Ryoji Yao

Lack of acrosome formation in mice lacking a Golgi protein, GOPC

The acrosome is a unique organelle that plays an important role at the site of sperm–zona pellucida binding during the fertilization process, and is lost in globozoospermia, an inherited infertility syndrome in humans. Although the acrosome is known to be derived from the Golgi apparatus, molecular mechanisms underlying acrosome formation are largely unknown. Here we show that Golgi-associated PDZ- and coiled-coil motif-containing protein (GOPC), a recently identified Golgi-associated protein, is predominantly localized at the trans-Golgi region in round spermatids, and male mice in which GOPC has been disrupted are infertile with globozoospermia. The primary defect was the fragmentation of acrosomes in early round spermatids, and abnormal vesicles that failed to fuse to developing acrosomes were apparent. In later stages, nuclear malformation and an abnormal arrangement of mitochondria, which are also characteristic features of human globozoospermia, were observed. Interestingly, intracytoplasmic sperm injection (ICSI) of such malformed sperm into oocytes resulted in cleavage into blastocysts only when injected oocytes were activated. Thus, GOPC provides important clues to understanding the mechanisms underlying spermatogenesis, and the GOPC-deficient mouse may be a unique and valuable model for human globozoospermia.

Oligo-astheno-teratozoospermia in mice lacking Cnot7, a regulator of retinoid X receptor beta

Spermatogenesis is a complex process that involves cooperation of germ cells and testicular somatic cells. Various genetic disorders lead to impaired spermatogenesis, defective sperm function and male infertility. Here we show that Cnot7−/− males are sterile owing to oligo-astheno-teratozoospermia, suggesting that Cnot7, a CCR4-associated transcriptional cofactor, is essential for spermatogenesis. Maturation of spermatids is unsynchronized and impaired in seminiferous tubules of Cnot7−/− mice. Transplantation of spermatogonial stem cells from male Cnot7−/− mice to seminiferous tubules of Kit mutant mice (KitW/W-v) restores spermatogenesis, suggesting that the function of testicular somatic cells is damaged in the Cnot7−/− condition. The testicular phenotypes of Cnot7−/− mice are similar to those of mice deficient in retinoid X receptor beta (Rxrb). We further show that Cnot7 binds the AF-1 domain of Rxrb and that Rxrb malfunctions in the absence of Cnot7. Therefore, Cnot7 seems to function as a coregulator of Rxrb in testicular somatic cells and is thus involved in spermatogenesis.

Specific Activation of a c-Jun NH2-terminal Kinase Isoform and Induction of Neurite Outgrowth in PC-12 Cells by Staurosporine

Staurosporine, a protein kinase inhibitor, is known to mimic the effect of nerve growth factor (NGF) in promoting neurite outgrowth. To elucidate the mechanism by which staurosporine induces neurite outgrowth in PC-12 cells, we performed an in-gel kinase assay using myelin basic protein as a substrate, and found that staurosporine induced the activation of a kinase with an apparent molecular mass of 57 kDa. The dose of staurosporine required to activate this kinase was consistent with that required to induce neurite outgrowth. Interestingly, the staurosporine-activated kinase was immunoprecipitated by anti-c-Jun NH2-terminal kinase (JNK) isoforms antibody, but not by anti-JNK1-specific antibody or anti-ERK1 antibody, raising the possibility that this kinase is a novel JNK isoform. The substrate specificity of the kinase was distinct from those of osmotic shock-activated JNKs and NGF-activated ERK1. The kinase phosphorylates transcription factors including c-Jun, Elk-1, and ATF2, as well as myelin basic protein, suggesting that it plays a role in gene induction. Furthermore, staurosporine induced immediate-early genes including Nur77 and fos, but notjun. The activation of the staurosporine-activated kinase, as well as the induction of neurite outgrowth, did not require Ras function, while Ras was required for the activation of ERKs and neurite outgrowth induced by NGF. Taken together, these results indicate staurosporine specifically activates a JNK isoform, which may contribute to biological activities including neurite outgrowth.

The selective continued linkage of centromeres from mitosis to interphase in the absence of mammalian separase

Separase is an evolutionarily conserved protease that is essential for chromosome segregation and cleaves cohesin Scc1/Rad21, which joins the sister chromatids together. Although mammalian separase also functions in chromosome segregation, our understanding of this process in mammals is still incomplete. We generated separase knockout mice, reporting an essential function for mammalian separase. Separase-deficient mouse embryonic fibroblasts exhibited severely restrained increases in cell number, polyploid chromosomes, and amplified centrosomes. Chromosome spreads demonstrated that multiple chromosomes connected to a centromeric region. Live observation demonstrated that the chromosomes of separase-deficient cells condensed, but failed to segregate, although subsequent cytokinesis and chromosome decondensation proceeded normally. These results establish that mammalian separase is essential for the separation of centromeres, but not of the arm regions of chromosomes. Other cell cycle events, such as mitotic exit, DNA replication, and centrosome duplication appear to occur normally. We also demonstrated that heterozygous separase-deficient cells exhibited severely restrained increases in cell number with apparently normal mitosis in the absence of securin, which is an inhibitory partner of separase.

MAGI-3 is involved in the regulation of the JNK signaling pathway as a scaffold protein for frizzled and Ltap.

A seven-transmembrane protein, frizzled, has been implicated in a planar cell polarity (PCP) pathway as well as the canonical Wnt signaling pathway. Although both pathways require a cytoplasmic protein, dishevelled, the molecular mechanism by which frizzled regulates intracellular signaling remains to be elucidated. In the mouse, nine frizzled family members have been identified and six of them contain a PDZ-binding motif at their carboxyl-termini. In this study, we show that a multi-PDZ containing protein, MAGI-3, specifically binds to frizzled-4 and -7. Furthermore, we also demonstrate that MAGI-3 interacts with Ltap, a mouse homolog of the Drosophila PCP protein, stbm, and that these three molecules can form a ternary complex. In epithelial cells, MAGI-3, frizzled-4, and Ltap colocalized at cell contact sites, indicating that these molecules form a physiologically significant complex. Finally, we found that MAGI-3 strongly activated JNK in conjunction with frizzled-4 and Ltap, and that this activation required the small GTPase, Rac. These results indicate that MAGI-3 functions as a scaffold protein for frizzled-4 and Ltap and regulates the JNK signaling cascade.

Factors Maintaining Normal Sperm Tail Structure During Epididymal Maturation Studied in Gopc−/− Mice

Abstract Gopc (Golgi-associated PDZ- and coiled-coil motif-containing protein)−/− mice are infertile, showing globozoospermia, coiled tails, and a stratified mitochondrial sheath. Transmission electron microscope (TEM) images of the spermatozoa were studied quantitatively to analyze disorganization processes during epididymal passage. Factors maintaining straight tail and normal mitochondrial sheath were also studied by TEM and immunofluorescent microscopy. Sperm tails retained a normal appearance in the proximal caput epididymidis. Tail disorganization started between the proximal and the middle caput epididymidis, and the latter is the major site for it. The tail moved up through the defective posterior ring and coiled around the nucleus to various degrees. Tail coiling occurred in the caput epididymidis suggesting it was triggered by cytoplasmic droplet migration. SPATA19/spergen-1, a candidate mitochondrial adhesion protein, remained on the stratified mitochondria, while GPX4/PHGPx, a major element of the mitochondrial capsule, was unevenly distributed on them. From these findings, we speculate GPX4 is necessary to maintain normal sheath structure, and SPATA19 prevents dispersal of mitochondria, resulting in a stratified mitochondrial sheath formation in Gopc−/− spermatozoa. The epididymal epithelium was normal in structure and LRP8/apoER2 expression suggesting that tail abnormality is due to intrinsic sperm factors. Three cell structures are discussed as requisite factors for maintaining a straight tail during epididymal maturation: 1) a complete posterior ring to prevent invasion of the tail into the head compartment, 2) stable attachment of the connecting piece to the implantation fossa, and 3) a normal mitochondrial sheath supported by SPATA19 and supplied with sufficient and normally distributed GPX4.

TACC3 is required for the proper mitosis of sclerotome mesenchymal cells during formation of the axial skeleton

Transforming acidic coiled‐coil‐containing (TACC) family members regulate mitotic spindles and have essential roles in embryogenesis. However, the functions of TACC3 in mitosis during mammalian development are not known. We have generated and characterized three mutant alleles of mouse Tacc3 including a conditional allele. Homozygous mutants of a hypomorphic allele exhibited malformations of the axial skeleton. The primary cause of this defect was the failure of mitosis in mesenchymal sclerotome cells. In vitro, 36% of primary mouse embryo fibroblasts (MEF) obtained from mutants homozygous for the hypomorphic allele and 67% of MEF from Tacc3 null mutants failed mitosis. In cloned immortalized MEF, Tacc3 depletion destabilized spindles and prevented chromosomes from aligning properly. Furthermore, chromosome separation and cytokinesis were also severely impaired. Chromosomes were moved randomly and cytokinesis initiated but the cleavage furrow eventually regressed, resulting in binucleate cells that then yielded aneuploid cells in the next cell division. Thus, in addition to spindle assembly, Tacc3 has critical roles in chromosome separation and cytokinesis, and is essential for the mitosis of sclerotome mesenchymal cells during axial formation in mammals. (Cancer Sci 2007; 98: 555–562)

Disruption of Tacc3 function leads to in vivo tumor regression

The formation of the bipolar spindle is responsible for accurate chromosomal segregation during mitosis. The dynamic instability of microtubules has an important role in this process, and has been shown to be an effective target for cancer chemotherapy. Several agents that target non-microtubule mitotic proteins, including the motor protein Eg5, Aurora kinases and Polo-like kinases, are currently being developed as chemotherapeutic drugs. However, because the efficacies of these drugs remain elusive, new molecular targets that have essential roles in tumor cells are desired. Here, we provide in vivo evidence that transforming acidic coiled-coil-3 (Tacc3) is a potential target for cancer chemotherapy. Using MRI, we showed that Tacc3 loss led to the regression of mouse thymic lymphoma in vivo, which was accompanied by massive apoptosis. By contrast, normal tissues, including the thymus, showed no overt abnormalities, despite high Tacc3 expression. in vitro analysis indicated that Tacc3 depletion induced multi-polar spindle formation, which led to mitotic arrest, followed by apoptosis. Similar responses have been observed in Burkitts lymphoma and T-ALL. These results show that Tacc3 is a vulnerable component of the spindle assembly in lymphoma cells and is a promising cancer chemotherapy target.

A small compound targeting TACC3 revealed its different spatiotemporal contributions for spindle assembly in cancer cells.

The mitotic spindle is assembled by the coordinated action of centrosomes and kinetochore microtubules. An evolutionally conserved protein family, transforming acidic coiled-coil (TACC), has been shown to be involved in this process. In humans, TACC3 is aberrantly expressed in a variety of human cancers, but its biological significance remains to be elucidated. Here, using a novel compound targeting TACC3, spindlactone (SPL), we show that the perturbation of TACC3 selectively inhibited the nucleation of centrosome microtubules in ovarian cancer cells. In contrast to centrosome microtubules, the kinetochore microtubules were robustly assembled, forming ectopic spindle poles that resulted in multipolar spindles. Interestingly, the extensive inhibition of TACC3 partially suppressed the nucleation of kinetochore microtubules. These dose-dependent effects of SPL were consistent with the results observed by the depletion of TACC3 and its binding partner, colonic and hepatic tumor overexpressed gene protein (TOGp). Although these proteins both have roles in the assembly of centrosome and kinetochore microtubules, their contributions were spatiotemporally different. Notably, SPL did not affect spindle assembly in normal cells. Furthermore, the oral administration of SPL significantly suppressed tumor growth in vivo. The unique mechanism of action of SPL not only enables it to be used as a tool to dissect the molecular basis of spindle assembly but also to provide a rationale for the use of TACC3 as a molecular target for cancer treatment. This rationale offers an opportunity to develop new strategies for cancer chemotherapy that overcome the limitations of microtubule toxins and expand their scope and clinical efficacy.

Appearance of an oocyte activation-related substance during spermatogenesis in mice and humans

BACKGROUND Recently we reported that an oocyte activation ability in human and mouse sperm is associated with head flatness or the presence of perinuclear theca (PT) substance, MN13, which is an oocyte activation-related protein localized on the post-acrosomal sheath (PAS). As such, we hypothesize that the appearance of oocyte activation ability is stage-specifically regulated and depends on the formation of the acrosome or PAS/PT in spermatids. METHODS We monitored the appearance and movement of MN13 as a PT-specific molecule during spermatogenesis and analysed how the MN13 localization is affected in mouse and human globozoospermic acrosomeless sperm. RESULTS MN13 was first detected on the surface of acrosomic vesicles, i.e. on the nascent outer acrosomal membrane of step 5-6 round spermatids (Sb1 spermatids in human), and it was then translocated via the outer acrosomal membrane surface to the most distal region of the acrosome in step 7 round spermatids (Sb2 spermatids). As spermatids elongated, MN13 was translocated via the cytoplasmic space between the nuclear envelope and the overlying plasma membrane towards the post-acrosomal region, and it was organized on the top of the nascent PAS that was typically found in step 14 elongated spermatids (Sd1 spermatids). In contrast, MN13 was not found in any GOPC-deficient spermatids, which completely lack the acrosome but have manchettes (microtubule bundles), nor in mouse and human acrosomeless sperm. CONCLUSIONS The MN13 appearance or the MN13-related PAS/PT formation is highly dependant on acrosome formation; the MN13-related oocyte activation factor/ability is stage-specifically acquired in elongating/elongated spermatids.