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. 2018 Feb;35(2):339-343.
doi: 10.1007/s10815-017-1073-5. Epub 2017 Oct 27.

Follicle dynamics: visualization and analysis of follicle growth and maturation using murine ovarian tissue culture

Tomohiko Murase  1 , Akira Iwase  2   3 , Kouji Komatsu  4 , Bayasula  1 , Tomoko Nakamura  1 , Satoko Osuka  1   5 , Sachiko Takikawa  1 , Maki Goto  1 , Tomomi Kotani  1 , Fumitaka Kikkawa  1
Affiliations

Follicle dynamics: visualization and analysis of follicle growth and maturation using murine ovarian tissue culture

Tomohiko Murase et al. J Assist Reprod Genet. 2018 Feb.

Abstract

Purpose: To visualize and analyze follicle development in ovarian tissue culture using physiological concentrations of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in order to establish an ovarian tissue culture system that enables efficient in vitro growth of follicles.

Methods: Ovarian tissues from 4-week-old female ICR mice were sliced and cultured. Images of ovarian tissues in culture were obtained at 24-h or 30-min intervals by using a microscope. The area of each follicle observed in the ovarian tissue slices was tracked and analyzed in association with oocyte maturation.

Results: We were able to track the development of each follicle using this culture system. Follicle growth was associated with oocyte maturation. Meiotically matured oocytes (MII) were obtained from 33% of all follicles investigated. Approximately, a quarter of follicles (24%) did not grow and resulted in atresia.

Conclusion: Follicle dynamics were successfully visualized and analyzed in murine ovarian tissue culture. We were able to obtain mature oocytes from the fully grown follicles in vitro. This culture system would be helpful for efficient in vitro culturing of ovarian tissues.

Keywords: Follicular development; Granulosa cells; Oocyte maturation; Ovary; Time-lapse imaging.

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Conflict of interest statement

Statement on the welfare of animals

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

Competing interests

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Evaluation of follicle growth in ovarian tissue culture. a Culture condition of murine ovarian slices for 14 days with physiological concentration of constant FSH and LH with periodic surge-like loading. b Outlined areas were determined as follicular area of growing follicles. Some oocytes were extruded from the ovary during culture (arrow). c Sizes of each follicle during in vitro culture are shown. The numbers of released oocytes of each meiotic phase (GV, MI, MII, or degeneration) are embedded in the panel. Similar results were obtained from three independent experiments. The graph showed the representative data. d Approximately 55.4% follicle extrusion occurred within 24 h after each LH surge. Italicized numbers indicate the P values calculated using one-way ANOVA with Bonferroni’s pairwise comparison. All values are indicated as the mean ± SD
Fig. 2
Fig. 2
Correlation of follicular area, diameter and meiotic stage of oocytes. a Oocyte diameter was positively correlated with follicular area in ovarian tissue culture (r = 0.714, P < 0.001). b Meiotic maturation of oocytes was correlated with follicular area. Follicular area of released oocytes was significantly different between GV and MII, and MI and MII. Italicized numbers show P values in One-way ANOVA with Bonferroni’s pairwise comparison. NS, no significance. Bar, median
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