Critical roles of the guanylyl cyclase B receptor in endochondral ossification and development of female reproductive organs

Abstract

Guanylyl cyclase B is the receptor for a small peptide (C-type natriuretic peptide) produced locally in many different tissues. To unravel the functions of the receptor, we generated mice lacking guanylyl cyclase B through gene targeting. Expression of the receptor mRNA in tissues such as bone and female reproductive organs was evident, and significant phenotypes associated with each of these tissues were apparent in null mice. A dramatic impairment of endochondral ossification and an attenuation of longitudinal vertebra or limb-bone growth were seen in null animals. C-type natriuretic peptide-dependent increases of guanylyl cyclase B activity, but not basal enzyme activity, appeared to be required for the progression of endochondral ossification. Female mice were infertile, but male mice were not. This result was due to the failure of the female reproductive tract to develop. Thus, the guanylyl cyclase B receptor is critical for the development of both bone and female reproductive organs.

Fig. 1.

No detectable GC-B mRNA expression or enzyme activity in mice with targeted disruption of the Npr2 gene. (a) Npr2 gene expression detected in the whole brain, growth-plate cartilage, and primary culture of dermal fibroblasts by Northern blot analysis (20 μg of total RNA per lane). Gapdh mRNA is shown in Lower as an internal control. (b) CNP-dependent increases of intracellular cGMP contents in dermal fibroblasts isolated from WT (▪) and null (•) mice (n = 3). On the x axis, “–10” means vehicle-treated. *, P < 0.001 vs. null, by two-way ANOVA with Bonferroni's post hoc test.

Fig. 2.

Dwarfism and early mortality of GC-B-null mice. (a) The longevity of Npr2^–/– mice (○, n = 48) is shorter than that of Npr2^+/+ (□, n = 56) or Npr2^+/– (▵, n = 107) mice (P < 0.0001, by the Kaplan–Meier method). (b) Gross appearance of 90-day-old WT and null females. (Scale bar, 2.5 cm.) (c) Male growth curves in nasoanal length: Npr2^+/+, □, n = 9; Npr2^+/–, ▵, n = 10; Npr2^–/– surviving beyond 100 days (group A), ○, n = 7; Npr2^–/– dying before 100 days (group B), •, n = 8. *, P < 0.0001 vs. Npr2^+/+;†, P < 0.05; ‡, P < 0.01 vs. Npr2^–/– (group A), by two-way ANOVA. (d) Female growth curves in nasoanal length: Npr2^+/+, □, n = 11; Npr2^+/–, ▵, n = 9; Npr2^–/– (group A), ○, n = 5; Npr2^–/– (group B), •, n = 7. (e and f) Growth curves of males (e) and females (f) in body weight. (g and h) Plain x-ray analysis of 30-day-old WT and GC-B-null mice. Shown are photos of representative cases (g) and bone dimensions of Npr2^+/+ (open bars) and Npr2^–/– (filled bars) mice (h)(n = 3). CL, nasooccipital length of the skull; CW, interparietal width of the skull; HL, humeral length; UL, ulnar length; FL, femoral length; TL, tibial length; VL, vertebral length; VW, vertebral width. The data of VL and VW are means of the first through fifth lumbar vertebrae. §, P < 0.05; ¶, P < 0.01 vs. Npr2^+/+, by Student's t test.

Fig. 3.

Growth-plate histology of 8-day-old WT and GC-B-null mice. (a) Hematoxylin/eosin staining of proximal growth-plate sections of tibiae. Periarticular (P), columnar proliferating (C), and prehypertrophic and hypertrophic (H) chondrocyte layers are indicated. (b) Immunohistochemical detection of PCNA in the tibial growth plate. I, with anti-PCNA rabbit serum; II, with nonimmune rabbit serum. (c) Detection of Npr2, Col10a1, and Ihh gene expression by in situ hybridization. (Scale bars, 0.1 mm.)

Fig. 4.

Tibial explant culture from neonates. (a) Tibial explants after 6-day culture with vehicle or 1 μM CNP. CP, cartilaginous primordium; OC, calcified ossification center. (b) Longitudinal growth of CPs (sum of CPs on both ends) and OC of tibial explants during the culture. The length of vehicle-treated WT (□) and null (○) explants and CNP-treated WT (▪) and null (•) explants are shown in Upper. Ratios of final to initial length in vehicle- and CNP-treated groups are shown by open and filled bars, respectively, in Lower. *, P < 0.05; †, P < 0.01 vs. day 0 in each group, by two-way ANOVA with Bonferroni's post hoc test (n = 5); ‡, P < 0.05 vs. vehicle-treated in each genotype, by ANOVA with Tukey's post hoc test (n = 5).

Fig. 5.

Transgene expression and phenotype of GC-B2 TG mice. (a) GC-B1 and GC-B2 mRNA expression in the growth-plate cartilage detected by RT-PCR. Selected molecular weight (MW) markers are shown on the left in bp; bands of GC-B1 and GC-B2 are indicated by an arrowhead and an arrow, respectively. Reactions with (+) or without (–) a reverse transcriptase of RNA isolated from the growth-plate cartilage of WT and GC-B2 TG mice are used for PCR as templates. (b) Growth of GC-B2 TG mice in nasoanal length. Data of WT females (○), TG females (•), WT males (□), and TG males (▪) are shown (n = 5). TG mice are significantly (P < 0.0001, by two-way ANOVA) shorter than WT mice in each sex.

Fig. 6.

Phenotypes of reproductive organs. (a–f) Gross appearance of female reproductive organs (a and b) and histology of uteri (c and d) and ovaries (e and f) from WT (a, c, and e) and null (b, d, and f) mice with hematoxylin/eosin staining. A corpora luteum (cl) is indicated in e.(g–k) Gross appearance of male reproductive organs (g) and histology of testes (h and i) and epididymi (j and k) from WT (h and j) and null (i and k) mice. (Scale bars: a, b, and g, 1 cm; c–f and h–k, 0.2 mm.)