Hormonally responsive bovine oviductal organoids recapitulate native oviductal secretions and enhance sperm capacitation

Key results

1. 01

   ShownEstradiol and the progesterone-analog regimen each induce a distinct transcriptional program in the organoids. These programs match follicular- and luteal-phase markers and overlap with in vivo bovine oviduct expression data. The mP4 response is much broader than the E2 response.

   RNA-seq (n = 3 per group): E2 vs control gave 225 DEGs (155 up, 70 down) and mP4 vs control 1,330 (463 up, 867 down) at p ≤ 0.05, |log2FC| > log2(1.5). 142 follicular and 107 luteal genes were concordantly upregulated vs the Cerny et al. (2015) microarray. RT-qPCR (n = 5): E2 raised OVGP1, NTS, OXTR; mP4 raised HP, TGM2, ESR2. · Generation and hormonal responsiveness of bovine…; Estradiol and progesterone treatments elicit…; Figure legends

2. 02

   ShownLuminal secretions recovered from the organoids contain thousands of proteins, most of which are also found in native bovine oviductal fluid. Their composition shifts with hormone treatment: OVGP1, PIP and SELENOM rise with E2, while HP, TGM2, TIMP1 and CTSZ are enriched with mP4.

   LC-MS/MS identified 4,070 proteins. 83.7 to 83.9% of ODS proteins were shared with early-luteal in vivo oviductal fluid (qualitative Venn comparison). Vs control, E2 ODS had 148 up and 122 down proteins, and mP4 ODS had 223 altered (141 up, 82 down); n = 3 biological replicates; PCA separated treatments. · Proteomic LC-MS/MS; Hormonal stimulation mimics the proteomic…; Figure legends

3. 03

   ShownSecretions from estradiol-treated organoids promote capacitation in bull sperm, similar to preovulatory oviductal fluid. They raise membrane fluidity, the acrosome reaction rate and phosphorylation of PKA substrates and tyrosine residues. Secretions from progesterone-analog-treated organoids had minimal effect.

   After 2 hours with ODS-E2, sperm had higher membrane fluidity and a significantly larger acrosome-reacted fraction than controls (p < 0.05, ANOVA/Tukey). Total and progressive motility only trended upward; only the BOF-LF positive control reached significance. Blots showed stronger pPKA and pTyr signals. Semen came from five bulls, with at least three straws per bull pooled per replicate. · Sperm capacitation treatment; Flow cytometry; Estradiol-stimulated organoid secretions enhance…

4. 04

   ShownMulti-omics factor analysis (MOFA2) of the paired transcriptome and secretome data separates latent factors tied to each hormone. This links hormone-driven transcription to changes in secreted proteins.

   Five factors explained 42.6% of transcriptomic and 82.0% of proteomic variance. Factor 2 tracked mP4 (p = 0.004; top features included SELENOP, TGM2, SGK1, PDXK, GLUL). Factor 3 tracked E2 (p = 0.027; ESM1, IGFBP4, SEC23A, SUCLG2, DLST). · Bioinformatics analysis; Multi-omics factor analysis identifies coherent…

5. 05

   SuggestedThe organoids form apical-in, single-layer epithelial cysts dominated by ampullar secretory cells and lacking multiciliated cells, whatever the hormone treatment. This structure lets secreted factors accumulate in the lumen, where they can be collected.

   CDH1 outlined the organoids over a laminin-positive basement membrane. CIBERSORTx deconvolution with a mouse scRNA-seq signature (GSM5005819), mapped to bovine genes, ranked ampullar secretory cells as the main population. SOX17 staining was uniform, and acetyl-α-tubulin staining showed no multiciliated cells; no treatment differences were seen. · Bioinformatics analysis; Generation and hormonal responsiveness of bovine…; Estradiol and progesterone treatments elicit…; Discussion