Wiley

Spontaneous lipid peroxidation in washed human spermatozoa was induced by aerobic incubation at 32 C and measured by malonaldehyde production; loss of motility during the incubation was determined simultaneously. Malonaldehyde production at the point of complete loss of motility, defined as the lipoperoxidative lethal end‐point (LLE), was 0.10 ± 0.03 nmol/10⁸ cells (X̄ ± SD, n = 40), and was independent of the time to complete loss of motility. Human spermatozoa produced both H₂O₂ and O₂⁻. during aerobic incubation. Inhibition of superoxide dismutase in these cells with KCN showed that all the H₂O₂ production is due to action of the dismutase. The superoxide dismutase activity of individual human sperm samples varied between 1 and 10 U/10⁸ cells, variations between samples from a single donor being nearly as great as those between different donors. The time to complete motility loss (t_L) showed equal variation of 1 to 10 hours among samples. The rate of spontaneous lipid peroxidation, calculated as LLE/t_L, for a given sperm sample and the superoxide dismutase activity of the same sample, determined prior to aerobic incubation, gave a good linear correlation (r = 0.97). Glutathione reductase, glutathione peroxidase, and glutathione were found to be present in human spermatozoa, but showed little variation among samples. These results suggest that superoxide dismutase plays the major role in protecting human spermatozoa against lipid peroxidation. In addition, the superoxide dismutase activity of a fresh sperm sample appears to be a good predictor of the lifetime (up to the complete loss of motility) of that particular sample, and so may prove useful in semen analysis.

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.

ABSTRACT:Oxidative stress results from the production of oxygen radicals in excess of the antioxidant capacity of the stressed tissue. Many conditions or events associated with male infertility are inducers of oxidative stress. X‐irradiation, for example, or exposure to environmental toxicants and the physical conditions of varicocele and cryptorchidism have been demonstrated to increase testicular oxidative stress, which leads to an increase in germ cell apoptosis and subsequent hypospermatogenesis. Such stress conditions can cause changes in the dynamics of testicular microvascular blood flow, endocrine signaling, and germ cell apoptosis. Testicular oxidative stress appears to be a common feature in much of what underlies male infertility, which suggests that there may be benefits to developing better antioxidant therapies for relevant cases of hypospermatogenesis.

ABSTRACT: Sperm chromatin integrity is vital for successful pregnancy and transmission of genetic material to the offspring. We evaluated chromatin integrity in sperm from 60 infertile men and 7 fertile donors comparing the sperm chromatin structure assay (SCSA), TdT‐mediated‐dUTP nick end labeling (TUNEL), the sperm chromatin dispersion (SCD) test, and acridine orange staining technique (AOT). The TUNEL and SCD assays showed a strong relationship with the SCSA (r > .866; P < .001) for sperm DNA fragmentation, both in infertile men and donors of known fertility. AOT did not show any relationship with SCSA. The breakdown of the DNA fragmentation index (DFI) into 3 categories (≤15%, >15%‐<30%, and ≥30%) showed that the SCSA, TUNEL, and SCD test predict the same levels of DNA fragmentation. AOT consistently showed higher levels of DNA fragmentation for each DFI category. DNA fragmentation in sperm between infertile men and donor sperm was significantly different (P < .05) under SCSA (22.0 ± 1.6 vs 11.8 ± 1.4), TUNEL (19.5 ± 1.3 vs 11.1 ± 0.9) and SCD (20.4 ± 1.3 vs 10.8 ± 1.1), respectively. DNA fragmentation in sperm evaluated by AOT did not differ (P > .05) between infertile men (31.3 ± 2.4) and donors (32.7 ± 4.8). AOT showed extreme variations for sperm DNA fragmentation in semen from both infertile men and donors. The problems of indistinct colors, rapid fading, and the heterogeneous staining were also faced. In conclusion, SCSA, TUNEL, and SCD show similar predictive values for DNA fragmentation, and AOT shows variable and increased levels of DNA fragmentation, which makes it of questionable value in clinical practice.

ABSTRACT: The current study was designed to examine the actions of a model endocrine disruptor on embryonic testis development and male fertility. Pregnant rats (F0) that received a transient embryonic exposure to an environmental endocrine disruptor, vinclozolin, had male offspring (F1) with reduced spermatogenic capacity. The reduced spermatogenetic capacity observed in the F1 male offspring was transmitted to the subsequent generations (F2‐F4). The administration of vinclozolin, an androgen receptor antagonist, at 100 mg/kg/day from embryonic day 8–14 (E8‐E14) of pregnancy to only the F0 dam resulted in a transgenerational phenotype in the subsequent male offspring in the F1‐F4 generations. The litter size and male/female sex ratios were similar in controls and the vinclozolin generations. The average testes/body weight index of the postnatal day 60 (P60) males was not significantly different in the vinclozolin‐treated generations compared to the controls. However, the testicular spermatid number, as well as the epididymal sperm number and motility, were significantly reduced in the vinclozolin generations compared to the control animals. Postnatal day 20 (P20) testis from the vinclozolin F2 generation had no morphological abnormalities, but did have an increase in spermatogenic cell apoptosis. Although the P60 testis morphology was predominantly normal, the germ cell apoptosis was significantly increased in the testes cross sections of animals from the vinclozolin generations. The increase in apoptosis was stage‐specific in the testis, with tubules at stages IX—XIV having the highest increase in apoptotic germ cells. The tubules at stages I—V also had an increase in apoptotic germ cells compared to the control samples, but tubules at stages VI—VIII had no increase in apoptotic germ cells. An outcross of a vinclozolin generation male with a wild‐type female demonstrated that the reduced spermatogenic cell phenotype was transmitted through the male germ line. An outcross with a vinclozolin generation female with a wild‐type male had no phenotype. A similar phenotype was observed in outbred Sprague Dawley and inbred Fisher rat strains. Observations demonstrate that a transient exposure at the time of male sex determination to the antiandrogenic endocrine disruptor vinclozolin can induce an apparent epigenetic transgenerational phenotype with reduced spermatogenic capacity.

ABSTRACT: Experimental animal studies have demonstrated that exposure to some phthalates may be associated with altered endocrine function and adverse effects on male reproductive development and function, but human studies are limited. In the present study, urine and serum samples were collected from 425 men recruited through a US infertility clinic. Urinary concentrations of mono(2‐ethylhexyl) phthalate (MEHP), the hydrolytic metabolite of di(2‐ethylhexyl) phthalate (DEHP), and other phthalate monoester metabolites were measured, along with serum levels of testosterone, estradiol, sex hormone—binding globulin (SHBG), follicle‐stimulating hormone, luteinizing hormone, inhibin B, and prolactin. Two oxidized urinary metabolites of DEHP were also measured in urine from 221 of the men. In multiple regression models adjusted for potential confounders, MEHP was inversely associated with testosterone, estradiol, and free androgen index (FAI). An interquartile range increase in MEHP was associated with 3.7% (95% confidence interval [CI], −6.8% to −0.5%) and 6.8% (95% CI, −11.2% to −2.4%) declines in testosterone and estradiol, respectively, relative to the population median hormone levels. There was limited evidence for effect modification of the inverse association between MEHP and FAI by the proportion of DEHP metabolites in the urine measured as MEHP (MEHP%), which is considered a phenotypic marker of less efficient metabolism of DEHP to its oxidized metabolites. Finally, the ratio of testosterone to estradiol was positively associated with MEHP (P = .07) and MEHP% (P = .007), suggesting potential relationships with aromatase suppression. In conclusion, these results suggest that urinary metabolites of DEHP are inversely associated with circulating steroid hormone levels in adult men. However, additional research is needed to confirm these findings.

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 < 0.01) and by alternate tests of sperm quality, including motility, acrosome integrity, Sephadex filtration and morphology of spermatozoa (all significant at P < 0.05 to P < 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.

ABSTRACT: Androgen‐dependent male urogenital development occurs via mesenchymal‐epithelial interactions in which mesenchyme induces epithelial morphogenesis, regulates epithelial proliferation, and evokes expression of tissue‐specific secretory proteins. Mesenchymal‐epithelial interactions continue to be important into adulthood. For example, mesenchyme of the urogenital sinus (UGM) and seminal vesicle (SVM) induce dramatic morphologic and functional changes in various adult epithelia. Since adult epithelial cells are unquestionably responsive to mesenchymes that can elicit expression of alternative morphologic and functional phenotypes, established carcinomas might also be influenced by their connective tissue environment. In this regard, Dunning prostatic tumor has been induced by UGM or SVM to differentiate into tall columnar secretory epithelial cells. This change in cytodifferentiation is associated with a reduction in growth rate and loss of tumorigenesis. The role of soluble growth factors in the mechanism of mesenchymal‐epithelial interactions is discussed.