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Germ Cells (germ + cell)
Kinds of Germ Cells Terms modified by Germ Cells Selected AbstractsApplication of Laser-Assisted Microdissection for Gene Expression Analysis of Mammalian Germ CellsANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 3 2010R. Kenngott With 1 figure and 2 tables Summary Laser-assisted microdissection (LAM) is an important method to provide new significant insights into many embryological processes. To understand these processes, it is important to obtain specific populations of cells from complex tissue in an efficient and precise manner and to combine with many different molecular biological methods. During the last few years, the sophistication of the techniques of LAM has increased significantly and made the procedure easy to use. New micro-extraction protocols for DNA, RNA and proteins now allow broad downstream applications in the fields of genomics, transcriptomics and proteomics. In this review, we give a short overview of the application of LAM in combination with quantitative qPCR for the analysis of gene expression in mammalian germ cells. [source] Imprinting Status of G,S, NESP55, and XL,s in Cell Cultures Derived from Human Embryonic Germ Cells: GNAS Imprinting in Human Embryonic Germ CellsCLINICAL AND TRANSLATIONAL SCIENCE, Issue 5 2009Janet L. Crane M.D. Abstract GNAS is a complex gene that through use of alternative first exons encodes signaling proteins G,s and XL,s plus neurosecretory protein NESP55. Tissue-specific expression of these proteins is regulated through reciprocal genomic imprinting in fully differentiated and developed tissue. Mutations in GNAS account for several human disorders, including McCune-Albright syndrome and Albright hereditary osteodystrophy, and further knowledge of GNAS imprinting may provide insights into variable phenotypes of these disorders. We therefore analyzed expression of G,s, NESP55, and XL,s prior to tissue differentiation in cell cultures derived from human primordia germ cells. We found that the expression of G,s was biallelic (maternal allele: 52.6%± 2.5%; paternal allele: 47.2%± 2.5%; p= 0.07), whereas NESP55 was expressed preferentially from the maternal allele (maternal allele: 81.9%± 10%; paternal allele: 18.1%± 10%; p= 0.002) and XL,s was preferentially expressed from the paternal allele (maternal allele: 2.7%± 0.3%; paternal allele: 97.3%± 0.3%; p= 0.007). These results demonstrate that imprinting of NESP55 occurs very early in development, although complete imprinting appears to take place later than 5,11 weeks postfertilization, and that imprinting of XL,s occurs very early postfertilization. By contrast, mprinting of G,s most likely occurs after 11 weeks postfertilization and after tissue differentiation. [source] The germ plasm component vasa allows tracing of the interstitial stem cells in the cnidarian Hydractinia echinataDEVELOPMENTAL DYNAMICS, Issue 6 2008Nicole Rebscher Abstract Germ cells in hydrozoans arise lifelong from multipotent interstitial stem cells. To discover if a true germline-soma segregation exists in these species, we studied gametogenesis in Hydractinia echinata using in situ hybridization and immunohistochemistry for the germ cell marker Vasa. We could show that Hevas is a zygotic transcript, present in embryos from the gastrula stage onward. In the planula larva, Hevas is expressed in the interstitial stem cells located in the endoderm. During metamorphosis, Hevas -expressing cells appear in the ectoderm in the lower half of the polyp. While the Hevas transcript is not detectable in developing gametes, the protein accumulates during oogenesis. Vasa containing granules are detectable at the polar-body-forming pole after fertilization. These results suggest that, in Hydractinia, maternal Vasa protein, but not the mRNA, is a maternal constituent of a germ plasm and might be involved in the specification and maintenance of interstitial stem cells. Developmental Dynamics 237:1736,1745, 2008. © 2008 Wiley-Liss, Inc. [source] Reproductive stem cell research and its application to urologyINTERNATIONAL JOURNAL OF UROLOGY, Issue 2 2008Takehiko Ogawa Abstract: Germ cells are defined by their innate potential to transmit genetic information to the next generation through fertilization. Males produce numerous sperm for long periods to maximize chances of fertilization. Key to the continuous production of large numbers of sperm are germline stem cells and their immediate daughter cells, functioning as transit amplifying cells. Recently, it has become possible to expand germline stem cells of rodents in vitro. In addition, multipotent stem cells, which are functionally the same as embryonic stem cells, have been established from neonatal mouse testes. These stem cells derived from the testis should contribute to biological research and technologies. On the other hand, the nature of human spermatogenesis is largely unknown due to the lack of an appropriate experimental system. However, the prevailing testicular sperm extraction procedure unraveled hitherto unknown facets of human spermatogenesis. The establishment of a culturing method for human spermatogonial stem cells in hopefully the near future would be a great benefit for achieving further insight into human spermatogenesis and should lead to more sophisticated diagnostic and therapeutic clinical measures for male infertility. [source] Identification, isolation, and RT-PCR analysis of single stage-specific spermatogenetic cells obtained from portions of seminiferous tubules classified by transillumination microscopyMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 12 2009Chiara Vasco The protocol here described allows the analysis of gene expression in single specific mouse spermatogenetic cells. Germ cells were singularly isolated by microdissection of portions of seminiferous tubules classified, based on their transillumination pattern, into four distinct zones along their length. Single portions of a seminiferous tubule, corresponding to specific zones, were mechanically disaggregated into single cells that were (1) identified as spermatogonia, spermatocytes, round or elongated spermatids, (2) isolated using a micromanipulator, and (3) singularly transferred into a test tube for retro-transcription PCR analysis. On each single isolated cell, we have determined the quantitative profile of expression of Gapdh, an endogenous housekeeping gene known to be expressed throughout spermatogenesis. The protocol described allows an accurate analysis of the temporal and quantitative profile of gene expression throughout the whole male gamete differentiation process which so far has mainly been performed on enriched population of cells. Mol. Reprod. Dev. 76: 1173,1177, 2009. © 2009 Wiley-Liss, Inc. [source] Regional differences in maturation of germ cells of cryptorchid testes: role of environmentACTA PAEDIATRICA, Issue 8 2009Dragana Zivkovic Abstract Objective:, To investigate differences in maturation of germ cells in cryptorchid testes in three different regions. Patients and methods:, A total of 103 consecutive patients were operated for unilateral undescended testis in Vojvodina, from March 2006 until September 2007, and had a testicular biopsy performed. Germ cells were counted, and the presence of Ad spermatogonia was noted. Biopsies were compared to biopsies of similar patients from two different regions: Philadelphia, USA (130), and Liestal, Switzerland (55 patients). Results:, In Vojvodina, 84.5% of patients had Sertoli cells only, or some spermatogonia, but no Ad spermatogonia, and 15.5% had Ad spermatogonia. In Philadelphia, 59.3% of patients had poor testicular histology, and 40.7% had Ad spermatogonia. In Liestal, 61.8% of patients had no, or some, spermatogonia, but no Ad spermatogonia, and 38.2% had Ad spermatogonia. There was a difference (p = 0.000025) between the patients with normal testicular histology from Philadelphia and those from Vojvodina, as well as between the patients from Vojvodina and Liestal (p = 0.0027). Conclusion:, The reduction in the number of germ cells in patients with cryptorchidism from Vojvodina is more pronounced than patients from either Switzerland or USA. This is a unique observation, since such a study has not been published yet. [source] Centrioles to basal bodies in the spermiogenesis of Mastotermes darwiniensis (Insecta, Isoptera)CYTOSKELETON, Issue 5 2009Maria Giovanna Riparbelli Abstract In addition to their role in centrosome organization, the centrioles have another distinct function as basal bodies for the formation of cilia and flagella. Centriole duplication has been reported to require two alternate assembly pathways: template or de novo. Since spermiogenesis in the termite Mastotermes darwiniensis lead to the formation of multiflagellate sperm, this process represents a useful model system in which to follow basal body formation and flagella assembly. We present evidence of a possible de novo pathway for basal body formation in the differentiating germ cell. This cell also contains typical centrosomal proteins, such as centrosomin, pericentrin-like protein, ,-tubulin, that undergo redistribution as spermatid differentiation proceeds. The spermatid centrioles are long structures formed by nine doublet rather than triplet microtubules provided with short projections extending towards the surrounding cytoplasm and with links between doublets. The sperm basal bodies are aligned in parallel beneath the nucleus. They consist of long regions close to the nucleus showing nine doublets in a cartwheel array devoid of any projections; on the contrary, the short region close to the plasma membrane, where the sperm flagella emerge, is characterized by projections similar to those observed in the centrioles linking the basal body to the plasma membrane. It is hypothesized that this appearance is in connection with the centriole elongation and further with the flagellar axonemal organization. Microtubule doublets of sperm flagellar axonemes are provided with outer dynein arms, while inner arms are rarely visible. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Erasure of the paternal transcription program during spermiogenesis: The first step in the reprogramming of sperm chromatin for zygotic developmentDEVELOPMENTAL DYNAMICS, Issue 5 2008Junke Zheng Abstract Male germ cells possess a unique epigenetic program and express a male-specific transcription profile. However, when its chromatin is passed onto the zygote, it expresses an transcription/epigenetic program characteristic of the zygote. The mechanism underlying this reprogramming process is not understood at present. In this study, we show that an extensive range of chromatin factors (CFs), including essential transcription factors and regulators, remodeling factors, histone deacetylases, heterochromatin-binding proteins, and topoisomerases, were removed from chromatin during spermiogenesis. This process will erase the paternal epigenetic program to generate a relatively naive chromatin, which is likely to be essential for installation of the zygotic developmental program after fertilization. We have also showed that transcription termination in male germ cells was temporally correlated with CF dissociation. A genome-wide CF dissociation will inevitably disassemble the transcription apparatus and regulatory mechanism and lead to transcription silence. Based on data presented in this and previous studies (Sun et al., Cell Research [2007] 17:117,134), we propose that paternal-zygotic transcription reprogramming begins with a genome-wide CF dissociation to erase the existing transcription program in later stages of spermatogenesis. This will be followed by assembling of the zygotic equivalent after fertilization. The transcription/epigenetic program of the male germ cell is transformed into a zygotic one using an erase-and-rebuild strategy similar to that used in the maternal-zygotic transition. It is also noted that transcription is terminated long after meiosis is completed and before chromatin becomes highly condensed during spermatogenesis. The temporal order of these events suggests that transcription silence does not have to be coupled to meiosis or chromatin condensation. Developmental Dynamics 237:1463-1476, 2008. © 2008 Wiley-Liss, Inc. [source] Proteome analysis of the culture environment supporting undifferentiated mouse embryonic stem and germ cell growthELECTROPHORESIS, Issue 10 2007Nicolas Buhr Abstract The therapeutical interest of pluripotent cells and ethical issues related to the establishment of human embryonic stem cell (ESC) or embryonic germ cell (EGC) lines raise the understanding of the mechanism underlying pluripotency to a fundamental issue. Establishing a protein pluripotency signature for these cells can be complicated by the presence of unrelated proteins produced by the culture environment. Here, we have analyzed the environment supporting ESC and EGC growth, and established 2-D reference maps for each constituent present in this culture environment: mouse embryonic fibroblast feeder cells, culture medium (CM) and gelatin. The establishment of these reference maps is essential prior to the study of ESC and EGC specific proteomes. Indeed, these maps can be subtracted from ESC or EGC maps to allow focusing on spots specific for ESCs or EGCs. Our study led to the identification of 110 unique proteins from fibroblast feeder cells and 23 unique proteins from the CM, which represent major contaminants of ESC and EGC proteomes. For gelatin, no collagen-specific proteins were identified, most likely due to difficulties in resolution and low quantities. Furthermore, no differences were observed between naive and conditioned CM. Finally, we compared these reference maps to ESC 2-D gels and isolated 17 ESC specific spots. Among these spots, proteins that had already been identified in previous human and mouse ESC proteomes were identified but no apparent ESC-specific pluripotency marker could be identified. This work represents an essential step in furthering the knowledge of environmental factors supporting ESC and EGC growth. [source] JKT-1 is not a human seminoma cell lineINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 4 2007Jeroen de Jong Summary The JKT-1 cell line has been used in multiple independent studies as a representative model of human testicular seminoma. However, no cell line for this specific tumour type has been independently confirmed previously; and therefore, the seminomatous origin of JKT-1 must be proven. The genetic constitution of the JKT-1 cells was determined using flow cytometry and spectral karyotyping, as well as array comparative genomic hybridization and fluorescent in situ hybridization. Marker profiling, predominantly based on differentially expressed proteins during normal germ cell development, was performed by immunohistochemistry and Western blot analyses. Moreover, genome wide affymetrix mRNA expression and profiling of 157 microRNAs was performed, and the status of genomic imprinting was determined. A germ cell origin of the JKT-1 cells was in line with genomic imprinting status and marker profile (including positive staining for several cancer-testis antigens). However, the supposed primary tumour, from which the cell line was derived, being indeed a classical seminoma, was molecularly proven not to be the origin of the cell line. The characteristic chromosomal anomalies of seminoma, e.g. gain of the short arm of chromosome 12, as well as the informative marker profile (positive staining for OCT3/4, NANOG, among others) were absent in the various JKT-1 cell lines investigated, irrespective of where the cells were cultured. All results indicate that the JKT-1 cell line is not representative of human seminoma. Although it can originate from an early germ cell, a non-germ cell derivation cannot be excluded. [source] Are germ cell factors essential in the testicular enlargement after neonatal hypothyroidism recovery?INTERNATIONAL JOURNAL OF ANDROLOGY, Issue 1 2002A study using W/Wv mutant mice model We examined the issue of whether germ cell factors are required for testicular enlargement that occurs after recovery from neonatal hypothyroidism. Experiments were performed using W/Wv mutant mice (lacking germ cells) and normal mice (ICR). The pups in experimental group (neonatal hypothyroid) received 6 propyl 2-thio-uracil (PTU) treatment, administered by adding 0.1% (w/v) to the water provided to the mother from day 1 of birth through day 25 postpartum, while the pups of control group received drinking water only. Mice were sacrificed at the age of day 25, 50 and 90, in the case of ICR mice, or at day 25 and 90 in the case of W/Wv mutant mice. In both groups, early hypothyroidism caused a partial recoverable decrease in body growth and testicular development. Both ICR and W/Wv mutant mice, those recovered from neonatal hypothyroidism showed an increase in testis weights, the number of Sertoli cells, and the diameter of the semniferous tubules. This study demonstrates that neonatal hypothyroidism led recovery caused testicular enlargement not only in ICR mice but also in germ cell depleted W/Wv mutant mice. Hence these findings deny direct involvement of the germ cell factors in the process of testicular enlargement in recovered mice even in vivo, and reaffirm the notion that thyroid hormone directly regulates the dynamics of Sertoli cell maturation. [source] Haspin-like proteins: A new family of evolutionarily conserved putative eukaryotic protein kinasesPROTEIN SCIENCE, Issue 8 2001Jonathan M.G. Higgins Abstract Haspin (haploid germ cell,specific nuclear protein kinase) is reported to be a serine/threonine kinase that may play a role in cell-cycle cessation and differentiation of haploid germ cells. In addition, Haspin mRNA can be detected in diploid cell lines and tissues. Here, Haspin-like proteins are identified in several major eukaryotic phyla,including yeasts, plants, flies, fish, and mammals,and an extended group in Caenorhabditis elegans. The Haspin-like proteins have a complete but divergent eukaryotic protein kinase domain sequence. Although clearly related to one another and to other eukaryotic protein kinases, the Haspin-related proteins lack conservation of a subset of residues that are almost invariant in known kinases and possess distinctive inserted regions. In fact, phylogenetic analysis indicates that the Haspin-like proteins form a novel eukaryotic protein kinase family distinct from those previously defined. The identification of related proteins in model organisms provides some initial insight into their functional properties and will provide new experimental avenues by which to determine the function of the Haspin proteins in mammalian cells. [source] The Biology of the Development of the Genital Organs.ANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 2005A Multimedia Teaching Program In my presentation, I review the sexual differentiation from the genetic sex until the appearance of the external genitalia and the developmental anomalies to use an animated cartoon. The first critical stage of sexual differentiation occurs at the moment of fertilization, when the genetic sex of the zygote is determined by the nature of the sex chromosome contributed by the sperm. Although an XY zygote is destined to become a male, no distinctive differences between the early development of male and female embryos have been noted. This is accomplished after migration of the primordial germ cell into the early gonad. Because of the early commonality of genital structures, anomalies are the result of abnormal retention or loss of appropriate genital structures. Therefore, most genital anomalies are some form of intersex. During the early differentiation of the gonads, while the mesonephros is still the dominant excretory organ, the gonads arise as ridge like thickenings (gonadal ridge) on its ventromedial face. Differentiation of the indifferent gonads into ovaries or testes occurs after the arrival of the primordial germ cells. The primordial germ cells arise from the endodermal cells of the yolk. The principal function of the Y chromosome is to direct the differentiation of the presented indifferent gonad into a testis from the sixth week, while two X chromosome are presented the ovaries start to develop, from the 12th week. The next and most obvious phase in sexual differentiation of the embryo is the differentiation of the somatic sex. The early embryo develops a dual set of potential genital ducts, one is the original mesonephric (Wolff ) ducts, which persists after degeneration of the mesonephros as an excretory organ, and the another is newly formed pair of ducts called the paramesonephric (Müllerian) ducts. Under the influence of testosterone secreted by the testes, the mesonephric ducts develop into the duct system through which the spermatozoa are conveyed from the testes to the urethra. The potentially female paramesonephric ducts regress under the influence of another product of the embryonic testes, the Müllerian inhibitory factor, a glycoprotein secreted by the Sertoli cells. In genetically female embryos, neither testosterone nor Müllerian inhibitory factor are secreted by the gonads. In the absence of testosterone the mesonephric ducts regress and lack of Müllerian inhibitory factor permits the paramesonephric ducts to develop into oviducts, the uterus and part of the vagina. The next stage is the development of the external genitalia. In very young embryos, a vaguely outlined elevation known as the genital eminence can be seen in the midline, just cephalic to the proctodeal depression. This is soon differentiated into a central prominence (genital tubercle) closely flanked by a pair of folds (genital folds) extending toward the proctodeum. Somewhat farther to either side are rounded elevation known as the genital swellings. From this common starting point the external genitalia of both sex differentiate. If the individual is to develop into a male the genital tubercle, under the influence of dihydrotestosterone, becomes greatly elongated to form the penis and the genital swellings become enlarged to form the scrotal pouches. During the growth of the penis a groove develops along the entire length of its caudal face and is continuous with the slit-like opening of the urogenital sinus. This groove later becomes closed over by a ventral fusion of the genital folds, establishing the penile portion of the urethra. The portion of the urogenital sinus between the neck of the bladder and the original opening of the urogenital sinus becomes the prostetic urethra. In the female, the genital tubercle becomes the clitoris, the genital folds become the labia minora, and the genital swellings become the labia majora. The urethra in the female is derived from the urogenital sinus, being homologous with the prostatic portion of the male urethra. [source] Use of a highly sensitive quantitative telomerase assay in intracytoplasmic sperm injection programmes for the treatment of 47,XXY non-mosaic Klinefelter men,ANDROLOGIA, Issue 4 2002Y. Yamamoto Summary. We evaluated the role of the sensitive quantitative telomerase assay (SQTA) in the management of men with non-mosaic Klinefelter's syndrome (KS). Diagnostic testicular biopsy (DTB) was performed in 24 men with KS. A part of the DTB was stained and the remaining fragment was processed for the SQTA. After 3,18 months, a therapeutic testicular biopsy (TTB) was performed in the same testicle and the recovered specimens were processed to identify spermatozoa. Men with a SQTA outcome equal to 0.00 Units ,g,1 protein (n=7) demonstrated therapeutic testicular biopsy material that was negative for spermatogenic cells. In five men with a SQTA outcome of 8.11,38.03 Units ,g,1, the most advanced germ cell was the spermatogonium/primary spermatocyte. In the remaining 12 men, the most advanced spermatogenic cell in the TTB was the spermatozoon. In these men, the SQTA outcome was equal to 25.76,92.68 Units ,g,1 protein. Using 39.00 Units ,g,1 protein as a cut-off value, the accuracy of the SQTA in identifying men positive for spermatozoa was 91.6%. It appears that the SQTA has a role for identifying non-mosaic KS men who have testicular spermatozoa. [source] Effects of 60 Hz 14 µT magnetic field on the apoptosis of testicular germ cell in miceBIOELECTROMAGNETICS, Issue 1 2009Yoon-Won Kim Abstract We recently reported that continuous exposure, for 8 weeks, of extremely low frequency (ELF) magnetic field (MF) of 0.1 or 0.5 mT might induce testicular germ cell apoptosis in BALB/c mice. In that report, the ELF MF exposure did not significantly affect the body weight or testicular weight, but significantly increased the incidence of testicular germ cell death. In the present study, we aimed to further characterize the effect of a 16-week continuous exposure to ELF MF of 14 or 200 µT on testicular germ cell apoptosis in mice. There were no significant effects of MF on body weight and testosterone levels in mice. In TUNEL staining (In situ terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling), germ cells showed a significantly higher apoptotic rate in exposed mice than in sham controls (P,<,0.001). TUNEL-positive cells were mainly spermatogonia. In an electron microscopic study, degenerating spermatogonia showed condensation of nuclear chromatin similar to apoptosis. These results indicate that apoptosis may be induced in spermatogenic cells in mice by continuous exposure to 60 Hz MF of 14 µT. Bioelectromagnetics 30:66,72, 2009. © 2008 Wiley-Liss, Inc. [source] Acute hyperammonaemic encephalopathy in a female newborn caused by a novel, de novo mutation in the ornithine transcarbamylase geneACTA PAEDIATRICA, Issue 5 2004D Valik A full-term female offspring of a first and uneventful pregnancy presented at 60 h of life with irritability, tachypnea and respiratory alkalosis progressing to deep coma with clinically dominant circulatory failure, tachycardia and hypotension. Diagnosis of ornithine transcarbamylase (OTC) deficiency was made on the basis of hyperammonaemia, hypocitrullinaemia and extreme hyper-excretion of orotic acid. The baby was treated with peritoneal dialysis, arginine hydrochloride and adequate energy supply. DNA analysis revealed an as of yet unidentified missense mutation in the 6th exon of the OTC gene, resulting in a change of lysine to glutamine at position 210 (K210Q). Her parents were not found to carry this mutation, implying that this mutation may have occurred either de novo in the patient or in a parental germ cell. Conclusion: An acute neonatal form of OTC deficiency should be considered in the differential diagnosis of coma in female newborns. [source] Epigenetic abnormality of SRY gene in the adult XY female with pericentric inversion of the Y chromosomeCONGENITAL ANOMALIES, Issue 2 2010Tomoko Mitsuhashi ABSTRACT In normal ontogenetic development, the expression of the sex-determining region of the Y chromosome (SRY) gene, involved in the first step of male sex differentiation, is spatiotemporally regulated in an elaborate fashion. SRY is expressed in germ cells and Sertoli cells in adult testes. However, only few reports have focused on the expressions of SRY and the other sex-determining genes in both the classical organ developing through these genes (gonad) and the peripheral tissue (skin) of adult XY females. In this study, we examined the gonadal tissue and fibroblasts of a 17-year-old woman suspected of having disorders of sexual differentiation by cytogenetic, histological, and molecular analyses. The patient was found to have the 46,X,inv(Y)(p11.2q11.2) karyotype and streak gonads with abnormally prolonged SRY expression. The sex-determining gene expressions in the patient-derived fibroblasts were significantly changed relative to those from a normal male. Further, the acetylated histone H3 levels in the SRY region were significantly high relative to those of the normal male. As SRY is epistatic in the sex-determination pathway, the prolonged SRY expression possibly induced a destabilizing effect on the expressions of the downstream sex-determining genes. Collectively, alterations in the sex-determining gene expressions persisted in association with disorders of sexual differentiation not only in the streak gonads but also in the skin of the patient. The findings suggest that correct regulation of SRY expression is crucial for normal male sex differentiation, even if SRY is translated normally. [source] Glutamylated tubulin: Diversity of expression and distribution of isoformsCYTOSKELETON, Issue 1 2003Marie-Louise Kann Abstract Glutamylation of , and , tubulin isotypes is a major posttranslational modification giving rise to diversified isoforms occurring mainly in neurotubules, centrioles, and axonemes. Monoglutamylated tubulin isoforms can be differentially recognized by two mAbs, B3 and GT335, which both recognize either polyglutamylated isoforms. In the present study, immunoelectron microscopy and immunofluorescence analyses were performed with these two mAbs to determine the expression and distribution of glutamylated tubulin isoforms in selected biological models whose tubulin isotypes are characterized. In mouse spermatozoa, microtubules of the flagellum contain polyglutamylated isoforms except in the tip where only monoglutamylated isoforms are detected. In spermatids, only a subset of manchette microtubules contain monoglutamylated tubulin isoforms. Cytoplasmic microtubules of Sertoli cells are monoglutamylated. Mitotic and meiotic spindles of germ cells are monoglutamylated whereas the HeLa cell mitotic spindle is polyglutamylated. Three models of axonemes are demonstrated as a function of the degree and extent of tubulin glutamylation. In lung ciliated cells, axonemes are uniformly polyglutamylated. In sea urchin sperm and Chlamydomonas, flagellar microtubules are polyglutamylated in their proximal part and monoglutamylated in their distal part. In Paramecium, cilia are bi- or monoglutamylated only at their base. In all cells, centrioles or basal bodies are polyglutamylated. These new data emphasize the importance of glutamylation in all types of microtubules and strengthen the hypothesis of its role in the regulation of the intracellular traffic and flagellar motility. Cell Motil. Cytoskeleton 55:14,25, 2003. © 2003 Wiley-Liss, Inc. [source] Ectopic germline cells in embryos of Xenopus laevisDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 7 2007Kohji Ikenishi Whether all descendants of germline founder cells inheriting the germ plasm can migrate correctly to the genital ridges and differentiate into primordial germ cells (PGCs) at tadpole stage has not been elucidated in Xenopus. We investigated precisely the location of descendant cells, presumptive primordial germ cells (pPGCs) and PGCs, in embryos at stages 23,48 by whole-mount in situ hybridization with the antisense probe for Xpat RNA specific to pPGCs and whole-mount immunostaining with the 2L-13 antibody specific to Xenopus Vasa protein in PGCs. Small numbers of pPGCs and PGCs, which were positively stained with the probe and the antibody, respectively, were observed in ectopic locations in a significant number of embryos at those stages. A few of the ectopic PGCs in tadpoles at stages 44,47 were positive in TdT-mediated dUTP digoxigenin nick end labeling (TUNEL) staining. By contrast, pPGCs in the embryos until stage 40, irrespective of their location and PGCs in the genital ridges of the tadpoles at stages 43,48 were negative in TUNEL staining. Therefore, it is evident that a portion of the descendants of germline founder cells cannot migrate correctly to the genital ridges, and that a few ectopic PGCs are eliminated by apoptosis or necrosis at tadpole stages. [source] Gonadal structure of the serial-sex changing gobiid fish Trimma okinawaeDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 1 2005Yasuhisa Kobayashi In order to obtain basic information about the role played by endogenous sex hormones in bringing about sex changes in the serial-sex changing gobiid fish Trimma okinawae, the gonadal structure of male and female phases were observed histologically. Steroid-producing cells (SPC; Leydig cells in a testis) were observed ultrastructurally in the ovaries and testes of both female-phase and male-phase fish. In addition, gonadal expression of P450 cholesterol side-chain-cleavage (scc) was examined immunohistochemically. Gonads of fish in female and male phases were observed to have both ovaries and testes simultaneously. Female-phase fish had matured with many developed vitellogenic oocytes, while male-phase individuals had immature ovaries with many numbers of previtellogenic oocytes at the perinucleolus stage. Testes of fish in different sexual phases had active spermatogenic germ cells. Organellae of SPC in the ovaries of female-phase fish had active structures of steroid production. In contrast, SPC in the ovaries of male-phase fish did not show active structures of steroid production. Immunopositive reactions against the scc antibody in the ovaries of female-phase fish were very strong, but immunoreactions in the ovaries of male-phase fish were very weak. In the testis, moderate immunopositive signals were obtained from dual-phase male/females. [source] Developmental delay and unstable state of the testes in the rdw rat with congenital hypothyroidismDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 4 2004Yasuhiro Sakai From the present study of the rdw rat, it is clear that the thyroid hormone is essential for the development and maintenance of the testes. In previous studies, the thyroid hormone has few serious effects on the testes except during the neonatal stage when the thyroid hormone receptor is mainly present. However, there is little knowledge concerning the prolonged effect of thyroid hormone deficiency throughout the rat's life span. In the present study, a morphological analysis was performed on the testes of rdw rats with congenital hypothyroidism. The rdw testes required a longer time to develop into the normal adult structure. Moreover, the developed, normal structure began to degenerate after full maturation. Specific characteristics of the rdw testes include: (i) a prolonged proliferation of Sertoli cells during postnatal development; (ii) a developmental delay in the appearance of spermatocytes and spermatid; (iii) direct contact with each other for both spermatocytes and spermatids, without Sertoli cell cytoplasm completely intervening between adjacent germ cells; (iv) subsequent apoptosis of germ cells after maturation; (v) reduction in the height of the seminiferous epithelium; and (vi) lower testosterone levels in the rdw rats, especially during old age. Thus, we conclude that the thyroid hormone plays an important role in developing and maintaining normal function of testes. [source] Cleavage-like cell division and explosive increase in cell number of neonatal gonocytesDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 1 2004Yasuhiro Sakai Based on previous conventional quantitative observations of rat testes, it was proposed that large numbers of gonocytes degenerate after birth and this notion was widely accepted. However, many studies show that neonatal gonocytes display high levels of mitotic activity. In order to resolve the apparent contradiction of increased mitotic activity in gonocytes despite a decrease in their numbers at the neonate stage, quantitative analysis using a marker of suitably higher resolution is required. It has been shown that the vasa protein could be used as a marker of germ cells. In this study, quantitative changes in gonocytes were re-examined using a germ-cell-specific marker in order to delineate more clearly the process of development from gonocytes to spermatogonia after birth. The vasa -positive cells, which correspond to gonocytes and spermatogonia, increased exponentially after birth. This observation suggests that all gonocyte divide actively after birth and do not degenerate as previously believed. Surprisingly, the cell volume of gonocytes decreased during their division. The largest population size was 2000,4000 µ3 at day 2, 1000,2000 µ3 at day 4 and 500,1000 µ3 at day 6. This finding suggests that gonocytes divide in a similar way to cleavage, which can be considered a special mode of fertilized eggs. Judging from the growth of seminiferous tubules and the degree of volume reduction, 60% of the contribution rate is estimated to be due to ordinal cell growth, and 40% due to volume reduction as in cleavage of a fertilized egg. This unique cleavage-like division may contribute to the supply of large numbers of spermatogonia. [source] Spatio-temporal expression of Xenopus vasa homolog, XVLG1, in oocytes and embryos: The presence of XVLG1 RNA in somatic cells as well as germline cellsDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 2 2000Kohji Ikenishi The expression of Xenopus vasa homolog or XVLG1 was examined in oocytes and embryos by whole-mount in situ hybridization and reverse transcription,polymerase chain reaction (RT-PCR). To confirm the results in embryos, both methods were also applied to explants of germ plasm-bearing cells (GPBC) from 32-cell embryos and to those of partial embryos deprived of GPBC. By hybridization, XVLG1 ribonucleic acid (RNA) was shown to be present throughout the cytoplasm in oocytes at stages I,III, except for the mitochondrial cloud. It was barely recognizable in a portion of germline cells of embryos at specific stages, notwithstanding that XVLG1 protein was present in those cells almost throughout their life-span. A weak signal for the RNA was detectable in some of the presumptive primordial germ cells (pPGC, descendants of GPBC from the gastrula stage onward) from the late gastrula (stage 12) to the hatching tadpole stage (stage 33/34), and in some of the PGC at stages 49,50. The results for pPGC were confirmed by the hybridization of explants of GPBC at equivalent stages in control embryos. In contrast, XVLG1 RNA was detected in certain somatic cells of embryos until stage 46. These observations were supported in part by the results of RT-PCR for embryos and explants. The possible role of the product of XVLG1 was reconsidered given its presence in both germline and somatic cells. [source] Medaka Oct4 is expressed during early embryo development, and in primordial germ cells and adult gonadsDEVELOPMENTAL DYNAMICS, Issue 2 2010Ana V. Sánchez-Sánchez Abstract Oct4 is a crucial transcription factor for controlling pluripotency in embryonic stem cells and the epiblast of mouse embryos. We have characterized the expression pattern of medaka (Oryzias latipes) Ol-Oct4 during embryonic development and in the adult gonads. Genomic analysis showed that Ol-Oct4 is the ortholog of zebrafish spg/pou2. However, their expression patterns are not the same, suggesting that Oct4 may play different roles in zebrafish and medaka. Using specific antibodies for the Ol-Oct4 protein, we showed that Ol-Oct4 is also expressed in primordial germ cells, in the spermatogonia (male germ stem cells), and during different stages of oocyte development. These results suggest that Ol-Oct4 plays a post-embryonic role in the maturing gonads and gametes. The Ol-Oct4 mRNA and protein expression patterns are similar to those of mammalian Oct4 and introduce medaka fish as a valid model for the functional and evolutionary study of pluripotency genes in vivo. Developmental Dynamics 239:672,679, 2010. © 2009 Wiley-Liss, Inc. [source] Sdmg1 is a component of secretory granules in mouse secretory exocrine tissuesDEVELOPMENTAL DYNAMICS, Issue 1 2009Diana Best Abstract Sdmg1 is a conserved eukaryotic transmembrane protein that is mainly expressed in the gonads where it may have a role in mediating signaling between somatic cells and germ cells. In this study we demonstrate that secretory exocrine cells in the pancreas, salivary gland, and mammary gland also express Sdmg1. Furthermore, we show that Sdmg1 expression is up-regulated during pancreas development when regulated secretory granules start to appear, and that Sdmg1 colocalizes with secretory granule markers in adult pancreatic acinar cells. In addition, we show that Sdmg1 co-purifies with secretory granules during subcellular fractionation of the pancreas and that Sdmg1 and the secretory granule marker Vamp2 are localized to distinct subdomains in the secretory granule membrane. These data suggest that Sdmg1 is a component of regulated secretory granules in exocrine secretory cells and that the developmental regulation of Sdmg1 expression is related to a role for Sdmg1 in post-Golgi membrane trafficking. Developmental Dynamics 238:223,231, 2009. © 2008 Wiley-Liss, Inc. [source] Erasure of the paternal transcription program during spermiogenesis: The first step in the reprogramming of sperm chromatin for zygotic developmentDEVELOPMENTAL DYNAMICS, Issue 5 2008Junke Zheng Abstract Male germ cells possess a unique epigenetic program and express a male-specific transcription profile. However, when its chromatin is passed onto the zygote, it expresses an transcription/epigenetic program characteristic of the zygote. The mechanism underlying this reprogramming process is not understood at present. In this study, we show that an extensive range of chromatin factors (CFs), including essential transcription factors and regulators, remodeling factors, histone deacetylases, heterochromatin-binding proteins, and topoisomerases, were removed from chromatin during spermiogenesis. This process will erase the paternal epigenetic program to generate a relatively naive chromatin, which is likely to be essential for installation of the zygotic developmental program after fertilization. We have also showed that transcription termination in male germ cells was temporally correlated with CF dissociation. A genome-wide CF dissociation will inevitably disassemble the transcription apparatus and regulatory mechanism and lead to transcription silence. Based on data presented in this and previous studies (Sun et al., Cell Research [2007] 17:117,134), we propose that paternal-zygotic transcription reprogramming begins with a genome-wide CF dissociation to erase the existing transcription program in later stages of spermatogenesis. This will be followed by assembling of the zygotic equivalent after fertilization. The transcription/epigenetic program of the male germ cell is transformed into a zygotic one using an erase-and-rebuild strategy similar to that used in the maternal-zygotic transition. It is also noted that transcription is terminated long after meiosis is completed and before chromatin becomes highly condensed during spermatogenesis. The temporal order of these events suggests that transcription silence does not have to be coupled to meiosis or chromatin condensation. Developmental Dynamics 237:1463-1476, 2008. © 2008 Wiley-Liss, Inc. [source] RNA expression microarray analysis in mouse prospermatogonia: Identification of candidate epigenetic modifiers,DEVELOPMENTAL DYNAMICS, Issue 4 2008Christophe Lefčvre Abstract The mammalian totipotent and pluripotent lineage exhibits genome-wide dynamics with respect to DNA methylation content. The first phase of global DNA demethylation and de novo remethylation occurs during preimplantation development and gastrulation, respectively, while the second phase occurs in primordial germ cells and primary oocytes/prospermatogonia, respectively. These dynamics are indicative of a comprehensive epigenetic resetting or reprogramming of the genome in preparation for major differentiation events. To gain further insight into the mechanisms driving DNA methylation dynamics and other types of epigenetic modification, we performed an RNA expression microarray analysis of fetal prospermatogonia at the stage when they are undergoing rapid de novo DNA remethylation. We have identified a number of highly or specifically expressed genes that could be important for determining epigenetic change in prospermatogonia. These data provide a useful resource in the discovery of molecular pathways involved in epigenetic reprogramming in the mammalian germ line. Developmental Dynamics 237:1082,1089, 2008. © 2008 Wiley-Liss, Inc. [source] Enhancer detection in the ascidian Ciona intestinalis with transposase-expressing lines of MinosDEVELOPMENTAL DYNAMICS, Issue 1 2008Yasunori Sasakura Abstract Germline transgenesis with a Tc1/mariner superfamily Minos transposon was achieved in the ascidian Ciona intestinalis. Transgenic lines that express transposases in germ cells are very useful for remobilizing transposon copies. In the present study, we created transposase-expressing lines of Minos in Ciona. A Ciona gene encoding protamine (Ci - prm) is expressed in the testes and sperm. Transgenic lines expressing Minos transposase in the testes and sperm were created with a cis -element of Ci - prm, and used for enhancer detection. Double-transgenic animals between transposase lines and a transgenic line with an enhancer detection vector passed on several independent enhancer detection events to subsequent progeny. This technique allowed us to isolate transgenic lines that express GFP in restricted tissues. This system provides an easy and efficient method for large-scale enhancer detection in Ciona intestinalis. Developmental Dynamics 237:39,50, 2008. © 2007 Wiley-Liss, Inc. [source] Sexual dimorphism of g-protein subunit Gng13 expression in the cortical region of the developing mouse ovaryDEVELOPMENTAL DYNAMICS, Issue 7 2007Akihiro Fujino Abstract In our search for genes required for the development and function of mouse gonads, we identified Gng13 (guanine nucleotide binding protein 13, gamma), a gene with an embryonic expression pattern highly restricted to the ovary. Based on reverse transcriptase-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization, Gng13 is expressed in both XX and XY gonads at embryonic day (E) 11.5, but becomes up-regulated in the XX gonad by E12.5. Expression is retained after treatment with busulfan, a chemical known to eliminate germ cells, pointing to the soma as a site of Gng13 transcription. In situ hybridization of embryonic ovarian tissue sections further localized the expression to the cortex of the developing XX gonad. Gng13 expression in the adult is also highly restricted. Northern blot analyses and Genomic Institute of the Novartis Research Foundation expression profiling of adult tissues detected very high expression in the cerebrum and cerebellum, in addition to, a weaker signal in the ovary. Gng13 belongs to a well-known family of signal transduction molecules with functions in many aspects of development and organ physiology. Here, we report that, in the developing mouse embryo, expression of Gng13 mRNA is highly restricted to the cortex of the XX gonad during sexual differentiation, suggesting a role for this gene during ovarian development. Developmental Dynamics 236:1991,1996, 2007. © 2007 Wiley-Liss, Inc. [source] GATA-4 is required for sex steroidogenic cell development in the fetal mouseDEVELOPMENTAL DYNAMICS, Issue 1 2007Malgorzata Bielinska Abstract The transcription factor GATA-4 is expressed in Sertoli cells, steroidogenic Leydig cells, and other testicular somatic cells. Previous studies have established that interaction between GATA-4 and its cofactor FOG-2 is necessary for proper Sry expression and all subsequent steps in testicular organogenesis, including testis cord formation and differentiation of both Sertoli and fetal Leydig cells. Since fetal Leydig cell differentiation depends on Sertoli cell,derived factors, it has remained unclear whether GATA-4 has a cell autonomous role in Leydig cell development. We used two experimental systems to explore the role of GATA-4 in the ontogeny of testicular steroidogenic cells. First, chimeric mice were generated by injection of Gata4,/, ES cells into Rosa26 blastocysts. Analysis of the resultant chimeras showed that in developing testis Gata4,/, cells can contribute to fetal germ cells and interstitial fibroblasts but not fetal Leydig cells. Second, wild-type or Gata4,/, ES cells were injected into the flanks of intact or gonadectomized nude mice and the resultant teratomas examined for expression of steroidogenic markers. Wild-type but not Gata4,/, ES cells were capable of differentiating into gonadal-type steroidogenic lineages in teratomas grown in gonadectomized mice. In chimeric teratomas derived from mixtures of GFP-tagged Gata4+/+ ES cells and unlabeled Gata4,/, ES cells, sex steroidogenic cell differentiation was restricted to GFP-expressing cells. Collectively these data suggest that GATA-4 plays an integral role in the development of testicular steroidogenic cells. Developmental Dynamics 236:203,213, 2007. © 2006 Wiley-Liss, Inc. [source] | ||||||||||||||||||||||||||||||||||