miR-212 specifically regulates bovine FIGLA <i>in vitro</i> in HeLa cells.

<p>(A) Western blot showing significant down-regulation of bovine FIGLA protein by ectopic expression of miR-212 in HeLa cells. β-Actin was used as a loading control. (B) Repression of luciferase activity due to specific interaction between miR-212 and the predicted MRE in the <i>FIGLA</i> 3`UTR cloned in the luciferase construct. Repression was abolished when the MRE was mutated. Relative firefly luciferase values were determined by the ratio of firefly to renilla luciferase with the negative control (cells transfected with native <i>FIGLA</i> 3`UTR construct alone) set at 1. Each group represents the mean ± SEM of four wells for an experiment repeated six times with similar results.</p

Prediction of a miR-212 binding site in the 3`UTR of bovine <i>FIGLA</i> mRNA.

<p>The prediction was performed using MicroInspector. Both <i>FIGLA</i> isoforms contain the binding site.</p

miR-212 represses endogenous FIGLA expression in early embryos.

<p>Effect of miR-212 mimic microinjection on abundance of FIGLA protein in 8-cell embryos as determined by immuofluorescent staining with anti-FIGLA antibody (n = 2 pools of 20 embryos per treatment). Uninjected embryos and embryos injected with a nonspecific miRNA (cel-miR-67N) mimic were used as controls. Nuclear DNA was stained with DAPI.</p

Analysis of bovine <i>FIGLA</i> mRNA expression.

<p>(A) Northern blot analysis of <i>FIGLA</i> transcript. (B) Expression of <i>FIGLA</i> mRNA in various bovine tissues. (C) Expression of <i>FIGLA</i> mRNA in bovine fetal ovaries of different developmental stages. (D) Quantitative expression of <i>FIGLA</i> mRNAs in granulosa cells, cumulus cells and GV oocytes. Data were normalized relative to abundance of endogenous control ribosomal protein S18 (RPS18) and are shown as mean <u>+</u> SEM (n=3).</p

Expression of <i>FIGLA</i> mRNA and protein during oocyte maturation and early embryonic development.

<p>(A) Analysis of steady-state levels of <i>FIGLA</i> mRNA in <i>in </i><i>vitro</i> produced bovine pre-implantation stage embryos. The oocyte and embryo samples used in this experiment included GV and MII stage oocytes, pronuclear (PN), 2-cell (2C), 4-cell (4C), 8-cell (8C), 16-cell (16C), morula (MO) and blastocyst (BL) stage embryos. Data were normalized relative to abundance of exogenous control (GFP) RNA and are shown as mean <u>+</u> SEM (n = 4 pools of 10 embryos per stage). Different letters indicate statistical difference (P < 0.05). (B) Western blot analysis of FIGLA protein expression in bovine oocytes and early embryos using antibodies specifically against bovine FIGLA (25 oocytes or embryos per lane).</p

Expression analysis of miR-212.

<p>(A) Tissue distribution of miR-212 analyzed by quantitative real time PCR. Data were normalized relative to abundance of endogenous control ribosomal protein S18 (RPS18) and abundance expressed as relative fold change using the sample with the lowest value as the calibrator (mean ± SEM; n = 4 per tissue). (B) Relative abundance of miR-212 in bovine oocytes and <i>in </i><i>vitro</i> produced bovine embryos. Data were normalized to abundance of miR-125b mRNA and expressed as relative fold change using the sample with the lowest value as the calibrator (mean ± SEM; n = 4 pools of five oocytes/embryos each). Different letters indicate statistical difference (P< 0.05).</p

Stage-specific effects of follistatin treatment on mRNA expression of genes involved in TE cell lineage determination (<i>CDX2</i>, <i>TFAP2C and BMP4</i>) and ICM pluripotency (<i>NANOG</i>) in bovine d7 blastocysts.

<p>Effect of exogenous follistatin supplementation during pre-compaction (d 1–3), peri-/post-compaction (d 4–7) and entire period (d 1–7) of <i>in vitro</i> embryo culture on; <b>(A)</b> <i>CDX2</i>, <b>(B)</b> <i>BMP4</i>, <b>(C)</b> <i>TFAP2C</i> and <b>(D)</b> <i>NANOG</i> transcript abundance as determined by real-time PCR in bovine blastocysts collected on d 7 after insemination. Values are shown as the mean ± SEM of the data collected from 6 replicates (n = 25–30 zygotes/treatment in each replicate). Values accompanied with different letters across the treatments indicate significant difference (<i>P</i><0.05).</p

Pre- and Peri-/Post-Compaction Follistatin Treatment Increases <i>In Vitro</i> Production of Cattle Embryos

<div><p>Our previous studies demonstrated that maternal (oocyte derived) follistatin (FST) expression is positively associated with bovine oocyte competence and exogenous follistatin treatment during the pre-compaction period of development (d 1–3 post insemination) is stimulatory to bovine early embryogenesis <i>in vitro</i> [blastocyst rates and cell numbers/allocation to trophectoderm (TE)]. In the present study, bovine embryos were treated with exogenous follistatin during d 1–3, d 4–7 and d 1–7 post insemination to test the hypothesis that embryotropic effects of exogenous follistatin are specific to the pre-compaction period (d 1–3) of early embryogenesis. Follistatin treatment during d 4–7 (peri-/post-compaction period) of embryo culture increased proportion of embryos reaching blastocyst and expanded blastocyst stage and total cell numbers compared to controls, but blastocyst rates and total cell numbers were lower than observed following d 1–3 (pre-compaction) follistatin treatment. Follistatin supplementation during d 1–7 of embryo culture increased development to blastocyst and expanded blastocyst stages and blastocyst total cell numbers compared to d 1–3 and d 4–7 follistatin treatment and untreated controls. A similar increase in blastocyst <i>CDX2</i> mRNA and protein (TE cell marker) was observed in response to d 1–3, d 4–7 and d 1–7 follistatin treatment. However, an elevation in blastocyst BMP4 protein (TE cell regulator) was observed in response to d 1–3 and d 1–7, but not d 4–7 (peri-/post-compaction) follistatin treatment. In summary, our study revealed the potential utility of follistatin treatment for increasing the success rate of <i>in vitro</i> embryo production in cattle. Such results also expand our understanding of the embryotropic actions of follistatin and demonstrate that follistatin actions on blastocyst development and cell allocation to the TE layer are not specific to the pre-compaction period.</p></div

Stage-specific effects of follistatin treatment on bovine blastocyst cell allocation.

<p>Effect of exogenous follistatin supplementation during pre-compaction (d 1–3), peri-/post-compaction (d 4–7) and entire period (d 1–7) of <i>in vitro</i> embryo culture on; <b>(A)</b> Total cell number. <b>(B)</b> Number of TE cells and <b>(C)</b> number of ICM cells as determined after differential staining of resulting blastocyst on d 7 after insemination. Values are shown as the mean ± SEM of the data collected from 6 replicates (n = 25–30 zygotes/treatment in each replicate). Values accompanied with different letters across the treatments indicate significant difference (p<0.05).</p

Stage-specific effects of follistatin treatment on protein abundance of TE cell lineage markers (CDX2 and BMP4) in bovine d7 blastocysts.

<p>Effect of exogenous follistatin supplementation during pre-compaction (d 1–3), peri-/post-compaction (d 4–7) and entire period (d 1–7) of <i>in vitro</i> embryo culture on <b>(A)</b> CDX2 and <b>(B)</b> BMP4 protein abundance as determined by Western blot analysis in bovine blastocysts collected on d 7 after insemination. Values are shown as the mean ± SEM of the data collected from 4 replicates (n = 10 blastocyst/treatment in each replicate). Values accompanied with different letters across the treatments indicate significant difference (<i>P</i><0.05).</p