Abstract
Background: Mammalian ovaries contain follicles containing an oocyte enclosed by layers of granulosa cells (GC). Follicle growth and oocyte maturation are largely dependent on GC numbers and viability, but there is no established, reliable method for assessing the number of viable GC within an isolated follicle. Methods: Centrifugation conditions and the Trypan Blue (TB) Exclusion assay were optimised for low cell densities compatible with the numbers of GC in follicles. Mouse ovarian follicles were disaggregated to produce a single cell suspension of GC which were examined by TB (n = 4), but also by crystal violet assay in a 96-well plate format after 24 h in vitro (n = 3). GC viability in vitro was characterised further by using enzyme-linked immunoassays to quantify GC production of anti-Mullerian hormone (AMH) and estrogen. Results: The centrifugation and low cell density TB protocol could accurately measure the viability of 78 GC in 10 μL, with an intra-assay coefficient of variation (CoV) 22%, and inter-assay CoV 7%. The best follicle disaggregation method (30 min 37°C exposure to 2 mg/mL collagenase prior to 30 min exposure to 0.025% hyaluronidase) yielded (656 ± 87) GC per antral follicle of which 82% ± 5% were viable. Culturing 312 - 20,000 GC per well for 24 hours and assessing viability by crystal violet assay generated a linear correlation between OD value and viable GC number (R2 = 0.98) and estrogen concentration per well (R2 = 0.92). 20,000 GC per well produced 143 ± 16 pg/mL estrogen during 24 hours in vitro, but no detectable AMH. Conclusion: This is the first report describing the isolation of viable, estrogen-producing GC from murine follicles, and their subsequent culture. These procedures are transferrable to other species including humans and can be applied to screening the reproductive toxicity of pharmaceutical agents.
Highlights
Follicles are isolated from ovarian tissue for many reasons [1]-[23] including in vitro maturation (IVM) to produce mature fertilizable oocytes [11] [20] [24] [25] [26] [27]
Mouse ovarian follicles were disaggregated to produce a single cell suspension of granulosa cells (GC) which were examined by Trypan Blue (TB) (n = 4), and by crystal violet assay in a 96-well plate format after 24 h in vitro (n = 3)
The KGN granulosa cell line derived from a human granulosa cell carcinoma [66] [67] was maintained in Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 supplemented with 1% insulin (5 ug/mL), transferrin (5 ug/mL) and selenium (5 ng/mL, ITS) and 10% FCS and 1% penicillin (10,000 units/mL) and streptomycin (10 mg/mL) and sub-cultured every 2 - 3 days as required using trypsin ethylene diamine tetra-acetic acid (EDTA) to detach cells
Summary
Follicles are isolated from ovarian tissue for many reasons [1]-[23] including in vitro maturation (IVM) to produce mature fertilizable oocytes [11] [20] [24] [25] [26] [27]. Isolated follicles are cultured in vitro in a number of different systems including 3D matrices [1] [24] [25] [28]-[36]. Conclusion: This is the first report describing the isolation of viable, estrogen-producing GC from murine follicles, and their subsequent culture. These procedures are transferrable to other species including humans and can be applied to screening the reproductive toxicity of pharmaceutical agents
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