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 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 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

Regulation and Regulatory Role of WNT Signaling in Potentiating FSH Action during Bovine Dominant Follicle Selection

<div><p>Follicular development occurs in wave like patterns in monotocous species such as cattle and humans and is regulated by a complex interaction of gonadotropins with local intrafollicular regulatory molecules. To further elucidate potential mechanisms controlling dominant follicle selection, granulosa cell RNA harvested from F1 (largest) and F2 (second largest) follicles isolated at predeviation (PD) and onset of diameter deviation (OD) stages of the first follicular wave was subjected to preliminary RNA transcriptome analysis. Expression of numerous WNT system components was observed. Hence experiments were performed to test the hypothesis that WNT signaling modulates FSH action on granulosa cells during follicular waves. Abundance of mRNA for WNT pathway members was evaluated in granulosa cells harvested from follicles at emergence (EM), PD, OD and early dominance (ED) stages of the first follicular wave. In F1 follicles, abundance of <i>CTNNB1</i> and <i>DVL1</i> mRNAs was higher and <i>AXIN2</i> mRNA was lower at ED versus EM stages and <i>DVL1</i> and FZD6 mRNAs were higher and <i>AXIN2</i> mRNA was lower in F1 versus F2 follicle at the ED stage. Bovine granulosa cells were treated in vitro with increasing doses of the WNT inhibitor IWR-1+/− maximal stimulatory dose of FSH. IWR-1 treatment blocked the FSH-induced increase in granulosa cell numbers and reduced the FSH-induced increase in estradiol. Granulosa cells were also cultured in the presence or absence of FSH +/− IWR-1 and hormonal regulation of mRNA for WNT pathway members and known FSH targets determined. FSH treatment increased <i>CYP19A1</i>, <i>CCND2</i>, <i>CTNNB1</i>, <i>AXIN2</i> and <i>FZD6</i> mRNAs and the stimulatory effect on <i>CYP19A1</i> mRNA was reduced by IWR-1. In contrast, FSH reduced <i>CARTPT</i> mRNA and IWR-1 partially reversed the inhibitory effect of FSH. Results support temporal and hormonal regulation and a potential role for WNT signaling in potentiating FSH action during dominant follicle selection.</p></div

Effect of FSH and WNT signaling inhibitor (IWR-1) treatment on CTNNB1 and AXIN2 protein abundance in bovine granulosa cells.

<p>Relative abundance of AXIN2 (A) and CTNNB1 (B) proteins (as determined by Western blot analysis and densitometry) in bovine granulosa cells cultured in the presence or absence of 0.5 ng/ml FSH plus 1.0 µM IWR-1. Results depict mean +/− SEM for four replicate experiments with relative protein abundance for CTNNB1 and AXIN2 normalized relative to ACTIN. ^A,B,C P<0.05.</p

Effect of IWR-1 mediated WNT signaling inhibition on FSH-induced regulation of FSH target gene (<i>CYP19A1</i>, <i>CCND2</i> and <i>CARTP</i>T) mRNA expression in bovine granulosa cells.

<p>Quantitative RT-PCR analysis was performed to determine the effects of treatment with 0 or 0.5 ng/ml FSH in the presence or absence of 1.0 µM IWR-1 on <i>CYP19A1 (A)</i>, <i>CCND2 (B) and CARTPT (C)</i> mRNA expression in cultured granulosa cells. Expression of mRNA for genes of interest was normalized relative to abundance of mRNA for <i>ACTIN</i> as internal control. Bars represent mean +/− SEM for four experiments. ^A,B P<0.05.</p

Effect of FSH and WNT signaling inhibitor (IWR-1) treatment on <i>FZD6</i>, <i>AXIN2</i>, <i>CTNNB1</i> and DVL1 <i>mRNA</i> abundance in bovine granulosa cells.

<p>Quantitative RT-PCR analysis was performed to determine the effects of treatment with 0 or 0.5 ng/ml FSH in the presence or absence of 1.0 µM IWR-1 on <i>FZD6 (A)</i>, <i>CCND2 (B) and CARTPT (C)</i> mRNA expression in cultured granulosa cells. Expression of mRNA for genes of interest was normalized relative to abundance of mRNA for <i>ACTIN</i> as internal control. Bars represent mean +/− SEM for four experiments. ^A,B P<0.05.</p

Effect of increasing concentrations of WNT signaling inhibitor IWR-1 on FSH stimulated estradiol production (A) and cell numbers (B) for bovine granulosa cells.

<p>Granulosa cells were treated with 0, 0.1 µM, 1.0 µM and 10 µM concentration of IWR-1 in the presence and absence of maximal stimulatory dose (0.5 ng/ml) of FSH. Each column represents mean± SEM for four independent experiments. ^a,b,c,d,e,f P<0.05.</p