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Polyspermy can be produced experimentally in pigs by delayed mating, which results in the fertilization of aged eggs (Pitkjanen, 1955; Thibault, 1959; Hancock, 1959; Dziuk & Polge, 1962; Hunter, 1967). A high incidence of polyspermy has also been observed in pigs following induced ovulation and insemination during the luteal phase of the oestrous cycle (Hunter, 1966). These observations suggest that the endogenous level of progesterone at the time of fertilization might affect the block to polyspermy in pig eggs, since, under both conditions cited, developing or fully functional corpora lutea would be present in the ovaries at the time of sperm penetration. The present study was conducted to determine the frequency of polyspermy in pigs injected with progesterone at various intervals before ovulation and fertilization.

The experimental animals were thirty-six mature, crossbred, Large White × Essex,

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.

The objective of these experiments was to evaluate factors affecting in vitro fertilization of bovine oocytes matured in vitro, and their subsequent development to blastocysts. In Expts 1 and 2, sperm concentration, spermatozoa and oocyte incubation time, motility enhancers and semen source were manipulated. Fluorescent microscopy of microtubules and chromatin was used to observe sperm penetration rate, sperm aster formation and chromatin decondensation. Oocyte penetration rates were affected by sperm concentration but not by spermatozoa and oocyte incubation time. The effect of sperm concentration was due primarily to changes in polyspermy and not monospermy. Motility enhancers had no effect on any parameter measured. In Expt 3, oocytes were matured for 17, 22, 28 and 34 h before fertilization and evaluated for fertilization rates, morphology of cortical granules and exocytosis and blastocyst development. A domain free of cortical granules that was associated with the metaphase chromatin was not observed in mature bovine oocytes. As oocytes matured from 17 to 34 h, the distribution of cortical granules progressed from clustered to diffuse. Although monospermic fertilization rates were similar and cortical granule exocytosis occurred in all groups, polyspermy increased with maturation time. Development to blastocysts increased from 17 to 22 h of maturation but decreased thereafter with increasing maturation time. These results suggest that polyspermy can be reduced by adjusting sperm concentration and spermatozoa and oocyte incubation time with little effect on monospermic fertilization. Increased polyspermy with increased maturation time was not due to a lack of cortical granule exocytosis.

The mechanisms for the prevention of polyspermy in mammalian eggs appear to be associated with the development of the so-called `zona reaction' (Braden, Austin & David, 1954) and with the development of a vitelline surface block to polyspermy (Austin & Braden, 1956). The time required for the development of the zona reaction has been estimated to be not less than 10 min and not more than 1½ to 2 hr (Braden et al., 1954).

Hamster eggs recovered from mated females show a low incidence (1·6%) of polyspermy (Austin & Braden, 1956) while eggs fertilized in vitro show a high incidence, often as high as 100% (Yanagimachi & Chang, 1964; Barros & Austin, 1967; Barros, 1968b; Yanagimachi, 1969).

It has not been established whether the high incidence of polyspermy in vitro is due to the penetration of several spermatozoa at the same time or to a continuous flow of spermatozoa

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.

Summary. A dose–response relationship between the incidence of polyspermy and sperm concentration was found by analysing chromosome preparations of first-cleavage mouse embryos derived from fertilizations in vitro with 3 concentrations of mouse spermatozoa (2 × 10⁴, 2 × 10 ⁶ and 1 × 10⁷/ml).

Summary. Female mice, induced to superovulate, were injected subcutaneously with progesterone or oestradiol near the time when hCG was given. The incidence of polyspermy in first-cleavage embryos following mating or in-vitro fertilization was then determined. There were no detectable differences in the incidence or degree of polyspermy between treated and control in either the in-vitro or in-vivo groups, although the mean incidence of polyspermy was higher in vitro than in vivo. Furthermore, there was no detectable acceleration of egg transport after administration of either hormone.

Incubation of hamster eggs with trypsin or chymotrypsin, but not with a series of glycosidases or lipases, prevents capacitated spermatozoa from binding to the zona pellucida so that fertilization is prevented (Hartmann & Gwatkin, 1971; Gwatkin, Williams & Andersen, 1973). Inactivation of the binding sites for spermatozoa on the zona pellucida by a trypsin-like protease released by discharge of cortical granules also appears to be responsible for the zona reaction which prevents polyspermy (Gwatkin, Williams, Hartmann & Kniazuk, 1973). Acrosin is also a trysin-like protease and it can be extracted from sperm acrosomes. We therefore investigated whether the fertilization of hamster eggs is blocked by incubation with soluble acrosin.

The pattern of changes in intracellular calcium concentration ([Ca²⁺]_i) in bovine oocytes after penetration by spermatozoa was determined. Dynamic video imaging, using Fura-2 as a probe for intracellular free calcium, showed that activation of oocytes by spermatozoa induced multiple transient increases in [Ca²⁺]_i with a spike interval of 24.2 ± 7.3 min, and that the early transient increases were propagated throughout the oocytes in the form of a wave. Calcium transients at fertilization are involved in the induction of cortical granule exocytosis and the resultant block to polyspermy. The hypothesis that the inhibition of Ca²⁺ release from inositol trisphosphate-sensitive stores would inhibit exocytosis and increase polyspermy was tested by injecting oocytes before fertilization with heparin, a potent inhibitor of inositol trisphosphate-activated Ca²⁺ release. There was no significant difference after fertilization in either [Ca²⁺]_i spikes or in polyspermy rates between control and experimental groups injected with low molecular mass heparin up to a final cytoplasmic concentration of 400 μmol l⁻¹. We conclude that inositol trisphosphate-independent Ca²⁺ stores may be mobilized during the fertilization of bovine oocytes.

Summary. Zona cutting and zona drilling of the mouse oocyte significantly increased the fertilization rate (3·8–90%) at low sperm concentrations (< 200 000/ml) compared with zona-intact controls (0–45%). More oocytes were fertilized after zona drilling. Zona cutting was associated with a low loss of oocytes (< 1%), no increase in polyspermy and normal development in vitro and in vivo after fertilization. There was a 4% oocyte loss rate after zona drilling, mostly due to extrusion of the oocyte from the zona during the procedure. Hatching of blastocysts occurred about 12 h earlier for zona-drilled than for zona-cut and zona-intact control oocytes. Zona drilling was associated with a higher, but not statistically significant, rate of polyspermy at all sperm concentrations tested.

The proportion of zygotes developing to the blastocyst stage was not different between the techniques (zona cut, 77%; zona drilled, 66%; control, 71%). Similarly, no difference was found in the percentage of embryos implanting after blastocyst transfer to the uterine horns of pseudopregnant female mice (zona cut, 67%; zona drilled, 68%; control, 77%).

Transmission electron microscopy demonstrated the induced defects in the zona with no damage to the oocyte or oolemma. Parthenogenetic activation was not seen after either of the micromanipulative techniques. Both techniques have promise for application to the human.

Keywords: in-vitro fertilization; oligospermia; zona drilling; zona cutting; polyspermy