Method of determining sperm capacitation
View Patent ↗This invention describes unique patterns of distribution of ganglioside G M1 in non-capacitated sperm and demonstrates that the pattern of distribution of G M1 undergoes changes that can be correlated with the process of capacitation and/or with acrosomal exocytosis. Accordingly, the present invention discloses a method for determining the ability of sperm to respond to capacitation and/or acrosomal exocytosis stimuli. The method comprises determination of distribution pattern for G M1 . The method can be used for both diagnostic and predictive purposes when assessing male reproductive fitness, and can also be used to assess the effects of any agent or environment on sperm including cryoprotective agents and protocols, and contraceptive agents.
1. A method of determining the effect of a test agent on the ability of sperm to respond to one or more stimuli associated with capacitation and/or acrosomal exocytosis comprising the steps of:
a) obtaining sperm which have been exposed to the test agent;
b) determining a G M1 distribution pattern in the sperm from step a) comprising;
i) exposing the sperm to the one or more stimuli;
ii) fixing the sperm in a fixative comprising paraformaldehyde, glutaraldehyde or combinations thereof;
iii) staining the fixed sperm to determine the G M1 distribution pattern in the fixed test sperm which had been exposed to the test agent
c) obtaining a G M1 distribution pattern of a fixed control sperm sample which has not been exposed to the test agent, but which has been exposed to the same one or more stimuli, fixed in the same fixative and stained in the same manner as the sperm in step b);
d) comparing the G M1 distribution pattern of the fixed test sperm and the fixed control sperm;
wherein a difference between the G M1 distribution patterns of the fixed test sperm and fixed control sperm is indicative of an effect of the test agent on the ability of sperm to respond to one or more stimuli associated with capacitation and/or acrosomal exocytosis.
2. The method of claim 1 , wherein the difference between the G M1 distribution patterns is an abnormal localization of G M1 within the fixed test sperm compared to the fixed control sperm.
3. The method of claim 1 , wherein the difference between the G M1 distribution patterns is a difference in the relative frequencies of different G M1 distribution patterns in the fixed test sperm compared to the frequencies of the same patterns in the fixed control sperm.
4. The method of claim 1 , wherein the one or more stimuli associated with capacitation is selected from the group consisting of calcium, bicarbonate, a mediator of sterol efflux and combinations thereof.
5. The method of claim 4 , wherein the mediator of sterol efflux is selected from the group consisting of 2-hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, serum albumin, high density lipoprotein, phospholipid vesicles, liposomes and combinations thereof.
6. The method of claim 1 , wherein the one or more stimuli associated with acrosomal exocytosis is selected from the group consisting of progesterone, calcium ionophore, zona pellucida proteins, lysophosphatidylcholine and combinations thereof.
7. The method of claim 1 , wherein the fixative comprises i) 0.004% paraformaldehyde; ii) 4% paraformaldehyde, 0.1% glutaraldehyde and 1 mM CaCl 2 ; or iii) 1.25% paraformaldehyde and 2.5% glutaraldehyde.
8. The method of claim 7 , wherein the fixative further comprises 100 nM sodium cacodylate and 0.5 mM CaCl 2 .
9. The method of claim 1 , wherein the sperm is from an individual selected from the group consisting of cat, cattle, dog, goat, guinea pig, horse, human, mouse, pig, rabbit, rat and sheep.
10. A method of determining the effect of a test agent on the ability of sperm to respond to one or more stimuli associated with capacitation and/or acrosomal exocytosis comprising the steps of:
a) obtaining sperm from an individual who has been exposed to the test agent;
b) determining a G M1 distribution pattern in the sperm from step a) comprising;
i) exposing the sperm to the one or more stimuli;
ii) fixing the sperm in a fixative comprising paraformaldehyde, glutaraldehyde or combinations thereof;
iii) staining the fixed sperm to determine the G M1 distribution pattern in the fixed test sperm;
c) obtaining a G M1 distribution pattern of a fixed control sperm sample which is obtained from an individual who has not been exposed to the test agent, but which has been exposed to the same one or more stimuli, fixed in the same fixative and stained in the same manner as the sperm in step b);
d) comparing the G M1 distribution pattern of the fixed test sperm and the fixed control sperm;
wherein a difference between the G M1 distribution patterns of the fixed test sperm and fixed control sperm is indicative of an effect of the test agent on the ability of sperm to respond to one or more stimuli associated with capacitation and/or acrosomal exocytosis.
11. The method of claim 10 , wherein the difference between the G M1 distribution patterns is an abnormal localization of G M1 within the fixed test sperm compared to the fixed control sperm.
12. The method of claim 10 , wherein the difference between the G M1 distribution patterns is a difference in the relative frequencies of different G M1 distribution patterns in the fixed test sperm compared to the frequencies of the same patterns in the fixed control sperm.
13. The method of claim 10 , wherein the test agent is one of the components of a pharmaceutical or neutraceutical.
14. The method of claim 10 , wherein the test agent is one of the components of a topical composition.
15. The method of claim 10 , wherein the one or more stimuli associated with capacitation is selected from the group consisting of calcium, bicarbonate, a mediator of sterol efflux and combinations thereof.
16. The method of claim 15 , wherein the mediator of sterol efflux is selected from the group consisting of 2-hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, serum albumin, high density lipoprotein, phospholipid vesicles, liposomes and combinations thereof.
17. The method of claim 10 , wherein the one or more stimuli associated with acrosomal exocytosis is selected from the group consisting of progesterone, calcium ionophore, zona pellucida proteins, lysophosphatidylcholine and combinations thereof.
18. The method of claim 10 , wherein the fixative comprises i) 0.004% paraformaldehyde; ii) 4% paraformaldehyde, 0.1% glutaraldehyde and 1 mM CaCl 2 ; or iii) 1.25% paraformaldehyde and 2.5% glutaraldehyde.
19. The method of claim 18 , wherein the fixative further comprises 100 nM sodium cacodylate and 0.5 mM CaCl 2 .
20. The method of claim 10 , wherein the sperm is from an individual selected from the group consisting of cat, cattle, dog, goat, guinea pig, horse, human, mouse, pig, rabbit, rat and sheep.