Characterization of ovine surface epithelium‑derived stem cells and the modulatory role of phenol red in their proliferation and differentiation

Alipour Choshali, Mahmoud; Shakeri, Malak; Zhandi, Mahdi; Moradi, Mehdi

doi:10.22092/ari.2026.372029.4028

Characterization of ovine surface epithelium‑derived stem cells and the modulatory role of phenol red in their proliferation and differentiation

Articles in Press

Document Type : Original Articles

Authors

¹ Department of Animal Science, University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran

² Department of Animal Science, Campus of Agriculture and Natural Resources, University of Tehran, Karaj, Iran

10.22092/ari.2026.372029.4028

Abstract

Introduction: The ovarian surface epithelium (OSE) is postulated to harbor stem cells in adults, yet direct evidence remains limited. Phenol red, a pH indicator with weak estrogenic activity, may influence estrogen‑sensitive stem cell differentiation, but its role is not well defined. This study examined the effect of phenol red on the isolation, characterization, and oocyte‑like differentiation of stem cells from sheep OSE.

Materials & Methods: OSE-derived cells were cultured for three weeks in media with or without phenol red. Isolated small round cells (2–4 µm) were expanded and assessed for pluripotency markers (SSEA-1, SSEA-3) and germ cell/oocyte markers (VASA (DDX4), ZP3) via immunocytochemistry. Expression of germline genes (c-Kit, ZP3, DDX4) was analyzed by quantitative real-time PCR and statistically compared between groups.

Results: The isolated cells showed high proliferative and colony‑forming capacity and expressed pluripotency and germ cell markers, confirming their stem and germline identity. These cells differentiated into oocyte‑like structures in vitro. Cultures with phenol red showed higher (P < 0.05) expression of c-Kit (9.5‑fold) and ZP3 (3.6‑fold), along with increased colony number (15%) and cellular density (70%), compared to phenol red‑free controls.

Conclusion: This study shows that ovine OSE contains putative stem cells capable of oocyte‑like differentiation. Phenol red enhanced this process, indicating its role as a supportive, estrogen‑mimicking factor in ovarian stem cell culture. This suggests that endogenous estrogenic signaling may regulate these cells in vivo. The findings contribute to the discussion on postnatal oogenesis and highlight the influence of culture medium composition on stem cell fate.

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