Chromosoma https://doi.org/10.1007/s00412-022-00777-0 ORIGINAL ARTICLE Time to match; when do homologous chromosomes become closer? M. Solé1 · J. Blanco1 · D. Gil2 · O. Valero3 · B. Cárdenas1 · G. Fonseka4 · E. Anton1 · Á. Pascual1 · R. Frodsham4 · F. Vidal1 · Z. Sarrate1 Received: 8 November 2021 / Revised: 12 May 2022 / Accepted: 14 July 2022 © The Author(s) 2022 Abstract In most eukaryotes, pairing of homologous chromosomes is an essential feature of meiosis that ensures homologous recom- bination and segregation. However, when the pairing process begins, it is still under investigation. Contrasting data exists in Mus musculus, since both leptotene DSB-dependent and preleptotene DSB-independent mechanisms have been described. To unravel this contention, we examined homologous pairing in pre-meiotic and meiotic Mus musculus cells using a three- dimensional fluorescence in situ hybridization-based protocol, which enables the analysis of the entire karyotype using DNA painting probes. Our data establishes in an unambiguously manner that 73.83% of homologous chromosomes are already paired at premeiotic stages (spermatogonia-early preleptotene spermatocytes). The percentage of paired homologous chromosomes increases to 84.60% at mid-preleptotene-zygotene stage, reaching 100% at pachytene stage. Importantly, our results demonstrate a high percentage of homologous pairing observed before the onset of meiosis; this pairing does not occur randomly, as the percentage was higher than that observed in somatic cells (19.47%) and between nonhomologous chromosomes (41.1%). Finally, we have also observed that premeiotic homologous pairing is asynchronous and independent of the chromosome size, GC content, or presence of NOR regions. Keywords Homologous chromosomes · Homologous pairing · Chromosome territories · FISH · Meiosis · Premeiotic cells Introduction of homologous centromeres towards opposite poles in the meiosis I spindle. In meiosis, germ cells are subjected to profound chromo- Bivalent formation begins at the early stages of meio- somal and morphological changes during the production of sis, where each chromosome approaches and aligns with its highly differentiated haploid cells. A key step in this process homolog in order to carry out pairing, synapsis, and recombi- is the segregation of homologous chromosomes at anaphase nation (for a review, see Zickler and Kleckner 2015). Although I. In most eukaryotes, homologous segregation depends on pairing and synapsis are somehow related processes, specific dif- the formation of bivalents in prophase I and the orientation ferences must be mentioned when referring to meiotic analysis; the approaching, juxtaposition, and overlapping of homologous chromosomes are commonly referred to as pairing, meanwhile, J. Blanco and Z. Sarrate contributed equally. the closer alignment and connection of homologous chromo- * Z. Sarrate somes by the synaptonemal complex is generally called synapsis zaida.sarrate@uab.cat (Zickler and Kleckner 2015). 1 The processes of synapsis and recombination have been Genetics of Male Fertility Group, Unitat de Biologia Cel·lular, Universitat Autònoma de Barcelona, extensively studied and described elsewhere (Zickler and Cerdanyola del Vallès, Spain Kleckner 1998, 1999; Champion and Hawley 2002; Page and 2 Computer Vision Center, Computer Science Department, Hawley 2003, 2004). In contrast, the mechanisms underly- Universitat Autònoma de Barcelona, Cerdanyola del Vallès, ing the regulation and timing of the pairing process remain Spain poorly understood, with studies demonstrating conflicting 3 Servei d’Estadística Aplicada, Universitat Autònoma de results and leaving many open questions. Barcelona, Cerdanyola del Vallès, Spain Two pieces of data suggest that pairing begins during 4 Cytocell Ltd, Cambridge Science Park, Milton Road, the leptotene stage of prophase I. First, there is a certain Cambridge CB4 0PZ, UK 13 Vol.:(0123456789) Chromosoma consensus that in many organisms, repair of the double- according to species, and it is important to highlight that strand breaks (DSBs) produced by the topoisomerase-like both models have been reported to occur in mice. This protein SPO11 during leptotene is the inducing factor for contrasting situation brought us to revaluate the timing homologous pairing in meiosis (reviewed by Baudat et al. of homologous meiotic pairing in mice germ cells via an 2013). Using various models, researchers have described experimental design based on the use of painting probes associations between the levels of or the correct develop- and a three-dimensional (3D) FISH strategy to analyze each ment of DSBs, and the extent of homologous coalignment, chromosome. successful pairing, and/or synapsis (Thorne and Byers 1993; Romanienko and Camerini-Otero 2000; Davis et al. 2001; Grelon et al. 2001; Peoples et al. 2002; Tessé et al. 2003; Methods Henderson and Keeney 2004; Kauppi et al. 2013; Rockmill et al. 2013). Secondly, during leptotene, the protein SUN1 Germ cell analysis tethers the chromosome ends to the inner nuclear enve- lope, where they cluster to form a structure that resembles The methodology used to process germ cells has been previ- a bouquet (Ding et al. 2007; Hiraoka and Dernburg 2009). ously described by our research group (Solé et al. 2021) and Bouquet formation have been described in many species it consists of several steps: (Zickler and Kleckner 1998, 2015; Scherthan 2001; Harper et al. 2004), suggesting that they ease homology searching • Cell obtainment by bringing the ends of chromosomes closer and aligning them. In fact, lack of SUN1 causes asynapsis and game- Testicular tissue was obtained from four 12-week-old togenesis disruption in mice (Ding et al. 2007; Chi et al. C57BL/6 J mice. The tissue was mechanically and enzymati- 2009). Accordingly, it has been postulated that, in the nuclei cally disaggregated following the procedure described by of premeiotic cells (before DSB and bouquet structure for- Garcia-Quevedo et al. (2012). Testicular cells were adhered mation), homologous chromosomes are spatially separated to customized polylysine-coated slides (1 mg/ml), fixed with from each other, as they are in somatic cells. This interpre- 4% paraformaldehyde, and subjected to a permeation treat- tation is experimentally supported by studies analyzing the ment with 0.1 N hydrochloric acid, 0.5% triton, liquid nitro- chromosome distribution of homologs in premeiotic cells gen, and 0.005% pepsin (protocol adapted from Cremer et al. using fluorescence in situ hybridisation (FISH)-based strate- 2008). Prior to application of the FISH procedure, slides gies (Scherthan et al. 1996; Scherthan and Schönborn 2001). were incubated in 50% formamide for a minimal period of Data from other studies suggests the existence of a mech- 2 months, with the purpose of achieving a prehybridization anism of homolog pairing that takes place before DSB for- slow DNA denaturation. mation. The DSB-independent pairing mechanism has been observed in species with specific chromosomal characteris- • Fluorescence in situ hybridization tics, such as dipterans, which exhibit somatic chromosome pairing (Wandall and Svendsen 1985), and wheat, which Three successive rounds of FISH were performed using a has a hexaploid karyotype that requires specific genes to custom designed Chromoprobe Multiprobe® OctoChrome ensure meiotic pairing (Martinez-Perez et al. 2001; Mar- Murine System ™ kit (Cytocell Ltd, Cambridge, UK). This tínez-Pérez et al. 1999; Prieto et al. 2004). However, this kit contains a multiprobe device that consists of three dif- mechanism has also been observed in species without any ferent coverslips with seven delimited independent regions. distinctive chromosomal features, such as worms (Dernburg Each of these regions presents a specific combination of et al. 1998; Phillips et al. 2009), flies (McKim et al. 1998), three painting labeled with a different fluorochrome (Aqua budding yeast (Burgess et al. 1999; Cha et al. 2000), fission DEAC, FITC, and Texas Red). Therefore, after sequential yeast (Scherthan et al. 1994; Nabeshima et al. 2001), and application of three coverslips, it is possible to identify up to mice (Boateng et al. 2013). Although no clear molecular nine chromosomes per region in the same nuclei. Analysis explanation has been established for this DSB-independent of all the coverslips and regions provides identification of all homolog recognition, research has suggested that hetero- chromosomes of the mouse karyotype as well as all possible chromatin aggregation, the SPO11 and SUN1 proteins, as combinations of chromosome pairs. well as some noncoding RNA and transcription factors, may In each round of FISH, the slide and the correspond- be involved in the process (Page and Hawley 2004; Barzel ing coverslip were mounted together in formamide solu- and Kupiec 2008; Dombecki et al. 2011; Ding et al. 2012; tion and then subjected to denaturation for 5 min at 75 °C Boateng et al. 2013). in a Hybridization Vysis HYBrite System. Hybridization Therefore, we cannot discard the coexistence of both was performed for 60 h at 37 °C (procedure adapted from DSB-dependent and -independent models of pairing Cytocell manufacturer instructions). After hybridization, 13 Chromosoma the coverslip was removed and slides were transferred • Identification of cell type to 1 × saline-sodium citrate (SSC) buffer, incubated for 2 min, and washed in 2 × SSC/0.05% Tween-20 at RT for An immunofluorescence procedure was designed to une- 30 s. Finally, hybridized areas were mounted in the anti- quivocally identify premeiotic cells in relation to somatic fade provided by the kit. After each FISH round, a washing cells and meiotic cells. With this purpose, three proteins step was performed with 0.0625 × SSC at 73 °C for 5 min were identified: synaptonemal complex protein 3 (SYCP3), in order to remove previous hybridization signals. which allowed identification of germ cells from mid-prel- eptotene to pachytene stages; testicle-specific histone H1 • Image capture (H1T), which enabled identification of germ cells ranging from the late pachytene stage to round spermatids; and a A Leica TCS-SP5 confocal microscope coupled to an germ cell-specific nuclear antigen recognized by the mono- image analysis system (LAS AF v.1.8.1) was used to cap- clonal antibody TRA98 (Tanaka et al. 1997), which allowed ture serial optical sections of nuclei after each hybridiza- discrimination between somatic cells and germ cells. Vari- tion round (Fig. 1). A hybrid detector (HyD) and HCX ous cell fractions were then identified based on the pres- PL APO lambda blue 63.0 × 1.40 OIL UV objectives ence or absence of these proteins as previously shown by were used. Specifically, lasers and excitation frequencies our research group (Solé et al. 2021): applied were a 405 nm UV diode laser, a 488 nm argon laser, and a 561 nm DPSS laser for the Aqua DEAC, FITC, I. Premeiotic cells. This category includes cells from and Texas Red fluorochromes, respectively. The HyD A-type spermatogonia to early preleptotene spermato- detector was configured at 415–470 nm for Aqua DEAC, cytes (the onset of the premeiotic S-phase in which DNA at 500–550 nm for FITC, and at 571–750 nm for Texas replication is performed). These nuclei stained positively Red. A high-speed resonant scanner module was used to for TRA98, with negative staining for SYCP3 and histone obtain serial optical sections on the X, Y, and Z axes, with H1T (Fig. 2). a distance between sections of 0.17 μm, a 512 × 256 pixel II. Mid preleptotene-zygotene spermatocytes. These format, and an optical zoom of 5X. The number of sections nuclei showed positive staining for TRA98 and for was different for each nucleus (i.e., according to the cor- SYCP3 protein and negative staining for histone H1T responding nuclear volume). All captures were associated (Fig. 2). Despite using the same fluorophores to detect with their coordinates in order to relocate and capture the SYCP3 and TRA98, both proteins were easily distin- same nuclei after each hybridization round. guishable because TRA98 showed a uniform labeling pattern that was distinct from SYCP3’s dotted or thread- • Image analysis like appearance (Fig. 2). III. Pachytene spermatocytes. Pachytene spermatocyte Image analysis was performed using customized devel- nuclei stained positive for TRA98, demonstrated a thread- oped scripts designed within the Fiji software environ- like staining pattern for SYCP3, and were either negative ment (Schindelin et al. 2012) and Matlab R2013b. The (early pachytene) or positive (late pachytene) for histone Fiji scripts permitted segmenting nuclei and chromosome H1T (Fig. 2). territories in different serial binary images as shown in IV. Round spermatids. Spermatid nuclei stained positive the animation Online resource 1. Following this, Matlab for TRA98 and histone H1T and were negative for SYCP3 scripts permitted 3D reconstruction of the nuclei and chro- (Fig. 2). mosome territories, enabling the extraction of numerical data concerning chromosome position and volume, as well The protein labeling process was carried out after chro- as nuclei volume. mosomal FISH analysis in two sequential rounds. In the first We considered that two homologous chromosomes round, detection of SYCP3 and histone H1T was performed were paired when all pixels of a specific signal formed a while, in the second round, samples were immunostained unique and continuous group of voxels (Online resource 2). with the monoclonal antibody against TRA98. Briefly, cells Conversely, homologous chromosomes were classified as were fixed in 3% paraformaldehyde for 10 min and permea- unpaired when two groups of voxels were observed as two bilized in a 1 × PBS-0.5% Triton-X100 solution for 5 min. separate entities, so sharing no voxel (Online resource 2). Next, samples were incubated for 15 min with blocking solu- Similarly, we considered that two nonhomologous chromo- tion (1% w/v bovine serum albumin). After blocking, the somes (also referred to as heterologous chromosomes) were cells were incubated at 4 °C with the primary antibodies, associated when their territories overlapped. The percent- specifically rabbit anti-SCP3 (Abcam, Cambridge, UK) plus age of overlapping was determined as the number of shared either guinea pig anti-H1T (The Jackson Laboratory, USA) voxels respect the voxels each chromosome territory occupy. (first round) or rat anti-TRA98 (Abcam, Cambridge, UK) 13 Chromosoma A A Spermatogonia– early preleptotene Chr. 3 Mid preleptotene– zygotene Chr. 1 (Premeiotic cells) FISH rounds FISH rounds A B C Chr. 7 A B C Chr. 4 Chr. 16 Chr. 14 B C B C Chr. 2 Chr. 10 Chr. X Chr. 3 Chr. 5 Chr. 9 Chr. 13 Chr. 7 Chr. 15 Chr. 18 Chr. Y Chr. 16 A A Pachytene Chr. 12 Round Spermatids Chr. 2 FISH rounds FISH rounds A B C A B C Chr. 5 Chr. 11 Chr. 19 Chr. 15 B C B C Chr. 10 Chr. 1 Chr. 1 Chr. 6 Chr. 9 Chr. 4 Chr. 4 Chr. 8 Chr. 18 Chr. 14 Chr. 14 Chr. 17 Fig. 1  3D-FISH confocal image captures of each round of hybridiza- intensity projections of confocal serial sections and 3D composite tion (a, b, and c) for four different mice germ cell nuclei (spermato- reconstructions using Imaris 9.3 software in both RGB merge and gonia-early preleptotene, mid-preleptotene-zygotene, pachytene, and split channels (to view the planes’ sequence of the maximum inten- round spermatids). Different combinations of three chromosomes sity confocal images and the 3D composite reconstruction of premei- (Chr.) displayed in FITC, Texas Red, or Aqua DEAC are observed otic cells, see Online resource 1) in each nucleus. For each hybridization round are shown maximum 13 Chromosoma Premeiosis Meiosis I Meiosis II Spermiogenesis Type A Type B Preleptotene Leptotene Zygotene Pachytene Early Mid Late Early Mid Late Early Mid Late S-phase DNA DSBs Bouquet (Zickler and Kleckner 1998; 2015; Ding (DNA replication) introduced by SPO11 et al. 2007; Hiraoka and Dernburg, 2009) (de Rooij, 1988; (Page and Hawley 2004) Sherthan et al 1996) SYCP3 H1T TRA98 Spermatogonia-early preleptotene Mid preleptotene-Zigotene Pachytene Round (premeiotic cells) spermatids Fig. 2  Graphical representation of the labeling pattern of synaptone- occur in primary spermatocytes. The colored bars indicate the pres- mal complex protein 3 (SYCP3), testicle-specific histone 1 (H1T), ence of each protein: SYCP3 (dark green bar), H1T (red bar), and and a testis-specific nuclear protein known as TRA98 during the sper- TRA98 (light green bar), throughout spermatogenesis (the top grey matogenesis process, along with some relevant meiotic events that bar) (second round). Next, samples were incubated with second- Table 1  Chromosome features used to test for possible condition- ary antibodies for 40 min at 37 °C. Specifically, the second- ing factors of homologous pairing. Data extracted from The Genome Reference Consortium, Mus musculus GRCm38.p6. aData extracted ary antibodies were goat anti-rabbit FITC (Jackson Immu- from The Genome Reference Consortium, Mus musculus GRCm38. noResearch Inc., Cambridge, UK) and goat anti-guinea pig p6. bData extracted from (Evans et al. 1974; Henderson et al. 1974; CY3 (Jackson ImmunoResearch Inc., Cambridge, UK) (first Atwood et al. 1976; Dev et al. 1977; Kurihara et al. 1994; Britton- round) or goat anti-rat FITC (Abcam, Cambridge, UK) (sec- Davidian et al. 2011). Crosses (x) indicate the presence of nucleolus organizer regions (NORs) ond round). Chr Size (Mb)a % ­GCa Gene ­densitya NORb • Data processing and statistical analysis 1 195.47 41.3 2,687 2 182.11 42.2 3,491 Results were analyzed and processed in the following 3 160.04 40.7 2,225 manner: data regarding chromosome position and volume, 4 156.51 42.5 2,622 which were extracted from the application of Matlab scripts, 5 151.84 42.7 2,507 permitted to calculate (1) the average percentage of paired 6 149.74 41.6 2,597 and unpaired homologous chromosomes; (2) the average 7 145.44 43.2 3,798 percentage of nonhomologous chromosomes sharing the 8 129.40 42.6 2,177 same chromosomal territory; (3) the average of nonho- 9 124.60 42.9 2,276 mologous overlapping percentage; and (4) the nuclear vol- 10 130.70 41.6 2,086 ume proportion occupied for each chromosomal territory 11 122.08 44.0 2,852 x analyzed. 12 120.13 42.0 2,002 x Pearson’s correlations were performed to evaluate the 13 120.42 41.9 2,127 degree of linear relationship between chromosome size, GC 14 124.90 41.4 2,111 content, and chromosome gene density (these parameters 15 104.04 42.2 1,620 x are detailed on Table 1) and the average rate of homolo- 16 98.21 41.2 1,367 x gous chromosome pairing. Besides, the percentage of paired 17 94.99 42.9 2,005 homologous chromosomes was compared between NOR- 18 90.70 41.7 1,218 x bearing-chromosomes and no NOR-bearing-chromosomes 19 61.43 43.1 1,283 x by a T-test. These analyses were only performed at the X 171,03 39.2 2,291 first two stages studied (i.e., spermatogonia-early prelep- Y 91,74 36.7 423 totene spermatocytes and mid preleptotene-zygotene sper- matocytes), as there were no unpaired chromosomes in the remaining stages. with an overlap of 0%, Wilsons’ score was used. Chromo- Concerning heterologous associations, the Wald's asymp- somes with a confidence interval above or below the overall totic method was used to calculate 95% confidence inter- weighted mean of associations were considered statistically vals for the overlap of each pair of chromosomes. For those significant. 13 Chromosoma Analyses were performed by SAS v9.4 software, SAS 4, 6, 7, 8, 9, 10, 11, 12, 14, 16, 17, 18, and 19. The analysis Institute Inc., Cary, NC, EEU and the significance level was was performed using an Olympus BX60 epifluorescence set to 0.05. microscope equipped with filter sets for FITC, Texas Red, Aqua DEAC, and DAPI/Texas Red/FITC. The same criteria Lymphocyte analysis applied in the germ cell analysis were then used in order to classify the homologous chromosomes as paired or unpaired. As somatic pairing does not occur in mice cells, we addition- ally analyzed the distribution of homologous chromosomes in lymphocytes, in order to obtain a basal level at which two Results homologous chromosomes are observed nearby due to non- pairing related causes. Accordingly, spleens were removed Spermatogenetic cells from two C57BL/6 J mice. After injecting 5 ml of RPMI into the spleens using a syringe and recovering the solution, lym- Application of the developed methodology allowed us to phocytes were isolated by Ficoll-Paque gradient separation, analyze the chromosome territories in a total of 147 spermat- fixed with 4% paraformaldehyde, and subjected to permea- ogonia-early preleptotene spermatocytes (premeiotic cells), tion treatment with 0.1 N hydrochloric acid, 0.5% triton, liq- 128 mid preleptotene-zygotene spermatocytes, 154 pachy- uid nitrogen, and 0.005% pepsin (protocol adapted from Cre- tene spermatocytes, and 321 round spermatids (Table 2). mer et al. 2008). Next, FISH was again performed using the Considering all four cell fractions together, we analyzed a custom designed Chromoprobe Multiprobe® OctoChrome total of 5631 chromosome territories. Murine System ™ kit (Cytocell Ltd, Cambridge, UK). In For each chromosome, the percentages of homologous this case, only one coverslip was used; specifically, this cov- chromosomes sharing the same territory (paired) or occupy- erslip permitted labeling the following chromosomes: 1, 3, ing different territories (unpaired) were represented for each Table 2  FISH results for each chromosome in the four analyzed meiotic intervals Spermatogonia-early preleptotene Mid preleptotene-zygotene (128) Pachytene (154) Round spermatids (321) (147) 2 signals 1 signal Total 2 signals 1 signal Total 2 signals 1 signal Total 2 signals 1 signal Total Chr n % n % n n % n % n n % n % n n % n % n 1 19 30.16% 44 69.84% 63 14 24.56% 43 75.44% 57 0 0 52 100% 52 0 0 113 100% 113 2 13 23.64% 42 76.36% 55 5 12.82% 34 87.18% 39 0 0 52 100% 52 0 0 108 100% 108 3 14 22.58% 48 77.42% 62 11 22.00% 39 78.00% 50 0 0 59 100% 59 0 0 100 100% 100 4 19 29.69% 45 70.31% 64 12 20.34% 47 79.66% 59 0 0 55 100% 55 0 0 121 100% 121 5 20 35.71% 36 64.29% 56 4 9.76% 37 90.24% 41 0 0 51 100% 51 0 0 109 100% 109 6 14 28.57% 35 71.43% 49 7 14.58% 41 85.42% 48 0 0 50 100% 50 0 0 130 100% 130 7 20 30.77% 45 69.23% 65 11 22.45% 38 77.55% 49 0 0 60 100% 60 0 0 100 100% 100 8 12 25.53% 35 74.47% 47 8 18.18% 36 81.82% 44 0 0 48 100% 48 0 0 126 100% 126 9 14 31.11% 31 68.89% 45 8 16.33% 41 83.67% 49 0 0 58 100% 58 0 0 123 100% 123 10 14 28.00% 36 72.00% 50 11 20.75% 42 79.25% 53 0 0 62 100% 62 0 0 123 100% 123 11 5 14.29% 30 85.71% 35 4 7.84% 47 92.16% 51 0 0 57 100% 57 0 0 113 100% 113 12 11 26.83% 30 73.17% 41 4 7.84% 47 92.16% 51 0 0 59 100% 59 0 0 118 100% 118 13 9 21.95% 32 78.05% 41 12 23.53% 39 76.47% 51 0 0 43 100% 43 0 0 104 100% 104 14 16 25.40% 47 74.60% 63 7 11.86% 52 88.14% 59 0 0 54 100% 54 0 0 122 100% 122 15 12 28.57% 30 71.43% 42 4 12.12% 29 87.88% 33 0 0 40 100% 40 0 0 77 100% 77 16 11 20.75% 42 79.25% 53 10 21.28% 37 78.72% 47 0 0 59 100% 59 0 0 85 100% 85 17 9 25.71% 26 74.29% 35 4 11.11% 32 88.89% 36 0 0 40 100% 40 0 0 103 100% 103 18 11 37.93% 18 62.07% 29 10 20.00% 40 80.00% 50 0 0 56 100% 56 0 0 97 100% 97 19 2 7.69% 24 92.31% 26 1 2.22% 44 97.78% 45 0 0 49 100% 49 0 0 76 100% 76 X 10 28.57% 25 71.43% 35 4 8.33% 44 91.67% 48 0 0 42 100% 42 0 0 52 100% 52 Y 10 25 35 4 44 48 0 0 42 42 0 0 52 52 Mean 26.17% 73.83% 15.40% 84.60% 0% 100% 0% 100% Std Dev 6.78 6.78 6.40 6.40 0 0 0 0 13 Chromosoma cell stage analyzed in Fig. 3 (i.e., spermatogonia-early prel- spermatogonia-early preleptotene spermatocyte stage eptotene spermatocytes, mid preleptotene-zygotene spermat- (p = 0.135) or at the mid preleptotene-zygotene spermato- ocytes, pachytene spermatocytes, and round spermatids). At cyte stage (p = 0.110). the spermatogonium-preleptotene stage, a mean of 73.83% The mean frequency of nonhomologous association was of homologous chromosomes was observed as a joint entity, 41.10% in spermatogonia-early preleptotene spermatocyte this percentage increased up to 84.60% at the mid prelep- stage. Data representing the frequencies at which nonhomol- totene-zygotene stage and reached 100% at the pachytene ogous chromosomes shared the same territory at this stage stage. As expected, round spermatids exhibited one signal are detailed in Table 4. Ten non-homologous chromosome per chromosome in 100% of the analyzed cells. Data showed pairs out of 190 showed statistically significant increases differences in the pairing levels between chromosomes. For with respect to the mean, while significant decreases were instance, chromosome 1 displayed one of the lowest pairing observed in 13 of them. Besides, data representing the rates in the first two stages studied, specifically 69.84% and degree of overlap between nonhomologous chromosomes 75.44% of chromosomes were observed as paired homolo- are detailed in Suppl. Table 1. On average, chromosomes gous chromosomes in the spermatogonia-early preleptotene overlap 2.60% of its volume with other chromosomes. In spermatocyte and mid preleptotene-zygotene spermatocyte the case of the ten nonhomologous chromosome pairs with stages, respectively. In contrast, chromosome 19 showed one significant increases associations, this percentage rises up of the highest pairing rates in these two stages; 92.31% and to 4.1%. This percentage decreases to 0.95% in the case of 97.78% respectively. nonhomologous chromosome pairs that interact less than On average, the nuclear volume occupied by chromo- the mean. somal territories classified as paired was higher than the nuclear volume occupied by unpaired signals, both in the Somatic cells spermatogonia-early preleptotene spermatocyte (1.79% for paired vs 1.11% for unpaired) and mid preleptotene-zygo- One hundred forty-seven nuclei of mouse lymphocyte cells tene spermatocyte (2.37% vs 1.30%) stages (Table 3). were analyzed, totalling 946 chromosome territories. A To investigate possible explanations for the differences mean value of 19.47% of homologous chromosomes was observed between chromosomes, Pearson’s correlation coef- observed as a joined entity (Fig. 3). Detailed data for each ficient testing and/or T-tests were performed between the chromosome are graphically represented in Fig. 4. pairing rates and various intrinsic chromosomal parameters (Table 1). Results revealed that none of the chromosomal parameters analyzed was associated with the timing of Discussion homologous pairing, neither at the spermatogonia-early prel- eptotene spermatocyte stage (size: r =  − 0.36, p = 0.117; % Two opposing hypotheses explain when homologous paring GC: r = 0.21, p = 0.365; gene density: r =  − 0.12, p = 0.616) begins in meiosis: the first proposes that homologous chro- nor at the mid preleptotene-zygotene spermatocyte stage mosomes approach one another during the leptotene stage (size: r =  − 0.41, p = 0.070; % GC: r = 0.12, p = 0.616; gene as a consequence of DSB formation and the establishment density: r =  − 0.24, p = 0.318). T-test analysis revealed of the bouquet structure. Conversely, the second theory pro- no significant differences between NOR-bearing-chro- poses that homologous chromosomes initiate pairing in the mosomes and the remaining chromosomes either at the early stages of meiosis, before DSB formation. Fig. 3  Percentage of homolo- Homologous chromosome pairing gous chromosome pairing 100% observed in different stages of 100% spermatogenesis: (I) spermato- 84.60% gonia-early preleptotene sper- 80% 73.83% matocytes I, (II) spermatocytes I at mid preleptotene-zygotene 60% stages, (III) spermatocytes I at pachytene stage, and (IV) 40% round spermatids, as well as in lymphocytes (Lym.). In round 20% 19.47% spermatids (IV), the paired value corresponds to cells with one signal per chromosome 0% Av XY e 10 12 13 14 15 16 17 18 19 1 2 3 4 5 6 7 8 9 11 ag er Lym. I II III IV 13 Chromosoma Table 3  Nuclear volume Spermatogonia-early preleptotene Mid preleptotene-zygotene proportion (NVP) occupied by one signal composed by One signal composed by: One signal composed by: one chromosome (unpaired chromosome) or one signal One chr Two chr One chr Two chr composed by two chromosomes Cr N NVP (%) N NVP (%) N NVP (%) N NVP (%) (paired chromosomes) in spermatogonia-early 1 38 1.98 44 1.85 28 1.94 43 2.44 preleptotene and in mid 2 26 1.09 42 2.14 10 1.24 34 3.59 preleptotene-zygotene germ cells. (Chr.) chromosomes, (N) 3 28 1.21 48 2.07 22 1.22 39 3.66 number of observations 4 38 1.67 45 1.96 24 1.22 47 3.00 5 40 0.78 36 2.23 8 0.94 37 2.47 6 28 1.11 35 1.84 14 1.76 41 2.76 7 40 0.91 45 2.04 22 1.76 38 2.74 8 24 0.97 35 1.87 16 0.78 36 1.97 9 28 0.53 31 1.21 16 0.86 41 1.91 10 28 1.21 36 1.82 22 1.70 42 2.91 11 10 1.08 30 1.62 8 1.22 47 1.87 12 22 1.23 30 2.63 8 1.58 47 3.02 13 18 1.68 32 2.14 24 0.65 39 2.44 14 32 1.28 47 1.83 14 1.08 52 2.29 15 24 0.64 30 1.18 8 0.99 29 1.36 16 22 1.16 42 1.48 20 1.59 37 2.07 17 18 0.56 26 1.31 8 1.00 32 1.53 18 22 0.78 18 1.21 20 1.18 40 1.68 19 4 0.38 24 0.84 2 1.46 44 1.38 X 10 1.62 25 1.72 4 1.05 44 2.84 Y 10 1.40 25 2.60 4 2.02 44 1.86 Average 1.11 1.79 1.30 2.37 Our results indicate that, in the murine model, there is a chromosome regions, but persist at telomeres, implying high percentage of homologous chromosomes already shar- that telomeric interactions are stabilized and essential for ing the same territory during the spermatogonium-prelep- future synapsis and recombination events. Unfortunately, our totene stage, prior to DSBs and bouquet formation Fig. 5. design, which relied on the employment of painting probes, Therefore, our data suggest that pairing, defined as the prevented us from confirming these findings. approaching, juxtaposition, and overlapping of homologous Our experimental design included various analyses whose chromosomes, is independent of both these processes. Our results supported our main conclusion. First, we analyzed results agree with a previous study in which homologous the homologous chromosome position in lymphocytes. paring for chromosome 3 in preleptotene spermatocytes Our results indicated that most homologous chromosomes were observed at a higher rate than heterologous interactions occupy two different chromosomal territories in somatic between chromosomes 3 and 7 (35% v.s 8% respectively; cells, in agreement with several previous studies (Lorenz Boateng et al. 2013). Although the percentage of pairing et al. 2003; Heride et al. 2010; Selvaraj et al. 2013; Rao in Boateng’s study is lower than the described by us for the et al. 2014; Joyce et al. 2016). Second, in this study, we same chromosomes (77.42% for homologous 3 chromosome determined that two homologous chromosomes were paired pairing and 36% for heterologous interactions between 3 and when they were overlapped and thus formed a unique entity. 7) (Tables 2 and 4), we have to keep in mind that a different However, it is important to mention that the experimental set of probes were used in both studies. The employment design did not allow us to distinguish between those asso- of a painting method that allows pairing-analysis along the ciations that resulted from pairing and those that resulted entire chromosome, rather than a method based in the use from the chromosomal topography of the nucleus. To clarify of a single interstitial probe (Boateng et al. 2013), predicts this point, we analyzed the frequency of nonhomologous an increase in the frequency of chromosomal-chromosome association (interaction between non-homologous chro- interactions. Boateng’s findings also revealed that premeiotic mosomes). On average, none of the chromosomes reaches homologous contacts are lost at leptotene in the interstitial the association rate of the homologous one. Specifically, 13 Chromosoma Table 4  Data representing the frequencies and number of observa- whereas significant decreases are only bolded. The average pairing tions (n) of nonhomologous associations between all pairs of chromo- for each chromosome with all other nonhomologous chromosomes somes at the spermatogonium-early preleptotene spermatocyte stage. is indicated in the last column of the table. The number of observa- At the top of the table (above the purple diagonal), the bluish tones tions made for each pair of chromosomes is indicated below the pur- indicate the frequency of overlap between all pairs of chromosomes, ple diagonal. Data in the mid-purple diagonal indicates the frequency the higher the frequency of pairing, the more intense the blue colora- of homologous pairing (the higher the frequency the more intense the tion. Significant increases of associations are bolded and asterisked, purple coloration) Spermatogonia - early preleptotene spermatocytes Chr. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 XY Average 1 0.70 0.42 0.24 0.56* 0.58 0.42 0.41 0.58 0.43 0.25 0.67 0.14 0.59 0.49 0.37 0.36 0.25 0.80* 0.00 0.28 0.41 2 26 0.76 0.40 0.39 0.39 0.42 0.47 0.39 0.23 0.38 0.40 0.14 0.38 0.44 0.41 0.41 0.44 0.36 0.50 0.38 0.39 3 17 15 0.77 0.28 0.40 0.57 0.36 0.54 0.29 0.39 0.19 0.65* 0.31 0.44 0.25 0.50 0.67 0.38 0.67 0.47 0.42 4 54 28 18 0.70 0.43 0.32 0.50 0.50 0.57 0.29 0.67 0.33 0.47 0.47 0.33 0.27 0.35 0.33 0.25 0.67* 0.42 5 26 54 15 28 0.64 0.42 0.44 0.35 0.21 0.18 0.20 0.29 0.13 0.52 0.56 0.18 0.39 0.09 0.33 0.50 0.35 6 24 24 14 25 24 0.71 0.21 0.43 0.38 0.25 0.36 0.29 0.75 0.28 0.28 0.55 0.23 0.43 0.30 0.50 0.39 7 17 17 59 18 18 14 0.69 0.69* 0.29 0.29 0.63 0.53 0.31 0.50 0.36 0.35 0.67 0.20 0.42 0.59 0.43 8 24 23 13 24 23 47 13 0.74 0.00 0.25 0.62 0.39 0.25 0.42 0.24 0.30 0.31 0.43 0.44 0.25 0.39 9 7 13 14 7 14 8 17 8 0.69 0.33 0.70* 0.27 0.43 0.14 0.33 0.31 0.33 0.48 0.40 0.25 0.34 10 8 16 18 7 17 8 21 8 45 0.72 0.40 0.42 0.71 0.29 0.20 0.26 0.50 0.59 0.60 0.13 0.35 11 6 5 16 6 5 14 16 13 10 10 0.86 0.54 0.57 0.33 0.50 0.46 0.33 0.00 0.40 0.57 0.45 12 7 7 17 6 7 14 17 13 11 12 35 0.73 0.20 0.17 0.80* 0.39 0.33 0.33 0.69* 0.20 0.37 13 17 8 16 17 8 4 16 4 7 7 7 10 0.78 0.53 0.40 0.46 0.25 0.40 0.50 0.61* 0.43 14 53 27 18 57 27 25 18 24 7 7 6 6 17 0.75 0.48 0.53 0.55 0.33 0.25 0.44 0.40 15 19 42 12 21 41 18 14 17 12 15 4 5 5 21 0.71 0.21 0.54 0.36 0.60 0.80* 0.42 16 14 17 50 15 17 11 52 10 16 19 13 13 13 15 14 0.79 0.44 0.30 0.46 0.36 0.37 17 20 18 9 20 18 35 9 35 6 6 9 9 4 20 13 9 0.74 0.20 0.50 0.75 0.42 18 5 11 8 6 11 7 10 7 29 29 6 6 5 6 11 10 5 0.62 0.40 0.60 0.37 19 3 6 12 4 6 10 12 9 5 5 25 26 8 4 5 11 8 5 0.92 0.38 0.43 XY 18 8 17 18 8 4 17 4 8 8 7 10 36 18 5 14 4 5 8 0.71 0.46 0.41 Fig. 4  Percentage of homolo- Somatic cells gous chromosomes occupying the same territory (one signal) 100% or occupying different territo- 80.53% ries (two signals) for each chro- 80% mosome in mice lymphocytes 60% 40% 19.47% 20% 0% ge 10 12 13 14 16 17 18 19 1 3 4 6 7 8 9 11 e ra Av 2 signals 1 signal nonhomologous chromosomes showed an average asso- proportion of nuclear volume occupied by the paired (seem- ciation of about 41.1%, practically half the value observed ingly formed by two chromosomes) and the unpaired signals in early meiotic stages Fig. 5. Third, we calculated the (each signal corresponding to one single chromosome) in 13 Chromosoma Fig. 5  Schematic representation of the dynamics of homologous arrowhead and the heterologous association by a blue arrowhead. The chromosome pairing and heterologous association from spermato- nuclear background color indicates the presence of TRA98 (green) gonia to pachytene spermatocytes of Mus musculus. There are rep- and H1T (red). The SYCP3 protein is represented in green dotted or resented three different homologous chromosomes in yellow, gar- continuous filaments net, and blue colors. The homologous pairing is indicated by a red premeiotic cells. Paired signals were, as a mean, 62% greater (data not shown). Another mechanism could be related to than those corresponding to unpaired signals, supporting the occurrence of interchromosomal contacts related to gene the interpretation that paired signals corresponded to two expression (Maass et al. 2019). Regarding this possibility, it homologous chromosomes. The fact that the ratio observed has been described in the murine meiosis, a preferred func- is not 2:1 can be interpreted into two different ways; chro- tional association between some heterologous chromosomes mosomes had a high percentage of overlap or paired chro- using conformation capture sequencing (Hi-C); studies that mosomes were more condensed. include the analysis of spermatogonia (Vara et al., 2019). Overall, these pieces of evidence suggest homologous The molecular mechanism that drives homologous chro- chromosomes begin pairing before the onset of meiosis mosomes to approach each other prior to DSB formation Fig. 5. The fact that there is a pairing of homologous chro- is still unknown. However, several pieces of data have mosomes in pre-meiotic cells does not preclude the exist- described different parameters that take part in this pro- ence of a homologous pairing associated with the forma- cess (Barzel and Kupiec 2008; Ding et al. 2012; Boateng tion of DSBs. It is possible that chromosome pairing is et al. 2013; Ishiguro et al. 2014). For instance, some stud- carried out in two steps: a weak premeiotic pairing with a ies in mice (Boateng et al. 2013; Ishiguro et al. 2014) have pre-juxtaposition function, and a robust late-meiotic pair- observed that the presence of Spo11 protein is involved ing that is crucial for the upcoming events of synapsis and in early pairing and synapsis. Additionally, Ishiguro et al. recombination. (2014) observed that disruption of RAD21L (a specific com- Our data also bring information about the associations ponent of some meiotic cohesin complexes) causes a defect between nonhomologous chromosomes before the onset of in early homolog pairing, bouquet stage arrest, and aberrant meiosis. As previously indicated, we identified a mean of synapsis and postulated that early homolog recognition is 41.10% non-homologous chromosome interactions, with achieved by the recognition of specific chromosome archi- certain pairs displaying significant increases or decreases, tecture rather than the homology of the DNA sequences. In also exhibiting higher or lower percentage of overlapping. other models, such as Schizosaccharomyces pombe, noncod- This result points out that in these particular cases, there ing RNA transcripts accumulate at their respective gene loci might be some mechanisms increasing or reducing the to promote pairing of homologous loci in early prophase likelihood of associations. Associations could be related (Ding et al. 2012). In any case, further studies will be needed to some intrinsic chromosome features such as size, %GC, to complete the puzzle of the molecular mechanisms related gene density, or presence of NOR (Table 1). Nevertheless, to pre-DSB homologous pairing and to determine whether the analysis of the chromosome pairs that showed differ- these are species-specific mechanisms or evolutionarily con- ences in comparison to the mean ruled out this scenario served processes. 13 Chromosoma Surprisingly, the high percentage of pairing observed in chromosomes move more easily than the larger ones and, our results is contradicted by other FISH studies. We pro- thus, complete the pairing process first. Alternatively, this pose two different reasons to explain this situation: first, the early small-chromosome pairing could be a consequence methodologies used in these studies are based on the use of of the association of the NOR-bearing chromosomes to locus-specific probes (Scherthan et al. 1996; Scherthan and form the nucleolus. However, our results do not indicate Schönborn, 2001). Therefore, they do not allow identify the significant associations between size and pairing neither entire territory of the chromosome and, thus, it is not possi- between NOR-bearing and non-NOR-bearing chromo- ble to state with precision if two homologous chromosomes somes and pairing. The fact that the mouse chromosome overlap. Secondly, only one or two chromosomes were ana- does not show large differences in size could be a reason lyzed in these studies and, therefore, the overall behavior of for the lack of these associations. all chromosomes cannot be known (Scherthan et al. 1996; In conclusion, our results confirm that there is a premei- Scherthan and Schönborn, 2001; Ishiguro et al. 2014). otic chromosome pairing in murine germ cells. Future stud- The main limitation of our experimental design is related ies will be needed to determine the molecular mechanisms to the impossibility of distinguishing between spermatogo- that regulate this process, as well as to elucidate any func- nia and the early-preleptotene stage. The initial spermatogo- tions that this has in the spermatogenic process. nium-preleptotene stage includes types A and B spermato- gonia, as well as spermatocytes I in the initial preleptotene Supplementary Information The online version contains supplemen- tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 00412-0​ 22-0​ 0777-0. stage. Nevertheless, it is important to mention that the expected ratio of type A spermatogonia, type B spermato- Acknowledgements We thank Dr. Ignasi Roig for his advice in opti- gonia, and preleptotene spermatocytes in the mice testis is mizing the technique of immunofluorescence and cell stage identifica- approximately 1: 1: 3 (Oakberg 1956; de Rooij 2001; Mar- tion. We thank M.A. Handel from Jackson Laboratories for providing chetti et al. 2018). Accordingly, it can be assumed that most us the H1T antibody. This manuscript has been proofread by Proof- Reading-Service.org. nuclei analyzed in this interval correspond to early prelep- totene spermatocytes, which are at the onset of the gradual Author contribution MS performed the scientific experiments, image approximation of homologous chromosomes in order to pair. analysis, and interpretation of the results. MS wrote the manuscript In fact, the presence of spermatogonia in this interval, as with the support of ZS and JB. ZS and JB conceived, designed, and well as the clear association of homologous chromosomes supervised the study. DG designed the customized developed scripts for Matlab analysis. OV conducted the statistical analyses. BC optimized observed, opens the possibility that this association rather the cell type identification methodology step. EA and FV helped shape starts before the onset of meiosis. In any case, additional the research and critically revised the manuscript. AP analyzed part studies that split the populations of premeiotic cells into of the heterologous associations by the supervision of MS, ZS, and various pure fractions will be needed to clarify this point. JB. GF and RF provided the Chromoprobe Multiprobe® OctoChrome Murine System kit. All authors reviewed and approved the final version Likewise, it has to be mentioned that the spermatocytes I of the manuscript. at mid preleptotene-zygotene interval also involved differ- ent stages. If our experimental design had been able to dis- Funding Open Access Funding provided by Universitat Autonoma tinguish among them, we would have probably observed a de Barcelona. This study was supported by CF-180034 (UAB), gradual increase of homologous pairing reaching up to 100% DPI2015-65286-R/SAF2016-77165-P/RTI2018-095209 (MINECO), 2017-SGR-1624, CERCA (Generalitat de Catalunya) and Instituto de at zygotene. In fact, in the case of pachytene spermatocytes Salud Carlos III, Ministerio de Ciencia e Innovación, Gobierno de (when full synapsis occurs), our results indicate that 100% España (PI21/00564). Mireia Solé was the recipient of a grant from of homologous chromosomes are joined. Reinforcing this UAB (PIF/2015). Debora Gil is a Serra Hunter Fellow. hypothesis, Scherthan et al. (1996) observe homologous chromosomes together in 100% zygotene and pachytene Data availability “Not applicable.” spermatocytes. Code availability “Not applicable.” Our results point out that DSB-independent pairing does not occur between all chromosomes at the same time but instead, it is asynchronous Fig. 5. Asynchronous pair- Declarations ing processes have also been observed in other species Ethics approval The Commission of Ethics in Animal and Human (Rasmussen and Holm 1978; Guitart et al. 1985; San- Experimentation of the Autonomous University of Barcelona declared tos et al. 1993). Some studies have observed a relation- that a formal consent is not required for this type of study. ship between the pairing timing and chromosome size. Consent to participate “Not applicable.” Specifically, smaller chromosomes initiate and complete their pairing earlier than the larger ones (Rasmussen and Consent for publication “Not applicable.” Holm 1978; Scherthan and Schönborn, 2001). In order to explain this association, it has been suggested that small 13 Chromosoma Conflict of interest The authors declare no competing interests. de Rooij DG (2001) Proliferation and differentiation of spermatogonial stem cells. Reproduction 121(3):347–354. https://d​ oi.o​ rg/1​ 0.1​ 530/​ Open Access This article is licensed under a Creative Commons Attri- rep.0.​12103​47 bution 4.0 International License, which permits use, sharing, adapta- Dev VG, Tantravahi R, Miller DA, Miller OJ (1977) Nucleolus organ- tion, distribution and reproduction in any medium or format, as long izers in Mus musculus subspecies and in the rag mouse cell line. as you give appropriate credit to the original author(s) and the source, Genetics 86:389–398 provide a link to the Creative Commons licence, and indicate if changes Ding X, Xu R, Yu J, Xu T, Zhuang Y, Han M (2007) SUN1 is required were made. The images or other third party material in this article are for telomere attachment to nuclear envelope and gametogenesis included in the article's Creative Commons licence, unless indicated in mice. Dev Cell. https://​doi.​org/​10.​1016/j.​devcel.​2007.​03.​018 otherwise in a credit line to the material. If material is not included in Ding DQ, Okamasa K, Yamane M, Tsutsumi C, Haraguchi T, Yama- the article's Creative Commons licence and your intended use is not moto M, Hiraoka Y (2012) Meiosis-specific noncoding RNA permitted by statutory regulation or exceeds the permitted use, you will mediates robust pairing of homologous chromosomes in meiosis. need to obtain permission directly from the copyright holder. To view a Science. https://​doi.​org/​10.​1126/​scien​ce.​12195​18 copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. Dombecki CR, Chiang AC, Kang HJ, Bilgir C, Stefanski NA, Neva BJ, Klerkx EP, Nabeshima K (2011) The chromodomain protein MRG-1 facilitates SC-independent homologous pairing during meiosis in Caenorhabditis elegans. Dev Cell. https://​doi.​org/​10.​ 1016/j.​devcel.​2011.​09.​019 References Evans HJ, Buckland RA, Pardue ML (1974) Location of the genes coding for 18S and 28S ribosomal RNA in the human genome. Atwood KC, Gluecksohn-Waelsch S, Yu MT, Henderson AS (1976) Chromosoma 48:405–426 Does the t-locus in the mouse include ribosomal DNA? Cytogenet Garcia-Quevedo L, Sarrate Z, Vidal F, Blanco J (2012) A sequential Cell Genet. https://​doi.​org/​10.​1159/​00013​0682 methodology that allows apoptotic cell sorting and FISH analysis Barzel A, Kupiec M (2008) Finding a match: how do homologous in human testicular cells. Syst Biol Reprod Med. https://​doi.​org/​ sequences get together for recombination? Nat Rev Genet. https://​ 10.​3109/​19396​368.​2012.​717163 doi.​org/​10.​1038/​nrg22​24 Grelon M, Vezon D, Gendrot G, Pelletier G (2001) AtSPO11-1 is Baudat F, Imai Y, de Massy B (2013) Meiotic recombination in mam- necessary for efficient meiotic recombination in plants. EMBO J. mals: localization and regulation. Nat Rev Genet. https://​doi.​org/​ https://​doi.​org/​10.​1093/​emboj/​20.3.​589 10.​1038/​nrg35​73 Guitart M, Coll MD, Ponsà M, Egozcue J (1985) Sequential study of Boateng KA, Bellani MA, Gregoretti IV, Pratto F, Camerini-Otero RD synaptonemal complexes in mouse spermatocytes by light and (2013) Homologous pairing preceding SPO11-mediated double- electron microscopy. Genetica. https://​doi.​org/​10.​1007/​BF024​ strand breaks in mice. Dev Cell. https://​doi.​org/​10.​1016/j.​devcel.​ 24457 2012.​12.​002 Harper L, Golubovskaya I, Cande WZ (2004) A bouquet of chromo- Britton-Davidian J, Cazaux B, Catalan J (2011) Chromosomal dynam- somes. J Cell Sci. https://​doi.​org/​10.​1242/​jcs.​01363 ics of nucleolar organizer regions (NORs) in the house mouse: Henderson KA, Keeney S (2004) Tying synaptonemal complex initia- micro-evolutionary insights. Heredity. https://​doi.​org/​10.​1038/​ tion to the formation and programmed repair of DNA double- hdy.​2011.​105 strand breaks. Proc Natl Acad Sci. https://​doi.​org/​10.​1073/​pnas.​ Burgess SM, Kleckner N, Weiner BM (1999) Somatic pairing of 04008​43101 homologs in budding yeast: existence and modulation. Genes Dev. Henderson AS, Eicher EM, Yu MT, Atwood KC (1974) The chromo- https://​doi.​org/​10.​1101/​gad.​13.​12.​1627 somal location of ribosomal DNA in the mouse. Chromosoma. Cha RS, Weiner BM, Keeney S, Dekker J, Kleckner N (2000) Progres- https://​doi.​org/​10.​1007/​BF003​48887 sion of meiotic DNA replication is modulated by interchromo- Heride C, Ricoul M, Kiêu K, von Hase J, Guillemot V, Cremer C, somal interaction proteins, negatively by Spo11p and positively Dubrana K, Sabatier L (2010) Distance between homologous by Rec8p. Genes Dev 14(4):493–503 chromosomes results from chromosome positioning constraints. Champion MD, Hawley RS (2002) Playing for half the deck: the molec- J Cell Sci. https://​doi.​org/​10.​1242/​jcs.​066498 ular biology of meiosis. Nat Cell Biol. Suppl:s50-6. https://​doi.​ Hiraoka Y, Dernburg AF (2009) The SUN rises on meiotic chromo- org/​10.​1038/​ncb-​nm-​ferti​lityS​50 some dynamics. Dev Cell. https://​doi.​org/​10.​1016/j.​devcel.​2009.​ Chi YH, Cheng LI, Myers T, Ward JM, Williams E, Su Q, Faucette L, 10.​014 Wang JY, Jeang KT (2009) Requirement for Sun1 in the expres- Ishiguro K, Kim J, Shibuya H, Hernández-Hernández A, Suzuki A, sion of meiotic reproductive genes and piRNA. Development. Fukagawa T, Shioi G, Kiyonari H, Li XC, Schimenti J, Höög C, https://​doi.​org/​10.​1242/​dev.​029868 Watanabe Y (2014) Meiosis-specific cohesin mediates homolog Cremer M, Grasser F, Lanctôt C, Müller S, Neusser M, Zinner R, recognition in mouse spermatocytes. Genes Dev. https://​doi.​org/​ Solovei I, Cremer T (2008) Multicolor 3D fluorescence in situ 10.​1101/​gad.​237313.​113 hybridization for imaging interphase chromosomes. Methods Mol Joyce EF, Erceg J, Wu C (2016) Pairing and anti-pairing: a balancing Biol. https://​doi.​org/​10.​1007/​978-1-​59745-​406-3_​15 act in the diploid genome. Curr Opin Genet Dev. https://​doi.​org/​ Davis L, Barbera M, McDonnell A, McIntyre K, Sternglanz R, Jin 10.​1016/j.​gde.​2016.​03.​002 Q, Loidl J, Engebrecht J (2001) The Saccharomyces cerevisiae Kauppi L, Barchi M, Lange J, Baudat F, Jasin M, Keeney S (2013) MUM2 gene interacts with the DNA replication machinery and Numerical constraints and feedback control of double-strand is required for meiotic levels of double strand breaks. Genetics breaks in mouse meiosis. Genes Dev. https://​doi.​org/​10.​1101/​ 157:1179–1189 gad.​213652.​113 Dernburg AF, McDonald K, Moulder G, Barstead R, Dresser M, Ville- Kurihara Y, Suh DS, Suzuki H, Moriwaki K (1994) Chromosomal neuve AM (1998) Meiotic recombination in C. elegans initiates by locations of Ag-NORs and clusters of ribosomal DNA in labora- a conserved mechanism and is dispensable for homologous chro- tory strains of mice. Mamm Genome 5:225–228. https://​doi.​org/​ mosome synapsis. Cell. https://​doi.​org/​10.​1016/​S0092-​8674(00)​ 10.​1007/​BF003​60550 81481-6 13 Chromosoma Lorenz A, Fuchs J, Bürger R, Loidl J (2003) Chromosome pairing does zygotene observations. Hereditas. https://​doi.​org/​10.​1111/j.​1601-​ not contribute to nuclear architecture in vegetative yeast cells. 5223.​1993.​00235.x Eukaryot Cell. https://​doi.​org/​10.​1128/​EC.2.​5.​856-​866.​2003 Scherthan H (2001) A bouquet makes ends meet. Nat Rev Mol Cell Maass PG, Barutcu AR, Rinn JL (2019) Interchromosomal interac- Biol. https://​doi.​org/​10.​1038/​35085​086 tions: a genomic love story of kissing chromosomes. J Cell Biol. Scherthan H, Schönborn I (2001) Asynchronous chromosome pairing https://​doi.​org/​10.​1083/​jcb.​20180​6052 in male meiosis of the rat (Rattus norvegicus). Chromosome Res. Marchetti F, Aardema M, Beevers C et al (2018) Simulation of mouse https://​doi.​org/​10.​1023/A:​10166​42528​981 and rat spermatogenesis to inform genotoxicity testing using Scherthan H, Bähler J, Kohli J (1994) Dynamics of chromosome organ- OECD test guideline 488. Mutat Res - Genet Toxicol Environ ization and pairing during meiotic prophase in fission yeast. J Cell Mutagen. https://​doi.​org/​10.​1016/j.​mrgen​tox.​2018.​05.​020 Biol. https://​doi.​org/​10.​1083/​jcb.​127.2.​273 Martinez-Perez E, Shaw P, Moore G (2001) The Ph1 locus is needed Scherthan H, Weich S, Schwegler H, Heyting C, Harle M, Cremer T to ensure specific somatic and meiotic centromere association. (1996) Centromere and telomere movements during early meiotic Nature. https://​doi.​org/​10.​1038/​35075​597 prophase of mouse and man are associated with the onset of pair- Martínez-Pérez E, Shaw P, Reader S, Aragón-Alcaide L, Miller T, ing. J Cell Biol. https://​doi.​org/​10.​1083/​jcb.​134.5.​1109 Moore G (1999) Homologous chromosome pairing in wheat. J Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pie- Cell Sci 112(Pt 11):1761–1769. https://​doi.​org/​10.​1242/​jcs.​112.​ tzsch T, Preibisch S, Rueden C, Saalfeld S et al (2012) Fiji - an 11.​1761 open source platform for biological image analysis. Nat Methods. McKim KS, Green-Marroquin BL, Sekelsky JJ, Chin G, Steinberg C, https://​doi.​org/​10.​1038/​nmeth.​2019 Khodosh R, Hawley RS (1998) Meiotic synapsis in the absence Selvaraj S, Dixon JR, Bansal V, Ren B (2013) Whole-genome haplo- of recombination. Science. https://​doi.​org/​10.​1126/​scien​ce.​279.​ type reconstruction using proximity-ligation and shotgun sequenc- 5352.​876 ing. Nat Biotechnol. https://​doi.​org/​10.​1038/​nbt.​2728 Nabeshima K, Kakihara Y, Hiraoka Y, Nojima H (2001) A novel mei- Solé M, Blanco J, Gil D, Valero O, Pascual Á, Cárdenas B, Fonseka G, osis-specific protein of fission yeast, Meu13p, promotes homolo- Anton E, Frodsham R, Vidal F, Sarrate Z (2021) Chromosomal gous pairing independently of homologous recombination. EMBO positioning in spermatogenic cells is influenced by chromosomal J. https://​doi.​org/​10.​1093/​emboj/​20.​14.​3871 factors associated with gene activity, bouquet formation and mei- Oakberg EF (1956) A description of spermiogenesis in the mouse and otic sex chromosome inactivation. Chromosoma. https://​doi.​org/​ its use in analysis of the cycle of the seminiferous epithelium 10.​1007/​s00412-​021-​00761-0 and germ cell renewal. Am J Anat. https://​doi.​org/​10.​1002/​aja.​ Tanaka H, Pereira LA, Nozaki M, Tsuchida J, Sawada K, Mori H, 10009​90303 Nishimune Y (1997) A germ cell-specific nuclear antigen recog- Page SL, Hawley RS (2003) Chromosome choreography: the meiotic nized by a monoclonal antibody raised against mouse testicular ballet. Science. https://​doi.​org/​10.​1126/​scien​ce.​10866​05 germ cells. Int J Androl 20(6):361–366. https://​doi.​org/​10.​1046/j.​ Page SL, Hawley RS (2004) The genetics and molecular biology of the 1365-​2605.​1998.​00080.x synaptonemal complex. Annu Rev Cell Dev Biol. https://​doi.​org/​ Tessé S, Storlazzi A, Kleckner N, Gargano S, Zickler D (2003) Locali- 10.​1146/​annur​ev.​cellb​io.​19.​111301.​155141 zation and roles of Ski8p protein in Sordaria meiosis and deline- Peoples TL, Dean E, Gonzalez O, Lambourne L, Burgess SM (2002) ation of three mechanistically distinct steps of meiotic homolog Close, stable homolog juxtaposition during meiosis in budding juxtaposition. Proc Natl Acad Sci. https://​doi.​org/​10.​1073/​pnas.​ yeast is dependent on meiotic recombination, occurs indepen- 20342​82100 dently of synapsis, and is distinct from DSB-independent pairing Thorne LW, Byers B (1993) Stage-specific effects of X-irradiation on contacts. Genes Dev. https://​doi.​org/​10.​1101/​gad.​983802 yeast meiosis. Genetics 134(1):29–42 Phillips CM, Meng X, Zhang L, Chretien JH, Urnov FD, Dernburg AF Vara C, Paytuví-Gallart A, Cuartero Y, Le Dily F, Garcia F, Salvà- (2009) Identification of chromosome sequence motifs that mediate Castro J, Gómez-H L, Julià E, Moutinho C, Aiese Cigliano R meiotic pairing and synapsis in C. elegans. Nat Cell Biol. https://​ et al (2019) Three-dimensional genomic structure and cohesin doi.​org/​10.​1038/​ncb19​04 occupancy correlate with transcriptional activity during spermato- Prieto P, Santos AP, Moore G, Shaw P (2004) Chromosomes associate genesis. Cell Rep. https://​doi.​org/​10.​1016/j.​celrep.​2019.​06.​037 premeiotically and in xylem vessel cells via their telomeres and Wandall A, Svendsen A (1985) Transition from somatic to meiotic centromeres in diploid rice (Oryza sativa). Chromosoma. https://​ pairing and progressional changes of the synaptonemal complex doi.​org/​10.​1007/​s00412-​004-​0274-8 in spermatocytes of Aedes aegypti. Chromosoma. https://​doi.​org/​ Rao SSP, Huntley MH, Durand NC, Stamenova EK, Bochkov ID, Rob- 10.​1007/​BF003​29808 inson JT, Sanborn AL, Machol I, Omer AD, Lander ES, Aiden Zickler D, Kleckner N (1998) The leptotene-zygotene transition of ELA (2014) A 3D map of the human genome at kilobase resolu- meiosis. Annu Rev Genet. https://​doi.​org/​10.​1146/​annur​ev.​genet.​ tion reveals principles of chromatin looping. Cell. https://​doi.​org/​ 32.1.​619 10.​1016/j.​cell.​2014.​11.​021 Zickler D, Kleckner N (1999) Meiotic chromosomes: integrating struc- Rasmussen SW, Holm PB (1978) Human meiosis II. Chromosome pair- ture and function. Annu Rev Genet. https://​doi.​org/​10.​1146/​annur​ ing and recombination nodules in human spermatocytes. Carls- ev.​genet.​33.1.​603 berg Res Commun. https://​doi.​org/​10.​1007/​BF029​06106 Zickler D, Kleckner N (2015) Recombination, pairing, and synapsis of Rockmill B, Lefrançois P, Voelkel-Meiman K, Oke A, Roeder GS, homologs during meiosis. Cold Spring Harb Perspect Biol. https://​ Fung JC (2013) High throughput sequencing reveals alterations doi.​org/​10.​1101/​cshpe​rspect.​a0166​26 in the recombination signatures with diminishing Spo11 activity. PLoS Genet. https://​doi.​org/​10.​1371/​journ​al.​pgen.​10039​32 Publisher's note Springer Nature remains neutral with regard to Romanienko PJ, Camerini-Otero RD (2000) The mouse Spo11 gene is jurisdictional claims in published maps and institutional affiliations. required for meiotic chromosome synapsis. Mol Cell. https://​doi.​ org/​10.​1016/​S1097-​2765(00)​00097-6 Santos JL, Del CAL, Díez M (1993) Spreading synaptonemal com- plexes from the grasshopper Chorthippus jacobsi: pachytene and 13