Difference in Developmental Kinetics of Y-Specific Monoclonal Antibody Sorted Male and Female In Vitro Produced Bovine Embryos

Abstract

Sex-related growth differences between male and female embryos remain an attractive subject for reproductive biologists. This study aimed to investigate the endogenous factors that play a crucial role in the pace of early development between male and female bovine embryos. Using sex pre-selected semen by Y-specific monoclonal antibodies for the production of bovine embryos, we characterized the critical endogenous factors that are responsible for creating the development differences, especially during the pre-implantation period between male and female embryos. Our results showed that at day seven, (57.8%) Y-sperm sorted in vitro cultured embryos reached the expanded blastocyst (BL) stage, whereas the X-sperm sorted group were only 25%. Y-BLs showed higher mRNA abundance of pluripotency and developmental competency regulators, such as Oct4 and IGF1-R. Interestingly, Y-sperm sorted BLs had a homogeneous mitochondrial distribution pattern, higher mitochondrial membrane potential (∆Ψ m ), efficient OXPHOS (oxidative phosphorylation) system and well-encountered production of ROS (reactive oxygen species) level. Moreover, Y-blastocysts (BLs) showed less utilization of glucose metabolism relative to the X-BLs group. Importantly, both sexes showed differences in the timing of epigenetic events. All these factors directly or indirectly orchestrate the whole embryonic progression and may help in the faster and better quality yield of BL in the Y-sperm sorted group compared to the X counterpart group.

Bovine Oviduct Epithelial Cell-Derived Culture Media and Exosomes Improve Mitochondrial Health by Restoring Metabolic Flux during Pre-Implantation Development

“…Approximately 18–20 h of post-insemination, the presumptive zygotes were cleared from the cumulus cells by repeated pipetting and completely denuded presumptive zygotes were cultured for up to 8 days in synthetic oviduct fluid (SOF) medium. To support the in vitro culture (IVC), SOF media supplemented with 44 μg/mL sodium pyruvate (C3H3NaO3), 14.6 μg/mL glutamine, 10 IU/mL penicillin, 0.1 mg/mL streptomycin, 3 mg/mL FBS, and 310 μg/mL glutathione were used for culturing of embryos [ 38 ]. The embryonic development proceeded to the BL stage under humidified atmospheric condition of 5% CO 2 at 38.5 °C were recorded and used for comparative analysis.…”

“…The total RNA from BLs ( n = 5 per group) was isolated with RNA isolation kit (PicoPure, Arcturus, ThermoFisher, Foster City, CA, USA) and used to synthesized cDNA with iScript reverse transcriptase (BioRad). The qRT-PCR analysis was performed as described by [ 38 ]. In a brief, the relative mRNA abundance of all genes was analyzed by real-time quantitative (q)RT-PCR with SYBR Green master mix using Cycler BioRad system.…”

“…The florescent probe 2′, 7′-dichlorodihydrofluorescein diacetate molecules (DCHDFA) from Sigma D-6883 Aldrich were used for the analysis of ROS (reactive oxygen species) production. The stock solution preparation and assay protocol was performed as previously described by [ 38 ]. In brief, day-8 BLs were collected, washed in PBS/polyvinyl pyrolidone (PVP) solution and incubated in a 10 mM/mL concentration of DCHDFA solution at 37 °C for 30 min.…”

Role of Wnt Signaling During In-Vitro Bovine Blastocyst Development and Maturation in Synergism with PPARδ Signaling

“…Oocyte aspiration and in-vitro maturation procedure was performed as described previously [22]. Briefly, abattoir collected ovaries were washed with Dulbecco's phosphate buffered saline (D-PBS).…”

“…Immunofluorescence staining was performed as described previously [22]. Briefly, different developmental stage embryos and BLs were fixed with 4% (w/v) PFA (paraformaldehyde) solution, and permeabilized with 0.5% Triton X-100.…”