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Relationship between seminal plasma levels of anandamide
Jun 20, 2018

Relationship between seminal plasma levels of anandamide congeners palmitoylethanolamide and oleoylethanolamide and semen quality 

Akwasi Atakora Amoako, B.Sc., M.B., Ch.B., M.R.C.O.G., Ph.D.,a Timothy Hywel Marczylo, Ph.D.,b Janine Elson, M.D., F.R.C.O.G.,c Anthony Henry Taylor, Ph.D.,a Jonathon M. Willets, Ph.D.,a and Justin Chi Konje, M.D., F.R.C.O.G.a 

a Endocannabinoid Research Group, Reproductive Science Section, Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester; b Centre for Radiation, Chemical and Environmental Hazards, Health Protection Agency, Didcot, Oxfordshire; and c London Women's Clinic, London, United Kingdom

Objective: To determine whether changes in seminal plasma concentrations of the endogenous lipid signaling molecules palmitoyle-thanolamide (PEA) and oleoylethanolamide (OEA) have significant effects on sperm quality.

Design: Biochemical and physiological studies of human seminal plasma and spermatozoa.

Setting: Academic tertiary care medical center.

Patient(s): Ninety men attending an infertility clinic for semen analysis.

Intervention(s): Palmitoylethanolamide and OEA extracted from seminal plasma were quantified by ultra high-performance liquid chromatography (HPLC)-tandem mass spectrometry. Patient sperm from semen with normal parameters were exposed in vitro to PEA or OEA to determine effects on sperm motility, viability, and mitochondrial activity. 

Main Outcome Measure(s): The relationship between seminal plasma concentrations of PEA and OEA and sperm quality and the effect of these compounds on sperm motility, viability, and mitochondria activity in vitro. 

Result(s): Palmitoylethanolamide and OEA concentrations in seminal plasma were lower in men with asthenozoospermia and oligoas-thenoteratozospermia compared with men with normal semen parameters. Palmitoylethanolamide and OEA rapidly and significantly improved sperm motility and maintained viability without affecting mitochondria activity in vitro. 

Conclusion(s): Maintenance of normal PEA and OEA tone in human seminal plasma may be necessary for the preservation of normal sperm function and male fertility. Exocannabinoids found in Cannabis, such as delta-9-tetrahydrocannabinol and 

cannabidiol, could compete with these endocannabinoids upsetting their finely balanced, normal functioning and resulting in male reproductive failure. (Fertil Steril 2014;102:1260–7. 2014 by American Society for

Reproductive Medicine.)

Key Words: Palmitoylethanolamide, oleoylethanolamide, anandamide, endocannabinoid system, seminal plasma

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Received January 20, 2014; revised and accepted July 9, 2014; published online September 8, 2014. 

A.A.A. has nothing to disclose. T.H.M. has nothing to disclose. J.E. has nothing to disclose. A.H.T. has nothing to disclose. J.M.W. has nothing to disclose. J.C.K. has nothing to disclose. 

Current address for A.A.A.: Leeds Centre for Reproductive Medicine, Leeds Teaching Hospitals NHS Trust, Seacroft Hospital, Leeds LS14 6UH, United Kingdom. 

Supported in part by miscellaneous educational funds from the University Hospitals of Leicester Na-tional Health Services Trust to support the Endocannabinoid Research Laboratory of University of Leicester. 

Reprint requests: Akwasi Atakora Amoako, B.Sc., M.B., Ch.B., M.R.C.O.G., Ph.D., and Justin Chi Konje, M.D., F.R.C.O.G., Endocannabinoid Research Group, Reproductive Science Section, Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester, United Kingdom (E-mail: akwasi.amoako@leedsth.nhs.uk).

Fertility and Sterility® Vol. 102, No. 5, November 2014 0015-0282/$36.00 

Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2014.07.767

Infertility   affects   one   in   six (15%–20%)   couples   trying   to achieving pregnancy (1–4). Female factors are responsible for half of cases of  infertility,  whereas  male  factors pure  or  in  association  with  female factors account for the remaining 50% (3,  5).  Male  factor  infertility  is  a complex and growing problem (6) with multifactorial  causes  and  usually present as qualitative or quantitative

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sperm defects in the form of either deficiency of sperm production, transportation, and/or morphology (7). Semen analysis remains the standard method of evaluation of male factor infertility but fails to elucidate the causes of sperm dysfunction. Recent studies have focused on a number of molecules with predictive significance of male reproductive potential. Among these are a group of lipid mediators, the endocannabinoids that are emerging as potential biomarkers of male reproductive health (8).

Endocannabinoid are endogenous bioactive lipid media- 

tors that bind to cannabinoid receptors and mimic the adverse reproductive effects of Cannabis. The endocannabinoids, their

molecular targets, synthetic and degradation enzymes, and protein transporters constitute the endocannabinoid system. The cannabinoid receptors were first identified as molecular targets for D9-tetrahydrocannabinol, the active principle of marijuana and later shown to be two well-characterized G-protein–coupled cannabinoid receptors, CB1 and CB2 (9, 10), which also bind endocannabinoids. 

N-arachidonoylethanolamide (Anandamide, AEA) is the most extensively studied member of this group of ligands and is widely distributed in most central and peripheral tissues including the reproductive system (11–13). The AEA and its congeners oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), commonly referred to as N-acylethanolamides, are hydrophobic molecules present at the low nanomolar range in many mammalian tissues and cells and produced from cell membrane phospholipid precursors in response to depolarizing agents, neurotr ansmitters, and hormones (14, 15). They seem to always occur together in tissues and biofluids, suggesting a common production pathway. 

Oleoylethanolamide and PEA do not activate the classic cannabinoid receptors CB1 and CB2 and their exact biolog-ical roles remain elusive. They are purported to act as ‘‘entourage compounds,’’ whereby they enhance the biolog-ical activity of AEA by inhibiting its degradation by the enzyme fatty acid amide hydrolase, acting as alternative substrates. Concentrations of AEA, PEA, and OEA have been detected at nanomolar levels in human reproductive fluids and tissues, such as midcycle oviductal fluid, follicular fluid (FF), and seminal plasma (16–18), and it has been shown that spermatozoa are sequentially exposed to declining levels of AEA as they swim from the ejaculate deposited in the vagina to the fertilization site in the oviductal ampulla (18). 

Several in vitro studies have shown a dose-dependent inhibition of mammalian sperm functions by AEA mediated by CB1 receptor activation (19–21). In mature human spermatozoa AEA reduces sperm motility, capacitation, and acrosomal exocytosis (21). Similarly AEA exerts an inhibitory role on sperm functions in several species (18, 19, 21, 22) through inhibition of mitochondrial activity that preserves energy and ensures gradual acquisition of sperm fertilizing capacity during ascent through the female reproductive tract (23). Similarly, D9-tetrahydrocannabinol interferes with downstream endogenous signaling pathways, modulating sperm functions, and male reproduction in invertebrates and mammals (18, 19, 21, 24).

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It is thought that a critical ‘‘endocannabinoid tone’’ is required for the maintenance of mammalian spermatozoa in the normal state and loss of these endocannabinoid protective functions results in sperm dysfunction and loss of fertilizing potential. Support for this comes from the observations that down-regulation of AEA tone and CB1 expression in seminal plasma and spermatozoa, respectively, occurs in men with abnormal semen parameters, indicating that aberrant endocannabinoid signaling in human sperma-tozoa plays a role relevant in the etiopathogenesis of human sperm dysfunction (25, 26). 

Palmitoylethanolamide and OEA concentrations in most human reproductive tracts and fluids are higher than those of AEA, with emerging evidence suggesting that PEA and OEA possess antioxidant, anti-inflammatory, and antimicro-bial activities that could protect sperm cells from oxidative damage, inflammation, and microbial activity. For example, PEA and OEA supplementation enhances sperm antioxidant activity, improves sperm kinematic parameters and hyperac-tivation, and protects cells from oxidative damage in some cases of idiopathic infertility (27–29). 

We proposed that seminal plasma levels of PEA and OEA may reflect sperm quality. The aims of this study were there-fore to explore possible relationships between PEA and OEA seminal plasma concentrations and semen quality and to determine the effects of these compounds on human sperma-tozoa in vitro. 

MATERIALS AND METHODS 

Chemicals 

Dimethyl sulfoxide (DMSO) and formic acid were from Sigma Aldrich. Palmitoylethanolamide, OEA, and their deuterated equivalents (PEA-d4 and OEA-d2), each of >98% purity (and >99% deuterated content), and 5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimidazolyl carbocyanine iodide (JC-1) were purchased from Cayman Chemicals. The high-performance liquid chromatography (HPLC) grade acetonitrile, chloroform, methanol, and ammonium acetate were purchased from Fisher Scientific and HPLC grade water was obtained us-ing a water purification system (Maxima ELGA, ELGA). Mobile phases were filtered through 0.2-mm, 47-mm diameter polyte-trafluoroethylene filters (Waters UK Ltd.) before use. Oasis hydrophilic-Lipophilic-Balanced (HLB) solid phase extraction cartridges (1 mL, 30 mg) were purchased from Waters. Live/ dead sperm viability kit was obtained from Invitrogen. 

Study Design 

This prospective study was approved and conducted accord-ing to the guidelines of the Leicestershire and Rutland local research ethics committee and all participants signed informed consent to take part. Semen was obtained from men who attended the Andrology Unit of the Leicester Royal Infirmary, an affiliated hospital of University of Leicester School of Medicine for routine semen analysis. Semen were collected from 90 consecutive patients by masturbation into a sterile plastic container after 2–5 days of sexual abstinence. Samples were allowed to liquefy at room temperature for

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1 hour. From an aliquot of the semen, standard seminal parameters were examined by a trained laboratory technician with due consideration of the department's internal quality control measure and in accordance with the World Health Organization criteria (30). World Health Organization refer-ence normal values were semen volume >2 mL; sperm con-centration >20 106/mL; sperm number per ejaculum of >40 106; and sperm motility of R50% with forward pro-gression (grades a and b) or R25% with progressive motility (grade a). Patients with previous or current use of recreational drugs, current use of any medication (except overcounter medication), presence of any systemic disease, or a history of vasectomy were excluded. The 90 participants were then characterized and placed into one of five groups as normo-zoospermia (n ¼ 45), asthenozoospermia (n ¼ 11), oligoasthe-noteratozoospermia (n ¼ 22), teratozoospermia (n ¼ 8), or azoospermia (n ¼ 4). 

Measurement of PEA and OEA in Human Seminal Plasma 

Semen was transported to the analytical laboratory on ice and processed within 2 hours of production. The fluid was transferred into a clean 7-mL Kimble scintillation vial (Kinesis) and centrifuged at 1,200 g for 30 minutes at 4 C to separate seminal plasma from spermatozoa and other contaminating cells. The supernatant was then transferred into a clean 7-mL Kimble scintillation vial and lipid extrac-tion performed immediately, as previously described (31, 32). A previously validated ultra HPLC-tandem electrospray ioni-zation mass spectrometry method (31, 32) was used for the analysis and quantification of PEA and OEA in the 90 semen samples. 

Sperm Preparation for In Vitro Experiments 

In vitro experiments were performed in modified Biggers, Whitten Whittingham (BWW) medium (5.6 mM D-glucose, 44 mM sodium lactate, 0.27 mM sodium pyruvate, 95 mM NaCl, 4.6 mM KCl, 1.7 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5 U/mL penicillin, 5 mg/mL streptomycin, buffered with 20 mM N-2-hydroxyethylpiperazine-N0-2-ethanesulfonic acid [HEPES], and supplemented with 0.3% bovine serum albumin [BSA], pH adjusted to 7.4). For these experiments, only semen samples donated by healthy volun-teers with normal World Health Organization parameters were used. After semen liquefaction for approximately 30 mi-nutes, motile spermatozoa were selected through the direct swim-up technique in BWW medium. Briefly, 1 mL of BWW buffered with HEPES (20 mM) and pH adjusted to 7.4 was underlayered with 0.3 mL of the liquefied sample in a polystyrene Falcon round-bottom tube (Becton Dickinson) and incubated for 1 hour at 37 C, 5% CO2, and at an angle of 45 . After incubation, 700 mL was carefully aspirated from the top layer of each tube, containing the motile cells, pooled into a 15-mL polystyrene Falcon tube, washed twice (700 g for 7 minutes), and resuspended in BWW medium (33). Sperm concentration was determined using a Neubauer counting chamber, in accordance with the World Health Or-

ganization methods (30) and adjusted to 10 million cells/mL with BWW medium. Stock solutions of PEA and OEA (1 mM in DMSO) stored at 20 C were diluted to the required concentration immediately before each experiment in 1 mL of BWW culture medium, such that the final DMSO concentra-tion was 0.2% (vol/vol), with the same volume of DMSO in BWW medium used in all control experiments (preliminary experiments showed that 0.2% DMSO had no significant ef-fects on sperm motility). 

Effect of PEA and OEA on Sperm Motility and Viability 

Aliquots of suspended sperm (10 mL) were taken 15, 30, 45, 60, and 90 minutes after the addition of PEA or OEA to examine the time-dependent effect of these on sperm motility. Dose-response curves were constructed by incu-bating aliquots of sperm suspension (10 106/mL) without (control) or with increasing concentrations of PEA or OEA (1 nM–10 mM) for 30 minutes. Sperm motility was assessed by placing 10 mL of sperm suspension onto a clean plain glass slide covered with a 22 mm 22 mm coverslip and mounted on a phase contrast microscope with a heated stage to maintain slide temperature at 37 C. A total of 200 cells were scored from different fields and the propor-tion of motile spermatozoa was expressed as a percentage of total motile sperm. Each sample was analyzed in tripli-cates and the average value taken as the percentage motility. A fourth sample was analyzed where there was >10% variation between the three samples. All analyses were done by the research team under the supervision of a trained laboratory technician. The effect of PEA or OEA on sperm viability was assessed after 6 hours of incubation using the LIVE/DEAD Sperm Viability Kit (Invitrogen), ac-cording to the manufacturer's instructions by viewing SYBR-14 (green) and PI (red) cellular fluorescence labeling patterns at 400 magnification on a Nikon Eclipse 300 mi-croscope. A total of 200 cells from eight randomly selected fields were examined for green and red fluorescence and sperm viability was expressed as a percentage of cells ex-hibiting green fluorescence. 

Flow Cytometric Evaluation of Mitochondrial Membrane Potential 

The lipophilic cationic dye JC-1 was used to assess mitochon-drial status by characterizing the mitochondrial membrane potential (Djm) of spermatozoa, as previously described (26). Briefly, 1-mL aliquots of spermatozoa (5 106/mL) were incubated with increasing concentrations of PEA or OEA (1 nM–1 mM) at 37 C under a 5% CO2 atmosphere in air for 15 minutes, after which 0.15 mmol/L JC-1 was added and the cells further incubated for 30 minutes. The green (monomeric low) and orange (multimeric high) JC-1 mito-chondrial membrane potentials were monitored by flow cy-tometry using a BD FACSAria II cell sorter (BD Biosciences) equipped with a 488-nm argon laser as excitation light source. A total of 10,000 gated events were collected per sam-ple at a rate of 500 events/s. Nonsperm activities were gated 1262 VOL. 102 NO. 5 / NOVEMBER 2014

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TABLE 1 

Semen characteristics and mean concentrations of NAEs in the study groups (expressed as median and 95% confidence interval). 

Variable

Volume (mL)

Sperm count (3106/mL)

Motility (%)

Normal forms (%)

Normozoospermia (n ¼ 45)

3.0

(2.9–3.9)

156

(129–187)

72 (67–73)

50

(44–51)

Asthenozoospermia (n ¼ 11)

2.0

(1.7–3.7)

80

(42–158)

32 (22–40)

24

(12–36)

Teratozoospermia (n ¼ 8)

3.3

(2.2–4.7)

49

(32–82)

54 (31–69)

5

(2–16)

Oligoasthenoteratozoospermia (n ¼ 22)

3.2

(1–5.9)

4

(3–10)

31 (22–40)

12

(8–18)

Azoospermia (n ¼ 4)

3.8

(3.2–4.7)


NA

NA


NA

Note: NA ¼ not available; NAE ¼ N-acylethanolamide. 

Amoako. Endocannabinoids in male reproduction. Fertil Steril 2014.

out from the cells of interest based on the forward scatter and side scatter of the sperm population recorded in the linear mode. The JC-1 green and orange fluorescences were detected at 530 nm and 610 nm, respectively. Flow cytometric data were analyzed with BD FACSDiVa Software (BD Biosciences) in the logarithmic mode. The population of orange stained cells was recorded as the percentage of cells with a high mito-chondrial membrane potential. 

Statistical Analysis 

Statistical analysis was performed using Graphpad (Instat version 3, Graphpad Software). Statistical comparison of endocannabinoid concentrations in men with normal and pathological seminal parameters was achieved using a Kruskal-Wallis one-way analysis of variance (ANOVA) with Dunn's ad hoc post test. For the in vitro experiments, compar-isons between treated and control samples was achieved by using one-way ANOVA with Dunnett's post test. In all cases, P<.05 was considered statistically significant.

RESULTS 

Relationship between Seminal Plasma Levels of PEA and OEA and Semen Quality 

World Health Organization criteria for sperm concentration, motility, and morphology were used to group the 90 men into 5 pathological and normal groups: asthenozoospermia, oligoasthenoteratozoospermia, teratozoospermia, azoo-spermia, or normozoospermia. The five groups had similar average age and their basic semen parameters are given in Table 1. 

The seminal plasma concentrations of PEA were signifi-cantly lower in men with asthenozoospermia (median, 6.23 nM; interquartile range [IQR], 2.88–7.58; 95% confidence interval [CI] 3.51–7.38) and oligoasthenoteratozoospermia (median, 3.02 nM; IQR, 2.17–8.44; 95% CI 3.29–7.25) compared with the normozoospermic controls (median, 11.94 nM; IQR, 7.81–15.17; 95% CI 10.44–16.08). No signifi-cant difference was seen between men with teratozoospermia (median, 10.62 nM; IQR, 5.64–21.39; 95% CI 3.51–7.38) or

FIGURE 1

image017.jpg

Relationship between seminal plasma levels of palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) and semen quality. Seminal plasma levels of (A) PEA and (B) OEA were quantified by ultra-performance-liquid chromatography-tandem electrospray-ionization-mass spectrometry in men with normozoospermia, asthenozoospermia, teratozoospermia, azoospermia, and oligoasthenoteratozoospermia. Data are reported as median and interquartile range and all samples were processed as duplicates. Comparison between groups were made using Kruskal-Wallis one-way analysis of variance (ANOVA) followed by Dunn's ad hoc post test. *P, **P, ***P represents data significantly (P<.05, P<.01, and P<.001 respectively) and ns represents data not significantly lower than normozoospermia. NOR ¼ normozoospermia (n ¼ 45); AST ¼ asthenozoospermia (n ¼ 11); TER ¼ teratozoospermia (n ¼ 8); AZO ¼ azoospermia (n ¼ 4); OAT ¼ oligoasthenoteratozoospermia (n ¼ 22).

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azoospermia (median, 5.77 nM; IQR, 3.55–16.49; 95% CI -3.55–20.76) compared with the normozoospermic controls (Fig. 1A). The OEA seminal plasma concentrations were signif-icantly lower in men with asthenozoospermia (median, 0.86 nM; IQR, 0.35–0.95; 95% CI 0.50–0.89), oligoasthenoter-atozoospermia (median, 0.52 nM; IQR, 0.29–0.87; 95% CI 0.47–0.74), and azoospermia (median, 0.29 nM; IQR, 0.24– 

0.39; 95% CI 0.19–0.41) when compared with the normozoo-spermic controls (median, 1.52 nM; IQR, 0.57–2.30; 95% CI 1.24–2.26), although no significant difference was seen with men with teratozoospermia (median, 1.02 nM; IQR, 0.46– 1.74; 95% CI 0.06–2.93) (Fig. 1B).

Time and Dose-dependent Effects of PEA and OEA on Sperm Motility 

After 15–90 minutes of incubation, PEA exerted a signifi-cant (P<.001) time-dependent increase in progressive sperm motility (77%, 81%, 81%, 80%, and 80% at 15, 30, 45, 60, and 90 minutes, respectively), whereas control sperm at the corresponding times showed a significant decrease (70%, 70%, 69%, 69%, and 67%) (Fig. 2A). Oleoylethanola-mide also induced a significant (P<.001) time-dependent increase in progressive sperm motility (74%, 77%, 79%, 79%, and 76%, respectively) compared with controls at the

FIGURE 2

image022.jpg

corresponding time (69%, 69%, 69%, 67%, and 67%) (Fig. 2B). Exposing sperm to increasing doses of PEA or OEA (10 nM–10 mM) for 30 minutes resulted in a significant dose-dependent increase in the percentage of progressive sperm motility for both PEA and OEA (one-way ANOVA, P>.05; n ¼ 6) (Fig. 2C and D). 

Effect of PEA and OEA on Sperm Viability and Mitochondria Activity 

Treating spermatozoa with increasing doses of PEA (10 nM– 10 mM) for 6 hours resulted in a significant dose-dependent maintenance of sperm viability compared with the control sperm (P<.001, one-way ANOVA; n ¼ 6) (Fig. 3A). The effect of OEA (10 nM–10 mM, 6 hours) on sperm viability also re-sulted in a significant dose-dependent, maintenance of sperm viability compared with the vehicle control (P<.001, one-way ANOVA; n ¼ 6) (Fig. 3B). Incubation with up to a concen-tration of 10 mM, PEA or OEA did not induce any significant changes in sperm mitochondrial activity as measured by mitochondria membrane potential (Fig. 3C and D). 

DISCUSSION

Male factor infertility is a global problem that is on the in-crease and although the precise etiopathology is unknown

Effect of palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) on sperm motility. Human spermatozoa were incubated in Biggers, Whitten Whittingham (BWW) medium at 37 C in a 5% CO2 incubator and stimulated with (blue) or without (red) (A) PEA or (B) OEA (1 mM) for up to 90 minutes. Aliquots of 10 mL were taken at 15, 30, 45, 60, and 90 minutes and sperm motility was evaluated by means of phase contrast microscopy. Data are expressed as mean SEM (n ¼ 4 separate experiments). Significant differences were determined by two-way analysis of variance (ANOVA). Pairwise comparison between treated and time-matched controls were determined by paired Student's t test; *P<.05, **P<.01, ***P<.001. Human spermatozoa were incubated in BWW medium at 37 C and stimulated with or without increasing concentrations of PEA (C) or OEA (D) for up to 30 minutes. Aliquots of 10 mL were taken and sperm motility was evaluated by means of phase contrast microscopy and expressed as a percentage of motile sperm. Columns are mean SEM of six independent experiments performed in duplicate. Significant differences were determined by one-way ANOVA and Dunnett's post hoc test; **P<.01, ***P<.001 versus control. 

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FIGURE 3

image025.jpg 

Effect of palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) on sperm viability and mitochondria activity. Human spermatozoa were incubated in Biggers, Whitten Whittingham (BWW) medium at 37 C in a 5% CO2 incubator and stimulated with or without increasing concentrations of (A) PEA or (B) OEA (10 nM–10 mM) for up to 6 hours. Sperm viability was measured by the Live/Dead sperm viability assay and fluorescence microscopy and expressed as percentage of viable sperm. Columns are mean SEM of six independent experiments performed in duplicate. Significant differences were determined by one-way analysis of variance (ANOVA) and Dunnett's post hoc test. **P<.01, ***P<.001 versus 0 nM PEA or OEA. Human spermatozoa were incubated in BWW medium at 37 C in a 5% CO2 incubator and stimulated with or without increasing concentrations of (C) PEA or (D) OEA (10 nM–1 mM) for up to 30 minutes. Aliquots of sperm suspension were stained with fluorescence dye JC-1 and mitochondria activity monitored by flow cytometry. The graphs indicate the percentage of sperm cells exhibiting orange fluorescence, indicating the higher mitochondrial activity, in the presence of the indicated doses of PEA or OEA. Data are expressed as mean SEM of four separate experiments, each run in duplicates. 

Amoako. Endocannabinoids in male reproduction. Fertil Steril 2014.

in most cases, defective sperm function from oxidative stress has been implicated (34). This results from either an increase in reactive oxygen species (ROS) or a decrease in the body's antioxidant capacity that results in peroxidative damage to the sperm plasma membrane. The ROS plays a significant role in postejaculatory sperm maturation events, such as capacitation and the acrosome reaction, but can cause irre-versible damage to the sperm plasma membrane at high con-centrations. This may result in loss of sperm motility and sperm DNA fragmentation causing sperm dysfunction and loss of fertilizing potential. Thus, it was suggested that a bal-ance between ROS generation and total antioxidant capacity plays a critical role in the pathophysiology of the disease state (35). Several antioxidant agents, enzymatic (superoxide dis-mutase, catalase, and glutathione peroxidase) and nonenzy-matic (e.g., a-tocopherol, b-carotene, ascorbate, urate), have been used as antioxidant supplementation to protect sperm cells (36). These agents are present in seminal plasma, and their use to enhance semen parameters remains controversial. Although earlier studies did not show any improvements in semen parameters after antioxidant supplementation (37),more recent studies have shown evidence of benefit (38–40). Emerging evidence suggest that N-acylethanolamides, especially PEA and OEA, have antioxidant, antimicrobial, and anti-inflammatory properties and can inhibit the radicals induced by in vitro oxidation of lipids (41, 42). These compounds have been shown in previous studies to inhibit nitric oxide production in macrophages (43) and protect isolated rat heart from ischemia (44) by their antioxidant effects. The presence of higher concentrations of these compounds in seminal plasma suggests that they may contribute to the antioxidant capacity of human reproductive fluids, which could explain their beneficial effects on sperm capacitation, motility, and viability (27–29). The source of PEA and OEA in seminal plasma remains unknown and has not been explored in this study. However, the spermatozoa themselves may be the source, as they possess the biochemical apparatus for the synthesis and degradation of PEA and OEA (26). Contribution from the epidydimis, prostate, and seminal vesicles is possible. In addition, cannabinoid receptors and endocannabinoid synthetic and degradation enzymes have been localized in

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fallopian tubal epithelium, which indicates exposure of spermatozoa to the endocannabinoids in the female genital tract (45–48). 

In the present study, the relationship between PEA and OEA seminal plasma concentrations and human sperm abnor-malities and functions showed remarkable differences in OEA and PEA concentrations in men with different pathological semen subtypes, with lower levels associated with decreased sperm count and abnormal sperm motility. This observation indicates that to maintain normal sperm count and motility, higher levels of PEA and OEA are required in the male genital tract. This suggests that PEA and OEA may play important physiological roles in the male genital tract and therefore loss of ‘‘PEA and/or OEA tone’’ in men with abnormal semen parameters may indicate aberrant endocannabinoid signaling in human spermatozoa. This may be relevant in the etiopatho-genesis of human sperm dysfunction and compromised male fertility. Thus, deregulation of the endogenous endocannabi-noid system during sperm differentiation and/or maturation probably results in the production of spermatozoa with abnormal morphology and higher levels of DNA fragmenta-tion. This is a common feature associated with spermatozoa from men with asthenozoospermia and oligoasthenoteratozo-spermia. The effects of PEA and OEA on human sperm motility, viability, and mitochondrial activity were examined to understand the potential role of these compounds on human sperm function. Both PEA and OEA significantly increased sperm motility in a time- and concentration-dependent manner and significantly maintained sperm viability in vitro during a 6-hour period in a concentration-dependent manner. The rapid effect of PEA and OEA on sperm motility supports the hypothesis of a protective action of PEA and OEA against ROS, which could explain its beneficial effects on in vitro ca-pacitated spermatozoa (27–29). Palmitoylethanolamide and OEA have been shown at physiologic concentrations, as detected in these men, to increase kinematic parameters, such as curvilinear velocity and amplitude of lateral head displacement and hyperactivation, in the presence and absence of oxidative stress (27–29). In vitro exposure of human spermatozoa to PEA and OEA conferred some protection against oxidative damage and protected these cells from oxidative damage in some cases of idiopathic infertility (27–29). Furthermore, the rapid effects of PEA and OEA on sperm suggest that these agents may exert their physiological role on human spermatozoa through other possible mechanisms not involving ROS, such as the one that does not require CB receptors, which could be contained in human sperm such as the GPR55, GPR119, and the peroxisome proliferator-activated receptors (18). 

The lipophilic nature of PEA and OEA suggest that these compounds may interact with sperm plasma membrane lipid bilayers and alter membrane polarity (49–51) to induce changes in plasma membrane composition and fluidity. This would increase membrane permeability to some molecules, such as calcium and bicarbonate, which are essential for the induction of sperm motility and capacitation (29). Decreased sperm plasma membrane fluidity and polarity are common features associated with spermatozoa from men with oligozoospermia and some idiopathic normozoospermic men

(52) and PEA and OEA are known to increase the rate of capacitation in sperm from men with oligozoospermia and idiopathic normozoospermic men displaying decreased membrane polarity (29). 

In conclusion, PEA and OEA levels in human seminal plasma may reflect the overall male reproductive health, including the testes, epididymides, and accessory reproduc-tive glands, and could be exploited as biomarkers for male factor infertility. Palmitoylethanolamide and OEA may there-fore play a crucial role in the preservation of normal sperm function and male fertility. Exocannabinoids, such as D9-tetrahydrocannabinol and cannabidiol, that compete with these lipid mediators and may upset their finely balanced, normal functioning and thus result in male reproductive fail-ure. Further investigations into the roles of endocannabioids and exocannabioids in male fertility are thus required. 

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