Journal: Frontiers in Cell and Developmental Biology

Article Title: Melatonin and agomelatine alleviate ivermectin-induced mouse spermatogonia apoptosis via suppression of oxidative stress and calcium overload

doi: 10.3389/fcell.2025.1713124

Figure Lengend Snippet: Effects of melatonin and agomelatine on proliferation loss in GC-1 spermatogonia (spg) treated with ivermectin (IVM) for 24 h (A) Chemical structure of IVM isomers. (B) Chemical structures of melatonin and its analogs, agomelatine and pinoline. (C) Schematic overview of the cell culture and experimental design. (D) Brightfield microscopy images showing cytoplasmic morphology. (E) Immunofluorescence images of Ki67 nuclear translocation. (F) Quantification of relative cell proliferation (n = 3). (G) Quantification of Ki67-positive nuclei (n = 3) in GC-1 spg treated with either control, melatonin, agomelatine, or pinoline with or without 16 μM IVM. IVM treatment conditions are denoted as positive (+) or negative (−). Scale bars: 100 µm. Data are presented as means ± SEM. Significant differences are indicated by different letters (a–d) at p < 0.05.

Article Snippet: Mouse type B GC-1 spermatogonial (spg) cells (CRL-2053, American Type Culture Collection, Manassas, VA, USA) were cultured in complete media using Dulbecco’s modified Eagle’s medium (DMEM, L0103-500; Biowest, Nuaillé, France) with 10% fetal bovine serum (FBS, S1480; Biowest) and penicillin/streptomycin (15140122, Gibco, Waltham, MA, USA) in 5% CO 2 at 37 °C.

Techniques: Cell Culture, Microscopy, Immunofluorescence, Translocation Assay, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Melatonin and agomelatine alleviate ivermectin-induced mouse spermatogonia apoptosis via suppression of oxidative stress and calcium overload

doi: 10.3389/fcell.2025.1713124

Figure Lengend Snippet: Inhibition of intracellular reactive oxygen species (ROS) generation by melatonin and agomelatine in GC-1 spermatogonia (spg) treated with ivermectin (IVM) for 3 h (A) Fluorescence microscopy images of GC-1 spg labeled with DCFDA. (B) Semi-quantification of DCFDA fluorescence intensity in GC-1 spg treated with either control, melatonin, agomelatine, or pinoline with or without 16 μM IVM (n = 5). IVM treatment conditions are denoted as positive (+) or negative (−). Scale bars: 100 µm. Data are shown as means ± SEM. Significant differences are indicated by different letters (a–d) at p < 0.05. Mel, melatonin; Ago, agomelatine; Pin, pinoline.

Article Snippet: Mouse type B GC-1 spermatogonial (spg) cells (CRL-2053, American Type Culture Collection, Manassas, VA, USA) were cultured in complete media using Dulbecco’s modified Eagle’s medium (DMEM, L0103-500; Biowest, Nuaillé, France) with 10% fetal bovine serum (FBS, S1480; Biowest) and penicillin/streptomycin (15140122, Gibco, Waltham, MA, USA) in 5% CO 2 at 37 °C.

Techniques: Inhibition, Fluorescence, Microscopy, Labeling, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Melatonin and agomelatine alleviate ivermectin-induced mouse spermatogonia apoptosis via suppression of oxidative stress and calcium overload

doi: 10.3389/fcell.2025.1713124

Figure Lengend Snippet: Melatonin and agomelatine inhibit cytoplasmic Ca 2+ accumulation in GC-1 spermatogonia (spg) treated with ivermectin (IVM) for 3 h (A) Fluorescence microscopy images of GC-1 spg cells labeled with Fluo-4, AM. (B) Semi-quantification of Fluo-4, AM fluorescence intensity in GC-1 spg treated with either control, melatonin, agomelatine, or pinoline with or without 16 μM IVM (n = 5). IVM treatment conditions are denoted as positive (+) or negative (−). Scale bars: 100 µm. Data are presented as means ± SEM. Significant differences are denoted by different letters (a–c) at p < 0.05. Mel, melatonin; Ago, agomelatine; Pin, pinoline.

Article Snippet: Mouse type B GC-1 spermatogonial (spg) cells (CRL-2053, American Type Culture Collection, Manassas, VA, USA) were cultured in complete media using Dulbecco’s modified Eagle’s medium (DMEM, L0103-500; Biowest, Nuaillé, France) with 10% fetal bovine serum (FBS, S1480; Biowest) and penicillin/streptomycin (15140122, Gibco, Waltham, MA, USA) in 5% CO 2 at 37 °C.

Techniques: Fluorescence, Microscopy, Labeling, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Melatonin and agomelatine alleviate ivermectin-induced mouse spermatogonia apoptosis via suppression of oxidative stress and calcium overload

doi: 10.3389/fcell.2025.1713124

Figure Lengend Snippet: Melatonin’s and agomelatine’s recovery of ΔΨm and mitochondrial mass in GC-1 spermatogonia (spg) treated with 16 μM ivermectin (IVM) for 3 h (A) Fluorescence microscopy images of GC-1 spg stained with tetramethylrhodamine ethyl ester (TMRE) and/or MitoTracker and Hoechst 33,342. (B) Semi-quantification of MitoTracker fluorescence intensity (n = 5), and (C) ratio of TMRE to MitoTracker fluorescence intensity in GC-1 spg treated with either control, melatonin, agomelatine, or pinoline with or without 16 μM IVM (n = 5). Scale bars: 100 µm. IVM treatment conditions are denoted as positive (+) or negative (−). Data are shown as means ± SEM. Significant differences are denoted by different letters (a–c) at p < 0.05. Mel, melatonin; Ago, agomelatine; Pin, pinoline

Article Snippet: Mouse type B GC-1 spermatogonial (spg) cells (CRL-2053, American Type Culture Collection, Manassas, VA, USA) were cultured in complete media using Dulbecco’s modified Eagle’s medium (DMEM, L0103-500; Biowest, Nuaillé, France) with 10% fetal bovine serum (FBS, S1480; Biowest) and penicillin/streptomycin (15140122, Gibco, Waltham, MA, USA) in 5% CO 2 at 37 °C.

Techniques: Fluorescence, Microscopy, Staining, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Melatonin and agomelatine alleviate ivermectin-induced mouse spermatogonia apoptosis via suppression of oxidative stress and calcium overload

doi: 10.3389/fcell.2025.1713124

Figure Lengend Snippet: Seahorse assay of GC-1 spermatogonia (spg) treated with or without melatonin analogs and/or ivermectin (IVM) for 3 h (A) Oxygen consumption rate (OCR) plot following sequential injections of oligomycin, FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone), and rotenone/antimycin A (R/AA). (B) Basal respiration, (C) ATP-linked respiration, (D) maximal respiration capacity, and (E) reserve capacity calculated from OCR data (n = 3). Data are presented as means ± SEM. Significant differences are indicated by different letters (a–d) at p < 0.05. Mel, melatonin; Ago, agomelatine; Pin, pinoline.

Article Snippet: Mouse type B GC-1 spermatogonial (spg) cells (CRL-2053, American Type Culture Collection, Manassas, VA, USA) were cultured in complete media using Dulbecco’s modified Eagle’s medium (DMEM, L0103-500; Biowest, Nuaillé, France) with 10% fetal bovine serum (FBS, S1480; Biowest) and penicillin/streptomycin (15140122, Gibco, Waltham, MA, USA) in 5% CO 2 at 37 °C.

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Melatonin and agomelatine alleviate ivermectin-induced mouse spermatogonia apoptosis via suppression of oxidative stress and calcium overload

doi: 10.3389/fcell.2025.1713124

Figure Lengend Snippet: Attenuation of apoptosis by melatonin and agomelatine in GC-1 spermatogonia (spg) treated with ivermectin (IVM) for 3 h (A) Brightfield microscopy images showing morphological changes in GC-1 spg following treatment with IVM and/or melatonin analogs. Scale bars: 100 µm. (B) Western blot analysis of apoptosis-related proteins, including cleaved caspases, BCL-2, BAX, Cytochrome c, and α-tubulin (used as a loading control), under the indicated treatment conditions. Mel, melatonin; Ago, agomelatine; Pin, pinoline (C) Quantification of apoptosis-related proteins (n = 3). Data are shown as means ± SEM. Significant differences are denoted by different letters (a–c) at p < 0.05. Mel, melatonin; Ago, agomelatine; Pin, pinoline.

Article Snippet: Mouse type B GC-1 spermatogonial (spg) cells (CRL-2053, American Type Culture Collection, Manassas, VA, USA) were cultured in complete media using Dulbecco’s modified Eagle’s medium (DMEM, L0103-500; Biowest, Nuaillé, France) with 10% fetal bovine serum (FBS, S1480; Biowest) and penicillin/streptomycin (15140122, Gibco, Waltham, MA, USA) in 5% CO 2 at 37 °C.

Techniques: Microscopy, Western Blot, Control

Journal: FEBS Open Bio

Article Title: The effect of salubrinal on the endoplasmic reticulum stress pathway in heat‐stressed spermatogonial cells in vitro

doi: 10.1002/2211-5463.70169

Figure Lengend Snippet: Determination of the IC50 values (μ m ) for GC1 and GC2 cell lines following 24 h SAL exposure. Representative phase‐contrast images demonstrate the typical epithelioid morphology of cells maintained under standard culture conditions and the reduced viability observed at the IC50 concentration of SAL. The cell images on the left side represent control (C) cells, while the images on the right side represent cells treated with IC50 concentrations of SAL (IC50 SAL). Black arrow: Living cell. White arrow: Dead cell. Scale bar: 100 μm.

Article Snippet: The mouse spermatogonial GC1 (ATCC® CRL‐2053TM Manassas, VA, USA) and GC2 (ATCC® CRL‐2196TM Manassas, VA, USA) cell lines used in the study were purchased commercially from ATCC (USA).

Techniques: Concentration Assay, Control

Journal: FEBS Open Bio

Article Title: The effect of salubrinal on the endoplasmic reticulum stress pathway in heat‐stressed spermatogonial cells in vitro

doi: 10.1002/2211-5463.70169

Figure Lengend Snippet: Inverted microscope images of GC1 and GC2 experimental groups. Quantitative analysis shows a decrease in viable cell numbers in the HSM‐applied groups compared to controls. Data are expressed as mean ± SD ( n = 3, 10 randomly selected fields per group). Statistical analysis: one‐way ANOVA, followed by Sidak's post hoc test. (** P < 0.05; statistical analysis was performed using graphpad Software). Scale bar: 100 μm.

Article Snippet: The mouse spermatogonial GC1 (ATCC® CRL‐2053TM Manassas, VA, USA) and GC2 (ATCC® CRL‐2196TM Manassas, VA, USA) cell lines used in the study were purchased commercially from ATCC (USA).

Techniques: Inverted Microscopy, Software

Journal: FEBS Open Bio

Article Title: The effect of salubrinal on the endoplasmic reticulum stress pathway in heat‐stressed spermatogonial cells in vitro

doi: 10.1002/2211-5463.70169

Figure Lengend Snippet: Representative fluorescent images (each image is an overlay with DAPI nuclear staining) and quantitative CTCF values for HSP70, GRP78, p‐IRE1α, p‐PERK, p‐eIF2α, and ATF6 in GC1 cells across all groups (C, SAL, HSM, HSMSAL). Composite images were generated by merging individual fluorescence channels. Data are expressed as mean ± SD ( n = 3, 10 fields/group). Statistical analysis: one‐way ANOVA, followed by Sidak's post hoc test. (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; statistical analysis was performed using graphpad Software). Scale bar: 50 μm.

Article Snippet: The mouse spermatogonial GC1 (ATCC® CRL‐2053TM Manassas, VA, USA) and GC2 (ATCC® CRL‐2196TM Manassas, VA, USA) cell lines used in the study were purchased commercially from ATCC (USA).

Techniques: Staining, Generated, Fluorescence, Software

Journal: FEBS Open Bio

Article Title: The effect of salubrinal on the endoplasmic reticulum stress pathway in heat‐stressed spermatogonial cells in vitro

doi: 10.1002/2211-5463.70169

Figure Lengend Snippet: Relative mRNA expression levels of GRP78 (A), PERK (B), and eIF2α (C) in GC1 and GC2 cells. Gene expression was quantified by qRT‐PCR and analyzed using the 2 − Δ Δ C t method. The control groups (GC1C and GC2C) were designated as calibrators and normalized to a fixed value of 1.0 by definition. Data are presented as mean ± SD from three independent biological replicates, each analyzed in technical triplicates. Individual data points are shown for all groups. Statistical evaluation was performed using the Kruskal–Wallis test, followed by Dunn's post hoc test (* P < 0.05; statistical analysis was performed using graphpad Software).

Article Snippet: The mouse spermatogonial GC1 (ATCC® CRL‐2053TM Manassas, VA, USA) and GC2 (ATCC® CRL‐2196TM Manassas, VA, USA) cell lines used in the study were purchased commercially from ATCC (USA).

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Control, Software