Journal: Reproductive Sciences

Article Title: The Effect of Thymoquinone on the TNF-α/OTULIN/NF-κB Axis Against Cisplatin-İnduced Testicular Tissue Damage

doi: 10.1007/s43032-024-01567-y

Figure Lengend Snippet: Effect of CS and\or TQ applications on reproductive hormones Serum testosterone and GnRH levels were similar in the control and TQ groups. GnRH levels increased while testosterone levels decreased in the CS group compared to the control group. In the CS + TQ group, it was determined that while testosterone levels increased compared to the CS group, GnRH levels decreased. *; compared to the control group ( p < 0.05), #; Compared with the CS group ( p < 0.05). CS; Cisplatin, TQ; Thymoquinone, GnRH; Gonadotropin-releasing hormone

Article Snippet: ELISA kits were used according to the manufacturer's instructions GnRH (Elabscience, USA), (OTULIN (SunRed, China), MDA, CAT, and SOD kits were purchased from Fine Test (China)).

Techniques: Control

Journal: Reproductive Sciences

Article Title: The Effect of Thymoquinone on the TNF-α/OTULIN/NF-κB Axis Against Cisplatin-İnduced Testicular Tissue Damage

doi: 10.1007/s43032-024-01567-y

Figure Lengend Snippet: Effect of CS and/or TQ applications on OTULIN levels in testicular tissues: A ; OTULIN immunoreactivity microphotographs and graph, B ; OTULIN ELISA levels, C ; OTULIN mRNA expression, D ; OTULIN gene protein level. OTULIN levels in testicular tissues of control and TQ groups were similar. OTULIN immunoreactivity, ELISA level, mRNA expression and gene protein level were decreased in the CS group compared to the control group. An increase in OTULIN levels was detected in the CS + TQ group compared to the CS group. *; compared to the control group ( p < 0.05), #; Compared to CS group ( p < 0.05). A; OTULIN immunohistochemical staining, scale bar; 100 μm. CS; Cisplatin, TQ; Thymoquinone

Article Snippet: ELISA kits were used according to the manufacturer's instructions GnRH (Elabscience, USA), (OTULIN (SunRed, China), MDA, CAT, and SOD kits were purchased from Fine Test (China)).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Control, Immunohistochemical staining, Staining

Journal: Endocrine Connections

Article Title: Role of KNDy neurons in puberty onset in male offspring following prenatal androgen exposure

doi: 10.1530/EC-25-0209

Figure Lengend Snippet: Effect of PNA on serum levels of GnRH, LH, FSH, and kisspeptin 1 in male offspring at the onset of puberty. No significant differences were observed between groups ( P > 0.05).

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits were used to quantify serum concentrations of GnRH (CSB-E08037r; Cusabio, China), LH (CSB-E12654r; Cusabio, China), FSH (CSB-E06869r; Cusabio, China), and kisspeptin 1 (CSB-E13434r; Cusabio, China).

Techniques:

Journal: iScience

Article Title: The E3 ubiquitin ligase RNF216/TRIAD3 is a key coordinator of the hypothalamic-pituitary-gonadal axis

doi: 10.1016/j.isci.2022.104386

Figure Lengend Snippet: Rnf216/Triad3 reduces GnRH and Ca 2+ transient frequency in GT1-7 cells (A) Generation of Rnf216/Triad3 hypothalamic GT1-7 knockout cells. Top , Representative immunoblot illustrating RNF216/TRIAD3 in CRISPR-Cas9 control (Ctrl) and knockout cells (A and B). Bottom , Mean RNF216/TRIAD3 in control and knockout cells. RNF216/TRIAD3 values were normalized to β-ACTIN. (F (2, 15) = 60.31, ∗∗∗∗p< 0.0001, One-way ANOVA). Bonferroni’s multiple comparisons test shows that CRISPR A (∗∗∗p = 0.0009) and B (∗∗∗∗p<0.0001) are significantly lower than control. N = 6 samples per condition. Error bars are +SEM. (B) Sanger sequencing demonstrates successful targeting of Rnf216/Triad3 in CRISPR A ( left ) and B ( right ) with arrows indicating break sites. The highlighted region represents gRNA targeting site/s. (C) qPCR demonstrating a significant decrease in Gnrh1 (F (2, 6) = 5.129, ∗p = 0.05, One-way ANOVA). Bonferroni’s multiple comparisons test showed a non-significant reduction in CRISPR A by 51.36% ± 0.1445%, p = 0.1820, and a significant reduction in CRISPR B by 80.82% ± 0.06998%, ∗p = 0.0389. N = 3 cDNA samples per condition. Error bars are +SEM. (D) GnRH ELISA depicting reduction in basal GnRH release in CRISPR A and B. F (2,8) =13.97, ∗∗p = 0.0025. One-way ANOVA with Dunnett’s multiple comparison test. Crispr A was significantly different from the control (∗∗p =0.0014). Crispr B was significantly different than the control (∗p = 0.0122). N = 3 samples for Ctrl, N = 4 samples for CRISPR A, and N = 4 samples for CRISPR B. Error bars are +SEM. (E) Left , Representative immunoblots demonstrating expression of GFP-RNF216/TRIAD3 Isoforms A and B transfected in Ctrl and CRISPR B. Right , qPCR of Gnrh1 with Ctrl and B transfected with GFP or GFP-tagged Crispr resistant RNF216/TRIAD3 A and B isoforms (Rescue). CRISPR B Rescue showed no significant differences compared to Ctrl GFP only. CRISPR B with GFP only (∗p = 0.0418) showed significant differences compared to Ctrl GFP only (F (5,18) = 8.063, ∗∗p = 0.0004). N = 4 cDNA samples per condition. Error bars are +SEM. (F) Calcium signaling in Ctrl and Rnf216/Triad3 knockout cells. Representative fluorescence intensity plots of CRISPR Ctrl, A, and B. Positive signals were measured as 2 standard deviations above the baseline mean indicated by (▼). Inset , Representative fluorescent images from each condition. Scale bars represent 10 μm. (G) Average amplitude of positive event transients. F (2, 81) = 5.690, ∗∗p = 0.0049. One-way ANOVA with Tukey post-hoc analysis. Crispr B was significantly different from Crispr A (∗∗p = 0.0038). Error bars are +SEM. (H) Frequency of event transients is counted as the total number of positive signals in 300 s. (F (2, 81) = 7.263, ∗∗p = 0.0013, One-way ANOVA) with Tukey post-hoc analysis. Crispr B was significantly different than the control (∗∗p = 0.0014) and from Crispr A (∗p = 0.0194). N = 28 cells for Ctrl, CRISPR A, and CRISPR B. Error bars are + SEM. See also Figure S1 .

Article Snippet: GnRH ELISA , Cusabio, Inc. , Cat#CSB-E08152m.

Techniques: Knock-Out, Western Blot, CRISPR, Control, Sequencing, Enzyme-linked Immunosorbent Assay, Comparison, Expressing, Transfection, Fluorescence

Journal: iScience

Article Title: The E3 ubiquitin ligase RNF216/TRIAD3 is a key coordinator of the hypothalamic-pituitary-gonadal axis

doi: 10.1016/j.isci.2022.104386

Figure Lengend Snippet: Loss of RNF216/TRIAD3 decreases GnRH soma size and GnRH production in both sexes and increases neuroinflammation in males (A) Representative confocal images of GnRH cells in the preoptic area of the hypothalamus in adult WT and KO male ( left ) and female ( right ) mice. GnRH neurons were imaged at 20× magnification. Scale bars represent 50 μm. (B) GnRH neurons were classified according to the number of dendrites protruding directly off the soma: none (zero dendrites), unipolar (1 dendrite), bipolar (2 dendrites), multipolar (>2 dendrites). Although KO animals had a higher percentage of none type compared to WT, this was not significantly different in males ( X 2 (3) = 1.709, p = 0.6349) or females (X 2 (3) = 5.198, p = 0.1579). Chi-square. For males per genotype, n= 12–17 cells for none, n= 40–45 cells for unipolar, n= 34–41 cells for bipolar, and n= 42–50 cells for multipolar. For females per genotype, n= 11–18 cells for none, n= 44–50 cells for unipolar, n= 31–50 cells for bipolar, and n= 19–27 cells for multipolar. (C) Significant differences in soma area in males ( t (50) =3.185, ∗∗p = 0.0025) and females ( t (48) =2.402, ∗p = 0.0202) compared to respective WT counterparts. There were also significant differences in the integrated density in KO males ( t (50) =2.637, ∗p = 0.0111) and females ( t (48) =2.061, ∗p = 0.0447) compared to respective WT; Unpaired t -test. N = 3 for males per genotype with 4–6 sections per animal represented in summary plots. N = 3 for females per genotype with across 3–4 sections per animal represented in summary plots. Error bars are ±SEM. (D) Top , representative images of microglia stained with Iba1 in the preoptic area of the hypothalamus in WT and KO males (left) and females (right) were imaged at 10x magnification. Scale bars represent 100 μm. Bottom , KO males show lower Iba1 total area (including processes) compared to WT (t (9) =5.280, ∗∗∗p = 0.0005; Unpaired t-test). N = 3 for males per genotype with one to two sections per animal represented in summary plots. No significant differences in females. N = 3 for females per genotype with two sections per animal represented in summary plots. No significant differences in cell density. Error bars are ±SEM. See also Figure S3 .

Article Snippet: GnRH ELISA , Cusabio, Inc. , Cat#CSB-E08152m.

Techniques: Staining

Journal: iScience

Article Title: The E3 ubiquitin ligase RNF216/TRIAD3 is a key coordinator of the hypothalamic-pituitary-gonadal axis

doi: 10.1016/j.isci.2022.104386

Figure Lengend Snippet: Rnf216/Triad3 CNS-specific knockout mice do not demonstrate reproductive deficits but KO males have altered microglia (A) Gonadal weights of Rnf216/Triad3 Nestin-CRE WT and KO mice at 16-, and 52-weeks. There were no significant differences at either age. N = 3–10 animals per genotype. (B) There were no significant differences in FSH levels in Rnf216/Triad3 Nestin-CRE KO male animals. N = 9–10 animals per genotype. (C) Representative confocal images of GnRH cells in the preoptic area of the hypothalamus in adult Rnf216/Triad3 Nestin-CRE WT and KO male ( left ) and female ( right ) mice. GnRH neurons were imaged at 20× magnification. Scale bars represent 50 μm. (D) GnRH neurons were classified according to the number of dendrites protruding directly off the soma. There were no significant differences between WT and KO in males or females. For males per genotype, n= 51–53 cells for none, n= 94–109 cells for unipolar, n= 68–79 cells for bipolar, and n= 37–44 cells for multipolar. For females per genotype, n= 54 cells for none, n= 76–103 cells for unipolar, n= 49–72 cells for bipolar, and n= 26–28 cells for multipolar. (E) There were no significant differences in the area of the soma or integrated density in Rnf216/Triad3 Nestin-CRE KO males or females. N = 3 for males with seven to eight sections per animal represented in summary plots. N = 3 for females with six to eight sections per animal represented in summary plots. (F) Top , representative images of microglia stained with Iba1 in the preoptic area of the hypothalamus in Rnf216/Triad3 Nestin-CRE WT and KO males (left) and females (right) imaged at 10x magnification. Scale bars represent 100 μm. Bottom , there were significant differences in Iba1 area (including processes) in males ( t (10) =3.584, ∗∗p = 0.005; Unpaired t-test). There were significant differences in cell density in males ( t (10) =2.595, ∗p = 0.0267; Unpaired t -test). N = 3 for males per genotype with two sections per animal represented in summary plots. No differences in females. N = 3 for females per genotype with two sections per animal represented in summary plots. Error bars are ±SEM. See also Figure S4 .

Article Snippet: GnRH ELISA , Cusabio, Inc. , Cat#CSB-E08152m.

Techniques: Knock-Out, Staining

Journal: iScience

Article Title: The E3 ubiquitin ligase RNF216/TRIAD3 is a key coordinator of the hypothalamic-pituitary-gonadal axis

doi: 10.1016/j.isci.2022.104386

Figure Lengend Snippet:

Article Snippet: GnRH ELISA , Cusabio, Inc. , Cat#CSB-E08152m.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Imaging, Recombinant, Modification, Saline, Blocking Assay, Electron Microscopy, Plasmid Preparation, Gel Extraction, Transfection, Polymerase Chain Reaction, Reverse Transcription, Staining, Ligation, Mutagenesis, CRISPR, Cloning, Sequencing, SYBR Green Assay, shRNA, Software

Journal: International Journal of Molecular Sciences

Article Title: Effect of Caffeine on the Inflammatory-Dependent Changes in the GnRH/LH Secretion in a Female Sheep Model

doi: 10.3390/ijms25052663

Figure Lengend Snippet: Effect of lipopolysaccharide (LPS; 400 ng/kg; iv.) and caffeine (30 mg/kg; iv.) injections on the concentration of gonadotropin-releasing hormone (GnRH) in the preoptic area of the hypothalamus. The data are presented as the mean value ± S.E.M. ( n = 6 animals per group). Significant differences marked with different capital letters were analyzed by a two-way ANOVA followed by a Fisher’s post hoc test. Statistical significance was stated when p < 0.05.

Article Snippet: The concentrations of GnRH in the POA homogenate prepared as previously described [ ] were determined using a commercial GnRH ELISA kit (cat. no. ESH0062, FineTest Biotech Inc., Boulder, CO, USA) appropriate for sheep.

Techniques: Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Effect of Caffeine on the Inflammatory-Dependent Changes in the GnRH/LH Secretion in a Female Sheep Model

doi: 10.3390/ijms25052663

Figure Lengend Snippet: The effect of caffeine on the relative gene expression (mean ± SEM; n = 6) of GnRH receptor and beta subunit of gonadotropins ( LHβ, FSHβ ) in the anterior pituitary under basal and lipopolysaccharide challenge conditions.

Article Snippet: The concentrations of GnRH in the POA homogenate prepared as previously described [ ] were determined using a commercial GnRH ELISA kit (cat. no. ESH0062, FineTest Biotech Inc., Boulder, CO, USA) appropriate for sheep.

Techniques: Gene Expression, Control

Journal: International Journal of Molecular Sciences

Article Title: Effect of Caffeine on the Inflammatory-Dependent Changes in the GnRH/LH Secretion in a Female Sheep Model

doi: 10.3390/ijms25052663

Figure Lengend Snippet: The effect of caffeine on the relative gene expression (mean ± SEM; n = 6) of GnRH in the hypothalamus under basal and lipopolysaccharide challenge conditions.

Article Snippet: The concentrations of GnRH in the POA homogenate prepared as previously described [ ] were determined using a commercial GnRH ELISA kit (cat. no. ESH0062, FineTest Biotech Inc., Boulder, CO, USA) appropriate for sheep.

Techniques: Gene Expression, Control

Journal: International Journal of Molecular Sciences

Article Title: Effect of Caffeine on the Inflammatory-Dependent Changes in the GnRH/LH Secretion in a Female Sheep Model

doi: 10.3390/ijms25052663

Figure Lengend Snippet: List of full names and abbreviations of all genes analyzed by RT-qPCR.

Article Snippet: The concentrations of GnRH in the POA homogenate prepared as previously described [ ] were determined using a commercial GnRH ELISA kit (cat. no. ESH0062, FineTest Biotech Inc., Boulder, CO, USA) appropriate for sheep.

Techniques: Amplification, Sequencing