Antifertility Effects of Pueraria tuberosa. Root Extract in Male Rats

Abstract

Oral administration of Pueraria tuberosa. D.C. root extract to male rats (100 mg/rat per day) for the period of 60 days did not cause body weight loss, whereas the weights of testes, epididymides, seminal vesicle, and ventral prostate were significantly reduced. The production of step-19 spermatids was reduced by 63.7% in P. tuberosa.–treated rats. The population of preleptotene spermatocytes and secondary spermatocytes were decreased by 61 and 72%, respectively. The seminiferous and Leydig cell nuclear area were reduced significantly (p < 0.001) when compared to controls. The number of mature Leydig cells was significantly reduced. Treated rats showed significant (p < 0.001) reduction in the sperm concentration of testes and cauda epididymides. The sperm motility of the cauda epididymides was also reduced significantly (p < 0.001). The Pueraria. treatment reduced the fertility of male rats by 100%. A significant fall in the total protein and sialic acid contents of the testes, epididymides, seminal vesicle, and ventral prostate as well as glycogen contents of testes was also observed. Fructose in seminal vesicle was lowered non-significantly, whereas testicular cholesterol was elevated in comparison to controls.

Keywords:

• Leydig cells
• Pueraria tuberosa
• sialic acid
• sperm motility
• testicular cell population

Introduction

Pueraria tuberosa. D.C. (Leguminosae) is commonly known as vidharikand in India and kudzu in China. The plant is distributed from West Himalaya to Sikkim, up to 4000 ft in Kumaon, the lower hills of Punjab, Mount Abu in Rajasthan (India), and South India. Pueraria. is used in the traditional system of medicine in India and China. In India, the flower of Pueraria tuberosa. is used as a medicine for the cure of leprosy, biliousness, disease of blood (vata.), burning sensation, urinary discharges, and indigestion; the root is used as a demulent and refrigerant in fever, to reduce swelling of joints (Kirtikar & Basu, 1935). Pueraria. species have shown cardiovascular effects (Fan et al.., 1982; Liu, 1990). Pueraria. root has shown antioxidant activity (Oh et al.., 1990), whereas Pueraria tuberosa. has been reported to be a potent antifertility plant (Shukla, 1995). Activity as a post control contraceptive agent, effects on the estrus cycle of adult rats, and induced vaginal cornification (Shukla et al.., 1987), antifertility behavior (Mathur & Shukla, 1987), and contraceptive potency (Prakash et al..,1985) of Pueraria tuberosa. and of other species have been reported. However, still no attention has been given to effects on male fertility, which led us to carry out work in this direction.

Materials and Methods

Extraction and isolation of the compounds

Pueraria tuberosa. root was harvested in June 2001 from the Mount Abu region (India) and authenticated by Dr. N.J. Sarana, Associate Professor, Department of Botany, University of Rajasthan (Jaipur, India). Shade-dried and powdered root part, approximately 1.5 kg, of Pueraria tuberosa. was extracted with methanol for about 48–52 h. The filtrate so obtained was concentrated under reduced pressure to a dark brown, semi solid mass. The brown mass was treated with petroleum ether for removal of the fatty part and then extracted with ethyl acetate. A part of this extract was fed to rats at 100 mg/rat per day, and 20 g was subjected to chromatographic separation over a Si-gel column; the following compounds were isolated and characterized with the help of physical and spectral methods. By eluting column with benzene: CHCl₃ (3:1), β-sitosterol was obtained. Further elutions of the column with benzene:CHCl₃ (2:2) gave daidzin, C₆H₆:CHCl₃(1:3) gave daidzein, and CHCl₃:ethylacetate (1:1) gave β-sitosterol-β − D-glucoside. In addition to these compounds, puerarine, puerarone, and puerarostan were eluted with methanol.

Animals

Twenty adult male Wistar rats weighing 150–160 g were used for the investigation. Animals were housed in standard rat cages and maintained under standard conditions (12 h light/12 h dark cycle: 25 ± 3°C; 35–60% relative humidity); water and food (commercial diet) were available ad libitum.. Drug and/or vehicle was administered to all animals by oral intubation.

Study protocol

Male rats of proven fertility were divided into 2 groups of 10 each.

• Group 1: Rats receiving vehicle (distilled water, 0.5 ml/day) for 60 days.

• Group 2: Rats treated with test extract (100 mg/rat per day) for 60 days.

On day 61, testes, epididymides, seminal vesicles, and ventral prostate were removed, cleared of fat and connective tissue, weighed, and kept at −20°C until assayed for total protein, sialic acid, and glycogen (Lowry et al.., 1951; Montogomery, 1957; Warren, 1959). Quantitative estimation of cholesterol was made according to the Lieberman–Burchard Method (Oser, 1965).

Histological preparations

Tissues were fixed in Bouin's fluid; paraffin sections were made and stained with hematoxylin and eosin or periodic acid-Schiff reagent (PAS). The classification of Lablond and Clermont (1952) was used for identification of the different stages of the spermatogenic cycle.

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's correcting factor was introduced (Berndtson, 1977) to correct for the better chance a big cell has to be counted. All types of interstitial cells, such as fibroblasts and mature and degenerating Leydig cells were estimated applying a differential count. The diameter of seminiferous tubules was determined by tracing and measuring an average of 100 selected seminiferous tubules. Thediameter of Leydig cell nuclei was measured at 800 × .

Mating studies

Toward the end of the treatment period, each male was housed individually with 3 proestrous females for 3–5 days. The females were scheduled for laparotomy 2 weeks later to determine the number and viability of fetuses. After mating, males were removed for autopsy.

Statistical analysis

Data were analyzed by the Student's t.-test to determine the level of significance between control and treated groups.

Results

Pueraria. root extract did not cause any significant change in the body weights of treated rats. However, the weights of testes, epididymides, seminal vesicle, and ventral prostate were reduced significantly (p < 0.001) in comparison with controls (Table 1).

Table 1.. Effect of Pueraria tuberosa. root extract on body weight, organ weights, and testicular histology in rats.^a.

		Organ weight (mg/100 g body weight)
	Body weight (g)	Testes	Epididymides	Seminal vesicle	Ventral prostate	Seminiferous tubule diameter	Leydig cell nuclear diameter	Abnormal seminiferous tubules (%)
Control (distilled water 0.5 ml)	240 ± 9.5	1345 + 4.7	529.5 ± 1.2	605.7 ± 9.5	308.5 ± 2.02	268 ± 9	11.1 ± 0.2	19.38 ± 0.02
P. tuberosa.  (100 mg/rat  per day)	220 ± 11^NS	920.90 ± 16.81**	397.87 ± 15.30**	311.20 ± 12.37**	172.57 ± 10.30**	204.3 ± 5.832**	5.766 ± 0.5278	81.5 ± 1.7**

NS, not significant.

^a.Values are mean±SEM. (n = 10).

*p < 0.001.

**p < 0.001 vs. control; Student's t.-test.

Cell population dynamics

As shown in Table 2, the number of step-19 spermatids was reduced by 63.7%; preleptotene and pachytene spermatocytes decreased by 60.8 and 53.8%, respectively. The total number of Sertoli cells was affected after Pueraria. root extract feeding. The total number of mature Leydig cells was significantly reduced by 30% in rats, whereas fibroblast Leydig cells remained unaffected. As shown in Table 1, the Leydig cell nuclear diameter and seminiferous tubule diameter were reduced significantly when compared to controls. Approximately 81% of tubules in this study were abnormal and degenerated.

Table 2.. Effect of Pueraria tuberosa. root extract on testicular cell population dynamics in rats.^a.

	Testicular cell counts/ (number/10 cross-section)					Leydig cell differential count (over 200 cells)
	Spermatogonia	Sertoli cell	Preleptotene	Pachytene	Sec spermatocyte	Step-19 spermatids	Fibroblast cells	Mature	Degenerating
Control (distilled water, 0.5 ml)	6.87 ± 0.02	2.81 ± 0.02	19.95 ± 1.9	29.29 ± 0.73	48.1 ± 0.6	35.1 ± 1.07	43.3 ± 3.6	99.7 ± 0.26	57.0 ± 2.8
P. tuberosa.  100 mg/rat per day	4.785 ± 1.62^NS	1.925 ± 0.1574** (−31.6)	7.812 ± 0.9** (−60.85)	13.539 ± 4.4 (−53.8)	13.63 ± 3.55** (−71.66)	12.74 ± 3.4**(−63.70)	63.17 31.918 ± 4.08 (+45.8)	70.54** 35.272 ± 4.08 (−30%)	66.29^NS 33.1465 ± 3.6 (+16.29)

NS, not significant.

*p < 0.01**p < 0.001 vs. control; Student's t.-test; percent variations vs. control in parentheses.

^a.Values are mean±SEM (n = 10).

Biochemical parameters

Protein content of testes, epididymides, ventral prostate, and seminal vesicle were reduced (p < 0.001) after Pueraria. treatment as compared with controls. Sialic acid contents of the testes, epididymides, and auxiliary gland (seminal vesicle and ventral prostate) were depleted. Glycogen contents of testes were decreased significantly (p < 0.01), whereas testicular cholesterol was elevated significantly (p < 0.001). Fructose in seminal vesicle was lowered non-significantly (Table 3).

Table 3.. Effect of Pueraria tuberosa. root extract on tissue biochemistry in rats.^a.

	Protein (mg/g)				Sialic acid (mg/g)				Glycogen (mg/g)	Cholesterol (mg/g)	Fructose (mg/g)
	Testes	Cauda epididymis	Seminal vesicle	Ventral prostate	Testes	Cauda epididymis	Seminal vesicle	Ventral prostate	Testes	Testes	Seminal vesicle
Control	178.8 ± 0.5	268.7 ± 3.6	186 ± 3.4	162.4 ± 0.18	4.64 ± 0.16	5.34 ± 0.15	5.26 ± 0.09	5.2 ± 0.23	4.85 ± 0.6	7.82 ± 0.11	4.24 ± 0.04
P. tuberosa. (100 mg/rat per day)	139.97 ± 1.2**	197.01 ± 1.9**	127.23 ± 4.74**	139.97 ± 1.4**	3.94 ± 0.04	3.80 ± 0.16**	3.86 ± 0.02**	3.76 ± 0.02**	2.026 ± 0.026	15.33 ± 0.66**	3.93 ± 0.13^NS

NS, not significant.

^a.Values are mean±SEM (n = 10),

*p < 0.01,**p < 0.001 vs. control (distilled water, 0.5 ml); Student's t.-test.

Sperm concentration/motility/fertility

As shown in Table 4, the Pueraria. treated rats showed a significant reduction (p < 0.001) in the sperm concentration of testes and cauda epididymides. The sperm motility of the cauda epididymides was also reduced significantly (p < 0.001). The Pueraria. treatment reduced the fertility of male rats by 100%.

Table 4.. Effect of Pueraria tuberosa. root extract on sperm motility, concentration, and fertility in rats.^a

	Sperm motility (%)	Sperm density (million/ml)
	cauda epididymides	Testes	Cauda epididymides	Fertility (%)
Control	74.1 ± 2.38	4.15 ± 0.21	52.2 ± 2.66	100(+ve)
P. tuberosa.	37.3 ± 2.1**	2.34 ± 0.12**	3.8 ± 1.5**	100(−ve)

^aAll figures ± SEM.

Levels of significance **p < 0.001.

Discussion

Oral feeding of Pueraria tuberosa. root extract for 60 days to male rats caused a significant loss in testes weight. Many authors analyzed the testes weight, which is mostly related to the number of spermatids and spermatozoa present in the testes. The reduction of DNA-synthesizing cells is related to the testes weight loss. The reduction in the weight of testes might be due to absence of spermatogenic elements (i.e., spermatocytes and spermatozoa) (Paul et al.., 1979).

The treatment with Pueraria tuberosa. root extract exerted strong inhibitory effect on epididymides, seminal vesicle, and prostate gland as evidenced by a decrease in their weights. Reduction in the weights of accessory reproductive organs directly supports the reduced availability of androgens (Mukherjee et al.., 1992). Rat prostate glands contain some androgen receptors that are a direct target of androgen action (Prins et al.., 1991) and mainly dependent on testicular androgens (Reiter et al.., 1995). Russell and Clermont (1977) found that the regression of seminiferous tubule epithelium after depletion of pituitary hormone was due to discriminate degeneration of mid-pachytene spermatocytes and step-19 spermatids in stage VII. In the mammalian spermatogenic cycle, normally step-19 spermatids are found in stage VII and VIII and particularly androgen dependent (Parvinen & Ruokonen, 1982). The reduced number of Sertoli cells adversely affects the spermatogenesis, as the Sertoli cells provide all or most of the nutritional and physical support for the developing germ cells (Ritzen et al.., 1981).

In the current study, treatment with Pueraria tuberosa. root extract caused a significant reduction in sperm density of cauda epididymides as well as testes.

The motility of sperms in cauda epididymides was reduced markedly after the administration of Pueraria tuberosa. root extract. Sperm motility is of importance with regard to sperm fertilizing capacity (Aitken et al.., 1984).

The total volume of Leydig cells is positively correlated with the onset of spermatogenesis. Therefore, shrinkage in Leydig cell nuclei of treated rats suggests a pronounced reduction of fertility. It is evident that testicular function would be altered by reduced protein content (Robaire & Hermo, 1988). Sialic acid acts as a “lubricant” to facilitate the downward movement of sperms and to reduce friction among spermatozoa (Riar et al.., 1973). Due to decreased sialic acid, it is likely that the structural integrity of acrosomal membrane is altered by the treatment, ultimately influencing sperm metabolism, motility, and fertilizing ability (Chinoy & Bhattacharya, 1997). Cholesterol is involved in steroidogenesis in the testes. An increased level of cholesterol in the testes is attributed to decreased androgen concentrations, which resulted in impaired spermatogenesis (Bedwal et al.., 1994). Depletion in testicular glycogen possibly attributed to a decreased number of postmeiotic germ cells (Gunaga et al.., 1972).

In conclusion, inhibition of spermatogenesis followed by changes in structure activity of Sertoli cells, Leydig cells, and meiotic germ cells did support the contraceptive-like activity of Pueraria. root extract.

Acknowledgments

The authors are thankful to the Head, Department of Zoology and Chemistry, to Prof. N.K. Lohiya, Coordinator SAP, Deptartment of Zoology, University of Rajasthan, Jaipur, and to the Principal, S.S. Jain Subodh PG College, Jaipur (India), for providing necessary facilities, as well as the University Grants Commission, Regional Office Bhopal (M.P.), India, for financial assistance.