Functional and ultrastructural features of DNA-fragmented human sperm.

The functional significance of deoxyribonucleic acid (DNA) fragmentation in ejaculated human sperm is unclear. In this study the extent of DNA strand breakage in swim-up selected spermatozoa was evaluated by terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL)-coupled flow cytometry and correlated with several functional and morphological sperm parameters. The extent of DNA fragmentation (mean = 11.07%+/-8.00%, range = 0.79%-42.64%, n = 140) was positively related to abnormal morphology and associated with defects of the sperm tail. A negative correlation was found between DNA breakage and progressive motility. When a stepwise multiple linear regression model was used to analyze the relationship between DNA fragmentation and the aforementioned parameters, only motility results were included in the model. The presence of spermatozoa showing submicroscopic characteristics resembling those of somatic apoptosis has been reported in human ejaculate. To verify whether sperm DNA fragmentation was associated with the presence of such apoptotic-like cells, we performed electron microscopy and TUNEL-coupled flow cytometry in a limited number of sperm samples (n = 24). Although we did not observe any significant relationship between DNA breakage and the characteristics that are suggestive of apoptosis, an association was found with several ultrastructural features, indicating an impaired motility. Hence, we conclude that in ejaculated sperm, DNA fragmentation does not correspond to the apoptosis-like phenomenon and that it is associated with defects of motility.

Uteroglobin and transglutaminase modulate human sperm functions.

During the process of capacitation, spermatozoa undergo significant changes in membrane composition, including removal of decapacitating factors (DFs), which are present in seminal plasma, that lead to increased sensitivity to physiological stimuli of the acrosome reaction. In the present study we investigated the presence, localization, and effects on human spermatozoa of 2 proteins of seminal plasma origin, uteroglobin (UG) and transglutaminase (TG). These 2 proteins interact with one another because TG promotes covalent links of UG to sperm surface proteins. We found that UG is localized around the entire surface of ejaculated human sperm, whereas TG is predominantly localized in the neck. FACScan analysis confirmed the surface localization of both antigens and demonstrated that swim-up selection of spermatozoa was associated with a significant reduction in the contents of the 2 substances when compared with unselected samples. Western blot analysis of UG in total sperm lysates confirmed the lower content of the protein in swim-up-selected sperm. Swim-up-selected sperm were characterized by their ability to undergo a spontaneous, time-dependent increase of capacitation-characteristic chlortetracycline pattern of fluorescence and increase in responsiveness to progesterone. Such changes were not observed in unselected sperm. Exogenous addition of TG, together with recombinant rabbit UG, prevented the spontaneous increase in responsiveness to progesterone (acrosome reaction and intracellular calcium) at 24 hours in swim-up-selected sperm, suggesting the occurrence of a capacitation-inhibiting activity of the 2 substances. In addition, we found that endogenous UG and TG contents, as determined by FACScan analysis, were negatively correlated (P < .0001) with sperm motility and that exogenous addition of the 2 substances resulted in a substantial reduction of progressive motility (P < .01). Collectively, these data indicate that TG and UG represent 2 DFs, and contribute to understanding the biochemical mechanisms that characterize the process of capacitation.

Progesterone-stimulated intracellular calcium increase in human spermatozoa is protein kinase C-independent.

Indirect studies suggested that protein kinase C (PKC) has a role in sperm motility and the acrosome reaction. Physiological inducers of the sperm acrosome reaction include progesterone, which can increase intracellular calcium ([Ca2+]i), tyrosine phosphorylation of proteins and chloride efflux in human spermatozoa. PKC may be involved in progesterone-stimulated acrosome reaction, although controversial results have been obtained concerning the effect of PKC inhibition on progesterone-stimulated [Ca2+]i increase. In the present study, we investigated the direct effect of progesterone on the activity of PKC, as well as the effect of a panel of PKC inhibitors on progesterone-stimulated [Ca2+]i increase and tyrosine phosphorylation of proteins. We found that progesterone stimulates sperm PKC activity and that PKC inhibition with staurosporine and bisindolylmaleimide partially reversed the effect of progesterone on acrosome reaction, indicating an involvement of the enzyme in the effect of the steroid. We next evaluated the effect of three different PKC inhibitors (sangivamycin, staurosporine and bisindolylmaleimide) on progesterone-stimulated [Ca2+]i increase. Neither short-term (15 min) nor long-term (90 min) preincubation with any of the three compounds had a substantial effect on the stimulatory effect of progesterone on sperm [Ca2+]i. Nor was responsiveness to progesterone affected by either short-term (determining activation of PKC) or long-term (determining down-regulation of PKC) incubation with the tumour promoter phorbol myristate acetate (PMA), a known non-physiological stimulator of PKC. These results indicate that progesterone-stimulated calcium influx is independent of PKC activation. In addition, we found that preincubation with PKC inhibitors had a stimulatory effect per se on tyrosine phosphorylation of sperm proteins. When compared with the appropriate control, the effect of progesterone on tyrosine phosphorylation was slightly (but not significantly) reduced by the inhibitors, sangivamycin, staurosporine and bisindolylmaleimide, but was significantly inhibited by calphostin C. These results do not permit a final conclusion on the involvement of PKC in progesterone-stimulated tyrosine phosphorylation of sperm proteins. However, the lack of effect of PMA on tyrosine phosphorylation indicates that PKC stimulation is not sufficient to induce this effect. In conclusion, our results indicate that PKC plays a role in progesterone-induced acrosome reaction and that progesterone-stimulated PKC activation is downstream to stimulation of calcium influx by the steroid.

Identification and characterization of functional nongenomic progesterone receptors on human sperm membrane.

The presence of functional nongenomic progesterone (P) receptors in human spermatozoa has been investigated by equilibrium binding studies in intact spermatozoa, ligand blot and Western blot analysis of sperm lysates, as well as determination of the effects of the steroid on sperm intracellular Ca2+ concentrations. Binding experiments were performed using progesterone-11alpha-glucuronide-[125I]iodotyramine as tracer. Computer analysis of competition curves using different steroids as competitors indicated the presence of two distinct binding sites for P. The high affinity site (Kd in the nanomolar range) appears to be specific for P, whereas the low affinity one (Kd in the micromolar range) binds with equal affinity 11beta-hydroxyprogesterone (11betaOHP) and 17alpha-hydroxyprogesterone (17alphaOHP). A significant correlation exists among affinity constants (as determined by binding studies) and EC50 values for the effects of P, 11betaOHP, and 17alphaOHP on intracellular Ca2+ in fura-2-loaded spermatozoa, strongly indicating the involvement of P-binding sites in the biological effect of the steroid. In particular, dose-response curves for P were biphasic, with an EC50 in the nanomolar range and another in the micromolar range. Conversely, curves for 11betaOHP and 17alphaOHP were monophasic, with an EC50 just in the micromolar range. Ligand blot analysis of sperm total lysates performed with peroxidase-conjugated P revealed the presence of two binding proteins of 54 and 57 kDa that were specific for P. Indeed, peroxidase-conjugated P binding was blocked by the simultaneous presence of the unconjugated steroid. Using alpha c262 antibody, which is directed against the P-binding domain of genomic receptor, we detected two proteins of similar molecular mass (54 and 57 kDa), whereas using antibodies directed against the DNA-binding and N-terminal domains of the genomic P receptors, the two proteins were not detected. In addition, p54 and p57 appear to be mostly localized in sperm membranes and virtually absent in the cytoplasm. The involvement of these proteins in the biological effects of P is indicated by the strong inhibitory effect of alpha c262 on P-induced acrosome reaction of capacitated human spermatozoa.