Abstract
Female reproductive gametes, known as oocytes, are encased within specialized ovarian follicles that provide an optimal environment for oocyte maturation. The process of folliculogenesis, involving the developmental progression of follicles through various stages, is crucial for the release of a mature oocyte at ovulation. Anti-Müllerian Hormone (AMH) serves as a pivotal regulator in this process. It is initially produced in an inactive form by granulosa cells of growing follicles, and the enzymes responsible for AMH activation have been identified in the vicinity of follicles. The regulatory role of AMH in the initial stages of folliculogenesis has been well-defined, however, its role in the intermediate stages remains unclear. Therefore, this study aims to explore the activation of AMH and its impact on the spatial arrangement of differently staged follicles within sheep ovaries.
The role of AMH in follicle development was examined by surveying follicles in different stages of folliculogenesis in control and AMH-immunised sheep ovaries. Interfollicular distances were measured to map the spatial distribution of different follicle types within the ovary. Western blot analyses on human and ovine follicular fluid samples was conducted to quantify active AMH levels to determine its localisation within the ovary.
The results of this study showed that AMH has a significant impact on late preantral follicles, with AMH-immunised ovaries showing significantly elevated 100-200 µm diameter follicle counts than controls. The three-dimensional ovarian reconstructions revealed that control ovaries exhibited clusters of large growing follicles, while this feature was less prominent in AMH-immunized ovaries. Follicular spatial analysis revealed that in control ovaries, late preantral 100-200 µm follicles were less abundant in proximity to antral 1000-2000 µm follicles compared to AMH-immunised ovaries. Western Blot analyses showed that AMH protein was not able to be detected in human and ovine follicular fluid samples, evidenced by the absence of bands.
To our knowledge, this study consists of the largest exploration of follicles in three-dimensional space to be documented in the literature, encompassing 58,534 follicles and 4,936,074 interfollicular distances. The findings of this study suggest that AMH has a predominant influence on late preantral 100-200 µm follicles, preventing their growth near small antral 1000-2000 µm follicles. This novel observation suggests that AMH prevents the excessive growth of follicles in the vicinity of large follicles to prevent overcrowding in the ovary. The results from the Western Blot suggested that AMH concentrations are too low to be readily detectable without higher sensitivity methods.