Lei Guo

Date of Award

January 2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Turk Rhen

Abstract

Many reptiles display temperature-dependent sex determination (TSD), in which the primary sex is determined by incubation temperatures rather than sex chromosomes. However, temperature is not the only factor that play critical roles in sex determination in the species with TSD. Previous studies in the snapping turtle, a species with TSD, showed that dihydrotestosterone (DHT) induces ovary development at temperatures that normally produce males or mixed sex ratios. In addition, the feminizing effect of DHT was found to be associated with increased expression of the ovary-determining gene Foxl2, suggesting a potential androgen-Foxl2 regulatory mechanism. This dissertation aims to clarify the molecular mechanisms underlying TSD in several aspects. First, determine the role of androgen in TSD; second, identify novel thermosensitive genes involved in TSD and lastly, reconstruct gene regulatory networks underlying sex determination.

To test the hypothetical androgen-Foxl2 interaction, I cloned the proximal promoter (1.6 kb) and coding sequence for snapping turtle Foxl2 (tFoxl2) in frame with mCherry, a red fluorescent protein. The tFoxl2-mCherry fusion plasmid or mCherry plasmid were stably transfected into mouse KK1 granulosa cells. Although expression of tFoxl2-mCherry was not affected by androgen treatment in KK1 cells, androgen inhibited expression of the endogenous mouse Foxl2 gene, suggesting the androgen-Foxl2 interaction does exist but it differs between species. We also found tFoxl2-mCherry potentiated low dose DHT effects on aromatase expression, which has not been reported in any other studies.

To identify novel sex-determining genes in TSD, I first de novo assembled and annotated the transcriptome of the snapping turtle using next-generation sequencing (NGS) and then performed RNA-seq analyses on the newly assembled reference transcriptome. With the differential gene expression analyses, I identified 293 thermosensitive genes. Among these genes, I find AEBP2, JARID2, and KDM6B of particular interest because these genes could influence expression of many other genes via epigenetic modifications.

To further investigate the molecular mechanisms underlying sex determination, I reconstructed gene regulatory networks using an entropy based network reconstructing algorithm – ARACNE with public microarray experiments in mouse gonads. The subsequent hub gene analyses revealed the basic molecular pathways underlying gonadal development and the master regulator analyses identified 110 candidate sex-determining genes including both known sex-determining genes and novel candidate genes.

My findings demonstrate that androgens can influence expression of key ovarian genes but further studies are needed to understand the androgen signaling in TSD. Furthermore, my study provides a first description of the snapping turtle transcriptome and the effects of temperature on transcriptome-wide patterns of gene expression during the TSP. In addition, hub genes and master regulators identified for mammalian gonad determination will guide the direction of future studies in the field of sex determination. However, additional studies are needed to validate the computational findings.

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