Serum albumin and HCO3- regulate separate pools of ATP in human spermatozoa.

STUDY QUESTION: Do the known capacitating agents HCO(3)(-) and serum albumin regulate the generation of ATP required for sperm motility and capacitation? SUMMARY ANSWER: Serum albumin and HCO(3)(-) seem to regulate two separate pools of ATP by different mechanisms in human spermatozoa. WHAT IS KNOWN ALREADY: Sperm capacitation is a maturation process that naturally occurs in the female reproductive tract preparing the sperm cell for fertilization. It is a highly energy-depending process as it involves hyperactive motility and substantial levels of protein phosphorylation. STUDY DESIGN, SIZE, DURATION: Human sperm cells from four (motility experiments) and three (all other experiments) healthy donors were used. Untreated cells were compared with cells treated with HCO(3)(-) and serum albumin for up to 4 h. PARTICIPANTS/MATERIALS, SETTING, METHODS: Changes in glycolysis and mitochondrial respiration rates upon treatment with serum albumin and HCO(3)(-) were analysed by metabolic tracing of (13)C-labelled substrates and respirometry studies, respectively. Levels of hyperactive spermatozoa and ATP content were measured during 4 h of incubation under capacitating conditions. MAIN RESULTS AND THE ROLE OF CHANCE: We found that HCO(3)(-) significantly (P < 0.05) increased glycolytic flux by >3-folds via a cAMP/PKA sensitive pathway. This was accompanied by an increase in hyperactive motility. In contrast, serum albumin significantly increased endogenous ATP levels by 50% without stimulating hyperactive motility or glycolysis, indicating that this pool of ATP is separately located from the HCO(3)(-)-induced ATP. The increase in ATP induced by albumin could be mimicked by treatment with the cholesterol acceptors 2-hydroxypropyl- and methyl-ß-cyclodextrin and counteracted by co-incubation with cholesterol sulphate to the level of the non-treated control (P < 0.05), pointing to cholesterol extraction from the sperm cell membrane as the main mechanism. However, the concentration of cyclodextrins needed to directly detect cholesterol extraction from the sperm cells was not compatible with maintenance of sperm viability. The increase in ATP seemed not to be dependent on the sperm-specific Ca(2+) channel CatSper. Finally, we demonstrated that neither HCO(3)(-) nor serum albumin stimulated mitochondrial respiration rates. However, serum albumin increased the respiratory capacity of mitochondria by >50%, an effect that was counteracted by HCO(3)(-). LIMITATIONS, REASONS FOR CAUTION: Great variation in motility and capacitation is observed between sperm cells from different species. Hence, caution should be taken when extrapolating the findings in this work on human spermatozoa to sperm from other species. WIDER IMPLICATIONS OF THE FINDINGS: It is already established that an efficient energy-generation is required to support sperm motility and capacitation. However, the mechanisms explaining how ATP production is regulated in spermatozoa are not fully understood. Our findings indicate that HCO(3)(-) stimulates hyperactive motility by increasing glycolytic flux and ATP production in a cAMP/PKA sensitive fashion. On the other hand, serum albumin seems to increase ATP concentration at a different location and by a mechanism different from glycolysis that involves extraction of cholesterol from the sperm cell membrane. These new insights into sperm metabolism may pave the way for both the development of new and improved male contraceptives and optimized assisted reproduction techniques. STUDY FUNDING: The work was funded by Spermatech AS, The University of Oslo and the Research Council of Norway. COMPETING INTEREST(S): T.H.H. and K.R.R. are employees at Spermatech. B.S.S is a shareholder in Spermatech.

Exogenous pyruvate accelerates glycolysis and promotes capacitation in human spermatozoa.

BACKGROUND: There has been an ongoing debate in the reproductive field about whether mammalian spermatozoa rely on glycolysis, oxidative phosphorylation or both for their energy production. Recent studies have proposed that human spermatozoa depend mainly on glucose for motility and fertilization but the mechanism behind an efficient glycolysis in human spermatozoa is not well understood. Here, we demonstrate how human spermatozoa utilize exogenous pyruvate to enhance glycolytic ATP production, motility, hyperactivation and capacitation, events that are crucial for male fertility. METHODS: Purified human spermatozoa from healthy donors were incubated under capacitating conditions (including albumin, bicarbonate and glucose) and tested for changes in ATP levels, motility, hyperactivation and tyrosine phosphorylation after treatment with pyruvate. The experiments were repeated in the presence of sodium cyanide in order to assess the contribution from mitochondrial respiration. The metabolism of (13)C labeled glucose and pyruvate was traced by a combination of liquid chromatography and mass spectrometry. RESULTS: The treatment of human spermatozoa with exogenous pyruvate increased intracellular ATP levels, progressive motility and hyperactivation by 56, 21 and 130%, respectively. In addition, added pyruvate induced a significant increase in tyrosine phosphorylation levels. Blocking of the electron transport chain did not markedly affect the results, indicating that the mechanism is independent of oxidative phosphorylation. However, the observed effects could be counteracted by oxamate, an inhibitor of lactate dehydrogenase (LDH). Metabolic tracing experiments revealed that the observed rise in ATP concentration resulted from an enhanced glycolytic flux, which was increased by more than 50% in the presence of exogenous pyruvate. Moreover, all consumed (13)C labeled pyruvate added was converted to lactate rather than oxidized in the tricarboxylic acid cycle. CONCLUSIONS: Human spermatozoa seem to rely mainly, if not entirely, on glycolysis as the source of ATP fueling the energy-demanding processes of motility and capacitation. The efficient glycolysis is dependent on exogenous pyruvate, which indirectly feeds the accelerated glycolysis with NAD(+) through the LDH-mediated conversion of pyruvate to lactate. Pyruvate is present in the human female reproductive tract at concentrations in accordance with our results. As seen in other mammals, the motility and fertility of human spermatozoa seem to be dictated by the available energy substrates present in the conspecific female.

A subdivision of strains of Staphylococcus aureus in the 94,96 complex by means of experimental phages.

In order to facilitate epidemiological investigations a subdivision of Staphylococcus aureus strains belonging to the 94,96 complex by means of two experimental phages, 16 and 47A, was performed. These phages were selected from the nine experimental phages initially examined because they gave the greatest discrimination. On the basis of reactions with these two phages, 2199 isolates which reacted with phages 94 and 96, and 773 isolates which reacted with phage 96 alone, were each subdivided into two major and two minor groups. Strains with different phage patterns were in a few cases (2/64) isolated from the same deep body site in a patient, and lysogenisation experiments suggested that differences in phage patterns were determined by the presence of prophages. Strains with the phage patterns 94/96 and 96 were found to be unevenly distributed throughout Denmark. This regional distribution suggested that particular strains might predominate in some areas. The extended phage patterns with the experimental phages did not give any retrospectively useful epidemiological information. It is proposed that in future phages 16 and 47A be used for specific investigations into the sources and relatedness of strains involved in small incidents.

Resistance to cadmium, arsenate and mercury among Danish strains of Staphylococcus aureus isolated from cases of bacteriaemia, 1957-74.

A collection of 3040 epidemiologically-unrelated strains of Staphylococcus aureus isolated from blood cultures during the period 1957-74 was examined for their resistance to cadmium (Cd), arsenate (As) and mercury (Hg); 98% of the strains had metal-resistance patterns of (a) sensitive to all these metals, (b) resistant to Cd, (c) resistant to Cd,As or (d) resistant to Cd,As,Hg. The percentage of Cd,As,Hg-resistant strains fell from 50 to 13 in the period of investigation and the percentage of Cd-resistant strains increased from 11 to 32. Strains resistant only to Cd were mainly P resistant only and a correlation was found between multiple antibiotic resistance and Cd,As,Hg resistance. Strains of phage types 80/81, 52/52A/80/81 and 52/52A/80 were never Cd resistant only, but mainly Cd,As,Hg resistant. Strains of the 94, 96 complex were mainly Cd resistant and were never resistant to Cd,As,Hg.

Unusual properties of Staphylococcus aureus strains of the new epidemic phage type 95.

Three hundred and seventy-one penicillin-resistant Staphylococcus aureus strains of phage type 95 isolated in the years 1977-1983 were investigated. They had characteristic resistance patterns to cadmium (Cd), arsenate (As) and mercury (Hg). Most were susceptible to all three heavy metals; 25 strains were resistant to As only and one to Cd and As. The susceptible strains had a uniform medium level of penicillinase production, whereas the As-resistant strains produced large amounts of penicillinase. In most of the strains, penicillin resistance was located on a very unstable penicillinase plasmid. The combination of rare properties found in Danish type-95 strains seems to point to the spread of one or two clones. Co-reactions with other phage groups or complexes and results of lysogenisation experiments suggest that the Danish type-95 strains are derived from strains of the 52, 52A, 80, 81 complex.

Correlation of penicillinase production with phage type and susceptibility to antibiotics and heavy metals in Staphylococcus aureus.

One hundred and thirty-nine bacteraemia strains of Staphylococcus aureus, representing different combinations of phage type and susceptibility to antibiotics and to cadmium (Cd), arsenate (As) and mercury (Hg), were investigated for penicillinase production. The determination of enzyme activity in induced and uninduced conditions was performed by iodometric titration. The amount of penicillinase produced could be correlated with phage pattern. Epidemically occurring strains of the 94,96 and the 83A complexes produced the largest amount of penicillinase, whereas strains of the 52,52A,80,81 complex were weaker producers. Group-II and group-III strains produced the smallest amount. Susceptibility to antibiotics and to Cd, As and Hg could not be correlated with enzyme activity, but strains resistant to penicillin plus tetracyclines and strains resistant only to Cd did produce less enzyme than strains with other resistance patterns. The percentage mean values than strains with other resistance patterns. The percentage mean values of extracellularity of the enzyme was highest amongst strains of the 94,96 complex and of type 95. Four strains had constitutive production, one being macro-constitutive and three micro-constitutive. All four strains represented rare combinations of the above properties but were susceptible to fusidic acid. The importance of penicillinase production by epidemically occurring strains is discussed.

Epidemiology of Pseudomonas aeruginosa infection and the role of contamination of the environment in a cystic fibrosis clinic.

In order to identify the possible reservoirs and routes of cross-infection with Pseudomonas aeruginosa, samples from patients, staff, and the environment of a cystic fibrosis centre and two control wards at an infectious disease clinic were collected during a two-week period in 1980. All the Ps. aeruginosa strains were phage and serotyped. Ps. aeruginosa was isolated from 90 (51%) of the cystic fibrosis patients and most belonged to the 0-3/9 complex, characteristic of strains from patients in the centre. Some of the patients were able to spread Ps. aeruginosa into the air and to their hands by coughing, and Ps. aeruginosa in dried sputum could survive for at least one week. Strains of the same epidemiological types as found in the cystic fibrosis patients were isolated from sinks, soap, baths, toys, tables, brushes, cloths, and air in the clinic. In contrast, Ps. aeruginosa of the same epidemiological types were only found in a few of the sinks in one of the control wards where a few cystic fibrosis patients were regularly treated in isolation cubicles. The precautions employed to prevent future cross-infection include segregation of Ps. aeruginosa-infected from non-infected patients in separate wards and arranging for visits on separate days in the out-patients clinic. The survival of cystic fibrosis patients treated in the centre is much longer than those treated outside the centre despite the problems of cross-infection.

Epidemiology of Pseudomonas aeruginosa infection in patients treated at a cystic fibrosis centre.

484 isolates of P. aeruginosa were obtained from the respiratory tract of 45 out of 70 cystic fibrosis (CF) patients of monthly examinations during one year at the Danish CF Centre. All isolates were serogrouped (O-antigens) and phage typed, and in this way 99 per cent of the isolates could be grouped and/or typed. The isolates from CF patients belonged to many different sero-groups, but 55 per cent were polyagglutinable, and most of these belonged to the 0-3/9 complex. This was significantly different from the distribution into O-groups of isolates obtained from non-CF patients. The results of the combined phage typing and sero-grouping of the isolates revealed the occurrence of 13 clusters of distinct epidemiological types of P. aeruginosa among small groups (2-10 individuals) of CF patients. The predominating endemic strain, which was isolated from 10 (22 per cent) of the patients, belonged to the 0-3/9 complex and was lysed by phage 109, either alone or in combination with a few other phages. Furthermore, in a few cases the eradication of another strain of P. aeruginosa by chemotherapy was followed by colonization of the lungs with the above-mentioned predominating strain, and this strain was associated with high lethality. According to these results, measures should be undertaken to identify and eliminate routes of cross-infection in CF centres in order to diminishe the prevalence of P. aeruginosa infection and thereby reduce the lethality of CF patients.

Studies on coagulase-negative staphylococci in patients undergoing cardiac surgery. Different hypotheses for the origin of multiply-resistant isolates.

Hypotheses for the origin of multiply-resistant coagulase-negative staphylococci from 146 patients undergoing cardiac surgery were tested. All received cephalothin per-operatively. Antibiotic susceptibility testing, phage-typing, bio-typing, and test for Tween-80-splitting enzyme were used to characterize 132 isolates from nose swabs. Seventy-five percent of the pre-operative susceptible isolates were of biotype 1, while biotypes 3 and 4 made up 59% of the post-operative, multiply-resistant isolates. Fifty-three percent of the isolates were typable by phage-typing. Typability of isolates of biotype 1 was high (56%) while almost 75% of biotype 4 were untypable. Susceptible isolates were more often typable than multiply-resistant ones. Of the 146 patients, 105 (72%) carried coagulase-negative staphylococci pre-operatively, only two of them carried multiply-resistant strains. Fifty-nine patients (41%) were colonized with multiply-resistant coagulase-negative staphylococci post-operatively. By combining the results of bio-typing, phage-typing, and test for Tween-splitting enzyme the study made it probable that a maximum of ten patients (6.8%) already carried multiply-resistant strains on admission to the hospital or were carriers of initially susceptible strains which developed multiple-resistance during administration of antibiotics. It therefore seemed likely that most of the patients were deprived of their natural bacterial flora by antibiotic treatment and subsequently colonized post-operatively with multiply-resistant coagulase-negative staphylococci from the environment.

Gentamicin-resistant Staphylococcus aureus strains isolated in Denmark in 1979.

In 1979 seven gentamicin (G)-resistant (res.) Staphylococcus aureus strains caused epidemic episodes in eight Danish hospitals. Furthermore, a total of 37 resistant strains were isolated from separate incidents. All the G-res. strains were resistant to kanamycin, sisomicin and tobramycin, five to amikacin and two to cephalothin. None were resistant to netilmicin. Twelve out of the total of 44 strains were multiply-res. (resistant to penicillin, streptomycin and tetracyclines), and 10 of these were also resistant to methicillin (M). All phage groups/complexes were represented, group III by 13, and the 83A complex by seven strains. Multiply-res. strains, resistant to both M and G were all resistant to mercury, but sensitive to arsenate, whereas such strains resistant to either M or G usually have been found resistant to both metals. Twenty-three strains lost resistance to G upon storage, among them only one multiply-res. Loss of resistance did not influence the metal resistance pattern. From one patient only, the various isolates (nine) differed in respect to bacteriological properties. It was concluded, however, that they all were descendants of the same unstable strain. In the majority of the cases treatment with G had preceded isolation of the resistant strain.

Frequency, phage types and antibiotic resistance of Staphylococcus aureus isolated from blood cultures in Denmark 1975-1981.

In the period 1975-1981, 4060 cases of Staphylococcus aureus bacteremia were recorded in Denmark, and the corresponding strains were examined. The percentage of strains, resistant to penicillin only, rose to 82, and the percentage of multiply-resistant strains fell to five. Newer phage types (94, 96 and 95) increased from 10% to 27% of the material. These strains were usually resistant only to penicillin, but produced large amounts of penicillinase. The ample penicillinase production has also been characteristic for previous epidemic strains, and it is furthermore correlated to mortality. The overall mortality of 34.6% was lower than that of the preceding period. Mortality rates were highest in elderly patients, nosocomial cases, patients with serious primary diseases and endocarditis cases.

Antibiotic policy and spread of Staphylococcus aureus strains in Danish hospitals, 1969-1974.

In the period 1969-1974 a decreasing number of Danish hospital departments recorded epidemic occurrences of Staphylococcus aureus strains, and the local spread of strains was less extensive. Multiple-resistant strains of the 83 A complex were succeeded by type 94 strains, resistant to penicillin only. The level of methicillin resistance fell from 19 to 6%. 12% of methicillin resistant strains are now sensitive to streptomycin and/or tetracyclines; they do not represent a few clones, but belong to a wide spectrum of phage types. The changes follow a reduction in the consumption of streptomycin and tetracyclines not of methicillin or other penicillins. As a contrast to the general reduction of combined resistance to streptomycin and tetracyclines, the strains in dermatological departments, where tetracyclines are commonly used, maintain a high degree of resistance to tetracyclines alone.

An epidemic spread of multiresistant Pseudomonas aeruginosa in a cystic fibrosis centre.

Early in 1983 an epidemic of a Pseudomonas aeruginosa resistant to aminoglycosides, carbenicillin, ureidopenicillins, ceftazidime, cefsulodin and imipenem occurred in a cystic fibrosis centre. Most of the epidemic could be attributed to a specific nosocomial strain by means of O-grouping and phage-typing. This strain was present in the centre at a low frequency in 1973 and developed resistance during courses of chemotherapy. The epidemic was stopped by isolating patients with the resistant strains. Restrictive and selective use of antibiotics have not been sufficient to eradicate the resistant strains, which persist in 42% of the patients. The extensive use of the third generation cephalosporins in the clinic is probably responsible for inducing and selecting for the resistant strains. Clustering of patients in the centre has facilitated the spread. First-line use of older beta-lactam antibiotics, close bacteriological monitoring and prompt isolation of patients with resistant strains are recommended.

Staphylococcus aureus strains isolated in Danish hospitals from January 1st, 1966, to December 31st, 1974.

During the years 1966-1974, 167,297 strains isolated from 167,297 patients or staff members in Danish hospitals were registered at Staten Seruminstitu. All the strains were phage-typed and examined for production of a 'Tween'-80-splitting enzyme and resistance to mercuric chloride. 158,236 strains were examined for resistance to antibiotics. Since 1968, a steep decrease in the number of strains resistant to three or more antibiotics (multiple-resistant) and in strains of the 83A complex was noticed. In recent years an increase in strains belonging to phage-group I and in those referred to as miscellaneous and non-identified was registered. The increase in the non-typable strains might be explained by the shift of the concentration of the typing phages from 1000 X RTD to 100 X RTD. It is concluded that at least two factors may have contributed to the reduction of the multiple-resistant strains: an altered antibiotic policy, restricting the use of streptomycin and tetracyclines, and an improved hospital hygiene, diminishing the spread of identical strains within the various departments. However, it is emphasized that the consumption of methicillin is still increasing.

Staphylococcus aureus bacteremia in patients with hematological malignancies and/or agranulocytosis.

A total of 6,253 cases of Staphylococcus aureus bacteremia, including 274 (4.4%) endocarditis cases, were registered in Denmark in the period 1975-1984. Patients with hematological malignancies and/or agranulocytosis accounted for 479 of the bacteremia cases. The incidence of endocarditis in this group of patients was only 0.4% as compared to 4.7% in other patients with staphylococcal bacteremia (p less than 0.01). The lower incidence of endocarditis complicating bacteremia in these patients may justify a shorter course of therapy than usually recommended for suspected endocarditis. Patients with hematological malignancies and other patients with agranulocytosis had a higher mortality (49 and 46%, respectively) than other patients with S. aureus bacteremia (33%). The highest mortality was found in patients with multiple myeloma (71%, p less than 0.01), the lowest in patients with acute lymphocytic leukemia (28%, p less than 0.01). The higher mortality in these patients may indicate that empiric antibiotic regimens in granulocytopenic patients should include a specific anti-staphylococcal agent.

Does centralized treatment of cystic fibrosis increase the risk of Pseudomonas aeruginosa infection?

Two hundred and forty Danish patients with cystic fibrosis (97% of the total CF population in Denmark) participated in a point-prevalence study of Pseudomonas aeruginosa infection. One hundred and ninety-two patients were treated at the Danish CF centre and 48 patients were treated in other places. The age distribution was significantly different as no patients older than 19 years were found in the non-centre group. Pathogenic bacteria were isolated from the sputum of 96% of the patients. P. aeruginosa was more prevalent in patients from the centre, whereas Staphylococcus aureus was more prevalent in the non-centre group. No difference in serogroup and phage pattern of P. aeruginosa was found. There was a tendency that non-centre treated patients acquired chronic broncho-pulmonary P. aeruginosa infection later, but at the age of 16 years 90% of all patients will be chronically infected. Chronic P. aeruginosa infection was significantly more common in the age group 10-14 years at the centre than outside the centre. It is not possible to prevent chronic P. aeruginosa infection in CF patients treated in small groups and because of the better prognosis of centralized treatment the latter must be recommended.

Staphylococcus aureus endocarditis in Denmark 1976-1981.

Clinical and bacteriological information on Staphylococcus aureus endocarditis from hospitals all over Denmark in the period 1976-1981 was reviewed in 119 cases, 61 females and 58 males. Patient ages ranged from 16 days to 85 years, with a median age of 63 years. The overall mortality was 71%. The mortality correlated significantly with such factors as age, hospital-acquired infections and resistance to penicillin in infecting strains. Hospital-acquired infections occurred in 38% of the patients. The distribution of phage types among strains isolated from blood cultures from patients with endocarditis corresponded to that of strains from other septicaemia cases. Group I and group III strains and strains of the 94, 96 complex comprised 74% of the phage types of the present material. Infections of the skin were the most common portal of entry for the infecting strains. Apart from drug addicts, of which 11 cases were included, mortality did not correlate with the presence of any underlying diseases.

Chemotherapy against Pseudomonas aeruginosa in cystic fibrosis. A study of carbenicillin, azlocillin or piperacillin in combination with tobramycin.

A comparative study was made on tobramycin combined with either carbenicillin (500 mg/kg/day) or one of the new penicillins: azlocillin or piperacillin (both 300 mg/kg/day) in 50 cystic fibrosis patients with chronic Pseudomonas aeruginosa infection. Average 2-h levels of penicillins in serum were 46 micrograms/ml (piperacillin), 88 micrograms/ml (azlocillin) and 66 micrograms/ml (carbenicillin). The Pseudomonas strains were significantly more sensitive to piperacillin and azlocillin than to carbenicillin (minimal inhibitory concentrations 1.9, 2.3 and 4.2 micrograms/ml). In 21 of 54 treatment course temporary eradication of Pseudomonas was achieved. Improved ventilatory capacity and diminished proteolytic activity in sputum were seen in most patients with or without bacteriological treatment success. Resistant strains - often belonging to other types - appeared in the patients with treatment failure. With increased number of precipitating antibodies against Pseudomonas and with increased minimal inhibitory concentrations, the chance of eradication was smaller. Seven out of 20 treated with azlocillin and 14 out of 30 treated with piperacillin developed fever and exanthema by the end of treatment. Our experience suggests caution in the use of the new penicillins.