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. 2024 May 22;9(10):e167598. doi: 10.1172/jci.insight.167598

Figure 5. Madd is expressed in mouse pituitary gonadotropes; dex30 leads to reduced expression and secretion of LH.

Figure 5

(A and B) Representative RNAscope mRNA in situ hybridizations using probes against Madd (blue) and (A) Lhb or (B) Fshb (red) in adult (P45) mouse pituitary, coronal section. Arrows indicate examples of double-positive cells. Scale bar: 200 μm, ROI scale bar: 20 μm. AL, anterior lobe. (C) A representative RNAscope mRNA in situ hybridization using probes against Madd (red) combined with immunostaining using antibodies against LH+FSH (green) in adult (P45) mouse anterior pituitary. Arrows indicate examples of double-positive cells. Scale bar: 50 μm, ROI scale bar: 15 μm. RNAscope mRNA in situ hybridizations (A–C) were repeated 5–8 times. (D) A schematic of the genome-editing strategy used for generating dex30 LβT2 clones. (E) An RT-PCR around Madd exon 30 showing truncated transcript in dex30 LβT2 clones. (F) mRNA expression of Lhb in WT and dex30 LβT2 cells (n = 15). (G) LH content in WT and dex30 LβT2 cells normalized to total protein (n = 5 for WT, n = 10 for dex30). (H) Spontaneous LH secretion in WT and dex30 LβT2 cells during 24 hours, normalized to total protein (n = 5 for WT, n = 10 for dex30). (I and J) LH secretion from WT and dex30 LβT2 clone 1 during 20 minutes of static incubations with no stimuli, 50 nM GnRH, and 60 mM KCl (n = 5). Normalized to total protein (I) or LH content (J). ****P < 0.0001, and *P < 0.05 analyzed by Student’s t test (F–H) or multiple t tests (I and J).