CYD-1 maintains oocyte quality and reproductive fidelity in wild-type worms

Since cyd-1 KD renders daf-2 worms sterile, we first asked how CYD-1 may influence WT germline where insulin signaling may be considered normal. The C. elegans hermaphrodite gonad has two U-shaped tubular arms where the germ line stem cell (GSC) pool resides near the distal end and divides mitotically. On moving away from the distal end, the germ cells enter meiotic prophase. The sperm formation takes place at L4 stage (sperms are stored in the spermatheca), after which a switch from sperm-to-oocyte fate takes place. Subsequently, germ cells are committed to developing into oocytes [54–56]. The quality of oocytes is ensured by programmed cell death of defective germ cells at the end of pachytene region. A common uterus that carries the fertilized eggs (until they are laid) connects both the gonadal arms [57] (Fig 1A; only one gonad arm is shown).

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Fig 1. CYD-1 ensures optimal oocyte quality in wild-type.

(A) A diagrammatic representation of the right arm of C. elegans gonad. Schematics in Fig 1A were created using BioRender.com. (B,C) DIC images showing oocyte morphology of WT (day-3 adult) grown on control or cyd-1 RNAi. White arrows mark normal oocytes while yellow arrows mark poor-quality oocytes that have cavities or are misshapen (B). Oocyte quality scores (based on morphology) (C). The quality was categorized as normal, or mild or severe based on its morphology (cavities, shape, organization) as shown in S1G Fig. Combined data from three biological replicates (n ≥ 85) is plotted. Chi-square analysis was used to compare between groups. (D) Percentage of WT eggs (day-3 adult) that hatched on control or cyd-1 RNAi. Average of four biological replicates (n ≥ 50 for each replicate). Unpaired t-test with Welch’s correction. (E,F) Oocyte morphology in unmated or mated day-3 adult rrf-3(pk1426) worms grown on control or cyd-1 RNAi (E). Oocyte quality scores (based on morphology as shown in S1G Fig) (F). Combined data from three biological repeats (n ≥ 48) is plotted. Chi-square analysis was used to compare between groups. (G) The reproductive span of WT (selfed and mated worms) on control or cyd-1 RNAi. Data from three biological replicates is shown. P-value for mated worms on control versus cyd-1 RNAi at day-2 and day-3 is shown. Unpaired t-test with Welch’s correction. (H) The total number of hatched progenies in WT (selfed and mated worms) grown on control or cyd-1 RNAi. Average of four biological repeats. Unpaired t-test with Welch’s correction. (I,J) Representative DAPI-stained gonads of WT (day-3 adult) worms grown on control or cyd-1 RNAi. White arrows mark normal eggs while yellow arrows mark endomitotic oocytes (emos) (I). Quantification of percent endomitotic oocytes (J). Average of three biological replicates (n ≥ 25 for each experiment). Unpaired t-test with Welch’s correction. (K,L) Representative fluorescent images of unc-119::gfp worms (day-3 adult) grown on control or cyd-1 RNAi. White arrows mark normal eggs that do not show GFP expression, while yellow arrows mark endomitotic oocytes that express GFP (K). Quantification of unc-119::GFP expression positive endomitotic oocytes (L). Average of four biological replicates (n ≥ 25 for each experiment). Unpaired t-test with Welch’s correction. Scale bars: 20 μm. Error bars are s.d. Experiments were performed at 20°C. Source data are provided in S1 Table.

https://doi.org/10.1371/journal.pgen.1011453.g001

WT worms were grown from larval stage 1 (L1) onwards on control or cyd-1 RNAi, DAPI stained at day-1 and imaged to observe the germ cell number (S1C and S1D Fig). Interestingly, we did not observe changes in the WT mitotic or transition zone germ cell number but found a significant reduction in the pachytene cell numbers (S1D Fig). We questioned whether a decrease in the pachytene germ cell number could be due to an increase in germ cell apoptosis. Indeed, we observed an increase in apoptotic germ cell numbers marked as CED-1::GFP-expressing foci [CED-1 is a transmembrane protein on the surface of sheath cells that engulfs the apoptotic germ cells, thereby marking the dying germ cells [58]] in the gonad of cyd-1 RNAi worms (S1E and S1F Fig). Normally, as the pachytene cells transition to diplotene, most of the defective/damaged germ cells are culled in the turn region of the gonad to ensure that only healthy oocytes mature [59]. The increase in apoptosis upon cyd-1 KD suggested an increase in damaged germ cells. We therefore evaluated the oocyte quality as a readout of germ cell quality. Firstly, the oocytes were examined morphologically by using differential interference contrast (DIC) microscopy (magnification of 400 X) on day-3 of adulthood. The oocytes were assigned a score (normal, mild or severely defective morphology) based on the presence of cavities, abnormal shape/size, and/or organization (S1G Fig). On day-3 of adulthood, WT worms grown on control RNAi showed the proper arrangement of stacked oocytes without any cavities (Fig 1B and 1C). However, upon cyd-1 KD, the oocytes became significantly more misshapen, and the gonads were disorganized, with cavities between oocytes (Fig 1B and 1C). Further, we asked if the observed poor oocyte morphology also translates into poor quality of these oocytes. Poor quality oocytes often lead to a reduction in the hatching efficiency of the eggs. In the case of the wild-type worms, the percentage of hatched eggs is known to decrease with age as the oocyte quality declines [59]. Noteworthily, we found a reduction in the percentage of eggs that hatched upon cyd-1 KD (Fig 1D). These observations indicate that CYD-1 is required in wild-type worms to maintain the quality of germ cells and oocytes.

The C. elegans self-fertilizing hermaphrodite contains both sperms and oocytes. To assess if the poor oocyte quality upon cyd-1 KD is dependent on sperm/sperm signals, we mated the cyd-1 KD hermaphrodites to males grown on control RNAi. Interestingly, both selfed and mated hermaphrodites displayed poor oocyte morphology upon cyd-1 depletion, implying that the poor oocyte quality is sperm-independent (Fig 1E and 1F).

The reproductive span (the number of days the worms can lay live eggs) of the cyd-1 KD worms was not reduced (in both selfed as well as mated worms) compared to control RNAi-grown worms (Fig 1G) although an overall reduction in the brood size was observed (Fig 1H). We also observed a greater than 3-fold increase in the incidences of endomitotic oocytes (emos) at day-3 of adulthood in cyd-1 KD WT worms (Fig 1I and 1J). Such emos are often found in the uterus of aged worms, in the post-reproductive phase [60]. We asked if these emos have lost their germ cell fate and acquired somatic fate, similar to ovarian teratomas. To address this, we examined the expression of unc-119::GFP, which is expressed pan-neuronally but not in oocytes [61]. Depletion of cyd-1 resulted in unc-119p::GFP expressing emos (Fig 1K and 1L), thereby indicating trans-differentiation of these germ cells. We also found the presence of these densely DAPI-stained emos upon cyd-1 KD in the feminized worms, indicating these are sperm-independent in origin (S1H Fig). Interestingly, cyd-1 KD post late-L4 larval stage does not lead to poor oocyte quality (S1I Fig) or endomitotic oocytes (S1J Fig), indicating a role of CYD-1 during development, that may later affect the oocyte quality in adulthood. Results here demonstrate that CYD-1 is required to maintain optimal oocyte quality in wild-type worms.

CYD-1 is required in the somatic gonad (uterus) for oocyte quality maintenance

Previously, the IIS and the TGF-β signaling in the somatic tissues (hypodermis, muscle and intestine) have been shown to regulate reproductive aging [18]. The somatic tissues crosstalk with the germline, not only to regulate aging [62–64] but also to influence germline development [65] and the oocyte quality [18]. Keeping this in mind, we wanted to test the tissue-specific requirement of CYD-1 in the regulation of oocyte quality. For this, we employed a tissue-specific RNAi system where in the rde-1 (gene encoding an Argonaute protein) mutant background, a tissue-specific promoter is used to drive RDE-1 expression, providing an elegant system to selectively knock-down a gene in that specific tissue alone [66]. Surprisingly, in contrast to the whole-body RNAi of cyd-1, KD of cyd-1 in the germ cells [using a transgenic line where sun-1 promoter drives the expression of RDE-1 only in the germ cells of the rde-1 mutant [67]] alone was unable to deteriorate the oocyte quality (Fig 2A and 2B), increase the percentage of endomitotic oocytes (Fig 2C and 2D) or affect the brood size (S2A Fig). Interestingly, somatic tissue-specific KD using a ppw-1 [a PAZ/PIWI protein required for efficient germline RNAi [68]] mutant resulted in poor oocyte quality (Fig 2A and 2B) with increased emos (Fig 2C and 2D) and a concomitant reduction in brood size (S2B Fig). The germline and somatic RNAi specificity of these mutants was validated by egg-5 RNAi [69] (which leads to the laying of dead eggs when RNAi is functional in the germline) and dpy-7 RNAi [70] (which leads to dumpy phenotype when RNAi is functional in the soma) (S2C Fig).

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Fig 2. CYD-1 regulates oocyte quality cell non-autonomously from the somatic gonad (uterus).

(A,B) DIC images showing oocyte morphology of WT, rde-1(mkc36);sun-1p::rde-1 (germline-specific RNAi) and ppw-1(pk1425) (soma-specific RNAi) (day-3 adult) grown on control or cyd-1 RNAi. White arrows- normal oocytes, yellow arrows- oocytes with abnormalities (A). Quantification of oocyte quality score (B). The quality of oocytes was categorized as normal, mild or severe based on their morphology (cavities, shape, organization) as shown in S1G Fig. Combined data from three biological replicates (n ≥ 29) is plotted. Chi-square analysis was used to compare between groups. (C,D) DIC images showing the presence of eggs or endomitotic oocytes (emos) in the uterus of WT, rde-1(mkc36);sun-1p::rde-1 (germline-specific RNAi) and ppw-1(pk1425) (soma-specific RNAi) (day-3 adult) grown on control or cyd-1 RNAi. White arrows mark normal eggs while yellow arrows mark endomitotic oocytes (emos) (C). Quantification for endomitotic oocytes (D). Combined data from three biological replicates (n ≥ 25) is plotted. Chi-square analysis was used to compare between groups. (E,F) DIC images showing oocyte morphology of rrf-3(pk1426) (whole body RNAi) or rrf-3(pk1426);rde-1(ne219);fos-1ap::rde-1(genomic) (uterine tissue-specific RNAi) worms (day-3 adult) grown on control or cyd-1 RNAi. White arrows mark normal oocytes while yellow arrows mark oocytes with severe abnormalities (E). Oocyte quality scores (based on morphology as shown in S1G Fig) (F). Combined data from three (whole body KD) and four (uterine specific KD) biological replicates (n ≥ 58) are plotted. Chi-square analysis was used to compare between groups. Scale bars: 20 μm. Experiments were performed at 20°C. Source data are provided in S1 Table.

https://doi.org/10.1371/journal.pgen.1011453.g002

To further investigate the somatic tissue where CYD-1 may function to influence the germline quality, we used the tissue-specific strains in the rrf-3 mutant background. Intestine or muscle-specific KD of cyd-1 resulted in no changes in the oocyte morphology (S2D and S2E Fig). Surprisingly, uterine-specific KD of cyd-1 [using a transgenic line where fos1a promoter drives the expression of RDE-1 only in the uterine cells of the rde-1 mutant [71]] led to compromised oocyte quality (Fig 2E and 2F) with an increase in the emos (S2F and S2G Fig) and a reduction in the total progeny number (S2H Fig). The strain was confirmed to have RNAi machinery specifically in the uterine cells as it showed a protruded vulva phenotype upon egl-43 KD [EGL-43 is essential for cell fate specification and development of vulva and uterus [72]] and no dead eggs or dumpy phenotype on egg-5 and dpy-7 RNAi, respectively (S2I Fig). Thus, CYD-1 is important in the somatic gonad (uterine tissue) for oocyte quality maintenance in the WT germline. This exemplifies a soma-to-germline crosstalk that ensures the quality of the germline.

Depletion of cyd-1 leads to a DAF-16a isoform-dependent pachytene arrest in daf-2

While the RNAi-mediated KD of cyd-1 accelerates the decline in oocyte quality in WT, RNAi depletion of cyd-1 under low insulin signaling (daf-2 worms) leads to complete sterility, as mentioned earlier (Figs 3A–3C and S1A). Interestingly, similar to stress resistance [11–14] and longevity [73,74], this germline arrest also was mediated by the downstream FOXO TF DAF-16, such that the fertility is rescued in the daf-16;daf-2;cyd-1 RNAi worms (Fig 3A–3C). To determine the effect of cyd-1 KD on germline development of daf-2, the gonads of day-1 adults were stained with DAPI. Interestingly, the cyd-1 KD did not lead to a reduction in the mitotic zone germ cell number. Importantly, however, no oocytes were formed upon cyd-1 KD in daf-2 and the oocyte formation was restored in the daf-16;daf-2 double mutants (Fig 3D and 3E). Upon quantification of the germ cell numbers, we found that cyd-1 KD led to small but significant changes in the transition zone germ cells, but drastically reduced the pachytene germ cell number in daf-2; this was restored in daf-16;daf-2 (Fig 3D and 3E). We measured cyd-1 mRNA levels and confirmed similar efficiency of cyd-1 KD in daf-2 and daf-16;daf-2 worms, implying that the germline arrest is not due to a differential cyd-1 RNAi efficiency (S3A Fig). We further speculated if KD of cyd-1 in daf-2 worms resulted in critically low levels of CYD-1 levels below a presumptive threshold, thereby causing sterility. To rule out this, we depleted cyd-1 (by RNAi) in a balanced cyd-1 mutant, cyd-1(he112)/+ that possesses a truncated (C-terminal) CYD-1 protein due to the presence of a nonsense mutation [75]. Interestingly, we observed deterioration of oocyte quality (S3B and S3C Fig) and increased formation of endomitotic oocytes (S3D and S3E Fig), but no sterility, implying the arrest is specific to daf-2. Thus, upon depletion of cyd-1 in daf-2, activated DAF-16 plays a decisive role in arresting the germ cells at the pachytene stage of meiosis and in preventing oocyte formation.

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Fig 3. Depletion of cyd-1 leads to FOXO/DAF-16-dependent germline arrest under low insulin signaling.

(A,B) Representative images showing that cyd-1 RNAi results in sterility in daf-2(e1370) worms that are rescued in daf-16(mgdf50);daf-2(e1370). White arrowheads show eggs while yellow arrowheads show gonads with no eggs (sterile worms) (A). The percentage of fertile worms (B). Average of three biological replicates (n ≥ 30 for each experiment). Two-way ANOVA-Tukey’s multiple comparisons test. (C) The total number of hatched progenies in daf-2(e1370) and daf-16(mgdf50);daf-2(e1370) worms grown on control or cyd-1 RNAi. Average of three biological repeats. Two-way ANOVA-Tukey’s multiple comparisons test. (D,E) Representative fluorescent images of DAPI-stained gonads of daf-2(e1370) and daf-16(mgdf50);daf-2(e1370) (day-1 adult) worms grown on control or cyd-1 RNAi. Oocytes are boxed for clarity. White arrows point towards oocytes while yellow shows the absence of oocytes. Sp denotes sperms (D). Quantification of DAPI-stained germ cell nuclei. n = 17 (daf-2); n = 16 (daf-16;daf-2). (E) Each point represents the number of mitotic (MT), transition (TS) or pachytene zone (PZ) cells. Unpaired t-test with Welch’s correction. (F,G) Isoform requirement of DAF-16 to mediate germline arrest in daf-2(e1370). Representative fluorescent images of DAPI-stained germ line of daf-16(mgdf50);daf-2(e1370);daf-16a(+) worms grown on control or cyd-1 RNAi. Oocytes are boxed for clarity. White arrows point towards oocytes while yellow shows the absence of oocytes. Sp denotes sperms (F). Percentage of the fertile worms (G). The cyd-1 was knocked down in daf-2(e1370), daf-16(mgdf50);daf-2(e1370) as well as in strains where different DAF-16 isoforms were rescued in daf-16(mgdf50);daf-2(e1370). Average of three biological replicates (n ≥ 30 for each replicate). Two-way ANOVA-Tukey’s multiple comparisons test. Scale bars: 20 μm. Error bars are s.d. Experiments were performed at 20°C. Source data are provided in S1 Table.

https://doi.org/10.1371/journal.pgen.1011453.g003

Since the RNAi depletion of cyd-1 phenocopies the sterility (pachytene arrest) due to cdk-12 KD in daf-2, we asked if similar to cdk-12 depletion, cyd-1 KD too weakened the DNA damage response (DDR). For this, we first checked for the mRNA levels of genes involved in the DDR. Wild-type late-L4 staged worms grown on control or cyd-1 RNAi showed no down-regulation of DDR genes on cyd-1 KD (S3F Fig). Additionally, both control and cyd-1 RNAi-fed WT worms showed a similar percentage of egg hatching post gamma exposure at late-L4 stage (S3G Fig). This hinted towards the existence of different mechanisms underlying the sterility upon cyd-1 and cdk-12 KD in daf-2.

One of the causes of sterility in daf-2;cyd-1 RNAi worms could be elevated levels of germ cell apoptosis. For measuring apoptosis, we employed the CED-1::GFP transgenic strain as described above. Elevated levels of apoptosis were observed for both daf-2 and daf-16;daf-2 worms grown on cyd-1 RNAi (S3H and S3I Fig), implying that the sterility in daf-2 upon cyd-1 KD may not be solely due to an increase in apoptosis. We wondered if the oocytes that bypass the pachytene arrest in daf-16;daf-2;cyd-1 RNAi worms would also exhibit poor quality. Indeed, we observed that cyd-1 KD in daf-16;daf-2 led to severe defects in oocyte quality (S4A and S4B Fig) as well as an increased occurrence of emo in the uterus of these animals (S4C and S4D Fig).

Different isoforms of DAF-16 exist due to alternative splicing events as well as alternate promoter usage. The different isoforms have distinct tissue-specific expression profiles and the long life span of daf-2 depends on each isoform to a different extent [76]. We next asked which isoform of DAF-16 was necessary for inducing arrest in daf-2 following KD of cyd-1. To address this, we used transgenic strains, wherein, different isoforms of DAF-16 were rescued in the daf-16;daf-2 double mutants [76]. Interestingly, we found that the cyd-1 KD in the daf-16;daf-2;daf-16a(+) strain led to sterility comparable to the daf-2 (Fig 3F and 3G), signifying a predominant role of DAF-16a isoform in mediating this germline pachytene arrest.

Additionally, the sterility due to cyd-1 KD in daf-2 involves the canonical IIS pathway components as a similar arrest at the pachytene stage of meiosis was observed upon cyd-1 KD in age-1(hx546) [mutant in mammalian PI3K ortholog [77, 78]] and pdk-1(sa680) [mutant in mammalian PDK ortholog [79]] (S4E and S4F Fig). Reducing the levels of the canonical binding partner of CYD-1, CDK-4 [80] also led to sterility in daf-2;rrf-3 (hypersensitive RNAi background in daf-2) (S4G Fig), hinting towards a CDK-4-dependent function of CYD-1 in oogenesis.

Depletion of cyd-1 in the somatic gonad of daf-2 results in germline arrest at the pachytene stage

Since we found a role for somatic CYD-1 in regulating WT germline quality assurance, we asked if somatic cyd-1 KD would lead to germline arrest in daf-2. We first checked if the germline arrest was mediated cell-autonomously by cyd-1 depletion in the germ cells. Interestingly, cyd-1 depletion in the germ cells alone did not lead to sterility in daf-2 (Fig 4A and 4B). However, cyd-1 KD specifically in the uterine cells per se resulted in the germ cell arrest at the pachytene stage (Fig 4A and 4B) and also caused a reduction in the pachytene germ cell number (Fig 4C). This somatic requirement of CYD-1 in germline arrest reiterates a non-canonical role of CYD-1 in oogenesis. Depletion of cyd-1 in other somatic tissues like muscle, neurons, intestine, hypodermis, Distal Tip Cell (DTC) and the vulva did not cause sterility in daf-2 (S5A Fig). Interestingly, we observed vulval defects (protruded vulva/vulvaless) upon cyd-1 KD in daf-2 while daf-16;daf-2 showed no such defects (S5B and S5C Fig).

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Fig 4. Depletion of cyd-1 in the somatic gonad (uterus) alone is sufficient to cause germline arrest under low insulin signaling.

(A,B) Representative images of DAPI-stained gonads showing no pachytene arrest in day-1 adult daf-2(e1370);rde-1(mkc36);sun-1p::rde-1 (germline-specific RNAi) worms upon cyd-1 KD. However, pachytene arrest ensues upon cyd-1 KD in daf-2(e1370);rrf-3(pk1426);rde-1(ne219);fos-1ap::rde-1 (uterine tissue-specific RNAi). Oocytes are boxed for clarity. White arrows point towards oocytes while yellow shows the absence of oocytes. Sp denotes sperms. Scale bar 20 μm (A). The percentage of fertile worms upon germline-specific and uterine-specific cyd-1 KD in daf-2(e1370) (B). Average of three biological replicates (n ≥ 25 per condition for each experiment). Unpaired t-test with Welch’s correction. (C) Quantification of DAPI-stained daf-2(e1370);rrf-3(pk1426);rde-1(ne219);fos-1ap::rde-1 (uterine tissue-specific RNAi) (day-1 adult) germ cell nuclei of different stages (mitotic (MT), transition (TS) or pachytene zones (PZ)); n = 11. Each point represents the number of mitotic (MT), transition (TS) or pachytene zone (PZ) cells. Unpaired t-test with Welch’s correction. (D) Schematic showing the egg laying apparatus of an adult C. elegans hermaphrodite. The egg-laying apparatus consists of the uterus, the uterine muscles, the vulva, the vulval muscles, and the egg-laying neurons. A stack of seven nonequivalent epithelial toroids (rings) forms the vulva. The anterior and posterior lobes of uterus each comprises of four uterine toroid epithelial cells (ut1–ut4). Adherens junctions connect the neighboring uterine toroids or vulval toroids, respectively. The uterine seam cell (utse) is an H shaped cell that connects the uterus to seam cells and thus holds the uterus in place. uv1-3 are interfacial cells between uterus and vulva (uv1 is a neuroendocrine cell). The process of egg-laying is made possible through the contraction of sex muscles, namely the vulval muscles (VM1,2) connected to the vulva lips and the uterine muscles (UM1,2) encircling the uterus. The activity of these muscles is under the regulation of motor neurons, specifically VCn (VC1-6) and HSNL/R, which form synapses with each other and with the vulval muscle arms. A = anterior, P = posterior, D = dorsal, V = ventral. Adapted from Wormbook (doi:10.3908/wormatlas.1.24). (E) Schematic showing the vulval cell divisions. The vulval precursor cells (VPCs) P5.p, P6.p and P7.p are induced to divide in the early L3 stage. Three rounds of cell division yield a total of 22 vulval cells by the end of L3 stage. The adult vulva is then formed through subsequent patterning and morphogenesis processes. Schematics in Fig 4D and 4E were created using BioRender.com. (F,G) Representative fluorescent and DIC merged images of gonads showing AJM-1::GFP (marks cell junctions) expression in daf-2(e1370) and daf-16(mgdf50);daf-2(e1370) worms (day-1 adult) grown on control and cyd-1 RNAi. Yellow arrows point towards abnormal expression. Scale bar 10 μm (F). Quantification of the normal or abnormal expression of AJM-1::GFP (G). Average of three biological replicates (n ≥ 24 for each experiment). Two-way ANOVA-Tukey’s multiple comparisons test. (H,I) Representative fluorescent and DIC merged images of gonads showing cdh-3::GFP expression in daf-2(e1370) and daf-16(mgdf50);daf-2(e1370) worms (late-L3 stage) on control, cyd-1 or egl-43 RNAi. Yellow arrows point towards abnormal expression. Scale bar 5 μm (H). Quantification for normal, reduced or missing cdh-3::GFP expression (I). Combined data from three biological replicates (n ≥ 36) is plotted. Chi-square analysis was used to compare between groups. Error bars are s.d. Experiments were performed at 20°C. Source data are provided in S1 Table.

https://doi.org/10.1371/journal.pgen.1011453.g004

The egg-laying apparatus consists of the uterus, the uterine muscles, the vulva, the vulval muscles, and the egg-laying neurons. The uterus and vulva are connected by the uterine seam cell [81] (Fig 4D). The vulva formation starts at the L3 larval stage and vulval cell-divisions are completed by the late-L3 stage (Fig 4E). We investigated if cyd-1 KD caused defects in the vulva-uterine morphology. We visualized the vulval muscle architecture by Phalloidin staining (that marks the F-actin) that revealed a distorted vulva muscle structure following the depletion of cyd-1 in daf-2 (S5D Fig). To further dissect the underlying cause of the vulval defects, we employed the AJM-1::GFP-expressing transgenic strain (which marks the adherens junction) [82]. Vulval development involves the formation of a tubular structure from only 22 epithelial cells. It encompasses an array of steps including vulval cell induction and differentiation, cell division, cell rearrangements, invagination, toroid formation and cell-cell fusion and attachment with other tissues (hypodermis, muscle, uterus) [83]. Normally, in the vulva, the AJM-1::GFP marks the symmetrical toroid; however, upon cyd-1 depletion in daf-2, the AJM-1::GFP was mis-localized and the expression was diminished. In contrast, cyd-1 KD in daf-16;daf-2 worms did not exhibit such abnormalities (Fig 4F and 4G). This highlights the crucial role of activated DAF-16 in causing vulval impairments consequent to cyd-1 KD. Next, we asked if these defects in vulva were due to impairment in the vulva development (vulval cell divisions or cell fate acquisition). We observed the vulval precursor cell (VPC) fate of late-L3 staged worms using the transgenic reporter strain, cdh-3::GFP. CDH-3 belongs to the cadherin family of proteins and is expressed in the cells of the egg laying apparatus (vulva, uterine cells, HSN neurons) during development [84]. In daf-2 worms fed control RNAi, we observed the correct expression of cdh-3::GFP in vulval cells, however, the expression was abnormal or absent upon cyd-1 RNAi, suggesting incomplete or aberrant vulval development. Notably, the daf-16;daf-2;cyd-1 RNAi animals exhibited a restoration of normal cdh-3::GFP expression in vulval cells (Fig 4H and 4I). Furthermore, we utilized the depletion of the egl-43 gene [EGL-43 is essential for vulval morphogenesis [72,85]] as a control, and observed analogous abnormalities in cdh-3::GFP expression (Fig 4H). Interestingly, in this context as well, we observed a DAF-16-dependence such that the cdh-3::GFP expression was restored in the daf-16;daf-2 worms (Fig 4H). Additionally, egl-43 KD in daf-2 resulted in endomitotic oocytes and sterility, which was partially rescued in the daf-16;daf-2 worms (S5E and S5F Fig). These observations allude to the possibility that activated DAF-16 may amplify deficiencies in the egg-laying apparatus, potentially hindering normal oogenesis.

Depletion of cyd-1 results in DAF-16-dependent defects in the somatic gonad of daf-2 worms

To gain deeper insights into the changes underlying the germline arrest due to cyd-1 KD in daf-2, we performed transcriptomics analysis of the late-L4 staged WT and daf-2 worms with cyd-1 KD only in the somatic gonad [using a transgenic strain where unc-62 promoter drives the expression of RDE-1 only in the egg-laying apparatus in the rde-1 mutant [86]]. Interestingly, the genes important for cell cycle/cell-fate, oogenesis and reproduction were significantly downregulated in daf-2;cyd-1 RNAi worms (S6A Fig), but not in cyd-1 KD in the WT worms (S6B Fig). Importantly, the genes involved in vulva-uterine [lin-11 [87], lim-6 [88], unc-59 [89]] and sheath cell development [lim-7 [90]] were also down-regulated in the daf-2 gonad-specific cyd-1 KD (Fig 5A and S2 Table) but not in WT (S6C Fig and S3 Table). The gonadal sheath cells are a crucial component of the worm somatic gonad that plays an important role in pachytene exit and oogenesis [56, 91–93]. The 5 pairs of sheath cells surround each gonadal arm (Fig 5B). We tested if cyd-1 KD could also lead to defects in the sheath cells. For this, we used the sheath cell marker strain lim-7p::GFP [92] [LIM-7 is one of the LIM-homeodomain transcription factor [90]]. Interestingly, we found that the cyd-1 KD in daf-2 led to the complete abrogation of the lim-7p::GFP expression, which was restored in the daf-16;daf-2 (Fig 5C). We stained the gonads of day-1 adults with Phalloidin (labels F-actin) to investigate potential alterations in sheath cell structure in daf-2;cyd-1 RNAi worms. Strikingly, we observed a significant disruption in the cytoskeletal organization of sheath cells in daf-2;cyd-1 RNAi worms, which was ameliorated in daf-16;daf-2 worms subjected to cyd-1 RNAi (Figs 5D and S6D). We inquired if knocking down genes essential for sheath cell development (e.g., lim-7, lim-6) would also lead to germline arrest in daf-2 but found no such sterility (S7 Fig). However, similar to cyd-1 RNAi, we observed a DAF-16-dependent pachytene arrest and sterility (Fig 5E) and repression of lim-7p::GFP expression (S6E and S6F Fig) upon KD of sys-1. SYS-1/β-catenin transcription factor acts downstream of the WNT signaling pathway and is known to play a key role in the development of the somatic gonad [94, 95]. This suggests that activated DAF-16 may amplify somatic gonad defects upon perceiving potential threats in the somatic reproductive tissue (such as CYD-1 or SYS-1 depletion), thereby halting oogenesis progression to ensure poor quality eggs are not laid.

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Fig 5. Depletion of cyd-1 under low insulin signaling leads to DAF-16-dependent defects in sheath cells.

(A) Volcano plot showing the magnitude [log₂(FC)] and significance [−log₁₀(P value)] of the genes that are differentially expressed in L4-staged daf-2(e1370);rrf-3(pk1426);rde-1(ne219);unc-62p::rde-1(genomic) worms, grown on control or cyd-1 RNAi. Y axis [−log₁₀(P value)] is restricted to a value of 10. (B) Schematic showing one of the two gonad arms of an adult C. elegans hermaphrodite. Sheath cells are colored green. Schematic in Fig 5B were created using BioRender.com. (C) Representative fluorescent and DIC merged images of gonads showing lim-7p::GFP (that marks the sheath cells) expression in daf-2(e1370) and daf-16(mgdf50);daf-2(e1370) worms (day-1 adult) grown on control and cyd-1 RNAi. The gonadal arm is outlined for clarity. Scale bar 20 μm. Quantification of the normal or missing expression of lim-7p::GFP is given below the figure. Combined data from three biological replicates (n ≥ 65) is plotted. Chi-square analysis was performed to compare between groups. (D) Representative fluorescent images of Phalloidin-stained (that marks the F-actin) gonads of daf-2(e1370) and daf-16(mgdf50);daf-2(e1370) worms (day-1 adult) grown on control and cyd-1 RNAi. Arrows point towards defects in the sheath cells. Scale bar 10 μm. Quantification of the normal or defective sheath cell structure (as per scoring scheme in S6D Fig) is shown on the right. Combined data from three biological replicates (n ≥ 12) is plotted. Chi-square analysis was performed to compare between groups. (E) Representative fluorescent images of DAPI-stained gonads of daf-2(e1370) and daf-16(mgdf50);daf-2(e1370) (day-1 adult) worms grown on control or sys-1 RNAi. Oocytes are boxed for clarity. White arrows point towards oocytes while yellow shows endomitotic oocytes or lack of oocytes. Scale bar 20 μm. The percentage of fertile worms is shown on the right. Average of three biological replicates (n ≥ 25 per condition for each experiment). Two-way ANOVA-Tukey’s multiple comparisons test. (F) Representative fluorescent images of gonads showing lim-7p::GFP (that marks the sheath cells) expression in WT worms (day-1 and day-3 adult) grown on control and cyd-1 RNAi. Arrows point towards defective/missing lim-7p::GFP expression in the sheath cells. The gonadal arm is outlined for clarity. Scale bar 20 μm. Quantification of the normal/defective or missing lim-7::GFP expression is given below the figure. Combined data from three biological replicates (n ≥ 39) is plotted. Chi-square analysis was performed to compare between groups. Error bars are s.d. Experiments were performed at 20°C. Source data are provided in S1 Table.

https://doi.org/10.1371/journal.pgen.1011453.g005

Next, we investigated if cyd-1 RNAi can alter the expression of another adult hermaphrodite gonad-specific marker (FKH-6::GFP). In the hermaphrodite gonad, the forkhead transcription factor, FKH-6 expresses in the adult spermatheca and is important for gonadal cell differentiation [96]. Similar to lim-7p::GFP expression, we found a DAF-16-dependent suppression of FKH-6::GFP expression upon cyd-1 depletion in daf-2 (S6G and S6H Fig). Interestingly, while the WT worms showed no obvious structural defects in the sheath cell upon cyd-1 depletion (S6I and S6J Fig), lim-7p::GFP expression was significantly reduced in day-3 cyd-1 KD WT worms compared to age-matched control worms (no difference in day-1 adults) (Fig 5F).

Overall, these data imply that cyd-1 depletion elicits a DAF-16-dependent distortion of sheath cell structure and suppression of hermaphrodite gonad marker expression in daf-2. The amplification of somatic gonadal abnormalities when activated DAF-16 is present may, in turn, influence oogenesis.

Lowering CYD-1 levels may cause defective sperm-to-oocyte fate switch in daf-2

In the hermaphrodites, a fixed number of sperms are formed in the L4 larval stage, after which the fate is switched from sperm to oocyte and adult worms make only oocytes from then onwards [97]. Upon cyd-1 KD in daf-2, we observed only sperms but no oocytes in the adult hermaphrodite worms (Fig 6A). Moreover, we showed above that lowering CYD-1 levels in daf-2 suppresses the hermaphrodite gonad-specific marker expression (Fig 5C, S6G and S6H). We supposed that either the germline has been masculinized (male mode of germline: continuous sperm production) or there may be a failure in the sperm-to-oocyte fate switch. However, we did not observe any ectopic expression of the male gonad marker (K09C8.2p::GFP) [98] in daf-2;cyd-1 RNAi worms (S8 Fig). Also, the sperm number remained unchanged in the cyd-1-depleted daf-2 young adult worms in comparison to control RNAi grown worms (Fig 6B) and the sperm number did not increase on day-2 or day-3 of adulthood, indicating that the germline had not been masculinized (Fig 6C). Alternatively, cyd-1 depletion in daf-2 may cause a defect in the sperm-to-oocyte cell fate switch and a consequent arrest of oogenesis at the pachytene stage. In line with this assumption, the depletion of cyd-1 led to a DAF-16-dependent downregulation of genes essential for the sperm-to-oocyte fate switch (Fig 6D and 6E). Interestingly, we also observed the transcripts of these sperm-to-oocyte switch genes to be downregulated upon KD of cyd-1 only in the uterine cells of the daf-2 (Fig 6F). Since the proximal-most sheath cell pairs play an important role in the pachytene exit [56,65], the deformation of sheath cells in the daf-2;cyd-1 RNAi worms may be the driver for the defective sperm-to-oocyte fate switch, leading to sterility in these animals.

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Fig 6. A defective sperm-to-oocyte switch may underlie germline arrest upon cyd-1 KD under low insulin signaling.

(A,B) Representative fluorescent images of DAPI stained daf-2(e1370) worms (early day-1 adult) grown on control and cyd-1 RNAi. Sperms are encircled for clarity. Scale bar 10 μm (A). Quantification of the sperm count (n = 21) (B). Each point represents the number of sperms per gonadal arm per worm. Unpaired t-test with Welch’s correction. (C) Quantification of the sperm count in daf-2(e1370) worms (day-2 and day-3 adults) grown on control and cyd-1 RNAi. Each point represents the number of sperms per gonadal arm per worm (n = 16). Two-way ANOVA-Tukey’s multiple comparisons test. (D) Quantitative RT-PCR analysis of sperm-to-oocyte switch genes in daf-2(e1370) worms (late-L4 stage) grown on control or cyd-1 RNAi. Expression levels were normalized to actin. Average of three biological replicates are shown. Unpaired t-test with Welch’s correction. (E) Quantitative RT-PCR analysis of sperm-to-oocyte switch genes in daf-16(mgdf50);daf-2(e1370) worms (late-L4 stage) grown on control or cyd-1 RNAi. Expression levels were normalized to actin. Average of three biological replicates are shown. Unpaired t-test with Welch’s correction. (F) Quantitative RT-PCR analysis of sperm-to-oocyte switch genes in daf-2(e1370);rrf-3(pk1426);rde-1(ne219);fos-1ap::rde-1 (uterine tissue-specific RNAi) worms (late-L4 stage) grown on control or cyd-1 RNAi. Expression levels were normalized to actin. Average of three biological replicates are shown. Unpaired t-test with Welch’s correction. Error bars are s.d. Experiments were performed at 20°C. Source data are provided in S1 Table.

https://doi.org/10.1371/journal.pgen.1011453.g006

C. elegans strain maintenance

Unless otherwise mentioned, all the C. elegans strains were maintained and propagated at 20°C on E. coli OP50 using standard procedures (Stiernagle, 2006). C. elegans strains were obtained from Caenorhabditis Genetics Center (CGC) (Table 1) or generated in-house using standard cross over techniques (Table 2).

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Table 1. The list of strains obtained from Caenorhabditis Genetics Center (CGC) for use in the study.

https://doi.org/10.1371/journal.pgen.1011453.t001

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Table 2. The strains listed below were generated in-house using standard cross-over techniques.

https://doi.org/10.1371/journal.pgen.1011453.t002

Preparation of RNAi plates

RNAi plates were poured using an autoclaved nematode growth medium supplemented with 100 μg/ml ampicillin and 2 mM IPTG. Plates were allowed to dry at room temperature for 1 day. Bacterial culture harboring a particular RNAi construct was grown in Luria Bertani (LB) media supplemented with 100 μg/ml ampicillin and 12.5 μg/ml tetracycline, overnight at 37°C in a shaker incubator. Saturated primary cultures were re-inoculated the next day (1/50^th volume) in fresh LB media containing 100 μg/ml ampicillin and grown in a 37°C shaker until OD₆₀₀ reached 0.5–0.6. The bacterial cells were pelleted down by centrifuging the culture at 3214 g for 5 minutes at 4°C and resuspended in 1/10^th volume of M9 buffer containing 100 μg/ml ampicillin and 1 mM IPTG. Around 300 μl of this suspension was seeded onto RNAi plates and left at room temperature for 2 days for drying, followed by storage at 4°C till further use.

Hypochlorite treatment to obtain synchronize worm population

Gravid adult worms, initially grown on E. coli OP50 bacteria, were collected using M9 buffer in a 15 ml falcon tube. Worms were washed twice by first centrifuging at 652 g for 60 seconds followed by resuspension of the worm pellet in 1X M9 buffer. After the second wash, the worm pellet was resuspended in 3.5 ml of 1X M9 buffer and 0.5 ml 5N NaOH and 1 ml of 4% Sodium hypochlorite solution was added. The mixture was vortexed for 4–5 minutes until the entire worm body dissolved, leaving behind the eggs. The eggs were washed 5–6 times, by first centrifuging at 1258 g, the 1X M9 was aspirated out, followed by resuspension in fresh 1X M9 buffer to remove traces of bleach and alkali. After the final wash, eggs were kept in 15 ml falcons with ~ 10 ml of 1X M9 buffer and kept on rotation ~15 r.p.m for 17 hours to obtain L1 synchronized worms for all strains. The L1 worms were obtained by centrifugation at 805 g followed by resuspension in approximately 200–300 μl of M9 and added to respective RNAi plates.

RNA isolation

Worms grown on respective RNAi were collected using 1X M9 buffer and washed thrice to remove bacteria. Trizol reagent (200 μl; Takara Bio, Kusatsu, Shiga, Japan) was added to the 50 μl worm pellet and subjected to three freeze-thaw cycles in liquid nitrogen with intermittent vortexing for 1 minute to break open the worm bodies. The samples were then frozen in liquid nitrogen and stored at -80°C till further use. Later, 200 μl of Trizol was again added to the worm pellet and the sample was vigorously vortexed for 1 minute. To this, 200 μl of chloroform was added and the tube was gently inverted several times followed by 5 minutes of incubation at room temperature. The sample was then centrifuged at 12000 g for 15 minutes at 4°C. The RNA containing the upper aqueous phase was gently removed into a fresh tube without disturbing the bottom layer and interphase. To this aqueous solution, an equal volume of isopropanol was added and the reaction was allowed to sit for 10 minutes at room temperature followed by centrifugation at 12000 g for 15 minutes at 4°C. The supernatant was carefully discarded without disturbing the RNA-containing pellet. The pellet was washed using 1 ml 70% ethanol solution followed by centrifugation at 8000g for 10 minutes at 4°C. The RNA pellet was further dried at room temperature and later dissolved in autoclaved MilliQ water followed by incubation at 65°C for 10 minutes with intermittent tapping. The concentration of RNA was determined by measuring absorbance at 260 nm using a NanoDrop UV spectrophotometer (Thermo Scientific, Waltham, USA) and the quality was checked using denaturing formaldehyde-agarose gel.

Gene expression analysis using quantitative real-time PCR (QRT-PCR)

First-strand cDNA synthesis was carried out using the Iscript cDNA synthesis kit (Biorad, Hercules, USA) following the manufacturer’s guidelines. The prepared cDNA was stored at -20°C. Gene expression levels were determined using the Brilliant III Ultra-Fast SYBR Green QPCR master mix (Agilent, Santa Clara, USA) and Agilent AriaMx Real-Time PCR system (Agilent, Santa Clara, USA), according to manufacturer’s guidelines. The relative expression of each gene was determined by normalizing the data to actin expression levels. The list of primers is summarized in S4 Table.

Measurement of cell corpses using CED-1::GFP

The analysis of engulfed cell corpses was performed by utilizing transgenic worms expressing CED-1::GFP, a transmembrane protein found on surrounding sheath cells that are responsible for engulfing cell corpses. ced-1p::ced-1::GFP(smIs34) worms were bleached and their eggs were allowed to hatch in 1X M9 buffer for 17 hours to obtain L1 synchronized worms. Approximately 100 L1 worms were placed onto control or cyd-1 RNAi in triplicates. On the day-1 of adulthood, worms were imaged in Z-stack using 488 nm laser to excite GFP on LSM980 confocal microscope (Carl Zeiss, Oberkochen, Germany). The images were converted into a maximum intensity projection (MIP). The number of cell corpses per gonadal arm was quantified manually.

DAPI staining

Worms were cultured on specific RNAi plates from the L1 stage onward. On day 1 of adulthood, worms were collected in a 1.5 ml Eppendorf tube with 1X M9 buffer and allowed to settle. Using a glass Pasteur pipette, the 1X M9 buffer was carefully removed, leaving approximately 100 μl of worm suspension. Subsequently, 1 ml of ice-cold 100% methanol was added to the worm pellet, and the mixture was incubated at -20°C for 30 minutes. The methanol-fixed worm pellet was then placed on a glass slide, and after the methanol had evaporated, Fluoroshield with DAPI (from Invitrogen, Carlsbad, USA) was applied for staining. A coverslip was carefully placed on top (avoiding bubbles) and sealed with transparent nail paint.

To stain dissected gonads, worms were positioned on a glass slide in 1X M9, and the gonads were obtained by carefully cutting the pharynx or tail end of the worm using a sharp 25G needle. After this 500 μl of chilled 100% methanol was added to the slide and allowed to evaporate. Subsequently, Fluoroshield with DAPI (Invitrogen, Carlsbad, USA) was added for staining and a coverslip was carefully placed on top (avoiding bubbles) and sealed with transparent nail paint. Finally, the images were captured using a 405 nm laser excitation DAPI on LSM980 confocal microscope (Carl Zeiss in Oberkochen, Germany).

Brood size, reproductive span, and egg hatching

Reproductive span

The number of progenies per worm was plotted for each day to monitor the reproductive span. For the mated reproductive span, the hermaphrodites were mated on 35 mm cross plates with fresh males (males were grown on control RNAi) (1:3 ratio of hermaphrodite:male) each day for 4 h and shifted back to 60 mm RNAi plates. The rest was performed according to the reproductive span of selfed worms. The total number of days when worms were capable of producing viable offspring was recorded as their reproductive span.

Egg hatching

The quality of eggs was assessed by determining the percentage of hatched eggs. Almost 50 adult worms per condition were allowed to lay eggs for an hour. The mother worms were then sacrificed and the number of eggs on respective plates were counted. After 48 hours the number of hatched progenies were counted, and the percentage of hatched eggs were calculated for each condition.

Analysis of oocyte morphology

Worms were grown from L1 onwards on control or cyd-1 RNAi. Microscopic images of oocytes on the third day of adulthood (except for S3B and S3C Fig where day-1 oocytes were imaged) were captured using differential interference contrast (DIC) settings (Carl Zeiss, Oberkochen, Germany). These oocytes were classified into three groups based on various characteristics such as cavities, shape, size, and organization. Depending on the severity of the observed features, oocytes were classified as either normal, mild, or severe. Oocytes without cavities, small size, or disorganization were labeled as normal. Oocytes exhibiting the presence of either two small oocytes, two cavities, or two disorganized oocytes were categorized as having a mild phenotype. Those with more than two cavities, small oocytes, or misshapen oocytes were considered to have a severe phenotype.

Quantification of fertile worms

Approximately 100 synchronized L1 worms were placed onto different RNAi plates, in triplicates. On the day-1 adult stage, bright-field images were captured (Carl Zeiss, Oberkochen, Germany). Worms carrying over five eggs in their uterus were categorized as fertile.

Germ cell count

Around 100 L1-staged worms were poured onto control or cyd-1 RNAi plates in triplicate. Utilizing a 405 nm laser to excite DAPI in a confocal microscope (Carl Zeiss, Oberkochen, Germany), images of DAPI stained day-1 adult germline were acquired. Z stacked images (0.13 μM sections between the starting plane and end plane) of germline were obtained followed by Maximum Intensity projection (MIP) of each z-stack in the XY plane to create a superimposed image. Quantification of germ cells per gonadal arm was done using Fiji (National Institute of Health) in different regions based on their morphology, namely the Mitotic Zone (spanning from the distal tip to the transition zone), the Transition zone (extending from the beginning to the end of crescent-shaped nuclei), and the pachytene zone (from the end of the transition zone to the turn region).

Sperm count

DAPI stained early day-1, day-2 or day-3 adult gonads were imaged using 405 nm laser to excite DAPI on a confocal microscope (Carl Zeiss, Oberkochen, Germany). Z stacked images (0.13 μM sections between the starting plane and end plane) of germline were obtained followed by Maximum Intensity projection (MIP) of each z-stack in the XY plane to create a superimposed image. Sperms per gonadal arm were quantified using Fiji (National Institute of Health).

Scoring of Endomitotic oocyte (emo)

Approximately 50 L1-staged worms were placed onto control or cyd-1 RNAi plates in triplicate. Day-3 adult worm gonads (except for S3D and S3E Fig where day-1 worm gonads were imaged) were imaged using differential interference contrast (DIC) settings in a microscope (Carl Zeiss, Oberkochen, Germany). Endomitotic oocytes in the uterus were identified by following features: a lack of distinct nucleus, nuclei may appear disorganized, lobulated, or enlarged [56]. Alternatively, day-3 adults were DAPI stained and subsequently imaged in a Z-stack using 405 nm laser to excite DAPI on an LSM980 confocal microscope (Carl Zeiss, Oberkochen, Germany). Nuclei that appeared enlarged and exhibited dense DAPI staining [123], were identified as endomitotic oocytes (emo). For the purpose of scoring, the images were converted into a maximum intensity projection (MIP).

unc-119::GFP positive emo

Approximately 50 L1-staged unc-119::gfp (neuronal fate marker) transgenic worms were placed onto control or cyd-1 RNAi plates in triplicates. Day-3 adult worm gonads were imaged using 488 nm laser to excite GFP as well as differential interference contrast (DIC) settings in a fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Endomitotic oocytes in the uterus were identified by following features: a lack of distinct nucleus, nuclei may appear disorganized, lobulated or enlarged [56]. The number of worms having endomitotic oocytes as well as that expressed unc-119::GFP in the emo were counted. Percentage of worms expressing GFP in emo was calculated for each condition.

Imaging of transgenic reporter strains

Synchronized L1 populations of transgenic reporter strains were grown on respective RNAi plates. For imaging, the worms were mounted on agar pads with 20 mM sodium azide and a coverslip was placed on the paralyzed worms. The coverslip was secured with a transparent nail paint. All images were acquired in a Z-stack using 488 nm laser to excite GFP on LSM980 confocal microscope (Carl Zeiss, Oberkochen, Germany). The images were converted into a maximum intensity projection (MIP). To qualitatively quantify lim-7p::GFP (sheath cell marker) and FKH-6::GFP (hermaphrodite spermatheca) expression, the day-1 or day-3 adult gonads were scored as having normal expression, reduced expression (less intensity than normal) or missing (no GFP expression). In the case of cdh-3::GFP (a marker for vulval cells), we used late-L3 staged worms and scored their gonads based on whether they displayed normal expression, abnormal expression (distorted pattern), or missing (no GFP expression). Similar categorization was utilized to assess the AJM-1::GFP (adherens junction marker) expression in early day-1 adult gonads.

Phalloidin staining and sheath cell structure

Worms were grown on control and cyd-1 RNAi L1 onwards. Day-1 or day-3 adult worms were collected in a 1.5 ml Eppendorf tube with 1X M9 buffer and allowed to settle. Using a glass Pasteur pipette, excess 1X M9 buffer was carefully removed, and worms were transferred to a glass slide. Using a 25G needle, worms were excised at the pharynx or near tail region, causing gonad arms to extrude from the body. The dissected gonad arms were transferred to 5 ml glass tubes and fixed in 4% formaldehyde for 20 minutes at room temperature and then washed thrice with 0.1% 1X PBST. After washing, fixed gonad arms were transferred to 1 ml glass tubes and stained with Oregon Green 488 Phalloidin (Molecular Probes, Invitrogen, Life Technologies, Grand Island, NY, USA) at a final concentration of 0.4 units/ml and placed in the dark at 4°C overnight followed by 30 minutes incubation at RT. Gonad arms were then washed twice with PBST, and extra liquid was removed and gonad arms were transferred onto slides with a drop of Fluoroshield with DAPI (Invitrogen, Carlsbad, USA). Coverslip was placed on the mounted worms and sealed using nail-paint. Phalloidin marks the actin structure. To analyze sheath cell structure gonads were imaged in a Z-stack using 488 nm laser to excite GFP on LSM980 confocal microscope (Carl Zeiss, Oberkochen, Germany). The images were converted into a maximum intensity projection (MIP). Sheath cell structure was scored as normal or mild defect (slight clustering or loosening of actin filaments) or severe defect (highly clustered or loosened actin filaments). To examine vulval morphology, the images were captured with a focus on the vulva-uterine region.

Vulva morphology

Worms were grown from L1 onwards on control or cyd-1 RNAi. Microscopic images of vulva on the early day-1 of adulthood were captured using differential interference contrast (DIC) settings on a microscope (Carl Zeiss, Oberkochen, Germany). These vulvas were classified as normal or abnormal (protruding vulva/vulvaless) based on its structure and organization.

RNA sequencing

Synchronized late-L4 worms grown on control or cyd-1 RNAi were collected using 1X M9 buffer, after washing it thrice to remove bacteria. Total RNA was isolated from these worm pellets using the Trizol method. The concentration of RNA was determined by measuring absorbance at 260 nm using a NanoDrop UV spectrophotometer (Thermo Scientific, Waltham, USA) and RNA quality was checked using RNA 6000 NanoAssay chip on a Bioanalyzer 2100 machine (Agilent Technologies, Santa Clara, USA). RNA above RNA integrity number = 9 was included in the study. A 1.5 μg aliquot of RNA samples was used for polyA mRNA isolation from total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module (Catalog no-E7490L) according to the maufacturer’s protocol. The NEBNext Ultra II Directional RNA Library Prep kit (Catalog no-E7760L) was used to construct libraries according to the manufacturer’s instructions. NovaSeq6000 NGS platform (Illumina Inc., San Diego, California, USA) with 2X150 paired end chemistry was used to generate 30 million paired end reads corresponding to 9 Gb data per sample. The RNA sequencing data are available at the https://www.ebi.ac.uk/biostudies/arrayexpress with accession number E-MTAB-13172.

RNA-seq Analysis

Sequencing reads were subjected to quality control using the Fastp kit [124]. The quality of reads was assessed and visualized by FASTQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc). Adapter sequence was removed by Trimmomatic tool [125]. For each of our samples, reads were aligned to the WBcel235 cell genome using STAR-2.7.10b [126] with an average 94.4% alignment rate. Gene counts were obtained with the quantMode GeneCounts option. Differential Gene expression analysis was performed using DeSeq2 [127] package in R. Differentially expressed genes were defined as those with padj values below 0.06. Genes with a cut-off of log₂(fold change) > 0.6 and log₂(fold change) < −0.6 were considered as upregulated and downregulated genes, respectively. For downstream analysis, the function variance stabilizing transformations (VST) in the DeSeq2 package was implemented. Wormbase GeneIDs were mapped to their gene symbols by AnnotationaDbi and C. elegans database (org.Ce.eg.db) packages in R. Counts function was used to normalize the gene counts. Gene Set Enrichment analysis was performed using gseGO function of clusterProfiler v4.6.2 [128] in R. Volcano plots and dot plots were plotted with ggplot2 (https://ggplot2.tidyverse.org) in R. For volcano plots, differentially expressed genes (padj <0.1 and |log₂(fold change)|>0.6) were indicated in green and purple colors. Dot plots were made with Enriched GO terms on y-axis and Normalized Enrichment Score (NES) on x-axis and size and color as padj value.

Statistical tests

All statistical tests were performed utilizing the built-in functions of GraphPad Prism 10.1.0. In instances where we compared two conditions (continuous data), we applied a Student’s t-test with Welch’s correction, making no assumptions about consistent standard deviations. When comparing categorical data, we used the chi-square test. When comparing multiple conditions (continuous), we employed a Two-way ANOVA with Tukey’s multiple comparison test.