scholarly journals MALDI-IMS combined with shotgun proteomics identify and localize new factors in male infertility

2021 ◽  
Vol 4 (3) ◽  
pp. e202000672
Author(s):  
Shibojyoti Lahiri ◽  
Wasim Aftab ◽  
Lena Walenta ◽  
Leena Strauss ◽  
Matti Poutanen ◽  
...  
Keyword(s):  
Male Infertility ◽  
Imaging Mass Spectrometry ◽  
Shotgun Proteomics ◽  
Cell Types ◽  
The Novel ◽  
Proteomic Approach ◽  
Maldi Ims ◽  
Tissue Proteomics ◽  
Different Cell Types

Spermatogenesis is a complex multi-step process involving intricate interactions between different cell types in the male testis. Disruption of these interactions results in infertility. Combination of shotgun tissue proteomics with MALDI imaging mass spectrometry is markedly potent in revealing topological maps of molecular processes within tissues. Here, we use a combinatorial approach on a characterized mouse model of hormone induced male infertility to uncover misregulated pathways. Comparative testicular proteome of wild-type and mice overexpressing human P450 aromatase (AROM+) with pathologically increased estrogen levels unravels gross dysregulation of spermatogenesis and emergence of pro-inflammatory pathways in AROM+ testis. In situ MS allowed us to localize misregulated proteins/peptides to defined regions within the testis. Results suggest that infertility is associated with substantial loss of proteomic heterogeneity, which define distinct stages of seminiferous tubuli in healthy animals. Importantly, considerable loss of mitochondrial factors, proteins associated with late stages of spermatogenesis and steroidogenic factors characterize AROM+ mice. Thus, the novel proteomic approach pinpoints in unprecedented ways the disruption of normal processes in testis and provides a signature for male infertility.

scholarly journals Imaging mass spectrometry and shotgun proteomics reveal dysregulated pathways in hormone induced male infertility

2020 ◽  
Author(s):  
Shibojyoti Lahiri ◽  
Lena Walenta ◽  
Wasim Aftab ◽  
Leena Strauss ◽  
Matti Poutanen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Male Infertility ◽  
Imaging Mass Spectrometry ◽  
Shotgun Proteomics ◽  
Cell Types ◽  
The Novel ◽  
Proteomic Approach ◽  
Tissue Proteomics ◽  
Different Cell Types

AbstractSpermatogenesis is a complex multi-step process involving intricate interactions between different cell types in the male testis. Disruption of these interactions results in infertility. Combination of shotgun tissue proteomics with MALDI imaging mass spectrometry is markedly potent in revealing topological maps of molecular processes within tissues. Here, we use a combinatorial approach on a characterized mouse model of hormone induced male infertility to uncover misregulated pathways. Comparative testicular proteome of wildtype and mice overexpressing human P450 aromatase (AROM+) with pathologically increased estrogen levels unravels gross dysregulation of spermatogenesis and emergence of proinflammatory pathways in AROM+ testis. In situ MS allowed us to localize misregulated proteins/peptides to defined regions within the testis. Results suggest that infertility is associated with substantial loss of proteomic heterogeneity, which define distinct stages of seminiferous tubuli in healthy animals. Importantly, considerable loss of mitochondrial factors, proteins associated with late stages of spermatogenesis and steroidogenic factors characterise AROM+ mice. Thus, the novel proteomic approach pinpoints in unprecedented ways the disruption of normal processes in testis and provides a signature for male infertility.

scholarly journals In situ differentiation of iridophore crystallotypes underlies zebrafish stripe patterning

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Dvir Gur ◽  
Emily J. Bain ◽  
Kory R. Johnson ◽  
Andy J. Aman ◽  
H. Amalia Pasoili ◽  
...  
Keyword(s):  
Skin Color ◽  
Cell Types ◽  
Color Pattern ◽  
Single Type ◽  
Sequencing Analysis ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cryogenic Scanning Electron Microscopy ◽  
Different Cell Types

AbstractSkin color patterns are ubiquitous in nature, impact social behavior, predator avoidance, and protection from ultraviolet irradiation. A leading model system for vertebrate skin patterning is the zebrafish; its alternating blue stripes and yellow interstripes depend on light-reflecting cells called iridophores. It was suggested that the zebrafish’s color pattern arises from a single type of iridophore migrating differentially to stripes and interstripes. However, here we find that iridophores do not migrate between stripes and interstripes but instead differentiate and proliferate in-place, based on their micro-environment. RNA-sequencing analysis further reveals that stripe and interstripe iridophores have different transcriptomic states, while cryogenic-scanning-electron-microscopy and micro-X-ray diffraction identify different crystal-arrays architectures, indicating that stripe and interstripe iridophores are different cell types. Based on these results, we present an alternative model of skin patterning in zebrafish in which distinct iridophore crystallotypes containing specialized, physiologically responsive, organelles arise in stripe and interstripe by in-situ differentiation.

scholarly journals Gnrh1-induced responses are indirect in female medaka Fsh cells

2019 ◽  
Author(s):  
Kjetil Hodne ◽  
Romain Fontaine ◽  
Eirill Ager-Wick ◽  
Finn-Arne Weltzien
Keyword(s):  
Patch Clamp ◽  
Adult Female ◽  
Reproductive Function ◽  
Cell Types ◽  
Gonadal Development ◽  
Pituitary Cells ◽  
Tissue Slices ◽  
Lh Cells ◽  
Different Cell Types

ABSTRACTReproductive function in vertebrates is stimulated by gonadotropin-releasing hormone (GnRH) that controls the synthesis and release of the two pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). FSH and LH, which regulates different stages of gonadal development, are produced by two different cell types in the fish pituitary, in contrast to mammals and birds, thus allowing the investigation of their differential regulation. In the present work, we show by fluorescentin situhybridization that Lh cells in adult female medaka express Gnrh receptors, whereas Fsh cells do not. This is confirmed by patch clamp recordings and cytosolic Ca2+measurements on dispersed pituitary cells, where Lh cells, but not Fsh cells, respond to Gnrh1 by increased action potential frequencies and cytosolic Ca2+levels. In contrast, both Fsh and Lh cells are able to respond electrically and by elevating the cytosolic Ca2+levels to Gnrh1 in brain-pituitary tissue slices. Using Ca2+uncaging in combination with patch clamp recordings and cytosolic Ca2+measurements, we show that Fsh and Lh cells form homo- and heterotypic networks in the pituitary. Taken together, these results show that the effects of Gnrh1 on Fsh release in adult female medaka is indirect, likely mediated via Lh cells.

Differential expression of glutamate receptor genes (GluR1-5) in the rat retina

1992 ◽  
Vol 8 (1) ◽  
pp. 49-55 ◽  
Author(s):  
Thomas E. Hughes ◽  
Irm Hermans-Borgmeyer ◽  
Steve Heinemann
Keyword(s):  
Glutamate Receptor ◽  
Amacrine Cells ◽  
Cell Types ◽  
Bipolar Cells ◽  
Inner Nuclear Layer ◽  
Dissociated Cells ◽  
Different Cell Types ◽  
The Brain ◽  
Overlapping Sets

AbstractThe recent isolation of at least five different cDNAs encoding functional subunits of glutamate receptors (GluR1 to GluR5) has revealed a diversity whose function is not understood. To learn more about how these different receptor subunits are used in the brain, we undertook an in situ hybridization study of the retina to define how the different glutamate receptor genes are expressed. We chose the retina because the glutamate sensitivities of its different cell types have been characterized, and these different neurons reside in different laminae.Hybridization of [35S]UTP-labeled cRNA probes with transverse sections and freshly dissociated cells reveals that all five receptor subunits are expressed in the retina. Hybridization signal is detected in different, but overlapping, sets of cells in the retina. GluR1, GluR2, and GluR5 are expressed by many somata, and GluR4 by a few, in the outer third of the inner nuclear layer, where the horizontal cells reside. Transcripts for GluR1, GluR2, and GluR5 are found in the somata within the middle third of the inner nuclear layer, which is where the bipolar cell somata are located, and GluR2 probes label freshly dissociated rod bipolar cells. All of the probes produce labeling over the cells at the inner edge of the inner nuclear layer, which are probably amacrine cells, as well as over the cell bodies in the ganglion cell layer.

scholarly journals Subcellular Localization of a Novel Alternative Splicing of IIIG9 and Colocalization with PPP1gamma Isoforms

2008 ◽  
Vol 14 (S3) ◽  
pp. 141-143
Author(s):  
C. Sousa ◽  
A.P. Vintém ◽  
M. Fardilha ◽  
O. da Cruz e Silva ◽  
E. da Cruz e Silva
Keyword(s):  
Alternative Splicing ◽  
Male Infertility ◽  
Cell Types ◽  
Sperm Cells ◽  
Regulatory Subunits ◽  
Specific Antibodies ◽  
Fluorescent Microscope ◽  
Bovine Sperm ◽  
Spermatogonia Cells ◽  
Different Cell Types

In testis we find mainly PPP1gamma2 isoform. We hypothesize that in different cell types we can find different regulatory subunits that may constitute targets for therapeutics of diseases such as male infertility, cancer and Alzheimer's disease. We identified a novel alternative splicing isoform of IIIG9 in testis, a known regulator of PPP1, IIIG9sT, and the aim of this study was its further characterization. We used a specific antibody for IIIG9sT in order to characterize its localization in bovine sperm cells. We also transfected IIIG9sT-GFP construct in mouse spermatogonia cells (GC-1 cells) and we used specific antibodies for each PPP1 isoform for the colocalization studies. We observed them under a fluorescent microscope and a LSM and quantified a high co-localization with PPP1gamma1 and 2 isoforms.

Involvement of PKCδ and PKD in pulmonary microvascular endothelial cell hyperpermeability

2004 ◽  
Vol 286 (1) ◽  
pp. C105-C111 ◽  
Author(s):  
John H. Tinsley ◽  
Nicole R. Teasdale ◽  
Sarah Y. Yuan
Keyword(s):  
Cell Types ◽  
Microvascular Endothelial Cell ◽  
The Novel ◽  
Membrane Translocation ◽  
Microvascular Endothelium ◽  
Pharmacological Agents ◽  
Kinase Substrate ◽  
Pulmonary Microvascular Endothelial Cell ◽  
Microvascular Endothelial ◽  
Different Cell Types

The involvement of PKC, the isoforms of which are categorized into three subtypes: conventional (α, βI, βII, and γ), novel [δ, ϵ, η, and μ (also known as PKD),θ], and atypical (ζ and ι/λ), in the regulation of endothelial monolayer integrity is well documented. However, isoform activity varies among different cell types. Our goal was to reveal isoform-specific PKC activity in the microvascular endothelium in response to phorbol 12-myristate 13-acetate (PMA) and diacylglycerol (DAG). Isoform activity was demonstrated by cytosol-to-membrane translocation after PMA treatment and phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein after PMA and DAG treatment. Specific isoforms were inhibited by using both antisense oligonucleotides and pharmacological agents. The data showed partial cytosol-to-membrane translocation of isoforms α, βI, and ϵ and complete translocation of PKCδ and PKD in response to PMA. Furthermore, antisense treatment and pharmacological studies indicated that the novel isoform PKCδ and PKD are both required for PMA- and DAG-induced MARCKS phosphorylation and hyperpermeability in pulmonary microvascular endothelial cells, whereas isoforms α, βI, and ϵ were dispensable with regard to these same phenomena.

scholarly journals Discovering new lipidomic features using cell type specific fluorophore expression to provide spatial and biological specificity in a multimodal workflow with MALDI IMS

2020 ◽  
Author(s):  
Marissa A. Jones ◽  
Sung Hoon Cho ◽  
Nathan Heath Patterson ◽  
Raf Van de Plas ◽  
Jeffrey Spraggins ◽  
...  
Keyword(s):  
Imaging Mass Spectrometry ◽  
Fluorescence Emission ◽  
Ether Lipid ◽  
Cell Types ◽  
Tissue Cell ◽  
Specific Cell ◽  
Cell Type ◽  
Emission Data ◽  
Maldi Ims ◽  
Lipid Species

<p></p><div> <p>Identifying the spatial distributions of biomolecules in tissue is crucial for understanding integrated function. Imaging Mass Spectrometry (IMS) allows simultaneous mapping of thousands of biosynthetic products such as lipids but has needed a means of identifying specific cell-types or functional states to correlate with molecular localization. We report here advances starting from identity marking with a genetically encoded fluorophore. The fluorescence emission data were integrated with IMS data through multimodal image processing with advanced registration techniques and data-driven image fusion. In an unbiased analysis of spleens, this integrated technology enabled identification of ether lipid species preferentially enriched in germinal centers. We propose that this use of genetic marking for microanatomical regions of interest can be paired with molecular information from IMS for any tissue, cell-type, or activity state for which fluorescence is driven by a gene-tracking allele and ultimately with outputs of other means of spatial mapping.</p> </div><br><p></p>

scholarly journals Term Human Placental Trophoblasts Express SARS-CoV-2 Entry Factors ACE2, TMPRSS2, and Furin

mSphere ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Yingshi Ouyang ◽  
Tarique Bagalkot ◽  
Wendy Fitzgerald ◽  
Elena Sadovsky ◽  
Tianjiao Chu ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Human Placenta ◽  
Cell Types ◽  
Maternal Blood ◽  
Transmission Risk ◽  
Rna Seq ◽  
Angiotensin Converting Enzyme 2 ◽  
Real Time Quantitative Pcr ◽  
Different Cell Types

ABSTRACT The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has had a massive impact on human lives worldwide. While the airborne SARS-CoV-2 primarily affects the lungs, viremia is not uncommon. As placental trophoblasts are directly bathed in maternal blood, they are vulnerable to SARS-CoV-2. Intriguingly, the human fetus is largely spared from SARS-CoV-2 infection. We tested whether the human placenta expresses the main SARS-CoV-2 entry factors angiotensin-converting enzyme 2 (ACE2), transmembrane protease serine 2 (TMPRSS2), and furin and showed that ACE2 and TMPRSS2 are expressed in the trophoblast rather than in other placental villous cells. While furin is expressed in the main placental villous cell types, we surveyed, trophoblasts exhibit the highest expression. In line with the expression of these entry factors, we demonstrated that a SARS-CoV-2 pseudovirus could enter primary human trophoblasts. Mechanisms underlying placental defense against SARS-CoV-2 infection likely involve postentry processing, which may be germane for mitigating interventions against SARS-CoV-2. IMPORTANCE Pregnant women worldwide have been affected by COVID-19. As the virus is commonly spread to various organs via the bloodstream and because human placental trophoblasts are directly bathed in maternal blood, feto-placental infection by SARS-CoV-2 seems likely. However, despite the heightened risk to pregnant women, thus far the transmission risk of COVID-19 to the feto-placental unit seems extremely low. This has been recently attributed to a negligible expression of SARS-CoV-2 entry factors in the human placenta. We therefore sought to explore the expression of the entry factors ACE2 and TMPRSS2 in the different cell types of human placental villi. Using a combination of transcriptome sequencing (RNA-seq), real-time quantitative PCR (RT-qPCR), in situ hybridization, and immunofluorescence, we found that trophoblasts, but not the other main villous cell types, express ACE2 and TMPRSS2, with a broad expression of furin. Correspondingly, we also showed that primary human trophoblasts are permissive to entry of SARS-CoV-2 pseudovirus particles.

scholarly journals Aromatase is abundantly expressed by neonatal rat penis but downregulated in adulthood

2004 ◽  
Vol 33 (2) ◽  
pp. 343-359 ◽  
Author(s):  
S Jesmin ◽  
C N Mowa ◽  
I Sakuma ◽  
N Matsuda ◽  
H Togashi ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Corpus Cavernosum ◽  
Cell Types ◽  
Neonatal Rat ◽  
Estrogen Synthesis ◽  
Aromatase Mrna ◽  
Reproductive Events ◽  
Different Cell Types

Although synthesis of estrogen by male gonads has been well documented for over half a century, it is only recently that the role of estrogen in male reproductive events has gained appreciation. We recently reported abundant expression of estrogen receptor (ER)-α and -β in different cell types of the rat penis, whose levels diminished with advancing age. The present study, which builds on data from the ER study, was designed to determine whether the penis is capable of generating its own local estrogen by examining evidence of the expression of aromatase, a microsomal enzymatic complex which irreversibly converts androgens to estrogens, using immunohistochemistry, Western blotting, in situ hybridization and real-time PCR analyses. Secondly, the effects of sex steroid hormones on penile aromatase were examined. Discrete aromatase immunoreactive cells were localized in primordial corpus cavernosum, corpus spongiosus and os penis, blood vessels and sensory corpuscle of glans penis. In situ hybridization signals corresponded with immunohistochemical findings. Western blot, enzyme immunoassay and real-time PCR analyses of rat penile samples revealed an age-dependent expression of aromatase and estrogen, with levels at week 1 almost resembling those of the ovary, but they decreased sharply by week 8, and decreased further by week 35. This expression pattern was strikingly similar to that of ER-α reported previously. Testosterone and diethylstilbesterol administered prenatally upregulate levels of aromatase mRNA and protein, and estrogen postnatally. Dihydrotestosterone upregulated aromatase mRNA and protein, but not estrogen. We conclude that estrogen acts via ER in a paracrine and/or autocrine manner to regulate penile events, particularly during development, and that estrogen synthesis is regulated by estrogen and androgens.

scholarly journals Expression of Membrane-associated Mucins MUC1 and MUC4 in Major Human Salivary Glands

2002 ◽  
Vol 50 (6) ◽  
pp. 811-820 ◽  
Author(s):  
Bing Liu ◽  
Jessica R. Lague ◽  
David P. Nunes ◽  
Paul Toselli ◽  
Frank G. Oppenheim ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Salivary Glands ◽  
Acinar Cells ◽  
Cell Types ◽  
Northern Blotting ◽  
Submandibular Glands ◽  
Human Genes ◽  
Mucin Layer ◽  
Different Cell Types

Mucins are high molecular weight glycoproteins secreted by salivary glands and epithelial cells lining the digestive, respiratory, and reproductive tracts. These glyco-proteins, encoded in at least 13 distinct human genes, can be subdivided into gel-forming and membrane-associated forms. The gel-forming mucin MUC5B is secreted by mucous acinar cells in major and minor salivary glands, but little is known about the expression pattern of membrane-associated mucins. In this study, RT-PCR and Northern blotting demonstrated the presence of transcripts for MUC1 and MUC4 in both parotid and submandibular glands, and in situ hybridization localized these transcripts to epithelial cells lining striated and excretory ducts and in some serous acinar cells. The same cellular distribution was observed by immunohistochemistry. Soluble forms of both mucins were detected in parotid secretion after immunoprecipitation with mucin-specific antibodies. These studies have shown that membrane-associated mucins are produced in both parotid and submandibular glands and that they are expressed in different cell types than gel-forming mucins. Although the function of these mucins in the oral cavity remains to be elucidated, it is possible that they both contribute to the epithelial protective mucin layer and act as receptors initiating one or more intracellular signal transduction pathways.

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