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Anti-spermatogenic Activity of Leptadenia hastata (Pers.) Decne Leaf Stems Aqueous Extracts in Male Wistar Rats



B. Bayala, P.B. Telefo, I.H.N. Bassole, H.H. Tamboura, R.G. Belemtougri, L. Sawadogo, B. Malpaux and J.L. Dacheux
 
ABSTRACT

Testis histology and sperm parameters were used to evaluate the antispermatogenic effect of Leptadenia hastata aqueous extract in male Wistar rats. 100, 200, 400 and 800 mg kg-1 of Leptadenia hastata aqueous extracts were orally administered during 60 consecutive days. L. hastata aqueous extract did not have significant (p>0.05) effect on body, testis and epididymis weights. Testes histology of rats treated with the plant extract showed the decrease of Leydig cells number and the spermatogenesis was been influenced with high doses of L. hastata aqueous extracts. Some sperm parameters as path velocity, progressive velocity, straightness, linearity and motility of spermatozoa were been significantly (p<0.05) decreased. The treated rats with the different amount of L. hastata aqueous extracts showed a significant (p<0.01) reduction in the number of sperm in the testis and the cauda epididymis. Even if its not significant, the extract showed a decrease of testes, epididymis weights and a rarity of Leydig cells. These results confirm the antiandrogenic effect of L. hastata and the claims of breeders that the consumption of the leaves of L. Hastata reduced the fertility of their animals.

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B. Bayala, P.B. Telefo, I.H.N. Bassole, H.H. Tamboura, R.G. Belemtougri, L. Sawadogo, B. Malpaux and J.L. Dacheux, 2011. Anti-spermatogenic Activity of Leptadenia hastata (Pers.) Decne Leaf Stems Aqueous Extracts in Male Wistar Rats. Journal of Pharmacology and Toxicology, 6: 391-399.

DOI: 10.3923/jpt.2011.391.399

URL: https://scialert.net/abstract/?doi=jpt.2011.391.399
 
Received: November 01, 2010; Accepted: January 28, 2011; Published: May 07, 2011

INTRODUCTION

Traditional medicine is a significant element of the African cultural heritage and for a large majority of people a resort for treating health problems. This knowledge is passed down from generation to the next in oral form but very little written informations are available on the active, safe and effective effects of this medicine. Leptadenia hastata (Pers.) Decne is an African traditional plant belongs to the family Asclepediaceae. Different parts of the plant have been reported to have several medicinal values. Its leaves and latex have trypanocydal, antimalarial, antibacterial, anti-inflammatory and sterility properties (Nikiema et al., 2001; Bizimana et al., 2006; Magassouba et al., 2007; Kaou et al., 2008; Aderbauer et al., 2008). In the majority of western Sahel country, the young leaves of L. hastata is an important staple when cereal harvests are inadequate to support populations (Sena et al., 1998; Glew et al., 2005; Hassan et al., 2007). However, little attention was paid to the examination of the action of L. hastata on the reproductive system of mammals since in northern Burkina Faso, the breeders and local people traditionally believe that L. hastata can reduced the fertility of their animals (Arbonnier, 2000).

In previous study, the aqueous extracts of L. hastata exhibited an antiandrogenic effect on immature castrated rats by Hershberger assay (Bayala et al., 2011). The present study deals with the effect of the aqueous leaf stems extract of L. hastata on the male reproductive organs of the wistar rat. We have evaluated various male reproductive end points such as organs weight, sperm parameters and testis histology.

MATERIALS AND METHODS

Plant collection and preparation of extract: The leaves and stems of L. hastata were collected from Kamboinsè (25 km in the North of Ouagadougou), during April and May 2009. The plant was authenticated by the Department of Botany at Ouagadougou University. Herbaria are made and their voucher specimen was deposited in the Department.

The leaf stems were first washed with large amount of water then dried in a ventilated room, away from dust and direct sunlight. 150 g of dried material were coarsely powdered and macerated in distilled water at 40°C. The obtained macerated product was then filtrated, run through Rotavapor (Buchi/R-114), lyophilisedand kept in a drier until ready for use. Yield of the extraction was 24.6%.

Animal model: Wistar male rats from Charles River Laboratories (France) were checked without signs of illness and anomalies. Animals were acclimatized to the laboratory environment for 7 days before use. During the experiment, rats were housed six animals per cage in polycarbonate cages under controlled environmental conditions, including a temperature of 22°C, a relative humidity of 55% and a 12 h light cycle/12 h dark cycle. Pellet rodent diet and drinking water were available ad libitum. All the experiments have been carried out under approval of institutional ethics committee.

Methodology: Thirty male rats of 25 days old were divided into five groups of six animals each. The control group received distilled water and the other groups received respectively 100, 200, 400 and 800 mg kg-1 of the aqueous extracts of L. hastata leaf stems. All the groups were treated by oral gavage during 60 consecutive days. The amount of L. hastata extracts was prepared daily prior to administration.

Histology of the testis: During the autopsy, the testis were removed and immediately fixed in Bouins liquid. The testis were remained in Bouin during 48 h and placed in gauze. They were washed with tap water during 2 h and passed into different amounts of alcohol. The testes were cut with paraffin at the thickness of 5 μM and stained with Mayers haematoxylin and eosin for the discrimination of the stages of spermatogenesis. The observations were made with the X40 enlargement (Ocular, X10) with the OLYMPUS BX51 Microscope. The images were captured using the Software SPOT BASIC, Version 4.0.8.

Sperm parameters: Epididymides were removed, isolated, rinsed and dilacerated in order to release the spermatozoa in 500 μL Phosphate Medium Bicarbonate (PMB): 90 mM NaCl, 30 mM NaHCO3, 11 mM KCl, 0,8 mM MgSO4, 7 H2O, 0,4 mM KH2PO4, 0,6 mM Na-acétate, 3,4 mM lactate (1,7 mM Ca2+), 2,3 mM Na-pyruvate, 6,6 mM glucose and 20 mM HEPES Buffer with 1 g/100 mL of BSA and adjusted to PH: 7,7. The osmolarity of PBM was 300 mosm kg-1. The maintained in PBM at 37°C was favourable for the survival of spermatozoa. The homogenate was diluted at 1/10 to facilitate the observations. Two drops from 5 to 10 μL of the diluted solution were examined with the sperm analyzer HTM-IVOS (Hamilton Thorne Research, Beverly, MA). The HTM-IVOS permitted the analysis of the following parameters: Path Velocity (VAP), Progressive Velocity (VSL), Track Speed (VCL), Elongation (El), Lateral Displacement (ALH), Beat Cross Frequency (BCF), Straightness (STR), Linearity (L) and the motility of the spermatozoa.

Density of the spermatozoa in the testicles and the cauda epididymis: The testicle and the cauda epididymis were removed, mashed and placed into 5mL NaCl (0.9%). Homogenates were kept refrigerated at 4°C for 24 h to allow sperm to be released from the walls. Then, 5 mL of eosine (2%) was added and vortexed. One milliliter of this mixture is diluted with 2 mL eosine (2%) and a sample is placed in a Neubauer chamber and head sperm counted in 25 squares.

Statistical analyses: Data were analyzed using the Statistical Package SYSTAT (Version 10) and were presented as Mean±standard error (n = 6) of the mean (SEM). If variances were homogeneous, differences between groups were assessed by one-way analysis of variance. Differences between pair of means were assessed by the LSD test. A value of p<0.05 was considered as statistically significant.

RESULTS

Testis and epididymis weight: Sixty days after treatment with L. hastata aqueous extracts, there were no significant (p>0.05) differences in the weight of bodies, testes and epididymides in the four treated-groups of rats compared to those in the control group (Fig. 1, 2). However, the different treatment of L. hastata aqueous extracts showed the decrease of testis and epididymis weight.

Testis histology: Histological analysis of the testes of control group showed a normal spermatogenesis with a full complement of germinal, Sertoli and Leydig cells (Fig. 3). After 60 days treatment with L. hastata aqueous extracts, the number of Leydig cells in intertubellar space decreased significantly (p<0.05) (Fig. 4-7). Seminiferous tubules were found to contain spermatozoa in abundance in control (Fig. 3) and treated groups (Fig. 4-7).

Fig. 1: Effect of L.hastata aqueous extract on body weight after sixty day of treatment

Fig. 2: Effect of L. hastata aqueous extract treatment on testis and epididymis weight

Fig. 3: Testis of rat control group treated distilled water during 60 consecutive days. Intertubular space of untreated testis presenting normal Leydig cells (arrow) (Mag. X400)

Fig. 4: Testis of rat group treated with 100 mg kg-1 of L. hastata aqueous extracts during 60 consecutive days (Mag. X400)

Spermatic parameters and sperm density in testis and cauda epididymis: After sixty days of treatment, the different amounts of L. hastata aqueous extracts showed a significant (p<0.05) decrease of path velocity, progressive velocity, track speed, straightness, linearity and motility of spermatozoa compared to control group, elongation, lateral displacement and beat cross did not showed a significant (p>0.05) difference compared to control group (Table 1).

Fig. 5: Testis of rat group treated with 200 mg kg-1 of L. hastata aqueous extracts during 60 consecutive days (Mag. X400)

Fig. 6: Testis of rat group treated with 400 mg kg-1 of L. hastata aqueous extracts during 60 consecutive days. Intertubular space of treated testis presenting the rarety of Leydig cells (Mag. X400)

Fig. 7: Testis of rat group treated with 800 mg kg-1 of L. hastata aqueous extracts during 60 consecutive days. Intertubular space of treated testis presenting the rarety of Leydig cells (Mag. X400)

Table 1: Effect of L. hastata aqueous extracts on some sperm parameters
Lh: Leptadenia hastata, VAP: Path velocity, VSL: Progressive velocity, VCL: Track speed, ELO: Elongation, ALH: Lateral displacement, BCF: Beat Cross Frequency, STR: Straightness, LIN: Linearity, DW: Distilled water, *: p<0.05 ; **:p<0.001

Fig. 8: Effect of L. hastata aqueous extract on sperm number in testis and cauda epididymis *: p<0.05 ; **: p<0.001

The treated rats with the different amounts of L. hastata aqueous extracts showed a significant (p<0.01) reduction in the sperm concentration of testis and cauda epididymis compared to control group. The decreased is significantly (p<0.05) important in cauda epididymis compared to testis (Fig. 8).

DISCUSSION

The results of the present study indicate that L. hastata treatment did not cause alterations in body weight of the treated animals, suggesting that the treatment had no systemic toxic effect in rat. These results confirm the works of Tamboura et al. (2004) which showed on mice the non toxic effect of L. hastata aqueous extracts. Furthermore the present results indicated that the treatment caused a non significant decrease of testis and epididymis weights. In the testis, though the histology showed the decrease of the number of Leydig cells after the treatment, the spermatogenesis was been influenced with high doses of L. hastata aqueous extracts (Homady, 2001; Al-Majed, 2007; Nejad et al., 2008).

In this study, treatment with the extracts resulted in a decrease of the number of sperm. It is well established that androgens and gonadotrophins are essential for the spermatogenesis and the function of male reproductive system (Leidl et al., 1982). The decrease of the number of sperm in rats treated with plant extracts may be derived from a hormonal imbalance including serum levels of testosterone, prolactin and Luteinising Hormone (LH). Low cauda epididymal and testis sperm count and a reduction in the epididymal and testis weight imply that L. hastata aqueous extract effect might be caused by several factors. One factor may be that L. hastata extract interferes with enzymatic reactions including the oxidative phosphorylation uncoupling (Abou-Donia and Dieckert, 1974; Ke and Tso, 1982). It is a well-known and widely accepted concept that LH is basically responsible for testosterone production (Ewing et al., 1980; Arafa, 2010). It is probable that the primary step in the mechanism of the effects on testis induced by the L. hastata extract was the suppression of LH. At the testicular level, the absence of stimulation by LH would secondarily cause Leydig cell dysfunction, thereby resulting in decline in testosterone secretion which is responsible for diminished spermatogenesis and hence, reduction in sperm counts (Chase et al., 1992; Shan and Hardy, 1992; Verhoeven, 1992; Wanichacheewa et al., 2001).

It is known that the structure and function of the epididymis are dependent on androgens (Cooper, 1992). In the present investigation, a dose-related suppression of cauda epididymal sperm motility in treatment groups suggests an undersupply of testosterone to epididymis and therefore, an impaired epididymal function. The impaired epididymal function may also be due to reduced activity of the testis which affects the normal passage of testicular fluid into the epididymis (Lohiya et al., 1992; Gupta et al., 1993; Ansari et al., 1998). This is also confirmed by reduced epididymal weight even if it’s not significant.

CONCLUSION

In conclusion, the oral administration of aqueous extract of L. hastata leaf stems to male rats produced dose related effects on reproduction. The effects may have an inhibitory influence on gonadotrophin release which may be responsible for the decline in testosterone production, leading to changes in spermatogenesis. Further long term studies are in progress for the evaluations of antifertility with this extract.

ACKNOWLEDGMENTS

This study is financially supported by IFS (International Foundation for Science). We would like to thank all the members of research team Neurobiologie et Maîtrise des Fonctions Saisonnières of INRA/Nouzilly/France.

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