This research was carried out to define in vitro conditions that would enable ejaculated rabbit sperm to fertilize ova in vitro. During 20 mm exposure of sperm cells to defined media of increasing NaCI concentrations, progressive removal or alteration of sperm surface seminal plasma antigenic components was initiated and completed as reflected by an immunological assay at approximately 300 and 380 mOsm/kg, respectively. Hypertonic (380 mOsm/kg) medium effected complete removal or alteration of these components as determined by the immunological assay during the first 5 mm of sperm treatment. Isotonic (305 mOsm/kg) treatment was shown to have effected removal or alteration of some but not all of the antigenic sperm coating seminal plasma components detectable by this means during 20 mm of exposure. Sperm within the perivitelline apace, presence of pronuclei. and 2- and 4-cell cleavage stages were taken as evidence for fertilization. None of 34 ova incubated for 27 h in vitro with once-washed sperm showed evidence of fertilization. Following treatment of sperm with hypertonic (380 mOsm/kg) medium prior to in vitro insemination, proportions of ova showing evidence of fertilization ranged from 8.1 percent (12 to 148 ova) to 72.2 percent (52 of 72 ova); and, following treatment of sperm with isotonic (305 mOsm/kg) medium, fertilization of ova ranged from 0 percent (0 to 19 ova) to 73.9 percent (SI of 69 ova) when results were considered according to individual male sperm donors. No large differences were found by the immunological assay that could be linked to variability of fertilizing ability of sperm from different bucks. Differences in metabolic characteristics of sperm cells was suggested as a likely reason for differences in fertilizing ability of sperm from different bucks. In experiments using sperm and ova from the same sources, no differences in fertilization results were apparent following 305 and 380 mOsm/kg sperm treatments, Initiation, but not completion, of removal or alteration of sperm surface seminal plasma antigenic components, then, was a necessary prerequisite to in vitro insemination for successful achievement of fertilization in vitro. Sperm penetration into ova occurred in some cases before 7-10 h after insemination (4 of 54 ova penetrated), but actively motile sperm were found within the perivitelline apace of other ova as late as 25 h after insemination. One hundred and seventy-six 2- and 4-cell stage embryos were transferred to 14 recipient does. Twenty-four embryos (13.1 percent) implanted in 6 recipients but most were resorbed. Three embryos resulting from in vitro fertilization with in vitro capacitated ejaculated rabbit sperm were carried successfully to term by 2 recipients and 2 of the offspring were males. This provides documentation for the authenticity of in vitro capacitation of ejaculated sperm of a mammalian species and represents the initial successful combination of in vitro sperm capacitation with in vitro fertilization and embryo transfer in the rabbit.

Capacitation of Rabbit Spermatozoa in vitro

Structure and function of the zona pellucida: identification and characterization of the proteins of the mouse oocyte's zona pellucida.

Aequorin-injected eggs of the medaka (a fresh water fish) show an explosive rise in free calcium during fertilization, which is followed by a slow return to the resting level. Image intensification techniques now show a spreading wave of high free calcium during fertilization. The wave starts at the animal pole (where the sperm enters) and then traverses the egg as a shallow, roughly 20 degrees-wide band which vanishes at the antipode some minutes later. The peak free calcium concentration within this moving band is estimated to be about 30 microM (perhaps 100-1,000 times the resting level). Eggs activated by ionophore A23187 may show multiple initiation sites. The resulting multiple waves never spread through each other; rather, they fuse upon meeting so as to form spreading waves of compound origin. The fertilization wave is nearly independent of extracellular calcium because it is only slightly slowed (by perhaps 15%) in a medium containing 5 mM ethylene glycol-bis[beta-aminoethyl ether]N,N9-tetraacetic acid (EGTA) and no deliberately added calcium. It is also independent of the large cortical vesicles, which may be centrifugally displaced. Normally, however, it distinctly precedes the well-known wave of cortical vesicle exocytosis. We conclude that the fertilization wave in the medaka egg is propagated by calcium-stimulated calcium release, primarily from some internal sources other than the large cortical vesicles. A comparison of the characteristics of the exocytotic wave in the medaka with that in other eggs, particularly in echinoderm eggs, suggests that such a propagated calcium wave is a general feature of egg activation.

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A free calcium wave traverses the activating egg of the medaka, Oryzias latipes.

WE have previously exposed sea urchin eggs to micromolar amounts of the divalent transporting ionophore A23187, and observed every parameter of normal fertilisation. The cortical reaction with elevation of the fertilisation membrane, the plasma membrane conductance changes, the respiratory burst, and increases in protein and DNA synthesis were all initiated in the usual fashion1. These activations of Lytechinus pictus and Strongylocentrotus purpuratus eggs were independent of the ionic composition of the external solutions1. A23187 seemed to act by releasing intracellular Ca2+, for eggs preloaded with 45Ca showed a twentyfold increase in 45Ca-efflux when activated by ionophore or fertilisation1. Measurements of free and bound calcium and magnesium in homogenates of unfertilised eggs showed that most of the Mg2+ was already available in soluble form, whereas the Ca2+ was sequestered but available for release1. After we found that 50 µM A23187 could activate eggs of the mollusc Acmaea insessa and the protochordate Ciona intestinalis to undergo several abnormal cleavages, we wondered whether ionophore activation might be a general phenomenon. To our knowledge artificial parthenogenesis has not been obtained with Acmaea and Ciona eggs by other methods. We have now found that calcium ionophore activation is common to several species widely separated phylogenetically.

Is calcium ionophore a universal activator for unfertilised eggs

Interest in pattern regulation arose with the first experiments of embryology almost a century ago. The hypotheses of His and Weismann (reviewed by Wilson, 1896) predicted that the fertilized egg behaves as a mosaic of autonomously differentiating parts, due to either the invisible promorphology of its cytoplasm (His) or the preexisting architecture of its chromosomes (Weismann). Roux’s first experiments in 1888 seemed to support these hypotheses: he pricked a frog egg (Rana esculenta) at the two-cell stage with a hot needle so as to inactivate one cell, hence half the egg, and found the other cell continuing to divide and in many cases developing into a half-embryo, usually a right or left half. Thus, the active cell failed to adjust its development to compensate for its defective neighbor, at least under these experimental conditions. The frog egg appeared to meet the definition of a mosaic egg, that is, one showing no pattern regulation.

Mechanisms Regulating Pattern Formation in the Amphibian Egg and Early Embryo

Formation and structure of the fertilization envelope in Xenopus laevis

A molecular approach to fertilization. II. Viability and artificial fertilization of Xenopus laevis gemetes.

As a step toward defining in molecular terms the sperm-triggered block to polyspermy reaction established by the egg at fertilization, vitelline (VE) and fertilization (FE) envelopes were isolated from eggs of the Sounth African clawed toad Xenopus laevis and some of their physicochemical properties determined. Envelopes were isolated after lysis of the fertilized or unfertilized eggs by sieving techniques; isolated envelopes retained their in situ morphology as determined by electron microscopy. The isolated envelopes had different solubility properties and, in general, VE was more readily dissolved by aqueous solvents than FE, although both could be completely dissolved by detergents or chaotropic agents. Changes in envelope solubility correlated with the progression of the cortical reaction implicating a role for cortical granule material in modifying the solubility properties of the envelope. The VE and FE were composed of protein and carbohydrate with no lipid components detected. As determined by immunodiffusion experiments, the FE contained the same antigens as the VE plus components derived from the cortical granules and the innermost jelly layer, J. The macromolecular composition of the envelopes was determined by sodium dodecyl sulfate gel electrophoresis. The VE contained at least 11 glycoproteins with molecular weights ranging from 125 000 to less than 16 000 with two components (40 000 and 33 000) accounting for almost two-thirds of the total stainable material. The FE contained ten glycoproteins that had the same molecular weights as those in the VE. One glycoprotein component underwent a reduction in molecular weight from 77 000 to 67 500 when the VE was converted to the FE. This molecular weight change was interpreted as the probable result of limited proteolysis. In addition, the FE gel electrophoresis patterns contained macromolecular components derived from the cortical granules and jelly layer, J, consistent with the immunodiffusion experiments. These components were absent when the FE was prepared in the absence of Ca2+, suggesting a role for Ca2+ in binding the VE, cortical granules, and J components together. We concluded that the conversion of the glycoproteinaceous VE to FE at fertilization is caused by interaction of the VE with components from the cortical granules and jelly layer J. These interactions are of both a chemical and physical nature.

Isolation, physicochemical properties, and the macromolecular composition of the vitelline and fertilization envelopes from Xenopus laevis eggs.

A molecular approach to fertilization: III. Development of a bioassay for sperm capacitation☆

[“SI-sulfate injected into ovulating Xeno pus laevi.s was incorporated into eggs and egg jelly coats. The extent of [“SI incorporation was dependent on the dose administered and time the eggs were ovulated after injection. The radioactivity was incorporated predominately into the innermost jelly coat layer J1; J1 contained the majority if not all of the sulfate in the jelly coat as determined by chemical analysis for sulfate. The [“SI incorporated was chemically in the form of sulfate esters as determined by chromatography of acid hydrolysates. Hydrolysis rates the [“SI-sulfate from the jelly coat macromolecules and the infrared absorption spectra of jelly suggested that sulfate was present as sugar ester of a secondary axial hydroxyl group, most likely on the C4 position of Gal, Ga1NAc, or Fuc residues. The sulfated jelly coat macromolecules were not incorporated into the egg during its passage through the oviduct nor into the embryo during development up to the hatching stage.

Don P. Wolf

Papers

Formation and structure of the fertilization envelope in Xenopus laevis

A molecular approach to fertilization. II. Viability and artificial fertilization of Xenopus laevis gemetes.

Isolation, physicochemical properties, and the macromolecular composition of the vitelline and fertilization envelopes from Xenopus laevis eggs.

A molecular approach to fertilization: III. Development of a bioassay for sperm capacitation☆

The Incorporation and Fate of [35S]-Sulfate in the Jelly Coat of Xenopus laevis Eggs