Abstract
<title>Abstract</title> <p> <bold>Background</bold> Granulosa cell survival and steroidogenic activity are essential for follicular development, endocrine function, and oocyte support in cattle. Negative energy balance during the transition period is associated with altered metabolic signaling and impaired reproductive performance in dairy cattle. Growth differentiation factor 15 (GDF15) is a stress-responsive cytokine whose abundance increases under metabolic challenges; however, its direct effects on bovine ovarian granulosa cells remain poorly understood. This study aimed to investigate whether sustained GDF15 exposure affects bovine granulosa cell function and to elucidate the underlying cellular mechanisms. <bold>Results</bold> GDF15 and GFRAL were detected in bovine ovarian follicles and primary granulosa cells. Exposure of primary bovine granulosa cells to recombinant GDF15 reduced cell viability and proliferation, suppressed the expression of steroidogenic genes and the production of estradiol and progesterone, and increased apoptosis. Transcriptomic analysis implicated apoptosis, calcium signaling, protein processing in the endoplasmic reticulum, and PI3K/AKT signaling. Mechanistically, prolonged GDF15 exposure progressively reduced the pre-labeled cell-surface GFRAL pool and subsequently decreased total GFRAL abundance. Pharmacological inhibition of endocytosis or lysosomal degradation preserved GFRAL abundance and partially restored RET phosphorylation. GFRAL neutralization attenuated GDF15-induced suppression of RET, PI3K, and AKT phosphorylation and alleviated endoplasmic reticulum stress and apoptosis. Activation of PI3K partially restored endoplasmic reticulum Ca²⁺ levels, reduced cytosolic Ca²⁺ accumulation, and attenuated endoplasmic reticulum stress-associated apoptosis, whereas PI3K inhibition exacerbated these alterations. Thapsigargin further aggravated GDF15-induced endoplasmic reticulum stress and apoptosis, while 4-phenylbutyric acid partially reversed these responses. <bold>Conclusions</bold> Prolonged GDF15 exposure impairs bovine granulosa cell proliferation, steroidogenic function, and survival through a mechanism involving GFRAL internalization and lysosomal degradation, attenuation of RET–PI3K/AKT signaling, intracellular Ca²⁺ dyshomeostasis, and endoplasmic reticulum stress-associated apoptosis. These findings identify GFRAL trafficking as a regulatory event in bovine granulosa cell homeostasis and provide a cellular framework for understanding how sustained stress-responsive signals may influence follicular function in cattle. Further in vivo studies are required to determine the physiological relevance of this pathway. </p>