Journal: International Journal of Molecular Sciences

Article Title: Gonadotropin-Releasing Hormone in Regulation of Thymic Development in Rats: Profile of Thymic Cytokines

doi: 10.3390/ijms20164033

Figure Lengend Snippet: Ontogenetic pattern of the gonadotropin-releasing hormone (GnRH) receptor expression in the developing thymus. ( A ) mRNA of GnRH receptor revealed by RT-PCR in the rat thymus on embryonic days (ED) 16, 17, 18, 19, 21 and postnatal day 3 (PND3). ( B ) Western blot assay of GnRH receptor in the rat thymus on ED17, ED18, ED19, ED20, ED21, and PND3. The anterior pituitary (PND3) was used as a positive control (+). ( C ) Protein expression of GnRH receptor in thymocytes and thymic stromal elements on ED18. Plots represent the optic density of the corresponding bands. Bars indicate the means ± SEM of three independent experiments; * p < 0.05 using one-way ANOVA test.

Article Snippet: Separated proteins were transferred to a nitrocellulose membrane in transfer buffer (25 mM Tris-HCl, pH 7.5, 192 mM glycine, 20% ethanol) and blots were incubated overnight at 4 °C with antibodies to GnRH receptor (1:1000, Alomone Labs, Jerusalem, Israel) or actin (1:10,000, Sigma, St. Louis, Mo, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Gonadotropin-Releasing Hormone in Regulation of Thymic Development in Rats: Profile of Thymic Cytokines

doi: 10.3390/ijms20164033

Figure Lengend Snippet: Schematic representation of GnRH effects on the thymus development in fetal and early postnatal rats. During prenatal development up to ED20–21, before the establishment of the HPG endocrine regulations, hypothalamic GnRH can be released to general circulation and provide a direct effect on the morphogenesis of thymus. Since the development of blood–brain barrier and the establishment of the HPG axis in early postnatal life GnRH is involved in the bidirectional programming of both neuroendocrine and immune functions via gonadotropins and sex steroids. In addition to the neuroendocrine regulation, the effects of GnRH synthesized in thymus can be realized by autocrine/paracrine mechanisms. The developmental pattern of the GnRH receptor expression in the thymus is in favor of this assumption. ED—embryonic day, PND—postnatal day, HP—hypophysis.

Article Snippet: Separated proteins were transferred to a nitrocellulose membrane in transfer buffer (25 mM Tris-HCl, pH 7.5, 192 mM glycine, 20% ethanol) and blots were incubated overnight at 4 °C with antibodies to GnRH receptor (1:1000, Alomone Labs, Jerusalem, Israel) or actin (1:10,000, Sigma, St. Louis, Mo, USA).

Techniques: Synthesized, Expressing

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: Figure 3 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: 2.5 Hormone assay 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: Journal of animal science and biotechnology

Article Title: Effect of CD14/TLR4 antagonist on GnRH/LH secretion in ewe during central inflammation induced by intracerebroventricular administration of LPS.

doi: 10.1186/s40104-018-0267-8

Figure Lengend Snippet: Fig. 1 Mean (± SEM) concentration of plasma LH (panel A) and GnRH in the ME (panel B) after icv administration of CD14/TLR4 antagonist during central inflammation induced by icv administration of LPS. Different lowercase letters (a, b) indicate significant differences between groups at P < 0.05 (one-way ANOVA followed by Tukey’s post hoc test)

Article Snippet: ELISA assay for the GnRH concentration in the ME Concentrations of GnRH in the ME were determined with a commercial GnRH ELISA kit dedicated for sheep (CUSABIO BIOTECH Co., Ltd., China).

Techniques: Concentration Assay, Clinical Proteomics

Journal: Journal of animal science and biotechnology

Article Title: Effect of CD14/TLR4 antagonist on GnRH/LH secretion in ewe during central inflammation induced by intracerebroventricular administration of LPS.

doi: 10.1186/s40104-018-0267-8

Figure Lengend Snippet: Fig. 4 Mean (± SEM) relative GnRH mRNA expression in four hypothalamic structures (POA, AHA, MBH, ME) after icv administration of CD14/TLR4 antagonist during central inflammation induced by icv administration of LPS. Different lowercase letters (a, b) indicate significant differences between groups at P < 0.05 (one-way ANOVA followed by Tukey’s post hoc test)

Article Snippet: ELISA assay for the GnRH concentration in the ME Concentrations of GnRH in the ME were determined with a commercial GnRH ELISA kit dedicated for sheep (CUSABIO BIOTECH Co., Ltd., China).

Techniques: Expressing

Journal: Journal of animal science and biotechnology

Article Title: Effect of CD14/TLR4 antagonist on GnRH/LH secretion in ewe during central inflammation induced by intracerebroventricular administration of LPS.

doi: 10.1186/s40104-018-0267-8

Figure Lengend Snippet: Fig. 5 Mean (± SEM) relative GnRH-R mRNA expression in the ME and in the AP after icv administration of CD14/TLR4 antagonist during central inflammation induced by icv administration of LPS. Different lowercase letters (a, b) indicate significant differences between groups at P < 0.05 (one-way ANOVA followed by Tukey’s post hoc test)

Article Snippet: ELISA assay for the GnRH concentration in the ME Concentrations of GnRH in the ME were determined with a commercial GnRH ELISA kit dedicated for sheep (CUSABIO BIOTECH Co., Ltd., China).

Techniques: Expressing

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: Heliyon

Article Title: Brucella abortus RB51 Δ leuB expressing Salmonella FliC conjugated gonadotropins reduces mouse fetal numbers: A possible feral swine brucellosis immunocontraceptive vaccine

doi: 10.1016/j.heliyon.2021.e06149

Figure Lengend Snippet: Anti-his-tag Western blot. Fluorescent anti-his-tag antibodies were applied to blotted protein extracts. Lane-1 consists of the BioRad Precision Plus Protein Ladder. Lane-2 consists of protein extract from strain RB51L, showing no significant bands. Lane-3 consists of protein extract from strain RB51LGnRH isolated from the spleens of BABLB/c mice 14 days post inoculation, with a his-tag protein between 37 kDa and 50 kDa. This corresponds with the theoretical size of the his-tagged FliC-GnRH protein (≈40kDa). The original pictograph is represented in Supplemental Figure 4 .

Article Snippet: The presence of GnRH and FSH antibodies was confirmed via absorbance values with the following protocol: Antigens (Provided by Boster and BEI Resources, VA, USA) were diluted in PBS to a concentration of 20 μg/ml.

Techniques: Western Blot, Isolation

Journal: Heliyon

Article Title: Brucella abortus RB51 Δ leuB expressing Salmonella FliC conjugated gonadotropins reduces mouse fetal numbers: A possible feral swine brucellosis immunocontraceptive vaccine

doi: 10.1016/j.heliyon.2021.e06149

Figure Lengend Snippet: GnRH and porcineFSHβ IgG antibody ELISAs from male and female BALB/c mice. (a) Fifty microliters of serum collected 81 days post vaccination from both male and female mice were tested in duplicate for detection of anti-GnRH IgG antibodies. All group values were compared via One-way ANOVA, and the statistically significant differences are shown. The OD 450 values from the serum of mice in the strain RB51LGnRH immunized group were significantly higher than those from the PBS control group with a p value of <0.0001. The OD 450 values from the serum of mice in the Improvest® immunized group were significantly higher than those from the control group PBS, with the p value of <0.0001. The OD 450 values from the serum of mice in the strain RB51LGnRH immunized group were significantly higher than those from the RB51L control group with a p value of 0.02. (b) Fifty microliters of serum collected 81 days post vaccination from both male and female mice were tested in duplicate for detection of porcineFSHβ IgG antibodies. All group values were compared via One-way ANOVA, and the statistically significant differences are shown. The OD 450 values from the serum of mice in the strain RB51LF immunized group were significantly higher than those from the PBS control group with a p value of <0.0001 and the control RB51L group with a p value of 0.0011. This shows that there was an immune response amounted to the vaccines, which can be further seen by the other data sets. There were no significant differences between groups without brackets and p values.

Article Snippet: The presence of GnRH and FSH antibodies was confirmed via absorbance values with the following protocol: Antigens (Provided by Boster and BEI Resources, VA, USA) were diluted in PBS to a concentration of 20 μg/ml.

Techniques: Control, Vaccines