Local analysis of the edge dislocation core in BaTiO(3) thin film by STEM-EELS.

The a <100> edge dislocation core formed in an epitaxial BaTiO(3) (BTO) thin film grown on a substrate was investigated by scanning transmission electron microscopy combined with electron energy-loss spectroscopy. Elemental analysis using core-loss spectrum indicates that the atomic ratios of O/Ti and Ba/Ti are decreased at the dislocation core. The near-edge fine structure of the oxygen K-edge recorded from the dislocation core differs slightly from that of relaxed BTO region, which suggests that Ba-O bonding is decreased at the dislocation core. The structure of the dislocation core is discussed using a high-angle annular dark-field image and the electron energy-loss spectroscopy results.

Possible presence of O-linked carbohydrate in the human male reproductive tract CD52.

Male reproductive tract CD52 (mrtCD52) is an antigen recognized by a complement-dependent sperm-immobilizing monoclonal antibody (SI-Abs) derived in an infertile patient. The molecule has been shown to contain a unique N-linked carbohydrate that does not cross-react with other tissues. In this study, we have investigated whether O-linked carbohydrate as well as N-linked carbohydrate is present in mrtCD52 using specific lectins and anti-CD52 core peptide antiserum. The lectin PNA, which recognizes O-linked carbohydrate [Galbeta1-3GalNAc], reacted with mrtCD52 and showed a similar polymorphic reaction pattern to that of the anti-peptide antiserum in western blotting analysis on two-dimensional SDS-PAGE. The PNA-reactive spots disappeared after removal of O-linked carbohydrate, but not after removal of N-linked carbohydrate. These results suggest that O-linked carbohydrate is present in mrtCD52. The moiety may possibly contribute to a specific antigenic epitope of mrtCD52.

Screening for deletions in interval D16-22 of the Y chromosome in azoospermic and oligozoospermic Japanese men.

The genetic basis of infertility remains unclear in a majority of infertile men. In this study, the Y chromosome long arm involving the DAZ (deleted in azoospermia) gene was screened in order to evaluate the occurrence of microdeletion in Japanese infertile men. One hundred and fifty-seven infertile Japanese men with azoospermia and oligozoospermia were analyzed for microdeletions in interval D16-22 of the Y chromosome, using polymerase chain reaction with sequence-tagged site markers. Sixteen sets of oligonucleotide primers were synthesized for the polymerase chain reaction, and Southern blot analysis was also performed. The men were divided into five categories on the basis of sperm concentration: functional azoospermia (A; n = 24), azoospermia caused by obstruction (AO; n = 20), oligozoospermia I (OI, sperm concentration less than I x 10(5)/ml; n = 33), oligozoospermia II (OII, sperm concentration less than 1 x 10(6)/ml; n = 30), and oligozoospermia III (OIII, sperm concentration less than 1 x 10(7)/ml; n = 50). Thirty fertile men with a sperm concentration of more than 2 x 10(7)/ml were also analyzed as controls. Microdeletions were identified, in 12 (7.6%) of the 157 infertile men, as follows: 1 man in category A, 1 in category AO, 5 in category OI, 4 in category OII, and 1 in category OIII. No deletion was identified in the fertile men. One common region around sY240 was identified in 11 of the infertile men with microdeletions. This locus may contain specific genes for spermatogenesis. The sperm concentration in the ten oligozoospermic men with microdeletions was below 1 x 10(6)/ml. There were no correlations between the severity of spermatogenic defects and the extent of the microdeletions. These results suggested that genes in the interval D16-22 of the Y chromosome might have important roles in spermatogenesis.

Assisted reproduction for infertile patients with 9 + 0 immotile spermatozoa associated with autosomal dominant polycystic kidney disease.

We investigated the clinical feature of patients with totally immotile spermatozoa due to 9 + 0 ultrastructural flagellar defects and polycystic kidney disease. We also tried to establish the feasibility of applying modern assisted reproduction technology (ART) in these patients. During 6-year interval a total of 1956 Japanese men were referred to the male infertility clinic. Of them, 16 were diagnosed to have immotile spermatozoa and four of them exhibited axonemal 9 + 0 defects in the sperm flagella. These four also had autosomal dominant polycystic kidney disease (ADPKD). Intrauterine insemination (IUI) and conventional in-vitro fertilization and embryo transfer failed to achieve fertilization. Intracytoplasmic sperm injection (ICSI) with 100% immotile spermatozoa was performed in all four cases. Two-pronuclear fertilization was obtained in 27 of the 70 (38.6%) of the successfully injected oocytes, but no pregnancy resulted. In one case, a few motile spermatozoa were present at the second cycle of ICSI, a pregnancy was successfully achieved using these spermatozoa. While immotile spermatozoa from patients with the axonemal 9 + 0 defect achieved fertilization by ICSI, the embryos failed to develop. Our results indicate that the central microtubules may play a role in fetal development. Since the 4 patients with 9 + 0 defects also had ADPKD, the genetic linkage between these two conditions should be studied by molecular biological methods so as to aid our ability to counsel such patients.

Assisted reproduction technology for patients with congenital bilateral absence of vas deferens.

PURPOSE: We investigate the frequency of cystic fibrosis transmembrane conductance regulator gene mutations in Japanese patients with congenital bilateral absence of the vas deferens, and assess treatment outcomes of assisted reproduction interventions. MATERIALS AND METHODS: In 10 Japanese patients with bilateral congenital absence of the vas deferens genetic analysis was performed for known frequent mutations of the cystic fibrosis transmembrane conductance regulator gene using polymerase chain reaction amplification followed by dot-blot hybridization with the allele-specific oligonucleotide probes and direct sequencing. Intracytoplasmic sperm injection using spermatozoa retrieved from the testes was performed in 7 of the couples. RESULTS: No known mutations of the gene were detected in the patients. However, analysis of the polythymidine tract polymorphism in intron 8 revealed 30% allele frequency of 5T. Pregnancy was achieved in 7 cycles of intracytoplasmic sperm injection using spermatozoa retrieved from the testes. CONCLUSION: The 5T variant in intron 8 polythymidine tract was identified with high allelic frequency in Japanese patients with congenital bilateral absence of the vas deferens, suggesting that the disease in Japan is also partially caused by this particular mutation of the cystic fibrosis transmembrane conductance regulator gene. Modern assisted reproduction technology offers an important option for patients with congenital bilateral absence of the vas deferens.

The operation of a superconducting wiggler at TERAS.

A superconducting wiggler has been successfully installed at the ETL 800 MeV electron storage ring facility (TERAS). The operation of the wiggler at magnetic field strengths of 5 T with electron beam energy of 750 MeV has been accomplished. The wiggler has been designed and constructed to produce synchrotron radiation with critical photon energy around 3 keV for scientific, industrial and medical applications. We report here experiments that demonstrate the possibility of stable operation of a superconducting wiggler in a small storage ring.

Characterization of fertilization-blocking monoclonal antibody 1G12 with human sperm-immobilizing activity.

A mouse hybridoma (1G12) producing sperm-immobilizing MoAb to human sperm was established and characterized in order to study the antigens relevant to sperm immobilization by antibodies. MoAb 1G12 had strong sperm-immobilizing and agglutinating activities and also showed a fertilization-blocking activity on in vitro fertilization tests. The antibody absorption experiments showed that MoAb 1G12 reacted not only to ejaculated sperm but also human seminal plasma, suggesting that the corresponding antigen might be a sperm coating antigen. The MoAb also reacted with peripheral blood lymphocytes. In histochemical studies, the epithelia of corpus epididymis were most strongly stained. Ejaculated sperm were stained with a granular pattern for their entire surface by immunofluorescence. MoAb 1G12 recognized polymorphic glycoproteins of 15-25 kD in the ejaculated sperm extract in Western blot analysis. After deglycosilation of the sperm extract, only a single staining band of under 15 kD was detected by MoAb 1G12. This suggests that the antigen epitope recognized by MoAb 1G12 might be a peptide of the core portion of the glycoprotein. MoAb 1G12 might be a useful tool for studying the mechanism of egg-sperm interaction, and also be applied to identifying the corresponding antigen by using gene technology.

Diversity of the blocking effects of antisperm antibodies on fertilization in human and mouse.

The blocking effects of complement-dependent sperm immobilizing antibodies in the sera of infertile women and monoclonal antisperm antibodies against humans and mice on fertilization were investigated. The hemizona assay (HZA) and sperm penetration assay (SPA) were used to study the inhibitory effects of sera from 22 infertile patients positive for sperm immobilizing antibodies. Use of these tests allowed us to differentiate whether the antibody blocked sperm-zona pellucida tight binding and/or sperm penetration into the ooplasm. The zona pellucida penetration assay (ZPA) was also used to study the effects of four monoclonal antibodies (mAbs) on human sperm penetration into the zona pellucida. Seven mAbs against murine spermatozoa were tested for their inhibitory effects on in-vitro fertilization (IVF) and HZA in mice. Of 22 patient sera with sperm immobilizing antibodies, 21 (95.5%) inhibited HZA attachment and penetration, whereas this did not occur in any of 13 patient sera without these antibodies. However, 19 of 22 (86.4%) patient sera with sperm immobilizing antibodies and eight of 13 (61.5%) patient sera without these antibodies inhibited the SPA. Two (2C6, 1G12) of four mAbs against human spermatozoa showed strong inhibitory effects in all the assays (HZA, ZPA and SPA). One mAb (3B10) did not inhibit HZA but blocked ZPA and SPA. Another mAb (H6-3C4) seemed to have no inhibitory effects on fertilization. Two (Vx 5 and Vx 8) of seven mAbs against murine spermatozoa inhibited IVF in mice but did not block mouse HZA. These findings suggest that antisperm antibodies block fertilization at specific stages. Some of them may inhibit sperm capacitation and thus prevent all processes of fertilization that follow. Some other antibodies may not affect capacitation and sperm binding to zona pellucida but inhibit the acrosome reaction, followed by the blocking of sperm penetration through zona pellucida and ooplasm.

Analysis of an epitope sequence recognized by a monoclonal antibody MAb-5H4 against a porcine zona pellucida glycoprotein (pZP4) that blocks fertilization.

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.

Direct production of the Fab fragment derived from the sperm immobilizing antibody using polymerase chain reaction and cDNA expression vectors.

PROBLEM: Sperm immobilizing antibodies cause infertility mainly through complement dependent sperm immobilization. To analyze any effect of sperm immobilizing antibody on fertilization, we had already established cell lines that secrete IgM monoclonal antibody (MAb H6-3C4) and IgG monoclonal antibody (MAb EnBCMGS). The latter was a class-switched recombinant IgG antibody that shares the same variable region as MAb H6-3C4. The biological effects of the IgG antibody were also reported previously to eliminate sperm immobilizing or sperm agglutinating activities. However, the method of chemical digestion of IgG had some disadvantage to prepare the purified Fab fragment stably and in large quantities. This time we report a unique method to obtain the recombinant Fab fragments (Fab EnBCMGS) using polymerase chain reaction (PCR) and cDNA expression vectors. METHOD: Two kinds of PCR primers were designed to make a truncated heavy chain (Fd) gene of MAb EnBCMGS. The amplified Fd gene and light chain gene were ligated into cDNA expression vectors and then transfected into mammalian cells. RESULTS: Expression of the Fd gene and light chain gene were confirmed by Northern blotting. Secretion of the recombinant Fab fragment from mammalian cells was also confirmed by Western blotting. The Fab fragment showed biological activity as is expected by FACS analysis. CONCLUSION: This method enables the stable production of genuine Fab fragments of IgG in mammalian cells without any chemical treatment that may be time consuming and affect the quality of the Fab fragments.

Progesterone treatment decreases sulfate carbohydrate antigen on endometrial carcinoma cells and inhibits the cell binding to laminin.

To investigate the effects of progesterone on endometrial carcinoma, sulfated carbohydrate antigen was studied using a monoclonal antibody, termed E8, that reacts with sulfatide. The reactivity of monoclonal antibody (MAb) E8 with endometrial carcinoma tissues decreased when patients were treated with medroxyprogesterone acetate (MPA). A reduction of reactivity of MAb E8 with endometrial carcinoma cells was also observed in in vitro examinations using an endometrial carcinoma cell line, Ishikawa cells incubated in MPA-containing medium. Sulfated carbohydrate, such as sulfatide, is the site of binding with laminin. The binding of cell surface components with laminin is thought to be an initial step in the invasion and metastasis of carcinoma cells. The MPA-treated Ishikawa cells bound to laminin-coated dishes less [corrected] efficiently than nontreated cells. MPA treatment of endometrial carcinoma reduces the sulfatide carbohydrate on the carcinoma cell surface and may be clinically more effective in reducing cell binding to laminin.

The effects of patients' sera with sperm-immobilizing antibodies on sperm of the Japanese monkey.

In order to study the interspecies cross-reactivity of sperm-immobilizing antibodies found in sera of women who were infertile for unknown reasons, we used the sperm-immobilization test to examine the effects of patients' sera with or without sperm-immobilizing antibodies on the sperm of the Japanese monkey (Macaca fuscata). Fourteen of 17 antibody-positive sera and 7 of 14 antibody-negative sera showed sperm-immobilizing activity with regard to monkey sperm. The sperm-immobilizing antibody that was active against both human and monkey sperm could be absorbed only by sperm from each corresponding species. The periodate treatment of human or monkey sperm markedly diminished their antibody-absorbing capabilities. Human and mouse monoclonal antibodies having sperm-immobilizing activity with regard to human sperm showed no sperm-immobilizing activity with regard to monkey sperm. These results indicate that the sperm-immobilizing activity of the sera of infertile women against human and monkey sperm might be due to antibodies with different specificities, which recognize a unique carbohydrate antigen epitope expressed in the sperm of each species.

Identification and characterization of a 27 kDa acrosome protein of human sperm defined by a monoclonal antibody with fertilization-blocking effect.

A monoclonal antibody, MAb 1G1, possessing a strong fertilization-blocking activity was prepared by immunizing a BALB/c mouse with capacitated human sperm in order to study the molecular nature of sperm antigens relevant to fertilization. MAb 1G1 inhibited human sperm penetration into zona-free hamster eggs. It reacted to the apical portion of acrosome-reacted human sperm, but did not react to live sperm before the acrosome reaction as demonstrated by immunofluorescence staining. In paraffin-embedded testis sections, the round spermatids, spermatocytes and spermatozoa were stained with MAb 1G1, but the spermatogonia were not stained. Neither Sertoli cells, Leydig cells nor other somatic tissues were stained. The sperm of Japanese monkey, bull, boar, hamster and mouse were not stained. Therefore, the staining of sperm was species specific. The antigen corresponding to MAb 1G1 showed a band at 27 kDa by immunoblotting. The reactivity of the antigenic component was not destroyed by periodic acid treatment. From the results obtained, it was postulated that the antigenic molecule might be a polypeptide. These results indicated that this MAb might be a useful tool for studying the mechanism of human sperm-egg fertilization and the development of a contraceptive vaccine.

Amino acid sequence of a porcine zona pellucida glycoprotein ZP4 determined by peptide mapping and cDNA cloning.

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.

Effects of sperm-immobilizing antibodies on sperm-zona pellucida tight binding.

OBJECTIVE: To evaluate the in vitro effects of sperm-immobilizing antibodies on sperm-zona pellucida (ZP) tight binding. DESIGN: The hemizona assay (HZA) was used to study the inhibitory effects of infertile women's sera with and without sperm-immobilizing antibodies on sperm ZP tight binding. These results were compared with those of monoclonal sperm-immobilizing antibodies. SETTING: The patients were collected from a university hospital infertility clinic. PATIENTS: Sera from 40 infertile women (24 with and 16 without sperm-immobilizing antibodies) and 2 postpartum women as control were used. RESULTS: Of 24 patients' sera with sperm-immobilizing antibodies, 23 (96%) showed significant inhibitory effect, whereas none of 16 patient's sera without sperm-immobilizing antibodies exhibited any inhibitory effect. However, there was no correlation between the antibody titers of sperm-immobilizing antibody and the hemizona index. Among four monoclonal sperm-immobilizing antibodies tested, only one showed a significant inhibitory effect on the sperm-zona tight binding. A human monoclonal antibody derived from an infertile woman with sperm-immobilizing antibodies, whose serum showed an inhibitory effect on HZA, did not inhibit the HZA. CONCLUSIONS: There are at least two kinds of sperm-immobilizing antibodies, one with both activities of sperm immobilization and blocking of sperm-zona tight binding and another with the former activity alone. The vast majority of sperm-immobilizing antibodies reduce zona binding even without the presence of complement.

Determination of immunoglobulin classes and subclasses of sperm immobilizing antibodies in infertile women and the inhibitory effect of IgG4 subclass on the sperm immobilizing antibody activity.

Immunoglobulin classes and subclasses of sperm immobilizing antibodies (SI-Abs) in the sera of sterile women were determined by the absorption of patients' sera with Staphylococcus aureus and immunoadsorbents bound with class or subclass-specific anti-human immunoglobulin antibodies. Among 18 patients' sera tested, 16 had the IgG-dominant SI-Abs and the remaining 2 sera contained IgM-dominant SI-Abs. From the former patients' sera, 9 were further studied to determine the IgG subclasses; 6 of them had IgG1-dominant SI-Abs, one IgG2-dominant, and in the other two IgG1 and IgG2 were equally dominant. Interestingly, SI-Ab activities in 6 of the 9 patients' sera increased after absorption of IgG4 subclass and the addition of IgG4 purified from an SI-positive patient to the same patient's serum diminished the SI-Ab activities.

Single nucleotide substitution of the androgen receptor gene in a case with receptor-positive androgen insensitivity syndrome (complete form).

Complete androgen insensitivity syndrome is caused by X chromosome linked disorder resulting in a target organ insensitivity to androgen. Two variants have been described in this syndrome. In the first, the binding of [3H]dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) to the androgen receptor is undetectable (receptor-negative), whereas in the second variant normal levels of androgen receptor are detectable but the binding of [3H] dihydrotestosterone to the androgen receptor is significantly thermolabile under certain conditions (receptor-positive). In receptor-negative cases, genetic disorders of the androgen receptor gene have been demonstrated. On the other hand, the genetic disorder of androgen receptor in receptor-positive cases is little known. In this study, the gene structure of androgen receptor in a receptor-positive case using a polymerase chain reaction technique is studied in the fibroblasts cultured from genital skin. The results demonstrate that the substitution of nucleotide (guanine-->cytosine) in exon G of the androgen receptor causes the replacement of an amino acid in position 820 (glycine-->alanine) which occurs in the hormone-binding domain of the androgen receptor. The substitution of nucleotide may explain the thermolability of the androgen receptor in a case with receptor-positive androgen insensitivity syndrome.

Stable production of recombinant human sperm immobilizing antibody using cDNA expression vectors.

PROBLEM: Sperm immobilizing antibodies present in sterile women may be one of the principal causes of immunological infertility. We already established cell lines that secrete recombinant human IgG sperm immobilizing antibody using class-switched (from IgM to IgG) genomic immunoglobulin genes. However, these transfectants produced a small quantity of antibody and required continuous use of a medium with selective reagents. We have now constructed cell lines that stably secrete the antibody in large quantities using immunoglobulin cDNAs and cDNA expression vectors. METHOD: The immunoglobulin heavy chain cDNA was cloned from transfectants that secrete the class-switched human IgG sperm immobilizing antibody. The light chain cDNA, which had already been cloned, and the heavy chain cDNA were inserted into the modified bovine papilloma virus-based cDNA expression vectors BCMGSNeo and BCMGSHyg, respectively. These constructs were sequentially transfected into a mouse myeloma cell line by electroporation. RESULTS: The established transfectants produced recombinant antibody that retained human sperm immobilizing activity in nonselective medium for at least 30 days. Moreover, the production of the antibody was increased three times over that of the previous cell lines. CONCLUSION: We have established an unique method that improves the production of sperm immobilizing antibody stably and in large quantities.

Sperm immobilizing antibodies react to the 3-O-sulfated galactose residue of seminolipid on human sperm.

It is well known that very few women who possess sperm immobilizing antibodies in their sera can conceive naturally even though there are no abnormalities in their reproductive organs on routine medical examination. A monoclonal antibody (MAb), designated 2H12, was produced by immunizing a BALB/c mouse with the human choriocarcinoma cell line JEG-3. MAb 2H12 showed strong sperm immobilizing activities and reacted to sulfatide and seminolipids. The sperm immobilizing activities of 2H12 were clearly absorbed with sulfatide or seminolipid whilst several other sperm immobilizing MAbs that were made by immunization with human sperm or seminal plasma could not be absorbed with the same sulfoglycolipids. The sperm immobilizing antibodies in the sera of infertile women with unknown cause were also clearly absorbed with sulfatide or seminolipid. MAb 2H12-conjugated immunobeads (MAb 2H12-IMBs) bound to motile sperm. This binding of 2H12-IMBs to sperm was competitively inhibited either by 2H12 or women's sera containing sperm immobilizing antibodies, but not by normal women's sera or several other sperm immobilizing MAbs to human sperm. These results suggest that the sperm immobilizing antibody in women's sera is directed against the 3-O-sulfogalactose residue of seminolipid on the sperm membrane.

Antifertility effect of active immunization with ZP4 glycoprotein family of porcine zona pellucida in hamsters.

Female golden hamsters were immunized with solubilized porcine zona pellucida (s-PZP) or ZP4 glycoprotein family isolated from s-PZP by preparative SDS-PAGE. Both antigen preparations induced production of antibodies which reacted not only with porcine zona pellucida but also with the hamster zona pellucida. The hamsters immunized with solubilized porcine zona pellucida mainly produced antibodies reactive to ZP3, while the hamsters immunized with ZP4 mainly produced antibodies reactive to ZP4. The former animals became permanently infertile but the infertility in the latter animals was temporary and they became pregnant later. Histological studies revealed that the ovarian follicles in hamsters immunized with s-PZP were completely destroyed leaving only atrophic follicle-like cell clusters, while in the ovaries of hamsters immunized with ZP4 a number of small follicles with oocytes remained intact. These observations are encouraging for the further characterization of the ZP4 antigens as candidates for the development of a contraceptive vaccine.