1174 Doaa M. Mokhtar et al.


adrenaline and ~20% of noradrenaline (Tank & Lee Wong, 2015). Although adrenaline is secreted exclusively from the adrenal medulla, the major source of circulating noradrenaline is from sympathetic nerve endings (Goldstein et al., 2003). Plasma dopamine comes from the adrenal medulla, sympathetic nerve endings, and the brain. The adrenal tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of catecholamine from the tyrosine amine acid, which is taken up by chromaffin cells in the medulla and converted to dopamine, noradrenaline, and adrenaline (Hiremagalur & Sabban, 1995; Jahng et al., 1997). The adrenal chromaffin cells secrete their granule con-


tents by exocytosis in response to the secretory stimuli. Released substances can act locally in a paracrine fashion or enter the bloodstream to act on distant targets in an endo- crine fashion (Kobayashi & Coupland, 1993). Adrenal chromaffin cells are innervated by preganglionic sympathetic nerve fibers in a similar way to sympathetic ganglion cells (Kesse et al., 1988). The adrenal medulla always contains nerve cells in addition to chromaffin cells (Unsicker et al., 1978).


The ultrastructure of only a few species has been


studied, and much work has been concentrated on the adrenal glands of the mouse (Kobayashi & Coupland, 1977), the laboratory rat (Rebuffat et al., 1988), and guinea pig (Kobayashi et al., 1985). Melatonin is a hormone secreted by the pineal gland in


the brain and involved in the regulation of multiple functions such as metabolism and reproduction. It is a hormone with endocrine, paracrine, and autocrine actions (Chen et al., 2011). In addition to its relevant antioxidant activity, mela- tonin exerts many of its physiological actions by interacting with membrane MT1 and MT2 receptors and intracellular proteins such as quinone reductase 2, calmodulin, and tubulin (Bandyopadhyay et al., 2011). Studies on melatonin effect on the adrenal gland that


represents an important endocrine organ controlling essential physiological functions are still deficient. Therefore, the present study was carried out to investigate the effect of exogenous melatonin treatment on the adrenal cortex and medulla using light and electron microscopy and immunohistochemistry.


MATERIALS AND METHODS


Animals Rams of Soay breed (Ovis aries) were obtained from specia- list breeders in Scotland (Mokhtar et al., 2016). The animals were kept outdoors grass paddocks at Dryden field station of animal breeding research organization near Edinburgh dur- ing long days (summer); 16 h light–8 h darkness and fed a standardized diet of grass pellets (500 g/animal; Vitagrass, Cumbria, UK) given daily. Soay rams were used because they are a short-day breeder and have pronounced seasonal reproductive cycles; with a rutting season (period of sexual activity) in autumn and a sexually quiescent period in


summer and spring and these cycles are responsive to changes in photoperiod (Lincoln & Short, 1980).


Experimental Design


Experiments were conducted in accordance with the UK Animals (Scientific Procedures) Act of 1986. The adrenal glands of 15 adult Soay rams (aged 1.5 years) were used in this study. At the end of May, eight animals were given a subcutaneous implant containing melatonin (treated group), while another group of seven animals was provided by implants devoid of melatonin to act as a control group. The melatonin implants were made of silastic sheeting (500-1 Dow Corning, Midland, MI, USA) containing 1 g melatonin (Sigma Chemical, Poole, Dorset, UK), previously known to produce a relatively constant concentration of melatonin in peripheral blood in daylight of about 200–500 pg/mL plasma (Lincoln et al., 1984). The implants were placed sub- cutaneously above the rib cage using local anesthesia and were left in place for 11 weeks. In mid-August, i.e., 11 weeks after the onset of the


experiment, all ramswere sacrificed by intravenous barbiturate administration and the adrenal glands were carefully excised and small samples were processed for light and electron microscopic examination. Both light and electronmicroscopic examinations were done at Assiut University, Egypt.


Light Microscopy


The samples were taken from adrenal glands of both the control and treated groups, were then dissected at 1×1×0.5 cm and were immediately fixed in Bouin’s fluid for 22 h. The fixed samples were dehydrated in an ascending series of ethanol dilutions, cleared in methyl benzoate, and then embedded in paraffin wax. Samples at 5–8 μmin thickness were cut transversely and longitudinally (Bancroft et al., 2013).


Immunohistochemistry


To elucidate the effect of exogenous melatonin administra- tion on the adrenal gland of Soay rams, we determined the colocalization (expression) of TH (the enzyme involved in the rate-limiting steps of catecholamines synthesis) and Synaptophysin (SYP) (the protein which expressed in the presynaptic vesicles and neuroendocrine cells) in paraffin section of these animals.


Antibodies and Reagents Mouse monoclonal anti-TH (MAB318) was purchased from Merck-Millipore (Darmstadt, Germany) and rabbit poly- clonal anti-Synptophysin1 (SySY101002) were used as pri- mary antibodies for immunofluorescence on tissue sections. Donkey anti-mouse (Dam) 488 and donkey anti-rabbit (Dar) IgG Alexa 568 from Dianova (Hamburg, Germany) were used as secondary antibodies. Fluoromount-G was from Biozol (Eching, Germany).


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