Figure 6. Uterine expansion correleates strongly with Anterior Lateral Microtubule cell (ALMR) exophergenesis regardless of whether eggs, oocytes, or debris are retained.
(A) Despite cbd-1 RNAi mediated disruption of eggshell formation and earliest embryonic cell divisions, exopher levels are high in the sem-2(rf) egg retention background. The percentage of ALMR exopher events among 50 Ad2 wild-type (left) or Ad1 sem-2(rf) hermaphrodite C. elegans that are treated with either empty vector control RNAi or RNAi targeting cbd-1 in each trial (total of 3 independent trials). sem-2 mutants bag extensively at Ad2 and cannot be tested. Diagram indicates uterine filling status of test sem-2(rf) + cbd-1 RNAi. Strain ZB4757: bzIs166[Pmec-4::mCherry] II vs. strain ZB4902: sem-2(n1343) I; bzIs166[Pmec-4::mCherry] II. ****p<0.0001 in Cochran–Mantel–Haenszel test. (B) Uterine length remains long in the egg-laying defective sem-2(rf) + cbd-1 RNAi. Uterus length of strain ZB4757: bzIs166[Pmec-4::mCherry] II vs. ZB4902: sem-2(n1343) I; bzIs166[Pmec-4::mCherry] II treated with RNAi against cbd-1. n = ~20 hermaphrodites from one trial, Ad2, ****p<0.0001 in two-tail unpaired t-test. (C) Blocking sperm maturation in a sem-2(rf) mutant, which fills the uterine space with oocytes, induces exophers in the absence of eggs. The percentage of ALMR exopher events among 50 sem-2(rf) hermaphrodite C. elegans that express the SPE-44 AID system (‘control’ is treated with 0.25% ethanol vehicle and ‘no sperm’ is treated with 1 mM auxin in 0.25% ethanol from egg to adult day 2) in each trial (total of 3 independent trials). L4 stage is the last larval stage before adult. Diagram indicates uterine oocyte filling status of test sem-2(rf) + SPE-44 AID. Strain ZB4953: sem-2(n1343) fxIs1[Ppie-1::TIR1::mRuby] I; bzIs166[Pmec-4::mCherry] II; spe-44(fx110[spe-44::degron]) IV. (D) When sperm maturation is disrupted in mutants blocked for egg laying, leaving oocytes to occupy reproductive structures, the uterus expands as oocytes accumulate. Uterus length of strain ZB4749: fxIs1[Ppie-1::TIR1::mRuby] zdIs5[Pmec-4::GFP] I; bzIs166[Pmec-4::mCherry] II; spe-44(fx110[spe-44::degron]) IV vs. ZB4953: sem-2(n1343) fxIs1[Ppie-1::TIR1::mRuby] I; bzIs166[Pmec-4::mCherry] II; spe-44(fx110[spe-44::degron]) IV. 1 mM auxin treatment induces the no sperm status to both strains. n = ~20 hermaphrodites from one trial, Ad2. ****p<0.0001 in two-tail unpaired t-test. (E) Disrupting sperm maturation in lin-39 RNAi animals blocked for egg-laying fills the uterine space with oocytes, and induces exophers in the absence of eggs. The percentage of ALMR exopher events among 50 adult day 1 or 2 SPE-44 AID no sperm hermaphrodite C. elegans treated with either control RNAi or lin-39 RNAi in each trial (total of 3 independent trials). Diagram indicates uterine oocyte filling status of test lin-39 RNAi + SPE-44 AID. ZB4749: fxIs1[Ppie-1::TIR1::mRuby] zdIs5[Pmec-4::GFP] I; bzIs166[Pmec-4::mCherry] II; spe-44(fx110[spe-44::degron]) IV. ****p<0.0001 in Cochran–Mantel–Haenszel test. (F) When sperm maturation is blocked, leaving oocytes to occupy reproductive structures, the uterus length is short; but if oocytes cannot be laid in the lin-39 RNAi background, the uterus expands as oocytes accumulate. Uterus length of strain ZB4749: fxIs1[Ppie-1::TIR1::mRuby] zdIs5[Pmec-4::GFP] I; bzIs166[Pmec-4::mCherry] II; spe-44(fx110[spe-44::degron]) IV +1 mM auxin treatment to eliminate sperm maturation. n = ~20 hermaphrodites. ****p<0.0001 in two-tail unpaired t-test. (G) Blocking oocyte production in the background of lin-39 RNAi-mediated disruption of the egg-laying apparatus eliminates early adult exophergenesis. We used SPE-44 AID to block sperm maturation and fem-3(q20) to prevent oocyte production; lin-39 RNAi to disrupt egg-laying capacity. Diagram indicates empty uterus status of test fem-3(q20); spe-44 AID; lin-39(RNAi) strain. Exopher scoring of Ad2 ZB4749: fxIs1[Ppie-1::TIR1::mRuby] zdIs5[Pmec-4::GFP] I; bzIs166[Pmec-4::mCherry] II; spe-44(fx110[spe-44::degron]) IV +1 mM auxin or ZB5042: bzIs166[Pmec-4::mCherry] II; fem-3(q20)ts IV, treated with either control empty vector (EV) RNAi or lin-39 RNAi at 25 °C. Total of three trials (50 worms per trial). ****p<0.0001 in Cochran–Mantel–Haenszel test. (H) Blocking oocyte production in the background of sem-2(rf)-mediated disruption of the egg-laying apparatus eliminates early adult exophergenesis. We used cbd-1 RNAi to disrupt eggshell and fem-3(q20) to prevent oocyte production; sem-2(n1343) to disrupt egg-laying capacity. Exopher scoring of adult day 2 hermaphrodites, treated with either control empty vector (EV) RNAi or cbd-1 RNAi at 25 °C. Total of three trials (50 worms per trial). Diagram indicates empty uterus status of test fem-3(q20); cbd-1 RNAi; sem-2(rf) strain. (I) The uterus length is correlated with ALMR exophergenesis. Data shown are the mean of uterus length (X-aixs) and percentage ALMRs with exopher (Y-axis) for different genotypes/treatments measured in this study. The correlation line is based on a linear fit model and the Pearson r and p value is based on the correlation assay. Uterus length from short to long: SPE-44 AID; cbd-1 RNAi; wild-type (adult day 1); mex-3 RNAi; gad-1 RNAi; wild-type (adult day 2); egl-3(Δ); egl-9(Δ); sem-2(rf); SPE-44 AID + lin-39 RNAi; SPE-44 AID + sem-2(rf).
Figure 6—figure supplement 1. Representative pictures of oocytes retention (red rectangle) in the uterus of Adult day 2 hermaphrodite.
