Modifications Of Zona Pellucida Research Articles

The impact of laser-assisted hatching on the outcome of frozen human embryo transfer cycles.

Biochemical modifications of zona pellucida (ZP) result in zona hardening. Zona hardening (ZH) is induced by several factors such as advancing maternal age, in vitro culture conditions and cryopreservation and adversely effects implantation. The objective of the clinical study was to determine whether or not laser-assisted hatching (LAH) applied on day 3 frozen embryos improves the outcome of frozen embryo transfer (FET) cycles in patients with recurrent implantation failure and/or advanced female age. In total, 413 patients of different ages with recurrent implantation failure (maximum three cycles) were involved into the study. Patients were allocated randomly into LAH and control groups. On the day of FET, after thawing and just before FET, the ZP was thinned using a laser system. In the control group no treatment was applied on frozen embryo before transfer. The main outcome measures were clinical pregnancy rate. Overall, the results indicate a tendency that LAH increased (P = 0.08) clinical pregnancy. However, for patients older than 37 years, LAH increased pregnancy rates significantly (P = 0.03). In the LAH and control groups, the age of patients and the number of transferred embryos influenced pregnancy rates (P = 0.01). For patients older than 37 years, no effect of number of transferred embryos was detected (P = 0.14). The incidence of multiple pregnancies also increased in the LAH group (P = 0.01). In conclusion, in older woman, to overcome the negative effect of zona hardening, LAH could be performed on frozen embryos as a routine strategy before FET in frozen cycles in order to increase the possibility of pregnancy formation.

Wild-Derived Inbred Mice No Longer ART-Resistant1

Wild-Derived Inbred Mice No Longer ART-Resistant1

Raman microspectroscopy as a non-invasive tool to assess the vitrification-induced changes of ovine oocyte zona pellucida

Cryopreservation-induced modifications of zona pellucida (ZP) have been explored to a lesser extent compared to other oocyte compartments. Different methods have been applied to identify ZP changes, but most of them are invasive and measure only few properties of ZP.Raman microspectroscopy (RMS) is a powerful technique for studying the molecular composition of cells but to date few studies have been performed on the oocytes using this method. The aim of the present study is to investigate the structural modifications of ZP of vitrified/warmed in vitro matured ovine oocytes by means of RMS. Cumulus–oocyte complexes were recovered from the ovaries of slaughtered adult sheep, matured in vitro and vitrified following the Minimum Essential Volume method using cryotops. ZPs of vitrified/warmed oocytes (VITRI), were exposed to vitrification solutions but not cryopreserved (CPA-exp) and untreated oocytes (CTR) were analyzed by RMS. We focused our analysis on the ZP protein and carbohydrate components by analyzing the 1230–1300cm−1 amide III region and the 1020–1140cm−1 spectral range in RMS spectra, respectively.The spectral profiles in the ranges of proteins and carbohydrates were comparable between CTR and CPA-exp ZPs, whereas VITRI ZPs showed a significantly altered protein secondary structure characterized by an increase in β-sheet content and a decrease in the α-helix content. A significant modification of the carbohydrate components was also observed. This study demonstrates that vitrification of ovine oocytes induces biochemical changes of ZP related to the secondary structure of proteins and carbohydrate residues. Cryoprotectants do not strongly alter the molecular composition of ZP which is affected mainly by cooling. Raman technology offers a powerful and non-invasive tool to assess molecular modifications induced by cryopreservation in oocytes.

What controls polyspermy in mammals, the oviduct or the oocyte?

A block to polyspermy is required for successful fertilisation and embryo survival in mammals. A higher incidence of polyspermy is observed during in vitro fertilisation (IVF) compared with the in vivo situation in several species. Two groups of mechanisms have traditionally been proposed as contributing to the block to polyspermy in mammals: oviduct-based mechanisms, avoiding a massive arrival of spermatozoa in the proximity of the oocyte, and egg-based mechanisms, including changes in the membrane and zona pellucida (ZP) in reaction to the fertilising sperm. Additionally, a mechanism has been described recently which involves modifications of the ZP in the oviduct before the oocyte interacts with spermatozoa, termed "pre-fertilisation zona pellucida hardening". This mechanism is mediated by the oviductal-specific glycoprotein (OVGP1) secreted by the oviductal epithelial cells around the time of ovulation, and is reinforced by heparin-like glycosaminoglycans (S-GAGs) present in oviductal fluid. Identification of the molecules contributing to the ZP modifications in the oviduct will improve our knowledge of the mechanisms of sperm-egg interaction and could help to increase the success of IVF systems in domestic animals and humans.

Biological Modifications of Zona Pellucida Affecting Resistance to Proteases Digestion, Sperm Binding, and Monospermy Are Mediated by Oviduct-Specific Glycoprotein in Pig and Cow.

Biological Modifications of Zona Pellucida Affecting Resistance to Proteases Digestion, Sperm Binding, and Monospermy Are Mediated by Oviduct-Specific Glycoprotein in Pig and Cow.

Zona Pellucida Modifications and Fertilization Following Metaphase II Oocyte Vitrification and Warming

Oocyte cryopreservation holds promise for preserving fertility in women undergoing cancer therapies, and can be achieved by slow-rate freezing or vitrification. Oocyte slow-rate freezing compromises zona pellucida (ZP) function, necessitating insemination/fertilization by intracytoplasmic sperm injection (ICSI). Whether oocyte vitrification causes similar ZP modifications is unknown. Objectives were to evaluate effects of vitrification/warming on oocyte fertilization by conventional in vitro insemination, ZP solubility, cortical granule (CG) release, fertilization by ICSI, and ability of fetuin to prevent ZP modifications.

Anti-hyaluronidase oligosaccharide derived from chondroitin sulfate a effectively reduces polyspermy during in vitro fertilization of porcine oocytes.

The present study was conducted to examine the effects of chondroitin sulfate A-derived oligosaccharide (ChSAO) on hyaluronidase activity and in vitro fertilization (IVF) parameters. The activity of hyaluronidase extracted from preincubated boar sperm was completely blocked by ChSAO at concentrations of 10 microg/ml or higher. After in vitro maturation of porcine cumulus-oocyte complexes, some oocytes were freed from their cumulus cells, and cumulus-intact or cumulus-free oocytes were inseminated with sperm in IVF medium containing various concentrations of ChSAO (0.1-100 microg/ml). In cumulus-intact oocytes, the penetration and the polyspermy rates (39% and 28%, respectively) were significantly decreased by treatment with 100 microg/ml ChSAO compared with those of oocytes treated without ChSAO (63% and 52%, respectively). On the contrary, in cumulus-free oocytes, the addition of 10-100 microg/ml ChSAO significantly reduced the polyspermy rate compared with the control (25-30% versus 53%, respectively), whereas ChSAO had no effect on sperm penetration. Interestingly, ChSAO added to IVF medium significantly decreased the number of sperm bound to the zona pellucida (ZP) of cumulus-free oocytes in a concentration-dependent manner between 0.1 and 100 microg/ml. However, ChSAO had no effect on the time course change in ZP modification after oocyte activation by electrostimulation and the incidence of the acrosome-reacted sperm. Treatment with 100 microg/ml ChSAO during IVF of cumulus-free oocytes significantly increased the proportion of development to the blastocyst stage after in vitro insemination. Therefore, the present findings indicate that hyaluronidase-inhibitory ChSAO is an efficient probe for promoting normal fertilization process in terms of an effective decrease in the incidence of polyspermy during IVF of porcine oocytes.

Analysis of zona pellucida modifications due to cortical granule exocytosis in single porcine oocytes, using enhanced chemiluminescence

The present study was carried out to determine whether modification of zona pellucida (ZP) of a single oocyte following the cortical granule (CG) exocytosis induced by electrical stimulation could be analyzed using enhanced chemiluminescent (ECL) detection of the biotinylated ZP in a porcine oocyte. When a biotinylated ZP derived from a single oocyte matured in vitro was subjected to SDS-PAGE, 3 major bands (ZP1, ZP2 and ZP3) were observed following ECL detection. In these oocytes, CGs staining with fluorescein isothiocyanate (FITC)labeled peanut agglutinin (FITC-PNA) had formed a monolayer underlying the plasma membrane. Electrical stimulation to induce artificial activation caused a decline in the fluorescent intensity of the CGs with a concomitant decrease in the amounts of ZP1 and ZP2 bands. However, the mobility changes of ZP1 and ZP2 on SDS-PAGE were not found under the inhibitory condition of the CG exocytosis in which oocytes were treated with ethylene glycol-bis(β-aminoethyl ether) N, N, N′, N′-tetraacetic acid (EGTA) or 1, 2-bis(2-aminophenoxy)ethane-N, N, N′, N′-tetraacetic acid tetrakis(acetoxymethyl) ester (BAPTA/AM). In addition, when a time-dependent decrease in amounts of ZP1 and ZP2 bands on SDS-PAGE was observed in a single oocyte during activation, a maximum decrease in these bands was detected in oocytes incubated for at least 3.5 h after electrical stimulation. These results show that the method employed, ECL detection of the biotinylated ZP of a single oocyte, is a valuable tool for the analysis of ZP modification resulting from a decrease in amounts of ZP1 and ZP2 glycoproteins in combination with exocytosis of CGs, and that the prolonged period after activation is required for complete ZP modification in porcine oocytes.

Time Course Analyses of Cortical Granule Exocytosis and Zona Pellucida Modifications after Artificial Activation in In-Vitro Matured Porcine Oocytes.

The present study was carried out to determine the time-dependent changes in the cortical granule (CG) distribution and the glycoprotein compositions of zona pellucida (ZP) after egg activation by electrostimulation in porcine oocytes matured in vitro. CG exocytosis was observed by staining with fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA) and laser confocal microscopy, and ZP modification was analyzed by using enhanced chemiluminescent (ECL) detection of the biotinylated ZP derived from a single oocyte. In the oocytes matured in vitro, CGs staining with FITC-PNA had formed a continuous monolayer underlying the oolemma, and three major bands (ZP1, ZP2 and ZP3) were observed in a biotinylated ZP subjected to SDS-PAGE followed by ECL detection. Electrostimulation to induce artificial activation caused a decline in the fluorescent intensity of the CGs with a concomitant decrease in the amounts of ZP1 and ZP2 bands, but CG exocytosis according to oocyte activation occurred slowly, and the incidence of oocytes with complete CG exocytosis was first observed 1.0 h after electrostimulation (9%). Similarly, the complete ZP modification, i.e. a maximum decrease in amounts of ZP1 and ZP2 bands, required more than 3.5 h after artificial activation, and the ZP dissolution time caused by 0.1% protease action increased apparently as incubation time was prolonged after electrostimulation. Results of these experiments suggest that the slower ZP modification due to the delayed CG exocytosis in porcine oocytes may reflect the remarkable increase in polyspermy frequency in in-vitro fertilization.

Spontaneous Activation of Ovulated Mouse Eggs: Time-dependent Effects on M-phase Exit, Cortical Granule Exocytosis, Maternal Messenger Ribonucleic Acid Recruitment, and Inositol 1,4,5-trisphosphate Sensitivity1

With increasing time after ovulation, mammalian eggs become more sensitive to agonists of activation in vitro or may undergo spontaneous activation in vivo. We have tested the hypothesis that postovulatory eggs undergo time-dependent cell cycle and cytoplasmic changes that result in a partially activated state, accounting for their time-dependent susceptibility to activate. In vivo changes in key activation markers in mouse eggs were quantified at 13, 16, and 22 h post-hCG (1, 4, and 10 h postovulation). Spontaneous activation was first detected at 16 h, with a 20-25% decrease in the activities of histone H1 and mitogen-activated protein (MAP) kinases and with 3% of eggs undergoing both anaphase onset and a partial loss of cortical granules. By 22 h, more than 60% of eggs were in anaphase, H1 and MAP kinase activities had decreased 40-50%, the extent of zona pellucida modification had increased, and proteins normally synthesized after fertilization had appeared. Pronuclear formation in response to inositol 1,4,5-trisphosphate injection increased dramatically from 10% at 13 h to about 40% and 90% at 16 h and 22 h, respectively. The partial decreases (less than those after fertilization) in H1 and MAP kinase activities provide a likely biochemical basis for the increased sensitivity of eggs to agonists, seen over time, that results in pronuclear formation. Also, all of these time-dependent changes caution against the use of mouse eggs > 16 h after hCG administration in studying the mechanism of normal fertilization and have implications for animal and human in vitro fertilization.

Modifications of the mouse zona pellucida during oocyte maturation: inhibitory effects of follicular fluid, fetuin, and alpha 2HS-glycoprotein.

Mouse oocyte maturation in vivo or in vitro is accompanied by a precocious release of cortical granules. When oocytes are matured in vitro in the absence of serum, cortical granule exocytosis results in modifications of the zona pellucida (ZP) that are known to constitute the zona pellucida block to polyspermy. Fetuin, a glycoprotein found in newborn calf serum, can inhibit these maturation-associated changes in the zona pellucida. In this report we demonstrate that the human fetuin homologue, alpha 2-HS-glycoprotein, is present in human follicular fluid. Human follicular fluid, or purified human alpha 2-HS-glycoprotein, inhibits the maturation-associated conversion of mouse ZP2 to ZP2f in a concentration-dependent manner. Although the concentration of alpha 2HS-glycoprotein in human follicular fluid is high enough to account for the inhibitory effect of human follicular fluid on this maturation-associated conversion, human follicular fluid immunodepleted of alpha 2HS-glycoprotein retains its full inhibitory activity. Thus, other inhibitory molecules are likely to be present in follicular fluid and contribute to the inhibition of the maturation-associated conversion of ZP2.

Regulation of the Polyspermy Block in the Mouse Egg: Maturation-Dependent Differences in Cortical Granule Exocytosis and Zona Pellucida Modifications Induced by Inositol 1,4,5-Trisphosphate and an Activator of Protein Kinase C1

Germinal vesicle (GV)-intact fully grown mouse oocytes do not undergo cortical granule (CG) exocytosis in response to A23187 treatment, whereas metaphase II (MII)-arrested eggs do. This differential response may reflect the development of the ability of the egg to undergo CG exocytosis, which is responsible for the biochemical modification of the glycoprotein ZP2 in the zona pellucida. Accordingly, we compared in these two stages the ability of 12-O-tetradecanoyl phorbol 13-acetate (TPA) or inositol 1,4,5-trisphosphate (IP3) to promote CG exocytosis and/or the ZP2 to ZP2f conversion; these agents are known to stimulate early events of mouse egg activation. TPA (10 ng/ml) treatment for 60 and 120 min resulted in a 25% and 52% CG loss in GV-intact oocytes and a 38% and 76% loss in MII eggs, respectively; fertilization resulted in a CG loss of approximately 70-80%. Although a similar extent of ZP2 to ZP2f conversion was observed in oocytes and eggs after a 120-min TPA treatment (approximately 70-80%), a greater extent of conversion was observed in oocytes after a 60-min treatment (80% for oocytes, 50% for eggs). Microinjection of IP3 (final concentration 1 microM) into MII eggs resulted in an extent of ZP2 conversion similar to that observed following fertilization, whereas little conversion occurred in GV-intact oocytes similarly injected. These results indicate that a protein kinase C sensitivity develops prior to meiotic maturation, whereas responsiveness to IP3 develops after maturation has resumed. We propose that the regulatory mechanism involving an IP3-mediated calcium release is deficient in GV-stage oocytes.

Role of G proteins in mouse egg activation: Stimulatory effects of acetylcholine on the ZP2 to ZP2 f conversion and pronuclear formation in eggs expressing a functional m1 muscarinic receptor

Sperm-mediated egg activation may be analogous to ligand-mediated signal transduction through G protein-coupled receptors. We investigated this possibility in the mouse egg by microinjecting mouse oocytes with an m1 muscarinic receptor mRNA. Following oocyte maturation in vitro, the metaphase II-arrested eggs were treated with acetylcholine and its effect was examined on zona pellucida modifications and pronuclear formation, which are end points of early and late egg activation, respectively. Treatment of these eggs with acetylcholine reveals that both the ZP2 to ZP2 f conversion and pronuclear formation occur. Atropine and microinjected GDPβS block the acetylcholine-induced ZP2 conversion, suggesting that the acetylcholine effects are mediated via a functional G protein-coupled m1 receptor. The acetylcholine-induced ZP2 conversion, however, is not inhibited by pertussis toxin under conditions in which greater than 90% of the endogenous G i is inactivated by ADP ribosylation. The presence of a pertussis toxin-insensitive G protein, G q, is detected by immunoblotting; this G protein could be a candidate to mediate the pertussis toxin-insensitive effects of acetylcholine. Results of these experiments are consistent with the hypothesis that receptor-mediated G protein activation may play a role in egg activation.

Modifications of the Mouse Zona Pellucida during Oocyte Maturation and Egg Activation: Effects of Newborn Calf Serum and Fetuin1

A precocious but limited loss of cortical granules (CG) occurs during mouse oocyte maturation both in vivo and in vitro. Although CG loss during maturation in vivo is not associated with changes in the zona pellucida (ZP), a maturation-associated conversion of ZP2 to ZP2f occurs during oocyte maturation in vitro in serum-free medium. We now demonstrate that a maturation-associated change of ZP3 to ZP3f, as assessed by a reduction in sperm binding, also occurs during maturation in vitro in serum-free medium, and that both newborn calf serum (NCS) and fetuin, each of which inhibits the ZP2 conversion, also inhibit the ZP3 conversion. The concentration-dependence of the NCS- and fetuin-mediated inhibition of the ZP2 conversion, coupled with the concentration of fetuin present in NCS, is consistent with fetuin being the component present in NCS that is primarily responsible for this inhibition. Although NCS can inhibit the ZP modifications that occur during oocyte maturation in vitro, ionophore treatment of eggs, which results in an extensive release of CGs over a short period of time, overcomes the inhibitory effect of NCS on the ZP2 conversion. Results of these studies suggest a potential regulatory function of serum-derived components in the formation of a fertilizable egg.

Zona pellucida modifications in the mouse in the absence of oocyte activation

Mouse oocytes arrested in metaphase II exhibit zona hardening and a reduced fertilization rate after exposure to the cryoprotectant dimethylsulfoxide (Johnson J, In Vitro Fertil Embryo Transfer 6:168-175, 1989) but do not undergo parthenogenetic activation (Johnson and Pickering, Development 100:313-324, 1987). This paper shows that dimethylsulfoxide causes proteolytic modification of the zona pellucida glycoprotein ZP2 and inhibition of sperm binding. These effects of dimethylsulfoxide are caused by premature exocytosis of the cortical granules, a process that is initiated usually on fertilization. A model for the mechanism of action of dimethylsulfoxide is proposed based on the combined effects of cytoskeletal modification and osmotic shock. The presence of serum before and during the exposure to dimethylsulfoxide was found to reduce significantly these deleterious effects on the mouse zona pellucida without inhibiting the cortical granule release. These results highlight the suitability of dimethylsulfoxide as a tool to study the mechanisms leading to cortical granule release. Use of dimethylsulfoxide allows the separation of oocyte parthenogenetic activation from cortical granule release, and addition of serum allows separation of cortical granule release from the action of the cortical granule contents. Their use allows a dissection of the mechanisms underlying each of these three related events.

Precocious loss of cortical granules during mouse oocyte meiotic maturation and correlation with an egg-induced modification of the zona pellucida

Fertilization results in cortical granule exocytosis, which is thought to be involved in modifications of the zona pellucida that constitute the zona pellucida block to polyspermy. A previous report demonstrated that a decrease in the number of Lens culinaris agglutinin-staining granules, which are likely to be cortical granules, occurred during in vivo mouse oocyte maturation with arrest at metaphase II, as well as the formation of a cortical granule-free domain in the area of the metaphase II spindle ( T. Ducibella, E. Anderson, D. F. Albertini, J. Aalberg, and S. Rangarajan, 1988, Dev. Biol. 130, 184–197). We extend these observations by reporting here that germinal vesicle-intact oocytes matured in vitro to metaphase II in either the absence or the presence of serum develop a cortical granule-free domain and have reduced numbers of cortical granules when compared to germinal vesicle-intact oocytes; these changes are similar to those of oocytes matured in vivo. The reduction in the number of cortical granules requires germinal vesicle breakdown, since it is prevented by dibutyryl cAMP, which inhibits germinal vesicle breakdown in vitro. The ability of oocytes to respond to the calcium ionophore A23187 with a reduction in the number of cortical granules is also associated with meiotic maturation and develops between 7 and 12 hr after initiation of maturation. The maturation-associated reduction in the number of cortical granules is likely to represent cortical granule exocytosis, since this reduction is accompanied by the formation of a cortical granule-free domain and a conversion of ZP2 to ZP2 f when the oocytes are matured in vitro in serum-free medium; this zona pellucida modification occurs following fertilization and is thought to be due to cortical granule exocytosis. In contrast, the loss of cortical granules and development of the cortical granule-free domain of oocytes matured in vitro in the presence of serum is not accompanied by the modification of ZP2. The inhibitory effect of serum on the ZP2 modification may afford in vivo a physiological mechanism to prevent a precocious modification of the zona pellucida that could result in a premature block to polyspermy and hence inhibit fertilization.

Egg-induced modifications of the zona pellucida of mouse eggs: Effects of microinjected inositol 1,4,5-trisphosphate

Mouse eggs microinjected with physiological concentrations of inositol 1,4,5-trisphosphate (IP 3) do not emit the second polar body, form a pronucleus, or display a fertilization-associated set of changes in the pattern of protein synthesis. IP 3-injected eggs, however, display a conversion of the zona pellucida glycoprotein ZP2 to ZP2 f. The effect is concentration-dependent with an EC 50 (effective concentration, 50%) of 5–10 n M and also occurs in the presence of reduced levels of extracellular calcium. The egg-induced zona pellucida modification is not elicited by several other inositol phosphates that are not able to release calcium from intracellular stores in other systems. Analysis of individual eggs microinjected with IP 3 reveals a strong correlation between a reduced binding of sperm to the zona pellucida and the ZP2 to ZP2 f conversion. In addition, solubilized zonae pellucidae isolated from IP 3-injected eggs possess reduced levels of acrosome reaction-inducing activity. These egg-induced modifications of the zona pellucida—reduced sperm receptor and acrosome reaction-inducing activities and the ZP2 to ZP2 f conversion—elicited by microinjected-IP 3 are similar to those that occur following fertilization. Results of these experiments suggest that IP 3 generated in response to fertilization may play a role in the egg-induced modifications of the zona pellucida that result in the polyspermy block.