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.

Follistatin treatment modifies DNA methylation of the CDX2 gene in bovine preimplantation embryos

“…Follistatin transcripts were higher in BCB positive (BCB+) high‐quality oocytes compared to BCB− oocytes (Ashry et al, 2015). Functional studies revealed that maternally derived follistatin is essential for early development in bovine preimplantation embryos and that exogenous follistatin supplementation during the first 72 hr of in vitro embryo culture improves the developmental capacity of bovine (Ashry et al, 2015; Ashry, Lee, Folger, Rajput, & Smith, 2018; Ashry, Rajput, et al, 2018; Lee, Bettegowda, Wee, Ireland, & Smith, 2009; Patel et al, 2007; Zhenhua et al, 2017) and Rhesus macaque embryos (VandeVoort, Mtango, Lee, Smith, & Latham, 2009). Interestingly, in vitro‐produced (IVP) blastocysts derived from embryos treated with follistatin exhibit higher trophectoderm (TE) cell numbers and increased expression of caudal‐type homeobox 2 ( CDX2 ) messenger RNA (mRNA; Lee et al, 2009; Zhenhua et al, 2017).…”

Follistatin supplementation induces changes in CDX2 CpG methylation and improves in vitro development of bovine SCNT preimplantation embryos

“…A growing body of evidence supports an embryotrophic role of follistatin in IVF embryos [ 11 – 15 , 27 , 28 ]. Exogenous follistatin supplementation (10ng/ml) during IVC1, improved blastocyst rates of bovine SCNT embryos [ 9 ].…”

“…Numerous studies in our laboratory showed that exogenous follistatin supplementation during in vitro embryo culture improves the developmental competence of bovine preimplantation embryos produced under different conditions. These include conventional IVF [ 12 – 14 ], IVF using poor quality oocytes [ 11 ], and IVF using sex sorted semen [ 15 ]. In general, follistatin treatment increases blastocyst rates, CDX2 expression and trophectoderm (TE) cell number on day 7 of development.…”