BIOCHEMICAL INVESTIGATION ON THE MATURATION OF SPERM IN EPIDIDYMIS

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.