2019 123 Age-Dependent Mapping of Somatotropic Hormone in Rat

SCVMJ, XXIV (2) 2019 123 Age-Dependent Mapping of Somatotropic Hormone in Rat Pituitary Gland; Histological, Histochemical and Immunohistochemical Studies Abdel-Hamid Kamel Osman ([email protected]) Hussein Eidaroos Hussein ([email protected]) Amal Arfaat Mokhtar Ahmed (amal_ahmed122yahoo.com) Fatma Khodary Mohamed Ali ([email protected]) Department of cytology and histology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt. Abstract The current study was performed to investigate age-dependent immunoexpression and localization of somatotropin in the pituitary gland. Twenty adult Wistar rats (fifteen females and five males) were housed; four animals per cage with observation of pregnancy. Samples from pituitary gland were collected from different ages of male offsprings. Tissue specimens were processed, and stained with Hematoxylin and Eosin (H&E), special stain and immunohistochemical technique. Sections were divided into four stages. H&E results showed that at stage I, acidophils were the most distinguished cells; but basophils were difficult to be seen then appeared at stage II. Special stain results showed increasing in somatotrophs number from stage I to stage III then decreased at the last stage. Immunoexpression of somatotropin in pars distalis showed that somatotrophs were numerous and uniformly distributed at the first three stages then they were restricted in clusters at the last stage. Key words: somatotropin- immunoexpression- pituitary gland- rat. Introduction hormones, a stimulatory growth Somatotrophic hormone has hormone releasing hormone functional and biological effects as (GHRH), and an inhibitory one of the chief anabolic hormones somatostatin hormone whose functional role is vital for (SST)(Tannenbaum et al., 2003). growth and metabolism throughout The immunostained somatotrophs prenatal and postnatal development showing a granular cytoplasmic (Campbell, 1997, Lecomte et al., pattern and distributed 2018,Zhang et al., 2018). It is a homogeneously in the rat pars polypeptide hormone produced by distalis of different postnatal ages the pituitary somatotrophs, under (Jurado et al., 1998). Thus, the aim the control of two hypothalamic of this work is to study the 124 Osman et al. immunohistochemical localization paraformaldehyde (pH 7.4) for 7 of somatotropin in the pituitary in days. male rat and to study the III- Histological procedures relationship between the Dehydration of samples were immunoexpression and the age- conducted by using ascending dependent changes of the ethanol series, cleared in xylene and immunoreactive cells. then embedded in paraffin wax. Paraffin sections were cut at 5-7 um Materials and Methods thick. Routine staining procedures I-Animals and housing were conducted by Harri's Twenty adult Wistar rats weighting Hematoxylin and Eosin (H&E) 200-250 g (fifteen females and five stain; slides were mounted in DPX. males) were housed under standard IV- Histochemical procedures conditions. All animals received Histochemical procedure aimed to professional care in accordance differentiate between chromophilic with “The guide for the care use of cells; this was done by using laboratory animals” published by Peracetic acid -Alcian blue –PAS The National Institute of Health. Orang G stain (PAA- AB-PAS This was carried out in accordance Orange G stain) according to with guidelines of the animal care (Adams and Swettenham, 1958, El- and use committee at Faculty of Sakhawy et al., 2012). Veterinary Medicine, Suez Canal V- Immunohistochemical University (approval no. 2019006). procedures Tissue samples were fixed in 4 % II-Experimental design paraformaldehyde at pH (7.4) for 2 Animals were separated into four days; and then processed and animals per cage (three females and mounted on positively charged one male) with observation of slides for immunohistochemical pregnancy. Each suspected pregnant detection of somatotrophic hormone female was isolated in a sporadic (Carson, 1997). Tissue slides were breeding cage. Recording of deparaffinized in xylene, rehydrated offspring's ages was performed in and incubated in citrate buffer (ph. controlled documents.Samples of 6) in a microwave oven.Then, pituitary gland were collected from vectastain rabbit blocking reagent male offsprings at the following was used. After that, polyclonal different ages (30, rabbit anti-HGH (cat# AR707-SR, 40,50,70,80,150,160,170, and 180 Thermo Fisher Scientific, Co., days).Five male rats from each age Manor Park, UK) was used as were sacrificed through cervical primary antibody; it was dilated in dislocation under ketamine PBS (1:300), then added to the anesthesia (80 mg\Kg I.p). Then, slides and incubated for 30 samples were immersed in 4 % min.Sections were washed in PBS SCVMJ, XXIV (2) 2019 125 three times for 5 min. each; sections of each age with scale bar biotinylated polyvalent secondary 20µm. antibody (Thermo scientific co., B- Counting of somatotrophs UK) was applied and incubated for using pituitary sections stained 30 min.After rinsing with PBS two with special stain. times for 3 min. each, they were Pituitary gland sections stained with incubated with Avidin-biotin PAA- AB- PAS -Orange G stain complex (ABC Kit, Vector were examined for somatotrophs Laboratories). Then, slides were counting; somatotrophs were incubated in 3,3´-diaminobenzidine demonstrated by their yellow color. peroxidase enzyme substrate This was performed using ten (DAB) for 30 min. and sections of each age with scale bar counterstained with Mayer´s 20µm. hematoxylin. Finally, slides were C- Measurement of dehydrated, cleared and mounted immunostaining intensity of with DPX. For negative control, somatotropin. Sections were treated with similar For the detection of the percentage steps with the exception of the of the immunostained area of the primary antibody.All procedures Somatotrophic hormone in pituitary were done according to Vectastain gland, liver, lung, and testis; Images Elite ABC kit (Vector laboratories. of selected parts were analyzed California, USA). using the image J software at scale VI- Quantitative parameters used bar 50µm. for histological, histochemical and VII- Statistical analysis immunohistochemical Data were collected as mean ± SE investigations: for statistical analysis, one way All images were analyzed using ANOVA followed by Duncan test image J software developed by The was applied. Data analysis was National Institute of Health performed employing the statistical (Betheda, Maryland. USA). They package for social science, version were performed as following: - 20 (SPSS software, SPSS Inc. A- Measurement of Chicago, USA). The level of somatotroph´s size using significance was set at P value immunohistochemical sections. <0.05. Measurement of somatotroph size was performed on the Results immunostained cross sections of I- Histological, pituitary gland; somatotrophs histochemical, and boundaries were demonstrated by immunohistochemicalfindings. their affinity to the brown color of A- General Hematoxylin & chromogen. Ten immunostained Eosin staining results: 126 Osman et al. The pituitary gland was made up of central nucleolus. The second one, the three common regions; Pars was larger in size, lighter and more distalis, Pars intermedia and Pars vesicular (Fig. 1E). Basophils were nervosa (Fig. 1 A) from the earliest obviously increased in their number (30 days old) examined rats. In this and size than the last stage. They study, we focused on the cell contained spherical or oval nuclei components of the pars distalis had with granular basophilic cytoplasm the somatotrophs features. Sections (Fig. 1 E). Stage IV (150-180 were divided into four stages days), there was an increase in according to the histological acidophil’s number (Fig. 1 F). The findings of the pituitary granulated appearance of the gland.Stages I (30 - 40 days), the basophil's cytoplasm was more distinguished cells at this stage were prominent (Fig. 1 F). chromophobes and acidophils; it B- Histochemical staining was difficult to identify basophils properties of chromophiles using (Fig. 1 B). Chromophobes had per acetic acid alcian blue – PAS- unstainable or faintly acidophilic Orange G (PAA- AB- PAS- cytoplasm and large, spherical, Orange G) special stain: euchromatic nuclei (Fig. 1 B). Staining of the Pars distalis with Acidophils were sporadically PAA- AB- PAS- Orange G stain distributed or arranged in small had displayed three types based on clumps; they had small, dark and the staining affinity of their polymorphic nuclei which were cytoplasmic granules as follows; surrounded with a homogenous cells tinged with yellow color were eosinophilic cytoplasm (Fig. 1 B). representing somatotrophs, orange- Stage II (50 days), Pars distalis was reddish color were representing formed from chromophobes, lactotrophs, and finally the magenta acidophils and basophils (Fig 1 C). red colored cells were representing There was increasing in acidophils basophils (Fig.2 A). At the first number whose nuclei were small three stages (stage I, II & III), and spherical with condensed somatotrophs were arranged into chromatin at the periphery (Fig. 1 clusters while lactotrophs were D). Furthermore, basophils could be distributed sporadically (Fig. 2 B, C seen at this stage, they were slightly & D). Basophils were difficult to be small in size with spherical, distinguished at stage I (Fig. 2 B) vesicular nuclei and finely granular but they were seen in a few number basophilic cytoplasm (Fig. 1 C, D). at stage II (Fig. 2 C) and somewhat

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