Anethum graveolens Linn. (dill) extract enhances the mounting frequency and level of testicular tyrosine protein phosphorylation in rats

Anethum graveolens Linn. (dill) extract enhances the mounting frequency and level of testicular tyrosine protein phosphorylation in rats 1 2 first-author †E-mail:iamsaard_sitt@yahoo.com †E-mail:iamsaard_sitt@yahoo.com iamsaard_sitt@yahoo.com 2 1 2 3 2 1 2 2 3 2 3 Department of Anatomy, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand Department of Anatomy, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand Integrative Complementary Alternative Medicine Research and Development Group, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand Integrative Complementary Alternative Medicine Research and Development Group, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand Department of Physiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand Department of Physiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand Objective: 2 To investigate the effect of Anethum graveolens (AG) extracts on the mounting frequency, histology of testis and epididymis, and sperm physiology. Methods: 2 Male rats induced by cold immobilization before treating with vehicle or AG extracts [50, 150, and 450 mg/kg body weight (BW)] via gastric tube for consecutive 1, 7, and 14 d were examined for mounting frequency, testicular phosphorylation level by immunoblotting, sperm concentration, sperm acrosome reaction, and histological structures of testis and epididymis, respectively. Results: 2 AG (50 mg/kg BW) significantly increased the mounting frequency on Days 1 and 7 compared to the control group. Additionally, rat testis treated with 50 mg/kg BW AG showed high levels of phosphorylated proteins as compared with the control group. In histological analyses, AG extract did not affect the sperm concentration, acrosome reaction, and histological structures of testis and epididymis. Conclusions: 2 AG extract enhances the aphrodisiac activity and is not harmful to sperm and male reproductive organs. Anethum graveolens Linn. (dill) Mounting frequency Testicular phosphorylation Sperm acrosome reaction Introduction 1 Sexual dysfunction in males results from various stresses and is an important problem in developed and developing countries, including Thailand. In the Western world, a major reason for divorce is the decreased frequency of male mating behaviors (Amato and Previti, 2003 2003 ). Currently, drugs against erectile dysfunction, including sildenafil citrate (also known as Viagra), are commercially available, but there are some side effects on other vital organ and they are expensive (Mirone et al., 2009 2009 ; Aliferis et al., 2012 2012 ). Therefore, the search for alternative remedies, especially medicinal plants that are effective, non-toxic, and inexpensive in treating sexual dysfunction, is required. The physical arousal of males is a very complex mechanism via the neuro-gonadal pathway. At the protein posttranslational level, the increments of protein phosphorylation in the male reproductive system are found before and during arousal (Wang et al., 2007 2007 ). Basically, protein phosphorylation is essential for sperm and testosterone production in testis (Fardilha et al., 2011 2011 ; Yamashita et al., 2011 2011 ). In fertile males, these phosphorylation events are involved in sperm capacitation and acrosome reaction (Morales et al., 2007 2007 ; Bailey, 2010 2010 ). Anethum graveolens (AG) Linn. (also called dill or “Pakchee Lao” in Thais), a vegetable, is commonly used for cooking in Southeast Asia. In addition, AG has been demonstrated to possess significant mucosal protective, antimicrobial, antigastric irritation, and antioxidant activities (Chaurasia and Jain, 1978 1978 ; Möhle et al., 1985 1985 ; Mahran et al., 1992 1992 ; Faber et al., 1997 1997 ; Yazdanparast and Alavi, 2001 2001 ; Hosseinzadeh et al., 2002 2002 ; Delaquis et al., 2002 2002 ). Moreover, previous studies have shown that AG could stimulate milk production and change the estrous cycles (Monsefi et al., 2006 2006 ). Indeed, these reports suggest that AG affects the hypothalamo-gonadal axis in females. However, the effects of AG on the male reproductive system have never been documented. Possibly, AG may also affect such axis on the male system that may result in enhancing male sexual behaviors or affect male reproductive organs. The objective of this investigation is to study the effect of AG on male mounting frequency and its toxicity to sperm morphology and function. Materials and methods 1 Animals and mounting screening 2 Forty adult male rats (200–250 g) provided by the Experimental Animal Unit of Medicine Faculty, Khon Kean University, Thailand were determined for mounting frequency using an active estrous female induced with estrogen as a libido inducer. These animals were divided into four groups. The control or vehicle group was fed with distilled water, while the AG-treated groups received the solution of AG extracts [50, 150, and 450 mg/kg body weight (BW)] via gastric tube. The administration was performed for 14 consecutive days. This study was approved by the Animal Ethics Committee of Khon Kaen University, based on the Ethics of Animal Experimentation of National Research Council of Thailand. Treatment and determination of mounting frequency 2 All animals were stressed by cold immobilization at 4 °C for 30 min to reduce their mounting behaviors before treating with vehicle (polyethylene glycol; PEG diluent) or AG extracts provided by Dr. Panee SRISAARD (Department of Pharmacology, Faculty of Medicine, Chiang Mai University, Thailand). On Days 1, 7, and 14 after AG administration for 30 min, the rats in all groups were examined for the mounting frequency within 1 h after libido stimulation by facing with an active estrous female rat. Histopathologic examinations of testis and epididymis 2 At the end of the experiment, all animals were sacrificed and right testis and epididymis were isolated. The tissues were dissected out and cleaned up to be free from fat pads. They were observed for gross lesions and fixed with 10% formalin in phosphate buffered saline (PBS) (pH 7.4) followed by routine histological processing (5 μm in thickness of the section) and observed under a Nikon ECLIPSE E200 Microscope. Western blot analysis 2 To study the effect of AG extract on the level of testicular protein phosphorylation, the left testis of the mounting-positive group was rapidly kept at 4 °C. Then, the testicular total protein lysate was prepared by homogenization of testis with cocktail protease inhibitors added-RIPA buffer (Cell Signaling Technology Inc., USA). The testicular homogenate was centrifuged at 12 000 r/min for 10 min and the supernatant (called testicular total protein lysate) was collected to measure the total protein concentration by NANO drop (NanoDrop ND-1000 Spectrophotometer V3.5 User’s Manual, NanoDrop Technologies Inc., USA). Fifty micrograms of total testicular proteins pooled from triplicate samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto nitrocellulose membrane to detect the intensity of phosphorylation by using the 4G10 primary antibody [1:3 000 (v/v), Millipore Corporation, USA]. In immuno Western blotting, bovine serum albumin (BSA; AMRESCO®, USA) and epidermal growth factors (EGFs; Millipore Corporation, USA) were used as the negative control and positive control, respectively. To detect the phosphorylated proteins, the enhanced chemiluminescence (ECL) substrate was used before visualization under Gel Doct 4 (ImageQuant 400, GH Healthcare, USA). Sperm concentration and acrosome reaction analyses 2 Sperm from the right epididymis and vas deferens were dipped into 1 ml KSOM medium (Millipore). Sperm suspension was diluted (1:20, v/v) with PBS before double counting on hematocytometer under light microscope. In the acrosome reaction assay (Bendahmane et al., 2002 2002 ; Iamsaard et al., 2011 2011 ), the sperm suspension was centrifuged at 500× g for 5 min, and the pellet (composed of sperm cells) was collected. The sperm pellet was fixed with 4% (0.04 g/ml) para form aldehyde-PBS, pH 7.4, at room temperature for 15 min and washed twice by PBS. The sperm suspension was dropped and air-dried on a 0.25% (2.5 g/L) gelatin-coated slide. The dried sperm cells were stained by 0.22% (2.2 g/L) Coomassie blue G-250 (containing 50% CH 3 OH, 10% glacial acetic acid, 40% H 2 O) for 5 min. The slide was mounted with PBS-glycerol, and observed under a light microscope. Sperm with an intact acrosome showed blue staining at the sperm head, and the acrosomes reacted sperm had no acrosome staining. To determine the percentage of acrosome-reacted sperm, 300 sperm were observed and counted from each slide. Statistical analysis 2 The data were statistically analyzed using the Sigma stat package. One-way analysis of variance (ANOVA) was used to test the hypothesis and the significant difference was considered where P -values were < 0.05. The data were expressed as mean±standard deviation (SD). Results 1 Effects of AG extract on mounting frequency 2 Male rats that received AG extract had increased mounting frequency (Fig. 1 1 ). On Day 1, AG extracts increased the mounting frequency in all doses. The highest mounting frequency was achieved at the dose of 50 mg/kg BW. This effect was reduced on Day 7 but only the 50 mg/kg BW dose induced a significant difference in the mounting frequency. On Day 14, no effect of the extract was shown (Fig. 1 1 ). Each bar represents the mean±SD ( n =10) in each group. ** P < 0.01, * P < 0.05, compared to the vehicle-treated controls Testicular weight and histology of testis and epididymis 2 Toxicity effects of AG extract on testes and epididymis were examined by observing both gross and histopathological structures. After treatment for consecutive 14 d, AG did not change the testicular weights (both right and left sides) as compared to the control (Fig. 2 2 ). In histological results, the control and AG-treated rats showed mostly testicular epithelium with an orderly arrangement of spermatogenic cells and Sertoli cells (Fig. 3 3 ). In addition, no histopathologic alterations (normal epididymal cell arrangement and sperm density) were observed in ductus epididymis of the control or AG-treated rats (Fig. 4 4 ). Data were presented as mean±SD ( n =10) in each group. No significant difference between vehicle-treated control group and AG-treated groups was detected No histological alterations are observed in testes of the control [(a) untreated and (b) vehicle] and AG-treated male rats [(c) 50, (d) 150, and (e) 450 mg/kg BW, respectively] No histological alterations are observed in the epididymis of the control [(a) untreated and (b) vehicle] and AG-treated male rats [(c) 50, (d) 150, and (e) 450 mg/kg BW, respectively] Effects of AG extract on testicular protein phosphorylation 2 As shown in Fig. 1 1 , 50 mg/kg BW AG extract significantly increased the mounting frequency on Days 1 and 7. The testes in this group were selected to investigate the level of protein phosphorylation. The testicular lysate of rats treated with 50 mg/kg BW AG extract showed some phosphorylated protein broad bands as compared to those of the control (Fig. 5 5 ). Bovine serum albumin (BSA) and epidermal growth factor-like growth factor (EGF) were used as negative and positive controls for anti-phosphotyrosine, respectively Sperm concentration and acrosome reaction (AR) status 2 The sperm concentration and AR status were assessed to investigate the direct effect of AG extract on sperm physiology. The results show that rats that received AG extracts are not significantly different in both sperm concentration and AR status (Fig. 6 6 ; Table 1 1 ) as compared to the control. (a) Acrosome intact sperm stained with Coomassie blue; (b) Acrosome-reacted sperm unstained Sperm concentrations were presented as mean±SD ( n =10) in each group Group Sperm concentration (×10 6 cells/ml) Acrosome reaction (%) Control 30±8.4556 4.12±2.4598 Dill (50 mg/kg BW) 31±7.3546 3.17±1.3696 Dill (450 mg/kg BW) 39±9.5539 5.16±1.2697 Discussion 1 The result in Fig. 1 1 suggests that low concentration of AG extract could effectively stimulate the mounting frequency with short term treatment. Herein whether a high dose of AG extract and increasing days could not stimulate mounting frequency remains unexplained. It has been suggested that vasodilatation, generation of nitric oxide, and/or elevation of androgens and gonadotropins are a major mechanism involved in aphrodisiac activity of many medicinal plants (Yakubu and Akanji, 2011 2011 ; Prabsattroo et al., 2012 2012 ). Basically, sexual behaviors are the result of stimulation of the central nervous system (CNS). Previously, Monsefi et al. ( 2006 2006 ) demonstrated that AG extract changed the estrous cycle and increased milk production during lactation in female rats, suggesting that dill stimulated some regions of CNS, especially via the hypothalamic axis (Benelli et al., 1995 1995 ; Cruz-Casallas et al., 1999 1999 ). In the same vein, AG extract may also stimulate the part of the CNS that is particularly responsible for the mounting behavior in male rats, as shown in the present study (Fig. 1 1 ). Similar to Moringa oleifera leaves extract activity (Prabsattroo et al., 2012 2012 ), our results also showed a significant increase of mounting frequency with a low dose of AG on Day 1 and declining on Day 14, as compared with the control group (Fig. 1 1 ). Since this frequency was not a dose-dependent phenomenon, it is possible that the mounting frequency in rats might be responsible for AG administration at the low dose and short time period. However, the actual mechanism of mounting frequency stimulated by low AG is being investigated. At the end of experiment (Day 14), the testes and epididymis of all groups were examined to observe the morphologic alterations. In Figs. 2 2 – 4 4 , the normal gross and histological results suggested that AG extracts were not harmful to any of the tissues studied (i.e., testes and epididymis). The increasing levels of posttranslational proteins are involved in sperm maturation and testosterone production (Jones et al., 2008 2008 ; Fardilha et al., 2011 2011 ; Yamashita et al., 2011 2011 ). In the present study, AG extract enhanced the protein phosphorylation level in testicular lysate (Fig. 5 5 ), suggesting that AG might stimulate tyrosine phosphorylation of testicular proteins involved in spermatogenesis and androgen synthesis. Although sperm concentration in the AG-treated group did not increase (Table 1 1 ), it is possible that they may be involved in testosterone synthesis. However, the testosterone levels in this study have not been measured. We assumed that the androgen levels in the serum of rats treated with AG extracts existed at a very small quantity, since there were no differences in mounting frequency on Day 14 (Fig. 1 1 ). If the androgen serums in this group were collected on Day 1 or 4, the testosterone levels responsible for male libido would be significantly increased. Similar to the morphological results, Fig. 6 6 and Table 1 1 revealed that AG extract did not affect the sperm physiology, especially sperm acrosome reaction status, suggesting that it may not cause male infertility. Taken together, AG extract is harmless to sperm production and acrosome exocytosis. In conclusion, Anethum graveolens (AG) Linn. extract can enhance the mounting frequency and testicular protein phosphorylation in male rats. Indeed, this vegetable extract is not toxic to testes, epididymis, sperm production, or sperm physiology. References K Aliferis , IK Petropoulos , B Farpour , MA Matter , AB Safran . Should central serous chorioretinopathy be added to the list of ocular side effects of phosphodiesterase 5 inhibitors? . Ophthalmologica , 2012 . 227 ( 2 ): 85 - 89 . 22156704 DOI: 10.1159/000333824 http://doi.org/10.1159/000333824 . Should central serous chorioretinopathy be added to the list of ocular side effects of phosphodiesterase 5 inhibitors? PR Amato , D Previti . People’s reasons for divorcing gender, social class, the life course, and adjustment . J Fam Issues , 2003 . 24 ( 5 ): 602 - 626 . DOI: 10.1177/0192513X03254507 http://doi.org/10.1177/0192513X03254507 . People’s reasons for divorcing gender, social class, the life course, and adjustment JL Bailey . Factors regulating sperm capacitation . Syst Biol Reprod Med , 2010 . 56 ( 5 ): 334 - 348 . 20849222 DOI: 10.3109/19396368.2010.512377 http://doi.org/10.3109/19396368.2010.512377 . Factors regulating sperm capacitation M Bendahmane , HT Zeng , DRP Tulsiani . Assessment of acrosomal status in rat spermatozoa: studies on carbohydrate and non-carbohydrate agonists . Arch Biochem Biophys , 2002 . 404 ( 1 ): 38 - 47 . 12127067 DOI: 10.1016/S0003-9861(02)00278-3 http://doi.org/10.1016/S0003-9861(02)00278-3 . Assessment of acrosomal status in rat spermatozoa: studies on carbohydrate and non-carbohydrate agonists A Benelli , A Bertolini , R Poggioli , E Cavazzuti , L Calza , L Giardino , R Arletti . Nitric oxide is involved in male sexual behavior of rats . Eur J Pharmacol , 1995 . 294 ( 2-3 ): 505 - 510 . 8750712 DOI: 10.1016/0014-2999(95)00578-1 http://doi.org/10.1016/0014-2999(95)00578-1 . Nitric oxide is involved in male sexual behavior of rats SC Chaurasia , PC Jain . Antibacterial activity of essential oils of four medicinal plants . Ind J Hosp Pharm , 1978 . 15 166 - 168 . . Antibacterial activity of essential oils of four medicinal plants PE Cruz-Casallas , AG Nasello , EE Hucke , LF Felicio . Dual modulation of male sexual behavior in rats by central prolactin: relationship with in vivo striatal dopaminergic activity . Psychoneuroendocrinology , 1999 . 24 ( 7 ): 681 - 693 . 10451905 DOI: 10.1016/S0306-4530(99)00021-9 http://doi.org/10.1016/S0306-4530(99)00021-9 . Dual modulation of male sexual behavior in rats by central prolactin: relationship with in vivo striatal dopaminergic activity PJ Delaquis , K Stanich , B Girard , G Mazza . Antimicrobial activity of individual and mixed fractions of dill, cilantro, coriander and eucalyptus essential oils . Int J Food Microbiol , 2002 . 74 ( 1-2 ): 101 - 109 . 11929164 DOI: 10.1016/S0168-1605(01)00734-6 http://doi.org/10.1016/S0168-1605(01)00734-6 . Antimicrobial activity of individual and mixed fractions of dill, cilantro, coriander and eucalyptus essential oils B Faber , K Bangert , A Mosandl . GC-IRMS and enantioselective analysis in biochemical studies in dill ( Anethum graveolens L.) . Flav Frag J , 1997 . 12 ( 5 ): 305 - 314 . DOI: 10.1002/(SICI)1099-1026(199709/10)12:5<305::AID-FFJ659>3.3.CO;2-Z http://doi.org/10.1002/(SICI)1099-1026(199709/10)12:5<305::AID-FFJ659>3.3.CO;2-Z . GC-IRMS and enantioselective analysis in biochemical studies in dill (Anethum graveolens L.) M Fardilha , SL Esteves , L Korrodi-Gregório , AP Vintém , SC Domingues , S Rebelo , N Morrice , PT Cohen , OA da Cruze Silva , EF da Cruz e Silva . Identification of the human testis protein phosphatase 1 interactome . Biochem Pharmacol , 2011 . 82 ( 10 ): 1403 - 1415 . 21382349 DOI: 10.1016/j.bcp.2011.02.018 http://doi.org/10.1016/j.bcp.2011.02.018 . Identification of the human testis protein phosphatase 1 interactome H Hosseinzadeh , GR Karimi , M Ameri . Effects of Anethum graveolens L. seed extracts on experimental gastric irritation models in mice . BMC Pharmacol , 2002 . 2 21 12493079 DOI: 10.1186/1471-2210-2-21 http://doi.org/10.1186/1471-2210-2-21 . Effects of Anethum graveolens L. seed extracts on experimental gastric irritation models in mice S Iamsaard , R Vanichviriyakit , G Hommalai , A Saewu , N Srakaew , B Withyachumnarnkul , A Basak , N Tanphaichitr . Enzymatic activity of sperm proprotein convertase is important for mammalian fertilization . J Cell Physiol , 2011 . 226 ( 11 ): 2817 - 2826 . 21302280 DOI: 10.1002/jcp.22626 http://doi.org/10.1002/jcp.22626 . Enzymatic activity of sperm proprotein convertase is important for mammalian fertilization R Jones , PS James , D Oxley , J Coadwell , F Suzuki-Toyota , EA Howes . The equatorial subsegment in mammalian spermatozoa is enriched in tyrosine phosphorylated proteins . Biol Reprod , 2008 . 79 ( 3 ): 421 - 431 . 18448843 DOI: 10.1095/biolreprod.107.067314 http://doi.org/10.1095/biolreprod.107.067314 . The equatorial subsegment in mammalian spermatozoa is enriched in tyrosine phosphorylated proteins GH Mahran , HA Kadry , CK Thabet , MM El-Olemy , MM Al-Azizi , PL Schiff , LK Wong , N Liv . GC/MS analysis of volatile oil of fruits of Anethum graveolens . Pharm Biol , 1992 . 30 ( 2 ): 139 - 144 . DOI: 10.3109/13880209209053978 http://doi.org/10.3109/13880209209053978 . GC/MS analysis of volatile oil of fruits of Anethum graveolens V Mirone , F Fusco , A Rossi , R Sicuteri , F Montorsi . Tadalafil and vardenafil vs. sildenafil: a review of patient-preference studies . BJU Int , 2009 . 103 ( 9 ): 1212 - 1217 . 19154480 DOI: 10.1111/j.1464-410X.2008.08267.x http://doi.org/10.1111/j.1464-410X.2008.08267.x . Tadalafil and vardenafil vs. sildenafil: a review of patient-preference studies B Möhle , W Heller , E Wellmann . UV-induced biosynthesis of quercetin 3- O -β- D -glucuronide in dill Anethum graveolens cell cultures . Phytochemistry , 1985 . 24 ( 3 ): 465 - 468 . DOI: 10.1016/S0031-9422(00)80748-7 http://doi.org/10.1016/S0031-9422(00)80748-7 . UV-induced biosynthesis of quercetin 3-O-β-D-glucuronide in dill Anethum graveolens cell cultures M Monsefi , M Ghasemi , A Bahaoddini . The effects of Anethum graveolens L. on female reproductive system . Phytother Res , 2006 . 20 ( 10 ): 865 - 868 . 16835877 DOI: 10.1002/ptr.1959 http://doi.org/10.1002/ptr.1959 . The effects of Anethum graveolens L. on female reproductive system P Morales , ES Díaz , M Kong . Proteasome activity and its relationship with protein phosphorylation during capacitation and acrosome reaction in human spermatozoa . Soc Reprod Fertil Suppl , 2007 . 65 269 - 273 . 17644968 . Proteasome activity and its relationship with protein phosphorylation during capacitation and acrosome reaction in human spermatozoa T Prabsattroo , J Wattanathorn , S Iamsaard , S Muchimapura , W Thukhammee . Moringa oleifera leaves extract attenuates male sexual dysfunction . Am J Neurosci , 2012 . 3 ( 1 ): 17 - 24 . DOI: 10.3844/amjnsp.2012.17.24 http://doi.org/10.3844/amjnsp.2012.17.24 . Moringa oleifera leaves extract attenuates male sexual dysfunction D Wang , Y Noda , H Tsunekawa , Y Zhou , M Miyazaki , K Senzaki , T Nabeshima . Behavioural and neurochemical features of olfactory bulbectomized rats resembling depression with comorbid anxiety . Behav Brain Res , 2007 . 178 ( 2 ): 262 - 273 . 17261334 DOI: 10.1016/j.bbr.2007.01.003 http://doi.org/10.1016/j.bbr.2007.01.003 . Behavioural and neurochemical features of olfactory bulbectomized rats resembling depression with comorbid anxiety MT Yakubu , MA Akanji . Effect of aqueous extract of Massularia acuminate stem on sexual behavior of male Wistar rats . Evid Based Complement Alternat Med , 2011 . ID738103 1 - 10 . DOI: 10.1155/2011/738103 http://doi.org/10.1155/2011/738103 . Effect of aqueous extract of Massularia acuminate stem on sexual behavior of male Wistar rats S Yamashita , P Tai , J Charron , C Ko , M Ascoli . The Leydig cell MEK/ERK pathway is critical for maintaining a functional population of adult Leydig cells and for fertility . Mol Endocrinol , 2011 . 25 ( 7 ): 1211 - 1222 . 21527500 DOI: 10.1210/me.2011-0059 http://doi.org/10.1210/me.2011-0059 . The Leydig cell MEK/ERK pathway is critical for maintaining a functional population of adult Leydig cells and for fertility R Yazdanparast , M Alavi . Antihyperlipidaemic and antihypercholesterolaemic effects of Anethum graveolens leaves after the removal of furocoumarins . Cytobios , 2001 . 105 185 - 191 . 11409638 . Antihyperlipidaemic and antihypercholesterolaemic effects of Anethum graveolens leaves after the removal of furocoumarins We thank the Complementary and Alternative Medicine Research Group, Faculty of Medicine, Khon Kaen University for providing AG extract. 10.1631/jzus.B1200287 Project (No. 53-03-2-00-008) supported by the Office of Research Administration, Khon Kaen University, Thailand 2012-10-19 2013-02-01 2012-12-04 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2013 2013 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 03 14