Journal: Biofabrication

Article Title: Collagen hydrogel tube microbioreactors for cell and tissue manufacturing

doi: 10.1088/1758-5090/ae2718

Figure Lengend Snippet: Fabricating seminiferous tubules with coltubes. (A) Phase-contrast images of coltubes containing leydig cells in the collagen shell and sertoli cells inside the tube at days 0, 1, and 3. (B) Live/Dead staining of the tubules on day 5 shows minimal cell death. (C) Confocal images showing leydig cells (green) in the collagen shell and sertoli cells (red) inside the tube.

Article Snippet: TM3 Leydig cells (#CRL-1714, ATCC) and TM4 Sertoli cells (#CRL-1715, ATCC) were cultured according to ATCC instructions.

Techniques: Staining

Journal: Research

Article Title: Environmental Fluoride Compromises Male Fertility: Differentially Modulated miR-34a-5p Targets REST to Regulate Autophagy in Testicular Somatic Cells

doi: 10.34133/research.1113

Figure Lengend Snippet: Fluoride impairs male reproductive capacity by differentially regulating autophagy of testicular somatic cells in mice. (A to F) Six-week-old male mice freely drank distilled water with or without 100 mg/l NaF for 18 weeks. (A) Representative images of H&E staining on testis. Scale bars, 100 μm (left) and 50 μm (right). (B) Representative microscopic views of sperm morphology. Scale bars, 50 μm. (C) Sperm quality. (D and E) Representative transmission electron microscopy images of autophagosomes in (D) Leydig cells and (E) Sertoli cells [scale bars, 2 μm (bottom) and 1 μm (top)], with red arrows indicating autophagosomes. (F) Immunohistochemistry was used to detect the protein expression of LC3B and p62 in Leydig cells and Sertoli cells. (G and H) Testicular somatic cells were treated with various concentrations of NaF for 24 h. The relative expression of LC3B and p62 proteins in (G) TM3 cells, (H) TM4 cells, and (I) TM4 cells cotreated with 20 μM chloroquine. Cell viability of (J) TM3 and (K) TM4 cells that were treated with 0.25 mM NaF and 20 μM chloroquine for 24 h or pretreated with 20 nM rapamycin for 1 h. All values in the figure are means ± SEM. n ≥ 3. * P < 0.05, ** P < 0.01 versus control; # P < 0.05, ## P < 0.01 versus chloroquine.

Article Snippet: Murine Leydig cell line (TM3) and murine Sertoli cell line (TM4) were purchased from the American Type Culture Collection (ATCC) (Manassas, USA).

Techniques: Staining, Transmission Assay, Electron Microscopy, Immunohistochemistry, Expressing, Control

Journal: Research

Article Title: Environmental Fluoride Compromises Male Fertility: Differentially Modulated miR-34a-5p Targets REST to Regulate Autophagy in Testicular Somatic Cells

doi: 10.34133/research.1113

Figure Lengend Snippet: miR-34a-5p was selected as the key miRNA in the differential regulation of autophagy in testicular somatic cells by fluoride. (A) Network toxicological analysis results of miRNAs related to fluoride-induced male reproductive injury. (B) HAMDB database analysis results of autophagy-related candidate miRNAs. (C and D) qRT-PCR revealed autophagy-related candidate miRNA expression changes in (C) TM3 and (D) TM4 cells after fluoride treatment. (E) FISH of miR-34a-5p in the testis of control and fluoride-treated mice (dashed areas indicate Leydig cells, and white arrows indicate Sertoli cells). All values in the figure are means ± SEM. n = 3. * P < 0.05, ** P < 0.01.

Article Snippet: Murine Leydig cell line (TM3) and murine Sertoli cell line (TM4) were purchased from the American Type Culture Collection (ATCC) (Manassas, USA).

Techniques: Quantitative RT-PCR, Expressing, Control

Journal: Research

Article Title: Environmental Fluoride Compromises Male Fertility: Differentially Modulated miR-34a-5p Targets REST to Regulate Autophagy in Testicular Somatic Cells

doi: 10.34133/research.1113

Figure Lengend Snippet: Fluoride differentially regulates miR-34a-5p to modulate autophagy in testicular somatic cells. (A to F) Testicular somatic cells were treated with lentiviruses expressing miR-34a-5p/miR-NC and Inhibitor-miR-34a-5p/Inhibitor-NC. (A and D) Relative expression of miR-34a-5p. (B, C, E, and F) Relative expression of LC3B and p62 proteins. (G to I) miR-34a-5p overexpression rescued fluoride-treated TM3 cells. (G) Relative expression of miR-34a-5p. (H and I) Relative expression of LC3B and p62 proteins. (J to L) miR-34a-5p knockdown rescued fluoride-treated TM4 cells. (J) Relative expression of miR-34a-5p. (K and L) Relative expression of LC3B and p62 proteins. All values in the figure are means ± SEM. n = 3. * P < 0.05, ** P < 0.01 versus control, miR-NC, or Inhibitor-NC; # P < 0.05, ## P < 0.01 versus 0.25 mM NaF.

Article Snippet: Murine Leydig cell line (TM3) and murine Sertoli cell line (TM4) were purchased from the American Type Culture Collection (ATCC) (Manassas, USA).

Techniques: Expressing, Over Expression, Knockdown, Control

Journal: Research

Article Title: Environmental Fluoride Compromises Male Fertility: Differentially Modulated miR-34a-5p Targets REST to Regulate Autophagy in Testicular Somatic Cells

doi: 10.34133/research.1113

Figure Lengend Snippet: miR-34a-5p targets REST in testicular somatic cells exposed to fluoride. (A) Expression changes of p62 mRNA in TM3 and TM4 cells after fluoride treatment and overexpression or knockdown of miR-34a-5p. (B) Prediction results of miR-34a-5p target genes by miRWalk combined with ChEA3 database. (C and F) Differential expression of the top 15 predicted target genes after fluoride treatment in (C) TM3 and (F) TM4 cells. (D, E, G, and H) Relative mRNA expression of Elf1 , Foxo1 , Foxp1 , Klf10 , Mef2a , Rest , Smad5 , and Yy1 . (I) Schematic showing the targeted binding sites of miR-34a-5p to FOXO1 and REST 3′ UTR. (J and K) The histogram shows the relative fluorescence intensity of each group. (L) FISH of miR-34a-5p and Rest mRNA in testicular tissue from control mice (white arrow indicates the positive area within the Sertoli cells or Leydig cells). (M and N) Differential expression of REST protein in testicular somatic cells after overexpression or knockdown of miR-34a-5p. All values in the figure are means ± SEM. n = 3. * P < 0.05, ** P < 0.01.

Article Snippet: Murine Leydig cell line (TM3) and murine Sertoli cell line (TM4) were purchased from the American Type Culture Collection (ATCC) (Manassas, USA).

Techniques: Expressing, Over Expression, Knockdown, Quantitative Proteomics, Binding Assay, Fluorescence, Control

Journal: Research

Article Title: Environmental Fluoride Compromises Male Fertility: Differentially Modulated miR-34a-5p Targets REST to Regulate Autophagy in Testicular Somatic Cells

doi: 10.34133/research.1113

Figure Lengend Snippet: miR-34a-5p targets REST to regulate autophagy in testicular somatic cells. (A to F) TM3 and TM4 cells were treated with lentivirus expressing OE-REST, si-REST, OE-NC, or si-NC. (A and D) Relative Rest mRNA expression. (B, C, E, and F) Relative LC3B, p62, and REST protein expression. (G to K) REST overexpression rescued TM3 and TM4 cells overexpressing miR-34a-5p. (G and H) Relative miR-34a-5p, Rest , and p62 mRNA expression. (I to K) Relative REST, LC3B, and p62 protein levels. All values in the figure are means ± SEM. n = 3. ns indicates not significant; * P < 0.05, ** P < 0.01 versus control; # P < 0.05, ## P < 0.01 versus miR-34a-5p.

Article Snippet: Murine Leydig cell line (TM3) and murine Sertoli cell line (TM4) were purchased from the American Type Culture Collection (ATCC) (Manassas, USA).

Techniques: Expressing, Over Expression, Control

Journal: Research

Article Title: Environmental Fluoride Compromises Male Fertility: Differentially Modulated miR-34a-5p Targets REST to Regulate Autophagy in Testicular Somatic Cells

doi: 10.34133/research.1113

Figure Lengend Snippet: Fluoride differentially regulates REST to modulate autophagy in testicular somatic cells. (A) Representative image of Rest mRNA FISH in testes from control and fluoride-treated mice. (B) Representative immunofluorescence staining of REST proteins in testes from control and fluoride-treated mice. Dashed areas indicate Leydig cells, and white arrows indicate Sertoli cells. (C and D) Relative REST protein expression in testicular somatic cells treated with fluoride. (E to G) REST knockdown rescued fluoride-treated TM3 cells. (E) Relative expression of Rest mRNA. (F and G) Relative expression of REST, p62, and LC3B proteins. (H to J) REST overexpression rescued fluoride-treated TM4 cells. (H) Relative expression of Rest mRNA. (I and J) Relative expression of REST, p62, and LC3B proteins. All values in the figure are means ± SEM. n = 3. * P < 0.05, ** P < 0.01 versus control, si-NC, or OE-NC; # P < 0.05, ## P < 0.01 versus 0.25 mM NaF.

Article Snippet: Murine Leydig cell line (TM3) and murine Sertoli cell line (TM4) were purchased from the American Type Culture Collection (ATCC) (Manassas, USA).

Techniques: Control, Immunofluorescence, Staining, Expressing, Knockdown, Over Expression

Journal: ACS Omega

Article Title: Biocompatibility of Nickel Ferrite Nanoparticles on Systemic and Testicular Cells

doi: 10.1021/acsomega.5c09491

Figure Lengend Snippet: Viability of kidney, liver, Leydig, and germ cells exposed to nickel ferrite nanoparticles. Viability of VERO cells (A), AML-12 cells (B), TM3 cells (C), and GC-1 cells (D) exposed to FeNi NPs at different concentrations (6.25, 12.5, 25, 50, 100, 250, and 500 μg/mL) and at different time points (24, 48, and 72 h). Statistical analyses used one-way ANOVA with Dunnett’s test for parametric data and Kruskal–Wallis with Dunn’s post hoc test for nonparametric data. Results are presented as mean ± SD ( n = 6). Significant differences are noted as (*) p ≤ 0.05, (**) p ≤ 0.01, (***) p ≤ 0.001, (****) p < 0.0001. The red line indicates the 70% viability threshold per ISO 10993–5.

Article Snippet: For biological assays, the following immortalized cell lines were used: hepatic cells (AML-12, ATCC CRL-2254), originating from hepatocytes isolated from the liver of a 3 month-old normal mouse; renal cells (VERO, ATCC CCL-81), derived from renal epithelial cells of an African green monkey; Leydig cells (TM3, ATCC CRL-1714), isolated from a male mouse; and germ cells (GC-1, ATCC CRL-2053), isolated from the testis of a 10 day-old male mouse.

Techniques:

Journal: Frontiers in Endocrinology

Article Title: Molecular characterization of the murine Leydig cell lines TM3 and MLTC-1

doi: 10.3389/fendo.2025.1715307

Figure Lengend Snippet: Ultrastructural analysis of TM3 cells. (A–F) TEM demonstrates the heterogeneous appearance (polygonal, rounded or elongated shape) of the TM3 Leydig cell line. The cells contain a prominent nucleus ( N ) with electron-dense heterochromatin ( H ) and visible nucleoli ( n ). TM3 cells exhibit numerous mitochondria ( M ) of tubular type with cristae and visible endoplasmic reticulum ( ER ) of smooth ( sER ) or rough ( rER ) type. Lytic vesicles ( Ly ) of various sizes and glycogen ( G ) can be found in the cytoplasm ( Cy ) of the cells. TM3 cells also contain some lipid droplets ( LD ) which appear as dark, circular areas with electron-dense dark frames. TEM images were captured at 3,597× (A) , 4,646× (B) , 21,560× (C–F) with scale bars representing 2500 nm or 500 nm.

Article Snippet: TM3 cells are distributed by the American Type Culture Collection (ATCC) as CRL-1714 TM and have been used in almost 400 studies according to the PubMed database as of end of September 2025.

Techniques:

Journal: Frontiers in Endocrinology

Article Title: Molecular characterization of the murine Leydig cell lines TM3 and MLTC-1

doi: 10.3389/fendo.2025.1715307

Figure Lengend Snippet: Markers associated with LDs in TM-3 and MLTC-1 LCs. (A) Basal mRNA expression of various LD-associated markers ( Plin1 , Plin2 , Plin3 , Plin4 , Plin5 ) and lipolysis genes ( Mgll , Pnpla2 , Lipe ) was compared between TM-3 and MLTC-1 cells using RT-qPCR. Relative mRNA expression was normalized to Rps6 expression, and values are presented relative to TM3 cells. Data (from n=3 independent experiments) are shown as mean ± SD. Statistical analysis was performed using a Student’s t -test. Significant differences between the cell lines are denoted with asterisks: *** p < 0.001. (B) Expression of LD-associated and lipolytic proteins was compared between MLTC-1 and TM3 cells through Western blot analysis. Cyclophilin A expression was used as an internal loading control for protein analysis. TM3 and MLTC-1 cells were either stimulated with 0.25 mM oleic acid (OA) for 24 hours or left unstimulated before LD staining with Oil Red O (ORO) (C) or with the fluorescence probe BODIPY 493/503 (D) . LD staining was combined with nuclear counterstain using either haematoxylin or DAPI. Images in C and D were captured at 600× magnification, and scale bars represent 25 µm.

Article Snippet: TM3 cells are distributed by the American Type Culture Collection (ATCC) as CRL-1714 TM and have been used in almost 400 studies according to the PubMed database as of end of September 2025.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Staining, Fluorescence

Journal: Frontiers in Endocrinology

Article Title: Molecular characterization of the murine Leydig cell lines TM3 and MLTC-1

doi: 10.3389/fendo.2025.1715307

Figure Lengend Snippet: Marker of steroidogenesis in TM-3 and MLTC-1 LCs. (A) Basal mRNA expression of key enzymes of steroidogenesis was compared between TM-3 and MLTC-1 cells by RT-PCR with testis tissue serving as a control. (B) Data was confirmed by RT-qPCR. Respective genes are shown as fold change normalized to Rps6 . The data are presented as mean ± SD. The statistical analysis used either an unpaired Student’s t -test or a Mann-Whitney test. Significant differences between groups are indicated by asterisks, *** p < 0.001. (C) Protein expression of selected steroidogenic markers was detected in TM3 and MLTC-1 cells with testis tissue as a control by Western blot analysis. All membranes were probed with antibodies to detect HSP90, GAPDH, Connexin 43 or Cyclophilin A to ensure equal protein loading. (D) 3β-HSD activity was visualized in TM3 and MLTC-1 cells using an established histological staining procedure. Before staining, cells were either treated with 5 IU/ml human chorionic gonadotropin (hCG) or left untreated for 24 hours. Blue precipitates within the cytoplasm of MLTC-1, but not in TM3 cells, indicate a positive reaction with and without hCG treatment. Images were captured at 600× magnification and scale bars equal 25 µm.

Article Snippet: TM3 cells are distributed by the American Type Culture Collection (ATCC) as CRL-1714 TM and have been used in almost 400 studies according to the PubMed database as of end of September 2025.

Techniques: Marker, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Quantitative RT-PCR, MANN-WHITNEY, Western Blot, Activity Assay, Staining

Journal: Frontiers in Endocrinology

Article Title: Molecular characterization of the murine Leydig cell lines TM3 and MLTC-1

doi: 10.3389/fendo.2025.1715307

Figure Lengend Snippet: Induction of steroidogenesis by hCG stimulation in TM-3 and MLTC-1 LCs. (A) Basal mRNA expression of Lhcgr was studied in TM-3 and MLTC-1 cells by RT-PCR with testis tissue as a control. (B) Data was confirmed by RT-qPCR. Lhcgr expression was normalized to Rps6 and data are displayed in relation to TM3 cells. Student’s t -test was used for statistical analysis. There was a significant difference in Lhcgr expression between TM3 and MLTC-1 cells, indicated by asterisks, *** p < 0.001. (C) Both cell lines were treated with or without hCG (0.5 IU/mL or 5 IU/mL) for 6 hours and RT-qPCR was used to detect changes in Lhcgr, Star or Hsd17b3 expression. Relative mRNA expressions are normalized to Rps6 and displayed in relation to untreated cells. The data in C and D are presented as mean ± SD. For statistical analysis in C, one-way ANOVA was used. (D) TM3 and MLTC-1 cells were treated with different concentrations of hCG (0, 0.25, 0.5, 1.0, 5.0 or 25 IU/mL) for 6 hours, and protein expression of different markers (Phospho-p42/44, total p42/44, StAR, phospho or CREB1) was detected by Western blot analysis. Please note that the phospho-CREB1 antibody detects phosphor-ATF1 as well (illustrated in grey letters). HSP90 expression was used to ensure equal protein loading. MLTC-1 cells but not TM3 cells show induction of steroidogenic markers upon hCG treatment. ** p < 0.01.

Article Snippet: TM3 cells are distributed by the American Type Culture Collection (ATCC) as CRL-1714 TM and have been used in almost 400 studies according to the PubMed database as of end of September 2025.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Endocrinology

Article Title: Molecular characterization of the murine Leydig cell lines TM3 and MLTC-1

doi: 10.3389/fendo.2025.1715307

Figure Lengend Snippet: Ultrastructural analysis of TM3 and MLTC-1 cells stimulated with hCG. (A–F) Following a 24-hour treatment with hCG, TEM was performed on TM3 (A–C) and MLTC-1 cells (D–E) . The ultrastructural analysis revealed the presence of lytic vesicles ( Ly ) in TM3 cells. In hCG-treated MLTC-1 cells, a significant amount of smooth endoplasmic reticulum ( sER ) was observed in the form of bundles or whorls consisting of ribosomal-free membranes with expanded lumens (white). Mitochondria ( M ) were found in in close proximity to both the sER and rough endoplasmic reticulum ( rER ). Abbreviations: nucleus ( N ). TEM images were taken at 10,000× (A, C) and 27,800× (B, C, E, F) with scale bars representing 1000 nm or 500 nm.

Article Snippet: TM3 cells are distributed by the American Type Culture Collection (ATCC) as CRL-1714 TM and have been used in almost 400 studies according to the PubMed database as of end of September 2025.

Techniques: