Production and characterization of monoclonal antibodies to the major protein of boar outer dense fibers.

Outer dense fibers (ODF) are structural elements in the mammalian sperm tail which surround the axoneme in the midpiece and principal piece and probably may help to maintain the elastic structures and elastic recoil of the sperm tail. In the present study, we have generated and characterized and describe a series of monoclonal antibodies (mAbs) against the 30 kDa major protein from boar ODF. For antibody screening an ELISA was developed using a newly developed method to fix the ODF proteins to the solid phase. A total of seven mAbs were selected and characterized by ELISA, Western blot and immunofluorescence. The mAbs recognize the major protein component of boar ODF on preparative Western blot and mark the mid- and principal piece of demembranated flagella. These mAbs also recognize the mid- and principal piece of demembranated human spermatozoa from normozoospermic patients, but not from those with asthenozoospermia. For the first time, we succeeded in obtaining hybridoma cell lines that secrete mAbs of class IgM, which react with the 30 kDa protein of boar ODF.

Inhibition of angiotensin-converting enzyme--a new concept of medical treatment of male infertility?

OBJECTIVES: To evaluate the effectiveness of systemic captopril therapy. DESIGN: Randomized double-blind study. SETTING: Andrology Unit at the Department of Dermatology, University of Munich, Munich, Germany. PATIENTS: Infertile men suffering from oligozoospermia (5-20 x 10(6) spermatozoa/mL) and/or asthenozoospermia. INTERVENTIONS: Captopril was given orally; samples of seminal plasma were collected twice before treatment and 4 and 12 weeks after captopril administration. MAIN OUTCOME MEASURE: Semen parameters, pregnancy rate, ACE activity, and kinin levels. RESULTS: After 4 weeks of therapy, significant differences between verum group and placebo group were found concerning ACE activity and kinin levels. Sperm density improved significantly after 12 weeks of captopril therapy. All other semen parameters remained unchanged. The pregnancy rate was not improved. CONCLUSIONS: The suitability of captopril in the therapy of oligozoospermia and/or asthenozoospermia for improvement of male infertility seems to be limited.

Relevance of zinc in human sperm flagella and its relation to motility.

OBJECTIVE: To measure the zinc content of human sperm flagella and to analyze its relation to sperm motility. DESIGN: Prospective study. SETTING: Center of Dermatology and Andrology. PATIENT(S): Semen samples collected from 90 andrology patients and healthy donors after 3-5 days of sexual abstinence. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sperm morphology after Shorr staining, sperm motility, and patient age were recorded. In addition, zinc concentrations in the seminal plasma, sperm heads, and flagella were determined with the use of atomic absorption spectrometry. RESULT(S): The mean zinc concentration was 144.3 mg/L in the seminal plasma and 146.9 mg/L in the whole ejaculate and was significantly correlated with parameters of motility. The sperm heads contained only 6.7% of the zinc that was present in the whole spermatozoon. The zinc concentration in the flagella was negatively correlated with sperm motility and velocity. In addition, it was positively correlated with the percentage of abnormally blue-stained flagella and the age of the patients. CONCLUSION(S): Our results clearly demonstrate the importance of zinc elimination during epididymal sperm maturation for functional competence of the outer dense fibers and, therefore, generation of motility.

Release of angiotensin-converting enzyme (ACE) from human spermatozoa during capacitation and acrosome reaction.

The present study examines the release of angiotensin-converting enzyme (ACE) from human spermatozoa during capacitation conditions and in correlation to acrosome reaction and cell death. The ACE content of spermatozoa was measured by treating the cells with different detergents. Glass wool-filtered and washed human spermatozoa were incubated for 3 hours at 37 degrees C. Percentages of acrosome-reacted and dead spermatozoa did not change significantly, but the ACE release increased from 0 to 2.93 +/- 0.44 mU/100 x 10(6) spermatozoa after 180 minutes (P < 0.001). In order to study the influence of acrosome reaction on ACE release, the acrosome reaction of noncapacitated spermatozoa was induced by 10 microM calcium ionophore A23187. The percentages of acrosome reaction and viability in noncapacitated spermatozoa as well as the ACE release were compared to corresponding data from experiments using capacitated spermatozoa (3 hours, 37 degrees C) from the same donors. Although the number of living acrosome-reacted spermatozoa after ionophore treatment (30.5 +/- 4.0%) was significantly higher than after capacitation (13.3 +/- 2.8%, P < 0.001), ACE release from ionophore-treated, noncapacitated spermatozoa was lower (P < 0.05). The data indicate that ACE release from human spermatozoa during capacitation is independent of acrosome reaction. Measurement of ACE release may be a clinically useful assay for human sperm capacitation.

Enzymatic digestion of bradykinin by rat Sertoli cell cultures.

Sertoli cells play a key role in spermatogenesis. To study the involvement of the kallikrein-kinin system in the testis, the pattern of bradykinin-inactivating kininases in rat Sertoli cells was investigated. Exogenous bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) is cleaved at Pro7-Phe8, Phe5-Ser6, and Gly4-Phe5, as demonstrated by high performance liquid chromatography analysis. Degradation of bradykinin was strongly inhibited by phosphoramidon and thiorphan, which are specific inhibitors of neutral metalloendopeptidase-24.11. The kininase type II-specific inhibitors, captopril and enalapril, were only partially effective in preventing peptidolysis. This indicates that the main kininases responsible for rapid bradykinin inactivation are neutral metalloendopeptidase and, to a lesser extent, kininase type II. Neutral metalloendopeptidase and kininase type II were shown to be located on Sertoli cell membranes. A low degree of bradykinin degradation was detected by simultaneous inhibition of neutral metalloendopeptidase-24.11 and kininase II, pointing out the involvement of further peptidases with minor activities. This remaining activity is probably not due to the action of kininase type I or cysteine proteases, as shown by specific inhibitors. The data presented indicate the occurrence of membrane-bound kininases, which are an important part of the kallikrein-kinin system, in rat Sertoli cell cultures.

Acrosin activity of human spermatozoa by means of a simple gelatinolytic technique: a method useful for IVF.

Acrosin activity was determined using a gelatinolysis technique in 100-microliter semen aliquots of 114 patients (normozoospermia, n = 90; asthenozoospermia, n = 12; oligozoospermia, n = 10; polyzoospermia, n = 2) attending an in vitro fertilization (IVF) program. Halo diameter, halo formation rate, and a calculated acrosin activity index correlated significantly with the IVF rates (P = 0.0054, r = 0.396; P = 0.0009, r = 0.401; and P = 0.0003, r = 0.428, respectively). In cases where the halo diameter was < 10 microns and halo formation rate was < 60%, all patients were subfertile or infertile, that is, they showed poor or no fertilization in vitro, respectively. The assay demonstrated a relatively low sensitivity: 25.7% for halo diameter, 37.1% for halo formation rate, and 25.7% for acrosin activity index, respectively. This might be attributed to other sperm functional aspects, such as disturbed acrosome reaction or impaired zona binding.

Influence of gossypol on the secretory function of cultured rat sertoli cells.

Cottonseed gossypol is a potent male contraceptive in several mammalian species including man. Sertoli cells play a crucial role in spermatogenesis. Therefore, the antifertility competence of gossypol may reflect a change in Sertoli cell function. Rat primary cultures were used to examine the effect of gossypol on cell viability, mitochondrial dehydrogenase function, lactate production and secretion of the Sertoli cell-specific protein inhibin. Exposure for 24 h to gossypol (3-6 microM) significantly enhance secretion of lactate but reduce secretion of inhibin without affecting cell viability. At 9-15 microM, the observed decrease of both lactate and inhibin accumulation apparently resulted from Sertoli cell degeneration and death, because viability and mitochondrial function were also reduced. The results suggest that mitochondria of Sertoli cells are a possible target for gossypol-induced infertility.

Protease-protease inhibitor interactions in Sertoli cell-germ cell crosstalk.

Peritubular cells, Sertoli cells, and germ cells of the seminiferous tubule synthesize and secrete several proteases and protease inhibitors. Experimental evidence suggests that the complex network of proteolytic enzyme activity and their regulation by protease inhibitors play an important role in male reproduction. Interaction between protease and protease inhibitors seems to play an important role in remodeling and restructuring of the seminiferous tubule during spermatogenesis. Controlled proteolytic activity is also involved in the migration of germ cells from the basal compartment to the lumen of the seminiferous epithelium, and in the release of spermatids during spermiation. The recently reported occurrence of Sertoli cell membrane-associated proteases indicate the possible involvement of regulatory peptide systems within the testis. This view is supported by the detection of all components of one of these paracrine systems, the kallikrein-kinin system, in cells of the seminiferous tubule.

Evaluation of the acrosomal membrane in bovine spermatozoa: effects of proteinase inhibitors.

Thawed bovine spermatozoa are characterized by a lack of homogeneity in the acrosomal membrane. Therefore, it is difficult to visualize the acrosome to assess morphology. Synthetic proteinase inhibitors were tested on thawed bovine semen for their effect on the integrity of acrosomal membranes. The proteinase inhibitors 4-nitrophenyl-4-guanidinobenzoate (NPGB) and N-L-p-tosyl-L-lysine-chloromethylketone (TLCK) were added to a medium containing spermatozoa separated on a percoll gradient. After incubation for 30 min at 38 degrees C in 5% CO(2), 95% air (final concentration 1 mM), the action of these inhibitors was controlled by measuring the activity of acrosome proteinases. The acrosomal membrane was evaluated by means of a dual stain procedure (trypan blue, Giemsa). In contrast to spermatozoa that had been incubated with proteinase inhibitor-free solution, samples that had been incubated with TLCK showed homogeneity in 90% of the acrosomal membranes and excellent visualization of the acrosome itself; in the NPGB-treated samples, homogeneous staining was observed in 83% of spermatozoa (P < 0.0005). It is concluded that alteration of the acrosomal membrane in thawed semen is not directly caused by freezing-thawing, but may be due to activation of acrosomal proteinases, which is increased during staining procedures. The addition of proteinase inhibitors before staining offers a new possibility for improved assessment of the acrosome in bovine spermatozoa.

Determination of soluble interleukin-2 receptor in human seminal plasma.

Following lymphocyte activation a soluble form of TAC antigen is released. This soluble molecule (soluble interleukin-2 receptor, SIL-2R) seems to corresponds to a truncated extracellular part of the membrane-bound TAC antigen, being smaller than its cellular counterpart. SIL-2R was determined and correlated with PMN elastase levels in the seminal plasma of 79 adult men having different concentrations of PMN elastase (0-700 micrograms/L). The normal level of SIL-2R was detected in the seminal plasma of 15 healthy men. The mean +/- SEM was 101 +/- 29 units/mL. The relation between PMN elastase in human seminal plasma as an index for the state of lymphocyte activity and the sperm motility is also investigated.

Synthesis and characterization of luciferin derivatives for use in bioluminescence enhanced enzyme immunoassays. New ultrasensitive detection systems for enzyme immunoassays, I.

Derivatives of luciferin, D-luciferin methyl ester, D-luciferyl-L-phenylalanine, D-luciferyl-L-N alpha-arginine, D-luciferin-O-sulphate and D-luciferin-O-phosphate, were synthesized for use as highly sensitive substrates for enzyme assays. The luciferin derivatives were characterized by ultraviolet and fluorescence spectrophotometry, by amino acid analysis and by fast atom bombardement mass spectrometry. Enzymatic cleavage of the compounds by enzymes leading to the release of D-luciferin was demonstrated. Kinetic constants were determined for the following enzyme/substrate pairs: D-luciferin methyl ester/carboxylic esterase, D-luciferyl-L-phenylalanine/carboxypeptidase A, D-luciferyl-L-N alpha-arginine/carboxypeptidase B, D-luciferin-O-sulphate/arylsulphatase, D-luciferin-O-phosphate/alkaline phosphatase. All compounds proved to be acceptable substrates for the respective enzymes, D-luciferin-O-phosphate being accompanied by an especially high turnover number (kcat = 1010 s-1) with alkaline phosphatase.

Bioluminescence enhanced enzyme immunoassay. New ultrasensitive detection systems for enzyme immunoassays, II.

Ultrasensitive bioluminescence immunoassays for the determination of peptides and proteins (illustrated with human urinary kallikrein, bradykinin and the determination of human urinary kallikrein antibody titers) have been developed. The usable ranges of the standard curves are from 5 pg to 5000 pg per liter. The relative intra-assay coefficients of variation of the tests were between 2 and 6%, and the interassay coefficients of variation between 4 and 12%.

A new type of ultrasensitive bioluminogenic enzyme substrates. I. Enzyme substrates with D-luciferin as leaving group.

Derivatives of D-luciferin, D-luciferin methyl ester, D-luciferin O-sulfate, D-luciferin O-phosphate, D-luciferyl-L-N alpha-arginine and D-luciferyl-L-phenylalanine were used as highly sensitive substrates for carboxylic esterase, arylsulfatase, alkaline phosphatase and carboxypeptidases A, B and N. Enzymatic cleavage of the compounds by enzymes leading to the release of D-luciferin was demonstrated. Kinetic constants have been determined for D-luciferin methyl ester and carboxylic esterase, for D-luciferin O-sulfate and arylsulfatase, for D-luciferin O-phosphate and alkaline phosphatase, for D-luciferyl-L-phenylalanine and carboxypeptidase A, and for carboxypeptidases B and N and D-luciferyl-L-N alpha-arginine. All compounds proved to be highly sensitive substrates for the respective enzymes, permitting a limit of detection for enzymes between 10 and 500 fg per assay.