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Effect of Murraya paniculata (LINN) stem extract on testicular cell population dynamics and histology of testes on albino rats.

INTRODUCTION

Plant Murraya paniculata (Linn.) belongs to family Rutaceae, and is commonly known as Orange jasmine or China box. It has a native range from India to China and Malaysia to Northern Australia. It is a large, multi-trunked evergreen shrub. The leaves are used as a stimulant and decongestant. Phytochemical studies showed that the plant M. paniculata is rich in flavones (Feraccin et al., 1998; Padmavati et al., 2002). The plant is considered beneficial for joint pain and general body aches (Edward & Gillman, 1999). The plant is also used as an anti fertility drug in females (Kong et a,l 1985), but no attention has been paid its effect on male reproductive system therefore, present study was an attempt to carry out the effects of Murraya paniculata on the fertility of male rats.

MATERIALS AND METHODS

Animals Five proven fertile male albino rats of the Wistar-strain (4-5 months old, 150-200g) were used in the present investigation. The animals were maintained under complete veterinary supervision in the Departmental Animal Facility, animals were acclimatized in standard conditions i.e. 12:12 hours light dark cycle, 30-70% relative humidity, 25 [+ or -] 5[degrees]C during the experimentation. Rat's pellets diet (Aashirwad Ind and co, Chandigarh) and water were provided ad libitum.

Extraction and Fractionation of test material

Stem of plant M. paniculata was collected locally from Jaipur City, identified and authenticated by Dr. Rakesh Gautam, Director, Plants Medical Laboratory Pvt. Ltd. Jaipur. Stem of M. paniculata was dried, crushed, powdered and its 70% methanol extract was collected. After washing with petroleum ether and benzene extract was chromatographed over silica gel column with solvent CHCl3:CH3OH (50:50). NMR spectral studies revealed that the isolated fraction has two compounds, which are flavones in nature, these are 5-hydrox-3, 7, 8, 3', 4'5'-hexamethox flavone (1) and 3', 4', 5", 3, 5, 6, 7-heptamethoxy flavonol (2).

Experimental design

Experimental design: The animals were divided into two groups.

Group I-Vehicle treated or control 0.5ml/dist. water/rat/day for 60 days.

Group II-Treated with CHCl3:CH3OH (50:50) fraction of M. paniculata stem extract orally at a dose level of 50mg/rat/day for 60 days.

After 24 hours of last dose on day 61st the final body weights of animals, were recorded and animals were autopsied by using ether anesthesia.

Sperm dynamics

Sperm motility of cauda epididymis and density of cauda epididymis and testicular sperm were assessed by the method of Prasad et al., 1972.

Fertility test

Successful mating was carried out with all experimental animals (male female ratio 1:2), 5 days prior to autopsy. The mated females were allowed to complete the gestation. The number of pups delivered were recorded and percent fertility was calculated (WHO, 1983).

Serum hormonal assay

Testosterone level was assayed by radio immuno assay (Belanger et al., 1980)

Testicular cell population dynamics

Percentage of normal and abnormal seminiferous tubules were calculated. Evaluations were made of 100 tubules/cross section. Interstitial cell types were counted in 200 seminiferous tubules and statistically verified by the binomial distribution (Dixon and Massey, 1957). All the testicular cell types were counted in 10 seminiferous tubules per cross section. The group count of all these cell types were designated as crude count and corrected according to the formula suggested by Abercrombie (1946).

Histology

Histopathological studies were carried out in the testes. The tissues were fixed into Bouin's fixative and cut into pieces. Tissues were passed through ethanol-xylene series. After processing they were embedded in paraffin wax (melting point 58-60[degrees]C.), cut at 5 ? thickness and stained with haematoxylin and eosin.

Statistical analysis

Data are expressed as mean [+ or -] SEM and analyzed for statistical significance by using Student's "t" test. The data are considered as significant and highly significant at P<0.01 and P<0.001 levels respectively.

RESULTS

Sperm dynamics

Sperm motility in cauda epididymides was significantly (P<0.01) reduced by 84.98%. Sperm density in cauda epididymides and testes declined by 90.36% and 91.16%, respectively. [Table-1].

Fertility test

The mating exposure test of the control or vehicle treated group showed 100% positive fertility, whereas the treated group showed 100% negative fertility [Table-1].

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Testicular cell population dynamics

The diameter and number of normal seminiferous tubules decreased by 21.10% and 16.16%, respectively, whereas number of abnormal tubules increased by 270%. The number of degenerating Leydig cells increased by 97.54%. However, numbers of mature Leydig cell decreased by 58.13% [Table-2]. Number of spermatogonia, primary spermatocytes, secondary spermatocytes and round spermatids decreased significantly [P<0.001] [Table-3]. Number of Sertol cell also declined by 43.20% in comparison to control group.

Histology

The seminiferous tubules of vehicle treated control animals depicted active spermatogenesis. Well-developed Leydig cells can be seen (Fig. 1&3). Whereas the shrunken seminiferous tubules of treated group were noticed. Process of spermatogenesis is disrupted. Few secondary spermatocytes with karyolytic nuclei can be seen. Lumen with devoid of sperm confirms the arrest of spermatogenesis (Fig. 2 &4).

DISCUSSION

Treatment with M. paniculata CHCl3CH3OH (50:50) fraction is found to be effective in suppressing the male reproductive potential by causing decline in sperm motility and density. Administration of drug might possibly inhibit the activity of adenosine triphosphate in the spermatozoa by uncoupling with oxidative phosphorylation from the respiratory chain and prevent phosphorylation of adenosine diphosphate to adenosine triphosphate and, thus renders the spermatozoa immotile (Kalla and Vasudev, 1981). Reduction in sperm counts in testes and cauda epididymides may also be due to suppression of gonadotropic hormone after treatment (Bastias et al., 1993). Inadequate concentration and sluggishly motile or immotile spermatozoa could not penetrate the cervical mucus and thus failed to fertilize the ova (Sharma et al., 1999) and resulted in infertility.

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 (Lefuente et al., 2001). The process of spermatogenesis depends on somatic testicular Leydig cells and Sertoli cells that control germ cell development (Sharpe et al., 2003). Reduction in number and degenerative changes in the Leydig cells indicate effects on functional ability of these cells to synthesize testosterone (Rama Swami and Marshall, 2003), which is required for the maintenance of the spermatogenesis (Sarkar et al 1997). FSH in combination with testosterone regulates Sertoli cells nursing function by increasing the lactate production, which is used by germ cell as an energy substance (Khan and Rai, 2004).

Decreased testosterone level will affect the Sertoli cells function as seen in our experiment. Number and functional status of Sertoli cells are crucial for the successful completion of spermatogenesis (Huleihel and Lunenfeld, 2004). Increase in percentage of abnormal seminiferous tubules along with the decline in number of spermatogonia, primary spermatocytes, secondary spermatocytes 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). Reduction of testosterone in treated animals resulted in progressive loss of round spermatids while spermatogonial cells appear to be less affected. In conclusion M. Paniculata stem fraction brought about the inhibition of spermatogenesis, which resulted in a total sterile state in male rats.

REFERENCES

(1.) Abercrombie, M. (1946). Estimation of nuclear populations from microtome section. Anat. Rec. 94: 238-248.

(2.) Bastias, M.C.; Kamijo, H. and Pavlou, S.N. (1993). Sperm motion parameters after suppression of spermatogenesis with a gonadotropin releasing hormone antagonist plus testosterone supplementation. Fertil. Steril. 69(6): 1261-1265

(3.) Belanger, A.; Caron, S. and Picvard, V. (1980). Simultaneous radio-immuno assay of progestine androgens and estrogens in the rat testis. J. Steroid. Biochem. 13(2): 185-190.

(4.) Dixon, W. and Massy, F.J. (1957). Introduction to statistical analysis. McGraw Hill Book Company. U.B.E., New York, pp. 228.

(5.) Edward, F. and Gillman, D.E. (1999). Murraya Paniculata. IFAS. Cooperative Extension Service, University of Florida.

(6.) Ferracin, R.J.; Das, M.F.; Dasilva, G.F.; Ferandes, J.B. and Vieira, P.C.(1998). Flavanoids from the fruits of Murraya Paniculata. Phytochem. 47: 393-396.

(7.) Huleihel, M. and Lunenfeld, E.(2004). Regulation of spermetogenasis by paracrine/autocrine testicular factors. Asian. J. Androl. 6: 259-268.

(8.) Kalla, N.R. and Vasudev, M. (1981). Studies on the male antifertility agent, gossypol acetic acid. II. Effect of gossypol acetic acid on the motility and ATPase activity of human spermatozoa. Andrologia. 13: 95-98.

(9.) Khan, U.W. and Rai, V. (2004). In vitro effect of FSH and testosterone on Sertoli cell nursing function in wall ligard. Gen. Comp. Endocrinol. 132: 225-231.

(10.) Kong, Y.C,; Ng, K.H.; Wat, K.H.; Wong, A.; Saxena, J.F.; Cheng, K.F.; But, P.P.H. and Chang, H.T. (1985). Yachchunke a novel anti-implantation indole alkaloid from Murraya paniculata. Planta. Med. 4: 304-307.

(11.) Lufuente, A.; Marquez, N.; Perez-Larezo, M.; Pazo, D. and Esquinfino, A.L.(2001). Cadmium effect on hypothalamic pituitary testicular axis in male rats. Expt. Biol. Med. 226(6): 605-611.

(12.) Padmavathi, P.; Kavitha, J.; Kanumuri, R.V. and Subbaraju, G.V. (2002): Synthesis of methyl 2-methoxy-5-hydroxycin-namate, a metabolite of Murraya paniculata. Ind. J. Chem. Sect. B 41B(3): 673.

(13.) Prasad, M.R.N; Chinoy, N.T. and Kadam, K.M.(1972).Changes in succinate dehydrogenoase levels in rat epididymis under normal and altered physiological conditions. Fertil. Steril. 23:189-190.

(14.) Raji, U. and Bolarinwa, A.F. (1997). Antifertility activity of Quassia amara in male rats in vivo study. Life Sci. 61 (11): 1067-1074.

(15.) Ramaswami. S. and Marshall, G.R.(2003) . Inhibitory and stimulatory regulation of testiculear inhibin B secretion by leutinizing hormone and follicle stimulating hormone, respectively in rhesus monkey. Endocrinol. 144: 1175-1185.

(16.) Russel, L.D. and Russel, J.A. (1991). Short term morphological response of the rat testis to administration of five chemotherapeutic agents. Am. J. Anat. 192: 142-168.

(17.) Sarkar, S.N.; Majumdar, A.C. and Chattopadhyay, S.K. (1997). Effect of Isoproturon on male reproductive system: Clinical, histological and histoenzymological studies in rats. Ind. J. Expt. Biol. 35: 133-138.

(18.) Sharma, S.; Kumar, M.; Goyal, R.B.; Manivannan, B. and Lohiya, N.K.(1999). Reversible antispermatogenesis effect of gossypol in langur monkey (Presbytis entllus entellus). Adv. Contracep. 15: 27-32.

(19.) Sharpe, R.M.; McKinnell, C.; Kivlin, C. and Fisher, J.S.(2003). Proliferation and functional maturation of Sertoli cells, and their relevance to disorders of testis function in adulthood. J. Reprod. 125: 769-784.

(20.) World Health Organization .(1983). Protocol MB-50, A method for examining the effect of the plant extract administered orally on the fertility of male rats. APF/IP 9914E. Geneva.

R. S. Gupta , M. Kanwar , H. Rehwani and J. B. S. Kachhawa

Reprodutive Physiology Section, Center of Advanced Studies, Department of Zoology, University of Rajasthan, Jaipur--302 004, India.

* Correspondence to: Dr. R.S. Gupta, TR-3, Teacher's Hostel, University of Rajasthan, Jaipur-302 004, Ph. No. +91-141-2711228, E-mail: gupta_rs@hotmail.com
Table-1 : Sperm dynamics & fertility after the treatment of
M. paniculata 50:50 CH[Cl.sub.3]:C[H.sub.3]OH fraction

Treatment Sperm Sperm density Ferti- Testos-
 Motility (million/ml) lity terone
 (%) (%) (ng/dl)

 Cauda Testes Cauda
 epididy- epididy-
 mis mis

Group-I 7.65 4.67 45.97 100 4.25
 control [+ or -] [+ or -] [+ or -] (+ve) [+ or -]
 (Vehicle 0.35 0.18 1.19 0.09
 treated)
Group-II 10.61 ** 0.45 ** 4.06 ** 100 0.85 **
M. Paniculata [+ or -] [+ or -] [+ or -] (-ve) [+ or -]
 stem fraction 0.85 0.07 0.09 0.04
 (50:50)
 CH[Cl.sub.3]:
 C[H.sub.3]OH
 50 mg/rat/day
(Percent (-) (-) (-) (-)
 deviation) 84.98% 90.36% 91.16% 80.00%
 (c)

Values are in mean [+ or -] SEM (n = 5)

Level of significant P [less than or equal to] 0.001 **
highly significant when compared with group 1

(C) values in parentheses are percentage reduction in
particular cell type.

Table-2 : Testicular cell population dynamics after the
treatment of M. paniculata 50:50 CH[Cl.sub.3]:C[H.sub.3]OH
fraction

Treatment Seminiferous Tubules
 Diameter Normal Abnormal
 ([micro]m) Tubules Tubules
 (%) (%)

Group-I 264.58 79.54 22.31
 control [+ or -] [+ or -] [+ or -]
 (Vehicle 5.33 1.01 1.73
 treated)

Group-II 208.76 ** 18.96 ** 82.55 **
 M. Paniculata [+ or -] [+ or -] [+ or -]
 stem fraction 5.76 2.09 1.55
 (50:50)
 CH[Cl.sub.3]:
 C[H.sub.3]OH
 50 mg/rat/day

(Percent (-) (-) (+)
 deviation) 21.10% 16.16% 270%
 (c)

Treatment Leydig Cell
 Nuclear Differential Counts
 Area
 ([micro] Mature Degenerated Fibroblast
 [m.sup.2])

Group-I 19.58 99.13 46.12 53.74
 control [+ or -] [+ or -] [+ or -] [+ or -]
 (Vehicle 1.03 2.33 2.09 2.76
 treated)

Group-II 9.73 ** 41.50 ** 91.11 ** 53.32 (ns)
 M. Paniculata [+ or -] [+ or -] [+ or -] [+ or -]
 stem fraction 0.48 2.44 0.43 2.66
 (50:50)
 CH[Cl.sub.3]:
 C[H.sub.3]OH
 50 mg/rat/day

(Percent (-) (-) (+) (-)
 deviation) 50.30% 58.13% 97.54% 6.66%
 (c)

Value are in mean [+ or -] SEM (n=5)

Level of significant P [less than or equal to] 0.001

** highly significant

ns = Non Significant

C values in parentheses are percentage reduction in
particular cell type.

Table-3 : Testicular cell population dynamics following the
treatment of M. paniculata 50:50 CH[Cl.sub.3]:C[H.sub.3]OH
fraction

Treatment Testicular cell counts
 (number/10 cross section)

 Sertoli cell Spermatogina

Group-I 2.94 7.03
 control [+ or -] [+ or -]
 (Vehicle 0.19 0.40
 treated)

Group-II 1.62 ** 4.44 **
 M. [+ or -] [+ or -]
 Paniculata 0.10 0.13
 stem fraction
 (50:50)
 CH[Cl.sub.3]:
 C[H.sub.3]OH
 50 mg/rat/day
(Percent (-44.89%) (-36.84%)
 deviation)
 (c)

 Testicular cell counts
 (number/10 cross section)

 Primary spermatocytes

 Preleptotene Pachytene

Group-I 19.79 34.62
 control [+ or -] [+ or -]
 (Vehicle 1.95 1.98
 treated)

Group-II 3.92 ** 3.39 **
 M. [+ or -] [+ or -]
 Paniculata 0.31 0.31
 stem fraction
 (50:50)
 CH[Cl.sub.3]:
 C[H.sub.3]OH
 50 mg/rat/day
(Percent (-80.18%) (-90.20%)
 deviation)
 (c)

 Testicular cell counts Sertoli cell
 (number/10 cross section) area
 ([micro]
 [m.sup.2])
 Secondary Rounded
 spermato- spermatide
 cytes

Group-I 47.55 34.78 53.77
 control [+ or -] [+ or -] [+ or -]
 (Vehicle 0.93 2.51 2.89
 treated)

Group-II 17.11 ** 15.51 ** 30.54
 M. [+ or -] [+ or -] [+ or -]
 Paniculata 2.01 0.39 0.58
 stem fraction
 (50:50)
 CH[Cl.sub.3]:
 C[H.sub.3]OH
 50 mg/rat/day
(Percent (-64.00%) (-64.05%) (-43.20%)
 deviation)
 (c)

Value are in mean [+ or -] SEM (n=5)

Level of significant P[less than or equal to] 0.001

** highly significant when compared with group 1

(C) values in parentheses are percentage reduction in
particular cell type.
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Author:Gupta, R.S.; Kanwar, M.; Rehwani, H.; Kachhawa, J.B.S.
Publication:Bulletin of Pure & Applied Sciences-Zoology
Date:Jan 1, 2006
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