gene yolkless (yl). yl RNA and protein are both expressed very early during the development of the oocyte, long before vitellogenesis begins. RNA in situ hybridization and lacZ reporter analyses show that yl RNA is synthesized by the germ line nurse cells and then transported to the oocyte. Yl protein is evenly distributed throughout the oocyte during the previtellogenic stages of oogenesis, demonstrating that the failure to take up yolk in these early stage oocyte is not due to the absence of the receptor. The transition to the vitellogenic stages is marked by the accumulation of yolk via clathrin-coated vesicles. After this transition, yolk protein receptor levels increase markedly at the cortex of the egg. Consistent with its role in yolk uptake, immunogold labeling of the receptor reveals Yl in endocytic structures at the cortex of wild-type vitellogenic oocytes. In addition, shortly after the inception of yolk uptake, we find multivesicular bodies where the yolk and receptor are distinctly partitioned. By the end of vitellogenesis, the receptor localizes predominantly to the cortex of the oocyte. However, during oogenesis in yl mutants that express full-length protein yet fail to incorporate yolk proteins, the receptor remains evenly distributed throughout the oocyte.     

The dynamics of yolk deposition in the desert locust Schistocerca gregaria

Injection of the protein dye Fast Green or the fluid-phase probe fluorescein dextran into the haemolymph of vitellogenic female desert locusts (Schistocerca gregaria) resulted in their incorporation into oocytes. We used Fast Green to study the physical dynamics of yolk deposition during vitellogenesis. Timed maternal injections of Fast Green reveal that yolk deposition and oocyte growth are inextricably linked during vitellogenesis, and that little or no yolk movement occurs within oocytes prior to embryogenesis. The yolk granules laid down early during vitellogenesis lie at the centre of the egg, with yolk granules deposited later packed around these, such that they lie progressively closer to the eventual egg surface. In contrast, during early embryogenesis yolk granules migrate in a manner that closely resembles the movement of early cleavage nuclei. We find fluorescein dextran to be a clear, robust and developmentally inert marker for the timing of maternal injections relative to vitellogenesis in S. gregaria, and we propose its use in parental RNAi or morpholino knockdown experiments. With such experiments in mind, we show that fluorescein-labelled DNA oligonucleotides are internalized within oocytes during vitellogenesis. However, neither Fast Green, fluorescein dextran nor fluorescein-labelled DNA oligonucleotides are detectably transferred from yolk granules to embryonic cells during embryogenesis, and our initial attempts at parental RNAi using maternal injections of dsRNA targeted to late vitellogenesis have proved unsuccessful.     

Connexin40, a component of gap junctions in vascular endothelium, is restricted in its ability to interact with other connexins.

The cellular distribution of connexin40 (Cx40), a newly cloned gap junction structural protein, was examined by immunofluorescence microscopy using two different specific anti-peptide antibodies. Cx40 was detected in the endothelium of muscular as well as elastic arteries in a punctate pattern consistent with the known distribution of gap junctions. However, it was not detected in other cells of the vascular wall. By contrast, Cx43, another connexin present in the cardiovascular system, was not detected in endothelial cells of muscular arteries but was abundant in the myocardium and aortic smooth muscle. We have tested the ability of these connexins to interact functionally. Cx40 was functionally expressed in pairs of Xenopus oocytes and induced the formation of intercellular channels with unique voltage dependence. Unexpectedly, communication did not occur when oocytes expressing Cx40 were paired with those expressing Cx43, although each could interact with a different connexin, Cx37, to form gap junction channels in paired oocytes. These findings indicate that establishment of intercellular communication can be spatially regulated by the selective expression of different connexins and suggest a mechanism that may operate to control the extent of communication between cells.     

Expression of a connexin 43/beta-galactosidase fusion protein inhibits gap junctional communication in NIH3T3 cells

Gap junctions contain membrane channels that mediate the cell-to-cell movement of ions, metabolites and cell signaling molecules. As gap junctions are comprised of a hexameric array of connexin polypeptides, the expression of a mutant connexin polypeptide may exert a dominant negative effect on gap junctional communication. To examine this possibility, we constructed a connexin 43 (Cx43)/beta-galactosidase (beta-gal) expression vector in which the bacterial beta-gal protein is fused in frame to the carboxy terminus of Cx43. This vector was transfected into NIH3T3 cells, a cell line which is well coupled via gap junctions and expresses high levels of Cx43. Transfectant clones were shown to express the fusion protein by northern and western analysis. X-Gal staining further revealed that all of the fusion protein containing cells also expressed beta-gal enzymatic activity. Double immunostaining with a beta-gal and Cx43 antibody demonstrated that the fusion protein is immunolocalized to the perinuclear region of the cytoplasm and also as punctate spots at regions of cell-cell contact. This pattern is similar to that of Cx43 in the parental 3T3 cells, except that in the fusion protein expressing cells, Cx43 expression was reduced at regions of cell-cell contact. Examination of gap junctional communication (GJC) with dye injection studies further showed that dye coupling was inhibited in the fusion protein expressing cells, with the largest reduction in coupling found in a clone exhibiting little Cx43 localization at regions of cell-cell contact. When the fusion protein expression vector was transfected into the communication poor C6 cell line, abundant fusion protein expression was observed, but unlike the transfected NIH3T3 cells, no fusion protein was detected at the cell surface. Nevertheless, dye coupling was inhibited in these C6 cells. Based on these observations, we propose that the fusion protein may inhibit GJC by sequestering the Cx43 protein intracellularly. Overall, these results demonstrate that the Cx43/beta-gal fusion protein can exert a dominant negative effect on GJC in two different cell types, and suggests that it may serve as a useful approach for probing the biological function of gap junctions.     

Oogenesis in the bluefin tuna,Thunnus thynnus L.: a histological and histochemical study

Histology and histochemistry are useful tools to study reproductive mechanisms in fish and they have been applied in this study. In the bluefin tuna, Thunnus thymus L., oocyte development can be divided into 4 principal phases based on the morphological features of developing oocytes and follicles. The primary growth phase includes oogonia and basophilic or previtellogenic oocytes classified as chromatin-nucleolus and perinucleolus stages. The secondary growth phase is represented by vitellogenic oocytes at early (lipid globule and yolk granule 1), mid (yolk granule 2) and late (yolk granule 3) vitellogenesis stages. The maturation phase involves postvitellogenic oocytes undergoing maturation process. During the spawning period, both postovulatory follicles, which indicate spawning, and atretic follicles can be distinguished in the ovary. Carbohydrates, lipids, proteins and specially those rich in tyrosine, tryptophan, cystine, arginine, lysine and cysteine, as well phospholipids and/or glycolipids and neutral glycoproteins were detected in yolk granules. Moreover, affinity for different lectins (ConA, WGA, DBA and UEA) was detected in vitellogenic oocytes (yolk granules, cortical alveoli, follicular layer and zona radiata), indicating the presence of glycoconjugates with different sugar residues (Mannose- Man- and/or Glucose -Glc-; N-acetyl-D-glucosamine- GlcNAc- and/or sialic acid- NANA-; N-acetyl-D-galactosamine- GalNAc-; L-Fucose -Fuc-). Histochemical techniques also demonstrated the presence of neutral lipids in globules (vacuoles in paraffin sections) and neutral and carboxylated mucosubstances in cortical alveoli. By using anti-vitellogenin (VTG) serum, immunohistochemical positive results were demonstrated in yolk granules, granular cytoplasm and follicular cells of vitellogenic oocytes. Calcium was also detected in yolk granules and weakly in follicular envelope. In females, the gonadosomatic index (GSI) increased progressively from May, during early vitellogenesis, until June during mid and late vitellogenesis, where the highest values were reached. Subsequently, throughout the maturation-spawning phases (July), GSI decreased progressively reaching the minimal values during recovering-resting period (October).     

Changes in gap junction distribution and connexin expression pattern during human fetal skin development.

Gap junctions are intercellular channels composed of connexin subunits that mediate cell-cell communication. The functions of gap junctions are believed to be associated with cell proliferation and differentiation and to be important in maintaining tissue homeostasis. We therefore investigated the expression of connexins (Cx)26 and 43, the two major connexins in human epidermis, and examined the formation of gap junctions during human fetal epidermal development. By immunofluorescence, Cx26 expression was observed between 49 and 96 days' estimated gestational age (EGA) but was not present from 108 days' EGA onwards. Conversely, Cx43 expression was observed from 88 days' EGA onwards. Using electron microscopy, the typical structure of gap junctions was observed from 120 days' EGA. The number of gap junctions increased over time and they were more common in the upper layers, within the periderm and intermediate keratinocyte layers rather than the basal layer. Immunoelectron microscopy revealed Cx43 labeling on the gap junction structures after 105 days' EGA. Formation of gap junctions increased as skin developed, suggesting that gap junctions may play an important role in fetal skin development. Furthermore, the changing patterns of connexin expression suggest that Cx26 is important for early fetal epidermal development.     

Changes of gap junctional cell-cell communication in overactive detrusor in rats

To evaluate the changes in intercellular communication through gap junctions in detrusor overactivity (DO), we studied 23 adult female Wistar rats with DO after partial outflow obstruction (DO group) and 13 sham-operated rats (control group). The two groups were compared by means of urodynamics, light and electron microscopy, expression of Cx40, Cx43, and Cx45 mRNA genes with RT-PCR, Cx43 protein with Western blot analysis, and functional intercellular communication with scrape loading dye transfer (SLDT) and fluorescence recovery after photobleaching (FRAP). The number of gap junctions and the expression of connexin mRNA and Cx43 protein were increased in DO rats, and intercellular communication through gap junctions increased after 6 wk of partial outflow obstruction as assessed with SLDT and FRAP techniques. The findings provide a theoretical rationale for using Cx43 antagonists and gap junction inhibitors in the treatment of patients with overactive detrusor secondary to partial bladder outflow obstruction.     

山溪鲵卵黄发生的显微与超微结构

用光镜和透射电镜观察了山溪鲵（Batrachuperus pinchonii)不同发育时期卵母细胞的显微与超微结构,特别注意了与卵黄发生相关的细胞器变化。结果表明,类核周体为线粒体、高尔基体、内质网等膜性细胞器聚集的场所;线粒体自身也可演变成卵黄前颗粒,参与构成卵黄小板的成分;吞饮泡和髓样小体是卵母细胞利用外源卵黄物质的中间媒介。经过与其他动物卵黄发生过程相比较,认为非哺乳脊椎动物卵黄发生是卵母细胞在多种细胞器参与下整体活动的结果,不是经由单一的模式或途径形成,因此呈现发生上的多元化;不同物种在卵黄发生中分别采取与各自相适应的模式或途径。     

Cathepsin D Activity in the Vitellogenesis of Xenopus laevis

An ovarian extract of Xenopus laevis exhibited in SDS-PAGE analyses an activity cleaving vitellogenin to lipovitellins under mildly acidic conditions. This activity was pepstatin-sensitive and inhibited by monospecific anti-rat liver cathepsin D antibody and thus identified as cathepsin D. Immunoblot analysis showed that two proteins of 43 kDa and 36 kDa immunoreacted with the antibody.
Immunocytochemical staining revealed that the enzyme was located in the cortical cytoplasm of stage I and II oocytes and in small yolk platelets and nascent forms of large yolk platelets in the cortical cytoplasm of stage III oocytes. In stage IV and V oocytes, small yolk platelets retained the immuno-staining but large yolk platelets decreased it. No immuno-positive signals were observed in oocytes at stage VI. When examined by immunoelectron microscopy, gold particles indicated that cathepsin D was located on dense lamellar bodies in the cortical cytoplasm of stage I and II oocytes. The particles were located on primordial yolk platelets and on the superficial layer of small yolk platelets in stage III oocytes, while they were sparse or not present at all on large yolk platelets in stage IV and V oocytes. These results indicate that cathepsin D plays a key role in vitellogenesis by cleaving endocytosed vitellogenin to yolk proteins in developing oocytes.     

N-cadherin and Cx43alpha1 gap junctions modulates mouse neural crest cell motility via distinct pathways

Our previous studies showed an essential role for connexin 43 or alpha1 connexin (Cx43alpha1) gap junctions in the modulation of neural crest cell motility. Cx43alpha1 gap junctions and N-cadherin containing adherens junctions are expressed in migrating cardiac neural crest cells. Analysis of the N-cadherin knockout (KO) mouse model revealed that N-cadherin is essential for gap junction mediated dye coupling but not for expression of Cx43alpha1 gap junctions in neural crest cells. Time lapse videomicroscopy and motion analysis showed that the motility of N-cadherin KO neural crest cells were altered, but the motility changes differed compared to Cx43alpha1 KO neural crest cells. These observations suggest that the role of N-cadherin in cell motility is not simply mediated via the modulation of Cx43alpha1 mediated cell-cell communication. This was confirmed by a parallel analysis of wnt-1 deficient neural crest cells, which also showed a reduction in dye coupling, and yet no change in cell motility. Analysis of p120 catenin (p120ctn), an Amardillo family protein known to play a role in cell motility, showed that it is colocalized with N-cadherin and Cx43alpha1 in migrating neural crest cells. This subcellular distribution was altered in the N-cadherin and Cx43alpha1 KO neural crest cells. Given these results, we propose that N-cadherin and Cx43alpha1 may modulate neural crest cell motility by engaging in a dynamic cross-talk with the cell's locomotory apparatus through p120ctn signaling.     

Connexin 30 Expression and Frequency of Connexin Heterogeneity in Astrocyte Gap Junction Plaques Increase with Age in the Rat Retina

We investigated age-associated changes in retinal astrocyte connexins (Cx) by assaying Cx numbers, plaque sizes, protein expression levels and heterogeneity of gap junctions utilizing six-marker immunohistochemistry (IHC). We compared Wistar rat retinal wholemounts in animals aged 3 (young adult), 9 (middle-aged) and 22 months (aged). We determined that retinal astrocytes have gap junctions composed of Cx26, -30, -43 and -45. Cx30 was consistently elevated at 22 months compared to younger ages both when associated with parenchymal astrocytes and vascular-associated astrocytes. Not only was the absolute number of Cx30 plaques significantly higher (P<0.05) but the size of the plaques was significantly larger at 22 months compared to younger ages (p<0.05). With age, Cx26 increased significantly initially, but returned to basal levels; whereas Cx43 expression remained low and stable with age. Evidence that astrocytes alter connexin compositions of gap junctions was demonstrated by the significant increase in the number of Cx26/Cx45 gap junctions with age. We also found gap junctions comprised of 1, 2, 3 or 4 Cx proteins suggesting that retinal astrocytes use various connexin protein combinations in their gap junctions during development and aging. These data provides new insight into the dynamic and extensive Cx network utilized by retinal astrocytes for communication within both the parenchyma and vasculature for the maintenance of normal retinal physiology with age. This characterisation of the changes in astrocytic gap junctional communication with age in the CNS is crucial to the understanding of physiological aging and age-related neurodegenerative diseases.