Sperm-borne glutathione-S-transferase omega 2 accelerates the nuclear decondensation of spermatozoa during fertilization in mice†

The postacrosomal sheath (PAS) of the perinuclear theca (PT) is the first compartment of the sperm head to solubilize into the ooplasm upon sperm-oocyte fusion, implicating its constituents in zygotic development. This study investigates the role of one such constituent, glutathione-S-transferase omega 2 (GSTO2), an oxidative-reductive enzyme found in the PAS and perforatorial regions of the PT. GSTO2 uses the conjugation of reduced glutathione, an electron donor shown to be compulsory in sperm disassembly within the ooplasm. The proximity of GSTO2 to the condensed sperm nucleus led us to hypothesize that this enzyme may facilitate nuclear decondensation by reducing disulfide bonds before the recruitment of GSTO enzymes from within the ooplasm. To test this hypothesis, we utilized a cell permeable isozyme-specific inhibitor, which fluoresces when bound to the active site of GSTO2, to functionally inhibit spermatozoa before performing intracytoplasmic sperm injections (ICSI) in mice. The technique allowed for targeted inhibition of solely PT-residing GSTO2, as all that is required for complete zygotic development is the injection of the mouse spermatozoon head. ICSI showed that inhibition of PT-anchored GSTO2 caused a delay in sperm nuclear decondensation, and further resulted in untimely embryo cleavage, and an increase in fragmentation beginning at the morula stage. The confounding effects of these developmental delays ultimately resulted in decreased blastocyst formation. This study implicates PT-anchored GSTO2 as an important facilitator of nuclear decondensation and reinforces the notion that the PAS-PT is a critical sperm compartment harboring molecules that facilitate zygotic development.

Defective sperm head decondensation undermines the success of ICSI in the bovine

The efficiency of intracytoplasmic sperm injection (ICSI) in the bovine is low compared to other species. It is unknown whether defective oocyte activation and/or sperm head decondensation limit the success of this technique in this species. To elucidate where the main obstacle lies, we used homologous and heterologous ICSI and parthenogenetic activation procedures. We also evaluated whetherin vitromaturation negatively impacted the early stages of activation after ICSI. Here we showed that injected bovine sperm are resistant to nuclear decondensation by bovine oocytes and this is only partly overcome by exogenous activation. Remarkably, when we used heterologous ICSI,in vivo-matured mouse eggs were capable of mounting calcium oscillations and displaying normal PN formation following injection of bovine sperm, althoughin vitro-matured mouse oocytes were unable to do so. Together, our data demonstrate that bovine sperm are especially resistant to nuclear decondensation byin vitro-matured oocytes and this deficiency cannot be simply overcome by exogenous activation protocols, even by inducing physiological calcium oscillations. Therefore, the inability of a suboptimal ooplasmic environment to induce sperm head decondensation limits the success of ICSI in the bovine. Studies aimed to improve the cytoplasmic milieu ofin vitro-matured oocytes and to replicate the molecular changes associated within vivocapacitation and acrosome reaction will deepen our understanding of the mechanism of fertilization and improve the success of ICSI in this species.

255 SPERM NUCLEAR DECONDENSATION ABILITY OF IN VITRO MATURED CANINE OOCYTES

The sperm chromatin decondensation occurs when a spermatozoon enters into an oocyte during fertilization, and the effectiveness of this process is connected to the grade of oocyte cytoplasmic maturation. In this study chromatin sperm decondensation was evaluated after IVF of canine oocytes matured in vitro (IVM), comparing different durations of maturation. Cumulus–oocytes complexes (COC) for IVM were obtained from bitch ovaries after ovariohysterectomy, selecting those COC with compact cumulus cells and a homogeneous dark cytoplasm. The COC were matured in vitro for 0, 48, 72, and 96 h in TCM-199 with Earle’s salt supplemented with 25 mM HEPES, 10% FCS, 0.25 mM pyruvate, 10 IU mL–1 of hCG, 300 IU mL–1 of penicillin, and 20 mg mL–1 of streptomycin at 38.5°C and 5% CO2. Fresh ejaculates from 3 adult dogs were centrifuged, and the sperm pellet was resuspended in fert-TALP medium. In each replicate, 100-μL fert-TALP drops containing 10 to 12 IVM oocytes after each culture time were co-culture with 2.5 × 106 spermatozoa mL–1 for 24 h under culture conditions. Soon after co-culture, all oocytes were denuded from cumulus cells and fixed in 3% paraformaldehyde. The nuclear stage of the oocytes and the appearance of the sperm nucleus were determined by 4′,6-diamidino-2-phenylindole staining under a fluorescence inverted microscope. For each treatment, at least 4 replicates were performed, and the data were compared statistically by chi-square test, using InfoStat Professional Program. A total of 800 oocytes were evaluated, and the percentages of oocytes with sperm penetration were 58% (138/238), 61% (108/177), 72% (118/165), and 70% (153/220) at 0, 48, 72, and 96 h of IVM, respectively. The percentage of sperm nuclear decondensation at each time point significantly increased up to 72 h of culture, showing 12, 34, 81, and 85% of sperm nuclei deconsated, respectively. The percentages of nuclear maturation also increased (P ≤ 0.05) with time, showing 0, 8, 20, and 27% of oocytes at second metaphase (MII) stage at 0, 48, 72, and 96 h of culture. The percentage of MII stage was much lower than that of chromatin decondensation in all maturing groups. These results suggest that canine oocytes matured in vitro are able to decondense the sperm chromatin during IVF, and this ability increases up to 72 h of culture. Nevertheless, cytoplasmic maturation, as evaluated by sperm chromatin decondensation, in canine oocytes matured in vitro may not be completely connected with nuclear development. This work was supported by Grant FONDECYT 1080618.

227 THE EFFECTS OF SEXED SEMEN ON EMBRYONIC DEVELOPMENT TO THE BLASTOCYST STAGE

Sexed semen (SS) exhibits approximately 80% of the fertilizing ability of conventional semen (CS), and studies have shown that this continues through the 8-cell stage of bovine embryo development. At the time of this study, no information could be found that, when used for IVF and intracytoplasmic sperm injection (ICSI) development, had been carried to the blastocyst stage. In addition, questions have arisen regarding which of the measured sperm parameters are responsible for the difference between the SS and CS and contribute to this decline in fertility. The goals of this project were to evaluate the effects of using sexed sperm as it relates to embryonic development and to determine if any of the differences in sperm parameters affect embryonic development. A preliminary project evaluated SS and CS from 5 bulls for IVF and ICSI. One bull was selected to provide the sperm (both SS and CS) for the trial, and 1752 oocytes were assigned to either IVF or ICSI. The SS and CS were divided among the available oocytes used for IVF and ICSI. Straws were thawed for 30 s at 37°C, and sperm were then evaluated for motility (provided by CASA, SpermVision, MiniTube of America, Verona, WI), morphology, acrosomal integrity (Coomassie and Pope stains), viability, and nuclear decondensation (SYBR Green and HALO). Results for SS v. CS were as follows: motility, 8 v. 26%; viability, 40.6 v. 30%; nuclear decondensation, 40 v. 30%; normal morphology and acrosomal integrity, no differences. Oocytes were obtained from Applied Reproductive Technologies, LLC (Madison, WI). The fertilization rate was consistently lower (Table 1) for both IVF and ICSI when SS were used (Z = 3.65; P = 0.0003), and there was no evidence that this decline in fertilization rate differed for the 2 methods (Z = 0.18; P = 0.86). Nor was there any evidence that the method affected the fertilization rate in general (Z = 0.75; P = 0.45). Thus, the difference was specific for fertilization rate and had no effect on Day 3 cells or Day 7 blastocysts. A higher fertility rate using ICSI would have indicated that a surface membrane factor may have been decreasing the fertility rate with SS because of the elimination of binding factors associated with ICSI. Thus, it may not be the sperm surface membrane that is distorted in the sexing procedure, but likely the integrity of the spermal DNA, as indicated by the increased nuclear decondensation of SS. Table 1.Comparison of sexed sperm with conventional sperm when used for IVF and intracytoplasmic sperm injection (ICSI) Partial funding for this project was made available by the California State University Agricultural Research Institute (ARI). Appreciation is extended to Sexing Technologies, Inc. (Navasota, TX) for donating the semen.

Possible participation of calmodulin in the decondensation of nuclei isolated from guinea pig spermatozoa

SummaryThe guinea pig spermatozoid nucleus contains actin, myosin, spectrin and cytokeratin. Also, it has been reported that phalloidin and/or 2,3-butanedione monoxime retard the sperm nuclear decondensation caused by heparin, suggesting a role for F-actin and myosin in nuclear stability. The presence of an F-actin/myosin dynamic system in these nuclei led us to search for proteins usually related to this system. In guinea pig sperm nuclei we detected calmodulin, F-actin, the myosin light chain and an actin-myosin complex. To define whether calmodulin participates in nuclear-dynamics, the effect of the calmodulin antagonists W5, W7 and calmidazolium was tested on the decondensation of nuclei promoted by either heparin or by a Xenopus laevis egg extract. All antagonists inhibited both the heparin- and the X. laevis egg extract-mediated nuclear decondensation. Heparin-mediated decondensation was faster and led to loss of nuclei. The X. laevis egg extract-promoted decondensation was slower and did not result in loss of the decondensed nuclei. It is suggested that in guinea pig sperm calmodulin participates in the nuclear decondensation process.