CENP-A is diluted during bovine spermatogenesis and is maintained at internally positioned centromere clusters in mature bull sperm

CENP-A is diluted during bovine spermatogenesis and is maintained at internally positioned centromere clusters in mature bull sperm | 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 CENP-A is diluted during bovine spermatogenesis and is maintained at internally positioned centromere clusters in mature bull sperm Miriama Štiavnická, Anna Ní Nualláin, Caitríona M Collins, Elaine M Dunleavy This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6929623/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Sep, 2025 Read the published version in Chromosome Research → Version 1 posted 9 You are reading this latest preprint version Abstract During spermatogenesis, chromatin structure is remodelled by the incorporation of distinct histone variants and associated posttranslational modifications, followed by the almost complete replacement of histones by protamines in sperm. However, the dynamics of the centromere-specific histone H3 variant CENP-A have not yet been elucidated during spermatogenesis in mammals. Here we investigate CENP-A localisation dynamics in cattle ( Bos taurus ). In bovine testis tissue sections, we quantify CENP-A intensity in key germ cell types; spermatogonia (pre-meiotic), primary spermatocytes (meiotic) and spermatids (post-meiotic). Our quantitation shows that spermatogonia harbour the highest amount of CENP-A compared to all other germ cell types. Spermatids have approximately one quarter the amount of CENP-A of spermatogonia indicating that overall, it is reduced and maintained through the two meiotic divisions. Yet, we also observed some unexpected dynamics. CENP-A is asymmetrically distributed such that undifferentiated spermatogonia harbour more CENP-A that differentiated spermatogonia that enter meiosis. We also noted an increase in CENP-A intensity in primary spermatocytes during meiotic prophase I, which is indicative of centromere assembly at this time. We also confirm the specific maintenance of CENP-A, and the absence of the centromeric DNA binding protein CENP-B, on mature bull sperm nuclei that have completed histone-to-protamine exchange. Finally, we present a model for centromere positioning in mature sperm nuclei and propose that centralised clustering of centromeres may serve a protective function during histone-to-protamine exchange. CENP-A centromeres bull sperm spermatogonia stem cells meiosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Centromeres are primary sites of chromosome constriction, which are essential for kinetochore assembly and the attachment of microtubules during cell division. This process ensures the equal distribution of genetic material to daughter cells. Rather than a particular DNA sequence, the presence of the histone H3 variant Centromere Protein A (CENP-A) specifies centromere identity and function in an epigenetic fashion (McKinley and Cheeseman 2015 ; Miga and Alexandrov 2021 ; Altemose et al. 2022 ). The functionality of human centromeres relies on the interaction between CENP-A-containing nucleosomes and members of the constitutive centromere-associated network (CCAN), as well as CENP-B that binds to centromeric DNA (Foltz et al. 2006 ; Kixmoeller et al. 2020 ; Yatskevich et al. 2023 ). During the cell cycle, CENP-A-containing nucleosomes are diluted as centromeric DNA is replicated. Therefore, CENP-A must be replenished each cell cycle to ensure continued centromere function and to maintain centromere identity. In somatic cells undergoing mitosis, the deposition of newly synthesised CENP-A occurs at early G1 phase (Jansen et al. 2007 ). This timing is conserved in most mitotic cell types that have been examined so far (Stellfox et al. 2013 ). Germ cells located in the gonads undergo unique cellular divisions including meiosis, followed by differentiation into haploid gametes (Bolcun-Filas and Handel 2018 ; Zickler and Kleckner 2023 ). Distinct CENP-A assembly dynamics have been observed in germ cells, mostly from studies conducted in the fruit fly Drosophila melanogaster (Ranjan et al. 2019 ; Dattoli et al. 2020 ; Kochendoerfer et al. 2023 ). In Drosophila , germline stem cells (GSCs) undergo asymmetric cell division to replenish the stem cell pool and to generate daughter cells that differentiate and undergo meiosis. Upon GSC division, CENP-A is asymmetrically distributed, with a higher CENP-A level observed in the stem cell compared to the daughter cell (Ranjan et al. 2019 ; Dattoli et al. 2020 ; Kochendoerfer et al. 2023 ). Based on this, a model of mitotic drive is proposed to occur in stem cells such that chromosomes with stronger centromeres, having more CENP-A, kinetochore proteins and earlier microtubule attachments, are preferentially segregated to the stem cell (Lampson and Black 2017 ; Akera et al. 2019 ; Ranjan and Chen 2022 ). With respect to the timing of CENP-A replenishment in meiosis, CENP-A is deposited during prophase I in Drosophila spermatocytes (Dunleavy et al. 2012 ; Raychaudhuri et al. 2012 ). This deposition timing differs from mitotic deposition after chromosome segregation at G1 phase, occurring prior to homologous chromosome segregation in meiosis. Observations in Drosophila have also confirmed that CENP-A is retained at mature sperm centromeres despite extensive chromatin remodelling and global histone removal occurring during histone-to-protamine exchange (Dunleavy et al. 2012 ; Raychaudhuri et al. 2012 ). Moreover, experiments using CENP-A-depleted fly sperm demonstrated that a critical amount of CENP-A must be retained at centromeres to ensure the fidelity of the mitotic divisions following fertilization (Raychaudhuri et al. 2012 ). Whether the unusual CENP-A dynamics observed in Drosophila are conserved in mammals has not yet been addressed. Early studies in the 1990’s identified centromeres in mouse, bull, and human sperm using CREST serum, recognising three centromeric proteins: CENP-A, -B, and -C (del Mazo et al. 1987 ; Palmer et al. 1990 ; Zalensky et al. 1993 ). Direct detection of CENP-A using monoclonal antibodies has only been achieved recently in mouse and human sperm (Manske et al. bioRxiv; Mudrak et al. 2009 ; Das et al. 2022 ). Investigations into the spatial positioning of chromosomes based on centromere and telomere sequences in mouse and human mature sperm have generated models for how these regions are uniquely packaged after protamination(Zalensky et al. 1993 ; Wiland et al. 2016 ; Ioannou et al. 2017 ; Champroux et al. 2018 ; Xu et al. 2025 ). Studies in bull mature sperm have investigated preferred positions of chromosomes and certain satellite repeats (Powell et al. 1990 ; Chagin et al. 2018 ), yet a detailed spatial coordinate analysis of centromere organisation in bull sperm nuclei is lacking. The non-random organisation of centromeres in mature sperm could be functionally important for chromosome capture and segregation in early embryo development. In this study, we investigate CENP-A localisation dynamics during spermatogenesis in cattle and probe CENP-A retention and centromere organisation in mature bull sperm. Bos taurus serves as an appropriate model given similarities between cattle and human sperm in terms of morphology, the length of spermatogenesis (Johnson et al. 2000 ; Thompson et al. 2018 ; Gu et al. 2019 ), and the frequency of aneuploidies observed in early embryogenesis (Ménézo and Hérubel 2002 ; Brooks et al. 2022 ), in addition to the ease of accessibility of bull testes and semen samples. Material and methods All chemicals were purchased from Sigma Aldrich (Arklow, Co Wicklow, Ireland) unless otherwise stated. All animal protocols were in accordance with the Cruelty to Animals Act (Ireland 1876, as amended by European Communities regulations 2002 and 2005) and the European Community Directive 2010/63/EU. Semen samples collected during commercial production were donated to this project and the study was deemed exempt from ethical approval. Testicular tissue was obtained from three fertile bulls with an average age of 22 months. Immunofluorescence of bull testes Bull testes were fixed in 4% formaldehyde for 24 hours and stored in 70% ethanol for another 48 hours. Testes tissue was paraffin embedded and 5 µm sections were prepared. For immunofluorescence, sections were deparaffinized in xylene and rehydrated through an ethanol series (100, 90, 70, 50%). After antigen retrieval (10 mM Sodium citrate, pH 6 at 98°C for 20 minutes), slides were allowed to cool down. Thereafter slides with sections were washed three times in permeabilization solution PBST (0.3% Triton X-100 in PBS, phosphate buffered saline) for 5 minutes and blocked with 5% bovine serum albumin in PBST at room temperature for 2 hours. Incubation with primary antibodies was done at 4 o C overnight. Primary antibodies: mouse anti-CENP-A (1:100; Enzo, Brussels, Belgium; ADI-KAM-CC006-E), rabbit anti-SYCP3 (1:200; Abcam, Amsterdam, Netherland; ab15093), rabbit anti-ZBTB16 (1:400; Invitrogen, Walthman, MA, USA; PA5-29213) and rabbit anti-TNP1 (1:200; Abcam; ab73135). Following three washing steps in PBS with 0.3% Triton X-100, sections were incubated with secondary antibodies anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 546 (1:200; Invitrogen) at room temperature for 1 hour. After three washes, slides were stained with Hoechst 33342 (1:1000; Invitrogen; 11544876) for 20 minutes, washed three times and mounted in Slow Fade Gold antifade reagent (Invitrogen; S36936). Immunofluorescence of bull sperm Frozen bull semen was thawed, and the content of the straw was released into a tube and washed with PBS three times. Sperm were diluted in PBS and briefly sonicated to detach the flagellum. Sperm heads were centrifuged and stored in ice cold methanol:acetic acid (3:1). For immunofluorescence, the sperm suspension was applied to slides and allowed to dry. Chromatin decondensation was performed in a solution of 3M NaOH at 37℃ for 3 minutes and immediately neutralized with distilled water. Slides were blocked in PBS with 5% normal goat serum and 0.3% Triton X-100 for 1 hour and incubated with anti-CENP-A (1:100), anti-CENP-B antibodies (1:100; Abcam, ab259855) or IgG control at 4℃ overnight. After three washes with PBS with 1% normal goat serum and 0.3% Triton X-100, slides were incubated with Alexa Fluor secondary antibodies and DAPI (1:200; 4′,6-diamidino-2-phenylindole). After 1 hour, three washes were performed and sperm on slides were mounted with Slow Fade Gold antifade reagent. Fluorescence in situ hybridization (FISH) of bull testes and sperm Probe for centromere satellite 1.723 and Y-specific centromere repeats were designed based on Escudeiro et al. 2019 and Olagunju et al. 2024 . Subsequently they were labelled by nick translation (Abbott Molecular, Illinois, USA). Telomere probe (TTAGGG) 6 was synthesised commercially based on Moens et al. ( 1990 ). FISH on bull testes Bull testes sections were prepared as for IF including antigen retrieval, after which slides were washed with PBS and processed for FISH. Slides with embedded tissue sections were washed twice with 2X Saline Sodium Citrate (SSC) buffer containing 0.1% Tween (SSCT). This was followed by two additional washes with 25% formamide in SSCT. Slides were incubated with 50% formamide in SSCT for 1 hour at room temperature (RT) and an additional hour at 37°C. A pre-hybridization step was conducted using hybridization buffer (3X SSC, 50% formamide, 10% dextran sulphate) for 1 hour at 37°C. Hybridization step was at 37°C for 2 hours. Thereafter, samples were denatured at 90°C for 5 minutes and incubated at 37°C for 48 hours. Post-incubation, the sections were washed three times in SSCT with 50% formamide (2x at 37°C and 1x at RT). Then sections were washed twice with SSCT at RT, followed by a single wash with 1X PBS at RT. Then we continued with IF staining as described above starting with blocking and co-staining with rabbit anti-SYCP3 primary antibody, washed and stain with anti-rabbit Alexa Fluor 647. Finally, the slides were stained with Hoechst (1:1000), washed twice with PBS, and mounted with Slow Fade Gold antifade reagent. FISH on bull sperm To enable accessibility of the probe to DNA, chromatin decondensation was performed based on previously published protocols (Li et al. 2008 ; De Oliveira et al. 2013 ). Briefly, frozen thawed sperm were washed 3x with PBS with 6 mM EDTA, pipetted onto coverslips and fixed with 0.5% PFA for 10 min. After washing with PBS, sperm were decondensed in the solution of 10 mM DTT and 5 ug/ml Heparin at RT for 15 min, washed in PBS, fixed in 4 % PFA, washed and stored in PBS. The covrslips were let to dry and dehydrate through an ethanol series of 50%, 75%, and 95%, respectively. FISH was performed with slight modifications. Cover slips with sperm were washed twice with SSCT at RT. This was followed by two additional washes with 50% formamide at RT. For the hybridization step, probe diluted in hybridization buffer was pipetted onto a microscope slide. The coverslip with sperm was inverted onto the slide and sealed with rubber cement. The slides were then incubated in humidified chamber at 37°C for 1 hour. Subsequently, the slides were denatured at 90°C for 5 minutes on a hot plate and incubated at 37°C overnight. After incubation, the rubber cement glue was removed, and the coverslips were returned to a 6-well plate. Coverslips were washed twice at 37°C in SSCT with 50% formamide, followed by a third wash at RT. Further washing steps included once in SSCT at RT and a final wash in 1X PBS at RT. To stain the nuclei, DAPI at a 1:1000 dilution was added and incubated for half an hour. The coverslips were washed twice with PBS and mounted. Widefield microscopy Images from immunofluorescent staining of testes or sperm were acquired using a DeltaVision Elite microscope system (Imaging Solutions Inc) equipped with a 40x, 60x and 100x oil immersion UPlanS-Apo objective (NA 1.4). Images were acquired as z-stacks with a step size of 0.2 µm or 0.1 µm for sperm nuclei analyses in Fig. 5 . Fluorescence passed through a 435/48 nm; 525/48 nm; 597/45 nm; 632/34 nm band-pass filter for detection of respectively DAPI, Hoechst, Alexa Fluor 488, Alexa Fluor 547 in sequential mode. Images were deconvolved using Softworx. Image quantification The quantitation of CENP-A including image preprocessing was done in Fiji ImageJ software. For each quantitation, one cell was considered. Images from a single cell (nucleus) were projected (maximum intensity) to capture all the centromeres present in the cell. The pictures were converted to 8-bit format and the threshold was adjusted using commands called max entropy. Integrated density of individual centromere foci was measured and then sum per nucleus to obtain information about total amount of CENP-A per cell. Western analysis Bull sperm, human hRPE1 and bovine MDBK cells were washed 3x in PBS and lysed in 2x SDS Laemmli buffer with protease and phosphatase inhibitor for 1 hour on ice. Prior to loading on the gel samples were incubated with Benzonase nuclease for 30 minutes and then boiled for 5 minutes. Proteins were separated by SDS-PAGE and transferred onto a PVDF membrane. Membranes were blocked with 5% milk in TBS with 0.1% Tween 20 (TBST) followed by overnight incubation at 4°C with primary antibody: mouse anti-CENPA, rabbit anti CENP-B; mouse anti-α-tubulin, mouse anti-histone H4, rabbit anti-human HJURP (Abcam, ab224076), sheep anti-bovine HJURP raised against peptide 683–693 (MRC PPU Reagents and Services, University of Dundee,UK) diluted in blocking solution at a concentration of 1:1000. After three washes with TBST, the membrane was incubated with HRP-conjugated secondary antibodies (1:5000, Invitrogen) in blocking solution for an hour at room temperature. To visualise stained protein, ECL Western Blot Substrates (Invitrogen) was used, and images were acquired with Fusion FX (Vilber) imager. Centromere organisation in mature sperm Foci coordinates were extracted using the 3D Object Counter plugin in FIJI/ImageJ (Bolte and Cordelières 2006 ). Manual thresholds were applied to each channel to capture all visible foci while minimizing background noise. Foci with volumes below 0.005 µm³ were excluded, based on telomere size estimates (~ 0.08 µm³;(Hazzouri et al. 2000 )). To remove artefacts, foci located beyond one standard deviation outside the average nuclear dimensions (width = 78.89 µm, height = 143.54 µm) were also discarded. Spatial coordinates (X, Y) were normalized to the centroid of each DAPI-stained nucleus to correct for positional variation introduced during imaging. Cleaned and normalised datasets, including coordinates and focus type (centromere or telomere), were imported into RStudio (v2025.5.0.496) for data visualisation and analysis (Auguie 2010 ; Wickham 2016 ; Posit Team 2025 ; R Core Team 2025 ). To correct for image orientation, Y-axis values were inverted. Foci abundance per nucleus was visualised using histograms. Spatial distributions were explored using 2D heatmaps generated with geom_bin2d(), overlaid on an ellipse representing average nuclear shape (width = 73.74 µm, height = 137.52 µm), centred at (0, 0). Consistent axis limits allowed direct comparisons between focus types. For radial analysis, normalised radial distances from the nucleus centre were calculated based on the average elliptical dimensions (major = 143.54 µm, minor = 78.89 µm). Foci were classified into three concentric rings (inner, middle, outer), each covering one-third of the radius, with outliers labelled "Outside." The proportion of centromere and telomere foci per ring was calculated. Final figures were prepared in Adobe Illustrator (v29.5.1). Statistical analysis Statistical analyses were performed in GraphPad Prism 10.3.(505). All obtained data were assessed for normality of distribution using the Shapiro-Wilk test and processed accordingly. Data were analysed either with Kruskal-Wallis ANOVA or Mann-Whitney U test. Differences between individual groups were further assessed with multiple pairwise-comparison tests. For the centromere organisation in mature sperm (Fig. 5 ), statistical analyses were performed in R using statistical packages (Wickham et al. 2019 ; Agostinelli and Lund 2024 ). To analyse angular distributions of foci, 2D XY coordinates were converted to polar angles (radians) using the atan2 function. Rayleigh tests for circular uniformity were conducted for centromeres and telomeres. To assess radial distribution patterns, observed counts in inner, middle, and outer rings were compared using chi-squared tests for independence. Fisher’s exact tests were used to assess enrichment or depletion of foci in each ring versus all others. The results were considered statistically significant if P < 0.05. Results CENP-A at centromeres is diluted during spermatogenesis. Spermatogenesis in bulls starts with the asymmetric division of spermatogonial stem cells into undifferentiated spermatogonia (spermatogonia A) giving rise to differentiated spermatogonia (spermatogonia B) that are committed to meiosis (Fig. 1 a). Spermatogonia A and B undergo approximately six rounds of mitosis entering a brief interphase before differentiating into primary spermatocytes, which then proceed into meiosis I (Staub and Johnson 2018 ). During prophase of meiosis I, homologous chromosomes pair and undergo genetic recombination. Primary spermatocytes complete homologous chromosome segregation in the first meiotic division resulting in haploid secondary spermatocytes. During meiosis II, sister chromatids are separated into daughter cells called spermatids. Spermatids undergo extensive morphological changes, including a global chromatin remodelling, in which most histones are replaced by protamines after histone-to-protamine exchange (Gaspa-Toneu and Peters 2023 ). Through DNA labelling of testicular sections, we identified spermatogonia, primary spermatocytes and spermatids in the seminiferous tubule based on nuclear morphology and spatial positioning (Fig. 1 b, c). Myoid cells constituting the border of individual seminiferous tubules were used as somatic cell controls. Secondary spermatocytes that had completed the first meiotic division were not included in our analysis, due to the rapid succession of the second meiotic division and the lack of specific markers for their identification. To label centromeres in each of these cell types, we stained testis sections with an anti-CENP-A monoclonal antibody (Fig. 2 a). To control for antibody accessibility in different cell types, we performed an anti-histone H4 staining that showed a similar pattern to DNA staining (Fig. S1). Co-staining for Synaptonemal Complex Protein 3 (SYCP3) enabled us to specifically mark primary spermatocytes undergoing synapsis in prophase of meiosis I. Furthermore, the SYCP3 staining pattern allowed us to mark cells with condensed chromosomes and either weak or absent SYCP3 staining, which we deemed to be in early prophase I (EP), or cells with more extensive SYCP3 staining, which we deemed to be later prophase I (LP) (Fig. 2 b). We then quantified CENP-A intensity in spermatogonia, primary spermatocytes in early and late prophase I, and spermatids, as well as in control myoid cells (nuclei pooled from N = 3 bulls) (Fig. 2 b, c). Initially, we observed that as cell division progresses from the basal lamina toward the lumen of the seminiferous tubule, CENP-A is reduced in intensity. Our quantitation showed that spermatogonia bear the highest amount of CENP-A when compared to all other germ and somatic cells in the tubule. Spermatids that have completed both meiotic divisions, have approximately a quarter of the amount of CENP-A present in spermatogonia, suggesting a proportional dilution of CENP-A during two successive meiotic divisions. Yet, spermatids contain only half the CENP-A of primary spermatocytes also suggesting that some CENP-A deposition might occur during meiosis. Indeed, in primary spermatocytes we measured a significantly higher amount of CENP-A in late prophase I compared to early prophase I (P = 0.0086), indicative of centromere assembly at this cell cycle time. We also noted a cell-type specific number and distribution of CENP-A foci within nuclei (Fig. 2 b, d). In spermatogonia, approximately 21 ± 0.57 CENP-A foci were detected that were dispersed throughout the nucleus. The highest number of CENP-A foci (34 ± 0.93) was detected in primary spermatocytes at late prophase I that were mostly located at the nuclear periphery. In spermatids, the number of foci decreased to 14 ± 0.42 in accordance with ploidy and centromeres adopted a more central position. To validate the CENP-A localisation pattern, we performed fluorescence in situ hybridization (FISH) using a centromeric satellite 1.723 probe which corresponds to the CENP-A-associated region in cattle (Escudeiro et al. 2019 ) and a (TTAGGG) 6 probe for mammalian telomere repeats (Moens et al. 1990 ) (Fig. S2). Centromere 1.723 FISH patterns closely resembled those observed by anti-CENP-A antibody detection, validating the specificity of antibody staining patterns. CENP-A is asymmetrically distributed between undifferentiated and differentiating spermatogonia. Our initial analysis indicated that a population of spermatogonia displayed the highest level of CENP-A amongst all other cells in the seminiferous tubule (Fig. 2 c). Using the stem cell specific marker Zinc Finger and BTB Domain Containing 16 (ZBTB16) we confirmed that these cells were undifferentiated spermatogonia/spermatogonia A (Fig. 3 a, b). Quantitation of CENP-A intensity in ZBTB16-positive and -negative spermatogonia revealed that undifferentiated spermatogonia have approximately 1.5-fold more CENP-A than differentiated spermatogonia/spermatogonia B. The quantity of CENP-A in undifferentiated spermatogonia was also higher than in somatic myoid cells (Fig. 3 c). Although centromere localisation was more central in undifferentiated spermatogonia and more dispersed in differentiated spermatogonia, the number of CENP-A foci did not differ between the two cell types (Fig. 3 d). Therefore, the 1.5-fold difference in intensity is not likely to be due to differences in centromere detection between cell types. CENP-A is maintained in mature bull sperm. We next wanted to determine whether CENP-A is specifically retained on mature bull sperm nuclei that have completed histone-to-protamine exchange. Immunostaining with antibodies recognising Transition Protein 1 (TPN1) enabled the identification of spermatids at the initial stages of histone-to-protamine exchange (Fig. S3a, b). However, we noticed that although TNP1 and CENP-A were both detectable in round spermatids, as the spermatids elongated the number of foci decreased. We concluded that we could not reliably detect CENP-A at this stage due to enhanced nuclear compaction and the occlusion of antibody epitopes. Hence, we examined CENP-A localisation in decondensed mature sperm nuclei in ejaculated semen samples. Upon decondensation to permit antibody accessibility, we stained mature sperm nuclei for CENP-A and observed distinct nuclear foci that were not detected using an IgG control (Fig. 4 a). The presence of CENP-A in mature bull sperm was validated by western analysis in which a single band corresponding to the molecular weight of bovine CENP-A (17 kDa) was detected (Fig. 4 b). Extracts from human RPE1 and bovine MDBK tissue culture cell lines served as positive controls for CENP-A antibody recognition and anti-alpha-tubulin served as a loading control. In parallel, we immunostained decondensed mature sperm with an anti-CENP-B antibody, however CENP-B was not detectable (Fig. 4 a). This result was confirmed by western analysis of bull sperm extracts (Fig. 4 b). As expected, CENP-B was detectable in human RPE1 cells and blotting of bovine MDBK cells confirmed that the antibody can recognise bovine CENP-B. For these experiments, histone H4 is used as the loading control. Finally, we tested for the presence of the CENP-A assembly factor HJURP on mature sperm (Fig. S4). We performed western analysis using two different antibodies, one raised against human HJURP, and another raised against bovine HJURP. We detected a band at the expected molecular weight for HJURP in bovine MDBK cells with both antibodies. In sperm, we did not detect this band, indicating the absence of HJURP. However, a higher molecular weight band at approximately 90 kDa was visible using both antibodies. Spatial organisation of centromeres in sperm is non-random. To complement our investigation into CENP-A maintenance on mature sperm, we performed a detailed examination of centromere positioning in mature sperm using FISH for the CENP-A-associated satellites 1.723 (autosomal centromeres), and a Y-specific centromeric repeat(Escudeiro et al. 2019 ; Olagunju et al. 2024 ). In parallel, we labelled telomeres by FISH to determine overall chromosome organisation in protamine-bound mature sperm nuclei (Fig. 5 a). Our analysis revealed that both centromeres and telomeres formed distinct clusters, with an average of 10–11 centromere and 17–19 telomere foci per nucleus (n = 300 nuclei) (Fig. 5 b). These counts were significantly lower than the expected 30 centromeres and 60 telomeres in the haploid bovine genome, suggesting that multiple loci (~ 3 on average) co-localise within each focus. To capture different spatial patterns within the nucleus, angular (directional) and radial (distance-based) distributions were assessed separately. The angular distribution (tested using Rayleigh’s test) evaluates whether foci are directionally biased or randomly oriented around the nuclear center. In contrast, the radial distribution reflects how concentrated foci are toward the inner, middle, or outer regions of the nucleus, regardless of direction. Rayleigh’s test for uniformity demonstrated that centromere positions were non-randomly distributed within the nucleus, while telomeres were randomly distributed (P < 0.01), i.e. telomeres were dispersed without directional bias across the nucleus. The heatmap in (Fig. 5 c) shows that centromeres were more internally positioned, with telomeres commonly found throughout the entirety of the nucleus. To further investigate radial organisation, each nucleus was modelled as an ellipse and divided into three concentric rings: inner, middle, and outer. Centromeres were predominantly located in the middle ring (59.7%), with markedly fewer found in the inner (19.8%) and outer (20.5%) rings. In contrast, telomeres showed a broader distribution across all regions, with the highest proportion observed in the middle ring (49.2%), followed by the outer (30.6%) and inner (18.1%) rings. Percentages do not total exactly 100% due to the exclusion of a small fraction of foci localised outside the elliptical nuclear boundary. To illustrate consistency across animals, the percentage and standard deviation of telomere and centromere localisation within each ring (calculated across the three bulls) are reported (Fig. 5 d). Fisher’s exact tests showed that centromeres were significantly enriched in the middle ring compared to telomeres P < 0.01), and telomeres were significantly enriched in the outer ring relative to centromeres (P < 0.01). This organisation is illustrated in a representative model of the average mature sperm nucleus (Fig. 5 e), with centromeres clustering more internally and telomeres extending toward the nuclear periphery. In sperm nuclei, internally localised centromeres were frequently in contact with telomeres, most likely because cattle autosomes are acrocentric. Discussion In this study we investigate the dynamics of the centromeric histone CENP-A and centromere localisation across different stages of spermatogenesis in the Bos taurus animal model. Overall, our findings reveal that haploid spermatids contain approximately one quarter the amount of CENP-A compared to differentiated spermatogonia (Fig. 6 a). This result indicates that CENP-A is proportionally diluted and maintained during the two successive meiotic divisions. However, we also noted that the level of CENP-A in spermatids is approximately half that found in primary spermatocytes in late prophase I. This is not expected as after two cell divisions, this number should be one quarter. Indeed, we measured an increase in CENP-A in spermatocytes, between early and late prophase I that can explain an elevated CENP-A level in spermatids. Comparable dynamics have been observed in Drosophila spermatogenesis in which CENP-A is replenished during prophase I (Dunleavy et al. 2012 ; Raychaudhuri et al. 2012 ). It is possible that CENP-A loading prior to chromosome segregation is required for kinetochore assembly such that homologous chromosome segregate in meiosis I. The measured increase in CENP-A coincides with the relocalisation of centromeres to the nuclear periphery in late prophase I, which is presumably linked to the processes of homolog pairing and synapsis and reflecting the bouquet configuration of bovine acrocentric chromosomes (Scherthan et al. 1996 ; Pfeifer et al. 2001 ; Scherthan 2001 ). Telomere tethering to the periphery prior to chromosome pairing explains this relocalisation (Fig. S2). However, whether CENP-A assembly occurs before, after or during the pairing of homologues chromosomes requires further investigation and more detailed identification of individual stages. Another possibility is that additional CENP-A assembly occurs in secondary spermatocytes that are not captured in our quantitation. We also report that spermatogonia contain the highest amount of CENP-A of any cell type in the testis seminiferous tubule. Using ZBTB16 to specifically mark undifferentiated spermatogonia, we found that CENP-A is asymmetrically distributed between undifferentiated and differentiated spermatogonia. This finding is very much in line with observations in Drosophila GSCs that carry the highest levels of CENP-A compared to other meiotic stages and show an asymmetric distribution of CENP-A between stem and daughter cells (Ranjan et al. 2019 ). In bulls, undifferentiated spermatogonia have 1.5-fold more CENP-A than differentiating spermatogonia, which is remarkably comparable to the 1.4-1.6-fold enrichment measured in Drosophila GSCs (Ranjan et al. 2019 ; Dattoli et al. 2020 ). In flies, a model of mitotic drive is proposed suggesting that the centromere of one sister chromatid harbours 1.5-fold more CENP-A and this biases chromosome segregation with strong centromeres more likely to end up in the stem cell. Our data indicates that mitotic drive might also operate in mammalian germline. A similar phenomenon was recently described during mouse spermatogenesis (Manske et al. bioRxiv) and in additional stem cell types (Behnan et al. 2016 ; Evano et al. 2020 ). Indeed, as CENP-A level can potentially mark cell fate, it has broad prognostic and diagnostic potential in the context of cancer and aging (Renaud-Pageot et al. 2022 ; Yang et al. 2022 ; Sikder et al. 2025 ). Moreover, CENP-A could be exploited as a marker of undifferentiated spermatogonial stem cells in mammals. Our study also confirmed the maintenance of CENP-A, and the absence of CENP-B, in mature bull sperm. This result aligns with initial findings from 40 years ago, reporting the presence of CENP-A on bull sperm using CREST serum (Štiavnická et al. 2025 ). However, in these experiments neither CENP-B nor CENP-C was detected in the bovine thymus, therefore it remained possible that human CREST serum did not detect bovine centromere proteins. Our results now confirm the maintenance of CENP-A on sperm using a monoclonal antibody and we also validated that the anti-human CENP-B antibody utilised can recognise bovine CENP-B. Unfortunately, we have not been able to clearly elucidate presence or absence of CENP-C, because of technical difficulties related to specificity of anti-CENP-C antibody and its ability to recognise the bovine antigen. In Drosophila , the sole presence of CENP-A and lack of CENP-C (the only CCAN component in flies) on mature sperm centromeres was previously demonstrated (Raychaudhuri et al. 2012 ; Dunleavy et al. 2012 ). Given that the maintenance of paternal CENP-A at centromeres on mature sperm is critical for fly embryo development (Raychaudhuri et al. 2012 ), it is possible that a critical amount of paternal CENP-A is vital for the mitotic divisions after fertilization in mammals too (Manske et al. bioRxiv). In contrast to flies, in which CAL1 (the Drosophila HJURP equivalent) was not present on sperm, HJURP has been recently detected in human and mouse sperm (Manske et al. bioRxiv). This suggests a role for paternal HJURP in the deposition of new CENP-A after fertilization. While we could not detect HJURP in mature bull sperm, we noted a higher molecular weight band that might represent a modified version or isoform in cattle. Lastly, we examined the spatial organisation of centromeres and the CENP-A-associated satellite during spermatogenesis and in mature bull sperm (Fig. 6 b). Centromeres showed a distinct, cell-type-specific distribution within nuclei, initially localising at the nuclear periphery in primary spermatocytes and shifting toward the nuclear interior in spermatids. Our detailed analysis of centromere and telomere positioning in mature sperm showed that centromeres are non-randomly distributed, forming distinct clusters toward the nuclear interior, while telomeres are more randomly dispersed and significantly enriched at the periphery in comparison to centromeres (Fig. 5 e). It is important to note that spatial organisation in mature sperm was assessed following DTT/Heparin-induced decondensation, which is required for the FISH protocol in line with several landmark studies (Zalensky et al. 1993 , 1995 ; Hazzouri et al. 2000 ; Ioannou et al. 2017 ; Chagin et al. 2018 ). Although DTT treatment results in uniform swelling while preserving gross nuclear shape, we acknowledge it may subtly affect chromatin architecture. This caveat should be considered when interpreting fine-scale spatial relationships. Our combination of random angular dispersion and peripheral radial enrichment indicates that telomeres are broadly distributed in all directions but significantly localize nearer the nuclear edge. This spatial separation suggests that centromeres and telomeres are organised under different biological constraints during spermatogenesis. The degree of clustering for both centromeres and telomeres is comparable to a study in human sperm, with the number of centromere clusters being more consistent and telomere clusters more variable across cells (Ioannou et al. 2017 ). While telomeres occupied similar positions throughout the nucleus in both bulls and humans, centromeres were found in both intermediate and peripheral locations in humans, whereas in bulls, they were predominantly enriched in the intermediate zone (Ioannou et al. 2017 ; Xu et al. 2025 ). One explanation for this difference may relate to the acrocentric nature of bovine chromosomes: because autosomes in cattle are acrocentric, one telomere is inherently positioned near the centromere. Future work using genome-wide spatial sequencing would help clarify whether the p-arm telomere (proximal to the centromere) localises internally and the q-arm telomere more peripherally. Interestingly, the internal positioning of centromeres seems to be already established by the spermatid stage, with a similar number of centromere foci detected (Fig. 2 d), pointing to a nuclear architecture that is maintained despite the histone-to-protamine exchange during sperm maturation. A likely function of clustering is CENP-A preservation, potentially safeguarding centromere identity during spermiogenesis and enabling rapid kinetochore assembly post-fertilization. The drivers of centromere clustering are not known, but heterochromatin interactions, structural constraints, or epigenetic factors may be involved. While certain chromosomes occupy conserved nuclear positions in bovine sperm (Chagin et al. 2018 ), whether specific centromeres co-cluster consistently remains to be determined. In summary, our findings in bulls validate previous observations in Drosophila melanogaster males and suggest a conservation of unusual CENP-A dynamics, mechanisms and functions in mammalian spermatogenesis. Future research should focus on investigating the functional significance of CENP-A's asymmetric distribution in stem cells and its potential as a marker of stem cell identity in spermatogenesis. Additionally, exploring the molecular mechanisms governing CENP-A deposition in meiosis I is likely to reveal unexpected results given that it differs from mitotic assembly in G1 phase. Determining CENP-C dynamics, as well as the functional significance of CENP-A and/or HJURP maintenance on sperm for fertility and chromosomal inheritance in the embryo is also critical. Given the similarity in sperm centromere organisation between bulls and humans, this system may offer valuable insights into centromere dynamics and CENP-A behaviour during human spermatogenesis. While access to early stages of spermatogenesis in humans is challenging, studying bulls may serve as a powerful model to infer conserved mechanisms of centromere regulation with potential implications for understanding male fertility. Our results are also important given ongoing efforts to generate telomere to telomere assemblies of centromeres in cattle and other ruminants (Kalbfleisch et al. 2024 ) that will allow studies of chromosome evolution and speciation. Declarations Acknowledgement We would like to thank Dr. Mark Canney from Human Biology at the University of Galway for help with preparation of tissue sections, the National Cattle Breeding Centre in Ireland for donation of frozen-thawed samples of bull semen and Kepak Ltd. Athleague for the donation of testes tissues. Funding This project was supported by Research Ireland (grant number 20/FFP-A/8519). Contribution E.M. Dunleavy, C.M. Collins and M. Štiavnická conceived and designed the study. M. Štiavnická and A. Ní Nualláin performed experiments, fluorescence microscopy imaging, quantitation and data analysis. M. Štiavnická, A. Ní Nualláin and E.M. Dunleavy wrote the manuscript. Ethical approval Not applicable. Competing interests Authors declare no competing interests. Clinical trial number Not applicable. References Agostinelli C, Lund U (2024) circular: Circular Statistics Akera T, Trimm E, Lampson MA (2019) Molecular Strategies of Meiotic Cheating by Selfish Centromeres. 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Springer-Verlag Zalensky AO, Brencman JW, Zalenskaya A, et al (1993) Organization of centromeres in the decondensed nuclei of mature human sperm. Springer-Verlag Zickler D, Kleckner N (2023) Meiosis: Dances Between Homologs. Annu Rev Genet 57:1–63. https://doi.org/10.1146/ANNUREV-GENET-061323-044915 Additional Declarations No competing interests reported. Supplementary Files FiguresStiavnickaetalFINAL7.png Fig. S1 Immunostaining of bull testes with histone H4. Staining of this canonical histones H4 colocalises with nuclear staining of individual stages and shows permeability of nuclear membrane and accessibility to nuclear proteins. H4 is not detectable in elongated and mature sperm due to chromatin compaction. Scale bar 20 µm. FiguresStiavnickaetalFINAL8.png Fig. S2 Spatial organization of centromeres in bull testes. For localization of centromeres FISH was performed with probe for centromere satellite 1.723 (Escudeiro et al. 2019) and telomere repeats (TTAGGG) 6 (Moens et al. 1990). a Cross section of a seminiferous tubule in bull testes. Scale bar 20 µm b Zoom of individual cell types/stages. Scale bar 5 µm. FiguresStiavnickaetalFINAL9.png Fig. S3CENP-A detection during spermiogenesis. a Cross section of a seminiferous tubule in bull testes displaying different stages of spermiogenesis immunostained for CENP-A and the transition protein TNP. Scale bar 20 µm. b Individual stages of spermiogenesis identified based on nuclear staining and presence or absence of transition proteins. Round spermatids display CENP-A foci, later stages of elongated spermatids do not display consistent staining patterns with the CENP-A antibody due to increased chromatin compaction. Scale bar 5 µm. FiguresStiavnickaetalFINAL10.png Fig. S4 Western blot detection of HJURP in mature bull sperm. HJURP was detected either with anti-human HJURP (upper panel) or anti-bovine HJURP (lower panel) antibodies. Human RPE1 (hRPE1) cells and bovine MDBK cells extracts were used as positive controls. Histone H4 was used as a loading control. Samples were loaded in duplicates (lanes 1, 2). Cite Share Download PDF Status: Published Journal Publication published 16 Sep, 2025 Read the published version in Chromosome Research → Version 1 posted Editorial decision: Revision requested 27 Jul, 2025 Reviews received at journal 26 Jul, 2025 Reviews received at journal 08 Jul, 2025 Reviewers agreed at journal 07 Jul, 2025 Reviewers agreed at journal 25 Jun, 2025 Reviewers invited by journal 23 Jun, 2025 Editor assigned by journal 20 Jun, 2025 Submission checks completed at journal 20 Jun, 2025 First submitted to journal 19 Jun, 2025 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. 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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-6929623","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":475352673,"identity":"332f45cb-04c2-4e9b-b00c-48c17b25543a","order_by":0,"name":"Miriama Štiavnická","email":"","orcid":"","institution":"University of Galway","correspondingAuthor":false,"prefix":"","firstName":"Miriama","middleName":"","lastName":"Štiavnická","suffix":""},{"id":475352674,"identity":"732c8b74-7677-476f-8b2c-13eb88c0467d","order_by":1,"name":"Anna Ní Nualláin","email":"","orcid":"","institution":"University of Galway","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"Ní","lastName":"Nualláin","suffix":""},{"id":475352675,"identity":"936b1526-8649-4c10-a892-0dedbc8f3e8f","order_by":2,"name":"Caitríona M Collins","email":"","orcid":"","institution":"Technological University of the Shannon","correspondingAuthor":false,"prefix":"","firstName":"Caitríona","middleName":"M","lastName":"Collins","suffix":""},{"id":475352676,"identity":"0b14d7fb-dfd5-435e-9d4f-64651689a427","order_by":3,"name":"Elaine M Dunleavy","email":"data:image/png;base64,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","orcid":"","institution":"University of Galway","correspondingAuthor":true,"prefix":"","firstName":"Elaine","middleName":"M","lastName":"Dunleavy","suffix":""}],"badges":[],"createdAt":"2025-06-19 09:23:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6929623/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6929623/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10577-025-09781-3","type":"published","date":"2025-09-16T15:57:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85379921,"identity":"88fb1d1d-a33e-49c5-ba99-8aa033f3b54a","added_by":"auto","created_at":"2025-06-25 09:07:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1499742,"visible":true,"origin":"","legend":"\u003cp\u003eSpermatogenesis in \u003cem\u003eBos taurus\u003c/em\u003e. \u003cstrong\u003ea\u003c/strong\u003e Cartoon of spermatogenesis indicating key stages and cell types. \u003cstrong\u003eb\u003c/strong\u003e Nuclear staining (Hoechst) of a testis cross section, showing three seminiferous tubules and indicating the position of myoid somatic cells at the tubule border. \u003cstrong\u003ec\u003c/strong\u003e Zoom into the individual seminiferous tubule depicting the typical morphology and position of germ cell types analysed (Spermatogonia; Primary spermatocytes; Spermatids; Mature sperm). Scale bar 20 µm.\u003c/p\u003e","description":"","filename":"FiguresStiavnickaetalFINAL1.png","url":"https://assets-eu.researchsquare.com/files/rs-6929623/v1/ffe4d182e402f5480551205e.png"},{"id":85381174,"identity":"d368046d-1c46-4645-ba90-4b508f608cf3","added_by":"auto","created_at":"2025-06-25 09:15:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1683736,"visible":true,"origin":"","legend":"\u003cp\u003eCENP-A is progressively diluted during spermatogenesis in bull. \u003cstrong\u003ea \u003c/strong\u003eCross section of a seminiferous tubule in bull testis displaying major stages of spermatogenesis immunostained for CENP-A (red) and SYCP3 (green) and DNA is stained with DAPI (cyan). Scale bar 20 µm. \u0026nbsp;\u003cstrong\u003eb \u003c/strong\u003eZoom of individual cell types immunostained for CENP-A (red) and SYCP3 (green). Scale bar 5 µm. \u0026nbsp;\u003cstrong\u003ec \u003c/strong\u003eCENP-A quantitation was performed based on the sum of integrated density of individual foci per nucleus for Spermatogonia (n=156), spermatocytes in early prophase I (Spermatocytes_EP, n=136), spermatocytes in late prophase I (Spermatocytes_LP) and Spermatids (n=172). Myoid cells (n=96) were used as control. \u003cstrong\u003ed \u003c/strong\u003eNumber of CENP-A foci per nucleus in myoid cells, spermatogonia, spermatocytes in early prophase I (Spermatocytes_EP), spermatocytes in late prophase I (Spermatocytes_LP) and Spermatids. Data are presented as median with interquartile range and were considered significant when P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"FiguresStiavnickaetalFINAL2.png","url":"https://assets-eu.researchsquare.com/files/rs-6929623/v1/f017dd300abd2e81da668e1c.png"},{"id":85379927,"identity":"548448bb-b6e4-44b3-8254-e983421a70be","added_by":"auto","created_at":"2025-06-25 09:07:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1015775,"visible":true,"origin":"","legend":"\u003cp\u003eCENP-A is asymmetrically distributed between undifferentiated spermatogonia and differentiating spermatogonia. \u003cstrong\u003ea \u003c/strong\u003eCross section of a seminiferous tubule in bull testes immunostained for CENP-A and ZBTB16 that labels undifferentiated spermatogonia/spermatogonia A located at the border of tubule. Scale bar 20 µm. \u003cstrong\u003eb\u003c/strong\u003e Undifferentiated and differentiated spermatogonia immunostained for CENP-A and ZBTB16. Scale bar 5 µm. \u003cstrong\u003ec\u003c/strong\u003e CENP-A quantitation was performed based on the sum of integrated density of individual foci per nucleus for ZBTB16-positive undifferentiated spermatogonia (n=78) and ZBTB16-negative differentiated spermatogonia (n=122). Myoid cells (n=96) were used as control. \u003cstrong\u003ed\u003c/strong\u003e Number of CENP-A foci per nucleus in undifferentiated and differentiated spermatogonia and in myoid cells. Data are presented as median with interquartile range and were considered significant when P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"FiguresStiavnickaetalFINAL3.png","url":"https://assets-eu.researchsquare.com/files/rs-6929623/v1/1aae9368c92038495d514244.png"},{"id":85381568,"identity":"09c091ad-2d45-4c63-9f40-91f7a860bd25","added_by":"auto","created_at":"2025-06-25 09:23:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1168388,"visible":true,"origin":"","legend":"\u003cp\u003eCENP-A, but not CENP-B, is maintained in mature bull sperm. \u003cstrong\u003ea \u003c/strong\u003eMature bull sperm stained with anti-CENP-A antibody, anti-CENP-B antibody or IgG control. Scale bar 5 µm. \u0026nbsp;\u003cstrong\u003eb \u003c/strong\u003ePresence of CENP-A and absence of CENP-B in bull sperm was confirmed by western blot. Human RPE1 (hRPE1) cells and bovine MDBK cells extracts were used as positive controls. α-tubulin or histone H4 were used as loading controls. Samples were loaded in duplicate (lanes 1, 2).\u003c/p\u003e","description":"","filename":"FiguresStiavnickaetalFINAL4.png","url":"https://assets-eu.researchsquare.com/files/rs-6929623/v1/006a6f2551ab82c0e4e2dbaf.png"},{"id":85379929,"identity":"429abad0-7a44-4477-80eb-6dc0e9399fe1","added_by":"auto","created_at":"2025-06-25 09:07:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":408548,"visible":true,"origin":"","legend":"\u003cp\u003eSpatial organisation of centromeres in mature bull sperm. \u003cstrong\u003ea\u003c/strong\u003e Representative DNA FISH image showing centromeres (red) and telomeres (green) in a sperm nucleus. Scale bar = 5 μm. \u003cstrong\u003eb\u003c/strong\u003eNumber of centromere and telomere foci per nucleus across 300 sperm nuclei (100 per bull), indicating clustering of both elements. \u003cstrong\u003ec\u003c/strong\u003e Heatmap of all foci positions normalised to nucleus centroids, revealing non-random centromere localization and uniform telomere distribution (Rayleigh’s test, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). \u003cstrong\u003ed\u003c/strong\u003e Radial analysis based on elliptical nuclear models divided into three concentric rings. Numerical labels denote mean ± standard deviation. Centromeres are enriched in the middle ring, while telomeres are more peripheral (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, Fisher’s exact test). \u003cstrong\u003ee\u003c/strong\u003eRepresentative model showing the average sperm nucleus (centromeres in red and telomeres in purple).\u003c/p\u003e","description":"","filename":"FiguresStiavnickaetalFINAL5.png","url":"https://assets-eu.researchsquare.com/files/rs-6929623/v1/e8e29d39b03a5f9fa87adf12.png"},{"id":85381569,"identity":"cb21092b-0cc3-4686-966e-e59842bbaacb","added_by":"auto","created_at":"2025-06-25 09:23:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":281552,"visible":true,"origin":"","legend":"\u003cp\u003eSummary of CENP-A dynamics during bull spermatogenesis. \u003cstrong\u003ea\u003c/strong\u003e Integrated density of CENP-A is normalised to differentiated spermatogonia/spermatogonia B (black bar). Undifferentiated spermatogonia (spermatogonia A) have 1.5 times more CENP-A than differentiated spermatogonia (spermatogonia B), most likely due to asymmetric division and the retention of more CENP-A in the stem cell. A lower CENP-A level was detected in somatic myoid cells compared to undifferentiated spermatogonia. CENP-A is diluted upon cell division with spermatids having approximately quarter of CENP-A from differentiated spermatogonia. CENP-A level is lower than expected in primary spermatocytes entering meiosis, suggesting incomplete replenishment of CENP-A during the previous mitoses. This reduction is partially compensated for in late primary spermatocytes, when CENP-A increases, enabling successful completion of both meiotic divisions without affecting centromere function. \u003cstrong\u003eb\u003c/strong\u003e Diagram of spermatogenesis depicting the typical pattern of CENP-A foci within nuclei of individual stages as well as in mature sperm which is not part of quantitation. Throughout spermatogenesis, individual stages show distinct patterns of centromere localisation and clustering. While the number of centromere clusters reflect ploidy, the spatial organisation of foci probably reflects functional events ongoing at time, such as pairing of homologues chromosomes in prophase I of primary spermatocytes and arrangement prior to protamination in spermatids.\u003c/p\u003e","description":"","filename":"FiguresStiavnickaetalFINAL6.png","url":"https://assets-eu.researchsquare.com/files/rs-6929623/v1/f0ac02c9bd862b02276e70fc.png"},{"id":91890056,"identity":"3d8a201f-81c2-4714-88fa-decd276ced1d","added_by":"auto","created_at":"2025-09-22 16:03:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6911103,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6929623/v1/6825aa9e-3d4f-4d73-b81e-274d298a9f41.pdf"},{"id":85379922,"identity":"5f551dcf-7bd9-45ed-8ff6-e91157f2047c","added_by":"auto","created_at":"2025-06-25 09:07:03","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":871312,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S1\u003cstrong\u003e \u003c/strong\u003eImmunostaining of bull testes with histone H4. Staining of this canonical histones H4 colocalises with nuclear staining of individual stages and shows permeability of nuclear membrane and accessibility to nuclear proteins. H4 is not detectable in elongated and mature sperm due to chromatin compaction. Scale bar 20 µm.\u003c/p\u003e","description":"","filename":"FiguresStiavnickaetalFINAL7.png","url":"https://assets-eu.researchsquare.com/files/rs-6929623/v1/39c8e13ab744f3204a00bfe1.png"},{"id":85379920,"identity":"df734234-57a2-492c-9f82-fe8d3d4e609e","added_by":"auto","created_at":"2025-06-25 09:07:03","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2355678,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S2 Spatial organization of centromeres in bull testes. For localization of centromeres FISH was performed with probe for centromere satellite 1.723 (Escudeiro et al. 2019) and telomere repeats (TTAGGG)\u003csub\u003e6\u003c/sub\u003e (Moens et al. 1990). \u003cstrong\u003ea\u003c/strong\u003e Cross section of a seminiferous tubule in bull testes. Scale bar 20 µm \u003cstrong\u003eb\u003c/strong\u003e Zoom of individual cell types/stages. Scale bar 5 µm.\u003c/p\u003e","description":"","filename":"FiguresStiavnickaetalFINAL8.png","url":"https://assets-eu.researchsquare.com/files/rs-6929623/v1/502d8b8430641ff49901cdbd.png"},{"id":85379925,"identity":"97f97853-0893-45b8-8789-ca83ecc67286","added_by":"auto","created_at":"2025-06-25 09:07:03","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1592493,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S3CENP-A detection during spermiogenesis.\u003cstrong\u003e a\u003c/strong\u003e Cross section of a seminiferous tubule in bull testes displaying different stages of spermiogenesis immunostained for CENP-A and the transition protein TNP. Scale bar 20 µm. \u003cstrong\u003eb\u003c/strong\u003e Individual stages of spermiogenesis identified based on nuclear staining and presence or absence of transition proteins. Round spermatids display CENP-A foci, later stages of elongated spermatids do not display consistent staining patterns with the CENP-A antibody due to increased chromatin compaction. Scale bar 5 µm.\u003c/p\u003e","description":"","filename":"FiguresStiavnickaetalFINAL9.png","url":"https://assets-eu.researchsquare.com/files/rs-6929623/v1/df97653b4ea0b9b60070276f.png"},{"id":85379930,"identity":"ef10d73a-5c8b-47d5-a6f8-ff71e8abffc9","added_by":"auto","created_at":"2025-06-25 09:07:03","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":852158,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S4\u003cstrong\u003e \u003c/strong\u003eWestern blot detection of HJURP in mature bull sperm. HJURP was detected either with anti-human HJURP (upper panel) or anti-bovine HJURP (lower panel) antibodies. Human RPE1 (hRPE1) cells and bovine MDBK cells extracts were used as positive controls. Histone H4 was used as a loading control. Samples were loaded in duplicates (lanes 1, 2).\u003c/p\u003e","description":"","filename":"FiguresStiavnickaetalFINAL10.png","url":"https://assets-eu.researchsquare.com/files/rs-6929623/v1/bdaced6f0439bb318dd28960.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"CENP-A is diluted during bovine spermatogenesis and is maintained at internally positioned centromere clusters in mature bull sperm","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCentromeres are primary sites of chromosome constriction, which are essential for kinetochore assembly and the attachment of microtubules during cell division. This process ensures the equal distribution of genetic material to daughter cells. Rather than a particular DNA sequence, the presence of the histone H3 variant Centromere Protein A (CENP-A) specifies centromere identity and function in an epigenetic fashion (McKinley and Cheeseman \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Miga and Alexandrov \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Altemose et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The functionality of human centromeres relies on the interaction between CENP-A-containing nucleosomes and members of the constitutive centromere-associated network (CCAN), as well as CENP-B that binds to centromeric DNA (Foltz et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Kixmoeller et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yatskevich et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDuring the cell cycle, CENP-A-containing nucleosomes are diluted as centromeric DNA is replicated. Therefore, CENP-A must be replenished each cell cycle to ensure continued centromere function and to maintain centromere identity. In somatic cells undergoing mitosis, the deposition of newly synthesised CENP-A occurs at early G1 phase (Jansen et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). This timing is conserved in most mitotic cell types that have been examined so far (Stellfox et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Germ cells located in the gonads undergo unique cellular divisions including meiosis, followed by differentiation into haploid gametes (Bolcun-Filas and Handel \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zickler and Kleckner \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Distinct CENP-A assembly dynamics have been observed in germ cells, mostly from studies conducted in the fruit fly Drosophila melanogaster (Ranjan et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Dattoli et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kochendoerfer et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In \u003cem\u003eDrosophila\u003c/em\u003e, germline stem cells (GSCs) undergo asymmetric cell division to replenish the stem cell pool and to generate daughter cells that differentiate and undergo meiosis. Upon GSC division, CENP-A is asymmetrically distributed, with a higher CENP-A level observed in the stem cell compared to the daughter cell (Ranjan et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Dattoli et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kochendoerfer et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Based on this, a model of mitotic drive is proposed to occur in stem cells such that chromosomes with stronger centromeres, having more CENP-A, kinetochore proteins and earlier microtubule attachments, are preferentially segregated to the stem cell (Lampson and Black \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Akera et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ranjan and Chen \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). With respect to the timing of CENP-A replenishment in meiosis, CENP-A is deposited during prophase I in \u003cem\u003eDrosophila\u003c/em\u003e spermatocytes (Dunleavy et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Raychaudhuri et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). This deposition timing differs from mitotic deposition after chromosome segregation at G1 phase, occurring prior to homologous chromosome segregation in meiosis. Observations in \u003cem\u003eDrosophila\u003c/em\u003e have also confirmed that CENP-A is retained at mature sperm centromeres despite extensive chromatin remodelling and global histone removal occurring during histone-to-protamine exchange (Dunleavy et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Raychaudhuri et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Moreover, experiments using CENP-A-depleted fly sperm demonstrated that a critical amount of CENP-A must be retained at centromeres to ensure the fidelity of the mitotic divisions following fertilization (Raychaudhuri et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhether the unusual CENP-A dynamics observed in Drosophila are conserved in mammals has not yet been addressed. Early studies in the 1990\u0026rsquo;s identified centromeres in mouse, bull, and human sperm using CREST serum, recognising three centromeric proteins: CENP-A, -B, and -C (del Mazo et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Palmer et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Zalensky et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Direct detection of CENP-A using monoclonal antibodies has only been achieved recently in mouse and human sperm (Manske et al. bioRxiv; Mudrak et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Das et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Investigations into the spatial positioning of chromosomes based on centromere and telomere sequences in mouse and human mature sperm have generated models for how these regions are uniquely packaged after protamination(Zalensky et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Wiland et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ioannou et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Champroux et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Studies in bull mature sperm have investigated preferred positions of chromosomes and certain satellite repeats (Powell et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Chagin et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), yet a detailed spatial coordinate analysis of centromere organisation in bull sperm nuclei is lacking. The non-random organisation of centromeres in mature sperm could be functionally important for chromosome capture and segregation in early embryo development. In this study, we investigate CENP-A localisation dynamics during spermatogenesis in cattle and probe CENP-A retention and centromere organisation in mature bull sperm. \u003cem\u003eBos taurus\u003c/em\u003e serves as an appropriate model given similarities between cattle and human sperm in terms of morphology, the length of spermatogenesis (Johnson et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Thompson et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Gu et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and the frequency of aneuploidies observed in early embryogenesis (M\u0026eacute;n\u0026eacute;zo and H\u0026eacute;rubel \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Brooks et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), in addition to the ease of accessibility of bull testes and semen samples.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003eAll chemicals were purchased from Sigma Aldrich (Arklow, Co Wicklow, Ireland) unless otherwise stated. All animal protocols were in accordance with the Cruelty to Animals Act (Ireland 1876, as amended by European Communities regulations 2002 and 2005) and the European Community Directive 2010/63/EU. Semen samples collected during commercial production were donated to this project and the study was deemed exempt from ethical approval. Testicular tissue was obtained from three fertile bulls with an average age of 22 months.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence of bull testes\u003c/h2\u003e \u003cp\u003eBull testes were fixed in 4% formaldehyde for 24 hours and stored in 70% ethanol for another 48 hours. Testes tissue was paraffin embedded and 5 \u0026micro;m sections were prepared. For immunofluorescence, sections were deparaffinized in xylene and rehydrated through an ethanol series (100, 90, 70, 50%). After antigen retrieval (10 mM Sodium citrate, pH 6 at 98\u0026deg;C for 20 minutes), slides were allowed to cool down. Thereafter slides with sections were washed three times in permeabilization solution PBST (0.3% Triton X-100 in PBS, phosphate buffered saline) for 5 minutes and blocked with 5% bovine serum albumin in PBST at room temperature for 2 hours. Incubation with primary antibodies was done at 4\u003csup\u003eo\u003c/sup\u003eC overnight. Primary antibodies: mouse anti-CENP-A (1:100; Enzo, Brussels, Belgium; ADI-KAM-CC006-E), rabbit anti-SYCP3 (1:200; Abcam, Amsterdam, Netherland; ab15093), rabbit anti-ZBTB16 (1:400; Invitrogen, Walthman, MA, USA; PA5-29213) and rabbit anti-TNP1 (1:200; Abcam; ab73135). Following three washing steps in PBS with 0.3% Triton X-100, sections were incubated with secondary antibodies anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 546 (1:200; Invitrogen) at room temperature for 1 hour. After three washes, slides were stained with Hoechst 33342 (1:1000; Invitrogen; 11544876) for 20 minutes, washed three times and mounted in Slow Fade Gold antifade reagent (Invitrogen; S36936).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunofluorescence of bull sperm\u003c/h3\u003e\n\u003cp\u003eFrozen bull semen was thawed, and the content of the straw was released into a tube and washed with PBS three times. Sperm were diluted in PBS and briefly sonicated to detach the flagellum. Sperm heads were centrifuged and stored in ice cold methanol:acetic acid (3:1). For immunofluorescence, the sperm suspension was applied to slides and allowed to dry. Chromatin decondensation was performed in a solution of 3M NaOH at 37℃ for 3 minutes and immediately neutralized with distilled water. Slides were blocked in PBS with 5% normal goat serum and 0.3% Triton X-100 for 1 hour and incubated with anti-CENP-A (1:100), anti-CENP-B antibodies (1:100; Abcam, ab259855) or IgG control at 4℃ overnight. After three washes with PBS with 1% normal goat serum and 0.3% Triton X-100, slides were incubated with Alexa Fluor secondary antibodies and DAPI (1:200; 4\u0026prime;,6-diamidino-2-phenylindole). After 1 hour, three washes were performed and sperm on slides were mounted with Slow Fade Gold antifade reagent.\u003c/p\u003e\n\u003ch3\u003eFluorescence in situ hybridization (FISH) of bull testes and sperm\u003c/h3\u003e\n\u003cp\u003eProbe for centromere satellite 1.723 and Y-specific centromere repeats were designed based on Escudeiro et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e and Olagunju et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e. Subsequently they were labelled by nick translation (Abbott Molecular, Illinois, USA). Telomere probe (TTAGGG)\u003csub\u003e6\u003c/sub\u003e was synthesised commercially based on Moens et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1990\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eFISH on bull testes\u003c/h3\u003e\n\u003cp\u003eBull testes sections were prepared as for IF including antigen retrieval, after which slides were washed with PBS and processed for FISH. Slides with embedded tissue sections were washed twice with 2X Saline Sodium Citrate (SSC) buffer containing 0.1% Tween (SSCT). This was followed by two additional washes with 25% formamide in SSCT. Slides were incubated with 50% formamide in SSCT for 1 hour at room temperature (RT) and an additional hour at 37\u0026deg;C. A pre-hybridization step was conducted using hybridization buffer (3X SSC, 50% formamide, 10% dextran sulphate) for 1 hour at 37\u0026deg;C. Hybridization step was at 37\u0026deg;C for 2 hours. Thereafter, samples were denatured at 90\u0026deg;C for 5 minutes and incubated at 37\u0026deg;C for 48 hours. Post-incubation, the sections were washed three times in SSCT with 50% formamide (2x at 37\u0026deg;C and 1x at RT). Then sections were washed twice with SSCT at RT, followed by a single wash with 1X PBS at RT. Then we continued with IF staining as described above starting with blocking and co-staining with rabbit anti-SYCP3 primary antibody, washed and stain with anti-rabbit Alexa Fluor 647. Finally, the slides were stained with Hoechst (1:1000), washed twice with PBS, and mounted with Slow Fade Gold antifade reagent.\u003c/p\u003e\n\u003ch3\u003eFISH on bull sperm\u003c/h3\u003e\n\u003cp\u003eTo enable accessibility of the probe to DNA, chromatin decondensation was performed based on previously published protocols (Li et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; De Oliveira et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Briefly, frozen thawed sperm were washed 3x with PBS with 6 mM EDTA, pipetted onto coverslips and fixed with 0.5% PFA for 10 min. After washing with PBS, sperm were decondensed in the solution of 10 mM DTT and 5 ug/ml Heparin at RT for 15 min, washed in PBS, fixed in 4 % PFA, washed and stored in PBS. The covrslips were let to dry and dehydrate through an ethanol series of 50%, 75%, and 95%, respectively. FISH was performed with slight modifications. Cover slips with sperm were washed twice with SSCT at RT. This was followed by two additional washes with 50% formamide at RT. For the hybridization step, probe diluted in hybridization buffer was pipetted onto a microscope slide. The coverslip with sperm was inverted onto the slide and sealed with rubber cement. The slides were then incubated in humidified chamber at 37\u0026deg;C for 1 hour. Subsequently, the slides were denatured at 90\u0026deg;C for 5 minutes on a hot plate and incubated at 37\u0026deg;C overnight. After incubation, the rubber cement glue was removed, and the coverslips were returned to a 6-well plate. Coverslips were washed twice at 37\u0026deg;C in SSCT with 50% formamide, followed by a third wash at RT. Further washing steps included once in SSCT at RT and a final wash in 1X PBS at RT. To stain the nuclei, DAPI at a 1:1000 dilution was added and incubated for half an hour. The coverslips were washed twice with PBS and mounted.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWidefield microscopy\u003c/h2\u003e \u003cp\u003eImages from immunofluorescent staining of testes or sperm were acquired using a DeltaVision Elite microscope system (Imaging Solutions Inc) equipped with a 40x, 60x and 100x oil immersion UPlanS-Apo objective (NA 1.4). Images were acquired as z-stacks with a step size of 0.2 \u0026micro;m or 0.1 \u0026micro;m for sperm nuclei analyses in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Fluorescence passed through a 435/48 nm; 525/48 nm; 597/45 nm; 632/34 nm band-pass filter for detection of respectively DAPI, Hoechst, Alexa Fluor 488, Alexa Fluor 547 in sequential mode. Images were deconvolved using Softworx.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImage quantification\u003c/h3\u003e\n\u003cp\u003eThe quantitation of CENP-A including image preprocessing was done in Fiji ImageJ software. For each quantitation, one cell was considered. Images from a single cell (nucleus) were projected (maximum intensity) to capture all the centromeres present in the cell. The pictures were converted to 8-bit format and the threshold was adjusted using commands called max entropy. Integrated density of individual centromere foci was measured and then sum per nucleus to obtain information about total amount of CENP-A per cell.\u003c/p\u003e\n\u003ch3\u003eWestern analysis\u003c/h3\u003e\n\u003cp\u003eBull sperm, human hRPE1 and bovine MDBK cells were washed 3x in PBS and lysed in 2x SDS Laemmli buffer with protease and phosphatase inhibitor for 1 hour on ice. Prior to loading on the gel samples were incubated with Benzonase nuclease for 30 minutes and then boiled for 5 minutes. Proteins were separated by SDS-PAGE and transferred onto a PVDF membrane. Membranes were blocked with 5% milk in TBS with 0.1% Tween 20 (TBST) followed by overnight incubation at 4\u0026deg;C with primary antibody: mouse anti-CENPA, rabbit anti CENP-B; mouse anti-α-tubulin, mouse anti-histone H4, rabbit anti-human HJURP (Abcam, ab224076), sheep anti-bovine HJURP raised against peptide 683\u0026ndash;693 (MRC PPU Reagents and Services, University of Dundee,UK) diluted in blocking solution at a concentration of 1:1000. After three washes with TBST, the membrane was incubated with HRP-conjugated secondary antibodies (1:5000, Invitrogen) in blocking solution for an hour at room temperature. To visualise stained protein, ECL Western Blot Substrates (Invitrogen) was used, and images were acquired with Fusion FX (Vilber) imager.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCentromere organisation in mature sperm\u003c/h2\u003e \u003cp\u003eFoci coordinates were extracted using the 3D Object Counter plugin in FIJI/ImageJ (Bolte and Cordeli\u0026egrave;res \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Manual thresholds were applied to each channel to capture all visible foci while minimizing background noise. Foci with volumes below 0.005 \u0026micro;m\u0026sup3; were excluded, based on telomere size estimates (~\u0026thinsp;0.08 \u0026micro;m\u0026sup3;;(Hazzouri et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2000\u003c/span\u003e)). To remove artefacts, foci located beyond one standard deviation outside the average nuclear dimensions (width\u0026thinsp;=\u0026thinsp;78.89 \u0026micro;m, height\u0026thinsp;=\u0026thinsp;143.54 \u0026micro;m) were also discarded. Spatial coordinates (X, Y) were normalized to the centroid of each DAPI-stained nucleus to correct for positional variation introduced during imaging. Cleaned and normalised datasets, including coordinates and focus type (centromere or telomere), were imported into RStudio (v2025.5.0.496) for data visualisation and analysis (Auguie \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Wickham \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Posit Team \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; R Core Team \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). To correct for image orientation, Y-axis values were inverted. Foci abundance per nucleus was visualised using histograms. Spatial distributions were explored using 2D heatmaps generated with geom_bin2d(), overlaid on an ellipse representing average nuclear shape (width\u0026thinsp;=\u0026thinsp;73.74 \u0026micro;m, height\u0026thinsp;=\u0026thinsp;137.52 \u0026micro;m), centred at (0, 0). Consistent axis limits allowed direct comparisons between focus types. For radial analysis, normalised radial distances from the nucleus centre were calculated based on the average elliptical dimensions (major\u0026thinsp;=\u0026thinsp;143.54 \u0026micro;m, minor\u0026thinsp;=\u0026thinsp;78.89 \u0026micro;m). Foci were classified into three concentric rings (inner, middle, outer), each covering one-third of the radius, with outliers labelled \"Outside.\" The proportion of centromere and telomere foci per ring was calculated. Final figures were prepared in Adobe Illustrator (v29.5.1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed in GraphPad Prism 10.3.(505). All obtained data were assessed for normality of distribution using the Shapiro-Wilk test and processed accordingly. Data were analysed either with Kruskal-Wallis ANOVA or Mann-Whitney U test. Differences between individual groups were further assessed with multiple pairwise-comparison tests. For the centromere organisation in mature sperm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003e), statistical analyses were performed in R using statistical packages (Wickham et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Agostinelli and Lund \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). To analyse angular distributions of foci, 2D XY coordinates were converted to polar angles (radians) using the atan2 function. Rayleigh tests for circular uniformity were conducted for centromeres and telomeres. To assess radial distribution patterns, observed counts in inner, middle, and outer rings were compared using chi-squared tests for independence. Fisher\u0026rsquo;s exact tests were used to assess enrichment or depletion of foci in each ring versus all others. The results were considered statistically significant if P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCENP-A at centromeres is diluted during spermatogenesis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSpermatogenesis in bulls starts with the asymmetric division of spermatogonial stem cells into undifferentiated spermatogonia (spermatogonia A) giving rise to differentiated spermatogonia (spermatogonia B) that are committed to meiosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Spermatogonia A and B undergo approximately six rounds of mitosis entering a brief interphase before differentiating into primary spermatocytes, which then proceed into meiosis I (Staub and Johnson \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). During prophase of meiosis I, homologous chromosomes pair and undergo genetic recombination. Primary spermatocytes complete homologous chromosome segregation in the first meiotic division resulting in haploid secondary spermatocytes. During meiosis II, sister chromatids are separated into daughter cells called spermatids. Spermatids undergo extensive morphological changes, including a global chromatin remodelling, in which most histones are replaced by protamines after histone-to-protamine exchange (Gaspa-Toneu and Peters \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Through DNA labelling of testicular sections, we identified spermatogonia, primary spermatocytes and spermatids in the seminiferous tubule based on nuclear morphology and spatial positioning (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, c). Myoid cells constituting the border of individual seminiferous tubules were used as somatic cell controls. Secondary spermatocytes that had completed the first meiotic division were not included in our analysis, due to the rapid succession of the second meiotic division and the lack of specific markers for their identification.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo label centromeres in each of these cell types, we stained testis sections with an anti-CENP-A monoclonal antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). To control for antibody accessibility in different cell types, we performed an anti-histone H4 staining that showed a similar pattern to DNA staining (Fig. S1). Co-staining for Synaptonemal Complex Protein 3 (SYCP3) enabled us to specifically mark primary spermatocytes undergoing synapsis in prophase of meiosis I. Furthermore, the SYCP3 staining pattern allowed us to mark cells with condensed chromosomes and either weak or absent SYCP3 staining, which we deemed to be in early prophase I (EP), or cells with more extensive SYCP3 staining, which we deemed to be later prophase I (LP) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). We then quantified CENP-A intensity in spermatogonia, primary spermatocytes in early and late prophase I, and spermatids, as well as in control myoid cells (nuclei pooled from N\u0026thinsp;=\u0026thinsp;3 bulls) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c). Initially, we observed that as cell division progresses from the basal lamina toward the lumen of the seminiferous tubule, CENP-A is reduced in intensity. Our quantitation showed that spermatogonia bear the highest amount of CENP-A when compared to all other germ and somatic cells in the tubule. Spermatids that have completed both meiotic divisions, have approximately a quarter of the amount of CENP-A present in spermatogonia, suggesting a proportional dilution of CENP-A during two successive meiotic divisions. Yet, spermatids contain only half the CENP-A of primary spermatocytes also suggesting that some CENP-A deposition might occur during meiosis. Indeed, in primary spermatocytes we measured a significantly higher amount of CENP-A in late prophase I compared to early prophase I (P\u0026thinsp;=\u0026thinsp;0.0086), indicative of centromere assembly at this cell cycle time.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also noted a cell-type specific number and distribution of CENP-A foci within nuclei (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, d). In spermatogonia, approximately 21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57 CENP-A foci were detected that were dispersed throughout the nucleus. The highest number of CENP-A foci (34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93) was detected in primary spermatocytes at late prophase I that were mostly located at the nuclear periphery. In spermatids, the number of foci decreased to 14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42 in accordance with ploidy and centromeres adopted a more central position. To validate the CENP-A localisation pattern, we performed fluorescence in situ hybridization (FISH) using a centromeric satellite 1.723 probe which corresponds to the CENP-A-associated region in cattle (Escudeiro et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and a (TTAGGG)\u003csub\u003e6\u003c/sub\u003e probe for mammalian telomere repeats (Moens et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) (Fig. S2). Centromere 1.723 FISH patterns closely resembled those observed by anti-CENP-A antibody detection, validating the specificity of antibody staining patterns.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCENP-A is asymmetrically distributed between undifferentiated and differentiating spermatogonia.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur initial analysis indicated that a population of spermatogonia displayed the highest level of CENP-A amongst all other cells in the seminiferous tubule (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Using the stem cell specific marker Zinc Finger and BTB Domain Containing 16 (ZBTB16) we confirmed that these cells were undifferentiated spermatogonia/spermatogonia A (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). Quantitation of CENP-A intensity in ZBTB16-positive and -negative spermatogonia revealed that undifferentiated spermatogonia have approximately 1.5-fold more CENP-A than differentiated spermatogonia/spermatogonia B. The quantity of CENP-A in undifferentiated spermatogonia was also higher than in somatic myoid cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Although centromere localisation was more central in undifferentiated spermatogonia and more dispersed in differentiated spermatogonia, the number of CENP-A foci did not differ between the two cell types (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Therefore, the 1.5-fold difference in intensity is not likely to be due to differences in centromere detection between cell types.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCENP-A is maintained in mature bull sperm.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe next wanted to determine whether CENP-A is specifically retained on mature bull sperm nuclei that have completed histone-to-protamine exchange. Immunostaining with antibodies recognising Transition Protein 1 (TPN1) enabled the identification of spermatids at the initial stages of histone-to-protamine exchange (Fig. S3a, b). However, we noticed that although TNP1 and CENP-A were both detectable in round spermatids, as the spermatids elongated the number of foci decreased. We concluded that we could not reliably detect CENP-A at this stage due to enhanced nuclear compaction and the occlusion of antibody epitopes. Hence, we examined CENP-A localisation in decondensed mature sperm nuclei in ejaculated semen samples. Upon decondensation to permit antibody accessibility, we stained mature sperm nuclei for CENP-A and observed distinct nuclear foci that were not detected using an IgG control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The presence of CENP-A in mature bull sperm was validated by western analysis in which a single band corresponding to the molecular weight of bovine CENP-A (17 kDa) was detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Extracts from human RPE1 and bovine MDBK tissue culture cell lines served as positive controls for CENP-A antibody recognition and anti-alpha-tubulin served as a loading control. In parallel, we immunostained decondensed mature sperm with an anti-CENP-B antibody, however CENP-B was not detectable (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). This result was confirmed by western analysis of bull sperm extracts (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). As expected, CENP-B was detectable in human RPE1 cells and blotting of bovine MDBK cells confirmed that the antibody can recognise bovine CENP-B. For these experiments, histone H4 is used as the loading control. Finally, we tested for the presence of the CENP-A assembly factor HJURP on mature sperm (Fig. S4). We performed western analysis using two different antibodies, one raised against human HJURP, and another raised against bovine HJURP. We detected a band at the expected molecular weight for HJURP in bovine MDBK cells with both antibodies. In sperm, we did not detect this band, indicating the absence of HJURP. However, a higher molecular weight band at approximately 90 kDa was visible using both antibodies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSpatial organisation of centromeres in sperm is non-random.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo complement our investigation into CENP-A maintenance on mature sperm, we performed a detailed examination of centromere positioning in mature sperm using FISH for the CENP-A-associated satellites 1.723 (autosomal centromeres), and a Y-specific centromeric repeat(Escudeiro et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Olagunju et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In parallel, we labelled telomeres by FISH to determine overall chromosome organisation in protamine-bound mature sperm nuclei (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Our analysis revealed that both centromeres and telomeres formed distinct clusters, with an average of 10\u0026ndash;11 centromere and 17\u0026ndash;19 telomere foci per nucleus (n\u0026thinsp;=\u0026thinsp;300 nuclei) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). These counts were significantly lower than the expected 30 centromeres and 60 telomeres in the haploid bovine genome, suggesting that multiple loci (~\u0026thinsp;3 on average) co-localise within each focus. To capture different spatial patterns within the nucleus, angular (directional) and radial (distance-based) distributions were assessed separately. The angular distribution (tested using Rayleigh\u0026rsquo;s test) evaluates whether foci are directionally biased or randomly oriented around the nuclear center. In contrast, the radial distribution reflects how concentrated foci are toward the inner, middle, or outer regions of the nucleus, regardless of direction. Rayleigh\u0026rsquo;s test for uniformity demonstrated that centromere positions were non-randomly distributed within the nucleus, while telomeres were randomly distributed (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), i.e. telomeres were dispersed without directional bias across the nucleus. The heatmap in (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003ec) shows that centromeres were more internally positioned, with telomeres commonly found throughout the entirety of the nucleus. To further investigate radial organisation, each nucleus was modelled as an ellipse and divided into three concentric rings: inner, middle, and outer. Centromeres were predominantly located in the middle ring (59.7%), with markedly fewer found in the inner (19.8%) and outer (20.5%) rings. In contrast, telomeres showed a broader distribution across all regions, with the highest proportion observed in the middle ring (49.2%), followed by the outer (30.6%) and inner (18.1%) rings. Percentages do not total exactly 100% due to the exclusion of a small fraction of foci localised outside the elliptical nuclear boundary. To illustrate consistency across animals, the percentage and standard deviation of telomere and centromere localisation within each ring (calculated across the three bulls) are reported (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Fisher\u0026rsquo;s exact tests showed that centromeres were significantly enriched in the middle ring compared to telomeres P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and telomeres were significantly enriched in the outer ring relative to centromeres (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). This organisation is illustrated in a representative model of the average mature sperm nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003ee), with centromeres clustering more internally and telomeres extending toward the nuclear periphery. In sperm nuclei, internally localised centromeres were frequently in contact with telomeres, most likely because cattle autosomes are acrocentric.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study we investigate the dynamics of the centromeric histone CENP-A and centromere localisation across different stages of spermatogenesis in the \u003cem\u003eBos taurus\u003c/em\u003e animal model. Overall, our findings reveal that haploid spermatids contain approximately one quarter the amount of CENP-A compared to differentiated spermatogonia (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). This result indicates that CENP-A is proportionally diluted and maintained during the two successive meiotic divisions. However, we also noted that the level of CENP-A in spermatids is approximately half that found in primary spermatocytes in late prophase I. This is not expected as after two cell divisions, this number should be one quarter. Indeed, we measured an increase in CENP-A in spermatocytes, between early and late prophase I that can explain an elevated CENP-A level in spermatids. Comparable dynamics have been observed in Drosophila spermatogenesis in which CENP-A is replenished during prophase I (Dunleavy et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Raychaudhuri et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). It is possible that CENP-A loading prior to chromosome segregation is required for kinetochore assembly such that homologous chromosome segregate in meiosis I. The measured increase in CENP-A coincides with the relocalisation of centromeres to the nuclear periphery in late prophase I, which is presumably linked to the processes of homolog pairing and synapsis and reflecting the bouquet configuration of bovine acrocentric chromosomes (Scherthan et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Pfeifer et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Scherthan \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Telomere tethering to the periphery prior to chromosome pairing explains this relocalisation (Fig. S2). However, whether CENP-A assembly occurs before, after or during the pairing of homologues chromosomes requires further investigation and more detailed identification of individual stages. Another possibility is that additional CENP-A assembly occurs in secondary spermatocytes that are not captured in our quantitation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also report that spermatogonia contain the highest amount of CENP-A of any cell type in the testis seminiferous tubule. Using ZBTB16 to specifically mark undifferentiated spermatogonia, we found that CENP-A is asymmetrically distributed between undifferentiated and differentiated spermatogonia. This finding is very much in line with observations in \u003cem\u003eDrosophila\u003c/em\u003e GSCs that carry the highest levels of CENP-A compared to other meiotic stages and show an asymmetric distribution of CENP-A between stem and daughter cells (Ranjan et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In bulls, undifferentiated spermatogonia have 1.5-fold more CENP-A than differentiating spermatogonia, which is remarkably comparable to the 1.4-1.6-fold enrichment measured in \u003cem\u003eDrosophila\u003c/em\u003e GSCs (Ranjan et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Dattoli et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In flies, a model of mitotic drive is proposed suggesting that the centromere of one sister chromatid harbours 1.5-fold more CENP-A and this biases chromosome segregation with strong centromeres more likely to end up in the stem cell. Our data indicates that mitotic drive might also operate in mammalian germline. A similar phenomenon was recently described during mouse spermatogenesis (Manske et al. bioRxiv) and in additional stem cell types (Behnan et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Evano et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Indeed, as CENP-A level can potentially mark cell fate, it has broad prognostic and diagnostic potential in the context of cancer and aging (Renaud-Pageot et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sikder et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Moreover, CENP-A could be exploited as a marker of undifferentiated spermatogonial stem cells in mammals.\u003c/p\u003e \u003cp\u003eOur study also confirmed the maintenance of CENP-A, and the absence of CENP-B, in mature bull sperm. This result aligns with initial findings from 40 years ago, reporting the presence of CENP-A on bull sperm using CREST serum (Štiavnick\u0026aacute; et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, in these experiments neither CENP-B nor CENP-C was detected in the bovine thymus, therefore it remained possible that human CREST serum did not detect bovine centromere proteins. Our results now confirm the maintenance of CENP-A on sperm using a monoclonal antibody and we also validated that the anti-human CENP-B antibody utilised can recognise bovine CENP-B. Unfortunately, we have not been able to clearly elucidate presence or absence of CENP-C, because of technical difficulties related to specificity of anti-CENP-C antibody and its ability to recognise the bovine antigen. In \u003cem\u003eDrosophila\u003c/em\u003e, the sole presence of CENP-A and lack of CENP-C (the only CCAN component in flies) on mature sperm centromeres was previously demonstrated (Raychaudhuri et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Dunleavy et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Given that the maintenance of paternal CENP-A at centromeres on mature sperm is critical for fly embryo development (Raychaudhuri et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), it is possible that a critical amount of paternal CENP-A is vital for the mitotic divisions after fertilization in mammals too (Manske et al. bioRxiv). In contrast to flies, in which CAL1 (the Drosophila HJURP equivalent) was not present on sperm, HJURP has been recently detected in human and mouse sperm (Manske et al. bioRxiv). This suggests a role for paternal HJURP in the deposition of new CENP-A after fertilization. While we could not detect HJURP in mature bull sperm, we noted a higher molecular weight band that might represent a modified version or isoform in cattle.\u003c/p\u003e \u003cp\u003eLastly, we examined the spatial organisation of centromeres and the CENP-A-associated satellite during spermatogenesis and in mature bull sperm (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Centromeres showed a distinct, cell-type-specific distribution within nuclei, initially localising at the nuclear periphery in primary spermatocytes and shifting toward the nuclear interior in spermatids. Our detailed analysis of centromere and telomere positioning in mature sperm showed that centromeres are non-randomly distributed, forming distinct clusters toward the nuclear interior, while telomeres are more randomly dispersed and significantly enriched at the periphery in comparison to centromeres (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). It is important to note that spatial organisation in mature sperm was assessed following DTT/Heparin-induced decondensation, which is required for the FISH protocol in line with several landmark studies (Zalensky et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1993\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Hazzouri et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Ioannou et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Chagin et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Although DTT treatment results in uniform swelling while preserving gross nuclear shape, we acknowledge it may subtly affect chromatin architecture. This caveat should be considered when interpreting fine-scale spatial relationships. Our combination of random angular dispersion and peripheral radial enrichment indicates that telomeres are broadly distributed in all directions but significantly localize nearer the nuclear edge. This spatial separation suggests that centromeres and telomeres are organised under different biological constraints during spermatogenesis. The degree of clustering for both centromeres and telomeres is comparable to a study in human sperm, with the number of centromere clusters being more consistent and telomere clusters more variable across cells (Ioannou et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). While telomeres occupied similar positions throughout the nucleus in both bulls and humans, centromeres were found in both intermediate and peripheral locations in humans, whereas in bulls, they were predominantly enriched in the intermediate zone (Ioannou et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). One explanation for this difference may relate to the acrocentric nature of bovine chromosomes: because autosomes in cattle are acrocentric, one telomere is inherently positioned near the centromere. Future work using genome-wide spatial sequencing would help clarify whether the p-arm telomere (proximal to the centromere) localises internally and the q-arm telomere more peripherally. Interestingly, the internal positioning of centromeres seems to be already established by the spermatid stage, with a similar number of centromere foci detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), pointing to a nuclear architecture that is maintained despite the histone-to-protamine exchange during sperm maturation. A likely function of clustering is CENP-A preservation, potentially safeguarding centromere identity during spermiogenesis and enabling rapid kinetochore assembly post-fertilization. The drivers of centromere clustering are not known, but heterochromatin interactions, structural constraints, or epigenetic factors may be involved. While certain chromosomes occupy conserved nuclear positions in bovine sperm (Chagin et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), whether specific centromeres co-cluster consistently remains to be determined.\u003c/p\u003e \u003cp\u003eIn summary, our findings in bulls validate previous observations in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e males and suggest a conservation of unusual CENP-A dynamics, mechanisms and functions in mammalian spermatogenesis. Future research should focus on investigating the functional significance of CENP-A's asymmetric distribution in stem cells and its potential as a marker of stem cell identity in spermatogenesis. Additionally, exploring the molecular mechanisms governing CENP-A deposition in meiosis I is likely to reveal unexpected results given that it differs from mitotic assembly in G1 phase. Determining CENP-C dynamics, as well as the functional significance of CENP-A and/or HJURP maintenance on sperm for fertility and chromosomal inheritance in the embryo is also critical. Given the similarity in sperm centromere organisation between bulls and humans, this system may offer valuable insights into centromere dynamics and CENP-A behaviour during human spermatogenesis. While access to early stages of spermatogenesis in humans is challenging, studying bulls may serve as a powerful model to infer conserved mechanisms of centromere regulation with potential implications for understanding male fertility. Our results are also important given ongoing efforts to generate telomere to telomere assemblies of centromeres in cattle and other ruminants (Kalbfleisch et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) that will allow studies of chromosome evolution and speciation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eWe would like to thank Dr. Mark Canney from Human Biology at the University of Galway for help with preparation of tissue sections, the National Cattle Breeding Centre in Ireland for donation of frozen-thawed samples of bull semen and Kepak Ltd. Athleague for the donation of testes tissues.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis project was supported by Research Ireland (grant number 20/FFP-A/8519).\u003c/p\u003e\n\u003ch2\u003eContribution\u003c/h2\u003e\n\u003cp\u003eE.M. Dunleavy, C.M. Collins and M. \u0026Scaron;tiavnick\u0026aacute; conceived and designed the study. M. \u0026Scaron;tiavnick\u0026aacute; and A. N\u0026iacute; Nuall\u0026aacute;in performed experiments, fluorescence microscopy imaging, quantitation and data analysis. M. \u0026Scaron;tiavnick\u0026aacute;, A. N\u0026iacute; Nuall\u0026aacute;in and E.M. Dunleavy wrote the manuscript.\u003c/p\u003e\n\u003ch2\u003eEthical approval\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAgostinelli C, Lund U (2024) circular: Circular Statistics\u003c/li\u003e\n\u003cli\u003eAkera T, Trimm E, Lampson MA (2019) Molecular Strategies of Meiotic Cheating by Selfish Centromeres. Cell 178:1132\u0026ndash;1144.e10. https://doi.org/10.1016/J.CELL.2019.07.001\u003c/li\u003e\n\u003cli\u003eAltemose N, Logsdon GA, Bzikadze A V., et al (2022) Complete genomic and epigenetic maps of human centromeres. 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Annu Rev Genet 57:1\u0026ndash;63. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/ANNUREV-GENET-061323-044915\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"chromosome-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"chrs","sideBox":"Learn more about [Chromosome Research](http://link.springer.com/journal/10577)","snPcode":"10577","submissionUrl":"https://submission.nature.com/new-submission/10577/3","title":"Chromosome Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"CENP-A, centromeres, bull sperm, spermatogonia stem cells, meiosis","lastPublishedDoi":"10.21203/rs.3.rs-6929623/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6929623/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDuring spermatogenesis, chromatin structure is remodelled by the incorporation of distinct histone variants and associated posttranslational modifications, followed by the almost complete replacement of histones by protamines in sperm. However, the dynamics of the centromere-specific histone H3 variant CENP-A have not yet been elucidated during spermatogenesis in mammals. Here we investigate CENP-A localisation dynamics in cattle (\u003cem\u003eBos taurus\u003c/em\u003e). In bovine testis tissue sections, we quantify CENP-A intensity in key germ cell types; spermatogonia (pre-meiotic), primary spermatocytes (meiotic) and spermatids (post-meiotic). Our quantitation shows that spermatogonia harbour the highest amount of CENP-A compared to all other germ cell types. Spermatids have approximately one quarter the amount of CENP-A of spermatogonia indicating that overall, it is reduced and maintained through the two meiotic divisions. Yet, we also observed some unexpected dynamics. CENP-A is asymmetrically distributed such that undifferentiated spermatogonia harbour more CENP-A that differentiated spermatogonia that enter meiosis. We also noted an increase in CENP-A intensity in primary spermatocytes during meiotic prophase I, which is indicative of centromere assembly at this time. We also confirm the specific maintenance of CENP-A, and the absence of the centromeric DNA binding protein CENP-B, on mature bull sperm nuclei that have completed histone-to-protamine exchange. Finally, we present a model for centromere positioning in mature sperm nuclei and propose that centralised clustering of centromeres may serve a protective function during histone-to-protamine exchange.\u003c/p\u003e","manuscriptTitle":"CENP-A is diluted during bovine spermatogenesis and is maintained at internally positioned centromere clusters in mature bull sperm","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-25 09:06:59","doi":"10.21203/rs.3.rs-6929623/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-27T12:04:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-26T15:51:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-08T11:17:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4235960835679582588652838169595583534","date":"2025-07-07T15:35:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"330364007320300435583214819441034150344","date":"2025-06-25T12:49:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-23T12:11:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-20T15:28:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-20T12:47:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Chromosome Research","date":"2025-06-19T09:13:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"chromosome-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"chrs","sideBox":"Learn more about [Chromosome Research](http://link.springer.com/journal/10577)","snPcode":"10577","submissionUrl":"https://submission.nature.com/new-submission/10577/3","title":"Chromosome Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"815148ed-80ae-4b03-8e2a-82a55dbe2f47","owner":[],"postedDate":"June 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-22T16:03:50+00:00","versionOfRecord":{"articleIdentity":"rs-6929623","link":"https://doi.org/10.1007/s10577-025-09781-3","journal":{"identity":"chromosome-research","isVorOnly":false,"title":"Chromosome Research"},"publishedOn":"2025-09-16 15:57:17","publishedOnDateReadable":"September 16th, 2025"},"versionCreatedAt":"2025-06-25 09:06:59","video":"","vorDoi":"10.1007/s10577-025-09781-3","vorDoiUrl":"https://doi.org/10.1007/s10577-025-09781-3","workflowStages":[]},"version":"v1","identity":"rs-6929623","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6929623","identity":"rs-6929623","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}