Stress disturbs cellular homeostasis. During stress, the hypothalamo–pituitary–adrenal (HPA) axis is activated. Thus, corticotropin-releasing hormone (CRH) released from the hypothalamus activates pituitary corticotrophs to secrete adrenocorticotrophic hormone (ACTH) that enters the circulation and stimulates the adrenal glands to secrete glucocorticoids (corticosterone in the rat; cortisol in the human and sheep). The steroid products of the adrenal cortex provide feedback control of ACTH secretion. The action of glucocorticoids is primarily directed towards modulation of energy metabolism and affects the immune system, allowing the organism adaptive responses to stress stimuli.

During fetal life, the corticotroph is the first type of differentiated pituitary hormone-producing cell to appear. The first cells that are immunopositive for ACTH can be found on fetal day 13 in the pars tuberalis anlage, whereas ACTH immunostaining is found 1 day later in the pars distalis. The pars intermedia of the fetal rat hypophysis is the last part to display ACTH staining. ACTH controls adrenal growth and development, as well as steroidogenic maturation of the fetal adrenal glands.

Stress during pregnancy is known to have long-lasting consequences on fetal development. Exposure to glucocorticoids during the fetal period or prenatal stress targets the main mediator of hormonal responses to stress, the fetal HPA axis, modifying its activity. Moreover, exposure to glucocorticoids during critical periods of rapid cell division, such as fetal development, may lead to permanent changes in various biological systems and functions, subsequently affecting organ structures by controlling the kinetics of cell division and their distribution, as well as altering hormonal feedback mechanisms and metabolic activity. These changes during fetal life may have pathological consequences in adulthood by increased predisposition to chronic illnesses such as cardiovascular disease and hypertension, diabetes, autoimmune disease and even behavioral changes such as defensive behavior or anxiety. The association between fetal events and subsequent disorders in adult life is known as programming.

As the effects of glucocorticoid treatment on the fetal HPA axis underlie long-term pathophysiological changes, we investigated interruptions of the fetal HPA axis at the level of ACTH-producing cells after maternal treatment with dexamethasone (Dx). Dx is a synthetic analog of the stress hormone and is used to study stress-induced effects of glucocorticoids.

In the present study, effects of maternal Dx administration during gestational days 16–18 were examined in fetal pituitary ACTH cells on day 21 using immunohistochemical and morphometrical methods.

Adult virgin Wistar female rats, weighing approximately 250 g, were housed in groups (3 rats per cage) in the presence of a sexually experienced male rat, weighing 400 g, for one night (24 h or less). The day when females were sperm positive was designed as gestational day 1. Pregnant rats were assigned randomly to prenatal control (C) or Dx-treated groups (Dx).

Pregnant rats were individually housed in plastic breeding cages, allowed ad libitum access to food and water, and maintained on a 12:12 light/dark cycle, at constant room temperature. Pregnant females in the experimental group received subcutaneous injections of 0.5, 0.5 and 1.0 mg Dx/kg body weight/day during days 16–18 of pregnancy. Control mothers were injected with the same amount of 0.9% saline. On day 21 of pregnancy, the dams and their fetuses were sacrificed under ether anesthesia and the fetuses were referred to as 21-day-old fetuses.

The pituitary glands were excised with part of the sphenoid bone, fixed in Bouin’s solution for 48 h and embedded in paraffin. Serial 5-?m thick tissue sections were deparaffinized in xylol and decreasing series of ethanol. Pituitary hormones were localized immunohistochemically using the peroxidase–anti-peroxidase complex (PAP) method of. Endogenous peroxidase activity was blocked by incubation in 9 mM hydrogen peroxide solution in methanol for 30 min at ambient temperature. Before application of specific primary antibodies, nonspecific background staining was prevented by incubation of the sections with nonimmune, i.e. normal, porcine serum diluted in phosphate-buffered saline (PBS; pH 7.4) for 60 min. Sections were then overlaid with the appropriate dilution of specific rabbit anti-human ACTH antibodies (Dako, Glostrup, Denmark) for 24 h at room temperature. After washing in PBS, sections were incubated for another 60 min with the secondary antibody (swine–anti-rabbit IgG; Dako) for 45 min, rinsed again with PBS for 10 min and then incubated with rabbit PAP complex for 45 min. Antibody localization was visualized by immersing the sections in Tris-HCl-buffered saline (0.5 mol/l; pH 7.4) supplemented with 3,3-diaminobenzidine tetrachloride (Serva, Heidelberg, Germany) and 9 mM hydrogen peroxide. Sections were thoroughly washed under running tap water, counterstained with hematoxylin and mounted in Canada balsam (Alkaloid, Skopje, Macedonia). Control sections were incubated without primary antibodies or by substituting the primary antibodies with nonimmune rabbit serum.

Figure 1. Serial sections of a pituitary gland of a 21-day-old fetus from the control group in the dorsal position in relation to the developing sphenoid bone after immunohistochemical staining (a) or control staining with omission of the primary antibody (negative control) (b); pd, pars distalis; pi, pars intermedia; nl, neural lobe; rl, residual lumen; sb, sphenoid bone. Magnification, ×112.

Volume densities (V_v) of the nuclei and the cytoplasm of ACTH-immunopositive cells, as well as the numerical density (N_a) of their nuclei per ?m³ were measured in 50 test areas per pituitary gland at a magnification of ×1000, using the M₄₂ multipurpose test system.

On the basis of earlier karyometric studies, the shape coefficient ? for pituitary cells was estimated to be 1.382. It relates N_v (number of cells counted per unit volume) to N_a (number of cells counted per square millimeter) and V_v (volume density) and depends on the axial ratio of the nuclei. The volume density of ACTH-positive cells was expressed as the percentage of total pituitary cell volume in ?m³.

Digital images were made on a DM RB Photomicroscope (Leica, Wetzlar, Germany) with a JVC TK 1280E Video Camera (Leica) using the Qwin program (Leica) for the acquisition and analysis of the images.

Biochemical and morphometric data obtained from each rat were averaged per experimental group and mean and standard deviations were calculated. One-way analysis of variance, followed by Duncan^’s multiple range test, was used for statistical comparison of the groups. A probability value of 5% or less was considered as statistically significant.