Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1998-03-10
2003-09-02
Russel, Jeffrey E. (Department: 1654)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S021800, C530S395000
Reexamination Certificate
active
06613737
ABSTRACT:
FIELD OF INVENTION
The present invention relates to a new motility-promoting protein macromolecule and a process for the isolation of the said motility promoting protein from buffalo serum/plasma: a slaughter house waste.
BACKGROUND OF THE INVENTION
Forward motility-promoting protein isolated from buffalo blood serum has the potential for the treatment of human infertility (due to low sperm motility) a great social curse in all human races. The product isolated by the present invention has also the potential for improving farm animal breeding with special reference to buffalo a milch animal of great economic importance in many countries including India.
Human infertility is a great social stigma in all human races as it brings personal miseries to the infertile people. It is generally believed that approx. 15% of the couples are infertile. A rough estimation shows that approx. 20 million Indian couples are infertile. It is thus evident that human infertility is a social problem of immense dimension all over the world. One of the main reasons of human male infertility is due to low order of sperm motility with normal cell count or normal sperm motility with lower sperm population in the ejaculated semen. Although several sophisticated Assisted Reproductive Technologies (e.g. IVF in vitro fertilisation, ICSI intracytoplasmic sperm injection) are available to solve the problems of oligospermic and asthenozoospermic patients ( with low sperm motility), these technologies are highly expensive and the success rate is extremely low. To solve the problem of human infertility due to low order of sperm motility, limited studies have been carried out for the isolation of motility promoting proteins from reproductive fluids and blood serum which are known to have sperm forward motility promoting potential(Morita Z. and Chang M. C.
J Reprod Fertil
24, 247-254, 1971; Hoskins D. D., Brandt H and Acott T. S.
Fed Proc
37, 2534-2542, 1978; Morton B. E., Fraser C. F and Albagli L.
Fertil Steril
32, 99-106, 1979; Brown D. V. and Senger P. L.
Biol Reprod
23, 271-275, 1980; Mandal M., Banerjee S. and Majumdar G. C.
Biol Reprod
41, 983-989, 1989. Acott and Hoskins (Acott T. S. and Hoskins D. D.
J Biol Chem
253, 6744-6750, 1978) have partially purified a 37 KDa glycoprotein from bovine seminal plasma/epididymal plasma which is essential for in vitro motility initiation in the immature bovine caput epididymal sperm. Akerlof et al (Akerlof E. Fredricsson B., Gustafson O., Lunell N. O. Nylund L., Rosenborg L., Slotte H and Pousette A.,
Int J Androl
12, 124-130, 1989) have demonstrated that human blood serum contains a protein (about 200 KDa) that activates forward motility of human ejaculated sperm. They have also made a patent on this human serum motility promoting protein (Akerlof, E. & Pousette, A., Patent No.WO 90 12 032, Oct. 18, 1990). The motility activating protein (approx. mol. Wt 200 KDa) has an isoelectric point of 5.1 and it comprises albumin, apolipoprotein designated as Al and immunoglobulin. The motility activating protein is usable for sperm motility assay and for the treatment of infertility. A pure protein complex of apolipoprotein and immunoglobulin of mol. wt. 180-250 KDa, has been isolated from human serum (U.S. Pat. No. 5,304,464, Apr. 19, 1994; U.S. Pat. No. 5,453,354, Sep. 26, 1995). The macromolecule also comprises albumin in equimolar amount with apolipoprotein Al and immunoglobulin. It has an isoelectric point of 5.1. The product stimulates sperm motility. Antibodies can be raised against this protein complex human infertility caused by sperm immotility can be treated by administering the product. It can be applied for both diagnostic and therapeutic uses. This product is rather expensive as it is derived from human blood serum.
OBJECT OF THE INVENTION
The main object of the present invention is to provide a new motility-promoting protein macromolecule having a molecular mass of 66 kda and a process for the isolation of said promoting protein from buffalo serum/plasma, a slaughter house waste which has potentiality for use in the treatment of human infertility (due to low sperm motility); a social problem of immense dimension in all countries. A major advantage of our isolated product (forward motility stimulating factor) is that as it is a product of slaughter house waste, it is economically more viable as a commercial product than the one from human serum developed earlier (patent Nos. WO 9012032, Oct. 18, 1990; U.S. Pat. No. 5,304,464, Apr. 19, 1994, U.S. Pat. No. 5,453,354, Sep., 26, 1995). No data are available on the motility promoting efficacy of the human serum product on spermatozoa of other species. The product of this invention can activate motility of sperm derived from various species including buffalo, bovine and goat. Another advantage of our product is that it has also the potential for improving the breeding of farm animals with special reference to buffalo; a milch animal of great economic importance in many countries.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a new motility-promoting protein macromolecule and a process for the isolation of the said motility promoting protein from buffalo serum/plasma: a slaughter house waste, which comprises fractionating buffalo serum/plasma, purifying the motility promoting protein from the above fractionated serum/plasma by conventional chromatography and electrophoresis methods.
Further, the present investigation provides a new motility-promoting protein macromolecule and a process for the isolation of the said motility promoting protein from buffalo serum/plasma: a slaughter house waste. The invention comprises (a) purification and characterisation of a novel forward motility-stimulating protein from buffalo blood serum/plasma and (b) the methodologies for the purification of the motility-promoting protein from the buffalo serum/plasma. The motility-promoting factor is a heat stable protein. This invention reports for the first time the purification to apparent homogeneity of a motility-promoting protein from a biological fluid. It activates forward motility of ejaculated sperm of all the species tested (buffalo, bull goat rat hamster and human) indicating that there is no species specificity for its motility activating potential. The molecular mass of the motility promoter as estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis, high performance liquid chromatography and Sephadex G100 gel filtration is 66 KDa. The factor showed high protein specificity and affinity for activating sperm forward motility. The motility promoter at 0.5 &mgr;M level showed nearly maximal activity when 60-70% of spermatozoa were forward motility. It acts on the sperm cells with great rapidity to cause instant activation of sperm motility. It is a glycoprotein that binds with high affinity to concanavalin A. It is an acidic protein with isoelectric point of 3.7 (approx.) and its activity is dependent on Mg
++
. Motility promoting protein has markedly higher efficacy for activating sperm motility than theophylline or bicarbonate or their combination. The sperm motility induced by the factor is greatly stabilized by bicarbonate. Amino acid analysis shows that it is rich in aspartic acid, glutamic acid and leucine. Both the sugar and protein parts of the molecule are essential for its biological activity. Specific receptors of motility promoting protein have been demonstrated on the sperm surface that may mediate the action of the factor on sperm. It is strongly immunogenic. Studies with antisera against the buffalo motility promoting protein (raised in rabbit) show that the antisera cross react with sera derived from goat, bull, cow and human. The data implicate that the motility promoter or motility promoter like proteins are present in the sera of other species. Using the antibody against the motility stimulating factor, the motility promoter has been shown to be present in testis and epididymis although liver is the richest source of it. The product isolated by the present
Banerjee Saswati
Majumder Gopal Chandra
Mandal Mahitosh
Caesar Rivise Bernstein Cohen & Pokotilow Ltd.
Council of Scientific and Industrial Research
Russel Jeffrey E.
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