68 Magnetic 3D culture system: Is this new culture system suitable for evaluating hormone responsiveness in oviductal cells?

Three-dimensional (3D) cell culture systems have been under the spotlight in many fields, and it is not different in reproduction science, especially for the oviduct. Studying the oviducts is a peculiar matter both in in vivo and in vitro conditions. In these circumstances, the 3D systems have earned an unbeatable position when discussing the oviduct by overpassing many limitations. The magnetic 3D culture system (Greiner Bio-One CELLSTAR^®), to our best knowledge, has only been used for oviduct cells by our group. Briefly, we have demonstrated that bovine oviduct epithelial (OEC) and stromal (OSC) cells form spheroids of attached cells once the OEC and OSC are magnetised and placed under a magnetic force. At the size of 10,000 cells, the spheroid formation occurs in three days, it survives for 21 days, it presents primary cilia, and it has a stable and replicable structure, which we named “oviducal magnetic spheroids” (OMS). Following this, our current goal was to evaluate the OMS hormone responsiveness. Oestradiol (E2) and progesterone (P4) play key roles in the oviduct cells during follicular and luteal oestrous phases, respectively. Therefore, we cultured the OMS for 14 days (DMEM media, for cell re-differentiation), followed by seven days under three conditions: (1) control (vehicle, 1% ethanol); (2) 100 ng/mL P4; and (3) 300 pg/mL E2. Afterwards, the OMS was submitted to morphological and mRNA abundance analyses (n = 3 OMS/treatment/analyses). Data were analysed in the GraphPad Software, using ANOVA or t-test, with P < 0.05 as significance level. In general, high variability was observed in the outcome parameters, especially in the OMS from the control group, which might be due to the lack of hormone stimulation. Comparing only E2 vs P4 groups, the E2 was incapable of increasing the OVGP1 (P = 0.20) and cell ciliation (P = 0.93, anti-acetylated-a-tubulin) abundances when analysed by immunofluorescence. Checking the sperm-adhesion capacity of OMS, the sperm number per area of OMS was not higher in the E2 group, as expected (P = 0.47). Nonetheless, OMS treated with P4 presented higher cellular necrosis than the E2 group (respectively, 3.4 ± 0.3 vs 1.8 ± 0.3 of necrotic intensity; P = 0.02) and the mRNA abundance of DDIT3, a transcription factor related to cell stress and cellular apoptosis, was also higher in the P4 than E2 group (respectively, 1.4 ± 0.17 vs 0.01 ± 0.00; P = 0.0014). The higher P4 levels in the oviduct during the luteal phase have been associated with the cell death process and epithelial renewal. In short, P4 treatment, but not E2, seems to modify OMS functionality. In other words, the OMS might hold promising opportunities in being used as an in vitro model to study hormone responsiveness in the oviducal cells, whereas some modifications (i.e. treatment start-point and hormone levels) might be advisable.

This research was supported by FAPESP (19/25982-7, 20/02500-4).