ORIGINAL RESEARCH published: 07 December 2020 doi: 10.3389/fcell.2020.588621 Red LED Light Acts on the Mitochondrial Electron Chain of Donkey Sperm and Its Effects Depend on the Time of Exposure to Light Jaime Catalán 1,2,3 , Marion Papas 1 , Lina Trujillo-Rojas 1 , Olga Blanco-Prieto 1 , Sebastián Bonilla-Correal 1 , Joan E. Rodríguez-Gil 1 , Jordi Miró 1† and Marc Yeste 2,3* † Edited by: 1 Tomer Avidor-Reiss, Unit of Animal Reproduction, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Autonomous University of Toledo, United States University of Barcelona, Bellaterra, Spain, 2 Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, Girona, Spain, 3 Unit of Cell Biology, Department of Biology, Reviewed by: Faculty of Sciences, University of Girona, Girona, Spain Jorge G. Farias, University of La Frontera, Chile Alberto Darszon, This work aimed to investigate how stimulation of donkey sperm with red LED light National Autonomous University of Mexico, Mexico affects mitochondrial function. For this purpose, freshly diluted donkey semen was Ana Josefa Soler, stimulated with red light for 1, 5, and 10 min, in the presence or absence of oligomycin A University of Castilla La Mancha, (Omy A), a specific inhibitor of mitochondrial ATP synthase, or FCCP, a specific disruptor Spain Felipe Martínez-Pastor, of mitochondrial electron chain. The results obtained in the present study indicated that Universidad de León, Spain the effects of red LED light on fresh donkey sperm function are related to changes in *Correspondence: mitochondria function. In effect, irradiation of donkey sperm resulted in an increase in Marc Yeste marc.yeste@udg.edu mitochondrial membrane potential (MMP), the activity of cytochrome C oxidase and the † These authors share senior rate of oxygen consumption. In addition, in the absence of oligomycin A and FCCP, authorship light-stimulation augmented the average path velocity (VAP) and modified the structure of motile sperm subpopulations, increasing the fastest and most linear subpopulation. In Specialty section: This article was submitted to contrast, the presence of either Omy A or FCCP abolished the aforementioned effects. Cell Growth and Division, Interestingly, our results also showed that the effects of red light depend on the exposure a section of the journal Frontiers in Cell and Developmental time applied, as indicated by the observed differences between irradiation protocols. In Biology conclusion, our results suggest that exposing fresh donkey sperm to red light modulates Received: 29 July 2020 the function of their mitochondria through affecting the activity of the electron chain. Accepted: 17 November 2020 However, the extent of this effect depends on the irradiation pattern and does not Published: 07 December 2020 exclude the existence of other mechanisms, such as those related to thermotaxis. Citation: Catalán J, Papas M, Keywords: sperm, red light stimulation, mitochondrial function, donkey, oligomycin A, FCCP Trujillo-Rojas L, Blanco-Prieto O, Bonilla-Correal S, Rodríguez-Gil JE, Miró J and Yeste M (2020) Red LED Light Acts on the Mitochondrial INTRODUCTION Electron Chain of Donkey Sperm and Its Effects Depend on the Time In recent years, the use and development of artificial insemination (AI) in equine species has of Exposure to Light. grown considerably (Canisso et al., 2008; Crowe et al., 2008; Squires, 2009), and is currently Front. Cell Dev. Biol. 8:588621. being considered as the basis of modern equine reproduction worldwide (Pagl et al., 2006; doi: 10.3389/fcell.2020.588621 Kowalczyk et al., 2019). However, while a significant number of previous studies have described Frontiers in Cell and Developmental Biology | www.frontiersin.org 1 December 2020 | Volume 8 | Article 588621 Catalán et al. Mitochondria and Donkey Sperm Irradiation and characterized the semen of domestic horses, studies on and neuropsin) appear to work as thermosensitizers rather donkey sperm are scarce (Miró and Papas, 2018; Catalán et al., than light-sensitive intracellular proteins (photosensitizers), via 2020a). In addition to this, at present, the most of European canonical phospholipase C (PLC) and cyclic nucleotide pathways donkey breeds are in danger of extinction, which added to a (cAMP/cGMP; Pérez-Cerezales et al., 2015). In effect, opsins, mounting world demand for new donkey uses (milk, cosmetics especially melanopsin and rhodopsin, are involved, along with or skin production, oncotherapy, silviculture, rural tourism. . .). the aforementioned TRPV proteins, in the sperm response to For these reasons, the interest in developing studies to improve thermotaxis (Zheng, 2013; Pérez-Cerezales et al., 2015; Roy the reproductive performance in this species has increased (Miró et al., 2020). Interestingly, a recent study has demonstrated that et al., 2020). At this respect, it is worth noting that while both rhodopsin and melanopsin trigger a different signaling pathway horses and donkeys are phylogenetically close species, they show but, unlike vision, both types of opsin coexist in the same sperm important reproductive differences, and not only do their sperm cells (Roy et al., 2020). The third hypothesis considers the role of vary in motility and morphology but also on how they interact endogenous photosensitizers, particularly cytochromes residing with the female endometrium (Miró and Papas, 2018). Be that in the mitochondria (Yeste et al., 2018). It is widely known that as it may, the fact that AI has been largely developed in the horse cytochromes are an essential component of the mitochondrial over the last years has fostered the application of those techniques electron chain and control oxidative phosphorylation, generation in the donkey, which necessarily entails the need of developing of reactive oxygen species and apoptosis (Ortega-Ferrusola et al., methods, protocols or procedures aimed at optimizing sperm 2009). Cytochromes have a heme group, which not only accepts survival and fertilizing capacity, as in the horse (Loomis, 2006; and donates electrons (Kessel, 1982; Pottier and Truscott, 1986), Varner, 2016). but is sensitive to light. However, not all cytochromes react Irradiation of sperm with red light (laser and LED) has against the same wavelengths (Lynch and Copeland, 1992). been demonstrated to jointly increase sperm motility and Thus, whereas cytochrome P450, which is also present in the mitochondrial activity in sheep, cattle, pigs, donkeys and horses endoplasmic reticulum of somatic cells, has its highest absorption (Iaffaldano et al., 2016; Siqueira et al., 2016; Yeste et al., 2016; peak at 450 nm, cytochrome C, which forms the complex Catalán et al., 2020a,b,c). These effects seem, however, to rely IV of the mitochondrial electron chain, has two peaks at upon the light-stimulation pattern, species and other factors, 610–630 nm and 660–680 nm (Lynch and Copeland, 1992). since while it has also been found to increase the in vivo fertilizing However, and despite unpublished data supporting the relevance ability in pigs (Yeste et al., 2016; Blanco-Prieto et al., 2019), its use of mitochondria in pig sperm (Blanco-Prieto et al., personal in separate farms around the world brings significant differences communication), one should not rule out that more than one of (Blanco-Prieto et al., 2019). In addition, the literature remains these hypothetical mechanisms could be involved in the sperm inconsistent on how irradiation affects sperm function and, at response to red light, as well as that the potential influence of light present, the mechanisms underlying that impact on mammalian on the conformation of other proteins might also be implied in spermatozoa remain largely unknown (reviewed in Yeste et al., that response (Yeste et al., 2018). 2018). Hence, further studies aimed at addressing the machinery Against this background, the objective of this study was to that potentially guides the response of sperm to red light in determine if the previously reported effects of light stimulation in separate species are warranted. In this context, the donkey could donkey sperm (Catalán et al., 2020a) are related to changes caused be an interesting model since previous studies have demonstrated by the action of light on mitochondrial function, since this is one that irradiation affects some sperm parameters in this species of the hypothetical mechanisms of action. Our hypothesis is that (Catalán et al., 2020a). the effects of irradiating donkey sperm with red-light are driven At present, three mechanisms have been suggested as being by changes in mitochondrial function. For this reason, freshly related to the response of sperm to red light (Yeste et al., diluted donkey semen was irradiated with red light for 1, 5, and 2018). The first hypothesizes that irradiation is linked to 10 min, in the presence/absence of Omy A, a specific inhibitor thermotaxis, via its interaction with specific members of the of the mitochondrial ATP synthase activity, or FCCP, which acts Transient Receptor Potential (TRP) family. While the TRP as a disruptor of the electron chain function. If mitochondria are family is a highly heterogeneous class of plasma membrane involved in the sperm response to irradiation, utilizing these two receptors, only those belonging to vanilloid TRP (TRPV), ankyrin effectors should affect the impact from red light. In addition, the TRP (TRPA), and melastanin TRP (TRPM) subfamilies have use of both oligomycin A and FCCP should allow elucidating the been reported to be involved in the control of thermotaxis exact part of the mitochondrial electron chain that is involved in (Vriens et al., 2014). Remarkably, ion channels are crucial the sperm response to light, and should also contribute to shed during sperm capacitation (Lishko et al., 2012; Singh and light onto the different effects between species. Rajender, 2014; Sun et al., 2017; Mundt et al., 2018), and TRPV4 has been linked to the thermotactic response both in mouse (Hamano et al., 2016) and human sperm (Mundt et al., MATERIALS AND METHODS 2018). A second potential mechanism envisages the participation of opsins, which are coupled to a G-protein (transducin) Animals, Semen Samples, and Ethics and also reside in the plasma membrane of mammalian Eight ejaculates, each coming from a separate jackass, were used. sperm (Pérez-Cerezales et al., 2015). The different members of Animals were allocated to individual paddocks at the Equine this family of proteins (melanopsin, encephalopsin, rhodopsin, Reproduction Service, Autonomous University of Barcelona Frontiers in Cell and Developmental Biology | www.frontiersin.org 2 December 2020 | Volume 8 | Article 588621 Catalán et al. Mitochondria and Donkey Sperm Irradiation (Bellaterra, Cerdanyola del Vallès, Spain). This is an EU-approved Sperm Motility semen collection center (Authorization code: ES09RS01E) that As indicated above, sperm motility was evaluated using a CASA operates under strict protocols of animal welfare and health system (Integrated Sperm Analysis System V1.0; Proiser S.L.; control. Jackasses were adult, healthy and of proven fertility, and Valencia, Spain). For this purpose, samples were incubated in a were fed grain forage, straw and hay, with water being provided water bath at 38◦ C for 5 min prior to analysis. Five microliter ad libitum. of each sperm sample was placed onto a Makler chamber (Sefi Ejaculates were collected through a Hannover artificial vagina Medical Instruments; Haifa, Israel) previously warmed at 38◦ C. (Minitüb GmbH, Tiefenbach, Germany) and an in-line nylon Samples were observed under a 10 × negative phase-contrast mesh filter to remove the gel fraction. Upon collection, gel-free objective (Olympus BX41 microscope; Olympus Corporation, semen was diluted 1:5 (v:v) with Kenney extender (Kenney et al., Tokyo, Japan), and at least 1,000 sperm cells were counted per 1975), previously preheated to 37◦ C. Thereafter, a conventional analysis. In each evaluation, percentages of total (TMOT) and spermiogram, based on total ejaculate volume, sperm progressively motile spermatozoa (PMOT) were recorded along concentration (Neubauer chamber, Paul Marienfeld GmbH, and with the following kinetic parameters: curvilinear velocity (VCL, Co. KG; Lauda-Königshofen, Germany), motility (Computer µm/s), which is the mean path velocity of the sperm head along Assisted Semen Analysis, CASA), morphology (eosin-nigrosin its actual trajectory; straight-line velocity (VSL, µm/s), which staining) and plasma membrane integrity (SYBR14/PI), was is the mean path velocity of the sperm head along a straight performed. All samples were confirmed to be above the standard line from its first to its last position; average path velocity (VAP, thresholds (≥70% of total motility and SYBR14+ /PI− sperm µm/s), which is the mean velocity of the sperm head along its and ≥ 70% morphologically normal sperm). average trajectory; percentage of linearity (LIN,%), which is the All jackasses used in this study were semen donors and no quotient between VSL and VCL multiplied by 100; percentage manipulation to animals, apart from semen collection in the of straightness (STR,%), which is the quotient between VSL and authorized center, was made. The animal study was reviewed VAP multiplied by 100; percentage of oscillation (WOB,%), which and approved by the Ethics Committee for Animal and Human is the quotient between VAP and VCL multiplied by 100; mean Experimentation (CEEAH) of our institution (Autonomous amplitude of lateral head displacement (ALH, µm), which is the University of Barcelona; authorization code: CEEAH 1424). mean value of the extreme side-to-side movement of the sperm head in each beat cycle; and frequency of head displacement Experimental Design (BCF, Hz), which is the frequency at which the actual sperm Prior to light-stimulation, sperm concentration was adjusted trajectory crosses the average path trajectory (Hz). to 30 × 106 spermatozoa/mL with Kenney extender using Settings of the CASA system were those recommended by a Neubauer chamber (Paul Marienfeld GmbH & Co. KG; the manufacturer: frames/s: 25 images captured per second; Lauda-Königshofen, Germany). Following this, each semen particle area > 4 and < 75 µm2 ; connectivity: 6; minimum sample was split into separate aliquots of 1.5 mL that were number of images to calculate the ALH: 10. Cut-off values subjected to three red light irradiation protocols with an were VAP ≥ 10 µm/s for TMOT, and STR ≥ 75% for air-refrigerated red LED light system (PhastBlue , IUL, S.L.; R PMOT. In addition, individual kinematic parameters (VSL, VCL, Barcelona, Spain; wavelength range: 620–630 nm; intensity VAP, LIN, STR, and BCF) were used to determine motile per sample: 35.05 W/m2 ). These three protocols consisted of sperm subpopulations. Three replicates were evaluated before exposing sperm to red light for 1 min (P1), 5 min (P5) calculating the corresponding mean ± SD. or 10 min (P10). In all cases, the temperature within the PhastBlue system was maintained at 20◦ C ± 0.1◦ C. The R Flow Cytometry control consisted of 1.5 mL tubes kept at 20◦ C in the dark Flow cytometry was used to determine plasma membrane for 10 min. In addition to the aforementioned, semen samples integrity, MMP, and intracellular levels of superoxides, peroxides were also exposed to the same three protocols (i.e., P1, P5, and calcium, following the recommendations set by the and P10) or non-exposed (control), in the presence of either International Society for Advancement of Cytometry (ISAC; 5 µM oligomycin A or 5 µM FCCP. In order to achieve a Lee et al., 2008). In all analyses, sperm concentration was maximal effect of both Omy A and FCCP, these two molecules previously adjusted to 1 × 106 spermatozoa/mL in a final were added to semen samples 10 min prior to exposure to volume of 500 µL, and three technical replicates were evaluated. red light. Following exposure to red light, sperm motility was Samples were examined using a Cell Laboratory QuantaSC evaluated through a computer-assisted sperm analysis system cytometer (Beckman Coulter, Fullerton, CA, United States), (CASA); sperm membrane integrity, mitochondrial membrane and the sheath flow rate was set at 4.17 µL/min. Electronic potential (MMP), and intracellular ROS and calcium levels volume (EV) and side scatter (SS) were recorded in a log- were determined through flow cytometry; O2 consumption rate; linear mode (EV/SS dot plots) for 10,000 events per replicate. intracellular ATP levels and total cytochrome C oxidase (CCO) The analyzer threshold was adjusted on the EV channel to activity were also assessed. In the case of intracellular ATP exclude subcellular debris (particle diameter < 7 µm) and levels and total CCO activity, samples were first centrifuged cell aggregates (particle diameter > 12 µm). When required, at 2,000 × g and 20◦ C for 30 s; the resulting pellets were compensation was used to minimize fluorescence spill-over into immediately frozen by plunging them into liquid N2 . Samples a different channel. Information on all events was collected in were stored at −80◦ C until use. List-mode Data files (EV, SS, FL1, FL2, and FL3) and processed Frontiers in Cell and Developmental Biology | www.frontiersin.org 3 December 2020 | Volume 8 | Article 588621 Catalán et al. Mitochondria and Donkey Sperm Irradiation using the Cell Lab QuantaSC MPL Analysis Software (version diacetate (H2 DCFDA) and hydroethidine (HE), which detect 1.0; Beckman Coulter). In all assessments, data were corrected hydrogen peroxide (H2 O2 ) and superoxide anion (·O2 − ), using the procedure described by Petrunkina et al. (2010), respectively (Murillo et al., 2007). Sperm were counterstained based on the percentage of debris particles (SYBR14− /PI− ) with PI (H2 DCFDA) or YO-PRO-1 (HE), following a protocol determined through SYBR14/PI staining. Fluorochromes were modified from Guthrie and Welch (2006). purchased from Molecular Probes (Invitrogen , Thermo Fisher R R With regard to peroxides, spermatozoa were incubated Scientific, Waltham, MA, United States) and diluted with with H2 DCFDA (final concentration: 200 µM) and PI (final dimethyl sulfoxide (DMSO). concentration: 12 µM) at room temperature for 30 min in the dark. H2 DCFDA is a stable, cell-permeable, non-fluorescent Plasma Membrane Integrity probe that, in the presence of H2 O2 , is deesterified and Sperm membrane integrity was assessed using the LIVE/DEAD converted into 20 ,70 -dichlorofluorescein (DCF). Fluorescence of Sperm Viability Kit (SYBR14/PI; Molecular Probes, Thermo DCF and PI were measured through FL1 and FL3 detectors, Fisher Scientific), according to the protocol described by Garner respectively. Four sperm populations were distinguished: (i) and Johnson (1995) and adapted to donkey spermatozoa. viable spermatozoa with low levels of peroxides (DCF− /PI− ); (ii) In brief, samples were first incubated with SYBR14 (final viable spermatozoa with high levels of peroxides (DCF+ /PI− ); concentration: 100 nM) at 38◦ C for 10 min, and then with (iii) non-viable spermatozoa with low levels of peroxides PI (final concentration: 12 µM) at 38◦ C for 5 min. Three (DCF− /PI+ ); and (iv) non-viable spermatozoa with high levels sperm populations were distinguished: (i) viable spermatozoa of peroxides (DCF+ /PI+ ). Percentages of debris particles found emitting green fluorescence (SYBR14+ /PI− ), which appeared on in SYBR14/PI staining (SYBR14− /PI− ) were subtracted from the right side of the lower half of FL1/FL3 dot plots; (ii) non- those of viable spermatozoa with low levels of peroxides viable spermatozoa emitting red fluorescence (SYBR14− /PI+ ), (DCF− /PI− ), and percentages of all sperm populations were which appeared on the left side of the upper half of FL1/FL3 recalculated. Spill-over of FL1- into the FL3-channel was dot plots; and (iii) non-viable spermatozoa emitting both green compensated (2.45%). Data are shown as corrected percentages and red fluorescence (SYBR14+ /PI+ ), which appeared on the of viable spermatozoa with high levels of peroxides (DCF+ /PI− ), right side of the upper half of FL1/FL3 dot plots. Non-stained and geometric mean of DCF+ -fluorescence intensity in the particles (SYBR14− /PI− ), which appeared on the left side of the DCF+ /PI− sperm population. lower half of FL1/FL3 dot plots, had similar EV/SS distributions As far as superoxides are concerned, samples were incubated than spermatozoa and were considered as non-DNA, debris with HE (final concentration: 4 µM) and YO-PRO-1 (final particles. Percentages of non-stained particles were used to concentration: 25 nM) at room temperature for 30 min in correct the percentages of double-negative sperm populations in the dark (Guthrie and Welch, 2006). Hydroethidine diffuses the other assessments. Spill-over of FL1- into FL3-channel was freely through plasma membrane and converts into ethidium compensated (2.45%). (E+ ) in the presence of superoxide anions (·O2 − ) (Zhao et al., 2003). Fluorescence of ethidium (E) was detected through FL3 Mitochondrial Membrane Potential and that of YO-PRO-1 was detected through FL1. Four sperm Mitochondrial membrane potential was determined populations were distinguished: (i) viable spermatozoa with low through incubation with JC1 (5,50 ,6,60 -tetrachloro- levels of superoxides (E− /YO-PRO-1− ); (ii) viable spermatozoa 1,10 ,3,30 tetraethyl−benzimidazolylcarbocyanine iodide; final with high levels of superoxides (E+ /YO-PRO-1− ); (iii) non- concentration: 0.5 µM) at 38◦ C for 30 min in the dark. At low viable spermatozoa with low levels of superoxides (E− /YO- MMP, JC1 remains as a monomer (JC1mon ) emitting green PRO-1+ ); and (iv) non-viable spermatozoa with high levels of fluorescence, which is detected through FL1. At high MMP, superoxides (E+ /YO-PRO-1+ ). Percentages of debris particles JC1 forms aggregates (JC1agg ) emitting orange fluorescence, found in SYBR14/PI staining (SYBR14− /PI− ) were subtracted which is collected through FL2. Three sperm populations were from those of viable spermatozoa with low levels of superoxides distinguished: (i) spermatozoa with green-stained mitochondria (E− /YO-PRO-1− ), and percentages of all sperm populations (low MMP); (ii) spermatozoa with heterogeneous mitochondria were recalculated. Spill-over of FL3-into the FL1-channel was stained both green and orange in the same cell (intermediate compensated (5.06%). Data are shown as corrected percentages MMP); and (iii) spermatozoa with orange-stained mitochondria of viable spermatozoa with high levels of superoxides (E+ /YO- (high MMP). Ratios between JC1agg (FL2) and JC1mon (FL1) PRO-1− ), and geometric mean of E+ -fluorescence intensity in fluorescence for each of these sperm populations were also the E+ /YO-PRO-1− sperm population. evaluated. Spill-over of FL1- into FL2-channel was compensated (68.50%). Percentages of debris particles found in SYBR14/PI Intracellular Calcium Levels staining (SYBR14− /PI− ) were subtracted from those of Previous studies found that Fluo3 mainly stains mitochondrial spermatozoa with low MMP, and percentages of all sperm calcium in pig sperm (Yeste et al., 2015). For this reason, we populations were recalculated. combined this fluorochrome with PI (Fluo3/PI), as described by Kadirvel et al. (2009). Four sperm populations were identified: Analysis of Intracellular ROS Levels: H2 O2 and ·O2 − (i) viable spermatozoa with low levels of intracellular calcium Intracellular ROS levels were determined through two oxidation (Fluo3− /PI− ); (ii) viable spermatozoa with high levels of sensitive fluorescent probes: 20 ,70 -dichlorodihydrofluorescein intracellular calcium (Fluo3+ /PI− ); (iii) non-viable spermatozoa Frontiers in Cell and Developmental Biology | www.frontiersin.org 4 December 2020 | Volume 8 | Article 588621 Catalán et al. Mitochondria and Donkey Sperm Irradiation with low levels of intracellular calcium (Fluo3− /PI+ ); and centrifuged at 1,000 × g and 17◦ C for 30 s. The resulting (iv) non-viable spermatozoa with high levels of intracellular pellets were immediately plunged into liquid N2 and stored for calcium (Fluo3+ /PI+ ). FL3 spill-over into the FL1-channel 3 weeks. Pellets were resuspended in 500 µL ice-cold PBS and (28.72%) and FL1 spill-over into the FL3-channel (2.45%) sonicated (10 kHz, 20 pulses; Bandelin Sonopuls HD 2070). were compensated. Thereafter, 500 µL Percoll (concentration: 1.055 mg/mL in R PBS) at 4◦ C was placed onto each sperm homogenate. Samples Determination of Intracellular ATP Levels were centrifuged at 3,000 × g and 10◦ C for 45 min and the Intracellular ATP levels were determined following the protocol mitochondria-enriched fraction was carefully harvested with a set by Chida et al. (2012). Immediately after light-stimulation, micropipette and transferred into a new 1.5 mL tube. Samples 1-mL semen aliquots were centrifuged at 17◦ C for 30 s; were again centrifuged at 12,000 × g and 20◦ C for 2 min and pellets were plunged into liquid N2 , and frozen pellets were the resulting pellets were resuspended in 100 µL PBS at 20◦ C. subsequently stored at −80◦ C for a maximum of 3 weeks. These mitochondria-enriched suspensions were split into two Pellets were then resuspended in 300 µL ice-cold 10 mM 2- separate aliquots. The first one was used to determine CCO [4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) activity using a commercial kit (Cytochrome C Oxidase Assay Kit; buffer containing 250 mM sucrose (pH was adjusted to 7.4), Sigma-Aldrich; catalog number CYTOCOX1). Enzyme activity and subsequently sonicated (10 kHz, 20 pulses; Bandelin was normalized against the total protein content. Therefore, the Sonopuls HD 2070; Bandelin Electronic GmbH and Co., Berlin, other aliquot (10 µL) was used to determine total protein content Germany). During this process, tubes were kept on ice to through a commercial kit (Bio-Rad laboratories) based on the avoid specimen heating. Next, samples were centrifuged at Bradford method (Bradford, 1976). 1,000× g and 4◦ C for 10 min, supernatants were harvested for further use and pellets were discarded. Twenty µL of Statistical Analysis supernatant was used to determine total protein content, and Statistical analyses were conducted using a statistical package the remaining volume was mixed with 300 µL ice-cold 10% (SPSS Ver. 25.0 for Windows; IBM Corp., Armonk, NY, R (v:v) trichloroacetic acid and held at 4◦ C for 20 s. Samples United States). Data were first tested for normal distribution were subsequently centrifuged at 1,000 × g and 4◦ C for (Shapiro-Wilk test) and homogeneity of variances (Levene 30 s, and supernatants were carefully separated from the pellet test) and, if required, they were transformed with arcsin √ and again centrifuged at 1,000 × g and 4◦ C for 10 min. x. The effects of irradiating donkey with red light and Supernatants were mixed with two volumes of 1 M Tris- the presence of oligomycin A/FCCP were tested on sperm acetate buffer (pH = 7.75), and the resulting suspension was motility parameters, percentages of spermatozoa with an used to determine the ATP content using the Invitrogen R intact plasma membrane (SYBR14+ /PI− ), spermatozoa with ATP Determination Kit (Thermo Fisher Scientific, Waltham, high and intermediate MMP, viable spermatozoa with high United States; catalog number: A22066). Determinations of ATP intracellular calcium levels (Fluo3+ /PI− ), viable spermatozoa content were carried out with an Infinite F200 fluorimeter with high superoxide levels (E+ /YO-PRO-1− ), and viable (TECAN ), using 96-wells microplates for fluorescence-based R spermatozoa with high peroxide levels (DCF+ /PI− ); geometric assays (Invitrogen ). Data were normalized against the total R mean fluorescence intensities (GMFI) of JC1agg , Fluo3+ , E+ protein content determined with the Bradford method (Bradford, and DCF+ ; JC1agg /JC1mon GMFI-ratios; intracellular levels of 1976) using a commercial kit (Bio-Rad laboratories; Hercules, ATP; O2 consumption rate; and cytochrome C oxidase activity CA, United States). were evaluated through two-way analysis of variance (ANOVA; factor 1: irradiation protocol; factor 2: presence/absence of Determination of O2 Consumption Rate Omy A or FCCP) followed by post hoc Sidak test for pair- Determination of O2 consumption rate was performed through wise comparisons. the SensorDish Reader (SDR) system (PreSens Gmbh; R Motile sperm subpopulations were determined through the Regensburg, Germany). After light-stimulation, 1 mL of each protocol described in Luna et al. (2017). In brief, individual sperm sample was transferred onto an Oxodish OD24 plate R kinematic parameters (VCL, VSL, VAP, LIN, STR, WOB, ALH, (24 wells/plate). Plates were sealed with Parafilm , placed within R and BCF) recorded for each sperm cell were used as independent the SDR system, and incubated at 37◦ C (controlled atmosphere) variables in a Principal Component Analysis (PCA). Kinematic for 2 h. During that time, O2 concentration was recorded in parameters were sorted into PCA components and the obtained each well at a rate of one reading/min. Data were exported to matrix was subsequently rotated using the Varimax method an Excel file and final O2 consumption rate was normalized with Kaiser normalization. As a result, each spermatozoon against the total number of viable spermatozoa per sample, was assigned a regression score for each of the new PCA which was determined through flow cytometry (SYBR14+ /PI− components and these values were subsequently used to run a spermatozoa) as described above. two-step cluster analysis based on the log-likelihood distance and the Schwarz Bayesian Criterion. Four sperm subpopulations Determination of Cytochrome C Oxidase Activity were identified and each individual spermatozoon was assigned The CCO activity was determined in mitochondria-enriched to one of these subpopulations (SP1, SP2, SP3, or SP4). sperm fractions, as described in Mclean et al. (1993). Briefly, 1- Following this, percentages of spermatozoa belonging to each mL sperm aliquots, previously irradiated with red light, were subpopulation were calculated per sample and used to determine Frontiers in Cell and Developmental Biology | www.frontiersin.org 5 December 2020 | Volume 8 | Article 588621 Catalán et al. Mitochondria and Donkey Sperm Irradiation the effects of irradiation and Omy A/FCCP on the distribution Regarding the effects of light-stimulation on sperm kinetic of motile sperm subpopulations through two-way ANOVA parameters (Table 1), irradiation for 5 min and 10 min followed by Sidak test. significantly (P < 0.05) increased VAP, when Omy A and FCCP In all analyses, the level of significance was set at P ≤ 0.05. were absent. In contrast, the presence of Omy A or FCCP in non- Data are shown as mean ± standard deviation (SD), median and irradiated samples led to a significant (P < 0.05) decrease in VCL, interquartile range (i.e., Q1 and Q3). VSL, VAP, LIN, and WOB (Tables 1A,B). Moreover, treatments with Omy A and FCCP showed significantly (P < 0.05) higher values of BCF (Table 1C) and STR in non-irradiated and RESULTS irradiated samples. Finally, as shown in Table 2, four different motile sperm subpopulations were identified in all treatments (SP1, SP2, SP3, Effects of Red Light Stimulation on and SP4). Interestingly, irradiation was found to affect the Plasma Membrane Integrity in the proportions of sperm belonging to each motile subpopulation. Presence or Absence of Either Omy A or Light-stimulation of spermatozoa with red light for 1 min FCCP significantly increased (P < 0.05) the proportions of sperm Irradiating donkey sperm with red light did not affect sperm belonging to SP1 (Figure 3A), which was the subpopulation that membrane integrity, as no significant differences between the exhibited the highest kinematic parameters (Table 2). Regardless control and light-stimulation protocols were observed with of whether samples were or not irradiated, the presence of Omy regard to the percentages of membrane-intact spermatozoa A or FCCP significantly (P < 0.05) decreased the proportions (SYBR14+ /PI− ; Figure 1), neither did the presence of Omy A or of sperm belonging to SP1, SP2, and SP4 (Figures 3A,B,D) and FCCP alter that parameter. increased those of sperm belonging to SP3 (Figure 3C), which was the motile subpopulation that exhibited the lowest values in most kinematic parameters (Table 2). Effects of Red Light Stimulation on Sperm Motility in the Presence or Effects of Red Light Stimulation on Absence of Either Omy A or FCCP As shown in Figure 2, percentages of TMOT (Figure 2A) and Mitochondrial Membrane Potential in the PMOT (Figure 2B) did not differ between non-irradiated and Presence or Absence of Either Omy A or irradiated sperm samples, in the presence/absence of either FCCP Omy A or FCCP. Moreover, the presence of Omy A or FCCP, As shown in Figure 4A, irradiation for 1, 5, and 10 min irrespective of irradiation/non-irradiation, led to a significant significantly (P < 0.05) increased the percentages of spermatozoa (P < 0.05) decrease in total (Figure 2A) and progressive with high MMP in samples without Omy A or FCCP. (Figure 2B) sperm motility. In treatments containing Omy A, irradiation for 1 min or 5 min significantly (P < 0.05) augmented the percentages of spermatozoa with high MMP compared to non-irradiated samples. Furthermore, percentages of spermatozoa with high MMP in samples containing FCCP were significantly (P < 0.05) higher in samples irradiated for 10 min than in their non- irradiated counterparts. In addition to this, percentages of spermatozoa with high MMP in non-irradiated samples and samples irradiated for 1, 5, or 10 min were significantly lower (P < 0.05) when FCCP was present than in the absence of this disruptor (Supplementary Figure 1). As shown in Figure 4B, no significant differences in the percentages of sperm with intermediate MMP were observed between non-irradiated and irradiated samples, either in the presence or absence of Omy A and FCCP. In addition, no significant differences in the geometric mean of JC1agg intensity (orange, FL2) of sperm populations with high (Figure 4C) and intermediate MMP (Figure 4D) FIGURE 1 | Effects of light-stimulation on the percentages of membrane-intact sperm (SYBR14+ /PI− ) in the presence/absence of Omy A were observed between irradiated and non-irradiated or FCCP. No significant differences between non-irradiated and irradiated samples, either in the presence or absence of Omy A sperm in the presence/absence of oligomycin A or FCCP were observed, nor or FCCP. However, geometric mean of the intensity of between samples in the presence of Omy A or FCCP and the treatment JC1agg (orange, FL2) in the sperm population with high without the two disruptors for a given light-stimulation protocol (i.e., control, MMP was significantly (P < 0.05) lower in the non- P1, P5, or P10). Data are shown as mean ± SD from eight separate experiments. irradiated sample containing FCCP than in that without any inhibitor/disruptor (Figure 4C). Frontiers in Cell and Developmental Biology | www.frontiersin.org 6 December 2020 | Volume 8 | Article 588621 Catalán et al. Mitochondria and Donkey Sperm Irradiation FIGURE 2 | Effects of light-stimulation on the percentages of total (A) and progressive (B) sperm motility in the presence/absence of oligomycin A (Omy A) or FCCP. Different letters (a b) indicate significant differences (P < 0.05) between oligomycin A or FCCP with respect to the treatment without the two for a given protocol (i.e., control, P1, P5, or P10). No significant differences were found between the samples exposed to the different irradiation times with respect to non-irradiated sperm. Data are shown as mean ± SD from eight separate experiments. TABLE 1A | Kinetic parameters (VSL, VCL, and VAP) of donkey sperm in the control and the different light-stimulation patterns in the presence or absence of oligomycin A (Omy A) and FCCP. Treatments Kinetic parameters VCL (µm/s) VSL (µm/s) VAP (µm/s) Mean ± SD Q1, median, Q3 Mean ± SD Q1, median, Q3 Mean ± SD Q1, median, Q3 Control Control 130.7 ± 13.5Aa 110.0, 133.0, 147.5 102.1 ± 10.9Aa 89.0, 105.3, 111.6 117.6 ± 6, 9Aa 101.3, 121.0, 131.1 Omy A 66.0 ± 8.7Ab 55.6, 64.7, 77.1 42.4 ± 8.7Ab 30.8, 44.2, 47.6 46.5 ± 6.0Ab 36.0, 49.1, 53.7 FCCP 75.8 ± 9.8Ab 69.5, 75.9, 87.8 44.0 ± 9.5Ab 35.1, 46.4, 49.9 49.2 ± 6.9Ab 40.7, 52.2, 57.4 P1 Control 135.2 ± 15.6Aa 110.6, 137.4, 156.4 105.1 ± 13.3Aa 88.6, 107.3, 120.5 126.3 ± 9.9Aa 103.2, 129.0, 138.8 Omy A 68.4 ± 8.1Ab 59.4, 66.6, 77.1 43.2 ± 7.4Ab 35.2, 46.9, 50.7 47.7 ± 5.5Ab 39.2, 50.4, 55.0 FCCP 73.4 ± 11.8Ab 63.5, 70.1, 91.5 41.2 ± 10.6Ab 31.9, 45.7, 51.8 46.5 ± 8.9Ab 34.1, 50.8, 58.3 P5 Control 139.6 ± 11.6Aa 123.7, 141.5, 153.4 109.2 ± 8.9Aa 94.9, 108.2, 117.4 130.7 ± 6.2Ba 115.3, 131.8, 142.5 Omy A 69.2 ± 10.8Ab 55.8, 65.9, 82.6 44.5 ± 10.6Ab 35.1, 46.0, 54.4 49.5 ± 5.5Ab 38.9, 51.1, 60.7 FCCP 80.4 ± 12.3Ab 69.2, 81.8, 92.2 48.6 ± 11.5Ab 39.4, 49.6, 54.2 53.0 ± 6.4Ab 43.7, 54.8, 59.2 P10 Control 141.2 ± 10.7Aa 132.6, 142.2, 158.7 110.0 ± 11.6Aa 99.5.0, 111.2, 119.5 132.1 ± 6.4Aa 122.4, 133.9, 142.3 Omy A 66.9 ± 8.1Ab 57.4, 66.2, 76.8 41.9 ± 7.5Ab 34.5, 42.8, 49.8 46.1 ± 5.5Ab 38.4, 47.3, 54.0 FCCP 78.2 ± 11.7Ab 68.4, 76.9, 90.5 47.1 ± 10.6Ab 37.1, 49.1, 53.7 54.2 ± 6.2Ab 41.4, 53.7, 59.3 Different letters (A,B) indicate significant differences (P < 0.05) between the control and the different light stimulation patterns used in the presence or absence of oligomycin A (Omy A) or FCCP. Different letters (a,b) indicate significant differences (P < 0.05) between Omy A or FCCP with respect to treatment without the two inhibitors for a given protocol (i.e., control, P1, P5, or P10). Data are shown as mean ± SD, median and interquartile range from eight separate experiments. Finally, we also evaluated JC1agg /JC1mon ratios of sperm high levels of peroxides (DCF+ /PI− ). No significant differences populations with high (Figure 4E) and intermediate MMP between irradiated and non-irradiated samples were observed, (Figure 4F). No significant differences between non- either in the presence or absence of Omy A or FCCP. In addition, irradiated and irradiated samples were observed, either in the no significant differences were found within each irradiation presence/absence of Omy A/FCCP, or within the same irradiation pattern when samples with and without disruptors (Omy A and pattern comparing samples with and without disruptors. FCCP) were compared. Percentages of viable sperm with high levels of superoxides Effects of Red Light Stimulation on (% E+ /YO-PRO-1− spermatozoa; Figure 5C) and GMFI of E+ Intracellular ROS Levels in the Presence in the viable sperm population with high levels of superoxides or Absence of Either Omy A or FCCP (E+ /YO-PRO-1− ; Figure 5D) did not significantly differ between Figures 5A,B show the proportions of viable spermatozoa with irradiated and non-irradiated samples, or between treatments high intracellular levels of peroxides (% DCF+ /PI− spermatozoa) with and without disruptors (Omy A and FCCP) within each and the GMFI of DCF+ in the population of viable sperm with irradiation pattern. Frontiers in Cell and Developmental Biology | www.frontiersin.org 7 December 2020 | Volume 8 | Article 588621 Catalán et al. Mitochondria and Donkey Sperm Irradiation TABLE 1B | Kinetic parameters (LIN, STR and WOB) of donkey sperm in the control and the different light-stimulation patterns in the presence or absence of oligomycin A (Omy A) and FCCP. Treatments Kinetic parameters LIN (%) STR (%) WOB (%) Mean ± SD Q1, median, Q3 Mean ± SD Q1, median, Q3 Mean ± SD Q1, median, Q3 Control Control 77.8 ± 3.4Aa 69.6, 79.7, 84.8 85.2 ± 2.1Aa 78.5.6, 87.1, 90.7 91.3 ± 3.6Aa 88.7, 91.1, 96.2 Omy A 63.9 ± 7.7Ab 55.0, 64.6, 72.0 90.3 ± 2.4Ab 87.3, 91.7, 92.8 70.6 ± 8.0Ab 63.1, 70.4, 79.6 FCCP 56.2 ± 7.5Ab 49.3, 59.2, 63.1 88.0 ± 2.9Aa 87.8, 90.4, 90.8 62.9 ± 7.9Ab 56.1, 65.2, 69.6 P1 Control 78.3 ± 2.6Aa 72.9, 77.8, 84.2 84.9 ± 1.8Aa 79.7, 85.2, 90.3 92.1 ± 2.8Aa 91.2, 91.7, 93.3 Omy A 63.2 ± 6.9Ab 56.9, 62.0, 69.1 90.4 ± 1.4Ab 89.2, 90.5, 91.3 69.8 ± 7.9Ab 63.5, 68.9, 76.3 FCCP 56.0 ± 7.8Ab 49.4, 54.7, 61.6 88.3 ± 2.2Aa 87.3, 88.5, 90.1 63.3 ± 8.3Ab 56.7, 61.2, 69.8 P5 Control 78.1 ± 3.2Aa 67.7, 81.1, 86.2 84.2 ± 2.2Aa 78.9, 86.8, 90.7 93.0 ± 3.6Aa 89.1, 91.4, 95.8 Omy A 64.3 ± 6.2Ab 54.6, 67.5, 72.1 89.7 ± 2.4Ab 86.6, 89.8, 92.5 71.5 ± 8.6Ab 63.0, 74.1, 78.5 FCCP 59.5 ± 9.7Ab 50.1, 56.9, 69.9 90.3 ± 1.4Ab 88.6, 91.1, 91.6 65.7 ± 10.3Ab 55.9, 63.0, 75.5 P10 Control 78.3 ± 3.5Aa 71.0, 77.9, 86.4 83.8 ± 2.7Aa 80.3, 84.8, 90.6 93.1 ± 4.8Aa 88.4, 91.9, 96.0 Omy A 62.6 ± 8.3Ab 55.3, 60.1, 70.8 90.8 ± 1.6Ab 89.2, 90.9, 92.5 68.8 ± 8.7Ab 61.9, 65.3, 77.7 FCCP 60.2 ± 10.0Ab 50.9, 58.7, 68.5 90.4 ± 1.1Ab 89.6, 90.3, 90.9 66.5 ± 10.4Ab 57.0, 65.1, 74.3 Different letters (A,B) indicate significant differences (P < 0.05) between the control and the different light stimulation patterns used in the presence or absence of oligomycin A (Omy A) or FCCP. Different letters (a,b) indicate significant differences (P < 0.05) between Omy A or FCCP with respect to treatment without the two inhibitors for a given protocol (i.e., control, P1, P5, or P10). Data are shown as mean ± SD, median and interquartile range from eight separate experiments. Effects of Red Light Stimulation on nor between the presence/absence of disruptors within each Intracellular Calcium Levels in the irradiation pattern (Figure 6A). Similar results were obtained in the case for the GMFI of Fluo3+ in the viable sperm Presence or Absence of Either Omy A or population with high intracellular calcium levels (Fluo3+ /PI− ; FCCP Figure 6B). Percentages of viable sperm with high intracellular calcium levels did not differ between irradiated and non-irradiated samples in the presence/absence of Omy A and FCCP, Effects of Red Light Stimulation on Intracellular Levels of ATP and Oxygen Consumption in the Presence or TABLE 1C | Kinetic parameters (ALH and BCF) of donkey sperm in the control and the different light-stimulation patterns in the presence or absence of Absence of Either Omy A or FCCP oligomycin A (Omy A) and FCCP. Figure 7A shows the intracellular levels of ATP observed in non-irradiated and irradiated samples in the presence or Treatments Kinetic parameters absence of Omy A or FCCP. No significant differences were ALH (µm) BCF (Hz) observed between non-irradiated and irradiated samples were Mean ± SD Q1, median, Q3 Mean ± SD Q1, median, Q3 observed, either in the presence or absence of oligomycin A. In the presence of FCCP, intracellular ATP levels were Control Control 2.5 ± 0.7Aa 1.8, 2.6, 3.3 9.2 ± 1.0Aa 8.1, 9.0, 10.3 significantly (P < 0.05) higher in sperm irradiated for Omy A 2.4 ± 0.3Aa 2.1, 2.4, 2.7 12.9 ± 1.0Ab 10.4, 12.6, 13.5 1 min than in those non-irradiated. In addition, samples FCCP 2.8 ± 0.3Aa 2.5, 2.9, 3.0 13.3 ± 0.7Ab 12.3, 13.3, 13.8 irradiated for 1 min showed significantly (P < 0.05) higher P1 Control 2.6 ± 0.4Aa 1.9, 2.5, 3.2 9.1 ± 1.1Aa 7.7, 8.9, 11.0 intracellular levels of ATP in the presence than in the Omy A 2.5 ± 0.2Aa 2.3, 2.5, 2.7 12.7 ± 0.8Ab 11.0, 12.5, 13.1 absence of FCCP. FCCP 2.9 ± 0.4Aa 2.5, 2.8, 3.2 12.9 ± 0.9Ab 12.2, 12.7, 13.7 Figure 7B shows that light stimulation (P < 0.05) significantly P5 Control 2.6 ± 0.6Aa 2.0, 2.5, 3.4 8.9 ± 1.0Aa 7.7, 8.8, 10.4 increased the O2 consumption rate in all three protocols (i.e., Omy A 2.5 ± 0.4Aa 2.2, 2.4, 2.8 11.8 ± 0.9Ab 10.6, 12.0, 12.9 P1, P5, and P10) compared to the non-irradiated control. FCCP 2.9 ± 0.3Aa 2.6, 3.0, 3.1 13.8 ± 1.0Ab 11.8, 13.6, 14.2 In samples containing Omy A, no significant differences P10 Control 2.7 ± 0.6Aa 2.0, 2.8, 3.5 9.4 ± 0.9Aa 8.2, 9.4, 10.6 between non-irradiated and irradiated samples were observed. Omy A 2.5 ± 0.3Aa 2.2, 2.4, 2.7 12.3 ± 0.9Ab 11.0, 12.4, 13.3 However, O2 consumption rates in non-irradiated and irradiated FCCP 2.8 ± 0.4Aa 2.4, 2.8, 3.1 13.8 ± 0.8Ab 12.1, 13.7, 14.0 samples containing Omy A were significantly (P < 0.05) Different letters (A,B) indicate significant differences (P < 0.05) between the control lower than in the ones that did not contain this inhibitor. and the different light stimulation patterns used in the presence or absence of On the other hand, no significant differences between oligomycin A (Omy A) or FCCP. Different letters (a,b) indicate significant differences irradiated and non-irradiated samples were observed in (P < 0.05) between oligomycin A or FCCP with respect to treatment without the two inhibitors for a given protocol (i.e., P1, P5, or P10). Data are shown as mean ± SD, samples containing FCCP. However, samples containing FCCP median and interquartile range from eight separate experiments. showed significantly (P < 0.05) higher O2 consumption rates Frontiers in Cell and Developmental Biology | www.frontiersin.org 8 December 2020 | Volume 8 | Article 588621 Catalán et al. Mitochondria and Donkey Sperm Irradiation TABLE 2 | Descriptive parameters (mean ± SD, median and interquartile range) of the four sperm subpopulations (SP1, SP2, SP3, and SP4) identified in donkey fresh semen, in the presence or absence of oligomycin A (Omy A) or FCCP. SP1 SP2 SP3 SP4 N 7,611 9,455 16,551 6,694 Parameter Mean ± SD Q1, median, Q3 Mean ± SD Q1, median, Q3 Mean ± SD Q1, median, Q3 Mean ± SD Q1, median, Q3 VCL µm/s) 168.4 ± 13.6 153.1, 168.1, 182.0 114.2 ± 16.1 98.0, 116.4, 132.8 70.2 ± 14.9 54.5, 68.7, 86.3 151.4 ± 19.5 132.3, 155.7, 173.9 VSL µm/s) 132.9 ± 18.0 113.5, 132.9, 150.9 101.7 ± 15.6 84.6, 102.3, 119.7 41.8 ± 9.3 30.5, 43.1, 53.8 54.5 ± 17.8 35.7, 56.0, 74.2 VAP µm/s) 152.4 ± 14.9 137.0, 151.8, 166.8 109.2 ± 16.1 92.1, 111.0, 128.2 47.4 ± 7.6 36.2, 47.8, 58.4 115.0 ± 19.1 98.9, 117.8, 134.2 LIN (%) 78.9 ± 9.2 69.8, 80.7, 88.7 89.1 ± 5.7 85.4, 91.3, 94.7 59.4 ± 13.2 49.8, 61.3, 71.2 35.9 ± 12.1 24.3, 37.4, 48.2 STR (%) 87.1 ± 7.2 81.8, 88.7, 94.4 93.3 ± 4.2 90.5, 95.5, 97.9 86.5 ± 12.2 84.9, 92.6, 95.5 47.3 ± 16.5 33.3, 49.2, 61.8 WOB (%) 90.3 ± 4.3 84.6, 91.1, 95.0 95.1 ± 3.6 92.4, 96.4, 98.8 68.1 ± 10.5 59.5, 68.5, 73.3 75.9 ± 9.1 69.3, 77.9, 84.2 ALH (µm) 3.7 ± 0.6 3.1, 3.6, 4.2 2.1 ± 0.5 1.6, 2.0, 2.5 2.6 ± 0.5 2.1, 2.5, 3.0 4.6 ± 1.1 3.7, 4.6, 5.5 BCF (Hz) 10.9 ± 2.5 9.0, 11.0, 13.0 8.2 ± 2.0 6.8, 8.0, 10.0 11.6 ± 3.1 9.0, 12.0, 14.0 8.0 ± 2.4 6.0, 8.0, 10.0 These data were obtained after classifying sperm cells into motile subpopulations through principal component and cluster analyses. FIGURE 3 | Effects of light stimulation on the structure of the motile sperm subpopulation in the presence/absence of oligomycin A (Omy A) or FCCP. (A) Subpopulation 1 (SP1, the fastest and most linear); (B) Subpopulation 2 (SP2 presented intermediate values, lower than SP1 and SP4, but more linear than SP4; (C) Subpopulation 3 (SP3, the slowest, but more linear than SP4); and (D) Subpopulation 4 (SP4 presented intermediate values, higher than SP2, but was the least linear). Different letters (A,B) indicate significant differences (P < 0.05) between non-irradiated samples and the different light stimulation patterns used in the presence or absence of oligomycin A (Omy A) or FCCP. Different letters (a,b) indicate significant differences (P < 0.05) between oligomycin A (Omy A) or FCCP with respect to the treatment without the two disruptors for a given protocol (i.e., control, P1, P5, or P10). Data are shown as mean ± SD from eight separate experiments. Frontiers in Cell and Developmental Biology | www.frontiersin.org 9 December 2020 | Volume 8 | Article 588621 Catalán et al. Mitochondria and Donkey Sperm Irradiation FIGURE 4 | Effects of light stimulation on the mitochondrial membrane potential in the presence/absence of oligomycin A (Omy A) or FCCP. The results are presented as percentages of sperm with high mitochondrial membrane potential (MMP; JC1agg ++ ; (A) and with intermediate mitochondrial membrane potential (MMP; JC1agg + ; (B); geometric mean of the JC1agg fluorescence intensity (GMFI, FL2) in the sperm populations with high (C) and intermediate (D) MMP; and the JC1agg /JC1mon (GMFI FL2/GMFI FL1) ratios in sperm populations with high (E) and intermediate (F) MMP in control and irradiation patterns (P1, P5, or P10), in the presence/absence of Omy A. Different letters (A, B) indicate significant differences (P < 0.05) between non-irradiated samples and the different light-stimulation patterns used in the presence or absence of Omy A or FCCP. Different letters (a, b) indicate significant differences (P < 0.05) between Omy A or FCCP with respect to the treatment without the two disruptors for a given protocol (i.e., control, P1, P5, or P10). Data are shown as mean ± SD from eight separate experiments. Frontiers in Cell and Developmental Biology | www.frontiersin.org 10 December 2020 | Volume 8 | Article 588621 Catalán et al. Mitochondria and Donkey Sperm Irradiation FIGURE 5 | Effects of light-stimulation on the percentages of viable spermatozoa with high peroxide levels (DCF+ /PI- ; (A) geometric mean of DCF+ -intensity (GMFI, FL1 channel) in the population of viable spermatozoa with high peroxide levels (B) and percentages of viable spermatozoa with high superoxide levels (E+ /YO-PRO-1- ; (C) and geometric mean of E+ -intensity (GMFI, FL3 channel) in the population of viable spermatozoa with high superoxide levels (D) in the control and irradiated samples in the presence or absence of oligomycin A (Omy A) or FCCP. No significant differences were observed between non-irradiated sperm and the different light-stimulation patterns used in the presence/absence of oligomycin A or FCCP, nor between the results of the samples in the presence of Omy A or FCCP with respect to the treatment without the two disruptors for a given protocol (i.e., control, P1, P5, or P10). Data are shown as mean ± SD from eight separate experiments. than those without this disruptor, regardless of whether they (P < 0.05) lower CCO activity than when these two were irradiated. molecules were absent from non-irradiated sperm and sperm irradiated for 5 min and 10 min. In contrast, CCO Effects of Red Light Stimulation on activity in sperm irradiated for 1 min was significantly Cytochrome C Oxidase Activity in the (P < 0.05) lower when Omy A was present than when this inhibitor was absent. Presence or Absence of Either Omy A or FCCP As shown in Figure 8, irradiation for 1, 5, or 10 min induced DISCUSSION a significant increase (P < 0.05) in CCO activity compared to non-irradiated samples. Although these effects were observed in The results obtained in the present study with regard to the both the presence and absence of Omy A or FCCP, the highest potential of mitochondrial membrane, sperm kinetic parameters, CCO activity was observed in sperm irradiated for 5 min in the the structure of motile sperm subpopulations, CCO activity absence of Omy A/FCCP. and oxygen consumption rate support that irradiation with While neither Omy A nor FCCP affected the significant red LED light affects mitochondrial activity of donkey sperm. (P < 0.05) increase in CCO activity observed in Furthermore, the impact of red light has been found to rely irradiated sperm compared to non-irradiated sperm, upon the time of exposure (i.e., 1, 5, or 10 min), regardless of samples containing Omy A or FCCP showed significantly the presence of Omy A and FCCP, which is in agreement with Frontiers in Cell and Developmental Biology | www.frontiersin.org 11 December 2020 | Volume 8 | Article 588621 Catalán et al. Mitochondria and Donkey Sperm Irradiation FIGURE 6 | Effects of light-stimulation on percentages of spermatozoa with high intracellular calcium levels (Fluo3+ ); (A) and geometric mean intensity of Fluo3+ (B) in the presence/absence of either oligomycin A or FCCP. No significant differences were observed between non-irradiated sperm and the different light stimulation patterns used in the presence or absence of oligomycin A (Omy A) or FCCP, nor between the results of the samples in the presence of Omy A or FCCP with respect to the treatment without the two inhibitors for a given protocol (i.e., control, P1, P5, or P10). Data are shown as mean ± SD from eight separate experiments. FIGURE 7 | Effects of light-stimulation on intracellular ATP levels (A) and O2 consumption rate (B) in the presence/absence of either oligomycin A (Omy A) or FCCP. Different letters (A, B) indicate significant differences (P < 0.05) between non-irradiated sperm and the different light stimulation patterns used in the presence or absence of Omy A or FCCP. Different letters (a, b) indicate significant differences (P < 0.05) between Omy A or FCCP with respect to the treatment without the two disruptors for a given protocol (i.e., control, P1, P5, or P10). Data are shown as mean ± SD from eight separate experiments. previous studies conducted in other mammalian species, such as related to the time of exposure and intensity. Therefore, the pigs, dogs, buffalos, humans, donkeys and horses (Corral-Baqués exact level of energy provided to sperm through red light et al., 2009; Abdel-Salam et al., 2011; Salman Yazdi et al., 2014; stimulation and the overall sperm function status appear to Yeste et al., 2016; Catalán et al., 2020a,b,c). In this context, be on the basis of the different impact observed between it is worth emphasizing that a high individual variation was treatments and species. observed, which indicates that apart from the relevance of the With regard to the effects of red light on the mitochondrial stimulation pattern, time and intensity (Yeste et al., 2016), the electron chain observed in this work, it is reasonable to surmise sperm response to red light also depend on the functional status that this could be explained by the direct action of red light on of the cell (Catalán et al., 2020a). Although the mechanisms mitochondrial photosensitizers (reviewed in Yeste et al., 2018). underlying this different response have yet to be elucidated, Indeed, we observed that red light affected the activity of CCO, this work suggests that, in donkey sperm, red light acts on which is a crucial component of the mitochondrial electron mitochondrial photosensitizers and that the energy supplied chain and is sensitive to light at a wavelength ranging from to the mitochondrial electron chain following irradiation is 630 to 660 nm (Lynch and Copeland, 1992). These observed Frontiers in Cell and Developmental Biology | www.frontiersin.org 12 December 2020 | Volume 8 | Article 588621 Catalán et al. Mitochondria and Donkey Sperm Irradiation irradiation of fresh donkey sperm with red light modifies the structure of motile sperm subpopulations by increasing the proportions of faster and more linear sperm cells. In addition, our results concur with a previous study conducted on dog semen, in which stimulation with red light (laser) significantly increased the proportions of the fastest sperm subpopulation (Corral-Baqués et al., 2009) and another one carried out with fresh horse sperm where, in a similar fashion to this work, an increase in the most rapid and linear subpopulation was observed (Catalán et al., 2020c). These changes in the characteristics of the motile sperm subpopulations, together with those observed in sperm kinetic parameters, indicate that not only does irradiation with red light increase the speed but also alters the motility pattern of donkey sperm. While there is not, at present, a clear explanation about how red light stimulation affects sperm motility, the aforementioned impact FIGURE 8 | Effects of light-stimulation on cytochrome C oxidase activity in the presence/absence of either oligomycin A (Omy A) or FCCP. Different letters (A, on mitochondrial function could provide some clues. Related B) indicate significant differences (P < 0.05) between non-irradiated sperm to this, it is worth mentioning that the increase observed in and the different light stimulation patterns used in the presence or absence of VAP and in the proportions of spermatozoa belonging to SP1 Omy A or FCCP. Different letters (a, b) indicate significant differences was concomitant with a rise in the percentages of sperm with (P < 0.05) between Omy A or FCCP with respect to the treatment without the high MMP, determined through JC1. These findings agree with two inhibitors for a given protocol (i.e., control, P1, P5, or P10). Data are shown as mean ± SD from eight separate experiments. Siqueira et al. (2016), who found that irradiation with a He- Ne laser at a wavelength of 633 nm augmented the percentage of sperm with intermediate MMP. Similar results were found in pig, donkey and horse sperm, since irradiation with red increases in CCO activity are similar to those reported in LED light at a wavelength between 620 and 630 nm increased ram semen irradiated with He-Ne laser (632.8 nm; Iaffaldano the percentages of sperm with high MMP (Yeste et al., 2016; et al., 2016) and in liquid-stored pig semen irradiated with LED Catalán et al., 2020a,b,c). Therefore, the current study suggests (Blanco-Prieto et al., personal communication), which would also that stimulation with red light increases mitochondrial activity support the hypothesis that mitochondrial photosensitizers play through endogenous photosensitizers, such as cytochrome C a relevant role in the effects of red light upon sperm cells. On (Begum et al., 2013; Iaffaldano et al., 2016; Yeste et al., the other hand, our results showed that neither irradiation nor 2016; Catalán et al., 2020a,b,c), which could, in turn, lead the presence of Omy A or FCCP had any detrimental effect on to greater motility of sperm and higher fertilization potential sperm viability, which was evaluated through the SYBR14+ /PI− (Breitbart et al., 1996). test. These results are in agreement with Yeste et al. (2016) Recent studies indicate that oxygen consumption represents and Pezo et al. (2019), who conducted their studies in liquid- an alternative measure of mitochondrial activity, which could stored pig semen; Catalán et al. (2020a), who evaluated the be better than the use MMP markers, such as JC1 (Moscatelli effects of red light irradiation on fresh and cooled-stored donkey et al., 2017; Meyers et al., 2019). Oxygen consumption rate semen; and Catalán et al. (2020b,c) that used frozen-thawed and would also provide an indirect measure of ATP produced fresh horse sperm. by oxidative phosphorylation in mammalian sperm (Meyers As far as the effects of red light on sperm motility are et al., 2019). The results obtained in this study showed an concerned, it is worth mentioning that while no significant increase in oxygen consumption on all irradiated samples differences were observed in the percentages of TMOT and (namely, light-stimulated for 1, 5, or 10 min) in the absence PMOT between non-irradiated and samples irradiated with red of Omy A or FCCP compared to the non-irradiated control. light, light-stimulation for 5 min or 10 min in the absence While these results are in agreement with those obtained from of Omy A/FCCP significantly increased VAP, which is in the evaluation of some motility parameters, the structure of agreement with previous studies conducted in dogs (Corral- motile subpopulations, the percentages of viable sperm with Baqués et al., 2005, 2009), cattle (Siqueira et al., 2016), buffalos high MMP and the activity of cytochrome C oxidase, it is (Abdel-Salam et al., 2011), pigs (Yeste et al., 2016), donkeys surprising that no link to intracellular levels of ATP was (Catalán et al., 2020a), and horses (Catalán et al., 2020b,c). observed, since no differences in this parameter were found We also evaluated the effects of red light on motile sperm when non-irradiated and irradiated samples without oligomycin subpopulations and we identified four different subpopulations, A/FCCP were compared. Moreover, no relationship between which is in agreement with that previously reported in the intracellular ATP levels and other mitochondrial parameters donkey (Miró et al., 2005). Remarkably, we observed that was observed in samples irradiated for 1 min in the presence irradiation for 1 min increased the percentages of sperm of FCCP. The increase in the potential of the mitochondrial belonging to SP1, which was the one that included the fastest membrane is associated with changes in the consumption and most linear motile sperm. Thus, our data suggest that of ATP and in the activity of respiratory chain enzymes Frontiers in Cell and Developmental Biology | www.frontiersin.org 13 December 2020 | Volume 8 | Article 588621 Catalán et al. Mitochondria and Donkey Sperm Irradiation (Alberts et al., 2002; Zorova et al., 2018). Related with this, would allow sperm to maintain ROS levels in the event of Iaffaldano et al. (2016) observed that light stimulation of a temporarily induced stress like the one caused by light- frozen-thawed ram sperm with a He-Ne laser increased ATP stimulation. Nevertheless, further research should be conducted content and the activity and affinity of cytochrome C oxidase to confirm this hypothesis. for its substrate (cytochrome C). Interestingly, these authors Regarding our results obtained in samples containing Omy found that CCO activity and ATP content were positively A or FCCP, one should note that irradiation in the presence correlated with each other and with sperm motility, supporting of these two molecules increased the percentages of sperm the hypothesis that the effects of red light upon sperm are with high MMP, compared to the non-irradiated control in mediated by mitochondria. However, and as aforementioned, the presence of these molecules. Furthermore, non-irradiated we did not observe that correspondence in the case of donkey samples in the presence of FCCP exhibited lower intensity of sperm. An explanation about why irradiation did not result JC1agg fluorescence in the sperm population with high MMP, in a clear increase of intracellular ATP levels could be that which did not occur in irradiated samples. These findings also ATP in sperm is mainly synthesized through two routes, support that light-stimulation of sperm exerts its effects via glycolysis and mitochondrial oxidative phosphorylation, without MMP. At this point, it is worth mentioning that some studies dismissing other energy sources available to sperm such as have recently questioned the use of MMP probes to assess β-oxidation (Storey and Keyhani, 1974; Amaral et al., 2013). the potential of the mitochondrial membrane due to its non- The precise balance of ATP production from both sources specific nature (Meyers et al., 2019). These reports point up is very dynamic and multifactorial, undergoing rapid changes that MMP probes are cationic and their accumulation rate not only between species, but also within the same cell inside the mitochondria is inversely proportional to the potential under different physiological and environmental conditions. of the internal mitochondrial membrane; hence, MMP probes The intracellular changes that are related to the complex like JC1, which was the one used in this study, may not be relationship between mitochondrial oxidative phosphorylation reliable as a quantitative measure of MMP without adequate and glycolysis, and that could explain these effects will be later controls. Therefore, these previous studies suggest that high/low discussed in more detail. MMP controls, such as ATP synthase inhibitors like Omy A In addition to the aforementioned, the electron chain is known and MMP uncouplers like FCCP or DNP, are needed (Meyers to play a fundamental role in the generation of reactive oxygen et al., 2019). Remarkably, the results obtained in this work with species (ROS), since mitochondria are the most important Omy A/FCCP showed this controlling effect, since percentages source of ROS generation in eukaryotic cells (Zhao et al., of sperm with high MMP in samples without disruptors were 2019). A previously established hypothesis indicates that light- lower than those containing Omy A, and significantly higher than stimulation increases ROS production by sperm (Cohen et al., those containing FCCP. Regarding sperm motility parameters 1998; Zan-Bar et al., 2005). Furthermore, cytochrome complexes in the presence of these two molecules, they were similar to are also involved in the intrinsic apoptotic pathway (Cai et al., those found by Ramió-Lluch et al. (2014) and Nesci et al. (2020) 1998), and it has been surmised that both generation of ROS in previous studies conducted with pig semen. According to and modulation of apoptotic-like changes could be crucial Nesci et al. (2020), the reduced motility observed in treatments to elicit and modulate the achievement of capacitated status containing Omy A or FCCP could result from the decrease by sperm (Ortega-Ferrusola et al., 2009; Aitken et al., 2015). in ATP content caused by these two molecules. However, our Against this background, one could reasonably suggest that results indicate that the presence of Omy A and FCCP in the red light-induced changes in CCO activity observed herein irradiated and non-irradiated sperm reduces sperm motility could ultimately affect the lifespan and capacitation status of without causing a decrease on overall sperm ATP levels. These mammalian sperm. However, our results showed that light- findings observed in the presence of Omy A are similar to stimulation of fresh donkey sperm did not increase intracellular those seen by Ramió-Lluch et al. (2014), who reported that levels of ROS or calcium, which is a crucial secondary messenger the control exerted by Omy A-sensitive ATP synthase over pig involved in early and late capacitation events (Yeste, 2013; sperm motility does not seem to be related to its inhibiting effect Correia et al., 2015). Thus, our data differ from those observed upon ATP levels. In this context, it is worth mentioning that in donkeys and other species in which irradiation was found to a previous study reported that mitochondrial respiration of pig increase intracellular ROS (Cohen et al., 1998; Zan-Bar et al., sperm incubated in the presence of glucose only contributes 2005; Catalán et al., 2020a) and intracellular calcium levels to 5% of the total energy produced by the cell, the other 95% (Lubart et al., 1992; Cohen et al., 1998). These differences could being obtained from glycolysis (Marín et al., 2003). Therefore, be due to the high individual variability in the sperm response it is surprising that the relatively low levels of energy produced to red light, as well as to differences in the light source, intensity through ATP synthase affected sperm motility as much as they and irradiation pattern, as previous reports found for intracellular did in the study of Ramió-Lluch et al. (2014), especially if one levels of ROS (Catalán et al., 2020a) and calcium (Breitbart et al., bears in mind that these authors did not observe an alteration 1996). Moreover, the existence of a complex homeostasis system of overall sperm energy levels. Related with this, in a recent aimed at maintaining ROS levels within a physiological range, study, Nesci et al. (2020) observed that, despite pig sperm being which would include systems such as the glutathione peroxidase- regarded to rely upon glycolysis, the motility of these cells is glutathione reductase complex (GPX/GSR) and enzymes such highly dependent on the ATP produced through mitochondrial as intracellular peroxidases (see Peña et al., 2019 for review), oxidative phosphorylation. In other studies, such as those carried Frontiers in Cell and Developmental Biology | www.frontiersin.org 14 December 2020 | Volume 8 | Article 588621 Catalán et al. Mitochondria and Donkey Sperm Irradiation out by Iaffaldano et al. (2016) on ram sperm and Odet et al. (2013) DATA AVAILABILITY STATEMENT on mouse sperm, an association between overall ATP content and sperm motility has also been observed. In the case of mice, The raw data supporting the conclusions of this article will be one should keep in mind that their sperm have been reported made available by the authors, without undue reservation, to any to maintain their function through the ATP originated from qualified researcher. glycolysis or mitochondrial respiration indistinctly (Pasupuleti, 2007). In addition to this, a recent study published by Balbach et al. (2020) has demonstrated a functional link between these ETHICS STATEMENT two pathways during mouse sperm capacitation. All these data strongly point to the existence of species-specific differences on All jackasses used in this study were semen donors and no the mechanisms by which mammalian spermatozoa produce manipulation to animals, apart from semen collection in the ATP via glycolysis or oxidative phosphorylation (Rodríguez-Gil authorized center, was made. The animal study was reviewed and Bonet, 2016). Our results suggest that changes in intracellular and approved by the Ethics Committee for Animal and Human ATP levels cannot be taken as a direct indicator of changes Experimentation (CEEAH) of our institution (Autonomous affecting mitochondrial function, since glycolysis and probably University of Barcelona; authorization code: CEEAH 1424. other metabolic pathways present in sperm also produce ATP; thus, further metabolomics studies in donkey sperm are needed to understand the precise glycolysis/oxidative phosphorylation AUTHOR CONTRIBUTIONS balance in these cells. While the evidence reported in this and another study in MY, JR-G, and JM: conceptualization. JC, MP, LT-R, OB-P, pigs (Blanco-Prieto et al., personal communication) indicates JM, MY, and JR-G: methodology and investigation. JM, that the effects of red light on sperm would be related to the MY, and JR-G: validation, project administration, and direct effect on intracellular light-sensitive proteins, especially the supervision. JR-G: formal analysis and data curation. JC: impact on endogenous photosensitizers, such as mitochondrial writing—original draft preparation. JM and MY: writing— cytochromes, other pathways or factors that might modulate the review and editing, funding acquisition, and resources. All effects of red light on mammalian sperm could also be involved authors have read and agreed to the published version of (reviewed in Yeste et al., 2018). These pathways or factors include the manuscript. light-sensitive receptors, such as opsins (Pérez-Cerezales et al., 2015) and transient receptor potential proteins (TRP) (De Blas et al., 2009; Bahat and Eisenbach, 2010; Gibbs et al., 2011), whose FUNDING one of their most probable functions in mammalian sperm is the regulation of thermotaxis (Wu et al., 2010; Pérez-Cerezales et al., JC was funded by the National Agency for Research 2015). Thermotaxis could be, in fact, an important modulator and Development (ANID), Ministry of Education, Chile of light stimulation, since mammalian sperm are sensitive to (Scheme: Becas Chile Doctorado en el Extranjero, PFCHA; temperature changes as small as 0.0006◦ C (Bahat et al., 2012; Grant: 2017/72180128). The authors also acknowledge the Pérez-Cerezales et al., 2015). Therefore, it is reasonable to suggest support from the Ministry of Science and Innovation, that red light stimulation could also act through this pathway, Spain (Grants: RYC-2014-15581 and AGL2017-88329- especially if one takes into consideration that, in this study, R), and the Regional Government of Catalonia, Spain the effects of irradiation on MMP and ATP content, which (2017-SGR-1229). rely on glycolysis/oxidative phosphorylation balance, differed. In addition, one should not discard that signaling transduction pathways triggered when opsins and TRP receptors are activated could change ATP production via glycolysis as well as the balance ACKNOWLEDGMENTS between glycolysis and oxidative phosphorylation. We acknowledge the technical support from Estela Garcia- Bonavila, Sandra Recuero, Ariadna Delgado-Bermúdez, Marc Llavanera, and Yentel Mateo. CONCLUSION In conclusion, our results indicate that the effects induced by the stimulation of fresh donkey sperm with red LED light are SUPPLEMENTARY MATERIAL related to mitochondrial photosensitizers, such as CCO, which modify the activity of the mitochondrial electron chain; the The Supplementary Material for this article can be found effect of red light on these photosensitizers depends on the time online at: https://www.frontiersin.org/articles/10.3389/fcell.2020. of exposure, among other factors. However, these findings do 588621/full#supplementary-material not exclude that this mitochondrial mechanism could work in Supplementary Figure 1 | Representative flow-cytometry dot-plots observed in conjunction with other pathways, such as thermotaxis, via plasma non-irradiated samples and samples irradiated for 5 min, in the presence/absence membrane receptors. of FCCP and Omy A. 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(2003). Superoxide reacts with hydroethidine but forms a de Laboratori Geniul, SL (Barcelona, Spain). fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide. The remaining authors declare that the research was conducted in the absence of Free Radic. Biol. Med. 34, 1359–1368. doi: 10.1016/S0891-5849(03)00 any commercial or financial relationships that could be construed as a potential 142-4 conflict of interest. Zhao, R. Z., Jiang, S., Zhang, L., and Yu, Z. B. (2019). Mitochondrial electron transport chain, ROS generation and uncoupling (Review). Int J. Mol. Med. 44, Copyright © 2020 Catalán, Papas, Trujillo-Rojas, Blanco-Prieto, Bonilla-Correal, 3–15. doi: 10.3892/ijmm.2019.4188 Rodríguez-Gil, Miró and Yeste. This is an open-access article distributed under the Zheng, J. (2013). Molecular mechanism of TRP channels. Compr. Physiol. 3, terms of the Creative Commons Attribution License (CC BY). The use, distribution 221–242. doi: 10.1002/cphy.c120001 or reproduction in other forums is permitted, provided the original author(s) and Zorova, L. D., Popkov, V. A., Plotnikov, E. Y., Silachev, D. N., Pevzner, I. B., the copyright owner(s) are credited and that the original publication in this journal Jankauskas, S. S., et al. (2018). Mitochondrial membrane potential. Anal. is cited, in accordance with accepted academic practice. No use, distribution or Biochem. 552, 50–59. doi: 10.1016/j.ab.2017.07.009 reproduction is permitted which does not comply with these terms. Frontiers in Cell and Developmental Biology | www.frontiersin.org 18 December 2020 | Volume 8 | Article 588621