In vitro model of premature ovarian insufficiency based on cyclophosphamide-induced mitochondrial dysfunction in granulosa cells

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Abstract

BACKGROUND: Currently, there is no unified approach or effective method for treating premature ovarian insufficiency. The primary strategy is hormone replacement therapy aimed at mitigating estrogen deficiency and its associated complications. However, this therapy does not restore lost ovarian function or fertility. Thus, further research into the pathogenesis of premature ovarian insufficiency is crucial for developing alternative pathogenetically based therapies. Investigating the efficacy of various drugs in preclinical trials using cellular models holds significant promise. Experimental modeling of premature ovarian insufficiency, which closely replicates the origin and development mechanism of the human disease, can be effectively used to develop promising therapeutic approaches, in particular, for testing new drugs.

AIM: The aim of this study was to develop a new method for experimental modeling of premature ovarian insufficiency using cyclophosphamide in Wistar rats, the significant advantages of which are high reproducibility, ease of implementation, and cost-effectiveness.

MATERIALS AND METHODS: A culture of Wistar rat ovarian granulosa cells after five stages of subculturing was treated with the drug cyclophosphamide, ensuring a working concentration in the growth medium of 0.1 mg/ml, followed by incubation for six hours.

RESULTS: A cellular model of premature ovarian insufficiency has been created, which is characterized by 100% modeling efficiency, high manufacturability and environmental safety for modeling the pathological condition.

CONCLUSIONS: The model created will allow for testing the medicinal effectiveness of chemicals with a view to their further use in medicine.

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About the authors

Karina A. Zakuraeva

The Research Institute of Obstetrics, Gynecology and Reproductology named after D.O. Ott

Author for correspondence.
Email: zakuraevak@icloud.com
ORCID iD: 0000-0002-8128-306X
SPIN-code: 5215-7869

MD

Russian Federation, 3 Mendeleevskaya Line, Saint Petersburg, 199034

Maria I. Yarmolinskaya

The Research Institute of Obstetrics, Gynecology and Reproductology named after D.O. Ott

Email: m.yarmolinskaya@gmail.com
ORCID iD: 0000-0002-6551-4147
SPIN-code: 3686-3605

MD, Dr. Sci. (Medicine), Professor, Professor of the Russian Academy of Sciences

Russian Federation, 3 Mendeleevskaya Line, Saint Petersburg, 199034

Andrey Yu. Vinokurov

Orel State University named after I.S. Turgenev

Email: vinokurovayu@oreluniver.ru
ORCID iD: 0000-0001-8436-1353
SPIN-code: 5518-3107

Cand. Sci. (Engineering)

Russian Federation, Orel

Marina Yu. Pogonyalova

Orel State University named after I.S. Turgenev

Email: mpogonalova@gmail.com
ORCID iD: 0000-0001-6919-0728
SPIN-code: 1300-9791
Russian Federation, Orel

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Supplementary files

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2. Fig. 1. The isolated cell cultures are characterized by the presence of a calcium signal in response to stimulation with folliclestimulating hormone (FSH)

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3. Fig. 2. Representative confocal images of ovarian granulosa cell culture loaded with tetramethylrhodamine methyl ester

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4. Fig. 3. Statistical processing of tetramethylrhodamine methyl ester fluorescence data. FCCP — carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone

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5. Fig. 4. Experimental curves of the mitochondrial membrane potential maintenance mechanism evaluation in control cells and cells treated with cyclophosphamide. * р ≤ 0,05

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6. Fig. 5. Effect of cyclophosphamide concentration (0.015–0.100 mg/ml) on the degree of mitochondrial depolarization in response to rotenone

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7. Fig. 6. Experimental graphs of changes in the fluorescence intensity of tetramethylrhodamine methyl ester (TMRM) under the influence of oligomycin A, rotenone and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) in control cells and cells treated with different concentrations of cyclophosphamide

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8. Fig. 7. The effect of incubation time on the development of cyclophosphamide-associated (0.1 mg/ml) mitochondrial dysfunction. TMRM, tetramethylrhodamine methyl ester. * р ≤ 0,05

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9. Fig. 8. Experimental graphs of changes in the fluorescence intensity of tetramethylrhodamine methyl ester (TMRM) under the influence of oligomycin A, rotenone and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) in control cells and cells treated with cyclophosphamide for 2–8 h

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СМИ зарегистрировано Федеральной службой по надзору в сфере связи, информационных технологий и массовых коммуникаций (Роскомнадзор).
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