Procr-expressing progenitor cells are responsible for murine ovulatory rupture repair of ovarian surface epithelium

Abstract

Ovarian surface epithelium (OSE) undergoes recurring ovulatory rupture and repair. The OSE replenishing mechanism post ovulation remains unclear. Here we report that the expression of Protein C Receptor (Procr) marks a progenitor population in adult mice that is responsible for OSE repair post ovulation. Procr+ cells are the major cell source for OSE repair. The mechanism facilitating the rapid re-epithelialization is through the immediate expansion of Procr+ cells upon OSE rupture. Targeted ablation of Procr+ cells impedes the repairing process. Moreover, Procr+ cells displayed robust colony-formation capacity in culture, which we harnessed and established a long-term culture and expansion system of OSE cells. Finally, we show that Procr+ cells and previously reported Lgr5+ cells have distinct lineage tracing behavior in OSE homeostasis. Our study suggests that Procr marks progenitor cells that are critical for OSE ovulatory rupture and homeostasis, providing insight into how adult stem cells respond upon injury.

Procr-expressing granulosa cells are highly proliferative and are important for follicle development

“…Protein C receptor (Procr) marks adult stem/progenitor cells in various tissues, including the mammary gland (Wang et al, 2015a), vascular endothelial cells (Yu et al, 2016), pancreatic islet (Wang et al, 2020), ovarian surface epithelium (OSE) (Wang et al, 2019), and hematopoietic system (Balazs et al, 2006). Procr encodes a single-pass transmembrane protein (Fukudome and Esmon, 1994).…”

“…Upon binding to its ligand Protein C, Procr activates several intracellular signaling, resulting in increased proliferation and survival in various tissues (Cheng et al, 2003;Gramling et al, 2010;Wang et al, 2018). Indeed, Procr+ OSE cells proliferate faster than Procr-OSE cells (Wang et al, 2019) and Procr+ endothelial cells exhibited more robust proliferation than Procrendothelial cells (Yu et al, 2016).…”

“…To analyze the expression of Procr in the ovarian follicle, we utilized Procr-rtTA;TetO-H2B-GFP mice (Figure S1A) (Wang et al, 2019). Mice at postnatal day 10 were fed with doxycycline (Dox), and the ovaries were harvested 2 days later (Figure 1A).…”

“…Ovaries from 4-week old R26-mTmG mice were harvested, washed in cold PBS, and separated from ovarian bursa by microdissection under a stereomicroscope. Single cells were obtained following the protocol we described before (Wang et al, 2019). In general, minced ovarian pieces were digested with digest buffer (RPMI 1640 (Thermo Fisher, catalog #12633-012) with 5% fetal bovine serum (FBS, Hyclone), 1% penicillin-streptomycin (Thermo Fisher, catalog #15140122), 25 mM HEPES and 300U/ml collagenase IV (Worthington, catalog # LS004189)) at 37°C, 100 rpm for 1 hour, lysed with red blood cell lysis buffer (Sigma, catalog #R7757) , and then treated with 0.05%Trypsin-EDTA (Thermo Fisher, catalog # 25200056) and DNaseI (Sigma, catalog #D4263) before filtering through 70 µm cell strainers.…”

Lentiviral CRISPR-guided RNA library screening identified Adam17 as an upstream negative regulator of Procr in mammary epithelium

“…Procr, also known as endothelial protein C receptor (EPCR), is a single-pass transmembrane protein with reported function in anticoagulation, inflammation and hematopoiesis [1][2][3][4][5]. Our previous studies have identified Procr as an adult stem cell marker of multiple tissues, such as the mammary gland epithelium, blood vasculature, ovary epithelium and pancreatic islets [6][7][8][9]. Moreover, almost half of aggressive triple negative breast cancer (TNBC) cases have been shown to harbor robust PROCR expression [10], indicating that PROCR could be a useful biomarker as well as a potential treatment target.…”