Oxygen tension modulates extracellular vesicles and its miRNA contents in bovine embryo culture medium

Extracellular vesicles (EVs) play a possible role in cell–cell communication and are found in various body fluids and cell conditioned culture media. The aim of this study was to isolate and characterize EVs in culture medium conditioned by bovine embryos in group and to verify if these EVs are functionally active. Initially, ultracentrifuged bovine serum albumin (BSA) containing medium was selected as suitable EV-free embryo culture medium. Next, EVs were isolated from embryo conditioned culture medium by OptiPrepTM density gradient ultracentrifugation. Isolated EVs were characterized by nanoparticle tracking analysis, western blotting, transmission, and immunoelectron microscopy. Bovine embryo-derived EVs were sizing between 25–230 nm with an average concentration of 236.5 ± 1.27 × 108 particles/mL. Moreover, PKH67 EV pre-labeling showed that embryo-secreted EVs were uptaken by zona-intact bovine embryos. Since BSA did not appear to be a contaminating EV source in culture medium, EV functionality was tested in BSA containing medium. Individual embryo culture in BSA medium enriched with EVs derived from conditioned embryo culture medium showed significantly higher blastocyst rates at day 7 and 8 together with a significantly lower apoptotic cell ratio. In conclusion, our study shows that EVs play an important role in inter embryo communication during bovine embryo culture in group.

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80 Evaluation of extracellular vesicles from culture medium of human embryos as a possible method of pre-implantation genetic diagnosis

Reproduction Fertility and Development ◽

10.1071/rdv32n2ab80 ◽

2020 ◽

Vol 32 (2) ◽

pp. 166

Author(s):

C. Aguilera ◽

D. Veraguas ◽

C. Henriquez ◽

A. Velasquez ◽

F. O. Castro ◽

...

Keyword(s):

Extracellular Vesicles ◽

Embryo Culture ◽

Culture Medium ◽

Chromosomal Abnormalities ◽

Genetic Diagnosis ◽

Poor Quality ◽

Human Embryos ◽

Acgh Analysis ◽

Embryo Culture Medium ◽

Efficiency Of Selection

Noninvasive methods are the clue to increase the efficiency of invitro-derived embryo selection without decreasing their competence. Embryos selection based on their morphology is the most used method but only 40% of selected embryos are able to implant and develop correctly. In humans, pre-implantation genetic diagnosis increases the efficiency of selection by excluding embryos with chromosomal abnormalities. However, pre-implantation genetic diagnosis needs embryonic cells, which might compromise embryo viability. On the other hand, embryos release extracellular vesicles (EVs: microvesicles and exosomes) to the culture medium that contain biological cargo-like proteins and mRNA lipids, and might contain genomic DNA (gDNA). For this study we evaluated the culture medium from embryos generated by intracytoplasmic sperm injection in a certified fertility clinic. Embryos were cultured in Global Total serum-free medium. The embryos were assessed at Day 3 of development and classified in three categories: top, fair, and poor quality. Corresponding medium was collected for isolation of EVs. The nature of EVs was confirmed by their size and concentration using nanoparticle tracking analysis (NTA), presence of surface markers (CD9, CD63, CD81, and CD40L), and morphology using transmission electron microscopy. A correlation analysis between NTA results (EV size and concentration) and embryo quality was performed. To evaluate chromosomal abnormalities of gDNA present in isolated EVs from embryo culture medium, microarray-based comparative genomic hybridization (aCGH) assay was performed. In a second experiment, aCGH analysis was performed and compared between arrested embryos and EVs isolated from corresponding culture medium. Isolated nanoparticles from embryo culture medium were positive to all markers CD9 (30.9%), CD63 (27.2%), CD81 (31.7%), CD40L (8.7%) and had a morphology accordingly to exosomes. The analysis of NTA data indicated that top-quality embryos had EVs with higher diameter (mean: 112.17nm, mode: 91.74nm) than embryos classified as fair (mean: 108.02nm, mode: 89.67nm) and poor quality (mean: 102.78nm, mode: 88.17 nm; P<0.05). The aCGH analysis showed the representation of the 23 pairs of chromosomes in EVs from culture medium and the chromosomal abnormalities were detected in chromosome 4 (C4: 6/15 (40%)) and chromosome 13 (C13: 6/15 (40%)). In the second experiment, the aCGH assay also showed abnormalities in different chromosomes from samples of EVs from culture medium (24.9%) and were more frequent than those observed in the arrested embryos (8.7%; P=0.03). However, the rate of similitude in chromosomal abnormalities between EVs and their respective embryo was 70-80%. In conclusion, the size and gDNA of EVs from culture medium might be an alternative to evaluate the competence of human embryos. This research was supported by FONDECYT-1170310 and Corfo 17Cote-72437, Chile.

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71 Removal of hypotaurine from porcine embryo culture medium decreases message for pro-apoptotic genes but does not affect development at low oxygen tension

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab71 ◽

2019 ◽

Vol 31 (1) ◽

pp. 161

Author(s):

P. R. Chen ◽

E. C. Leffeler ◽

L. D. Spate ◽

R. S. Prather

Keyword(s):

Oxidative Stress ◽

Oxygen Tension ◽

Embryo Culture ◽

Culture Medium ◽

Atmospheric Oxygen ◽

Tunel Staining ◽

Blastocyst Development ◽

Embryo Culture Medium ◽

Porcine Embryo ◽

High O2

Hypotaurine (HT) is a routine additive to embryo culture medium, acting primarily as an oxygen radical scavenger. However, the practice of culturing embryos at ~5% O2 has been widely adopted because this is more physiologically relevant to the oxygen tensions detected in the oviduct and uterus. Thus, the utility of HT may be diminished as fewer oxygen radicals are produced during culture at 5% O2. The objective here was to determine the effects of removing HT from our porcine embryo culture medium (MU2) on the development of embryos incubated at a lower oxygen tension (5% O2) compared with atmospheric oxygen (~20% O2). Porcine cumulus-oocyte complexes were aspirated, matured, and fertilized with standard procedures from our laboratory. Presumptive zygotes were cultured in 1 of 4 conditions: MU2 with 5mM HT at 5% O2 (low O2 +HT; control), MU2 without HT at 5% O2 (low O2 −HT), MU2 with 5mM HT at 20% O2 (high O2 +HT), or MU2 without HT at 20% O2 (high O2 −HT). The percentage of presumptive zygotes that developed to the blastocyst stage on Day 6 and total number of nuclei in the blastocysts were determined. The RNA was extracted from pools of 30 blastocysts, and cDNA was synthesised for quantitative PCR for genes associated with HT synthesis, oxidative stress, and apoptosis. Damage to DNA was assessed by TUNEL staining of Day 6 blastocysts. Data were analysed for normality by using a Shapiro-Wilk test, and differences between means were detected by using 2-way ANOVA followed by Tukey’s honest significant difference test with P<0.05 considered significant. Embryos cultured with high O2 −HT had significantly decreased blastocyst development compared with all other groups (26.2±2.5% v. 36.9-41.7±3.3-4.3%). Embryos cultured with low O2 −HT had significantly more nuclei than those cultured with high O2 +HT (50.5±1.5v. 45.5±1.2). Notably, differences in blastocyst development (41.7±3.3% v. 36.9±3.3%) or total number of nuclei (50.0±1.8v. 50.5±1.5) were not detected between embryos cultured with low O2 +HT or low O2 −HT. The abundance of messages for genes involved in HT synthesis (cysteine sulfinic acid decarboxylase [CSAD]) and oxidative stress (superoxide dismutase 1 [SOD1] and glutathione peroxidase 6 [GPX6]) did not differ between groups. Contrarily, messages for 2 pro-apoptotic markers (BCL2 associated agonist of cell death [BAD] and caspase 3 [CASP3]) were significantly increased in embryos cultured with +HT regardless of oxygen tension; however, percentages of DNA-damaged nuclei in blastocysts after TUNEL staining were not different between groups (5.4-6.5±0.5-1.0%). These results indicate that HT is not necessary for porcine pre-implantation development at 5% O2 but is beneficial at atmospheric oxygen tension. Further investigation is required to confirm if HT promotes apoptosis in pre-implantation embryos. This research was supported by Food for the 21st Century at the University of Missouri and R01 HD080636.

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