(357) Unraveling Pyroptosis in the Mammalian Ovary: Insights into Ovulation and Follicular Atresia

Yu-Hsiang Liao¹; Hanxue Zhang¹; Madhav Mantri²; Iwijn De Vlaminck²; Yi Athena Ren¹

1. Department of Animal Science, Cornell University, Ithaca, United States

2. Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, United States

Abstract Text: Ovulation involves programmed cell death at the follicle rupture site and shares features with acute inflammation. Pyroptosis is a recently discovered inflammatory programmed cell death. It initiates from inflammasome activation, followed by the cleavage of pro-interleukin-1β and -18 by cleaved Caspase-1 (cl-Caspase-1), and is executed by cleaved Gasdermin D (cl-GSDMD) to form pores on the cell membrane, leading to the rupture of cell membrane and the release of cytokines. Before reaching the preovulatory stage, most ovarian follicles undergo atresia, a process that primarily involves apoptosis but may also include other forms of cell death, such as autophagic cell death. Recent research indicates that pyroptosis participates in ovulation in zebrafish. However, little is known about pyroptosis in the mammalian ovary. This study aims to address this knowledge gap and test whether pyroptosis is involved in follicular atresia and ovulation in mice. We first characterized the spatial and temporal dynamics of pyroptosis-related proteins during the preovulatory stage in immature mice superovulated by pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), with the latter mimicking the effect of the preovulatory luteinizing hormone surge to induce ovulation. Immunostaining and western blotting revealed that Caspase-1 and cl-Caspase-1 are mostly expressed in the ovarian stroma, with minimal cl-GSDMD and Caspase-1 signals in granulosa cells (GCs) post-hCG. The expression of Caspase-1 increased between 0 h and 11.5 h post-hCG in whole ovaries, and expression of cl-Caspase-1 in GCs increased at 11.5 h post-hCG, suggesting a role of Caspase-1 in ovulation. To test if pyroptosis is involved in ovulation, we intraperitoneally injected pyroptosis inhibitors into superovulated mice at the time of hCG stimulation. Among the tested inhibitors, Ac-FLTD-CMK, a cl-Caspase-1 inhibitor, significantly reduced ovulation rate. In contrast, GSDMD inhibitors (disulfiram and LDC7559) had no effect on ovulation, suggesting the involvement of cl-Caspase-1 but not GSDMD in ovulation. To test if Ac-FLTD-CMK reduced the rate of ovulation through its direct effect on the ovary, we employed an ex vivo ovulation system and successfully induced ovulation in cultured ovarian tissue pieces. We will apply this ex vivo system to test the direct effect of Ac-FLTD-CMK on ovulation as the next step. Overall, our data suggest that evidence supporting the direct involvement of GSDMD in mouse ovulation appears limited, but alternative mechanisms downstream of cl-Caspase-1 may be novel regulators of ovulation. We next tested whether pyroptosis is involved in follicular atresia. In ovaries from 21-23-day-old mice, cl-Caspase-1 and cl-GSDMD are present within the same atretic follicles marked by cl-Caspase-3 staining and pyknotic nuclei in GCs, suggesting that pyroptosis may participate in follicular atresia. As PMSG is known to prevent follicular atresia, we hypothesized that it may downregulate pyroptosis-related proteins. Indeed, protein levels of cl-GSDMD and cl-Caspase-1 decreased in whole-ovary tissues at 24 h post-PMSG, consistent with fewer cl-Caspase-3+ and cl-GSDMD+ cells identified by immunostaining on ovarian tissue sections. These data support the involvement of pyroptosis in follicular atresia. Taken together, our study provides evidence for pyroptosis and its related proteins in the normal physiology of mammalian ovaries.