STUDIES ON £-GALACTOSIDASE FROM THE GOAT EPIDIDYMIS

Abstract

Sperm produced in the testis spend varying periods of time in the epididymis during which they undergo significant changes collectively termed 'maturation'. Remodelling of sperm surface is one of the most prominent features observed. The sperm membrane like most other membranes is made up of a lipid bilayer with several glycoproteins tucked in. In the past few years, the role of glycoproteins in cell-cell interaction has been repeatedly emphasized. This concept has been extended to the interaction of egg and sperm too. The epididymis supports several glycosylating events such as addition, deletion and modification of carbohydrate moieties on the sperm surface. Several types of enzymes like glycosyltransferases and exoglycosidases responsible for these events are abundant in the epididymis. The action of these enzymes lead to a reorganized sperm surface which is reflected in the enhanced ability of sperm for forward progressive motility and interaction with ova. In animals like goat which have been used in the present study, reproduction is seasonal and so is epididymal sperm maturation. Roorkee experiences five distinct seasons: spring, summer, monsoon, autumn and winter. Several environmental, cues like termperature, light, humidity, rainfall and photoperiod ensure that breeding is restricted to the monsoon months of July, August and September. The excellent linear correlation between the weight of testis and epididymis together with information gathered from animal breeders in Roorkee confirmed that the monsoon season was the period of high sexual activity. In terms of monitoring the functional status of the epididymal •11- microenvironment, the activity of 6-galactosidase was measured in the various segments of the epididymis at all seasons. Epididymal 6-galactosidase is an exoglycosidase that influences sperm maturation by deletion of galactose moieties from sperm surface molecules. A spurt in 6-galactosidase activity in epididymal segments observed during the breeding season gave an indication that this enzyme may have a definite role to play in sperm maturation. Interest on this enzyme thus grew and an attempt was made to purify the enzyme from the goat epididymis. Purification was done using the technigues of acid precipitation, salt precipitation, dialysis, cation exchange chromatography, gel filtration and preparative electrophoresis. This resulted in a 137 fold purification of B-galactosidase. The purified preparation migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel subjected to electrophoresis. Discontinuous cathodic polyacrylamide gel electrophoresis followed by enzyme specific staining helped to prove that the homogeneous preparation was B-galactosidase. Antibodies raised against purified B-galactosidase gave a single precipitin line following double immunodiffusion on agarose gel, which stained for B-galactosidase activity with 6-bromo-2-naphthyl B-D-galactopyranoside. Thus, the homogeneity was also established immunologically. Once the enzyme was purified in sufficient amounts an attempt was made to study its properties. Molecular weight of the purified preparation was determined and found to be 64K by gel filtration

and 63K by SDS-PAGE. The purified enzyme was guite stable at acidic pH (pH 4.2-5.0). Temperatures of upto 40°C did not substantially affect the 6-galactosidase activity. At higher temperatures significant loss in activity was observed within a few minutes. The enzyme was otherwise very stable at low temperatures. Storage at 4°, 0° and -70° C did not have any impact on the activity of 6-galactosidase during the entire period of three months for which the enzyme activity was checked. Of the two specific substrates of 6-galactosidase tried, p-nitrophenyl 6-D-galactopyranoside was hydrolyzed 3.5 times faster (Km = 0.102 mM; Vmax = 1.084 juM/min/unit of enzyme) than o-nitrophenyl 6-D-galactopyranoside (Km = 0.8mM; Vmax =0.32 /xM/min/unit of enzyme) . In addition, the preparation was to a limited extent able to hydrolyze p-nitrophenyl N-acetyl- B-D-glucosaminide and p-nitrophenyl a-D-galactopyranoside indicating that it possesed N-acetyl-6-D-glucosaminidase and a-D-galactosidase activities as well. The preparation did not however hydrolyze the substrates of other glycosidases namely, the p-nitrophenyl derivatives of a-D-mannoside, and a-D-and 6-D-glucoside. V-D-Galactonolactone competitively inhibited the hydrolysis of p-nitrophenyl 6-D-galactopyranoside with a Ki value of 0.231 mM. Enzyme activity was also strongly inhibited by Tris and D-galactose. The inhibitory action of synthetic sugars methyl a-D-glucopyranoside and methyl a-D-mannopyranoside was very small.

are known to influence sperm maturation in a variety of ways including regulation of the catalytic activity of many enzymes. In order to know which of the ions are important, a survey was made of the ions present in the luminal fluid at various sites of the epididymal duct. Luminal fluid was collected and the composition determined using the techniques of Inductively Coupled Plasma Emission Spectroscopy and Flame Photometry. Cu2+ , Ca2+,Mg2\ Na\ K+ and Zn2+were detected in the samples, of the luminal fluid. m addition, trace levels of Ni2+, Cr2+ and Ti2+ were also detected. However, Mn2+, cd2+ and Co2+ were not found. An attempt was then made to ascertain which of the ions from the luminal fluid influenced the activity of 6-galactosidase. m recent years, industrialization has led to pollution of the environment particularly with heavy metals. Heavy metal toxicity is of immense concern to animal species. The particular impact on the reproductive processes is even more important since the quantity and quality of the offspring are critically affected. with this in mind, the activity of the purified 6-galactosidase was measured in the presence and absence of specific ions. it was found that the enzyme activity was strongly inhibited by Cu2+, Ni2+, Pb2+, Ag+ and Hg2+ . The matter embodied in the thesis is presented in four parts. in the first part seasonal changes in epididymal 6-galactosidase activity have been shown implicating the importance of the enzyme in male reproduction. i„ the second part, strategies used to purify this enzyme have been described. The third part highlights the properties of the purified preparation. The last -vpart is concerned with the ionic regulation of the enzyme. Thus the work carried out represents a detailed study of one epididymal exoglycosidase, B-galactosidase, which is known to play a role in sperm maturation.