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Yan C. Cheng - One of the best experts on this subject based on the ideXlab platform.
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Planar cell polarity protein Dishevelled 3 (Dvl3) regulates ectoplasmic specialization (ES) dynamics in the testis through changes in cytoskeletal organization
Nature Publishing Group, 2019Co-Authors: Baiping Mao, Ming Yan, Qingquan Lian, Yan C. ChengAbstract:Abstract In the mammalian testes, such as in rats, the directional alignment of polarized elongating/elongated Spermatids, in particular step 17–19 Spermatids, across the plane of seminiferous epithelium resembles planar cell polarity (PCP) found in hair cells of the cochlea. It is obvious that Spermatid PCP is necessary to support the simultaneous development of maximal number of elongating/elongated Spermatids to sustain the daily production of > 50 million sperm per adult rat. Studies have shown that the testis indeed expresses multiple PCP proteins necessary to support Spermatid PCP. Herein, using physiological and biochemical assays, and morphological analysis, and with the technique of RNA interference (RNAi) to knockdown PCP protein Dishevelled (Dvl) 1 (Dvl1), Dvl2, Dvl3, or Dvl1/2/3, Dvl proteins, in particular Dvl3, it was shown that Dvl3 played a crucial role of support Sertoli cell tight junction (TJ)-permeability barrier function through changes in the organization of actin- and microtubule (MT)-based cytoskeletons. More important, an in vivo knockdown of Dvl1/2/3 in the testis, defects of Spermatid polarity were remarkably noted across the seminiferous epithelium, concomitant with defects of Spermatid adhesion and Spermatid transport, leading to considerably defects in spermatogenesis. More important, Dvl1/2/3 triple knockdown in the testis also impeded the organization of actin- and MT-based cytoskeletons owing to disruptive spatial expression of actin- and MT-regulatory proteins. In summary, PCP Dishevelled proteins, in particular, Dvl3 is a regulator of Sertoli cell blood–testis barrier (BTB) and also Spermatid PCP function through its effects on the actin- and MT-based cytoskeletons in Sertoli cells
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ezrin is an actin binding protein that regulates sertoli cell and Spermatid adhesion during spermatogenesis
Endocrinology, 2014Co-Authors: Ece N Gungorordueri, Elizabeth I Tang, Ciler Celikozenci, Yan C. ChengAbstract:During spermatogenesis, the transport of Spermatids and the release of sperms at spermiation and the remodeling of the blood-testis barrier (BTB) in the seminiferous epithelium of rat testes require rapid reorganization of the actin-based cytoskeleton. However, the mechanism(s) and the regulatory molecule(s) remain unexplored. Herein we report findings that unfold the functional significance of ezrin in the organization of the testis-specific adherens junction at the Spermatid-Sertoli cell interface called apical ectoplasmic specialization (ES) in the adluminal compartment and the Sertoli cell-cell interface known as basal ES at the BTB. Ezrin is expressed at the basal ES/BTB in all stages, except from late VIII to IX, of the epithelial cycle. Its knockdown by RNA interference (RNAi) in vitro perturbs the Sertoli cell tight junction-permeability barrier via a disruption of the actin microfilaments in Sertoli cells, which in turn impeded basal ES protein (eg, N-cadherin) distribution, perturbing the BTB function. These findings were confirmed by a knockdown study in vivo. However, the expression of ezrin at the apical ES is restricted to stage VIII of the cycle and limited only between step 19 Spermatids and Sertoli cells. A knockdown of ezrin in vivo by RNAi was found to impede Spermatid transport, causing defects in spermiation in which Spermatids were embedded deep inside the epithelium, and associated with a loss of Spermatid polarity. Also, ezrin was associated with residual bodies and phagosomes, and its knockdown by RNAi in the testis also impeded the transport of residual bodies/phagosomes from the apical to the basal compartment. In summary, ezrin is involved in regulating actin microfilament organization at the ES in rat testes.
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actin binding proteins Spermatid transport and spermiation
Seminars in Cell & Developmental Biology, 2014Co-Authors: Xiaojing Qian, Will M. Lee, Dolores D Mruk, Yan Ho Cheng, Elizabeth I Tang, Daishu Han, Elissa W P Wong, Yan C. ChengAbstract:The transport of germ cells across the seminiferous epithelium is composed of a series of cellular events during the epithelial cycle essential to the completion of spermatogenesis. Without the timely transport of Spermatids during spermiogenesis, spermatozoa that are transformed from step 19 Spermatids in the rat testis fail to reach the luminal edge of the apical compartment and enter the tubule lumen at spermiation, thereby arriving the epididymis for further maturation. Step 19 Spermatids and/or sperms that remain in the epithelium beyond stage VIII of the epithelial cycle will be removed by the Sertoli cell via phagocytosis to form phagosomes and be degraded by lysosomes, leading to subfertility and/or infertility. However, the biology of Spermatid transport, in particular the final events that lead to spermiation remain elusive. Based on recent data in the field, we critically evaluate the biology of spermiation herein by focusing on the actin binding proteins (ABPs) that regulate the organization of actin microfilaments at the Sertoli-Spermatid interface, which is crucial for Spermatid transport during this event. The hypothesis we put forth herein also highlights some specific areas of research that can be pursued by investigators in the years to come.
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secreted frizzled related protein 1 sfrp1 regulates Spermatid adhesion in the testis via dephosphorylation of focal adhesion kinase and the nectin 3 adhesion protein complex
The FASEB Journal, 2013Co-Authors: Elissa W P Wong, Will M. Lee, Yan C. ChengAbstract:Development of spermatozoa in adult mammalian testis during spermatogenesis involves extensive cell migration and differentiation. Spermatogonia that reside at the basal compartment of the seminiferous epithelium differentiate into more advanced germ cell types that migrate toward the apical compartment until elongated Spermatids are released into the tubule lumen during spermiation. Apical ectoplasmic specialization (ES; a testis-specific anchoring junction) is the only cell junction that anchors and maintains the polarity of elongating/elongated Spermatids (step 8–19 Spermatids) in the epithelium. Little is known regarding the signaling pathways that trigger the disassembly of the apical ES at spermiation. Here, we show that secreted Frizzled-related protein 1 (sFRP1), a putative tumor suppressor gene that is frequently down-regulated in multiple carcinomas, is a crucial regulatory protein for spermiation. The expression of sFRP1 is tightly regulated in adult rat testis to control Spermatid adhesion and sperm release at spermiation. Down-regulation of sFRP1 during testicular development was found to coincide with the onset of the first wave of spermiation at approximately age 45 d postpartum, implying that sFRP1 might be correlated with elongated Spermatid adhesion conferred by the apical ES before spermiation. Indeed, administration of sFRP1 recombinant protein to the testis in vivo delayed spermiation, which was accompanied by down-regulation of phosphorylated (p)-focal adhesion kinase (FAK)-Tyr397 and retention of nectin-3 adhesion protein at the apical ES. To further investigate the functional relationship between p-FAK-Tyr397 and localization of nectin-3, we overexpressed sFRP1 using lentiviral vectors in the Sertoli-germ cell coculture system. Consistent with the in vivo findings, overexpression of sFRP1 induced down-regulation of p-FAK-Tyr397, leading to a decline in phosphorylation of nectin-3. In summary, this report highlights the critical role of sFRP1 in regulating spermiation via its effects on the FAK signaling and retention of nectin-3 adhesion complex at the apical ES.—Wong, E. W. P., Lee, W. M., Cheng, C. Y. Secreted Frizzled-related protein 1 (sFRP1) regulates Spermatid adhesion in the testis via dephosphorylation of focal adhesion kinase and the nectin-3 adhesion protein complex.
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regulation of spermiogenesis spermiation and blood testis barrier dynamics novel insights from studies on eps8 and arp3
Biochemical Journal, 2011Co-Authors: Yan C. Cheng, Dolores D MrukAbstract:Spermiogenesis in the mammalian testis is the most critical post-meiotic developmental event occurring during spermatogenesis in which haploid Spermatids undergo extensive cellular, molecular and morphological changes to form spermatozoa. Spermatozoa are then released from the seminiferous epithelium at spermiation. At the same time, the BTB (blood-testis barrier) undergoes restructuring to facilitate the transit of preleptotene spermatocytes from the basal to the apical compartment. Thus meiotic divisions take place behind the BTB in the apical compartment to form Spermatids. These germ cells enter spermiogenesis to transform into elongating Spermatids and then into spermatozoa to replace those that were released in the previous cycle. However, the mole-cular regulators that control spermiogenesis, in particular the dynamic changes that occur at the Sertoli cell-Spermatid interface and at the BTB, are not entirely known. This is largely due to the lack of suitable animal models which can be used to study these events. During the course of our investigation to develop adjudin [1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide] as a potential male contraceptive, this drug was shown to 'accelerate' spermiation by inducing the release of premature Spermatids from the epithelium. Using this model, we have identified several molecules that are crucial in regulating the actin filament network and the unique adhesion protein complex at the Sertoli cell-Spermatid interface known as the apical ES (ectoplasmic specialization). In the present review, we critically evaluate these and other findings in the literature as they relate to the restricted temporal and spatial expression of two actin regulatory proteins, namely Eps8 (epidermal growth factor receptor pathway substrate 8) and Arp3 (actin-related protein 3), which regulate these events.
Ryuzo Yanagimachi - One of the best experts on this subject based on the ideXlab platform.
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fourteen babies born after round Spermatid injection into human oocytes
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: A Tanaka, M Nagayoshi, Youichi Takemoto, I Tanaka, Hiroshi Kusunoki, Seiji Watanabe, Keiji Kuroda, Satoru Takeda, Masahiko Ito, Ryuzo YanagimachiAbstract:During the human in vitro fertilization procedure in the assisted reproductive technology, intracytoplasmic sperm injection is routinely used to inject a spermatozoon or a less mature elongating Spermatid into the oocyte. In some infertile men, round Spermatids (haploid male germ cells that have completed meiosis) are the most mature cells visible during testicular biopsy. The microsurgical injection of a round Spermatid into an oocyte as a substitute is commonly referred to as round Spermatid injection (ROSI). Currently, human ROSI is considered a very inefficient procedure and of no clinical value. Herein, we report the birth and development of 14 children born to 12 women following ROSI of 734 oocytes previously activated by an electric current. The round Spermatids came from men who had been diagnosed as not having spermatozoa or elongated Spermatids by andrologists at other hospitals after a first Micro-TESE. A key to our success was our ability to identify round Spermatids accurately before oocyte injection. As of today, all children born after ROSI in our clinic are without any unusual physical, mental, or epigenetic problems. Thus, for men whose germ cells are unable to develop beyond the round Spermatid stage, ROSI can, as a last resort, enable them to have their own genetic offspring.
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Human Reproduction vol.14 no.4 pp.1050–1056, 1999 Status of genomic imprinting in mouse Spermatids
2015Co-Authors: Fay L. Shamanski, Yasuyuki Kimura, Marie-cecile Lavoir, Roger A. Pedersen, Ryuzo YanagimachiAbstract:3To whom correspondence should be addressed The advent of human round Spermatid microinjection (ROSI) into oocytes as a treatment for severe male infertility raises the question of whether Spermatids have undergone all of the maturation processes necessary for normal development. It is particularly important to know whether Spermatids have undergone correct genomic imprinting, which results in the parent-of-origin-specific expression of only one allele of a gene. We assessed the imprinting status of three maternally and three paternally expressed genes in interspecific hybrid embryos generated by injecting Mus castaneus Spermatids into Mus musculus oocytes. We used the single nucleotide primer extension (SNuPE) assay to measure the relative expression of maternal and paternal alleles on the basis of sequence polymorphisms in the transcripts. Expression of imprinted genes in mouse embryos derived by ROSI did not differ from controls, indicating that paternal genes have undergone proper imprinting by the round Spermatid stage. Key words: genomic imprinting/infertility/ROSI/Spermatid
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mouse round Spermatids developed in vitro from preexisting spermatocytes can produce normal offspring by nuclear injection into in vivo developed mature oocytes
Biology of Reproduction, 2003Co-Authors: Joel Marh, Ryuzo Yanagimachi, Laura L. Tres, Yukiko Yamazaki, Abraham L. KierszenbaumAbstract:Abstract It has been shown that mature oocytes injected with nuclei from round Spermatids collected from mouse testis can generate normal offspring and that round Spermatids can develop in vitro. An undetermined issue is whether Spermatids developed in vitro are capable of generating fertile offspring by nuclear injection into oocytes. Herein, we report the production of normal and fertile offspring by nuclear injection using haploid Spermatid donors derived from mouse primary spermatocyte precursors cocultured with Sertoli cells. Cocultured spermatogonia and spermatocytes were characterized by their nuclear immunoreactive patterns determined by an antibody to phosphorylated histone H2AX (γ-H2AX), a marker for DNA double-strand breaks. Cocultured round Spermatid progenies display more than one motile flagellum, whose axonemes were recognized by antitubulin immunostaining. Flagellar wavelike movement and flagellar-driven propulsion of round Spermatids developed in vitro were documented by videomicroscopy (...
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behaviour of hamster and mouse round Spermatid nuclei incorporated into mature oocytes by electrofusion
Zygote, 1993Co-Authors: Atsuo Ogura, Ryuzo Yanagimachi, Noriko UsuiAbstract:Round Spermatids of the hamster and mouse were electrofused with homologous mature oocytes to examine the behaviour of their nuclei within the ooplasm. A single Spermatid was inserted in the perivitelline space of a mature oocyte and an electric fusion pulse given. In the hamster, the best Spermatid-oocyte fusion took place when the oocytes were pretreated with neuraminidase, subjected to 30 s AC (2 MHz, 20 V/cm) followed by a single fusion DC pulse (3000 V-cm, 10 microseconds) and another 30 s AC current. Inclusion of micromolar Ca2+ and Mg2+ in the fusion medium was essential for oocyte activation. Under these conditions all oocytes were activated and 20-40% fused with Spermatids. Of these fused oocytes only 5-10% had fully developed Spermatid (male) and oocyte (female) pronuclei. In the rest the Spermatid-derived pronuclei remained small throughout the pronuclear stage. However, nucleolus-like structures appeared de novo and DNA synthesis occurred in these small pronuclei. Regardless of the size of male pronuclei, chromosomes from the Spermatid and oocyte appeared to mingle and participate in the first cleavage. About 70% of the fused oocytes developed into the 2-cell stage. Electrofusion of mouse oocytes with Spermatids was less efficient. Even under the best conditions tested, less than 10% of the oocytes fused with Spermatids. Here again, most Spermatid nuclei remained small throughout the pronuclear stage.
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round Spermatid nuclei injected into hamster oocytes from pronuclei and participate in syngamy
Biology of Reproduction, 1993Co-Authors: Atsuo Ogura, Ryuzo YanagimachiAbstract:Round Spermatids are spermatogenic cells that have just completed meiosis. To discover whether nuclei of these haploid cells are genetically ready for fertilization, nuclei were individually injected into mature hamster oocytes via a microsurgical technique. In the majority of oocytes that were successfully injected and activated, Spermatid nuclei transformed into pronuclei and underwent DNA synthesis. Ultimately their chromosomes mingled with ootid chromosomes immediately prior to the first cleavage. Although we could not determine the developmental potential of these zygotes, Spermatid nuclei appear capable of participating in syngamy.
Abraham L. Kierszenbaum - One of the best experts on this subject based on the ideXlab platform.
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GMAP210 and IFT88 are present in the Spermatid golgi apparatus and participate in the development of the acrosome–acroplaxome complex, head–tail coupling apparatus and tail
Developmental dynamics : an official publication of the American Association of Anatomists, 2011Co-Authors: Abraham L. Kierszenbaum, Bradley K Yoder, Eugene Rivkin, Laura L. Tres, Courtney J. Haycraft, Michel Bornens, Rosa M. RiosAbstract:We describe the localization of the golgin GMAP210 and the intraflagellar protein IFT88 in the Golgi of Spermatids and the participation of these two proteins in the development of the acrosome–acroplaxome complex, the head–tail coupling apparatus (HTCA) and the Spermatid tail. Immunocytochemical experiments show that GMAP210 predominates in the cis-Golgi, whereas IFT88 prevails in the trans-Golgi network. Both proteins colocalize in proacrosomal vesicles, along acrosome membranes, the HTCA and the developing tail. IFT88 persists in the acrosome–acroplaxome region of the sperm head, whereas GMAP210 is no longer seen there. Spermatids of the Ift88 mouse mutant display abnormal head shaping and are tail-less. GMAP210 is visualized in the Ift88 mutant during acrosome–acroplaxome biogenesis. However, GMAP210–stained vesicles, mitochondria and outer dense fiber material build up in the manchette region and fail to reach the abortive tail stump in the mutant. In vitro disruption of the Spermatid Golgi and microtubules with Brefeldin-A and nocodazole blocks the progression of GMAP210- and IFT88-stained proacrosomal vesicles to the acrosome–acroplaxome complex but F-actin distribution in the acroplaxome is not affected. We provide the first evidence that IFT88 is present in the Golgi of Spermatids, that the microtubule-associated golgin GMAP210 and IFT88 participate in acrosome, HTCA, and tail biogenesis, and that defective intramanchette transport of cargos disrupts Spermatid tail development. Developmental Dynamics 240:723–736, 2011. V C 2011 Wiley-Liss, Inc.
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the acroplaxome is the docking site of golgi derived myosin va rab27a b containing proacrosomal vesicles in wild type and hrb mutant mouse Spermatids
Biology of Reproduction, 2004Co-Authors: Abraham L. Kierszenbaum, Eugene Rivkin, Laura L. Tres, Ningling Kangdecker, Jan M Van DeursenAbstract:Acrosome biogenesis involves the transport and fusion of Golgi-derived proacrosomal vesicles along the acroplaxome, an F-actin/keratin 5-containing cytoskeletal plate anchored to the Spermatid nucleus. A significant issue is whether the acroplaxome develops in acrosomeless mutant mice. Male mice with a Hrb null mutation are infertile and both Spermatids and sperm are round-headed and lack an acrosome. Hrb, a protein that contains several NPF motifs (Asn-Pro-Phe) and interacts with proteins with Eps15 homology domains, is regarded as critical for the docking and/or fusion of Golgi-derived proacrosomal vesicles. Here we report that the lack of an acrosome in Hrb mutant Spermatids does not prevent the development of the acroplaxome. Yet the acroplaxome in the mutant contains F-actin but is deficient in keratin 5. We also show that the actin-based motor protein myosin Va and its receptor, Rab27a/b, known to be involved in vesicle transport, are present in the Golgi and Golgi-derived proacrosomal vesicles in wild-type and Hrb mutant mouse Spermatids. In the Hrb mutant, myosin-Va-bound proacrosome vesicles tether to the acroplaxome, where they flatten and form a flat sac, designated pseudoacrosome. As spermiogenesis advances, round-shaped Spermatid nuclei of the mutant display several nuclear protrusions, designated nucleopodes. Nucleopodes are consistently found at the acroplaxome- pseudoacrosome site. Our findings support the interpretation that the acroplaxome provides a focal point for myosin-Va/ Rab27a/b-driven proacrosomal vesicles to accumulate, coalesce, and form an acrosome in wild-type Spermatids and a pseudoacrosome in Hrb mutant Spermatids. We suggest that nucleopodes develop at a site where a keratin 5-deficient acroplaxome may not withstand tension forces operating during Spermatid nuclear shaping.
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acroplaxome an f actin keratin containing plate anchors the acrosome to the nucleus during shaping of the Spermatid head
Molecular Biology of the Cell, 2003Co-Authors: Abraham L. Kierszenbaum, Eugene Rivkin, Laura L. TresAbstract:Nuclear shaping is a critical event during sperm development as demonstrated by the incidence of male infertility associated with abnormal sperm ad shaping. Herein, we demonstrate that mouse and rat Spermatids assemble in the subacrosomal space a cytoskeletal scaffold containing F-actin and Sak57, a keratin ortholog. The cytoskeletal plate, designated acroplaxome, anchors the developing acrosome to the nuclear envelope. The acroplaxome consists of a marginal ring containing keratin 5 10-nm-thick filaments and F-actin. The ring is closely associated with the leading edge of the acrosome and to the nuclear envelope during the elongation of the Spermatid head. Anchorage of the acroplaxome to the gradually shaping nucleus is not disrupted by hypotonic treatment and brief Triton X-100 extraction. By examining spermiogenesis in the azh mutant mouse, characterized by abnormal Spermatid/sperm head shaping, we have determined that a deformity of the Spermatid nucleus is restricted to the acroplaxome region. These findings lead to the suggestion that the acroplaxome nucleates an F-actin-keratin-containing assembly with the purpose of stabilizing and anchoring the developing acrosome during Spermatid nuclear elongation. The acroplaxome may also provide a mechanical planar scaffold modulating external clutching forces generated by a stack of Sertoli cell F-actin-containing hoops encircling the elongating Spermatid nucleus.
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mouse round Spermatids developed in vitro from preexisting spermatocytes can produce normal offspring by nuclear injection into in vivo developed mature oocytes
Biology of Reproduction, 2003Co-Authors: Joel Marh, Ryuzo Yanagimachi, Laura L. Tres, Yukiko Yamazaki, Abraham L. KierszenbaumAbstract:Abstract It has been shown that mature oocytes injected with nuclei from round Spermatids collected from mouse testis can generate normal offspring and that round Spermatids can develop in vitro. An undetermined issue is whether Spermatids developed in vitro are capable of generating fertile offspring by nuclear injection into oocytes. Herein, we report the production of normal and fertile offspring by nuclear injection using haploid Spermatid donors derived from mouse primary spermatocyte precursors cocultured with Sertoli cells. Cocultured spermatogonia and spermatocytes were characterized by their nuclear immunoreactive patterns determined by an antibody to phosphorylated histone H2AX (γ-H2AX), a marker for DNA double-strand breaks. Cocultured round Spermatid progenies display more than one motile flagellum, whose axonemes were recognized by antitubulin immunostaining. Flagellar wavelike movement and flagellar-driven propulsion of round Spermatids developed in vitro were documented by videomicroscopy (...
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ran a gtp binding protein involved in nucleocytoplasmic transport and microtubule nucleation relocates from the manchette to the centrosome region during rat spermiogenesis
Molecular Reproduction and Development, 2002Co-Authors: Abraham L. Kierszenbaum, Eugene Rivkin, Mara Gil, Laura L. TresAbstract:Ran, a Ras-related GTPase, is required for transporting proteins in and out of the nucleus during interphase and for regulating the assembly of microtubules. cDNA cloning shows that rat testis, like mouse testis, expresses both somatic and testis-specific forms of Ran–GTPase. The presence of a homologous testis-specific form of Ran–GTPase in rodents implies that the Ran–GTPase pathway plays a significant role during sperm development. This suggestions is supported by distinct Ran–GTPase immunolocalization sites identified in developing Spermatids. Confocal microscopy demonstrates that Ran–GTPase localizes in the nucleus of round Spermatids and along the microtubules of the manchette in elongating Spermatids. When the manchette disassembles, Ran–GTPase immunoreactivity is visualized in the centrosome region of maturing Spermatids. The circumstantial observation that fractionated manchettes, containing copurified centrin-immunoreactive centrosomes, can organize a three-dimensional lattice in the presence of taxol and GTP, points to the role of Ran–GTPase and associated factors in microtubule nucleation as well as the potential nucleating function of Spermatid centrosomes undergoing a reduction process. Electron microscopy demonstrates the presence in manchette preparations of Spermatid centrosomes, recognized as such by their association with remnants of the implantation fossa, a dense plate observed only at the basal surface of developing Spermatid and sperm nuclei. In addition, we have found importin β1 immunoreactivity in the nucleus of elongating Spermatids, a finding that, together with the presence of Ran–GTPase in the nucleus of round Spermatids and the manchette, suggest a potential role of Ran–GTPase machinery in nucleocytoplasmic transport. Our expression and localization analysis, correlated with functional observations in other cell systems, suggest that Ran–GTPase may be involved in both nucleocytoplasmic transport and microtubules assembly, two critical events during the development of functional sperm. In addition, the manchette-to-centrosome Ran–GTPase relocation, together with the similar redistribution of various proteins associated to the manchette, suggest the existence of an intramanchette molecular transport mechanism, which may share molecular analogies with intraflagellar transport. Mol. Reprod. Dev. 63: 131–140, 2002. © 2002 Wiley-Liss, Inc.
Atsuo Ogura - One of the best experts on this subject based on the ideXlab platform.
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birth of a marmoset following injection of elongated Spermatid from a prepubertal male
Molecular Reproduction and Development, 2019Co-Authors: Narumi Ogonuki, Yoko Kurotaki, Yukiko Abe, Erika Sasaki, Atsu Aiba, Kazuki Nakao, Atsuo OguraAbstract:The common marmoset is a small nonhuman primate in which the application of transgenesis and genetic knockout techniques allows the generation of gene-modified models of human diseases. However, its longer generation time than that of rodents is a major obstacle to the widespread use of gene-modified marmosets for biomedical research. In this study, we examined the feasibility of shortening the generation time by using prepubertal marmoset males as gamete donors. We collected late round stage Spermatids (Steps 5-7), elongated Spermatids, and testicular spermatozoa from the testis of a prepubertal 11-month-old male marmoset and injected them into in vitro-matured oocytes. After 7 days in culture, two embryos from elongated Spermatid injection and two embryos from sperm injection were transferred into two separate recipient females. The recipient female that received elongated Spermatid injection-derived embryos became pregnant and gave birth to one female infant. This is the first demonstration that a Spermatid from a prepubertal male primate can support full-term development. Using this method, we can expect to obtain offspring of gene-modified males 6 months to a year earlier than with natural mating.
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oocyte activating capacity of fresh and frozen thawed Spermatids in the common marmoset callithrix jacchus
Molecular Reproduction and Development, 2018Co-Authors: Narumi Ogonuki, Hiroki Inoue, Shogo Matoba, Yoko Kurotaki, Hidetoshi Kassai, Yukiko Abe, Erika Sasaki, Atsu Aiba, Atsuo OguraAbstract:The common marmoset (Callithrix jacchus) represents a promising nonhuman primate model for the study of human diseases because of its small size, ease of handling, and availability of gene-modified animals. Here, we aimed to devise reproductive technology for marmoset Spermatid injection using immature males for a possible rapid generational turnover. Spermatids at each step could be identified easily by their morphology under differential interference microscopy: thus, early round Spermatids had a round nucleus with a few nucleolus-like structures and abundant cytoplasm, as in other mammals. The Spermatids acquired oocyte-activating capacity at the late round Spermatid stage, as confirmed by the resumption of meiosis and Ca2+ oscillations upon injection into mouse oocytes. The Spermatids could be cryopreserved efficiently with a simple medium containing glycerol and CELL BANKER®. Late round or elongated Spermatids first appeared at 10-12 months of age, 6-8 months before sexual maturation. Marmoset oocytes microinjected with frozen-thawed late round or elongated Spermatids retrieved from a 12-month-old male marmoset developed to the 8-cell stage without the need for artificial oocyte activation stimulation. Thus, it might be possible to shorten the intergeneration time by Spermatid injection, from 2 years (by natural mating) to 13-15 months including gestation.
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birth of normal mice following round Spermatid injection without artificial oocyte activation
Journal of Reproduction and Development, 2011Co-Authors: Narumi Ogonuki, Atsuo Ogura, Kimiko InoueAbstract:For fertilization using round Spermatid injection (ROSI) in mice, oocytes need to be artificially preactivated because of the lack of oocyte-activating capacity in round Spermatids of this species. However, when round Spermatids were frozen-thawed before microinjection, 11-71% of injected oocytes developed into 2-cell embryos without any artificial activation. After being transferred into recipient females, 5-27% of these embryos reached term. At least some of the injected oocytes showed intracellular Ca2+ oscillations, which normally occur after fertilization by mature spermatozoa. Thus, these round Spermatids could transmit a sperm-borne oocyte-activating factor, which might have been released from spermatozoa and elongated Spermatids in the same suspension by freezing and thawing. This possibility was further supported by activation of intact oocytes following transplantation of the pronuclei from ROSI-generated embryos. Thus, one-step ROSI can be achieved in mice simply by injecting frozen-thawed round Spermatids into intact oocytes. Clearly, there is a need for careful interpretation of microinjection experiments when assessing the oocyte-activating capacity of spermatogenic cells, especially when they are derived from frozen-thawed stocks.
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behaviour of hamster and mouse round Spermatid nuclei incorporated into mature oocytes by electrofusion
Zygote, 1993Co-Authors: Atsuo Ogura, Ryuzo Yanagimachi, Noriko UsuiAbstract:Round Spermatids of the hamster and mouse were electrofused with homologous mature oocytes to examine the behaviour of their nuclei within the ooplasm. A single Spermatid was inserted in the perivitelline space of a mature oocyte and an electric fusion pulse given. In the hamster, the best Spermatid-oocyte fusion took place when the oocytes were pretreated with neuraminidase, subjected to 30 s AC (2 MHz, 20 V/cm) followed by a single fusion DC pulse (3000 V-cm, 10 microseconds) and another 30 s AC current. Inclusion of micromolar Ca2+ and Mg2+ in the fusion medium was essential for oocyte activation. Under these conditions all oocytes were activated and 20-40% fused with Spermatids. Of these fused oocytes only 5-10% had fully developed Spermatid (male) and oocyte (female) pronuclei. In the rest the Spermatid-derived pronuclei remained small throughout the pronuclear stage. However, nucleolus-like structures appeared de novo and DNA synthesis occurred in these small pronuclei. Regardless of the size of male pronuclei, chromosomes from the Spermatid and oocyte appeared to mingle and participate in the first cleavage. About 70% of the fused oocytes developed into the 2-cell stage. Electrofusion of mouse oocytes with Spermatids was less efficient. Even under the best conditions tested, less than 10% of the oocytes fused with Spermatids. Here again, most Spermatid nuclei remained small throughout the pronuclear stage.
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round Spermatid nuclei injected into hamster oocytes from pronuclei and participate in syngamy
Biology of Reproduction, 1993Co-Authors: Atsuo Ogura, Ryuzo YanagimachiAbstract:Round Spermatids are spermatogenic cells that have just completed meiosis. To discover whether nuclei of these haploid cells are genetically ready for fertilization, nuclei were individually injected into mature hamster oocytes via a microsurgical technique. In the majority of oocytes that were successfully injected and activated, Spermatid nuclei transformed into pronuclei and underwent DNA synthesis. Ultimately their chromosomes mingled with ootid chromosomes immediately prior to the first cleavage. Although we could not determine the developmental potential of these zygotes, Spermatid nuclei appear capable of participating in syngamy.
Dolores D Mruk - One of the best experts on this subject based on the ideXlab platform.
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actin binding proteins Spermatid transport and spermiation
Seminars in Cell & Developmental Biology, 2014Co-Authors: Xiaojing Qian, Will M. Lee, Dolores D Mruk, Yan Ho Cheng, Elizabeth I Tang, Daishu Han, Elissa W P Wong, Yan C. ChengAbstract:The transport of germ cells across the seminiferous epithelium is composed of a series of cellular events during the epithelial cycle essential to the completion of spermatogenesis. Without the timely transport of Spermatids during spermiogenesis, spermatozoa that are transformed from step 19 Spermatids in the rat testis fail to reach the luminal edge of the apical compartment and enter the tubule lumen at spermiation, thereby arriving the epididymis for further maturation. Step 19 Spermatids and/or sperms that remain in the epithelium beyond stage VIII of the epithelial cycle will be removed by the Sertoli cell via phagocytosis to form phagosomes and be degraded by lysosomes, leading to subfertility and/or infertility. However, the biology of Spermatid transport, in particular the final events that lead to spermiation remain elusive. Based on recent data in the field, we critically evaluate the biology of spermiation herein by focusing on the actin binding proteins (ABPs) that regulate the organization of actin microfilaments at the Sertoli-Spermatid interface, which is crucial for Spermatid transport during this event. The hypothesis we put forth herein also highlights some specific areas of research that can be pursued by investigators in the years to come.
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regulation of spermiogenesis spermiation and blood testis barrier dynamics novel insights from studies on eps8 and arp3
Biochemical Journal, 2011Co-Authors: Yan C. Cheng, Dolores D MrukAbstract:Spermiogenesis in the mammalian testis is the most critical post-meiotic developmental event occurring during spermatogenesis in which haploid Spermatids undergo extensive cellular, molecular and morphological changes to form spermatozoa. Spermatozoa are then released from the seminiferous epithelium at spermiation. At the same time, the BTB (blood-testis barrier) undergoes restructuring to facilitate the transit of preleptotene spermatocytes from the basal to the apical compartment. Thus meiotic divisions take place behind the BTB in the apical compartment to form Spermatids. These germ cells enter spermiogenesis to transform into elongating Spermatids and then into spermatozoa to replace those that were released in the previous cycle. However, the mole-cular regulators that control spermiogenesis, in particular the dynamic changes that occur at the Sertoli cell-Spermatid interface and at the BTB, are not entirely known. This is largely due to the lack of suitable animal models which can be used to study these events. During the course of our investigation to develop adjudin [1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide] as a potential male contraceptive, this drug was shown to 'accelerate' spermiation by inducing the release of premature Spermatids from the epithelium. Using this model, we have identified several molecules that are crucial in regulating the actin filament network and the unique adhesion protein complex at the Sertoli cell-Spermatid interface known as the apical ES (ectoplasmic specialization). In the present review, we critically evaluate these and other findings in the literature as they relate to the restricted temporal and spatial expression of two actin regulatory proteins, namely Eps8 (epidermal growth factor receptor pathway substrate 8) and Arp3 (actin-related protein 3), which regulate these events.
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restricted arp3 expression in the testis prevents blood testis barrier disruption during junction restructuring at spermatogenesis
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Pearl P Y Lie, Will M. Lee, Dolores D Mruk, Apple Y N Chan, Yan C. ChengAbstract:In epithelia, a primary damage of tight junctions (TJ) always leads to a secondary disruption of adherens junction (AJ), and vice versa. This response, if occurring in the testis, would disrupt spermatogenesis because the blood-testis barrier (BTB) must remain intact during the transit of Spermatids in the seminiferous epithelium, which is associated with extensive apical ectoplasmic specialization (apical ES, a testis-specific AJ type) restructuring. As such, apical ES restructuring accompanied with the transit of developing Spermatids during spermiogenesis must be segregated from the BTB to avoid an immunological barrier breakdown in all stages of the seminiferous epithelial cycle, except at stage VIII when spermiation and BTB restructuring take place concurrently. We report herein a mechanism involving restricted spatial and temporal expression of Arp2/3 complex and N-WASP, whose actin branching activity associated with apical ES and BTB restructuring in the seminiferous epithelium. High expression of Arp3 at the apical ES was shown to correlate with Spermatid movement and proper Spermatid orientation. Likewise, high Arp3 level at the BTB associated with its restructuring to accommodate the transit of preleptotene spermatocytes at stage VIII of the epithelial cycle. These findings were validated by in vitro and in vivo studies using wiskostatin, an inhibitor that blocks N-WASP from activating Arp2/3 complex to elicit actin branching. Inhibition of actin branching caused a failure of Spermatid transit plus a loss of proper orientation in the epithelium, and a "tightened" Sertoli cell TJ permeability barrier, supporting the role of Arp2/3 complex in segregating the events of AJ and BTB restructuring.
