The effect of C–C motif chemokine ligand 2 supplementation on in vitro maturation of porcine cumulus-oocyte complexes and subsequent developmental competence after parthenogenetic activation

Abstract

Porcine embryos are used for a variety of applications. However, the maturation rate in vitro remains low, and novel in vitro maturation (IVM) techniques that facilitate the collection of mature oocytes are necessary. C-C motif chemokine ligand 2 (CCL2) is a key periovulatory chemokine present in cumulus-oocyte complexes (COCs). We aimed to examine the effects of CCL2 supplementation during IVM on oocyte maturation and embryonic development. The CCL2 concentration was significantly higher in porcine follicular fluid (pFF) derived from follicles >8 mm in size than in pFF derived from smaller follicles. There was a significant increase in CCL2 mRNA levels in all follicular cells after IVM compared with that before IVM. We analyzed the localization of CCL2 and its receptor, the CCL2 receptor, in follicular cells. During IVM, different concentrations of CCL2 were added to COCs cultured in a maturation medium. After IVM, the group treated with 100 ng/mL CCL2 showed significantly higher metaphase II rates than the control group. All CCL2-treatment groups showed a significant increase in intracellular glutathione levels and a significant decrease in reactive oxygen species levels, compared to the control. In CCs treated with 100 ng/mL CCL2, the mRNA levels of BAX, CASP3, and NPR2 were significantly decreased. Furthermore, the mRNA levels of SOD1, SOD2, and CD44 were significantly increased. In oocytes treated with 10 ng/mL CCL2, mRNA levels of BAX and CASP3 were significantly decreased, whereas, NRF2 and NPM2 were significantly increased. ERK1 exhibited significantly increased mRNA expression in both CCs and oocytes treated with 10 ng/mL CCL2. The protein expression ratio of phosphorylated ERK1/2 to total ERK1/2 was significantly increased in CCs treated with 10 ng/mL CCL2. After parthenogenetic activation, cleavage rates were significantly improved in the 100 ng/mL CCL2 treatment group, and blastocyst formation rates were significantly enhanced in the 10 ng/mL CCL2 treatment group. Overall, our results suggest that IVM medium along with CCL2 improves porcine oocyte maturation and the development of parthenogenetically-activated embryos.

BDE-47 Induces Mitochondrial Dysfunction and Endoplasmic Reticulum Stress to Inhibit Early Porcine Embryonic Development

“…In this research, Parthenogenetic embryos were employed. Because parthenogenesis technology is mature, the embryo development state is good, a lot of embryos with diploid features are easy to obtain, and there is no bias of paternal contribution; porcine parthenogenetic embryos were used as an excellent model to detect early embryo development [36,37]. We found that exposure to 100 µM BDE-47 significantly decreased the blastocyst rate.…”

Supplementation with fibroblast growth factor 7 during in vitro maturation of porcine cumulus-oocyte complexes improves oocyte maturation and early embryonic development

“…Mature oocytes were gathered 42-44 h post IVM, and their internal levels of glutathione (GSH) and reactive oxygen species (ROS) were evaluated using a previously described method (30). Cell Tracker Blue 4-chloromethyl-6, 8-difluoro-7-hydroxycoumarin (CMF2HC) and the 2' , 7'dichlorodihydrofluorescein diacetate (H2DCFDA) (Invitrogen, Carlsbad, CA, USA) indicators were used to detect the GSH and ROS levels within the ooplasm, respectively.…”

“…After IVM, Metaphase II (MII) oocytes from each group were chosen and rinsed with a calcium-free TLH/PVA medium. The process of Parthenogenetic activation (PA) was carried out using a standard method (30). MII oocytes were rinsed twice in a 280 mM mannitol solution containing 0.01 mM CaCl 2 and 0.05 mM MgCl 2 .…”

“…The chamber was connected to an electrical pulse generator (LF101; Nepa Gene, Chiba, Japan), and the MII oocytes were stimulated by two direct-current pulses of 60 s at 120 V/mm each. Following PA, activated oocytes from all groups were immediately transferred to an in vitro culture (IVC) medium (30), composed of porcine zygote medium containing 5 µg/mL cytochalasin B. The oocytes were incubated for 4 h at 39 • C in a humid atmosphere composed of 5% CO 2 and 95% N 2 .…”

“…Developing embryos were divided into 2-3, 4-5 and 6-8 cell embryonic stages, one-cell embryos, and fragmented embryos. On the seventh day, the formation of blastocysts was evaluated (30). Based on their development, the blastocysts were divided into early blastocyst, expanded blastocyst, and hatched blastocyst stages.…”