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Although somatic cell nuclear transfer (SCNT) is a critical component of animal cloning, this approach has several issues. We previously introduced the cytoplasm injection cloning technology (CICT), which significantly improves the quality and quantity of cloned embryos. This study examined the residual status of fused cytoplasmic organelles, such as the endoplasmic reticulum (ER) and lysosomes, in the CICT group during early embryo development. We found that extra-cytoplasmic organelles stained using the ER-Tracker™ Green dye and LysoTracker™ Deep Red probe were fused and dispersed throughout the recipient oocyte and were still visible in day 8 blastocysts. We screened for ER stress, autophagy, and apoptosis-related genes to elucidate the association between the added organelles and improved embryo quality in CICT-cloned embryos. We found that CHOP, ATF4, ATG5, ATG7, and LC3 genes showed non-significantly up- or downregulated expression between CICT- and in vitro fertilization (IVF)-derived embryos but showed significantly (p < 0.05) upregulated expression in SCNT-cloned embryos. Surprisingly, a non-significant difference in the expression of some genes, such as ATF6 and caspase-3, was observed between the CICT- and SCNT-cloned embryos. Our findings imply that compared to conventional SCNT cloning, CICT-derived cloned embryos with additional cytoplasm have much higher organelle activity, lower autophagy, lower rates of apoptosis, and higher embryo development rates., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Cadmium (Cd) is a major environmental contaminant that has been linked to oocyte quality reduction and early embryo mortality in various in vivo studies. In this study, we investigated the mechanism of Cd-induced mitochondrial toxicity in bovine in vitro matured oocytes, primary cultured bovine cumulus cells, and in vitro developed bovine embryos. Cd significantly reduced PPARGC1A (PGC-1α) and nuclear respiratory factors, which leads to mitochondrial damage and hence reduction in oocyte maturation and embryo development. NAD-dependent deacetylase sirtuin-1 (SIRT1) is the upstream marker of PGC-1α and nuclear respiratory factors, and its activation significantly mitigated Cd-induced mitochondrial damage. For SIRT1 activation, we used Hesperetin (Hsp), a citrus flavonoid and a potent activator of SIRT1. The molecular docking approach was used to investigate the binding of hesperetin to bovine SIRT1, which revealed that hesperetin creates polar and non-polar interactions with residues that are reported essential for the activation of SIRT1. Furthermore, the SIRT1 enzymatic activity was measured in primary cultured bovine granulosa cells after hesperetin treatment. To further confirm the SIRT1-dependent effects of hesperetin we used a specific inhibitor of SIRT1 (EX527), which significantly (p < 0.05) reduced the effects of hesperetin on embryo mitochondria. Next, we treated hesperetin and Cd to early bovine embryos and discovered a significant (p 0.05) increase in PGC-1, NRF1, and NFE2L2 protein expression as well as embryo development recovery. Thus, we came to the conclusion that hesperetin can activate PGC-1 and nuclear respiratory factors via SIRT1, which can greatly reduce Cd-induced mitochondrial toxicity and promote mitochondrial biogenesis in early bovine embryos., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Kisspeptin (Kp), a multifunctional neuropeptide critical for initiating puberty and regulating ovulation, was reported to be expressed in mammalian ovaries. Fibronectin (FN), a major secretory product of granulosa cells, provided the extracellular environment for the cumulus cells during maturation. In the current study, we aimed to investigate the potential interplay between FN and Kp in bovine preantral follicles in the context of follicular development and quality. The results showed that Kp significantly reduced the follicular diameters after 14 days in culture, and this was prevented by the addition of FN. Follicles treated with Kp in the presence of FN showed lower levels of apoptotic cells compared to the Kp-treated group. The immunofluorescence analysis showed high levels of cyclooxygenase-2 (COX2), nuclear factor kappa B (NF-κB), and caspase 3, and low levels of sirtuin 1 (Sirt1) and Poly ADP-Ribose Polymerase 1 (PARP1) in the Kp-treated group compared to the control and FN-Kp co-treated groups. The protein expression levels of phosphoinositide 3 kinase (PI3K) increased significantly in the FN and FN-Kp combination treatment groups. Finally, we examined the signal pathway affecting the follicular development after Kp treatment. We detected a significant decrease in the mRNA levels of B-cell lymphoma 2 (BCL2), Sirt1, and PI3K, but the mRNA levels of NF-κB, Caspase3, COX2, P21, and P53 were significantly higher than in the control. Taken together, our results showed the importance of FN for preantral follicle developmental, and, for the first time, we reported that FN could neutralize the deleterious consequences of Kp, suggesting a potential role in the regulation of PI3K/Sirt1 signaling in bovine preantral follicle development., Competing Interests: Declaration of competing interest There are no declarations of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The mammalian Sirtuin family of seven enzymes, members of the NAD + -dependent histone deacetylase family that modify histones via direct deacetylation, is involved in the regulation of many antioxidant and oxidative stresses. In the present study, we explored the effects of nicotinamide (NAM)-induced oxidative stress on the in vitro development of bovine embryos, on the acetylation of histone H3 lysine 56 (H3K56ac) and on expression of apoptosis-related genes. Treatment with NAM (10, 20 or 40 mM for 24, 48 or 196 h) during IVC resulted in significantly decreased blastocyst formation (24 h: 38.8 vs. 33.1, 27.3 and 10.2%, with P > 0.05, P < 0.05 and P < 0.01, respectively; 48 h: 37.5 vs. 28.2, 13.4 and 0%, with P < 0.05 and P < 0.01, respectively; 196 h: 35.8 vs. 23.4, 0 and 0%, with P < 0.05, respectively). Treatment with NAM (20 and 40 mM for 24 h) resulted in increased intracellular reactive oxygen species (ROS) levels in 2-cell and blastocysts, and apoptotic cell numbers in blastocysts and decreased mitochondrial membrane potential (ΔΨ) in 2-cell embryos (P < 0.05). Polydatin (PD) and I-CBP112 rescued the 20 mM NAM-induced embryo developmental defects and reduced ROS levels and apoptotic cell numbers in blastocysts (P < 0.05). The gene expression of NF-κB, COX2 and p53 was significantly increased in the NAM-treated group. Immunofluorescence analysis confirmed that the protein levels of nuclear factor-kappa B (NF-κB) decreased significantly after PD and I-CBP112 treatment compared with the control (P < 0.05). High level of H3K56ac induced by NAM was decreased after PD and I-CBP112 treatment (P < 0.05). These findings suggest that NAM treatment induces high levels of H3K56 acetylation that may be involved in oxidative stress-induced bovine developmental defects, which can be tolerated by PD and I-CBP112 treatment., (Copyright © 2019 Elsevier Inc. All rights reserved.)

This study sought to modulate factors that reduce embryo quality in in vitro culture (IVC) systems. Over eight replicates, 3075 oocytes were cultured in in vitro maturation media containing various concentrations of lycopene, followed by in vitro fertilization and culture. The percentages of MII-stage oocytes, the presumptive zygotes that underwent cleavage and developed into blastocysts were significantly (P < 0.05) higher, the intracellular ROS concentrations reduced significantly (P < 0.05) in oocytes/blastocysts, TUNEL assay demonstrates reduced apoptosis and increased total cell number per blastocyst (P < 0.05), Immunocytochemistry confirmed that diminished protein expression of nuclear factor kappa B (NFκB), cyclooxygenase-2 (COX2), and 8-oxoguanine (indicated by ROS) and relative mRNA expression of the Caspase-3, NFκB, COX2, iNOS and BCL2-associated X (BAX) was significantly (P < 0.05) lower whereas the anti-apoptotic gene BCL2 was significantly (P < 0.05) higher in the 0.2 μM lycopene-supplemented group than the control. In conclusion, lycopene improves blastocyst quality by overcoming unfavorable conditions in in vitro culture systems., (Copyright © 2017 Elsevier Inc. All rights reserved.)