In these studies, we aimed to characterize the effects of the physiological, homologous agonists of the acrosome reaction, i.e. the zona pellucida (ZP) and progesterone/follicular fluid, on human sperm. The specific aims of our studies were: (i) to examine the dependency of the solubilized ZP-induced acrosome reaction on G(i) protein activation and presence of extracellular calcium; and (ii) to determine whether progesterone/follicular fluid exert a priming or synergist effect on the solubilized ZP-induced acrosome reaction. Highly motile sperm from fertile donors were exposed to the agonists in a microassay and the acrosomal status of live sperm was determined by indirect immunofluorescence using PSA-FITC/Hoechst double-staining. Pretreatment with pertussis-toxin (100 ng/ml) and EGTA (2.5 mmol/l) significantly inhibited the ZP-induced acrosome reaction without affecting the spontaneous rate of exocytosis. Progesterone (1.25 microg/ml) and human follicular fluid (10%) exerted a priming, time-dependent effect on the ZP-induced acrosome reaction. These studies demonstrated that: (i) acrosomal exocytosis of capacitated human sperm triggered by the homologous ZP is dependent on the activation of G(i) proteins (pertussis toxin-sensitive) and the presence of extracellular calcium; and (ii) progesterone and follicular fluid exert a priming effect on the ZP-induced acrosome reaction.

Considering that the final protection of the DNA against major assaults in terms of chromatin condensation is finalized in the epididymis, it is not known how sperm production of reactive oxygen species (ROS) and inflammatory processes can contribute to protamine deficiency that is predetermined in the testes. Therefore, this study aimed at investigating relationships between poor chromatin condensation, morphology, ROS production, DNA damage and the impact of the presence of leucocytes. In 70 patients, sperm DNA status was determined using TUNEL and chromomycin A(3) (CMA(3)) assays, and ROS-production by means of dihydroethidine. Morphology was evaluated according to strict criteria. The percentage of CMA(3)-positive spermatozoa and leucocyte concentration (r = 0.178, P = 0.0377) as well as percentage of ROS-positive spermatozoa (r = 0.3010; P = 0.012) correlated significantly. Particularly, patients with leucocyte counts >0.5 x 10(6) ml(-1) exhibited higher CMA(3) positivity. No association was found between CMA(3) positivity, TUNEL positivity and sperm morphology. While P- (poor prognosis: 0-4% normal morphology) and G-pattern (good prognosis: 5-14% normal morphology) morphology did not differ regarding chromatin condensation, P-pattern patients had a significantly higher percentage of DNA fragmentation (P = 0.0323). As oxidative stress is associated with disturbed chromatin condensation, results suggest that the idea that under-protamination of sperm DNA will automatically lead to DNA fragmentation might have to be revisited.

No abstract

Since the onset of intracytoplasmic sperm injection, researchers have intensified the search for the ideal spermatozoa to be used for injection. The aim of this study was to record the functional role of cumulus cell interaction with human spermatozoa as far as capacitation, acrosome reaction, morphology, zona binding and chromatin packaging quality are concerned. Using a previously described cumulus oophorus model, we recorded specific sperm functional aspects of sperm populations that transverse a cumulus cells mass. Control spermatozoa were kept under similar experimental conditions in the culture media only. Results indicated cumulus cells to be beneficial to spermatozoa as far as functional and capacitational events are concerned. The mean percentage of morphologically normal spermatozoa in the control sample was 6.9%, while the spermatozoa that traversed the cumulus oophorus (test) had a significantly higher percentage of normal forms (mean 9.5%; P < or = 0.01). We observed a decline in the percentage of CMA3-positive spermatozoa when we compared the control population (49.1%) to the test, i.e. 38.4%, (P = <0.05), thus implying that the spermatozoa with good chromatin condensation increased during cumulus penetration. Significantly more (P < or = 0.01) acrosome-reacted spermatozoa were found in the penetrated spermatozoa (mean 23%) than in the control spermatozoa (mean 11%). The test spermatozoa had a higher zona binding capacity with significantly more (P < or = 0.01) tightly bound spermatozoa on the hemizona (61 +/- 15) than the control spermatozoa (47 +/- 18). In the absence of sophisticated and expensive sperm selection products, the use of a cumulus model to select spermatozoa for intracellular sperm injection seems to be an alternative method.