ENZYMES IN THE SEMINAL PLASMA FROM AZOOSPERMIC MEN: CORRELATION WITH THE ORIGIN OF THEIR AZOOSPERMIA

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1 FERTILITY AND STERILITY Copyright C'1981 The American Fertility Society Vol. 36, No.3, September 1981 PrinU!d in UBA. ENZYMES IN THE SEMINAL PLASMA FROM AZOOSPERMIC MEN: CORRELATION WITH THE ORIGIN OF THEIR AZOOSPERMIA JEAN-FRANCOIS GUERIN, PH.D.*t JACQUESROLLET, M.D.* PAUL PERRIN, M.D.* YVES MENEZO, PH.D. ALINE ORGIAZZI, M.S. * JEAN-CLAUDE CZYBA, M.D.* Department of Histology, University of Medecine, Lyon, France Semi-quantitative estimations of the activities of65 enzymes were studied for cases of azoospermia in an attempt to correlate the seminal enzyme profile with the origin of the azoospermia. One single enzyme-a.-glucosidase-showed variation. All other specificities tested gave identical results for all the azoospermic samples and for normal controls. The a.-glucosidase activity that is present in normal semen was. absent from samples from patients with azoospermia associated with a complete ob.struction of the genital tract, and alsofrom all semen samples from vasectomized subjects. This enzyme appears to be secreted by the epididymis, and the activity present in azoospermic semen can provide information concerning the origin of the azoospermia and the functional state of the epididymis. Positive but reduced a.-glucosidase activity appears to be frequently associated with inflammatory disease. The consequences of these findings are discussed. Fertil Steril 36:368, 1981 The enzymes present in cell-free seminal plasma may be derived from several different sources. Some originate as secretions of the reproductive tract epithelium, others from the prostate and seminal vesicles, and yet others may be intracellularenzymes released from dead or damaged spermatozoa. In a previous study! we showed that the enzymatic composition ofseminal plasma was very similar to that of the cellular content of washed spermatozoa. In view of this, it was surprisingto find that there was very little difference in enzyme composition between normal seminal Received March 3,1981; revised and accepted May 26,1981. *Laboratoire d'histologie-embryologie, Faculte de Medecine, 8 Avenue Rockefeller, Lyon, France. treprint requests: Jean-Francois Guerin, Ph.D., Laboratoire d'histologie-embryologie, Faculte de Medecine, 8 Avenue Rockefeller, Lyon, France. *Department of Urology, H6pital de l'antiquaille, Lyon; France. Department of Biology, INSA, Villeurbanne, France. plasma and that from oligozoospermic or even azoospermic subjects. The condition of azoospermia results principally from one of two causes: a failure of spermatogenesis or an obstruction of the reproductive tract. In the latter case, it might be postulated that secretions from regions of the reproductive tract upstream from the blockage should be absent from the ejaculate. Some semen components are known to originate principally from the epididymis, and only minor contributions are made by the genital accessory glands, for example, glycerophosphoryl choline and carnitine 2-4 ; but at present little is known about the origins of the enzymes found in seminal plasma. We have therefore studied the enzymes in seminal plasmas from a group of clinically well-defined patients by a micromethod that allows simultaneous investigations of a large number of activities. In this report we confirm and extend our preliminary observation that a-glucosidase activity was absent from the semen of patients with congenital 368

2 Vol. 36, No.3 CORRELATION OF ENZYMES WITH AZOOSPERMIA 369 obstruction of the deferent canals, and from that of vasectomized subjects. 5 MATERIALS AND METHODS Azoospermic semen samples were obtained from 30 patients consulting for sterility, all of whom had a total absence of spermatozoa in their ejaculate. These patients were all submitted to careful clinical examination. Their blood serum levels of follicle-stimulating hormone (FSH) were measured, and in some cases the luteinizing hormone-releasing hormone (LH-RH) stimulation test was performed. Patients with normal FSH levels were in most cases further investigated by vesiculo-deferentography and surgical examination when an obstruction appeared evident. Testicular biopsies were taken during surgery as well as from patients with intermediate FSH levels or when spermatogenesis was uncertain. Further samples of azoospermic semen were obtained from 15 vasectomized subjects at least 1 month after surgery. In all cases the liquified ejaculates were centrifuged at 3000 x g for 15 minutes to eliminate residual blood cells and.debris. Samples of epididymal or deferent secretions were obtained by microcatheterization during surgery from 11 patients suffering from complete bilateral obstruction of the reproductive tracts of congenital, inflammatory, or undetermined origin. These samples were clarified by gentle centrifugation at 600 x g for 10 minutes. The API-ZYM (API System, 38, Montalieu, France) microtechnique was used as described previouslyl to estimate 65 different enzyme activities (mostlyhydrolases and dehydrogenases) simultaneously. Each microwell contained lyophilized specific substrate (50 nmoles) and buffer at the optimum ph for the corresponding enzyme. After incubation with seminal plasma (50 ~liwell) for 4 hours at 37 C in a moist atmosphere, the reaction is visualized by coupling the released napthol with a diazonium salt. The intensity of the color produced increases linearly with increasing enzyme activity up to 30 nmoles of substrate released. The sensitivity of the method is about 1.5 nm substrate/hr/50 ~l of seminal plasma. RESULTS Preliminary testing of 20 samples of azoospermic semen for all the enzymatic activities covered by the test galleries available to us showed that four galleries of 10 hydrolases each and one gallery of 8 dehydrogenases systematically gave identical results for all the samples and that these reactions were of the same intensity as those previously reported for normal semen samples. 1 Of these 48 activities, 18 were never present, and the 30 positive reactions were of the normal strength. For the rest of the study we therefore considered the results from a single gallery of 19 substrates, testing for various hydrolases and 8 glycosylases. Of these 19 activities, 18 showed only minor fluctuations within the test group or in comparison with normal values, but one activity-a-glucosidase-showed major variations in some of the azoospermic specimens. Three categories of a-glucosidase activity could be distinguished in the samples of azoospermic semen: normal activity as compared with normal seminal plasma was found in 10 of 30 samples studied; absence or only trace amounts of activity was found in 8 of 30 samples; and positive reactions at a much lower level than those of normal specimens were found in 12 of 30 samples. The clinical data concerning the donors of the azoospermic samples showed that the origins of their azoospermia were heterogeneous, frequently complex, and sometimes uncertain. We have, however, classified the patients into three groups, mainly according to the results of the measurement of plasma FSH and of the observations from testicular biopsy or surgical exploration, which were generally performed in cases with normal plasma FSH levels. It is our opinion that a frankly elevated plasma FSH level is clear evidence of a defect in spermatogenesis. The first group (7 patients) consisted of subjects with a straightforward failure of spermatogenesis without associated abnormalities of the excretory ducts. The second group (8 patients) consisted of subjects with demonstrated obstruction of the excretory ducts (congenital or not) but without associated failure of spermatogenesis. These patients had normal plasma FSH levels. For seven of these patients the obstruction was demonstrated by surgical investigation, and in one case the lesion was examined histologically; the corresponding testicular biopsies revealed no lesions. The third group (15 patients) consisted of subjects with multiple or uncertain causes of azoospermia; a total or partial failure of spermatogenesis associated with an inflammatory disease of the excretory ducts (epididymitis or deferentitis with previous infection). For eight of these subjects the plasma FSH level was frankly elevated, and four others had values

3 370 GUERIN ET AL. September 1981 TABLE 1. Relationship Between a-glucosidase Activity in the Ejaculates from Azoospermic Patients and the Origin of Their Azoospermia Origin of azoospermia Failure of spermatogenesis Obstruction of genital tract Mixed pathologic condition Vasectomy Total Normal o 10 a-glucosidase activity Low Undetectable Total at the superior limit of the normal. The three remaining subjects had normal plasma FSH levels but gave abnormally high responses after injection of LH-RH. Table 1 shows the relationship between the a glucosidase activity and the clinical data for the 30 patients with azoospermia. Of ten cases with normal levels of enzyme activity (column 1), nine had a failure of spermatogenesis, associated in five cases with some degree of involvement of the excretory ducts; however, in one case an apparently complete obstruction of the excretory ducts was present. In contrast, when the enzyme was not detected in the samples (column 3), all the subjects had obstruction (8 of 8), in three cases associated with a "secretory" disease. Samples with a reduced level of enzyme activity are frequently obtained from subjects with mixed causes involving an inflammatory obstruction of the excretory ducts (7 of 12). Reduced enzyme activity was also seen, however, in three cases with an apparently isolated failure of spermatogenesis and in two cases with presumably complete obstruction of the excretory ducts. In order to confirm the relation between the absence of enzyme activity and a complete obstruction of the excretory ducts, we examined seminal plasma from 15 vasectomized subjects at least 1 month after operation. The total obstruction of the excretory ducts is certain in these cases. No a-glucosidase activity was detected in any of these specimens, indicating that the enzyme is not produced by the accessory glands but derives specifically from the excretory ducts. We have attempted to localize the site of secretion of a-glucosidase more precisely by measuring its activity in specimens of fluid from the efferent ducts, the epididymis, and the canal deferens during surgical exploration of 11 patients. Only a single specimen was obtained from each individual. Table 2 shows that a-glucosidase activity was not found in a specimen from the efferent ducts nor in three specimens from the caput epididymis. The activity appears in three specimens from the corpus epididymis and is at normal levels in secretions taken from the cauda of the epididymis or from the canal deferens. We therefore conclude that a-glucosidase is probably secreted by the corpus epididymis. DISCUSSION The present results indicate that whereas the overall enzymatic profile of seminal plasma from azoospermic patients is remarkably similar to that of normal subjects, one enzyme-a-glucosidase-is regularly absent from the semen of vasectomized subjects and from that of patients with a complete obstruction of the genital tract suggesting that this enzyme is secreted by the upper portion of the genital tract. Examination ofsecretions from different regions indicates that it may a-glucosidase activitya TABLE 2. a-glucosidase Activity in Secretions from Different Regions of the Human Male Genital Tract of Patients with Obstructive Azoospermia Efferent tubules Epididymis Caput Corpus Tail Deferent canal Absent 1I1 b 3/3 Low 113 Normal 2/3 2/2 2/2 a As compared with the activity in normal seminal plasma. bnumber of specimens with stated activity/number of specimens from stated location.

4 Vol. 36, No.3 CORRELATION OF ENZYMES WITH AZOOSPERMIA 371 be produced by the epididymal epithelium of the corpus. These findings are in agreement with the studies of Chapdeleine et al. 6 and Tremblay et al.7 Our attempts to correlate this activity with the origin of azoospermia in our patients as either a failure of spermatogenesis or obstruction of the genital tract were complicated by the frequency of mixed pathologic conditions. Half the cases studied here (15 of 30) presented both abnormal plasmatic hormonal levels and evidence of inflammatory processes in the genital tract (e.g., epididymitis). It would be convenient if so simple a test could provide useful clinical data and even, in some cases, avoid the necessity for radiologic or hormonal investigations. It would appear that the absence of detectable a-glucosidase in the seminal plasma is a definitive indication of an obstruction in the genital tract which may, of course, be accompanied in addition by a defect of spermatogenesis. The enzyme may be present in the ejaculate if the obstruction of the genital tract is situated above the site of its secretion, that is, in the efferent tubules or the caput epididymis. Alternatively, the azoospermia in these cases may be due to a failure of spermatogenesis with no associated obstruction. In the present study we found three cases where a-glucosidase activity was detected in the ejaculate, despite an apparently obstructive cause of the azoospermia, because of normal values of plasmatic FSH; two of these subjects had lowered enzyme levels, but the third had a normal activity. This third case was further investigated by histologic examination of tissue surgically re- moved from the site of the obstruction. We observed a narrowing of the lumen of the deferent canal without total closure (Fig. 1). Below this inflammatory lesion a few spermatozoa could be found, but these were rapidly phagocytosed by the numerous spermiophages present (Fig. 2). Clearly, in this case, although no spermatozoa succeeded in passing into the ejaculate, there was no major obstacle to fluid flow and thus passage of the enzyme. Reduced levels of a-glucosidase activity appear to be frequently associated with an inflammatory condition usually due to infection. Increased resistance to the passage of fluid through the inflamed region might account for some of the reduction, but it is possible that there is functional damage to the epididymal epithelium. If so, the measurement of the activity of this enzyme in the ejaculate could furnish a useful indication of the functional state of the epididymis, as pointed out by Amelar et al. 8 Two nonenzymatic compounds, glycosylphosphoryl choline (GPC) and carnitine, have been studied in this respect. 2-4 The authors are not in full agreement concerning the relative importance of the epididymal origin of these compounds, i.e., from 70% to 100% for GPC 2 3 and from 58% to 95% for carnitine. 3 4 Our results suggest that the total a-glucosidase present in the ejaculate is of epididymal origin, so that this enzyme should constitute a reliable marker. Chapdelaine et al. 6 found that after vasectomy the average maltase activity was only about 15% of the initial activity in normal semen. This residual activity would originate mainly from prostatic secretions. In addition, the measurement of the FIG. 1. Section of human epididymis in the region of expected obstruction. A, Area with spermatozoa normally present in the lumen of tubules. B, Area with spermatozoa and numerous spermiophages in the lumen. C, Area with very rare spermatozoa phagocytosed by spermiophages. FIG. 2. Section of epididymis with details of the obstructive region. A, Portion which constitutes a fibrosed diaphragm of the lumen. B, Numerous spermatozoa and spermiophages in the lumen. C, Very rare spermatozoa in the lumen (black points), most of them included in spermiophages.

5 372 GUERIN ET AL. enzyme activity as described here is very quick and simple and requires only a very small volume of ejaculate. In conclusion, we believe that the measurement of a-glucosidase activity in seminal plasma can orientate the investigation of cases of azoospermia by giving good evidence of the presence of a total obstruction ofthe genital tract, and, in combination with clinical and other biological investigations, can provide information concerning the functional state of the epididymis, particularly in cases with inflammatory antecedents. Such simple additional investigatory techniques are still desirable, since the present tests do not allow a definitive diagnosis of the origin of azoospermia in all cases. For example, we recently studied three patients who, despite normal plasma FSH levels, had no spermatozoa in their efferent tubules. That means that no obstruction was detected in the genital tract; this fact could be predicted by the discovery of normal a-glucosidase activity in the semen of these patients. We would, however, like to obtain more precise measurements ofseminal a-glucosidase activity, and we are at present developing a spectrophotometric technique for the quantitation of this enzyme, in relation to the clinical data. REFERENCES September Guerin JF, Menezo Y, Czyba JC: Enzyme comparative study of spermatozoa and seminal plasma in normal and subfertile man. Arch Androl 3:251, Brown-Woodman DC, Morley PB, Morris S, Rodger JC, White IG: Origin of glycerophosphorylcholine, inositol, N-acetylamino-sugar, and prostaglandins in human seminal plasma and their effects on sperm metabolism. Arch Androl 4:149, Frenkel G, Peterson RN, Davis JE,.Freund M: Glycerophosphorylcholine and carnitine in normal semen and in postvasectomy semen: differences in concentration. Fertil Steril 25:848, Wetterauer U, Heite HJ: Carnitine in seminal plasma: its significance in diagnostic andrology. Arch Androl 4:137, Guerin JF, Czyba JC: Influence of seminal plasma quality on motility velocity and oxygen consumption of human spermatozoa. Presented at the Fifth Annual Meeting of the American Society of Andrology, Chicago, March Chapdelaine P, Tremblay RR, Dube JY, St Yves C, Mailhot J: Origin of maltase and variations in infertile men. Arch Androl 1:61, Tremblay RR, Chapdelaine P, Mailhot J: (X 1-4 glucosidase activity in human semen: variations with number and motility of spermatozoa. Fertil Steril 3:34, Amelar RD, Dubin L, Schoenfeld CY: Sperm motility. Fertil Steril 3:34, 1980