Fig. 3. Mating-induced shrinking and transcriptional changes require the functional piRNA pathway in the germline.
a–f, Body size measurements of mated and unmated hermaphrodites related to different small RNA pathways. a, Mutants that are defective in miRNA or siRNA pathways still shrink after mating. N2 unmated, 1,334 ± 81 μm (n = 30); N2 mated, 1,088 ± 122 μm (n = 31); ***P < 0.001. dcr-1(mg375) unmated, 1,347 ± 84 μm (n = 30); mated, 1,088 ± 122 μm (n = 30); ***P < 0.001. sid-1(qt9) unmated, 1,102 ± 78 μm (n = 20); mated, 898 ± 101 μm (n = 21); ***P < 0.001. fog-2(q71) unmated, 1,436 ± 54 μm (n = 30); mated, 1,099 ± 128 μm (n = 22); ***P < 0.001. alg-4(ok1041); alg-3(tm1155) unmated, 1,364 ± 78 μm (n = 25); mated, 1,060 ± 136 μm (n = 25); ***P < 0.001. b–f, Mutants in which multiple steps of the piRNA pathway are affected are resistant to mating-induced shrinking. b, piRNA pathway mutants do not shrink after mating. fog-2(q71) unmated, 1,546 ± 55 μm (n = 30); mated, 1,065 ± 127 μm (n = 30); ***P < 0.001. prg-1(n4357) unmated, 1,368 ± 100 μm (n = 30); mated, 1,369 ± 114 μm (n = 30); P = 0.9603. prde-1(mj207) unmated, 1,385 ± 106 μm (n = 30); mated, 1,376 ± 120 μm (n = 31); P = 0.7581. c, ekl-1 and drh-3 mutant alleles are maintained by chromosome balancers. Postmating body size of siblings with homozygous mutant alleles (–/–, no balancer) and heterozygous mutant allele (+/–, with the balancer) was measured in the same experiment. ekl-1(ok1197)[+/–] unmated, 1,306 ± 105 μm (n = 25); mated, 1,039 ± 140 μm (n = 20); ***P < 0.001. ekl-1(ok1197)[–/–] unmated, 1,464 ± 87 μm (n = 16); mated, 1,460 ± 111 μm (n = 16); P = 0.9063. drh-3(tm1217)[+/–] unmated, 1,247 ± 117 μm (n = 21); mated, 951 ± 58 μm (n = 16); ***P < 0.001. drh-3(tm1217)[–/–] unmated, 1,294 ± 104 μm (n = 16); mated, 1,321 ± 58 μm (n = 5); P = 0.5863. d, fog-2(q71) unmated, 1,422 ± 69 μm (n = 30); mated, 1,122 ± 125 μm (n = 30); ***P < 0.001. hpl-2(ok916) unmated, 1,215 ± 130 μm (n = 30); mated, 1,212 ± 164 μm (n = 30); P = 0.9286. MAGO12 unmated, 1,365 ± 65 μm (n = 30); mated, 1,361 ± 100 μm (n = 30); P = 0.8577. e, fog-2(q71) unmated, 1,349 ± 73 μm (n = 25); mated, 1,151 ± 89 μm (n = 25); ***P < 0.001. meg-3(tm4259) meg-4(ax2026) unmated, 1,312 ± 71 μm (n = 25); mated, 1,289 ± 161 μm (n = 25); P = 0.5179. meg-1(vr10) meg-3(tm4259) unmated, 1,298 ± 89 μm (n = 25); mated, 1,311 ± 104 μm (n = 25); P = 0.6330. f, N2 unmated, 1,191 ± 40 μm (n = 25); mated, 1,078 ± 69 μm (n = 25); ***P < 0.001. Cer1(–) (RNAi for three generations) unmated, 1,170 ± 46 μm (n = 25); mated, 1,081 ± 59 μm (n = 25); ***P < 0.001. TEI, transgenerational epigenetic inheritance. g, Mating induces shrinking in hermaphrodites with germline-specific rescue of the piRNA pathway. fog-2(q71) unmated, 1,462 ± 68 μm (n = 25); mated, 1,189 ± 131 μm (n = 25); ***P < 0.001. prg-1(n4357) unmated, 1,322 ± 96 μm (n = 20); mated, 1,363 ± 101 μm (n = 25); P = 0.1803. Germline-specific prg-1 rescue (CQ655) unmated, 1,431 ± 74 μm (n = 25); mated, 1,212 ± 152 μm (n = 25); ***P < 0.001. h, Summary of the genetic requirement for mating-induced shrinking. i, PCA of normalized read counts from the mRNA transcriptomes of the isolated germlines. Black, fog-2 unmated; red, fog-2 mated; gray, prde-1 unmated; pink, prde-1 mated. Full list of mating-induced significantly differentially-expressed genes in prde-1 germline is available as Source Data. j, Quantification of maximum value of Pdaf-9::daf-9::gfp expression in the spermatheca in wildtype (left) and prde-1 (right) background. All measurements were normalized to the DAF-9 expression in the XXX cells in the head, which is not affected by mating. Unmated control, 44.1 ± 17.0 a.u. (n = 11); mated control, 25.7 ± 11.7 a.u. (n = 13); P = 0.0049; unmated prde-1, 34.4 ± 7.4 a.u. (n = 13); mated prde-1, 31.7 ± 4.2 a.u. (n = 14); P = 0.2676. Data are presented as maximum values ± s.d.; n, number of biologically independent animals. Two-tailed t-test was used to determine statistical significance. **P < 0.01.