Introduction
Phytoestrogens are plant-derived compounds from e.g. red clover, which may elicit an oestrogenic response via the oestrogen receptor Markiewicz et al., 1993). Furlanetto et al.(1999) demonstrated a direct effect of oestradiol on thyroid follicular cell growth. The effect of phytoestrogens on thyroid gland morphology and localisation of oestrogen receptor alfa (ERa) was studied in ovariectomized ewes.
Methods
Eight long-term ovariectomized ewes were used, four were fed 3.5 kg of 100% red clover (Trifolium pratense) silage for 14 days and four served as control animals.
Immunohistochemistry. Thyroid tissue was fixed in Bouin's solution and embedded in paraffin. Sections (4 m) were deparaffinized and pretreated in a microwave oven. Endogenous peroxidase and biotin was blocked using blocking kit (Vector Laboratories, Inc., Burlingame, CA). The sections were incubated with a mouse monoclonal antibody against ERa (C-311, Santa Cruz Biotechnology, Inc., CA) diluted 1:25. According to the manufacturer this antibody does not react with ERb or other steroid receptors. After treatment with a biotinylated secondary antibody the sections were incubated with ABC-complex (Vectastain Elite ABC, Vector). The peroxidase activity was visualised with diaminobenzidine tetrachloride (Saveen Biotech AB, Sweden). Negative controls were obtained by exchanging the primary antibody for normal mouse IgG (Vectastain, Vector). The sections were dehydrated and mounted in Pertex, some of them after first having been counterstained with hematoxylin.
Image analysis. Tissue was fixed in 2.5% glutaraldehyde and embedded in water-soluble resin. Sections (2 m) were cut and stained with hematoxylin/eosin. The area of 400 - 600 thyroid follicles from each animal were measured by an image analysis system.
Results
The cross-section area of thyroid follicles tended to be larger in ewes fed red clover silage than in the control animals (P=0.07). The immunohistochemical localisation of ERa showed that the receptor was present in both groups of animals, but at varying frequencies and intensities. Individual differences were also seen between animals in the same group. Neighbouring follicles differed in number of stained cell nuclei as well as in staining intensity and, especially in the control animals, unlabelled follicles were common. However, as a whole the nuclear ERa immunoreactivity was stronger and more frequent in the thyroid glands from the ovariectomized ewes exposed to phytoestrogens than in the ewes fed hay. No specific nuclear staining was found in the negative control sections.
Conclusions
Daily ingestion of 81 - 95 mg phytoestrogens per kg body weight for 14 days tended to increase follicle size and ERa immunoreactivity of thyroid glands in ovariectomized ewes.
This work was supported financially by the Swedish Council for Forestry and Agricultural Research.
References
Furlanetto, T.W., Nguyen, L.Q. & Jameson, J.L. 1999. Endocrinology 140, 5705-5711.
Markiewicz, L., Garey, J., Adlercreutz, H. & Gurpid, E. 1993 J Steroid Biochem Mol Biol 45, 399-405.