Fig 2: 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.

Fig 3: 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 4: Male gonadal underdevelopment and reproductive deficits in Rnf216/Triad3 knockout mice(A) Left, representative images of male and female reproductive organs in WT and KO mice at 16 weeks old. Scale bar represents 25 mm. Right, Gonadal weights of WT and KO mice at 4-, 16-, and 52-weeks. Male KO (blue) mice show a significant reduction in testicular weights compared to WT at 4- (t (8) =4.321, **p = 0.0025), 16- (t (13) =13.96, ****p<0.0001), and 52-weeks (t (17) =5.065, ****p<0.0001). No significant differences in ovarian weights, which includes the fat pads surrounding the ovaries (pink). Unpaired t-test. N = 9–12 animals per group. Error bars are ±SEM.(B) Assessments of breeding viability in WT and KO mice at 6–7 weeks. Left, there were no differences in the number of days before the first litter was produced. There were significant differences in the number of litters (F (3, 13) =11.76, ***p = 0.0005) (middle) and number of pups per litter (right) (F (3, 24) = 8.213, ***p = 0.0006). One-way ANOVA with Tukey’s post-hoc analysis. *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001, N = 4-5 cages per genotype crossing pair and N = 8–12 pups per litter. Error bars are ±SEM.(C) Percentage of pup survival from WT and KO breeding pairs. n = 102 for pups that survived and n = 29 for pups that died. In deceased pups, KO mothers have a higher percentage compared to other genotypes (X2 (2) = 8.756, *p = 0.0126) Chi-square.(D) Left, No differences in LH serum levels in WT and KO mice at 16-weeks. Right, Male KO animals have elevated FSH (t (19) =2.521, *p = 0.0208). No significant differences in females. N = 10–12 animals per sex per genotype. Error bars are ±SEM.(E) Male KO animals show reductions in basal inhibin B levels (t (14) =6.608, ****p<0.0001); Unpaired t-test. N = 7–10 male mice. Error bars are ±SEM.(F) Days females spent in each estrous phase for a duration of 20 days. KO females spent significantly less time in proestrus compared to WT (t (15) =2.809, *p = 0.0132); Unpaired t-test. N= 8-9 female mice per genotype. Error bars are ±SEM.(G) Left, Representative estrous cycle of WT and KO females. KO females showed irregular cycling compared to WT females. Right, Arrests in each phase of the estrous cycle of Rnf216/Triad3 KO females for a duration of 20 days. Cycle arrests were measured as spending >1 day in proestrus, estrus, and metestrus or >2 days in diestrus. Female KO mice (Median = 1.000) significantly arrested in diestrus compared to WT (Median= 0.000). Mann-Whitney U, U= 16, *p= 0.0294. N= 8-9 female mice per genotype. Error bars are + SEM.See also Figure S2.