Fig 1.
Experimental design of this study.
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.
Fig 2.
Stage-specific effects of follistatin treatment on bovine early embryo development.
Effect of exogenous follistatin treatment during pre-compaction (d 1–3), peri-/post-compaction (d 4–7) and entire period (d 1–7) of in vitro embryo culture on; (A) First embryonic cleavage (30hpi). (B) Total cleavage rate (48hpi). (C) Proportion of embryos developing to the 8–16 cell stage on 3 d post insemination (pi). (D) Blastocyst rate (7 dpi). (E) Rate of expanded blastocysts (8 dpi). 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).
Fig 3.
Stage-specific effects of follistatin treatment on bovine blastocyst cell allocation.
Effect of exogenous follistatin supplementation during pre-compaction (d 1–3), peri-/post-compaction (d 4–7) and entire period (d 1–7) of in vitro embryo culture on; (A) Total cell number. (B) Number of TE cells and (C) 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).
Fig 4.
Stage-specific effects of follistatin treatment on mRNA expression of genes involved in TE cell lineage determination (CDX2, TFAP2C and BMP4) and ICM pluripotency (NANOG) in bovine d7 blastocysts.
Effect of exogenous follistatin supplementation during pre-compaction (d 1–3), peri-/post-compaction (d 4–7) and entire period (d 1–7) of in vitro embryo culture on; (A) CDX2, (B) BMP4, (C) TFAP2C and (D) NANOG 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 (P<0.05).
Fig 5.
Stage-specific effects of follistatin treatment on protein abundance of TE cell lineage markers (CDX2 and BMP4) in bovine d7 blastocysts.
Effect of exogenous follistatin supplementation during pre-compaction (d 1–3), peri-/post-compaction (d 4–7) and entire period (d 1–7) of in vitro embryo culture on (A) CDX2 and (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 (P<0.05).