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Reversible antifertility effect of aqueous rhizome extract of Curcuma longa L. in male laboratory mice
Contraception 79 (2009) 479 – 487
Original research article
Reversible antifertility effect of aqueous rhizome extract of Curcuma longa L. in male laboratory mice Raghav Kumar Mishra, Shio Kumar Singh⁎ Department of Zoology, Banaras Hindu University, Varanasi, Uttar Pradesh 221 005, India Received 23 August 2008; revised 1 January 2009; accepted 4 January 2009
Abstract Background: The purpose of the present study was to evaluate the antifertility potential of Curcuma longa L. in the male laboratory mouse. Study Design: Mice of the Parkes (P) strain were orally administered aqueous rhizome extract of C. longa (600 mg/kg body weight per day for 56 and 84 days), and effect of the treatment on various male reproductive endpoints and fertility was evaluated. Recovery studies were also performed. Results: Histologically, testes in mice treated with the plant extract showed nonuniform degenerative changes in the seminiferous tubules as both affected and normal tubules were observed in the same section; the affected tubules showed loosening of germinal epithelium, intraepithelial vacuolation and mixing of spermatids of different stages of spermatogenesis. Marked reductions in diameter of seminiferous tubules, height of germinal epithelium and number of germ cells in Stage VII tubules were also noted in testes of extract-treated mice. Epididymis and seminal vesicle also showed histological alterations. Furthermore, the treatment had adverse effects on motility, viability, morphology and number of spermatozoa in the cauda epididymidis, levels of sialic acid in the epididymis and fructose in the seminal vesicle, serum level of testosterone and on fertility and litter size. By 56 days of treatment withdrawal, however, the above parameters recovered to control levels. Conclusions: The results show that in P mice C. longa treatment causes reversible suppression of spermatogenesis and fertility, thereby suggesting the potential of this plant in the regulation of male fertility. © 2009 Elsevier Inc. All rights reserved. Keywords: Curcuma longa; Mice; Seminiferous tubules; Spermatozoa; Testosterone; Fertility
1. Introduction From times immemorial, humans have relied on plants and their products as sources of drugs and therapeutic agents. Thus, the quest for developing an antifertility agent from a plant source in the regulation of male fertility appears to be an attractive proposition. Further, such an approach is cost-effective and has relatively low toxicity [1,2]. There is therefore a need to explore plants for their antifertility potential in the male, with the hope of developing a contraceptive for use in men. We have previously carried out studies with gossypol tetra-acetic acid [3,4], Azadirachta indica [5], Allamanda cathartica
⁎ Corresponding author. Tel.: +91 542 6702532; fax: +91 542 2368174. E-mail address: [email protected] (S.K. Singh). 0010-7824/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.contraception.2009.01.001
[6] and Bacopa monnieri [7]. In this study, we report our findings with Curcuma longa L. The plant C. longa L. (family Zingiberaceae) is a perennial herb and is widely cultivated in India. Dried rhizome of C. longa is the source of turmeric (a symbol of prosperity in Hindu culture). In Ayurveda, rhizome of C. longa has been used in the treatment of a variety of diseases such as those associated with skin, liver and pulmonary and gastrointestinal systems [8]. Furthermore, the plant has also been shown to possess antimutagenic and anticarcinogenic properties [9]. However, potential of this plant in the regulation of male fertility has not been well studied. The present study deals with the effect of the aqueous rhizome extract of C. longa on the male reproductive organs and fertility of the Parkes (P) strain mouse, which we have been using for an animal model [3–7]. We have evaluated various male reproductive end points such as organs weight, sperm
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parameters, histopathology, hormone assay and fertility indices; recovery studies were also performed. 2. Materials and methods 2.1. Plant material and preparation of extract Fresh rhizomes of C. longa were locally collected from the field and were authenticated by experts from the Botany Department of the Banaras Hindu University. A voucher specimen (Z-ING-2008-21) was deposited in the herbarium of the Botany Department of the Banaras Hindu University. The shade-dried rhizomes were cut into small pieces and coarsely ground. The ground plant material was then processed for preparation of aqueous extract of the rhizomes with sterile distilled water [6,10]. Briefly, the ground plant material weighing 50 g was covered with sterile distilled water in a ratio of 1:20 (w/v) and boiled in a glass vessel for 1 h. It was then cooled to room temperature and filtered. The filtrate was evaporated to dryness in oven at 37°C, and the yield of the extract was approximately 4 g, i.e., about 8.0% of the raw material. The extract was suspended in sterile distilled water, and the dose was expressed as dry weight of the extract. 2.2. Animals Adult (age 12–15 weeks) male laboratory mice of the P strain weighing 32–34 g were used in the investigations. Mice were from closed and randomly bred colony maintained in the animal house of the Department of Zoology, Banaras Hindu University. Animals were housed in a wellventilated room at 23±2°C with 12-h photoperiod and relative humidity of 50±20% and were maintained on pellet food (Mona Laboratory Animal Feeds, Varanasi) and drinking water ad libitum. Animals in each group were housed separately in polypropylene cages (450 mm× 270 mm×150 mm) with dry rice husk as the bedding material. General health condition and body weight of the animals were monitored throughout the experimental period. Animals were maintained according to the Guidelines of Institutional Animal Ethics Committee. 2.3. Treatments Mice were randomly allocated to six groups (nine animals per group) and treated as follows: Groups
Treatment (dose and duration)
Necropsy (time after last treatment)
1 2 3 4 5 6
Untreated controls Distilled water-treated controls C. longa at 600 mg/kg BW for 56 days C. longa at 600 mg/kg BW for 84 days C. longa at 600 mg/kg BW for 84 days Distilled water-treated controls
24 h 24 h 24 h 56 days 56 days
BW, body weight.
Aqueous rhizome extract of C. longa was suspended in sterile distilled water and administered orally, with the help
of an oral feeding needle. The dosing material was made fresh each day and administered after stirring. Controls (Groups 2 and 6) were administered an equivalent volume of sterile distilled water (0.5 mL/100 g body weight per day) in a similar manner. The dose of C. longa was selected on the basis of a pilot study conducted in our laboratory in P mice. We used 56- and 84-day durations in order to assess the effect of the treatment for longer periods. 2.4. Necropsy After recording final body weights at the end of the treatment schedule, animals were sacrificed by decapitation. The testis, epididymis and seminal vesicle were dissected out, blotted free of blood and weighed. 2.5. Sperm analyses At the time of euthanasia, spermatozoa were obtained from cauda epididymidis of six mice in each group in physiological saline maintained at 37°C [11]. Motility, viability and number were assessed [12]. The preparations used for assessing sperm viability were also used for assessing sperm morphology. The criteria of Wyrobek and Bruce [13] and Zaneveld and Polakoski [14] were employed for evaluation of sperm abnormalities. 2.6. Histological studies For histological examination, the testis, epididymis and seminal vesicle were randomly excised from left or right sides of six mice in each group, fixed in Bouin's fluid, dehydrated in graded ethanol series, cleared in benzene and embedded in paraffin. Tissues were sectioned at 6 μm, and the sections were then stained with periodic acid-Schiff (PAS) and counterstained with hematoxylin. Stages of spermatogenesis in mouse testis were identified according to the criteria of Russell et al. [15]. For evaluation of the quantitative changes in spermatogenesis caused by C. longa treatment, germ cell number at Stage VII of the spermatogenic cycle was determined; Stage VII is the most frequent stage of spermatogenesis and contains spermatogonia A, preleptotene spermatocytes, pachytene spermatocytes and spermatids. For this purpose, 10 seminiferous tubules at Stage VII of the spermatogenic cycle were randomly selected from a section of the testis from six mice in each group. The crude count of different germ cells was corrected using Abercombie's formula [16]. The diameter of the seminiferous tubules and height of the germinal epithelium were also measured in Stage VII round or slightly oblique seminiferous tubules (n=10) [7]. Percentage of affected seminiferous tubules was also determined [3]. 2.7. Testosterone assay Serum level of testosterone was measured by radioimmunoassay using commercial kit, as per manufacturer's instruction (Immunotech, Marseille, France). The sensitivity of the assay was 0.025 ng/mL. All samples were quantified
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Table 1 Body weight and weights of the testis, epididymis and seminal vesicle in mice after C. longa treatment (600 mg/kg body weight) and following treatment withdrawal Group and treatment
1, Control (untreated) 2, Control (distilled water) 3, C. longa, 56 days 4, C. longa, 84 days 5a, C. longa recovery 6a, Control (distilled water) recovery
Body weight (g)
Sex organs weight (mg/100 g body weight)
Initial
Final
Testis
Epididymis
Seminal vesicle
32.00±0.00 32.00±0.00 36.00±0.00 36.00±0.00 34.00±0.00 32.00±0.00
32.00±0.70 32.00±0.86 35.00±1.32 34.00±1.22 34.00±1.22 34.00±1.50
326.03±15.65 362.04±23.89 249.54⁎±16.56 265.62⁎±24.79 306.66±25.23 339.90±16.95
123.60±10.52 122.53±9.99 80.13⁎±12.33 86.63⁎±8.12 90.08±12.27 98.97±10.47
394.26±20.51 373.63±18.20 241.70⁎±44.14 222.15⁎±25.85 306.34±20.55 346.77±22.46
Values are mean±S.D. for six animals. Sex organ weight refers to the weight of the unpaired organ. a Treatment was discontinued after 84 days, and animals were sacrificed 56 days after treatment withdrawal. ⁎ Significantly different from controls (pb.05) by ANOVA followed by Newman–Keuls' multiple range test.
in a single assay with intra- and interassay coefficient of variations being 14.8% and 15%, respectively.
becoming pregnant were then allowed to deliver at term, and the litter size was recorded [5].
2.8. Analyses of sialic acid and fructose contents
2.10. Statistical analysis
The concentration of sialic acid in the epididymis was determined according to Aminoff's thiobarbituric acid method [17], while that of fructose in the seminal vesicle was estimated by the method of Lindner and Mann [18].
All data, except for body weight and fertility test results, were analyzed by one-way analysis of variance (ANOVA) followed by Newman–Keuls' multiple range test for the comparison of group means. Body weight data were analyzed by the Student's t test. Data on male fertility and pregnancy in the females were analyzed by the chi-square test. Student's t test was used to analyze data on litter size. Values were considered significant at pb.05.
2.9. Fertility test Twelve adult (age 12–15 weeks) male mice of proven fertility were used in the fertility test. Mice were administered aqueous rhizome extract of C. longa or sterile distilled water (n=6 per treatment). C. longa extract was suspended in sterile distilled water and administered orally at a dose of 600 mg/kg body weight per day for 84 days, while the controls received sterile distilled water in a similar manner. One hundred twenty adult female mice were employed in the fertility test. The fertility of C. longa or sterile distilled water-treated males was assessed 24 h and 2, 4, 6, and 8 weeks after cessation of treatment. Fertility was evaluated at each interval by allowing each male to cohabit with two proestrus females for overnight. Females were checked the next morning for the presence of vaginal plug for an indication of mating. The females
3. Results 3.1. Body weight and organ weight No significant differences were found between the initial and final body weights of C. longa-treated mice and controls (Table 1). All animals maintained a healthy appearance throughout the period of investigation. On the other hand, significant reductions were noted in the weights of the testis, epididymis and seminal vesicle in extract-treated mice compared to controls; however, the organ weights recovered to control levels by 56 days after cessation of treatment (Table 1).
Table 2 Motility, viability, abnormal morphology, and number of spermatozoa in cauda epididymidis of mice after C. longa treatment (600 mg/kg body weight) and following treatment withdrawal Group and treatment
Motility (%)
Viability (%)
Morphologically abnormal spermatozoa (%)
Sperm number (×106)
1, Control (untreated) 2, Control (distilled water) 3, C .longa 56 days 4, C. longa 84 days 5a, C. longa recovery 6a, Control (distilled water) recovery
80.50±2.66 79.41 ±2.76 56.16⁎±8.24 44.83⁎±15.19 72.58±2.76 77.58±2.39
86.75±2.38 86.58±4.66 78.16⁎±2.82 66.00⁎±4.95 80.66±5.06 83.33±4.43
8.91±3.48 9.91±2.67 18.83⁎±4.02 25.75⁎±4.09 15.83±4.49 11.33±6.79
12.25±0.84 11.05±0.68 8.05⁎±1.53 6.33⁎±0.84 11.05±1.28 11.28±1.22
Values are mean±S.D. for six animals. a Treatment was discontinued after 84 days, and animals were sacrificed 56 days after treatment withdrawal. ⁎ Significantly different from controls (pb.05) by ANOVA followed by Newman–Keuls' multiple range test.
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Fig. 1. PAS-hematoxylin stained sections of mouse testis. A, B, and D×175; C×230. (A) Control showing normal appearance of the seminiferous tubules. (B) After C. longa treatment, 600 mg/kg body weight/day for 56 days and sacrificed 24 h after the last treatment. Note loosening of germinal epithelium and appearance of intraepithelial vacuoles (arrows) in the seminiferous tubules. A seminiferous tubule (asterisk) showing normal appearance is also seen. (C) After the same treatment as shown in (B) showing mixing of spermatids of different stages of spermatogenesis in a seminiferous tubule. Steps 9, 10, 12 and 15 spermatids are seen in the tubule in the center. (D) After C. longa treatment, 600 mg/kg body weight per day for 84 days and sacrificed 24 h after the last treatment. Note that the severely affected seminiferous tubules are lined only by Sertoli cells and a few germ cells.
Table 3 Diameter of seminiferous tubules, height of germinal epithelium, corrected count of germ cells in stage VII seminiferous tubules, and percentage of affected seminiferous tubules in mice after C. longa treatment (600 mg/kg body weight) and following treatment withdrawal Group and treatment
Diameter (μm)
Height (μm)
Corrected count of germ cells Spermatogonia A Preleptotene spermatocytes
1, Control (untreated) 215.10±8.02 51.71±1.74 1.75±0.21 2, Control (distilled water) 219.8±8.20 49.21±3.63 1.94±0.19 3, C. longa 56 days 162.80⁎±7.04 35.41⁎±3.49 1.32⁎±0.16 4, C. longa 84 days 145.38⁎±9.04 26.72⁎±2.08 1.24⁎±0.21 218.83±6.79 44.91±5.34 1.66±0.25 5a, C. longa recovery 6a, Control (distilled water) 213.38±5.36 49.44±2.37 1.83±0.31 recovery
9.17±1.11 10.01±1.64 7.33⁎±1.36 4.91⁎±0.48 7.61±0.99 8.43±1.07
Pachytene spermatocytes
Step 7 spermatids
15.57±2.44 14.09±0.65 12.44⁎±2.16 11.63⁎±1.22 13.05±1.18 14.06±0.86
51.34±5.55 49.91±4.29 29.73⁎±11.67 22.49⁎±7.18 39.86±3.29 43.18±4.02
Values are mean±S.D. for six animals. a Treatment was discontinued after 84 days, and animals were sacrificed 56 days after treatment withdrawal. ⁎ Significantly different from controls (pb.05) by ANOVA followed by Newman–Keuls' multiple range test.
Affected seminiferous tubules (%) 6.78±2.28 10.96±2.64 43.65⁎±28.37 45.10⁎±28.13 11.05±4.32 10.43±2.11
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3.2. Sperm analyses Significant reductions were found in motility, viability, and in the number of spermatozoa in caudae epididymidis of C. longa-treated mice compared to controls (Table 2).
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There was also a significant increase in the number of morphologically abnormal spermatozoa in extract-treated mice (Groups 3 and 4) than in controls (Table 2); abnormal spermatozoa were mainly those that showed cytoplasmic droplet attached to the tail; sometimes, head- and tail-less
Fig. 2. PAS–hematoxylin-stained sections through Segments I–V of mouse epididymis (original magnification ×175). Segments I (A), II (B), III (C), IV (D) and V (E) of a control mouse showing normal features. Segments I (F), II (G), III (H), IV (I) and V (J) of a mouse after C. longa treatment, 600 mg/kg body weight/ day for 84 days and sacrificed 24 h after the last treatment. In Segment I (F), the tubules show a decrease in size with a slight increase in stroma; the nuclei of the epithelial cells are irregularly arranged. In Segment II (G), the epithelium shows loss of stainability; the lumen is empty or contains only PAS-positive materials or PAS-positive materials and sperm. In Segments III (H), IV (I) and V (J), the epithelium shows a normal appearance, except that sometimes, vacuolelike spaces (arrows) are seen in Segment III (H); the lumen of the tubules in these segments contains PAS-positive materials, sperm and occasionally exfoliated germ cells.
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Fig. 3. PAS–hematoxylin stained sections of mouse seminal vesicle (original magnification ×175). (A) Control showing normal features. Note that the lumen is distended with secretion. (B) After C. longa treatment, 600 mg/kg body weight per day for 84 days and sacrificed 24 h after the last treatment. Note that the epithelium shows inward ramifications, and the lumen contains scanty secretory material. cf. (A).
spermatozoa were also observed. By 56 days of treatment withdrawal, however, the altered sperm parameters recovered to control levels (Table 2). 3.3. Histological studies 3.3.1. Testis The testes of untreated controls (Fig. 1A) and distilled water-treated controls exhibited normal histological features in nearly all the seminiferous tubules, except in a few (Group 1: 6.78%; Group 2: 10.96%) (Table 3) in which the tubules showed loosening of germinal epithelium or intraepithelial vacuolation. In contrast, marked degenerative changes were observed in the histoarchitecture of the testis in C. longatreated mice; the changes in the seminiferous tubules were, however, not uniform as both affected and normal tubules were observed in the same sections of the testis. The histological changes noticed in the affected seminiferous tubules in testes of mice treated with C. longa for 56 or 84
days were not much different, although the changes became more pronounced with the increasing duration of the treatment (Fig. 1B–D). The affected tubules showed loosening of germinal epithelium, intraepithelial vacuolation and mixing of spermatids of different stages of spermatogenesis (Fig. 1B and C); in severe cases, the affected tubules showed Sertoli cells and few germ cells (Fig. 1D). Significant reductions were also found in the diameter of the seminiferous tubules and in the height of the germinal epithelium in extract-treated mice compared to controls (Table 3). This was also true for the number of germ cells in Stage VII seminiferous tubules in testes of treated mice (Table 3). Fifty-six days after cessation of treatment, however, the alterations caused in the seminiferous tubules recovered to control levels. 3.3.2. Epididymis The epididymis of untreated controls (Fig. 2A–E) and distilled water-treated controls showed normal histological
Fig. 4. Serum level of testosterone in mice (n=6) after C. longa treatment (600 mg/kg body weight) and following treatment withdrawal. Values are mean±S.D. In recovery group, mice were treated with C. longa daily for 84 days, and thereafter, the treatment was discontinued and mice were sacrificed 56 days after treatment withdrawal. ⁎Significantly different from controls (pb.05) by ANOVA followed by Newman–Keuls' multiple range test.
R.K. Mishra, S.K. Singh / Contraception 79 (2009) 479–487 Table 4 Concentrations of sialic acid in the epididymis and of fructose in the seminal vesicle in mice after C. longa treatment (600 mg/kg body weight) and following treatment withdrawal Group and treatment
Sialic acid concentration Fructose concentration (μ moles/100 g tissue) (mcg/100 mg tissue)
1, Control (untreated) 2, Control (distilled water) 3, C. longa 56 days 4, C. longa 84 days 5a, C. longa recovery 6a, Control (distilled water) recovery
140.37±27.60 117.90±16.54 80.15⁎±21.61 30.33⁎±17.09 111.08±12.30 155.87±13.27
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drawal of treatment, epididymis presented a histology which was similar to that of controls. 3.3.3. Seminal vesicle The seminal vesicle of controls showed normal histological features (Fig. 3A). In contrast, in extract-treated mice, the gland showed marked histological alterations. The lumen of the gland contained scanty secretion, and the epithelium showed excessive inward ramification; the muscle layer surrounding the glandular epithelium was also considerably increased (Fig. 3B; cf. Fig. 3A). By 56 days of treatment withdrawal, however, the gland recovered and presented a normal histology.
221.74±22.64 218.53±23.72 167.39⁎±25.54 126.89⁎±50.93 199.15±22.60 212.36±29.76
Values are mean±S.D. for six animals. a Treatment was discontinued after 84 days, and animals were sacrificed 56 days after treatment withdrawal. ⁎ Significantly different from controls (pb.05) by ANOVA followed by Newman–Keuls' multiple range test.
3.4. Testosterone assay Significant reduction was noted in serum level of testosterone in extract-treated mice compared to controls. By 56 days of treatment withdrawal, however, the hormone level increased and the values even exceeded control level (Fig. 4).
features. On the other hand, in mice treated with C. longa extract for 56 or 84 days, detectable histological alterations were noticed in the epididymis (Fig. 2F-J). Furthermore, the alterations noticed at 56 or 84 days of treatment durations were nearly similar, and they are therefore described together. In Segment I (Fig. 2F), the size of the tubules appeared decreased with a slight increase in stroma, and the nuclei of the epithelial cells were sometimes irregularly arranged in the epithelium of the duct. In Segment II (Fig. 2G), the epithelium showed loss of stainability and a vacuolated appearance of the cells; the stroma also appeared slightly increased, and the lumen was either empty or contained only PAS-positive materials or PASpositive materials and sperm. In Segments III (Fig. 2H), IV (Fig. 2I) and V (Fig. 2J) of the epididymal duct, the epithelium presented a normal appearance, except that in Segment III, sometimes, vacuole-like spaces were noticed; the stroma also showed a little increase, and the lumen contained PAS-positive materials, a little less sperm and occasionally exfoliated germ cells. Fifty-six days after with-
3.5. Analyses of sialic acid and fructose contents Significant reductions were noted in the levels of sialic acid in the epididymis and that of fructose in the seminal vesicle, respectively, in extract-treated mice compared to controls; by 56 days of treatment withdrawal, however, the values recovered to control levels (Table 4). 3.6. Fertility test When C. longa-treated males were caged with females at 24 h and 2, 4, 6, and 8 weeks after withdrawal of treatment, mating was not affected at any interval. Fertility of the extract-treated males was affected at 24 h post withdrawal; thereafter, no effect on fertility of the treated males was found from 2 up to 8 weeks of treatment withdrawal, though only four of six mice had shown return of fertility at 2 weeks post withdrawal (Table 5). The litter size in females impregnated by extract-treated males was significantly
Table 5 Fertility of male mice after treatment with C. longa (600 mg/kg body weight/day for 84 days) or distilled water Time after treatment
Treatnent
24 h
Control C. longa Control C. longa Control C. longa Control C. longa Control C. longa
2 weeks 4 weeks 6 weeks 8 weeks
Number of males
Number of females
Litter size
Tested
Mated
Fertile
Tested
Mated
Fertile
6 6 6 6 6 6 6 6 6 6
6 6 6 6 6 6 6 6 6 6
6 2a 6 4 6 6 6 6 6 6
12 12 12 12 12 12 12 12 12 12
12 7 12 9 10 9 10 10 11 10
10 2a 12 4a 10 6 9 8 10 10
Values are mean±S.D. a Significantly different from controls by chi-square test. ⁎ Significantly (pb.001) different from controls by the Student's t test.
7.66±1.21 2.00⁎±1.41 7.08±0.73 4.75⁎±2.98 7.16±0.81 4.66⁎±1.75 7.16±0.81 6.00±1.22 7.00±0.89 7.41±0.73
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decreased compared to controls at 24 h and up to 4 weeks of treatment withdrawal; at 6 and 8 weeks after cessation of treatment, however, no differences could be detected in litter size in females impregnated by treated males compared to controls (Table 5).
4. Discussion The results of the present study indicate that the C. longa treatment did not cause alterations in body weight of the treated animals, suggesting that the treatment had no systemic toxic effect in P mice. Further, the present results also show that the treatment caused degenerative changes in the seminiferous tubules in mice testes with a marked reduction in the weight of the organ. The changes, however, were not uniform as both affected and normal seminiferous tubules were observed in the same section of the testis; the affected tubules showed loosening of germinal epithelium, intraepithelial vacuolation and mixing of spermatids of different stages of spermatogenesis. Similar histological changes have also been described in testes of P mice after treatment with aqueous leaf extracts of Azadirachta indica [5] and Allamanda cathartica [6], and hexane extract of flower buds of Syzygium aromaticum [19] and several other antispermatogenic agents such as gossypol tetra-acetic acid [3,4], nitrofurazone [20] and SC 12937 [11]. The observation that there were marked reductions in the number of spermatogonia A, preleptotene spermatocytes, pachytene spermatocytes and Step 7 spermatids in Stage VII seminiferous tubules in testes of extract-treated mice as reported here suggests that the C. longa treatment interferes with both mitotic and meiotic processes resulting in the suppression of spermatogenesis. Furthermore, the treatment also caused significant reductions in the diameter of the seminiferous tubules and in the height of the germinal epithelium in testes of extract-treated mice compared to controls; these observations are suggestive of the adverse effect of the treatment on spermatogenesis. In P mice in the present study, C. longa treatment caused marked reduction in the weight of the epididymis accompanied by detectable histological changes in the organ. The changes in the luminal content of the epididymis in treated mice appeared to be the outcome of the changes in the testis. The observation that there was a significant reduction in the level of sialic acid in the epididymis of the treated mice compared to controls suggests that the C. longa treatment interferes with the secretory function of the organ [21]. Furthermore, the treatment also had adverse effects on motility, morphology, viability, and on the number of spermatozoa in the cauda epididymidis. Purohit and Bhagat [22] have also reported reductions in density and motility of spermatozoa in cauda epididymidis of rats following treatment with aqueous and alcoholic extracts of rhizome of C. longa. The reduction in the number of spermatozoa in cauda epididymidis in treated mice in the present study
appeared to be due to the suppressive effect of C. longa treatment on spermatogenesis as the sperm number recovered to control levels after restoration of spermatogenic activity following cessation of treatment, while the alterations in sperm motility, viability and morphology might have resulted from disturbances in epididymal function [23]. Results of sialic acid level in the epididymis of the treated mice in the present study also lend support to the above view. The treatment also caused significant reduction in the weight of the seminal vesicle in treated mice compared to controls. Furthermore, the treatment also caused a marked reduction in the level of fructose in the seminal vesicle in treated mice, suggesting that the C. longa treatment had an adverse effect on the secretory function of the gland. The histological observations of the gland in treated mice also support this contention. In P mice in the present study, mating was not affected by the treatment. Marked reductions were, however, found in litter size in females impregnated by C. longa-treated males in the present study at 24 h and 2 and 4 weeks after cessation of treatment, and this may be attributed to the poor sperm quality recorded in treated males. The present results show that the C. longa treatment causes suppression of spermatogenesis in mice testis, though the mechanism of this suppression remains poorly understood. In immature male rat, C. comosa, another species of Curcuma, has been suggested to act directly on the testis or indirectly to inhibit gonadotropin secretion, which consequently reduces testosterone production, or to act at both levels [24]. In P mice in the present study, C. longa treatment caused significant reduction in serum level of testosterone in treated mice compared to controls. It is well known that testosterone plays a major role in the maintenance of spermatogenesis [25]. Thus, it is probable that the C. longainduced suppression of spermatogenesis in mice testes in the present study is caused due to deficiency of testosterone. In conclusion, our results in P mice suggest that the C. longa treatment causes marked alterations in the male reproductive organs and that the alterations are reversible after cessation of treatment. Thus, C. longa may have a potential in the regulation of fertility in the male. Acknowledgment This work was supported by funds from DST, and the University Grants Commission, New Delhi, through CAS in Zoology, Banaras Hindu University. References [1] Chaudhury RR. Plant contraceptives: translating folklore into scientific application. In: Jelliffe DB, Jelliffe EFP, editors. Advances in Maternal and Child Health. Oxford: Oxford University Press; 1985. p. 58–74. [2] Qureshi AA, Sanghai DB, Padgilwar SS. Herbal options for contraception: a review. Pharmacog Mag 2005;2:204–14. [3] Singh SK, Rath SK. Histologic changes in the mouse testis after treatment with gossypol tetra-acetic acid. Arch Histol Cytol 1990;53: 393–6.
R.K. Mishra, S.K. Singh / Contraception 79 (2009) 479–487 [4] Singh SK, Rath SK. Effect of gossypol tetra-acetic acid on the reproductive organs in male mice. In: Singh VK, Govil JN, editors. Recent progress in medicinal plants, vol. 25. Houston (TX): Studium Press LLC; 2009. p. 159–76. [5] Mishra RK, Singh SK. Effect of aqueous leaf extract of Azadirachta indica on the reproductive organs in male mice. Indian J Exp Biol 2005;43:1093–103. [6] Singh A, Singh SK. Reversible antifertility effect of aqueous leaf extract of Allamanda cathartica L. in male laboratory mice. Andrologia 2008;40:337–45. [7] Singh A, Singh SK. Evaluation of antifertility potential of Brahmi in male mouse. Contraception 2009;79:71–9. [8] Kirtikar KR, Basu BD. Indian medicinal plants. Delhi: Periodical Experts Book Agency; 1984. [9] Scartezzini P, Speroni E. Review on some plants of Indian traditional medicine with antioxidant activity. J Ethnopharmacol 2000;71:23–43. [10] World Health Organization. WHO Chemical Guidelines, CG-03, 1001A/ip. Geneva: WHO; 1986. [11] Singh SK, Chakravarty S. Antispermatogenic and antifertility effects of 20,25-diazacholesterol dihydrochloride in mice. Reprod Toxicol 2003;17:37–44. [12] World Health Organization. WHO Laboratory Manual for the Examination of Human Semen and Semen–Cervical Mucus Interaction. Cambridge: Cambridge University Press; 1999. [13] Wyrobek AJ, Bruce WR. Chemical induction of sperm abnormalities in mice. Proc Natl Acad Sci U S A 1975;72:4425–9. [14] Zaneveld L, Polakoski KL. Collection and physical examination of the ejaculate. In: Hafez ESE, editor. Techniques of human andrology. New York: Elsevier/North Holland Biomedical Press; 1977. p. 147–72. [15] Russell LD, Ettlin RA, Hikim APS, Clegg ED. Histological and histopathological evaluation of the testis. Clearwater: Cache River Press; 1990.
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[16] Abercombie M. Estimation of nuclear population from microtome sections. Anat Rec 1946;94:239–47. [17] Aminoff D. Methods for the quantitative estimation of N-acetyl neuraminic acid and their application to hydrolysates of sialomucoids. Biochem J 1961;81:384–92. [18] Lindner HR, Mann T. Relationship between the content of androgenic steroids in the testis and the secretory activity of the seminal vesicles in the bull. J Endocrinol 1960;21:341–60. [19] Mishra RK, Singh SK. Safety assessment of Syzygium aromaticum flower bud (clove) extract with respect to testicular function in mice. Food Chem Toxicol 2008;46:3333–8. [20] Singh SK, Chakravarty S. Effect of nitrofurazone on the reproductive organs in adult male mice. Asian J Androl 2001;3:39–44. [21] Hamilton DW. Structure and function of the epithelium lining the ductuli efferentes, ductus epididymidis, and ductus deferens in the rat. In: Hamilton DW, Greep RO, editors. Handbook of physiology, Section 7, vol. V. Washington (DC): American Physiological Society; 1975. p. 259–301. [22] Purohit A, Bhagat M. Contraceptive effect of Curcuma longa (L.) in male albino rat. Asian J Androl 2004;6:71–4. [23] Rajalakshmi M. Regulation of male fertility: epididymis as a potential extragonadal site. In: Ghosh D, Sengupta J, editors. Frontiers in reproductive physiology. New Delhi: Wiley Eastern Limited; 1992. p. 63–6. [24] Piyachaturawat P, Timinkul A, Chuncharunee A, Suksamrarn A. Growth suppressing effect of Curcuma comosa extract on male reproductive organs in immature rats. Pharmaceut Biol 1998;36:44–9. [25] O'Donell L, Meachem SJ, Stanton PG, McLachlan RI. Endocrine regulation of spermatogenesis. In: Neill JD, editor. Knobil and Neill's Physiology of Reproduction, 3rd ed., vol. 1. St Louis (MO): Elsevier Academic Press, St; 2006. p. 1017–69.
Original research article
Reversible antifertility effect of aqueous rhizome extract of Curcuma longa L. in male laboratory mice Raghav Kumar Mishra, Shio Kumar Singh⁎ Department of Zoology, Banaras Hindu University, Varanasi, Uttar Pradesh 221 005, India Received 23 August 2008; revised 1 January 2009; accepted 4 January 2009
Abstract Background: The purpose of the present study was to evaluate the antifertility potential of Curcuma longa L. in the male laboratory mouse. Study Design: Mice of the Parkes (P) strain were orally administered aqueous rhizome extract of C. longa (600 mg/kg body weight per day for 56 and 84 days), and effect of the treatment on various male reproductive endpoints and fertility was evaluated. Recovery studies were also performed. Results: Histologically, testes in mice treated with the plant extract showed nonuniform degenerative changes in the seminiferous tubules as both affected and normal tubules were observed in the same section; the affected tubules showed loosening of germinal epithelium, intraepithelial vacuolation and mixing of spermatids of different stages of spermatogenesis. Marked reductions in diameter of seminiferous tubules, height of germinal epithelium and number of germ cells in Stage VII tubules were also noted in testes of extract-treated mice. Epididymis and seminal vesicle also showed histological alterations. Furthermore, the treatment had adverse effects on motility, viability, morphology and number of spermatozoa in the cauda epididymidis, levels of sialic acid in the epididymis and fructose in the seminal vesicle, serum level of testosterone and on fertility and litter size. By 56 days of treatment withdrawal, however, the above parameters recovered to control levels. Conclusions: The results show that in P mice C. longa treatment causes reversible suppression of spermatogenesis and fertility, thereby suggesting the potential of this plant in the regulation of male fertility. © 2009 Elsevier Inc. All rights reserved. Keywords: Curcuma longa; Mice; Seminiferous tubules; Spermatozoa; Testosterone; Fertility
1. Introduction From times immemorial, humans have relied on plants and their products as sources of drugs and therapeutic agents. Thus, the quest for developing an antifertility agent from a plant source in the regulation of male fertility appears to be an attractive proposition. Further, such an approach is cost-effective and has relatively low toxicity [1,2]. There is therefore a need to explore plants for their antifertility potential in the male, with the hope of developing a contraceptive for use in men. We have previously carried out studies with gossypol tetra-acetic acid [3,4], Azadirachta indica [5], Allamanda cathartica
⁎ Corresponding author. Tel.: +91 542 6702532; fax: +91 542 2368174. E-mail address: [email protected] (S.K. Singh). 0010-7824/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.contraception.2009.01.001
[6] and Bacopa monnieri [7]. In this study, we report our findings with Curcuma longa L. The plant C. longa L. (family Zingiberaceae) is a perennial herb and is widely cultivated in India. Dried rhizome of C. longa is the source of turmeric (a symbol of prosperity in Hindu culture). In Ayurveda, rhizome of C. longa has been used in the treatment of a variety of diseases such as those associated with skin, liver and pulmonary and gastrointestinal systems [8]. Furthermore, the plant has also been shown to possess antimutagenic and anticarcinogenic properties [9]. However, potential of this plant in the regulation of male fertility has not been well studied. The present study deals with the effect of the aqueous rhizome extract of C. longa on the male reproductive organs and fertility of the Parkes (P) strain mouse, which we have been using for an animal model [3–7]. We have evaluated various male reproductive end points such as organs weight, sperm
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parameters, histopathology, hormone assay and fertility indices; recovery studies were also performed. 2. Materials and methods 2.1. Plant material and preparation of extract Fresh rhizomes of C. longa were locally collected from the field and were authenticated by experts from the Botany Department of the Banaras Hindu University. A voucher specimen (Z-ING-2008-21) was deposited in the herbarium of the Botany Department of the Banaras Hindu University. The shade-dried rhizomes were cut into small pieces and coarsely ground. The ground plant material was then processed for preparation of aqueous extract of the rhizomes with sterile distilled water [6,10]. Briefly, the ground plant material weighing 50 g was covered with sterile distilled water in a ratio of 1:20 (w/v) and boiled in a glass vessel for 1 h. It was then cooled to room temperature and filtered. The filtrate was evaporated to dryness in oven at 37°C, and the yield of the extract was approximately 4 g, i.e., about 8.0% of the raw material. The extract was suspended in sterile distilled water, and the dose was expressed as dry weight of the extract. 2.2. Animals Adult (age 12–15 weeks) male laboratory mice of the P strain weighing 32–34 g were used in the investigations. Mice were from closed and randomly bred colony maintained in the animal house of the Department of Zoology, Banaras Hindu University. Animals were housed in a wellventilated room at 23±2°C with 12-h photoperiod and relative humidity of 50±20% and were maintained on pellet food (Mona Laboratory Animal Feeds, Varanasi) and drinking water ad libitum. Animals in each group were housed separately in polypropylene cages (450 mm× 270 mm×150 mm) with dry rice husk as the bedding material. General health condition and body weight of the animals were monitored throughout the experimental period. Animals were maintained according to the Guidelines of Institutional Animal Ethics Committee. 2.3. Treatments Mice were randomly allocated to six groups (nine animals per group) and treated as follows: Groups
Treatment (dose and duration)
Necropsy (time after last treatment)
1 2 3 4 5 6
Untreated controls Distilled water-treated controls C. longa at 600 mg/kg BW for 56 days C. longa at 600 mg/kg BW for 84 days C. longa at 600 mg/kg BW for 84 days Distilled water-treated controls
24 h 24 h 24 h 56 days 56 days
BW, body weight.
Aqueous rhizome extract of C. longa was suspended in sterile distilled water and administered orally, with the help
of an oral feeding needle. The dosing material was made fresh each day and administered after stirring. Controls (Groups 2 and 6) were administered an equivalent volume of sterile distilled water (0.5 mL/100 g body weight per day) in a similar manner. The dose of C. longa was selected on the basis of a pilot study conducted in our laboratory in P mice. We used 56- and 84-day durations in order to assess the effect of the treatment for longer periods. 2.4. Necropsy After recording final body weights at the end of the treatment schedule, animals were sacrificed by decapitation. The testis, epididymis and seminal vesicle were dissected out, blotted free of blood and weighed. 2.5. Sperm analyses At the time of euthanasia, spermatozoa were obtained from cauda epididymidis of six mice in each group in physiological saline maintained at 37°C [11]. Motility, viability and number were assessed [12]. The preparations used for assessing sperm viability were also used for assessing sperm morphology. The criteria of Wyrobek and Bruce [13] and Zaneveld and Polakoski [14] were employed for evaluation of sperm abnormalities. 2.6. Histological studies For histological examination, the testis, epididymis and seminal vesicle were randomly excised from left or right sides of six mice in each group, fixed in Bouin's fluid, dehydrated in graded ethanol series, cleared in benzene and embedded in paraffin. Tissues were sectioned at 6 μm, and the sections were then stained with periodic acid-Schiff (PAS) and counterstained with hematoxylin. Stages of spermatogenesis in mouse testis were identified according to the criteria of Russell et al. [15]. For evaluation of the quantitative changes in spermatogenesis caused by C. longa treatment, germ cell number at Stage VII of the spermatogenic cycle was determined; Stage VII is the most frequent stage of spermatogenesis and contains spermatogonia A, preleptotene spermatocytes, pachytene spermatocytes and spermatids. For this purpose, 10 seminiferous tubules at Stage VII of the spermatogenic cycle were randomly selected from a section of the testis from six mice in each group. The crude count of different germ cells was corrected using Abercombie's formula [16]. The diameter of the seminiferous tubules and height of the germinal epithelium were also measured in Stage VII round or slightly oblique seminiferous tubules (n=10) [7]. Percentage of affected seminiferous tubules was also determined [3]. 2.7. Testosterone assay Serum level of testosterone was measured by radioimmunoassay using commercial kit, as per manufacturer's instruction (Immunotech, Marseille, France). The sensitivity of the assay was 0.025 ng/mL. All samples were quantified
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Table 1 Body weight and weights of the testis, epididymis and seminal vesicle in mice after C. longa treatment (600 mg/kg body weight) and following treatment withdrawal Group and treatment
1, Control (untreated) 2, Control (distilled water) 3, C. longa, 56 days 4, C. longa, 84 days 5a, C. longa recovery 6a, Control (distilled water) recovery
Body weight (g)
Sex organs weight (mg/100 g body weight)
Initial
Final
Testis
Epididymis
Seminal vesicle
32.00±0.00 32.00±0.00 36.00±0.00 36.00±0.00 34.00±0.00 32.00±0.00
32.00±0.70 32.00±0.86 35.00±1.32 34.00±1.22 34.00±1.22 34.00±1.50
326.03±15.65 362.04±23.89 249.54⁎±16.56 265.62⁎±24.79 306.66±25.23 339.90±16.95
123.60±10.52 122.53±9.99 80.13⁎±12.33 86.63⁎±8.12 90.08±12.27 98.97±10.47
394.26±20.51 373.63±18.20 241.70⁎±44.14 222.15⁎±25.85 306.34±20.55 346.77±22.46
Values are mean±S.D. for six animals. Sex organ weight refers to the weight of the unpaired organ. a Treatment was discontinued after 84 days, and animals were sacrificed 56 days after treatment withdrawal. ⁎ Significantly different from controls (pb.05) by ANOVA followed by Newman–Keuls' multiple range test.
in a single assay with intra- and interassay coefficient of variations being 14.8% and 15%, respectively.
becoming pregnant were then allowed to deliver at term, and the litter size was recorded [5].
2.8. Analyses of sialic acid and fructose contents
2.10. Statistical analysis
The concentration of sialic acid in the epididymis was determined according to Aminoff's thiobarbituric acid method [17], while that of fructose in the seminal vesicle was estimated by the method of Lindner and Mann [18].
All data, except for body weight and fertility test results, were analyzed by one-way analysis of variance (ANOVA) followed by Newman–Keuls' multiple range test for the comparison of group means. Body weight data were analyzed by the Student's t test. Data on male fertility and pregnancy in the females were analyzed by the chi-square test. Student's t test was used to analyze data on litter size. Values were considered significant at pb.05.
2.9. Fertility test Twelve adult (age 12–15 weeks) male mice of proven fertility were used in the fertility test. Mice were administered aqueous rhizome extract of C. longa or sterile distilled water (n=6 per treatment). C. longa extract was suspended in sterile distilled water and administered orally at a dose of 600 mg/kg body weight per day for 84 days, while the controls received sterile distilled water in a similar manner. One hundred twenty adult female mice were employed in the fertility test. The fertility of C. longa or sterile distilled water-treated males was assessed 24 h and 2, 4, 6, and 8 weeks after cessation of treatment. Fertility was evaluated at each interval by allowing each male to cohabit with two proestrus females for overnight. Females were checked the next morning for the presence of vaginal plug for an indication of mating. The females
3. Results 3.1. Body weight and organ weight No significant differences were found between the initial and final body weights of C. longa-treated mice and controls (Table 1). All animals maintained a healthy appearance throughout the period of investigation. On the other hand, significant reductions were noted in the weights of the testis, epididymis and seminal vesicle in extract-treated mice compared to controls; however, the organ weights recovered to control levels by 56 days after cessation of treatment (Table 1).
Table 2 Motility, viability, abnormal morphology, and number of spermatozoa in cauda epididymidis of mice after C. longa treatment (600 mg/kg body weight) and following treatment withdrawal Group and treatment
Motility (%)
Viability (%)
Morphologically abnormal spermatozoa (%)
Sperm number (×106)
1, Control (untreated) 2, Control (distilled water) 3, C .longa 56 days 4, C. longa 84 days 5a, C. longa recovery 6a, Control (distilled water) recovery
80.50±2.66 79.41 ±2.76 56.16⁎±8.24 44.83⁎±15.19 72.58±2.76 77.58±2.39
86.75±2.38 86.58±4.66 78.16⁎±2.82 66.00⁎±4.95 80.66±5.06 83.33±4.43
8.91±3.48 9.91±2.67 18.83⁎±4.02 25.75⁎±4.09 15.83±4.49 11.33±6.79
12.25±0.84 11.05±0.68 8.05⁎±1.53 6.33⁎±0.84 11.05±1.28 11.28±1.22
Values are mean±S.D. for six animals. a Treatment was discontinued after 84 days, and animals were sacrificed 56 days after treatment withdrawal. ⁎ Significantly different from controls (pb.05) by ANOVA followed by Newman–Keuls' multiple range test.
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Fig. 1. PAS-hematoxylin stained sections of mouse testis. A, B, and D×175; C×230. (A) Control showing normal appearance of the seminiferous tubules. (B) After C. longa treatment, 600 mg/kg body weight/day for 56 days and sacrificed 24 h after the last treatment. Note loosening of germinal epithelium and appearance of intraepithelial vacuoles (arrows) in the seminiferous tubules. A seminiferous tubule (asterisk) showing normal appearance is also seen. (C) After the same treatment as shown in (B) showing mixing of spermatids of different stages of spermatogenesis in a seminiferous tubule. Steps 9, 10, 12 and 15 spermatids are seen in the tubule in the center. (D) After C. longa treatment, 600 mg/kg body weight per day for 84 days and sacrificed 24 h after the last treatment. Note that the severely affected seminiferous tubules are lined only by Sertoli cells and a few germ cells.
Table 3 Diameter of seminiferous tubules, height of germinal epithelium, corrected count of germ cells in stage VII seminiferous tubules, and percentage of affected seminiferous tubules in mice after C. longa treatment (600 mg/kg body weight) and following treatment withdrawal Group and treatment
Diameter (μm)
Height (μm)
Corrected count of germ cells Spermatogonia A Preleptotene spermatocytes
1, Control (untreated) 215.10±8.02 51.71±1.74 1.75±0.21 2, Control (distilled water) 219.8±8.20 49.21±3.63 1.94±0.19 3, C. longa 56 days 162.80⁎±7.04 35.41⁎±3.49 1.32⁎±0.16 4, C. longa 84 days 145.38⁎±9.04 26.72⁎±2.08 1.24⁎±0.21 218.83±6.79 44.91±5.34 1.66±0.25 5a, C. longa recovery 6a, Control (distilled water) 213.38±5.36 49.44±2.37 1.83±0.31 recovery
9.17±1.11 10.01±1.64 7.33⁎±1.36 4.91⁎±0.48 7.61±0.99 8.43±1.07
Pachytene spermatocytes
Step 7 spermatids
15.57±2.44 14.09±0.65 12.44⁎±2.16 11.63⁎±1.22 13.05±1.18 14.06±0.86
51.34±5.55 49.91±4.29 29.73⁎±11.67 22.49⁎±7.18 39.86±3.29 43.18±4.02
Values are mean±S.D. for six animals. a Treatment was discontinued after 84 days, and animals were sacrificed 56 days after treatment withdrawal. ⁎ Significantly different from controls (pb.05) by ANOVA followed by Newman–Keuls' multiple range test.
Affected seminiferous tubules (%) 6.78±2.28 10.96±2.64 43.65⁎±28.37 45.10⁎±28.13 11.05±4.32 10.43±2.11
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3.2. Sperm analyses Significant reductions were found in motility, viability, and in the number of spermatozoa in caudae epididymidis of C. longa-treated mice compared to controls (Table 2).
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There was also a significant increase in the number of morphologically abnormal spermatozoa in extract-treated mice (Groups 3 and 4) than in controls (Table 2); abnormal spermatozoa were mainly those that showed cytoplasmic droplet attached to the tail; sometimes, head- and tail-less
Fig. 2. PAS–hematoxylin-stained sections through Segments I–V of mouse epididymis (original magnification ×175). Segments I (A), II (B), III (C), IV (D) and V (E) of a control mouse showing normal features. Segments I (F), II (G), III (H), IV (I) and V (J) of a mouse after C. longa treatment, 600 mg/kg body weight/ day for 84 days and sacrificed 24 h after the last treatment. In Segment I (F), the tubules show a decrease in size with a slight increase in stroma; the nuclei of the epithelial cells are irregularly arranged. In Segment II (G), the epithelium shows loss of stainability; the lumen is empty or contains only PAS-positive materials or PAS-positive materials and sperm. In Segments III (H), IV (I) and V (J), the epithelium shows a normal appearance, except that sometimes, vacuolelike spaces (arrows) are seen in Segment III (H); the lumen of the tubules in these segments contains PAS-positive materials, sperm and occasionally exfoliated germ cells.
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Fig. 3. PAS–hematoxylin stained sections of mouse seminal vesicle (original magnification ×175). (A) Control showing normal features. Note that the lumen is distended with secretion. (B) After C. longa treatment, 600 mg/kg body weight per day for 84 days and sacrificed 24 h after the last treatment. Note that the epithelium shows inward ramifications, and the lumen contains scanty secretory material. cf. (A).
spermatozoa were also observed. By 56 days of treatment withdrawal, however, the altered sperm parameters recovered to control levels (Table 2). 3.3. Histological studies 3.3.1. Testis The testes of untreated controls (Fig. 1A) and distilled water-treated controls exhibited normal histological features in nearly all the seminiferous tubules, except in a few (Group 1: 6.78%; Group 2: 10.96%) (Table 3) in which the tubules showed loosening of germinal epithelium or intraepithelial vacuolation. In contrast, marked degenerative changes were observed in the histoarchitecture of the testis in C. longatreated mice; the changes in the seminiferous tubules were, however, not uniform as both affected and normal tubules were observed in the same sections of the testis. The histological changes noticed in the affected seminiferous tubules in testes of mice treated with C. longa for 56 or 84
days were not much different, although the changes became more pronounced with the increasing duration of the treatment (Fig. 1B–D). The affected tubules showed loosening of germinal epithelium, intraepithelial vacuolation and mixing of spermatids of different stages of spermatogenesis (Fig. 1B and C); in severe cases, the affected tubules showed Sertoli cells and few germ cells (Fig. 1D). Significant reductions were also found in the diameter of the seminiferous tubules and in the height of the germinal epithelium in extract-treated mice compared to controls (Table 3). This was also true for the number of germ cells in Stage VII seminiferous tubules in testes of treated mice (Table 3). Fifty-six days after cessation of treatment, however, the alterations caused in the seminiferous tubules recovered to control levels. 3.3.2. Epididymis The epididymis of untreated controls (Fig. 2A–E) and distilled water-treated controls showed normal histological
Fig. 4. Serum level of testosterone in mice (n=6) after C. longa treatment (600 mg/kg body weight) and following treatment withdrawal. Values are mean±S.D. In recovery group, mice were treated with C. longa daily for 84 days, and thereafter, the treatment was discontinued and mice were sacrificed 56 days after treatment withdrawal. ⁎Significantly different from controls (pb.05) by ANOVA followed by Newman–Keuls' multiple range test.
R.K. Mishra, S.K. Singh / Contraception 79 (2009) 479–487 Table 4 Concentrations of sialic acid in the epididymis and of fructose in the seminal vesicle in mice after C. longa treatment (600 mg/kg body weight) and following treatment withdrawal Group and treatment
Sialic acid concentration Fructose concentration (μ moles/100 g tissue) (mcg/100 mg tissue)
1, Control (untreated) 2, Control (distilled water) 3, C. longa 56 days 4, C. longa 84 days 5a, C. longa recovery 6a, Control (distilled water) recovery
140.37±27.60 117.90±16.54 80.15⁎±21.61 30.33⁎±17.09 111.08±12.30 155.87±13.27
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drawal of treatment, epididymis presented a histology which was similar to that of controls. 3.3.3. Seminal vesicle The seminal vesicle of controls showed normal histological features (Fig. 3A). In contrast, in extract-treated mice, the gland showed marked histological alterations. The lumen of the gland contained scanty secretion, and the epithelium showed excessive inward ramification; the muscle layer surrounding the glandular epithelium was also considerably increased (Fig. 3B; cf. Fig. 3A). By 56 days of treatment withdrawal, however, the gland recovered and presented a normal histology.
221.74±22.64 218.53±23.72 167.39⁎±25.54 126.89⁎±50.93 199.15±22.60 212.36±29.76
Values are mean±S.D. for six animals. a Treatment was discontinued after 84 days, and animals were sacrificed 56 days after treatment withdrawal. ⁎ Significantly different from controls (pb.05) by ANOVA followed by Newman–Keuls' multiple range test.
3.4. Testosterone assay Significant reduction was noted in serum level of testosterone in extract-treated mice compared to controls. By 56 days of treatment withdrawal, however, the hormone level increased and the values even exceeded control level (Fig. 4).
features. On the other hand, in mice treated with C. longa extract for 56 or 84 days, detectable histological alterations were noticed in the epididymis (Fig. 2F-J). Furthermore, the alterations noticed at 56 or 84 days of treatment durations were nearly similar, and they are therefore described together. In Segment I (Fig. 2F), the size of the tubules appeared decreased with a slight increase in stroma, and the nuclei of the epithelial cells were sometimes irregularly arranged in the epithelium of the duct. In Segment II (Fig. 2G), the epithelium showed loss of stainability and a vacuolated appearance of the cells; the stroma also appeared slightly increased, and the lumen was either empty or contained only PAS-positive materials or PASpositive materials and sperm. In Segments III (Fig. 2H), IV (Fig. 2I) and V (Fig. 2J) of the epididymal duct, the epithelium presented a normal appearance, except that in Segment III, sometimes, vacuole-like spaces were noticed; the stroma also showed a little increase, and the lumen contained PAS-positive materials, a little less sperm and occasionally exfoliated germ cells. Fifty-six days after with-
3.5. Analyses of sialic acid and fructose contents Significant reductions were noted in the levels of sialic acid in the epididymis and that of fructose in the seminal vesicle, respectively, in extract-treated mice compared to controls; by 56 days of treatment withdrawal, however, the values recovered to control levels (Table 4). 3.6. Fertility test When C. longa-treated males were caged with females at 24 h and 2, 4, 6, and 8 weeks after withdrawal of treatment, mating was not affected at any interval. Fertility of the extract-treated males was affected at 24 h post withdrawal; thereafter, no effect on fertility of the treated males was found from 2 up to 8 weeks of treatment withdrawal, though only four of six mice had shown return of fertility at 2 weeks post withdrawal (Table 5). The litter size in females impregnated by extract-treated males was significantly
Table 5 Fertility of male mice after treatment with C. longa (600 mg/kg body weight/day for 84 days) or distilled water Time after treatment
Treatnent
24 h
Control C. longa Control C. longa Control C. longa Control C. longa Control C. longa
2 weeks 4 weeks 6 weeks 8 weeks
Number of males
Number of females
Litter size
Tested
Mated
Fertile
Tested
Mated
Fertile
6 6 6 6 6 6 6 6 6 6
6 6 6 6 6 6 6 6 6 6
6 2a 6 4 6 6 6 6 6 6
12 12 12 12 12 12 12 12 12 12
12 7 12 9 10 9 10 10 11 10
10 2a 12 4a 10 6 9 8 10 10
Values are mean±S.D. a Significantly different from controls by chi-square test. ⁎ Significantly (pb.001) different from controls by the Student's t test.
7.66±1.21 2.00⁎±1.41 7.08±0.73 4.75⁎±2.98 7.16±0.81 4.66⁎±1.75 7.16±0.81 6.00±1.22 7.00±0.89 7.41±0.73
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decreased compared to controls at 24 h and up to 4 weeks of treatment withdrawal; at 6 and 8 weeks after cessation of treatment, however, no differences could be detected in litter size in females impregnated by treated males compared to controls (Table 5).
4. Discussion The results of the present study indicate that the C. longa treatment did not cause alterations in body weight of the treated animals, suggesting that the treatment had no systemic toxic effect in P mice. Further, the present results also show that the treatment caused degenerative changes in the seminiferous tubules in mice testes with a marked reduction in the weight of the organ. The changes, however, were not uniform as both affected and normal seminiferous tubules were observed in the same section of the testis; the affected tubules showed loosening of germinal epithelium, intraepithelial vacuolation and mixing of spermatids of different stages of spermatogenesis. Similar histological changes have also been described in testes of P mice after treatment with aqueous leaf extracts of Azadirachta indica [5] and Allamanda cathartica [6], and hexane extract of flower buds of Syzygium aromaticum [19] and several other antispermatogenic agents such as gossypol tetra-acetic acid [3,4], nitrofurazone [20] and SC 12937 [11]. The observation that there were marked reductions in the number of spermatogonia A, preleptotene spermatocytes, pachytene spermatocytes and Step 7 spermatids in Stage VII seminiferous tubules in testes of extract-treated mice as reported here suggests that the C. longa treatment interferes with both mitotic and meiotic processes resulting in the suppression of spermatogenesis. Furthermore, the treatment also caused significant reductions in the diameter of the seminiferous tubules and in the height of the germinal epithelium in testes of extract-treated mice compared to controls; these observations are suggestive of the adverse effect of the treatment on spermatogenesis. In P mice in the present study, C. longa treatment caused marked reduction in the weight of the epididymis accompanied by detectable histological changes in the organ. The changes in the luminal content of the epididymis in treated mice appeared to be the outcome of the changes in the testis. The observation that there was a significant reduction in the level of sialic acid in the epididymis of the treated mice compared to controls suggests that the C. longa treatment interferes with the secretory function of the organ [21]. Furthermore, the treatment also had adverse effects on motility, morphology, viability, and on the number of spermatozoa in the cauda epididymidis. Purohit and Bhagat [22] have also reported reductions in density and motility of spermatozoa in cauda epididymidis of rats following treatment with aqueous and alcoholic extracts of rhizome of C. longa. The reduction in the number of spermatozoa in cauda epididymidis in treated mice in the present study
appeared to be due to the suppressive effect of C. longa treatment on spermatogenesis as the sperm number recovered to control levels after restoration of spermatogenic activity following cessation of treatment, while the alterations in sperm motility, viability and morphology might have resulted from disturbances in epididymal function [23]. Results of sialic acid level in the epididymis of the treated mice in the present study also lend support to the above view. The treatment also caused significant reduction in the weight of the seminal vesicle in treated mice compared to controls. Furthermore, the treatment also caused a marked reduction in the level of fructose in the seminal vesicle in treated mice, suggesting that the C. longa treatment had an adverse effect on the secretory function of the gland. The histological observations of the gland in treated mice also support this contention. In P mice in the present study, mating was not affected by the treatment. Marked reductions were, however, found in litter size in females impregnated by C. longa-treated males in the present study at 24 h and 2 and 4 weeks after cessation of treatment, and this may be attributed to the poor sperm quality recorded in treated males. The present results show that the C. longa treatment causes suppression of spermatogenesis in mice testis, though the mechanism of this suppression remains poorly understood. In immature male rat, C. comosa, another species of Curcuma, has been suggested to act directly on the testis or indirectly to inhibit gonadotropin secretion, which consequently reduces testosterone production, or to act at both levels [24]. In P mice in the present study, C. longa treatment caused significant reduction in serum level of testosterone in treated mice compared to controls. It is well known that testosterone plays a major role in the maintenance of spermatogenesis [25]. Thus, it is probable that the C. longainduced suppression of spermatogenesis in mice testes in the present study is caused due to deficiency of testosterone. In conclusion, our results in P mice suggest that the C. longa treatment causes marked alterations in the male reproductive organs and that the alterations are reversible after cessation of treatment. Thus, C. longa may have a potential in the regulation of fertility in the male. Acknowledgment This work was supported by funds from DST, and the University Grants Commission, New Delhi, through CAS in Zoology, Banaras Hindu University. References [1] Chaudhury RR. Plant contraceptives: translating folklore into scientific application. In: Jelliffe DB, Jelliffe EFP, editors. Advances in Maternal and Child Health. Oxford: Oxford University Press; 1985. p. 58–74. [2] Qureshi AA, Sanghai DB, Padgilwar SS. Herbal options for contraception: a review. Pharmacog Mag 2005;2:204–14. [3] Singh SK, Rath SK. Histologic changes in the mouse testis after treatment with gossypol tetra-acetic acid. Arch Histol Cytol 1990;53: 393–6.
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