UC Berkeley

For over a century, Xenopus laevis, also known as the African clawed frog, has been used as an experimental model for studies of vertebrate development and endocrinology in biological research. Xenopus laevis has genetic sex determination, using a ZZ/ZW system. The heterogametic sex, animals with ovaries, is defined as ZW. In Xenopus laevis, DM-W is a key gene critical for animals to develop ovaries. However, a line of Xenopus laevis in the laboratory no longer carried the DM-W gene, yet could develop ovaries. This dissertation explored potential mechanistic hypotheses as to how ovary development in ZZ animals could occur.

In Chapter 1, I reviewed sex determination and differentiation in vertebrates, and discussed mechanisms and pathways for testes and ovary development.

In Chapter 2, I established a method to measure relative gene expression in the gonads of Xenopus laevis tadpoles. This chapter introduced a novel method of adjusting a feature in qPCR: the standard curve. This method (Curve-Standardized qPCR) creates a standard curve via PCR Product Purification to measure genes with low expression. Additionally, this chapter proposed techniques for dissecting gonads from Xenopus laevis tadpoles without contamination by other tissues and methods for maximizing RNA extraction yield to improve the reliability of results. Key findings include the minimum number of gonads needed to yield reliable results and the use of PCR to create a usable standard.

In Chapter 3, I used DNA sequencing and the Curve-Standardized qPCR method discussed in Chapter 2 to explore the role of the Xenopus laevis testis development gene, DMRT1. If DMRT1 was not functional in ZZ ovary-developing animals (ZZ OD), it could potentially lead to ovary development. No significant differences in the coding region of DMRT1 were observed between ZZ OD and ZZ non-ovary-developing animals (ZZ non-OD). Additionally, no differences were observed in the gene expression of DMRT1 in ZZ OD and ZZ non-OD animals during the critical stage of sex determination.

In Chapter 4, I explored the genome of ZZ OD using Double Digest Restriction Associated DNA sequencing (ddRADseq) and identified a region of interest on Chromosome 5L. Key sex differentiation genes known on Chromosome 5L include Foxl2, Fshr, Gata4-a and Gata4-b. However, none of these genes are in the region of interest. Four out of five genes regulated by estrogen found on Chromosome 5L are in the region of interest. These genes are crim1, tm4sf1, myom2 and serpini2. One gene (SYNE1: spectrin repeat containing nuclear envelope protein 1) regulated by aryl hydrocarbon receptor (AHR) is found on Chromosome 5L in the region of interest. There are SNPs in these genes that differ between ZZ OD and ZZ non-OD. None of these genes are currently recognized to have a role in sex differentiation or steroidogenesis.

In the final chapter, I concluded with future directions, including additional experiments, ideas, and discussion derived from findings presented in the previous chapters.