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Effects of plumieride, an iridoid on spermatogenesis in male albino rats.

Summary

Oral feeding of male rats with plumieride (15 mg/rat/day) for the period of 60 days did not cause any significant change in the body weight of treated rats. However, the weights of testes, epididymides, seminal vesicle and ventral prostate were significantly reduced when compared to control values. The production of step-19 spermatids was reduced by 87.26% in plumieride treated rats. The population of preleptotene and pachytene spermatocytes were decreased by 64.26% and 55.13% respectively. Spermatogonia and sertoli cell population was also affected. Plumieride treatment resulted in an arrest of spermatogenesis without any systemic side effect. Sperm motility as well as sperm density was reduced significantly. The number of mature Leydig cells was decreased and complete suppression of fertility was observed. A significant fall in the protein and sialic acid contents of the testes, epididymides, seminal vesicle and ventral prostate as well as glycogen content of testes was also noticed. Fructose in seminal vesicle was lowered whereas testicular cholesterol was elevated. There was no significant change in RBC and WBC count, haemoglobin, haematocrit and sugar in the whole blood and total protein, cholesterol, phospholipid and triglycerides in the serum. Conclusion: Plumieride administration arrests spermatogenesis in male rats without noticeable side effects. For the clinical use more experiments should be carried out in a phased programme.

Key words: Plumieride, testicular cell population, sperm motility, sialic acid, Sertoli cell, Leydig cells

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Introduction

Plumeria bicolor, a plant of the Apocynaceae family is commonly known as "Champa" in India. Plumeria is grown as an ornamental plant in India, Indonesia, Philippines and South Africa. Various species of this plant are used as medicine for the cure of many diseases and used as antipsychotic, diuretic or antitumour agents. Some iridoids and triterpenoids obtained from this plant are reported to have algicidal (Coppen and Cobb, 1983; Coppen, 1983), antibacterial (Sandujza et al. 1985) and cytotoxic activity (Anderson et al. 1988; Adam et al. 1979). Plumieride, an iridoid has also been reported to show the antidermantophytic activity (Tiwari et al. 2002). The plant has also been reported to have antifertility and anticancerous activity. Some species of this plant are used for the cure of rheumatism, diarrhoea, blennorhea, venereal disease and leprosy (Perry and Metzer, 1980; Watt and Brreyer, 1962; Kardono et al. 1990).

Major constituent of chloroform extract of bark is plumieride, an iridoid. Two reasons, availability of plumieride approximately 10% of extract and reported antifertility activity of plumeria species, prompted us to evaluate the active principle i.e. antifertility activity of plumieride. Since very less attention has been given to male antifertility, we started work with male rats.

Plant and Material

The plant was collected from the campus of University of Rajasthan, Jaipur and identified by the Department of Botany, University of Rajasthan, Jaipur.

Isolation of Plumieride

The bark of plant was shade dried and grinded to powder. 4 kg. of powdered bark was extracted by methanol extensively for approximately 72 hrs. The extract was distilled and brown mass obtained was treated with acetonitrile to remove fatty parts and acetonitrile was removed under reduced pressure. The brown mass obtained was approximately 100 gm which was re-extracted by chloroform. Approximately 40 gm extract was obtained on removal of chloroform by distillation process. This chloroform extract was subjected to column chromatography for separation and isolation of pure compounds. For this purpose a column of height 1.2 meter with diameter 5 cm filled with 900 gm of Si gel was taken. The column was chromatographed with various solvents and mixture of solvents in the order of increasing polarity. When column was eluted with chloroform and ethyl acetate in ratio of 1:1, plumieride was isolated. Plumieride (I) was recrystallised by methanol to obtain pure plumieride, which was used in the experiment of the antifertility activity. Its mp was 225-228 [degrees]C. The structure was established by HNMR and 13C NMR data (Abe et al. 1984).

Animals

Male albino rats of wistar strain were used in the present investigation. Rats weighing between 150-170 g were housed in polypropylene cages under controlled environmental conditions. They were fed with rat feed pellets and free access to water.

Study Protocol

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

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

Group-2: Rats treated with Plumieride orally (15 mg/rat/day) for 60 days.

After 24 hrs. of last dosing of 60 days duration, the animals were autopsied under ether anaesthesia. The testes, epididymides, seminal vesicles, ventral prostate and liver were removed, cleared off fat and connective tissue, weighed and kept at -20 [degrees]C until assayed for protein (Lowry et al. 1951), glycogen (Montogomery, 1957), sialic acid (Warren, 1959) and cholesterol (Oser, 1965).

Sperm motility and Sperm density

The motility of cauda epididymal spermatozoa was recorded. Sperm density was assessed in testes and cauda epididymis by the method of Prasad (Prasad et al. 1972).

Blood and serum analysis

Whole blood was analysed for RBC, WBC count, haemoglobin (Crossby et al. 1954) haematocrit and sugar (Astoor and King, 1954) and serum was analysed to estimate total protein, cholesterol (Zlatkis et al. 1953), triglyceride (Gottfried and Rosenberg, 1973) and phospholipids (Zelversmit et al. 1950).

Testicular cell dynamics

Tissues were fixed in Bouin's fluid, Paraffin sections were made at the thickness of 5 [micro]m and stained with hematoxylin and eosin. The evaluation of cell population was based on the counts of each cell type per cross-tubular sections. Various cell components were quantitatively analysed using spherically appearing sections. These counts were designated as crude counts and these crude counts were corrected by Abercrombie's formula (Berndtson, 1977). Fibroblast, mature and degenerating Leydig cells were counted. Mean tubular diameters were determined by tracing and measuring an average of 100 selected seminiferous tubules. Diameters of Leydig cell nuclei were measured at 800X.

Statistical analysis

All the values of body/organ weight, biochemical estimation, histometry and testicular dynamics were expressed in terms of mean [+ or -] standard error. The treated groups were compared to control using the Student's t-test (Ipstein and Poly, 1970).

Results

The oral administration of plumieride to male rats for 60 days did not cause any significant change in the body weight of treated rats. However the weights of testes, epididymides, seminal vesicle and ventral prostate were significantly (p < 0.001) reduced when compared to control values (Table 1).

[FIGURE 1 OMITTED]

Cell population dynamics

The number of step-19 spermatids was reduced by 87.26%; spermatogonia and preleptotene spermatocytes were decreased by 59.49 and 64.26% respectively. Pachytene spermatocytes reduced by 55.1%. The number of sertoli cells were reduced by 29.18%. The number of mature Leydig cells was decreased significantly (p < 0.001, Table 2). The seminiferous tubule diameter and Leydig cell nuclear diameter were reduced significantly (p < 0.001, Table 1).

Biochemical parameters

The protein contents of testes, epididymides, seminal vesicles and ventral prostate were reduced significantly (p < 0.001) in comparison with controls (Table 3). Sialic acid contents of the testes, cauda epididymides, seminal vesicle and ventral prostate were depleted (Table 3). Glycogen contents in the testes were decreased significantly following treatment with plumieride. Seminal vesicular fructose was decreased significantly, whereas, testicular cholesterol was elevated significantly (p < 0.001).

Sperm concentration/motility/fertility

As shown in Table 4, the 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 plumieride treatment reduced the fertility of male rats by 100%.

Blood and serum analysis

Blood variables i.e. RBC and WBC counts, haemoglobin, haematocrit and sugar were within the normal range. Protein, cholesterol, phospholipid and triglycerides did not change significantly after plumieride treatment to rats (Table 5).

Discussion

Oral administration of plumieride to male rats for 60 days brought about a significant loss in testes weight, which is known to be mostly related to the number of spermatids and spermatozoa present in the tissue. The reduced testicular weights and shrunken seminiferous tubular dimensions indicate wide spread damage (Keel and Abney, 1980). The treatment with plumieride exerted strong inhibiting effect on epididymides, seminal vesicle and prostate gland as evinced by decrease in their weight. Reduction in the weights of accessory reproductive organs directly supports the reduced availability of androgens (Mukerjee et al. 1992). 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 (Ritzen et al. 1981; Parvinen and Ruokonen, 1982).

The number of sertoli cells are reduced by 29.18% which adversely affects the spermatogenesis since sertoli cells provide all or most of nutritional and physical support for the developing germ cells. Sperm count shows statistically significant correlation with antifertility (Bostofte et al. 1984). In the present study, treatment with plumieride caused a significant reduction in sperm density of cauda epididymis as well as testes. The motility of sperms in cauda epididymides was reduced significantly (p < 0.001) after the administration of plumieride. Complete inhibition of fertility may be due to suppressed sperm motility and decreased sperm count, since fast and progressive sperms in a sufficient number are necessary for fertilization (Aitken et al. 1984).

The number of mature Leydig cells was decreased significantly (p < 0.001). The impairment of Leydig cell function was evinced by its reduced nuclear area and lower number of mature Leydig cells. The number of mature Leydig cells has a direct bearing on spermatogenesis (Gupta et al. 2000).

The protein contents of testes, epididymides, seminal vesicles and ventral prostate were reduced significantly. It is evident that testicular function would be altered by reduced protein content. The principal cells of epididymis synthesize proteins which have important role for maturation of spermatozoa (Kasturi et al. 1995). Sialic acid acts as 'lubricant' to facilitate the downward movement of sperms and to reduce friction among spermatozoa (Robaine and Hermo, 1988). Mukherjee et al. (1992) studied the effects of flutamide on testes and accessory sex organs of male rats and also found reduction in sialic acid contents. Depletion in testicular glycogen possibly attributed to decreased number of post meiotic germ cells. Increased level of testicular cholesterol may be due to decreased androgen production, which resulted in accumulation of cholesterol in testes, hence impaired spermatogenesis (Bedwal et al. 1994). Depletion in testicular glycogen possibly attributed to decreased number of post meiotic germ cells (Gunaga, 1972). Seminal vesicular fructose was decreased significantly, the reduction might be due to decreased secretory activity.

It has been observed that the blood and serum parameters were within the normal range indicating nontoxicity of plumieride on general body metabolism. Conclusion: Our results reflect anti spermatogenic effect of plumieride in male albino rats.
Table 1. Effect of Plumieride on body weight, organ weights together
with Seminiferous tubule and Leydig cell nuclear diameter. (a)

 Body Weight Organ weight (mg/100 g body wt.)
 (g) Testes

Control 245 [+ or -] 20 1345 [+ or -] 4.7
Plumieride 230 [+ or -] 18 (ns) 1009.99** [+ or -] 20.5
15 mg/rat/day

 Organ weight (mg/100 g body wt.)
 Epididymis Seminal vesicle

Control 529.5 [+ or -] 11.2 605 [+ or -] 9.5
Plumieride 312.1** [+ or -] 18 312.1** [+ or -] 15
15 mg/rat/day

 Organ weight (mg/100 g body wt.) Seminiferous
 Ventral Prostate Tubule diameter
 [micro]m

Control 308.5 [+ or -] 2.02 268 [+ or -] 9
Plumieride 177.10** [+ or -] 10.8 182.4** [+ or -] 0.28
15 mg/rat/day

 Leydig cell
 nuclear diameter
 [micro]m

Control 11.1 [+ or -] 0.02
Plumieride 5.6** [+ or -] 0.168
15 mg/rat/day

(a) Values are mean [+ or -] SEM (n = 10); ns: non significant.
** p < 0.001 vs control.

Table 2. Effect of Plumieride on testicular cell population in rats. (a)

 Testicular cell counts (number/10 cross section)
 Spermatogonia Sertoli cell

Control 6.87 [+ or -] 0.02 2.81 [+ or -] 0.02
Plumieride 2.783** [+ or -] 0.368 1.99* [+ or -] 0.18)
 (-59.49) (-29.18)

 Testicular cell counts (number/10 cross section)
 Preleptotene Pachytene

Control 19.95 [+ or -] 1.9 29.29 [+ or -] 0.73
Plumieride -7.13** [+ or -] 0.9 13.14** [+ or -] 1.02
 (-64.26) (-55.13)

 Testicular cell counts (number/10 cross section)
 Secondary Step-19
 spermatocytes spermatid

Control 48.1 [+ or -] 0.6 35.1 [+ or -] 1.07
Plumieride 19.8** [+ or -] 3.7 4.47** [+ or -] 1.86
 (-58.83) (-87.26)

 Leydig cell differential count
 Fibroblast Mature

Control 43.3 [+ or -] 3.6 99.7 [+ or -] 0.26
Plumieride 59.903 (ns) [+ or -] 9.70 63.47* [+ or -] 7.83
 (-36.33)

 Leydig cell differential count
 Degenerated

Control 57.0 [+ or -] 2.8
Plumieride 66.61 (ns) [+ or -] 3.16

(a) Values are mean [+ or -] SEM (n = 10); ns: non significant
(b) *p < 0.01; **p < 0.001 vs control. Percent variations vs control in
parentheses.

Table 3. Effect of Plumieride on tissue biochemistry in rats. (a)

 Protein (mg/g)
 Testes Cauda Seminal
 epididymis vesicle

Control 178 [+ or -] 0.5 268 [+ or -] 3.6 186 [+ or -] 3.4
Plumieride 113.4** 119.9** 122.1**
15 mg/rat/day [+ or -] 4.4 [+ or -] 4.5 [+ or -] 3

 Protein (mg/g) Sialic acid (mg/g)
 Ventral Testes Cauda
 prostate epididymis

Control 162 [+ or -] 0.18 4.62 [+ or -] 0.16 5.3 [+ or -] 0.15
Plumieride 131** 3.11** 3.84**
15 mg/rat/day [+ or -] 5.7 [+ or -] 0.03 [+ or -] 0.15

 Sialic acid (mg/g)
 Seminal Ventral Glycogen (mg/g)
 vesicle prostate Testes

Control 4.2 [+ or -] 0.09 5.2 [+ or -] 0.23 4.85 [+ or -] 0.6
Plumieride 2.99** 3.78** 2.8*
15 mg/rat/day [+ or -] 0.03 [+ or -] 0.1 [+ or -] 0.05

 Cholesterol (mg/g) Fructose (mg/g)
 Testes Seminal vesicle

Control 7.3 [+ or -] 0.2 4.52 [+ or -] 0.19
Plumieride 14.99** 3.11**
15 mg/rat/day [+ or -] 0.5 [+ or -] 0.11

(a) Values are mean [+ or -] SEM (n = 10); ns: non significant
*p < 0.01; **p < 0.001 vs control.

Table 4. Effect of Plumieride on Sperm motility concentration and
fertility in rats. (a)

 Sperm motility (%) Sperm density (million/ml)
 Cauda epididymides Testes

Control 70.25 [+ or -] 0.53 4.2 [+ or -] 0.06
Plumieride treatment 17.59** [+ or -] 1.72 1.225** [+ or -] 0.075

 Sperm density (million/ml) Fertility
 Cauda epididymides

Control 50.04 [+ or -] 0.59 100% ([+ or -] ve)
Plumieride treatment 5.55** [+ or -] 0.35 100% (-ve)

(a) All Figures [+ or -] SEM; level of significance ** p < 0.001

Table 5. Effect of Plumieride on some components in whole blood and
serum of rats. (a)

 RBC's WBC's Haemoglobin
 million/[mm.sup.3] no/[mm.sup.3] gm%

Control 5.1 [+ or -] 0.16 8516 [+ or -] 201 14.4 [+ or -] 0.2
Plumieride 5.62 (ns) 8176 (ns) 13.8 (ns)
15 mg/rat/ [+ or -] 0.085 [+ or -] 100 [+ or -] 0.2
 day

 Haematocrit Blood sugar Total protein
 (%) mg/dl mg/dl

Control 39.7 [+ or -] 2.0 89.1 [+ or -] 2.5 12160 [+ or -] 407
Plumieride 32.46 (ns) 99.99 (ns) 12333.32
15 mg/rat/ [+ or -] 2.11 [+ or -] 4.165 111.11
 day

 Total cholesterol Phospholipid Triglyceride
 mg/dl mg/dl mg/dl

Control 106 [+ or -] 5.2 150.05 [+ or -] 10.4 82.9 [+ or -] 3.87
Plumieride 122.22 (ns) 170.5 (ns) 94.4
15 mg/rat/ 11.11 6.945 6.67
 day

(a) Values are mean [+ or -] SEM (n = 10); ns: non significant
vs control.


Acknowledgements

The authors are thankful to the Head, Dept. of Zoology and Chemistry; Prof. N. K. Lohiya, Coordinator, SAP, Dept. of Zoology, University of Rajasthan and Principal, S. S. Jain Subodh P.G. College, Jaipur (India) for providing necessary facilities and University Grants Commission, Regional Office, Bhopal (MP) India for financial support.

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R. S. Gupta (1), A. K. Bhatnager (2), Y. C. Joshi (3), R. Sharma (1), and A. Sharma (1)

(1) Reproduction Physiology section, Department of Zoology, University of Rajasthan, Jaipur, India

(2) Department of Chemistry, S.S. Jain Subodh P.G. College, Jaipur, India

(3) Department of Chemistry, University of Rajasthan, Jaipur, India

Address

R. S. Gupta, Reproduction Physiology section, Department of Zoology, University of Rajasthan, Jaipur-302004, India

Tel.: +91-141-701137; Fax: +91-141-2395-453, 2711-158; e-mail: atulbhatnager@rediffmail.com
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Author:Gupta, R.S.; Bhatnager, A.K.; Joshi, Y.C.; Sharma, R.; Sharma, A.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:9INDI
Date:Feb 1, 2004
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