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Tuesday 25 April 2017

2015 In-vitro effects of Thymus munbyanus essential oil and thymol on human sperm motility and function

Reproductive BioMedicine Online
Volume 31, Issue 3, September 2015, Pages 411–420
Article


  • a Département des Sciences Alimentaires, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, Bejaia 06000, Algeria
  • b Laboratoire d'Ecologie Microbienne (LEM), Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, Bejaia 06000, Algeria
  • c INSERM U1016, Institut Cochin, Paris 75014, France
  • d CNRS UMR8104, Paris 75014, France
  • e Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris 75014, France
  • f Laboratoire Associé en Ecosystèmes Marins et Aquacoles, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, Bejaia 06000, Algeria
  • g Département de Technologie des Industries Agricoles et Alimentaires, Ecole Nationale Supérieure Agronomique (ENSA), El-Harrach, Alger 16200, Algeria
  • h GH Cochin Broca Hôtel Dieu. Laboratoire d'Histologie Embryologie – Biologie de la Reproduction, Assistance Publique-Hôpitaux de Paris, Paris 75014, France
Received 23 December 2014, Revised 1 June 2015, Accepted 2 June 2015, Available online 23 June 2015

http://doi.org/10.1016/j.rbmo.2015.06.011
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Abstract

Traditional medicine has been used worldwide for centuries to cure or prevent disease and for male or female contraception. Only a few studies have directly investigated the effects of herbal compounds on spermatozoa. In this study, essential oil from Thymus munbyanus was extracted and its effect on human spermatozoa in vitro was analysed. Gas chromatography and Gas chromatography–mass spectrometry analyses identified 64 components, accounting for 98.9% of the composition of the oil. The principal components were thymol (52.0%), γ-terpinene (11.0%), ρ-cymene (8.5%) and carvacrol (5.2%). Freshly ejaculated spermatozoa was exposed from control individuals to various doses of the essential oil for different time periods, and recorded the vitality, the mean motility, the movement characteristics (computer-aided sperm analysis), the morphology and the ability to undergo protein hyperphosphorylation and acrosomal reaction, which constitute two markers of sperm capacitation and fertilizing ability. In vitro, both the essential oil extracted from T. munbyanus and thymol, the principal compound present in this oil, impaired human sperm motility and its capacity to undergo hyperphosphorylation and acrosome reaction. These compounds may, therefore, be of interest in the field of reproductive biology, as potential anti-spermatic agents.

Keywords

  • acrosome reaction;
  • motility;
  • protein hyperphosphorylation;
  • spermatozoa;
  • thymol;
  • Thymus munbyanus

Introduction

Traditional medicine, fundamentally defined as the use of plants, herbs and spices (in infusions or cataplasms) to cure or prevent diseases and attenuate symptoms, has been practised worldwide for centuries. The benefits of traditional medicine for targeting specific diseases have been recognized (Craig, 1999). More recently, interest has focused on the essential oils extracted from the plants, owing to the high lipophilic compound content of these oils. Remarkably, in vivo, the treatment of rodents with essential oils extracted from various plants such as Myrtus communis L, marjoram, lavender, Pelargonium graveolens L'Her, has been shown to protect against the oxidative stress induced by alcohol, anticancer alkylating agents and formaldehyde pesticides ( Ben Slima et al, 2013; El-Ashmawy et al, 2007; Kose et al, 2011; Mimica-Dukic et al, 2010 ;  Rezvanfar et al, 2008).
Traditional medicines have also been used as contraceptive agents in both men and women and as an abortifacient since the 7th century, but only a few studies have directly investigated the effects of herbal compounds on sperm functionality. Furthermore, only a few of these studies focused on essential oils. The administration of essential oils from various plants (Satureja khuzestanica, lavender, geranium or fennel) has been shown to protect male fertility against the reproductive damage induced in vivo by anticancer alkylating agents or pesticides ( Ben Slima et al, 2013; Kose et al, 2011; Rezvanfar et al, 2008 ;  Tripathi et al, 2013). In the physiological context, only one study has been conducted in vivo to date ( Abdollahi et al., 2003). This study reported a positive effect of the administration of herbal essential oil to healthy, adult male rats. Hence a stimulatory effect was reported for essential oil extracted from Satureja khuzestanica and administrated orally to male rats over a period of 45 days at doses of 75, 150, and 225 mg/kg/day. The weight of the reproductive organs (testes, prostate, seminal vesicles), sperm counts and the concentrations of FSH and testosterone were significantly higher in the treated groups than in the control groups ( Abdollahi et al, 2003 ;  Haeri et al, 2006). Cinnamon oil was also found to improve sperm parameters of Wistar rats when administered by gavage at a dose of 100 mg/kg/day during 10 weeks (Yuce et al., 2013). No such stimulatory properties, however, were observed when adult male rats were treated with 375–1500 mg/kg/day of essential oil from Schinus terebinthifolius administered orally during 60 days and no reproductive toxicity was reported ( Affonso et al., 2012), suggesting a specific pro-spermatic effect of the compounds from Satureja khuzestanica and cinnamon.
In contrast to the above studies conducted in vivo, all the studies conducted in vitro to date have reported a deleterious effect of essential oil when directly applied to spermatozoa. Hence, a preliminary study conducted by Buch et al. in 1988 revealed a spermicidal effect of volatile oils extracted from cinnamon, basil, clove and peppermint, whereas no such effect was observed for fixed oils ( Buch et al., 1988). Riar et al. (1990) also showed that the essential oil extracted from neem (fraction NIM-76) decreased the motility of spermatozoa from rats and humans, at doses of 0.25 and 25 mg/ml, respectively. This fraction was subsequently shown to induce damage to the cell membrane in association with the spermicidal effect (Sharma et al., 1996). More recently, Paul, Kang, 2011 ;  Paul, Kang, 2012 showed a spermicidal effect of ajowan fruits (Trachyspermum ammi) collected in India. The essential oil of T. ammi was found to immobilize human spermatozoa instantly in vitro, at very low concentrations, given that the minimum effective dose in this study was 125 µg/ml. The effect of T. ammi essential oil was also shown to be irreversible.
In the context of increasing demands for the use of traditional medicine in many countries, and in particular in developing countries where it could constitute an effective solution in meeting the public health demands and the financial costs of development, there is a clear need to characterize these compounds and their biological properties both in vivo and in vitro. We focus here on Thymus munbyanus Boiss. & Reut., a plant commonly used in North Africa and several other countries, in the form of infusions or syrups, to treat coughs; the biological properties of which remain poorly defined. Previous studies with essential oils extracted from Thymus have shown this plant to have antioxidant and anti-microbial properties in vitro ( Benchabane et al, 2012 ;  Hazzit et al, 2006). As part of a comprehensive study aiming to define the properties of T. munbyanus in various cell types and organs, the effect of T. munbyanus essential oil on human sperm integrity and functionality in vitro was analysed to determine the potential utility of these oils in the field of reproductive biology, as pro- or anti-spermatic agents.

Materials and methods

Plant material

Aerial parts (leaves and inflorescences) of T. munbyanus Boiss. Reut. were collected in July 2012 at Ouzellaguen (800–1000 m above sea level) in the Kabylie region. The taxonomic identity of the plants collected was confirmed by comparison with voucher specimens of known identity already deposited in the Herbarium of the Botany Department of the National Superior School of Agronomy (ENSA, Algiers) and authenticated by Professor H Abdelkrim.

Essential oil isolation

Leaves and inflorescences (100 g) were subjected to hydrodistillation for 3 h with a Clevenger-type apparatus, as described by the European Pharmacopoeia (European Pharmacopoeia Commission. European Pharmacopoeia 5th Ed. Council of Europe: Strasbourg Cedex, France, 2004). At the end of the distillation procedure, the oil was collected, dried with anhydrous Na2SO4, quantified and transferred to a glass flask that was filled to the top with the oil and kept at a temperature of 4°C for further analysis.

Essential oil analyses

Gas chromatography

Gas chromatography analysis was carried out on a Chrompack CP 9002 gas chromatograph equipped with an HP5 MS (cross-linked with 5% phenyl methyl siloxane) capillary column (30 m × 0.25 mm, 0.25 mm film thickness). Column temperature was initially maintained at 60°C for 8 min, then gradually increased to 280°C at a rate of 2°C/min, and the column was maintained at this final temperature for 30 min. The injector and flame ionization detector temperatures were 250°C and 280°C, respectively. Nitrogen was used as the carrier gas, at a flow rate of 1 ml/min, and 0.2 µl of the sample was injected in split mode. The percentage composition of the oil was calculated from gas chromatography peak areas without the use of correction factors.

Gas chromatography–mass spectrometry

Gas chromatography–mass spectrometry (GC-MS) analyses was carried out with an Agilent 6890 series apparatus GC system (Agilent Technologies, Santa Clara, CA) interfaced with a quadrupole mass spectrometer (model HP 5973) equipped with an HP5 MS (cross-linked with 5% phenyl methyl siloxane) capillary column (30 m × 0.25 mm, 0.25 mm film thickness). The conditions for GC-MS were as follows: helium as the carrier gas, at a flow rate of 0.5 ml/min; split mode (1:20); 0.2 µl without dilution as the injected volume; 250°C as the injection temperature. The oven temperature programme is described in the gas chromatography section. An ionization mode with electronic impact was used at 70 eV over a scan range of 30–550 atomic mass units. The temperature of the ion source was 175°C. Oil constituents were identified by comparing their gas chromatography Kovats retention indices, determined with reference to a homologous series of C-C n-alkanes (C8-C17), with those of available authentic standards and published data (Babushok et al., 2011). The identification of the constituents was confirmed by comparing their mass spectral fragmentation patterns with those stored in the mass spectrometry database (NIST 2005 and Wiley 7N libraries) and with published mass spectra.

Semen samples

The study was conducted in accordance with ethical guidelines, and informed consent was obtained from all individuals included in the study. Semen samples were obtained by masturbation, after 2–5 days of abstinence, and were allowed to liquefy for 30 min before examination. All samples were prepared and evaluated in line with the recommendations and current values of the World Health Organization (WHO) (Cooper et al., 2010).

Sperm vitality and motility

Semen samples from five individuals with normal sperm parameters according to WHO values (Cooper et al., 2010) were incubated with an equal volume of T. munbyanus dilutions of essential oil or thymol.
Solutions of T. munbyanus essential oil were prepared in 5% dimethyl sulfoxide (DMSO) at concentrations of 100, 250, 500, 750 and 1000 µg/ml; control samples were incubated with 5% DMSO.
Solutions of synthetic thymol (Biochem Chemopharma, Montreal, Quebec, Canada) at concentrations of 100, 200, 300, 400 and 500 µg/ml were prepared in ethanol; control samples were simultaneously prepared and tested in parallel by incubating semen samples with solutions of 2, 4, 6, 8 and 10% ethanol (corresponding to the concentrations of pure ethanol present in each dilutions of thymol).
The percentages of motile and viable spermatozoa were assessed for each dilution, immediately after the addition of the essential oil (t = 0 min) and 30 min later (t = 30 min). The graphs show the statistics for the total sperm population, for the five individuals. All the experiments were conducted in duplicate.
Sperm vitality was assessed according to WHO values (Cooper et al., 2010) by eosin–nigrosin staining (Vita Eosine, RAL Instruments) on sperm smears (100 spermatozoa were analysed for each individual, each time point and each dilution). Sperm motility was assessed by computer-aided sperm analysis with Hamilton Thorne version 10 HTM IVOS Analyzer (Hamilton-Thorne Biosciences, Beverly, MA USA). The total number of spermatozoa analysed for T. munbyanus essential oil was 54,701 (27,590 for t = 0; 27,111 for t = 30; 10,491 for 0 µg/ml; 10,759 for 100 µg/ml; 9236 for 250 µg/ml; 9978 for 500 µg/ml, 9677 for 750 µg/ml and for 4560 for 1000 µg/ml). The total number of spermatozoa analysed for the thymol was 59,660 (30,948 for t = 0; 28,712 for t = 30; 13,759 for 0 µg/ml; 13,005 for 100 µg/ml; 11,736 for 200 µg/ml; 10,242 for 300 µg/ml; for 7658 for 400 µg/ml and for 3260 for 500 µg/ml). The percentage of motile and progressive spermatozoa was recorded together with the average path velocity (VAP), straight line velocity (VSL) and curvilinear velocity (VCL). The settings of the analyzer were as follows: frame rate, 60 Hz; frame acquired, 30; straighness threshold, 80%; medium VAP cut-off, 25 µm/s; temperature, 37°C.
The effective concentrations of essential oil and thymol causing 50% of the spermatozoa to become immobilized, EC50, were determined from the motility assay.

Statistical analysis

Unpaired Student's t-tests were carried out on the global population of spermatozoa. The significance of the effect of the essential oil on sperm vitality and motility, taking the individual into account, was also determined by analysis of variance. P < 0.05 was considered statistically significant.

Capacitation assay

Capacitation assay was conducted as previously described (Lhuillier et al., 2009). Spermatozoa 4 × 106 were washed from fresh ejaculate, and then suspended in 200 µl of Ferticult-IVF medium (FertiPro NV, Beernem, Belgium). The resulting 200 µl suspension was mixed with an equal volume of one of the following solutions: Ferticult-IVF medium, 5% DMSO solution or a solution of 1000 µg/ml essential oil diluted in 5% DMSO. The samples were then incubated for 3 h at 37°C in 5% CO2 (t = 3h); control samples (t = 0) were immediately processed for protein hyperphosphorylation and acrosomal reaction detection.

Detection of hyperphosphorylation by western blotting

Sperm samples collected at t = 0 and t = 3 h were washed with cold phosphate-buffered saline supplemented with a cocktail of phosphatase inhibitors (PhosphoSTOP, Roche-Boehringer) and centrifuged at 11000 g for 10 min. Protein extracts were obtained by lysing the pellet in Laemmli buffer. They were subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE; 1 × 106 cells/lane) and the gel was then transferred to nitrocellulose membranes for anti-phosphotyrosine immunoblotting with the 4G10 antibody (1:1000; Millipore).

Detection of the acrosomal reaction by immunofluorescence assays

Sperm collected at t = 0 and t = 3 h of capacitation were first stained with SYBR 14 dye for 10 min at 37°C to detect live spermatozoa (LIVE-DEAD sperm viability kit, Molecular Probes), then spread onto Superfrost Plus slides (Menzel Glasbearbeitungswerk, GmbH & Co. KG, Braunschweig, Germany). The slides were briefly dried and fixed by incubation for 5 min in 4% paraformaldehyde solution. The slides were then incubated in a 5 µg/ml solution of rhodamine-labeled Pisum sativum agglutinin (PSA; Vector Laboratories, Burlingame, USA) for 15 min, washed in distilled water and mounted in Vectashield medium (Vector Laboratories, Burlingame, USA) supplemented with 0.5 mg/ml 40,6-diamidino-2-phenylindole (DAPI). An average of 50 SYBR14-stained spermatozoa (live sperm) were counted and the percentage of spermatozoa undergoing the acrosome reaction was determined by the proportion of Rhodamine-PSA negative spermatozoa (acrosome reacted) for each conditions.

Transmission electron microscopy (TEM)

Spermatozoa 2 × 107 was inclubated from fresh ejaculate at 37°C for 30 min, with an equal volume of one of the following solutions: 5% DMSO solution or a 1000 µg/ml solution of essential oil in 5% DMSO. The spermatozoa were then washed with M2 medium (Sigma-Aldrich Co. Ltd, Irvine, UK), centrifuged at 300 g for 10 min and fixed by incubation in 0.1 M phosphate buffer pH 7 supplemented with 3% glutaraldehyde (Grade I; Sigma-Aldrich Co.) for 1.5 h at room temperature. The samples were then washed twice in phosphate-buffered saline and resuspended in 0.2 mol/l sodium cacodylate buffer. The samples were then post-fixed by incubation with 1% osmium tetroxide (Electron Microscopy Sciences, Hatfield, UK), after which they were dehydrated by immersion in a graded series of alcohol solutions and embedded in Epon resin (Polysciences Inc., Warrington, USA). Semi-thin sections were cut and stained with toluidine blue-Azur II. Ultra-thin sections (90 nm) were cut with a Reichert Ultracut S ultramicrotome (Reichert-Jung AG, Wien, Austria) and were then stained with uranyl acetate and lead citrate. Sections were analysed with a JEOL 1011 microscope, and digital images were acquired with a Gatan Erlangshen CCD camera and Digital Micrograph software.

Results

Isolation and characterization of the essential oil of Thymus munbyanus

The essential oil isolated by hydrodistillation from the aerial parts (leaves and inflorescences) of T. munbyanus was a yellow liquid; a yield of 2.8% (v/w) dry weight was achieved. The percentage composition and modes of identification of the principal oil components (>1%) are presented in Table 1, in order of elution from the HP-5MS column. Gas chromatography and gas chromatography-mass spectrometry analyses resulted in the identification of 64 components, accounting for 98.9% of the composition of the oil. Forty of these components were quantified, the others being present only in trace amounts (<0.1%). The oil contained large amounts of oxygen-containing monoterpenes (63.1%), the next most frequent component being monoterpene hydrocarbons (29.0%). The predominant components detected were thymol (52%), γ- terpinene (11 %), ρ-cymene (8.5%) and carvacrol (5.2%).
Table 1. Percentage composition of Thymus munbyanus oil. a
NumberCompoundRetention indexRelative peak area (%)Identification
1α-Pinene9351.9RI, MS, co-GC
2β-Myrcene9923.7RI, MS, co-GC
3α-Terpinene10171.4RI, MS, co-GC
4ρ-Cymene10268.5RI, MS, co-GC
5Limonene10291.5RI, MS, co-GC
6γ-Terpinene106111.0RI, MS, co-GC
7Linalool11042.3RI, MS, co-GC
8Thymyl methyl ether12352.5RI, MS
9Thymol130252.0RI, MS, co-GC
10Carvacrol13185.2RI, MS, co-GC
11β-Caryophyllene14152.8RI, MS, co-GC
a
Components present at more than 1% are listed in order of elution from the HP 5MS column. Identification: co-GC = co-injection with an authenticated compound; MS = comparison of mass spectra with MS libraries; RI = comparison of retention index with published data. The values shown are the means of three measurements of peak area for a constituent.
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Effects of Thymus munbyanus essential oil on sperm motility and vitality

Sperm motility and vitality were recorded after exposure to the essential oil diluted in DMSO immediately (t = 0) or 30 min after the incubation (t = 30). A significant immobilizing effect of the essential oil was observed at doses of 500 µg/ml and above for all the individuals tested (P < 0.05 to P < 0.001). This effect was dose-dependent and independent of the time of exposure. The immobilizing effect was more pronounced at 30 min of exposure, when a significant immobilizing effect was observed with a lower concentration of essential oil, 250 µg/ml (P < 0.05) ( Figure 1A). Sperm vitality was assessed in parallel and found to be decreased significantly after the addition of 1000 µg/ml essential oil (P < 0.01) (t = 0 min) ( Figure 1B).
Effect of Thymus munbyanus essential oil on human sperm motility and vitality. ...
Figure 1. 
Effect of Thymus munbyanus essential oil on human sperm motility and vitality. Dilutions of T. munbyanus essential oil were prepared in 5% dimethyl sulfoxide (DMSO), at the following concentrations: 100, 250, 500, 750 and 1000 µg/ml. Sperm motility and vitality were recorded immediately (t = 0 min; blue) or 30 min after the addition of the oil (t = 30 minutes; red). All the percentages of motile (panel A) and of live (panel B) spermatozoa are reported as a ratio with respect to the values for the control sample diluted in DMSO alone. Data are expressed as means ± SD (n = 5). The asterisks indicate the significance of P-values: *(P < 0.05), **(P < 0.01), ***(P < 0.001), compared with control (DMSO 5%). EO = essential oil. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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The statistical significance of the results was confirmed in an analysis of variance. Dose (P = 9.5 × 10−124) and the duration of exposure (P = 3.33 × 10−9) to T. munbyanus essential oil affected sperm motility, and dose also affected sperm vitality (P = 2.18 × 10−30). The potency of the sperm-immobilizing effect of exposure to the oil differed between individuals (P = 7.5 × 10−130); hence it was found that sperm motility of all individuals was affected upon exposure to 500 µg/ml (and all concentrations above) of T. munbyanus essential oil but some individuals gave a response to concentrations below 500 µg/ml, consistent with differences in sensitivity between individuals.
Altogether, these results indicate that essential oil from T. munbyanus impairs sperm motility when incubated with sperm at concentrations of 250 µg/ml and above, and is spermicidal at higher dose (1000 µg/ml).

Kinematic analysis of sperm exposed to Thymus munbyanus essential oil

Seman samples were incubated with various concentrations of T. munbyanus essential oil, and computer-aided sperm analysis was carried out to assess the movement of spermatozoa. For each sample, VAP, VSL and VCL was evaluated for each sample, immediately (t = 0 min) or 30 min (t = 30 min) after the addition of the essential oil.
The VAP recorded for the controls at 0 and 30 min, revealed the presence of two major sperm populations corresponding to the rapid and slowly progressive sperm populations (Figure 2A). In these two populations, most of the spermatozoa had a VAP of 25 µm/s (slow spermatozoa) or 95 µm/s (rapid spermatozoa). The addition of the essential oil principally affected the population of rapid spermatozoa, for which a significant decrease in VAP was observed (P < 0.05); in particular for 750 µg/ml essential oil in DMSO, which decreased the VAP from 95 to 65 µm/s, almost eliminating the difference in speed between the two populations of spermatozoa ( Figure 2B).
Sperm kinematics on exposure to Thymus munbyanus essential oil. Computer-aided ...
Figure 2. 
Sperm kinematics on exposure to Thymus munbyanus essential oil. Computer-aided sperm analysis was conducted on human semen incubated with T. munbyanus essential oil diluted in dimethyl sulfoxide (DMSO). The number of spermatozoa with a average path velocity (VAP) from 0 to 150 µm/s is indicated. Control samples treated with 5% DMSO (panel A) contained two populations of spermatozoa corresponding to the slow and rapid populations. The addition of 750 µg/ml essential oil decreased the VAP of the rapid spermatozoa (panel B). EO = essential oil.
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A similar pattern was observed for VSL. The controls displayed two populations of spermatozoa with VSL values of 25 µm/s of 85 µm/s. The addition of 750 µg/ml essential oil abolished the rapid progressive population of spermatozoa (not shown).

Effects of Thymus munbyanus essential oil on sperm capacitation

Protein hyperphosphorylation was assessed along with the percentage of spermatozoa undergoing the acrosomal reaction upon induction of capacitation in a medium containing bicarbonate, calcium and albumin and in the presence of the essential oil. Protein hyperphosphorylation was strongly diminished by the addition of 1000 µg/ml of essential oil to the capacitating medium (Figure 3A), compared with untreated spermatozoa and spermatozoa treated with DMSO only. In addition, by carrying out a double staining with Rhodamine-PSA and SYBR14 dye (Figure 3B; SYBR14 staining not shown), the proportion of spermatozoa performing the acrosome reaction was decreased in the presence of 1000 µg/ml of essential oil. Hence, after induction of capacitation during 3 h, while the percentage of acrosome reacted spermatozoa was of 46,7% for untreated spermatozoa, it was of 30% and 10,9% when the spermatozoa was incubated with DMSO and 1000 µg/ml of essential oil, respectively. Altogether, these findings suggest that T. munbyanus essential oil is deleterious for both the flagellum protein hyperphosphorylation and the potential to perform acrosomal reaction, which are two events essential for sperm capacitation and fertilizing ability.
Effect of Thymus munbyanus essential oil on sperm protein hyperphosphorylation ...
Figure 3. 
Effect of Thymus munbyanus essential oil on sperm protein hyperphosphorylation and acrosomal reaction. Hyperphosphorylation (panel A) and acrosomal reaction (panel B) were analysed after incubating the semen with 1000 µg/ml T. munbyanus essential oil diluted in dimethyl sulfoxide (DMSO) in capacitation medium and compared to untreated spermatozoa and spermatozoa treated with DMSO. Hyperphosphorylation was detected by western blotting with an anti-phosphotyrosine antibody and Hexokinase staining was used as a loading control (panel A). The proportion of spermatozoa with reacted acrosome was determined by double staining with SYBR 14 dye (not shown) and rhodamine-labelled Pisum sativum agglutinin (PSA) (panel B). An average of 50 SYBR14-stained spermatozoa (live sperm) for each condition were analyzed: untreated spermatozoa, spermatozoa treated with DMSO and spermatozoa treated with the essential oil. Among them, the number of spermatozoa with non-reacted acrosome, which were stained with the PSA (strong staining, ++ and intermediate staining, +) and the number of acrosome reacted spermatozoa (no PSA staining, –) were counted and the proportion of acrosomal reacted spermatozoa was calculated. EO = essential oil; t = time.
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Morphological analysis of the spermatozoa exposed to Thymus munbyanus essential oil by TEM

TEM was used to assess the morphology and ultrastructure of the spermatozoa in the presence of the essential oil. Consistent with the reduced percentage of acrosome reacted spermatozoa reported above, a dilation of the acrosome after the incubation of the spermatozoa with 1000 µg/ml T. munbyanus essential oil was observed ( Figure 4C and 4D). No such dilation was observed in the control samples (Figure 4A and 4B). In addition, flagellum defects were not observed, and the plasma membrane seemed normal and intact in the sperm treated with the essential oil (Figure 4F), compared with the control samples (Figure 4E).
Effect of Thymus munbyanus essential oil on sperm morphology and ultrastructure. ...
Figure 4. 
Effect of Thymus munbyanus essential oil on sperm morphology and ultrastructure. Transmission electron microscopy (TEM) was performed on semen samples incubated with 5% dimethyl sulfoxide (DMSO) (A, B and E) or 1000 µg/ml T. munbyanus essential oil diluted in 5% DMSO (C, D and F), over a period of 30 min. Asterisks (*) indicate abnormal sperm heads, displaying dilation of the acrosome vesicle. Arrows indicate the plasma membrane, the acrosome and the inner and outer membrane of the acrosome, the mitochondrial sheath and the fibrous sheath.
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Effects of thymol on sperm motility, vitality and morphology

The effects of the thymol, the major component of the oil, were assesed on sperm motility and vitality as previously conducted with the total extract of T. munbyanus essential oil. The percentage of total progressive spermatozoa was significantly decreased by concentrations of 200 µg/ml (P < 0.001) and above. This effect was dose-dependent and independent of exposure time ( Figure 5A). The percentage of viable spermatozoa decreased similarly, in a dose-dependent manner, beginning at a concentration of 200 µg/ml (P < 0.01) ( Figure 5B).
Effect of thymol on human sperm motility and vitality. Thymol was resuspended in ...
Figure 5. 
Effect of thymol on human sperm motility and vitality. Thymol was resuspended in ethanol and dilutions of thymol were prepared in water at the following concentrations: 100, 200, 300, 400 and 500 µg/ml. For each dilution of thymol, a corresponding ethanol–water control was performed in parallel (2, 4, 6, 8 and 10% ethanol dilutions in water). Sperm motility and vitality were recorded immediately (t = 0 min; blue) or 30 min after addition of the thymol (t = 30 min; red). All the percentages are reported as a ratio with respect to the values for the control sample diluted in pure water. Data are expressed as means ± SD (n = 5). The asterisks indicate the significance of the P-values: **(P < 0.01), ***(P < 0.001) compared with control (water). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Analysis of variance confirmed these results and showed an effect of thymol dose on sperm motility and vitality (P = 4.6 × 10−38; 4.6 × 10−16, respectively), together with an effect of duration of exposure on sperm vitality, as significant differences were observed between the values obtained after 0 and 30 min of exposure to thymol (P = 8.98 × 10−07).
Ultrastructural analysis by TEM revealed the same abnormalities of the acrosome observed with the essential oil, at a thymol concentration of 500 µg/ml (data not shown). Again, the plasma membrane of the flagellum did not appear to be altered (data not shown). Finally, the EC50 value was calculated for thymol and the T. munbyanus essential oil. This value is defined as the effective concentration causing the immobilization of 50% of the spermatozoa in the population. The EC50 value was 223 µg/ml for thymol and 781 µg/ml for T. munbyanus oil. As thymol accounts for 52% of the composition of the essential oil, this finding suggests that the effects of thymol are partly masked by the effects of the other compounds present in the oil.

Discussion

In this study, essential oil from T. munbyanus was extracted by hydrodistillation of the aerial parts and incubated in various concentrations of the oil with freshly ejaculated semen, to investigate its effects on sperm vitality, motility and ability to undergo protein hyperphosphorylation and acrosomal reaction, which constitute two markers of the sperm fertilizing potential.
The essential oil we obtained was composed predominantly of phenolic compounds and contained 52% thymol, consistent with the findings of a previous study by Hazzit et al. (2006) on a sample of T. munbyanus collected from the same geographical location, in which thymol was found to be a rich chemotype (37.7%). These findings, however, conflict with those of Benchabane et al. (2012), who found that carvacrol (35.2%) was more abundant than thymol (18.5%) in a sample collected from Azzazga in northern central Algeria. These differences may be accounted for by the existence of several chemotypes of this species.
The essential oil was diluted in DMSO in a range of concentrations from 100 to 1000 µg/ml and incubated with equal volumes of freshly ejaculated human sperm. It was anticipated that the solvent by itself, and the change of osmolarity induced by the addition of essential oil, could also affect sperm motility and functionality. Hence, from the data collected, we observed that both the DMSO and the addition of pure water significantly decreased sperm motility compared with untreated spermatozoa (P < 0.001 at 750 µg/ml; data not shown). All the results presented are compared with spermatozoa treated with the solvent, in order to take into account both the effect of the DMSO and the change of osmolarity, and to precisely determine the effect of the different dilutions of essential oil.
T. munbyanus oil was found to decrease sperm motility significantly when added at concentrations above 500 µg/ml for all the individuals tested. Sperm hyperactivation and acrosomal reaction was also analysed, which constitute two markers of sperm capacitation. Lower levels of spermatozoon hyperactivation were observed, as indicated by the lack of protein hyperphosphorylation upon incubation in capacitating medium. In addition, the potential to undergo the acrosomal reaction was decreased, in consistence with the abnormal acrosomal dilatation observed by electron microscopy analysis.
Similar results on sperm motility and ultra-structure were obtained when the semen samples were incubated with thymol, the major compound present in T. munbyanus oil. Interestingly, a comparison of the EC50 values of thymol and T. munbyanus essential oil suggested that other compounds present in the essential oil might antagonize or inhibit the intrinsic activity of the thymol. The EC50 value is defined as the effective concentration causing the immobilization of 50% of the spermatozoa in the population. From our experimental data, the EC50 value was 223 µg/ml for thymol and 781 µg/ml for T. munbyanus oil. As thymol accounts for 52% of the composition of the essential oil, we expected the EC50 value of T. munbyanus oil to be lower than what we found (around 446 µg/ml). These finding suggests that the effects of the thymol could be partly masked by the effects of other compounds present in the oil. Further studies of the other compounds, although present in smaller amounts than thymol, may lead to the identification of potent pro-spermatic agents.
The antioxidant, antimicrobial activities of T. munbyanus, or both, have been reported before. A preliminary study carried out by Hazzit et al. in 2006 and aiming to compare the antioxidant and antilisterial effects of volatile oils from Algerian Thymus and from oregano, indicated that T. munbyanus essential oil was the most effective at preventing lipid peroxidation, with efficiency values even higher than those for synthetic antioxidants ( Hazzit et al., 2006). Similarly, Benchabane et al. (2012) recently reported T. munbyanus oil to have potent antioxidant activity when used at high concentrations (600 to 1000 mg/l). Our results indicate that T. munbyanus essential oil and thymol have no protective effects on spermatozoa, instead behaving as potent immobilizing and spermicidal agents.
Components with phenolic structures, such as carvacrol, thymol and eugenol, are also known to inhibit microbial growth (Gallucci et al., 2009). These hydrophobic compounds were shown to be highly active despite their poor solubility in water. In particular, thymol has been shown to interact with cell membranes and phospholipids and to affect membrane permeability, membrane potential and potassium fluxes. It has also been shown to interact directly with several proteins (Hyldgaard et al., 2012).
Sperm motility and capacitation are induced after a series of biochemical and electrophysiological modifications to the plasma membrane and cytoplasm (Fraser, 2010 ;  Visconti et al, 2002). Cholesterol depletion is known to promote the reorganization of membrane phospholipids and complex multiple ion movements, resulting in hyperpolarization of the sperm plasma membrane, intracellular alkalinization and protein hyperphosphorylation (Visconti et al., 1995). Overall, these modifications lead to sperm motility and hyperactivation. Given the hydrophobic properties of essential oils and of thymol, and the described interaction of thymol with the plasma membrane, T. munbyanus essential oil probably interferes with the membrane lipids of the spermatozoa, thereby affecting the cell signalling events normally required to induce sperm motility and hyperactivation.
In conclusion, our results indicate that T. munbyanus oil and thymol do not have protective effects on spermatozoa in vitro, instead constituting potent sperm-immobilizing agents. T. munbyanus oil and thymol could therefore be of potential use in the field of reproductive biology, as vaginal anti-spermatic or spermicidal agents, on condition that they would not be deleterious to the vaginal mucosa. Our analysis also suggests that T. munbyanus oil contains other compounds that antagonize or reduce the efficiency of the thymol and might therefore constitute pro-spermatic agents to further investigate.

Acknowledgements

We thank all the technicians from the Service de Biologie de la Reproduction (Hôpital Cochin) for routine semen sample evaluation. We thank the Cellular Imaging Facility of the Institut Cochin (INSERM U1016, CNRS UMR8104, Université Paris Descartes) for electron microscopy analysis. We thank Denise Escalier for scientific discussions.
This work was supported by Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Université Paris Descartes, Agence Nationale de la Recherche (ANR-12-BSV1-0011-01MUCOFERTIL).

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Corresponding author.

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Aminata Touré is a Research Director (DR2, CNRS) at the Cochin Institute in Paris. She received her PhD in 2000 and worked as a post-doctoral fellow in P. Burgoynes's lab on the role of X and Y multi-copy genes during mouse spermiogenesis. Her current research programmes focus on the physiology and pathophysiology of human spermatozoa.
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