María Jiménez-Movilla

Ovastacin, a cortical granule protease, cleaves ZP2 in the zona pellucida to prevent polyspermy

After fertilization, the metalloendoprotease ovastacin is released by cortical granule exocytosis and cleaves the zona pellucida glycoprotein ZP2, an essential step to block sperm binding to an already fertilized egg.

FIGLA, a Basic Helix-Loop-Helix Transcription Factor, Balances Sexually Dimorphic Gene Expression in Postnatal Oocytes

ABSTRACT Maintenance of sex-specific germ cells requires balanced activation and repression of genetic hierarchies to ensure gender-appropriate development in mammals. Figla (factor in the germ line, alpha) encodes a germ cell-specific basic helix-loop-helix transcription factor first identified as an activator of oocyte genes. In comparing the ovarian proteome of normal and Figla null newborn mice, 18 testis-specific or -enhanced proteins were identified that were more abundant in Figla null ovaries than in normal ovaries. Transgenic mice, ectopically expressing Figla in male germ cells, downregulated a subset of these genes and demonstrated age-related sterility associated with impaired meiosis and germ cell apoptosis. Testis-associated genes, including Tdrd1, Tdrd6, and Tdrd7, were suppressed in the transgenic males with a corresponding disruption of the sperm chromatoid body and mislocalization of MVH and MILI proteins, previously implicated in posttranscriptional processing of RNA. These data demonstrate that physiological expression of Figla plays a critical dual role in activation of oocyte-associated genes and repression of sperm-associated genes during normal postnatal oogenesis.

ZP2 and ZP3 Traffic Independently within Oocytes prior to Assembly into the Extracellular Zona Pellucida

ABSTRACT The extracellular zona pellucida surrounds mammalian eggs and mediates taxon-specific sperm-egg recognition at fertilization. In mice, the zona pellucida is composed of three glycoproteins, but the presence of ZP2 and ZP3 is sufficient to form a biologically functional structure. Each zona pellucida glycoprotein is synthesized in growing oocytes and traffics through the endomembrane system to the cell surface, where it is released from a transmembrane domain and assembled into the insoluble zona pellucida matrix. ZP2 and ZP3 colocalize in the endoplasmic reticulum and in 1- to 5-μm post-Golgi structures comprising multivesicular aggregates (MVA), but a coimmunoprecipitation assay does not detect physical interactions. In addition, ZP2 traffics normally in growing oocytes in the absence of ZP3 or if ZP3 has been mutated to prevent incorporation into the zona pellucida matrix, complementing earlier studies indicating the independence of ZP3 secretion in Zp2 null mice. N glycosylation has been implicated in correct protein folding and intracellular trafficking of secreted proteins. Although ZP3 contain five N-glycans, enhanced green fluorescent protein-tagged ZP3 lacking N glycosylation sites is present in MVA and is incorporated into the zona pellucida matrix of transgenic mice. Thus, ZP2 secretion is seemingly unaffected by ZP3 lacking N-glycans. Taken together, these observations indicate that ZP2 and ZP3 traffic independently through the oocyte prior to assembly into the zona pellucida.

Oocytes use the plasminogen-plasmin system to remove supernumerary spermatozoa

BACKGROUND The role of the plasminogen-plasmin (PLG-PLA) system in fertilization is unknown, although its dysfunction has been associated with subfertility in humans. We have recently detected and quantified plasminogen in the oviductal fluid of two mammals and showed a reduction in sperm penetration during IVF when plasminogen is present. The objective of this study was to describe the mechanism by which PLG-PLA system regulates sperm entry into the oocyte. METHODS AND RESULTS By combining biochemical, functional, electron microscopic, immunocytochemical and live cell imaging methods, we show here that (i) plasminogen is activated into the protease plasmin, by gamete interaction; (ii) urokinase-type and tissue-type plasminogen activators are present in oocytes, but they are not of cortical granule origin; (iii) sperm binding to oocytes triggers the releasing of plasminogen activators and (iv) the generated plasmin causes sperm detachment from the zona pellucida. CONCLUSIONS Our results describe a novel mechanism for the success or failure of fertilization in mammals, by which molecules present in the oviductal environment are activated by molecules originating within the gametes. We anticipate that therapeutic up- or down-regulation of this physiological mechanism may be used to help in conception or as a contraceptive tool. Since components of the PLG-PLA system are already available as drugs for heart attacks or cancer therapies, basic research on this novel function would be rapidly transferable for clinical application.

ZP2 and ZP3 cytoplasmic tails prevent premature interactions and ensure incorporation into the zona pellucida

The zona pellucida contains three proteins (ZP1, ZP2, ZP3), the precursors of which possess signal peptides, ‘zona’ domains and short (9–15 residue) cytoplasmic tails downstream of a transmembrane domain. The ectodomains of ZP2 and ZP3 are sufficient to form the insoluble zona matrix and yet each protein traffics through oocytes without oligomerization. ZP2 and ZP3 were fluorescently tagged and molecular interactions were assayed by fluorescent complementation in CHO cells and growing oocytes. ZP2 and ZP3 traffic independently, but colocalize at the plasma membrane. However, protein–protein interactions were observed only after release and incorporation of ZP2 and ZP3 into the extracellular matrix surrounding mouse oocytes. In the absence of their hydrophilic cytoplasmic tails, ZP2 and ZP3 interacted within the cell and did not participate in the zona pellucida. A heterologous GPI-anchored ‘zona’ domain protein fused with the cytoplasmic tails was integrated into the zona matrix. We conclude that the cytoplasmic tails are sufficient and necessary to prevent intracellular oligomerization while ensuring incorporation of processed ZP2 and ZP3 into the zona pellucida.

ZP2 peptide beads select human sperm in vitro, decoy mouse sperm in vivo, and provide reversible contraception

Sperm bind to ZP2 peptide beads, which can select superior sperm for assisted reproduction but can also be used for contraception. Two sides of the same peptide The zona pellucida, a matrix that surrounds ovulated eggs, is the site of sperm recognition and binding, which precede sperm penetration and fertilization. Avella et al. identified a peptide from the zona pellucida called ZP2, which the authors attached to agarose beads to facilitate infertility treatment or, conversely, contraception. On the one hand, the beads with ZP2 peptide were combined with sperm in vitro to successfully select the best sperm for assisted reproductive technologies. On the other hand, the same beads placed into the uterus of fertile mice bound any incoming sperm and prevented them from reaching the eggs, thus providing a long-acting method of contraception. A related podcast discusses these findings. Gamete recognition in the female reproductive tract occurs at the surface of the zona pellucida surrounding ovulated eggs. The acellular zona matrix is composed of three (mouse) or four (human) proteins (ZP1 to ZP4), and the amino terminus of ZP2 is the primary sperm-binding ligand. Mouse and human sperm bind, respectively, to recombinant moZP235–149 and huZP239–154 peptides attached to agarose beads. Mouse ZP2 peptide beads markedly inhibited fertilization of ovulated mouse eggs inseminated in vitro and incubated overnight. Similarly, human ZP2 peptide beads prevented sperm binding and penetration of transgenic ZP2Rescue zonae pellucidae, in which human ZP2 replaced mouse ZP2. When mouse ZP2 peptide beads were transcervically deposited into the uterus, there was no change in mating behavior and copulatory plugs were present, but bound sperm did not progress into the oviduct and female mice were infertile. On average, contraception lasted >10 estrus cycles but was reversible with no detectable pathology in the reproductive tract. Despite the long-term contraceptive effect, initial sperm binding to the peptide beads was reversible in vitro. We exploited this observation to select human sperm that were better able to penetrate the zonae of human ZP2Rescue eggs, and the approach holds promise for identifying superior sperm for human assisted reproductive technologies (ART). We conclude that the amino-terminal ZP2 peptide supports sperm binding, which is initially reversible but, with time, becomes irreversible. Short-term, reversible binding may be useful in selecting sperm for ART, and long-term binding decoys sperm and results in effective contraception in mice.

Calreticulin from suboolemmal vesicles affects membrane regulation of polyspermy.

This study was designed to determine whether calreticulin (CRT), a chaperone protein, is present in in vitro-matured (IVM) pig oocytes and to study its potential role in the block to polyspermy. Western blot analysis, using an anti-CRT antibody, of oocyte lysate showed an immunoreactive band of ∼60  kDa. Simultaneous labeling of IVM oocytes with anti-CRT antibody and peanut agglutinin lectin (PNA lectin, a porcine cortical granules (CG)-specific binding lectin) revealed localization of CRT in the subplasmalemmal region with a 27.7% colocalization with PNA staining. After IVF, PNA labeling was not observed and anti-CRT labeling decreased significantly in zygotes and disappeared in two-cell embryos. Western blot analysis of oocyte exudate obtained from zona pellucida (ZP)-free oocytes activated with calcium ionophore confirmed the presence of a band that reacted with an anti-CRT antibody. Anti-CRT antibody and PNA labeling were not observed in activated oocytes despite being detectable in non-activated oocytes. The presence of CRT in vesicles located under the oolemma was demonstrated using immunogold cytochemistry at the ultrastructural level. To study the role of CRT in fertilization, ZP-enclosed and ZP-free oocytes were incubated with exogenous CRT and then inseminated. Whereas ZP-free oocytes showed fewer penetrating sperm and lower polyspermy rates than untreated oocytes, the opposite effect was observed in ZP-enclosed oocytes. In conclusion, CRT is confined to subplasmalemmal vesicles partially overlapping with CG contents. Its exocytosis after the oocyte activation seems to participate in the membrane block to polyspermy in pigs but is not involved in the ZP block.

The C-terminal region of OVGP1 remodels the zona pellucida and modifies fertility parameters

OVGP1 is the major non-serum glycoprotein in the oviduct fluid at the time of fertilization and early embryo development. Its activity differs among species. Here, we show that the C-terminal region of recombinant OVGP1 regulates its binding to the extracellular zona pellucida and affects its activity during fertilization. While porcine OVGP1 penetrates two-thirds of the thickness of the zona pellucida, shorter OVGP1 glycoproteins, including rabbit OVGP1, are restricted to the outer one-third of the zona matrix. Deletion of the C-terminal region reduces the ability of the glycoprotein to penetrate through the zona pellucida and prevents OVGP1 endocytosis. This affects the structure of the zona matrix and increases its resistance to protease digestion. However, only full-length porcine OVGP1 is able to increase the efficiency rate of in vitro fertilization. Thus, our findings document that the presence or absence of conserved regions in the C-terminus of OVGP1 modify its association with the zona pellucida that affects matrix structure and renders the zona matrix permissive to sperm penetration and OVGP1 endocytosis into the egg.

Cytoplasmic cleavage of DPPA3 is required for intracellular trafficking and cleavage-stage development in mice

Degradation of maternal proteins by the ubiquitin-proteasome system (UPS) accompanies the maternal-to-zygotic transition. DPPA3/Stella/PGC7, encoded by a maternal effect gene, is present in the nucleus and cytoplasm of zygotes and has been associated with protecting the female pronucleus from TET3-mediated demethylation. We now report that cytoplasmic DPPA3 is partially cleaved by the ubiquitin-proteasome system and an N-terminus fragment remains in the cytoplasm where it associates with early and re-cycling endosomes. If DPPA3 is absent or if cleavage is prevented, multiple vesicles coalesce/aggregate and markers of lysosomes are decreased. Fertilized eggs develop poorly into blastocysts, which results in significantly decreased fecundity of Dppa3R60A transgenic mice. This phenocopies aspects of Lamp1/2 knockdowns and Dppa3KO embryos can be partially rescued in vitro by DPPA31–60 and to a lesser extent by LAMP1/2. Thus, the N-terminus of DPPA3 has a significant role in cytoplasmic vesicular trafficking in addition to its previously reported nuclear function.During the maternal-to-zygotic transition there is degradation of maternal proteins via the ubiquitin-proteasome system (UPS). Here, the authors show that in the embryo the maternally inherited epigenetic factor DPPA3/Stella/PGC7 is cleavage by the UPS and controls intracellular trafficking.

Effects of recombinant OVGP1 protein on in vitro bovine embryo development

Previously, our group demonstrated that recombinant porcine oviductin (pOVGP1) binds to the zona pellucida (ZP) of in vitro-matured (IVM) porcine oocytes with a positive effect on in vitro fertilization (IVF). The fact that pOVGP1 was detected inside IVM oocytes suggested that this protein had a biological role during embryo development. The aim of this study was to evaluate the effects of pOVGP1 on bovine in vitro embryo development. We applied 10 or 50 µg/ml of pOVGP1 during IVF, embryonic in vitro culture (IVC), or both, to evaluate cleavage and embryo development. Blastocyst quality was assessed by analyzing the expression of important developmental genes and the survival rates after vitrification/warming. pOVGP1 was detected in the ZP, perivitelline space, and plasma membrane of blastocysts. No significant differences (P > 0.05) were found in cleavage or blastocyst yield when 10 or 50 µg/ml of pOVGP1 was used during IVF or IVC. However, when 50 µg/ml pOVGP1 was used during IVF + IVC, the number of blastocysts obtained was half that obtained with the control and 10 µg/ml pOVGP1 groups. The survival rates after vitrification/warming of expanded blastocysts cultured with pOVGP1 showed no significant differences between groups (P > 0.05). The use of pOVGP1 during IVF, IVC, or both, increased the relative abundance of mRNA of DSC2, ATF4, AQP3, and DNMT3A, the marker-genes of embryo quality. In conclusion, the use of pOVGP1 during bovine embryo in vitro culture does not affect embryo developmental rates but produces embryos of better quality in terms of the relative abundance of specific genes.