QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 24 December 2003
doi: 10.1242/dev.00885

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Development Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rodeheffer, C. Articles by Shur, B. D. Search for Related Content PubMed PubMed Citation Articles by Rodeheffer, C. Articles by Shur, B. D.

Sperm from ß1,4-galactosyltransferase I-null mice exhibit precocious capacitation

Department of Cell Biology, Graduate Program in Biochemistry, Cell and Developmental Biology, Emory University School of Medicine, 615 Michael Street, Atlanta, GA 30322, USA

View larger version (12K): [in a new window]   Fig. 1. Experimental protocols used to assay for capacitation in wild-type and GalT I-null sperm. Sperm were isolated, collected by centrifugation, and capacitated for various periods of time (starting at time 0) in either complete medium or medium deficient for specific components as described in the text. At the indicated time, aliquots of sperm were incubated either with eggs for 30 minutes (to monitor for zona binding activity), or with A23187 for 10 minutes (to monitor induction of the acrosome reaction).

View larger version (17K): [in a new window]   Fig. 2. GalT I-null sperm do not require capacitation in vitro to achieve maximal binding to the zona pellucida. Wild-type (gt +/+) and GalT I-null (gt –/–) sperm were capacitated for varying amounts of time before incubation with ovulated eggs. The binding of GalT I-null sperm to the zona pellucida is independent of capacitation in vitro, whereas wild-type sperm show a capacitation-dependent increase in their ability to bind the egg. Each bar represents the mean±s.e.m.; n=3 experiments. In a single experiment, the value for each data point represents the average of three determinations. Thus, each bar reflects data from nine assays, each containing 30-40 eggs. The average number of sperm bound to eggs at 0 minutes was: wild type, 8±0; GalT I-null, 25±2; at 15 minutes: wild-type, 14±2; GalT I-null, 25±4; at 30 minutes: wild-type, 19±5; GalT I-null 25±2; and at 60 minutes: wild type, 19±2; GalT I-null, 21 ± 2. In each experiment, this value was normalized to fully capacitated sperm (60 minutes of capacitation is equivalent to 100% bound), so that results from replicate assays could be compared. *P<0.01.

View larger version (18K): [in a new window]   Fig. 3. GalT I-null sperm are more sensitive to Ca2+ ionophore than wild-type sperm during in vitro capacitation. Wild-type (gt +/+) and GalT I-null (gt –/–) sperm were capacitated for varying periods of time before incubation with 10 µM A23187 or an equal volume of DMSO. GalT I-null sperm show higher basal levels of sensitivity to ionophore-induced acrosome reactions, as well as greater sensitivity during capacitation in vitro. Each point represents the mean±s.e.m.; n=3 experiments. In a single experiment, the value of each data point represents three determinations. Thus, each data point reflects data collected from nine assays, each containing 200 sperm counted. *P<0.01.

View larger version (14K): [in a new window]   Fig. 4. Removing epididymal decapacitation factors increases the sensitivity of wild-type sperm, but not GalT I-null sperm, to Ca2+ ionophore. Wild-type (gt +/+, A) and GalT I-null (gt –/–, B) sperm were isolated from the cauda epididymis by trituration and resuspended in fresh media, centrifuged and resuspended again (washed). Control sperm were triturated and centrifuged in parallel but resuspended in the original supernatant (unwashed). The sperm were then capacitated for varying periods of time before incubation with 10 µM A23187 or an equal volume of DMSO (data not shown). Each point represents the mean±s.e.m.; n=3 experiments. In a single experiment, the value of each data point represents three determinations for a total of nine individual assays. A minimum of 200 sperm was counted for each determination. *P<0.01.

View larger version (16K): [in a new window]   Fig. 5. GalT I-null sperm do not require albumin, Ca2+, or HCO3– during capacitation to undergo an ionophore-induced acrosome reaction. Wild-type (gt +/+) and GalT I-null (gt –/–) sperm were capacitated for varying periods of time in complete media (unbroken line) or in media lacking (broken line)either albumin (A), Ca2+ (B) or HCO3– (C). Sperm were subsequently incubated with 10 µM A23187 and the appropriate co-factor (to reconstitute complete media) and the percentage of acrosome-reacted sperm was determined. Each point represents the mean±s.e.m.; n=3 experiments. In a single experiment, the value of each data point represents three determinations for a total of nine assays. A minimum of 200 sperm was counted for each of the assays.

View larger version (13K): [in a new window]   Fig. 6. GalT I-null sperm do not require Ca2+ or HCO3– during capacitation to achieve maximal binding to the zona pellucida. Wild-type (gt +/+) and GalT I-null sperm (gt –/–) were capacitated in either albumin-free (A), Ca2+-free (B), HCO3– -free (C) or complete media (control) for varying amounts of time before incubation with ovulated eggs in complete media. Each bar represents the mean±s.e.m.; n=3 experiments. In a single experiment, the value for each data point represents the average of three determinations (nine assays in total). The average number of sperm bound to eggs at each time point was normalized to fully capacitated sperm (60 minutes of capacitation in complete media is equivalent to 100% bound).

View larger version (18K): [in a new window]   Fig. 7. GalT I-null sperm exhibit constitutively increased levels of cAMP during capacitation in HCO3– -containing or HCO3– -free media. Wild-type (gt +/+) and GalT I-null (gt –/–) were capacitated for varying periods of time in HCO3– -containing or HCO3– -free media. The cAMP in 1x106 sperm was extracted with ice-cold ethanol and detected by enzyme immunoassay. Each bar represents the mean±s.e.m.; n=4 experiments. For a single experiment, the value for each data point represents the average of two determinations (for a total of eight assays).

View larger version (36K): [in a new window]   Fig. 8. Wild-type and GalT I-null sperm show similar profiles of capacitation-dependent protein tyrosine phosphorylation. Wild-type (gt +/+) and GalT I-null (gt –/–) sperm lysates were fractionated by SDS-PAGE, transferred to nitrocellulose and probed with anti-phosphotyrosine and anti--tubulin antibodies. The arrow indicates a protein of 200 kDa that is present only in GalT I-null sperm lysates. This blot is representative of at least six experiments. (C) Protein kinase A activity in wild-type (gt +/+) and GalT I-null (gt –/–) sperm capacitated for varying periods of time in HCO3– -containing or HCO3– -free media. Each point represents the mean±standard error; n=3 experiments. In a single experiment, the value of each data point represents three determinations. Thus, each data point reflects data collected from nine assays.

View larger version (15K): [in a new window]   Fig. 9. Wild-type and GalT I-null sperm exhibit similar motility during in vitro capacitation. Wild-type (gt +/+) and GalT I-null sperm (gt –/–) were incubated in capacitating media for either 10 (A) or 60 (B) minutes before CASA. Each bar represents the mean±s.e.m.; n=3 experiments for each time point. Each experiment was carried out in duplicate.