dc.description.abstract |
The sperm produced in the testis are initially non functional,
lacking the ability for forward progressive motility and
fertilizing capacity. But they acquire these traits after spending
considerable periods of time in the epididymis. The epididymis
nurtures, nourishes and provides the right ingredients that
transform the sperm in a way it can accomplish its task of
successful fertilization. This transformation is reflected in the
form of morphological, biochemical and physiological changes in
sperm. The sperm surface too undergoes remarkable remodelling
during epididymal transit. The current study tries to identify
some of the sperm surface changes and understand the mechanism(s)
by which they are brought about.
To accomplish this, goat epididymal sperm has been used as model
for two main reasons. First, the goat epididymis is large sized
and therefore sperm can be recovered from multiple sites of the
duct. Second, the study material is easily available since goats
are routinely slaughtered at the local abattoir for meat
consumption.
So far, most investigators have studied the epididymal tissue,
luminal fluid and sperm as a single unit making it difficult to
interpret data. The lack of appropriate methods for the separation
of these three entities has been solely the reason for such an
approach. But over the years techniques such as micropuncture and
retrograde flushing have become available. But these methods have
limited practical utility when more segments of the epididymis are
involved. In the present study a novel approach has been used to
separate tissue, fluid and sperm. Essentially the method involves
subjecting 1mm slices of epididymis in desired buffer to an
evacuation pressure of 200mm Hg for 2 minutes and then filtering
the suspension through cheese cloth. Tissue is retained on the
cheese cloth while the fluid and sperm pass through. Simple
centrifugation of the filtrate yields luminal fluid as the
supernatant and sperm as a pellet. Thus, sperm from 12 segments
of goat epididymis were obtained.
Since the main interest was in studying changes on the sperm
surface it was essential to obtain pure sperm plasma membranes.
This was done by sonication and centrifugat ion at high speeds in
sucrose gradients. The identity of the membrane fraction obtained
was checked by electron microscopic studies employing
phosphotungstic acid as a negative stain. Adenylate cyclase ser ed
as an enzyme marker for final confirmation.
Since lipids, carbohydrates and proteins form important
constituents of membranes, changes in these constituents were
monitored in membrane preparations of sperm collected from twelve
different sites of the epididymis. The results indicate that sperm
membranes tend to loose substantial amounts of phospholipids,
cholesterol, neutral sugars, amino sugars, sialic acid and
proteins during epididymal transit. The ratio of cholesterol to
phospholipid, however, rose from 0.41 to 0.51. The significance of
these findings has been analysed in the light of events happening
in the epididymis.
Of the six membrane constituents, sperm surface proteins were
selected for detailed examination. These biological molecules
exhibit the greatest heterogeneity and some on account of being
acquired or modified in the epididymis are suspected to perform
unique functions. In order to study the protein profiles of sperm
surface it is essential to release and isolate these molecules
from the membrane preparations. To achieve this, two ionic
detergents (sodium dodecyl sulfate and sodium deoxycholate) and
two nonionic detergents (Triton X-100 and Nonidet P-40) were used.
Optimal solubilization conditions for each of these detergents
were determined worked out.
Analysis by polyacrylamide gel electrophoresis revealed that when
ionic detergents were used, the staining intensity of low
molecular weight proteins was high. On the other hand, the use of
non ionic detergents resulted in better staining of proteins
having a molecular weight in the intermediate range. On the whole
the solubilization of membrane proteins by sodium dodecyl sulfate
was found to be superior in terms of the higher quantity and the
number of proteins that could be obtained.
When solubilized proteins from membrane preparations of sperm
isolated from 12 sites of the goat epididymal duct were analyzed
on SDS-PAGE, several differences could be observed. For a more
detailed examination there was need to isolate and purify specific
proteins of interest. Commercially available equipment are able to
elute proteins from polyacrylamide gels but these methods are not
useful for separating a mixture of proteins from these gels.
Therefore an effort was made to design and fabricate an apparatus
that could elute proteins during an electrophoretic separation.
The assembly made resembled a disc electrophoretic apparatus
except that a single large sized gel tube is used with provision
of elution towards the bottom. Both low and high molecular weight
proteins could be resolved and eluted by altering the percentage
of gel. In addition, the performance of the apparatus was governed
by the strength of electric field and flow rate of buffer used for
elution.
Sperm surface proteins that were solubilized by sodium dodecyl
sulfate were initially electrophoresed on 10% slabs of SDS
polyacrylamide. When electrophoresis was complete, the slab was
cut horizontally such that the top half containing the high
molecular weight proteins could be separated from the lower half
containing low molecular weight proteins. Each half of the gel was
minced separately in appropriate buffer and used as sample to be
loaded on the special apparatus designed and tested for elution of
proteins from polyacrylamide gel.
From the elution prdfiles significant observations were made. A
124kD protein was detected from the surface of the caput sperm
which was not present among the proteins solubilized from cauda
sperm membranes. Instead, an additional protein of 31kD was found
in cauda sperm membrane. Polyclonal antibody to the 31kD protein
specifically interacted with the 124kD protein, but not any other
high molecular weight proteins solubilized from caput sperm.
Further, limited proteolysis of both the 124kD and 31kD protein by
cyanogen bromide and chymotrypsin treatment followed subsequently
by analysis on polyacrylamide gel showed that certain fragments
were common to both these proteins.
These results suggest that the proteolytic cleavage of the 124kD
protein results in the formation of the 31kD protein. This
transformation is seen as one of the mechanisms involved in the
post. translational modification of sperm membrane protein.
Numerous instances have been reported in the literature where
proteins become biologically activated when cleaved. It is
proposed from the present study that the 31kD protein might be an
activated form that participates in sperm maturation and storage. |
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