Superoxide dismutase, catalase, and glutathione peroxidase during

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Superoxide dismutase, catalase, and glutathione peroxidase during epididymal maturation and prolonged storage of spermatozoa in the Mexican big-eared bat ...

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Superoxide dismutase, catalase, and glutathione peroxidase during epididymal maturation and prolonged storage of spermatozoa in the Mexican big-eared bat (Corynorhinus mexicanus) E. Arenas-Ríos, M.A. León-Galván, P.E. Mercado, and A. Rosado

Abstract: We studied the activities of reactive oxygen species (ROS) scavenging enzymes during epididymal spermatozoon maturation and storage in Corynorhinus mexicanus (G.M. Allen, 1916), a vespertilionid bat that stores spermatozoa in the epididymides for several months after regression of the testes. Depending on the phase of the epididymal reproductive cycle, two different patterns of antioxidant enzyme activities were observed in C. mexicanus. Catalase activity is clearly present in both caput and cauda epididymides throughout the entire annual reproductive cycle, being particularly high during the post-testicular phase of epididymal function. Superoxide dismutase (SOD) activity, present during the testicular phase of epididymal transport and maturation of spermatozoa, is almost completely absent or inhibited in both epididymal segments during the post-testicular epididymal storage period. GPx activity is low during the testicular phase of epididymal spermatozoon maturation and is high in both epididymal segments during the storage phase of epididymal function. From our results, we postulate that (i) the pattern of epididymal antioxidant enzyme activities in C. mexicanus is significantly different from the pattern that is proposed to be unique for mammals; (ii) epididymal function in these species of bats can be clearly divided into two phases, a testicular-dependent phase that is related to the spermatozoon maturation function of the epididymides and a testicular-independent phase that is related to the long-term spermatozoon storage function observed in these mammals; (iii) the study of the regulation of the redox potential of the microenvironment, associated with mammalian spermatozoa as they transit through the epididymides, must be particularly focused on the anatomical region where ROS generation scavenging and spermatozoon maturation storage processes take place. Résumé : Nous avons étudié les activités des enzymes 1565 protectrices des formes réactives de l’oxygène (ROS) durant la maturation et le stockage des spermatozoïdes dans les épididymes chez Corinorhinus mexicanus (G.M. Allen, 1916), une chauve-souris de la famille des vespertilionidés, qui emmagasine les spermatozoïdes dans les épididymes pendant plusieurs mois après la régression des testicules. Selon la phase du cycle reproducteur dans les épididymes, deux patrons différents d’activité des enzymes anti-oxydantes s’observent chez C. mexicanus. L’activité de la catalase est clairement évidente, tant dans le segment apical que dans le segment caudal des épididymes, pendant tout le cycle reproducteur annuel et elle est particulièrement élevée durant la phase post-testiculaire de la fonction des épididymes. L’activité de la superoxyde dismutase (SOD), présente durant la phase testiculaire du transport et de la maturation des spermatozoïdes dans les épididymes, est presque complètement absente ou inhibée dans les deux segments des épididymes durant la période post-testiculaire de stockage dans les épididymes. L’activité de la GPx est faible durant la phase testiculaire de maturation des spermatozoïdes dans les épididymes et elle est forte dans les deux segments des épididymes durant la phase de stockage de la fonction des épididymes. Nos résultats nous permettent d’avancer que (i) le patron d’activité des enzymes anti-oxydantes des épididymes chez C. mexicanus est significativement différent du patron proposé comme particulier aux mammifères, (ii) la fonction des épididymes chez ces chauves-souris se divise clairement en deux phases, une phase dépendante des testicules qui est reliée à la fonction de maturation des spermatozoïdes des épididymes et une phase indépendante des testicules reliée à la fonction de stockage à long terme des spermatozoïdes observée chez ces mammifères et (iii) l’étude de la régulation du potentiel redox du microenvironnement associé aux spermatozoïdes de mammifères lors de leur traversée des épididymes doit se concentrer de façon particulière sur la région anatomique où se déroulent la génération et la collecte des ROS et les processus de stockage et de maturation des spermatozoïdes. [Traduit par la Rédaction]

Arenas-Ríos et al.

Received 18 May 2005. Accepted 13 October 2005. Published on the NRC Research Press Web site at http://cjz.nrc.ca on 1 December 2005. E. Arenas-Ríos,1 P.E. Mercado, and A. Rosado. Laboratorio de Reproducción Animal Asistida, Departamento de Biología de la Reproducción, Universidad Autónoma Metropolitana, Unidad Iztapalapa, Avenida San Rafael Atlixco No. 186, Colonia Vicentina, CP 09340, México DF, México. M.A. León-Galván. Laboratorio de Biología y Ecología de Mamíferos, Departamento de Biología, Universidad Autónoma Metropolitana, Unidad Iztapalapa, Avenida San Rafael Atlixco No. 186, Colonia Vicentina, CP 09340, México DF, México. 1

Corresponding author (e-mail: [email protected]).

Can. J. Zool. 83: 1556–1565 (2005)

doi: 10.1139/Z05-152

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Introduction

ence and activities of ROS-scavenging enzymes. For these reasons antioxidant strategies that protect spermatozoa during epididymal transit are of great importance in ensuring the capacity of these cells to fertilize the oocyte. In general, mammalian epididymal spermatozoa contain important ROS-scavenger enzymatic mechanisms. Three enzymes have been proposed as being important in this function: enzymes related to glutathione, mainly glutathione peroxidase and their isozymes (GPx, E.C. 1.11.1.9; PHGPx; E.C. 1.11.1.19) (Alvarez and Storey 1989; Godeas et al. 1997); superoxide dismutase (SOD, E.C. 1.15.1.1) (Zini et al. 1993); and catalase (CAT; E.C. 1.11.1.6) (Bauchè et al. 1994; Gu and Hecht 1996; Bilodeau et al. 2000). Since ROS are both necessary and deleterious to the spermatozoa, we postulate that the redox potential of the microenvironment associated with mammalian spermatozoa as they transit through the epididymides must be carefully regulated. This regulation must be focused not only on ROSgeneration processes (enzymatic and nonenzymatic), but also on the anatomical region in which the ROS generation scavenging and spermatozoon maturation storage processes take place. Occurrence of prolonged epididymal spermatozoon storage has been reported recently in the Mexican big-eared bat (Corynorhinus mexicanus (G.M. Allen, 1916)), a vespertilionid which belongs to the group that only experiences torpor (López-Wilchis 1989; Geiser and Ruf 1995). This makes C. mexicanus a good model to study the ROS role in the process of spermatozoon maturation and storage in the epididymides. The aim of this work is to study the specific activity of the three antioxidant enzymes (CAT, GPx, and SOD) that have been proposed as being fundamental in the protection of spermatozoa during epididymal maturation and storage in C. mexicanus. Since our interest focused on the role that ROS-scavenging enzymes may play in epididymal spermatozoon maturation and storage, we decided to use the whole isolated caput and cauda epididymides, including the contained spermatozoa, in samples obtained throughout the annual reproductive cycle of P. mexicanus.

Vespertilionid and rhinolophid bats frequently show an important temporal asynchrony in the development and function of male reproductive organs (Krutzsch 2000; León-Galvan et al. 2005). Characteristically, development of the testes and spermatogenesis take place mainly in summer, whereas full development of accessory sex glands, expression of libido, and breeding activity take place in autumn. This asynchrony results in an unusually long period of storage of mature spermatozoa in the epididymides, which may extend for several months after the testes have totally regressed (Racey and Entwistle 2000). Initial attempts to explain this important reproductive strategy supported the idea that prolonged storage of spermatozoa might be a natural consequence of hibernating hypothermia. However, it is now known that there are several species of nonhibernating tropical bats that show epididymal spermatozoon storage (Gopalakrishna and Bhatia 1980; Karim and Banerjee 1985; Singh and Krishna 1995). In addition, many species of hibernating bats that store spermatozoa wake up periodically during the hibernating period (Avery 1985). The prolonged epididymal spermatozoon storage becomes more interesting if we keep in mind that some of the most important physiological properties of mammalian spermatozoa that are necessary for fertilization (the potential to move, to undergo capacitation, and to interact with the zona pellucida of the oocyte) develop gradually as they progress from the caput down to the cauda regions of the epididymides in a species-specific way. These functional changes are known as epididymal maturation. Epididymal maturation in human and other mammalian species takes approximately 10–20 days (Moore and Akhondi 1996), and is usually completed before the spermatozoa reach the cauda epididymides, at which point the spermatozoa will remain in storage until ejaculation. During their epididymal journey (transit + storage), spermatozoa are seriously at risk. Mammalian spermatozoa have been described as highly susceptible to the negative effects produced by reactive oxygen species (ROS) (Shekarriz et al. 1995; McKinney et al. 1996). DNA breakage (Kodama et al. 1997; Aitken et al. 1998), protein denaturation (Freeman et al. 1997), and lipid peroxidation (Alvarez and Storey 1982, 1984, 1985) that are due to persistent ROS effects (oxidative stress) eventually critically impair the fertilizing capacity and genetic integrity of the spermatozoa (Aitken 1999). ROS generation has been frequently associated with subfertility or even infertility in human patients (Aitken et al. 1996; Griveau and Le Lannou 1997). Interestingly, ROS may be produced by spermatozoa and by some nonphagocytic cell types that may be present in the epididymal tissue (Matsubara and Ziff 1986; Plante et al. 1994). Despite these adverse effects, production of regulated concentrations of ROS by the spermatozoa themselves and (or) by the epididymal environment is required during the epididymal maturation of the spermatozoa to achieve complete functional competence. Generation of adequate ROS levels has been related to tyrosine–protein phosphorylation, an important process in the development of coordinated spermatozoon movement (de Lamirande et al. 1997b; Leclerc et al. 1997) and in the ability of undergoing spermatozoon capacitation (Ford 2004). Oxidative stress can be regarded as the consequence of an imbalance between ROS-generating systems and the pres-

Material and methods Adult male C. mexicanus were captured monthly (LeónGalván et al. 2005) from June 2003 to May 2004 in México (19°37′N, 98°02′W). Bats were always captured before or as they left their roost. Individuals were sexed, weighed using an Ohaus® portable electronic balance (±0.01 g), and their forearm length measured with a Vernier caliper (±0.1 mm). To ensure adult status of bats, only those animals with complete ossification of the cartilaginous epiphyseal growth plates of the fourth metacarpal–phalangeal joint were selected (Kunz and Anthony 1982). Animals for each monthly determinations were captured on the same day, except for two bats that were captured in the same week. After decapitation of the specimens, testes and epididymides were isolated, cleared of fat and connective tissue, and weighed in a Mettler® model AB204 balance (±0.01 mg). Caput and cauda segments of the epididymides (after ligation at the level of deferent-cauda, cauda-corpus, corpus-caput, and caput-testis junctions) were removed, cleared of fat and connective tissue, rinsed in © 2005 NRC Canada

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Biggers, Whitten, and Whittingham medium (BWW) (Biggers et al. 1971), weighed in a Mettler® model AB204 balance (±0.01 mg), and frozen in liquid nitrogen (−170° C). Isolation of caput from the corpus was done by cutting at the level where the walls of the epididymides were clearly parallel. The cauda epididymides, flattened in appearance in nonbreeding bats, are easily differentiated in breeding bats because they become swollen and bulbous. In nonbreeding bats, cauda epididymides were isolated by cutting at the site where the deferens had clearly separated from the epididymides. Epididymal segments were thawed to 4 °C in 10 volumes of cold HEPES buffer (0.1 mol/L, pH 7.4) and homogenized in a Potter-Elvehjem type, glass–glass homogenizer. In all cases, corresponding segments of both epididymides of each animal were homogenized together. In some occasions, it was necessary to pool the tissues from 2 to 3 individuals to obtain the amounts of tissues necessary to do all the indicated assays. In these occasions tissues of animals captured at the same time and showing similar characteristics were used. The obtained homogenates were centrifuged for 10 min at 5000g, the precipitates were discarded, and all enzymatic activities were measured in the supernatants. Assay of GPx activity GPx activity was determined at 25 °C using Ransel kits (catalogue No. RS 505; Randox Laboratories Limited, Crumlin, Northern Ireland). This kit uses the standard indirect method of Paglia and Valentine (1967) with cumene hydroperoxide (Sigma) as a substrate for total GPx activity (Zhang et al. 1989). Enzyme activity was monitored on a Shimadzu UV–VIS 1601PC spectrophotometer at 340 nm for 5 min. The specific activity of GPx was expressed in nanomoles per minute per milligram of protein. Assay of SOD activity SOD activity was measured with RANSOD kits (catalogue No. SD 125; Randox Laboratories Limited, Crumlin, Northern Ireland). In this system SOD activity is measured using xanthine and xanthine oxidase to generate superoxide radicals (O2⋅ – ) that react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5phenyltetrazolium chloride (INT) to form a red formazan dye. Inhibition of this reaction by the presence of SOD in the sample is then used to calculate the activity of the enzyme. The activity was measured at 37 °C in the UV–VIS spectrophotometer, and absorbance was monitored at 505 nm for 3 min. The unit of activity was defined (Bogdanska et al. 2003) as the amount of enzyme that inhibits the rate of formazan dye formation by 50% using standard curves obtained with diluted solutions of commercial SOD (RANSOD kits, catalogue No. SD 125, Randox Laboratories Limited, Crumlin, Northern Ireland). Specific activity was then expressed as SOD units per milligram of protein. Assay of CAT activity CAT activity was determined at 25 °C using the method proposed by Cohen et al. (1970) for the measurement of this enzyme in tissue extracts. This method required pretreatment of the homogenate samples with ethanol (0.01 mL EtOH/mL homogenate) and Triton X-100 (1.0% (v/v) final concentration) into an ice bath to decompose complex II (Chance 1950) and solubilize the enzyme. After the incubation period,

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the amount of remaining H2O2 in the incubation mixture is determined by the addition of an excess of KMnO4, followed by the measurement of the non-reacted KMnO4 at 25 °C in the UV–VIS spectrophotometer at 480 nm. The specific activity is expressed as the rate constant k of the firstorder reaction (Cohen et al. 1970) per milligram of protein. Some preliminary enzyme determinations were done to ensure appropriate kinetic conditions. Additionally, to ensure zero-order kinetics, all enzyme assays were run in duplicate using single and double amounts of homogenate. All assays where duplicates did not produce comparable results were rerun with smaller amounts of homogenate. Total protein content for the determination of the specific activities of the enzymes was established using a commercially available bicinchoninic acid protein assay kit (Pierce, Rockford, Illinois). Statistical analysis Monthly changes in the size of the studied organs were examined using cross-correlation analysis (Hamilton 1994). Comparisons among groups were made with one-way ANOVA (Sokal and Rohlf 1995), which was followed by the Bonferroni post hoc test. Homogeneity of variances was tested by Bartlett and Levene tests (Sokal and Rohlf 1995). Linear relationships between tissue mass and enzyme activities across time were tested with an ANCOVA, using tissue mass as the continuous variable. In all cases, the ANCOVA showed no significant relationships between the studied variables across time. Statistical analyses were performed using Stata® release 7.0 (StataCorp LP 2001). ANCOVAs were performed using SPSS® (SPSS Inc. 1986). Differences were considered statistically significant when P < 0.05.

Results In total 44 adult male bats were used. Mean body mass was 7.5 g (range 7.2–8.0 g) and mean forearm length was 41.8 mm (range 41.5–43.1 mm). Testes were permanently inguinal, their recrudescence occurred in May, with a peak mass in August. Afterwards, testes underwent a profound involution and were totally regressed in November (Fig. 1a). Epididymal growth started in June, maximal size was reached in September, and then a gradual involution occurred, with the smallest size reached in February (Fig. 1a). The cauda epididymides were flattened in appearance in nonbreeding bats and had become swollen and bulbous in autumn when they became engorged with spermatozoa (LeónGalvan et al. 1999). The caput epididymides reached its growth peak in September, whereas the caudal region showed maximal mass in October (Fig. 1b). Involution of caput epididymides occurred also at least 1 month before the associated regression of the cauda (Fig. 1b). Figure 2 indicates the specific activities of GPx, CAT, and SOD in caput and cauda epididymides during the annual reproductive cycle of C. mexicanus. The changes in specific activity of SOD during the annual cycle of C. mexicanus were very similar in the caput and cauda epididymides (Fig. 2a). Specific activity of SOD increased from February to March, and remained more or less constant until July. Then a sharp decrease occurred that brought the activity to almost zero values from August to February. Although the differences be© 2005 NRC Canada

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Fig. 1. Changes in reproductive organs of adult male Corynorhinus mexicanus: (a) testicular and epididymal mass and (b) epididymal segment (caput and tail) mass. The epididymal segments were obtained after ligation of the epididymides at the deferent-cauda, caudacorpus, corpus-caput, and caput-testis junctions; see text for more details. For simplicity, we report only the mean + SE mass of one reproductive organ or epididymal region per bat, using all bats in the sample, and the values in parentheses indicate the number of bats. Different letters indicate statistically significant differences (P < 0.05) between values in the same trace (ANOVA plus Bonferroni post hoc test applied to the testis data).

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in July and then increased sharply in August, reaching its highest value, and decreased steadily throughout the rest of the year. However, the activity of GPx was important in the period between January and April. During the months of highest activity, August to October, the activity of GPx was significantly higher in caput than in cauda epididymis.

Discussion Transport of the spermatozoa through the epididymides is not a passive function. During this journey spermatozoa must actively interact with the epididymal environment to achieve epididymal maturation, a process that makes them fully capable of undergoing all the necessary processes (progressive motility, capacitation, hyperactivation, and acrosomal reaction) to accomplish the fertilization of the oocyte. Afterwards, spermatozoa must stay in viable and fertile conditions during the storage periods of a few days to © 2005 NRC Canada

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Fig. 2. Reactive oxygen species (ROS) scavenging enzymes in caput and cauda epididymides of C. mexicanus bats captured monthly during the annual reproductive cycle (see Fig. 1): (a) superoxide dismutase expressed as SOD unit, where one unit of activity is defined as the amount of enzyme that inhibits the rate of formazan dye formation by 50%; (b) catalase expressed as changes in k, the constant rate of the first-order reaction (Cohen et al. 1970); and (c) glutathion peroxidase expressed as GPX activity (nmol NADPH oxidized/min). Values are means + SE for the caput epididymal region and means – SE for the cauda epididymal region, and numbers in parentheses indicate the number of bats. In all cases, there were no significant relationships between the studied variables across time (ANCOVA; SODcaput: F[1,30] = 0.43, P = 0.519 and SODcauda: F[1,29] = 1.56, P = 0.221; kcaput: F[1,29] = 1.75, P = 0.215 and kcauda: F[1,29] = 0.78, P = 0.383; GPXcaput: F[1,28] = 1.19, P = 0.285 and GPXcauda: F[1,26] = 1.01, P = 0.323). Different letters indicate statistically significant differences (P < 0.05) between values in the same trace (ANOVA plus Bonferroni post hoc test). The asterisks indicate statistically significant differences (P < 0.05) between the values obtained from both epididymal regions (Student’s t test).

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several weeks, depending on the species, until ejaculation. In most mammals both epididymal functions depend on testicular function. However, in vespertilionid and rhinolophid bats, a considerable asynchrony may exist between functions of the testes and epididymides. It has been shown in several species of mammals, using the isolated cauda epididymal system in which both extremes of the cauda epididymides have been ligated (Lubicz-Nawrocki 1974), that the spermatozoa stored in the isolated cauda of intact non-orchidectomized animals degenerate within several weeks but do not decrease noticeably in numbers (Jones 2004). If bilateral orchidectomy is practised at the same time that the cauda epididymides are isolated, spermatozoa die rapidly and practically disappear (>90%) in a few days. This effect of bilateral castration could be prevented by the administration of testosterone (Jones 2004). As the levels of testosterone decline in seasonally breeding males, the number of spermatozoa in the epdidymides decrease and finally disappear (Millar 1972). Our observations in C. mexicanus indicate that testes are totally regressed at the time when the spermatozoon storage properties of the epididymides are completely functional. These observations allow us to propose that the epididymides in this species, and in all other vespertilionid and rhinolophid bats which show the same temporal asynchrony in the development and function of male reproductive organs (Krutzsch 2000; León-Galvan et al. 2005), go through a particular functional cycle: an initial testicular phase, probably dependent on testicular function, and a second post-testicular phase, in which epididymal functions are, apparently, independent of testicular function. In accordance with this proposal, we must mention that the pattern of antioxidant enzyme activities presented herewithin for C. mexicanus appears to be significantly different than the pattern proposed by Gu and Hecht (1996), which they suggest is unique for mammalian male gametes. In fact, two quite different patterns can be observed depending on the phase of the epididymal reproductive cycle of C. mexicanus. CAT activity is clearly present in both epididymal segment throughout the entire annual reproductive cycle, but it is particularly high during the post-testicular phase of epididymal function. SOD activity is present during the testicular phase of epididymal transport and maturation of spermatozoa, but is almost completely absent or inhibited, in both caput and cauda epididymides, during the post-testicular epididymal storage period. On the other hand, GPx activity is low during the testicular phase of epididymal spermatozoon maturation and high in both epididymal segments during the storage phase of the epididymal function. Published values for ROS-scavenging enzymes in the different regions of the epididymides are scarce; most authors use the entire epididymis for their determinations. In general, our results are similar to those published in the rat by Chitra et al. (2001) and by Sujatha et al. (2001) and lower, but in the range, with those published for GPX in the mouse (Vernet et al. 1999) and for CAT in the aging brown Norway rat (Zubkova and Robaire 2004). There is growing evidence that regulation of redox pathways through ROS generation and ROS-scavenging systems, in both intra- and extra-cellular environments of the spermatozoa, must be considered a central feature of epididymal

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maturation (Aitken and Vernet 1998). Recent research has clearly shown that a series of important structural and physiological events of the maturing spermatozoa are susceptible to the detrimental actions of ROS, including DNA integrity (Aitken et al. 1998; Aitken and Krausz 2001), competence to undergo capacitation and the acrosome reaction (de Lamirande et al. 1997a; Ecroyd et al. 2003; Ford 2004; Rivlin et al. 2004), and disruption of the capacity to engage in spermatozoon– oocyte fusion (Aitken et al. 1998). However, recent research has shown that the presence of controlled levels of ROS in the epididymal environment is required by maturing spermatozoa to accomplish functional competence. The increasing experimental evidence suggesting that low and controlled concentrations of ROS participate in signal transduction mechanisms (Bauskin et al. 1991; Fialkow et al. 1993) is, surely, of the greatest importance for our understanding of the complex process, or related processes that participate, in epididymal maturation and storage. At least two signal transduction cascades have been proposed to be regulated by ROS in the spermatozoa: a redox-regulated cAMP-mediated induction of tyrosine phosphorylation (Aitken et al. 1998) and the extra-cellular signal-regulated kinase cascade (ERK) (de Lamirande and Gagnon 2002). The presence and concentration of ROS in the epididymal environment during maturation and storage of spermatozoa may be due to several factors. Spermatozoa are not the only cells capable of efficiently generating ROS (Aitken et al. 1989; Aitken and West 1990; Plante et al. 1994); some nonphagocytic types of cells that are present in the spermatozoon epididymal environment may also produce ROS, including endothelial cells (Matsubara and Ziff 1986), fibroblasts (Meier et al. 1991), and vascular smooth muscle (Ushio-Fukai et al. 1996). In addition, the production of ROS in biological systems is not only the result of normal physiological processes, but also a consequence of cell activation (e.g., immunological responses or apoptosis). Excessive generation of ROS, particularly O2⋅ – and H2O2, as a result of leukocyte contamination within the semen has been associated with subfertility or even infertility in patients (Aitken et al. 1996; Griveau and Le Lannou 1997). Although our knowledge of the process of epididymal maturation in the case of spermatozoon-storing species of bats is almost nonexistent, it is possible that the initial aspects of this process may be similar to that observed in many other mammals. Many important tasks related to this process, such as nuclear condensation, mitochondrial stabilization, capacity for progressive motility, preparation for hyperactivation, and spermatozoon–oocyte fusion, appear to be under redox control (de Lamirande et al. 1997a; Leclerc et al. 1997; Lewis and Aitken 2001; Ford 2004). In C. mexicanus, all three ROS-related enzymes studied in this work are present in both cauda and caput epididymides during the testicular phase of spermatozoon maturation, but interestingly, SOD activity is absent or inhibited in both epididymal segments throughout the post-testicular phase of spermatozoon storage function. It is important to consider that the ratio of ROS generation/ ROS scavenging may have different effects on spermatozoa, which are not only dependent on the specific environments like the cauda and caput epididymides where they reside, but © 2005 NRC Canada

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also on the stage of the process, maturation–storage, that they have reached. Unfortunately, experimental results about this topic are very limited in all mammalian species, and practically nonexistent in spermatozoon-storing species of bats. The presence of important concentrations of ROS in the spermatozoa system during epididymal spermatozoon maturation is required to produce axonemal protein phosphorylation (de Lamirande and Gagnon 1992a, 1992b). Insufficient axonemal protein phosphorylation may result in abnormalities in spermatozoon motility that decrease the potential of spermatozoa to acquire hyperactivated motility when properly stimulated, and therefore are unable to successfully fertilize the oocyte (Griveau et al. 1995). Although the superoxide anion (O2⋅ – ) can be generated by actively metabolizing spermatozoa, which is probably dependent on the activity of a NADPH oxidase, this type of ROS is not particularly toxic because of their relatively short life span (1 ms) and because it dismutates spontaneously or enzymatically (SOD activity) to form H2O2. On the contrary, H2O2 has a higher oxidant potential, is relatively stable, and being uncharged can be freely diffusible. As a result of these properties, H2O2 has been recognized as the most active oxidizing species for spermatozoa (de Lamirande et al. 1997a). Our results, showing the presence of significant levels of SOD activity during the testicular phase of spermatozoon maturation in the epididymides, are in harmony with this requirement, particularly if we notice that high levels of SOD in the caput epididymides correspond, generally, with low levels of catalase in this epididymal region. Furthermore, generation of suitable amounts of H2O2 through the activity of SOD is also necessary during epididymal maturation to produce stabilization of the mitochondrial capsule by thiol oxidation (Godeas et al. 1997; Pfeifer et al. 2001; Roveri et al. 2001). It has been shown that reduced glutathione (GSH) is an important regulator of intracellular redox status (Barres et al. 1992; Watson 2002). Therefore, the conjugated enzyme system glutathione peroxidase/glutathione reductase must be very important in the regulation of the equilibrium between the oxidized and the reduced forms of glutathione during epididymal maturation and, particularly in vespertilionid and rhinolophid species of bats, during the long periods of spermatozoon epididymal storage. Activity of GPx depends on the presence of at least four isozymes (Faure et al. 1991; Zachara 1992): classical cellular GPx, plasma glutathione peroxidase, phospholipid–hydroperoxide glutathione peroxidase, and an epididymal-specific, androgen-regulated isozyme that is expressed only in the caput epididymides (Vernet et al. 1999). The intracellular level of GSH participates in several important ways in the normal spermatozoon maturation process. It has been shown that generation of O2⋅ – reduces the intracellular levels of GSH (Spencer et al. 1995). Reduced glutathione levels may be important in chromatin compaction and DNA stabilization. Under these conditions GPx isozymes, particularly PHGPx, would prefer to utilize protamine thiol groups as substrates instead of glutathione. However, decreased levels of glutathione can induce apoptosis in target cells either directly or indirectly (Spencer et al. 1995). This effect may be very important when spermatozoon stor-

Can. J. Zool. Vol. 83, 2005

age is as prolonged as it has been observed in vespertilionid and rhinolophid bats. In addition, the conservation of adequate levels of reduced glutathione might be important in ensuring the process of chromatin decondensation once the spermatozoon has penetrated the oocyte (Reyes et al. 1989). It has been shown that the presence of low levels of ROS produced a significant reduction in DNA fragmentation. Godeas et al. (1997) have suggested that the beneficial effects of low-level oxidative stress on DNA damage is mediated through the activity of phospholipid–glutathione peroxidase (PHGPx or glutathione peroxidase 4), which is the predominant isozyme of GPx found in mammalian spermatozoa for both H2O2 and lipid peroxide reduction (Imai and Nakagawa 2003). Mammalian spermatozoa PHGPx in addition to glutathione, and the usual electron acceptors, can use as substrate the thiol groups associated with the protamines of caput epididymal spermatozoa (Godeas et al. 1997). Thus, it is possible to propose that the presence of high activities of GPx in both epididymal regions, particularly during the post-testicular phase of storage of spermatozoa, participates in the protection of the spermatozoon nuclear integrity both stimulating the required process of spermatozoon chromatin compaction and at the same time protecting the spermatozoon genome from excess oxidative attack (Manicardi et al. 1998). Sustained pro-oxidant action of even low H2O2 concentrations may cause lipid peroxidation of the cell membranes of stored epididymal spermatozoa (Alvarez and Storey 1982, 1984, 1985). Damage to spermatozoon plasma membranes may produce important deficiencies in the susceptibility to capacitation and acrosomal reaction during the post-ejaculatory phase of spermatozoon function (Aitken et al. 1989; Aitken 1994). GPx and CAT are the only two enzymes that specifically and effectively destroy H2O2 (Cohen and Hochstein 1963). Our results show that during the post-testicular phase of sperm epididymal storage, while the generation of H2O2 by SOD is almost completely absent, high activities of CAT and GPx are present in the epididymal environment. Crichton et al. (1994) proposed that hypertonic conditions of the epididymal environment, decreasing spermatozoon metabolism, was the key promoter of epididymal storage. Although this hypothesis does not explain the occurrence of prolonged spermatozoon storage in the female reproductive tract of C. mexicanus (López-Wilchis 1989; León-Galván et al. 1999), as well as in many other bat species (Son et al. 1988), it is possible that hypertonic environments may participate in regulating the activity of ROS-producing enzymes, particularly SOD. Redox equilibrium of the microenvironments associated with the milieus by which mammalian spermatozoa must progress during their transit through the epididymides must be specifically and differentially controlled to ensure the adequate realization of a function that is so important for reproductive success. Our data on ROS-related enzyme activity stress the existence of a carefully regulated equilibrium between the assayed enzymes in the cauda and the caput epididymides that is dependent on the specific maturation/storage function. The real significance of the differential enzyme activities in the diverse segments of the epididymides must await further investigation. © 2005 NRC Canada

Arenas-Ríos et al.

Acknowledgements This work was supported partially by the Consejo Nacional de Ciencia y Tecnología (Beca crédito No. 169578 and convenio No. 400200-5-31743-N).

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