Reprod Dom Anim 49, 657–664 (2014); doi: 10.1111/rda.12343 ISSN 0936–6768
Caspase Activation, Hydrogen Peroxide Production and Akt Dephosphorylation Occur During Stallion Sperm Senescence JM Gallardo Bola~nos1, C Balao da Silva1, P Martın Mun˜oz1, M Plaza Da´vila1, J Ezquerra2, IM Aparicio3, JA Tapia3, C Ortega Ferrusola1 and FJ Pen˜a1 1 Laboratory of Equine Reproduction and Equine Spermatology, Faculty of Veterinary Medicine, University of Extremadura; 2Faculty of Veterinary Medicine, Veterinary Teaching Hospital, University of Extremadura; 3Department of Physiology, Faculty of Veterinary Medicine, University of Extremadura, C aceres, Spain
Contents To investigate the mechanisms inducing sperm death after ejaculation, stallion ejaculates were incubated in BWW media during 6 h at 37°C. At the beginning of the incubation period and after 1, 2, 4 and 6 h sperm motility and kinematics (CASA), mitochondrial membrane potential and membrane permeability and integrity were evaluated (flow cytometry). Also, at the same time intervals, active caspase 3, hydrogen peroxide, superoxide anion (flow cytometry) and Akt phosphorylation (flow cytometry) were evaluated. Major decreases in sperm function occurred after 6 h of incubation, although after 1 h decrease in the percentages of motile and progressive motile sperm occurred. The decrease observed in sperm functionality after 6 h of incubation was accompanied by a significant increase in the production of hydrogen peroxide and the greatest increase in caspase 3 activity. Additionally, the percentage of phosphorylated Akt reached a minimum after 6 h of incubation. These results provide evidences that sperm death during in vitro incubation is largely an apoptotic phenomena, probably stimulated by endogenous production of hydrogen peroxide and the lack of prosurvival factors maintaining Akt in a phosphorylated status. Disclosing molecular mechanisms leading to sperm death may help to develop new strategies for stallion sperm conservation.
Introduction Most of the ejaculated sperm are committed to dye except the one that finally fertilizes an oocyte. This biological fact implies the existence of sperm subpopulations and the need of mechanisms to effectively and silently remove dead sperm from the female genitalia (Aitken et al. 2014). The study of the mechanisms involved in the regulation of sperm survival after ejaculation arises as an interesting area of research with implications for sperm biotechnology and male factor infertility. In humans, defective sperm function is a major cause of infertility (Agarwal et al. 2003), and recent research reveals that oxidative stress and a truncated form of apoptosis are mechanisms that may be responsible of sperm malfunction in humans (Aitken and Koppers 2011; Aitken et al. 2011, 2012b). Recent research in our laboratory has identified both factors in subfertile stallions and also in relation to sperm deterioration after biotechnological procedures such as refrigeration, freezing and sex sorting (Ortega Ferrusola et al. 2009b; Gallardo Bolanos et al. 2012; Balao da Silva et al. 2013). The existence of complete apoptosis in spermatozoa is still a controversial issue. On one side, the terminal nature of the male germinal cell and the fact that the spermatozoa are transcriptionally silent © 2014 Blackwell Verlag GmbH
cells will argue against a complete form of apoptosis in sperm. However, a growing body of evidence indicates that post-translational modification of proteins is a major part of sperm physiology (Gonzalez-Fernandez et al. 2009) and that ejaculated spermatozoa depict many characteristics of apoptotic cells. This is especially evident in subfertile individuals, and after biotechnological procedures in humans and animals. The theory of abortive apoptosis (Sakkas et al. 2003; Cayli et al. 2004) states that these ‘apoptotic spermatozoa’ represent cells ‘marked’ to die but that scape from the final steps of this process and appear in the ejaculate. However, some technological processes induce or accelerate the apparition of apoptotic markers in sperm (Said et al. 2010), creating an apparent contradiction between both theories. It is proposed that all the spermatozoa in the ejaculate are programed to die; however, different sperm subpopulations are in a more or less advanced stage in their way to death. Moreover, some circumstances, such oxidative stress during sperm malfunction or osmotic stresses imposed by biotechnologies, may accelerate this process. Hydrogen peroxide is reported to be a major inductor or apoptosis (Gautam et al. 2006; Kotwicka et al. 2008). Prosurvival pathways acting through the inhibition of an ‘apoptotic-like’ process may be present in the spermatozoa (Aitken et al. 2014) and may be modified after ejaculation; in addition, increased levels of hydrogen peroxide may occur as a result of the intrinsic sperm metabolism after ejaculation. The PI3 kinase-Akt pathway is associated with cell survival (Hers et al. 2011), and recent evidences suggest that also plays a role in sperm regulation (Aparicio et al. 2005; Pujianto et al. 2010). To identify changes in prosurvival mechanisms and their role in sperm senescence and death after ejaculation, firstly, we identified the presence of phosphorylated Akt in stallion spermatozoa; then, we monitored the dynamic of their phosphorylation status during incubation at 37°C. At the same time intervals, we monitored sperm motility, viability, caspase 3 activation, mitochondrial membrane potential, hydrogen peroxide and superoxide anion.
Material and Methods Reagents and media Ethidium homodimer, Mitrotracker deep Red, YO-PRO-1, Caspase 3 and 7 detection kit, Hoechst 33342, Hydroethidine and dichlorodihydrofluorescein diacetate were from Molecular Probes (Leiden, The
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JM Gallardo Bola~ nos, C Balao da Silva, P Martın Mu~ noz, M Plaza D avila, J Ezquerra, IM Aparicio, JA Tapia, C Ortega Ferrusola and FJ Pe~ na
Netherlands), antiphospho-Akt (pSer473) was purchased from Cell Signalling Technology (Danvers, MA, USA), and antiprolactin receptor was from abcam (Cambridge UK). Semen collection and processing Semen was obtained from 7 Pure Spanish horses (PRE) (three ejaculates each) that are individually housed at the Veterinary Teaching Hospital of the University of Extremadura, C aceres, Spain. Stallions were maintained according to institutional and European regulations. Ejaculates were collected on a regular basis (two collections/week) during the 2012 breeding season using a pre-warmed, lubricated Missouri model artificial vagina with an inline filter to eliminate the gel fraction. The semen samples were immediately transported to the laboratory for evaluation and processing. The ejaculates were extended 1 : 1 in INRA-96, centrifuged (600 9 g 9 10 min) and resuspended in Biggers-Whitten–Whittingham (BWW) medium supplemented with 1% PVA (Balao da Silva et al. 2013) to obtain a final concentration of 50 9 106 spermatozoa/ml. Sperm motility Sperm motility and kinematics were assessed using a CASA system (ISASâ Proiser, Valencia, Spain) (Pen˜a et al. 2005a; Nun˜ez-Martinez et al. 2007a; GonzalezFernandez et al. 2009). Semen was loaded in a 20 lm depth Leja chamber (Leja, Amsterdam, The Netherlands) and placed on a warmed (37°C) stage. The analysis was based on the examination of 60 consecutive digitalized images (60 Hz) obtained from each field using 910 negative phase contrast objective. At least three independent fields were recorded to ensure a minimum of 200 individual cells per sample analysed. Objects incorrectly identified as spermatozoa were excluded by observation using the playback function. With respect to the setting parameters for the program, spermatozoa with a VAP 15 lm/s were considered motile. Spermatozoa deviating 45 lm/s were designated rapid sperm. The absolute and recalculated kinematic parameters measured by CASA included the following: circular velocity (VCL) lm/s, measures the sequential progression along the true trajectory; linear velocity (VSL) lm/s, measures the straight trajectory of the spermatozoa per unit time; mean velocity (VAP) lm/s, measures the mean trajectory of the spermatozoa per unit of time. Flow cytometry Flow cytometric analyses were conducted using a MACSQUANT Analyzer 10 (Miltenyi Biotech) flow cytometer equipped with three lasers emitting at 405, 488 and 635 nm and 10 photomultiplier tubes (PTMs): V1 (excitation (Ex) 405 emission (Em) 450/50), V2 (Ex 405 filter 525/50), B1 (Ex 488 filter 525/50), B2 (Ex 488 filter 585/40), B3 (Ex 488 filter 655–730 (655LP+ split
730)), B4 (Ex 499 filter 750 LP), R1 (Ex 635 filter 655– 730 (655LP+ split 730)) and R2 (Ex 635 filter 750 LP). The system was controlled with the MACSQUANTIFY software. Sperm subpopulations were divided by quadrants, determined using unstained and single-stained controls, and the frequency of each subpopulation was quantified. Forward and sideways light scatter were recorded for a total of 50 000 or 100 000 events per sample. Non-sperm events were eliminated gating the sperm population after Hoechst 33342 staining. The instrument was calibrated daily using specific calibration beads provided by the manufacturer, and compensation overlap performed before each experiment using unstained and single-stained controls. Flow cytometric detection of phosphorylated Akt (Ser 473) in stallion spermatozoa Spermatozoa (1 9 106/ml) were washed with salineHepes medium and fixed in 2% paraformaldehyde in phosphate buffer saline (PBS) at room temperature (RT) for 15 min. After fixation, the cells were washed twice with PBS and once with PBS/1% BSA, permeabilized for 30 min using 0.1% saponin in PBS containing 1% BSA and incubated in the same buffer with 2 ll/ ml of phospho-Akt (Ser 473)–Alexa Fluor 488 conjugate (Cat number 4071, Cell signaling) for 30 min in the dark at 22°C. Samples were then washed in PBS, and the pellets resuspended in 500 ll of PBS and analysed using a MACSQUANTâ Flow cytometer (Miltenyi Biotech, Madrid, Spain) and a Bio-Rad MRC1024 confocal laser microscope with an X60 oil-immersion objective. Hoechst 33342 was added to restrict analysis to spermatozoa and to exclude debris. Flow cytometric assessment of subtle membrane changes, and viability The following stock solutions were prepared in DMSO: Yo-Pro-1 (25 lM) and ethidium homodimer-1 (1.167 mM). Hoechst 33342 (1.62 mM in water) was used to identify spermatozoa and eliminate debris from the analysis. One ml of a sperm suspension containing 5 9 106 spermatozoa/ml was stained with 1 ll of Yo-Pro-1 and 0.3 ll of Hoechst 33342. After thorough mixing, the sperm suspension was incubated at RT in the dark for 25 min. Then, 0.3 ll of ethidium homodimer was added, and spermatozoa incubated further 5 min before reading in the flow cytometer. This staining is modified after previous protocols (Pen˜a et al. 2005b; Nun˜ez-Martinez et al. 2007b; Ortega Ferrusola et al. 2009a) and distinguishes four sperm subpopulations. The first is the subpopulation positive for only Hoechst 33 342 and was considered to be alive and without any membrane alteration. Another subpopulation is the Yo-Pro-1 positive cells emitting green fluorescence as membranes become slightly permeable during the first steps of damage, enabling Yo-Pro-1 but not ethidium homodimer to cross the plasma membrane. Neither probe enters intact cells. Finally, two subpopulations of dead spermatozoa were easily detected. These were either apoptotic (spermatozoa stained both with Yo-Pro-1 and ethidium homodimer, © 2014 Blackwell Verlag GmbH
Stallion Sperm Senescence
emitting both green and red fluorescence) or necrotic spermatozoa (cells stained only with ethidium homodimer, emitting red fluorescence). Simultaneous flow cytometric detection of active caspases 3 and 7 and active mitochondria CellEventTM (Molecular Probes, Leiden, the Netherlands) caspase 3/7 green detection reagent is a fluorogenic substrate for activated caspases 3 and 7. The reagent consists of a four amino acid peptide (DEVD) conjugated to a nucleic acid-binding dye. This cellpermeant substrate is intrinsically non‑fluorescent because the DEVD peptide inhibits the ability of the dye to bind to DNA. After activation of caspase-3 and caspase-7 in apoptotic cells, the DEVD peptide is cleaved, enabling the dye to bind to DNA and produce a bright fluorogenic response with an absorption/emission maxima of ~502/530 nm. One important advantage of this assay is that no wash steps are required avoiding cell losses during washing. The stock solutions of CellEvent (2 mM in DMSO), ethidium homodimer (1.167 mM in DMSO), Mitotracker deep red (0.5 lM in DMSO) and Hoechst 33342 (1.62 mM in water) were prepared. Spermatozoa (5 9 106/ml) in 1 ml of PBS were stained with 1 ll of cell event, 0.3 ll of Hoechst 33342 and 0.3 ll of Mitotracker deep red and incubated in the dark at RT for 25 min. Next, 0.3 ll of ethidium homodimer was added to each sample. After incubation for 5 min, the samples were immediately run in the flow cytometer. As cryopreservation induces caspase activity in stallion sperm (Ortega-Ferrsula et al. 2008), cryopreserved samples were used as positive controls for caspase 3 and 7. Determination of anion superoxide (O2 ) and hydrogen peroxide (H2O2) production Stallion sperm was stained with 9 ll of Hydroethidine (HE, stock solution 40 lM) for detection of superoxide anion (O2 ) and 9 ll of dichlorodihydrofluorescein diacetate (DCFA, stock solution 2 mM) for detection of H202. Following protocols previously validated for stallion sperm (Macias-Garcia et al. 2012). To restrict the analysis to spermatozoa, 0.3 ll of Hoechst 33342 (stock solution 1.62 mM in water) was added. The samples were incubated for 30 min at 38°C before analysing 100 000 events in the flow cytometer. HE and DCFA were excited at 488 nm, and fluorescence recorded at 530 and 610 nm, respectively. Hoechst 33 342 was excited at 405 nm, and fluorescence recorded at 450 nm. Western blotting Stallion semen was centrifuged and washed twice with PBS. After washing, sperm cells were sonicated for 5 s at 4°C in 100 ll of lysis buffer consisting in 50 mM Tris/ HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 1 mM EGTA, 0.4 mM EDTA, a protease inhibitor cocktail (Complete, EDTA-free) and 0.2 mM Na3VO4. The homogenates were clarified by centrifugation at 10 000 9 g (15 min, 4°C), and the supernatant was used for analysis of protein concentration followed by dilution with 4 9 SDS sample buffer. Proteins © 2014 Blackwell Verlag GmbH
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(25 lg/well) from stallion sperm lysates were fractionated by SDS-PAGE using 4–20% polyacrylamide gradient gels and transferred to nitrocellulose membranes. After blocking, membranes were incubated overnight at 4°C with anti-pAkt Ser 473(1 : 1000) or antiprolactin receptor (1 : 1000). The following day, membranes were washed twice and incubated for 45 min at 25°C with anti-rabbit IgG -HRP conjugated secondary Ab. Membranes were then washed again, incubated with enhanced chemiluminescence detection reagents and finally exposed to Hyperfilm ECL films (Amersham, UK). Positive controls for pAkt Ser 473 were pancreatic acini lysates, and positive controls for prolactin receptor were rat ovary lysates. The intensity and molecular weight of appearing bands were quantified using the software SCION IMAGE for Windows, version 4.02 (Scion Corp., Frederick, MD, USA), normalized to b actin values. Immunocytochemistry Spermatozoa were washed and suspended in PBS, with an adjustment of the cell concentration to 1 9 106 cells/ ml. Fifteen microlitres of the sperm suspension was spread on poly-l lysine-coated slides and allowed to attach for 10 min. Cells were then fixed with 3% formaldehyde in PBS for 15 min and permeabilized with 0.2 Triton X-100 in PBS for 5 min. Slides were washed 3 times for 10 min each with PBS and incubated in PBS supplemented with 5% bovine serum albumin (BSA) (w/v) for 90 min to block non-specific sites. After blocking, slides were incubated overnight at 4°C with antiphospho-Akt Ser 473 (1 : 50) diluted in PBS containing 5% BSA (w/v). On the next day, samples were extensively washed with PBS and further incubated with an Alexa 488 conjugated goat anti-rabbit antibody for 45 min at room temperature. Finally, slides were washed with PBS and examined with a Bio-Rad MRC1024 confocal microscope with a 609 objective in oil immersion. Samples were excited at 488 nm with an argon laser, and emission recorded using a 515 nm long pass filter set. Samples without any primary antibody were assessed to confirm the absence of non-specific staining. Statistical analysis All experiments were repeated at least four times in independent samples, and the results analysed using ANOVA with the (IBM SPSS Statistics for Windows, Version 21.0; IBM Corp., Armonk, NY, USA) for Mac. Differences with a p < 0.05 were regarded as significant.
Results Changes in sperm motility and kinematics Sperm motility, both progressive and total, experienced major changes during the incubation period. A major decrease both in progressive and total motility occurred after 1 h of incubation (p < 0.01). After 6 h of incubation, a further decrease occurred (Fig. 1). Sperm velocities decreased after 1 h of incubation (p < 0.01); however, in contrast to progressive and total motility, no further decreases were detected along the rest of the incubation period (Fig. 2).
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JM Gallardo Bola~ nos, C Balao da Silva, P Martın Mu~ noz, M Plaza D avila, J Ezquerra, IM Aparicio, JA Tapia, C Ortega Ferrusola and FJ Pe~ na
(YoPro+/Eth+) spermatozoa (Fig. 3). YoPro+ sperm increased significantly after 2 and 6 h of incubation, and YoPro+/Eth+ sperm increased significantly only after 6 h of incubation at 37°C.
Changes in the percentage of membrane intactness Sperm membranes remained intact until the sixth hour of incubation, when the percentage of spermatozoa with intact membranes decreased significantly (p < 0.01). This decrease was accompanied by an increase in the percentage of early (YoPro+) and late apoptotic
Changes in caspases 3 and 7 activity Caspases 3 and 7 activity was normalized to values at the beginning of the incubation period. After 2 h of incubation, caspase activity doubled the values at the beginning of the incubation period (p < 0.01); however, the greatest increase was observed after 6 h of incubation, when caspases 3 and 7 activity increased to 600% of initial values (p < 0.001) (Fig. 4a).
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Changes in the percentage of active mitochondria During the first 4 h of incubation, mitochondrial membrane potential remained unchanged. However, after 6 h of incubation, the mitochondrial membrane potential decreased significantly (p < 0.01) (Fig. 4b).
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Akt is phosphorylated at Ser 473 in stallion spermatozoa, and its phosphorylation status changes during in vitro incubation Akt was present in stallion spermatozoa; flow cytometry, western blotting and immunocytochemistry confirmed its presence. Phosphorylated Akt was present in the post-acrosomal region and in the mid-piece, supporting its putative role inhibiting an apoptotic-like sperm death (Fig. 5). Akt dephosphorylated spontaneously during in vitro incubation (Fig. 6), but with an increase after 4 h of incubation and a further decrease after 6 h.
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Fig. 1. Changes in sperm motility (total motility MOT, and progressive motility PM) during in vitro incubation of stallion sperm at 37° C up to 6 h in BWW media. (n = 7 stallions) *p < 0.05, **p < 0.01. Results are given as means SD
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Fig. 2. Changes in sperm velocities during in vitro incubation of stallion sperm at 37°C in BWW media. VCL = circular velocity (lm/s), VSL = straight line velocity (lm/s), VAP = average path velocity (lm/s). *p < 0.05 (n = 7 stallions). Results are given as means SD
© 2014 Blackwell Verlag GmbH
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Fig. 3. Changes in sperm membrane permeability and integrity 6 h of incubation at 37°C in BWW media. Intact%, percentage of spermatozoa with completely intact membranes; YoPro+, percentage of spermatozoa with increased permeability in their membranes; YoPro+/Eth+, percentage of apoptotic spermatozoa; Eth+, percentage of dead spermatozoa. Results are given as means SD. *p < 0.05. n = 7 stallions
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Fig. 4. Changes in the percentage of increase of caspase activity normalized to initial values (a) and percentage of mitochondrial membrane potential (b) during in vitro incubation of stallion spermatozoa at 37°C in BWW media. (n = 7 stallions) *p < 0.05, **p < 0.01. Results are given as means SD. n = 7 stallions
Changes in superoxide anion and hydrogen peroxide production during in vitro incubation Stallion spermatozoa produced both superoxide anion and hydrogen peroxide thorough all the incubation © 2014 Blackwell Verlag GmbH
period; while significant changes in O2 were not detected, H2O2 increased significantly after 6 h of incubation (p < 0.01) (Fig. 7).
Discussion In the present study, we investigated changes associated with stallion sperm senescence during in vitro incubation in a defined media. Major changes occurred after 6 h of incubation and were accompanied with the expression of markers related to apoptotic cell death, such as caspase activation, oxidative stress, loss of mitochondrial membrane potential and dephosphorylation of Akt. Results obtained in this study indicate that during in vitro incubation the time-dependent loss of motility and viability of stallion sperm may be due to caspase 3 activation and endogenous overproduction of H202. This study supports with previous findings from our laboratory (Moran et al. 2008; Ortega Ferrusola et al. 2009b; Gallardo Bolanos et al. 2012) and others (Thomson et al. 2009; Aitken et al. 2012a) indicating that oxidative damage inducing an apoptotic cascade, probably sperm specific, is involved in sperm death during biotechnological procedures such as cooling and freezing and thawing. Our results also confirm previous findings in human sperm indicating that sperm survival after ejaculation is tightly regulated through a balance of pro survival factors, such as prolactin, that maintain sperm Akt in a phosphorylated state on one side and the production of ROS on the other (Pujianto et al. 2010). Noteworthy the prolactin receptor was also identified in stallion spermatozoa (Fig. 5d). Interestingly, changes in sperm quality followed the same temporal pattern as caspase activation and increases in hydrogen peroxide
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JM Gallardo Bola~ nos, C Balao da Silva, P Martın Mu~ noz, M Plaza D avila, J Ezquerra, IM Aparicio, JA Tapia, C Ortega Ferrusola and FJ Pe~ na
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Fig. 5. Subcellular localization of phospho-Akt in stallion spermatozoa. Ejaculated stallion spermatozoa were washed and stained using specific antiphosphoAkt antibodies and visualized using confocal laser microscopy, as described in the materials and methods. (a) Transmission image (b) fluorescence recorded using the 540/30 emission filter (c) merged images, n = 7 stallions (d) identification of the prolactin receptor in stallion sperm lysates
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pAkt Ser 473 Fig. 6. Changes in Akt phosphorylation (Ser473) during in vitro incubation of stallion spermatozoa at 37°C in BWW media up to six hours. Akt phosphorylation was determined flow cytometrically (a) and using western blotting (b), as described in material and methods. Quantification was performed after flow cytometry data. *p < 0.01. Events in the upper right quadrant represent spermatozoa depicting phosphorylated Akt; events in upper left quadrant represent spermatozoa (Hoechst 33342 positive)
production. This suggests a direct effect of caspase activation and oxidative stress on sperm senescence during in vitro incubation. Also this mechanism has been proposed as responsible of human sperm
senescence, suggesting that this is well conserved and responsible for sperm selection (as spermatozoa in a more advanced state of apoptotic death rapidly lose motility) and silent removal of redundant spermatozoa © 2014 Blackwell Verlag GmbH
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Fig. 7. Changes in the production of superoxide O2 (HE) and H2O2 (H2DFDA) during in vitro incubation of stallion spermatozoa at 37°C in BWW media up to 6 h. Results are given as means SD *p < 0.05 n = 7 stallions
from the female genitalia (Aitken et al. 2012a,b; Aitken et al. 2014). Motility decreased after 1 h of incubation and had a further decrease after 6 h; however, sperm velocities only decreased after 6 h of incubation. Caspase activation followed a similar temporal trend with initial increases after 2 h of incubation and a major increase after 6 h of incubation, as did hydrogen peroxide production that reached a maximum at 6 h, when significant decreases in the percentage of intact spermatozoa and in the mitochondrial membrane potential occurred. These results further support the idea that a sperm-specific apoptotic phenomenon plays a major role sperm death during in vitro incubation. Especially supporting this assumption, percentage of intact sperm and mitochondrial membrane potential reached the lowest values as the same moment as caspase activation reached the maximum increase. Akt was evaluated flow cytometrically allowing the identification of the phosphorylation status at an individual cell level. Phosphorylation at Ser 473 decreased initially but showed an increase after 4 h of
References Agarwal A, Saleh RA, Bedaiwy MA, 2003: Role of reactive oxygen species in the pathophysiology of human reproduction. Fertil Steril 79, 829–843. Aitken RJ, Koppers AJ, 2011: Apoptosis and DNA damage in human spermatozoa. Asian J Androl 13, 36–42. Aitken RJ, Findlay JK, Hutt KJ, Kerr JB, 2011: Apoptosis in the germ line. Reproduction 141, 139–150. Aitken RJ, De Iuliis GN, Gibb Z, Baker MA, 2012a: The Simmet lecture: new horizons on an old landscape–oxidative stress, DNA damage and apoptosis in the male germ line. Reprod Domest Anim 47 (Suppl 4), 7–14. Aitken RJ, Jones KT, Robertson SA, 2012b: Reactive oxygen species and sperm function–in sickness and in health. J Androl 33, 1096–1106. Aitken RJ, Lambourne S, Gibb Z, 2014: The John Hughes Memorial Lecture: aspects of sperm physiology oxidatie stress and
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incubation and then decreased again. These changes may be the result of oxidative stress originated due to the intrinsic metabolic activity of the spermatozoa; for this reason, we evaluated endogenous reactive oxygen species production, anion superoxide O2 and H202 over the time of incubation. While no significant changes occurred in O2 , H202 increased significantly after 6 h of incubation, suggesting that this ROS is related to the loss of sperm viability in view of the temporal relationship of both processes. In short, apoptotic mechanisms occur in stallion spermatozoa during in vitro senescence and at the same time that changes in hydrogen peroxide production and dephosphorylation of Akt. This findings suggest that sperm survival after ejaculation is regulated through the interaction of prosurvival factors yet to be identified in the equine species and pro death factors, apparently overproduction of H202 being a major one. Although descriptive, our findings may open new clues to study molecular mechanisms behind male factor infertility and new strategies for sperm conservation. Acknowledgements The authors received financial support for this study from Ministerio de Ciencia e Innovaci on-FEDER, Madrid, Spain, grants AGL201343211-R, AGL 2010-20758 (GAN), BFU2011-30261, Junta de Extremadura-FEDER (GR 10010 and PCE1002). JMGB holds a PhD grant from the Basque Government/Eusko Jaurlaritzak.
Conflict of interest None of the authors have any conflict of interest to declare.
Author contributions FP designed the paper, analysed the data and wrote the paper, CMC and JGB performed most of the experiments, JAT and IA performed the WB and all the rest of authors collaborated in the analysis of the data and approved the drafted manuscript.
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relationship? Reprod Biomed Online 16, 657–663. Macias-Garcia B, Gonzalez-Fernandez L, Gallardo-Bolanos JM, Pen˜a FJ, Johannisson A, Morrell JM, 2012: Androcoll-E large selects a subset of live stallion spermatozoa capable of producing ROS. Anim Reprod Sci 132, 74–82. Moran JM, Madejon L, Ortega Ferrusola C, Pen˜a FJ, 2008: Nitric oxide induces caspase activity in boar spermatozoa. Theriogenology 70, 91–96. Nun˜ez-Martinez I, Moran JM, Pen˜a FJ, 2007a: Identification of sperm morphometric subpopulations in the canine ejaculate: do they reflect different subpopulations in sperm chromatin integrity? Zygote 15, 257–266. Nun˜ez-Martinez I, Moran JM, Pen˜a FJ, 2007b: Sperm indexes obtained using computer-assisted morphometry provide a forecast of the freezability of canine sperm. Int J Androl 30, 182–189. Ortega Ferrusola C, Gonzalez Fernandez L, Macias Garcia B, Salazar-Sandoval C, Morillo Rodriguez A, Rodriguez Martinez H, Tapia JA, Pen˜a FJ, 2009a: Effect of cryopreservation on nitric oxide production by stallion spermatozoa. Biol Reprod 81, 1106–1111. Ortega Ferrusola C, Gonzalez Fernandez L, Morrell JM, Salazar Sandoval C, Macias
Garcia B, Rodriguez-Martinez H, Tapia JA, Pen˜a FJ, 2009b: Lipid peroxidation, assessed with BODIPY-C11, increases after cryopreservation of stallion spermatozoa, is stallion-dependent and is related to apoptotic-like changes. Reproduction 138, 55–63. Ortega-Ferrsula C, Sotillo-Galan Y, Varela-Fernandez E, Gallardo-Bolanos JM, Muriel A, Gonzalez-Fernandez L, Tapia JA, Pen˜a FJ, 2008: Detection of “apoptosis-like” changes during the cryopreservation process in equine sperm. J Androl 29, 213–221. Pen˜a FJ, Saravia F, Garcia-Herreros M, Nunez-martinez I, Tapia JA, Johannisson A, Wallgren M, Rodriguez-Martinez H, 2005a: Identification of sperm morphometric subpopulations in two different portions of the boar ejaculate and its relation to postthaw quality. J Androl 26, 716–723. Pen˜a FJ, Saravia F, Johannisson A, Walgren M, Rodriguez-Martinez H, 2005b: A new and simple method to evaluate early membrane changes in frozen-thawed boar spermatozoa. Int J Androl 28, 107–114. Pujianto DA, Curry BJ, Aitken RJ, 2010: Prolactin exerts a prosurvival effect on human spermatozoa via mechanisms that involve the stimulation of Akt phosphorylation and suppression of caspase acti-
vation and capacitation. Endocrinology 151, 1269–1279. Said TM, Gaglani A, Agarwal A, 2010: Implication of apoptosis in sperm cryoinjury. Reprod Biomed Online 21, 456–462. Sakkas D, Seli E, Bizzaro D, Tarozzi N, Manicardi GC, 2003: Abnormal spermatozoa in the ejaculate: abortive apoptosis and faulty nuclear remodelling during spermatogenesis. Reprod Biomed Online 7, 428–432. Thomson LK, Fleming SD, Aitken RJ, De Iuliis GN, Zieschang JA, Clark AM, 2009: Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis. Hum Reprod 24, 2061–2070.
Submitted: 1 Jan 2014; Accepted: 3 May 2014 Author’s address (for correspondence): FJ Pe~ na, Laboratory of Equine Spermatology and Reproduction, Faculty of Veterinary Medicine, Veterinary Teaching Hospital, University of Extremadura, Avd de la Universidad s/n 10003, Caceres, Spain. E-mail: [email protected]
© 2014 Blackwell Verlag GmbH
Caspase Activation, Hydrogen Peroxide Production and Akt Dephosphorylation Occur During Stallion Sperm Senescence JM Gallardo Bola~nos1, C Balao da Silva1, P Martın Mun˜oz1, M Plaza Da´vila1, J Ezquerra2, IM Aparicio3, JA Tapia3, C Ortega Ferrusola1 and FJ Pen˜a1 1 Laboratory of Equine Reproduction and Equine Spermatology, Faculty of Veterinary Medicine, University of Extremadura; 2Faculty of Veterinary Medicine, Veterinary Teaching Hospital, University of Extremadura; 3Department of Physiology, Faculty of Veterinary Medicine, University of Extremadura, C aceres, Spain
Contents To investigate the mechanisms inducing sperm death after ejaculation, stallion ejaculates were incubated in BWW media during 6 h at 37°C. At the beginning of the incubation period and after 1, 2, 4 and 6 h sperm motility and kinematics (CASA), mitochondrial membrane potential and membrane permeability and integrity were evaluated (flow cytometry). Also, at the same time intervals, active caspase 3, hydrogen peroxide, superoxide anion (flow cytometry) and Akt phosphorylation (flow cytometry) were evaluated. Major decreases in sperm function occurred after 6 h of incubation, although after 1 h decrease in the percentages of motile and progressive motile sperm occurred. The decrease observed in sperm functionality after 6 h of incubation was accompanied by a significant increase in the production of hydrogen peroxide and the greatest increase in caspase 3 activity. Additionally, the percentage of phosphorylated Akt reached a minimum after 6 h of incubation. These results provide evidences that sperm death during in vitro incubation is largely an apoptotic phenomena, probably stimulated by endogenous production of hydrogen peroxide and the lack of prosurvival factors maintaining Akt in a phosphorylated status. Disclosing molecular mechanisms leading to sperm death may help to develop new strategies for stallion sperm conservation.
Introduction Most of the ejaculated sperm are committed to dye except the one that finally fertilizes an oocyte. This biological fact implies the existence of sperm subpopulations and the need of mechanisms to effectively and silently remove dead sperm from the female genitalia (Aitken et al. 2014). The study of the mechanisms involved in the regulation of sperm survival after ejaculation arises as an interesting area of research with implications for sperm biotechnology and male factor infertility. In humans, defective sperm function is a major cause of infertility (Agarwal et al. 2003), and recent research reveals that oxidative stress and a truncated form of apoptosis are mechanisms that may be responsible of sperm malfunction in humans (Aitken and Koppers 2011; Aitken et al. 2011, 2012b). Recent research in our laboratory has identified both factors in subfertile stallions and also in relation to sperm deterioration after biotechnological procedures such as refrigeration, freezing and sex sorting (Ortega Ferrusola et al. 2009b; Gallardo Bolanos et al. 2012; Balao da Silva et al. 2013). The existence of complete apoptosis in spermatozoa is still a controversial issue. On one side, the terminal nature of the male germinal cell and the fact that the spermatozoa are transcriptionally silent © 2014 Blackwell Verlag GmbH
cells will argue against a complete form of apoptosis in sperm. However, a growing body of evidence indicates that post-translational modification of proteins is a major part of sperm physiology (Gonzalez-Fernandez et al. 2009) and that ejaculated spermatozoa depict many characteristics of apoptotic cells. This is especially evident in subfertile individuals, and after biotechnological procedures in humans and animals. The theory of abortive apoptosis (Sakkas et al. 2003; Cayli et al. 2004) states that these ‘apoptotic spermatozoa’ represent cells ‘marked’ to die but that scape from the final steps of this process and appear in the ejaculate. However, some technological processes induce or accelerate the apparition of apoptotic markers in sperm (Said et al. 2010), creating an apparent contradiction between both theories. It is proposed that all the spermatozoa in the ejaculate are programed to die; however, different sperm subpopulations are in a more or less advanced stage in their way to death. Moreover, some circumstances, such oxidative stress during sperm malfunction or osmotic stresses imposed by biotechnologies, may accelerate this process. Hydrogen peroxide is reported to be a major inductor or apoptosis (Gautam et al. 2006; Kotwicka et al. 2008). Prosurvival pathways acting through the inhibition of an ‘apoptotic-like’ process may be present in the spermatozoa (Aitken et al. 2014) and may be modified after ejaculation; in addition, increased levels of hydrogen peroxide may occur as a result of the intrinsic sperm metabolism after ejaculation. The PI3 kinase-Akt pathway is associated with cell survival (Hers et al. 2011), and recent evidences suggest that also plays a role in sperm regulation (Aparicio et al. 2005; Pujianto et al. 2010). To identify changes in prosurvival mechanisms and their role in sperm senescence and death after ejaculation, firstly, we identified the presence of phosphorylated Akt in stallion spermatozoa; then, we monitored the dynamic of their phosphorylation status during incubation at 37°C. At the same time intervals, we monitored sperm motility, viability, caspase 3 activation, mitochondrial membrane potential, hydrogen peroxide and superoxide anion.
Material and Methods Reagents and media Ethidium homodimer, Mitrotracker deep Red, YO-PRO-1, Caspase 3 and 7 detection kit, Hoechst 33342, Hydroethidine and dichlorodihydrofluorescein diacetate were from Molecular Probes (Leiden, The
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Netherlands), antiphospho-Akt (pSer473) was purchased from Cell Signalling Technology (Danvers, MA, USA), and antiprolactin receptor was from abcam (Cambridge UK). Semen collection and processing Semen was obtained from 7 Pure Spanish horses (PRE) (three ejaculates each) that are individually housed at the Veterinary Teaching Hospital of the University of Extremadura, C aceres, Spain. Stallions were maintained according to institutional and European regulations. Ejaculates were collected on a regular basis (two collections/week) during the 2012 breeding season using a pre-warmed, lubricated Missouri model artificial vagina with an inline filter to eliminate the gel fraction. The semen samples were immediately transported to the laboratory for evaluation and processing. The ejaculates were extended 1 : 1 in INRA-96, centrifuged (600 9 g 9 10 min) and resuspended in Biggers-Whitten–Whittingham (BWW) medium supplemented with 1% PVA (Balao da Silva et al. 2013) to obtain a final concentration of 50 9 106 spermatozoa/ml. Sperm motility Sperm motility and kinematics were assessed using a CASA system (ISASâ Proiser, Valencia, Spain) (Pen˜a et al. 2005a; Nun˜ez-Martinez et al. 2007a; GonzalezFernandez et al. 2009). Semen was loaded in a 20 lm depth Leja chamber (Leja, Amsterdam, The Netherlands) and placed on a warmed (37°C) stage. The analysis was based on the examination of 60 consecutive digitalized images (60 Hz) obtained from each field using 910 negative phase contrast objective. At least three independent fields were recorded to ensure a minimum of 200 individual cells per sample analysed. Objects incorrectly identified as spermatozoa were excluded by observation using the playback function. With respect to the setting parameters for the program, spermatozoa with a VAP 15 lm/s were considered motile. Spermatozoa deviating 45 lm/s were designated rapid sperm. The absolute and recalculated kinematic parameters measured by CASA included the following: circular velocity (VCL) lm/s, measures the sequential progression along the true trajectory; linear velocity (VSL) lm/s, measures the straight trajectory of the spermatozoa per unit time; mean velocity (VAP) lm/s, measures the mean trajectory of the spermatozoa per unit of time. Flow cytometry Flow cytometric analyses were conducted using a MACSQUANT Analyzer 10 (Miltenyi Biotech) flow cytometer equipped with three lasers emitting at 405, 488 and 635 nm and 10 photomultiplier tubes (PTMs): V1 (excitation (Ex) 405 emission (Em) 450/50), V2 (Ex 405 filter 525/50), B1 (Ex 488 filter 525/50), B2 (Ex 488 filter 585/40), B3 (Ex 488 filter 655–730 (655LP+ split
730)), B4 (Ex 499 filter 750 LP), R1 (Ex 635 filter 655– 730 (655LP+ split 730)) and R2 (Ex 635 filter 750 LP). The system was controlled with the MACSQUANTIFY software. Sperm subpopulations were divided by quadrants, determined using unstained and single-stained controls, and the frequency of each subpopulation was quantified. Forward and sideways light scatter were recorded for a total of 50 000 or 100 000 events per sample. Non-sperm events were eliminated gating the sperm population after Hoechst 33342 staining. The instrument was calibrated daily using specific calibration beads provided by the manufacturer, and compensation overlap performed before each experiment using unstained and single-stained controls. Flow cytometric detection of phosphorylated Akt (Ser 473) in stallion spermatozoa Spermatozoa (1 9 106/ml) were washed with salineHepes medium and fixed in 2% paraformaldehyde in phosphate buffer saline (PBS) at room temperature (RT) for 15 min. After fixation, the cells were washed twice with PBS and once with PBS/1% BSA, permeabilized for 30 min using 0.1% saponin in PBS containing 1% BSA and incubated in the same buffer with 2 ll/ ml of phospho-Akt (Ser 473)–Alexa Fluor 488 conjugate (Cat number 4071, Cell signaling) for 30 min in the dark at 22°C. Samples were then washed in PBS, and the pellets resuspended in 500 ll of PBS and analysed using a MACSQUANTâ Flow cytometer (Miltenyi Biotech, Madrid, Spain) and a Bio-Rad MRC1024 confocal laser microscope with an X60 oil-immersion objective. Hoechst 33342 was added to restrict analysis to spermatozoa and to exclude debris. Flow cytometric assessment of subtle membrane changes, and viability The following stock solutions were prepared in DMSO: Yo-Pro-1 (25 lM) and ethidium homodimer-1 (1.167 mM). Hoechst 33342 (1.62 mM in water) was used to identify spermatozoa and eliminate debris from the analysis. One ml of a sperm suspension containing 5 9 106 spermatozoa/ml was stained with 1 ll of Yo-Pro-1 and 0.3 ll of Hoechst 33342. After thorough mixing, the sperm suspension was incubated at RT in the dark for 25 min. Then, 0.3 ll of ethidium homodimer was added, and spermatozoa incubated further 5 min before reading in the flow cytometer. This staining is modified after previous protocols (Pen˜a et al. 2005b; Nun˜ez-Martinez et al. 2007b; Ortega Ferrusola et al. 2009a) and distinguishes four sperm subpopulations. The first is the subpopulation positive for only Hoechst 33 342 and was considered to be alive and without any membrane alteration. Another subpopulation is the Yo-Pro-1 positive cells emitting green fluorescence as membranes become slightly permeable during the first steps of damage, enabling Yo-Pro-1 but not ethidium homodimer to cross the plasma membrane. Neither probe enters intact cells. Finally, two subpopulations of dead spermatozoa were easily detected. These were either apoptotic (spermatozoa stained both with Yo-Pro-1 and ethidium homodimer, © 2014 Blackwell Verlag GmbH
Stallion Sperm Senescence
emitting both green and red fluorescence) or necrotic spermatozoa (cells stained only with ethidium homodimer, emitting red fluorescence). Simultaneous flow cytometric detection of active caspases 3 and 7 and active mitochondria CellEventTM (Molecular Probes, Leiden, the Netherlands) caspase 3/7 green detection reagent is a fluorogenic substrate for activated caspases 3 and 7. The reagent consists of a four amino acid peptide (DEVD) conjugated to a nucleic acid-binding dye. This cellpermeant substrate is intrinsically non‑fluorescent because the DEVD peptide inhibits the ability of the dye to bind to DNA. After activation of caspase-3 and caspase-7 in apoptotic cells, the DEVD peptide is cleaved, enabling the dye to bind to DNA and produce a bright fluorogenic response with an absorption/emission maxima of ~502/530 nm. One important advantage of this assay is that no wash steps are required avoiding cell losses during washing. The stock solutions of CellEvent (2 mM in DMSO), ethidium homodimer (1.167 mM in DMSO), Mitotracker deep red (0.5 lM in DMSO) and Hoechst 33342 (1.62 mM in water) were prepared. Spermatozoa (5 9 106/ml) in 1 ml of PBS were stained with 1 ll of cell event, 0.3 ll of Hoechst 33342 and 0.3 ll of Mitotracker deep red and incubated in the dark at RT for 25 min. Next, 0.3 ll of ethidium homodimer was added to each sample. After incubation for 5 min, the samples were immediately run in the flow cytometer. As cryopreservation induces caspase activity in stallion sperm (Ortega-Ferrsula et al. 2008), cryopreserved samples were used as positive controls for caspase 3 and 7. Determination of anion superoxide (O2 ) and hydrogen peroxide (H2O2) production Stallion sperm was stained with 9 ll of Hydroethidine (HE, stock solution 40 lM) for detection of superoxide anion (O2 ) and 9 ll of dichlorodihydrofluorescein diacetate (DCFA, stock solution 2 mM) for detection of H202. Following protocols previously validated for stallion sperm (Macias-Garcia et al. 2012). To restrict the analysis to spermatozoa, 0.3 ll of Hoechst 33342 (stock solution 1.62 mM in water) was added. The samples were incubated for 30 min at 38°C before analysing 100 000 events in the flow cytometer. HE and DCFA were excited at 488 nm, and fluorescence recorded at 530 and 610 nm, respectively. Hoechst 33 342 was excited at 405 nm, and fluorescence recorded at 450 nm. Western blotting Stallion semen was centrifuged and washed twice with PBS. After washing, sperm cells were sonicated for 5 s at 4°C in 100 ll of lysis buffer consisting in 50 mM Tris/ HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 1 mM EGTA, 0.4 mM EDTA, a protease inhibitor cocktail (Complete, EDTA-free) and 0.2 mM Na3VO4. The homogenates were clarified by centrifugation at 10 000 9 g (15 min, 4°C), and the supernatant was used for analysis of protein concentration followed by dilution with 4 9 SDS sample buffer. Proteins © 2014 Blackwell Verlag GmbH
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(25 lg/well) from stallion sperm lysates were fractionated by SDS-PAGE using 4–20% polyacrylamide gradient gels and transferred to nitrocellulose membranes. After blocking, membranes were incubated overnight at 4°C with anti-pAkt Ser 473(1 : 1000) or antiprolactin receptor (1 : 1000). The following day, membranes were washed twice and incubated for 45 min at 25°C with anti-rabbit IgG -HRP conjugated secondary Ab. Membranes were then washed again, incubated with enhanced chemiluminescence detection reagents and finally exposed to Hyperfilm ECL films (Amersham, UK). Positive controls for pAkt Ser 473 were pancreatic acini lysates, and positive controls for prolactin receptor were rat ovary lysates. The intensity and molecular weight of appearing bands were quantified using the software SCION IMAGE for Windows, version 4.02 (Scion Corp., Frederick, MD, USA), normalized to b actin values. Immunocytochemistry Spermatozoa were washed and suspended in PBS, with an adjustment of the cell concentration to 1 9 106 cells/ ml. Fifteen microlitres of the sperm suspension was spread on poly-l lysine-coated slides and allowed to attach for 10 min. Cells were then fixed with 3% formaldehyde in PBS for 15 min and permeabilized with 0.2 Triton X-100 in PBS for 5 min. Slides were washed 3 times for 10 min each with PBS and incubated in PBS supplemented with 5% bovine serum albumin (BSA) (w/v) for 90 min to block non-specific sites. After blocking, slides were incubated overnight at 4°C with antiphospho-Akt Ser 473 (1 : 50) diluted in PBS containing 5% BSA (w/v). On the next day, samples were extensively washed with PBS and further incubated with an Alexa 488 conjugated goat anti-rabbit antibody for 45 min at room temperature. Finally, slides were washed with PBS and examined with a Bio-Rad MRC1024 confocal microscope with a 609 objective in oil immersion. Samples were excited at 488 nm with an argon laser, and emission recorded using a 515 nm long pass filter set. Samples without any primary antibody were assessed to confirm the absence of non-specific staining. Statistical analysis All experiments were repeated at least four times in independent samples, and the results analysed using ANOVA with the (IBM SPSS Statistics for Windows, Version 21.0; IBM Corp., Armonk, NY, USA) for Mac. Differences with a p < 0.05 were regarded as significant.
Results Changes in sperm motility and kinematics Sperm motility, both progressive and total, experienced major changes during the incubation period. A major decrease both in progressive and total motility occurred after 1 h of incubation (p < 0.01). After 6 h of incubation, a further decrease occurred (Fig. 1). Sperm velocities decreased after 1 h of incubation (p < 0.01); however, in contrast to progressive and total motility, no further decreases were detected along the rest of the incubation period (Fig. 2).
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(YoPro+/Eth+) spermatozoa (Fig. 3). YoPro+ sperm increased significantly after 2 and 6 h of incubation, and YoPro+/Eth+ sperm increased significantly only after 6 h of incubation at 37°C.
Changes in the percentage of membrane intactness Sperm membranes remained intact until the sixth hour of incubation, when the percentage of spermatozoa with intact membranes decreased significantly (p < 0.01). This decrease was accompanied by an increase in the percentage of early (YoPro+) and late apoptotic
Changes in caspases 3 and 7 activity Caspases 3 and 7 activity was normalized to values at the beginning of the incubation period. After 2 h of incubation, caspase activity doubled the values at the beginning of the incubation period (p < 0.01); however, the greatest increase was observed after 6 h of incubation, when caspases 3 and 7 activity increased to 600% of initial values (p < 0.001) (Fig. 4a).
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Akt is phosphorylated at Ser 473 in stallion spermatozoa, and its phosphorylation status changes during in vitro incubation Akt was present in stallion spermatozoa; flow cytometry, western blotting and immunocytochemistry confirmed its presence. Phosphorylated Akt was present in the post-acrosomal region and in the mid-piece, supporting its putative role inhibiting an apoptotic-like sperm death (Fig. 5). Akt dephosphorylated spontaneously during in vitro incubation (Fig. 6), but with an increase after 4 h of incubation and a further decrease after 6 h.
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Fig. 1. Changes in sperm motility (total motility MOT, and progressive motility PM) during in vitro incubation of stallion sperm at 37° C up to 6 h in BWW media. (n = 7 stallions) *p < 0.05, **p < 0.01. Results are given as means SD
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Fig. 2. Changes in sperm velocities during in vitro incubation of stallion sperm at 37°C in BWW media. VCL = circular velocity (lm/s), VSL = straight line velocity (lm/s), VAP = average path velocity (lm/s). *p < 0.05 (n = 7 stallions). Results are given as means SD
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Fig. 3. Changes in sperm membrane permeability and integrity 6 h of incubation at 37°C in BWW media. Intact%, percentage of spermatozoa with completely intact membranes; YoPro+, percentage of spermatozoa with increased permeability in their membranes; YoPro+/Eth+, percentage of apoptotic spermatozoa; Eth+, percentage of dead spermatozoa. Results are given as means SD. *p < 0.05. n = 7 stallions
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Fig. 4. Changes in the percentage of increase of caspase activity normalized to initial values (a) and percentage of mitochondrial membrane potential (b) during in vitro incubation of stallion spermatozoa at 37°C in BWW media. (n = 7 stallions) *p < 0.05, **p < 0.01. Results are given as means SD. n = 7 stallions
Changes in superoxide anion and hydrogen peroxide production during in vitro incubation Stallion spermatozoa produced both superoxide anion and hydrogen peroxide thorough all the incubation © 2014 Blackwell Verlag GmbH
period; while significant changes in O2 were not detected, H2O2 increased significantly after 6 h of incubation (p < 0.01) (Fig. 7).
Discussion In the present study, we investigated changes associated with stallion sperm senescence during in vitro incubation in a defined media. Major changes occurred after 6 h of incubation and were accompanied with the expression of markers related to apoptotic cell death, such as caspase activation, oxidative stress, loss of mitochondrial membrane potential and dephosphorylation of Akt. Results obtained in this study indicate that during in vitro incubation the time-dependent loss of motility and viability of stallion sperm may be due to caspase 3 activation and endogenous overproduction of H202. This study supports with previous findings from our laboratory (Moran et al. 2008; Ortega Ferrusola et al. 2009b; Gallardo Bolanos et al. 2012) and others (Thomson et al. 2009; Aitken et al. 2012a) indicating that oxidative damage inducing an apoptotic cascade, probably sperm specific, is involved in sperm death during biotechnological procedures such as cooling and freezing and thawing. Our results also confirm previous findings in human sperm indicating that sperm survival after ejaculation is tightly regulated through a balance of pro survival factors, such as prolactin, that maintain sperm Akt in a phosphorylated state on one side and the production of ROS on the other (Pujianto et al. 2010). Noteworthy the prolactin receptor was also identified in stallion spermatozoa (Fig. 5d). Interestingly, changes in sperm quality followed the same temporal pattern as caspase activation and increases in hydrogen peroxide
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JM Gallardo Bola~ nos, C Balao da Silva, P Martın Mu~ noz, M Plaza D avila, J Ezquerra, IM Aparicio, JA Tapia, C Ortega Ferrusola and FJ Pe~ na
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Fig. 5. Subcellular localization of phospho-Akt in stallion spermatozoa. Ejaculated stallion spermatozoa were washed and stained using specific antiphosphoAkt antibodies and visualized using confocal laser microscopy, as described in the materials and methods. (a) Transmission image (b) fluorescence recorded using the 540/30 emission filter (c) merged images, n = 7 stallions (d) identification of the prolactin receptor in stallion sperm lysates
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pAkt Ser 473 Fig. 6. Changes in Akt phosphorylation (Ser473) during in vitro incubation of stallion spermatozoa at 37°C in BWW media up to six hours. Akt phosphorylation was determined flow cytometrically (a) and using western blotting (b), as described in material and methods. Quantification was performed after flow cytometry data. *p < 0.01. Events in the upper right quadrant represent spermatozoa depicting phosphorylated Akt; events in upper left quadrant represent spermatozoa (Hoechst 33342 positive)
production. This suggests a direct effect of caspase activation and oxidative stress on sperm senescence during in vitro incubation. Also this mechanism has been proposed as responsible of human sperm
senescence, suggesting that this is well conserved and responsible for sperm selection (as spermatozoa in a more advanced state of apoptotic death rapidly lose motility) and silent removal of redundant spermatozoa © 2014 Blackwell Verlag GmbH
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Fig. 7. Changes in the production of superoxide O2 (HE) and H2O2 (H2DFDA) during in vitro incubation of stallion spermatozoa at 37°C in BWW media up to 6 h. Results are given as means SD *p < 0.05 n = 7 stallions
from the female genitalia (Aitken et al. 2012a,b; Aitken et al. 2014). Motility decreased after 1 h of incubation and had a further decrease after 6 h; however, sperm velocities only decreased after 6 h of incubation. Caspase activation followed a similar temporal trend with initial increases after 2 h of incubation and a major increase after 6 h of incubation, as did hydrogen peroxide production that reached a maximum at 6 h, when significant decreases in the percentage of intact spermatozoa and in the mitochondrial membrane potential occurred. These results further support the idea that a sperm-specific apoptotic phenomenon plays a major role sperm death during in vitro incubation. Especially supporting this assumption, percentage of intact sperm and mitochondrial membrane potential reached the lowest values as the same moment as caspase activation reached the maximum increase. Akt was evaluated flow cytometrically allowing the identification of the phosphorylation status at an individual cell level. Phosphorylation at Ser 473 decreased initially but showed an increase after 4 h of
References Agarwal A, Saleh RA, Bedaiwy MA, 2003: Role of reactive oxygen species in the pathophysiology of human reproduction. Fertil Steril 79, 829–843. Aitken RJ, Koppers AJ, 2011: Apoptosis and DNA damage in human spermatozoa. Asian J Androl 13, 36–42. Aitken RJ, Findlay JK, Hutt KJ, Kerr JB, 2011: Apoptosis in the germ line. Reproduction 141, 139–150. Aitken RJ, De Iuliis GN, Gibb Z, Baker MA, 2012a: The Simmet lecture: new horizons on an old landscape–oxidative stress, DNA damage and apoptosis in the male germ line. Reprod Domest Anim 47 (Suppl 4), 7–14. Aitken RJ, Jones KT, Robertson SA, 2012b: Reactive oxygen species and sperm function–in sickness and in health. J Androl 33, 1096–1106. Aitken RJ, Lambourne S, Gibb Z, 2014: The John Hughes Memorial Lecture: aspects of sperm physiology oxidatie stress and
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incubation and then decreased again. These changes may be the result of oxidative stress originated due to the intrinsic metabolic activity of the spermatozoa; for this reason, we evaluated endogenous reactive oxygen species production, anion superoxide O2 and H202 over the time of incubation. While no significant changes occurred in O2 , H202 increased significantly after 6 h of incubation, suggesting that this ROS is related to the loss of sperm viability in view of the temporal relationship of both processes. In short, apoptotic mechanisms occur in stallion spermatozoa during in vitro senescence and at the same time that changes in hydrogen peroxide production and dephosphorylation of Akt. This findings suggest that sperm survival after ejaculation is regulated through the interaction of prosurvival factors yet to be identified in the equine species and pro death factors, apparently overproduction of H202 being a major one. Although descriptive, our findings may open new clues to study molecular mechanisms behind male factor infertility and new strategies for sperm conservation. Acknowledgements The authors received financial support for this study from Ministerio de Ciencia e Innovaci on-FEDER, Madrid, Spain, grants AGL201343211-R, AGL 2010-20758 (GAN), BFU2011-30261, Junta de Extremadura-FEDER (GR 10010 and PCE1002). JMGB holds a PhD grant from the Basque Government/Eusko Jaurlaritzak.
Conflict of interest None of the authors have any conflict of interest to declare.
Author contributions FP designed the paper, analysed the data and wrote the paper, CMC and JGB performed most of the experiments, JAT and IA performed the WB and all the rest of authors collaborated in the analysis of the data and approved the drafted manuscript.
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Submitted: 1 Jan 2014; Accepted: 3 May 2014 Author’s address (for correspondence): FJ Pe~ na, Laboratory of Equine Spermatology and Reproduction, Faculty of Veterinary Medicine, Veterinary Teaching Hospital, University of Extremadura, Avd de la Universidad s/n 10003, Caceres, Spain. E-mail: [email protected]
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