title: Validation and use of a HPLC-MS/MS method for the analysis of multiple steroid hormones in humpback whale blubber
Creators: name: Dalle Luche, Greta
Publisher: name: Griffith University
Year: 2020
Subjects: subject: multiple steroid hormone analysis
subject: endocrinology
subject: reproductive biology
subject: Southern Hemisphere humpback whale
subject: Megaptera novaeangliae

description: This thesis describes the novel use of multiple steroid hormone analysis to investigate and gain understanding of previously unknown aspects of the endocrinology and reproductive biology of the Southern Hemisphere humpback whale (Megaptera novaeangliae). Steroid hormones are a class of structurally related molecules that function as messengers in a variety of important physiological processes (e.g. salt homeostasis, immune system, sexual development and reproductive cycles). A number of endogenous steroid hormones have been identified as critical for reproduction (e.g. androgens, progestogens, oestrogens) and stress response (e.g. corticosteroids) in mammals. Understanding the species-specific roles and baseline levels of endogenous steroid hormones enable these compounds to be used as biomarkers of reproductive status and stress exposure. The use of steroid biomarkers for these purposes promises to be extremely advantageous in cetacean species, since other approaches often require increased time and resources. However, the collection and interpretation of steroid hormone data in the humpback whale is currently limited by methodological issues (e.g. challenging sampling, difficult comparison between sample types, analytical approach restricted to single hormone analysis), and by a general lack of detailed knowledge of the humpback whale endocrine system. Collection of biological samples from cetaceans is inherently difficult. In particular, free-swimming individuals of baleen whale species, including humpback whales, cannot be kept in captivity, nor they can be immobilised for sampling. Contemporary studies have demonstrated that steroid hormones analysis is viable in baleen species through the use of remotely collected tissues and fluids (i.e. faeces, blow, blubber). The seasonal migration of the humpback whales from the Antarctic feeding areas to the tropical breeding grounds, however, imposes further restrictions in regard to sample types and timing of collection. To date, blubber is the only matrix employed for steroid analysis in this specie and comprehensive seasonal steroid hormone changes have not being studied. Blubber is a lipid-rich tissue, able to sequester traces of steroid hormones from circulation. This is advantageous as multiple types of steroid hormones are likely to be present in this tissue. However, steroid hormones in blubber, particularly those at trace levels, can only be quantified by using sensitive techniques that need to accommodate the presence of lipids in the matrix. Enzyme immunoassay (EIA) is typically used for steroid hormone analysis in complex matrices, as it requires minimal sample preparation. Although EIA provides high sensitivity, its precision can suffer due to the indirect nature of the measurements and by the possibility of cross-reactivity. A significant disadvantage of employing EIAs for the investigation of species-specific endocrinology is that each EIA quantifies only a single steroid hormone, or a single class of steroid metabolites in its ensemble. Single steroid hormone measurements can be misleading, as steroid hormones act in concert. The same steroid hormone can have different roles and increase or decrease in concentration to accommodate different biosynthetic pathways (e.g. acting as an active metabolite, or as a precursor to or degradation product from other steroids). Liquid chromatography coupled with tandem mass-spectrometry (LC-MS/MS) represents an alternative to EIAs, as it can provide accurate and precise quantification of multiple steroid hormones from the same sample portion. Major issues in LC-MS/MS methods include the coelution of multiple analytes or the interference of matrix components, which can sometimes be resolved only through a trade-off between analyte resolution and sensitivity. These challenges can however be identified prior to the analysis, and overcome, at least partially, by optimising the sample extraction and cleanup. This thesis aimed to adapt and validate a LC-MS/MS method for extracts of humpback whale blubber, and to evaluate use of multiple steroid hormone measurements in relation to reproductive status and possible stress exposure from free-ranging individuals. Blubber samples from stranded humpback whale carcasses were initially employed to assess the applicability, scope, and repeatability of a LC-MS/MS method for the analysis of multiple steroid hormones (Chapter 2). The method, initially developed for bottlenose dolphin (Tursiops truncatus) blubber, proved to be applicable to the comparatively lipid-rich humpback whale outer blubber. Levels of five corticosteroids and six reproductive steroids (including androgens, progestagens and oestrogens) were determined in humpback whale blubber, and ten of these analytes could be quantified with high accuracy (error on amended samples &lt; 15%) and repeatability (percent standard deviation &lt; 15%). The multi-steroid hormone profiles obtained by the stranded carcasses were also investigated as potentially indicative of the endocrinological responses of some of the whales to stress exposure. Subsequently, the LC-MS/MS method was applied to biopsy extracts from freeswimming humpback whales collected at two time points of the whale breeding season over multiple years. The application of this methodology to live biopsies resulted in a smaller number of analytes detected, predominantly as an effect of the corticosteroid analytes being lower in concentration or undetectable in these samples. The quantified steroid hormones provided, however, novel insights into endocrinology and reproductive biology of female (Chapter 3) and male (Chapter 4) humpback whales. In Chapter 3, we show how the use of a previously validated pregnancy concentration threshold for the single hormone progesterone failed to detect any pregnant females based on LC-MS/MS analysis of blubber extracts from 23 females sampled while approaching the breeding grounds. However, by considering multiple steroid profiles including androgens and cortisol, we suggest that relatively high blubber concentrations of androstenedione may distinguish pregnant individuals during the last month of gestation. This hypothesis is supported by reference to steroid hormone measurements through pregnancy in evolutionarily related mammal species, although further work (e.g combining photo identification) is required to provide conclusive evidence. In addition, the same previously validated progesterone threshold resulted in an unexpectedly low proportion of pregnant females (3%) from among 29 females departing the breeding grounds. This prompted the suggestion of a new lower progesterone concentration threshold for assignment of early pregnancy status. In Chapter 4, the seasonal variations in blubber steroid hormone concentration in males are examined through the breeding season. Lowering of testosterone during the expected peak in reproductive activity suggests asynchronicity between blubber testosterone levels and male fertility. Correlation analyses among multiple hormone couples are used to postulate the changes in biosynthetic pathways behind the large interseasonal variations in steroid concentrations. These large intra-season variations in blubber androgens and a contrasting relationship with cortisol across the migration, encourages further investigation on the effects of age and sexual maturity on concentrations of these two hormones. This is necessary before they can be used as biomarkers for reproductive status or stress exposure. An application of how some of the multi-hormone information can be used to improve humpback whale monitoring is provided in Chapter 5. Early and late pregnancy diagnoses as formulated in Chapter 3, are combined with body condition information simultaneously measured in the same female individuals. Superior body condition among late-pregnant female corroborated previous observations that females would increase their feeding opportunities before parturition in order to support, as capital breeders, the combined energetic cost of migration and lactation. In contrast, no relationship was found between whales identified as early pregnant and their body condition compared to the rest of the migrating females. These results suggest that body condition and fertility might not be directly related during the breeding season. Instead, annual winter-feeding efficiency might be more important than accumulated energy stores in guaranteeing the annual reproductive success in females of this species. Overall, by analysing for a larger suite of steroid compounds and focusing on a comparatively less studied period of the migration, this work revealed some limitations of the current methodology used to measure steroid hormones in humpback whales. These findings enhance our knowledge of the humpback whale endocrine system and provide new approaches for the diagnostic use of steroid hormones profiles in free-swimming humpback whales.