Abstract
In the ovary, eggs develop inside a cellular structure called an ovarian follicle. Follicles comprise of an egg, granulosa cells and an outer layer of theca cells. Follicle development or folliculogenesis is crucial to the growth and maturation of the egg. It is an extended process under complex hormonal regulation which works to maintain fertility in females. During folliculogenesis, follicles transition through many developmental stages, characterised by a drastic increase in size and altered hormonal responses. Follicle-stimulating hormone (FSH) is a dominant regulator of folliculogenesis. FSH stimulation results in the upregulation of Cyp19a1, which encodes for aromatase. Aromatase catalyses the production of estrogen in granulosa cells which promotes the growth and survival of follicles. Anti-müllerian hormone (AMH) is another important regulator of folliculogenesis. AMH can downregulate aromatase expression and estrogen production in granulosa cells. This opposes the function of FSH, but it is unknown if AMH acts independently of FSH signalling. FSHmediated estrogen production is associated with follicle growth, suggesting AMH inhibits growth. However, research has also shown that AMH can promote follicle growth. Therefore, this project aimed to clarify the role of AMH in follicle development using an AMH-knock-out model.
The effect of AMH on follicle growth rate was investigated by culturing follicles 100-170µm in diameter from Amh–/– and Amh+/+ mice. Amh–/– follicles from 100-135µm in diameter exhibited a significantly reduced growth rate compared to Amh+/+ follicles. This indicated that AMH promotes the growth of small preantral follicles. This project also aimed to investigate the expression of Fshr and Cyp19a1 in individual follicles across development using RT-qPCR. However, inconsistent amplification of RNA was observed and a series of troubleshooting experiments were undertaken. Quantification of mRNA expression in individual follicles was unsuccessful but a protocol was developed for quantification in pooled samples. This provides a method to investigate the impact of AMH on transcription in granulosa cells and reveal the mechanism behind AMH-mediated growth. AMH promoting the growth of follicles in mice is a novel observation and suggests previous claims that AMH attenuates FSH-sensitivity are incorrect. Instead, this shows AMH to be a dominant regulator of early folliculogenesis and that ovarian follicles exhibit an altered hormonal response to AMH with the progression of development.