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Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) pre-exposure ensures follicle integrity during in vitro culture of ovarian tissue but not during cryopreservation in the domestic cat model

  • Fertility Preservation
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Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

Temporary and reversible downregulation of metabolism may improve the survival of tissues exposed to non-physiological conditions during transport, in vitro culture, and cryopreservation. The objectives of the study were to (1) optimize the concentration and duration of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP—a mitochondrial uncoupling agent) exposures for biopsies of domestic cat ovarian tissue and (2) examine the effects of FCCP pre-exposures on follicle integrity after tissue culture and/or cryopreservation.

Methods

Biopsies of cat ovarian tissue were first treated with various concentrations of FCCP (0, 10, 40, or 200 nM) for 10 or 120 min to determine the most suitable pre-exposure conditions. Based on these results, tissues were pre-exposed to 200 nM FCCP for 120 min for the subsequent studies on culture and cryopreservation. In all experiments and for each treatment group, tissue activity and integrity were measured by mitochondrial membrane potential (relative optical density of rhodamine 123 fluorescence), follicular viability (calcein assay), follicular morphology (histology), granulosa cell proliferation (Ki-67 immunostaining), and follicular density.

Results

Ovarian tissues incubated with 200 nM FCCP for 120 min led to the lowest mitochondrial activity (1.17 ± 0.09; P < 0.05) compared to control group (0 nM; 1.30 ± 0.12) while maintaining a constant percentage of viable follicles (75.3 ± 7.8 %) similar to the control group (71.8 ± 11.7 %; P > 0.05). After 2 days of in vitro culture, percentage of viable follicles (78.8 ± 8.9 %) in similar pre-exposure conditions was higher (P < 0.05) than in the absence of FCCP (61.2 ± 12.0 %) with percentages of morphologically normal follicles (57.6 ± 17.3 %) not different from the fresh tissue (70.2 ± 7.1 %; P > 0.05). Interestingly, percentages of cellular proliferation and follicular density were unaltered by the FCCP exposures. Based on the indicators mentioned above, the FCCP-treated tissue fragments did not have a better follicle integrity after freezing and thawing.

Conclusions

Pre-exposure to 200 nM FCCP during 120 min protects and enhances the follicle integrity in cat ovarian tissue during short-term in vitro culture. However, FCCP does not appear to exert a beneficial or detrimental effect during ovarian tissue cryopreservation.

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Acknowledgements

The authors would like to thank Asst. Prof. Dr. Sayamon Srisuwatanasagul for histologic technical assistance, Dr. Paweena Thuwanut for histologic interpretation advice and Assoc. Prof. Dr. Padet Tummaruk for helping with statistical analysis.

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Authors and Affiliations

  1. Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, 10330, Thailand

    Nae Tanpradit & Kaywalee Chatdarong

  2. Smithsonian Conservation Biology Institute, National Zoological Park, Washington, DC, 20008, USA

    Pierre Comizzoli

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  1. Nae Tanpradit
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  2. Kaywalee Chatdarong
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  3. Pierre Comizzoli
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Correspondence to Pierre Comizzoli.

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Funding

Financial support for this work was provided by the Royal Golden Jubilee Ph.D. Program (PHD/0199/2552), the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund), and Research Unit of Obstetrics and Reproduction in Animals, Chulalongkorn University.

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Capsule Uncoupling the oxidative phosphorylation of the domestic cat ovarian tissue with carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) is beneficial to the viability and morphology of follicles during ovarian tissue culture. However, tissue pre-exposure to FCCP does not have a protective or detrimental effect during cryopreservation.

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Tanpradit, N., Chatdarong, K. & Comizzoli, P. Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) pre-exposure ensures follicle integrity during in vitro culture of ovarian tissue but not during cryopreservation in the domestic cat model. J Assist Reprod Genet 33, 1621–1631 (2016). https://doi.org/10.1007/s10815-016-0810-5

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  • DOI: https://doi.org/10.1007/s10815-016-0810-5

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