Reduced gonadotroph stimulation by ethanolamine plasmalogens in old bovine brains

Ethanolamine plasmalogens (EPls), unique alkenylacyl-glycerophospholipids, are the only known ligands of G-protein-coupled receptor 61—a novel receptor co-localised with gonadotropin-releasing hormone receptors on anterior pituitary gonadotrophs. Brain EPl decreases with age. Commercial EPl—extracted from the cattle brain (unidentified age)—can independently stimulate FSH secretion from gonadotrophs. We hypothesised that there exists an age-related difference in the quality, quantity, and ability of bovine brain EPls to stimulate bovine gonadotrophs. We compared the brains of young (about 26 month old heifers) and old (about 90 month old cows) Japanese Black bovines, including EPls obtained from both groups. Additionally, mRNA expressions of the EPl biosynthesis enzymes, glyceronephosphate O-acyltransferase, alkylglycerone phosphate synthase, and fatty acyl-CoA reductase 1 (FAR1) were evaluated in young and old hypothalami. The old-brain EPl did not stimulate FSH secretion from gonadotrophs, unlike the young-brain EPl. Molecular species of EPl were compared using two-dimensional liquid chromatography-mass spectrometry. We identified 20 EPl molecular species of which three and three exhibited lower (P < 0.05) and higher (P < 0.05) ratios, respectively, in old compared to young brains. In addition, quantitative reverse transcription-polymerase chain reaction detected higher FAR1 levels in the POA, but not in the ARC&ME tissues, of old cows than that of fertile young heifers. Therefore, old-brain EPl may be associated with age-related infertility.


Large-scale EPl extraction from whole brains for evaluation with cultured anterior pituitary cells
Five young or old whole brains were minced in a food processor (DLC-NXJ2PS, Conair Japan G. K., Tokyo, Japan), pooled, frozen at −80°C, and vacuum-dried (ADP200, Yamato Scientific Co. Ltd., Tokyo, Japan). Vacuum-dried brain tissue was extracted by incubation in ethanol (brain tissue/ethanol, 1:10, v/v) at 40 °C for 8 h with shaking.
After centrifugation at 10,800 × g for 1 hour at 25 °C, the supernatant was collected and dried using a rotary evaporator (N2110; Tokyo Rikakikai Co. LTD., Tokyo, Japan). The remaining lipids were collected in several 50-mL glass centrifuge tubes and dissolved in diluted acetone (acetone/water, 2:1, v/v; lipids/diluted acetone, 1:10, v/v). After storing at 4 °C for 1 hour, the solutions were centrifuged at 1,200 × g for 20 min at 4 °C, and the supernatant was subsequently removed. The remaining precipitates were dissolved in diluted acetone (acetone/water, 1:1 v/v; precipitate/diluted acetone, 1:10 v/v). The solutions were centrifuged at 1,200 × g for 20 min at 4 °C to collect the precipitates, which were subsequently mixed with cold acetone (precipitate/acetone, 1:10 v/v), and stored at -20 °C overnight. Thereafter, the acetone-treated precipitates were centrifuged at 1,200 × g for 20 min at 4 °C and the supernatant was removed. The remaining precipitates were dissolved in a hexane and acetone mixture (hexane/acetone, 7:3 v/v; precipitate/mixture, 1:10 v/v). This solution was subjected to centrifugation at 1,200 × g for 30 min at 4 °C, and the supernatant was collected into a flask and dried using a rotary evaporator. After evaporation, the remaining lipids were treated with 20 mg/mL phospholipase A1 dissolved in 0.1 M citric acid buffer (pH 4.5) at 50 °C, in a volume ratio of 1:10, under a low oxygen atmosphere (air pressure, 50 kPa), in a rotary evaporator flask for 2 h. Subsequently, the enzyme-treated sample was mixed with a 1:1 (v/v) mixture of hexane and acetone (sample/mixture, 1:6 v/v) and transferred to a separating funnel for collection of the upper layer. This extraction was repeated two more times. The upper layer was transferred to a flask for drying using a rotary evaporator. After evaporation, the residual lipids were dissolved in acetone (lipids/acetone, 1:10 v/v) in glass tubes, and stored at -20 °C overnight. Subsequently, the solutions were centrifuged at 1,200 × g for 20 min at 4 °C to collect the precipitates, which were washed with acetone (precipitate/acetone, 1:10 v/v), and recentrifuged at 1,200 × g for 20 min at 4 °C. The resulting precipitates were dissolved in a hexane/acetone mixture (hexane/acetone, 7:3 v/v; precipitate/mixture, 1:10 v/v). The solutions were centrifuged at 1,200 × g for 20 min at 4 °C and the supernatant was 5 collected for evaporation. The remaining lipids after evaporation were dissolved in a mixture of hexane, acetone, and water (hexane/acetone/water, 3:3:1 v/v; lipids/mixture, 1:10 v/v), and transferred to a separating funnel. After shaking the separating funnel, the upper layer was collected. This extraction was repeated two more times. The combined upper layer extracts were evaporated using a rotary evaporator to obtain EPl-rich lipids.
Aliquots of these lipids were vacuum-packed and stored at -30 °C prior to analysis.

Small-scale EPl extraction from the hypothalamus for 2D LC-MS analysis
All organic solvents were prepared to a 0.01% (w/v) concentration in 2,6-ditertbutyl-4-methylphenol. Briefly, frozen hypothalamus samples were thawed and homogenised in twice the volume of methanol, and stored overnight at -20 °C. The homogenate was subsequently vortexed for re-suspension. Next, 1 mL of the homogenate was transferred to a 10-mL glass centrifuge tube, mixed with 2 mL of methanol and 6 mL of chloroform, and vortexed at room temperature for 10 min. It was subsequently centrifuged at 1,200 × g for 15 min at 25 °C. The upper layer was collected in a glass tube for heat drying (45 ℃) under a gentle stream of N2 gas. This extraction was repeated thrice for each sample. After drying, 3 mL of chloroform/methanol (2:1, v/v) was added into the tube to dissolve the residue. This mixture was centrifuged at 1,200 × g for 15 min at 25 °C, and the supernatant was collected into another 10 mL glass centrifuge tube for heat drying (45 ℃) under a N2 gas stream. This extraction was repeated two more times for each sample. After drying, 1.5 mL of 20 mg/mL phospholipase A1 (dissolved in 0.1 M citric acid buffer, pH 4.5) was added to the tube, which was filled with N2 gas before capping, and the sample was incubated at 45 ℃ for 2 h. When the sample became milky, 10 mL of acetone/hexane (2:1 v/v) was added. The sample was vortexed and centrifuged at 1,200 × g for 15 min 7 at 25 °C to collect the upper layer into another glass centrifuge tube for heat drying (45 ℃) under a N2 gas stream. This extraction was repeated two more times for each sample. After drying, 10 mL of cold acetone was added, and the sample was vortexed and incubated overnight at -20 °C. Thereafter, the sample was centrifuged at 1,200 × g for 15 min at 25 °C to remove the supernatant. This washing step was repeated two more times. After the supernatant was removed, the remaining precipitate was dissolved in 10 mL of hexane/acetone (7:3 v/v), and centrifuged at 1,200 × g for 15 min at 25 °C to collect the supernatant into a glass tube for heat drying (45 ℃) under a N2 gas stream.
After drying, 6 mL of a hexane/acetone mixture (7:3 v/v) was added, the sample was vortexed, and 0.9 mL of water was added. After shaking, the sample was centrifuged at 1,200 × g for 15 min at 25 °C to collect the upper layer into a glass tube of predetermined weight. This extraction was repeated two more times. After heat drying (45 ℃) under a N2 gas stream, the tube was weighed to calculate the weight of the obtained lipids. The tube was filled with N2 gas, capped, vacuum-packed, and transported on dry ice to the 2D LC-MS system.

2D LC-MS analysis
This system, as previously reported 6 , consists of: (1) normal-phase HPLC, to separate targeted phospholipids (phosphatidylethanolamines, in this study) from other lipid classes, in the first column (first-dimensional HPLC separation); (2) a switching valve, trapping column, and make-up pump, to trap the target lipid class; (3) reversephase HPLC to separate the target lipid classes, in the second column (seconddimensional HPLC separation); (4) a charged aerosol detector and electrospray ionization-mass spectrometer to identify and quantify EPl molecular species. The components of the 2D LC-MS system, such as the columns, pumps, autosampler, detectors, electrospray ionization-mass spectrometer, and the software, were the same as those previously reported 6 . First-dimensional separation was performed using a YMC-Pack PVA-Sil [250 mm length (L) × 4.6 mm internal diameter (I.D.), 5-μm column; YMC Co. Ltd., Kyoto, Japan]. The HPLC separation temperature and flow rate were set to 30 °C and 1.0 mL/min, respectively. The lipid sample was prepared at a concentration of 5 mg/mL in chloroform/methanol (2:1 v/v), and a 0.02-mL aliquot was injected into the 2D-HPLC system. Τhe mobile phases, A, B, and C, were hexane, 2methoxy-2-methylpropane, and methanol, respectively. The solvent gradient program was as follows: 0-7 min A/B/C (v/v/v%) 88/10/2; 7-12 min A/B/C (v/v/v%) 2/88/10; The mobile phase was acetonitrile/methanol/20 mM ammonium acetate (25/68.5/6.5, v/v/v%) at a flow rate of 1 mL/min. The eluent from the second column was split into a charged aerosol detector and electrospray ionization-mass spectrometer. The acquisition range and N2 gas pressure of the charged aerosol detector were 500 pA and 241.3 kPa, respectively. An electrospray ionization-mass spectrometer was used to identify the species (positive ion mode; N2 sheath gas flow rate: 5 units; capillary temperature: Each sample was analysed in duplicate, and each relative standard deviation of the retention time and peak area was less than 0.05% and 0.93%, respectively.