Modification of embryonic resistance to heat shock in cattle by melatonin and genetic variation in HSPA1L.
The objectives were to test whether (1) melatonin blocks inhibition of embryonic development caused by heat shock at the zygote stage, and (2) the frequency of a thermoprotective allele for HSPA1L is increased in blastocysts formed from heat-shocked zygotes as compared with blastocysts from control zygotes. It was hypothesized that melatonin prevents effects of heat shock on development by reducing accumulation of reactive oxygen species (ROS) and that embryos inheriting the thermoprotective allele of HSPA1L would be more likely to survive heat shock. Effects of 1 µM melatonin on ROS were determined in experiments 1 and 2. Zygotes were cultured at 38.5 or 40°C for 3 h in the presence of CellROX reagent (ThermoFisher Scientific, Waltham, MA). Culture was in a low [5% (vol/vol)] oxygen (experiment 1) or low or high [21% (vol/vol)] oxygen environment (experiment 2). Heat shock and high oxygen increased ROS; melatonin decreased ROS. Development was assessed in experiments 3 and 4. In experiment 3, zygotes were cultured in low oxygen ± 1 µM melatonin and exposed to 38.5 or 40°C for 12 h (experiment 1) beginning 8 h after fertilization. Melatonin did not protect the embryo from heat shock. Experiment 4 was performed similarly except that temperature treatments (38.5 or 40°C, 24 h) were performed in a low or high oxygen environment (2×2 × 2 factorial design with temperature, melatonin, and oxygen concentration as main effects), and blastocysts were genotyped for a deletion (D) mutation (C→D) in the promoter region of HSPA1L associated with thermotolerance. Heat shock decreased percent of zygotes developing to the blastocyst stage independent of melatonin or oxygen concentration. Frequency of genotypes for HSPA1L was affected by oxygen concentration and temperature, with an increase in the D allele for blastocysts that developed in high oxygen and following heat shock. 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 and high oxygen. Perhaps, embryonic survival during heat stress could be improved by selecting for thermotolerant genotypes.
Department of Animal Sciences, D.H. Barron Reproductive and Perinatal Biology Research Program and Genetics Institute, University of Florida, Gainesville 32611-0910.
M Sofia
MS
Ortega
School of Veterinary Medicine, Laboratory of Reproductive Physiology, Universidade Estadual Paulista, Araçatuba, SP 16050-680, Brazil; Graduate Program in Veterinary Medicine, School of Agrarian and Veterinary Sciences, Department of Animal Reproduction, Universidade Estadual Paulista, Jaboticabal, SP 16050-680, Brazil.
Nathália A S
NAS
Rocha-Frigoni
School of Veterinary Medicine, Laboratory of Reproductive Physiology, Universidade Estadual Paulista, Araçatuba, SP 16050-680, Brazil; Graduate Program in Veterinary Medicine, School of Agrarian and Veterinary Sciences, Department of Animal Reproduction, Universidade Estadual Paulista, Jaboticabal, SP 16050-680, Brazil.
Gisele Zoccal
GZ
Mingoti
Department of Animal Sciences, Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University, Rehovot 76100, Israel.
Zvi
Z
Roth
Department of Animal Sciences, D.H. Barron Reproductive and Perinatal Biology Research Program and Genetics Institute, University of Florida, Gainesville 32611-0910. Electronic address: pjhansen@ufl.edu.
Peter J
PJ
Hansen
D000818:Animals; D001755:Blastocyst; D002417:Cattle; D047108:Embryonic Development; D005260:Female; D014644:Genetic Variation; D018869:Heat-Shock Response; D006358:Hot Temperature; D008550:Melatonin
D008550:Melatonin
HSPA1L; heat shock; melatonin; reactive oxygen species