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Exposure to dexamethasone reduces pituitary volume and gonadotropic cell number in rat fetuses
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Exposure to dexamethasone reduces pituitary volume and gonadotropic cell number in rat fetuses ´ Milica Manojlovic-Stojanoski, ´ ´ Nataˇsa Ristic´ ∗ , Nataˇsa Nestorovic, Ivana Medigovic, ˇ sic-Jurjevi ´ Branka Soˇ ´ ´ Verica Miloˇsevic´ Svetlana Trifunovic, c, University of Belgrade, Institute for Biological Research “Siniˇsa Stankovi´c”, Department of Cytology, Belgrade, Serbia
a r t i c l e
i n f o
Article history: Received 14 February 2014 Received in revised form 13 March 2014 Accepted 1 April 2014 Available online xxx Keywords: Pituitary Gonadotropic cells Rat fetuses Physical disector
a b s t r a c t Overexposure to glucocorticoids during the fetal period induces changes in developmental processes in various fetal tissues. The aim of this study was to investigate the effects of the synthetic glucocorticoid, dexamethasone (Dx), on pituitary volume and gonadotropic cells during a critical period of pituitary development. The effects of Dx on stereological parameters of the pituitary gland and FSH and LH cells were investigated in 19 and 21-day old fetuses. On day 16 of pregnancy, the experimental dams received 1.0 mg Dx/kg b.w. subcutaneously, followed by 0.5 mg Dx/kg b.w./day on days 17 and 18 of gestation. The control gravid females received the same volume of saline. FSH and LH cells were stained immunohistochemically by the peroxidase–antiperoxidase method (PAP). In 19-day old fetuses, exposure to Dx caused a significant decrease of pituitary volume, estimated by Cavalieri’s principle. Also, the total number of FSH and LH cells per pituitary, determined by physical fractionator counting technique, was significantly reduced. These changes persisted until fetal day 21. Volume densities and numerical densities of FSH and LH cells after exposure to Dx in 19 and 21-day old fetuses remained unaffected. Our results suggest that altered stereological parameters in pituitary gland after exposure to dexamethasone in fetal period could be long-lasting. © 2014 Elsevier GmbH. All rights reserved.
Introduction Glucocorticoids play a crucial role in the final maturation of fetal organ systems, acting on the tissue morphology and function in preparation for extrauterine life and promoting nervous, respiratory, cardiovascular and immune system differentiation and biochemical maturation (Bakker et al., 1997; Flagel et al., 2002; Arima et al., 2008). Transplacental glucocorticoid diffusion is normally limited by placental 11-hydroxysteroid dehydrogenase type 2 (11-HSD2), an enzyme that forms the placental glucocorticoid barrier (Yang, 1997). This enzyme plays a key role in regulating glucocorticoid concentrations in the fetal circulation by inactivating maternal glucocorticoids at the level of the placenta and protects fetuses from glucocorticoid overexposure. During fetal development, glucocorticoid overexposure retards fetal growth and may program postnatal development in glucocorticoid sensitive physiological systems in early life (glucocorticoid
∗ Corresponding author at: Institute for Biological Research “Siniˇsa Stankovic”, ´ Department of Cytology, 142 despota Stefana Blvd., 11060 Belgrade, Serbia. ´ E-mail address: [email protected] (N. Ristic).
programming) (Seckl, 2001). Some of the most commonly used experimental models in the study of programming are maternal undernutrition, maternal stress, maternal treatment with synthetic glucocorticoids and inhibition of placental 11-HSD2. The majority of these experimental models ultimately result in fetal glucocorticoid overexposure, with intrauterine growth retardation (IUGR) and programming of endocrine axes with long-term effects as consequences (Manojlovic-Stojanoski et al., 2012). Antenatal dexamethasone (Dx) therapy is often used in obstetric practice when risks of preterm delivery persist, as a highly effective approach for reducing the frequency of respiratory complications and perinatal death (Roberts and Dalziel, 2006). Dexamethasone as synthetic glucocorticoid passes through the placenta and promotes differentiation and maturation of fetal tissues (Seckl, 2004), but the effects on pituitary growth and gonadotropic cells during the critical period of pituitary development are largely unknown. In the developing mammalian anterior pituitary gland, the gonadotropic cells follicle stimulating (FSH) and luteinizing (LH) are the last hormone-producing cell types to reach maturation. Both types of gonadotropes express a common ␣-glycoprotein subunit (␣-GSU) which combines with the hormone-specific  subunits, FSH and LH, to form biologically active dimeric FSH and LH
http://dx.doi.org/10.1016/j.acthis.2014.04.003 0065-1281/© 2014 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Ristic´ N, et al. Exposure to dexamethasone reduces pituitary volume and gonadotropic cell number in rat fetuses. Acta Histochemica (2014), http://dx.doi.org/10.1016/j.acthis.2014.04.003
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glycoprotein hormones. However, Wen et al. (2010) revealed a striking dichotomy in the gonadotrope population of the developing pituitary. At fetal day 16.75 in mice only LH-expressing gonadotropes, express the gonadotropin-releasing hormone receptor (GnRHR) and at this stage, the fetal pituitary gland becomes GnRH responsive. Also, embryonic increase in LH secretion is needed to promote proper development of FSH-expressing gonadotropes, which might be mediated through paracrine interactions within the fetal pituitary gland. From that moment intrapituitary LH and FSH levels rise dramatically until the end of gestation. These observations suggest that the hypothalamicpituitary-gonadal axis might already be functional during fetal development. Exposure to a compound that affects pituitary cell proliferation and differentiation may be critical for pituitary development and function. The aim of this study was to investigate the effects of exposure to tapering doses of Dx on the pituitary volume and gonadotropic cells during the critical period of pituitary development. Exposure to tapering doses, i.e., reduction of Dx doses minimize adverse effects of glucocorticoid administration, such as damage during neuronal development, and suppression of the HPA axis in fetuses and newborn infants (Flagel et al., 2002; Nahaczewski et al., 2004). The best way to identify the changes caused by Dx treatment was to determine the volume of the pituitary gland and total number of FSH and LH cells per pituitary gland in 19 and 21-day old fetuses after exposure to Dx using unbiased stereological methods. Material and methods
these antisera were assessed by the NIDDK. Sections were incubated in primary antibodies for 45 min at room temperature. After washing in phosphate-buffered saline (PBS; pH 7.4) sections were incubated for 1 h with polyclonal swine anti-rabbit immunoglobulins conjugated with horseradish peroxidase (IgG/HRP, Dako A/S, Glostrup, Denmark). The antigen-antibody complex was visualized by incubating the sections with a chromogen substrate, 0.05% 3,3diaminobenzidine (DAB; Dako A/S, Glostrup, Denmark) and 0.03% H2 O2 . The incubated sections were counterstained with hematoxylin. Control sections were incubated with PBS without primary antisera. Stereological measurements All stereological analyses were carried out using a workstation comprising a microscope (Olympus, BX-51) equipped with a microcator (Heidenhain MT1201) to control movements in the z-direction (0.2-m accuracy), a motorized stage (Prior) for stepwise displacement in the x–y directions (1-m accuracy), and a CCD video camera (PixeLink) connected to a 19 PC monitor (Dell). The whole system was controlled by the new-CAST stereological software package (VIS – Visiopharm Integrator System, version 3.2.7.0; Visiopharm, Horsholm, Denmark). The objectives used 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 rectangular unbiased disector frames) were provided by new-CAST software running on a Dell computer. Pituitary volume estimation (Vpt – mm3 )
Animals Female and male Wistar strain rats, weighing approximately 250 g and 400 g respectively, were mated in the vivarium of the Institute for Biological Research, Belgrade, at night. The mornings in which sperm-positive vaginal smears were obtained were declared gestation day 1. Pregnant females were housed individually, under standard conditions (12:12 h light-dark cycle at 22 ± 2 ◦ C) and offered food and water ad libitum. Dams were randomized into two groups: control and experimental group, each consisting of six animals. On day 16 of pregnancy, experimental dams received subcutaneously 1.0 mg Dx (Dexamethasone phosphate, Krka FARMA d.o.o. Belgrade, Serbia, dissolved in 0.9% saline)/kg b.w., followed by 0.5 mg Dx/kg b.w./day on day 17 and 18 of gestation. The control gravid females received the same volume of saline. Female fetuses from control and experimental dams were sacrificed under ether narcosis on day 19 and 21 of gestation and they are referred to as 19 and 21-day old fetuses. The experimental protocols were approved by the Local Animal Care Committee in conformity with the recommendations provided in the European Convention for Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (ETS no. 123, Appendix A). Tissue preparation The pituitary glands were excised with part of the sphenoid bone, fixed in Bouin’s solution for 48 h and processed for embedding in Histowax (Histolab Product AB, Göteborg, Sweden). Serial 3 m thick tissue sections were deparaffinized in xylene and rehydrated through a decreasing series of ethanol. Gonadotropes were localized immunohistochemically using the peroxidaseantiperoxidase method. Polyclonal rabbit anti-rat FSH (1:300, v/v) and polyclonal rabbit anti-rat LH (1:500, v/v) served as primary antibodies (National Hormone Peptide Program (NHPP), Harbor-UCLA Medical Center, Carson, CA, USA). The specificities of
Pituitary volumes were estimated 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 a known distance apart. The total volume of the object was estimated by summing the areas over all sections and multiplying the results by the section thickness. To ensure a random position for the first section from each tissue block, a random number table was used. As every 10th section from each tissue block was analyzed (the same sections were used in the subsequent estimation of FSH/LH cell numbers by the physical disector method), a number between 1 and 10 was obtained from a random number table for sampling the first section. On the monitor, a final magnification of 300× allowed easy and accurate recognition of tissue boundaries. Mean section thickness was estimated using the block advance (BA) method (Dorph-Petersen et al., 2001), and we found that there was no variation from 3.0 m, as set in the microtome, i.e., there was no difference between nominal and true section thickness. The volume of the pituitary gland (Vpt ) was estimated as the sum of the total volume of immunohistochemically labeled FSH/LH cells (VFSH/LH ) and the volume of uncolored phase of adenohypophysis (Vua ), which gives the volume of the entire adenohypophysis (Vahy ), and volume of neurohypophysis and pars intermedia (Vni ): Vpt = Vua + VFSH/LH + Vni The volume of individual phases of the pituitary gland was calculated using the following formula: V¯ = a(p) · BA ·
n
Pi
i=1
where a(p) is the area associated with each sampling point; BA (the block advance) is the mean distance between two consecutively studied sections (real section thickness 3 m × 10); n is the number
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Fig. 1. Unbiased counting frame on reference and look-up sections. In this position, only cell marked A was counted, because its nucleus appeared within the counting frame of the reference section, but did not appear in that of the look-up section. Also this cell was intersected with acceptance (green) lines of frame. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
of sections studied for each pituitary; and hitting a given target.
Pi is the sum of points
Volume densitiy of FSH/LH cells (VV – %) The percentage of FSH/LH cells per unit volume of adenohypophysis i.e. the volume density is equivalent to the total volume occupied by FSH/LH cells divided by volume of adenohypophysis and multiplied by 100.
VV =
VFSH/LH Vahy
× 100
Quantification of FSH/LH cell number (No) A fractionator/physical disector design with two levels of sampling was used to estimate the number of FSH/LH cells in 19 and 21-day old control and fetuses exposed to Dx. Sampling was systematically uniform from a random start (Dorph-Petersen et al., 2001; Gundersen and Jensen, 1987). For the first level of sampling, every 10th and 11th section was at random selected all along the serial sections. The random number for each block being analyzed was chosen by using the random number table. Sections designated as section pairs were first captured into a super-image. After defining and linking, the pairs were aligned by translation and rotation using the montage option in the new-CAST software. For the cell counting, a 100× oil-immersion objective was
used. One section in the pair was designated the reference section and the other the “look-up” section. Subsequently, the analysis was performed in both directions with the reference section also becoming the look-up section. This doubled the first sampling fraction from 1/10th to 1/5th [sampling fraction 1(f1) = 1/5 = 0.2]. For estimation of FSH and LH cell number, an unbiased counting frame measuring 40 m × 40 m (1600 m2 ) was used to ensure 150–200 cells per animal. After defining tissue boundaries, meander sampling was set to analyze 100% of the tissue. Therefore, sampling fraction 2 for FSH/LH cell number was f 2 = 1. The fields of vision were randomly selected and the percentage of tissue analyzed was controlled by the software. Additionally new-CAST software facilitated matching of fields between the reference and the look-up section. When the field of vision was selected at the reference section, the matching position at the consecutive look up section was scanned and a matching field of vision was generated from four images. FSH/LH cells were counted if their nuclei appeared within the unbiased counting frame applied to the reference section, they were not intersected by exclusion boundaries (Gundersen, 1986) and did not appear in the look-up section (Fig. 1). Raw counts (Q− ) of FSH/LH cell number were multiplied by the reciprocals of the sampling fractions to estimate the total number of FSH and LH cells per pituitary. Q− ranged between 171 and 177 for FSH, and 214 and 245 for LH in control 19-day old fetuses. In 19-day old fetuses exposed to Dx Q− ranged between 100 and 124 for FSH, and 104 and 115 for LH. In 21-day old control fetuses Q− ranged between 312 and 348 for FSH, and 322 and 364 for LH. In 21-day old fetuses exposed to Dx Q− ranged between 217 and 292 for FSH cells and 259 and 294 for LH. The
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Table 1 Body weight, volume of pituitary gland and volume of adenohypophysis in 19 and 21-day old control fetuses (C19 and C21) and fetuses exposed to Dx (Dx19 and Dx21).
Body weight (g) Volume of pituitary gland (mm3 ) Volume of adenohypophysis (mm3 )
C19
Dx19
C21
Dx21
1.4 ± 0.07 0.07 ± 0.01 0.06 ± 0.007
1.1 ± 0.2b 0.04 ± 0.004b 0.04 ± 0.008b
4.4 ± 0.7a 0.15 ± 0.02a 0.1 ± 0.01a
3.1 ± 0.2c , d 0.09 ± 0.003c , d 0.07 ± 0.0006c , d
All results are given as mean ± SD, n = 6, p < 0.05. a C21 vs C19. b Dx19 vs C19. c Dx21 vs C21. d Dx21 vs Dx19.
number of FSH/LH cells = Q− (FSH/LH cells) × (1/f1) × (1/f2) = Q¯ (FSH/LH cells) × 1/0.2 × 1/1. Mean FSH/LH cell volume (Vc – m3 ) The mean volume of a single FSH/LH cell is equivalent to the total volume occupied by FSH/LH cells divided by their number (de Lima et al., 2007). Vc =
VFSH/LH No
groups. They were polygonal, oval or polyhedral in shape, with large, prominent often eccentrically located nuclei and a thin layer of surrounding cytoplasm. Gonadotropes were single or in groups, often in close contact with blood capillaries. FSH and LH cells were more numerous in the adenohypophysis of 21-day old than in 19day old fetuses (Figs. 2 and 3). After exposure to dexamethasone FSH and LH cells were less numerous, smaller in size and irregularly shaped in 19-day old fetuses. A striking reduction in the number of gonadotropes was retained to fetal day 21 (Figs. 2 and 3).
Numerical densities of FSH/LH cells (NV )
Stereological analysis
Numerical density depicts the number of FSH and LH cells per adenohypophysis volume unit, and was calculated as the ratio of the absolute number of FSH and LH cells and the volume of adenohypophysis. Numerical density has an exponent of −3.
Stereological analysis showed that the volume of the pituitary gland and the volume of adenohypophysis increased from fetal day 19–21 by 2 (p < 0.05) and 1.7 fold (p < 0.05), respectively (Table 1). Increased volume of pituitary gland and adenohypophysis was followed by increase in the number of FSH and LH cells by 2.2 fold (p < 0.05) and 1.7 fold (p < 0.05), respectively (Fig. 4a). At the same time the volume of FSH and LH cells was significantly decreased by 38% (p < 0.05) and 34% (p < 0.05), respectively (Fig. 4b). Volume density and numerical density remained unchanged (Fig. 4c and d). Exposure to dexamethasone caused a decrease in the volume of the pituitary gland and the volume of the adenohypophysis by 43% (p < 0.05) and 33% (p < 0.05), respectively in 19-day old fetuses. Reduction of these parameters was also detected in 21-day old fetuses, by 40% (p < 0.05) and 30% (p < 0.05), respectively in comparison with the control groups. In Dx exposed fetuses the volume of pituitary gland and the volume of adenohypophysis increased from fetal day 19–21 by 2.3 (p < 0.05) and 1.8 fold (p < 0.05), respectively (Table 1). The volume of the pituitary gland correlated to the body weight was significantly decreased in Dx exposed fetuses when compared with the control values by 27% (p < 0.05) and 24% (p < 0.05), respectively in 19 and 21-day old fetuses (Table 2). Exposure to Dx during the last week of pregnancy led to a 37% (p < 0.05) decrease in the number of FSH cells, and 51% (p < 0.05) decrease in the number of LH cells in 19-day old fetuses in comparison to the control group. A reduced number of gonadotropes was still present in 21-day old fetuses, where the number of FSH and LH cells was decreased by 30% (p < 0.05) and 31% (p < 0.05), respectively in comparison to controls (Fig. 4a). In Dx exposed fetuses number of FSH and LH cells increased from fetal day 19–21 by 2.5 fold (p < 0.05). The number of FSH and LH cells was correlated to the pituitary volume and body weight. When expressed as number per pituitary volume unit, no changes were observed between control and Dx exposed fetuses at both time points. But, when expressed as number per body weight unit, the number of FSH and LH cells was significantly decreased by 26% (p < 0.05) and 41% (p < 0.05), respectively, in 19-day old fetuses, and by 9% (p < 0.05) and 9.2% (p < 0.05), respectively, in 21-day old fetuses exposed to Dx when compared with control values (Table 3).
NV =
No Vahy
Statistical analysis All results are expressed as mean values for six animals per group ± SD. Data were tested for normality of distribution by the Kolgomorov–Smirnov test. Data were analyzed by 2-way analysis of variance (ANOVA), with the age of fetus (19 and 21 day) and applied treatment (C and Dx) as factors. To determine significant differences between the groups, the post hoc Bonferroni test was used. A probability value of 5% or less was considered statistically significant.
Results Body weight Body weight of rat fetuses in the control group physiologically increased from fetal day 19 to 21 by 3 fold (p < 0.05). Exposure to Dx during the last week of fetal development induced a significant decrease in body weight in 19 and 21-day old fetuses by 21% (p < 0.05) and 30% (p < 0.05), respectively. In Dx exposed fetuses body weight increased from fetal day 19–21 by 2.8 fold (p < 0.05) (Table 1). Histological analysis In 19-day old fetuses the pituitary gland already had definite histological organization. FSH and LH cells were strongly immunohistochemically stained in the pars distalis in fetuses of both age
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Fig. 2. Immunohistochemically labeled FSH and LH cells in the pituitary pars distalis of 19-day old control fetuses (C FSH and C LH) and fetuses exposed to Dx (Dx FSH and Dx LH); * blood vessels; scale bar = 10 m.
Fig. 3. Immunohistochemically labeled FSH and LH cells in the pituitary pars distalis of 21-day old control fetuses (C FSH and C LH) and fetuses exposed to Dx (Dx FSH and Dx LH); scale bar = 10 m.
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Fig. 4. Stereological parameters of FSH and LH cells in 19 and 21-day old control and fetuses exposed to Dx; (a) Number of FSH/LH cells expressed per pituitary, (b) volume of FSH/LH cells, (c) volume densities of FSH/LH cells, (d) numerical densities of FSH/LH cells. Results are given as mean ± SD, n = 6, p < 0.05, a: C21 vs C19, b: Dx vs corresponding C, c: Dx21 vs Dx19. Table 2 Volume of pituitary gland expressed per body weight unit in 19 and 21-day old control fetuses (C19 and C21) and fetuses exposed to Dx (Dx19 and Dx21). Volume of pituitary gland per body weight unit (mm3 /g) C19
Dx19
C21
Dx21
0.052 ± 0.009
0.038 ± 0.002*
0.037 ± 0.0069
0.028 ± 0.002*
All results are given as mean ± SD, n = 6. * p < 0.05.
After exposure to Dx the volume of FSH and LH cells was decreased compared with the control values by 16% (p < 0.05) and 39% (p < 0.05), respectively in 19-day old fetuses, but no changes were observed in the volume of gonadotropes in 21-day old fetuses (Fig. 4b). Volume densities of FSH and LH cells in fetuses of both age groups did not change significantly after exposure to Dx (Fig. 4c). When the number of FSH and LH cells per adenohypophysis volume unit was calculated, there were no statistically significant differences between 19 and 21-day old fetuses from control and experimental group (Fig. 4d). Discussion Our results demonstrate that exposure to Dx during the last third of pregnancy has an inhibitory effect on the volume of the pituitary gland, number and volume of FSH and LH cells in 19-day old fetal rats. Reduced volume of pituitary gland and number of FSH
and LH cells are still present in 21-day old fetuses, but the volume of gonadotropic cells was normalized. In our experimental conditions rat fetuses were exposed to Dx in a period of intensive proliferation of precursor and differentiated pituitary cells, i.e. from fetal day 16–18 (Taniguchi et al., 2002). Rapid pituitary growth during the last third of pregnancy is interrupted in Dx exposed fetuses, and the volume of fetal pituitary gland was significantly lower in 19 and 21-day old fetuses. Reduction of pituitary volume probably is the result of reducing the number of all types of pituitary cells, including ACTH, TSH (Manojlovic-Stojanoski et al., 2006, 2008, 2010) and as shown in this study, FSH and LH cells. Dx may act indirectly on the pituitary cell number by diminution of the stimulatory hypothalamic contribution to pituitary cell synthetic and proliferative activity. In the rat, GnRH neurons develop and migrate during the fetal period (Daikoku and Koide, 1998). Dx exposure at this time induces neuronal apoptosis and reduction of the number of GnRH neurons accompanied by reduced synthesis of GnRH (Reagan and McEwen, 1997). Also, Dx application caused inhibition of thyrotropin-releasing hormone (TRH) (Alkemade et al., 2005) and corticotropin-releasing hormone (CRH) synthesis and release (Dupouy et al., 1987). By direct action, expressing strong antiproliferative and proapoptotic effect (Nolan et al., 1998, 2004; Nolan and Levy, 2006), Dx caused reduction of pituitary cell number, including a gonadotropic cell, which also contributes to reducing the volume of the entire gland. During development of the pituitary gland, precursors of gonadotropic cells intensively divide and differentiate
Table 3 The number of FSH and LH cells (No) expressed per pituitary volume unit and per body weight unit in 19 and 21-day old control fetuses (C19 and C21) and fetuses exposed to Dx (Dx19 and Dx21). No per pituitary volume unit (No/mm3 )
FSH LH
No per body weight unit (No/g)
C19
Dx19
C21
Dx21
C19
Dx19
12,308 ± 1659 16,114 ± 3755
12,633 ± 134 12,729 ± 955
13,341 ± 2428 13,675 ± 2463
14,720 ± 101 14,825 ± 1242
630 ± 23 821 ± 31
464 ± 44 486 ± 48* *
C21
Dx21
482 ± 23 490 ± 34
439 ± 25* 445 ± 19*
All results are given as mean ± SD, n = 6. * p < 0.05.
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contributing to the increase of FSH and LH cell number. Pope et al. (2006) suggested that as soon as FSH or LH genes are switched on, the gonadotropes becomes terminally differentiated in human fetuses. This observation is in agreement with reports demonstrating that in rodent gonadotropes, the most intense proliferation occurs before the terminal differentiation (Khar et al., 1978; Pawlikowski et al., 1978; Levy, 2002). Therefore, the number of gonadotropes in the pituitary is mostly set during fetal life, and there is little replacement during the rest of life. In the postnatal period mitoses of gonadotropes are infrequent, but can become active in some physiological and experimental conditions (Taniguchi et al., 2002). The decreased number of FSH and LH cells in 19 and 21-day old fetuses indicate that Dx inhibits proliferation of gonadotrope precursors. Despite accelerated pituitary growth and a larger increase in gonadotropic cell number from fetal day 19–21 in Dx exposed fetuses, control values are not reached. Considering the inhibitory effect of Dx on pituitary volume and FSH and LH cell number, as well as unchanged FSH and LH cell volume in 21-day old fetuses, it can be concluded that these results could represent a maturational effect of glucocorticoids. Glucocorticoids promote maturation in a wide range of fetal tissues including lungs, liver, kidneys, muscle, fat and gut, by switching cell cycle from proliferation to differentiation (Fowden et al., 1998). Unchanged volume density and numerical density of fetal gonadotropes results from a proportional decrease of the total volume occupied by FSH and LH cells, as well as their absolute number, in relation to volume of the adenohypophysis after exposure to Dx. Also, when the number of FSH and LH cells was correlated to the pituitary volume, no changes were observed between control and Dx exposed fetuses at both time points. These results indicate that the structure of the fetal pituitary gland after Dx exposure remained unchanged. The present study shows that exposure to Dx reduces fetal growth, resulting in a decrease of body weight of 19 and 21-day old fetuses, in accord with previous observations (Seckl, 1997; Fowden et al., 1998; Smith and Waddell, 2003; Manojlovic-Stojanoski et al., 2010). IUGR is associated with programming of endocrine axis, with long-lasting consequences. Reduction of the pituitary volume and gonadotropic cell number in our experimental conditions may be part of the programming process of the gonadotropic axis. The volume of the pituitary gland and number of gonadotropes expressed per body weight unit was significantly decreased in Dx exposed fetuses. This indicates that the changes in the pituitary gland are not only a reflection of a reduced rate of growth of the whole body, but rather indicate a specific effect of Dx on pituitary gland during fetal development. We assumed that exposure to Dx, during the most vulnerable period for inducing changes in pituitary cell proliferation, differentiation and maturation, may be an important determinant of postnatal pituitary development and function. Such observations confirm the hypothesis of “glucocorticoid programming” that is the action of glucocorticoids during the fetal period on the development and maturation of fetal tissues producing effects that can persist throughout life.
Acknowledgments This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, Grant number 173009. We would like to thank Company Krka FARMA d.o.o. Belgrade, Serbia for the donation of Dexamethasone and Dr. Walter Severs for the kind donation of FSH and LH primary antibodies. The authors also thank Mrs. Milce Mitic for language correction of the manuscript.
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Exposure to dexamethasone reduces pituitary volume and gonadotropic cell number in rat fetuses ´ Milica Manojlovic-Stojanoski, ´ ´ Nataˇsa Ristic´ ∗ , Nataˇsa Nestorovic, Ivana Medigovic, ˇ sic-Jurjevi ´ Branka Soˇ ´ ´ Verica Miloˇsevic´ Svetlana Trifunovic, c, University of Belgrade, Institute for Biological Research “Siniˇsa Stankovi´c”, Department of Cytology, Belgrade, Serbia
a r t i c l e
i n f o
Article history: Received 14 February 2014 Received in revised form 13 March 2014 Accepted 1 April 2014 Available online xxx Keywords: Pituitary Gonadotropic cells Rat fetuses Physical disector
a b s t r a c t Overexposure to glucocorticoids during the fetal period induces changes in developmental processes in various fetal tissues. The aim of this study was to investigate the effects of the synthetic glucocorticoid, dexamethasone (Dx), on pituitary volume and gonadotropic cells during a critical period of pituitary development. The effects of Dx on stereological parameters of the pituitary gland and FSH and LH cells were investigated in 19 and 21-day old fetuses. On day 16 of pregnancy, the experimental dams received 1.0 mg Dx/kg b.w. subcutaneously, followed by 0.5 mg Dx/kg b.w./day on days 17 and 18 of gestation. The control gravid females received the same volume of saline. FSH and LH cells were stained immunohistochemically by the peroxidase–antiperoxidase method (PAP). In 19-day old fetuses, exposure to Dx caused a significant decrease of pituitary volume, estimated by Cavalieri’s principle. Also, the total number of FSH and LH cells per pituitary, determined by physical fractionator counting technique, was significantly reduced. These changes persisted until fetal day 21. Volume densities and numerical densities of FSH and LH cells after exposure to Dx in 19 and 21-day old fetuses remained unaffected. Our results suggest that altered stereological parameters in pituitary gland after exposure to dexamethasone in fetal period could be long-lasting. © 2014 Elsevier GmbH. All rights reserved.
Introduction Glucocorticoids play a crucial role in the final maturation of fetal organ systems, acting on the tissue morphology and function in preparation for extrauterine life and promoting nervous, respiratory, cardiovascular and immune system differentiation and biochemical maturation (Bakker et al., 1997; Flagel et al., 2002; Arima et al., 2008). Transplacental glucocorticoid diffusion is normally limited by placental 11-hydroxysteroid dehydrogenase type 2 (11-HSD2), an enzyme that forms the placental glucocorticoid barrier (Yang, 1997). This enzyme plays a key role in regulating glucocorticoid concentrations in the fetal circulation by inactivating maternal glucocorticoids at the level of the placenta and protects fetuses from glucocorticoid overexposure. During fetal development, glucocorticoid overexposure retards fetal growth and may program postnatal development in glucocorticoid sensitive physiological systems in early life (glucocorticoid
∗ Corresponding author at: Institute for Biological Research “Siniˇsa Stankovic”, ´ Department of Cytology, 142 despota Stefana Blvd., 11060 Belgrade, Serbia. ´ E-mail address: [email protected] (N. Ristic).
programming) (Seckl, 2001). Some of the most commonly used experimental models in the study of programming are maternal undernutrition, maternal stress, maternal treatment with synthetic glucocorticoids and inhibition of placental 11-HSD2. The majority of these experimental models ultimately result in fetal glucocorticoid overexposure, with intrauterine growth retardation (IUGR) and programming of endocrine axes with long-term effects as consequences (Manojlovic-Stojanoski et al., 2012). Antenatal dexamethasone (Dx) therapy is often used in obstetric practice when risks of preterm delivery persist, as a highly effective approach for reducing the frequency of respiratory complications and perinatal death (Roberts and Dalziel, 2006). Dexamethasone as synthetic glucocorticoid passes through the placenta and promotes differentiation and maturation of fetal tissues (Seckl, 2004), but the effects on pituitary growth and gonadotropic cells during the critical period of pituitary development are largely unknown. In the developing mammalian anterior pituitary gland, the gonadotropic cells follicle stimulating (FSH) and luteinizing (LH) are the last hormone-producing cell types to reach maturation. Both types of gonadotropes express a common ␣-glycoprotein subunit (␣-GSU) which combines with the hormone-specific  subunits, FSH and LH, to form biologically active dimeric FSH and LH
http://dx.doi.org/10.1016/j.acthis.2014.04.003 0065-1281/© 2014 Elsevier GmbH. All rights reserved.
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glycoprotein hormones. However, Wen et al. (2010) revealed a striking dichotomy in the gonadotrope population of the developing pituitary. At fetal day 16.75 in mice only LH-expressing gonadotropes, express the gonadotropin-releasing hormone receptor (GnRHR) and at this stage, the fetal pituitary gland becomes GnRH responsive. Also, embryonic increase in LH secretion is needed to promote proper development of FSH-expressing gonadotropes, which might be mediated through paracrine interactions within the fetal pituitary gland. From that moment intrapituitary LH and FSH levels rise dramatically until the end of gestation. These observations suggest that the hypothalamicpituitary-gonadal axis might already be functional during fetal development. Exposure to a compound that affects pituitary cell proliferation and differentiation may be critical for pituitary development and function. The aim of this study was to investigate the effects of exposure to tapering doses of Dx on the pituitary volume and gonadotropic cells during the critical period of pituitary development. Exposure to tapering doses, i.e., reduction of Dx doses minimize adverse effects of glucocorticoid administration, such as damage during neuronal development, and suppression of the HPA axis in fetuses and newborn infants (Flagel et al., 2002; Nahaczewski et al., 2004). The best way to identify the changes caused by Dx treatment was to determine the volume of the pituitary gland and total number of FSH and LH cells per pituitary gland in 19 and 21-day old fetuses after exposure to Dx using unbiased stereological methods. Material and methods
these antisera were assessed by the NIDDK. Sections were incubated in primary antibodies for 45 min at room temperature. After washing in phosphate-buffered saline (PBS; pH 7.4) sections were incubated for 1 h with polyclonal swine anti-rabbit immunoglobulins conjugated with horseradish peroxidase (IgG/HRP, Dako A/S, Glostrup, Denmark). The antigen-antibody complex was visualized by incubating the sections with a chromogen substrate, 0.05% 3,3diaminobenzidine (DAB; Dako A/S, Glostrup, Denmark) and 0.03% H2 O2 . The incubated sections were counterstained with hematoxylin. Control sections were incubated with PBS without primary antisera. Stereological measurements All stereological analyses were carried out using a workstation comprising a microscope (Olympus, BX-51) equipped with a microcator (Heidenhain MT1201) to control movements in the z-direction (0.2-m accuracy), a motorized stage (Prior) for stepwise displacement in the x–y directions (1-m accuracy), and a CCD video camera (PixeLink) connected to a 19 PC monitor (Dell). The whole system was controlled by the new-CAST stereological software package (VIS – Visiopharm Integrator System, version 3.2.7.0; Visiopharm, Horsholm, Denmark). The objectives used 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 rectangular unbiased disector frames) were provided by new-CAST software running on a Dell computer. Pituitary volume estimation (Vpt – mm3 )
Animals Female and male Wistar strain rats, weighing approximately 250 g and 400 g respectively, were mated in the vivarium of the Institute for Biological Research, Belgrade, at night. The mornings in which sperm-positive vaginal smears were obtained were declared gestation day 1. Pregnant females were housed individually, under standard conditions (12:12 h light-dark cycle at 22 ± 2 ◦ C) and offered food and water ad libitum. Dams were randomized into two groups: control and experimental group, each consisting of six animals. On day 16 of pregnancy, experimental dams received subcutaneously 1.0 mg Dx (Dexamethasone phosphate, Krka FARMA d.o.o. Belgrade, Serbia, dissolved in 0.9% saline)/kg b.w., followed by 0.5 mg Dx/kg b.w./day on day 17 and 18 of gestation. The control gravid females received the same volume of saline. Female fetuses from control and experimental dams were sacrificed under ether narcosis on day 19 and 21 of gestation and they are referred to as 19 and 21-day old fetuses. The experimental protocols were approved by the Local Animal Care Committee in conformity with the recommendations provided in the European Convention for Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (ETS no. 123, Appendix A). Tissue preparation The pituitary glands were excised with part of the sphenoid bone, fixed in Bouin’s solution for 48 h and processed for embedding in Histowax (Histolab Product AB, Göteborg, Sweden). Serial 3 m thick tissue sections were deparaffinized in xylene and rehydrated through a decreasing series of ethanol. Gonadotropes were localized immunohistochemically using the peroxidaseantiperoxidase method. Polyclonal rabbit anti-rat FSH (1:300, v/v) and polyclonal rabbit anti-rat LH (1:500, v/v) served as primary antibodies (National Hormone Peptide Program (NHPP), Harbor-UCLA Medical Center, Carson, CA, USA). The specificities of
Pituitary volumes were estimated 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 a known distance apart. The total volume of the object was estimated by summing the areas over all sections and multiplying the results by the section thickness. To ensure a random position for the first section from each tissue block, a random number table was used. As every 10th section from each tissue block was analyzed (the same sections were used in the subsequent estimation of FSH/LH cell numbers by the physical disector method), a number between 1 and 10 was obtained from a random number table for sampling the first section. On the monitor, a final magnification of 300× allowed easy and accurate recognition of tissue boundaries. Mean section thickness was estimated using the block advance (BA) method (Dorph-Petersen et al., 2001), and we found that there was no variation from 3.0 m, as set in the microtome, i.e., there was no difference between nominal and true section thickness. The volume of the pituitary gland (Vpt ) was estimated as the sum of the total volume of immunohistochemically labeled FSH/LH cells (VFSH/LH ) and the volume of uncolored phase of adenohypophysis (Vua ), which gives the volume of the entire adenohypophysis (Vahy ), and volume of neurohypophysis and pars intermedia (Vni ): Vpt = Vua + VFSH/LH + Vni The volume of individual phases of the pituitary gland was calculated using the following formula: V¯ = a(p) · BA ·
n
Pi
i=1
where a(p) is the area associated with each sampling point; BA (the block advance) is the mean distance between two consecutively studied sections (real section thickness 3 m × 10); n is the number
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Fig. 1. Unbiased counting frame on reference and look-up sections. In this position, only cell marked A was counted, because its nucleus appeared within the counting frame of the reference section, but did not appear in that of the look-up section. Also this cell was intersected with acceptance (green) lines of frame. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
of sections studied for each pituitary; and hitting a given target.
Pi is the sum of points
Volume densitiy of FSH/LH cells (VV – %) The percentage of FSH/LH cells per unit volume of adenohypophysis i.e. the volume density is equivalent to the total volume occupied by FSH/LH cells divided by volume of adenohypophysis and multiplied by 100.
VV =
VFSH/LH Vahy
× 100
Quantification of FSH/LH cell number (No) A fractionator/physical disector design with two levels of sampling was used to estimate the number of FSH/LH cells in 19 and 21-day old control and fetuses exposed to Dx. Sampling was systematically uniform from a random start (Dorph-Petersen et al., 2001; Gundersen and Jensen, 1987). For the first level of sampling, every 10th and 11th section was at random selected all along the serial sections. The random number for each block being analyzed was chosen by using the random number table. Sections designated as section pairs were first captured into a super-image. After defining and linking, the pairs were aligned by translation and rotation using the montage option in the new-CAST software. For the cell counting, a 100× oil-immersion objective was
used. One section in the pair was designated the reference section and the other the “look-up” section. Subsequently, the analysis was performed in both directions with the reference section also becoming the look-up section. This doubled the first sampling fraction from 1/10th to 1/5th [sampling fraction 1(f1) = 1/5 = 0.2]. For estimation of FSH and LH cell number, an unbiased counting frame measuring 40 m × 40 m (1600 m2 ) was used to ensure 150–200 cells per animal. After defining tissue boundaries, meander sampling was set to analyze 100% of the tissue. Therefore, sampling fraction 2 for FSH/LH cell number was f 2 = 1. The fields of vision were randomly selected and the percentage of tissue analyzed was controlled by the software. Additionally new-CAST software facilitated matching of fields between the reference and the look-up section. When the field of vision was selected at the reference section, the matching position at the consecutive look up section was scanned and a matching field of vision was generated from four images. FSH/LH cells were counted if their nuclei appeared within the unbiased counting frame applied to the reference section, they were not intersected by exclusion boundaries (Gundersen, 1986) and did not appear in the look-up section (Fig. 1). Raw counts (Q− ) of FSH/LH cell number were multiplied by the reciprocals of the sampling fractions to estimate the total number of FSH and LH cells per pituitary. Q− ranged between 171 and 177 for FSH, and 214 and 245 for LH in control 19-day old fetuses. In 19-day old fetuses exposed to Dx Q− ranged between 100 and 124 for FSH, and 104 and 115 for LH. In 21-day old control fetuses Q− ranged between 312 and 348 for FSH, and 322 and 364 for LH. In 21-day old fetuses exposed to Dx Q− ranged between 217 and 292 for FSH cells and 259 and 294 for LH. The
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Table 1 Body weight, volume of pituitary gland and volume of adenohypophysis in 19 and 21-day old control fetuses (C19 and C21) and fetuses exposed to Dx (Dx19 and Dx21).
Body weight (g) Volume of pituitary gland (mm3 ) Volume of adenohypophysis (mm3 )
C19
Dx19
C21
Dx21
1.4 ± 0.07 0.07 ± 0.01 0.06 ± 0.007
1.1 ± 0.2b 0.04 ± 0.004b 0.04 ± 0.008b
4.4 ± 0.7a 0.15 ± 0.02a 0.1 ± 0.01a
3.1 ± 0.2c , d 0.09 ± 0.003c , d 0.07 ± 0.0006c , d
All results are given as mean ± SD, n = 6, p < 0.05. a C21 vs C19. b Dx19 vs C19. c Dx21 vs C21. d Dx21 vs Dx19.
number of FSH/LH cells = Q− (FSH/LH cells) × (1/f1) × (1/f2) = Q¯ (FSH/LH cells) × 1/0.2 × 1/1. Mean FSH/LH cell volume (Vc – m3 ) The mean volume of a single FSH/LH cell is equivalent to the total volume occupied by FSH/LH cells divided by their number (de Lima et al., 2007). Vc =
VFSH/LH No
groups. They were polygonal, oval or polyhedral in shape, with large, prominent often eccentrically located nuclei and a thin layer of surrounding cytoplasm. Gonadotropes were single or in groups, often in close contact with blood capillaries. FSH and LH cells were more numerous in the adenohypophysis of 21-day old than in 19day old fetuses (Figs. 2 and 3). After exposure to dexamethasone FSH and LH cells were less numerous, smaller in size and irregularly shaped in 19-day old fetuses. A striking reduction in the number of gonadotropes was retained to fetal day 21 (Figs. 2 and 3).
Numerical densities of FSH/LH cells (NV )
Stereological analysis
Numerical density depicts the number of FSH and LH cells per adenohypophysis volume unit, and was calculated as the ratio of the absolute number of FSH and LH cells and the volume of adenohypophysis. Numerical density has an exponent of −3.
Stereological analysis showed that the volume of the pituitary gland and the volume of adenohypophysis increased from fetal day 19–21 by 2 (p < 0.05) and 1.7 fold (p < 0.05), respectively (Table 1). Increased volume of pituitary gland and adenohypophysis was followed by increase in the number of FSH and LH cells by 2.2 fold (p < 0.05) and 1.7 fold (p < 0.05), respectively (Fig. 4a). At the same time the volume of FSH and LH cells was significantly decreased by 38% (p < 0.05) and 34% (p < 0.05), respectively (Fig. 4b). Volume density and numerical density remained unchanged (Fig. 4c and d). Exposure to dexamethasone caused a decrease in the volume of the pituitary gland and the volume of the adenohypophysis by 43% (p < 0.05) and 33% (p < 0.05), respectively in 19-day old fetuses. Reduction of these parameters was also detected in 21-day old fetuses, by 40% (p < 0.05) and 30% (p < 0.05), respectively in comparison with the control groups. In Dx exposed fetuses the volume of pituitary gland and the volume of adenohypophysis increased from fetal day 19–21 by 2.3 (p < 0.05) and 1.8 fold (p < 0.05), respectively (Table 1). The volume of the pituitary gland correlated to the body weight was significantly decreased in Dx exposed fetuses when compared with the control values by 27% (p < 0.05) and 24% (p < 0.05), respectively in 19 and 21-day old fetuses (Table 2). Exposure to Dx during the last week of pregnancy led to a 37% (p < 0.05) decrease in the number of FSH cells, and 51% (p < 0.05) decrease in the number of LH cells in 19-day old fetuses in comparison to the control group. A reduced number of gonadotropes was still present in 21-day old fetuses, where the number of FSH and LH cells was decreased by 30% (p < 0.05) and 31% (p < 0.05), respectively in comparison to controls (Fig. 4a). In Dx exposed fetuses number of FSH and LH cells increased from fetal day 19–21 by 2.5 fold (p < 0.05). The number of FSH and LH cells was correlated to the pituitary volume and body weight. When expressed as number per pituitary volume unit, no changes were observed between control and Dx exposed fetuses at both time points. But, when expressed as number per body weight unit, the number of FSH and LH cells was significantly decreased by 26% (p < 0.05) and 41% (p < 0.05), respectively, in 19-day old fetuses, and by 9% (p < 0.05) and 9.2% (p < 0.05), respectively, in 21-day old fetuses exposed to Dx when compared with control values (Table 3).
NV =
No Vahy
Statistical analysis All results are expressed as mean values for six animals per group ± SD. Data were tested for normality of distribution by the Kolgomorov–Smirnov test. Data were analyzed by 2-way analysis of variance (ANOVA), with the age of fetus (19 and 21 day) and applied treatment (C and Dx) as factors. To determine significant differences between the groups, the post hoc Bonferroni test was used. A probability value of 5% or less was considered statistically significant.
Results Body weight Body weight of rat fetuses in the control group physiologically increased from fetal day 19 to 21 by 3 fold (p < 0.05). Exposure to Dx during the last week of fetal development induced a significant decrease in body weight in 19 and 21-day old fetuses by 21% (p < 0.05) and 30% (p < 0.05), respectively. In Dx exposed fetuses body weight increased from fetal day 19–21 by 2.8 fold (p < 0.05) (Table 1). Histological analysis In 19-day old fetuses the pituitary gland already had definite histological organization. FSH and LH cells were strongly immunohistochemically stained in the pars distalis in fetuses of both age
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Fig. 2. Immunohistochemically labeled FSH and LH cells in the pituitary pars distalis of 19-day old control fetuses (C FSH and C LH) and fetuses exposed to Dx (Dx FSH and Dx LH); * blood vessels; scale bar = 10 m.
Fig. 3. Immunohistochemically labeled FSH and LH cells in the pituitary pars distalis of 21-day old control fetuses (C FSH and C LH) and fetuses exposed to Dx (Dx FSH and Dx LH); scale bar = 10 m.
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Fig. 4. Stereological parameters of FSH and LH cells in 19 and 21-day old control and fetuses exposed to Dx; (a) Number of FSH/LH cells expressed per pituitary, (b) volume of FSH/LH cells, (c) volume densities of FSH/LH cells, (d) numerical densities of FSH/LH cells. Results are given as mean ± SD, n = 6, p < 0.05, a: C21 vs C19, b: Dx vs corresponding C, c: Dx21 vs Dx19. Table 2 Volume of pituitary gland expressed per body weight unit in 19 and 21-day old control fetuses (C19 and C21) and fetuses exposed to Dx (Dx19 and Dx21). Volume of pituitary gland per body weight unit (mm3 /g) C19
Dx19
C21
Dx21
0.052 ± 0.009
0.038 ± 0.002*
0.037 ± 0.0069
0.028 ± 0.002*
All results are given as mean ± SD, n = 6. * p < 0.05.
After exposure to Dx the volume of FSH and LH cells was decreased compared with the control values by 16% (p < 0.05) and 39% (p < 0.05), respectively in 19-day old fetuses, but no changes were observed in the volume of gonadotropes in 21-day old fetuses (Fig. 4b). Volume densities of FSH and LH cells in fetuses of both age groups did not change significantly after exposure to Dx (Fig. 4c). When the number of FSH and LH cells per adenohypophysis volume unit was calculated, there were no statistically significant differences between 19 and 21-day old fetuses from control and experimental group (Fig. 4d). Discussion Our results demonstrate that exposure to Dx during the last third of pregnancy has an inhibitory effect on the volume of the pituitary gland, number and volume of FSH and LH cells in 19-day old fetal rats. Reduced volume of pituitary gland and number of FSH
and LH cells are still present in 21-day old fetuses, but the volume of gonadotropic cells was normalized. In our experimental conditions rat fetuses were exposed to Dx in a period of intensive proliferation of precursor and differentiated pituitary cells, i.e. from fetal day 16–18 (Taniguchi et al., 2002). Rapid pituitary growth during the last third of pregnancy is interrupted in Dx exposed fetuses, and the volume of fetal pituitary gland was significantly lower in 19 and 21-day old fetuses. Reduction of pituitary volume probably is the result of reducing the number of all types of pituitary cells, including ACTH, TSH (Manojlovic-Stojanoski et al., 2006, 2008, 2010) and as shown in this study, FSH and LH cells. Dx may act indirectly on the pituitary cell number by diminution of the stimulatory hypothalamic contribution to pituitary cell synthetic and proliferative activity. In the rat, GnRH neurons develop and migrate during the fetal period (Daikoku and Koide, 1998). Dx exposure at this time induces neuronal apoptosis and reduction of the number of GnRH neurons accompanied by reduced synthesis of GnRH (Reagan and McEwen, 1997). Also, Dx application caused inhibition of thyrotropin-releasing hormone (TRH) (Alkemade et al., 2005) and corticotropin-releasing hormone (CRH) synthesis and release (Dupouy et al., 1987). By direct action, expressing strong antiproliferative and proapoptotic effect (Nolan et al., 1998, 2004; Nolan and Levy, 2006), Dx caused reduction of pituitary cell number, including a gonadotropic cell, which also contributes to reducing the volume of the entire gland. During development of the pituitary gland, precursors of gonadotropic cells intensively divide and differentiate
Table 3 The number of FSH and LH cells (No) expressed per pituitary volume unit and per body weight unit in 19 and 21-day old control fetuses (C19 and C21) and fetuses exposed to Dx (Dx19 and Dx21). No per pituitary volume unit (No/mm3 )
FSH LH
No per body weight unit (No/g)
C19
Dx19
C21
Dx21
C19
Dx19
12,308 ± 1659 16,114 ± 3755
12,633 ± 134 12,729 ± 955
13,341 ± 2428 13,675 ± 2463
14,720 ± 101 14,825 ± 1242
630 ± 23 821 ± 31
464 ± 44 486 ± 48* *
C21
Dx21
482 ± 23 490 ± 34
439 ± 25* 445 ± 19*
All results are given as mean ± SD, n = 6. * p < 0.05.
Please cite this article in press as: Ristic´ N, et al. Exposure to dexamethasone reduces pituitary volume and gonadotropic cell number in rat fetuses. Acta Histochemica (2014), http://dx.doi.org/10.1016/j.acthis.2014.04.003
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contributing to the increase of FSH and LH cell number. Pope et al. (2006) suggested that as soon as FSH or LH genes are switched on, the gonadotropes becomes terminally differentiated in human fetuses. This observation is in agreement with reports demonstrating that in rodent gonadotropes, the most intense proliferation occurs before the terminal differentiation (Khar et al., 1978; Pawlikowski et al., 1978; Levy, 2002). Therefore, the number of gonadotropes in the pituitary is mostly set during fetal life, and there is little replacement during the rest of life. In the postnatal period mitoses of gonadotropes are infrequent, but can become active in some physiological and experimental conditions (Taniguchi et al., 2002). The decreased number of FSH and LH cells in 19 and 21-day old fetuses indicate that Dx inhibits proliferation of gonadotrope precursors. Despite accelerated pituitary growth and a larger increase in gonadotropic cell number from fetal day 19–21 in Dx exposed fetuses, control values are not reached. Considering the inhibitory effect of Dx on pituitary volume and FSH and LH cell number, as well as unchanged FSH and LH cell volume in 21-day old fetuses, it can be concluded that these results could represent a maturational effect of glucocorticoids. Glucocorticoids promote maturation in a wide range of fetal tissues including lungs, liver, kidneys, muscle, fat and gut, by switching cell cycle from proliferation to differentiation (Fowden et al., 1998). Unchanged volume density and numerical density of fetal gonadotropes results from a proportional decrease of the total volume occupied by FSH and LH cells, as well as their absolute number, in relation to volume of the adenohypophysis after exposure to Dx. Also, when the number of FSH and LH cells was correlated to the pituitary volume, no changes were observed between control and Dx exposed fetuses at both time points. These results indicate that the structure of the fetal pituitary gland after Dx exposure remained unchanged. The present study shows that exposure to Dx reduces fetal growth, resulting in a decrease of body weight of 19 and 21-day old fetuses, in accord with previous observations (Seckl, 1997; Fowden et al., 1998; Smith and Waddell, 2003; Manojlovic-Stojanoski et al., 2010). IUGR is associated with programming of endocrine axis, with long-lasting consequences. Reduction of the pituitary volume and gonadotropic cell number in our experimental conditions may be part of the programming process of the gonadotropic axis. The volume of the pituitary gland and number of gonadotropes expressed per body weight unit was significantly decreased in Dx exposed fetuses. This indicates that the changes in the pituitary gland are not only a reflection of a reduced rate of growth of the whole body, but rather indicate a specific effect of Dx on pituitary gland during fetal development. We assumed that exposure to Dx, during the most vulnerable period for inducing changes in pituitary cell proliferation, differentiation and maturation, may be an important determinant of postnatal pituitary development and function. Such observations confirm the hypothesis of “glucocorticoid programming” that is the action of glucocorticoids during the fetal period on the development and maturation of fetal tissues producing effects that can persist throughout life.
Acknowledgments This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, Grant number 173009. We would like to thank Company Krka FARMA d.o.o. Belgrade, Serbia for the donation of Dexamethasone and Dr. Walter Severs for the kind donation of FSH and LH primary antibodies. The authors also thank Mrs. Milce Mitic for language correction of the manuscript.
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