Detection of apoptosis-inducing factor in involuting

Micron 35 (2004) 307–310 www.elsevier.com/locate/micron

Short communication

Detection of apoptosis-inducing factor in involuting mammary tissue by immunoelectron microscopy M. Colittia,*, R. Musettib, B. Stefanona b

a Department of Scienze della Produzione Animale, University of Udine, via delle Scienze, Udine 208-33100, Italy Department of Biologia Applicata alla Difesa delle Piante, University of Udine, via delle Scienze, Udine 208-33100, Italy

Received 8 April 2003; revised 28 July 2003; accepted 1 August 2003

Abstract Immunoelectron microscopy was used to study the subcellular localisation of apoptosis-inducing factor (AIF) in involuting bovine mammary tissue. AIF was detected using a polyclonal antibody and secondary anti-rabbit antibody conjugated to colloidal gold particles under optimised conditions. The polyclonal antibody appeared specific for the bovine antigen and immunocytological examination identified specific localisation of AIF in mitochondria, cytoplasm and nucleus of mammary epithelial cells in involuting mammary tissue, suggesting a subcellular translocation in cells undergoing apoptosis. q 2003 Elsevier Ltd. All rights reserved. Keywords: Cow; Mammary gland; Apoptosis-inducing factor; Immunogold technique; Transmission electron microscopy

1. Introduction Apoptosis-inducing factor (AIF) is a flavoprotein containing an oxidoreductase domain, normally confined to mitochondria, but responsible for nuclear chromatinolysis and mitochondrial membrane permeabilization, if stimulated by apoptotic factors (Zamzami et al., 1996; Susin et al., 1999; Crompton, 1999). Under normal circumstances, AIF is contained in the mitochondrial intermembrane space, but, on induction of apoptosis, the protein is transported to the cytosol and the nucleus, where it triggers the classical signs of apoptosis (Susin et al., 1999, 2000). The release of AIF occurs before that of cytochrome c and before caspase activation; therefore, DNA fragmentation induced by AIF, is caspase-independent (Daugas et al., 2000). It is known that apoptosis plays an important role in mammary development (Strange et al., 2001), lactation (Wilde et al., 1997; Li et al., 1999) and involution after lactation (Capuco and Akers, 1999; Wilde et al., 1999). In a recent paper (Colitti et al., 2003), we reported the nucleotide sequence of bovine mammary AIF, that was identified and its expression examined in mammary tissue by in situ immunocytochemistry. At peak lactation, AIF expression, together with that * Corresponding author. Tel.: þ 39-432558583; fax: þ 39-432558585. E-mail address: [email protected] (M. Colitti). 0968-4328/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2003.08.002

of other apoptosis-related genes (bcl-2 and bax), appeared to favour a shift in the dynamic balance in the cell population from proliferation towards apoptosis. However, AIF expression is, by itself, no indicator of apoptogenic signalling, inasmuch it is expressed ubiquitously in healthy human tissues (Daugas et al., 2000). To assess whether AIF is likely to have a role in mammary involution, the subcellular localisation of AIF was examined in involuting bovine mammary tissue at a time when tissue remodelling and apoptosis was occurring.

2. Materials and methods Four Holstein Friesian cows were housed in a free-stall for the duration of the study. Clinical examination showed that the cows were healthy and not affected by mastitis. Mammary tissue was sampled 10 days after termination of lactation by cessation of milking. Tissue samples (10 –20 mg) were obtained using a Bard biopsy instrument, mounted with a 12 gauge needle (Magnum Biopsy System, Bard, Convington, GA 30209, USA) under subcutaneous anaesthesia from the right-rear quarter of the udder. The biopsy site was treated with antiseptic liquid and antibiotic powder. The experiment was carried out in accordance with state and local laws and ethical regulations.

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Tissue was processed for AIF detection and for identification of apoptotic cells by the TUNEL method. Accordingly, samples were fixed, respectively, in glutaraldehyde at 1 and 0.5% in 0.1 M phosphate buffer solution (PBS) pH 7.4 and kept for 2 h at 4 8C. They were then rinsed with shaking in PBS pH 7.4 for 30 min. Tissue pieces were dehydrated in a series of graded ethanol solution (25, 50 and 75%; 30 min per step), and finally placed in 100% ethanol for 1 h. Samples were then infiltrated with LR White (LRW) resin, first for 30 min in resin:ethanol (1:2, v/v), then for 30 min in resin:ethanol (2:1, v/v) and finally overnight at 4 8C in resin alone, with two changes. Samples were embedded in fresh LRW resin in BEEM capsules and polymerised at 60 8C for 24 h. Grids were then placed on droplets of 0.05 M Tris-buffered saline (TBS), pH 7.6, containing 1/30 normal goat antiserum (NGS) for 10 min to block non-specific binding sites. Samples were incubated overnight at 4 8C with primary rabbit polyclonal antibody (RAb) against AIF (Chemicon Int., Temecula, CA, USA) at antibody concentrations in the range 1– 50 mg/ml, and in the presence of NGS. Grids were then washed in TBS and treated with secondary anti-rabbit antibody coated with colloidal 5 nm gold particles (GAR 5) (EY Lab. Inc., San Mateo, CA, USA), diluted 1:20, 1:50, 1:100 in TBS. Sections were placed in 2% glutaraldehyde for 5 min, then 1% OsO4 for 15 min, and were stained with 3% uranyl acetate for 1 h at 40 8C and rinsed in water. The samples were then observed with a Philips CM 10 transmission electron microscope (TEM) operated at 80 kV.

3. Results and discussion The amino acid sequence of bovine AIF identified in a previous study had 93% homology with that of the human protein (residues 501 – 567) (Colitti et al., 2003). A rabbit anti-AIF (internal region) polyclonal antibody, raised against a peptide corresponding to amino acids 517 – 531 of human AIF, was used in immunoelectron microscopy. Microscopy showed that AIF was present in the mitochondria, cytoplasm and nucleus of mammary epithelial cells, while background staining was absent in other areas of the sections. The intensity of the reaction changed according to the concentration of both the primary Rab and the GAR, best results being obtained with a dilution of 25 mg/ml of the Rab and 1:10 of the GAR (results not shown). No reaction was observed when primary antibody was omitted (Fig. 1). Cell apoptosis is an integral part of bovine mammary tissue’s repertoire during the whole lactation cycle (Stefanon et al., 2002) and ranges from 2.4 ^ 0.5 to 4.8 ^ 0.6% of total cells during lactation and involution, respectively (Wilde et al., 1997). In the present paper, the average percentage of apoptotic cells in mammary tissue of the four cows, measured with TUNEL method, was equal to 4.38 ^ 1.69% of the total cell population. Apoptotic cell

Fig. 1. Negative control. Mitochondrion in mammary gland epithelial cell showed no AIF labelling. TEM. Scale bar ¼ 0.5 mm.

death in ruminant mammary gland is confined to individual cells in the alveolar epithelium (Colitti et al., 1999). Light microscopic evaluation of tissue samples confirmed that apoptotis was localised in individual cells, while their neighbours survived (data not shown). Under TEM, we identified normal and apoptotic cells based on their structural modification or chromatin condensation in nuclei (Colitti, 1999, Fig. 2) and, therefore, were able to compare the localisation of AIF in dying and surviving cells. In non-apoptotic cells, AIF was only detected close to the outer mitochondrial membrane (Fig. 3a), whilst in cells showing typical signs of early apoptosis AIF present in the cytoplasm were concomitantly detected (Fig. 3b), along the nuclear membrane (Fig. 3c) and in cell nuclei (Fig. 3d). Subcellular translocation of AIF has been observed in many models of apoptosis-induced in cell culture, i.e.

Fig. 2. The nucleus of an apoptotic epithelial cell. The coiled chromatin threads of the supranucleosomal level of organisation are seen (heads arrows). TEM. Scale bar ¼ 0.5 mm.


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Fig. 3. (a) Mitochondrion in non-apoptotic mammary epithelial cell showing AIF close to the outer membrane. TEM. Scale bar ¼ 0.5 mm. (b) Mitochondrion in apoptotic mammary epithelial cell. AIF-associated gold particles are present in mitochondrion and are widely distributed in cell cytoplasm (heads arrows). TEM. Scale bar ¼ 0.1 mm. (c) Gold particles detecting AIF presence outside and on the nuclear membrane of an epithelial cell during apoptosis. TEM. Scale bar ¼ 0.2 mm. (d) Gold particles detecting AIF presence outside (open arrows) and in the nucleus of an epithelial cell during apoptosis (arrows). TEM. Scale bar ¼ 0.5 mm.

mouse fibroblasts, smooth muscle cells and is a feature of apoptosis in diseased tissues in vivo, including retinal degeneration, brain damage and myocard infarction (Cande´ et al., 2002). The results of the present research suggest that AIF subcellular translocation also occurs in vivo in mammary cells stimulated to undergo apoptosis as part of the tissue’s normal physiological remodelling.

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