There is scanty information on the biochemistry

Transcription

1 FERTILITY AND STERILITY Copyright 1973 by The Williams & Wilkins Co. Vol. 24, No.4, April 1973 Printed in U.S.A. ACTIVITY AND LOCALIZATION OF ISOCITRIC DEHYDROGENASE, ASPARTATE AMINOTRANSFERASE, ALANINE AMINOTRANSFERASE, ALKALINE AND ACID PHOSPHATASE OF THE HUMAN VAS DEFERENS* A. T. GREGOIRE,t M. J. MORAN, AND A. CUADROS Margaret Sanger Research Bureau, Incorporated, New York, New York 10011, and The Population Council, New York, New York There is scanty information on the biochemistry and histochemistry of the human vas deferens. The recent increase in the use of vasectomy as a method of contraception has provided abundant material for investigation of the current methods, and various medical, physiologic, and psychologic effects of vasectomy have been published. 1 A brief description of the ultrastructure of the vas has also been reported. 2 The present study deals with the quantitative activity and histochemical localization of enzymes present in the human vas deferens related to their possible role in the transport of materials. Isocitric dehydrogenase, ICD (E.C ), aspartate aminotransferase, SGOT (E.C ), and alanine aminotransferase, SGPT (E.C ) are enzymes of the tricarboxylic cycle. Alkaline phosphatase, ALP (E.C ) and acid phosphatase, ACP (E.C ) are ubiquitous in biologic systems and are essential in the catalysis of transphosphorylation in a number of biochemical reactions and metabolic changes of various compounds. MATERIALS AND METHODS A total of 30 aliquots of vas deferens tissue was obtained at vasectomy at the Received October 5, Supported by The Population Council, New York, N. Y., and by National Institutes of Health Grant POIHD t Present address: Battelle Columbus Laboratories. 505 King Ave., Columbus, Ohio Vasectomy Service of the Margaret Sanger Research Bureau, Inc. One aliquot was weighed, homogenized with cold saline, and centrifuged at 3000 r.p.m. for 20 min., and the enzyme activity immediately determined, while a second aliquot was immediately frozen for histochemical analysis. The isocitric dehydrogenase (ICD) activity was determined quantitatively by measuring the a-ketoglutarate formed after incubation of d-isocitrate with triphospho pyridine nucleotide (TPN). The a-ketoglutarate was measured by the color produced when it reacted with 2, 4-dinitrophenylhydrazine to form a hydrozone. 3 A Sigma unit is standardized and will produce 1 nmole of TPNH per hour at ph 7.5. The aspartate aminotransferase (SGOT) and alanine aminotransferase (SGPT) were both determined colorimetrically by determining the hydro zone formed with the appropriate substrate. 4 One theoretical Sigma Frankel unit of SGOT or SGPT transaminase will form 4.82 x 10-4 #Lm. of glutamate per minute at ph 7.5. The determination of both acid, ACP, and alkaline phosphatase, ALP, is based upon the hydrolysis of the phosphate group in the compound p-nitrophenyl phosphate with the liberation of the yellow salt, p-nitrophenol, which is measured colorimetrically.5 A Sigma unit of phosphatase will liberate 1 #Lm. of p-nitrophenol per hour under specified conditions. All samples were incubated at the speci-

2 "' 310 GREGOIRE ET AL. Vol. 24 fied temperature C for the required time intervals and the enzyme activity expressed as Sigma units per milligrams of tissue. Frozen sections of vasa deferentia were used for the demonstration of alkaline and acid phosphatase by the method of Gomori. 5, 6 Sections were also fixed in Bouin's solution and stained with H & E. RESULTS The quantitative activity of the enzymes is listed in Table 1 and the histochemical localization of alkaline and acid phosphatases is shown in Figs. 1 and 2. There was a great range in the content of isocitric dehydrogenase. The levels of both transaminases, SGOT and SGPT, were significantly different (p < 0.05), the activity of the former enzyme being about 16-fold greater than that of the latter. There was significantly greater acid phosphatase activity than alkaline phosphatase activity in the vas tissue (p < 0.05). Histologic localization revealed that, while alkaline phosphatase activity TABLE 1. The Enzyme Activity of Human Vas Deferens Tissue (Sigma Units per Milligram of Tissue) ) Transaminases Isocitric dehydrogenase Glutamic Glutamic oxalacetic pyruvic Alkaline Phosphatases Acid No. of specimens Mean value ± S.E ± ± ± ± ± 0.17 Range FIG. 1. Histologic localization of alkaline phosphatase activity of the vas deferens. The activity is found in the basal membrane in areas surrounding the ductal epithelium and the layers of smooth muscle. x 43.

3 April 1973 ENZYMES IN HUMAN VAS DEFERENS 311 FIG. 2. Histologic localization of acid phosphatase. This enzyme is localized mostly in the epithelium, in the apical area and stereocilia of the luminal epithelium. x 356. was negative in the epithelium (Fig. 1), it was evident below the basal membrane in the capillaries surrounding the ductal epithelium as well as in the three layers of smooth muscle. The acid phosphatase activity was observed in the epithelium localized mostly in the apical area into the stereocilia and lumen (Fig. 2). The muscle layers were negative for the localization of this enzyme. The epithelium of the duct deferens appeared pseudostratified (Fig. 3). It showed a basal cell layer com posed of cuboidal cells containing large round nuclei and scanty cytoplasm. The rest of the epithelium was composed of tall columnar cells with large ovoidal nuclei and long stereocilia at the apical border. In some cells the cytoplasm formed projections into the lumen. There was a certain degree of pseudostratification and formation of folds which gave a festooned outline to the luminal duct. This appearance may be considered as an artifact, perhaps as a result of the muscular fibers shortening at the time of the surgical resection. There were three well-defined layers of smooth muscle. The inner layer was separated from. the epithelium by the basal membrane and connective tissue; its fibers ran longitudinally to the duct. The middle layer was the circular layer, and the outer layer had its fibers running longitudinally to the vas. On the periphery there was abundant connective tissue where numerous blood vessels were observed, including the deferential artery. DISCUSSION Isocitric dehydrogenase (led) occurs in the Krebs cycle in two forms, one dependent on diphosphopyridine nucleotide

4 312 GREGOIRE ET AL. Vol. 24 FIG. 3. Human vas deferens. The epithelium is pseudostratified and has a basal layer of cuboidal cells possessing large round nuclei. There are three well-defined layers of smooth muscle. H & E section x 86. as a coenzyme and the other on TPN. The latter was measured. ICD catalyzes the conversion of d-isocitrate to a-ketoglutarate by the reduction of TPN to TPNH. Activity of this enzyme has been described in the prostate lobes of the rat, which secrete citric acid. B The average quantity of ICD in vas tissue was 17 -fold greater than that found in the semen of various types of human seminal plasma,9 and less than that present in human sera. 3 The transaminases are also part of the tricarboxylic acid cycle, and in the presence of the amino acids, aspartic acid or alanine, catalyze the formation of either oxalacetic or pyruvic and glutamic, which are then further metabolized. In the male, transaminases are synthesized in the prostate gland and found in seminal plasma.9 The SGOT activity is correlated with the sperm concentration, while the SGPT is not. In the vas tissue, as in semen, the amount of SGPT is less than SGOT. The amounts of SGOT activity observed are in the same range but slightly greater than previously reported. 10 The enzyme acid phosphate is believed to be a secondary sex characteristic in the male reproductive system. It occurs in large quantities in semen and exhibits variation with various conditions of infertility.11 The localization of the acid phosphate is limited to the luminal epithelium, with no activity in the muscular layer, and is similar to that found in the vas deferens of the domestic rooster, 12

5 April 1973 ENZYMES IN HUMAN VAS DEFERENS 313 where highest quantities of the enzymes of the entire reproductive tract were found. 13 These latter findings in the rooster and the human suggest a synthesis and secretion of acid phosphatase by the vas tissue. Unlike the human, the alkaline phosphatase activity of the bull vas deferens is localized in the luminal and glandular epithelium. 14 The increased alkaline phosphatase activity in the human vas deferens in highly vascular muscular tissue areas and its limitation to the muscular layer suggest a role in hydrolysis and transport of materials into the vascular system. These results suggest that the human vas contains the necessary biochemical components to carryon metabolism by the tricarboxylic acid pathway. SUMMARY Enzymes of the tricarboxylic acid cycle and alkaline and acid phosphatases were measured quantitatively. Alkaline and acid phosphatase activities were localized histochemically. Alkaline phosphatase was negative in the ductal epithelium and positive in the smooth muscle. Acid phosphatase activity was present in the apical areas of the epithelium and in the stereocilia and lumen, suggesting a role in hydrolysis and transport of substances into the vascular system. Levels of isocitric dehydrogenase were greater than those found in semen, while the amounts of transaminase were less. Acknowledgments. We would like to acknowledge the valuable technical assistance of Mr. E. Tovar and Mrs. M. Melendez. REFERENCES 1. HULKA, J. F., AND DAVIS, J. E. Vasectomy and reversible vasocclusion. Fertil SteriI23:683, McLEOD, D. G., POPOVIC, N. A., AND BOSK!, A. A. Electron microscopic examination of human vas deferens (abstr. 4055). Fed Proc 972, SIGMA TECHNICAL BULLETIN No The colorimetric determination of isocitric dehydrogenase SIGMA TECHNICAL BULLETIN No The colorimetric determination of glutamic-oxalacetic and glutamic-pyruvic transaminases SIGMA TECHNICAL BULLETIN No The colorimetric determination of acid and alkaline phosphatase GOMORI, G. "Calcium Cobalt Method for Alkaline Phosphatase." In Microscopic Histochemistry: Principle and Practice, Gomori, G., Ed. Univ. Chicago Press, Chicago, 1952, p GOMORI, G. An important histochemical technic for acid phosphatase. Stain Tech 25:81, MANN, T. The Biochemistry of Semen and of the Male Reproductive Tract. Wiley, New York, GREGOIRE, A. T., AND MORAN, M. J. The enzyme activity, protein and fructose content of normal, oligospermic, postvasectomy, and infertile azoospermic men. Fertil Steril. 24:202, JOEL, C. A., AND HERZBERG, M. Glutamic-oxaloacetic transaminase in human semen and reproductive organs. J Reprod Fertil 10:185, NUN, S., MUSACCHIO, I., EpSTEIN, J. A. Variations in seminal plasma constituents from fertile, subfertile, and vasectomized azoospermic men. Fertil Steril 23:357, WILCOX, F. Phosphatases in chicken semen. J Reprod Fertil 2:148, LAKE, P. F. Histochemical demonstration of phosphomonoesterase secretion in the genital tract of the domestic cock. J Reprod Fertil 3:356, STALLCUP, O. T., AND GRIFFON, E. N. Histochemical localization of some enzymes in the ampullae of the bull ductus deferens (abstr.). J Reprod Fertil 18:180, 1969.