Extracellular vesicles from oviductal and uterine fluids supplementation in sequential in vitro culture improves bovine embryo quality

Abstract

Background: In vitro production of bovine embryos is a well-established technology, but the in vitro culture (IVC) system still warrants improvements, especially regarding embryo quality. This study aimed to evaluate the effect of extracellular vesicles (EVs) isolated from oviductal (OF) and uterine fluid (UF) in sequential IVC on the development and quality of bovine embryos. Zygotes were cultured in SOF supplemented with either BSA or EVs-depleted fetal calf serum (dFCS) in the presence (BSA-EV and dFCS-EV) or absence of EVs from OF (D1 to D4) and UF (D5 to D8), mimicking in vivo conditions. EVs from oviducts (early luteal phase) and uterine horns (mid-luteal phase) from slaughtered heifers were isolated by size exclusion chromatography. Blastocyst rate was recorded on days 7-8 and their quality was assessed based on lipid contents, mitochondrial activity and total cell numbers, as well as survival rate after vitrification. Relative mRNA abundance for lipid metabolism-related transcripts and levels of phosphorylated hormone-sensitive lipase (pHSL) proteins were also determined. Additionally, the expression levels of 383 miRNA in OF- and UF-EVs were assessed by qRT-PCR.

Results: Blastocyst yield was lower (P < 0.05) in BSA treatments compared with dFCS treatments. Survival rates after vitrification/warming were improved in dFCS-EVs (P < 0.05). EVs increased (P < 0.05) blastocysts total cell number in dFCS-EV and BSA-EV compared with respective controls (dFCS and BSA), while lipid content was decreased in dFCS-EV (P < 0.05) and mitochondrial activity did not change (P > 0.05). Lipid metabolism transcripts were affected by EVs and showed interaction with type of protein source in medium (PPARGC1B, LDLR, CD36, FASN and PNPLA2, P < 0.05). Levels of pHSL were lower in dFCS (P < 0.05). Twenty miRNA were differentially expressed between OF- and UF-EVs and only bta-miR-148b was increased in OF-EVs (P < 0.05).

Conclusions: Mimicking physiological conditions using EVs from OF and UF in sequential IVC does not affect embryo development but improves blastocyst quality regarding survival rate after vitrification/warming, total cell number, lipid content, and relative changes in expression of lipid metabolism transcripts and lipase activation. Finally, EVs miRNA contents may contribute to the observed effects.

Keywords: Cattle; Cryopreservation; Embryo development; Exosomes; Lipid metabolism; Oviduct; Uterus; miRNAs.

Fig. 1

Experimental design. A EVs isolation and characterization. Oviducts (n = 5) and uterine horns (n = 5), ipsilateral to a Stage 1 (oviduct) or Stage 2 (uterus) corpus luteum, were flushed with PBS (1 and 2 mL, respectively), and after centrifugation to remove cells and cellular debris, samples were filtered (0.22 μm) and EVs isolated by size exclusion chromatography. EVs samples were concentrated by ultracentrifugation and pellets resuspended in 100 μL PBS. Thirty microliters from each of the five EVs suspensions from oviduct (150 μL) and uterus (150 μL) were pooled and from this, 5 μL were used for EVs characterization by nanoparticle tracking analysis (NTA) and 5 μL for transmission electron microscopy (TEM), and the rest was frozen at − 20 °C and used in IVC. The remaining volume of the original five EVs samples (70 μL) was used for miRNA analysis. To have enough protein amount (35 μg) for EV biomarkers to be detectable by western blot, a pool of 10 oviducts (Stage 1) and 10 uterine horns (Stage 2) flushings was obtained and prepared the same way. B Embryo culture in vitro. At approximately 20 h after insemination, presumptive zygotes were cultured in 4 treatments: BSA: SOF with 0.3% BSA (3 mg/mL, w/v); dFCS: SOF with 5% dFCS; BSA-EV: SOF with 0.3% BSA supplemented with 3 × 10⁵ EVs/mL from OF (D1 to D4) and 3 × 10⁵ EVs/mL from UF (D4 to D9); and dFCS-EV: SOF with 5% dFCS supplemented with 3 × 10⁵ EVs/mL from OF (D1 to D4) and 3 × 10⁵ EVs/mL from UF (D4 to D9). BSA and dFCS treatments underwent media renewal at 96 hpi (day 4). Blastocyst development was assessed on days 7, 8 and 9. A representative number of day 7–8 blastocysts from each group was assessed for quality either by vitrification/warming (survival rate at 24, 48 and 72 h) or fixed and stained for total cell number, mitochondrial activity and lipid content and analysis. In addition, day 7–8 blastocysts were frozen in liquid nitrogen in groups of 10 and stored at − 80 °C for gene expression or western blot for protein analyses. C miRNA contents in EVs. Total RNA in oviducts and uteri EVs samples (n = 3) was extracted with a miRNeasy mini kit, reversed transcribed with the miScript PCR System and relative miRNA levels determined by qRT-PCR

Fig. 2

OF and UF-EVs characterization. A Averaged finite track length adjustment Concentration/Size graphs for NTA of particles in fluid isolated from oviducts (left) and uterine horns (right). B Representative images from electron microscopy analyses of OF (left) and UF (right) samples. Some EVs and their sizes are indicated in the images. C Representative images of membranes treated for detection of EVs marker-proteins (CD9, HSP70 and ALIX) and EVs negative protein (CANX) in samples isolated from oviduct (OF-EVs) and uterus (UF-EVs). PC = positive controls (CD9 = blood, HSP70 = pancreas, ALIX = lung, CANX = urethra). MW = molecular weight marker. Numbers indicate the MW. Expected MW: CD9 = 22, 24 and 35 kDa, HSP70 = ~ 70 kDa, ALIX = 90 kDa and CANX = 90 kDa

Fig. 3

Representative image of embryo on day 3 of IVC with EVs from OF and UF. A Representative image of embryo on day 3 of culture in vitro with EVs from OF and C on day 8 with EVs from UF. Nuclei in blue indicate cells (blastomeres) and EVs labelled with PHK67 (green). B Negative control for OF in D3 embryo and D for UF in D8 embryo. 63 ×

Fig. 4

Total cell number in blastocysts IVC. Total cell number in blastocysts cultured in vitro with BSA or dFCS supplemented or not with OF-EVs (D1–4) and UF-EVs (D5–8) (BSA = 57, BSA-EV = 55, dFCS = 58, dFCS-EV = 56). Data are the means ± SEM. Different letters show statistically significant differences between treatments (P < 0.05)

Fig. 5

Representative fluorescence images of mitochondrial activity in bovine blastocysts. A Representative fluorescence images of mitochondrial activity in bovine blastocysts cultured in vitro in medium with BSA, BSA-EV, dFCS and dFCS-EV. Images captured on 63 × objective. B Quantification of mitochondria fluorescence intensity in arbitrary unites (au) in blastocysts cultured in vitro with BSA or dFCS supplemented or not with OF-EVs (D1–4) and UF-EVs (D5–8). (BSA = 29, BSA-EV = 29, dFCS = 33, dFCS-EV = 27). Data are the means ± SEM. (P > 0.05)

Fig. 6

Representative fluorescence images of lipid droplets in bovine blastocysts. A Representative fluorescence images of lipid droplets in bovine blastocysts cultured in vitro in medium with BSA, BSA-EV, dFCS and dFCS-EV. Images captured on 63 × objective. B Quantification of total area of lipids in D7 - 8 blastocysts cultured in vitro with BSA or dFCS supplemented or not with OF-EVs (D1–4) and UF-EVs (D5–8) (BSA = 28, BSA-EV = 26, dFCS = 25, dFCS-EV = 29). Data are the means ± SEM. Different letters show statistically significant differences between treatments (P < 0.05)

Fig. 7

Relative mRNA abundance of lipid metabolism-related genes in blastocysts. Relative mRNA abundance of lipid metabolism-related genes in blastocysts cultured in vitro with BSA or dFCS only or supplemented with OF-s (D1–4) and UF-EVs (D5–8) (BSA, BSA-EV, dFCS and dFCS-EV). Bars represent the relative abundance of the transcripts analysed and normalized to H2AFZ and ACTB as housekeeping genes. The experimental treatments are represented by columns. Data are the means ± SEM. Different letters show statistically significant differences between treatments (P < 0.05)

Fig. 8

pHSL phosphorylation levels in bovine blastocysts. Effect of in vitro culture medium with BSA or dFCS supplemented or not with OF-EVs (D1–4) followed by UF-EVs (D5–8) on pHSL phosphorylation levels in bovine blastocysts. A pHSL phosphorylation level. Expression levels were normalized to the abundance of endogenous control actin. Phosphorylation level was expressed as pHSL/HSL. Data are expressed as means ± SEM. Different letters show statistically significant differences between treatments (P < 0.05). B Representative images of membranes treated for detection of lipolysis protein, total HSL and phosphorylated p-HSL, and β-actin. MW = molecular weight marker. Numbers indicate the MW. Expected MW: pHSL and HSL = 81 kDa and β-actin = 42 kDa

Fig. 9

Relative abundance of miRNAs in EVs from OF and UF in bovine embryos. Relative abundance of miRNAs in EVs from OF and UF used in the in vitro culture of bovine embryos. From 20 differentially expressed miRNAs, 19 were upregulated in UF-EVs and one* in OF-EVs (P < 0.05)