Involvement of microRNA Lethal-7a in the Regulation of Embryo Implantation in Mice

MicroRNAs interact with multiple mRNAs resulting in their degradation and/or translational repression. This report used the delayed implantation model to determine the role of miRNAs in blastocysts. Dormant blastocysts in delayed implanting mice were activated by estradiol. Differential expression of 45 out of 238 miRNAs examined was found between the dormant and the activated blastocysts. Five of the nine members of the microRNA lethal-7 (let-7) family were down-regulated after activation. Human blastocysts also had a low expression of let-7 family. Forced-expression of a family member, let-7a in mouse blastocysts decreased the number of implantation sites (let-7a: 1.1±0.4; control: 3.8±0.4) in vivo, and reduced the percentages of blastocyst that attached (let-7a: 42.0±8.3%; control: 79.0±5.1%) and spreaded (let-7a: 33.5±2.9%; control: 67.3±3.8%) on fibronectin in vitro. Integrin-β3, a known implantation-related molecule, was demonstrated to be a target of let-7a by 3′-untranslated region reporter assay in cervical cancer cells HeLa, and Western blotting in mouse blastocysts. The inhibitory effect of forced-expression of let-7a on blastocyst attachment and outgrowth was partially nullified in vitro and in vivo by forced-expression of integrin-β3. This study provides the first direct evidence that let-7a is involved in regulating the implantation process partly via modulation of the expression of integrin-β3. (200 words)

Forced-expression of integrin-β3 partially nullifies the inhibitory action of let-7a on implantation.

<p>Eight-cell embryos were electroporated with let-7a precursor either with or without integrin β3 (Itgb3) RNA. (a) Expression of Itgb3 protein after electroporation. Each lane contained protein lysate from 40 blastocysts. The experiment was repeated 3 times. (b) Semi-quantitative comparison of the Itgb3 protein expression. (c) Attachment and (d) spreading of the resulting blastocysts <i>in vitro</i>. Forced-expression of integrin β3 partially reduced the inhibitory effects of let-7a on embryo attachment and outgrowth. Each datum point consisted of 25–30 embryos. The data represent the mean and standard errors (n = 3). (e) Implantation of the electroporated blastocyst after transfer to the uterine horn on day 3 of pseudopregnancy. The number of implanted embryos was counted on Day 8 of pregnancy. Five blastocysts were transferred to each horn (n = 14). Forced-expression of integrin β3 partially reduced the inhibitory effects of let-7a on implantation <i>in vivo</i>. *denotes P<0.05 (One way ANOVA).</p

MicroRNAs differentially expressed between dormant and activated blastocysts (p<0.05).

<p>MicroRNAs differentially expressed between dormant and activated blastocysts (p<0.05).</p

Expression of let-7 family members (in Ct values) in dormant and activated blastocysts.

<p>BD =  below detection limit.</p

Electroporation of RNA at the 8-cell embryo stage.

<p>(a) Electroporation of Cy3-labeled scramble RNA. Fluorescence was detected in all the electroporated embryos. (b) Relative level of mature let-7a in embryos after electroporation of pre-let-7a at the 8-cell embryo stage. Each experiment contained 5 embryos and the experiment was repeated at least 3 times. (*P<0.05, One-way ANOVA).</p

Precursor of let-7 inhibits embryo implantation <i>in vivo</i>.

<p>Eight-cell embryos were electroporated with precursor of let-7a or control RNA, and transferred into opposite uterine horns of mice on Day 3 of pseudopregnancy (5 embryos for each horn). Let-7a reduced the number of embryos implanted in the uterus. Statistical significant difference (P<0.05, Chi square) in the number of implantation sites was found on Day-8 of pregnancy (n = 18).</p

Pathways of differentially-regulated miRNAs.

<p>Pathways of differentially-regulated miRNAs.</p

Biological processes of the differentially-regulated miRNAs.

<p>Biological processes of the differentially-regulated miRNAs.</p

MicroRNA profiling of dormant and activated mouse blastocysts.

<p>(a) Supervised hierarchical clustering of the expression of 238 miRNAs in mouse dormant and activated blastocysts. Heat map shows normalized, log-transformed relative intensities of the studied miRNA. Red, green and black color represent low, high and mean expression levels of miRNAs, respectively. Genes with similar expression patterns are grouped together. (b) Validation of expression of miR-34a, -20a, -140, and -16 by qPCR without pre-amplification. The expression patterns of these miRNAs in dormant and activated blastocyst were similar to those determined by miRNA profiling. All values are calculated against the C_T values of dormant blastocysts and are presented as relative fold-change. (c) Relative expression of let-7a and let-7e in dormant, activated and normal Day 4 blastocysts as determined by qPCR without pre-amplification (^{a–b, c–d} denote P<0.05 between corresponding groups). (d) The expression of let-7 in human embryos.</p