| gap junction protein alpha 1 | OKDB#: 348 |
| Symbols: | GJA1 | Species: | human | ||
| Synonyms: | HSS, CMDR, CX43, EKVP, GJAL, ODDD, AVSD3, EKVP3, HLHS1, PPKCA | Locus: | 6q22.31 in Homo sapiens |
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| General Comment |
Gap junctions are conduits that allow the direct cell-to-cell passage of small cytoplasmic molecules, including ions, metabolic intermediates, and second messengers, and thereby mediate intercellular metabolic and electrical communication. Gap junction channels consist of connexin protein subunits, which are encoded by a multigene family. More insights into the CCN3/Connexin43 interaction complex and its role for signaling. Gellhaus A et al. Connexin43 (Cx43) forms gap junction channels but also serves as a signaling center by binding to proteins via its C-terminus. Here, we combined fluorescence resonance energy transfer (FRET), co-immunoprecipitation and proliferation and expression assays to characterize the interaction complex of Cx43 and CCN3. FRET measurements confirmed the interaction of CCN3 with wild-type Cx43 (amino acids 1-382) and with mutants of Cx43 truncated at the C-terminus resulting in Cx43 proteins of amino acids 1-374, 1-273, 1-264, 1-257 in 293T cells. These results matched the co-immunoprecipitation data. Interestingly, although FRET revealed distinct efficiencies in interaction of Cx43 with CCN3 for all deletion constructs only wild-type Cx43 and one deletion construct (1-374) led to increased CCN3 expression. Only these interactions which were associated with increased CCN3 expression resulted in a reduced cell proliferation. Our study provides evidence that only defined binding properties between Cx43 and CCN3 leading to an upregulation of CCN3 are needed for signaling. Furthermore, the data obtained by FRET analysis allowed us to model the 3D structure of the C-terminus of Cx43 interacting with CCN3.
NCBI Summary: This gene is a member of the connexin gene family. The encoded protein is a component of gap junctions, which are composed of arrays of intercellular channels that provide a route for the diffusion of low molecular weight materials from cell to cell. The encoded protein is the major protein of gap junctions in the heart that are thought to have a crucial role in the synchronized contraction of the heart and in embryonic development. A related intronless pseudogene has been mapped to chromosome 5. Mutations in this gene have been associated with oculodentodigital dysplasia, autosomal recessive craniometaphyseal dysplasia and heart malformations. [provided by RefSeq, May 2014] |
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| General function | Channel/transport protein | ||||
| Comment | The ovarian follicle in mammals is a functional syncytium, with the oocyte being coupled with the surrounding cumulus granulosa cells, and the cumulus cells being coupled with each other and with the mural granulosa cells, via gap junctions. The gap junctions coupling granulosa cells in mature follicles contain several different connexins (gap junction channel proteins), including connexins 32, 43, and 45. Connexin43 immunoreactivity can be detected from the onset of folliculogenesis just after birth and persists through ovulation. | ||||
| Cellular localization | |||||
| Comment | miR-130b regulates gap junctional intercellular communication through connexin 43 in granulosa cells from patients with polycystic ovary syndrome. Jiang L et al. (2020) MicroRNAs (miRNAs) are small, noncoding RNAs that negatively regulate gene expression post-transcriptionally. We explored whether connexin 43 (Cx43) was differently expressed in luteinized granulosa cells from women with polycystic ovary syndrome (PCOS) compared with luteinized granulosa cells from women with a normal menstrual cycle, and whether certain miRNAs regulate the Cx43 level and gap junctional intercellular communication. The miRNA profile was investigated in ovarian cortex tissues from five women with PCOS and five women without PCOS using a miRNA microarray. The levels of miR-130b and Cx43 mRNA were measured using real-time PCR in human luteinized granulosa cells from 20 women with PCOS and 25 women without PCOS. Protein and mRNA expression analysis and luciferase assays were conducted to confirm the substrate of miR-130b. PCOS ovarian cortex showed differential expression of miRNAs compared with non-PCOS ovarian cortex. Furthermore, miR-130b levels were increased in PCOS ovarian cortex and in luteinized granulosa cells compared with those in women with normal menstrual cycles, whereas the level of Cx43 mRNA, the identified target of miR-130b, was decreased in granulosa cells from patients with PCOS. Overexpression of miR-130b in a granulosa cell line resulted in reduced Cx43 protein levels and inhibited gap junctional intercellular communication using scrape loading and dye transfer assay. Meanwhile, inhibition of miR-130b increased the Cx43 level. In conclusion, miR-130b was increased in PCOS granulosa cells, where it targets Cx43 to affect gap junctional intercellular communication. The results of the present study suggested that miR-130b, via post-transcriptional regulation of Cx43, is involved in the pathophysiology of PCOS, which provides new insight into the pathological mechanism of PCOS.////////////////// | ||||
| Ovarian function | Follicle development, Antral follicle growth, Cumulus expansion, Follicle atresia, Ovulation, Luteinization, Oogenesis, Oocyte maturation | ||||
| Comment | Involvement of GJA1 and Gap Junctional Intercellular Communication between Cumulus Cells and Oocytes from Women with PCOS. Liu Q et al. (2020) Polycystic ovary syndrome (PCOS) is a common female endocrine system disease that affects 17.8% of women of reproductive age and leads to infertility, obesity, glucose metabolic disorders, cardiovascular disease, and body-mind problems. However, the etiology of PCOS remains unclear. Follicular growth is disrupted as a result of ovarian hyperandrogenism and distorted intraovarian paracrine signaling in women with PCOS. Microcommunication between oocytes and cumulus cells plays a critical role in folliculogenesis. Gap junction alpha 1 (GJA1) plays a crucial role in the developing follicles by forming communication channels between cumulus cells and oocytes, but this has not yet been reported in women with PCOS. Therefore, we aimed to study the role of GJA1 in the microcommunication between oocytes and cumulus cells in women with PCOS. In our study, cumulus cell-oocyte complexes (COCs) from women were isolated via ultrasound-guided vaginal puncture, and oocytes were selected from COCs and categorized based on 3 oocyte maturation stages. Then, RT-qPCR and immunofluorescence analysis were performed to detect both the gene expression and protein of GJA1 in oocytes from women with and without PCOS. There was no statistically significant difference in age and BMI (body mass index), but patients with PCOS had a higher ratio of basic LH/FSH (luteinizing hormone/follicle-stimulating hormone), androstenedione, and total ovarian volume. The qRT-PCR results showed higher gene expression of GJA1 in oocytes without PCOS at the germinal vesicle (GV) stage compared with that of oocytes from women with PCOS. Immunofluorescence analysis showed that the expression level of GJA1 in oocytes from women with PCOS was very weak compared with that of oocytes from women without PCOS. In conclusion, GJA1 may play a critical role in the development of oogenesis arrest in women with PCOS throughout the oogenesis processes, including oogenesis and oocyte maturation.//////////////////Gap junctions are essential for murine primordial follicle assembly immediately before birth. Teng Z et al. (2015) The reserve of primordial follicles determines the reproductive ability of the female mammal over its reproductive life. The primordial follicle is composed of two types of cells, the oocyte and the surrounding pre-granulosa cells. However, the underlying mechanisms regulating primordial follicle assembly is largely undefined. In this study, we found that gap junction communication (GJC) established between the ovarian cells in the perinatal mouse ovary may be involved in the process. First, gap junction structures between the oocyte and surrounding pre-granulosa cells appear at around 19.0 dpc (days post coitum). As many as 12 gap junction-related genes were upregulated at birth, implying that a complex communication may exist between ovarian cells, because specifically silencing the genes of individual gap junction proteins, such as Gja1, Gja4 or both, has no influence on primordial follicle assembly. On the other hand nonspecific blockers of GJC, such as carbenoxolone (CBX) and 18-alpha-glycyrrhetinic acid (AGA), significantly inhibit mouse primordial follicle assembly. We proved that the temporal window for GJC establishment in the fetal ovary is from 19.5 dpc to 1 dpp (days postpartum). In addition, the expression of ovarian somatic cell (OSC)-specific genes, such as Notch2, Foxl2 and Irx3, were negatively affected by GJC blockers, whereas oocyte-related genes, such as Ybx2, Nobox and Sohlh1, were hardly affected, implying that the establishment of GJC during this period may be more important to OSCs than to oocytes. In summary, our results indicated that GJC involves in the mouse primordial follicle assembly process at a specific temporal window which needs Notch signaling cross talking.////////////////// Correlation of cumulus gene expression of GJA1, PRSS35, PTX3, and SERPINE2 with oocyte maturation, fertilization, and embryo development. Li SH et al. (2015) GJA1 and PTX3 were proposed as gene markers for oocyte and embryo developmental competence, while SERPINE2 was reported to be associated with pregnancy outcome. PRSS35, which is exclusively expressed in the ovary, may be correlated with oocyte competence. This study was conducted to evaluate the correlation of cumulus GJA1, PRSS35, PTX3, and SERPINE2 gene expression levels with oocyte maturation, fertilization, and early embryo development. In total, 308 cumulus cell samples separated from individual cumulus-oocyte complex were obtained from 40 patients undergoing the intracytoplasmic sperm injection treatment procedure. Gene expression levels (mRNA levels) in cumulus cells were assessed using quantitative real-time polymerase chain reaction. Gene expression levels of GJA1 and SERPINE2 in cumulus cells surrounding mature oocytes were significantly lower than those in cumulus cells enclosing immature oocytes. PRSS35 mRNA levels in cumulus cells of fertilized oocytes were significantly higher than those in cumulus cells of unfertilized oocytes. GJA1 and SERPINE2 seemed to express higher mRNA levels, while PRSS35 showed lower expression in cumulus cells of oocytes that developed into embryos with good morphology; however, the expression levels of all three genes and PTX3 showed no significant differences between embryos with good or poor morphology. GJA1 and SERPINE2 represent potential gene markers associated with oocyte maturation. PRSS35 may be correlated with oocyte fertilization potential. However, GJA1, PRSS35, PTX3, and SERPINE2 may not be considered as marker genes for predicting embryo morphology.////////////////// Prophase I Arrest of Mouse Oocytes Mediated by Natriuretic Peptide Precursor C Requires GJA1 (connexin-43) and GJA4 (connexin-37) Gap Junctions in the Antral Follicle and Cumulus-Oocyte Complex. Richard S 2014 et al. Fully grown germinal vesicle stage mouse oocytes remain arrested in meiotic prophase I until ovulation. This arrest is maintained by cGMP produced in cumulus granulosa cells surrounding the oocyte. Recently, it was found that cGMP production in cumulus cells depends on NPR2 guanylate cyclase activated by its ligand natriuretic peptide precursor C (NPPC). It is assumed that cGMP reaches the oocyte through gap junctions that couple cumulus granulosa cells to each other and to the oocyte. Previous work identified two main types of gap junctions in the follicle, connexin-43 gap junctions (GJA1 protein) between granulosa cells and connexin-37 gap junctions (GJA4) between cumulus cells and the oocyte. However, it had not been established that both types are required for meiotic arrest mediated by NPPC/NPR2 signaling. To investigate this, we used connexin mimetic peptides (CMP) that specifically disrupt gap junction isoforms within cumulus-oocyte complexes (COCs) and isolated antral follicles in culture. We furthermore developed a punctured antral follicle preparation to permit CMP access to the antral cavity in an otherwise intact follicle. CMP directed against connexin-43 (Cx43 CMP) overcame NPPC-mediated meiotic arrest in both isolated COCs and antral follicles. Cx37 CMP, in contrast, had no effect when present in the medium, but released oocyte arrest in the presence of NPPC when microinjected into the perivitelline space near the oocyte surface in COCs. This is consistent with both connexin isoforms being required for meiotic arrest, and with the reported localization of connexin-43 throughout the cumulus cells and connexin-37 at the oocyte surface. ///////////////////////// FSH induces the development of circadian clockwork in rat granulosa cells via a gap junction protein Cx43-dependent pathway. Chen H et al. The present study was designed to assess the relationship between gap junctions and the maturation of a clock system in rat granulosa cells stimulated by FSH. Immature and mature granulosa cells were prepared by puncturing the ovaries of diethylstilbestrol- and equine chorionic gonadotropin (eCG)-treated mouse Period2 (Per2)-dLuc reporter gene transgenic rats, respectively. Mature granulosa cells exposed to dexamethasone (DXM) synchronization displayed several Per2-dLuc oscillations and a rhythmic expression of clock genes. Intriguingly, we observed clear evidence that the FSH stimulation significantly increased the amplitude of Per2 oscillations in the granulosa cells, which was confirmed by the elevation of the Per2 and Rev-erba (Nr1d1) mRNA levels. FSH also induced a major phase-advance shift of Per2 oscillations. The mature granulosa cells cultured for 2 d with FSH expressed higher mRNA level of Per2, Rev-erba, Bmal1 (Arnt1), Lhcgr, and Cx43 (Gja1) compared to the immature granulosa cells. Consistently, our immunofluorescence results revealed abundant Cx43 protein in antral follicles stimulated with eCG, and weak or no fluorescence signal of Cx43 in primary and preantral follicles. Similar results were confirmed by western blotting analysis. Two gap junction blockers, lindane and carbenoxolone (CBX), significantly decreased the amplitude of Per2 oscillations, which further adhered significant decreases in Per2 and Rev-erba transcript levels. In addition, both lindane and CBX induced a clear phase-delay shift of Per2 oscillations. These findings suggest that FSH induces the development of the clock system by increasing the expression of Cx43. Phosphorylation of Serine Residues in the C-terminal Cytoplasmic Tail of Connexin43 Regulates Proliferation of Ovarian Granulosa Cells. Dyce PW et al. Connexin43 (Cx43) forms gap junctions that couple the granulosa cells of ovarian follicles. In Cx43 knockout mice, follicle growth is restricted as a result of impaired granulosa cell proliferation. We have used these mice to examine the importance of specific Cx43 phosphorylation sites in follicle growth. Serines at residues 255, 262, 279, and 282 are MAP kinase substrates that, when phosphorylated, reduce junctional conductance. Mutant forms of Cx43 were constructed with these serines replaced with amino acids that cannot be phosphorylated. These mutants were transduced into Cx43 knockout ovarian somatic cells that were combined with wild-type oocytes and grafted into immunocompromised female mice permitting follicle growth in vivo. Despite residues 255 or 262 being mutated to prevent their being phosphorylated, recombinant ovaries constructed with these mutants were able to rescue the null phenotype, restoring complete folliculogenesis. In contrast, Cx43 with serine to alanine mutations at both residues 279 and 282 or at all four residues failed to rescue folliculogenesis; the mutant molecules were largely confined to intracellular sites, with few gap junctions. Using an in vitro proliferation assay, we confirmed a decrease in proliferation of granulosa cells expressing the double mutant construct. These results indicate that Cx43 phosphorylation by MAP kinase at serines 279 and 282 occurs in granulosa cells of early follicles and that this is involved in regulating follicle development. Furger et al. (1996) determined that connexin 43 may play a role in the regulation of both follicular development and oocyte status. Intercellular communication provided via the gap junctions may play a role in coordinating the process of atresia was discovered by Wiesen et al. (1994). Regulation of gap junctions in porcine cumulus-oocyte complexes: Contributions of granulosa cell contact, gonadotropins and lipid rafts. Sasseville M et al. Gap-junctional communication (GJC) plays a central role in oocyte growth. However, little is known about the regulation of connexin 43 (Cx43)-based gap-junction channels in cumulus-oocyte complexes (COC) during in vitro maturation (IVM). We show that rupture of COC from mural granulosa cells up-regulates Cx43-mediated GJC and that gonadotropins signal GJC breakdown by recruiting Cx43 to lipid rafts when oocyte meiosis resumes. Oocyte calcein uptake through gap-junctions increases during early in vitro oocyte maturation and remains high until 18 hours, when it falls simultaneously with the oocyte germinal vesicle breakdown. Immunodetection of Cx43, and fluorescence recovery after photobleaching assays, revealed that the increase of GJC is independent of gonadotropins but requires RNA transcription, RNA polyadenylation and translation. GJC rupture, in contrast, is achieved by a gonadotropin-dependent mechanism involving recruitment of Cx43 to clustered lipid rafts. These results show that GJC up-regulation in COCs in in vitro culture is independent of gonadotropins and transcriptionally regulated. However, GJC breakdown is gonadotropin-dependent and mediated by the clustering of Cx43 in lipid raft microdomains. In conclusion, this study supports a functional role of lipid raft clustering of Cx43 in GJC breakdown in the COC during in vitro maturation. | ||||
| Expression regulated by | FSH, LH, Steroids, Growth Factors/ cytokines, wnt2, BMP15, vitamine D, retinoic acid , TGFb | ||||
| Comment | Effects of androgen on immunohistochemical localization of androgen receptor and Connexin 43 in mouse ovary. Yang M et al. (2015) Androgens have essential roles in the regulation of follicular development and female fertility. Androgen excess is the leading defect in polycystic ovary syndrome (PCOS) patients and involved in the ovarian dysfunction. The aim of this study was to elucidate the regarding regulatory role of androgen in the follicular development of female mouse. Immunohistochemical staining and Western blot analyses were performed to detect androgen receptor (AR) and Connexin 43 (Cx43) expression in ovaries from both control and testosterone-treated group mice. In this study, localizations of AR and Cx43 were dramatically altered in testosterone-treated mouse ovaries. In addition, AR expression was significantly increased, whereas Cx43 expression was markedly decreased after testosterone treatment. Alterations of AR and Cx43 expression by testosterone with concomitant reduction of MII oocytes. Overall, these results suggest the involvement of androgen in the regulation of AR and Cx43 localizations in mouse ovary. Alterations of AR and Cx43 expression by testosterone may affect normal folliculogenesis. Together these findings will enable us to begin understanding the important roles of AR and Cx43 actions in the regulation of follicular development, as well as providing insights into the role of AR and Cx43 actions in the androgen-associated reproductive diseases such as PCOS.////////////////// Transforming growth factor-β1 up-regulates connexin43 expression in human granulosa cells. Chen YC et al. (2015) Does transforming growth factor-β1 (TGF-β1) up-regulate connexin43 (Cx43) to promote cell-cell communication in human granulosa cells? TGF-β1 up-regulates Cx43 and increases gap junction intercellular communication activities (GJIC) in human granulosa cells, and this effect occurs via the activin receptor-like kinase (ALK)5-mediated Sma- and Mad-related protein (SMAD)2/3-SMAD4-dependent pathway. TGF-β1 and its receptors are expressed in human granulosa cells, and follicular fluid contains TGF-β1 protein. In human granulosa cells, Cx43 gap junctions play an important role in the development of follicles and oocytes. This is an experimental study which was performed over a 1-year period. Immortalized human granulosa cells (SVOG cells) and primary human granulosa-lutein cells obtained from women undergoing IVF in an academic research center were used as the study models. Cx43 mRNA and protein expression levels were examined after exposure of SVOG cells to recombinant human TGF-β1. An activin/TGF-β type I receptor inhibitor, SB431542, and small interfering RNAs targeting ALK4, ALK5, SMAD2, SMAD3 and SMAD4 were used to verify the specificity of the effects and to investigate the molecular mechanisms. Real-time-quantitative PCR and western blot analysis were used to detect the specific mRNA and protein levels, respectively. GJIC between SVOG cells were evaluated using a scrape loading and dye transfer assay. Results were analyzed by one-way analysis of variance. TGF-β1 treatment increased phosphorylation of SMAD2/3 (P < 0.0001) and up-regulated Cx43 mRNA and protein levels (P < 0.001) in SVOG cells and these stimulatory effects were abolished by the TGF-β type I receptor inhibitor SB431542. In addition, the up-regulatory effect of TGF-β1 on Cx43 expression (mRNA and protein) was confirmed in primary cultures of human granulosa-lutein cells (P < 0.05). The small interfering RNA-mediated knockdown of ALK5, but not ALK4, abolished the TGF-β1-induced phosphorylation of SMAD2/3 and the up-regulation of Cx43. Furthermore, knockdown of SMAD2/3 or the common SMAD, SMAD4, abolished the stimulatory effects of TGF-β1 on Cx43 expression in SVOG cells. The TGF-β1-induced up-regulation of Cx43 contributed to the increase of GJIC between SVOG cells (P < 0.001). The results of this study were generated from in vitro system and may not reflect the intra-ovarian microenvironment in vivo. Our studies represent the first comprehensive research of molecular mechanisms of TGF-β1 in the regulation of Cx43 expression and GJIC in human granulosa cells and demonstrate that TGF-β1 may play a crucial role in the local modulation of cell-cell communication. Deepening our understanding of the molecular determinants will offer important insights into ovarian physiology and lead to the development of potential therapeutic methods for fertility regulation. This research was supported by an operating grant from the Canadian Institutes of Health Research to P.C.K.L. There are no conflicts of interest to declare. NA.////////////////// A role for retinoids in human oocyte fertilization: regulation of connexin 43 by retinoic acid in cumulus granulosa cells. Best MW et al. (2015) Retinoids are essential for ovarian steroid production and oocyte maturation in mammals. Oocyte competency is known to positively correlate with efficient gap junction intercellular communication (GJIC) among granulosa cells in the cumulus-oocyte complex. Connexin 43 (Cx43) is the main subunit of gap junction channels in human cumulus granulosa cells (CGC) and is regulated by all-trans retinoic acid (ATRA) in other hormone responsive cell types. The objectives of this study were to quantify retinoid levels in human CGC obtained during IVF oocyte retrievals, to investigate the potential relationship between CGC ATRA levels and successful oocyte fertilization, and to determine the effects of ATRA on Cx43 protein expression in CGC. Results showed that CGC cultures actively metabolize retinol to produce ATRA. Grouped according to fertilization rate tertiles, mean ATRA levels were 2-fold higher in pooled CGC from women in the highest versus the lowest tertile (P < 0.05). ATRA induced a rapid dephosphorylation of Cx43 in CGC and granulosa cell line (KGN) cultures resulting in a >2-fold increase in the expression of the functional non-phosphorylated (P0) species (P < 0.02). Similar enhancement of P0 by ATRA was shown in CGC and KGN cultures co-treated with LH or hCG which, by themselves, enhanced the protein levels of Cx43 without altering its phosphorylation profile. Correspondingly, the combination of ATRA+hCG treatment of KGN caused a significant increase in GJIC compared with single agent treatments (P < 0.025) and a doubling of GJIC from that seen in untreated cells (P < 0.01). These findings indicate that CGC are a primary site of retinoid uptake and ATRA biosynthesis. Regulation of Cx43 by ATRA may serve an important role in folliculogenesis, development of oocyte competency, and successful fertilization by increasing GJIC in CGC.////////////////// //////////////////,25-Dihydroxyvitamin D3 increases testosterone-induced 17beta-estradiol secretion and reverses testosterone-reduced connexin 43 in rat granulosa cells. Lee CT 2014 et al. BACKGROUND Aromatase converts testosterone into 17beta-estradiol in granulosa cells, and the converted 17beta-estradiol contributes to follicular maturation. Additionally, excessive testosterone inhibits aromatase activity, which can lead to concerns regarding polycystic ovary syndrome (PCOS). Generally, 1,25-dihydroxyvitamin D3 (1,25D3) supplements help to improve the symptoms of PCOS patients who exhibit low blood levels of 1,25D3. Therefore, this study investigated the interaction effects of 1,25D3 and testosterone on estrogenesis and intercellular connections in rat granulosa cells. METHODS Primary cultures of granulosa cells were treated with testosterone or testosterone plus 1,25D3, or pre-treated with a calcium channel blocker or calcium chelator. Cell lysates were subjected to western blot analysis to determine protein and phosphorylation levels, and 17beta-estradiol secretion was examined using a radioimmunoassay technique. Cell viability was evaluated by MTT reduction assay. Connexin 43 (Cx43) mRNA and protein expression levels were assessed by qRT-PCR, western blot, and immunocytochemistry. RESULTS Testosterone treatment (0.1 and 1 microg/mL) increased aromatase expression and 17beta-estradiol secretion, and the addition of 1,25D3 attenuated testosterone (1 microg/mL)-induced aromatase expression but improved testosterone-induced 17beta-estradiol secretion. Furthermore, testosterone-induced aromatase phosphotyrosine levels increased at 10 min, 30 min and 1 h, whereas 1,25D3 increased the longevity of the testosterone effect to 6 h and 24 h. Within 18-24 h of treatment, 1,25D3 markedly enhanced testosterone-induced 17beta-estradiol secretion. Additionally, pre-treatment with a calcium channel blocker nifedipine or an intracellular calcium chelator BAPTA-AM reduced 1,25D3 and testosterone-induced 17beta-estradiol secretion. Groups that underwent testosterone treatment exhibited significantly increased estradiol receptor beta expression levels, which were not affected by 1,25D3. Neither testosterone nor 1,25D3 altered 1,25D3 receptor expression. Finally, at high doses of testosterone, Cx43 protein expression was decreased in granulosa cells, and this effect was reversed by co-treatment with 1,25D3. CONCLUSIONS These data suggest that 1,25D3 potentially increases testosterone-induced 17beta-estradiol secretion by regulating aromatase phosphotyrosine levels, and calcium increase is involved in both 1,25D3 and testosterone-induced 17beta-estradiol secretion. 1,25D3 reverses the inhibitory effect of testosterone on Cx43 expression in granulosa cells. ///////////////////////// Oocyte-derived BMP15 but not GDF9 down-regulates connexin43 expression and decreases gap junction intercellular communication activity in immortalized human granulosa cells. Chang HM 2014 et al. In the ovary, connexin-coupled gap junctions in granulosa cells play crucial roles in follicular and oocyte development as well as in corpus luteum formation. Our previous work has shown that theca cell-derived bone morphogenetic protein (BMP)4 and BMP7 decrease gap junction intercellular communication (GJIC) activity via the down-regulation of connexin43 (Cx43) expression in immortalized human granulosa cells. However, the effects of oocyte-derived growth factors on Cx43 expression remain to be elucidated. The present study was designed to investigate the effects of oocyte-derived growth differentiation factor (GDF)9 and BMP15 on the expression of Cx43 in a human granulosa cell line, SVOG. We also examined the effect relative to GJIC activity and investigated the potential mechanisms of action. In SVOG cells, treatment with BMP15 but not GDF9 significantly decreased Cx43 mRNA and protein levels and GJIC activity. These suppressive effects, along with the induction of Smad1/5/8 phosphorylation, were attenuated by co-treatment with a BMP type I receptor inhibitor, dorsomorphin. Furthermore, knockdown of the central component of the TGF-?superfamily signaling pathway, Smad4, using small interfering RNA reversed the suppressive effects of BMP15 on Cx43 expression and GJIC activity. The suppressive effects of BMP15 on Cx43 expression were further confirmed in primary human granulosa-lutein cells obtained from infertile patients undergoing an in vitro fertilization procedure. These findings suggest that oocyte-derived BMP15 decreases GJIC activity between human granulosa cells by down-regulating Cx43 expression, most likely via a Smad-dependent signaling pathway. ///////////////////////// The Canonical WNT2 Pathway and FSH Interact to Regulate Gap Junction Assembly in Mouse Granulosa Cells. Wang HX 2013 et al. WNTs are extracellular signaling molecules that exert their actions through receptors of the frizzled (FZD) family. Previous work indicated that WNT2 regulates cell proliferation in mouse granulosa cells acting through CTNNB1 (beta-catenin), a key component in canonical WNT signaling. In other cells, WNT signaling has been shown to regulate expression of connexin43 (CX43), a gap junction protein, as well as gap junction assembly. Since previous work had demonstrated that CX43 is also essential in ovarian follicle development, the objective of this study was to determine if WNT2 regulates CX43 expression and/or gap junctional intercellular communication (GJIC) in granulosa cells. WNT2 knockdown via siRNA markedly reduced CX43 expression and GJIC. CX43 expression, the extent of CX43-containing gap junctional membrane, and GJIC were also reduced by CTNNB1 transient knockdown. CTNNB1 is mainly localized in the membranes between granulosa cells but disappeared from this location after WNT2 knockdown. Furthermore, CTNNB1 knockdown interfered with the ability of FSH to promote the mobilization of CX43 into gap junctions. We propose that the WNT2/CTNNB1 pathway regulates CX43 expression and GJIC in granulosa cells by modulating CTNNB1 stability and localization in adherens junctions, and that this is essential for FSH stimulation of GJIC. ///////////////////////// Granot et al. (1994) showed that Phosphorylation and expression of connexin-43 ovarian gap junction protein are regulated by luteinizing hormone. Kalma Y, et al reported that Luteinizing hormone-induced connexin 43 down-regulation is mediated by inhibition of translation. Luteinizing hormone causes MAP kinase-dependent phosphorylation and closure of connexin 43 gap junctions in mouse ovarian follicles: one of two paths to meiotic resumption. Norris RP et al. Luteinizing hormone (LH) acts on ovarian follicles to reinitiate meiosis in prophase-arrested mammalian oocytes, and this has been proposed to occur by interruption of a meioisis-inhibitory signal that is transmitted through gap junctions into the oocyte from the somatic cells that surround it. To investigate this idea, we microinjected fluorescent tracers into live antral follicle-enclosed mouse oocytes, and we demonstrate for the first time that LH causes a decrease in the gap junction permeability between the somatic cells, prior to nuclear envelope breakdown (NEBD). The decreased permeability results from the MAP kinase-dependent phosphorylation of connexin 43 on serines 255, 262 and 279/282. We then tested whether the inhibition of gap junction communication was sufficient and necessary for the reinitiation of meiosis. Inhibitors that reduced gap junction permeability caused NEBD, but an inhibitor of MAP kinase activation that blocked gap junction closure in response to LH did not prevent NEBD. Thus, both MAP kinase-dependent gap junction closure and another redundant pathway function in parallel to ensure that meiosis resumes in response to LH. The coordinated function of the different compartments of the follicle, the oocyte and the somatic cumulus/granulosa cells, is enabled by the presence of a network of cell-to-cell communication generated by gap junctions. Connexin 43 (Cx43) is the most abundant gap junction protein expressed by the ovarian follicle. The expression of Cx43 is subjected to the control of gonadotropins as follows: FSH up-regulates, whereas LH down-regulates its levels. The aim of this study was to explore the mechanism by which LH reduces the levels of Cx43 and to identify the signal transduction pathway involved in this process. The effect of LH was studied in vitro using isolated intact ovarian follicles. The possible mediators of LH-induced Cx43 down-regulation were examined by incubating the follicles with LH in the presence or absence of inhibitors of protein kinase A (PKA) and of MAPK signaling pathways. Our experiments revealed a 3-h half-life of Cx43 in both control and LH-treated follicles, suggesting that LH did not affect the rate of Cx43 degradation. We further demonstrated that the level of Cx43 mRNA was not significantly influenced by this gonadotropin. However, upon LH administration, (35)S-methionine incorporation into Cx43 protein was remarkably reduced. The LH-induced arrest of Cx43 synthesis was counteracted by inhibitors of both the PKA and the MAPK cascades. We show herein that LH inhibits Cx43 expression by reducing its rate of translation and that this effect is mediated by both PKA and MAPK. Identification of Down-regulated Messenger RNAs in Bovine Granulosa Cells of Dominant Follicles Following Stimulation with Human Chorionic Gonadotropin Ndiaye K, et al . Molecular determinants and mechanisms involved in ovarian follicular growth, ovulation and luteinization are not well understood. The objective of this study was to identify genes expressed in bovine granulosa cells (GC) of dominant follicles (DF) and down-regulated after hCG-induced ovulation, using the suppression subtractive hybridization (SSH). GC were collected from DF at day 5 of the estrous cycle and from ovulatory follicles (OF) obtained 23 h following injection of hCG. A subtracted cDNA library (DF-OF) was generated and screened using unsubtracted (DF, OF) and subtracted (DF-OF, OF-DF) cDNAs as complex (32)P-probes. A total of 32 non-redundant cDNAs were identified: 23 cDNAs matched with sequences of known biological function and 9 cDNAs with complete or partial sequences of undefined biological function. Detection of genes known to be down-regulated during the periovulatory period in the bovine species, such as CPD, CYP11A1, CYP19A1, FSHR, LRP8/ApoER2 and SERPINE2, validated the physiological model and analytical techniques used. For a subset of genes such as ARFGAP3, CYP11A1, CYP19A1, FSHR, FST, GJA1, IDH3, INHBA, LHCGR, LHCGR lacking exon 10, PRC1, PRG1, RPA2, SCD and TRIB2, gene expression profiles were compared by virtual Northern blot or reverse transcriptase-polymerase chain reaction from follicles obtained at different developmental stages. Results confirmed a down-regulation of the respective mRNAs in GC of OF compared with that of DF. We conclude that we have identified novel genes that are down-regulated by hCG in bovine GC of DF during the periovulatory period, which may contribute to follicular growth, ovulation and/or luteinization. Gene expression decreased. Luteinization of porcine preovulatory follicles leads to systematic changes in follicular gene expression. Agca C et al. The LH surge initiates the luteinization of preovulatory follicles and causes hormonal and structural changes that ultimately lead to ovulation and the formation of corpora lutea. The objective of the study was to examine gene expression in ovarian follicles (n = 11) collected from pigs (Sus scrofa domestica) approaching estrus (estrogenic preovulatory follicle; n = 6 follicles from two sows) and in ovarian follicles collected from pigs on the second day of estrus (preovulatory follicles that were luteinized but had not ovulated; n = 5 follicles from two sows). The follicular status within each follicle was confirmed by follicular fluid analyses of estradiol and progesterone ratios. Microarrays were made from expressed sequence tags that were isolated from cDNA libraries of porcine ovary. Gene expression was measured by hybridization of fluorescently labeled cDNA (preovulatory estrogenic or -luteinized) to the microarray. Microarray analyses detected 107 and 43 genes whose expression was decreased or increased (respectively) during the transition from preovulatory estrogenic to -luteinized (P<0.01). Cells within preovulatory estrogenic follicles had a gene-expression profile of proliferative and metabolically active cells that were responding to oxidative stress. Cells within preovulatory luteinized follicles had a gene-expression profile of nonproliferative and migratory cells with angiogenic properties. Approximately, 40% of the discovered genes had unknown function. | ||||
| Ovarian localization | Oocyte, Cumulus, Granulosa | ||||
| Comment | Furger et al. (1996) found that numerous gap junctions exist between granulosa cells, between cumulus cells and between cumulus cells and the oocyte. Their results show that human granulosa cells in culture exhibited functional gap junctions. Connexin 43 was present and the permeability of the gap junctions was up-regulated by cyclic AMP, an important modulator of human granulosa cell function. | ||||
| Follicle stages | Secondary, Antral, Preovulatory | ||||
| Comment | |||||
| Phenotypes |
PCO (polycystic ovarian syndrome) |
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| Mutations |
4 mutations
Species: mouse
Species: mouse
Species: mouse
Species: mouse
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| Genomic Region | show genomic region | ||||
| Phenotypes and GWAS | show phenotypes and GWAS | ||||
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| created: | Dec. 13, 1999, midnight | by: |
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| last update: | June 23, 2020, 11:23 a.m. | by: | hsueh email: |
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