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

Abstract

Purpose Temporary and reversible downregulation of metabolism may improve the survival of tissues exposed to nonphysiological 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 preexposures 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.

Influence of warming and reanimation conditions on seminiferous tubule morphology, mitochondrial activity, and cell composition of vitrified testicular tissues in the domestic cat model

“…Similar result in the mitochondrial activity after warming and culture was found in a study with vitrification of ovine oocyte [ 52 ]. However, in other research evaluating the mitochondrial membrane potential in frozen-thawed ovaries from domestic cats, their results showed that all treatments groups were lower than fresh group [ 35 ]. Cellular energy production is performed in the mitochondria.…”

“…Measured area was the same for all samples and the relative intensity was determined by subtracting the tissue Rhodamine 123 fluorescence emission of the background of each slide. We evaluated 30 randomly selected areas in each experimental group and high mitochondrial membrane potential was considered the tissue with high intensity of Rhodamine 123 [ 35 ].…”

Influence of microwave-assisted dehydration on morphological integrity and viability of cat ovarian tissues: First steps toward long-term preservation of complex biomaterials at supra-zero temperatures

“…Although once considered experimental, it is increasingly integrated into routine human fertility preservation [5]. Despite various degree of success reported in different species, morphological, cytological and molecular alterations are observed in cryopreserved tissue in many species studied, including mouse [9, 10], cat [11, 12], sheep [13], pig [14], cattle [14, 15], monkey [16], and human [17, 18]. Although cryopreserved biomaterials can be stably stored at cryogenic temperatures, the dependence on liquid nitrogen storage generates high demand on long-term maintenance and financial sustainability.…”

Influence of different warming temperatures on the vitrified testicular fragments from pre‐pubertal cat

“…Animal reproduction biotechnology seeks to increasingly develop and improve methodologies in order to enhance techniques, which ensure the conservation of the biological material to the fertility preservation (Tanpradit et al., 2016) with minimum interference over the genetic material. (Moreira, 2017).…”

“…(Moreira, 2017). Obtaining the biological material is important in adverse situations as early death, especially in the collection of genetic material of endangered species (Comizzoli, 2017; Thuwanut et al., 2013) or those that will be previously exposed to gonadotoxic factors (Oliveira, 2015; Lima and Silva, 2017), whether for animal models (Tanpradit et al., 2016).…”