Endocannabinoids and ReproductionView this Special Issue
Research Article | Open Access
Brain Levels of Prostaglandins, Endocannabinoids, and Related Lipids Are Affected by Mating Strategies
Background. Endogenous cannabinoids (eCBs) are involved in the development and regulation of reproductive behaviors. Likewise, prostaglandins (PGs) drive sexual differentiation and initiation of ovulation. Here, we use lipidomics strategies to test the hypotheses that mating immediately activates the biosynthesis and/or metabolism of eCBs and PGs and that specific mating strategies differentially regulate these lipids in the brain. Methods. Lipid extractions and tandem mass spectrometric analysis were performed on brains from proestrous rats that had experienced one of two mating strategies (paced or standard mating) and two nonmated groups (chamber exposed and home cage controls). Levels of PGs (PGE2 and PGF2alpha), eCBs (AEA and 2-AG, N-arachidonoyl glycine), and 4 related lipids (4 N-acylethanolamides) were measured in olfactory bulb, hypothalamus, hippocampus, thalamus, striatum, midbrain, cerebellum, and brainstem. Results. Overall, levels of these lipids were significantly lower among paced compared to standard mated rats with the most dramatic decreases observed in brainstem, hippocampus, midbrain, and striatum. However, chamber exposed rats had significantly higher levels of these lipids compared to home cage controls and paced mated wherein the hippocampus showed the largest increases. Conclusions. These data demonstrate that mating strategies and exposure to mating arenas influence lipid signaling in the brain.
Decades of studies on mating behavior in laboratory rats (typically Rattus norvegicus) provide a wealth of knowledge about developmental and motivational roles of various neurotransmitter systems in sexual differentiation and/or manifestation of reproductive behaviors . Standard mating procedures for rats in laboratory environments typically involve placing a sexually experienced male rat in a testing chamber (these vary in size but are usually around 60 × 50 × 40 cm aquaria) with a female in behavioral estrus (the time of ovulation and sexual receptivity in female rats). In this situation, typically rats mate with the timing of sexual contacts being driven by the male until he ejaculates. When mating chambers are large enough, males often participate in a common preintercourse sequence of sniffing, hopping, and then mounting . In this context, these behaviors are both initiated and regulated by the male in that the male is able to “pace” his interactions with the female, which may engender intrinsic reward, associating the females as a conditioned incentive . Although this standard procedure for mating is rewarding for males, female rats that cannot pace their sexual interactions typically do not develop a conditioned place preference as opposed to their female counterparts that “pace” their sexual contacts in a mating strategy called paced mating [2, 3]. In the laboratory, “paced mating boxes” are larger mating arenas, which have a divider with a small hole. This apparatus allows females to engage and withdraw from males and have some control over the receipt of copulatory stimuli. This paradigm has been used to ascertain neurophysiological mechanisms associated with paced mating and/or standard mating (partition is removed).
Endocannabinoids are endogenous lipid neurotransmitters that activate cannabinoid receptors and play a role in regulating motivated behaviors, such as feeding, anxiety, drug seeking, pain, and reproduction [4, 5]. The most studied of the endogenous ligands are N-arachidonoyl ethanolamine (anandamide; AEA), 2-arachidonoyl glycerol (2-AG) , and more recently the endogenous metabolite of AEA N-arachidonoyl glycine (NAGly) was shown to activate the GPR18, which is a putative cannabinoid receptor [7–9]. Cannabinoid agonist, WIN 55,212-2 (WIN), administered to male rats reduced intromission frequency and increased intervals between ejaculations . Injecting proestrus, but not hormone primed, rats with a CB1 (cannabinoid receptor 1) antagonist/inverse agonist and GPR18 antagonist, AM251 facilitated sexual motivation . Levels of endocannabinoid ligands (AEA, NAGly, and 2-AG) change significantly in rodent brain with the estrous cycle and show sex differences, suggesting a preparatory role for mating . Indeed, progesterone can also upregulate CB1 receptor activity in the hypothalamus . Together these findings suggest a mutual regulation between the endocrine system and endocannabinoid system which may play a role in the neuronal control of mating and its rewarding properties.
A structurally similar lipid signaling system to the endocannabinoids and the prostaglandins, specifically PGE2, may act in the hypothalamus by inhibiting release of a prolactin-secretion-inhibiting factor during mating, which could contribute to induction of prolactin surges . PGE2 can facilitate lordosis in response to mounting among estrogen primed, ovariectomized, and adrenalectomized rats . In paced mating paradigms, the interval between sexual contacts is directly related to how much stimulation females receive and increases with each encounter . Among female guinea pigs (also spontaneous ovulators), prostaglandin release in response to mating may disrupt hypothalamus stimulatory norepinephrine signaling, which leads to the postmating inhibition of sexual behavior . Prostaglandins are key components in the mechanism leading up to the follicular rupture involved in ovulation at the site of the ovary . Their influence is demonstrated at the level of the hypothalamus and pituitary by releasing luteinizing hormone (LH), a gonadotropin essential for the onset of ovulation [15, 16].
Lipidomics techniques, in which lipid extracts from tissues are analyzed using tandem mass spectrometry, allow us to measure multiple different lipids from the same tissue and determine relative amounts of lipid between brain areas and treatment groups. Here, we test the hypothesis that production of the prostaglandins PGE2 and PGF2α, as well as the endocannabinoid ligands AEA, 2-AG, and NAGly, and structurally related lipids and signaling molecules N-palmitoyl ethanolamine (PEA), N-oleoyl ethanolamine (OEA), N-docosahexaenoyl ethanolamine (DHEA), and N-stearoyl ethanolamine (SEA) are differentially regulated acutely by mating strategies in the female rodent brain. Brains from female rats that were either paced or standard mated and two control groups (chamber exposed and home cage control) were analyzed using high-performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS) for production levels of the lipids listed above in eight different brain regions (olfactory bulb, hypothalamus, hippocampus, thalamus, striatum, midbrain, cerebellum, and brainstem). Overall, levels of these lipids were significantly lower among paced compared to standard mated rats in the majority of brain areas with the most dramatic decreases observed in brainstem, hippocampus, midbrain, and striatum. However, chamber exposed rats had significantly higher levels of these lipids than did home cage controls wherein the hippocampus showed the largest increases. These data demonstrate that mating strategies and exposure to mating arenas influence lipid signaling in the brain and imply that eCBs, N-acylethanolamines, and PGs are involved in driving the neurophysiological outcomes of mating behaviors.
Arachidonoyl ethanolamide-d4 (d4-AEA) was purchased from Tocris Bioscience (St. Louis, MO). AEA, PEA, SEA, OEA, DHEA, and 2-AG were purchased from Cayman Chemical (Ann Arbor, MI). NAGly was purchased from Biomol (Plymouth Meeting, PA). HPLC-grade water and methanol were purchased from VWR International (Plainview, NY). HPLC-grade acetic acid and ammonium acetate were purchased from Sigma-Aldrich (St. Louis, MO).
2.1.1. Animals—Animal Subjects Used in This Experiment Were Housed at the State University of New York (SUNY) Albany
Age-matched and littermate female Long-Evans rats (4–6 months old) in behavioral estrus ( per group) and sexually experienced male rats were maintained on a 12 : 12 h reversed dark-light cycle (08:00 dark and 18:00 light). Food and water were available ad libitum. Vaginal cytology of rats was obtained daily to assess phase of the estrous cycle. On the day of testing, all females (even control groups) with a proestrous smear were vaginally masked and were behaviorally assessed with a male to make sure they were sexually receptive, which was determined by responding to one male mount with lordosis. Only sexually receptive females were used as test subjects. They were then returned to their home cages for a minimum of 2.5 hours before testing.
Experimental Conditions. This protocol was according to Erskine, 1985 . It was performed at the SUNY Albany in the Laboratory of Cheryl Frye.
Animals were tested during the dark phase of the cycle between the hours of 08:00 and 16:00. The animals were transported from the animal housing room to the testing area in their home cages, where they were placed outside the testing room on a rack until testing began. Experimental subjects had vaginal masks affixed to the perineum to minimize mating-induced changes. Behavioral analyses and manipulations were taken by an observer who was unaware of the hypotheses and experimental conditions.
2.2. Paced Mating
Testing was conducted in a white melamine chamber (37.5 × 75 × 30 cm) that was divided into two compartments via a Plexiglas divider that had been cleaned with quatricide and allowed to dry. This apparatus was also constructed and tested to confirm it was functioning properly before testing began. The Plexiglas divider had a small (5 cm) hole in the bottom center that was large enough for a female to pass through but not large enough for a male. The males had also been previously conditioned to stay away from the hole. This allowed the females to self-administer or “pace” their mating by controlling the frequency of mating contacts and the amount of time between mounts, intromissions, and ejaculation. The males were habituated to the testing chamber first, followed by the female in the opposite side of the chamber. Their sexual interaction was observed for 15 minutes or until the first ejaculatory emission was reached. Lordosis quotients, aggression quotients, proceptivity, and percent exits were measured. Once the fifteen minutes or the first ejaculatory emission was reached, the female was immediately removed from the chamber and decapitated. The brain was then removed and immediately frozen on dry ice. The chamber was then cleaned with quatricide and allowed to dry before the next trial was done.
2.3. Standard Mating
The same 37.5 × 75 × 30 cm melamine chamber that was used for paced mating was also used for this test group. The Plexiglas divider was removed for this experiment. This allowed the male, instead of the female, to control how often mating was administered. The females were vaginally masked to prevent pregnancy and other mating-induced changes.
2.4. Chamber Exposed
A female was placed in the 37.5 × 75 × 30 cm melamine chamber with the Plexiglas divider (same as paced mating chamber design) inserted, for 15 minutes. She was then immediately removed from the chamber and decapitated. The brain was removed and immediately flash-frozen on dry ice. The chamber was then cleaned with quatricide and allowed to dry before the next trial was done.
2.5. Home Cage Control
Females were taken from their housing chambers and decapitated. The brains were removed and immediately flash-frozen on dry ice. They had no social exposure (to males) and no chamber exposure.
2.6. Tissue Dissection
After all the tissues were collected, the tissue was sent overnight on dry ice from Albany, NY, to Bloomington, IN, where it was stored in a −80°C freezer until brains were dissected and processed. Tissue dissection and storage were performed as previously described by Bradshaw et al., 2006 . In brief, the frozen brains were thawed for approximately 5 minutes on an ice cold tin foil covered dissection plate. Once thawed, brains were dissected into the following regions: olfactory bulb, hypothalamus, striatum, thalamus, hippocampus, midbrain, brainstem, and cerebellum. Each region was then placed in a 1.5 mL microfuge tube and flash-frozen with liquid nitrogen. They were stored in the −80°C freezer until used for lipid extractions.
2.7. Lipid Extraction
Each brain area was processed separately and all tissues from a specific brain area were processed together, although the order of processing was randomized, as previously described . The samples were removed from the −80°C freezer. After being shocked with liquid nitrogen, they were weighed and placed in centrifuge tubes on ice. Furthermore, 40 : 1 volumes of methanol were added to each tube followed by 10 μL of 1 uM d4-AEA. d4-AEA was added to act as an internal standard to determine the recovery of the compounds of interest. The tubes were then covered with parafilm and left on ice and in darkness for approximately 2 hours. Remaining on ice, the samples were then homogenized using a polytron for approximately 1 minute on each sample. The samples were then centrifuged at 19,000 ×g at 24°C for 20 minutes. The supernatants were then collected and placed in polypropylene tubes (15 or 50 mL), and HPLC-grade water was added making the final supernatant/water solution 25% organic. To isolate the compounds of interest, partial purification of the 25% solution was performed on a Preppy apparatus (Sigma-Aldrich) assembled with 500 mg C18 solid-phase extraction columns (Agilent Technologies, Santa Clara, CA). The columns were conditioned with 5 mL of HPLC-grade methanol immediately followed by 2.5 mL of HPLC-grade water. The supernatant/water solution was then loaded onto the C18 column and then washed with 2.5 mL of HPLC-grade water followed by 1.5 mL of 40% methanol. The prostaglandins were then collected with a 1.5 mL elution of 70% methanol, NAGly with a 1.5 mL elution of 85% methanol, and the ethanolamides with a 1.5 mL elution of 100% methanol. All were collected in individual autosampler vials and then stored in a −20°C freezer until mass spectrometer analysis.
2.8. LC/MS/MS Analysis and Quantification
Samples were removed from the −20°C freezer and allowed to warm to room temperature and then vortexed for approximately 1 minute before being placed into the autosampler and held at 24°C (Agilent 1100 series autosampler, Palo Alto, CA) for LC/MS/MS analysis. Also 10–20 μL of eluants was injected separately for each sample to be rapidly separated using a C18 Zorbax reversed-phase analytical column (Agilent Technologies, Santa Clara, CA) to scan for individual compounds (mobile phase A: 20% HPLC methanol, 80% HPLC water, and 1 mM ammonium acetate; mobile phase B: 100% HPLC methanol and 1 mM ammonium acetate). Gradient elution (200 μL/min) then occurred under the pressure created by two Shimadzu 10AdVP pumps (Columbia, MD). Next, electrospray ionization was accomplished using an Applied Biosystems/MDS Sciex (Foster City, CA) API3000 triple quadrupole mass spectrometer. A multiple reaction monitoring (MRM) setting on the LC/MS/MS was then used to analyze levels of each compound present in the sample injection. Synthetic standards were used to generate optimized MRM methods and standard curves for analysis. Figure 1 shows a flowchart of the extraction process starting after the animals had been mated.
2.9. Data Analyses
The amount of analyte in each sample was calculated by using a combination of calibration curves of the synthetic standards and deuterium-labeled internal standards obtained from the Analyst software. The standards provided a reference for the retention times by which the analytes could be compared. They also helped to identify the specific precursor ion and fragment ion for each analyte which enabled their isolation. These processes provide confidence in the claim that the compounds measured were, in fact, the compounds of interest. The amount of each compound in each tissue was then converted to moles per gram tissue, which is how it was statistically analyzed.
The current study had 4 treatment groups (/grp) and profiled 9 lipids in 8 different brain regions generating over 1700 data points. In an effort to consider the relatedness between analytes in each brain region, general linear models (GLM) were used to consider the experimental conditions between subjects variables and analytes as nested variables across brain regions. Table 1 summarizes the values from the GLM analysis for each brain region. Using this analysis, it was shown that there were significant interactions between analyte and treatment group in the brainstem (BS), hippocampus (HIPP), midbrain (MB), and striatum (STR). Therefore, post hoc analyses of each individual group to each other were performed using ANOVA, described below. Using this criterion, additional post hoc analyses were not performed on the remaining four brain regions analyzed. Those values are presented in Table 2.
|. Significant differences are shown in bold black.|
Data from the BS, HIPP, MB, and STR were subsequently analyzed for each brain using the nontested group as the control value compared to the chamber exposed, standard mated, and paced mated group. Follow-up analyses considered the chamber exposed group as the experimental control compared to standard and paced mated groups. Finally, the standard mated group data was compared to the paced mated group. Each comparison was a one-way ANOVA with post hoc Fisher’s LSD with a 95% confidence interval for the mean using SPSS software. Data in Tables 3–6 are presented as means ± SE of the means, where was considered statistically significant.
|(a) Comparisons of home cage to each group|
|(b) Comparisons of chamber exposed to standard or paced mating|
|(c) Comparisons of standard mating to paced mating|