Biology of Sex Determination and Sexual Development in the Cane Toad (Bufo marinus)

To date, the majority of studies into sex determination and sexual development have focused on the mammalian system due to the mouse model being an excellent tool for developmental biology as well as obvious implications to human health and development. However, the focus on the mammalian system has caused a large fraction of other vertebrate groups to be overlooked. The last decade has seen an array of studies into various non-mammalian organisms like fishes, birds, reptiles, and amphibians. These studies have unveiled a remarkably conserved molecular background utilized for sexual differentiation amongst all vertebrates, ranging from the alligator which employs temperature dependent sex determination to the mouse, which employs genotypic sex determination In this project, I implemented molecular methods traditionally used to study model organisms, to investigate an amphibian species, the cane toad (Bufo marinus). The cane toad was chosen due to its invasive status in Australia, as well as being a representative of one of the most successful and specious families of vertebrates, Bufonidae. Since, its introduction, this species has rapidly spread across the continent, adversely effecting native species throughout its introduced range. Recent studies have identified the disruption of the sex-determination pathway as a realistic goal in an otherwise, seemingly futile, effort to curtail their expansion. We decided to approach the study of cane toad sex determination and development on three levels, in order to have a broader understanding of the similarities between toads and other groups, and additionally, to better assess the feasibility of utilizing molecular means to control cane toads in non-native habitats. Firstly, I used a candidate gene approach to clone and characterize five genes which were previously known to be involved in both mammalian as well as amphibian sex determination: Sox9, Dmrt1, p450arom, Sf1, and Dax1. I chose Sox9 and Dmrt1 due to their known involvement in the male-specific pathway of mammals. Dmrt1 was also known to be male specific in other groups, including amphibians. Conversely, I also decided to investigate p450arom and Dax1, due to their affiliation with the female pathway, while Sf1 has a role in both sexes. All of these genes were expressed in the gonads of both sexes of cane toads. However, Sox9 exhibited strong transcriptional up-regulation in testes at the time of sexual differentiation, similar to mammals. Thus, using the candidate gene approach, I was successful in identifying a sex-specific marker which could be utilized in the manipulation of the sex determination pathway (e.g. female to male sex reversal). VIIThe second approach involved the study of sex determination on a cytogenetic level. Traditionally, Bufonids have been assumed to utilize a ZZ/ZW (male homogametic/female heterogametic) sex chromosome system, yet only one recent study has been successful in the identification of sex chromosomes in a toad species. Moreover, the authors identified the sex chromosomes in only one population (of 18 studied), highlighting the scarcity of discernible sex chromosomes in toads. When we began to investigate the karyotype of cane toads, we were able to identify a female-specific length polymorphism in the nucleolus organizer region (NOR) of chromosome 7, making this chromosome pair a strong candidates for the Z and W sex chromosomes. In order to verify our hypothesis, we also performed chromomycin A3 staining to reveal a differential signal between the chromosome pair, indicative of heterochromatin accumulation on the brighter NOR. This study lent strong support to the observed difference between the chromosomes, yet we required further investigation in order to identify the W and the Z chromosomes, specifically. By utilizing comparative genomic hybridization (CGH), I was able to identify a female specific region on the chromosome with the larger NOR, identifying it as the W chromosome. This study was successful in identifying the sex chromosomes in the cane toad. Moreover, it allows us to make a more accurate prediction of the possible sex determination method utilized: either a dosage dependent male determination system, or a female-specific gene in a female determination system. The third approach focused on the Bidder’s organ and its role in sexual development of cane toads. The Bidder’s organ is a gonadal structure comprised of ovary-like tissue, found in both males and females, with no known function. Since our study was focused on female to male sex-reversal of cane toads, we decided to investigate the potential of the Bidder’s organ to interrupt this process. By utilizing the candidate genes, which I had previously cloned, I was able to identify the Bidder’s organ as having an entirely distinct transcriptional pattern in comparison to the gonads of either sex. Additionally, the Bidder’s organ showed significantly higher levels of p450arom expression than the gonads, identifying it as a possible key player in the production of aromatase enzyme for oestrogen production. Moreover, gene expression patterns in the Bidder’s organ strongly correlated with the significant developmental time points in the sexual development of the toads, implying a possible function in the development process of toads. In conclusion, I have performed the first study of the molecular, cytogenetic and anatomical aspects of sexual development in a toad. I was able to verify that cane toads utilize a ZZ/ZW chromosome system and furthermore, likely utilize the mammalian male-linked gene, Sox9, in male development. I was also able to show that the Bidder’s organ is transcriptionally active at key time points, likely indicative of a functional role during development