Choline

Antibiotics were purchased from Carl Roth (Karlsruhe, Germany), United States Biochemical Corp. (Cleveland, OH, United States), Sigma-Aldrich (Steinheim, Germany), and InvivoGen (San Diego, CA, United States). Choline, glycine betaine and the chromogenic substrate for the TreA enzyme assays, para-nitrophenyl-α-D-glucopyranoside (α-PNPG), were purchased from Sigma-Aldrich (Steinheim, Germany). Anhydrotetracycline-hydrochloride (AHT), Strep-Tactin Superflow chromatography material, and desthiobiotin were obtained from IBA GmbH (Göttingen, Germany). Marker proteins to standardize size-exclusion chromatography columns were purchased from GE Healthcare (München, Germany) and from Sigma-Aldrich (Steinheim, Germany), respectively.

Calcium lactate and inulin were purchased (Agnex, Białystok, Poland), as were pumpkins sourced from domestic organic farming. Experimental research and field studies on plants, including the collection of plant material, complied with relevant national, institutional, and international guidelines and legislation. Permission to collect the plant material (pumpkin) was obtained from the landowner. The ingredients of the animal feed—vitamins, minerals, l-cysteine, and choline—were purchased from Sigma-Aldrich (Darmstadt, Germany), while casein, corn starch, dextrin, cellulose, sucrose, and rapeseed oil were purchased from Hortimex (Konin, Poland). ELISA (enzyme-linked immunosorbent assay) kits were purchased from Mediagnost (Reutlingen, Germany).

The following chemicals were purchased from Sigma-Aldrich (St. Louis, MO): methanol and acetonitrile (MS grade), ammonium acetate, ascorbic acid, β-mercaptoethanol, formic acid and standards, 5-adenosyl-methionine, 5-adenosyl-homocysteine, ATP, betaine, choline, cyanocobalamin, cystathionine, cysteine, dihydrofolate, dimethylglycine, dUMP, folic acid, folinic acid, glycine, homocysteine, methionine, methylcobalamine, methyl-tetrahydrofolate, NADPH, pyridoxal 5-phosphate, riboflavin, serine, taurine, tetrahydrofolate, thymidine 5-phosphate and Dulbecco's modified Eagle's medium mixed 1:1 with Ham's F-12 (DMEM/F12). Ultrapure type 1 water was obtained from a Milli Q water system (Merck Millipore, Darmstadt, Germany). Matrigel (growth factor-reduced) was purchased from BD Biosciences (San Jose, CA). Recombinant basic fibroblast growth factor (bFGF) was purchased from Mylteni (San Diego, USA).

Oocytes were secured in a Perspex chamber which was perfused continuously with SOS at a constant flow rate (4 ml min⁻¹) via a gravity-fed system (Buckingham et al., 1994 (link)). Atropine (0.5 μM) was included in the saline to suppress responses from endogenous muscarinic AChRs (Lupu-Meiri et al., 1990; Blake et al., 1993 ). Membrane currents were measured using the two-electrode voltage clamp method (as described in Buckingham et al., 2006 (link)) where the oocyte membrane was clamped at −100 mV. Nicotine, ACh, choline, imidacloprid and spinosad (all Sigma–Aldrich) were diluted with SOS containing 0.5 μM atropine immediately prior to experimentation. Oocytes were challenged for 5 s with increasing concentrations of ACh at intervals of 3–5 min to minimise the effects of desensitisation. The maximum amplitude of the current recorded for each challenge was normalised to the response to 300 μM ACh. Using GraphPad Prism version 4.0 (GraphPad Software Inc. USA), normalised data were fitted to the following equation: Y = I_min + (I_max − I_min)/1 + 10(^logEC₅₀^−X)ⁿ_H where Y is the normalised response amplitude to a compound applied at concentration X, I_max and I_min are the maximum and minimum normalised responses respectively. EC₅₀ is the concentration giving half the maximum normalised response and n_H is the Hill co-efficient (Hosie and Sattelle, 1996 (link)).

Methanol (HPLC grade) and acetonitrile (HPLC grade) were purchased from Merck (Darmstadt, Germany). Formic acid (HPLC grade), 2-chloro-L-phenylalanine (as an internal standard), xanthine, L-phenylalanine, D9-phenylalanine, choline, hypoxanthine, L-tryptophan, succinic acid, and L-tyrosine standards were purchased from Sigma (Saint Louis, MO, USA). A Milli-Q system (Millipore, Bedford, MA, USA) was applied to produce ultrapure water.
The animal treatment used corn oil (GLBIO, Montclair, CA, USA) and testosterone (GLBIO, Montclair, CA, USA). An 80% paraformaldehyde stationary liquid (Labgic Technology, Beijing, China) was applied for sample preservation.
For the decalcification of bone tissue, EDTA-2Na and sodium hydroxide were purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China., We applied the same pretreatment process for Hematoxylin-eosin (HE) and tartrate-resistant acid phosphatase (TRAP) staining. Anhydrous ethanol was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). An environmentally friendly dewaxing agent (alkane mixture), a transparent agent (alkane mixture, xylene-free), and other dye tools were purchased from Tonsen tech, Wuhan, China. For further staining, the HE dye kit was purchased from Biossci (Beijing, China), model BP092. The TRAP dye kit was purchased from Biossci (Beijing, China), model BP088.