Quantification of viable granulosa cells in murine ovarian follicles

ABSTRACT

Ovarian follicle growth and oocyte maturation depend on the viability of granulosa cells (GC). We quantified GC in whole mouse follicles. Single follicles were isolated from adult mouse ovaries and stained with DAPI or Live-Dead stain before fixation. An objective image analysis protocol for counting fluorescent labeled GC was developed that used Image J software to measure GC cytoplasmic and nuclear areas. These data were compared to the number of GC obtained by disaggregating 96 follicles with enzymes to produce a suspension of GC, which then was stained with trypan blue and assessed using a hemocytometer. We found a linear relation between GC/follicle and follicle diameter. Viability of GC/follicle ranged from 40 ± 11 to 72 ± 7%. The coefficient of variation for image analysis of DAPI stained GC by different assessors was 4%, but the number of GC obtained from image analysis was approximately 50% less than from disaggregated follicles. The number of GC in intact mouse follicles was greater than the number reported earlier for fixed ovarian sections. We found that the number of GC was less in fluorescence labeled follicles; it is possible that the three-dimensional structure of the intact follicles obscured the fluorescent signal. Direct quantification of viable GC isolated from follicles appears to be the most accurate method.

KEYWORDS:

• collagenase
• follicle
• folliculogenesis
• granulosa cells
• hyaluronidase
• mouse
• oocyte
• ovary

Disclosure statement

Flinders Fertility company supported some of our research with a grant administered by the University; the company had no oversight or influence on the research or results. The Flinders Animal Welfare Committee approved our use of cull animals on the condition that no requests were made for specific strains or ages. The data sets used and/or analyzed during the current study are available from the contact author upon request. The authors report no competing interests in the research.

Additional information

Funding

We acknowledge financial assistance from the publicly owned company, Flinders Fertility.