Successful in vitro culture of pre-antral follicles derived from vitrified murine ovarian tissue: oocyte maturation, fertilization, and live births

Wang, Xiaoqian; Catt, Sally; Pangestu, Mulyoto; Temple-Smith, Peter

doi:10.1530/rep-10-0383

Cited by 65 publications

(44 citation statements)

References 63 publications

(74 reference statements)

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“…A mouse model is useful for this purpose. Several culture methods were applied to preantral mouse follicles with diameters of 110-140 mm (Cortvrindt & Smitz 1998, Kreeger et al 2005, Wang et al 2011. However, these methods are still insufficient for earlier preantral stages.…”

Section: Discussionmentioning

confidence: 99%

Live births from isolated primary/early secondary follicles following a multistep culture without organ culture in mice

et al. 2013

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Although the ovary has a large store of germ cells, most of them do not reach mature stages. If a culture system could be developed from early growing follicles to mature oocytes, it would be useful for biological research as well as for reproductive medicine. This study was conducted to establish a multistep culture system from isolated early growing follicles to mature oocytes using a mouse model. Early growing follicles with diameters of 60-95 mm corresponding to primary and early secondary follicles were isolated from 6-day-old mice and classified into three groups by diameter. These follicles contained oocytes with diameters of w45 mm and one or a few layered granulosa cells on the basal lamina. Embedding in collagen gel was followed by first-step culture. After 9-day culture, the growing follicles were transferred onto collagen-coated membrane in the second step. At day 17 of the culture series, the oocyte-granulosa cell complexes were subjected to in vitro maturation. Around 90% of the oocytes in follicles surviving at day 17 resumed second meiosis (metaphase II oocytes: 49.0-58.7%), regardless of the size when the follicle culture started. To assess developmental competence to live birth, the eggs were used for IVF and implantation in pseudopregnant mice. We successfully obtained two live offspring that produced next generations after puberty. We thus conclude that the culture system reported here was able to induce the growth of small follicles and the resultant mature oocytes were able to develop into normal mice.

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Section: Discussionmentioning

confidence: 99%

Live births from isolated primary/early secondary follicles following a multistep culture without organ culture in mice

et al. 2013

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“…Specifically, the addition of a non-permeable sugar, such as sucrose, to the vitrification solution can mitigate osmotic shock, protect membranes, and improve the glass-forming capabilities of permeable CPAs (e.g., ethylene glycol, dimethylsulfoxide, glycerol) [11]. Furthermore, the assessment of damage caused by vitrification should be investigated over time after warming, either in a culture system [12,13] or following tissue grafting [14], rather than immediately after warming as the effect of CPA exposure and vitrification on cell metabolism and function may not be detectable yet and may be mitigated over time.…”

Section: Introductionmentioning

confidence: 99%

Damage to fetal bovine ovarian tissue caused by cryoprotectant exposure and vitrification is mitigated during tissue culture

Mouttham

Fortune

Comizzoli

2015

J Assist Reprod Genet

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Purpose The objective of this study is to characterize the impact of exposure to cryoprotectants followed by vitrification on primordial follicle survival and activation using a fetal bovine model. Methods In the first study, fetal bovine cortical pieces were exposed to cryoprotectants with or without sucrose and cultured up to 7 days in the presence or absence of insulin. In the second study, cortical pieces were exposed to cryoprotectants with or without sucrose, vitrified, and cultured up to 7 days after warming in the presence or absence of insulin. Viability and morphology of follicles, as well as proliferation and/or DNA repair in ovarian tissue were analyzed.Results When compared to non-exposed controls, normal follicular morphology was affected in groups exposed to cryoprotectants only immediately post-exposure and after 1 day of culture, but improved by day 3 and did not significantly differ by day 7. Similarly, normal follicular morphology was compromised in vitrified groups after warming and on day 1 compared to controls, but improved by days 3 and 7. Proliferation and/or DNA repair in ovarian tissue was not affected by vitrification in this model. Cryoprotectant exposure and vitrification of ovarian tissue did not impair the activation of primordial follicles in response to insulin, although activation was delayed relative to non-exposed controls. Interestingly, sucrose had no noticeable protective effect. Conclusion Vitrified fetal bovine ovarian tissue has the intrinsic capacity to mitigate the immediate damage to primordial follicles' morphology and retains the capacity to activate. These findings provide a basis for a successful cryopreservation protocol for ovarian cortical tissue in other species including humans.

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“…Specifically, 2,4-dinitrophenol-a proton ionophorewas able to lower serological glucose, triglyceride, and insulin levels in serum and decrease reactive oxygen species levels and tissue DNA oxidation [28]. Here, we examined the mitigation of freezing stress on the ovarian tissue and the enclosed follicles that are critical for fertility preservation strategies [29][30][31]. Knowing that slow freezing procedures have resulted in most of the reports of live birth in human ovarian tissue cryopreservation followed by grafting [2,32,33], we investigated the cryopreservation method by using a protocol previously published [16].…”

Section: Discussionmentioning

confidence: 99%

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

Tanpradit

Chatdarong

Comizzoli

2016

J Assist Reprod Genet

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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.

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Successful in vitro culture of pre-antral follicles derived from vitrified murine ovarian tissue: oocyte maturation, fertilization, and live births

Cited by 65 publications

References 63 publications

Live births from isolated primary/early secondary follicles following a multistep culture without organ culture in mice

Live births from isolated primary/early secondary follicles following a multistep culture without organ culture in mice

Damage to fetal bovine ovarian tissue caused by cryoprotectant exposure and vitrification is mitigated during tissue culture

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

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