In-vitro effects of Thymus munbyanus essential oil and thymol on human sperm motility and function  Amirouche Chikhoune, Laurence Stouvenel, Mokrane Iguer-Ouada, Mohamed Hazzit, Alain Schmitt, Patrick Lorès, Jean Philippe Wolf, Kamel Aissat, Jacques Auger, Daniel Vaiman, Aminata Touré  Reproductive BioMedicine Online  Volume 31, Issue 3, Pages 411-420 (September 2015) DOI: 10.1016/j.rbmo.2015.06.011 Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

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.) Reproductive BioMedicine Online 2015 31, 411-420DOI: (10.1016/j.rbmo.2015.06.011) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

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. Reproductive BioMedicine Online 2015 31, 411-420DOI: (10.1016/j.rbmo.2015.06.011) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

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. Reproductive BioMedicine Online 2015 31, 411-420DOI: (10.1016/j.rbmo.2015.06.011) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

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. Reproductive BioMedicine Online 2015 31, 411-420DOI: (10.1016/j.rbmo.2015.06.011) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

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.) Reproductive BioMedicine Online 2015 31, 411-420DOI: (10.1016/j.rbmo.2015.06.011) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions