Abstract

We have previously demonstrated a positive association of follistatin mRNA abundance with bovine oocyte competence. Furthermore, exogenous follistatin supplementation during the early stages of in vitro bovine embryo development (before embryonic genome activation) can reduce time to first cleavage, increase proportion of embryos developing to the blastocyst stage, and increase trophectoderm cell numbers, suggesting a potential role for follistatin in bovine early embryonic development. However, the requirement of endogenous follistatin for early embryogenesis in cattle has not been directly tested. Thus, the aim of the present study was to determine the requirement of follistatin for early embryonic development using small interfering RNA (siRNA)- based knockdown procedures. Small interfering RNA corresponding to exons 2 (siRNA 2) and 3 (siRNA 3) of the bovine follistatin gene were synthesized, and the optimal dose of each siRNA resulting in maximal reduction in follistatin mRNA (at the 4-cell stage) following microinjection into presumptive zygotes was determined. Injection of follistatin siRNA 2 or siRNA 3 resulted in a >80% decrease in follistatin mRNA abundance in 4-cell embryos, but mRNA abundance for 5 housekeeping genes and the oocyte-specific gene JY-1 was not affected. Effects of follistatin siRNA injection on follistatin protein abundance were evaluated by immunofluorescence staining of 16-cell embryos. Follistatin immunoreactivity was dramatically reduced in siRNA-treated v. uninjected embryos. Upon validation, the effects of follistatin siRNA on early embryonic development were investigated. Cumulus–oocyte complexes were harvested from ovaries obtained from a local abattoir, matured and fertilized in vitro. Sixteen to 18 h following fertilization, denuded presumptive zygotes (25–30 per treatment, n = 4 replicates) were microinjected with (1) follistatin siRNA 2, (2) negative control (nonspecific) siRNA, (3) sham (water), or (4) served as uninjected controls. After injections, embryos were cultured in KSOM medium supplemented with 0.3% BSA. Proportions of embryos reaching the 2-cell stage within 30 h (early cleaving), 30–36 h (late cleaving), and within 48 h post-fertilization (total cleavage rate) were recorded. Number of embryos reaching the 8–16-cell stage was recorded 72 h after fertilization, and embryos were cultured in fresh KSOM medium supplemented with 0.3% BSA and 10% fetal bovine serum until day 7. Injection of follistatin siRNA 2 did not affect proportion of early and late cleaving embryos (21 v. 19% and 41 v. 37%) and total cleavage rate (80 v. 81%). However, injection of follistatin siRNA 2 decreased the proportion of embryos reaching the 8–16-cell stage (41 v. 59%) and percentage blastocyst development (12 v. 27%, P < 0.05). Experiments were repeated, and effects of follistatin siRNA 3 determined (25–30 embryos per treatment, n = 4 replicates). Similar results were obtained as for follistatin siRNA 2 injection. Results support a requirement of endogenous follistatin for bovine early embryogenesis.