Morphometric analysis of the sperm midpiece during capacitation

Morphometric analysis of the sperm midpiece during capacitation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Morphometric analysis of the sperm midpiece during capacitation Maria Fernanda Skowronek, Santiago Pietroroia, Diego Silvera, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4145928/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background In mammalian sperm, mitochondria are very densely packed and form a helical sheath located in the midpiece of the flagellum. Mitochondria play multiple roles in the cell and can rapidly change shape to adapt to environmental conditions. During capacitation, mammalian spermatozoa undergo morphological and physiological changes to acquire fertilization ability. This is evidenced by changes in sperm motility patterns (hyperactivation) and the ability to perform the acrosome reaction. Whether there are changes in sperm mitochondrial shape or dimensions during capacitation is unknown. This work aimed to quantify morphometric changes in the sperm midpiece during capacitation based on computational analysis and image processing. Results Using mitochondrial fluorescent probes and a combination of freely available software, we quantified the dimensions and fluorescence intensity of the midpiece of the sperm flagellum. After capacitation, the area occupied by the mitochondria decreased. This decrease was due to a reduction in the width but not the length of the midpiece. A reduction in the area and width of the midpiece occurred in spermatozoa that underwent the acrosome reaction, suggesting a shrinkage of the mitochondria during the process of capacitation. Conclusion These results suggest that the flagellar structure is remodeled during sperm capacitation and the acrosome reaction, which is consistent with the observed changes in mitochondrial organization. The application of image processing to fluorescence microscopy images may help to identify morphological changes during capacitation. sperm midpiece mitochondria image processing capacitation acrosome reaction Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Background Mitochondria play several roles in the cell, including oxidative phosphorylation, the coupled production of ATP, lipid metabolism, the control of programmed cell death, calcium buffering, and the production of a variety of signaling metabolites ( 1 , 2 ). On the one hand, the shape of the mitochondria significantly influences these functions; on the other hand, cells can rapidly change their mitochondrial shape to adapt to environmental conditions ( 3 ). Furthermore, in different cell types, mitochondria exhibit dynamic behaviors such as fusion, fission, or movement within the cell ( 4 ). In mammalian spermatozoa, the mitochondria are unique and form a noticeable helical mitochondrial sheath located in the midpiece of the flagellum ( 5 , 6 ). This capsule-like structure provides mechanical stability, protection and resistance to osmotic changes ( 5 ). Furthermore, sperm mitochondria are connected to their neighbors via inter-mitochondrial linkers and to the underlying cytoskeleton through conserved protein arrangements on the outer mitochondrial membrane (OMM) ( 7 ). Therefore, sperm mitochondria are prevented from undergoing the same dynamic changes observed in other cell types. Despite these properties, some changes in mitochondrial sperm morphology have been described in both normal and pathological situations ( 8 , 9 ). Mammalian sperm acquire their fertilization ability during their passage to the female reproductive tract, a process known as capacitation ( 10 , 11 ). At the cell biological level, sperm capacitation is recognized by changes in the motility pattern of sperm, known as hyperactivation, and by the preparation of the sperm to undergo the acrosome reaction ( 12 ). Mature sperm carry a secretory vesicle on the apical head surface: the acrosome. Prior to fertilization, the acrosome and the plasma membrane of the sperm fuse through a calcium-dependent event of exocytosis, the acrosome reaction (AR). Both hyperactivation and ARs are required to penetrate the oocyte and achieve fertilization ( 12 , 13 ). In the first electron microscopy studies of the 1960s, the consensus was that capacitation did not cause any morphological changes, with the exception of those caused by the loss of the acrosome ( 14 ). However, later analysis showed that the morphology of mitochondria in human spermatozoa modifies during capacitation, possibly due to increased mitochondrial volume ( 8 ). There is a growing interest in understanding mitochondrial structure, accompanied by the use of a wealth of new reagents and computational tools to study mitochondrial structure and function. The application of image processing to fluorescence microscope images, as well as the advent of fluorescent probes, can reveal subtle changes in mitochondrial morphology during functional processes. In this work, we developed a tool for analyzing the size, shape and fluorescence intensity of mitochondria-labeled midpiece of capacitated (CAP) and non-capacitated (NC) mouse sperm. The tool is based on the analysis and quantification of fluorescence images performed with a custom image processing method based on ImageJ/Fiji. We detected a decrease in the area of the midpiece of CAP sperm. 2. Materials and methods 2.1. Animals and sperm preparation Sperm were obtained from male B6Bc/JF1 mice (12–18 weeks old). All animals were maintained on a 12-hour/12-hour dark-light cycle at a constant temperature of 22 ± 1°C with free access to food and water. The mice were killed by cervical dislocation. Sperm were collected from the epididymis of the mice after 15 minutes in 1.5 ml of medium (swim-out). The "Comisión Honoraria de Experimentación Animal" (Uruguay-CHEA) approved the protocol for these experiments. Global Total GT® medium (Fertilization/LifeGlobal Europe, Brussels Belgium, Ref: LGTF-100) was used for capacitation. GT® was previously placed in a CO 2 incubator to equilibrate the pH according to the manufacturer’s instructions. The sperm suspensions were transferred to preheated CELL-VU chambers (with a depth of 20 µm) (DRM-600, Millennium Sciences, Inc., CELL-VU®, NY). The sperm concentration and motility were analyzed using a computer-assisted sperm analysis (CASA) system (SCA, Microptic, Barcelona, Spain) under a Nikon (Japan) Eclipse E200 with 100X phase contrast system equipped with a Basler acA780-75gc camera (Germany). The settings used were as follows: acquisition, 30 frames/second; frequency, 60 Hz; head size, 5–70. Sperm cells with hyperactivated motility were classified using the following sperm kinetic parameters: ALH > 8 µM, VCL > 180 µm/s, and LIN < 50 µm. At least 500 sperm cells were analyzed in each assay. After motility analysis, the samples were divided into three tubes. One of them was processed immediately and was considered the non-capacitated condition (NC). The sperm in the other tubes were incubated for 90 minutes at 37°C in Global Total GT® medium to induce capacitation. In one tube, progesterone (100 µM) was added to induce AR. 2.2. Evaluation of the acrosome reaction (AR) by Coomassie blue The percentage of acrosome-reacted sperm was determined by placing 15 µl of each sample on glass slides, fixing it in 4% paraformaldehyde for 30 min, and washing it twice with phosphate-buffered saline (PBS). After washing, the slides were incubated with 0.22% Coomassie blue (Coomassie Blue G-250; Thermo Scientific, Massachusetts), 50% methanol, 10% glacial acetic acid, and 40% water for 2 min. Excess dye was removed by thorough washing with distilled water. Slides were air-dried, and coverslips were placed on the slides using mounting medium. The stained spermatozoa were examined under a bright field microscope at 400X (Nikon E100, Japan) to verify the percentage of sperm that had undergone AR. At least 200 spermatozoa were evaluated in each experiment. AR was expressed as the percentage of spermatozoa that underwent AR relative to the total number of spermatozoa counted. 2.3. In vitro fertilization (IVF) In brief, female B6Bc/JF1 mice (4 weeks old) were superovulated by intraperitoneal injections of 7.5 IU of PMSG (Syntex, Argentina) or 7.5 IU of HCG (Intervet International B. V-Holanda) 48 hours later. After 24 h, the oviducts were removed, and the cumulus–oocyte complexes (COCs) were transferred to a dish containing a 200-µL drop of GT® (1 M). Sperm was added to IVF drops containing COCs and incubated in 5% CO 2 at 37°C. Twenty-four hours later, the fertilization rates were recorded ( 15 ). A portion of the cleaved oocytes was transferred into GT® (Ref: LGGT-060) with CO 2 to reach a more advanced stage of embryonic development, confirming the capacitation of the sperm fraction used. 2.4. Sperm mitochondria and AR preparation for epifluorescence microscopy For morphometric analysis of the sperm midpiece, spermatozoa were incubated with 200 nM MitoTracker™ Red CMXRos-M7512 (Invitrogen, USA) for 30 minutes at 37°C, immediately after swim-out or after the capacitation period (90 minutes). Then, 50 µl of each sperm sample was added to a glass slide. The sperm were fixed with 4% PAF for 30 min and washed three times in PBS. After fixation, the sperm were incubated with 50 µg/ml PSA lectin (Pisum Sativum Agglutinin, Biotinylated B-1055-5), an acrosomal marker that allows differentiation between reacted and non-reacted spermatozoa. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). 2.5. Microscopy Slides were observed using an epifluorescent Nikon Eclipse E400 microscope with a 100X, 1.4 NA oil objective (excitation: λ = 488 nm and λ = 543 nm). Fluorescence images of at least 100 sperm for each condition were taken and merged with transmitted light microscopy photographs to verify that the head and midpiece were included. Digital photographs were taken with a Nikon DS-Fi3 de 5.9 megapixel digital camera and subsequently processed. 2.6. Image processing Between 80–100 images were analyzed for each condition and each mouse. Images acquired under the epifluorescence microscope were processed using ImageJ/Fiji ( 16 ) routines implemented in Python ( 17 ). The images were processed as 8-bit images Gaussian filtered for noise reduction, and automatic segmentation of spermatozoa midpieces was obtained from the MitoTracker channel using the MaxEntropy thresholding method ( 18 ). The obtained regions of interest (ROIs) were user validated and analyzed to obtain only isolated and well-defined midpieces. Mathematical morphology operations were performed to remove sharp peaks and smooth the ROIs. This procedure allows well-defined ROI segmentation of sperm midpieces without the need to know in advance whether they are reacted or not. The results of this process were also validated by the user (see Supplementary Fig. 1. a). Several descriptors for shape and fluorescence intensity were automatically calculated for each ROI. These parameters quantify morphological and intensity descriptors such as area, perimeter, circularity, integrated density and mean fluorescence intensity values. In addition, the average length and width of the SMP was estimated by averaging the distances of selected points in the skeleton of the SMP to its contour (Supplementary Fig. 1. b). The following libraries were also used: scikit-image ( 19 ), OpenCV (Bradski 2000), and SimpleITK ( 20 ). Spermatozoa were manually classified as reacted (R) by visual inspection if the green PNA label was absent and as non-reacted (NR) if a clear green cap was present in the sperm head. The morphometric measurements obtained from the SMP were grouped and compared between the R and NR sperm. The means and standard deviations of the measures in each condition were computed and compared (Supplementary Fig. 1. c). Finally, to objectively quantify the fluorescence intensity of the SMPs and normalize the data given by the ROI, a script was created to automatically determine the fluorescence intensity of the background of each image. For this purpose, the sperm structure was isolated from the image, and the average background fluorescence intensity was calculated. This average value was then subtracted from each pixel in the original images to obtain the real fluorescence intensity value of the SMP. The mean fluorescence intensity of each SMP detected in the background was analyzed and compared under the different conditions investigated (Supplementary Fig. 1. b and c). Supplementary Fig. 1 2.7. Statistical analysis After checking the normal distribution of the data using the Shapiro‒Wilk normality test, the fluorescence and morphometric values of the ROIs of the midpieces of the NC, CAP, and acrosome-reacted and non-reacted sperm were compared using paired t tests. The effect of progesterone on NC and CAP sperm was analyzed via ANOVA. p < 0.05 indicated statistical significance. GraphPad Prism version 9 for Windows (GraphPad Software, La Jolla California, USA http://www.graphpad.com ) was used for all analyses. 3. Results 3.1 Capacitation-associated events after incubation in capacitative media Sperm capacitation is characterized by several events that enable the cell to reach and fertilize the oocytes ( 21 ). To test sperm capacitation, we first examined three hallmarks of capacitation after 90 minutes of incubation in BSA- and bicarbonate-enriched medium (CAP condition) (see Supplementary Fig. 2). A greater percentage of hyperactivated cells were detected in spermatozoa incubated under CAP conditions than in those incubated in NC medium (Supplementary Fig. 2. a) (mean ± SD in NC: 1.3 ± 0.8 vs in CAP: 12.2 ± 1.8, n = 4, p = 0.0012, paired t test). The mean ± SD of the percentage of reacted-acrosome spermatozoa increased in CAP spermatozoa (Supplementary Fig. 2. b) (NC: 6.6 ± 1.0, CAP: 38.3 ± 15.9. and after induction with progesterone (CAPi): 49.0 ± 18.9, n = 9, p < 0.001, ANOVA). In addition, embryo viability was also assessed by obtaining two-cell embryos after 24 hours of FIV (arrows in Supplementary Fig. 2. c) and blastocysts after 96 hours (Supplementary Fig. 2. c, image below). Based on these results, we confirmed that the sperm fraction incubated for 90 minutes in medium enriched with BSA and bicarbonate represented capacitated spermatozoa (CAP). 3.2. The sperm mitochondrial area is reduced under capacitation conditions Most sperm midpieces were labeled with MitoTracker under both CAP and NC conditions (Fig. 1 a and b). The red fluorescent labeling was confined to the midpiece region of each sperm, consistent with the beginning of the tail in the transmitted light images (Fig. 1 . a). The specificity of the labeling allowed us to precisely match the ROIs to the region of the flagellum where the mitochondria are located (Fig. 1 . c). Morphometric analysis of the ROIs provided by the Fiji script showed that the mean ± SD of the area of NC sperm was greater than that of CAP sperm (22.47 ± 1.37 vs. 21.44 ± 2.26 µm 2 , n = 10, p = 0.01), suggesting shrinkage of the midpiece during the process of capacitation (Fig. 1 . d). 3.3. The area of the sperm midpiece is larger in non-acrosome-reacted sperm than in reacted sperm. To test whether the remodeling of the midpiece is related to the changes that occurred simultaneously in the head of the sperm during capacitation, the area of the midpiece was measured in acrosome-reacted and non-reacted sperm. For this purpose, the population of PSA-labeled spermatozoa was classified by the observer into intact and acrosome-reacted spermatozoa. The latter was performed based on the presence or absence of the green comma-shaped structure covering the sperm head (Fig. 1 . b). ROIs were measured in both situations: non-acrosome-reacted sperm (ROI 1 in Fig. 2 . c) and acrosome-reacted sperm (ROI 2 in Fig. 2 . c). The area of the midpiece in acrosome-reacted sperm decreased by 8.5% compared to that in non-acrosome-reacted spermatozoa (mean ± SD of non-AR: 24.33 ± 0.47 vs. AR: 22.25 ± 0.21 µm 2 , p < 0.0001, paired t test). A decrease was observed in spontaneous AR occurring in NC spermatozoa (non-AR: 24.27 ± 0.66 vs. AR: 22.08 ± 0.56, p < 0.001, n = 6; paired t test) and after the incubation of spermatozoa in CAP medium (non-AR: 24.31 ± 0.39 vs. AR: 22.43 ± 0.29, p < 0.0001, paired t test) or in P4-induced ARs (non-AR: 24.44 ± 0.29 vs. AR: 22.25 ± 0.26, p < 0.0001, paired t test) (Fig. 2 d). We then examined which of the dimensions of the midpiece could explain the observed decrease in the area of acrosome-reacted sperm. Skeletonization of the ROIs allowed us to measure the width and length of the labeled regions (Fig. 3 . a - d). The mean ± SD of the width decreased in the reacted acrosome sperm (non-AR: 0.54 ± 0.014 µm vs. AR: 0.50 ± 0.13 µm, n = 16, p < 0.001, paired t test), while the length remained similar (non-AR: 19.50 ± 0.49 µm vs. AR:19.36 ± 0.68 µm, n = 16, p = 0.3, paired t test). A decrease in the width of the midpiece was observed in the three conditions analyzed: NC (non-AR: 0.53 ± 0.02 µm vs. AR: 0.51 ± 0.01 µm, n = 6, p = 0.2); CAP (non-AR: 0.53 ± 0.00 µm vs. AR:4.9 ± 0.00 µm, n = 6, p < 0.001); and CAP + P4 (non-AR: 0.52 ± 0.01 µm vs. AR: 0.49 ± 0.01 µm, n = 4, p < 0.001) (Fig. 3 . f and g). 3.4 Analysis of the mean fluorescence value of the sperm midpiece The ROIs of the images obtained in sections 3.2 and 3.3 were reanalyzed after background subtraction (see section M&M and Supplementary Fig. 1) to obtain the mean fluorescence value of the sperm midpieces. There were no significant differences between the mean ± SD of fluorescence intensity under CAP and non-CAP conditions (NC: 15.29 ± 5.64 vs. under CAP: 19.23 ± 7.86, n = 10, p = 0.2, paired t test; Fig. 4 . a. When spermatozoa were classified as non-acrosome reacted or AR, as described in section 3.3 , the mean fluorescence intensity of MitoTracker-labeled flagella was similar between non-acrosome reacted and acrosome-reacted spermatozoa (mean ± SD of non-AR: 31.54 ± 10.47 vs. AR:31.04 ± 13.91, paired t test; Fig. 4 . b) regardless of sperm condition (Fig. 4 . c). 4. Discussion The quantification of sperm morphology is important for the assessment of sperm quality and can determine fertility ( 22 – 25 ). In particular, variations in the size and morphology of the midpiece and mitochondria have been associated with male infertility in men with asthenozoospermia ( 9 , 25 ). In addition, midpiece length affects sperm motility and competitiveness in other mammalian species ( 26 – 28 ). In this context, we performed a morphometric analysis of fluorescently labeled mouse sperm mitochondria. We found differences in the dimensions of the middle piece between CAP and NC sperm. Fluorescence imaging has been used extensively to quantitatively assess mitochondrial morphology in other cell types ( 2 ). Computational tools have been used to study mitochondrial network dynamics and changes in mitochondrial area under different conditions ( 1 , 29 ). Processing software that converts large amounts of complex multichannel image data into quantitative information is available through image processing and analysis tools such as Fiji ( 16 ). Fiji is a distribution of the open-source software ImageJ, which includes several of the most commonly used plugins that facilitate scientific image analysis ( 16 ). However, in regard to a specific analysis, the built-in plugins usually do not fulfill all the user requirements. A customized procedure based on the building blocks provided by the software platforms is often required. This is particularly important in the case of the sperm midpiece, where the singular characteristics of mitochondrial organization force the creation of specific tools to analyze the organelle. Due to their size and distribution as a spiral structure (6), mitochondria in sperm are more difficult to distinguish as separate entities. Instead, we have developed tools that generate ROIs that encompass the entire midpiece. MitoTracker® diffuses passively through the plasma membrane and accumulates in active mitochondria. The dye is permanently bound to the mitochondria and therefore remains after the cell has died or been fixed ( 30 , 31 ). Since the probe targets mitochondria, modifications in the midpiece accurately reflect changes in the organelle. The pipeline shown here allowed us to semi-automatically process all collected data with a systematic and objective procedure that minimizes or completely avoids human interaction and bias. Although the quantification of sperm characteristics is important for understanding the physiology of these cells, few studies have analyzed the morphological changes in flagella during capacitation. When live cells were imaged during in vitro fertilization, a decrease in the diameter of the midpiece was observed over time after acrosomal exocytosis ( 32 ). This observation is consistent with our results in fixed cells. We observed a decrease in the area of the midpiece and especially its width in the population of capacitated sperm (Fig. 3 ). A decrease in SMP was also observed in spontaneous ARs or ARs induced by a physiological agonist such as progesterone (Fig. 2 d and Fig. 3 f), which is also consistent with the results of the mentioned authors ( 32 ). Sperm capacitation involves several cellular changes that are associated with increased energy requirements. Despite the high efficiency of oxidative phosphorylation in mitochondria, glycolysis is considered the favored energy metabolic pathway for capacitation and hyperactivation ( 33 – 35 ). Consequently, the source of ATP in this process is under constant debate. In this context, our group has already shown that mitochondrial activity (i.e., oxygen consumption, membrane potential and ATP/ADP exchange) increases during sperm capacitation ( 15 ) meaning that changes in mitochondrial morphology could be accompanied by an increase in mitochondrial function. The mitochondrial membrane potential (MMP) depends on mitochondrial function and dysfunction and has already been monitored in spermatozoa with different cationic fluorescent probes using flow cytometry, epifluorescence or confocal microscopy ( 36 ). For example, Moscatelli et al. used MitoTracker Green to distinguish between sperm with active and non-active mitochondria ( 37 ). In our study, labeling sperm mitochondria with MitoTracker® Red., no differences between the mean fluorescence of mid-piece CAP sperm and NC sperm were found. These data may be considered contradictory with those of other studies ( 38 ), including ours ( 15 ), which revealed an increase in the MMP level in CAP sperm compared to that in NC sperm. Each of the chemicals in the “mitotracker” family has different properties, and there is not complete consistency in the use of all these probes to determine the functional status of the cells. For microscopy images of fixed spermatozoa, Amaral and Ramalho-Santos suggested monitoring the MMP of sperm while coincubating the cells with viability probes and expressing the results as a percentage of live spermatozoa ( 36 ). Although the lack of specificity in monitoring the MMP of the chosen probe (without the combination with other functional markers) may be considered a weakness of this work, the fact that we were able to quantify the mitochondrial morphology and fluorescence simultaneously is proof of the instrument’s function. In the future, the developed tools should be tested with other probes that can monitor both the form and function of sperm mitochondria. We have not investigated the mechanisms that cause the reduction in the width of the sperm midpiece during capacitation. However, our data indicate that this reduction is associated with the process of the acrosome reaction (Fig. 2 d and 3 f). Changes in the cytoskeleton are known to occur during the acrosome reaction ( 39 ), but little information is available on possible changes in the flagellar cytoskeleton during capacitation ( 32 , 40 , 41 ). The cytoskeleton of the flagellum consists mainly of the axoneme, which is composed of 9 doublets and a central pair of microtubules ( 42 ). Recent findings have shown that a double helix arrangement of polymerized actin accompanies the mitochondria in the midpiece of the mouse sperm flagellum. This spatial distribution does not extend to the principal piece, where actin is evenly distributed between the axoneme and the plasma membrane ( 43 ). The regulation of acrosomal exocytosis involves changes in the actin cytoskeleton in addition to other signaling pathways ( 40 , 41 , 44 ). The actin cytoskeleton of flagella is a plausible candidate for involvement in the observed constraint of the midpiece during AR. Further studies will be conducted to test this hypothesis. If there are links between the head and the mid-piece remodeling, these could potentially alter the metabolic status of sperm mitochondria, which are thought to play a crucial role in sperm function. 5. Conclusions On the one hand, we propose a tool that allows the morphofunctional analysis of sperm mitochondria by measuring the midpiece of the flagellum. On the other hand, with this instrument, we detected changes in the size of this part of the sperm, suggesting that during capacitation and the acrosome reaction, a process of remodeling of the sperm cell occurs that involves the midpiece and the mitochondria. Abbreviations AR acrosome reaction COCs Cumulus–oocyte complexes IVF In vitro fertilization OMM Outer mitochondrial membrane SMP Sperm midpiece CAP Capacitation NC Non-capacitated PMSG: equine chorionic gonadotropin ROI Region of interest R Reacted NR Non-reacted MMP Mitochondrial membrane potential Declarations Ethics approval and consent to participate No human data were included in this manuscript, so is “Not applicable” Animal data were obtained under protocols that were approved by "Comisión Honoraria de Experimentación Animal" CHEA as is stated in M&M: The "Comisión Honoraria de Experimentación Animal" (Uruguay-CHEA) approved the protocol for these experiments. Consent for publication Not applicable. Availability of data and materials The main generated data are presented in the manuscript. The datasets generated during image processing analysis will be available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests. Funding Financial support for this work was received from: Comisión Sectorial de Investigación Científica (CSIC I+D 2020, ID23), Universidad de la República (UdelaR), UdelaR Espacio Interdisciplinario, Núcleos 2015, UdelaR, Programa de Desarrollo de las Ciencias Básicas (PEDECIBA). Chan Zuckerberg Initiative Expanding Global Access to Bioimaging Bioimage Acquisition and Processing Core: Building Skills in Biomedicine Authors' contributions S.P. collected the samples. M.F. evaluated the acrosome reaction by Coomassie blue and IVF. M.F.S. performed the sperm mitochondria and acrosome reaction preparation for epifluorescence microscopy and microscopy. S.P., D.S., F.L. and M.F.S. performed image processing. R.S., F.L. and M.F.S. drafted the manuscript. R.S., M.F.S., F.L., and A.C. revised the manuscript. R.S. conceived and supervised the project.. All the authors have read and approved the final manuscript. 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Reorganization of the Flagellum Scaffolding Induces a Sperm Standstill During Fertilization. bioRxiv [Internet]. 2023 Oct 18; Available from: http://dx.doi.org/10.1101/2023.06.22.546073 Williams a. C, Ford WC. The role of glucose in supporting motility and capacitation in human spermatozoa. J Androl. 2001;22(4):680–95. Goodson SG, Qiu Y, Sutton KA, Xie G, Jia W, O’Brien DA. Metabolic substrates exhibit differential effects on functional parameters of mouse sperm capacitation. Biol Reprod. 2012 Sep;87(3):75. Tourmente M, Sansegundo E, Rial E, Roldan ERS. Capacitation promotes a shift in energy metabolism in murine sperm. Front Cell Dev Biol. 2022 Aug 23;10:950979. Amaral A, Ramalho-Santos J. Assessment of mitochondrial potential: Implications for the correct monitoring of human sperm function. Int J Androl [Internet]. 2010;33(1). Available from: http://dx.doi.org/10.1111/j.1365-2605.2009.00987.x Moscatelli N, Spagnolo B, Pisanello M, Lemma ED, De Vittorio M, Zara V, et al. Single-cell-based evaluation of sperm progressive motility via fluorescent assessment of mitochondria membrane potential. Sci Rep [Internet]. 2017;7(1). Available from: http://dx.doi.org/10.1038/s41598-017-18123-1 Giaccagli MM, Gómez-Elías MD, Herzfeld JD, Marín-Briggiler CI, Cuasnicú PS, Cohen DJ, et al. Capacitation-induced mitochondrial activity is required for sperm fertilizing ability in mice by modulating hyperactivation. Front Cell Dev Biol. 2021 Oct 26;9:767161. Schiavi-Ehrenhaus LJ, Romarowski A, Jabloñski M, Krapf D, Luque GM, Buffone MG. The early molecular events leading to COFILIN phosphorylation during mouse sperm capacitation are essential for acrosomal exocytosis. J Biol Chem. 2022 Jun;298(6):101988. Brener E, Rubinstein S, Cohen G, Shternall K, Rivlin J, Breitbart H. Remodeling of the actin cytoskeleton during mammalian sperm capacitation and acrosome reaction. Biol Reprod. 2003 Mar;68(3):837–45. Romarowski A, Velasco Félix ÁG, Torres Rodríguez P, Gervasi MG, Xu X, Luque GM, et al. Super-resolution imaging of live sperm reveals dynamic changes of the actin cytoskeleton during acrosomal exocytosis. J Cell Sci [Internet]. 2018 Nov 8;131(21). Available from: http://dx.doi.org/10.1242/jcs.218958 Fawcett DW. The mammalian spermatozoon. Dev Biol. 1975 Jun;44(2):394–436. Gervasi MG, Xu X, Carbajal-Gonzalez B, Buffone MG, Visconti PE, Krapf D. The actin cytoskeleton of the mouse sperm flagellum is organized in a helical structure. J Cell Sci [Internet]. 2018 Jun 11;131(11). Available from: http://dx.doi.org/10.1242/jcs.215897 Romarowski A, Luque GM, La Spina FA, Krapf D, Buffone MG. Role of Actin Cytoskeleton During Mammalian Sperm Acrosomal Exocytosis. Adv Anat Embryol Cell Biol. 2016;220:129–44. Additional Declarations No competing interests reported. Supplementary Files supfig1conrayitas.tif Supplementary files Supplementary Figure 1. Image processing workflow. a. Images are pre-processed and validated by the user to improve quality prior the analysis. Region of interest are obtained. b. The area occupied by mitochondrial structures is calculated from the binarized image prior to skeletonizing. Parameters of the ROIs and the average width and length of the midpiece is estimated by averaging the distances of selected points in the skeleton of the midpiece to its contour c. The mean and standard deviation of the measures in each condition were computed and compared ROI: Region of interest Sfigsup2.jpg Supplementary Figure 2. Markers of mouse sperm capacitation. a. Percentage of hyperactivated spermatozoa measured with CASA, expressed as the mean ± SD, p<0.05, n=4, paired t test. b. Percentage of acrosome-reacted spermatozoa measured with the Coomassie blue technique, expressed as the mean ± SD, n=9, p<0.05, ANOVA. c. Representative images of embryos obtained after sperm incubation in capacitating medium. White arrows indicate 2 cells of mouse embryos obtained after 24 hs of FIV. The lower images show embryos in the blastocyst stage after 96 hs of FIV. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4145928","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":285949188,"identity":"1dc1ba46-1dd3-47fb-b08d-b62bb68cc0ac","order_by":0,"name":"Maria Fernanda Skowronek","email":"","orcid":"","institution":"Unidad Académica Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Fernanda","lastName":"Skowronek","suffix":""},{"id":285949191,"identity":"22323070-60e0-420d-b48a-f08b6420e0fe","order_by":1,"name":"Santiago Pietroroia","email":"","orcid":"","institution":"Unidad Académica Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo","correspondingAuthor":false,"prefix":"","firstName":"Santiago","middleName":"","lastName":"Pietroroia","suffix":""},{"id":285949193,"identity":"b2ee4183-a091-41d5-8c61-2e3c8aa47db1","order_by":2,"name":"Diego Silvera","email":"","orcid":"","institution":"Departamento de Procesamiento de Señales, Facultad de Ingeniería, Universidad de la República, Montevideo, Uruguay.","correspondingAuthor":false,"prefix":"","firstName":"Diego","middleName":"","lastName":"Silvera","suffix":""},{"id":285949195,"identity":"b2f065dd-d568-4921-bb38-1e676335e342","order_by":3,"name":"Mariana Ford","email":"","orcid":"","institution":"Unidad Académica Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo","correspondingAuthor":false,"prefix":"","firstName":"Mariana","middleName":"","lastName":"Ford","suffix":""},{"id":285949197,"identity":"002af0cf-2824-4dc0-83bb-6aa3d13c9452","order_by":4,"name":"Adriana Cassina","email":"","orcid":"","institution":"Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo","correspondingAuthor":false,"prefix":"","firstName":"Adriana","middleName":"","lastName":"Cassina","suffix":""},{"id":285949198,"identity":"5c0f707a-8d5f-465d-9382-ada046ab5cd1","order_by":5,"name":"Federico Lecumberry","email":"","orcid":"","institution":"Departamento de Procesamiento de Señales, Facultad de Ingeniería, Universidad de la República, Montevideo, Uruguay.","correspondingAuthor":false,"prefix":"","firstName":"Federico","middleName":"","lastName":"Lecumberry","suffix":""},{"id":285949199,"identity":"6d092f1f-d918-4c6b-8e94-d80a32e3fe1e","order_by":6,"name":"Rossana Sapiro","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYHADxgYGhgogzczcQIqWMyAtjERrAelqg+nFA8zb2x9/+LnDzp5/dnPbh4/zaqP524FaflRsw6lF5swZM8neM8mJM+4cbJ45c9vx3BmHGRsYe87cxqlFQiKHjYG3jTmB4UZiMzPvtmO5DUAtzIxt+LSkP/74t63eXh6sZc6x3PmEtSQYSPO2HWbcANbSUJO7gaAWnjNm0rJtxxM3Av3COOPYgdyNQC0H8fqFvf3xx7dt1fZyt9sfM3yoqcudd/7wwQc/KnBrQdIMJg+DyQNEqIdrqSNO8SgYBaNgFIwoAAA40lx+0GLz9QAAAABJRU5ErkJggg==","orcid":"","institution":"Unidad Académica Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo","correspondingAuthor":true,"prefix":"","firstName":"Rossana","middleName":"","lastName":"Sapiro","suffix":""}],"badges":[],"createdAt":"2024-03-21 22:14:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4145928/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4145928/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54107044,"identity":"a335c2e1-f6a4-4c57-89da-eec882562bef","added_by":"auto","created_at":"2024-04-04 17:25:52","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4081413,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImage processing and morphometric analysis of mouse sperm midpieces under non-capacitated and capacitated conditions.\u003c/strong\u003e a. Mouse spermatozoa in a bright field. b. Mouse spermatozoa were loaded with MitoTracker Red, and the sperm midpieces are shown in red. c. Segmentation of the sperm midpiece and determination of ROIs. d. Morphometric parameters (area) in comparison between non-capacitated and capacitated conditions. *p\u0026lt;0.05\u003c/p\u003e","description":"","filename":"floatimage1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4145928/v1/d6cbf6e37f096b0a32b1afd2.jpg"},{"id":54107046,"identity":"6fdf1327-a6e7-4885-9285-5921d3198794","added_by":"auto","created_at":"2024-04-04 17:25:52","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3203887,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImage processing and morphometric analysis of the mouse sperm midpiece and acrosome reaction.\u003c/strong\u003e a. Mouse spermatozoa in a bright field. b. Mouse sperm loaded with MitoTracker Red (sperm midpieces are shown in red) and PSA lectin revealing the state of the acrosomes (acrosomes are shown in green = *non-reacted, ** reacted). c. Segmentation of sperm midpieces and determination of ROIs. d. Morphometric parameters (area) corresponding to the acrosome state of the spermatozoa at the different stages of sperm capacitation analyzed. *p\u0026lt;0.05\u003c/p\u003e","description":"","filename":"floatimage2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4145928/v1/d32ef054aaba45ababfcf47d.jpg"},{"id":54107049,"identity":"11089e21-a63c-4e1b-9a1f-7f5a46f7d573","added_by":"auto","created_at":"2024-04-04 17:25:53","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5131099,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImage processing and analysis of the dimensions of MitoTracker®-labeled sperm midpiece. \u003c/strong\u003ea - e: Skeletonization of the ROIs of the sperm midpiece of acrosome-reacted and non-reacted sperm, allowing the determination of the d-width and e-length of the sperm midpiece. Statistical analysis of sperm midpiece width (f) and length (g) comparing reacted and non-reacted spermatozoa under different capacitation conditions. **p\u0026lt;0.001, ns: not statistically significant, paired t test. Clear bars: non-reacted, red bars: reacted sperm.\u003c/p\u003e","description":"","filename":"floatimage3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4145928/v1/948cdbd110930bd8c51e990a.jpg"},{"id":54107048,"identity":"c3aaced3-7085-46a5-b11f-2da406a91bc5","added_by":"auto","created_at":"2024-04-04 17:25:53","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2365153,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of the fluorescence of the Mitotracker®-labeled sperm midpiece. \u003c/strong\u003ea. Mean+/-SD of the mean fluorescence values of the sperm midpiece under CAP and non-CAP conditions b. Sum of the mean fluorescence in AR and non-AR sperm c. Mean fluorescence after CAP condition. ns= not statistically significant; paired t test.\u003c/p\u003e","description":"","filename":"floatimage4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4145928/v1/771259bc492be11f8314fa23.jpg"},{"id":55537723,"identity":"a340f1ad-3e68-44ea-85f5-bb65930e308d","added_by":"auto","created_at":"2024-04-29 16:45:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":902485,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4145928/v1/5921f252-110e-452b-ae31-aa55e7070dc1.pdf"},{"id":54107045,"identity":"f71b87cf-c35e-460c-9e12-b044bce4ea68","added_by":"auto","created_at":"2024-04-04 17:25:52","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":877452,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary files\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1.\u003c/strong\u003e Image processing workflow. a. Images are pre-processed and validated by the user to improve quality prior the analysis. Region of interest are obtained. b. The area occupied by mitochondrial structures is calculated from the binarized image prior to skeletonizing. Parameters of the ROIs and the average width and length of the midpiece is estimated by averaging the distances of selected points in the skeleton of the midpiece to its contour c. The mean and standard deviation of the measures in each condition were computed and compared ROI: Region of interest\u003c/p\u003e","description":"","filename":"supfig1conrayitas.tif","url":"https://assets-eu.researchsquare.com/files/rs-4145928/v1/a19294684047e055089035ad.tif"},{"id":54107043,"identity":"cc290b73-a0ef-4c9d-86b9-5370b387b336","added_by":"auto","created_at":"2024-04-04 17:25:52","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":373610,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2. \u0026nbsp;Markers of mouse sperm capacitation.\u003c/strong\u003e a. Percentage of hyperactivated spermatozoa measured with CASA, expressed as the mean ± SD, p\u0026lt;0.05, n=4, paired t test. b. Percentage of acrosome-reacted spermatozoa measured with the Coomassie blue technique, expressed as the mean ± SD, n=9, p\u0026lt;0.05, ANOVA. c. Representative images of embryos obtained after sperm incubation in capacitating medium. \u0026nbsp;White arrows indicate 2 cells of mouse embryos obtained after 24 hs of FIV. The lower images show embryos in the blastocyst stage after 96 hs of FIV.\u003c/p\u003e","description":"","filename":"Sfigsup2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4145928/v1/30c9764a26db458229fa25c5.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Morphometric analysis of the sperm midpiece during capacitation","fulltext":[{"header":"1. Background","content":"\u003cp\u003eMitochondria play several roles in the cell, including oxidative phosphorylation, the coupled production of ATP, lipid metabolism, the control of programmed cell death, calcium buffering, and the production of a variety of signaling metabolites (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). On the one hand, the shape of the mitochondria significantly influences these functions; on the other hand, cells can rapidly change their mitochondrial shape to adapt to environmental conditions (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Furthermore, in different cell types, mitochondria exhibit dynamic behaviors such as fusion, fission, or movement within the cell (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn mammalian spermatozoa, the mitochondria are unique and form a noticeable helical mitochondrial sheath located in the midpiece of the flagellum (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). This capsule-like structure provides mechanical stability, protection and resistance to osmotic changes (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Furthermore, sperm mitochondria are connected to their neighbors via inter-mitochondrial linkers and to the underlying cytoskeleton through conserved protein arrangements on the outer mitochondrial membrane (OMM) (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Therefore, sperm mitochondria are prevented from undergoing the same dynamic changes observed in other cell types. Despite these properties, some changes in mitochondrial sperm morphology have been described in both normal and pathological situations (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMammalian sperm acquire their fertilization ability during their passage to the female reproductive tract, a process known as capacitation (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). At the cell biological level, sperm capacitation is recognized by changes in the motility pattern of sperm, known as hyperactivation, and by the preparation of the sperm to undergo the acrosome reaction (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Mature sperm carry a secretory vesicle on the apical head surface: the acrosome. Prior to fertilization, the acrosome and the plasma membrane of the sperm fuse through a calcium-dependent event of exocytosis, the acrosome reaction (AR). Both hyperactivation and ARs are required to penetrate the oocyte and achieve fertilization (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the first electron microscopy studies of the 1960s, the consensus was that capacitation did not cause any morphological changes, with the exception of those caused by the loss of the acrosome (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). However, later analysis showed that the morphology of mitochondria in human spermatozoa modifies during capacitation, possibly due to increased mitochondrial volume (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere is a growing interest in understanding mitochondrial structure, accompanied by the use of a wealth of new reagents and computational tools to study mitochondrial structure and function. The application of image processing to fluorescence microscope images, as well as the advent of fluorescent probes, can reveal subtle changes in mitochondrial morphology during functional processes. In this work, we developed a tool for analyzing the size, shape and fluorescence intensity of mitochondria-labeled midpiece of capacitated (CAP) and non-capacitated (NC) mouse sperm. The tool is based on the analysis and quantification of fluorescence images performed with a custom image processing method based on ImageJ/Fiji. We detected a decrease in the area of the midpiece of CAP sperm.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Animals and sperm preparation\u003c/h2\u003e \u003cp\u003eSperm were obtained from male B6Bc/JF1 mice (12\u0026ndash;18 weeks old). All animals were maintained on a 12-hour/12-hour dark-light cycle at a constant temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C with free access to food and water. The mice were killed by cervical dislocation. Sperm were collected from the epididymis of the mice after 15 minutes in 1.5 ml of medium (swim-out). The \"Comisi\u0026oacute;n Honoraria de Experimentaci\u0026oacute;n Animal\" (Uruguay-CHEA) approved the protocol for these experiments.\u003c/p\u003e \u003cp\u003eGlobal Total GT\u0026reg; medium (Fertilization/LifeGlobal Europe, Brussels Belgium, Ref: LGTF-100) was used for capacitation. GT\u0026reg; was previously placed in a CO\u003csub\u003e2\u003c/sub\u003e incubator to equilibrate the pH according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003eThe sperm suspensions were transferred to preheated CELL-VU chambers (with a depth of 20 \u0026micro;m) (DRM-600, Millennium Sciences, Inc., CELL-VU\u0026reg;, NY). The sperm concentration and motility were analyzed using a computer-assisted sperm analysis (CASA) system (SCA, Microptic, Barcelona, Spain) under a Nikon (Japan) Eclipse E200 with 100X phase contrast system equipped with a Basler acA780-75gc camera (Germany). The settings used were as follows: acquisition, 30 frames/second; frequency, 60 Hz; head size, 5\u0026ndash;70. Sperm cells with hyperactivated motility were classified using the following sperm kinetic parameters: ALH\u0026thinsp;\u0026gt;\u0026thinsp;8 \u0026micro;M, VCL\u0026thinsp;\u0026gt;\u0026thinsp;180 \u0026micro;m/s, and LIN\u0026thinsp;\u0026lt;\u0026thinsp;50 \u0026micro;m. At least 500 sperm cells were analyzed in each assay.\u003c/p\u003e \u003cp\u003eAfter motility analysis, the samples were divided into three tubes. One of them was processed immediately and was considered the non-capacitated condition (NC). The sperm in the other tubes were incubated for 90 minutes at 37\u0026deg;C in Global Total GT\u0026reg; medium to induce capacitation. In one tube, progesterone (100 \u0026micro;M) was added to induce AR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Evaluation of the acrosome reaction (AR) by Coomassie blue\u003c/h2\u003e \u003cp\u003eThe percentage of acrosome-reacted sperm was determined by placing 15 \u0026micro;l of each sample on glass slides, fixing it in 4% paraformaldehyde for 30 min, and washing it twice with phosphate-buffered saline (PBS). After washing, the slides were incubated with 0.22% Coomassie blue (Coomassie Blue G-250; Thermo Scientific, Massachusetts), 50% methanol, 10% glacial acetic acid, and 40% water for 2 min. Excess dye was removed by thorough washing with distilled water. Slides were air-dried, and coverslips were placed on the slides using mounting medium. The stained spermatozoa were examined under a bright field microscope at 400X (Nikon E100, Japan) to verify the percentage of sperm that had undergone AR. At least 200 spermatozoa were evaluated in each experiment. AR was expressed as the percentage of spermatozoa that underwent AR relative to the total number of spermatozoa counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. In vitro fertilization (IVF)\u003c/h2\u003e \u003cp\u003eIn brief, female B6Bc/JF1 mice (4 weeks old) were superovulated by intraperitoneal injections of 7.5 IU of PMSG (Syntex, Argentina) or 7.5 IU of HCG (Intervet International B. V-Holanda) 48 hours later. After 24 h, the oviducts were removed, and the cumulus\u0026ndash;oocyte complexes (COCs) were transferred to a dish containing a 200-\u0026micro;L drop of GT\u0026reg; (1 M). Sperm was added to IVF drops containing COCs and incubated in 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. Twenty-four hours later, the fertilization rates were recorded (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). A portion of the cleaved oocytes was transferred into GT\u0026reg; (Ref: LGGT-060) with CO\u003csub\u003e2\u003c/sub\u003e to reach a more advanced stage of embryonic development, confirming the capacitation of the sperm fraction used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Sperm mitochondria and AR preparation for epifluorescence microscopy\u003c/h2\u003e \u003cp\u003eFor morphometric analysis of the sperm midpiece, spermatozoa were incubated with 200 nM MitoTracker\u0026trade; Red CMXRos-M7512 (Invitrogen, USA) for 30 minutes at 37\u0026deg;C, immediately after swim-out or after the capacitation period (90 minutes).\u003c/p\u003e \u003cp\u003eThen, 50 \u0026micro;l of each sperm sample was added to a glass slide. The sperm were fixed with 4% PAF for 30 min and washed three times in PBS. After fixation, the sperm were incubated with 50 \u0026micro;g/ml PSA lectin (Pisum Sativum Agglutinin, Biotinylated B-1055-5), an acrosomal marker that allows differentiation between reacted and non-reacted spermatozoa. The nuclei were counterstained with 4\u0026prime;,6-diamidino-2-phenylindole dihydrochloride (DAPI).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Microscopy\u003c/h2\u003e \u003cp\u003eSlides were observed using an epifluorescent Nikon Eclipse E400 microscope with a 100X, 1.4 NA oil objective (excitation: λ\u0026thinsp;=\u0026thinsp;488 nm and λ\u0026thinsp;=\u0026thinsp;543 nm). Fluorescence images of at least 100 sperm for each condition were taken and merged with transmitted light microscopy photographs to verify that the head and midpiece were included. Digital photographs were taken with a Nikon DS-Fi3 de 5.9 megapixel digital camera and subsequently processed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Image processing\u003c/h2\u003e \u003cp\u003eBetween 80\u0026ndash;100 images were analyzed for each condition and each mouse. Images acquired under the epifluorescence microscope were processed using ImageJ/Fiji (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) routines implemented in Python (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe images were processed as 8-bit images Gaussian filtered for noise reduction, and automatic segmentation of spermatozoa midpieces was obtained from the MitoTracker channel using the MaxEntropy thresholding method (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). The obtained regions of interest (ROIs) were user validated and analyzed to obtain only isolated and well-defined midpieces. Mathematical morphology operations were performed to remove sharp peaks and smooth the ROIs. This procedure allows well-defined ROI segmentation of sperm midpieces without the need to know in advance whether they are reacted or not. The results of this process were also validated by the user (see Supplementary Fig.\u0026nbsp;1. a). Several descriptors for shape and fluorescence intensity were automatically calculated for each ROI. These parameters quantify morphological and intensity descriptors such as area, perimeter, circularity, integrated density and mean fluorescence intensity values. In addition, the average length and width of the SMP was estimated by averaging the distances of selected points in the skeleton of the SMP to its contour (Supplementary Fig.\u0026nbsp;1. b). The following libraries were also used: scikit-image (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), OpenCV (Bradski 2000), and SimpleITK (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSpermatozoa were manually classified as reacted (R) by visual inspection if the green PNA label was absent and as non-reacted (NR) if a clear green cap was present in the sperm head. The morphometric measurements obtained from the SMP were grouped and compared between the R and NR sperm. The means and standard deviations of the measures in each condition were computed and compared (Supplementary Fig.\u0026nbsp;1. c).\u003c/p\u003e \u003cp\u003eFinally, to objectively quantify the fluorescence intensity of the SMPs and normalize the data given by the ROI, a script was created to automatically determine the fluorescence intensity of the background of each image. For this purpose, the sperm structure was isolated from the image, and the average background fluorescence intensity was calculated. This average value was then subtracted from each pixel in the original images to obtain the real fluorescence intensity value of the SMP.\u003c/p\u003e \u003cp\u003eThe mean fluorescence intensity of each SMP detected in the background was analyzed and compared under the different conditions investigated (Supplementary Fig.\u0026nbsp;1. b and c).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Statistical analysis\u003c/h2\u003e \u003cp\u003eAfter checking the normal distribution of the data using the Shapiro‒Wilk normality test, the fluorescence and morphometric values of the ROIs of the midpieces of the NC, CAP, and acrosome-reacted and non-reacted sperm were compared using paired t tests. The effect of progesterone on NC and CAP sperm was analyzed via ANOVA. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicated statistical significance. GraphPad Prism version 9 for Windows (GraphPad Software, La Jolla California, USA \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.graphpad.com\u003c/span\u003e\u003cspan address=\"http://www.graphpad.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e was used for all analyses.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Capacitation-associated events after incubation in capacitative media\u003c/h2\u003e \u003cp\u003eSperm capacitation is characterized by several events that enable the cell to reach and fertilize the oocytes (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). To test sperm capacitation, we first examined three hallmarks of capacitation after 90 minutes of incubation in BSA- and bicarbonate-enriched medium (CAP condition) (see Supplementary Fig.\u0026nbsp;2). A greater percentage of hyperactivated cells were detected in spermatozoa incubated under CAP conditions than in those incubated in NC medium (Supplementary Fig.\u0026nbsp;2. a) (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD in NC: 1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 vs in CAP: 12.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8, n\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;=\u0026thinsp;0.0012, paired t test). The mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of the percentage of reacted-acrosome spermatozoa increased in CAP spermatozoa (Supplementary Fig.\u0026nbsp;2. b) (NC: 6.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0, CAP: 38.3\u0026thinsp;\u0026plusmn;\u0026thinsp;15.9. and after induction with progesterone (CAPi): 49.0\u0026thinsp;\u0026plusmn;\u0026thinsp;18.9, n\u0026thinsp;=\u0026thinsp;9, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ANOVA). In addition, embryo viability was also assessed by obtaining two-cell embryos after 24 hours of FIV (arrows in Supplementary Fig.\u0026nbsp;2. c) and blastocysts after 96 hours (Supplementary Fig.\u0026nbsp;2. c, image below). Based on these results, we confirmed that the sperm fraction incubated for 90 minutes in medium enriched with BSA and bicarbonate represented capacitated spermatozoa (CAP).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2. The sperm mitochondrial area is reduced under capacitation conditions\u003c/h2\u003e \u003cp\u003eMost sperm midpieces were labeled with MitoTracker under both CAP and NC conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and b). The red fluorescent labeling was confined to the midpiece region of each sperm, consistent with the beginning of the tail in the transmitted light images (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. a). The specificity of the labeling allowed us to precisely match the ROIs to the region of the flagellum where the mitochondria are located (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. c). Morphometric analysis of the ROIs provided by the Fiji script showed that the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of the area of NC sperm was greater than that of CAP sperm (22.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37 vs. 21.44\u0026thinsp;\u0026plusmn;\u0026thinsp;2.26 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e, n\u0026thinsp;=\u0026thinsp;10, p\u0026thinsp;=\u0026thinsp;0.01), suggesting shrinkage of the midpiece during the process of capacitation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3. The area of the sperm midpiece is larger in non-acrosome-reacted sperm than in reacted sperm.\u003c/h2\u003e \u003cp\u003eTo test whether the remodeling of the midpiece is related to the changes that occurred simultaneously in the head of the sperm during capacitation, the area of the midpiece was measured in acrosome-reacted and non-reacted sperm. For this purpose, the population of PSA-labeled spermatozoa was classified by the observer into intact and acrosome-reacted spermatozoa. The latter was performed based on the presence or absence of the green comma-shaped structure covering the sperm head (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. b). ROIs were measured in both situations: non-acrosome-reacted sperm (ROI 1 in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. c) and acrosome-reacted sperm (ROI 2 in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. c).\u003c/p\u003e \u003cp\u003eThe area of the midpiece in acrosome-reacted sperm decreased by 8.5% compared to that in non-acrosome-reacted spermatozoa (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of non-AR: 24.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47 vs. AR: 22.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, paired t test). A decrease was observed in spontaneous AR occurring in NC spermatozoa (non-AR: 24.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66 vs. AR: 22.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, n\u0026thinsp;=\u0026thinsp;6; paired t test) and after the incubation of spermatozoa in CAP medium (non-AR: 24.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39 vs. AR: 22.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, paired t test) or in P4-induced ARs (non-AR: 24.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 vs. AR: 22.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, paired t test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then examined which of the dimensions of the midpiece could explain the observed decrease in the area of acrosome-reacted sperm. Skeletonization of the ROIs allowed us to measure the width and length of the labeled regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. a - d). The mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of the width decreased in the reacted acrosome sperm (non-AR: 0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014 \u0026micro;m vs. AR: 0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 \u0026micro;m, n\u0026thinsp;=\u0026thinsp;16, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, paired t test), while the length remained similar (non-AR: 19.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 \u0026micro;m vs. AR:19.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68 \u0026micro;m, n\u0026thinsp;=\u0026thinsp;16, p\u0026thinsp;=\u0026thinsp;0.3, paired t test). A decrease in the width of the midpiece was observed in the three conditions analyzed: NC (non-AR: 0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;m vs. AR: 0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u0026micro;m, n\u0026thinsp;=\u0026thinsp;6, p\u0026thinsp;=\u0026thinsp;0.2); CAP (non-AR: 0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 \u0026micro;m vs. AR:4.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 \u0026micro;m, n\u0026thinsp;=\u0026thinsp;6, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001); and CAP\u0026thinsp;+\u0026thinsp;P4 (non-AR: 0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u0026micro;m vs. AR: 0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u0026micro;m, n\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. f and g).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Analysis of the mean fluorescence value of the sperm midpiece\u003c/h2\u003e \u003cp\u003eThe ROIs of the images obtained in sections \u003cspan refid=\"Sec12\" class=\"InternalRef\"\u003e3.2\u003c/span\u003e and \u003cspan refid=\"Sec13\" class=\"InternalRef\"\u003e3.3\u003c/span\u003e were reanalyzed after background subtraction (see section M\u0026amp;M and Supplementary Fig.\u0026nbsp;1) to obtain the mean fluorescence value of the sperm midpieces. There were no significant differences between the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of fluorescence intensity under CAP and non-CAP conditions (NC: 15.29\u0026thinsp;\u0026plusmn;\u0026thinsp;5.64 vs. under CAP: 19.23\u0026thinsp;\u0026plusmn;\u0026thinsp;7.86, n\u0026thinsp;=\u0026thinsp;10, p\u0026thinsp;=\u0026thinsp;0.2, paired t test; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. a.\u003c/p\u003e \u003cp\u003eWhen spermatozoa were classified as non-acrosome reacted or AR, as described in section \u003cspan refid=\"Sec13\" class=\"InternalRef\"\u003e3.3\u003c/span\u003e, the mean fluorescence intensity of MitoTracker-labeled flagella was similar between non-acrosome reacted and acrosome-reacted spermatozoa (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of non-AR: 31.54\u0026thinsp;\u0026plusmn;\u0026thinsp;10.47 vs. AR:31.04\u0026thinsp;\u0026plusmn;\u0026thinsp;13.91, paired t test; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. b) regardless of sperm condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe quantification of sperm morphology is important for the assessment of sperm quality and can determine fertility (\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). In particular, variations in the size and morphology of the midpiece and mitochondria have been associated with male infertility in men with asthenozoospermia (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). In addition, midpiece length affects sperm motility and competitiveness in other mammalian species (\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). In this context, we performed a morphometric analysis of fluorescently labeled mouse sperm mitochondria. We found differences in the dimensions of the middle piece between CAP and NC sperm.\u003c/p\u003e \u003cp\u003eFluorescence imaging has been used extensively to quantitatively assess mitochondrial morphology in other cell types (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Computational tools have been used to study mitochondrial network dynamics and changes in mitochondrial area under different conditions (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Processing software that converts large amounts of complex multichannel image data into quantitative information is available through image processing and analysis tools such as Fiji (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Fiji is a distribution of the open-source software ImageJ, which includes several of the most commonly used plugins that facilitate scientific image analysis (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). However, in regard to a specific analysis, the built-in plugins usually do not fulfill all the user requirements. A customized procedure based on the building blocks provided by the software platforms is often required. This is particularly important in the case of the sperm midpiece, where the singular characteristics of mitochondrial organization force the creation of specific tools to analyze the organelle. Due to their size and distribution as a spiral structure (6), mitochondria in sperm are more difficult to distinguish as separate entities. Instead, we have developed tools that generate ROIs that encompass the entire midpiece. MitoTracker\u0026reg; diffuses passively through the plasma membrane and accumulates in active mitochondria. The dye is permanently bound to the mitochondria and therefore remains after the cell has died or been fixed (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Since the probe targets mitochondria, modifications in the midpiece accurately reflect changes in the organelle. The pipeline shown here allowed us to semi-automatically process all collected data with a systematic and objective procedure that minimizes or completely avoids human interaction and bias.\u003c/p\u003e \u003cp\u003eAlthough the quantification of sperm characteristics is important for understanding the physiology of these cells, few studies have analyzed the morphological changes in flagella during capacitation. When live cells were imaged during in vitro fertilization, a decrease in the diameter of the midpiece was observed over time after acrosomal exocytosis (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). This observation is consistent with our results in fixed cells. We observed a decrease in the area of the midpiece and especially its width in the population of capacitated sperm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). A decrease in SMP was also observed in spontaneous ARs or ARs induced by a physiological agonist such as progesterone (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef), which is also consistent with the results of the mentioned authors (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSperm capacitation involves several cellular changes that are associated with increased energy requirements. Despite the high efficiency of oxidative phosphorylation in mitochondria, glycolysis is considered the favored energy metabolic pathway for capacitation and hyperactivation (\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Consequently, the source of ATP in this process is under constant debate. In this context, our group has already shown that mitochondrial activity (i.e., oxygen consumption, membrane potential and ATP/ADP exchange) increases during sperm capacitation (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) meaning that changes in mitochondrial morphology could be accompanied by an increase in mitochondrial function. The mitochondrial membrane potential (MMP) depends on mitochondrial function and dysfunction and has already been monitored in spermatozoa with different cationic fluorescent probes using flow cytometry, epifluorescence or confocal microscopy (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). For example, Moscatelli et al. used MitoTracker Green to distinguish between sperm with active and non-active mitochondria (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). In our study, labeling sperm mitochondria with MitoTracker\u0026reg; Red., no differences between the mean fluorescence of mid-piece CAP sperm and NC sperm were found. These data may be considered contradictory with those of other studies (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e), including ours (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), which revealed an increase in the MMP level in CAP sperm compared to that in NC sperm. Each of the chemicals in the \u0026ldquo;mitotracker\u0026rdquo; family has different properties, and there is not complete consistency in the use of all these probes to determine the functional status of the cells. For microscopy images of fixed spermatozoa, Amaral and Ramalho-Santos suggested monitoring the MMP of sperm while coincubating the cells with viability probes and expressing the results as a percentage of live spermatozoa (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Although the lack of specificity in monitoring the MMP of the chosen probe (without the combination with other functional markers) may be considered a weakness of this work, the fact that we were able to quantify the mitochondrial morphology and fluorescence simultaneously is proof of the instrument\u0026rsquo;s function. In the future, the developed tools should be tested with other probes that can monitor both the form and function of sperm mitochondria.\u003c/p\u003e \u003cp\u003eWe have not investigated the mechanisms that cause the reduction in the width of the sperm midpiece during capacitation. However, our data indicate that this reduction is associated with the process of the acrosome reaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). Changes in the cytoskeleton are known to occur during the acrosome reaction (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e), but little information is available on possible changes in the flagellar cytoskeleton during capacitation (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). The cytoskeleton of the flagellum consists mainly of the axoneme, which is composed of 9 doublets and a central pair of microtubules (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Recent findings have shown that a double helix arrangement of polymerized actin accompanies the mitochondria in the midpiece of the mouse sperm flagellum. This spatial distribution does not extend to the principal piece, where actin is evenly distributed between the axoneme and the plasma membrane (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). The regulation of acrosomal exocytosis involves changes in the actin cytoskeleton in addition to other signaling pathways (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). The actin cytoskeleton of flagella is a plausible candidate for involvement in the observed constraint of the midpiece during AR. Further studies will be conducted to test this hypothesis. If there are links between the head and the mid-piece remodeling, these could potentially alter the metabolic status of sperm mitochondria, which are thought to play a crucial role in sperm function.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eOn the one hand, we propose a tool that allows the morphofunctional analysis of sperm mitochondria by measuring the midpiece of the flagellum. On the other hand, with this instrument, we detected changes in the size of this part of the sperm, suggesting that during capacitation and the acrosome reaction, a process of remodeling of the sperm cell occurs that involves the midpiece and the mitochondria.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAR acrosome reaction\u003c/p\u003e\n\u003cp\u003eCOCs Cumulus\u0026ndash;oocyte complexes\u003c/p\u003e\n\u003cp\u003eIVF In vitro fertilization\u003c/p\u003e\n\u003cp\u003eOMM Outer mitochondrial membrane\u003c/p\u003e\n\u003cp\u003eSMP Sperm midpiece\u003c/p\u003e\n\u003cp\u003eCAP Capacitation\u003c/p\u003e\n\u003cp\u003eNC Non-capacitated\u003c/p\u003e\n\u003cp\u003ePMSG: equine chorionic gonadotropin\u003c/p\u003e\n\u003cp\u003eROI Region of interest\u003c/p\u003e\n\u003cp\u003eR Reacted\u003c/p\u003e\n\u003cp\u003eNR Non-reacted\u003c/p\u003e\n\u003cp\u003eMMP Mitochondrial membrane potential\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo human data were included in this manuscript, so is \u0026ldquo;Not applicable\u0026rdquo;\u003c/p\u003e\n\u003cp\u003eAnimal data were obtained under protocols that were approved by \u0026quot;Comisi\u0026oacute;n Honoraria de\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExperimentaci\u0026oacute;n Animal\u0026quot; CHEA as is stated in M\u0026amp;M:\u003c/p\u003e\n\u003cp\u003eThe \u0026quot;Comisi\u0026oacute;n Honoraria de Experimentaci\u0026oacute;n Animal\u0026quot; (Uruguay-CHEA) approved the protocol for these experiments.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eThe main generated data are presented in the manuscript. The datasets generated during image processing analysis will be available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eFinancial support for this work was received from:\u003c/p\u003e\n\u003cp\u003eComisi\u0026oacute;n Sectorial de Investigaci\u0026oacute;n Cient\u0026iacute;fica (CSIC I+D 2020, ID23), Universidad de la Rep\u0026uacute;blica (UdelaR), UdelaR Espacio Interdisciplinario, N\u0026uacute;cleos 2015, UdelaR, Programa de Desarrollo de las Ciencias B\u0026aacute;sicas (PEDECIBA).\u003c/p\u003e\n\u003cp\u003eChan Zuckerberg Initiative Expanding Global Access to Bioimaging Bioimage Acquisition and Processing Core: Building Skills in Biomedicine\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.P. collected the samples. M.F. evaluated the acrosome reaction by Coomassie blue and IVF. M.F.S. performed the sperm mitochondria and acrosome reaction preparation for epifluorescence microscopy and microscopy. S.P., D.S., F.L. and M.F.S. performed image processing. R.S., F.L. and M.F.S. drafted the manuscript. R.S., M.F.S., F.L., and A.C. revised the manuscript. R.S. conceived and supervised the project.. All the authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRohani A, Kashatus JA, Sessions DT, Sharmin S, Kashatus DF. Mito Hacker: a set of tools to enable high-throughput analysis of mitochondrial network morphology. Sci Rep. 2020 Nov 3;10(1):18941.\u003c/li\u003e\n\u003cli\u003eSprenger HG, Langer T. The Good and the Bad of Mitochondrial Breakups. Trends Cell Biol. 2019 Nov;29(11):888\u0026ndash;900.\u003c/li\u003e\n\u003cli\u003eZemirli N, Morel E, Molino D. Mitochondrial Dynamics in Basal and Stressful Conditions. Int J Mol Sci [Internet]. 2018 Feb 13;19(2). Available from: http://dx.doi.org/10.3390/ijms19020564\u003c/li\u003e\n\u003cli\u003eMishra P, Chan DC. Metabolic regulation of mitochondrial dynamics. J Cell Biol. 2016 Feb 15;212(4):379\u0026ndash;87.\u003c/li\u003e\n\u003cli\u003eAmaral A, Louren\u0026ccedil;o B, Marques M, Ramalho-Santos J. Mitochondria functionality and sperm quality [Internet]. Vol. 146, Reproduction. 2013. Available from: http://dx.doi.org/10.1530/REP-13-0178\u003c/li\u003e\n\u003cli\u003eHo HC, Wey S. Three dimensional rendering of the mitochondrial sheath morphogenesis during mouse spermiogenesis. Microsc Res Tech. 2007;70(8):719\u0026ndash;23.\u003c/li\u003e\n\u003cli\u003eLeung MR, Zenezini Chiozzi R, Roelofs MC, Hevler JF, Ravi RT, Maitan P, et al. 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Redox Biol. 2021 Nov 1;48:102176.\u003c/li\u003e\n\u003cli\u003eSchindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676\u0026ndash;82.\u003c/li\u003e\n\u003cli\u003eVan Rossum G, Drake FL. Python 3 Reference Manual: (Python Documentation Manual Part 2). CreateSpace; 2009. 242 p.\u003c/li\u003e\n\u003cli\u003eKapur JN, Sahoo PK, Wong AKC. A new method for gray-level picture thresholding using the entropy of the histogram. Computer Vision, Graphics, and Image Processing. 1985;29(1):140.\u003c/li\u003e\n\u003cli\u003evan der Walt S, Sch\u0026ouml;nberger JL, Nunez-Iglesias J, Boulogne F, Warner JD, Yager N, et al. scikit-image: image processing in Python. PeerJ. 2014 Jun 19;2:e453.\u003c/li\u003e\n\u003cli\u003eBeare R, Lowekamp B, Yaniv Z. Image Segmentation, Registration and Characterization in R with SimpleITK. J Stat Softw [Internet]. 2018 Aug;86. Available from: http://dx.doi.org/10.18637/jss.v086.i08\u003c/li\u003e\n\u003cli\u003eStival C, Puga Molina L del C, Paudel B, Buffone MG, Visconti PE, Krapf D. Sperm Capacitation and Acrosome Reaction in Mammalian Sperm. Adv Anat Embryol Cell Biol. 2016;220:93\u0026ndash;106.\u003c/li\u003e\n\u003cli\u003eVawda AI, Gunby J, Younglai EV. Andrology: Semen parameters as predictors of in-vitro fertilization: the importance of strict criteria sperm morphology. Hum Reprod. 1996 Jul 1;11(7):1445\u0026ndash;50.\u003c/li\u003e\n\u003cli\u003eBenedetti S, Tagliamonte MC, Catalani S, Primiterra M, Canestrari F, Stefani SD, et al. Differences in blood and semen oxidative status in fertile and infertile men, and their relationship with sperm quality. Reprod Biomed Online. 2012;25(3):300\u0026ndash;6.\u003c/li\u003e\n\u003cli\u003eWyrobek AJ, Gordon LA, Burkhart JG, Francis MW, Kapp RW, Letz G, et al. An evaluation of the mouse sperm morphology test and other sperm tests in nonhuman mammals: A report of the U.S. environmental protection agency Gene-Tox program. Mutation Research/Reviews in Genetic Toxicology. 1983 May 1;115(1):1\u0026ndash;72.\u003c/li\u003e\n\u003cli\u003eMundy AJ, Ryder TA, Edmonds DK. Asthenozoospermia and the human sperm mid-piece. Hum Reprod. 1995 Jan;10(1):116\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eFirman RC, Simmons LW. Sperm midpiece length predicts sperm swimming velocity in house mice. Biol Lett. 2010 Aug 23;6(4):513\u0026ndash;6.\u003c/li\u003e\n\u003cli\u003eBoguenet M, Bouet PE, Spiers A, Reynier P, May-Panloup P. Mitochondria: their role in spermatozoa and in male infertility. Hum Reprod Update. 2021 Jun 22;27(4):697\u0026ndash;719.\u003c/li\u003e\n\u003cli\u003eGu NH, Zhao WL, Wang GS, Sun F. Comparative analysis of mammalian sperm ultrastructure reveals relationships between sperm morphology, mitochondrial functions and motility. Reprod Biol Endocrinol. 2019 Aug 15;17(1):66.\u003c/li\u003e\n\u003cli\u003eValente AJ, Maddalena LA, Robb EL, Moradi F, Stuart JA. A simple ImageJ macro tool for analyzing mitochondrial network morphology in mammalian cell culture. Acta Histochem. 2017 Apr;119(3):315\u0026ndash;26.\u003c/li\u003e\n\u003cli\u003ePoot M, Zhang YZ, Kr\u0026auml;mer JA, Wells KS, Jones LJ, Hanzel DK, et al. Analysis of mitochondrial morphology and function with novel fixable fluorescent stains. J Histochem Cytochem. 1996 Dec;44(12):1363\u0026ndash;72.\u003c/li\u003e\n\u003cli\u003eChazotte B. Labeling mitochondria with MitoTracker dyes. Cold Spring Harb Protoc. 2011 Aug 1;2011(8):990\u0026ndash;2.\u003c/li\u003e\n\u003cli\u003eJablo\u0026ntilde;ski M, Luque GM, G\u0026oacute;mez-El\u0026iacute;as MD, Sanchez-Cardenas C, Xu X, de la Vega-Beltran JL, et al. Reorganization of the Flagellum Scaffolding Induces a Sperm Standstill During Fertilization. bioRxiv [Internet]. 2023 Oct 18; Available from: http://dx.doi.org/10.1101/2023.06.22.546073\u003c/li\u003e\n\u003cli\u003eWilliams a. C, Ford WC. The role of glucose in supporting motility and capacitation in human spermatozoa. J Androl. 2001;22(4):680\u0026ndash;95.\u003c/li\u003e\n\u003cli\u003eGoodson SG, Qiu Y, Sutton KA, Xie G, Jia W, O\u0026rsquo;Brien DA. Metabolic substrates exhibit differential effects on functional parameters of mouse sperm capacitation. Biol Reprod. 2012 Sep;87(3):75.\u003c/li\u003e\n\u003cli\u003eTourmente M, Sansegundo E, Rial E, Roldan ERS. Capacitation promotes a shift in energy metabolism in murine sperm. Front Cell Dev Biol. 2022 Aug 23;10:950979.\u003c/li\u003e\n\u003cli\u003eAmaral A, Ramalho-Santos J. Assessment of mitochondrial potential: Implications for the correct monitoring of human sperm function. Int J Androl [Internet]. 2010;33(1). Available from: http://dx.doi.org/10.1111/j.1365-2605.2009.00987.x\u003c/li\u003e\n\u003cli\u003eMoscatelli N, Spagnolo B, Pisanello M, Lemma ED, De Vittorio M, Zara V, et al. Single-cell-based evaluation of sperm progressive motility via fluorescent assessment of mitochondria membrane potential. Sci Rep [Internet]. 2017;7(1). Available from: http://dx.doi.org/10.1038/s41598-017-18123-1\u003c/li\u003e\n\u003cli\u003eGiaccagli MM, G\u0026oacute;mez-El\u0026iacute;as MD, Herzfeld JD, Mar\u0026iacute;n-Briggiler CI, Cuasnic\u0026uacute; PS, Cohen DJ, et al. Capacitation-induced mitochondrial activity is required for sperm fertilizing ability in mice by modulating hyperactivation. Front Cell Dev Biol. 2021 Oct 26;9:767161.\u003c/li\u003e\n\u003cli\u003eSchiavi-Ehrenhaus LJ, Romarowski A, Jablo\u0026ntilde;ski M, Krapf D, Luque GM, Buffone MG. The early molecular events leading to COFILIN phosphorylation during mouse sperm capacitation are essential for acrosomal exocytosis. J Biol Chem. 2022 Jun;298(6):101988.\u003c/li\u003e\n\u003cli\u003eBrener E, Rubinstein S, Cohen G, Shternall K, Rivlin J, Breitbart H. Remodeling of the actin cytoskeleton during mammalian sperm capacitation and acrosome reaction. Biol Reprod. 2003 Mar;68(3):837\u0026ndash;45.\u003c/li\u003e\n\u003cli\u003eRomarowski A, Velasco F\u0026eacute;lix \u0026Aacute;G, Torres Rodr\u0026iacute;guez P, Gervasi MG, Xu X, Luque GM, et al. Super-resolution imaging of live sperm reveals dynamic changes of the actin cytoskeleton during acrosomal exocytosis. J Cell Sci [Internet]. 2018 Nov 8;131(21). Available from: http://dx.doi.org/10.1242/jcs.218958\u003c/li\u003e\n\u003cli\u003eFawcett DW. The mammalian spermatozoon. Dev Biol. 1975 Jun;44(2):394\u0026ndash;436.\u003c/li\u003e\n\u003cli\u003eGervasi MG, Xu X, Carbajal-Gonzalez B, Buffone MG, Visconti PE, Krapf D. The actin cytoskeleton of the mouse sperm flagellum is organized in a helical structure. J Cell Sci [Internet]. 2018 Jun 11;131(11). Available from: http://dx.doi.org/10.1242/jcs.215897\u003c/li\u003e\n\u003cli\u003eRomarowski A, Luque GM, La Spina FA, Krapf D, Buffone MG. Role of Actin Cytoskeleton During Mammalian Sperm Acrosomal Exocytosis. Adv Anat Embryol Cell Biol. 2016;220:129\u0026ndash;44.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"sperm midpiece, mitochondria, image processing, capacitation, acrosome reaction","lastPublishedDoi":"10.21203/rs.3.rs-4145928/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4145928/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn mammalian sperm, mitochondria are very densely packed and form a helical sheath located in the midpiece of the flagellum. Mitochondria play multiple roles in the cell and can rapidly change shape to adapt to environmental conditions. During capacitation, mammalian spermatozoa undergo morphological and physiological changes to acquire fertilization ability. This is evidenced by changes in sperm motility patterns (hyperactivation) and the ability to perform the acrosome reaction. Whether there are changes in sperm mitochondrial shape or dimensions during capacitation is unknown. This work aimed to quantify morphometric changes in the sperm midpiece during capacitation based on computational analysis and image processing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing mitochondrial fluorescent probes and a combination of freely available software, we quantified the dimensions and fluorescence intensity of the midpiece of the sperm flagellum. After capacitation, the area occupied by the mitochondria decreased. This decrease was due to a reduction in the width but not the length of the midpiece. A reduction in the area and width of the midpiece occurred in spermatozoa that underwent the acrosome reaction, suggesting a shrinkage of the mitochondria during the process of capacitation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese results suggest that the flagellar structure is remodeled during sperm capacitation and the acrosome reaction, which is consistent with the observed changes in mitochondrial organization. The application of image processing to fluorescence microscopy images may help to identify morphological changes during capacitation.\u003c/p\u003e","manuscriptTitle":"Morphometric analysis of the sperm midpiece during capacitation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-04 17:25:46","doi":"10.21203/rs.3.rs-4145928/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e43536c1-72c9-4602-be1a-58b4c02c63bc","owner":[],"postedDate":"April 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-29T15:12:34+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-04 17:25:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4145928","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4145928","identity":"rs-4145928","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}