Development and Application of an Ex Vivo Adipose Tissue-based Model from Cetacean Blubber Biopsies to Study Impacts of Stress Hormones

Abstract

Cetaceans serve as ecosystem engineers and sentinel species for ocean and human health. They are exposed to various anthropogenic stressors, including pollution, noise, human activities, and climate change which contribute to high levels of stress hormones such as glucocorticoids (e.g. cortisol). While stress response mechanisms are vital in the short term, prolonged exposure to stress can have serious health implication as elevated glucocorticoid levels have been associated with lower immunity, impaired reproduction, and stranding. However, the overall impact of stress on cetaceans remains unclear, as in vivo exposure studies are non-feasible, research rely on correlative approaches without establishing cause-effect relationships between stressors and health. To address this knowledge gap, this study developed an ex vivo adipose tissue model using cetacean blubber biopsies to investigate the effects of stress hormones on genes involved in stress and immune responses. Precision-cut adipose tissue slices (PCATS) were made from blubber biopsies of sperm whale (Physeter macrocephalus) and humpback whale (Megaptera novaeangliae).

Viability of PCATS from sperm whale was evaluated through the release of lactate dehydrogenase (LDH), reflecting damages in PCATS, but resulted in incomplete responses. A novel viability test was applied on PCATS from humpback whale by measuring oxygen saturation in the culture medium, as a proxy for mitochondrial respiration, which confirmed viability of PCATS.

Messenger RNA was used to study stress-induced changes on gene expression, however, trial samples revealed RNA content in PCATS to be a limitation. Thus, an optimized RNA extraction protocol was developed and applied on blubber biopsies from five cetacean species, alongside histological analysis, to investigate species differences in RNA content and blubber composition. The findings suggest that investigating gene expression in the PCATS model is suitable for pilot whales, killer whales, humpback whales, and fin whales, and less suitable for sperm whales due to their lower adipose tissue content and RNA yields. Additionally, RNA yield and integrity in humpback whale blubber were compared across tissue prior to slicing, non-cultured PCATS, and PCATS cultured for 48 hours. Results showed that sampling and slicing blubber biopsies caused RNA degradation. Yet, sufficient high-integrity RNA found in PCATS cultured for 48 hours enables analyses such as RT-qPCR and transcriptomics. Notably, RNA degradation ceased once biopsies were placed in the sampling medium, supporting the potential for remote sampling, and also stopped after slicing, enabling studies extending beyond 48 hours, pending viability.

Lastly, PCATS from humpback whales were cultured for 48 hours with 400 nM cortisol introduced every 12 hours to mimic chronic stress response alone or combined with 10 µM epinephrine, another stress hormone involved in the immediate stress response, in the final 12 hours to simulate an acute stress response. RT-qPCR analysis revealed that cortisol alone and combined with epinephrine upregulated PPARG and downregulated TNFα and TLR4 expression, suggesting that cortisol contributes to an anti-inflammatory state in humpback whale PCATS. These findings highlight a novel tool to investigate the impact of anthropogenic stressors on cetacean blubber physiology.