Intraoviductal concentrations of steroid hormones during in vitro culture changed phospholipid profiles and cryotolerance of bovine embryos

Abstract

The objective of this study was to evaluate the effect of progesterone (P4), estradiol (E2), and cortisol (CO) at intraoviductal concentrations on bovine embryo development and quality in vitro. After fertilization of in vitro matured oocytes, zygotes were cultured for 8 days in synthetic oviductal fluid, supplemented with 55 ng/ml P4, 120 pg/ml E2, 40 ng/ml CO, or their combination (ALL). Control embryos were cultured with vehicle (0.1% ethanol). Exposure to steroids did not affect the embryo developmental rate nor the mean number of cells per blastocyst. However, at 24 hr after vitrification‐warming, exposure to P4 improved the proportion of embryos that re‐expanded and were viable while exposure to CO decreased the proportion of viable embryos. By intact cell MALDI‐TOF mass spectrometry, a total of 242 phospholipid masses of 400–1000 m/z were detected from individual fresh blastocysts. Exposure to ALL induced the highest and most specific changes in embryo phospholipids, followed by P4, E2, and CO. In particular, the m/z 546.3 and 546.4 attributed to lysophosphatidylcholines were found less abundant after exposure to P4. In conclusion, exposure of bovine embryos to intraoviductal concentrations of steroid hormones did not affect in vitro development but changed blastocyst quality in terms of cryotolerance and phospholipid profiles.

Oviduct Fluid Extracellular Vesicles Change the Phospholipid Composition of Bovine Embryos Developed In Vitro

“…The embryos exposed to oEVs displayed a shift toward PC, PE, and SM of higher molecular weight, i.e., possessing fatty acids of longer chains, whereas PC and PE species of lower molecular weight and LPC were detected among less abundant phospholipids compared to control embryos. Phospholipids are important components of the bovine oviductal [33][34][35] and uterine [36][37][38] fluids and may serve as an energy source and as signaling precursors for the gametes and developing embryos. However, there is currently very few information on their roles for the establishment of pregnancy in ruminants.…”

“…However, the mechanisms by which oviduct fluid components enhance the quality of developing embryos are poorly known. We previously showed that the steroid hormones progesterone, estradiol, and cortisol added at intraoviductal concentrations to the culture medium altered the phospholipid composition and cryosurvival of bovine embryos [35]. Several phospholipids higher than 700 m/z that were more abundant in embryos exposed to physiological concentrations of steroid hormones-including PC (34:2), PC (36:3), SM (d40:2), PC (36:2), and PC (38:4) at m/z 758.56, 784.56, 785.58, 786.6, and 810.6, respectively-were also found overabundant in blastocysts exposed to oEVs, whereas PC (29:2) or PE (32:2) at m/z 688.43 was found less abundant in both conditions [35].…”

“…We previously showed that the steroid hormones progesterone, estradiol, and cortisol added at intraoviductal concentrations to the culture medium altered the phospholipid composition and cryosurvival of bovine embryos [35]. Several phospholipids higher than 700 m/z that were more abundant in embryos exposed to physiological concentrations of steroid hormones-including PC (34:2), PC (36:3), SM (d40:2), PC (36:2), and PC (38:4) at m/z 758.56, 784.56, 785.58, 786.6, and 810.6, respectively-were also found overabundant in blastocysts exposed to oEVs, whereas PC (29:2) or PE (32:2) at m/z 688.43 was found less abundant in both conditions [35]. Two previous studies compared the phospholipid composition of bovine blastocysts conceived in vivo, which have a high likelihood of pregnancy after uterine transfer to a recipient cow; and those produced in vitro, which have lower developmental potential [18,42].…”

“…Bovine ovaries were collected at a local slaughterhouse, transported to the laboratory at 32-35 • C, then cumulus oocyte complexes (COCs) were collected by aspirating follicles of 2-8 mm in diameter. The in-vitro maturation (IVM)/in-vitro fertilization (IVF) protocols used in the present study have been described previously [35]. Briefly, groups of 40-50 COCs with more than three compact layers of cumulus cells were matured in 25 mM bicarbonate-buffered TCM 199 supplemented with 10% fetal calf serum (FCS; GIBCO BRL), 10 µg/mL follicle-stimulating hormone (FSH), 10 µg/mL luteinizing hormone (LH; Stimufol; SPRL Reprobiol), 1 µg/mL oestradiol 17β, and 50 µg/mL gentamycin for 22 h at 38.5 • C with 5% CO 2 .…”

Composing the Early Embryonic Microenvironment: Physiology and Regulation of Oviductal Secretions

“…At the expected day of embryo presence in the oviduct (day 4 after ovulation), distinct phospholipid profiles were evidenced between oviducts from beef females with contrasted embryo receptivity [32]. Furthermore, the addition of progesterone at intra-oviductal concentrations during in vitro culture changed the phospholipid composition of bovine embryos with a parallel improvement of their survival rate after vitrification [34].…”