Abstract
Sex is fixed from birth in most organisms, where individuals develop as either female or male and retain their sex through life. Traditionally, sex has been considered to be determined by genetic elements or by environmental factors. However, we now know numerous examples of plant and animal species that challenge this false dichotomy and where sexual fate is influenced by both strategies. One of the most fascinating examples of sexual plasticity is found in sequentially hermaphroditic fishes, in which species begin life as one sex, but can change to the other sometime thereafter.
Considerable advances have been made in the understanding of the behavioural, morphological and molecular basis underlying this fascinating transformation. However, how cues received by fish (e.g. social cues, attainment of a certain size, etc.) trigger sex change at a physiological level is still not fully understood, and current research is biased towards tropical species. It has been hypothesised that this trigger is mediated by cross-talk between the hypothalamic-pituitary-gonadal (HPG) and -interrenal (HPI) axes, which regulate reproduction and stress, respectively. Cortisol has emerged as a potential key element in the transduction of environmental signals that can initiate sex change. A more comprehensive knowledge of this process is of considerable value, as not only will it shed light on our understanding of vertebrate sexual development, but may also be applied for sex control in aquaculture settings.
This thesis aimed to characterise sex change in the temperate New Zealand spotty wrasse (Notolabrus celidotus), and highlight its potential to become a model for the study of sequential hermaphroditism. Spotties exhibit protogynous (female-to-male) sex change based on social context. Removal of the dominant male from a social group can induce sex change in the largest female. This thesis explored the physiological and genetic cascade underlying sex change in spotties, paying special attention to the role of cortisol, while also highlighting differences between tropical fishes that breed throughout the year and temperate fishes that breed seasonally.
Experiments involving aromatase inhibition or social manipulation, within or outside the breeding season, were used to characterise the chemical and social induction of spotty sex change between 2014 – 2018. Alterations in gonadal morphology, steroid profile and gonadal candidate gene expression were explored. Measurement of plasma sex steroids 17β-estradiol (E2) and 11-ketotestosterone (11KT) across experiments showed a general trend of decreasing E2 and increasing 11KT towards maleness. A higher number of transitioning individuals were found among spotties manipulated outside their breeding season, when sex change occurs naturally. Timing of experiments relative to the breeding season also affected the steroid profile, and a drop in E2 concentrations at the onset of sex change, characteristic of tropical fish, was not observed in spotties during the post-spawning period, when sex steroids are expected to already be low. This suggested that arrested E2 production may not be a key event at the initiation of sex change in spotties, and potentially other temperate species.
A panel of 21 genes were selected to explore the role of the sex determination and stress response pathways in the process of sex change using the nanoString platform. Principal Component Analysis of the patterns of gene expression revealed by our sex and stress gene panel in gonadal samples from fish socially induced to change sex revealed strong clustering of samples by sex-change stage, validating the histological classification of stages based on gonadal morphology. The expression patterns for many of these steroidogenic and sex determination and differentiation genes was strongly conserved between spotties and other species. Interestingly, expression of the masculinising gene amh (anti-Müllerian hormone) was upregulated at the onset of sex change, suggesting a potential early regulatory role of amh as a proximate trigger of sex change. Some major male-pathway (e.g. sox9a, znrf3) and female-pathway (e.g. cyp19a1a, rspo1) genes and also some epigenetic-related genes (e.g. kdm6bb) exhibited unexpected expression patterns based on mammalian and reptilian models. This suggests that the genetic cascade orchestrating sex change in spotty is regulated by a genetic network that is broadly conserved among protogynous sex-changing fish, albeit with some variations in the roles of specific genes. It also seems likely that epigenetic factors play an important role in the regulation of sex change.
The role of cortisol as a potential trigger of sex change was tested through in vivo administration of cortisol (5000 µg) to female spotties for 71 days. Although this treatment did not induce sex change, upregulation of amh expression was observed, implying a physiological effect on its expression by cortisol, but this was obviously not strong enough to set off the full genetic cascade triggering sex change. Several technical issues, such as the relative rapid release of cortisol from the implants used (8 – 10 days) or the seasonality of this study potentially impacted the likelihood of sex change due to cortisol treatment.
For the first time in a sequentially hermaphroditic fish, the patterns of expression for key steroidogenic and epigenetic regulatory genes were also evaluated in the head kidney, the organ functionally analogous to the adrenal gland in mammals and the site of most stress hormone production. Spotties implanted with cortisol, showed upregulation of hsd11b2 (11β-hydroxysteroid dehydrogenase type 2) expression confirming the physiological effect of cortisol previously observed. However, significant sex-biased expression of major sex-pathway genes was generally not observed in the head kidney of spotties socially induced to change sex.
Finally, an in vitro culture system for fish ovaries was successfully developed, enabling research of the effect of E2, 11KT and cortisol in sex change to be investigated in spotties and potentially other sex changing fish. Oocyte degeneration associated with tissue culture was observed across treatments, but doses of E2 higher than 10 ng/mL were found to minimise this effect. Neither 11KT nor cortisol was observed to induce sex change of spotty ovaries. This outcome was hypothesised to be affected by the duration of the culture (i.e. 21 days), which was substantially shorter than the time needed for sex change to be completed in vivo (i.e. 70 days). Nonetheless, the valuable organ culture system developed here creates new opportunities to couple in vitro manipulative studies with promising techniques such as single-cell sequencing.
Successful induction of sex change in captive spotties, the ability to precisely stage transitioning gonads, the development of a gene panel that accurately captures the ovary-to-testis genetic transition, and the establishment of an ovarian culture system reinforce the utility of spotty as an exceptional model for the study of fish sequential hermaphroditism. These experiments and resources set the baseline for further exploration of the role of stress during sex change at the level of both the gonad and the head kidney in this and other systems.