Stress Fiber Formation and Migration Are Novel PGR-Dependent Pathways Initiated in the Granulosa Cells of the Ovulating Follicle

Ovulation is the release of the mature oocyte from the ovary, a pivotal event for female reproduction. For ovulation to be successful, a series of intercellular events must be spatially and temporally coordinated throughout the ovarian follicle, culminating in rupture at the follicle apex and subsequent oocyte release. The nuclear steroid receptor, progesterone receptor (PGR) is a master-regulator of these ovulatory events. Mice null for progesterone receptor (PRKO) fail to ovulate, however the pathways initiated by PGR that are essential to ovulation are not comprehensively understood. The overarching aim of this thesis is to identify the underlying mechanisms that lead to successful ovulation. In particular, discovering PGR-dependent pathways will identify mechanisms that are most crucial to oocyte release and provide valuable insight for development of non-hormonal female contraception. Previously, PGR isoform-specific PGR-A (PRAKO) and PGR-B (PRBKO) null mice were used to establish that PGR-A alone is essential for ovulation. However, in studying these anovulatory PRAKO mice, this thesis found that PRAKO are not isoform-specific KOs but are in fact null for both isoforms. Subsequentially, a genuine PGRA-specific KO model was generated, ensuring PGR-B expression was maintained. These newly developed PGR-A null mice ovulated normally and were fertile, revising long-held dogma of PGR-isoform action and establishing redundancy of PGR isoforms for critical reproduction events. Next, the PGR-dependent transcriptome across ovarian compartments was analysed to identify novel PGR-regulated effector pathways in ovulation. Analysis of 154 key PGR-dependent genes in granulosa cells identified ‘formation of actin filaments’ and ‘formation of stress fibers’ as primary pathways. In contrast, RNA-seq analysis of the ovarian stromal compartment found minimal PGR-dependent regulation of stromal genes. It was then established that in response to ovulatory stimulus, thick actin stress fibers do indeed form in granulosa cells in a PGR-dependent manner. Further characterisation of function in ovulating follicles, observed activation of mechanoresponsive element Yap1 at the follicle base and identified a gain in migratory capacity in granulosa cells. Concurrently, the basal granulosa cell layer thickened immediately prior to ovulation. Each of these events was PGR-dependent. This data demonstrates granulosa cell stress fiber function and migration as novel PGR-dependent pathways occurring during ovulation. Two key PGR-regulated genes, Jam3 and vinculin were investigated further to establish a role in oocyte release. Vinculin, a stress fiber protein important for cell-ECM connection and collective cell migration, was upregulated following the LH surge and localised to leading edge-like protrusions from the antral-most granulosa cells. Jam3 tight junction protein, important for cell-cell connectivity and collective migration, was strongly regulated by PGR and localised within the basal granulosa cell layer. Using a Cyp19a1-Cre line, granulosa cell conditional ‘knockout’ mouse models for both Jam3 and vinculin were established. Surprisingly, neither model showed loss of ovulatory function. However, further investigation found evidence that the Cyp19a1-Cre line, did not adequately delete gene function. Cumulatively, this thesis provides new information on the intercellular regulation of ovulation by progesterone receptor isoforms; in particular, establishing stress fiber formation and migration in granulosa cells as novel effector pathways initiated in the ovulatory follicle.

Author dc:creator

• Smith, Kirsten Mary

Advisors dc:contributor.advisor

• Robker, Rebecca
• Russell, Darryl

• Progesterone Receptor
• PGR
• PRKO
• PRAKO
• Ovulation
• Stress Fiber
• Migration
• Granulosa Cells
• JAM3
• Vinculin
• Fertility

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/2440/144613

OAI identifier oai:identifier

oai:digital.library.adelaide.edu.au:2440/144613