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 on sperm of cryoprotective agents and protocols, and contraceptive agents.
1. A method of determining the effect of storage media on the ability of sperm to respond to one or more stimuli associated with capacitation and/or acrosomal exocytosis, comprising the steps of:
storing the sperm in a storage media;
removing the stored sperm from the storage media;
determining a G M1 distribution pattern in the stored sperm comprising:
i) exposing the stored sperm to the one or more stimuli;
ii) fixing the stored sperm in a fixative comprising paraformaldehyde, glutaraldehyde or combinations thereof;
iii) staining the fixed stored sperm to determine the G M1 distribution pattern in the fixed stored sperm;
obtaining a G M1 distribution pattern of a fixed control sperm sample which has not been stored in the storage media, 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 fixed stored sperm; and
comparing the G M1 distribution patterns of the fixed stored sperm and the fixed control sperm;
wherein a difference between the G M1 distribution patters of the fixed stored sperm and fixed control sperm is indicative of an effect of the storage media 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 stored 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 stored 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 pellucids proteins, lysophosphatidylcholine and combinations thereof.
7. The method of claim 1 , wherein the storage medium is a cryopreservation medium.
8. The method of claim 1 , wherein the storage medium is a semen extender.
9. The method of claim 1 , wherein the fixative comprises 0.00400 paraformaldehyde.
10. The method of claim 1 , wherein the fixative comprises 4% paraformaldehyde, 0.1% glutaraldehyde and 1 mM CaCl 2 .
11. The method of claim 1 , wherein the fixative comprises 1.25% paraformaldehyde and 2.5% glutaraldehyde.
12. The method of claim 11 , wherein the fixative further comprises 100 nM sodium cacodylate and 0.5 mM CaCl 2 .
13. The method of claim 1 , wherein the sperm sample is from an individual selected from the group consisting of cat, cattle, dog, goat, guinea pig, horse, human, mouse, pig, rabbit, rat and sheep.