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Leydig Cells

Leydig cells are a type of interstitial cell found in the testes that produce testosterone, the primary male sex hormone.
These cells play a crucial role in male sexual development and function.
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Most cited protocols related to «Leydig Cells»

Single-Cell Transcriptomics of Hydra

Hydra vulgaris AEP and Hydra vulgaris transgenic lines were dissociated into single cells and were prepared for Drop-seq (49 (link)); FACS was used to enrich for neurons. Sequencing reads were mapped to a de novo assembled transcriptome and a Hydra genome reference and clustering was performed. Subclustering was performed on the following subsets of the data: epithelial ectodermal cells, epithelial endodermal cells, interstitial cells, and neurons and neuronal progenitors. The in situ location of neuron subclusters was determined using in situ hybridization and differential gene expression analysis of separated epithelial layers. URD (24 (link)) was used to build differentiation trajectories for the interstitial and male germline lineages and to analyze the spatial expression of genes in the ectodermal, endodermal, and gland lineages. To analyze regulatory regions, co-expression modules were identified using NMF, ATAC-seq was performed to identify regions of open chromatin, and motif enrichment analysis was used to identify candidate regulators of the gene modules. Colorimetric in situ hybridization, fluorescent in situ hybridization, immunohistochemistry, and generation of transgenic lines was performed and used to validate biomarkers and cell states. For complete methods see supplementary material and methods.

Siebert S., Farrell J.A., Cazet J.F., Abeykoon Y., Primack A.S., Schnitzler C.E, & Juliano C.E. (2019). Stem cell differentiation trajectories in Hydra resolved at single-cell resolution. Science (New York, N.Y.), 365(6451), eaav9314.

Publication 2019

Animals, Transgenic ATAC-Seq Biological Markers Cells Chromatin Colorimetry Ectoderm Endoderm Epithelial Cells Fluorescent in Situ Hybridization Gene Expression Gene Expression Profiling Genes, Regulator Genome Germ Line Hydra Immunohistochemistry In Situ Hybridization Leydig Cells Males Neurons Regulatory Sequences, Nucleic Acid Transcriptome

Testis Cell Dissociation Protocol

Testis dissociation was based on a recently described method (5 ) with modifications. Our final protocol was conducted based on six consecutive steps described below.

Testis digestion: After the removal of tunica albuginea, each testis was placed in a 15 ml conical tube on ice with 6 ml “Collagenase I/Dnase I” solution. The tube was sealed with parafilm, shaken in horizontal position at 150 rpm for 10 min at 35°C. The temperature and agitation speed were the same for all subsequent incubation steps. Halfway into the 10 min incubation, the testis were gently pipetted, up and down twice to help tubule dispersion. This and all other pipetting steps were done using disposable transfer pipets. By the end of this step, tubules appeared thin and dispersed.

Somatic cell removal: Tubules were allowed to settle for 2 min at room temperature (RT) by standing the tube vertically. The supernatant, enriched in interstitial testicular cells was removed, leaving just enough liquid to cover the settled tubules.

Seminiferous tubule digestion: 6 ml of pre-heated “Collagenase I/Dnase I/Trypsin” solution was added to the tube and the tubules were gently pipetted up and down 10 times. Halfway into the 25 min digestion period, 60 μl of 2.5% Trypsin was added, and the tubules were pipetted again 10 times. At the end of the incubation time, pipetting was repeated 10 times. The tubules appeared fragmented and solution dense with cells. The resulting suspension was passed through a 100 μm nylon cell strainer. At this point, a 100 μl aliquot was removed to be processed for cell counting and viability estimation (see Materials and Methods below) while the rest of filtered cell suspension was pre-stained with Hoechst dye (during step 4 below).

Pre-staining with 100 μg Hoechst dye: To the resulting filtered cell suspension, 10 μl of DNAse I (1 mg/ml) and 10 μl of Hoechst dye (10 mg/ml) were added. The suspension was pipetted up and down 10 times and incubated for 20 min. Halfway into the 20 min period, the suspension was pipetted again. At the end of incubation, 600 μl of NCS was added to inactivate trypsin and the suspension was pipetted up and down 5 times.

Staining with Hoechst: After determining the cell number, the suspension was spiked with 10 μl of DNAse I (1 mg/ml), and stained with Hoechst dye for the final 6 μg Hoechst/million cells. The suspension was pipetted up and down 10 times and incubated for 25 min. Halfway into the incubation period, the solution was pipetted again, and then once more at the end of the incubation. Finally, the suspension was passed through a 40 μm nylon cell strainer. The suspension was kept on ice until sorting, which usually proceeded within 30 min to one hour after the completion of step 5.

Staining with PI: Immediately prior to sorting, 2 ml cell suspension was removed into a 5 ml polypropylene culture tube, the cells were stained with 10 μl of PI at room temperature and filtered into a tube with 35 μm cell strainer cap.

Gaysinskaya V., Soh I.Y., van der Heijden G.W, & Bortvin A. (2014). Optimized flow cytometry isolation of murine spermatocytes. Cytometry. Part A : the journal of the International Society for Analytical Cytology, 85(6), 556-565.

Publication 2014

Cells Collagenase Collagenase, Clostridium histolyticum Deoxyribonuclease I Digestion Diploid Cell Leydig Cells Nylons One-Step dentin bonding system Polypropylenes PRSS2 protein, human Seminiferous Tubule Testis Trypsin

Thrombotic and Anti-Thrombotic Markers in Porcine Aortic Valve Aging

AV leaflets were dissected from fresh porcine hearts acquired from commercial abattoirs and assigned into one of three age groups: YNG (6 weeks old), ADT (6 month old), or OLD (2 years old). Immunostains for numerous thrombotic and anti-thrombotic markers (Table 1) were performed on the AV leaflets of different ages, and the proportion of the tissue stained was quantified. The same hemostatic markers were also assessed using qRT-PCR and immunocytochemistry on porcine aortic valve endothelial cell (PAVEC) cultures of the three age groups, human umbilical vein endothelial cell (HUVEC) and porcine pulmonary artery endothelial cell (PPAEC) cultures. A sandwich ELISA was performed to quantify levels of VWF protein release and cleavage from histamine stimulated PAVECs, HUVECs, and PPAECs. To assess the effects of VEC-released VWF on calcific nodule formation in vitro, conditioned culture mediums consisting of 3% (v/v) PAVEC stimulation medium supernatant (via histamine stimulation) from the three age groups (YNG, ADT, OLD) were mixed with low serum VIC culture medium, and exposed to porcine aortic valve interstitial cells PAVICs) for 10 days. For study controls, PAVICs were also cultured in VIC low serum medium only, low serum VIC medium with 3% fresh PAVEC stimulation medium and no histamine, and low serum PAVIC medium with 3% fresh PAVEC stimulation medium with 8.4 nM histamine. PAVICs were stained with Alizarin Red S to dye calcified nodules red, and the number nodules and nodule sizes were measured. A full description of the materials and methods is available in the online-only Data Supplement.

Balaoing L.R., Post A.D., Liu H., Minn K.T, & Grande-Allen K.J. (2013). Age-related changes in aortic valve hemostatic protein regulation. Arteriosclerosis, thrombosis, and vascular biology, 34(1), 72-80.

Publication 2013

Age Groups Alizarin Red S Cell Culture Techniques Culture Media, Conditioned Cytokinesis Dietary Supplements Endothelial Cells Endothelium Enzyme-Linked Immunosorbent Assay Heart Hemostasis Histamine Human Umbilical Vein Endothelial Cells Immunocytochemistry Leydig Cells Pigs Proteins Pulmonary Artery Serum Tissues Valves, Aortic

Purification of Primary Sertoli Cells

This step is performed 36–48 h after plating Sertoli cells on 100-mm dishes, multiwell dishes or glass coverslips to remove residual germ cells from Sertoli cells, so that these Sertoli cells will be contaminated with negligible germ cells and Leydig cells (see Note 4.1). For hypotonic treatment:

Prepare 20 mM Tris, pH 7.4 (100–500 ml), depending on the number of dishes/plates that need to be treated.

Filter the above buffer to sterilize.

Buffer must be sterilized by filtration through a 0.2-μm filtering unit.

Aspirate media with a Pasteur pipette.

Add an appropriate volume of the above buffer per dish (e.g., ~10 ml per 100-mm dish; ~ 2 ml per well in a six-well dish) for exactly 2.5 min to lyse contaminating germ cells.

Aspirate buffer with a Pasteur pipette.

Wash two times with F12/DMEM to remove residual Tris buffer.

Add F12/DMEM supplemented with four factors as described above.

Incubate cells in a humidified CO₂ incubator at 35°C with 95% air and 5% CO₂ (v/v).

Feed cells every 3–4 days (low cell density) or every 1–2 days (high cell density) with growth factor supplemented-F12/DMEM.

Mruk D.D, & Cheng C.Y. (2011). An In Vitro System to Study Sertoli Cell Blood-Testis Barrier Dynamics. Methods in molecular biology (Clifton, N.J.), 763, 237-252.

Publication 2011

Buffers Cells Filtration Germ Cells Growth Factor Hyperostosis, Diffuse Idiopathic Skeletal Leydig Cells Sertoli Cells Strains Tromethamine

Isolation and Characterization of Human Aortic Valve Cells

Primary VEC and VIC were harvested from non‐calcified cusps (or portions of the cusp) of human aortic valves obtained from 3 patients who underwent valve replacement surgery (according to Dr Carol Davila Central Military Emergency University Hospital protocol) for severe calcific aortic valve stenosis. The investigation was carried out according to the principles outlined in the Declaration of Helsinki for experiments involving human samples.²² Participants gave their written informed consent by signing the appropriate paperwork and respecting their anonymity and privacy rights. The Ethics Committee of the Institute of Cellular Biology and Pathology ‘Nicolae Simionescu’ has approved the study.
Calcific deposits or thickened lesions were removed from the leaflet, and the remaining non‐calcified tissue was cut into smaller pieces that were enzymatically digested for 5‐10 minutes with collagenase I (Biochrom/Merck) at 37°C. Released VEC were cultured in endothelial cell growth medium with 20% FBS (Gibco) and 100 U/mL penicillin, 100 μg/mL streptomycin and 50 μg/mL neomycin (Sigma‐Aldrich, Germany). VEC used in this work were isolated from three different patients (characterized in Table S1). For VIC isolation, enzymatic digestion of valve pieces was continued for 4‐5h at 37°C, using Liberase (Roche, Sigma). The resulted VIC were cultured in DMEM with 15% FBS and antibiotics. To limit the inter‐individual variability, we performed the majority of experiments using commercial VIC (cryo‐preserved valvular Interstitial cells/ P10462—Innoprot), except for VIC used in Figure 1B and Figure S1B, that were obtained from a single patient.
Following the first cell passage, CD31‐positive VEC were purified using magnetic beads conjugated to monoclonal anti‐human CD31 antibody (Miltenyi Biotec). VEC phenotype was confirmed by contact inhibited growth and expression of endothelial‐specific markers such as PECAM1 (CD31) and von Willebrand factor (vWF). VIC phenotype was established by expression of vimentin and alpha‐smooth muscle actin (α‐SMA).

Vadana M., Cecoltan S., Ciortan L., Macarie R.D., Tucureanu M.M., Mihaila A.C., Droc I., Butoi E, & Manduteanu I. (2020). Molecular mechanisms involved in high glucose‐induced valve calcification in a 3D valve model with human valvular cells. Journal of Cellular and Molecular Medicine, 24(11), 6350-6361.

Publication 2020

alpha-Actin Antibiotics Antibodies, Anti-Idiotypic Aortic Stenosis, Calcific CD31 Antigens Collagenase, Clostridium histolyticum Cultured Cells Culture Media Digestion Emergencies Endothelium Enzymes Ethics Committees Factor VIII-Related Antigen Germ Cells Homo sapiens isolation Leydig Cells Liberase Military Personnel Neomycin Operative Surgical Procedures Patients Penicillins Phenotype Smooth Muscles Streptomycin Tissues TP63 protein, human Valves, Aortic Vimentin

Most recents protocols related to «Leydig Cells»

Single-cell Analysis of Cardiac and Liver Cells

The single-cell dataset of mouse cardiac interstitial cells at 3 and 7 days after sham operation or MI was downloaded from ArrayExpress under accession E-MTAB-7376 and that of mouse liver cells treated with corn oil or CCl₄ was downloaded from GEO under accession GSE171904. The data were processed using the Seurat package. We removed all cells with fewer than 200 genes, and all genes were expressed in fewer than three cells. In addition, cells with more than 10% or 20% mitochondrial genes were filtered out. Subsequently, gene expression measurements were normalized, and 2000 variable genes were selected for dimensionality reduction. The data were then scaled, and linear dimensional reduction was performed. The cells were then clustered and visualized using UMAP. Genes that were differentially expressed between the two groups were identified using the Seurat package, and a heat map was generated using the ggplot2 package. The gene abundance in Dbn1-positive cells (log2 (fold change) > 0.2) was analyzed using DAVID Bioinformatics Resources, version 6.8.

Hironaka T., Takizawa N., Yamauchi Y., Horii Y, & Nakaya M. (2023). The well-developed actin cytoskeleton and Cthrc1 expression by actin-binding protein drebrin in myofibroblasts promote cardiac and hepatic fibrosis. The Journal of Biological Chemistry, 299(3), 102934.

Publication 2023

CCL4 protein, human Corn oil Gene Expression Genes Genes, Mitochondrial Heart Hepatocyte Leydig Cells Mus Surgery, Day

Comparative analysis of embryonic and adult cardiac progenitor cells

Embryonic murine heart cells were used from our previous studies and other research groups^{23 (link),37 (link)} for the purpose of comparing our in vitro embryonic and adult cardiac cell CPC transcriptomes. Embryonic FACS-sorted CPC on days 7 and 9 in vitro differentiation and adult CPC were collected via FACS sorting and further analyzed using the Fluidigm C1 machine and workflow according to the manufacturer’s protocol. We examined a total of 20 cells derived from embryonic heart between E9.5-E10.5, 76 cells derived from P5 CPC, 22 Pdgfra⁺ interstitial adult cardiac cells and 240 YFP⁺ cells from D7 injured ventricles.
Sequence data have already been submitted to NCBI Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo) under the accession numbers GSE63796 and at CNCB with accession number PRJCA013789.

Siatra P., Vatsellas G., Chatzianastasiou A., Balafas E., Manolakou T., Papapetropoulos A., Agapaki A., Mouchtouri E.T., Ruchaya P.J., Korovesi A.G., Mavroidis M., Thanos D., Beis D, & Kokkinopoulos I. (2023). Return of the Tbx5; lineage-tracing reveals ventricular cardiomyocyte-like precursors in the injured adult mammalian heart. NPJ Regenerative Medicine, 8, 13.

Publication 2023

Adult Cells Embryo Gene Expression Heart Heart Ventricle Leydig Cells Mus Transcriptome

Histopathological Assessment of Organ Responses

After 28 days of the treatment, the heart left ventricle, skin, lung, spleen, and kidney of each group were fixed in a 10% neutral formaldehyde solution for 1 day, dehydrated in a conventional gradient, embedded in paraffin, sectioned with a thickness of 5 μm, and stained with HE. The morphology of myocardial tissue, skin, and organs at the site of application of QS was observed under the microscope, and the films were taken. Evaluation criteria of cardiac tissue lesions were as follows: myocardial fiber degeneration necrosis, myocardial fibrosis, and interstitial inflammatory cell infiltration were observed and rated as 0–4 points according to the degree of lesions from mild to severe. Normal was scored 0, mild or minimal amount was scored 0.5, mild or small amount was scored 1, moderate or more was scored 2, severe or large amount was scored 3, and a very severe or large amount was scored 4. All scores were accumulated, and the mean score “X ± SD” was calculated for each group. An independent sample t-test analysis was performed.

Zhao N., Ma Y., Liang X., Zhang Y., Hong D., Wang Y, & Bai D. (2023). Efficacy and Mechanism of Qianshan Huoxue Gao in Acute Coronary Syndrome via Regulation of Intestinal Flora and Metabolites. Drug Design, Development and Therapy, 17, 579-595.

Publication 2023

Aftercare Fibrosis Formalin Heart Inflammation Kidney Left Ventricles Leydig Cells Lung Microscopy Myocardium Myocytes, Cardiac Necrosis Paraffin Embedding Skin Spleen Tissues

Histopathological Assessment of Renal Tissue

The tissues were fixed in a solution of 10% formaldehyde for at least 24 h. Renal tissue was embedded in paraffin. Tissue sections (5 µm) were prepared using a microtome and mounted on slides. Masson’s trichrome stain (Merck KGaA, Darmstadt, Hesse, Germany) was used to detect interstitial fibrosis. In addition, hematoxylin–eosin staining (Merck KGaA, Darmstadt, Hesse, Germany) was also carried out to detect tubular injury (blebbing of the apical membrane into the tubular lumen, cell fragments within the tubular lumen, flattening of the tubular epithelium, or loss of nuclei), tubular atrophy, and interstitial infiltrate of inflammatory cells [44 ]. Hematoxylin–eosin and Masson’s trichrome staining were performed according to standard procedures. Slices were evaluated via images captured with a Moticam 1080 digital camera (Motic China Group Co Ltd, Xiamen, Fujian, China) attached to a Moticam BA310E optical microscope (Motic China Group Co Ltd, Xiamen, Fujian, China) with 10× and 40× objectives. All images were captured under the same conditions of light and exposure.
Renal histopathologic lesions were calculated as the percentage of the total area observed under the microscope [45 (link)]. The total area of the renal tissue cut was considered 100%, and the percentage of the cortical area affected by tubular injury, interstitial fibrosis, tubular atrophy, and interstitial infiltration of inflammatory cells was quantified. Additionally, the total percentage of the renal cortex tissue altered histopathologically was calculated and was the result of the sum of the percentages of tubular injury, tubular atrophy, fibrosis, and inflammatory infiltrate in the renal cortex. Tubular injury was defined as the flattening of the tubular epithelium with calcified or noncalcified cellular fragments within their lumens, blebbing of the apical membrane into the tubular lumen, or loss of nuclei [44 ]. Interstitial fibrosis was defined as increased extracellular matrix separating tubules in the cortical area [46 (link)], demonstrated as the blue-stained areas on Masson’s trichrome stains [47 (link)]. Tubular atrophy was defined by thick, irregular tubular basement membranes, with decreased diameters of tubules [46 (link)]. Interstitial infiltrate of inflammatory cells was defined as an excess of inflammatory cells within the cortical interstitium [46 (link)]. The evaluations were carried out blindly by two anatomopathologic experts.

Aurelien-Cabezas N.S., Paz-Michel B.A., Jacinto-Cortes I., Delgado-Enciso O.G., Montes-Galindo D.A., Cabrera-Licona A., Zaizar-Fregoso S.A., Paz-Garcia J., Ceja-Espiritu G., Melnikov V., Guzman-Esquivel J., Rodriguez-Sanchez I.P., Martinez-Fierro M.L, & Delgado-Enciso I. (2023). Protective Effect of Neutral Electrolyzed Saline on Gentamicin-Induced Nephrotoxicity: Evaluation of Histopathologic Parameters in a Murine Model. Medicina, 59(2), 397.

Publication 2023

Atrophy Cell Nucleus Cells Cortex, Cerebral Eosin Epithelium Extracellular Matrix Fibrosis Fingers Formalin Hematoxylin Inflammation Injuries Kidney Kidney Cortex Leydig Cells Light Light Microscopy Membrane, Basement Microscopy Microtomy Nephritis Paraffin Embedding Tissue, Membrane Tissues trichrome stain

Isolation and Purification of Rat Spermatogenic Cells

RSs were isolated from KO, KI and WT mice testes (10 to 12-week-old), as per the protocol described previously, with minor modifications [44 ]. Briefly, testes from three individual mice for each group were decapsulated, seminiferous tubules were mildly dispersed and digested using collagenase (1 mg/mL in 1× Krebs buffer; Worthington, NJ, USA) at 37 °C for 3 min to remove Leydig cells. After two washes with Krebs buffer, the tubules were digested with trypsin (0.6 mg/mL in 1× Krebs buffer; Sigma-Aldrich, St. Louis, MO, USA) containing DNase I (ThermoFisher Scientific, Waltham, MA, USA) at 34 °C for 15 min (~15 rpm). The obtained cell suspension was pre-chilled on ice and filtered with 40 μm filter (Millipore). The cells were centrifuged and cell pellet was washed with ice-cold Krebs buffer and mixed with 0.5% BSA, filtered again with 40 μm filter to obtain a single cell suspension. The germ cells (in 0.5% BSA-Krebs Buffer) were loaded onto the prepared BSA gradient (1% to 5% BSA-Krebs Buffer) and allowed to sediment for 90 min on ice. After sedimentation, the cell fractions (1 mL) were collected, washed in ice-cold Krebs buffer and the viability was measured by cell counters (Thermo Scientific). The purity of the RS fractions was verified with DAPI staining (Thermo Scientific) followed by microscopic examination (EVOS M-5000, Thermo Scientific).

Kavarthapu R., Anbazhagan R., Pal S, & Dufau M.L. (2023). Single-Cell Transcriptomic Profiling of the Mouse Testicular Germ Cells Reveals Important Role of Phosphorylated GRTH/DDX25 in Round Spermatid Differentiation and Acrosome Biogenesis during Spermiogenesis. International Journal of Molecular Sciences, 24(4), 3127.

Publication 2023

Buffers Cells Collagenase Common Cold DAPI Deoxyribonuclease I Germ Cells Leydig Cells Microscopy Mus Seminiferous Tubule Testis Trypsin

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More about "Leydig Cells"

Leydig cells are a vital component of the male reproductive system, playing a crucial role in the production of testosterone, the primary male sex hormone.
These interstitial cells, found within the testes, are responsible for the development and maintenance of male sexual characteristics and function.
Leydig cells are involved in a complex process that starts with the conversion of cholesterol to pregnenolone, a precursor for testosterone synthesis.
This process is regulated by the pituitary gland, which releases luteinizing hormone (LH) to stimulate Leydig cells to produce testosterone.
Testosterone produced by Leydig cells is essential for the proper development of male reproductive organs, the maturation of sperm, and the maintenance of secondary sexual characteristics, such as facial hair and a deepened voice.
Disruptions in Leydig cell function can lead to various reproductive and endocrine disorders, making them a key target for research and clinical interventions.
In Leydig cell research, researchers often utilize various cell culture media and reagents to maintain and study these cells.
For example, Fetal Bovine Serum (FBS) and Dulbecco's Modified Eagle Medium (DMEM) or DMEM/F12 are commonly used to culture Leydig cells.
Additionally, Penicillin/streptomycin is often added to the culture medium to prevent bacterial contamination.
Hyaluronidase may be used to dissociate Leydig cells from the testicular tissue, while TRIzol reagent is employed for RNA extraction and analysis.
To further enhance the reproducibility and accuracy of Leydig cell research, the PubCompare.ai platform can be utilized.
This AI-driven protocol comparison tool helps researchers locate the best research protocols from literature, preprints, and patents, enabling them to improve their experimental design and product selection.
By leveraging the power of PubCompare.ai, researchers can take their Leydig cell studies to the next level, ensuring robust and reliable results.