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Aging and sperm signals alter DNA break formation and repair in the C. elegans germline [1]

['Erik Toraason', 'University Of Oregon', 'Department Of Biology', 'Institute Of Molecular Biology', 'Eugene', 'Oregon', 'United States Of America', 'Victoria L. Adler', 'Diana E. Libuda']

Date: 2022-12 

Female reproductive aging is associated with decreased oocyte quality and fertility. The nematode Caenorhabditis elegans is a powerful system for understanding the biology of aging and exhibits age-related reproductive defects that are analogous to those observed in many mammals, including dysregulation of DNA repair. C. elegans germline function is influenced simultaneously by both reproductive aging and signals triggered by limited supplies of sperm, which are depleted over chronological time. To delineate the causes of DNA repair defects in aged C. elegans germlines, we assessed both DNA double strand break (DSB) induction and repair during meiotic prophase I progression in aged germlines which were depleted of self-sperm, mated, or never exposed to sperm. We find that germline DSB induction is dramatically reduced only in hermaphrodites which have exhausted their endogenous sperm, suggesting that a signal due specifically to sperm depletion downregulates DSB formation. We also find that DSB repair is delayed in aged germlines regardless of whether hermaphrodites had either a reduction in sperm supply or an inability to endogenously produce sperm. These results demonstrate that in contrast to DSB induction, DSB repair defects are a feature of C. elegans reproductive aging independent of sperm presence. Finally, we demonstrate that the E2 ubiquitin-conjugating enzyme variant UEV-2 is required for efficient DSB repair specifically in young germlines, implicating UEV-2 in the regulation of DNA repair during reproductive aging. In summary, our study demonstrates that DNA repair defects are a feature of C. elegans reproductive aging and uncovers parallel mechanisms regulating efficient DSB formation in the germline.

Aging leads to a decline in the quality of the female reproductive cells, known as oocytes. Oocytes subjected to reproductive aging experience an increase in both infertility and aneuploidies that cause miscarriages and birth defects. The nematode Caenorhabditis elegans is a classic model system used to determine the mechanisms of aging. Old C. elegans oocytes accrue many defects which may contribute to their reduced quality, including dysregulation of DNA repair. C. elegans fertility and germline function are also regulated oocyte-independently by sperm-dependent signals. To determine how aging and sperm may independently impact DNA repair in aging C. elegans oocytes, we control oocyte aging and sperm presence independently to evaluate their effects on DNA break formation and repair. We find that running out of sperm reduces the levels of DNA breaks which are produced, but the efficiency of DNA repair declines during aging, independent of sperm effects. We also identify a protein which specifically promotes DNA repair in the oocytes of young animals, suggesting that this protein may regulate DNA repair in the germline during aging. Taken together, our research defines aging-specific and aging-independent mechanisms which regulate the genome integrity of oocytes.

Funding: This work was supported by the National Institutes of Health T32GM007413 and Achievement Rewards for College Scientists (ARCS) Foundation Award to E.T. and National Institute of General Medical Sciences (R35GM128890) to D.E.L. D.E.L. is also a recipient of a March of Dimes Basil O’Connor Starter Scholar award and Searle Scholar Award. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

To define DNA repair defects which are specific to reproductive aging, we assayed levels of DSB formation and repair in the meiotic oocytes of aged mated and unmated C. elegans hermaphrodites, as well as feminized germline mutants that do not produce sperm (fog-2 mutants). We demonstrate that while the depletion of sperm downregulates DSB induction in aged germlines, delayed DSB repair is a shared feature of aging germlines independent of sperm presence. Finally, we identify the E2 ubiquitin-conjugating enzyme variant UEV-2 as a putative regulator of DNA repair during germline aging. Taken together, our work distinguishes DNA repair defects specific to reproductive aging and identifies parallel mechanisms regulating gamete quality in the immortal germline.

Sperm also regulate C. elegans germline physiology and reproduction. C. elegans hermaphrodites produce sperm only during a late stage in larval development [ 20 ]. By the third to fourth day of adulthood, these sperm are depleted, which leads to a premature cessation of reproduction in C. elegans hermaphrodites [ 4 ]. Sperm depletion also induces broad transcriptional remodeling independent of aging processes, resulting in a ‘female-like’ transcriptional profile [ 21 ]. Mating extends the hermaphrodite reproductive span on average to the sixth day of adulthood, after which declining oocyte quality limits fertility [ 4 ]. Mating and even exposure to males, however, also induces deleterious responses in hermaphrodites leading to premature demise [ 22 , 23 ]. It remains unknown how reproductive aging, signaling induced by the presence or depletion of sperm, and mating intersect to regulate meiotic processes in aged C. elegans germlines.

Multiple lines of evidence suggest that preservation of genome integrity is important for the maintenance of oocyte quality during reproductive aging. Human females carrying DNA repair protein variants exhibit extended fertility [ 19 ]. C. elegans mutants with extended reproductive periods are also resilient to exogenous DNA damage and upregulate genes associated with DNA repair [ 4 ]. Further, recent evidence demonstrated that DNA damage and repair are altered in aged C. elegans germlines [ 8 , 10 ]. By the fourth day of adulthood, C. elegans oocyte nuclei exhibit fewer programmed DSBs, delayed loading of recombination proteins, and increased engagement of error-prone repair mechanisms [ 8 , 10 ].

The nematode Caenorhabditis elegans is a key model system for the study of aging biology, including age-related infertility [ 11 ]. C. elegans hermaphrodites (which produce oocytes as adults) undergo reproductive senescence due to declining oocyte quality and incur many of the defects observed in the aging mammalian ovary [ 4 , 5 , 10 , 12 ]. Unlike many mammalian systems, however, which generate oocytes in utero and hold them in dictyate arrest until ovulation, C. elegans hermaphrodites continuously produce new oocytes during their adult reproductive period [ 13 ]. Mitotic proliferation and ovulation of oocytes is dependent upon signals from sperm, which are stored at the end of the germline in a specialized compartment called the ‘spermatheca’ [ 14 , 15 ]. “Obligate female” mutants, which do not produce sperm, therefore exhibit dramatically slowed germline proliferation and progression [ 14 – 18 ]. The C. elegans germline is organized in a spatial temporal gradient wherein oocytes mitotically proliferate at the distal tip and move proximally through the germline as they progress through meiotic prophase I [ 13 ]. Thus, oocyte nuclei at all stages of meiotic prophase I are simultaneously present in the adult germline and enable assessment of meiotic events which are dynamic across prophase, such as the induction and repair of DSBs.

Genome integrity must be preserved during gamete development, as any genetic defects incurred may have detrimental effects on progeny or fertility. Meiosis, the specialized cell division that generates haploid gametes such as eggs and sperm, utilizes specific DNA repair pathways to both ensure accurate chromosome segregation and preserve genomic integrity. During early meiotic prophase I, DNA double-strand breaks (DSBs) are intentionally induced across the genome by the conserved topoisomerase-like protein Spo11 [ 1 , 2 ]. A specific subset of these breaks must be repaired by recombination as crossovers, creating the physical connections between homologous chromosomes required for accurate chromosome segregation. Failure to repair meiotic DSBs accurately and efficiently can contribute to infertility or risk the formation of de novo germline mutations.

All data are fully available without restriction. The spreadsheets delineating the raw numerical data used in this study are included in supporting information. These files include our COSA-1 counts in fog-2 mutants, the brood viability counts in fog-2 mutants, the brood viability and incidence of males counts in wild type and uev-2 mutants, DAPI body counts in wild type and uev-2 mutants, RAD-51 counts in rad-54 mutants, and RAD-51 counts in wild type, uev-2, fog-2, and pie-1p::uev-2 mutants. The gonad linearization algorithm is available on the Libuda Lab GitHub <github.com/libudalab/Gonad-Analysis-Pipeline> and on the Libuda Lab website < libudalab.org >.

All statistics were calculated in R (v4.0.3). Data wrangling was performed using the Tidyverse package (v1.3.0) [ 34 ]. Specific statistical tests used are denoted in the Fig legends and text. P values were adjusted for multiple comparisons when appropriate. If 3 pairwise comparisons were being performed, Bonferroni correction was applied. If >3 pairwise comparisons were performed, Holm-Bonferroni correction was instead applied to reduce the risk of type II statistical errors.

L4 hermaphrodites were isolated on NGM plates seeded with OP50 ~20 hours before DAPI body quantification and were maintained at 20°C. These worms were then picked to a SuperFrost Plus slide (VWR) in M9 buffer and were fixed 3x in 95% EtOH. 20μL of a solution containing 50% VECTASHIELD mounting media (Vector laboratories) and 50% 2μg/mL DAPI in ddH 2 O was applied to each slide, and the slides were mounted with a No 1.5 coverslip (VWR) and sealed with nail polish. DAPI staining bodies were scored the same day as the slides were made on a Leica DM5500B microscope using a 60x objective. Only oocytes in the -1 to -3 positions were included in this analysis.

C. elegans strains were maintained at 20°C during fertility assays. n = 5 L4 wildtype and uev-2 hermaphrodites were isolated and ~20 hours later were singled to individual NGM plates seeded with OP50. ~24 hours after singling, the parent hermaphrodites were discarded. ~24 hours after the parent hermaphrodites were removed, the plates were scored for unhatched eggs. ~48 hours after the hermaphrodites were removed, the hatched F1 progeny were scored as hermaphrodites or males. Brood viability was calculated as (hatched progeny) / (hatched progeny + dead eggs). Incidence of males was calculated as (male progeny) / (male progeny + hermaphrodite progeny).

During the course of the brood viability assays, some mated fog-2 females exhibited matricidal hatching. This phenotype was more pronounced in aged worms, consistent with previous work which showed that matricidal hatching is exacerbated with maternal age [ 33 ]. Only eggs which were successfully ovulated were scored in the assay.

C. elegans strains were maintained at 20°C during fertility assays. Feminized fog-2 mutants were synchronized in age by placing gravid mated CB4108 females onto an NGM plate seeded with OP50 for one hour. Hatched female progeny were isolated as L4s from these plates and were kept in isolation from males to prevent mating. At adult day 1, 2, 3, 4, or 5, these isolated fog-2 females were then placed on individual plates with n = 2 young adult N2 males each. Mated fog-2 females were then subsequently transferred to new NGM plates seeded with OP50 with young adult N2 males at either 6hr, 12hr, 18hr, 24hr, and 48hr after the first mating, or at 24hr and 48hr after the first mating. 72hr after the first mating, adult females were discarded. Plates were scored ~24hr after the parent female was removed for hatched progeny, dead eggs, and unfertilized oocytes. Brood viability was calculated as (hatched progeny) / (hatched progeny + dead eggs). Fertility assays were replicated twice with n = 5 females of each age group assayed per replicate.

GFP::COSA-1 foci were quantified manually from late pachytene nuclei from 3D z-stacks using Fiji. Nuclei that were completely contained within the image stack were quantified in the last few rows of the late pachytene region in which all nuclei displayed bright GFP::COSA-1 foci (last ~3–6 rows of late pachytene in old fog-2 germlines, last ~6–8 rows of late pachytene in young fog-2 germlines).

Germline DSB-2 staining was analyzed in Imaris using germlines stitched in Fiji as described above. The length of the germline was defined using the Imaris Measurements tool. Specific points were placed at the beginning of the transition zone, end of the transition zone, beginning of the DSB-2 zone (defined as the row of nuclei in which most nuclei had DSB-2 staining), the end of the DSB-2 zone, the final position of one or more nuclei which had DSB-2 staining, and the end of pachytene. The measured distances were then normalized to pachytene, where the beginning of pachytene is position 0 and the end of pachytene is position 1.

Nuclei positions were transformed from 3D coordinates to a linear order using the Gonad Linearization Algorithm implemented in R [ 32 ]. Gonad length in germlines which lacked a defined transition zone (e.g. fog-2 mutants, S3 Fig ) was normalized to the distance from the premeiotic tip to the end of pachytene, where the premeiotic tip begins at position 0 and the end of pachytene is at position 1. In all other germlines, the gonad length was normalized to pachytene, where the beginning of pachytene is position 0 and the end of pachytene is position 1. The accumulation of RAD-51 foci in rad-54 mutant germlines ( S1 Fig ) was scored manually in 3D germlines using Imaris. Nuclei were rotated in 3D to ensure the identification of all RAD-51 foci. A RAD-51 focus was counted if the observed signal: 1) was associated with DAPI signal; and, 2) was brighter and larger than internuclear background staining, if any was present.

Images were analyzed as described in [ 32 ]. Image quantification was performed using Imaris software (Bitplane). Individual nuclei within stitched gonads were identified as Surface objects (Smooth 0.1–0.15, Background 3–4, Seed Point Diameter 3–4) based on DAPI staining intensity. Manual thresholding of specific values were used per gonad to generate surfaces which represented the nuclei observed. Defined surfaces were then split to designate individual nuclei using the Imaris Surfaces Split module. Nuclei which were partially imaged or overlapped with other nuclei were eliminated from the analysis. We previously demonstrated that a minority nuclei (~23%) in the total population must be eliminated in this manner and that the inclusion of multiple germlines in a dataset enables thorough sampling of nuclei across the course of prophase I for analysis [ 32 ]. RAD-51 foci were defined as Spot objects (Estimated XY Diameter 0.1, Model PSF-elongation 1.37, Background Subtraction enabled). To determine the number of RAD-51 foci per nucleus, we either utilized the “Find Spots Close to Surface” MATLAB module (Threshold value 0.1) or utilized the “Closest Distance to Surface” statistic calculated by Imaris to find the number of Spots ≤0.1μm distant from nuclei. The length of each germline was defined using the Imaris Measurements tool. Measurement points were specifically placed at the beginning of the premeiotic tip and the end of pachytene. For germlines which had a defined transition zone by DAPI morphology, points were also placed at the start and end of the transition zone.

Immunofluorescence images were acquired at 512x512 or 1024x1024 pixel dimensions on an Applied Precision DeltaVision microscope with a 60x lens and a 1.5x optivar. All images were acquired in 3 dimensions with Z-stacks at 0.2μm intervals. In a minority of aged unirradiated germlines, we noted that most nuclei in mid-late pachytene exhibited high levels of RAD-51 and condensed DNA morphology characteristic of apoptosis. These aberrant gonads were not included in our analyses. Images were deconvolved with Applied Precision softWoRx software and individual image tiles were stitched into a single image for analysis using the Grid/Collection Stitching module in Fiji with regression threshold 0.7 [ 28 ] or using Imaris Stitcher software (Bitplane).

Immunofluorescence samples were prepared as in [ 27 ]. Nematodes were dissected in 1x Egg Buffer (118 mM NaCl, 48 mM KCl 2 , 2 mM CaCl 2 , 2 mM MgCl 2 , 25 mM HEPES pH7.4, 0.1% Tween20) and were fixed in 1x Egg Buffer with 1% paraformaldehyde for 5 min on a SuperFrost Plus slide (VWR). Slides were then flash frozen in liquid nitrogen and the cover slip was removed before the slides were placed in ice cold methanol for 1 minute. Slides were washed in 1xPBST (1x PBS, 0.1% Tween20) 3x for 10 minutes before they were placed in Block (1xPBST with 0.7% bovine serum albumin) for a minimum of one hour. 50μL of primary antibody diluted in PBST (see below for specific antibody dilutions) was then placed on each slide and samples were incubated for 16-18hrs in a dark humidifying chamber with parafilm coverslips. Slides were then washed 3x in 1xPBST for 10 minutes. 50μL of secondary antibody diluted 1:200 in PBST were then added to each sample and slides were incubated for 2hr in a dark humidifying chamber with parafilm coverslips. Slides were washed 3x in 1xPBST for 10 minutes, and then 50μL of 2μg/mL DAPI was applied to each slide. Samples were incubated in a dark humidifying chamber with parafilm coverslips for 5 minutes, then were washed 1x in PBST for 5 minutes. Slides were mounted in VECTASHIELD mounting media (Vector laboratories) with a No 1.5 coverslip (VWR) and sealed with nail polish. All slides were maintained at 4°C until imaging.

The candidate insertion identified among progeny from the above injected hermaphrodites contained an undesired additional 43bp of sequence between the 5’ 3xFLAG tag of the edited wbmIs60 landing site and the start codon of the uev-2 coding sequence. The strain carrying this insertion allele was backcrossed 3x to N2 and was CRISPR/Cas9 edited again to remove the undesired 5’ sequence. Worms were injected with 0.25 μg/μL Cas9 (IDT), 100ng/μL tracrRNA (IDT), 28ng/μL gRNA DLR022 (5’-GAUCUUUAUAAUCACCGUCA-3’), 28ng/μL gRNA DLR023 (5’-UGUUGCUACGUCUUCGCAUC-3’), 25ng/μL ssODN donor DLO1173 (5’-AACAATTAAAAATCAAATTTTCTTTTCCAGATGCGGAGGCGAAGTAATAGACAATATGTTGATCTCTCATATTTTCGCGAAAC-3’), and 40ng/μL purified pRF4 plasmid. Successful removal of the 3xFLAG sequence and undesired 43bp inserted sequence were confirmed by PCR and Sanger sequencing (Sequetech).

For experiments in which fog-2 worms were transiently mated once in order to assess the effects of sperm depletion on feminized germlines, fog-2 females were transferred to a new NGM plate seeded with OP50 1 day post-L4 and young adult N2 males were also added at a ratio of ~2–3 males per female. The animals were allowed to mate for 6–8 hours, and then the females were transferred to a new NGM plate seeded with OP50 and the males were discarded. The fog-2 mated females were then transferred to new NGM plates seeded with OP50 every ~48-60hrs to prevent their progeny from overconsuming the available food.

In experiments with aged animals, L4 hermaphrodites were isolated and maintained on NGM plates seeded with OP50 in the absence of males. Strains which produced self progeny were transferred to new NGM plates seeded with OP50 2 days post-L4 to prevent overconsumption of food from F1 progeny. At this transfer, if the experimental cohort was to be mated in order to prevent sperm depletion, young adult male N2 worms were additionally added to these plates at a ratio of ~1.5–2 males per hermaphrodite. Mated hermaphrodites were again transferred to new NGM plates with OP50 ~20–26 hours after males were added and the male animals were discarded.

Strain DLW135 was generated by backcrossing VC30168 to N2 10 times. VC30168 was created by the Million Mutations Project [ 24 ] and carried many mutations in addition to the uev-2(gk960600) allele of interest. Following backcrossing, mutations on Chromosomes I, II, IV, V, and X were assumed to have been eliminated. To determine the success of backcrossing on removing undesired mutations in cis with uev-2 on Chromosome III, we assessed the presence of known flanking mutations to uev-2(gk960600gk429008gk429009). Presence of the upstream most proximal genic mutation to uev-2, pho-9(gk429005), was assessed via PCR amplification using OneTaq 2x Master Mix (forward primer DLO1142 5’-ACCCATTTCCCATTCAATCA-3’ reverse primer DLO1143 5’-TTGTAATCTGCCCCAAAAGG-3’) and subsequent HpaII restriction digest (New England Biolabs). DLW135 carried a wild type allele of pho-9, indicating that the region of Chromosome III upstream of uev-2 was successfully reverted to wild-type sequence by recombination. However, the closely linked (~1 cM) downstream allele rgr-1(gk429013) was preserved in DLW135, as confirmed by Sanger sequencing (Sequetech) of a PCR amplified region of the rgr-1 locus using OneTaq 2x Master Mix (forward primer DLO1140 5’-TGGAATGGGACTTCCTCTTG-3’ reverse primer DLO1141 5’-TTTCCAAAAGCCAGGACATC-3’) isolated using a GeneJET PCR Purification kit (ThermoFisher). The rgr-1(gk429013) allele is a single base pair substitution resulting in a S360N missense mutation. RGR-1 is a Mediator complex subunit involved in transcriptional activation that is required for embryonic viability [ 25 ]. S360N does not disrupt a predicted functional domain, and mutants carrying rgr-1(gk429013) survive embryogenesis and are fertile, indicating that this mutation does not severely disrupt function of the RGR-1 protein. As RGR-1 is not known to play a role in DNA damage repair, and uev-2 has been previously demonstrated to modulate germline sensitivity to DNA damage [ 4 ], the phenotypes we observed using DLW135 in this manuscript are not best explained by the presence of the rgr-1(gk429103) mutation. For simplicity, DLW135 mutants are referred to as ‘uev-2 mutants’ in the text of this manuscript.

Results

UEV-2 is required for ‘youthful’ germline DSB repair To identify proteins which may regulate DSB repair in the aging C. elegans germline, we looked to candidate genes upregulated in long-reproductive sma-2 mutant oocytes, which exhibit DNA damage resilience in addition to delayed reproductive senescence [4]. The sma-2 DNA damage resilience phenotype requires upregulation of the E2 ubiquitin-conjugating enzyme variant UEV-2, suggesting that this protein may promote efficient germline DNA repair [4]. UEV proteins lack a catalytic cysteine residue conserved in E2 enzymes [40] but have been shown to form heterodimeric complexes with other E2 enzymes to influence their function, implying regulatory roles for this protein class [41,42]. To assess the influence of UEV-2 on DSB repair during germline aging, we utilized a strain carrying the putative null allele uev-2(gk960600), which ablates the translation initiation site and second exon boundary of the gene (S7 Fig; see Methods). We determined that uev-2 is not required for successful meiosis, as uev-2 mutants exhibited normal progeny viability (wildtype 99.5% viable progeny n = 954, uev-2 99.3% viable progeny n = 914, Chi Square test p = 0.690), no evidence of elevated X chromosome nondisjunction (wildtype 0.1% male progeny n = 950, uev-2 0.1% male progeny n = 909, Chi Square test p = 0.975), and normal chiasma formation (diakinesis nuclei with 6 DAPI bodies wild type 100% n = 55, uev-2 100% n = 60, Fisher’s exact test p = 1.000). With the uev-2 mutant strain, we examined the number of RAD-51 foci in germline nuclei derived from young (1 day post-L4) or aged (4 days post-L4) animals (Fig 5A). Aged uev-2 mutants were also mated to avoid the DSB induction defects associated with sperm depletion (Fig 5A). If UEV-2 functions to promote efficient DSB repair in young gonads but becomes dysregulated or loses function during aging, then we would expect uev-2 mutants to exhibit defects in DSB repair in young germlines but minimal additional defects in aged germlines. Indeed, when we compared the levels of RAD-51 observed in young and aged mated wildtype and uev-2 germlines, we observed DSB repair defects that did not accumulate with age. In early pachytene, young and aged uev-2 mutants exhibited similar levels of RAD-51 to young wildtype germlines (Fig 5B and 5C, Bin 2 young wildtype 3.5±2.8, young uev-2 3.0±2.6, aged uev-2 4.0±3.2 Mann-Whitney U test p>0.05; Bin 3 young wildtype 6.9±3.9, young uev-2 6.6±4.1, aged uev-2 7.0±4.7 Mann-Whitney U test p>0.05), indicating that UEV-2 is not required for meiotic DSB induction nor RAD-51 loading. In contrast, at mid pachytene, young uev-2 mutant germlines maintained elevated RAD-51 foci relative to young wildtype germlines (Fig 5B and 5C, Bin 4 young wildtype 5.0±4.1, young uev-2 5.3±3.3, aged uev-2 6.3±5.9 Mann-Whitney U test p<0.05; Bin 5 young wildtype 2.4±2.0, young uev-2 3.8±4.0, aged uev-2 5.0±7.3 Mann-Whitney U test p<0.05). The specific levels of DSBs at mid pachytene in young uev-2 mutants were also indistinguishable from aged wildtype germlines (Fig 5B and 5C, Bin 4 young uev-2 5.3±3.3, aged wildtype 5.8±4.5 Mann-Whitney U test p>0.05; Bin 5 young uev-2 3.8±4.0, aged wildtype 4.2±5.2 Mann-Whitney U test p>0.05). These results at mid pachytene indicate that DSB repair is delayed in young uev-2 mutants to an extent which recapitulates the effect we observe during wildtype aging. Aged uev-2 germline RAD-51 levels at mid pachytene were statistically indistinguishable from either young or aged mated wildtype germlines (Fig 5B and 5C, Bin 4 young wildtype 5.0±4.1, aged wildtype 5.8±4.5, aged uev-2 6.3±5.9 Mann-Whitney U test p>0.05; Bin 5 young wildtype 2.4±2.0, aged wildtype 4.2±5.2, aged uev-2 5.0±7.3 Mann-Whitney U test p>0.05), suggesting that the uev-2 mutation does not grossly exacerbate DSB repair defects with age. PPT PowerPoint slide

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TIFF original image Download: Fig 5. UEV-2 is required for ‘youthful’ DNA repair. A) Schemes used to isolate young (1 day post-L4) and aged (4 days post L4) uev-2 mutant worms for experiments. Days count ~18–24 hour periods after hermaphrodites were isolated as L4 larvae and are separated by alternating grey shaded boxes. B) Representative images of RAD-51 foci in meiotic nuclei of young uev-2, aged mated uev-2, young wildtype, and aged mated wildtype germlines. Scale bars represent 5μm. C) RAD-51 foci per nucleus in oocytes. Line plots represent the mean RAD-51 foci per nucleus along the length of the germline in a sliding window encompassing 0.1 units of normalized germline distance with a step size of 0.01 germline distance units. Mean RAD-51 foci were calculated from nuclei analyzed in n = 9 total germlines derived from ≥3 experimental replicates within each age and genotype group. Shaded areas around each line represent ± SEM. Total nuclei analyzed (Bins 1/2/3/4/5/6/7) Young Wildtype: 185/117/146/107/117/97/83; Aged Wildtype Mated: 234/205/192/173/154/177/96; Young uev-2: 186/135/134/113/112/132/95; Aged uev-2 Mated: 129/161/155/158/167/142/120. Germlines distances were normalized to the start (0) and end (1) of pachytene based on DAPI morphology (see Methods). For analysis, the germline was divided into 7 bins encompassing the transition zone (Bin 1), early pachytene (Bins 2–3), mid pachytene (Bins 4–5), and late pachytene (Bins 6–7). The germline positions at which each bin start and end are marked on the X axis as vertical grey lines. Heat maps below each bin display the p values of pairwise comparisons of RAD-51 foci per nucleus counts within that bin. P values were calculated using Mann-Whitney U tests with Holm-Bonferroni correction for multiple comparisons. Blue tiles indicate significant differences (adjusted p value <0.05) and grey tiles indicate nonsignificant effects (adjusted p value >0.05). Young and aged mated wild type data is shared with Figs 1 and 6. Numerical data associated with this figure is presented in S1 Data. https://doi.org/10.1371/journal.pgen.1010282.g005 In late pachytene, the specific rates of DSB resolution diverged slightly between young and aged uev-2 and wildtype germlines (Fig 5B and 5C, Bin 6 young wildtype 1.6±2.2, aged wildtype 1.9±4.3, young uev-2 1.9±1.9, aged uev-2 2.3±6 Mann-Whitney U test p<0.05; Bin 7 young wildtype 0.9±1.4, aged wildtype 0.5±1.9, young uev-2 0.7±1.0, aged uev-2 0.7±3.2 Mann-Whitney U test p<0.05), suggesting that UEV-2-independent and age-specific effects may contribute to DSB resolution at this meiotic stage. Taken together, our results indicate that loss of uev-2 in young germlines is sufficient to phenocopy the mid-pachytene patterns of DSB repair observed in an aged wildtype context. This observation supports a model in which UEV-2 functions in young germlines specifically to promote efficient DSB repair.

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010282

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