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
View Archive Info| Field | Value | |
| Title |
A novel method of sperm motility analysis
and characterisation of a sperm motility
promoting protein from goat blood serum
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| Creator |
Saha, Sudipta
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| Subject |
Cell Biology & Physiology
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| 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.
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| Date |
2008
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| Type |
Thesis
NonPeerReviewed |
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| Format |
application/pdf
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| 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. |
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| Relation |
http://www.eprints.iicb.res.in/440/
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