Assay of Hyaluronidase in Human Semen

Transcription

1 Assay of Hyaluronidase in Human Semen Leo Wilson, M.D., and William Aronson, M.D. RECENT STUDIES have revealed the probable physiological role of hyal uronidase in facilitating sperm penetration and fertilization of the mammalian ovum The testicular enzyme apparently is carried by the spermatozoa to the vicinity of the freshly ovulated egg where it disperses the enveloping cumulus cells. This occurs by virtue of the specific ability of the enzyme to liquefy the viscid intercellular cement substance which binds the cumulus cells together. There is now general agreement that the hyaluronidase content of a semen specimen is roughly proportional to its sperm count and that the enzyme is absent in azoospermia It has been claimed that the enzyme content of some specimens may be considerably below average in the presence of a normal (or even high) sperm count 6 but this has been denied by others. 17 What is not yet understood is the extent, if any, to which human infertility can be ascribed to hyaluronidase deficiency. To assume a causal relationship solely on the basis of a reduced sperm count would be hazardous indeed since the count by itself is no index of fertility unless it falls to extremely low levels (less than 2 million spermatozoa per mi. ).8 Not much information has been gained thus far from attempts to use the enzyme therapeutically. The reported results are contradictory and prove only that hyaluronidase is no panacea for sterility. Until we are able to determine those cases in which deficiency of the enzyme exists, it will be impossible to assess properly the value of hyaluronidase therapy. What is needed to clarify the relationship of hyaluronidase to infertility is an accu- From the Departments of Obstetrics-Gynecology and Pathology, Morrisania City Hospital, New York, N.Y. 254

2 Vol. 1, No. 3, 195] HYALURONIDASE IN HUMAN SEMEN 255 rate method of enzyme assay of the semen. If, by this means, it becomes possible to set a minimum hyaluronidase level for fertile men, it should be a simple matter to detect enzyme deficiency irrespective of the sperm count. With this objective in mind, we examined various methods of hyaluronidase assay. Only the viscosimetric method has proven satisfactory in our hands."' The original method of Madinaveitia and Quibell has undergone a number of modifications/ ' all based on the fact that hyaluronidase reduces the viscosity of a solution of its substrate (hyaluronic acid or one of its salts ) 2 in direct proportion to the concentration of the enzyme. 13 This action is independent of the concentration of the substrate 13 when the latter exceeds.7 per cent. 16 The measurement is made in an Ostwald viscosity pipette under standard conditions of volume, ph, temperature and sodium chloride concentration. Swyer and Emmens recently investigated the fundamental principles involved in the viscosimetric assay of hyaluronidase and were able to devise an improved technic with a standard error of less than 1 per cent. The chief features of this method are: 1) incubation of the buffered hyaluronatesemen mixture for a fixed time ( 2 minutes) which _avoids the tedious repeated flow-time readings of the earlier "time to half-viscosity" technics and 2) use of the flow-time indgx (V), which corrects for individual viscosimeter differences and permits comparison of assays made in different viscosimeters. The flow-time index is: (fs- fe) V = 1 (fs _ fo) where fs is the flow-time of the substrate plus the viscosity-equivalent of the semen, fo is the flow-time of all solvents without hyaluronate and fe is the flow-time of the semen-substrate mixture after 2 minutes incubation. Then fs- fo represents the viscosity of the substrate and fs- fe is the amount of substrate rendered non-viscous by the action of the enzyme. The constant, 1, eliminates the decimal points. TECHNIC We have adopted the principles of the Swyer~Emmens method but have modified some of its details for greater convenience and accuracy. The The mucin-clot-prevention ( M.C.P.) test as modified by Kurzrok6 we found too insensitive. The turbidimetric method (Karl Meyer modification) 14, though excellent for the assay of purified enzyme preparations, proved completely unsatisfactory for human semen.

3 256 WILSON & ARONSON [Fertility & Sterility semen (a minimum of one ml. is required) is diluted 1:4 with Mcilvaine's buffer" and refrigerated for 24 hours to permit maximum release of the enzyme. (The assay may be made later the same day or may be postponed several days if the specimen is refrigerated.) The diluted semen, buffer and substrate solutions must be filtered before starting the assay to remove any particles which could obstruct the viscosimeters. The substrate solution (.16%) consists of 8 mg. potassium hyaluronatet dissolved in 5 ml. Mcilvaine's buffer. Refrigerated substrate solution will keep for one or two weeks. The slight but progressive reduction in its viscosity does not affect the accuracy of the assay. Three tubes are prepared for each specimen as shown in Table 1. Duplicate assays are made when the quantity of filtrate permits. All reagents are warmed to 37 C. in a constant-temperature water bath. The Ostwald viscosimeter is suspended in the water bath for 1 minutes before adding the semen-substrate or semen-buffer mixture. The semen-filtrate in tube #1 (fs) must be inactivatedf for 3 minutes at 6o C. and then cooled down Table 1 tube# semen filtrate substrate buffer fs I I mi." 2 mi. fe 2 I mi. 2 mi. fo 3 I mi. 2 mi. Heat-inactivated. to 37 before adding the substrate. The semen-substrate mixture in tube #2 (fe) is incubated for 2 minutes at 37 and its flow-time is measured immediately thereafter. More than 8 semen specimens, the majority from partners in childless marriages, have been assayed thus far. In this study only men whose wives are pregnant at the time of the assay are considered of proven fertility. The M/6, ph 7. (containing NaCl) 12 consists of: Na2PH4 (anhydrous) Gm., citric acid 2.2 Gm., NaCl 3.5 Gm., water to 1 liter. vve use this buffer to dilute the semen and as the solvent for the hyaluronate. f We are extremely grateful to Prs. Erwin Schwenk and D. Roy McCullagh of the Schering Corporation for generous supplies of hyaluronate. t Swyer and Emmens substitute.5% gum arabic solution for the semen filtrate in the fs measurement, the same solution having been used as the diluent of the semen. This method is not quite accurate because there is considerable variation in the viscosities of different semen specimens. The use of heat-inactivated semen is a more precise method.

4 Vol. 1, No. 3, 195] HYALURONIDASE IN HUMAN SEMEN 257 Va y 8 A L u 7 R N I SO.Q A s oo 5 E I N 8 D 4 E X to ~ r- eo Oo i 3~.. og e 2~ o88 1 o8 5~ MILLION I SPERMATOZOA PER ML. FIGURE 1. Showing the relation of the hyaluronidase index ( V) to the sperm count per ml. in 116 assays including 16 on men of proven fertility (solid circles). Semi-logarithmic graph. relation of the flow-time index to the sperm count, which is more or less proportional, is shown in Figure 1. Most of the points on the graph fall within the range of two to ten times the sperm count (in millions per ml.) with the heaviest concentration between three and four times the sperm count. Azoospermic specimens gave very low readings ranging up to 44 V. \Vhile these values are within the limits of experimental error, it is possible that minute quantities of hyaluronidase may be present. 15 A detailed analysis of our findings will be reported in a separate communication.

5 258 WILSON & ARONSON [Fertility & Sterility SUMMARY An improved technic for the viscosimetric assay of hyaluronidase in human semen is described. It is a modification of the method of Swyer and Emmens, in which the buffered semen-hyaluronate mixture is incubated for a fixed time and the results are expressed in terms of the flow-time index, V. This technic is now being. employed to determine the minimum level of hyaluronidase in the semen of fertile men in order to clarify the role of this enzyme in human infertility. REFERENCES 1. Bergenstal, D. M., and Scott, W. W.: J.A.M.A. 137:157, Chain, E., and Duthie, E. S.: Nature 133:977, Fekete, E., and Duran-Reynals, F.: Proc. Soc. Exper. Bioi. & Med. 52:119, Haas, E.: J. Bioi. Chern. 163:63, Joel, C. A., and Eichenberger, E.: Schweiz. med. Wschr. 27:61, Kurzrok, R., Leonard, S. L., and Conrad, H.: Amer. J. Med. 1:491, Kurzrok, R.: Amer. J. Clin. Path. 18:491, Lane-Roberts, C. L., Sharman, A., Walker, K., Wiesner, B. P., and Barton, M.: Sterility and Impaired Fertility, 2nd ed., Roeber, 1948, p Leonard, S. L., and Kurzrok, R.: Endocrinology 37:171, McClean, D., and Hale, C. W.: Biochem. J. 35:159, McClean, D., and Rowlands, I. W.: Nature 15:627, Mcilvaine, T. C.: J. Bioi. Chern. 49:183, Madinaveitia, J., and Quibell, T. H.: Biochem. J. 34:625, Meyer, K.: Physiol. Rev. 27:335, Sherber, D. A., Birnberg, C. H., and Kurzrok, R.: Endocrinology 42:2, Swyer, G. I. M., and Emmens, C. W.: Biochem. J. 41:29, Swyer, G. I. M.: Biochem. J. 41:49, Tafel, R. E., Titus, P., and Wightman, W. W.: Amer. J. Obst. & Gynec. 55:123, Werthessen, N. T., Berman, S., Greenberg, B. E., and Cargill, S. L.: J. Urol. 54:565, 1945.