Harmful algal bloom toxin microcystin-LR interferes with PP1-mediated PI3K/AKT/FOXO1 signaling in granulosa cells to disrupt ovarian follicle maturation and ovulation

Harmful algal blooms (HABs) arise from the rapid growth of algae in both marine and freshwater ecosystems due to the continuous global temperature rise and anthropogenic eutrophication. Humans are at a great risk of exposure to toxins released from HABs through drinking water, food, and recreational activities, making HAB toxins contaminants of emerging concern (CECs). The impact of HAB toxins on women’s reproductive health, however, remains poorly understood. Here, we investigated the effects of microcystin-LR (MC-LR), the most common HAB toxin, on the ovary, the female gonad, and associated ovarian functions. The results of a chronic daily oral mouse exposure model and an acute exposure using the mouse superovulation model revealed that MC-LR accumulated in the ovary and environmentally-relevant exposure to MC-LR perturbed follicle-stimulating hormone (FSH)-dependent follicle maturation to disrupt ovulation and luteinization. Using an ex vivo mouse follicle maturation and ovulation assay and in vitro culture of human primary granulosa cells, mechanistic studies such as the single-follicle RNA sequencing analysis and others elucidated that MC-LR inhibited protein phosphatase 1 (PP1) and interfered with PP1-mediated PI3K/AKT/FOXO1 signaling in granulosa cells, which suppressed follicle maturation, ovulation, luteinization, and progesterone secretion. Together, our study demonstrates that environmentally-relevant exposure to MC-LR acts a PP1 inhibitor to interfere with the PI3K/AKT/FOXO1 signaling in granulosa cells, which disrupts follicle maturation and results in adverse female reproductive outcomes. As a newly identified ovarian endocrine disrupting chemical (EDC), exposure to MC-LR poses a serious threat to women’s reproductive health and fertility. Ovaries were removed from 16-day-old CD-1 female mice and multilayered secondary follicles were enzymatically isolated using enzymatic solution. Next, morphologically normal follicles with diameters of 130 to 160 µm were selected, encapsulated with alginate hydrogel for eIVFG. To investigate the effects of MC-LR on the whole follicular transcriptome, antral follicles treated vehicle or 10 µM MC-LR were collected from day 6 of eIVFG for single-follicle RNA sequencing.