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1
Iwao, Yasuhiro. "Egg activation in physiological polyspermy." REPRODUCTION 144, no. 1 (July 2012): 11–22. http://dx.doi.org/10.1530/rep-12-0104.
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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.
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Funahashi, Hiroaki. "Polyspermic penetration in porcine IVM - IVF systems." Reproduction, Fertility and Development 15, no. 3 (2003): 167. http://dx.doi.org/10.1071/rd02076.
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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.
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Oterino, J., G. Sánchez Toranzo, L. Zelarayán, J. N. Valz-Gianinet, and M. I. Bühler. "Cortical granule exocytosis in Bufo arenarum oocytes matured in vitro." Zygote 9, no. 3 (August 2001): 251–59. http://dx.doi.org/10.1017/s0967199401001277.
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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.
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Galli, C., G. Crotti, P. Turini, I. Lagutina, and G. Lazzari. "295 INFLUENCE OF FERTILIZATION MEDIUM ON THE INCIDENCE OF POLYSPERMY IN THE PIG." Reproduction, Fertility and Development 19, no. 1 (2007): 263. http://dx.doi.org/10.1071/rdv19n1ab295.
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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).
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Schmidt, K., A. Clark, A. Mello, C. Durfey, A. Buck, K. Boyd, and B. D. Whitaker. "The effects of glucuronic acid and N-acetyl-D-glucosamine on in vitro fertilisation of porcine oocytes." Reproduction, Fertility and Development 28, no. 8 (2016): 1223. http://dx.doi.org/10.1071/rd14226.
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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.
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Gonçalves, Paulo Bayard, and Charles Graves. "Fetuin: a tool to study the block to polyspermy." Ciência Rural 27, no. 1 (March 1997): 127–31. http://dx.doi.org/10.1590/s0103-84781997000100022.
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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.
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Snook, Rhonda R., David J. Hosken, and Timothy L. Karr. "The biology and evolution of polyspermy: insights from cellular and functional studies of sperm and centrosomal behavior in the fertilized egg." REPRODUCTION 142, no. 6 (December 2011): 779–92. http://dx.doi.org/10.1530/rep-11-0255.
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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.
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Park, C. H., B. S. Koo, J. I. Yun, M. G. Kim, S. G. Lee, H. Y. Son, and C. K. Lee. "281REDUCTION OF POLYSPERMY IN PORCINE IN VITRO FERTILIZATION BY MODIFIED SWIM-UP METHOD." Reproduction, Fertility and Development 16, no. 2 (2004): 260. http://dx.doi.org/10.1071/rdv16n1ab281.
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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<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<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.
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Oterino, J., G. Sánchez Toranzo, L. Zelarayán, and M. I. Bühler. "Polyspermy in Bufo arenarum oocytes matured in vitro." Zygote 5, no. 3 (August 1997): 267–71. http://dx.doi.org/10.1017/s0967199400003713.
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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.
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WEBSTER, BOBBY W., ANNE COLSTON WENTZ, KEVIN G. OSTEEN, B. JANE ROGERS, and WILLIAM K. VAUGHN. "Hormonal Correlates with Polyspermy." Annals of the New York Academy of Sciences 442, no. 1 In Vitro Fert (May 1985): 332–35. http://dx.doi.org/10.1111/j.1749-6632.1985.tb37537.x.
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Eckberg, William R., and Winston A. Anderson. "Blocks to polyspermy inChaetopterus." Journal of Experimental Zoology 233, no. 2 (February 1985): 253–60. http://dx.doi.org/10.1002/jez.1402330213.
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Tatemoto, Hideki, Isao Tokeshi, Satoshi Nakamura, Norio Muto, and Tadashi Nakada. "Inhibition of boar sperm hyaluronidase activity by tannic acid reduces polyspermy during in vitro fertilization of porcine oocytes." Zygote 14, no. 4 (November 2006): 275–85. http://dx.doi.org/10.1017/s0967199406003819.
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SummaryThe present study was conducted to examine the effects of three polyphenols (tannic acid, apigenin and quercetin) on hyaluronidase activity and in vitro fertilization (IVF) parameters. Among them, tannic acid showed by far the strongest potency for blocking hyaluronidase activity extracted from preincubated boar sperm, causing a dose-dependent inhibition over the range of 2–10 μg/ml. When cumulus-intact and cumulus-free oocytes were inseminated in IVF medium containing tannic acid, the penetration and the polyspermy rates were significantly decreased in the presence of 10 μg/ml tannic acid compared with those in the absence of tannic acid, and the addition of 5 μg/ml tannic acid significantly reduced the polyspermy rate (p < 0.05) compared with that of the control while maintaining the high penetration rate. However, apigenin and quercetin had no effect on the rate of polyspermy. Interestingly, the incidence of polyspermy was significantly reduced in oocytes inseminated with sperm pretreated with 5 μg/ml tannic acid (p < 0.05), although the pretreatment of oocytes had no effect against the polyspermy after insemination with untreated sperm. Treatment with tannic acid caused neither a protective proteolytic modification of the zona pellucida matrix before fertilization, nor a reduction of the proteolytic activity of acrosomal contents or the number of zona-bound spermatozoa. These data suggest that an appropriate concentration of tannic acid prevents polyspermy through the inhibition of sperm hyaluronidase activity during IVF of porcine oocytes.
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Gardner, Allison J., and Janice P. Evans. "Mammalian membrane block to polyspermy: new insights into how mammalian eggs prevent fertilisation by multiple sperm." Reproduction, Fertility and Development 18, no. 2 (2006): 53. http://dx.doi.org/10.1071/rd05122.
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To inhibit fertilisation by more than one sperm (a condition known as polyspermy), eggs have developed preventative mechanisms known as blocks to polyspermy. The block at the level of the egg extracellular coat (the zona pellucida in mammals, the vitelline envelope in non-mammals) has been well characterised in many different animal species and the block at the level of the egg plasma membrane is understood in some non-mammalian species. However, virtually nothing is known about the membrane block to polyspermy in mammalian eggs, despite data dating back 50–90 years that provide evidence for its existence. In the present review, we will discuss the background on blocks to polyspermy used by animal eggs and then focus on the membrane block to polyspermy in mammalian eggs. This will include a summary of classical studies that provide evidence for this block in mammalian eggs, assays used to study the mammalian membrane block and what has been elucidated from recent experimental studies about the cellular signalling events that lead to membrane block establishment and the mechanism of how the membrane block may prevent additional fertilisation.
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Talevi, R. "Polyspermic eggs in the anuran Discoglossus pictus develop normally." Development 105, no. 2 (February 1, 1989): 343–49. http://dx.doi.org/10.1242/dev.105.2.343.
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Fertilization and development in 400 eggs of the anuran Discoglossus pictus has been followed. In these eggs successful sperm interaction is restricted to a small area of the animal dimple called D1 and causes a rapid depolarization. A high incidence of polyspermy (36%) was detected by in vivo observations of fertilization cone formation. Polyspermic eggs gave rise to fertilization potentials comparable to monospermic eggs and developed normally. By using current-injection technique it is shown that sperm penetration is independent of membrane potential. The role of the egg envelopes in regulating sperm-egg interaction is discussed.
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Sun, Jing-Tao, Jia-Hui Liu, Xi-Qing Jiang, Xin Luo, Jin-Dong Yuan, Qi Zhang, Xin-Yue Qi, Sanghoon Lee, Zhong-Hua Liu, and Jun-Xue Jin. "Tannin Reduces the Incidence of Polyspermic Penetration in Porcine Oocytes." Antioxidants 11, no. 10 (October 14, 2022): 2027. http://dx.doi.org/10.3390/antiox11102027.
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Tannin (TA) improves porcine oocyte cytoplasmic maturation and subsequent embryonic development after in vitro fertilization (IVF). However, the mechanism through which TA blocks polyspermy after IVF remains unclear. Hence, the biological function of organelles (cortical granule [CG], Golgi apparatus, endoplasmic reticulum [ER], and mitochondria) and the incidence of polyspermic penetration were examined. We found no significant difference in oocyte nuclear maturation among the 1 µg/mL, 10 µg/mL TA, and control groups. Moreover, 100 μg/mL TA significantly reduced 1st polar body formation rate compared to the other groups. Additionally, 1 and 10 μg/mL TA significantly increased the protein levels of GDF9, BMP15, and CDK1 compared to the control and 100 μg/mL TA groups. Interestingly, 1 and 10 μg/mL TA improved the normal distribution of CGs, Golgi, ER, and mitochondria by upregulating organelle-related gene expression and downregulating ER stress (CHOP) gene expression. Simultaneously, 1 and 10 μg/mL TA significantly increased the proportion of normal fertilized oocytes (2 pronuclei; 2 PN) and blastocyst formation rate compared to the control, as well as that of 100 μg/mL TA after IVF by upregulating polyspermy-related genes. In conclusion, TA during IVM enhances 2PN and blastocyst formation rates by regulating organelles’ functions and activities.
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Shimizu, K. K., and K. Okada. "Attractive and repulsive interactions between female and male gametophytes in Arabidopsis pollen tube guidance." Development 127, no. 20 (October 15, 2000): 4511–18. http://dx.doi.org/10.1242/dev.127.20.4511.
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Sexual reproduction in plants, unlike that of animals, requires the action of multicellular haploid gametophytes. The male gametophyte (pollen tube) is guided to a female gametophyte through diploid sporophytic cells in the pistil. While interactions between the pollen tube and diploid cells have been described, little is known about the intercellular recognition systems between the pollen tube and the female gametophyte. In particular, the mechanisms that enable only one pollen tube to interact with each female gametophyte, thereby preventing polysperm, are not understood. We isolated female gametophyte mutants named magatama (maa) from Arabidopsis thaliana by screening for siliques containing half the normal number of mature seeds. In maa1 and maa3 mutants, in which the development of the female gametophyte was delayed, pollen tube guidance was affected. Pollen tubes were directed to mutant female gametophytes, but they lost their way just before entering the micropyle and elongated in random directions. Moreover, the mutant female gametophytes attracted two pollen tubes at a high frequency. To explain the interaction between gametophytes, we propose a monogamy model in which a female gametophyte emits two attractants and prevents polyspermy. This prevention process by the female gametophyte could increase a plant's inclusive fitness by facilitating the fertilization of sibling female gametophytes. In addition, repulsion between pollen tubes might help prevent polyspermy. The reproductive isolations observed in interspecific crosses in Brassicaceae are also consistent with the monogamy model.
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Anzalone, D., M. Czernik, L. Palazzese, Y. Ressaissi, P. Scapolo, and P. Loi. "119 Short spermatozoa-oocyte co-incubation improves outcomes of IVF in sheep." Reproduction, Fertility and Development 32, no. 2 (2020): 186. http://dx.doi.org/10.1071/rdv32n2ab119.
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The assisted reproductive technique IVF is routinely applied in humans and large animals, both to boost reproductive performance and also for basic research. Despite its value, IVF has seen very little progress in the last two decades and relies on established paradigms, such as overnight sperm-egg co-incubation. However, the long exposure of oocytes to spermatozoa in a dish increases the risk of polyspermy and could be detrimental for early stages of embryonic development. We identified a time window within which fertilization occurs, in order to reduce the length of sperm-egg co-incubation and optimize the procedure, comparing polyspermy rate and embryo development after short (shIVF) and overnight (o/nIVF) spermatozoa-oocyte co-incubation. A total of 666 invitro-matured sheep oocytes were co-incubated with spermatozoa in IVF medium (synthetic oviductal fluid (SOF) with 20% oestrus sheep serum and 16 µM isoproterenol). First, small batches of oocytes were collected every 30min to check for the presence of a fertilizing spermatozoon. To assess this, cumulus cells were removed and presumptive fertilized oocytes were fixed and stained with propidium iodide for nuclei and Pisum sativum agglutinin for zona pellucida (ZP) detection, respectively. Then, pronuclear formation (PN) and embryo development were evaluated after 16h (PN), 24h (2 cells), and 7 days of culture (blastocyst). The oocytes that were not cleaved at 24h were stained for DNA content with Hoechst 33342. Furthermore, we evaluated embryo quality by counting cells of 8-day blastocysts after differential staining of inner cell mass (ICM) and trophectoderm (TE). We found that spermatozoa reach the ZP no earlier than 90min from the beginning of co-incubation and achieve fertilization within 4h. Polyspermic fertilization (>2PN) was lower in shIVF (6.5%) than in o/nIVF (17.8%; P=0.006). This proportion of polyspermy was maintained between groups in noncleaved oocytes at 24h from fertilization. Likewise, cleavage and blastocyst rate were higher in shIVF compared with the o/n-IVF group (2-cells: 48.3% vs. 31.6%, P=0.001; blastocyst: 29.4% vs. 20.5%, P=0.046, respectively). Differential staining of blastocysts revealed no significant difference in cell number between the blastocysts of the two groups. This work demonstrates that 4h of sperm-egg interaction are sufficient to achieve fertilization, reduce polyspermy, and improve the rate of embryos reaching blastocyst stage without compromising embryo quality.
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Angell, R. R., A. A. Templeton, and I. E. Messinis. "Consequences of polyspermy in man." Cytogenetic and Genome Research 42, no. 1-2 (1986): 1–7. http://dx.doi.org/10.1159/000132242.
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Dale, Brian, and Louis DeFelice. "Polyspermy prevention: facts and artifacts?" Journal of Assisted Reproduction and Genetics 28, no. 3 (November 23, 2010): 199–207. http://dx.doi.org/10.1007/s10815-010-9513-5.
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Tekleyohans, Dawit G., Yanbo Mao, Christina Kägi, York-Dieter Stierhof, and Rita Groß-Hardt. "Polyspermy barriers: a plant perspective." Current Opinion in Plant Biology 35 (February 2017): 131–37. http://dx.doi.org/10.1016/j.pbi.2016.11.012.
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VIGFÚSSON, EINAR. "On polyspermy in the sunflower." Hereditas 64, no. 1 (September 2, 2009): 1–52. http://dx.doi.org/10.1111/j.1601-5223.1970.tb02272.x.
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Gómez, Marta I., and Marcelo O. Cabada. "Amphibian cross-fertilization and polyspermy." Journal of Experimental Zoology 269, no. 6 (September 1, 1994): 560–65. http://dx.doi.org/10.1002/jez.1402690609.
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Gould, Meredith C., and Jose Luis Stephano. "Polyspermy prevention in marine invertebrates." Microscopy Research and Technique 61, no. 4 (June 10, 2003): 379–88. http://dx.doi.org/10.1002/jemt.10351.
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Lambert, C. C. "Ascidian eggs release glycosidase activity which aids in the block against polyspermy." Development 105, no. 2 (February 1, 1989): 415–20. http://dx.doi.org/10.1242/dev.105.2.415.
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To ensure normal development, most animals have evolved a number of mechanisms to block polyspermy including prevention of binding to surface coats as well as sperm-egg fusion. Ascidian sperm bind to vitelline coat (VC) glycosides. In the genus Ascidia, N-acetylglucosamine (GlcNAc) is the ligand to which sperm bind. The number of sperm bound to the VC is biphasic following fertilization; sperm binding increases through the first minute or so, then abruptly declines. At fertilization, the eggs of Ascidia callosa, A. ceratodes, A. mentula, A. nigra and Phallusia mammillata release N-acetylglucosaminidase into the sea water (SW). This has been shown to inactivate VC GlcNAc groups, blocking the binding of supernumerary sperm and polyspermy in A. nigra. This block to polyspermy is inactivated by GlcNAc (2mM) or 150 mM-Na+ (choline substituted) SW. These treatments are not additive and therefore probably affect the same process. In A. callosa, fertilization in low Na+ SW causes a 60% decline in enzyme release and a similar increase in the number of sperm remaining on the VC at 4 min as well as a great increase in polyspermy. Thus the principal block to polyspermy in ascidian eggs involves the release of N-acetylglucosaminidase which appears to be Na+ dependent. Enzyme activity is found in the supernatant SW by 15 s after fertilization, suggesting that it is stored very near the egg surface. Histochemical staining of whole eggs and embryos shows loss of surface-associated enzyme activity following fertilization. Like other lysosomal enzymes this N-acetylglucosaminidase is mannosylated and has an acidic pH optimum.
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Limatola, Nunzia, Filip Vasilev, Jong Tai Chun, and Luigia Santella. "Sodium-mediated fast electrical depolarization does not prevent polyspermic fertilization in Paracentrotus lividus eggs." Zygote 27, no. 4 (August 2019): 241–49. http://dx.doi.org/10.1017/s0967199419000364.
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SummaryDuring sea urchins fertilization, the activating spermatozoon triggers a series of physiological changes that transforms the quiescent egg into a dynamic zygote. It has been suggested that several of these egg activation events, e.g. sperm-induced plasma membrane depolarization and the Ca2+-linked cortical reaction, play additional roles to prevent the entry of supernumerary spermatozoa. In particular, the abrupt shift in egg membrane potential at fertilization, which is sustained by a Na+ influx, has been considered as a fast mechanism to block polyspermy. To test the relevance of the Na+-mediated fast electrical block to polyspermy, we fertilized sea urchin eggs in artificial seawater with a low concentration of Na+; nearly all the eggs were still monospermic, as judged by the number of Hoechst 33422-stained sperm. When fertilized in normal seawater, eggs that were pre-incubated in the low Na+ medium exhibited impaired elevation of the fertilization envelope. Nevertheless, these eggs manifested entry of a single spermatozoon, suggesting that the fertilization envelope was not the primary determinant of the block to polyspermy. Furthermore, we showed that the abnormal cleavage patterns displayed by eggs pre-incubated in low Na+, which were often considered a hallmark of polyspermy, were due to the alterations in the cortical actin filaments dynamics following fertilization, and not to the formation of multipolar spindles associated with supernumerary sperm centrosomes. Hence, our results suggested that Paracentrotus lividus eggs do not utilize Na+ to rapidly prevent additional spermatozoa from entering the egg, at variance with the hypothesis of an electrical fast block to polyspermy.
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Wozniak, Katherine L., Maiwase Tembo, Wesley A. Phelps, Miler T. Lee, and Anne E. Carlson. "PLC and IP3-evoked Ca2+ release initiate the fast block to polyspermy in Xenopus laevis eggs." Journal of General Physiology 150, no. 9 (July 16, 2018): 1239–48. http://dx.doi.org/10.1085/jgp.201812069.
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The prevention of polyspermy is essential for the successful progression of normal embryonic development in most sexually reproducing species. In external fertilizers, the process of fertilization induces a depolarization of the egg’s membrane within seconds, which inhibits supernumerary sperm from entering an already-fertilized egg. This fast block requires an increase of intracellular Ca2+ in the African clawed frog, Xenopus laevis, which in turn activates an efflux of Cl− that depolarizes the cell. Here we seek to identify the source of this intracellular Ca2+. Using electrophysiology, pharmacology, bioinformatics, and developmental biology, we explore the requirement for both Ca2+ entry into the egg from the extracellular milieu and Ca2+ release from an internal store, to mediate fertilization-induced depolarization. We report that although eggs express Ca2+-permeant ion channels, blockade of these channels does not alter the fast block. In contrast, insemination of eggs in the presence of Xestospongin C—a potent inhibitor of inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release from the endoplasmic reticulum (ER)—completely inhibits fertilization-evoked depolarization and increases the incidence of polyspermy. Inhibition of the IP3-generating enzyme phospholipase C (PLC) with U73122 similarly prevents fertilization-induced depolarization and increases polyspermy. Together, these results demonstrate that fast polyspermy block after fertilization in X. laevis eggs is mediated by activation of PLC, which increases IP3 and evokes Ca2+ release from the ER. This ER-derived Ca2+ then activates a Cl− channel to induce the fast polyspermy block. The PLC-induced cascade of events represents one of the earliest known signaling pathways initiated by fertilization.
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Okamoto, Takashi, Yukinosuke Ohnishi, and Erika Toda. "Development of polyspermic zygote and possible contribution of polyspermy to polyploid formation in angiosperms." Journal of Plant Research 130, no. 3 (March 8, 2017): 485–90. http://dx.doi.org/10.1007/s10265-017-0913-9.
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Alcântara-Neto, A. S., M. Fernandez-Rufete, E. Corbin, G. Tsikis, R. Uzbekov, A. S. Garanina, P. Coy, C. Almiñana, and P. Mermillod. "Oviduct fluid extracellular vesicles regulate polyspermy during porcine in vitro fertilisation." Reproduction, Fertility and Development 32, no. 4 (2020): 409. http://dx.doi.org/10.1071/rd19058.
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High polyspermy is one of the major limitations of porcine invitro fertilisation (IVF). The addition of oviductal fluid (OF) during IVF reduces polyspermy without decreasing the fertilisation rate. Because extracellular vesicles (EVs) have been described as important OF components, the aim of this study was to evaluate the effect of porcine oviductal EVs (poEVs) on IVF efficiency compared with porcine OF (fresh and lyophilised). OF was collected from abattoir oviducts by phosphate-buffered saline flush, and poEVs were isolated by serial ultracentrifugation. Four IVF treatments were conducted: poEVs (0.2mgmL–1), OF (10%), lyophilized and reconstituted pure OF (LOF; 1%) and IVF without supplementation (control). Penetration, monospermy and IVF efficiency were evaluated. Transmission electron microscopy showed an EVs population primarily composed of exosomes (83%; 30–150nm). Supplementation with poEVs during IVF increased monospermy compared with control (44% vs 17%) while maintaining an acceptable penetration rate (61% vs 78% respectively) in a similar way to OF and LOF. Western blotting revealed poEVs proteins involved in early reproductive events, including zona pellucida hardening. In conclusion, our finding show that poEVs are key components of porcine OF and may play roles in porcine fertilisation and polyspermy regulation, suggesting that supplementation with poEVs is a reliable strategy to decrease porcine polyspermy and improve invitro embryo production outcomes.
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Sullivan, Robert, and Pierre Couillard. "Effet du vieillissement sur la défense rapide contre la polyspermie chez l'ovule d'oursin." Biochemistry and Cell Biology 65, no. 12 (December 1, 1987): 1064–69. http://dx.doi.org/10.1139/o87-139.
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Aging of unfertilized ova of Echinoids is marked by a lowered fertilizability and an increased susceptibility to polyspermy. Since the fast block to polyspermy is electrically mediated in these species, we have studied the electrophysiological events triggered by fertilization in aging ova of Strongylocentrotus purpuratus. Aging of unfertilized ova is accompanied by a decrease in the resting potential. This could be explained by a decreased activity of membrane ionic pumps and could account for the gradual unfertilizability of aged eggs. Similarly, the depolarization plateau, which follows fertilization in freshly shed eggs, is abolished in eggs older than 80 min. Such aged eggs become vulnerable to polyspermy until the definitive, complete block is established by the cortical reaction. Finally, irrespective of age, fertilized ova repolarize at the same average membrane potential of −72 mV. This could reflect the general metabolic activation that follows fertilization.
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Limatola, Nunzia, Filip Vasilev, Luigia Santella, and Jong Tai Chun. "Nicotine Induces Polyspermy in Sea Urchin Eggs through a Non-Cholinergic Pathway Modulating Actin Dynamics." Cells 9, no. 1 (December 25, 2019): 63. http://dx.doi.org/10.3390/cells9010063.
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While alkaloids often exert unique pharmacological effects on animal cells, exposure of sea urchin eggs to nicotine causes polyspermy at fertilization in a dose-dependent manner. Here, we studied molecular mechanisms underlying the phenomenon. Although nicotine is an agonist of ionotropic acetylcholine receptors, we found that nicotine-induced polyspermy was neither mimicked by acetylcholine and carbachol nor inhibited by specific antagonists of nicotinic acetylcholine receptors. Unlike acetylcholine and carbachol, nicotine uniquely induced drastic rearrangement of egg cortical microfilaments in a dose-dependent way. Such cytoskeletal changes appeared to render the eggs more receptive to sperm, as judged by the significant alleviation of polyspermy by latrunculin-A and mycalolide-B. In addition, our fluorimetric assay provided the first evidence that nicotine directly accelerates polymerization kinetics of G-actin and attenuates depolymerization of preassembled F-actin. Furthermore, nicotine inhibited cofilin-induced disassembly of F-actin. Unexpectedly, our results suggest that effects of nicotine can also be mediated in some non-cholinergic pathways.
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Scott, Rod J. "Polyspermy in apomicticCrataegus: yes and no." New Phytologist 173, no. 2 (December 19, 2006): 227–30. http://dx.doi.org/10.1111/j.1469-8137.2006.01958.x.
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Luis Stephano, José, and Meredith Gould. "Avoiding polyspermy in oyster (Crassostrea gigas)." Aquaculture 73, no. 1-4 (October 1988): 295–307. http://dx.doi.org/10.1016/0044-8486(88)90063-4.
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Koyanagi, Sayaka, Hiroko Hamasaki, Satoshi Sekiguchi, Kenshiro Hara, Yoshiyuki Ishii, Shigeru Kyuwa, and Yasuhiro Yoshikawa. "Effects of ubiquitin C-terminal hydrolase L1 deficiency on mouse ova." REPRODUCTION 143, no. 3 (March 2012): 271–79. http://dx.doi.org/10.1530/rep-11-0128.
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Maternal proteins are rapidly degraded by the ubiquitin–proteasome system during oocyte maturation in mice. Ubiquitin C-terminal hydrolase L1 (UCHL1) is highly and specifically expressed in mouse ova and is involved in the polyspermy block. However, the role of UCHL1 in the underlying mechanism of polyspermy block is poorly understood. To address this issue, we performed a comprehensive proteomic analysis to identify maternal proteins that were relevant to the role of UCHL1 in mouse ova using UCHL1-deficientgad. Furthermore, we assessed morphological features ingadmouse ova using transmission electron microscopy. NACHT, LRR, and PYD domain-containing (NALP) family proteins and endoplasmic reticulum (ER) chaperones were identified by proteomic analysis. We also found that the ‘maternal antigen that embryos require’ (NLRP5 (MATER)) protein level increased significantly ingadmouse ova compared with that in wild-type mice. In an ultrastructural study,gadmouse ova contained less ER in the cortex than in wild-type mice. These results provide new insights into the role of UCHL1 in the mechanism of polyspermy block in mouse ova.
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Maluchnik, Darek, and Ewa Borsuk. "Sperm entry into fertilised mouse eggs." Zygote 2, no. 2 (May 1994): 129–31. http://dx.doi.org/10.1017/s096719940000188x.
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SummaryWe have investigated the oolemma block to polyspermy in the mouse. Zona-free and zona-intact eggs were fertilised and subsequently re-inseminated (the latter following zona pellucida removal). The ‘perivitelline’ block to polyspermy in zona-intact eggs renders motile sperm in the perivitelline space unable to bind to the oolemma. This is not connected with irreversible changes in the egg plasma membrane, because freshly added sperm can still fuse with such eggs freed from the zona. Fertilised eggs eventually lose the ability to fuse with sperm within 1 h, while still being able to bind many sperm.
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Jaffe, Laurinda A. "The fast block to polyspermy: New insight into a century-old problem." Journal of General Physiology 150, no. 9 (August 6, 2018): 1233–34. http://dx.doi.org/10.1085/jgp.201812145.
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Stepińska, Urszula, and Bozenna Olszańska. "Detection of deoxyribonuclease I and II activities in Japanese quail oocytes." Zygote 9, no. 1 (February 2001): 1–7. http://dx.doi.org/10.1017/s0967199401001010.
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Birds exhibit physiological polyspermy, i.e. numerous spermatozoa enter the germinal disc of an oocyte and form pronuclei during fertilisation. However, only one of them unites with the female pronucleus to form a zygote nucleus; the supernumerary spermatozoal nuclei degenerate at the early cleavage stages. To establish a factor responsible for spermatozoal degeneration, the presence of DNase activity was studied in vitro in extracts of Japanese quail oocytes using λ DNA/HindIII as a substrate. The experimental conditions were designed to reveal the presence of either DNase I or DNase II activities, separately. Degradation of the substrate DNA was evaluated by electrophoresis on agarose gels stained with ethidium bromide. High activities of DNase I and DNase II were found in the germinal discs of the largest vitellogenic oocytes. DNase I activity was estimated to be about 3 × 10−3 Kunitz units and DNase II about 4 × 10−2 Kunitz units per germinal disc. DNase I activity in an oocyte seems to increase during oogenesis since DNA degradation by the extracts from the germinal discs of the largest vitellogenic oocytes was much higher than by those from previtellogenic and small vitellogenic oocytes. The presence of high DNase I and II activities in the largest vitellogenic oocytes would point to their role in degradation of DNA from supernumerary spermatozoa entering the ovum during polyspermic fertilisation in birds. The enzymes could be a factor, or one of the factors, in the late block to polyspermy in the cytoplasm of avian eggs. It is suggested here that the DNase activities might also be responsible for poor efficiency in obtaining transgenic birds by microinjection of exogenous DNA into the fertilised chick ovum.
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Que, Emily L., Francesca E. Duncan, Amanda R. Bayer, Steven J. Philips, Eric W. Roth, Reiner Bleher, Sophie C. Gleber, Stefan Vogt, Teresa K. Woodruff, and Thomas V. O’Halloran. "Zinc sparks induce physiochemical changes in the egg zona pellucida that prevent polyspermy." Integrative Biology 9, no. 2 (2017): 135–44. http://dx.doi.org/10.1039/c6ib00212a.
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Luttikhuizen, Pieternella C., and Laas P. Pijnacker. "Mosaic haploiddiploid embryos and polyspermy in the tellinid bivalve Macoma balthica." Genome 45, no. 1 (February 1, 2002): 59–62. http://dx.doi.org/10.1139/g01-128.
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We investigated meiosis, fertilization, and early development in eggs of the tellinid bivalve Macoma balthica (L.), which has external fertilization. Meiosis is standard but polyspermy is found to be very common. In all eight crosses examined, mosaic embryos consisting of a mixture of diploid (2n = 38) and haploid cells occur at a frequency ranging from 2.7 to 29.1%. The earliest mosaic found is in the two-cell stage. We propose that an androgenic haploid cell lineage can originate from one supernumerary sperm that decondenses into a functional haploid nucleus, starts mitosis, and is incorporated in the developing embryo.Key words: bivalves, fertilization, embryos, polyspermy, mosaicism.
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Ferré, Luis B., Yanina Bogliotti, James L. Chitwood, Cristóbal Fresno, Hugo H. Ortega, Michael E. Kjelland, and Pablo J. Ross. "Effect of spermatozoa motility hyperactivation factors and gamete coincubation duration on in vitro bovine embryo development using flow cytometrically sorted spermatozoa." Reproduction, Fertility and Development 29, no. 4 (2017): 805. http://dx.doi.org/10.1071/rd15289.
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The aim of the present study was to evaluate the effects of sperm motility enhancers and different IVF times on cleavage, polyspermy, blastocyst formation, embryo quality and hatching ability. In Experiment 1, sex-sorted X chromosome-bearing Bos taurus spermatozoa were incubated for 30 min before 18 h fertilisation with hyperactivating factors, namely 10 mM caffeine (CA), 5 mM theophylline (TH), 10 mM caffeine and 5 mM theophylline (CA + TH); and untreated spermatozoa (control). In Experiment 2, matured B. taurus oocytes were fertilised using a short (8 h) or standard (18 h) fertilisation length, comparing two different fertilisation media, namely synthetic oviducal fluid (SOF) fertilisation medium (SOF-FERT) and M199 fertilisation medium (M199-FERT). Cleavage and blastocyst formation rates were significantly higher in the CA + TH group (77% and 27%, respectively) compared with the control group (71% and 21%, respectively). Cleavage rates and blastocyst formation were significantly lower for the shortest fertilisation time (8 h) in M199-FERT medium (42% and 12%, respectively). The SOF-FERT medium with an 8 h fertilisation time resulted in the highest cleavage rates and blastocyst formation (74% and 29%, respectively). The SOF-FERT medium produced the highest embryo quality (50% Grade 1) and hatching rate (66%). Motility enhancers did not affect polyspermy rates, whereas polyspermy was affected when fertilisation length was extended from 8 h (3%) to 18 h (9%) and in M199-FERT (14%) compared with SOF-FERT (6%). We conclude that adding the motility enhancers CA and TH to sex sorted spermatozoa and Tyrode’s albumin lactate pyruvate (TALP)-Sperm can improve cleavage and embryo development rates without increasing polyspermy. In addition, shortening the oocyte–sperm coincubation time (8 h) resulted in similar overall embryo performance rates compared with the prolonged (18 h) interval.
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Gadella, B. M. "002. PIG SPERM EGG INTERACTION AND FORMATION OF A ZONA PELLUCIDA BINDING COMPLEX." Reproduction, Fertility and Development 22, no. 9 (2010): 2. http://dx.doi.org/10.1071/srb10abs002.
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In order to achieve fertilization sperm cells first need to successfully interact with the zona pellucida. Before reaching the zona pellucida the sperm cell undergoes extensive remodeling both in the male and female genital tract. These changes serve to mediate optimal recognition of the zona pellucida in the oviduct (primary zona pellucida binding). Optimal sperm-zona interactions are crucial for porcine oocyte fertilization: The zona pellucida- attached sperm cell is triggered to undergo the acrosome reaction and will also become hypermotile. Together these two responses allow the sperm cell to drill through the zona pellucida (secondary zona pellucida binding) this coincides with local sperm zona drilling so that a few sperm can reach the perivitellin space. This delaying strategy allows only one sperm cell in a given time-point to bind and fuse with the oocyte (fertilization) and thus minimizes the risk of polyspermy. The polyspermy block is essentially executed by the fertilized oocyte that immediately secretes its cortical granule content into the perivitellin space. This content blocks sperm-oocyte fusion either by sticking to the oolemma or by the induction of a biochemical reaction of the zona pellucida (zona pellucida hardening). The cortical reaction thus blocks sperm-zona pellucida binding and/or sperm-zona pellucida drilling. Note that zona pellcuida interactions under pig IVF conditions relatively frequently result in polyspermy. It is not clear whether this is also the case in vivo after natural mating. More importantly we do not know how other artificial reproductive technologies affect polyspermy rates. This is especially relevant for new sperm treatments and insemination technologies in which sperm are activated by capacitation media essentially mimicking the IVF media. Therefore, better understanding of sperm activation and of the arrangement of proteins involved in zona pellucida interactions are relevant for designing strategies to further improve pig reproduction.
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Grossniklaus, Ueli. "Polyspermy produces tri-parental seeds in maize." Current Biology 27, no. 24 (December 2017): R1300—R1302. http://dx.doi.org/10.1016/j.cub.2017.10.059.
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VIGFÚSSON, EINAR. "On polyspermy in charlock (Sinapis arvensis L.)." Hereditas 70, no. 1 (February 12, 2009): 23–38. http://dx.doi.org/10.1111/j.1601-5223.1972.tb00991.x.
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Toda, Erika, and Takashi Okamoto. "Formation of triploid plants via possible polyspermy." Plant Signaling & Behavior 11, no. 9 (August 11, 2016): e1218107. http://dx.doi.org/10.1080/15592324.2016.1218107.
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Nicotra, Antonietta, and Gerald Schatten. "Propranolol induces polyspermy during sea urchin fertilization." Molecular Reproduction and Development 43, no. 3 (March 1996): 387–91. http://dx.doi.org/10.1002/(sici)1098-2795(199603)43:3<387::aid-mrd13>3.0.co;2-z.
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Wang, Wei-Hua, Billy N. Day, and Guang-Ming Wu. "How does polyspermy happen in mammalian oocytes?" Microscopy Research and Technique 61, no. 4 (June 10, 2003): 335–41. http://dx.doi.org/10.1002/jemt.10346.
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Pongsuthirak, Pallop. "Comparison of short and long co-incubation time of gametes for in vitro fertilization." International Journal of Reproduction, Contraception, Obstetrics and Gynecology 8, no. 11 (October 23, 2019): 4366. http://dx.doi.org/10.18203/2320-1770.ijrcog20194548.
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Background: The short and long co-incubation time of gametes for in vitro fertilization are still debatable issues. This study aims to investigate the effects of short and long co-incubation time of gametes on fertilization, polyspermy, embryonic developmental potential, and clinical outcomes.Methods: Sixty-five patients undergoing IVF treatment were invited to participate in the study between May 2017 and March 2019. Ovarian hyperstimulation was prescribed and oocytes were obtained by trans-vaginal aspiration under ultrasound guidance. Sibling oocytes were randomly allocated to short co-incubation for 4 hours (Group I) in 352 oocytes and long co-incubation for 16-18 hours in 363 oocytes (Group II). Rescue ICSI was carried out if total fertilization failure was documented. Fertilization, embryonic development, and pregnancy outcomes were determined.Results: No significant differences between short and long co-incubation were found in fertilization, polyspermy, cleavage, blastocyst, implantation, clinical pregnancy, and live birth rates.Conclusions: The present study showed that short co-incubation of gametes had no significant difference in fertilization, polyspermy, embryo development, and pregnancy outcomes when compared to long co-incubation. The short co-incubation with early cumulus cell removal and rescue ICSI may have the potential to help a couple who had total fertilization failure.
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Pongsuthirak, Pallop. "Comparison of embryonic competence and clinical outcomes between early and late cumulus cell removal for in vitro fertilization." Clinical and Experimental Reproductive Medicine 48, no. 4 (December 1, 2021): 362–67. http://dx.doi.org/10.5653/cerm.2021.04497.
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Objective: The impact of early mechanical removal of cumulus cells on fertilization and embryonic development is not yet precisely known. This study aimed to investigate the effects of early and late cumulus cell removal on fertilization, polyspermy, embryonic development potential, blastocyst development, and clinical outcomes.Methods: A prospective study was conducted of patients who underwent in vitro fertilization between September 2019 and October 2020. Sibling oocytes were randomly allocated after insemination to early cumulus cell removal at 6 hours (group I) and late cumulus cell removal at 16–18 hours (group II). If total fertilization failure (TFF) was determined to have occurred at early cumulus cell removal, rescue intracytoplasmic sperm injection (ICSI) was performed. Fertilization, embryonic development, and pregnancy outcomes were compared.Results: A total of 912 oocytes were assigned to group I (458 oocytes) and group II (454 oocytes). Fertilization, polyspermy, embryo quality, and pregnancy outcomes were not significantly different between both groups. Rescue ICSI enabled fertilization of 79.2% of the TFF oocytes.Conclusion: Early cumulus cell removal at 6 hours had no significant difference in fertilization, polyspermy, embryo development, or obstetric and perinatal outcomes compared to late removal. Early cumulus cell removal combined with early rescue ICSI may have the potential to help couples with TFF.
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Tokeshi, I., H. Tatemoto, N. Muto, T. Yoshimoto, S. Nakamura, and T. Nakada. "317 ANTI-HYALURONIDASE ACTION OF ELLAGIC ACID EFFECTIVELY PREVENTS POLYSPERMY THROUGH SUPPRESSION OF THE ACROSOME REACTION INDUCED BY THE SPERM - ZONA INTERACTION DURING PORCINE IN VITRO FERTILIZATION." Reproduction, Fertility and Development 19, no. 1 (2007): 274. http://dx.doi.org/10.1071/rdv19n1ab317.
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We previously reported that the anti-hyaluronidase agents oligosaccharide and tannic acid (TA) were efficient probes for promoting the normal fertilization process in terms of an effective decrease in the incidence of polyspermy, not only in cumulus-enclosed but also in denuded oocytes in pigs. It was unclear, however, why the polyspermic penetration into the zona pellucida (ZP) was directly prevented by the anti-hyaluronidase action. The present study was conducted to examine the effects of 3 tannin relatives [TA, gallic acid (GA), and ellagic acid (EA)] on IVF parameters and the acrosome reaction induced by the sperm–ZP interaction. The anti-hyaluronidase and radical-scavenging activities of tannin relatives were measured by the colorimetric and the DPPH methods, respectively. Porcine cumulus–oocyte complexes (COCs) were cultured for 44 h in 0.1 mL of TCM-199 supplemented with 0.6 mM cysteine, 40 mU mL-1 of FSH, 20 mU mL-1 of LH, and 10% porcine follicular fluid. After in vitro maturation (IVM), the COCs were freed from their cumulus cells and inseminated by frozen-thawed ejaculated sperm in modified Tris-buffered medium (IVF medium) containing 0 (control) or 5 �g mL-1 of tannin relatives. After 2 h of co-incubation, the oocytes were gently pipetted to remove loosely bound sperm and stained with Hoechst 33342 to count the number of ZP-bound sperm, or stained with fluorescein isothiocyanate (FITC)-PNA, PI, and 422,6-diamidino-2-phenylindole to evaluate the acrosomal status. At 10 h post-insemination, IVF parameters were examined by lacmoid staining. The data were analyzed by ANOVA and the Tukey-Kramer test. None of the tannin relatives caused a protective proteolytic modification of the ZP matrix or a reduction of the acrosomal proteolytic activity or the number of ZP-bound sperm. There was no difference in the sperm penetration rate even in the presence of tannin relatives (73-82%). However, the incidence of polyspermy was remarkably prevented by TA (32%; 31/98) and EA (21%; 20/94) compared with the control (58%; 58/100; P < 0.05), resulting from their strong anti-hyaluronidase actions, whereas GA without the anti-hyaluronidase action had no effect on the prevention of polyspermy (51%; 43/84). The rate of acrosome reaction induced by the sperm–ZP interaction was decreased by TA (15%; 123/833) and EA (16%; 110/708) compared with the control (25%; 238/939; P < 0.05), and a similar result was found in sperm binding to the pretreated ZP with 500 U of hyaluronidase for 2 h (18%; 351/1959). Interestingly, when sperm were incubated in IVF medium with 10 �g mL-1 of progesterone for 0.5 h to induce a compulsory acrosome reaction instead of the ZP, EA never disturbed the acrosome reaction (23%; 98/424) as control (23%; 102/437), although this reaction was blocked by TA (13%; 57/427) and GA (13%; 50/375), which possessed higher levels of radical-scavenging activity than EA (P < 0.05). These results indicate that the anti-hyaluronidase action of TA and EA effectively prevented polyspermy during porcine IVF as a consequence of suppression of the acrosome reaction functionally induced by the sperm–ZP interaction requiring the hyaluronidase intervention.
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Men, H., W. Si, and J. K. Critser. "305 INCREASED MONOSPERMIC PENETRATION OF PORCINE OOCYTES USING A VISCOUS IN VITRO FERTILIZATION SYSTEM." Reproduction, Fertility and Development 19, no. 1 (2007): 268. http://dx.doi.org/10.1071/rdv19n1ab305.
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The high incidence of polyspermy remains a major problem in porcine IVF systems. The high number of sperm bound to the oocyte is one of the major causes for pathological polyploidy (Funahashi 2003 Reprod. Fertil. Dev. 15, 167–177). In this experiment, we tested the hypothesis that limiting the number of competent sperm able to participate in fertilization during IVF will result in a reduction in polyspermic penetration. Combinations of various sperm (3 levels) and Percoll concentrations (3 levels) were used in a modified Tris-buffered medium (mTBM) with 3% polyvinylpyrrolidone supplementation to find the optimal combination that could result in a significant reduction in polyspermy rate compared with that resulting from the current porcine IVF system (Abeydeera and Day 1997 Theriogenology 48, 537–544). In vitro-matured gilt oocytes (n = 844) were randomly allocated into 1 of 10 treatment groups in groups of 30. Motile sperm were selected from frozen–thawed samples by density gradient centrifugation using a discontinuous Percoll gradient consisting of 3 mL of 54% Percoll in PBS on top of 1 mL of 72% Percoll in a 15-mL conical tube at 600g for 15 min. After centrifugation, the supernatant was removed as much as possible without disturbing the sperm pellet. Sperm stock solutions were made in a manner that resulted in the desired final concentration of sperm when 5 µL of the sperm solution were added to 95 µL of fertilization medium. During IVF, the sperm stock solutions were placed at one side of an IVF droplet so that most sperm were trapped inside the viscous Percoll solution and only a limited number of sperm, with high progressive motility, were capable of fertilizing the oocytes. A group of oocytes fertilized using our standard IVF protocol with a sperm concentration of 5 × 105 sperm mL−1 in mTBM served as a control. Pronuclear formation after 12 h of fertilization was used to assess sperm penetration. The data from 3 replicates were analyzed by standard ANOVA procedures using the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). The treatment group of 5 × 105 sperm mL−1 + 10% Percoll resulted in a significantly higher monospermy rate than the control or the treatment group of 1 × 106 sperm mL−1 + 30% Percoll (Table 1; P < 0.05). These data support the hypothesis that the polyspermy rate in a porcine IVF system can be reduced by limiting the number of competent sperm fertilizing the oocytes. Table 1.Monospermy rates in a viscous IVF system (mean ± SEM%) This project was supported by NIH U42 RR 018877.
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Kreysing, U., T. Nagai, and H. Niemann. "Male-dependent variability of fertilization and embryo development in two bovine in vitro fertilization systems and the effects of casein phosphopeptides (CPPs)." Reproduction, Fertility and Development 9, no. 4 (1997): 465. http://dx.doi.org/10.1071/r96097.
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This study investigated the effects of semen from five different bulls and two different ejaculates of the same bull on penetration, cleavage, blastocyst formation, and cell allocation in bovine blastocysts produced in vitro. Casein phosphopeptides (CPPs) were tested for their ability to enhance fertilization and minimize variability among bulls and ejaculates. In Experiment 1, the BO-fertilization system was employed. Penetration and polyspermy both displayed great variation among bulls and between ejaculates, whereas no significant differences were observed in cleavage and blastocyst-formation rates. Similar variability was observed in penetration, polyspermy, cleavage, blastocyst-formation rates and cell allocation and distribution when the two fertilization systems, TALP and BO, were compared in Experiment 2. The BO-system supported penetration and polyspermy better (P < 0·05) than the TALP-system, whereas the TALP-system was superior (P < 0·05) in supporting cleavage and blastocyst formation. Significant interactions existed between bulls and the fertilization system employed. It is concluded that the success of in vitrofertilization is markedly dependent on individual bulls as well as on ejaculates from the same bull. CPPs are able to enhance penetration and embryo development in certain bulls or ejaculates and thus contribute to reducing the degree of individual variability, but they do not generally improve the success of bovine embryo production in vitro.