In vitro development of IVF‐derived bovine embryos following cytoplasmic microinjection for the episomal expression of the IGF2 gene

Abstract

Important genomic imprinting changes usually occur following the in vitro production (IVP) of bovine embryos, especially in the imprinting pattern of components of the IGF system. This study aimed to evaluate the effects of a transient episomal overexpression of the IGF2 gene in bovine IVP embryos following embryo cytoplasmic microinjection (CMI) at the 1‐cell stage on embryo survival, early and late developmental kinetics and morphological quality up to Day 7 of development. Selected cumulus–oocyte complexes (COCs) were matured and fertilized in vitro and subsequently segregated into six experimental groups: non‐CMI control group and five CMI groups at increasing doses (0, 10, 20, 40 and 80 ng/μl) of a GFP vector built for the episomal expression of bovine IGF2. Zygote CMI was effective in delivering the expression vector into the ooplasm, irrespective of the groups, with 58% of positive GFP fluorescence in Day 7 blastocysts. Considering developmental rates and late embryo kinetics, the 10‐ng/μl CMI vector dose promoted a lower blastocyst rate (10.4%), but for blastocysts at more advanced stages of development (93.0% blastocysts and expanded blastocysts), and higher number of cells (116.0 ± 3.0) than non‐CMI controls (23.3%, 75.0% and 75.0 ± 6.8 were obtained, respectively). In conclusion, CMI at the 1‐cell stage did not compromise subsequent in vitro development of surviving embryos, with the 10‐ng/μl group demonstrating a possible growth‐promoting effect of the IGF2 gene on embryo development, from the 1‐cell to the blastocyst stage.

Promoter‐specific expression of the imprinted IGF2 gene in bovine oocytes and pre‐implantation embryos

“…Four independent replicates for the in vitro production (IVP) of bovine embryos were performed, according to our established procedures (Campagnolo et al, 2020). Bovine ovaries from Bos taurus var.…”

“…A solution containing 10 5 IU/mL sodium penicillin, 10 mg/ml streptomycin sulphate and 25 mg/ml amphotericin B (GIBCO-BRL1, Life Technologies, Grand Island, NY, USA) was also added to the medium in the proportion of 1% (v/v) of the final IVM medium (1:100). Procedures for in vitro sperm capacitation and IVF were based on Parrish et al, (1986), modified by Campagnolo et al, (2020). Briefly, frozen-thawed bovine sperm cells were segregated by Percoll ® gradient with Sperm-TALP medium.…”