Cellular responses and microRNA profiling in bovine spermatozoa under heat shock.

In brief: Elevated temperatures disturbed sperm physiology. Bovine sperm cells exposed to heat shock led to diminished mitochondrial activity, fertilizing ability, increased oxidative stress and caspase activity concomitant with a delay in embryonic developmental kinetics and modulation of sperm-borne microRNAsmiRNAs. Abstract: 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, 38.5, 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 cleavage 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. Moreover, miR-181d was enriched in sperm cells exposed to higher temperatures. Hence, elevated temperature altered the physiology of mature sperm cells by perturbing cellular processes and the miR profile, which collectively led to lower fertilizing ability and preimplantation development.

Follicular fluid exosomes act on the bovine oocyte to improve oocyte competence to support development and survival to heat shock.

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.

Thermoprotective effect of insulin-like growth factor 1 on in vitro matured bovine oocyte exposed to heat shock.

The 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 TUNEL-positive 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 influence of stimulus phase duration on discomfort and electrically induced torque of quadriceps femoris

BACKGROUND: Although a number of studies have compared the influence of different electrical pulse parameters on maximum electrically induced torque (MEIT) and discomfort, the role of phase duration has been poorly investigated. OBJECTIVE: To examine the variation in muscle torque and discomfort produced when electrically stimulating quadriceps femoris using pulsed current with three different phase durations in order to establish whether there are any advantages or disadvantages in varying the phase duration over the range examined. METHOD: This is a two repeated-measures, within-subject study conducted in a research laboratory. The study was divided into 2 parts with 19 healthy young adults in each part.In part 1, MEIT was determined for each phase duration (400, 700, and 1000 µs), using a biphasic pulsed current at a frequency of 50 Hz. In part 2, stimulus amplitude was increased until the contractions reached 40% of maximum voluntary isometric contraction (MVIC) and the associated discomfort produced by each phase duration was measured. RESULTS: In part 1 of the study, we found that the average MEITs generated with each phase duration (400, 700, and 1000 µs) were 55.0, 56.3, and 58.0% of MVIC respectively, but the differences were not statistically significant (p=.45). In part 2, we found a statistically significant increase in discomfort over the same range of phase durations. The results indicate that, for a given level of torque production, discomfort increases with increasing phase duration (p=.008). CONCLUSIONS: Greater muscle torque cannot be produced by increasing the stimulus phase duration over the range examined. Greater discomfort is produced by increasing the stimulus phase duration. .

The influence of stimulus phase duration on discomfort and electrically induced torque of quadriceps femoris.

BACKGROUND: Although a number of studies have compared the influence of different electrical pulse parameters on maximum electrically induced torque (MEIT) and discomfort, the role of phase duration has been poorly investigated. OBJECTIVE: To examine the variation in muscle torque and discomfort produced when electrically stimulating quadriceps femoris using pulsed current with three different phase durations in order to establish whether there are any advantages or disadvantages in varying the phase duration over the range examined. METHOD: This is a two repeated-measures, within-subject study conducted in a research laboratory. The study was divided into 2 parts with 19 healthy young adults in each part.In part 1, MEIT was determined for each phase duration (400, 700, and 1000 µs), using a biphasic pulsed current at a frequency of 50 Hz. In part 2, stimulus amplitude was increased until the contractions reached 40% of maximum voluntary isometric contraction (MVIC) and the associated discomfort produced by each phase duration was measured. RESULTS: In part 1 of the study, we found that the average MEITs generated with each phase duration (400, 700, and 1000 µs) were 55.0, 56.3, and 58.0% of MVIC respectively, but the differences were not statistically significant (p=.45). In part 2, we found a statistically significant increase in discomfort over the same range of phase durations. The results indicate that, for a given level of torque production, discomfort increases with increasing phase duration (p=.008). CONCLUSIONS: Greater muscle torque cannot be produced by increasing the stimulus phase duration over the range examined. Greater discomfort is produced by increasing the stimulus phase duration.