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1.
Commun Biol ; 5(1): 1320, 2022 12 13.
Article in English | MEDLINE | ID: mdl-36513809

ABSTRACT

Selection for system-wide morphological, physiological, and metabolic adaptations has led to extreme athletic phenotypes among geographically diverse horse breeds. Here, we identify genes contributing to exercise adaptation in racehorses by applying genomics approaches for racing performance, an end-point athletic phenotype. Using an integrative genomics strategy to first combine population genomics results with skeletal muscle exercise and training transcriptomic data, followed by whole-genome resequencing of Asian horses, we identify protein-coding variants in genes of interest in galloping racehorse breeds (Arabian, Mongolian and Thoroughbred). A core set of genes, G6PC2, HDAC9, KTN1, MYLK2, NTM, SLC16A1 and SYNDIG1, with central roles in muscle, metabolism, and neurobiology, are key drivers of the racing phenotype. Although racing potential is a multifactorial trait, the genomic architecture shaping the common athletic phenotype in horse populations bred for racing provides evidence for the influence of protein-coding variants in fundamental exercise-relevant genes. Variation in these genes may therefore be exploited for genetic improvement of horse populations towards specific types of racing.


Subject(s)
Genome-Wide Association Study , Genome , Horses/genetics , Animals , Phenotype , Genomics , Sequence Analysis, DNA
2.
Article in English | MEDLINE | ID: mdl-33803108

ABSTRACT

Pollution of the environment is increasing and threatens the health and wellbeing of adults and children around the globe. The impact of air pollution on pulmonary and cardiovascular disease has been well documented, but it also has a deleterious effect on reproductive health. Ulaanbaatar, the capital city of Mongolia, has one of the highest levels of air pollution in the world. During the extreme winters when temperatures routinely fall below -20 °C the level of air pollution can reach 80 times the WHO recommended safe levels. Heating mainly comes from coal, which is burned both in power stations, and in stoves in the traditional Ger housing. We studied the impact of air pollution on conception rates and birth outcomes in Ulaanbaatar using a retrospective analysis of health data collected from the Urguu Maternity hospital in Ulaanbaatar, Mongolia. Daily levels of SO2, NO2, PM10, and PM2.5 were collected from the government Air Quality Monitoring Stations in Ulaanbaatar for the same period as the study. In January, the month of highest pollution, there is a 3.2-fold decrease in conceptions that lead to the successfully delivered infants compared to October. The seasonal variations in conceptions resulting in live births in this study in Ulaanbaatar are shown to be 2.03 ± 0.20 (10-sigma) times greater than those in the Denmark/North America study of Wesselink et al., 2020. The two obvious differences between Ulaanbaatar and Europe/North America are pollution and temperature both of which are extreme in Ulaanbaatar. The extreme low temperature is mitigated by burning coal, which is the main source of domestic heat especially in the ger districts. This drives the level of pollution so the two are inextricably linked. Infants conceived in the months of June-October had the greatest cumulative PM2.5 pollution exposure over total gestation, yet these were also the pregnancies with the lowest PM2.5 exposure for the month of conception and three months prior to conception. The delivered-infant conception rate shows a markedly negative association with exposure to PM2.5 prior to and during the first month of pregnancy. This overall reduction in fecundity of the population of Ulaanbaatar is therefore a preventable health risk. It is of great consequence that the air pollution in Ulaanbaatar affects health over an entire lifespan including reproductive health. This could be remedied with a clean source of heating.


Subject(s)
Air Pollutants , Air Pollution , Adult , Air Pollutants/analysis , Air Pollution/adverse effects , Air Pollution/analysis , Child , Coal , Environmental Exposure/analysis , Environmental Monitoring , Europe , Female , Fertility , Heating , Humans , Infant , Mongolia , North America , Particulate Matter/analysis , Pregnancy , Retrospective Studies , Seasons
3.
Development ; 143(19): 3632-3637, 2016 10 01.
Article in English | MEDLINE | ID: mdl-27702788

ABSTRACT

In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences.


Subject(s)
In Situ Hybridization/methods , RNA, Messenger/metabolism , Animals , Drosophila , Embryo, Nonmammalian/metabolism , Humans , Zebrafish
4.
Methods Mol Biol ; 1448: 95-106, 2016.
Article in English | MEDLINE | ID: mdl-27317176

ABSTRACT

Most transgenic technologies rely on the oocyte as a substrate for genetic modification. Transgenics animals are usually generated by the injection of the gene constructs (including lentiviruses encoding gene constructs or modified embryonic stem cells) into the pronucleus of a fertilized egg followed by the transfer of the injected embryos into the uterus of a foster mother. Male germ cells also have potential as templates for transgenic development. We have previously shown that mature sperm can be utilized as template for lentiviral transduction and as such used to generate transgenic mice efficiently with germ line capabilities. We provide here a detailed protocol that is relatively simple, to establish transgenic mice using lentivirally transduced spermatozoa. This protocol employs a well-established lentiviral gene delivery system (usual for somatic cells) delivering a variety of transgenes to be directly used with sperm, and the subsequent use of these modified sperm in in vitro fertilization studies and embryo transfer into foster female mice, for the establishment of transgenic mice.


Subject(s)
Genetic Vectors , Lentivirus/genetics , Mice, Transgenic/genetics , Transduction, Genetic/methods , Animals , Embryo Transfer/methods , Embryonic Stem Cells/cytology , Fertilization in Vitro/methods , Gene Transfer Techniques , Male , Mice , Oocytes/growth & development , Spermatozoa/growth & development
5.
Cold Spring Harb Protoc ; 2015(3): 259-68, 2015 Mar 02.
Article in English | MEDLINE | ID: mdl-25734068

ABSTRACT

Multiplexed fluorescent hybridization chain reaction (HCR) and advanced imaging techniques can be used to evaluate combinatorial gene expression patterns in whole mouse embryos with unprecedented spatial resolution. Using HCR, DNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled DNA HCR hairpins self-assemble into tethered fluorescent amplification polymers. Each target mRNA is detected by a probe set containing one or more DNA probes, with each probe carrying two HCR initiators. For multiplexed experiments, probe sets for different target mRNAs carry orthogonal initiators that trigger orthogonal DNA HCR amplification cascades labeled by spectrally distinct fluorophores. As a result, in situ amplification is performed for all targets simultaneously, and the duration of the experiment is independent of the number of target mRNAs. We have used multiplexed fluorescent in situ HCR and advanced imaging technologies to address questions of cell heterogeneity and tissue complexity in craniofacial patterning and anterior neural development. In the sample protocol presented here, we detect three different mRNA targets: Tg(egfp), encoding the enhanced green fluorescent protein (GFP) transgene (typically used as a control); Twist1, encoding a transcription factor involved in cell lineage determination and differentiation; and Pax2, encoding a transcription factor expressed in the mid-hindbrain region of the mouse embryo.


Subject(s)
Embryo, Mammalian , Gene Expression Profiling/methods , Gene Expression Regulation, Developmental , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Hybridization/methods , RNA, Messenger/analysis , Animals , Brain/embryology , Mice
6.
FEBS Open Bio ; 4: 266-75, 2014.
Article in English | MEDLINE | ID: mdl-24918038

ABSTRACT

Spermatozoa and lentiviruses are two of nature's most efficient gene delivery vehicles. Both can be genetically modified and used independently for the generation of transgenic animals or gene transfer/therapy of inherited disorders. Here we show that mature spermatozoa can be directly transduced with various pseudotyped lentiviral vectors and used in in vitro fertilisation studies. Lentiviral vectors encoding Green Fluorescent Protein (GFP) were shown to be efficiently processed and expressed in sperm. When these transduced sperm were used in in vitro fertilisation studies, GFP expression was observed in arising blastocysts. This simple technique of directly transducing spermatozoa has potential to be a powerful tool for the study of early and pre-implantation development and could be used as a technique in transgenic development and vertical viral transmission studies.

7.
FASEB J ; 28(2): 569-76, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24297703

ABSTRACT

Transgenic technologies conventionally rely on the oocyte as a substrate for genetic modification. Owing to their accessibility, however, male germ cells, including mature sperm, have material advantages for use in transgenesis. Here we have exploited lentiviruses to generate transgenic animals via the male germline. When pseudotyped lentiviral vectors encoding green fluorescent protein (GFP) were incubated with mouse spermatozoa, these sperm were highly successful in producing transgenics. Lentivirally transduced mouse spermatozoa were used in in vitro fertilization (IVF) studies, and when followed by embryo transfer, ≥ 42% of founders were found to be transgenic for GFP. Inverse PCR strategy for integration site analysis demonstrated integration of at least 1 or 2 copies of GFP in the transgenics, mapping to different chromosomes. GFP expression was detected in a wide range of murine tissues, including testis and the transgene was stably transmitted to a third generation of transgenic animals. This relatively simple, yet highly efficient, technique for generating transgenic animals by transducing spermatozoa with lentiviral vectors in vitro is a powerful tool for the study of fertilization/preimplantation development, vertical viral gene transmission, gene function and regulation, and epigenetic inheritance.


Subject(s)
Lentivirus/genetics , Spermatozoa/metabolism , Animals , Fertilization in Vitro , Germ Cells/metabolism , Male , Mice , Mice, Transgenic
8.
Invest Ophthalmol Vis Sci ; 49(11): 5055-9, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18566457

ABSTRACT

PURPOSE: To test the hypothesis that a novel phase-contrast optical coherence tomography (OCT) system can image retinal and choroidal vessels in the living mouse. METHODS: A high-speed spectral domain optical coherence tomography (SDOCT) system, which measures the reflections for the entire depth of the retina at once with each axial scan (A-scan), was developed for mouse retinal imaging. Acquiring multiple A-scans over a transverse line across the mouse retina offers a two-dimensional cross-sectional image (B-scan); several neighboring B-scans can be assembled into a three-dimensional OCT image. To visualize mobility and transverse flow in retinal vessels, the statistical variance of phase for each location was calculated from multiple B-scans acquired successively for the same retinal cross-section. Such measures of phase variance offer a direct measure of motions over a large dynamic range of flow velocities. RESULTS: Three-dimensional phase-contrast images of the live mouse retina were created using multiple two-dimensional cross-sectional image slices through the retina. For the data presented here, each cross-sectional phase-contrast slice resulted from five images of 100 or 200 transverse pixels, acquired over 25 ms or 50 ms, respectively. The approach offered clear identification of motion regions at different depths, including flow in the retinal microvasculature and in the choroidal vessels. CONCLUSIONS: Phase-contrast OCT enables three-dimensional visualization of retinal and choroidal vasculature in vivo.


Subject(s)
Blood Flow Velocity/physiology , Choroid/blood supply , Retina/anatomy & histology , Retinal Vessels/anatomy & histology , Tomography, Optical Coherence/methods , Animals , Choroid/physiology , Imaging, Three-Dimensional/methods , Mice , Reproducibility of Results , Retina/physiology , Retinal Vessels/physiology
9.
Asian J Androl ; 9(6): 739-50, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17968459

ABSTRACT

AIM: To analyze the functional interactions of Cyclin with p53 and Atm in spermatogenesis and DNA double-strand break repair. METHODS: Two lines of double knockout mice were generated. Spermatogenesis and double strand break repair mechanisms were analyzed in Cyclin A1 (Ccna1); p53- and Ccna1; Atm-double knockout mice. RESULTS: The block in spermatogenesis observed in Cyclin A1-/- (Ccna1-/-) testes at the mid-diplotene stage is associated with polynucleated giant cells. We found that Ccna1-deficient testes and especially the giant cells accumulate unrepaired DNA double-strand breaks, as detected by immunohistochemistry for phosphorylated H2AX. In addition, the giant cells escape from apoptosis. The development of giant cells occurred in meiotic prophase I, because testes lacking ATM, which are known to develop spermatogenic arrest earlier than prophase I, do not develop giant cells in the absence of cyclin A1. Cyclin A1 interacted with p53 and phosphorylated p53 in complex with CDK2. Interestingly, p53-deficiency significantly increased the number of giant cells in Ccna1-deficient testes. Gene expression analyses of a panel of DNA repair genes in the mutant testes revealed that none of the genes examined were consistently misregulated in the absence of cyclin A1. CONCLUSION: Ccna1-deficiency in spermatogenesis is associated with defects in DNA double-strand break repair, which is enhanced by loss of p53.


Subject(s)
Cell Cycle Proteins/genetics , Cyclin A/genetics , DNA-Binding Proteins/genetics , Protein Serine-Threonine Kinases/genetics , Spermatogenesis/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Proteins/genetics , Animals , Ataxia Telangiectasia Mutated Proteins , Cell Cycle/genetics , Cell Cycle/physiology , Cell Cycle Proteins/metabolism , Cyclin A/metabolism , Cyclin A1 , Cyclin B , Cyclin B2 , DNA/genetics , DNA Repair/genetics , DNA Repair/physiology , DNA-Binding Proteins/metabolism , Gene Expression Regulation/genetics , Gene Expression Regulation/physiology , Male , Mice , Mice, Knockout , Protein Serine-Threonine Kinases/metabolism , Spermatogenesis/physiology , Testis/cytology , Testis/metabolism , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/metabolism
10.
Neurochem Res ; 32(2): 159-65, 2007 Feb.
Article in English | MEDLINE | ID: mdl-16927171

ABSTRACT

Our objective was to follow the course of a dysmyelinating disease followed by partial recovery in transgenic mice using non-invasive high-resolution (117 x 117 x 70 microm) magnetic resonance (microMRI) and evoked potential of the visual system (VEP) techniques. We used JOE (for J37 golli overexpressing) transgenic mice engineered to overexpress golli J37, a product of the Golli-mbp gene complex, specifically in oligodendrocytes. Individual JOE transgenics and their unaffected siblings were followed from 21 until 75-days-old using non-invasive in vivo VEPs and 3D T2-weighted microMRI on an 11.7 T scanner, performing what we believe is the first longitudinal study of its kind. The microMRI data indicated clear, global hypomyelination during the period of peak myelination (21-42 days), which was partially corrected at later ages (>60 days) in the JOE mice compared to controls. These microMRI data correlated well with [Campagnoni AT (1995) "Molecular biology of myelination". In: Ransom B, Kettenmann H (eds) Neuroglia--a Treatise. Oxford University Press, London, pp 555-570] myelin staining, [Campagnoni AT, Macklin WB (1988) Cellular and molecular aspects of myelin protein gene-expression. Mol Neurobiol 2:41-89] a transient intention tremor during the peak period of myelination, which abated at later ages, and [Lees MB, Brostoff SW (1984) Proteins in myelin. In: Morell (ed) Myelin. Plenum Press, New York and London, pp 197-224] VEPs which all indicated a significant delay of CNS myelin development and persistent hypomyelination in JOE mice. Overall these non-invasive techniques are capable of spatially resolving the increase in myelination in the normally developing and developmentally delayed mouse brain.


Subject(s)
Evoked Potentials, Visual/physiology , Myelin Basic Protein/deficiency , Animals , Brain/growth & development , Central Nervous System Diseases/physiopathology , Longitudinal Studies , Magnetic Resonance Imaging , Mice , Mice, Neurologic Mutants , Mice, Transgenic , Nerve Tissue Proteins/physiology , Transcription Factors/physiology
11.
J Neurosci Res ; 84(8): 1716-26, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17044038

ABSTRACT

Visually evoked potentials (VEPs) and micro magnetic resonance imaging (micro MRI) are widely used as noninvasive techniques for diagnosis of central nervous system (CNS) diseases, especially myelin diseases, such as multiple sclerosis. Here we use these techniques in tandem to validate the in vivo data in mouse models. We used the shiverer mutant mouse, which has little or no CNS myelin, as a test model. These data show that shiverer (MBP(shi)/MBP(shi)) has a VEP latency that is 30% longer than that of its wild-type sibling. Surprisingly, the heterozygous (MBP(shi)/+) mouse, with apparently normal myelin, nevertheless has a 7% increase in its VEP latency vs. wild type. The micro MRIs of the same animals show that myelinated white matter is hypointense compared with gray matter as a result of the shorter T2 in myelinated regions of the CNS. T2-weighted images of wild-type and heterozygous shiverer mice show regions of hypointensity corresponding to the major myelinated tracts, including the optic nerve and the optic tract of the CNS, whereas shiverer mice have no regions of low intensity and therefore no detectable myelinated areas. In shiverer mice, micro MRI can discern hypomyelination throughout the brain, including the optic tract, and these changes correlate with longer VEP latencies. In addition, VEPs can also detect changes in the molecular make up of myelin that are not discernible with histology or micro MR. These data show the potential of using micro MRI in combination with VEPs to follow changes in both the quality and the quantity of myelin in vivo. These combined methods would be useful for longitudinal studies and therapy testing.


Subject(s)
Evoked Potentials, Visual/genetics , Magnetic Resonance Imaging , Mice, Neurologic Mutants , Myelin Basic Protein/deficiency , Animals , Evoked Potentials, Visual/physiology , Female , Immunohistochemistry/methods , Mice , Mice, Transgenic/genetics , Reaction Time/genetics
12.
Neuroimage ; 29(4): 1058-65, 2006 Feb 15.
Article in English | MEDLINE | ID: mdl-16213163

ABSTRACT

Shiverer is an important model of central nervous system dysmyelination characterized by a deletion in the gene encoding myelin basic protein with relevance to human dysmyelinating and demyelinating diseases. Perfusion fixed brains from shiverer mutant (C3Fe.SWV Mbp(shi)/Mbp(shi)n = 6) and background control (C3HeB.FeJ, n = 6) mice were compared using contrast enhanced volumetric diffusion tensor magnetic resonance microscopy with a nominal isotropic spatial resolution of 80 mum. Images were accurately coregistered using non-linear warping allowing voxel-wise statistical parametric mapping of tensor invariant differences between control and shiverer groups. Highly significant differences in the tensor trace and both the axial and radial diffusivity were observed within the major white matter tracts and in the thalamus, midbrain, brainstem and cerebellar white matter, consistent with a high density of myelinated axons within these regions. The fractional anisotropy was found to be much less sensitive than the trace and eigenvalues to dysmyelination and associated microanatomic changes.


Subject(s)
Brain/pathology , Diffusion Magnetic Resonance Imaging/statistics & numerical data , Hereditary Central Nervous System Demyelinating Diseases/pathology , Image Processing, Computer-Assisted/instrumentation , Mice, Neurologic Mutants/genetics , Myelin Sheath/pathology , Animals , Anisotropy , Bias , Blood-Brain Barrier/physiology , Brain Mapping , Hereditary Central Nervous System Demyelinating Diseases/genetics , Male , Mice , Mice, Congenic , Mice, Inbred C3H , Myelin Basic Protein , Nerve Tissue Proteins/genetics , Neural Pathways/pathology , Transcription Factors/genetics
13.
J Neurosci ; 25(30): 7004-13, 2005 Jul 27.
Article in English | MEDLINE | ID: mdl-16049176

ABSTRACT

The myelin basic protein (MBP) gene encodes two families of proteins, the classic MBP constituents of myelin and the golli-MBPs, the function of which is less well understood. In this study, targeted ablation of the golli-MBPs, but not the classic MBPs, resulted in a distinct phenotype unlike that of knock-outs (KOs) of the classic MBPs or other myelin proteins. Although the golli KO animals did not display an overt dysmyelinating phenotype, they did exhibit delayed and/or hypomyelination in selected areas of the brain, such as the visual cortex and the optic nerve, as determined by Northern and Western blots and immunohistochemical analysis with myelin protein markers. Hypomyelination in some areas, such as the visual cortex, persisted into adulthood. Ultrastructural analysis of the KOs confirmed both the delay and hypomyelination and revealed abnormalities in myelin structure and in some oligodendrocytes. Abnormal visual-evoked potentials indicated that the hypomyelination in the visual cortex had functional consequences in the golli KO brain. Evidence that the abnormal myelination in these animals was a consequence of intrinsic problems with the oligodendrocyte was indicated by an impaired ability of oligodendrocytes to form myelin sheets in culture and by the presence of abnormal Ca2+ transients in purified cortical oligodendrocytes studied in vitro. The Ca2+ results reported in this study complement previous results implicating golli proteins in modulating intracellular signaling in T-cells. Together, all these findings suggest a role for golli proteins in oligodendrocyte differentiation, migration, and/or myelin elaboration in the brain.


Subject(s)
Myelin Sheath/pathology , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/physiology , Oligodendroglia/pathology , Optic Nerve/pathology , Transcription Factors/genetics , Transcription Factors/physiology , Visual Cortex/pathology , Animals , Calcium/metabolism , Female , Homeostasis , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Microscopy, Electron , Myelin Basic Protein , Nerve Fibers, Myelinated/pathology , Nerve Fibers, Myelinated/ultrastructure , Oligodendroglia/metabolism
14.
Hum Reprod ; 20(1): 108-16, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15539445

ABSTRACT

BACKGROUND: A proliferation marker, proliferating cell nuclear antigen (PCNA), a Sertoli cell specific transcription factor, GATA-1 and the male germ cell specific, RNA binding motif (RBM), were used to identify different cellular populations during postnatal development of the mouse testis. METHODS: Immunohistochemistry, RT-PCR and real-time quantitative RT-PCR (QRT-PCR) were used. RESULTS: PCNA was expressed in pre-Sertoli and germ cells on the day of birth. Both pre-meiotic germ cells and spermatocytes expressed RBM throughout postnatal development. RBM-positive cell counts and QRT-PCR of RBM showed that average level of RBM per cell is highest in juvenile males between 14 and 21 days. From 42 days onward, there was a dramatic decrease in RBM expression in individual pre-meiotic and meiotic germ cells. CONCLUSIONS: These markers were used to correlate cell proliferative capability, gene expression profile and anatomic location within the developing mouse testis. The majority of germ cells start active proliferation once they have migrated to the basement membrane or immediately before. RBM is more highly expressed during the first wave of spermatogenesis versus subsequent waves, suggesting that there may be a change in the activity of RBM.


Subject(s)
Spermatozoa/metabolism , Testis/growth & development , Animals , Animals, Newborn , Base Sequence , Biomarkers/metabolism , DNA, Complementary/genetics , DNA-Binding Proteins/metabolism , Erythroid-Specific DNA-Binding Factors , GATA1 Transcription Factor , Gene Expression Regulation, Developmental , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Nuclear Proteins , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sertoli Cells/metabolism , Spermatogenesis/genetics , Spermatogenesis/physiology , Spermatozoa/cytology , Testis/cytology , Testis/metabolism , Transcription Factors/metabolism
15.
Magn Reson Imaging ; 21(8): 913-21, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14599542

ABSTRACT

Three dimensional Magnetic Resonance Imaging (MRI) datasets are becoming increasingly important in clinical and research applications because of their inherent signal to noise (SNR) advantages, high resolution and isotropic voxels. Despite SNR advantages, some 3D acquisitions may be SNR-limited, particularly in MR microscopy. Historically, both classic filtering and wavelet-based denoising techniques have been performed on a slice-by-slice basis. In principle, adaptive techniques such as best- basis wavelet-packet denoising might offer inherent advantages when performed in 3D, instead of 2D, by tracking through plane "structure" and suppressing noise "pseudostructure." This hypothesis was tested in 10 volumetric MR microscopy datasets from several different MR microscopy atlas projects. 3D wavelet-packet denoised images consistently yielded lower minimum mean-square error and subjectively perceived noise power than corresponding 2D denoised images using otherwise identical algorithms and parameters. MR microscopy researchers preferred the denoised images to the unprocessed images for their atlas projects.


Subject(s)
Imaging, Three-Dimensional , Magnetic Resonance Imaging/methods , Microscopy , Animals , Image Processing, Computer-Assisted , Mice , Quail
16.
Int J Mol Med ; 11(3): 311-5, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12579332

ABSTRACT

The human cyclin A1 gene is highly expressed in pachytene spermatocytes and is essential for spermatogenesis. To analyze mechanisms of cyclin A1 gene expression in vivo, we cloned a 1.3 kb fragment of the promoter upstream of the cDNA of enhanced green fluorescent protein (EGFP). Four lines of transgenic mice were generated that carried the transgene. Cyclin A1 promoter activity in the organs of the transgenic mice was analyzed using fluorescence microscopy and flow cytometry. Expression of EGFP was seen in male germ cells of all four murine lines. Spermatogonia at the basal membrane expressed low levels of EGFP, but bright green fluorescence was present in spermatocytes entering meiosis. Interestingly, a further sharp increase in EGFP expression was found in spermatocytes approximately at the stage of the first meiotic division. EGFP levels stayed high thereafter and EGFP was present in mature spermatozoa. A portion of c-kit expressing cells in the testis also expressed EGFP indicating cyclin A1 promoter activity in a subpopulation of spermatogonia. These data suggest that cyclin A1 is active not only in pachytene spermatocytes but also in earlier phases of spermatogenesis.


Subject(s)
Cyclin A/genetics , Cyclin A/metabolism , Spermatogenesis , Animals , Cyclin A1 , Gene Expression Regulation , Humans , Male , Meiosis , Mice , Mice, Transgenic , Promoter Regions, Genetic , Proto-Oncogene Proteins c-kit/metabolism , Spermatocytes/metabolism , Spermatogonia/metabolism , Spermatozoa/metabolism , Testis/cytology
17.
Reprod Biomed Online ; 4 Suppl 1: 52-7, 2002.
Article in English | MEDLINE | ID: mdl-12470336

ABSTRACT

The development of the mammalian germ line has been well studied, from the designation of primordial germ cells and their migration in the embryo to their progression through gametogenesis. The pattern of germ cell development, as established through classical studies, is now being overlaid with molecular, genetic and epigenetic data. Eventually, proteonomics will lead to a deeper understanding of the function of these genes. Through knowledge of germ cell gene expression patterns, it is now possible to develop transgenic molecular tools for the isolation of germ cells at different stages of development. By linking stage-specific germ cell promoter regions to the green fluorescent protein (GFP) reporter gene it is possible to tag these cells genetically for histological identification and cell sorting. Our long-term goal is to develop male germ cells as stem cells for therapeutic purposes. It is hoped that this goal will be achieved by purifying germ cells at different stages in development and gaining a deeper understanding of them by studying their gene expression patterns, potency and plasticity, both in vivo and in vitro.


Subject(s)
Genes/physiology , Spermatozoa/physiology , Animals , Cell Polarity , Gene Expression , Male , Meiosis/physiology , Zygote/physiology
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