Cellular Senescent Markers Increase During the Follicular to Luteal Cell Transition

Robyn M. Moses¹; Michele R. Plewes^1,2; Corrine F. Monaco^1,3; Chloe M. Jones¹; Ailenn Castillo¹; Brooke E. Rudloff³; Renee M. McFee⁴; Jennifer. R. Wood³; Andrea S. Cupp³; John S. Davis^1,2

1. Department of Obstetrics and Gynecology, University of Nebraska Medical Center, Omaha, NE

2. US Department of Veterans Affairs Nebraska Western Iowa Health Care System, Omaha, NE

3. Department of Animal Sciences, University of Nebraska-Lincoln, Lincoln, NE

4. School of Veterinary Medicine and Biosciences, University of Nebraska-Lincoln, Lincoln, NE

Abstract Text:

The corpus luteum (CL) is a transient structure on the ovary, formed from the transition of granulosa (GC) and theca cells (TC) to large (LLC) and small (SLC) luteal cells, respectively. This transition is necessary to produce large amounts of progesterone required for maintenance of pregnancy. Luteal cells exhibit several characteristics of cellular senescence, a state marked by morphological changes, loss of proliferative capacity, and an inflammatory secretory profile. This study aimed to characterize senescence markers in follicular and luteal cells and test whether the senescence-like phenotype can be induced by mimicking luteinizing hormone (LH) signaling in follicular cells. To access transcriptional changes associated with cellular senescence, previously published microarray data (NCBI GSE83524) from purified antral follicular cells (GCs and TCs) and mid-luteal phase LLCs and SLCs was analyzed using Ingenuity Pathway Analysis (IPA). IPA identified the senescence pathway as one of the most significantly upregulated pathways during the transition from GC to LLC (z-score = 3.975; P < 0.0001) and TC to SLC (z-score = 3.787; P < 0.0001). Further analysis involved aligning the transcriptional data to the CellAge Database of Senescence Genes, followed by principal component analysis (PCA). The PCA revealed distinct clustering between follicular cells (GC/TC) and luteal cells (SLC/LLC) along PC1 (49.34%) and PC2 (14.84%), indicating significant transcriptional divergence during luteal differentiation. Subsequent gene expression analysis identified a transcriptional profile consistent with cellular senescence. Specifically, expression of cyclin dependent kinases (CDK1, CDK2, CDK4, CDK6), proliferation markers (PCNA, MK167, MCM2), and lamin B1 (LMNB1) were significantly down-regulated (P < 0.05) during the follicular-to-luteal transition, whereas transcripts encoding cyclin-dependent kinase inhibitors (CDKN1A, CDKN1B, CDKN2A, CDKN2B), lysosomal makers (GLB1, LAMP1, LAMP2), cytokines and chemokines (IL6, IL1B, TNF, CXCL10, CCL2), senescence-associated transcription factors (ETS1, ETS2), and p38 (MAPK14) were significantly upregulated (P < 0.05). To validate these findings at the protein level, proteins were isolated from GCs of early-stage follicles, SLCs and LLCs from mid-cycle CLs, and whole mid-cycle CL tissue. Full-length LMNB1 protein levels were 130-fold greater in GCs than in whole CL lysates (P < 0.05), whereas the cleaved form of LMNB1, associated with apoptosis, was predominant in LLCs compared to GCs (P < 0.05). CDK2 protein levels were also greater in GCs than in whole CL lysates (P < 0.05). However, ETS1, p27^Kip1, CDK4, and p38 showed no significant differences across cell types or whole CL (P > 0.05). To test if LH/PKA signaling may induce the senescence-like phenotype, antral follicle-derived GCs were luteinized in vitro for 96 hours. Compared to untreated GCs, those stimulated with a cAMP activator to mimic LH surge exhibited a significant increase in p27^Kip1 protein levels (P < 0.05), while no significant changes in MAPK14 or ETS1 protein were observed (P > 0.1). Collectively, these findings indicate that the follicular-to-luteal cell transition shares key molecular features with cellular senescence, suggesting that luteal cells may utilize senescence-like signaling to regulate CL function and maintenance. Further studies are needed to elucidate how this senescence-like phenotype regulates progesterone production and the luteal lifespan. These studies were funded by the USDA-AFRI-NIFA Competitive Grant No. 2023-67015-40795 (JSD, MRP, ASC) and the U.S Department of Veterans Affairs Grant No. IK2 BX004911-01A1 (MRP) and IK6 BX005797 (JSD).