Modification of embryonic resistance to heat shock in cattle by melatonin and genetic variation in HSPA1L

Abstract Text:

Seeking for new methods to reduce the effect of heat stress on fertility we examined 1) whether melatonin blocks inhibition of embryonic development caused by heat shock and 2)whether frequency of a thermoprotective allele for HSPA1L is increased in blastocysts formed from heat-shocked zygotes as compared to blastocysts from control zygotes. It was hypothesized that melatonin prevents effects of heat shock on development by reducing accumulation of reactive oxygen species (ROS).  Effects of 1 µM melatonin on ROS were determined in Exp.1 and 2.  Zygotes were cultured at 38.5 or 40°C for 3 h in the presence of CellROX® reagent. Culture was in a low [5% (v/v)] oxygen (Exp.1) or low or high [21% (v/v)] oxygen environment (Exp.2). Heat shock and high oxygen increased ROS; melatonin decreased ROS. In Exp.1, for example, fluorescent intensity at 38.5°C  was 346±54 and 451±51 and 769±47 and 361±50 at 40°C for control and melatonin-treated embryos (interaction, P<0.0001). Development was assessed in Exp.3-5. In Exp.3 and 4, zygotes were cultured in low oxygen + 1 µM melatonin and exposed to 38.5 or 40°C for 12 (Exp.1) or 24 h (Exp.2) beginning 8 h after fertilization. Melatonin was not thermoprotective in either experiment. Exp.5 was performed similarly except that temperature treatments (38.5 or 40°C, 24 h) were performed in a low or high oxygen environment (2x2x2 factorial design with temperature, melatonin and oxygen concentration as main effects). Heat shock decreased (P=0.003) percent of zygotes developing to the blastocyst stage (26±1.5 vs 20±1.5%) independent of melatonin or oxygen concentration. For Exp.5, blastocysts were genotyped for a deletion (D) mutation (C®D) in the promoter region of HSPA1L associated with thermotolerance. Genotype was affected by temperature (P=0.002). The percent of blastocysts CC, CD or DD was 43.3, 28.5 and 28.2% for blastocysts from control zygotes and 32.4, 36.0, and 31.6% for blastocysts from heat-shocked zygotes.  It was concluded that 1)lack of effect of melatonin or oxygen concentration on embryonic development means that the negative effects of heat shock on the zygote are not mediated by ROS, 2)previously reported effect of melatonin on fertility of heat-stressed cows might involve actions independent of the antioxidant properties of melatonin, and 3)the deletion mutation in the promoter of HSPA1L confers protection to the zygote from heat shock.  Perhaps, embryonic survival during heat stress could be improved by selecting for thermotolerant genotypes (Support: BARD US-4719-14).

Keywords: heat shock, melatonin, reactive oxygen species, HSPA1L