Anandamide Capacitates Bull Spermatozoa through CB1 and TRPV1 Activation

Anandamide (AEA), a major endocannabinoid, binds to cannabinoid and vanilloid receptors (CB1, CB2 and TRPV1) and affects many reproductive functions. Nanomolar levels of anandamide are found in reproductive fluids including mid-cycle oviductal fluid. Previously, we found that R(+)-methanandamide, an anandamide analogue, induces sperm releasing from bovine oviductal epithelium and the CB1 antagonist, SR141716A, reversed this effect. Since sperm detachment may be due to surface remodeling brought about by capacitation, the aim of this paper was to investigate whether anandamide at physiological concentrations could act as a capacitating agent in bull spermatozoa. We demonstrated that at nanomolar concentrations R(+)-methanandamide or anandamide induced bull sperm capacitation, whereas SR141716A and capsazepine (a TRPV1 antagonist) inhibited this induction. Previous studies indicate that mammalian spermatozoa possess the enzymatic machinery to produce and degrade their own AEA via the actions of the AEA-synthesizing phospholipase D and the fatty acid amide hydrolase (FAAH) respectively. Our results indicated that, URB597, a potent inhibitor of the FAAH, produced effects on bovine sperm capacitation similar to those elicited by exogenous AEA suggesting that this process is normally regulated by an endogenous tone. We also investigated whether anandamide is involved in bovine heparin-capacitated spermatozoa, since heparin is a known capacitating agent of bovine sperm. When the spermatozoa were incubated in the presence of R(+)-methanandamide and heparin, the percentage of capacitated spermatozoa was similar to that in the presence of R(+)-methanandamide alone. The pre-incubation with CB1 or TRPV1 antagonists inhibited heparin-induced sperm capacitation; moreover the activity of FAAH was 30% lower in heparin-capacitated spermatozoa as compared to control conditions. This suggests that heparin may increase endogenous anandamide levels. Our findings indicate that anandamide induces sperm capacitation through the activation of CB1 and TRPV1 receptors and could be involved in the same molecular pathway as heparin in bovines

Epididymis Response Partly Compensates for Spermatozoa Oxidative Defects in snGPx4 and GPx5 Double Mutant Mice

We report here that spermatozoa of mice lacking both the sperm nucleaus glutathione peroxidase 4 (snGPx4) and the epididymal glutathione peroxidase 5 (GPx5) activities display sperm nucleus structural abnormalities including delayed and defective nuclear compaction, nuclear instability and DNA damage. We show that to counteract the GPx activity losses, the epididymis of the double KO animals mounted an antioxydant response resulting in a strong increase in the global H2O2-scavenger activity especially in the cauda epididymis. Quantitative RT-PCR data show that together with the up-regulation of epididymal scavengers (of the thioredoxin/peroxiredoxin system as well as glutathione-S-transferases) the epididymis of double mutant animals increased the expression of several disulfide isomerases in an attempt to recover normal disulfide-bridging activity. Despite these compensatory mechanisms cauda-stored spermatozoa of double mutant animals show high levels of DNA oxidation, increased fragmentation and greater susceptibility to nuclear decondensation. Nevertheless, the enzymatic epididymal salvage response is sufficient to maintain full fertility of double KO males whatever their age, crossed with young WT female mice

Can the SCD test and terminal uridine nick-end labeling by flow cytometry technique (TUNEL/FCM) be used interchangeably to measure sperm DNA damage in routine laboratory practice?

Background: Numerous tests have been proposed to evaluate sperm DNA integrity. To assess the sperm chromatin dispersion (SCD) test in an andrology laboratory, twenty-five men attending Clermont-Ferrand (France) University Hospital's Center for Reproductive Medicine were recruited. Sperm DNA damage was measured in the same semen samples using the SCD test and the Terminal Uridine Nick-end Labeling by flow cytometry technique (TUNEL/FCM) after density gradient centrifugation. Results: SCD test reliability between readings, readers or slides was clearly established with very high agreement between measurements (Intraclass correlation coefficient (ICC) at 0.97, 0.95 and 0.98 respectively). Despite very good agreement between the SCD test and TUNEL/FCM (ICC at 0.94), the SCD test tended to slightly but significantly underestimate DNA damage compared with TUNEL (p = 0.0127). This systematic difference between the two techniques was - 3.39 +/- 1.45% (mean +/- SE). Conclusions: Andrology laboratories using the SCD test to measure sperm DNA damage need to know that it appears to give slightly underestimated measurements compared to TUNEL/FCM. However, this systematic underestimation is very small in amplitude. Both techniques give almost perfectly congruent results. Our study underlines the importance for each laboratory to validate its method to assess sperm DNA damage before implementing it in routine andrology lab practice

Levels of liver X receptors in testicular biopsies of patients with azoospermia.

International audienceOBJECTIVE: To determine whether the transcription factors liver X receptors (LXRs) and their downstream genes, which are involved in the regulation of several testicular functions in mouse models, are differentially expressed in testes of men with nonobstructive azoospermia (NOA) or obstructive azoospermia (OA). DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): Patients with various types of NOA (n=22) and with OA (n=5). INTERVENTION(S): Human testicular biopsies. MAIN OUTCOME MEASURE(S): Transcript levels were measured in testicular biopsies with the use of quantitative polymerase chain reaction. Correlations of LXR mRNA levels with the number of germ cells, the expression of proliferation and apoptosis markers, and the amount of intratesticular lipids and testosterone were evaluated. The localization of LXRalpha was analyzed by immunofluorescence. RESULT(S): LXR mRNA levels were decreased by 49%-98% in NOA specimens and positively correlated with germ cell number. Accumulations of IDOL and SREBP1c (LXR targets involved in lipid homeostasis) were 1.8-2.1 times lower in NOA samples and mRNA levels of the SREBP1c target gene ELOVL6 were increased 1.9-2.4-fold. Interestingly, the amount of triglycerides and free fatty acids were higher in NOA testes (3.4-12.2-fold). LXRalpha was present in Leydig cells. Accumulations of LXR downstream genes encoding the steroidogenic proteins StAR and 3betaHSD2 were higher in NOA testes (5.9-12.8-fold). CONCLUSION(S): Knowledge of changes in the transcript levels of LXRs and some of their downstream genes during altered spermatogenesis may help us to better understand the physiopathology of testicular failure in azoospermic patients

Accuracy of human sperm DNA oxidation quantification and threshold determination using an 8-OHdG immuno-detection assay

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Indoleamine 2,3-dioxygenase 1 (Ido1) is involved in the control of mouse caput epididymis immune environment

The epididymis maintains a state of immune tolerance towards spermatozoa while also protecting them and itself against infection and acute inflammation. The immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (Ido1) participates in this delicate local equilibrium. Using the mouse Ido1−/− model, we show here that the absence of IDO1 expression leads in the epididymis but not in serum to (1) an increase in the inflammatory state as evidenced by changes in the content of cytokines and chemokines, (2) the engagement of a Th1-driven inflammatory response as evidenced by changes in the Th17/Treg as well as Th1/Th2 equilibria, as well as (3) differences in the content of lipid intermediates classically involved in inflammation. Despite this more pronounced inflammatory state, Ido1−/− animals succeed in preserving the local epididymal immune situation due to the activation of compensatory mechanisms that are discussed

Analysis of the inflammatory status of the epididymis.

<p><b>A:</b> KYN:TRP ratios in caput epididymis and blood plasma, respectively, of 6 month-old <i>wt</i> and <i>IDO1−/−</i> animals. The Y-axis is a semi logarithmic representation in µmole/mmole. <b>B:</b> Histograms show the levels of INF-γ and TNF-α in caput epididymidis extracts and plasma from 6 month-old <i>wt</i> and <i>IDO1−/−</i> animals. <b>C:</b> Histogram shows the levels of the soluble TNF receptors (sTnfRI and sTnfRII) in caput epididymidis extracts from 6 month-old <i>wt</i> and <i>IDO1−/−</i> animals. *<i>P</i>≤0.05, **<i>P</i>≤0.01, ***<i>P</i>≤0.001.</p