Yonago Acta medica 1999;42:87–94

Protective Effects of Antioxidants on Testicular Functions of Varicocelized Rats
Nobuo Suzuki and Nikolaos Sofikitis
Department of Urology, Faculty of Medicine, Tottori University, Yonago 683-0826, Japan
Left varicocele detrimentally affects spermatogenesis and the epididymal sperm maturation process bilaterally. We analyzed the effect of antioxidants on sperm production
and quality in rats which were varicocelized. Non-varicocelized rats served as controls(group A). Left varicocele was done in 5 groups (B, C , D , E and F) of rats of the sameage. Antioxidants taurine, catalase or superoxide dismutase were administered intraabdominally twice a week in groups C, D or E, respectively. Group F received taurine,
catalase and superoxide dismutase. Groups A and B received a vehicle dose of saline
equally. Eight weeks after varicocele induction bilateral epididymal caudal sperm content, motility and fertilizing ability were analyzed. All the parameters were significantly
lower in group B than in groups A, C, D, E and F, although the values in group A werehigher than in the other groups. Bilateral testicular weight and testosterone responses
to human chorionic gonadotropin stimulation were significantly higher in groups A, C,
D, E and F than in the group B. There were no significant differences in serum testosterone basal profiles among groups A, B, C, D, E and F. These results indicated theeffectiveness of antioxidants for the preservation of testicular function and the epididymal sperm maturation process in varicocelized subjects.
Key words: antioxidants; reactive oxygen species; sperm quantity and quality; varicocele

uncontroled production of ROS is considered as
an important factor in the tissue damage induced by several pathophysiologies.
Since human sperm plasma membranes
have a high content of polyunsaturated fatty
acids, the membranes are sensitive to damage
by free radicals (Alvarez et al., 1987). As a result of the lipid peroxidation induced by free
radicals, spermatozoa may not be able to undergo acrosomal reaction and may be impaired for
motility (Rao et al., 1989; Aitken and West,1990). Spermatozoa and infiltrating leucocytes
are considered to be the major sources of ROS
in semen samples (Aitken and West, 1990;
Kessopoulou et al., 1992). Spermatozoa andseminal plasma have their own anti-oxidative
mechanisms to protect ROS-induced cellular
damage. Several studies have demonstrated thepresence of enzymatic antioxidants such as

Reactive oxygen species (ROS) are cytotoxic
and cause tissue injury in several pathophysiologies, such as cancer, glomerular nephritis,
atherosclerosis and arthritis. In addition, hyperoxia, ischemia/reperfusion, chemotherapy andradiotherapy result in the production of ROS(Sie, 1991). The generation of ROS is linked toprofessional phagocytes which utilize these
pernicious molecules during the destruction of
invading cells and organisms. However, many
non-phagocytic cell types also have the capacity to produce ROS. Thus, ROS produced byendothelial cells, mesangial cells, fibroblasts,
thyroid cells, Leydig cells and oocytes can playa positive, functional role in the electron transfer reactions that regulate cell activity. Although the controlled generation of these highly
reactive molecules may serve as a second messenger system in many different cell types, theAbbreviations: hCG, human chorionic gonadotropin; IVF, in vitro fertilization; LTV, left testicular vein;
ROS, reactive oxygen species; SOD, superoxide dismutase

87

N. Suzuki and N. Sofikitis

left renal vein ligation was performed at a point
medial to the junction of the adrenal and spermatic veins, so as to reduce the renal vein to an
external diameter of 1 mm. Then the anastomotic branch between the left testicular vein(LTV) and the left common iliac vein was ligated, and all the small anastomotic branches of
the LTV were carefully ligated (Saypol et al.,
1981; Sofikitis and Miyagawa, 1992). Nine rats
were similarly anesthesized and only underwent dissection of the LTV and the left renalvein medial to the sites of the entry of the adrenal and spermatic veins. These rats were consider as the controls (group A) . Groups B, C,
D, E and F included 7, 9, 10, 9 and 8 rats, respectively. Each group received various medical treatments by intraabdominal injections
from the first week after operation for 8 weeks.
All drugs were administered via the intraabdominal route (Beaman et al., 1987), since
this route is less time consuming and lessinvasive than that of intravenous injection. A
vehicle dose of 0.5 mL of saline (0.85% w/v)
was administered to groups A and B twice aweek. Taurine (100 mg), catalase (10 µg) or
SOD (150 IU) (Sigma Chemical Co. St Louis,MO) was diluted in 0.5 mL of saline and given
to groups C, D or E, respectively, twice a week.
A mixture of taurine (100 mg), catalase (10 µg)
and SOD (150 IU) diluted in 0.5mL of saline
was given to group F equally. We adopted theabove doses of antioxidants for testicular dysfunction with experimentally induced varicoceles according to our preliminary studies.

catalase, superoxide dismutase (SOD), andglutathione peroxidase/reductase in humansemen (Alvarez et al., 1987; Jeulin et al., 1989;
Kobayashi et al., 1991). Besides these enzymatic systems, other compounds with antioxidant properties have been found in humansemen. Albumin (Halliwell and Gutteridge,1987), taurine and hypotaurine (Alvarez andStorey, 1983), pyruvate (de Lamirande andGagnon, 1992), urate (Grootveldt and Halliwell,1987), vitamins E and C (Chow, 1991; Thiele etal., 1995) and β-carotene (Burton and Ingold,
1984), play an important role in the protectionof tissues against free radical attack.
Varicoceles are the main cause of primaryand secondary male infertility. The large
incidence of varicoceles in men with secondaryinfertility and the common observation thatvaricoceles generally develop at the time ofpuberty suggest that the presence of varicoceles
can cause a progressive decline in fertility. Although varicocelectomy is considered as ahighly effective treatment of infertility due tovaricoceles a significant number of men remaininfertile after varicocelectomy (Sofikitis andMiyagawa, 1992). Therefore there is an increasing interest to develop conservative modesof therapy of infertility due to varicoceles.
Considering that oxygen radical scavengers
provide significant restoration of testicularfunction after testicular vascular diseases(Henry and Turner, 1996), we studied theeffects of the administration of antioxidants on
testicular spermatogenic and steroidogenic
functions in rats with varicoceles.

Determination of testicular temperature
Materials and Methods

Testicular and intraabdominal temperatures
were measured by percutaneous insertion of a
29-gauge needle probe attached to a digital
thermometer (Unique Medical, PTC 201
model, Tokyo, Japan). The difference between
the intraabdominal and intratesticular temperatures on each side was recorded.

Grouping of rats, induction of varicoceles
and administration of antioxidants
Four-week-old male Wistar rats (Shimizu Experimental Co., Kyoto, Japan) weighing 90 to
100 g were divided into 6 groups (A, B, C, D, E
and F). Groups B, C, D, E and F were anesthetized with intraperitoneal nembutal (25 mg/kg)
and left varicocele was induced as previously
described (Saypol et al., 1981). In brief, partial

Assessment of fertility
Eight weeks after operation (experimental period), each rat from each group was placed in the88

Effects of antioxidants of varicocelized rats

mined using a Makler Counting Chamber (Sefi
Medical Instruments, Haifa, Israel). Subsequently, 10 droplets of the filtrate were counted
to calculate the percentage of motile spermatozoa. The proportion of motile spermatozoa wasdetermined by counting 100 cells in randomly
selected fields. Samples for motility evaluation
were kept at 35˚C.

same cage with three 10-week-old female
Wistar rats for 5 days. Twenty-one to 27 days
after the beginning of the mating experiment,
the females were checked daily for parturition.
The male rats were considered as fertile if its
matings resulted in at least one pregnancy.

Determination of serum testosterone concentration

Sperm processing for in vitro fertilization (IVF)

Serum testosterone concentration was evaluated twice; prior to intraabdominal injection ofhuman chorionic gonadotropin (hCG; 1,500 IU/
animal; Teikoku Zohki Co., Tokyo) and 3 hafter injection. Serum was transferred to theJapanese Special Reference Laboratory and thetestosterone concentration was determined byradioimmunoassay using kits from Nihon DPC
Corporation (Tokyo) as previously described(Coyotupa et al., 1973). The intra- and interassay coefficients of variation were 5% and 9%,
respectively. The sensitivity of the assay was
0.1 ng.

Samples containing 2 × 106 spermatozoa/mL
were prepared from the sperm suspensions as
described. These samples were incubated at
37˚C under 5% CO2 for 12 h. Then, 0.1 mL of
the sperm suspensions was introduced to 0.9
mL of the Toyoda and Chang medium containing the oocytes in a different dish by calibration
with a Pasteur pipette as previously described(Toyoda and Chang, 1974).

Collection of oocytes and insemination
Immature female rats were injected subcutaneously with 30 IU pregnant mare’s serum gonadotropin (Sigma Chemical Co.) 54 h prior to
an intraabdominal injection of 30 IU hCG(Teikoku Zohki Co.). Rats were killed 19 h
after hCG injection. The oviducts were removed and the ampullar portion was put into a plastic dish containing Toyoda and Chang medium.
The eggs in cumulus masses were dissected outof the oviducts and introduced into the medium.
Oocytes were inseminated with 2 × 105 spermatozoa per oocyte. After 24 h of insemination,
the number of oocytes with 2 pronuclei plus a
second polar body was assessed. Five male rats
from each group were processed for the IVF
analysis. Ten oocytes were inseminated byspermatozoa from the left epididymal cauda ofeach male rat. Ten oocytes were inseminatedby spermatozoa from the right epididymal
cauda of each male rat.

Determination of the weight of the testicles and seminal vesicles
The rats were sacrificed. The testicles and seminal vesicles were excised free of surrounding
tissue and weighed on a Mettler Basbal scale(Delta Range, Tokyo).

Epididymal sperm content and quantitative sperm motility
Epididymides were separated carefully fromthe testicles under a 10-time magnification
provided by a stereo zoom microscope (model
TL2, Olympus Co., Tokyo). The epididymis
was divided into 3 segments; head, body andtail. The epididymal tail was trimmed andminced in 5 mL of Toyoda and Chang medium(Toyoda and Chang, 1974). The minced epididymal tissue was separated from the liberated
spermatozoa by filtration through a stainlesssteel wire mesh with a pore size of 60 µm(Tomoda, Matsue, Japan). Five drops of the filtrate were used for assessing the sperm count(number of spermatozoa/mL) which was deter-

Statistical analysis
Statistical analysis was performed on all datausing the paired t-test to compare quantitative89

N. Suzuki and N. Sofikitis

Table 1. Testicular weight and the differences between the intraabdominal and testiculartemperatures
Group

Left testis
Weight (mg)
∆ (˚C)

Right testis
Weight (mg)
∆ (˚C)

A:Control[9]1328 ± 174*
4.3 ± 0.3*1401 ± 157*
4.5 ± 0.3*
B:Saline[7]613 ± 108**2.5 ± 0.4**824 ± 82**2.7 ± 0.4**
C:Taurine[9]801 ± 79***
2.6 ± 0.3**
1061 ± 64***2.8 ± 0.3**
D:Catalase[10]824 ± 89***2.9 ± 0.4**1102 ± 75***
3.0 ± 0.3**
E:SOD
[9]
811 ± 85***
2.8 ± 0.4**
1098 ± 73***
3.0 ± 0.3**
F:Mix
[8]
846 ± 97***2.8 ± 0.3**1106 ± 82***2.9 ± 0.4**[ ], number of animals in each group; ∆, differences between intraabdominal and testicular temperatures;Mix, taurine + catalase + SOD; SOD, superoxide dismutase.
Within the same column, * versus **; ** versus ***; * versus ***: P < 0.05.

However, group A showed significantly higher
values to the varicocelized groups B, C, D, E
and F.

parameters referring to paired organs within agroup and one-way analysis of variance (P <0.05) followed by the Duncan’s multiple range
test (P < 0.05) to analyze intergroup differencesof quantitative parameters. All values were expressed as the mean ± SD. Qualitative parameters were analyzed by chi-square test. A
probability of < 0.05 was considered to be statistically significant.

Testicular weight
Left testicular weights were significantly lower
than right testicular weight in groups B, C, D, E
and F, except group A (Table 1). The left andright testicular weights in group B weresignificantly lower than in all other groups.

Results

Weights of the seminal vesicles
Testicular temperature

The weights of seminal vesicles are summarized in Table 2. The weight was significantly
lower in group B than in all other groups. The
weights in groups C, D, E and F were right inbetween groups A and B.

The differences between intraabdominal andintratesticular temperatures are summarized inTable 1. There were no significant differences
between the left and right testes in all groups.
Table 2. Weights of the seminal vesicles
Group

Epididymal sperm content, motility andfertilization rate

Weight†

The epididymal caudal sperm content, motility
and fertilization rate are summarized in Table 3.
The sperm contents on the left or right side ingroup A were significantly higher than in allother groups. Left and right epididymal sperm
contents were significantly lower in group B
than in all other groups. In group B sperm
content was significantly lower on the left sidethan on the right side. The percentage of motile
sperm on left and right sides in group B was
significantly lower than in all other groups. The

A: Control[9]781 ± 82*
B: Saline[7]426 ± 77**
C: Taurine[9]579 ± 61***
D: Catalase [10]601 ± 58***
E: SOD[9]608 ± 53***
F: Mix[8]640 ± 61***[ ], number of animals in each groups; Mix,taurine + catalase + SOD; SOD, superoxide dismutase.
† Weight (mg) of the left and right seminal vesicles.
Within the column, * versus **; ** versus ***;* versus ***: P < 0.05.

90Effects of antioxidants of varicocelized rats

Table 3. Epididymal sperm content, motility and IVF outcome
Group

Left epididymis
Sperm content Motile Oocytes with(millions ofsperm
2-PN +
sperma2nd PB
tozoa/mL)(%)(FO/IO)

Right epididymis
Sperm content Motile Oocytes with(millions ofsperm
2-PN +
sperma2nd PB
tozoa/mL)(%)(FO/IO)

A: Control[9]74 ± 8*78 ± 8*43/50*
71 ± 6*75 ± 7*44/50*
B: Saline[7]
13 ± 8**19 ± 7**
4/50**
33 ± 8**26 ± 6**
8/50**
C: Taurine[9]40 ± 7***41 ± 6***21/50***
48 ± 6***49 ± 7*** 27/50***
D: Catalase [10]46 ± 5***45 ± 6***25/50***
53 ± 7***55 ± 8*** 27/50***
E: SOD[9]45 ± 6***45 ± 5***27/50***
51 ± 5***56 ± 6*** 28/50***
F: Mix[8]48 ± 6***49 ± 8***29/50***
55 ± 7***58 ± 6*** 29/50***[ ], number of animals in each group.
FO, fertilized oocytes; IO, inseminated oocytes; IVF, in vitro fertilization; Mix, taurine + catalase + SOD; 2PN + 2nd PB, 2 pronuclei plus a second polar body; SOD, superoxide dismutase.
Within the same column, * versus **; ** versus ***; * versus ***: P < 0.05.

percentages of motile spermatozoa on the left
and right sides were significantly higher ingroup A than in all other groups. The proportion of fertilized oocytes to inseminated oocytes
on both the left and right sides was significantly
smaller in group B than in all other groups. The
largest values were observed in group A.

than the other groups. The frequency of fertile
rats was increased in antioxidant treated groups
C, D, E and F, although they were not extended
to group A.

Discussion
The present study confirms previous studies in
our laboratory (Sofikitis and Miyagawa, 1992)
that the induction of left varicocele in Wistar
rats results in a bilateral decrease in testicularversus intraabdominal temperature difference.
The increase in testicular temperature may be
the cause of the impaired Leydig cell secretory
function (Table 4) in this study since it is known

hCG test and mating test
Basal testosterone serum profiles were notsignificantly different among all groups (Table4). However testosterone responses weresignificantly lower in group B. The highest
values were found in group A. The proportionof fertile rats in group B was significantly lower

Table 4. Serum concentrations of testosterone, testosterone responses to hCG and theresults of the mating testGrouphCG (ng/mL)
Basal testosterone
Testosterone response

Fertile rats/tested rats

A: Control[9]120 ± 45*857 ± 51*
9/9****
B: Saline[7]
119 ± 32*501 ± 61**
1/7*****
C: Taurine[9]116 ± 31*692 ± 85***
3/9
D: Catalase[10]
111 ± 39*701 ± 97***
4/10
E: SOD[9]120 ± 36*708 ± 86***
3/9
F: Mix[8]123 ± 22*728 ± 65***
4/8[ ], number of animals in each group; hCG, human chorionic gonadotropin; Mix, taurine + catalase + SOD;
SOD, superoxide dismutase.
Within the same column, * versus **; ** versus ***; * versus ***; **** versus *****: P < 0.05.

91

N. Suzuki and N. Sofikitisthat an increase in testicular temperature resultsin Leydig cell secretory deficiency (Sofikitis
and Miyagawa, 1992). The significantly lower
testosterone responses in varicocelized rats
indicated a defect in Leydig cell secretory function. The secretory defect detrimentally influenced the spermatogenesis and epididymal
sperm maturation process in varicocelized rats
in this study. This hypothesis is supported by
the reduced epididymal caudal sperm content
and motility profiles (Tables 3 and 4). It is
known that optimal Leydig cell function is necessary for the activation and maintenance of
spermatogenesis and epididymal sperm maturation process (Saypol et al., 1981; Hurt et al.,1986). The lighter seminal vesicular weight invaricocelized rats may be attributed to the impaired Leydig cell secretory function since asmall alteration in androgen production canaffect seminal vesicular weight in rats (Sofikitis
and Miyagawa, 1992). The absence of significant difference in serum basal testosterone
concentration between varicocelized rats andcontrol rats does not indicate an absence ofdifference in Leydig cell secretory function
between the 2 groups since delicate alterations
in Leydig cell secretory function may not be
accompanied by alterations in peripheral serum
androgen levels (Saypol et al., 1981).
The present study also confirm previous
studies (Hurt et al., 1986; Takihara et al., 1990)
providing evidence of a reduction of spermatogenesis in varicocelized rats by cytometric
DNA analysis or by calculation of sperm concentration directly in the epididymal fluid. We
revealed a bilateral detrimental effect of left
varicocele on sperm fertilizing capacity. This is
supported by the significantly smaller IVF trial
outcome bilaterally in varicocelized rats. The
diminished sperm fertilizing ability may be due
to defects in the epididymal sperm maturation
process since it is known that failure of spermatozoa to complete the cascade of events thatcharacterize the epididymal sperm maturation
process resulted in impaired sperm fertilizing
ability (Hurt et al., 1986). Development ofspermatozoal ability to expose forward motility, undergo capacitation, and penetrate thezona pellucida of the oocyte are examples of theseveral important properties spermatozoa acquire during epididymal sperm passage.
ROS are regularly formed during the process of normal respiration. However, the production is kept at physiologically low levels byintracellular free radical scavengers (Southorn
and Powis, 1988). While eukaryotic life is impossible without adequate oxygen, exposure toexcess concentrations of oxygen results in progressive and lethal damage in several tissues.
Several studies have shown that a large production of ROS in semen was detected in
more than 25% of infertile men (Aitken and
Clarkson, 1987b; Iwasaki and Gagnon, 1992).
Aitken and West (1990) and Kessopoulou andco-workers (1992) have demonstrated that the
major sources of ROS in semen were derived
from the spermatozoa and infiltrating leucocytes. Kessopoulou and co-workers (1995)
reported that oral administration of vitamin E
significantly improved the in vitro function ofhuman spermatozoa of men with a high ROS
production. We showed a benefical effect of
the administration of antioxidants on testicularspermatogenic and steroidogenic functions invaricocelized rats. Detrimental effects inducedby the administration of drugs were not observed in all rats during this experimental period(data not shown). The significantly larger
epididymal caudal sperm content and theenhanced testosterone responses were observedin varicocelized rats treated with antioxidants
rather than in sham-treated varicocelized rats
(Tables 3 and 4). Furthermore, treatment withantioxidants improved bilateral testicularfunctions and ameliorated the epididymal
sperm maturation process as indicated by thesignificantly higher epididymal caudal sperm
motility and fertilizing ability. The number of
fertile rats was increased in varicocelized rats
treated with antioxidants than in sham-treatedvaricocelized rats. The improvement in testicular functions in varicocelized rats treatedwith antioxidants could not be attributed to thetesticular countercurrent heat exchange systemsince the alterations in testicular temperature
was not observed. However, it should be emphasized that the values of quantitative andqualitative sperm parameters remained signif92

Effects of antioxidants of varicocelized rats

Acknowledgments: We wish to thank Prof. Takeshi
Sairenji, Prof. Eikichi Hashimoto and Prof. Ikuo
Miyagawa, Depts. of Biosignaling, Pathological
Biochemistry and Urology, respectively, Faculty of
Medicine, Tottori University for their advice andcomments on the manuscript.

icantly lower than the control values, although
the antioxidants improved testicular and epididymal parameters in varicocelized rats.
SOD specifically scavenges the superoxide
radicals by catalyzing their dismutation tohydrogen peroxide and oxygen. Hydrogen peroxide has been proposed to be the most toxic
ROS for human spermatozoa (Alvarez et al.,
1987; Aitken et al., 1993). Catalase enhances
the degradation of hydrogen peroxide to oxygen
and water. Treatment with SOD and catalase
reduced the degree of testicular damage inexperimental acute torsion (Henry and Turner,
1996). Taurine is known to reduce the cellular
production of ROS (Alvarez and Storey, 1983).
Morsny and co-workers (1979) have reported
that taurine stimulated hamster sperm motility.
In our study each of the administered antioxidants had benefical effects on sperm parameters. Although the testicular function ingroup F was higher compared with that ingroups C, D and E, the difference was not
significant. The small number of animals ineach group might have been responsible for thelack of significant difference. This implies that
there was an increased intracellular testicular
production of ROS that exceeds the capacity ofthe testicular tissue by removing oxidants andrestoring oxidized components in varicocelized
rats. The overall result may be disorganizationof intracellular enzymes and membranes. The
inflammatory response that generally follows
tissue damage due to ROS (Aitken and Clarkson,
1987a; Southorn and Powis, 1988; Ahotupa andHuhtaniemi, 1992; Mackenna and Kessopoulou,
1993) may amplify the primary result. Administration of antioxidants may support the natural
antioxidant mechanism of testicular tissue andpartially protects the tissue damage due to the
induction of varicoceles.
The present study demonstrated a protective
effect of antioxidants on testicular spermatogenic and steroidogenic functions and raised thepossibility of administering antioxidants to men
with varicoceles who have failed to improvesperm qualitative and quantitative parameters
after varicocelectomy.

References
1 Ahotupa M, Huhtaniemi I. Impaired detoxification of reactive oxygen and consequent oxidative stress in experimentally cryptochid rat
testis. Biol Reprod 1992;46:1114–1118.
2 Aitken RJ, Buckingham DW, Harkiss D. Use ofa xanthine oxidase free radical generating system
to investigate the cytotoxic effects of reactive
oxygen species on human spermatozoa. J Reprod
Fertil 1993;97:441–450.
3 Aitken RJ, Clarkson JS. Cellular basis of defective sperm function and its association with thegenesis of reactive oxygen species by humanspermatozoa. J Reprod Fertil 1987a;81:459–469.
4 Aitken RJ, Clarkson JS. Generation of reactive
oxygen species by human spermatozoa. In
Dormandy T, Rice-Evans C, eds. Free radicals:recent developments in lipid chemistry, experimental pathology and medicine. London:
Richelieu Press; 1987b. p. 333–335.
5 Aitken RJ, West KM. Analysis of the relationship between reactive oxygen species production
and leukocyte infiltration in fractions of humansemen separated on Percoll gradients. Int J
Androl 1990;13:433–451.
6 Alvarez JG, Storey BT. Taurine, hypotaurine,
epinephrine and albumin inhibit lipid peroxidation in rabbit spermatozoa and protect against
loss of motility. Biol Reprod 1983;29:548–555.
7 Alvarez JG, Touchstone JC, Blasco L Storey BT.
Spontaneous lipid peroxidation and production ofhydrogen peroxide and superoxide in humanspermatozoa. J Androl 1987;8:338–348.
8 Beaman M, Birtwistle R, Howie AJ, Michael J,
Adu D. The role of superoxide anion and hydrogen peroxide in glomerular injury induced by
puromycin aminonucleoside in rats. Clin Sci
1987;73:329–332.
9 Burton GW, Ingold KU. β-Carotene: an unusual
type of lipid antioxidant. Science 1984; 224:569573.
10 Chow CK. Vitamin E and oxidative stress. Free
Radic Biol Med 1991;11:215–232.
11 Coyotupa J, Parlow AF Kovacic N. Serumtestosterone and dihydrotestosterone levels
following orchiectomy in the adult rat. Endocrinology 1973;92:1579–1581.
12 de Lamirande E, Gagnon C. Reactive oxygen
species and human spermatozoa. II. Depletion of93

N. Suzuki and N. Sofikitis

adenosinetriphosphate (ATP) plays an importantrole in the inhibition of sperm motility. J Androl
1992;13:379–386.
13 Grootveldt M, Halliwell B. Measurement ofallantoin and uric acid in human body fluids.
Biochem J 1987;243:803–808.
14 Halliwell B, Gutteridge JMC. Free radicals inbiology and medicine. 2nd ed. Oxford: Clarendon
Press; 1987.
15 Henry MP, Turner TT. Rescue of testicularfunction following acute experimental torsion.
Proc AUA 1996;155:442A.
16 Hurt GS, Howards SS, Turner TT. Repair ofexperimental varicoceles in the rat. Long-term
effects on testicular blood flow and temperatureand cauda epididymal sperm concentration andmotility. J Androl 1986;7:271–276.
17 Iwasaki A, Gagnon C. Formation of reactive
oxygen species in spermatozoa of infertile patients. Fertile Sterile 1992;57:409–416.
18 Jeulin C, Soufir JC, Weber P, Laval-Martin D,
Calvayrac R. Catalase activity in human spermatozoa and seminal plasma. Gamete Res 1989;
24:185–196.
19 Kessopoulou E, Powers HJ, Sharma KK, Pearson
MJ, Russell JM, Cooke ID, et al. A double-blind
randomized placebo cross-over controlled trial
using the antioxidant vitamin E to treat reactive
oxygen species associated male infertility. Fertil
Steril 1995;64:825–831.
20 Kessopoulou E, Tomlinson MJ, Barratt CLR,
Bolton AE, Cooke ID. Origin of reactive oxygen
species in semen;spermatozoa or leukocytes? J
Reprod Fertil 1992;94:463–470.
21 Kobayashi T, Miyazaki T, Nator M, Nozawa S.
Protective role of superoxide dismutase in humansperm motility: superoxide dismutase activity
and lipid peroxide in human seminal plasma andspermatozoa. Hum Reprod 1991;6:987–991.
22 Mackenna A, Kessopoulou E. The contributionof a hidden male factor to unexplained infertility.
Fertil Steril 1993;59:405–411.
23 Morsny RJ, Waxman L, Meizel S. Taurine
maintains and stimulates motility of hamster
sperm during capacitation in vivo. J Exp Zool
1979;210:123–128.
24 Rao B, Soufir JC, Martin M, David G. Lipid peroxidation in human spermatozoa as related to
midpiece abnormalities and motility. Gamete
Res 1989;24:127–134.
25 Saypol DC, Howards SS, Turner TT, Miller ED.
Influence of surgically induced varicocele on
testicular blood flow, temperature, and histology
in adults rats and dogs. J Clin Invest 1981;68:39–
45.
26 Sie H. Oxidative stress: from basic research toclinical application. Am J Med 1991;91:31–38.
27 Sofikitis N, Miyagawa I. Experimental models
for the study of varicocele:A selected review. Jpn
J Fertil Steril 1992;38:168–177.
28 Southorn PA, Powis G. Free radicals in medicine. I. Chemical nature and physiologic reaction. Mayo Clin Proc 1988;63:3881–3889.
29 Takihara H, Ishizu K, Ueno T, Isoyama R, Baba
Y, Sakatoku J. Pathogenesis of varicocele: experimental study using flow cytometric DNA
analysis. Andrologia 1990;22:137–143.
30 Thiele JJ, Freisleben HJ, Fuchs J, Ochsendorf
FR. Ascorbic acid and urate in human seminal
plasma: determination and interrelationships
with shemiluminescence in washed semen. Hum
Reprod 1995;10:110–115.
31 Toyoda Y, Chang MC. Fertilization of rat eggs
in vitro by epididymal spermatozoa and thedevelopment of eggs following transfer. J
Reprod Fertil 1974;36:9–22.

(Received December 18, Accepted January 13, 1998)

94