Evaluation of stemness marker expression in bovine ovarian granulosa cells.

Son Nghia Hoang,Diem Hong Tran,Long Thanh Le,Chung Chinh Doan,Chi Nguyen Quynh Ho,Thao Thi Phuong Nguyen

doi:10.21451/1984-3143-ar2018-0083

Abstract

The objective of this study was to assess the stemness marker expressions (Oct4, Nanog, and Sox2) of granulosa cells (GCs) collected from bovine ovarian follicles and in vitro expansion. The single bovine ovarian follicles were isolated and categorized into 4 groups according to their diameter including group A (<2 mm), group B (2-3 mm), group C (3-4 mm), and group D (>4 mm). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and immunostaining were applied to evaluate the stemness marker expression of bovine GCs from ovarian follicles. We also estimated the stemness marker transcript expressions of GCs during in vitro expression by qRT-PCR. qRT-PCR analysis demonstrated that fresh GCs from bovine ovarian follicles expressed the stemness markers (Oct4, Nanog, Sox2). These markers were down-regulated during antral stage follicular development. We also estimated stemness marker transcript expressions of GCs which were isolated and in vitro expanded from ovarian follicles of group A. The qRT-PCR results showed that Oct4 and Sox2 transcript expressions were reduced during in vitro expansion while Nanog transcript was not expressed.

Highlights

The mammalian ovarian follicle development is begun from the primordial follicle, which contains an immature small egg and a single undifferentiated granulosa cell layer (Gougeon, 1996)
The qRTPCR showed that the transcript expression of stemness markers (Oct4, Nanog and Sox2) were detected in fresh granulosa cells (GCs) from ovarian follicles with the specific bands in electrophoresis (Fig. 1)
The Oct4, Nanog and Sox2 transcript expression were highest in group A which was smaller than 2 mm in diameter

Summary

Introduction

The mammalian ovarian follicle development is begun from the primordial follicle, which contains an immature small egg and a single undifferentiated granulosa cell layer (Gougeon, 1996). This cell layer is surrounded by a follicular wall (Gougeon, 1996). Many studies have provided evidence that GCs carry the characteristics of stem cells (Chronowska, 2012). Following the study of Kossowska-Tomaszczuk on luteinizing GCs isolated from the ovarian follicles of infertile patients, OCT4, a typical stem cell marker, was expressed in long-term culture with the presence of the leukemiainhibiting factor (Kossowska-Tomaszczuk et al, 2009)

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