Necroptosis in primate luteolysis: a role for ceramide

Konstantin Bagnjuk,Rüdiger Behr,Ulrike Berg,Georg Josef Arnold,Cecily Bishop,Jing Xu,Jan Bernd Stöckl,Lars Kunz,Thomas Fröhlich,Dieter Berg,Artur Mayerhofer

doi:10.1038/s41420-019-0149-7

Abstract

The corpus luteum (CL) is a transient endocrine organ, yet molecular mechanisms resulting in its demise are not well known. The presence of phosphorylated mixed lineage kinase domain-like pseudokinase pMLKL(T357/S358) in human and nonhuman primate CL samples (Macaca mulatta and Callithrix jacchus) implied that necroptosis of luteal cells may be involved. In M. mulatta CL, pMLKL positive staining became detectable only from the mid-late luteal phase onwards, pointing to necroptosis during regression of the CL. Cell death, including necroptosis, was previously observed in cultures of human luteal granulosa cells (GCs), an apt model for the study of the human CL. To explore mechanisms of necroptotic cell death in GCs during culture, we performed a proteomic analysis. The levels of 50 proteins were significantly altered after 5 days of culture. Interconnectivity analysis and immunocytochemistry implicated specifically the ceramide salvage pathway to be enhanced. M. mulatta CL transcriptome analysis indicated in vivo relevance. Perturbing endogenous ceramide generation by fumonisin B1 (FB1) and addition of soluble ceramide (C2-CER) yielded opposite actions on viability of GCs and therefore supported the significance of the ceramide pathway. Morphological changes indicated necrotic cell death in the C2-CER treated group. Studies with the pan caspase blocker zVAD-fmk or the necroptosis blocker necrosulfonamid (NSA) further supported that C2-CER induced necroptosis. Our data pinpoint necroptosis in a physiological process, namely CL regression. This raises the possibility that the primate CL could be rescued by pharmacological inhibition of necroptosis or by interaction with ceramide metabolism.

Highlights

The corpus luteum (CL) forms after ovulation.Upon the ovulatoryluteinizing hormone (LH) surge granulosa and theca cells differentiate into large and small luteal cells, stop dividing and produce progesterone[1,2]
While 3- and 7-day-old CL samples were negative for pMLKL(T357/S358) (Fig. 1a, b), cells positive for pMLKL(T357/S358) were evident in the 14 day-old CL (Fig. 1c)
LC-MS/MS data from cultured granulosa cells (GCs) and literature data of human CL and in vivo-developed primate luteal cells stemming from the mid to late luteal phase were compared

Summary

Introduction

The corpus luteum (CL) forms after ovulation.Upon the ovulatoryluteinizing hormone (LH) surge granulosa and theca cells differentiate into large and small luteal cells, stop dividing and produce progesterone[1,2]. Knowledge about the molecular events leading to functional and structural regression of the primate CL is limited. A fraction of the luteal cells undergo apoptosis in humans[4,5], and involvement of autophagocytosis was suggested[6,7,8]. Both are immunologically silent events, yet other forms of cell death attract immune cells. For example, macrophages, appear to play an indispensable role in ovarian functions[9] and CD11b positive macrophages invade the nonhuman primate CL during its regression and produce various cytokines and chemokines[10]

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Results

Conclusion