Defining the impacts of oestrogen on cell fate decisions in the gonad

Abstract

The increasing incidence of testicular dysgenesis syndrome-related conditions and decline in fertility has been linked to the prevalence of oestrogenic endocrine disrupting chemicals (EDCs) in the environment. Ectopic activation of oestrogen signalling by EDCs in the gonad can impact the latter’s function and development in both males and females, but little is understood about the processes behind this. In non-mammalian vertebrates, oestrogen is the critical driver of ovarian differentiation, while in mammals, oestrogen is not required for ovarian determination but is essential for its maintenance. In marsupials, exogenous oestrogen signalling causes cytoplasmic retention of the key testis factor SOX9, leading to a switch from testicular to ovarian developmental programs. SOX9 nuclear import is critical for testis differentiation; several key processes regulate its subcellular localisation, including the microtubule cytoskeleton, post-translational modifications of SOX9, and binding to importin-b and calmodulin.

In this thesis, I demonstrated that exogenous oestrogen similarly causes cytoplasmic retention of SOX9, suppression of pro-testis genes and activation of pro-ovarian genes in the human testis-derived cell line NT2/D1. Utilising this established system, I investigated the effects of oestrogen on SOX9 nuclear import requirements. Immunofluorescence studies showed that exogenous oestrogen treatment of NT2/D1 cells led to the nuclear accumulation of importin-b, decreased phosphorylation of SOX9, and promoted the stabilisation of the microtubule network. ERK1/2 has a role in mediating gonad developmental pathways, is responsive to oestrogen and is known to affect the microtubule network—thus, ERK1/2 presented as a candidate for regulating the microtubule network and SOX9 in response to oestrogen. To further understand the non-genomic regulation of microtubules and role of ERK1/2 in this process, I assessed the rapid response of these factors to brief oestrogen treatment. Oestrogen rapidly stabilised microtubules and caused the cytoplasmic retention of SOX9, while treatment with an ERK1/2 inhibitor prevented these effects of oestrogen. From these experiments, I demonstrated that oestrogen rapidly activates ERK1/2 to facilitate stabilisation of microtubules and the cytoplasmic retention of SOX9. This established that oestrogen acts in a non-genomic manner to influence SOX9 bioavailability, which is reinforced later by genomic changes that promote a switch to ovarian somatic cell fate.

Using proteomic and phosphoproteomic analyses, I examined the response of the MAP3K1 and MAP3K4 cascades—which have a role in mediating gonad differentiation and act upstream of ERK1/2—to brief or prolonged oestrogen treatment. The MAP3K1 cascade appeared upregulated following EE2 treatment and b-catenin was activated by phosphorylation at Ser552, while the MAP3K4 pathway remained unchanged. This demonstrated that oestrogen can target this crucial pathway to promote a shift from testicular (pro-SOX9) to ovarian (pro-b-catenin) fate. From these analyses, I further showed that oestrogen treatment can impact proteins involved in SOX9 post-translational modification, the nuclear import pathway and the mTOR pathway. These studies also demonstrated that oestrogen can impact microtubule regulation through hypophosphorylation of MAPs, remodel the actin cytoskeleton through ARP2/3- and mDia2-mediated actin polymerisation, and increase the abundance of the intermediate filament vimentin. Together, these analyses provide an unparalleled investigation of the mechanisms behind the cytoplasmic retention of SOX9 and the widespread impacts of oestrogen on a testis-derived cell line.

Finally, I assessed the impact of exogenous oestrogen on the localisation of SOX9 in mouse gonads. In an in vitro gonad culture system, exogenous oestrogen caused cytoplasmic retention of SOX9, suppression of the testis gene Dhh and activation of the ovarian gene Fst. Treatment of mice in utero to the oestrogenic EDC diethylstilbestrol at a concentration known to cause hypospadias and reduced anogenital distance similarly increased cytoplasmic SOX9, demonstrating an association between the suppression of SOX9 and development of hypospadias and reduced anogenital distance.

In this thesis, I have demonstrated that the cytoplasmic retention of SOX9 is a conserved effect of oestrogen in mammals, suggesting this mechanism may be a remnant of oestrogen-driven sex determination in non-mammalian vertebrates. Furthermore, I have revealed some of the processes involved in the cytoplasmic retention of SOX9, which are directly relevant for our understanding of how exogenous oestrogen can regulate gonad somatic cell fate. These findings are not only important for understanding the impact of oestrogenic EDCs on male fertility, but also ovarian regulation and other diseases associated with misregulation of SOX9.