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Study Objective: To evaluate the effects of Cassia fistula seeds extract on the reproductive system and fertility using adult male albino rats with special emphasis on testicular cell population dynamics.
Design: Aqueous extract (CFSAq) was prepared from seeds of Cassia fistula. The effect of CFSAq on reproductive system of male albino rats were investigated at three different doses viz., 100, 200 and 300 mg-1 kg -1 rat-1 day for 60 days recovery was also investigated after the withdrawal of 120 days.
Results: All major accessory sex organs shed weight post- administration of CFSAq. Marked reduction in motility and density of the sperms derived from cauda epididymis of the treated animals was observed. Cassia fistula reduced fertility of rats by 100% at 300 mg dose level. Serum testosterone levels also dwindled significantly in all the experimental groups. The extract brought about an interference with spermatogenesis. The number of normal tubules and the height of epithelial cells of caput and cauda were reduced significantly. The cross sectional surface area of Sertoli cells and mature Leydig cell number were significantly reduced. The population of Preleptotene, Pachytene and secondary Spermatogonia along with spermatocytes was significantly reduced in a dose dependent manner.
Conclusions: Cassia fistula (CFSAq) suppresses fertility in male rats. Antifertility effects of Cassia fistula seemed to be mediated by disturbances in structure and function in testicular somatic cell including Leydig and Sertoli cells resulting alteration in physio-morphological events of spermatogenesis. Withdrawal of extract restored all the altered parameters to control levels after 120 days.
Keywords: Cassia fistula, testicular cell population dynamics, recovery, male albino rats, testosterone, Leydig Cells, Histopathology.
At present time, one of the social problems regarding world health is the stability of population growth. The study of contraception is very important, but is a secondary issue. Contraceptive methods are indicated for birth control; so safe and effective methods of contraception that suit the needs of different couples necessary for successful population control. Many methods have been devised for the female, while the male has not received enough attention in this respect (Shafik 1994). Globally, men have not shared equally with women the responsibility for fertility regulation; the lack of male involvement may also reflect the limited options available to men (Ringheim 1993).
Many studies have been done regarding male contraception based on hormonal mechanisms. But there are many other compounds that have some action on the male reproductive system. In this regard, plants reported to decrease male fertility can be studied for a male contraceptive drug.
Native to India, the Amazon and Sri Lanka, Cassia fistula Linn., a semi-wild Indian Labernum also known as the Golden Shower, has become extensively diffused in various countries including Mauritius, India, South Africa, Mexico, China, West Indies, East Africa and Brazil as an 'Ornamental tree' for its beautiful bunches of yellow flowers. Recognized by British Pharmacopoeia (Mukhopadhyay et al 1998), Cassia fistula, a member of the Leguminosae family, is widely used for its medicinal properties, its main property being that of a mild laxative suitable for children (Bahorun et al 2005).
The Ayurvedic system of medicine describes that its seeds are used in the treatment of biliousness and to improve the appetite (Yadava and Verma 2003). This plant is widely used by tribal people to treat various ailments including ringworm and other fungal skin infections (Rajan et al. 2001). Antitumor (Gupta et al. 2000), hepatoprotective (Bhakta et al. 1999), antifertility in female rats (Yadav and Jain 1999) and antioxidant (Luximon-Ramma et al. 2002) properties of Cassia fistula as well as its actions on the central nervous system (Mazumdar et al. 1998) have also been suggested.
Earlier studies in our laboratory have shown that extract of Cassia fistula seeds possess anti-spermatogenic activity in rats, as shown by the decline in the weights of reproductive organs along with a decline in sperm motility and density (Chauhan and Agarwal, 2009). In this article, we wish to report the antifertility effect of the Cassia fistula aqueous extract of seeds (CFSAq) in male albino rats with special reference to testicular cell population dynamics.
Materials and Methods
Plant material used
Cassia fistula plant seeds were collected from the hillocks near University Campus, Jaipur and were authenticated in the Herbarium, Department of Botany, University of Rajasthan under the specimen voucher number RUBL 20103.
2.2 Preparation of Plant Extract:
The seeds were shade dried at room temperature, crushed and extracted with water at room temperature. The filtrate was concentrated at dryness under vacuum to obtain a dark brownish powder i.e. drug or CFSAq. The dry crude extract yield was 5.34% (5.34 g extract/100 g raw material) (W. H. O. 1983).
2.3 Animal model used:
50 adult male wistar rats weighing 180-200 gms were used in the present investigation. Animals were housed in standard rat cages and maintained under standard conditions (12-h light/dark cycle; 25±3°C temperature; 35-60 relative humidity), provided a standard laboratory chow (Aashirwad Food Industries Ltd., Chandigarh, India) and water ad libitum. Drug and/or vehicle were administered to all animals by oral intubation.
2.4 Dose and Duration of treatment
Male rats of proven fertility were divided in to five groups of 10 rats each. The daily dose of the plant extract was freshly dissolved in 0.5 ml of distilled water and orally administered to each experimental animal every morning for 60 days.
Group A: Control rats received 0.5 ml day-1 of the vehicle i.e. distilled water.
Group B: Rats treated with Cassia fistula Extract (CFSAq) at 100 mg-1 kg -1 rat-1 day.
Group C: Rats treated with Cassia fistula Extract (CFSAq) at 200 mg-1 kg -1 rat-1 day.
Group D: Rats treated with Cassia fistula Extract (CFSAq) at 300 mg-1 kg -1 rat-1 day.
Group E: Recovery of 120 days after treatment from Group D.
2.6 Autopsy Schedule
The animals were weighed and autopsied under light ether anesthesia 24 hours after last dose of the treatment.
2.7 Body and Organ Weight Measurements
Initial and final body weights of the animals were recorded. The animals were dissected and various organs of reproductive tract viz., testes, epididymis, seminal vesicle, ventral prostate and vas deferens along with some vital organs like liver, kidney, adrenal and heart were excised, cleared of adhering fat and connective tissue and weighed to the nearest mg on a top pan balance.
2.12 Histological Studies of the Testis:
The effects of CFSAq on the reproductive system of male rats could be assessed by the histological examination of the reproductive tissues mainly testis; as testis is the site of sperm production. For histology the tissues of all the groups were fixed in Bouin's fluid, dehydrated and embedded in paraffin for sections at 5m. These sections were stained with Harris hematoxylene and eosin (Lablond and Clermont 1952).
2.13 Quantitative analysis:
The evaluation of cell population dynamics was based on the counts of each cell type per cross-tubular sections. Various cell components were quantitatively analyzed using spherically appearing sections (Abercrombie 1946, Berndtson 1977). All types of interstitial cells were counted. Diameter of Leydig cell nuclei and Seminiferous tubules were determined at 800 X and 80 X respectively. Mean tubular diameters were determined by tracing and measuring an average of 100 selected seminiferous tubules.
2.14 Ethical Aspects
The study was approved by ethical committee of the Centre for Advanced Studies, University Department of Zoology, Jaipur, Rajasthan, India. Indian National Science Academy, New Delhi (INSA, 2000) Guidelines were followed for maintenance and use of the experimental animals.
2.13 Statistical Analysis
Data are expressed as mean ± standard error of mean (SEM) The student's t-test was used to ascertain which mean was statistically significant. The level of statistical significance was set at p≤0.01 and p≤0.001.
3.1. Changes in Testicular cell population:
Testicular cell components showed a marked decline after oral administration of Cassia fistula extract (CFSAq), a decrease in the diameter and number of abnormal tubules was highly significant (p≤0.001), while the enhancement in number of abnormal seminiferous tubules was highly significant (p≤0.001). A highly significant decline (p≤0.001) in the height of epithelial cell of caput and cauda and nuclear area of Sertoli cells noticed. Withdrawal of drug for 120 days resulted in the restoration of all the altered values to the control counterparts (Table-1).
The decline in the nuclear diameter and the cytoplasmic and nuclear area of Leydig cell was highly significant (p≤0.001) with respect to the control values. The decline in the differential cell count of the Leydig cell was highly significant (p≤0.001), along with highly significant increase (p≤0.001) in the number of degenerating Leydig cells. The counts of fibroblast like cells were not altered in all the experimental and recovery groups. The above changed parameters were restored to the normal value after the withdrawal of CFSAq, showing a marked recovery (Table-2).
The testicular/seminiferous cell components were decreased after the oral administration of Cassia fistula extract (CFSAq). Sertoli cells and Spermatogonia were reduced markedly (p≤0.001) in the present study. A highly significant decline (p≤0.001) in the number of Preleptotene, Pachytene and secondary spermatocytes was observed. The number of round and elongated spermatids was also highly significantly declined (p≤0.001) with respect to the control counterparts. The recovery phase of 120 days showed a marked restoration in the counts of all the spermatogenic elements to the control level (Table-3).
3.2. Changes in Testicular histopathology:
The section of testes of control rat, consisted of seminiferous tubules which were lined with germinal epithelium. The successive stages of spermatogenesis i.e. spermatogonia, primary spermatocyte, secondary spermatocytes, spermatids (round and elongated) and spermatozoa were present. Sertoli cells were elongated and present in the outermost layer among the spermatogonia. Inside the stroma and between the seminiferous tubules, groups of Leydig cells were present. Leydig cells were large and polyhedral with an eccentric nucleus (Fig.1).
100 mg of Cassia fistula treated showed testicular lesions. Tunica albuginea was normal and composed of collagenous connective tissues. Cassia treatment showed a reduction in the seminiferous tubules diameter. Lumen of seminiferous tubules was filled with debris of broken tail and dead spermatozoa. Interstitial cells were normal and present between the seminiferous tubules. Sertoli cells were also normal in appearance (Fig. 2).
200 mg treated testes was small and edematous. There was no hemorrhage of blood vessels. Most of the tubules showed arrest of spermatogenesis at spermatid stage. The tubules were contained Sertoli cells, spermatogonia, primary and secondary spermatocytes and few spermatids. Lumen was devoid of spermatozoa. Leydig cells nucleus were normal (Fig. 3).
The administration of 300 mg Cassia (CFSAq) caused moderate to complete necrosis in the major part of testes. Some secondary spermatocytes showed pyknosis of their nuclei and cytoplasm. Lumen of seminiferous tubules was devoid of spermatozoa. The Leydig cells were present in the treated testes and showed a vacuolated cytoplasm (Fig. 4).
The section of seminiferous tubule of recovery group animals showed all the successive stages of spermatogenic elements. Lumen was full with sperms. Leydig Cells were also normal in appearance (Fig. 5).
Oral administration of Cassia fistula extract (CFSAq) caused reduction in the testosterone level and in the weights of testes and other accessory sex organs of the experimental rats; the effects produced by CFSAq were almost similar for all the rats within the same group. The epididymis, seminal vesicle, ventral prostate and vas deferens are androgen dependent, relying on testosterone for their growth and function (Klinefelter and Hess 1998); their weight loss reflects a decline in bioavailability and production of androgens (Peter et al, 1999).
Treatment with Cassia fistula is found to be effective in suppressing the male reproductive potential by causing decline in sperm motility and density. Reduction in sperm counts in testes and cauda epididymis may be due to suppression of gonadotropic hormone after treatment (Bastias et al. 1993). Inadequate concentration and sluggishly motile and immotile spermatozoa could not penetrate the cervical mucus and thus failed to fertilize the ova and resulted in infertility (Gupta et al. 2006). A significant decline in serum testosterone might be due to adverse effect of treatment on hormonal milieu of the testes. The hormonal milieu originates in the hypothalamus which release gonadotropin releasing hormone in a pulsatile manner (Lufente et al. 2001).
Histopathological results showed the reduction in sizes of the seminiferous tubules might be due to contractile activities of the fibroblasts which aid the movement of the non-motile spermatozoa into the epididymal tubule where the final maturation of motile spermatozoa takes place (Peter et al. 1999). This might be the reason for the increase in the interstitial spaces.
The seminiferous tubule diameters were decreased due to absence of spermatogenic cells. Shrinkage in seminiferous tubular diameter may be attributed possible due to decline in testosterone production (Raji and Bolarinwa 1997). Structural changes (reduction in area) of Sertoli cell nuclei may be due to lack of androgens and exogenous testosterone. Reduced testicular weight and decreased serum levels of testosterone are associated with the malfunction of Sertoli cells.
The process of spermatogenesis depends on somatic testicular Leydig and Sertoli cells that control germ cell development (Sharpe et al. 2003). The impairment of Leydig cell function was evidenced by its reduced area, nuclear dimensions and fewer number of mature Leydig cells. Reductions in volume and number of mature Leydig cells indicate low production of androgen, affecting the fertility. Reduction in the number and degenerative changes in the Leydig cells indicate effects on functional ability of these cells to synthesize testosterone (Ramaswami and Marshall 2003), which is required for the maintenance of the spermatogenesis.
Decline in the number of Spermatogonia, primary and secondary spermatocyte and spermatids probably correspond to the decrease in testosterone production or inhibition of pituitary gonadotropin secretion, which resulted in disruption of spermatogenesis (Raji and Bolarinwa 1997). Germ cell transformations particularly at meiotic and post meiotic levels are highly sensitive to the availability of testosterone (Russel and Russel 1991).
In conclusion, Cassia fistula can lead to a reversible sterile state in the male rat, due to interference on the testicular androgen levels altering structure, function, viability and concentration of spermatozoa in the cauda epididymis. Cassia fistula treatment brought about the inhibition of spermatogenesis, which resulted in a total sterile state in male rats. The effects thus produced were reversible after a withdrawal period of 120 days, thus forming Cassia fistula a potential reversible contraceptive. The herb might be useful for men also. However, the extrapolation should be made with caution, since the real human risk can not be assessed on the basis of present study.
The authors are thankful to the Center for Advanced Studies, Department of Zoology, University of Rajasthan, Jaipur for providing necessary facilities.