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 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

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

Experimental design of this study.

<p>After 20 hrs of fertilization, presumptive zygotes were cultured from day (d) 1–3 (pre-compaction period) in the presence and absence of 10 ng/ml follistatin (FST). At day 3, 8–16 cell embryos from control and follistatin treatment group were further cultured from day 4–7 (peri-/post-compaction period) in the absence and presence of follistatin (10 ng/ml). Early cleavage, total cleavage, 8–16 cell, d7 blastocyst and expanded blastocyst stages of pre-implantation embryo development were recorded at in all the treatment group including untreated control, FST d 4–7, FST d 1–3, and FST d1-7. Blastocysts from all the treatment groups were collected at day 7 and analyzed for cell number (total, TE, ICM) and mRNA and protein abundance for select markers/determinants of blastocyst cell lineage.</p