Recombinant Human Bone Morphogenetic Protein 6 Enhances Oocyte Reprogramming Potential and Subsequent Development of the Cloned Yak Embryos

Abstract

This study investigated the effects of bone morphogenetic protein 6 (BMP6) supplementation in the medium during in vitro maturation (IVM) on the developmental potential of oocytes and in the subsequent development of cloned yak embryos. Cumulus-oocyte complexes (COCs) were aspirated from the antral follicles of yak ovaries and cultured with different concentrations of recombinant human BMP6 in oocyte maturation medium. Following maturation, the metaphase II (MII) oocytes were used for somatic cell nuclear transfer (SCNT), and these were cultured in vitro. The development of blastocysts and cell numbers were detected on day 8. The apoptosis and histone modifications of yak cloned blastocysts were evaluated by detecting the expression of relevant genes and proteins (Bax, Bcl-2, H3K9ac, H3K18ac, and H3K9me3) using relative quantitative RT-PCR or immunofluorescence. The presence of 100 ng/mL BMP6 significantly enhanced the oocyte maturation ratios (66.12 -2.04% vs. 73.11 -1.38%), cleavage rates (69.40 -1.03% vs. 78.16 -0.93%), and blastocyst formation rates (20.63 -1.32% vs. 28.16 -1.67%) of cloned yak embryos. The total blastocysts (85.24 -3.12 vs. 103.36 -5.28), inner cell mass (ICM) cell numbers (19.59 -2.17 vs. 32.20 -2.61), and ratio of ICM to trophectoderm (TE) (22.93 -1.43% vs. 31.21 -1.62%) were also enhanced ( p < 0.05). The ratio of the Bax to the Bcl-2 gene was lowest in the SCNT + BMP6 groups ( p < 0.05). The H3K9ac and H3K18ac levels were increased in SCNT + BMP6 groups ( p < 0.05), whereas the H3K9me3 level was decreased; the differences in blastocysts were not significant ( p > 0.05). These study results demonstrate that addition of oocyte maturation medium with recombinant BMP6 enhances yak oocyte developmental potential and the subsequent developmental competence of SCNT embryos, and provides evidence that BMP6 is an important determinant of mammalian oocyte developmental reprogramming.

Sperm-borne small RNAs regulate α-tubulin acetylation and epigenetic modification of early bovine somatic cell nuclear transfer embryos

“…We also observed that the level of H3K9me3 in the C-NT group was significantly higher than that in the IVF group at the 2PN and two-cell stages. This finding is consistent with the previous finding in cloned bovine embryos (Pan, He et al 2015),…”

“…These epigenetic markers on chromatin were thought to be at the core of genome reprograming after fertilization or SCNT (Zhao, Whyte et al 2010). To clarify the underlying mechanisms, we focused on the expression level of H3K9me3, which is associated with the repression of chromatin, and the formation of heterochromatin (Pan, He et al 2015). Our data showed that H3K9me3 was detected only in female pronuclei during the pronuclear period in IVF embryos.…”

Oocyte maturation-related gene expression in the canine oviduct, cumulus cells, and oocytes and effect of co-culture with oviduct cells on in vitro maturation of oocytes

“…BMP6, part of the TGF-β superfamily, plays a critical role in ovarian function and fertility through bidirectional communication between granulosa cells and oocytes [38]. To date, very few studies have investigated effects of BMP6 on mammalian oocytes and embryos; however, recent research demonstrated that treatment of the culture medium with BMP6 enhanced the maturation rate of oocytes and developmental competence of embryos [39]. We demonstrated that BMP6 expression was markedly increased in CCs and OOs derived from the estrus stage, while OCs showed significantly high gene expression of BMP6 in the diestrus stage.…”

Low oxygen concentration improves yak oocyte maturation and inhibits apoptosis through HIF‐1 and VEGF

“…Immature COCs were washed three times with tissue culture medium, consisting of TCM‐199 (Gibco B.R.L. ), 50 μg/ml gentamicin, 200 mM pyruvate, 1 μg/ml β‐oestradiol (E2), 100 μg/ml follicle‐stimulating hormone (FSH) and 10% FBS (Pan, He, et al., 2015). Then, the COCs were randomly divided into four groups, and approximately 40–50 COCs were cultured in 400 µl of IVM medium in four‐well dishes (Nunc, Roskilde, Denmark) and cultured with mineral oil overlay.…”

“…Immunofluorescence staining was described previously (Pan, He, et al., 2015; Wen et al., 2017) with some modifications. Briefly, different groups of COCs were fixed in PBS with 4% paraformaldehyde for 1 h at RT, and COCs were washed 3 times with PBS containing 2% BSA.…”