Showing papers on "Semen published in 1988"

Significance of Reactive Oxygen Species and Antioxidants in Defining the Efficacy of Sperm Preparation Techniques

Abstract: The mechanisms responsible for mediating the influence of sperm preparation protocols on human sperm function have been investigated. Techniques that involved the separation of motile spermatozoa prior to centrifugation were found to yield sperm suspensions of highest quality. If the spermatozoa were centrifuged prior to isolation of the motile cells, sperm function was impaired. The detrimental effects of centrifugation were associated with a sudden burst of reactive oxygen species production by a discrete subpopulation of cells (characterized by significantly diminished motility and fertilizing capacity) that could be separated from normal functional spermatozoa on Percoll gradients. If unfractionated sperm suspensions were subjected to centrifugation, the reactive oxygen species generated by this subpopulation impaired the functional competence of normal spermatozoa in the same suspension. Assessment of the ability of the antioxidants, butylated hydroxytoluene, and vitamin E, to curtail the peroxidative damage inflicted by such cells in response to centrifugation revealed a significant improvement of sperm function in the presence of vitamin E.

Relationships between computerized measurements of motion of frozen-thawed bull spermatozoa and fertility.

Abstract: A computerized system (CellSoft, CRYO Resources, Ltd.) was validated using video tapes of frozen-thawed bull spermatozoa diluted in filtered (0.2 micron) egg yolk-citrate extender (8 X 10(6) spermatozoa/ml) and analyzed at 30 frames/sec for the percentage of motile spermatozoa (greater than or equal to 20 microns/sec) and linear velocity of motile spermatozoa. Virtually all motile spermatozoa were detected and debris rarely were classified as immotile spermatozoa if the extender had been filtered. Variation about the mean for percent motile cells was similar when only 12 rather than 20 or 30 frames/field were analyzed. Use of 20 frames/field was adequate to determine the percentage of motile bull spermatozoa. Five mixtures of live and killed spermatozoa were analyzed (four bulls) to evaluate accuracy. Percent motile spermatozoa was correlated (r = 0.97) with the ratio of live:killed spermatozoa. Mean linear velocity of motile spermatozoa was similar for each mixture (P greater than 0.05). To further evaluate accuracy, percent motile spermatozoa was determined by computer and by "track motility" (20 samples; 0 to 63% motile spermatozoa); values were correlated (r = 0.95). The system was precise (CV of 6% based on triplicate analyses of the same samples) and reasonably accurate for evaluating bull sperm motility if the extender had been filtered and 20 to 25 fields (greater than or equal to 200 spermatozoa) were evaluated. Correlations between measurements of sperm motion and fertility were studied using cryopreserved semen from two fertility trials. For the first, 75-day nonreturn rate data for 20 samples of bull semen (10 bulls) were not significantly correlated with evaluations made by CellSoft. For the second fertility trial, the competitive fertility index (a measure of relative fertility) for nine bulls was correlated (r greater than or equal to 0.68; P less than 0.05) with percent motile spermatozoa, linear velocity and straight-line velocity. Multiple correlations based on six characteristics evaluated by CellSoft, at 0 or 1.5 hours, and the competitive fertility index were greater than or equal to 0.94. Based on the latter data, the system may facilitate prediction of the relative fertility of bull spermatozoa.

The sperm chromatin structure assay. Relationship with alternate tests of semen quality and heterospermic performance of bulls.

Abstract: Data obtained by the sperm chromatin structure assay (SCSA) on spermatozoa from nine bulls were correlated with fertility, measured by heterospermic performance (-0.94, P less than 0.01) and by alternate tests of sperm quality, including motility, acrosome integrity, Sephadex filtration and morphology of spermatozoa (all significant at P less than 0.05 to P less than 0.01). The SCSA uses flow cytometry to determine the susceptibility of nuclear DNA to low pH-induced denaturation in situ as measured by the ratio of acridine orange binding to double- or single-stranded DNA. The error associated with multiple SCSA measurements was relatively low. The primary finding is that the assay of chromatin structure stability performed on killed spermatozoa was as highly correlated with the heterospermic performance of semen as the best of the classical tests for semen quality. The SCSA may therefore be a highly useful technique for evaluation of sperm quality.

Induction of potential for sperm motility by bicarbonate and pH in rainbow trout and chum salmon.

Abstract: Spermatozoa of rainbow trout and chum salmon, which have no potential for motility in the testis, acquire that potential in the sperm duct. This paper demonstrates that there is little difference between the levels of sodium, potassium, calcium, magnesium, chloride and osmolality of the seminal plasma in the testis and in the sperm duct. However, the bicarbonate concentration of the seminal plasma and the pH value of semen were higher in the sperm duct than in the testis. When immotile spermatozoa obtained from the testis were incubated in artificial seminal plasma with a high pH and containing HCO3-, spermatozoa became motile within 1 h. These results suggest that spermatozoa of salmonid fish acquire the potential for motility as a result of the increase in seminal bicarbonate concentration and pH that occurs as spermatozoa pass from the testis to the sperm duct.

Gonadotropin therapy in men with isolated hypogonadotropic hypogonadism: the response to human chorionic gonadotropin is predicted by initial testicular size.

Abstract: This study was designed to determine whether exogenous hCG alone can complete spermiogenesis in men with isolated hypogonadotropic hypogonadism (IHH). hCG was administered to 22 men with IHH until maximal testicular growth was achieved. Their mean testicular volume increased from 5.5 ± 1.1 (±se) mL (pretreatment) to 10.8 ± 1.6 mL (maximum) during treatment (P < 10−6). The maximum mean testicular volume was highly positively correlated with initial volume (r = 0.84; P < 10−6). All men attained normal serum testosterone levels, and 7 of 22 men achieved supraphysiological serum estradiol levels. During hCG treatment, 14 of the 22 men had sperm appear in their semen. Six of 11 men with complete gonadotropin deficiency, defined as an initial mean testicular volume less than 4 mL, became sperm positive during hCG treatment. In contrast, 9 of 11 men with partial gonadotropin deficiency (initial mean testicular volume of 4 mL or more) produced sperm during treatment (P < 0.001). Sperm concentration was highly pos...

Temporal changes in motility parameters related to acrosomal status: identification and characterization of populations of hyperactivated human sperm.

Abstract: The occurrence and time course of capacitation, acrosomal loss, and hyperactivated motility require quantitative definition in order to characterize fertile human sperm. In this study, video microscopy and digital image analysis were used to measure curvilinear (VCL) and straight line (VSL) velocity, average linearity of progression (UN [100 x VSL/VCUJ), maximum and mean amplitude of lateral head displacement (ALH), beat-cross-frequency (BCF), DANCE (VCL x meanALH) and DANCEMEAN (meanALH/(UIN/100)). These parameters were measured for sperm in semen and in the swim-up fraction of washed cells during incubation for up to 24 h under in vitro fertilization (IVF) conditions. Acrosomal loss was monitored in the same population of washed cells by an immunofluorescence end-point assay. The greatest increase in mean values of motility parameters was observed when seminal sperm were washed free of seminal plasma. Increases continued for up to 6 h of incubation. Two subpopulations of hyperactivated sperm were identified; one type, not found in semen, showed star-spin trajectories, and constituted 3.0, 3.8, 4.5, and 4.1% of the swim-up population after 0, 3, 6 and 24 h of incubation. The second type, termed transitional showed a more progressive trajectory and constituted less than 1% in semen. In total, hyperactivated cells constituted 0.8% of cells in semen, 14.5% of the swim-up population with no incubation, and 23.1, 22.7, and 19.4% after 3, 6, and 24 h of incubation, respectively. Acrosomal loss in the swim-up population was delayed during the first 3 h of incubation, then increased from near 5% at 3 h to 7 and 12% at 6 and 24 h, respectively. The kinetics of change in the extent of hyperactivation and in acrosomal loss, although measured in different cell populations, are consistent with an association between these two events.

Freezing and thawing alter chromatin stability of ejaculated human spermatozoa: fluorescence acridine orange staining and Feulgen-DNA cytophotometric studies.

Abstract: Cryopreservation and freezing-thawing effects on the fertilizing ability of human spermatozoa commonly are evaluated by post-thaw motility. Various studies have depicted the ultrastructural changes caused by freezing-thawing, yet the chromatin alterations have been studied very limitedly. Our aim was to determine the extent to which freezing-thawing alters the chromatin of human spermatozoa, using two analytical methods: acridine orange staining and Feulgen-DNA cytophotometric studies. Both methods revealed a dramatic effect of freezing-thawing on sperm chromatin: the native DNA content decreased as did the Feulgen-DNA content, and sperm surface area was reduced. These results indicate an effect on DNA, diminished accessibility for Feulgen, and a decrease in nuclear surface area and prompt us to hypothesize a relationship between an "overcondensation" state for sperm chromatin after freezing-thawing and a lower conception rate for human semen after cryostorage.

Detection of human immunodeficiency virus in cell-free seminal fluid.

Abstract: Human immunodeficiency virus (HIV) was detected by assay of reverse transcriptase activity in a "virus pellet" obtained by differential sucrose density centrifugation of cell-free semen from three patients with the acquired immune deficiency syndrome (AIDS), one individual with AIDS-related complex (ARC), and in an asymptomatic homosexual male. Reverse transcriptase assays indicated virus concentrations in the range of 10(8) particles/ml of semen, an accumulation substantiated by electron microscopic visualization of cell-free virus. This is the first description of cell-free retrovirus in seminal fluid and at a greater concentration than reported for blood or other body fluids or tissues. These results suggest that the male reproductive tract of humans may be a reservoir of HIV expression, and raises the possibility that the cells lining the epididymal lumen could be chronically infected with HIV. These are important considerations in formulating treatment and preventive strategies.

Selenium and glutathione peroxidase in seminal plasma of men and bulls

Abstract: High levels of selenium and glutathione peroxidase (GSH-Px) were found in bull seminal plasma but low concentrations in human seminal plasma. In man the seminal plasma selenium was associated with two macromolecules separable by gel filtration, but no GSH-Px was found in the same fractions. Selenium in bull seminal plasma was associated with two proteins, which could be separated by gel filtration and anion exchange chromatography. Both macromolecules coeluted with GSH-Px activity and had identical optima at pH 7.0. Their responses to thermal treatment, however, differed. Seminal vesicle secretory fluid in the bull contained both these proteins, while the larger molecule was also found in fractionations of ampulla, prostate and Cowper's glands. The larger enzyme form is evidently a tetramer of the smaller one. Both enzyme forms were extremely sensitive to heavy metals and some divalent metal ions. GSH caused an activation while other reducing agents were suppressive. Triton X-100 had no effect, while sodium deoxycholate was inhibitory. These properties are typical for a phospholipid hydroperoxide GSH-Px. It is concluded that this selenium-dependent enzyme may be important in the protection of bovine spermatozoa against damage caused by oxygen radicals, while in man such a mechanism is not functional.

Concurrent zidovudine levels in semen and serum determined by radioimmunoassay in patients with AIDS or AIDS-related complex.

Abstract: Zidovudine was present in the semen and serum of six patients with acquired immunodeficiency syndrome or the related complex who were receiving 200 mg of the drug orally every four to six hours. Mean semen zidovudine levels (as measured by a new radioimmunoassay) in samples collected 0.75 to 1.25 hours after oral dosing were 3.63 to 7.19 μmol/L. Levels in semen samples collected 3.0 to 4.5 hours after oral dosing were 1.68 to 6.43 μmol/L. These values are above the in vitro minimum inhibitory concentration for the human immunodeficiency virus type 1 (HIV-1). Mean serum concentrations at the early and late times after oral dosing were 0.22 to 3.07 μmol/L and 0.10 to 1.42 μmol/L, respectively. Ratios of semen/serum zidovudine levels ranged from 1.3 to 20.4. It is possible that a pH-dependent trapping mechanism, which has been described in the prostate for other antibiotics, was responsible for the relatively high semen levels observed. ( JAMA 1988;259:3023-3026)

Interaction of human immunodeficiency virus with human sperm in vitro.

Abstract: Transmission of human immunodeficiency virus (HIV) in semen by sexual intercourse is believed to be a major etiological factor in the spread of the disease. In order to explore the role of sperm in this transmission, cell-free HIV-1(IIIB) was coincubated with human sperm for 5 h. After incubation, unattached HIV-1 was extensively washed off the cells and the presence and location of HIV-1 in or on the sperm was determined by electron microscopic (EM) and immunofluorescence methods. Examination of sperm at the EM level showed that HIV-1 attaches to the sperm surface and enters the sperm through the intact cell membrane. Immunofluorescence studies, using polyvalent anti-HIV antibodies, demonstrated the presence of small round fluorescent particles in 5-10% of sperm heads after 5 h of incubation. A search for HIV-1 receptors (CD4 epitopes) revealed the presence of OKT4 epitopes, including OKT4A, B, D, and F on 5-10% of sperm by epifluorescence microscopy and on 10-20% of sperm by flow-cytometric analysis. These in vitro data suggest that human sperm may be the primary cellular element in the transmission of HIV by semen.

Diagnostic value of sperm function tests and routine semen analyses in fertile and infertile men

Abstract: The results of routine semen analyses, the zona-free hamster oocyte penetration test, the hypoosmotic swelling test, and semen adenosine triphosphate levels were studied in 66 fertile and 130 infertile men. Multivariate discriminant analysis demonstrated that routine semen parameters including semen volume, sperm count, percent sperm motility, and percent normal spermatozoa in combination could predict the fertility of these patients with 70.4% accuracy. Of the three sperm function tests evaluated, the zona-free hamster oocyte penetration test and the hypoosmotic swelling test were selected by the multivariate discriminant analysis as variables capable of providing significant information on the fertility status of the patients. However, the addition of the results of these two tests to the routine semen analysis did not significantly improve the predictability of fertility. The overall correct prediction rate was 77.6% after incorporation of the results of these two sperm function tests. In this group of subjects, the presently available sperm function tests did not predict the fertility status of a patient with a high degree of accuracy.

Fertilizing potential of acrosome-defective sperm following microsurgical injection into eggs.

Abstract: Ejaculates from three infertile men were examined ultrastructurally and found to include a high number of amorphous acrosomeless spermatozoa. Two of the patient's spermatozoa exhibited the typical characteristics of round-head syndrome--spherical-shaped heads completely absent of acrosome and postacrosomal sheath. The semen of the third patient was found to contain a mixture of round-headed and irregularly shaped acrosomeless sperm and a small percentage of normal acrosome-intact sperm. Previous studies have shown that acrosomeless sperm do not have the ability to bind or penetrate zona-free hamster eggs (Weissenberg et al., Syms et al.). In an attempt to determine if such amorphous sperm are capable of decondensation and pronuclear formation, sperm of all three men were microsurgically injected into zona-intact hamster eggs. All of the sperm injected were found to be capable of decondensation or pronuclear formation, suggesting that if the inability to penetrate an egg is bypassed, the sperm of these infertile men are capable of participating in the early events of fertilization.

Abnormal sperm morphology and other semen parameters related to the outcome of the hamster oocyte human sperm penetration assay.

Abstract: A new method for evaluation of sperm morphology using strict criteria is currently used in the andrology laboratory at the Eastern Virginia Medical School. A prospective study was designed to evaluate the following semen parameters in samples of all patients over a set period of time: sperm concentration and motility, and normal sperm morphology. These factors were correlated with results of the hamster zona-free oocyte/human sperm penetration assay (SPA). One hundred patients with a sperm concentration ranging from 2 to 219 X 10(6)/ml, a motile sperm fraction ranging from 6.9 to 87%, and normal sperm morphology ranging from 1 to 39%, were evaluated. The statistical analysis system general linear model was used to judge the influence of the different variables. There was a statistically significant relationship between the per cent of sperm with normal morphology and penetration rate in the SPA (P = 0.001). Outcome of the SPA was also correlated with in vitro fertilization, retrospectively, in 84 patients. Thirty-eight patients had an SPA less than 10%, with no fertilization in vitro in 13 patients (33.3%) and fertilization in 25 (66.7%). Forty-five had an SPA greater than 10% with fertilization in 37 (82.2%) and no fertilization in eight (17.8%) patients.

Motility of rat spermatozoa at the site of fertilization.

Abstract: This study was undertaken to examine the factors that may affect the numbers and motility patterns of spermatozoa at the site of fertilization. The contents of the oviductal amp ullae of previously mated cycling or superovulated immature rats were examined microscopically. We determined whether spermatozoa were free or associated with cells and whether they exhibited hyperactivated motility, forward progressive motility, or were immo tile. These data were correlated with the percentage of fertilized eggs. In addition, the beat pattern of hyperactivated spermatozoa was characterized by using high-speed video microscopy. At the time when half of the eggs were fertilized, ampullae of cycling rats contained an average of less than one motile spermatozoon per ampulla. Most of these motile spermatozoa were hyperactivated. About half of these were free in the ampulla and about half were in the cumulus or zona pellucida. Hyperactivated spermatozoa displayed a nonprogressive whiplash wave form with a high amplitude recovery stroke similar to that described in hamster and guinea pig spermatozoa capacitated in vitro. In addition to motile sperinatozoa, we counted about three immotile spermatozoa for each motile spermatozoon. In superovulated, immature female rats, we found about ten times as many spermatozoa in each category as in cycling rats. From our observations, it is clear that very few spermatozoa reach the ampulla of the oviduct. Furthermore our observations suggest that in cycling rats progressively swimming spermatozoa may become hyperactivated shortly after entering the amp ulla of the oviduct. They probably enter the cumulus mass within a short time or become immotile. The fact that nearly half of the spermatozoa in the ampulla reside in the cumulus or zona suggests that it may take a longer period to traverse these investments than has been estimated in the hamster. Superovula ted females contain ten times as many spermatozoa as cycling rats. This may be an effect of the hormone level as a result of induced ovulation. Alternatively, it may be a consequence of the younger age of the superovulated animals.

Comparison of electrostimulation methods for semen recovery in the rhesus monkey (Macaca mulatta).

Abstract: Electroejaculation of 17 rhesus monkeys was performed at intervals during a 15-month period using both penile and rectal probe stimulation. The semen quality was compared for the two stimulation methods. Both methods were effective in approximately 90% of attempts, and there was no difference in fertilizing capacity of the sperm. A seasonal difference in semen quality was detected, and samples recovered by penile stimulation showed higher sperm count.

The relationship between the motility and morphology of spermatozoa in human semen.

Abstract: High-speed videomicrography was used to assess simultaneously the morphology and motility of seminal spermatozoa from 10 fertile donors and 10 patients being evaluated for infertility. In both donors and patients, morphologically normal spermatozoa were more likely to be motile and had significantly higher straight line velocity, greater rolling frequency and flagellar beat frequency than abnormally shaped cells. For donors and patients there were highly significant, linear correlations (R = 0.7 to R = 0.98) between the movement characteristics of morphologically normal and abnormal spermatozoa within an ejaculate. A greater percentage of normal donor spermatozoa were motile than were the normal spermatozoa from patients (56% vs. 28%, respectively, P less than 0.005) and normal donor spermatozoa also swam faster than normal patient spermatozoa (49.1 +/- 3.2 microns/sec vs. 37.4 +/- 4.3 microns/sec, mean +/- sem, respectively, P less than 0.05). Overall, a multivariate analysis of variance, including straight line velocity, rolling frequency, beat frequency, and flagellar beat amplitude, demonstrated that these movement characteristics were significantly greater for the normal cells from donors than for the normal spermatozoa from patients. These biologic distinctions notwithstanding, the discrimination between semen from donors and patients was not improved when only morphologically normal cells were analyzed for motility.