title: Monitoring preantral follicle survival and growth in bovine ovarian biopsies by repeated use of neutral red and cultured in vitro under low and high oxygen tension.
abstract: The development and optimization of preantral follicle culture methods are crucial in fertility preservation strategies. As preantral follicle dynamics are usually assessed by various invasive techniques, the need for alternative noninvasive evaluation tools exists. Recently, neutral red (NR) was put forward to visualize preantral follicles in situ within ovarian cortical fragments. However, intense light exposure of NR-stained tissues can lead to cell death because of increased reactive oxygen species production, which is also associated with elevated oxygen tension. Therefore, we hypothesize that after repeated NR staining, follicle viability and dynamics can be altered by changes in oxygen tension. In the present study, we aim (1) to determine whether NR can be used to repeatedly assess follicular growth, activation, and viability and (2) to assess the effect of a low (5% O2) or high (20% O2) oxygen tension on the viability, growth, and stage transition of preantral follicles cultured in vitro by means of repeated NR staining. Cortical slices (n = 132; six replicates) from bovine ovaries were incubated for 3 hours at 37 °C in a Leibovitz medium with 50 μg/mL NR. NR-stained follicles were evaluated in situ for follicle diameter and morphology. Next, cortical fragments were individually cultured in McCoy's 5A medium for 6 days at 37 °C, 5% CO2, and 5% or 20% O2. On Days 4 and 6, the fragments were restained by adding NR to the McCoy's medium and follicles were reassessed. In both low and high oxygen tension treatment groups, approximately 70% of the initial follicles survived a 6-day in vitro culture, but no significant difference in follicle survival on Day 4 or 6 could be observed compared with Day 0 (P > 0.05). A significant decrease in the number of primordial and increase in primary and secondary follicles was observed within 4 days of culture (P < 0.001). In addition, a significant increase of the mean follicle diameter in NR-stained follicles was observed (P < 0.001), resulting in an average growth of 11.82 ± 0.81 μm (5% O2) and 11.78 ± 1.06 μm (20% O2) on Day 4 and 20.94 ± 1.24 μm (5% O2) and 19.12 ± 1.36 μm (20% O2) on Day 6 compared with Day 0. No significant differences in follicle growth rate or stage transition could be observed between 5% and 20% O2 (P > 0.05). In conclusion, after repeated NR staining, we could not find a difference between low and high oxygen tension in terms of follicle viability, stage transition, or growth. Therefore, under our culture conditions follicle dynamics are not determined by the oxygen tension in combination with quality assessment protocols using repeated NR staining.
authors.affiliation: Laboratory of Veterinary Physiology, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Wilrijk, Belgium. Electronic address: Ellen.Jorssen@ua.ac.be.
authors.forename: Ellen P A
authors.initials: EP
authors.lastname: Jorssen
authors.affiliation: Laboratory of Veterinary Physiology, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Wilrijk, Belgium.
authors.forename: An
authors.initials: A
authors.lastname: Langbeen
authors.affiliation: StatUa Center for Statistics, University of Antwerp, Antwerp, Belgium.
authors.forename: Erik
authors.initials: E
authors.lastname: Fransen
authors.affiliation: Laboratory of Veterinary Physiology, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Wilrijk, Belgium; CIMAGT Centro de Investigaciones para el Mejoramiento Animal de la Ganaderia Tropical, Loma de Tierra, Cotorro, Ciudad de la Habana, Cuba.
authors.forename: Emilia L
authors.initials: EL
authors.lastname: Martinez
authors.affiliation: Laboratory of Veterinary Physiology, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Wilrijk, Belgium.
authors.forename: Jo L M R
authors.initials: JL
authors.lastname: Leroy
authors.affiliation: Laboratory of Veterinary Physiology, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Wilrijk, Belgium.
authors.forename: Peter E J
authors.initials: PE
authors.lastname: Bols
mesh_terms: D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques / Q000662:veterinary*; D005260:Female; D009499:Neutral Red / Q000633:toxicity*; D006080:Ovarian Follicle / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D010100:Oxygen; D017382:Reactive Oxygen Species / Q000378:metabolism; D018675:Toxicity Tests / Q000662:veterinary
chemical_list: D017382:Reactive Oxygen Species; D009499:Neutral Red; D010100:Oxygen
keywords: Follicular culture; Neutral red; Ovarian follicle survival in vitro; Preantral follicle