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D. P. L. Green

Summary. Hamster zonae pellucidae were labelled with fluorescein isothiocyanate and placed under thin polyacrylamide sheets polymerized on the floor of an observation chamber. The integrity of these FITC-labelled zonae pellucidae was monitored continuously by fluorescence microscopy using a silicon-intensified target video-camera during exposure to a 0·1% sodium dodecyl sulphate solution diffusing through the polyacrylamide sheet. Complete dissolution of zonae pellucidae was observed at the threshold level of detection (estimated as 0·2% of the signal from a labelled zona pellucida). Similar behaviour was shown by zonae pellucidae isolated from hamster eggs 5, 15 and 30 min after a cortical reaction induced by the ionophore A23187. These findings suggest that the hamster zona pellucida contains no underlying covalent matrix before the cortical reaction, and that the cortical reaction does not produce covalent inter-molecular cross-linking of the zona pellucida.

Keywords: zona pellucida; cortical reaction; fluorescent labelling; SDS; hamster

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D. P. L. Green

Summary. A method has been developed for establishing from electron micrographs the radius of curvature of rabbit and guinea-pig sperm nuclei in head-to-tail section (the major axis), and of guinea-pig spermatozoa with intact acrosomes in transverse section (the minor axis). Radii of curvature of head-to-tail sections lie, for the most part, between 7 and 100 μm in both species. Guinea-pig sperm nuclei in transverse section show a range of radii between 3 and 10 μm, and approximate, therefore, caps of prolate spheroids. The method is not applicable to rat and mouse spermatozoa but Nomarski light microscopy suggests that these nuclei are straight in the longitudinal direction. The distribution of the radii of curvature in rabbit sperm nuclei is similar to that of the radii of curvature of penetration slits through the zona pellucida of the rabbit egg. It is suggested that longitudinal curvature of sperm heads is correlated with any curvature of the penetration slit.

Keywords: mammalian spermatozoa; head shape; penetration slit; zona pellucida

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D. P. L. Green

Summary. Guinea-pig spermatozoa were induced to undergo an acrosome reaction with the ionophore A23187. A time course for the activation of protease activity was established. Acid treatment of fully activated spermatozoa at pH 2·35 for 30 min exposed additional activity. This was attributed to the acid dissociation of a protein inhibitor from acrosin. The acrosin content of fully activated and acid-dissociated sperm extracts was measured using a sensitive active-site titrant for serine proteases. The number of acrosin molecules per spermatozoon, calculated on the basis of the sperm count, was approximately 2 × 106, of which half were available without dissociation of the inhibitor.

Keywords: guinea-pig; spermatozoa; acrosin; active-site titration

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D. P. L. Green

Summary. Hamster oocytes were loaded with the DNA dyes Hoechst 33342 or propidium iodide. Oocytes incubated in 10 μmol Hoechst 33342 l−1 showed intracellular fluorescence within 10–20 s of exposure, as did hamster and guinea-pig spermatozoa. Impaled oocytes to which acrosome-intact hamster spermatozoa were bound before injection of Hoechst 33342 showed dye transfer to adhering spermatozoa within 2 min of injection. Oocytes loaded passively with Hoechst 33342 showed dye transfer to bound, acrosome-intact hamster spermatozoa within 10 min. On ultrastructural examination, no bound, acrosome-intact hamster spermatozoa (n=311) were found to be fused. By contrast, oocytes incubated with 10 μmol propidium iodide l−1 showed no intracellular fluorescence after 2 h, although in approximately 50% of oocytes, fluorescence developed rapidly in the first polar body. Oocytes injected with propidium iodide showed intracellular fluorescence but no dye transfer to bound, acrosome-intact hamster spermatozoa. Oocytes impaled on pipettes containing propidium iodide showed no dye transfer to unlabelled oocytes with which they were brought into contact, whereas in similar experiments using Hoechst 33342 detectable dye transfer to an adjacent oocyte occurred within 10 min. Oocytes loaded with propidium iodide transferred propidium iodide to fusion-competent guinea-pig spermatozoa during in vitro fertilization. Normally, between 20 and 40 spermatozoa bound per oocyte, and the percentage of spermatozoa showing dye transfer varied between 0 and 41%. Dye transfer occurred within 5–45 min. Only those nuclei that showed propidium iodide transfer subsequently decondensed, suggesting that dye transfer is correlated with fusion. The presence of fused spermatozoa was confirmed by ultrastructural examination of oocytes. In separate experiments, hamster and guinea-pig spermatozoa showed detectable fluorescence from propidium iodide within 20 s of osmotic rupture or membrane stripping by detergent, suggesting the lag in dye transfer to sperm nuclei during fertilization reflects a delay in sperm–oocyte fusion following adhesion.

This evidence suggests that Hoechst 33342 could be an unreliable marker for sperm–oocyte fusion in fertilization because of its capacity for passive movement from oocyte to spermatozoon. This problem can be overcome using oocytes injected with propidium iodide. With this technique, it was possible to show that fusion-competent guinea-pig spermatozoa that are held in pipettes will fuse with hamster oocytes when placed mechanically against the oocyte surface.

Keywords: spermatozoa; oocyte; cell fusion; fluorescent dye; mammalian fertilization

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J. K. Hodges, D. I. Green, P. G. Cottingham, M. J. Sauer, C. Edwards and S. L. Lightman

Summary. Doses of 100 or 200 μg of a novel GnRH antagonist ([N-acetyl-dβNall-d-pCl-Phe2-d-Phe3-d-Arg6-Phe7-Arg8-d-Ala10]NH2 GnRH) (4 animals/dose) were administered on Days 10/11 of the luteal phase and induced a marked suppression of circulating bioactive LH and progesterone concentrations within 1 day of treatment (P < 0·01). Thereafter, progesterone concentrations remained low or undetectable until after the next ovulation. Similar results were obtained when 200 μg antagonist were given on Days 5/6 of the luteal phase (N = 4). The interval from injection of antagonist (200 pg but not 100 μg to ovulation (based on a rise in progesterone above 10 ng/ml) was significantly longer than that from prostaglandin-induced luteal regression to ovulation in control cycles (N = 4/treatment) (range, 13–15 days after antagonist vs 8–10 days after prostaglandin, P < 0·01). This delay of 4–5 days was equivalent to the duration for which LH concentrations were significantly suppressed by 200 μg antagonist when administered to ovariectomized animals (N = 3). Corpus luteum function during the cycle after GnRH antagonist treatment appeared normal according to the pattern of circulating progesterone. These results show that corpus luteum function and preovulatory follicular development in the marmoset monkey are dependent on pituitary gonadotrophin secretion.

Keywords: GnRH antagonist; monkey; corpus luteum