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A novel method of sperm motility analysis and characterisation of a sperm motility promoting protein from goat blood serum

IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata

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Title A novel method of sperm motility analysis and characterisation of a sperm motility promoting protein from goat blood serum
Creator Saha, Sudipta
Subject Cell Biology & Physiology
Description Sperm motility (specially velocity) is an important characteristic for evaluating the quality of semen, which is essential for fertilization in vivo. Different available techniques including CASA measure the "horizontal" velocity only. There is not a single instrument available for measuring the "vertical" velocity of spermatozoa whereas selecting sperm by swim-up technique is a routine practice in IVF. Inspired by this idea, a computerized automated vertical motility analyzer has been developed for the first time using goat sperm as the model system. Highly motile spermatozoa are known to move upward against the gravity when layered at the bottom of a cuvette placed in a spectrophotometer. To measure the vertical velocity of motile cells, it is necessary to measure the absorbance of vertically motile spermatozoa at different heights of the cuvette with respect to time. For this purpose the cuvette had to be moved vertically up and down within the spectrophotometric light path. There are different types of spectrophotometers available in the market, in which cuvettes are vertically static, but movable in lateral or circular directions. Thus, to make the system adaptable to any spectrophotometer the vertical movement of the cuvette was arranged in two types of spectrophotometers, one with static cuvette holders which is laterally movable manually and another with circular moving or rotating cuvette holders. The development has been accomplished by designing an electromechanical system comprising a modified cuvette holder and a stepper motor tailor made to fit inside the small area of the standard spectrophotometer. Introduction of this electromechanical system permitted us to analyze vertically moving sperm cells at different heights with respect to time due to the controlled upward / downward movement of the cuvette holder. Another important component of this innovation was three custom designed softwares developed for cuvette movement, data acquisition at different heights of the cuvette and data analysis purposes. Visual Basic, Studio 6.0, Enterprise Edition (VB), was used to develop the analytical software and the user interface. Report generation software was developed with Seagate Crystal 8.0, Developer Edition, which generated the necessary reports on sperm motility as required. Online data was acquired and stored into MS-Access database system in the computer. Once the data are acquired, the analytical software performs all the mathematical calculations required for the determination of sperm vertical velocity. As the positions of different batches of sperm cells (in terms of absorbance) at different time intervals were obtained, then a group of sperm cells can be traced for its upward movement throughout the journey. Thus, the time required by a particular batch to complete its journey from the base of the cuvette to different known heights and in between different heights can be easily determined from the Absorbance Vs Time plot. Then vertical velocity of different groups of sperm cells could easily be calculated just by using the simple formula: Vertical Velocity = Vertical distance traversed by a particular group of sperm / Time elapsed. Average of velocities from base of the cuvette to different heights and in between different heights were calculated. Average sperm vertical velocity is obtained from these two vertical velocities. It is evident from the results that weak sperm cells possessing significant horizontal movement (microscopic assay) fail to register any appreciable vertical movement (spectrophotometric assay). This is because, the cells cannot take up vertical motility against the gravity due to weak “health”. Average vertical velocity (VAV) of the same sample has been found to be comparatively lesser than its corresponding average path (horizontal) velocity (VAP) measured by CASA. This also supports the importance of vertical velocity as an index to measure the “health” of a motile cell. Early investigators reported the occurrence of multiple protein factors in biological fluids that may regulate sperm motility essential for fertility potential. But most of these factors were not adequately purified and characterized. A forward motility stimulating protein (FMSF) was purified to apparent homogeneity from buffalo blood serum and its physical and biochemical characteristics were established using goat cauda sperm. However, the molecular identity of the buffalo serum FMSF could not be elucidated due to unavailability of its N-terminal amino acid sequencing data. This study reports for the first time purification of a forward motility stimulating factor (FMSF) to apparent homogeneity from goat blood serum and some of its physical, biochemical and physiological characteristics have been established using the homologous sperm system. N-terminal sequencing of goat and buffalo FMSF(s) have also been done to establish their molecular identities. Goat FMSF has been purified from goat blood serum using several purification steps such as boiling of serum, ammonium sulphate precipitation, CM-Cellulose cation exchanger column chromatography, Sephacryl S-200 gel filtration column chromatography and non-denaturing polyacrylamide gel electrophoresis (PAGE). It is a heat-stable 66 kDa protein. Its purity and molecular weight was confirmed by SDS-PAGE, Sephacryl S-200 gel filtration and high performance liquid chromatography (HPLC). FMSF is a Mg2+ - dependent monomeric protein. Mg2+ at 0.8 mM level caused maximal activation of FMSF activity. Goat FMSF showed high degree of immunospecificity as evident from the Western Blotting test. It is present in testis in comparatively good amount although goat blood is the richest source from where it was purified. The observation that FMSF is present in testis and epididymal plasma, as determined by ELISA, suggests that the motility factor has a regulatory role on sperm physiology. FMSF antibody at lower levels causes significant inhibition of sperm motility. Spermatozoa undergo head to head agglutination when treated with higher-level anti-sera against FMSF, demonstrating thereby the localization of the motility-promoter on the outer surface of sperm head. The data are compatible with the view that FMSF binds with its specific receptors localized on the sperm head surface, although it is not clear as to how the FMSF-receptor interaction triggers the flagellar motility. At saturating concentration (0.9 μM), FMSF is a much powerful activator of sperm motility than the combined action of both bicarbonate and theophylline, suggests that FMSF acts by a mechanism, which is largely different from those of theophylline/ bicarbonate. This view is supported by the kinetic data showing that FMSF acts much more rapidly (30 sec – 1 min) than bicarbonate and theophylline. Theophylline together with bicarbonate showed significant activity for stabilizing FMSF – dependent sperm forward progression. The derived N-terminal amino acid sequence of goat FMSF was DTHKSEIAHRFNDLGEE (upto 17th amino acid). N-terminal sequence of the FMSF purified from buffalo serum was also done for comparison and confirming the uniqueness of the proteins. The derived N-terminal amino acid sequence of buffalo FMSF was derived as DTHKSEIAHRFKDL (upto 14th amino acid). Both the sequences are almost similar with only a single variation at the 12th amino acid residue. The sequences showed high sequence homology with serum albumin precursors of several species including BSA. But, FMSF is clearly different from BSA as the derived FMSF sequence showed maximum homology with amino acid residues located from the 25th position of the N-terminal end of BSA but it did not at all match with the Nterminal of BSA. Molecular weight of BSA and FMSF are similar but they differ markedly in several physical and biochemical properties. The Nterminal sequence of both the FMSF(s) match partially with the N-terminal of different proteins such as a 66 kDa Seroreactive Protein from Mycobacterium tuberculosis (92%), a 49 kDa protein and a 70 kDa seizure activity-linked albumin-like glycoprotein both from Rattus sp. (85%), Alpha 1-proteinase inhibitor from Ostrich (83%), Antagonist protein from rabbit (76%). The sequence of the first 10 amino acid residues of FMSF exactly matches with the N-terminal of an unknown protein NF041 from 2D-PAGE from Naegleria fowleri. This study thus reveals that FMSF is a novel motility promoting protein. Pilot/preliminary studies were done to find the effect of FMSF antibody on in vitro fertilization of mice. The goat FMSF antibody did not inhibit fertilization at 1:100 dilution but markedly inhibited fertilization to an extent of 50% at 1:50 dilution and 100% at 1:25 dilution. So, a dose-dependent reduction in fertilization was found with the FMSF antibody compared to the control studies. This study shows that FMSF has a direct role in fertilization and its antibody may have contraceptive potential. Now concluding on the entire studies, we have developed for the first time a novel vertical velocity-measuring instrumental system for more objective evaluation of semen quality. We have also established for the first time the uniqueness of a blood serum motility-promoting protein (FMSF) through a plethora of systematic studies on its purification, characterization (physical, biochemical and immunological) and N-terminal amino acid sequencing data. Our data clearly show that FMSF activates “horizontal” as well as “vertical” movement of spermatozoa. The above-mentioned novel computer-assisted vertical velocity – measuring instrument together with FMSF, may form the basis for new approaches of cattle breeding, conservation of endangered species, human contraception and treatment of male infertility: a social problem of immense dimension all over the world.
Date 2008
Type Thesis
Format application/pdf
Identifier http://www.eprints.iicb.res.in/440/1/Sudipta_Saha_%2D_Ph.D._Thesis.pdf
Saha, Sudipta (2008) A novel method of sperm motility analysis and characterisation of a sperm motility promoting protein from goat blood serum. PhD thesis, Jadavpur University.
Relation http://www.eprints.iicb.res.in/440/