Effects of chemosynthetic choline plasmalogens on gonadotropin secretion from bovine gonadotrophs

Abstract

Ethanolamine plasmalogens (EPls) and choline plasmalogens (CPls), unique glycerophospholipids may play important roles in milk production and reproduction in postpartum dairy cows. While CPls are more abundant in bovine blood, EPls are predominant in the brain. Brain EPls are the only recognized ligands of G protein-coupled receptor 61 (GPR61), a receptor that co-localizes with GnRH receptors on gonadotrophs. We hypothesized that chemosynthetic CPls stimulate gonadotropin secretion from bovine gonadotrophs, similar to the reported effects of chemosynthetic EPls. Anterior pituitary cells from healthy, post-pubertal heifers, were cultured for 3.5 days and then treated with increasing concentrations (0, 0.7, 7, 70, or 700 pM) of EPl with vinyl-ether-bonded stearic acid and ester-bonded oleic acid (C18:0-C18:1EPl) as a positive control, or CPls with vinyl-ether-bonded stearic acid and ester-bonded oleic acid (C18:0-C18:1CPl), arachidonic acid (C18:0-C20:4CPl), or docosahexaenoic acid (C18:0-C22:6CPl). After 2 h, the medium samples were harvested for FSH and LH assays. C18:0-C18:1EPl (7-700 pM) stimulated basal FSH and LH secretion (P < 0.01). None of the tested CPl concentrations stimulated LH secretion. Only 700 pM of C18:0-C18:1CPl, but not lower concentrations, stimulated FSH secretion (P < 0.05), an effect that was inhibited by a SMAD pathway inhibitor. However, both C18:0-C18:1CPl and C18:0-C20:4CPl synergized with GnRH to stimulate FSH secretion. In silico molecular-docking simulations using the deep-learning algorithm ColabFold revealed that CPls bind to the three-dimensional structural model of GPR61. In conclusion, C18:0-C20:4CPl stimulated FSH secretion exclusively in the presence of GnRH, whereas C18:0-C18:1CPl weakly stimulated FSH secretion and showed potential interaction with the GnRH signaling pathways.

Keywords: ColabFold; Drug discovery; Follicle-stimulating hormone; Gonadotropin-releasing hormone receptor; Luteinizing hormone.

Fig. 1.

Effect of addition of numerous concentrations of a chemosynthetic ethanolamine plasmalogen (EPl) or three chemosynthetic choline plasmalogen (CPl) in medium lacking GnRH on hormone secretion from cultured anterior pituitary cells. The FSH and LH concentrations in control cells (cultured in medium lacking EPls, CPls, and GnRH) were averaged and set as 100%; the mean FSH and LH concentrations in each treatment group are expressed as percentages of the control value. Bars labeled with distinct letters (A, B, and C): significantly different stimulatory effects (P < 0.05); bars labeled with same letter: similar stimulatory effect. C18:0-C18:1EPl: 1-(1Z-octadecenyl)-2-oleoyl-sn-glycero-3-phosphoethanolamine (syn. PLAPE, or phosphatidylethanolamine-plasmalogen-oleic acid). C18:0-C18:1CPl: 1-(1Z-octadecenyl)-2-oleoyl-sn-glycero-3-phosphocholine (syn. phosphatidylcholine-plasmalogen-oleic acid). C18:0-C20:4CPl: 1-(1Z-octadecenyl)-2-arachidonoyl-sn-glycero-3-phosphocholine (syn. phosphatidylcholine-plasmalogen-arachidonoic acid). C18:0-C22:6CPl: 1-(1Z-octadecenyl)-2-docosahexaenoyl-sn-glycero-3-phosphocholine (syn. phosphatidylcholine-plasmalogen- docosahexaenoic acid).

Fig. 2.

Effect of addition of various concentrations of the chemosynthetic EPl or three CPl in medium containing GnRH on hormone secretion from cultured anterior pituitary cells. The FSH and LH concentrations in control cells (cultured in medium lacking EPls, CPls, and GnRH) were averaged and set as 100%; the mean FSH and LH concentrations in each treatment group are expressed as percentages of the control value. Bars labeled with distinct letters (A, B, and C): significantly different stimulatory effects (P < 0.05); bars labeled with same letter: similar stimulatory effect.

Fig. 3.

Effect of the SMAD pathway inhibitor, LDN212854 on C18:0-C18:1CPl-induced FSH secretion from cultured AP cells of post-pubertal heifers. The mean FSH or LH concentration for each treatment group is expressed as a percentage of the control value. Different letters indicate statistically significant differences (P < 0.05).

Fig. 4.

Three-dimensional structure of bovine GPR61 modeled using AlphaFold2. The three binding sites detected as targets in the docking simulation, the extracellular (site 1), cytoplasmic (site 2), and transmembrane (site 3) sites (indicated by colored spheres). TM, transmembrane region.

Fig. 5.

Docking pose of C18:0-C18:1EPl (A, E, I), C18:0-C18:1CPl (B, F, J), C18:0-C20:4CPl (C, G, K), and C18:0-C22:6CPl (D, H, L) in the extracellular site (site 1) (A, B, C, D), the cytoplasmic site (site 2) (E, F, G, H), or the transmembrane site (site 3) (I, J, K, L) of three-dimensional (3D) structure of bovine GPR61 modeled using ColabFold. The conformations shown feature the smallest docking scores. Red, blue, and yellow surfaces represent regions of the binding site that are suitable for ligand hydrogen-bond acceptors, ligand hydrogen-bond donors, and hydrophobic groups, respectively. Red arrows represent the head group and purple and brown side-chains represent fatty alcohols bonded to a glycerol backbone at sn-1 position through a vinyl-ether bond and fatty acids bonded to sn-2 position through an ester bond, respectively. TM, transmembrane region.

Fig. 6.

Distributions of docking scores (A, B, and C) and the binding free energy based on the MM-GBSA method (D, E, and F), for C18:0-C18:1EPl, C18:0-C18:1CPl, C18:0-C20:4CPl, and C18:0-C22:6CPl across three binding sites: extracellular (site 1) (A, D), cytoplasmic (site 2) (B, E), and transmembrane (site 3) (C, F). The 3D structure of bovine GPR61 was modeled using ColabFold. All ligands used in docking were converted to three‑dimensional, ionized structures, and these prepared structures were consistently employed across all calculations. The docking score represents the estimated binding free energy for each molecular species, reflecting the free energy difference between the bound and unbound states. For each molecular species, 100 poses were generated during docking onto the sites. The bolded and italicized values represent the mean and population variance in the docking scores and the binding free energy across the 100 poses.