scholarly journals Multiplex shRNA Screening of Germ Cell Development by in vivo Transfection of Mouse Testis

2016 ◽  
Author(s):  
Nicholas R. Y. Ho ◽  
Abul R. Usmani ◽  
Yan Yin ◽  
Liang Ma ◽  
Donald F. Conrad
Keyword(s):  
Germ Cell ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Gene Knockout ◽  
Mouse Testis ◽  
Separate Experiment ◽  
Great Promise ◽  
Cell Physiology ◽  
Functional Screening

AbstractSpermatozoa are one of the few mammalian cells types that cannot be fully derived in vitro, severely limiting the application of modern genomic techniques to study germ cell biology. The current gold standard approach of characterizing single gene knockout mice is slow as generation of each mutant line can take 6-9 months. Here, we describe an in vivo approach to rapid functional screening of germline genes based on a new non-surgical, non-viral in vivo transfection method to deliver nucleic acids into testicular germ cells. By coupling multiplex transfection of short hairpin RNA constructs with pooled amplicon sequencing as a readout, we were able to screen many genes for spermatogenesis function in a quick and inexpensive experiment. We transfected nine mouse testes with a pilot pool of RNAi against well-characterized genes to show that this system is highly reproducible and accurate. With a false negative rate of 18% and a false positive rate of 12%, this method has similar performance as other RNAi screens in the well-described Drosophila model system. In a separate experiment, we screened 26 uncharacterized genes computationally predicted to be essential for spermatogenesis and found numerous candidates for follow up studies. Further, by characterizing the effect of both libraries on neuronal N2a cells, we show that our screening results from testis are tissue-specific. Our calculations indicate that the current implementation of this approach could be used to screen thousands of protein-coding genes simultaneously in a single mouse testis. The experimental protocols and analysis scripts provided will enable other groups to use this procedure to study diverse aspects of germ cell biology ranging from epigenetics to cell physiology. This approach also has great promise as an applied tool for validating diagnoses made from medical genome sequencing, or designing synthetic biological sequences that act as potent and highly specific male contraceptives.

scholarly journals A cellular endolysosome-modulating pore-forming protein from a toad is negatively regulated by its paralog under oxidizing conditions

2020 ◽  
Vol 295 (30) ◽  
pp. 10293-10306 ◽  
Author(s):  
Qiquan Wang ◽  
Xianling Bian ◽  
Lin Zeng ◽  
Fei Pan ◽  
Lingzhen Liu ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Cell Biology ◽  
Biochemical Properties ◽  
Bacterial Toxin ◽  
Cell Physiology ◽  
Dependent Manner ◽  
Membrane Pore ◽  
Intracellular Vesicles

Endolysosomes are key players in cell physiology, including molecular exchange, immunity, and environmental adaptation. They are the molecular targets of some pore-forming aerolysin-like proteins (ALPs) that are widely distributed in animals and plants and are functionally related to bacterial toxin aerolysins. βγ-CAT is a complex of an ALP (BmALP1) and a trefoil factor (BmTFF3) in the firebelly toad (Bombina maxima). It is the first example of a secreted endogenous pore-forming protein that modulates the biochemical properties of endolysosomes by inducing pore formation in these intracellular vesicles. Here, using a large array of biochemical and cell biology methods, we report the identification of BmALP3, a paralog of BmALP1 that lacks membrane pore-forming capacity. We noted that both BmALP3 and BmALP1 contain a conserved cysteine in their C-terminal regions. BmALP3 was readily oxidized to a disulfide bond-linked homodimer, and this homodimer then oxidized BmALP1 via disulfide bond exchange, resulting in the dissociation of βγ-CAT subunits and the elimination of biological activity. Consistent with its behavior in vitro, BmALP3 sensed environmental oxygen tension in vivo, leading to modulation of βγ-CAT activity. Interestingly, we found that this C-terminal cysteine site is well conserved in numerous vertebrate ALPs. These findings uncover the existence of a regulatory ALP (BmALP3) that modulates the activity of an active ALP (BmALP1) in a redox-dependent manner, a property that differs from those of bacterial toxin aerolysins.

Society for Reproductive Biology Founders' Lecture 2007. Insights into germ cell biology: from the bench to the clinic

2007 ◽  
Vol 19 (7) ◽  
pp. 783 ◽  
Author(s):  
Angshumoy Roy ◽  
Martin M. Matzuk
Keyword(s):  
Germ Cell ◽  
Cell Biology ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Signalling Pathways ◽  
Male And Female ◽  
Paracrine Signalling ◽  
Fertility Clinic ◽  
Female Germ Cell

The germline is unique among tissues in being the only lineage that is transmitted through generations. The gonadal somatic cells that interact with male and female germ cells are equally important for their juxtacrine and paracrine signalling pathways that lead to the formation of functionally mature gametes and healthy progeny. The present review summarises exciting new studies that our group and others have achieved at the frontier of male and female germ cell biology and in studying transforming growth factor-β signalling pathways in oocyte–somatic cell interactions and gonadal growth and differentiation. In the process, we have produced over 70 transgenic and knockout models to study reproduction in vivo. These models have helped us identify novel and unexplored areas of germ cell biology and translate this work into the fertility clinic.

Micro- and Nanofluidics for Cell Biology, Cell Therapy, and Cell-Based Drug Testing

2009 ◽  
Author(s):  
Shuichi Takayama ◽  
Dongeun Huh ◽  
Jonathan Song ◽  
Wansik Cha ◽  
Yunseok Heo
Keyword(s):  
Cell Biology ◽  
Mammalian Cells ◽  
Cancer Metastasis ◽  
Dna Analysis ◽  
The Body ◽  
Biological Information ◽  
Screening Methods ◽  
Biological Studies

Many biological studies, drug screening methods, and cellular therapies require culture and manipulation of living cells outside of their natural environment in the body. The gap between the cellular microenvironment in vivo and in vitro, however, poses challenges for obtaining physiologically relevant responses from cells used in basic biological studies or drug screens and for drawing out the maximum functional potential from cells used therapeutically. One of the reasons for this gap is because the fluidic environment of mammalian cells in vivo is microscale and dynamic whereas typical in vitro cultures are macroscopic and static. This presentation will give an overview of efforts in our laboratory to develop microfluidic systems that enable spatio-temporal control of both the chemical and fluid mechanical environment of cells. The technologies and methods close the physiology gap to provide biological information otherwise unobtainable and to enhance cellular performance in therapeutic applications. Specific biomedical topics that will be discussed include, in vitro fertilization on a chip, microfluidic tissue engineering of small airway injuries, breast cancer metastasis on a chip, electrochemical biosensors, and development of tuneable nanofluidic systems towards applications in single molecule DNA analysis.

scholarly journals Increased expression of Polδ does not alter the canonical replication program in vivo

2021 ◽  
Vol 6 ◽  
pp. 44
Author(s):  
Róbert Zach ◽  
Antony M. Carr
Keyword(s):  
Cell Biology ◽  
Rare Event ◽  
Replication Initiation ◽  
Cell Physiology ◽  
Moderate Increase ◽  
Sequencing Analysis ◽  
Phase Duration ◽  
Leading Strand ◽  
The Impact

Background: In vitro experiments utilising the reconstituted Saccharomyces cerevisiae eukaryotic replisome indicated that the efficiency of the leading strand replication is impaired by a moderate increase in Polδ concentration. It was hypothesised that the slower rate of the leading strand synthesis characteristic for reactions containing two-fold and four-fold increased concentration of Polδ represented a consequence of a relatively rare event, during which Polδ stochastically outcompeted Polε and, in an inefficient manner, temporarily facilitated extension of the leading strand. Inspired by this observation, we aimed to determine whether similarly increased Polδ levels influence replication dynamics in vivo using the fission yeast Schizosaccharomyces pombe as a model system. Methods: To generate S. pombe strains over-expressing Polδ, we utilised Cre-Lox mediated cassette exchange and integrated one or three extra genomic copies of all four Polδ genes. To estimate expression of respective Polδ genes in Polδ-overexpressing mutants, we measured relative transcript levels of cdc1+, cdc6+ (or cdc6L591G), cdc27+ and cdm1+ by reverse transcription followed by quantitative PCR (RT-qPCR). To assess the impact of Polδ over-expression on cell physiology and replication dynamics, we used standard cell biology techniques and polymerase usage sequencing. Results: We provide an evidence that two-fold and four-fold over-production of Polδ does not significantly alter growth rate, cellular morphology and S-phase duration. Polymerase usage sequencing analysis further indicates that increased Polδ expression does not change activities of Polδ, Polε and Polα at replication initiation sites and across replication termination zones. Additionally, we show that mutants over-expressing Polδ preserve WT-like distribution of replication origin efficiencies. Conclusions: Our experiments do not disprove the existence of opportunistic polymerase switches; however, the data indicate that, if stochastic replacement of Polε for Polδ does occur in vivo, it represents a rare phenomenon that does not significantly influence canonical replication program.

scholarly journals Bring it back, bring it back, don't take it away from me – the sorting receptor RER1

2020 ◽  
Vol 133 (17) ◽  
pp. jcs231423
Author(s):  
Wim Annaert ◽  
Christoph Kaether
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Future Directions ◽  
Binding Motifs ◽  
Sorting Receptor ◽  
Intermediate Compartment ◽  
The Brain

ABSTRACTThe quote “bring it back, bring it back, don't take it away from me” from Queen's Love of my life describes the function of the sorting receptor RER1, a 23 kDa protein with four transmembrane domains (TMDs) that localizes to the intermediate compartment and the cis-Golgi. From there it returns escaped proteins that are not supposed to leave the endoplasmic reticulum (ER) back to it. Unique about RER1 is its ability to recognize its ligands through binding motifs in TMDs. Among its substrates are ER-resident proteins, as well as unassembled subunits of multimeric complexes that are retrieved back into the ER, this way guarding the full assembly of their respective complexes. The basic mechanisms for RER1-dependent retrieval have been already elucidated some years ago in yeast. More recently, several important cargoes of RER1 have been described in mammalian cells, and the in vivo role of RER1 is being unveiled by using mouse models. In this Review, we give an overview of the cell biology of RER1 in different models, discuss its controversial role in the brain and provide an outlook on future directions for RER1 research.

Cell Junction Dynamics in the Testis: Sertoli-Germ Cell Interactions and Male Contraceptive Development

2002 ◽  
Vol 82 (4) ◽  
pp. 825-874 ◽  
Author(s):  
C. Yan Cheng ◽  
Dolores D. Mruk
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Cell Biology ◽  
Cell Communication ◽  
Seminiferous Epithelium ◽  
Cell Junction ◽  
In Vivo Models ◽  
Blood Testis Barrier

Spermatogenesis is an intriguing but complicated biological process. However, many studies since the 1960s have focused either on the hormonal events of the hypothalamus-pituitary-testicular axis or morphological events that take place in the seminiferous epithelium. Recent advances in biochemistry, cell biology, and molecular biology have shifted attention to understanding some of the key events that regulate spermatogenesis, such as germ cell apoptosis, cell cycle regulation, Sertoli-germ cell communication, and junction dynamics. In this review, we discuss the physiology and biology of junction dynamics in the testis, in particular how these events affect interactions of Sertoli and germ cells in the seminiferous epithelium behind the blood-testis barrier. We also discuss how these events regulate the opening and closing of the blood-testis barrier to permit the timely passage of preleptotene and leptotene spermatocytes across the blood-testis barrier. This is physiologically important since developing germ cells must translocate across the blood-testis barrier as well as traverse the seminiferous epithelium during their development. We also discuss several available in vitro and in vivo models that can be used to study Sertoli-germ cell anchoring junctions and Sertoli-Sertoli tight junctions. An in-depth survey in this subject has also identified several potential targets to be tackled to perturb spermatogenesis, which will likely lead to the development of novel male contraceptives.

scholarly journals Early Postnatal Death and Motor Disorders in Mice Congenitally Deficient in Calnexin Expression

2002 ◽  
Vol 22 (21) ◽  
pp. 7398-7404 ◽  
Author(s):  
Angela Denzel ◽  
Maurizio Molinari ◽  
Cesar Trigueros ◽  
Joanne E. Martin ◽  
Shanti Velmurgan ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Gene Knockout ◽  
Nerve Fibers ◽  
Motor Disorders ◽  
Type I ◽  
Myelinated Nerve ◽  
Gene Knockout Mice ◽  
Deficient Mice ◽  
Insight Into

ABSTRACT Calnexin is a ubiquitously expressed type I membrane protein which is exclusively localized in the endoplasmic reticulum (ER). In mammalian cells, calnexin functions as a chaperone molecule and plays a key role in glycoprotein folding and quality control within the ER by interacting with folding intermediates via their monoglucosylated glycans. In order to gain more insight into the physiological roles of calnexin, we have generated calnexin gene-deficient mice. Despite its profound involvement in protein folding, calnexin is not essential for mammalian-cell viability in vivo: calnexin gene knockout mice were carried to full term, although 50% died within 48 h and the majority of the remaining mice had to be sacrificed within 4 weeks, with only a very few mice surviving to 3 months. Calnexin gene-deficient mice were smaller than their littermates and showed very obvious motor disorders, associated with a dramatic loss of large myelinated nerve fibers. Thus, the critical contribution of calnexin to mammalian physiology is tissue specific.

233. A role for activin/inhibin in mouse gonocyte relocation and proliferation

2008 ◽  
Vol 20 (9) ◽  
pp. 33
Author(s):  
L. Tubino ◽  
B. Barakat ◽  
S. Prahbu ◽  
S. Meachem ◽  
A. Nagaraja ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Germ Cell ◽  
Cell Development ◽  
Tumour Suppressor Gene ◽  
Mouse Testis ◽  
Nuclear Antigen ◽  
Spatiotemporal Pattern ◽  
Newborn Mouse

Gonocytes in the testis resume proliferation after birth and relocate to contact the basement membrane of the seminiferous cords where they become spermatogonia. A previous in vitro study indicated that activin can increase gonocyte numbers on day 3 postpartum (dpp) rat testis, while the activin antagonist, follistatin, together with FSH, increased the number of spermatogonia (Meehan et al. 2000). The aim of this study was to understand how FSH, activin and inhibin, a potent activin antagonist, interact to influence gonocyte proliferation and relocation in the newborn mouse testis using in vivo and in vitro approaches. Two mouse models were analysed, the inhibin α knock out (inh a −/−) mouse and the InhbaBK mouse. The Inh a −/− mouse lacks inhibin, and thus activin acts unopposed by its most potent antagonist (Matzuk et al. 1992). The InhbaBK mouse has the Inhbb allele inserted into the Inhba locus, thus directing the expression of the less bioactive activin βB, in the spatiotemporal pattern of activin βA (Brown et al. 2000). In addition, an in vitro model was developed in which 1dpp wild type testis fragments were cultured in hanging drops for 24 h with the addition of combinations of activin, inhibin and FSH. Gonocyte proliferation in inh a −/− was assessed using proliferating cell nuclear antigen (PCNA). A significant increase in germ cell proliferation and relocation to the basement membrane was measured in 0dpp inh a −/−, while no difference was observed at 4dpp. The opposite was observed in InhbaBK mice, with reduced gonocyte migration in mutant animals at 0dpp. In vitro, inhibin seemed to inhibit proliferation and reduce the percentage of relocated gonocytes while FSH showed a tendency for the opposite effect on gonocyte migration. These findings show that inhibin levels affect germ cell development during early postnatal development in mouse testis influencing both cell maturation and proliferation. (1) Meehan T, Schlatt S, O'Bryan MK, de Kretser DM, Loveland KL. Regulation of germ cell and Sertoli cell development by activin, follistatin, and FSH. Dev Biol. 2000 Apr 15;220(2):225–37. (2) Matzuk MM, Finegold MJ, Su JG, Hsueh AJ, Bradley A. Alpha-inhibin is a tumour-suppressor gene with gonadal specificity in mice. Nature. 1992 Nov 26;360(6402):313–9. (3) Brown CW, Houston-Hawkins DE, Woodruff TK, Matzuk MM. Insertion of Inhbb into the Inhba locus rescues the Inhba null phenotype and reveals new activin functions. Nat Genet. 2000 Aug;25(4):453–7.

scholarly journals Human UDP-galactose 4′-epimerase (GALE) is required for cell-surface glycome structure and function

2019 ◽  
Vol 295 (5) ◽  
pp. 1225-1239
Author(s):  
Alex Broussard ◽  
Alyssa Florwick ◽  
Chelsea Desbiens ◽  
Nicole Nischan ◽  
Corrina Robertson ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Antigen ◽  
Mammalian Cells ◽  
Death Receptor ◽  
Cell Physiology ◽  
Total Sialic Acid ◽  
Death Receptor Signaling ◽  
Induced Apoptosis ◽  
And Function

Glycan biosynthesis relies on nucleotide sugars (NSs), abundant metabolites that serve as monosaccharide donors for glycosyltransferases. In vivo, signal-dependent fluctuations in NS levels are required to maintain normal cell physiology and are dysregulated in disease. However, how mammalian cells regulate NS levels and pathway flux remains largely uncharacterized. To address this knowledge gap, here we examined UDP-galactose 4′-epimerase (GALE), which interconverts two pairs of essential NSs. Using immunoblotting, flow cytometry, and LC-MS–based glycolipid and glycan profiling, we found that CRISPR/Cas9-mediated GALE deletion in human cells triggers major imbalances in NSs and dramatic changes in glycolipids and glycoproteins, including a subset of integrins and the cell-surface death receptor FS-7-associated surface antigen. In particular, we observed substantial decreases in total sialic acid, galactose, and GalNAc levels in glycans. These changes also directly impacted cell signaling, as GALE−/− cells exhibited FS-7-associated surface antigen ligand-induced apoptosis. Our results reveal a role of GALE-mediated NS regulation in death receptor signaling and may have implications for the molecular etiology of illnesses characterized by NS imbalances, including galactosemia and metabolic syndrome.

scholarly journals Identification of the Functions of Liver X Receptor-β in Sertoli Cells Using a Targeted Expression-Rescue Model

Endocrinology ◽  
2015 ◽  
Vol 156 (12) ◽  
pp. 4545-4557 ◽  
Author(s):  
Salwan Maqdasy ◽  
Fatim-Zohra El Hajjaji ◽  
Marine Baptissart ◽  
Emilie Viennois ◽  
Abdelkader Oumeddour ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Germ Cell ◽  
Germ Cells ◽  
Sertoli Cells ◽  
Lipid Accumulation ◽  
Smooth Muscle Actin ◽  
Lipid Homeostasis ◽  
Seminiferous Tubules ◽  
Cell Physiology

Liver X receptors (LXRs) are key regulators of lipid homeostasis and are involved in multiple testicular functions. The Lxrα−/−;Lxrβ−/− mice have illuminated the roles of both isoforms in maintenance of the epithelium in the seminiferous tubules, spermatogenesis, and T production. The requirement for LXRβ in Sertoli cells have been emphasized by early abnormal cholesteryl ester accumulation in the Lxrβ−/− and Lxrα−/−;Lxrβ−/− mice. Other phenotypes, such as germ cell loss and hypogonadism, occur later in life in the Lxrα−/−;Lxrβ−/− mice. Thus, LXRβ expression in Sertoli cells seems to be essential for normal testicular physiology. To decipher the roles of LXRβ within the Sertoli cells, we generated Lxrα−/−;Lxrβ−/−:AMH-Lxrβ transgenic mice, which reexpress Lxrβ in Sertoli cells in the context of Lxrα−/−;Lxrβ−/− mice. In addition to lipid homeostasis, LXRβ is necessary for maintaining the blood-testis barrier and the integrity of the germ cell epithelium. LXRβ is also implicated in the paracrine action of Sertoli cells on Leydig cells to modulate T synthesis. The Lxrα−/−;Lxrβ−/− and Lxrα−/−;Lxrβ−/−:AMH-Lxrβ mice exhibit lipid accumulation in germ cells after the Abcg8 down-regulation, suggesting an intricate LXRβ-dependent cooperation between the Sertoli cells and germ cells to ensure spermiogenesis. Further analysis revealed also peritubular smooth muscle defects (abnormal lipid accumulation and disorganized smooth muscle actin) and spermatozoa stagnation in the seminiferous tubules. Together the present work elucidates specific roles of LXRβ in Sertoli cell physiology in vivo beyond lipid homeostasis.

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