To understand the relationship between the sexually dimorphic aspects of meiosis and the SC, we used the model system C. elegans where oocyte and spermatocyte development can be easily accessed and analyzed at both the same time and developmental stage. Adult male worms undergo spermatogenesis, while adult hermaphrodite worms undergo oogenesis (Figure 1A). The germline for both sexes is organized as a spatial-temporal gradient along the distal-proximal axis, thereby allowing for easy and simultaneous access to all stages of meiotic prophase I (Gartner and Engebrecht, 2022; Hillers et al., 2017). The SC initiates assembly late in the transition zone (leptotene/zygotene) and is fully assembled by pachytene (MacQueen et al., 2002). While many proteins have been identified within the C. elegans SC, we focused our analyses here on the central region proteins, which are all called SYP (SYnaPsis protein) and, to date, six SYP proteins (SYP-1–SYP-6) span the gap between the homologous chromosomes (Figure 1B; Colaiácovo et al., 2003; Hurlock et al., 2020; MacQueen et al., 2002; Smolikov et al., 2007a; Smolikov et al., 2009; Zhang et al., 2020).

To assess if SC dynamics differ between spermatocytes and oocytes, we performed fluorescent recovery after photobleaching (FRAP) assays with two SYP proteins endogenously tagged with fluorescent proteins: (1) GFP::SYP-2 from Gao et al., 2016, and (2) mCherry::SYP-3 that we generated using CRISPR/Cas9 (see ‘Methods’; Figure 1C, Figure 1—figure supplements 1 and 2). Both SYP-2 (213 amino acids) and SYP-3 (224 amino acids) are the smallest SYP proteins in the C. elegans SC and have very little sequence similarity between themselves and the other SYP proteins (Kursel et al., 2021). However, structurally SYP-2 and SYP-3 are similar to the other SYP proteins with all the SYP proteins having predicted coiled-coil protein domains (Kursel et al., 2021). We found that both SYP-2 and SYP-3 have sex-specific differences in protein turnover, with SYP-2 dynamics higher in spermatocytes and SYP-3 dynamics higher in oocytes.

For GFP::SYP-2, spermatocytes displayed significantly higher recovery dynamics throughout pachytene compared to oocytes (Figure 1C, Figure 1—figure supplements 1 and 2; p<0.001, Bonferroni–Dunn adjusted, Mann–Whitney). In contrast, mCherry::SYP-3 recovered more quickly in oocytes during early pachytene compared to spermatocytes (early pachytene p=0.0015; Bonferroni–Dunn adjusted, Mann–Whitney). During mid and late pachytene, SYP-3 recovery rates were similar between the sexes. Notably, we observed that the progressive stabilization of spermatocyte SYP-3 was less pronounced and more subtle than that of oocyte SYP-3, suggesting that spermatocytes may not modulate SYP-3 stability via the same mechanisms as oocytes (Figure 1C). Collectively, these data indicate that SYP dynamics are not uniformly regulated throughout prophase and instead exhibit both SYP-specific and sex-specific dynamics.

Overall, both sexes showed the same overall trend of progressive stabilization of SYP-2 and SYP-3 throughout pachytene, matching previous observations with the transgene GFP::SYP-3 (Figure 1, Figure 1—figure supplements 1 and 2; see ‘Methods’; Nadarajan et al., 2017; Pattabiraman et al., 2017; Rog et al., 2017). Upon comparing SYP mobilization between the sexes, we found that SYP-2 and SYP-3 displayed differences in mobility when compared to each other within each sex. In oocytes, GFP::SYP-2 turnover was significantly reduced compared to mCherry::SYP-3 turnover (Figure 1C, Figure 1—figure supplements 1 and 2; p<0.01, Bonferroni–Dunn adjusted, Mann–Whitney). Comparatively in spermatocytes, only early pachytene displayed significant differences in GFP::SYP-2 and mCherry::SYP-3 turnover (Figure 1C; p=0.0237, Bonferroni–Dunn adjusted, Mann–Whitney). These results demonstrate that SYP-2 and SYP-3 can be independently regulated during pachytene.

To determine whether differential regulation of SYP-2 and SYP-3 might be influenced by chromosome length, we measured SC length during early, mid, and late pachytene in both sexes. Our results found that SC length was similar throughout pachytene in each sex (Figure 1—figure supplement 3). The slightly shorter SC lengths in late pachytene spermatocytes were likely due to differences in chromosome compaction between the sexes (Rourke and Jaramillo-Lambert, 2022; Samson et al., 2014). These results suggest that independent regulation of SYP-2 and SYP-3 within an assembled SC is not due to changes in chromosome length, but rather by other factors.

The differences in SYP-2 and SYP-3 mobility between oogenesis and spermatogenesis suggest that the abundance or concentration of each protein within the SC may also be sexually dimorphic. To compare the relative assembled SYP compositions within the SC between sexes during meiotic prophase I, we calculated the mean fluorescence intensity per cubic micrometer of assembled SC for individual nuclei throughout pachytene in oogenesis and spermatogenesis, and then calculated the average SYP intensity among nuclei in a sliding window across the normalized germline length (see ‘Methods’). We found that the accumulation of GFP::SYP-2 in the SC is sexually dimorphic (Figure 2A and B). Wild-type oocytes progressively accumulated SYP-2 up until the early to mid pachytene transition (early pachytene mean intensity 153,292.27 ± SD 13,983.85; early/mid pachytene mean peak intensity 161,878.14± SD 13,930.43; mid pachytene mean intensity 148,430.1± SD 13,058.02) and then reduce the amount of SYP-2 slightly before maintaining a relatively constant level of SYP-2 through late pachytene. In contrast to oocytes, wild-type spermatocytes progressively loaded SYP-2 into the SC until the onset of late pachytene at which point the amount of SYP-2 began to stabilize (Figure 2A and B). Notably, early pachytene spermatocytes had less SYP-2 loaded than oocytes, but late pachytene spermatocytes had near or slightly more amounts of SYP-2 in the SC than oocytes (p<0.001, Bonferroni adjusted, Mann–Whitney). Additionally, slightly past the early/mid pachytene transition of SYP-2 intensity, we noted only oocytes display a few persisting bright nuclei in nearly all germlines examined (eight out of nine germlines with persisting bright nuclei; Figure 2B). Thus, the incorporation of SYP-2 throughout pachytene differs between sexes.

In contrast to SYP-2, mCherry::SYP-3 progressively accumulated throughout pachytene in both sexes, matching previous observations using a GFP::SYP-3 transgene (Figure 2C and D; Pattabiraman et al., 2017). Therefore, differences in fluorescent tags on SYP-3 do not appear to largely influence the incorporation of SYP-3 into the SC during pachytene (see ‘Methods’). In comparison to oocytes, spermatocytes incorporated less SYP-3 throughout pachytene (p<0.001, Bonferroni adjusted, Mann–Whitney), which was also observed in the early regions of pachytene with SYP-2. Thus, spermatocytes have less SYP-3 and SYP-2 in the SC than oocytes, specifically within the early and mid regions of pachytene. Moreover, unlike with SYP-2, we did not observe in either sex any bright SYP-3 nuclei that were not surrounded by other nuclei of similar intensity, thereby suggesting that these bright SYP-2 nuclei may have defects or changes that only trigger a response with SYP-2 levels (Figure 2D). Taken together, these data demonstrate that SYP-2 and SYP-3 are differentially incorporated in the SC both over the course of meiotic prophase I and between sexes.

During pachytene, one of the main functions of the SC is to facilitate and regulate recombination. In C. elegans, SPO-11-induced DSBs are formed in the context of fully assembled SC in early pachytene, and these breaks get repaired as the nuclei traverse through pachytene (Gartner and Engebrecht, 2022). By the transition to late pachytene, crossover recombination events are designated and marked by the pro-crossover protein COSA-1 (Yokoo et al., 2012). Notably, in C. elegans oocytes, the SC is modified in response to recombination and alterations of the SC influences and/or impairs recombination (Almanzar et al., 2023; Cahoon et al., 2019; Colaiácovo et al., 2003; Gao et al., 2016; Gordon et al., 2021; Köhler et al., 2020; Libuda et al., 2013; Nadarajan et al., 2016; Rog et al., 2017; Sato-Carlton et al., 2018; Schild-Prüfert et al., 2011). To determine whether the changes we observed in SYP-2 and SYP-3 accumulation during pachytene were caused by recombination, we assessed how the absence of recombination influenced the incorporation of each protein within the SC. To achieve this, we inhibited the formation of crossovers at different stages of recombination using two mutants: (1) spo-11(me44) mutants, which cannot form DSBs; and (2) cosa-1(tm3298) mutants, which cannot designate DSBs for crossover formation.

The amount of both GFP::SYP-2 and mCherry::SYP-3 loaded into the SC was significantly increased in spo-11 mutant oocytes, but not spermatocytes (Figure 3A and C, Figure 3—figure supplement 1). The pattern of SYP-2 accumulation was altered in spo-11 oocytes, in which SYP-2 amounts continued to increase within the SC displaying a 1.27-fold increase in SYP-2 amounts during early pachytene, 1.62-fold increase during mid pachytene, and 1.57-fold increase during late pachytene (Figure 3A). While the overall pattern of SYP-3 accumulation in spo-11 oocytes remains very similar to wild-type with a progressive accumulation throughout pachytene, the total amounts of SYP-3 significantly increase within the SC (Figure 3C). In contrast, spo-11 spermatocytes displayed mild changes in amounts of SYP-2 and SYP-3 in the SC compared to wild-type and the trend of SYP incorporation through pachytene was unaltered (Figure 3A and C). Additionally, western blot whole worm analysis of GFP::SYP-2 protein showed a slight, but not statistically significant, increase in SYP-2 protein levels in only oocytes (Figure 3—figure supplement 2). Thus, oocytes and spermatocytes differentially regulate the amount of SYP-2 protein in the absence of DSBs. Taken together, these results demonstrate that the incorporation of SYPs is regulated in a recombination-dependent and sex-specific manner during prophase I.

This sex-specific regulation of the SYPs in response to DSB formation was also observed in the crossover-deficient cosa-1 mutants, albeit to a different degree in comparison to spo-11 mutant oocytes (Figure 3B and D, Figure 3—figure supplement 1). Specifically, cosa-1 oocytes did not increase the levels of SYP-2 and SYP-3 to the same amounts as observed in spo-11 oocytes (Figure 3). In early pachytene, cosa-1 oocytes showed a 0.8-fold decrease in SYP-2 amounts that changed in mid and late pachytene with SYP-2 amounts increasing by 1.16-fold and 1.22-fold over wild-type amounts, respectively (Figure 3B). Whereas spo-11 oocytes increased the amount of assembled SYP-2 to a greater degree than cosa-1 oocytes. Thus, SYP-2 and SYP-3 levels in oocytes can be differentially regulated depending on the specific recombination stage that is impeded. Similar to spo-11 mutant spermatocytes, cosa-1 mutant spermatocytes did not largely alter SYP-2 and SYP-3 levels (Figure 3). Even when recombination is hindered, the pattern of SYP-2 and SYP-3 incorporation throughout pachytene is not the same for each sex or between the SYPs. Overall, the SYPs within the SC of oocytes largely respond to alterations in recombination whereas the SC of spermatocytes do not significantly respond to recombination.

The SYP-specific changes in SC composition and in response to recombination defects raised the possibility that SYP protein dosage may regulate specific steps of recombination. To alter the dosage of each SYP, we used heterozygous null mutants for either syp-2(ok307) or syp-3(ok785) referred to as syp-2/+ or syp-3/+. In oocytes, reducing the dosage of SYP-1, SYP-2, or SYP-3 by 60–70% was sufficient to permit SC assembly and crossover designation (Libuda et al., 2013). It remained unclear, however, whether altering the dosage of the SYPs also influenced chromosome pairing or the timing of SC assembly, which can also influence downstream meiotic processes like recombination (Couteau et al., 2004; Couteau and Zetka, 2005; Goodyer et al., 2008; MacQueen et al., 2005; Martinez-Perez and Villeneuve, 2005; Mlynarczyk-Evans and Villeneuve, 2017; Nabeshima et al., 2004; Zhang et al., 2012).

In syp-2/+ and syp-3/+ mutants, the transition zone (determined by DAPI-stained DNA morphology) was not significantly extended in either sex, indicating that chromosome pairing is not impaired by SYP dosage (Figure 4—figure supplement 1). Since SYP-1, SYP-2, and SYP-3 are dependent on each other for assembly, we assessed SC assembly using SYP-1 staining in syp-2/+ and syp-3/+ oocytes and spermatocytes (Colaiácovo et al., 2003; MacQueen et al., 2002; Smolikov et al., 2007b). SC assembly and/or the maintenance of full-length SC were altered by reducing the dosage of SYP-2 only in oocytes (Figure 4). In early pachytene, syp-2/+ oocytes displayed more discontinuities in the SC along the chromosomes than both wild type and syp-3/+, suggesting that there is an SC assembly or maintenance defect in syp-2/+ (Figure 4A, yellow arrowheads). Notably, these SC defects in early pachytene caused a significant increase in the length of the SC assembly zone in syp-2/+ oocytes, but these mutants did maintain full-length SC after this zone (Figure 4C and E; p<0.001, Bonferroni p adjusted, Mann–Whitney). In contrast, syp-2/+ and syp-3/+ spermatocytes did not display any significant defects in SC assembly in early pachytene (Figure 4B and D). Additionally, syp-2/+ or syp-3/+ mutants in both sexes did not display any changes in SC disassembly or pachytene length, as indicated by DAPI morphology (Figure 4, Figure 4—figure supplement 1). We also checked SUN-1 S8 phosphorylation in both syp-2/+ and syp-3/+ to assess whether synapsis checkpoints were activated, but there was no significant change in the length of the SUN-1 S8 phosphorylation zone in either mutant or sex (Figure 4—figure supplement 1; Woglar et al., 2013). Thus, SC assembly and/or maintenance only in oocytes are sensitive to SYP-2 dosage during the early stages of pachytene and this defect is not severe enough to trigger synapsis checkpoint activation.

Since altering SYP dosage permitted assembly of full-length SC and did not impair chromosome pairing, we next assessed how reducing the dosage of SYP-2 and SYP-3 influenced recombination. To assess the mechanics of recombination, we used immunofluorescence to quantify and detect specific proteins that mark sites undergoing three different stages of recombination: (1) DSB formation with RAD-51 (Colaiácovo et al., 2003) (note: the subsequent removal of RAD-51 also indicates progression of a DSB down a repair pathway); (2) joint molecule formation with GFP::MSH-5 Janisiw et al., 2018; and (3) crossover designation with GFP::COSA-1 in oocytes (Yokoo et al., 2012) and OLLAS::COSA-1 in spermatocytes (Janisiw et al., 2018; see ‘Methods’). For simplicity, here we refer to the tagged versions of GFP::MSH-5 and GFP::COSA-1 or OLLAS::COSA-1 as only the protein name either MSH-5 or COSA-1. The average number of foci of each protein was determined by using a sliding window of 0.01 along the normalized germline length, which was divided into early, mid, and late pachytene (see ‘Methods).

Since we found that manipulating the dosage of SYP-2 and SYP-3 altered multiple steps of recombination and previous studies indicate that the SC can regulate crossover numbers (Colaiácovo et al., 2003; Gordon et al., 2021; Hayashi et al., 2010; Köhler et al., 2020; Libuda et al., 2013; MacQueen et al., 2002; Smolikov et al., 2007b), we wanted to determine whether SYP-2 and SYP-3 dosage influences the recombination landscape by changing where crossovers are positioned along the length of the chromosome. To assess the recombination landscape, we used established single-nucleotide polymorphism (SNP) recombination mapping between two C. elegans isolates (Bristol and Hawaiian) to identify crossovers (see ‘Methods’; Bazan and Hillers, 2011). For oocytes, we mapped recombination on both chromosome II and the X chromosome. Since male worms have an unpaired X chromosome and do not form crossovers on this sex chromosome, only recombination on chromosome II was mapped in spermatocytes.

On chromosome II, both syp-2/+ and syp-3/+ only altered the crossover landscape in oocytes (Figure 6, Table 1). In spermatocytes, both syp-2/+ and syp-3/+ displayed no significant changes in crossover frequencies across all of chromosome II. In contrast, both syp-2/+ and syp-3/+ oocytes displayed significant changes in the crossing over distribution on chromosome II (Figure 6; syp2/+ p=0.0343, syp3/+ p=0.0333, chi-squared). Specifically, both mutants increased crossing over in the first interval (A to B) on the left side of chromosome II by ~10 cM each (Figure 6; wild-type 14 cM, syp-2/+ 24 cM, syp-3/+ 25 cM; syp2/+ p=0.0449, syp3/+ p=0.0170, Fisher’s exact test). Intriguingly, this first interval is also where the pairing center is located on chromosome II. Thus, one possible explanation for the elevated crossing over in the interval is that by reducing the SYP dosage crossovers are now more often formed where the SC is first assembled at the pairing center (Hayashi et al., 2010; Rog and Dernburg, 2015).

On the X chromosome, syp-2/+ oocytes displayed a significant decrease in crossover frequency along the entire chromosome (Figure 6—figure supplement 1; wild-type 51 cM, syp-2/+ 32 cM; p=0.0343, chi-squared). This decrease was not observed in syp-3/+ oocytes, which showed no significant changes in crossover frequency to wild-type for X chromosome recombination (Figure 6—figure supplement 1; wild-type 51 cM, syp-3/+ 45 cM; p=0.5849, chi-squared). The large decrease in crossing over in syp-2/+ suggests that these mutants should have a significant amount of X chromosome nondisjunction or missegregation. In C. elegans, mutants with frequent X chromosome nondisjunction produce a high incidence of male (Him) phenotype as male worms are hemizygous for the X chromosome. Notably, syp-2/+ did not display an elevation in the frequency of male progeny nor did they have an increase in dead eggs (Figure 6—figure supplement 1; wild-type 0.4% dead eggs and 0.2% Him, syp-2/+ 0.4% dead eggs and 0.2% Him). Since the SNP recombination mapping experiment is performed in a hybrid Bristol/Hawaiian background, we also checked the hybrid background for male progeny and dead eggs. Due to known meiotic drive elements between these strains, the Bristol/Hawaiian hybrids produce more dead eggs (Seidel et al., 2011; Seidel et al., 2008). However, syp-2/+ Bristol/Hawaiian hybrids did not display a higher incidence of dead eggs or more male progeny than the wild-type Bristol/Hawaiian hybrid (Figure 6—figure supplement 1; wild-type 19.4% dead eggs and 0.2% Him; syp-2/+ 23.3% dead eggs and 0.1% Him). Thus, it remains unclear as to why reducing SYP-2 causes a significant decrease in recombination without a corresponding increase in X chromosome nondisjunction. One possible explanation is that syp-2/+ have a chromosome distortion event occurring in the later stages of meiosis that is removing recombinant X chromosomes. Future studies are needed to understand what is happening to the recombinant X chromosomes in syp-2/+.

In comparison to wild-type, the broods from syp-3/+ hermaphrodites exhibited both an increased lethality (more dead eggs; Figure 6—figure supplement 1, wild-type Bristol 0.4% and hybrid 19.4% dead eggs; syp-3/+ Bristol 1.2% and hybrid 27.0% dead eggs) as well as a higher incidence of male progeny than wild-type (Figure 6—figure supplement 1, wild-type Bristol 0.2% and hybrid 0.2% HIM; syp-3/+ Bristol 0.7% and hybrid 1.6% HIM, Bristol p<0.0001, hybrid P<0.0001, chi-squared). In addition, syp-3/+ also produced a higher rate of progeny with dumpy and/or uncoordinated mutant phenotypes than both wild-type and syp-2/+ (Figure 6—figure supplement 1, Bristol 0.06% mutant progeny, syp-2/+ Bristol 0.06% mutant progeny, syp-3/+ Bristol 0.47% mutant progeny; wild-type Bristol p=0.0207, syp-2/+ Bristol p=0.0388, Fisher’s exact test). SYP proteins have been implicated in regulating DSB repair pathway choice by preventing access to error-prone and sister chromatid repair (Láscarez-Lagunas et al., 2022; Lemmens et al., 2013; Macaisne et al., 2018; Rosu et al., 2011; Smolikov et al., 2007a; Yin and Smolikove, 2013). Additionally, SYP-3 directly interacts with the pro-crossover protein BRC-1 to promote crossover DSB repair (Janisiw et al., 2018). Thus, the mutant phenotypes observed in progeny from syp-3/+ hermaphrodites are likely due to error-prone repair using nonhomologous end joining, which is used by syp-3 nulls to repair persisting DSBs (Smolikov et al., 2007b). These results suggest that SYP-3 amounts are important for suppressing error-prone repair. Notably, in contrast to oocytes, spermatocyte fertility was unaffected in syp-2/+ or syp-3/+ (Figure 6—figure supplement 1). Thus, reducing the dosage of SYP-2 or SYP-3 has sexually dimorphic consequences on fertility.

The SYP dosage-dependent regulation of recombination (Figures 4 and 5), different patterns of SYP protein incorporation throughout pachytene (Figure 2), and independent regulation of SYP loading into the SC (Figures 2 and 3) suggest that SYP accumulation in the SC may be dynamically altered during pachytene to regulate the steps of recombination. Thus, reducing the gene dosage of each SYP may influence how the SYPs are accumulating within the SC, which could influence the regulation of recombination. To determine how syp gene dosage influences the incorporation of each SYP during pachytene, we used heterozygous null mutants of each syp gene with a wild-type fluorescently tagged GFP::SYP-2 or mCherry::SYP-3: syp-2(ok307)/gfp::syp-2 (referred to as syp-2/+) or syp-3(ok785)/mCherry::syp-3 (referred to as syp-3/+). Indeed, we found that the dosage of syp-2 and syp-3 influenced both SYP-2 and SYP-3 accumulation during pachytene in both sexes (Figure 7).

Altering the syp-2 gene dosage resulted in both sexes to initially have an ~0.7-fold decrease in SYP-2 amounts within the SC; however, SYP-2 levels progressively increased throughout pachytene until wild-type amounts were reached or pachytene ended (Figure 7A). Western blot analysis revealed that GFP::SYP-2 protein levels were not significantly increased in syp-2/+ oocytes and spermatocytes (Figure 3—figure supplement 2). Overall, these results suggest that both sexes attempt to compensate for the haploinsufficiency of losing a syp-2 gene by loading as much SYP-2 into the SC as possible during pachytene.

In contrast, SYP-3 accumulation in syp-2/+ mutants displayed sex-specific responses (Figure 7B). In oocytes, reducing the syp-2 gene dosage resulted in a slight decrease in SYP-3 levels in early pachytene that becomes more pronounced in late pachytene, which we noted was the same window when SYP-2 protein levels reached wild-type amounts in syp-2/+ mutants (Figure 7B; p<0.001, Bonferroni adjusted, Mann–Whitney). In spermatocytes, reducing the syp-2 gene dosage resulted in an increase in SYP-3 amount in early pachytene that are indistinguishable from wild-type by late pachytene (Figure 7B). Thus, while altering syp-2 gene dosage caused both sexes to respond similarly with reduced loading of SYP-2 into assembled SC, SYP-3 levels within assembled SC were inversely affected via sex-specific mechanisms.

We found that altering syp-3 gene dosage resulted in the same overall changes to SYP-2 and SYP-3 amounts in each sex as altering syp-2 gene dosage, albeit the degree to which each SYP changed was different (Figure 7). Specifically, reducing syp-3 gene dosage caused an initial reduction in SYP-2 levels in both sexes that was less than the reduction in syp-2/+ (Figure 7A and C; early pachytene syp-2/+ oocytes 0.72-fold change vs. syp-3/+ oocytes 0.87-fold change; early pachytene syp-2/+ spermatocytes 0.71-fold change vs. syp-3/+ spermatocytes 0.89-fold change). However, similar to syp-2/+, both sexes progressively increase SYP-2 amounts in syp-3/+ until wild-type levels were reached or pachytene ended (Figure 7A and C). Additionally, western blot analysis of GFP::SYP-2 protein levels showed that both sexes only slightly increased SYP-2 protein levels (Figure 3—figure supplement 2). Thus, both sexes respond to alterations in syp gene dosages with similar trends of SYP-2 accumulation within the SC.

In oocytes, reducing syp-3 gene dosage caused a stronger decrease in overall SYP-3 protein amounts compared to syp-2/+ (Figure 7B and D, early pachytene syp-2/+ 0.97-fold change vs. syp-3/+ 0.91-fold change, mid pachytene 0.99-fold change vs. syp-3/+ 0.90-fold change, late pachytene 0.91-fold change vs. syp-3/+ 0.85-fold change). In contrast, syp-3/+ spermatocytes displayed an increase in SYP-3 levels that were similar to the increase in SYP-3 observed in syp-2/+ spermatocytes (Figure 7B and D). Thus, the dosage of each SYP gene influences the accumulation of SYP-2 and SYP-3 within the assembled SC similarly for each sex. Oocytes respond to reduced syp gene dosage by decreasing both SYP-2 and SYP-3 within the SC, while spermatocytes respond to reduced syp gene dosage by decreasing SYP-2 and increasing SYP-3 in the SC. Overall, these differences are likely influencing and/or responding to the changes we observed in recombination for both syp-2/+ and syp-3/+ (Figure 5) and suggest that the dosage of each SYP throughout pachytene regulates recombination in a sex-specific manner (see ‘Discussion’).

The ability of both SYP-2 and SYP-3 to affect the incorporation of each other within the SC suggested that the dosage of each SYP may influence and/or regulate the incorporation of other SYP proteins in the SC. Since the inability to functionally tag SYP-1 and SYP-4 with fluorescent proteins precluded analysis of these two proteins, we assessed how the composition of SYP-5 and SYP-6 within the SC was influenced by syp-2 and syp-3 gene dosage in both oocytes and spermatocytes. Using endogenously CRISPR tagged lines containing either SYP-5::GFP or SYP-6::GFP (Zhang et al., 2020) combined with heterozygous null mutants of syp-2 (syp-2/+) or syp-3 (syp-3/+), we found that syp-2 and syp-3 gene dosage can influence SYP-5 and SYP-6 incorporation in the SC via sex-specific mechanisms (Figure 8).

One of the roles of the SC during meiosis is regulating recombination to promote the establishment of a crossover on each homolog pair. Our data here demonstrate that the SC in C. elegans regulates recombination via sexually dimorphic mechanisms. We show that the central region proteins SYP-2 and SYP-3 have sex-specific differences in protein turnover rates within the SC that may influence the sexually dimorphic composition of the SC. Specifically, SYP-2, SYP-3, SYP-5, and SYP-6 are incorporated into the SC at different levels via sex-specific mechanisms. Moreover, we find that SYP protein levels in the SC are dependent on recombination such that SYP protein dosage impacts the regulation of recombination. Our data support a model where SYP-2 and SYP-3 levels within the SC regulate the proper timing and execution of specific steps of recombination (Figure 9).

Our data suggest that the amount of SYP-2 within the SC of oocytes promotes the formation and/or maintenance of joint molecules (Figure 9A). syp-2/+ oocytes incorporate less SYP-2 in the SC during meiotic stages when MSH-5-stabilized joint molecules are formed (Figures 5 and 7) and exhibit a severe reduction in MSH-5 foci (Figure 5). This relationship between the SC and joint molecules is SYP-2 specific, as a reduction in SYP-3 levels within an assembled SC did not result in reduced MSH-5 foci (Figures 5 and 7). We therefore suggest that a specific threshold of SYP-2 in the SC ensures the formation and/or stabilization of joint molecules.

Similar to oocytes, spermatocyte SYP-2 dosage is required for the maintenance of joint molecules marked by MSH-5 (Figure 9B). Unlike oocytes, reducing SYP-2 levels in spermatocytes did not alter the assembly of the SC (Figure 4) and is likely why MSH-5 foci are able to initially form near wild-type levels in early pachytene (Figure 5). However, the rapid loss of MSH-5 in spermatocytes during mid pachytene suggests that these joint molecules are either being rapidly resolved and designated for crossover recombination or destabilized, resulting in MSH-5 removal. We favor a model where the rapid loss of MSH-5 foci is underpinned by destabilization of joint molecules as the crossover designation marker COSA-1 is not prematurely loaded when SYP-2 dosage is reduced. This function of SYP-2 in the maintenance of joint molecules appears conserved between the sexes as syp-2/+ spermatocytes initially form wild-type levels of MSH-5 foci that are rapidly lost (Figure 5). However, the specific threshold of SYP-2 required in spermatocytes to establish and/or stabilize joint molecules is significantly reduced compared to oocytes (Figures 2, 5,, 7). This discrepancy between the sexes suggests that there are mechanistic differences in how joint molecules are regulated between spermatocytes and oocytes. Thus, our work here adds to a growing body of work illustrating that spermatocytes process DSBs into crossovers differently than oocytes (Brick et al., 2018; Checchi et al., 2014; Durand et al., 2022; Jaramillo-Lambert et al., 2007; Jaramillo-Lambert and Engebrecht, 2010; Li et al., 2020).

We further propose that SYP-3 dosage in oocytes regulates the timing of recombination steps (Figure 9A). SYP-3 accumulates within the SC throughout pachytene as recombination intermediates are successively processed (Figure 2). Notably, MSH-5-marked joint molecules and COSA-1-marked crossover designation appear prematurely when the levels of SYP-3 in the SC are reduced (Figures 5 and 7). This acceleration in crossover designation coincide with a disproportionate formation of crossovers near the pairing center side of chromosome II (Figure 6). This change in crossover designation and positioning was also present in syp-2/+. However, we assert that this phenotype is likely underpinned by SYP-3 levels as SYP-3 incorporation is also reduced in syp-2/+ (Figure 7). Thus, the timing of recombination events in the germline appears to be sensitive to the amounts of SYP-3. We suggest that SYP-3 incorporation is dynamically regulated in response to meiotic stresses to ensure that recombination is completed by the end of pachytene, which is spatially and temporally limited by the length of the gonad.

In spermatocytes, SYP-3 also regulates the timing of recombination events during pachytene. However, spermatocytes’ SYP-3 incorporation is minimally impacted by syp-3/+ heterozygosity (Figure 9B). We suggest that the relatively normal levels of SYP-3 in the syp-3/+ spermatocyte SC explain the absence of crossover positioning defects (Figure 7). In fact, SYP-3 levels are subtly elevated in early pachytene in syp-3/+ spermatocytes and coincide with a delay in crossover designation (Figures 5 and 7). As spermatogenesis and oogenesis operate on very different timescales (Jaramillo-Lambert et al., 2007) and the amount of SYP-3 in the SC of spermatocytes and oocytes differs, we raise the possibility that titrating the level of SYP-3 incorporation may function to regulate the timing of recombination events between the sexes.

Sex-comparative studies are critical to understand the differences in egg and sperm development. Here we demonstrate that the SC in C. elegans is sexually dimorphic. Intriguingly, the sex-specific differences in SYP-2 and SYP-3 dynamics within the SC suggest a difference in protein regulation between the sexes (Figure 1). The progressive stabilization of SC during pachytene in oocytes has been linked with phosphorylation of specific SYP proteins (Nadarajan et al., 2017; Pattabiraman et al., 2017). Additionally, during oogenesis, many SYP proteins are known to be post-translationally modified, including SYP-2, in response to recombination and many of these modifications are critical for DSB formation and repair as well as the timing of SC assembly and disassembly (Garcia-Muse et al., 2019; Láscarez-Lagunas et al., 2022; Nadarajan et al., 2017; Nadarajan et al., 2016; Sato-Carlton et al., 2018). Since spermatocytes also displayed the same progressive stabilization of the SYP-2 and SYP-3 during pachytene as oocytes, it is possible that the same post-translational modifications of SYP proteins may also have similar functions in spermatocytes.

We found that SYP-2 was more dynamic in spermatocytes even at the ‘stabilized’ state in late pachytene (Figure 1). Thus, spermatocytes may not stabilize the SC to the same degree as oocytes. One reason for this could be to allow DSBs in late pachytene to be repaired with the homolog. In oocytes, the stabilization of the SC in late pachytene is thought to prevent the formation of more crossovers with the homolog. Spermatocytes have been shown to have differences in DNA repair, the number of crossovers, and the checkpoints that monitor repair events, and these differences may require a more dynamic SC (Checchi et al., 2014; Gabdank and Fire, 2014; Jaramillo-Lambert et al., 2007; Jaramillo-Lambert and Engebrecht, 2010; Jaramillo-Lambert et al., 2010; Li et al., 2020). Future studies are needed in spermatocytes to examine these relationships between SC dynamics, post-translational modification of the SC, and DNA repair outcomes.

The amount of SYP-2, SYP-3, and SYP-5 required to properly execute the same steps of recombination is significantly different between oocytes and spermatocytes (Figures 8 and 9). Oocytes require more of both SYP-2, SYP-3, and SYP-5 during early and mid pachytene than spermatocytes (Figures 2 and 8). One possibility for this difference is due to male worms having a hemizygous X chromosome, which does not normally assemble the SC (Jaramillo-Lambert and Engebrecht, 2010). This lack of SC on one chromosome should potentially reduce the amount of SC proteins we get from our analysis. If this were the case, then we would expect the SYP proteins to be reduced in spermatocytes along the entire length of pachytene. Instead, we observe SYP-2 levels in spermatocytes reach those of oocytes during late pachytene and SYP-6 levels are nearly identical between the sexes in early pachytene regions (Figures 2 and 9). Further, spermatocytes have increased SYP-2 turnover dynamics in late pachytene compared to oocyte SYP-2 (Figure 1), which may help facilitate the repair of DSBs in late pachytene since spermatocytes do not undergo checkpoint-mediated apoptosis (Jaramillo-Lambert et al., 2010). Thus, having a more dynamic SYP-2 may aid DSB repair when errors occur late in pachytene.

Our observation that reduced SYP-3 dosage triggers an increase in SYP-3 composition within the SC suggests that spermatocytes may be able to compensate for the loss of a functional copy of syp-3. Given that spermatocytes differ in the dynamics of protein turnover within the assembled SC, one way spermatocytes could alter SYP-3 levels is to alter the SYP-3 turnover rates within the SC. We demonstrate that spermatocytes do indeed have altered protein dynamics in the SC compared to oocytes (Figure 1). Therefore, sex-specific differences in SYP-2 and SYP-3 dynamics may regulate the composition of the SC during pachytene.

One common feature amongst SC proteins in nearly all SC-containing organisms is that most, if not all, of the central region proteins are dependent upon each other for assembly of the SC (reviewed in Cahoon and Hawley, 2016). In C. elegans, the SYP proteins are also dependent on each other for protein stability. Specifically, the result where depletion of one SYP leads to the degradation of the other SYPs has led to the assumption that the SYPs are all completely interdependent for assembly, stability, and function (Colaiácovo et al., 2003). Here we show that fluctuations in both SYP-2 and SYP-3 levels can not only differentially influence the amount of each other within the SC and other SYPs (Figures 7 and 8), but also each SYP protein can be regulated independently (Figures 2, 3, 7,, 8). Notably, we show that these differences in the proportion of SYP-2 and SYP-3 within the SC are directly involved in regulating specific steps of recombination. Additionally, some of the SYPs display similar accumulation patterns and respond similarly to changes in SYP protein levels during pachytene, such as SYP-5 and SYP-3 in oocytes (Figures 7 and 8). Thus, we hypothesize that each SYP in the SC maintains both SYP-dependent functions where they function together as a group (e.g., assembling the SC) and SYP-independent functions where they can individually or in smaller groups to influence other aspects of meiosis (e.g., regulating specific steps of recombination).

In worms and yeast, the SC appears to grow in width throughout pachytene, suggesting that the composition of proteins within the complex is highly dynamic (Pattabiraman et al., 2017; Voelkel-Meiman et al., 2012). Here we found that the incorporation of SYP-2 and SYP-3 is not proportional during pachytene (Figure 1). Further, our results suggest that recombination influences the pattern of SYP accumulation within the SC and that each of the SYPs is independently regulated within the SC (Figure 3). The ability to adjust the amount of SYP-2, SYP-3, SYP-5, and SYP-6 within the SC demonstrates flexibility in the requirements of SYPs to assemble the complex. SYP-2 is positioned in the very center of the SC and may be slightly more external on the complex to SYP-3, SYP-5, and SYP-6 (Köhler et al., 2020), which would facilitate regulation of SYP-2 levels without compromising the whole complex. Also, the proteins within this very central region of the SC play a critical role in recombination (Gordon et al., 2021; Voelkel-Meiman et al., 2019; Voelkel-Meiman et al., 2015; Voelkel-Meiman et al., 2022). Thus, these proteins in the center of the SC, such as SYP-2, may not have as strong of structural roles in the complex as those positioned broadly in the central region, such as SYP-3.

The sex-specific differences with SYP-6 localization and the sexually dimorphic response of SYP-5 and SYP-6 to SYP-2 and SYP-3 levels suggest that SYP-5 and SYP-6 have some individual sex-specific roles during meiosis. Null mutants of syp-5 and syp-6 in oocytes also indicate that there are shared and distinct functions between these proteins (Hurlock et al., 2020; Zhang et al., 2020). syp-5 mutants have stronger defects in fertility and crossing over than syp-6 mutants, thereby suggesting that SYP-5 may play a more significant role in oocyte recombination than that of SYP-6 (Hurlock et al., 2020; Zhang et al., 2020). However, the sex-specific retention of SYP-6 localization into late pachytene of spermatocytes suggests that SYP-6 has a spermatocyte-specific role in late pachytene (Figure 8). Since spermatocytes do not undergo checkpoint-mediated apoptosis (Jaramillo-Lambert et al., 2010), retaining SYP-6 late in pachytene may facilitate repair of any DSBs still present into late pachytene. Future studies examining the role of SYP-6 in spermatocyte recombination may reveal that the fertility defects in syp-6 mutants are caused by defective sperm rather than oocytes.

Not only are the patterns of SYP-2 and SYP-3 accumulation in the SC different, but the dosage of each SYP protein can influence the amount of the other SYP proteins. Regardless of which SYP dosage was altered, the same change in SYP-2 and SYP-3 accumulation occurred individually for each sex. In oocytes, both SYP-2, SYP-3, and SYP-5 amounts decreased in response to reduced SYP-2 or SYP-3 (Figure 7). Whereas in spermatocytes reduced SYP-2 or SYP-3 decreased SYP-2 amounts, increased SYP-3 amounts, and did not change SYP-5 amounts (Figures 7 and 8). The exception to this was SYP-6, which increased upon reduced SYP-2 dosage and did not change significantly in response to altered SYP-3 dosage (Figure 8). One explanation for these sex-specific differences is that spermatocytes require a different stoichiometry of proteins in the SC compared to oocytes. In mice, the SC in oocytes is narrower than the SC between spermatocytes due to structural differences in the organization of proteins within the central element and the chromosome axis (Agostinho et al., 2018). While C. elegans does not have a defined central element based on electron micrographs of the SC, SYP-2 is located in the very center of the SC where the central element proteins are located in other organisms (Cahoon and Hawley, 2016). Future studies to determine the stoichiometric ratios of chromosome axis proteins may reveal that spermatocytes assemble an SC that is structurally different from oocytes.

All strains were generated from the N2 background and were maintained and crossed at 20°C under standard conditions on nematode growth media (NGM) with lawns of Escherichia coli. InVivo Biosystems tagged the C-terminus of SYP-3 with a piRNA-optimized mCherry using CRISPR/Cas9. The CRISPR homology-directed repair template was constructed containing at least 500 base pairs of homology on either side of the insertion site at the SYP-3 locus. A small region of DNA was recoded section at the sgRNA site to avoid Cas9 cutting the template and mCherry was attached to SYP-3 with a glycine serine linker (GGSGGGGS). These repair constructs were synthesized into plasmids and injected into unc-119(ed3) mutant worms with two sgRNAs. Successful CRISPR/Cas9 integrations were screened using a loxP flanked unc-119 rescue transgene, which was inserted into an intron of syp-3 and removed following successful PCR confirmation of the integration by injecting Cre recombinase (Dickinson et al., 2013). All sequences and screening primers for the CRISPR/Cas9 tagging of SYP-3 are in Supplementary file 1. CRISPR/Cas9 worm lines were backcrossed to N2 worms three times, and loss of unc-119(ed3) mutation was confirmed by PCR before processing with any strain construction.

The following strains were used in this study:

Worms were mounted for all live imaging studies using our auxin-inducible conditional paralysis method, which is described in Cahoon and Libuda, 2021. Briefly, young adult worms (18–24 hr post L4) from a parental generation that was grown on NGM with either 1 mM auxin (for oocyte studies) or 10 mM auxin (for spermatocyte studies) were picked into 1 µL drop of live imaging media (M9 media with 25 mM serotonin [Sigma-Aldrich, Cat# H7752, Rog and Dernburg, 2015], 0.08% tricaine [ethyl 3-aminobenzoate methanesulfonate; Sigma-Aldrich, Cat# E10521-50G], 0.008% tetramisole hydrochloride [Sigma-Aldrich, Cat# T1512-10G]) and either 1 mM or 10 mM auxin (naphthaleneacetic acid [K-NAA], PhytoTechnology Laboratories, Cat# N610; Martinez et al., 2020) on a 22 × 40 mm (no. 1.5) coverslip. (Note: we found that poly-lysine treating the coverslips was not necessary for immobilization of the worms in most cases as long as the liquid under the agarose pad is minimal.) Also, 7–9% agarose pads (Invitrogen, Cat# 16500500) were gently placed over the top of the worms and excess liquid was wicked away using Whatman paper. A microscope slide was adhered to the agarose pad worm coverslip sandwich using a ring of Vaseline around the pad. Worms were then imaged using the setting described below. For both SC intensity and photobleaching studies, worms were imaged immediately following mounting and worms were only kept mounted for a max of 1 hr even though worms can survive being mounted for 2–3 hr (Cahoon and Libuda, 2021).

We did note that previous studies displayed higher fractions of SYP-3 recovery, which is likely caused by our studies not upshifting the worms to 25°C overnight (Nadarajan et al., 2017; Pattabiraman et al., 2017; Rog et al., 2017). Shifting the worms to 25°C is known to cause significant elevations in meiotic gene expression and is used to enhance expression of fluorescently tagged meiotic proteins (Pattabiraman et al., 2017; Song et al., 2010; Yokoo et al., 2012). Notably, we found that in recombination-deficient mutants, such as cosa-1, the elevated levels of GFP::SYP-2 protein in the SC with the elevation in protein expression that comes at 25°C caused large aggregates to form in mid/late pachytene that would persist into diakinesis (Figure 3—figure supplement 3). When the worms were only grown at 20°C without any 25°C upshift, these aggregates did not form. Thus, none of the worms for the studies in this paper were placed at 25°C overnight. Nevertheless, the trends appear the same for progressive stabilization with both GFP::SYP-3 and mCherry::SYP-3 (Figure 1, Figure 1—figure supplement 2), suggesting that mCherry and GFP fluorescent tags of SYP-3 do not appear to illicit differential dynamics (Nadarajan et al., 2017; Pattabiraman et al., 2017; Rog et al., 2017).

All live imaging studies of SYP-2::GFP, mCherry::SYP-3, SYP-5::GFP, and SYP-6::GFP were imaged on a Nikon CSU SoRa Spinning Disk Microscope with a ×60 water lens/N.A. 1.2 using a Z-step size of 0.3 µm. For SC intensity quantifications, the laser power and exposure times were kept consistent for all genotypes. All GFP::SYP-2, SYP-5::GFP, and SYP-6::GFP were imaged using the 488 laser at 16% power and 500 ms exposure time. All mCherry::SYP-3 imaging used the 561 laser at 25% power and 700 ms exposure time. Additionally, only the bottom half of the germline closest to the coverslip was imaged and germlines were not imaged if the position of the mounted worm caused the gut to cover germline or moved parts of the germline deeper into the worm.

The FRAP studies were performed as described in Pattabiraman et al., 2017 with minor changes. Briefly, a Z-stack was taken prior to photobleaching to obtain a pre-bleach image. Then, a region of interest defined by the point tool in Elements was photobleached. A timelapse was started immediately post-photobleaching with images captured every 5 min for 35 min to monitor the fluorescence recovery. Pilot experiments showed that the 35 min timepoint displayed the highest recovery fluorescence observed before the signal plateaued. So, to minimize photobleaching and phototoxicity effects, we concluded the recovery timelapses at 35 min. For photobleaching small regions of GFP::SYP-2 or mCherry::SYP-3, a 405 laser was used with 1–5% laser power and 10–30 ms exposure depending on the germline location of the nucleus and the tagged protein with GFP::SYP-2 requiring less laser power to photobleach than mCherry::SYP-3. Previous studies showed that immobilized worms without serotonin have significantly diminished or absent chromosome motion, and the loss of this motion does not impair SC recovery dynamics (Pattabiraman et al., 2017; Rog and Dernburg, 2015; Rog et al., 2017). Since the addition of chromosome motion makes the FRAP recovery analysis very challenging, we did not include serotonin to minimize the motion of the chromosomes and to allow for better tracking of the photobleached SC region within each nucleus during the recovery timelapse.

Immunofluorescence slides of fixed gonad were imaged on a GE DeltaVision microscope with a ×63/N.A. 1.42 lens and 1.5× optivar at 1024 × 1024 pixel dimensions. Images were acquired using 0.2 µm Z-step size and deconvolved with softWoRx deconvolution software.

Immunofluorescence was performed as described in Cahoon and Libuda, 2021; Libuda et al., 2013. Briefly, gonads were dissected in egg buffer with 0.1% Tween20 onto VWR Superfrost Plus slides from 18 to 24 hr post L4 worms. Dissected gonads were fixed in 5% paraformaldehyde for 5 min, flash frozen in liquid nitrogen, and fixed for 1 min in 100% methanol at –20°C. Slides were washed three times in PBS + 0.1% Tween20 (PBST) for 5 min each and incubated in block (0.7% bovine serum albumin in PBST) for 1 hr. Primary antibodies (chicken anti-RAD-51, 1:1500 [Kurhanewicz et al., 2020; Toraason et al., 2021]; rabbit anti-SYP-1, 1:1000 [gift from Silva Lab]; rabbit anti-DSB-2 [Rosu et al., 2013]; 1:5000; rabbit anti-OLLAS 1:1000 [GenScript, A01658]; guinea pig anti-SUN-1 S8P, 1:700 [Woglar et al., 2013]) were added and incubated overnight in a humid chamber with a parafilm cover. Slides were then washed three times in PBST for 10 min each and incubated with secondary antibodies (goat anti-rabbit Alexa Fluor 488, Thermo Fisher, Cat# A11034; goat anti-chicken Alexa Fluor488, Thermo Fisher, Cat# A11039; goat anti-rabbit Alexa Fluor 555, Thermo Fisher, Cat# A21428; GFP booster, Chromotek, gb2AF488-50; goat anti-guinea pig Alex Fluor 488, Thermo Fisher, Cat# A11073) at 1:200 dilution for 2 hr in a humid chamber with a parafilm cover. Slides were washed two times in PBST then incubated with 2 µg/mL DAPI for 15–20 min in a humid chamber. Prior to mounting, slides were washed once more in PBST for 10 min and mounted using Vectashield with a 22 × 22 mm coverslip (no. 1.5). Slides were sealed with nail polish and stored at 4°C until imaged. GFP::MSH-5 slides were imaged within 24–48 hr of mounting due to significant signal loss in the GFP::MSH-5 staining if the slide were stored longer.

For both male and hermaphrodite, 100 adult worms were picked and washed once with M9 before being boiled for 5–10 min in sample buffer with occasional vortexing. For all western blots, samples were run on SDS-PAGE and wet transferred to low fluorescence PVDF membranes (Thermo Fisher Cat# 22860). Membranes were blocked in 5% milk in TBS + 0.1% Tween20 (TBST) for 1 hr at room temperature. Primary antibodies (anti-SYP-2 rabbit 1:1000 [gift from Yumi Kim], anti-Tubulin mouse 1:1000 [Abcam Cat# ab7291]) were incubated and agitated on an orbital shaker overnight at 4°C in 5% milk. Blots were washed three times for 10 min with TBST and LI-COR secondary antibodies (IRDye 680 donkey anti-mouse [LI-COR Cat# 926-68072], IRDye 800CW donkey anti-rabbit [LI-COR Cat# 926-32213]) were incubated at 1:1000 in TBST at room temperature for 1 hr. Blots were washed twice for 10 min each and imaged using LI-COR Odyssey Fc.

Since male worms only have one germline, all hermaphrodite samples were diluted to adjust for the two germlines present in hermaphrodite worms. To determine the correct dilution factor, we performed a dilution series of the hermaphrodite sample and measured the sum intensity of the SYP-2 bands in each dilution using FIJI (Figure 3—figure supplement 2). Then, we subtracted the background intensity from each SYP-2 measurement and normalized each dilution by the average intensity of SYP-2 from two undiluted male samples (Figure 3—figure supplement 2). From the normalized dilution curve, we determined that a 40% dilution of the hermaphrodite sample equated to similar amounts of SYP-2 in the undiluted male samples. This 40% dilution was applied to all hermaphrodite samples in Figure 3—figure supplement 2. To quantify the amount of SYP-2 in each sample, we used FIJI to measure the sum intensity of the SYP-2 and loading control alpha-tubulin bands that were both subtracted from a background intensity. Then, the background-subtracted SYP-2 intensity was normalized by the background-subtracted loading control alpha-tubulin intensity to generate a normalized SYP-2 intensity (Figure 3—figure supplement 2).

To assess hermaphrodite fertility, L4 hermaphrodite worms were placed onto new NGM plates and were transferred every 24 hr for a total of 2 d. To assess male fertility, single L4 male worms were mated to single L4 fog-2 obligate females and transferred every 24 hr for a total of 2 d before permanently removing the parental worms. After 3 d of removing the parental worms, each plate was scored for dead eggs. Then, the following day (4 d post removing parental worms) each plate was scored for living hermaphrodite and male progeny (male progeny was not scored in male fertility assays). Additionally, any progeny with mutant Unc or Dpy phenotypes was also scored for five worms in each genotype. Also, 7–10 worms were assayed for fertility for each genotype.

SNP recombination mapping of chromosome II and X was performed as described in Bazan and Hillers, 2011 with minor changes. syp-2(ok307) and syp-3(ok785) were generated in Bristol (N2) backgrounds, and we PCR confirmed that both mutant strains carried all Bristol SNPs for both chromosomes assayed prior to mapping recombination. To generate Bristol/Hawaiian hybrids for mapping recombination, we crossed Bristol, syp-2(ok307) (DLW188), and syp-3(ok785) (DLW190) hermaphrodites to Hawaiian (CB4856) males. Then, 8–10 Bristol/Hawaiian hybrid L4 hermaphrodites were picked off the cross plates for oocyte recombination mapping and 10–15 Bristol/Hawaiian hybrid L4 males were picked off the cross plates for spermatocyte recombination. For oocyte recombination mapping, hybrid L4 hermaphrodites of each genotype were crossed to Bristol males, and male progeny were picked into 96-well plates for lysis and recombination PCR screening. For spermatocyte recombination mapping, hybrid L4 males of each genotype were crossed to Bristol hermaphrodites and male progeny were picked into 96-well plates for lysis and recombination PCR screening.

We used previously designed PCR primers and restriction digests to map six Bristol and Hawaiian SNPs on both chromosomes II and X (Bazan and Hillers, 2011). However, we were unable to get the primers to work for SNP E on chromosome II, so we redesigned new primers for this SNP that worked with the existing restriction digest for SNP identification at this genomic location. All SNP positions, PCR primers, restriction digests, and band sizes of the products for the Bristol or Hawaiian SNPs can be found in Supplementary file 2. The recombination frequency or map length in centiMorgans (cM) was calculated by taking the total number of crossovers identified in each interval divided by the total chromosomes scored multiplied by 100.

Statistical analysis of the FRAP data, SC length, SNP recombination mapping, and fertility assays was done using Prism. Mann–Whitney U tests adjusted for multiple comparisons using the Bonferroni–Dunn method were performed on the FRAP data. Kruskal–Wallis tests were performed on the SC lengths with corrections for multiple comparisons. Chi-squared tests were performed on the entire SNP recombination mapping distribution. Pairwise comparisons between recombination intervals were performed using Fisher’s exact test. Chi-squared and Fisher’s exact tests were performed on the fertility assays. Mann–Whitney U tests adjusted for multiple comparisons using the Bonferroni–Dunn method were performed on the SC intensity, quantification of RAD-51, MSH-5, and COSA-1, transition zone length, pachytene length, end of SYP-1 assembly zone, SUN-1 S8P zone, end of DSB-2 zone, and end of DSB-2 straggler zone using R. Each test used is indicated in the ‘Results’ section next to the reported p value, and all n values are reported in the figure legends.