Follistatin supplementation during in vitro embryo culture improves developmental competence of bovine embryos produced using sex-sorted semen

Abstract

Using sex-sorted semen to produce offspring of desired sex is associated with reduced developmental competence in vitro and lower fertility rates in vivo. The objectives of the present study were to investigate the effects of exogenous follistatin supplementation on the developmental competence of bovine embryos produced with sex-sorted semen and possible link between TGF-β regulated pathways and embryotrophic actions of follistatin. Effects of follistatin on expression of cell lineage markers (CDX2 and Nanog) and downstream targets of SMAD signaling (CTGF, ID1, ID2 and ID3) and AKT phosphorylation were investigated. Follistatin was supplemented during the initial 72 h of embryo culture. Exogenous follistatin restored the in vitro developmental competence of embryos produced with sex-sorted semen to the levels of control embryos produced with unsorted semen, and comparable results were obtained using sorted semen from three different bulls. The mRNA abundance for SMAD signaling downstream target genes, CTGF (SMAD 2/3 pathway) and ID2 (SMAD 1/5 pathway), was lower in blastocysts produced using sex-sorted versus unsorted semen, but mRNA levels for CDX2, NANOG, ID1 and ID3 were similar in both groups. Follistatin supplementation restored CTGF and ID2 mRNA in blastocysts produced using sex-sorted semen to levels of control embryos. Moreover, levels of phosphorylated (p)AKT (Ser-473 and Thr-308) were similar in embryos derived from sex-sorted and unsorted semen, but follistatin treatment increased pAKT levels in both groups. Taken together, results demonstrated that follistatin improves in vitro development of embryos produced with sex-sorted semen and such effects are associated with enhanced indices of SMAD signaling.

Keywords: AKT; Bovine; Follistatin; Oocytes quality; SMAD; Sorted semen; TGF-β.

Fig. 1.

Effects of follistatin supplementation during IVC1 on developmental progression of early bovine embryos produced with sex-sorted semen. Presumptive zygotes produced with unsorted or sorted semen (bull A, B and C) were cultured with or without 10 ng/ml recombinant human follistatin for 72 h, then 8–16- cell embryos were isolated, washed and cultured in fresh media without follistatin until d7 (n = 4 replicates, n = 25–30 zygote/group). Effects of follistatin on (A) early cleavage, (B) total cleavage, (C) development to 8–16 cell stage and (D) d7 blastocyst rate were determined. Data are expressed as mean ± SEM. Values with different superscripts across indicate treatment significant differences (P < 0.05).

Fig. 2.

Expression of cell lineage markers in bovine blastocysts produced with sex-sorted semen. The mRNA abundance for CDX2 (A) and Nanog (B) in d7 blastocyst produced with sorted or unsorted semen (n = 4 replicates, n = 10 blastocysts/pool) was determined by qRT−PCR. Expression was normalized relative to the abundance of RPS18 as a housekeeping gene. Data are expressed as mean ± SEM. Values with different superscripts across treatment indicate significant differences (P < 0.05).

Fig. 3.

Effects of follistatin treatment on AKT phosphorylation in early bovine embryos produced with sex-sorted semen. Presumptive zygotes produced with sorted or unsorted semen were cultured with or without 10 ng/ml follistatin for 10 h, then subjected to Western blot analysis for pAKT-Thr308, pAKT-Ser473, tAKT and actin (n = 6 replicates/phosphorylation site, n = 20 embryos/group). Data were normalized relative to the abundance of actin and are expressed as mean ± SEM. Values with different superscripts across treatments indicate significant differences (P < 0.05). Representative Western blot images are shown.

Fig. 4.

Expression of downstream targets of SMAD signaling in bovine blastocysts produced with sex-sorted semen. The mRNA abundance for CTGF (A), ID1 (B), ID2 (C) and ID3 (D) in d7 blastocyst produced with sorted or unsorted semen (n = 4 replicates, n = 10 blastocysts/pool) was determined by qRT-PCR. Expression was normalized relative to the abundance of RPS18 as a housekeeping gene. Data are expressed as mean ± SEM. Values with different superscripts across treatment indicate significant differences (P < 0.05).