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Comparative effects of retinoic acid, granulosa cells conditioned medium or forskolin in combination with granulosa cell co-culturing on mouse germ cell differentiation

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Abstract

Background

Devising of an appropriate in vitro culture method for germ cells differentiation in the presence of soluble factors has attracted considerable attention, which results will provide new insight into reproductive biology. In this study, we compared the effects of forskolin, retinoic acid (RA) or granulosa cell-conditioned medium in the presence or absence of granulosa cell co-culturing on germ cell differentiation from embryonic stem cells (ESCs).

Methods and Results

Embryonic stem cells were differentiated using embryoid bodies (EBs) for 5 days, and then EB-derived cells were co-cultured with or without adult mouse granulosa cells using monolayer protocol and treated with 50 µM forskolin, 1 µM RA and 50% granulosa cell-conditioned medium for 4 days. Granulosa cell-conditioned medium significantly increased the levels of Scp3, Rec8, Mvh and Gdf9 expression in the granulosa cell co-culture method compared to untreated cells. A significant elevation of Stra8, Rec8 and Mvh was observed after treatment with RA in the absence of granulosa cells and there was no significant increase in the levels of expression of germ cell-specific genes after treatment with forskolin compared to control. Furthermore, forskolin and RA significantly increased viability and proliferation of germ-like cells, compared with granulosa cell-conditioned medium.

Conclusions

Our study revealed that granulosa cell-conditioned medium and RA effectively can induce germ cell differentiation from ESCs, however combined application of granulosa cell-conditioned medium and co-culturing with granulosa cells had synergic effect on germ cell development in vitro as optimized protocol.

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Data Availability

All data generated or analyzed during this study are included in this published article.

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Acknowledgements

The work was done at the laboratory for stem cells research in Anatomy Department of Shiraz University of Medical Sciences, Shiraz, Iran. The authors would like to thank Mrs Salmannezhad and Sani for their technical assistances.

Funding

The project was assigned as grant number 7912 in the office of the Vice Chancellor of Research Affairs, Shiraz University of Medical Sciences.

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Authors and Affiliations

  1. Laboratory for stem cell research, Department of Anatomical Sciences, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

    Soghra Bahmanpour, Zia Moasses & Nehleh Zarei-Fard

  2. Histomorphometry and Stereology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

    Nehleh Zarei-Fard

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  1. Soghra Bahmanpour
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  2. Zia Moasses
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Contributions

Z.M. and N.Z. performed the experiments and was involved in the collection, analysis, and interpretation of data and manuscript drafting. S.B. and N.Z. conceived the original idea and supervised the project. In addition, S.B. and N.Z. interpreted the data and revised the manuscript. All authors read and approved the final manuscript.

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Correspondence to Nehleh Zarei-Fard.

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This study was approved by the Ethics Committee of Shiraz University of Medical Sciences.

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Bahmanpour, S., Moasses, Z. & Zarei-Fard, N. Comparative effects of retinoic acid, granulosa cells conditioned medium or forskolin in combination with granulosa cell co-culturing on mouse germ cell differentiation. Mol Biol Rep 50, 631–640 (2023). https://doi.org/10.1007/s11033-022-07920-1

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