Effects of the conditioned medium of mesenchymal stem cells on mouse oocyte activation and development

D.Q Feng,Y Zhou,H.M Wei,Y.Y Shi,T Gao,B Ling,Z.G Tian

doi:10.1590/s0100-879x2009000600006

Abstract

Mesenchymal stem cells (MSCs) have been reported to secrete a variety of cytokines and growth factors acting as trophic suppliers, but little is known regarding the effects of conditioned medium (CM) of MSCs isolated from femurs and tibias of mouse on the artificial activation of mouse oocytes and on the developmental competence of the parthenotes. In the current study, we investigated the effect of CM on the events of mouse oocyte activation, namely oscillations of cytosolic calcium concentration ([Ca(2)+]i), meiosis resumption, pronucleus formation, and parthenogenetic development. The surface markers of MSCs were identified with a fluorescence-activated cell sorter. The dynamic changes of the spindle and formation of pronuclei were examined by laser-scanning confocal microscopy. Exposure of cumulus-oocyte complexes to CM for 40 min was optimal for inducing oocyte parthenogenetic activation and evoking [Ca(2)+]i oscillations similar to those evoked by sperm (95 vs 100%; P > 0.05). Parthenogenetically activated oocytes immediately treated with 7.5 microg/mL cytochalasin B (CB), which inhibited spindle rotation and second polar body extrusion, were mostly diploid (93 vs 6%, P < 0.01) while CB-untreated oocytes were mostly haploid (5 vs 83%, P < 0.01). Consequently, the blastocyst rate was higher in the CB-treated than in the CB-untreated oocytes. There was no significant difference in developmental rate between oocytes activated with CM and 7% ethanol (62 vs 62%, P > 0.05), but the developmental competence of the fertilized oocytes was superior to that of the parthenotes (88 vs 62%, P < 0.05). The present results demonstrate that CM can effectively activate mouse oocytes, as judged by the generation of [Ca(2)+]i oscillations, completion of meiosis and parthenogenetic development.

Highlights

Mesenchymal stem cells (MSCs), widely distributed in a variety of tissues in the adult organism, secrete many kinds of cytokines and growth factors, such as monocyte chemotactic protein-1, vascular endothelial growth factorA, epidermal growth factor (EGF), fibroblast growth factor2, interleukin-6, leukemia inhibitory factor, transforming growth factor-β, and so on [1,2]
O’Donnell et al [10] reported that activation of EGF receptor (EGFR) by EGF or transforming growth factor-α induces both auto- and trans-phosphorylation of EGFRs, which result in cytosolic calcium concentration ([Ca2+]i) elevation and subsequent membrane permeabilization in mouse cumulus-oocyte complexes (COCs), but oocytes fail to respond to the EGF stimulus
Other batches of COC used as control were incubated with DMEM-HG containing 10% fetal calf serum for 0, 10, 40 min, 1, or 2 h followed by additional incubation in CZB medium containing 0.4% BSA after cumulus cells were removed by hyaluronidase

Summary

Introduction

Mesenchymal stem cells (MSCs), widely distributed in a variety of tissues in the adult organism, secrete many kinds of cytokines and growth factors, such as monocyte chemotactic protein-1, vascular endothelial growth factorA, epidermal growth factor (EGF), fibroblast growth factor, interleukin-6, leukemia inhibitory factor, transforming growth factor-β, and so on [1,2]. We thought it would be useful to assess the ability of CM to activate mouse oocytes parthenogenetically, and embryo development before implantation Some of these secreted bioactive factors have been reported to improve meiotic maturation in vitro and subsequently the embryo developmental potential directly or via cumulus cells [4,5,6]. Cytochalasin B (CB), an inhibitor of microfilament polymerization, prevents the release of the second polar body (Pb2) after oocyte activation, which would result in diploid development [17], and may help prevent embryo fragmentation [18,19] These artificial stimuli mimicking sperm-triggered events evoke a series of [Ca2+]i oscillations, which is critical for oocyte activation. We investigated the pattern of [Ca2+]i changes in mouse oocytes after treatment with CM, and the effects of time of exposure to CM and to a sequential combination of CM and CB in improving parthenogenetic activation and development of mouse oocytes

Material and Methods

Results

Findings

Discussion