Mutual adaptation between mouse transglutaminase 4 and its native substrates in the formation of copulatory plug.

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.

A proline rich acidic protein PRAP identified from uterine luminal fluid of estrous mice is able to enhance the estrogen responsiveness of Ishikawa cells.

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.

Identification of the major TG4 cross-linking sites in the androgen-dependent SVS I exclusively expressed in mouse seminal vesicle.

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.

Identification of a tyrosine-phosphorylated CCCTC-binding nuclear factor in capacitated mouse spermatozoa.

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.