Compositions and methods for human sperm activation and quantitative assessment of human sperm genome replication
First Claim
1. A composition comprising a purified cytoplasmic extract from eggs of Xenopus laevis which supports activation of human sperm and complete replication of a human sperm genome, said composition being prepared by a method comprising the steps of:
- a) providing a sexually mature female Xenopus laevis toad;
b) priming said toad for egg production by administering to said toad a gonadotropin hormone in an amount effective to stimulate said egg production;
said priming comprising;
i) a first priming step wherein a first aliquot of pregnant mare gonadotropin is administered two days prior to egg collection; and
ii) a second priming step wherein at least one additional aliquot of human chorionic gonadotropin is administered between about 12 and about 24 hours prior to egg collection;
c) collecting the eggs;
d) de-jellying the collected eggs;
e) removing from the de-jellied eggs substantially all eggs which appear damaged or of non-uniform character as compared with normal, intact eggs from Xenopus laevis;
f) activating the eggs to shift from mitotic phase to S phase in their cell cycle;
g) rinsing the activated eggs with a compatible rinsing solution;
h) packing the eggs by removing substantially all excess rinsing solution from the rinsed eggs;
i) preparing an initial cytoplasmic fraction from the packed eggs byi) subjecting the eggs to centrifugation under conditions resulting in crushing of the eggs and separation of the initial cytoplasmic fraction from other egg components; and
ii) collecting only the initial cytoplasmic fraction; and
j) preparing the purified cytoplasmic extract from the initial cytoplasmic fraction byi) subjecting the initial cytoplasmic fraction to centrifugation under conditions resulting in separation of the purified cytoplasmic extract from vesicular components remaining in the initial cytoplasmic fraction;
ii) collecting only said purified cytoplasmic extract; and
k) optionally freezing and thawing said cytoplasmic extract, said extract retaining biological activity following said freezing and thawing.
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Abstract
A composition is provided, which comprises a purified cytoplasmic extract from eggs of Xenopus laevis which is capable of supporting activation of human sperm and complete replication of a human sperm genome. Methods of using the extract to achieve complete replication of the sperm genome are disclosed. A flow cytometric method for quantitatively monitoring replication of human sperm genomes is also disclosed. This method can be used for clinical assessment of sperm quality in a test sample of human sperm from an infertile male donor.
51 Citations
21 Claims
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1. A composition comprising a purified cytoplasmic extract from eggs of Xenopus laevis which supports activation of human sperm and complete replication of a human sperm genome, said composition being prepared by a method comprising the steps of:
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a) providing a sexually mature female Xenopus laevis toad; b) priming said toad for egg production by administering to said toad a gonadotropin hormone in an amount effective to stimulate said egg production;
said priming comprising;i) a first priming step wherein a first aliquot of pregnant mare gonadotropin is administered two days prior to egg collection; and ii) a second priming step wherein at least one additional aliquot of human chorionic gonadotropin is administered between about 12 and about 24 hours prior to egg collection; c) collecting the eggs; d) de-jellying the collected eggs; e) removing from the de-jellied eggs substantially all eggs which appear damaged or of non-uniform character as compared with normal, intact eggs from Xenopus laevis; f) activating the eggs to shift from mitotic phase to S phase in their cell cycle; g) rinsing the activated eggs with a compatible rinsing solution; h) packing the eggs by removing substantially all excess rinsing solution from the rinsed eggs; i) preparing an initial cytoplasmic fraction from the packed eggs by i) subjecting the eggs to centrifugation under conditions resulting in crushing of the eggs and separation of the initial cytoplasmic fraction from other egg components; and ii) collecting only the initial cytoplasmic fraction; and j) preparing the purified cytoplasmic extract from the initial cytoplasmic fraction by i) subjecting the initial cytoplasmic fraction to centrifugation under conditions resulting in separation of the purified cytoplasmic extract from vesicular components remaining in the initial cytoplasmic fraction; ii) collecting only said purified cytoplasmic extract; and k) optionally freezing and thawing said cytoplasmic extract, said extract retaining biological activity following said freezing and thawing. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 18, 19, 20, 21)
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12. A method of preparing a purified cytoplasmic extract from eggs of Xenopus laevis which supports activation of human sperm and complete replication of a human sperm genome, said method comprising the steps of:
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a) providing a sexually mature female Xenopus laevis toad; b) priming said toad for egg production by administering to said toad a gonadotropin hormone in an amount effective to stimulate said egg production;
said priming comprising;i) a first priming step wherein a first aliquot of pregnant mare gonadotropin is administered two days prior to egg collection; and ii) a second priming step wherein at least one additional aliquot of human chorionic gonadotropin is administered between about 12 and about 24 hours prior to egg collection; c) collecting the eggs; d) de-jellying the collected eggs; e) removing from the de-jellied eggs substantially all eggs which appear damaged or of non-uniform character as compared with normal, intact eggs from Xenopus laevis; f) activating the eggs to shift from mitotic phase to S phase in their cell cycle; g) rinsing the activated eggs with a compatible rinsing solution; h) packing the eggs by removing substantially all excess rinsing solution from the rinsed eggs; i) preparing an initial cytoplasmic fraction from the packed eggs by i) subjecting the eggs to centrifugation under conditions resulting in crushing of the eggs and separation of the initial cytoplasmic fraction from other egg components; and ii) collecting only the initial cytoplasmic fraction; and j) preparing the purified cytoplasmic extract from the initial cytoplasmic fraction by i) subjecting the initial cytoplasmic fraction to centrifugation under conditions resulting in separation of the purified cytoplasmic extract from vesicular components remaining in the initial cytoplasmic fraction; ii) collecting only said purified cytoplasmic extract; and k) optionally freezing and thawing said extract said extract maintaining biological activity following said freezing and thawing. - View Dependent Claims (13, 14, 15, 16, 17)
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Specification