The major seminal vesicle secreted proteins in human semen, semenogelin I and semenogelin II, interact non-covalently and via disulphide bridges to instantly form a coagulum upon ejaculation. The coagulum is liquefied after a few minutes due to the action of a prostatic serine protease, prostate-specific antigen (PSA). In contrast to rat semen, which forms an insoluble plug within minutes of expulsion, no transglutaminase-mediated cross-linking has been demonstrated in ejaculated human semen. However, we here show that semenogelin I and semenogelin II, both in seminal vesicle fluid and purified from semen, are substrates for factor XIIIa, the fibrin cross-linking transglutaminase. The cross-linking of the semenogelins, which was conformation-dependent, and the incorporation of a fluorescence-labelled amine, were visualised by SDS/PAGE and Western blot. Purified semenogelin I and semenogelin II could be cross-linked separately into complexes. Moreover, digestion of semenogelin with PSA produced fragments, some of which were cross-linked into complexes by factor XIIIa. We also found that PSA was unable to release any semenogelin fragments during exposure of the high molecular-mass complexes of cross-linked semenogelin to active PSA.Keywords : Semenogelin ; seminal vesicle; semen ; transglutaminase ; prostate-specific antigen.Immediately after ejaculation, human semen form a coagu-ejaculation, these proteins are highly cross-linked and form an insoluble complex known as the copulatory plug, which blocks lum. This is mainly due to interactions between semenogelin I and semenogelin II, which are the major coagulating proteins in the vagina. SVS II is the major protein in the plug (see [13] for a review). The cross-linking is mediated by a prostate-specific human semen [1, 2]. The coagulum can be dissolved using urea transglutaminase, which is secreted from the coagulating gland and a reducing agent, indicating that the interactions are mainly in an apocrine fashion [14Ϫ16]. of non-covalent nature and via disulphide bridges [3]. SemenoTransglutaminases, which are Ca 2ϩ -dependent transferases, gelin I consists of 439 amino acid residues, appears at 52 kDa catalyse the acyl transfer reaction between glutamine residues in in SDS/PAGE, and is expressed exclusively in the seminal vesipeptides and primary amine groups [17Ϫ19]. Factor XIII cles [4Ϫ6]. Semenogelin II comprises 559 amino acid residues (FXIII) in human blood is probably the best characterised transand exists in a non-glycosylated and a glycosylated form glutaminase, consisting of two a and two b subunits. Upon (71 kDa and 76 kDa, respectively, in SDS/PAGE) [6Ϫ8]. Semecleavage by thrombin and binding of calcium to the a subunit, nogelin II is expressed primarily in the seminal vesicles, but also the b subunits dissociate, exposing the active site in the a subunit in the caudal region of the epididymis [5].(FXIIIa) [19]. Other human transglutaminases (i.e. keratinocyte, The coagulum formed in human semen upon ejaculation is tissue and epidermal transglutaminases...

Twelve research groups participated in the ISOBM TD-3 Workshop in which the reactivity and specificity of 83 antibodies against prostate-specific antigen (PSA) were investigated. Using a variety of techniques including cross-inhibition assays, Western blotting, BIAcore, immunoradiometric assays and immunohistochemistry, the antibodies were categorized into six major groups which formed the basis for mapping onto two- and three-dimensional (2-D and 3-D) models of PSA. The overall findings of the TD-3 Workshop are summarized in this report. In agreement with all participating groups, three main antigenic domains were identified: free PSA-specific epitopes located in or close to amino acids 86–91; discontinuous epitopes specific for PSA without human kallikrein (hK2) cross-reactivity located at or close to amino acids 158–163; and continuous or linear epitopes shared between PSA and hK2 located close to amino acids 3–11. In addition, several minor and partly overlapping domains were also identified. Clearly, the characterization of antibodies from this workshop and the location of their epitopes on the 3-D model of PSA illustrate the importance of selecting appropriate antibody pairs for use in immunoassays. It is hoped that these findings and the epitope nomenclature described in this TD-3 Workshop are used as a standard for future evaluation of anti-PSA antibodies.

Mouse seminal vesicles secrete four major protein components with estimated molecular masses of 95, 38, 17, and 16 kDa. Amino acid sequencing revealed that the 95-kDa component represents a protein with an unknown structure, while the 38-kDa component was identified as semenoclotin, the 17-kDa component as seminal-vesicle-secreted protein IV, and the 16-kDa component as seminal-vesicle-secreted protein V. Semenoclotin and the 95-kDa component were readily cross-linked by transglutaminase, suggesting that the two proteins are involved in the formation of the mouse copulatory plug. Treatment of mouse seminal vesicle fluid with human prostate-specific antigen rapidly degraded semenoclotin, indicating a structural resemblance of this protein to human semenogelins, despite the vast difference in primary structure. As previously reported for other seminal-vesicle-secreted proteins, the semenoclotin transcripts are shown to be under androgen control.Keywords: seminal vesicle ; semen ; androgen; transglutaminase ; coagulation.Mammalian semen is formed by the mixing of spermatozoarich fluid from epididymis with secretions from the accessory sex glands during ejaculation, that mainly consists of fluids from the seminal vesicles and the prostate (Mann and Lutwark-Mann, 1981). In most species, the major contribution is provided by the seminal vesicles, amounting to more than half of the semen volume. The newly ejaculated sperm is highly viscous or gellike, but within a short time it will either liquefy, as in man, or transform into a firm coagulum, yielding a copulatory plug through the action of a prostate-secreted transglutaminase, as seen in rodents.The proteins responsible for gel and clot formation are present at very high concentrations in the seminal vesicle fluid. Somewhat unexpectedly, the primary structure of these proteins are highly dissimilar between species (Lundwall and Lazure, 1995; Lundwall and Ulvsback, 1996). This is caused by a relatively recent evolution of an ancestral gene, involving duplications, exon expansion, and varying selection of splice sites. The genes constitute the REST gene family (abbreviation for rapidly evolving substrates for transglutaminase) as most members seem to encode substrates for transglutaminase (Lundwall, 1996 : Lundwall and Ulvsback, 1996).The rat seminal vesicle secretion consists of five predominant protein components, designated seminal-vesicle-secreted proteins I-V (SVS I-V) in order of size (Ostrowski et al., 1979). The SVS IV and V components are also referred to as S and F proteins by some authors (Higgins et al., 1976). The strucCorrespondence to A. Lundwall,

Monoclonal antibodies were raised against prostate-specific antigen (PSA) by immunization with purified free PSA, i.e. not in complex with any protease inhibitor (F-PSA) and PSA in complex with alpha1-anti-chymotrypsin (PSA-ACT). Epitope mapping of PSA using the established monoclonal antibody revealed a complex pattern of independent and partly overlapping antigenic domains in the PSA molecule. Four independent antigenic domains and at least three partly overlapping domains were exposed both in F-PSA and in the PSA-ACT complex, while one antigenic domain was specific for F-PSA. The different domains contained both continuous and discontinuous epitopes. The combination of antibodies recognizing antigenic domains exposed both in F-PSA and PSA-ACT made it possible to develop several highly sensitive sandwich immunoassays for determination of total PSA, i.e. F-PSA + PSA-ACT, with the same molar response for F-PSA and PSA-ACT. Assays specific for F-PSA (cross-reactivity between F-PSA and PSA-ACT < 1%) were developed by the combination of antibodies recognizing epitopes exposed only in F-PSA and antibodies recognizing epitopes exposed both in F-PSA and PSA-ACT. Images Figure 3

Antibodies submitted to the ISOBM TD-3 PSA Workshop were tested for reactivity with free prostate-specific antigen (PSA) and the PSA α₁-antichymotrypsin (ACT) complex, inhibition of proteolytic activity, recognition of continuous epitopes, and epitope specificity based on complete cross-inhibition studies. The antibodies could be separated into two categories: those recognizing hidden epitopes specific for free PSA, and those recognizing epitopes in both free PSA and the PSA-ACT complex. A large number of distinct antigenic domains were identified both in free PSA and the PSA-ACT complex.