Involvement of mitochondrial dysfunction in the adverse effect exerted by seminal plasma from men with spinal cord injury on sperm motility

Authors


Correspondence: Felice Francavilla, Andrology Unit, Department of Life, Health and Environment Sciences, University of L'Aquila, Via Vetoio, L'Aquila 67100, Italy. E-mail: francavi@cc.univaq.it

Summary

The aetiology of severe asthenozoospermia in men with spinal cord injury includes an adverse impact of seminal plasma (SP) on sperm motility. In this study we investigated the effect exerted by SP from men with SCI on donor sperm mitochondrial activity and its reflection on motility. Donor spermatozoa were exposed (1 h) to SP from 22 ejaculates of men with SCI. Only SP from samples exhibiting both a low fructose level and an inhibitory effect on mitochondrial membrane potential (ΔΨm), assessed at flow cytometry with JC-1, affected donor sperm motility when evaluated 1 h after co-incubation. This effect was reverted by washing from SP and sperm re-suspension in medium containing glucose, in spite of persistently depressed ΔΨm. In the same samples, sperm motility and vitality dramatically decreased when evaluated 6 h after washing and re-suspension in the glucose-containing medium. Seminal plasmas which induced a disruption of ΔΨm, also enhanced a mitochondrial ROS generation, as assessed by MitoSOX red. The enhanced mitochondrial ROS generation was associated with a late induction of sperm membrane lipid peroxidation, as assessed by BODIPY C11, when evaluated at 6 h, but not at 1 h, after washing from SP. Furthermore, activation of caspase-9 and caspase-3 accompanied the loss of ΔΨm. In conclusion, a double energetic blockage (glycolysis and mitochondrial respiration) can represent a metabolic determinant of the early adverse effect exerted by SP from men with SCI on sperm motility. Mitochondrial dysfunction-related oxidative/apoptotic mechanisms can account for later consequences on sperm motility/vitality.

Introduction

Spinal cord-injured men usually experience sexual-/reproductive-reduced health (Biering-Sørensen & Sønksen, 2001). Although effective treatments for erectile dysfunction are now available, the ability to procreate naturally is lost in the majority of males with spinal cord injury (SCI) because of ejaculatory failure, occurring in up to 90% of the cases. Methods of assisted ejaculation have been developed, including penile vibratory stimulation (PVS) and rectal electroejaculation; however, abnormal characteristics of semen specimens from men with SCI have been consistently reported, especially impaired sperm motility (Patki et al., 2008). This strongly hinders the effectiveness of the home vaginal deposition of semen obtained with PVS, thereby creating a need to undergo intracytoplasmic sperm injection (ICSI) in most cases.

Among many factors which have been suggested to play a role in SCI-related asthenozoospermia, including testicular hyperthermia, urinary tract infections and sperm stagnation in the seminal ducts owing to anejaculation (Biering-Sørensen & Sønksen, 2001; Patki et al., 2008), we focused on the reported adverse impact of seminal plasma (SP) from ejaculates of men with SCI on sperm motility (Brackett et al., 1996; Biering-Sørensen & Sønksen, 2001). A low concentration of fructose (the major glycolysable substrate in SP), often detected in ejaculates from spinal cord-injured men (Hirsch et al., 1991), has been pointed out as a metabolic factor in the adverse effect exerted by SP on sperm motility (Biering-Sørensen & Sønksen, 2001; Patki et al., 2008). This might be in accordance with some evidence suggesting the key role of adenosine triphosphate (ATP) generated by glycolysis, rather than by mitochondrial respiration, in maintaining sperm motility. Actually, whether glycolysis or oxidative phosphorylation should be considered as the major biochemical pathway supplying energy that supports motility in mammalian spermatozoa remains still controversial (Ford, 2006; Miki, 2007; Ruiz-Pesini et al., 2007). In favour of the role of glycolysis there is the experimental evidence that both in mouse (Mukai & Okuno, 2004) and human spermatozoa (Koppers et al., 2008; Barbonetti et al., 2010), flagellar glycolysis compensates for any lack of ATP production by mitochondria in maintaining sperm motility. However, mitochondrial gluconeogenesis, supported by pyruvate and lactate, may in turn maintain motility in the absence of extracellular glycolysable substrates (Mukai & Okuno, 2004; Barbonetti et al., 2010). In this scenario, a decrease in seminal fructose level would not explain the asthenozoospermia induced by SP from spinal cord-injured men, in the presence of normally functioning mitochondria. Intriguingly, a decrease in sperm mitochondrial activity has been reported in rat models of SCI (Nunez et al., 2004; Wang et al., 2007). Indeed, independently from the controversial role of mitochondria in supplying energy for sperm motility, there is some experimental evidence that sperm motility could be affected by mitochondrial dysfunction-related oxidative mechanisms (Koppers et al., 2008). Furthermore, in other cell types, the loss of mitochondrial membrane potential may be owing to the activation of the mitochondrial permeability transition pore with cytochrome c release and consequent caspase activation leading to apoptotic cell death (Hotchkiss et al., 2009).

With these assumptions, in this study we investigated the effect exerted by SP from men with SCI on donor sperm mitochondrial activity and its reflection on motility.

Materials and methods

Chemicals

The protease inhibitor cocktail and 55,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenimidazolyl carbocyanine iodide (JC-1) were purchased from Sigma-Aldrich S.r.l. (Milan, Italy). CaspGLOW Fluorescein Active Kits for caspase-9 and caspase-3 were obtained from BioVision (M-Medical S.r.l., Milan, Italy). MitoSOX red and BODIPY (581/591) C11 were purchased from Molecular Probes, Inc. (Life Technologies, Monza, MB, Italy).

Semen samples

Twelve patients, aged 32.5 ± 2.1 years, admitted to a rehabilitation programme for traumatic SCI at the Centre for Clinical Research San Raffaele of Sulmona and seeking the evaluation of their fertility potential, were included in the study. All patients had a documented history of neurologically stable cervical or thoracic SCI, with a mean time post-injury of 5.0 ± 2.3 years. All patients were requested to sign a written informed consent and the study was approved by the local ethics committee.

Antegrade semen specimens were collected by the standard method of PVS (Brackett, 1999), using the Ferti Care Personal vibrator (Multicept A/S, Albertslund, Denmark) with vibratory amplitude of 2.5 mm and frequency of 100 Hz. For all patients, but one, included in the study, this was the first ejaculation as SCI. On each specimen a semen analysis was performed according to the WHO criteria (World Health Organization, 2010) and a further ejaculate was retrieved from asthenozoospermic subjects with a mean interval of 15 ± 7 days between the specimen collections. Each semen sample was centrifuged at 400 × g for 10 min to obtain the SP fraction, which was filtered through a 0.2-μm Millipore filter (Millipore S.p.A., Milan, Italy). To ensure that the effects exerted by SP were not caused by residual bacteria, filtrated SP were cultured and negative results confirmed the absence of bacteria. After the addition of the protease inhibitor cocktail (1 : 100 v/v), SP was stored at −80 °C until use.

Ejaculates from 12 normozoospermic healthy donors were collected by masturbation following an abstinence period of 3–7 days. The donors were students or post-graduate students from the University of L'Aquila, who had no known prior male reproductive pathologies including varicocoele and infection. All samples were normozoospermic according to WHO criteria and did not show leukocytospermia. Each semen sample was centrifuged at 400 × g for 10 min to obtain the SP fraction, which was divided into aliquots and stored at −80 °C, after the addition of the protease inhibitor cocktail.

Measurement of fructose in seminal plasma

Fructose concentrations in SP recovered from ejaculates of subjects with SCI and donors were determined using an enzymatic UV method according to the instructions of the commercial kit manufacturer (AB Analitica, Padua, Italy). Briefly, the absorbance increase at 340 nm, induced by the enzymatic generation of nicotinamide adenine dinucleotide phosphate reduced (NADPH) from NADP and d-fructose, was measured with an UV/Visible spectrophotometer (Perkin-Elmer Lambda 25; Artisan Scientific Corporation, Champaign, IL, USA). Fructose concentration in each sample was stoichiometrically related to the NADPH generation and was calculated in micromoles per ejaculate, according to WHO (World Health Organization, 2010).

In vitro exposure of donor spermatozoa to seminal plasmas

Further ejaculates from the same donors were used to obtain motile sperm suspensions by swim-up procedure. Briefly, spermatozoa were washed twice (700 × g, 7 min) in Biggers, Whitten and Wittingham (BWW) medium. After the second centrifugation, supernatants were removed by aspiration, leaving 0.5 mL on the pellet and, after an incubation time of 30 min, supernatants, containing highly concentrated motile spermatozoa, were carefully aspirated and sperm concentration was properly adjusted. Motile sperm suspensions were divided into aliquots, centrifuged and re-suspended in thawed SP from ejaculates of spinal cord-injured men and from donors, in different setting and incubation times, according to experimental design described below. In each setting, the same fresh donor sperm suspension was exposed to a donor SP and to 1–3 SP from men with SCI.

Evaluation of sperm motility and vitality

Sperm motility was evaluated by Computer-Aided Semen Analysis (CASA) using ATS20 (JCD, Gauville, France). Ten microlitres of each SP-treated sperm suspension were placed into a pre-warmed (37 °C) Makler counting chamber (Sefi Medical Instruments, Haifa, Israel). At least 200 spermatozoa were evaluated for each sample. Setting parameters were as follows: analysis duration, 1 sec; minimum contrast, 80; minimum size, 3; low size gate, 0.7; high size gate, 2.6; low intensity gate, 0.34; light intensity gate, 1.40. Spermatozoa exhibiting an average pathway velocity >5 μm/sec were categorized by the software as motile spermatozoa.

Sperm vitality was evaluated under light microscope by the eosin technique, according to WHO (World Health Organization, 2010).

Flow cytometric evaluation of ΔΨm

The fluorescent lipophilic cationic dye JC-1 was used to evaluate the sperm ΔΨm, as previously described (Barbonetti et al., 2010). This probe possesses the ability to differentially label mitochondria with high and low ΔΨm, by forming multimeric aggregates or monomers, emitting orange-red light or green light, respectively, in the presence of high or low ΔΨm, when excited at 488 nm.

After 1-h incubation with SP from ejaculates of spinal cord-injured men or SP from healthy donors, donor sperm suspensions, each containing 5 × 106 spermatozoa, were diluted in 1 mL of phosphate-buffered saline (PBS) before staining with 0.5 μL of JC-1 stock solution (3 mm in dimethyl sulfoxide, DMSO). Samples were incubated at 37 °C in the dark for 60 min and then analysed using a flow cytometer (Beckman-Coulter Epics XL-4; Beckman Coulter, Inc., Fullerton, CA, USA) equipped with a 15 mW argon-ion laser for excitation. For each sample 10 000 events were recorded at a flow rate of 200–300 cells/sec. Compensation between FL1 and FL2 was carefully adjusted according to the manufacturer's instructions. Green fluorescence (480–530 nm) was measured in the FL-1 channel and orange-red fluorescence (580–630 nm) was measured in the FL-2 channel. The percentage of positive cells was evaluated on a 1 023 channel scale, using the flow cytometer System II Version 3.0 software (Beckman Coulter, Inc.).

Flow cytometric assessment of activated caspases

Activation of caspase-9 and caspase-3 was evaluated by using ApoptosisCaspGLOW Fluorescein Active Caspase-3 and -9 Staining Kits (BioVision). These assays use the caspase-9 or caspase-3 inhibitors, LEHD-FMK or DEVD-FMK, respectively, conjugated to fluorescein isothiocyanate (FITC), as fluorescent markers. These cell-permeable, non-toxic peptides covalently bind to activated caspases (Tzeng et al., 2005; Zaheen et al., 2009; Barbonetti et al., 2010). Briefly, donor sperm suspensions, each containing 0.3 × 106 spermatozoa, were exposed for 1 h to SP from ejaculates of spinal cord-injured men or SP from healthy donors. After dilution in 300 μL of PBS, spermatozoa were stained with FITC-LEHD-FMK or FITC-DEVD-FMK (1 μL) for 1 h at 37 °C in an atmosphere of 5% CO2. After three centrifugations (1 000 × g, 4 min), spermatozoa were re-suspended in 500 μL of PBS and analysed at flow cytometer.

Flow cytometric assessment of mitochondrial generation of ROS

Mitochondrial generation of ROS was evaluated using MitoSOX red (MSR), a lipid soluble cation that is selectively targeted to the mitochondrial matrix and emits red fluorescence in the presence of mitochondrial ROS generation (Koppers et al., 2008). For this assay, MSR stock solutions (5 mm in DMSO) were diluted in BWW and added to donor sperm suspensions (20 × 106/mL) to give a final concentration of 2 μm, and incubated for 15 min at 37 °C. After two centrifugations (600 × g for 5 min) in BWW, spermatozoa loaded with the MSR dye were exposed for 1 h to SP from ejaculates of spinal cord-injured men or SP from healthy donors and then analysed at flow cytometry.

Flow cytometric assessment of membrane lipid peroxidation

Lipid peroxidation was evaluated using the fluorophore BODIPY 581/591 C11. This probe is readily incorporated into biologic sperm membranes and responds to free radical attack with a spectral emission shift from red to green, which can be readily monitored and quantified by flow cytometry (Aitken et al., 2007; Barbonetti et al., 2011). Albumin-free BWW medium was used because it was found that bovine serum albumin (BSA) binds the lipophilic BODIPY C11. BODIPY C11 (5 μm) was added to donor sperm suspensions (10 × 106/mL), incubated for 30 min at 37 °C and washed twice (600 × g for 5 min) before the exposure to SP from ejaculates of spinal cord-injured men or SP from healthy donors.

Statistical analysis

Statistical analysis was performed using the sas statistical software (version 9.2; SAS Institute Inc., Cary, NC, USA). The normal distribution of values was assessed with Shapiro–Wilk normality test. When more than one SP from men with SCI were tested against one donor SP in different settings (unbalanced data), the results were subjected to two-way analysis of variance (general linear model procedure, PROC GLM) to separate variations because of donor spermatozoa in different settings from variations because of treatment (SP from men with SCI and donor SP). When one SP from men with SCI was tested against one donor SP in different settings, paired samples t-test was used. Correlations were analysed with the Spearman correlation test. Statistical significance was accepted when p ≤ 0.05. Data were expressed as mean ± SD.

Results

Semen analysis and seminal fructose concentrations

At the analysis of the first ejaculate, only 2 of the 12 subjects with SCI exhibited normal percentages of spermatozoa with progressive motility. Seminal fructose was in the normal range only in one of the two ejaculates with normal motility, whereas in all the other samples it was undetectable (seven cases) or dramatically reduced (four cases) (Fig. 1A).

Figure 1.

Progressive sperm motility and seminal fructose concentration in ejaculates of 12 spinal cord-injured men. In all subjects the first semen specimen (A) was collected by penile vibratory stimulation after an anejaculation period of 5.0 ± 2.3 years. In 10 asthenozoospermic subjects, a second ejaculate (B) was obtained with a mean interval of 15 ± 7 days between specimen collections. Horizontal lines show lower reference values for seminal fructose concentration (dotted line = 13 μmol/ejaculate) and percentage of progressive motility (solid line = 32%).

A second ejaculation was induced in all patients showing asthenozoospermia at the first semen analysis (= 10), with a mean interval of 15 ± 7 days between the specimen collections. In the second ejaculate, progressive motility was improved in seven specimens (up to the normal range), and in five of these cases an increase in fructose concentrations up to the normal range was also observed (Fig. 1B).

leukocytospermia, as evaluated with peroxidase staining test (World Health Organization, 2010), was revealed in 77.3% of samples (17/22).

In all the ejaculates from normozoospermic donors (= 12) seminal fructose was above the lower reference limit (World Health Organization, 2010), ranging from 16.7 to 161.2 μmol/ejaculate.

Effect of SP from men with SCI on donor sperm ΔΨm and its short- and long-term impact on sperm motility

To evaluate whether SP recovered from ejaculates of spinal cord-injured men could affect sperm ΔΨm and motility, motile sperm suspensions of normozoospermic donors were exposed for 1 h to SP of men with SCI or to SP from healthy donors. In 11 experimental settings, the effect of the 22 SP from men with SCI was compared with that of 11 donor SP.

As shown in Fig. 2A, sperm suspensions exposed to donor SP exhibited a high percentage of spermatozoa with high ΔΨm and with progressive motility, whereas, the same sperm suspensions exposed to SP from men with SCI exhibited a significantly lower percentage of spermatozoa with high ΔΨm and with progressive motility.

Figure 2.

(A) Mitochondrial membrane potential (ΔΨm) and progressive motility exhibited by 11 donor sperm suspensions after 1-h exposure to 11 seminal plasmas (SP) from donors and 22 SP from men with spinal cord injury (SCI); each donor sperm suspension was exposed to 1 SP of healthy donor and 2 SP from men with SCI; *p = 0.0004 (F = 24.44) vs. donor SP and **p = 0.006 (F = 12.44) vs. donor SP with PROC GLM. (B) Percentage of decrement in ΔΨm observed in each donor sperm suspension exposed to SP from men with SCI with respect to the same sperm suspension exposed to SP of healthy donor. (C) Decrement (%) in sperm motility observed in each donor sperm suspension exposed to SP from men with SCI with respect to the same sperm suspension exposed to SP of healthy donor.

As shown in Fig. 2B, ΔΨm was depressed by 18 SP from men with SCI, which produced variable percentages of decrement, ranging from 28.6 to 94%, in donor sperm ΔΨm, with respect to the same sperm suspensions exposed to SP of healthy donors; whereas 4 of the 22 SP from men with SCI did not affect donor sperm ΔΨm.

As shown in Fig. 2C, SP exhibiting both low fructose concentrations and inhibitory effect on sperm ΔΨm (13/22) greatly depressed donor sperm motility. On the contrary, sperm motility was not affected by SP (9/22) exhibiting normal fructose concentration and/or inability to affect ΔΨm.

In following settings, when donor sperm suspensions exposed to SP exhibiting both low fructose levels and ΔΨm-inhibiting effect were washed and re-suspended in glucose-containing medium for 1 h, they regained their motility but not their ΔΨm (Fig. 3). In the same donor sperm suspensions, the percentage of motile spermatozoa dramatically decreased with respect to controls when assessed at 6 h after washing and the loss of motility was associated to a decrease in the percentage of viable spermatozoa (Fig. 3).

Figure 3.

Short- and long-term impact of sperm mitochondrial inhibition induced by seminal plasmas (SP) from men with spinal cord injury (SCI) on donor sperm motility and viability. Six donor sperm suspensions were exposed for 1 h to 6 seminal plasmas (SP) from donors and 10 SP from men with SCI (each donor sperm suspension was exposed to 1 SP of healthy donor and 1–3 SP from men with SCI). After 1-h incubation, donor spermatozoa were re-suspended for up to 6 h in glucose-containing Biggers, Whitten and Wittingham (BWW) medium. Sperm mitochondrial membrane potential (ΔΨm) was evaluated at flow cytometry using JC-1, emitting red fluorescence in the presence of high ΔΨm. *p = 0.0008, F = 85.66 vs. donor SP; **p < 0.0001, F = 262.47 vs. donor SP; §p < 0.0001, F = 511.95 vs. donor SP; #p < 0.0001, F = 929.16 vs. donor SP; ##p < 0.0001, F = 1334.26 (PROC GLM).

Seminal plasma inhibiting ΔΨm also induced sperm mitochondrial ROS generation and caspase activation

Afterwards, we explored mechanisms potentially involved in the long-term negative impact of ΔΨm inhibition induced by SP from men with SCI on donor sperm motility and viability.

As dysfunctional mitochondria can represent intrinsic sources of free radicals in human spermatozoa (Koppers et al., 2008) and the loss of ΔΨm could reflect an early apoptotic stage, coinciding with caspase activation (Mancini et al., 1997; Salvioli et al., 1997; Wadia et al., 1998), in subsequent experiments, we checked the mitochondrial ROS generation, as well as the activation of caspase-9 (induced by mitochondrial apoptotic pathway) and caspase-3 (the downstream effector caspase) upon 1-h exposure of donor sperm suspensions to SP from spinal cord-injured men or SP from healthy donors, as control.

The incubation in ΔΨm-inhibiting SP from men with SCI stimulated a significant increase in the percentage of donor spermatozoa with ROS-generating mitochondria with respect to that observed in control SP (Fig. 4A).

Figure 4.

Effect of ΔΨm-inhibiting seminal plasma from spinal cord-injured men (SCI SP) and SP from healthy donors (donor SP) on (A) mitochondrial ROS generation evaluated with MitoSOX Red (MSR), (B) caspase-9 activation evaluated with FITC-LEHD-FMK and (C) caspase-3 activation evaluated with FITC-DEVD-FMK, on donor sperm suspensions. Top, Typical flow cytometric histograms of fluorescence; Bottom, percentages of fluoresceing spermatozoa. (A) Mean ± SD of seven experiments using seven donor sperm suspensions exposed to seven SCI SP and seven donor SP; *p = 0.005 vs. donor SP with paired samples t-test. (B) Mean ± SD of four experiments using four donor sperm suspensions exposed to 11 SCI SP and four donor SP; *p = 0.0098 (F = 10.64) vs. donor SP with PROC GLM. (C) Mean ± SD of four experiments using four donor sperm suspensions exposed to 11 SCI SP and four donor SP; *p = 0.027 (F = 7.3) vs. donor SP with PROC GLM.

The ΔΨm-inhibiting SP also produced a significant increase in percentage of spermatozoa with activated caspase-9 (Fig. 4B) and caspase-3 (Fig. 4C), with respect to control sperm suspensions incubated with donor SP.

On the contrary, SP with no effects on ΔΨm (n = 4) did not induce either mitochondrial ROS generation or caspase activation (data not shown).

Seminal plasma inhibiting ΔΨm induced sperm membrane lipid peroxidation

As mitochondrial ROS generation, triggered by ΔΨm disruptors, could be responsible for a late lipid peroxidation of sperm membrane (Koppers et al., 2008), we evaluated whether ΔΨm-inhibiting SP, which induced mitochondrial ROS generation (Fig. 4A), could also promote sperm lipid peroxidation.

Donor sperm suspensions were exposed for 1 h to SP from ejaculates of men with SCI or control SP. After two washes (600 × g for 5 min), samples were re-suspended for 1 and 6 h in standard BWW medium before evaluating membrane lipid peroxidation. By evaluating the percentages of spermatozoa emitting BODIPY C11 green fluorescence, sperm suspensions, which had been previously exposed to ΔΨm-inhibiting SP, exhibited a significant increase in lipid peroxidation at 6 h, but not at 1 h, after washing (Fig. 5). Membrane lipid peroxidation was not induced by SP exerting no effect on ΔΨm (data not shown).

Figure 5.

Effect of seminal plasma (SP) of men with spinal cord injury (SCI) on sperm membrane lipid peroxidation. BODIPY C11-loaded donor spermatozoa were exposed for 1 h to SCI SP with inhibitory effect on mitochondrial membrane potential (ΔΨm) or to control SP from healthy donors. Green BODIPY C11 fluorescence (indicating lipid peroxidation) was evaluated at flow cytometry 1 and 6 h after washing from SP and re-suspension in Biggers, Whitten and Wittingham (BWW) medium. Mean ± SD of four experiments using four donor sperm suspensions exposed to 11 SCI SP and four donor SP; *p = 0.006 (F = 14.69) vs. donor SP with PROC GLM.

Relationship between SP properties and semen features

As far as the relationship between SP properties and ejaculated spermatozoa from the same semen sample from men with SCI is concerned, SP from all asthenozoospermic samples (n = 13) exhibited both low fructose levels and ΔΨm-inhibiting effect on donor spermatozoa. Seminal plasmas from the remaining nine samples without asthenozoospermia exhibited only one of these features: ΔΨm-inhibiting effect (five samples), low fructose level (three samples) or neither (one sample). An inverse correlation was found between SP ΔΨm-inhibiting effect and motility (%) of ejaculated spermatozoa (r = −0.42, p = 0.048); SP ΔΨm-inhibiting effect was also correlated with semen leucocytes concentration (r = 0.54, p = 0.008).

Discussion

This study provides the evidence for an adverse effect exerted by SP from spinal cord-injured men on sperm mitochondrial activity and explores mechanisms by which mitochondrial dysfunction induced by seminal plasma factors may affect sperm motility.

According to literature data and clinical experience, spinal cord-injured men included in this study exhibited both a high prevalence of asthenozoospermia at the analysis of the first semen specimen, collected after an anejaculation period lasting as the occurrence of SCI, and a high prevalence of very low levels of fructose, the major glycolysable substrate in SP. The improvement of sperm motility observed at the analysis of the second semen specimen in 70% of patient, who were asthenozoospermic at the first evaluation, was associated, but not always, to an increase in fructose levels. To our knowledge, only in an old study, fructose levels were investigated after repeated penile vibrator stimulation, and they were higher than after the first stimulation (Siösteen et al., 1990). The reason of this poorly investigated finding remains to be elucidated.

The first original datum arising from this study is that only SP-exhibiting both extremely low fructose levels and a depolarizing effect on ΔΨm affected donor sperm motility, when evaluated soon after 1-h exposure. On the contrary, SP from ejaculates of men with SCI which exhibited normal fructose concentrations and/or inability to decrease sperm ΔΨm, thereby preserving the integrity of at least one metabolic pathway (glycolysis and/or mitochondrial respiration), did not affect donor sperm motility. Therefore, a double energetic blockage (glycolysis and mitochondrial respiration) may represent a metabolic determinant of the early adverse effect exerted by SP from men with SCI on sperm motility. A simple spermatozoa washing followed by sperm re-suspension in a glucose-containing medium completely abolished the inhibitory effect exerted by SP on donor sperm motility, in spite of persistently low ΔΨm. The observation that in the presence of glucose sperm motility was no longer affected by mitochondrial inhibition, suggests that ATP generated in loco by glycolysis fully supports flagellar movement. Accordingly, all SP exhibiting both low fructose levels and ΔΨm-inhibiting effect on donor spermatozoa were recovered from asthenozoospermic samples.

Another major datum arising from this study is that, although mitochondrial ATP production was not required for the maintenance of motility in the presence of glycolysable substrates, the disruption of ΔΨm was associated with a late loss of sperm motility. Seminal plasmas from ejaculates of men with SCI, which induced a disruption of ΔΨm, also enhanced a mitochondrial ROS generation and induced caspase activation. The enhanced mitochondrial ROS generation was associated with a late induction of lipid peroxidation, as assessed by BODIPY C11. This effect was produced only by SP from ejaculates of men with SCI, which enhanced a mitochondrial ROS generation and was observed only when evaluated at 6 h but not at 1 h after washing from SP. This suggests that peroxidative damage could only be induced once the production of ROS in the mitochondrial matrix had overwhelmed the intra-mitochondrial antioxidant defence enzymes (Koppers et al., 2008). Any possible peroxidative effect by contaminant bacteria was excluded as SP were filtered before co-incubation with donor sperm suspensions, filtrated SP were cultured and negative results confirmed the absence of bacteria.

The disruption of ΔΨm was also associated with the activation of caspase-9 and caspase-3, suggesting that SP factors could activate BAX and BAK proteins that cause loss of mitochondrial membrane permeability with subsequent release of cytochrome c. The cytochrome c release represents a pro-oxidative/pro-apoptotic event followed by the activation of caspase-9 and caspase-3 (Hotchkiss et al., 2009). Although definitive apoptosis in mature spermatozoa is still debated, the contribution of the activation of this cascade in the late loss of sperm motility/vitality is strongly suggested in the present model.

Although an inverse correlation was found between SP ΔΨm-inhibiting effect and motility (%) of ejaculated spermatozoa, ΔΨm-inhibiting effect was also exhibited by some SP from non-asthenozoospermic samples. As an explanation, mitochondrial dysfunction-related oxidative/apoptotic mechanisms may not be reflected on sperm motility evaluated soon after semen liquefaction, when seminal factors responsible for mitochondrial dysfunction are of prostate/seminal vesicles origin.

Seminal plasma factors in ejaculates of men with SCI which induce mitochondrial dysfunction have not been investigated in this study. High levels of inflammatory cytokines have been reported in seminal plasma from men with SCI (Basu et al., 2004; Cohen et al., 2004), related to a frequent leukocytospermia (Aird et al., 1999; Basu et al., 2002). In other cell types, tumour necrosis factor-α (TNFα), which triggers the death-receptor pathway through its transmembrane receptor TNFR1, by inducing the catalytic activity of caspase-8 (Hotchkiss et al., 2009; Parameswaran & Patial, 2010), can also cause mitochondrial dysfunction. The truncated form of the protein BID (tBID), produced by the caspase-8–mediated BID cleavage, interacts with BAX and BAK proteins and activates mitochondrial permeability transition pores for the cytochrome c release followed by the activation of caspase-9 and caspase-3 (Yin, 2000; Hotchkiss et al., 2009). Although no evidence has been produced for the presence of TNFR1 in mammalian mature spermatozoa, it has been reported that the exposure of human spermatozoa to TNF-α produced a loss of ΔΨm and phosphatidylserine externalization (Perdichizzi et al., 2007). A careful investigation of mediators with the potential to inhibit sperm mitochondrial activity in SP from men with SCI is in progress in our lab. In any case, a role for inflammatory mechanisms is suggested by the correlation between semen leukocytes concentration and SP ΔΨm-inhibiting effect, which was observed in this study.

In conclusion, a double energetic blockage of both glycolysis and mitochondrial respiration can account for the early adverse effect exerted by SP from spinal cord-injured men on sperm motility, thus reinforcing the hypothesis that glycolysis supports sperm motility, which, however, can be maintained by mitochondrial activity, when extracellular glycolysable substrates are lacking. However, dysfunctional mitochondria, whatever the cause may be, either intrinsic (Koppers et al., 2010) or extrinsic (Koppers et al., 2008; present data), could affect sperm motility by membrane lipid peroxidation induced by increased mitochondrial ROS generation and possibly by apoptotic mechanisms (Koppers et al., 2010). In this light, the reported correlation between ΔΨm (Paoli et al., 2011) or mitochondrial morphologic integrity (Pelliccione et al., 2011) and motility of ejaculated spermatozoa could be explained by mitochondrial dysfunction-related oxidative/apoptotic mechanisms rather than impaired mitochondrial ATP generation.

Acknowledgements

This work was supported by a grant from the Ministero dell'Università e Ricerca, PRIN 2009.

Conflicts of interest

None declared.

Authors' contribution

A.B. – conception and design, acquisition, analysis and interpretation of data, drafting the article, MRC Vassallo – acquisition, analysis and interpretation of data, A.D.R. – acquisition, analysis and interpretation of data, Y.L. – acquisition, analysis and interpretation of data, G.F. – critical revision for important intellectual content, L.G. – critical revision for important intellectual content, A.L. – critical revision for important intellectual content, S.N. – statistical analysis, S.F. – analysis and interpretation of data, critical revision for important intellectual content drafting the article, F.F. – conception and design, analysis and interpretation of data, critical revision for important intellectual content, final approval of the version to be published.

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