Zona Pellucida Digestion Research Articles

Addition of exogenous proteins detected in oviductal secretions to in vitro culture medium does not improve the efficiency of in vitro fertilization in pigs

This work was designed to study whether HSP70-1A, HSP90α, ezrin or PDI4, proteins previously identified in porcine oviductal secretions, have a role in zona pellucida (ZP) resistance to enzymatic digestion, in vitro fertilization (IVF) and sperm viability. In vitro matured porcine cumulus oocyte complexes were denuded and i) incubated for 1 h in TALP medium supplemented or not with each exogenous oviductal protein and in presence or absence of heparin to assess ZP digestion time by pronase; and ii) inseminated with fresh ejaculated boar spermatozoa in medium supplemented or not with each exogenous oviductal protein to assess their effect on fertilization results. Finally, spermatozoa were incubated in Tyrode’s medium (0, 1 and 20 h) supplemented or not with HSP-701A, HSP-90α or ezrin, to assess simultaneously sperm viability and acrosome status by means of flow cytometry. Although all proteins increased the ZP digestion time, this increase was lower than 1 min, being ezrin the protein with a stronger effect. Presence of heparin in the medium reinforced the ZP hardening effect of ezrin and HSP-701A up to one more min, but not HSP-90α nor PDI4. Sperm penetration, but not IVF efficiency, increased when gametes were cocultured in medium containing PDIA4 whereas sperm penetration and polyspermy rates decreased in presence of ezrin and HSP proteins. This reduction was not the result of a detrimental effect of proteins on sperm viability or acrosome reaction. In conclusion, addition of exogenous proteins detected in oviductal secretions to artificial media does not reproduce the effect of adding such secretions nor improve the final efficiency of the porcine IVF system.

Interaction of sperm with the zona pellucida during fertilization

In order to achieve fertilization sperm cells, first need to successfully interact with the zona pellucida. To this end, the sperm surface is extensively remodeled during capacitation and the resulting sperm cells also possess hyperactivated motility. Together, this serves to mediate optimal recognition of the zona pellucida in the oviduct or after in vitro fertilization incubations (primary zona pellucida binding). When the sperm cell attaches to the zona pellucida, it will be triggered to undergo the acrosome reaction which allows the hyperactivated motile sperm cell to drill through the zona pellucida (secondary zona pellucida binding coinciding with sequential local zona pellucida digestion and rebinding). After successful zona penetration, some sperm cells may enter the perivitelline space. This delaying strategy of the oocyte allows only one sperm cell at a given time to bind and fuse with the oocyte (fertilization) and thus minimizes the risk of polyspermy. The fertilization fusion between the oocyte and the first sperm cell is immediately followed by a polyspermic fertilization block, in which the content of the oocyte's cortical granules is released into the perivitelline space. The cortical reaction blocks further sperm-oocyte fusion either by sticking at the oolemma or by the induction of a biochemical reaction of the zona pellucida (zona pellucida hardening). The cortical reaction thus blocks sperm-zona pellucida binding and/or sperm-zona pellucida penetration. This review summarizes the current understanding of sperm-zona pellucida interactions in relation to mammalian fertilization. The lack of knowledge about sperm-zona pellucida binding in ruminants will be critically discussed.

Developmental competence of in vitro matured ovine oocytes vitrified in solutions with different concentrations of trehalose.

This study aimed to determine the optimum concentration of trehalose in solutions used for vitrification of in vitro matured (IVM) ovine oocytes. IVM oocytes were randomly divided into four experimental (vitrified) and one control (fresh) groups. Experimental groups were treated with different concentrations (0.0, 0.25, 0.5 and 1.0M) of trehalose. After warming, some viable oocytes were exposed to 0.25% pronase to test zona pellucida hardening, whereas the others were fertilized and cultured in vitro for 8days to evaluate their developmental competence. Blastocysts quality was assessed by differential staining and TUNEL test. Survival and developmental rates of oocytes vitrified in the presence of 0.5M trehalose were significantly higher than those of the other vitrified groups. Furthermore, there was a significant difference between fresh and vitrified groups in total blastocyst rate. Analysis of blastocysts quality also revealed a significant difference between the group treated with 0.5M trehalose and other groups in terms of apoptotic index. Furthermore,zona pellucida digestion time period was longer in trehalose-free (0.0M) group compared to other groups. In conclusion, we found that IVM ovine oocytes vitrified in solutions containing 0.5M trehalose are fertilization-competent and are able to produce good-quality blastocysts with an apoptotic index comparable to that of the fresh oocytes. Therefore, 0.5M may be considered the optimum concentration of trehalose to be used in solutions prepared for vitrification of oocytes.

Intracellular activation of ovastacin mediates pre-fertilization hardening of the zona pellucida.

How and where is pro-ovastacin activated and how does active ovastacin regulate zona pellucida hardening (ZPH) and successful fertilization? Ovastacin is partially active before exocytosis and pre-hardens the zona pellucida (ZP) before fertilization. The metalloproteinase ovastacin is stored in cortical granules, it cleaves zona pellucida protein 2 (ZP2) upon fertilization and thereby destroys the ZP sperm ligand and triggers ZPH. Female mice deficient in the extracellular circulating ovastacin-inhibitor fetuin-B are infertile due to pre-mature ZPH. We isolated oocytes from wild-type and ovastacin-deficient (Astlnull) FVB mice before and after fertilization (in vitro and in vivo) and quantified ovastacin activity and cleavage of ZP2 by immunoblot. We assessed ZPH by measuring ZP digestion time using α-chymotrypsin and by determining ZP2 cleavage. We determined cellular distribution of ovastacin by immunofluorescence using domain-specific ovastacin antibodies. Experiments were performed at least in triplicate with a minimum of 20 oocytes. Data were pre-analyzed using Shapiro-Wilk test. In case of normal distribution, significance was determined via two-sided Student's t-test, whereas in case of non-normal distribution via Mann-Whitney U-test. Metaphase II (MII) oocytes contained both inactive pro-ovastacin and activated ovastacin. Immunoblot and ZP digestion assays revealed a partial cleavage of ZP2 even before fertilization in wild-type mice. Partial cleavage coincided with germinal-vesicle breakdown and MII, despite the presence of fetuin-B protein, an endogenous ovastacin inhibitor, in the follicular and oviductal fluid. Upon exocytosis, part of the C-terminal domain of ovastacin remained attached to the plasmalemma, while the N-terminal active ovastacin domain was secreted. This finding may resolve previously conflicting data showing that ovastacin acts both as an oolemmal receptor termed SAS1B (sperm acrosomal SLLP1 binding protein; SLLP, sperm lysozyme like protein) and a secreted protease mediating ZP2 cleavage. For this study, only oocytes isolated from wild-type and ovastacin-deficient FVB mice were investigated. Some experiments involved oocyte activation by the Ca2+ ionophore A23187 to trigger ZPH. This study provides a detailed spatial and temporal view of pre-mature cleavage of ZP2 by ovastacin, which is known to adversely affect IVF rate in mice and humans. None. This work was supported by the Center of Natural Sciences and Medicine and by a start-up grant of the Johannes Gutenberg University Mainz to W.S., and by a grant from Deutsche Forschungsgemeinschaft and by the START program of the Medical Faculty of RWTH Aachen University to J.F. and W.J.D. There are no competing interests to declare.

Differential influence of ampullary and isthmic derived epithelial cells on zona pellucida hardening and in vitro fertilization in ovine

The central role of the oviduct, as the site of zona pellucida (ZP) maturation, fertilization and early embryogenesis, has been recognized. The objective of this study was to investigate whether ampullary and isthmic derived epithelial cells have different effects on in vitro ZP hardening, in vitro fertilization (IVF) and in vitro culture (IVC) of the resulting embryos. Cumulus oocyte complexes (COCs) were matured in a coculture system with ampullary/isthmic epithelial cells, TCM199 supplemented with insulin-like growth factor I (IGF-I) and epithelial derived growth factor (EGF) (GF treated group), conditioned media produced using ampullary (ACM), isthmic (ICM), COCs+ampullary, and COCs+isthmic epithelial cells, contactless culture system, oviductal fluid, GF+ACM/ICM, and drops of TCM199 (control), for 24h. The matured oocytes were randomly divided into two groups: Group I was subjected to ZP digestion; Group II underwent IVF. The duration of the ZP digestion, in a coculture system with ampullary epithelial cells (AE) was significantly increased (p<0.05), compared with other groups. Penetrated oocytes and monospermic fertilization were significantly increased (p<0.05) in the AE group. The mean number of spermatozoa per penetrated oocyte was reduced dramatically for the AE group (p<0.05). A significant increase (p<0.05) in the embryo development was observed in all treated groups, compared to the control. Results revealed that epithelial cells harvested from the ampullary segment of the oviduct had in vitro specialized role in ZP hardening and have subsequent IVF and IVC outcomes.

161 EFFECT OF OVIDUCTAL FLUID ON BOVINE OOCYTE ZONA PELLUCIDA HARDENING AND SPERM BINDING, AND ON EARLY EMBRYO DEVELOPMENT

This study was designed to evaluate the effects of oviductal fluid at different periovulatory times on oocyte maturation, modification of the zona pellucida (ZP), fertilization and embryo development. Bovine oviducts were collected at a slaughterhouse and classified as preovulatory (pre-ov: 1 pre-ov follicle and a regressing corpus luteum) or post-ovulatory (post-ov: a corpus haemorrhagicum or recent corpus luteum; n = 10 cows/stage). Both oviducts were flushed with 1 mL of sterile TCM-199, and oviductal flushes (OF) were aliquoted and stored at –80°C. Abattoir-derived bovine ovaries were aspirated and cumulus‐oocyte complexes (COC) with at least 3 cumulus layers and homogeneous oocyte cytoplasm were in vitro matured for 22 h in standard maturation medium (control group, n = 319) or in standard medium with 2× concentrated additives supplemented (50% v/v) with pre-ov OF (n = 255) or post-ov OF (n = 248). After in vitro maturation (IVM), subgroups of COC were denuded, and the time of digestion of the ZP by pronase 0.1% (v/v in TCM-199) was determined to evaluate ZP hardening. After IVM, COC were fertilised in vitro for 18–20 h at a final concentration of 1.106 million spermatozoa (spz)/mL. After in vitro fertilization (IVF), COC were denuded, washed twice and cultured for 8 days more under standard conditions. After IVM, IVF, and embryo culture, oocytes/embryos were fixed with ethanol, stained with Hoescht, and examined under fluorescence microscopy for determination of (1) maturation and developmental stages, (2) numbers of fertilised and polyspermic oocytes, and (3) spz bound to the ZP. Percentages were compared between groups by chi-square. Times of ZP digestion were compared by Kruskal‐Wallis test. Numbers of spz bound to the ZP were compared by ANOVA on normalised data followed by Newman-Keuls tests. Data are presented as mean ± SEM. A P &lt; 0.05 was considered significant. Addition of OF during IVM had no effect on maturation rates compared with the control. However, the digestion time of the ZP by pronase was reduced after IVM with pre-ov OF (313 ± 21 s; n = 26) compared with post-ov OF (459 ± 23 s; n = 23) but not with the control (416 ± 30 s; n = 25). After IVF, the number of spermatozoa bound to the ZP was increased after IVM with pre-ov OF (57 ± 5 spz/oocyte; n = 67) and decreased after IVM with post-ov OF (34 ± 3 spz/oocyte; n = 76) compared with the control (42 ± 5 spz/oocyte; n = 60). Addition of OF during IVM had no effect on rates of IVF and polyspermia. However, the rate of development to the blastocyst stage was less after IVM with post-ov OF (10%, n = 97 cleaved oocytes) compared with control (24%, n = 130) and pre-ov OF (29%, n = 101). In conclusion, the OF collected before ovulation decreased the resistance of the ZP to protease digestion and increased its ability to bind spz, whereas it was the opposite for the post-ov OF. Furthermore, the post-ov OF decreased the developmental competence of fertilised oocytes.

Nitric oxide synthase (NOS) inhibition during porcine in vitro maturation modifies oocyte protein S-nitrosylation and in vitro fertilization.

Nitric oxide (NO) is a molecule involved in many reproductive processes. Its importance during oocyte in vitro maturation (IVM) has been demonstrated in various species although sometimes with contradictory results. The objective of this study was to determine the effect of NO during IVM of cumulus oocyte complexes and its subsequent impact on gamete interaction in porcine species. For this purpose, IVM media were supplemented with three NOS inhibitors: NG-nitro-L-arginine methyl ester (L-NAME), NG-monomethyl-L-arginine (L-NMMA) and aminoguanidine (AG). A NO donor, S-nitrosoglutathione (GSNO), was also used. The effects on the cumulus cell expansion, meiotic resumption, zona pellucida digestion time (ZPdt) and, finally, on in vitro fertilization (IVF) parameters were evaluated. The oocyte S-nitrosoproteins were also studied by in situ nitrosylation. The results showed that after 42 h of IVM, AG, L-NAME and L-NMMA had an inhibitory effect on cumulus cell expansion. Meiotic resumption was suppressed only when AG was added, with 78.7% of the oocytes arrested at the germinal vesicle state (P<0.05). Supplementation of the IVM medium with NOS inhibitors or NO donor did not enhance the efficiency of IVF, but revealed the importance of NO in maturation and subsequent fertilization. Furthermore, protein S-nitrosylation is reported for the first time as a pathway through which NO exerts its effect on porcine IVM; therefore, it would be important to determine which proteins are nitrosylated in the oocyte and their functions, in order to throw light on the mechanism of action of NO in oocyte maturation and subsequent fertilization.

Timing of oviductal fluid collection, steroid concentrations, and sperm preservation method affect porcine in vitro fertilization efficiency

To determine optimal conditions for the inclusion of oviductal fluid (OF) in IVF protocols. Experimental prospective study. Mammalian reproduction research laboratory. Oviducts and ovaries from porcine females were collected at a slaughterhouse. A total of 30 oviducts and 1,285 oocytes were used. Boar-ejaculated spermatozoa were also used. In vitro-matured porcine oocytes were preincubated with OF collected from animals before or after ovulation and later fertilized in vitro. Zona pellucida digestion time in oocytes after preincubation in OF was assessed. Concentrations of E2 and P4 in OF were measured. IVF was performed, including within the culture media the E2 and P4 concentrations found in the preovulatory OF. The effect of preovulatory OF on IVF efficiency was compared between fresh and frozen-thawed spermatozoa. E2 and P4 concentrations in OF; penetration and monospermy rates; number of spermatozoa within the ooplasm and on the zona pellucida after IVF under different experimental conditions; zona pellucida resistance to protease digestion. Preincubation of oocytes in OF collected before ovulation enhances IVF efficiency in the pig compared with OF collected after ovulation (29.58 ± 3.84 vs. 11.03 ± 2.69). When frozen-thawed spermatozoa are used for the IVF of these OF-treated oocytes, their fertilization ability increases compared with fresh semen. OF collected before and after ovulation shows significantly different concentrations of E2 (99.00 ± 8.72 vs. <10 pg/mL) and P4 (2.53 ± 0.66 vs. 12.27 ± 2.33 ng/mL), respectively. Addition of E2 and P4 at concentrations similar to those in the OF before ovulation partially simulates the effect of the fluid on IVF outcome. Preincubation of oocytes in OF collected before ovulation is a suitable protocol for increasing the efficiency of IVF with fresh semen in the pig model and could be a useful tool to increase the fertilization ability of frozen-thawed spermatozoa in other species. E2 concentrations in preovulatory OF are higher than those reported in blood serum at the same phase of the estrous cycle.

The human is an exception to the evolutionarily-conserved phenomenon of pre-fertilization zona pellucida resistance to proteolysis induced by oviductal fluid

Is zona pellucida (ZP) resistance to proteolysis, induced by oviductal fluid (OF), a mechanism common to species other than the pig and cow? ZP resistance to proteolysis induced by OF was observed in the mouse, rat, hamster, rabbit, sheep, goat, pig and cow, but not in humans. Oviductal ZP resistance to proteolysis occurs in the pig and cow where it influences the incidence of fertilization and polyspermy. The effect is observed after incubation of ZP in OFs from pig (pOF), cow (cOF), rabbit (rOF) and sheep (sOF). Oocytes from nine different species, including ungulates, rodents, lagomorphs and primates were incubated in rOF, sOF, gOF, cOF, pOF and human oviductal fluid (hOF). ZP digestion times for the matured oocytes of these nine species, without any treatment or incubated in 5 (mouse, rat, hamster, rabbit, cow, ewe and goat) or 6 (pig and humans) of the OFs collected were compared using three replicates per treatment and at least three oocytes per replicate. In vivo matured oocytes from rat, hamster, mouse, rabbit and humans, in vitro matured oocytes from cow, goat, ewe and pig and rOF, cOF, gOF, sOF, pOF and human (hOF) were collected and processed for the study. Oocytes from each species were incubated in the different OFs for 30 min. The resistance of the ZP of the oocytes to enzymatic digestion in a pronase solution (0.5% in PBS) was measured and registered as ZP digestion time. rOF increased ZP resistance to proteolytic digestion in the range of between 96 and 720 h for any of the species tested, whereas the corresponding increase in human ZP was only 1 min. OFs from the remaining species also had a significant effect, with variations among the cross-species experiments (P < 0.05). hOF, which was only tested on human and porcine oocytes, had no effect on ZP chemical hardening. Measurements of ZP digestion times are not of extreme accuracy and errors of a few seconds can be assumed in the experimental data. However, when differences are in the range of hours among treatments, variations measured in seconds do not alter the robustness of the findings. Human oocytes and OF were of limited access, compared with oocytes from species collected in slaughterhouses. OFs from mouse, rat and hamster were not tested due to the small size of the genital tract in these species and the small volume of fluid available. Since oviductal modification of ZP resistance to proteolytic digestion has been demonstrated to influence fertilization and this pre-fertilization mechanism is considered to contribute to the control of polyspermy, the apparent absence of this mechanism in humans suggests that the regulation of polyspermy depends mainly on other mechanisms, most probably of cortical granule origin. Investigation into a possible relationship between the lack of oviductal ZP hardening in human oocytes and the existence of tubal ectopic pregnancies in this species is proposed. This work was supported by the Spanish Ministry of Science and Innovation and FEDER, Grant AGL2009-12512-C02-01-02. The authors declare no competing interest.

261 SUPPLEMENTATION OF SHEEP OOCYTE MATURATION MEDIA WITH NITRIC OXIDE SYNTHASE INHIBITORS MODIFIES THE ZONA PELLUCIDA AND DECREASES SPERM ZONA BINDING

Nitric oxide plays a critical role in several reproductive functions, including sperm and egg maturation. Nitric oxide has complex and sometimes antagonistic biological effects. At low and controlled levels, NO aids gamete maturation, but at high levels, it has detrimental effects and often causes cell death. Both the female tract and egg vestment contain endothelial NO synthase (eNOS) and inducible NOS (iNOS). The knockout mouse for specific NOS has been shown to impair egg maturation and ovulation. The aim of this work was to study the effect of NOS inhibitors on in vitro sheep oocyte maturation and function. We evaluated the cumulus cell expansion, sperm zona binding, and time required to fully digest the zona pellucida (ZP). The 3 different NOS inhibitors used were 1 mM l-NAME (N-ω-nitro-l-arginine methyl ester, an eNOS inhibitor), 1 mM l-NMMA (NG-monomethyl-l-arginine acetate, a competitive inhibitor of all three isoforms of NOS), and 1 mM aminoguanidine (eNOS and iNOS inhibitors). We also used a NO donor, S-nitroso-glutathione (GSNO; 50 µM). Oocytes were matured in TCM without supplementation or supplemented with GSNO or NOS inhibitors for 24 h at 38°C, 5% CO2. The effect of the inhibitors on the ZP was assessed by determining the time necessary to complete digestion of the ZP with pronase (0.1%). To evaluate the number of sperm attached to the ZP, oocytes matured as described above were denuded and co-incubated for 4 h (38°C, 5% CO2) with epididymal sheep sperm (1 × 106 spermatozoa mL–1 in TALP medium). At the end of the incubation, sperm unbound to the ZP were eliminated by several washings. The oocytes were fixed and stained with Hoechst to stain the sperm nucleus, and the number of sperm bound per zona was measured under an epifluorescence microscope. The times required for complete ZP digestion and spermatozoa attachment were analysed by one-way ANOVA. Mean digestion times (seconds) and numbers of spermatozoa bound to the ZP in the control, GSNO, l-NAME, aminoguanidine, and l-NMMA groups were 234.6a, 297.8a, 243.2a, 323.0ab, 398.4b, and 20.2a, 12.0ab, 11.1b, 7.7b, and 8.1b, respectively (a,bP &lt; 0.001). Our results showed that 1) egg maturation, as measured by cumulus expansion, was not affected by the NOS inhibitors or GSNO, 2) the time necessary to completely digest the ZP increased in the presence of aminoguanidine and l-NMMA, and 3) all three NOS inhibitors decreased the number of spermatozoa that could be attached to the ZP. The GSNO showed no effect because these levels represent physiological concentration observed during normal egg maturation. In conclusion, these results are in accordance with the importance of NO for egg maturation, and this study shows for the first time the possible involvement of NO in maintaining the structure of the ZP, which is important for sperm–egg binding and further fertilization. Such an effect on the ZP proteins could be due to a change in protein S-nitrosylation, which is a post-translational modification attributable to NO.

One-step collagenase method for zona pellucida removal in unfertilized eggs: easy and gentle method for large-scale preparation.

Zona pellucida (ZP)-free eggs are often used for studies such as evaluating the interaction of sperm-oolemma. To acquire ZP-free eggs, the most commonly used methods employ acidified Tyrode's solution, enzymatic digestion with a trypsin-like enzyme, or mechanical methods using micropipettes. However, acidified Tyrode's solution and trypsin-like enzymes often damage the oolemma, especially when many eggs are treated at once for mass sample analyses. The mechanical method requires skill, and it is time-consuming to prepare many ZP-free eggs. Therefore, in this study, to establish an easy, reliable method for preparing ZP-free eggs, we examined the ZP digestion method originally reported by Zuccotti et al. (J Reprod Fertil 93:515-520, 1991) that uses collagenase. Mouse unfertilized eggs were treated with collagenase and acidified Tyrode's solution to compare the ZP-free rates, the effect on the oolemma, and the two-cell development rates of ZP-free eggs by in vitro fertilization. The effects on the oolemma were gauged by observing the polarity of the transmembrane protein localization of enhanced green fluorescence protein tagged CD9 protein (CD9-EGFP) and using differential interference contrast microscopy. Collagenase removed the ZP and the cumulus cells from the cumulus oocyte complex. The collagenase method had no influence on the localization of CD9-EGFP, resulting in a high two-cell development rate. Additionally, the collagenase method could exclude low quality eggs with hardened ZP, since collagenase could not digest the hardened ZP. The one-step collagenase method is an easy preparation method for large numbers of high-quality ZP-free eggs.

Pre-fertilization zona pellucida hardening by different cross-linkers affects IVF in pigs and cattle and improves embryo production in pigs

Zona pellucida (ZP) hardening (resistance to proteolysis) has been classically identified as a post-fertilization event that contributes to the block to polyspermy. Di-(N-succinimidyl)-3,3'-dithiodipropionate (DSP), a permeable amine-reactive cross-linker, was recently shown to induce pre-fertilization ZP hardening and to improve porcine IVF productivity. The objectives of this study were to investigate i) how DSP affects pre-fertilization ZP hardening and IVF in cattle, ii) if a non-permeable amine-reactive cross-linker such as bis(sulfosuccinimidyl) suberate (BS3) affects ZP hardening and IVF in cattle and pigs, and iii) whether DSP or BS3, if improvement in IVF productivity was demonstrated in either species, affects in vitro embryo development. Bovine and porcine in vitro matured oocytes were incubated with the cross-linkers (0.06, 0.3, and 0.6 mg/ml) for 30 min. Then they were subjected to ZP digestion or IVF. In cattle, both DSP and BS3 induced ZP hardening and decreased the penetration rate, although monospermy, penetration, or male pronuclear formation was not affected. In pigs, BS3 treatment induced ZP hardening, decreased penetration and male pronuclear formation, and increased monospermy. IVF productivity only improved when porcine oocytes were exposed to DSP. When porcine zygotes derived from this treatment were further cultured in vitro, the cleavage and blastocyst formation rates increased. These results support the idea that mechanisms involved in the prevention of polyspermic fertilization in cattle and pigs have different efficiencies, and ZP hardening induced by DSP cross-linker may be useful for improving porcine embryo production.

Positive effect of partial zona pellucida digestion on in vitro fertilization of mouse oocytes with cryopreserved spermatozoa

Cryopreservation of mouse spermatozoa has been widely used; however, fertility of frozen spermatozoa in some strains, especially when inseminating cryopreserved oocytes, is low and may be improved by assisted fertilization techniques. The present study was performed to investigate the effect of partial zona pellucida (ZP) digestion on the in vitro fertilization (IVF) capacity of frozen mouse spermatozoa. Mouse oocytes were subjected to partial ZP digestion using acidic Tyrode's solution (pH 3.1). Fertilization rates in digestion groups (30 or 45 s) were higher (P < 0.05) than that of zona-intact control (78.3% or 86.3% vs. 52.5%). The recovery rate at 45 s was lower (P < 0.05) than that at 30 s (84.2% vs. 97.3%). Among vitrified oocytes, the fertilization rate in treatment group (digested for 30 s) was higher (P < 0.05) than that of zona-intact group (50.8% vs. 22.1%). After embryo transfer at the two-cell stage, 17.7% and 11.8% of transferred embryos derived from fresh and vitrified digested oocytes developed to term and showed no significant difference as compared with that from zona-intact oocytes (24.1%, P > 0.05). These results indicate that partial ZP digestion improves IVF efficiency of fresh and vitrified oocytes with frozen mouse spermatozoa, which can provide valuable information for in vitro assisted fertilization using cryopreserved gametes in the re-establishment of mouse colonies.

Vitrification solution containing DMSO and EG can induce parthenogenetic activation of in vitro matured ovine oocytes and decrease sperm penetration

This study was designed to examine the reduced incidence of normal fertilization in vitrified ovine oocytes. After in vitro maturation for 24 h, the oocytes were randomly allocated into three groups: (1) untreated (control), (2) exposed to vitrification solution (VS) without being plunged into liquid nitrogen (toxicity), or (3) vitrified by open-pulled straw method (vitrification). In experiment 1, the treated and control oocytes were matured for another 2 h, and the oocytes were then in vitro fertilized for 12 h to examine sperm penetration. The percentage of monospermy in toxicity group (29.3%) and vitrification group (28.2%) dramatically decreased compared to the control group (45.0%) ( P < 0.05). To find the mechanism that the VS decreased the monospermy, some treated and control oocytes were used to test the distribution of CG and the resistance of zona pellucida (ZP) to 0.1% pronase E immediately (IVM 24 h), after another 2 h of maturation (IVM 26 h), and after 12 h of in vitro fertilization (IVF 12 h) respectively. Others were used to examine female pronucleus formation after 12 h of culture in fertilization medium with the absence of sperm. The results showed that the percentage of CG completely release in the oocytes (IVM 24 and 26 h) of toxicity group (41.2% and 39.9%) and vitrification group (41.7% and 51.7%) was significantly higher than that of control group (7.1% and 18.4%) ( P < 0.05). The ZP digestion duration in the oocytes (IVM 26 h) of the toxicity group (435.6 s) and vitrification group (422.3 s) was longer than that of control group (381.6 s) ( P < 0.05). The percentage of female pronucleus formation in toxicity group (58.7%) and vitrification group (63.9%) was higher than that (8.2%) of control group ( P < 0.05). The data above demonstrated that the VS containing DMSO and EG could parthenogenetically activate in vitro matured ovine oocytes, resulting in ZP hardening and decreased sperm penetration.

204 AMINO-REACTIVE CROSSLINKER BIS(SULFOSUCCINIMIDYL)SUBERATE INDUCES ZONA PELLUCIDA HARDENING AND REDUCES PENETRATION IN PIG IN VITRO FERTILIZATION

Zona pellucida (ZP) hardening is considered to be the final step in the prevention of polyspermy during fertilization in mammals. However, unfertilized pig oviductal oocytes show a resistance of hours or days to pronase digestion (Broermann et al. 1989 J. Anim. Sci. 67, 1324–1329). We previously demonstrated that the amino-reactive crosslinker DSP is effective in inducing ZP hardening and improves the monospermy levels at pig IVF (Coy et al. 2007, Reprod. Dom. Anim., in press). In this study, a different chemical crosslinker, BS3 [bis(sulfosuccinimidyl) suberate], which also forms stable amide bonds among proteins, was used to evaluate its effect on ZP digestion time, penetration, male pronuclear formation and monospermy percentages, and the mean number of sperm per oocyte. In experiment 1, porcine in vitro-matured oocytes (n = 300) were incubated for 30 min at 0, 0.06, 0.30, or 0.60 mg mL–1 of BS3 in TALP medium and assessed for ZP digestion time in 0.5% pronase solution. The results (analyzed by ANOVA in all the experiments) showed a significant (P ≤ 0.01) dose-dependent increase in ZP hardening, from 69.0 s in the control to 426.3, 2028.3, and 2979.2 s, respectively, for the different BS3 concentrations. In experiment 2, oocytes (n = 473) were fertilized in vitro after no treatment or treatment with BS3 at 0.06, 0.30, and 0.60 mg mL–1. Fresh ejaculated spermatozoa were selected by Percoll� gradient 45:90. Oocytes were inseminated with 105 sperm mL–1, which resulted in high penetration and polyspermy percentages in the control group (83.1 and 89.9%, respectively). However, for the BS3-treated oocytes, significant differences compared with the control group (P ≤ 0.001) were observed in all 3 groups, showing penetration percentages of 22.2, 18.1, and 21.5%, respectively, and monospermy percentages of 100, 88.2, and 95.0%, respectively. The mean numbers of sperm per oocyte were 1.0, 1.1, and 1.05 for the BS3 groups, which were significantly different from 5.0 for the control group. In conclusion, BS3 can be used to induce ZP hardening in the pig and regulate polyspermy in IVF systems, although additional experiments are necessary to find the optimal concentration to improve the penetration percentages with high levels of monospermy. Granted by MEC and FEDER (AGL2006-03495).

198 FUNCTIONAL EVIDENCE FOR THE EXISTENCE OF AN OVIDUCTAL FACTOR THAT INDUCES ZONA PELLUCIDA HARDENING AND REGULATES POLYSPERMY IN THE PIG AND COW

Many differences between in vivo and in vitro fertilization (IVF) efficiency in mammals are related to the differences between IVF media and oviductal fluid. One of the best known examples is the frequency of polyspermy observed under in vitro conditions in cattle (Roh S et al. 2002 J. Vet. Med. Sci. 64, 667–671) and, in particular, in pigs (Coy P and Romar R 2002 Reprod. Fertil. Dev. 14, 275–286). Zona pellucida (ZP) resistance to pronase digestion (ZP hardening) has been considered as a postfertilization event contributing to the block of polyspermy in mammals (Green D 1997 Rev. Reprod. 2, 147–156). However, pig and cow unfertilized ovulated oocytes show a ZP hardening of hours or days (Katska L et al. 1999 Reprod. Dom. Anim. 34, 255–259; Kolbe T and Holtz W 2005 Theriogenology 63, 1695–1705) compared with the minutes or seconds observed in the in vitro-matured oocytes, even after fertilization (Coy P et al. 2002 Reproduction 124, 279–288; Coy P et al. 2005 Reproduction 129, 19–26). Consequently, we propose the existence of an oviductal factor that induces ZP hardening before any contact of the oocyte with the sperm, thus regulating polyspermy. Porcine and bovine oviductal fluid was obtained by aspiration of oviducts collected at the slaughterhouse and stored frozen. In vitro-matured porcine and bovine oocytes were incubated for 30 min in the oviductal fluid, washed thoroughly in fresh medium, and either assessed for ZP digestion time or in vitro fertilized. The results, analyzed by ANOVA, showed a very strong ZP hardening in oviductal-treated oocytes (2866.83 � 94.4 s in the pig and 4301.1 � 441.7 s in the cow) compared with control oocytes (63.5 � 2.9 s in the pig and 124.2 � 5.9 s in the cow). Moreover, the percentage of monospermy for the oviductal-treated oocytes was significantly higher in both species (50.0 � 10.0% in the pig and 91.7 � 3.0% in the cow) compared with the control groups (5.56 � 3.8% in the pig and 80.8 � 3.5% in the cow). Percentage penetration did not change in porcine oocytes but decreased in bovine oocytes (58.1 � 3.3 v. 38.4 � 3.3, P ≤ 0.001), whereas the mean number of sperm per oocyte decreased for the porcine-treated oocytes (2.7 � 0.2 v. 8.2 � 0.4, P ≤ 0.001) and did not change for the bovine oocytes. These results support the hypothesis that an oviductal factor induces ZP hardening, contributing to the control of polyspermy in the pig and cow, and that it could be used to improve the output of IVF. Supported by MEC and FEDER (AGL2006-03495).

A prospective randomized study to assess the benefit of partial zona pellucida digestion before frozen-thawed embryo transfers

Assisted hatching (AH) in fresh embryo transfer (ET) could be associated with increased implantation rates. However, very few prospective randomized studies have specifically addressed the issue of AH during frozen-thawed embryo transfers (FET) cycles, those that have reported controversial results. The aim of this study was to evaluate the benefit of an enzymatic zona pellucida treatment of frozen-thawed embryos before transfer. This was a prospective study including 125 non-donor FET cycles from 125 infertile couples. FETs were randomly allocated into AH group (n = 61, embryos pretreated with pronase 5 IU/ml for 1 min at 37 degrees C) or control group (n = 64, untreated embryos). Zona pellucida thickness was measured for each transferred embryo. The main outcome parameters were clinical pregnancy and implantation rates. The two groups were comparable regarding mean women's age, duration and indications of infertility, IVF outcome after fresh ETs, numbers and quality of fresh and frozen embryos, frozen-thawed embryo survival rates and blastomeres survival indexes. Despite a statistically significant decrease of zona pellucida thickness after pronase treatment [(mean +/- SD) 18.5 +/- 2.25 versus 14.5 +/- 2.75 microm; P < 0.0001], implantation (9.6 versus 9.2%) and clinical pregnancy rates (18.0 versus 17.2%) were not statistically different after FETs, with a similar mean number of embryos transferred between AH and control groups, respectively. Within the constraints of our protocol, partial enzymatic digestion of zona pellucida by pronase was not related with any benefit of the FET outcome especially concerning the implantation ability of frozen-thawed embryos.

Modifications to Improve the Efficiency of Zona-Free Mouse Nuclear Transfer

In the present study, some modifications were made to the zona-free nuclear transfer technique in the mouse in order to achieve greater efficiency. Firstly, a 1-h interval was allowed between cumulus removal and zona pellucida digestion. Secondly, acid Tyrode's was selected for zona pellucida removal, because contrary to pronase, it allows embryo survival during parthenogenic activation in the absence of calcium. Even when the exposure time to pronase was reduced to as little as 1 min or washed with fetal calf serum to inhibit the enzyme, the percentage of lysis during activation in the absence of calcium was still very high. Thirdly, electrofusion was performed at room temperature (21 degrees C), instead of 30 degrees C as in our previous experiments. Finally, embryos were cultured in groups of 12-15, instead of individually, using a "well of the wells" system during activation and culture. When compared, parthenogenic activated control embryos showed an increase in the development to blastocyst when cultured in pairs instead of individually. By the end of the experiments and using embryonic stem (ES) cells, there was a significant increase in fusion rate (1.5-fold increase) and in development to morula/blastocyst from cleaved reconstructed embryos (1.5-fold increase) when compared with the results before the modifications. A 2.4-fold increase in overall efficiency was achieved from the oocyte to morula/blastocyst stages.

Effect of oviductal and cumulus cells on zona pellucida and cortical granules of porcine oocytes fertilized in vitro with epididymal spermatozoa

The objective of this study was to evaluate the effects of porcine oviductal epithelial cell (POEC) monolayers and cumulus cells on the zona pellucida (ZP) and cortical granules (CG) of in vitro matured porcine oocytes. Denuded and cumulus-enclosed oocytes were exposed to POEC before or during in vitro fertilization (IVF). The functional effects of the co-culture system were the tested on the ZP resistance, measured by the time necessary to dissolve the ZP with 0.1% pronase, and the distribution and density of the cortical granules. CG density in the equator and cortex of each oocyte was evaluated by confocal microscopy after staining with fluorescein isothiocyanate-labelled peanut agglutinin (FITC-PNA). Both variables were assessed immediately after an in vitro maturation period (IVM group), 3 and 6 h after culture with or without (Control) oviductal cells (Experiment 1) and 3 h after insemination with frozen-thawed epididymal spermatozoa in the presence or absence (Control) of oviductal cells (Experiment 2). The time to dissolve the ZP of oocytes from IVM group was 440.4 ± 61.7 s and no difference was observed among groups in Experiment 1. In contrast, the density of CG was affected; oocytes pre-incubated for 6 h had a higher density than those pre-incubated for 3 h ( P < 0.001). Oocytes fertilized in vitro in the presence of POEC (Experiment 2) had a similar ZP digestion time as control oocytes 3 h after insemination. The presence of POEC during IVF as well as the presence of cumulus cells had no effect on the density and distribution of CG. However, a significant decrease in the density of CG was observed in the fertilized oocytes compared to in vitro matured oocytes ( P < 0.001). It is concluded that under the conditions employed the oviductal and cumulus cells in the perifertilization period had no effect on ZP hardening and CG density. However, an increase in CG density was observed when oocytes were maintained in culture. In addition, no hardening of ZP was observed after IVF, and denuded and cumulus-enclosed oocytes showed similar cortical reactions after insemination with epididymal spermatozoa regardless of the presence of POEC.

Derivation of a diploid human embryonic stem cell line from a mononuclear zygote.

IVF occasionally produces aneuploid zygotes with one or three pronuclei (PN). Routinely, these zygotes are discarded. The aim of this work was to establish human embryonic stem cell (hESC) lines from blastocysts resulting from abnormal fertilization. Abnormally fertilized zygotes were cultured to the blastocyst stage and, following zona pellucida digestion, zona-free blastocysts were placed on a mouse feeder layer. Culture of hESCs was carried out as described earlier. Six out of the nine developing blastocysts attached to the feeder layer. One hESC line, originating from a mononuclear zygote following ICSI, was successfully derived. This line displayed typical phenotype and embryonic surface markers, and exhibited the potential to develop into all three embryonic germ layers both in vitro (by embryoid body formation) and in vivo (teratoma generation). Genetic examination revealed normal diploid karyotype and heterozygotic appearance for metachromatic leukodystrophy (MLD). This method, which requires neither immuno nor mechanical removal of the trophectoderm, may facilitate the derivation of hESC lines in general, and those from abnormal embryos in particular. Furthermore, it is shown that aneuploid zygotes can be used as a source for normal hESC derivation and hold the potential to generate aneuploid hESC lines for research purposes.