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1.
Development ; 147(2)2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31969357

RESUMO

The development and maintenance of the correct morphology of sperm is important for their functions. Cellular morphogenesis of sperm occurs during the post-meiotic developmental stage; however, little is known about what coordinates this process. In the present study, we investigated the role of A-kinase anchoring protein 3 (AKAP3) during mouse spermiogenesis, using both mouse genetics and proteomics. It was found that AKAP3 is essential for the formation of the specific subcellular structure of the sperm flagellum, motility of sperm and male fertility. Additionally, lack of AKAP3 caused global changes of the sperm proteome and mislocalization of sperm proteins, including accumulation of RNA metabolism and translation factors and displacement of PKA subunits in mature sperm, which may underlie misregulated PKA activity and immotility in sperm. Interestingly, sperm lacking a complete fibrous sheath from both Akap3 and Akap4 null mice accumulated F-actin filaments and morphological defects during post-testicular maturation in the epididymis. These results suggest that the subcellular structures of sperm could be formed via independent pathways, and elucidate the roles of AKAP3 during the coordinated synthesis and organization of the sperm proteome and sperm morphology.

2.
Stem Cells Dev ; 28(20): 1365-1375, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31580778

RESUMO

Glaucoma is characterized by retinal ganglion cell (RGC) degeneration and is the second leading cause of blindness worldwide. However, current treatments such as eye drop or surgery have limitations and do not target the loss of RGC. Regenerative therapy using embryonic stem cells (ESCs) holds a promising option, but ethical concern hinders clinical applications on human subjects. In this study, we employed spermatogonial stem cells (SSCs) as an alternative source of ESCs for cell-based regenerative therapy in mouse glaucoma model. We generated functional RGCs from SSCs with a two-step protocol without applying viral transfection or chemical induction. SSCs were first dedifferentiated to embryonic stem-like cells (SSC-ESCs) that resemble ESCs in morphology, gene expression signatures, and stem cell properties. The SSC-ESCs then differentiated toward retinal lineages. We showed SSC-ESC-derived retinal cells expressed RGC-specific marker Brn3b and functioned as bona fide RGCs. To allow in vivo RGC tracing, Brn3b-EGFP reporter SSC-ESCs were generated and the derived RGCs were subsequently transplanted into the retina of glaucoma mouse models by intravitreal injection. We demonstrated that the transplanted RGCs could survive in host retina for at least 10 days after transplantation. SSC-ESC-derived RGCs can thus potentially be a novel alternative to replace the damaged RGCs in glaucomatous retina.

3.
Exp Cell Res ; 373(1-2): 71-79, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30266657

RESUMO

During spermatogenesis, a group of undifferentiated spermatogonia undergoes an essential transition to a differentiating stage, which involves gain of Kit receptor. In the current study, we showed that a small non-coding RNA, miRNA-26b could induce transition from Kit- to Kit+ and inhibit proliferation of spermatogonia. A key transcriptional factor for undifferentiated spermatogonia, Plzf, was proven as a direct target of miR-26b. When undifferentiated spermatogonia were treated with Retinoic acid (RA), miR-26b was increased, further promoting RA-induced differentiation of spermatogonia. In addition, miR-26b could repress 5-hydroxymethylcytosine (5hmC) via repression of Tet3 in spermatogonia. These findings demonstrate that miR-26b might play a role in promoting the transition from Kit- to Kit+ SSCs.


Assuntos
MicroRNAs/fisiologia , Espermatogênese , Espermatogônias/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Apoptose , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Masculino , Camundongos , MicroRNAs/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-kit/análise , Espermatogônias/citologia , Espermatogônias/efeitos dos fármacos , Tretinoína/farmacologia
4.
Biol Reprod ; 99(4): 773-788, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29701755

RESUMO

Translational regulation plays a central role during post-meiotic development of male germ cells. Previous studies suggested that P-element induced wimpy testis like 1 (PIWIL1), a PIWI-interacting RNA (piRNA) binding protein that is critical for sperm development, participates in the maintenance and translational regulation of post-meiotic mRNAs in haploid spermatids. However, how PIWIL1 regulates protein translation remains largely unclear. Using biochemical assays, we show here that PIWIL1 utilizes different domains to interact with post-meiotic mRNAs and Poly-A binding protein cytoplasmic 1 (PABPC1), a general protein translation regulator. PIWIL1 binds 3'-untranslated regions (3'-UTRs) of several spermiogenic mRNAs via its N-terminal domain, whereas its interactions with PABPC1 are mediated through its N- and C-terminal domains in an RNA-dependent manner. Using a heterologous cell system, we analyzed its effects on protein translation via luciferase reporter assay and sucrose gradient sedimentation. It was found that PIWIL1 augments protein translation with PABPC1 in the presence of 3'-UTRs of post-meiotic mRNAs. While both the N-terminal RNA recognition motif (RRM) domain and the central linker region of PABPC1 stimulate translation, only the PIWI Argonaute and Zwille (PAZ) domain of PIWIL1 positively affects translation of reporter mRNAs. Interestingly, the PAZ domain was found absent from polysomal fractions, in contrast to the N- and C-terminal domains of PIWIL1. Taken together, the results suggest that PIWIL1 interacts with various partners using different domains and participates in translational regulation partly through 3'-UTRs. It will be of interest to further explore how PIWIL1 elicit its versatile functions, including translational regulation of post-meiotic mRNAs through intrinsic structural changes and extrinsic signals during mouse spermiogenesis under more physiological settings.


Assuntos
Proteínas Argonauta/química , Proteínas Argonauta/metabolismo , Proteína I de Ligação a Poli(A)/química , Proteína I de Ligação a Poli(A)/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espermatogênese/genética , Regiões 3' não Traduzidas , Animais , Proteínas Argonauta/genética , Células HEK293 , Humanos , Masculino , Meiose/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteína I de Ligação a Poli(A)/genética , Biossíntese de Proteínas , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Testículo/metabolismo
5.
Cell Regen (Lond) ; 5: 1, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26839690

RESUMO

Adult stem cells that reside in particular types of tissues are responsible for tissue homeostasis and regeneration. Cellular functions of adult stem cells are intricately related to the gene expression programs in those cells. Past research has demonstrated that regulation of gene expression at the transcriptional level can decisively alter cell fate of stem cells. However, cellular contents of mRNAs are sometimes not equivalent to proteins, the functional units of cells. It is increasingly realized that post-transcriptional and translational regulation of gene expression are also fundamental for stem cell functions. Compared to differentiated somatic cells, effects on cellular status manifested by varied expression of RNA-binding proteins and global protein synthesis have been demonstrated in several stem cell systems. Through the cooperation of both cis-elements of mRNAs and trans-acting RNA-binding proteins that are intimately associated with them, regulation of localization, stability, and translational status of mRNAs directly influences the self-renewal and differentiation of stem cells. Previous studies have uncovered some of the molecular mechanisms that underlie the functions of RNA-binding proteins in stem cells in invertebrate species. However, their roles in adult stem cells in mammals are just beginning to be unveiled. This review highlights some of the RNA-binding proteins that play important functions during the maintenance and differentiation of mouse male germline stem cells, the adult stem cells in the male reproductive organ.

6.
Reproduction ; 151(4): 351-67, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26762401

RESUMO

Cell lineage determination during early embryogenesis has profound effects on adult animal development. Pre-patterning of embryos, such as that of Drosophila and Caenorhabditis elegans, is driven by asymmetrically localized maternal or zygotic factors, including mRNA species and RNA binding proteins. However, it is not clear how mammalian early embryogenesis is regulated and what the early cell fate determinants are. Here we show that, in mouse, mitochondrial ribosomal RNAs (mtrRNAs) are differentially distributed between 2-cell sister blastomeres. This distribution pattern is not related to the overall quantity or activity of mitochondria which appears equal between 2-cell sister blastomeres. Like in lower species, 16S mtrRNA is found to localize in the cytoplasm outside of mitochondria in mouse 2-cell embryos. Alterations of 16S mtrRNA levels in one of the 2-cell sister blastomere via microinjection of either sense or anti-sense RNAs drive its progeny into different cell lineages in blastocyst. These results indicate that mtrRNAs are differentially distributed among embryonic cells at the beginning of embryogenesis in mouse and they are functionally involved in the regulation of cell lineage allocations in blastocyst, suggesting an underlying molecular mechanism that regulates pre-implantation embryogenesis in mouse.


Assuntos
Blastocisto/citologia , Blastômeros/citologia , Linhagem da Célula/genética , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/genética , RNA/genética , Animais , Blastocisto/metabolismo , Blastômeros/metabolismo , Diferenciação Celular , Células Cultivadas , Embrião de Mamíferos/metabolismo , Feminino , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mitocondrial , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
Mol Reprod Dev ; 81(7): 595-607, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24687590

RESUMO

cAMP-dependent protein kinase A (PKA) plays important regulatory roles during mouse spermatogenesis. PKA-mediated signaling has been shown to regulate gene expression, chromatin condensation, capacitation, and motility during sperm development and behavior, although how PKA is regulated in spatiotemporal manners during spermatogenesis is not fully understood. In the present study, we found that PKA subunit isoforms are expressed and localized differently in meiotic and post-meiotic mouse spermatogenic cells. Regulatory subunit I alpha (RIα) is expressed in spermatocytes and round spermatids, where it is localized diffusely throughout the cytoplasm of cells. During late spermiogenesis, RIα abundance gradually decreases. On the other hand, RIIα is expressed constantly throughout meiotic and post-meiotic stages, and is associated with cytoskeletal structures. Among several A kinase anchoring proteins (AKAPs) expressed in the testis, sperm-specific AKAP3 can be found in the cytoplasm of elongating spermatids and interacts with RIα, as demonstrated by both in vivo and in vitro experiments. In mature sperm, AKAP3 is exclusively found in the principal piece of the flagellum, coincident with only RIIα. Mutagenesis experiments further showed that the preferential interactions of AKAP3 with PKA regulatory subunits are mediated by two highly conserved amphipathic peptides located in the N-terminal region of AKAP3. Thus, AKAP3 is a dual-specificity molecule that modulates PKA isotypes in a spatiotemporal manner during mouse spermatogenesis.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Espermátides/química , Espermátides/metabolismo , Proteínas de Ancoragem à Quinase A/análise , Proteínas de Ancoragem à Quinase A/química , Sequência de Aminoácidos , Animais , AMP Cíclico/química , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/química , Células HEK293 , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Alinhamento de Sequência
8.
Biol Reprod ; 90(6): 119, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24648398

RESUMO

Mammalian spermatogenesis is regulated by coordinated gene expression in a spatiotemporal manner. The spatiotemporal regulation of major sperm proteins plays important roles during normal development of the male gamete, of which the underlying molecular mechanisms are poorly understood. A-kinase anchoring protein 3 (AKAP3) is one of the major components of the fibrous sheath of the sperm tail that is formed during spermiogenesis. In the present study, we analyzed the expression of sperm-specific Akap3 and the potential regulatory factors of its protein synthesis during mouse spermiogenesis. Results showed that the transcription of Akap3 precedes its protein synthesis by about 2 wk. Nascent AKAP3 was found to form protein complex with PKA and RNA binding proteins (RBPs), including PIWIL1, PABPC1, and NONO, as revealed by coimmunoprecipitation and protein mass spectrometry. RNA electrophoretic gel mobility shift assay showed that these RBPs bind sperm-specific mRNAs, of which proteins are synthesized during the elongating stage of spermiogenesis. Biochemical and cell biological experiments demonstrated that PIWIL1, PABPC1, and NONO interact with each other and colocalize in spermatids' RNA granule, the chromatoid body. In addition, NONO was found in extracytoplasmic granules in round spermatids, whereas PIWIL1 and PABPC1 were diffusely localized in cytoplasm of elongating spermatids, indicating their participation at different steps of mRNA metabolism during spermatogenesis. Interestingly, type I PKA subunits colocalize with PIWIL1 and PABPC1 in the cytoplasm of elongating spermatids and cosediment with the RBPs in polysomal fractions on sucrose gradients. Further biochemical analyses revealed that activation of PKA positively regulates AKAP3 protein synthesis without changing its mRNA level in elongating spermatids. Taken together, these results indicate that PKA signaling directly participates in the regulation of protein translation in postmeiotic male germ cells, underscoring molecular mechanisms that regulate protein synthesis during mouse spermiogenesis.


Assuntos
Proteínas de Ancoragem à Quinase A/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/fisiologia , Espermátides/fisiologia , Espermatogênese/fisiologia , Proteínas de Ancoragem à Quinase A/metabolismo , Animais , Proteínas Argonauta/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Masculino , Camundongos Endogâmicos , Proteínas de Ligação a Poli(A)/metabolismo , Biossíntese de Proteínas/fisiologia , Processamento Pós-Transcricional do RNA/fisiologia , RNA Mensageiro/metabolismo , Cauda do Espermatozoide/fisiologia , Espermátides/citologia
9.
Gene Expr Patterns ; 14(1): 30-41, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24140495

RESUMO

Nucleosomes are basic chromatin structural units that are formed by DNA sequences wrapping around histones. Global chromatin states in different cell types are specified by combinatorial effects of post-translational modifications of histones and the expression of histone variants. During mouse spermatogenesis, spermatogonial stem cells (SSCs) self-renew while undergo differentiation, events that occur in the company of constant re-modeling of chromatin structures. Previous studies have shown that testes contain highly expressed or specific histone variants to facilitate these epigenetic modifications. However, mechanisms of regulating the epigenetic changes and the specific histone compositions of spermatogenic cells are not fully understood. Using real time quantitative RT-PCR, we examined the dynamic expression of replication-dependent histone genes in post-natal mouse testes. It was found that distinct sets of histone genes are expressed in various spermatogenic cells at different stages during spermatogenesis. While gonocyte-enriched testes from mice at 2-dpp (days post partum) express pre-dominantly thirteen histone variant genes, SSC-stage testes at 9-dpp highly express a different set of eight histone genes. During differentiation stage when testes are occupied mostly by spermatocytes and spermatids, another twenty-two histone genes are expressed much higher than the rest, including previously known testis-specific hist1h1t, hist1h2ba and hist1h4c. In addition, histone genes that are pre-dominantly expressed in gonocytes and SSCs are also highly expressed in embryonic stem cells. Several of them were changed when embryoid bodies were formed from ES cells, suggesting their roles in regulating pluripotency of the cells. Further more, differentially expressed histone genes are specifically localized in either SSCs or spermatocytes and spermatids, as demonstrated by in situ hybridization using gene specific probes. Taken together, results presented here revealed that different combinations of histone variant genes are expressed in distinct spermatogenic cell types accompanying the progression of self-renewal and differentiation of SSCs, suggesting a systematic regulatory role histone variants play during spermatogenesis.


Assuntos
Cromatina/fisiologia , Células-Tronco Embrionárias/metabolismo , Histonas/genética , Histonas/metabolismo , Espermatogênese/genética , Espermatogônias/citologia , Animais , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Células NIH 3T3 , Espermatogênese/fisiologia , Espermatogônias/fisiologia , Testículo/metabolismo
10.
Genesis ; 51(2): 83-96, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23175476

RESUMO

Mammalian spermatogenesis originates from spermatogonial stem cells (SSCs), which undergo mitosis, meiosis and spermiogenesis in order to generate mature spermatozoa. SSCs are adult stem cells that can both self-renew and differentiate. To maintain pluripotency, SSCs are regulated by both extrinsic factors secreted from surrounding somatic cells and intrinsic factors including specific gene expression programs. Using fluorescent labeled germ line stem cells, mouse gonocytes and SSCs were purified up to 97% by improved FACS method. Through microarray analyses, global gene expression profiles of gonocytes, SSCs, and differentiated cells were compared. A large number of distinctive genes were found to be enriched in respective cell populations, indicating different functional requirements of each cell type. Functional clustering analyses revealed that while gonocytes and SSCs preferentially express genes implicated in gene expression regulation and epigenetic modifications, differentiated cells including somatic cells are enriched with genes encoding proteins involved in various cellular activities. Further in situ hybridization and RT-PCR experiments confirmed SSC specific expression of several genes of which functions have not been characterized in SSCs. The comparative gene expression profiling provides a useful resource for gene discovery in relation to SSC regulation and opens new avenues for the study of molecular mechanisms underlying SSC self-renewal and differentiation.


Assuntos
Perfilação da Expressão Gênica , Espermatogônias/citologia , Espermatogônias/fisiologia , Células-Tronco/fisiologia , Animais , Animais Recém-Nascidos , Diferenciação Celular/genética , Citometria de Fluxo/métodos , Hibridização In Situ , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Espermatogênese/genética
12.
Soc Reprod Fertil Suppl ; 63: 187-201, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17566273

RESUMO

For sperm to fertilize eggs, they must bind to and penetrate the zona pellucida (ZP) that surrounds the plasma membrane of all mammalian eggs. The ZP first appears during oocyte growth and increases in thickness as oocytes increase in diameter. The ZP is an extracellular matrix composed of long, crosslinked filaments. In mice, three glycoproteins, called mZP1-3, are synthesised and secreted by growing oocytes and assembled into a thick (-6.5 microm) extracellular coat over a 2-3 week period. Recently, we identified several regions of nascent ZP glycoproteins that affect their secretion and incorporation into the ZP (assembly) by growing oocytes. Among these are the ZP domain, the consensus furin cleavage site (CFCS) and the C-terminal propeptide (CTP) with its transmembrane domain (TMD), external hydrophobic patch (EHP), charged patch (CP), conserved cysteine (Cys) residue, and short cytoplasmic tail (CT). Particularly important is the ZP domain, a approximately 260 amino acid region with 8 conserved Cys residues that is common to a variety of extracellular proteins of diverse functions found in a wide range of multicellular eukaryotes. Our results show that the ZP domain functions as a polymerisation module and that its N-terminal half, including 4 conserved Cys residues, is largely responsible for this role. Additionally, two conserved hydrophobic sequences, one within the ZP domain (internal hydrophobic patch; IHP) and another within the CTP (EHP), apparently regulate polymerisation of nascent ZP glycoproteins. Collectively, our findings suggest a general mechanism for assembly of all ZP domain proteins based on coupling between proteolytic processing and polymerisation.


Assuntos
Proteínas do Ovo/metabolismo , Mamíferos/metabolismo , Glicoproteínas de Membrana/metabolismo , Oócitos/metabolismo , Oogênese/fisiologia , Zona Pelúcida/metabolismo , Motivos de Aminoácidos , Animais , Proteínas do Ovo/química , Feminino , Humanos , Masculino , Glicoproteínas de Membrana/química , Camundongos , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Interações Espermatozoide-Óvulo/fisiologia , Zona Pelúcida/ultraestrutura , Glicoproteínas da Zona Pelúcida
13.
Proc Natl Acad Sci U S A ; 104(4): 1219-23, 2007 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-17227845

RESUMO

Mammalian spermatozoa become motile at ejaculation, but before they can fertilize the egg, they must acquire more thrust to penetrate the cumulus and zona pellucida. The forceful asymmetric motion of hyperactivated spermatozoa requires Ca2+ entry into the sperm tail by an alkalinization-activated voltage-sensitive Ca2+-selective current (ICatSper). Hyperactivation requires CatSper1 and CatSper2 putative ion channel genes, but the function of two other related genes (CatSper3 and CatSper4) is not known. Here we show that targeted disruption of murine CatSper3 or CatSper4 also abrogated ICatSper, sperm cell hyperactivated motility and male fertility but did not affect spermatogenesis or initial motility. Direct protein interactions among CatSpers, the sperm specificity of these proteins, and loss of ICatSper in each of the four CatSper-/- mice indicate that CatSpers are highly specialized flagellar proteins.


Assuntos
Canais de Cálcio/fisiologia , Fertilidade/fisiologia , Isoformas de Proteínas/fisiologia , Motilidade Espermática/fisiologia , Animais , Masculino , Camundongos , Dados de Sequência Molecular
14.
Mol Cell Endocrinol ; 234(1-2): 95-103, 2005 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-15836958

RESUMO

Mammalian fertilization has been the subject of intensified research in recent times. Application of recombinant DNA, transgenic and gene targeting technology, in particular, to issues in mammalian fertilization has revolutionized the field. Here, we present some of the latest results coming from application of these and other technologies to four aspects of mammalian fertilization: 1. formation of the egg zona pellucida (ZP) during oocyte growth; 2. species-specific binding of sperm to the egg zona pellucida; 3. induction of the sperm acrosome reaction (AR) by the egg zona pellucida 4. binding of sperm to and fusion with egg plasma membrane. In virtually every instance, new information and new insights have come from relatively recent investigations.


Assuntos
Reação Acrossômica/fisiologia , Fertilização/fisiologia , Espermatozoides/fisiologia , Zona Pelúcida/fisiologia , Animais , Membrana Celular/fisiologia , Feminino , Humanos , Masculino , Mamíferos , Oócitos/crescimento & desenvolvimento , Oócitos/fisiologia
15.
Eur J Obstet Gynecol Reprod Biol ; 115 Suppl 1: S57-60, 2004 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15196717

RESUMO

Mammalian eggs are surrounded by a zona pellucida (ZP) that regulates egg-sperm teractions during fertilization. The ZP consists of long filaments composed of two glycoproteins, ZP2 and ZP3, that are crosslinked by a third glycoprotein, ZP1. The presence of both ZP2 and ZP3 is essential for assembling a ZP around growing oocytes, as well as for fertility of females. Acrosome-intact sperm recognize and bind to O-linked oligosaccharides linked to Ser residues at the sperm combining-site of ZP3. Structural differences in oligosaccharides on ZP3 from different species may account for whether or not sperm are able to bind to the ZP. Bound sperm undergo the acrosome reaction, penetrate the ZP, and can then fuse with egg plasma membrane. Following fertilization, sperm are unable to bind to either ZP3 or the ZP of one-cell embryos.


Assuntos
Interações Espermatozoide-Óvulo/fisiologia , Animais , Fertilização/fisiologia , Humanos , Camundongos
16.
Proc Natl Acad Sci U S A ; 101(16): 5922-7, 2004 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-15079052

RESUMO

Many secreted eukaryotic glycoproteins that play fundamental roles in development, hearing, immunity, and cancer polymerize into filaments and extracellular matrices through zona pellucida (ZP) domains. ZP domain proteins are synthesized as precursors containing C-terminal propeptides that are cleaved at conserved sites. However, the consequences of this processing and the mechanism by which nascent proteins assemble are unclear. By microinjection of mutated DNA constructs into growing oocytes and mammalian cell transfection, we have identified a conserved duplicated motif [EHP (external hydrophobic patch)/IHP (internal hydrophobic patch)] regulating the assembly of mouse ZP proteins. Whereas the transmembrane domain (TMD) of ZP3 can be functionally replaced by an unrelated TMD, mutations in either EHP or IHP do not hinder secretion of full-length ZP3 but completely abolish its assembly. Because mutants truncated before the TMD are not processed, we conclude that the conserved TMD of mammalian ZP proteins does not engage them in specific interactions but is essential for C-terminal processing. Cleavage of ZP precursors results in loss of the EHP, thereby activating secreted polypeptides to assemble by using the IHP within the ZP domain. Taken together, these findings suggest a general mechanism for assembly of ZP domain proteins.


Assuntos
Proteínas do Ovo/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas do Ovo/química , Proteínas do Ovo/genética , Humanos , Hidrólise , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Homologia de Sequência de Aminoácidos , Glicoproteínas da Zona Pelúcida
17.
Nat Cell Biol ; 4(6): 457-61, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12021773

RESUMO

Many eukaryotic extracellular proteins share a sequence of unknown function, called the zona pellucida (ZP) domain. Among these proteins are the mammalian sperm receptors ZP2 and ZP3, non-mammalian egg coat proteins, Tamm-Horsfall protein (THP), glycoprotein-2 (GP-2), alpha- and beta-tectorins, transforming growth factor (TGF)-beta receptor III and endoglin, DMBT-1 (deleted in malignant brain tumour-1), NompA (no-mechanoreceptor-potential-A), Dumpy and cuticlin-1 (refs 1,2). Here, we report that the ZP domain of ZP2, ZP3 and THP is responsible for polymerization of these proteins into filaments of similar supramolecular structure. Most ZP domain proteins are synthesized as precursors with carboxy-terminal transmembrane domains or glycosyl phosphatidylinositol (GPI) anchors. Our results demonstrate that the C-terminal transmembrane domain and short cytoplasmic tail of ZP2 and ZP3 are not required for secretion, but are essential for assembly. Finally, we suggest a molecular basis for dominant human hearing disorders caused by point mutations within the ZP domain of alpha-tectorin.


Assuntos
Proteínas do Ovo/química , Proteínas do Ovo/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Mucoproteínas/química , Mucoproteínas/metabolismo , Receptores de Superfície Celular , Sequência de Aminoácidos , Animais , Células CHO , Sequência Conservada , Cricetinae , Proteínas do Ovo/genética , Evolução Molecular , Espaço Extracelular/metabolismo , Deleção de Genes , Humanos , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Mucoproteínas/genética , Mutagênese Sítio-Dirigida/fisiologia , Oócitos/fisiologia , Polímeros/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Uromodulina , Glicoproteínas da Zona Pelúcida
18.
Mol Biol Cell ; 13(2): 530-41, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11854410

RESUMO

The zona pellucida (ZP) is a highly organized extracellular coat that surrounds all mammalian eggs. The mouse egg ZP is composed of three glycoproteins, called mZP1-3, that are synthesized, secreted, and assembled into a ZP exclusively by growing oocytes. Here, we microinjected epitope-tagged (Myc and Flag) cDNAs for mZP2 and mZP3 into the germinal vesicle (nucleus) of growing oocytes isolated from juvenile mice. Specific antibodies and laser scanning confocal microscopy were used to follow nascent, recombinant ZP glycoproteins in both permeabilized and nonpermeabilized oocytes. When such cDNAs were injected, epitope-tagged mZP2 (Myc-mZP2) and mZP3 (Flag-mZP3) were synthesized, packaged into large intracellular vesicles, and secreted by the vast majority of oocytes. Secreted glycoproteins were incorporated into only the innermost layer of the thickening ZP, and the amount of nascent glycoprotein in this region increased with increasing time of oocyte culture. Consistent with prior observations, the putative transmembrane domain at the C terminus of mZP2 and mZP3 was missing from nascent glycoprotein incorporated into the ZP. When the consensus furin cleavage site near the C terminus of mZP3 was mutated, such that it should not be cleaved by furin, secretion and assembly of mZP3 was reduced. On the other hand, mZP3 incorporated into the ZP lacked the transmembrane domain downstream of the mutated furin cleavage site, suggesting that some other protease(s) excised the domain. These results strongly suggest that nascent mZP2 and mZP3 are incorporated into only the innermost layer of the ZP and that excision of the C-terminal region of the glycoproteins is required for assembly into the oocyte ZP.


Assuntos
Proteínas do Ovo/biossíntese , Proteínas do Ovo/metabolismo , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/metabolismo , Oócitos/fisiologia , Receptores de Superfície Celular , Zona Pelúcida/fisiologia , Animais , DNA Complementar , Proteínas do Ovo/genética , Epitopos/genética , Feminino , Genes myc , Glicoproteínas de Membrana/genética , Camundongos , Microinjeções , Oligopeptídeos , Oócitos/ultraestrutura , Peptídeos/genética , Glicoproteínas da Zona Pelúcida
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