Autophagy is a pro-survival adaptive response to heat shock in bovine cumulus-oocyte complexes

Abstract

Autophagy is a physiological mechanism that can be activated under stress conditions. However, the role of autophagy during oocyte maturation has been poorly investigated. Therefore, this study characterized the role of autophagy on developmental competence and gene expression of bovine oocytes exposed to heat shock (HS). Cumulus-oocyte-complexes (COCs) were matured at Control (38.5 °C) and HS (41 °C) temperatures in the presence of 0 and 10 mM 3-methyladenine (3MA; autophagy inhibitor). Western blotting analysis revealed that HS increased autophagy marker LC3-II/LC3-I ratio in oocytes. However, there was no effect of temperature for oocytes matured with 3MA. On cumulus cells, 3MA reduced LC3-II/LC3-I ratio regardless of temperature. Inhibition of autophagy during IVM of heat-shocked oocytes (3MA-41 °C) reduced cleavage and blastocyst rates compared to standard in vitro matured heat-shocked oocytes (IVM-41 °C). Therefore, the magnitude of HS detrimental effects was greater in the presence of autophagy inhibitor. Oocyte maturation under 3MA-41 °C reduced mRnA abundance for genes related to energy metabolism (MTIF3), heat shock response (HSF1), and oocyte maturation (HAS2 and GREM1). In conclusion, autophagy is a stress response induced on heat shocked oocytes. inhibition of autophagy modulated key functional processes rendering the oocyte more susceptible to the deleterious effects of heat shock. Autophagy is a programmed lysosomal process that degrades damaged or unnecessary cellular proteins, lipids, DNA, RNA, and organelles for recycling amino acids, fatty acids, and nucleosides to act as cellular building blocks for anabolic processes 1-3. This event is involved in development, differentiation, immunity, aging, and cell death 2. The autophagy pathway can be triggered by different stress conditions in order to restore homeostasis as a prosurvival adaptive response to stress. For example, autophagy can be induced by diseases 4-6 and environmental factors such as nutrient deprivation 7,8 , high temperature 9-11 , oxidative stress 12,13 , hypoxia 14,15 , and toxins 16-18. There are three autophagy types described in mammalian cells: chaperone-mediated autophagy, macroautophagy, and microautophagy 1. In the chaperone-mediated autophagy, heat shock cognate 70 complex (Hsc70 complex) directs abnormal proteins that have KFERQ-like motifs exposed to lysosome-mediated degradation. On the other hand, during microautophagy the lysosome membrane invaginates, thus capturing a small portion of the cytoplasm to be degraded 2. Macroautophagy is the best-understood type of autophagy, which is mediated by double-membrane vesicles that transport abnormal proteins and organelles through the cytoplasm (i.e., autophagosomes). The autophagosome membrane fuses to lysosomes to breakdown the transported material 2. During embryonic development, autophagy is first induced after fertilization to clear sperm-born material that is not necessary for further embryogenesis 19-21. In turn, autophagy is also associated with abundance of maternal...

Contextualizing Autophagy during Gametogenesis and Preimplantation Embryonic Development

“…Nonetheless, BECLIN1 protein also correlated with corpus luteum viability, thus marking theca-lutein and granulosa-lutein cells [101]. In turn, autophagy-related protein 8 (ATG8, also known as light-chain 3-LC3) was detected at the protein level in cumulus/granulosa cells of several species [62,98], irrespective of follicle size and developmental stage [102]. Exposure to FSH increased LC3 protein accumulation in granulosa cells both in vitro and in vivo [102].…”

“…Required for mouse oogenesis (except for oocyte maturation) [46,60] and for bovine and pig oocyte maturation [61,62] Activated during oocyte aging [63] and environmental stress (e.g., heat stress) [62,64] Contributes to oocyte survival after vitrification [65][66][67] but rapamycin was detrimental to developmental competence after warming [68] Spermatogenesis Required for testosterone production, sperm and acrosome formation, motility, and fertility [69][70][71][72][73] Activated during environmental stress [74][75][76][77] Activated by sperm cooling and freezing [78,79]. Autophagy inhibition improved sperm viability [80,81] Fertilization…”

“…Porcine cumulus-oocyte complexes (COCs) subject to IVM with the type III PI3K inhibitor 3-methyladenine (3MA) displayed lower cumulus cell expansion and oocyte arrest at the germinal vesicle stage [99]. Recently, maturation of bovine COCs with 3MA during IVM diminished oocyte competence as revealed by lower cleavage rate and development to the blastocyst stage [62]. Alternatively, the autophagy inducer rapamycin enhanced porcine oocyte maturation rates and embryonic development to the blastocyst stage.…”

“…Exposure to FSH increased LC3 protein accumulation in granulosa cells both in vitro and in vivo [102]. Under bovine oocyte IVM conditions, cumulus cells showed higher LC3-I/LC3-II conversion compared to oocytes [62]. It would be exciting to generate theca/cumulus/granulosa specific Atg deletions to determine the magnitude of oogenesis abrogation.…”

The Impact of Aging on Macroautophagy in the Pre-ovulatory Mouse Oocyte

“…For instance, elevated levels of GFP-LC3 puncta, indicative of autophagy pathway activation, have been detected in mouse oocytes subjected to cold stress ( Bang et al, 2014 ). Activation of autophagy has also been observed in bovine oocytes in response to heat shock ( Latorraca et al, 2020 ). Similarly, the ovulated oocytes of obese mice present with increased levels of autophagy accompanied by mitochondrial dysfunction compared to those harvested from lean mice ( Wu et al, 2015 ).…”