Do IRX3 and IRX5 Direct Cytoplasm Traffic Between Oocytes During Germline Cyst Breakdown?

Abstract

Poor oocyte quality and a diminished ovarian reserve contribute significantly to female infertility. The ovarian reserve is determined by the finite supply of primordial follicles a woman has at birth. Evidence suggests primordial follicles form as a result of germline cyst breakdown wherein many oocytes surrender their cytoplasmic contents to selected oocytes that are ultimately surrounded by pre-granulosa cells to form the intimate unit. We previously discovered that Iroquois homeobox transcription factors, IRX3 and IRX5 (IRX3/5), support follicle survival via intercellular communication between oocytes and their supporting granulosa cells. We observed that IRX3 and IRX5 colocalize to pre-granulosa cells and oocytes during the time of germline cyst breakdown; however, the means by which they promote follicle integrity are unknown. We hypothesized that IRX3/5 direct cytoplasmic content transfer between oocytes during germline cyst breakdown to ensure healthy oocytes within new primordial follicles. We tested this hypothesis using embryonic ovaries from wild type and Irx3/5 double knockout (Irx3- Irx5EGFP/Irx3-

Irx5EGFP, Irx3/5 DKO) mice collected at E14.5 and cultured in vitro for 6 days. Ovaries were harvested, processed, and every 5th section was collected for H&E staining. Ovary sections were imaged and analyzed using ImageJ to trace oocytes in germline cysts, primordial and primary follicles to determine their areas, perimeter, and quantity. If the hypothesis was true, we would expect that Irx3/5 DKO ovaries would harbor more, smaller oocytes; however, results showed no significant difference in oocyte structural measurements or quantities at any follicle stage when wild type and Irx3/5 DKO ovaries were compared using a t-test. Based on these findings, we turn our attention to determine the molecular mechanisms that facilitate intercellular communication during and after primordial follicle formation and examine when significant structural differences arise at later stages of development.