Antispermatogenic and hormonal effects of Crotalaria juncea Linn. seed extracts in male mice
B. Vijaykumar, I. Sangamma, A. Sharanabasappa, Saraswati B. Patil
Laboratory of Reproductive Biology, Department of Post-Graduate Studies and Research in Zoology, Gulbarga University, Gulbarga-585 106, India
Asian J Androl 2004 Mar; 6: 67-70
Keywords: Crotalaria juncea; testis; accessory reproductive organs; antispermatogenic effect; antiandrogenic effect
Aim: To evaluate the antifertility activity of various extracts of Crotalaria juncea seeds in male mice. Methods: Adult male mice were gavaged the petroleum ether, benzene and ethanol extracts of C. juncea seeds, 25 mg·(100g)-1·day-1 for 30 days. On day 31 the animals were sacrificed by cervical dislocation and the testes, epididymis, vas deferens, seminal vesicles, prostate gland, bulbourethral gland and levator ani were dissected out and weighed. The organs were processed for biochemical and histological examination. Results: In petroleum ether, benzene and ethanol extracts treated rats, there was a decrease in the weights of testis and accessory reproductive organs. The diameters of the testis and seminiferous tubules were decreased. Spermatogonia, spermatocytes and spermatids in the testis and the sperm count in cauda epididymis were also decreased. There was a significant reduction in the protein and glycogen contents and an increase in the cholesterol content in the testis, epididymis and vas deferens. Of the 3 extracts, the ethanol extract appeared to be the most potent in antispermatogenic activity. When the ethanol extract was tested in immature male mice, there was an antiandrogenic effect as the weights of accessory organs were reduced. Conclusion: The various extracts of C. juncea seeds arrest spermatogenesis and are likely to have an antiandrogenic activity.
Plants have served as a natural source of antifertility substances. Henshaw  has listed many plants used by primitive people in different countries to control fertility. Though many indigenous plants have been shown to prevent birth, only a few have so far been investigated for antispermatogenic activity [2-5]. Crotalaria juncea Linn. (Papilionaceae), commonly known as Sunn hemp, is cultivated throughout India and Indian Ayurvedic medicine indicated that various parts of C. juncea have analgesic, astringent, emmenagogue and abortifacient activities and is also used for the treatment of skin diseases. The seeds of this plant have been reported to possess contraceptive activity , however, no detailed study is available so far on its possible male antifertility activity. The present study was designed to clarify this point in albino mice.
2 Materials and methods
2.1 Plant material
The fresh seeds of C. juncea were obtained from a local source during October and November 2000 and were authenticated by Dr. Y. N. Sitharam, Department of Botany, Gulbarga University, Gulbarga (HGUG No. 71), India, where voucher specimens were deposited.
2.2 Extract preparation
The seeds were shade-dried, powdered and subjected to Soxhlet extraction successively and separately with petroleum ether (B. P. 60-80 ), benzene and ethanol (95 %). The decoctions produced were evaporated under reduced pressure below 45 . The residual extracts thus obtained (500 g of C. juncea seed produces: 3.8 g of petroleum ether extract, 1.2 g of benzene extract and 2.73 g of ethanol extract) were screened for their antifertility activity in mice. Before use, the extract was suspended in Tween-80 (1 %) normal saline with a final concentration of 25 mg/mL.
Adult, healthy and virgin Swiss strain male albino mice (Mus musculus), 60-70 days old and 35-40 g body weight, were selected from the inbred animal colony for experimental use. The animals were maintained under uniform husbandary conditions of light and temperature and were given pellet diet (Central Food and Technological Research Institute, Mysore, India) and tap water ad libitum. The animals were divided into 4 groups of 6 each.
After preliminary trials, a dose of 25 mg/100 g body weight per day ip for 30 days was determined for evaluating the antifertility effect. Group 1 served as the control and received the vehicle. Group 2, 3 and 4 received the petroleum ether, the benzene and the ethanolic extract in Tween-80, respectively.
The animals were sacrificed 24 h after the last treatment. The testes, epididymis, seminal vesicles, vas deferens, prostate, bulbourethral gland, and levator ani were excised, blotted free of blood, carefully freed from the surrounding fat and connective tissue and weighed up to the nearest mg on an electronic balance. Fresh tissues from testis, epididymis and vas deferens were processed for the estimation of glycogen, protein and cholesterol by conventional methods. Besides, they were fixed in Bouin's fluid, embedded in paraffin, sectioned at 5 mm and stained with haematoxylin-eosin for histological examination. The testicular and seminiferous tubular diameters were calculated by the method described by Deb et al . Spermatogenic element count was made from randomly chosen twenty round cross-sections. The cauda epididymal sperm suspension was prepared in normal saline and epididymal sperm count was estimated by the method of Kempinas and Lamano Carvalho .
2.6 Antiandrogenic activity
Swiss strain immature mice of 25 days old were used to assess the possible antiandrogenic activity. Among the 3 extracts, the ethanolic extract showed the maximum changes in 2.5 (Observation). Thus it was used in this experiment with 3 groups of 6 animals each: Group A: Vehicle control, Group B: 20 mg testosterone ip per animal per day for 5 days, and Group C: Ethanol extract 25 mg/100 g body weight per day ip for 5 days.
All the animals were sacrificed on day 6 by cervical dislocation and the testes, epididymis, seminal vesicles, vas deferens, prostate, bulbourethral gland and levator ani were excised, blotted free of blood, carefully freed from surrounding fat and connective tissue and weighed up to the nearest mg.
2.7 Statistical analysis
The data were expressed as meanSE and Student's t test was used to assess the statistical significance.
3.1 Changes in testis
3.1.1 Gravimetric and histometric changes
A significant (P<0.01) reduction in the weight of testis was observed in the ethanol, petroleum ether and benzene extracts groups. Histometric data were similar to those of weight changes: a significant (P<0.01) reduction in the diameters of the testis and seminiferous tubules with all the 3 extracts (Table 1).
Table 1. Effect of various extracts of C. juncea seeds on testicular parameters (meanSE). cP<0.01, compared with controls.
3.1.2 Histological changes
The number of spermatogonia, spermatocytes and spermatids are significantly reduced (P<0.01) with the ethanol extract, while the petroleum ether and benzene extracts significantly reduced the number of spermatogonia and spermatocytes (P<0.01). In all treated groups, there were pyknosis in the primary and secondary spermatocytes and degeneration of Leydig cells, spermatozoa were completely absent from the seminiferous tubular lumen and the sperm density was significantly reduced in the cauda epididymis (P<0.01) (Table 1).
3.1.3 Biochemical changes
The cholesterol content of the testis was significantly (P<0.01) increased in the ethanol and benzene extract groups, while the protein content was significantly reduced in the ethanol extract group (P<0.01) and in the petroleum ether and benzene extract groups (P<0.05). The glycogen content was significantly reduced with the benzene and ethanol extracts (P<0.01), but not with the petroleum ether extract (Table 1).
3.2 Changes in accessory organs
The weight of epididymis was significantly reduced in the ethanol and benzene groups (P<0.01), but not in the petroleum ether group. The cholesterol content of the epididymis was significantly increased (P<0.01) and the protein and glycogen contents of epididymis were significantly reduced (P<0.01) with all the three extracts (Tables 2 & 3).
Table 2. Effect of various extracts of C. juncea seeds on accessory organ weights (mg/100 g body weight, meanSE). cP<0.01, compared with controls.
Table 3. Effect of various extracts of C. juncea seeds on epididymal and vasal biochemistry (meanSE). cP<0.01, compared with controls.
3.2.2 Vas deferens
A significant reduction in the weight of vas deferens (P<0.01) was observed in the ethanol extract group, but not in the other 2 groups. The protein content of vas deferens was significantly reduced (P<0.01) with the ethanol extract, whereas insignificant with petroleum ether and benzene extracts. Though all the 3 extracts reduced the glycogen content of vas deferens, it was significant (P<0.01) only with the ethanol extract (Tables 2 & 3).
3.2.3 Seminal vesicles and prostate
The weight of seminal vesicle was significantly (P<0.01) reduced with all the three extracts. The weight of prostate was significantly reduced (P<0.01) with the ethanol extract, but not with the petroleum ether and benzene extracts (Table 2).
3.2.4 Bulbourethral gland and levator ani
The weights of the bulbourethral gland and levator ani showed a significant (P<0.01) reduction in the benzene, ethanolic and petroleum ether groups (Table 2).
3.3 Antiandrogenic activity
Testosterone administration caused a highly significant (P<0.01) increase in the wet weights of epididymis, vas deferens, seminal vesicles, prostate, bulbourethral gland and levator ani. On the contrary, the ethanol extract significantly decreased the wet weights of these organs (P<0.01) compared to the controls, indicating an antiandrogenicity of the ethanol extract (Table 4).
Table 4. Effect of ethanolic extract of C.juncea seeds on reproductive organ weights (mg/100 g body weight, meanSE) in immature mice. cP<0.01, compared with controls.
Administration of the petroleum ether, benzene and ethanol extracts of C. juncea reduces the testicular weight, which may be due to a decreased production of seminiferous tubular fluid that contributes to the weight of testis . The reduced protein content may be another reason as the growth rate of any organ is proportional to its protein content. The observed reduction in the number of spermatogonia, spermatocytes and spermatids may indicate lowered availability of FSH and LH/ICSH, which are essential for initiation and maintenance of spermatogenesis. For this reason, the seminiferous tubular lumen is devoid of spermatozoa.
The glycogen content in the cell represents the energy storage. The Sertoli cells and spermatogonia contain glycogen and provide nourishments to the seminiferous tubular cells and the glycogen content is found to be directly proportional to the steroid hormone levels . A decrease in the glycogen content of the testis reduces the energy source for spermatogenic activity.
The increased cholesterol content of testis may reflect a reduced conversion of cholesterol to androgens, which is dependent on the availability of LH/ ICSH. The reduction or non-availability of androgens is further supported by the reduction in the weight of accessory organs. All these organs play important roles in sperm maturation and mobility and the formation of semen. The ethanol extract when tested in immature mice also showed antiandrogenic effect. In conclusion, various extracts of C. juncea seeds arrest spermatogenesis and are likely to have an antiandrogenic activity.
PS. Physiologic control of fertility. Science 1953; 117: 572-82.
Prof. (Ms.) Saraswati B. Patil, Department
of Zoology, Gulbarga University, Gulbarga- 585 106, India.