Connexin hemichannels and cell death as measures of bovine COC vitrification success

in Reproduction
Correspondence should be addressed to K J Szymańska or L Leybaert; Email: katarzyna.jsv@gmail.com or luc.leybaert@ugent.be
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Vitrification of immature germinal vesicle-stage oocytes is a promising method in assisted reproduction but is associated with reduced developmental potential and low birth rates. Cumulus-oocyte complexes (COCs) express several connexins that form hexameric hemichannels, which interact head to head to create a gap junction or exist as unopposed free hemichannels. The latter are normally closed but open under stress conditions and may exert detrimental effects. We determined whether minimizing hemichannel opening and cell death during vitrification could improve COC quality. Bovine immature COCs underwent vitrification, storage and warming, followed by dye uptake to assess hemichannel opening and TUNEL staining to detect cell death. Based on these scores, we optimized the procedure by tuning the equilibration time, temperature, cryoprotectant concentration and extracellular Ca2+ concentration and assessed its impact on maturation, cleavage and blastocyst formation after parthenogenetic activation. We found that the major stressor resides in the cooling/warming phase of the vitrification procedure and observed that hemichannel opening and cell death in cumulus cells measure different aspects of cell stress. Optimization of the hemichannel and cell death readouts demonstrated that combined minimal hemichannel opening/cell death gave the highest cleavage rates but had no effect on maturation and blastocyst formation. Neither hemichannel nor cell death optimization performed better than the non-optimized protocol, leading to the conclusion that cell stress factors other than those detected by hemichannel dye uptake or TUNEL positivity are involved.

 

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    Overview of the experimental design and primary vitrification and warming protocol. Immature oocytes were collected from ovaries from the slaughterhouse. The non-vitrified control group was kept in the basic medium (BM) medium during the vitrification time, and then directly divided into groups: (1) hemichannel dye uptake assay, (2) cell death TUNEL assay or (3) for development test. For the latter, COCs were in vitro matured for 26 h and then parthenogenetically activated with 5 µM ionomycin. Maturation was based on Hoechst staining, cleavage was assessed after 45 h of culture. Vitrified groups were vitrified, stored in liquid nitrogen (LN2) and warmed. The process of vitrification and warming is described in detail in the gray shaded panel. The primary protocol consisted of two equilibration steps: for equilibration STEP 1, COCs were kept in a 50 μL droplet of BM solution for 1 min. This droplet was then merged with an adjacent 50 μL droplet of ES 1 for 2 min; then again merged with a third 50 μL ES 1 droplet for 2 min. Next, oocytes were transferred to a fourth ES 1 droplet for 10 min. During STEP 2 oocytes were sequentially transferred to three 25 µL droplets of ES2 solution, for 5 s, 5 s and 10 s, respectively. The fourth drop was used to load 8–9 COCs on the HSV straw – STEP 3. Then, oocytes were loaded on the straws (STEP 3), submerged in LN2 (STEP 4) and stored for 12 h (STEP 5). Warming consisted of transfer to a 500 μL WS droplet of 39°C for 1 min (STEP 6) and subsequent washing in the 50 µL droplets of decreasing sucrose concentrations: 2 × 2 min in DS 1 and 3 × 3 min in DS 2 (STEP 7). All the steps were performed at RT (24–27°C) except the first droplet of the warming step (39°C). After warming COCs were placed in the BM medium until all the oocytes from each group were thawed and then were divided into groups based on the performed experiment. The figure was created using Servier Medical ART.

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    Stepwise guide through protocol optimization process. 1Time in the last drop of equilibration 1 – STEP 1. 2RT–RT during the whole procedure; RT/39°C – RT throughout except 1st drop of warming step; 39°C–39°C during the whole procedure. 3Low: 5% DMSO/5% EG in STEP 1; 7.5% DMSO/7.5% EG in STEP 2; Standard: 7.5% DMSO/7.5% EG in STEP 1; 15% DMSO/15% EG in STEP 2; High: 10% DMSO/10% EG in STEP 1; 20% DMSO/20% EG in STEP 2. 4Zero Ca2+: 4 mM EGTA added to the Ca2+ containing solution; Low Ca2+: 0.65 mM EGTA added to the Ca2+ containing solution, giving 0.61 mM free Ca2+; Normal 1.26 mM Ca2+.

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    Localization of CX43 in the membrane of cumulus cells and CX37 in oocytes. (A and B) Confocal images show the presence of CX43 (A) and CX37 (B) in bovine COCs in green. Both connexins are present in transzonal projections and are indicated by white arrows. Blue represents nuclear Hoechst staining and red shows transzonal projections by F-actin staining with phalloidin-rhodamine. Scale bar measures 50 µm.

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    Vitrification triggers hemichannel opening measured with PI dye uptake (A) and apoptotic cell death, measured by TUNEL staining (B) in COCs. COCs were vitrified and warmed with or without peptide Gap26 and afterward, either PI dye uptake assay or TUNEL staining were performed. (A) Gap26 significantly blocked PI uptake triggered by vitrification. The positive control group was exposed to medium without Ca2+ and Mg2+. Oocytes were never PI-positive. (B) Cryopreservation significantly increased the number of TUNEL-positive cells which is only partially inhibited by Gap26. Oocytes were never TUNEL positive. The graphs illustrate summary data of the percentage of the PI-positive cell area or TUNEL-positive area relative to the total nuclei (Hoechst) area per COC. All data are presented as mean ± s.e.m., results were analyzed with a non-parametric ANOVA, Kruskal–Wallis test; ***P < 0.001.

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    Hemichannel-mediated dye uptake after each step of vitrification process. Dye uptake in COCs was performed after each step of vitrification (equilibration 1 – STEP 1, two equilibration steps 1 and 2 – STEP 1 and 2, equilibration 1 and 2 and loading on a vitrification straw – STEP 1, 2 and 3, two equilibration steps 1 and 2, loading on a straw, warming and wash-out procedure STEP 1, 2, 3, 6 and 7 or full vitrification procedure (includes cooling in LN2) – vitrified to assess the trigger for hemichannel opening. HC opening was observed only after full vitrification procedure (LN2 cooling and warming)). Exposure to media with a high concentration of CPs, warming from RT to 39°C or mechanical stimulation during handling did not increase HC opening. The graph shows summary data of the percentage of the PI-positive area relative to the total nuclei (Hoechst) area per COC. Data are presented as mean ± s.e.m., results were analyzed with a non-parametric ANOVA, Kruskal–Wallis test; ***P < 0.001 – difference vs all other groups.

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    First step of protocol optimization: influence of the equilibration time on the probability of hemichannel opening (A) and apoptotic cell death (B). Testing the duration of the equilibration in the last equilibration drop. (A) 20 min equilibration in the last Equilibration Solution 1 droplet of STEP 2 showed the lowest dye uptake and is further referred as protocol A. Oocytes were never PI-positive. #Difference of control vs vitrified (P < 0.05 for non-vitrified vs vitrified 20 min; P < 0.001 for non-vitrified vs 5, 10 and 30 min), *difference vs vitrified 20 min (P < 0.001 for 20 vs 5; P < 0.05 for 20 vs 10 and 20 vs 30); (B) 30 min equilibration in the last droplet of STEP 2 showed the lowest TUNEL positivity and this condition is further referred as protocol B. Oocytes were never TUNEL-positive. The graphs illustrate summary data of the percentage of the PI-positive cell area or TUNEL-positive area relative to the total nuclei (Hoechst) area per COC. ***Difference of 30 min condition vs all vitrified groups P < 0.001. All data are presented as mean ± s.e.m., results were analyzed with a non-parametric ANOVA, Kruskal–Wallis test.

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    Second step of optimization process: influence of temperature on the probability of hemichannel opening (A1–B1) and apoptotic cell death (A2–B2). We tested the response of hemichannels to different vitrification temperature. COCs were vitrified with different optimized protocols (A: 20 min residence in the last equilibration drop and B: 30 min residence in the last equilibration drop). Best vitrification conditions were chosen based on the results of the previous experiment and are indicated with a pattern for lowest HC opening and for the lowest apoptosis (A1–A2 shows results of protocol A, and B1–B2 shows results of protocol B). The graphs illustrate summary data of the percentage of the PI-positive cell area or TUNEL-positive area relative to the total nuclei (Hoechst) area per COC. All data are presented as mean ± s.e.m., results were analyzed with a non-parametric ANOVA, Kruskal–Wallis test; ###P < 0.001, #P < 0.05 difference of control vs vitrified; *P < 0.05, ***P < 0.001 difference vs other vitrified groups.

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    Third step of protocol optimization: influence of the CP concentration (equilibration steps) on the probability of hemichannel opening (A1–B1) and apoptotic cell death (A2–B2). COCs were vitrified with different optimized protocols (A: 20 min residence in the last equilibration drop, RT throughout the process except the 39°C warming drop and B: 30 min residence in the last equilibration drop and RT throughout the procedure). Best vitrification conditions were chosen based on the results of the previous experiment and are indicated with a pattern or a black box for lowest HC opening and for the lowest apoptosis (A1–A2 shows results of protocol A, and B1–B2 shows results of protocol B). The graphs illustrate summary data of the percentage of the PI-positive cell area or TUNEL-positive area relative to the total nuclei (Hoechst) area per COC. All data are presented as mean ± s.e.m., results were analyzed with a non-parametric ANOVA Kruskal–Wallis test; ***P < 0.001 in A1 and B1 graph show difference of low CP concentration vs all other groups.

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    Fourth step of protocol optimization: influence of the extracellular Ca2+ concentration on the probability of hemichannel opening (A1–B1) and apoptotic cell death (A2–B2). COCs were vitrified with different optimized protocols and different extracellular Ca2+ concentrations of the media were tested (A: 20 min residence in the last equilibration drop, RT throughout the process except the 39°C warming drop, standard CP concentration: 7.5% DMSO/7.5% EG in STEP 1 and 15% DMSO/15% EG in STEP 2 and B: 30 min residence in the last equilibration drop and RT throughout, high concentration: 10% DMSO/10% EG in STEP 1 and 20% DMSO/20% EG in STEP 2). Best vitrification conditions were chosen based on the previous results and are indicated with a pattern or a black box for lowest HC opening and for the lowest TUNEL positivity (A1–A2 shows results of protocol A, and B1–B2 shows results of protocol B). The graphs illustrate summary data of the percentage of the PI-positive cell area or TUNEL-positive area relative to the total nuclei (Hoechst) area per COC. All data are presented as mean ± s.e.m., results were analyzed with a non-parametric ANOVA, Kruskal–Wallis test; ###P < 0.001 difference of control vs vitrified; *P < 0.05, ***P < 0.001.

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