Granulosa cell-derived extracellular vesicles mitigate the detrimental impact of thermal stress on bovine oocytes and embryos

Abstract

Climate change-induced global warming results in rises in body temperatures above normal physiological levels (hyperthermia) with negative impacts on reproductive function in dairy and beef animals. Extracellular vesicles (EVs), commonly described as nano-sized, lipid-enclosed complexes, harnessed with a plethora of bioactive cargoes (RNAs, proteins, and lipids), are crucial to regulating processes like folliculogenesis and the initiation of different signaling pathways. The beneficial role of follicular fluid-derived EVs in inducing thermotolerance to oocytes during in vitro maturation (IVM) has been evidenced. Here we aimed to determine the capacity of in vitro cultured granulosa cell-derived EVs (GC-EVs) to modulate bovine oocytes' thermotolerance to heat stress (HS) during IVM. Moreover, this study tested the hypothesis that EVs released from thermally stressed GCs (S-EVs) shuttle protective messages to provide protection against subsequent HS in bovine oocytes. For this, sub-populations of GC-EVs were generated from GCs subjected to 38.5°C (N-EVs) or 42°C (S-EVs) and supplemented to cumulus-oocyte complexes (COCs) matured in vitro at the normal physiological body temperature of the cow (38.5°C) or HS (41°C) conditions. Results indicate that S-EVs improve the survival of oocytes by reducing ROS accumulation, improving mitochondrial function, and suppressing the expression of stress-associated genes thereby reducing the severity of HS on oocytes. Moreover, our findings indicate a carryover impact from the addition of GC-EVs during oocyte maturation in the development to the blastocyst stage with enhanced viability.

Keywords: cumulus-oocyte complex; embryo development; extracellular vesicles; granulosa cells; heat stress; oocyte maturation.

FIGURE 1

Morphological and molecular characterization of GC-EVs. The relative size of EVs isolated from cultured granulosa cells spent culture medium was determined using nanoparticle tracking analysis (NTA). The median size of N-EVs was 137.75 nm (blue) compared to S-EVs 147.95 nm (red). Granulosa cells subjected to thermal stress released a significantly higher quantity of particles (red) and had tendencies to be larger in size (nm) compared to those subjected to normal ambient temperatures (blue) (A). Immunoblotting analysis for EV-specific protein markers (CD63, TSG101, and CD81) and cellular-specific protein marker (CYCS) were verified to determine protein lysate composition in N-EVs, S-EVs, and GCs as a positive control. Full-length blots are presented in Supplementary Figure S1 (B). Transmission electron microscopy (TEM) representative images from N-EVs and S-EVs (marked with red arrows) show the morphology of the lipid bilayer membrane of EV particles. Scale bar, 100 nm (C).

FIGURE 2

Effects of GC-EV supplementation on receptivity of follicular cells EV uptake following maturation at 38.5°C (blue) and 41°C (red) and cumulus expansion. Confocal images of intact cumulus-oocyte complexes co-incubated with PKH26 labeled GC-EVs for 24 h during in vitro maturation. Scale bar, 50 µM (A). Follicular cell uptake of PKH26 labeled EVs is shown in the surrounding cumulus cells (CC), in relation to the oocyte (OO) and respective zona pellucida (ZP). Measurements for cumulus expansion were obtained before and after maturation using the Olympus Cellsens software. The increase in diameter was determined by subtracting the mean diameter before maturation from the mean diameter after maturation (B). Results were carried out in four replicates (a total of 113-252 COCs per treatment). Data among treatments represent the mean±SEM and the differences between means were analyzed using Two-way ANOVA followed by Tukey’s Multiple Comparisons Test. Bars with different letters (38.5°C; uppercase) (41°C; lowercase) indicate statistically significant differences of at least (p < 0.05) between treatments under the same culture conditions, while (*) indicate statistical differences between the same treatment under different culture environments during IVM (****p < 0.0001). The expression of cumulus expansion marker genes was assessed using qRT-PCR from cumulus cells isolated from oocytes in triplicates (a total of 50 COCs per treatment) cultured under thermoneutral (C) and heat stress (D) conditions. Data among treatments cultured under the same environment represent the mean ± SEM and the differences between means were analyzed using One-way ANOVA followed by Tukey’s Multiple Comparisons Test. (*p < 0.05), (**p < 0.01), (***p < 0.001), (****p < 0.0001).

FIGURE 3

The impact of GC-EV supplementation on transcript levels of stress-associated genes in cumulus cells from cumulus-oocyte complexes following maturation at 38.5°C (blue) and 41°C (red). The expression of stress-associated genes was significantly diminished in cumulus cells from oocytes supplemented with S-EVs and matured at 38.5°C (A) and 41°C (B). Results are from cumulus cells removed from a total of three pools of 50 oocytes per pool for each treatment. Data represent the mean ± SEM and the differences between means were analyzed using One-way ANOVA followed by Tukey’s Multiple Comparisons Test. (*p < 0.05), (**p < 0.01), (***p < 0.001).

FIGURE 4

The impact of GC-EV supplementation on transcript levels of stress-associated genes in oocytes following maturation at 38.5°C (blue) and 41°C (red). The expression of stress-associated genes were significantly diminished in oocytes supplemented with N-EVs and cultured at 38.5°C (A), and similar decreases in gene expression were visualized in oocytes supplemented with S-EVs and exposed to HS (B). Results are from a total of three pools of 50 oocytes per pool for each treatment. Data represent the mean ± SEM and the differences between means were analyzed using One-way ANOVA followed by Tukey’s Multiple Comparisons Test. (*p < 0.05), (**p < 0.01), (***p < 0.001), (****p < 0.0001).

FIGURE 5

The beneficial effects of GC-EVs on oocyte function under in vitro normal physiological (38.5°C; blue) and HS (41°C; red) conditions. Fluorescence images of in vitro matured bovine oocytes treated with 2ʹ,7ʹ-Dichlorofluorescin Diacetate (H₂DCFDA) were captured for the measurement of intracellular ROS levels in single oocytes. Scale bar, 50 µM [38.5°C; (A)] [41°C; (B)]. Quantification of the relative fluorescence intensity in single oocytes were carried out in triplicates (total = of 15–50 oocytes per treatment) (C). Fluorescence images of in vitro matured bovine oocytes treated with 5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimidazolylcarbocynanine iodide (JC-1) for the measurement of MMP (∆Ψm), an indicator of mitochondrial activity in single oocytes. Scale bar, 50 µM [38.5°C; (D)] [41°C; (E)]. Quantification of the relative fluorescence intensity (red/green ratio) in single oocytes were carried out in duplicates (total = of 12–22 oocytes per treatment) (F). Data among treatments represent the mean ± SEM and the differences between means were analyzed using Two-way ANOVA followed by Tukey’s Multiple Comparisons Test. Bars with different letters (38.5°C; uppercase) (41°C; lowercase) indicate statistically significant differences of at least (p < 0.05) between treatments under the same culture conditions, while (*) indicate statistical differences between the same treatment under different culture environments during IVM (***p < 0.001).

FIGURE 6

The effect of GC-EVs on the developmental capacity of oocytes matured at 38.5°C (blue) and 41°C (red). Results are from a total of six replicates (281–299 total oocytes per treatment). Oocytes supplemented with N-EVs and cultured under 38.5°C showed significant improvements in cleavage percentage compared to the NC, while no visual differences were observed among treatments under HS conditions (A). The percentage of oocytes developed into blastocysts was significantly affected by maturation temperature with significant increases in developmental competence in those treatments supplemented with GC-EVs (B). Similarly, the percentage of cleaved embryos developed into blastocysts was linearly affected by maturation temperature with significant protection from inherent stress visualized in those treatments supplemented with GC-EVs (C). Data among treatments represent the mean ± SEM and the differences between means were analyzed using Two-way ANOVA followed by Tukey’s Multiple Comparisons Test. Bars with different letters (38.5°C; uppercase) (41°C; lowercase) indicate statistically significant differences of at least (p < 0.05) between treatments under the same culture conditions, while (*) indicate statistical differences between the same treatment under different culture environments during IVM (***p < 0.001) (****p < 0.0001).

FIGURE 7

The impact of GC-EVs on transcript levels of stress-associated genes in blastocysts derived from oocytes treated with GC-EVs during oocyte maturation at 38.5°C (blue) and 41°C (red). The expressions of stress-associated genes were significantly diminished in blastocysts derived from oocytes supplemented with N-EVs and cultured at 38.5°C (A), and similar decreases in gene expression were visualized in blastocysts derived from oocytes supplemented with S-EVs and exposed to HS (B). Results are from a total of four pools of 5 blastocysts per pool for each treatment. Data represent the mean ± SEM and the differences between means were analyzed using One-way ANOVA followed by Tukey’s Multiple Comparisons Test. (*p < 0.05), (**p < 0.01), (***p < 0.001), (****p < 0.0001).

FIGURE 8

Functional alterations of GC-EV supplementation during in vitro maturation on blastocyst quality under in vitro normal physiological (38.5°C; blue) and HS (41°C; red) conditions. Fluorescence images of in vitro cultured bovine blastocysts treated with 2ʹ,7ʹ-Dichlorofluorescin Diacetate (H₂DCFDA) were captured for the measurement of intracellular ROS levels in single blastocysts. Scale bar, 50 µM [38.5°C; (A)] [41°C; (B)]. Quantification of the relative fluorescence intensity in single blastocysts were carried out in triplicates (a total of 6-9 blastocysts per treatment) (C). Fluorescence images of in vitro cultured bovine blastocysts treated with 5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimidazolylcarbocynanine iodide (JC-1) for the measurement of MMP (∆Ψm), an indicator of mitochondrial activity in single blastocysts. Scale bar, 50 µM [38.5°C; (D)] [41°C; (E)]. Quantification of the relative fluorescence intensity (red/green ratio) in single blastocysts were carried out in duplicates (a total of 7–11 blastocysts per treatment) (F). Data among treatments represent the mean ± SEM and the differences between means were analyzed using Two-way ANOVA followed by Tukey’s Multiple Comparisons Test. Bars with different letters (38.5°C; uppercase) (41°C; lowercase) indicate statistically significant differences of at least (p < 0.05) between treatments under the same culture conditions, while (*) indicate statistical differences between the same treatment under different culture environments during IVM (**p < 0.01).

FIGURE 9

The beneficial effect of GC-EV supplementation during in vitro maturation on blastocyst quality under in vitro normal physiological (38.5°C; blue) and HS (41°C; red) conditions. Fluorescence images of in vitro cultured bovine blastocysts treated with Hoechst 33342/DAPI were used to determine total cell numbers in preimplantation embryos. Scale bar, 50 µM [38.5°C; (A)] [41°C; (B)]. Quantification of total cell numbers in single blastocysts were carried out in triplicates (a total of 18–30 blastocysts per treatment) (C). Fluorescence images of in vitro cultured bovine blastocysts subjected to a TUNEL assay were captured for specific detection of apoptotic cells. Scale bar, 50 µM [38.5°C; (D)] [41°C; (E)]. Quantification of TUNEL + cells in single blastocysts were carried out in triplicates (a total of 19–41 blastocysts per treatment) (F). Data among treatments represent the mean ± SEM and the differences between means were analyzed using Two-way ANOVA followed by Tukey’s Multiple Comparisons Test. Bars with different letters (38.5°C; uppercase) (41°C; lowercase) indicate statistically significant differences of at least (p < 0.05) between treatments under the same culture conditions, while (*) indicate statistical differences between the same treatment under different culture environments during IVM (*p < 0.05), (**p < 0.01), (***p < 0.001), (****p < 0.0001).

FIGURE 10

Graphical Synopsis. Schematic illustration showing EVs secreted from follicular granulosa cultured in vitro under 38.5°C (N-EVs) and 42°C (S-EVs), preferentially uptaken in the cumulus cells in intact cumulus-oocyte complexes following co-incubation during in vitro oocyte maturation. Results indicate that S-EVs effectively modulate the thermotolerance of bovine oocytes to elevated temperatures shown through the functional analysis conducted in the cumulus cells, oocytes, and developed blastocysts.