Zona Pellucida Of Oocytes Research Articles

Deleted in malignant brain tumor 1 is secreted in the oviduct and involved in the mechanism of fertilization in equine and porcine species

Oviductal environment affects preparation of gametes for fertilization, fertilization itself, and subsequent embryonic development. The aim of this study was to evaluate the effect of oviductal fluid and the possible involvement of deleted in malignant brain tumor 1 (DMBT1) on IVF in porcine and equine species that represent divergent IVF models. We first performed IVF after pre-incubation of oocytes with or without oviductal fluid supplemented or not with antibodies directed against DMBT1. We showed that oviductal fluid induces an increase in the monospermic fertilization rate and that this effect is canceled by the addition of antibodies, in both porcine and equine species. Moreover, pre-incubation of oocytes with recombinant DMBT1 induces an increase in the monospermic fertilization rate in the pig, confirming an involvement of DMBT1 in the fertilization process. The presence of DMBT1 in the oviduct at different stages of the estrus cycle was shown by western blot and confirmed by immunohistochemical analysis of ampulla and isthmus regions. The presence of DMBT1 in cumulus-oocyte complexes was shown by western blot analysis, and the localization of DMBT1 in the zona pellucida and cytoplasm of equine and porcine oocytes was observed using immunofluorescence analysis and confocal microscopy. Moreover, we showed an interaction between DMBT1 and porcine spermatozoa using surface plasmon resonance studies. Finally, a bioinformatic and phylogenetic analysis allowed us to identify the DMBT1 protein as well as a DMBT1-like protein in several mammals. Our results strongly suggest an important role of DMBT1 in the process of fertilization.

Biomolecule screening for efficient attachment of biofunctionalized microparticles to the zona pellucida of mammalian oocytes and embryos

Individual tagging of oocytes and embryos through the attachment of micrometer-sized polysilicon barcodes to their zona pellucida (ZP) is a promising approach to ensure their correct identification and traceability in human assisted reproduction and in animal production programs. To provide barcodes with the capacity of binding to the ZP, they must be first biofunctionalized with a biomolecule capable of binding to the ZP of both oocytes and embryos. The aim of this work was to select, among an anti-ZP2 antibody and the two lectins wheat germ agglutinin (WGA) and phytohemagglutinin-L, the most optimal biomolecule for the eventual biofunctionalization of barcodes, using mouse oocytes and embryos and commercially available microspheres as a model. Despite the anti-ZP2 antibody showed the highest number of binding sites onto the ZP surface, as determined by field emission scanning electron microscopy, the binding of anti-ZP2-biofunctionalized microspheres to the ZP of cultured oocytes and embryos was less robust and less stable than the binding of lectin-biofunctionalized ones. WGA proved to be, among the three candidates tested, the most appropriate biomolecule to biofunctionalize microparticles with the aim to attach them to the ZP of both oocytes and embryos and to maintain them attached through oocyte activation (zona reaction) and in vitro culture up to the blastocyst stage. As saccharides recognized by WGA are highly abundant in the ZP of most mammalian species, WGA-biofuncionalized microparticles would be able to attach to the ZP of oocytes/embryos of species other than the mouse, such as humans and farm animals.

Boar seminal plasma exosomes: Effect on sperm function and protein identification by sequencing

Mammalian seminal plasma contains membranous vesicles (exosomes), with a high content of cholesterol and sphingomyelin and a complex protein composition. Their physiological role is uncertain because sperm stabilization and activation effects have been reported. To analyze a putative modulatory role for semen exosomes on sperm activity in the boar, the effects of these vesicles on several sperm functional parameters were examined. Additionally, boar exosome proteins were sequenced and their incorporation into sperm was explored. Boar sperm were incubated under conditions that induce capacitation, manifested as increased tyrosine phosphorylation, cholesterol loss and greater fluidity in apical membranes, and the ability to undergo the lysophosphatidylcholine-induced acrosome reaction. After establishing this cluster of capacitation-dependent functional parameters, the effect produced by exosomes when present during or after sperm capacitation was analyzed. Exosomes inhibited the capacitation-dependent cholesterol efflux and fluidity increase in apical membranes, and the disappearance of a 14-kD phosphorylated polypeptide. In contrast, the acrosome reaction (spontaneous and lysophosphatidylcholine-induced) was not affected, and sperm binding to the oocyte zona pellucida was reduced only when vesicles were present during gamete coincubation. Liposomes with a lipid composition similar to that present in exosomes mimicked these effects, except the one on zona pellucida binding. Interaction between exosomes and sperm was confirmed by transfer of aminopeptidase activity. In addition, the major exosome protein, identified as actin, appeared to associate with sperm after coincubation. Exosome composition had a predominance for structural proteins (actin, plastin, ezrin, and condensin), enzymes, and several porcine seminal plasma-specific polypeptides (e.g., spermadhesins). Transfer of proteins from exosome to sperm and their ability to block cholesterol efflux supports a direct interaction between these vesicles and sperm, whereas inhibition of some capacitation-dependent features suggests a stabilizing function for exosomes in boar semen.

Immunogenicity and Contraceptive Efficacy ofEscherichia coli-Expressed Recombinant Porcine Zona Pellucida Proteins

To overcome availability of the purified native zona pellucida (ZP) glycoproteins for immunocontraception, porcine ZP3, and ZP4 were expressed in E.coli. Purified recombinant proteins were characterized by SDS-PAGE and Western blot, and immunogenicity and contraceptive efficacy determined in FvB/J female mice. Purified ZP3, ZP3 with promiscuous T-cell epitope of tetanus toxoid, ZP4 and ZP4 incorporating promiscuous T-cell epitope of bovine RNase revealed ~44-, ~49-, ~53-, and ~55-kDa bands by SDS-PAGE and Western blot, respectively. Immunization of female mice with recombinant proteins elicited high antibody titers as well as T-cell responses. Immune sera recognized mouse oocyte ZP and also inhibited in vitro fertilization. Immunized mice showed significant decrease in fertility. Recombinant proteins were able to recall memory antibody response in female mice primed with porcine native ZP. Availability of recombinant porcine proteins will be useful in the development of contraceptive vaccine.

Identification of sperm head proteins involved in zona pellucida binding

Which human sperm proteins interact with zona pellucida (ZP) glycoproteins, ZPA/2, ZPB/4 and ZPC/3? Co-precipitation experiments with recombinant human ZP (rhZP) coated beads demonstrated interactions with various proteins, including glutathione S-transferase M3 (GSTM) with ZPB/4 and voltage-dependent anion channel 2 (VDAC2) with ZPA/2 and ZPC/3. Regarding sperm-ZP binding, several target spot/proteins have been detected in several species, but not all have been characterized. The limit of these studies was that a mixture of the different ZP glycoproteins was used and did not allow the identification of the specific ZP glycoprotein (ZPA/2, ZPC/3 or ZPB/4) involved in the interaction with the sperm proteins. To identify the human sperm proteins interacting with the oocyte ZP, we combined two approaches: immunoblot of human spermatozoa targeted by antisperm antibodies (ASAs) from infertile men and far western blot of human sperm proteins overlayd by each of the rhZP proteins. We used rhZP expressed in Chinese hamster ovary (CHO) cells and ASA eluted from infertile patients undergoing IVF failure. Sperm proteins separated by two-dimensional (2D) electrophoresis recognized by both sperm-eluted ASAs from infertile patients and rhZP were identified by mass spectrometry (MALDI-MS/MS). Some of these proteins were further validated by co-precipitation experiments with rhZP and functional zona binding tests. We identified proteins that are glycolytic enzymes such as pyruvate kinase 3, enolase 1, glyceraldehyde-3-phosphate dehydrogenase, aldolase A, triosephosphate isomerase, detoxification enzymes such as GSTM or phospholipid hydroperoxide glutathione peroxidase, ion channels such as VDAC2 and structural proteins such as outer dense fibre 2. Several of the proteins were localized on the sperm head. However, these proteins have also been described to exert other functions in the flagellum. Co-precipitation experiments with rhZP-coated beads confirmed the direct interaction of GSTM with ZP4 and of VDAC2 with ZP2 and ZP3. We used recombinant ZP in place of native ZP. Thus, the post-translational modifications of the proteins, such as glycosylations, can be different and can influence their function. However, CHO cell-expressed rhZP are functional, e.g. can bind human spermatozoa and induce the acrosome reaction. Moreover, the identification of relevant proteins was limited by the need for sufficient amounts of proteins on the preparative 2D-gel to be subsequently analysed in MALDI-TOF MS/MS. Our results bring new insights on the ability of sperm proteins to exert several functions depending on their sub-cellular localization, either the head or flagellum. Their multiple roles suggest that these sperm proteins are multifaceted or moonlighting proteins. This work was supported by the grant ReproRio (CNRS, INRA, INSERM and CEA) and the Société d'Andrologie de Langue Française. Not applicable.

The Zona Pellucida Porosity: Three-Dimensional Reconstruction of Four Types of Mouse Oocyte Zona Pellucida Using a Dual Beam Microscope

In the last decade, the applicability of focus ion beam-field emission scanning electron microscopy (FIB-FESEM) in the biological field has begun to get relevance. Among the possibilities offered by FIB-FESEM, high-resolution three-dimensional (3D) reconstruction of biological structures is one of the most interesting. Using this tool, the 3D porosity of four different types of mouse oocyte zona pellucida (ZP) was analyzed. A surface analysis of the mouse oocyte ZP was first performed by SEM. Next, one oocyte per ZP type was selected, and an area of its ZP was completely milled, using the cut and view mode, in the FIB-FESEM. Through a 3D reconstruction of the milled area, a map of the distribution of the pores across the ZP was established and the number and volume of pores were quantified, thus enabling for the first time the study of the inner porosity of the mouse ZP. Differences in ZP porosity observed among the four types analyzed allowed us to outline a model to explain the changes that the ZP undergoes through immature, mature, predegenerative, and degenerative stages.

Viscoelastic Characterization of Mouse Zona Pellucida

The viscoelastic properties of the zona pellucida (ZP), which is the extracellular coat surrounding an oocyte/embryo, are evaluated in this study. Previous studies demonstrate that ZP mechanical properties change during oocyte maturation, fertilization, and early embryo development, but linear pure elastic models currently being used do not satisfy the time-dependent mechanical behavior of the ZP. In this paper, nonlinear viscoelastic characterization was performed using the Hunt-Crossley model and the newly developed vision-based nanoforce estimation method. The results show that viscoelasticity is a physical property of the ZP that exhibits hysteresis. The stiffness and viscosity parameters simultaneously increase following fertilization, causing the stiffness and viscosity of the embryo ZP (ten samples) to be 2.57-fold and 4.44-fold greater, respectively, than that of the oocyte ZP (eleven samples). This behavior well describes the noncovalently cross-linked filamentous structure of the ZP, supporting zona hardening during fertilization as a mechanically relevant event.

Genetic and physiological study of morphologically abnormal human zona pellucida

To investigate genetic, molecular and functional aspects of human zona pellucida (ZP) in oocytes with an abnormal appearance. The study included three women with unexplained infertility whose oocytes had an abnormal ZP appearance and the mother and fertile sister of one of them. The coding exons and their flanking intron regions of the four ZP genes and the regulatory element for the ZP3 gene were sequenced. Immunofluorescence staining of discarded oocytes using monoclonal antibodies against recombinant human ZP glycoproteins and a hemizona assay were performed. No new mutations were observed in the ZP1 (12 exons), ZP2 (19 exons), ZP3 (9 exons), ZP4 (12 exons) genes or in the ZP3 regulatory element of the three studied women. Sequencing of the genes revealed eight synonymous and non-synonymous reported polymorphisms only in ZP1, ZP2 and ZP3. Immunofluorescence staining of the discarded oocytes of two women showed clear and strong staining of the ZP1, ZP2 and ZP4 proteins, but weak staining of the ZP3 protein, although their ZP displayed normal sperm binding ability in the hemizona assay. Intracytoplasmic sperm injection yielded good pregnancy outcomes, even though few injected oocytes developed normally up to day 3. The abnormal oocyte ZP appearance in the three study women may not have been due to the genetic changes in the ZP genes. Moreover, sperm binding was normal despite low ZP3 staining observed, suggesting that ZP3 profile may play a subordinate role in the reported cases. Our findings support previous studies which claim that abnormal oocyte morphology is not associated with a decrease in fertilization rates or birth outcomes in couples undergoing intracytoplasmic sperm injection.

Absence of Dpy19l2, a new inner nuclear membrane protein, causes globozoospermia in mice by preventing the anchoring of the acrosome to the nucleus

Sperm-head elongation and acrosome formation, which take place during the last stages of spermatogenesis, are essential to produce competent spermatozoa that are able to cross the oocyte zona pellucida and to achieve fertilization. During acrosome biogenesis, acrosome attachment and spreading over the nucleus are still poorly understood and to date no proteins have been described to link the acrosome to the nucleus. We recently demonstrated that a deletion of DPY19L2, a gene coding for an uncharacterized protein, was responsible for a majority of cases of type I globozoospermia, a rare cause of male infertility that is characterized by the exclusive production of round-headed acrosomeless spermatozoa. Here, using Dpy19l2 knockout mice, we describe the cellular function of the Dpy19l2 protein. We demonstrate that the protein is expressed predominantly in spermatids with a very specific localization restricted to the inner nuclear membrane facing the acrosomal vesicle. We show that the absence of Dpy19l2 leads to the destabilization of both the nuclear dense lamina (NDL) and the junction between the acroplaxome and the nuclear envelope. Consequently, the acrosome and the manchette fail to be linked to the nucleus leading to the disruption of vesicular trafficking, failure of sperm nuclear shaping and eventually to the elimination of the unbound acrosomal vesicle. Finally, we show for the first time that Dpy19l3 proteins are also located in the inner nuclear envelope, therefore implying that the Dpy19 proteins constitute a new family of structural transmembrane proteins of the nuclear envelope.

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.

101 LIPID FINGERPRINTING OF OOCYTES AND PRE-IMPLANTATION MOUSE EMBRYOS BY DESORPTION ELECTROSPRAY IONIZATION MASS SPECTROMETRY

Lipids and metabolites can be directly detected from biological samples by desorption electrospray ionization mass spectrometry (DESI-MS). The introduction of morphologically friendly solvent systems allows nondestructive analysis of samples and because of its extractive nature, ion intensities obtained are adequate for profiling microscopic samples. This work reports the individual chemical profiling of mouse oocytes and diverse pre-implantation developmental stages of embryos by DESI-MS. Mice were superovulated with 5 IU pregnant mare serum gonadotropin (PMSG) IP followed 46 h later by 5 IU hCG IP and mated or not for embryo and oocyte recovery, respectively. Mice were killed and their oviducts removed and flushed at different intervals post-hCG to collect oocytes and embryos at different stages of development. Two-cell embryos were also cultured in vitro to obtain additional samples. For MS analysis, samples (n = 63) were taken from the flushing or culture medium, washed briefly in distilled water and placed on a glass slide for same-day analysis. Mass spectra were acquired in the negative ion mode at the mass/charge range of m/z 150 to 1000 using 1:1 (v/v) acetonitrile/DMF as a solvent system in an LTQ linear ion trap mass spectrometer (ThermoFisher Scientific, Pittsburgh, PA). Mass spectra from oocytes (n = 11) as well as zygotes (n = 9), 2-cell (n = 11), 4-cell (n = 1), 8- to 16-cell (n = 19) embryos and blastocysts (n = 12) showed the presence of deprotonated free fatty acids such as palmitic (m/z 255.2), linoleic (m/z 281.2), arachidonic (m/z 303.3) and docosahexanoenic (m/z 327.3), as well as prominent fatty acid dimers in the region of m/z 500 to 650, such as m/z 563.5 (oleic acid dimer) and glycerophospholipids in the m/z 700 to 1000 range. These complex lipids were represented by chlorinated phosphocholines such as PC 34:1, m/z 794.7; phosphatidylinositols such as PI 38:4, m/z 885.8; phosphatidylethanolamines such as PE 38:3, m/z 768.7; and phosphatidylserines such as PS 38:4, m/z 810.7. After analysis, samples observed on the glass slide displayed evident dehydration caused by the action of the organic solvent. Chemical profiling was more difficult for oocytes and blastocysts compared with other developmental stages, probably because of the presence of the intact zona pellucida in oocytes, whereas blastocysts were more prone to lysis during the rapid water wash, leading to a decrease in signal intensity. Optimization of conditions, comparison of MS spectra from a greater number of samples of different developmental stages by multivariate statistics and the influence of in vitro vs in vivo conditions are currently being investigated. Although small molecules have been shown to play a role in the direction of cell fate, the mechanisms involved are poorly understood. Perspectives for the application of this novel DESI-MS technique include the ability to study chemical signatures of hierarchical pluripotency observed during early embryonic development including embryonic stem cell differentiation. Support from the Purdue University Center for Cancer Research Small Grants Program is gratefully acknowledged.

Zona pellucida birefringence correlates with developmental capacity of bovine oocytes classified by maturational environment, COC morphology and G6PDH activity

In the present study we aimed to analyse structural changes during in vitro maturation of the bovine zona pellucida (ZP) by scanning electron microscopy (SEM) ands zona pellucida birefringence (ZPB). Here we show that alterations during in vitro maturation invasively analysed by SEM are reflected in ZPB. In vivo-matured oocytes displayed significantly lower birefringence parameters and significantly higher blastocyst rates compared with in vitro-derived oocytes (39.1% vs 21.6%). The same was observed for in vitro-matured oocytes with cumulus-oocyte complex (COC) Quality 1 (Q1) compared with Q3-COCs with respect to zona birefringence and developmental capacity. Immature oocytes with Q1-COCs displayed higher ZPB values and a higher developmental capacity to the blastocyst stage (27.7% vs 16.9%) compared with immature Q3-COCs. Considering in vitro-matured oocytes, only those with Q1-COC showed a trend for ZPB similar to in vivo-matured oocytes. Therefore, a decreasing trend for ZPB during in vitro maturation seems to be typical for high-quality oocytes and successful cytoplasmic maturation. In accordance, fully-grown immature oocytes reached significantly higher blastocyst rates (32.0% vs 11.5%) and lower ZPB values compared with still-growing ones. In conclusion, we successfully evaluated the applicability of zona imaging to bovine oocytes: alterations during in vitro maturation invasively analysed by scanning electron microscopy were reflected in the birefringence of the zona pellucida of bovine oocytes affecting developmental capacity at the same value. Therefore ZPB measurement by live zona imaging has potential to become a new tool to assess correctness of in vitro maturation and to predict developmental competence.

The relative contributions of propulsive forces and receptor–ligand binding forces during early contact between spermatozoa and zona pellucida of oocytes

When reaching the zona pellucida (ZP) of the oocyte, spermatozoa apply propulsive forces produced by the motion of their flagella, which push against the ZP and theoretically should contribute to their penetration into the ZP. Additionally, specific receptors on the spermatozoon head bind to ZP3 ligands located on the surface of the ZP, which locks the sperm's head onto the oocyte. Both mechanisms are important cofactors in the initial sperm penetration into the ZP, which is required for successful fertilization of the oocyte, but it is unclear which forces—mechanical thrust or biochemical binding—are more influential at this stage. To address this question, we developed a biomechanical sperm–oocyte contact model, which is based on the Johnson–Kendall–Roberts model adopted from the contact mechanics theory. The modeling predicted that during the early stage of penetration into the ZP, biochemical binding forces acting on spermatozoa, which are swimming at a (normal) velocity of 100 μm/s are ∼4.2-times to ∼16.7-times less than the mechanically-generated propulsive forces. In a simulated pathology of a low number of properly functioning receptors (50 out of 300 receptors/μm 2), the biochemical binding forces are ∼63-times less than the propulsive forces for the normally swimming sperm. It is suggested that such dominance of the propulsive forces over the biochemical binding forces can prevent efficient binding of spermatozoa to the ZP of the oocyte due to continuous movement of the sperm (which is not necessarily perpendicular to the ZP surface, and can cause sliding of sperm over the ZP). Thus, our theoretical analysis indicates that a sufficiently large density of receptors to ZP3 ligands on the sperm head is critical at the stage of early sperm–oocyte contact, in order to allow an efficient acrosome reaction to follow, so that the spermatozoon can start penetrating into the ZP.

Ubiquitin‐activating enzyme (UBA1) is required for sperm capacitation, acrosomal exocytosis and sperm–egg coat penetration during porcine fertilization

Protein ubiquitination is a stable, covalent post-translational modification that alters protein activity and/or targets proteins for proteolysis by the 26S proteasome. The E1-type ubiquitin-activating enzyme (UBA1) is responsible for ubiquitin activation, the initial step of ubiquitin-protein ligation. Proteasomal proteolysis of ubiquitinated spermatozoa and oocyte proteins occurs during mammalian fertilization, particularly at the site of sperm acrosome contact with oocyte zona pellucida. However, it is not clear whether the substrates are solely proteins ubiquitinated during gametogenesis or if de novo ubiquitination also occurs during fertilization supported by ubiquitin-activating and -conjugating enzymes present in the sperm acrosome. Along this line of inquiry, UBA1 was detected in boar sperm-acrosomal extracts by Western blotting (WB). Immunofluorescence revealed accumulation of UBA1 in the nuclei of spermatogonia, spermatocytes and spermatids, and in the acrosomal caps of round and elongating spermatids. Thiol ester assays utilizing biotinylated ubiquitin and isolated sperm acrosomes confirmed the enzymatic activity of the resident UBA1. A specific UBA1 inhibitor, PYR-41, altered the remodelling of the outer acrosomal membrane (OAM) during sperm capacitation, monitored using flow cytometry of fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Although viable and motile, the spermatozoa capacitated in the presence of PYR-41, showed significantly reduced fertilization rates during in vitro fertilization (IVF; p < 0.05). Similarly, the fertilization rate was lowered by the addition of PYR-41 directly into fertilization medium during IVF. In WB, high Mr bands, suggestive of protein ubiquitination, were detected in non-capacitated spermatozoa by antibodies against ubiquitin; WB with anti-phosphotyrosine antibodies and antibodies against acrosomal proteins SPINK2 (acrosin inhibitor) and AQN1 (spermadhesin) revealed that the capacitation-induced modification of those proteins was altered by PYR-41. In summary, it appears that de novo protein ubiquitination involving UBA1 contributes to sperm capacitation and acrosomal function during fertilization.

Maskless Gray Scale Lithography and its 3D Microfluidic Applications

This paper presents the novel three-dimensional fabrication using maskless exposure equipment and threedimensional (3D) microfluidic cell manipulation uses grayscale data to directly control the exposed photoresist height without using a mask. The 3D microchannel and microvalve were fabricated simply using lowcost exposure and height ranging from 0 to 200 µm. The 3D microvalve prevents liquid leakage when the membrane is closed – difficult to do using conventional 2D photolithography. We removed the oocyte zona pellucida passing through the 3D microchannel whose cross-section is gradually restricted along the path to provide mechanical stimulation omnidirectionally on the oocyte surface. The microfluidic chip may contribute to make high peeled-oocyte throughput effective without damaging the oocytes.

Contraceptive Vaccines: Success, Status, and Future Perspective

Contraceptive Vaccines: Success, Status, and Future Perspective

Mathematical modeling of calcium signaling during sperm hyperactivation

Mammalian sperm must hyperactivate in order to fertilize oocytes. Hyperactivation is characterized by highly asymmetrical flagellar bending. It serves to move sperm out of the oviductal reservoir and to penetrate viscoelastic fluids, such as the cumulus matrix. It is absolutely required for sperm penetration of the oocyte zona pellucida. In order for sperm to hyperactivate, cytoplasmic Ca(2+) levels in the flagellum must increase. The major mechanism for providing Ca(2+) to the flagellum, at least in mice, are CatSper channels in the plasma membrane of the principal piece of the flagellum, because sperm from CatSper null males are unable to hyperactivate. There is some evidence for the existence of other types of Ca(2+) channels in sperm, but their roles in hyperactivation have not been clearly established. Another Ca(2+) source for hyperactivation is the store in the redundant nuclear envelope of sperm. To stabilize levels of cytoplasmic Ca(2+), sperm contain Ca(2+) ATPase and exchangers. The interactions between channels, Ca(2+) ATPases, and exchangers are poorly understood; however, mathematical modeling can help to elucidate how they work together to produce the patterns of changes in Ca(2+) levels that have been observed in sperm. Mathematical models can reveal interesting and unexpected relationships, suggesting experiments to be performed in the laboratory. Mathematical analysis of Ca(2+) dynamics has been used to develop a model for Ca(2+) clearance and for CatSper-mediated Ca(2+) dynamics. Models may also be used to understand how Ca(2+) patterns produce flagellar bending patterns of sperm in fluids of low and high viscosity and elasticity.

Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization

To fuse with oocytes, spermatozoa of eutherian mammals must pass through extracellular coats, the cumulus cell layer, and the zona pellucida (ZP). It is generally believed that the acrosome reaction (AR) of spermatozoa, essential for zona penetration and fusion with oocytes, is triggered by sperm contact with the zona pellucida. Therefore, in most previous studies of sperm-oocyte interactions in the mouse, the cumulus has been removed before insemination to facilitate the examination of sperm-zona interactions. We used transgenic mouse spermatozoa, which enabled us to detect the onset of the acrosome reaction using fluorescence microscopy. We found that the spermatozoa that began the acrosome reaction before reaching the zona were able to penetrate the zona and fused with the oocyte's plasma membrane. In fact, most fertilizing spermatozoa underwent the acrosome reaction before reaching the zona pellucida of cumulus-enclosed oocytes, at least under the experimental conditions we used. The incidence of in vitro fertilization of cumulus-free oocytes was increased by coincubating oocytes with cumulus cells, suggesting an important role for cumulus cells and their matrix in natural fertilization.

Oocytic expression of zona pellucida protein ZP4 in Japanese quail (Coturnix japonica)

The avian perivitelline layer, an extracellular matrix homologous to the zona pellucida (ZP) of mammalian oocytes, is composed mainly by zona pellucida gene family glycoproteins. Our previous studies in Japanese quail have demonstrated that the matrix's components, ZP3 and ZPD, are synthesized in ovarian granulosa cells. Another component, ZP1, is synthesized in the liver. Recently, we demonstrated that another minor constituent, ZP2 is produced in the oocytes of the immature follicles. In the present study, we report the isolation of complementary DNA encoding quail ZP4 and its expression and origin in the female birds. By ribonuclease protection assay and in situ hybridization, we demonstrated that ZP4 transcripts were transcribed in the oocytes of small white follicles. The expression level of ZP4 decreased dramatically during follicular development, and the highest expression was observed in the small white follicles. Western blot analysis using the specific antibody against ZP4 indicated that the immunoreactive 58.2 kDa protein was present in the lysates of the small white follicles. These results demonstrate for the first time that the avian ZP4 is expressed in the oocyte, and that the expression pattern of the gene is similar to that of ZP2.

Zona pellucida glycoprotein 3 (pZP3) and integrin β2 (ITGB2) mRNA and protein expression in porcine oocytes after single and double exposure to brilliant cresyl blue test

Brilliant cresyl blues (BCB) staining test is a useful tool in assessing the competence of cumulus-oocyte-complexes (COCs) in several mammalian species. It is mostly used to select gametes after they are recovered from the ovary or before and after IVM to isolate those oocytes that reach developmental competency. However, there is evidence that double exposure to BCB test may lead to impaired fertilization or even have a toxic effect on cells. The aim of the present study was to investigate the expression pattern of sperm-egg interaction molecules in oocytes after single and double exposure to BCB test. Follicles were dissected from porcine ovaries after slaughter and aspirated COCs were cultured in standard porcine IVM culture medium (TCM 199) for 44 h. The BCB test was applied to COCs before and after IVM. In developmentally competent oocytes, assessed by determining the activity of glucose-6-phosphate dehydrogenase (G6PDH; BCB test), real-time quantitative PCR reaction methods, western blot and confocal microscopy analysis were applied to determine the transcript levels of porcine zona pellucida glycoprotein 3 (pZP3), and integrin beta 2 (ITGB2), as well as the levels of pZP3 and ITGB2 proteins. In the control group, assessment of the expression of the investigated genes was performed before and after IVM without BCB test. We observed a significantly higher level of pZP3 mRNA in oocytes after single exposure to BCB test compared to control before and after IVM (P < 0.001), and to double staining (P < 0.05). The level of ITGB2 mRNA was also increased in gametes after single exposure to BCB test as compared to control before and after IVM (P < 0.001, P < 0.01, respectively), and double staining (P < 0.05). Western blot analysis demonstrated a higher level of pZP3 protein in oocytes after single staining with BCB as compared to control both before and after IVM (P < 0.001, P < 0.05, respectively) and double staining (P < 0.05). Confocal microscopic observations have revealed the same pattern of increased level of pZP3 and ITGB2 expression after single exposure to BCB test. In both cases we detected specific cytoplasmic localization of both proteins. The ITGB2 protein has zona pellucida and membrane localization in control oocytes before IVM. After IVM and after single exposure to BCB, ITGB2 was also strongly detected in the cytoplasm. In both cases, after double exposure to BCB both proteins were detected only partially in the cytoplasm. Our results suggest that (i) single exposure to BCB increased the expression of sperm-oocyte interaction genes, (ii) double exposure to BCB leads to only partial expression of pZP3 and ITGB2 in oocyte cytoplasm, (iii) the BCB staining test itself may be a cause of specific pZP3 translocation from the zona pellucida to the cytoplasm, and that (iv) in vitro maturation of oocytes may increase ITGB2 expression and translocation from the zona pellucida to the cytoplasm.