Effect of thyroxin on cell morphology and hormone secretion of pituitary grafts in rats

Journal Pre-proof Effect of thyroxin on cell morphology and hormone secretion of pituitary grafts in rats ´ Quintanar-Stephano, Fabio Rotondo, Matilde Lombardero, Andres Eva Horvath, Kalman Kovacs

PII:

S0940-9602(20)30029-7

DOI:

https://doi.org/10.1016/j.aanat.2020.151486

Reference:

AANAT 151486

To appear in:

Annals of Anatomy

Received Date:

4 October 2019

Revised Date:

30 January 2020

Accepted Date:

3 February 2020

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ORIGINAL RESEARCH PAPER

Effect of Thyroxin on cell morphology and hormone secretion of pituitary grafts in rats.

Matilde Lombarderoa, Andrés Quintanar-Stephanob, Fabio Rotondoc, Eva Horvathc, and Kalman

Department of Anatomy, Animal Production and Clinical Veterinary Sciences, Faculty of

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a

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Kovacsc

b

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Veterinary Sciences, Campus of Lugo, University of Santiago de Compostela, Spain Department of Physiology, Center of Basic Sciences, Autonomous University of Aguascalientes,

c

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Aguascalientes, Mexico (E-mail: [email protected])

Department of Laboratory Medicine, Division of Pathology, St. Michael’s Hospital, University of

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Toronto, Toronto, Ontario, Canada (E-mails: [email protected] ;

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[email protected]; [email protected])

*Corresponding

author:

Matilde Lombardero

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Department of Anatomy, Animal Production and Clinical Veterinary Sciences Faculty of Veterinary Sciences University of Santiago de Compostela, Campus of Lugo 27002 Lugo- SPAIN Email: [email protected] Tel +34 982 822 333 1

Highlights

   

Thyroidectomy cells were found in pituitary grafts disconnected from the hypothalamus Initially, T4 could not reach the graft, probably due to lack of vascularization Later, T4 treatment reduced the presence of thyroidectomy cells in 2/3 of the samples T4 treatment increased GH & PRL blood levels, as well as GH cell number in all groups

Abstract

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Introduction: Growth hormone and prolactin secretion is affected by thyroid hormones. To see if this

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influence is subsidiary to the hyptothalamus, we investigated the effects of thyroxin (T4) on hormone

(autografted or allografted under the kidney capsule).

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secretion and histology of sellar pituitaries and pituitary grafts detached from the hypothalamus

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Materials and methods: Male Wistar rats were divided into eight groups: control, thyroidectomised, pituitary autografted, pituitary allografted, and four additional groups that were injected with T4 for

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two weeks, starting four weeks after surgery. At sacrifice, adenohypophysial hormone blood levels were assessed, and tissue from sellar and grafted pituitaries were investigated by histology and

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electron microscopy.

Results: Growth hormone and prolactin blood levels, as well as the number of growth hormone immunopositive cells increased in T4- treated groups. Both pituitary auto- and allo-grafts showed lactotroph hyperplasia and displayed spongiform areas containing cells with vesicles in their

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cytoplasm resembling thyroidectomy cells. This phenomenon was minimized in their respective T4treated group. Thyroidectomy cells were identified in pituitary grafts, indicating that hypothalamic control was not essential to induce them. Discussion and conclusion: It is intriguing that the pituitary allografted group, even maintaining normal T4 blood levels, developed thyroidectomy cells in their grafts, suggesting that a long- term deficit of vascularization (>4 weeks) prevented T4 from reaching the graft. After 6 weeks, post T4 2

treatment of two weeks seemed to be the determining factor to minimize thyroidectomy cells in both pituitary autografted+T4 and pituitary allografted+T4 grafts compared to the untreated groups, although more time and/or higher T4 doses may be required to fully restore the euthyroid morphology.

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Keywords: electron microscopy; light microscopy; pituitary grafts; pituitary hormones; thyroxin

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1. Introduction 1.1. Pituitary cell types and hormones The pituitary gland is composed of several cell types that secrete a variety of hormones which regulate the function of various endocrine glands including the thyroid. Thyrotroph cells are the least abundant cell type in the anterior pituitary, comprising

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approximately 5-8% of all cells (Villalobos et al., 2004) producing thyroid-stimulating hormone (TSH) under control of the hypothalamic thyrotrophin-releasing hormone (TRH). The effect of TRH

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on TSH gene regulation is mediated by several nuclear proteins, such as Pit-1 which is essential for the development of several pituitary cell lines, and it is expressed in somatotrophs, lactotrophs and

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thyrotrophs. TSH is the main regulator of thyroid development and growth, and stimulates all of the

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steps involved in the synthesis and secretion of thyroid hormones (Ortiga-Carvalho et al., 2016). Thyroxin (T4) is the main secretory product of thyroid follicular epithelial cells, but has low

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biological activity. Through the process of deiodination, T4 is converted to the active form 3,5,3’triiodothyronine (T3) (Brook and Marshal, 2001). The final cellular physiological response to thyroid

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hormones is a result of a complex mechanism involving both genomic and non-genomic actions (Bargi-Souza et al.,2017).

In rats and humans, growth hormone (GH) is synthesized in somatotroph cells of the adenohypophysis. TRH, as well as thyroid hormones, can stimulate GH synthesis and release from

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the somatotroph cells (Harvey, 1990; Giustina and Wehrenberg,1995) upregulating Gh transcription. T3-mediated activation of rat Gh gene expression presented a synergism between transcriptional factor Pit-1 and T3, increasing the Gh mRNA content (Bargi-Souza et al.,2017). In rodents, hypothyroidism conspicuously reduced the expression of GH, while increasing the expression of both TSH subunits (Bargi-Souza et al.,2017),. Additionally, thyroid hormones play an important role in 4

the maintenance of prolactin (PRL) cell function and its reactivity to TRH. In the presence of thyroid hormones, TRH stimulates PRL release from lactotrophs in a dose-dependent manner, both in vitro and in vivo (Tashjian et al.,1971; Blake, 1974). TRH is secreted into the bloodstream of the hypophysial stalk and it binds to two types of receptors TRH-R1/R2. TRH-R1 mediates neuroendocrine hormonal functions, while TRH-R2, which is highly expressed in the brain region, likely mediates neurotransmitter effects (Deflorian et al., 2008). TRH-R1 is most important for

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activity in thyrotropes, although it is also expressed in lactotropes and a fraction of somatotropes

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(Ortiga-Carvalho et al., 2016). Hypothyroidism results in elevation of TRH that, contrary to the expected increase in PRL secretion, induces a deficiency in lactotrophs (Stahl et al., 1999).

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Low levels of thyroid hormones after thyroidectomy lead not only to the proliferation of

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thyrothroph cells and their hypertrophy, but also to a decrease in GH secretion and a reduction in somatotroph quantity (Quintanar-Stephano and Valverde, 1997; Stahl et al., 1999; Nolan et al., 2004).

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It can also lead to decreased PRL secretion and morphological changes in lactotroph cells, therefore, when in hypothyroid state, there is a complex hormonal dysfunction rather than a single hormonal

2004).

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defect. These effects are reversible by T4 administration (Gómez Dumm et al., 1985; Nolan et al.,

1.2. Pituitary grafts

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Since the hypothalamus plays a major role in regulating pituitary hormone secretion, pituitary

grafts have been used as experimental models to study the effects of different substances on pituitary cells not connected with the hypothalamus. In pituitary grafts, PRL hypersecretion is induced by the lack of hypothalamic inhibition, while the failure of the hypophysiotropic releasing hormones to reach the pituitary graft causes low secretion of the remaining adenohypophysial hormones. Hence,

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pituitary grafted rats are not only valid models of excessive and abnormal PRL secretion, but may also be useful for distinguishing between stimuli requiring an intact hypothalamic-pituitary axis and agents which act directly on the pituitary (Alder, 1986). Autotransplantation of the rat anterior pituitary under the kidney capsule results in chronic hyperprolactinemia and hypopituitarism where GH secretion and other anterior pituitary functions, as well as growth, are diminished. In contrast, in non-hypophysectomised rats implanted with anterior pituitary glands from littermate donors, GH

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secretion, growth and thyroid function remain normal even though they have high levels of

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circulating PRL (Adler, 1986).

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1.3. Aim of the study

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Herein, we studied the effect of T4 on pituitary hormone secretion and the functional morphology of pituitary secretory cells autografted (AUTO) or allografted (ALLO) under the kidney capsule, with

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no hypothalamic influence under an integrative point of view, including body and endocrine organ

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weights, hormonal levels, functional morphology and cell proliferation.

2. Materials and Methods 2.1. Animals

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Male Wistar rats of three months of age weighing 250 grams (g) were divided into eight groups of five to nine animals each (Table 1): 1) intact untreated controls (CO); 2) thyroidectomised (TX); 3) pituitary autografted under the kidney capsule (AUTO); 4) rats with intact pituitaries and an isogenic female pituitary allografted under the kidney capsule (ALLO); the remaining four groups were similar but injected intramuscularly (i.m.) with T4 (6μg/100g of body weight) daily for two weeks. Injections started four weeks after surgeries. All groups were sacrificed six weeks after surgeries (Fig. 6

1). All surgical and experimental procedures were approved by the Institutional Animal Care and Use Committee of the Universidad Autónoma of Aguascalientes and are compatible with the Institute for Laboratory Animal Research (USA) guidelines, 2011.

2.1.1. Surgical Procedures surgical

procedures

thyroparathyroidectomy)

employed (TX),

in

our

study

hypophysectomy,

included

pituitary

thyroidectomy autograft

(actually,

(AUTO),

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The

and

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allotransplantation (ALLO) have all been described in previous publications (Waynforth and Flecknell, 1992; Quintanar-Stephano and Quintanar, 1994). Surgeries were performed by one of the

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authors (A.Q-S.). Hypophysectomy and pituitary grafts were carried out under Ketamin-Xilasin

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anesthesia. The right kidney was exposed via a dorsal approach and a pouch was prepared under the kidney capsule prior to sacrificing the donor rats. For the pituitary autografts, the kidney capsule

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pouch was prepared prior to the hypophysectomy. Removal of the pituitary was carried out through the parapharyngial approach under a dissecting microscope (Quintanar-Stephano and Quintanar,

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1994). A hole was drilled at the center of the sphenoid–occipital joint, until the pituitary was seen at the bottom. The dura mater was cut and the pituitary removed by gentle aspiration via a bent Pasteur pipette with a wide-open end. The opposite end of the pipette was equipped with a nylon trap to retain the excised pituitary. The pituitary was placed into isotonic saline solution, the neurointermediate

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lobe was discarded and the anterior pituitary lobe cut into two pieces and placed deep under the kidney capsule pouch. The kidneys were replaced to their fossa and the dorsal wound closed. For pituitary allografts, isogenic female donors from the same litter were used. They were anaesthetized with sodium pentobarbital, decapitated, and their pituitary was quickly removed.

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The graft time for allografts generally did not exceed 60 seconds whereas for pituitary autografts did not exceed five min. Full recovery of the operated animals occurred within 20-30 minutes. After surgery, the animals were injected with penicillin (Procaine penicillin; 5000 IU i.m., Likeside, Mexico) daily for three days. Refer to the Supplementary material section for detailed surgical procedures. Animals’ diet consisted of standard Purina rat chow and drinking water ad libitum. For AUTO

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group, the diet was supplemented with sweet hard cookies, apple slices, and 5% glucose in the

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drinking water in order to avoid hypoglycaemia whereas, to avoid hypocalcaemia in the TX group, drinking water was supplemented with 1% calcium lactate. The animals were habituated to their

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housing conditions for at least seven days before surgery. Body weights were assessed before surgery

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and once a week until sacrifice.

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2.1.2. Sacrifice

Six weeks after surgery, animals from all groups were anesthetized with sodium pentobarbital (40

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mg×Kg-1 body weight) and bled through the abdominal aorta. The blood was centrifuged and the plasma stored at -80 °C until hormone measurement. Sellar pituitaries and kidney AUTO- or ALLO- grafted pituitaries containing a fragment of the kidney were quickly removed, fixed in 10% buffered formalin and embedded in paraffin for light

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microscopic study, or fixed in 4% paraformaldehyde–1% glutaraldehyde in Sörensen buffer (pH 7.4) and embedded in Epon for electron microscopic investigation. Sellar adenohypophysis, thyroid, adrenal glands and left testicle were dissected and weighed.

Body weight (B.W.) was expressed in grams (g), whereas relative weights of endocrine glands were expressed in mg ×100 g−1 of B.W.

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2.2. Hormone blood levels GH and PRL serum levels were assessed by ELISA method, whereas adrenocorticotropic hormone (ACTH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were assessed by radioimmunoassay (RIA). Inter- and intra-assay coefficients of variance were 8.2 and 7.8% for PRL and 7.8 and 6.6% for GH respectively, 9.74 and 8.59% for ACTH, 9.21 and 7.89% for FSH,

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8.54 and 7.56% for LH, respectively. Results were expressed in ng×mL−1 for all hormones except

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ACTH, which was expressed as pg×mL−1.

T4 serum levels were not assessed at sacrifice owing to the fact that there were some intact

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animals, others were thyrectomized, some auto- or allo-grafted, and others treated with T4. The

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animals were considered euthyroid (CO, TX+T4, AUTO+T4, and ALLO), hyperthyroid (CO+T4 and

2.3. Morphologic study

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ALLO+T4) or hypothyroid (TX and AUTO).

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For routine light microscopy, 5-μm sections of sellar pituitaries and pituitary grafts were stained with hematoxylin-eosin (H&E) and periodic acid-Schiff (PAS). Immunohistochemical stainings for adenohypophysial hormones were performed using the streptavidin-biotin-peroxidase complex method. Sections were pretreated for antigen retrieval by microwaving with 0.01 M citric

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buffer, pH 6.0. After heating, slides were removed from the microwave and allowed to cool for 15 minutes at room temperature then rinsed in distilled water and in TRIS-buffered saline (TBS) for five minutes. Specific polyclonal antibodies were directed against the complete spectrum of pituitary hormones, including GH (rabbit anti-rat GH, 1:5000, ab126882; Abcam, Cambridge, MA, USA), PRL (anti-rat PRL, 1:4000; NIDDK, Bethesda, MA, USA), ACTH (anti-rat ACTH, 1:4000, sc-

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57021; Santa Cruz Biotechnology; Dallas, TX, USA), FSH (anti-rat β-FSH, 1:4000, AB928; Millipore Sigma, Massachusetts, USA), LH (anti-rat β-LH, 1:3000, MCA5837G; Bio-Rad, Hercules, CA, USA) and TSH (anti-human β-TSH, 1:3000, ab155958; Abcam, Cambridge, MA, USA). Slides were incubated for one hour with biotinylated secondary antibody and for one hour with AvidinPeroxidase Complex (Vectastain ABC kit; Vector Laboratories, Inc., Burlingame, CA, USA). The final reaction was achieved by incubating the sections in a solution of 3,3’-diaminobenzidine (DAB)

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and H2O2, pH 7.4. To ensure specificity, control testing included replacement of the primary antibody

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with TBS; this served as a negative control. We also used tissues that were known to express the particular antigen as a positive control for each antibody; this served as a positive control. Sections

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were then counterstained with haematoxylin, dehydrated, cleared and coverslipped.

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For electron microscopic study, samples were routinely processed in 2.5% glutaraldehyde and

2.4. Cell proliferation index

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thin sections were studied on a Hitachi H7650 digital transmission electron microscope.

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In order to visualize replicating DNA in the adenohypophysis cells, a thymidine analogue 5bromo-2-deoxyuridine (BrdU, Sigma-Aldrich) was injected to each rat in a single dose (50 mg×kg-1 of body weight) one hour prior to sacrifice. After DNA denaturation, BrdU incorporation was assessed using the immunohistochemistry method employing a monoclonal antibody anti-BrdU

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(Boehringer, dilution 1:1000) and including a positive control from a previous project. Unfortunately, the BrdU incorporation into the replicating DNA failed. The Feulgen procedure was subsequently used to identify DNA in our cell specimens since it remains the gold standard for precise DNA image nuclear staining (Biesterfeld et al., 2011). In the visible light spectrum, only the Feulgen reaction has been accepted as a stoichiometric procedure for exclusive staining of nuclear DNA in a reproducible,

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standardized manner (Schulte and Wittekind, 1989; Chieco and Derenzini, 1999). According to the procedure by García del Moral (1993), the DNA in the samples were denatured using 1N HCl for 12 minutes at 60° C prior to being stained with Schiff’s reagent for 55 minutes at room temperature (García del Moral, 1993). DNA present in the samples stained red.

2.5. Densitometric measurements

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In each sample, a mean of 10 randomly selected fields were photographed at high magnification

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(600x) and investigated. The necrotic area in the central part of the grafts was avoided. Nuclear area, density mean, color density (blue, green and red), and the integrated optical density (IOD) of

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approximately 2000-3500 nuclei per group were assessed using a semi-automatic computer image

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analysis (MicroImage; Media Cybernetics, Silver Spring, MD, USA). The IOD median value of approximately 400 lymphocytes served as a measurement-specific internal standard for the normal

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diploid value (G0/1-phase) that, once multiplied by the corrective factor allowed rescaling of IOD values into DNA content (following the guidelines of EUROQUANT for DNA image cytometry)

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(EUROQUANT). Values >2n were set at IOD>8.17. Only non-distorted and non-overlapping nuclei with easily discernible borders were suitable for analysis. All quantitative evaluations were performed blindly by one of the authors (M.L.). Approximately 25,000 nuclei were investigated and results were expressed as percentage of proliferating cells (G2/M-

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phase).

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2.6. Statistical Analysis Statistical analysis of body weight, as well as adenohypophysis, thyroid, adrenal and left testicle weights, along with hormone serum levels were performed on all samples. Two-way analysis of variance (ANOVA) and Tukey post test were conducted to estimate the significant differences in body weight and percentage of body weight weekly between groups. Oneway analysis of variance (ANOVA) and Dunn´s post- test were conducted to estimate the differences

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between groups at 95% CI. All statistical analyses and graphics were performed using the Graph Pad

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Prism 6.0 software. Differences with a P- value less than 0.05 was considered significant. Results

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were expressed as mean±SEM. 3. Results

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3.1. Body weights

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Body weights were assessed weekly for six weeks Fig. 2A. The percentage of body weight variation is summarized in Fig. 2B.

The body weights of CO animals increased until the end of the experiment (average of 48.5g),

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representing 19.5% of increment in their body weight. In contrast, CO+T4 animals increased in weight an average of only 4.9%, which was four times less than the CO group. The TX group body weight decreased 3.3% and their final body weights were 18% lower than those in CO group. The

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TX+T4 group increased an average of 6.2% of their body weight while the AUTO and AUTO+T4 groups had lost weight during the same period, -22.9% and -13.8%, respectively. On the other hand, the ALLO group showed an increase in body weight of 15.8% while the ALLO+T4 had a very low gain of 1.6%, showing a trend similar to CO and CO+T4.

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3.2. Endocrine organ relative weights The relative weights of all the endocrine glands were summarized in Table 2. The relative weight of the adenohypophyses was not assessed in AUTO and AUTO+T4 animals since their pituitaries were autografted under the kidney capsule. The relative weights of the thyroids showed no statistical differences between the groups studied. TX and TX+T4 had no thyroid at sacrifice due to their removal.

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Regarding adrenal glands, in comparison with its respective nontreated group, T4 injections

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induced a small and statistically non-significant increase in adrenal weights; except in AUTO+T4, whose adrenals were smaller than AUTO. In addition, adrenals from AUTO and AUTO+T4 are

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significantly smaller than in other groups (except in CO and AUTO, the second lowest values).

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The relative weights of the testes between the respective untreated and T4-treated groups were similar. The highest values corresponded to CO+T4 and TX, and the lowest value was achieved by

3.3. Serum Hormone levels

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AUTO+T4, with statistically significant differences.

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The serum levels of GH, PRL, ACTH, FSH and LH are documented in Table 3. The highest GH serum value was noted in the ALLO+T4 and GH serum levels from TX were the lowest values, with statistically significant differences between both groups. An increase in GH levels were noted in all T4-treated groups with no statistically significant differences, except between

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the TX and TX+T4.

An increase in serum PRL levels in all T4-treated groups were also noted, but no statistically

significant differences were seen between the untreated and T4-treated groups. The lowest PRL values corresponded to TX and CO groups. The highest PRL values were obtained from AUTO+T4 and ALLO+T4 groups. PRL hypersecretion is characteristic in rats with pituitary grafts as we could

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demonstrate in the AUTO (10 times the CO values) and ALLO (approximately 14 times the CO values) and their respective T4-treated groups.In regards to serum ACTH levels, an increase in all T4-treated groups was noted, but no statistically significant differences were found between the untreated and T4-treated groups. Significant differences were apparent between the highest values from ALLO and ALLO+T4, and the lowest values achieved by AUTO and AUTO+T4. The effect of T4 treatment on LH serum levels affected each group differently. T4 treatment

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decreased LH levels in CO+T4 and ALLO+T4 compared to the untreated groups. However, T4

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treatment increased LH levels in TX+T4 and AUTO+T4, in comparison to TX and AUTO, respectively. Serum FSH levels were consistently lower in all T4-treated groups except in the

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ALLO+T4 group. The highest serum FSH levels were seen in the TX group with statistically

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significant differences (P<0.0001) over the lowest serum FSH levels noted in the AUTO and the AUTO+T4 groups. There are also statistically significant differences between the FSH levels of TX

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3.4. Morphologic study

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and CO groups.

Intrasellar pituitaries and grafts showed histologic changes in different pituitary cell types. H&E staining of intrasellar pituitaries exhibited an increase in acidophilia in T4-treated groups: CO+T4, TX+T4 and ALLO+T4 groups. In grafts (AUTO and ALLO), no changes were noted in terms of

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acidophilia/basophilia, with or without T4 treatment. 3.4.1. CO and CO+T4 3.4.1.1. Light microscopy Comparing the CO and CO+T4 groups, an increase in the average cell number was noted in CO+T4 group (153.20±3.19 nuclei/field) compared to CO (139.19±2.80 nuclei/field) (Fig. 3). T4

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treatment induced a reduction of cell size, possibly because some cells were less active. The histology of the adenohypophyses in the CO group showed the presence of all the various hormone producing cell types (Fig. 4). Their nuclei were euchromatic with very scarse dark nuclei, while in the CO+T4 group, there was an increase of dark nuclei surrounded of eosinophilic cytoplasm. Somatotroph cells were evenly distributed in the adenohypophyses of CO, but CO+T4 displayed more intense immunopositive GH cells. The lactotrophs were irregular in shape and randomly

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distributed in the pituitaries of CO rats. Lactotroph cell number increased in the CO+T4 but stained

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slightly weaker than in CO group. A number of PRL immunopositive cells resembled somatotrophs in morphology (Fig. 4).

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The number of TSH cells appeared constant in CO and CO+T4, although there was a slight

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increase in TSH immunopositivity, indicating that, as there was an exogenous T4 source, TSH granules were not released and were stored in the cytoplasm (Fig. 4).

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3.4.1.2. Transmission electron microscopy

By electron microscopy, the “typical” pituitary cells, possessing their morphologic markers, are

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easily defined. It was however noted that there is a wide overlap of granule sizes of the various cell types, and many cells did not have markers (such as characteristic granule morphology), making their recognition difficult or impossible at times. Such cells can be identified only by immunoelectron microscopy. The normal appearance of the rat pituitary ultrastructure includes somatotrophs (GH)

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which are ovoid or polyhedral in shape with a slightly eccentric, spherical euchromatic nucleus, usually with a prominent, dense nucleolus. The well-developed rough endoplasmic reticulum (RER) is normally at the preriphery of cells. Their Golgi apparatus contains developing secretory granules. The cytoplasmic storage granules are spherical with high electron density, measuring 300–400 nm. Lactrotroph cells (PRL) in the male rat have an eccentric nucleus, with abundant membranous

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organelles and mainly ovoid to pleomorphic secretory granules, generally small in males (200–300 nm) while in the female, lactotrophs are large cells, with granules measuring up to 800 nm. Thyrotrophs (TSH) are the least common pituitary cells with varying cell size. While some are small, with sparse, small secretory granules (50–100 nm), the more predominant thyrotroph cells are fairly large, elongated cells that are stellate or angular in shape. The amount of RER varies greatly, and the

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Golgi apparatus is usually prominent containing developing secretory granules. In addition, in sparsely granulated TSH cells, the secretory granules often form a single layer under the plasma

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membrane, whereas in the well-granulated cells, the granules are distributed throughout the cell, measuring 100–200 nm. Gonadotroph cells (GTH) are middle-sized or large, ovoid or polyhedral

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cells with a spherical nucleus and a prominent Golgi apparatus. There are two variants, the ‘LH cell’

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which contains only small granules (200–250 nm); and the ‘FSH cell’, most prevalent in males, which harbours both small and large spherical granules (400 nm and up). Lastly, corticotroph cells (ACTH)

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are elongated, angular or stellate, with a central nucleus often elongated or flattened. Their cytoplasm is pale and poor in rough endoplasmic reticulum and their Golgi complex is at one pole of the nucleus.

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They produce spherical or slightly irregular secretory granules, different in size and electron density from each other, measuring up to 250 nm, and placed mainly at the periphery of the cell. In the adenohypophyses of the CO group, lactotrophs, somatotrophs, thyrotrophs, corticotrophs and gonadotrophs were identified. These cells contained rounded or ovoid nuclei, variably developed

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endoplasmic reticulum membranes, Golgi apparatus, mitochondria and secretory granules of sizes appropriate for the cell types. Scarce dark cells, representing degenerating cells, with increased electron density were noted in the parenchyma (Fig. 4). Pituitaries from the T4-treated group showed an increase in the number of secretory granules, in accordance with an increase of hormone synthesis

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and secretion of GH, PRL and ACTH. In contrast, gonadotrophs seemed to have reduced numbers of secretory granules (Fig. 4).

3.4.2. TX and TX+T4 3.4.2.1. Light microscopy The morphology in the pituitaries from TX group were very similar. The TX group had an

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average of 127.15±2.63 cells/high power field, while the TX+T4 had an average of 128.83±3.70

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cells/field (Fig. 3). There were approximately 10-12 cells less in each high-power field (compared to CO). This is consistent with an increase in cell size (hypertrophy) of TSH cells, becoming

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“thyroidectomy” cells. Few mitotic figures were also noted in TX group.

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Sections stained with H&E showed an increase in basophilia in TX pituitaries. In contrast, those from TX+T4 displayed strong eosinophilia. The nuclei of the TX group showed mainly decondensed

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chromatin, with few (1-2) nucleoli. The TX+T4 contained less prominent nucleoli, and a higher number of dark nuclei. Plenty of vesicles filled with a light homogeneous content were noted in both

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TX and TX+T4 adenohypophyses. It is well known that hypophyses of thyrectomized animals were characterized by the existence of the so called “thyroidectomy cells” (Table 4). In the pituitaries of the TX group, the thyrotrophs exhibited both hypertrophy and hyperplasia, and their large vesicles were weakly immunopositive for TSH. In TX+T4 adenohypophyses, many thyrotrophs exhibited

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smaller vesiculated cytoplasm (Table 4). However, immunopositivity for TSH was apparent mainly in the perimeter of the vesicles (Fig. 5). The somatotrophs showed a reduction in the number and immunopositivity in TX pituitaries compared to CO (Fig. 4). Their cytoplasm was reduced, losing their typical round/ovoid shape. An interesting finding was that TX+T4 adenohypophyses displayed more GH cells than TX (similar to CO –Fig. 4–), although weakly immunopositive. In addition, some

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somatotrophs were similar to thyroid deficient cells, displaying large cytoplasmic vesicles exhibiting immunopositivity for GH (Fig. 5b). As they are gradually acquiring the morphologic features of thyrotrophs, they undergo transdifferentiation to somatothyrotroph –an intermediate step into the transdifferentiation to a thyrotroph cell. In TX pituitaries, lactotrophs were strongly immunopositive for PRL but underwent a reduction in number (Fig. 4). Some PRL immunopositive cells had a round shape, which was more characteristic of a somatotroph rather than of a lactotroph. However, in

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simultaneous with an increase in PRL synthesis and release (Table 3).

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TX+T4 there was an increase in PRL cell number (Fig. 4), but a reduction of immunopositivity,

3.4.2.2. Transmission electron microscopy

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Pituitaries from the TX group displayed a mosaic of cells, with euchromatic nuclei and some

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had a prominent nucleolus. These cells were difficult to identify because they were smaller in size and lacked secretory granules. At six weeks after thyroidectomy, these cells were almost completely

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degranulated (Fig. 5). They were intermingled with thyrotrophs that underwent hypertrophy and contained dilated endoplasmic reticulum displaying the typical morphology of “thyroidectomy cell”

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(Fig. 5). The thyrotrophs had a spherical nuclei and abundant cytoplasm with progressive accumulation, dilation of rough endoplasmic reticulum and large Golgi complex with scarce small, usually intracisternal, secretory granules. This morphology revealed that the thyroidectomy cells had undergone hypertrophy, and were hyper-functioning TSH cells affected by the lack of negative

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feedback of thyroid hormone and an increase of hypothalamic TRH secretion. In addition, some somatotrophs appeared to transform into thyroidectomy cells, showing dilated rough endoplasmic reticulum and displaying both large and small secretory granules (analogous to both GH and TSH granules). Pituitaries from TX+T4 animals displayed apparent cell granulation and showed similar thyroidectomy cells (Fig. 5j), although with less apparent cytoplasmic vesicles than in TX. . Some

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somatotrophs resembled thyroidectomy cells with enlarged RER and two size granules (Fig. 5j), as there was an extra apport of T4, they were recovering their GH-secreting-cell morphologic features, possibly undergoing reverse transdifferentiation from somatothyrotroph to somatotroph, In summary, TX samples had a significant loss of secretory granules in all cell types. The TSH cells displayed hypertrophy and hyperplasia whereas lactotrophs and somatotrophs seemed to be reduced in number. They also showed an altered morphology post-thyroidectomy, suggesting they

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were undergoing trandifferentiation to enhance TSH secretion. In TX+T4, granulation of the cells

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was evident and TSH cell alterations were less apparent and dissimilar from the euthyroid

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morphology.

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3.4.3. AUTO and AUTO+T4 3.4.3.1. Light microscopy

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The number of cells per high power field was 141.10±5.51 for AUTO and 152.10±3.85 for AUTO+T4. There was a similar trend of AUTO and AUTO+T4 compared to CO and CO+T4,

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respectively (Fig. 3).

Autografts displayed connective tissue in the central portion which replaced the necrotic area that was previously present. Histologically, the adenohypophysial architecture seemed to be well preserved (Fig. 6). No difference was seen between AUTO and AUTO+T4 tissue stained with H&E.

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However, in the AUTO group, some areas‒ mainly between the connective tissue and the renal parenchyma‒ contained “spongiform” cytoplasms displaying small vesicles filled with a clear material. The thyrotrophs in AUTO showed “spongy” cytoplasm with several small vesicles that appeared smaller in size when compared to thyroidectomised animals. This finding was identified in

19

all samples of the AUTO grafts. However, these vesicles were seen only in 1/3 of the AUTO+T4 samples (Table 4, Fig. 6). The pituitary grafts of the AUTO+T4 group showed a higher number of somatotrophs displaying more intense immunopositivity than in AUTO. Lactotroph hyperplasia was evident with an intense and scattered PRL immunopositivity in both AUTO and AUTO+T4 groups. The number of TSH cells was greatly reduced in AUTO+T4 and displayed weak immunpositivity (Fig. 6).

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3.4.3.2. Transmission electron microscopy

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In the AUTO grafts, tissue was composed of a mosaic of cells with different cytoplasmic electron density (Fig. 6). Almost all cells were depleted of secretory granules. The lactotrophs were dominant

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in the surviving portions of the grafts, and displayed a rounded or ovoid euchromatic nuclei with a

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low electron dense cytoplasm and very few secretory granules. Somatotrophs, corticotrophs, thyrotrophs, and gonadotrophs were not as numerous and were interspersed with lactotrophs. Their

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nuclei contained some heterochromatin, mainly in the proximity of the nuclear envelope. Some thyrotroph cells displayed a vesiculated cytoplasm. Grafts from AUTO+T4 showed conspicuous

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granulation compared to the AUTO group. It was noted that some cells had abundant secretory granules of variable diameter intermingled with dilated sacs of endoplasmic reticulum filled with low electron dense material. Their nuclei were irregular in shape with euchromatin (Fig. 6). These cells were comparable to hypertrophic thyrotrophs with secretory granules as seen in TX+T4, although

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considerably less apparent and with smaller ER vesicles.

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3.4.4. ALLO and ALLO+T4 3.4.4.1. Light microscopy The sellar pituitaries contained an average of 158.50±3.09 cells for ALLO and 155.71±4.48 for ALLO+T4 per high power field. In the grafts, the cell number per high-power field was 129.20±5.93 for ALLO and 131.17±2.97 for ALLO+T4 (Fig. 3). There were no major differences between untreated and T4-treated groups. The grafts showed a slight reduction in number of cells compared

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to the sellar pituitaries.

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Homotopic pituitaries of rats bearing an allograft did not show any alteration from the normal histology (Fig. 7). No thyroid deficient cells were observed in any of the sellar pituitaries. An increase

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in acidophilia was displayed in the sellar pituitaries of ALLO+T4, similar to the findings in the

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adenohypophyses of CO+T4. ALLO hypophyses displayed some dark nuclei with condensed chromatin, and surrounded by acidophil cytoplasms. This occurrence increased in the ALLO+T4

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sellar pituitares, but some contained a basophilic cytoplasm (Fig. 7). Allografts of ALLO and ALLO+T4 groups (Fig. 8) displayed a central area of connective tissue

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replacing the previous necrosis. In most grafts, there were many inflammatory cells, mainly lymphocytes, widespread or in a mass, located on the border of the graft in contact with the renal cortex. No apparent differences were seen in the H&E stained sections between both groups in terms of acidophilia/basophilia. In ALLO (Fig. 8), the central areas of the grafts showed spongiform

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cytoplasms (Table 4), similar to AUTO (Fig. 6). This alteration was more apparent in the allografts that displayed less intense inflammatory reaction. In contrast, this alteration (although less intense) was present in only 1/3 of the samples of the ALLO+T4 group (Fig. 8, Table 4). ALLO grafts displayed an abundance of somatotrophs (Fig. 8), but were not as numerous as in the ALLO+T4 group. This is concurrent with a dramatic elevation of GH serum levels in ALLO+T4

21

(Table 3). PRL hyperplasia was more apparent in ALLO+T4 concurrently with an increase of PRL serum levels in this group. However, thyrotrophs were scant in the allografts, especially in ALLO+T4 (Fig. 8). 3.4.4.2. Transmission electron microscopy In both the ALLO and ALLO+T4 groups, the surviving portions of the grafts were composed mainly of lactotrophs. The grafts consisted of fibroblasts and collagen fibers interspersed with groups of

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lactotrophs and other cells with different electron density (Fig. 8). Some cells stored plenty of

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secretory granules while others had a cytoplasm completely devoid of them. Marked lactotroph hyperplasia with signs of endocrine activity was noted. Most of the nuclei had decondensed

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chromatin with some heterochromatin in their periphery. Some cells (as in Fig. 8j) have morphologic

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features of GH secreting cells (high electron-dense secretion granules), but displayed dilated RER, as if they were undergoing transdifferentiation to the intermediate step of mammosomatotroph.

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3.5. Cell proliferation

Results of cell proliferation are shown in Fig. 9.

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T4 treatment marginally increased cell proliferation in CO+T4 and induced a small increase in the grafts of ALLO+T4 compared to the untreated groups. In the other groups, T4 treatment reduced the number of replicating cells compared to the untreated groups. The highest cell proliferation rates were seen in the TX pituitaries (1.852%), compared to the

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CO group (0.311%). A marked reduction of cell proliferation was seen in TX+T4 (0.851%). The second highest proliferation index was noted in the grafts of AUTO (1.452%), decreasing in AUTO+T4 (1,003%). The percentage of proliferating cells was 2.8 times higher in the sellar pituitaries from the ALLO group (0.876%) than in CO.

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4. Discussion 4.1. Occurrence of thyroid deficient cells in pituitary grafts In normal animals, thyroidectomy results in the development of thyroidectomy cells in the pituitaries (Fig. 5). It is well-known that the feedback mechanism between the thyroid gland and the

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pituitary is affected from reduced thyroid function. Thyroidectomy cells are supposed to be very

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active TSH cells activated by the increased hypothalamic TRH level, attempting to compensate the missing T3 and T4. In our experiment, there is no TRH nearby the explanted pituitaries.

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Consequently, the most intriguing finding was that thyroidectomy cells developed in the pituitary grafts (Fig. 6). This is a novel and interesting finding because the grafts had no direct connection with

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the hypothalamus, then thyroidectomy cells are not induced by TRH. In explanted pituitaries there is

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no TRH nearby, in that case what activates TSH cell under this condition should be the lack of T3 and T4 negative feedback. To our knowledge, this study is the first to document this occurrence in ectopic or non-sellar pituitaries (although not as extensive as in TX adenohypophyses). In addition,

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it seems that hypothalamic control is not essential to induce thyroidectomy cells, as suggested by their presence in pituitaries grafted under the kidney capsule. In explanted pituitaries TSH secretion is supposed to be lower than normal resulting in a reduction of thyroid hormones being secreted,

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thereby inducing hypothyroidism and thyroidectomy cells. Administration of the thyroid hormones T4 and T3 prevents the development of thyroidectomy cells. Accordingly, T4 treatment in AUTO+T4 (Fig. 6) showed a lower degree of alteration in thyroptrophs (appearance of the clear spongiform cytoplasm) present only in 1/3 of the samples, indicating that T4 reduces it occurrence. It is interesting to note that this state could possibly be overridden by increasing T4 doses and/or exposure times. The same situation was also apparent, although to a lesser extent, in the grafts from ALLO and ALLO+T4 23

groups (Fig. 8). This phenomenon is very intriguing because these rats kept their sellar pituitaries intact, thus they had no hypothyroid condition. However, the allografts developed vesiculated cytoplasms analogous to thyroidectomy cells. This finding is difficult to explain, but it is logical to assume that the allografted tissue shared similar conditions to the autografts, since after transplantation the grafts experienced hypoxia and complete disconnection from hypothalamic control. It is also reasonable to suggest that T4 could not reach the graft parenchyma due to lack of

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adequate vascularization. Vesiculated cytoplasms were less apparent in ALLO (Fig. 8) than in AUTO

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(Fig.6). This is due to the fact that once the vascularization was established (after an initial “hypoxic phase”), circulating T4 could finally reach the graft, reducing the cytoplasmic vesicles in the ALLO

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grafts which was noted by the dramatic reduction of cytoplasmic vesiculation seen in ALLO+T4. In

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previous experiments studying pituitary autografts under the kidney capsule in rats no thyroidectomy cells were observed, possibly due to the shorter survival period in those experiments (three and four

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4.2. Transdifferentiation

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weeks, respectively) (Lombardero et al., 2006; Lombardero et al., 2009).

Regarding sellar pituitaries, it is well known that thyroidectomised rats show an increase in the number of thyrotrophs, as well as in cell size (hypertrophy), and T4 treatment induces an increase in TSH immunopositivity attributed to an increment in TSH storage due to the neoformation of secretory

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granules (Ozawa,1991).

Replicating nuclei (based on IOD, Fig. 9) increased in our hypothyroid animals (TX and AUTO

groups), and T4 treatment induced a decrease in their values, although not to euthyroid levels. Surks and DeFesi (1977) determined the percentage of each adenohypophysial cell type in hypothyroid rats, reporting that the major changes in TX rats were a marked increase in percentage

24

of thyrotrophs, and a decrease in percentage of somatotrophs. In accordance to other publications, authors reported a decrease in the number of pituitary GH and PRL cells in their TX animals (Kimura and Furudate, 1996) or in their experimental TSH deficient mice (Stahl et al.,1999). However, no massive cell death or dramatic mitotic increment were reported suggesting that the increase in some cell types must occur at the expense of other cell types to maintain equilibrium. Since thyrotrophs, somatotrophs, and lactotrophs derive from a common precursor, hyperplasia of thyrotrophs may

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deplete the precursor pool available for differentiation to the other cell types (Stahl et al., 1999). The

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change in the number of a specific cell population is determined not only by cell proliferation and apoptosis, but also by transdifferentiation, a phenotypic switch between mature cell types without

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cell division (Vidal et al., 2000). In our TX samples, some cells immunopositive for GH were similar

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to thyroidectomy cells and vice versa, possibly due to transdifferentiation of some somatotroph into thyrosomatotrophs, regarded as intermediate cells in the transdifferentiation of somatotrophs to

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thyrotrophs. Our findings are in concordance with those reported in rats (Horvath et al.,1990; Villalobos et al., 2004), and humans (Vidal et al., 2000; Radian et al., 2003), indicating that

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thyrosomatotroph formation should be specifically favoured by the pressure of low plasma thyroid hormone levels in hypothytoidism.

On the contrary, in the pituitary grafts (AUTO and ALLO, and their respective T4-treated groups), there was an extensive PRL hyperplasia (Fig. 6, 8) resulting from the fact that dopamine

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cannot exert its negative feedback on lactotrophs (PRL is the only pituitary hormone that is under inhibitory rather than stimulatory control). In contrast, percentages of replicating nuclei in the pituitary grafts were not as high as it would have been expected (Fig. 9). This data led to the hypothesis that this massive increase in lactotroph cell number might also be attributable to transdifferentiation, as it does not require cell division. There were some studies regarding

25

transdifferentiation from diverse pituitary cell types to PRL cells in homotopic pituitaries (Vidal et al., 2001; Jentoft et al., 2012; Tun et al., 2019), that reported the existence of bi-hormonal cells as intermediates. It is interesting that pituitary graft cells disrupted from the hypothalamus-pituitary axis, may also undergo transdifferentiation. This fact suggests that transdifferentiation is not under hypothalamic control and might be up- or down-regulated by diverse factors produced by the pituitary tissue itself. According to our results, it seemed that T4-treatment induced an extensive PRL

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hyperplasia in the pituitary grafts from ALLO+T4 when compared to ALLO (Fig. 8), although no

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differences were seen in terms of PRL immunostaining between AUTO+T4 and AUTO (Fig.6). Further research will be required to elucidate the main mechanisms involved in transdifferentiation

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into PRL-producing cells in explanted pituitaries. In addition, owing to the extensive PRL hyperplasia

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spontaneously induced, pituitary grafts might be appropriate experimental models to investigate the

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mechanisms of transdifferentiation of the diverse hormone producing cell types to lactotrophs.

4.3. Cell proliferation index and densitometric measurements

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In addition, there were many other changes that occurred within the various groups as well. Sellar pituitaries from CO+T4, ALLO and ALLO +T4 showed an increase in cell number per high power field (Fig. 3), apparently because as there was an extra hormone supply (T4-treatment and the allograft, or both, respectively), cells from sellar pituitaries became less active and showed a

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reduction in the size of their cytoplasm. Surprisingly, these sellar pituitaries showed an increment of IOD (Fig. 9), that was translated in an increase of replicating nuclei when compared to CO group. This data was completely unexpected because they had reduced secretory activity. This change may be due to condensed chromatin which could be interpreted as an increment in the density of the nucleus and, consequently in IOD. Feulgen stain could be an outstanding method for quantifying

26

DNA in cancer cells with very high metabolic activity (Schulte and Wittekind, 1989; Haroske et al., 2001; Biesterfeld et al., 2011), but might be less accurate in cells with low metabolic activity. In our experiment, the incorporation of Bromo-deoxyuridine (BrdU) into the nuclei was unsuccessful, consequently Feulgen stain was chosen as a second option. BrdU labeling index has many advantages (more accurate and much less time consuming) but it also has disadvantages compared to Feulgen

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stain, mainly, being restricted to research with laboratory animals.

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4.4. Body- and endocrine gland relative- weights

Regarding body weight (Fig. 1), it seems that T4 administration to euthyroid animals (CO and

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ALLO) causes a decrease in body weight. In contrast, when T4 is supplemented to hypothyroid

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animals (TX and AUTO), their body weight increased compared to the non-treated group. Data on body weights of our samples showed a similar trend to other authors: CO achieved the highest values,

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while it was diminished in TX animals, yet slightly increased in T4- treated (TX+T4). However, there is controversy about the effect of T4 administrated to euthyroid animals (CO). Our results gave values

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in between CO and TX (Fig. 1), while another study by Kimura and Furudate (1996) reported that their CO+T4 animals reached similar body weights to their CO group. Referred to endocrine gland relative weight (Table 2), T4 treatment induced an increase in the pituitary relative weights of CO+T4 and ALLO+T4 which could be attributable to their lower body

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weight compared to the untreated group. The increase in the adenohypophysis relative weights of TX pituitaries was most likely due to hyperplasia and hypertrophy of TSH cells, along with their low body weight increase. No significant variations were noted in the thyroid relative weights between untreated and T4-treated groups.

27

With the exception of the AUTO and AUTO+T4 groups, the adrenal gland weights from all the T4-treated groups were higher than their counterparts (Table 2). AUTO and AUTO+T4 had the lowest adrenal weight values and left testis relative weights, compatible with pituitary hypofunction since pituitary hormones are not properly synthesized and released by the grafts. These results are in accordance with those reported. There is no significant difference in testicular weight between TX and control rats, in accordance

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with Tohei et al. (1998). In addition, regarding adrenal relative weights, CO and TX had also similar

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values.

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4.5. Serum hormone levels

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It is generally accepted that deprived of releasing hormones (isolated from the hypothalamus), the pituitary trophic hormones stop being produced, except for PRL. T4 treament induced an increase

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in GH and PRL serum levels in all groups compared to the untreated groups (Table 3). Our findings support the theory behind the importance of T4 for normal GH production. This finding is in

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accordance with those reported by Kimura and Furudate (1996) in euthyroid rats. As shown in Table 3, the changes in the hormone levels in the AUTO groups meets the expectation. However, the ALLO group values, which are expected to be near the sum of the CO and AUTO groups differ significantly, especially in the case of GH. This could not be attributed to the putative crossreaction of the GH

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antibodies used (otherwise in AUTO+T4, with the highest figures of PRL levels, GH levels should have been similar, nonetheless this does not occur). The abrupt increase of GH serum levels in the ALLO and ALLO+T4 is difficult to explain and further research will be required to identify which factors or conditions act synergistically to produce such GH augment.

28

T4 administration had nominal impact on serum levels of the other adenohypophysial hormones (Table 3), unless in AUTO and AUTO+T4 regarding LH (T4 increased LH), and TX and TX+T4 concerning FSH (T4 decreased FSH). In addition, it is intriguing that hypothyroid goups (TX and AUTO) had so dissimilar levels of FSH: AUTO got one of the lowest values of FSH serum levels, while TX got the highest figures. Maybe it is due to AUTO group has a complex hormonal dysfunction rather than a single hormonal defect, as in TX.The same comment is applicable regarding

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the left testis relative weights. This was, however, partially in contrast to a study by Tohei (2004)

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who stated that hypothyroidism is associated with gonadal dysfunctions and decreased plasma concentrations of LH and FSH. Divergent findings were also reported by Ai et al. (2007) who stated

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that the serum concentrations of FSH and LH were not significantly different among their hypo-,

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hyper- and euthyroid rats (equivalent to our TX and AUTO; CO+T4 and ALLO+T4; and CO,

5. Conclusion

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respectively).

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In summary, thyroid hormones (T4) induced changes in pituitary cell morphology, as seen by light microscopy and TEM. The development of thyroidectomy cells in pituitaries grafted under the kidney capsule (disconnected from hypothalamus) is a remarkable phenomenon that was not previously reported. A long term hypothyroid state (six weeks) induces the development of

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thyroidectomy cells, even in the pituitary grafts, denoting that hypothalamic control is not necessary for its occurrence. In addition, T4-treated groups showed lower alteration in their morphology, although longer exposure times or higher doses are required to fully restore the euthyroid morphology. Pituitary grafts developed PRL hyperplasia through transdifferentiation of the hormoneproducing cells to lactotrophs. T4 treatment enhanced PRL hyperplasia in pituitary grafts from those

29

rats that kept their homotopic pituitaries intact. Our study also shows that T4 has an effect on cell size, reflected on an increase of cell count (up to ten cells per high power field) in CO+T4 and AUTO+T4. Additionally, T4 had diverse influence on serum hormone levels, increasing GH and PRL levels, as well as on the endocrine organ relative weights. Our study supports that T4 plays an important role regulating the activity and morphology, not only of thyrotrophs, but also of

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somatotrophs and lactotrophs.

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ETHICAL STATEMENT

All surgical and experimental procedures were approved by the Institutional Animal Care and Use Committee of the

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Universidad Autónoma of Aguascalientes (Mexico) and are compatible with the Institute for Laboratory Animal Research

Ethical approval

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(USA) guidelines, 2011.

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All procedures performed in this animal study were in accordance with the ethical standards of the institution or practice at which the study was conducted.

CONFLICT OF INTEREST

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The authors declared that they had no conflict of interests with respect to their authorship or the publication of this article.

Declarations of interest: none

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Acknowledgments This work was supported by CONACYT 221262 grant in México (A.Q-S). Authors are also grateful to the Jarislowsky and Lloyd Carr-Harris Foundations for their support.

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Figure captions Fig. 1 Timeline of our experiment. A schematic time course of the experimental procedures.

Fig. 2 Body weight variation charts. Filled charts with discontinuous lines correspond to T4-treated groups.

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(A) Weekly body weight assessment for six weeks. (B) Percentage of body weight variation in six

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weeks. The P values calculated in a two-way ANOVA at the end of the experiment (6W) are included

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showing statistically significant differences.

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Fig. 3

Average of number of cells per high power field. Graphic showing the average of nucleus count per

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high power field (600x) in each group.

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Fig. 4

Light microphotographs of CO and CO+T4 pituitaries stained with hematoxylin-eosin, and immunostained for GH, PRL and TSH. (a, f): differences between the groups of male rat pituitaries are clearly visible in regards to cell types present and hormone production. In the CO+T4 group (f),

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more acidophilic cells as well as dark nuclei () were present. (b, g): somatotophs were the most prevalent cell type in the CO male rat pituitary (b) (). CO+T4 (g) showed more cells with stronger GH immunopositivity (); PRL-producing cells in CO pituitary (c) ( >), more numerous in CO+T4 (h); (d, i): thyrotrophs () were scarce, randomly distributed, and irregular in shape in both groups. Transmission electron micrographs (e, j) showing the ultrastructure of the pituitary. CO group (e)

displaying different cell types with normal appearance, CO+T4 (j) showing an increment of stored granules in the cytoplasm as well as differences in granule size between cell types (GH: high electrondensity granules, measuring 300–400nm; PRL: pleomorphic, electron-dense granules; TSH: sparse, small secretory granules measuring 50–100 nm; GTH: contained two granule populations: small granules measuring 200–250 nm and large, spherical granules of 450 nm–visible in j (). Scale bars:

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25 μm (a, f), 10 μm (b–d, g–i), or 2 μm (e, j).

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Fig. 5

Light micrographs of TX and TX+T4 pituitaries stained with hematoxylin-eosin, and immunostained

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for GH, PRL and TSH. (a,f): many large vesicles () and dark nuclei () present in the cytoplasm

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of some cells corresponding to “thyroidectomy cells”; (b): somatotrophs lost their typical shape and size () as well as their number in TX; N.B. immunopositive GH cell (#), similar to a thyroid

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deficient cell, with a large cytoplasmic vesicle. (g): TX+T4 showing an increase in GH cells but not in immunopositivity (). N.B. a GH cell (#) resembling a thyroidectomy cell with apparent

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cytoplasmic vesicles; (c): scarce, but strongly immunopositive PRL cells (>) in TX, (h) TX+T4 lactotrophs (>) more abundant, but less intense PRL immunospositivity. (d): TSH immunopositive thyrotrophs displaying typical thyroidectomy cell appearance, with large vesicles filled with a light homogeneous content () and exhibiting hypertrophy and hyperplasia, TX+T4 (i) many TSH

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immunopositive () thyrotrophs with smaller vesicles. Transmission electron microphotographs (e, j) showing the ultrastructure of TX and TX+T4 cell pituitaries. (e): thyroidectomy cells displaying characteristic dilated RER forming vesicles filled with clear content () with intracisternal granules (), scarce secretion granules, most forming a single layer below the plasma membrane () and spherical euchromatic nucleus with prominent Golgi complex (#). (j): TX+T4 pituitary with a TSH

36

cell displaying an euchromatic nucleus with some heterochomatin, cytoplasmic scarce granules forming a single layer under the plasma membrane (), Golgi complex (#), and less dilated RER (). N.B. the GH cell undergoing reverse transdifferentiation from somatothyrotroph to somatotroph with an euchromatic nucleus, well-developed RER with dilated cisternae (&) and with heterogeneous secretory granules varying in size and electron density () corresponding to TSH content. C,

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capillary (j). Scale bars: 25 μm (a, f), 10 μm (b–d, g–i), or 2 μm (e, j).

Fig. 6

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Light micrographs of pituitary grafts from AUTO and AUTO+T4 groups stained with hematoxylin-

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eosin and immunostained for GH, PRL and TSH. (a): AUTO cases contained “spongiform” cytoplasm displaying small vesicles (), characterized by light staining, (f): AUTO+T4 containing

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“spongy” cytoplasm in only a few vesicles (); (b,c,d,g,h,i): The presence of certain cell types and immunopositivity of hormones between groups varied as well. N.B. somatotrophs with scarce

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cytoplasm (), TSH cells with “spongy” cytoplasm with some small vesicles () (d). Thyrotrophs () were dramatically reduced in AUTO+T4 (i), presenting less intense immunopositivity compared to AUTO. Transmission electron microphotographs (e, j) showing the ultrastructure of the autograft tissue. (e): Pituitary graft from AUTO displaying different cell types with dissimilar electrondensity

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and prevalence of lactotrophs (PRL), AUTO+T4 tissue (j) showing the typical morphology from “thyroid deficient cells” when treated with T4: dilated RER, vesicles filled with electronlucent content (), an increase of stored granules in the cytoplasm (), and a well-developed Golgi complex (#). C, capillary (e). Scale bars are equal to 10 μm (a–d, f–i), or 2 μm (e, j).

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Fig. 7 Light micrographs of sellar-pituitary tissue stained with hematoxylin-eosin and immunostained for GH, PRL, and TSH. (a): ALLO hypophyses displaying dark nuclei with condensed chromatin, and surrounded by acidophil cytoplasms (),in the ALLO+T4 sellar pituitaries (e), many dark nuclei were noted, most of them displaying acidophilic () or basophilic cytoplasms (). An increase of

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acidophilia in ALLO+T4 (e) –similar to CO+T4 (Fig. 4) was also noted. (b, f): somatotrophs were

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less abundant than in similar groups of CO and CO+T4 (Fig. 4), displaying mild immunostaining for GH () in both groups. (c) lactotrophs () were stellate and irregular in shape in ALLO, (g)

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lactotrophs showing reduced size and cytoplasmic processes in ALLO+T4; (d): ALLO group

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containing angular shaped thyrotrophs with mild TSH immunopositivity (), (h) ALLO+T4 with

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less TSH immunostaining () than ALLO group. Scale bars: 10 μm.

Fig. 8

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Light micrographs of allografted tissue from ALLO and ALLO+T4 stained with hematoxylin-eosin and immunostained for GH, PRL, and TSH. (a): in ALLO, grafts showed spongiform cytoplasm ()–similar to AUTO (Fig. 6)–, (f) the ALLO+T4 group showing less extensive and intense spongiform cytoplasms (). Numerous GH cells () in ALLO grafts (b) that increased in number

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in ALLO+T4 (g), also displaying stronger GH immunopositivity; (c) due to the lack of hypothalamic inhibition, most of the secretory cells were PRL immunopositive both in ALLO (c) and ALLO+T4 (h) groups; (d): thyrotrophs () were scarce in the allografts, TSH cell number and immunopositivity were dramatically reduced in ALLO+T4 (i). Seen here is a “thyroidectomy cell” (), and two TSH cells with dark nuclei (); (e, j): transmission electron microphotographs showing

38

the ultrastructure of the ALLO graft (e) displaying different cell types, some of them are TSH cells with plenty of secretion granules in their cytoplasm (), whereas other cells () are depleted of granules; (j) ALLO+T4 tissue displaying one of the few samples showing TSH cells with a “thyroidectomy cell” appearance after being treated with T4. Dilated RER () with vesicles of variable size filled with low electron-dense content (), secretion granules (), and Golgi complex

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(#). C, capillary (j). Scale bars: 10 μm (b–d, g–i), or 2 μm (e, j).

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Fig. 9

Percentage of replicating cells based on IOD. Bar diagram showing the percentage of nuclei in a G2-

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M phase in each group based on the Integrated Optical Density (IOD) assessed in sections stained

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with the Feulgen method, a stoichiometric staining for DNA.

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Table 1. Animal groups in our experiment.

Untreated

T4-treated

Intact

CO

CO+T4

Thyroidectomized

TX

TX+T4

Autografted pituitary

AUTO

AUTO+T4

- Sellar pituitary

ALLO pit

ALLO+T4 pit

- Allograft

ALLO graft

ALLO+T4 graft

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Animal group

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Allografted pituitary:

Organ n

Adenohypo

Thyroid

physis

Group

CO+T4 TX

Adrenals

n

Left Testis

n

6

1.9 ± 0.5 ab *

7

6.2 ± 1.0

7

13.4 ±2.4 abc

7

503.4 ± 33.6 ab

5

2.4 ± 0.9 ab

7

6.4 ± 1.1

7

17.5 ± 5.1 a

7

517.2 ± 55.0 b

4

3.2 ± 0.7 a

NA

7

13.6 ± 2.4 abc

7

518.4 ± 153.5 b

4

2.8 ± 0.7 ab

NA

7

16.2 ± 2.3 ac

7

512.4 ± 55.9 b

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TX+T4

n

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CO

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Table 2. Endocrine gland relative weights expressed in mg×100g body weight-1

AUTO

NA

7

6.5 ± 0.6

7

8.5 ± 1.4 b

7

117.9 ± 14.6 a

AUTO+T4

NA

5

7.5 ± 0.9

5

7.2 ± 1.5 b

5

102.6 ± 14.7 a

1.7 ± 0.2 b

7

6.4 ± 0.9

7

17.6 ± 3.3 a

7

486.1 ± 48.5 ab

ALLO

4

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ALLO+T4

4

P value

7

1.8 ± 0.4 ab 0.0102

6.5 ± 1.5

7

7

21.3 ± 4.0 a

0.3197

495.8 ± 61.2 ab

< 0.0001

< 0.0001

One-way ANOVA, * Means with different letters in the same column differ statistically using the Dunn’s

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group comparison test at P < 0.05

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Table 3. Serum hormone levels expressed in ng×ml-1, except for ACTH, expressed in pg×ml-1 Hormone

TX

7

7

7

7 ab

5.4 ± 5.1 a

7 bc

16.4 ± 18.3

AUTO

6 abc

45.2 ± 43.7 AUTO+ 5 abc T4 ALLO

103.9 ± 48.7 ab

7 ab

83.6 ± 41.9

TX+T4

27.5± 17.8

6 abc

7

161.6 ± 185.2 abc

430.6 ± ALLO+ 7 493.7 b T4

67.3 ± 36.9

9.6 ± 5.6 b 28.5 ± 10.3

6 ab 6

119.8 ± 163.1 ab

5

269.5 ± 259.7 a 153.9 ± 68.7

7 a 7

0.02 ± 0.01

7 abc

107.7 ± 51

251.8 ± 205.6 a

0.04 ± 0.04

6 ab 5

0.01 ± 0.02

7 abc

4

4.6 ± 1.5

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CO+T4

10.9 ± 7.3 b

5 bcd

12.9 ± 3.9

7 a

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53.5 ± 27.0

7

7.2 ± 2.2

6 bc

6 cd

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7 ac

6 b

14.9 ± 5.0 b

5

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ACTH LH FSH n n -1 -1 (ng×ml ) (ng×ml-1) (pg×ml ) 71.7 ± 21.6 0.03 ± 0.02 6 ab 7 abc 6 8.6 ± 3.1 c n

15.4 ± 5.5

b

5

7

130.1 ± 39.1 a

6

5

200.2 ± 115.3 a

6

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Group

PRL (ng×ml-1)

n GH (ng×ml n 1) 12.4 ± 13.2

1.6 ± 0.7

0.007 ± 0 ab 0.07 ± 0

0.1 ± 0.1

4 b

c

0.01 ± 0.008 ab

4.1 ± 2.1

7 bd

4.6 ± 1.8

0.007 ± 0

a

6 bdc

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< 0.0001 0.0002 < 0.0001 0.0001 < 0.0001 P value One-way ANOVA, * Means with different letters in the same column differ statistically using

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the Dunn’s group comparison test at P < 0.05

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Table 4. Occurrence of thyroidectomy cells. ALLO Presence (+) or

CO

TX

AUTO

absence ( ̶ ) of thyroidectomy ̶

+++

++

CO+T4

TX+T4

AUTO+T4

Pit*

graft

̶

+ 100%

of

cells

+≈33%

++

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* Sellar pituitaries

Pit*

graft

̶

+≈33%

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ALLO+T4

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