Academic literature on the topic 'Polyspermy'

Fertilization is indispensable not only for restoring diploid genomes but also for the initiation of early embryonic cell cycles in sexual reproduction. While most animals exhibit monospermy, which is ensured by polyspermy blocks to prevent the entry of extra sperm into the egg at fertilization, several animals exhibit physiological polyspermy, in which the entry of several sperm is permitted but only one sperm nucleus participates in the formation of a zygote nucleus. Polyspermy requires that the sperm transmit the egg activation signal more slowly, thus allowing the egg to accept several sperm. An increase in intracellular Ca2+ concentration induced by the fertilizing sperm is both necessary and sufficient for egg activation in polyspermy. Multiple small Ca2+ waves induced by several fertilizing sperm result in a long-lasting Ca2+ rise, which is a characteristic of polyspermic amphibian eggs. We introduced a novel soluble sperm factor for egg activation, sperm-specific citrate synthase, into polyspermic newt eggs to cause Ca2+ waves. Citrate synthase may perform dual functions: as an enzyme in mitochondria and as a Ca2+-inducing factor in egg cytoplasm. We also discuss the close relationship between the mode of fertilization and the Ca2+ rise at egg activation and consider changes in this process through evolution in vertebrates.

Although techniques for in vitro production of porcine embryos have proceeded very rapidly during the past decade, polyspermic penetration still remains a persistent obstacle to porcine in vitro fertilization (IVF) systems. Considerable research on in vitro polyspermic penetration in porcine in vitro-matured (IVM) oocytes has been undertaken to try to solve this problem. In the current paper, recent advancements in overcoming the problems of polyspermy in porcine IVF systems are reviewed. Partial induction of the acrosome reaction of boar spermatozoa in IVF media that contain caffeine is likely to be one of the major causes of polyspermy. A reduction in the number of incompletely acrosome-reacted spermatozoa, which can bind tightly to the zona pellucida and mask free sperm receptors of the zona pellucida, could reduce the incidence of polyspermic penetration; however, morphological differences in the reaction of the zona pellucida have been observed between IVM and ovulated oocytes, which suggests that altered zona morphology may be another cause of polyspermic penetration. It has been shown that the developmental ability of polyspermic porcine embryos to the blastocyst stage is similar to that of normal embryos but that developmental competence to term is much lower. To overcome the current problems of polyspermy, it is suggested that future efforts should be focused on controlling boar sperm function and/or sperm–zona binding to achieve the final maturation associated with normal zona modifications of porcine oocytes at fertilization.

Denuded Bufo arenarum oocytes matured in vitro by progesterone treatment exhibited abnormal segmentation due to the penetration of more than one sperm. These oocytes were able to respond to activation stimuli and exhibited the external signs characteristic of activation. However, the prevention of polyspermy was not effective in these oocytes, which exhibited numerous sperm in their cytoplasm. The aim of this work was to analyse the cortical reaction in polyspermic Bufo arenarum oocytes matured in vitro. The result indicate that the cortical reaction of these oocytes seems to occur with a chronological sequence similar to that described for ovoposited oocytes of this species. In addition, when, 1 min after pricking, cortical granule exocytosis occurred, the oocytes became refractory to sperm entry, suggesting that they are able to establish a slow block to polyspermy.

The in vitro production of embryos is well established in most domestic species including cattle, pigs, sheep, and goats. However, a major problem of IVF in the pig is the high incidence of polyspermy. In our laboratory, we investigated the effect of 2 different media, TALP and SOFaa, on the rate of fertilization and polyspermy of pig oocytes. Preliminary experiments indicated that TALP provided the highest fertilization but also the highest polyspermy rates, as reported in the literature (Coy et al. 2002 Reproduction 124, 279–288). By contrast, much lower polyspermy rates but also much lower fertilization rates were obtained in SOFaa. Therefore, we made a direct comparison between the 2 media and a third medium prepared by mixing TALP and SOFaa equally (1 : 1 TALP–SOF) and using 2 different boars for IVF. Porcine oocytes were recovered from slaughtered donors and matured in vitro for 40 to 44 h in DMEM-F12 supplemented with 10% FCS, 0.05 IU of LH and FSH (Menogon; Ferring, Milan, Italy), 0.3 mM cystine, 0.5 mM cysteamine, 50 ng mL−1 of long-EGF, 100 ng mL−1 of long-IGF1, and 5 ng mL−1 of bFGF (Sigma-Aldrich, Milan, Italy) in 5% CO2 at 38.5°C. Boar frozen–thawed semen was separated on a Percoll gradient (45–90%) and diluted in TALP or in SOFaa with PHE (penicillamine, hypotaurine, epinefrine) and heparin (1 µg mL−1) to concentrations ranging from 0.01 to 0.15 million sperm/mL. The concentration was optimized for each boar and medium: For boar A, the concentration was 0.015 million sperm/mL for medium TALP and TALP–SOF and 0.15 million sperm/mL for medium SOF; for boar B, the concentration was 0.1 million sperm/mL for medium TALP and TALP–SOF and 0.15 million sperm/mL for medium SOF. The oocytes were co-incubated with the sperm suspension for 18 h and then were denuded of the surrounding cumulus and fixed in acetic acid–ethanol (1 : 3) for 48 h. Finally, they were stained with lacmoid and observed under phase-contrast microscopy. The data are shown in Table 1 and were compared by a chi-squared test. Our results indicated that TALP was the most efficient medium for pig IVF but over 50% of the oocytes were polyspermic. By contrast, very low polyspermy, but also very low fertilization, was observed in SOF medium for both boars A and B. Interestingly, the empirical approach of mixing the 2 media 50% each provided a dramatic reduction of the polyspermy rate while maintaining the fertilization rate at over 60% in both boars. At present, experiments are ongoing to clarify the role of specific components of the 2 media on the fertilization and polyspermy rates of pig oocytes. Table 1.Effect of different media on fertilization and polyspermy rates with 2 different boars This work was supported by grants from EUROSTELLS-ESF (ERAS-CT-2003-980409).

High incidences of polyspermic penetration continue to challenge researchers during porcine in vitro fertilisation (IVF). The aim of this study was to reduce the incidence of polyspermy by increasing the perivitelline space thickness with glucuronic acid and N-acetyl-D-glucosamine (GlcNAc) supplementation during oocyte maturation. After maturation, zona pellucida and perivitelline space thicknesses, intracellular glutathione concentrations and fertilisation kinetics were measured, in addition to embryonic cleavage and blastocyst formation at 48 h and 144 h after IVF, respectively. There were no significant differences between the treatments for zona pellucida thickness, penetration rates, male pronuclear formation or cortical granule exocytosis. Glucuronic acid supplementation significantly increased (P < 0.05) the perivitelline space thickness and significantly lowered the incidence (P < 0.05) of polyspermy. GlcNAc supplementation significantly increased (P < 0.05) intracellular glutathione concentrations. Supplementation with 0.005 mM glucuronic acid plus 0.005 mM GlcNAc during oocyte maturation produced significantly higher rates (P < 0.05) of cleavage and blastocyst formation by 48 and 144 h after IVF compared with all other groups. These results indicate that supplementing with 0.005 mM glucuronic acid and 0.005 mM GlcNAc during oocyte maturation decreases the incidence of polyspermic penetration by increasing perivitelline space thickness and improving embryo development in pigs.

The effect of fetuin, an a1-glycoprotein that has protease inhibitor activity, on the biock to potyspermy was determined. Cumulus-oocyte complexes from eCG primed mice were matured in vitro in the presence of 0, 0.01, 0.1, l and 10mg/ml of fetuin in modifica TCM 199. Both in vivo and in vitro matured oocytes were fertilized in the presence of fetuin and incubated for 6 and 24h. Fetuin present in a concentration of 1 mg/ml in the ferlilization but not in the maturation medium was able to induce polyspermy in 52.4% of the eggs. There was a positive relationship between concentration of fetuin in the fertilization medium and the proportion of polyspermic eggs (p<0.05). A significant interaclion between 0.1 and l mg/ml of fetuin during maturation and fertilization was observed (p<0.05). The results of these experiments demonstrate the inhibition of the block to polyspermy using a protease inhibitor during the fertilization.

Recent studies of centrosome biogenesis, microtubule dynamics, and their management point to their role in mediating conditions such as aging and cancer. Centrosome dysfunction is also a hallmark of pathological polyspermy. Polyspermy occurs when the oocyte is penetrated by more than one sperm and can be pathological because an excess of centrosomes compromises development. However, in some taxa, multiple sperm enter the egg with no apparent adverse effect on zygote viability. Thus, some taxa can manage excess centrosomes and represent cases of non-pathological polyspermy. While these two forms of polyspermy have long been known, we argue that there is limited understanding of the proximate and ultimate processes that underlie this taxonomic variation in the outcome of polyspermy and that studying this variation could help uncover the control and role(s) of centrosomes during fertilization in particular, but also mitosis in general. To encourage such studies we: 1) describe taxonomic differences in the outcome of polyspermy, 2) discuss mechanistic aspects of reproductive biology that may contribute to the different consequences of polyspermy, and 3) outline the potential selective events that could lead to the evolution of variation in polyspermy outcomes. We suggest that novel insights into centrosome biology may occur by cooperative studies between reproductive and evolutionary biologists focusing on the mechanisms generating variation in the fitness consequences of polyspermy, and in the taxonomic distribution of all these events. The consequent discoveries of these studies may lead to informative insights into cancer and aging along with other centrosome-related diseases and syndromes.

In vitro production (IVP) of porcine embryos facilitates research related to biotechnology and biomedicine. Even though many attempts have been made to optimize the IVP of porcine embryos, the outcome is still unsatisfactory compared to other species, such as mouse and cattle. The high incidence of polyspermic fertilization is one of the major causes lowering the overall efficiency of porcine IVF. The common procedure for fertilization in vitro involves the co-culture of both gametes in the medium drop, which increases sperm concentration and incidence of polyspermy. Therefore, the present study was carried out to increase the efficiency of porcine IVF by reducing polyspermy using a modified swim-up method. This method modifies conventional swim-up washing by placing oocytes directly at the time of washing. Porcine oocytes were aspirated from ovaries and matured. Sperm pellet was prepared in the tube and mature oocytes were placed on a cell strainer with 70-μm pore size (Falcon 2350) at the top of the tube. After fertilization, the oocytes were fixed and stained for examination. Also, the developmental potential of fertilized embryos was measured to evaluate for the feasibility of this method. While penetration rates were similar in both methods (86.67±2.36% to 83.33±1.36%), there was a significant reduction of polyspermy in the modified swim-up method (17.50±1.60%) compared to the control (44.1±3.70%) (P&lt;0.05). Subsequent culture showed higher rate of blastocyst formation in the modified swim-up method (20.44±0.99%) than in the control (15.73±3.26%) (P&lt;0.05), even though the difference was not significant. These results suggest that, by controlling the number of spermatozoa reaching the oocytes, porcine oocytes might be protected from polyspermy in vitro. Also, the developmental potential of the fertilized embryos using this method could be improved by increasing the pool of spermatozoa with better quality. Further optimization of the procedure is required to impliment this method in routine porcine IVF.

SummaryFull-grown ovarian oocytes of the amphibian Bufo arenarum were induced to mature in vitro by removing the follicular layers (spontaneous maturation) or by treatment with progesterone (hormone-induced maturation). These oocytes were then treated with trypsin and inseminated with homologous spermatozoa. Oocytes matured in vivo that had not undergone any influence of the oviducts (coelomic oocytes), inseminated under the same experimental conditions, were used as controls. The results show that oocytes induced to mature in vitro and exhibiting apparently normal signs of activation were polyspermic. In fact, 2 h after insemination numerous functioning pronuclei could be observed in the animal hemisphere. These results suggest that even though the oocytes which matured in vitro were able to undergo activation after insemination, they were unable to establish an effective block to polyspermy.

El objetivo de este trabajo consistió en describir el papel del sistema plasminógeno/plasmina (PLG/PLA) en la fecundación bovina y porcina. Mediante fecundación in vitro, demostramos que la presencia de PLG ó PLA en el medio de coincubación de los gametos disminuía la penetración de los espermatozoides en los ovocitos y su unión a la zona pelúcida (ZP). Esta disminución no se debía a alteraciones de la funcionalidad espermática ni a cambios en la resistencia de la ZP a la proteolisis, sino a que la PLA provocaba la liberación de los espermatozoides adheridos a la ZP. Mediante inmunofluorescencia indirecta detectamos la presencia de PLG y sus activadores en la ZP y en el oolema de los ovocitos antes de la fecundación. Tras la fecundación, dicha presencia disminuyó o desapareció por completo, por lo que proponemos que el sistema PLG/PLA se activa durante la interacción espermatozoide-ovocito y contribuye a regular la polispermia.
The aim of this study was to describe the role of the plasminogen/plasmin system (PLG/PLA) in bovine and porcine fertilization. Through in vitro fertilization, we demonstrated that the presence of PLG or PLA in the incubation medium of gametes decreased penetration of oocytes and sperm binding to the zona pellucida (ZP). This decrease was not due to alterations in sperm function or changes in the ZP resistance to proteolysis, but the PLA caused the release of sperm previously bound to the ZP. By indirect immunofluorescence we detected the presence of PLG and its activators in the ZP and oolema of the oocytes before fertilization. After fertilization, this presence diminished or disappeared completely, so we propose that the PLG/PLA system is activated during sperm-oocyte interaction and contributes to the regulation of polyspermy.

Con el presente trabajo se ha pretendido investigar la influencia de diversos factores relacionados con el cocultivo de los gametos porcinos, sobre los resultados de la fecundación" in vitro" (AV), fundamentalmente con la intención de mejorar la eficacia actual del sistema de AV en cuanto a la consecución de embriones viables (fecundaciones monospérmicas). Para ello, se han utilizado 129 hembras porcinas prepúberes a las que se indujo la ovulación mediante un tratamiento con 1250 U.I. de PMSG y 750 U.I. de HCG. El tratameinto empleado resultó eficaz para los fines perseguidos en un 80'62% de las hembras y el número medio de ovocitos recogidos fue de 19'07 + 1'52 por animal, utilizándose un total de 1984 ovocitos.En relación a las condiciones del sistema de fecundación, el primer factor investigado ha sido el tiempo de cocultivo, entendido como tiempo de contacto entre los gametos. En las dos experiencias realizadas, utilizando tiempos de 4, 6 u 8 horas (experiencia la), o de 1, 2, 3 ó 4 horas (experiencia lb), los mejores resultados se obtuvieron tras 4 horas de cocultivo, ya que los porcentajes de penetración de mantuvieron altos con respecto al máximo alcanzado a las 8 horas (82,68 vs. 93,96%), mientras que los de monospermia no disminuyeron excesivamente con respecto a los obtenidos con tiempos de cocultivo menores, teniendo en cuenta que la concentración de espermatozoides empleada fue intencionadamente elevada (12 x 105 esp/ml). El segundo factor analizado fue la concentración espermática. Se utilizaron concentraciones de 3, 6 y 12 x 105 esp vivos/ml, deduciéndose de los resultados que la mayor efectividad en nuestro sistema correspondía a la concentración de 6 x lO 5 esp/ml, ya que los porcentajes de penetración fueron significativamente diferentes a los obtenidos con la concentración espermática más alta (71'62% vs. 76'83%), y los porcentajes de monospermia tampoco se diferenciaron de los obtenidos con la concentración espermática más baja (62'26 vs. 68'08%). El tercer factor estudiado ha sido la influencia de la presencia o ausencia en el medio de cocultivo del "cumulus" expandido que acompaña al ovocito en la ovulación. Por los resultados obtenidos, se puede pensar que la presencia estas células junto con la correspondiente matriz intrecelular de ácido hialúrico es altamente beneficiosa para la mejora del rendimiento de la FIV debido a que los porcentajes de penetración en los ovocitos denudados (53'69 fueron menores (p<0'0l) que los obtenidos en los ovocitos con "cumulus" (69'10%), mientras que los porcentajes de monospermia fueron superios (p < 0'01) en el segundo caso (39'45% vs. 60'97%). Por último, se ha investigado el efecto de la reducción del volumen medio de cocultivo más comúnmente utilizado (2 mI) a otro menor (0'4 n obteniéndose resultados equivalentes en ambos casos para los porcentajes penetración, pero mayores porcentajes de monospermia (p<0'05) con volumen de 0'4 mI (57'53% vs. 78'12%). Del conjunto de los resultados se deduce que los porcentajes de penetración y polispermia en la AV porcina son consecuencia de la influencia de diferentes factores, entre los que se encuentran el tiempo de cocultivo, concentración espermática, la presencia del "cumulus oophorus" y el volumen de medio de cocultivo utilizados.
In the present work, we have investigated the influence of differen factors, related to porcine gametes coculture, on the results of "in vitro" fertilization (IVF). We have try to improve the efficiency of the current system to get viable embryos (monospermic fertilizations). 129 prepuberal gilts have been used after the induction of ovulation by administration of 1250 I.U. of PMSG followed, 55 hours later, by 750 I.U. of HCG. The results showed that the best moment for the recovery of oocytes was 44 h after HCC administration. In the same way, the treatment followed was effective for the required objectives in 80.62% of the studied females and the medium number of recovered oocytes was 19.07 + 1.52 per animal, giving a total number 01 1984 oocytes used. In relation with the conditions of the fertilization system, the first investigated factor was the coculture time, understanding it as contact time between gametes. To study the effect of this factor, two experiences were realized; fot the first one, 4, 6 or 8 hours of coculture time were used (experience lb) The best results were obtained at 4 hours of coculture, because the percentage of penetration was maintained high (82.68%) and, at the same time, the percentage of monospermy increased (p