Regulation of TGFβ/Smad Signalling During Early Follicle Development in the Mouse Ovary

The molecular events responsible for the activation and growth of gonadotropin-independent stages of follicles are not well understood. This study is interested on the role of TGFβ signalling based on preliminary findings from our laboratory suggesting that this pathway is important in this context. Specifically, nuclear expression of the TGFβ signalling mediators and transcription factor Smad2/3 were more evident in the granulosa cells of primordial follicles but appeared to be excluded from nuclei in cells of early growing follicles. The overall aim of this thesis was to identify factors that have the potential to inhibit the Smad2/3 pathway and potentially determine their impact on early follicle development in the mouse ovary. Therefore, the first part of the study evaluated the expression of a selected of candidate genes (known to inhibit Smad signalling) during early follicle development.
Many Smad inhibitors were detectable, although serine-threonine kinase receptor associated protein Strap was further evaluated as its protein expression in granulosa cells of small follicles coincided with Smad2/3 staining. Neonatal mouse ovary fragments and preantral follicle culture models were employed to evaluate the function of Strap. Inhibition of Strap caused a significant reduction in the proportion of primordial follicles, leading to an increase in the proportion and size of growing follicles, while Strap supplementation promoted the growth of preantral follicles. Therefore, it is indicated that Strap can regulate the early follicle development in a stage-dependent manner and its function can be employed to expand our knowledge regarding several reproductive disorders, such as premature ovarian failure.
The expression of another Smad inhibitor, Tmepai, was also assessed and was found to be specifically localised in small preantral follicles that had just initiated growth. Since this coincided with the aforementioned reduction in nuclear Smad2/3, attempts were then made to determine whether TGFβ signalling regulated its expression. Inhibition of TGFβRI in cultured preantral follicles caused a significant decrease in Tmepai expression level. Thus, it is suggested that Tmepai can regulate TGFβ signalling in a negative feedback mechanism and consequently a relevant role in follicle growth. Finally, considering that TGFβ requires processing for their activity, and considering the evidence in this thesis and elsewhere that TGFβ signalling is important throughout the early stages of follicle development, we looked at the expression of the latent TGFβ binding proteins (Ltbp) in mouse ovaries.
Transcripts of Ltbp1-4 were expressed in the immature and adult ovaries. Ltbp1 protein appeared to be more evident in the ovary surface, while Ltbp4 mainly detected in blood vessels. These distinct expression patterns might indicate that each Ltbp member functions in a diverse way.
In conclusion, this thesis presents a series of studies that show the essential role of TGFβ/Smad2/3 signalling in the regulation of early follicle development in the mouse ovary. The expression of many Smad2/3 inhibitors indicates that Smad pathway is regulated by a complex mechanism. Strap can regulate early follicle growth in a stage-specific manner. Tmepai is detectable in follicles with specific growth stages and regulated through TGFβRI receptor. The extracellular modulator of TGFβ ligands (Ltbp1-4) are expressed in the ovary, where their relative proteins localised in distinct compartments of the ovary.