Deciphering the influence of mare oviductal organoid secretions on equine spermatozoa

Abstract

The oviductal milieu assists fertilization by modulating sperm survival, supporting capacitation, and facilitating transport of early embryos. In many mammals, the direct relationship between the spermatozoa and the oviductal epithelial cells prolong sperm survival by inducing quiescence and suppressing metabolic activity. However, the detailed mechanisms remain unknown. In this study we sought to develop an equine organoid model to examine sperm-oviduct interactions. We aimed to determine whether oviductal secretions alone can induce quiescence, or alternatively, drive capacitation in preparation for fertilization. This was done by exposing spermatozoa to oviductal organoid-conditioned media and assessing sperm motility, viability, mitochondrial function, and capacitation. Oviductal organoids were cultured from single cells derived from the enzymatic digestion of freshly collected mare (n=3) infundibulum. Mature organoids (>21 days) with ciliated and secretory cells were cultured for 24 h with either fresh oviductal epithelial (OE) culture medium (containing Advanced DMEM-F12, Glutamax, HEPES, penicillin-streptomycin and WNT3A-RSPO3-NOGGIN) or Biggers-Whitten-Whittingham medium (BWW). Thereafter, culture media were collected and centrifuged (conditioned medium). Spermatozoa were collected from a stallion of known fertility, isolated using discontinuous gradient centrifugation and diluted to 20 × 106 cells/ml. Spermatozoa were then co-incubated with conditioned and non-conditioned (control) OE, and conditioned and non-conditioned BWW medium. Total (TM) and progressive (PM) motility were assessed at 1h and 24h; viability (via Live-Deadfluorescent stain/flow cytometry), mitochondrial membrane potential (MMP; via JC-1 probe/flow cytometry) and capacitation (via anti-tyrosine phosphorylation immunocytochemistry) were assessed at 24 h. Spermatozoa incubated with conditioned OE media showed significantly reduced motility at 24h (TM: conditioned vs control; 17.1±2.6% vs 43.2±5.5%; p=.025. and PM: conditioned vs control; 3.7±0.4% vs 27.1±2.8%; p=.008). Similar results were seen for BWW. Spermatozoa viability was not significantly affected (conditioned vs control; OE: 58.2±5.8% vs 84.6±5.9%; p>.05) nor was MMP (conditioned vs control; OE: 63.8±6.1% vs 66.9±7.5% of live cells with high MMP; p>.05). For BWW, similar results were recorded for viability (conditioned vs control; 84.5±6.2% vs 75.6±0.05%; p>.05), and MMP (conditioned vs control; 31.3±10.2% vs 56.2±13.5% of live cells with high MMP; p>.05). Observationally, MMP was suppressed in organoid-conditioned BWW exposed spermatozoa in 2 of the 3 replicates (from 41.3% to 6.3%, and from 89.1% to 42% cells with high MMP, concurrent with higher viability in exposed samples). Tyrosine phosphorylation, indicative of capacitation, showed no difference between organoid-conditioned medium exposed spermatozoa and controls. In summary, spermatozoa exposed to oviductal organoid-conditioned medium respond with suppressed motility but remain viable. These findings are consistent with the hypothesis that oviductal epithelial secretions can induce quiescence in the spermatozoa reservoir awaiting arrival of the oocyte.