Leptin and leptin receptor mRNA and protein expression in the murine fetus and placenta

Abstract

Leptin is a 167-aa protein that is secreted from adipose tissue and is important in the regulation of energy balance. It also functions in hematopoiesis and reproduction. To assess whether leptin is involved in fetal growth and development we have examined the distribution of mRNAs encoding leptin and the leptin receptor (which has at least six splice variants) in the 14.5-day postcoitus mouse fetus and in the placenta using reverse transcription-PCR and in situ hybridization. High levels of gene expression for leptin, the leptin receptor, and the long splice variant of the leptin receptor with an intracellular signaling domain were observed in the placenta, fetal cartilage/bone, and hair follicles. Receptor expression also was detected in the lung, as well as the leptomeninges and choroid plexus of the fetal brain. Western blotting and immunocytochemistry, using specific antibodies, demonstrated the presence of leptin and leptin receptor protein in these tissues. These results suggest that leptin may play a role in the growth and development of the fetus, both through placental and fetal expression of the leptin and leptin receptor genes. In the fetus, leptin may be multifunctional and have both paracrine and endocrine effects.

Figure 1

RT-PCR expression analysis of leptin and alternatively spliced OB-Rs in mouse placentae (P1-P3) and mouse adipose tissue (W). The expression of leptin (OB) was compared with the common extracellular domain of the OB-R, the short splice variant (OB-Ra), and the long form of the OB-R (OB-Rb). β-actin amplification was used as a control. The tissues all were extracted and reverse-transcribed at the same time. No bands were observed with the mock cDNA (data not shown) or in the absence of cDNA (C). Molecular markers (100-bp ladder) and size of RT-PCR products are shown.

Figure 2

(A) Western blot of leptin protein in mouse (M), rat (R), human (H), and ovine (O) placentae compared with mouse white adipose tissue (WAT). A major band was observed at 16 kDa. (B) Western blot of OB-R protein in two mouse placentae. Markers are as shown. Ten micrograms of protein was loaded onto each lane, and Western blotting was performed as described in Experimental Procedures.

Figure 3

In situ hybridization to sections of murine fetus and placenta hybridized with a [³⁵S] antisense (Upper) and sense (Lower) riboprobes to (A) leptin and (B) common extracellular domain of the OB-R mRNA. PL, placenta; L, lung; LM, leptomeninges; V, vertebrae; R, ribs; F, femur; HL, hindlimb; PI, primordium of incisor teeth; NT, nasal turbinate; CD, cochlear duct and HF, hair follicle. (×2.7.)

Figure 4

Dark-field high-power images of in situ hybridization to adjacent sections of the murine fetal fifth rib with antisense or sense riboprobes to (A) leptin (B) OB-R, and (C) OB-Rb mRNA. (Bar = 100 μm.) Dark-field high-power images of in situ hybridization to sections of (D) murine placenta hybridized with antisense or sense riboprobes to OB-Rb mRNA and murine fetal hindlimb digits hybridized with riboprobes to OB-R (E) and OB-Rb (F) mRNA. (Bar = 100 μm in D and 250 μm in E and F.)

Figure 5

Murine fetal tissue sections of rib (A–F), placenta (G and J), vertebrae (H and K), and hind limb digits (I and L) were incubated with leptin (A and G–I) and OB-R (D) antiserum. The immunoreaction is visualized by diaminobenzidine, positive cells giving a brown color at the site of reaction. The specificity of the immunoreaction of leptin (B and J–L) and OB-R (E) was confirmed by omission of the primary antibody. In the case of leptin, specificity was further confirmed by preabsorption of the leptin antiserum with synthetic leptin (C) and for the OB-R by incubation with human Ig G antiserum instead of the primary antibody (F). Sections were counterstained with neutral red (D and E) and toluidine blue (all other sections). (Bar = 50 μm in A, D, and H, 100 μm in (G), and 250 μm in I.)