Fungal metabolites are known to have potent and diverse properties such as antiviral, antidiabetic, antitumour, antioxidant, free radical scavenging, and antibacterial effects which can be utilized to treat diseases. In this study, we investigated the functional activity of stereumamide A (StA) derived from a culture broth of Trichaptum fuscoviolaceum during the in vitro fertilization (IVF) of pig oocytes, to determine its effects on sperm penetration. Oocytes matured in vitro were fertilized in the absence or presence of varying concentrations of StA (0-50 μg/ml StA). When StA was directly added into the IVF medium, significantly lower fertilization rates were seen with the 20 or 50 μg/ml StA (2.0–17.5%) treatments compared with those of 10 μg/ml StA or the controls (60.9–62.3%), whereas StA had no influence on the survival of oocytes and spermatozoa throughout the IVF process. For evaluating the control of sperm entry, mature oocytes were pre-incubated in a medium containing 20 μg/ml StA for 1 h, and then IVF was subsequently performed. The incidence of polyspermy was significantly reduced when oocytes were pre-incubated with StA (15.0% vs. 50.4–57.5% in controls). In conclusion, sperm penetration was inhibited in the medium in the presence of StA during IVF, while StA did not affect sperm motility and fertility competence. Fertilization was controlled when mature oocytes were incubated with StA prior to IVF, suggesting the possible use of the fungal metabolite in assisted reproductive technology for humans and animals.

The present study was designed to evaluate the effect of the combination of oviduct fluid flush (OFF) and oviduct epithelial cells (OEC) in modulating the incidence of polyspermy in pigs. Therefore, for in vitro fertilization (IVF), oocyte and sperm were co-cultured in Tris-buffered medium (TBM) either supplemented with 10% OFF (OFFD group), or in the presence of a bovine OEC monolayer (OEC group), or the oocytes were exposed to OFF for 30 min before IVF (OFFB group), or in the presence of an OEC monolayer (OFFB + OEC group). Regardless of sperm concentration used (0.5, 1.5, and 4.5 × 105 cells/ml), supplementation of IVF medium with 10% OFF led to an increased (P < 0.05) monospermy rate, without alteration (P > 0.05) of the penetration rate in comparison with the control and OEC groups. When the IVF medium was supplemented with heparin, an overall increase (P < 0.05) of the final output of the IVF system in terms of zygotes with two pronuclei (2PN) was observed in the OFFD group, compared with the control and OEC groups, at a sperm concentration of 4.5 × 105 cells/ml. At this concentration, OFFB improved the monospermy rate but decreased the penetration rate, resulting in low efficiency of monospermic zygotes production. Despite this, no major effect was observed in the developmental competence of the presumed zygotes up to the blastocyst stage. The combination of OFFB with OEC improved the penetration rate, while maintaining the high monospermic rate induced by OFFB. In conclusion, the combination of treatment of oocytes by diluted OFF 30 min before IVF, followed by IVF in the presence of OEC, improved monospermic zygote production without reducing the penetration rate, when the IVF medium was supplemented with heparin.

During 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.

Preovulatory binding of viable spermatozoa in the oviduct isthmus is widely accepted as a preliminary to fertilization, but details of physiological events associated with epithelial binding and release from binding are themselves little understood. Important questions include the potential number, distribution and stability of such sites in the caudal isthmus, whether multiple molecular forms of binding exist within a single-mated individual, and whether some sites are more favourable than others for the maintenance of preovulatory sperm viability. Also to be resolved is whether spermatozoa interact with the first available binding sites in the isthmus, whether spermatozoa from second or subsequent matings bind closer to the site of fertilization, and whether the first spermatozoa entering the oviduct are those that will be released first with impending ovulation. Ideally, future research needs to focus on a fertilizing spermatozoon monitored in vivo and not on spermatozoa destined to remain on or in the zona pellucida or in the lower reaches of the oviduct.

The objective of this study was to determine the effects of oviductal fluid and heparin on sperm penetration and the characteristics of spermatozoa. The addition of oviductal fluid and heparin to the fertilisation medium decreased sperm penetration and the mean number of spermatozoa in penetrated eggs. The number of spermatozoa firmly bound to zona pellucida was also decreased in the presence of oviductal fluid and heparin. Chlortetracycline (CTC) fluorescence patterns were used to determine the incidence of capacitation and the acrosome reaction. The proportion of capacitated and acrosome-free spermatozoa increased when spermatozoa were exposed for 1.5 and 3 h to oviductal fluid and heparin. In contrast heparin alone did not increase the number of capacitated spermatozoa at these time points. These results suggest that factor(s) in oviductal secretions reduce polyspermic fertilisation and the number of spermatozoa that will penetrate porcine oocytes. The reduction of polyspermic penetration by oviductal secretions may be due to a reduced number of spermatozoa in the fertilisation medium with an intact acrosome.

Full-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.

The 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.

During activation of amphibian eggs, cortical granule exocytosis causes elaborate ultrastructural changes in the vitelline envelope. These changes involve modifications in the structure of the vitelline envelope and formation of a fertilization envelope (FE) that can no longer be penetrated by sperm. In Bufo arenarum, as the egg traverses the oviduct, the vitelline envelope is altered by a trypsin-like protease secreted by the oviduct, which induces an increased susceptibility of the vitelline envelope to sperm lysins. Full-grown oocytes of B. arenarum, matured in vitro by progesterone, are polyspermic, although cortical granule exocytosis seems to occur within a normal chronological sequence. These oocytes can be fertilized with or without trypsin treatment, suggesting that the vitelline envelope is totally sperm-permeable. Vitelline envelopes without trypsin treatment cannot retain either gp90 or gp96. This suggests that these glycoproteins are involved in the block to polyspermy and that trypsin treatment of matured in vitro oocytes before insemination is necessary to enable vitelline envelopes to block polyspermy. The loss of the binding capacity in vitelline envelopes isolated from B. arenarum oocytes matured in vitro with trypsin treatment and activated by electric shock suggests that previous trypsin treatment is a necessary step for sperm block to occur. When in vitro matured oocytes were incubated with the product of cortical granules obtained from in vitro matured oocytes (vCGP), vitelline envelopes with trypsin treatment were able to block sperm entry. These oocytes exhibited the characteristic signs of activation. These results support the idea that B. arenarum oocytes can be activated by external stimuli and suggest the presence of unknown oocyte surface receptors linked to the activation machinery in response to fertilization. Electrophoretic profiles obtained by SDS-PAGE of solubilized vitelline envelopes from oocytes matured in vitro revealed the conversion of gp40 (in vitro matured oocytes, without trypsin treatment) to gp38 (ascribable to trypsin activity or cortical granule product activity, CGP) and the conversion of gp70 to gp68 (ascribable to trypsin activity plus CGP activity). Taking into account that only the vitelline envelopes of in vitro matured oocytes with trypsin treatment and activated can block sperm entry, we may suggest that the conversion of gp70 to gp68 is related to the changes associated with sperm binding.

During polyspermic fertilisation in birds numerous spermatozoa enter the eggs, in contrast to the situation in mammals where fertilisation is monospermic. However, in birds only one of the spermatozoa which have entered an egg participates in zygote nucleus formation, while the supernumerary spermatozoa degenerate at early embryogenesis. Our previous work has demonstrated the presence in preovulatory quail oocytes of DNase I and II activities able to digest naked λDNA/HindIII substrate in vitro. In the present studies, the activities of both DNases in quail oocytes at different stages of oogenesis and in ovulated mouse oocytes were assayed in vitro using the same substrate. Degradation of quail spermatozoa by quail oocyte extracts was also checked. Digestion of the DNA substrate was evaluated by electrophoresis on agarose gels. The activities of DNase I and II in quail oocytes increased during oogenesis and were the highest in mature oocytes. The activities were present not only in germinal discs but also in a thin layer of cytoplasm adhering to the perivitelline layer surrounding the yolk. At all stages of oogenesis the activity of DNase II was much higher than that of DNase I. DNA contained in spermatozoa was also degraded by the quail oocyte extracts under conditions optimal for both DNases. In contrast to what is observed in quail oocytes, no DNase activities were detected in ovulated mouse eggs; this is logical as they would be useless or even harmful in monospermic fertilisation. The possible role of DNase activities in avian oocytes, in degradation of accessory spermatozoa during polyspermic fertilisation, is discussed.

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.

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.

Our previous study indicated that thimerosal is one of the most effective artificial activators to mimic sperm-induced increases in the intracellular free calcium concentration ([Ca2+]i) and other activation events in pig oocytes (Macháty et al., 1997). The present study was conducted to examine the temporal relationship between intracellular calcium transients, cortical granule (CG) exocytosis and the zona reaction induced by thimerosal. When pig oocytes matured in vitro were exposed to 200 μM thimerosal the first intracellular calcium transient, with a mean peak ratio of 4.97 ± 1.14, was observed 509.64 ± 122.03 s after addition of thimerosal. The density of CGs fell significantly from 63.3 ± 11.7 CGs/100 μm2 of cortex in control oocytes to 25.7 ± 19.2 CGs/100 μm2 of cortex (59.4% release) at 2 min after the first intracellular calcium transient. At 5 min after the calcium transient the residual CG density had been reduced to 10.7 ± 10.4 CGs/100 μm2 of cortex (83.1% release). This degree of CG exocytosis was the same as that in oocytes penetrated by sperm (9.5 ± 5.1 CGs/100 μm2 of cortex). No further decrease in residual CG density was observed at 10 min (10.3 ± 14.8 CGs/100 μm2 of cortex). Whereas 77.4% (120/155) of control oocytes were penetrated by spermatozoa only 1.4% (2/144) of thimerosal-treated oocytes were penetrated. Further experimental results obtained by in vitro fertilisation of oocytes with preincubated (capacitated) spermatozoa suggested that the zona block to sperm penetration in thimerosal-treated oocytes occurred within 35 min after CG exocytosis and 40 min after the first calcium transient. These results indicate that polyspermic penetration of pig oocytes inseminated in vitro is not due to delayed or incomplete CG exocytosis but more likely to a delayed zona reaction and/or simultaneous sperm penetration.

In the red alga Bostrychia moritziana, release of spermatia is triggered by slight osmotic shock; they emerge under pressure apparently generated by swelling of the mucilaginous sheath. Spermatia adhere tenaciously to trichogynes of the carpogonium. Adhesion triggers spermatial mitosis, which is complete in about 30–45 min; there is no cytokinesis and the binucleate spermatium becomes vacuolated. The delicate, dynamic trichogyne cytoplasm contains complex membranous components and vacuoles. At the contact zone, the trichogyne and spermatial wall erode, forming a pore, and cytoplasmic continuity (plasmogamy) is achieved after about 50–70 min. Many trichogynes rupture during these events because of inadequate structural connection with the spermatia. Normally, both spermatial nuclei enter the trichogyne in sequence; rarely, both nuclei enter together. Entrance is rapid, and the nuclei often become thin and greatly elongated as each squeezes through the narrow pore into the trichogyne. Once inside, each nucleus resumes its normal shape as it starts to move steadily along the trichogyne, often with irregular pauses. One nucleus of each pair (not necessarily the first out of the spermatium) migrates along the trichogyne towards the carpogonium base to fertilize the female nucleus; the other moves in the opposite direction, away from the entry site, and it often ends up near the tip of the trichogyne. This same scenario was observed for each of several spermatia contributing nuclei to one trichogyne. Thus, our observations indicate that the two nuclei in each spermatium are differentiated so that only one is capable of fertilization, differentiation being visibly expressed in the direction that the nucleus moves as it enters the trichogyne.