CatSper-null mutant spermatozoa are unable to ascend beyond the oviductal reservoir

2009 ◽  
Vol 21 (2) ◽  
pp. 345 ◽  
Author(s):  
Katharine Ho ◽  
Collin A. Wolff ◽  
Susan S. Suarez
Keyword(s):  
Zona Pellucida ◽  
High Amplitude ◽  
Null Mouse ◽  
Null Mutant ◽  
Wild Type ◽  
Timely Manner ◽  
Oviductal Epithelium ◽  
Sperm Reservoir

Sperm hyperactivation is characterised by high-amplitude, asymmetrical flagellar bending and is required to penetrate the oocyte zona pellucida. It was proposed that hyperactivation also enables spermatozoa to reach the oocyte by assisting escape from the oviductal sperm reservoir. To test this hypothesis, the behaviour of CatSper-null mouse spermatozoa in the oviduct was compared with that of spermatozoa from heterozygotes. CatSper–/– males are infertile because their spermatozoa fail to hyperactivate, whereas spermatozoa from CatSper+/– males have normal amounts of CatSper proteins and can hyperactivate. Males were mated with wild-type females on the morning of ovulation. Oviducts were obtained 1 or 4 h later, and behaviour of spermatozoa was examined using transillumination. At 1 h, null mutant spermatozoa remained attached by their heads to oviductal epithelium in the reservoir, whereas spermatozoa from heterozygotes detached from the oviductal epithelium after performing deep asymmetrical flagellar bends. At 4 h, 50 to 200 CatSper+/– spermatozoa were still seen in the oviducts; in contrast, only one CatSper–/– spermatozoon was found. CatSper–/– spermatozoa were lost from the oviducts after failing to detach from the epithelium in a timely manner, thus demonstrating that hyperactivation is required by spermatozoa to ascend beyond the oviductal reservoir.

scholarly journals Haploid induction by a maize cenh3 null mutant

2021 ◽  
Vol 7 (4) ◽  
pp. eabe2299 ◽  
Author(s):  
Na Wang ◽  
Jonathan I. Gent ◽  
R. Kelly Dawe
Keyword(s):  
Histone H3 ◽  
Null Mutation ◽  
Null Mutant ◽  
Wild Type ◽  
Haploid Induction ◽  
Cell Divisions ◽  
Gamete Formation ◽  
Simplified Method ◽  
Single Cross ◽  
Subsequent Loss

The production of haploids is an important first step in creating many new plant varieties. One approach used in Arabidopsis involves crossing plants expressing different forms of centromeric histone H3 (CENP-A/CENH3) and subsequent loss of genome with weaker centromeres. However, the method has been ineffective in crop plants. Here, we describe a greatly simplified method based on crossing maize lines that are heterozygous for a cenh3 null mutation. Crossing +/cenh3 to wild-type plants in both directions yielded haploid progeny. Genome elimination was determined by the cenh3 genotype of the gametophyte, suggesting that centromere failure is caused by CENH3 dilution during the postmeiotic cell divisions that precede gamete formation. The cenh3 haploid inducer works as a vigorous hybrid and can be transferred to other lines in a single cross, making it versatile for a variety of applications.

scholarly journals CodY Is a Global Regulator of Virulence-Associated Properties for Clostridium perfringens Type D Strain CN3718

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Jihong Li ◽  
Menglin Ma ◽  
Mahfuzur R. Sarker ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Null Mutant ◽  
Intestinal Infection ◽  
Wild Type ◽  
Global Regulator ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Type D ◽  
Epsilon Toxin ◽  
Virulence Properties

ABSTRACT CodY is known to regulate various virulence properties in several Gram-positive bacteria but has not yet been studied in the important histotoxic and intestinal pathogen Clostridium perfringens. The present study prepared an isogenic codY-null mutant in C. perfringens type D strain CN3718 by insertional mutagenesis using the Targetron system. Western blot analysis indicated that, relative to wild-type CN3718 or a complementing strain, this isogenic codY mutant produces reduced levels of epsilon toxin (ETX). Using supernatants from cultures of the wild-type, codY-null mutant, and complementing strains, CodY regulation of ETX production was shown to have cytotoxic consequences for MDCK cells. The CodY regulatory effect on ETX production was specific, since the codY-null mutant still made wild-type levels of alpha-toxin and perfringolysin O. Sialidase activity measurements and sialidase Western blot analysis of supernatants from CN3718 and its isogenic derivatives showed that CodY represses overall exosialidase activity due to a reduced presence of NanH in culture supernatants. Inactivation of the codY gene significantly decreased the adherence of CN3718 vegetative cells or spores to host Caco-2 cells. Finally, the codY mutant showed increased spore formation under vegetative growth conditions, although germination of these spores was impaired. Overall, these results identify CodY as a global regulator of many C. perfringens virulence-associated properties. Furthermore, they establish that, via CodY, CN3718 coordinately regulates many virulence-associated properties likely needed for intestinal infection. IMPORTANCE Clostridium perfringens is a major human and livestock pathogen because it produces many potent toxins. C. perfringens type D strains cause intestinal infections by producing toxins, especially epsilon toxin (ETX). Previous studies identified CodY as a regulator of certain virulence properties in other Gram-positive bacteria. Our study now demonstrates that CodY is a global regulator of virulence-associated properties for type D strain CN3718. It promotes production of ETX, attachment of CN3718 vegetative cells or spores to host enterocyte-like Caco-2 cells, and spore germination; the last two effects may assist intestinal colonization. In contrast, CodY represses sporulation. These results provide the first evidence that CodY can function as a global regulator of C. perfringens virulence-associated properties and that this strain coordinately regulates its virulence-associated properties using CodY to increase ETX production, host cell attachment, and spore germination but to repress sporulation, as would be optimal during type D intestinal infection.

Defective zonae pellucidae in Zp2-null mice disrupt folliculogenesis, fertility and development

Development ◽  
2001 ◽  
Vol 128 (7) ◽  
pp. 1119-1126 ◽  
Author(s):  
T.L. Rankin ◽  
M. O'Brien ◽  
E. Lee ◽  
K. Wigglesworth ◽  
J. Eppig ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Embryonic Stem ◽  
Single Copy ◽  
Targeted Mutagenesis ◽  
Developmental Competence ◽  
Normal Male ◽  
Null Mouse ◽  
Developmental Potential ◽  
Early Embryos

All vertebrate eggs are surrounded by an extracellular matrix. This matrix is known as the zona pellucida in mammals and is critically important for the survival of growing oocytes, successful fertilization and the passage of early embryos through the oviduct. The mouse zona pellucida is composed of three glycoproteins (ZP1, ZP2 and ZP3), each encoded by a single copy gene. Using targeted mutagenesis in embryonic stem cells, Zp2-null mouse lines have been established. ZP1 and ZP3 proteins continue to be synthesized and form a thin zona matrix in early follicles that is not sustained in pre-ovulatory follicles. The abnormal zona matrix does not affect initial folliculogenesis, but there is a significant decrease in the number of antral stage follicles in ovaries isolated from mice lacking a zona pellucida. Few eggs are detected in the oviduct after stimulation with gonadotropins, and no two-cell embryos are recovered after mating Zp2-null females with normal male mice. The structural defect is more severe than that observed in Zp1-null mice, which have decreased fecundity, but not quite as severe as that observed in Zp3-null mice, which never form a visible zona pellucida and are sterile. Although zona-free oocytes matured and fertilized in vitro can progress to the blastocyst stage, the developmental potential of blastocysts derived from either Zp2- or Zp3-null eggs appears compromised and, after transfer to foster mothers, live births have not been observed. Thus, in addition to its role in fertilization and protection of early embryos, these data are consistent with the zona pellucida maintaining interactions between granulosa cells and oocytes during folliculogenesis that are critical to maximize developmental competence of oocytes.

scholarly journals The sperm protein SPACA4 is required for efficient fertilization in mice

2021 ◽  
Author(s):  
Sarah Herberg ◽  
Yoshitaka Fujihara ◽  
Andreas Blaha ◽  
Karin Panser ◽  
Kiyonari Kobayashi ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Knockout Mice ◽  
Mammalian Species ◽  
Wild Type ◽  
Sperm Protein ◽  
Mammalian Sperm ◽  
Fundamental Process ◽  
Mammalian Fertilization

Fertilization is the fundamental process that initiates the development of a new individual in all sexually reproducing species. Despite its importance, our understanding of the molecular players that govern mammalian sperm-egg interaction is incomplete, partly because many of the essential factors found in non-mammalian species do not have obvious mammalian homologs. We have recently identified the Ly6/uPAR protein Bouncer as a new, essential fertilization factor in zebrafish (Herberg et al., 2018). Here, we show that Bouncer's homolog in mammals, SPACA4, is also required for efficient fertilization in mice. In contrast to fish, where Bouncer is expressed specifically in the egg, SPACA4 is expressed exclusively in the testis. Male knockout mice are severely sub-fertile, and sperm lacking SPACA4 fail to fertilize wild-type eggs in vitro. Interestingly, removal of the zona pellucida rescues the fertilization defect of Spaca4-deficient sperm in vitro, indicating that SPACA4 is not required for the interaction of sperm and the oolemma but rather of sperm and zona pellucida. Our work identifies SPACA4 as an important sperm protein necessary for zona pellucida penetration during mammalian fertilization.

scholarly journals Methylglyoxal detoxification by an aldo-keto reductase in the cyanobacterium Synechococcus sp. PCC 7002

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2013-2021 ◽  
Author(s):  
Dongyi Xu ◽  
Xianwei Liu ◽  
Cong Guo ◽  
Jindong Zhao
Keyword(s):  
Enzyme Activity ◽  
Carbon Source ◽  
Toxic Metabolite ◽  
Heterotrophic Growth ◽  
Null Mutant ◽  
Wild Type ◽  
Activity Profile ◽  
Aldehydes And Ketones ◽  
Synechococcus Sp ◽  
Ph Dependent

Aldo-keto reductases (AKRs) are a superfamily of enzymes that reduce aldehydes and ketones, and have a broad range of substrates. An AKR gene, sakR1, was identified in the cyanobacterium Synechococcus sp. PCC 7002. A mutant strain with sakR1 inactivated was sensitive to glycerol, a carbon source that can support heterotrophic growth of Synechococcus sp. PCC 7002. It was found that the sakR1 null mutant accumulated more toxic methylglyoxal than the wild-type when glycerol was added to growth medium, suggesting that SakR1 is involved in the detoxification of methylglyoxal, a highly toxic metabolite that can damage cellular macromolecules. Enzymic analysis of recombinant SakR1 protein showed that it can efficiently reduce methylglyoxal with NADPH. Based on immunoblotting, SakR1 was not upregulated at an increased cellular methylglyoxal concentration. A pH-dependent enzyme-activity profile suggested that SakR1 activity could be regulated by cellular pH in Synechococcus sp. PCC 7002. The broad substrate specificity of SakR1 implies that SakR1 could play other roles in cellular metabolism.

scholarly journals Wild-Type Circadian Rhythmicity Is Dependent on Closely Spaced E Boxes in the Drosophila timelessPromoter

2001 ◽  
Vol 21 (4) ◽  
pp. 1207-1217 ◽  
Author(s):  
Michael J. McDonald ◽  
Michael Rosbash ◽  
Patrick Emery
Keyword(s):  
Transcription Factor ◽  
Transcriptional Regulation ◽  
Locomotor Activity ◽  
Circadian Rhythms ◽  
High Amplitude ◽  
Wild Type ◽  
Activity Rhythms ◽  
E Box ◽  
E Boxes ◽  
Circadian Transcription

ABSTRACT Transcriptional regulation plays an important role inDrosophila melanogaster circadian rhythms. The period promoter has been well studied, but the timeless promoter has not been analyzed in detail. Mutagenesis of the canonical E box in the timelesspromoter reduces but does not eliminate timeless mRNA cycling or locomotor activity rhythms. This is because there are at least two other cis-acting elements close to the canonical E box, which can also be transactivated by the circadian transcription factor dCLOCK. These E-box-like sequences cooperate with the canonical E-box element to promote high-amplitude transcription, which is necessary for wild-type rhythmicity.

Evidence for increased myofibrillar mobility in desmin-null mouse skeletal muscle

2002 ◽  
Vol 205 (3) ◽  
pp. 321-325
Author(s):  
Sameer B. Shah ◽  
Fong-Chin Su ◽  
Kimberly Jordan ◽  
Derek J. Milner ◽  
Jan Fridén ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Quantitative Estimation ◽  
Horizontal Displacement ◽  
Radial Force ◽  
Null Mouse ◽  
Force Transmission ◽  
Wild Type ◽  
Functional Roles ◽  
Fifth Toe ◽  
Knockout Animals

SUMMARY Quantitative electron microscopy was used to characterize the longitudinal mobility of myofibrils during muscle extension to investigate the functional roles of skeletal muscle intermediate filaments. Extensor digitorum longus fifth toe muscles from wild-type (+/+) and desmin-null (des –/–) animals were passively stretched to varying lengths, and the horizontal displacement of adjacent Z-disks in neighboring myofibrils (Δxmyo) and average sarcomere length (SL) were calculated. At short SL (<2.20 μm), wild-type and desmin-null Δxmyo were not significantly different, although there was a trend towards greater Z-disk misalignment in muscles from knockout animals (Δxmyo 0.34±0.04 μm versus 0.22±0.09 μm; P>0.2; means ± s.e.m.). However, at higher SL (>2.90 μm), muscles from knockout animals displayed a dramatically increased Δxmyo relative to wild-type muscles (0.49±0.10 μm versus 0.25±0.07 μm; P<0.05). The results, which establish a maximum extension of the desmin network surrounding the Z-disk, provide what we believe to be the first quantitative estimation of the functional limits of the desmin intermediate filament system in the presence of an intact myofibrillar lattice. The existence of a limit on the extension of desmin suggests a mechanism for the recruitment of desmin into a network of force transmission, whether as a longitudinal load bearer or as a component in a radial force-transmission system.

scholarly journals Muscles of mice deficient in α-sarcoglycan maintain large masses and near control force values throughout the life span

2005 ◽  
Vol 22 (2) ◽  
pp. 244-256 ◽  
Author(s):  
Christina M. Consolino ◽  
Franck Duclos ◽  
Jane Lee ◽  
Roger A. Williamson ◽  
Kevin P. Campbell ◽  
...  
Keyword(s):  
Connective Tissue ◽  
Life Span ◽  
Structure And Function ◽  
Null Mouse ◽  
Control Force ◽  
Wild Type ◽  
Cross Sectional ◽  
Muscle Structure ◽  
And Function

α-Sarcoglycan-deficient ( Sgca-null) mice provide potential for elucidating the pathogenesis of limb girdle muscular dystrophy type 2D (LGMD 2D) as well as for studying the effectiveness of therapeutic strategies. Skeletal muscles of Sgca-null mice demonstrate an early onset of extensive fiber necrosis, degeneration, and regeneration, but the progression of the pathology and the effects on muscle structure and function throughout the life span are not known. Thus the phenotypic accuracy of the Sgca-null mouse as a model of LGMD 2D has not been fully established. To investigate skeletal muscle structure and function in the absence of α-sarcoglycan throughout the life span, we analyzed extensor digitorum longus and soleus muscles of male and female Sgca-null and wild-type mice at 3, 6, 12, and 18 mo of age. Maximum isometric forces and powers were measured in vitro at 25°C. Also determined were individual myofiber cross-sectional areas and numbers, water content, and the proportion of the cross section occupied by connective tissue. Muscle masses were 40–100% larger for Sgca-null compared with age- and gender-matched wild-type mice, with the majority of the increased muscle mass for Sgca-null mice attributable to greater connective tissue and water contents. Although the greater mass of muscles in Sgca-null mice was primarily noncontractile material, absolute forces and powers were maintained near control levels at all ages, indicating a successful adaptation to the deficiency in α-sarcoglycan not observed at any age in LGMD 2D patients.

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