SEARCH

SEARCH BY CITATION

Keywords:

  • uropygial gland;
  • chicken;
  • apoptosis;
  • survivin;
  • Bax;
  • PCNA;
  • holocrine

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. LITERATURE CITED

This study was designed to elucidate the presence of apoptosis and the localization of apoptosis-related Bax and survivin proteins and proliferating cell nuclear antigen (PCNA) within the chicken uropygial gland, a specialized holocrine secretory gland. In day-old chicks, survivin and Bax immunoreactivities were observed in the cell cytoplasm of the germinative and secretory layers of the luminal epithelium and tubules. During this period, the TUNEL reaction, an indication of apoptosis, was only sporadically positive in the tubules. From the 7th day to the 150th day of posthatching, survivin was detected in the cytoplasm of cells in the germinative layer and in the nuclei of some cells in the secretory layers of the gland. The germinative layer cells showed weak homogeneous cytoplasmic staining for Bax, whereas the cells of the secretory and intermediate layers of luminal epithelium and tubules exhibited granular cytoplasmic staining. After day 7, TUNEL-positive cells were observed in the secretory and degenerative layers of the luminal epithelium and central tubules. After day 12, some TUNEL-positive cells were also seen in the peripheral tubules. At all posthatch ages, the cytoplasm and nucleus of the germinative layers of luminal epithelium and tubules reacted with PCNA, whereas only a small number of cell nuclei in the secretory layers were immunopositive. These results support the theory that specific PCNA/Bax/survivin expression patterns could reflect particular cell differentiation states in the uropygial gland and that holocrine secretion in the gland is realized mainly by way of apoptosis. Anat Rec, 296:504–520, 2012. © 2012 Wiley Periodicals, Inc.


Abbreviations used
APES

3-aminopropyl-ethoxy-silane

Bax

bcl-2-associated X protein

Bcl-2

B-cell lymphoma 2

Blimp1

B lymphocyte-induced maturation protein

DAB

3,3′-diaminobenzidine tetrahydrochloride

IAP

inhibitor of apoptosis protein

p21/Waf1

cyclin-dependent kinase inhibitor 1

PBS

phosphate buffered saline

PCNA

proliferating cell nuclear antigen

PPAR-γ

peroxisome proliferator-activated receptor gamma

S-IR

survivin immunoreactivity

SG

sebaceous gland

TdT

terminal deoxynucleotidyl transferase

TUNEL

terminal deoxynucleotidyl transferase (TdT) mediated dUTP Nick End Labeling

UG

uropygial gland

INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. LITERATURE CITED

The uropygial gland (UG), also referred to as the preen gland, is a significant skin gland found in many bird species and is located at the base of tail. The gland exhibits a striking morphological diversity in size, shape and presence/absence of tufts of feathers (Salibián and Montalti, 2009). Despite great differences in size and location, the UG is similar in structure and function to mammalian sebaceous glands (SG) and secretes an oily, waxy substance called preen oil, or sebum, which lubricates the skin by a holocrine process (Jacob and Ziswiler, 1982; Thody and Shuster, 1989; Zouboulis, 2003) to protect it against bacterial and fungal infections (Bandyopadhyay and Bhattacharyya, 1999; Shawkey et al., 2003; Georgel et al., 2005; Reneerkens et al., 2008). Salibián and Montalti (2009) showed that acidic mucins, neutral lipids, glycolipids and phospholipids are normal components of secretion. Recently, we determined that the chicken UG contains both αβ and ββ dimers of S100 protein, a calcium-binding protein (Liman et al., 2009).

Sebum is produced by sebocytes, specialized epithelial cells in the UG (Jenik et al., 1987; Goodridge et al., 1989; Smith and Thiboutot 2008; Schneider and Paus, 2010). Sebocytes are constantly undergoing a process of cell division, differentiation, and cell death (Jenkinson et al., 1985; Jenik et al., 1987; Suzuki, 1994; Wróbel et al., 2003). Both the avian UG (Jenik et al., 1987; Goodridge et al., 1989) and mammalian SGs (Tamada et al., 1994; Kishimoto et al., 1999; Zouboulis, 2000; Wróbel et al., 2003; Smith and Thiboutot 2008; Schneider and Paus, 2010) consist of two types of sebocytes: peripheral, immature cells and central, differentiated cells. The immature cells have been characterized as the undifferentiated and mitotically active population of the glands; they are cubodial or flattened and lack lipid. The holocrine process of sebum production begins with the proliferation of these cells (Piérard-Franchimont et al., 2010). As they grow, they accumulate lipid, undergo nuclear degeneration and move centrally. All of these are phenomena indicating the terminal differentiation of sebocytes (Jenik et al., 1987; Goodridge et al., 1989; Tamada et al., 1994; Kishimoto et al., 1999; Zouboulis, 2000; Wróbel et al., 2003; Smith and Thiboutot, 2008; Schneider and Paus, 2010). It has been reported that, in mammalian SGs, this differentiation state can lead to the induction of apoptosis and bursting of the cell (holocrine secretion) (Tamada et al., 1994; Kishimoto et al., 1999; Zouboulis, 2000; Wróbel et al., 2003; Smith and Thiboutot, 2008; Schneider and Paus, 2010). Although differentiation of peripheral basal cells into mature sebocytes in the UG is accompanied by the accumulation of large amounts of fatty acid synthase and malic enzyme, (Jenik et al., 1987; Goodridge et al., 1989), the key molecular mechanisms that regulate the proliferation and differentiation process in UG cells remain unknown.

Appropriate cell number and organ size in a multicellular organism are intimately linked to the balance between cell proliferation, differentiation, and apoptosis (reviewed by Conlon and Raff, 1999; Hipfner and Cohen, 2004). Cell proliferation results in an increase in the number of cells as a result of growth and division, whereas apoptosis results in controlled self-destruction and can be initiated by a myriad of different mechanisms in different cell types. Apoptotic regulation seems to involve multiple pathways, resulting in either induction or inhibition of apoptosis. Bcl-2 (B-cell lymphoma 2) and the inhibitor of apoptosis protein (IAP) families have emerged as some of the most important regulators of apoptosis, playing a crucial role in the balance between cell survival and cell death (Jaattela, 1999; Yang and Li, 2000; Salvesen and Duckett, 2002; Rumble and Duckett, 2008). The Bax (bcl-2-associated X protein) gene was the first identified pro-apoptotic member of the Bcl-2 protein family (Oltvai et al., 1993). Bax is expressed in normal epidermis and its appendages, with the suprabasal compartment being stained more strongly than basal keratinocytes. Bax protein is also expressed in SGs (Tomkova et al., 1998). Bax has also been shown to mediate differentiation and apoptosis in cultured keratinocytes (Song et al., 1996).

Among the recently described IAP family of proteins, survivin is highly expressed in the developing fetus and neoplastic tumors; in adults its expression is restricted to highly proliferating normal tissues, including skin (reviewed by Li, 2003). In the skin, it is mostly detected in a subpopulation of basal keratinocytes interpreted as keratinocyte stem cells (Chiodino et al, 1999; Marconi et al., 2007; Bongiovanni et al., 2009a, 2009b; Dallaglio et al., 2012), but it is also expressed in melanocytes and fibroblasts (Dallaglio et al., 2012). Survivin expression in adnexa of the skin (sebaceous and sweat glands, follicular outer root sheath) is also a debated question. The main objectives of this study were to assess whether the processes of cellular differentiation in the UG occur through apoptosis and to investigate the apoptotic phenomena involved by the immunohistochemical detection of apoptosis-related proteins (Bax and survivin) and DNA fragmentation.

Another objective of the current study was to investigate the localization of Proliferating Cell Nuclear Antigen (PCNA), one of the central molecules responsible for decisions of life and death of the cell (Paunesku et al., 2001; Moldovan et al., 2007), and to define the relationship between its expression and that of survivin and Bax.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. LITERATURE CITED

Animals and Tissue Preparation

Thirty Hy-Line W-36 chickens were obtained from a local breeder (Kaytas, Kayseri, Turkey) and were reared from between day 1 and day 150 posthatching. The animals were housed on a farm in individual cages, with free access to water and feed, with a lighting period of 16 h of light and 8 h of dark. In the first week posthatching, the temperature of the environment where the animals were housed was maintained at 31 to 34 °C. Subsequently, the pen temperature was reduced 2 °C/wk until a level of 20 to 22 °C was reached. The relative humidity in the pens, as measured with a thermo-hygrometer, was maintained at 60 to 70%. The pens were ventilated naturally, with the entrance of fresh air through windows, panels, and low gate wings. This study was approved by the Ethics Committee of the Faculty of Veterinary Medicine of Erciyes University (approval number 2004-38-48). Five females each, aged 1, 7, 12, 30, 60, and 150 days, were sacrificed under ether anesthesia and their UGs were quickly removed. All tissue samples were fixed in a 10% formol-alcohol solution for 18 h, and were subsequently dehydrated, cleared, and embedded in Paraplast. For histological and immunohistochemical analyses and the terminal deoxynucleotidyl transferase (TdT) mediated dUTP Nick End Labeling (TUNEL) method, six slides were prepared from each sample. At least two 5 µm thick tissue sections, cut with a Leica RM2125RT microtome, were mounted on each glass slide that had been coated with 3-aminopropyl-ethoxy-silane (APES) (Sigma-Aldrich Chemicals, St. Louis, MO) and were then dried at 37°C. The first slides containing sections of gland were stained with Crossmon's method (Crossmon, 1937) for the histological evaluation of the gland, and the following adjacent sections were immunostained for survivin, Bax and PCNA, employing primary antibodies. Furthermore, the TUNEL method was applied to other sections for detecting cells with nuclear DNA fragmentation, suggestive of apoptosis (Gavrieli et al., 1992).

Antibodies

The following antibodies were used: (a) Survivin: ab469 rabbit polyclonal antiserum raised against recombinant full-length human survivin (Abcam Ltd, Cambridge, UK). (b) Survivin (C-19): sc-8807 goat polyclonal antibody raised against a peptide mapping at the C-terminus of survivin of human origin (Santa Cruz Biotechnology, Inc.). (c) Bax (P-19): sc526 rabbit polyclonal antibody raised against a peptide mapping near the N-terminus of Bax of mouse origin (Santa Cruz Biotechnology, Inc.).(d) Bax (Δ 21): sc-6236 rabbit polyclonal antibody raised against amino acids 1-171 representing all but the C-terminal 21 amino acids of Baxα of mouse origin (Santa Cruz Biotechnology, Inc.). (e) PCNA (FL-261): sc-7907 rabbit polyclonal antibody raised against amino acids 1-261 representing full length PCNA of human origin (Santa Cruz Biotechnology, Inc.).

Immunohistochemistry

Immunohistochemistry was performed in accordance with the previously reported immunostaining protocol of Thermo Scientific. Sections were deparaffinized in xylene (two rinses, 5 min each) and rehydrated through a graded series of ethanol. To block any endogenous peroxidase activity, the sections were treated with 3% H2O2 in methanol for 15 min at room temperature and rinsed thoroughly (3 × 5 min each) in phosphate-buffered saline (PBS; pH 7.4). Antigen retrieval was performed in citrate buffer (pH 6) for 30 min at 95 °C with cooling for 30 min before immunostaining. Subsequently, the sections were washed in PBS, and nonspecific binding of primary antibodies was blocked for 5 min using a blocking solution which was a PBS solution, pH 7.6, containing 0.5% BSA, 0.5% casein and less than 0.1% sodium azide (Ultra V Block®, Thermo Fisher Scientific, LabVision Corporation, Fremont, CA). Blocking serum was removed by tapping the slides, which were then incubated with primary antibodies for 60 min at room temperature or incubated overnight at 4°C (PCNA). Optimized dilutions were: survivin 1:400; survivin (C-19) 1:100; Bax (P-19) 1:200; Bax (Δ 21) 1:50; and PCNA (FL-261) 1:100. Sections were washed in PBS, incubated with biotinylated anti-rabbit antibody [when primary antibody recognized survivin (ab469), Bax (sc-526 and sc-6236), and PCNA (sc-7907)] or biotinylated polyvalent antibody [when the primary antibody recognized survivin (sc-8807)] (Thermo Fisher Scientific, Immunon Detection Systems, CadenzaTags) for 20 min at room temperature. After three further 5 min washes in PBS, sections were incubated for 20 min with streptavidin peroxidase and subsequently washed with PBS. Finally, sections were incubated with 3,3-diaminobenzidine (DAB; Thermo Fisher Scientific Lab Vision Corporation, Fremont) for 5 min. Sections were counterstained with Gill's hematoxylin for 3 min, dehydrated through an alcohol series, cleared in xylene, and mounted in entellan under a cover slip.

The specificity of the immunohistochemical procedures was checked by using negative and positive control sections. As positive controls, sections of human spleen, lymph node, breast, and colon carcinoma were incubated with primary antibodies. Morphologically, chicken epidermis and UG epithelium are similar in that both gland and skin cells of the translational layer show hyperthrophy and cytoplasmic vacuoles containing sebaceous secretion. In view of this fact, tail skin samples with the UG were taken from chickens and used as positive controls to ensure the specificity of the reaction in the UG. Additionally, some sections were incubated with PBS instead of primary antibody as negative controls to verify the specificity of the immunoreactions.

All samples were treated in accordance with the same protocol. Tissue sections from different posthatching days were examined by conventional light microscopy (BX51; Olympus, Tokyo, Japan) and were evaluated for protein localizations.

TUNEL Method

The TACS-XL-DAB (R&D Systems Minneapolis, MN) in situ apoptosis detection kit was used according to the manufacturer's instructions. The procedures were performed at room temperature if not stated otherwise. Tissue sections (5 μm) were incubated for 5 min at 58 °C, deparaffinized in xylene, and hydrated in a graded series of ethanol solution before incubation with PBS, pH 7.4 for 10 min. The membranes were permeabilized with proteinase K for 30 min. After washing in deionized, sterile water, the endogenous peroxidase activity was quenched using 10% H2O2 in methanol for 5 min and rinsed in PBS for 1 min. Additional tissue sections were immersed in Terminal deoxynucleotidyl Transferase (TdT) labeling buffer for 5 min. Labeling reaction mix TdT was added to each sample and incubated for 1 h at 37 °C. The reaction was stopped with TdT stop buffer for 5 min and rinsed in PBS. The samples were then incubated for 10 min with streptavidin-conjugated horseradish peroxidase, rinsed in PBS, and incubated with 3,3′-diaminobenzidine tetrahydrochloride (DAB) working solution for 7 min followed by washing in several changes of deionized water. To aid the morphological assessment of apoptosis, the sections were counterstained with 1% methyl green. The slides were dehydrated in a series of ethanol solutions and cleared with xylene. Coverslips were placed over mounting medium for evaluation by light microscopy. The specificity of the TUNEL procedures was checked by using negative (unlabeled experimental control sample) and positive (TACS-Nuclease-treated control) control sections.

Unlabeled experimental control sample: one sample was treated with the labeling rection mix without the TdT enzyme. TACS-nuclease-treated control: after incubation in proteinase K solution and washing in deionized water, one sample was treated with TACS-nuclease to generate DNA breaks in every cell, thus confirming that the permeabilization and DNA labeling reaction worked.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. LITERATURE CITED

The chicken uropygial gland was embedded beneath the dorsal skin on the base of the tail. It contained two heart-shaped lobes and was surrounded by a connective tissue capsule. Each lobe was composed of numerous secretory tubules that opened into a central cavity or lumen, that collects the secretion from tubules and communicates with a main excretory duct opening unto the surface of the skin. The loose connective tissue septa extending from the capsule were observed between the secretory tubules. The secretory tubules and the lumen of the central cavity and excretory duct were lined with glandular epithelium. In all ages studied, the histological organization of the UG corresponded to that of a sebaceous gland (Fig. 1A–D). In a 1-day-old chicken, the gland had not yet reached the adult form. Each glandular lobe was composed of secretory tubules, each being terminated by an end-bulb, or end-bud. The epithelium linings of the secretory tubules, central lumen and excretory ducts of the each lobe were composed of three layers: (a) a germinative layer consisting of one stratum of flat or cuboidal basophilic cells, (b) a secretory layer consisting of one or two strata of polygonal cells, and (c) a degenerative layer composed of cells with pyknotic nuclei (Fig. 1A). In a 7-day-old chicken, the UG had two distinct zones: a peripheral zone consisting of externally situated “peripheral tubules” and a central zone consisting of interiorly situated “central tubules” (Fig. 1B). The structure of the tubules of the peripheral zone was similar to this region in a 1-day-old chicken, whereas the tubular epithelium of the central zone was made up of four layers: (a) a germinative layer, (b) an intermediate layer consisting of polygonal cells with a basophilic cytoplasm, (c) a secretory layer formed by polygonal cells that increase in size toward the central lumen, and (d) a degenerative layer. Generally, these characteristics of the peripheral and central tubules were similar for all age groups studied, except in the 1-day-old chicken. However, the number of tubules and the thickness of the tubular epithelium increased in direct proportion to the age and growth of the UG. In the UG studied from the 12th day after hatching until day 150, the peripheral tubules exhibited a pale staining with Crossmon's method, except for the germinative layer; however, the central tubules and luminal epithelium showed a basophilic staining similar to that of the germinative layer of the peripheral tubules. Therefore, the zones were easily distinguished (Fig. 1C–D). In the 150-day-old chicken, the germinative layer of both the peripheral and central tubules consisted of two strata of cuboidal cells (data not shown).

image

Figure 1. Structure of the chicken uropygial gland at different posthatching periods. (A) 1-day-old; (B) 7-day-old; (C), 30-day-old; (D), 60-day-old. (A) In the 1-day-old chick, each glandular lobe is surrounded by a connective tissue capsule (Ct) and consists of secretory tubules (St) that open into a central lumen (L) and terminal end bulbs (Eb) at the tips of the tubules. The epithelium lining the central lumen (Le) and the secretory tubule epithelium consist of three layers: germinative layer (gl) consisting of one stratum of flat or cuboidal basophilic cells, secretory layer (sl) consisting of one stratum of polygonal cells and degenerative layer (dl) composed of cells with pyknotic nuclei. (B–D) After day 7, the gland has two distinct zones: a peripheral zone (PZ) consisting of externally situated “peripheral tubules” (PSt) and a central zone (CZ) consisting of interiorly situated “central tubules” (CSt). The structure of the peripheral tubules is similar to this region in a 1-day-old chicken, whereas the epithelia lining the central lumen and central tubules are composed of four layers: germinative layer (gl), intermediate layer (il), secretory layer (sl) and degenerative layer (dl). Scale bars, 50 μm (A, B, C),100 μm (D).

Download figure to PowerPoint

Apoptosis

In situ TUNEL analysis was used to detect the presence of apoptotic activity in UG sections. The positive control, in which TACS nuclease was used to generate DNA fragments with free 3′-OH end, showed positive staining in all of the nuclei, whereas the negative control, in which the labeling buffer was used instead of TdT, did not show any considerable positive staining (data not shown). In the 1-day-old chicks, TUNEL-labeled (brown) nuclei were found only sporadically in the epithelium of secretory tubules (Fig. 2A). The number of TUNEL-labeled nuclei increased with increasing age and glandular growth. On day 7 posthatching, the labeled nuclei were mainly observed among the glandular cells of the secretory and degenerative layers of the central tubules, as well as the luminal epithelium; in contrast, the cells of the peripheral tubules did not exhibit any labeling (Fig. 2B). Following day 12 after hatching, some sporadic TUNEL-positive labeled nuclei were also present in the peripheral tubules (data not shown). However, the number of these cells in the secretory and degenerative layers of the peripheral and central tubules increased with the advance of age and glandular growth (Fig. 2C,D) and peaked at 150 days (data not shown).

image

Figure 2. TUNEL-positive cells in the uropygial gland at different posthatching periods. (A) In a 1-day-old chick, TUNEL-positive cells (arrowhead) are rare. L, central lumen; Le, luminal epithelium; St, secretory tubule. (B) In a 7-day-old chick, TUNEL-positive cells are mainly observed in the secretory (sl) and degenerative layers (dl) of the luminal epithelium (Le) and central tubules (CSt) in the central zone (CZ). (C and D) In a 30-day-old chick, TUNEL-positive cells are seen in the secretory (sl) and degenerative layers (dl) in both the peripheral (PSt) (C) and central tubules (CSt) (D). Ct, Capsule; gl, germinative layer; v, blood vessel. Scale bars, 20 μm (A), 50 μm (B–D).

Download figure to PowerPoint

Apoptotic Proteins: Survivin and Bax

There was no immunostaining in the sections that were incubated without the primary antibody, indicating the specificity of the immunohistochemical procedures (Fig.3A–D). Furthermore, immunohistochemical staining indicated that survivin and Bax proteins were present in chicken skin. In normal tail skin of chicken, positive immunoreactions for survivin and Bax were detected in most epidermal keratinocytes as well as in skin appendages, including the peripheral cells of the feather sheath (data not shown).

image

Figure 3. Sections at different ages incubated with PBS (negative controls) did not exhibit any immunoreaction for survivin or Bax antibodies. (A) 1-day-old; (B) 7-day-old; (C), 12-day-old; (D), 30-day-old. L, central lumen; Le, luminal epithelium; Ct, capsule; St, secretory tubules. Eb, terminal end bulbs; gl, germinative layer; sl, secretory layer; dl, degenerative layer; il, intermediate layers; PZ, peripheral zone; PSt, peripheral secretory tubule; CZ, central zone; CSt, central secretory tubule; v, blood vessels; e: endothelial cells, s; connective tissue cells in the septa; m, mitotic figure. Scale bars: 20 μm (A–D).

Download figure to PowerPoint

In this study, Avidin-Biotin Peroxidase staining was performed using a panel of two commercial survivin-specific polyclonal antibodies, one of which was prepared against the recombinant full-length protein survivin and one against the C-terminus. The investigation of different epitopes of survivin showed that their tissue localizations were the same, demonstrating that immunoreactions were specific (Figs. 4A–E, 5A–E).

image

Figure 4. Immunohistochemical localization of the recombinant full-length protein survivin. (A) 1 day old; (B) 7 days old; (C), 12 days old; (D), 60 days old; (E), 150 days old. Survivin immunoreactivity is observed in the cytoplasm of cells in the germinative layers (gl) of both peripheral (PSt) and central tubules (CSt) and the intermediate layer (il) of the central tubules, but is not observed in the cells of the degenerative layer (dl). Survivin protein is also seen in the nuclei of certain cells in the secretory layers (arrowheads). L, central lumen; Le, luminal epithelium; Ct, capsule; St, secretory tubules. PZ, peripheral zone; CZ, central zone; v, blood vessels; e: endothelial cells, s; connective tissue cells in the septa; m, mitotic figure. (B, D, E). Scale Bars, 20 μm (E), 50 μm (A, B, D). 100 μm (C).

Download figure to PowerPoint

image

Figure 5. Immunohistochemical localization of survivin (C-19) protein. (A) 1 day old; (B) 7 days old; (C), 12 days old; (D), 30 days old; (E), 60 days old. Survivin (C-19) immunoreactivity is observed in the cytoplasm of cells in the germinative layers (gl) of both peripheral (PSt) and central tubules (CSt) and the intermediate layer (il) of the central tubules, but is not observed in the cells of the degenerative layer (dl). (B) Interestingly, certain cells in the germinative layer of the central tubules exhibit a strong positive reaction for survivin (C-19) (arrowheads). Nuclear immunostaining for survivin (C-19) is also apparent (n). L, central lumen; Le, luminal epithelium; Ct, capsule; Eb, end-bulbs; St, secretory tubules. PZ, peripheral zone; CZ, central zone; v, blood vessels; s; connective tissue cells in the septa; m, mitotic figure. Scale bars, 20 μm (A), 50 μm (B-E).

Download figure to PowerPoint

In the frontal sections of the UG of 1-day-old chicks, both survivins (S-IRs) were detected in the cytoplasm of the end bulb cells and the peripheral cuboidal basophilic cells of the germinative layer in the luminal epithelium and the secretory tubules. Although the cells of the degenerative layer did not show any positive reaction with survivin antibodies, the cells of the secretory layer were immunopositive (Figs. 4A, 5A).

In the UGs studied between days 7 and 150 posthatching, S-IRs were observed in the cytoplasm of cells in the germinative layers of the luminal epithelium and of both the peripheral and central tubules. The intermediate layers of the central tubules and luminal epithelium were also stained with both survivin antibodies (Figs. 4B–E and 5B–E). Although all cells in the germinative layer of the luminal epithelium and the central tubules were strongly or moderately positive for recombinant full-length survivin (Fig. 4B–E), certain cells in the germinative layer of the luminal epithelium and the central tubules exhibited a strong positive reaction for epitope mapping at the C-terminus of survivin (Fig. 5B–E). Furthermore, due to the increase in the amount of secretory tubules and the growth of the gland, a clear increase in the number of positive cells for recombinant full-length survivin was also observed between days 12 and 150 posthatching. Occasionally, the immunoreactive nuclei for both survivins were detected in the secretory layers of the luminal epithelium and of both the peripheral and central tubules (Figs. 4B–E and 5B,D).

Positive immunoreactivity for survivin antibodies was also seen in several connective tissue cells and endothelial cells of blood vessels in the connective tissue septa of glands (Figs. 4A–C and 5A–D).

Subsequently, we performed similar experiments with commercial antibodies against Bax protein: Bax (P-19) and Bax (Δ 21). In the 1-day-old chick, immunoreactions for both Bax antibodies (Bax-IRs) were detected as a granular staining pattern in the cell cytoplasm of the germinative and the secretory layers in the luminal epithelium and in the secretory tubules and end bulb (Figs. 6A and 7A). In the UGs from the 7th day of hatching until day 150, the observed cytoplasmic staining in the the germinative layer of the peripheral tubules for Bax antibodies was more homogeneous; however, microscopic examination at magnifications of ×400 or ×1,000 (objective: ×40 or ×100, eye piece: ×10) and oil immersion revealed that immunostaining for Bax antibodies was present as a few small brown granules near the plasma membrane or in the cytoplasm of the intermediate and secretory cells of the luminal epithelium and central tubules (Figs. 6B,C and 7B,C). In the 150-day-old chicken, it was observed that Bax protein immunoreactivity was greater in the intermediate and secretory layers of the central tubules and luminal epithelium (Fig. 6D).

image

Figure 6. Immunohistochemical localization of Bax protein. (A) 1 day old; (B) 7 days old; (C), 30 days old; (D) 150 days old. At the subcellular level, Bax protein is expressed as a few small brown granules (arrowheads) near the cell membrane or in the cytoplasm of cells in the secretory (sl) and intermediate (il) layers of both the peripheral (PSt) and central tubules (CSt). L, central lumen; Le, luminal epithelium; Ct, capsule; St, secretory tubules; gl, germinative layer; dl, degenerative layer; PZ, peripheral zone; CZ, central zone. Scale bars, 50 μm (A, B, C), 20 μm (D).

Download figure to PowerPoint

image

Figure 7. Immunohistochemical localization of Bax (Δ 21) protein. (A) 1 day old; (B) 12 days old; (C), 60 days old. Bax (Δ21) protein is seen as a few small brown granules (arrowheads) near the cell membrane or in the cytoplasm of the secretory (sl) and intermediate (il) layers of both the peripheral (PSt) and central tubules (CSt). L, central lumen; Le, luminal epithelium; Ct, capsule; St, secretory tubules. Eb, terminal end bulbs; gl, germinative layer; dl, degenerative layer; PZ, peripheral zone; CZ, central zone. Scale bars, 20 μm (A–C).

Download figure to PowerPoint

Cell Proliferation

In the 1-day-old chicken, remarkable PCNA staining was found in the cytoplasm and nucleus of end bulb cells and peripheral cuboidal basophilic cells of the germinative layer in the luminal epithelium and the secretory tubules (Fig. 8A). From the 7th day of hatching until day 150, expression of PCNA was detected in the cytoplasm and nuclei of the germinative layer in the luminal epithelium and in both the peripheral and central tubules, similar to that of the 1-day-old chicken. In addition, some cell nuclei of the intermediate layers of the luminal epithelium and central tubules were immunopositive for PCNA. Generally, in all ages studied, the degenerative layer cells showed a negative reaction to PCNA antibody, although a few cell nuclei in the secretory layers of the luminal epithelium and the tubules exhibited a positive reaction (Fig. 8A–H). After day 30, the density of PCNA positive nuclei in the secretory layer of the peripheral tubules was more than that of the central tubules and luminal epithelium (Fig. 8E–H). Positive immunoreactivity for PCNA was also seen in connective tissue and blood vessel endothelial cells in the connective tissue septa of glands (Fig. 8A,D,H).

image

Figure 8. PCNA-positive cells at different posthatching periods. (A) 1 day old; (B) 7 days old; (C, D) 12 days old; (E, F) 30 days old; (G, H) 150 days old. In a 1-day-old chick, the cytoplasm and nucleus of the end-bulb cells (Eb) and the germinative layer cells (gl) in the luminal epithelium (Le) and the secretory tubules (St) are immunopositive for PCNA. However, only very few cell nuclei in the secretory layers (sl) exhibit a positive reaction to PCNA antibody. The degenerative layer cells (dl) show a negative reaction to PCNA antibody. In addition, in other age groups, some cell nuclei of the intermediate layers (il) of the luminal epithelium and central tubules (CSt) are immunopositive for PCNA. (E–H) After day 30, the amount of the PCNA positive nuclei in the secretory layer of the peripheral tubules (PSt) is more than that of the central tubules (CSt) and luminal epithelium. Ct, Capsule; L, Lumen; Le, Luminal epithelium; PZ, peripheral zone; CZ, central zone; v, blood vessels; e: endothelial cells, s; connective tissue cells in the septa; arrowheads, PCNA-negative cells. Scale Bars, 20 μm (A–H).

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. LITERATURE CITED

SGs secrete by a holocrine mechanism, which results in death of the secreting cell. This process is an example of programmed cell death, or apoptosis (Henrikson et al., 1997). Cell death can occur by either of two distinct mechanisms, necrosis or apoptosis. Necrosis is a pathological process that occurs when cells are exposed to a serious physical or chemical insult. Apoptosis is a physiological and controlled process by which unwanted or useless cells are eliminated during development and other normal biological processes. Various assays, such as Annexin V binding, caspase enzyme activity, TUNEL method and DNA gel electrophoresis have been developed to detect or quantitate apoptotic cells. Measurement of DNA fragmentation using the TUNEL method is one of the most common methods of detecting apoptosis (Gavrieli et al., 1992). However several reports suggest that some forms of non-apoptotic DNA fragmentation (such as necrotic cells containing damaged DNA) are labeled also by the TUNEL assay and that therefore, results from this assay should not be considered a specific marker of apoptosis (Grasl-Kraupp et al., 1995; Riss, 2001). However, the TUNEL assay has become an accepted method for assessing the apoptosis of SG cells (Selleri et al., 2006; Nelson et al., 2008, 2011; Jeong et al., 2011). Since a single apoptosis assay does not work in all cell models it should be paired with another assay, such as cleaved caspase 3 activation or annexin V. Unfortunately, since commercially produced antibodies have been developed primarily for use in mammalian species, there are no available specific primary antibodies for cleaved caspase 3 or annexin V that react positively with poultry tissues. Therefore, in this study, we were restricted to the use of the TUNEL assay and immunohistochemistry for survivin and Bax to investigate the presence of apoptosis and the localization of apoptosis-related proteins in the chicken UG.

Our investigation revealed that TUNEL-labeled apoptotic cells were present in the degenerative layer of the luminal epithelium of the UG in day-old chicks, while between days 7 and 150 posthatching, apoptotic cells were evident in the secretory and degenerative layers of both the peripheral and central zones and luminal epithelium. Furthermore, our data demonstrated that on day 7, cells in the peripheral zone did not exhibit any labeling, whereas after day 12, some sporadic TUNEL-positive labeled nuclei were present in the secretory and degenerative layers of the peripheral zone, but not the germinative layer.

Haake and Polakowska (1995) showed that, in human epidermis, apoptosis is operational in the suprabasal layers, whereas basal keratinocytes seem to maintain anti-apoptotic defense. Indeed, it has been shown that endonuclease-induced DNA fragmentation is observed suprabasally, but not in basal keratinocytes (McCall and Cohen, 1991). In the present study, the localization of TUNEL-positive apoptotic cells in the UG suggests that apoptosis is operational in the suprabasal layers, including the secretory and degenerative layers, whereas basal layers, including germinative layers, seem to maintain anti-apoptotic defense as in the human epidermis (McCall and Cohen, 1991; Haake and Polakowska, 1995).

Survivin, the smallest member of the IAP family, is a bifunctional protein that acts as a suppressor of cell death and plays a key role in cell division (Altieri, 2010). Survivin expression is controlled at the transcriptional level in a cell cycle dependent manner (Li et al., 1998; for reviews see Wheatley and McNeish, 2005; Altieri, 2006). As an IAP the survivin protein may inhibit the activation of caspase, thereby acting as a negative regulator of apoptosis (O'Connor et al., 2000) induced by Fas and Bax (Ambrosini et al., 1997; Tamm et al., 1998) and is a mitotic regulator (Wheatley and McNeish, 2005; Lens et al., 2006; Altieri, 2010). Survivin is undetectable in most nonproliferating adult tissues (Ambrosini et al., 1997) except for CD34+ hematopoietic stem cells (Fukuda and Pelus, 2001), placenta (Lehner et al., 2001), basal cells of the colonic epithelium (Gianani et al., 2001), gastric mucosa (Chiou et al., 2003), keratinocyte stem cells, melanocytes and fibroblasts (Dallaglio et al., 2012), thymus (Kobayashi et al., 2002), and cyclic endometrium (Konno et al, 2000; Liman et al., 2012).

When reviewing the literature, no information was found about the developmental changes and the localization of survivin protein in the UG of birds. In this study, we found for the first time that survivin was expressed in the sebocytes of the chicken UG during the posthatching period. Our results showed that immunoreactivities for two commercial survivin-specific antibodies in the UG were restricted to the cytoplasm of the germinative and secretory layers of the luminal epithelium and secretory tubules in day-old chicks. Between days 7 and 150 posthatching, S-IRs were evident in the cytoplasm of cells in the germinative layers of the luminal epithelium and of both the peripheral and central tubules and in the intermediate layer of the luminal epithelium and the central tubules. Given that germinative layer cells along the basement membrane of tubules in the UG are dividing progenitor cells (Jenik et al., 1987; Suzuki, 1994), the localization of survivin proteins in the UG supports previous studies indicating that survivin is absent in most normal, terminally differentiated tissues (Johnson and Howerth, 2004), but is abundantly expressed in proliferating cells (Li et al., 1998). These observations, together with the presence of survivin immunoreactivity in the germinative layers, support the hypothesis that survivin plays an important role in the anti-apoptotic mechanisms of the proliferative basal cell compartment of the chicken UG.

Several studies have proposed that survivin is an epithelial stem cell marker in interfollicular epidermis (Marconi et al., 2007; Bongiovanni et al., 2011) and have been shown to potentially play an important role in hair follicle growth and differentiation (Botchkareva et al., 2007; Bongiovanni et al., 2009b). Bongiovanni et al. (2011) have determined that the immunolabeling of survivin and Ki67 is overlapped in canine epidermis, hair follicles and sebaceous glands (Bongiovanni et al., 2011). Recently, Bongiovanni et al. (2012) have found that survivin is expressed only in scattered reserve cells of normal sebaceous glands, where stem cells are supposed to reside. These authors have hypothesized that survivin could represent a marker for “sebaceous” stem cells or progenitor cells and they have suggested that survivin could possibly play a similar prosurvival role in sebocytes as in keratinocytes, guaranteeing the long life of stem cells in their niche (Bongiovanni et al., 2012). In the present study, we observed that certain cells in the germinative layer of the luminal epithelium and central tubules exhibited a strong positive reaction for epitope mapping at the C-terminus of survivin. Since our data are limited to an immunohistochemical study, we could not determine if survivin is a marker for “sebaceous” stem cells or progenitor cells, although our results support those of Bongiovanni et al. (2012). However, more research on this topic needs to be undertaken.

Survivin exists in a number of subcellular locations such as the mitochondria (Dohi et al., 2004), cytoplasm, nucleus (Fortugno et al., 2002), and the extracellular space (Khan et al., 2009, 2011). Recent evidence suggests that the subcellular localization of survivin may designate its role. In normal tissue survivin is not seen in mitochondria, suggesting that it is notedly associated with tumor transformation (Dohi et al., 2004). In response to cell death stimulation in tumor cells, mitochondrial survivin is rapidly discharged into the cytosol, where it exerts cytoprotection by preventing the activation of initiator caspase-9, inhibiting apoptosis (Dohi et al., 2004), while extracellular survivin is able to enhance cellular proliferation, survival and tumor cell invasion (Khan et al., 2009). Nuclear survivin is thought to control cell division, whereas cytoplasmic survivin appears to be predominantly responsible for its cytoprotective activity (Colnaghi et al., 2006). During interphase, under normal circumstances, survivin exhibits a predominantly cytoplasmic localization (Rodriguez et al., 2002; Knauer et al., 2007; Stauber et al., 2007). During the early phases of apoptosis, it relocates from the cytoplasm into the nucleus. Survivin cannot prevent apoptosis when compartmentalized in the nucleus (Colnaghi et al., 2006; Connell et al., 2008; Temme et al., 2007). Endogenous survivin has limited cytoprotectivity in the nucleus as it is prevented from reaching and interacting with its protein targets (such as the caspases) in the cytoplasm during apoptosis (Chan et al., 2010). It is also degraded rapidly if forced to localise to the nucleus (Connell et al., 2008). In the present study, we also determined that S-IRs existed only in the nuclei of some cells in the secretory layer of both peripheral and central tubules. This nuclear survivin immunoreactivity was similar to the TUNEL-labeled apoptotic cells. Based on these findings and the previous reports from Connell et al., (2008) and Chan et al. (2010), we speculate that the prosurvival protein survivin relocates from the cytoplasm into the nucleus in the cells of secretory layers, thus acting as a physiological switch to commit the preen gland cells to apoptosis.

Our findings, which showed survivin immunoreactivity in blood vessels in the connective tissue trabeculae of glands, are in agreement with reports (O'Connor et al., 2000; Blanc-Brude et al., 2002; Pasquier et al., 2006) indicating that survivin is present in the endothelial cells of newly formed capillaries and large blood vessels, and suggest that it is important for the proliferation and survival of vascular endothelial cells of the UG. The levels of survivin are low in resting endothelial cells and could be up-regulated to proliferate on activation.

Bax is a recently identified member of the bcl-2 family and one of the principal inducers of apoptosis (Oltvai et al., 1993; Krajewski et al., 1994; Tomkova et al.,1998). Our study appears to be the first description of the presence and localization of Bax protein in the UG of chickens during the posthatching period. We observed that, in day-old chicks, Bax proteins were seen in all cell layers of the luminal epithelium and secretory tubules. However, after day 7, we determined positive staining for both Bax antibodies in a cytosolic punctuate pattern in the proximity of the cell membrane of the secretory cell layer and throughout the cytoplasm of cells in the intermediate layer. These findings revealed that the localization of Bax in the UG has characteristics similar to those of the skin epidermis (Tomkova et al., 1998; Chao and Korsmeyer, 2001; Cho et al., 2001; Batinac et al., 2007). The subcellular location of Bax protein has been found to be important in apoptotic processes. In healthy mammalian cells, the majority of Bax is found in the cytosol, but upon initiation of apoptotic signalling, Bax undergoes a conformational shift, and inserts into organelle membranes, primarily the outer mitochondrial membrane (Hsu et al., 1997; Wolter et al., 1997). Bax translocation into mitochondria targets the mitochondrial intermembrane contact sites and releases cytochrome c (Gao et al., 2001; De Giorgi et al., 2002). The release of cytochrome c from the mitochondria cleaves and activates caspase-3 and caspase-9, resulting in the sequence of apoptotic processes (Capano and Crompton, 2002). Based on our results and previous studies, we suggest that Bax activity seems to be in a mitochondrial membrane-bound form in cells undergoing apoptosis in the UG. Further studies are needed to elucidate these details.

Apoptosis and cell proliferation are balanced throughout the life of multicellular organisms. Apoptosis, especially in the adult, must be balanced by cell renewal. SGs are constantly renewed throughout life. But how the SG is formed during development or is renewed throughout adult life has been a matter of debate, One group of scientists (Panteleyev et al., 2000; Taylor et al., 2000, Morris et al., 2004) has proposed that skin stem cells generate the gland and a second group (Ghazizadeh and Taichman, 2001; Horsley et al., 2006; Blanpain and Fuchs, 2009) has suggested that the SG has its own stem cells that are residing unipotent progenitor cells located at the periphery of the gland. Horsley et al. (2006) have found that late in embryonic development, near birth, a population of transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp1)-expressing sebocyte progenitor cells is able to generate and maintain the SG. These authors have also reported that these cells are the progenitors of the cells within the gland, including the proliferative, i.e., transiently amplifying, sebocytes that subsequently differentiate to form the sebum-secreting cells. By contrast, the Blimp1+ sebaceous gland cells are negative for both proliferation and differentiation markers, including Ki67 and Peroxisome proliferator-activated receptor gamma (PPAR-γ), respectively. In the present study, the progenitors of the cells within the UG have not been investigated. However, the presence and localization of proliferative sebocytes within the UG was evaluated by immunohistochemistry using PCNA. PCNA is synthesized in proliferative cells and has been identified as an auxiliary protein of DNA polymerases δ and ε, which are required for DNA synthesis during replication (Kurki et al., 1986; Bravo et al., 1987; Prelich et al., 1987; Maga and Hübscher, 1995; Wood and Shivji, 1997; Moldovan et al., 2007). To our knowledge, this is the first report of the role of PCNA as an indicator of the proliferative activity of sebocytes in the chicken UG. In our study we observed that almost all cells in the germinative layer and certain cells in the secretory layer of the gland exhibited PCNA immunoreactivity. This finding is in agreement with previous studies reporting that cells expressing Ki67 are predominantly situated towards the periphery of the SGs in normal human skin (Wu et al., 2000; McBride et al., 2002). Furthermore, these authors have demonstrated that differentiated sebocytes are negative for Ki67 but show nuclear staining for p21/Waf1 (cyclin-dependent kinase inhibitor 1), a nuclear protein that regulates cell cycle progression.

Previous studies have shown that PCNA is expressed predominantly in G1/S (Kurki et al., 1986; Bravo et al., 1987; Morris and Mathews, 1989), while survivin is found at the G2/M phase of the cell cycle (Li et al., 1998). In our study, survivin-positive cells in the germinative layers of the luminal epithelium and of both the peripheral and central tubules exhibited a significantly greater labeling with PCNA, thereby suggesting that survivin can be expressed in cytoplasm of proliferating sebocytes throughout all phases of the cell cycle and plays an important role linking sebocyte survival and proliferation (Yang et al., 2004). This finding further supports the idea that the germinative layer cells are proliferative sebocytes and are responsible for maintenance of the UG. Our findings, which showed scarcity or absence of PCNA in the secretory or degenerative layers, support studies showing that if PCNA is rendered nonfunctional, or is absent or present in low quantities in the cell, apoptosis occurs (Paunesku et al.,2001; Moldovan et al., 2007).

Until recently, PCNA was known only for its essential nuclear functions, namely DNA replication and reparation. Recently, Witko-Sarsat et al. (2010) have identified that PCNA is expressed and located exclusively in the cytosol of neutrophils. They have also shown that it prevents activation of procaspases and thus has an antiapoptotic role. Furthermore, these authors have determined that cytosolic PCNA abundance decreases in apoptotic neutrophils. In the present study, both cytoplasmic and nuclear localization of PCNA was observed only in the germinative layer cells, while the secretory layer cells exhibited only nuclear localization of PCNA. It is currently unknown whether or not cytoplasmic PCNA is associated with the survival of sebocytes in the UG. However, based on previous studies (Witko-Sarsat et al., 2010) and our findings, we speculate that, in the chicken uropygial gland, cytosolic PCNA in the germinative layer sebocytes has an antiapoptotic role, and that cytosolic PCNA is associated with the survival of undifferentiated cells and decreases in apoptotic sebocytes. This study confirms that PCNA is one of the central molecules responsible for decisions of life and death of the cell in the UG (Paunesku et al., 2001).

In summary, our study has shown that apoptosis and proliferation occur in topologically distinct compartments in the UG. The germinative layer cells undergo mitosis and proliferate, whereas the suprabasal layers, including the secretory and degenerative layers, undergo apoptosis. The presence of apoptotic cells in the UG suggests that UG cells (sebocytes) in chickens are naturally eliminated by apoptosis on their way to terminal differentiation before their death and by holocrine secretion, as in human sebaceous glands (Wróbel et al., 2003). Survivin and Bax proteins are differentially expressed in the chicken UG during the posthatching period. Therefore, we hypothesize that specific Bax/survivin expression patterns could reflect particular cell differentiation states in the chicken UG, and that survivin-positive cells, in part, could represent proliferating cells, whereas Bax-positive cells may represent cells undergoing apoptosis. Consequently, the combined functions of Bax, a member of the proapoptotic Bcl-2 family, and survivin, a member of the inhibitors of apoptosis (IAP) gene family, are essential for the normal development of the UG. Furthermore, the expression of PCNA detected by immunohistochemistry may serve as a reliable tool to detect the proliferative activity of sebocytes in the chicken UG.

ACKNOWLEDGEMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. LITERATURE CITED

In Situ Apoptosis detection Kit (TACS-XL) kit, survivin and Bax antibodies used in this study belong to a research project.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. LITERATURE CITED