Immunocytochemical Localization of Bovine Serum Albumin (BSA) in the Liver and Testis of Rats injected with Testosterone-BSA, Hydrocortisone-BSA or Corticosterone-BSA.

Abstract

ABSTRACT. Through observations of colloidal gold with silver enhancement, we have demonstrated that 2-nm colloidal gold labeled-testosterone-bovine serum albumin (BSA) conjugate or hydrocortisone-BSA conjugate injected intravenously enters the hormone-target cell nuclei of rats (Nishimura and Ichihara, 1997; Nakano, 1997, 1999). To confirm immunocytochemically whether the nature of BSA in the steroid hormone-BSA conjugates (steroid-BSAs) remains intact in the hormone-target cell nuclei, testosterone-BSA, hydrocortisone-BSA or corticosterone-BSA was injected into the vascular system of rats, then the liver and testes of rats killed 2 h postinjection were reacted with FITC-conjugated anti-BSA antibody, and examined under fluorescence microscopy and confocal laser scanning microscopy. In the liver of rat injected with testosterone-BSA, the fluorescence was observed in the nuclei of endothelial cells, but not in the nuclei of hepatocytes, hepatic stellate cells and Kupffer cells. In the liver of rat injected with hydrocortisone-BSA, intense fluorescence was seen in the nuclei of hepatic stellate cells, but did not seem to be present in the nuclei of the other three kinds of cells. In the liver of rat injected with corticosterone-BSA, the fluorescence seemed to be in a few nuclei of hepatic stellate cells, and appeared as speckles in a few nuclei of the hepatocytes and Kupffer cells. In some seminiferous tubules of rat injected with testosterone-BSA, fluorescence was observed in the nuclei of spermatocytes and spermatids. These results suggest that BSA conjugated with steroid hormone can enter the hormone-target cell nuclei with its antigenicity kept intact, and that the fate of steroid-BSAs is decided at the cell membrane level.Key words: testosterone-BSA/hydrocortisone-BSA/corticosterone-BSA/target cell nuclei/ in vivo/immunocytochemistry Steroid hormones circulate in blood plasma in three physical states: free, albumin-bound, and bound to serum steroid binding proteins such as sex hormone-binding globulin (SHBG) and corticosteroid-binding globulin (CBG) (Kuhn,

Nongenomic Steroid Action: Controversies, Questions, and Answers

“…Often, the potency of steroid conjugates to evoke effects is 30-to 100-fold lower than free steroid, which correlates well with the amount of free steroid present in many preparations (1-3%). Second, recent reports have shown that the presumably cell-impermeable conjugates have been detected inside the cell (307,335), so physiological actions exerted by steroid conjugates pose some difficulties for interpretation. However, studies that demonstrate exclusive membrane localization of fluorescently labeled steroid conjugates [e.g., confocal techniques (44)] still give strong evidence for steroid receptors located in the membrane.…”

Steroid Hormones as Transporters to Carry Exogenous Macromolecules into the Target Cell Nuclei in Vivo

“…BSA conjugated with steroid hormones could enter the hormone-target cell nuclei while maintaining its antigenicity. These results suggest the possibility that steroid hormone can transport the macromolecule to nucleus [31]. IgG antibodies introduced into the cells are functionally stable in the cytoplasm.…”

Nuclear Localization of Bovine Immunoglobulin G (bIgG) in the Liver of Rats Injected with Hydrocortisone-bIgG Conjugate Intravenously

“…After 2 or 3 h, the rats were perfused, fixed, frozen and sliced as described previously (Nishimura and Nakano, 2000).…”

“…30 min, then with the antibody for 2 h at room temperature, washed with PBS-, stained with 0.01% Evans blue as described previously (Nishimura and Nakano, 2000), and observed by differential interference contrast and immunofluorescence using a confocal laser scanning microscope (LSM-GB200: Olympus, Japan).…”

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Summary: Bovine serum albumin conjugated with steroid hormone injected intravenously into rats can enter the hormone-target cell nuclei with its antigenicity kept intact (Nishimura and Nakano, 2000). Antinuclear antibodies (ANA) such as immunoglobulin G (IgG) are found in some autoimmune diseases.

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