Romanian Biotechnological Letters Vol.19, No3, 2014 Copyright © 2014 University of Bucharest Printed in Romania. All rights reserved

ORIGINAL PAPER

Romanian Biotechnological Letters, Vol. 19, No. 3, 2014 9437

Research regarding the role of TRPV1 and capsaicin (CPS) implication for capacitation and acrosome reaction

Received for publication, February 07, 2014 Accepted, May 20, 2014

CARSTEA CLAUDIA1, STRASSER HORATIU1, IPATE IUDITH1, BACILA VASILE1 STRASSER CONSTANTA2

1 Romanian Academy- INCE-CSCBA

2Faculty of Veterinary Science Bucharest

Email: Ipate Iudith1- ipate.iudith@gmail.com, Carstea Claudia2- clau_genetic@yahoo.com

Strasser Constanta2- strasserconstanta@yahoo.com,

Abstract

In this study we want the tested the capsaicin (CPS) effect in activation the TRPV1 receptor in mouse sperm- acrosomial reaction. The role of vanilloid (TRPV1) in sperm functions involved in fertilization will be studied by evaluating the effect of capsaicin selective antagonists of TRPV1, on spontaneous and induced acrosome reaction (AR). The vanilloid (TRPV1) receptors, plays a crucial role in controlling functionality of sperm, with the impact on reproduction. The TRPV1 binding was detected in fertile sperm and this is reactive of the capsaicin caused the sperm membrane depolarization due to the Na+ influx and calcium channels (VGCC) opening.

Keywords: acrosome, sperm, capacitation,capsaicin Introduction The capsaicin (CPS), the active ingredient in chilli pepper was first isolated over a century ago. In 1919 the exact chemical structure of capsaicin was determined and the complete synthesis of the compound was achieved a decade later. Capsaicin has been used as an analgesic for decades, but the therapeutic potential of capsaicin was first recognized as early as 1850. The effects of the pungent chemical, capsaicin, is mediated through the ligand gated ion channel TRPV1. The vanilloid TRPV1 receptor is part of a family of transient receptor potential (TRP) channels (see Benham et al., 2002) [2].This receptor is a nonselective cation channel that integrates multiple noxious stimuli and is associated with the pathophysiology of various major diseases (Szallasi, 2002)[7]. It is activated by the naturally occurring vanilloids, and resiniferatoxin (RTX), noxious heat and acid [3]. The TRPV1 receptor is associated with the pathophysiology of various major diseases (Szallasi, 2002) and male fertility. Alterations in the expression and sensitivity of TRPV1 receptors is concurrent with the development of chronic pain (see Di Marzo et al., 2002a) [4] and inflammation (Amaya et al., 2003) [1]. The expression of TRPV1 is dramatically increased in colonic nerve fibres of patients with active inflammatory bowel disease (Yiangou et al., 2001[8]).

Research regarding the role of TRPV1 and capsaicin (CPS) implication for capacitation and acrosome reaction

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Materials and Methods For obtaining the mouse sperm we euthanized the male C57BL6 male mouse (10–

15 wk old) by cervical dislocation, dissect the mouse using a transverse cut through the posterior ventral abdominal wall. At room temperature, expose right and left caudale epididymides with attached vas deferentia. Remove to the central well of an organ culture dish (Falcon, cat. no. 3037) containing 1.0 mL of M-HTF; isolated each vas deferens and milk sperm from it by means of a watchmaker’s forceps. Make several punching using a 26 gauge needle, across each cauda epididymis to release sperm. Allow sperm to disperse from the epididymis for about 15 min before removing all of the organ tissues from the dish. Pipette the sperm suspension into a capped polystyrene tube (Falcon, cat. no. 2054) and allow debris to sediment for 5 min. Decant the supernatant into a second tube. Repeat three times to clarify the sperm suspension. Centrifuge the sperm at 2000 rpm, for 5 min and that re suspend it in 100 µl of HTF medium. The following dishes will be prepared one day before experiment: 2 dishes of 1 ml of HTF medium containing 100 nM Capsaicin (one for spontaneous –AR and one for AR-induced by Ca-ionophore treatment); 2 dishes of 1 ml of HTF medium containing 500 nM Capsaicin (one for spontaneous –AR and one for AR-induced by Ca-ionophore treatment); 2 dishes of 1 ml of HTF medium for control groups (one for spontaneous –AR and one for AR-induced by Ca-ionophore treatment). In each of the sperm dishes will be added 20 µl of sperm suspension.

Experimental plan: Capsaicin group

spontan-AR induced-AR by Ca-ionophore wild-type 100nM 500nM Control 100nM 500nM Control

TRPV1KO 100nM 500nM Control 100nM 500nM Control

It was incubate the sperm suspensions undisturbed for 1 h at 37°C to attain

capacitation. Remove the upper two-thirds of the incubate and place in a separate tube to obtain sperm of high motility.

Calcium ionophore treatment, after 1 h, the spermatozoa are treated with 5 μM of calcium ionophore for 30 min. (stock solution 1mM) at 37°C.; spermatozoa are mixed with the staining solution and incubated at 37°C for 5 -10 min. Staining solution for FACS: 1 ml HTF, 10µl FITC-PNA (stock solution 1mg/ml), 2 µl PI (stock solution 1,5mM).Spermatozoa are washed twice with normal HTF by centrifugation at 2000 RPM (500g) for 5 min. and immediately used staining and FACS. At a final concentration of 10 µg/ml of FITC-PNA working solution, (1 mg/ml of stock solution diluted in PBS) and 12 µM PI (8 µl/ml) (Propidium Iodide) stock solution) are added to 1 ml of diluted semen. Stained sperm suspensions subsequently are run through a flow cytometer. The two dyes are excited in the flow cytometer using a 488-nm argon excitation laser. Nonviable cells become PI positive, and their red fluorescent signal is detected using fluorescence detector 3 (detects emitted photons of a wavelength .670 nm). Acrosome-damaged cells are stained positive for FTC-PNA, and its and its green fluorescent signal is detected using fluorescence detector 1 (detects emitted photons in the wavelength of 515–545 nm).

Immunocytological staining of acrosome

CARSTEA CLAUDIA, STRASSER HORATIU, IPATE IUDITH, BACILA VASILE STRASSER CONSTANTA

Romanian Biotechnological Letters, Vol. 19, No. 3, 2014 9439

Responsiveness of the acrosome to Capsaicin o will examine after treatment for 1 h. Acrosome status of spermatozoa will be examined immediately, under the two different sperm incubation conditions for each antagonist.

The procedure for staining the acrosome is basically performed: in brief, the sperm suspension will be put in a eppendor tube with 1 ml of HTF containing PI +FITC-PNA [(1 ml HTF+ 10µl FITC-PNA (stock solution 1mg/ml) + 2 µl PI (stock solution 1,5mM)]

The staining reaction will be continued for 5-10 min at 37°C to distinguish live spermatozoa from dead spermatozoa and the AR and the percentage of spermatozoa not uniformly fluorescing at the anterior region of the head (reacted spermatozoa). After washing twice with HTF, spermatozoa are smeared on a slide glass. The slides are covered with covers slips and exanimate immediately under the microscope. At least 200 spermatozoa will be counted in each smear.

Results and Discussion Experiment optimization and progress: Preliminary experiments on immunocytological staining of acrosome by using PI-FITC-

PNA gave us good results. The staining is specific, FITC-PNA green signal it is detected only at the level of the reacted acrosome (Fig.1, Fig.2)

Fig.1 Fig.2

Specific staining with FITC-PNA -green signal detected only at the level of the reacted acrosome The assessment for the images was realized using digital images obtained with a Nikon TE-300 Eclipse microscope with standard visible and epi-fluorescence (xenon lamp). Illustrations represent the images obtained under fluorescence (Fl) or transmitted light illumination (Ph, phase contrast; DIC, differential interference contrast). Experiments were repeated at least three times; representative sperm are shown. Original magnification X40.JPG Images.Dimension:1388 pixels

Preliminary experiments on FACS analyses The FACS detected: the population of nonviable cells PI positive, and their red

fluorescent signal; the acrosome-damaged cells stained positive for FTC-PNA, and its and its green fluorescent signal.

In the table 1 are presented the % of acrosome damaged acrosome reaction (AR) sperm cells from each of the analyzed groups: in case of spontaneous acrosome reaction AR groups

Research regarding the role of TRPV1 and capsaicin (CPS) implication for capacitation and acrosome reaction

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(the sperm cells were capacitated in HTF medium for 1 h in the presence of Capsaicin 100nM, 500 nM or just HTF, in case of control group).

In case of induced acrosome reaction AR (the sperm cells were capacitated in HTF medium for 1 h in the presence of Capsaicin 100nM, 500 nM or just HTF, in case of control group and than the AR was induced by using Calcium ionophore - 5µl for 30 min., after capacitation)

Table 1 Capsaicin group

C57BL/6♂ spontan-AR induced-AR by Ca-ionophore

(wild-type) 100nM 500nM Control 100nM 500nM Control No. of sperm

analyzed 8305 8129 8182 8492 8341 8215

% acrosome reactions detected

30.6% AR 33.9% AR 29.2% AR 54.8% 57.9% 56.3%

AR acrosome reaction - acrosome damaged sperm cells, detected by flow cytometer

based on their GFP signal. Statistical analysis Results are expressed as means ± standard deviation (SD). Statistical analysis was

performed using the Student’s t test. Differences were considered significant for p < 0.05.

Conclusions The results from the FACS analysis show that: the exposure of the sperm cells, during

the capacitation time, to the Capsaicin (dosage of 100nM and 500 nM) it doesn’t show, a directly effect on the acrosome reaction (AR) in the spontaneous and induced groups; the differences detected by FACS analyze, between the treated groups and the control groups are not showed to be significant; the capsaicin activated the receptors specific TRPV1. The CPS caused the sperm membrane depolarization due to the Na+ influx and the consequent voltage gated calcium channels (VGCC) opening. In conclusion it was suggested that TRPV1 channels modulate the major pathways involved in capacitation Acknowledgment "This work was sported by a grant of the Romanian National Authority for Scientific Research, CNDI-UEFISCDI, project number 120/2012" and the Romanian Academy Research Theme XI.4-"Research regarding the traceability in livestock eco-systems of animal’s products in socio-economic conditions of Romania and Moldova."

CARSTEA CLAUDIA, STRASSER HORATIU, IPATE IUDITH, BACILA VASILE STRASSER CONSTANTA

Romanian Biotechnological Letters, Vol. 19, No. 3, 2014 9441

References: 1.AMAYA F., OH-HASHI K., NARUSE Y., IIJIMA N., UEDA M., SHIMOSATO G., TOMINAGA M., TANAKA Y., TANAKA M. Local inflammation increases vanilloid receptor-1 expression within distinct subgroups of DRG neurones. Brain Res. 2003;963:190–196.[PubMed] 2. BERNABÒ N, PISTILLI MG, FALASCA G, CURINI V, GAROFALO ML, TURRIANI M, MATTIOLI M, BARBONI B. Role of TRPV1 channels during the acquisition of fertilizing ability in boar spermatozoa., [Mol Cell Endocrinol. 2010]. 3. BENHAM C.D., DAVIS J.B., RANDALL A.D. Vanilloid and TRP channels: a family of lipid-gated cation channels. Neuropharmacology. 2002;42:873–888.[PubMed] 4. DI MARZO V. Foreword-endocannabinoids in the new millennium. Prostaglandins Leukot. Essent. Fatty Acids. 2002;66:91–92.[PubMed] 5. DI MARZO V., BISOGNO T., DE PETROCELLIS L. Anandamide: some like it hot. Trends Pharmacol. Sci. 2001;22:346–349.[PubMed] 6. DI MARZO V., FONTANA A., CADAS H., SCHINELLI S., CIMINO G., SCHWARTZ J.C., PIOMELLI D. Formation and inactivation of endogenous cannabinoid anandamide in central neurons. Nature. 1994;372:686–691.[PubMed] 7.SZALLASI A. Vanilloid (capsaicin) receptors in health and disease. Am. J. Clin. Pathol. 2002;118:110–121.[PubMed] 8.YIANGOU Y., FACER P., DYER N.H., CHAN C.L., KNOWLES C., WILLIAMS N.S., ANAD P. Vanilloid receptor 1 immunoreactivity in inflamed human bowel. Lancet. 2001;357:1338–1339.[PubMed]