This web only provides the extract of this article. If you want to read the figures and tables, please reference the PDF full text on Blackwell Synergy. Thank you.

- Original Article -

Destabilization of acrosome and elastase influence mediate the release of secretory phospholipase A₂ from human spermatozoa

Jacqueline Lebig¹, Uta Reibetanz¹, Jürgen Arnhold¹, Hans-Jürgen Glander²

¹Institute for Medical Physics and Biophysics, Medical Faculty, University of Leipzig, Leipzig D-04107, Germany
²Department of Dermatology/Andrological Training Centre of the European Academy of Andrology, University of Leipzig, Leipzig D-04103 , Germany

Abstract

Aim: To determine the cellular distribution of secretory phospholipase A₂ (sPLA₂) in dependence on the acrosomal state and under the action of elastase released under inflammatory processes from leukocytes. Methods: Acrosome reaction of spermatozoa was triggered by calcimycin. Human leukocyte elastase was used to simulate inflammatory conditions. To visualize the distribution of sPLA₂ and to determine the acrosomal state, immunofluorescence techniques and lectin binding combined with confocal laser scanning fluorescence microscopy and flow cytometry were used.  Results: Although sPLA₂ was detected at the acrosome and tail regions in intact spermatozoa, it disappeared from the head region after triggering the acrosome reaction. This release of sPLA₂ was associated with enhanced binding of annexin V-fluoroscein isothiocyanate (FITC) to spermatozoa surfaces, intercalation of ethidium-homodimer I, and binding of FITC-labelled concanavalin A at the acrosomal region. Spermatozoa from healthy subjects treated with elastase were characterized by release of sPLA₂, disturbance of acrosome structure, and loss of vitality. Conclusion: The ability of spermatozoa to release secretory phospholipase A₂ is related to the acrosomal state. Premature destabilization of the acrosome and loss of sPLA₂ can occur during silent inflammations in the male genital tract. The distribution pattern of sPLA₂ in intact spermatozoa might be an additional parameter for evaluating sperm quality. (Asian J Androl 2008 Nov; 10: 829_836)

Keywords: acrosome reaction; elastase; human spermatozoa; inflammation; secretory phospholipase

Correspondence to: Dr Jacqueline Leßig, University of Leipzig, Medical Faculty, Institute for Medical Physics and Biophysics, Haertelstrasse 16-18, Leipzig D-04107, Germany.
Tel: +49-341-9715-737 Fax: +49-341-9715-709
E-mail: jacqueline.lessig@medizin.uni-leipzig.de
Received 2008-04-14 Accepted 2008-06-30

DOI: 10.1111/j.1745-7262.2008.00440.x

1 Introduction

The Ca²⁺-dependent, membrane-bound enzyme secretory phospholipase A₂ (sPLA₂, EC 3.1.1.4) is thought to play a key role in membrane structure alterations and fusion events during the acrosome reaction and spermatozoa-egg fusion [1]. There are two further main groups of phospholipases A₂, cytosolic PLA₂ and calcium-independent PLA₂, that differ considerably in occurrence, calcium binding and other properties. The 14-kDa sPLA₂ is calcium-dependent in the millimolar range, whereas cytoplasmatic PLA₂ is a significantly heavier protein and depends on Ca²⁺ in the micromolar range. sPLA₂ is known to possess a catalytic dyad (histidine_aspartic acid), contrary to the catalytic triad of cytosolic PLA₂ containing a serine-residue in the active site [2, 3].

sPLA₂ is mainly detected in the acrosomal region of resting spermatozoa [4]. When sPLA₂ is released during the acrosome reaction, it initiates the formation of lysophospholipids (LP) and free fatty acids, either in the plasma membrane [5] or in the seminal plasma, resulting in an altered plasma membrane bilayer structure and increased membrane fluidity [4, 6]. The generated LP are well known as fusiogenic molecules implicated in membrane fusion events [7]. For successful fertilization of oocytes, spermatozoa have to reach the site of fertilization, in order to perform capacitation, to undergo the acrosome reaction on the surface of the oocyte and, subsequently, to complete membrane fusion. The LP lysophospha-tidylcholine (LPC) may induce changes in the zona pellucida and in the oolemma, promoting spermatozoa-egg fusion. Based on these findings, it is suggested that sperm sPLA₂ and its product, LPC, may contribute to membrane fusion events in mammalian fertilization. Thus, sPLA₂ may be functionally relevant for the fertilizing capacity of spermatozoa. Therefore, the localization of sPLA₂ in the acrosome region can provide information about the functional quality of human spermatozoa. A disturbance of the sPLA₂ supply, such as premature or reduced release from the acrosome, may contribute to infertility because such spermatozoa are incapable of lysing egg envelopes. This situation is also imaginable as a result of genital tract inflammations, which may lead to an impaired acrosome reaction either by decreasing the inducibility of the acrosome reaction or by increasing the spontaneous acrosome reaction [8]. Therefore, we investigated the effect of elastase as one of the mediators of inflammation [9], as well as of the acrosome status, on sPLA₂ localization and on sPLA₂-release by confocal laser scanning fluorescence microscopy and fluorescence-activated cell sorting (FACS).

2 Materials and methods

2.1 Selection criteria of semen samples

Semen samples, which were within semen parameters for normal values according to the World Health Organisation (WHO) reference ranges [10], were obtained from healthy volunteers (donors) following a period of 3_5 days of sexual abstinence and after their written informed consent, in accordance with the ethical standard guidelines of the University of Leipzig (Leipzig, Germany). The approval for this study was obtained from the ethical committee of the University of Leipzig. Semen samples with a sperm concentration of more than 20 × 10⁶/mL, more than 15% morphologically normal cells and at least 50% progressive sperm motility were selected for the study. The lower limit for the percentage of spermatozoa with normal morphology was set at 15% according to the recommendations of the German Society of Andrology. The semen samples were collected by masturbation into sterile plastic Petri dishes. Semen samples with a positive mixed antiglobulin reaction test, i.e. >10% spermatozoa with adherent particles, were excluded. The computer-aided sperm motion analysis (CASA) was performed by Mika cell motion analysis (Version 2.0 for Windows NT 4.0, Mika Medical GmbH, Montreux, Switzerland). Aliquots of semen samples (5 µL) were placed into 10 µm deep disposable counting chambers (Stroemberg-Mika, Montreux, Switzerland) on a 36ºC microscope stage warmer. A minimum of 100 spermatozoa from at least four different fields were analyzed from each specimen [11].

The donors were free of clinical symptoms of genital inflammation such as urethritis, abnormal swelling of the epididymis, or painful prostate and epididymis or pelvic pain. No individual had been treated with antibiotics or anti-inflammatory drugs within 3 months before examination.

2.2 Semen preparation and processing

Spermatozoa were isolated with the density gradient medium Sil Select Plus (Santa Ana, CA, USA) with centrifugation at 400 × g for 20 min. The sediment was washed twice in human tubal fluid (HTF) at 400 × g for 8 min. The supernatants were discarded and the pellets were used for further experiments.

2.3 Acrosome reaction

To trigger the acrosome reaction, spermatozoa (15 × 10⁶ cells) were incubated in 0.14 mol/L NaCl, 4 mmol/L KCl, 10 mmol/L glucose, 4 mmol/L HEPES, 1 mmol/L CaCl₂, pH 7.4 at 37ºC for 60 min. The calcium_ionophore calcimycin A23187 (2 × 10^_5 mol/L, Sigma-Aldrich, Taufkirchen, Germany) was added and the samples were incubated at 37ºC for 60 min. The method varies from the standard method recommended by the WHO [10] in order to obtain a higher number of acrosome-reacted cells [12].

2.4 Incubation of human spermatozoa with elastase

To simulate inflammatory conditions in the human genital tract, human spermatozoa (15 × 10⁶ cells) were incubated with elastase from human polymorphonuclear leukocytes (PMN; 3 µg/mL; Calbiochem, Merck, Schwalbach, Germany) at 37ºC for 2 h.

2.5 Detection of sPLA₂ in human spermatozoa

Immunohistochemical techniques were used to visua-lize secretory phospholipase A₂ in human spermatozoa. Spermatozoa were incubated with the primary antibody mouse-anti-human-secretory PLA₂ mIgG1 (200 µg/mL, 1 : 100, Research Diagnostics, RDI, Flanders, NJ, USA) for 1 h. After washing (three times with 10 mmol/L phosphate-buffered saline [PBS], pH 7.4, 400 × g, 5 min), spermatozoa were incubated with the secondary antibody (goat-anti-mouse-CY3 conjugated, 1 : 100, Dianova, Hamburg, Germany) for 45 min followed by three washing steps (10 mmol/L PBS, pH 7.4, 400 × g, 5 min). Controls to specify specific/unspecific binding were performed by only using the secondary antibody. Pre-immune serum was also used as control followed by the incubation with the secondary antibody. Using intact spermatozoa, we could not observe any staining.

The distribution of sPLA₂ was detected by confocal laser scanning fluorescence microscopy (Leica DM IRBE, Leica, Wetzlar, Germany) using the software Leica Microsystems TCS SP2, and by flow cytometry (FACS) using a FACScan (Becton Dickinson, San Joes, CA, USA) and CellQuest software (Becton Dickinson). The flow cytometer device used an argon laser emitting at 488 nm. The following settings were acquired to obtain the data (10 000 events): forward scatter, voltage E00; side scatter, 288 V; fluorescence channel 1 for fluoroscein isothiocyanate (FITC)-fluorescence and channel 2 for Cy-3-fluorescence, 628 V. The influence of FITC in channel 2 was removed using a compensation of 25%. The primary sPLA₂ antibodies were validated by dot blot techniques and the mean fluorescence intensity for antibody binding were compared with the Cy-3 labelled second antibody conjugate controls using the CellQuest Software.

2.6 Detection of annexin V binding to phosphatidylserine (PS) of the spermatozoa

The binding of annexin V to PS epitopes was used to detect dead and apoptotic spermatozoa. Cells with externalized PS or deteriorated plasma membrane bind annexin V, whereas vital cells with intact membranes do not bind this protein. Spermatozoa samples were centrifuged (400 × g, 5 min) and resuspended in binding buffer (ApoAlert Annexin V-FITC apoptosis kit, Clontech Lab., Mountain View, CA, USA). After addition of 5 µL FITC-conjugated annexin V solution, samples were incubated in the dark at room temperature [13]. The samples were used for FACS analyses and fluorescence microscopy after washing and resuspending in PBS. A formalin-treated concanavalin A_FITC-marked sample (100 mg/mL; Biomeda, Foster City, CA, USA) was used as positive control.

2.7 Evaluation of the acrosomal state

The ability of concanavalin A-FITC to bind to glycoconjugates of the inner acrosomal membrane of spermatozoa [14] was used to determine the acrosomal state of the cells. Although spermatozoa with deteriorated plasma and acrosomal membranes have been reported to become fluorescently labelled over the anterior sperm head and equatorial regions, spermatozoa with intact membranes were impermeable to this lectin and did not produce fluorescence signal after exposure [14]. This feature has allowed the use of concanavalin A (Sigma-Aldrich, Taufkirchen, Germany; 100 mg/mL in PBS, pH 7.4, 30 min at room temperature, two washing steps, with distilled water) for the evaluation of acrosomal integrity in spermatozoa by fluorescence microscopy [14] as well as by flow cytometry [15]. Spermatozoa were not permeabilized at all.

2.8 Vitality assay with ethidium-homodimer I

Avital spermatozoa were labelled with ethidium-homodimer I [16]. Fifty µL of sperm suspension were mixed with 50 µL of a freshly prepared 2 mmol/L solution of ethidium-homodimer I (Sigma-Aldrich, Taufkirchen, Germany). After addition of 15 µL of the fixation solution (1 mmol/L Tris, 1.25 mol/L glutaraldehyde, pH 7.0), 10 µL of the suspension was investigated by fluorescence microscopy and also assessed by FACS.

3 Results

3.1 Effects of acrosome status and sperm viability on the localization of sPLA₂

To determine the relationship between the acrosome reaction and sPLA₂ release, we examined the localization of the enzyme in freshly isolated, non-acrosome-reacted and chemically acrosome-reacted, human spermatozoa from healthy donors by means of immunocytochemistry and confocal laser scanning fluorescence microscopy.

A freshly ejaculated spermatozoa population from healthy volunteers produced with our modified method of detection of acrosome state only a weak fluorescence (Figure 1A, E, G; fluorescence classes 10_500). Thirty-nine percent of this population was recognized as acrosome reacted or as destabilized acrosome (Figure 1A, E, G; fluorescence classes 500_10 000). Nearly half of these cells also contained sPLA₂ (47%, Figure 1C, F, G; fluorescence classes 10_10 000). The secretory PLA₂ was localized at the acrosome and tail regions in approximately 75% of the cells. Figure 2A shows a typical distribution pattern in red fluorescence (Cy-3 staining) by the secondary sPLA₂ antibody. In all other cells, sPLA₂ was found only at the tail region, not inside the acrosome, only in small patches (Figure 2D). The tail staining of spermatozoa seems to be specific since controls only using the secondary antibody did not show any significant fluorescence labelling. Viability at freshly isolated cells amounted to 75%, which was slightly decreasing during the immunohistochemical procedure. The negative control with only the Cy-3 conjugated secondary antibody generated an insignificant fluorescence signal between 0 and 10 (autofluorescence). The concomitant analysis of these cells with annexin V-FITC revealed that the sPLA₂-positive cells were annexin V-FITC negative, whereas annexin V-FITC was able to bind to cells that did not contain sPLA₂ in the acrosome region (data not shown). Concanavalin A shows slight staining for cells containing sPLA₂ and bright staining for cells with only small patches of the enzyme at the tail region (Figure 2B, E). The phase contrast pictures are added for better recognition.

The induction of the acrosome reaction in human spermatozoa by calcimycin induced the release of sPLA₂ from the acrosome. In acrosome-reacted spermatozoa, sPLA₂ was only detected at the tail region, partially at middle portion and in small patches at the plasma membrane in the head region of cells. Interestingly, the acrosome region was free of sPLA₂ (Figure 3A, B, C, D). Acrosome-reacted spermatozoa were also annexin-V positive, as revealed by co-staining with FITC-labelled annexin-V (Figure 3B) and concanavalin A-FITC at the acrosomal region, which reveals the availability of the inner acrosomal membrane (Figure 3C). This change during the course of the acrosome reaction could also be verified by quantitative detection with flow cytometry. With initiation of the acrosome reaction by calcimycin, the concanavalin A-FITC fluorescence increased dramatically (92%, Figure 1B, F, G), whereas the relative number of sPLA₂ -containing spermatozoa decreased to 23% (Figure 1D, F, G). These results suggest a strong association of sPLA₂ release with the acrosome reaction.

Ethidium-homodimer I verifies the vitality loss of cells subsequent to the acrosome reaction (Figure 3D, false colour staining, turquoise).

3.2 Effect of elastase on sPLA₂ release

A premature sPLA₂ loss seems to occur during a premature acrosome reaction, which takes place in the presence of elastase that is used as a model for inflammatory conditions in the male genital tract (Figure 4A, concanavalin A-FITC staining, immunofluorescence techniques for sPLA₂). Numerous polymorphonuclear leukocytes (PMN) are known to accumulate at inflammatory loci and to secrete elastase [16]. Incubation of human spermatozoa with PMN elastase (3 µg/mL) caused a premature triggering of the acrosome reaction. The ability of concanavalin A able to bind to the inner acrosomal membrane (Figure 4A, concanavalin A-FITC staining) indicated the loss of the outer acrosomal membrane. sPLA₂ could no longer be detected at the acrosome region (Figure 4A). Figure 4B shows the phase contrast picture. The quantitative assessment of the correlation of sPLA₂ release from the spermatozoal cells to the initiation of the acrosome reaction was confirmed by flow cytometry (Figure 1).

4 Discussion

In the present study, we demonstrated the concomitance of sPLA₂ release with chemically induced deterioration of the acrosome with a calcium ionophore, as well as under inflammatory conditions. The enzyme disappeared from the acrosome region in acrosome-reacted spermatozoa as well as in spermatozoa after treatment with elastase. On the other hand, some portion of sPLA₂ is localized at the tail region of spermatozoa. Apparently, this portion is only hardly changed during acrosome reaction. It remains unknown to us, during which stage of sperm maturation, sPLA₂ appears on these epitopes.

sPLA₂ is known to induce the formation of LP, which results in increased membrane fluidity [4, 6] and mediation of membrane fusion events [7]. Thus, sPLA₂, provided at the appropriate time might promote the fertilization process and, on the other hand, premature sPLA₂ release could contribute to impaired fertilizing capabilities.

Successful fertilization of the oocyte can only take place with spermatozoa that are capable of passing through capacitation, acrosome reaction, binding and penetration through the oocyte surrounding complex. This sequence of fertilization events may be impaired by silent genital tract inflammations, which are often not recognized because of the mostly asymptomatic course of the disease [17], but may be associated with reduced sperm motility and vitality as well as with sperm apoptosis [17, 18]. Increased levels of the leukocyte protease elastase as well as cytokines as TNFα, IL 1β or IL 8 are often found at areas of inflammation [16, 19, 20, 21]. Since we chose elastase as model for inflammatory processes, it is crucial that elevated elastase concentrations in infertile men are frequently associated with the number of peroxidase-positive cells and negatively correlated with sperm vitality, motility and intact DNA. Therefore, the influence of elastase on the acrosome and PLA₂ release was of practical interest. Besides the leukocytes, bacteria themselves, as well as bacterial endotoxins, may also have a damaging effect on spermatozoa by stimulating the production of reactive oxygen species (ROS) in leukocytes [22]. Oxidative stress increases the risk of lipid peroxidation and impairs the biological function of sperm membranes [23]. These two inflammatory mediators, bacteria and leukocytes, play a mutual role in deepening the harmful effect of oxidative stress on spermatozoa [22]. Inflammations in the male genital tract may affect two pathways critical for fertilization, intracellular apoptosis associated signals [24] and premature acrosome desta-bilization. Apoptotic spermatozoa are characterized by scramblase-activation [25], increased amounts of ROS and sPLA₂ activation [26]. A premature acrosome reaction or deterioration of the acrosome during silent inflammations results in a loss of lytic ability before contact with the egg, as well as impairment of fertilizing ability, since the capability of these spermatozoa to fuse with oocyte membranes is highly reduced. These cells are removed from the pool of fertilizing spermatozoa and the consequence may be reduced fertility. The concomitance of the acrosome reaction and sPLA₂ release suggests that the sPLA₂-distribution pattern can be regarded as one indicator for the ability of spermatozoa to proceed through a successful fertilization sequence.

The calcimycin-induced acrosome reaction in human spermatozoa led to sPLA₂ release from the sperm head, as evidenced by immunohistochemical techniques and visualization by confocal laser-scanning fluorescence microscopy and flow cytometry. This chemically induced situation can be used as model for the natural occurring incidences in order to determine the relationship between acrosome status and sPLA₂ effects [1, 27]. The monoclonal sPLA₂ antibody specifically detects sPLA₂ mostly in the acrosome and in the tail regions of intact spermatozoa, whereas acrosome-reacted populations are characterized by the presence of only small patches of sPLA₂ in the head region demonstrating the localization of the enzyme at the acrosomal membranes released by the membrane loss. Since permeabilization of cells was not performed it can be evidenced that sPLA₂ is localized at the sperm surface but not in the acrosomal content.

The determination of the acrosomal state of spermatozoa by chlorotetracycline, pisum sativum agglutinin or concanavalin A as well as the distribution of specified molecules with confocal laser scanning fluorescence microscopy provides the ability to scan objects in slices and therefore establishes the possibility of extremely accurately determining the localization of specific molecules or attributes [28, 29, 30]. The high precision of the performed visualization of the estrogen receptor β, the androgen receptor and binding of myeloperoxidase at spermatozoa [31, 32] induced us to use this method to estimate the location of sPLA₂ during different acrosomal states in the cells.

Our suggested conclusion is that the concomitance of disintegration of acrosome and sPLA₂ release represents a functional relationship and may allow sPLA₂-distribution patterns to be regarded as indicators for the ability of spermatozoa to fuse with the oocyte. The existence of invading polymorphonuclear leukocytes at inflammatory loci and specific PMN products, such as elastase, might prevent spermatozoa from supplying sPLA₂ for the fertilization process.

Acknowledgment

This work was supported by the German Research Foundation (Project No. GL 199/4-3). The technical assistance of Mrs Kersten is gratefully acknowledged.

References

1 Dominguez L, Yunes RMF, Fornes MW, Burgos M, Mayorga LS. Calcium and phospholipase A₂ are both required for the acrosome reaction mediated by g-proteins stimulation in human spermatozoa. Mol Reprod Dev 1999; 52: 297_302.

2 Gelb MH, Valentin E, Ghomashchi F, Lazdunski M, Lambeau G. Cloning and recombinant expression of a structurally novel human secreted phospholipase A₂. J Biol Chem 2000; 275: 39823_6.

3 Six DA, Dennis EA. The expanding superfamiliy of phospholipase A₂ enzymes: classification and characterization. Biochim Biophys Acta 2000; 1488: 1_19.

4 Roldan ER. Role of phospholipases during sperm acrosomal exocytosis. Front Biosci 1998; 3: d1109_19.

5 Lessig J, Glander H-J, Schiller J, Petkovic M, Paasch U, Arnhold J. Destabilization of the acrosome results in release of phospholipase A₂ from human spermatozoa and subsequent formation of lysophospholipids. Andrologia 2006; 38: 69_75.

6 Yuan YY, Chen WY, Shi QX, Mao LZ, Yu SQ, Fang X, et al. Zona pellucida induces activation of phospholipase A₂ during acrosomal exocytosis in guinea pig spermatozoa. Biol Reprod 2003; 68: 904_13.

7 Poole AR, Howell JI, Lucy JA. Lysolecithin and cell fusion. Nature 1970; 227: 810_4.

8 Henkel R, Ludwig M, Schuppe HC, Diemer T, Schill WB, Weidner W. Chronic pelvic pain syndrome/chronic prostatitis affect the acrosome reaction in human spermatozoa. World J Urol 2006; 24: 39_44.

9 Henkel R, Maass G, Jung A, Haidl G, Schill WB, Schuppe HC. Age-related changes in seminal polymorphonuclear elastase in men with asymptomatic inflammation of the genital tract. Asian J Androl 2007; 9: 299_304.

10 World Health Organization. Laboratory manual for the examination of human semen and sperm-cervical mucus interaction. UK: Cambridge University Press; 1999.

11 Paasch U, Glander HJ. Conventional and standardized computer-assisted sperm motion analysis (CASA) in 3731 semen samples. Adv Reprod 1998; 1: 57_67.

12 Kitiyanant Y, Chaisalee B, Pavasuthipaisit K. Evaluation of the acrosome reaction and viability in buffalo spermatozoa using two staining methods: the effects of heparin and calcium ionophore A23187. Int J Androl 2002; 25: 215_22.

13 Glander HJ, Schaller J. Binding of annexin V to plasma membranes of human spermatozoa: a rapid assay for detection of membrane changes after cryostorage. Mol Hum Reprod 1999; 5: 109_15.

14 Holden CA, Hyne RV, Sathananthan AH, Trounson AO. Assessment of the human sperm acrosome reaction using concanavalin A lectin. Mol Reprod Dev 1990; 25: 247_57.

15 Sahu BR, Mohapatra AD, Majumder A, Das PK, Ravindran B. A flow cytometry based method for studying embryogenesis and immune reactivity to embryogenic stages in filarial parasites. Filaria J 2005; 4: 11_24.

16 Markovits J, Roques BP, de Pecq JP. Ethidium dimer: a new reagent for the fluorimetric determination of nucleic acids. Anal Biochem 1979; 94: 259_64.

17 Micic S, Macura M, Lalic N, Dotlic R. Elastase as an indicator of silent genital tract infection in infertile men. Int J Androl 1989; 12: 423_9.

18 Zorn B, Sesek-Briski A, Osredkar J, Meden-Vrtovec H. Semen polymorphonuclear neutrophil leukocyte elastase as a diagnostic and prognostic marker of genital tract inflammation—a review. Clin Chem Lab Med 2003; 41: 2_12.

19 Eggert-Kruse W, Boit R, Rohr G, Aufenanger J, Hund M, Strowitzki T. Relationship of seminal plasma interleukin (IL)-8 and IL-6 with semen quality. Hum Reprod 2001; 16: 517_28.

20 Eggert-Kruse W, Kiefer I, Beck K, Demirakca T, Strowitzki T. Role for tumor necrosis factor alpha (TNF-a) and interleukin 1-beta (IL-1β) determination in seminal plasma during infertility investigation. Fertil Steril 2007; 87: 810_23.

21 Sanocka D, Jedrzejczak P, Szumala-Kakol A, Fraczek M, Kurpisz M. Male genital tract inflammation: The role of selected interleukins in regulation of pro-oxidant and anti-oxidant enzymatic substances in seminal plasma. J Androl 2003; 24: 448_55.

22 Fraczek M, Szumala-Kakol A, Jedrzejczak P, Kamieniczna M, Kurpisz M. Bacteria trigger oxygen radical release and sperm lipid peroxidation in in vitro model of semen inflammation. Fertil Steril 2007; 88: 1076_85.

23 Zalata AA, Hafez T, Comhaire F. Evaluation of the role of reactive oxygen species in male infertility. Hum Reprod 1995; 10: 1444_51.

24 Grunewald S, Paasch U, Said TM, Sharma RK, Glander HJ, Agarwal A. Caspase activation in human spermatozoa in response to physiological and pathological stimuli. Fertil Steril 2005; 83: 1106_12.

25 Diaz C, Schroit AJ. Role of translocases in the generation of phosphatidylserine asymmetry _ topical review. J Membrane Biol 1996; 151: 1_9.

26 Baldi E, Falsetti C, Krausz C, Gervasi G, Carloni V, Casano R, et al. Stimulation of platelet-activating factor synthesis by progesterone and A23187 in human spermatozoa. Biochem J 1993; 292: 209_16.

27 Garde J, Roldan ERS. Stimulation of Ca²⁺-dependent exocytosis of the sperm acrosome by cAMP acting downstream of phosholipase A₂. J Reprod Fertil 2000; 118: 57_68.

28 Kaul G, Sharma GS, Singh B, Gandhi KK. Capacitation and acrosome reaction in buffalo bull spermatozoa assessed by chlorotetracycline and pisum sativum agglutinin fluorescence assay. Theriogenology 2000; 55: 1457_68.

29 Merino Ruiz M, Luna Aranda HM, Morales Martinez A, Saldivar Rodriguez D, Vidal Gutierrez O. Acrosome reaction of human spermatozoid demonstrated with chlortetracycline. Ginecol Obstet Mex 2004; 72: 239_46.

30 Leβig J, Gey C, Schiller J, Süβ R, Paasch U, Grunewald S, et al. Hypochlorous acid-induced stress on human spermatozoa. A model for inflammation in the male genital tract. Chem Phys Lipids 2005;135: 201_11.

31 Solakidi S, Psarra A-MG, Nikolaropoulos S, Sekeris CE. Estrogen receptors α and β (ERα and ERβ) and androgen receptor (AR) in human sperm: localization of ERβ and AR in mitochondria of the midpiece. Hum Reprod 2005; 20: 3481_7.

32 Leβig J, Spalteholz H, Reibetanz U, Salavei P, Fischlechner M, Glander HJ, et al. Myeloperoxidase binds to non-vital spermatozoa on phosphatidylserine epitopes. Apoptosis 2007; 12: 1803_12.