Development of an in vitro assay to detect endocrine disrupting compounds (EDCs) using cryopreserved ovine luteal cells

The blue-green algal toxin, cylindrospermopsin (CYN), inhibited basal and hCG-stimulated progesterone (P) production by luteinised human granulosa cells, independently of cytotoxicity. CYN was therefore likely to disrupt endocrine function of ovine luteal cells. The EDC ethinyl estradiol (EE2) acts via estrogen receptors, and should not affect ovine luteal cells. Cryopreservation of luteal cells would avoid seasonal unavailability of corpora lutea (CL) and improve applicability of the proposed EDC activity assay.

Aims were to examine effects of cryopreservation on responsiveness to LH, hCG, dbcAMP, 22OH cholesterol and P production, and characterise effects of EDCs.

1xCL preparation comprised ~40 abbatoir-derived CL disaggregated in 400U/ml collagenase. Half were stored at -80oC in 10% DMSO+DMEM+10%FCS. 10000 fresh or thawed luteal cells/well were added to 96-well plates in DMEM+10%FCS for 24h before replenishment with medium supplemented with 5 to 200 ng/ml hLH, or 0.1 to 1000IU/ml hCG or 2 to 50uM 22OHChol or 0.1 to 10mM dbcAMP, or combinations of these, (n≥3) preparations. Cryopreserved cells (n=3) additionally cultured with 0.1 to 3uM CYN or 10-8 to 10-4M EE2. After 24h, media were collected for P measurement, and photomicrographs of cells taken before the MTT cell viability assay was performed.

None of the supplements affected ‘fresh' or cryopreserved viable cell numbers. Both ‘fresh' and thawed cells were seeded at 10000 viable cells/well, but 48h later numbers of cryopreserved cells were lower than ‘fresh' for all supplements. For example, freeze-thawed viable cell numbers were 37% and 41% of ‘fresh' after 24h with 0mM or10mM dbcAMP respectively. All 4 supplements caused dose-dependent increases in P production, but the increase was attenuated in freeze-thawed cells; 10mM dbcAMP-stimulated P production by cryopreserved cells was 27% that of ‘fresh' cells. CYN caused dose-dependent cytotoxicity as expected, so did EE2. Surprisingly, CYN did not inhibit basal P production. Conclude that EDC activity assay requires further characterisation.