Antizyme inhibitor 2 hypomorphic mice. New patterns of expression in pancreas and adrenal glands suggest a role in secretory processes

Abstract

The intracellular levels of polyamines, polycations implicated in proliferation, differentiation and cell survival, are regulated by controlling their biosynthesis, catabolism and transport. Antizymes and antizyme inhibitors are key regulatory proteins of polyamine levels by affecting ornithine decarboxylase, the rate-limiting biosynthetic enzyme, and polyamine uptake. We recently described the molecular function of a novel antizyme inhibitor (AZIN2). However, the physiological function of AZIN2 in mammals is mostly unknown. To gain insight on the tissue expression profile of AZIN2 and to find its possible physiological role, we have generated, transgenic mice with severe Azin2 hypomorphism. This mouse model expresses transgenic bacterial β-D-galactosidase as a reporter gene, under the control of the Azin2 endogenous promoter, what allows a very sensitive and specific detection of the expression of the gene in the different tissues of transgenic mice. The biochemical and histochemical analyses of β-D-galactosidase together with the quantification of Azin2 mRNA levels, corroborated that AZIN2 is mainly expressed in testis and brain, and showed for the first time that AZIN2 is also expressed in the adrenal glands and pancreas. In these tissues, AZIN2 was not expressed in all type of cells, but rather in specific type of cells. Thus, AZIN2 was mainly found in the haploid germinal cells of the testis and in different brain regions such as hippocampus and cerebellum, particularly in specific type of neurons. In the adrenal glands and pancreas, the expression was restricted to the adrenal medulla and to the Langerhans islets, respectively. Interestingly, plasma insulin levels were significantly reduced in the transgenic mice. These results support the idea that AZIN2 may have a role in the modulation of reproductory and secretory functions and that this mouse model might be an interesting tool for the progress of our understanding on the role of AZIN2 and polyamines in specific mammalian cells.

Figure 1

A: Generation of the Azin2 reporter mice by gene-trap insertional mutagenesis. The recombinant ES clone harbours the cassette between the coding exons 4 and 5. Azin2F/Azin2R genotyping primer pairs hybridize on intron 4 at either side of the insertion point resulting in amplification only from the wild-type allele, whereas the Azin2F/V76R pairs result in amplification from the targeted allele. The recombinant gene product conserves 92 amino terminal residues of the native Azin2. B: Characteristic genotyping PCR bands of the resulting phenotypes. C: Real-time RT-PCR analysis of the expression of Azin2 and β-gal reporter mRNA in Azin2 expressing-tissues. Relative expression with respect to the values in testis.

Figure 2. Real-time RT-PCR analysis of Azin2 mRNA levels in different tissues of wild type and homozygous transgenic mice (Azin2^βGeo/βGeo).

Results in the transgenic mice are expressed as percent of wild type values. The absolute values in wild type mice normalized against beta-actin (expressed as mean±SE, n = 6–8) were: 0.590±0.031(testis), 0.0231±0.0032 (brain), 0.0134±0.0028 (adrenal glands), 0.151±0.030 (pancreas). Statistical significance (*) P<0.001 vs wild type.

Figure 3. β-D-galactosidase activity in different mouse tissues.

A: Comparison of enzyme activity in wild type (Azin2^+/+), heterozygous (Azin2^+/βGeo) and homozygous (Azin2^βGeo/βGeo) mice. Results are expressed as mean±SE of 4–6 animals per group. Activity is expressed as ?A₄₂₀ per h and g wet tissue. Statistical significance (*) P<0.05 vs wild type and heterozygous mice. B: Relative β-D-galactosidase activity in the soluble and particulate cell fractions obtained from different tissues of homozygous transgenic mice, as described in the Materials and methods section.

Figure 4. X-gal staining of testis and brain sections from Azin2^+/βGeo mice (blue) counterstained with Neutral Red.

A: Cross section of a seminiferous tubule, showing blue staining in the inner part of the tubule, where spermatids and spermatozoa are located. B: Cross section of the epididymis, showing blue staining in the lumen, where spermatozoa are stored. C: Brain hippocampus, showing blue staining in the dentate gyrus. D: Cerebellum with neurons showing vesicular-like structures with intense blue staining. Endogenous β-D-galactosidase activity at experimental conditions was only detectable in proximal epithelium of the epididymis.

Figure 5. X-gal staining of pancreas and adrenal gland sections from Azin2^+/βGeo mice (blue) counterstained with Neutral Red.

A: Adrenal gland showing high Azin2 expression throughout the medulla. B: Magnification of the adrenal medulla depicting areas of cytosolic and granular localization of X-gal staining; v, blood vessel. C: Section of the pancreas showing Azin2 expressing islets (arrows). D: Langerhans islet showing sparse staining within the islet cell population. At the experimental conditions X-gal staining was not detected in pancreas or adrenal glands of wild type mice.

Figure 6. Immunofluorescence analysis of insulin expression combined with X-gal histochemical staining on a pancreatic islet.

A: Conventional X-gal staining of an islet. Note both the cytosolic and granular staining. B: Electronic transformation of the histochemical staining into a fluorescence-like red staining (RGB-color images from bright field microscopy were converted to gray scale 8-bit images with ImageJ, inverted, and after that, red color was assigned to replace gray). C: Immunostaining with an insulin antibody (green). The vast majority of cells within the islet are insulin-expressing β-cells. D: Merge of X-gal staining (red) and insulin staining (green).