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Cynomorium songaricum induces spermatogenesis with glial cell-derived neurotrophic factor (GDNF) enhancement in rat testes
Journal of Ethnopharmacology 128 (2010) 693–696
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Ethnopharmacological communication
Cynomorium songaricum induces spermatogenesis with glial cell-derived neurotrophic factor (GDNF) enhancement in rat testes Woong Mo Yang a , Ha Young Kim a , Soo Yeon Park b , Hyung-Min Kim c , Mun Seog Chang a , Seong Kyu Park a,∗ a b c
Department of Prescriptionology, College of Oriental Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea Sports Medicine Center, East-West Neo Medical Center, Kyung Hee University, Seoul 130-701, Republic of Korea Department of Pharmacology, College of Oriental Medicine, Institute of Oriental Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 13 November 2009 Received in revised form 16 February 2010 Accepted 18 February 2010 Available online 26 February 2010 Keywords: Cynomorium songaricum Glial cell-derived neurotrophic factor Spermatogenesis
a b s t r a c t Aim of the study: Cynomorium songaricum Ruprecht has been used in traditional Korean medicine to treat male infertility, including sexual dysfunction, by improving kidney function. Glial cell-derived neurotrophic factor (GDNF) produced by Sertoli cells induces the proliferation of undifferentiated spermatogonia. We investigated the effects of Cynomorium songaricum on sperm parameters and GDNF expression in rat testes. Materials and methods: Sperm analysis, RT-PCR, and Western blotting assays were performed after administration of CS to 8-week-old male Wistar rats for 56 consecutive days (1.0 g/kg/day, p.o.), the period of sperm formation in the rat. Results and conclusions: The CS-treated animals showed significant increases in epididymal sperm count and absolute testes weights compared to the control group. CS also increased the expression of GDNF at both the mRNA and protein levels. These results suggest that CS may improve male fertility by enhancing spermatogenesis and GDNF expression. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The incidence rates of male infertility are increasing worldwide, the leading cause of which is low sperm count. It has been reported that sperm counts in Western countries have decreased by 1% annually over the last 50 years (Carlsen et al., 1992). Although there have been many clinical trials aim at improving the number and quality of sperm, they have met with only limited success. Recent studies on rats have indicated that male fertility, including sexual dysfunction, can be improved by medicinal herbs (Yeh et al., 2008). Glial cell-derived neurotrophic factor (GDNF) is an important growth factor that facilitates communication between Sertoli cells and spermatogonia. GDNF induces the proliferation of undifferentiated spermatogonia under in vivo and in vitro conditions (Meng et al., 2000). In the testis, the GDNF dosage controls the cell fate of undifferentiated spermatogonia (Airaksinen and Saarma, 2002). Gene-targeted mice with one GDNF-null allele show partial depletion of spermatogenic stem cells, whereas those overexpressing GDNF show clusters of undifferentiated spermatogonia. GDNF stimulates DNA synthesis in spermatogonia (Viglietto et al., 2000).
∗ Corresponding author. 2Tel.: +82 2 961 0330; fax: +82 2 961 0536. E-mail address: [email protected] (S.K. Park). 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.02.020
Cynomorium songaricum (CS) is used in traditional Korean medicine as an herbal remedy for kidney and intestinal ailments, as well as impotence (Nickrent et al., 2005) which is a fleshy, monoecious or dioecious, holoparasitic herb that often produces swollen tuberous haustorial root connections to its host plants (Kuijt, 1969). Its chemical constituents include steroids, triterpenes, fructosides, flavonoids, and condensed tannins (Jiang et al., 2001). Several studies have examined the effects of CS extracts on mammalian reproductive cells. In addition, a water extract of CS induced a significant increase in sperm count, improved the percentage of live sperm and their motility, and decreased the number of abnormal sperm (Abdel-Rahman et al., 1999), and there was a marked increase in testicular weight in animals treated with the extracts, which also had a direct spermatogenic influence on the seminiferous tubules of immature rats, presumably by exerting a testosterone-like effect (Abdel-Magied et al., 2001). However, the relationship between CS and its effects on male reproductive tract malfunction associated with GDNF gene expression and spermatogenesis in vivo have not been elucidated. To investigate the effects of CS on male reproductive system in rats, we performed sperm analysis, including testicular weight, sperm count, and sperm motility. In addition, we assessed GDNF expression at the mRNA and protein levels.
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W.M. Yang et al. / Journal of Ethnopharmacology 128 (2010) 693–696
2. Materials and methods 2.1. Preparation of CS extract Cynomorii Herba is the dried fleshy stem of Cynomorium songaricum Ruprecht (Fam. Cynomoiaceae). CS, produced in China, was purchased from Wonkwang Herbal Dug Co. Ltd. (Seoul, Korea). A 300 g sample of dried CS was boiled in 6 L water at 100 ◦ C for 2 h and the suspension was filtered. The filtrate was concentrated under reduced pressure and then lyophilised. The final yield was 26% (dry weight 76.60 g), and the sample was stored at 4 ◦ C. A voucher specimen (SK003) was deposited in our laboratory. Before each experiment, the dried extract was dissolved in distilled deionised water and vortexed for 2 min at room temperature. 2.2. Animals Eight-week-old male Wistar rats were purchased from SLC Inc. (Shizuoka, Japan). The animals were housed in a specific pathogenfree environment with a 12/12 h light/dark cycle at the Center for Laboratory Animal Care and Use of Kyung Hee University. Animal care and experimental procedures were performed in accordance with the “Guide for the Care and Use of Laboratory Animals” (Department of Health, Education and Welfare, NIH publication #78-23, 1996). Animals had free access to standard rodent pellets (Purina Inc., Korea) and water. After 7 days of acclimation to the environment, the rats were administered CS extract (1.0 g/kg/day, p.o.) dissolved in water for 56 days. The same volume of vehicle was administered to control rats (n = 8). The animals were weighed weekly to adjust the gavage volume and monitor their general health. 2.3. Sperm analysis
bromide using i-MAX gel image analysis system (Core Bio Systems, Seoul, Korea) and analyzed using AlphaEase FC software (Alpha Innotech, San Leandro, CA). The results from at least three separate experiments were used for statistical analysis. 2.5. Western blotting assay for detection of GDNF Proteins from homogenised testes were separated using a nuclear extract kit according to the manufacturer’s protocol with minor modifications (Active Motif, Carlsbad, CA). Equivalent amounts (50 g) of protein extracts were separated in 10% Tris–glycine gels by SDS-PAGE and transferred electrophoretically onto nitrocellulose membranes using 25 mM Tris and 250 mM glycine containing 20% methanol, pH 8.3. Transfer was performed at a constant voltage of 20 V for 1 h. After blotting, nonspecific binding was blocked with 5% skimmed milk in TPBS and the membranes were incubated with anti-GDNF (1:1000; rabbit polyclonal IgG; Abcam, Cambridge, UK) and anti--tubulin (1:2000; rabbit polyclonal IgG; Santa Cruz Biotechnology, Santa Cruz, CA) antibodies overnight at 4 ◦ C, washed, and incubated with secondary horseradish peroxidase-conjugated anti-rabbit IgG antibodies (1:1000; Jackson ImmunoResearch, West Grove, PA) for 2 h. The membranes were then washed and developed with Western blotting chemiluminescent reagents (Thermo Scientific, Rockford, IL), and then exposed to X-ray films (Agfa, Mortsel, Belgium). The films were analyzed with AlphaEase FC software (Alpha Innotech). 2.6. Statistical analysis The results are expressed as the means ± SD. Differences between groups were analyzed using Student’s t-test with P < 0.001, P < 0.01, and P < 0.05 taken to indicate significance as shown in the figures (Fig. 1).
Epididymal sperm count and motility were evaluated as described by Connolly et al. (2005), with some modifications. To determine the sperm count, the entire epididymis from the rats was minced in M199 medium (CureBio, Seoul, Korea) containing 0.5% bovine serum albumin (BSA) and incubated for 5 min at 37 ◦ C. The sperm concentration was determined manually using a hemocytometer (Neubauer, Germany). To assess sperm motility, sperm were recovered from the excised cauda epididymides and allowed to capacitate for 5 min in M199 medium containing 0.5% BSA at 37 ◦ C. Sperm were scored as motile if any movement was detected. The total number of sperm and the number that were motile were determined.
None of the animals died during the study and no noticeable adverse effects were observed in any of the animals after administration of the extract or vehicle. Rats treated with CS showed a slight change in body weight compared to the control group (429.2 ± 34.5 and 420.5 ± 26.9 g, respectively). The absolute weights of the testes of the CS-treated group were slightly but significantly greater than those of the control group (1.64 ± 0.05 and 1.52 ± 0.18 g, respectively; 7.89% increase, P < 0.05).
2.4. RT-PCR for detection of GDNF mRNA
3.2. Effects of CS on sperm counts and sperm motility
Total RNA was extracted from the rat testes. First-strand cDNA synthesis with 2 g total RNA was performed using MMLV reverse transcriptase and oligo dT primer for 1 h at 42 ◦ C. Then PCR amplification was performed. First, 5 L cDNA was added to 2.5 L 10× PCR buffer, 1 L 25 mM MgCl2 , 1 L 2.5 mM dNTP, 0.5 L polymerase (1 U), 1 L each primer (4 pmol), and DEPCtreated distilled water (DEPC-DW) to give a final volume of 25 L. The PCR protocol consisted of an initial 5-min denaturation step at 95 ◦ C followed by 35 cycles of the following: annealing for 1 min at 58 ◦ C, extension at 70 ◦ C for 1 min, followed by denaturation for 45 s 95 ◦ C. The run was concluded by a final extension step at 70 ◦ C for 10 min. The sequences of rat GDNF primers were as follows: 5 -ATGAAGTTATGGGATGTCGT-3 (forward) and 5 -CAGGGTCAGATACATCCACA-3 (reverse). Those for rat -actin were 5 -AGGCATCCTGACCCTGAAGTAC-3 (forward) and 5 -TCTTCATGAGGT AGTCTGTCAG-3 (reverse). PCR products were separated on 1.5% agarose gels, visualised by staining with ethidium
After 56 days of treatment with CS, the epididymal sperm counts were significantly higher (37.8% higher than those in the control group, P < 0.01). However, there was no change in sperm motility compared to the control group.
3. Results 3.1. Effects of CS on body and testicular weights
3.3. Effect of CS on GDNF mRNA and protein levels RT-PCR analysis and Western blotting assays were performed to determine the effects of CS on GDNF gene and protein expression in rat testes. The GDNF fragment was detected as a band of 416 base pairs (bp). GDNF mRNA levels were significantly elevated up to 157.09% in testes from the rats treated with CS (P < 0.05). The level of GDNF mRNA was normalised according to the -actin signal. The expression of GDNF protein in the PG-treated group was significantly higher than that in the control group (119.17%; P < 0.05). The level of GDNF protein was normalised according to the -tubulin signal. These results suggest that CS treatment may improve male
W.M. Yang et al. / Journal of Ethnopharmacology 128 (2010) 693–696
695
Fig. 1. Effects of CS treatment on sperm count and GDNF expression in rat testes. Sperm count was significantly increased (A). Expression of GDNF mRNA in CS-treated group was significantly increased compared to control group (B) and (C), and confirmed by Western blotting assay (D). Analysis of PCR reaction on RNA isolated from rat testes using GDNF and -actin primers. Results are presented as mean ±SD. *The mean differs significantly between control group and CS-treated group (P < 0.05).
reproductive ability by inducing GDNF gene expression and protein biosynthesis. 4. Discussion and conclusion Infertility is an increasing worldwide concern. The leading cause of male infertility is a low sperm count, and sperm counts in Western countries have decreased by 1% annually over the last 50 years (Carlsen et al., 1992). Although many clinical trials have been performed to improve the number and quality of sperm, they have had limited success. We believe that some medicinal herbs may improve male fertility with relatively few side effects. CS has been used in traditional Korean medicine to tone the kidneys, replenish the vital essence and blood, strengthen the yang, benefit the semen, and lubricate the intestines, in the treatment of indications such as impotence, sterility, constipation due to blood deficiency, weakness of the loins and knees, haematuria, and spermatorrhoea (Bensky, 1986). The Chinese name for the herb Cynomorium means “lock the Yang” and refers to its ability to lock up the kidney essence, assuring male potency. In a previous study, CS has been used for management of menopausal symptoms (Zhang et al., 2005). We investigated the effects of CS on spermatogenesis in rats treated with CS for 56 days. The epididymal sperm count of rats in the treatment group increased significantly compared to that of the control group. In addition, the absolute weights of testes in the CS-treated group exhibited slight but significant increases over the control group, suggesting that CS may stimulate the reproductive organs. Sertoli cells, the somatic cells in the seminiferous epithelium, are a major component of the stem cell niche for sperm stem cells (SSCs) (Spradling et al., 2001). Sertoli cells provide physical support for SSCs and also express proteins and other factors that regulate all facets of germ cell development (Ogawa et al., 2005). Among these proteins, GDNF regulates the cell fate of undifferentiated spermatogonial cells, which include the stem cells for spermatogenesis (Meng et al., 2000). To investigate the effects of
CS on GDNF expression, RT-PCR and Western blotting assays were performed. The testes of rats treated with CS exhibited a significant increase in GDNF mRNA levels compared to the control group, and Western blotting revealed a significant increase in the GDNFimmunoreactive band in CS-treated rats compared to the control group. Cynomorium songaricum may have a specific effect on sperm concentration and reproductive ability by inducing GDNF expression. These results confirm the traditional use of CS for male reproductive function. In this study, no noticeable adverse effects arose from administering the extract or vehicle, including aspartate aminotransferase and alanine aminotransferase levels (data not shown). The enhancing effect of CS on spermatogenesis may lead to the development of new drugs and food adducts to prevent or treat male infertility. Acknowledgement This research was supported by the Kyung Hee University Research Fund in 2008. (KHU-20081565) References Abdel-Rahman, H.A., El-Badry, A.A., Mahmoud, O.M., Harraz, F.A., 1999. The effect of the aqueous extract of Cynomorium cocconieum on the epydidymal sperm pattern of the rat. Phytotherapy Research 13, 248–250. Abdel-Magied, E.M., Abdel-Rahman, H.A., Harraz, F.M., 2001. The effect of aqueous extracts of Cynomorium coccineum and Withania somnifera on testicular development in immature Wistar rats. Journal of Ethnophamacology 75, 1–4. Airaksinen, M.S., Saarma, M., 2002. The GDNF family: signalling, biological functions and therapeutic value. Nature Reviews Neuroscience 3, 383–394. Bensky, D., 1986. Chinese Herbal Medicine. Eastland Press, pp. 489–490. Carlsen, E., Giwercman, A., Keiding, N., Skakkebaek, N.E., 1992. Evidence for decreasing quality of semen during past 50 years. British Medical Journal 305, 609–613. Connolly, C.M., Dearth, A.T., Braun, R.E., 2005. Disruption of murine Tenr results in teratospermia and male infertility. Developmental Biology 278, 13–21. Jiang, Z.H., Tanaka, T., Sakamoto, M., Jiang, T., Kouno, I., 2001. Studies on a medicinal parasitic plant: lignans from the stems of Cynomorium songaricum. Chemical & Pharmaceutical Bulletin 49, 1036–1038.
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Kuijt, J., 1969. The Biology of Parasitic Flowering Plants. University of California Press, Berkeley, CA, p. 246. Meng, X., Lindahl, M., Hyvonen, M.E., et al., 2000. Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 287, 1489–1493. Nickrent, D.L., Der, J.P., Anderson, F.E., 2005. Discovery of the “Maltese mushroom” Cynomorium. BMC Evolutionary Biology 5, 38. Ogawa, T., Ohmura, M., Ohbo, K., 2005. The niche for spermatogonial stem cells in the mammalian testis. International Journal of Hematology 82, 381–388. Spradling, A., Drummond-Barbosa, D., Kai, T., 2001. Stem cells find their niche. Nature 414, 98–104.
Viglietto, G., Dolci, S., Bruni, P., et al., 2000. Glial cell line-derived neutrotrophic factor and neurturin can act as paracrine growth factors stimulating DNA synthesis of RET-expressing spermatogonia. International Journal of Oncology 16, 689– 694. Yeh, K.Y., Pu, H.F., Kaphle, K., et al., 2008. Ginkgo biloba extract enhances male copulatory behavior and reduces serum prolactin levels in rats. Hormones and Behavior 53, 225–231. Zhang, C.Z., Wang, S.X., Zhang, Y., Chen, J.P., Liang, X.M., 2005. In vitro estrogenic activities of Chinese medicinal plants traditionally used for the management of menopausal symptoms. Journal of Ethnophamacology 98, 295–300.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Ethnopharmacological communication
Cynomorium songaricum induces spermatogenesis with glial cell-derived neurotrophic factor (GDNF) enhancement in rat testes Woong Mo Yang a , Ha Young Kim a , Soo Yeon Park b , Hyung-Min Kim c , Mun Seog Chang a , Seong Kyu Park a,∗ a b c
Department of Prescriptionology, College of Oriental Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea Sports Medicine Center, East-West Neo Medical Center, Kyung Hee University, Seoul 130-701, Republic of Korea Department of Pharmacology, College of Oriental Medicine, Institute of Oriental Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 13 November 2009 Received in revised form 16 February 2010 Accepted 18 February 2010 Available online 26 February 2010 Keywords: Cynomorium songaricum Glial cell-derived neurotrophic factor Spermatogenesis
a b s t r a c t Aim of the study: Cynomorium songaricum Ruprecht has been used in traditional Korean medicine to treat male infertility, including sexual dysfunction, by improving kidney function. Glial cell-derived neurotrophic factor (GDNF) produced by Sertoli cells induces the proliferation of undifferentiated spermatogonia. We investigated the effects of Cynomorium songaricum on sperm parameters and GDNF expression in rat testes. Materials and methods: Sperm analysis, RT-PCR, and Western blotting assays were performed after administration of CS to 8-week-old male Wistar rats for 56 consecutive days (1.0 g/kg/day, p.o.), the period of sperm formation in the rat. Results and conclusions: The CS-treated animals showed significant increases in epididymal sperm count and absolute testes weights compared to the control group. CS also increased the expression of GDNF at both the mRNA and protein levels. These results suggest that CS may improve male fertility by enhancing spermatogenesis and GDNF expression. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The incidence rates of male infertility are increasing worldwide, the leading cause of which is low sperm count. It has been reported that sperm counts in Western countries have decreased by 1% annually over the last 50 years (Carlsen et al., 1992). Although there have been many clinical trials aim at improving the number and quality of sperm, they have met with only limited success. Recent studies on rats have indicated that male fertility, including sexual dysfunction, can be improved by medicinal herbs (Yeh et al., 2008). Glial cell-derived neurotrophic factor (GDNF) is an important growth factor that facilitates communication between Sertoli cells and spermatogonia. GDNF induces the proliferation of undifferentiated spermatogonia under in vivo and in vitro conditions (Meng et al., 2000). In the testis, the GDNF dosage controls the cell fate of undifferentiated spermatogonia (Airaksinen and Saarma, 2002). Gene-targeted mice with one GDNF-null allele show partial depletion of spermatogenic stem cells, whereas those overexpressing GDNF show clusters of undifferentiated spermatogonia. GDNF stimulates DNA synthesis in spermatogonia (Viglietto et al., 2000).
∗ Corresponding author. 2Tel.: +82 2 961 0330; fax: +82 2 961 0536. E-mail address: [email protected] (S.K. Park). 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.02.020
Cynomorium songaricum (CS) is used in traditional Korean medicine as an herbal remedy for kidney and intestinal ailments, as well as impotence (Nickrent et al., 2005) which is a fleshy, monoecious or dioecious, holoparasitic herb that often produces swollen tuberous haustorial root connections to its host plants (Kuijt, 1969). Its chemical constituents include steroids, triterpenes, fructosides, flavonoids, and condensed tannins (Jiang et al., 2001). Several studies have examined the effects of CS extracts on mammalian reproductive cells. In addition, a water extract of CS induced a significant increase in sperm count, improved the percentage of live sperm and their motility, and decreased the number of abnormal sperm (Abdel-Rahman et al., 1999), and there was a marked increase in testicular weight in animals treated with the extracts, which also had a direct spermatogenic influence on the seminiferous tubules of immature rats, presumably by exerting a testosterone-like effect (Abdel-Magied et al., 2001). However, the relationship between CS and its effects on male reproductive tract malfunction associated with GDNF gene expression and spermatogenesis in vivo have not been elucidated. To investigate the effects of CS on male reproductive system in rats, we performed sperm analysis, including testicular weight, sperm count, and sperm motility. In addition, we assessed GDNF expression at the mRNA and protein levels.
694
W.M. Yang et al. / Journal of Ethnopharmacology 128 (2010) 693–696
2. Materials and methods 2.1. Preparation of CS extract Cynomorii Herba is the dried fleshy stem of Cynomorium songaricum Ruprecht (Fam. Cynomoiaceae). CS, produced in China, was purchased from Wonkwang Herbal Dug Co. Ltd. (Seoul, Korea). A 300 g sample of dried CS was boiled in 6 L water at 100 ◦ C for 2 h and the suspension was filtered. The filtrate was concentrated under reduced pressure and then lyophilised. The final yield was 26% (dry weight 76.60 g), and the sample was stored at 4 ◦ C. A voucher specimen (SK003) was deposited in our laboratory. Before each experiment, the dried extract was dissolved in distilled deionised water and vortexed for 2 min at room temperature. 2.2. Animals Eight-week-old male Wistar rats were purchased from SLC Inc. (Shizuoka, Japan). The animals were housed in a specific pathogenfree environment with a 12/12 h light/dark cycle at the Center for Laboratory Animal Care and Use of Kyung Hee University. Animal care and experimental procedures were performed in accordance with the “Guide for the Care and Use of Laboratory Animals” (Department of Health, Education and Welfare, NIH publication #78-23, 1996). Animals had free access to standard rodent pellets (Purina Inc., Korea) and water. After 7 days of acclimation to the environment, the rats were administered CS extract (1.0 g/kg/day, p.o.) dissolved in water for 56 days. The same volume of vehicle was administered to control rats (n = 8). The animals were weighed weekly to adjust the gavage volume and monitor their general health. 2.3. Sperm analysis
bromide using i-MAX gel image analysis system (Core Bio Systems, Seoul, Korea) and analyzed using AlphaEase FC software (Alpha Innotech, San Leandro, CA). The results from at least three separate experiments were used for statistical analysis. 2.5. Western blotting assay for detection of GDNF Proteins from homogenised testes were separated using a nuclear extract kit according to the manufacturer’s protocol with minor modifications (Active Motif, Carlsbad, CA). Equivalent amounts (50 g) of protein extracts were separated in 10% Tris–glycine gels by SDS-PAGE and transferred electrophoretically onto nitrocellulose membranes using 25 mM Tris and 250 mM glycine containing 20% methanol, pH 8.3. Transfer was performed at a constant voltage of 20 V for 1 h. After blotting, nonspecific binding was blocked with 5% skimmed milk in TPBS and the membranes were incubated with anti-GDNF (1:1000; rabbit polyclonal IgG; Abcam, Cambridge, UK) and anti--tubulin (1:2000; rabbit polyclonal IgG; Santa Cruz Biotechnology, Santa Cruz, CA) antibodies overnight at 4 ◦ C, washed, and incubated with secondary horseradish peroxidase-conjugated anti-rabbit IgG antibodies (1:1000; Jackson ImmunoResearch, West Grove, PA) for 2 h. The membranes were then washed and developed with Western blotting chemiluminescent reagents (Thermo Scientific, Rockford, IL), and then exposed to X-ray films (Agfa, Mortsel, Belgium). The films were analyzed with AlphaEase FC software (Alpha Innotech). 2.6. Statistical analysis The results are expressed as the means ± SD. Differences between groups were analyzed using Student’s t-test with P < 0.001, P < 0.01, and P < 0.05 taken to indicate significance as shown in the figures (Fig. 1).
Epididymal sperm count and motility were evaluated as described by Connolly et al. (2005), with some modifications. To determine the sperm count, the entire epididymis from the rats was minced in M199 medium (CureBio, Seoul, Korea) containing 0.5% bovine serum albumin (BSA) and incubated for 5 min at 37 ◦ C. The sperm concentration was determined manually using a hemocytometer (Neubauer, Germany). To assess sperm motility, sperm were recovered from the excised cauda epididymides and allowed to capacitate for 5 min in M199 medium containing 0.5% BSA at 37 ◦ C. Sperm were scored as motile if any movement was detected. The total number of sperm and the number that were motile were determined.
None of the animals died during the study and no noticeable adverse effects were observed in any of the animals after administration of the extract or vehicle. Rats treated with CS showed a slight change in body weight compared to the control group (429.2 ± 34.5 and 420.5 ± 26.9 g, respectively). The absolute weights of the testes of the CS-treated group were slightly but significantly greater than those of the control group (1.64 ± 0.05 and 1.52 ± 0.18 g, respectively; 7.89% increase, P < 0.05).
2.4. RT-PCR for detection of GDNF mRNA
3.2. Effects of CS on sperm counts and sperm motility
Total RNA was extracted from the rat testes. First-strand cDNA synthesis with 2 g total RNA was performed using MMLV reverse transcriptase and oligo dT primer for 1 h at 42 ◦ C. Then PCR amplification was performed. First, 5 L cDNA was added to 2.5 L 10× PCR buffer, 1 L 25 mM MgCl2 , 1 L 2.5 mM dNTP, 0.5 L polymerase (1 U), 1 L each primer (4 pmol), and DEPCtreated distilled water (DEPC-DW) to give a final volume of 25 L. The PCR protocol consisted of an initial 5-min denaturation step at 95 ◦ C followed by 35 cycles of the following: annealing for 1 min at 58 ◦ C, extension at 70 ◦ C for 1 min, followed by denaturation for 45 s 95 ◦ C. The run was concluded by a final extension step at 70 ◦ C for 10 min. The sequences of rat GDNF primers were as follows: 5 -ATGAAGTTATGGGATGTCGT-3 (forward) and 5 -CAGGGTCAGATACATCCACA-3 (reverse). Those for rat -actin were 5 -AGGCATCCTGACCCTGAAGTAC-3 (forward) and 5 -TCTTCATGAGGT AGTCTGTCAG-3 (reverse). PCR products were separated on 1.5% agarose gels, visualised by staining with ethidium
After 56 days of treatment with CS, the epididymal sperm counts were significantly higher (37.8% higher than those in the control group, P < 0.01). However, there was no change in sperm motility compared to the control group.
3. Results 3.1. Effects of CS on body and testicular weights
3.3. Effect of CS on GDNF mRNA and protein levels RT-PCR analysis and Western blotting assays were performed to determine the effects of CS on GDNF gene and protein expression in rat testes. The GDNF fragment was detected as a band of 416 base pairs (bp). GDNF mRNA levels were significantly elevated up to 157.09% in testes from the rats treated with CS (P < 0.05). The level of GDNF mRNA was normalised according to the -actin signal. The expression of GDNF protein in the PG-treated group was significantly higher than that in the control group (119.17%; P < 0.05). The level of GDNF protein was normalised according to the -tubulin signal. These results suggest that CS treatment may improve male
W.M. Yang et al. / Journal of Ethnopharmacology 128 (2010) 693–696
695
Fig. 1. Effects of CS treatment on sperm count and GDNF expression in rat testes. Sperm count was significantly increased (A). Expression of GDNF mRNA in CS-treated group was significantly increased compared to control group (B) and (C), and confirmed by Western blotting assay (D). Analysis of PCR reaction on RNA isolated from rat testes using GDNF and -actin primers. Results are presented as mean ±SD. *The mean differs significantly between control group and CS-treated group (P < 0.05).
reproductive ability by inducing GDNF gene expression and protein biosynthesis. 4. Discussion and conclusion Infertility is an increasing worldwide concern. The leading cause of male infertility is a low sperm count, and sperm counts in Western countries have decreased by 1% annually over the last 50 years (Carlsen et al., 1992). Although many clinical trials have been performed to improve the number and quality of sperm, they have had limited success. We believe that some medicinal herbs may improve male fertility with relatively few side effects. CS has been used in traditional Korean medicine to tone the kidneys, replenish the vital essence and blood, strengthen the yang, benefit the semen, and lubricate the intestines, in the treatment of indications such as impotence, sterility, constipation due to blood deficiency, weakness of the loins and knees, haematuria, and spermatorrhoea (Bensky, 1986). The Chinese name for the herb Cynomorium means “lock the Yang” and refers to its ability to lock up the kidney essence, assuring male potency. In a previous study, CS has been used for management of menopausal symptoms (Zhang et al., 2005). We investigated the effects of CS on spermatogenesis in rats treated with CS for 56 days. The epididymal sperm count of rats in the treatment group increased significantly compared to that of the control group. In addition, the absolute weights of testes in the CS-treated group exhibited slight but significant increases over the control group, suggesting that CS may stimulate the reproductive organs. Sertoli cells, the somatic cells in the seminiferous epithelium, are a major component of the stem cell niche for sperm stem cells (SSCs) (Spradling et al., 2001). Sertoli cells provide physical support for SSCs and also express proteins and other factors that regulate all facets of germ cell development (Ogawa et al., 2005). Among these proteins, GDNF regulates the cell fate of undifferentiated spermatogonial cells, which include the stem cells for spermatogenesis (Meng et al., 2000). To investigate the effects of
CS on GDNF expression, RT-PCR and Western blotting assays were performed. The testes of rats treated with CS exhibited a significant increase in GDNF mRNA levels compared to the control group, and Western blotting revealed a significant increase in the GDNFimmunoreactive band in CS-treated rats compared to the control group. Cynomorium songaricum may have a specific effect on sperm concentration and reproductive ability by inducing GDNF expression. These results confirm the traditional use of CS for male reproductive function. In this study, no noticeable adverse effects arose from administering the extract or vehicle, including aspartate aminotransferase and alanine aminotransferase levels (data not shown). The enhancing effect of CS on spermatogenesis may lead to the development of new drugs and food adducts to prevent or treat male infertility. Acknowledgement This research was supported by the Kyung Hee University Research Fund in 2008. (KHU-20081565) References Abdel-Rahman, H.A., El-Badry, A.A., Mahmoud, O.M., Harraz, F.A., 1999. The effect of the aqueous extract of Cynomorium cocconieum on the epydidymal sperm pattern of the rat. Phytotherapy Research 13, 248–250. Abdel-Magied, E.M., Abdel-Rahman, H.A., Harraz, F.M., 2001. The effect of aqueous extracts of Cynomorium coccineum and Withania somnifera on testicular development in immature Wistar rats. Journal of Ethnophamacology 75, 1–4. Airaksinen, M.S., Saarma, M., 2002. The GDNF family: signalling, biological functions and therapeutic value. Nature Reviews Neuroscience 3, 383–394. Bensky, D., 1986. Chinese Herbal Medicine. Eastland Press, pp. 489–490. Carlsen, E., Giwercman, A., Keiding, N., Skakkebaek, N.E., 1992. Evidence for decreasing quality of semen during past 50 years. British Medical Journal 305, 609–613. Connolly, C.M., Dearth, A.T., Braun, R.E., 2005. Disruption of murine Tenr results in teratospermia and male infertility. Developmental Biology 278, 13–21. Jiang, Z.H., Tanaka, T., Sakamoto, M., Jiang, T., Kouno, I., 2001. Studies on a medicinal parasitic plant: lignans from the stems of Cynomorium songaricum. Chemical & Pharmaceutical Bulletin 49, 1036–1038.
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