Human Seminal Plasma Proteins Research Articles

Rapid detection of human seminal plasma proteins by membrane aspiration test (MAT)

A new dot-immunobinding method, called the membrane aspiration test (MAT), was developed for the detection of human seminal plasma (HSP) antigens. Biological materials, such as macerated vaginal swabs or extracts from seminal stains, were dotted on a nitrocellulose membrane in a disposable aspiration kit. After the drying and aspiration of a quenching solution, a suspension of gold particles (GP) coated with an immune serum to HSP was aspirated through the nitrocellulose membrane. The dotted portion on the membrane turned red after several repeated aspirations, if HSP antigen was present. Results can be obtained within a few minutes. This test has broad applicability because of its rapidity and simplicity.

訂正とおわび

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Analytical studies on cobalamin-binding proteins of human seminal plasma.

Analytical studies on cobalamin-binding proteins of human seminal plasma.

Evaluation and treatment of localized vaginal immunoglobulin E-mediated hypersensitivity to human seminal plasma

Localized vaginal inflammation after contact with seminal plasma has been previously described but without a clear understanding of the underlying immunopathogenesis. The purpose of this report is to describe three women who presented with localized postcoital vaginal reactions that were successfully treated with rapid immunotherapy using human seminal plasma proteins. One atopic and two nonatopic women with histories of immediate onset of localized postcoital vaginal symptoms and immediate skin test reactions to human seminal plasma were treated with rapid immunotherapy using their spouse's purified seminal plasma protein fractions. One of the women had decreased cutaneous reactivity post-therapy and detectable circulating specific immunoglobulin (Ig) G and IgE antibodies to her spouse's fractionated seminal plasma proteins. Increased IgG, IgE, and IgA antibodies were found in these patients' sera to whole seminal plasma of a normal male control and to fractionated proteins of different men, indicating that one or more homologous seminal plasma proteins may be responsible for these reactions. Their localized vaginal symptoms resolved completely after rapid immunotherapy treatment. Two of the women have remained symptom-free, whereas the third woman had recurrent localized vaginal symptoms after failing to maintain frequent sexual intercourse after treatment. Rapid immunotherapy immediately alleviated vaginal reactions to seminal plasma in all patients. These cases indicate that the pathogenesis of some localized vaginal inflammatory reactions may involve IgE-mediated or other immune responses to human seminal plasma proteins.

Transforming Growth Factor β as an Immunosuppressive Protein in Human Seminal Plasma

Human seminal plasma is known to exhibit immunosuppressive activity in seminal plasma. The purpose was to characterize immunosuppressive proteins in seminal plasma. Gel filtration fractions of 100 to > 440 kDa were identified that inhibited DNA synthesis and killing activity of interleukin-2 stimulated lymphocytes. The fractions exhibiting immunosuppression also inhibited DNA synthesis in a mink lung cell bioassay commonly used to measure the activity for transforming growth factor beta (TGF-beta). The negative growth activity was diminished by a TGF-beta neutralizing monoclonal antibody. TGF-beta was further detected in the active fractions by Western immunoblot. These results identified TGF-beta as an immunosuppressive protein in human seminal plasma and may provide insight into the role of immunosuppression played by seminal plasma, such as in reproduction and neoplasia.

Variations in 3H‐diisopropylfluorophosphate binding proteins in human seminal plasma

We have characterized the electrophoretic pattern and variations in 3H-diisopropylfluorophosphate (DFP)-binding proteins in human seminal plasma from normal men and from 103 patients attending the infertility clinic of our hospital. This study shows that 34 kDa prostate-specific antigen (PSA) is the major 3H-DFP-binding protein and that two other ubiquitous bands of 100 and 60 kDa are also present in seminal plasma from all the men studied. Additional bands of 92, 50-54 (doublet) and 38 kDa were also observed in some patients. The 38 kDa band was shown to be a highly glycosylated form of PSA. Further complexity was demonstrated by two-dimensional gel electrophoresis in the 27-30 kDa range of the gels since at least 10 major spots and rows of spots were seen. The concentration of these spots, including PSA, was extremely variable, as was their pattern of inhibition by various active site inhibitors of serine-proteases; these variations were not correlated with any specific sperm characteristics. With the exception of PSA, the proteins have not been identified. Their distribution suggests that most of them are exclusively of prostatic origin although a few could also derive from the seminal vesicles or blood. Future studies will be aimed at determining the nature of these proteins and their potential usefulness in andrology.

Relative ratios of lactoferrin, albumin, and acid phosphatase seminal levels as sperm quality markers in fertile and infertile men.

Human seminal plasma proteins from fertile and infertile men were fractionated by electrophoresis. The amounts of three Coomassie-stained protein bands were measured by densitometry. Their relative ratios were constant in normospermic men but varied in the infertile patients. Laboratory manipulation of the semen was shown not to affect the protein ratios as observed after liquefaction, incubation at various temperatures, and storage at -20 degrees C. The three proteins were purified by chromatographic techniques and identified as lactoferrin, albumin, and acid phosphatase by electrophoresis, high-pressure liquid chromatography, and enzyme assays. The use of these proteins to evaluate the contributions of different fluids to seminal plasma is discussed.

Presence of a 16 kd protein in human seminal plasma counteracts the effects of the anti-fertility agent, gossypol

Gossypol inhibited lactate dehydrogenase (LDH) noncompetitively in human spermatozoa. The inhibitory effect of gossypol on LDH was cancelled by the addition of human serum albumin, human gamma-globulin, bovine serum albumin or human seminal plasma. Seminal plasma was at least 10 times more effective than the other three proteins, when expressed on a per mg protein basis. Attempts were made to purify the active fraction from human seminal plasma. The purification steps included gel filtration, ammonium sulphate precipitation, centrifugal microconcentration and fast-performance liquid chromatography. A single active protein of Mr = 16,000 was purified to a final yield of 0.18%. The 16 kd protein was not observed in male blood plasma. The protein was found to be heat-stable and leucine-rich (16% of the molecule), and has been designated 'gossact'. The inhibitory effect of gossypol on the LDH reaction was completely blocked by the addition of gossact (5 micrograms/ml); human blood plasma (25 micrograms/ml) and human serum albumin (200 micrograms/ml) were far less potent in this assay. In addition, gossact bound 1.4 mol of gossypol/mol of protein with the dissociation constant (Kd) = 3.06 x 10(-5) M. The role of gossact in the protection of LDH from gossypol is discussed.

Identification of IgG and Fc-Binding Proteins in Human Seminal Plasma and Sperm

Human seminal plasma contains two novel soluble proteins capable of binding IgG and Fc, but not Fab. The IgG- and Fc-binding proteins were identified by immunoblotting using IgG of various species, Fc and Fab fragments. Their estimated molecular sizes are 16-kD and 20-kD. The monoclonal antibody (MAb), Leu 11b, raised against Fc gamma RIII interacts with the 16-kD protein, whereas other mAbs (32.2, IV.3, and 3G8) raised against FcR did not. The 16-kD protein is capable of binding IgG of several species (human, mouse, rabbits, and goat), whereas the 20-kD protein interacted only with human IgG-Fc fragment. The 16-kD IgG and the 20-kD Fc-binding proteins were found in the PBS extract of human sperm. Human seminal plasma/sperm contain an IgG- and a Fc-binding protein with estimated Mr of 16 and 20 kD, respectively.

ヒト精しょう蛋白の二次元電気泳動法による分析（第３報）

従来の二次元電気泳動を改良したデュアルミクロ二次元電気泳動と名づけた方法で, 精漿蛋白について泳動を行い, クマシー染色したゲルについて市販のレーザー画像処理装置により画像解析を行った. デュアルミクロ二次元電気泳動は, 一次元目で泳動した二つの試料を二次元目の濃度勾配ゲル1枚で泳動し, 染色するため, 泳動および染色条件が同一となり, 異なる二つの試料のスポットの比較や画像解析が容易であった.精漿蛋白の画像解析の結果, pI 5.7, MW5Kに位置するスポットの濃度は4.4%と測定された. また, このスポットについてアミノ酸分析を行った.

Identification of a Potential FSH Modulatory Protein in Human Testis and Seminal Plasma

A variety of factors capable of inhibiting the in vitro binding of FSH to its receptor have been identified in gonadal tissues from males and females. Interest in these factors has been stimulated because of their potential role as local modulators of gonadotropin action. Studies reported here were undertaken to determine if proteins having antigenic homologies with human FSH or an "FSH-like" protein isolated from porcine follicular fluid were present in human testicular tissue or seminal plasma. Polyclonal antibodies were generated against fractions of porcine follicular fluid containing FSH receptor binding inhibitory activity, FSH agonist activity in vitro, and a 58,000 Mr protein recognized by human FSH antiserum. Antiserum against this fraction of porcine follicular fluid and antiserum against human FSH were used to probe Western blots of proteins from human testis homogenates or seminal plasma. A 58,000 Mr protein was identified in both human testis extract and seminal plasma. This protein appears to be related antigenically to both human FSH and the 58,000 Mr "FSH-like" protein in porcine follicular fluid. It does not appear to be a metabolic degradatory product of human FSH since the protein is larger than FSH, does not dissociate into subunits under reducing conditions and is recognized by the antiserum to FSH-like protein that does not recognize human FSH. These data identify a 58,000 Mr protein in human testis and seminal plasma that may represent a local modulator of FSH action in the testis.

Immunologically reactive albumin-like protein in human testis and seminal plasma.

An immunologically reactive albumin-like protein (albumin) was localized, by an immunostaining technique, in the testis of infertile men (normal spermatogenesis, obstructive azoospermia) at the level of the Sertoli cells and in some cells of the germinal epithelium (secondary spermatocytes and early spermatids). No positive reaction was detectable in prepubertal testis. In vasectomized men, mean seminal albumin values were drastically reduced (by about 80%) in comparison to fertile controls, indicating a probable testicular origin. Mean seminal albumin values were also decreased in patients affected by azoospermia due to a seminiferous tubular lesion (about 40%) and in oligozoospermic patients (about 30%). In the same seminal samples transferrin, an index of Sertoli cell function, was also measured. Albumin and transferrin results were well correlated in the seminal plasma of each group (with the exception of vasectomized subjects), including a group of men with abnormally high concentrations of seminal transferrin. A weak correlation was found between seminal albumin and sperm count. We suggest that the presence of albumin in the human adult testis and in seminal plasma could be related to its ability to transport androgens.

A ram epididymal secretory protein shares common antigenic determinants with rat epididymal proteins and human seminal plasma proteins

A ram epididymal secretory protein shares common antigenic determinants with rat epididymal proteins and human seminal plasma proteins

Possible significance of transferrin levels in seminal plasma of fertile and infertile men.

Human seminal plasma contains large amounts of transferrin, which is a protein secreted mostly by Sertoli cells. It has been suggested that the concentration of transferrin may serve as a possible clinical marker of Sertoli cell function. Therefore the concentration of this protein in human seminal plasma from fertile and infertile men has been evaluated in order to find a relationship between transferrin concentrations and human semen parameters and plasma FSH levels. Findings show that seminal transferrin in subjects with oligozoospermia or azoospermia is significantly lower than in controls, and that it is strongly related to sperm count. Results also indicate that transferrin secretion can be impaired when plasma FSH levels are still normal, suggesting that seminal transferrin is an early and specific marker of Sertoli cell function. These results, however, do not clarify whether impairment of transferrin secretion by Sertoli cells is due to an organic dysfunction or to an organic secretory alteration.

Amino acid sequence of the predominant basic protein in human seminal plasma

The predominant basic protein in liquefied human seminal plasma is the major degradation product of the gel-forming protein secreted by the seminal vesicles. The amino acid sequence of this basic protein is presented. The basic protein contains 52 amino acid residues. It is devoid of cysteine, methionine, tryptophan, and leucine, but contains seven histidine residues located in the NH 2-terminal half of the molecule. The calculated M r of 5753 is in close agreement with that obtained from gel filtration in guanidine-HCl on Sephacryl S-200( M r = 6000).

Characterization of a seminal plasma-associated inhibitor of human seminal plasma protein kinase.

Human sperm-free seminal plasma (HSP) contains inhibitors (I) of the seminal plasma histone kinase activity (HK). One I is dialyzable and the other I is nondialyzable and precipitable by dialysis of HSP against a hypotonic buffer. When the nondialyzable, precipitable I fraction is resolubilized, it inhibits HK in a concentration-dependent manner. Sephadex G-25 column chromatography of whole HSP resolves I in both the void (Vo) and inclusion (Vi) volumes. Rechromatography of the VoI resolves I solely in the Vo. These and other data suggest that the ViI does not originate from the VoI, and that both I activities represent separate molecular entities. VoI was further characterized and found to be heat labile, trypsin and neuraminidase insensitive, and alpha-chymotrypsin sensitive. VoI is not soluble in CHCl3 or CHCl3:CH3OH (2:1) and is not adsorbed by charcoal. Chromatography of VoI on Sephadex G-100 yields a broad peak of I that migrates just past the Vo. VoI has no detectable cyclic AMP (cAMP) binding activity and VoI activity is not affected by coincubation of VoI and HK with cAMP. VoI also does not bind to zinc-chelate or phenothiazine affinity columns. These data suggest that VoI is protein in nature with properties distinct from the class of previously described protein kinase inhibitors. Although the identity of VoI is not known, it does not appear to be the regulatory subunit of a cAMP-dependent protein kinase, calsemin or a zinc binding protein.

Characterization of the predominant basic protein in human seminal plasma, one cleavage product of the major seminal vesicle protein.

From liquefied human seminal plasma, we purified the predominant basic protein which appears following liquefaction of coagulated semen. The protein was purified in the presence of di-isopropylfluorophosphate to retard its degradation. Heparin-Sepharose chromatography was followed by gel filtration (Biogel P 60) and by fast performance liquid chromatography on a reversed phase column (C8). The basic protein is a single polypeptide chain with an apparent molecular mass of 12.8 kDa, and has a pI value between that of trypsinogen (9.3) and cytochrome C (10.3). The protein contains no carbohydrate, is rich in histidine, glutamate, and lysine, but is devoid of both cysteine and methionine. The amino-terminal portion of the protein sequence is unique: H N K Q E G R D H D K S K G H F H R V V I H H K G G K A H R G-. A specific rabbit antiserum was raised against the 12.8 kDa basic protein. The protein was found to be unique to seminal plasma among all extracellular fluids examined. Three immunologically related 52 kDa, 71 kDa, and 76 kDa proteins were identified in seminal vesicle secretion when it had been reduced. Prostatic enzyme(s) degraded these proteins to the 12.8 kDa basic protein and several other basic proteins with apparent molecular masses below 18 kDa.

A thin‐layer isoelectric focusing method for the separation of proteins in follicular fluid and seminal plasma

AbstractA thin‐layer electrofocusing method has been developed for the separation of proteins in human follicular fluid, seminal plasma, and blood plasma/serum. The method has a number of unique features. It is simple and rapid, and it produces distinct, undistorted bands that can be readily quantitated. Any samples solubilized by standard methods can be analyzed, and it can be adapted to favor specific regions of the pH spectrum. The gels used are composed of agarose and sorbitol, and they are formed between a plastic template and a Mylar film. Sample volumes ranging from 0.5 to 5 μl can be used. Protein separation is usually completed in 30–40 min at constant power of 1 or 1.5 W. The use of low power ensures that little heat is generated during the run and hence only minimal temperature control is needed. Carbon rod electrodes are a special feature as they allow the inverted gel to rest in direct contact with the electrodes, eliminating the necessity for electrode solutions. The inverted gel position allows fluid to drain from the surface of the gel and so prevents distortion of protein patterns. This overcomes the problems inherent in conventional horizontal electrofocusing of unpurified biologic fluids such as plasma, follicular fluid, and seminal plasma. Rapid protein staining is carried out with fixed and dried gels. The excellent resolution and the lack of distortion make this method suitable for reliable quantitative analysis of differences between samples by means of laser beam densitometry. This has enabled us to assess differences in specific follicular fluid proteins between individual follicles in the human, and to correlate these differences with fertilizability and cleavage potential of oocytes derived from such follicles.

Immunonephelometry of specific proteins in human seminal plasma.

Using immunonephelometry, we measured albumin, transferrin, alpha 1-antitrypsin, orosomucoid, hemopexin, IgG, IgA, and secretory IgA in seminal plasma of men who were attending a fertility clinic. Seminal plasma, obtained by centrifugation of semen, is a turbid liquid producing high blank values that were not diminished by ultracentrifugation, filtration, or treatment with protamine, dextran sulfate, or various lipidophilic gels. Pretreatment of seminal plasma with polyethylene glycol 6000 (final concentration, 40 g/L) considerably decreased the blank values without appreciably changing the relative concentration of the eight proteins studied. Our investigations illustrate the feasibility of this application of immunonephelometry. This approach can be a valuable diagnostic tool in the laboratory evaluation of the male partner in infertile unions.

Immunologic Determination of Seminal Secretions by a Latex Microtiter Technique

The presence of human seminal plasma proteins in biological stains was demonstrated by an absorption test using anti-human seminal protein rabbit serum decreased degree of antibody activity was titrated on a microtiter plate by the agglutination of latex particles coated with human seminal plasma proteins. This method of test was sensitive and highly specific. The latex reagent for this test could be preserved in a refrigerator for over one year without the loss of reactivity.