Zona Pellucida Hardening Research Articles

Role of the Oviduct in Early Embryo Development

This review highlights the role of the oviduct in early embryo development, which has to fulfil many aligned and well-tuned tasks during early embryogenesis. The oviductal lining is subjected to dynamic changes to timely accomplish gamete transport, fertilization and embryo development and to deliver a competent and healthy conceptus to the endometrium which can implant and develop to term. Although knowledge about the role of the oviduct is limited, we know that embryos are very sensitive to the environment in which they develop. The success of in vitro embryo production techniques demonstrates that it is possible to bypass the oviduct during early development and, to a certain extent, replicate the conditions in vitro. However, comparative studies show that embryos developed in vivo are superior to their in vitro produced counterparts, underlining our relatively poor knowledge of the biology of the oviduct. Oviduct activity is orchestrated by various factors, depending on cyclic dynamics, which crucially affect the success of tubal transfer and/or (re-)collection of embryos in embryo transfer studies. This paper reviews data which demonstrate that in vivo culture of embryos in the bovine oviduct is a useful tool for the assessment of embryos developed under various conditions (e.g. superovulation vs single ovulation, lactating dairy cows vs non-lactating cows). It is concluded that more work in the field of early embryo development within the oviduct would contribute to improved ART protocols leading to healthy pregnancies and offspring.

Nanoscale characterization of the biomechanical hardening of bovine zona pellucida

The zona pellucida (ZP) is an extracellular membrane surrounding mammalian oocytes. The so-called zona hardening plays a key role in fertilization process, as it blocks polyspermy, which may also be caused by an increase in the mechanical stiffness of the ZP membrane. However, structural reorganization mechanisms leading to ZP's biomechanical hardening are not fully understood yet. Furthermore, a correct estimate of the elastic properties of the ZP is still lacking. Therefore, the aim of the present study was to investigate the biomechanical behaviour of ZP membranes extracted from mature and fertilized bovine oocytes to better understand the mechanisms involved in the structural reorganization of the ZP that may lead to the biomechanical hardening of the ZP. For that purpose, a hybrid procedure is developed by combining atomic force microscopy nanoindentation measurements, nonlinear finite element analysis and nonlinear optimization. The proposed approach allows us to determine the biomechanical properties of the ZP more realistically than the classical analysis based on Hertz's contact theory, as it accounts for the nonlinearity of finite indentation process, hyperelastic behaviour and material heterogeneity. Experimental results show the presence of significant biomechanical hardening induced by the fertilization process. By comparing various hyperelastic constitutive models, it is found that the Arruda-Boyce eight-chain model best describes the biomechanical response of the ZP. Fertilization leads to an increase in the degree of heterogeneity of membrane elastic properties. The Young modulus changes sharply within a superficial layer whose thickness is related to the characteristic distance between cross-links in the ZP filamentous network. These findings support the hypothesis that biomechanical hardening of bovine ZP is caused by an increase in the number of inter-filaments cross-links whose density should be higher in the ZP inner side.

The involvement of N‐glycosylation of zona glycoproteins during meiotic maturation in sperm–zona pellucida interactions of porcine denuded oocytes

The present study was conducted to delineate whether N-glycosylation of zona pellucida (ZP) glycoproteins occurred during meiotic maturation and whether this N-glycosylation played a role in sperm-ZP interactions of porcine cumulus denuded oocytes (DOs). After mechanical removal of cumulus cells from cumulus oocyte complexes (COCs), DOs were cultured for 44 h in in vitro maturation (IVM) culture. The experiments were carried out to determine the effects of tunicamycin, a specific N-glycosylation inhibitor, for various intervals during IVM on sperm-ZP interactions in porcine DOs. The results determined that DOs could induce meiotic maturation, although the maturation rate of DOs was earlier than that of COCs. In addition, N-glycosylation of ZP glycoproteins occurred during meiotic maturation and was crucial in sperm-ZP interactions, was responsible for sperm penetration, sperm binding to ZP and induction of acrosome reaction in ZP-bound sperm. However, the inhibition of N-glycosylation by tunicamycin during IVM did not influence ZP hardness and male pronuclear formation, indicating that this N-glycosylation was involved in the initial stage of fertilization. We conclude that 24-44 h of N-glycosylation of ZP glycoproteins during meiotic maturation was crucial in sperm penetration and sperm binding to ZP and the induction of acrosome reaction in sperm bound to ZP of porcine DOs.

Coculturing denuded oocytes during the in vitro maturation of bovine cumulus oocyte complexes exerts a synergistic effect on embryo development

The present study examined the effect of coculturing cumulus oocyte complexes (COCs) and denuded oocytes (DOs) during in vitro maturation (IVM) on nuclear and cytoplasmic maturation, zona pellucida (ZP) hardening, the pattern of fertilization and glutathione peroxidase 1 (GPX1) gene expression in the oocyte. Furthermore, the rate of embryonic development and the quality of blastocysts were examined for both COCs and DOs. Three IVM conditions were studied: 1) the coculture of 12 COCs and 60 DOs, 2) COC control with 12 COCs, and 3) DO control with 60 DOs. The IVM was performed in a 120-μl droplet of TCM199-based IVM medium. Following IVM, in vitro fertilization (IVF) and in vitro culture (IVC) were conducted separately for the COCs and DOs (DO coculture) from the IVM coculture group. Coculturing COCs and DOs increased the percentage of oocytes reaching the blastocyst stage and the total number of cells per blastocyst in both the COC coculture (44.4 ± 8.6 vs 26.7 ± 9.7%, P < 0.01, and 137.9 ± 24.9 vs 121.7 ± 21.1, P < 0.05) and the DO coculture (20.5 ± 5.0 vs 11.1 ± 2.5%, P < 0.01, and 121.9 ± 27.5 vs 112.3 ± 33.2, P < 0.05) compared to their respective control groups. The synergistic effects of coculturing were detected as increased nuclear and cytoplasmic maturation, the prevention of ZP hardening, increased monospermic fertilization and increased expression of GPX1 in the oocytes in response to endogenous oocyte-secreted factors. In conclusion, coculturing COCs and DOs may be an effective culture system for both intact COCs and immature DOs.

87 EFFECT OF CRYOPROTECTANT CONCENTRATION IN THE VITRIFICATION SOLUTION ON THE ZONA PELLUCIDA HARDENING AND SPERMATOZOA PENETRATION OF BOVINE OOCYTES

In the murine model, it has been shown that the high concentration of cryoprotectants required for vitrification can activate the oocytes through a process mediated by calcium influx. This activation induces the zona pellucida (ZP) hardening and affects the sperm penetration. This study aimed to evaluate the effect of exposure of bovine oocytes to the vitrification solutions (VS1 and VS2) in calcium-free medium with 3 concentrations of etilenglycol (EG) and dimetylsulfoxide (DMSO) on the oocyte activation. Cumulus oocyte complexes (COC) were matured in vitro (22 h), partially denuded through pipetting in medium with hyaluronidase, and subject to four treatments: T1, untreated (control); T2, exposed to 20% EG+0% DMSO (VS1) and then 40% EG+0% DMSO (VS2); T3, 10% EG+10% DMSO (VS1) and then 20% EG+20% DMSO (VS2); and T4, 0% EG+20% DMSO (VS1) and then 0% EG+40% DMSO (VS2). The contact with each VS was 3 min and 30 s, respectively. After this, the COC were matured up to 24 h. In Expt. 1, COC were denuded and placed in a solution of pronase E in PBS (1 mg mL–1) to determine the number of oocytes with ZP digested after 9 min of exposure to the enzyme. In Expt. 2, COC were fertilized in TALP medium with 50 mg mL–1 heparin and 1 million mL–1 sperm. After 12 h, COC were denuded and stained with bisbenzimide (Hoechst 33342) and examined under epi-fluorescence. The number of oocytes indicating spermatic penetration was determined by presence of intact sperm heads, spermatic pro-nucleus, or 2 polar bodies. Data were analysed by the PROC GENMOD (SAS; see Table 1). In Expt. 1, there were no differences in the percentage of oocytes without ZP after pronase treatment in groups T1, T2, and T3. The T4 group had the lowest percentage of digestion, and T3 was not different from T4. In Expt. 2 there were no differences in the percentage of sperm penetration between T2, T3, and T4. All treatments had lower values than T1. In conclusion, bovine oocytes undergo hardening of the ZP when put in contact with the cryoprotectants, and this effect was significantly increased with the use of DMSO. Moreover, there was a decrease in sperm penetration in all treated groups, indicating that the natural blocking of polyspermy depends not only on the hardening of the ZP, but another process that could act at the plasma membrane. It is possible that cryoprotectants, regardless of their concentration, may trigger this early block through a mechanism that would be independent of calcium. Table 1.Effect of EG and DMSO concentration in the VS on the ZP hardening and sperm penetration of bovine oocytes exposed to these solutions Acknowledgment: the National Research Agency through the grant PICT 2007/1205.

Alteration in the Mechanical Properties of Human Ovum Zona Pellucida Following Fertilization: Experimental and Analytical Studies

The unfertilized oocyte is surrounded by a spherical layer called the zona pellucida (ZP). The physical hardness of this layer plays a crucial role in fertilization and it is largely unknown because of the lack of appropriate measuring and modeling methods. Recently, considerable biomedical attentions have concentrated on determination of the mechanical properties of oocytes as a single cell. In order to investigate the biophysical characteristics of mammalian oocytes, a change in the elasticity of human ZP has been quantitatively evaluated before and after fertilization. Young’s modulus of ZP of metaphase-II (MII) and pronuclear (PN) stages have been estimated using two different protocols of the micropipette aspiration, step-by-step and continuous increase in pressure, in combination with proportional theoretical models. Experimental results clearly demonstrated that after fertilization the mean Young’s modulus of the ZP calculated from the step-by-step aspiration test (MII: 7.34 ± 1.36 kPa vs PN: 13.18 ± 1.17 kPa.) and continuous aspiration test (MII: 2.41 ± 0.75 kPa vs. PN: 4.43 ± 1.66 kPa) significantly increased, (p < 0.05). Mathematical Evaluation of the results shows that although the results of the two methods are different but both confirm that the hardening of ZP will increase following fertilization. As can be seen, different experimental methods can influence the choice of the models and this in turn will lead the mechanical properties to be found.

002. PIG SPERM EGG INTERACTION AND FORMATION OF A ZONA PELLUCIDA BINDING COMPLEX

In order to achieve fertilization sperm cells first need to successfully interact with the zona pellucida. Before reaching the zona pellucida the sperm cell undergoes extensive remodeling both in the male and female genital tract. These changes serve to mediate optimal recognition of the zona pellucida in the oviduct (primary zona pellucida binding). Optimal sperm-zona interactions are crucial for porcine oocyte fertilization: The zona pellucida- attached sperm cell is triggered to undergo the acrosome reaction and will also become hypermotile. Together these two responses allow the sperm cell to drill through the zona pellucida (secondary zona pellucida binding) this coincides with local sperm zona drilling so that a few sperm can reach the perivitellin space. This delaying strategy allows only one sperm cell in a given time-point to bind and fuse with the oocyte (fertilization) and thus minimizes the risk of polyspermy. The polyspermy block is essentially executed by the fertilized oocyte that immediately secretes its cortical granule content into the perivitellin space. This content blocks sperm-oocyte fusion either by sticking to 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 drilling. Note that zona pellcuida interactions under pig IVF conditions relatively frequently result in polyspermy. It is not clear whether this is also the case in vivo after natural mating. More importantly we do not know how other artificial reproductive technologies affect polyspermy rates. This is especially relevant for new sperm treatments and insemination technologies in which sperm are activated by capacitation media essentially mimicking the IVF media. Therefore, better understanding of sperm activation and of the arrangement of proteins involved in zona pellucida interactions are relevant for designing strategies to further improve pig reproduction.

Estimating Young's modulus of zona pellucida by micropipette aspiration in combination with theoretical models of ovum.

The zona pellucida (ZP) is the spherical layer that surrounds the mammalian oocyte. The physical hardness of this layer plays a crucial role in fertilization and is largely unknown because of the lack of appropriate measuring and modelling methods. The aim of this study is to measure the biomechanical properties of the ZP of human/mouse ovum and to test the hypothesis that Young's modulus of the ZP varies with fertilization. Young's moduli of ZP are determined before and after fertilization by using the micropipette aspiration technique, coupled with theoretical models of the oocyte as an elastic incompressible half-space (half-space model), an elastic compressible bilayer (layered model) or an elastic compressible shell (shell model). Comparison of the models shows that incorporation of the layered geometry of the ovum and the compressibility of the ZP in the layered and shell models may provide a means of more accurately characterizing ZP elasticity. Evaluation of results shows that although the results of the models are different, all confirm that the hardening of ZP will increase following fertilization. As can be seen, different choices of models and experimental parameters can affect the interpretation of experimental data and lead to differing mechanical properties.

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.

Recombinant Mouse Sperm ZP3-binding Protein (ZP3R/sp56) Forms a High Order Oligomer That Binds Eggs and Inhibits Mouse Fertilization in Vitro

Many candidates have been proposed as zona pellucida-binding proteins. Without precluding a role for any of those candidates, we focused on mouse sperm protein ZP3R/sp56, which is localized in the acrosomal matrix. The objective of this study was to analyze the role of ZP3R/sp56 in mouse fertilization. We expressed recombinant ZP3R/sp56 as a secreted protein in HEK293 cells and purified it from serum-free, conditioned medium. In the presence of reducing agents, the recombinant ZP3R/sp56 exhibited a molecular weight similar to that observed for the native ZP3R/sp56. Reminiscent of the native protein, recombinant ZP3R/sp56 formed a high molecular weight, disulfide cross-linked oligomer consisting of six or more monomers under non-reducing conditions. Recombinant ZP3R/sp56 bound to the zona pellucida of unfertilized eggs but not to 2-cell embryos, indicating that the changes that take place in the zona pellucida at fertilization affected the interaction of this protein with the zona pellucida. The extent of in vitro fertilization was reduced in a dose-dependent manner when unfertilized eggs were preincubated with recombinant ZP3R/sp56 (74% drop at the maximum concentrations assayed). Eggs incubated with the recombinant protein showed an absence of or very few sperm in the perivitelline space, suggesting that the reduction in the fertilization rate is caused by the inhibition of sperm binding and/or penetration through the zona pellucida. These results indicate that sperm ZP3R/sp56 is important for sperm-zona interactions during fertilization and support the concept that the acrosomal matrix plays an essential role in mediating the binding of sperm to the zona pellucida.

Hardening of the zona pellucida of unfertilized eggs can reduce polyspermic fertilization in the pig and cow

One of the proposed mechanisms of polyspermy block is an increased resistance of the zona pellucida (ZP) to proteolytic digestion (ZP hardening) as a consequence of cortical granule exocytosis that occurs soon after fertilization. However, evidence is available that the zonae pellucidae of freshly ovulated pig and cow oocytes harden considerably before fertilization. It was thought that such pre-fertilization ZP hardening could be involved in the control of polyspermy, and its lack in the oocytes matured in vitro could be one of the reasons for the extremely high incidence of polyspermy in pig in vitro fertilization (IVF). To test this hypothesis, two different types of cross-linking reagents were employed and their effects on ZP hardening and IVF efficiency were examined. The sulfhydryl-reactive cross-linkers produced a slight hardening of ZP (P<0.001) of treated oocytes compared with control oocytes, and totally inhibited sperm penetration into pig oocytes after IVF. In the cow, sperm penetration into eggs was reduced to 10%. It is proposed that formation of disulfide bonds in ZP or blocking of SH groups in the oocyte plasma membrane proteins prevents sperm penetration. An amine-reactive cross-linker was then assayed and produced strong ZP hardening, increasing the incidence of monospermy in both pig and cow oocytes after fertilization. When the cross-linker concentration was optimized, a 45% improvement for pig IVF efficiency was reached. It is proposed that the observed physiological ZP hardening is a mechanism to control polyspermy, differentially affecting various mammalian species and can be imitated by the use of amine-reactive cross-linkers during IVF.

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).

P-197: Assisted hatching using either acidic tyrode solution or laser pluse improves the implantation of vitrified human blastocysts

Recent attention has been focused on vitrification as a convenient method for cryopreservation of human embryos. However, steps of cryopreservation including exposure to cryoprotectants (CPA) are proven to cause the hardening of zona pellucida, which might disturb the hatching and implantation process. Therefore, it will be possible to improve the implantation rate with AH on vitrified human BLs, prior to transfer.

O-154: The effect of chelating cytosolic calcium on the viability of vitrified mouse oocytes

Vitrification is an alternative to cryopreserve MII oocytes, however, the embryonic potential of oocytes vitrified by choline chloride DMSO-EG-sucrose vitrification system is still low. Recently, it has been reported that DMSO and EG may induce the increase of intracellular calcium. This increase in calcium may have detrimental effects: premature cortical granule release, zona pellucida hardening, metaphase-to-anaphase transition, and/or egg activation. Preventing the increase of intracellular calcium by calcium-free media combined with a cell membrane permeable calcium chelator was carried out to assess the viability of mouse oocytes exposed to a choline chloride DMSO-EG-sucrose based vitrification system. Prospective study using mouse oocytes at a University-based research laboratory. MII oocytes were collected from 6-8 weeks old CB6F1 mice by hyperstimulation of 5IU PMSG and 5IU hCG. MII oocytes were denuded by brief exposure to 100iu/ml hyaluronidase, and then cultured in vitro less than 1hour prior to exposure of choline chloride DMSO-EG-sucrose vitrification system. Oocytes were loaded in pulled straw and heat-sealed just before plunging directly into liquid nitrogen. The experimental groups include presence or absence of calcium in the vitrification system, and concentration of BAPTA AM, a cell permeant calcium chelator. Group 1, calcium-based PBS; Group 2: calcium-free PBS; Groups 3 - 7: calcium-free PBS + varying concentrations (0.1μM, 0.5μM, 5μM, 50μM and 500μM) of BAPTA AM. After thawing, eggs were allowed to recover for one hour. Surviving oocytes were inseminated by capacitated sperm. Embryos were cultured in sequential Quinn’s Media (Sage) supplemented with 6% plasmanate, and monitored at day 1 and day 5(Table 1). Day 5 blastocysts were graded and corroborated by double staining of Oct 3/4 (detect functional inner cell mass) and of Hoechst 33342 (detect total embryonic cell number). Mouse oocytes, in response to 0.1μM BAPTA based vitrification system, display high rate of survival (97%) and cleavage (71%), comparable to that response to calcium based and calcium-free based vitrification system. However, raising the concentration of BAPTA to 0.5μM and above, the rate of survival (<82%) and cleavage (<55%) declined dramatically. Furthermore, more embryos in group of 0.1μM BAPTA develop into good (45% ≥3BB) and excellent (32% ≥4AA) blastocyst than in other groups. The group of 0.5μM BAPTA yielded less blastocysts, but still generated a number of good and excellent blastocysts comparable to the group of 0.1μM BAPTA. BAPTA influences the viability of mouse oocytes in a concentration dependant manner. BAPTA, at a concentration of 0.1μM (equivalent to cytosolic Ca2+ concentration -10-7 M = 0.1μM), exhibits the maximum benefit to improve the viability of vitrified oocytes. The effect of BAPTA on post-implantation embryos and full-term offspring needs to be addressed in further study.

1,2-propanediol and the type of cryopreservation procedure adversely affect mouse oocyte physiology

The aim of this work was to examine the effect of 1,2-propanediol (PrOH) and type of cryopreservation procedure (slow freezing and vitrification) on oocyte physiology. Intracellular calcium of mouse metaphase II (MII) oocytes was quantified by fluorescence microscopy. The effect of PrOH on cell physiology was further assessed through analysis of zona pellucida hardening and cellular integrity. Protein profiles of cryopreserved oocytes were generated by time-of-flight mass spectrometry (TOF-MS). PrOH caused a protracted increase in calcium, which was sufficient to induce zona pellucida hardening and cellular degeneration. Using 'nominally calcium free' media during PrOH exposure significantly reduced the detrimental effects. Proteomic analysis identified numerous up- and down-regulated proteins after slow freezing when compared with control and vitrified oocytes. Using such approaches to assess effects on cellular physiology is fundamental to improving assisted reproduction techniques (ART). This study demonstrates that PrOH causes a significant rise in intracellular calcium. Using calcium-free media significantly reduced the increase in calcium and the associated detrimental physiological effects, suggesting that calcium-free media should be used with PrOH. In addition, analysis of the oocyte proteome following cryopreservation revealed that slow freezing has a significant effect on protein expression. In contrast, vitrification had a minimal impact, indicating that it has a fundamental advantage for the cryopreservation of oocytes.

Do age and extended culture affect the architecture of the zona pellucida of human oocytes and embryos?

Advanced female age and extended in vitro culture have both been implicated in zona pellucida (ZP) hardening and thickening. This study aimed to determine the influence of (i) the woman's age and (ii) prolonged in vitro culture of embryos on ZP thickness and density using non-invasive polarized light (LC-PolScope) microscopy. ZP thickness and density (measured as retardance) were determined in oocytes, embryos and blastocysts in women undergoing intracytoplasmic sperm injection (ICSI) in two age groups (older, > 38 years; younger, < or = 38 years). A total of 193 oocytes from 29 patients were studied. The younger group contained 100 oocytes and the older group 93 oocytes. The ZP was significantly thicker in metaphase II oocytes in the older group compared with the younger group (mean +/- SD: 24.1 +/- 2.5 microm vs 23.1 +/- 3.3 microm; p = 0.01) but ZP density was equal (2.8 +/- 0.7 nm). By day 2 of culture, embryos from the two groups had similar ZP thickness (22.2 +/- 2.2 microm vs 21.7 +/- 1.6 microm; p = 0.28) and density (2.9 +/- 0.7 nm vs 2.8 +/- 0.8 nm; p = 0.57). For the embryos cultured to blastocyst (older: n = 20; younger: n = 18) ZP thickness was similar in the two groups (19.2 +/- 2.7 microm vs 19.1 +/- 5.0 microm; p = 0.8) but thinner than on day 2. The older group had significantly denser ZP than the younger group (4.2 +/- 0.5 nm vs 3.3 +/- 1.0 nm, p < 0.01). Blastocysts from both groups had significantly denser ZP than their corresponding day 2 embryos (older: 4.2 +/- 0.5 nm vs 2.9 +/- 0.7 nm, p < 0.001; younger: 3.3 +/- 1.0 nm vs 2.8 +/- 0.8 nm, p = 0.013). It is concluded that there is little relationship between ZP thickness and its density as measured by polarized light microscopy. While ZP thickness decreases with extended embryo culturing, the density of the ZP increases. ZP density increases in both age groups with extended culture and, interestingly, more in embryos from older compared with younger women.

181 DIFFERENCES EXIST IN ZONA PELLUCIDA HARDNESS BETWEEN IN VIVO- AND IN VITRO-GENERATED BOVINE EMBRYOS

The zona pellucida (ZP) of mammalian ova plays an important role during maturation, fertilization, and early embryonic development. The hardness of ZP of mammalian ova has been mainly evaluated by a biochemical method, such as a difference in ova dissolution speed with an enzyme or an acid reagent treatment. However, the physical hardness of ZP in bovine embryos is largely unknown. Recently, we developed a system measuring the physical hardness of ZP of mammalian ova with a micro bio-sensor (MBS). The objective of this study was to examine the physical ZP hardness of in vivo- and in vitro-derived bovine embryos using an MBS. In the measurement system, the thin glass needle was connected at the tip of a piezo-electric ceramic tactile sensor, and the sample was pushed into a sensor at fixed speed under the computer-controlled micromanipulation system. The change in frequency at the time of displacement of the sample into the sensor was recorded using the computer program. Measurement of gelatin samples of known concentration (4 to 8%) was used to determine the basic characteristic of a sensor and to make a standard curve. In vivo-generated embryos were recovered from 6 superovulated Japanese Black cows with multiple injection of FSH. On Day 7 after insemination, morulae (M), early blastocysts (EB), and blastocysts (BL) were recovered by nonsurgical flushing of uterine horns. In vitro-generated embryos were produced as described earlier (Yoshida et al. 1998 J. Vet. Med. Sci. 60, 549–554). The M, EB, and BL at Days 5 to 7 of post-insemination in vitro were used for measurement of ZP hardness. When a sensor made contact with a harder gelatin sample, the change in frequency was large; the change in frequency has the characteristic of being small for a softer gelatin sample. By comparison with a standard curve, ZP hardness converted into gelatin concentration for each stage of bovine embryos generated in vivo was 3.95% (M: n = 9), 4.14% (EB: n = 32), and 3.92% (BL: n = 14), respectively. On the other hand, ZP hardness of bovine embryos generated in vitro was 3.42% (M: n = 56), 3.33% (EB: n = 36), and 3.25% (BL: n = 23), respectively. There was a significant difference (P &amp;lt; 0.01) in the hardness of ZP between in vivo- and in vitro-generated bovine embryos.

Calcium-free vitrification reduces cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mouse oocytes

Despite the success of embryo cyropreservation, routine oocyte freezing has proved elusive with only around 200 children born since the first reported birth in 1986. The reason for the poor efficiency is unclear, but evidence of zona pellucida hardening following oocyte freezing indicates that current protocols affect oocyte physiology. Here we report that two cryoprotectants commonly used in vitrification procedures, dimethyl sulfoxide (DMSO) and ethylene glycol, cause a large transient increase in intracellular calcium concentration in mouse metaphase II (MII) oocytes comparable to the initial increase triggered at fertilization. Removal of extracellular calcium from the medium failed to affect the response exacted by DMSO challenge, but significantly reduced the ethylene glycol-induced calcium increase. These results suggest that the source of the DMSO-induced calcium increase is solely from the internal calcium pool, as opposed to ethylene glycol that causes an influx of calcium across the plasma membrane from the external medium. By carrying out vitrification in calcium-free media, it was found that zona hardening is significantly reduced and subsequent fertilization and development to the two-cell stage significantly increased. Furthermore, such calcium-free treatment appears not to affect the embryo adversely, as shown by development rates to the blastocyst stage and cell number/allocation. Since zona hardening is one of the early activation events normally triggered by the sperm-induced calcium increases observed at fertilization, it is possible that other processes are negatively affected by the calcium rise caused by cryoprotectants used during oocyte freezing, which might explain the current poor efficiency of this technique.