Mitochondria are key organelles that regulate energy production, oxidative stress, and steroidogenesis. They are highly dynamic, continuously undergoing fusion and fission. While disruptions in these processes affect follicular development and fertility in mammals, little is known about their role in avian species. This study investigated the expression and functional roles of mitochondrial fusion in hen granulosa and theca cells isolated from dominant (F1) and smaller (F3/F4) follicles. Gene expression analysis revealed that fusion-related genes ( MFN1, MFN2 , and OMA1 ) are more highly expressed in theca cells than in granulosa cells. Furthermore, MFN1 and MFN2 levels significantly increase as follicles mature from the F3/F4 to the F1 stage, suggesting that mitochondrial fusion is closely linked to follicular development. To explore the functional impact, cells were treated with Mdivi-1 and leflunomide; both compounds successfully promoted mitochondrial fusion, as evidenced by elongated mitochondrial morphologies. However, their effects on mitochondrial homeostasis differed: leflunomide, but not Mdivi-1, significantly increased fusion gene expression and reduced mitochondrial DNA (mtDNA) copy number ( ND4, ND6, ATP6 ). Inducing mitochondrial hyperfusion generally impaired cell viability and proliferation. High doses of both inhibitors suppressed proliferation across both cell types, molecularly confirmed by the downregulation of the pro-proliferative gene CCND1 and the upregulation of the cell cycle inhibitor P21 . Regarding steroidogenesis, mitochondrial fusion exerted cell-specific effects. In granulosa cells, both treatments enhanced progesterone secretion, supported by increased expression of STAR, 3BHSD , and CYP11A1 . Conversely, in theca cells, fusion led to decreased testosterone secretion and reduced expression of CYP11A1 and CYP19A1 . Finally, while Mdivi-1 did not alter reactive oxygen species (ROS) or ATP levels, leflunomide significantly reduced oxidative stress and increased ATP production. In conclusion, mitochondrial dynamics play a critical, cell-specific role in regulating hen ovarian physiology, influencing the balance between proliferation, metabolism, and steroidogenesis.
Couty et al. (Sun,) studied this question.