Naoyuki Takuma

Requirement of nitric oxide for murine oocyte maturation, embryo development, and trophoblast outgrowth in vitro.

We investigated the extent to which NO participates in the developmental competence (oocyte maturation, fertilization and embryo development to blastocyst) using an in vitro culture system adding sodium nitroprusside (SNP), NO donor, and NOS inhibitor (N‐omega‐nitro‐L‐arginine methyl ester, L‐NAME). We also assessed the effects of NO/NOS system on blastocyst implantation using an in vitro trophoblast outgrowth assay. The treatment of low concentrations of SNP (10−7 M) significantly stimulated meiotic maturation to metaphase II stages in cumulus enclosed oocytes. In contrast, 10−3 and 10−5 M L‐NAME demonstrated a significant suppression in resumption of meiosis. This inhibition was reversed by the addition of SNP. No development beyond the four‐cell stage was observed by the addition of high concentration of SNP (10−3 M). Inhibition of embryo development, especially the conversion of morulae to blastocysts, was also observed in the treatment of lower doses of SNP (10−5 and 10−7 M). Similarly, inhibition of NO by NOS inhibitor resulted in the dose‐dependent inhibition of embryo development and hatching rates, but the concomitant addition of SNP with L‐NAME reversed the inhibitory effect by each SNP or L‐NAME treatment. Furthermore, low concentration of SNP (10−7 M) but not high concentration of SNP (10−3 M) significantly stimulated trophoblast outgrowth, whereas the addition of L‐NAME suppressed the spreading of blastocysts in a dose‐dependent manner. These results suggest that NO may have crucial roles in oocyte maturation and embryogenesis including the process of implantation. The observed differences in required amount of NO and the sensitivity to cytotoxicity of NO in each developmental stage embryos may also suggest that NO/NOS system is tightly regulated in developmental stage specific manner. Mol. Reprod. Dev. 58:262–268, 2001.

NKX2 gene expression in neuroectoderm but not in mesendodermally derived structures depends on sonic hedgehog in mouse embryos

Abstract NKX2 genes in vertebrates encode a sub- family of homeodomain-containing transcription factors which regulate morphogenetic events and cell differentiation during embryogenesis. In mouse embryos several NKX2 genes are expressed in the ventral midline domains of the neuroectoderm, while other NKX2 genes are primarily expressed in the mesendoderm and mesendodermally derived organs, such as heart and gut. Within several patterning centers for tissue organization sonic hedgehog (Shh) is an important signal in the formation of ventral midline structures in vertebrate embryos. Here, we investigated the role of Shh in the embryonic expression of six different but closely related NKX2 genes in Shh null mutant mice. We found that expression of NKX2.1, NKX2.2, and NKX2.9 in neural domains requires Shh signaling, whereas NKX2.3, NKX2.5 and NKX2.6 expression in endoderm and mesoderm is independent of Shh.

Treatment of Climacteric Symptoms with Herbal Formulas of Traditional Chinese Medicine

Hormone replacement therapy (HRT) is not successful or is contraindicated for the treatment of climacteric symptoms in some patients. To investigate whether certain herbal formulas of traditional Chinese medicine (Kampo in Japanese) could be used as an alternative treatment, a longitudinal ‘before and after’ comparative study was carried out in 18 Japanese women, and the results were compared with those of 16 women who underwent HRT. Kampo improved all the climacteric symptoms. In contrast, improvement of cold limbs, sleeping disorders, shoulder stiffness/lumbago, and fatigue in the HRT group was either not significant or of limited extent. In addition, the serum level of estradiol in postmenopausal women was raised by the combined use of two Kampo formulas. These results suggest that Kampo may be considered an alternative to HRT for the treatment of climacteric symptoms, but vigorous monitoring for potential side effects of increased estrogen levels in some postmenopausal patients is needed.

Isolation and Expression Analysis of the Testis-Specific Gene, STRA8, Stimulated by Retinoic Acid Gene 8

Retinoids are known to be required for vertebrate reproduction, and in the male, for the maintenance of normal testicular structure and function. Previously several novel retinoic acid responsive genes, collectively designated as the Stra genes, had been isolated in the mouse. The Stra8 gene encodes a cytoplasmic protein and is expressed specific to the developing male gonad during mouse embryogenesis. In adult mouse, its expression is restricted to the premeiotic germ cells. Thus it has been suggested that the mouse Stra8 protein may play a role in the premeiotic phase of spermatogenesis.Recently a lot of genes that are expressed only in male germ cells have been isolated in the mouse. The mouse Stra8, Rnh2, Piwil2, Tex17, and Tuba7 were identified as testis-specific expressed genes. In addition, the Figla was known to be a testis- and ovary-specific gene. Recently we had reported the isolation of the human RNH2 cDNA and its expression, which is limited to the human testis. In the present study, we have isolated full-length cDNA of STRA8 and partial cDNAs of PIWIL2, FIGLA, TEX17, and TUBA7, and analyzed their expression patterns in human tissues.

Isolation and expression analysis of the human testis-specific gene, SPERGEN-1, a spermatogenic cell-specific gene-1.

Spermatogenesis in testis is an excellent model system to study regulation of gene expression during cell differentiation. Previously more than 100 cDNA fragments, which include several novel as well as already identified genes with developmentally upregulated expression in rat testis, were cloned by using differential display method. One of them is the gene encoding iba1, an ionized calcium-binding adapter molecule-1, which was expressed in haploid spermatids, but not in other germ cells, in rat testis (1). Recently another gene, spergen-1, a spermatogenic cell-specific gene-1, was isolated by the same procedure (2). The rat spergen-1 is highly expressed in testis; however, it is undetectable in other organs examined. It was first detectable at 4 weeks of postnatal development and its expression increased thereafter. In situ hybridization analysis demonstrated that spergen-1 mRNA is expressed specifically in spermatids of steps 5–11 in the seminiferous epithelium of the rat testis. Genes specifically expressed in haploid spermatids are very interesting, because such genes might be involved in spermatid differentiation or spermiogenesis. For example, haploid-specific genes, such as hapsin, hsp70t, and oaz-t, have provided valuable clues to understand the mechanism regulating spermiogenesis (3–5). In addition, spergen-1 protein has a mitochondriatargeting signal at the N terminus in rat (2). Previously we have reported that isolation of the human RNH2 and STRA8 cDNA and their expressions, which are limited to the human testis (6,7). In this study, we have isolated the human SPERGEN-1 cDNA and analyzed its expression patterns in human tissues.

Laparoscopic Treatment of Endometrioma-Associated Infertility and Pregnancy Outcome

Ovarian endometriomas do not respond well to medical treatment with hormonal suppression, and surgical removal of the endometriomas is usually required. In this study, we attempt to identify the optimal laparoscopic procedures in laparoscopic treatment of ovarian-endometrioma-associated infertility. Among cases in which patients received no IVF-ET after the laparoscopic treatment, the pregnancy rate after complete cystectomy of endometriomas was statistically lower than that after fenestration with electrocoagulation of the cyst wall. Among cases in which patients received IVF-ET, there was no difference in ovarian response between patients that had complete cystectomy and fenestration with electrocoagulation of the cyst wall. However, the pregnancy rate in patients who had aspiration alone was statistically lower than that in patients who had aspiration followed by ethanol fixation. Thus, it appears that for patients who do not require follow-up IVF-ET, fenestration with electrocoagulation of the cyst wall is suitable, whereas for patients who need follow-up IVF-ET, ethanol fixation may be a better choice.

Differential expression of heparin-binding epidermal growth factor-like growth factor in the rat ovary

We investigated the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and its receptors in the rat ovary to define the role of HB-EGF in the ovarian function. The expression pattern of HB-EGF mRNA and protein were studied by semi-quantitative RT-PCR and immuno-histochemistry using an antibody that was specifically stained for the precursor form of HB-EGF in naturally cycling rats and immature pseudo-pregnant rat models. The immuno-histochemical study showed that in naturally cycling rats, HB-EGF was expressed in most granulosa cells of early follicles and all the developing follicles but not in preovulatory follicles. This was supported by the semi-quantitative RT-PCR results in that the lowest level of HB-EGF mRNA during the estrous cycle was found in the evening of proestrous when the HB-EGF negative preovulatory follicles were most prominent. The results suggest that HB-EGF might be a mitogen for granulosa cells and down regulation of its expression may be necessary for the final maturation of follicles. In corpora lutea, luteal cells of older generation stained stronger than those of younger generation. Moreover, luteal cells of late luteal phase stained stronger than those of the mid and early luteal phases in the immature pseudo-pregnant rat models, indicating that the precursor form may be associated with death of luteal cells. Finally, of the two cognate receptors for HB-EGF, erbB1 was expressed in the rat ovary, but erbB4 was specifically not expressed in this organ. The spatial and temporal pattern of HB-EGF expression suggest that HB-EGF may an important local regulator of ovarian function and structure.

Nuclear Dynamics of Parthenogenesis of Human Oocytes: Effect of Oocyte Aging in vitro

We investigated in detail the nuclear kinetics of oocyte activation of aged human oocytes following combined activation treatment with calcium ionophore and puromycin. Two types of oocytes were used: (a) 1-day-old oocytes after 20–24 h retrieval, and (b) 2-day-old oocytes after 44–50 h retrieval. A total of 185 unfertilized aged oocytes, 91 1-day-old and 94 2-day-old oocytes, were fixed at 1, 2, 4, 6 and 8 h after activation treatment and then metaphase II (MII), anaphase or telophase II (A/T II) or pronuclear stage were recorded. We demonstrated that a combined calcium ionophore and puromycin treatment induced a high activation rate in both 1-day-old (95.6%) and 2-day-old oocytes (95.2%). Our results also demonstrated that the nuclear progression was faster in 2-day-old oocytes than in 1-day-old oocytes, although nuclear progression in parthenogenetically activated human oocytes requires the longer time periods compared with ICSI fertilization. It is concluded that combined treatment of the calcium ionophore and puromycin allows a high rate of parthenogenetic activation and the nuclear kinetics of parthenogenetically activated human oocytes appears to be more rapid in in vitro aging oocytes.

Short Communication: Isolation and Expression Analysis of the Testis-Specific Gene, Human OPPO1

AbstractPurpose: To investigate human spermatogenesis, we isolated human testis-specific genes. Methods: Using mouse amino acid sequences, we found the region including homology in amino acid level in the human genome sequences. The primers encompassing introns were made and RT-PCR and RACE were carried out. The resultant PCR products were sequenced. Results: The full-length cDNA of human OPPO1 was isolated. It encodes 257 amino acid residues. The expression of the human OPPO1 was predominantly in the testis. On the other hand, partial cDNAs of ZNF8, GR194, GR219, GR093, GR046, GR163, and GR200 were expressed in the various tissues. Conclusions: Our data suggests that the human OPPO1 may play important roles in human spermatogenesis.

The human transcript induced in spermatogenesis 50

Background and AimsRecently, a number of genes that are expressed specifically in the testis have been identified in rat and mouse. In 2002, 80 transcript induced in spermatogenesis (Tisp) genes with this specific expression were isolated in mice. In the human, however, the number of such genes isolated is much lower. The aim of this study therefore was the isolation of human genes specifically expressed in testis.MethodsWe searched for human genome region with homology to the mouse Tisp gene family at the amino acid level using GenBank. The primers were made in human homologous regions, and polymerase chain reaction analysis was performed with templates using cDNA libraries of a range of human tissues. The cDNA specifically expressed in testis were isolated and detailed expression analysis was performed.ResultsThe 28 human TISP related genes were analyzed. Five of these genes were not expressed in testis and only three, TISP50, TISP15 and TISP43 related gene, were expressed specifically in testis. The cDNA of these three genes were isolated.ConclusionExpression analysis demonstrated that there is some discrepancy between human and mouse for the TISP gene family. From expression patterns and amino acid sequences, it is suggested that the human TISP50, TISP15 and TISP43 related genes play some critical roles in spermatogenesis.