Oocyte response to heat stress

Author(s)

Lais Barbosa Latorraca

• Document type: Master's Dissertation
• Press: Botucatu. 2019-04-12.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Botucatu
• Defense date: 2019-02-27
• Advisor: Fabíola Freitas de Paula Lopes; Weber Beringui Feitosa

Abstract

The series of events triggered during the oocyte maturation is very susceptible to environmental stress. Adverse conditions such as pro-oxidant agents and environmental temperature compromise oocyte function, reducing fertilization capacity and subsequent embryonic development. The negative effect of heat stress on bovine fertility has been well characterized. Among the cellular effects of heat stress on bovine oocytes, one can highlight cytoskeletal disorganization, increased production of reactive oxygen species (ROS), mitochondrial damage, and activation of cell death by apoptosis. A conserved effect of heat shock on different cell types is protein denaturation, activating protection mechanisms in the cytoplasm and endoplasmic reticulum (ER) to maintain proteostasis. The ER acts as an environmental stress sensor activating Unfolded Protein Response (UPR), which can trigger autophagy and/or apoptosis, depending on the intensity of the stress. Despite the importance of ER for oocyte function, the effects of heat shock on this organelle have never been investigated. Therefore, the general objectives of this dissertation were to determine the role of ER stress on function of bovine oocytes submitted to heat shock (Chapter 2) and the role of autophagy on gene expression and developmental competence of bovine oocytes submitted to heat shock during in vitro maturation (IVM) (Chapter 3). For the ER studies, cumulus-oocyte complexes (COCs) were initially distributed in the following groups: 0 h (immature oocytes), Control (IVM at 38.5°C for 22 h) and Heat Shock (IVM at 41°C for 16 h followed by 6 h at 38.5°C). After IVM, denuded oocytes were stained for meiotic progression and ER localization evaluated by confocal microscopy. Exposure of bovine oocytes to heat shock reduced maturation rate and negatively altered the localization pattern of ER compared to control. In the second series of experiments, the oocytes were submitted to Control and Heat Shock temperatures in IVM medium, vehicle control [0.00096% (v/v) DMSO], and Salubrinal medium (400 nM Salubrinal - SA: ER stress inhibitor) to determine overall protein synthesis, abundance of ER stress protein markers and oocyte function. In some models, the ER stress inducer (5 μg/mL Tunicamycin - TM) was also used as a positive control. Exposure of COCs to heat shock in IVM medium did not reduce the ability of oocyte to synthesise proteins. However, inhibition of ER stress during heat shock increased de novo synthesis of proteins relative to control group (IVM at 38.5°C). In addition, TM reduced protein synthesis compared to the other groups. Western blotting analysis showed no difference in the abundance of sXBP1 (ER stress marker) and ubiquitin (protein degradation marker) between groups. However, Caspase 3 protein (apoptosis marker) was increased in TM-matured oocytes compared to inhibition of ER stress during heat shock, demonstrating that ER stress can induce cell death. Functional evaluation indicated heat shock in IVM medium did not change normal fertilization pattern (2 pro-nuclei-PN). However, supplementation with DMSO had a protective effect on stressed oocytes, increasing the percentage of oocytes with 2 PN. Oocyte exposure to heat shock caused a delay in embryonic developmental kinetics by reducing the percentage of 2-cell embryos at 29, 35 and 41 hours after insemination (hai), as well as cleavage at 32-48 hai. On days 3 and 8 after insemination, cleavage and blastocyst rates were also reduced in stressed oocytes. Addition of DMSO recovered some of the deleterious effects of heat shock on early embryonic kinetics and blastocyst development, masking the actual effect of ER inhibition. Chapter 3 determined the impact of autophagy inhibition on developmental competence and gene expression of bovine oocytes exposed to heat shock during IVM. Experiment 1 determined the effect of autophagy inhibition on developmental competence of bovine oocytes submitted to heat shock. COCs were matured at Control (38.5ºC for 22 h) and Heat Shock (41ºC for 16 h followed by 38.5ºC for 6 h) temperatures in the presence of 0 and 10mM 3MA (3-methyladenine, autophagy inhibitor). After IVM, oocytes were submitted to IVF and IVC for 8 days. Inhibition of autophagy during IVM of oocytes exposed to heat shock reduced cleavage and blastocyst rates compared to the other groups. Therefore, the magnitude of the deleterious effects of heat shock on oocyte developmental competence was greater when autophagy was inhibited. Experiment 2 was conducted with the same 2 x 2 factorial design to determine the effect of autophagy inhibition on gene expression of oocytes exposed to heat shock. Inhibition of autophagy during heat shock reduced mRNA abundance of genes related to oocyte maturation (BMP15, HAS2, and GREM1), lipid and energy metabolism (SREBF2 and MTIF3), cell growth (IGF2), and heat shock response (HSF1 and HSPA1A). In conclusion, heat shock during IVM adversely affected meiotic progression, distribution of ER, and retarded kinetics of embryonic development, reducing cleavage and blastocyst rates. Inhibition of autophagy modulated important functional processes in bovine oocyte, turning the oocyte more susceptible to the deleterious effects of heat shock. Therefore, autophagy is a pro-survival oocyte response under stress conditions. However, additional experiments are needed to demonstrate the importance of ER stress on oocytes submitted to heat shock. (AU)

• FAPESP's process: 17/13082-6 - Oocyte responses to heat stress
• Grantee: Lais Barbosa Latorraca
• Support type: Scholarships in Brazil - Master