https://orcid.org/0000-0003-1746-3012
Figures & data
Table 1. Bestkeeper, NormFinder and geNorm analysis on three different candidate miRNA internal controls.
Table 2. Bestkeeper, NormFinder and geNorm analysis on three different candidate miRNA internal controls.Geo Mean = geometric Mean of Ct values are Mean = Arithmetic mean of Ct values, stdev = standard deviation of Ct values, CV= coefficient variation.
Table 3. Bestkeeper, NormFinder and geNorm analysis on three different candidate miRNA internal controls. Geo Mean = geometric Mean of Ct values are Mean = Arithmetic mean of Ct values, stdev = standard deviation of Ct values, CV= coefficient variation.
Table 4. Bestkeeper, NormFinder and geNorm analysis on three different candidate miRNA internal controls. Geo Mean = geometric Mean of Ct values are Mean = Arithmetic mean of Ct values, stdev = standard deviation of Ct values, CV= coefficient variation.
Table 5. Bestkeeper, NormFinder and geNorm analysis on three different candidate miRNA internal controls. Geo Mean = geometric Mean of Ct values are Mean = Arithmetic mean of Ct values, stdev = standard deviation of Ct values, CV= coefficient variation.
Table 6. Bestkeeper, NormFinder and geNorm analysis on three different candidate miRNA internal controls. Geo Mean = geometric Mean of Ct values are Mean = Arithmetic mean of Ct values, stdev = standard deviation of Ct values, CV= coefficient variation.
Figure 1. Transgenic mouse model for detecting involvement of altered preimplantation miRNA content in transmitting traits across generations. a) timeline describing transient doxycycline (dox) introduction into WT female mice. Dox (40 mg/kg) was added to female mouse chow 48 hrs. before mating and then removed at mating. Its concentration in animals estimated (grey triangle based on known 48 hr t ½ of dox). b) scheme for how mating CSI-stressed, heterozygous transgenic, males with females pretreated with doxycycline can test whether reduced preimplantation embryo miR-34/449 levels participate in generating stress-related behaviours in female and suppressed sperm miR-34/449 levels in male offspring. WT: wild-type.
Figure 2. Induction of miR-34c levels in preimplantation embryos derived from mating CSI-stressed, tet-inducible miR-34c transgenic males. a) relative miR-34c levels in morula stage embryos derived from mating either unstressed WT males to females fed normal chow (first bar) or CSI-stressed WT males each mated to both females fed regular chow (second bar) or Dox (third bar). n = 11 for control WT, n = 6 for CSI WT No Dox and n = 5 for CSI WT Dox. Dox: doxycycline. Data are expressed as mean ± S.E.M. One-way Anova, multiple comparisons. N.S.= not significant. b) miR-34c level in morula stage embryos derived from mating either unstressed transgenic males to females fed normal chow (first bar) or CSI-stressed transgenic (Tg) males each mated to both females fed normal chow (second bar) or Dox (third bar). N = 8 for control WT, n = 5 for CSI WT No Dox and n = 5 for CSI WT Dox. Dox: doxycycline. Data are expressed as mean ± S.E.M. One-way Anova, multiple comparisons, N.S.= not significant. c) 5 independently derived CSI-stressed transgenic (tg) males were mated to both WT unstressed females pre-fed doxycycline (Dox) or regular chow (no Dox) and the fold-change in miR-34c level were measured in morula stage embryos derived from each mating. Data are expressed as mean ± S.E.M; Mann-Whitney test two-tailed, N.S.= not significant. c) miR-34c induction from the 2–4 cells stage through the blastocyst stage of preimplantation embryos derived from CSI stressed, tet-inducible transgenic males mated to both females pre-fed a control or dox-containing chow. Pools of 40–60 embryos were isolated at the indicated times. n = 3 for 4 cells and blastocysts and n = 5 for morula. Data are expressed as mean ± S.E.M; Mann-Whitney test two-tailed, N.S.= not significant. In all cases, miR-192 is used as an internal assay control because we found its ct values did not change significantly after any of the perturbations tested (see Supplemental Fig. S5).
Figure 3. Induction of miR-34c levels in morula stage mouse embryos derived from mating CSI stressed transgenic but not WT males, increases the levels of miR-449a/b and miR-34b in them. a-c) 5 independently derived CSI-stressed transgenic (Tg) males were mated with both WT unstressed females pre-fed regular chow (no Dox) or doxycycline (Dox) and the fold change in levels of miR-34/449 family members levels were measured in morula stage embryos derived from these mating. Data are expressed as mean ± S.E.M; Mann-Whitney test two-tailed, d) miR-34/449 family levels in morula stage embryos derived from mating CSI stressed WT males with WT unstressed females fed with regular chow or doxycycline. Data are expressed as mean ± S.E.M; two-way ANOVA multiple comparison, N.S.= not significant.
Figure 4. Restoring miR-34c/449 levels in PIEs derived from mating CSI-stressed males suppresses stress -associated phenotypes, and at least partially prevents reduced miR-34c and miR-449a levels in sperm normally found when these embryos mature into female and male adults, respectively. a) timeline for assessing stress-related phenotypes in F1 wild-type and transgenic female and male littermates of control and CSI stressed males. b and d) measurement of the % time mice spent in the open arms of the EPM for WT and transgenics (Tg) littermates derived from control or CSI-stressed transgenic males mated to either control-fed or dox pre-fed females. c and e) interaction time in social interaction with juvenile assay for WT and transgenics (Tg). Control WT n = 50, CSI WT No dox n = 39, CSI WT dox n = 44, control Tg n = 20, CSI Tg No Dox n = 19 and CSI Tg Dox n = 24. Data are expressed as mean ± S.E.M. Ordinary one-way Anova with multiple comparisons (Tukey correction). N.S.= not significant. d, e) measurement of miR-34c and miR-449a in sperm of male WT and transgenic (f, g) littermates of females analysed in B and C above. Control WT n = 8, CSI WT No Dox n = 9, CSI WT Dox n = 7, control Tg n = 8, CSI Tg No Dox n = 10 and CSI Tg Dox n = 9. Expression was normalized to that for miR-192. Data are expressed as mean ± S.E.M; Kruskal-Wallis test with multiple comparisons, dox: doxycycline, tg: transgenic, WT: wild-type. N.S.= not significant.
Figure 5. miR-34c alters the level of miR-449a,b in morula stage mouse embryos at the post-transcriptional level. a) levels of Pri-34bc and Pri-449ab were measured in morula stage embryos derived from the mating of unstressed and stressed WT, Mann-Whitney test, control WT n = 8, CSI WT No dox n = 6 for Pri-34bc and control WT n = 8, CSI WT No Dox n = 6 for Pri-449ab, WT: wild-type, CSI: chronic social instability. b) levels of Pri-34bc and Pri-449ab were measured in morula stage embryos derived from the mating of unstressed, stressed and stressed inducible miR-34c transgenic males. In the experiments, the CSI F0 fathers are the same between no dox and dox groups. Expression was normalized to that for GAPDH. Data are expressed as mean ± S.E.M, Kruskal-Wallis test with multiple comparisons. Dox: doxycycline, Tg: transgenic, CSI: chronic social instability. Control Tg n = 8, CSI Tg No Dox n = 5 and CSI Tg Dox n = 5. N.S.= not significant.
Figure 6. Elevation of miR-34c levels in human embryonic stem cells, increases the levels of miR-34b and miR-449a and b in them, but not at the transcriptional level. a) CHB-04 human embryonic stem cells were transfected with a random control miRNA (control) or miR-34c mimic 48 hrs. later the levels of miRnas indicated were assayed. n = 4. Data are expressed as mean ± S.E.M; Mann-Whitney test two-tailed, b) Pri-34bc and Pri-449ab levels were measure 48 hrs after transfecting CHB-04 human embryonic stem cells, expression was normalized to that for ACTB. Data are expressed as mean ± S.E.M. Mann-Whitney test two tailed, n = 3 N.S.= not significant.
