The extragenomic action of progesterone on human spermatozoa: evidence for a ubiquitous response that is rapidly down-regulated.

Image analysis techniques have been used to demonstrate that progesterone induces a rapid calcium transient in the acrosomal domain of greater than 90% of human spermatozoa (n = 2354). These results are at variance with previous reports, suggesting that progesterone receptors are only expressed on a small subpopulation of these cells, by virtue of their ability to bind fluorescent probes incorporating progesterone 3- (O-carboxymethyl) oxime conjugated to BSA. In the present study, we could confirm that such probes only bound to a small proportion of human spermatozoa (3.01 +/- 0.29%; n = 7557) although 91.79 +/- 1.8% of the same sperm populations exhibited a calcium transient in response to progesterone. These results indicate that the binding of labeled progesterone conjugates to human spermatozoa does not reflect the size of the progesterone responsive population; the response elicited by this steroid is essentially ubiquitous. Progesterone action was shown to involve an influx of extracellular calcium via mechanisms that did not involve voltage sensitive- or second messenger operated-channels, phospholipase C, or G proteins. Despite previous evidence suggesting that progesterone action might involve a GABAA receptor/chloride channel, neither GABA nor the GABA agonist muscimol had any effect on intracellular calcium concentrations in human spermatozoa or influenced their functional competence. The only factor that disrupted the responses of human spermatozoa to progesterone was this steroid itself. Progesterone exposure induced a prolonged period of refractoriness to further stimulation that influenced the capacity of these cells to generate calcium transients, and their ability to exhibit a biological response to changes in intracellular calcium. There are implications in these results for our understanding of the extragenomic action of progesterone on human spermatozoa and the clinical manipulation of this system for the assessment and suppression of human sperm function.

Superoxide dismutase in human sperm suspensions: relationship with cellular composition, oxidative stress, and sperm function.

Sensitive techniques have been developed for monitoring superoxide dismutase (SOD) activities in human sperm preparations. In contradiction to the protective role normally assigned to SOD, populations of defective spermatozoa recovered from the low density region of Percoll gradients were found to have three times more SOD than functionally competent preparations pelleting in high density Percoll. SOD activity was negatively correlated with the movement characteristics of human spermatozoa and their capacity for oocyte fusion, and positively associated with the induction of peroxidative damage. SOD activity was also highly correlated with other markers of the cytoplasmic space, creatine kinase (CK), and glucose-6-phosphate dehydrogenase (G-6-PDH). We conclude that while SOD may play a physiological role in maintaining a balance between O2.- and H2O2, high levels of this enzyme are associated with impaired sperm function because (a) the human spermatozoon is highly susceptible to the cytotoxic effects of H2O2, (b) O2.- is an important mediator of normal sperm function, and (c) high SOD activities reflect errors in spermatogenesis associated with germ cell exfoliation and the retention of excess residual cytoplasm by the spermatozoa.

The extragenomic action of progesterone on human spermatozoa is influenced by redox regulated changes in tyrosine phosphorylation during capacitation.

Capacitation had no effect on the ability of progesterone to elicit a rapid calcium transient in the acrosomal domain of human spermatozoa but had a marked influence of the ability of this steroid to induce a biological response. The development of this responsiveness to progesterone appeared to be redox regulated in that it was promoted by the stimulation of reactive oxygen species generation and inhibited by the presence of antioxidants, including catalase and membrane permeant thiols. The ability of redox conditions to influence the biological responsiveness of human spermatozoa did not involve changes in the dynamics of the calcium transients induced by progesterone but was causally linked with clear differences in tyrosine phosphorylation. We conclude that the ability of human spermatozoa to respond to the calcium transients induced by progesterone depends on a background of phosphotyrosine expression that can be profoundly influenced by the redox status of the spermatozoa during capacitation.

Analysis of the responses of human spermatozoa to A23187 employing a novel technique for assessing the acrosome reaction.

Protocols for the use of A23187 in assessing the ability of human spermatozoa to acrosome-react and exhibit sperm-oocyte fusion have been developed and the results compared in two independent cohorts of infertile patients. Both bioassays were found to depend upon such factors as the dose and formulation of A23187, the duration of exposure, and the amount and type of protein used to supplement the medium. An optimal protocol for the hamster oocyte penetration test comprised a 3-hour exposure to 1.25-2.5 microM ionophore and gave penetration rates of 93.2 +/- 3.2% (11.26 +/- 1.27 sperm/egg) for a group of 33 fertile donors compared with 63.0 +/- 5.4% (4.73 +/- 0.81 sperm/egg) for a cohort of 56 patients consulting for infertility (P < 0.001). Higher doses (5.0-10.0 microM) of A23187 caused an inhibition of sperm-oocyte fusion in association with a loss of motility, although the integrity of sperm plasma membrane did not appear to be compromised and high rates (approximately 80%) of acrosome reaction were observed. A protocol for assessing the ability of viable human spermatozoa to acrosome-react in response to A23187 was developed, employing a fluorescein-conjugated lectin in concert with the hypoosmotic swelling test, which gave values of 20.1 +/- 2.6% and 13.6 +/- 1.6% for groups of fertile donors (n = 29) and infertile patients (n = 32) respectively (P < 0.05). Although only acrosome-reacted spermatozoa were capable of fusing with zona-free hamster oocytes, there was no significant correlation between the proportion of acrosome-reacted cells and the levels of sperm-oocyte fusion observed in two independent groups of patients, indicating that these bioassays are measuring different aspects of human sperm function. These results have implications for the way in which the responses of human spermatozoa to ionophore treatment are quantified and interpreted.

Development of an image analysis system to monitor the retention of residual cytoplasm by human spermatozoa: correlation with biochemical markers of the cytoplasmic space, oxidative stress, and sperm function.

A method has been developed for quantifying the residual cytoplasm present in the midpiece of human spermatozoa, based upon the imaging of NADH oxidoreductase activity. This procedure used NADH and nitroblue tetrazolium as electron donor and acceptor, respectively, and resulted in the discrete staining of the entire midpiece area, including the residual cytoplasm. Image analysis techniques were then used to generate binary images of the midpiece, from which objective measurements of this cellular domain could be undertaken. Such data were found to be highly correlated with biochemical markers of the cytoplasmic space, such as creatine kinase (CK) and glucose-6-phosphate dehydrogenase (G-6-PDH), in sperm populations depleted of detectable leukocyte contamination. Morphometric analysis of the sperm midpiece was also found to reflect semen quality in that it predicted the proportion of the ejaculate that would be recovered from the high-density region of Percoll gradients and was negatively correlated with the movement and morphology of the spermatozoa in semen. Variation in the retention of excess residual cytoplasm was also associated with differences in the functional competence of washed sperm preparations, both within and between ejaculates. Thus, within-ejaculate comparisons of high- and low-density sperm subpopulations revealed a relative disruption of sperm function in the low-density fraction. This disruption was associated with the presence of excess residual cytoplasm in the midpiece, high concentrations of cytoplasmic enzymes, and the enhanced-generation reactive oxygen species (ROS). Functional differences between individual high-density Percoll preparations were also negatively correlated with the area of the midpiece and the corresponding capacity of the spermatozoa to generate ROS. These findings suggest that one of the factors involved in the etiology of defective sperm function is the incomplete extrusion of germ cell cytoplasm during spermiogenesis as a consequence of which the spermatozoa experience a loss of function associated with the induction of oxidative stress.

Reactive oxygen species and human spermatozoa: analysis of the cellular mechanisms involved in luminol- and lucigenin-dependent chemiluminescence.

We have shown that human spermatozoa generate and release reactive oxygen species that can be detected by chemiluminescence techniques. Analysis of the cellular mechanisms responsible for this activity suggests that the probe, luminol, undergoes an intracellular dioxygenation reaction mediated by hydrogen peroxide and a sperm peroxidase located within the acrosome. Support for this model included the following observations: (1) the luminol-dependent signal could be suppressed with peroxidase inhibitors, phenylhydrazine and sodium azide; (2) this suppression could be reversed by the addition of an azide-insensitive peroxidase, horse radish peroxidase (HRP); (3) inhibition of intracellular superoxide dismutase (SOD) with potassium cyanide (KCN) suppressed the luminol signal; (4) peroxidase activity could be detected in purified populations of human spermatozoa with 3,3',5,5' tetramethylbenzidine (TMB); (5) this peroxidase was active at the pH prevailing within the acrosomal vesicle; and (6) peroxidase activity and luminol-dependent chemiluminescence were minimal in spermatozoa exhibiting a congenital absence of acrosomes. Human spermatozoa could also generate lucigenin-dependent chemiluminescent signals that could neither be suppressed with peroxidase inhibitors nor enhanced by the addition of peroxidase. However, these signals could be enhanced by suppression of intracellular SOD with KCN or inhibited by exogenous SOD, suggesting that lucigenin was responding to superoxide anion released into the extracellular space. The ability of chemiluminescent techniques to detect and discriminate the production of superoxide and hydrogen peroxide by spermatozoa should facilitate the further analysis of reactive oxygen species as mediators of normal and abnormal human sperm function.

Analysis of lipid peroxidation mechanisms in human spermatozoa.

The mechanisms by which ferrous ion promoters induce malondialdehyde generation by human spermatozoa have been investigated in order to provide a rational basis for the quantification and interpretation of lipid peroxidation assays. Incubation of human spermatozoa with a ferrous ion promoter in the presence of thiobarbituric acid (TBA) led to the generation of the bone fide malondialdehyde-TBA adduct. The importance of iron in the stimulation of lipid peroxidation was emphasized by the ability of Desferal and EDTA to suppress malondialdehyde generation. Paradoxically, when the concentration of EDTA relative to iron was equimolar or greater, the suppression of malondialdehyde formation was accompanied by the generation of hydroxyl radicals. These results suggested that the addition of promoter did not effect the first-chain initiation of lipid peroxidation but favored an alternative mechanism involving the catalytic decomposition of pre-existing lipid peroxides. This conclusion was reinforced by the inability of reagents that would limit the formation (superoxide dismutase and/or catalase) or availability (mannitol, formate) of hydroxyl radicals, to influence malondialdehyde generation. While hydroxyl radicals were not directly involved in Fe(2+)-promoted malondialdehyde generation, the existence of significant correlations between reactive oxygen species production and the outcome of the TBA assay, suggested that Fenton chemistry might be important in the initiation of peroxidative damage. It is proposed that the impeded propagation of peroxidation initiated by Fenton or Haber Weiss reactions would lead to the accumulation of lipid peroxides in the spermatozoa and it is these peroxides that are induced to decompose during the Fe(2+)-promoted TBA assay, stimulating a lipoperoxidative chain reaction and malondialdehyde formation.

Redox regulation of tyrosine phosphorylation in human spermatozoa and its role in the control of human sperm function.

The redox status of human spermatozoa was found to have a profound influence on the fertilizing potential of these cells in association with qualitative and quantitative changes in the patterns of tyrosine phosphorylation. In general, oxidizing conditions enhanced tyrosine phosphorylation and stimulated sperm function, whereas reducing conditions had the opposite effect. Unstimulated human spermatozoa exhibited low levels of spontaneous acrosomal exocytosis and sperm-oocyte fusion and minimal reactive oxygen species generation, while phosphotyrosine expression was largely confined to a single protein of 116 kDa. However, if the spermatozoa were exposed to oxidizing conditions through the addition of exogenous H2O2, or the stimulation of endogenous NADPH-dependent reactive oxygen species generation, then a dramatic increase in tyrosine phosphorylation was observed (major phosphotyrosyl bands at 222 kDa, 200 kDa, 159 kDa, 133 kDa, 116 kDa and 82 kDa) in concert with the functional activation of the spermatozoa. A causal association between reactive oxygen species generation, tyrosine phosphorylation and sperm function was indicated by studies with the ionophore, A23187, which induced high rates of spermoocyte fusion together with enhanced rates of reactive oxygen species production and the increased expression of phosphotyrosyl proteins. This functional response to A23187 could be abrogated, without any concomitant change in sperm motility or viability, by using membrane permeant thiols or catalase to suppress the reactive oxygen species-induced increase in phosphotyrosine expression. The fact that the biological responses of human spermatozoa to biological agonists (recombinant human ZP3 and progesterone) could also be inhibited by catalase indicated the general relevance of these findings.(ABSTRACT TRUNCATED AT 250 WORDS)

On the use of paramagnetic beads and ferrofluids to assess and eliminate the leukocytic contribution to oxygen radical generation by human sperm suspensions.

PROBLEM: To develop a methodology to determine a) the leukocytic contribution to reactive oxygen species generation by human sperm suspensions and b) the therapeutic value of removing these cellular contaminants. METHODS: Leukocytes were removed with paramagnetic beads or colloidal ferrofluids coated with anti-CD45 antibody. The sperm suspensions were monitored for oxidant generation by chemiluminescence, leukocyte contamination by immunocytochemistry, and fertilizing potential using zona-free hamster oocytes. RESULTS: Percoll -prepared human sperm suspensions exhibited a competence for PMA-induced reactive oxygen species generation which was significantly correlated with leukocyte contamination. However, the purified spermatozoa remaining after paramagnetic bead treatment, also demonstrated an intrinsic capacity for PMA-responsive reactive oxygen species generation and, freed from the oxidative stress created by the leukocytes, exhibited a significantly enhanced capacity for sperm-oocyte fusion. CONCLUSIONS: Although human spermatozoa can generate reactive oxygen species, sperm function is inhibited by the additional oxidative stress created by contaminating leukocytes. Removal of these cells with paramagnetic beads enhances fertilizing potential.

Analysis of sperm movement in relation to the oxidative stress created by leukocytes in washed sperm preparations and seminal plasma.

The addition of luminol to unprocessed semen samples resulted in the generation of chemiluminescent signals, the intensity of which was highly correlated with the level of leukocyte contamination. Despite the spontaneous oxidant-generating capacity of seminal leukocytes, no correlations were observed between leukocyte contamination and the fertility status of the subjects or any aspect of the semen profile, including the motility of the spermatozoa or their performance in a hyaluronate penetration assay. Luminol-dependent chemiluminescence and leukocyte contamination were also correlated in washed sperm suspensions prepared either by repeated centrifugation or on discontinuous Percoll gradients. However, in such sperm suspensions, the spontaneous generation of oxidants by contaminating leukocytes (> 2 x 10(4) leukocytes/ml) was invariably associated with a decreased capacity for movement. Moreover, causative associations between leukocyte contamination, reactive oxygen species generation, lipid peroxidation and impaired sperm motility were revealed by experiments involving the selective addition or removal of activated leukocytes. From these observations we can conclude that low concentrations of leukocytes are a common feature of the human ejaculate and can impair sperm function, particularly in the absence of seminal plasma. These findings have implications for our understanding of the importance of leukocytospermia in defining the fertility of human spermatozoa in vivo and in vitro.