The measurement of E and 19-hydroxy E prostaglandins in human seminal plasma

A method is described which measures the four main prostaglandins of human semen (PGE₁, E₂, 19-hydroxy PGE₁, and 19-hydroxy PGE₂). For routine measurements E₁ and E₂ are measured together as are 19-OH E₁ and 19-OH E₂. These are measured by forming oximes in aqueous solution. extraction, methylation and trimethyl silylation followed by gas chromatography. The method has sufficient sensitivity to measure the levels found in the majority of semen samples. The normal range in men with proven fertility was 90 to 260 μg/ml of 19-hydroxy Es and 30–200 μg/ml of Es.     

The measurement of E and 19-hydroxy E prostaglandins in human seminal plasma

A method is described which measures the four main prostaglandins of human semen (PGE₁, E₂, 19-hydroxy PGE₁, and 19-hydroxy PGE₂). For routine measurements E₁ and E₂ are measured together as are 19-OH E₁ and 19-OH E₂. These are measured by forming oximes in aqueous solution. extraction, methylation and trimethyl silylation followed by gas chromatography. The method has sufficient sensitivity to measure the levels found in the majority of semen samples. The normal range in men with proven fertility was 90 to 260 μg/ml of 19-hydroxy Es and 30–200 μg/ml of Es.     

The measurement of E and 19-hydroxy E prostaglandins in human seminal plasma.

A method is described which measures the four main prostaglandins of human semen (PGE1, E2, 19-hydroxy PGE1, and 19-hydroxy PGE2). For routine measurements E1 and E2 are measured together as are 19-OH E1 and 19-OH E2. These are measured by forming oximes in aqueous solution extraction, methylation and trimethyl silylation followed by gas chromatography. The method has sufficient sensitivity to measure the levels found in the majority of semen samples. The normal range in men with proven fertility was 90 to 260 mug/ml of 19-hydroxy Es and 30-200 mug/ml of Es.     

Ca 125 and Ca 19-9: two cancer-associated sialylsaccharide antigens on a mucus glycoprotein from human milk

The cancer-associated antigens Ca 125 and Ca 19-9 were demonstrated by radioimmunoassay to form structural units of a mucus glycoprotein in human milk taken from healthy women four days after parturition. The glycoprotein precipitated with the casein fraction at pH 4.6 and was completely absent in the whey as judged from Ca 19-9 assay. It could be effectively enriched by phenol-saline extraction from soluble milk proteins and further purified by gel filtration on Sephacryl S300 and Sephacryl S400. The active component with a bouyant density of 1.41 g/ml in isopycnic density gradient centrifugation (CsCl) shared common physico-chemical and chemical characteristics of mucus glycoproteins. Carbohydrates representing about 68% by weight were conjugated to protein by alkali-labile linkages, exclusively and were essentially free of D-mannose. Activities of Ca 125 and Ca 19-9 were both destroyed by treatment with periodate, mild alkali or neuraminidase suggesting the antigens are sialylated saccharides bound to protein by alkali-labile linkages. The fraction of monosialylated saccharide alditols isolated after reductive beta-elimination from the mucus glycoprotein was shown to inhibit monoclonal antibodies anti-(Ca 125) and anti-(Ca 19-9) in radioimmunoassay.     

Structure of tumor-associated carbohydrate antigen Ca 19-9 on human seminal-plasma glycoproteins from healthy donors

The monoclonal antibody-defined, tumor-associated antigen Ca 19-9, chemically identical with the sialylated Lewisa-carbohydrate determinant of a monoganglioside and a mucin, was demonstrated by radioimmunoassay to be present in large amounts as component of fucose-rich sialoglycoproteins, which had been extracted from human seminal plasma of healthy donors. The carbohydrate antigen of these glycoproteins (m greater than 205 kDa and m 115 kDa), which are presumably secreted by the prostatic gland, was absent in seminal plasma from blood-group-Lewis-negative men. The Ca 19-9 active sialyl-oligosaccharide was cleaved from the proteins by mild alkaline borohydride treatment and was shown to chromatograph on gradient elution from DEAE-Sephadex with the fraction of monosialylated saccharide alditols (MS-SP). The asialo derivative of the major saccharide alditol in this fraction was composed of L-fucose, D-galactose, N-acetyl-D-glucosamine and N-acetyl-D-galactosaminitol in the molar proportions 1:2:1:1 and chromatographed on Bio-Gel P2 according to approximately seven hexose units. A methylation analysis of the sialylated saccharide alditol in fraction MS-SP, which had been purified by high-pressure liquid chromatography, revealed the presence of terminal, non-reducing L-fucose, 3-O-substituted D-galactose, 3,4 di-O-substituted N-acetyl-D-glucosamine and 3-O-substituted N-acetyl-D-galactosaminitol. The presented data and the fragmentation pattern obtained on direct probe EI and FAB+ mass spectrometry of the permethylated asialo derivative are in accordance with the structure of a sialylated pentasaccharide alditol (formula; see text).     

Lucigenin chemiluminescence in human seminal plasma

Seminal plasma protects spermatozoa from the detrimental effects of reactive oxygen species such as hydrogen peroxide. We investigated the lucigenin-dependent chemiluminescence in cell-free seminal plasma from andrological patients. The seminal plasma was separated from cells by centrifugation. In all seminal plasmas studied lucigenin-dependent chemiluminescence (LCL) was detected. The LCL showed a strong pH-dependence. The signal was stable if samples were stored at +4°C for up to 4 days or up to 8 days at -80°C. Filtration of the samples (0.45 and 0.22 μm pore size) did not lower their luminescence. The addition of superoxide dismutase (SOD) and ascorbic acid oxidase (AAO) lowered LCL nearly to baseline values while trolox and desferal showed moderate effect, whereas allopurinol had no effect. Electron paramagnetic resonance spectroscopy demonstrated ascorbyl radicals in seminal plasma. Physiological concentrations of ascorbic acid yielded SOD-inhibitable lucigenin-chemiluminescence. The nitroblue-tetrazolium assay showed that ascorbic acid in buffer solution produced formazan. Superoxide-anion radicals were not detected in seminal plasma by the spin-trap DEPMPO due to their low steady state concentration. It is concluded that in seminal plasma ascorbate reacts with molecular oxygen yielding ascorbyl radicals and superoxide anion. If lucigenin is added to seminal plasma, reducing substances present, such as ascorbate, reduce lucigenin to the corresponding radical; this radical reacts with molecular oxygen and also forms O₂^-2.. So LCL in human seminal plasma results from the autoxidation of ascorbate and the oxidation of the reduced lucigenin. While the physiological relevance of the former mechanism is unknown, the latter is an artifact.     

Fibronectin fragments in human seminal plasma

The study has revealed the presence of fibronectin (FN) fragments and a lack of intact FN in 72 seminal plasma samples. The FN fragmentation was examined by immunoblotting with a monoclonal antibody specific to the central cellular FN domain and was confirmed with a monoclonal antibody directed to the C-terminal domain of FN. Nine FN fragments between 60 and 200 kDa and five fragments of 60-150 kDa were identified in seminal plasma samples of normozoospermic and of terato-, oligoterato-, and oligoasthenoterato-spermic groups, respectively. The relative amounts of the 60, 90 and 100 kDa FN fragments were 2-3 times higher in seminal plasmas with abnormal semen characteristics than in the normozoospermic group. The results suggest that seminal plasma FN fragments may contribute to fertilization and the analysis of FN fragmentation may have a diagnostic value in andrological investigations.     

The clinical relevance of tumor marker CEA, CA 19-9 in regional chemotherapy of hepatic metastases of colorectal carcinoma

Up to December 1986, 50 patients with documented hepatic metastases from colorectal carcinoma were treated with 5-fluoro-2-deoxyuridine (FUDR) using Infusaid pumps. The response of liver metastases to regional chemotherapy was studied by computerized tomography (CT) and carcino-embryonal antigen (CEA), and/or CA 19-9 antigen serum assays. Preoperative CEA values were pathological in 94% of the patients but only 48% had a pathological concentration of the antigen CA 19-9 of over 37 U/ml. The course of CEA and CA 19-9 in combination with the arterial angio-CT reflected the response of liver metastases to regional chemotherapy. A decrease or normalisation of CEA and CA 19-9 after the beginning of therapy is an indication of partial or complete remission of metastases (68% of the patients showed lowered CEA serum values). If the marker continues to rise in serum this is a danger signal of progression of liver metastases or of extrahepatic tumor spread if the tumor stage in the liver remains unchanged.     

Lactoferrin binding molecules in human seminal plasma

During ejaculation, the iron binding protein lactoferrin binds to sperm and forms a major component of sperm-coating antigens. Physicochemical properties of lactoferrin in seminal plasma (SP) and on sperm differ from those of purified lactoferrin. These differences have been attributed to the binding of unknown seminal macromolecules to lactoferrin. We have studied lactoferrin binding molecules in SP. The SP samples were coated onto microtiter plates and tested for binding of biotinylated lactoferrin. SP was found to specifically bind biotinylated lactoferrin. This binding was competitively inhibited by coincubation with unlabeled lactoferrin but was not affected by control incubations done with human IgG or transferrin. Lactoferrin binding molecules in SP were biochemically characterized by using SDS-PAGE and ligand blotting. Biotinylated lactoferrin bound to SP molecules of approximately 120, 60 and 30 kDa. No binding was observed with biotinylated transferrin. The presence of molecules that associate with lactoferrin in SP was further studied by using crossed immunoelectrophoresis. Lactoferrin in SP immunoprecipitated as two peaks, one of which corresponded to purified lactoferrin. These results suggest that some lactoferrin molecules in SP are free and that others are associated with lactoferrin binding molecules. Binding of lactoferrin to lactoferrin binding molecules appears to change its physicochemical properties and thus could influence its biologic activity and its affinity to sperm.     

Gelatinolytic proteinase activities in human seminal plasma

Proteinase activities in human seminal plasma were detected using gelatin-containing sodium dodecyl sulphate-polyacrylamide gel electrophoresis zymography. Three prominent bands of activity of Mr 60,000, 66,000 and 90,000 were observed as well as 9 other bands of less intensity (34,000-158,000). These proteinases were dependent upon calcium for optimal activity, did not hydrolyse casein, and were predominantly in the soluble portion of seminal plasma. Examination of seminal plasma of men with different sperm concentrations, split ejaculates, and prostatic secretions indicated that the prostate gland was a source of most of these activities. Proteinase activities of Mr 34,000, 37,000, 82,000 and 120,000 were expressed more frequently in seminal plasma from normozoospermic men than from seminal plasma of oligo- or azoospermic men, indicating that they may also arise from spermatozoa in the semen sample. The proteinases of Mr 60,000 and 66,000 were found in all seminal plasmas whereas there was variation in the expression of the other molecular forms of enzyme, even in the normozoospermic samples. There are multiple forms of gelatinolytic proteinase activities in human seminal plasma which appear to arise from multiple sources in the reproductive tract including the Cowper's/urethral glands, the prostate gland, seminal vesicle and/or spermatozoa. Their function(s) in semen remains to be established.     

Epidermal growth factor in human seminal plasma

In the present study, we have partially purified a characterized epidermal growth factor (EGF)-like substance(s) from human seminal plasma, and determined the concentrations of immunoreactive (IR)-hEGF in seminal plasma from normal and infertile males. Competitive binding curves of seminal plasma extracts were parallel to those of standard hEGF in both radioimmunoassay and receptor assay. Seminal IR-hEGF was similar to standard hEGF by gel exclusion chromatography, isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The concentrations of IR-hEGF in normal seminal plasma (48 +/- 9 ng/ml) did not differ from those of infertile males (41 +/- 3 ng/ml); the concentrations of seminal plasma IR-hEGF did not correlate with density, motility or morphology of sperm. These data clearly demonstrate the presence of hEGF in human seminal plasma indistinguishable from hEGF of urinary origin, and suggest that it may not play an important role in the sperm function. The tissue(s) of its origin and its physiological function in the male reproductive organs remain undetermined.     

Quantification studies in human seminal plasma samples identify prolactin inducible protein as a plausible marker of azoospermia

Background: Prolactin inducible protein (PIP) is a ~17?kDa protein, which is known to play vital roles in immunoregulation, fertility, antimicrobial activity, apoptosis and tumour progression. Objectives: This study reports quantification of PIP concentration in human seminal plasma (SP) samples. Methodology: PIP was purified by immunoprecipitation and its concentration in human SP samples was quantified by ELISA method. Results: Average concentration of PIP in normozoospermia, oligozoospermia and azoospermia was 290.3?±?71.5 μg/mL, 306.4?±?71.2 μg/mL and 60.5?±?23.6 μg/mL respectively. Conclusion: There was no significant variation in PIP levels in normozoospermia and oligozoospermia while its expression was down-regulated in azoospermia, indicating that PIP may be a plausible marker of azoospermia.     

Quantification studies in human seminal plasma samples identify prolactin inducible protein as a plausible marker of azoospermia

Background: Prolactin inducible protein (PIP) is a ~17?kDa protein, which is known to play vital roles in immunoregulation, fertility, antimicrobial activity, apoptosis and tumour progression.

Objectives: This study reports quantification of PIP concentration in human seminal plasma (SP) samples.

Methodology: PIP was purified by immunoprecipitation and its concentration in human SP samples was quantified by ELISA method.

Results: Average concentration of PIP in normozoospermia, oligozoospermia and azoospermia was 290.3?±?71.5 µg/mL, 306.4?±?71.2 µg/mL and 60.5?±?23.6 µg/mL respectively.

Conclusion: There was no significant variation in PIP levels in normozoospermia and oligozoospermia while its expression was down-regulated in azoospermia, indicating that PIP may be a plausible marker of azoospermia.     

Identification of calmodulin-like activity in human seminal plasma

Human seminal plasma was found to contain relatively high levels of a heat stable proteinaceous factor with properties similar to that of the calcium-binding protein calmodulin. The seminal plasma factor increases the (Ca²⁺ + Mg²⁺)-ATPase activity found in human red blood cell plasma membranes by 370% and the activation was completely abolished by chlorpromazine, amitriptyline and theophylline. A similar calmodulin-activated Ca²⁺ pump, has been found in the plasma membrane of ram sperm tails. The existence of calmodulin in mammalian seminal plasma may be responsible for some of the metabolic changes associated with sperm maturation.