Irene Mondéjar

Oviduct-specific glycoprotein and heparin modulate sperm–zona pellucida interaction during fertilization and contribute to the control of polyspermy

Polyspermy is an important anomaly of fertilization in placental mammals, causing premature death of the embryo. It is especially frequent under in vitro conditions, complicating the successful generation of viable embryos. A block to polyspermy develops as a result of changes after sperm entry (i.e., cortical granule exocytosis). However, additional factors may play an important role in regulating polyspermy by acting on gametes before sperm–oocyte interaction. Most studies have used rodents as models, but ungulates may differ in mechanisms preventing polyspermy. We hypothesize that zona pellucida (ZP) changes during transit of the oocyte along the oviductal ampulla modulate the interaction with spermatozoa, contributing to the regulation of polyspermy. We report here that periovulatory oviductal fluid (OF) from sows and heifers increases (both, con- and heterospecifically) ZP resistance to digestion with pronase (a parameter commonly used to measure the block to polyspermy), changing from digestion times of ≈1 min (pig) or 2 min (cattle) to 45 min (pig) or several hours (cattle). Exposure of oocytes to OF increases monospermy after in vitro fertilization in both species, and in pigs, sperm–ZP binding decreases. The resistance of OF-exposed oocytes to pronase was abolished by exposure to heparin-depleted medium; in a medium with heparin it was not altered. Proteomic analysis of the content released in the heparin-depleted medium after removal of OF-exposed oocytes allowed the isolation and identification of oviduct-specific glycoprotein. Thus, an oviduct-specific glycoprotein–heparin protein complex seems to be responsible for ZP changes in the oviduct before fertilization, affecting sperm binding and contributing to the regulation of polyspermy.

Fertilization outcome could be regulated by binding of oviductal plasminogen to oocytes and by releasing of plasminogen activators during interplay between gametes

OBJECTIVE To detect plasminogen and plasminogen activators (PA) in oviduct and oocytes and to clarify the role of the plasminogen/plasmin system on mammalian fertilization. DESIGN Experimental prospective study. SETTING Mammalian reproduction research laboratory. ANIMAL(S) Oviducts and ovaries from porcine and bovine females were collected at slaughterhouse. A total of 52 oviducts and 2,292 oocytes were used. Boar and bull ejaculated spermatozoa were also used. INTERVENTION(S) Plasminogen concentration in oviductal fluid (OF) through the cycle was measured. Immunolocalization of plasminogen and PAs in oocytes was carried out before and after fertilization. Porcine and bovine oocytes were in vitro fertilized, with plasminogen and plasmin added to the culture medium at different concentrations. MAIN OUTCOME MEASURE(S) Plasminogen concentration in OF. Plasminogen and PAs immunolocalization in oocytes. Penetration and monospermy rates, number of spermatozoa in the ooplasma and on the zona pellucida (ZP) after IVF. RESULT(S) Oviductal fluid contains about 92 μg/mL of plasminogen. The mature oocyte shows immunoreactivity toward plasminogen and toward PAs on its oolemma and ZP. After fertilization, plasminogen and PAs immunolabeling decreases in the oocyte, suggesting its conversion into plasmin. When exogenous plasminogen is added to the IVF medium, sperm entry into the oocyte is hampered, suggesting that the role of plasminogen activation during fertilization is to reduce the number of (or to select) penetrating spermatozoa. CONCLUSION(S) The plasminogen/plasmin system is activated during gamete interaction and regulates the sperm entry into the oocyte.

The Oviduct: Functional Genomic and Proteomic Approach

The mammalian oviduct is an anatomical part of the female reproductive tract, which plays several important roles in the events related to fertilization and embryo development. This review examines and compares several studies related to the proteomic and transcriptomic profile of the oviduct in different domestic animals. This information could be important for clarifying the role of oviductal factors in different events regulating fertilization and early embryo development, as well as for improving synthetic media for in vitro maturation/in vitro fertilization/embryo culture techniques (IVM/IVF/EC).

Identification of Potential Oviductal Factors Responsible for Zona Pellucida Hardening and Monospermy During Fertilization in Mammals

ABSTRACT Oviduct fluid increases the time required for digestion of the zona pellucida (ZP) by proteolytic enzymes (ZP hardening). This effect has been associated with levels of monospermy after in vitro fertilization (IVF) in the pig and cow, but the possible existence of a directly proportional relationship between hardening and monospermy remains unknown. To investigate whether variations in hardening of different oviductal fluids (OFs) are correlated with variations in levels of monospermy after IVF, porcine oocytes were incubated with three batches of OFs known to produce different ZP hardening effects (3, 7, and 25 min); after IVF, monospermy levels were 0%, 14.58% ± 5.14%, and 35.14% ± 7.95%, respectively. These results could partially explain the lack of polyspermy found during in vivo fertilization in pigs (with a hardened oviductal ZP) compared with levels found during IVF (with no hardened ZP). Using the bovine model, OF was fractionated by heparin affinity chromatography, and the hardening effect on the ZP was tested for each fraction obtained from a linear gradient of sodium chloride concentration. The highest effect was obtained with the fraction eluted with 0.4 M sodium chloride. Fractions with high-level or low-level effects were processed by on-chip electrophoresis and high-performance liquid chromatography-tandem mass spectrometry. A list of potential proteins responsible for this effect includes OVGP1 and members of the HSP and PDI families.

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.

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

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

How Is Plasminogen/Plasmin System Contributing to Regulate Sperm Entry Into the Oocyte?:

Plasminogen is present in the oviduct, on the zona pellucida (ZP) and on oolemma, and reduces the number of sperm penetrating the oocyte during in vitro fertilization in pig and cow. It is unknown how this reduction occurs. We tested whether plasminogen (1) changed the ZP resistance to enzymatic digestion thus making the passage of the spermatozoa across it difficult; (2) reduced the sperm functionality, assessed by sperm viability, motility, spontaneous acrosome reaction and membrane lipid disorder; or (3) affected the sperm–ZP binding before or after sperm–ZP interaction. The mechanism by which plasminogen/plasmin system contributes to regulate sperm entry into the oocyte is not inducing a ZP hardening or a decrease in sperm functionality but detaching more than 50% of sperm bound to the ZP. It is suggested that the fertilizing spermatozoon activates plasminogen into plasmin at the oocyte surface and that plasmin removes additional spermatozoa attached to the ZP.

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.

Total urokinase-type plasminogen activator (uPA) levels in seminal plasma are associated with positive assisted reproductive technology outcomes

PurposeThe plasminogen/plasmin system is an important extracellular protease system whose function has been implicated in male reproductive function. However, its clinical relevance to fertility in human assisted reproduction technologies has not been systematically investigated. Here, we examined whether total and active populations of urokinase-type plasminogen activator (uPA) in human seminal plasma and spermatozoa are predictive of pregnancy outcome in couples undergoing insemination or intracytoplasmic sperm injection (ICSI).MethodsSeminal samples from 182 men, 5 donors, 21 patients attending the clinic for infertility screening, and 156 for assisted reproduction technology (ART) treatment (insemination and ICSI), were evaluated. Total uPA in seminal plasma and spermatozoa as well as active uPA in seminal plasma were measured by ELISA. Sperm quality parameters and fertility outcomes following insemination or ICSI were correlated with the uPA values.ResultsActive uPA in seminal plasma was positively correlated to the volume of the ejaculate, total number of spermatozoa in the ejaculate, and total motility. However, these values were not prognostic of fertility outcomes. Total uPA in spermatozoa was inversely related to sperm concentration, total sperm in ejaculate, morphology, and total and progressive motility, and this measure was not related to fertility. Importantly, however, higher values of total uPA in seminal plasma were detected in cases that resulted in pregnancy compared to those that did not follow insemination and ICSI treatment.ConclusionsTaken together, these findings lay the foundation for further understanding the mechanism by which total uPA in seminal plasma affects fertility and how this marker can be used as a predictor of ART outcomes.