Poster Presentation ESA-SRB Conference 2015

Quantification of granulosa cells in mouse ovarian follicles (#296)

Mohammad Asaduzzaman 1 2 , John Drummond 1 2 , David Kennedy 1 2 , Fiona Young 1 2
  1. Flinders Fertility, Flinders Medical Centre, Adelaide, South Australia, Australia
  2. Flinders University, Bedford Park, SA, Australia

Chemotherapy often decreases antimullerian hormone levels and fertility in vivo, but effects on follicular granulosa cells are poorly characterized. Granulosa cell (GC) numbers in freshly isolated follicles can be used as baseline control values to develop less gonadotoxic chemotherapeutics. A review of 3 published reports indicated that GC/follicle numbers calculated from fixed histological sections of mouse ovaries were 21-60 (primary), 61-200 (secondary) and 201->600 (antral). We aimed to confirm these numbers using freshly isolated whole mouse follicles.

Mouse ovaries (n=5) were disaggregated with 2mg/mL collagenase IV. Isolated follicles were stained with DAPI or Calcein AM & Ethidium Homodimer-1 (‘Live-Dead’ stain) before fixation. The diameters and GC numbers of  M1 (viable regular morphology) or M2 (viable irregular morphology)[1] DAPI-stained follicles (n=215) were determined using fluorescent microscopy, Image J software and nuclear area. These data were validated by confocal microscopy, in which additional DAPI or Live-Dead stained M1 folliclesfrom each size cohort were examined. Confidence intervals were calculated and data subjected to 1-way ANOVA with Tukey post-test.

The area of Homodimer-1 stained GC nuclei (n=60) was 15.68±3.26 µm2. Follicle diameters were 63±13µm (n=65), 120±20µm (n=87) and 196±32µm (n=63), and GC/follicle numbers were 73±33 (p<0.05), 197±67 (p<0.05) and 431±163 for high quality viable M1&M2 primary, secondary and antral follicles respectively.

We found that GC/follicle numbers were slightly higher than values obtained previously; 65-82 v 21-60 in primary, 183-211 v 61-200 in secondary and 390-472 v 201->600 GC in antral mouse follicles. This might be because follicles are not perfect spheres but have a polar orientation with irregular GC distribution[2], meaning that GC counted in the histological section containing the oocyte nucleus would underestimate GC/follicle numbers. Our method incorporates fluorescence from all DAPI or Homodimer-1 stained GC nuclei, irrespective of location in the follicle, and is therefore likely to be more accurate.

  1. Dolmans, M.M., et al., Evaluation of Liberase, a purified enzyme blend, for the isolation of human primordial and primary ovarian follicles. Hum Reprod, 2006. 21(2): p. 413-20.
  2. Wezel, I.L.v. and R.J. Rodgers, Morphological characterization of bovine primordial follicles and their environment in vivo. Biology of Reproduction, 1996. 55: p. 1003-1011.