Alteration of transporter activities in the epididymides of infertile initial segment-specific Pten knockout mice†

Abstract A fully functional initial segment, the most proximal region of the epididymis, is important for male fertility. Our previous study generated a mouse model to investigate the importance of initial segment function in male fertility. In that model, phosphatase and tensin homolog (Pten) was conditionally removed from the initial segment epithelium, which resulted in epithelial de-differentiation. When spermatozoa progressed through the de-differentiated epithelial duct, they developed angled flagella, suggesting compromised sperm maturation, which eventually resulted in male infertility. To understand the molecular mechanisms, by which PTEN regulates epididymal sperm maturation, we compared the transcriptome profile of the initial segment between controls and initial segment-specific Pten knockouts and revealed that water, ion, and organic solute transporter activities were one of the top molecular and cellular functions altered following loss of Pten. Alteration in protein levels and localization of several transporters following loss of Pten were also observed by immunofluorescence analysis. Epithelial cells of the initial segment from knockouts were more permeable to fluorescein isothiocyanate–dextran (4000 Da) compared to controls. Interestingly, conditional deletion of Pten from other organs also resulted in changes in transporter activity, suggesting a common role of PTEN in regulation of transporter activity. Taken together, our data support the hypothesis that loss of Pten from the initial segment epithelium results in changes in the transporting and permeability characteristics of the epithelium, which in turn altered the luminal fluid microenvironment that is so important for sperm maturation and male fertility.

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Lycopene Inhibits Urotensin-II-Induced Cardiomyocyte Hypertrophy in Neonatal Rat Cardiomyocytes

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2014/724670 ◽

2014 ◽

Vol 2014 ◽

pp. 1-9 ◽

Cited By ~ 5

Author(s):

Hung-Hsing Chao ◽

Li-Chin Sung ◽

Cheng-Hsien Chen ◽

Ju-Chi Liu ◽

Jin-Jer Chen ◽

...

Keyword(s):

Molecular Mechanisms ◽

Glycogen Synthase ◽

Activity Level ◽

Neonatal Rat ◽

Cardiomyocyte Hypertrophy ◽

Urotensin Ii ◽

Rat Cardiomyocytes ◽

Protein Levels ◽

Neonatal Rat Cardiomyocytes ◽

Tensin Homolog

This study investigated how lycopene affected urotensin-II- (U-II-) induced cardiomyocyte hypertrophy and the possible implicated mechanisms. Neonatal rat cardiomyocytes were exposed to U-II (1 nM) either exclusively or following 6 h of lycopene pretreatment (1–10 μM). The lycopene (3–10 μM) pretreatment significantly inhibited the U-II-induced cardiomyocyte hypertrophy, decreased the production of U-II-induced reactive oxygen species (ROS), and reduced the level of NAD(P)H oxidase-4 expression. Lycopene further inhibited the U-II-induced phosphorylation of the redox-sensitive extracellular signal-regulated kinases. Moreover, lycopene treatment prevented the increase in the phosphorylation of serine-threonine kinase Akt and glycogen synthase kinase-3beta (GSK-3β) caused by U-II without affecting the protein levels of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN). However, lycopene increased the PTEN activity level, suggesting that lycopene prevents ROS-induced PTEN inactivation. These findings imply that lycopene yields antihypertrophic effects that can prevent the activation of the Akt/GSK-3βhypertrophic pathway by modulating PTEN inactivation through U-II treatment. Thus, the data indicate that lycopene prevented U-II-induced cardiomyocyte hypertrophy through a mechanism involving the inhibition of redox signaling. These findings provide novel data regarding the molecular mechanisms by which lycopene regulates cardiomyocyte hypertrophy.

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A rapidly evolving actin mediates fertility and developmental tradeoffs in Drosophila

10.1101/2020.09.28.317503 ◽

2020 ◽

Cited By ~ 1

Author(s):

Courtney M. Schroeder ◽

Sarah A. Tomlin ◽

John R. Valenzuela ◽

Harmit S. Malik

Keyword(s):

Heat Stress ◽

Early Development ◽

Male Fertility ◽

Sperm Maturation ◽

Cellular Functions ◽

Male Germline ◽

Term Retention ◽

Long Term Retention ◽

Related Proteins

AbstractMost actin-related proteins (Arps) are highly conserved in eukaryotes, where they carry out well-defined cellular functions. Drosophila and mammals also encode divergent non-canonical Arps in their male-germline whose roles remain unknown. Here, we show that Arp53D, a rapidly-evolving Drosophila Arp, localizes to fusomes and actin cones, two male germline-specific actin structures critical for sperm maturation, via its non-canonical N-terminal tail. Although we expected that Arp53D loss would reduce male fertility, we instead find that Arp53D-KO males are more fertile, both in isolation and in competition with wildtype males. Upon investigating why evolution would retain a gene that negatively impacts male fertility, we unexpectedly found that Arp53D-KO females are less fertile. Moreover, KO embryos exhibit reduced viability, which worsens under heat stress. We conclude that ‘testis-specific’ Arp53D is detrimental to male fertility, but is required for female fertility and early development, leading to its long-term retention and recurrent adaptation in Drosophila.

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Tubulin glycylation controls axonemal dynein activity, flagellar beat, and male fertility

Science ◽

10.1126/science.abd4914 ◽

2021 ◽

Vol 371 (6525) ◽

pp. eabd4914

Author(s):

Sudarshan Gadadhar ◽

Gonzalo Alvarez Viar ◽

Jan Niklas Hansen ◽

An Gong ◽

Aleksandr Kostarev ◽

...

Keyword(s):

Posttranslational Modifications ◽

Male Fertility ◽

Electron Tomography ◽

Structural Basis ◽

Cellular Functions ◽

Flagellar Beat ◽

Cryo Electron Tomography ◽

Axonemal Dynein ◽

Dynein Arms ◽

Cilia And Flagella

Posttranslational modifications of the microtubule cytoskeleton have emerged as key regulators of cellular functions, and their perturbations have been linked to a growing number of human pathologies. Tubulin glycylation modifies microtubules specifically in cilia and flagella, but its functional and mechanistic roles remain unclear. In this study, we generated a mouse model entirely lacking tubulin glycylation. Male mice were subfertile owing to aberrant beat patterns of their sperm flagella, which impeded the straight swimming of sperm cells. Using cryo–electron tomography, we showed that lack of glycylation caused abnormal conformations of the dynein arms within sperm axonemes, providing the structural basis for the observed dysfunction. Our findings reveal the importance of microtubule glycylation for controlled flagellar beating, directional sperm swimming, and male fertility.

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Mitochondrial Dynamics, ROS, and Cell Signaling: A Blended Overview

Life ◽

10.3390/life11040332 ◽

2021 ◽

Vol 11 (4) ◽

pp. 332

Author(s):

Valentina Brillo ◽

Leonardo Chieregato ◽

Luigi Leanza ◽

Silvia Muccioli ◽

Roberto Costa

Keyword(s):

Molecular Mechanisms ◽

Mitochondrial Dynamics ◽

Eukaryotic Cells ◽

Therapeutic Approaches ◽

Cellular Functions ◽

Lipid Trafficking ◽

Mitochondrial Ros ◽

Intracellular Organelles ◽

Proliferation Regulation ◽

Dynamic Plasticity

Mitochondria are key intracellular organelles involved not only in the metabolic state of the cell, but also in several cellular functions, such as proliferation, Calcium signaling, and lipid trafficking. Indeed, these organelles are characterized by continuous events of fission and fusion which contribute to the dynamic plasticity of their network, also strongly influenced by mitochondrial contacts with other subcellular organelles. Nevertheless, mitochondria release a major amount of reactive oxygen species (ROS) inside eukaryotic cells, which are reported to mediate a plethora of both physiological and pathological cellular functions, such as growth and proliferation, regulation of autophagy, apoptosis, and metastasis. Therefore, targeting mitochondrial ROS could be a promising strategy to overcome and hinder the development of diseases such as cancer, where malignant cells, possessing a higher amount of ROS with respect to healthy ones, could be specifically targeted by therapeutic treatments. In this review, we collected the ultimate findings on the blended interplay among mitochondrial shaping, mitochondrial ROS, and several signaling pathways, in order to contribute to the dissection of intracellular molecular mechanisms involved in the pathophysiology of eukaryotic cells, possibly improving future therapeutic approaches.

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Carfilzomib in Combination with Bortezomib Enhances Apoptotic Cell Death in B16-F1 Melanoma Cells

Biology ◽

10.3390/biology10020153 ◽

2021 ◽

Vol 10 (2) ◽

pp. 153

Author(s):

Min Seung Lee ◽

So Hyun Lim ◽

Ah-Ran Yu ◽

Chi Yeon Hwang ◽

Insug Kang ◽

...

Keyword(s):

Er Stress ◽

Dose Reduction ◽

Molecular Mechanisms ◽

Apoptotic Cell ◽

Proteasome Inhibitors ◽

Annexin V ◽

Fluorescein Isothiocyanate ◽

Melanoma Cells ◽

Eif2α Phosphorylation ◽

Pharmacological Interactions

Proteasome inhibitors, such as bortezomib (BZ) and carfilzomib (CFZ), have been suggested as treatments for various cancers. To utilize BZ and/or CFZ as effective therapeutics for treating melanoma, we studied their molecular mechanisms using B16-F1 melanoma cells. Flow cytometry of Annexin V-fluorescein isothiocyanate-labeled cells indicated apoptosis induction by treatment with BZ and CFZ. Apoptosis was evidenced by the activation of various caspases, including caspase 3, 8, 9, and 12. Treatment with BZ and CFZ induced endoplasmic reticulum (ER) stress, as indicated by an increase in eIF2α phosphorylation and the expression of ER stress-associated proteins, including GRP78, ATF6α, ATF4, XBP1, and CCAAT/enhancer-binding protein homologous protein. The effects of CFZ on ER stress and apoptosis were lower than that of BZ. Nevertheless, CFZ and BZ synergistically induced ER stress and apoptosis in B16-F1 cells. Furthermore, the combinational pharmacological interactions of BZ and CFZ against the growth of B16-F1 melanoma cells were assessed by calculating the combination index and dose-reduction index with the CompuSyn software. We found that the combination of CFZ and BZ at submaximal concentrations could obtain dose reduction by exerting synergistic inhibitory effects on cell growth. Moreover, this drug combination reduced tumor growth in C57BL/6 syngeneic mice. Taken together, these results suggest that CFZ in combination with BZ may be a beneficial and potential strategy for melanoma treatment.

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Deficiency of the onco-miRNA cluster, miR-106b∼25, causes oligozoospermia and the cooperative action of miR-106b∼25 and miR-17∼92 is required to maintain male fertility

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gaaa027 ◽

2020 ◽

Vol 26 (6) ◽

pp. 389-401

Author(s):

Alicia Hurtado ◽

Rogelio Palomino ◽

Ina Georg ◽

Miguel Lao ◽

Francisca M Real ◽

...

Keyword(s):

Male Fertility ◽

Molecular Mechanisms ◽

Mirna Cluster ◽

Knock Out ◽

Cooperative Action ◽

Mouse Mutants ◽

New Genes ◽

Mirna Clusters ◽

Testicular Histology ◽

And Function

Abstract The identification of new genes involved in sexual development and gonadal function as potential candidates causing male infertility is important for both diagnostic and therapeutic purposes. Deficiency of the onco-miRNA cluster miR-17∼92 has been shown to disrupt spermatogenesis, whereas mutations in its paralog cluster, miR-106b∼25, that is expressed in the same cells, were reported to have no effect on testis development and function. The aim of this work is to determine the role of these two miRNA clusters in spermatogenesis and male fertility. For this, we analyzed miR-106b∼25 and miR-17∼92 single and double mouse mutants and compared them to control mice. We found that miR-106b∼25 knock out testes show reduced size, oligozoospermia and altered spermatogenesis. Transcriptomic analysis showed that multiple molecular pathways are deregulated in these mutant testes. Nevertheless, mutant males conserved normal fertility even when early spermatogenesis and other functions were disrupted. In contrast, miR-17∼92+/−; miR-106b∼25−/− double mutants showed severely disrupted testicular histology and significantly reduced fertility. Our results indicate that miR-106b∼25 and miR-17∼92 ensure accurate gene expression levels in the adult testis, keeping them within the required thresholds. They play a crucial role in testis homeostasis and are required to maintain male fertility. Hence, we have identified new candidate genetic factors to be screened in the molecular diagnosis of human males with reproductive disorders. Finally, considering the well-known oncogenic nature of these two clusters and the fact that patients with reduced fertility are more prone to testicular cancer, our results might also help to elucidate the molecular mechanisms linking both pathologies.

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Hypoxia-Induced Epithelial-Mesenchymal Transition Is Involved in Bleomycin-Induced Lung Fibrosis

BioMed Research International ◽

10.1155/2015/232791 ◽

2015 ◽

Vol 2015 ◽

pp. 1-10 ◽

Cited By ~ 11

Author(s):

Liang Guo ◽

Jun-mei Xu ◽

Lei Liu ◽

Su-mei Liu ◽

Rong Zhu

Keyword(s):

P38 Mapk ◽

Lung Fibrosis ◽

Molecular Mechanisms ◽

Epithelial Mesenchymal Transition ◽

Severe Disease ◽

Progressive Increase ◽

Lung Epithelial Cells ◽

Cellular Mechanisms ◽

Mesenchymal Transition ◽

Protein Levels

Pulmonary fibrosis is a severe disease that contributes to the morbidity and mortality of a number of lung diseases. However, the molecular and cellular mechanisms leading to lung fibrosis are poorly understood. This study investigated the roles of epithelial-mesenchymal transition (EMT) and the associated molecular mechanisms in bleomycin-induced lung fibrosis. The bleomycin-induced fibrosis animal model was established by intratracheal injection of a single dose of bleomycin. Protein expression was measured by Western blot, immunohistochemistry, and immunofluorescence. Typical lesions of lung fibrosis were observed 1 week after bleomycin injection. A progressive increase in MMP-2, S100A4,α-SMA, HIF-1α, ZEB1, CD44, phospho-p44/42 (p-p44/42), and phospho-p38 MAPK (p-p38) protein levels as well as activation of EMT was observed in the lung tissues of bleomycin mice. Hypoxia increased HIF-1αand ZEB1 expression and activated EMT in H358 cells. Also, continuous incubation of cells under mild hypoxic conditions increased CD44, p-p44/42, and p-p38 protein levels in H358 cells, which correlated with the increase in S100A4 expression. In conclusion, bleomycin induces progressive lung fibrosis, which may be associated with activation of EMT. The fibrosis-induced hypoxia may further activate EMT in distal alveoli through a hypoxia-HIF-1α-ZEB1 pathway and promote the differentiation of lung epithelial cells into fibroblasts through phosphorylation of p38 MAPK and Erk1/2 proteins.

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Deleterious effects of obesity upon the hormonal and molecular mechanisms controlling spermatogenesis and male fertility

Human Fertility ◽

10.3109/14647273.2015.1070438 ◽

2015 ◽

Vol 18 (3) ◽

pp. 184-193 ◽

Cited By ~ 42

Author(s):

Lien M. Davidson ◽

Kate Millar ◽

Celine Jones ◽

Muhammad Fatum ◽

Kevin Coward

Keyword(s):

Male Fertility ◽

Molecular Mechanisms

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Physical dissociation of the TCR-CD3 complex accompanies receptor ligation.

Journal of Experimental Medicine ◽

10.1084/jem.182.6.1997 ◽

1995 ◽

Vol 182 (6) ◽

pp. 1997-2006 ◽

Cited By ~ 40

Author(s):

H Kishimoto ◽

R T Kubo ◽

H Yorifuji ◽

T Nakayama ◽

Y Asano ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Surface ◽

Molecular Mechanisms ◽

Signal Transduction Pathway ◽

Surface Expression ◽

Cell Surface Expression ◽

Protein Levels ◽

Before And After ◽

Physical Dissociation ◽

Receptor Ligation

Recent studies indicate that there may be functional uncoupling of the TCR-CD3 complex and suggest that the TCR-CD3 complex is composed of two parallel signal-transducing units, one made of gamma delta epsilon chains and the other of zeta chains. To elucidate the molecular mechanisms that may explain the functional uncoupling of TCR and CD3, we have analyzed their expression by using flow cytometry as well as immunochemical means both before and after stimulation with anti-TCR-beta, anti-CD3 epsilon, anti-CD2, staphylococcal enterotoxin B, and ionomycin. We present evidence that TCR physically dissociates from CD3 after stimulation of the TCR-CD3 complex. Stimulation with anti-CD3 resulted in down-modulation of TCR within 45 min whereas CD3 epsilon was still expressed on the cell surface as detected by flow cytometry. However, the cell surface expression of TCR and CD3 was not affected when cells were stimulated with anti-TCR-beta under the same conditions. In the case of anti-CD3 treatment of T cells, the TCR down-modulation appeared to be due to the internalization of TCR, as determined by immunoelectron microscopy. Immunochemical analysis of cells after stimulation with either anti-TCR or anti-CD3 mAbs revealed that the overall protein levels of TCR and CD3 were similar. More interestingly, the dissociation of the TCR-CD3 complex was observed with both treatments and occurred in a manner that the TCR and the associated TCR-zeta chain dissociated as a unit from CD3. These results provide the first report of physical dissociation of TCR and CD3 after stimulation through the TCR-CD3 complex. The results also suggest that the signal transduction pathway triggered by TCR may differ from that induced by CD3.

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Newly synthesized benzimidazoles inhibits vascular endothelial growth factor and matrix metalloproteinase-2 and -9 levels in prostate cancer cells

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520621666210924114856 ◽

2021 ◽

Vol 21 ◽

Author(s):

Suleyman Ilhan ◽

Gamze Dilekci ◽

Adem Guner ◽

Hakan Bektas

Keyword(s):

Prostate Cancer ◽

Cancer Cells ◽

Molecular Mechanisms ◽

Umbilical Vein ◽

Imidazole Ring ◽

Matrix Metalloproteinase 2 ◽

Benzimidazole Derivatives ◽

Prostate Cancer Cells ◽

Protein Levels ◽

Umbilical Vein Endothelial Cells

Background: Investigating the effects of newly synthesized agents on various molecular mechanisms to understand their mechanism of action is an important step of pre-clinical screening. Benzimidazoles are composed of a unique fused benzene and imidazole ring and have attracted great attention due to their broad bioactivities, including antitumor. Objective: In the current study, we reported the synthesis of novel benzimidazole derivatives and investigated the possible cytotoxic and anti-angiogenic effects on human prostate cancer and umbilical vein endothelial cells (HUVECs). Methods: MTT assay was used to assess cell viability. A scratch assay was conducted to monitor the migration of cells. mRNA expression levels of VEGF, MMP-2, and MMP-9 were evaluated using qPCR. Changes in protein levels were evaluated by western blotting. Results: Compound G1, having a chlorine moiety, showed a potent cytotoxic activity on both prostate cancer cells and HUVECs, and inhibited cell migration via decreasing the mRNA and protein levels of key angiogenesis-related molecules such as VEGF, MMP-2, and MMP-9. Conclusion: These results suggest that newly synthesized G1 may be a novel anti-angiogenic agent for prostate cancer treatment.

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