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Pyroglutamyl peptidase I and prolyl endopeptidase in human semen: increased activity in necrozoospermia
Regulatory Peptides 122 (2004) 79 – 84 www.elsevier.com/locate/regpep
Pyroglutamyl peptidase I and prolyl endopeptidase in human semen: increased activity in necrozoospermia Asier Valdivia a,*, Jon Irazusta a, David Ferna´ndez a, Juan Mu´gica b, Carmen Ochoa c, Luis Casis a a
Department of Physiology, Faculty of Medicine and Dentistry, University of the Basque Country, P.O. Box 699 Bilbao, Bizkaia, Spain b Department of Nursing, Nursing School, University of the Basque Country, P.O. Box 699 Bilbao, Bizkaia, Spain c Laboratory of Seminology and Clinical Embryology, Euskalduna Clinic, c/Euskalduna Nj 10-3j, 48080 Bilbao, Bizkaia, Spain Received 20 January 2004; received in revised form 21 May 2004; accepted 21 May 2004 Available online 4 July 2004
Abstract Thyrotropin-releasing hormone (TRH) and its analogues have been reported to have important functions in human semen. In the present paper, we have characterized the activity of the TRH-degrading enzymes pyroglutamyl peptidase I and prolyl endopeptidase in the fluid and prostasomes of human semen and in subcellular fractions of the corresponding sperm. Enzymatic activities were measured fluorimetrically using h-naphthylamine derivatives as substrate. Activity associated with both enzymes was detected in seminal fluid and in the prostasome fraction, as well as in soluble and particulate sperm subcellular fractions. Pyroglutamyl-peptidase I activity presented highest levels in the particulate sperm fraction, whereas the activity of prolyl endopeptidase was maximal in the soluble sperm fraction. In addition, we compared the activity of both enzymes in different seminal fractions in normozoospermic, fertile men and in subfertile patients with different abnormalities revealed by spermiogram analysis (astenozoospermia, necrozoospermia and teratozoospermia). The activities of pyroglutamyl peptidase I and prolyl endopeptidase in necrozoospermia were found to be higher in the corresponding soluble and particulate sperm fractions, respectively, with respect to those measured in normozoospermic semen. The results of the present study indicate that these enzymes may participate in regulating the levels of seminal TRH analogues and in mediating sperm death associated with necrozoospermia. D 2004 Elsevier B.V. All rights reserved. Keywords: Peptidase; FPP; TRH; Semen; Spermatozoa; Prostasome
1. Introduction Thyrotropin-releasing hormone (TRH), a tripeptide with the amino acid sequence pGlu-His-Pro-NH2, is a principal regulator of thyroid system function [1]. This tripeptide was first isolated from the hypothalamus and characterized by its ability to stimulate thyroid-stimulating hormone (TSH) secretion. However, TRH has been identified in many other regions of the brain [2] and also in the gastrointestinal tract [3]. In addition, high levels of TRH immunoreactivity, which appeared to be chromatographically distinct from canonical TRH, have been found in male prostate tissue [4] and semen [5]. In this regard, the tripeptides pGlu-GluPro-NH2 [6], also called fertilization promoting peptide or
* Corresponding author. Tel.: +34-94-601-5671; fax: +34-94-6015662. E-mail address: [email protected] (A. Valdivia). 0167-0115/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2004.05.005
FPP [7], pGlu-Phe-Pro-NH2 and pGlu-Gln-Pro-NH2 [8] have been localized in the male reproductive system. FPP has been reported to be a possible regulator of sperm function in vivo [9], increasing capacitation and fertilizing ability in mammalian spermatozoa [10,11]. Although the presence of a receptor for FPP or other TRH analogs in human spermatozoa has not been directly demonstrated, recent evidence suggests that a putative FPP receptor is expressed, at least in mouse spermatozoa [12,13]. A homologous human gene has also been reported [14]. The activity of peptides can be controlled by their enzymatic hydrolysis. Two main pathways for the enzymatic degradation of TRH, and possibly its analogues, have been characterized [15]. Prolyl endopeptidase (EC 3. 4.21.26), an enzyme which is mainly present in the cytosol [16] of cells from different tissues, such as the brain, testis [17], kidney [18] and lung [19], can cleave the Pro-NH2 bond of TRH and FPP in seminal plasma [20]. The pGluHis bond can be cleaved by two pyroglutymyl (pGlu)-
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peptidases: pGlu-peptidase I (EC 3.4.9.14), a widely expressed enzyme which actively degrades a variety of pGlu-peptides such as TRH and its analogues, LHRH, neurotensin or bombesin [15]; and pGlu-peptidase II (EC 3.4.19.-), which is localized to synaptosomal membranes in brain tissue [21,22]. The presence pGlu-peptidase II activity, which is very similar to that found in blood plasma [23], does not seem to be very relevant in semen, because inhibitors of this enzyme accelerated the degradation of TRH and FPP [5]. Few studies have characterized the presence and activity of these enzymes in semen [16,24]. To the best of our knowledge, no studies have been carried out to characterize their distribution in human semen fractions. Therefore, the aim of the present study was to measure the activity of pGlu-peptidase I and prolyl endopeptidase in human seminal fractions and to evaluate if altered activity was associated with male reproductive pathologies. To this end, we analyzed the activity of both enzymes in different fractions of normozoospermic semen, and in seminal fractions from patients presenting astenozoospermia, teratozoospermia and necrozoospermia.
2. Materials and methods 2.1. Materials All chemicals, which were of reagent grade, were obtained from the Sigma (St. Louis, MO, USA). 2.2. Human semen collection and diagnosis Normozoospermic semen was obtained from healthy donors who were known to be fertile. Samples of semen from subfertile males were obtained from patients whose semen had been characterized and diagnosed in the Laboratory of Seminology and Clinical Embryology of the Euskalduna Clinic (Bilbao, Spain). The study was fully approved by the Clinical Research Ethical Committee of the Hospital of Cruces, and informed consent of patients was obtained. The criteria of the World Health Organization were used to define normozoospermia and the studied semen pathologies. Semen was considered as astenozoospermic when less than 50% of the spermatozoa had A + B type motility (range in this study: 0– 51.8%) or less than 25% had A type motility (range in this study: 0– 27.3%) during the first hour after ejaculation. Teratozoospermia was diagnosed when more than 85% of spermatozoa from an ejaculate had abnormal shape (range in this study: 85 –96%).
which more than 50% of spermatozoa were dead (range in this study: 51 –97%). Following these criteria samples used in this study were divided in Nz (n = 24), Az (n = 16), Az + T (n = 8) and Az + Ne; (n = 24). 2.4. Preparation of seminal fractions Samples of semen were diluted (1:1, v/v) in Tris buffered saline (TBS: Tris 30 mM, NaCl 130 mM, pH 7.4) and centrifuged at 600 g for 10 min. The resulting supernatant contains seminal fluid and prostasomes, while the spermatozoa were located in the pellet. To avoid seminal plasma contamination, these pellets were washed, resuspended fully in 4 ml TBS and centrifuged again at 600 g for 10 min. After discarding the supernatants, the pellets were washed, resuspended and centrifuged at 1000 g for 15 min. Once again, the supernatants were discarded while the resulting pellets, containing pure spermatozoa, were washed with TBS and vigorously homogenized in hypotonic Tris buffer solution (10 mM Tris – HCl) to rupture the cells. Finally, these homogenates were stored at 30 jC until use. In order to obtain soluble and particulate (membranebound) sperm fractions, the purified homogenates were thawed and incubated on ice with hypotonic Tris buffer solution for 1 h. After a brief sonication (6 bursts of 30 s with standby intervals of 15 s), samples were centrifuged at 100,000 g for 35 min. The resulting supernatants were centrifuged again (100,000 g, 35 min) and the soluble sperm fraction was obtained from the new supernatants. In order to obtain the particulate sperm fraction, pellets from centrifugation of the sonicated samples mentioned above were washed and homogenized in hypotonic Tris buffer solution. This homogenate was centrifuged again at 100,000 g for 35 min and the resulting pellet was washed and homogenized with hypotonic Tris buffer solution. In order to obtain seminal fluid and prostasome fractions, we employed the method described by Ronquist [26]. The supernatants obtained after the first centrifugation (600 g, 10 min), containing seminal fluid and prostasomes, were centrifuged at 1000 g for 15 min to eliminate cell debris and residual spermatozoa. The resulting pellets were discarded while the new supernatants were frozen at 30 jC until use. In all cases, samples were maintained frozen for no more than 2 weeks before use. Thawed supernatants were centrifuged at 100,000 g for 2 h. Resulting supernatants were centrifuged again (100,000 g, 2 h) and the resulting prostasome-free supernatants were used as the seminal fluid fraction. Pellets obtained from the previous centrifugation (100,000 g, 2 h) were washed and resuspended with TBS and they were used as the prostasome fraction.
2.3. Viability determination 2.5. Measurement of peptidase activities Necrozoospermia was determined by microscopic quantification of eosin-positive (death) and negative sperm cells [25]. Necrozoospermic semen was considered as that in
pGlu-peptidase I activity was fluorimetrically measured using pGlu-h-naphthylamide as a substrate [27]. In contrast,
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Z-Gly-Pro-h-naphthylamide was employed as the substrate to measure prolyl endopeptidase activity [17]. This assay is based on the fluorescence of h-naphthylamine generated from the hydrolysis of the substrate by the enzyme. The assay buffer (in a total volume of 1 ml) consisted of the following: 50 mM sodium phosphate buffer (pH 7.4), 0.1 mg bovine serum albumin, 32 mM dithiothreitol (DTT) and 0.5 mM substrate. The reaction was initiated by adding 10 Al of sample to the assay mixture. After a 30-min incubation at 37 jC, 1 ml of 0.1 M sodium acetate buffer (pH 4.2) was added to the mixture to terminate the reaction. The sample volume and time of incubation were selected after having carried out determinations to ensure that under these conditions, the enzyme reaction is in the linear phase (results not shown). The released h-naphthylamine was determined by measuring the intensity of fluorescence at 412 nm with excitation at 345 nm. Tubes without samples were used to determine background fluorescence. Relative fluorescence was converted into picomoles of product using a standard curve, constructed with increasing concentrations of hnaphthylamine. For activation assays, the reaction was carried out using varying concentrations of DTT in the assay medium. In both cases, one unit of enzyme activity was defined as the amount of enzyme which hydrolyzes 1 pmol of substrate per minute under the conditions described above. Protein concentration was measured in triplicate by the method of Bradford [28] using bovine serum albumin as a standard. Activities were expressed as units of peptidase activity per milligram protein and in the case of the sperm fractions, the activities were also given as units of peptidase per 106 sperm.
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100%, the activity of the other fractions was as follows: seminal fluid 6.2%, particulate sperm fraction 4.5% and prostasomes 2.2%. Thus, the highest LDH activity values were recorded in the soluble sperm fraction, these being 20fold higher than in the particulate sperm fraction. 3.2. Effect of DTT on peptidase activities The effect of DTT on pGlu-peptidase and prolyl endopeptidase activity is shown in Fig. 1. The activity of both enzymes in normozoospermic human semen increased in a dose-related manner with DTT concentration. This experiment was performed to determine if the activities detected were really due to the studied enzymes, pGlu-peptidase I and Prolyl Endopeptidase, which are both activated by DTT. 3.3. Peptidase activity in normozoospermic semen fractions The activity of the peptidases in the seminal fluid and prostasome fractions, and in the soluble and particulate sperm fractions from normozoospermic male semen, are illustrated in Table 1. pGlu-peptidase I (Table 1a) activity was detected in all of these fractions, but it was heterogeneously distributed. Thus, the particulate sperm fraction contained approximately 36% of the sum of specific activ-
2.6. Measurement of lactate dehydrogenase (LDH) activity Lactate dehydrogenase activity was determined spectrophotometrically by following NADH oxidation after the addition of pyruvate [29]. 2.7. Statistics Results were analyzed using the ANOVA test, followed by the PLSD test to compare 2 2 diagnoses.
3. Results 3.1. Lactate dehydrogenase activity Since pGlu-peptidase I and prolyl endopeptidase are mainly cytosolic enzymes in most tissues, we measured the activity of lactate dehydrogenase (LDH), a cytosolic marker, in cellular and non-cellular fractions in order to assess the possible contamination of the fractions under study. Taking the soluble sperm enzyme activity level as
Fig. 1. Effect of increasing concentrations (0 – 32 mM) of DTT on the activity of pGlu-peptidase I (a) and prolyl endopeptidase (b). The activity of both enzymes increased with higher concentrations of DTT.
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Table 1 Distribution of pGlu-peptidase I (a) and prolyl endopeptidase (b) activities in different fractions of normozoospermic human semen
in the soluble (55%) and particulate (21%) sperm fractions. Lower specific activities were found in the prostasome (13%) and seminal fluid (11%) fractions.
Seminal fraction
Activity distribution (a) pGlutamyl peptidase I (%)
(b) Prolyl endopeptidase (%)
3.4. Peptidase activities in fractions of subfertile semen
SsP PsP SF P
16 36 24 24
55 21 11 13
The specific activity of pGlu-peptidase I was found to be higher ( p < 0.05) in the soluble sperm fraction of necrozoospermic semen compared to normozoospermic semen (Fig. 2a). Significant differences were not found when the activity of other fractions was compared with the corresponding fraction from subfertile semen. Similar results were obtained when enzymatic activity was expressed per 106 sperm (Fig. 2b). The activity of prolyl endopeptidase in fractions of semen from patients with different pathologies is shown in Fig. 3. In comparison to normozoospermic samples, higher prolyl endopeptidase activity was measured in the particulate sperm fraction obtained from necrozoospermic semen, when measured as either per milligram of protein (Fig. 3a) or per 106 sperm (Fig. 3b) ( p < 0.05). No other statistically significant differences were observed when the activity of a given fraction was compared with that pertaining to a
Values are expressed as the percentage of specific activity in each fraction with regard to the total specific activity in all fractions determined by the sum of their specific activities. Abbreviations: sSp, soluble sperm fraction; pSp; particulate sperm fraction; SF, seminal fluid; P, prostasome fraction.
ities. Intermediate levels of activity (24%) were found in the prostasome and seminal fluid fractions. The soluble sperm fraction had the lowest level of pGlu-peptidase activity, representing 16% of the sum of the specific activities. Prolyl endopeptidase activity (Table 1b) was more heterogeneously distributed and the highest activity was found
Fig. 2. Specific pGlu-peptidase I activity (a) in normozoospermic (N; n = 24), astenozoospermic (A; n = 16), necrozoospermic (Ne; n = 24) and astenozoospermic + teratozoospermic (AT; n = 8) seminal fractions. Values are expressed as mean (pmol of h-naphthylamine released. min 1 mg prot 1) F standard error of the mean. Abbreviations: sSp, soluble sperm fraction; pSp, particulate sperm fraction; SF, seminal fluid and P, prostasome fraction. Values corresponding to the sperm fractions (soluble and particulate) are also expressed as activity per 106 sperm (b).
Fig. 3. Specific prolyl endopeptidase activity (a) in normozoospermic (N, n = 24), astenozoospermic (A; n = 16), astenozoospermic + teratozoospermic (AT; n = 8) and necrozoospermic (Ne; n = 24) seminal fractions. Values are expressed as mean (pmol of h-naphthylamine released min 1 mg prot 1) F standard error of the mean. Abbreviations: sSp, sperm soluble fraction; pSp, sperm particulate fraction; SF, seminal fluid; P, prostasome fraction. Values in sperm fractions (soluble and particulate) are also expressed as activity per 106 sperm (b).
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different pathology. However, higher enzymatic activities were also measured in the soluble sperm fractions of necrozoospermic semen although these increased levels were not found to be statistically significant.
4. Discussion In this work, we have studied the activity of prolyl endopeptidase and pyroglutamyl peptidase I in various fractions of human semen. Both enzymes were found to be present in all of the seminal fractions examined, although to varying degrees. The results of enzyme activity assays in the presence of DTT indicated that the pGlu-peptidase in human semen is type I, since DTT is an activator of type I but an inhibitor of type II pGlu-peptidase [15]. This result is consistent with the fact that type I pGlu-peptidase can hydrolyze FPP or pGluGlu-Pro-NH2 [15,21], the most abundant TRH related peptide in semen [5]. In contrast, pGlu-peptidase II cannot, since it hydrolyzes only the pGlu-His bond of TRH or very closely related peptides. Moreover, it has been reported that the degradation of TRH and FPP in human semen is accelerated in the presence of DTT [5], further corroborating the presence of pGlu-peptidase I in human semen. The pGlu-peptidase I enzyme is predominately cytosolic in most tissues [15,17], although a membrane-associated form has also been described [17]. In the present study, we report for first time the distribution of this enzyme in various fractions of human semen. Surprisingly, in human semen the activity was predominantly found in membrane-associated (particulate sperm and prostasome) fractions. This distribution is not likely due to contamination by cytosolic enzymes, because the activity of the cytosolic marker LDH was found to be low in membrane associated fractions (4.5% and 2.2% of total activity, respectively). These results indicate that membrane-associated pGlu-peptidase I could have important novel functions in reproductive physiology and/or pathology. Prostasomes are extracellular organelles, which are components of semen [30,31]. Being present in prostasome and sperm membranes, pGlu-peptidase I may act by regulating, through hydrolysis, the activity of peptides released to seminal fluid, such as TRH, FPP or other analogues [5], thereby modulating different aspects of sperm physiology such as capacitation [10,11]. Prolyl endopeptidase is also mainly cytosolic in most tissues [16]. This was also found to be the case in human semen, because the soluble sperm fraction was found to be the most active fraction. Thus, this enzyme may play a predominantly intracellular role in sperm cells. However, the activity of this enzyme was also found in the other fractions, indicating that it may also participate in the degradation of active peptides in seminal fluid, as proposed by Siviter and Cockle [20]. The activity of both pGlu-peptidase I and prolyl endopeptidase was found to be higher in semen fractions from
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necrozoospermic patients. However, no other significant differences were observed when the activity of a given fraction was compared with the corresponding activity pertaining to a different pathology. The association of higher enzyme activity specifically with necrozoospermia, but not with other subfertile conditions raises the possibility that the higher enzyme activity may participate in inducing sperm cell death. Indeed, this association has previously been demonstrated in other tissues. Thus in the brain, the administration of selective inhibitors of prolyl endopeptidase protected cells from death by inhibiting the degradation of TRH [32], and murine T cells with high levels of activity of prolyl endopeptidase are susceptible to activation-induced cell death [33]. Since prolyl endopeptidase is more active in the cytosolic fraction, it is unlikely that this enzyme plays a predominant role in regulating peptides binding to external receptors. In contrast, cytosolic peptidases seem to be essential to maintain cell viability [34]. High peptidase activity may endow sperm cells with a higher susceptibility to cell death, as occurs with murine cells with high prolyl endopeptidase activity [33], possibly by decreasing the levels of peptides which protect the cell from death, such as TRH (or their analogs) [35]. However, the possibility that an increase in peptidase activity may be due to the death of the cell in order to hydrolyze the remaining material should also be considered. Nevertheless, if increased enzymatic activity were an effect of cell death, then significantly increased activity could be expected in all fractions, rather than in specific fractions as observed in this study. Further studies will be required to clarify if the increased levels of peptidase activity associated with necrozoospermia are a cause or consequence of sperm cell death. Finally, it should be noted that the standard error of the mean of enzyme activities was quite high in all the groups under study, indicating that in addition to the pathologies herein analyzed, other factors might be important in determining seminal peptidase activity. In summary, we have measured pGlu-peptidase I and prolyl endopeptidase activities in different fractions of normal and sub-fertile human semen. The results of this study point to a possible role of these enzymes in subfertility associated with necrozoospermia.
Acknowledgements We thank Raquel Villares for her technical assistance. This work was supported by a grant from the University of the Basque Country (UPV/EHU, 00081.327-EA-4512/ 1998). The authors would like to thank the agency Academic Consulting and Translating Services (ACTS, http://www.euskalnet.net/acts) for having improved the English of this manuscript. Acknowledgement of financial support: This work was supported by two grants from the University of the Basque Country (UPV/EHU, 00081.327-
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EA-4512/1998 and 1/UPV 00081.327-E-14891/2002). A. Valdivia holds a fellowship from the University of the Basque Country.
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Pyroglutamyl peptidase I and prolyl endopeptidase in human semen: increased activity in necrozoospermia Asier Valdivia a,*, Jon Irazusta a, David Ferna´ndez a, Juan Mu´gica b, Carmen Ochoa c, Luis Casis a a
Department of Physiology, Faculty of Medicine and Dentistry, University of the Basque Country, P.O. Box 699 Bilbao, Bizkaia, Spain b Department of Nursing, Nursing School, University of the Basque Country, P.O. Box 699 Bilbao, Bizkaia, Spain c Laboratory of Seminology and Clinical Embryology, Euskalduna Clinic, c/Euskalduna Nj 10-3j, 48080 Bilbao, Bizkaia, Spain Received 20 January 2004; received in revised form 21 May 2004; accepted 21 May 2004 Available online 4 July 2004
Abstract Thyrotropin-releasing hormone (TRH) and its analogues have been reported to have important functions in human semen. In the present paper, we have characterized the activity of the TRH-degrading enzymes pyroglutamyl peptidase I and prolyl endopeptidase in the fluid and prostasomes of human semen and in subcellular fractions of the corresponding sperm. Enzymatic activities were measured fluorimetrically using h-naphthylamine derivatives as substrate. Activity associated with both enzymes was detected in seminal fluid and in the prostasome fraction, as well as in soluble and particulate sperm subcellular fractions. Pyroglutamyl-peptidase I activity presented highest levels in the particulate sperm fraction, whereas the activity of prolyl endopeptidase was maximal in the soluble sperm fraction. In addition, we compared the activity of both enzymes in different seminal fractions in normozoospermic, fertile men and in subfertile patients with different abnormalities revealed by spermiogram analysis (astenozoospermia, necrozoospermia and teratozoospermia). The activities of pyroglutamyl peptidase I and prolyl endopeptidase in necrozoospermia were found to be higher in the corresponding soluble and particulate sperm fractions, respectively, with respect to those measured in normozoospermic semen. The results of the present study indicate that these enzymes may participate in regulating the levels of seminal TRH analogues and in mediating sperm death associated with necrozoospermia. D 2004 Elsevier B.V. All rights reserved. Keywords: Peptidase; FPP; TRH; Semen; Spermatozoa; Prostasome
1. Introduction Thyrotropin-releasing hormone (TRH), a tripeptide with the amino acid sequence pGlu-His-Pro-NH2, is a principal regulator of thyroid system function [1]. This tripeptide was first isolated from the hypothalamus and characterized by its ability to stimulate thyroid-stimulating hormone (TSH) secretion. However, TRH has been identified in many other regions of the brain [2] and also in the gastrointestinal tract [3]. In addition, high levels of TRH immunoreactivity, which appeared to be chromatographically distinct from canonical TRH, have been found in male prostate tissue [4] and semen [5]. In this regard, the tripeptides pGlu-GluPro-NH2 [6], also called fertilization promoting peptide or
* Corresponding author. Tel.: +34-94-601-5671; fax: +34-94-6015662. E-mail address: [email protected] (A. Valdivia). 0167-0115/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2004.05.005
FPP [7], pGlu-Phe-Pro-NH2 and pGlu-Gln-Pro-NH2 [8] have been localized in the male reproductive system. FPP has been reported to be a possible regulator of sperm function in vivo [9], increasing capacitation and fertilizing ability in mammalian spermatozoa [10,11]. Although the presence of a receptor for FPP or other TRH analogs in human spermatozoa has not been directly demonstrated, recent evidence suggests that a putative FPP receptor is expressed, at least in mouse spermatozoa [12,13]. A homologous human gene has also been reported [14]. The activity of peptides can be controlled by their enzymatic hydrolysis. Two main pathways for the enzymatic degradation of TRH, and possibly its analogues, have been characterized [15]. Prolyl endopeptidase (EC 3. 4.21.26), an enzyme which is mainly present in the cytosol [16] of cells from different tissues, such as the brain, testis [17], kidney [18] and lung [19], can cleave the Pro-NH2 bond of TRH and FPP in seminal plasma [20]. The pGluHis bond can be cleaved by two pyroglutymyl (pGlu)-
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peptidases: pGlu-peptidase I (EC 3.4.9.14), a widely expressed enzyme which actively degrades a variety of pGlu-peptides such as TRH and its analogues, LHRH, neurotensin or bombesin [15]; and pGlu-peptidase II (EC 3.4.19.-), which is localized to synaptosomal membranes in brain tissue [21,22]. The presence pGlu-peptidase II activity, which is very similar to that found in blood plasma [23], does not seem to be very relevant in semen, because inhibitors of this enzyme accelerated the degradation of TRH and FPP [5]. Few studies have characterized the presence and activity of these enzymes in semen [16,24]. To the best of our knowledge, no studies have been carried out to characterize their distribution in human semen fractions. Therefore, the aim of the present study was to measure the activity of pGlu-peptidase I and prolyl endopeptidase in human seminal fractions and to evaluate if altered activity was associated with male reproductive pathologies. To this end, we analyzed the activity of both enzymes in different fractions of normozoospermic semen, and in seminal fractions from patients presenting astenozoospermia, teratozoospermia and necrozoospermia.
2. Materials and methods 2.1. Materials All chemicals, which were of reagent grade, were obtained from the Sigma (St. Louis, MO, USA). 2.2. Human semen collection and diagnosis Normozoospermic semen was obtained from healthy donors who were known to be fertile. Samples of semen from subfertile males were obtained from patients whose semen had been characterized and diagnosed in the Laboratory of Seminology and Clinical Embryology of the Euskalduna Clinic (Bilbao, Spain). The study was fully approved by the Clinical Research Ethical Committee of the Hospital of Cruces, and informed consent of patients was obtained. The criteria of the World Health Organization were used to define normozoospermia and the studied semen pathologies. Semen was considered as astenozoospermic when less than 50% of the spermatozoa had A + B type motility (range in this study: 0– 51.8%) or less than 25% had A type motility (range in this study: 0– 27.3%) during the first hour after ejaculation. Teratozoospermia was diagnosed when more than 85% of spermatozoa from an ejaculate had abnormal shape (range in this study: 85 –96%).
which more than 50% of spermatozoa were dead (range in this study: 51 –97%). Following these criteria samples used in this study were divided in Nz (n = 24), Az (n = 16), Az + T (n = 8) and Az + Ne; (n = 24). 2.4. Preparation of seminal fractions Samples of semen were diluted (1:1, v/v) in Tris buffered saline (TBS: Tris 30 mM, NaCl 130 mM, pH 7.4) and centrifuged at 600 g for 10 min. The resulting supernatant contains seminal fluid and prostasomes, while the spermatozoa were located in the pellet. To avoid seminal plasma contamination, these pellets were washed, resuspended fully in 4 ml TBS and centrifuged again at 600 g for 10 min. After discarding the supernatants, the pellets were washed, resuspended and centrifuged at 1000 g for 15 min. Once again, the supernatants were discarded while the resulting pellets, containing pure spermatozoa, were washed with TBS and vigorously homogenized in hypotonic Tris buffer solution (10 mM Tris – HCl) to rupture the cells. Finally, these homogenates were stored at 30 jC until use. In order to obtain soluble and particulate (membranebound) sperm fractions, the purified homogenates were thawed and incubated on ice with hypotonic Tris buffer solution for 1 h. After a brief sonication (6 bursts of 30 s with standby intervals of 15 s), samples were centrifuged at 100,000 g for 35 min. The resulting supernatants were centrifuged again (100,000 g, 35 min) and the soluble sperm fraction was obtained from the new supernatants. In order to obtain the particulate sperm fraction, pellets from centrifugation of the sonicated samples mentioned above were washed and homogenized in hypotonic Tris buffer solution. This homogenate was centrifuged again at 100,000 g for 35 min and the resulting pellet was washed and homogenized with hypotonic Tris buffer solution. In order to obtain seminal fluid and prostasome fractions, we employed the method described by Ronquist [26]. The supernatants obtained after the first centrifugation (600 g, 10 min), containing seminal fluid and prostasomes, were centrifuged at 1000 g for 15 min to eliminate cell debris and residual spermatozoa. The resulting pellets were discarded while the new supernatants were frozen at 30 jC until use. In all cases, samples were maintained frozen for no more than 2 weeks before use. Thawed supernatants were centrifuged at 100,000 g for 2 h. Resulting supernatants were centrifuged again (100,000 g, 2 h) and the resulting prostasome-free supernatants were used as the seminal fluid fraction. Pellets obtained from the previous centrifugation (100,000 g, 2 h) were washed and resuspended with TBS and they were used as the prostasome fraction.
2.3. Viability determination 2.5. Measurement of peptidase activities Necrozoospermia was determined by microscopic quantification of eosin-positive (death) and negative sperm cells [25]. Necrozoospermic semen was considered as that in
pGlu-peptidase I activity was fluorimetrically measured using pGlu-h-naphthylamide as a substrate [27]. In contrast,
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Z-Gly-Pro-h-naphthylamide was employed as the substrate to measure prolyl endopeptidase activity [17]. This assay is based on the fluorescence of h-naphthylamine generated from the hydrolysis of the substrate by the enzyme. The assay buffer (in a total volume of 1 ml) consisted of the following: 50 mM sodium phosphate buffer (pH 7.4), 0.1 mg bovine serum albumin, 32 mM dithiothreitol (DTT) and 0.5 mM substrate. The reaction was initiated by adding 10 Al of sample to the assay mixture. After a 30-min incubation at 37 jC, 1 ml of 0.1 M sodium acetate buffer (pH 4.2) was added to the mixture to terminate the reaction. The sample volume and time of incubation were selected after having carried out determinations to ensure that under these conditions, the enzyme reaction is in the linear phase (results not shown). The released h-naphthylamine was determined by measuring the intensity of fluorescence at 412 nm with excitation at 345 nm. Tubes without samples were used to determine background fluorescence. Relative fluorescence was converted into picomoles of product using a standard curve, constructed with increasing concentrations of hnaphthylamine. For activation assays, the reaction was carried out using varying concentrations of DTT in the assay medium. In both cases, one unit of enzyme activity was defined as the amount of enzyme which hydrolyzes 1 pmol of substrate per minute under the conditions described above. Protein concentration was measured in triplicate by the method of Bradford [28] using bovine serum albumin as a standard. Activities were expressed as units of peptidase activity per milligram protein and in the case of the sperm fractions, the activities were also given as units of peptidase per 106 sperm.
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100%, the activity of the other fractions was as follows: seminal fluid 6.2%, particulate sperm fraction 4.5% and prostasomes 2.2%. Thus, the highest LDH activity values were recorded in the soluble sperm fraction, these being 20fold higher than in the particulate sperm fraction. 3.2. Effect of DTT on peptidase activities The effect of DTT on pGlu-peptidase and prolyl endopeptidase activity is shown in Fig. 1. The activity of both enzymes in normozoospermic human semen increased in a dose-related manner with DTT concentration. This experiment was performed to determine if the activities detected were really due to the studied enzymes, pGlu-peptidase I and Prolyl Endopeptidase, which are both activated by DTT. 3.3. Peptidase activity in normozoospermic semen fractions The activity of the peptidases in the seminal fluid and prostasome fractions, and in the soluble and particulate sperm fractions from normozoospermic male semen, are illustrated in Table 1. pGlu-peptidase I (Table 1a) activity was detected in all of these fractions, but it was heterogeneously distributed. Thus, the particulate sperm fraction contained approximately 36% of the sum of specific activ-
2.6. Measurement of lactate dehydrogenase (LDH) activity Lactate dehydrogenase activity was determined spectrophotometrically by following NADH oxidation after the addition of pyruvate [29]. 2.7. Statistics Results were analyzed using the ANOVA test, followed by the PLSD test to compare 2 2 diagnoses.
3. Results 3.1. Lactate dehydrogenase activity Since pGlu-peptidase I and prolyl endopeptidase are mainly cytosolic enzymes in most tissues, we measured the activity of lactate dehydrogenase (LDH), a cytosolic marker, in cellular and non-cellular fractions in order to assess the possible contamination of the fractions under study. Taking the soluble sperm enzyme activity level as
Fig. 1. Effect of increasing concentrations (0 – 32 mM) of DTT on the activity of pGlu-peptidase I (a) and prolyl endopeptidase (b). The activity of both enzymes increased with higher concentrations of DTT.
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Table 1 Distribution of pGlu-peptidase I (a) and prolyl endopeptidase (b) activities in different fractions of normozoospermic human semen
in the soluble (55%) and particulate (21%) sperm fractions. Lower specific activities were found in the prostasome (13%) and seminal fluid (11%) fractions.
Seminal fraction
Activity distribution (a) pGlutamyl peptidase I (%)
(b) Prolyl endopeptidase (%)
3.4. Peptidase activities in fractions of subfertile semen
SsP PsP SF P
16 36 24 24
55 21 11 13
The specific activity of pGlu-peptidase I was found to be higher ( p < 0.05) in the soluble sperm fraction of necrozoospermic semen compared to normozoospermic semen (Fig. 2a). Significant differences were not found when the activity of other fractions was compared with the corresponding fraction from subfertile semen. Similar results were obtained when enzymatic activity was expressed per 106 sperm (Fig. 2b). The activity of prolyl endopeptidase in fractions of semen from patients with different pathologies is shown in Fig. 3. In comparison to normozoospermic samples, higher prolyl endopeptidase activity was measured in the particulate sperm fraction obtained from necrozoospermic semen, when measured as either per milligram of protein (Fig. 3a) or per 106 sperm (Fig. 3b) ( p < 0.05). No other statistically significant differences were observed when the activity of a given fraction was compared with that pertaining to a
Values are expressed as the percentage of specific activity in each fraction with regard to the total specific activity in all fractions determined by the sum of their specific activities. Abbreviations: sSp, soluble sperm fraction; pSp; particulate sperm fraction; SF, seminal fluid; P, prostasome fraction.
ities. Intermediate levels of activity (24%) were found in the prostasome and seminal fluid fractions. The soluble sperm fraction had the lowest level of pGlu-peptidase activity, representing 16% of the sum of the specific activities. Prolyl endopeptidase activity (Table 1b) was more heterogeneously distributed and the highest activity was found
Fig. 2. Specific pGlu-peptidase I activity (a) in normozoospermic (N; n = 24), astenozoospermic (A; n = 16), necrozoospermic (Ne; n = 24) and astenozoospermic + teratozoospermic (AT; n = 8) seminal fractions. Values are expressed as mean (pmol of h-naphthylamine released. min 1 mg prot 1) F standard error of the mean. Abbreviations: sSp, soluble sperm fraction; pSp, particulate sperm fraction; SF, seminal fluid and P, prostasome fraction. Values corresponding to the sperm fractions (soluble and particulate) are also expressed as activity per 106 sperm (b).
Fig. 3. Specific prolyl endopeptidase activity (a) in normozoospermic (N, n = 24), astenozoospermic (A; n = 16), astenozoospermic + teratozoospermic (AT; n = 8) and necrozoospermic (Ne; n = 24) seminal fractions. Values are expressed as mean (pmol of h-naphthylamine released min 1 mg prot 1) F standard error of the mean. Abbreviations: sSp, sperm soluble fraction; pSp, sperm particulate fraction; SF, seminal fluid; P, prostasome fraction. Values in sperm fractions (soluble and particulate) are also expressed as activity per 106 sperm (b).
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different pathology. However, higher enzymatic activities were also measured in the soluble sperm fractions of necrozoospermic semen although these increased levels were not found to be statistically significant.
4. Discussion In this work, we have studied the activity of prolyl endopeptidase and pyroglutamyl peptidase I in various fractions of human semen. Both enzymes were found to be present in all of the seminal fractions examined, although to varying degrees. The results of enzyme activity assays in the presence of DTT indicated that the pGlu-peptidase in human semen is type I, since DTT is an activator of type I but an inhibitor of type II pGlu-peptidase [15]. This result is consistent with the fact that type I pGlu-peptidase can hydrolyze FPP or pGluGlu-Pro-NH2 [15,21], the most abundant TRH related peptide in semen [5]. In contrast, pGlu-peptidase II cannot, since it hydrolyzes only the pGlu-His bond of TRH or very closely related peptides. Moreover, it has been reported that the degradation of TRH and FPP in human semen is accelerated in the presence of DTT [5], further corroborating the presence of pGlu-peptidase I in human semen. The pGlu-peptidase I enzyme is predominately cytosolic in most tissues [15,17], although a membrane-associated form has also been described [17]. In the present study, we report for first time the distribution of this enzyme in various fractions of human semen. Surprisingly, in human semen the activity was predominantly found in membrane-associated (particulate sperm and prostasome) fractions. This distribution is not likely due to contamination by cytosolic enzymes, because the activity of the cytosolic marker LDH was found to be low in membrane associated fractions (4.5% and 2.2% of total activity, respectively). These results indicate that membrane-associated pGlu-peptidase I could have important novel functions in reproductive physiology and/or pathology. Prostasomes are extracellular organelles, which are components of semen [30,31]. Being present in prostasome and sperm membranes, pGlu-peptidase I may act by regulating, through hydrolysis, the activity of peptides released to seminal fluid, such as TRH, FPP or other analogues [5], thereby modulating different aspects of sperm physiology such as capacitation [10,11]. Prolyl endopeptidase is also mainly cytosolic in most tissues [16]. This was also found to be the case in human semen, because the soluble sperm fraction was found to be the most active fraction. Thus, this enzyme may play a predominantly intracellular role in sperm cells. However, the activity of this enzyme was also found in the other fractions, indicating that it may also participate in the degradation of active peptides in seminal fluid, as proposed by Siviter and Cockle [20]. The activity of both pGlu-peptidase I and prolyl endopeptidase was found to be higher in semen fractions from
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necrozoospermic patients. However, no other significant differences were observed when the activity of a given fraction was compared with the corresponding activity pertaining to a different pathology. The association of higher enzyme activity specifically with necrozoospermia, but not with other subfertile conditions raises the possibility that the higher enzyme activity may participate in inducing sperm cell death. Indeed, this association has previously been demonstrated in other tissues. Thus in the brain, the administration of selective inhibitors of prolyl endopeptidase protected cells from death by inhibiting the degradation of TRH [32], and murine T cells with high levels of activity of prolyl endopeptidase are susceptible to activation-induced cell death [33]. Since prolyl endopeptidase is more active in the cytosolic fraction, it is unlikely that this enzyme plays a predominant role in regulating peptides binding to external receptors. In contrast, cytosolic peptidases seem to be essential to maintain cell viability [34]. High peptidase activity may endow sperm cells with a higher susceptibility to cell death, as occurs with murine cells with high prolyl endopeptidase activity [33], possibly by decreasing the levels of peptides which protect the cell from death, such as TRH (or their analogs) [35]. However, the possibility that an increase in peptidase activity may be due to the death of the cell in order to hydrolyze the remaining material should also be considered. Nevertheless, if increased enzymatic activity were an effect of cell death, then significantly increased activity could be expected in all fractions, rather than in specific fractions as observed in this study. Further studies will be required to clarify if the increased levels of peptidase activity associated with necrozoospermia are a cause or consequence of sperm cell death. Finally, it should be noted that the standard error of the mean of enzyme activities was quite high in all the groups under study, indicating that in addition to the pathologies herein analyzed, other factors might be important in determining seminal peptidase activity. In summary, we have measured pGlu-peptidase I and prolyl endopeptidase activities in different fractions of normal and sub-fertile human semen. The results of this study point to a possible role of these enzymes in subfertility associated with necrozoospermia.
Acknowledgements We thank Raquel Villares for her technical assistance. This work was supported by a grant from the University of the Basque Country (UPV/EHU, 00081.327-EA-4512/ 1998). The authors would like to thank the agency Academic Consulting and Translating Services (ACTS, http://www.euskalnet.net/acts) for having improved the English of this manuscript. Acknowledgement of financial support: This work was supported by two grants from the University of the Basque Country (UPV/EHU, 00081.327-
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EA-4512/1998 and 1/UPV 00081.327-E-14891/2002). A. Valdivia holds a fellowship from the University of the Basque Country.
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