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The steroid binding properties of the human pancreatic estrogen binding protein (hEBP) in cytosol were studied by equilibrium dialysis. A high ligand specificity of the protein was revealed. hEBP in cytosol binds unconjugated steroid estrogens with a medium affinity (Kd = 10−7M) but does not bind conjugated estrogens or unconjugated androgens, gestagens, glucocorticoids or cholesterol. Quantitation of hEBP by radioimmunoassay in subcellular fractions of human pancreatic tissue indicated that the protein is translocated into different subcellular compartments. Duodenal juice taken from patients following stimulation of pancreatic secretion by food ingestion (Lund's test) showed high hEBP concentrations, and the levels of hEBP changed concomitantly with the levels of pancreatic isoamylase, indicating that hEBP secretion was stimulated by food ingestion. The levels of hEBP in peripheral serum from healthy subjects showed no sex difference, but were positively correlated to age. Highly elevated (10–20-fold) hEBP levels were found in serum from patients wih acute pancreatitis, while normal serum hEBP values were found in other abdominal diseases. It is speculated that hEBP might have a specific role in the transport of estrogens from the peripheral circulation via the pancreas to the duodenum. The elevated hEBP levels in patients with acute pancreatitis indicate that this protein may be used as a marker of cellular damage in the pancreas.

Steroid metabolizing enzyme activities in rat liver may be classified according to their regulation. One group consists of enzymes, the activities of which are irreversibly determined at birth by androgens, a second group encompasses enzymes that are reversibly affected by androgens and a third group is made up of enzymes essentially independent of androgens. The mechanisms by which androgens influence hepatic enzyme activities are still not clear but the hypophysis seems to be essential for the sexual differentiation of liver metabolism in the rat. Possibly this control is exerted by a hypophyseal feminizing factor that feminizes a basal masculine enzyme pattern in rat liver. Some kind of direct androgen action on the liver cell may also be postulated since a radioactively labelled complex between 4-androstene-3,17-dione and a high affinity protein can be extracted from liver cytosol and nuclei after intrapertitoneal administration of [1,2,6,7- 3 H]-testosterone to castrated male and female rats. This androgen “receptor” moves as a 10 S protein when analysed by sucrose density gradient centrifugation, has a pI of about H5 when analysed by isoelectric focusing and is selectively concentrated in unlabelled liver nuclei when these are incubated with liver cytosol containing labelled androgen-protein complex.

A radioimmunoassay for quantitation of the human pancreatic estrogen binding protein (hEBP) was developed using polyclonal rabbit hEBP antiserum and iodinated purified hEBP. Parallel dose-response curves were obtained when serial dilutions of human serum and of cytosols obtained from human pancreas, prostate and colon were analyzed simultaneously with serial dilutions of purified hEBP standard. Very high levels of hEBP (500–1000 mg/kg wet weight) were found in normal pancreas. High as well as medium levels were found in pancreatic carcinoma tissue and medium values (0.1–1 mg/kg wet weight) in prostate, colon and ovarian tissue. Other tissues and serum from healthy volunteers showed low values, usually below 0.1 mg/kg. When serial dilutions of rat pancreatic cytosol were analyzed in the hEBP assay, [ 125 I]hEBP was displaced by the rat preparation, but the dose-response curves were not parallel to the standard curves, indicating similarity but non-identity between the estrogen binding proteins in human and in rat pancreas.

At birth testicular androgens irreversibly programme hypothalamic centres involved in hypothalamopituitary control of hepatic sex-dependent steroid and drug metabolism. This imprinting process results in activation of a hypothalamic ‘feminostatin’—secreting centre that is turned on just before puberty. Feminostatin inhibits pituitary secretion of ‘feminotropin’, a novel pituitary hormone that feminizes the basal type of metabolism characterizing the autonomous liver, i.e. the liver of hypophysectomized and gonadectomized rats. Consequently, female rats that are devoid of feminostatin will secrete feminotropin from the pituitary, leading to a feminine type of hepatic metabolism. Male rats, on the other hand, have an active feminostatin-secreting centre and the inhibition of pituitary feminotropin release results in an autonomous type of liver metabolism. Full ‘masculinity’ of hepatic metabolism in male rats is induced by testicular androgens that act on the hypothalamo-pituitary axis to stimulate release of a pituitary ‘masculinizing factor’. Female rats show a relative androgen unresponsiveness and seem incapable of releasing masculinizing factor after treatment with androgens. A possible explanation for this is the absence of androgen receptor proteins in female brain as contrasted to the presence of androgen receptors in male pituitary, hypothalamus and pineal gland. It seems reasonable to assume on the basis of present knowledge that the level of androgen receptors in the central nervous system is programmed at the time of birth by testicular androgens. The hypothesis presented above has been based on results obtained from experiments involving hypophysectomy, pituitary transplants under the kidney capsule, electrothermic lesions in the hypothalamus, neonatal and postpubertal castrations, treatment with androgens and estrogens at various times during development and transplantations with different lines of pituitary tumour cells. The novel pituitary hormone, feminotropin, has also been studied in vitro using hepatoma (HTC) cells or isolated hepatocytes in tissue culture. Feminotropin increases the apparent 5α-reductase activity in HTC cells at subsaturation concentrations of substrate (androstenedione) by decreasing the K m value of the enzyme. Furthermore, feminotropin increases the apparent 5α/16α ratio in isolated hepatocytes, i.e. the ratio between total 5α-reduced and 16α-hydroxylated metabolites formed from androstenedione. Feminotropin has been shown to be different from any of the known pituitary hormones. It is stored in pituitary granules with a density of 1.13–1.17 g/cm 3 and has a molecular weight of about 20,000 daltons. Feminotropin is produced by isolated pituitary cells in primary culture and by certain lines of pituitary tumour cells, e.g. C 8 11RAP cells. Tissue culture medium from cultures of this cell line serves as source in our purification programme for feminotropin.

Hybridomas producing antibodies against rat prostatic estramustine binding protein (rEMBP) have been obtained by fusion of spleen lymphocytes from Balb/c mice, immunized with rEMBP, and SP2/0 Ag 14 mouse myeloma cells. Anti-rEMBP IgG producing cultures were identified in a solid phase ELISA using alkaline phosphatase conjugated sheep antimouse IgG. Cultures producing specific antibodies were subcloned and expanded. The produced immunoglobulins were characterized according to interaction with subunits of rEMBP. No interaction was found with [3H]estramustine-human estramustine binding protein. Following development of a radioimmunoassay, tissue distribution of rEMBP in the rat was studied. Despite high specificity, shown by selective interaction with rEMBP (F and S subunits), macromolecules interacting with anti-rEMBP were found in high concentrations in the prostrate and also in low concentrations, in other tissues of the male genital tract, and further in the adrenals, pancreas and submaxillary gland. The high specificity also makes it possible to study the F and S subunit selectively. These results show that macromolecules very similar to rEMBP are present in hormone-sensitive tissues in the rat.

When castrated male rats were injected intraperitoneally with [1,2,6,7- 3 H]-testosterone, liver nuclei specifically retained [ 3 H]-androstenedione. Treatment of labelled nuclei with 0.4 M KC1 resulted in extraction of an androstenedione-protein complex with a pI of 5.1. An androstenedione-protein complex with identical pI was also found in liver cytosol; this complex had a sedimentation coefficient of 3.3 s and was eluted at 0.55 column vol. when chromatographed on a Sephadex G-100 column. The 3.3 s protein bound androstenedione with a limited number of binding sites. In reconstitution experiments where labelled liver cytosol was incubated with unlabelled nuclei from castrated male rats it was possible to demonstrate specific nuclear uptake of the androstenedione-protein complex from the cytosol; it could also be shown that this process was saturable and that the uptake increased with time. It is suggested that the androstenedione-binding protein in rat liver cytosol plays a role in the intranuclear transfer of androstenedione.

The estrogen binding protein in human pancreas has been purified from pancreatic cytosol by chromatography on Concanavalin-A-Sepharose and hydroxyl-apatite followed by ion-exchange chromatography carried out using a fast-protein liquid chromatography apparatus (FPLC). The purified protein, still able to bind labelled [3H]estradiol, appeared as one single band corresponding to 31 K in SDS-gel electrophoresis. Total amino acid analysis revealed high levels of histidine, glutamic acid and leucine. The capacity of the purified protein to bind estrogens could be increased more than 4-fold by addition of a cytosolic factor, probably being a small peptide, that is present in crude cytosol, but lost during the purification procedure. The iodinated protein does not bind to DNA-cellulose or phosphocellulose, and shows no similarities to estrogen receptor proteins.

An estramustine binding protein, in many aspects similar to the prostatic secretion protein (PSP), has partly been characterized in the submaxillary gland of the male rat. The [3H]estramustine-macromolecule complex is found in the void volume of a Sephadex G 200 column, indicating a Stokes radius larger than 52 A. The estramustine binding protein is bound to Concanavalin-A, indicating a glycoprotein structure. Like PSP, the macromolecule complex that is bound to Concanavalin-A inhibits the binding of the androgen-receptor complex to DNA-cellulose. The concentration of the protein is decreased following testectomy or estrogen treatment but can be restored to normal values following testosterone administration. These results strongly indicate that the estramustine binding macromolecule in the submaxillary gland belongs to the same group of proteins as PSP. We have earlier proposed a role for PSP as an intracellular regulator of androgen activity. Based on these new results it is tempting to speculate that androgen sensitive glycoproteins may act in the same way in all androgen sensitive tissues.