Dexamethasone exposure affects paraventricular nucleus and pituitary corticotrophs in female rat fetuses: An unbiased stereological and immunohistochemical study

Dexamethasone exposure affects paraventricular nucleus and pituitary corticotrophs in female rat fetuses: An unbiased stereological and immunohistochemical study

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ARTICLE IN PRESS

YTICE-1022; No. of Pages 8

Tissue and Cell xxx (2016) xxx–xxx

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Dexamethasone exposure affects paraventricular nucleus and pituitary corticotrophs in female rat fetuses: An unbiased stereological and immunohistochemical study ∗ ´ ´ Svetlana Trifunovic, ´ Nataˇsa Ristic, ´ Milica Manojlovic-Stojanoski , Nataˇsa Nestorovic, ´ Branko Filipovic, ´ Verica Miloˇsevic´ Ivana Jaric, Institute for Biological Research “Siniˇsa Stankovi´c”, University of Belgrade, 142 despota Stefana Blvd., 11060 Belgrade, Serbia

a r t i c l e

i n f o

Article history: Received 9 March 2016 Received in revised form 27 June 2016 Accepted 27 June 2016 Available online xxx Keywords: Paraventricular nucleus Fetus Dexamethasone Programming CRH ACTH

a b s t r a c t The hypothalamic paraventricular nucleus (PVN) drives the stress response by activating the hypothalamo-pituitary-adrenal (HPA) axis, particularly vulnerable to glucocorticoid exposure during development. To evaluate the effects of fetal dexamethasone (Dx) exposure on the stereological features of PVN and HPA axis activity in female rat fetuses, pregnant rats received 0.5 mg Dx/kg/b.w./day on days 16, 17 and 18 of pregnancy and 21-day-old fetuses were obtained; controls received the same volume of saline. In an unbiased stereological approach, Cavalieri’s principle and an optical fractionator were used for estimating volume and total cell number of the PVN, respectively. The intensity of corticotropinreleasing hormone (CRH) immunoreactivity in the median eminence (ME) was determined by CRH optical density and the adrenocorticotropic hormone (ACTH) relative fluorescence signal intensity (RIF) in pituitary corticotrophs was measured using Image J. Significant reductions (p < 0.05) in PVN volume and cell number were found in fetuses exposed to Dx. Additionally, CRH optical density in the ME and ACTH RIF (p < 0.05) in the corticotrophs were decreased. The established results suggest that the reduced number of cells in the PVN after maternal Dx administration negatively affects the CRH content in the ME and the ACTH quantity in pituitary corticotrophs in near-term fetuses. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Development of the specific structural organization of the hypothalamus that controls a spectrum of vital physiological functions begins in utero and continues during the postnatal period. The paraventricular nucleus (PVN) integrates essential endocrine and autonomic responses that sustain homeostasis, energetic balance and behavioral reactions (Altman and Bayer, 1986). The PVN achieves its multiple functions due to the presence of three distinct neuronal populations: magnocellular neuroendocrine neurons that project directly to the posterior pituitary, parvicellular neuroendocrine neurons that project to the median eminence (ME) controlling anterior pituitary hormone secretion and nonneuroendocrine neurons which create connections with multiple CNS regions responsible for autonomic and behavioral responses (Swanson, 2009). In fetuses, the proliferative neuroepithelium

∗ Corresponding author. ´ E-mail address: [email protected] (M. Manojlovic-Stojanoski).

of the third ventricle generates the majority of cells from the mentioned populations between embryonic days (E) 12 and 14 (Markakis and Swanson, 1997). Markakis and Swanson (1997) and Markakis (2002) suggest that newborn neurons probably remain near the periventricular zone before taking their final position i.e. a form of ‘arrested-delayed migration’ may be necessary in order to complete functional differentiation. The first corticotrophin releasing hormone (CRH) immunoreactive neurons in the PVN are detected at E 16 and their number significantly increases during E 17. Additionally, CRH immunopositive axonal projections to the ME are recognized during E 17.5, indicating the beginning of CRH release into the hypophyseal portal system (Daikoku et al., 1984). When it reaches the pituitary, CRH positively regulates adrenocorticotropic hormone (ACTH) synthesis and secretion in corticotrophs which further stimulate steroidogenesis in the adrenal gland. Regulation of the hypothalamo-pituitary-adrenal (HPA) axis activity begins in utero in order to sustain appropriate glucocorticoid levels which are crucial for numerous glucocorticoid actions, including maintaining homeostasis, timely maturation of vital organs and initiation

http://dx.doi.org/10.1016/j.tice.2016.06.012 0040-8166/© 2016 Elsevier Ltd. All rights reserved.

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of parturition (Boudouresque et al., 1988; Reichardt and Schütz, 1996). Also, maternal glucocorticoids that reach the fetus are inactivated by placental enzyme 11␤-hydroxysteroid dehydrogenase type 2 (11␤HSD2) (cortisol in humans or corticosterone in rodents) that represents a protective shield from environmental disturbances during gestation (van Beek et al., 2004). But, if adverse conditions present during pregnancy are severe and long lasting, such as maternal undernutrition or stress exposure, fetal tissues are exposed to elevated glucocorticoid levels affecting the neurodevelopment of the fetus, particularly the HPA axis, in parallel with growth retardation (Fowden et al., 1998; Lesage et al., 2001; Reynolds 2013). The programming concept links alterations in the fetal maturational pathway caused by glucocorticoid excess during critical developmental stages with an increased incidence of later diseases including metabolic, cardiovascular and behavioral disorders (Barker 1994; Seckl and Meaney, 2004; Harris and Seckl, 2011; ´ 2013). Kundakovic, Maternal treatment with synthetic glucocorticoids (dexamethasone (Dx) or bethamethasone), which are poor substrates for 11␤HSD2, leads to increased glucocorticoid levels in the fetal circulation (Roberts and Dalziel, 2006). Synthetic glucocorticoids exert rapid maturational effects on the lungs, brain, liver and kidney in near-term fetuses and their application in high-risk pregnancies has thus become a widely used approach that decreases newborn mortality and has beneficial effects on complications such as the neonatal respiratory distress syndrome (Liggins and Howie, 1972; Roberts and Dalziel, 2006). In parallel, an increasing amount of evidence from animal and human studies shows that reduced fetal growth caused by glucocorticoid overexposure is followed by numerous adverse health consequences expressed in adulthood (Barker, 1994; Seckl and Meaney, 2004; Shoener et al., 2006). Moreover, developmental programming of the HPA axis function, as a consequence of excessive glucocorticoid exposure, might predispose the fetus to specific diseases such as hypertension, type II diabetes, obesity and emotional and cognitive impairments (Fowden et al., 1998; Kapoor et al., 2008; Waffarn and Davis, 2012; ´ 2013). Kundakovic, Identification of the structural and functional changes that occur at all levels of the fetal HPA axis under the influence of glucocorticoids during the fetal period enables better understanding of the causes underlying the programming phenomena. Thus, the aim of this study was to establish the influence of maternal Dx application during pregnancy on the levels of PVN morphometric parameters in 21-day-old fetuses. The PVN volume and total cell number were estimated using an unbiased stereological approach by combining a fractionator sampling method and an optical disector counting technique. In order to assess the HPA axis activity in near-term fetuses, intensity of the immunohistochemical appearance of CRH in the ME by optical density and relative intensity of ACTH fluorescent signal (RIF) in pituitary corticotrophs were determined. According to the literature data, female fetuses show higher susceptibility to programming effects and were thus selected as the subjects of this research (Matthews 2002; Tobe et al., 2005). The presented data show that structural changes in the fetal PVN, including significant reductions in PVN volume and total cell number after maternal Dx application, are associated with lower CRH content in the ME and reduced ACTH intracellular protein quantity in pituitary corticotrophs.

2. Material and methods 2.1. Animals Adult female Wistar rats, weighing 230 ± 10 g, bred in the laboratory of the Institute for Biological Research, Belgrade, were

housed with free access to food and water, at a controlled room temperature (22 ± 2 ◦ C), under a 12: 12 h light dark cycle (light on at 8.00 a.m., off at 8.00 p.m.). Vaginal smears were examined daily and only the females showing a regular 4-day estrus cycle were included in the experimental procedure. The morning after caging with a fertile male, the presence of spermatozoa in vaginal smears on the night of proestrus was indicative of pregnancy and this day was considered as day 0 of gestation. Dams were randomized into a control and an experimental group, each consisting of six animals. On days 16, 17 and 18 of pregnancy, experimental dams received 0.5 mg Dx (Dexamethasone phosphate – Krka, Novo Mesto, Slovenia, dissolved in 0.9% saline) per kg/b.w. subcutaneously. Control gravid females received the same volume of saline vehicle. On day 21 of pregnancy, fetuses in each group were obtained by Cesarean section and they are referred to as 21-day-old fetuses. All animal procedures complied with the EEC Directive (86/609/EEC) on the protection of animals used for experimental and other scientific purposes and were approved by the Ethical Committee for the Use of Laboratory Animals of the Institute for Biological Research ‘Siniˇsa ´ University of Belgrade. Stankovic’, 2.2. Tissue preparation, immunohistochemistry and immunofluorescence Selection of female fetuses from control and Dx-treated dams was based on ano-genital distance. Fetal brains and pituitary glands with sphenoid bone were removed and fixed in paraformaldehyde solution for 24 h. The fetal brains were dehydrated in 10–30% sucrose solution, cryoprotected and stored at −80 ◦ C. Serial coronal sections of fetal brain were cut on cryocat (Leica, CM1850, Wetzlar, Germany) set to 25 ␮m thickness and mounted on silica-coated glass slides (SuperFrost Plus, Prohosp, Denmark). Additionally, the fetal brains and pituitary glands were dehydrated in an increasing gradient of ethanol and xylene, embedded in Histowax (Histolab Product AB, Göteborg, Sweden), then serially sectioned at 3 ␮m thickness on a rotary microtome (RM 2125RT Leica, Wetzlar, Germany) after which the sections were placed on silica-coated glass slides (SuperFrost Plus, Prohosp, Denmark). Routine histological staining with hematoxylin/eosin as well as with cresyl violet was applied and selected sections of fetal brains were chosen in order to implement stereological measurements. Using the Atlas of Prenatal Brain Development as a guide (Altman and Bayer, 1995), the PVN was identified as a clearly delineated triangular cell group of the anterior hypothalamus located adjacent to the third ventricle. CRH immunoreactivity in the ME region of the hypothalamus was detected using the peroxidase–antiperoxidase (PAP) method. After hydration, sections were exposed to microwaves (700 W) in 0.05 M citrate buffer (pH 6.0) for 15 min for antigen unmasking. After cooling, the sections were rinsed in 0.01 M phosphatebuffered saline (PBS). Endogenous peroxidase activity was blocked by incubation with methanol containing 0.3% H2 O2 and the sections were preincubated in normal swine serum (dilution 1:10, Dako, Glostrup, Denmark). Primary rabbit anti-CRH antibody (dilution 1:500 in PBS; ab8901-100 Abcam) was applied overnight at 4 ◦ C. The secondary antibody, polyclonal swine anti-rabbit IgG/HRP (dilution 1:100 in PBS; Dako, Glostrup, Denmark) was applied for another 60 min, then rinsed again with PBS for 10 min. Antibody localization was visualized with 0.05% 3,3-diaminobenzidine tetrachloride (DAB) liquid substrate chromogen system (Dako, Glostrup, Denmark). Sections were thoroughly washed under running tap water, counterstained with hematoxylin and mounted on DPX. The pituitary ACTH cells were determined by immunofluorescence. Dewaxing, hydration and rinsing in 0.01 M PBS were followed by incubation in normal donkey serum (1:10 Dako, Glostrup, Denmark). Primary rabbit anti-rat ACTH antibody (1:500; AFP-156102789, NIDDK) was applied overnight at 4 ◦ C. After wash-

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ing in PBS, the sections were incubated with donkey anti-rabbit Alexa Fluor 488 IgG (1:300; Invitrogen) for 60 min, then washed in PBS and covered with Mowiol mounting medium (Mowiol 4-88, Aldrich Chemistry). 2.3. Stereological measurements All stereological analyses were carried out using a workstation comprising a microscope (Olympus, BX-51) equipped with a microcator (Heidenhain MT1201, Heidenhain, Traunreut, Germany) to control movement in the z – direction (0.2 ␮m accuracy), a motorized stage (Prior Scientific, Inc. Rockland, U.S.A.) for stepwise displacement in the x–y directions (1 ␮m accuracy) and a CCD video camera (PixeLink Ottawa, ON, Canada) connected to a 19” PC monitor (Dell). The whole system was controlled by newCAST stereological software package (VIS – Visiopharm Integrator System, version 2.12.1.0; Visiopharm; Denmark). The main objectives were planachromatic 10× dry lenses and a 100× oil lens. Control of the stage movements and the interactive test grids (uniformly spaced points test grids and a rectangular unbiased disector frame) were provided by newCAST software running on a Dell computer.

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while meander sampling was set to analyze 10% of the PVN. The X–Y step of meander sampling movements was 126.49 ␮m (step x,y 126.49 ␮m). The last level of sampling, the tissue sampling fraction (tsf) is the thickness of tissue at each sampling location where cells were counted and reflected as the ratio between the height (h) of the dissector, that was chosen to be 12 ␮m, and the actual section thickness, that was estimated to be 22 ␮m (t) (from the measurements of section thickness made at every tenth disector). Raw counts (Q) of PVN cell number were multiplied by the reciprocals of sampling fractions to estimate the total number of PVN cells. Raw counts (Q) ranged between 510 and 580, i.e. 385 and 437 for the PVN cells of 21-day-old fetuses from control and Dx-treated mothers, respectively. The average number of 33 and 23 disectors was used to count cells in the control and experimental group. The total number of cells was then calculated as: N=

n i=1

Q−

1 1 1 ssf asf tsf

tsf =

h t

where Q – total number of cells counted in all disectors; t – actual section thickness; h – disector height.

2.4. Estimation of PVN volume

2.6. Volume density

The PVN volumes were determined using Cavalieri’s principle (Gundersen and Jensen, 1987). The Cavalieri principle is an unbiased way of estimating the volume of an object, obtained by dividing it into a series of parallel planes with known distances between them. The total volume of an object is estimated by summing up the areas across all sections and multiplying the results by section thickness. In order to ensure random position for the first section, a random number table was used. From the first selected section, every 3rd section that contained PVN was analyzed to enable systematic uniform random sampling. The number of points falling within the PVN boundaries was counted bilaterally, separately for both sides of the ventricle. Mean section thickness was estimated by the block advance (BA) method (Dorph-Petersen et al., 2001) using a Heidenhain microcator attached to a microscope stage with 100× oil objective. True section thickness was found to be 22 ␮m, while nominal section thickness was set at 25.0 ␮m. The volume of PVN (Vpvn ) was then estimated as:

Volume density estimation was used to determine the percentage of tissue occupied by neuronal cell bodies and parenchyma that includes axonal and dendrite processes in the PVN. Four central paraffin sections with PVN were analyzed per animal and six animals were analyzed per group. The test points hitting the cell bodies (A) and parenchyma (B) were counted. Volume density (VV ) was calculated as the ratio of the number of points hitting each tissue component (Pp) divided by the number of points hitting the reference space (Pt) (delineated PVN):

Vpvn = a(p) · BA ·

n 

VV (%) = Pp/Pt × 100. Firstly, average values per animal were calculated and then volume density for each analyzed component was estimated per group. 2.7. The intensity of CRH immunoreactivity

Pi

i=1

where a(p) is the area associated with each sampling point (5377.31 ␮m2 ); BA is the block advance i.e. mean distance between two consecutively studied sections (66 ␮m); n is the number of sections studied for each PVN, and Pi is the sum of points hitting a given target. 2.5. Determination of the absolute number of PVN cells The optical disector counting technique was used in combination with a fractionator sampling design i.e. an optical fractionator was used to estimate total cell number in the PVN. Combination of these two stereological principles enables accurate and unbiased estimation of the absolute cell number. Since every third section from each tissue block was analyzed, the section sampling fraction (ssf) was 1/3, and 8 sections for control fetuses and 7 for experimental fetuses were analyzed on average. The next level of sampling, the area sampling fraction (asf), represents the area of the disector counting frame (40 × 40 ␮m) in relation to the area associated with each movement in the x–y direction, comprising 1/10 of the delineated PVN area where the cells were counted directly. An unbiased counting frame measuring 40 ␮m x 40 ␮m (1600 ␮m2 ) was used,

The intensity of CRH immunoreactivity in the ME was analyzed using sections from three different levels of the ME per animal (rostral, medial and caudal). Captured images with an image size of 1079 × 863 pixels were used (taken at 20× objective magnification). Analysis was performed using Windows based Image J program (Image J, Version 1.50f). First, the spectral deconvolution method of DAB/Hematoxylin color spectra was applied by using optimized optical density (OD) vectors of the color deconvolution plug-in for proper separation of DAB color spectra. To determine the OD for the RGB channel of Hematoxylin and DAB, we followed the protocol previously described by Varghese et al. (2014). Since the OD is proportional to the concentration of the stain, the amount of stain present will be the factor determining the OD at the wavelength specific to the stain, according to the formula: OD = −log10 (IC /I0.C ), where I is the transmitted light, IC represents the intensity of detected light after passing through a specimen and I0.C is the intensity of light entering the specimen. After calculating the average OD value at three different ME levels per animal, the OD was also averaged per group (control or experimental).

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Fig. 1. Representative micrographs of PVN in control (a) and Dx exposed fetuses (b) positioned in proximity of the third ventricle, at the level of optic chiasma, scale bar 160 ␮m. The volume of PVN and the total number of PVN cells; graphic C-control fetuses; Dx- Dx exposed fetuses; left (L) or right (R) PVN side (*p < 0.05 versus C).

2.8. Relative intensity of fluorescence (RIF) in the cytoplasm of pituitary ACTH cells of 21-day-old fetuses For the evaluation of ACTH intracellular protein (hormonal) content, measurements of the relative intensity of fluorescent signal (RIF) were performed with Image J, as described previously (Jensen, 2013). In order to ensure unbiased selection of corticotrophs, 100 cells with nuclei per animal were measured at three different pituitary levels, taking into account cells located in the central part as well as in the marginal regions of the adenohypophysis. The formula: RIF = Integrated Density – (Area of selected cell X Mean fluorescence of background readings) was used. The sections were examined and photographed using a Zeiss Axiovert fluorescence microscope, equipped with a camera and EC Plan-Apochromat. The RIF of ACTH cells was measured in 100 cells per animal, followed by calculating the average value per animal, and finally the average per group.

2.9. Statistical analysis All results are expressed as means for six animals per group ± SD. Data were tested for normality of distribution by the Kolmogorov–Smirnov test, whereas homogeneity of variances was evaluated by Levene’s test. Data related to the stereological parameters of the hypothalamus (the PVN volume, number of cells, and tissue percentage occupied by neuronal cell bodies and parenchyma) were analyzed by two-way analysis of variance (ANOVA), with treatment (control and Dx treatment) and hemi-

sphere (left and right PVN) as factors. To determine significant differences between groups, the post hoc Bonferroni test was used. Student’s t-test was used to compare mean values between control and experimental groups when the CRH OD in the ME and the RIF of pituitary ACTH cells were analyzed. A level of significance a = 0.05 was used for all statistical tests.

3. Results The body weight of 21-day-old female fetuses exposed to Dx (4.31 ± 0.19 g) was significantly decreased (p < 0.05) in comparison to control fetuses (4.82 ± 0.17 g).

3.1. Histological analysis The PVN was bilaterally traced in its entirety. The PVN was positioned in the proximity of the third ventricle of the anterior hypothalamus, occupying in both groups of examined fetuses a triangular space extending across the rostro-caudal axis of the anterior hypothalamus. No differences were observed in terms of magnocellular and parvicellular neurons or their further grouping into subdivisions. The euchromatic neuronal nuclei, which occupied the majority of cells’ bodies, were either spherical or ovoid in shape. Dendritic and axonal processes were projected from neuronal cell bodies to the surrounding parenchyma (Fig. 1).

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Fig. 2. Neuronal cell bodies and parenchyma in control (a) and Dx exposed fetuses (b), scale bar 150 ␮m; graphic C-control fetuses; Dx- Dx exposed fetuses; LA and RA percentage of PVN occupied by neuronal cell bodies on left (L) or right (R) PVN side; LB and RB percentage of PVN occupied by neuronal processes on left (L) or right (R) PVN side (*p < 0.05 versus C).

Fig. 3. CRH immunoreactivity in the median eminence in 21-day-old control fetuses (a) and fetuses from Dx treated mothers (b). 3V-third ventricle, scale bar 100 ␮m.

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Fig. 4. ACTH relative intensity of fluorescent signal (RIF) in pituitary corticotrophs of control fetuses (a) and fetuses from Dx treated mothers (b), scale bar 50 ␮m; graphic C-control fetuses; Dx- Dx exposed fetuses (*p < 0.05 versus C).

3.2. Stereological parameters of the PVN As shown by the two-way ANOVA, a significant main effect of treatment was found regarding PVN volume (F = 196.06, df = 1, p < 0.001) and cell number (F = 160.227, df = 1, p < 0.001), but no differences were seen between the left and right PVN considering PVN volume (F = 0.07, df = 1, p < 0.79) and number of PVN cells (F = 0.174, df = 1, p < 0.68). Subsequent post hoc analysis revealed that PVN volume was significantly decreased (p < 0.05) in the fetuses from Dx treated mothers compared to control group (Fig. 1). Following maternal Dx treatment, the absolute number of cells positioned in the PVN was significantly reduced (p < 0.05) in comparison to control values, namely by 24% (Fig. 1). No significant differences in the PVN volume and cell number were detected between the left and right side of PVN in control and Dx exposed fetuses (Fig. 1). There were no statistically significant differences in volume density of neuronal cell bodies and parenchyma i.e. axonal and dendritic projections in the PVN between control and experimental fetuses as well as left and right hemisphere (Fig. 2).

3.3. Intensity of CRH and ACTH immunohistochemical staining The ME is a structure at the base of the hypothalamus where hypothalamic releasing and inhibiting hormones converge onto the portal capillary system that vascularizes the anterior pituitary gland. CRH immunoreactivity in the ME was found in both experimental groups, along the entire structure, with lower CRH immunoreactivity presence in the medial part of the ME compared to the lateral (Fig. 3). Also, lower CRH immunoreactive intensity was noted in the ME of Dx exposed fetuses compared with control (Fig. 3). This qualitative histological result was confirmed by measurement of OD values, namely OD was significantly decreased in experimental group (p < 0.05) comparing to the control (Fig. 3).

Image J analysis showed lower (p < 0.05) ACTH fluorescence intensity in the pituitary corticotrophs of Dx exposed fetuses (41%) in comparison to control fetuses (Fig. 4).

4. Discussion The presented results show that fetal glucocorticoid exposure during E 16, 17 and 18 causes a reduction in the PVN volume and total number of PVN cells in 21-day-old fetuses. This is associated with reductions in the OD values of CRH immunohistochemical staining in the ME and ACTH immunofluorescence in pituitary corticotrophs, i.e. CRH quantity in the ME and the ACTH intracellular protein content were diminished in near-term fetuses exposed to Dx. The timing of maternal Dx application, between E 16 and 18, coincides with the period of brain development when progressive enlargement of the brain parenchyma takes place (Altman and Bayer, 1995). This is also the time specific window for Dx action, as the onset of glucocorticoid receptor (GR) gene expression in the PVN neurons was recorded on embryonic day 16, followed by robust GR mRNA increase verified on E 18 (Yi et al., 1994). Furthermore, the GR has been suggested to have a crucial role in CRH neuronal differentiation (Yi et al., 1994), as GR mRNA was present prior to CRH mRNA, first detected on E 17 (Grino et al., 1989). In 21-day-old fetuses, significant reduction in body weight after exposure to Dx is a consequence of the potent influence of glucocorticoids on tissue maturation and is in accordance with the results presented in numerous reports that have investigated the glucocorticoid influence during development (Dupouy et al., 1987; Bloom ´ et al., 2001; Lesage et al., 2001; Haris and Seckl, 2011; ManojlovicStojanoski et al., 2012). After the GR becomes expressed in the tissue derivatives of all three germ layers during the early steps of fetal development (Kitraki et al., 1997), glucocorticoids act to promote differential processes by inhibiting cell division (Diaz et al.,

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1998). By activating the expression of specific genes in nervous tissue, lung, liver and adipose tissue, glucocorticoids enable the establishment of the definitive structure and function of tissues (Trejo et al., 1995; Fowden et al., 1998). Additionally, the rate of programmed cell death is enhanced under glucocorticoid influence (Fujioka et al., 1999; Tobe et al., 2005). Application of synthetic glucocorticoids during the last third of fetal rat development redirects the developmental trajectory in order to enhance chances of survival after birth, with decreased body weight as the consequence (Haris and Seckl, 2011). Although the PVN can be identified unequivocally on E 17, adult compartmentalization of PVN to magnocellular and parvicellular subdivisions and to further areas was difficult to distinguish in fetuses, which is in accordance with the results of others (Grino et al., 1989; Markakis, 2002). Mature glia in the hypothalamus develops considerably later than neurons. The glial markers, glial fibrillary acidic protein (GFAP) and galactosylceramidase (GalC), revealed relatively few marked cells in the PVN before postnatal day 8, followed by an increase in their number during the subsequent period (Chang et al., 2009), demonstrating that Dx exposure in utero has the greatest impact on the development of neurons. Detailed quantitative information on the structural parameters of neural tissue, including volume and number, can be obtained using unbiased sampling strategies such as systematic-random sampling in combination with simple stereological probes, without any assumptions about the geometrical properties of structures (Dockery, 2014). The volume of PVN in 21-day-old fetuses was significantly decreased after maternal Dx application due to the decreased number of cells, as the results show, and retarded dendritic and axonal growth within this structure. Reduction in total length of the processes of Golgi-impregnated neurons in the fetal PVN was demonstrated under the influence of increased fetal exposure to glucocorticoids (Fujioka et al., 1999). In fact, the influence of glucocorticoid overexposure during development results in reduced brain weight throughout the perinatal period, particularly of the hippocampus in terms of its volume and neurogenesis (Sousa et al., 1998; Whitelaw and Thoresen, 2000). The results show that the ratio between neural cell bodies and the parenchyma made of axons and dendrites within the PVN did not differ between control and experimental fetuses, indicating that maternal Dx treatment affected both components to the same extent. To our knowledge, no reports so far have obtained the absolute number of cells in the fetal PVN. In fetuses, cells were counted in the defined 2D area, on several representative sections (Tobe et al., 2005) that failed to take into account spatial differences in cell positions across the whole PVN volume and do not represent the exact cell number. The optical disector counting technique in combination with the fractionator sampling method showed decreased numbers of PVN cells in 21-day-old fetuses exposed to Dx, as reported, most likely a consequence of apoptotic cell death. Literature data show an increased number of apoptotic cells in 18-day-old fetal PVN caused by the excessive amount of glucocorticoids entering fetal blood circulation as a consequence of severe maternal stress (Fujioka et al., 1999). Also, due to repeated maternal stress, reduction in the total number of PVN cells caused by increased apoptotic cell death is reported in 21-day-old fetuses, suggesting great vulnerability of PVN neurons to excessive glucocorticoid exposure in utero (Tobe et al., 2005). Morphological modification of fetal PVN neurons, shorter, less complex processes and decreased arborization represent other potential neurotoxic actions of glucocorticoids during their differentiation (Fujioka et al., 1999). It has been shown that Dx induces a significant increase in the incidence of apoptotic cells in mice fetal hippocampus as well as in the rat dentate gyrus during their life cycle (Hassan et al., 1996; Noorlander et al., 2014). While basal levels of glucocorticoids are essential for neuronal development, their plasticity and

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survival (Trejo et al., 1995), it is assumed that the dosing and timing of applied Dx lead to neuronal loss. Although the majority of neuroendocrine neurons are generated between E 12 and 14, very few non-neuroendocrine neurons become post-mitotic during the subsequent days (Markakis and Swanson 1997; Markakis, 2002) suggesting that the antiproliferative Dx impact is not dominant. Thus, it is indicated that reduced neuronal survival rather than inhibition of cell proliferation is responsible for the decreased PVN cell number under Dx influence. As mentioned above, the presence of GR within CRH neurons exposes these cells to Dx that acts by inhibiting CRH synthesis (Sawchenko, 1987; Dupouy et al., 1987; McCabe et al., 2001) and decreases CRH amount in the ME of 21-day-old fetuses as presented in our results based on OD values. Decreased level of CRH mRNA was also demonstrated in newborn rats overexposed to maternal glucocorticoids due to maternal undernutrition during late pregnancy (Lesage et al., 2001). The reduced number of CRH parvicellular neurons and their axonal projections to the ME, among other PVN cells, additionally leads to the weakening of CRH staining intensity, as recorded in the literature (Bakker et al., 1995). The presented immunohistochemical results indicate that the quantity of released CRH that reaches adenohypophyseal cells via the hypophyseal portal system is reduced. This speculation is supported by the decreased ACTH fluorescence intensity in pituitary corticotrophs which means that the amount of ACTH in individual cells is decreased. The reduction in positive CRH drive not only affects the synthetic activity of corticotrophs of 21-day-old Dx exposed fetuses as presented here, but also leads to reduction in individual ACTH cell volume and number in near-term fetuses, ´ as established in our earlier reports (Manojlovic-Stojanoski et al., 2006, 2012). Additionally, CRH in synergy with vasopressin and other PVN neuropeptides promotes the maturation and functioning of fetal corticotrophs, regulating CRH type 1 receptor expression (Myers et al., 1999). The decreased amount of neuropeptides mentioned above caused by the reduced total PVN cell number also may negatively affect ACTH protein content in the corticotrophs of nearterm Dx exposed fetuses. The presented result is in accordance with Dupouy and coworkers (1987) who demonstrated a significant reduction in pituitary ACTH content and drastic decrease in plasma ACTH levels in 21-day-old fetuses from mothers given Dx. Also, a direct Dx influence on the corticotrophs where GR is expressed cannot be excluded (Shoener et al., 2006). 5. Conclusion In summary, the presented data suggest that the decreased PVN volume in 21-day-old fetuses that is a result of the reduced number of cells in the PVN after maternal Dx administration negatively affects the CRH content in the ME and the ACTH quantity in pituitary corticotrophs. Changes in the stereological characteristics of fetal PVN reported in this study imply the existence of a structural basis that at least in part underlies the perinatal decrease of HPA axis activity following maternal Dx exposure described by others (Dupouy et al., 1987; Lesage et al., 2001; Waffarn and Davis, 2012), with implications on the dysregulation of HPA axis during the lifespan (Matthews, 2002; Kapoor et al., 2006; Shoener et al., 2006). Thereby, glucocorticoids represent signals that transfer internal and external environmental influences, programming the developmental trajectory and postnatal health outcome. Funding This work was supported by the Ministry of Education and Science of Serbia, Grant number 173009. The Ministry’s involvement was only of financial nature.

´ Please cite this article in press as: Manojlovic-Stojanoski, M., et al., Dexamethasone exposure affects paraventricular nucleus and pituitary corticotrophs in female rat fetuses: An unbiased stereological and immunohistochemical study. Tissue Cell (2016), http://dx.doi.org/10.1016/j.tice.2016.06.012

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ARTICLE IN PRESS M. Manojlovi´c-Stojanoski et al. / Tissue and Cell xxx (2016) xxx–xxx

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´ Please cite this article in press as: Manojlovic-Stojanoski, M., et al., Dexamethasone exposure affects paraventricular nucleus and pituitary corticotrophs in female rat fetuses: An unbiased stereological and immunohistochemical study. Tissue Cell (2016), http://dx.doi.org/10.1016/j.tice.2016.06.012