Mechanism of the adverse effect of hyaluronidase used for oocyte denudation on early development of bovine embryos

Summary Hyaluronidase is widely used in animal and human assisted reproductive technologies (ARTs) to remove cumulus cells around oocytes. However, adverse effects of hyaluronidase treatment, such as increased rates of degeneration and parthenogenesis, have been found after treatment of human and mouse oocytes. Currently, the mechanism(s) of the detrimental effects are unclear. The present study was initiated to identify the mechanism of adverse responses to hyaluronidase treatment in bovine oocytes and early embryos. Cumulus cells were removed from cumulus–oocyte complexes (COCs) with or without hyaluronidase and the oocytes were subjected to intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Significantly lower rates of blastocyst formation were obtained in the hyaluronidase treatment group after ICSI (22.4%) and IVF (21.2%) compared with the non-hyaluronidase control groups: 36.1% after ICSI and 30.4% after IVF. Next, we examined the effect of hyaluronidase on parthenogenetic development rates and on the cytoplasmic levels of free calcium ions (Ca2+), reactive oxygen species (ROS) and reduced glutathione (GSH). No differences in parthenogenesis rates were found between treated and untreated groups. Ca2+ levels in oocytes from the hyaluronidase treatment group indicated using mean fluorescence intensity were significantly higher (68.8 ± 5.3) compared with in the control group (45.0 ± 2.5). No differences were found in the levels of ROS or GSH between the treated and untreated groups. We conclude that hyaluronidase might trigger an increase in Ca2+ levels in oocytes, resulting in a decreased potential for normal embryonic development.

The influence of the duration of in vitro maturation and incubation with an activating agent on the hatching capacity of bovine parthenotes - short communication

The period of both in vitro maturation (IVM) and incubation with oocyte activators affects the blastocyst yield following parthenogenetic activation (PA). Nevertheless, it is still unknown how these conditions impact the expansion and hatching rates of bovine parthenogenetic blastocysts. The objective of this study was to assess the influence of the duration of IVM and exposure to the activating agent, 6-dimethylaminopurine (6-DMAP), on a number of developmental parameters in bovine parthenotes, including: Cleavage, blastocyst formation, expansion, and hatching. Slaughterhouse oocytes were subjected to different periods of IVM. Subsequently, eggs were first parthenogenetically activated for five minutes with ionomycin and then incubated for distinct lengths of time with a second activator, 6-DMAP. The treatments were: a) Control: 22 h IVM/4 h 6-DMAP; b) 22 h IVM/5 h 6-DMAP; c) 24 h IVM/4 h 6-DMAP; and d) 24 h IVM/5 h 6-DMAP. Developmental stages were evaluated at day 4 and day 8 of in vitro culture (IVC). No differences were detected in most developmental parameters. However, the duration of IVM and incubation with 6-DMAP significantly affected (P<0.05) hatching capacity considering the number of blastocysts (Hatch./Blast.). Also, this same variable was higher (P<0.05) in group b) 22 h IVM/5 h 6-DMAP (45.89 ± 12.59%), as compared to c) 24 h IVM/4 h 6-DMAP (6.67 ± 6.67%). In conclusion, the length of IVM and incubation with 6-DMAP influenced parthenogenetic development, where 22 h IVM/5 h 6-DMAP was the condition producing the highest Hatch./Blast. rate in bovine parthenotes.

Bovine somatic cell nuclear transfer using mitomycin C-mediated chemical oocyte enucleation

SummaryChemical oocyte enucleation holds the potential to ease somatic cell nuclear transfer (SCNT), although high enucleation rates remain limited to micromanipulation-based approaches. Therefore, this study aimed to test mitomycin C (MMC) for use in bovine functional chemical oocyte enucleation. Incubation of denuded eggs in 10 µg ml−1 MMC for different periods did not affect most maturation rates (0.5 h: 85.78%A, 1.0 h: 72.77%B, 1.5 h: 83.87%A, and 2.0 h: 82.05%A) in comparison with non-treated controls (CTL; 85.77%A). Parthenogenetic development arrest by MMC was efficient at cleavage (CTL: 72.93%A, 0.5 h: 64.92%A,B, 1.0 h: 60.39%B,C, 1.5 h: 66.35%A,B, and 2.0 h: 53.84%C) and blastocyst stages (CTL: 33.94%A, 0.5 h: 7.58%B, 1.0 h: 2.47%C, 1.5 h: 0.46%C, and 2.0 h: 0.51%C). Blastocysts were obtained after nuclear transfer (NT) using MMC enucleation [NT(MMC): 4.54%B] but at lower rates than for the SCNT control [NT(CTL): 26.31%A]. The removal of the meiotic spindle after MMC incubation fully restored SCNT blastocyst development [NT(MMC+SR): 24.74%A]. Early pregnancies were obtained by the transfer of NT(MMC) and NT(MMC+SR) blastocysts to synchronized recipients. In conclusion, MMC leads to functional chemical oocyte enucleation during SCNT and further suggests its potential for application towards technical improvements.

DNA methylation analysis and editing in single mammalian oocytes

Mammalian oocytes carry specific nongenetic information, including DNA methylation to the next generation, which is important for development and disease. However, evaluation and manipulation of specific methylation for both functional analysis and therapeutic purposes remains challenging. Here, we demonstrate evaluation of specific methylation in single oocytes from its sibling first polar body (PB1) and manipulation of specific methylation in single oocytes by microinjection-mediated dCas9-based targeted methylation editing. We optimized a single-cell bisulfite sequencing approach with high efficiency and demonstrate that the PB1 carries similar methylation profiles at specific regions to its sibling oocyte. By bisulfite sequencing of a single PB1, the methylation information regarding agouti viable yellow (Avy)-related coat color, as well as imprinting linked parthenogenetic development competency, in a single oocyte can be efficiently evaluated. Microinjection-based dCas9-Tet/Dnmt–mediated methylation editing allows targeted manipulation of specific methylation in single oocytes. By targeted methylation editing, we were able to reverse Avy-related coat color, generate full-term development of bimaternal mice, and correct familial Angelman syndrome in a mouse model. Our work will facilitate the investigation of specific methylation events in oocytes and provides a strategy for prevention and correction of maternally transmitted nongenetic disease or disorders.

Improved functional oocyte enucleation by actinomycin D for bovine somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) allows animal cloning but remains technically challenging. This study investigated limitations to functional oocyte enucleation by actinomycin D (AD) as a means of making SCNT easier to perform. Denuding oocytes or inhibiting transcription before AD treatment revealed that the toxicity of this compound during bovine oocyte maturation is mediated by cumulus cells. Exposure of denuded oocytes to higher concentrations of AD (5–20μgmL−1) and stepwise reductions of the incubation period (from 14.0 to 0.25h) led to complete inhibition of parthenogenetic development. Bovine SCNT using this improved AD enucleation protocol (NT(AD)) restored cleavage rates compared with rates in the parthenogenetic and SCNT controls (P(CTL) and NT(CTL) respectively). However, NT(AD) was associated with increased caspase-3 activity in cleavage stage embryos and did not recover blastocyst rates. The removal of AD-treated oocyte spindle before reconstruction (NT(AD+SR)) improved embryo development and reduced caspase-3 activity to levels similar to those in the P(CTL) and NT(CTL) groups. Furthermore, mid-term pregnancies were achieved using NT(AD+SR) blastocysts. In conclusion, improvements in AD functional enucleation for bovine SCNT circumvents most cellular roadblocks to early embryonic development and future investigations must focus on restoring blastocyst formation.