Nichani P, Lazer T, Power J, Behrouzi B, Gauthier-Fisher A, Kenigsberg S, Librach CL
Sunnybrook Research Institute Research Day, Toronto, Canada
Publication year: 2015

Abstract

Impact Statement: Leukaemia inhibitory factor receptor alpha (LIFRα) is present in human granulosa cells (GC). This may improve our understanding of GC-mediated regulation of folliculogenesis in different patient groups and the causes for poor oocyte maturation rates in patients. It may also allow for the possible development of in vitro follicle maturation protocols for fertility restoration strategies and have implications for regenerative medicine.

Introduction: GCs play an important role in follicular development and ovulation, they undergo dramatic morphological and phenotypical changes, and they can also differentiate into mesenchymal lineages. It has been postulated that GCs consist of subpopulations of differentiated and less differentiated cells that express the stem cell marker OCT-4 and thus have multipotent potential. In addition, these multipotent GCs survive and divide in response to leukaemia inhibitory factor (LIF). LIF is a member of the interleukin 6 (IL-6) cytokines, specifically involved in cellular differentiation, proliferation, and survival in the embryo. We hypothesize that patients with high ovarian reserve (OR) have a higher proportion of undifferentiated cells that will be reflected in high expression of the LIFR-α transcript and protein.

Methods: This study was approved by the University of Toronto REB and informed consent was obtained from all patients (age: 24-42, BMI: 19.5-33) involved. Aspirated follicular cells (AFC) were collected after transvaginal oocyte aspiration (low OR: n = 3, normal OR: n = 6, high OR: n = 6). GCs were obtained via differential centrifugation and haemolysis was performed to eliminate erythrocytes, leukocytes, and neutrophils from cell populations. Various enzymatic reactions and cell disruption protocols were used to break apart highly viscous GC primary cell populations. LIFRα (+) cells were detected using flow cytometry with the control CD45. LIFRα expression validated using western blot and immunocytochemistry (ICC) analysis. LIFRα gene expression analysis was detected using qPCR with the RotorGene 6000 after preparing RNA using the Total RNA Purification Kit (Norgen BioTek), assessing RNA quality with the NanoVue, and the synthesis of DNA using the RNA to cDNA EcoDry Premix (Double Primed) Kit (Clontech). ICC was performed to evaluate the expression of LIFRα (+) GCs and and cumulus cells (CMC) and the localization of LIFRα within the GCs and CMCs.

Results: A large population of LIFRα (+) cells was found in flow cytometric analysis, which were not positive for CD45 and therefore not leukocytes or neutrophils. CD45 (+) cells represented ~3% of the cell population. A large proportion of LIFRα (+) cells were found in HOR patients. Cultured GCs and CMCs for ICC, co-stained with LIFRα, FSHR, and DAPI, expressed LIFRα. Western blot analysis revealed LIFRα expression in CMCs and in FFs but not in GCs. qPCR results showed a 6.5 fold-change increase in HOR-patient LIFRα expression as compared to NOR patients.

Conclusion: LIFRα was detected in ovarian somatic cells (GCs, CMCs, and FFs). Western blot analysis showed that GCs may be shedding their LIFRα into the FFs constrained in a closed environment. Our exciting qPCR results provide the first experimental evidence showing differential gene expression in LOR vs. NOR vs. HOR patients; however, further protocol optimization and analyses are required to strengthen our findings.

Keywords: granulosa cells; cumulus cells; leukaemia inhibitory factor receptor alpha (LIFRα); pluripotent stem cell markers

Disclosures: This project was funded by the CReATe Fertility Centre and the D+H Sunnybrook Research Institute Studentship Award. There are no conflicts of interest to disclose.

Presentations

  1. Nichani P, Lazer T, Power J, Behrouzi B, Gauthier-Fisher A, Kenigsberg S, Librach CL. (Aug 2015). Leukaemia inhibitory factor receptor (LIFR-α) expression in human ovarian granulosa cells as an indication of undifferentiated cells. Sunnybrook Research Institute Research Day, Toronto, ON (poster). View poster here.