RESUMEN
SAYSVFN domain-containing protein 1 (SAYSD1) is an evolutionarily conserved membrane protein that has recently been identified as a ubiquitin-fold modifier 1 (UFM1)-conjugated ribosome-recognition protein that plays a critical role in translocation-associated quality control (TAQC). However, its expression and roles in mammals in vivo remain largely unknown. We found that SAYSD1 is predominantly expressed in round and elongating spermatids and localizes in the endoplasmic reticulum (ER) of mouse testes, but not in differentiated spermatozoa. Mice deficient in Saysd1 developed normally post-partum. Furthermore, Saysd1-deficient mice were fertile, with no apparent differences in sperm morphology or motility compared with wild-type sperm, although the cauda epididymis contained slightly less sperm. Expression of the ER stress markers spliced X-box binding protein 1s (XBP1s) and CCAAT/enhancer binding protein (C/EBP)-homologous protein (CHOP) in the testes was comparable between Saysd1-deficient and wild-type mice. These results suggested that SAYSD1 is involved in sperm production in mice but is dispensable for their development and fertility.
Asunto(s)
Proteínas de la Membrana , Espermatogénesis , Animales , Masculino , Ratones , Fertilidad , Mamíferos , Ratones Noqueados , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas , Semen , Espermátides/metabolismo , Espermatozoides/metabolismo , Testículo/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismoRESUMEN
Background: The spermatozoon has a complex molecular apparatus necessary for fertilization in its head and flagellum. Recently, numerous genes that are needed to construct the molecular apparatus of spermatozoa have been identified through the analysis of genetically modified mice. Methods: Based on the literature information, the molecular basis of the morphogenesis of sperm heads and flagella in mice was summarized. Main findings Results: The molecular mechanisms of vesicular trafficking and intraflagellar transport in acrosome and flagellum formation were listed. With the development of cryo-electron tomography and mass spectrometry techniques, the details of the axonemal structure are becoming clearer. The fine structure and the proteins needed to form the central apparatus, outer and inner dynein arms, nexin-dynein regulatory complex, and radial spokes were described. The important components of the formation of the mitochondrial sheath, fibrous sheath, outer dense fiber, and the annulus were also described. The similarities and differences between sperm flagella and Chlamydomonas flagella/somatic cell cilia were also discussed. Conclusion: The molecular mechanism of formation of the sperm head and flagellum has been clarified using the mouse as a model. These studies will help to better understand the diversity of sperm morphology and the causes of male infertility.
RESUMEN
Solute carrier 22a member 14 (SLC22A14) plays a critical role in male infertility in mice. We previously revealed that one of the causes of infertility is impaired capacitation. However, the molecular mechanism remained unclear. Here, we show that the influx of HCO3-, a trigger of capacitation, is impaired and intracellular pH (pHi) is decreased in the sperm of Slc22a14 knockout (KO) mice. While intracellular cAMP concentration did not increase during capacitation in Slc22a14 KO spermatozoa, HCO3--dependent soluble adenylate cyclase activity was normal, and the addition of 8-bromo cAMP rescued the decreased protein tyrosine phosphorylation. In addition, the pHi of Slc22a14 KO sperm was lower than that of WT sperm and did not increase after the addition of HCO3-. Although its relationship to the regulation of pHi is unknown, transmembrane protein 225, a possible protein phosphatase inhibitor, was found to be decreased in Slc22a14 KO sperm. The decreased in vitro fertilization rate of Slc22a14 KO sperm was partially rescued by an increase in the pHi and the addition of 8-bromo cAMP. These results suggest that SLC22A14 is involved in capacitation through the regulation of HCO3- transport and pHi.
Asunto(s)
Infertilidad Masculina , Capacitación Espermática , Animales , Concentración de Iones de Hidrógeno , Infertilidad Masculina/etiología , Infertilidad Masculina/metabolismo , Masculino , Ratones , Ratones Noqueados , Fosforilación , Capacitación Espermática/fisiología , Espermatozoides/metabolismoRESUMEN
Deleted in lung and esophageal cancer 1 (DLEC1) is a tumour suppressor gene that is downregulated in various cancers in humans; however, the physiological and molecular functions of DLEC1 are still unclear. This study investigated the critical role of Dlec1 in spermatogenesis and male fertility in mice. Dlec1 was significantly expressed in testes, with dominant expression in germ cells. We disrupted Dlec1 in mice and analysed its function in spermatogenesis and male fertility. Dlec1 deletion caused male infertility due to impaired spermatogenesis. Spermatogenesis progressed normally to step 8 spermatids in Dlec1-/- mice, but in elongating spermatids, we observed head deformation, a shortened tail, and abnormal manchette organization. These phenotypes were similar to those of various intraflagellar transport (IFT)-associated gene-deficient sperm. In addition, DLEC1 interacted with tailless complex polypeptide 1 ring complex (TRiC) and Bardet-Biedl Syndrome (BBS) protein complex subunits, as well as α- and ß-tubulin. DLEC1 expression also enhanced primary cilia formation and cilia length in A549 lung adenocarcinoma cells. These findings suggest that DLEC1 is a possible regulator of IFT and plays an essential role in sperm head and tail formation in mice.
Asunto(s)
Infertilidad Masculina/genética , Espermatozoides/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Células A549 , Animales , Sistemas CRISPR-Cas , Eliminación de Gen , Células HEK293 , Humanos , Infertilidad Masculina/metabolismo , Canales Iónicos/metabolismo , Masculino , Ratones , Especificidad de Órganos , Espermatogénesis , Tubulina (Proteína)/metabolismoRESUMEN
G-protein-coupled receptors (GPCRs) participate in diverse physiological functions and are promising targets for drug discovery. However, there are still over 140 orphan GPCRs whose functions remain to be elucidated. Gpr62 is one of the orphan GPCRs that is expressed in the rat and human brain. In this study, we found that Gpr62 is also expressed in male germ cells in mice, and its expression increases along with sperm differentiation. GPR62 lacks the BBXXB and DRY motifs, which are conserved across many GPCRs, and it was able to induce cAMP signaling in the absence of a ligand. These structural and functional features are conserved among mammals, and the mutant analysis of GPR62 has revealed that lacking of these motifs is involved in the constitutive activity. We also found that GPR62 can homooligomerize, but it is not sufficient for its constitutive activity. We further investigated its physiological function by using Gpr62 knockout (Gpr62-/-) mice. Gpr62-/- mice were born normally and did not show any abnormality in growth and behavior. In addition, both male and female Gp62-/- mice were fertile, and the differentiation and motility of spermatozoa were normal. We also found that Gpr61, the gene most similar to Gpr62 in the GPCR family shows a constitutive activity and an expression pattern similar to those of Gpr62 Our results suggest that GPR62 constitutively activates the cAMP pathway in male germ cells but is dispensable for male fertility, which is probably due to its functional redundancy with GPR61.
Asunto(s)
AMP Cíclico/metabolismo , Fertilización , Infertilidad Masculina , Receptores Acoplados a Proteínas G/fisiología , Espermatozoides/metabolismo , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos ICR , Ratones Noqueados , Transducción de Señal , Motilidad Espermática , Espermatogénesis , Espermatozoides/citologíaRESUMEN
We previously identified solute carrier 22a14 (Slc22a14) as a spermatogenesis-associated transmembrane protein in mice. Although Slc22a14 is a member of the organic anion/cation transporter family, its expression profile and physiological role have not been elucidated. Here, we show that Slc22a14 is crucial for sperm motility and male fertility in mice. Slc22a14 is expressed specifically in male germ cells, and mice lacking the Slc22a14 gene show severe male infertility. Although the overall differentiation of sperm was normal, Slc22a14-/- cauda epididymal spermatozoa showed reduced motility with abnormal flagellar bending. Further, the ability to migrate into the female reproductive tract and fertilise the oocyte were also impaired in Slc22a14-/- spermatozoa. The abnormal flagellar bending was thought to be partly caused by osmotic cell swelling since osmotic challenge or membrane permeabilisation treatment alleviated the tail abnormality. In addition, we found structural abnormalities in Slc22a14-/- sperm cells: the annulus, a ring-like structure at the mid-piece-principal piece junction, was disorganised, and expression and localisation of septin 4, an annulus component protein that is essential for the annulus formation, was also impaired. Taken together, our results demonstrated that Slc22a14 plays a pivotal role in normal flagellar structure, motility and fertility in mouse spermatozoa.
Asunto(s)
Infertilidad Masculina/patología , Transportadores de Anión Orgánico/metabolismo , Motilidad Espermática/fisiología , Espermatozoides/metabolismo , Animales , Secuencia de Bases , Sistemas CRISPR-Cas/genética , Epidídimo/metabolismo , Epidídimo/patología , Femenino , Fertilización , Flagelos/fisiología , Células Germinativas/citología , Células Germinativas/metabolismo , Infertilidad Masculina/metabolismo , Masculino , Ratones , Ratones Noqueados , Mutagénesis , Transportadores de Anión Orgánico/deficiencia , Transportadores de Anión Orgánico/genética , Testículo/metabolismo , Testículo/patologíaRESUMEN
Tmem225 encodes a putative four-transmembrane domain protein that has an RVxF motif, which is known to be a consensus site for interacting with serine/threonine protein phosphatase 1 (PP1). We previously identified Tmem225 as one of 53 spermatogenesis-associated transmembrane protein genes, with no known physiological function. In this study, we investigated the expression and molecular characteristics of TMEM225 in mice. Tmem225 production was found to be specific to testicular germ cells, with expression increasing during spermatogenesis. In mature spermatozoa, TMEM225 is localized to the equatorial segment of the acrosome but not to the midpiece or tail. TMEM225 appears to be an outer and/or inner acrosomal membrane protein that is lost from the dorsal region of the acrosome after the acrosome reaction. TMEM225 interacts with PP1 in vivo, and a pull-down assay revealed that the carboxy-terminal region of TMEM225 can bind to PP1γ2, the predominant isoform of PP1 in male germ cells. In addition, TMEM225 inhibited PP1γ2 activity in vitro via its RVxF motif. Our results suggest that in mice, TMEM225 is involved in the differentiation and function of spermatozoa through the regulation of PP1γ2 activity, which is necessary for normal spermatogenesis as well as spermatozoa capacitation and motility.
Asunto(s)
Proteínas de la Membrana/metabolismo , Ratones/genética , Proteína Fosfatasa 1/metabolismo , Espermatozoides/metabolismo , Acrosoma/metabolismo , Animales , Cartilla de ADN/genética , Regulación de la Expresión Génica/fisiología , Vectores Genéticos/genética , Células Germinativas/metabolismo , Inmunohistoquímica , Masculino , Ratones/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espermatogénesis/fisiología , Testículo/metabolismoRESUMEN
Mouse embryonic stem cells (ESCs) have the potential to differentiate into germ cells (GCs) in vivo and in vitro. Interestingly, XY ESCs can give rise to both male and female GCs in culture, irrespective of the genetic sex. Recent studies showed that ESC-derived primordial GCs contributed to functional gametogenesis in vivo; however, in vitro differentiation techniques have never succeeded in generating mature oocytes from ESCs due to cryptogenic growth arrest during the preantral follicle stages of development. To address this issue, a mouse ESC line, capable of producing follicle-like structures (FLSs) efficiently, was established to investigate their properties using conventional molecular biological methods. The results revealed that the ESC-derived FLSs were morphologically similar to ovarian primary-to-secondary follicles but never formed an antrum; instead, the FLSs eventually underwent abnormal development or cell death in culture, or formed teratomas when transplanted under the kidney capsule in mice. Gene expression analyses demonstrated that the FLSs lacked transcripts for genes essential to late folliculogenesis, including gonadotropin receptors and steroidogenic enzymes, whereas some other genes were overexpressed in FLSs compared to the adult ovary. The E-Cadherin protein, which is involved in cell-to-cell interactions, was also expressed ectopically. Remarkably, it was seen that oocyte-like cells in the FLSs exhibited androgenetic genomic imprinting, which is ordinarily indicative of male GCs. Although the FLSs did not express male GC marker genes, the DNA methyltransferase, Dnmt3L, was expressed at an abnormally high level. Furthermore, the expression of sex determination factors was ambiguous in FLSs as both male and female determinants were expressed weakly. These data suggest that the developmental dysfunction of the ESC-derived FLSs may be attributable to aberrant gene expression and genomic imprinting, possibly associated with uncertain sex determination in culture.
Asunto(s)
Células Madre Embrionarias/citología , Regulación del Desarrollo de la Expresión Génica , Impresión Genómica , Células Germinativas/citología , Oocitos/citología , Animales , Proteína Morfogenética Ósea 4/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Cadherinas/metabolismo , Medios de Cultivo Condicionados/farmacología , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Femenino , Perfilación de la Expresión Génica , Riñón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Folículo Ovárico/metabolismo , Receptores de Gonadotropina/metabolismo , Factores SexualesRESUMEN
MARCH11, a RING-finger transmembrane ubiquitin ligase, is predominantly expressed in spermatids and localized to the trans-Golgi network (TGN) and multivesicular bodies (MVBs). Because ubiquitination acts as a sorting signal of cargo proteins, MARCH11 has been postulated to mediate selective protein sorting via the TGN-MVB pathway. However, the physiological substrate of MARCH11 has not been identified. In this study, we have identified and characterized SAMT1, a member of a novel 4-transmembrane protein family, which consists of four members. Samt1 mRNA and its expression product were found to be specific to the testis and were first detected in germ cells 25 days after birth in mice. Immunohistochemical analysis further revealed that SAMT1 was specifically expressed in haploid spermatids during the cap and acrosome phases. Confocal microscopic analysis showed that SAMT1 co-localized with MARCH11 as well as with fucose-containing glycoproteins, another TGN/MVB marker, and LAPM2, a late endosome/lysosome marker. Furthermore, we found that MARCH11 could increase the ubiquitination of SAMT1 and enhance its lysosomal delivery and degradation in an E3 ligase activity-dependent manner. In addition, the C-terminal region of SAMT1 was indispensable for its ubiquitination and proper localization. The other member proteins of the SAMT family also showed similar expression profile, intracellular localization, and biochemical properties, including ubiquitination by MARCH11. These results suggest that SAMT family proteins are physiological substrates of MARCH11 and are delivered to lysosomes through the TGN-MVB pathway by a ubiquitin-dependent sorting system in mouse spermatids.
Asunto(s)
Proteínas de la Membrana/metabolismo , Espermátides/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Humanos , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos ICR , ARN Mensajero/genética , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Relaxin-like factor (RLF), also known as insulin-like factor 3 (INSL3), is produced by testicular Leydig cells, but its specific receptor LGR8 (leucine-rich repeat family of G-protein-coupled receptor 8) has not been identified in goats. This study aimed to identify complementary DNA (cDNA) sequences of goat LGR8, and characterize the expression of both RLF and LGR8 in goat testes by RT-PCR and immunohistochemistry. Testes were collected from immature (3-month-old) and mature (24-month-old) Saanen goats, and partial cDNA sequences of the goat homologue of human LGR8 were identified. The sequence encoded a reduced peptide sequence of 167 amino acids, which corresponded to transmembrane regions 2 through 5, followed by the beginning of intracellular loop 3 of human LGR8. Expression of both LGR8 and RLF genes was drastically increased in mature testes compared with immature ones. Although RLF protein was restricted to Leydig cells, LGR8 protein was detected in both Leydig cells and seminiferous epithelial cells (possibly germ cells and Sertoli cells). These results reveal a possible existence of the RLF-LGR8 ligand-receptor system within the goat testis, suggesting that RLF may play a role in testicular function through LGR8 on Leydig cells and seminiferous epithelial cells in an autocrine and/or paracrine manner.
Asunto(s)
Cabras/fisiología , Insulina/genética , Insulina/metabolismo , Proteínas/genética , Proteínas/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Análisis de Secuencia de ADN , Testículo/metabolismo , Testículo/fisiología , Animales , Comunicación Autocrina , Secuencia de Bases , Células Epiteliales/fisiología , Expresión Génica , Humanos , Inmunohistoquímica , Insulina/fisiología , Células Intersticiales del Testículo/metabolismo , Células Intersticiales del Testículo/fisiología , Masculino , Datos de Secuencia Molecular , Comunicación Paracrina , Proteínas/fisiología , Receptores Acoplados a Proteínas G/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Epitelio Seminífero/citología , Testículo/citologíaRESUMEN
Although the physiological role of relaxin (RLN) in males remains largely unknown, there is limited evidence that the testis might be a candidate source and target of RLN in boars, as RLN transcripts are detected in the boar testis and it contains RLN-binding sites. To determine whether the boar testis acts as a source and target tissue of RLN, we characterised the expression pattern and cellular localisation of both RLN and its own receptor LGR7 (RXFP1) in boar testes during postnatal development by molecular and immunological approaches. Testes were collected from Duroc boars, and partial cDNA sequences of the boar homologue of human RXFP1 were identified. RLN expression increased through puberty onwards, while RXFP1 expression changed little during development. RLN mRNA and protein expression were restricted to the Leydig cells, whereas both Leydig cells and seminiferous epithelial cells expressed RXFP1 mRNA and protein. Interestingly, RLN was expressed in the testis as an 18âkDa form (the expected size of proRLN), but not as the 6âkDa mature form, during development because of a lack of the enzyme required for proRLN processing. In contrast, RXFP1 was detected at all stages as specific bands of 75 and 91-95âkDa (likely non-glycosylated and glycosylated RXFP1 respectively). Thus, we provide evidence for expression of RLN-RXFP1 ligand-receptor system in the boar testis, suggesting that the testis act as a source and possible target tissue of RLN.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismo , Relaxina/metabolismo , Porcinos/fisiología , Testículo/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Masculino , Datos de Secuencia Molecular , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores de Péptidos/química , Receptores de Péptidos/genética , Relaxina/química , Relaxina/genética , Alineación de SecuenciaRESUMEN
Osteoclasts are multinucleated giant cells with bone resorbing activity. We previously reported that the expression of the transcription factor NFAT2 (NFATc1) induced by receptor activator of NF-kappaB ligand (RANKL) is essential for the formation of multinucleated cells. We subsequently identified L-Ser in the differentiation medium as necessary for the expression of NFAT2. Here we searched for serine analogs that antagonize the function of L-Ser and suppress the formation of osteoclasts in bone marrow as well as RAW264 cells. An analog thus identified, H-Ser(tBu)-OMe x HCl, appeared to suppress the production of 3-ketodihydrosphingosine by serine palmitoyltransferase, and the expression and localization of RANK, a cognate receptor of RANKL, in membrane lipid rafts was down-regulated in the analog-treated cells. The addition of lactosylceramide, however, rescued the osteoclastic formation. When administered in vivo, the analog significantly increased bone density in mice and prevented high bone turnover induced by treatment with soluble RANKL. These results demonstrate a close connection between the metabolism of L-Ser and bone remodeling and also the potential of the analog as a novel therapeutic tool for bone destruction.
Asunto(s)
Huesos/metabolismo , Osteoclastos/metabolismo , Serina/análogos & derivados , Serina/química , Animales , Antígenos CD/metabolismo , Densidad Ósea , Células de la Médula Ósea/citología , Ésteres/química , Femenino , Regulación de la Expresión Génica , Células Gigantes/citología , Técnicas In Vitro , Lactosilceramidos/metabolismo , Microdominios de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Factores de Transcripción NFATC/metabolismo , Ligando RANK/metabolismo , Serina/farmacología , Serina C-Palmitoiltransferasa/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMEN
The expression and cellular localization of relaxin and its own receptor, LGR7/RXFP1, were demonstrated in the boar testis, where relaxin was produced by the Leydig cells as 18-kDa pro-relaxin and LGR7/RXFP1 was detected in both Leydig cells and seminiferous epithelial cells, suggesting that a functional relaxin-LGR7/RXFP1 hormone-receptor network operates within the boar testis.
Asunto(s)
Relaxina/genética , Relaxina/metabolismo , Porcinos/fisiología , Testículo/metabolismo , Animales , Células Epiteliales/metabolismo , Inmunohistoquímica , Hibridación in Situ , Células Intersticiales del Testículo/metabolismo , Masculino , Receptores Acoplados a Proteínas G/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Testículo/citologíaRESUMEN
Unmodified [60]fullerenes (C60) were solubilised with high stability using various type of poly(ethylene glycol) (PEG) based block copolymer micelles. Block copolymer micelle-incorporated C60 fullerenes were studied in cultures for biological activities using human cervical cancer HeLa cells. As a result, the cationic block copolymer micelles delivered C60 into the cells depending on their surface densities and showed cytotoxicity under photoirradiation.
Asunto(s)
Fulerenos/química , Micelas , Fármacos Fotosensibilizantes/química , Polietilenglicoles/química , Polímeros/química , Neoplasias del Cuello Uterino/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Cationes , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Fulerenos/farmacología , Células HeLa , Humanos , Luz , Estructura Molecular , Fármacos Fotosensibilizantes/farmacología , Polímeros/síntesis química , Polímeros/farmacocinética , Solubilidad , Neoplasias del Cuello Uterino/metabolismoAsunto(s)
Diferenciación Celular/genética , Osteoclastos/citología , Proteínas Tirosina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Adaptadoras Transductoras de Señales/fisiología , Animales , Proteína Tirosina Quinasa CSK , Humanos , Inositol Polifosfato 5-Fosfatasas , Ratones , Osteoclastos/fisiología , Monoéster Fosfórico Hidrolasas/fisiología , Fosforilación , Sistemas de Mensajero Secundario , Familia-src QuinasasRESUMEN
We developed an in situ hybridization method for detecting relaxin mRNA in the porcine corpus luteum (CL) by employing a non-radioactive probe and microwave fixation. We subsequently examined the expression and cellular patterns of relaxin mRNA in the CL during pregnancy and then evaluated whether relaxin mRNA was a factor limiting hormone production by the CL. Digoxigenin (DIG)-labeled RNA probes complementary to porcine relaxin mRNA were produced by in vitro transcription. The specificity was validated by showing, by Northern analysis, that the anti-sense probe hybridized to a 1.0-kb relaxin transcript in the CL. Microwave fixation (2-min irradiation in a conventional microwave oven) combined with DIG-labeled cRNA probes allowed precise and reliable analysis of relaxin mRNA, with superior retention of the mRNA and a higher resolving power. Application of this method to the porcine CL during pregnancy demonstrated that the relaxin mRNA level per cell and the percentage of mRNA-expressing cells increased as gestation progressed, with a marked decline near term. Northern analysis revealed the cellular pattern of relaxin mRNA localization, showing that the increase of relaxin mRNA with advancing pregnancy was attributable to an increase of both the cellular mRNA level and the percentage of mRNA-expressing cells. The present findings, taken together with known relaxin levels in the CL, reveal that changes of relaxin mRNA are correlated with changes of the hormone in the CL during pregnancy, suggesting that the relaxin level is determined by the amount of mRNA available for translation.
Asunto(s)
Cuerpo Lúteo/metabolismo , Células Epiteliales/metabolismo , Ovario/metabolismo , ARN Mensajero/metabolismo , Relaxina/genética , Animales , Northern Blotting , Cuerpo Lúteo/citología , Células Epiteliales/citología , Femenino , Regulación de la Expresión Génica/genética , Hibridación in Situ , Ovario/citología , Embarazo , Biosíntesis de Proteínas/genética , ARN Complementario/genética , ARN Mensajero/análisis , Relaxina/biosíntesis , Sus scrofaAsunto(s)
Regulación hacia Abajo/fisiología , Osteoclastos/fisiología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Ligando RANK/antagonistas & inhibidores , Transducción de Señal/fisiología , Animales , Resorción Ósea/metabolismo , Proteína Tirosina Quinasa CSK , Humanos , Osteoclastos/metabolismo , Proteínas Tirosina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Ligando RANK/fisiología , Regulación hacia Arriba/fisiología , Familia-src QuinasasRESUMEN
Intracellular uptake of a lipid-membrane-incorporated C(60) with a cationic surface into HeLa cells was found to induce cell death under visible light irradiation in high efficiency.
Asunto(s)
Fulerenos/farmacología , Luz , Membranas Artificiales , Fotoquimioterapia/métodos , Neoplasias del Cuello Uterino/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Muerte Celular/efectos de la radiación , Femenino , Fulerenos/química , Fulerenos/farmacocinética , Células HeLa , Humanos , Liposomas/síntesis química , Liposomas/química , Estructura Molecular , Solubilidad , Relación Estructura-Actividad , Propiedades de Superficie , Neoplasias del Cuello Uterino/metabolismo , Agua/químicaRESUMEN
Osteoclast precursor cells (OPCs) have previously been established from bone marrow cells of SV40 temperature-sensitive T antigen-expressing transgenic mice. Here, we use retrovirus-mediated gene transfer to conditionally immortalize OPCs by expressing temperature-sensitive large T antigen (tsLT) from wild type bone marrow cells. The immortalized OPCs proliferated at the permissive temperature of 33.5 degrees C, but stopped growing at the non-permissive temperature of 39 degrees C. In the presence of receptor activator of NFkappaB ligand (RANKL), the OPCs differentiated into tartrate-resistant acid phosphatase (TRAP)-positive cells and formed multinucleate osteoclasts at 33.5 degrees C. From these OPCs, we cloned two types of cell lines. Both differentiated into TRAP-positive cells, but one formed multinucleate osteoclasts while the other remained unfused in the presence of RANKL. These results indicate that the established cell lines are useful for analyzing mechanisms of differentiation, particularly multinucleate osteoclast formation. Retrovirus-mediated conditional immortalization should be a useful method to immortalize OPCs from primary bone marrow cells.
Asunto(s)
Diferenciación Celular , Osteoclastos/citología , Retroviridae/genética , Células Madre/citología , Animales , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular , Ratones , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Ligando RANK/farmacología , Células Madre/efectos de los fármacos , TemperaturaRESUMEN
Multinucleated cell formation is crucial for osteoclastogenesis, and the expression of nuclear factor of activated T cells (NFAT)2 (NFATc1) is essential for this process. We previously found, using mouse RAW264 cells, that culture at high cell density blocked progression to the multinucleated cell stage induced by stimulation with receptor activator of nuclear factor kappaB ligand (RANKL). Here, we have confirmed this finding in a bone marrow cell system and extended the analysis further. A high cell density appeared to cause a change in the composition of the culture medium accompanying downregulation of NFAT2 expression, and we identified L-serine (LSer) as essential for the expression of NFAT2 induced by RANKL. Namely, culture at high cell density caused a depletion of LSer in the medium. Consequently, L-Ser appeared to exert its effect at an early stage under the regular conditions used for inducing the expression of c-Fos, an upstream regulator of NFAT2. D-Ser, an enantiomer of L-Ser, showed no NFAT2-inducing activity. The expression of NFAT2, using a retrovirus vector, could compensate for the depletion of L-Ser and resume the progression to the multinucleated cell stage. These results demonstrate a novel role for L-Ser in RANKL-induced osteoclastogenesis in vitro.