Effects of ovarian disaggregation on adult murine follicle yield and viability

Fiona M. Young, John Stephen Drummond, Emma Akers, Louise Bartle, David Thomas Kennedy, Mohammad Asaduzzaman

    Research output: Contribution to journalArticle

    3 Citations (Scopus)

    Abstract

    Follicles are isolated from ovaries for numerous reasons, including IVM, but adult murine yields are <2 folliclesmg-1. The aim of the present study was to optimise ovarian disaggregation and develop methods applicable to the rapid screening of follicle viability. Ovaries from adult mice (n≤7) were halved and disaggregated mechanically, or by using collagenase IV (Col-IV; 590UmL-1) or animal origin-free collagenase IV (AOF) at 590 or 1180UmL-1. Isolated follicles were stained with 4′,6′-diamidino-2-phenylindole (DAPI; nuclei), chloromethyl-X-rosamine (CMXRos; mitochondria) or fluorescein isothiocyanate-conjugated anti-α-tubulin antibody. Follicle diameters and staining were measured and analysed using ImageJ, and data analysed using GraphPad Prism. Col-IV disaggregation yielded the highest number of follicles (17±10 folliclesmg-1 ovarian tissue). All disaggregation methods released more secondary follicles (86±20 per ovary; P<0.05) than any other size cohort. Mechanical and Col-IV disaggregation yielded similar numbers of morphologically intact follicles, whereas AOF disaggregation caused more damage (P<0.01). As the morphological disruption increased, DAPI and CMXRos staining decreased (P<0.05), and tubulin localisation became more heterogeneous. Col-IV disaggregation gave the best yield of morphologically intact follicles containing viable granulosa cells. In conclusion, we improved adult murine follicle yields and applied molecular markers to assess follicle morphology, cellular cytoskeleton and mitochondrial function.

    Original languageEnglish
    Pages (from-to)2400-2410
    Number of pages11
    JournalReproduction Fertility and Development
    Volume29
    Issue number12
    DOIs
    Publication statusPublished - Jun 2017

    Keywords

    • Mitochondrial activity
    • alpha-tubulin
    • follicle morphology

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  • Cite this

    Young, F. M., Drummond, J. S., Akers, E., Bartle, L., Kennedy, D. T., & Asaduzzaman, M. (2017). Effects of ovarian disaggregation on adult murine follicle yield and viability. Reproduction Fertility and Development, 29(12), 2400-2410. https://doi.org/10.1071/RD16398