Biol Reprod
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tramer, F.
Right arrow Articles by Panfili, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tramer, F.
Right arrow Articles by Panfili, E.
Agricola
Right arrow Articles by Tramer, F.
Right arrow Articles by Panfili, E.
Biology of Reproduction 59, 753-758 (1998)
©Copyright 1998 Society for the Study of Reproduction, Inc.

Antioxidant Systems in Rat Epididymal Spermatozoa1

Federica Tramera, Francesca Roccoa, Fulvio Micalia, Gabriella Sandria, , and Enrico Panfili2,a

a Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Trieste, Italy


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian caput and cauda epididymidal spermatozoa exhibit diverse stages of maturation, and their plasma membrane shows diverse composition and stability levels, thus enabling these spermatozoa to undergo the acrosomal reaction after transit through the epididymis. As a result, the study of antiperoxidative mechanisms is quite relevant, since epididymal spermatozoa must be properly protected against agents such as reactive oxygen species, which can impair the complex maturation process. We considered activities of certain enzymes (glutathione peroxidase [GPx], phospholipid hydroperoxide glutathione peroxidase [PHGPx], glutathione reductase [GR], superoxide dismutase [SOD], and catalase [CAT]) and the vitamin E content in isolated rat caput and cauda epididymidal spermatozoa. The results indicate that caput epididymidal sperm have significantly greater PHGPx (3.5x), GPx (2.4x), and SOD (1.7x) activities, as well as a greater amount of vitamin E (3.8x). There were no detectable differences in the GR and CAT activities of caput and cauda epididymidal spermatozoa. The substantial drop in PHGPx activity during epididymal transit is discussed in relation to an additional function of this enzyme: the use of caput sperm protamines as a sulfhydryl substrate. In vitro peroxidation of the two sperm populations by the free radical generator (azo-initiator) 2,2'-azobis(2-amidinopropane) dihydrochloride revealed that only about 13% of the vitamin E content of the caput epididymidal spermatozoa was consumed, which contrasts with the greater consumption (about 70%) of the vitamin in cauda epididymidal spermatozoa. Selective inhibition of PHGPx, SOD, or CAT did not change this picture. The higher susceptibility of cauda epididymidal spermatozoa to radicals is discussed in relation to the diverse enzymatic activities, vitamin E content, and peroxidative response. These factors are correlated with the different stages of sperm cell maturation, which are characterized—from caput to cauda epididymidis—by progressive destabilization of the plasma and acrosomal membranes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Epididymal spermatozoa undergo a rather complex maturation process as they move from caput to cauda. Maturation is principally mediated by the extracellular environment, given their limited metabolic activity [1]. Epididymal sperm are transformed from nonmotile, nonfertile cells into potentially fertile cells that are capable, if stimulated, of undergoing the acrosomal reaction (see [1–3] for review). This transformation mainly involves chemical and physical alterations that take place in the lipid assemblage and composition of the plasma membrane and the outer acrosomal membrane [1, 3, 4–6]. These changes result in a membrane structure that is potentially ready to fuse ("fusogenic") but is still prevented from doing so. Epididymal spermatozoa are not at this point subjected to peroxidative damage caused by exogenous agents such as reactive oxygen substances (ROS) and/or neutrophils present in semen [7–9], in which there are, however, specific protective mechanisms. This condition does not exclude the need for a protective mechanism against (lipo)peroxidation, which may also occur in the epididymal lumen. Protective mechanisms are indeed present in the sperm cells [10–14] as well as in the epididymal fluid [15]. ROS production has been studied in semen and in epididymal spermatozoa [10, 16–19], in which the presence of O2-· and H2O2 is partially due to mitochondrial activity and, in the case of H2O2, to superoxide dismutase [10, 18, 19]. These molecules, together with exogenous ROS and pro-oxidants, may cause lipid peroxidation of sperm cell membranes, damage to the midpiece and axonemal structure, malfunctioning of capacitation and acrosomal reaction, and loss of motility, and may ultimately result in infertility. In any event, all the above phenomena must be evaluated differently in the two specific environments (epididymis and seminal plasma), because of the dissimilar equilibria between the accumulation of ROS and its depletion by scavenging mechanisms. Study of this topic has been more limited in epididymal sperm cells than in sperm cells in the ejaculate because the latter are also examined from a clinical point of view in the case of human specimens. Data available for epididymal spermatozoa demonstrate that they are equipped with enzymatic mechanisms that can dispose of H2O2 and other potentially dangerous ROS. Glutathione peroxidase (GPx; E.C. 1.11.1.9) [10, 12, 13], glutathione reductase (GR; E.C.1.6.4.2) [12], and superoxide dismutase (SOD; E.C. 1.15.1.1) [11, 12, 20, 21] have been found in epididymal spermatozoa, whereas the presence of catalase (CAT; E.C. 1.11.1.6) is still controversial [11, 14, 19, 22–24]. The presence of reduced and oxidized glutathione (GSH, GSSG) is also under debate [10, 12, 25–27], which thus raises a puzzling question concerning the substrate requirement for both GPx and GR. Another important enzyme in the field of ROS detoxification is the phospholipid hydroperoxide glutathione peroxidase (PHGPx; E.C. 1.11.1.19), which we recently monitored in epididymal sperm cells [28] after an extensive description of its activity in testis [29–31]. In the field of nonenzymatic activity capable of inhibiting peroxidative damage, vitamin E is of primary importance in hydrophobic domains. No information is available on the amount, localization, and role of this vitamin in mammalian spermatozoa, although there are ample references concerning its possible therapeutic use in mammalian infertility [32–34].

We present data on the activities of the above-mentioned enzymes in isolated rat caput and cauda epididymidal spermatozoa, on vitamin E content and distribution, and on vitamin consumption elicited under conditions of native or inhibited enzymatic activities by the free radical generator 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), which allows milder and more controlled peroxidative conditions based on the peroxyl radical-induced consumption of antioxidants. The oxidation of lipids (LH) in the membrane proceeds schematically by the following mechanism [35, 36]:chain initiation:

chain propagation:

The generation of lipid peroxyl radicals (LOO·) produces lipid hydroperoxides (LOOH) and carbon-centered lipid radicals (L·), eventually causing membrane damage.

The results indicate the existence of different enzymes and vitamin E equipment for caput epididymidal sperm as compared to cauda, with different behaviors regarding peroxidation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation and Fractionation of Epididymal Spermatozoa

All procedures were carried out at 0–4°C unless otherwise indicated. Milli-Ro and Milli-Q-grade water (Millipore Co., Bedford, MA) was used for all media. All chemicals were of the highest reagent grade and were obtained from Sigma Chemical Co. (St. Louis, MO).

Wistar adult rats (3-mo-old, Harlan-Nossan, Italy) were maintained on a 12L:12D cycle, fed a standard diet (Harlan-Nossan, Italy) and tap water ad libitum, and housed according to the guidelines of European Community laws. They were killed by a guillotine, and the epididymides were rapidly removed and placed in a medium containing 0.01 M Tris-Cl (pH 7.5), 0.12 M NaCl, 1 mM PMSF, and 0.1 mM desferrioxamine mesylate (desferal) (afterwards indicated as BSPD: buffer-saline-PMSF-desferal). Spermatozoa from whole epididymis or from separate caput and cauda portions were collected essentially as described in Seligman et al. [5]. The final sediment was suspended in a small amount of BSPD, and the spermatozoa were counted by using a hemocytometer with Neubauer ruling (400 squares/mm2). Further purification was performed by layering the suspended sperm cells (about 1 x 106 cells in 10 ml BSPD) on 13 ml 1.8 M sucrose in BSPD. The two layers were spun down (91 000 x g, swinging bucket rotor, 45 min), and the sperm pellet was washed in BSPD (about 3 x 106 cells/ml) and centrifuged (4500 x g, 15 min). The final sediment was suspended in a small amount of BSPD and counted.

Sperm tails and heads from epididymal spermatozoa were purified as described by San Augustin and Witman [37], after brief sonication.

Sperm plasma and acrosomal membranes were isolated from purified epididymal sperm according to the method of Ward et al. [38]. The final pellet was resuspended in a small volume of 50 mM Tris-Cl (pH 7.4) containing 1 mM EDTA and 0.1 mM desferal.

Assays

The activity of the following enzymes was measured spectrophotometrically in accordance with the cited references: PHGPx [39], GPx [40], GR [41], and SOD [42]. CAT was assayed with the Clark oxygen electrode according to the method of Del Rio et al. [43].

Aliquots of purified epididymal spermatozoa, resuspended and briefly sonicated in BSPD (about 70 x 106 cells/ml), were subjected to peroxidation (20 min, 37°C, under ample stirred air) in the presence of the free-radical generator (AAPH) in the range of concentrations indicated in the figures. Before treatment with AAPH, certain enzymes were inhibited by incubating the sperm suspension with the following compounds (15–30 min at room temperature): 2 mM NaN3 for inhibiting CAT, 20 mM KCN for SOD, and 2 mM bromosulfophthalein for PHGPx. One nanomole per 106 cells of filipin III (from Streptomyces filipinensis) was added to render the plasma membrane permeable [44].

The vitamin E assay was carried out on the treated sperm cell aliquots after lyophilization by repeated pentane extraction and HPLC monitoring (C18 column 0.46 x 15 cm: System Gold, Beckman Instruments Inc., Fullerton, CA; and spectrofluorometric detector RF-551: Shimadzu, Kyoto, Japan; Ex 286nm, Em 330nm), as already described [45].

GSH was assayed on aliquots of purified epididymal spermatozoa (about 70 x 106 cells/ml) by derivatization with N-(1-pyrenyl)maleimide (Sigma) and HPLC monitoring (C18 column 0.46 x 15 cm; spectrofluorometric detector RF-551, Ex 330nm, Em 380nm) according to the technique of Winters et al. [46].

A malondialdehyde (MDA) assay was performed on aliquots of purified epididymal sperm (about 40 x 106 cells/ml in the final assay volume) according to the method of Vatassery et al. [47].

The bicinchoninic acid method was used for protein evaluation [48].

Light Microscopy

The epididymal sperm suspension in BSPD was photographed with a contrast-phase Light Labolux 20 microscope (Ernst Leitz Wetzlar GmbH, Wetzlar, Germany).

Statistical Analysis

Results are reported as means ± SD of separate experiments (n = 4–12). Differences between caput and cauda epididymidal spermatozoa were analyzed using Student's t test for paired data; in all cases, p < 0.05 was selected as the minimum criterion for statistically significant differences.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Figure 1 shows the sperm population obtained from epididymis treated as indicated in Materials and Methods. No cells other than intact spermatozoa could be detected; in fact, blood cells contaminating the first sperm sediment from squeezed epididymis had been removed on the 1.8 M sucrose cushion used for purification.



View larger version (151K):
[in this window]
[in a new window]
  		FIG. 1. Purified rat epididymal spermatozoa. Bar = 30 µm.

Table 1 reports the specific activities of PHGPx, GPx, GR, CAT, and SOD on sperm cells isolated from caput and cauda epididymidis, expressed as activity over 106 cells. There was a significant statistical difference between caput and cauda epididymidal spermatozoa for PHGPx, GPx, and SOD: the activity of these enzymes was in fact higher in the caput. With regard to GR and CAT, no difference was noted between the two sperm types.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Enzyme activities in epididymal spermatozoa.

With regard to the presence of glutathione in epididymal spermatozoa, which will be discussed later, we found the amount of HPLC-detectable GSH to be about 35 pmol/106 cells, and no appreciable differences were encountered between caput and cauda epididymidal spermatozoa. The value was similar (or no greater than one order of magnitude for some specimens) to those reported by Ting-Kai Li [25] for rabbit, dog, boar, ram, goat, and human sperm cells.

Table 2 reports the mean values obtained for vitamin E content in isolated whole-epididymis spermatozoa, in their subcellular fractions (heads, tails, and plasma plus acrosomal membrane), and in separated caput and cauda epididymidal sperm. The vitamin was present, as expected, in the membranes of the sperm heads. Its presence in the tail could be ascribed to midpiece mitochondria, as demonstrated by our data already reported [45] regarding the vitamin E content of rat testis mitochondrial membranes. The difference in vitamin content between epididymal caput and cauda sperm was statistically significant.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Vitamin E content in epididymal spermatozoa.

Figure 2A shows the amount of MDA produced and of vitamin E consumed by isolated epididymis spermatozoa subjected to controlled peroxidation by the free-radical generator AAPH. A 10-mM concentration resulted in the consumption of about 50% of the vitamin E contained in the isolated sperm cells. A 50-mM concentration produced a level of peroxidation resulting in the total loss of vitamin E. Since these experiments were performed in the presence of desferal, no Fenton-like reactions had to be taken into account. If Fe2+ was used, or if desferal was omitted, a more marked vitamin E consumption was noted (data not shown).



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 2. Vitamin E and MDA content of AAPH-peroxidated A) rat epididymal spermatozoa or B) caput (circles) and cauda (squares) epididymidal spermatozoa. Vitamin E values are expressed as percentage of a nonperoxidated control.

Figure 2B shows the different behavior of epididymal caput and cauda spermatozoa with regard to MDA accumulation and vitamin E consumption. The graphs clearly indicate that the caput sperm population, despite the higher MDA production, retained much more vitamin E (up to 30 mM AAPH) than did cauda spermatozoa.

Figure 3 shows the mean values of vitamin E consumed in epididymal caput and cauda spermatozoa treated with 12 mM AAPH, under conditions of native enzymatic activity and separate inhibition (95–100%) of the SOD, PHGPx, and CAT enzymes. The histograms are expressed as percentage of vitamin E remaining after treatment, as compared to control spermatozoa incubated without AAPH and inhibitors. The data clearly indicate that, in addition to the higher vitamin E consumption already reported for peroxidated epididymal cauda spermatozoa (Fig. 2B), none of the enzymatic inhibitors used was capable of further diminishing the level of vitamin consumption, either in the caput population or in the cauda population.



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 3. Vitamin E content of caput (A) and cauda (B) epididymidal spermatozoa. Vitamin E values are expressed as percentage of a nonperoxidated, noninhibited control. AAPH: 12 mM; SODI, PHGPxI, and CATI indicate inhibition of the enzymes in the sample. Open bars: vitamin E content after enzyme inhibitions; black bars: vitamin E content after peroxidation (AAPH) or after peroxidation plus enzyme inhibition (SODI + AAPH, PHGPxI + AAPH, CATI + AAPH).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Mammalian spermatozoa exhibit differing characteristics as they pass from caput to cauda epididymidis, which lead to capability of the mature cell for the acrosome reaction when stimulated. Several such characteristics have been extensively described [1, 3, 49, 50], whereas information is scarce or contradictory on the enzymatic protection systems and the vitamin E content in epididymal spermatozoa. The latter is relevant to the claimed therapeutic benefit of vitamin E supplements for human sterility [33]. Our data on the enzymes SOD, GPx, and GR for whole epididymal sperm cells generally agree with those already reported [10–13, 20, 21, 51]. With exception of our own data [28], no information is available on PHGPx in sperm cells or on other enzymatic activities of epididymal caput and cauda spermatozoa. Moreover, the presence of catalase in sperm cells is still uncertain [11, 14, 19, 22–24] and may depend on contamination by blood cells. We detected catalase activity in purified epididymis sperm cells, and no differences were found between caput and cauda populations. Our data lie in the wide range of activity values reported for human [23], hamster [24], and rabbit [52] spermatozoa. PHGPx, GPx, and SOD activity was about 2- to 3-fold higher for epididymal caput sperm cells, which are still immature. The data on the presence of GR, GPx, and PHGPx raise the question of whether the GSH/GSSG coupling is present in mammalian sperm cells. Contradictory data are found on this subject. The data range from complete absence [12] to highly variable amounts in different specimens [25]. Furthermore, some authors maintain that the production of GSH in spermatogenetic cells is dependent upon Sertoli cells and is based on an interaction between different cell types [26, 27]. In theory, it would be difficult to explain the presence of GSH/GSSG-dependent enzymes in the complete absence of this type of substrate, although some other thiol-containing molecules may represent alternative substrates. The limited amount and sensitivity of the assay could explain why it is difficult to evaluate this molecule. Our data show that a small amount of GSH is indeed present in epididymal rat spermatozoa.

The significant difference in the specific activity of PHGPx found in epididymal caput spermatozoa as compared with cauda (Table 1) deserves additional discussion. We demonstrated [28] that PHGPx in rat sperm cells is able to draw upon a source of thiols other than GSH; i.e., the reduced protamines of the caput sperm. On the other hand, this enzyme is unable to catalyze the protamines of cauda sperm cells. When one also considers the greater amount of the enzyme in epididymal caput sperm cells, it could be suggestive of a key function of PHGPx in the oxidation of the protamine thiol groups, which is essential in the condensation of chromatin. These two phenomena are no longer present in the cauda [53]. To complete this picture, we found that about 60% of the PHGPx was bound to chromatin in isolated testis nuclei [31]. The presence and the different activity of PHGPx in epididymal spermatozoa can therefore provide new perspectives on disulfide bond formation during epididymal transit.

We have reported the vitamin E content in rat testis mitochondria (0.603 ± 0.04 nmol/mg proteins) [45]. The present data on epididymal spermatozoa and their subfractions (Table 2) indicate that the sperm tails contain a considerable amount of the vitamin, thus suggesting that it may be present in the mitochondria even after transformation into the midpiece sheath. The vitamin content in the isolated sperm heads is explained by its great amount in isolated plasma plus acrosomal membranes. Interesting in this regard is the fact that there is more of the vitamin in epididymal caput spermatozoa than in cauda spermatozoa, which indicates that the vitamin content of the sperm cells decreases as they mature. This fact and a similar situation for the specific activity of PHGPx (Table 1) may suggest a functional difference in the protection processes offered by PHGPx activity and vitamin E content native to the two different spermatozoa populations.

Other evidence indicates that caput and cauda epididymidal spermatozoa respond to peroxidative damage in different ways. A valid alternative to Fe2+ and ascorbate as pro-oxidants [54] is the free-radical initiator AAPH [35, 36]. The decrease of vitamin E content induced by AAPH is always more marked in cauda epididymidal sperm cells (Figs. 2B and 3), in which both the vitamin content and the PHGPx specific activity are lower than in caput cells. The differing MDA production in the two isolated sperm populations (Fig. 2B) is rather puzzling; in effect, the lower level of MDA formation in cauda epididymidal sperm cells, in which a higher content of unsaturated fatty acids has been demonstrated [55], is difficult to explain. Such reduced formation could suggest a greater stability (or inaccessibility) of the potentially peroxidizable species, despite the inferior protective potential (vitamin E, enzymes) inherent in cauda epididymidal spermatozoa. PHGPx inhibition does not additionally affect vitamin E consumption after peroxidation—either in caput or in cauda sperm cells. This result is similar to what we have already reported for mitochondrial membranes in rat testis [45]. The same behavior is also noted for both SOD and CAT inhibition (Fig. 3). The entire picture seems to indicate that the endogenous increase of ROS (O2-·, H2O2, and phospholipid hydroperoxides, induced by inhibition of SOD, CAT, and PHGPx, respectively) does not represent per se an additional factor that leads to vitamin E consumption or that there is still a capacity for sustaining damage by ROS—provided that no Fe ions are available in the sample medium.

Our results thus indicate that a stronger protection is built up in epididymal caput cells, which are still immature, and that this system weakens at the cauda level. This situation can be correlated with the changes that occur in the plasma membrane from caput to cauda, which are characterized by higher polyunsaturated fatty acid and cholesterol content, as well as higher asymmetry [1]. The sum total of these conditions renders the membranes more susceptible to alteration, provided that the "safety device," represented by cholesterol and membrane proteins, is removed. The resulting condition of the sperm may be defined as "potentially fusogenic," e.g., potentially capable of undergoing the acrosomal reaction. The lower protection capability inherent in cauda epididymal sperm cells, which is revealed by our data, is consistent with this picture.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Cristiana Godeas (Oncologic Reference Center, CRO, Aviano, Italy) for critical discussion and Dr. Daniel Gold (Instructor of English, University of Trieste) for his help with the text.


   FOOTNOTES
 
1 Financial support by MURST (Rome) and by University of Trieste. Back

2 Correspondence: Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Via Giorgieri 1, 34127 Trieste, Italy. FAX: 39 40 676 3691; panfili{at}bbcm.univ.trieste.it Back

Accepted: May 11, 1998.

Received: February 17, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Nolan JP, Hammerstedt RH. Regulation of membrane stability and the acrosome reaction in mammalian sperm. FASEB J 1997; 11:670–682.[Abstract]
  2. Williams RM, Graham JK, Hammerstedt RH. Determination of the capacity of ram epididymal and ejaculated sperm to undergo the acrosome reaction and penetrate ova. Biol Reprod 1991; 44:1080–1091.[Abstract]
  3. Seligman J, Kosower NS, Weissenberg R, Shalgi R. Thiol-disulfide status of human sperm proteins. J Reprod Fertil 1994; 101:435–443.[Abstract]
  4. Yanagimachi R. Mammalian fertilization. In Knobil E, Neill J, Ewing LL, Greenwald GS, Market C, Pfaff W (eds.), The Physiology of Reproduction. New York: Raven Press; 1988: 135–185.
  5. Seligman J, Shalgi K, Oschry Y, Kosower NS. Sperm analysis by flow cytometry using the fluorescent thiol labeling agent monobromobimane. Mol Reprod Dev 1991; 29:276–281.[CrossRef][Medline]
  6. Eddy EM. The spermatozoon. In: Knobil E, Neill J, Ewing LL, Greenwald GS, Market C, Pfaff W (eds.), The Physiology of Reproduction. New York: Raven Press; 1988: 27–68.
  7. Gagnon C, Iwasaki A, de Lamirande E, Kowalski N. Reactive oxygen species and human spermatozoa. Ann NY Acad Sci 1991; 637:436–444.[Medline]
  8. Sikka SC, Rajasekaran M, Hellstrom WJG. Role of oxidative stress and antioxidants in male infertility. J Androl 1995; 16:464–468.[Abstract/Free Full Text]
  9. Baker HWG, Brindle J, Irvine DS, Aitken RJ. Protective effect of antioxidants on the impairments of sperm motility by activated polymorphonuclear leukocytes. Fertil Steril 1996; 65:411–419.[Medline]
  10. Alvarez JG, Storey BT. Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation. Gamete Res 1989; 23:77–90.[CrossRef][Medline]
  11. Zini A, de Lamirande E, Gagnon C. Reactive oxygen species in semen of infertile patients: levels of superoxide dismutase- and catalase-like activities in seminal plasma and spermatozoa. Int J Androl 1993; 16:183–188.[Medline]
  12. Bauchè F, Fouchard MH, Jégou B. Antioxidant systems in rat testicular cells. FEBS Lett 1994; 349:392–396.[CrossRef][Medline]
  13. Pavlova SK, Kanchev LN, Alexandrov MT. Peroxidase activities in bull spermatozoa. Mol Reprod Dev 1994; 37:204–209.[CrossRef][Medline]
  14. Gu W, Hecht NB. Developmental expression of glutathione peroxidase, catalase and manganese superoxide dismutase mRNAs during spermatogenesis in the mouse. J Androl 1996; 17:256–262.[Abstract/Free Full Text]
  15. Hinton BT, Palladino MA, Rudolph D, Labus JC. The epididymis as protector of maturating spermatozoa. Reprod Fertil Dev 1995; 7:731–745.[CrossRef][Medline]
  16. Tosic J. Mechanism of hydrogen peroxide formation by spermatozoa and the role of amino acids in sperm motility. Nature 1947; 159:544–545.
  17. Tosic J, Walton A. Metabolism of spermatozoa. Formation of hydrogen peroxide by spermatozoa and its effect on motility and survival. Biochem J 1950; 47:199–206.
  18. Holland MK, Storey BT. Oxygen metabolism of mammalian spermatozoa. Generation of hydrogen peroxide by rabbit epididymal spermatozoa. Biochem J 1981; 198:273–280.[Medline]
  19. Bize I, Santander G, Cabello P, Driscoll D, Sharpe C. Hydrogen peroxide is involved in hamster sperm capacitation in vitro. Biol Reprod 1991; 44:398–403.[Abstract]
  20. Faraone Mennella MR, Jones R. Properties of spermatozoa superoxide dismutase and lack of involvement of superoxides in metal-ion-catalyzed lipid-peroxidation reactions in semen. Biochem J 1980; 191:289–297.[Medline]
  21. Alvarez JG, Touchstone JC, Blasco L, Storey BT. Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicity. J Androl 1987; 8:338–348.[Abstract/Free Full Text]
  22. Mann T. The Biochemistry of Semen and the Male Reproductive Tract. London: Methuen; 1964: 130–131.
  23. Jeulin C, Soufir JC, Weber P, Laval-Martin D, Calvayrac R. Catalase activity in human spermatozoa and seminal plasma. Gamete Res 1989; 24:185–196.[CrossRef][Medline]
  24. Oberley TD, Oberley LW, Slattery AF, Lauchner LJ, Elwell JH. Immunohistochemical localization of antioxidant enzymes in adult Syrian hamster tissues and during kidney development. Am J Physiol 1990; 137:199–214.
  25. Li T-K. The glutathione and thiol content of mammalian spermatozoa and seminal plasma. Biol Reprod 1975; 12:641–646.[Abstract]
  26. Den Boer PJ, Mackenbach P, Grootegoed JA. Glutathione metabolism in cultured Sertoli cells and spermatogenic cells from hamster. J Reprod Fertil 1989; 87:391–400.[Abstract]
  27. Li L, Seddon AP, Meister A, Risley MS. Spermatogenic cell-somatic cell interaction are required for maintenance of spermatogenic cell glutathione. Biol Reprod 1989; 40:317–331.[Abstract]
  28. Godeas C, Tramer F, Micali F, Soranzo MR, Sandri G, Panfili E. Distribution and possible novel role of phospholipid hydroperoxide glutathione peroxidase in rat epididymal spermatozoa. Biol Reprod 1997; 57:1502–1508.[Abstract]
  29. Roveri A, Casasco A, Maiorino M, Dalan P, Calligaro A, Ursini F. Phospholipid hydroperoxide glutathione peroxidase of rat testis: gonadotropin dependence and immunocytochemical identification. J Biol Chem 1992; 267:6142–6146.[Abstract/Free Full Text]
  30. Godeas C, Sandri G, Panfili E. Distribution of phospholipid hydroperoxide glutathione peroxidase in rat testis mitochondria. Biochim Biophys Acta 1994; 1191:147–150.[Medline]
  31. Godeas C, Tramer F, Micali F, Roveri A, Maiorino M, Nisii C, Sandri G, Panfili E. Phospholipid hydroperoxide glutathione peroxidase (PHGPx) in rat testis nuclei is bound to chromatin. Biochem Mol Med 1996; 59:118–124.[CrossRef][Medline]
  32. Wu ASH, Oldfield JE, Shull LR, Cheeke PR. Specific effect of selenium deficiency on rat sperm. Biol Reprod 1979; 20:793–798.[Abstract]
  33. Kessopoulou E, Powers HJ, Sharma KK, Pearson MJ, Russell JM, Cooke ID, Barrat CL. A double-blind randomized placebo cross-over controlled trial using the antioxidant vitamin E to treat reactive oxygen species associated male infertility. Fertil Steril 1995; 64:825–831.[Medline]
  34. Suleiman SA, Ali ME, Zaki ZM, el-Malik EM, Nasr MA. Lipid peroxidation and human sperm motility: protective role of vitamin E. J Androl 1996; 17:530–537.[Abstract/Free Full Text]
  35. Miki M, Tamai H, Mino M, Yamamoto Y, Niki E. Free-radical chain oxidation of rat red blood cells by molecular oxygen and its inhibition by {alpha}-tocopherol. Arch Biochem Biophys 1987; 258:373–380.[CrossRef][Medline]
  36. Ingold KU, Bowry VW, Stocker R, Walling C. Autoxidation of lipids and antioxidation by {alpha}-tocopherol and ubiquinol in homogeneous solution and in aqueous dispersions of lipids: unrecognized consequences of lipid particle size as exemplified by oxidation of human low density lipoprotein. Proc Natl Acad Sci USA 1993; 90:45–49.[Abstract/Free Full Text]
  37. San Augustin JT, Witman GB. Isolation of ram sperm flagella. Methods Cell Biol 1995; 47:31–36.[Medline]
  38. Ward CR, Storey BT, Kopf GS. Activation of a Gi protein in mouse sperm membranes by solubilized proteins of the zona pellucida, the egg's extracellular matrix. J Biol Chem 1992; 267:14061–14070.[Abstract/Free Full Text]
  39. Maiorino M, Gregolin C, Ursini F. Phospholipid hydroperoxide glutathione peroxidase. Methods Enzymol 1990; 186:448–457.[Medline]
  40. Flohè L, Günter WA. Assays of glutathione peroxidase. Methods Enzymol 1984; 105:114–121.[Medline]
  41. Goldberg DM, Spooner RJ. Glutathione reductase. In: Bergmayer HU, Bergmayer J, Graessl M (eds.), Methods in Enzyme Analysis vol III. Basel: Verlag Chemie; 1983: 258–265.
  42. Flohè L, Ötting F. Superoxide dismutase assays. Methods Enzymol 1984; 105:93–104.[Medline]
  43. Del Rio LA, Ortega MG, Lopez AL, Gorgè JL. A more sensitive modification of the catalase assay with the Clark oxygen electrode. Anal Biochem 1977; 80:409–415.[CrossRef][Medline]
  44. Breitbart H, Wehbie R, Lardy HA. Calcium transport in bovine sperm mitochondria: effect of substrates and phosphate. Biochim Biophys Acta 1990; 1026:57–63.[Medline]
  45. Godeas C, Tramer F, Sandri G, Panfili E. Rat testis mitochondrial phospholipid hydroperoxide glutathione peroxidase does not protect endogenous vitamin E against Fe2+-induced (lipo)peroxidation. Biochem Mol Med 1996; 58:221–226.[CrossRef][Medline]
  46. Winters RA, Zukowski J, Ercal N, Matthews RH, Spitz DR. Analysis of glutathione, glutathione disulfide, cysteine, homocysteine, and other biological thiols by high performance liquid chromatography following derivatization by N-(1-pyrenyl)maleimide. Anal Biochem 1995; 227:14–21.[CrossRef][Medline]
  47. Vatassery GT, Smith WE, Quach HT, Lai JCK. In vitro oxidation of vitamin E, vitamin C, thiols and cholesterol in rat brain mitochondria incubated with free radicals. Neurochem Int 1995; 26:527–535.[CrossRef][Medline]
  48. Smith PK, Krahn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Kleink DC. Measurement of protein using bicinchoninic acid. Anal Biochem 1985; 150:76–85.[CrossRef][Medline]
  49. Skudlarek MD, Tulsiani DRP, Nagdas SK, Orgebin-Crist MC. ß-D-galactosidase of rat spermatozoa: subcellular distribution, substrate specificity and molecular changes during epididymal maturation. Biol Reprod 1993; 49:204–213.[Abstract]
  50. Amann RP, Hammerstedt RH, Veeramachaneni DNR. The epididymis and sperm maturation: a perspective. Reprod Fertil Dev 1993; 5:361–381.[CrossRef][Medline]
  51. Alvarez JG, Storey BT. Lipid peroxidation and the reaction of superoxide and hydrogen peroxide in mouse spermatozoa. Biol Reprod 1984; 30:833–841.[Abstract]
  52. Stambaugh R, Smith M. A comparison of several extraction procedures for rabbit acrosomal enzymes. J Reprod Fertil 1973; 35:127–130.[Medline]
  53. Balhorn R, Corzett M, Mazrismas J, Watkins B. Identification of bull protamine disulfide. Biochemistry 1991; 30:175–181.[CrossRef][Medline]
  54. Jones R, Mann T. Damage to ram spermatozoa by peroxidation of endogenous phospholipids. J Reprod Fertil 1977; 50:261–268.[Abstract]
  55. Parks JE, Hammerstedt RH. Developmental changes occurring in the lipids of ram epididymal spermatozoa plasma membrane. Biol Reprod 1985; 32:653–656.[Abstract]


This article has been cited by other articles:


Home page
 J AndrolHome page
C. P. Weir and B. Robaire
Spermatozoa Have Decreased Antioxidant Enzymatic Capacity and Increased Reactive Oxygen Species Production During Aging in the Brown Norway Rat
J Androl, March 1, 2007; 28(2): 229 - 240.
[Abstract] [Full Text] [PDF]

Home page
 J AndrolHome page
S. Wang, G. Wang, B. E. Barton, T. F. Murphy, and H. F. S. Huang
Beneficial Effects of Vitamin E in Sperm Functions in the Rat After Spinal Cord Injury
J Androl, March 1, 2007; 28(2): 334 - 341.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
L. M. Wallock-Montelius, J. A. Villanueva, R. E. Chapin, A.J. Conley, H. P. Nguyen, B. N. Ames, and C. H. Halsted
Chronic Ethanol Perturbs Testicular Folate Metabolism and Dietary Folate Deficiency Reduces Sex Hormone Levels in the Yucatan Micropig
Biol Reprod, March 1, 2007; 76(3): 455 - 465.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
A Glover and S J Assinder
Acute exposure of adult male rats to dietary phytoestrogens reduces fecundity and alters epididymal steroid hormone receptor expression.
J. Endocrinol., June 1, 2006; 189(3): 565 - 573.
[Abstract] [Full Text] [PDF]

Home page
 J AndrolHome page
J. Seligman, G. L. Newton, R. C. Fahey, R. Shalgi, and N. S. Kosower
Nonprotein Thiols and Disulfides in Rat Epididymal Spermatozoa and Epididymal Fluid: Role of {gamma}-Glutamyl-Transpeptidase in Sperm Maturation
J Androl, September 1, 2005; 26(5): 629 - 637.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
E. V. Zubkova and B. Robaire
Effect of Glutathione Depletion on Antioxidant Enzymes in the Epididymis, Seminal Vesicles, and Liver and on Spermatozoa Motility in the Aging Brown Norway Rat
Biol Reprod, September 1, 2004; 71(3): 1002 - 1008.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. K. Rao, C. M. Wayne, and M. F. Wilkinson
Pem Homeobox Gene Regulatory Sequences That Direct Androgen-dependent Developmentally Regulated Gene Expression in Different Subregions of the Epididymis
J. Biol. Chem., December 6, 2002; 277(50): 48771 - 48778.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
H. Imai, K. Suzuki, K. Ishizaka, S. Ichinose, H. Oshima, I. Okayasu, K. Emoto, M. Umeda, and Y. Nakagawa
Failure of the Expression of Phospholipid Hydroperoxide Glutathione Peroxidase in the Spermatozoa of Human Infertile Males
Biol Reprod, February 1, 2001; 64(2): 674 - 683.
[Abstract] [Full Text]

Home page
 FASEB J.Home page
R. BRIGELIUS-FLOHÉ and M. G. TRABER
Vitamin E: function and metabolism
FASEB J, July 1, 1999; 13(10): 1145 - 1155.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tramer, F.
Right arrow Articles by Panfili, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tramer, F.
Right arrow Articles by Panfili, E.
Agricola
Right arrow Articles by Tramer, F.
Right arrow Articles by Panfili, E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS