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David W. Silversides - One of the best experts on this subject based on the ideXlab platform.

  • Porcine SRY Promoter Is a Target for Steroidogenic
    2016
    Co-Authors: Robert S. Viger, David W. Silversides
    Abstract:

    To study the process of mammalian sex determination and in particular to further understand the mechanisms of transcrip-tional regulation of the SRY gene, we have isolated a 4.5-kilo-base (kb) pig SRY 59 flanking sequence. To facilitate the in vitro analysis of these sequences, we have generated a porcine Genital Ridge (PGR) cell line (9E11) that expresses SRY as well as SOX9, steroidogenic factor-1 (SF-1), and DAX1. Via primer extension analysis on RNA from this cell line, a transcription start site for porcine SRY was identified at 2661 base pairs (bps) 59 from the translation initiation site. Deletion studies of the SRY 59 flanking sequences in PGR 9E11 cells demonstrated that 21.4 kb of 59 flanking sequences retained full transcriptional activity com-pared with the 24.5 kb fragment, but that transcriptional activ-ity fell when further deletions were made. Sequences down

  • Transgenic mouse analysis of Sry expression during the pre- and peri-implantation stage
    Developmental dynamics : an official publication of the American Association of Anatomists, 2012
    Co-Authors: David W. Silversides, Diana L. Raiwet, Ouliana Souchkova, Robert S. Viger, Nicolas Pilon
    Abstract:

    Silversides DW, Raiwet DL, Souchkova O, Viger RS, Pilon N. Dev Dyn. 2012 Jul ;241(7):1192-204. doi : 10.1002/dvdy.23798. Epub 2012 May 18. BACKGROUND : The SRY/Sry gene is expressed in pre-Sertoli cells of the male Genital Ridge and functions as the mammalian testis determining factor (TDF). In addition, expression of SRY/Sry outside the Genital Ridge has been reported, including preimplantation embryos, although the functional significance of this is not well understood. RESULTS : Using (...)

  • Human and pig SRY 5' flanking sequences can direct reporter transgene expression to the Genital Ridge and to migrating neural crest cells.
    Developmental dynamics : an official publication of the American Association of Anatomists, 2006
    Co-Authors: Alexandre Boyer, Nicolas Pilon, Diana L. Raiwet, Jacques G. Lussier, David W. Silversides
    Abstract:

    Mechanisms for sex determination vary greatly between animal groups, and include chromosome dosage and haploid-diploid mechanisms as seen in insects, temperature and environmental cues as seen in fish and reptiles, and gene-based mechanisms as seen in birds and mammals. In eutherian mammals, sex determination is genetic, and SRY is the Y chromosome located gene representing the dominant testes determining factor. How SRY took over this function from ancestral mechanisms is not known, nor is it known what those ancestral mechanisms were. What is known is that SRY is haploid and thus poorly protected from mutations, and consequently is poorly conserved between mammalian species. To functionally compare SRY promoter sequences, we have generated transgenic mice with fluorescent reporter genes under the control of various lengths of human and pig SRY 5' flanking sequences. Human SRY 5' flanking sequences (5 Kb) supported reporter transgene expression within the Genital Ridge of male embryos at the time of sex determination and also supported expression within migrating truncal neural crest cells of both male and female embryos. The 4.6 Kb of pig SRY 5' flanking sequences supported reporter transgene expression within the male Genital Ridge but not within the neural crest; however, 2.6 Kb and 1.6 Kb of pig SRY 5' flanking sequences retained male Genital Ridge expression and now supported extensive expression within cells of the neural crest in embryos of both sexes. When 2 Kb of mouse SRY 5' flanking sequences (-3 to -1 Kb) were placed in front of the 1.6 Kb of pig SRY 5' flanking sequences and this transgene was introduced into mice, reporter transgene expression within the male Genital Ridge was retained but neural crest expression was lost. These observations suggest that SRY 5' flanking sequences from at least two mammalian species contain elements that can support transgene expression within cells of the migrating neural crest and that additional SRY 5' flanking sequences can extinguish this expression.

  • Pre-sertoli specific gene expression profiling reveals differential expression of Ppt1 and Brd3 genes within the mouse Genital Ridge at the time of sex determination.
    Biology of reproduction, 2004
    Co-Authors: Alexandre Boyer, Jacques G. Lussier, Andrew H. Sinclair, Peter J. Mcclive, David W. Silversides
    Abstract:

    In mammals, testis determination is initiated when the SRY gene is expressed in pre-Sertoli cells of the undifferentiated Genital Ridge. SRY directs the differentiation of these cells into Sertoli cells and initiates the testis differentiation pathway via currently ill-defined mechanisms. Because Sertoli cells are the first somatic cells to differentiate within the developing testis, it is likely that the signals for orchestrating testis determination are expressed within pre-Sertoli cells. We have previously generated a transgenic mouse line that expresses green fluorescent protein under the control of the pig SRY promoter, thus marking pre-Sertoli cells via fluorescence. We have now used suppression-subtractive hybridization (SSH) to construct a normalized cDNA library derived from fluorescence-activated cell sorting (FACS) purified pre-Sertoli cells taken from 12.0 to 12.5 days postcoitum (dpc) fetal transgenic mouse testes. A total of 35 candidate cDNAs for known genes were identified. Detection of Sf1, a gene known for its role in sex determination as well as Vanin-1, Vcp1, Sparc, and Aldh3a1, four genes previously identified in differential screens as gene overexpressed in developing testis compared with ovary, support the biological validity of our experimental model. Whole-mount in situ hybridization was performed on the 35 candidate genes for qualitative differential expression between male and female Genital Ridges; six were upregulated in the testis and one was upregulated in the ovary. The expression pattern of two genes, Ppt1 and Brd3, were examined in further detail. We conclude that combining transgenically marked fluorescent cell populations with differential expression screening is useful for cell expression profiling in developmental systems such as sex determination and differentiation.

  • the porcine sry promoter is transactivated within a male Genital Ridge environment
    Genesis, 2002
    Co-Authors: Isabelle Daneau, Robert S. Viger, Nicolas Pilon, Jacques G. Lussier, Alexandre Boyer, Ramin Behdjani, Paul A Overbeek, David W. Silversides
    Abstract:

    Daneau I, Pilon N, Boyer A, Behdjani R, Overbeek PA, Viger R, Lussier J, Silversides DW. In mammals the SRY gene functions as a dominant genetic switch for testis determination (Gubbay et al. : Nature 346:1128-1135, 1990 ; Koopman et al. : Nature 351:117-121, 1991 ; Sinclair et al. : Nature 346:240-244, 1990). To study SRY transcriptional regulation within an evolutionary context, we have generated transgenic mice that express green fluorescent protein (GFP) under the control of 4.5 kb of pig (...)

Blanche Capel - One of the best experts on this subject based on the ideXlab platform.

  • Concerted morphogenesis of Genital Ridges and nephric ducts in the mouse captured through whole embryo imaging.
    Development (Cambridge England), 2021
    Co-Authors: Corey Bunce, Jennifer Mckey, Blanche Capel
    Abstract:

    During development of the mouse uroGenital complex, the gonads undergo changes in three-dimensional structure, body position, and spatial relationship with the mesonephric ducts, kidneys, and adrenals. The complexity of Genital Ridge development obscures potential connections between morphogenesis and gonadal sex determination. To characterize the morphogenic processes implicated in regulating gonad shape and fate, we used whole embryo tissue clearing and light sheet microscopy to assemble a time course of gonad development in native form and context. Analysis revealed that gonad morphology is determined through anterior-to-posterior patterns as well as increased rates of growth, rotation, and separation in the central domain that may contribute to regionalization of the gonad. We report a close alignment of gonad and mesonephric duct movements and delayed duct development in a gonad dysgenesis mutant, which together support a mechanical dependency linking gonad and mesonephric duct morphogenesis.

  • Three-dimensional structure of the developing mouse Genital Ridge
    Philosophical transactions of the Royal Society of London. Series B Biological sciences, 1995
    Co-Authors: Jeannie Karl, Blanche Capel
    Abstract:

    We are interested in understanding how the field of cells which forms the gonad arises, and how the testis-determining gene, Sry , controls morphogenesis of a testis within this field of cells. To appreciate changes in the three-dimensional structure of the mouse Genital Ridge at this time in development, whole-mount Genital Ridges taken from male and female embryos over the developmental period when the initiation of testis cord morphogenesis takes place, were stained with an antibody against laminin. Samples were visualized using confocal microscopy. Anti-laminin illuminates the elaborate array of mesonephric duct and tubules which occupy the cranial two-thirds of the mesonephros at the earliest timepoint. This complex structure gradually regresses as testis cords form in male gonads. No structural organisation is recognized by this antibody in the female gonadal region during this period. Confocal sections in the Z-plane reveal continuous cellular connections between 3-6 mesonephric tubules and the gonadal primordium. These cellular bRidges are present in male and female gonads, so they do not depend on the expression of Sry . We consider the possibility that these bRidges constitute the pathways of the founder cells of the gonadal primordium.

  • expression of sry the mouse sex determining gene
    Development, 1995
    Co-Authors: Adam Hacker, Blanche Capel, Peter N Goodfellow, Robin Lovellbadge
    Abstract:

    In the mouse, Sry is expressed by germ cells in the adult testis and by somatic cells in the Genital Ridge. Transcripts in the former exist as circular RNA molecules of 1.23 kb, which are unlikely to be efficiently translated. We have used RNase protection to map the extent of the less abundant Sry transcript in the developing gonad. We demonstrate that it is a linear mRNA derived from a single exon. This begins in the unique region 5′ of the protein coding region and extends several kilobases into the 3′ arm of the large inverted repeat which bounds the Sry genomic locus. Knowledge of this transcript, which is very different from that of the human SRY gene, allows us to predict its protein product and reveals several features which may be involved in translational control. Our data is also consistent with there being two promoters for the Sry gene, a proximal one that gives functional transcripts in the Genital Ridge and a distal promoter used in germ cells in the adult testis. As RNase protection is a quantitative technique, a detailed timecourse of Sry expression was carried out using accurately staged samples. Sry transcripts are first detectable just after 10.5 days post coitum, they reach a peak at 11.5 days and then decline sharply so that none are detected 24 hours later. This was compared with anti-Mullerian hormone gene expression, an early marker of Sertoli cells and the first known downstream gene of Sry. Amh expression begins 20 hours after the onset of Sry expression at a time when Sry transcripts are at their peak. While this result does not prove a direct interaction between the two genes, it defines the critical period during which Sry must act to initiate Sertoli cell differentiation.

  • circular transcripts of the testis determining gene sry in adult mouse testis
    Cell, 1993
    Co-Authors: Blanche Capel, Peter Koopman, Adam Hacker, Peter N Goodfellow, Amanda Swain, Silvia K Nicolis, Michael A Walter, Robin Lovellbadge
    Abstract:

    Sry is expressed at higher levels in the adult testis, where no function has been determined, than in the Genital Ridge, its critical site of action. cDNA and 5' RACE clones isolated from testis or from Sry-transfected cell lines have an unusual structure, with 3' sequences located in a 5' position. RNAase protection assays and reverse transcription polymerase chain reactions confirmed that these unusual RNA molecules represent the most abundant transcript in testis. Furthermore, oligonucleotide hybridization and RNAase H digestion proved that these Sry RNA molecules are circular. Similar transcripts were detected in the testes of mice with Mus musculus musculus, Mus musculus domesticus, and Mus spretus Sry genes. The circular RNA is found in the cytoplasm but is not substantially bound to polysomes. We suggest that the circles arise from normal splicing processes as a consequence of the unusual genomic structure surrounding the Sry locus in the mouse.

Peter Koopman - One of the best experts on this subject based on the ideXlab platform.

  • widespread expression of the testis determining gene sry in a marsupial
    Nature Genetics, 1995
    Co-Authors: Jenny L Harry, Peter Koopman, Francine E Brennan, Jennifer Marshall A Graves, Marilyn B. Renfree
    Abstract:

    There is compelling evidence from mutation analysis and transgenesis that the SRY gene isolated from human and mouse encodes the testis-determining factor on the mammalian Y chromosome. However, how SRY achieves this function is unclear. Although marsupials have been separated from eutherian mammals for approximately 100 million years, homologues of SRY have been localised to the Y chromosome of two unrelated marsupial species, the tammar wallaby and the Darling Downs dunnart. Gonadal development is fundamentally similar in eutherian and marsupial mammals, but the timing of morphological events is different. Fetal Sry transcripts are confined to somatic cells of the male mouse Genital Ridge between 10.5-12.5 days post coitum, corresponding with the onset of testis differentiation. Analysis of Sry gene expression in the Genital Ridge of normal and germ cell-deficient fetal mice has established that this gene acts in the somatic cell lineage, and is presumed to induce the formation of Sertoli cells. This assumption can be tested more critically in the tammar, where the equivalent stages of testis differentiation are observed over a 7-day period. We have examined the relationship of SRY expression to testis differentiation in the tammar wallaby. We show the marsupial SRY gene cannot be exclusively coupled to Sertoli cell differentiation, as this gene is expressed in the male fetus from several days before Genital Ridge formation until 40 days after birth. SRY transcripts are also present in a variety of extra-gonadal tissues in the developing young and adult male, a pattern of SRY expression similar to that observed in humans. These data indicate that, in addition to a role in testis determination, SRY may have other functions [corrected].

  • Expression of a linear Sry transcript in the mouse Genital Ridge
    Nature genetics, 1995
    Co-Authors: Y. Jeske, Josephine Bowles, Andy Greenfield, Peter Koopman
    Abstract:

    Sry is the Y-chromosomal gene that is pivotal in the determination of sex in mammals, however the structure of the Sry transcript produced in the embryo has not been determined. We show here that the transcript expressed in the developing mouse gonad at the sex determining stage of development is linear, polyadenylated and encoded by a single exon, in contrast to the circular, apparently untranslated transcript produced in adult testes. The linear transcript was not detected in any other fetal tissue nor in any adult tissue tested, and was expressed only in the Genital Ridge portion of the uroGenital Ridge. The spatial and temporal profile of Sry expression suggests that its role in the mouse fetus is limited to initiating Sertoli cell development during testis determination.

  • circular transcripts of the testis determining gene sry in adult mouse testis
    Cell, 1993
    Co-Authors: Blanche Capel, Peter Koopman, Adam Hacker, Peter N Goodfellow, Amanda Swain, Silvia K Nicolis, Michael A Walter, Robin Lovellbadge
    Abstract:

    Sry is expressed at higher levels in the adult testis, where no function has been determined, than in the Genital Ridge, its critical site of action. cDNA and 5' RACE clones isolated from testis or from Sry-transfected cell lines have an unusual structure, with 3' sequences located in a 5' position. RNAase protection assays and reverse transcription polymerase chain reactions confirmed that these unusual RNA molecules represent the most abundant transcript in testis. Furthermore, oligonucleotide hybridization and RNAase H digestion proved that these Sry RNA molecules are circular. Similar transcripts were detected in the testes of mice with Mus musculus musculus, Mus musculus domesticus, and Mus spretus Sry genes. The circular RNA is found in the cytoplasm but is not substantially bound to polysomes. We suggest that the circles arise from normal splicing processes as a consequence of the unusual genomic structure surrounding the Sry locus in the mouse.

Goro Yoshizaki - One of the best experts on this subject based on the ideXlab platform.

  • Primordial germ cell migration in the yellowtail kingfish (Seriola lalandi) and identification of stromal cell-derived factor 1
    General and comparative endocrinology, 2015
    Co-Authors: J.a. Fernández, Erin J. Bubner, Yutaka Takeuchi, Goro Yoshizaki, Tianfang Wang, Scott F. Cummins, Abigail Elizur
    Abstract:

    Primordial germ cells (PGCs) are progenitors of the germ cell lineage, giving rise to either spermatogonia or oogonia after the completion of gonadal differentiation. Currently, there is little information on the mechanism of PGCs migration leading to the formation of the primordial gonad in perciform fish. Yellowtail kingfish (Seriola lalandi) (YTK) (order Perciforms) inhabit tropical and temperate waters in the southern hemisphere. Fundamental details into the molecular basis of larval development in this species can be easily studied in Australia, as they are commercially cultured and readily available. In this study, histological analysis of YTK larvae revealed critical time points for the migration of PGCs to the Genital Ridge, resulting in the subsequent development of the primordial gonad. In YTK larvae at 3, 5, 7 and 10 days post hatch (DPH), PGCs were not yet enclosed by somatic cells, indicating the primordial gonad had not yet started to form. While at 15, 18 and 20 DPH PGCs had already settled at the Genital Ridge and started to become enclosed by somatic cells indicating the primordial gonad had started to develop. A higher number of PGCs were observed in the larvae at 15 and 18 DPH indicating PGCs proliferation, which corresponds with them becoming enclosed by the somatic cells. Directional migration of PGCs toward the Genital Ridge is a critical event in the subsequent development of a gonad. In zebrafish, mouse and chicken, stromal-cell derived factor (SDF1) signalling is one of the key molecules for PGC migration. We subsequently isolated from YTK the SDF1 (Slal-SDF1) gene, which encodes for a 98-residue precursor protein with a signal peptide at the N-terminus. There is spatial conservation between fish species of four cysteine residues at positions C9, C11, C34 and C49, expected to form disulphide bonds and stabilize the SDF structure. In YTK, Slal-SDF1 gene expression analyses shows that this gene is expressed in larvae from 1 to 22 DPH and demonstrates distinct spatial localisation in the larvae at 7 DPH. These results provide a platform for further studies into the molecular machinery of PGC migration in yellowtail kingfish, as well as other perciform fish species.

  • Short-term in vitro culturing improves transplantability of type A spermatogonia in rainbow trout (Oncorhynchus mykiss).
    Molecular reproduction and development, 2013
    Co-Authors: Shinya Shikina, Kazue Nagasawa, Makoto Hayashi, Maki Furuya, Yoshiko Iwasaki, Goro Yoshizaki
    Abstract:

    Continuous production of sperm within the testes is supported by spermatogonial stem cells capable of both self-renewal and the production of numerous differentiated germ cells. We previously demonstrated that a subpopulation of trout type A spermatogonia transplanted into the body cavity of a recipient embryo incorporated into the Genital Ridge, where they produced functional gametes within the gonads. Various cell-surface proteins could have played a role in the incorporation of spermatogonia into recipient Genital Ridges. During the preparation of cell suspensions for transplantation in our experimental protocol, however, dissociation of testis by strong proteases was unavoidable. This was problematic as cell-surface proteins may have been at least partially digested by protease activity. In the present study, recovery of spermatogonial surface proteins using short-term culture prior to transplantation was attempted. It was found that spermatogonia cultured in vitro could be harvested by ethylenediaminetetraacetic acid (EDTA) instead of protease treatment. Furthermore, when cultured spermatogonia collected by EDTA treatment were maintained for 24 hr in vitro, they exhibited high adhesiveness. These cultured spermatogonia also possessed higher survival of transplantation compared to spermatogonia newly dispersed by trypsin treatment. These results indicated that spermatogonia possess a reduced ability to migrate toward, adhere to, and/or be incorporated into the recipient Genital Ridge immediately after protease treatment. Short-term in vitro culturing, however, could allow spermatogonia to recover the surface proteins required for successful incorporation into the recipient Genital Ridge.

  • A novel transforming growth factor-β superfamily member expressed in gonadal somatic cells enhances primordial germ cell and spermatogonial proliferation in rainbow trout (Oncorhynchus mykiss)
    Developmental biology, 2006
    Co-Authors: Etsuko Sawatari, Shinya Shikina, Toshio Takeuchi, Goro Yoshizaki
    Abstract:

    Our understanding of the molecular mechanisms of primordial germ cell (PGC) proliferation in fish is rudimentary, but it is thought to be controlled by the surrounding somatic cells. We assumed that growth factors that are specifically involved in PGC proliferation are expressed predominantly in the surrounding Genital Ridge somatic cells. In order to isolate these growth factors, we compiled a complementary DNA (cDNA) subtractive library using cDNA from the Genital Ridges of 40-dpf rainbow trout embryos as the tester and cDNA from embryos without Genital Ridges as the driver. This approach identified a novel cytokine, designated gonadal soma-derived growth factor (GSDF), which is a member of the transforming growth factor (TGF)-beta superfamily. GSDF was expressed in the Genital Ridge somatic cells surrounding the PGCs during embryogenesis, and in both the granulosa and Sertoli cells at later stages. Inhibition of GSDF translation by antisense oligonucleotides suppressed PGC proliferation. Moreover, isolated testicular cells that were cultured with recombinant GSDF demonstrated dose-dependent proliferation of type-A spermatogonia; this effect was completely blocked by antiserum against GSDF. These results denote that GSDF, a novel member of the TGF-beta superfamily, plays an important role for proliferation of PGC and spermatogonia.

Michael Clinton - One of the best experts on this subject based on the ideXlab platform.