Addition of follicular fluid to oocyte maturation medium can affect cumulus cell function, increase competence of the oocytes to be fertilised and develop to the blastocyst stage and protect the oocyte from heat shock. Here, it was tested whether exosomes in follicular fluid are responsible for the effects of follicular fluid on the function of the cumulus–oocyte complex (COC). This was accomplished by culturing COCs during oocyte maturation at 38.5°C (body temperature of the cow) or 41°C (heat shock) with follicular fluid or exosomes derived from follicular fluid and evaluating various aspects of function of the oocyte and the embryo derived from it. Negative effects of heat shock on cleavage and blastocyst development, but not cumulus expansion, were reduced by follicular fluid and exosomes. The results support the idea that exosomes in follicular fluid play important roles during oocyte maturation to enhance oocyte function and protect it from stress.

a b s t r a c tThe role of insulin-like growth factor 1 (IGF1) on cellular function and developmental capacity of heat-shocked oocytes has not been completely understood. Therefore, the objective of this study was to determine the effect of IGF1 on apoptosis, mitochondrial activity, cytoskeletal changes, nuclear maturation, and developmental competence of bovine oocytes exposed to heat shock. Cumulus-oocyte complexes were submitted to control (38.5 C for 22 hours) and heat shock (41 C for 14 hours followed by 38.5 C for 8 hours) in the presence of 0 or 100 ng/mL IGF1 during IVM. Heat shock increased the percentage of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling)-positive oocyte and reduced oocyte mitochondrial activity. However, addition of 100 ng/mL IGF1 minimized these deleterious effects of temperature. Caspase activity was affected neither by heat shock nor IGF1. Exposure of bovine oocytes to 41 C during the first 14-hour IVM affected cortical actin localization and microtubule organization at the meiotic spindle and reduced the percentage oocytes that reached the metaphase II stage. However, in the presence of IGF1, cortical actin and percentage of metaphase II oocytes were not different between control and heat-shocked oocytes, suggesting a partial beneficial effect of IGF1. There was no effect of IGF1 on microtubule organization. Heat shock also reduced the percentage of oocytes that reached the blastocyst stage, blastocyst cell number, and increased the percentage of TUNELpositive blastomeres. However, there was no effect of 100 ng/mL IGF1 on oocyte development to the blastocyst stage and blastocyst quality. Therefore, 100 ng/mL IGF1 prevented some heat shock-induced cellular damage in bovine oocytes but had no effect on oocyte developmental competence. In contrast, a low IGF1 concentration (25 ng/mL) had a thermoprotective effect on oocyte developmental competence to the blastocyst stage. In conclusion, IGF1 prevented part of the damage induced by heat shock on oocyte function. This effect was modulated by IGF1 concentration.

The cellular mechanisms induced by elevated temperature on oocytes are not fully understood. However, there is evidence that some of the deleterious effects of heat shock are mediated by a heat-induced increase in reactive oxygen species (ROS). In this context, carotenoid antioxidants might have a thermoprotective effect. Therefore, the objective of this study was to determine the role of astaxanthin (AST) on oocyte ROS production and on the redox profile and developmental competency of cumulus-oocyte complexes (COCs) after 14h heat shock (41°C) during in vitro maturation (IVM). Exposure of oocytes to heat shock during IVM increased ROS and reduced the ability of the oocyte to cleave and develop to the blastocyst stage. However, 12.5 and 25nM astaxanthin rescued these negative effects of heat shock; astaxanthin counteracted the heat shock-induced increase in ROS and restored oocyte developmental competency. There was no effect of astaxanthin on maturation medium lipid peroxidation or on glutathione peroxidase and catalase activity in oocytes and cumulus cells. However, astaxanthin stimulated superoxide dismutase (SOD) activity in heat-shocked cumulus cells. In conclusion, direct heat shock reduced oocyte competence, which was restored by astaxanthin, possibly through regulation of ROS and SOD activity in oocytes and COCs.

Elevated temperature can compromise the ability of the mammalian oocyte to develop to the blastocyst stage after fertilization. The microenvironment of the oocyte is determined by the cellular and non-cellular components of the follicle including cumulus cells and follicular fluid. Here we tested whether follicular fluid contains molecules that can protect the bovine oocyte from heat shock during maturation, and if so, whether some of these protective molecules are present in exosomes. The experiments utilised ovaries from Bos taurus and admixtures of B. taurus and Bos indicus. Four separate pools of follicular fluid were prepared by aspiration of follicles from 48 to 70 slaughterhouse ovaries. Exosomes were isolated from follicular fluid by a series of centrifugation, filtration, and ultracentrifugation steps before being reconstituted in PBS. Each of the 4 exosome preparations was subject to particle size and concentration analysis. The experiments were designed as 2 × 3 factorial to test the effect of temperature and supplementation. Cumulus-oocyte complexes (COC) obtained from slaughterhouse ovaries were matured at 38.5°C for 22 h (control) or 41°C for 14 h followed by 38.5°C for 8 h (heat shock). Maturation was performed in the presence of vehicle (PBS), 10% (v/v) follicular fluid, or exosomes (16 × 109 particles/mL). Data were analysed by least-squares ANOVA. Orthogonal contrasts and the mean separation test pdiff were used to compare means. Effects of treatment on cumulus cell expansion (change in diameter after maturation) were replicated 5 times using 119 to 122 COC per treatment. Effects of treatment on embryonic development after fertilization of treated COC was determined in 6 replicates using 244 to 286 embryos per replicate. Expansion was reduced by heat shock (P < 0.001), and affected by treatment (P < 0.05), with both follicular fluid and exosomes preventing the decrease in expansion caused by heat shock. Cleavage was reduced by heat shock (P < 0.001) and affected by treatment (P < 0.05) and the interaction between temperature × supplementation (P < 0.05). Although heat shock reduced the cleavage rate for vehicle-treated oocytes (77 v. 67%), there was no effect of heat shock for oocytes treated with follicular fluid FF (78 v. 74%) or exosomes (79 v. 78%; SEM = 1.4%). Heat shock also reduced the percent of cleaved embryos becoming blastocysts for the vehicle group (27 v. 17%; P < 0.05) but had no effect on percent of cleaved embryos becoming blastocysts for the follicular fluid (31% v. 26%) or exosome groups (28 v. 26%). Uptake of exosomes into isolated cumulus cells and oocytes cultured at 38.5°C for 0.5, 1, 14 and 22 h was examined using labelling of exosomes with 10 µM BODIPY® Ceramide TR (Thermo Fisher Scieintific, Waltham, MA, USA) and confocal microscopy. Exosomes were taken up by cumulus cells after culture for 1 h or later but were not taken up by oocytes. In conclusion, follicular fluid exosomes protected the oocytes from heat shock and this effect seems to be mediated by cumulus cells. Study supported by BARD US-4719-14.

Sperm function is susceptible to adverse environmental conditions. It has been demonstrated that in vivo and in vitro exposure of bovine sperm to elevated temperature reduces sperm motility and fertilizing potential. However, the cascade of functional, cellular and molecular events triggered by elevated temperature in the mature sperm cell remains not fully understood. Therefore, the aim of this study was to determine the effect of heat shock on mature sperm cells. Frozen-thawed Holstein sperm were evaluated immediately after Percoll purification (0 h non-incubation control) or after incubation at 35°C, 38.5°C, and 41°C for 4 h. Heat shock reduced sperm motility after 3 - 4 h at 41°C while mitochondrial activity was reduced by 38.5 and 41°C when compared to the control. Heat shock also increased sperm reactive oxygen species production and caspase activity. Heat-shocked sperm had lower fertilizing ability, which led to diminished cleaved and blastocyst rates. Preimplantation embryo developmental kinetics was also slowed and reduced by sperm heat shock. The microRNA (miR) profiling identified >300 miRs in bovine sperm. Among these, three and seven miRs were exclusively identified in sperm cells exposed to 35 and 41°C, respectively.