Activated-farnesoid X receptor (FXR) expressed in human sperm alters its fertilising ability

in Reproduction
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The farnesoid X receptor alpha (FXR) is a bile acid sensor activated by binding to endogenous bile acids including chenodeoxycholic acid (CDCA). Although, FXR is expressed in male reproductive tissue, the relevance of the receptor on reproduction is scarcely known. Here, we demonstrated the FXR presence and its action on several human sperm features. Western blot and immunofluorescence assays evidenced the FXR expression in human spermatozoa and the localisation in the middle piece. CDCA increasing concentrations and GW4064, synthetic ligand of FXR, were used to study the FXR influence on sperm motility, survival, capacitation, acrosome reaction and on glucose as well as lipid metabolism. Interestingly, our data showed that increasing concentrations of CDCA negatively affected sperm parameters, while the receptor blockage by (Z)-Guggulsterone and by the anti-FXR Ab reversed the effects. Intriguingly, elevated CDCA levels increased triglyceride content, while lipase and G6PDH activities were reduced with respect to untreated samples, thus impeding the metabolic reprogramming typical of the capacitated sperm. In conclusion, in this study, we demonstrated for the first time a novel target for FXR and that the activated receptor alters the acquisition of sperm fertilising ability. We showed that sperm itself express the FXR and it is responsive to specific ligands of the receptor; therefore, bile acids influence this cell both in male and in female genital tracts. It might be hypothesized that bile acid levels could be involved in infertility with idiopathic origin as these compounds are not systematically measured in men undergoing medically assisted procreation.

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    Society for Reproduction and Fertility

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    FXR expression in human spermatozoa. (A) Representative experiment of pooled sperm samples (lanes N1, N2); MCF7 breast cancer cells as positive control (MCF7), (C−) negative control; (A1) negative pre-absorption control. Actin serves as a loading control. The experiments were repeated at least four times and images show the results of one representative experiment. FXR immunolabelling of human sperm. (B) Phase contrast; (B1) expression of FXR in human spermatozoa showed a red immunofluorescence labelling in the sperm mid piece; (B2) negative pre-absorption control. Pictures are representative of four similar experiments. Scale bars: 5 µM.

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    FXR influences on sperm motility and survival. (A) Sperm motility, (B) sperm vitality after CDCA (0.5, 2, 10, 50 and 100 µM), GW (6 µM), Gug (10 µM) with or without 50 µM CDCA, + FXR Ab with or without 50 µM CDCA, NRS, BSA and EtOH. NC, non-capacitated sperm. Columns represent mean ± s.e.m. of six independent experiments each done in duplicate, *P < 0.05 vs control, **P < 0.001 vs control.

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    pAKT e pMAPK activation in human spermatozoa. Western analyses of pAKT, and pMAPK were performed on 70 µg of total protein. (A) Representative experiment of pooled sperm samples (lane 1 untreated sperm cells); (lanes 2, 3 sperm cells incubated with CDCA 2 and 50 µM respectively); (lane 4 sperm incubated with GW 6 µM); (lane 5 and 6 sperm cell treated with Gug 10 µM without/with CDCA 50 µM). (B) Columns are mean of four independent experiments in which band intensities were evaluated in terms of optical density arbitrary units. Actin serves as a loading control.

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    FXR affects sperm capacitation and acrosome reaction. (A) Sperm cholesterol efflux after the previous indicated (Fig. 2) treatments. NC, non-capacitated sperm. Columns represent mean ± s.e.m. of six independent experiments each done in duplicate, *P < 0.05 vs control, **P < 0.001 vs control. (B) Acrosome reaction staining pattern with FITC–PNA + P: A and B dead non-reacted spermatozoa, C dead reacted sperm, D and E live non-reacted spermatozoa, F live reacted sperm. (B1) Dead reacted sperm after CDCA treatment. Columns represent mean percentage ± s.e.m. of six independent experiments. *P < 0.05 vs control, **P < 0.001 vs control.

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    FXR alters sperm lipid metabolism. (A) Sperm tryglicerides levels; (B) lipase activity after the previous indicated treatments (Fig. 2), furthermore, the lipase activity was determined with three control media: one without the substrate (-Su), another without the colipase (-Co) and the third without either substrate or co-enzyme (-Su and Co). NC, non-capacitated sperm. Columns represent mean ± s.e.m. of six independent experiments each done in duplicate. *P < 0.05, **P < 0.001 vs control.

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    FXR reduces sperm glucose metabolism through the PPP. (A) Sperm G6PDH activity after the previous indicated treatments (Fig. 2). NC, non-capacitated sperm. The enzymatic activity was determined with three control media: one without glucose-6-phosphate as substrate (G1), another without the coenzyme (NADP+) (G2) and the third without either substrate or coenzyme (G3). Columns represent mean ± s.e.m. of six independent experiments each done in duplicate, *P < 0.05 vs control, **P < 0.001 vs control.

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