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Biomedical subjects

Koji Koyama

Publications and source records attributed to Koji Koyama.

At least 19 recordsLinked to original sources
PubMed2007

Relationship between level of serum sperm immobilizing antibody and its inhibitory effect on sperm migration through cervical mucus in immunologically infertile women.

PROBLEM: Sperm immobilizing antibodies often interfere with the penetration of sperm through the cervical mucus. However, the relationship between sperm immobilizing antibody titer and the result of the post-coital test (PCT) has not yet been clarified. The aim of this study was to investigate whether the 50% sperm immobilization unit (SI50) titer, a quantitative measure of sperm immobilizing antibody, in patients' sera was correlated with the result of PCT. METHOD OF STUDY: The sperm immobilization test (SIT) was performed for 2834 infertile women in two university hospitals. The SI50 titers were evaluated by quantitative SIT. In some cases with sperm immobilizing antibody, PCT was carried out before ovulation and assessed according to the criteria of World Health Organization (1992). Infertile couples with abnormal semen characteristics were excluded. RESULTS: Seventy-four of 2834 women had sperm immobilizing antibodies in their sera, giving a positive rate of 2.6%. Twenty-four (77.4%) of 31 women with sperm immobilizing antibodies and 28 (20.4%) of 137 women without the antibody had abnormal PCT results. There was a significant difference between the two groups (P<0.0001). When patients with sperm immobilizing antibodies were divided into two groups according to the SI50 titers, the abnormal result of PCT was obtained in all 10 patients with high (>10) SI50 titers, while that was 14 (66.7%) in 21 patients with low (10) SI50 titers. There was a significant difference between the two groups (P=0.04). CONCLUSION: The SI50 titer in the serum can predict inhibitory effects on sperm migration through cervical mucus in immunologically infertile women. Evaluation of the SI50 titers in patients' sera seems to be useful for decision-making in infertile women with sperm immobilizing antibodies regarding whether they have the possibility of conceiving by timed intercourse.

Antibodies↗
PubMed2006

Pup birth from mouse oocytes in preantral follicles derived from vitrified and warmed ovaries followed by in vitro growth, in vitro maturation, and in vitro fertilization.

OBJECTIVE: To determine an optimum condition for vitrification of various animal species and to examine the developing capacity into pups of mouse oocytes in preantral follicles originating from cryopreserved ovaries. DESIGN: Experimental animal study. SETTING: Laboratory environment. ANIMAL(S): Normal (C57BL/6 x DBA2) F1 mice. INTERVENTION(S): Vitrification of animal ovaries using polyester sheets as a storage device; collection of oocyte-granulosa cell complexes by enzymatic treatment; and in vitro growth (IVG), in vitro maturation (IVM), and IVF, with embryo transplantation to pseudopregnant mice. MAIN OUTCOME MEASURE(S): Histological analysis of vitrified and warmed ovaries from several animal species; measurement of successful rates in IVG, IVM, and IVF of mouse oocytes in oocyte-granulosa cell complexes collected from the vitrified and warmed ovaries; and achievement of pup birth. RESULT(S): The vitrification method used was effective for storage of various animal ovaries. The oocytes enclosed in preantral follicles that were yielded from the vitrified-warmed ovaries preserved the capacity for developing into pups after IVG, IVM, and IVF. CONCLUSION(S): The findings indicated that ovarian cryopreservation by vitrification and subsequent IVG, IVM, and IVF are promising methods for restoring young cancer patients' fertility. The practical information presented here is applicable to human ovarian tissues.

Animals↗
PubMed2006

Upregulation of macrophage migration inhibitory factor and calgizzarin by androgen in TM4 mouse Sertoli cells.

AIM: To identify proteins induced by androgen in Sertoli cells during spermatogenesis. METHODS: We analyzed protein profiles in TM4 Sertoli cells treated with dihydrotestosterone (DHT) using surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS). RESULTS: We found increases in the expression of a 5.0-kDa protein at 15 min, an 11.3-kDa protein at 24 h and 4.3 kDa, 5.7 kDa, 5.8 kDa, 9.95 kDa and 9.98 kDa proteins at 48 h after the treatment. In contrast, the expression of 6.3 kDa and 8.6 kDa proteins decreased at 30 min, and 4.9 kDa, 5.0 kDa, 12.4 kDa and 19.8 kDa proteins at 48 h after the treatment. The 11.3-kDa protein was identified as macrophage migration inhibitory factor (MIF) known to having various functions. The 9.98-kDa protein was identified as calgizzarin related to calcium channels. The timing of their expression suggests that MIF and calgizzarin are involved in late regulation of spermatogenesis in Sertoli cells by androgen. CONCLUSION: MIF and calgizzarin are two important androgen-responsive proteins produced by Sertoli cells and they might play a role in regulating spermatogenesis.

Androgens↗
PubMed2006

Bone morphogenetic protein-2 counterregulates interleukin-18 mRNA and protein in MC3T3-E1 mouse osteoblastic cells.

Fibroblast growth factors-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2) are two of the main factors that regulate differentiation of osteoblasts. Interleukin-18 (IL-18), originally cloned as an interferon gamma-inducing factor, has been reported to inhibit maturation of osteoclasts by upregulation of osteoprotegerin secreted from osteoblasts. Little is known about the functional relationship between IL-18 and the two growth factors in osteoblast differentiation. To better understand this relationship, we analyzed the effect of BMP-2 and FGF-2 on the mRNA expression levels of IL-18, as well as IL-1alpha and IL-6, in MC3T3-E1 mouse osteoblastic cells. Following this, the effects of BMP-2 on the expression of IL-18 protein and caspase-1 protein were analyzed by immunofluorescence staining. Real-time PCR and immunofluorescence staining analysis showed that FGF-2 had no effect on the expression of IL-18 mRNA and protein, but while BMP-2 reduced IL-18 mRNA levels, increased immunostaining of both IL-18 protein and caspase-1 protein was detected in BMP-2-treated MC3T3-E1 cells. Although the significance and mechanisms of this counterregulation of IL-18 mRNA and protein were not determined in this study, the increase of IL-18 protein suggested that BMP-2 may induce an active form of IL-18.

Alkaline Phosphatase↗
PubMed2006

Pig zona pellucida 2 (pZP2) protein does not participate in zona pellucida formation in transgenic mice.

The zona pellucida, an extracellular matrix surrounding mammalian oocytes, is composed of three or four glycoproteins. It is well known that the zona pellucida plays several critical roles during fertilization, but there is little knowledge about its formation. The purpose of this study is to examine whether a pig zona pellucida glycoprotein 2 (pZP2) would assemble with mouse zona pellucida. A transgene construct was prepared by placing a minigene encoding pZP2 downstream from the promoter of mouse ZP2. The result showed that the transgenic protein was synthesized in growing oocytes but not incorporated into the zona pellucida. Furthermore, the pZP2 transgene did not rescue the phenotype in ZP2-knockout zona-deficient mice. These results indicate that pZP2 does not participate in mouse zona pellucida formation and the zona pellucida is constituted from its component proteins in a molecular species-specific manner between mice and pigs.

Animals↗
PubMed2005

Immunogenicity and contraceptive potential of recombinant human sperm associated antigen (SPAG9).

Human sperm-associated antigen 9 (hSPAG9) is a potential target for sperm-based contraceptive vaccine in lieu of its location on the sperm acrosomal compartment and its implication in sperm-egg interaction. SPAG9 is an acrosomal molecule which is not only restricted to a specific region (domain) of the acrosome but also undergoes relocation to the equatorial region in a stage-specific manner during acrosome reaction, demonstrating its potential role in sperm-egg binding. Human SPAG9 nucleotide sequence revealed 94% identity with macaque SPAG9 and 96.8% with baboon SPAG9 over the entire sequence. The amino acid sequence comparison of human SPAG9 with macaque and baboon revealed an overall homology of 84.9% and 90.6%, respectively. The presence of a high level of homology at the amino acid and nucleotide levels indicates that SPAG9 is conserved in macaque, baboon and human sharing common function and common origin in the biological past. Immunogenicity studies were carried in rats, which demonstrated that recombinant hSPAG9 protein adsorbed on alum is highly immunogenic. Antibodies thus generated after immunization reacted with recombinant human SPAG9 (rhSPAG9) and native SPAG9 protein from human sperm in Western blot analysis. In an in vitro assay, anti-rhSPAG9 antibodies inhibited sperm adherence to or penetration in zona-free hamster egg penetration test. Further, anti-SPAG9 antibodies inhibited the binding of human sperm to intact human oocyte as well as to matched hemi-zonae, indicating that the recombinant protein is a suitable contraceptive vaccinogen. Together these results demonstrate that the rhSPAG9 adsorbed on alum is immunogenic in nature, which is a permissible adjuvant for immunogenicity and fertility trials in non-human primates.

Acrosome Reaction↗
PubMed2005

Characterization of a novel human sperm-associated antigen 9 (SPAG9) having structural homology with c-Jun N-terminal kinase-interacting protein.

We report a novel SPAG9 (sperm-associated antigen 9) protein having structural homology with JNK (c-Jun N-terminal kinase)-interacting protein 3. SPAG9, a single copy gene mapped to the human chromosome 17q21.33 syntenic with location of mouse chromosome 11, was earlier shown to be expressed exclusively in testis [Shankar, Mohapatra and Suri (1998) Biochem. Biophys. Res. Commun. 243, 561-565]. The SPAG9 amino acid sequence analysis revealed identity with the JNK-binding domain and predicted coiled-coil, leucine zipper and transmembrane domains. The secondary structure analysis predicted an alpha-helical structure for SPAG9 that was confirmed by CD spectra. Microsequencing of higher-order aggregates of recombinant SPAG9 by tandem MS confirmed the amino acid sequence and mono atomic mass of 83.9 kDa. Transient expression of SPAG9 and its deletion mutants revealed that both leucine zipper with extended coiled-coil domains and transmembrane domain of SPAG9 were essential for dimerization and proper localization. Studies of MAPK (mitogenactivated protein kinase) interactions demonstrated that SPAG9 interacted with higher binding affinity to JNK3 and JNK2 compared with JNK1. No interaction was observed with p38alpha or extracellular-signal-regulated kinase pathways. Polyclonal antibodies raised against recombinant SPAG9 recognized native protein in human sperm extracts and localized specifically on the acrosomal compartment of intact human spermatozoa. Acrosome-reacted spermatozoa demonstrated SPAG9 immunofluorescence, indicating its retention on the equatorial segment after the acrosome reaction. Further, anti-SPAG9 antibodies inhibited the binding of human spermatozoa to intact human oocytes as well as to matched hemizona. This is the first report of sperm-associated JNK-binding protein that may have a role in spermatozoa-egg interaction.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Efficacy of antibodies against a chimeric synthetic peptide encompassing epitopes of bonnet monkey (Macaca radiata) zona pellucida-1 and zona pellucida-3 glycoproteins to inhibit in vitro human sperm-egg binding.

Immunocontraception achieved by immunization with zona pellucida (ZP) glycoproteins is invariably associated with ovarian dysfunction. Use of ZP glycoprotein-based synthetic peptides as immunogens has been proposed to overcome adverse side effects on ovaries. In the present study, a chimeric peptide encompassing the epitopes of bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1; amino acid residues 251-273) and ZP glycoprotein-3 (bmZP3; amino acid residues 324-347), separated by a tri-glycine spacer, was synthesized and conjugated to diphtheria toxoid (DT). Immunization of female BALB/cJ mice and bonnet monkeys with the chimeric peptide led to generation of antibodies that reacted with the chimeric peptide, individual bmZP1 & bmZP3 peptides, and also recombinant bmZP1 and bmZP3 proteins expressed by E. coli in an ELISA. Indirect immunofluorescence studies revealed that the immune serum also recognized human as well as bonnet monkey ZP. A significant inhibition of human sperm binding to ZP was observed with antibodies generated against the chimeric peptide in mice (P = 0.0001) as well as monkeys (P = 0.0002) in a hemizona assay (HZA). The inhibition efficacy was significantly higher than that observed by using antibodies against the individual bmZP1 and bmZP3 peptides. Interestingly, no ovarian pathology was observed in female bonnet monkeys immunized with the chimeric peptide. These studies have demonstrated that the chimeric peptide encompassing peptides of multiple ZP glycoproteins may be a promising candidate antigen for designing immunocontraceptive vaccines.

Amino Acid Sequence↗
PubMed2005

Target antigens for sperm-immobilizing antibodies found in infertile women.

Complement-dependent sperm-immobilizing antibodies (SI-Abs) have frequently been detected in the sera of infertile women. Analyses using monoclonal antibodies as well as patients' antisperm antibodies have shown that the carbohydrate moieties of sperm and seminal plasma are major epitopes of these antibodies. In the present study, we characterized the antigen molecules recognized by human monoclonal SI-Ab (MAb H6-3C4) derived from a patient with strong SI-Ab activity. Immunofluorescent staining showed that MAb H6-3C4 reacted with ejaculated human sperm and the epididymis but not with the testes and other somatic organs including the spleen. In Western blot analysis, MAb H6-3C4 reacted to antigen molecules of Mr 15-27 kDa present in the methanol-chloroform extracts of either ejaculated sperm or seminal plasma. Amino acid sequence analysis of the reactive molecule revealed that the amino acid sequence was identical to the core peptide of CD52, a GPI-anchored protein on lymphocytes. Furthermore, the epitope for MAb H6-3C4 was found to be the specific N-linked carbohydrate for the male reproductive tract. These results suggest that SI-Abs in some infertile women target a sperm-specific carbohydrate antigen on CD52 molecules which are secreted from the epididymis and coat the sperm surface.

Animals↗
PubMed2005

Follicular dysfunction induced by autoimmunity to zona pellucida.

The mammalian zona pellucida is an extracellular matrix that occurs in growing oocytes, ovulated eggs and pre-implantation embryos, and is known to be involved in several important events during ovarian folliculogenesis and fertilization. Since the zona pellucida is formed at an early stage of oocyte growth, circulating antibodies against zona pellucida may impair ovarian function. In this article we discuss whether anti-zona antibodies cause ovarian dysfunction and infertility. The discussion is based on clinical examination and animal experiments including the following approaches: 1/ immunological method using solubilized human zona pellucida detected anti-zona antibody with a high frequency in infertile patients, especially premature ovarian failure syndrome; 2/ in vivo experiment using hamsters showed that some, but not all, animals experienced ovarian failure after immunization with hamster recombinant zona proteins; 3/ in vitro experiment using mouse isolated ovarian follicles showed significant inhibitory effects on follicular growth and oocyte development. We concluded that anti-zona antibody may be involved in causing ovarian failure.

Animals↗
PubMed2004

Synthesis and preliminary biological evaluation of truncated zoanthenol analogues.

Zoanthamines are a family of marine alkaloids that have complex heptacyclic structures and are reported to be interleukin-6 modulators. While the structure of zoanthamines, especially the ABC-ring portion, is similar to that of steroids, the CDEFG-ring portion, composed of aminoacetal and lactone core, is a unique structural element. In this report, we designed and synthesized ABC-ring 6 and CDEFG-ring 7, which are truncated analogues of the northern and southern hemispheres of zoanthenol 5, respectively, and which incorporate all of the functionality of each hemisphere. A preliminary SAR study suggested that the hydrochloride of the CEFG-ring portion is an active pharmacophore for suppressing the growth of interleukin-6-dependent MH60 cells.

Alkaloids↗
PubMed2004

Radiographic and genetic diagnosis of sporadic hypochondroplasia early in the neonatal period.

Hypochondroplasia is an autosomal dominant skeletal dysplasia expressing postnatal onset of short stature with mild rhizomelic shortening of the limbs. This manifestation leads to restricted prenatal diagnosis of the disorder. We report here on a sporadic case of a hypochondroplastic baby, whose prenatal sonographic measurements were serially recorded from 19 weeks of gestation. Mild shortening of the limbs became manifest after 26 weeks of gestation. Biparietal diameter was within the normal range throughout gestation. Both parents were of average stature. A tentative diagnosis of a nonlethal short-limb skeletal dysplasia was made. At birth, the clinical manifestations of the neonate were not characteristic, but the radiographic features raised the possibility of hypochondroplasia. Molecular analyses revealed a C to G mutation at nucleotide 1659 of the fibroblast growth factor receptor 3 (FGFR3) gene, a common mutation in hypochondroplasia.

Adult↗
PubMed2004

In vitro growth and maturation as well as fertilization of mouse preantral oocytes from vitrified ovaries.

OBJECTIVE: To examine the capacity for fertilization and development of preantral oocytes in oocyte-granulosa cell complexes (OGC) originating from vitrified ovaries. DESIGN: Experimental animal study. SETTING: University-based research laboratory. ANIMAL(S): Normal (C57BL/6xDBA2) F1 mice in a laboratory environment. INTERVENTION(S): Vitrification of mouse ovaries using polyester sheets as a storage device; collection of OGC by enzymatic treatment; in vitro growth (IVG), in vitro maturation (IVM), and in vitro fertilization (IVF). MAIN OUTCOME MEASURE(S): We performed histologic analysis of vitrified and warmed ovaries, and measured the successful rate in IVG, IVM, and IVF of oocytes in the OGC collected from the ovaries. RESULT(S): The cortical region of ovaries maintained good morphologic structure after vitrification and warming. Upon IVG and IVM, 75.9% of oocytes in OGC matured to the metaphase II (MII) stage. The fertilization rate of these oocytes was 57.5% as compared with 69.5% for fresh ovaries. CONCLUSION(S): The vitrification method used was effective for storage of ovaries. The oocytes enclosed in preantral follicles from the ovaries preserved capacity for fertilization and development to preimplantation embryos.

Animals↗
PubMed2004

Birth of healthy neonates after intracytoplasmic injection of ejaculated or testicular spermatozoa from men with nonmosaic Klinefelter's syndrome: a report of 2 cases.

BACKGROUND: Klinefelter's syndrome is one of the major causes of azoospermia, cryptozoospermia and severe oligozoospermia with either a nonmosaic (47,XXY) or mosaic (47,XXY/46,XY) ICSI treatment with cryopreserved testicular spermatozoa failed, but after the third attempt, 6 of 8 oocytes injected with cryopreserved sperm were fertilized and karyotype. Men with Klinefelter's syndrome generally have difficulty having children. CASES: Patient 1 had motile spermatozoa in the ejaculate, which were injected into 3 oocytes, resulting in fertilization and cleavage. Two good-quality embryos were transferred into his wife's uterine cavity. She conceived and, following a normal pregnancy, delivered a healthy female infant. Two years later she conceived for the second time with motile spermatozoa in the ejaculate and delivered a healthy male infant uneventfully. To our knowledge, this was the first case in which a nonmosaic Kleinefelter's syndrome patient fathered 2 children through intracytoplasmic sperm injection (ICSI) using motile spermatozoa in the ejaculate. Patient 2, with azoospermia, was subjected to testicular biopsy to collect spermatozoa. The first 2 attempts at ICSI treatment with cryopreserved testicular spermatozoa failed, but after the third attempt, 6 of 8 oocytes injected with cryopreserved sperm were fertilized and cleaved. Two of these embryos were transferred into the wife's uterine cavity. She conceived and, following a normal pregnancy, delivered a healthy male infant. In all cases, amniocentesis followed by genetic analysis showed a normal karyotype. CONCLUSION: Two infertile men with nonmosaic Klinefelter's syndrome successfully fathered normal children after intracytoplasmic injection of ejaculated or testicular spermatozoa.

Adult↗
PubMed2003

Prevention of multiple pregnancies by restricting the number of transferred embryos: randomized control study.

INTRODUCTION: The high incidence of multiple pregnancies is a major concern in the treatment of infertility by in vitro fertilization (IVF). Risks and costs are associated with multiple pregnancies and it is important to find a way to decrease multiple pregnancies. The standard protocol for IVF includes the transfer of two or three embryos. METHODS: In this study, we compared the rate of single and multiple pregnancies between patients who received two and three embryos. One hundred and sixty-nine patients were randomly divided into two groups, with 106 cycles of IVF each. Group 1 received two embryos, and group 2 three. RESULTS: Group 1 had total 40 (37.8%) pregnancies, while group 2 had 29 (27.4%). The number of ongoing pregnancies was 33 (31.1%) in group 1 and 26 (24.5%) in group 2. The number of baby-take-home was 32 (30.1%) in group 1 and 26 (24.5%) in group 2. These results show that there was no significant difference between the two groups in both the total and ongoing pregnancies. On the other hand, the number of twin pregnancies was 6 (15.0%) in group 1 and 12 (41.4%) in group 2, showing a significant difference. Group 1 had no triplet pregnancies, while Group 2 had two. CONCLUSION: We conclude that the transfer of two embryos reduces the incidence of multiple pregnancies while maintaining an acceptable rate of pregnancy in IVF.

Embryo Transfer↗