Ex vivo medaka pituitary culture reveals differential CCK and GnRH regulation of Fsh and Lh cells and selective disruption by estradiol and bisphenol A

Ex vivo pituitary culture sits between in vivo exposure and dissociated cell culture, but its suitability for studying gonadotrope regulation and endocrine disruption is unestablished. In teleosts, luteinizing hormone (Lh) and follicle-stimulating hormone (Fsh) are produced by separate cells, allowing their regulation to be studied independently. We characterised intact cultured medaka (Oryzias latipes) pituitaries and applied the preparation to bisphenol A (BPA) and 17β-estradiol (E2). Pituitaries remained viable throughout a 10-day culture period, with metabolic activity peaking between days 2 and 6 before decreasing. Basal lhb transcript levels fell significantly by day 2 and fshb by day 4, consistent with loss of hypothalamic input, and both were restored by mdGnRH and CCK8 respectively, with responsiveness persisting until day 6 for lhb and day 8 for fshb . Using transgenic lines expressing a genetically encoded Ca²⁺ indicator in Lh or Fsh cells, agonist-evoked responses persisted to day 4 in Lh cells and throughout the 6-day period tested in Fsh cells. Unexpectedly, CCK8 also increased lhb transcript levels. Single-cell transcriptomic data showed that, among the CCK receptor paralogs, only cck2rb was detected in the pituitary, in 40.9% of fshb ⁺/ lhb ⁻ and 15.3% of lhb ⁺/ fshb ⁺ gonadotropes but only 1.6% of lhb ⁺/ fshb ⁻ cells. Together with the known plasticity of medaka gonadotropes in culture, this suggests the lhb response to CCK8 reflects a small bi-hormonal population rather than a major regulatory pathway. E2 attenuated the CCK8-induced fshb increase, whereas BPA did not, and neither altered mdGnRH-induced lhb transcript levels. Agonist-evoked Ca²⁺ responses were not significantly affected by acute, 48-h ex vivo or 48-h in vivo exposure. Ex vivo culture is therefore a viable alternative to live-animal exposure for studying gonadotrope regulation and its disruption.