Abstract
Despite an incredible improvement in our understanding of the genetic mechanisms regulating mammalian reproductive development and ovarian function, there remain an abundance of reproductive disorders for which there are no identified genetic cause. In this thesis we aim to expand the lens through which we view genes involved in reproductive development in a research context. Extending our understanding of the molecular systems involved in normal ovarian development and physiology will undoubtedly improve our ability to understand clinical abnormalities when they arise.
The transcription factor LIM Homeobox 9 (Lhx9) is a critical factor in the development of the mammalian ovary progenitor structure, the genital ridge. In the absence of Lhx9 the epithelial cells that form the genital ridge fail to proliferate, resulting in absent gonads and infertility. Despite this, the function of Lhx9 in the reproductive system has not been expanded upon beyond the embryonic period, nor have the regulatory mechanisms acting on Lhx9 during gonadal development been explored. This research project has taken a broad approach to analysing the role of Lhx9 in the mouse ovary. For this I utilised an established Lhx9 heterozygous knockout mouse line where the gonads develop until maturity. Despite no obvious changes in ovarian development, the Lhx9+/- mice exhibit subfertility, with heterozygous mice of either sex producing significantly less pups per litter compared to wildtype littermates beginning at around 4 months of age.
In the first chapter, an analysis of Lhx9 in the bi-potential gonad was undertaken. Here I investigated the potential function of methylation mediated isoform expression as a method of fine-tuning Lhx9 expression in a sex dimorphic manner. This was done to understand the mechanisms behind previously observed sex-dimorphic expression of Lhx9 in the bi-potential gonad prior to Sry gene expression. Lhx9 has several isoforms with differing 5’ regions that overlap with regions of high CG content i.e., CG islands. Targeted analysis of these CG islands in male and female bipotential gonads revealed a significantly greater level of methylation in the male gonad compared to female littermates. The high level of proposed transcription factor activity at these differentially methylated CG islands led me to explore a functional role for methylation in transcription factor binding activity. To investigate this, I undertook ChIP-qPCR analysis of known co-factor WT1 binding near these regions in the male and female bipotential gonad. This revealed a significantly enriched binding of WT1 in the female consistent with the reduced methylation of CG di-nucleotides near proposed WT1 binding sites.
In the second chapter I aimed to expand the characterised function of Lhx9 beyond embryonic development to the neonatal period. This dynamic time in the ovary is critical for establishing the primordial follicle pool through tightly regulated interactions between germ cells and differentiating somatic populations. Through analysis of recently published single-cell sequencing data I was able to characterise Lhx9 as a marker of an epithelial progenitor population critical for the differentiation of somatic cells in the neonatal ovary. Aligning with these results, for the first time expression of Lhx9 has been shown in the neonatal mouse ovary. To determine the functional impact of reduced Lhx9 expression during this time I analysed changes in the follicle nest structures of the Lhx9+/- postnatal day 4 ovary. While there were no differences in the number of follicles present, there was a significant change in the proportion of oocytes existing within nest structures compared to primordial follicles. RT-qPCR expression analysis of granulosa marker genes revealed significantly greater expression of markers in the Lhx9+/- ovaries. I hypothesised this may be due to altered differentiation of cells from the Lhx9-positive progenitor population as a result of Lhx9 haploinsufficiency. This chapter provided functional evidence of a role for Lhx9 during primordial follicle formation in mice.
Lastly, I aimed to determine whether the subfertility exhibited by the Lhx9+/- mice could be due to Lhx9 function in the adult ovary. To my knowledge I have characterised the first expression pattern for Lhx9 in the mature mouse ovary. Expression in a wide range of somatic cell types, both follicular and stromal, was characterised through in situ hybridisation and immunohistochemistry. Histological analysis revealed no obvious differences in the structure of the ovary in Lhx9+/- mice. Thus, I looked to RNA-sequencing to determine what the functional role of Lhx9 may be in the adult ovary. Differential gene expression analysis revealed altered expression of several genes in response to Lhx9 haploinsufficiency. Ontological analysis showed an overrepresentation of the differentially expressed genes in terms related to steroidogenesis, epithelial regulation, and cancer. Given the expression of Lhx9 observed in the ovarian epithelium, and the altered expression of key marker genes, I carried out a closer analysis of the epithelium in the heterozygous ovary revealing a highly abnormal structure.
This thesis acts to characterise the function of Lhx9 beyond its established role in the proliferation of the earliest gonadal progenitor the genital ridge. It provides evidence of Lhx9 function in the neonatal and mature mouse ovary. Together the results presented here point toward a functional role of Lhx9 as a regulator of cell differentiation with implications for fertility and other reproductive pathologies such as cancer.