Urea changes oocyte competence and gene expression in resultant bovine embryo in vitro

Abstract

SummaryNutrition influences the microenvironment in the proximity of oocyte and affects early embryonic development. Elevated blood urea nitrogen, even in healthy dairy cows, is associated with reduced fertility and there is high correlation between blood urea levels and follicular fluid urea levels. Using a docking calculation (in silico), urea showed a favorable binding activity towards the ZP-N domain of ZP3, that of ZP2, and towards the predicted full-length sperm receptor ZP3. Supplementation of oocyte maturation medium with nutrition-related levels of urea (20 or 40 mg/dl as seen in healthy dairy cows fed on low or high dietary protein, respectively) dose-dependently increased: (i) the proportion of oocytes that remained uncleaved; and (ii) oocyte degeneration; and reduced cleavage, blastocyst and hatching rates. High levels of urea induced shrinkage in oocytes, visualised using scanning electron microscopy. Urea downregulated NANOG while dose-dependently upregulating OCT4, DNMT1, and BCL2 expression. Urea at 20 mg/dl induced BAX expression. Using mathematical modelling, the rate of oocyte degeneration was sensitive to urea levels; while cleavage, blastocyst and hatching rates exhibited negative sensitivity. The present data imply a novel role for urea in reducing oocyte competence and changing gene expression in the resultant embryos.

Epidermal growth factor alleviates the negative impact of urea on frozen-thawed bovine sperm, but the subsequent developmental competence is compromised

“…For example, we reported that high levels of urea disrupted the oviduct epithelial cell monolayer and reduced the viability of bovine cumulus cells [15][16][17] . We also documented that urea-incubated oocytes had a lower rate of developmental competence and higher more amino acid depletion during in vitro maturation (IVM) 17,18 . It has been shown that urea uses EGFR transactivation and induces the expression of immediate-early genes 19 and, as a toxic molecule, can induce ROS production in a variety of cells, such as oocytes 20 .…”

“…Cumulus-oocyte complexes (COCs) with at least five cell layers were selected using a stereomicroscope (Olympus, Tokyo, Japan) and transferred to HEPES-TCM-199 supplemented by 50 mg/ml of kanamycin and 50 mg/ml of heparin. Intact COCs were then washed twice in HEPES-TCM199 and randomly put in groups of 10 in 50-µl droplet of maturation medium containing bicarbonate-buffered TCM199 supplemented by 10% 17,18 . After IVM, approximately 600 COCs (from 60 cows) were assigned to different treatments: (1) the control group in which oocytes were inseminated by untreated sperm cells (n = 140 oocytes, through 10 replicates, each replication (drop) containing 10-15 oocytes); (2) the EGF group in which oocytes were inseminated by sperm cells pre-treated with 10 ng/ml EGF for 30 min (n = 150 oocytes, 10 replicates, each replicate (drop) containing 10-15 oocytes); (3) the urea group in which oocytes were inseminated by sperm cells pre-treated with 40 mg/dl urea (equivalent to 18.7 mg/dl BUN) for 30 min (n = 150 oocytes in 10 replicates, each replicate (drop) containing 10-15 oocytes); and (4) EGF-urea group in which oocytes were inseminated by sperm cells pre-treated with a mixture of 40 mg/dl of urea and 10 ng/ml of EGF for 30 min (n = 160 oocytes in 10 replicates, each replicate (drop) containing 10-15 oocytes).…”

A multilevel analysis identifies the different relationships between amino acids and the competence of oocytes matured individually or in groups

“…We have previously reported that high urea levels not only increased the percentage of oocyte degeneration, but also decreased the in vitro viability of CCs and oviduct epithelial cells 16,17,39 . In this study, the addition of urea to the maturation medium increased the percentage of oocyte degeneration by day 3 after insemination.…”

“…www.nature.com/scientificreports/ RNA extraction, reverse transcription, quantitative analysis of transcripts using qRT-PCR. RNA extraction and reverse transcription were conducted as reported earlier 16 . In short, total RNA was extracted from day-7 blastocysts using RNeasy Micro kit (Qiagen, Mississauga, Ontario, Canada) and then treated with DNase I (Ambion, Streetsville, Ontario, Canada) according to the manufacturer's manual.…”

“…Blood urea nitrogen (BUN) levels greater than 19 mg/dl are directly associated with decreased reproductive efficiency in healthy dairy cows 14 . A growing body of evidence supports the detrimental effect of high levels of urea on the competence of bovine oocytes, in vivo and in vitro [15][16][17] . Moreover, urea increases the consumption of AAs by CCs, denuded oocytes (DOs) and COCs 17 .…”