scholarly journals A disease-causing variant of COL4A5 in a Chinese family with Alport syndrome: a case series

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Jing Wu ◽  
Jun Zhang ◽  
Li Liu ◽  
Bo Zhang ◽  
Tomohiko Yamamura ◽  
...  
Keyword(s):  
Alport Syndrome ◽  
Wide Spectrum ◽  
Case Series ◽  
Pcr Analysis ◽  
Chinese Family ◽  
Compound Heterozygous ◽  
Rt Pcr ◽  
Col4a5 Gene ◽  
Minigene Assay

Abstract Background Alport syndrome (AS), which is a rare hereditary disease caused by mutations of genes including COL4A3, COL4A4 and COL4A5, has a wide spectrum of phenotypes. Most disease-causing variants of AS are located in the exons or the conservative splicing sites of these genes, while little is known about the intronic disease-causing variants. Methods A Chinese AS family was recruited in this study. All the clinical data of AS patient were collected from medical records. After pedigree analysis, the pathogenic variants were studied by the whole exome sequencing (WES). Minigene assay and in vivo RT-PCR analysis were performed to validate the functions of the variants. Results Renal biopsy showed a typical histopathology changes of AS. WES revealed compound heterozygous substitution, NM_033380 c.991–14(IVS17) A > G, in the intron 17 of the COL4A5 gene, which were confirmed by Sanger sequencing. Moreover, the variant was co-segregated with the phenotype in this family. Minigene assay in cultured cell lines showed that a splicing error was induced by this intronic variant, which further confirmed by in vivo RT-PCR analysis. Conclusion A novel intronic disease-causing variant in COL4A5 gene was identified by WES, which was the molecular pathogenic basis of AS.

In Vivo Modulation of T Cell and Monocyte Function Following Infusion of Mesenchymal Stromal Cells (MSC)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4739-4739
Author(s):  
Cristina Castilla-LLorente ◽  
Mineo Iwata ◽  
Marco Mielcarek ◽  
V. Kraig Abrams ◽  
Billanna Hwang ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Lymph Nodes ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Mononuclear Cells ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Blood Mononuclear Cells ◽  
Post Infusion

Abstract Mesenchymal stromal cells (MSCs) expanded ex vivo from aspirated marrow, have been used clinically with variable success to facilitate repair of infarcted hearts, treat graft versus host disease, and facilitate marrow reconstitution after radiation damage. While it is now generally acknowledged that these benefits are not the result of engraftment and differentiation of MSC into the target tissues, the mechanism by which these beneficial effects are achieved is not clear. We hypothesize that MSCs mediate their effect by activating an endogenous cell population which in turn modulates the immune response and/or homes to damaged tissue and participates in repair. To begin to test this hypothesis immortalized and cloned populations of canine MSC were generated to provide a consistent product for in vivo testing. One line, designated DS-1, has been evaluated in vivo by infusion into two normal dogs. Blood samples were taken pre infusion, immediately following infusion and at 1, 6, 24, 48, 72, 96 hours, and 7, 14, 21, and 28 days post infusion. Following infusion there was no consistent change in the number of WBC, however by day 3 there was a marked decrease in the % of CD3+ cells expressing FOXP3 and TGFβ in the blood, which did not recover to pre-infusion levels during the period of observation. At autopsy there was an increased number of these cells in the lymph nodes and spleen, whereas there was an overall decrease in the number of TH1 cells in these tissues. Quantitative RT- PCR analysis of cDNA prepared from blood mononuclear cells indicated an upregulation in the expression of CD133, Tie-2, and MARCO between 1–24 hours post infusion, and an increase in LOX1/OLR1 between 2–4 days. However the % of monocytes and the expression levels of CD14, CD68, CD45, and CD105/Endoglin were constant at all time points. Samples taken at 6 hours, 4 and 7 days post infusion were also analyzed for the presence of DS-1 cells by PCR and in vitro out growth assays. Results indicated that the DS-1cells were detectable up to 6 hours post infusion, but not thereafter. Adherent cells grown from blood mononuclear cells at days 4 and 7, displayed macrophage and endothelial cell morphologies. RT-PCR analysis of these cultures detected expression of macrophage associated markers CD14+/CD68+/MARCO+/LOX1+, as well as endothelial cell associated markers CD34+/CD144/VECAD+. These data indicate that a single infusion of DS-1 cells results in activation of circulating monocytes and a shift of regulatory T cells from the periphery to lymph nodes and spleen which persists for at least 28 days. We speculate that these changes may contribute to the immunomodulatory effects reported for some preparations of MSC.

Real-Time RT-PCR Analysis of Individual Osteolineage Cells within the Hematopoietic Stem Cell Niche

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2389-2389
Author(s):  
Lev Silberstein ◽  
Masatake Osawa ◽  
Charles Lin ◽  
Peter Kharchenko ◽  
Cristina Lo Celso ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Pcr Analysis ◽  
Rna Seq ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Chi Squared ◽  
The Individual ◽  
Osteolineage Cells ◽  
Angiopoietin 1

Abstract Abstract 2389 Osteolineage cells (OLCs) have been shown to participate in a regulatory bone marrow microenvironment for the hematopoietic stem and progenitor cells (HSPCs) – the endosteal niche. Our previous experiments using live animal imaging have demonstrated that single transplanted HSPCs preferentially home in close proximity to the individual OLCs. We hypothesized that these HSPC-proximal cells represent a distinct subpopulation of OLCs, which is specifically involved in a non-cell autonomous regulation of HSPC quiescence and self-renewal. To test this hypothesis, we developed a novel experimental platform, which allows visualization of HSPC-OLC cell pairs in-vivo and retrieval of the individual OLCs for molecular analysis. We intravenously injected DiI labeled adult bone marrow-derived FACS-sorted Lin−Sca1+c-kit+CD34−Flk2− HSPCs into irradiated newborn collagen 2.3GFP mouse recipients; in this transgenic strain, the majority of the OLCs are labeled with green fluorescent protein (GFP). 48 hours later, we sacrificed the animals and obtained fresh unfixed sections of femoral trabecular bone. Using a combination of differential interference contrast fluorescent microscopy, in-situ enzymatic digestion and micromanipulation, we harvested individual GFP-positive OLCs located within 2 cell diameters (“niche” OLCs) or greater than 5 cell diameters (“control” OLCs) from single DiI-bright HSPCs. Following reverse transcription and cDNA amplification with 29 cycles of PCR, as per the single cell RNA-Seq protocol (Tang et al, Nature Protocols 2010), we performed real-time RT-PCR analysis of 31 samples – 15 niche cells and 16 controls - for the OLC signature genes (osteocalcin, osterix) and for the genes implicated in playing a functional role in the HSPC-OLC cell interaction (osteopontin, CXCL12, angiopoietin 1). Transcripts for GAPDH, collagen 1 and GFP served as positive controls for the amplification. As expected, all cells were positive for GFP and over 85% cells expressed collagen 1. Osteopontin and CXCL12 were expressed at a similar level and frequency in the niche and control OLCs. However, we found that angiopoietin 1 transcripts were detected exclusively in the niche OLCs (3/15 versus 0/16, p <0.05 by Chi-squared). Moreover, niche OLCs were enriched for the osterix-positive cells (7/15 versus 2/16, p <0.05 by Chi-squared) and expressed a lower level of osteocalcin, as normalized for GAPDH expression (1.13 vs. 0.97, p< 0.05 by t-test). Our results suggest that niche OLCs may have a distinct molecular signature and reside within a population of very immature OLCs, as evidenced by the osterix + osteocalcin low phenotype. Further unbiased transcriptome characterization of these cells using genome-wide RNA-Seq assay is therefore likely to provide more evidence in support of our hypothesis and reveal novel non-cell autonomous regulators of HSPC quiescence. To our knowledge, this approach represents the first attempt to define molecular heterogeneity in-vivo at a single cell level using the micro-anatomical relationship between two heterologous cell types. Disclosures: Scadden: Fate Therapeutics: Equity Ownership.

scholarly journals The Effect of Hydroxyapatite Nanocrystals on Osseointegration of Titanium Implants: AnIn VivoRabbit Study

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Karin Breding ◽  
Ryo Jimbo ◽  
Mariko Hayashi ◽  
Ying Xue ◽  
Kamal Mustafa ◽  
...  
Keyword(s):  
Surface Chemistry ◽  
Calcium Phosphates ◽  
Surface Characteristics ◽  
Healing Time ◽  
Titanium Implants ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Implant Surface ◽  
Hydroxyapatite Nanocrystals

Osseointegration is dependent on implant surface characteristics, including surface chemistry and topography. The presence of nanosized calcium phosphates on the implant surface is interesting to investigate since they affect both the nanotopography and surface chemistry, forming a bone mineral resembling surface. In this work, the osseointegration of titanium implants with and without the presence of hydroxyapatite (HA) nanocrystals has been evaluatedin vivo. The integration was examined using removal torque measurements and real-time polymerase chain reaction (RT-PCR) analysis. The study was performed using two healing time points, 3 and 12 weeks. The results showed that the torque needed to remove the implants was insignificant between the non- and HA-coated implants, both at weeks 3 and 12. The RT-PCR, however, showed significant differences for osteoblast, osteoclast, and proinflammation markers when HA nanocrystals were present.

Human CB-Derived Cells Give Rise to Insulin-Producing-Cells in Xenogeneic Hosts.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3603-3603
Author(s):  
Shuro Yoshida ◽  
Fumihiko Ishikawa ◽  
Noriaki Kawano ◽  
Hua Zhang ◽  
Yuan Kong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Human Chromosome ◽  
Human Insulin ◽  
Mononuclear Cells ◽  
Pcr Analysis ◽  
Fish Analysis ◽  
Rt Pcr ◽  
Post Transplantation ◽  
Insulin Producing Cells

Abstract (Purpose) We examine the capacity of human cord blood (CB)-derived cells to generate insulin-producing cells and other lineages of cells in pancreatic tissue in vivo. (Method) Ten millions of human CB-derived T-cell-depleted mononuclear cells were intravenously transplanted into conditioned newborn NOD/SCID/b2-microglobulinnull mice with or without chemical injury by an intraperitoneal injection of streptozotocin (STZ) at dose of 100 mg/g body weight. At 1–3 months post-transplantation, pancreatic tissues of the recipient mice were analyzed for the presence of human CB-derived cells by performing immunofluorescence study (insulin, amylase, or CD45) and FISH analysis for human chromosomes on the same specimens. RNA was isolated from pancreatic tissues of recipient mice, and RT-PCR analysis using human insulin specific primer was performed to examine human insulin at RNA level. Finally, double FISH analysis for human- and murine chromosomes was performed to get an insight into the mechanism for the generation of human CB-derived insulin-producing cells in vivo. (Results) At 1–3 months post-transplantation, human CB-derived T-cell-depleted mononuclear cells gave rise to both myeloid and lymphoid progeny (CD33+, CD19+, and CD3+ cells) in bone marrow and peripheral blood of the recipient mice. In recipient pancreatic tissues, human CB-derived cells were identified inside and outside islets. Outside pancreatic islets, the vast majority of human chromosome+ cells were CD45+ hematopoietic cells, while human chromosome+ amylase+ acinar cells were also identified. Inside islets, human chromosome+ cells accounted for 1.01 +/− 0.73 % (n=6) without STZ treatment. Among them, human CB-derived insulin-producing cells were identified at a frequency of 0.65 +/− 0.64 % (n=6) of total insulin+ cells in xenogeneic hosts. RT-PCR analysis demonstrated the presence of human insulin, whose sequence was fully identical to that of already-known human insulin cDNA. Chemical injury with STZ treatment led to the significant destruction of islet tissue and reduction of cell numbers in islets. In STZ-treated recipient mice, however, human insulin-producing cells were identified at a frequency of 0.23 +/− 0.27 % (n=4) in islets, which was lower than the mice without STZ treatment. Finally, double FISH analyses using species-specific probes demonstrated the presence of human chromosome+ murine chromosome+ insulin-producing cells and human chromosome+ murine chromosome- insulin-producing cells in recipient islets. (Conclusion) It is concluded that human CB cells contain the progenitor cells to generate the insulin-producing cells in vivo. The mechanism of CB-derived insulin-producing cells includes both fusion-dependent and independent mechanisms. Although the capacity of CB-derived cells needs to be compared with other stem cell sources such as tissue stem cells or embryonic stem cells, the present study suggests the possibility of CB cells as new source for future regenerative medicine for diabetes mellitus.

Identification of Genes Associated with Resistance and Response in Vivo to Therapy with Rituximab, Fludarabine and Cyclophosphamide in Patients with Chronic Lymphocytic Leukemia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1622-1622 ◽  
Author(s):  
Medhat Shehata ◽  
Dita Demirtas ◽  
Stefanie Tauber ◽  
Susanne Schnabl ◽  
Martin Bilban ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Signature ◽  
Poor Response ◽  
Response To Therapy ◽  
Lymphocytic Leukemia ◽  
Pcr Analysis ◽  
Poor Responders ◽  
Rt Pcr ◽  
Number Of Patients

Abstract Fludarabine and cyclophosphamide are the backbone of therapy for patients with CLL. The addition of rituximab leads to further improvement of response rates and progression free survival (results from the randomized CLL8 study of the GCLLSG). However, a considerable number of patients have insufficient or short responses to FC or RFC and there is a need to identify factors influencing response or resistance to therapy. The aims of this study are to identify gene expression associated with response or resistance before the start of therapy, to investigate changes in the expression of specific genes or pathways associated with response or resistance during the first cycle of FC and RFC, to provide a rationale for the additional use of novel drugs to improve remission and overcome resistance. We investigated peripheral blood samples from 20 patients receiving FC (n=10) or RFC (n=10) by gene expression profiling, flow cytometry, RT-PCR and western blotting before and during therapy. Sixteen patients received FC or RFC as first line (8 within the CLL8 study) and 4 as second line treatment. All patients were in stage Binet B or C. Gene expression was analyzed and correlated to good (CR or PR) or poor clinical response (SD or PD) at the end of therapy based on NCI-WG/IWCLL criteria. CD19+ cells were harvested by cell sorting before therapy, 24 hours after FC (FC arm), 24 hours after rituximab, and 24 hours after FC (RFC arm). Microarray analysis was performed using Affymetrix U133A gene chips. Genes with a consistent pattern of expression (high or low) in the majority of samples in the good or poor response group were further analyzed. Overall, 9 patients responded adequately to therapy (3 CR, 7 PR), while 11 did not (7 SD, 3PD). Unmutated IgVH status and poor risk cytogenetics were more frequent in poor responders. Gene expression signature before treatment showed that overexpression of 39 genes strongly correlated with response, while overexpression of 20 genes (including HSPA1B, IFI6, APP, CEACAM1, CD9, GAB1, INPP5F) was associated with resistance. Changes in expression after initiation of treatment was also analyzed. Seven genes (including CENTD1, HBA2, COL9A2 and APRIN) were significantly upregulated after rituximab in non-responders. Upregulation of 13 genes (including PMAIP1, SFRS11, CLK1, EFHC1, MRPL39, TUG1, TBRG1, CD49d, PTPRC) after R-FC and 7 genes (including ITPKB, LOC641298, CD44, TAF5) after FC was associated with poor response (resistance) to RFC and FC respectively. Many of these genes are involved in regulation of apoptosis, cell cycle, integrin and PI3-K signaling Therapeutic antibodies or inhibitors against some of these targets are already available. RT-PCR analysis demonstrated a significant downregulation of Akt1 mRNA 24 hours after rituximab infusion in RFC group but no significant changes were observed in patients receiving FC alone. In vitro exposure to rituximab confirmed its in vivo effect and resulted in a significant downregulation of Akt1 and PI3-K-p85 mRNA expression. FACS analysis demonstrated a decrease in the percentage and mean fluorescence intensity (MFI) of surface CD20 after rituximab infusion. This effect was associated with a significant change in total amount and phosphorylation state of CD20 in the RFC group. There was also a decrease in the MFI of CD44 and CD23 after rituximab in the majority of patients in the RFC group but this effect was not consistent in the FC group. In conclusion, we have identified a set of markers associated with good or poor response to FC or RFC before therapy and during the first cycle of treatment. The data provide a rationale for targeted drug combinations to overcome resistance and improve response to therapy in CLL.

scholarly journals Transgenic Mice Overexpressing the Complement Inhibitor Crry as a Soluble Protein Are Protected from Antibody-induced Glomerular Injury

1998 ◽  
Vol 188 (7) ◽  
pp. 1321-1331 ◽  
Author(s):  
Richard J. Quigg ◽  
Chun He ◽  
Alice Lim ◽  
Dawn Berthiaume ◽  
Jessy J. Alexander ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Complex Disease ◽  
Alternative Pathway ◽  
Disease Model ◽  
Fractional Excretion ◽  
Transgenic Animals ◽  
Pcr Analysis ◽  
Glomerular Injury ◽  
Rt Pcr

Complement receptor 1–related gene/protein y (Crry) is a potent murine membrane complement regulator that inhibits classical and alternative pathway C3 convertases. In nephrotoxic serum (NTS) nephritis, injected antibodies (Abs) bind to glomeruli, leading to complement activation and subsequent glomerular injury and albuminuria. To study the phenotypic effects of continuous complement pathway blockade, transgenic mice were created that express recombinant soluble (rs) Crry directed by the broadly active and heavy metal-inducible metallothionein-I promoter. One transgenic line expressing high levels of rsCrry was propagated. Serum rsCrry levels were 18.7 ± 2.7 μg/ml (n = 5) at basal level and increased to 118.1 ± 20.6 μg/ml 4 d after addition of zinc to the drinking water. By reverse transcription polymerase chain reaction (RT-PCR), transgene messenger (m)RNA was present in liver, kidney, brain, lung, and spleen, but not in heart. By in situ RT-PCR analysis of kidneys, transgene mRNA was widely expressed both in renal glomeruli and tubules. Urinary excretion of rsCrry was 113.4 ± 22.4 μg/ml with a fractional excretion relative to creatinine of 13.2 ± 2.7%, consistent with local renal production of rsCrry and secretion into urine. The founder and all transgene positive adult animals have remained healthy with no mortality or apparent phenotypic abnormalities, including infection or immune complex disease. To determine whether rsCrry blocked complement-mediated injury, NTS nephritis was induced by injection of NTS immunoglobulin (Ig)G, followed by an 18-h urine collection to quantitate the excretion of albumin as a measure of glomerular injury. In transgene-negative littermates (n = 15), transgene-positive animals (n = 10), and transgene-positive animals fed zinc (n = 10), albuminuria was 4,393 ± 948, 1,783 ± 454, and 1,057 ± 277 μg/mg creatinine, respectively (P &lt; 0.01 by ANOVA). Glomerular C3 was evident by immunofluorescence staining in 12/15 transgene-negative animals, but in none of the transgene-positive animals fed zinc. Thus, we have produced the first transgenic animals that overexpress a soluble C3 convertase inhibitor. rsCrry expression markedly ameliorates an Ab-induced disease model in vivo. These results support the hypothesis that continuous complement inhibition at the C3 convertase step is feasible and effective in complement-mediated injury states.

scholarly journals Expression and Localization of Plasma Transglutaminase Factor XIIIA in Bone

2007 ◽  
Vol 55 (7) ◽  
pp. 675-685 ◽  
Author(s):  
Yukiko Nakano ◽  
Hadil F. Al-Jallad ◽  
Aisha Mousa ◽  
Mari T. Kaartinen
Keyword(s):  
Transglutaminase 2 ◽  
Pcr Primers ◽  
Cell Types ◽  
Proteolytic Processing ◽  
Factor Xiiia ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Osteoblast Cell Line

Transglutaminases (TGs) are protein crosslinking enzymes involved in cell adhesion and signaling and matrix stabilization and maturation, in many cell types and tissues. We previously described that in addition to transglutaminase 2 (TG2), cultured MC3T3-E1 osteoblasts also express the plasma TG Factor XIIIA (FXIIIA). Here we report on the expression and localization of FXIIIA in bone in vivo and provide confirmatory in vitro data. Immuno-histochemistry and in situ hybridization demonstrated that FXIIIA is expressed by osteoblasts and osteocytes in long bones formed by endochondral ossification (femur) and flat bones formed primarily by intramembranous ossification (calvaria and mandible). FXIIIA immuno-reactivity was localized to osteoblasts, osteocytes, and the osteoid. RT-PCR analysis revealed FXIIIA expression by both primary osteoblasts and by the MC3T3-E1 osteoblast cell line. Western blot analysis of bone and MC3T3-E1 culture extracts demonstrated that FXIIIA is produced mainly as a small, 37-kDa form. Sequential RT-PCR analysis using overlapping PCR primers spanning the full FXIIIA gene showed that the entire FXIIIA gene is expressed, thus indicating that the 37-kDa FXIIIA is not a splice variant but a product of posttranslational proteolytic processing. Forskolin inhibition of osteoblast differentiation revealed that FXIIIA processing is regulated by the protein kinase A pathway.

scholarly journals The Gonococcal Fur-Regulated tbpA and tbpB Genes Are Expressed during Natural Mucosal Gonococcal Infection

2005 ◽  
Vol 73 (7) ◽  
pp. 4281-4287 ◽  
Author(s):  
Sarika Agarwal ◽  
Carol A. King ◽  
Ellen K. Klein ◽  
David E. Soper ◽  
Peter A. Rice ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Iron Concentration ◽  
Pcr Analysis ◽  
Positive Trend ◽  
Gonococcal Infection ◽  
Rt Pcr ◽  
Male Subjects

ABSTRACT Iron is limiting in the human host, and bacterial pathogens respond to this environment by regulating gene expression through the ferric uptake regulator protein (Fur). In vitro studies have demonstrated that Neisseria gonorrhoeae controls the expression of several critical genes through an iron- and Fur-mediated mechanism. While most in vitro experiments are designed to determine the response of N. gonorrhoeae to an exogenous iron concentration of zero, these organisms are unlikely to be exposed to such severe limitations of iron in vivo. To determine if N. gonorrhoeae expresses iron- and Fur-regulated genes in vivo during uncomplicated gonococcal infection, we examined gene expression profiles of specimens obtained from male subjects with urethral infections. RNA was isolated from urethral swab specimens and used as a template to amplify, by reverse transcriptase PCR (RT-PCR), gonococcal genes known to be regulated by iron and Fur (tbpA, tbpB, and fur). The constitutively expressed gonococcal rmp gene was used as a positive control. RT-PCR analysis indicated that gonorrhea-positive specimens where rmp expression was seen were also 93% (51/55) fbpA positive, 87% (48/55) tbpA positive, and 86% (14 of 16 tested) tbpB positive. In addition, we detected a fur transcript in 79% (37 of 47 tested) of positive specimens. We also measured increases in levels of immunoglobulin G antibody against TbpA (91%) and TbpB (73%) antigens in sera from infected male subjects compared to those in uninfected controls. A positive trend between tbpA gene expression and TbpA antibody levels in sera indicated a relationship between levels of gene expression and immune response in male subjects infected with gonorrhea for the first time. These results indicate that gonococcal iron- and Fur-regulated tbpA and tbpB genes are expressed in gonococcal infection and that male subjects with mucosal gonococcal infections exhibit antibodies to these proteins.

scholarly journals Novel Expression of Type 1 Corticotropin-Releasing Hormone Receptor in Multiple Endocrine Cell Types in the Murine Anterior Pituitary

Endocrinology ◽  
2008 ◽  
Vol 150 (1) ◽  
pp. 260-267 ◽  
Author(s):  
Nicole J. Westphal ◽  
Ryan T. Evans ◽  
Audrey F. Seasholtz
Keyword(s):  
Hormone Receptor ◽  
Anterior Pituitary ◽  
Cell Types ◽  
Pituitary Cell ◽  
Pcr Analysis ◽  
Corticotropin Releasing Hormone ◽  
Rt Pcr ◽  
Releasing Hormone ◽  
Anterior Pituitary Cell

The CRH family of ligands signals via two distinct receptors, CRH-R1 and CRH-R2. Previous studies localized CRH-R1 and CRH-R2 to a subset of anterior pituitary corticotropes and gonadotropes, respectively. However, numerous studies have indicated that stress and CRH activity can alter the secretion of multiple anterior pituitary hormones, suggesting a broader expression of the CRH receptors in pituitary. To examine this hypothesis, the in vivo expression of CRH-R1 and CRH-R2 mRNA was further characterized in adult mouse pituitary. Quantitative RT-PCR analysis demonstrated that CRH-R1 mRNA is greater than 100-fold more abundant than CRH-R2 mRNA in male and female mouse pituitaries. Dual in situ hybridization analysis identified cell-specific CRH-R1 expression in the anterior pituitary. At least half of the CRH-R1-positive cells expressed proopiomelanocortin-mRNA (50% in females; 70% in males). In females, a significant percentage of the cells expressing CRH-R1 also expressed transcript for prolactin (40%), LHβ (10%), or TSH (3%), all novel sites of CRH-R1 expression. Similarly in males, a percentage of CRH-R1-positive cells expressed prolactin (12%), LHβ (13%), and TSH (5%). RT-PCR studies with immortalized murine anterior pituitary cell lines showed CRH-R1 and/or CRH-R2 expression in corticotropes (AtT-20 cells), gonadotropes (αT3-1 and LβT2 cells), and thyrotropes (αTSH cells). Whereas CRH-R1 expression in corticotropes is well established, the presence of CRH-R1 mRNA in a subset of lactotropes, gonadotropes, and thyrotropes establishes these cell types as novel sites of murine CRH-R1 expression and highlights the pituitary as an important site of interaction between the hypothalamus-pituitary-adrenal and multiple endocrine axes. Corticotropin-releasing hormone receptor 1 mRNA is detected in multiple mouse anterior pituitary cell types, including corticotropes, lactotropes, and gonadotropes, in a sexually dimorphic pattern.

scholarly journals Successful transplantation of spermatogonial stem cells into the seminiferous tubules of busulfan-treated mice

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Hossein Azizi ◽  
Amirreza Niazi Tabar ◽  
Thomas Skutella
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Spermatogonial Stem Cells ◽  
Pcr Analysis ◽  
Seminiferous Tubules ◽  
Flow Cytometry Analysis ◽  
Rt Pcr ◽  
Expression Levels ◽  
Successful Transplantation

Abstract Background Spermatogonial stem cells (SSCs) in the testis are crucial for transferring genetic information to the next generation. Successful transplantation of SSCs to infertile men is an advanced therapeutic application in reproductive biology research. Methods In this experimental research, both in vitro and in vivo characterization of undifferentiated and differentiated SSCs were performed by morphology—immunocytochemistry (ICC), immunohistochemistry (IMH), Fluidigm Real-Time polymerase chain reaction (RT-PCR) and flow cytometry analysis. The isolated SSCs were finally microinjected into the rete testis of busulfan-treated mice. The compact undifferentiated and more loosely connected round differentiated SSCs were isolated during testicular cell expansion from their specific feeder layer. Results ICC analysis indicated high and low expression levels of Zbtb16 in undifferentiated and differentiated germ cells. Also, IMH analysis showed different expression levels of Zbtb16 in the two different germ stem cell populations of the testicular tissue. While Fluidigm RT-PCR analysis indicated overexpression of the TAF4B germ cell gene, the expression of DAZL, VASA, and Zbtb16 were down-regulated during the differentiation of SSCs (P < 0.05). Also, flow cytometry analysis confirmed the significant downregulation of Itgb1 and Itga4 during differentiation. By transplantation of SSCs into busulfan-treated NOD/SCID mice, GFP-labeled sperm cells developed. Conclusions In the current study, we performed a transplantation technique that could be useful for the future microinjection of SSCs during infertility treatment and for studying in vivo differentiation of SSCs into sperm.

Sign in / Sign up

Export Citation Format

Share Document