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Connexin expression and gap junctional coupling in human cumulus cells: contribution to embryo quality.

Wang HX, Tong D, El-Gehani F, Tekpetey FR, Kidder GM - J. Cell. Mol. Med. (2008)

Bottom Line: Multiple connexins (Cx), the subunits of gap junction channels, have been found within ovarian follicles in several species but little is known about the connexins in human follicles.The strength of gap junctional conductance varied between patients and was significantly and positively correlated with Cx43 level, but neither was correlated with patient age.Interestingly, Cx43 level and intercellular conductance were positively correlated with embryo quality as judged by cleavage rate and morphology, and were significantly higher in patients who became pregnant than in those who did not.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, ON, Canada.

ABSTRACT
Gap junctional coupling among cumulus cells is important for oogenesis since its deficiency in mice leads to impaired folliculogenesis. Multiple connexins (Cx), the subunits of gap junction channels, have been found within ovarian follicles in several species but little is known about the connexins in human follicles. The aim of this study was to determine which connexins contribute to gap junctions in human cumulus cells and to explore the possible relationship between connexin expression and pregnancy outcome from in vitro fertilization (IVF). Cumulus cells were obtained from IVF patients undergoing intra-cytoplasmic sperm injection (ICSI). Connexin expression was examined by RT-PCR and confocal microscopy. Cx43 was quantified by immunoblotting and gap junctional coupling was measured by patch-clamp electrophysiology. All but 5 of 20 connexin mRNAs were detected. Of the connexin proteins detected, Cx43 forms numerous gap junction-like plaques but Cx26, Cx30, Cx30.3, Cx32 and Cx40 appeared to be restricted to the cytoplasm. The strength of gap junctional conductance varied between patients and was significantly and positively correlated with Cx43 level, but neither was correlated with patient age. Interestingly, Cx43 level and intercellular conductance were positively correlated with embryo quality as judged by cleavage rate and morphology, and were significantly higher in patients who became pregnant than in those who did not. Thus, despite the presence of multiple connexins, Cx43 is a major contributor to gap junctions in human cumulus cells and its expression level may influence pregnancy outcome after ICSI.

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Relationship between Cx43 level and clinical data. (A) Comparison of Cx43 level, determined with reference to vimentin, and clinical outcome. Oocytes were evaluated for nuclear maturity and graded as metaphase II (MII), metaphase I, or prophase I. Fertilization was considered to have occurred when two clear pro-nuclei were present after 16–18 hrs insemination. Embryo transferability was estimated on day 3 after insemination according to a grading system, with embryos having more than five blastomeres and good morphology being considered as transferable. Implantation rate is the ratio of foetuses (determined by day 40 ultrasound) to embryos transferred. (B) Comparison of Cx43 level, determined with reference to GAPDH, and clinical outcome. (C) Confirmation that cumulus cell vimentin and GAPDH levels are comparable between pregnant and non-pregnant patients. Lysis buffer volumes were adjusted to account for differing cell numbers obtained from different patients. The mean vimentin or GAPDH level for each sample was determined from at least two measurements. (D) Relationship between Cx43 level and pregnancy outcome (determined by day 40 ultrasound): difference between cumulus cell sample groups, partitioned by pregnancy outcome, in relative level of Cx43 (relative band intensity as determined by reference to either vimentin or GAPDH). In all cases, different letters above the bars indicate significant differences (P< 0.05 by one-way anova). In this study 35 patients became pregnant while 46 did not.
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fig08: Relationship between Cx43 level and clinical data. (A) Comparison of Cx43 level, determined with reference to vimentin, and clinical outcome. Oocytes were evaluated for nuclear maturity and graded as metaphase II (MII), metaphase I, or prophase I. Fertilization was considered to have occurred when two clear pro-nuclei were present after 16–18 hrs insemination. Embryo transferability was estimated on day 3 after insemination according to a grading system, with embryos having more than five blastomeres and good morphology being considered as transferable. Implantation rate is the ratio of foetuses (determined by day 40 ultrasound) to embryos transferred. (B) Comparison of Cx43 level, determined with reference to GAPDH, and clinical outcome. (C) Confirmation that cumulus cell vimentin and GAPDH levels are comparable between pregnant and non-pregnant patients. Lysis buffer volumes were adjusted to account for differing cell numbers obtained from different patients. The mean vimentin or GAPDH level for each sample was determined from at least two measurements. (D) Relationship between Cx43 level and pregnancy outcome (determined by day 40 ultrasound): difference between cumulus cell sample groups, partitioned by pregnancy outcome, in relative level of Cx43 (relative band intensity as determined by reference to either vimentin or GAPDH). In all cases, different letters above the bars indicate significant differences (P< 0.05 by one-way anova). In this study 35 patients became pregnant while 46 did not.

Mentions: Since Cx43 level in cumulus cells correlates with gap junctional conductance, and given the demonstrated importance of the latter for folliculogenesis in mutant mice, we explored the possibility that clinical outcome from ICSI is related to Cx43 level. Patients were partitioned into two groups based on whether their cumulus cell Cx43 expression was above or below the mean for all patients (Fig. 8A and B). Oocyte maturation (MII) rate and fertilization rate in the high Cx43 group were not different from those in the low Cx43 group, although the MII rate in the high Cx43 group was slightly higher than that in the low Cx43 group using GAPDH as the standard. On the other hand, higher Cx43 level was significantly associated with higher transferable rate and implantation rate. Comparison of vimentin and GAPDH band intensities on the Western blots did not reveal any difference between patients who became pregnant and those who did not (Fig. 8C). Pregnancy outcome was then used to partition the 81 patients for which we had determined relative Cx43 level into two groups, and the mean relative intensity of the Cx43 band, normalized to vimentin or GAPDH, for the two groups was compared. Regardless of which protein was used as the internal standard, the mean relative Cx43 level was significantly higher for samples taken from patients who became pregnant (P< 0.01) (Fig. 8D). Correspondingly, for vimentin-normalized samples, the pregnancy rate in the higher Cx43 group (more than the mean) was 57.1% while the pregnancy rate in the lower Cx43 group (less than the mean) was only 28.2%. For GAPDH-normalized samples the corresponding difference was 71.9%versus 24.5%.


Connexin expression and gap junctional coupling in human cumulus cells: contribution to embryo quality.

Wang HX, Tong D, El-Gehani F, Tekpetey FR, Kidder GM - J. Cell. Mol. Med. (2008)

Relationship between Cx43 level and clinical data. (A) Comparison of Cx43 level, determined with reference to vimentin, and clinical outcome. Oocytes were evaluated for nuclear maturity and graded as metaphase II (MII), metaphase I, or prophase I. Fertilization was considered to have occurred when two clear pro-nuclei were present after 16–18 hrs insemination. Embryo transferability was estimated on day 3 after insemination according to a grading system, with embryos having more than five blastomeres and good morphology being considered as transferable. Implantation rate is the ratio of foetuses (determined by day 40 ultrasound) to embryos transferred. (B) Comparison of Cx43 level, determined with reference to GAPDH, and clinical outcome. (C) Confirmation that cumulus cell vimentin and GAPDH levels are comparable between pregnant and non-pregnant patients. Lysis buffer volumes were adjusted to account for differing cell numbers obtained from different patients. The mean vimentin or GAPDH level for each sample was determined from at least two measurements. (D) Relationship between Cx43 level and pregnancy outcome (determined by day 40 ultrasound): difference between cumulus cell sample groups, partitioned by pregnancy outcome, in relative level of Cx43 (relative band intensity as determined by reference to either vimentin or GAPDH). In all cases, different letters above the bars indicate significant differences (P< 0.05 by one-way anova). In this study 35 patients became pregnant while 46 did not.
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Related In: Results  -  Collection

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fig08: Relationship between Cx43 level and clinical data. (A) Comparison of Cx43 level, determined with reference to vimentin, and clinical outcome. Oocytes were evaluated for nuclear maturity and graded as metaphase II (MII), metaphase I, or prophase I. Fertilization was considered to have occurred when two clear pro-nuclei were present after 16–18 hrs insemination. Embryo transferability was estimated on day 3 after insemination according to a grading system, with embryos having more than five blastomeres and good morphology being considered as transferable. Implantation rate is the ratio of foetuses (determined by day 40 ultrasound) to embryos transferred. (B) Comparison of Cx43 level, determined with reference to GAPDH, and clinical outcome. (C) Confirmation that cumulus cell vimentin and GAPDH levels are comparable between pregnant and non-pregnant patients. Lysis buffer volumes were adjusted to account for differing cell numbers obtained from different patients. The mean vimentin or GAPDH level for each sample was determined from at least two measurements. (D) Relationship between Cx43 level and pregnancy outcome (determined by day 40 ultrasound): difference between cumulus cell sample groups, partitioned by pregnancy outcome, in relative level of Cx43 (relative band intensity as determined by reference to either vimentin or GAPDH). In all cases, different letters above the bars indicate significant differences (P< 0.05 by one-way anova). In this study 35 patients became pregnant while 46 did not.
Mentions: Since Cx43 level in cumulus cells correlates with gap junctional conductance, and given the demonstrated importance of the latter for folliculogenesis in mutant mice, we explored the possibility that clinical outcome from ICSI is related to Cx43 level. Patients were partitioned into two groups based on whether their cumulus cell Cx43 expression was above or below the mean for all patients (Fig. 8A and B). Oocyte maturation (MII) rate and fertilization rate in the high Cx43 group were not different from those in the low Cx43 group, although the MII rate in the high Cx43 group was slightly higher than that in the low Cx43 group using GAPDH as the standard. On the other hand, higher Cx43 level was significantly associated with higher transferable rate and implantation rate. Comparison of vimentin and GAPDH band intensities on the Western blots did not reveal any difference between patients who became pregnant and those who did not (Fig. 8C). Pregnancy outcome was then used to partition the 81 patients for which we had determined relative Cx43 level into two groups, and the mean relative intensity of the Cx43 band, normalized to vimentin or GAPDH, for the two groups was compared. Regardless of which protein was used as the internal standard, the mean relative Cx43 level was significantly higher for samples taken from patients who became pregnant (P< 0.01) (Fig. 8D). Correspondingly, for vimentin-normalized samples, the pregnancy rate in the higher Cx43 group (more than the mean) was 57.1% while the pregnancy rate in the lower Cx43 group (less than the mean) was only 28.2%. For GAPDH-normalized samples the corresponding difference was 71.9%versus 24.5%.

Bottom Line: Multiple connexins (Cx), the subunits of gap junction channels, have been found within ovarian follicles in several species but little is known about the connexins in human follicles.The strength of gap junctional conductance varied between patients and was significantly and positively correlated with Cx43 level, but neither was correlated with patient age.Interestingly, Cx43 level and intercellular conductance were positively correlated with embryo quality as judged by cleavage rate and morphology, and were significantly higher in patients who became pregnant than in those who did not.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, ON, Canada.

ABSTRACT
Gap junctional coupling among cumulus cells is important for oogenesis since its deficiency in mice leads to impaired folliculogenesis. Multiple connexins (Cx), the subunits of gap junction channels, have been found within ovarian follicles in several species but little is known about the connexins in human follicles. The aim of this study was to determine which connexins contribute to gap junctions in human cumulus cells and to explore the possible relationship between connexin expression and pregnancy outcome from in vitro fertilization (IVF). Cumulus cells were obtained from IVF patients undergoing intra-cytoplasmic sperm injection (ICSI). Connexin expression was examined by RT-PCR and confocal microscopy. Cx43 was quantified by immunoblotting and gap junctional coupling was measured by patch-clamp electrophysiology. All but 5 of 20 connexin mRNAs were detected. Of the connexin proteins detected, Cx43 forms numerous gap junction-like plaques but Cx26, Cx30, Cx30.3, Cx32 and Cx40 appeared to be restricted to the cytoplasm. The strength of gap junctional conductance varied between patients and was significantly and positively correlated with Cx43 level, but neither was correlated with patient age. Interestingly, Cx43 level and intercellular conductance were positively correlated with embryo quality as judged by cleavage rate and morphology, and were significantly higher in patients who became pregnant than in those who did not. Thus, despite the presence of multiple connexins, Cx43 is a major contributor to gap junctions in human cumulus cells and its expression level may influence pregnancy outcome after ICSI.

Show MeSH
Related in: MedlinePlus