Failure of the expression of phospholipid hydroperoxide glutathione peroxidase in the spermatozoa of human infertile males

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) was intensely expressed in mitochondria in the midpiece of human spermatozoa by immunostaining with anti-PHGPx monoclonal antibodies. The PHGPx not only reduced phospholipid hydroperoxide but also scavenged hydrogen peroxide in human spermatozoa. We found a dramatic decrease in the level of expression of PHGPx in the spermatozoa of some infertile males by immunoblotting with anti-PHGPx monoclonal antibodies. These individuals accounted for about 10% of the group of 73 infertile males that we examined. All seven patients with PHGPx-defective spermatozoa were classified as suffering from oligoasthenozoospermia, a defect in which both the number and the motility of spermatozoa are significantly below normal. Males with PHGPx-defective spermatozoa accounted for 26% of the 27 infertile males with oligoasthenozoospermia. No defects in expression of PHGPx in spermatozoa were observed in 31 fertile volunteers. After a 3-h incubation, the relative number of motile spermatozoa with low-level expression of PHGPx was significantly lower than that of spermatozoa with normal expression of PHGPx. The PHGPx-defective spermatozoa failed to incorporate rhodamine 123, revealing a loss of mitochondrial membrane potential. Ultrastructual analysis of mitochondria by electron microscopy demonstrated that the morphology of mitochondria in PHGPx-defective spermatozoa was abnormal. The results suggest that failure of the expression of mitochondrial PHGPx in spermatozoa might be one of the causes of oligoasthenozoospermia in infertile men.     

Dmbx1, a novel evolutionarily conserved paired-like homeobox gene expressed in the brain of mouse embryos.

To identify novel homeobox genes expressed during mouse embryogenesis, we searched the databases and found a novel mouse paired-like homeobox gene, Dmbx1(diencephalon/mesencephalon-expressed brain homeobox gene 1), that is also conserved in zebrafish and human. Linkage analysis mapped mouse Dmbx1 to the mid-portion of chromosome 4 that is the homologous gene cluster region of human chromosome 1, where human DMBX1 is located. Both mouse and human Dmbx1/DMBX1 have four coding exons and their gene structures are conserved. Whole-mount in situ hybridization revealed that Dmbx1 expression is detected in 7.5-9.5 dpc mouse embryos. At 7.5 and 8.5 dpc, Dmbx1 is expressed in a sub-region of the anterior head folds. At 9.5 dpc, expression is observed in the caudal diencephalon as well as in the mesencephalon and is restricted to the neuroepithelium. Expression in adult tissues was detected in brain, stomach, and testis. Dmbx1 provides a unique marker of the developing anterior nervous system and should provide a useful molecular resource to elucidate the mechanisms that pattern the vertebrate brain.     

Embryonic fibroblasts from Gpx4+/- mice: a novel model for studying the role of membrane peroxidation in biological processes

A previous study using mice null for Gpx4 indicates that PHGPx plays a critical role in antioxidant defense and is essential for the survival of the mouse. In the present study, we further analyzed the stress response of MEFs (murine embryonic fibroblasts) derived from mice heterozygous for the Gpx4 gene (Gpx4(+/-) mice). MEFs from Gpx4(+/-) mice have a 50% reduction in PHGPx expression without any changes in the activities of other major antioxidant defense enzymes. Compared to MEFs from Gpx4(+/+) mice, MEFs from Gpx4(+/-) mice were more sensitive to exposure to the oxidizing agent t-butyl hydroperoxide (t-BuOOH), and t-BuOOH exposure induced increased apoptosis in MEFs from Gpx4(+/-) mice. When cultured at low cell density, MEFs from Gpx4(+/-) mice also showed retarded growth under normal culture conditions (20% oxygen) that was reversed by culturing under low oxygen (2% oxygen). In addition, oxidative damage was increased in the MEFs from the Gpx4(+/-) mice, as indicated by increased levels of F(2)-isoprostanes and 8-oxo-2-deoxyguanosine in these cells. Our data demonstrate that MEFs from Gpx4(+/-) mice are more sensitive to oxidative stress because of reduced expression of PHGPx.     

The ter mutation first causes primordial germ cell deficiency in ter/ter mouse embryos at 8 days of gestation

The ter (teratoma) mutation causes primordial germ cell (PGC) deficiency in ter/ter embryos at 9.5–12.5 days of post-coitum (dpc) in mouse strains 129/Sv- ter and LTXBJ- ter . To study the effects of the ter mutation on the PGC development more precisely, we examined the PGC number and distribution in 7.5–12.5 dpc embryo of ter congenic C57BL/6J- ter strain using their complete serial sections. The ter genotypes of embryos were identified by the polymerase chain reaction (PCR) polymorphisms of the microsatellite DNA of the Grl -1 locus mapped near the ter locus. Results showed that: (i) the PGC number in ter/ter embryos was similar to those of + / ter and + / + embryos at 7.5 dpc, and did not increase at 8.0–12.5 dpc, although those of normal littermates did usually; (ii) the PGC migration to genital ridges was never affected in all embryos; and (iii) the ter genotype difference in the PGC numbers was not recognized between + / ter and + / + embryos. We concluded that the ter mutation does not affect the PGC appearance around 7.5 dpc, but first causes PGC deficiency around 8.0 dpc at the beginning of their migration and proliferation, suggesting that the normal function of the ter gene may be essential for the proliferation or survival mechanisms of PGC.     

Phospholipid hydroperoxide glutathione peroxidase: expression pattern during testicular development in mouse and evolutionary conservation in spermatozoa

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein belonging to the family of glutathione peroxidases and has been implicated in antioxidative defense and spermatogenesis. PHGPx accounts for almost the entire selenium content of mammalian testis. In an attempt to verify the expression pattern of PHGPx, testes of mouse mutants with arrest at different stages of germ cell development and testes of mice at different ages were subjected to immunostaining with a monoclonal anti-PHGPx antibody. PHGPx was detected in Leydig cells of testes in all developmental stages. In the seminiferous tubuli, the PHGPx staining was first observed in testes of 21-day-old mice which is correlated with the appearance of the first spermatids. This result was confirmed when the testes of mutant mice with defined arrest of germ cell development were used. An immunostaining was observed in the seminiferous tubuli of olt/olt and qk/qk mice which show an arrest at spermatid differentiation. In Western blot analysis of proteins extracted from testes of mutant mice and from developing testes, two signals at 19- and 22-kDa were observed which confirm the existence of two PHGPx forms in testicular cells. In mouse spermatozoa, a subcellular localization of PHGPx and sperm mitochondria-associated cysteine-rich protein (SMCP) was demonstrated, indicating the localization of PHGPx in mitochondria of spermatozoa midpiece. For verifying the midpiece localization of PHGPx in other species, spermatozoa of Drosophila melanogaster, frog, fish, cock, mouse, rat, pig, bull, and human were used in immunostaining using anti-PHGPx antibody. A localization of PHGPx was found in the midpiece of spermatozoa in all species examined. In electronmicroscopical analysis, PHGPx signals were found in the mitochondria of midpiece. These results indicate a conserved crucial role of PHGPx during sperm function and male fertility.     

Embryonic expression profile of phospholipid hydroperoxide glutathione peroxidase

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) is indispensable for murine embryonic development; yet, the cellular mechanisms leading to embryonic death around gastrulation are still unclear. To investigate PHGPx expression patterns during embryogenesis, we performed a detailed analysis that revealed a complex expression profile. Up to embryonic day 9.5, PHGPx was ubiquitously expressed, which was, albeit to a lower extent, maintained throughout later stages of embryogenesis. Notably, strong expression was frequently observed in epithelial tissue. A transient increase in PHGPx expression was detected in developing tissues, suggesting a crucial role for PHGPx in proliferation and differentiation. By semi-quantitative RT-PCR analysis we observed that the cytosolic form of PHGPx was present in embryonic and somatic tissues whereas the mitochondrial and nuclear forms were detectable only in testicular tissue. This strongly suggests that it is the cytosolic form of PHGPx that is indispensable for embryonic development.     

Isolation and characterization of a novel PHGPx gene in Raphanus sativus

A full-length cDNA encoding putative phospholipid hydroperoxide glutathione peroxidase (PHGPx) was cloned from Raphanus sativus. The cDNA, designated RsPHGPx, includes an open reading frame which encodes 197 amino acid residues. The alignment of amino acid sequences showed that RsPHGPx had the highest sequence homology to plant PHGPx and contained an N-terminal extension characteristic of a mitochondrial targeting peptide. Northern blot analysis indicated that RsPHGPx was constitutively and ubiquitously expressed during radish development, and its expression was differently regulated by various stress conditions. The expression of RsPHGPx in a yeast PHGPx-deletion mutant significantly rescued the mutant sensitivity to oxidation-sensitive linolenic acid, just as the yeast PHGPx3 gene did. This suggested that RsPHGPx encodes a functional PHGPx protein.     

The targeted disruption of the MYPT1 gene results in embryonic lethality

Myosin phosphatase (MP) is a major phosphatase responsible for the dephosphorylation of the regulatory light chain of myosin II. MYPT1, a target subunit of smooth and nonmuscle MP, is responsible for activation and regulation of MP. To identity the physiological roles of MP, we have generated MYPT1-deficient mice by gene targeting. The heterozygous mice showed no changes in expression levels of MYPT1 and no distinct phenotype compared to wild-type mice was observed. None of the F2 mice were homozygous for the MYPT1 deletion, indicating that the targeted disruption of the MYPT1 gene resulted in embryonic lethality. The point of embryonic lethality is before 7.5 dpc. These findings indicate that MYPT1 is essential for mouse embryogenesis.     

Expression of smoothened in mouse embryonic maxillofacial development

Abstract

Hedgehog (Hh) signaling plays many key roles in the development of Drosophila and vertebrate embryos including regulation of craniofacial development. The seven-transmembrane protein, smoothened (Smo) transduces the Hh signal across the plasma membrane as an essential receptor of PTCHED1/2. There are few studies that evaluate the detailed expression of Smo in mouse embryonic craniofacial development. We investigated the expression patterns of Smo during murine embryonic craniofacial development using in situ hybridization (ISH), studies of whole-mounts and sections, immunohistochemistry, quantitative real time PCR, and Western blot analysis. We found that Smo mRNA was expressed in the face of mouse embryos at 11 and 12.5 days post coitum (dpc). After 13.5 dpc, the expression decreased to a low level and was faintly detected after birth. Smo protein could be detected also in embryos at 11, 12.5, and 14.5 dpc. After 15.5 dpc, the expression was very faint and paralleled the gene expression studies. No expression was detected in whisker follicle during facial development and faint signal was detected in Meckel's cartilage. These findings concerning Smo expression should guide further investigation of sonic Hh signaling pathway gene function during maxillofacial development.     

Molecular cloning and functional expression of nucleolar phospholipid hydroperoxide glutathione peroxidase in mammalian cells

We cloned a full-length cDNA for phospholipid hydroperoxide glutathione peroxidase (PHGPx) including exon Ib from rat and mouse testis. The nuclear signal sequence of the N terminal of rat nuclear PHGPx possessed a different sequence from that previously reported for rat sperm nuclei GPx (SnGPx). Expression of this PHGPx-YFP (yellow fluorescent protein) fusion protein including a novel nuclear signal sequence was exclusively localized in nucleolus; although YFPs fused with only a novel nuclear signal sequence were distributed in the whole nucleus, indicating that preferential translocation of nucleolar PHGPx into nucleoli was required for the nuclear signal sequence and internal sequence of PHGPx. Low level expression of nucleolar PHGPx was detected in several tissues, but the expression of nucleolar PHGPx was extensively high in testis. Immunohistochemical analysis with anti-nucleolar PHGPx indicated that expression of nucleolar PHGPx was observed in the nucleoli in the spermatogonia, spermatocyte, and spermatid. Overexpression of 34kDa nucleolar PHGPx in RBL2H3 cells significantly suppressed cell death induced by actinomycin D and doxorubicin that induced damage in the nucleolus. These results indicated that nucleolar PHGPx plays an important role in prevention of nucleolus from damage in mammalian cells.     

Preferential expression of Mdm2 oncogene during the development of neural crest and its derivatives in mouse early embryogenesis

The Mdm2 oncoprotein acts as the principal negative regulator of p53 activities and is essential for its control during mouse early development, at least before implantation. We analyzed Mdm2 expression between 7.5 and 9 days post-coitum (dpc) by whole-mount in situ hybridization and report here a novel expression pattern during neural crest development. At 7.5 dpc Mdm2 becomes preferentially expressed at the top of the neural folds. Between 8 and 9 dpc, this preferential expression is also observed in neural crest cells migrating from the closing brain towards craniofacial regions and the first three branchial arches. It persists in the craniofacial mesenchyme and the first branchial arch in 9 dpc embryos. Migrating neural crest cells in the tail region are also preferentially labeled at this stage. At day 9.5 Mdm2 becomes more ubiquitously expressed throughout the embryo as reported before.     

萝卜磷脂氢谷胱甘肽过氧化物酶基因结构及其调控序列分析

磷脂氢谷胱甘肽过氧化物酶 (PHGPx) 是谷胱甘肽过氧化物酶 (GPx) 家族的重要一员,是目前已知能直接保护生物膜免受过氧化损伤的唯一酶类 . 此前的研究表明,萝卜磷脂氢谷胱甘肽过氧化物酶基因 (RsPHGPx) 编码一个有生理功能的过氧化物酶 , 并且 RsPHGPx 基因的表达可能受发育和环境胁迫信号的复杂调控 . 要深入了解该基因的表达调控机制首先必须阐明 RsPHGPx 基因的结构及其上游调控序列 . DNA 印迹表明萝卜 RsPHGPx 基因以单拷贝的形式存在于基因组中 . 以基因组 DNA 为模板,通过常规 PCR 与染色体步行相结合的方法克隆到了一段 3.3 kb 长的 RsPHGPx 基因组序列 . 分析发现,该基因由 7 个外显子和 6 个内含子组成,所有内含子的剪切位点均符合真核生物 GT-AG 规则 . 另外还发现该基因的上游基因是生物素合成酶基因;位于 RsPHGPx 基因上游的调控序列只有不足 300 bp. 这些结构特征与拟南芥 AtGPX3 基因极其相似 . 顺式作用元件的数据库搜索发现 RsPHGPx 基因的上游调控序列含有多个响应激素 ( 如 E-Box 和 W-Box) 、胁迫 ( 如转录因子 MYB 和 MYC 的结合位点 ) 和光 ( 如 Box Ⅱ和Ⅰ -Box) 信号的元件 . RNA 印迹分析表明 RsPHGPx 基因的表达受到脱落酸 (ABA) 和连续光照 ( 在黄化苗中 ) 处理的负调控,受到冷胁迫 (4℃ ) 的正调控,这暗示了预测的顺式作用元件的调控作用 . 然而,除草剂 paraquat 对该基因表达的正调控作用,暗示了某些与氧化胁迫相关的未知元件的存在 . 这些结果进一步印证了 RsPHGPx 基因的表达受发育和环境胁迫信号复杂调控的推测 . 这是迄今为止首个关于植物 PHGPx 基因结构和上游调控序列的系统报道,为今后全面认识植物 PHGPx 基因的表达调控机制奠定了必要基础 .     

Physiological role of phospholipid hydroperoxide glutathione peroxidase in mammals

The redox enzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) has emerged as one of the most significant selenoenzymes in mammals, corroborated by early embryonic lethality of PHGPx null mice. PHGPx is one of five selenium-dependent glutathione peroxidases and the second glutathione peroxidase to be discovered in 1982. PHGPx has a particular position within this family owing to its peculiar structural and catalytic properties, its multifaceted roles during male gametogenesis, and its necessity for early mouse development. Interestingly, mice devoid of endogenous glutathione die at the same embryonic stage as PHGPx-deficient mice compatible with the hypothesis that a similar phenotype of embryonic lethality may be provoked by PHGPx deficiency and lack of its reducing substrate glutathione. Various gain- and loss-of-function approaches in mice have provided some insights into the physiological functions of PHGPx. These include a protective role for PHGPx in response to irradiation, increased resistance of transgenic PHGPx mice to toxin-induced liver damage, a putative role in various steps of embryogenesis, and a contribution to sperm chromatin condensation. The expression of three forms of PHGPx and early embryonic lethality call for more specific studies, such as tissue-specific disruption of PHGPx, to precisely understand the contribution of PHGPx to mammalian physiology and under pathological conditions.     

Genomic characterization and embryonic expression of the mouse Bigh3 (Tgfbi) gene

Mutations in human BIGH3 (TGFB1), a gene identified after treatment of an adenocarcinoma cell line with TGF-beta, have been observed in patients with granular Groenouw type I, Reis-Bücklers, Thiel-Behnke, Avellino, and Lattice type I and IIIa, six autosomal dominant corneal dystrophies linked to chromosome 5q. In order to gain insight into the physiological role of this gene, we characterized the genomic structure of the mouse Bigh3 and its expression in murine embryos. The gene spans 30 kb on mouse chromosome 13 and has 17 exons. Embryonic expression of Bigh3 is observed in the mesenchyme of the first and second branchial arches as early as dpc 11.5 and is particularly strong in the mesenchyme of numerous tissues throughout all the development stages. In fetal eye, the expression is first seen at 11.5 dpc in the mesenchyme surrounding the optic stalk, extends toward the sclera and choroid by 14.3 dpc and reaches the cornea by 17.5 dpc. Because the physiological role of BIGH3/Bigh3 is still largely unknown, embryonic expression in organs like heart, vessels, and intestine may help to identify new functions which could be searched for in patients and in knock-out animal models. The characterization of the murine structure is a prerequisite for the making of such models.     

Cytokeratins 8 and 19 in the mouse placental development

To investigate the expression and biological roles of cytokeratin 19 (K19) in development and in adult tissues, we inactivated the mouse K19 gene (Krt1-19) by inserting a bacterial beta-galactosidase gene (lacZ) by homologous recombination in embryonic stem cells, and established germ line mutant mice. Both heterozygous and homozygous mutant mice were viable, fertile, and appeared normal. By 7.5-8.0 days post coitum (dpc), heterozygous mutant embryos expressed lacZ in the notochordal plate and hindgut diverticulum, reflecting the fact that the notochord and the gut endoderm are derived from the axial mesoderm-originated cells. In the adult mutant, lacZ was expressed mainly in epithelial tissues. To investigate the possible functional cooperation and synergy between K19 and K8, we then constructed compound homozygous mutants, whose embryos died approximately 10 dpc. The lethality resulted from defects in the placenta where both K19 and K8 are normally expressed. As early as 9. 5 dpc, the compound mutant placenta had an excessive number of giant trophoblasts, but lacked proper labyrinthine trophoblast or spongiotrophoblast development, which apparently caused flooding of the maternal blood into the embryonic placenta. These results indicate that K19 and K8 cooperate in ensuring the normal development of placental tissues.