Search

Formation of copulatory plugs by male animals is a common means of reducing competition with rival males. In mice, copulatory plugs are formed by the coagulation of seminal vesicle secretion (SVS), which is a very viscous and self-clotting fluid containing high concentration of proteins. In its native state, mouse SVS contains a variety of disulfide-linked high-molecular-weight complexes (HMWCs) composed of mouse SVS I-III, which are the major components of mouse SVS. Further, mouse SVS I-III are the substrates for transglutaminase 4 (TGM4), a cross-linking enzyme secreted from the anterior prostate. According to activity assays, mouse TGM4 prefers a mild reducing and alkaline environment. However, under these conditions, the activity of mouse TGM4 toward SVS I-III was much lower than that of a common tissue-type TGM, TGM2. On the other hand, mouse TGM4 exhibited much higher cross-linking activity than TGM2 when native HMWCs containing SVS I-III were used as substrates under non-reducing condition. By the action of TGM4, the clot of SVS became more resistant to proteolysis. This indicates that the activity of TGM4 can further rigidify the copulatory plug and extend its presence in the female reproductive tract. Together with the properties of TGM4 and the nature of its disulfide-linked SVS protein substrates, male mice can easily transform the semen into a rigid and durable copulatory plug, which is an important advantage in sperm competition.

Background and Purpose: As prognosis for patients with metastatic ovarian cancer is generally poor, advances in treatment are needed. Here, we studied the mechanism of action of a recombinant viral capsid protein (rVP1) and explored its effect against ovarian tumour growth and metastasis in vivo., Experimental Approach: The human ovarian cancer cell line SKOV3 and BALB/cAnN-Foxn1 female nude mice were used. Effects of rVP1 on the viability, invasive ability, matrix metalloproteinase (MMP)-2 activity and cancer cell proliferation and metastasis were determined by cell proliferation assay, Matrigel invasion assay, gelatin zymographic analysis, as well as bioluminescence imaging and immunohistological analysis in xenograft mouse models respectively. Levels of total and phosphorylated focal adhesion kinase (FAK), PKB/Akt, phosphatase and tensin homologue (PTEN) and glycogen synthase kinase-3β (GSK-3β) were detected by Western blotting., Key Results: rVP1 promoted apoptosis and decreased invasion of human ovarian cancer cells. This effect of rVP1 was accompanied by activation of PTEN and GSK-3β as well as down-regulation of FAK, Akt and MMP-2. Anti-integrin antibodies or overexpression of constitutively active Akt reversed the cellular effects of rVP1. Orthotopic and intraperitoneal xenograft mouse models demonstrated that rVP1 attenuated survival and metastasis of human ovarian cancer SKOV3 cell line in vivo through selective regulation of Akt and GSK-3β activity as shown by bioluminescence imaging of mice and immunohistochemical analysis., Conclusion and Implications: These results indicate that negative regulation of Akt signalling and MMP-2 by rVP1 may have the potential to suppress ovarian tumour growth and metastasis in vivo., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Using mice as experimental animals, proteins in the uterine luminal fluid (ULF) from both adults and diethylstilbestrol dipropionate (DES)-treated immature animals were resolved by 2D gel electrophoresis. Two of the protein spots, (a) and (b) around the positions of 18-20 kDa, in the adult ULF were not found in the DES-treated ULF. Automated Edman degradation established the same N-terminal sequences of AHQVPVKTKGKHVFP for the two protein spots. Two trypsin digests of spot (a) were analyzed using CID MS/MS to establish the peptide sequences DNQLGPLLPEPK and RPDAMTWVETEDILSHLR. These partial sequences were confirmed in the cDNA-deduced mouse proline rich acidic protein (PRAP). Using human Ishikawa cell line as a surrogate endometrial model, we demonstrated rapid entrance of exogenous PRAP into the cells and its ability to enhance alkaline phosphatase activity of the E(2) -stimulated cells. Further, the transcripts of five estrogen-responsive genes, including ALPP (Placental alkaline phosphtase), ALPPL (placental alkaline phosphatase-like 2), TGF (transforming growth factor), PR (progesterone receptor), and Wnt7a, were measured after the cell incubation in modified Eagle medium containing 0.1 nM E(2) , or 0-25 µM PRAP, or both together at 37°C for 48 h. As compared with the control, E(2) alone increased the transcripts of ALPP, ALPPL, TGF-α, and PR, and reduced the transcript of Wnt7a, whereas PRAP alone had a slight impact on their expression. E(2) together with PRAP greatly increased the E(2) -stimulated transcriptions of ALPP, ALPPL, TGF-α, and PR, and markedly reduced the E(2) -suppressed transcription of Wnt7a., (Copyright © 2011 Wiley Periodicals, Inc.)

Syncytin 2 is a newly identified placental membrane protein with fusogenic and immunosuppressive activities. Major facilitator superfamily domain containing 2A (MFSD2A) is the cognate receptor for syncytin 2-mediated cell-cell fusion. Both syncytin 2 and MFSD2A are highly expressed in placenta. In this study to understand the regulation of syncytin 2 and MFSD2A expression in placenta, we found that syncytin 2 gene is epigenetically silenced in nonplacental cells by cytosine-phosphate-guanine (CpG) dinucleotide methylation and that expression of syncytin 2 and MFSD2A genes are regulated by the placental transcription factor GCM1 in placental cells. Functional GCM1-binding sites were identified in syncytin 2 and MFSD2A promoters based on electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Because GCM1 activity is decreased in hypoxic placental cells, we further confirmed that expression of MFSD2A is downregulated in hypoxic BeWo choriocarcinoma cells. Interestingly, ectopic expression of GCM1 activated syncytin 2 and MFSD2A expression in MCF-7 breast cancer cells and facilitated MCF-7 cell fusion. The expression of syncytin 2 in MCF-7 cells was partly attributed to CpG demethylation in the syncytin 2 promoter in the presence of GCM1. Our results suggest that GCM1 is a critical factor in controlling placental cell fusion through transcriptional regulation of syncytin 2 and MFSD2A gene expression in placenta. In addition, GCM1 may also play an important role in the epigenetic regulation of syncytin 2 gene expression.

We identified a testis-specific protease-like protein tentatively named TESPL and a pancreatic trypsinogen Prss2 from the clones of a yeast two-hybrid screen against a mouse testicular cDNA library using the trypsin inhibitor Spink3 from male accessory sexual glands as bait. The enzymatic motifs and the cysteine patterns in serine proteases are highly conserved in these two proteins. Based on the phylogenetic analysis, Prss2 duplicated recently and TESPL underwent distant evolution without gene duplication from the progenitor of trypsin-like and chymotrypsin-like proteases. We found that TESPL transcription was restricted to the testis and that the level of transcription was positively correlated with animal maturation. In contrast, Prss2 was constitutively expressed in many tissues including testis. Alignment of the cDNA-deduced sequences of serine proteases showed the replacement of an essential serine residue in the catalytic triad of serine proteases by a proline residue in TESPL, which was demonstrated to be a membrane-bound protein devoid of proteolytic activity. The immunohistochemical staining patterns of seminiferous tubules in the testis revealed TESPL mainly on postmeiotic cells such as spermatids and spermatozoa. On the mouse sperm from caudal epididymis, TESPL was localized mainly on the plasma membrane overlaying the acrosomal region. Further, orthology group for mouse TESPL was identified in the conserved gene family of eutherian testis serine protease 5., ((c) 2010 Wiley-Liss, Inc.)

SVS I was exclusively expressed in seminal vesicle in which the protein was immunolocalized primarily to the luminal epithelium of mucosal folds. The developmental profile of its mRNA expression was shown to be androgen-dependent, manifesting a positive correlation with the animal's maturation. There are 43 glutamine and 43 lysine residues in one molecule of SVS I, which is one of the seven major monomer proteins tentatively assigned on reducing SDS-PAGE during the resolution of mouse seminal vesicle secretion. Based on the fact that SVS I-deduced protein sequence consists of 796 amino acid residues, we produced 7 recombinant polypeptide fragments including residues 1-78/F1, residues 79-259/F2, residues 260-405/F3, residues 406-500/F4, residues 501-650/F5, residues 651-715/F6, and residues 716-796/F7, and measured the covalent incorporation of 5-(biotinamido)pentylamine (BPNH(2)) or biotin-TVQQEL (A25 peptide) to each of F1-to-F7 by type 4 transglutaminase (TG(4)) from the coagulating gland secretion. F2 was active to a greater extent than the other fragments during the BPNH(2)-glutamine incorporation, and a relatively low extent of A25-lysine cross link was observed with all of the seven fragments. The MS analysis of BPNH(2)-F2 conjugate identified Q(232) and Q(254) as the two major TG(4) cross-linking sites. This was substantiated by the result that much less BPNH(2) was cross-linked to any one of the three F2 mutants, including Q232G and Q254G obtained from single-site mutation, and Q232G/Q254G from double-site mutation., ((c) 2009 Wiley-Liss, Inc.)

A 75-kDa protein secreted from mouse coagulating gland was purified to homogeneity by a series of isolation steps including ion exchange chromatography on a DEAE-Sephacel column and ion exchange high-performance liquid chromatography on a sulfopropyl column. It was identified to be Type IV transglutaminase (TG(4)), based on the establishment of N-terminal sequences by automated Edman degradation together with partial sequences by MS analysis. Its cross-linking activity was tested on the reduced sample of mouse seminal secretion which contained seven major monomer proteins tentatively designated as SVS I-VII. The enzyme was able to cross-link any of SVS I-III but failed to cross-link the other SVS proteins with a M(r) value less than 14 kDa. SVS I and SVS III showed comparable substrate activity, but were much weaker than SVS II during the TG(4) catalysis.

Mammalian sperm gain the ability to fertilize an egg successfully by the capacitation process. An unregulated capacitation process causes sperm to undergo a spontaneous acrosome reaction (AR) and resulting in loss of their fertilization activity. Thus, functional sperm activation is tightly regulated by a capacitation and suppression (decapacitation) mechanism. Factors, such as platelet-activating factor (PAF) present in both sperm and the female genital tract, are able to stimulate sperm capacitation. Seminal plasma is thought to have the ability to suppress sperm capacitation; however, the regulatory mechanisms of seminal plasma protein on sperm capacitation are not well understood. Recently, we demonstrated that seminal vesicle autoantigen (SVA), a major seminal vesicle secretory protein, is able to suppress mouse sperm capacitation. To further study the suppression spectra of SVA on sperm capacitation, we investigated the effect of SVA on PAF-induced mouse sperm capacitation-related signals. Here, we demonstrate that SVA decreases the [Ca(2+)](i) to suppress the PAF's effects on [Ca(2+)](i), the cAMP level, protein tyrosine phosphorylation, and capacitation. The inhibition of PAF-induced protein tyrosine phosphorylation and capacitation by SVA can be reversed by cAMP agonists. Characterization of the interactions of SVA with PAF by TLC overlay and tryptophan fluorescence spectrum analyses indicates that SVA is capable of binding PAF with an apparent dissociation constant K(d) > 50 microM. Together with these results, we demonstrate that SVA deceases [Ca(2+)](i) and cross-talks with PAF-induced intracellular signals to regulate mouse sperm capacitation.

P12 is a Kazal-type trypsin inhibitor that has been purified from mouse seminal vesicle secretion. We observed a slight impact of P12 on sperm capacitation, and demonstrated the removal of plasma membrane overlaying the acrosome region by immunoaggregation of P12 on mouse sperm. Further, we compared the immunoreactivity of P12 antibody to ten P12 variants, including six single-site mutated mutants (R19L, Y21V, D22G, R43G, K44S, and R45T), two multisite mutated mutants (R43G/K44S/R45T and L50H/R52G/K53A), and two deletion mutants (Nd10 and Cd8) in which 10 and 8 residues were deleted from the N- and C-terminals, respectively. We found that the N-terminal region, 43RKR45, and the C-terminal region, but not R19, Y21, and D22, are involved in the three epitopes that reside on one side and are three-dimensionally distant from R19, Y21, and D22 on the P12 molecule. Based on the epitope topology, we elucidated the structural basis by which P12 antibody immunoaggregated P12 on sperm head.

The molecular basis of mammalian sperm capacitation, either in vivo in the female reproductive tract, or in vitro, is poorly understood. It is well known that sperm capacitation is associated with an increase in tyrosine phosphorylation of a subset of proteins. We resolved the phosphoproteins in the cell lysate of mouse sperm after capacitation by 2-DE. One tyrosine-phosphorylated 130-kDa spot was trypsin-digested, and six oligopeptide sequences were established from the MS data. These were confirmed in a CCCTC-binding nuclear factor (CTCF), a widely expressed and highly conserved protein. Further, both an anti-phosphotyrosine antibody and an anti-CTCF antibody showed immunoreactivity to a 130-kDa component in the immunoprecipitates obtained after incubation of the cell lysate from the capacitated sperm using another anti-CTCF antibody. The data support the presence of a tyrosine-phosphorylated CTCF in the capacitated sperm. Immunolocalization of the CTCF revealed fluorescent staining in the acrosome region in both capacitated and incapacitated sperm. The electrophoretic mobility shift assay, using a CTCF target sequence 5'-GGCGGCGCCGCTAGGGGTCTCTCT-3' found in the promoter of the amyloid beta-protein precursor, manifested that, relative to CTCF in the incapacitated sperm, the tyrosine-phosphorylated protein in the capacitated sperm had stronger affinity to the CTCF target sequence.

Capacitation is the prerequisite process for sperm to gain the ability for successful fertilization. Unregulated capacitation will cause sperm to undergo a spontaneous acrosome reaction and then fail to fertilize an egg. Seminal plasma is thought to have the ability to suppress sperm capacitation. However, the mechanisms by which seminal proteins suppress capacitation have not been well understood. Recently, we demonstrated that a major seminal vesicle secretory protein, seminal vesicle autoantigen (SVA), is able to suppress bovine serum albumin (BSA)-induced mouse sperm capacitation. To further identify the mechanism of SVA action, we determine the molecular events associated with SVA suppression of BSA's activity. In this communication, we demonstrate that SVA suppresses the BSA-induced increase of intracellular calcium concentration ([Ca2+]i), intracellular pH (pH(i)), the cAMP level, PKA activity, protein tyrosine phosphorylation, and capacitation in mouse sperm. Besides, we also found that the suppression ability of SVA against BSA-induced protein tyrosine phosphorylation and capacitation could be reversed by dbcAMP (a cAMP agonist).

CEACAM10 was purified from mouse seminal vesicle secretions by a series of purification steps that included ion exchange chromatography on a DEAE-Sephacel column and ion exchange high-performance liquid chromatography on a sulfopropyl column. It was shown to be a 36-kDa glycoprotein with an N-linked carbohydrate moiety. The circular dichromoism spectrum of CEACAM10 in 50 mM phosphate buffer at pH 7.4 appeared as one negative band arising from the beta form at 217 nm. CEACAM10 was expressed predominantly in seminal vesicles of adult mice. Both CEACAM10 and its mRNA were demonstrated on the luminal epithelium of the mucosal folds in the seminal vesicle. The amount of Ceacam10 mRNA in the seminal vesicle was correlated with the stage of animal maturation. Castration of adult mice resulted in cessation of Ceacam10 expression, while treatment of castrated mice with testosterone propionate in corn oil restored Ceacam10 expression in the seminal vesicle. During the entire course of pregnancy, Ceacam10 might be silent in the embryo. A cytochemical study illustrated the presence of the CEACAM10 binding region on the entire surface of mouse sperm. CEACAM10-sperm binding greatly enhanced sperm motility in vitro.

Among the major proteins in the rat seminal vesicle secretion, transglutaminase catalyzed the cross-links among RSVS I-III. Six peptide sequences determined from the trypsin digests of RSVS III were confirmed in the protein sequence derived from two paralogs, RSVS III(alpha) and RSVS III(beta) gene, in rat 3q42. Their transcription units are organized with the first exon encoding a signal peptide, and the second a secreted protein, whereas the third encompasses a 3'-non-translated nucleotide that shares common features of rapidly evolving substrates of transglutaminase (REST) gene family. These two genes have 99% identity in their coding region and both express in adult rats with the same transglutaminase cross-linking site, manifesting functional preservation. All of REST genes reported thus far for human and Muridae were mapped in a chromosomal locus between KCNS1 and SLPI suggesting the locus as an active evolving region. The molecular evolution of this gene family is discussed.

Six variants of P12, a Kazal-type trypsin inhibitor in the secretion of male mouse accessory sexual glands, were made using single-site mutations including R19L, Y21V, D22G, R43G, K44S, and R45T, based on one-letter-code mutation of amino acids. The other two variants, Nd10 and Cd8, were made using the deletion of 10 and 8 residues from the N- and C-terminals, respectively. Their CD profiles revealed maintenance of the P12 conformation in the seven variants, excluding Cd8, which became unfolded. Only R19L entirely lost the ability while the other variants were as strong as P12 in inhibiting the trypsin digestion of N-benzoyl-Phe-Val-Arg 7-amido-4-methylcoumarin. The immunocytochemical results demonstrated that D22G and Cd8 failed to bind to sperm, Y21V very weakly did so, and the other variants retained their sperm-binding abilities. Concomitantly, the immunocytochemical stainability of each ligand was parallel to its inhibitory effect on 125I-P12-sperm binding, and a synthetic oligopeptide corresponding to residues 18-24 of P12 was able to inhibit P12-sperm binding. The data together concluded that R19 was essential for protease inhibition and D22 and/or Y21 mainly being responsible for the binding of P12 to sperm. The steric arrangement of R19, Y21, and D22 on the tertiary structure of P12 is discussed.

The leading cause of food poisoning in both Taiwan and Japan is Vibrio parahaemolyticus infection, whose mechanism of enteropathogenesis is still unclear. To evaluate whether surface components are responsible for the intestinal adhesion of V. parahaemolyticus, we have developed a novel method for isolating the capsular polysaccharide (CPS) from V. parahaemolyticus (serotype O4:K8). We found that culturing of V. parahaemolyticus in broth for 1 week or more changed the colony form of the bacteria on an agar plate from opaque to translucent. The translucent colonies of V. parahaemolyticus contained little CPS and exhibited a much lower level of adherence to epithelial cells (Int-407) than the opaque colonies of the bacteria. Incubation of V. parahaemolyticus in medium supplemented with bile increased the levels of CPS and adherence. Treatment of V. parahaemolyticus with anti-CPS but not anti-LPS serum decreased the level of bacterial adherence. In addition, purified CPS bound to epithelial cells in a dose-dependent manner. Intranasal administration of CPS to mice in the presence of adjuvants such as immunostimulatory sequence oligodeoxynucleotides or cholera toxin elicited CPS-specific mucosal and systemic immune responses. These results indicate that CPS plays an important role in the adherence of V. parahaemolyticus to its target cells and may be considered a potential target for the development of a vaccine against this pathogen.

The nucleotide sequence of MpSv-1, a novel androgen-regulated gene exclusively expressed in mouse seminal vesicle, was analyzed to establish a 5'-flanking region of 2123 bp, three exons of 95, 765, and 330 bp, and two introns of 222 and 811 bp. The transcription unit is organized with the first exon encoding a signal peptide, and the second a secreted protein, whereas the third encompasses a 3'-non-translated nucleotide that shares common features of rapid evolving substrates of transglutaminase gene family. The protein sequence deduced from this gene contains 265 amino acid residues in which the central part, residues 116-145, is a region composed of five short tandem repeats, consisting of four amino acid residues, QXK(S/T), where X is an aliphatic amino acid residue. Among the mouse seminal vesicle secretory proteins that could be resolved by SDS-PAGE into seven major components, SVS I-VII, the antiserum against residues 77-109 of the MpSv-1-translated protein only reacted with SVS III. Matrix-assisted laser desorption/ionization-time of flight mass spectral analysis from a trypsin digest of SVS III supported this protein as derived from MpSv-1. SVS III was immunolocalized to the epithelium of both the primary and secondary folds of the seminal vesicle and the copulatory plug. All of mouse SVS I-III were proven to be substrates of transglutaminase and could be cross-linked readily after the enzyme reaction. The transglutaminase cross-linking site of SVS III was identified to be the tandem repeats of QXK(S/T) in the central part of this protein molecule.

We demonstrate the presence of complement factor B (Bf) and complement C3 in uterine luminal fluid collected from estrogen-stimulated immature and adult female mice. We examined the synthesis and secretion of these two proteins in mouse endometrium at various stages of the natural estrous cycle and during the pregnancy period. The mRNA levels of these two proteins increased markedly in proestrus and estrus and declined sharply in metestrus to an undetectable level. The Bf mRNA remained undetectable, whereas a readily detectable C3 mRNA level reappeared, in diestrus. Meanwhile, these two proteins were immunolocalized to the apical cytoplasm of glandular and luminal epithelial cells of the endometrium during the estrous cycle. Administration of an estrogenic steroid to immature or ovariectomized adult mice markedly stimulated the expression of Bf, C3, and their RNA messages in the endometrium, whereas injection of progesterone alone to ovariectomized animals did not stimulate their expression. Expression of C3 was remarkably enhanced, whereas that of Bf changed only slightly, after injection of combined estrogen and progesterone to ovariectomized animals. In pregnant mice (Day [D] 1 = day of vaginal plug), Bf mRNA was at a high level on D1 and D2, dropped to an almost undetectable level from D3 to D8, and then increased to a low level thereafter until delivery. The C3 mRNA was at a high level on D1, dropped on D2 to an almost undetectable level from D3 to D9, increased to a very high level from D10 to D18, and then declined sharply before delivery. Immunohistochemical patterns of both proteins in the endometrium during preimplantation were positively correlated with changes in their mRNA levels.