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Effects
of tripchlorolide on the epididymides and testes of rats
Zuo-Peng
WANG, Zhi-Ping GU, Lin CAO, Yang XU Gen-Di YOU, Bai-Yong MAO, Shao-Zhen
QIAN
Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, Shanghai
200031, China
Asian J Androl 1999 Sep; 1: 121-125
Keywords:
tripchlorolide;
epididymis; testis; carnitine; glucosidases;
lactate
dehydrogenase; hyaluronidase
Abstract
Aim: To further evaluate the antifertility effects of tripchlorolide, a derivative of triptolide produced at the extraction procedure of Tripterygium wilfordii Hook. f., in male rats and to investigate its sites and possible mechanisms of action. Methods: In male rats, tripchlorolide was given by oral garage at a dose of 50 gkg-1d-1 for 5 weeks, fertility was assessed by mating tests, and biochemical indices and light microscopic observation of the epididymides and testes were also performed. Results: Administration of tripchlorolide at 50 gkg-1d-1 for 3 weeks did not influence the fertility of male rats, but 5-week treatment rendered the rats infertile. The density and motility of spermatozoa collected from cauda epididymides were reduced significantly. The epididymal weights, as well as the L-carnitine concentration and -glucosidase content in the epididymal fluid were decreasd. There were no significant differences in -glucosidase and acid phosphatase (ACP) in caput epididymal homogenates between the control and the experimental rats. Obvious morphological changes were observed in the epididymal spermatozoa, mainly including head and tail separation or acrosome curving. Sloughed spermatids were found in the seminifeous and epididymal tubules. In testicular homogenates, tripchlorolide had no influence on the lactate dehydrogenase-C4 (LDH-C4) and hyaluronidase activities. No apparent lesions were observed in the seminiferous and epididymal epithelium. Conclusion: At the dose level employed, tripchlorolide has a significant effect on the fertility in male rats and the primary sites of action may be spermatids and testicular and epididymal spermatozoa.1 Introduction
Tripterygium
wilfordii Hook. F. is a traditional Chinese medicine plant. A crude
extract from the root bark of the plant, the multiglycosides of Tripterygium
wilfordii (GTW), have been applied clinically in the treatment of autoimmune
diseases. In 1986, it was found to possess a reversible male antifertility
effect in rats[1]. Hereafter, an isolation and screening programme
was launched to search for effective antifertility compounds. Tripchlorolide
(Figure 1) is
a derivative of triptolide, one of the active ingredients thus obtained.
The antifertility potency of tripchlorolide is 200 times stronger than
that of GTW[2]. At its antifertility dose level, there was
neither genetic influence nor
immunosuppressive effect on the experimental animals, and the serum
testosterone and sex behavior were unaffected[3,4].
The aim of this study was to further evaluate its antifertility
effect and to investigate its sites and possible mechanisms of action.
2 Materials and methods
2.1
Drugs
Tripchlorolide was kindly provided by Prof. Guo-Wei QIN of this Institute; DTNB(5,5-dithiobis-2-nitrobenzoic acid), AcCOA (acetyl-coenzyme A), PNPG (p-nitrophenyl--D-glucoside), L-carnitine and CAT (carnitine acetyirase) were purchased from the Sigma Chemical Co. PNP (p-Nitrophenyl) was purchased from the Merck Co.
2.2
Fertility estimation
Sixteen
male adult Sprague-Dawley rats (grade II, supplied by Shanghai experimental
animal center, Chinese Academy of Sciences) were randomly divided into
experimental and control
groups, respectively. Tripchlorolide, freshly suspended in 1% CMC, was
given by gastric gavage at a dose of 50 gkg-1d-1,
6 d a week for 5 weeks. The controls received equal volume of vehicle.
All rats were allowed free access to food and water. Body weight was assessed
weekly and the dose was adjusted accordingly. Starting from d 21 and d
35 of treatment, each rat was caged with 2 female SD rats for 4 d. Vaginal
smears were then examined every morning until
the presence of spermaroza, which was considered the sign of successful
mating and the date, d 1 of gestation. The female rats were killed on
d 13 and the pregnancy
rate, and the numbers of corpora lutea and viable fetuses were recorded.
After successful mating, the male rats were sacrificed, and the motility
and density of sperm recovered from caudal epididymides were assessed.
Reproductive organs were dissected and weighed.
2.3
Epididymal biochemistry
One
cauda epididymide in each male rat was cut into pieces in a 10-mL beaker
containing 2 mL 0.9% saline. After standing for 15 min, the sperm suspension
was filtered through a 200-mesh stainless steel screen. The residue was
then washed twice with
2 mL 0.9 % saline, and the suspensions were collected and centrifuged.
The supernatants of one group were combined and used for biochemical analysis.
The caput epididymidis was homogenized in citric acid-phosphoric acid
buffer (pH 6.2, 0.4 % Triton X-100 solution) and centrifuged at 20000
rpm for 30 min. The supernatant was used for biochemical analysis.
2.3.1
Total protein
Protein
concentrations were determined according to the method of Lowry et
al[5].
2.3.2
-Glycosidase
The -glycosidase activity was measured by calorimetric method[6]. The reaction system contained 69 mmol/L phosphoric acid buffer solution (pH 6.8) 1.2 mL, 23 mmol/L PNPG 0.2 mL and supernatant 0.2 mL (the same volume buffer as the blank control). The reaction medium was incubated at 37 for 4 h and 0.1 mol/L Na2CO3 was added to stop the reaction. The absorbance was measured at 400 nm with a spectrophotometer and the PNP content was estimated in reference to the PNP standard curve. The -glycosidase activity was expressed as mg PNP/mg protein.
2.3.3
L-carnitine
L-carnitine
was determined by the spectrophotometric method[7]. A 0.1 mL
supernatant was added into reaction medium containing 0.1
mol/L Tris/HCl, AcCOA 0.05 mol/L, DTNB 0.1 mol and CAT 0.05 mL (19
U/mL). The mixture was incubated at 37 for 30 min, then 2
mL Tris buffer was added, and its absorbance was measured at 412 nm with
a spectrophotometer. The L-carnitine content in caudal epididymal fluid
was expressed as mol/g protein.
2.3.4
Acid phosphatase (ACP)
The
ACP activity was assayed by calorimetric method[8]. A medium
containing 0.1 mL supernatant, 4-PNP 0.05 mL and 0.05 mol/L citrate buffer
0.5 mL was incubated at 37 for 30 min. NaOH 0.2 mol/L 2.5 mL was added
to stop the reaction, spectrophotometric determination was recorded at
405 nm. The PNP standard curve was obtained by the same method. The activity of
ACP was expressed as mg PNP/mg protein.
2.4
Testis biochemistry
2.4.1 Lactate dehydrogenase-C4 (LDH-C4)
Testis
tissue 0.4-0.6 g in each male rat was dissected and homogenized in 0.25
mol/L sucrose solution, and then centrifuged at 10000g for 30 min (4).
The supernatant was used for measurement of lactate dehydrogenase-C4 (LDH-C4)
by an ultraviolet method[9]. Briefly, the final concentrations
of the components of the reaction media were 0.1 mol/L sodium phosphate
buffer (pH 7.3) 0.9 mL, 0.2 mmol/L NADH 2.5 mL, 25 mmol/L -ketoglutaric
acid 0.5 mL, and testis supernatant 0.1 mL. The reaction was proceeded
at 37, and the OD value was read at 340 nm every 30 s for 2.5 min since
-ketoglutaric acid was added into the mixture.
2.4.2
Hyaluronidase
Approximately
0.2 g testes from each male rat was homogenized in 0.1 mol/L citric acid-phosphoric
acid buffer (pH 4.5, 0.1% Triton X-100 was added just before used),
and then centrifuged at 105000g for 60 min. The supernatant was used
for the measurement of hyaluronidase[10]. The reaction system
contained buffer 200 L, 6 mg/mL hyaluronidase 50 L and supernatant
50 L. The reaction mixture was incubated at 37 for 1 h, and then 0.8
mol/L sodium bicarbonate solution (pH 8.9, 90) 50 L was added and
heated in a boiling water for 5 min. After cooling, 1.8 mL of 1% p-dimethylaminobenzaldehyde
in acid was added, and then incubated at 37 for 20 min, cooled and the
absorbence was measured at 585 nm. Standard N-acetylglucosamine was used
to convert absorbence to the absolute content.
2.5
Histology and histochemistry observation
Portions
of testes and epididymides from each male rat were fixed in neutral methanol,
embedded with paraffin wax, sectioned 5 m, and stained with haematoxylon
and eosin.
2.6
Statistical analysis
3 Results
3.1
General health, body and reproductive organ weights
During
the 5 weeks of tripchlorolide treatment, the rats kept healthy, growing
at a normal rate. Their body weight gain was similar to that of the controls
(Table 1).
Table
1. Body weight (g) changes after 5 weeks of tripchlorolide (50 gkg-1d-1).
n=8. means.
|
Time |
Control |
Treated |
| 0 |
265.023.7 |
252.510.4 |
| 3
wk |
330.027.5 |
340.020.0 |
| 5
wk |
358.031.2 |
382.532.8 |
A
slight but statistically significant decrease in epididymal weight was
seen in the experimental group compared with the control group. The weights
of other reproductive organs remained unchanged (Table 2).
Table
2. Reproductive organ weights (g/kg body weight) after 5 weeks of tripchlorolide
(50 gkg-1d-1). n=8. means. bP<0.05
vs control.
|
Organ |
Control |
Treated |
| Pituitary |
28.254.65 |
25.791.53 |
| Testes |
8.121.16 |
7.510.78 |
| Epididymides |
2.810.18 |
2.510.18b |
| Seminal
vesicles |
4.120.89 |
3.880.50 |
| Prostate |
1.360.18 |
1.440.20 |
3.2
Fertility
Tripchlorolide,
at a dose of 50 gkg-1d-1, 6 d a week for 3
weeks did not affect the fertility of male rats, the pregnancy rate and
numbers of fetuses and corpora lutea per pregnant rat being similar to
those of the controls. However, after 5 weeks of administration, all the
rats became infertile, the pregnancy rate of the mated females dropped
to 0 and no viable fetuses were found. (Table 3)
Table
3. Effects of tripchlorolide (50 gkg-1d-1)
on fertility of male rats after oral administration for 3 and 5 weeks.
means.
|
Group |
Course(wk) |
Female
rats cohabited with the males |
||
|
Pregnancy
rate |
No.
Viable fetuses |
No.
Corpora lutea |
||
|
Treated |
3 |
8/8 |
12.62.1 |
13.72.4 |
|
Control |
3 |
8/8 |
12.92.01 |
2.82.4 |
|
Treated |
5 |
0/8 |
0 |
13.01.3 |
|
Control |
5 |
8/8 |
11.92.0 |
13.11.4 |
3.3
Sperm density and motility
At
the 5th week, the sperm counts and motilities were reduced significantly
compared with the control (Table 4).
Table
4. Epididymal sperm density and motility after 5 weeks of tripchlorolide
(50 gkg-1d-1). cP<0.01
vs control group.
|
Group |
Daily
dose (g/kg) |
No.
of rats |
Density(106/mL) |
Motility |
| Treated |
50 |
6 |
11.57.7c |
+ |
| Control |
- |
8 |
32.53.5 |
+++ |
3.4
Biochemical measurement
In
the experimental group, the carnitine levels and the -glycosidase activity
in supernatants of cauda epididymides were significantly lower than those
of the control, while the -glycosidase activity in homogenates of caput
epididymidis did not show significant difference between the two groups.
Tripchlorolide did not change ACP activity in caput epididymal homogenates
(Table 5), nor the LDH-C4 and hyaluronidase activity in the
testicular homogenate (Table 6).
Table
5. Changes in epididymal biochemistry after 5 weeks of tripchlorolide
(50 gkg-1d-1). means. bP<0.05,
cP<0.01 vs control.
|
|
Index |
Treated
(n=7) |
Control
(n=8) |
| Cauda |
L-carnitine(mmol/mg
pro) |
6.782.79c |
14.093.35 |
| -Glucosidase(mg
PNP/mg pro) |
25.705.58 |
30.563.36 |
|
| Caput |
-Glucosidase(mg
PNP/mg pro) |
44.7611.01 |
42.3510.29 |
| ACP(mg
PNP/mg pro) |
12.183.47 |
12.632.64 |
Table
6. Changes in tecticular LDH-C4 and hyaluronidase after 5 weeks
of tripchlorolide
(50 gkg-1d-1). n=8. means.
|
Group |
Hyaluronidase(mg/mg
pro) |
LDH-C4(mg
PNP/mg pro) |
| Treated |
120.0415.22 |
47.029.50 |
| Control |
127.3210.98 |
46.054.27 |
3.5
Histology
In
the experimental rats, histological observations under light microscope
did not show apparent changes, but in most epididymal tubules spermatozoa
were wrapped together and their morphology significantly altered: the
majority were fractured and deformed with head and tail separation and/or
acrosome curving. Sloughed spermatids were found in the epididymal tubules
(Figure 2). In the seminiferous epithelium
only insignificant lesions were seen, and the germ cells were arranged
practically in normal order. However, detached spermatids were seen in
some seminferous tubules and in a number of stage IX and X seminferous
tubules, detained spermatozoa could be found (Figure
3).
Figure
2. Epididymal tubules in tripchlorolide-treated rats, showing anamorphic
sperm and sloughed spermatids (H.E.400).
Figure 3. Seminferous tubules
in tripchlorolide-treated rats, showing detained
sperms (H.E.400)
4 Discussion
A number of diterpene epoxides have been isolated from Tripterygium wilfordii Hook.f. and reported to cause infertility in male rats; it was also indicated that they possessed immunosuppressive effect, but the effect was only apparent at dosage levels 5-12 times their antifertility doses[4]. Tripchlorolide is not a naturally occurring compound, but a derivative of triptolide obtained during the process of extraction[11]. It also had a male antifertity effect of similar potency as its mother compound, triptolide[12]. Although it was reportedly shown that triptolide might be a potential candidate for a male contraceptive agent[13], preliminary evaluaton showed that the toxicity of triptochloride was much less than that of triptolide[12]. Thus, we first selected trichlorolide for further evaluation. Previous studies have demonstrated that it exerted an antifertility activity at 50-100 gkg-1d-1 for 6-7 weeks in rats, but the exact sites and mechanism of action remain unclarified[2,14].
The present study confirmed that tripchlorolide at a dose of 50 gkg-1d-1 caused infertility at the 5th week of administration, but not at the 3rd. Theoretically, in rats if the spermatocyte was the primary target cell of an antifertility agent, infertility should occur at the 6th or 7th week of administration; if it is spermatid, at the 3rd to 5th week, and if epididymis, at the 2nd week. It seems that the primary target cell for triptochlorolide is spermatid.
In
the experimental rats, significant changes were also found in the epididymis:
(1) the epididimal carnitine content, an important marker of epididymal
function related to fertility[15], was
reduced, (2) the caudal glucosidase level, another marker related
to spermatozoa storage[16], was diminished, and (3) a reduction
in epididymal weight. Thus,
the authors believe that the primary target of action of tripchlororide
is the spermatids with a resultant decrease in the sperm density and motillity
in the cauda epididymidis; Besides, disturbance in epididymal function
may also contribute to the antifertility mechanism of tripchlororide.
LDH-C4
was a marker of the cytoplasmic compartment taking part in the glucolysis
of spermatids and spermatocytes. LDH-C4 was the target site
for another antispermatogenic agent, gossypol[18]. It is interesting
to note that tripchlorolide, though
also acting on spermatid, had no significant effect on this enzyme. Testicular
hyaluronidase activity was shown to decrease after 7 weeks of tripchlorolide
administration at 100 gkg-1d-1[19]. However,
at the present dose regime, the enzyme level did not show apparent changes,
indicating that it may not play a significant role in tripchlorolide-induced
infertility.
References
[1]
Qian SZ, Zhong CQ, Xu Y. Effect of Tripterygium wilfordii on
the fertility in rats. Contraception 1986; 33: 105-10.
[2] Ye WS, Deng YC, Huang YL, Xue SP. Antispermatogenic effect of Tripterygium
wifordii and tripchlorolide (T4) on rat gametogenesis and spermatozoa.
Chin Med Sci J 1994; 9: 110-3.
[3] Ye WS, Deng YC, Huang YQ, Xue SP. Genetic effects of tripchlorolide
on germ cells in rats. Chin J Androl 1991; 5: 130-2.
[4] Qian SZ, Ye X, Wei ZJ. Recent progress in research on Tripterygium:
a male antifertility plant. Contraception 1995; 51: 121-9.
[5] Lowry OH, Rosebrough
NJ, Farr AL, Rendall RJ. Protein measurement with the Folin phenol reagent.
J Biol Chem 1951; 193: 265-75.
[6] Wang CN, Xie WY, Hang PZ. Study on the relationship between
neutral alpha-glucosidase in seminal plasma and sperm motility. Chin J
Androl 1993; 7: 217-9.
[7] Marquis NR, Fritz IB. Enzymological determination of
free carnitine concentrations in rat tissues. J Lipid Res 1964; 5:
184-7.
[8] Hang PZ, Li YH. Male infertility. Beijing: Science-Technology Document
Publishing House; 1990. p 337-8.
[9] Lillian S, Harold LS. Regulation of -ketoglutarate by
homogeneous lactic dehydrogenase X of testicular tissue. J Biol Chem 1969;
244: 4393-7.
[10] Males JL, Turkington RW. Hormonal regulation of hyaluronidase during
spermatogenesis in the rat. J Biol Chem 1970; 245: 6329-34.
[11] Matlin SA, Belenguer A, Stacey VE, Qian SZ, Xu Y, Zhang JW, et
al. M
[12] Yu DQ, Zhang DM, Wang HB, Liang XT. Structure modification of triptolide,
a diterpenoid from Tripterygium wifordii. Acta Pharm Sin 1992; 27: 830-6.
[13] Lue Y, Hikim APS, Wang C, Leung A, Baravarian S, Reutrakul V,
et al. Triptolide: a potential male contraceptive. J Androl 1998;
19: 479-86.
[14] Qian SZ, Xu Y, Wang Y, Zhang ZT, Lin N, Wang SM, et al. Screening
and pharmacodynamic study of male antifertility compounds isolated from
Tripterygium Wilfordii. Chin J Androl 1989; 3: 207-10.
[15] Cooper TG, Yeung CH, Nashan D, Nieschlag I. Epididymal markers in
human infertility. J Androl 1988; 9: 91-101.
[16] Yeung CH, Cooper JG. Study of the role of epididymal alpha-glucosidase
in the fertility of male rats by the administration of the enzyme inhibitor
castanospermine. J Reprod Fert 1994; 102: 401-10.
[17] Lu XQ, Chen XH, Liu P, Cheng H, Shen XM, Wang BJ, et al.
Comparative studies on antifertility mechanism and toxicology of Tripterygium
wifordii monomer T4 and Gossypol. Acta Acad Med Sin 1990; 12: 441-4.
[18] Soufir JC, Radigue C, Dantec MC, Garnier D, Jegou B. Gossypol-induced modifications
in the microenvironment of rat epididymal spermatozoa. J Reprod Fert 1989;
86: 427-34.
[19] Zuo X, Wang N. Study on antifertility mechanisms of tripchlorolide.
Ac
Correspondence
to Prof. Zhi-Ping GU.
Tel +86-21-6431 1833, ext 310 Fax +86-21-6437 0269
E-mail zpgu@server.shcnc.ac.cn
Received
1999-04-29 Accepted 1999-08-26
