An immunoaffinity liquid chromatography-tandem mass spectrometry assay for detection of endogenous aggrecan fragments in biological fluids: Use as a biomarker for aggrecanase activity and cartilage degradation.

The degradation of articular cartilage by aggrecanases (ADAMTS-4 and ADAMTS-5) plays a significant role in the pathology of osteoarthritis (OA). To monitor aggrecanase activity in OA, we have developed a sensitive, accurate, and versatile assay for detection of two specific cleavage sites on aggrecan. The assay uses an immunoaffinity-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect cleavage at the (374)ARGS site and the (1820)AGEG site. The dynamic range of the assay is more than three orders of magnitude, with interassay precision less than 15%. It has been successfully applied to various biological fluids and species, including rat, bovine, dog, and human. The assay has been analytically qualified for use in human urine and synovial fluid (SF). The limits of detection (LODs) for ARGS in urine and SF are 2.5 and 10 pg/ml, respectively, whereas the LOD for AGEG is 20 pg/ml in SF. Analysis of these biomarkers from OA subjects and normal healthy volunteers revealed a significant elevation of both markers in OA. Similarly, in a rat model of cartilage degradation, both ARGS and AGEG were elevated, demonstrating the utility of these biomarkers for translational research. These data suggest that the ARGS and AGEG biomarkers developed have potential as measures of aggrecanase activity in OA and may contribute to our understanding of OA pathology.

Interaction between human cervical mucus and sperm surface antibodies.

The immunobead test was used to study the immunoglobulin class of antibodies on sperm before and after penetration through a microcolumn of cervical mucus. Sixteen men with positive sperm antibodies (positive sperm immobilization test) and sperm that penetrated cervical mucus in prior tests were selected for study. However, at the time of study, sperm from seven subjects could not be recovered from the microcolumn. The nine subjects from whom motile sperm were obtained after passage through the column had better sperm mucus penetration tests, lower proportions of sperm binding to anti-IgA immunobeads, and higher proportions of sperm with tail-tip-only binding. Sperm recovered after penetration through the mucus microcolumn displayed a greatly reduced binding to anti-IgA immunobeads in all nine subjects, whereas similar reductions in anti-IgG binding occurred only in four subjects. These results confirm that IgA and sperm-head-directed antibodies are more important than IgG and sperm tail-tip-directed antibodies in impairing sperm penetration of cervical mucus.

Correlation between defective motility (asthenospermia) and ATP reactivation of demembranated human spermatozoa.

Human spermatozoa treated with 0.05% Triton X-100 to remove the cell membranes became immotile but flagellar movement was reinitiated by addition of 0.06 to 1 mM adenosine triphosphate (ATP). The percentage of spermatozoa showing flagellar movement 2 minutes after addition of 1 mM ATP from men with idiopathic asthenospermia (mean +/- SD, 34 +/- 15%), oligozoospermia (17 +/- 21%), sperm autoimmunity (17 +/- 12%), vasoepididymostomy (20 +/- 22%), or idiopathic zero motility (0%) was significantly lower than with spermatozoa from normal men (54 +/- 12%). The percentage of reactivated spermatozoa was correlated with the proportion of live cells (Eosin Y stain, r = 0.602, P less than 0.001), percentage of motile cells (r = 0.761, P less than 0.001), and motility index (r = 0.759, P less than 0.001) in the same semen samples. When expressed relative to initial sperm motility, the proportion reactivated was similar for idiopathic asthenospermia (85%) and normospermia (82%). Thus, failure to produce ATP does not appear to be a frequent cause of low sperm motility in man.

Is conventional bacteriology useful in the management of male infertility?

An aetiological role for non-specific genital tract infection (GTI) in male infertility has long been postulated and has led to routine bacteriologic culture of semen in some laboratories. To assess the value of examination of various semen characteristics and conventional culture of semen as diagnostic criteria for GTI, frequencies of bacterial isolation and relationships with leukocytospermia and other abnormalities of semen were determined in 806 samples from 459 men seen over a 2 year period. Combinations of features suggestive of GTI were present in 19.6%. High leucocyte counts were found in 13.5% of specimens and evidence of possible accessory sex organ dysfunction in 57.7%. Significant numbers of bacteria were isolated from 19.5% of samples. Of the few pathogens cultured (46 specimens), 53% were Staphylococcus aureus, a possible contaminant. Furthermore, only one specimen had more than 10(6) pathogenic bacteria per ml, also Staph. aureus. There was a lack of correlation between the presence of bacteriospermia, leukocytospermia and semen changes possibly attributable to accessory sex organ dysfunction, shedding doubt on their relevance to identification of GTI. This was the case even in repeatedly culture-positive specimens. Conventional bacteriological examination of semen specimens is not merited as a routine procedure.

Immunoglobulins on human sperm: validation of a screening test for sperm autoimmunity.

The immunobead test (IBT) is a new method of detecting antisperm antibodies with the advantages of enabling determination of the presence of antisperm antibodies during routine semen analysis, immunoglobulin isotypes and antibody specificity for binding to sperm structures such as head or tail. The IBT is compared with the conventional methods of detecting antisperm antibodies in serum; the tray agglutination test (TAT) and the sperm immobilization test (SIT). Positive IBT (50% or more of washed motile sperm binding to anti-IgG and/or anti-IgA immunobeads) were found in 6.8% of 689 men being investigated for infertility. Both IgG and IgA were found to be present in all cases. The IBT correlated closely with the serum antisperm antibody tests; of the 202 men tested by all three methods, 200 (99%) were either positive for circulating antibodies and IBT or negative for both. No men with serum antisperm antibodies lacked local antisperm antibodies as detected by IBT, and only two men (1%) were IBT positive in the absence of either one or both types of circulating antibodies. Individually, the TAT and the SIT correlated equally well with the IBT; 97% and 98% of men tested by TAT and IBT, and SIT and IBT respectively, were either positive by both or negative by both. It is concluded that the IBT is an excellent procedure for the detection of sperm antibodies.

Are conventional sperm morphology and motility assessments of predictive value in subfertile men?

Sperm morphology and motility are believed to be important prognostic factors for fertility. Results of a group of 67 men investigated for primary infertility who had mean sperm concentrations greater than 5 million per ml and who later produced pregnancies, were compared with those of 67 matched controls who remained infertile. All female partners were potentially fertile. The groups were matched for other known prognostic factors for fertility, namely, wife's age, the duration of infertility, sperm concentration and varicocele size. There were no significant differences between the two groups overall in the (mean +/- SEM)% of sperm with normal morphology (58.3 +/- 2.1; 58.5 +/- 2.2), or motility (40.6 +/- 1.8; 37.0 +/- 2.0). However, among oligospermic men from the two groups, sperm motility was significantly higher (P less than 0.05) in the subsequently fertile group (43.1 +/- 2.6%) than in the persistently infertile group (33.3 +/- 3.7). These results indicate that sperm morphology as currently assessed may not be important in predicting fertility in subfertile men with a mean sperm concentration over 5 million/ml and the % sperm motility may only be a relevant predictor in oligospermic men.

Structural characterization of the two refold dimers of recombinant bovine somatotropin (bST).

Two major dimers are generated during the folding/oxidation of inclusion bodies of recombinant bovine somatotropin (bST). These dimers represent the major part of the inactive high molecular weight species that are formed in this process. The structures of the two dimers are unambiguously determined by peptide mapping using trypsin, thrombin cleavage, and selective DTT reduction experiments. Results indicate that the formation of both dimers involves the large disulfide loop cysteines. The latter-eluting dimer from RP-HPLC, previously reported as a large loop concatenated dimer, was revised to be an antiparallel disulfide-linked dimer. On the other hand, the first eluting dimer is a concatenane in which two monomers are held together by the interlocking of the two large disulfide loops.

Patterns of human seminal plasma proteins on polyacrylamide gel electrophoresis.

Seminal plasma proteins were separated by sodium dodecylsulphate polyacrylamide gel electrophoresis and the patterns for different conditions compared by densitometry. The changes in pattern with time after ejaculation because of proteolysis could be largely prevented by addition of bacitracin. Patterns were similar within the one man and little influenced by duration of abstinence from ejaculation. There were marked variations in pattern between individuals and no specific differences could be detected between samples from men with normal semen analyses, vasectomy, or azoospermia from seminiferous tubule failure. However, there were distinct patterns with congenital absence of the vasa and seminal vesicles and with androgen deficiency. Methods for identification of testicular and epididymal proteins in semen would expand the clinical usefulness of examination of seminal plasma proteins.

Immobilization of sperm by condoms and their components.

Condoms and other rubber products immobilize sperm, but the mechanism is unknown. Sperm motility and eosin Y exclusion were measured following exposure of samples of semen to latex condoms of various types. Sperm motility was markedly reduced by exposure to all latex condoms, but eosin Y exclusion was unchanged. While there appeared to be some variability between semen samples and in time to complete immobilization of all sperm, condoms from one batch were similar. The immobilizing effect was not altered by prewashing the condom with buffer, chelating agent or acid. After immobilization, motility was not recovered by sperm washed in buffer, mixed with normal mid-cycle cervical mucus, or exposed to caffeine or adenosine triphosphate after demembranation with detergent. Studies with components of condoms indicated that raw latex and zinc dimethyl- and dibutyl-dithiocarbamate accelerators immobilized sperm within 30 min. In contrast, silicone rubber had no effect on sperm motility for up to 4 h. Enhancement of the sperm immobilizing effect might improve the contraceptive efficacy of condoms, while a useful non-toxic sperm collecting device for semen analysis, artificial insemination or in vitro fertilisation might be developed by omitting the toxic components from the rubber.

Patterns of human epididymal fluid proteins on polyacrylamide gel electrophoresis.

Proteins were analysed by sodium dodecylsulphate polyacrylamide gel electrophoresis in fluid taken from six epididymal regions in 10 men undergoing microsurgery to bypass epididymal obstructions resulting from various disorders. Some major proteins common to most samples were identified with apparent molecular weights of 95, 67, 56, and 44 kilodaltons. A degree of regional specificity in the synthesis and secretion of epididymal proteins was indicated. There appeared to be no correlation between protein pattern or the abundance of individual proteins and the cause of obstruction, although methodological constraints may have partially obscured any such relationship.

Destripeptide insulin-like growth factor-I in milk from bovine somatotropin-treated cows.

Total somatomedins from milk of bovine somatotropin-treated cows were isolated and characterized to determine the relative amount of the three amino acid N-terminally truncated form of IGF-I (destripeptide IGF-I). The somatomedin fraction was isolated using organic solvent and solid-phase extractions followed by preparative reverse phase HPLC and affinity chromatography. The overall yield of IGF-I was 28%, and destripeptide IGF-I was recovered with similar efficiency. The isolated somatomedins were resolved by capillary zonal electrophoresis and identified using recombinant somatomedin standards. The concentration of destripeptide IGF-I relative to full length IGF-I was determined by amino terminal sequencing and by bioassay. Results from these experiments indicated that the level of destripeptide IGF-I in milk from somatotropin-treated cows was less than 3% of the IGF-I concentration. Destripeptide IGF-I is therefore a minor component of the somatomedins present in milk from treated cows and does not contribute significantly to the proliferative activity of this milk.

Cholesterol oxidase: a potent insecticidal protein active against boll weevil larvae.

The discovery of proteins that control insects is critical for the continued growth of the agricultural biotechnology industry. A highly efficacious protein that killed boll weevil (Anthonomus grandis grandis Boheman) larvae was discovered in Streptomyces culture filtrates. The protein was identified as cholesterol oxidase (E.C. 1.1.3.6). Purified cholesterol oxidase was active against boll weevil larvae at a concentration (LC50 = 20.9 micrograms/ml) comparable to the bioactivity of Bacillus thuringiensis proteins against other insect pests. Histological studies demonstrated that cholesterol oxidase lysed the boll weevil midgut epithelium, suggesting that this is the primary mechanism of lethality.

Cell-type-specific and site-specific N-glycosylation of type I and type II human tissue plasminogen activator.

Tissue plasminogen activator (t-PA) is an important initiator of fibrinolysis. The t-PA polypeptide has four potential N-glycosylation sites of which three are occupied in type I (Asn-117, -184, and -448) and two in type II (Asn-117 and -448). In an effort to elucidate the factors controlling the expression of N-linked oligosaccharides on this polypeptide, we have used a combination of sequential exoglycosidase digestion, methylation analysis, and controlled acetolysis to determine the oligosaccharide structures at each of the N-glycosylation sites of type I and type II t-PA when isolated from a human colon fibroblast cell strain and from a Bowes melanoma cell line. Our results suggest the following: (i) type I and type II t-PA are N-glycosylated in an identical way at Asn-117 and Asn-448, when isolated from the same cell line; (ii) Asn-117 is predominantly associated with oligomannose-type structures in all cases; (iii) Asn-184 and Asn-448 are predominantly associated with complex-type structures when t-PA is isolated from fibroblast cells, but with both complex- and oligomannose-type structures when isolated from melanoma cells; (iv) fibroblast cell derived t-PA is associated with both neutral and sialylated oligosaccharides, while melanoma cell derived t-PA is also associated with sulfated oligosaccharides, which are located exclusively at Asn-448 of type II t-PA; (v) no complex-type structures occur in common between t-PA from the two cell lines. These results indicate that the t-PA glycoprotein is secreted by each cell line as a set of glycoforms, each glycoform being unique with respect to the nature and disposition of oligosaccharides on a common polypeptide. Further, the two cell lines express no glycoform in common, despite expressing the same t-PA polypeptide. The implications of these results for both the control of oligosaccharide processing in different cell lines and the genetic engineering of mammalian glycoproteins are discussed.