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Effect of NMDA receptor agonist and antagonist on spermatogonial stem cells proliferation in 2- and 3- dimensional culture systems

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Abstract

Background

The main purpose of this study was to investigate the effect of D-serine (DS) and Dizocilpine (MK-801) on the proliferation of spermatogonial stem cells (SSCs) in two-dimensional (2D) and three-dimensional (3D) culture systems.

Methods and results

The SSCs of male NMRI mice were isolated by enzymatic digestion and cultured for two weeks. Then, the identity of SSCs was validated by anti-Plzf and anti-GFR-α1 antibodies via immunocytochemistry (ICC). The proliferation capacity of SSCs was evaluated by their culture on a layer of the decellularized testicular matrix (DTM) prepared from mouse testis, as well as two-dimensional (2D) with different mediums. After two weeks of the initiation of proliferation culture on 3D and 2D medium, the pre-meiotic at the mRNA and protein levels were evaluated via qRT-PCR and flow cytometry methods, respectively. The results showed that the proliferation rate of SSCs in 3D culture with 50 mM glutamic acid and 20 mM D-serine was significantly different from other groups after 14 days treatment. mRNA expression levels of promyelocytic leukemia zinc finger (Plzf) in 3D cultures supplemented by 20 mM D-serine and 50 mM glutamic acid were considerably higher than the 3D control group (p < 0.001). The flow cytometry analysis revealed that the amount of Plzf in the 2D-culture groups of SSCs with 20 mM MK-801 was considerably lower compared to the 2D-culture control group (p < 0.001).

Conclusions

This study indicated that decellularized testicular matrix supplemented with D-serine and glutamic acid could be considered a promising vehicle to support cells and provide an appropriate niche for the proliferation of SSCs.

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Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on request.

Code availability

Not applicable' for that section.

Abbreviations

2D:

Two-dimensional

3D:

Three-dimensional

NMDARs:

N-methyl d-aspartate type glutamate receptors

SSCs:

Spermatogonia Stem Cells

DTM:

Decellularized testicular matrix

PLZF:

Promyelocytic leukemia zinc finger

GFRα1:

GDNF family receptor alpha-1

qRT-PCR:

Quantitative Reverse Transcription Polymerase Chain Reaction:

ICC:

Immunocytochemistry

SEM:

Scanning electron microscopy

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Acknowledgements

This study was financially supported by the research deputy of the University of Tabriz, Tabriz, Iran. This manuscript is written based on the Ph.D thesis of Amirhessam Eskafi Noghani

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Authors and Affiliations

  1. Department of Clinical Science, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran

    Amirhessam Eskafi Noghani, Reza Asadpour & Adel Saberivand

  2. Basic Medical Science Research Center, Histogenotech Company, Tehran, Iran

    Zohreh Mazaheri

  3. Department of Basic Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran

    Gholamreza Hamidian

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  1. Amirhessam Eskafi Noghani
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  2. Reza Asadpour
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Contributions

Amirhessam Eskafi Noghani: Performed the research and prepared manuscript. Reza Asadpour: Contributed to study design, wrote the draft, and interpreted the data. Adel Saberivand: Contributed in performing study and revised the manuscript. Zohreh Mazaheri: Contributed in the construction of 3D culture system, interpret the data, Gholamreza Hamidian: Revise the manuscript and interpret histology data; all authors approved the submitted version.

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Correspondence to Reza Asadpour.

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This study was approved by Ethics Committee of University of Tabriz (Code No: IR.TABRIZU.REC.1399.045), and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

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Noghani, A.E., Asadpour, R., Saberivand, A. et al. Effect of NMDA receptor agonist and antagonist on spermatogonial stem cells proliferation in 2- and 3- dimensional culture systems. Mol Biol Rep 49, 2197–2207 (2022). https://doi.org/10.1007/s11033-021-07041-1

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