Peptidase Activities in Crotalus Durissus Terrificus Semen

REPRODUCTIONRESEARCH

Peptidase activities in Crotalus durissus terriﬁcus semen

Camila Eduardo Marinho1,4, Selma Maria Almeida-Santos2, Sylvia Mendes Carneiro3, Simone Cristina Yamasaki1 and Paulo Flavio Silveira1 1Laboratory of Pharmacology, 2Laboratory of Ecology and Evolution and 3Laboratory of Cellular Biology, Instituto Butantan, Av. Vital Brasil, 1500, Saõ Paulo SP 05503-900, Brazil and 4Department of Physiology, Instituto de Biocieˆncias, Universidade de Saõ Paulo, Saõ Paulo SP 05508-900, Brazil Correspondence should be addressed to P F Silveira; Email: [email protected]

Abstract

To understand the role of peptidases in seminal physiology of Crotalus durissus terriﬁcus, activity levels of representative enzymes in semen and their sensitivities to inhibitors, cofactors, and peptide hormones were evaluated. The existence of seminal fractions and the association of peptidases with these fractions were also characterized for the ﬁrst time in snakes. The prominent inhibitors of aminopeptidases (APs) were amastatin for acid, basic, and neutral; bestatin for basic; and diprotin A for dipeptidyl-IV. Cystyl and prolyl- imino APs were similarly susceptible to the majority of these inhibitors. The basic and neutral were characterized as metallo-peptidases,

acid AP was activated by MnCl2, and cystyl, prolyl-imino, and type I pyroglutamyl were characterized as sulphydryl-dependent APs. Angiotensin II, vasotocin, bradykinin, fertilization-promoting peptide, and TRH altered the majority of these peptidase activities; these peptides are possible substrates and/or modulators of these peptidases. Peptidase activities were found in all seminal fractions: seminal plasma (SP), prostasome-like (PR) structures, and soluble (S-) and membrane-bound fractions (MFs) of spermatozoa. The levels of activity of each peptidase varied among different seminal fractions. In SP, the higher activities were puromycin-insensitive neutral and basic APs. In PR, the higher activity was puromycin-insensitive neutral AP. In spermatozoa, the higher activity in subcellular SF was puromycin- sensitive neutral, while in MF both puromycin-sensitive and -insensitive neutral APs were equally higher than the other examined peptidases. Data suggested that these peptidases, mainly basic and neutral, have a high relevance in regulating seminal functions of C. d. terriﬁcus. Reproduction (2008) 136 767–776

Introduction enkephalin, BK, OXT, AVP, LHRH (Barret et al. 1998), TRH, and FPP (Siviter & Cockle 1995) are hydrolyzed by Enzyme hydrolysis leading to inactivation or processing POP. OXT and AVP are hydrolyzed by cystyl AP (CAP, EC of peptides has been assumed to be a limitation step for 3.4.11.3; Davison et al. 1993). LHRH and TRH are their biological activities. Many peptides, such as susceptible to type I pyroglutamyl AP (PAP-I, EC 3.4.19.3; angiotensins (Angs; Fraser et al. 2006), kinins (Blaukat 2003), substance P, enkephalins (Sastry et al. 1991), Cummins & O’Connor 1998), and FPP is a possible endorphin (El-Haggar et al.2006), oxytocin (OXT), substrate of this enzyme. BK, kallidin, met-enkephalin, vasopressin (AVP; Assinder et al. 2000), LHRH (Amory and somatostatin are hydrolyzed by basic AP (APB, EC & Bremner 2003), TRH, and fertilization-promoting 3.4.11.6; Barret et al. 1998). Substance P and endorphin- peptide (FPP; Green et al. 1996), as well some peptidases, 2 are substrates of dipeptidyl peptidase-IV (DPPIV, EC particularly aminopeptidases (APs) and oligopeptidases 3.4.14.5; Barret et al. 1998). have been related to seminal physiology in mammals The distribution of peptidases in seminal fractions is (Fernańdez et al. 2002, Irazusta et al. 2004, Valdivia et al. fundamental to the regulatory role of these enzymes. 2004, Subirań et al. 2008). In this sense, Ang I and II are Until the present, earlier studies of human seminal hydrolyzed by acid AP (APA; EC 3.4.11.7; Kugler 1982) fractions evaluated the distribution of only three of the and, also, by prolyl oligopeptidase (POP; EC 3.4.21.26; above-mentioned peptidases: APN (Fernańdez et al. Barret et al. 1998). The formation of Ang IV from Ang III 2002, Subirań et al. 2008), PAP, and POP (Valdivia et al. (Kugler 1982) and bradykinin (BK) from kallidin (Mizutani 2004). APN was detected in soluble (S-) and solubilized et al. 1993) is catalyzed by neutral AP (APN). Enkephalin membrane-bound fractions (MFs) of seminal plasma (SP) is hydrolyzed by puromycin-insensitive APN (APN-PI; and prostasomes (PRs; Fernańdez et al. 2002). In sperm, E.C 3.4.11.2) and puromycin-sensitive APN (APN-PS, E.C APN was detected in the equatorial segment of the upper 3.4.11.14; Fernańdez et al.2002). Substance P, post-acrosomal region of the head, in the neck, and

q 2008 Society for Reproduction and Fertility DOI: 10.1530/REP-08-0192 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/27/2021 10:41:54AM via free access 768 C E Marinho and others along the tail (Subirań et al. 2008). Alterations of APN 0.75 (23) levels in human semen were associated with different abnormalities in the sperm of subfertile patients (Irazusta 0.50 et al. 2004). PAP-I and POP activities were detected in SP ∗ (23) and in the PR fraction, as well as in soluble and 0.25 membrane-bound sperm subcellular fractions, and have higher activities in necrozoospermia than in normo- mM/min per mg protein 0.00 zoospermic semen (Valdivia et al.2004). AP and G amylase activities are also known to be the main agents Figure 1 LDH activity (means S.E.M.) in soluble (white bar) and solubilized membrane-bound (black bar) fractions of whole semen of in the liquefaction process of the ejaculated semen C. d. terrificus. Number of animals is given in parentheses. *P!0.0003 coagulum, in humans (Chatterjee et al. 1997). Moreover, (unpaired two-side Student’s t-test). AP inhibitors, bestatin and puromycin, are capable of diminishing cellular proliferation and viability in or indifferent with the adopted agents (Olivo et al. 2008). mammals (Takahashi et al. 1989, Constam et al. 1995). CAPactivity in SF,PAP-I, and PIP had a common pattern of C However, bestatin is capable of restoring and promoting decrease with BK, TRH, and FPP (with or without Ca2 ). follicular growth and ovulation after its suppression by Ang II and BK diminished APA activity in SFand MF.Ang II stress (Nakamura et al. 1998). and arginine vasotocin (AVT) decreased CAPactivity in SF To our knowledge, the fractionation of semen and and PIP activity. AVT also decreased CAP activity in MF. the presence of seminal APs and POP have not yet DL-dithiothreitol (DTT) improved CAP activity in SF and been studied in snakes. Furthermore, species possessing MF and PIP activity, and decreased APB activity. EDTA sperm with long lifespans, such as the rattlesnake Crotalus diminished the activities of APB and APN SF and MF. durissus terrificus, are interesting experimental models MnCl2 decreased the activities of CAP in MF, PAP, and to study the distribution and function of peptidases in DPPIV SF and MF, and increased APA activity. seminal fractions. In C. d. terrificus, spermatogenesis As shown in Fig. 2, a peak coincided with the dead volume begins in spring and has its peak in summer. The (Vo) of seminal fraction from gel filtration in Sephadex spermatozoa are maintained in the male tract until G-200. This peak was analyzed by transmission electron mating (middle of autumn; Almeida-Santos et al. 2004) microscopy and had numerous rounded or oval mem- and, subsequently, in the female tract until ovulation branous vesicles of the size 50–200 nm, shown in Fig. 3. (spring; Almeida-Santos & Salomaõ 1997). The goal of the Figure 4 shows AP activities in seminal fractions: SP, present study was to evaluate the existence of mamma- PR, and spermatozoa subcellular SF and MF. In SP, the lian-like seminal fractions (SP, PR, and sperm), and to higher activities were APN-PI and APB relative to other characterize the activity levels of soluble and membrane- examined peptidases. In PR, APN-PI activity also had the bound APA, APB, APN-PI, APN-PS, CAP, PAP-I, DPPIV, higher level, followed by APB and APN-PS. In sperma- POP, and prolyl iminopeptidase (PIP) and their sensi- tozoa subcellular SF, APN-PS activity was higher, while tivities to inhibitors, cofactors, and possible natural in spermatozoa subcellular MF, APN-PS and APN-PI substrates in whole semen, as well as their associations with different seminal fractions of C. d. terrificus. Table 1 Activity levels of aminopeptidases: acid (APA), basic (APB), puromycin-insensitive neutral (APN-PI), puromycin-sensitive neutral Results (APN-PS), dipeptidyl peptidase-IV (DPPIV), pyroglutamyl-I (PAP-I), prolyl-imino (PIP); and prolyl oligopeptidase (POP) in soluble (SF) and Figure 1 shows that lactate dehydrogenase (LDH) activity solubilized membrane-bound (MF) fractions of whole semen of in SF was threefold higher than in MF of whole semen. Crotalus durissus terrificus. Similar proportion was obtained in spermatozoa sub- Activities (U/mg protein) cellular SF and MF (data not shown). Table 1 shows values of peptidase activity in SF and Aminopeptidase SF MF MF of whole semen. APB, PIP, and POP activities were APA 691G180a (16) 838G229a (16) detected only in SF, while the others were detected in SF APB 18 549G3227b,* (15) Absent APN-PI 30 372G3107c (13) 23 866G1938b (13) and MF. APN-PS 9019G1133d,* (10) 12 637G1290c (11) Table 2 shows that APB activity was markedly CAP 31G14a (14) 30G13a (15) inhibited by amastatin and bestatin, APA and APN by DPPIV 234G57a,* (13) 560G153a (11) a a amastatin, and diprotin A had a stronger effect on DPPIV PAP-I 261G34 (14) 231G71 (16) PIP 257G66a,* (15) Absent activity in SF and MF. CAP and PIP activity decreased in POP 1777G252a,d,* (14) Absent the presence of majority of examined inhibitors. Bestatin and puromycin were more efficient to inhibit PAP. POP Values are meansGS.E.M. Number of animals is given in parentheses. Z activity was not evaluated by classical AP inhibitors, U picomoles of hydrolyzed substrate per minute. Different letters indicate different peptidase activity levels in the same fraction (One- since it is an endooligopeptidase (the only one under way ANOVA P!0.0001, Bonferroni P!0.05). *P!0.05 in comparison study here), and this activity was shown to be increased to MF (unpaired, two-side Student’s t-test).

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activities were higher than other examined peptidase -test). 22 22 32 20 20 21 24 30 18 activities. Considering activity levels of each peptidase t G G G G G G G G G among different seminal fractions, APB, PIP, and POP were higher in SP; APB, APN-PI, DPPIV, and PAP-I in PR; APN-PS in spermatozoa subcellular SF; and PAP-I and 28 117 22 – 9* – 5* – 7* – 7* 118 9* 140 4* 140 6* 149 2* 151 1* 161 211* 128 14 103 CAP in spermatozoa subcellular MF. APA was equally G G G G G G G G G G G G G distributed among all seminal fractions. It was note-

Crotalus durissus terrificus worthy that APN-PS activity was absent in SP. 16* 74 27 78 12* 16 24 67 9* 44 16 29 32 38 18* 7 9* 46 11* 15 19* 4 24 1302 21 110 G G G G G G G G G G G G G Discussion S), cystyl (CAP), dipeptidyl peptidase-IV The efficiency of the employed procedure to separate SF 14* 0 19 92 9* 45 21 92 13* 61 11 82 21 89 16* 34 12* 0 8* 37 13* 41 18 129 23 90 and MF was confirmed by the pronounced LDH activity G G G G G G G G G G G G G ons of whole semen of (cytosolic marker) in SF of all samples, which allowed de (FPP), fertilization-promoting peptide (FPP) plus the characterization of the activity of each peptidase in different fractions, and consequently the association of 4* 0 14 78 12 56 11* 97 11 56 19 94 21 199 14 59 14 0 18 17 17 63 18 114 981 intra- and extracellular peptides with the peptidase G G G G G G G G G G G G G activities under study. Overall, peptides involved in intracellular signaling are primarily hydrolyzed outside the cell, mainly by membrane-bound peptidases which 8* 73 7* 89 9* 91 9* 68 11* 91 11 85 13 94 12 92 15 91 8* 82 989 883 10 86 have, in general, the active sites in the extracellular side, G G G G G G G G G G G G G which interact with released peptides (O’Cuinn 1998). (% control) Soluble peptidases act mainly in intracellular processes, 0.05 in comparison with the respective control (unpaired, two-side Student’s

. but can also be exocytosed or act in recaptured peptides, 2 ! 2* 67 7* 51 5* 52 4* 51 4* 69 3* 89 6* 97 5* 91 6* 85 9* 72 6* 86 654* 88 56 95 P which are internalized as a part of the peptide-receptor G G G G G G G G G G G G G complex, as occurs with peptide hormones (Gibson et al. 1989). Under stimulation, cytosolic enzymes can be 100%. *

Z translocated to the cell surface and efficiently process 18 0 18 0 2* 0 5* 0 7* 11 9* 0 9* 0 6* 0 11* 0 3* 0 3* 0 193* 7587 24 236 extracellular substances (Albiston et al. 2004). In the G G G G G G G G G G G G G present study, APB, PIP, and POP activities were detected only in SF and the other activities were detected in SF and Remaining peptidase activity MF, following the same pattern found in tissues and 17 84 2* 83 5* 8 8* 48 9* 34 12 20 11 28 11 0 10 36 21 10 11 0 14 2510 3* 122 semen of mammals (Irazusta et al. 2001, 2004, Silveira G G G G G G G G G G G G G et al. 2001, Varona et al. 2003). The use of substances derived from naphthylamide has been the initial step to detect or confirm the involvement 870 4* 14 9* 21 12 54 11 45 884 880 784 18 88 12 87 11 84 11 88 9* 16 of APs and POP in physiological mechanisms. Variables G G G G G G G G G G G G G that could not be controlled might have influenced peptidase activities in the present study, e.g. intra-season and geographical and circadian variations. This is 14 107 2* 9 2* 69 23 100 11 93 18 104 17 105 9 102 8 105 9 104 15 102 10* 106 9* 74 possible considering the seasonality, since samples G G G G G G G G G G G G G were obtained in different days of the same season (austral autumn – the mating season for C. d. terrificus). However, the fertility potential of semen can be 51* 86 3* 5 7* 7 11 112 15 95 2* 105 11 112 9* 95 16 99 19 102 7 101 19 42 639 presumed effective and homogeneous along this season, G G G G G G G G G G G G G based on the existence of a high quality of seminal dynamic parameters of Boa constrictor occidentalis

APA APN(Tourmente CAPet al.2007 DPPIV), the peak PAP-I of testosterone . from triplicates of pool of 11 animals. Control (absence of any factor) 42* 452 1* 21 7* 33 884 13 75 10* 35 278 11* 65 998 11 84 11 95 971 992 (Zacariotti et al. 2005) and the decrease of spermatozoa M ]), TRH, DL-dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA) and MnCl . E G G G G G G G G G G G G G SF MF APB SF MF SF MF SF MF SF MF PIP POP . C

S defects (Zacariotti 2004)inC. d. terriﬁcus. Geographical G 356 variation was not a factor as snakes were captured Ca[2 C ]96 from locations with similar fauna and environmental

C conditions. Moreover, we can also exclude circadian Remaining activities of aminopeptidases: acid (APA), basic (APB), puromycin-insensitive neutral (APN-PI), puromycin-sensitive neutral (APN-P

Ca[2 variations, because the material was obtained in the 2

C same period of the day, with a maximum difference Table 2 (DPPIV), pyroglutamyl-I (PAP-I), prolyl-imino (PIP); and prolylwith oligopeptidase classical (POP) aminopeptidase in inhibitors soluble (SF) andcalcium and angiotensin (FPP solubilized II membrane-bound (MF) (AngII), fracti arginine vasotocin (AVT), bradykinin (BK), fertilization-promoting pepti Values are means Agents Amastatin 11 Bestatin 76 Diprotin A 99 Puromycin 97 AngII 40 AVT 99 BK 77 FPP 81 FPP TRH 92 DTT 86 EDTA 88 MnCl of 90 min. www.reproduction-online.org Reproduction (2008) 136 767–776

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0.6 Blue dextran the peritoneal membrane and manually distended, its 0.55 0.5 length is about six times the total body length, although 0.45 the right duct is always slightly longer than the left. Three 0.4 0.35 regions can be distinguished: proximal (testicular), 0.3 median, and distal (cloacal). In this snake, spermatozoa 0.25 0.2 become increasingly more motile from the proximal to 0.15 the distal region (Almeida-Santos et al. 2004). Regarding 0.1 0.05 the presence of examined peptidase activities in the 0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 spermatozoa, the identification of their anatomical sources need to be investigated; for example, in the Prostasome-like acrosomal complex, which is known in all vertebrate 0.55 0.5 taxa as an enzyme-rich organelle that facilitates Absorbance 280nm 0.45 spermatozoal penetration into the ovum (Subirań et al. 0.4 0.35 2008). In squamates, the acrosomal complex is highly 0.3 compartimentalized (Gribbins et al. 2007), and this 0.25 compartimentalization has been suggested as an aid to 0.2 0.15 the sequential release of acrosomal enzymes (Talbot 0.1 1991). Since the scope of the present study was not to 0.05 0 compare the seminal fractions and peptidase compo- 15 30 45 60 75 90 105 120 135 150 165 180 195 210 sition from different areas of the ductus deferens, semen Eluction (min) from the entire ductus deferens of C. d. terrificus was Figure 2 Gel filtration chromatography on Sephadex G-200. The taken systematically to assure similar seminal fluid column was equilibrated and washed with Tris–saline buffer (30 mM components for each analysis. Tris plus 130 mM NaCl), pH 7.4, flow rate 0.16 ml/min. The APs under study are known to be sensitive to amastatin, bestatin, diprotin A, and puromycin (Cadel The anatomy and morphology of the male reproduc- et al. 1995, Ronai et al. 1999, Sato 2003). POP is inhi- tive tract could also influence the features of the bited by Z-Pro-prolinal (Garcıá-Horsman et al. 2007) peptidase activities and their distribution in seminal and belonging to a distinct class of enzymes, it was not fractions of C. d. terrificus. In reptiles, the ductus included in the inhibition assay. deferens are recognized as the site of spermatozoa In the present study, APB susceptibility to bestatin storage in males and, differently from mammals, the distinguished this enzyme from APA and APN. By the epididymis does not participate in sperm maturation and susceptibility of DPPIV to diprotin A, and PAP-I, CAP, storage (Sever et al. 2002). The ductus deferens of C. d. and PIP to puromycin, it was possible to differentiate terrificus are convoluted and enter the cloaca indepen- them from APA and APB. The inhibition of DPPIV by dently of the ureters. As previously described by diprotin A is a known feature of this enzyme (CD26) in Almeida-Santos et al. (2004), the ductus deferens occupy mammals (Minelli et al. 1999). As distinguishing about one-third of the body length and when freed from features, APB and APN are metallo-peptidases, because of their inhibition by EDTA (Kawata et al. 1980). APA was marked as activated by MnCl2, which is a known characteristic of this enzyme in mammals (Ramı´rez et al. 1990). CAP and PAP-I were activated by DTT, a thiol-reducing agent, characterizing them as sulphydryl- dependent peptidases. DTT is an activator of the type I PAP and an inhibitor of the type II (O’Cuinn et al. 1990), and the degradation of FPP (possible substrate of PAP-I) is also increased by the presence of DTT (Cockle et al. 1994), which confirms that PAP-I is among the enzymes studied in the present work and FPP is one of its substrates. Overall, the present results show that seminal peptidase activities of C. d. terrificus have distinct biochemical properties, and therefore it is possible to attribute them to different proteins. It was notable that enzyme activities were affected by the evaluated 0.2µm peptides, which are their possible substrates (e.g. Ang Figure 3 Electron microphotography of prostasome-like structures II on APA, AVT on CAP, and PIP, and particularly TRH from semen collected from the ductus deferens of C. d. terrificus in and FPP with or without Ca[2C] on CAP, PIP, and PAP-I). austral autumn. These results indicate the competition among these

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∗ ∗ a 10000 Seminal plasma 83000 b Prostasome-like a

8000 43000 ∗ ∗ a a ∗ 6000 3000 1000 b 1300 ac 900 750 ∗ 500 ∗ c 500 100 150 b b 50

Activity (U/mg protein) 100 25 c c c 50 b b b b c 0 0 Spermatozoa subcellular SF Spermatozoa subcellular MF ∗ 20000 1200 b a

15000 1000 a

10000 3000 a 800 600 1800 a 300 b ∗ 600 b ∗ 400 a 200 b b Activity (U/mg protein) a 200 100 b b b c c cc 0 0

PIP APA APB CAP POP APA APB CAP PIP POP DPPIV PAP-I DPPIV PAP-I APN-PI APN-PS APN-PI APN-PS

Figure 4 Distribution of activity levels of aminopeptidases: acid (APA), basic (APB), puromycin-insensitive neutral (APN-PI), puromycin-sensitive neutral (APN-PS), cystyl (CAP), dipeptidyl peptidase-IV (DPPIV), pyroglutamyl-I (PAP-I), prolyl-imino (PIP); and prolyl oligopeptidase (POP) in seminal plasma, prostasome-like and spermatozoa subcellular soluble (SF) and solubilized membrane-bound (MF) fractions of C. d. terrificus. Values are meansGS.E.M. from triplicates of samples (see Materials and Methods). UZ picomoles of hydrolyzed substrate per min. Different letters indicate different peptidase activity levels in the same fraction (one-way ANOVA, P!0.0001, Bonferroni P!0.001). Asterisks indicate the higher activity of each peptidase when comparisons were made among all seminal fractions (one-way ANOVA, P!0.0003, Bonferroni P!0.05). peptides and the synthetic substrates, strongly suggesting cholesterol over phospholipids (Kravets et al. 2000). that those peptides are natural substrates of seminal PRs have been related to sperm motility, liquefaction, peptidases of C. d. terrificus. and immunosuppression (Kravets et al. 2000). Since PRs The semen of the rattlesnake contains SP and sperma- can be identified only after purification procedures it is tozoa, as well as vesicles resembling mammalian PRs. difficult to infer how frequent they are in C. d. terrificus. Although ulterior ultrastructural analyses are needed to Another important question that remains to be investi- determine peculiarities of these vesicles seen in C. d. gated is the origin of these vesicles in C. d. terrificus.In terrificus, compared with PRs of mammals, they could humans, PRs are secreted by the prostate gland (Kravets be considered as PR structures since they were obtained et al. 2000). In bovines, PRs are originated in the seminal by the same purification procedure employed to obtain vesicles (Agrawal & Vanha-Perttulla 1987). Seminal human PRs (Fernańdez et al. 2002, Vivacqua et al. 2004) vesicles secrete proteins, vitamin C, fructose, prosta- and had peptidase activities (Fernańdez et al. 2002), glandin, and fibrinogen, while the prostate gland shape (Fernańdez et al. 1997, Vivacqua et al. 2004), and secretes a whitish liquid with low viscosity which C size (Olsson & Ronquist 1990, Vivacqua et al. 2004) contains citrate and Ca2 (Minelli et al. 1998). It is similar to human PRs. Mammalian PRs are membranous noteworthy that C. d. terrificus possesses PR structures, vesicles (50–550 nm; 150–200 nm are the most fre- since squamates do not possess a prostate gland, seminal quent), which contain large amounts of cholesterol, vesicles, and bulbo-urethral and urethral glands (Sever sphingomyelin, calcium, and several enzymes (Kravets 2004). The renal sexual segment is known to act as an et al. 2000). They are surrounded by a bilaminal or accessory sex organ (Sever et al. 2002), from which multilaminar lipoprotein membrane with unusual lipid those structures could have originated. This renal sexual composition due to quantitative predominance of segment of snakes secretes a complex of glycogen, www.reproduction-online.org Reproduction (2008) 136 767–776

Downloaded from Bioscientifica.com at 09/27/2021 10:41:54AM via free access 772 C E Marinho and others mucopolysaccharides, lipids, mucoproteins, and phos- importance. Ang II, AVT, BK, FPP, and TRH altered the phatases (Ku¨hnel & Krisch 1974) in a pattern that can be majority of the peptidase activities under study, related to spermatogenic cycle and mating activity suggesting them as possible substrates and/or modulators (Sever et al. 2002). However, since in the present study of these peptidases. the semen was collected from the ductus deferens, it would not have mixed with fluids from the renal sexual segment but probably from other areas of major secretory activity in the snake urogenital system, e.g. Materials and Methods the ampulla ductus deferentis and the ductuli epididy- Animals mides of the epididymis (Volsoe 1944), from which the PR structures could have originated. The ductuli Adult male snakes (C. d. terrificus, Serpentes, Viperidae, Z epididymides or ductuli efferents have been observed Crotalinae; n 26) were captured from their natural environ- in the snake Seminatrix pygaea as efferent ducts from ment in the states of Saõ Paulo and Minas Gerais (Brazil) during seminiferous tubules of the testis that lead into the austral autumn, when testosterone is at its peak (Zacariotti et al. epididymis, from which sperm pass into the ductus 2005) and mating occurs (Almeida-Santos & Salomaõ 1997). deferens (Sever 2004). In several mammals, the terminal The animals were identified by the Laboratory of Herpetology of the Instituto Butantan and housed individually in wooden portion of the ductus deferens is differentiated into an cages (inside length!width!height of 35!26!22 cm) and ampulla, which is considered a multifaceted organ, acclimated to controlled conditions of temperature (25 8C), morphologically similar to the seminal vesicles (Riva humidity (65.3G0.9%), and photoperiod (12 h light:12 h et al. 1982). Among reptiles, the ampulla has been dark–lights on at 0600 h) in a restricted-access room for a reported only in squamates, being more prominent in period of 10 days. lizards than in snakes (Akbarsha et al. 2005). The After anesthesia with CO2 exposure for 3 h, the ductus ampulla of squamates and mammals shares the irregular deferens of the reproductive tract were removed by laparotomy narrow folded epithelium (Sever 2004). (Langlada et al. 1994), and semen was obtained from these The higher levels of the majority of the examined ducts. The animals were then destined for other studies after peptidase activities were found in SP and PR fractions. euthanizing by decapitation. APN and APB activities were more expressive in all The animal and research protocols used in this study are in seminal fractions. The conversion of kallidin to BK agreement with the Brazilian Council Directive (COBEA- (Mizutani et al. 1993) and inactivation of BK (Barret et al. BRAZIL) and were approved by the Ethics Committee of the 1998) are, respectively, actions of APN and APB, Instituto Butantan (193/04). indicating the importance of the regulation of these peptides to seminal function in C. d. terrificus. However, considering our results and the large range of peptides Collection of C. d. terrificus semen that are susceptible to these peptidases, it is not possible As previously described by Almeida-Santos et al. (2004), the to assure that the high levels of peptidases are restricted ductus deferens were stretched out on polystyrene plates for only to the kinin system. In other words, the relevance semen extraction. Pressure with a cell scraper (TPP – Techno of other evaluated peptidases cannot be ignored. For Plastic Products AG, Trasadingen, Switzerland) along the whole instance, DPPIV and POP activities were also highly extension of the two ducts was applied. The total amount expressed in PR and SP of C. d. terrificus respectively. of semen obtained was used for the following procedures. Considering their known hydrolytic effects over endorphin-2 and enkephalin, respectively (Barret et al. 1998), and the fact that endogenous opioid peptides Preparation of soluble and solubilized membrane- seem to have a marked role in seminal physiology bound fractions from whole semen of C. d. terrificus (O’Hara et al. 1994, Agirregoitia et al. 2006), DPPIV and In order to obtain SF and MF, individual samples of whole POP activities may have a relevant role in seminal semen were resuspended in 1 ml of 10 mM Tris–HCl buffer (pH physiology of C. d. terrificus. 7.4), homogenized with a Teflon pestle in a glass potter (2 min In conclusion, this is the first report of fractionation at 800 r.p.m.) and ultracentrifuged (100 000 g for 35 min; and peptidase composition of semen of reptiles, and Hitachi model HIMAC CP56GII). The resulting supernatants demonstrated the presence of acid, basic, neutral correspond to the SF of whole semen. To avoid contamination (puromycin-sensitive and -insensitive), cystyl, dipepti- with the SF, the resulting pellet was washed three times with the dyl-IV, type I pyroglutamyl, and prolyl-imino APs, as well same buffer and was then homogenized (2 min at 800 r.p.m.) in as POP activities in SP, spermatozoa, PR structures, and 1 ml of 10 mM Tris–HCl buffer plus 0.1% (v/v) Triton X-100 whole semen from ductus deferens of C. d. terrificus. (Calbiochem, San Diego, CA, USA), and then ultracentrifuged Metallo-dependent APs inhibited by amastatin and (100 000 g for 35 min). The resulting supernatants correspond bestatin (mainly those that act on neutral or basic to the MF of whole semen. All steps were carried out at 4 8C. SF amino acids) had marked levels in whole semen and in and MF were stored in polystyrene tubes at K80 8C until their all seminal fractions, indicating a high physiological use in LDH, protein, and peptidase activities assays.

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Fractionation of semen of C. d. terrificus Electron microscopy of PR fraction In order to obtain SP, PR, and spermatozoa subcellular SF and The PR pellet was fixed for 2 h in a solution of 1.5% MF of C. d. terrificus semen, the methodology described glutaraldehyde (Electron Microscopy Sciences-EMS, Hatfield, by Fernańdez et al.(2002)for human semen was adapted PA, USA) and 1% paraformaldehyde (Sigma) in 0.1 M cacodylate as follows. The pool of whole semen of five animals was buffer, pH 7.3, and post-fixed in 1% osmium tetroxide in the same homogenized in 1 ml Tris–saline buffer (30 mM Trizma (Sigma) buffer. After dehydration in ethanol graded series, the pellet was plus 130 mM NaCl (Merck), pH 7.4) and centrifuged (600 g embedded in Embed-812 resin (Electron Microscopy Sciences- for 20 min; Hitachi HIMAC CP56GII). The supernatant contained EMS). Silver ultrathin sections were stained with uranyl acetate SP and PR and was reserved for later use, while the pellet and lead citrate, examined and microphotographed (LEO 906E contained spermatozoa. To avoid contamination with SPand PR, Transmission Electron Microscope, Zeiss, Go¨ttingen, Germany). the pellet was washed with 1 ml Tris–saline buffer and subsequently resuspended in 2.5 ml of the same buffer and centrifuged (600 g for 10 min). The resulting supernatant was Protein discarded and the pellet was washed with 2.5 ml Tris–saline Protein content was measured at 630 nm in triplicates by the buffer and subsequently resuspended in 2.5 ml of the same buffer method of Bradford (1976; Bio-Rad protein assay). Protein and centrifuged (1000 g for 15 min). The supernatant was contents were extrapolated by comparison with standard discarded, and the resulting pellet contained only spermatozoa. curves of BSA (Sigma) in the same diluent. This pellet was homogenized in 2.5 ml 10 mM Tris–HCl buffer (pH 7.4; 2 min at 800 r.p.m.) and stored at K80 8C, until use for separation of subcellular SF and MF. Then, the homogenate was LDH sonicated (6 pulses of 30 s, with intervals of 15 s) and As a marker for the fractionation procedure, LDH activity was ultracentrifuged (100 000 g for 35 min). The pellet was reserved determined (Bergmeyer & Brent 1972) in samples of 3 mlofSFand and the supernatant was ultracentrifuged (100 000 g for 35 min), MF in triplicates incubated with 297 mlofNADH(b-NADH, and the resulting pellet was discarded while the supernatant was reduced form; Sigma), dissolved in 100 mM phosphate buffer, pH considered the spermatozoa subcellular SF. The reserved pellet 7.4, containing 1.6 mM sodium pyruvate (Sigma) and 200 mM from the penultimate ultracentrifugation was, then, homogenized NaCl. Absorbance was read in 96-well flat bottom microplates, at at 800 r.p.m. (2 min, in 1 ml 10 mM Tris–HCl buffer) and 0 and 10 min, in Bio-Tek PowerWave X spectrophotometer ultracentrifuged (100 000 g for 35 min). After discarding the resulting supernatant, the pellet was homogenized at 800 r.p.m. absorbance reader at 340 nm. Values of LDH activity were (2 min, in 1 ml 10 mM Tris–HCl buffer), and was considered as obtained by subtracting the absorbance reading at 10 min from 8 the spermatozoa subcellular MF. time zero of incubation at 37 C, and extrapolated by comparison The resulting supernatant from the first centrifugation (SP and with a standard curve of NADH dissolved in 100 mM phosphate PR) was centrifuged (1000 g for 20 min) in order to eliminate buffer, pH 7.4, containing 200 mM NaCl. LDH activity was debris and residual spermatozoa. The resulting pellet was expressed as mmol NADH oxidized/min/mg protein. discarded and the supernatant was stored at K80 8C. After defrost, this supernatant was ultracentrifuged (100 000 g for Peptidase activity assays 120 min). The obtained pellet was reserved and the supernatant was ultracentrifuged at the same conditions. The resulting pellet Peptidase activities were quantified on the basis of the amount was discarded and the resulting supernatant contained SP. The of 4-methoxy-b-naphthylamine (Sigma; for DPPIV and CAP) or reserved pellet from the penultimate ultracentrifugation was b-naphthylamine (Sigma; for all other peptidases) released washed with 0.6 ml Tris–saline buffer and subsequently resus- (Irazusta et al. 2001, Silveira et al. 2001, Gasparello-Clemente pended in the same volume. To obtain 0.6 ml more of this & Silveira 2002, Gasparello-Clemente et al. 2003, Varona et al. resuspension, all needful procedures were repeated with another 2003), which is the result of the enzyme activity of 20–50 ml pool of whole semen offive animals. Both the resuspensions were samples incubated with prewarmed substrate solution at mixed (1.2 ml), filtered in a membrane filter with a pore size concentrations of 0.125 mM (APA, APN-PS, APN-PI, CAP, 0.22 mm (Millipore, Bedford, MA, USA), and then submitted to PAP, PIP, and POP), 0.2 mM (DPPIV), and 0.5 mM (APB) in gel filtration in Sephadex G-200 (Sigma; 1.5!30 cm), at a flow respective 0.05 M buffers containing BSA 0.1 mg/ml in 96-well rate of 0.16 ml/min, in a column pre-equilibrated with Tris–saline flat bottom microplates for 30 min at 37 8C in a total volume of buffer. The dead volume (Vo) was determined by injecting a 300 ml. The content of naphthylamine was estimated fluor- solution of Blue dextran (Sigma). PR was not retained by the ometrically (microplate fluorescence reader Bio-Tek FL600FA) column (Fernańdez et al. 2002) and, then, was collected in the Vo at 460/40 nm emission wavelength and 360/40 nm excitation (fractions 60–120 min or 9.6–19.2 ml), in a volume of about wavelength. The fluorescence value obtained at time zero was 9.6 ml. The eluate with PR was ultracentrifuged (100 000 g for subtracted from values at time 30 min and the relative 120 min) and the resultant pellet (PR fraction) was homogenized fluorescence was then converted to picomoles of in 1 ml Tris–saline buffer. All procedures were performed at 4 8C. b-naphthylamine (Sigma) or 4-methoxy-b-naphthylamine After obtaining SP,PR, SF, and MF of spermatozoa, samples were (Sigma) by comparison with a correspondent standard curve, transferred to polystyrene tubes and maintained at K80 8Cuntil which was dissolved in the same diluent as the incubation. the measurements of protein and peptidase activities. Part of PR Peptidase activities were expressed as picomoles of hydro- fraction was destined to the electron microscopy. lyzed substrate per min (U) per milligram of protein, in which www.reproduction-online.org Reproduction (2008) 136 767–776

Downloaded from Bioscientifica.com at 09/27/2021 10:41:54AM via free access 774 C E Marinho and others the existence of a linear relationship between time of hydrolysis and/or MF pool of whole semen from 11 animals with: and protein content in the fluorometric assay was a previous 0.315 mM Ang II (Sigma); 0.314 mM AVT (reptilian analog of condition. Considering enzyme activity measures as a OXT and AVP; Sigma); 0.310 mM BK (Sigma); 0.94 mM FPP C comparative tool, the possible unspecific degradation during (Sigma); 0.94 mMFPP 1.8 mM CaCl2;0.911mMTRH homogenization was not considered. (Sigma); 0.97 mM DTT; 2 mM EDTA; 1 mM MnCl2.The The following substrates and conditions were used: samples were incubated in the presence or absence of these substances, for 30 min, at 25 8C, and peptidase activities were –APA,L-aspartic acid a-(b-naphthylamide; Sigma; solubilized in subsequently quantified. 0.012 M NaOH) in Tris–HCl buffer, pH 7.4, with 1 mM MnCl2; –APB,L-arginine b-naphthylamide (Sigma; solubilized in H2O) in phosphate buffer, pH 6.5, with 150 mM NaCl, and 0.02 mM Statistical analysis puromycin (Sigma); Data were analyzed statistically using GraphPad Prism and –APN,L-alanine-b-naphthylamide (Sigma; solubilized in Instat softwares (GraphPad Software Inc., San Diego, CA, USA). 0.012 M HCl) in phosphate buffer, pH 7.4, with 1 mM DTT Regression analysis was performed to obtain standard curves. (Sigma), with or without puromycin – APN-PI activity is the ANOVA, followed by Bonferroni test, compared values among result of the incubation with puromycin, while APN-PS is the all peptidase activities in the same seminal fraction. Student’s t- result of values of incubates without puromycin minus those test was performed to compare values of LDH between SF and with puromycin; MF, and peptidase activities in different incubation conditions – CAP, H-Cys-4-methoxy-b-naphthylamide (Bachem Bioscience relative to the respective controls. Differences were considered Inc., Torrance, CA, USA; solubilized in 0.012 M HCl) Tris- statistically significant at a minimum level of P!0.05. maleate, pH 5.9; – DPPIV, Glycil-Proline-4-methoxy-b-naphthylamide (Bachem Bioscience Inc.; solubilized in dimethyl sulfoxide, DMSO Declaration of interest (Sigma)) in Tris–HCl buffer, pH 8.3; –PAP-I,L-pyroglutamic acid-b-naphthylamide (Sigma; solubil- The authors declare that there is no conflict of interest that ized in DMSO) in phosphate buffer, pH 7.4, with 2 mM DTT could be perceived as prejudicing the impartiality of the (DTTinhibits PAP-II and actives PAP-I (O’Cuinn et al. 1990)) and research reported. 2mMEDTA(Merck); –PIP,L-proline-b-naphthylamide (Sigma; solubilized in DMSO) in phosphate buffer, pH 7.4; Funding – POP, Z-Gly-Pro-b-naphthylamide (Bachem Bioscience Inc.; This work was supported by Fundaçaõ de Amparo a` Pesquisa solubilized in DMSO) in phosphate buffer, pH 7.4, with do Estado de Saõ Paulo – FAPESP, Brazil (research grant no. 2mMDTT. 04/14971-9). C E M (05/03745-0) and S C Y (07/06829-6) were recipients of a FAPESP fellowship.

Characterization of peptidase activities in SF and MF of whole semen Acknowledgements The characteristics of peptidase activities with their classical The authors thank Valdir Jose´ Germano for his efficient inhibitors technical assistance. The susceptibilities of APA, APB, APN, CAP, DPPIV, PAP-I, and PIP peptidase activities were comparatively evaluated in a SF and/or MF pool of whole semen from 11 animals with: amastatin References ([(2S,3R)-3-Amino-2- -5-methylhexanoyl]-Val-Val-Asp-OH.HCl; Bachem Bioscience Inc.): 0.026 mM in acetic acid 8%; bestatin Agirregoitia E, Valdivia A, Carracedo A, Casis L, Gil J, Subiran N, Ochoa C & Irazusta J 2006 Expression and localization of delta-, kappa-, and ([(2S,3R)-3-Amino-2-hydroxy-4- phenylbutanoyl]-L-leucine; mu-opioid receptors in human spermatozoa and implications for sperm Bachem Bioscience Inc.): 0.026 mM in acetic acid 8%; diprotin motility. Journal of Clinical Endocrinology and Metabolism 91 A (H-Lle-Pro-Lle-OH; Bachem Bioscience Inc.): 0.026 mM in 4969–4975. deionized water; puromycin (30-[a-Amino-p-methoxyhydrocin- Agrawal Y & Vanha-Perttula T 1987 Effect of secretory particles in bovine namamido]-3-deoxy-N,N-dimethyladenosine): 0.026 mM in seminal vesicles secretion on sperm motility and acrosome reaction. Journal of Reproduction and Fertility 79 409–419. deionized water. The samples were incubated in the presence Akbarsha MA, Tamilarasan V, Kadalmani B & Daisy P 2005 Ultrastructural or absence of those substances, for 30 min, at 25 8C, and evidence for secretion from the epithelium of ampulla ductus deferentis peptidase activities were quantified. of the fan-throated lizard Sitana ponticeriana Cuvier. Journal of Morphology 266 94–111. Albiston AL, Fernando R, Ye S, Peck GR & Chai SY 2004 Alzheimer’s, The characteristics of peptidase activities with peptide angiotensin IV and an aminopeptidase. Biological & Pharmaceutical hormones, DTT, EDTA, and MnCl2 Bulletin 27 765–767. Almeida-Santos SM & Salomaõ MG 1997 Long-term sperm storage in the The susceptibilities of APA, APB, APN, CAP, DPPIV, PAP-I, and neotropical rattlesnake Crotalus durissus terrificus (Viperidae: Crotali- PIP peptidase activities were comparatively evaluated in a SF nae). Japanese Journal of Herpetology 17 46–52.

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