Yonago Acta medica 1998;41:23–29

Effect of Hypercholesterolaemia on Testicular Function and Sperm
Physiology
Kenji Shimamoto and Nikolaos Sofikitis
Department of Urology, Faculty of Medicine, Tottori University, Yonago 683-0826, JapanThe effect of hypercholesterolaemia on testicular endocrine and exocrine function was
evaluated. Furthermore, the influence of hypercholesterolaemia on sperm quality, quantity, and fertilizing potential was appreciated. Eight mature rabbits (group A) were
fed chow containing 3% cholesterol for 12 weeks. An additional six rabbits (group B)
were fed normal chow for the same period. At the end of the experimental period
hormonal profiles and sperm parameters were evaluated. In addition, the sperm reproductive potential was assessed by in vitro fertilization (IVF) procedures. Peripheral
serum responses to testicular stimulation with human chorionic gonadotropin, epididymal sperm content and motility, and IVF outcome were significantly lower in group A
than in group B. In contrast, serum cholesterol levels were significantly higher in group
A. It appears that hypercholesterolaemia has a detrimental effect on Leydig cell function,
spermatogenesis, the epididymal sperm maturation process, and the overall sperm fertilizing capacity.
Key words: cholesterol; infertility; sperm; temperature; testischanging their conformation (Yeagle, 1985).
Furthermore, serum albumin, known to serve asa capacitating factor, induces its effect by promoting the efflux of cholesterol from the humansperm plasma membrane (Langlais et al., 1988).
There is strong evidence that cholesterol modifies the fusion of the plasma membrane with theouter acrosomal membrane in the same way
other decapacitating factors do (Davis andHungund, 1976). Cholesterol provides rigidness to the membrane, whereas polyunsaturated
agents promote membrane fluidity.
A cholesterol enriched diet produces a decrease in the kinetics of the acrosome reaction
in live rabbit spermatozoa (Diaz-Fontdevillaand Buston-Obregon, 1993). This effect is consistent with the studies of Sebastian and colleagues (1987) showing that human male infertility might be associated with altered lipid
metabolism in seminal plasma. The decapacitating properties of cholesterol have also beenclearly demonstrated by Diaz-Fontdevilla andCholesterol is secreted in the seminal plasma by
the prostate and protects spermatozoa against
environmental shock (Sofikitis and Miyagawa,1991). High sexual satisfaction prior to/during
ejaculation is accompanied by an enhanced
prostatic secretion of cholesterol (Sofikitis andMiyagawa, 1993a).
Several reports refer to the role of cholesterol in sperm capacitation and the acrosome
reaction process (Benoff et al., 1993). Changes
in sperm cholesterol profiles appear to be involved in the asynchronism of capacitation(Benoff et al., 1993). During the sperm’s passage through the female reproductive tract, aloss of cholesterol from the spermatozoal membrane is involved in the mechanism of sperm
capacitation in mammals. Cholesterol has previously been demonstrated to limit protein
insertion into phospholipid bilayers, to restrict
lateral mobility of functional receptor sites embedded in the cell surface lipid core, and tomodulate the activity of membrane proteins byAbbreviations: DPBS, Dulbecco’s phosphate buffered saline; hCG, human chorionic gonadotropin; IVF, invitro fertilization

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K. Shimamoto and N. Sofikitis

Buston-Obregon (1993). In that study a cholesterol-enriched diet was found to alter the
filipin-sterol complexes in the plasma membrane of the acrosomal region, suggesting thatsperm membrane lipid domains induced byhypercholesterolaemia are the cause of themodification in sperm capacitation and acrosome reaction kinetics. However, there are noreports investigating the influence of cholesterol-enriched diets on spermatogenesis andLeydig cell function. Our objective was to estimate the effects of hypercholesterolaemia on
testicular endocrine and exocrine function, andon overall sperm fertilizing capacity.

Serum/tissue cholesterol and lipid evaluation
Cholesterol was extracted from testicular tissue
using the modified Bligh-Dyer method (Blighand Dyer, 1959; Kate,1986). Then, serum or
testicular cholesterol and serum total lipid were
evaluated by the Special Reference Laboratory(Hiroshima, Japan) as previously described(Sofikitis and Miyagawa, 1993a).

Serum testosterone assay
Testosterone levels were assessed by radioimmunoassay according to the method of
Coyotupa and coworkers (1972) using kits from
Nihon DPC Corporation (Tokyo, Japan).

Materials and Methods
Eight mature (6-month-old) New Zealand whitemale rabbits were fed chow containing 3% cholesterol for 12 weeks (group A). An additional
six rabbits were fed normal rabbit chow for thesame period (group B). The animals were
caged individually during the experimental
period and received water and feeding ad
libitum. At the end of the experimental period,
blood was aspirated from a peripheral vein (earvein) and cholesterol, total lipids and testosterone were evaluated. Then, all the animals received intramuscular administration of humanchorionic gonadotropin (hCG; 1,500 IU). Threehours later, peripheral blood was aspirated andtestosterone responses to hCG stimulation were
measured. One week later, all the animals wereanesthetized with intravenous sodium pentobarbital (30 mg/kg, Nembutal, Abbot Laboratories, Chicago, IL). Intravenous heparin sulfate (120 units/kg) was given for anticoagulation. Intra-abdominal and left testicular temperature were assessed. One-hundred milligramsfrom the left cauda epididymis (just prior to theorigin of the vas deferens) were resected, andthe epididymal sperm content, motility and fertilizing capacity were appreciated. The left
testis was resected, and the left testicular weight
was recorded. Cholesterol concentration in theleft testicular tissue was assessed.

Measurement of testicular temperature
Left testicular temperature was assessed by
percutaneous insertion of a 29-gauge needle
probe attached to a digital thermometer (Unique
Medical, PTC 201 model, Tokyo). Intraabdominal temperature was monitored with arectal probe, and body temperature was maintained between 36.7˚C and 37.3˚C with radiantheat throughout the procedure. The difference
between the intra-abdominal and left intratesticular temperature was recorded.

Testicular weight
The testes were dissected free of surrounding
tissue and weighed on a Mettler Basbal scale(Tokyo).

Epididymal caudal sperm content andmotility
Epididymal caudal sperm content was measured as previously described (Sofikitis andMiyagawa, 1993b). The epididymal fragment
was trimmed and minced in 5 mL of modified
Dulbecco’s phosphate buffered saline (DPBS;
Sigma Chem., St. Louis, MO) (Sofikitis et al.,1996). A stereo microscope facilitated cutting
the tissue into the smallest possible pieces. The

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Cholesterol and testicular function

minced epididymal tissue was separated fromthe liberated spermatozoa by filtration througha 20-µm-diameter paper filter (Whatman Co.,New York, NY). Six droplets (a volume of eachsample was 0.06 mL) of the filtrate were used
for the sperm count (number of spermatozoa/mL), and the average number was calculated. A
Makler Counting Chamber (Sefi Medical Instruments, Haifa, Israel) was placed on the slide ofan ordinary microscope, and a 20-power objective and 10-power eyepiece were used. Ten droplets were counted to calculate the percentage ofmotile spermatozoa in the filtrate immediately
after its preparation. The proportion of motile
spermatozoa in each drop was determined bycounting 300 cells in randomly selected fields.
Motility was also graded qualitatively and assigned a value from 0 to 4 as previously described(Sofikitis et al., 1991).

ples of a concentration equal to 2,000,000/mL
were prepared (final samples) and incubated inmodified Brackett medium at 37˚C under 5%
carbon dioxide for 3 h. Cumulus masses containing oocytes were recovered from female mature
rabbits as previously described (Sofikitis et al.,1996). Ten cumulus masses were inseminatedwith sperm samples from each male rabbit.
Each cumulus mass placed in 0.9 mL of RD
medium containing 10 mM of taurine [RDmedium, Dulbecco’s low-glucose Modified
Eagle’s medium (GIBCO Co., Grand Island,NY) mixed 1:1 (v/v) with RPMI 1640
(GIBCO)] (Carney et al., 1991) was inseminated with 0.1 mL of the final sperm sample andincubated at 37˚C under 5% carbon dioxide(Sofikitis et al., 1996). Oocytes were observed
at 18 and 36 h after insemination. The percentage of oocytes with two pronuclei at 18 h
and the percentage of cleaved oocytes at 36 h
were recorded.

Epididymal caudal sperm fertilizing capacity

Statistical analysis

The ability of rabbit spermatozoa to penetrateand fertilize ova was tested in vitro. Epididymal caudal rabbit sperm samples were centrifuged at 200 × g for 10 min. The sperm pellet
was transferred to modified Brackett medium(containing 3.2 g/L bovine albumin) (Brackett,
1970; Brackett and Oliphant, 1975). Then,
sperm samples underwent a standard swim-up
procedure. In brief, 1 mL-aliquots were incubated at a 45˚ angle at 37˚C under 5% carbon
dioxide for 30 min. Supernatants (0.5 mL) wererecovered. Spermatozoa in supernatants fromgroup B exhibited motility ranging from 81% to96%. Supernatants from group A rabbits showed sperm motility ranging from 53% to 70%.
To avoid differences of sperm motility between
both groups, spermatozoa from group A were
filtrated via Sperm Prep (ZBL, Lexington, KY)
as previously described (Sofikitis et al., 1993).
The filtrates recovered from Sperm Prep columns exhibited percentages of motile spermatozoa ranging from 73% to 91%. Post-swim-up
supernatants from group B rabbits and Sperm
Prep filtrates from group A rabbits were centrifuged at 200 × g for 15 min, and sperm sam-Wilcoxon’s test was used for statistical analysis. A probability of less than 5% (< 0.05) was
considered to be statistically significant. Values were expressed as mean ± SD. Measurements of hormonal and sperm parameters were
performed in blind procedure.

Results

Cholesterol assay
Peripheral serum cholesterol levels weresignificantly higher in group A than in group B(Table 1). In contrast, there were no significant
differences in cholesterol contents in testicular
tissue between groups A and B.

Total lipid profiles
Total lipid levels in peripheral serum weresignificantly higher in group A than in group B(Table 1).

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K. Shimamoto and N. Sofikitis

Table 1. Testicular and peripheral serum cholesterol/total lipid concentrations in rabbits fed
cholesterol-enriched chow and rabbits which received normal chow
Group

Chow

Cholesterol
Serum (mg/dL)
Testicular tissue(nmol/107 cells)

A
Cholesterol-enriched
1803 ± 113*
B
Control
36 ± 8
Values are expressed as mean ± SD.
* P < 0.05 compared with the control group.

15.2 ± 1.4
14.4 ± 1.1

Testosterone assay

Total lipids
Serum (mg/dL)
6986 ± 189*
184 ± 16

Epididymal sperm content, motility andfertilizing capacity

Basal peripheral serum testosterone profiles
were not significantly different between groups
A and B. In contrast, testosterone responses tohCG stimulation were significantly lower ingroup A (Table 2).

Epididymal sperm content, percentage of motile spermatozoa, and motility grade were significantly lower in group A than in group B(Table 3). Although differences in percentage
of motile spermatozoa between group A afterswim-up plus Sperm Prep filtration and group B
after swim-up were not significantly different(see Materials and Methods), the percentage ofoocytes with two pronuclei at 18 h after
insemination, and percentage of cleaved
oocytes at 36 h after insemination weresignificantly lower in group A than group B.

Testicular temperature
Left testicular versus intra-abdominal temperature difference was not significantly different
between groups A and B (3.9 ± 0.4˚C and 4.1 ±0.3˚C, respectively).

Testicular weight
Discussion

Differences in mean values of left testicularweight between groups A and B were not significantly different (3153 ± 101 mg and 3208 ±94 mg, respectively).

We created a hypercholesterolaemia model
similar to that described by Girerd and coworkers (1990) to illustrate the influence ofcholesterol-enriched diets on testicular
endocrine and exocrine function. Such a study
is of clinical importance because hypercholesterolaemia is a social problem in many developed countries and high-cholesterol products
are often advertised for commercial reasons.
Peripheral serum basal testosterone levels
between hypercholesterolaemic and control
rabbits showed no significant differences. Of
course a serum basal testosterone profile is agood indicator of Leydig cell function, but it
often fails to indicate small changes in the rateof testicular testosterone synthesis (Steinberger
et al., 1973). Therefore, evaluation of the possible potency of Leydig cell function caused by

Table 2. Peripheral blood testosterone profiles in hypercholesterolaemic and control
animals
Group Chow

Testosterone (ng/mL)
Basal
Response to hCGACholesterolenriched
2.4 ± 0.4
4.9 ± 0.7*
B
Control
2.2 ± 0.7
6.8 ± 0.9
Values are expressed as mean ± SD.
*P < 0.05 compared with the control group.

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Cholesterol and testicular function

Table 3. Epididymal caudal sperm content, motility and fertilizing capacity in hypercholesterolaemic and control rabbits
Hypercholesterolaemic group(Group A)

Control group(Group B)

Sperm content (× 10/mL medium)
413 ± 151*
734 ± 106
Motile sperm(%)
61 ± 7*79 ± 6
Motility grade
2.3 ± 0.4*
3.3 ± 0.3
Oocytes with two pronuclei (%)†
27 ± 4.3*
53 ± 7.6
Cleaved oocytes(%)†
23 ± 5.1*
47 ± 7.3
Values are expressed as mean ± SD.
* P < 0.05 compared with the control group.
† Sperm samples for IVF in groups A and B were used after preparation via the swim-up and Sperm Prep
methods, and the swim-up method, respectively. There were no significant differences in the percentage ofmotile sperm between groups A and B after above preparations.

hCG stimulation should be important. In fact,
the present study reveals secretory dysfunctionof stimulated Leydig cells in a hypercholesterolaemic testicular environment. We evaluatedtestosterone responses to hCG 3 h after stimulation because we has previously demonstrated
that the peak testosterone response to hCG
stimulation in rabbits occurs 3 h after injection(Sofikitis and Miyagawa, 1994). Optimal
Leydig cell function and testosterone secretion
are known to be prerequisites for i) normal
activation of spermatogenesis (Steinberger etal., 1973) and ii) maintenance of epididymal
sperm maturation process and development ofsperm capacity to exhibit a vigorous forward
progression (Turner, 1985). Thus, the significantly lower epididymal caudal sperm counts
and motility profiles in animals with hypercholesterolaemia may be attributable to a defect inthe secretory function of the Leydig cells
resulting in impaired spermatogenesis and an
impaired epididymal sperm maturation process.
In this study the proportion of cleaved oocytes to fertilized oocytes was significantly
lower in rabbits in the hypercholesterol groupcompared with the control group. Because
there were no significant differences in the percentage of sperm motility between the groups
after preparation of the sperm by Sperm Prep
and the swim-up method, these results may not
be attributable to the decreased sperm motility
in rabbits with hypercholesterolaemia.

These findings may suggest that hypercholesterolaemia results in quantitative or qualitative alterations in sperm membrane lipids
leading to an impaired sperm capacity for capacitation and acrosome reaction. In addition,
hypercholesterolaemia may have a harmful
effect on the final event of the sperm fertilizing
process [i.e., Zona binding penetration of thezona pellucida, fusion with the vitelimembrane,
and so on (Yamamoto et al., 1997)] and subsequently on the potential for transformation of
the sperm head to male pronuclei. Furthermore,
the significantly lower percentage of cleaved
oocytes in rabbits treated with cholesterol dietsclearly indicated that early embryonic development is impaired.
The absence of a significant difference intesticular tissue cholesterol content between thetwo groups is consistent with the results of previous reports (Diaz-Fontdevilla and BustonObregon,1992; Diaz-Fontdevilla et al., 1993)
showing that cholesterol content does not increase in the seminal plasma of animals treatedwith cholesterol-enriched diets. Our results andthe above previous studies taken together suggest the existence of a blood-testis or a bloodmale reproductive tract barrier for cholesterol.
It is difficult to explain why Leydig cellfunction is impaired in hypercholesterolaemic
animals without an elevation of testicular tissuecholesterol concentration. We measured testicular temperatures known to be regulated by a27

K. Shimamoto and N. Sofikitis

7 Davis BK, Hungund BJ. Effect of modified
membrane vesicles from seminal plasma on thefertilizing capacity of rabbit spermatozoa. Biochem Biophys Res Commun 1976;69:1004–
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M. Distribution of filipin-sterol complexes insperm membranes from hypercholesterolaemic
rabbits. Andrologia 1992;24:279–283.
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diet: Effects on kinetics of the acrosome reaction
in rabbit spermatozoa. Mol Reprod Dev 1993;
35:176–180.
10 Gired XJ, Hirsh AT, Cooke JP, Dzau VJ, Creager
M. L-arginine augments endothelium-dependent
vasodilation in cholesterol-fed rabbits. Circulation Research 1990;67:1301–1308.
11 Kates M. Laboratory techniques in biochemistry
and molecular biology. In: Kates M, ed. Technique of lipidology: isolation, analysis, and identification of lipids. 2nd ed. New York: Elsevier,1986:100–111.
12 Langlais J, Kan FWK, Granger L, Raymond L,
Bleau G, Roberts KD. Identification of sterol
acceptors that stimulate cholesterol efflux fromhuman spermatozoa during in vitro capacitation.
Gamete Res 1988;20:185-201.
13 Rubenstein SC, Green LS, Dewolf WC,
Morgentaler A. Temperature-dependent expression of a 95 kd protein in rat testis. Andrologia
1995;27:107–113.
14 Sebastian SM, Selvaraj S, Aruldhas M,
Govindarajulu P. Pattern of neutral phospholipids in the semen of normospermic, oligospermia and azoospermic men. Reprod Fertil
1987;79:373–378.
15 Sofikitis N, Miyagawa I. Secretory dysfunctionof the male accessory genital glands due to prostatic infections and fertility: a selected review ofthe literature. Jpn Fertil Steril 1991; 36:690–699.
17 Sofikitis N, Miyagawa I. Endocriological, biophysical, and biochemical parameters of semencollected via masturbation versus sexual intercourse. J Androl 1993a;14:366–373.
18 Sofikitis N, Miyagawa I. Left adrenalectomy invaricocelized rats dose not inhibit the development of varicocele related physiologic alterations. Int J Fertil 1993b;38:250–255.
20 Sofikitis N, Miyagawa I. Reproductive capacityof the male gamete after completion of meiosis. J
Assist Reprod Genet 1994;11:335–341.
16 Sofikitis N, Miyagawa I, Toda T, Harada T, Mio
Y, Terakawa N. The effect of kallikrein onhuman sperm membrane function. Tohoku J Exp
Med 1991;164:23–28.
19 Sofikitis N, Miyagawa I, Zavos PM. Selection of
single-stranded deoxyribonucleic acid spermatozoa via the Sperm Prep filtration column. Fertil

counter-current heat exchange testicular vasculature to evaluate whether vascular consequences of hypercholesterolaemia on the
counter-current heat exchange system are responsible for the testicular damage (Rubenstein
et al., 1995). However, the functionality of thecounter-current heat exchange system in hypercholesterolaemic animals appears to be intact.
Our hypotesis is that hypercholesterolaemia
may not exert a direct influence on cells in
seminiferous tubules, but other substances
induced by hypercholesterolaemia have a
detrimental effect on spermatogenesis.
The present study is the first report showing
an effect of cholesterol-enriched diets on testicular endocrine and exocrine function, the epididymal sperm maturation process, and theoverall sperm fertilizing capacity. The conceptthat there is a blood-testis barrier for cholesterol
is supported by the current findings and deserves further investigation.
Acknowledgments: The authors wish to thank Prof.
Ikuo Miyagawa of the Department of Urology,
Faculty of Medicine, Tottori University, for his kindadvice and valuable suggestions in this study.

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