SCVMJ, XXIV (2) 2019 123

Age-Dependent Mapping of Somatotropic Hormone in Rat ; 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:

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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, 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 Stage III (70-80 days), two increased at stage III (Fig. 2 D). At different sizes of acidophil's nuclei last stage (IV), somatotrophs and were observed; the first one, the lactotrophs were tended to be medium-sized vesicular nuclei with arranged in cords; lactotrophs were condensed edges of chromatin and SCVMJ, XXIV (2) 2019 127 mainly localized around the blood Morphometric analysis of capillaries (Fig. 2 E). somatotroph’s size at different C- Immunohistochemical stages showed a significant increase expression of somatotropin in between stages I and stage IV (Fig. somatotrophs: 4); but there was no significance Section of Pars distalis showed between the early two stages (I & negative somatotropin II) (Fig. 4). immunoreactivity; when primary B- Counting of somatotrophs: antibody was replaced by normal somatotrophs cell count at different goat serum (Fig. 3 A). At the first stages revealed significant increase three stages (stage I, II & III), at early stage I until the stage III somatotrophs were numerous and then significant dropat stage IV uniformly distributed (Fig. 3 B, C & (Fig. 5). D). At stage IV, they were reduced C- Mean % area of in number and restricted into Somatotropin immunoreactivity clusters (Fig. 3 E). in pars distalis: II- Quantitative analysis of Mean % area of somatotropin pituitary gland : immunoreactivity showed that the A- Morphometric analysis of most reduced intensity was at stage somatotroph´s size using I (Fig. 6) while the highest intensity immunostained sections : was at stage III (Fig. 6).

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Fig (1): Photomicrographs of pituitary gland showing histomorphological changes at different stages. (A) Pituitary gland showing three well-developed parts on day 30, Pars distalis (1), Pars intermedia (2) and Pars nervosa (3). Scale bar 100µm. (B) At stage I, pars distalis showed faint acidophilic cytoplasm of chromophobes (arrow head) and homogenous acidophilic cytoplasm of acidophils (arrow). (C) At stage II, Pars distalis consisted o acidophils (arrow), basophils (arrow head) and chromophobes (curved arrow). (D) At stage II, Condensed chromatin at the periphery of acidophil´s nuclei (arrow) and euchromatic nuclei of basophils (curved arrow). (E) At stageIII, basophil´s nuclei (curved arrow) were larger than those of acidophils. Note two sizes of acidophil´s nuclei; medium sized one (arrow) and large sized nuclei (arrow head). (F) At stage IV, notice increasing in number of acidophils (arrow) and the granulated cytoplasm of basophils (curved arrow). H&E stain, Scale bar 10µm. SCVMJ, XXIV (2) 2019 129

Fig (2): Photomicrographs of Pars distalis at different stages. (A)Pars distalis on day 70 showing the staining affinity of somatotroph to yellow color (arrow), lactotrophs to orange red color (curved arrow) and basophils to magenta red color (arrow head).Scale bar 10µm. (B) stage I, (c) Stage II and (D) Stage III showing the distribution of somatotrophs in clusters (arrow) and lactotrophs were distributed sporadically (curved arrow). Note few number of basophils (arrow head) at stage II and slight increase in basophils number (arrow head) at stage III. (E) Stage IV showing decreasing of somatotrophs (arrow) and distribution of lactotrophs around the blood sinusoids (curved arrow). PAA-AB-PAS-Orange G stain, Scale bar 20µm.

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Fig (3): Photomicrographs of immunostained Pars distalis at different stages.(A) Pars distalis stained as negative control for somatotropin immunoreactivity showing negative reaction. (B) Stage I showing the lowest intensity of somatotropin. (C) Stage II showing increasing the intensity of somatotropin. (D) Stage III showing the highest intensity of somatotropin. (E) Stage IV showinglower staining affinity of cytoplasmic granules than stage III.ABC stain, Scale bar 50µm. SCVMJ, XXIV (2) 2019 131

Size of somatotrophs 200 150 100 Size of somatotrophs 50 0 stage I stage II stage III satge IV

Fig (4): An illustration of the measurement of size of somatotrophs at different stages. Data were the mean ± SE (p< 0.05).

count of somatotrophs 150

100 count of 50 somatotrophs

0 stage I stage II stage III stage IV

Fig (5): An illustration of the count of somatotrophs at different stages. Data were mean ± SE (p <0.05).

Mean % area of somatotropin immunoreactivity in pars distalis

40 30 Mean % area of 20 somatotropin 10 immunoreactivity 0 in pars distalis stage I stage II stage III stage IV

Fig (6): An illustration of the measurement of mean percentage area of somatotropin immunoreactivity at different stages. Data represented the mean ±SE (p< 0.05).

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Discussion nuclei and homogenous acidophilic In this study, H&E sections of the cytoplasm. Also, basophils were pituitary gland at stage I (30 days) larger in size than acidophils with showed a well-differentiated Pars their granular basophilic cytoplasm distalis, Pars intermedia and Pars and vesicular eccentric nuclei. nervosa. This was supported by Some studies on the age-related many authors who reported that the changes of the pituitary gland three parts of the pituitary gland structure had supported these were completely differentiated in findings (Ibrahim et al., 2002, rat fetus on day 19 of pregnancy Badway et al., 2004). (Manojlović-Stojanoski et al., Our study demonstrated three cell 2007). At stage I, the distinguished types forming the pars distalis; cells in the Pars distalis were somatotrophs, lactotrophs, and Chromophobes and acidophils; basophils. This was based on the increased number of acidophils was staining affinity of their observed at stage II. The cytoplasmic granules to PAA- AB- predominance of acidophils in PAS- Orange G stain. young rats was also recorded by the Differentiation of basophils into previous study (Ibrahim et al., two sub-types by using this specific 2002). It was speculated that the stain was not noticed; a possible rapid growth rate of the young rats explanation for this point that the is correlated to a concomitant alcian blue was not reactive in rat increase of the acidophil cell pituitary gland. In a preceding fraction which is responsible for the study, researchers showed that synthesis of specific hormones alcian blue-PAS stain differentiated (Badway et al., 2004). The present two types of basophils in the human study has revealed that basophils pituitary gland, but it failed to do were not clearly seen at the sage I, the same action when applied to the whereas they could be seen at stage pituitary of rat (Swettenham, 1960). II; they were comparatively small in Counting of Somatotrophs at size with spherical, vesicular nuclei different stages revealed a and finely granular cytoplasm. A significant increase at the early significant increase in the size and three stages; the highest number number of basophils was observed was at stage III. But, a dramatic at stage III. Similar results were drop was recorded at stage IV. This also recorded in previous studies observation might be corresponding (Ibrahim et al., 2002). Our findings to the previous study which stated showed that at stage IV, that somatotrophs increased after 15 chromophils were well- days of gestation in rat and occupy differentiated with a pronounced a greater area of Pars distalis in increase in the number and size of young stage (2-3 month) than in the acidophils with their vesicular other stages of growth (Tauiguchi SCVMJ, XXIV (2) 2019 133 et al., 1989). The drop of the wings of the adenohypophysis of somatotrophs number might be adult male rat (Wilson and Wyatt, attributed to the concomitant 1988). Regarding the highest increase of the somatostatin intensity at stage III, this finding receptor expression in the pituitary came a line with the previous study gland with progression of postnatal which reported that the age (Reed et al., 1999). Growth immunoreactive somatotrophs were hormone- releasing hormone numerous in young rats suggesting receptor (GHRHR) mRNA levels a parallel increase in the synthesis were increased during the first 30 and accumulation of somatotropin days after birth and then declined until immediate release (Smets et with age (Korytko et al., 1996). The al., 1987). The decreasing of GH synthesis and secretion of the mRNA and GH immunoreactivity somatotrophic hormone is with age occurred as a result of controlled by GHRH and decreasing in GHRH neurons somatostatin, which produced its (Kuwahara et al., 2004). The level actions through their receptors of GH mRNA in the pituitary gland GHRHR and somatostatin receptor was increased with age in the male (SSTR) (Gunawardane et al., rat from day 7 postnataly then it 2015). Also, GHRHR mRNA was decreased markedly by day 90 days increased from Day 7 to Day 40 (Zhang et al., 2018). Morphometric postnataly then decreased on Day analysis of somatotrophs size at 90 (Zhang et al., 2018). In this different stages displayed a gradual study, the somatotropin increase from early to late stage; a immunoreactivity showed that the previous study has shown an most reduced immunoreactive increase in somatotroph's size with intensity was at stage I, while the age from suckling to adulthood on highest intensity was at stage III. the female mice (Sasaki, 1988). Then, it was reduced at the last stage (stage IV). Our findings were References: inconsistent with many data Adams, C. & Swettenham, K. (Hemming et al., 1986, 1958. 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المسح المعتمد على العمر لهرمون النمو فى الغدة النخامية للفئران؛ دراسات هستولوجية وهيستوكيميائية وهيستوكميائية مناعية

الملخص العربى

تم إجراء الدراسة الحالية لدراسة التعبيرالمناعي المعتمد على العمر و توطين هرمون النمو في الغدة النخامية. تم إيواء عشرون من الفئران )خمسةعشراناث وخمس ذكور(, بأربعة حيوانات لكل قفص مع مالحظة الحمل. جمعت العينات من الغدة النخامية من مختلف األعمار من ذرية الذكور. تم معالجة العينات النسيجية وصبغاتها بالهيماتوكسيلين وااليوسين والصبغة الخاصة و تقنية النسيجوكيميائية المناعية. قسمت القطاعات النسيجية الى اربعة مراحل على حسب النتائج الهيستولوجية للغدة النخامية. أظهرت نتائج الهيماتوكسيلين وااليوسين أن هناك تغيرات نسيجية في الفص االمامى للغدة النخامية في جميع المراحل. فى المرحلة االولى كانت الخاليا الحامضية هي الخاليا المميزة لكن كان من الصعب رؤية الخاليا القاعدية، ثم ظهرت في المرحلة الثانية. و باستخدام الصبغة الخاصة لوحظ زيادة اعداد الخاليا المفرزة لهرمون النمو من المرحلة االولى الى الثالثة ثم تنخفض فى المرحلة االخيرة. أظهرالتعبيرالمناعي لهرمون النمو في الفص االمامى للغدة النخامية أنا لخاليا المفرزة لهرمون النمو كانت عديدة وموزعة بشكل موحد في المراحل الثالث األولى. ولكن، في المرحلة األخيرة، انخفض عددها وتقيدت في مجموعات.