Figures and data
Analysis of embryo development corresponding to C1/C3 and C1/C4 DKO males
Sperm migration through the female reproductive tract.
A. Representative diagram showing sperm within the different regions of the female reproductive tract. UTJ, utero-tubal junction. Superovulated females were mated with Acrosine-GFP control or C1/C3 DKO males and, 4 hours after mating, sperm were analyzed inside the tract by fluorescence microscopy. B. Bright field (upper panel) and fluorescence (lower panel) images of the reproductive tract of a female mated with a control male (x40). C. Bright field (upper panel) and fluorescence (lower panel) images of the reproductive tract of a female mated with C1/C3 DKO male (x40). Figures are representative of at least 3 independent experiments.
In vivo fertilization and embryo development
A. Control and C1/C3 DKO males were mated with superovulated females and the percentage of fertilized eggs recovered from the ampulla 4 hours after mating was evaluated. Eggs were considered fertilized when they reached the two-cell embryo stage 24 h after in vitro incubation. B. Two-cell embryos from A were incubated in vitro for an additional 3 days, and the percentage reaching the blastocyst stage was determined. C. Control or C1/C3 DKO cauda epididymal sperm were inseminated into the uterus of superovulated females. After 15 h, eggs were recovered from the ampulla, incubated for 24 hours in vitro and were considered fertilized when reaching the two-cell embryo stage. D. Two-cell embryos from C were incubated in vitro for an additional 3 days and the percentage reaching the blastocyst stage was determined. Data are mean ± SEM; n=5, *p<0.05; **p<0.01. Percentages of two-cell embryos were determined by dividing the number of two-cell embryos by the total number of eggs examined and percentages of blastocysts as the number of eggs reaching the blastocyst stage divided by the total number of two cell embryos recovered.
In vitro fertilization and embryo development.
A, B. In vitro capacitated control or C1/C3 DKO cauda epididymal sperm were co-incubated with cumulus oocyte complexes (COC) for 3.5 hs. Eggs were either analyzed at that moment and considered fertilized when at least one decondensing sperm nucleus or two pronuclei were found in the ooplasm (n=3) (A) or incubated for additional 24 hs to determine the percentage reaching the two-cell embryo stage (n=7) (B). C. Two-cell embryos from B were incubated for 3 days in vitro and the percentage reaching 4/8 cells (day 1), morula (day 2) or blastocyst (day 3) stages determined (n=7). D. In vitro capacitated control or C1/C3 DKO epididymal sperm were co-incubated with ZP-free eggs for 1 h and fertilization was evaluated by DNA staining. Eggs were considered fertilized when at least one decondensing sperm nucleus was found in the ooplasm (n≥5). E. Fertilized ZP-free eggs obtained as in D were incubated for an additional 3 days in vitro and the percentage progressing to blastocysts was determined. (n=5). F. Fertilized eggs from D were analyzed for maternal DNA status and classified as arrested in Metaphase II (Met II) or exhibiting 2nd polar body (2PB) (n≥5). Data are the mean ± SEM; *p< 0.05; **p < 0.01. Percentages of fertilization were determined by dividing the number of fertilized eggs by the total number of eggs examined. Percentages of two-cell embryos were determined by dividing the number of two-cell embryos by the total number of eggs examined. Percentages of either 4/8 cell embryos, morula and blastocysts were calculated as the number of embryos reaching each of these stages divided by the total number of two cell embryos obtained.
Egg Ca2+ oscillations in in vitro fertilized eggs.
A. Representative traces of Ca2+ oscillation patterns following in vitro fertilization of ZP-free eggs with control or C1/C3 DKO sperm. Arrows indicate the time of sperm addition. B. Number of peaks within 90 minutes, C. Time to first peak, D. Oscillation frequency, E. First peak area under the curve and F. First transient duration. Values were normalized to basal Ca2+ levels recorded prior to sperm addition. Data are mean ± SEM of at least 9 oocytes from 3 independent experiments; A-F: ns.
Sperm DNA fragmentation and ROS levels.
A. DNA fragmentation of control or C1/C3 DKO cauda epididymal sperm was analyzed by SCD assay and both the area of DNA halo in each individual cell (left panel) and the percentage of sperm heads with small or no halo (right panel) determined. B. DNA fragmentation of cauda and caput epididymal control or C1/C3 DKO sperm was analyzed by SCD assay and the area of DNA halo in each individual cell determined. C. ROS levels in control and C1/C3 DKO cauda sperm analyzed by fluorescence confocal microscopy. Absence of probe and presence of Oligomycin were used as negative and positive controls, respectively. In all cases n=4. *p<0,05; ****p < 0,0001, Different letters indicate significant differences between treatments, p<0.0001.
Influence of epididymal fluid on sperm DNA integrity and intracellular Ca2+ levels.
A. DNA fragmentation of control or C1/C3 DKO cauda epididymal sperm incubated for one hour with their own or the other genotype epididymal fluid in the presence or absence of 10mM Ca2+ was analyzed by SCD assay and the area of DNA halo in each individual cell determined. Different letters indicate significant differences between treatments (p<0.05). B. Intracellular Ca2+ levels were evaluated by flow cytometry using Fluo-4-AM probe. Results are shown as normalized mean fluorescence intensity (rMFI) of Fluo-4-AM compared to the control condition in each experiment for non-capacitated (fresh) and capacitated (CAP) sperm. Data are mean ± SEM, n= 5; *p<0,05.
