A cell- and developmental stage-specific promoter drives the expression of a truncated c-kit protein during mouse spermatid elongation

Development ◽  
1996 ◽  
Vol 122 (4) ◽  
pp. 1291-1302 ◽  
Author(s):  
C. Albanesi ◽  
R. Geremia ◽  
M. Giorgio ◽  
S. Dolci ◽  
C. Sette ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Germ Cells ◽  
Transcriptional Start Site ◽  
Seminiferous Tubules ◽  
Tyrosine Kinase Receptor ◽  
Reading Frame ◽  
Nuclear Extracts ◽  
Kit Gene

In the postnatal testis, the c-kit transmembrane tyrosine-kinase receptor is expressed in type A spermatogonia, and its transcription ceases at the meiotic phase of spermatogenesis. Alternative, shorter c-kit transcripts are expressed in post-meiotic germ cells. These transcripts should encode a truncated version of the c-kit protein, lacking the extracellular, the transmembrane and part of the intracellular tyrosine-kinase domains. The 5′ end of the alternative c-kit transcripts maps within an intron of the mouse c-kit gene. We now show that this intron contains a promoter active in nuclear extracts of round spermatids, and that two discrete sequences upstream of the transcriptional start site bind spermatid-specific nuclear factors. Deletion of both these sequences abolishes activity of the promoter in vitro. We have also established that this promoter is functional in vivo, in a tissue-and cell-specific fashion, since intronic sequences drive the expression of the E. coli lacZ reporter gene in transgenic mice specifically in the testis. Transgene expression is confined to haploid germ cells of seminiferous tubules, starting from spermatids at step 9, and disappearing at step 13, indicating that cryptic promoter within the 16th intron of the mouse c-kit gene is active in a short temporal window at the end of the transcriptional phase of spermiogenesis. In agreement with these data, western blot experiments using an antibody directed against the carboxy-terminal portion of the mouse c-kit protein showed that a polypeptide, of the size predicted by the open reading frame of the spermatid-specific c-kit cDNA, accumulates in the latest stages of spermatogenesis and in epididymal spermatozoa. An immunoreactive protein of the same size can be produced in both eukaryotic and prokaryotic artificial expression systems.

scholarly journals The insulin sensitiser metformin regulates chicken Sertoli and germ cell populations

Reproduction ◽  
2016 ◽  
Vol 151 (5) ◽  
pp. 527-538 ◽  
Author(s):  
M Faure ◽  
E Guibert ◽  
S Alves ◽  
B Pain ◽  
C Ramé ◽  
...  
Keyword(s):  
Germ Cells ◽  
Sertoli Cells ◽  
Energy Content ◽  
Morphological Changes ◽  
Seminiferous Tubules ◽  
Reduce Food Intake ◽  
Testicular Weight

Abstract Metformin, an insulin sensitiser from the biguanide family of molecules, is used for the treatment of insulin resistance in type 2 diabetes individuals. It increases peripheral glucose uptake and may reduce food intake. Based on the tight link between metabolism and fertility, we investigated the role of metformin on testicular function using in vitro culture of Sertoli cells and seminiferous tubules, complemented by in vivo data obtained following metformin administration to prepubertal chickens. In vitro, metformin treatment reduced Sertoli cell proliferation without inducing apoptosis and morphological changes. The metabolism of Sertoli cells was affected because lactate secretion by Sertoli cells increased approximately twofold and intracellular free ATP was negatively impacted. Two important pathways regulating proliferation and metabolism in Sertoli cells were assayed. Metformin exposure was not associated with an increased phosphorylation of AKT or ERK. There was a 90% reduction in the proportion of proliferating germ cells after a 96-h exposure of seminiferous tubule cultures to metformin. In vivo, 6-week-old chickens treated with metformin for 3 weeks exhibited reduced testicular weight and a 50% decrease in testosterone levels. The expression of a marker of undifferentiated germ cells was unchanged in contrast to the decrease in expression of ‘protamine’, a marker of differentiated germ cells. In conclusion, these results suggest that metformin affects the testicular energy content and the proliferative ability of Sertoli and germ cells. Reproduction (2016) 151 527–538

scholarly journals Involvement of a membrane skeletal protein, 4.1G, for Sertoli/germ cell interaction

Reproduction ◽  
2010 ◽  
Vol 139 (5) ◽  
pp. 883-892 ◽  
Author(s):  
Nobuo Terada ◽  
Nobuhiko Ohno ◽  
Sei Saitoh ◽  
Yurika Saitoh ◽  
Masayuki Komada ◽  
...  
Keyword(s):  
Adhesion Molecule ◽  
Germ Cells ◽  
Cell Membranes ◽  
Cell Interaction ◽  
Seminiferous Tubules ◽  
Wild Type ◽  
Skeletal Protein ◽  
Deficient Mice

We previously reported that a membrane skeletal protein, 4.1G (also known as EPB41L2), is immunolocalized in mouse seminiferous tubules. In this study, the 4.1G immunolocalizaiton was precisely evaluated at various stages of the mouse seminiferous epithelial cycle with ‘in vivocryotechnique’ and also with pre-embedding immunoelectron microscopy in testicular tissues whose ultrastructures were well preserved with glycerol treatment before cryosectioning. In addition, 4.1G-deficient mice were produced, and the morphology of their seminiferous tubules was also evaluated. The 4.1G immunolocalization was different among stages, indicating that it was not only along cell membranes of Sertoli cells, but also those of spermatogonia and early spermatocytes. To confirm the 4.1G immunolocalization in germ cells,in vitroculture of spermatogonial stem cells (SSCs) was used for immunocytochemistry and immunoblotting analysis. In the cultured SSCs, 4.1G was clearly expressed and immunolocalized along cell membranes, especially at mutual attaching regions. In testicular tissues, cell adhesion molecule-1 (CADM1), an intramembranous adhesion molecule, was colocalized on basal parts of the seminiferous tubules and immunoprecipitated with 4.1G in the tissue lysate. Interestingly, in the 4.1G-deficient mice, histological manifestation of the seminiferous tubules was not different from that in wild-type mice, and the CADM1 was also immunolocalized in the same pattern as that in the wild-type. Moreover, the 4.1G-deficient male mice were fertile. These results were probably due to functional redundancy of unknown membrane skeletal molecules in germ cells. Thus, a novel membrane skeletal protein, 4.1G, was found in germ cells, and considering its interaction with CADM family, it probably has roles in attachment of both Sertoli–germ and germ–germ cells.

Differentiation of Human Male Germ Cells From Human Umbilical Cord-Derived Mesenchymal Stem Cells in Vitro and In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4581-4581
Author(s):  
Lian Ma ◽  
Xiaoying Wu ◽  
Limin Lin ◽  
Guangyu Lin ◽  
Qiuling Tang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Umbilical Cord ◽  
Germ Cells ◽  
Seminiferous Tubules ◽  
Human Umbilical Cord ◽  
Cell Markers ◽  
Human Male

Abstract Abstract 4581 Introduction Infertility affects 15% of couples, about 50% infertility caused by male and growing evidence suggested an increasing problems in male reproductive. Recent studies have demostrated that adult stem cells have more flexible potentials than expected, and possessed the plasticity and capacity to transdifferentiate into mutilineage cells, including germ cells. Human umbilical cord-derived mesenchymal stem cells (HUCMSCs) possess stem cell properties. In this study, we cultured HUCMSCs, and assessed the possibility of HUCMSCs differentiated into human male germ cells in vivo and in vitro, and find a new source of cells for the transplantation to the male infertility. Methods The ethics committee of our institution approved this study. HUCMSCs were isolated from the Wharton's jelly of the umbilical cord, clonally expanded. To investigate the capacity of differentiation in vitro, HUCMSCs were treated with human menopausal gonadotropinn (HMG) and retinoid acid (RA) in vitro. While investigate the capacity of differentiation in vivo, HUCMSCs were transplanted into the seminiferous tubules of busulfan-treated mice testes after labeled with pIRES2-EGFP or bromodeoxyuridine (BrdU). After induction in vitro, the morphologic changes of the differentiated cells were detected by phase contrast microscopy?Aelectron microscopy and transmission electron microscope?Gthe male germ cell markers were detected by immunohistochemistry, Western-blot and PT-PCR. HUCMSCs were also transplanted into the seminiferous tubules of the busulfan-treated mice by microinjection. To assess the fate of HUCMSCs in the testis, the survival?Amigration and germ cell markers of the HUCMSCs in the infertility mice testis were detected by immunohistochemistry?A immunofluorescence. Results HUCMSCs can express some some molecular markers of germ cells after induction. Immunohistochemistry revealed that HUCMSCs can survive in the mice testis at least 120 days, and they can migrate from the lumens to the basement membrane then to lumens again. Immunofluorescence showed that HUCMSCs can go further differentiation in the mice favorable testicular environment, and express the germ cell markers. Conclusions These suggested that HUCMSCs may differentiate into male germ cell-like cells after induced by HMG and RA in vitro; and it could survive also in a favorable testicular environment, may differentiate into germ cell lineages. This finding may provide a new strategy for the treatment of male infertility. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Novel Peptide CM 7 Targeted c-Met with Antitumor Activity

Molecules ◽  
2020 ◽  
Vol 25 (3) ◽  
pp. 451
Author(s):  
Chunlei Xia ◽  
Ying Wang ◽  
Chen Liu ◽  
Liwen Wang ◽  
Xinmei Gao ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Specific Binding ◽  
Biological Activities ◽  
Inhibitory Effect ◽  
Tyrosine Kinase Receptor ◽  
Tyrosine Kinase Signaling ◽  
Mesenchymal Epithelial Transition Factor ◽  
Factor C

Anomalous changes of the cell mesenchymal–epithelial transition factor (c-Met) receptor tyrosine kinase signaling pathway play an important role in the occurrence and development of human cancers, including gastric cancer. In this study, we designed and synthesized a novel peptide (CM 7) targeting the tyrosine kinase receptor c-Met, that can inhibit c-Met-mediated signaling in MKN-45 and U87 cells. Its affinity to human c-Met protein or c-Met-positive cells was determined, which showed specific binding to c-Met with high affinity. Its biological activities against MKN-45 c-Met-positive cells were evaluated in vitro and in vivo. As a result, peptide CM 7 exhibited moderate regulation of c-Met-mediated cell proliferation, migration, invasion, and scattering. The inhibitory effect of peptide CM 7 on tumor growth in vivo was investigated by establishing a xenograft mouse model using MKN-45 cells, and the growth inhibition rate of tumor masses for peptide CM 7 was 62%. Based on our data, CM 7 could be a promising therapeutic peptide for c-Met-dependent cancer patients.

scholarly journals The efficiency of use lentiviral vectors for the transformation of rooster spermatogenic cells in vivo

2019 ◽  
Vol 14 (2) ◽  
pp. 162-169
Author(s):  
Natalya Alexandrovna Volkova ◽  
Anastasia Nikolaevna Vetokh ◽  
Lyudmila Aleksandrovna Volkova ◽  
Anatolievna Zinovyeva Nataliya
Keyword(s):  
Genetic Transformation ◽  
Reporter Gene ◽  
Germ Cells ◽  
Lentiviral Vectors ◽  
Seminiferous Tubules ◽  
Target Cells ◽  
Spermatogenic Cells ◽  
Viral Preparation

Male gonad cells are considered as promising target cells for the introduction of recombinant DNA within obtaining genetically modified individuals with given characteristics. The use of testicular spermatogonial stem cells is of the greatest interest. In the process of differentiation, this type of cell gives rise to a significant population of mature male germ cells. In the case of their genetic transformation, differentiated cells can be used to inseminate females in order to produce transgenic progeny. The aim of the research was to study the efficiency of using lentiviral vectors for the local transformation of roosters’ testicular spermatogenic cells. We used a lentiviral vector containing the ZsGreen reporter gene under the control of the CMV promoter. In vitro transformation of rooster spermatogenic cells was carried out by infection with a viral preparation, in vivo through multiple injections of the viral preparation into the testicular parenchyma of roosters ( n = 5). The efficiency of transformation was assessed by expression of the reporter ZsGreen gene in transfected spermatogenic cells. The success of using lentiviral vectors for the genetic transformation of rooster spermatogenic cells was shown in experiments in vitro and in vivo . The transformation efficiency of this cells types in an in vitro culture varied from 45 to 57% and averaged 48 ± 4%. The expression of the ZsGreen reporter gene in the cells of the spermatogenic epithelium of the testes was established in almost all experimental roosters in the in vivo experiments. The number of seminiferous tubules with transformed spermatogenic cells varied in the studied experimental roosters from 10 to 22%. The effectiveness of genetic transformation of the testes spermatogenic cells was 1.8 ± 0.2%. The obtained results indicate to the success of using lentiviral vectors for the genetic transformation of spermatogenic cells of rooster testes in vivo in order to create individuals with genetically transformed germ cells for the further production of transgenic offspring with given characteristics.

Molecular iodine synergized and sensitized neuroblastoma cells to the antineoplastic effect of ATRA

2020 ◽  
Vol 27 (12) ◽  
pp. 699-710
Author(s):  
Irasema Mendieta ◽  
Gabriel Rodríguez-Gómez ◽  
Bertha Rueda-Zarazúa ◽  
Julia Rodríguez-Castelán ◽  
Winniberg Álvarez-León ◽  
...  
Keyword(s):  
Residual Disease ◽  
Neuroblastoma Cells ◽  
Survivin Expression ◽  
Body Weight Loss ◽  
Immunohistochemical Analysis ◽  
Molecular Iodine ◽  
Tyrosine Kinase Receptor ◽  
Adjuvant Effect

Neuroblastoma (NB) is the most common solid childhood tumor, and all-trans retinoic acid (ATRA) is used as a treatment to decrease minimal residual disease. Molecular iodine (I2) induces differentiation and/or apoptosis in several neoplastic cells through activation of PPARγ nuclear receptors. Here, we analyzed whether the coadministration of I2 and ATRA increases the efficacy of NB treatment. ATRA-sensitive (SH-SY5Y), partially-sensitive (SK-N-BE(2)), and non-sensitive (SK-N-AS) NB cells were used to analyze the effect of I2 and ATRA in vitro and in xenografts (Foxn1 nu/nu mice), exploring actions on cellular viability, differentiation, and molecular responses. In the SH-SY5Y cells, 200 μM I2 caused a 100-fold (0.01 µM) reduction in the antiproliferative dose of ATRA and promoted neurite extension and neural marker expression (tyrosine hydroxylase (TH) and tyrosine kinase receptor alpha (Trk-A)). In SK-N-AS, the I2 supplement sensitized these cells to 0.1 μM ATRA, increasing the ATRA-receptor (RARα) and PPARγ expression, and decreasing the Survivin expression. The I2 supplement increased the mitochondrial membrane potential in SK-N-AS suggesting the participation of mitochondrial-mediated mechanisms involved in the sensibilization to ATRA. In vivo, oral I2 supplementation (0.025%) synergized the antitumor effect of ATRA (1.5 mg/kg BW) and prevented side effects (body weight loss and diarrhea episodes). The immunohistochemical analysis showed that I2 supplementation decreased the intratumoral vasculature (CD34). We suggest that the I2 + ATRA combination should be studied in preclinical and clinical trials to evaluate its potential adjuvant effect in addition to conventional treatments.

scholarly journals Targeting USP47 overcomes tyrosine kinase inhibitor resistance and eradicates leukemia stem/progenitor cells in chronic myelogenous leukemia

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hu Lei ◽  
Han-Zhang Xu ◽  
Hui-Zhuang Shan ◽  
Meng Liu ◽  
Ying Lu ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Progenitor Cells ◽  
Chronic Myelogenous Leukemia ◽  
Drug Targets ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Myelogenous Leukemia ◽  
Tki Resistance

AbstractIdentifying novel drug targets to overcome resistance to tyrosine kinase inhibitors (TKIs) and eradicating leukemia stem/progenitor cells are required for the treatment of chronic myelogenous leukemia (CML). Here, we show that ubiquitin-specific peptidase 47 (USP47) is a potential target to overcome TKI resistance. Functional analysis shows that USP47 knockdown represses proliferation of CML cells sensitive or resistant to imatinib in vitro and in vivo. The knockout of Usp47 significantly inhibits BCR-ABL and BCR-ABLT315I-induced CML in mice with the reduction of Lin−Sca1+c-Kit+ CML stem/progenitor cells. Mechanistic studies show that stabilizing Y-box binding protein 1 contributes to USP47-mediated DNA damage repair in CML cells. Inhibiting USP47 by P22077 exerts cytotoxicity to CML cells with or without TKI resistance in vitro and in vivo. Moreover, P22077 eliminates leukemia stem/progenitor cells in CML mice. Together, targeting USP47 is a promising strategy to overcome TKI resistance and eradicate leukemia stem/progenitor cells in CML.

scholarly journals Integrin α6 mediates the drug resistance of acute lymphoblastic B-cell leukemia

Blood ◽  
2020 ◽  
Vol 136 (2) ◽  
pp. 210-223 ◽  
Author(s):  
Eun Ji Gang ◽  
Hye Na Kim ◽  
Yao-Te Hsieh ◽  
Yongsheng Ruan ◽  
Heather A. Ogana ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Kinase Inhibitor ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Underlying Mechanism ◽  
Conditional Knockout

Abstract Resistance to multimodal chemotherapy continues to limit the prognosis of acute lymphoblastic leukemia (ALL). This occurs in part through a process called adhesion-mediated drug resistance, which depends on ALL cell adhesion to the stroma through adhesion molecules, including integrins. Integrin α6 has been implicated in minimal residual disease in ALL and in the migration of ALL cells to the central nervous system. However, it has not been evaluated in the context of chemotherapeutic resistance. Here, we show that the anti-human α6-blocking Ab P5G10 induces apoptosis in primary ALL cells in vitro and sensitizes primary ALL cells to chemotherapy or tyrosine kinase inhibition in vitro and in vivo. We further analyzed the underlying mechanism of α6-associated apoptosis using a conditional knockout model of α6 in murine BCR-ABL1+ B-cell ALL cells and showed that α6-deficient ALL cells underwent apoptosis. In vivo deletion of α6 in combination with tyrosine kinase inhibitor (TKI) treatment was more effective in eradicating ALL than treatment with a TKI (nilotinib) alone. Proteomic analysis revealed that α6 deletion in murine ALL was associated with changes in Src signaling, including the upregulation of phosphorylated Lyn (pTyr507) and Fyn (pTyr530). Thus, our data support α6 as a novel therapeutic target for ALL.

scholarly journals The FMS like Tyrosine Kinase 3 (FLT3) Is Overexpressed in a Subgroup of Multiple Myeloma Patients with Inferior Prognosis

Cancers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2341
Author(s):  
Normann Steiner ◽  
Karin Jöhrer ◽  
Selina Plewan ◽  
Andrea Brunner-Véber ◽  
Georg Göbel ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tyrosine Kinase ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Tyrosine Kinase Receptor ◽  
Bone Marrow Biopsies ◽  
Flt3 Inhibitors ◽  
Flt3 Expression

Therapy resistance remains a major challenge in the management of multiple myeloma (MM). We evaluated the expression of FLT3 tyrosine kinase receptor (FLT3, CD135) in myeloma cells as a possible clonal driver. FLT3 expression was analyzed in bone marrow biopsies of patients with monoclonal gammopathy of undetermined significance or smoldering myeloma (MGUS, SMM), newly diagnosed MM (NDMM), and relapsed/refractory MM (RRMM) by immunohistochemistry (IHC). FLT3 gene expression was analyzed by RNA sequencing (RNAseq) and real-time PCR (rt-PCR). Anti-myeloma activity of FLT3 inhibitors (midostaurin, gilteritinib) was tested in vitro on MM cell lines and primary MM cells by 3H-tymidine incorporation assays or flow cytometry. Semi-quantitative expression analysis applying a staining score (FLT3 expression IHC-score, FES, range 1–6) revealed that a high FES (>3) was associated with a significantly shorter progression-free survival (PFS) in NDMM and RRMM patients (p = 0.04). RNAseq and real-time PCR confirmed the expression of FLT3 in CD138-purified MM samples. The functional relevance of FLT3 expression was corroborated by demonstrating the in vitro anti-myeloma activity of FLT3 inhibitors on FLT3-positive MM cell lines and primary MM cells. FLT3 inhibitors might offer a new targeted therapy approach in a subgroup of MM patients displaying aberrant FLT3 signaling.

scholarly journals Genetic deletion of trkB.T1 increases neuromuscular function

2012 ◽  
Vol 302 (1) ◽  
pp. C141-C153 ◽  
Author(s):  
Susan G. Dorsey ◽  
Richard M. Lovering ◽  
Cynthia L. Renn ◽  
Carmen C. Leitch ◽  
Xinyue Liu ◽  
...  
Keyword(s):  
Calcium Release ◽  
Contractile Activity ◽  
Muscle Tension ◽  
Neuromuscular Synapse ◽  
Neuromuscular Function ◽  
In Vitro Assays ◽  
Tyrosine Kinase Receptor ◽  
Akt Activation

Neurotrophin-dependent activation of the tyrosine kinase receptor trkB.FL modulates neuromuscular synapse maintenance and function; however, it is unclear what role the alternative splice variant, truncated trkB ( trkB.T1), may have in the peripheral neuromuscular axis. We examined this question in trkB.T1 null mice and demonstrate that in vivo neuromuscular performance and nerve-evoked muscle tension are significantly increased. In vitro assays indicated that the gain-in-function in trkB.T1 −/− animals resulted specifically from an increased muscle contractility, and increased electrically evoked calcium release. In the trkB.T1 null muscle, we identified an increase in Akt activation in resting muscle as well as a significant increase in trkB.FL and Akt activation in response to contractile activity. On the basis of these findings, we conclude that the trkB signaling pathway might represent a novel target for intervention across diseases characterized by deficits in neuromuscular function.

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