The zona pellucida glycoprotein that surrounds the mammalian oocyte has several target antigens that have potential use in the development of a contraceptive vaccine. In the present study, an epitope sequence recognized by a monoclonal antibody to the porcine zona pellucida glycoprotein ZP4 was determined. Three candidate peptides were synthesized, based on an epitope mapping by cDNA and an analysis of chain flexibility of porcine ZP4. Only one synthetic peptide, corresponding to amino acid positions 50–67, reacted with the monoclonal antibody; the other synthetic peptides, corresponding to positions 60–79 and 70–100, did not react. The reactive epitope was identified as CTYVLDPENL, corresponding to positions 50–59 of porcine ZP4. The peptide inhibited the reaction of the monoclonal antibody binding to native ZP4 in a dose-dependent manner. When the synthetic peptide 50–67 was used to immunize mice, the resultant antisera reacted not only with the synthetic peptide but also with native pig zona pellucida. In addition, anti-peptide 50–67 antibody inhibited porcine fertilization in vitro. It is thus concluded that the peptide identified as an epitope for the monoclonal antibody would be a promising candidate for the development of a contraceptive vaccine.
A. Hasegawa, N. Yamasaki, M. Inoue, K. Koyama, and S. Isojima
F Saji, Y Samejima, S Kamiura, and M Koyama
Transplacental transport of maternal immunoglobulin G (IgG) to the developing fetus is extremely important in the protection of the newborn from infection. Although the exact mechanisms of the selective and active transfer of IgG across the placental barrier are not fully understood, receptors for the Fc part of IgG (FcgammaRs) in the placenta are believed to play a key role. Several known Fc receptors, FcgammaRI, FcgammaRII, FcgammaRIII and FcRn (neonatal FcR), demonstrate heterogeneous expression patterns in placenta. Immunohistochemical analysis shows the expression of FcgammaRI on Hofbauer cells in stromal tissue, FcbetaRII on Hofbauer cells and fetal blood endothelium, FcgammaRIII on Hofbauer cells and trophoblasts, and FcRn on syncytiotrophoblasts and endothelial cells. Recent studies provide evidence for important associations among these receptors and transcytosis of IgG, as well as scavenger mechanisms for clearing immune complexes in the placenta during pregnancy.
A. Hasegawa, K. Koyama, Y. Okazaki, M. Sugimoto, and S. Isojima
Mammalian zona pellucida is an attractive target for developing a contraceptive vaccine. In this study, the amino acid sequence of a core protein of porcine zona pellucida glycoprotein ZP4 was determined by peptide mapping and cDNA cloning. Two kinds of ZP4 peptides were identified: one consisted of 128 amino acid residues and the other of 133 amino acid residues with an additional five amino acid sequence at the carboxy-terminal end of the 128 amino acid peptide. Both peptides had two potential N-linked glycosylation sites. The 128 amino acid peptide showed 39.1% similarity to the amino-terminal region of mouse ZP2 polypeptide. The positions of five cysteine residues were the same for porcine ZP4 and mouse ZP2. The cloned cDNA possessed an additional 195 nucleotides at the 3' end of the sequence corresponding to the 133 amino acid peptide. This additional sequence was found to encode the amino-terminal 10 amino acid sequence of porcine ZP2 polypeptide. These results suggest that porcine ZP4 and ZP2 are derived from a common parent polypeptide by proteolytic cleavage at the position between 133 and 134 residues.
Y. Yoshimura, Y. Nakamura, T. Oda, M. Ando, Y. Ubukata, M. Karube, N. Koyama, and H. Yamada
Summary. The involvement of cyclic adenosine monophosphate (cAMP) in mammalian oocyte maturation was assessed using cultures of rabbit cumulus–oocyte complexes and perfused rabbit ovaries. Rabbit cumulus–oocyte complexes were cultured in Brackett's medium with or without forskolin at 10−4, 10−5 or 10−6 mol l−1 for 3–6 h. At 3 or 4 h spontaneous meiotic maturation was significantly (P < 0·05) inhibited by forskolin at 10−4 mol l−1. With prolonged incubation, spontaneous maturation progressed despite exposure to forskolin. In the second experiment ovaries were perfused for 12 h with forskolin (10−4, 10−5 or 10−6mol l−1) or medium alone. Neither ovulation nor degeneration of follicular oocytes occurred in any perfused ovary. The percentage of follicular oocytes achieving germinal vesicle breakdown was significantly (P < 0·001) increased in response to forskolin in a dose-related manner. In an additional experiment, ovaries were perfused with forskolin at 10−4 mol l−1. A significant increase in the cAMP content in the follicle was observed within 30 min, but the ability to produce cAMP in response to forskolin decreased as the duration of perfusion was increased. Intraoocyte cAMP increased significantly within 30 min and reached its maximum 2 h after exposure to forskolin. Thereafter, cAMP levels in the oocytes decreased abruptly. This drop in intraoocyte cAMP concentration was followed by the resumption of meiosis. The alterations of intraoocyte cAMP contents following exposure to hCG in vivo paralleled those observed in the ovaries perfused with forskolin. These data suggest that a transient, but not continuous, increase in cAMP concentration after the gonadotrophin surge may be required to initiate oocyte maturation.
Keywords: oocyte maturation; cAMP; forskolin; gonadotrophin; rabbit