ABSTRACT

To investigate the participation of intracellular steroid hormone receptors in the sexual transformation process of the Harderian gland, a series of experiments were undertaken in adult golden hamsters. The in-vitro labelling of cytosolic steroid-binding sites with appropriate radioligands revealed the presence of androgen, oestrogen and glucocorticoid but not progestin receptors in the glands from animals of both sexes. The androgen receptor of the female gland was further characterized because it was found to be the predominant intracellular steroid receptor. Studies of binding kinetics using [³H]7α,17α-dimethyl-17β-hydroxy-4-oestren-3-one (DMNT) as ligand, demonstrated a high affinity androgen-binding site with an apparent dissociation constant (K _d) of 0·7 nmol/l and maximal saturation binding capacity of 84·0 ± 3·0 (s.d.) fmol/mg protein. Specificity of the androgen receptor was assessed by displacement analysis; DMNT, 5α-dihydrotestosterone, testosterone and 3α-androstanediol were efficient competitors for the androgen-binding site, while oestradiol-17β, progesterone and dexamethasone exhibited very little, if any, competitive potency. The sedimentation coefficient of the androgen receptor in sucrose density gradients was 8–9 S. These data indicate that the physicochemical characteristics of the androgen receptor from the female gland are similar to those previously described in the male gland. The striking observation of a complete lack of oestrogen-inducible and oestrogen-insensitive progestin receptors in glands cytosol, even after stimulation with cholera toxin, adds further support to the concept that the androgen receptor is the key molecule mediating the hormone-induced sexual transformation of the Harderian gland in this species.

Journal of Endocrinology (1989) 121, 149–156

Ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), the terminal enzyme of the haem biosynthetic pathway, catalyses the insertion of ferrous iron into protoporphyrin IX to form protohaem. The Syrian hamster Harderian gland (HG) is known for its ability to produce and accumulate large amounts of protoporphyrins. In this species, the female gland contains up to 120 times more porphyrin than the male gland. Data from biochemical studies suggest that this gland possesses the enzymatic complex for haem biosynthesis but lacks ferrochelatase activity. The abundance of intraglandular haem proteins does not support this idea. To gain more insight into this process, we isolated cDNA for ferrochelatase from hamster liver, using the 5′- and 3′- rapid amplification of complementary DNA ends (RACE), and investigated its expression in HG from males and females. The full-length cDNA comprises an open reading frame of 1269 bp encoding a polypeptide of 422 amino-acid residues. Hamster DNA sequence exhibits 92% identity to mouse and 87% identity to human sequences. The predicted hamster enzyme was shown to have structural features of mammalian ferrochelatase, including a putative NH₂- terminal presequence, a central core of about 330 amino-acid residues and an extra 30–50-amino-acid stretch at the carboxyl-terminus. RNA blotting experiments indicated that this cDNA hybridized to a liver mRNA of about 2.1 kb, while a weak hybridization signal was observed with mRNA from HG preparations. RT–PCR assays confirmed the expression of specific transcripts in both tissues. Male glands contained approximately twofold more enzyme mRNA than female glands. Likewise, the intraglandular content of mRNA varied during the oestrous cycle, with the highest levels found in the oestrous phase. These cyclic variations were less evident in liver. Ovariectomy plus treatment with progesterone or 17β-oestradiol plus progesterone increased ferrochelatase mRNA of the gland. In HG of short- or long-term castrated males, the administration of testosterone did not affect the ferrochelatase mRNA concentration. Based on mRNA expression levels, we conclude that Harderian ferrochelatase may play an active role in maintaining the physiological pool of haem required for processing cytochromes and other glandular haem proteins. Likewise, the sex-steroid hormones appear to have only a modest influence upon Harderian ferrochelatase.

ABSTRACT

Studies were conducted in castrated golden hamsters to assess whether sexual dimorphism and sensitivity to sex steroid hormones in the rodent Harderian gland are mediated by an interaction of androgens with specific intracellular receptors. Physical properties, binding kinetics and stereospecificity of the androgen receptor were analysed using [³H]mibolerone as the radioligand. The presence of [³H]mibolerone–androgen receptor complexes with a sedimentation coefficient of 7–8S was demonstrated in Harderian gland cytosol by a linear sucrose gradient ultracentrifugation technique using a vertical rotor. Kinetic analysis revealed an androgen-binding site with an apparent dissociation constant of 0·3±0·07 (s.d.) nmol/l and a saturation binding capacity of 113±15 fmol/mg protein. Displacement studies indicated that unlabelled mibolerone, methyltrienolone, 5α-dihydrotestosterone and testosterone were efficient competitors for the androgen-binding sites, while progesterone, 17β-oestradiol, dexamethasone, dehydroepiandrosterone, ethiocholanolone and 5α-16-androsten-3-one were not. Experiments in long-term castrated animals revealed that the Harderian gland androgen receptor concentration and sedimentation coefficient remained unmodified. The results of these studies were interpreted as demonstrating the presence of a specific high-affinity intracellular androgen receptor in the male hamster Harderian gland.

J. Endocr. (1987) 112, 3–8