Fig. 6: Lactating females require maternal brain CCN3 to sustain progeny.
a, Representative images of coronal brain sections from Esr1fl/fl females stained for ERα (magenta) and CCN3 (green) in the posterior medial basal ARC during pregnancy and postpartum stages (lactation and post weaning) (N ≥ 2 for each time point). Scale bar, 50 µm. b, Colocalization of Ccn3 (green), Kiss1 (red) and Esr1 (magenta and cyan) transcripts in the ARC of a lactating control (Esr1fl/fl) female at 7 days. Scale bar, 50 µm. c, Relative Ccn3 quantified from microdissected ARC tissue obtained from Esr1fl/fl virgin, Esr1Nkx2.1-cre mutant virgin and Esr1fl/fl lactating (7 DPP) female mice (N = 5, 3, 3). d, CCN3 (green) and vimentin (red, VIM) in the posterior ARC of virgin Esr1Nkx2.1-cre mutant and lactating or OVX Esr1fl/fl control mice (1 week after surgery). e–i, Ccn3 knockdown (KD) in the ARC of lactating female mice. e, Schematic of injection of shRNA Ccn3 (shCcn3) or shRNA control (shCtrl) vectors into the ARC of control Esr1fl/fl females with experimental timeline (left) and representative images of CCN3 at 12 DPP (right). Scale bar, 500 µm. LCD, low-calcium diet. f,g, Litter sizes (f) and BV of femurs (g) from mothers injected with shCtrl or shCcn3 (N = 5, 9) fed SD (0.8%Ca2+). h, Average pup weight (5–6 pups per litter) nursed by dams injected with shCtrl or shCcn3 and fed SD (N = 5,9) or LCD (0.01% Ca2+, N = 4,2). i, Litters at 8 DPP nursed by dams injected with shCtrl or shCcn3 and fed a LCD, with survival values in parentheses. Scale bar, 1 cm. j, Brain-derived MBH (that is, CCN3) replaces E2 as an osteoanabolic hormone during lactation and counteracts the catabolic actions of mammary-gland PTHrP to promote healthy bone formation, thereby ensuring adequate calcium supplies for milk and maternal skeleton integrity during lactation. One-way ANOVA for c (Šidák’s multiple-comparisons test). Unpaired Student’s t-test, two-tailed for f, and one-tailed for g. Three-way ANOVA for h (Tukey’s multiple-comparisons test). *P < 0.05, **P < 0.01, ****P < 0.0001. Error bars ± s.e.m. Graphics in j (mammary, bone and calcium) were reproduced or adapted from BioRender (https://www.biorender.com). Graphic in j (dam and litter) was reproduced from Mind the Graph (https://mindthegraph.com) under a Creative Commons licence CC BY-SA 4.0.
