TYPE Original Research PUBLISHED 07 October 2022 DOI 10.3389/fcell.2022.930855 The effects of red LED light on pig OPEN ACCESS sperm function rely upon EDITED BY Jitendra Kumar Sundaray, Central Institute of Freshwater mitochondrial electron chain Aquaculture (ICAR), India REVIEWED BY activity rather than on a Gayatri Mohanty, University of Massachusetts Amherst, United States PKC-mediated mechanism Debora Cohen, Consejo Nacional de Investigaciones Olga Blanco-Prieto 1,2†, Carolina Maside 3,4†, Àlex Peña 1, Científicas y Técnicas (CONICET), Argentina Júlia Ibáñez-Príncep 1, Sergi Bonet 3,4, Marc Yeste 3,4,5*‡ and *CORRESPONDENCE Joan E. Rodríguez-Gil 1*‡ Marc Yeste, 1 marc.yeste@udg.edu Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Autonomous University Joan E. Rodríguez-Gil, of Barcelona, Barcelona, Spain, 2Department of Veterinary Medicine Science, University of Bologna, juanenrique.rodriguez@uab.cat Bologna, Italy, 3Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, Girona, Spain, 4Unit of Cell Biology, Department of † These authors share first authorship Biology, Faculty of Sciences, University of Girona, Girona, Spain, 5Catalan Institution for Research and ‡ These authors share senior authorship Advanced Studies (ICREA), Barcelona, Spain SPECIALTY SECTION This article was submitted to Molecular and Cellular Reproduction, a section of the journal While irradiation with red LED light has been reported to modulate sperm Frontiers in Cell and Developmental Biology function in different mammalian species, the mechanisms underlying their response are poorly understood. This work sought to provide new insights RECEIVED 28 April 2022 ACCEPTED 12 September 2022 into whether this effect relies on a direct action upon mitochondrial electron PUBLISHED 07 October 2022 chain and/or on PKC-linked mechanisms such as those related to opsins. For CITATION this purpose, pig semen was light-stimulated for 1, 5 or 10 min in the presence/ Blanco-Prieto O, Maside C, Àlex Peña, absence of antimycin A, an inhibitor of the mitochondrial electron chain, or PKC Ibáñez-Príncep J, Bonet S, Yeste M and ® Rodríguez-Gil JE (2022), The effects of 20–28 (PKCi), a PKC inhibitor. Antimycin A completely blocked the effects of red LED light on pig sperm function rely light at all the performed irradiation patterns. This effect was linked to a upon mitochondrial electron chain activity rather than on a PKC- complete immobility of sperm, which was accompanied with a significant mediated mechanism. (p < 0.05) drop in several markers of mitochondrial activity, such as JC-1 Front. Cell Dev. Biol. 10:930855. staining and O2 consumption rate. Antimycin A, however, did not affect doi: 10.3389/fcell.2022.930855 intracellular ATP levels, intramitochondrial calcium, total ROS, superoxides or COPYRIGHT © 2022 Blanco-Prieto, Maside, Àlex cytochrome C oxidase (CCO) activity. In the case of PKCi, it did also counteract Peña, Ibáñez-Príncep, Bonet, Yeste and the effects of light on motility, O2 consumption rate and CCO activity, but not to Rodríguez-Gil. This is an open-access the same extent than that observed for antimycin A. Finally, the effects observed article distributed under the terms of the Creative Commons Attribution License when sperm were co-incubated with antimycin A and PKCi were similar to (CC BY). The use, distribution or those observed with antimycin A alone. In conclusion, red LED light acts on reproduction in other forums is sperm function via a direct effect on mitochondrial electron chain. Additionally, permitted, provided the original author(s) and the copyright owner(s) are light-activated PKC pathways have a supplementary effect to that observed in credited and that the original the electron chain, thereby modulating sperm parameters such as motility and publication in this journal is cited, in accordance with accepted academic CCO activity. practice. No use, distribution or reproduction is permitted which does KEYWORDS not comply with these terms. sperm, mitochondria, antimycin A, PKC, light irradiation Frontiers in Cell and Developmental Biology 01 frontiersin.org Blanco-Prieto et al. 10.3389/fcell.2022.930855 Introduction contribute to explain why irradiating sperm with red light can increase their fertilizing ability, despite this impact not being In the last years, there has been increasing evidence about observed in all farms (Yeste et al., 2016; Blanco-Prieto et al., the effects of red light irradiation on sperm function, including 2019). in vivo fertilizing ability, in species from separate phyla such as Finally, a third mechanism would be related to the effect of birds, fishes and mammals (see Yeste et al., 2018 for review). red light on transmembrane proteins, particularly on ion Although, under a practical point of view, getting new insights channels pertaining to the Transient Receptor Potential into how light exerts the aforementioned effects could improve (TRP) family (Vriens et al., 2014). The TRP family includes in vivo reproductive efficiency, the mechanisms underlying a fairly big, heterogeneous number of transmembrane proteins that response remain largely unknown. In addition, previous that are involved in the control of thermotaxis in eukaryotic research has demonstrated that the sperm response to light cells (Vriens et al., 2014). In mammalian sperm, the depends on other factors, including the species, nature of TRPV4 vanilloid type of TRP receptor has been identified semen (i.e., fresh, frozen-thawed), wavelength range and as the most important temperature-sensitive ion channel color of the recipient/straw (Catalán et al., 2020a; Catalán (Mundt et al., 2018). This is of interest, because et al., 2020b; Blanco-Prieto et al., 2020; Catalán et al., 2020c; thermotaxis has been described as a very important Catalán et al., 2020d; Catalán et al., 2021). In the case of pigs, mechanism in the modulation of mammalian sperm different studies reported inconsistent results, as whereas function inside the female genital tract (Lottero-Leconte Luther et al. (2019) found an impairment of sperm et al., 2017). Additionally, considering the extreme function, others observed a positive impact on sperm sensitivity of mammalian sperm to temperature changes as quality and fertilizing ability (Yeste et al., 2016; Blanco- small as 0.0006°C (Hunter, 2012), a tight control of Prieto et al., 2019; Pezo et al., 2019). thermotaxis-modulating systems such as TRPV4 could be Thus far, irradiating sperm with a wavelength ranging from important for the regulation of sperm function after 610 nm to 640 nm (which corresponds to orange-to-red light) ejaculation. In fact, the relevance of thermotaxis is also has the most positive effects (see Rodríguez-Gil, 2019 for review). evidenced by the fact that, apart from TRPs, there are other The mechanisms through which red light could exert these transmembrane temperature-sensitive proteins, such as outcomes have been suggested to combine different pathways opsins, that are related to this mechanism (Pérez-Cerezales that would act simultaneously. On this respect, there are at least et al., 2015; Rodríguez-Gil, 2019). One of the most three separate mechanisms that could explain light effects. The conspicuous opsins detected in mammalian sperm is first mechanism would be related with a direct action of light on rhodopsin, which reaches its maximal sensitivity when separate cell structures through the modification of covalent stimulated by red light (Foster et al., 1991). For this reason, bonds. In effect, red light irradiation has been reported to it could be hypothesized that the action of red LED light on induce an increase in the energy stored in phosphodiester sperm rhodopsin could be related to thermotaxis. In addition, bonds present in molecules such as ATP, thus increasing the it is worth noting that TRPV4 and rhodopsin could have a energy stored by cells without modifying the total number of common pathway via which light would affect sperm. At this synthesized ATP molecules (Deng et al., 2020). While this direct respect, the transduction pathways triggered by both effect is not well understood, the light-induced additional energy TRPV4 and rhodopsin are related to the Ca2+ and stored in the covalent bonds of a great number of molecules in calmodulin-dependent protein kinase C (PKC; see Kelleher addition to ATP should have an impact on the overall cell and Johnson, 1986; Newton and Williams, 1991; Fan et al., function. In any case, more in-depth studies are needed to get 2009; Mundt et al., 2018). Because sperm mitochondrion plays further insights into this mechanism. a crucial role in calcium homeostasis (Rossi et al., 2019), the The second mechanism would be related to a direct action on impact of light on TRPV4 and rhodopsin could ultimately mitochondrial function, particularly, on different locations of the affect that organelle. electron chain. Indeed, both cytochrome C and cytochrome In the light of all the aforementioned, this work sought to P450 complexes are sensitive to light at 450 nm and determine to which extent the mitochondrial electron-chain and 610–630 nm/660–680 nm, respectively (Lynch and Copeland, the transduction pathways involving PKC, such as those 1992). Furthermore, it has been described that red LED light has activated by TRPV4 and rhodopsin, are involved in the sperm a direct effect on the mitochondrial electron chain of mammalian response to red light. For this purpose, pig sperm were irradiated sperm through the modulation of cytochrome C oxidase (CCO) with red LED light for 1, 5 or 10 min in the absence or presence of activity (Blanco-Prieto et al., 2020; Catalán et al., 2020d). Not antimycin A, a specific inhibitor of the cytochrome C reductase only could the light-induced impact on sperm mitochondria activity and thus mitochondrial electron chain (Alexandre and affect their energy levels, but also other important cell events such Lehninger, 1984), and/or PKC 20–28®, a specific blocker of PKC. as those related to the apoptotic-like changes occurring during Following this, plasma membrane and acrosome integrity, sperm capacitation (Aitken et al., 2015). This effect could motility, mitochondrial function (JC-1), O2 consumption, Frontiers in Cell and Developmental Biology 02 frontiersin.org Blanco-Prieto et al. 10.3389/fcell.2022.930855 CCO activity, and levels of ROS, calcium and ATP were In the case of intracellular ATP levels and activities of PKC evaluated. and CCO, samples were first centrifuged at 2,000×g and 20°C for 30 s and the resulting pellet was frozen in liquid N2. Subsequently, samples were stored at -80°C until use, which Materials and methods did not last more than 1 month. For the evaluation of O2 consumption, samples were also incubated with either 5 µM Animals and semen samples oligomycin A, a specific inhibitor of ATP synthase activity (Grüber et al., 1994), or 5 µM carbonyl cyanide-4 Sixteen semen samples coming from separate healthy (trifluoromethoxy) phenylhydrazone (FCCP), an uncoupling Piétrain boars (2–3 years old) were provided by a agent of the electron chain (Grasmick et al., 2018), alone commercial farm (Servicios Genéticos Porcinos, S.L. Roda (control) or together with PKCi and/or antimycin A. The use de Ter, Spain). Animals were housed under climate- of oligomycin A and FCCP aimed to gain further insights into controlled conditions, fed with a standard diet and how light stimulation affects O2 consumption rate. provided with water ad libitum. Ejaculates were collected through the gloved-hand method by specialized staff from the farm. The obtained sperm-rich fractions were immediately Determination of protein kinase C activity diluted at 17°C to a final concentration of 2×107 sperm/mL in a commercial extender (Duragen®, Magapor, S.L.; Ejea de los A first step was to determine total PKC activity to ascertain Caballeros, Zaragoza, Spain). Diluted samples were then split the ability of PKCi to induce its inhibitory effect. The description into 90-ml artificial insemination doses; two of these doses of this technique is provided in Supplementary File S1. were transported at 17°C to our laboratory. Transport of samples from the commercial farm to our laboratory lasted as much as 60 min and experimental procedures started upon Evaluation of sperm motility arrival. Because motility is one of the most relevant features of sperm, a careful analysis of this parameter was needed. As in the case of Experimental design PKC activity, the description of this technique is shown in Supplementary File S1. All experiments were performed using eight independent biological samples, each resulting from two separate seminal doses. These samples came from eight Flow cytometry different boars, all from the Pietrain breed. Samples were first checked to fulfil the following quality standards: ≥80% Flow cytometry analysis was performed using a CytoFLEX membrane-intact spermatozoa (SYBR14 + /PI − ), ≥85% cytometer (Beckman Coulter, California, United States). morphologically normal spermatozoa, and ≥80% total Sperm viability (SYBR-14/PI), mitochondrial membrane motile spermatozoa. Thereafter, samples were split into potential (JC-1), total intracellular ROS levels (H2DCFDA/ four separate 1.5-ml aliquots and were either incubated PI), superoxides (HE/YO-PRO-1), acrosome membrane for 10 min at 20 °C in the dark (Control) or irradiated integrity (PNA-FITC/PI) and intracellular calcium levels with red LED light at a wavelength range of 620–630 nm (Fluo3-AM/PI) were evaluated. In all cases, sperm were (PhastBlue ®, IUL, S.L.; Barcelona, Spain) for 1 min (1′), previously diluted with pre-warmed PBS to a final 5 min (5′) or 10 min (10’). In all cases, the temperature concentration of 1×106 sperm per mL before they were within the PhastBlue ® system was maintained at 20 °C. In stained with the corresponding protocol. SYBR-14, Fluo3- addition to the aforementioned, samples were exposed to AM, YO-PRO-1, PNA-FITC, H2DCFDA and JC-1 the same four protocols (i.e., control, or irradiation for 1, monomers were excited with the 488 nm laser and their 5 or 10 min) in the presence of 100 nM of PKC 20–28 ® fluorescence was detected by the FITC channel (525/40). (PKCi; Sigma Aldrich; St. Louis, MO, United States, HE and JC-1 aggregates were excited with the 488 nm laser catalog number 476480), 1 µM antimycin A (Sigma and its fluorescence was detected through the PE channel (585/ Aldrich, catalog number A8674), or both inhibitors 42). PI was excited with the 488 nm laser and its fluorescence together. Following this, motility; plasma membrane and was detected by the PC5.5 channel (690/50). acrosome integrity; mitochondrial membrane potential; Sperm viability was determined by analyzing plasma intracellular levels of ROS, calcium and ATP; O 2 membrane integrity following incubation with SYBR-14 consumption rate; and activities of PKC and cytochrome (final concentration: 31.8 nM) and PI (final concentration: C oxidase (CCO) were determined. 7.6 µM) at 38°C for 10 min, as described by Garner and Frontiers in Cell and Developmental Biology 03 frontiersin.org Blanco-Prieto et al. 10.3389/fcell.2022.930855 Johnson (1995). Sperm with an intact plasma membrane Evaluation of mitochondrial membrane (SYBR14+/PI−) were distinguished from those showing potential by confocal microscopy different degrees of plasma membrane alterations (SYBR14-/PI+ and SYBR14+/PI+). Not only were sperm stained with JC-1 analyzed Mitochondrial membrane potential was evaluated through flow cytometry, but they were also observed following a modified protocol from Ortega-Ferrusola et al. under a Leica TCS 4D confocal laser scanning microscope (2007). All samples were incubated with JC-1 (final (Leica Lasertechnik; Wertrieb, Germany) adapted to an concentration: 750 nM) at 38°C in the dark for 30 min. inverted Leitz DMIRBE microscope at 63× (NA = 1.4 oil) High mitochondrial membrane potential leads to the Leitz Plan-Apo Lens (Leitz; Stuttgarts, Germany) in which formation of JC-1 aggregates that emit orange fluorescence, the light source was an argon/krypton laser whereas low mitochondrial membrane potential keeps JC-1 as (74 mW). Observations were carried out in 4-well plates, monomers that emit green fluorescence. each well containing 20 µL of sample. Fluorescence was Total ROS levels were determined through co-staining excited at 488 nm and detected at 530 nm for green with H2DCFDA (final concentration: 100 µM) and PI (final emission and at 590 nm for the orange/red one. In addition, concentration: 6 µM) at 38°C for 20 min, as described by motility of stained sperm was recorded through sequential Guthrie and Welch (2006). The percentage and geometric tracking at a velocity of one capture/s for 20 s (Blanco- mean of fluorescence intensity of DCF+ in the subpopulation Prieto et al., 2020). All biological replicates (n = 8) and of viable sperm with high ROS levels (DCF+/PI−) were treatments were examined, with a minimum of 200 sperm recorded. per sample. The assessment of intracellular superoxide levels was performed by co-staining with HE (final concentration: 5 µM) and YO-PRO-1 (final concentration: 31.25 nM) at 38°C in the Determination of cytochrome C oxidase dark for 20 min, following a modification of the protocol of (CCO) activity Guthrie and Welch (2006). The percentage and geometric mean of fluorescence intensity of E+ in the subpopulation of viable Activity of COO was analyzed in mitochondria-enriched sperm with high superoxide levels (E+/YO-PRO-1-) were sperm fractions, as described in Mclean et al. (1993). For this recorded. purpose, frozen pellets were solubilized in 500 µl of ice-cold Acrosome membrane integrity was evaluated following a PBS and then sonicated under the same conditions as those procedure modified from Nagy et al. (2003). Sperm samples described for PKC activity. Thereafter, 500 µl of a 1.055 mg/ml were incubated with PNA-FITC (final concentration: 1.17 µM) Percoll® solution in PBS at 4°C was placed onto each and PI (final concentration: 5.6 µM) at 38°C in the dark for homogenate. Samples were subsequently centrifuged at 10 min. This procedure allowed the identification of four 3,000×g and 10°C for 45 min. As a result, a mitochondria- subpopulations. One of these subpopulations corresponded to enriched fraction appeared as a tight cloudy band that viable sperm with an intact acrosome membrane (PNA-FITC-/ separated the aqueous phase from the Percoll®-enriched PI−), and the other three comprised those sperm cells that had one. This band was carefully collected with a micropipette different degrees of alteration in plasma membrane and and transferred into 1.5-ml tubes, which were centrifuged at acrosome membranes (PNA-FITC+/PI−, PNA-FITC-/PI+ and 12,000×g and 20°C for 2 min. As a final step, resulting pellets PNA-FITC+/PI+). were resuspended in 100 µL PBS at 20°C. Mitochondria- Intracellular calcium levels of sperm were determined enriched suspensions were finally split into two separate following the protocol described by Harrison et al. (1996) aliquots. The first aliquot was used to determine CCO as modified by Yeste et al. (2015). Samples were incubated activity through spectrophotometry with a commercial kit 10 min at 38°C with Fluo3-AM (final concentration: (Cytochrome C Oxidase Assay Kit; Sigma-Aldrich; 1.2 µM) and PI (final concentration: 5.6 µM) at 38°C catalogue number CYTOCOX1). The other aliquot, which in the dark for 10 min. Four sperm subpopulations had a volume of 10 μL, was used to determine the total were identified: 1) viable sperm with low calcium levels protein content through a commercial kit based on the (Fluo3-/PI−); 2) viable sperm with high calcium levels Bradford method (Bradford, 1976), as described above. (Fluo3+/PI−); 3) non-viable sperm with low calcium levels Enzyme activity in each sample was calculated after (Fluo3-/PI+); and 4) non-viable sperm with high calcium normalizing the obtained spectrophotometric values against levels (Fluo3+/PI+). the total protein content. Frontiers in Cell and Developmental Biology 04 frontiersin.org Blanco-Prieto et al. 10.3389/fcell.2022.930855 of ice-cold 10 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (HEPES) buffer containing 250 mM sucrose (pH 7.4), following the protocol set by Chida et al. (2012). Solubilized pellets were then sonicated with a Bandelin Sonopuls HD 2070 system (Bandelin Electronic GmbH and Co.) at a frequency of 10 kHz (20 pulses); samples were kept on ice at 4°C to avoid specimen heating. Following this, samples were centrifuged at 1,000×g and 4°C for 10 min and supernatants were collected. Twenty µl of each supernatant were used to determine the total protein content through the Bradford method (Bradford, 1976), using a commercial kit (Bio-Rad laboratories). The remaining volume was thoroughly mixed with 300 µl of ice-cold 10% (v:v) trichloroacetic acid, and then incubated at 4°C for 20 s. Subsequently, samples were centrifuged at 1,000×g and 4°C for 30 s, and the resulting supernatants were carefully separated from the pellet. These supernatants were again centrifuged at 1,000×g and 4°C for 10 min, obtaining a small, white pellet. The pellet was discarded, and the supernatant was mixed with two volumes of 1 M Tris-acetate buffer (pH = 7.75). Concentration of ATP in these resulting supernatants was determined through an Invitrogen® ATP Determination Kit (ThermoFisher Bioscientific; Waltham, United States; catalogue number: A22066), following the manufacturer’s instructions. Samples were transferred into a 96-well microplate for fluorescence-based assays (Invitrogen®), and ATP levels were measured in an Infinite F200 fluorimeter (TECAN®; Männedorf, Switzerland). Data were normalized against the total protein content determined by the Bradford method. Determination of O2 consumption rate FIGURE 1 Proportions of motile sperm subpopulations following incubation with or without PKCi and further irradiation of sperm The O2 consumption rate was determined with a with red LED light. Sperm were not irradiated (No Photo) or SensorDish® Reader (SDR) system (PreSens Gmbh; irradiated for 1 min (1 min), 5 min (5 min) or 10 min (10 min) in Regensburg, Germany), as described in Blanco-Prieto et al. the absence (CONTROL) or presence of 100 nM PKCi (PKCi). White sections: percentages of sperm included in Subpopulation 1. Grey (2020). For this purpose, 1-ml aliquots were transferred into a sections: percentages of sperm included in Subpopulation 2. Black specifically-designed Oxodish® OD24 plates (24 wells/plate). sections: percentages of sperm included in Subpopulation 3. Total number of analyzed sperm was 10,237. Different superscript letters Plates were sealed with Parafilm® and then placed into the indicate significant (p < 0.05) differences between samples that SDR device, which was introduced in an incubator at 38°C. were light-irradiated with different patterns (i.e., 1, 5 or 10 min) and The O2 consumption was evaluated once a min for 2 h and, those that were not irradiated (i.e., 0 min), regardless of whether they were previously incubated with PKCi. Different superscript when finished, data were exported to an Excel file. The O2 numbers indicate significant (p < 0.05) differences between consumption rate was calculated as the decrease of O2 samples incubated with PKCi and those non-incubated with the concentration in each well during the 2-h incubation period, inhibitor, and irradiated with the same pattern (i.e., 1, 5 or 10 min). Eight biological replicates involving eight independent samples normalized against the percentage of viable sperm (SYBR14+/ were examined. PI−) determined by flow cytometry. As mentioned in the Experimental design section and to delve further into how irradiation with red light in the presence of antimycin A and/or PKCi affects O2 consumption, sperm were Determination of ATP levels also incubated with 5 µM oligomycin A, a specific ATPase inhibitor, or 5 µM FCCP, a specific disruptor of the electron For determination of ATP levels, pellets prepared as chain, as described in Blanco-Prieto et al. (2020). The inclusion of described previously were thawed and resuspended in 300 µL these additional samples allowed us to determine the following Frontiers in Cell and Developmental Biology 05 frontiersin.org Blanco-Prieto et al. 10.3389/fcell.2022.930855 parameters (Supplementary Figure S1): 1) non-mitochondrial O2 consumption (NMC), which corresponded to O2 consumption recorded in the presence of 1 µM antimycin A; 2) basal respiratory rates (BR), which were the values obtained in the absence of any inhibitor/disruptor; 3) oligomycin-resistant O2 consumption (OC), which corresponded to samples incubated with 5 µM oligomycin A; 4) Maximal respiration (MR), which corresponded to samples incubated with 5 µM FCCP; 5) H+ leakage (HL): OC-NMC; 6) ATP turnover (AT): BR-HL; and 7) Spare respiratory capacity (SRC): MR-BR. Total O2 consumption rate resulted from the sum of NMC, BR and SRC, and, for all experimental conditions, the percentage of each of these components was calculated. Statistical analyses Statistical analyses were conducted with a statistical package (SPSS® Ver. 25.0 for Windows; IBM corp., Armonk, NY, United States). The first step was to test normality and homogeneity of variances through Shapiro- Wilk and Levene tests, respectively. When required, data were linearly transformed with √x or arcsin √x. The effects of light- stimulation and the presence/absence of antimycin A and PKCi on all variables (motility, plasma membrane and acrosome integrity, MMP, calcium levels, total ROS, superoxides, activities of PKC and CCO, ATP, and O2 consumption rate, including NMC, BR and SRC) were determined through a two-way analysis of variance (ANOVA) followed by post-hoc Sidak test. In this model, factors were: 1) light-stimulation pattern (i.e. non-irradiation, irradiation for 1 min, irradiation for 5 min, and irradiation for 10 min); and 2) presence/absence of inhibitors (control, FIGURE 2 Box-and-whisker plot of the effects of antimycin A and PKCi on antimycin A and PKCi, as well as oligomycin A and FCCP mitochondrial membrane potential. Sperm were subject to separate in the case of O2 consumption rate analysis). red LED light irradiation patterns in the presence of 1 µM antimycin A, 100 nM PKCi or both together, and parameters related to MMP On the other hand, motile sperm subpopulations were also were analyzed as described in Materials and Methods section. Sperm determined as described in Luna et al. (2017). Kinematic were not irradiated (white boxes) or irradiated with red LED light for parameters corresponding to each sperm cell (VCL, VSL, 1 min (light grey boxes), 5 min (dark grey boxes) or 10 min (black boxes) in the absence (C) or presence of 1 µM antimycin A (AA), VAP, BCF, ALH, DNC, absMAD and algMAD) were used to 100 nM PKCi (P) or 1 µM antimycin A and 100 nM PKCi together (AA + run a Principal Component Analysis (PCA). The obtained matrix P). (A) percentages of sperm with high MMP. (B) Mean values of the JC-1agg/JC-1mon ratio in the sperm population with high MMP. Figure was rotated through the Varimax method and normalized with shows means ± S.E.M. for eight biological replicates involving eight the Kaiser approach. The regression scores assigned to each independent samples. Asterisks inside boxes indicate means, sperm cell for the new PCA components were used to run a whereas lines inside represent medians. Different superscript numbers indicate significant (p < 0.05) differences between samples two-step cluster analysis (log-likelihood distance; Schwarz’s pre-incubated with the same inhibitor (i.e., Control, antimycin A, PKCi Bayesian Criterion). Three motile sperm subpopulations were or antimycin A + PKCi) but subject to distinct irradiation patterns identified (SP1, SP2 or SP3) and the proportions of each of these (i.e., 0, 1, 5 or 10 min). Different superscript letters indicate significant (p < 0.05) differences between samples incubated with separate subpopulations were used to run a two-way ANOVA and post- inhibitors (Control, antimycin A, PKCi, or antimycin A + PKCi) and hoc Sidak test. irradiated with the same pattern. In all analyses, the level of significance was set at p ≤ 0.05. Frontiers in Cell and Developmental Biology 06 frontiersin.org Blanco-Prieto et al. 10.3389/fcell.2022.930855 Results Total PKC activity Neither light-stimulation, regardless of the pattern, nor the presence of antimycin A modified PKC activity. More details are provided in Supplementary File S1. Sperm motility Total and progressive sperm motility were not affected by irradiation with red light; these results are further described in Supplementary File S1. Regarding the structure of motile sperm subpopulations, three sperm subpopulations (SP) were identified, and their descriptive parameters are shown in Supplementary Table S2. These SPs were named in a descending order according to their VCL values. Proportions of SP1 (the fastest, based on VCL), SP2 and SP3 (the slowest) in FIGURE 3 non-irradiated samples without any inhibitor were 28.6% ± 8.1%, Box-and-whisker plot of the effects of antimycin A and PKCi 51.4% ± 6.7% and 20.1% ± 8.1%, respectively (Figure 1). While on intracellular levels of total ROS and superoxides. Sperm were subject to separate red LED light irradiation patterns in the these proportions remained unaffected when samples were presence of 1 µM antimycin A, 100 nM PKCi or both together, irradiated for 1 min, those of SP1 increased and those of and parameters related to ROS were analyzed as described in SP2 decreased when they were exposed to red light for 5 min Materials and Methods section. Sperm were not irradiated (white boxes) or irradiated with red LED light for 1 min (light grey boxes), or 10 min (p < 0.05; Figure 1). Remarkably, pre-incubation of 5 min (dark grey boxes) or 10 min (black boxes) in the absence (C) sperm with PKCi abolished the aforementioned increase in the or presence of 1 µM antimycin A (AA), 100 nM PKCi (P) or 1 µM proportions of SP1 observed when samples were irradiated for antimycin A and 100 nM PKCi together (AA + P). (A) percentages of viable sperm with high ROS levels. (B) Fluorescence intensity of 5 min or 10 min (Figure 1). DCF+ in the population of viable sperm with high ROS levels (DCF+/ PI−). (C) percentages of viable sperm with high superoxide levels. (D) Fluorescence intensity of E+ in the population of viable sperm with high superoxide levels (E+/YO-PRO-1-). Figure shows Plasma membrane and acrosome integrity means ± S.E.M. for eight biological replicates involving eight independent samples. Asterisks inside boxes indicate means, whereas lines inside represent medians. Irradiation of sperm with red LED light did not affect plasma membrane or acrosome integrity. More information about the evaluation of these two sperm variables is provided in Supplementary File S1. sperm population with high MMP significantly (p < 0.05) decreased after irradiation for 1 min (non-irradiated, control Mitochondrial membrane potential samples: 0.56 ± 0.01 vs. irradiation for 1 min: 0.77 ± 0.03; see Figure 2B). In both non-irradiated and irradiated samples, pre- The percentage of sperm with high MMP, which in non- incubation with antimycin A, either alone or together with PKCi, irradiated control samples was 71.0% ± 4.6%, did not vary when also induced a significant (p < 0.05) decrease in this ratio sperm were pre-incubated with PKCi (Figure 2A). Light (Figure 2B). In contrast, pre-incubation with PKCi had no irradiation induced a significant (P˂0.05) decrease in the effect, either in non-irradiated or irradiated samples (Figure 2B). percentage of sperm with high MMP at all tested irradiation When sperm stained with JC-1 were examined under a times (Figure 2A). Preincubation with PKCi did not significantly confocal laser scanning microscope, orange-stained modified the light-induced effect. Otherwise, pre-incubation mitochondria were mainly located at the two mid-piece poles with antimycin A, either alone or together with PKCi, led to in control samples and in those pre-incubated with PKCi an almost complete reduction of the percentage of sperm with (Supplementary Figure S4). Conversely, sperm pre-incubated high MMP in both control and irradiated samples, regardless of with antimycin A did not show orange-stained mitochondria, the irradiation time (Figure 2A). but all were uniformly stained in green (Supplementary Figure In the absence of inhibitors, the ratio between JC-1 S4). No irradiation pattern modified these specific distributions aggregates and JC-1 monomers (JC-1agg/JC-1mon) in the (data not shown). Frontiers in Cell and Developmental Biology 07 frontiersin.org Blanco-Prieto et al. 10.3389/fcell.2022.930855 Total ROS The percentage of viable sperm with high total ROS levels in non-irradiated, control samples was 15.1% ± 8.4% (DCF+/ PI−; Figure 3A). No significant differences in this percentage or in the geometric fluorescence intensity of DCF+ in the subpopulation of viable sperm with high ROS were observed following irradiation or when samples were pre- incubated with PKCi. Sperm pre-incubated with antimycin A, however, and irradiated for 5 min or 10 min showed a significantly (p < 0.05) lower geometric mean fluorescence of DCF+-intensity than those irradiated for 1 min (Figure 3B). Superoxides The percentage of viable sperm with high superoxide levels was very low in non-irradiated, control sperm, and lower than that of viable sperm with total ROS (1.6% ± 0.6%; Figure 3C). While irradiation did not induce a significant change in this parameter, percentages of viable sperm with high superoxide levels in samples pre-incubated with antimycin A were FIGURE 4 significantly (p < 0.05) lower after irradiation for 1 min or Box-and-whisker plot of the effects of antimycin A and PKCi 10 min than when not exposed to light (Figure 3C). Finally, on intracellular ATP levels and total cytochrome C oxidase activity. Sperm were subject to separate red LED light irradiation patterns in the geometric mean fluorescence of E+-intensity in the the presence of 1 µM antimycin A, 100 nM PKCi or both subpopulation of viable sperm with high superoxide levels together, before intracellular ATP levels and cytochrome C (E+/YO-PRO-1-) was not affected by irradiation patterns or oxidase activity were evaluated as described in Materials and Methods section. Sperm were not irradiated (white boxes) or the presence of antimycin A or PKCi (Figure 3D). irradiated with red LED light for 1 min (light grey boxes), 5 min (dark grey boxes) or 10 min (black boxes) in the absence (C) or presence of 1 µM antimycin A (AA), 100 nM PKCi (P) or 1 µM antimycin A and 100 nM PKCi together (AA + P). (A) intracellular ATP levels. (B) Cytochrome C oxidase activity and cytochrome C oxidase activity. Figure shows means ± S.E.M. for intracellular ATP levels eight biological replicates involving eight independent samples. Asterisks inside boxes indicate means, whereas lines inside represent medians. In Figure 4A, different superscript numbers As shown in Figure 4A, irradiation of sperm for 5 min indicate significant (p < 0.05) differences between samples pre- significantly (p < 0.05) increased CCO activity (irradiation for incubated with the same inhibitor (i.e., Control, antimycin A, PKCi or antimycin A + PKCi) but subject to distinct irradiation patterns 5 min: 65.2 mIU/mg protein±19.1 mIU/mg protein vs. non- (i.e., 0, 1, 5 or 10 min). Different superscript letters indicate irradiated, control sperm: 28.1 mIU/mg protein±9.6 mIU/mg significant (p < 0.05) differences between samples incubated with separate inhibitors (Control, antimycin A, PKCi, or antimycin A + protein). Yet, this increase was abolished when samples were PKCi) and irradiated with the same pattern. In the case of Figure 4B, previously incubated with PKCi. Moreover, no changes in superscripts were not needed as no significant differences were CCO activity were observed in samples pre-incubated with found. antimycin A. Otherwise, neither light-stimulation nor antimycin A or PKCi altered intracellular ATP levels, which in non-irradiated, control samples were 15.0 nmol/mg Intracellular calcium levels protein±2.7 (Figure 4B). As shown in Supplementary Figure S5, the percentage of viable sperm with high calcium levels was low in non-irradiated, O2 consumption rate control samples (0.55% ± 0.20%). Neither irradiation nor antimycin A or PKCi affected that percentage or the Irradiation of sperm for either 5 min or 10 min induced a geometric mean intensity of Fluo3+ in the subpopulation of significant (p < 0.05) increase in total O2 consumption rate (non- viable sperm with high calcium (Fluo3+/PI−; Supplementary irradiated, control samples. 1.11 nmol O2×h/106 viable Figure S5B). sperm±0.06 nmol O2×h/106 viable sperm vs. irradiation for Frontiers in Cell and Developmental Biology 08 frontiersin.org Blanco-Prieto et al. 10.3389/fcell.2022.930855 FIGURE 5 Box-and-whisker plot of the effects of red LED light irradiation on O2 consumption rate. Sperm were subject to separate red LED light irradiation patterns before total O2 consumption rate was analyzed and divided into its components as described in Materials and Methods section. Sperm were not irradiated (white boxes) or irradiated with red LED light for 1 min (light grey boxes), 5 min (dark grey boxes) or 10 min (black boxes). (A) total consumption rate, defined as the sum of non-mitochondrial O2 consumption, basal respiratory rate and spare respiratory capacity. (B) basal respiration. (C) maximal respiration. (D) spare respiratory capacity. (E) ATP turnover. (F) H+ leak. (G) non-mitochondrial O2 consumption. Figure shows means ± S.E.M. for 8 separate experiments. Figure shows means ± S.E.M. for eight biological replicates involving eight independent samples. Asterisks inside boxes indicate means, whereas lines inside represent medians. Different superscript letters indicate significant (p < 0.05) differences between irradiation patterns (i.e., 0, 1, 5 or 10 min). Superscripts were not needed in Figure 5G, as no significant differences were found. 10 min: 2.09 nmol O2×h/106 viable sperm±0.04 nmol O2×h/106 rate, which were concomitant with a significant (p < 0.05) viable sperm; Figure 5A, Figure 6). This increase in total O2 decrease in the reserve capacity (Figures 5B–D). In contrast, consumption rate was based upon significant (p < 0.05) increases light-stimulation did not affect H+ leakage or non-mitochondrial in basal and maximal levels of respiration, and ATP turnover O2 consumption (Figure 5E). On the other hand, while pre- Frontiers in Cell and Developmental Biology 09 frontiersin.org Blanco-Prieto et al. 10.3389/fcell.2022.930855 Discussion Our results support that, whereas the mitochondrial electron chain is instrumental in explaining the effects of red light effects on sperm, those related to PKC only have a minor modulating role (mainly restricted to motility and CCO activity). Related with this, it is worth mentioning that previous research already suggested that indirect inhibition of the mitochondrial electron chain through blocking of ATP synthase induces effects compatible to those observed herein (Blanco-Prieto et al., 2020; Catalán et al., 2020d). The cytochrome C complex appears to be the most important component of the electron FIGURE 6 Box-and-whisker plot of the effects of antimycin A, PKCi, chain to explain the sperm response to red light, as it can be oligomycin A and FCCP on total O2 consumption rate. Sperm excited at 610–630 nm and 660–680 nm (Hollis et al., 2003). This were subject to separate red LED light irradiation patterns in the reactivity could explain the light-induced activation of CCO presence of 1 µM antimycin A, 100 nM PKCi, 1 µM antimycin A+100 nM PKCi, 5 µM oligomycin A or 5 µM FCCP. Total observed after 5 min of irradiation. Furthermore, the fact that O2 consumption rate, defined as the sum of non-mitochondrial the activity of CCO was significantly increased after 5 min, but O2 consumption, basal respiratory rate and spare respiratory capacity, was analyzed as described in Materials and Methods not after 1 min and 10 min, would indicate that the specific section. Sperm were not irradiated (white boxes) or irradiated with amount of energy absorbed by the cytochrome C complex red LED light for 1 min (light grey boxes), 5 min (dark grey boxes) or follows the Arndt-Schulz curve (Lubart et al., 2006). 10 min (black boxes) in the absence (C) or presence of 1 µM antimycin A (AA), 100 nM PKCi (P) or 1 µM antimycin A+100 nM It is worth mentioning that antimycin A, which was used to PKCi (AA + P). Figure shows means ± S.E.M. for eight biological inhibit the mitochondrial electron chain, acts through binding replicates involving eight independent samples. Asterisks inside boxes indicate means, whereas lines inside represent medians. the Qi site of the cytochrome C reductase (CCR), a component of Different superscript numbers indicate significant (p < 0.05) the cytochrome C complex included in Complex III (Huang differences between samples pre-incubated with the same et al., 2005). Inhibition of CCR activity by antimycin A blocks the inhibitor (i.e., Control, antimycin A, PKCi or antimycin A + PKCi) but exposed to distinct irradiation patterns (i.e., 0, 1, 5 or 10 min). oxidation of ubiquinol into ubiquinone, which, in turn, stops the Different superscript letters indicate significant (p < 0.05) entire electron chain (Huang et al., 2005). The fact that antimycin differences between samples incubated with separate inhibitors A blocked CCR would agree with the absence of response to red (Control, antimycin A, PKCi, antimycin A + PKCi, oligomycin A or FCCP) and irradiated with the same pattern. Incubation with light when samples were previously incubated with this inhibitor. oligomycin A and FCCP was needed to determine basal Moreover, not only was the abolishment of sperm response to red respiration, maximal respiration, spare respiratory capacity, ATP turnover, H+ leak and non-mitochondrial O2 consumption. light in the presence of antimycin A apparent in the analysis of CCO activity, but also on MMP and intracellular superoxide levels. Interestingly, superoxide levels were more affected than total ROS when sperm were pre-incubated antimycin A. Total incubation with antimycin A, either alone or in combination with ROS include other reactive species, such as hydrogen peroxide PKCi, almost suppressed mitochondrial O2 consumption, this and hydroxyl radical. While superoxide anion is known to be inhibition was not counteracted by red light irradiation generated in complexes I and III of the electron chain (Brand (Figure 6). Pre-incubation with PKCi significantly (p < 0.05) et al., 2004), hydrogen peroxide results from the conversion of increased O2 consumption rate in non-irradiated samples (PKCi: the previously formed superoxides by the superoxide dismutase 1.54 nmol O2×h/106 viable sperm±0.04 nmol O2×h/106 viable complex (Boveris and Cadenas, 2000). Therefore, while the sperm vs. control: 1.11 nmol O2×h/106 viable sperm±0.06 nmol generation of superoxides is directly linked to the activity of O2×h/106 viable sperm; Figure 6). Pre-incubation with PKCi, complex III, which is inhibited by antimycin A, total ROS levels nevertheless, prevented the aforementioned increase in total O2 are only related indirectly. Additionally, whereas the effect of consumption observed after irradiation for 5 min or 10 min antimycin A on the electron chain was expected, that of the PKC (Figure 6). This effect occurred together with a decrease in inhibitor was less clear. One could indicate, on this respect, that SRC without affecting AT (Supplementary Figure S6A). When ROS are known to regulate PKC activity, which is in turn linked sperm were pre-incubated with PKCi and were subsequently to the modulation of mitochondria-controlled functions as irradiated for 1 min or 5 min, a decrease in BR and an increase in relevant as apoptosis (see Cosentino-Gomes, 2012 for review). SRC were observed (p < 0.05; Supplementary Figure S6B). These In fact, a direct relationship between the activity of PKC, which is effects were less apparent when samples were irradiated for also present inside mitochondria, and mitochondrial ROS, which 10 min, as only the increase in SRC was significant (p < 0.05; is a by-product of the electron chain, has been established Supplementary Figure S6A). Finally, neither PKCi nor antimycin (Pakrash Mandal et al., 2021). Yet, while the present study A had an impact on NMC (Supplementary Figure S6C). supports the existence of a link between PKC and the electron Frontiers in Cell and Developmental Biology 10 frontiersin.org Blanco-Prieto et al. 10.3389/fcell.2022.930855 chain, the exact mechanism of such a connection remains the achievement of in vitro capacitation of pig sperm, including unknown. the achievement of hyperactivated motility patterns (Martínez- Samples pre-incubated with antimycin A and irradiated for Abad et al., 2017). In both cases, capacitation is accompanied 5 min or 10 min altered the integrity of plasma membrane, with a hike of intra-mitochondrial Ca2+ levels (Martínez-Abad although no effects on that of the acrosome were observed. A et al., 2017; Sun et al., 2017). This increase was not observed in potential explanation could be the accumulation of energy in our conditions, but this is reasonable considering that sperm sperm incubated for 5 min or 10 min when the cytochrome C were maintained in basal, non-capacitating conditions. The complex was blocked by antimycin A. Because red light has been electron chain arrest induced by antimycin A, nonetheless, reported to increase the percentage of sperm that reach the would cause a severe change in mitochondria membrane capacitated status (Yeste et al., 2016), it is reasonable to electric polarity, due to the lack of H+ flux (Alberts et al., suggest that this accumulation of energy could elicit some 2002). This alteration would affect the activity of para-apoptotic signal/s that would ultimately cause these mitochondrial voltage channels, such as VDAC2, and, in turn, alterations in the plasma membrane. In fact, cytochrome C is the sperm functional parameters that depend on proper cation released upon permeabilization of mitochondrial matrix in one trafficking, such as motility (Lishko et al., 2012), which would of the earliest apoptotic-like changes (Martinou and Green, 2001; ultimately lead to immobility. Thus, while the overall intra- Waterhouse et al., 2002). Because sperm capacitation shares mitochondrial calcium levels would not be affected, calcium some molecular mechanisms with apoptosis (Aitken et al., trafficking through mitochondrial membranes would be 2015) and red light has been reported to affect the ability of altered, thus affecting the potential needed for the sperm to reach the capacitated status, it could be that these maintenance of motility (Bulkeley et al., 2021). changes in plasma membrane integrity would result from an According to our results, whereas PKCi did not affect sperm activation of a para-apoptotic mechanism, which other authors motility, CCO activity or O2 consumption in non-irradiated have named ‘spermptosis’ (Ortega-Ferrusola et al., 2017). samples, it did counteract the increase observed in the Changes observed in sperm motility also deserve an irradiated ones. A strong relationship between PKC activity explanation. The effects of irradiation with red light observed and sperm motility has previously been reported (Naor and herein were similar to those reported previously (Yeste et al., Breitbart, 1997). In our experiment, however, PKC inhibition 2016; Pezo et al., 2019; Blanco-Prieto et al., 2020) and, as was not complete, which would suggest that, in basal conditions, expected, pre-incubation with antimycin A led to sperm sperm motility would only require low PKC activity. This would immobilization. In spite of this, this immobilization was not be in accordance with previous reports, where sperm motility was linked to a remarkable drop in intracellular energy, as ATP levels found to be maintained at low PKC activity (Naor and Breitbart, were not significantly altered by antimycin A. These results were 1997). In this way, this basal PKC activity, which would partially similar to those observed when sperm were incubated with remain in the presence of PKCi, would be enough to maintain the oligomycin A, before and after inducing in vitro sperm observed basal motion characteristics. capacitation (Ramió-Lluch et al., 2013; Blanco-Prieto et al., Analysis of O2 consumption rate in non-irradiated, control 2020), even though the extent of that immobilization was sperm indicated that this parameter can be much increased, as its larger in the case of antimycin A than in that of oligomycin rise after light-stimulation demonstrated. In this context, A. The fact that ATP levels were maintained even when electron interrogating on the role of this increase with regard to the chain (antimycin A) or ATP synthase (oligomycin A) were lifespan of pig sperm is relevant. Previous studies demonstrated blocked is not surprising, because most of the ATP produced that, while O2 consumption rate is low - even after achieving the in non-capacitated pig sperm comes from glycolysis, and only 3% capacitated status (Ramió-Lluch et al., 2013; Blanco-Prieto et al., at the most is generated by oxidative phosphorylation (Marín 2020) -, a transient increase occurs upon triggering the acrosome et al., 2003). These data, therefore, support that the regulation of reaction (Ramió-Lluch et al., 2013). This peak could explain the sperm motility by mitochondria is not entirely related to the ATP high SRC observed in pig sperm, which require a great spare produced by oxidative phosphorylation. Rather, other factors capacity to cover the sudden need for energy linked to acrosome such as cation trafficking and intracellular redox balance could be exocytosis, as indicated by the O2 consumption peak (Ramió- involved in the regulation of sperm motility. In fact, voltage- Lluch et al., 2013). This process could be different in species dependent Ca2+ channels, such as CatSper and mitochondrial where oxidative phosphorylation provides more ATP than in the VDAC2, are involved in the regulation of sperm motility (Ren pig, such as mice or humans (Mukai and Okuno, 2004; et al., 2001). This is important because mitochondria are the most Nascimento et al., 2008). Remarkably, pre-incubation of important storage place for Ca2+ in sperm (Costello et al., 2009). sperm with PKCi counteracted the increase in O2 Hence, the achievement of hyperactivated motility during consumption rate observed in irradiated samples. On this capacitation requires a proper flux of Ca2+ through CatSper respect, it is important to highlight that PKC depends on that would increase mitochondria activity (Sun et al., 2017). mitochondrial-related proteins, such as the mitochondrial, Likewise, mitochondria voltage channel VDAC2 is involved in ATP-sensitive K+ channel mitoKATP, involved in the Frontiers in Cell and Developmental Biology 11 frontiersin.org Blanco-Prieto et al. 10.3389/fcell.2022.930855 regulation of MMP and redox homeostasis (Queliconi et al., funding acquisition, project administration, data curation, 2011). Based on these findings and given that the response of writing of original draft and supervision. sperm to irradiation mediated by TRPV4and rhodopsin is related to PKC, one could suggest that the inhibition of PKC could ultimately affect the mitochondrial activity via modulation of Funding proteins such as mitoKATP. In summary, our results indicated that the response of sperm This work was supported by the Ministry of Science and to red light is largely reliant upon the mitochondrial electron Innovation, Spain (grants: AGL2017-88329-R and PID2020- chain, since the complete blockage of that chain by antimycin A 113320RB-I00), the Catalan Agency for Management of prevented light action and was not countered by irradiation. This University and Research Grants, Regional Government of response would be related to a direct action of light on Catalonia, Spain (Grant: 2017-SGR-1229), and the Catalan mitochondrial photosensitizers, especially those of the electron Institution for Research and Advanced Studies (ICREA). chain. Moreover, other indirect mechanisms related to PKC, like those of TRPV4 and rhodopsin, would be less relevant for the response of pig sperm to red light. Yet, and because the inhibition Acknowledgments of PKC counteracted the changes in motile sperm subpopulations induced by irradiation, one could suggest that The authors acknowledge the support from Manel Bosch these PKC-related mechanisms could have an effect on specific (Confocal Microscopy Facility, University of Barcelona, Spain). sperm function variables, such as CCO activity and motility. Conflict of interest Data availability statement JR-G, SB and MY are inventors of a patent entitled “Method The raw data supporting the conclusions of this article will be and apparatus improving the quality of mammalian sperm” made available by the authors, without undue reservation. (European Patent Office No. 16199093.2; EP-3-323-289-A1), which is owned by Intruments Utils de Laboratori Geniul, S.L. (Barcelona, Spain) and an indirect relationship with the Ethics statement described experiments. The remaining authors declare that the research was This study uses boar diluted semen samples purchased as conducted in the absence of any commercial or financial commercial AI doses from the firm Gepork (Roda de Ter; Spain). relationships that could be construed as a potential conflict of Both Ethical Committees of the Autonomous University of interest. Barcelona and the University of Girona did not require the study to be reviewed or approved by an ethics committee because semen samples used in this study were not collected Publisher’s note for the sole research purpose, but for their commercial use. The farm thus provided us with some doses, whereas the remaining All claims expressed in this article are solely those of the ones were used for AI elsewhere. authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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