DEVELOPMENT OF A METHOD TO STUDY SEQUENTIAL PROLIFERATION WITHIN HUMAN OVARIAN FOLLICLES UTILIZING THYMIDINE ANALOGUE INCORPORATION

To date, methods for quantifying cellular proliferation within the human ovary have relied primarily on static measurements at time of tissue recovery, such as labeling ki67 and phosphohistone-H3. Here, we describe a method based consecutive injection two thymidine analogues, ethynyl deoxyuridine (EdU) 5-chloro-2′-deoxyuridine (CldU), in ovarian xenograft model, allowing resolution division discrete temporal windows follicle development observation previously undescribed influence lead follicles proliferative activity surrounding stroma preantral follicles. NSG mice were bilaterally transplanted with cortical pieces fibrin matrix endothelial cells (n=4). After 12 weeks, sequentially injected Edu (100mg/kg 2 days) then CldU 24 hrs apart. A day later, sacrificed, xenografts recovered cryosectioned EdU incorporation. The percentage EdU+, CldU+, or EdUCldU+ nuclei quantified granulosa (GC) each using ZEN colocalization software. Stromal primordial (n=6) primary (n=4) showed higher levels compared to GCs, potentially suggesting early follicle–stroma interactions. Beginning secondary stage, (n=17) from containing leading (>3 mm, n=2 xenografts) significant increase GC (p=0.007) (p=0.01) when grafts without antral (n=2). Preantral (n=5) small (n=2) condition an increased presence rapidly dividing (p<0.005), while control condition, few identified (n=1) exhibited no limited proliferation, respectively. Comparison follicles, 3-mm 7-mm periovulatory follicle, revealed that is similar between these stages, theca/stroma compartment decreases size. We applied utilizes sequential incorporation analogues construct timeline xenograft-resident different stages. observed stromal preceded activation peaked stroma- theca-surrounding was reduced size approaching ovulatory competence. Finally, augmented pace paracrine and/or growth this experimental setting.