Characterization of the effects of heat stress on autophagy induction in the pig oocyte

Abstract

Background: Heat stress (HS) occurs when body heat accumulation exceeds heat dissipation and is associated with swine seasonal infertility. HS contributes to compromised oocyte integrity and reduced embryo development. Autophagy is a potential mechanism for the oocyte to mitigate the detrimental effects of HS by recycling damaged cellular components.

Methods: To characterize the effect of HS on autophagy in oocyte maturation, we utilized an in vitro maturation (IVM) system where oocytes underwent thermal neutral (TN) conditions throughout the entire maturation period (TN/TN), HS conditions during the first half of IVM (HS/TN), or HS conditions during the second half of IVM (TN/HS).

Results: To determine the effect of HS on autophagy induction within the oocyte, we compared the relative abundance and localization of autophagy-related proteins. Heat stress treatment affected the abundance of two well described markers of autophagy induction: autophagy related gene 12 (ATG12) in complex with ATG5 and the cleaved form of microtubule-associated protein 1 light chain 3 beta (LC3B-II). The HS/TN IVM treatment increased the abundance of the ATG12-ATG5 complex and exacerbated the loss of LC3B-II in oocytes. The B-cell lymphoma 2 like 1 protein (BCL2L1) can inhibit autophagy or apoptosis through its interaction with either beclin1 (BECN1) or BCL2 associated X, apoptosis regulator (BAX), respectively. We detected colocalization of BCL2L1 with BAX but not BCL2L1 with BECN1, suggesting that apoptosis is inhibited under the HS/TN treatment but not autophagy. Interestingly, low doses of the autophagy inducer, rapamycin, increased oocyte maturation.

Conclusions: Our results here suggest that HS increases autophagy induction in the oocyte during IVM, and that artificial induction of autophagy increases the maturation rate of oocytes during IVM. These data support autophagy as a potential mechanism activated in the oocyte during HS to recycle damaged cellular components and maintain developmental competence.

Keywords: Autophagy; Heat stress; Oocyte; Pig.

Fig. 1

Heat stress during the first half of IVM increases the abundance of ATG12 in complex with ATG5. Oocytes collected from aspirated pig follicular fluid underwent in vitro maturation (IVM) in either TN conditions throughout the entire 42-h maturation period (TN/TN), HS conditions during the second half of IVM (TN/HS), or HS conditions during the first half of IVM (HS/TN). Representative western blots for BECN1, ATG12-ATG5 complex, BCL2L1, LC3B, and α-tubulin (A) from oocytes collected before IVM (GV) and after 42 h of IVM. There was increased abundance of ATG12 in complex with ATG5 after 42 h of IVM under the HS/TN temperature treatment compared to the TN/TN and TN/HS (B). Different superscripts within the same protein denotes significant difference (P < 0.05)

Fig. 2

Heat stress exacerbates the decrease of LC3B-II in the oocyte. Oocytes collected from aspirated pig follicular fluid underwent in vitro maturation (IVM) in either TN conditions throughout the entire 42 h maturation period or HS conditions during the first half of IVM (HS/TN). Representative western blot for LC3B from oocytes having undergone TN or HS conditions in IVM after 21 or 42 h or collected before IVM (A). There was decreased abundance of cleaved LC3B-II at 21 h of HS IVM compared with 21 h of TN IVM (B). Asterisks denotes significant difference (P < 0.05) from control

Fig. 3

Colocalization of BAX and BCL2L1. Oocytes that underwent either TN/TN IVM or HS/TN IVM were fixed and used for IHC to determine colocalization. Neither the time point during IVM nor the temperature treatment affected colocalization of antibody-labeled BCL2L1 and BAX in the oocyte. Based on level of fluorescence, there appeared to be a qualitative increase in BAX (red) at 21 h of IVM under HS compared to 21 h of IVM at TN conditions, though there appeared to be no effect on the abundance of BCL2L1 (green; A). The comparison between 21-h TN (B), 21-h HS (C), or negative control (D) shows that there was punctate yellow signal representing colocalization in both TN and HS oocytes. For A, the white bar in images in the left and middle columns represent 50 μm, and the white bar in images in the right column represents 20 μm. The white arrows point to areas of colocalization. The images in the right column are magnified 5× images from the same oocyte from the middle column

Fig. 4

Colocalization of BECN1 and BCL2L1. Oocytes that underwent either TN/TN IVM or HS/TN IVM were fixed and used for IHC to determine co-localization. Colocalization of BECN1 (green) and BCL2L1 (red) was low regardless of time point during IVM or temperature treatment. There appeared to be more punctate intense spots of BECN1 staining in oocytes having undergone 21 h of IVM under HS compared to the oocytes that underwent 21 h of IVM at TN conditions (A). The comparison between 21-h TN (B), 21-h HS (C), or negative control (D) shows that there was punctate green signal representing BECN1 but no colocalization in both TN and HS oocytes. For A, the white bar in images in the left and middle columns represent 50 μm, and the white bar in images in the right column represents 20 μm. The white arrows point to areas of punctate BECN1 signal but no colocalization. The images in the right column are magnified 5× images from the same oocyte from the middle column

Fig. 5

Low Concentration of Rapamycin Increases Maturation Rate. Oocytes underwent IVM with the HS/TN temperature treatment in the presence of vehicle control, 100 nM, 10 nM, or 1 nM rapamycin. The inclusion of 100 nM or 10 nM rapamycin in the IVM media had no effect on maturation rate after HS/TN IVM compared to vehicle control, though oocytes matured in the presence of 1 nM rapamycin had increased maturation rate compared to vehicle control