First Responders to Hyperosmotic Stress in Murine Astrocytes: Connexin 43 Gap Junctions Are Subject to an Immediate Ultrastructural Reorganization

In a short-term model of hyperosmotic stress, primary murine astrocytes were stimulated with a hyperosmolar sucrose solution for five minutes. Astrocytic gap junctions, which are mainly composed of Connexin (Cx) 43, displayed immediate ultrastructural changes, demonstrated by freeze–fracture replica immunogold labeling: their area, perimeter, and distance of intramembrane particles increased, whereas particle numbers per area decreased. Ultrastructural changes were, however, not accompanied by changes in Cx43 mRNA expression. In contrast, transcription of the gap junction regulator zonula occludens (ZO) protein 1 significantly increased, whereas its protein expression was unaffected. Phosphorylation of Serine (S) 368 of the Cx43 C–terminus has previously been associated with gap junction disassembly and reduction in gap junction communication. Hyperosmolar sucrose treatment led to enhanced phosphorylation of Cx43S368 and was accompanied by inhibition of gap junctional intercellular communication, demonstrated by a scrape loading-dye transfer assay. Taken together, Cx43 gap junctions are fast reacting elements in response to hyperosmolar challenges and can therefore be considered as one of the first responders to hyperosmolarity. In this process, phosphorylation of Cx43S368 was associated with disassembly of gap junctions and inhibition of their function. Thus, modulation of the gap junction assembly might represent a target in the treatment of brain edema or trauma.

The Role of Steroids, Growth Factors and CAMP Stimulators on the Gap Junction Activity in Cumulus Oocyte Complexes in the Rat

<p>Bidirectional communication between mammalian oocytes and their surrounding somatic cells is essential for oocyte maturation. Gap junctions promote the transfer of essential metabolites, nucleotides, amino acids and ions from cumulus cells to the oocyte that are crucial for oocyte growth and development. However, the range of factors present in the microenvironment of the developing antral follicle, which modulate gap junction activity of the cumulus-oocyte complexes (COCs), is largely unknown. The primary objective of this study was to determine the effects of various steroids, growth factors and cAMP stimulators on the gap junction activity in rat COCs. The gap junction activity was measured in presence or absence of different treatments using a fluorescence dye and in the presence of milrinone, a phosphodiesterase type 3 inhibitor. The major findings of this study were that cAMP stimulators increased the rate of dye transfer from cumulus cells to the oocyte. Under in vitro conditions it was established that neither steroids nor IGF1 by themselves were able to modulate gap junction activity in rat COCs. Furthermore, forskolin, a potent cAMP stimulator; caused a relative increase in Cx37 gene expression levels following a four hours incubation period. The outcomes from the present study may help to provide new insights into developing suitable in vitro conditions, for the in vitro maturation of mammalian oocytes.</p>

The Role of Steroids, Growth Factors and CAMP Stimulators on the Gap Junction Activity in Cumulus Oocyte Complexes in the Rat

<p>Bidirectional communication between mammalian oocytes and their surrounding somatic cells is essential for oocyte maturation. Gap junctions promote the transfer of essential metabolites, nucleotides, amino acids and ions from cumulus cells to the oocyte that are crucial for oocyte growth and development. However, the range of factors present in the microenvironment of the developing antral follicle, which modulate gap junction activity of the cumulus-oocyte complexes (COCs), is largely unknown. The primary objective of this study was to determine the effects of various steroids, growth factors and cAMP stimulators on the gap junction activity in rat COCs. The gap junction activity was measured in presence or absence of different treatments using a fluorescence dye and in the presence of milrinone, a phosphodiesterase type 3 inhibitor. The major findings of this study were that cAMP stimulators increased the rate of dye transfer from cumulus cells to the oocyte. Under in vitro conditions it was established that neither steroids nor IGF1 by themselves were able to modulate gap junction activity in rat COCs. Furthermore, forskolin, a potent cAMP stimulator; caused a relative increase in Cx37 gene expression levels following a four hours incubation period. The outcomes from the present study may help to provide new insights into developing suitable in vitro conditions, for the in vitro maturation of mammalian oocytes.</p>

Development of an in Vitro Assay to Assess Gap Junction Activity in Cumulus-Oocyte Complexes (COC) in the Rat

<p>The capacity of an oocyte to mature during ovarian follicular development is a key process in reproductive biology. Bidirectional communication between mammalian oocytes and their associated follicular somatic cells (cumulus-cells) is essential for oocyte maturation. Historically, studies examining the control of ovarian follicular development focused mainly on the endocrine (external) signalling but recently intraovarian (paracrine) regulation has also been shown to be important. In addition, signalling via gap junctions between follicular cells had also been crucial for oocyte maturation and follicular development. In antral follicles, gap junction activity between the oocyte and adjacent cumulus cells first increase during follicular growth and shortly before ovulation they decrease as the oocyte resumes meiosis once more before ovulation. The range of factors that modulate gap junction activity of oocyte-cumulus cell complexes (COC) is largely unknown. The aims of these studies were to develop an assay to assess the rate of transfer of low molecular weight materials from cumulus cells to the oocyte via gap junctions. The first objective was to validate a bioassay by which to test the effects of hormones, second messengers, and growth factors on gap junction activity in rat cumulus-oocyte complexes. In this study, COCs were collected from antral follicles of untreated post-pubertal Sprague Dawley rats. Gap junction activity was measured in the presence or absence of different treatments using the fluorescence dye, Calcein-AM and in the presence of a phosphodiesterase type 3 inhibitor (PDE3) milrinone. Transfer of the calcein dye from cumulus cells into the oocyte was measured at various times using CRAIC fluorescence system. The results showed that removal of the COCs from their follicular environments disrupted the gap junction activity which recovered over time in culture media. COC were sensitive to changes in pH concentration and gap junction activity could be blocked with 8 ocatnol-1 but not carbenoxolone. Treating rat COCs with dibutyryl cAMP or agents that maintained or increased intracellular cAMP levels like milrinone or forskolin were unable to modulate gap junction activity. Further, the combined effect of the oocyte-derived growth factors: growth differentiating factor 9 (GDF9) with bone morphogenetic protein 15 (BMP15) was also unable to modulate the rate of calcein dye transfer from cumulus cells to the oocyte. Ovarian steroids such as oestradiol and testosterone by themselves were unable to modulate the gap junction activity of rat COC but the combined treatment of testosterone plus forskolin or testosterone plus forskolin plus insulin-like growth factor 1 (IGF-1) increased the rate of dye transfer from cumulus cells to the oocyte. In conclusion, a fluorescence dye transfer assay was developed to measure the effects of different treatments on gap junction activity in rat COC. Under in vitro conditions, it was established that the combination of steroid and cAMP stimulators or a steroid, cAMP stimulator with IGF1 but not these reagents individually could enhance the recovery of gap junction function in rat COC. The outcomes of these experiments may help to provide new insights into developing suitable in vitro conditions, for the in vitro maturation of mammalian oocytes. Also, the newly developed assay may serve as a useful in vitro model to evaluate the effects of hormones, nutritional supplements and other factors on COC functions.</p>

Development of an in Vitro Assay to Assess Gap Junction Activity in Cumulus-Oocyte Complexes (COC) in the Rat

<p>The capacity of an oocyte to mature during ovarian follicular development is a key process in reproductive biology. Bidirectional communication between mammalian oocytes and their associated follicular somatic cells (cumulus-cells) is essential for oocyte maturation. Historically, studies examining the control of ovarian follicular development focused mainly on the endocrine (external) signalling but recently intraovarian (paracrine) regulation has also been shown to be important. In addition, signalling via gap junctions between follicular cells had also been crucial for oocyte maturation and follicular development. In antral follicles, gap junction activity between the oocyte and adjacent cumulus cells first increase during follicular growth and shortly before ovulation they decrease as the oocyte resumes meiosis once more before ovulation. The range of factors that modulate gap junction activity of oocyte-cumulus cell complexes (COC) is largely unknown. The aims of these studies were to develop an assay to assess the rate of transfer of low molecular weight materials from cumulus cells to the oocyte via gap junctions. The first objective was to validate a bioassay by which to test the effects of hormones, second messengers, and growth factors on gap junction activity in rat cumulus-oocyte complexes. In this study, COCs were collected from antral follicles of untreated post-pubertal Sprague Dawley rats. Gap junction activity was measured in the presence or absence of different treatments using the fluorescence dye, Calcein-AM and in the presence of a phosphodiesterase type 3 inhibitor (PDE3) milrinone. Transfer of the calcein dye from cumulus cells into the oocyte was measured at various times using CRAIC fluorescence system. The results showed that removal of the COCs from their follicular environments disrupted the gap junction activity which recovered over time in culture media. COC were sensitive to changes in pH concentration and gap junction activity could be blocked with 8 ocatnol-1 but not carbenoxolone. Treating rat COCs with dibutyryl cAMP or agents that maintained or increased intracellular cAMP levels like milrinone or forskolin were unable to modulate gap junction activity. Further, the combined effect of the oocyte-derived growth factors: growth differentiating factor 9 (GDF9) with bone morphogenetic protein 15 (BMP15) was also unable to modulate the rate of calcein dye transfer from cumulus cells to the oocyte. Ovarian steroids such as oestradiol and testosterone by themselves were unable to modulate the gap junction activity of rat COC but the combined treatment of testosterone plus forskolin or testosterone plus forskolin plus insulin-like growth factor 1 (IGF-1) increased the rate of dye transfer from cumulus cells to the oocyte. In conclusion, a fluorescence dye transfer assay was developed to measure the effects of different treatments on gap junction activity in rat COC. Under in vitro conditions, it was established that the combination of steroid and cAMP stimulators or a steroid, cAMP stimulator with IGF1 but not these reagents individually could enhance the recovery of gap junction function in rat COC. The outcomes of these experiments may help to provide new insights into developing suitable in vitro conditions, for the in vitro maturation of mammalian oocytes. Also, the newly developed assay may serve as a useful in vitro model to evaluate the effects of hormones, nutritional supplements and other factors on COC functions.</p>

Connexin-Based Channel Activity Is Not Specifically Altered by Hepatocarcinogenic Chemicals

Connexin-based channels play key roles in cellular communication and can be affected by deleterious chemicals. In this study, the effects of various genotoxic carcinogenic compounds, non-genotoxic carcinogenic compounds and non-carcinogenic compounds on the expression and functionality of connexin-based channels, both gap junctions and connexin hemichannels, were investigated in human hepatoma HepaRG cell cultures. Expression of connexin26, connexin32, and connexin43 was evaluated by means of real-time reverse transcription quantitative polymerase chain reaction analysis, immunoblot analysis and in situ immunostaining. Gap junction functionality was assessed via a scrape loading/dye transfer assay. Opening of connexin hemichannels was monitored by measuring extracellular release of adenosine triphosphate. It was found that both genotoxic and non-genotoxic carcinogenic compounds negatively affect connexin32 expression. However, no specific effects related to chemical type were observed at gap junction or connexin hemichannel functionality level.

Analytical Methods for Assessing Retinal Cell Coupling Using Cut-Loading

Electrical coupling between retinal neurons contributes to the functional complexity of visual circuits. “Cut-loading” methods allow simultaneous assessment of cell-coupling between multiple retinal cell-types, but existing analysis impedes direct comparison with gold standard direct dye injection techniques. Therefore cut-loading was used to assess coupling strength in a-type horizontal cells in dark-adapted Guinea pig retinae (n=29) using the standard protocol (Method 1) compared with two non-linear methods. Method 1 describes the distance of dye-diffusion (space constant), while Method 2 extracted the coupling coefficient (kj) and Method 3 measured the diffusion coefficient (De). Dye transfer was measured after one of five diffusion times (1-20 mins), or with a coupling inhibitor, meclofenamic acid (MFA) (50–500µM after 20 mins diffusion). Method 1 includes background fluorescence, producing less accurate coupling estimates than measuring the fluorescence of individual cell-soma (p<0.001). The space constant (Method 1) increased with diffusion time (p<0.01), whereas Methods 2 (p=0.54) and 3 (p=0.63) produced consistent results across all diffusion times. Method 1 was less sensitive to detecting changes induced by MFA than Methods 2 (p<0.01) and 3 (p<0.01). Comparatively, Methods 2 and 3 proved more sensitive and generalisable; allowing for detailed assessment of the coupling between different populations of gap-junction linked cell networks.

Androgen Regulates the Expression and Function of Connexin 43 in Granulosa Cells via the PKC Signalling Pathway

Background: Poor ovarian response (POR) is one of the most challenging issues in assisted reproductive technology. Given the importance of androgens in follicle recruitment, growth and development, domestic and foreign reproductive centers have added androgen as an adjuvant to in vitro fertilization protocols for patients with POR to improve the ovarian response. The mechanism underlying the effects of androgens is unclear. The aim of this research was to study the effects of testosterone on granulosa cells and the underlying mechanisms.Methods: The human ovarian granulosa cell line KGN was treated with testosterone at various concentrations, an androgen receptor antagonist (flutamide) and a protein kinase C (PKC) inhibitor (GF109203X). The protein levels of PKC and phosphorylated connexin 43 (p-Cx43) were measured using Western blot analysis. The location and distribution of Cx43 and p-Cx43 were determined by immunofluorescence. Gap junction intercellular communication (GJIC) was monitored using the scrape loading/dye transfer method.Results: The results showed that treatment with testosterone at a physiological level significantly increased the PKC and p-Cx43 levels and GJIC activity in a concentration-dependent manner. However, the expression of PKC, p-Cx43 levels and GJIC activity in the cells treated with testosterone above physiological concentrations (10-5 M) decreased compared with those in the cells treated with 10-7 M testosterone. Compared to the 10-7 M testosterone-treated cells, the KGN cells treated with flutamide had much lower levels of PKC and p-Cx43. The levels of p-Cx43 and GJIC activity in the GF109203X-treated group were decreased compared to those in the 10-7 M testosterone group. Immunofluorescence showed that testosterone treatment had no effect on the localization and distribution of gap junction proteins.Conclusions: This study suggests that androgen can increase GJIC activity in granulosa cells by phosphorylating Cx43 via the PKC signalling pathway to promote oocyte development. The results of this study might provide a theoretical basis for androgen pretreatment of patients with POR.

Applicability of Scrape Loading-Dye Transfer Assay for Non-Genotoxic Carcinogen Testing

Dysregulation of gap junction intercellular communication (GJIC) is recognized as one of the key hallmarks for identifying non-genotoxic carcinogens (NGTxC). Currently, there is a demand for in vitro assays addressing the gap junction hallmark, which would have the potential to eventually become an integral part of an integrated approach to the testing and assessment (IATA) of NGTxC. The scrape loading-dye transfer (SL-DT) technique is a simple assay for the functional evaluation of GJIC in various in vitro cultured mammalian cells and represents an interesting candidate assay. Out of the various techniques for evaluating GJIC, the SL-DT assay has been used frequently to assess the effects of various chemicals on GJIC in toxicological and tumor promotion research. In this review, we systematically searched the existing literature to gather papers assessing GJIC using the SL-DT assay in a rat liver epithelial cell line, WB-F344, after treating with chemicals, especially environmental and food toxicants, drugs, reproductive-, cardio- and neuro-toxicants and chemical tumor promoters. We discuss findings derived from the SL-DT assay with the known knowledge about the tumor-promoting activity and carcinogenicity of the assessed chemicals to evaluate the predictive capacity of the SL-DT assay in terms of its sensitivity, specificity and accuracy for identifying carcinogens. These data represent important information with respect to the applicability of the SL-DT assay for the testing of NGTxC within the IATA framework.

Androgen regulates the expression and function of connexin 43 in granulosa cells via the PKC signalling pathway

Background Poor ovarian response (POR) is one of the most challenging issues in assisted reproductive technology. Given the importance of androgens in follicle recruitment, growth and development, domestic and foreign reproductive centers have added androgen as an adjuvant to in vitro fertilization protocols for patients with POR to improve the ovarian response. The mechanism underlying the effects of androgens is unclear. The aim of this research was to study the effects of testosterone on granulosa cells and the underlying mechanisms. Methods The human ovarian granulosa cell line KGN was treated with testosterone at various concentrations, an androgen receptor antagonist (flutamide) and a protein kinase C (PKC) inhibitor (GF109203X). The protein levels of PKC and phosphorylated connexin 43 (p-Cx43) were measured using Western blot analysis. The location and distribution of Cx43 and p-Cx43 were determined by immunofluorescence. Gap junction intercellular communication (GJIC) was monitored using the scrape loading/dye transfer method. Results The results showed that treatment with testosterone at a physiological level significantly increased the PKC and p-Cx43 levels and GJIC activity in a concentration-dependent manner. However, the expression of PKC, p-Cx43 levels and GJIC activity in the cells treated with testosterone above physiological concentrations (10− 5 M) decreased compared with those in the cells treated with 10− 7 M testosterone. Compared to the 10− 7 M testosterone-treated cells, the KGN cells treated with flutamide had much lower levels of PKC and p-Cx43. The levels of p-Cx43 and GJIC activity in the GF109203X-treated group were decreased compared to those in the 10− 7 M testosterone group. Immunofluorescence showed that testosterone treatment had no effect on the localization and distribution of gap junction proteins. Conclusions This study suggests that androgen can increase GJIC activity in granulosa cells by phosphorylating Cx43 via the PKC signalling pathway to promote oocyte development. The results of this study might provide a theoretical basis for androgen pretreatment of patients with POR.