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  • Author: R. B. L. GWATKIN x
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R. B. L. GWATKIN

Summary.

The zona pellucida of the early mouse embryo was permeable to Mengovirus at the morula as well as at the 2-cell stage of development.

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R. B. L. GWATKIN

Summary.

A survey was made of the effectiveness of acid media and of twelve different enzyme preparations in dissolving the zona pellucida of the mouse egg. It was found necessary to lower the pH of the medium to nearly 4 in order to dissolve the zona pellucida. Purified papain, crude ficin and crude Streptomyces griseus protease (`pronase') were found to be effective enzymes for zona pellucida removal. A crude preparation of β-glucuronidase also dissolved the zona pellucida, but its activity was shown not to be due to its β-glucuronidase content.

A difference in response between fertilized and unfertilized eggs was noted with the crude β-glucuronidase preparation : it dissolved the zona pellucida of unfertilized eggs more rapidly than that of fertilized eggs. Purified elastase did not dissolve the zona pellucida of either group, but caused the zona pellucida of some unfertilized eggs to swell.

The following enzyme preparations had no apparent effect on the mouse zona pellucida: purified trypsin, α-chymotrypsin and carboxypeptidase, crude preparations of trypsin, pancreatic lipase, lecithinase D, β-1,4-glucosidase and lysozyme.

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A. A. HAIDRI and R. B. L. GWATKIN

Oocytes explanted from the ovaries of normally cycling mice mature to polarbody formation and the second metaphase stage in the absence of a fixed N2 source. Optimum maturation occurs with a Po2 of 5% in the gas phase, although a high proportion will do so even under an air atmosphere (Haidri, Miller & Gwatkin, 1971). By contrast, oocytes of the golden hamster, Mesocricetus auratus, require isoleucine, glutamine, phenylalanine and methionine for maturation. These amino acids have been incorporated in medium GH-1, specifically developed for the culture of the oocytes explanted from the follicles of the cycling hamster (Gwatkin & Haidri, 1973). Hamster oocytes, have also been found to be extremely sensitive to O2 and rapidly become necrotic under an air-atmosphere (Gwatkin & Haidri, 1973).

In this communication, we report that golden hamster

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R. B. L. GWATKIN and D. T. WILLIAMS

Summary.

Before fertilization, the protease present within the cortical granules of the hamster egg is relatively stable to heat. Once discharged into the medium, however, it is extremely heat sensitive, undergoing inactivation even at 37°C. Thus, body temperature would be expected to prevent the zona reaction of one egg from rendering neighbouring eggs infertile.

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R. B. L. GWATKIN and O. F. ANDERSEN

Rapid transport of spermatozoa to the site of fertilization in the oviduct has been demonstrated in several species (Howe & Black, 1963; Yanagimachi & Chang, 1963; Harper, 1973). In the mouse, the uterotubal junction was shown to close a few minutes after mating (Zamboni, 1972). These findings suggest that, at least in some species, normal physiological capacitation of the spermatozoa takes place in the oviduct rather than in the uterus. Using hamster gametes in vitro, we earlier demonstrated that the cumulus oophorus and not the oviducal fluid is responsible for the capacitating action of the postovulatory contents of the oviduct (Gwatkin, Andersen & Hutchison, 1972).

When hamster spermatozoa are incubated with cumulus cells in vitro, the spermatozoa bind to a neuraminidase-sensitive site on the cell surface and detach spontaneously 3 to 4 hr later (Gwatkin et al., 1972). In the presence of a dialysable factor from the cumulus matrix, spermatozoa

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R. B. L. GWATKIN and A. A. HAIDRI

Summary.

Hamster oocytes exhibited a sharply defined optimum of 5% O2 for maturation to metaphase II in vitro. Without the addition of O2, all of the oocytes were arrested during chromatin condensation. Under 10% O2, most of the oocytes were arrested at several phases of meiosis up to and including telophase I.

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R. B. L. Gwatkin and D. T. Williams

Summary. Capacitated golden hamster spermatozoa bound to the inner as well as to the outer surface of the zona pellucida, suggesting that the receptor-for-spermatozoa may occur throughout this egg envelope. When solutions of hamster zonae pellucidae were prepared by heating zonae in an aqueous buffer, receptor activity was retained and was stable to boiling. The addition of such solutions to capacitated spermatozoa (5 zonae/μl) prevented them from binding to eggs and fertilizing them. Solubilized mouse zonae were also partly effective, as would be predicted from the limited cross-binding binding between mouse and hamster gametes. Receptor activity was lost following the zona reaction.

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J. C. Conover and R. B. L. Gwatkin

Summary. Mouse oocytes exposed to 1 μg Hoechst 33342 (H-33342)/ml and then fertilized in vitro developed normally into blastocysts and blastocyst outgrowths. After penetration of the zona, the fertilizing spermatozoon showed intense fluorescence upon fusion with the vitelline membrane. Due to fluorochrome leakage from the perivitelline space a faint fluorescence was detected in zona-bound spermatozoa. This fluorescence of zona-bound spermatozoa intensified with increased fluorochrome concentration (10μg/ml), obscuring the fluorescence of the fertilizing spermatozoa. Spermatozoa added to zona-free mouse oocytes (pre-loaded with 1 or 10 μg H-33342/ml) fluoresced within 10 min of insemination, provided the zonae were removed mechanically. Removal by protease digestion induced leakage of fluorochrome, so that all spermatozoa in the vicinity of an oocyte pre-loaded with 10 pg H-33342/ml became labelled. This leakage was not visibly apparent when protease-treated oocytes were exposed to only 1 μg H-33342/ml. The technique could not be applied to zona-free hamster oocytes under our conditions, since the fluorochrome leaked freely from the oocytes whether the zona was removed mechanically or enzymically.

Keywords: gamete fusion; fluorochrome; in-vitro fertilization; mammals

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H. Hyatt and R. B. L. Gwatkin

Summary. The acrosomal matrix of hamster spermatozoa was enriched and characterized. Acrosomal matrices were released from spermatozoa with shaking in a pH 5·2 buffer containing Triton X-100 and protease inhibitors, and enriched on a glass-bead column. Phase-contrast microscopy indicated that 70–80% of the acrosomal matrices were released from the spermatozoa and only minor contamination from sperm heads was detected. Transmission electron microscopy confirmed the low level of contamination in the preparation and revealed a bilaminar structure similar but not identical to that of guinea-pig acrosomal matrix. One- and two-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed the acrosomal matrix to be a complex structure enriched for several polypeptides. Proteinase activity was demonstrated by gelatin–SDS-PAGE. The major activity corresponded to bands of relative molecular masses (M r) of 56 000, 51 000 and 48 000 with two minor bands of M r 30 000 and 28 000. The lectin Pisum sativum agglutinin (PSA) bound to the anterior head of spermatozoa and isolated acrosomal matrix as judged by fluorescence microscopy using FITC–PSA. Western blots of spermatozoa and acrosomal matrices followed by overlay with biotinylated PSA indicated that there are at least two PSA-binding glycoproteins of M r 60 000 and 72 000.

Keywords: acrosome; acrosomal matrix; hamster spermatozoa; acrosin; proacrosin

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R. B. L. GWATKIN, O. F. ANDERSEN and D. T. WILLIAMS

Summary.

Capacitation of mouse spermatozoa in vitro is brought about by epididymal secretions released into the medium at the time of sperm collection. Inhibition by glucaro(1→4)lactone indicates that an essential component of these secretions is β-glucuronidase. In the absence of the secretions, capacitation can be induced by components of the cumulus oophorus.