Figure 1.
Genomic organization and protein structure of the porcine Igf1.
The core transcript, consisting of exons 3 and 4 (yellow and blue), is either spliced to exon 1 (orange) or exon 2 (green). IGF1-Ea is joined to exon 6 (violet). IGF1-Eb is joined to exon 5 (red). Mgf consists of the exons 3, 4, the N-terminal part of exon 5, consisting of 49 or 52 nucleotides (depending on the species) and exon 6 (striated violet). Exon 5 evokes a frame shift in exon 6 resulting in an altered amino acid sequence at the C-terminal end. The two dark grey regions symbolize the promoter regions of the corresponding transcript. The colours of the exons were chosen to match a corresponding figure of the human IGF gene in Goldspink G Physiology 2005;20∶232–238.
Table 1.
Primer sequences for real-time PCR.
Table 2.
MGF Peptide sequences.
Figure 2.
Peptide sequences of MGF in different species.
MGF sequences are highly conserved and always consist of 25 amino acids with the exception of the human MGF peptide, which consists of only 24 amino acids.
Figure 3.
Mgf and Igf1 mRNA expression profile (normalized to 18S rRNA) of various porcine tissue samples (A) and within different zones of the growth plate separated by laser capture microdissection (B) as quantified by real-time PCR.
(n≥3 per tissue, One-way ANOVA of log-transformed data (a) or Kruskal-Wallis with Bonferroni-adjusted post-hoc tests (b) were used to detect differences between groups: ** p<0.01; ***p<0.001 vs proliferative chondrocytes; ###p<0.001 vs hypertrophic chondrocytes. Data are presented as mean + SEM).
Figure 4.
Immunohistochemical detection of MGF and IGF-1 in the porcine growth plate (overview, magnification 10x).
MGF and IGF-1 was detected in chondrocytes of the resting (rz), proliferating (pz) and hypertrophic (hz) zone of porcine growth plate. We found the MGF peptide in the cytoplasm and in some chondrocytes also in the nucleus of resting and hypertrophic chondrocytes, although some cells were completely negative. More negative cells were detected in the proliferating zone (magnification 50×).
Figure 5.
Immunofluorescent staining of GP cartilage (rz) for MGF and IGF1 shows co-localisation of green signal (white arrows) with DAPI stained nuclei (blue) in few cells.
Some cells are negative for MGF as well as IGF1.
Figure 6.
Immunohistochemical detection of MGF in porcine liver (a) tendon (c) and articular cartilage (d). Only weak staining was seen in porcine skeletal muscle (b), (magnification 40×).
Figure 7.
Proliferation effects of MGF/IGF-1.
Effects of IGF-1, different MGF peptides and their combination on proliferation of porcine growth plate chondrocytes as measured by BrdU staining. (n = 5–11 per group; One-way ANOVA of log-transformed data, Games-Howell post-hoc analyses: * p<0.05; ** p<0.01; *** p<0.001 vs respective untreated control; Data are shown as mean + SEM).