Fig 1: 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 (X2(3) = 1.709, p = 0.6349) or females (X2(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.
Fig 2: Rnf216/Triad3 reduces GnRH and Ca2+ 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 ß-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.
Fig 3: 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.
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