The Effects of Calcitonin on the Development of and Ca²⁺ Levels in Heat-shocked Bovine Preimplantation Embryos In Vitro

Abstract

Intracellular calcium homeostasis is essential for proper cell function. We investigated the effects of heat shock on the development of and the intracellular Ca²⁺ levels in bovine preimplantation embryos in vitro and the effects of calcitonin (CT), a receptor-mediated Ca²⁺ regulator, on heat shock-induced events. Heat shock (40.5 C for 10 h between 20 and 30 h postinsemination) of in vitro-produced bovine embryos did not affect the cleavage rate; however, it significantly decreased the rates of development to the 5- to 8-cell and blastocyst stages as compared with those of the control cultured for the entire period at 38.5 C (P < 0.05). The relative intracellular Ca²⁺ levels at the 1-cell stage (5 h after the start of heat shock), as assessed by Fluo-8 AM, a fluorescent probe for Ca²⁺, indicated that heat shock significantly lowered the Ca²⁺ level as compared with the control level. Semiquantitative reverse transcription PCR and western blot analyses revealed the expression of CT receptor in bovine preimplantation embryos. The addition of CT (10 nM) to the culture medium ameliorated the heat shock-induced impairment of embryonic development beyond the 5- to 8-cell stage. The Ca²⁺ level in the heat-shocked embryos cultured with CT was similar to that of the control embryos, suggesting that heat shock lowers the Ca²⁺ level in fertilized embryos in vitro and that a lower Ca²⁺ level is implicated in heat shock-induced impairment of embryonic development. Intracellular Ca²⁺-mobilizing agents, e.g., CT, may effectively circumvent the detrimental effects of heat shock on early embryonic development.