Antifertility effects of crude ethanol extracts of Tripterygium hypoglaucum (Levl.) Hutch in male Mongolian gerbils (Meriones unguiculatus)

Abstract

To evaluate the antifertility effects of crude ethanol extracts of Tripterygium hypoglaucum (Levl.) Hutch in Mongolian gerbils. Crude ethanol extracts of T. hypoglaucum root (ETH_R) was administrated orally at a dose of 160 mg/kg for 30 days. Sperm density, sperm motility levels and the percentage of misshapen sperm in the cauda epididymides were assessed. The fertility rate of mated female gerbils was calculated. The results indicate that ETH_R has an effect in Mongolian gerbils. Oral administration of ETH_R caused an increase of the percentage of misshapen sperm significantly. Testis and epididymis weights, the sperm density and motility were significantly reduced. Compared to the control group, there was a distinct reduction in the number of litters produced, and the average number of pups per male decreased from 3.4 to 0.87. Reversibility tests showed that testis and epididymis weights, sperm indices, and fertility started to gradually recover after 30-days cessation of medication. The ETH_R brought a significant decrease in birth rate of Mongolian gerbils.

Keywords:

• Tripterygium hypoglaucum
• ethanol extracts
• male contraceptives
• Mongolian gerbils (Meriones unguiculatus)

Introduction

There has been considerable interest in the use of contraceptive methods for rodent control (Wang and Tai 2003; Zhang et al. 2004; Huo et al. 2006; Zhang et al. 2009). In the course of our studies on development of the sterilants, we focused on traditional Chinese medicinal plants. Tripterygium hypoglaucum (Levl.) Hutch is perennial climbing vines, which are commonly found to the south of Yangtze River Valley and in southern China (Sun and Liu 2008). It is a traditional Chinese herb used for the treatment of rheumatoid arthritis, chronic nephritis, and other immunological disorders (Yang et al. 2005; Zeng and Chang 2005). However, when using these herbs, some patients experience oligospermia, azoospermia, a decrease in testicular size, and other signs of infertility during the period of treatment (Yu 1983; Wang et al. 1992). The active ingredients extracted from T. wilfordii or T. hypoglaucum are referred to as multi-glycosides of T. wilfordii (GTW), and these have a marked effect on fertility regulation in rats and mice (Zhang 2002; Li and He 2006). Most of the studies on plant extract that have been conducted to investigate the antifertility effects of T. wilfordii or T. hypoglaucum in human males have used rats or mice experimental models (Ma et al. 2000; Li and He 2006; Chen et al. 2010). But no literatures contain a report of the antifertility effect in the field rodent. In the present study, Tripterygium hypoglaucum (Levl.) Hutch was focused for its effectiveness as a botanic sterilant for rodent control. We examined the antifertility activity of crude ethanol extracts of T. hypoglaucum on Mongolian gerbils (Meriones unguiculatus), which is a pest rodent in the farming-pastoral zone of northwest China (Luo et al. 2000).

Materials and methods

Plant materials and preparation of extracts

The T. hypoglaucum plants were collected in the Jiuhua Mountain area of Anhui Province, China (30°27′13.09′′N, 117°47′ 57.17?E) in June 2008 and were identi?ed by Dr. Shao Jianwen (College of Life Sciences, Anhui Normal University). The plants were divided into roots, stems, and leaves, oven-dried at 80°C, then pulverised to a powder of 0.85-mm sieve. The powder of the dried parts (2000 g each) was blended three times with 95% ethanol for 20 h (volume to weight ratio, 1:10; g millilitre⁻¹). The extract was then concentrated to 1000 ml at a temperature of less than 50°C under vacuum, and the residual solution was extracted with an equal volume chloroform (4×250 ml) in order to obtain crude extract. The extracts were pooled and decolourised using saturated lead acetate solution. The lead acetate was subsequently evaporated in vacuo to give residues that were crude ethanol extracts of T. hypoglaucum roots, stems, and leaves (named ETH_R, ETH_S, and ETH_L respectively). The high-performance liquid chromatographic (HPLC) was performed on each extract in order to determine the triptolide content, Triptolide (purity, 99.31%) obtained from the National Institute for the Control of Pharmaceutical and Biological Products, China was used as a reference material. Crude extracts with a high content of triptolide were selected in order to test their sterilant properties.

Test material administration and statistical analysis

Mongolian gerbils were trapped in the fields of Kangbao County, Hebei Province (41°51′22.04′′N, 114°36′49.07′′E). In all experiments, animals were maintained in conventional animal houses consisting of 25×10×25 cm vitreous cages, under conditions of 14:10 light: dark illumination, a temperature of 25±2°C, and continuous once-through air exchange. Mature male gerbils were randomly divided into two groups (average body weight, 66.46±0.98 g). One group (n = 45, consisting of three subgroup [groups I, II, and III], each of 15 animals) received ETH_S daily for 30 days uninterruptedly. The second group is the control consisting of 15 untreated animals. There was no significant difference in the body weights of treatment and control animals (F[3,56] = 1.748, p=0.168).

The ETHs were suspended in carboxymethyl cellulose. The doses we used were based on that used by Wang Shimin and colleagues (300 mg/kg in male rats; 1992), the LD₅₀ values reported by Lipsky and Tao (160 mg/kg in mice; 1996), and our results from a previous study. The treatment group (n = 45) received a dose of 160 mg/kg daily for 30 days, whereas the control group (n = 15) received 1% carboxymethyl cellulose suspension for 30 days. One-third of the treated animals (Group I) and the control group animals were allowed to mate with mature fertile female gerbils for five days. These animals were then sacrificed and autopsied. During the 60-days recovery phase, a further 30 treated animals (groups II and III) were mated with untreated females at 30-day intervals after the cessation of treatment. Testes and epididymis weights as percentages of total body weight were then determined.

The spermatozoa density of the cauda epididymides was determined using a white blood cell (WBC) counting Neubauer chamber of a hemocytometre and were expressed as million sperm per mL suspension. Epididymal sperm counts and motility were determined as described previously (Lue et al. 1998; Umesiobi 2010). Spermatozoa motility was determined according to the method described in the WHO laboratory manual (WHO 2001). The movement of every encountered sperm was graded as a, b, c, or d. The sperm were stained with eosin-nigrosin stain in order to determine the ratio of dead sperm to living sperm in a total of 200 sperm. The number of misshapen sperm in a total 200 was determined by staining with Wright-Giemsa.

The fertility rate of mated female gerbils was calculated according to the WHO Protocol MB-50 (1983). All animals mated successfully five days prior to sacrifice (male:female ratio, 1:2). Mated females were allowed to complete gestation. The number of litters was determined after the completion of one gestation period (30 d) in all experimental groups. The number of pups was recorded, and litter size and percentage fertility were calculated.

Data are expressed as means±SEM and were analysed for statistical significance by using one-way ANOVA and an LSD test. The fertility rate data were analysed by a crosstab test using the SigmaStat program (SPSS, Inc., Chicago, IL). The data were considered as significant and highly significant at p<0.05 and p<0.001, respectively. During the study, one animal from group II and two animals from group III died on the 63rd day and 92nd day (they were bitten to death when they were paired with females); these animals were accordingly excluded from the analysis.

Results

Details of the extracts derived from the different plant parts are shown in Table 1. The results showed that the ethanol extract of stems contained more triptolide per gram of extract (44 mg/g) than the remaining two extracts. The ETH_R could be responsible for the antifertility effect test.

Table 1. The percentage of extracts to the plants and the contents of triptolide in different extracts.

Content	Root	Stems	Leaves
Extracts (%)	0.5	0.12	0.31
Triptolide/extract (mg/g)	38	44	12
Note: The quantitative analysis of triptolide was conducted by HPLC.

The 160 mg/kg ETH_R daily for 30 days caused significant reduction (p<0.01) in testis and epididymis weights (Figure 1) compared with those of the control and recovery groups (p=0.00). During the recovery period, the ratio of testis to body weight differed in groups II (n = 14) and III (n = 13) but did not differ significantly from that of the control group (p=0.16 and p=0.84, respectively). In contrast, epididymis weight was significantly lower in all the treated groups (Group I, p=0.00; Group II, p=0.00; Group III, p=0.03) compared with the control. No significant differences in the ratio were found between controls and the extract-treated gerbils 30 days after the cessation of treatment (p=0.87). However, the treated animals made a remarkable recovery at 60 days after the treatment was stopped (p=0.001).

Figure 1.  Testis and epididymis weight as a percentage of total body weight of the different groups of Mongolian gerbils. (A) testis weight as a percentage of total body weight in all groups. (B) epididymis weight as a percentage of total body weight in all groups. Group I (n=15) received extract for 30 days, Groups II (n=14), and III (n=13) are the withdrawal animals for 30 and 60 days, respectively. The same letters indicate no significant difference, whereas different letters indicate a significant difference (p<0.05)

After oral administration uninterrupted for 30 days, the treated group had a significant decrease in sperm count (p=0.00), sperm motility levels (p=0.00) and the percentage living sperm (p=0.00). Level b, c, and d sperm motility did not exhibit a notable difference (level b, p=0.066; level c, p=0.105; level d, p=0.091); and the percentage misshapen sperm increased significantly (p=0.00). Thirty days after the withdrawal of treatment with ETH_S, the sperm parameters (group II) begin have a recovery compared with group I, but the levels are no obvious exception for percentage misshapen sperm (p=0.01). With an increase after a 60-day recovery period (group III), a perfect recovery was observed in sperm motility (level a, p=0.004; level b, p=0.049; level c, p=0.066; level d, p=0.32) and percentage living sperm (p=0.00), there was a significant decrease in the percentage misshapen sperm (p=0.011) in comparison to group II. In this stage, there was no significant difference in sperm characters with the exception of sperm density when compared with the control group (Table 2).

Table 2. The testing of cauda epididymal sperms parameters.

		Sperm motility (%)
Group	Sperm density (million/mm^3)	a	b	c	d	Living sperm (%)	Misshapen sperm (%)
I	242±61^a	1.34±0.29^a	1.16±0.20^a	1.88±0.23^a	89.00±6.38^a	17.00±4.69^a	70.70±5.87^a
II	337±52^a	2.55±0.67^a	2.57±1.66^a	3.52±0.94^ab	84.21±6.83^a	25.54±3.91^a	43.88±7.68^b
III	753±63^b	10.14±2.39^b	7.27±2.77^b	7.00±2.28^bc	75.67±6.01^a	60.82±9.12^b	22.93±5.50^c
Control	1173±56^c	14.93±2.44^b	5.32±0.84^ab	5.32±0.84^ac	75.14±3.86^a	76.23±2.98^b	9.48±0.82^c
Note: Sperm motility were defined in levels: ^arapid progressive motility; ^bslow progressive motility; ^cnon-progressive motility, and ^dshows immotility (WHO 2001). Data in the table represent average values for the LSD test. Values are expressed as the mean±SEM. Values with the same letters are not significantly different, whereas those with different letters are significantly different (p<0.05).

The ETH_S caused a significant decrease in the number of litters produced by females. The litter numbers decreased to eight (Group I; Figure 2A). The treatment caused a significant (p=0.00) reduction (0.87) in the average number of pups per male (Group I; Figure 2B). This indicates that the antifertility action of the extracts is effective. Furthermore, one abnormal foetus was found among the offspring of the gerbils that received continued ETH_S for 30 days. Fertility started to recover 60 days after the cessation of medication, and the percentage of litters produced increased by 9.04% and 37.18% at 30-day and 60-day in the recovery groups II and III, and the average number of pups per male increased to 1.5 and 2.46, respectively. All pups born in these groups were normal and healthy (Figure 2).

Figure 2.  Comparison of the reproduction among treated, recovering, and control groups. (A) The numbers of reproductive females. (B) The average number of pups per male in each group. The same letters indicate no significant difference, whereas different letters indicate a significant difference (p<0.05).

Discussion

Fertility control is an effective measure to manage rodent pests. In identifying the best fertility control methods, many studies have been conducted with a view to selecting appropriate contraceptives for the rodent management [Zhang et al. 1997, 2001, 2004). Oral administration of triptolide at a dosage of 100 µg/kg body weight daily for 70 days completely inhibited fertility in male rats (Lue et al. 1998). Some triptolide was converted into tripchlorolide during the isolation of male antifertility compounds from the plants or by reaction with hydrochloric acid (Zhang et al. 1996). In the present study, the HPLC results indicated that the highest triptolide content was in the ethanol extract of stems (Table 1). Thus, ETH_S was selected for the assessment of antifertility effects in male Mongolian gerbils.

Daily administration of ETH_S for 30 days resulted in a 46.66% reduction in the number of litters produced, and that the average number of pups per male decreased from 3.4 to 0.87 (Figure 2). But the birth rate did not decline to zero in the treatment group, indicating that the extracts were unable to suppress spermatogenesis completely. All sperm and birth rate indices of the 60-day recovery group, recovered to levels similar to the control indexes, almost increased to the level of control group. The results have shown that ETH_S causes reversible infertility in male Mongolian gerbils.

Although we were unable to determine the mechanisms by which the extracts exert their antispermatogenic effects, a decrease in the weights of testes and epididymis as percentages of total body weight were found. After administration of the extracts for 30 days, no obvious changes were noticed on the surface of the treated male gonads. With the increase of recovery time, the epididymis weight to body weight ratio became closer to that of the control group (Figure 1). It may be due to the site of action of the active component of the extract is post-testicular and confined to the sperm in the cauda epididymides as the effect of Foeniculum vulgare (Dehghani et al. 2005). That is in agreement with the decrease in epididymal sperm density in group I (n=15). Furthermore, the administration of the extract, may be like the triptolide, does not interfere with the spermatogenic status of the animals (Sinha Hikim et al. 2000; Liu et al. 2001; Zha et al. 2008) and the antifertility effect is reversible (Xu et al. 2007). Following withdrawal of the compound, it is speculated that testicular tissue structure initially recovers, and that spermatogonia then begins to produce normal spermatids. Thereafter, the number of sperm in the epididymis gradually increases to the normal level.

As the results demonstrate, the crude ethanol extracts of T. hypoglaucum can cause a marked decrease in the birth rate of male Mongolian gerbils; it can be useful for the management of populations of this animal. Further studies on the mechanisms of action and safety of T. hypoglaucum extracts, including gene mutation in animals, are required before these extracts can be considered for use in controlling rodent pests in the field.

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (No.30800727) and the National Basic Research Program of China (No. 2007CB109105). We thank the staff (Kangbao Grassland Work Stations, Hebei Province, China) for providing excellent facilities during our trapping samples. We also thank Dr. Qinghong Liu for providing technical advice on the preparation of extracts.