Chemistry: molecular biology and microbiology – Maintaining blood or sperm in a physiologically active state...
Patent
1991-11-27
1994-04-19
Wityshyn, Michael G.
Chemistry: molecular biology and microbiology
Maintaining blood or sperm in a physiologically active state...
524520, 524531, 514 8, 514 21, A01N 102, A61K 3710
Patent
active
053044649
DESCRIPTION:
BRIEF SUMMARY
FIELD OF INVENTION
The present invention relates to a macromolecule of proteinaceous nature which activates sperm motility, a process for the preparation thereof by purification of the macromolecule from extracellular fluids, pharmaceutical preparations comprising the macromolecule and antibodies directed against determinants specific to said macromolecule.
BACKGROUND ART
It is previously known that only spermatozoa with a good ability to move and with a relatively straightlined progressive motility have the possibility to achieve fertilization and that only the spermatozoa with the best morphology enter the cervical mucus (Fredricsson B. and Bjork G. (1977): Morphology of postcoital spermatozoa in the cervical secretion and its clinical significance. Fertil. Steril. 28:841-845). It is also previously known that serum support sperm motility /Austin R. (1985) "Sperm maturation in the male and female genital tracts." In: Biology of Fertiliztion, vol. 2 (eds C Metz and A Monroy) pp. 121-147, Academic Press, New York/ and is therefore used in connection with sperm separation for fertilization in vitro.
However, there are problems inherent in the use of a fluid comprising thousands of components and a specific active component would give a safer and more predictible effect than such a fluid. An active factor would also give the effect without dangerous concomittant actions e.g. immunologic factors or infectious diseases.
DISCLOSURE OF INVENTION
During work with fertilization, in vitro standardized model systems were developed for e.g. sperm motility test, to obtain more homogeneous populations of motile and viable spermatozoa and to analyze the adenosinetriphosphate (ATP) content of sperm and its specific progressive motility (SPM): .ANG.kerlof E., Fredricsson B., Gustafson O., Lundin A., Lunell N-O., Nylund L., Rosenborg L. and Pousette .ANG. (1987) Int. J. Androl. 10, 663-669 and Pousette .ANG.., .ANG.kerlof E., Lundin A., Rosenborg L. and Fredricsson B. (1986) Int. J. Androl. 9, 331-340.
Said standardized model systems became the tools for the further investigation of serum to find the factor or factors giving the motility activity to the sperm necessary for the fertilization to take place. Through an extensive research work the present inventors have now found that sperm motility is activated by a macromolecule having a molecular weight of about 200,000 Daltons.
Said macromolecule is of proteinaceous nature, essentially pure and activates sperm motility. The molecular weight of said protein is according to a preferred embodiment of the invention about 180,000 Daltons.
A further preferred embodiment of the invention discloses a proteinaceous macromolecule having an isoelectric point of about 5.1 and comprising albumin. The molecular weight of said macromolecule is preferably about 250,000 Daltons.
The macromolecule according to the invention is substantially homogeneous and obtainable from extracellular fluids, particularly from serum of animals or human beings.
The proteinaceous macromolecule is purified from extracellular fluids from animals or human beings, preferably from serum and according to a preferred embodiment from serum of human beings.
According to a further preferred embodiment a proteinaceous macromolecule having an isoelectric point of about 5.1 and comprising apolipoprotein Al, immunoglobulin and albumin is purified through a three step process comprising Ion-exchange Chromatography, Chromatofocusing and Fast Protein Liquid Chromatography (FPLC a system from Pharmacia LKB Biotechnology).
According to another preferred embodiment a proteinaceous macromolecule having a molecular weight of about 180,000 Daltons and comprising apolipoprotein Al and immunoglobulin is prepared by a four step purification process comprising Ion-exchange Chromatography, Chromatofocusing, Fast Protein Liquid Chromatography and Affinity Chromatography.
According to a yet further preferred embodiment the sperm motility activity is checked after every single purification step.
The invention is also relating to ph
REFERENCES:
patent: 4511558 (1985-04-01), Shur
patent: 4804537 (1989-02-01), Bergman
Scopes Robert R, Protein Purification, Springer-Verlag N.Y. 1982 pp. 101-105, 111-113.
Chemical Abstracts, vol. 111, No. 7, Aug. 1989, (Columbus, Ohio, US), Eva Aakerloef et al.: "Serum factors stimulate the motility of human spermatozoa", see p. 497, abstract 54821x, & Int. J. Androl. 1989, 12 (2), 124-130.
Akerlof Eva
Pousette Ake
Akerlof Eva
Applied Research Systems ARS Holding N.V.
Gitomer Ralph
Pousette Ake
Wityshyn Michael G.
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