Simultaneous assessment of sperm chromatin condensation and morphology before and after separation procedures: effect on the clinical outcome after in vitro fertilization

OBJECTIVE: To look for correlations between acridine orange (AO) staining and semen parameters before and after sperm separation procedures and to assess whether the AO test predicts fertilization or pregnancy outcomes after standard IVF and intracytoplasmic sperm injection. DESIGN: Prospective study that simultaneously assesses sperm morphology and nuclear protein maturity on a cell-by-cell basis before and after preparative procedures. SETTING: University teaching hospital. PATIENT(S): Men (n = 140) undergoing diagnostic semen analysis. MAIN OUTCOME MEASURE(S): Acridine orange fluorescence of sperm nuclei, semen parameters, IVF outcome. RESULT(S): In unprocessed samples, 90% of sperm with normal heads displayed green fluorescence (mature nuclear protein); significantly lower percentages of green fluorescence were observed in sperm with abnormal heads. The percentage of mature normal sperm in the specimen correlated with motility. Sperm maturity after swim-up or Percoll gradient was significantly improved for sperm with normal or abnormal heads. The percentage of mature normal sperm correlated with motility after either Percoll or swim-up. Neither the percentages of mature nuclei nor mature normal nuclei correlated with fertilization or pregnancy outcome. CONCLUSION(S): Nuclear protein maturation correlates with sperm motility and morphology. Because morphologically normal and motile sperm are more mature, separation procedures should generate a population of sperm with the highest fertilization capacity. Acridine orange staining, however, did not predict fertilization efficiency or pregnancy outcome in IVF cycles.     

A logistic regression model including DNA status and morphology of spermatozoa for prediction of fertilization in vitro

To determine predictive values of routine semen analysis, sperm morphology evaluation using strict criteria and DNA status for in-vitro fertilization (IVF), 66 consecutive couples undergoing IVF in a university hospital IVF programme were prospectively investigated. Semen samples from 66 men were evaluated by routine semen analysis, morphology evaluation using strict criteria and acridine orange staining for determination of DNA status. A new technique is described for acridine orange scoring which consisted of evaluation of two smears per case, with and without heat treatment. Resistance to heat-provoked denaturation was determined by the difference between two evaluations. A logistic regression model was built and receiver operating characteristic curves were constructed to determine the threshold values and to compare diagnostic properties. Morphology evaluation using strict criteria and concentration of progressively motile spermatozoa were found to be the principal parameters determining the sperm fertilizing capacity in vitro. The logistic regression model composed of morphology evaluation using strict criteria and acridine orange score had a powerful diagnostic capability for prediction of fertilization in vitro.     

Chromatin structural changes in sperm after scrotal insulation of Holstein bulls

The reported effects on semen quality ascribed to testicular heat stress generally relate to traits impacting sperm transport and fertilizing ability but not to the genetic material contained by the sperm. To characterize the effects of testicular heat stress on sperm chromatin, susceptibility of DNA in sperm nuclear chromatin to in situ acid denaturation was measured by flow cytometry after staining with acridine orange using the sperm chromatin structure assay (SCSA). Semen was collected from Holstein bulls at 3-day intervals, before and after 48-hour scrotal insulation, until the morphologically abnormal sperm content in raw semen exceeded 50%. After cryopreservation in egg yolk-citrate extender, semen was thawed and sampled during incubation in vitro at 38.5 degrees C. Overall, SCSA results showed that chromatin susceptibility to denaturation was increased for sperm collected post- vs. preinsulation and was more pronounced for sperm presumably in the testes during insulation than for those sperm presumably in the epididymides. Increased susceptibility was detected as early as the first collection postinsulation; however, chromatin of sperm presumably in the proximal epididymis during insulation did not appear to have been detrimentally affected. Chromatin susceptibility to denaturation increased with increased incubation time in vitro, but the rate of change in susceptibility during incubation did not differ among pre- vs. postinsulation specimens. We conclude that elevated scrotal temperatures adversely affect both epididymal and testicular sperm by reducing sperm chromatin stability. The effects of heat stress on the chromatin of epididymal sperm were more subtle than those exhibited by testicular sperm but detectable within close proximity to the heat stress event.     

Environmental, management, and genetic factors affecting semen production in Holstein bulls

The objective of this study was to evaluate the importance of environment, management, physiological status, and genetics on semen quality (volume of the ejaculate, sperm concentration, sperm motility, number of sperm, and number of motile spermatozoa per ejaculate) of Canadian Holstein bulls. For this purpose, semen production data from 198 bulls were analyzed using mixed linear models. Young bulls (up to 30 mo old) and mature bulls (between 4 and 6 yr old) were analyzed separately. Semen characteristics generally improved significantly with age of young bulls. Season significantly affected all semen traits in young bulls but did not significantly affect volume and sperm motility of mature bulls. Performance was better in winter than in summer. The highest numbers of motile spermatozoa per ejaculate were obtained with intervals of at least 4 to 5 d between collections. Although the bull handler and semen collector caused less than 10% of the variance, the collection team significantly affected semen volume, number of sperm, and number of motile sperm per ejaculate for both growing and mature bulls. Heritabilities for volume, concentration, sperm motility, number of sperm, and number of motile sperm per ejaculate were, respectively, 0.24, 0.52, 0.31, 0.38, and 0.49 for young bulls and 0.44, 0.36, 0.01, 0.54, and 0.64 for mature bulls. Repeatability of semen traits varied from 0.41 to 0.64. Genetics, management, and environmental factors clearly contribute to semen production in Holstein bulls.     

The canine as an animal model of human aging and dementia

Cytochemical defects in chromatin were examined by transmission electron microscopy (TEM) after the staining by alcoholic phosphotungstic acid (PTA) of normal and malformed ejaculated spermatozoa from 35 male partners of infertile couples, and in six sperm samples retrieved from the caput epididymidis of men affected by obstructive azoospermia. PTA staining was also analysed in normal ejaculates of fertile men after incubation of the washed spermatozoa with dithiothreitol (DTT) to reduce disulfides to thiols, or with DTT followed by iodoacetamide, a blocking agent for thiol groups. PTA stained 63 (27-100)% of malformed heads and 25 (10-100)% of normal sperm heads (median (range) n = 35; P = 0.0001, Wilcoxon matched pairs test). The percentage of normal heads stained by PTA was negatively correlated with the percentage of heads of normal form, with condensed chromatin and a normal acrosome (Spearman r = 0.75; P = 0.0001), and positively correlated with the percentage of malformed heads after conventional TEM analysis (Spearman r 0.60; P = 0.0001). Staining with PTA in normal heads was not correlated with the presence of non-condensed chromatin in otherwise normal sperm heads evaluated by conventional TEM analysis. In spermatozoa recovered from the caput epididymidis, 15% of normal heads were stained with PTA, significantly fewer than in ejaculated sperm samples (P = 0.014). The reduction of disulfides to thiols was associated with PTA staining of all normal heads, and this was prevented by incubation with iodoacetamide. We conclude that PTA staining of the nuclei of human ejaculated spermatozoa may indicate a defect of chromatin condensation, owing to an excess of free thiol groups. The lower percentage of normal epididymal sperm heads that stained with PTA in cases of obstructive azoospermia compared with ejaculated sperm may be related to an overoxidation of thils owing to the ageing of spermatozoa.     

Glucose-stimulated expressed sequence tags from rat pancreatic islets

The ability of seminal plasma to influence the fertility of ejaculated bull spermatozoa was examined using a sperm penetration assay for zona-free bovine oocytes. Washed, ejaculated spermatozoa from bulls of below (low) or above average (high) fertility were mixed with seminal plasma from the same bull, or with seminal plasma from a bull of contrasting fertility. Treated spermatozoa were stained with different fluorochromes and competed to penetrate oocytes after heterospermic insemination in vitro. Washed spermatozoa exposed to seminal plasma from bulls of high fertility penetrated more oocytes than those spermatozoa mixed with seminal plasma from bulls of low fertility (P < 0.01). Mixing low fertility spermatozoa with high fertility seminal plasma generally improved penetrating ability compared with low fertility spermatozoa mixed with low fertility seminal plasma (P = 0.05). Washed spermatozoa from a bull of low fertility mixed with his own seminal plasma had greater ability to penetrate oocytes than did washed spermatozoa from a bull of high fertility mixed with seminal plasma from a bull of low fertility (P < 0.01). The bias associated with using low fertility seminal plasma from the bull providing the spermatozoa was removed by repeating this experiment using pooled seminal plasma from different subfertile bulls. After combination with pooled seminal plasma from bulls of low fertility spermatozoa from bulls of high or low fertility penetrated oocytes in a similar way, but high fertility spermatozoa had a slightly higher penetration rate (P = 0.12). In conclusion, the penetration of zona-free oocytes by ejaculated spermatozoa from bulls of low fertility was marginally improved by seminal plasma from bulls of high fertility, but penetration by high fertility spermaotoza was decreased by exposure to low fertility seminal plasma. Seminal plasma from bulls of low fertility similarly affected the penetrating ability of high and low fertility spermatozoa if the seminal plasma used was foreign to the spermatozoa being tested.     

Mitochondrial membrane potential and DNA stainability in human sperm cells: a flow cytometry analysis with implications for male infertility

Sperm cells from control donors of proven fertility and men from barren couples were studied by conventional procedures, i.e., light microscopy as well as flow cytometry. Light microscopy analysis of semen included the measurement of spermatozoa concentration, morphology, and motility. All the men from barren couples were asthenozoospermic at the conventional analysis of semen samples. Flow cytometry was applied to study two important parameters of sperm cells: mitochondrial membrane potential (MMP) assessed by the cationic dye JC-1 and DNA stainability with propidium iodide (PI). JC-1 staining was more reliable than the classical procedure used for this purpose, i.e., rhodamine 123 (Rh123) staining, and allowed us to show a positive correlation between MMP and spermatozoa motility. Regarding DNA analysis, a higher relative percentage of immature spermatozoa, showing a high accessibility of DNA to the intercalating PI fluorochrome, was found in men from barren couples compared to donors of proven fertility. The relative percentage of immature spermatozoa was significantly higher in semen from oligoasthenozoospermic subjects. Moreover, a positive correlation was found between immature spermatozoa, as evaluated by PI staining, and cells with depolarized mitochondria, as evaluated by JC-1 staining, suggesting that spermatozoa defective for nuclear maturity could be functionally defective cells. No correlation between immature spermatozoa determined by FCM and immature spermatozoa determined by light microscopy was found, suggesting that these two techniques assess sperm cell maturity at different levels.     

Escherichia coli and Lactobacillus plantarum responses to osmotic stress

Artificial insemination using cryopreserved semen is a common management tool of the contemporary livestock producer. However, cryopreservation is detrimental to sperm function and fertility, killing some 50% of the spermatozoa during the process. Prediction of cryopreservation damage from prefreeze samples remains elusive. Computer-automated sperm head morphometry was used in this study to determine the effects of cryopreservation on bovine sperm head morphometry. Semen was collected from 18 bulls and was divided. One portion was extended to 200 x 10(6) sperm/ml and a microscope slide was prepared, while the remaining portion was cryopreserved in a Triscitrate-yolk extender. After thawing, the cryopreserved samples were prepared on microscope slides. All slides were air dried and were stained with hematoxylin and rose bengal. The morphometric dimensions for length, width, width/length, area, and perimeter for a minimum of 200 sperm heads were analyzed from each slide by computer-aided sperm head morphometry analysis, and the mean measurements were recorded. Bull sperm heads were significantly (P < 0.01) smaller in cryopreserved spermatozoa than in the companion extended samples for length (8.56+/-0.07 vs. 8.63+/-0.08 microm), width (4.39+/-0.05 vs. 4.48+/-0.05 microm), area (28.42+/-0.07 vs. 29.14+/-0.08 microm), and perimeter (23.33+/-0.21 vs. 23.70+/-0.23 microm) for all bulls. Width/length was also different (0.513 vs. 0.519). In addition, differences (P < 0.05) were found within 14 of 18 bulls for at least four of the morphometric parameters. The percent change in measures after cryopreservation were correlated (P < or = 0.05) to the variability of the extended sample. Variations in sperm head measurements were lower (P < or = 0.05) in extended samples of the four bulls in which no changes occurred than in extended samples of the remaining 14 bulls. These data suggest that the variability in sperm head measurements of individual bulls, or ejaculates, may be an indicator of sperm cryosurvivability.     

The relationship between the number of spermatozoa inseminated and the reproductive efficiency of individual dairy bulls

The semen of 20 mature, evaluated bulls was split-sample diluted and contained 2.1 x 10(6) to 17.3 x 10(6) total spermatozoa per 0.25-ml French straw. The number of viable inseminated spermatozoa ranged from 1.1 x 10(6) to 11.8 x 10(6). Each bull had 2430 to 5330 first or second inseminations performed. The nonreturn rate at 56 d after AI was estimated for every dilution. The daily nonreturn rates to 180 d were used to estimate conception and calving rates at a given concentration. The relationship was determined between these estimations and the number of spermatozoa that were actually inseminated. The bulls differed significantly in their maximal nonreturn rate at high sperm numbers per AI and in the rate at which they approached this maximum. There was no correlation between the maximum nonreturn at high sperm numbers and the rate of approach, which implies that the ranking of the bulls for nonreturn rate 56 d after AI changes with the number of spermatozoa inseminated. Multiphasic analysis of reproductive efficiency revealed bull differences in estimated conception and calving rates. The estimated calving rate after conception was 82 to 90% and was independent of the number of spermatozoa that were inseminated. The sperm numbers needed to obtain 95% of the maximal conception rate ranged from 1 x 10(6) to 11 x 10(6).     

Investigation of epididymal sperm maturation in the golden hamster

During passage of hamster spermatozoa through the epididymis their maturation is shown to involve changes in the sperm head, midpiece (mitochondria) and tail. The sum of these changes results in a dramatic increase in the fertilizing potential of the spermatozoa. When comparable numbers of spermatozoa from the caput or corpus epididymis were injected into one uterine horn of mature females, following ovulation induction, and spermatozoa from the cauda epididymis were injected into the contralateral horn, no fertilization was observed with caput epididymal spermatozoa, 1.7% of oocytes were fertilized by corpus epididymal spermatozoa, whereas 79.5% fertilization was obtained with cauda epididymal spermatozoa. Total sperm numbers increased from caput to corpus to cauda [28.3 +/- 12.2, 40.6 +/- 20.8, 144 [corrected] +/- 62 million, respectively]. The percentage of progressively motile spermatozoa increased from 27.9 +/- 6.4 to 33.8 +/- 4.8 to 70 +/- 10.7 during this passage. Viability, measured by exclusion of the dye, propidium iodide, was significantly less in spermatozoa from the cauda than from the proximal or mid-caput epididymis. The percentage of the live cells that were stained intensely by rhodamine-123 (a measure of mitochondrial membrane potential) increased during epididymal passage from 22.8 +/- 7.8% in the proximal caput epididymis to 57.2 +/- 16.5% in the cauda epididymis. Staining with acridine orange (a measure of DNA packaging in the sperm head) indicated an increase in chromatin condensation in cauda epididymal spermatozoa, when compared to those obtained from the caput or corpus.     

Sperm numbers inseminated in dairy cattle and nonreturn rates revisited

Three experiments were conducted to test fertility when sperm numbers per insemination ranged from 10 x 10(6) to 40 x 10(6) total sperm. All semen was from Holstein bulls that were on a regular schedule of semen collection. The semen was extended with heated homogenized whole milk, cooled, glycerolated, and frozen according to standard procedures. Semen was distributed to a large group of inseminators to minimize differential field effects on treatment. All experiments were a randomized block design, including a split plot in Experiment 2. In Experiment 1, data for 31,399 first inseminations distributed among treatments of 20 x 10(6), 25 x 10(6), 30 x 10(6), and 40 x 10(6) total sperm resulted in 69.8, 70.0, 70.1, and 70.1% nonreturns at 59 d, respectively. In Experiment 2, data for 18,197 first inseminations divided over treatments of 12 x 10(6), 16 x 10(6), and 20 x 10(6) total sperm resulted in 70.2, 72.4, and 70.8% nonreturns at 59 d, respectively. In Experiment 3, 38,890 first inseminations distributed over treatments of 10 x 10(6), 13 x 10(6), 16 x 10(6), and 20 x 10(6) total sperm resulted in 70.5, 72.2, 73.1, and 71.5% nonreturns at 59 d, respectively. Bull nonreturns ranged from 64 to 76% in the three trials. These results indicate that, under good conditions, total sperm numbers per straw can be reduced to 10 x 10(6) total sperm with a reduction of nonreturn rates at 59 d, for most bulls, of about 1 percentage unit from the maximum when professional inseminators are use.     

Detection of bovine herpesvirus 1 (BHV-1) genomic sequences in bovine semen inoculated with BHV-1 by polymerase chain reaction

Polymerase chain reaction (PCR) technique was applied to detect BHV-1 in bovine semen inoculated with BHV-1. The technique was found to be 10(6) times more sensitive than a non-isotopic dot-blot hybridization method in detecting viral genomic DNA. Of the three primer pairs used, the one chosen from glycoprotein gC appeared to be most sensitive as it could detect up to 0.01 TCID50 of BHV-1 in the semen. The technique could be useful in screening breeding bulls or samples of frozen semen prior to use in artificial insemination.     

Prevalence of morphologic defects in spermatozoa from beef bulls

OBJECTIVE: To determine the overall prevalence of morphologic defects in spermatozoa from beef bulls and to determine whether prevalence varies with the age of the bull. DESIGN: Cross-sectional observational study. ANIMALS: 2,497 beef bulls that were evaluated for breeding soundness in 1994 by 29 practicing veterinarians in a 5-state geographic region. PROCEDURE: Slides of spermatozoa from each bull were made and submitted by practicing veterinarians for morphologic evaluation. One hundred spermatozoa per slide were examined, and each was classified as having 1 of 9 morphologic defects or as normal. RESULTS: 63% of bulls evaluated were 10 to 12 months old, and 20% were 13 to 18 months old. A mean of 70.6% of spermatozoa was classified as normal. Most common defects were proximal droplets (8.4%), distal midpiece reflexes (6.7%), separated heads (5.5%), and distal droplets (3.8%). Other defects were seen < 2% of the time. Bulls 10 to 12 months of age had a higher prevalence of proximal and distal droplet defects than older bulls. CLINICAL IMPLICATIONS: Practitioners conducting breeding soundness evaluations in beef bulls must be aware of common spermatozoal defects. Bulls that are evaluated at a young age will have more defects than older bulls and should be reevaluated, particularly for those defects for which prevalence decreases with age.     

Selective capacity of glass-wool filtration for the separation of human spermatozoa with condensed chromatin: a possible therapeutic modality for male-factor cases?

PURPOSE: The aim of this study was to evaluate chromatin condensation of human spermatozoa following swim-up compared to glass-wool separation. Semen aliquots from men attending an andrological outpatient clinic were processed by means of a swim-up procedure and glass-wool filtration. Chromatin condensation was recorded using aniline blue staining and results were reported according to color intensity of stained sperm heads. Morphometric measurements of sperm heads were performed on stained sperm samples. RESULTS: Glass-wool filtration resulted (i) in a significantly higher total motile sperm count (P < 0.0005) compared to swim-up and (ii) in a significantly higher percentage of normal chromatin-condensed spermatozoa compared to the ejaculate (P < 0.01). CONCLUSION: In contrast, comparing swim-up to the ejaculate, the percentage of matured nuclei (unstained spermatozoa) retrieved following swim-up was significantly lower (P < 0.005). Glass-wool filtration separates human spermatozoa according to motility and size of the sperm head. The size of the sperm head closely correlated with the chromatin condensation quality.     

Effect of accessory sex gland fluid from bulls of differing fertilities on the ability of cauda epididymal sperm to penetrate zona-free bovine oocytes

The ability of accessory sex gland fluid to affect the fertility of cauda epididymal sperm was evaluated for 10 bulls that ranged in fertility from 6.2% below to 6.0% above the average fertility of bulls at artificial breeding cooperatives. Cauda epididymal sperm collected from indwelling vasa deferentia catheters and cauda epididymal sperm exposed to accessory sex gland fluid from the same bull were compared on the basis of their rates of in vitro penetration of zona-free oocytes after heterospermic insemination. Incubation of cauda epididymal sperm with accessory sex gland fluid significantly enhanced the ability to penetrate oocytes, and bull fertility affected the magnitude of this improvement. For bulls of average and higher fertility, the positive influence of accessory sex gland fluid on penetrating ability of sperm was highly significant (p < 0.0001). Accessory sex gland fluid from bulls of below-average fertility also improved the penetrating ability of cauda epididymal sperm, although not significantly (p = 0.07). Heterospermic competitions compared the penetrating ability of cauda epididymal sperm exposed to homologous accessory sex gland fluid with a portion of the same sperm population incubated in heterologous accessory sex gland fluid from a bull of contrasting fertility. In experiments involving sperm from 12 different bulls, paired in 42 fertile/subfertile combinations, samples of cauda epididymal sperm mixed with accessory sex gland fluid from the higher-fertility bulls had greater oocyte-penetrating ability than when aliquots of that sample were mixed with accessory gland fluid from lower-fertility bulls (p < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)     

The Acridine Orange test: a clinically relevant screening method for sperm quality during infertility investigation?

To determine the clinical usefulness of Acridine Orange (AO) staining of spermatozoa as a screening test for the evaluation of semen quality during basic infertility investigation, semen smears from 103 randomly chosen males of subfertile couples were examined. The median duration of infertility was 4.5 years (range 1-15) and the median age was 33 years (range 21-43). The outcome of AO staining ranged from 5 to 81%, with a median of 24%, green fluorescent spermatozoa. Results were not significantly related to the parameters of semen analysis (sperm count, motility, standard morphology, viability, pH and volume, as well as fructose concentration and number of found cells) or to local sperm antibody testing and semen cultures. Fluorescence after AO staining was also not related to sperm functional capacity (evaluated using sperm-mucus interaction tests in vitro and in vivo), or the medical history of the patient. No significant differences in the AO test outcome were seen in patients with explained and unexplained infertility, or with regard to subsequent fertility [with a median value of 21% (range 5-46) green fluorescence in the fertile group, compared with a median value of 28% (range 9-81) green fluorescence in the other men]. The results of this prospective study indicate that under the usual conditions of conception, the AO test is not clinically useful as a screening procedure to determine semen quality during basic infertility investigation.     

Effect of lysoplatelet-activating factor on human sperm fertilizing ability

OBJECTIVE: To evaluate the penetration rates in the hamster zona-free oocyte sperm penetration assay (SPA) after exposure of spermatozoa to lysoplatelet-activating factor (LPAF) and lysophosphatidyl choline (LPC). DESIGN: Washed human spermatozoa were exposed to 100 microM of LPAF or LPC, followed by the assessment of their fertilizing ability using the SPA. The percentage of penetration, the sperm binding in the SPA, the percentage of motile spermatozoa, and the acrosome reaction rates were quantified. SETTING: Private research and university laboratories. PATIENTS, PARTICIPANTS: Fresh and frozen semen samples from fertile donors with proven fertility were used as well as fresh semen from infertile patients attending a fertility clinic. All the infertile patients had abnormal semen analysis. INTERVENTIONS: Human spermatozoa were incubated for 90 minutes in the presence or absence of LPAF or LPC at 100 microM with 0.3% albumin in Ham's F-10 (GIBCO, Dorval, Quebec, Canada), and their fertilizing ability was evaluated using the SPA. The effect of these lysophospholipids on the percentage of acrosome reaction was evaluated with a fluorescent microscopy technique. RESULTS: The penetration rates of the SPA in male factor increased significantly from 3% +/- 6% with controls to 19% +/- 9% and 34% +/- 22% after incubation with LPC and LPAF, respectively. Sperm-oocyte binding was not significantly increased in this group. Sperm penetration assay penetration rates were also increased in fertile cryopreserved spermatozoa with LPC and LPAF. In this group, the acrosome reaction was significantly increased from 2% +/- 1% in controls to 10% +/- 6% and 8% +/- 3% after incubation with LPC and LPAF, respectively. CONCLUSION: Lysoplatelet-activating factor and LPC independently increased the penetration rate of spermatozoa and the percentage of acrosome reaction. Lysophosphatidylcholine and LPAF may be beneficial in the treatment of spermatozoa with male factor infertility and may increase fertilization rates in IVF.     

The applicability of the flow cytometric sperm chromatin structure assay in epidemiological studies. Asclepios

The impact of demographic, lifestyle, and seminal factors on the sperm chromatin structure assay (SCSA) parameters was evaluated in a population of 277 healthy Danish men. This cohort was established within the framework of a European Concerted Action on occupational hazards to male reproductive capability in order to examine the possible reproductive effects of exposure to styrene or pesticides. The SCSA measures the susceptibility of sperm DNA to in-situ acid-induced denaturation, by multiparameter flow cytometric analysis after staining with the DNA-specific fluorescent dye acridine orange. The green versus red bivariate cytogram patterns were quite variable among donors, showing a wide heterogeneity of sperm DNA denaturability. Nevertheless, in those cases where we had the possibility to measure two semen samples from the same donor, the cytogram pattern remained stable over time (0.64 < r < 0.78). Analysis of variance demonstrated that the SCSA results can be influenced by the age of the donor (P < 0.0001), smoking habits (P < 0.05), the presence of leukocytes and immature germ forms in the ejaculate (P < 0.0001), and the duration of sexual abstinence (P < 0.0001). Furthermore, the relationship between the SCSA data and sperm concentration, morphology, and vitality was weak (-0.22 < r < -0.46). Therefore, the SCSA provides independent and complementary measurements of semen quality and is thus a useful tool for epidemiological studies, but the effects of some confounders should be accounted for in the survey design and analysis.     

Immunoexpression of testis-specific histone 2B in human spermatozoa and testis tissue

During mammalian spermatogenesis, the chromatin of the spermatogenic cells is profoundly reorganized. Somatic histones are partly replaced by testis-specific histones. These histones are then replaced by transition proteins and finally by protamines. This series of nucleoprotein rearrangements results in a highly condensed sperm cell nucleus. In contrast to spermatozoa from other species, human spermatozoa still contain a significant amount of histones, including testis-specific histone 2B (TH2B). In the present study it is shown that an antibody targeting tyrosine hydroxylase, which has been found previously to cross-react with rat TH2B, also specifically immunoreacts with human TH2B on Western blots, in immunohistochemistry of human testis tissue, and in immunocytochemistry of decondensed human spermatozoa. In human testis tissue, TH2B immunostaining first apparent in spermatogonia, shows marked variation, especially at the pachytene spermatocyte stage, and then reaches an intense signal in round spermatids. Shortly before spermatid elongation, a portion of the spermatid nucleus, corresponding to the acrosomal region, loses its immunoreactivity. During condensation of the spermatid nucleus, the immunodetectability of TH2B disappears gradually, from the anterior region of the nucleus onwards. At the final stages of spermiogenesis, the immunostaining is completely absent. Immunocytochemical staining of spermatozoa revealed no TH2B immunosignal, but immunostaining was observed when spermatozoa obtained from semen were decondensed to make nuclear proteins accessible to the antibody. There was, however, a striking intercellular variability in the intensity of staining of spermatozoa within an ejaculate. In a population of 35 men attending our Andrology Clinic, we observed interindividual differences in total sperm TH2B content, which showed a significant, although not very pronounced, negative correlation with normal morphology (P = 0.05).     

"Non-condensed" and "condensed" chromain in transitional cell carcinoma of the human urinary bladder

The area and content of "non-condensed" and "condensed" chromatin in smeared Feulgen-stained malignant urothelial cells were determined by means of scanning-cytophotometry. The results were compared with those from similar measurements of benign human transitional epithelial cells. There was no difference between the relative area and content of "non-condensed" and "condensed" chromatin in cancer nuclei and normal urothelial nuclei as far as nuclei of the same size and ploidy class were considered. Within the same ploidy class the relative area and content of "non-condensed" chromatin increased with increasing nuclear size. As increased nuclear size within the same ploidy class is typical for most cancer cells, cancer specimens therefore contained relatively more "non-condensed" chromatin than normal urothelium. Analogously the relative values of "condensed" chromatin decreased in cancer specimens. Only in high-polyploid cancer cells, which occurred more frequently in undifferentiated tumours, a slight decrease of the relative area and content of "non-condensed" chromatin was observed as compared with well differentiated diploid tumour cells. It was in polyploid tumours that the absolute area and content of "condensed" chromatin was increased as compared with diploid normal urothelium. This means that the changes in "non-condensed" and "condensed" chromatin were primarily dependent on nuclear size and total chromatin content and were not found to be a characteristic of cancer nuclei as compared with control nuclei of the same size and ploidy. These findings differ from the results of biochemical analyses of heterochromatin both in cells during carcinogenesis and also in cancer cells, but are in agreement with qualitative and quantitative morphological studies of smeared cancer nuclei.