Department of Urology, Tottori University School of Medicine, 36 Nishimachi, Yonago 683, Japan
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Abstract |
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Key words: cholesterol/infertility/spermatozoa/temperature/testis
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Introduction |
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A cholesterol-enriched diet leads to a decrease in the kinetics of the acrosome reaction in live rabbit spermatozoa (Diaz-Fontdevila and Bustos-Obregon, 1993). This effect is consistent with previous studies (Sebastian et al., 1987) showing that human male infertility might be associated with altered lipid metabolism in seminal plasma. Although several studies have evaluated the effect of hypercholesterolaemia on the mechanisms modulating sperm capacitation and acrosome reaction (Diaz-Fontdevila et al., 1992; Diaz-Fontdevila and Bustos-Obregon, 1993), there are no reports concerning the influence of cholesterol-enriched diets on Leydig and Sertoli cell function, spermatogenesis, the epididymal sperm maturation process, and sperm fertilizing capacity. Our objective was to estimate the effects of hypercholesterolaemia on testicular endocrine and exocrine function, on sperm quality and quantity, and on the ability of spermatozoa to penetrate/fertilize oocytes, and to initiate early embryonic development.
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Materials and methods |
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In addition, sperm cholesterol content, seminal plasma cholesterol and lipid concentrations, and morphometric sperm parameters were estimated in the first, second, and third sample, respectively, of each rabbit. Then all the animals were anaesthetized with i.v. sodium pentobarbital (30 mg/kg, Nembutal; Abbot Laboratories, Chicago, IL, USA). Intravenous heparin sulphate (120 units/kg) was given for anticoagulation. The intra-abdominal and left testicular temperature were assessed. One hundred milligrams of tissue from the left cauda epididymis (just proximal to the origin of the vas deferens) was resected and the epididymal sperm motility and fertilizing capacity were measured. The left testis was resected and the left testicular weight was recorded. Cholesterol and total lipid concentrations in the left testicular tissue were assessed. Androgen-binding protein (ABP) activity in right testicular cytosols was measured.
Serum/seminal plasma/testicular tissue cholesterol and total lipid evaluation
Cholesterol was extracted from spermatozoa, testicular tissue and seminal plasma samples using the modified BlighDyer method (Bligh and Dyer, 1959; Kates, 1986
; Sugkraroek et al., 1991
). Spermatozoa cholesterol content, serum cholesterol and total lipids, testicular tissue cholesterol and total lipids, and seminal plasma cholesterol and total lipids were evaluated by the Japanese Special Reference Laboratory (Hiroshima, Japan) as previously described (Kates, 1986
).
Collection of semen samples
Semen collection was achieved by using an artificial vagina. A doe was placed in the same cage with the buck. Each male was allowed three false mounts prior to collecting the ejaculate (Sofikitis et al., 1996a). Semen volume was recorded after liquefaction and the sperm concentration and sperm motility were evaluated in all samples as previously described (Sofikitis et al., 1996a
). Both quantitative sperm motility (percentage of motile spermatozoa) and qualitative sperm motility (motility grade; from 0 to 4; Sofikitis et al., 1991
) were assessed. Following assessment of semen volume and sperm concentration and motility the first semen sample was diluted with an equal volume of Brackett medium (Brackett, 1970
) and divided into two equal aliquots. Both were centrifuged at 1000 g for 45 min. The pellets (spermatozoa) were pooled and processed for cholesterol assay. The supernatants (diluted seminal plasma) were also pooled, and processed for cholesterol and total lipid assay. Microscopic evaluation of drops of the supernatants demonstrated the absence of spermatozoa and round cells. Three droplets of the second semen sample of each animal were put on the stage of a confocal scanning laser microscope and morphometric parameters of >300 undisturbed non-stained by trypan blue randomly selected spermatozoa were calculated in each droplet as previously described (Sofikitis et al., 1994
, 1996a
). The maximal length of the sperm head (MLH), the maximal width of the sperm head (MWH), and the length of the midpiece (LMP) were measured.
Following assessment of the standard parameters of semen analysis, the third semen sample from each rabbit was centrifuged at 1000 g for 45 min. The pellet (spermatozoa) and the upper 1 ml of the supernatant (seminal plasma) from each rabbit were isolated. The recovered spermatozoa from each one rabbit of group A were resuspended in the isolated seminal plasma from a rabbit of group B. In a similar fashion, recovered spermatozoa from each rabbit of group B were resuspended to the recovered seminal plasma from a rabbit of group A. The 10 samples of group A spermatozoagroup B seminal plasma and the 10 samples of group B spermatozoagroup A seminal plasma were incubated at 37°C for 3 h. Quantitative and qualitative sperm motility were assessed at the beginning (0 h) and at the end (3 h) of the incubation period.
Serum testosterone assay
Testosterone concentrations were assessed in triplicate by radioimmunoassay according to previously published methods (Coyotupa et al., 1972) using kits from Nihon DPC Corporation (Tokyo, Japan). The intra-assay and interassay coefficients of variation were 4% and 8%, respectively, and the lower limit of detection was 0.1 ng.
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, Japan). Intra-abdominal temperature was monitored with a rectal probe and body temperature was maintained between 36.7°C and 37.3°C with radiant heat throughout the procedure (Sofikitis and Miyagawa, 1994b). The difference between the intra-abdominal and left intratesticular temperature was recorded.
Testicular weight
Each left testis was dissected carefully free of surrounding tissue using a stereo microscope (SZ-STS, Olympus, Tokyo, Japan) and weighed on a Mettler Basbal scale (Tokyo, Japan).
ABP activity in testicular cytosols
ABP activity was measured in testicular cytosols by The Japanese Special Reference Laboratory using established methods (Ritzen 1974). In brief, testicular cytosols were prepared, treated with a charcoal suspension in TEMG medium (Ritzen, 1974
) containing TrisHCl, EDTA, mercaptoethanol, glycerol, and dextran, and incubated with radiolabelled dihydrotestosterone. The samples were then electrophoresed and the gels were sliced. The slices were placed into counting vials containing scintillation fluid and measurements of radioactivity were translated into concentrations of ABP (see for review Ritzen, 1974
).
Caudal epididymal sperm motility
Sperm motility in the caudal epididymis was measured as we previously described (Sofikitis et al., 1993a). The epididymal fragment was trimmed and minced in 5 ml of modified Dulbecco's phosphate-buffered saline (DPBS; Sigma, St Louis, MO, USA) (Sofikitis et al., 1996a
,b
). A stereo microscope facilitated cutting the tissue into small pieces.
The minced epididymal tissue was separated from the liberated spermatozoa by filtration through a paper filter (Whatman Co., New York, NY, USA) of pore diameter approximately equal to 20 µm. Ten droplets were counted to calculate the percentage of motile spermatozoa in the filtrate immediately after its preparation. The proportion of motile spermatozoa in each drop was determined by counting 300 cells in randomly selected fields. Motility was also graded qualitatively (Sofikitis et al., 1991).
Epididymal caudal sperm fertilizing capacity
The ability of rabbit spermatozoa to penetrate and fertilize oocytes was assessed. Samples of epididymal caudal spermatozoa were centrifuged at 200 g for 15 min. The sperm pellet was resuspended in modified Brackett medium (containing 3.2 g/l bovine albumin; Brackett, 1970; Brackett and Oliphant, 1975
). Sperm samples were then subjected to a swim up procedure. In brief, 0.3 ml aliquots of epididymal caudal spermatozoa in modified Brackett medium were prepared. A volume of 0.7 ml modified Brackett medium was gently layered onto each aliquot. After incubation at a 45° angle at 37°C under 5% carbon dioxide for 30 min, supernatants (0.5 ml) were recovered. Spermatozoa in supernatants from group B exhibited motility equal to 8196%. Supernatants from group A rabbits showed sperm motility equal to 5370%. To avoid statistically significant differences in sperm motility between groups A and B prior to performance of IVF techniques, supernatants from group A animals were filtered via a SpermPrep column (ZBL, Lexington, KY, USA) as previously described (Sofikitis et al., 1993b
). Filtrates recovered from SpermPrep columns exhibited a percentage of motile spermatozoa equal to 7391%. During SpermPrep column filtration of the supernatants of group A, post-swim-up supernatants from group B were maintained at room temperature. Aliquots of post-swim-up supernatants from group B rabbits and aliquots of SpermPrep filtrates from group A rabbits were then centrifuged at 200 g for 20 min and sperm pellets were collected and resuspended in modified Brackett medium. Sperm samples containing 2x106 spermatozoa/ml were incubated at 37°C under 5% carbon dioxide for 3 h. Cumulus masses containing oocytes were recovered from mature female rabbits (Sofikitis et al., 1996a
,b
). Ten cumulus masses were inseminated with sperm samples from each male rabbit. Each cumulus mass in 0.9 ml of modified (RD; RPMI medium plus Dulbecco's medium; received from Hayashi company, Matsue, Japan) medium (RD medium plus 10 mM of taurine; Sofikitis et al., 1996a
) was inseminated with 0.1 ml of sperm sample and incubated at 37°C under 5% carbon dioxide. Oocytes were observed 18, 36 and 80 h post-insemination. Medium was changed at 18 h and 36 h post-insemination. The percentage of oocytes with two pronuclei plus second polar body (normally fertilized oocytes), the percentage of cleaved oocytes, and the percentage of morulae/blastocysts at 18, 36 and 80 h respectively, post-insemination were calculated.
Statistical analysis
Wilcoxon's test was used for statistical analysis of quantitative parameters. Differences in the normally fertilized oocytes, percentage of cleaved oocytes, and morulae/blastocysts were analysed using the 2-test. P < 0.05 was considered to be statistically significant. Values of quantitative parameters were expressed as means ± SD. Measurements of biochemical, hormonal and sperm parameters were performed in a blinded fashion.
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Results |
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Testosterone assay
Basal peripheral serum testosterone profiles were not significantly different between groups A and B. In contrast, testosterone responses to HCG stimulation were significantly lower in group A (Table II).
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Testicular temperature
Left testicular versus intra-abdominal temperature difference was not significantly different between groups A and B (4.0 ± 0.4°C versus 4.2 ± 0.4°C respectively).
Testicular weight
The difference in the mean values of left testicular weight between groups A and B was not significant (3098 ± 112 versus 3181 ± 98 mg respectively).
Semen parameters
The difference in semen volume between groups A and B was not significant (Table III). Sperm concentration, percentage of motile spermatozoa, and the motility grade were significantly higher in group B than group A (Table III
). Differences in MLH or MWH between groups A and B were not significant. The LMP was significantly longer in group B.
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Discussion |
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However, the effects of hypercholesterolaemia on testicular function and overall sperm fertilizing capacity have not yet been evaluated. We created a hypercholesterolaemia model similar to that previously described (Girerd et al., 1990; Diaz-Fontdevilla et al., 1992
; Diaz-Fontdevilla and Bustos-Obregon 1993
) to assess the influence of cholesterol-enriched diets on testicular endocrine and exocrine function. Such a study is of clinical importance since hypercholesterolaemia is a social problem in Western developed countries. However, the peripheral serum cholesterol concentrations demonstrated in rabbits treated with cholesterol are higher that those usually observed in hypercholesterolaemic humans. Additional studies are necessary to investigate the effects of small or moderate alterations in serum cholesterol on the human male reproductive potential and to clarify whether there is a threshold in the increases of peripheral serum cholesterol above which the effects of hypercholesterolaemia on testicular function and sperm physiology are more severe.
The absence of significant difference in peripheral serum basal testosterone values between the two rabbit groups does not imply that normal testicular testosterone biosynthesis is occurring in the hypercholesterolaemic animal population because peripheral serum basal testosterone profiles are not a sensitive indicator of Leydig cell function (Steinberger et al., 1973). Basal peripheral serum testosterone concentration often fails to indicate small changes in the rate of testicular testosterone biosynthesis (Steinberger et al., 1973
). Testosterone responses to HCG were evaluated 3 h post-stimulation because a previous report demonstrated that the peak testosterone response to HCG stimulation in the rabbit occurs at this time (Sofikitis and Miyagawa, 1994
). This study is the first to suggest a defect in the secretory function of Sertoli cells in hypercholesterolaemic subjects. This is supported by the significantly smaller profiles of ABP in testicular cytosols of hypercholesterolaemic rabbits. Optimal Sertoli and Leydig cell secretory functions are known to be a prerequisite for activation and maintenance of spermatogenesis (Steinberger et al., 1973
) and maintenance of the epididymal sperm maturation process and the ability of spermatozoa to exhibit a vigorous forward progression (Turner, 1979
). Thus, the significant reduction in sperm concentration, percentage of motile spermatozoa, and motility grade in ejaculated semen samples and epididymal caudal sperm motility profiles in animals with hypercholesterolaemia may be attributable to defects in the secretory function of the Sertoli and Leydig cells resulting in impaired spermatogenesis and the epididymal sperm maturation process.
Epididymal dysfunction in hypercholesterolaemic animals may have detrimental effects on the cytostructural modifications and biochemical changes that occur during sperm epididymal maturation and may result both in decreased sperm motility and in the sperm morphometric abnormality (in the midpiece) found in hypercholesterolaemic animals. The significantly lower quantitative and qualitative sperm motility observed in hypercholesterolaemic rabbits may also result from the shorter length of the sperm midpiece region (Sofikitis et al., 1994). The lower sperm motility profiles in hypercholesterolaemic rabbits may not be a direct result of the influence of cholesterol or another component of prostatic/seminal vesicular secretions since (i) epididymal spermatozoa also demonstrated an impaired motility; (ii) control spermatozoa did not demonstrate lower motility parameters when placed in seminal plasma from hypercholesterolaemic rabbits; (iii) motility of spermatozoa from hypercholesterolaemic rabbits did not improve significantly when placed in control seminal plasma; (iv) there were no significant differences in the sperm or seminal plasma cholesterol content between control and hypercholesterolaemic rabbits.
The present study also suggests that spermatozoa from hypercholesterolaemic rabbits have diminished sperm capacity to penetrate and fertilize oocytes. Percentage of normally fertilized oocytes (per inseminated oocyte), percentage of cleaved oocytes (per inseminated oocyte), and percentage of morulae/blastocysts (per inseminated oocyte) were all significantly lower in the cholesterol-fed animals. This may not be due to the decrease in sperm motility because an additional step (i.e. SpermPrep filtration) was added to the technique of sperm preparation for IVF in the hypercholesterolaemic animals to eliminate differences in sperm motility between the two groups. The epididymal sperm maturation process involves a cascade of biophysical and biochemical events that results in the development of sperm capacity to penetrate and fertilize the oocyte. Hypercholesterolaemia may have a detrimental effect on this process either by interfering with the cascade during epididymal passage, or through alterations to the sperm membrane lipids. The lower fertilizing capacity of epididymal spermatozoa from the hypercholesterolaemic rabbits may not result from the direct influence of cholesterol or a substance of prostatic or seminal vesicular origin on the spermatozoa, since these spermatozoa are not exposed to prostatic or seminal vesicular secretions. Further, there were no significant differences in either testicular cholesterol content or in epididymal caudal sperm cholesterol content (data not shown).
The percentage of normally fertilized oocytes that developed to morula/blastocyst stage was significantly lower in hypercholesterolaemic rabbits, suggesting a defect in their developmental capacity. This is consistent with previous reports suggesting that the male gamete not only activates and fertilizes the oocyte but also contributes to the ability of the zygote to undergo the first mitotic divisions (Janny and Ménézo, 1994). Hypercholesterolaemia may exert an adverse effect on the sperm factors which mediate the paternal influence on early embryonic development.
The absence of a significant difference in seminal plasma cholesterol concentration between the two groups is consistent with previous reports (Diaz-Fontdevila et al., 1992; Diaz-Fontdevila and Bustos-Obregon, 1993) showing that cholesterol does not increase in the seminal plasma of animals on a cholesterol-enriched diet. The present study also suggests that there is no increase in testicular tissue cholesterol of hypercholesterolaemic animals. This may imply the existence of either a bloodseminiferous tubule (since the testicular volume mainly comprises seminiferous tubules) and/or a bloodreproductive tract selective barrier against high concentrations of cholesterol. Additional studies are necessary to clarify whether there is a high concentration of cholesterol in the interstitial testicular tissue, since harmful consequences within the seminiferous tubules may be secondary to the secretory deficiency of the Leydig cells. Alternatively, other compounds induced by hypercholesterolaemia may exert a direct detrimental action outside of the seminiferous tubule (i.e. on Leydig cells) or within the seminiferous tubule (on spermatogenic cells and Sertoli cells).
Previous studies have shown that vasodilated vessels in hypercholesterolaemic rabbits do not relax normally in response to pharmacological stimuli (Girerd et al., 1990). This may be due to impaired synthesis or release of endothelium-derived relaxing factor. Testicular temperatures are known to be regulated by a counter-current heat exchange testicular vasculature. In hypercholesterolaemic animals the functionality of this system appears to be intact, suggesting that vascular consequences of hypercholesterolaemia on the counter-current heat exchange system are not responsible for the development of testicular damage.
The present study is the first to suggest an adverse effect of cholesterol-enriched diets on Leydig and Sertoli cell secretory function, spermatogenesis and sperm cytoskeleton, epididymal sperm maturation process, and the overall sperm fertilizing capacity and ability to initiate further embryonic development. Further studies are necessary to discover the factors induced by hypercholesterolaemia that are responsible for the latter effects.
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Notes |
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References |
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Submitted on August 11, 1998; accepted on February 2, 1999.