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In sexually reproducing organisms, germ cells faithfully transmit the genome to the next generation by forming haploid gametes, such as eggs and sperm. Although most meiotic proteins are conserved between eggs and sperm, many aspects of meiosis are sexually dimorphic. The mechanisms regulating recombination display sex-specific differences in multiple organisms such that the same proteins in each sex are being utilized in different ways to produce sexually dimorphic outcomes. The synaptonemal complex (SC), a large ladder-like structure that forms between homologous chromosomes, is essential for regulating meiotic chromosome organization and promoting recombination. To assess whether sex-specific differences in the SC underpin sexually dimorphic aspects of meiosis, we examined twoCaenorhabditis elegansSC central region proteins, SYP-2 and SYP-3, in oogenesis and spermatogenesis and uncovered sex-specific roles for the SYPs in regulating meiotic recombination. We find that SC composition is regulated by sex-specific mechanisms throughout meiotic prophase I. During pachytene, both oocytes and spermatocytes differentially regulate the stability of SYP-2 and SYP-3 within an assembled SC, with increased SYP-2 dynamics in spermatocytes and increased SYP-3 dynamics in oocytes. Further, we uncover that the relative amount of SYP-2 and SYP-3 within the SC is independently regulated in both a sex-specific and a recombination-dependent manner. Specifically, we find that SYP-2 regulates the early steps of recombination in both sexes, while SYP-3 controls the timing and positioning of crossover recombination events across the genomic landscape in only oocytes. Taken together, we demonstrate dosage-dependent regulation of individual SC components with sex-specific functions in recombination. These sexual dimorphic features of the SC provide insights into how spermatogenesis and oogenesis adapted similar chromosome structures to differentially regulate and execute recombination.

The pairing of homologous chromosomes (homologs) in meiosis is essential for distributing the correct numbers of chromosomes into haploid gametes. In budding yeast, pairing depends on the formation of 150-200 Spo11-mediated double-strand breaks (DSBs) that are distributed among 16 homolog pairs, but it is not known if all, or only a subset of these DSBs, contribute to the close juxtaposition of homologs. Having established a system to measure the position of fluorescently tagged chromosomal loci in 3D space over time, we analyzed locus trajectories to determine how frequently, and how long, loci spend colocalized or apart. Continuous imaging revealed highly heterogeneous cell-to-cell behavior of foci, with the majority of cells exhibiting a “mixed” phenotype where foci move into and out of proximity, even at late stages of prophase, suggesting that the axial structures of the synaptonemal complex may be more dynamic than anticipated. The observed plateaus of the mean-squared change in distance (MSCD) between foci informed the development of a biophysical model of two diffusing polymers that captures the loss of centromere linkages as cells enter meiosis, nuclear confinement, and the formation of Spo11-dependent linkages. The predicted number of linkages per chromosome in our theoretical model closely approximates the small number (~2-4) of estimated synapsis-initiation sites, suggesting that excess DSBs have negligible effects on the overall juxtaposition of homologs. These insights into the dynamic in-terchromosomal behavior displayed during homolog pairing demonstrate the power of combining time-resolvedin vivoanalysis with modeling at the granular level.Significance StatementEssential for sexual reproduction, meiosis is a specialized cell division required for the production of haploid gametes. Critical to this process is the pairing, recombination, and segregation of homologous chromosomes (homologs). While pairing and recombination are linked, it is not known how many linkages are sufficient to hold homologs in proximity. Here, we reveal that random diffusion and the placement of a small number of linkages are sufficient to establish the apparent “pairing” of homologs. We also show that colocalization between any two loci is more dynamic than anticipated. Our study is the first to provide observations of live interchromosomal dynamics during meiosis and illustrates the power of combining single-cell measurements with theoretical polymer modeling.

The visualization of biological processes using fluorescent proteins and dyes in living organisms has enabled numerous scientific discoveries. The nematode Caenorhabditis elegans is a widely used model organism for live imaging studies since the transparent nature of the worm enables imaging of nearly all tissues within a whole, intact animal. While current techniques are optimized to enable the immobilization of hermaphrodite worms for live imaging, many of these approaches fail to successfully restrain the smaller male worms. To enable live imaging of worms of both sexes, we developed a new genetic, conditional immobilization tool that uses the auxin-inducible degron (AID) system to immobilize both adult and larval hermaphrodite and male worms for live imaging. Based on chromosome location, mutant phenotype, and predicted germline consequence, we identified and AID-tagged three candidate genes (unc-18, unc-104, and unc-52). Strains with these AID-tagged genes were placed on auxin and tested for mobility and germline defects. Among the candidate genes, auxin-mediated depletion of UNC-18 caused significant immobilization of both hermaphrodite and male worms that was also partially reversible upon removal from auxin. Notably, we found that male worms require a higher concentration of auxin for a similar amount of immobilization as hermaphrodites, thereby suggesting a potential sex-specific difference in auxin absorption and/or processing. In both males and hermaphrodites, depletion of UNC-18 did not largely alter fertility, germline progression, nor meiotic recombination. Finally, we demonstrate that this new genetic tool can successfully immobilize both sexes enabling live imaging studies of sexually dimorphic features in C. elegans.

Errors during meiosis are the leading cause of birth defects and miscarriages in humans. Thus, the coordinated control of meiotic events is critical for the faithful inheritance of the genome with each generation..., Crossovers (COs) between homologous chromosomes are critical for meiotic chromosome segregation and form in the context of the synaptonemal complex (SC), a meiosis-specific structure that assembles between aligned homologs. During Caenorhabditis elegans meiosis, central region components of the SC (SYP proteins) are essential to repair double-strand DNA breaks (DSBs) as COs. Here, we investigate the relationships between the SYP proteins and conserved pro-CO factors by examining the immunolocalization of these proteins in meiotic mutants where SYP proteins are absent, reduced, or mislocalized. Although COs do not form in syp null mutants, pro-CO factors COSA-1, MSH-5, and ZHP-3 nevertheless colocalize at DSB-dependent sites during late prophase, reflecting an inherent affinity of these factors for DSB repair sites. In contrast, in mutants where SYP proteins are present but form aggregates or display abnormal synapsis, pro-CO factors consistently track with SYP-1 localization. Further, pro-CO factors usually localize to a single site per SYP-1 structure, even in SYP aggregates or in mutants where the SC forms between sister chromatids, suggesting that CO regulation occurs within these aberrant SC structures. Moreover, we find that the meiotic cohesin REC-8 is required to ensure that SC formation occurs between homologs and not sister chromatids. Taken together, our findings support a model in which SYP proteins promote CO formation by promoting the localization of pro-CO factors to recombination events within an SC compartment, thereby ensuring that pro-CO factors identify a recombination event within an SC structure and that CO maturation occurs only between properly aligned homologous chromosomes.

The visualization of biological processes using fluorescent proteins and dyes in living organisms has enabled numerous scientific discoveries. The nematode Caenorhabditis elegans is a widely used model organism for live imaging studies since the transparent nature of the worm enables imaging of nearly all tissues within a whole, intact animal. While current techniques are optimized to enable the immobilization of hermaphrodite worms for live imaging, many of these approaches fail to successfully restrain the smaller male worms. To enable live imaging of worms of both sexes, we developed a new genetic, conditional immobilization tool that uses the auxin inducible degron (AID) system to immobilize both hermaphrodites and male worms for live imaging. Based on chromosome location, mutant phenotype, and predicted germline consequence, we identified and AID-tagged three candidate genes (unc-18, unc-104, and unc-52). Strains with these AID-tagged genes were placed on auxin and tested for mobility and germline defects. Among the candidate genes, auxin-mediated depletion of UNC-18 caused significant immobilization of both hermaphrodite and male worms that was also partially reversible upon removal from auxin. Notably, we found that male worms require a higher concentration of auxin for a similar amount of immobilization as hermaphrodites, thereby suggesting a potential sex-specific difference in auxin absorption and/or processing. In both males and hermaphrodites, depletion of UNC-18 did not largely alter fertility, germline progression, nor meiotic recombination. Finally, we demonstrate that this new genetic tool can successfully immobilize both sexes enabling live imaging studies of sexually dimorphic features in C. elegans.ARTICLE SUMMARYC. elegans is a powerful model system for visualizing biological processes in live cells. In addition to the challenge of suppressing the worm movement for live imaging, most immobilization techniques only work with hermaphrodites. Here, we describe a new genetic immobilization tool that conditionally immobilizes both worm sexes for live imaging studies. Additionally, we demonstrate that this tool can be used for live imaging the C. elegans germline without causing large defects to germline progression or fertility in either sex.

Arranged in a spatial-temporal gradient for germ cell development, the adult germline of Caenorhabditis elegans is an excellent system for understanding the generation, differentiation, function, and maintenance of germ cells. Imaging whole C. elegans germlines along the distal-proximal axis enables powerful cytological analyses of germ cell nuclei as they progress from the pre-meiotic tip through all the stages of meiotic prophase I. To enable high-content image analysis of whole C. elegans gonads, we developed a custom algorithm and pipelines to function with image processing software that enables: (1) quantification of cytological features at single nucleus resolution from immunofluorescence images; and (2) assessment of these individual nuclei based on their position within the germline. We show the capability of our quantitative image analysis approach by analyzing multiple cytological features of meiotic nuclei in whole C. elegans germlines. First, we quantify double-strand DNA breaks (DSBs) per nucleus by analyzing DNA-associated foci of the recombinase RAD-51 at single-nucleus resolution in the context of whole germline progression. Second, we quantify the DSBs that are licensed for crossover repair by analyzing foci of MSH-5 and COSA-1 when they associate with the synaptonemal complex during meiotic prophase progression. Finally, we quantify P-granule composition across the whole germline by analyzing the colocalization of PGL-1 and ZNFX-1 foci. Our image analysis pipeline is an adaptable and useful method for researchers spanning multiple fields using the C. elegans germline as a model system.

Arranged in a spatial-temporal gradient for germ cell development, the adult germline ofCaenorhabditis elegansis an excellent system for understanding the generation, differentiation, function, and maintenance of germ cells. Imaging wholeC. elegansgermlines along the distal-proximal axis enables powerful cytological analyses of germ cell nuclei as they progress from the pre-meiotic tip through all the stages of meiotic prophase I. To enable high-throughput image analysis of wholeC. elegansgonads, we developed a custom algorithm and pipelines to function with image processing software that enables: 1) quantification of cytological features at single nucleus resolution from immunofluorescence images; and, 2) assessment of these individual nuclei based on their position within the germline. We demonstrate the capability of our quantitative image analysis approach by analyzing multiple cytological features of meiotic nuclei in wholeC. elegansgermlines. First, we quantify double strand DNA breaks (DSBs) per nucleus by analyzing DNA-associated foci of the recombinase RAD-51 at the single-nucleus resolution in the context of whole germline progression. Second, we quantify the DSBs that are licensed for crossover repair by analyzing foci of MSH-5 and COSA-1 when they associate with the synaptonemal complex during meiotic prophase progression. Finally, we quantify P-granule composition across the whole germline by analyzing the colocalization of PGL-1 and ZNFX-1 foci. Our image analysis pipeline is an adaptable and useful method for researchers spanning multiple fields utilizing theC. elegansgermline as a model system.

During meiosis, diploid organisms reduce their chromosome number by half to generate haploid gametes. This process depends on the repair of double strand DNA breaks as crossover recombination events between homologous chromosomes, which hold homologs together to ensure their proper segregation to opposite spindle poles during the first meiotic division. Although most organisms are limited in the number of crossovers between homologs by a phenomenon called crossover interference, the consequences of excess interfering crossovers on meiotic chromosome segregation are not well known. Here we show that extra interfering crossovers lead to a range of meiotic defects and we uncover mechanisms that counteract these errors. Using chromosomes that exhibit a high frequency of supernumerary crossovers in Caenorhabditis elegans, we find that essential chromosomal structures are mispatterned in the presence of multiple crossovers, subjecting chromosomes to improper spindle forces and leading to defects in metaphase alignment. Additionally, the chromosomes with extra interfering crossovers often exhibited segregation defects in anaphase I, with a high incidence of chromatin bridges that sometimes created a tether between the chromosome and the first polar body. However, these anaphase I bridges were often able to resolve in a LEM-3 nuclease dependent manner, and chromosome tethers that persisted were frequently resolved during Meiosis II by a second mechanism that preferentially segregates the tethered sister chromatid into the polar body. Altogether these findings demonstrate that excess interfering crossovers can severely impact chromosome patterning and segregation, highlighting the importance of limiting the number of recombination events between homologous chromosomes for the proper execution of meiosis., Author summary Meiosis is a process that ensures developing eggs and sperm contain the correct number of chromosomes. Failure to accurately segregate chromosomes during meiosis is one of the leading causes of birth defects and miscarriages. During meiosis, crossover events must form between the homologous chromosomes to ensure proper chromosome segregation. Although crossover events are required for proper chromosome segregation in most organisms, crossover numbers are limited even when the meiotic cell is overloaded with DNA breaks, the initiating events for crossovers. This stringent limitation of crossovers in multiple organisms suggests that there are negative consequences to having too many crossovers, but this has not been formally tested. In this study, we find that increasing crossover number negatively impacts chromosome segregation during meiosis by altering chromosome-associated structures, which leads to misalignment of the chromosomes on the meiotic spindle. Moreover, chromosomes with excess crossovers often have large chromatin bridges during the chromosome segregation process, but we find that these bridges can be corrected by at least two mechanisms. Our results thus highlight the importance of limiting crossover numbers to enable faithful chromosome segregation during sperm and egg development.

The synaptonemal complex (SC) is a conserved meiotic structure that regulates the repair of double-strand breaks (DSBs) into crossovers or gene conversions. The removal of any central-region SC component, such as the Drosophila melanogaster transverse filament protein C(3)G, causes a complete loss of SC structure and crossovers. To better understand the role of the SC in meiosis, we used CRISPR/Cas9 to construct 3 in-frame deletions within the predicted coiled-coil region of the C(3)G protein. Since these 3 deletion mutations disrupt SC maintenance at different times during pachytene and exhibit distinct defects in key meiotic processes, they allow us to define the stages of pachytene when the SC is necessary for homolog pairing and recombination during pachytene. Our studies demonstrate that the X chromosome and the autosomes display substantially different defects in pairing and recombination when SC structure is disrupted, suggesting that the X chromosome is potentially regulated differently from the autosomes.

A multiplexed approach to DNA FISH experiments has been used to visualize the three-dimensional organization of chromosomes and specific chromosomal regions in C. elegans.

Meiosis is a conserved cell division process that is used by sexually reproducing organisms to generate haploid gametes. Males and females produce different end products of meiosis: eggs (females) and sperm (males). In addition, these unique end products demonstrate sex-specific differences that occur throughout meiosis to produce the final genetic material that is packaged into distinct gametes with unique extracellular morphologies and nuclear sizes. These sexually dimorphic features of meiosis include the meiotic chromosome architecture, in which both the lengths of the chromosomes and the requirement for specific meiotic axis proteins being different between the sexes. Moreover, these changes likely cause sex-specific changes in the recombination landscape with the sex that has the longer chromosomes usually obtaining more crossovers. Additionally, epigenetic regulation of meiosis may contribute to sexually dimorphic recombination landscapes. Here we explore the sexually dimorphic features of both the chromosome axis and crossing over for each stage of meiotic prophase I in Mus musculus, Caenorhabditis elegans, and Arabidopsis thaliana. Furthermore, we consider how sex-specific changes in the meiotic chromosome axes and the epigenetic landscape may function together to regulate crossing over in each sex, indicating that the mechanisms controlling crossing over may be different in oogenesis and spermatogenesis.

The synaptonemal complex (SC) is a meiosis-specific scaffold that links homologous chromosomes from end to end during meiotic prophase and is required for the formation of meiotic crossovers. Assembly of SC components is regulated by a combination of associated nonstructural proteins and post-translational modifications, such as SUMOylation, which together coordinate the timing between homologous chromosome pairing, double-strand-break formation and recombination. In addition, transcriptional and translational control mechanisms ensure the timely disassembly of the SC after crossover resolution and before chromosome segregation at anaphase I.

Crossovers (COs) between homologous chromosomes are critical for meiotic chromosome segregation and form in the context of the synaptonemal complex (SC), a meiosis-specific structure that assembles between aligned homologs. DuringCaenorhabditis elegansmeiosis, central region components of the SC (SYP proteins) are essential to repair double-strand DNA breaks (DSBs) as COs, but the roles of these SYP proteins in promoting CO formation are poorly understood. Here, we investigate the relationships between the SYP proteins and conserved CO-promoting factors by examining the immunolocalization of these factors in meiotic mutants where SYP proteins are absent, reduced, or mis-localized. Although COs do not form insypnull mutants, CO-promoting proteins COSA-1, MSH-5, and ZHP-3 nevertheless become co-localized at a variable number of DSB-dependent sites during late prophase, reflecting an inherent affinity of these factors for DSB repair sites. In contrast, in mutants where SYP proteins are present but form aggregates or display abnormal synapsis, CO-promoting proteins consistently track with SYP-1 localization. Moreover, CO-promoting proteins usually localize to a single site per SYP-1 structure, even in SYP aggregates or in mutants where SC forms between sister-chromatids, suggesting that CO regulation occurs within these structures. Further, we find that sister chromatids in the meiotic cohesin mutantrec-8require both CO-promoting proteins and the SC to remain connected. Taken together, our findings support a model in which SYP proteins promote CO formation by directing and constraining the localization of CO-promoting factors to ensure that CO maturation occurs only between properly aligned homologous chromosomes.Article SummaryErrors during meiosis are the leading cause of birth defects and miscarriages in humans. Thus, the coordinated control of meiosis events is critical for the faithful inheritance of the genome each generation. The synaptonemal complex (SC) is a meiosis-specific structure that assembles between homologs chromosomes and is critical for the establishment and regulation of crossovers, which ensure the accurate segregation of the homologous chromosomes at meiosis I. Here we show that the SC proteins function to regulate crossovers by directing and constraining the localization of proteins involved in promoting the formation of crossovers.

Multiple meiosis-specific cohesion proteins act to facilitate homolog segregation at the first meiotic division. A recent paper demonstrates that meiotic cohesins can be separated into two complexes, one that establishes and maintains intersister cohesion and one that promotes interhomolog adhesion by regulating synaptonemal complex assembly.

In Drosophila, meiotic recombination is initiated by the formation of programmed DNA doublestrand breaks (DSBs), which occur within the context of the synaptonemal complex (SC). We created an in-frame deletion mutant in c(3)G (deleting amino acids L340 to N550, denoted as c(3)GccΔ1), which encodes the major transverse filament protein of the SC. Although c(3)GccΔ1 oocytes assemble full-length SC and exhibit normal DSB formation, euchromatic SC in both c(3)GccΔ1 heterozygotes and homozygotes precociously disassembles in early to mid pachytene. Centromeric SC, however, is unaffected in both genotypes. Thus, c(3)GccΔ1 is a separation-offunction mutant that establishes different functional and structural requirements between euchromatic and centromeric SC. Additionally, the chromosomes differ in their sensitivity to c(3)GccΔ1-induced perturbations in the SC. The X chromosome is distinctly sensitive to these perturbations, such that euchromatic pairing and crossing over are altered in c(3)GccΔ1 heterozygotes and severely reduced in c(3)GccΔ1 homozygotes. On the autosomes, crossovers are shifted to centromere-proximal regions and crossover interference is defective in both c(3)GccΔ1 homozygotes and heterozygotes. However, only c(3)GccΔ1 homozygotes display a progressive loss of euchromatic pairing in distal autosomal regions, suggesting that discontinuity in the euchromatic SC—rather than failed pairing—might cause the altered crossover distribution. These phenotypes reveal that different chromatin states or regions have differing requirements to maintain both the SC and homologous pairing. Furthermore, c(3)GccΔ1 is the first mutant in Drosophila to demonstrate that the SC appears to facilitate the regulation of recombination frequency and distribution differently on each chromosome.

The synaptonemal complex (SC), a structure highly conserved from yeast to mammals, assembles between homologous chromosomes and is essential for accurate chromosome segregation at the first meiotic division. In Drosophila melanogaster, many SC components and their general positions within the complex have been dissected through a combination of genetic analyses, superresolution microscopy, and electron microscopy. Although these studies provide a 2D understanding of SC structure in Drosophila, the inability to optically resolve the minute distances between proteins in the complex has precluded its 3D characterization. A recently described technology termed expansion microscopy (ExM) uniformly increases the size of a biological sample, thereby circumventing the limits of optical resolution. By adapting the ExM protocol to render it compatible with structured illumination microscopy, we can examine the 3D organization of several known Drosophila SC components. These data provide evidence that two layers of SC are assembled. We further speculate that each SC layer may connect two nonsister chromatids, and present a 3D model of the Drosophila SC based on these findings.

Homologous recombination drives structural reorganization of the nucleus in early meiosis. In order to investigate the connection between homolog pairing, meiotic progression, and the dynamics of the underlying chromatin, we tracked flourescently labeled homolog pairs in synchronized S. cerevisae. Various previously unreported statistics of the anomalous inter-loci motion correlate with meiotic progression and can be quantitatively reproduced by a simple polymer model of the sister chromatids. The first of these is the distribution of waiting times for the homologous loci to come into and out of contact with each other (loosely, inter-locus “looping” and “unlooping” times). The full shape of the looping time distribution can be quantitatively reproduced by a simple model of two polymers diffusing independently in a spherical confinement. This finding suggests a dominant role for diffusion-limited, undirected search in homolog pairing in early meiosis. This is in sharp contrast with the intuition that a heavy-tailed search process could never drive such a critical cell-cycle stage. We further show that the inter-locus velocity-velocity correlation (VVC) quantitatively matches analytical results for the inter-locus VVC of our polymer model, allowing us to leverage our analytical theory to extract the time scale of stress communication between the labeled loci along the chromosome. We show that stresses can take tens of minutes to propagate between loci on paired chromosomes, and that the increasing connectivity between the chromosomes as the cell progresses through meiosis can be quantified by the shortening of this communication time. Our study highlights the power of coarse-grained polymer models to analyze dynamic structural properties of the nucleus in vivo and the importance of analytical theory for uncovering intracellular connections that might be obscured by lag times of many minutes.

Homolog pairing and crossing over during meiosis I prophase is required for accurate chromosome segregation to form euploid gametes. The repair of Spo11-induced double-strand breaks (DSB) using a homologous chromosome template is a major driver of pairing in many species, including fungi, plants, and mammals. Inappropriate pairing and crossing over at ectopic loci can lead to chromosome rearrangements and aneuploidy. How (or if) inappropriate ectopic interactions are disrupted in favor of allelic interactions is not clear. Here we used an in vivo “collision” assay in budding yeast to test the contributions of cohesion and the organization and motion of chromosomes in the nucleus on promoting or antagonizing interactions between allelic and ectopic loci at interstitial chromosome sites. We found that deletion of the cohesin subunit Rec8, but not other chromosome axis proteins (e.g. Red1, Hop1, or Mek1), caused an increase in homolog-nonspecific chromosome interaction, even in the absence of Spo11. This effect was partially suppressed by expression of the mitotic cohesin paralog Scc1/Mdc1, implicating Rec8's role in cohesion rather than axis integrity in preventing nonspecific chromosome interactions. Disruption of telomere-led motion by treating cells with the actin polymerization inhibitor Latrunculin B (Lat B) elevated nonspecific collisions in rec8Δ spo11Δ. Next, using a visual homolog-pairing assay, we found that the delay in homolog pairing in mutants defective for telomere-led chromosome motion (ndj1Δ or csm4Δ) is enhanced in Lat B–treated cells, implicating actin in more than one process promoting homolog juxtaposition. We suggest that multiple, independent contributions of actin, cohesin, and telomere function are integrated to promote stable homolog-specific interactions and to destabilize weak nonspecific interactions by modulating the elastic spring-like properties of chromosomes., Author Summary Meiosis is the key stage of gametogenesis, when the diploid genome complement is reduced by one half to form haploid gametes for sexual reproduction. Accurate chromosome segregation requires that homologous chromosomes pair, recombine by crossing over, and segregate from one another during the first meiotic division. Missegregation of homologs leads to the formation of aneuploid gametes, while erroneous crossing over between ectopic chromosomal loci can lead to chromosomal rearrangements such as translocations and deletions. We found that nonspecific interactions between interstitial chromosomal sites can be enabled or prevented through multiple, independent mechanisms during meiosis in budding yeast. These include organization of chromosomes in the nucleus, integrity of the chromosome axis structure, and actin-led chromosome movement. Acting together, these processes can reinforce strong chromosome interactions that promote pairing, while acting in opposition they can eliminate weak nonspecific interactions. These data provide an integrated view of how homologous chromosome pairing is achieved.

Few distinctions in biology are as clearly drawn as the one between mitosis and meiosis. The function of mitosis is to produce two identical daughter cells, while the purpose of the first division of meiosis is to ensure the segregation of homologous chromosomes, and the second division is to create haploid gametes. These meiotic segregations usually rely on meiosis-specific processes such as the induction of preprogramed double-strand breaks, homolog pairing, synaptonemal complex (SC) formation, synapsis, and recombination between homologs to form crossovers. While not all meiotic systems function identically, enough commonalities exist to allow us to draw clear distinctions between meiosis and mitosis. Mitotic cells simply do not build SC, and the process of full-length homolog pairing is almost always a prerogative of cells entering meiosis, not mitosis. Vexingly for those of us who like our distinctions made clearly, all this has changed, at least in Drosophila, as a result of two papers in this issue of PLOS Genetics [1], [2]. These authors show that although homologs are neither paired nor synapsed in germline stem cells (GSCs) of Drosophila females, both homolog pairing and the accumulation of SC components in pericentromeric regions initiate during the five mitotic divisions preceding meiosis. While the mechanisms and consequences of these interactions remain to be elucidated, it is clear that pairing and synapsis begin earlier than previously imagined—indeed, they initiate during the premeiotic mitotic divisions.