Immunohistochemical Evaluation of Bcl-2 Oncoprotein in Buffalo’s Adrenal Gland

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Introduction
The adrenal gland consists of cortex and medulla. The adrenal cortex is composed of three concentric layers: the zona glomerulosa, the zona fasciculata, and the zona reticularis, all of which present different morphological and functional properties. Zona glomerulosa is specialized in the production of aldosterone while, zona fasciculata/ reticularis synthesize cortisol in humans and bovine, and corticosterone in rodents [1].
The protein product of Bcl-2 is associated with mitochondria [6] and located on outer mitochondrial membranes, the endoplasmic reticulum, and the nuclear envelope [5].
The Bcl-2 oncoprotein is thought to regulate programmed cell death and to facilitate cell survival [7]. It is topographically restricted to cells in proliferating zones and cells with long life spans, and it is downregulated in terminally differentiating cells.
The main function of the Bcl-2 family is likely to protect mitochondrial integrity and to regulate the release into the cytoplasm of mitochondrial proteins that are involved in the apoptotic pathway, such as cytochrome c [8].
The expression pattern of the Bcl-2 family differs depending on the cell type or stage of differentiation [9]. Moreover, their relative abundance may influence the cellular sensibility to undergo apoptosis in response to a given stimulus (i.e. glucocorticoids). On the other hand, these proteins would be the target for external signals that regulate programmed cell death.
The current study, therefore aimed primary to investigate the immunohistochemical expression of Bcl-2 oncoprotein in buffalo's adrenal gland at different ages.

Material and Methods
Animals and tissues 14 (7 males and 7 females), buffaloes (Bubalus bubalis) adrenal glands were collected from Toukh abattoir in kalyobia governorates, Egypt. Their ages were ranged from 1-12 years and their weight ranges from 100-350 kgs. Specimens were immersed overnight in Bouin's fluid, dehydrated in a graded series of ethanol, cleared in xylene, embedded in Paraplast wax (Sigma-Aldrich, St. Louis, MO, USA) and sectioned at 4 µm thickness. Tissue sections were mounted on positively charged, coated slides (Thermo Scientific, Menzel-Gläser, Braunschweig, Germany).

Materials
Rabbit polyclonal antibody (Sigma Laboratories). Universal kit used avidin biotin peroxidase system produced by (NovaCastra Laboratories Ltd), UK. It was purchased from WAKO Pharmaceutical Company, Tokyo.

Immunohistochemical localization
Immunohistochemical staining for detection of Bcl-2 protein (antiapoptotic marker) was done according to [10].
Immunohistochemical reaction was carried using avidin biotin peroxidase system. The primary antibody used was a rabbit polyclonal antibody (Sigma Laboratories) at a dilution of 1:100 was used. Universal kit used avidin biotin peroxidase system produced by NovaCastra Laboratories Ltd, UK. The same method was applied to prepare negative control sections but the primary antibody was not added. Mayer's hematoxilin was added as counter stain. Tonsil was used as positive control tissue. Finally, the Bcl-2 cytoplasmic site of reaction was stained brown and nuclei stained blue. This technique was done in the institute of tumours, Cairo University. Digital photomicrographs were taken using an imaging system consisting of light microscope (Leica DM LB, Leica Microsystems, Wetzlar, Germany) and digital camera (Leica EC3, Leica Microsystems).
The immunostained cells in the adrenal cortex and medulla were counted in 5 different tissue sections for each age group. For each section, cells were counted in 10 fields (x10) and the mean values for each section were calculated. The numbers (means ± SD) of cells were then calculated for each age group (Table 1).

Results
Immunohistochemical analysis with polyclonal antibodies to Bcl-2 oncoprotein in paraffin-embedded tissue sections revealed that a higher percentage of immunoreactivity was detected in the cells of the buffalo's adrenal gland. The adrenal cortex consisted of, zona glomuralis (zona arcuata), zona fasicuolata and zona reticularis.
At young ages (1-4 years old), positive immunoreactivity for Bcl-2 was detected in the cells of buffaloes adrenal cortex. The degree of immunoreactivity intensity was strong in zona reticularis then zona fasciculata and lastly in zona glomuralis (zona arcuata). While, negative immunoreactivity was detected in the cells of the adrenal medulla ( Figure 1 and Table 1).
At old ages (10-12 years old), moderate immunoreactivity for Bcl-2 was detected in the cells of the cortex of buffalo's adrenal gland, while negative immunoreactivity in the cells of the adrenal medulla ( Figure  2 and Table 1).

Discussion
The Bcl-2 gene, located at chromosome 18q21. It was originally identified by chromosome analysis of the t(14;18) translocation in human follicular lymphoma [4]. The Bcl-2 gene product is a mitochondrial membrane protein. It found also in the nuclear envelope and to a lesser degree in the cell membrane [11], which prevents programmed cell death (apoptosis) [6]. It can be detected by immunostaining in primary lymphoid follicles and in the mantle zone of secondary follicles that largely constitute the homing sites of long-lived memory B-cells [12].
Prominent Bcl-2 staining was observed in the adrenal cortex with a bright intense staining pattern in the zona reticularis and a prominent stippled positive staining in the fasciculate, while the adrenal medulla did not stained positive for Bcl-2 [13]. These findings augmented our findings in the buffalo's adrenal gland however, [14] showed positivity of Bcl-2 staining in both benign adrenal medulla and pheochromocytomas. The authors observed numerous cells of normal adrenal medulla stained positive for Bcl-2 and depict heochromocytomas with perivascular Bcl-2 staining pattern. In addition, the authors reported occasional Bcl-2 positive cells in the adrenal cortex.
Bcl-2 protein also found and expressed in non-lymphoid haematopoietic precursors, such as the early identifiable myeloid cells [15], duct cells of exocrine glands, uterine smooth muscle, endometrial glands, thyroid follicles, prostatic glands and breast lobules [16].
The detection of Bcl-2 protein in fetal epithelial germinative regions, in placental trophoblast, as well as endocrine cells and neurons, suggests   that this protein may play a regulatory role in normal homeostasis of both developing and established tissues. Expression of Bcl-2 protein apparently serves to maintain cells in the tissues, by avoiding cellular senescence and death.
In addition, Bcl-2 protein is involved in morphogenesis by inducing interactions between epithelia and mesenchyme, contributing to the formation of condensations of cells that are "commited" to develop more differentiated structures [16].
Bcl-2 has also been found to promote survival in response to a wide variety of cell stresses and cytotoxic chemicals, including heat shock, ionizing radiation, excess calcium influx and a range of chemotherapeutic drugs [17,18].
Apoptosis is a tightly regulated and a highly efficient process that requires the interplay of multiple factors. It can be triggered by various stimuli of diverse origin that converge into common cell death machinery involving the activation of cysteine-dependent aspartatespecific proteases, called caspases [19]. The apoptotic process is regulated at several checkpoints. One of them is mainly controlled by members of the Bcl-2 family [20,21]. Bcl-2 homologues can be defined by the presence of conserved sequence motifs known as Bcl-2 homologous domains (BH1-BH4).
In mammals, more than 30 relatives have been described; some of them belong to the group of anti-apoptotic members (i.e. Bcl-2, Bcl-xL, and Mcl-1 [22], which possess the four BH domains, while other belong to the pro-apoptotic group, such as the Bax-subfamily (Bax, Bak and Bcl-xS [21], which possesses three out of four domains (BH1, BH2 and BH3), and the BH3-only members such as Bid, Bim and Bad which have only the BH3 domain, necessary and sufficient for the killing effect [8,23].
The Bcl-2 protein is a regulator of the apoptotic process, and elevated levels can provide resistance to cell death [24,25]. The Bcl-2 protein is expressed in the interior of cells, frequently localized to the mitochondrial membrane [26,27].
The expression pattern of the Bcl-2 family differs depending on the cell type or stage of differentiation [9]. Moreover, their relative abundance may influence the cellular sensibility to undergo apoptosis in response to a given stimulus (i.e. glucocorticoids). On the other hand, these proteins would be the target for external signals that regulate programmed cell death.
Glucocorticoids control apoptosis in a tissue-specific manner. For instance, these steroids trigger thymocyte apoptosis in a complex process that involves the enhancement of cyclin-dependent kinase 2 activity, the down-regulation of antiapoptotic Bcl-2 protein expression and the up-regulation of the pro-apoptotic Bcl-2 proteins [28], such as the BH3-subfamily member Bad [29].
The adrenal gland, as an organ responding to stress, is subject to dynamic structural changes, including both proliferation as well as cell death. A balance between these two processes is essential for integrity and functionality of this organ. Regulatory systems of the organism and the autocrine and paracrine local networks determine the cell cycle balance [30].
In rat adrenal cortex, the cells composing each zone are continuously renewed by migration of new cells derivate from progenitor cells that differentiate and die. Thus, apoptosis regulation in adrenal gland may be essential for the definition of the functional zonal architecture [31].
Three theories to explain the zonalization of adrenal cortex. The migration theory describes cell proliferation in the cortex outer part, their migration and differentiation from glomerular zone to fascicular zone and from this to reticular zone where the cells degenerate and die. On the other hand, the transformation theory proposes two transformation directions considering the replacement of zonal tissue by proliferant cells. These transformation zones are placed between the glomerular zone and the fascicular zone from which migration takes place in two opposite directions: on one side towards the medulla and on the other towards the capsule. Finally, the zonal theory proposes an equal proliferation in the three zones. Since the apoptosis also takes place in the three cortex zones, each zone could be locally regulated without being affected by other zones. All these theories intend to explain the phenomenon of maintaining structural homeostasis and the relationship between proliferation and programmed cell death [30,32].
Apoptosis might represent a regulatory mechanism of tissue integrity which might allow the organism to face stressing situations as well as functional demands required for replication, differentiation and cellular migration. Thus, the occurrence of the apoptosis in the three adrenal cortex zones might guarantee the independent regulation of all stages of cellular development in the different cytophysiopathologic situations [33].
The cell proliferation indices are the highest in zona glomuralis and in the outer part of zona fasiculata but the lowest in zona reticularis [34]. In contrast, the apoptotic index is the highest in zona reticularis [35]. Although the majority of these studies were performed in rodents, the similar observations concern the human adrenal gland [36]. It is suggested that adrenocortical cells renew from the progenitor cells located in zona glomuralis/zona fasiculata boundary and/or in subcapsular layer.
Suppresses apoptosis in a variety of cell systems including factordependent lymphohematopoietic and neural cells. It regulates cell death by controlling the mitochondrial membrane permeability. It appears to function in a feedback loop system with caspases. It inhibits caspase activity either by preventing the release of cytochrome c from the mitochondria and/or by binding to the apoptosis-activating factor (APAF-1) [37].
The Bcl-2 gene product prevents programmed cell death (apoptosis) and possibly promotes tumour development. This protein has mainly been demonstrated in the cytoplasm of various normal and neoplastic cells [38]. This protein also may be involved in adrenal insufficiency in acute necrotizing pancreatitis (ANP). The adrenal injury is attributable to hemorrhagic inflammation, necrosis, and apoptosis of the adrenal cortex [39].
Apoptosis, which plays a central role in both normal cell cycle and tumor biology, is controlled by a family of related proteins. Bcl-2 protein is the main representative of this family, and its role in preventing apoptosis is well known. However, alternative apoptotic pathways have also been found, which are regulated by Bcl-2-like proteins. Some of these proteins, such as Bcl-xL, Mel-1, Murine Al, Ced 9 and Bag, inhibit apoptotic cell death, whereas others, such as Bcl-xS, Bax, Bad and Bak accelerate apoptosis [40,41].
The expression of Bcl-2 was prominent in the buffalo's adrenal cortical cells of the young ages (incidence of apoptosis was lower), while the expression of Bcl-2 was moderate in the buffalo's adrenal cortical cells of the old ages (incidence of apoptosis was higher).