Seminal Plasma Prostate-specific Antigen Research Articles

Association of decreased sperm motility and increased seminal plasma IGF-I, IGF-II, IGFBP-2, and PSA levels in infertile men.

Previous studies have suggested the involvement of serum insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in the regulation of the female reproductive system. Little is known of these peptides in the seminal plasma (SP) of men and their potential effects on fertility. We assessed SP levels of these peptides in infertile men with low sperm motility (asthenozoospermic; AZ) and low sperm counts (oligozoospermic; OZ), its effects on in vivo sperm motility, and whether there is a correlation with aging. Twenty-eight infertile men (AZ; n = 18 and OZ; n = 10) and 20 fertile normozoospermic (NZ) men were studied. Seminal plasma IGF-I, IGF-II, IGFBP-2, IGFBP-3, and prostate-specific antigen (PSA) levels were measured, and spermatozoa mRNA transcript patterns were examined. Asthenozoospermic men had higher SP IGF-I, IGF-II, IGFBP-2, and PSA levels than NZ and OZ men, whereas SP IGFBP-3 levels were similar between the three groups. Sperm count positively correlated with SP IGF-I, IGF-II, and IGFBP-2; sperm motility negatively correlated with SP IGF-II and IGFBP-2; and age correlated positively with SP IGF-II. The expression of IGF-I and IGF-II mRNA and mRNA receptors was detectable, but no variations in transcript levels were noted. Decreased sperm motility, but not sperm count, in infertile AZ men is associated with increased SP IGF-I, IGF-II, IGFBP-2, and PSA levels. Changes in SP IGFs and their interactions with IGFBPs and IGF receptors, and PSA levels suggest a role of these SP peptides in modulating sperm motility and possibly prostate disease development in aging men.

Development of glycan specific lectin based immunoassay for detection of prostate specific antigen

We describe an analytical approach for the detection and verification of glycosylation patterns of prostate specific antigen (PSA), a key biomarker currently used for understanding the onset and prognosis of prostate cancer. PSA has been purified from the human seminal plasma and total PSA from prostate cancer sera. PSA is a monomeric glycoprotein with an apparent molecular mass 28040.467Da, which exhibits a characteristic protease activity against casein and gelatin. Its optimal protease activity is centered on neutral pH. Peptide mass fingerprint analysis of the purified PSA has yielded peptides that partially match with known database sequences (Uniprot ID P07288). Tryptic digestion profile of isolated PSA, infer the exclusive nature of PSA and may be additive molecule in the dictionary of seminal proteins. Surface plasmon resonance and lectin immunoassay revealed direct interaction between a newly developed anti-PSA monoclonal antibody (C4E6) and PSA. A lectin based immunoassay is reported here which was achieved with the C4E6 anti-PSA antibody and biotinylated plant lectins. This investigation provides an alternative method to isolate and quantify PSA with altered glycosylation which might be seen in the prostate cancer and developing a lectin based immunoassay to detect PSA in serum of prostate cancer patients.

Genetic Variation in KLK2 and KLK3 Is Associated with Concentrations of hK2 and PSA in Serum and Seminal Plasma in Young Men

Genetic variants in KLK2 and KLK3 have been associated with increased serum concentrations of their encoded proteins, human kallikrein-related peptidase 2 (hK2) and prostate-specific antigen (PSA), and with prostate cancer in older men. Low PSA concentrations in seminal plasma (SP) have been associated with low sperm motility. To evaluate whether KLK2 and KLK3 genetic variants affect physiological prostatic secretion, we studied the association of SNPs with hK2 and PSA concentrations in SP and serum of young, healthy men. Leukocyte DNA was extracted from 303 male military conscripts (median age 18.1 years). Nine SNPs across KLK2-KLK3 were genotyped. We measured PSA and hK2 in SP and serum using immunofluorometric assays. The association of genotype frequencies with hK2 and PSA concentrations was tested with the Kruskal-Wallis test. Four KLK2 SNPs (rs198972, rs198977, rs198978, and rs80050017) were strongly associated with hK2 concentrations in SP and serum, with individuals homozygous for the major alleles having 3- to 7-fold higher concentrations than the intermediate concentrations found in other homozygotes and heterozygotes (all P < 0.001). Three of these SNPs were significantly associated with percentage of free PSA (%fPSA) in serum (all P < 0.007). Three KLK3 SNPs showed associations with PSA in SP, and the rs1058205 SNP was associated with total PSA in serum (P = 0.001) and %fPSA (P = 0.015). Associations observed in young, healthy men between the SP and serum concentrations of hK2 and PSA and several genetic variants in KLK2 and KLK3 could be useful to refine models of PSA cutoff values in prostate cancer testing.

Molecular microheterogeneity of prostate specific antigen in seminal fluid by mass spectrometry

Prostate specific antigen (PSA) is a widely used and clinically valuable marker for prostate disease. In order to enable the development of new PSA assays and progress the understanding of the biology of PSA we have analyzed PSA in seminal plasma. PSA in seminal plasma from men attending a fertility clinic and healthy controls was analyzed using SDS-PAGE, Western blotting and mass spectrometry. Using mass spectrometry, different forms of PSA could be identified in 1-9 bands seen on SDS-PAGE analysis of the respective sample. However, a majority of these molecular forms of PSA were not observed on Western blots. Enzymatic activity of PSA isoforms was demonstrated by sequencing data in zymogram gels. Multivariate analysis of clinical data revealed well-separated patient groups. We demonstrated that PSA in seminal plasma occurs in several isoforms, yet not all were detectable using an antibody based clinical routine method. The heterogeneity of PSA expression might be of clinical significance, by an improved patient phenotyping.

Thiophilic-interaction chromatography of enzymatically active tissue prostate-specific antigen (T-PSA) and its modulation by zinc ions

Prostate-specific antigen (PSA) is a serine protease secreted both by normal prostate glandular epithelial cells and prostate cancer cells. We explored “thiophilic-interaction chromatography” (TIC) to isolate tissue prostate-specific antigen (T-PSA) from fresh human prostate cancer tissue harvested by radical prostatectomy for the purpose to characterize T-PSA for its enzymatic activity and sensitivity to zinc ions. We have shown, for the first time, that T-PSA has strong affinity for the thiophilic gel (T-gel). The average recovery of T-PSA from T-gel is over 87%. The presence of PSA in the column eluate was confirmed by ELISA and SDS/PAGE. Western blot developed with monoclonal antibody to PSA revealed that T-PSA was predominantly in the “free” form having a molecular weight of 33 kDa. Furthermore, T-PSA was found to be enzymatically active. T-PSA was found to be less enzymatically active as compared to seminal plasma PSA. The inhibition of enzymatic activity of both f-PSA and T-PSA over a wide range of concentrations of Zn 2+ ions (10 nM to 50 μM) was comparable. In contrast, the enzymatic activity of chymotrypsin, another serine-protease, was affected differently. At higher concentrations of Zn 2+ (10 μM and higher) the enzymatic activity of chymotrypsin was inhibited, whereas, at lower concentrations of Zn 2+ (5 μM and lower), the enzymatic activity was enhanced.

Oligosaccharide Profiles of the Prostate Specific Antigen in Free and Complexed Forms from the Prostate Cancer Patient Serum and in Seminal Plasma: a Glycopeptide Approach

The oligosaccharide structures of prostate specific antigen (PSA) are expected to be useful in discriminating prostate cancer from benign conditions both accompanied by increased serum PSA levels. A large proportion of PSA forms a covalent complex with a glycoprotein, alpha(1)-antichymotrypsin, in human blood. In the present study, the glycan profiles of free and complexed forms of PSA from cancer patient serum and of seminal plasma PSA were compared by analyzing the glycopeptides obtained by lysylendopeptidase digestion of the electrophoretically separated PSA with mass spectrometry. The profiles of the PSA N-glycans from the free and complexed molecules were quite similar to each other and consisted of fucosylated biantennary oligosaccharides as the major class. They were mostly sialylated, and a considerable sialic acid fraction was alpha2,3-linked as determined by Streptococcus pneumoniae neuraminidase digestion of the glycopeptides. In the seminal plasma PSA, high-mannose and hybrid types of oligosaccharides were predominant, and the sialic acids attached to the latter as well as to biantennary oligosaccahrides were exclusively alpha2,6-linked because they were removed by Arthrobacter ureafaciens neuraminidase but resistant to S. pneumoniae neuraminidase. Complex-type oligosaccharides from other sources were found in the seminal plasma sample, indicating that analysis of released glycans carries a risk of being misleading. The results suggest that identification of alpha2,3-linked sialic acids on PSA potentially discriminates malignant from benign conditions, if the analysis is applied to oligosaccharides specifically attached to the N-glycosylation site of PSA in either a free or a complexed form in the serum.

Different glycan structures in prostate-specific antigen from prostate cancer sera in relation to seminal plasma PSA

Prostate-specific antigen (PSA), the tumor marker currently used for prostate cancer (PCa), is not specific enough to distinguish between PCa and benign prostate hyperplasia (BPH). Glycan processing is normally perturbed in tumors, therefore we investigated whether changes in glycosylation of PSA could be useful diagnostic indicators. Previously we determined that the glycosylation of PSA secreted by the tumor prostate cell line LNCaP differs significantly from that of PSA from seminal plasma (normal control). We therefore undertook a detailed glycan analysis of PSA derived from sera from PCa patients and, importantly, established that the glycosylation of the PCa serum PSA was significantly different from the PSA from the LNCaP cell line. In comparison with seminal plasma PSA, the fucose content of PSA from the PCa patient serum was significantly lower and there was a decrease in alpha2,3-linked sialic acid. Differences in the glycosylation of PSA derived from PCa patients' sera, seminal plasma, and LNCaP cells were further established by lectin detection, glycosylation immunosorbent assay, and two-dimensional electrophoresis. We also investigated whether the impact of glycosylation changes initiated by the tumor was reflected in the serum glycome. By comparing the glycans released from the total glycoproteins in PCa patient serum with those of normal serum we found an increase in the proportion of sialyl-Lewis x structures. Further analysis of the glycosylation of PSA from PCa and BPH sera will be required in order to determine the utility of these glycan differences to discriminate specifically between benign and malignant prostate states.

Separation of enzymatically active and inactive prostate-specific antigen (PSA) by peptide affinity chromatography.

Prostate-specific antigen (PSA) is a serine protease with highly prostate-specific expression and an important marker for prostate cancer. We have previously identified novel PSA-binding peptides that enhance the enzymatic activity of PSA when produced as fusion proteins. PSA-binding peptides and derivatives with a spacer were chemically synthesized and used to prepare an affinity column, which was used to fractionate PSA in seminal plasma, serum, and LNCap cell culture medium. Approximately 67% of seminal plasma PSA bound to the peptide affinity column and was eluted under mild conditions. Eluted PSA was intact and enzymatically active while the unbound fraction mainly contained various nicked forms. ProPSA from LNCap cells bound to the peptide column only after activation by trypsin. PSA-binding peptides can be used to separate enzymatically active and inactive forms of PSA. Thus the peptides are potentially useful as ligands for development of methods for specific detection of active free PSA.

Expression, purification, and characterization of active recombinant prostate-specific antigen in Pichia pastoris (yeast).

Prostate-specific antigen (PSA), a member of the kallikrein family of serine proteases, is a chymotrypsin-like glycoprotein produced by the prostate epithelium. Elevated serum PSA (> 4 ng/ml) is a tumor marker for prostatic cancer and benign prostatic hypertrophy; increasing serum PSA over time is indicative of metastatic disease. It has been suggested that PSA may contribute to tumor metastasis through degradation of extracellular matrix glycoproteins, as well as cleavage of IGF binding protein-3, a modulator of IGF-1. To elucidate the role of PSA in the development and progression of prostatic cancer, it is necessary to have a reliable, cost-effective source of enzymatically active protein. Previous efforts to express recombinant PSA (rPSA) produced inactive proPSA, or mixtures of active and inactive PSA requiring activation by removal of the propeptide. We describe the expression of active recombinant mature PSA in yeast. Stable chromosomal integration of a construct consisting of the yeast alpha-factor signal sequence preceding the mature PSA sequence resulted in secretion of rPSA. The rPSA was purified from the yeast cell culture supernatant to homogeneity by strong cation-exchange chromatography, and characterized by SDS-PAGE, Western analysis, electrospray mass spectrometry, N-glycanase digestion, N-terminal amino acid sequencing, and inactivation by a PSA-specific inhibitor. We report the production of active, mature rPSA in Pichia pastoris. Two forms of rPSA varying slightly in glycosylation were identified. The specific activity of the rPSA was equal to that of human seminal plasma PSA (0.56 micromol/min mg) as determined using a chromogenic substrate. Large-scale production of active rPSA will be useful in the exploration of PSA effects on tumor cell proliferation, migration and metastasis. In addition, a large supply of enzyme should facilitate the discovery of novel inhibitors for in vitro and in vivo evaluation, and may provide a reproducible source of rPSA for use as a standard in diagnostic testing.

Seminal plasma contains “BPSA,” a molecular form of prostate‐specific antigen that is associated with benign prostatic hyperplasia

BACKGROUND We previously reported that levels of BPSA, a modified form of prostate-specific antigen (PSA), are significantly elevated in prostate transition-zone tissue exhibiting nodular hyperplastic changes associated with the presence of benign prostatic hyperplasia (BPH). BPSA was purified and found to contain a characteristic clip between Lys182 and Ser183. We now describe the identification of BPSA in seminal plasma. METHODS PSA was purified from seminal plasma by immunoaffinity chromatography. The purified PSA was further resolved by hydrophobic interaction chromatography, and the individual PSA forms were analyzed by gel electrophoresis and N-terminal amino-acid sequencing. RESULTS BPSA comprised about 8% of the PSA in pooled seminal plasma, and was identical to BPSA purified from prostate tissues. BPSA was cleanly resolved from all active and inactive forms of PSA. Other inactive forms of PSA in seminal plasma consisted largely of PSA clipped at Lys145, though about 30% of the inactive seminal plasma PSA was intact, mature PSA. CONCLUSIONS BPSA represents a distinct form of inactive PSA in the seminal plasma that may represent a specific marker for the biochemical changes associated with nodular development in the prostate transition zone found in patients with BPH. Prostate 45:271–276, 2000. © 2000 Wiley-Liss, Inc.

Inactive free : total prostate specific antigen ratios in ejaculate from men with suspected and known prostate cancer, compared with young control men.

To measure free : total prostate specific antigen (PSA) ratios in ejaculate from men with suspected and known prostate cancer, and in young control men, to determine if this ratio might be useful in discriminating benign from malignant prostatic conditions. Patients, subjects and methods Forty-seven men with prostate cancer (positive biopsies), 52 men with suspected prostate cancer but who had negative biopsies and 28 young men (< 30 years old) and with no family history of cancer, provided either a single ejaculate specimen (total 59) or multiple specimens (total 193) on subsequent occasions. Free and total PSA were measured using appropriate assays. All specimens were diluted in a PSA-negative female serum pool. The median free : total PSA ratios were 0.76-0.81 among the patient groups and control men, and there was no statistical difference between the groups. These data presumably only reflect the inactive component of free PSA, given that any alpha2-macroglobulin or alpha1-antichymotrypsin in the assay serum diluent was likely to have bound the active free PSA component in these samples. Similar results were obtained from those providing single and multiple samples, suggesting that a single specimen is sufficient to reflect the seminal plasma free : total PSA ratio over that period. There was no relationship between seminal plasma free : total PSA ratio and age for the controls or the positive biopsy group, although there was a negative relationship (i.e. a decline with age) that almost reached significance in those with negative biopsies (P = 0.058, R2 = 0.07). This is the first report of free : total PSA ratios in the ejaculate of men with suspected and known prostate cancer compared with young control men. Although no significant changes were detected in the free : total PSA ratios in ejaculate, these results may be confounded by differences in ratios with age, as is the case for serum PSA or different molecular forms of PSA. Indeed, these data suggest that a large proportion of free PSA in seminal plasma may be inactive. Further studies are needed to determine the potential utility of measuring free : total PSA, or other candidate markers, in ejaculate to better discriminate benign from malignant prostate disease.

Seminal plasma contains "BPSA," a molecular form of prostate-specific antigen that is associated with benign prostatic hyperplasia.

We previously reported that levels of BPSA, a modified form of prostate-specific antigen (PSA), are significantly elevated in prostate transition-zone tissue exhibiting nodular hyperplastic changes associated with the presence of benign prostatic hyperplasia (BPH). BPSA was purified and found to contain a characteristic clip between Lys182 and Ser183. We now describe the identification of BPSA in seminal plasma. PSA was purified from seminal plasma by immunoaffinity chromatography. The purified PSA was further resolved by hydrophobic interaction chromatography, and the individual PSA forms were analyzed by gel electrophoresis and N-terminal amino-acid sequencing. BPSA comprised about 8% of the PSA in pooled seminal plasma, and was identical to BPSA purified from prostate tissues. BPSA was cleanly resolved from all active and inactive forms of PSA. Other inactive forms of PSA in seminal plasma consisted largely of PSA clipped at Lys145, though about 30% of the inactive seminal plasma PSA was intact, mature PSA. BPSA represents a distinct form of inactive PSA in the seminal plasma that may represent a specific marker for the biochemical changes associated with nodular development in the prostate transition zone found in patients with BPH.

SERUM AND SEMEN PROSTATE SPECIFIC ANTIGEN CONCENTRATIONS ARE DIFFERENT IN YOUNG SPINAL CORD INJURED MEN COMPARED TO NORMAL CONTROLS

Purpose Recent investigations have indicated that factors within the seminal plasma may contribute to the condition of low sperm motility in men with spinal cord injury. To determine whether the prostate gland functions normally in these men we chose prostate specific antigen (PSA) as a marker of prostatic function, and compared serum and semen concentrations in spinal cord injured and healthy noninjured men. Materials and Methods The study included 21 spinal cord injured men (mean age 33.3 +/- 1.2 years) and 22 noninjured normal men (mean age 30.3 +/- 1.5 years). Blood was obtained from subjects following at least 24 hours of abstinence from ejaculation and serum PSA was determined by modified enzyme immunoassay. Antegrade ejaculates from all subjects were frozen to -80C, exactly 15 minutes after collection. Seminal plasma PSA was determined using Hybritech Tandem [dagger] MP assay. Results Mean serum PSA concentration was 1.20 +/- 0.19 ng./ml. in spinal cord injured and 0.69 +/- 0.07 ng./ml. in noninjured men (p <0.02). Mean seminal plasma PSA concentration was 0.59 +/- 0.11 mg./ml. in spinal cord injured and 1.29 +/- 0.15 mg./ml. in noninjured men (p <0.001). Conclusions Our findings of elevated serum and decreased seminal plasma PSA concentrations indicate that prostatic secretory dysfunction is present in men with spinal cord injury.

Prostate-specific antigen (PSA) in human seminal plasma.

Prostate-specific antigen (PSA) was measured in seminal plasma and serum of 113 men under investigation for involuntary barrenness. PSA in seminal plasma was 0.4 +/- 0.3 (median 0.35) mg/mL and in serum 0.6 +/- 0.5 (median 0.6) ng/mL. The concentrations in these 2 compartments were independent of each other. The determination of PSA in neither blood serum nor seminal plasma brought new essentials for fertility status.

Two-dimensional gel electrophoresis detects prostate-specific antigen–α1-antichymotrypsin complex in serum but not in prostatic fluid

We investigated the interaction between prostate-specific antigen (PSA) and 1-antichymotrypsin (ACT) in prostatic secretions, identifying PSA and ACT in human serum, prostatic fluid, and seminal plasma by two-dimensional gel electrophoresis (2-D PAGE). Both PSA and ACT were detected in all three body fluids, but PSA-ACT complex was detected only in serum. Moreover, the 2-D PAGE Western blot staining profile for ACT from serum differed from that for prostatic fluid or seminal plasma. Incubation of prostatic fluid with purified ACT led to formation of PSA-ACT complex. Incubation of prostatic fluid with purified PSA, however, failed to form the complex, suggesting that the ACT in prostatic fluid was inactive or inhibited. Given that physiological concentrations of zinc inhibited the formation of PSA-ACT complex, we consider zinc a possible physiological inhibitor of the formation of the PSA-ACT complex. These results indicate that the failure to detect the PSA-ACT complex in prostatic fluid could be related to the inactivation of ACT, the presence of inhibitors (e.g., zinc), or simply the PSA:ACT ratio in the fluid.

The Influence of Ejaculation on Serum Levels of Prostate Specific Antigen

While prostatic manipulation and surgery have been shown to increase serum prostate specific antigen (PSA), the influence of ejaculation on serum PSA remains controversial. We examined the effect of ejaculation on serum PSA levels. We evaluated 100 men 25 to 35 years old with no history of surgery or inflammatory disease of the urogenital tract. Serum PSA was evaluated 1 and 24 hours after ejaculation, and serum testosterone and seminal fluid PSA levels were determined. In all men a baseline PSA level was detected. There were no statistically significant changes in serum PSA and testosterone 1 and 24 hours after ejaculation. Mean PSA concentration was significantly higher in seminal plasma than in serum. However, we did not observe a correlation between serum and seminal plasma PSA levels. Based on our data ejaculation does not affect serum PSA concentration in young men, and there seems to be no physiological relationship between ejaculation and PSA level.

Insulin-like growth factor-I and alpha 2-macroglobulin in seminal plasma correlate with semen quality.

Insulin-like growth factor-I (IGF-I) and alpha 2-macroglobulin (alpha 2-M) are believed to be involved in the development of germ cells. IGF-I is mainly controlled by concentrations of human growth hormone (HGH), influences cell proliferation and differentiation and its action is mediated by insulin-like growth factor-binding proteins (IGFBP), placental protein 14 (PP14) and prostate-specific antigen (PSA). alpha 2-M acts as a broad spectrum proteinase inhibitor and a binding protein for many cytokines and hormones, e.g. inhibin and activin. This study was designed to identify concentrations of these molecules in seminal plasma in normal semen samples of healthy men, correlations with semen quality, the relationship of IGF-I and alpha 2-M with factors affecting male fertility, and whether vasectomy influences the concentrations of these molecules. Concentrations of IGF-I and alpha 2-M in human seminal plasma were related to semen quality, basal concentrations of HGH, testosterone, IGFBP-3, soluble fibronectin receptor (sFNR), PSA and PP14 in seminal plasma and to serum concentrations of luteinizing hormone (LH) and follicle stimulating hormone (FSH). Commercially available assays were used to analyse 69 semen samples of various quality and 11 post-vasectomy samples. IGF-I concentrations in seminal plasma were significantly correlated with the percentage of morphologically normal spermatozoa (r = 0.748, P = 0.00001) and sperm concentration (r = 0.301, P = 0.011), but negatively correlated with serum FSH (r = -0.506, P = 0.00006) and PSA in seminal plasma (r = -0.388, P = 0.00009). Total alpha 2-M was significantly correlated with sperm count (r = 0.423, P = 0.0005), percentage of progressively motile spermatozoa (r = 0.444, P = 0.00019), quality of motility (sperm motile efficiency, r = 802, P = 0.00001) and straight line velocity (r = 0.411, P = 0.0013). Correlation between the sperm concentration and HGH in seminal plasma was weak (r = 0.287, P = 0.015). Vasectomy reduced the concentration of total alpha 2-M (P = 0.00008) and HGH (P = 0.0068) in the seminal plasma; IGF-I was also reduced after vasectomy when the total ejaculate amount was considered. Thus IGF-I and alpha 2-M are significant for the germ cell development: IGF-I in the maturation of spermatozoa and alpha 2-M in progressive motility.