In the distal part of the pituitary gland of Rana ridibunda, cells are found that synthesize and secrete a variety of hormones. These cells have been distinguished in mature and larval stages of various amphibians, using immunocytochemical methods. These studies, however, are limited to only a certain number of amphibian species.

Highest concentrations of thyrotropin-releasing hormone (TRH) in amphibians is found in the hypothalamus and the extrahypothalamic parts of the brain. In early studies, TRH was believed not to stimulate thyrotropin (thyroid stimulating hormone—TSH) secretion in amphibian pituitary glands. In subsequent studies, TRH was demonstrated to have a stimulating effect on TSH secretion. In addition, TRH was found to stimulate secretion of prolactin (PRL), growth hormone, and gonadotropin in the amphibian pituitary gland, in both in vitro and in vivo studies. Yet, our understanding of physiological functions of TRH in lower vertebrates is limited. TRH, a tripeptide amide (pGlu–His–Pro NH₂), has not only a hypophysiotropic effect, but also functions as neurotransmitter and neuromodulator in various neural and extraneural tissues.

Thyroxin (T₄) has recently been shown to have a negative feedback effect on secretion of bioactive TSH. Thyroid hormones have a potent feedback control on synthesis and storage of TSH and PRL. This regulatory effect is reported to take place either directly in the distal lobe, or indirectly via TRH or any other hypothalamic factor. It has been shown that exogenous T₄ extends the light–dark cycle and affects the rate of metamorphosis, and is directly effective on genes that encode for proteins that have positive and/or negative regulatory effects on thyroid hormones of tadpoles. Thyroid hormones inhibit the human PRL gene promoter, and play an important role in maintaining functions of PRL-producing cells.

The aim of the present study was to discriminate TSH- and PRL-producing cells in the pituitary of mature R. ridibunda by using light microscopical and electron microscopical immunocytochemical methods to detect the localization of these cells in the anterior pituitary gland, and to compare the effects of treatment with TRH and T₄ in different doses on these cells.

R. ridibunda, an anura amphibian species (weighing 27–65 g, n=18) was used. The animals were fed on chicken liver and earthworms and were divided at random into six groups, each containing frogs of both gender, and were placed individually in plastic boxes with lids.

Group 1 (2 animals) was treated 6 times every other day with 0.5 mg -T₄ (Sigma, St. Louis, MO, USA) per ml of 0.64% NaCl. Group 2 (2 animals) was treated 6 times every other day with 0.4 mg -T₄ (Sigma) per ml of 0.64% NaCl. Group 3 (2 animals) was treated 6 times every other day with 0.2 mg -T₄ (Sigma) per ml of 0.64% NaCl. To the animals in each experimental group, 1 ml solution with the specified concentration was given by intravenous injection in the groin between 10.00 and 11.00 am. Group 4 (2 animals) was treated with 200 ?g TRH (Sigma) per 0.5 ml of 0.64% NaCl once a week for 4 weeks. Group 5 (2 animals) served as control group that did not receive injections. Group 6 (6 animals) served as control for groups 1, 2 and 3, that received only 1 ml of 0.64% NaCl 6 times every other day. Group 7 (2 animals) served as control for group 4 and received once a week 0.5 ml of 0.64% NaCl for 4 weeks.

Anterior pituitary glands were removed immediately after decapitation, fixed in Hollande–Bouin fixative for 48 h, then kept in running tap water for 24 h, and then placed in 70% alcohol. Following dehydration, the tissue samples were embedded in paraffin wax before sectioning.

Sections, 4–5-?m thickness, were collected onto slides covered with poly -lysine and stained with anti-TSH and anti-PRL antibodies and the streptavidin–biotin–peroxidase method. In order to block endogenous peroxidase in the sections, 3% H₂O₂ was applied for 10 min. For the localization of PRL, samples were incubated overnight with rabbit anti-PRL antibodies (Harlan, Crawley Down, Sussex, UK) in a dilution of 1:2000 at 4°C, and rabbit anti-TSH antibodies (Harlan) in a dilution of 1:1000. The specific reactivity was determined by using biotinylated secondary antibodies (goat anti-rabbit-IgG) and the streptavidin–biotin–peroxidase complex and 3-amino-9-ethyl carbazole (AEC) (Biogenex, San Ramon, CA, USA). Detection procedures were carried out following guidelines of the manufacturer (Biogenex). Hematoxylin was used for counterstaining. Sections were rinsed in running tap water, then rinsed in phosphate-buffered saline and distilled water and finally mounted in glycerin. TSH- and PRL-producing cell counts were determined in the 20 areas (each area is 0.26 mm²) of the four different regions (anterior, posterior, ventral, dorsal) of the pituitary from five sections taken from the two animals in each group using a light microscope. For counting, a ×10 ocular and a ×40 objective magnification were used. Specificity of the immunohistochemical staining was checked by omission of the primary antibody or by using an irrelevant antibody (against somatostatin) in the same dilutions. Control pituitary sections were used as positive control.

Anterior pituitary glands were fixed in 2.5% glutaraldehyde in Millonig’s phosphate buffer, pH 7.4, for 3 h. After dehydration in a graded series of ethanol and propylene oxide, tissue was embedded in Araldite MCY212 (Fluka, Buchs, Switzerland). For ultrastructural localization of PRL and TSH, the protein A-gold method was applied to thin sections placed on nickel grids. Protein A-gold complex with a particle size of 15 nm was obtained from Aurion, (Wageningen, The Netherlands). Rabbit anti-PRL was applied in a dilution of 1:2000 and rabbit anti-TSH was applied in a dilution of 1:1000. Sections were stained with uranyl acetate and lead citrate, and were examined with a 9S-2 electron microscope (Zeiss, Oberkochen, Germany).

To confirm specificity of immunocytochemical labelling, the following control incubations were performed: (1) omission of the primary antibody and application of the protein A-gold solution alone; (2) replacement of the antibodies by nonimmune serum in the same dilutions.

Distribution of TSH- and PRL-producing cells and differences between groups were determined by using the one-way ANOVA approach. Differences in cell numbers in areas examined of the anterior pituitary were compared between control and test groups.

Distribution patterns of TSH- and PRL-producing cells in the anterior pituitary gland of animals in the control group were as follows. TSH-producing cells were mainly present in the dorsal part of the pituitary, their occurrence decreased gradually towards anterior and ventral areas and were nearly absent in the posterior region. TSH-producing cells were mainly located in the margins of anterior pituitary islands but sometimes in the center of the islands, usually forming clusters of 2 or 3 cells, and occasionally as individual cells. They were usually polarized towards capillaries.

Figure 1. Immunohistochemical staining of TSH (arrowheads) in the pituitary gland of control R. ridibunda. Magnification, ×578.

View Within ArticlePRL-producing cells were abundantly present, being prominent in the anterior part, whereas they were less numerous in dorsal, ventral, and posterior regions. PRL-producing cells showed a polyhedral, spherical or triangular shape. They were usually polarized towards capillaries, like TSH-producing cells.

TRH treatment caused dilation of sinusoids in anterior pituitary glands of frogs. Besides, TSH-producing cells were enlarged and their numbers were increased. However, differences with the control group, that was treated with physiological salt, were not significant. The PRL-producing cells were also enlarged, and their numbers were increased when compared to untreated group, but the differences were not statistically significant.