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. 2017 Apr 1;158(4):815-830.
doi: 10.1210/en.2016-1788.

TRH Action Is Impaired in Pituitaries of Male IGSF1-Deficient Mice

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Free PMC article

TRH Action Is Impaired in Pituitaries of Male IGSF1-Deficient Mice

Marc-Olivier Turgeon et al. Endocrinology. .
Free PMC article

Abstract

Loss-of-function mutations in the X-linked immunoglobulin superfamily, member 1 (IGSF1) gene cause central hypothyroidism. IGSF1 is a transmembrane glycoprotein of unknown function expressed in thyrotropin (TSH)-producing thyrotrope cells of the anterior pituitary gland. The protein is cotranslationally cleaved, with only its C-terminal domain (CTD) being trafficked to the plasma membrane. Most intragenic IGSF1 mutations in humans map to the CTD. In this study, we used CRISPR-Cas9 to introduce a loss-of-function mutation into the IGSF1-CTD in mice. The modified allele encodes a truncated protein that fails to traffic to the plasma membrane. Under standard laboratory conditions, Igsf1-deficient males exhibit normal serum TSH levels as well as normal numbers of TSH-expressing thyrotropes. However, pituitary expression of the TSH subunit genes and TSH protein content are reduced, as is expression of the receptor for thyrotropin-releasing hormone (TRH). When challenged with exogenous TRH, Igsf1-deficient males release TSH, but to a significantly lesser extent than do their wild-type littermates. The mice show similarly attenuated TSH secretion when rendered profoundly hypothyroid with a low iodine diet supplemented with propylthiouracil. Collectively, these results indicate that impairments in pituitary TRH receptor expression and/or downstream signaling underlie central hypothyroidism in IGSF1 deficiency syndrome.

Figures

Figure 1.
Figure 1.
Generation of Igsf1 loss-of-function mice using CRISPR-Cas9. (a) PCR amplification of the targeted region in exon 18 of Igsf1 in the founder female (CRISPR.2, lane 1) and five of her progeny (3.1.1 to 3.1.5, lanes 2 to 6). Arrows at the right indicate the wild-type (top) or Δ312 (bottom) alleles. (b) Genomic organization (top) and RNA splicing (bottom) around exons 17 to 20 of the wild-type (left) and Δ312 alleles (right). Exons are presented as boxes and are numbered. Intervening introns are shown as lines. The deleted parts of exon 18 and intron 18 in the Δ312 allele are shown with a line at the top of the wild-type schematic (left). The novel exon in the Δ312 allele, which is derived from the 5′ end of exon 18, the 3′ end of intron 18 (labeled 18i), and all of exon 19, is shown with broken lines (right). Dark boxes denote translated sequences, whereas the pale boxes reflect untranslated regions. Genomic structure 5′ of exon 17 is not pictured. WT, wild-type.
Figure 2.
Figure 2.
Reduced pituitary IGSF1 protein and mRNA expression in Igsf1Δ312/y mice. (a) Proteins extracted from four adult wild-type (lanes 1 to 4) and six Δ312 males (lanes 5 to 10) were subjected to SDS-PAGE and immunoblotting using an anti–IGSF1-CTD antibody. Arrows mark the mature and immature glycoforms in wild-type and the truncated protein in Δ312 males. *, nonspecific bands. (b) Protein extracts from two wild-type (lanes 1 to 6) and two Δ312 males (lanes 7 to 12) were treated with EndoH or PNGaseF prior to analysis as in (a). (c) RNA was extracted from pituitaries of adult wild-type (circles) or Δ312 males (squares). mRNA expression of Igsf1 was analyzed by reverse transcription qPCR using primers common to both transcripts. Group means are indicated with solid horizontal lines. The data were analyzed by a two-tailed t test (t = 19.95, df = 12.64, ****P < 0.0001). IB, immunoblotting; WT, wild-type.
Figure 3.
Figure 3.
TSH synthesis is reduced in Igsf1Δ312/y mice. Serum (a) TSH, (b) T4, (c) T3 levels, and (d) pituitary TSH content in 8-week-old wild-type and Igsf1Δ312/y males. (e) Number of thyrotrope cells counted per unit area on immunohistochemistry-stained sections. (f–h) Tshb, Cga, and Trhr1 mRNA levels in pituitaries of 10-week-old wild-type and Igsf1Δ312/y males. The data were analyzed by a two-tailed unpaired t test: (a) t = 0.7735, df = 20.40, N.S. P = 0.4481; (b) t = 0.3515, df = 22.00, N.S. P = 0.7285; (c) t = 0.4876, df = 20.88, N.S. P = 0.6309; (d) t = 4.645, df = 21.78, ***P = 0.0001; (e) t = 0.5445, df = 9.263, N.S. P = 0.5990; (f) t = 5.316, df = 15.68, ****P < 0.0001; (g) t = 2.644, df = 23.04, *P = 0.0145; (h) t = 4.353, df = 17.82, ***P = 0.0004. N.S., not significant.
Figure 4.
Figure 4.
TSH release is blunted in Igsf1Δ312/y mice. Ten-week-old male wild-type (black line with circles) and Igsf1Δ312/y (black line with squares) mice were placed on a LoI/PTU diet for 3 weeks. (a) Serum TSH was measured immediately before, and then at weekly intervals after, mice were placed on the diet. The data were analyzed by hybrid two-way ANOVA [interaction: F(3, 75) = 40.6, ****P < 0.0001; time on diet: F(3, 75) = 184.0, ****P < 0.0001; genotype: F(1, 25) = 77.68, ****P < 0.0001] and post hoc t tests. n = 14 for wild-type and n = 13 for Igsf1Δ312/y. Data at the 3-week time point are expanded to show individual data points (two-tailed t test: t = 7.322, df = 15.16, ****P < 0.0001). (b) T4 levels were measured from the same serum samples as in (a). The data were analyzed by two-way ANOVA: interaction: F(3, 97) = 2.148, N.S. P = 0.0992; time on diet: F(3, 97) = 349.2, ****P < 0.0001; genotype: F(1, 97) = 0.004213, N.S. P = 0.9484). (c–e) Pituitary RNA from the same animals as in (a) was extracted after 3 weeks on the LoI/PTU diet and (c) Tshb, (d) Cga, and (e) Trhr1 mRNA levels were measured by reverse transcriptase qPCR. The data were analyzed by two-tailed t tests: (c) t = 4.039, df = 14.31, **P = 0.0012; (d) t = 4.531, df = 18.73, ***P = 0.0002; (e) t = 2.614, df = 15.66, *P = 0.0190. N.S., not significant.
Figure 5.
Figure 5.
Thyroid hormone feedback mechanisms appear to be intact in Igsf1-deficient mice. (a) Representative ISH images of Trh mRNA expression in the PVN of brains from 8-week-old Igsf1+/y and Igsf1Δ312/y mice on a normal diet. (b) Quantification of average Trh ISH signal in Igsf1+/y and Igsf1Δ312/y brains. The data were analyzed by two-tailed t test: t = 5.861, df = 21.87, ****P < 0.0001. (c) Representative ISH images of Trh mRNA expression in the PVN of brains from 11-week-old Igsf1+/y and Igsf1Δex1/y mice on a LoI/PTU diet for 3 weeks. On the last 4 days of the experiment the mice were injected with one of four treatments (saline, low T3, high T3, high T4). (d) Quantification of average Trh ISH signal in Igsf1+/y and Igsf1Δex1/y brains in each condition. The data were analyzed by two-way ANOVA [interaction: F(3, 19) = 0.4753, N.S. P = 0.7032; treatment: F(3, 19) = 72.83, ****P < 0.0001; genotype: F(1, 19) = 0.02785, N.S. P = 0.8692]. N.S., not significant.
Figure 6.
Figure 6.
TRH-induced TSH release is blunted in Igsf1-deficient mice. (a) Eight-week-old male wild-type and Igsf1Δ312/y mice were injected with TRH (i.p. 10 μg/kg). Blood samples were collected before the TRH injection as well as 15 minutes, 1 hour, and 2 hours after injection. Plasma TSH levels were measured by ELISA. Data were analyzed by two-way ANOVA [interaction: F(3, 99) = 30.31, ****P < 0.0001; time after injection: F(3, 99) = 160.9, ****P < 0.0001; genotype: F(1, 99) = 37.67, ****P < 0.0001] and post hoc t test, n = 13 for each genotype. The data from the 15-minute time point were expanded to show individual data points (two-tailed t test: t = 5.627, df = 22.72, ****P < 0.0001). (b) Male 8-week-old wild-type and Igsf1Δex1/y mice were injected with TRH (i.p. 10 μg/kg). Blood samples were collected prior to and 15 minutes after TRH injection. Serum TSH levels were measured by RIA. Data were analyzed by two-way ANOVA [interaction: F(1, 20) = 11.01, **P = 0.0034; time: F(1, 20) = 79.65, ****P < 0.0001; genotype: F(1, 20) = 13.88, **P = 0.0013] and post hoc t test (t = 3.531, df = 8.466, **P = 0.0071), n = 6 per genotype.

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