Journal of steroid biochemistry最新文献

The relative binding affinities of a series of twelve para-hydroxylated triphenylethylenes (TPEs) for the estradiol receptor (ER) of calf uterus cytosol were measured by a competition method. The results obtained under equilibrium conditions support the hypothesis of the additivity of the energies corresponding to each of the hydrogen-bond type interactions of di- or tri-hydroxylated TPEs with the estradiol binding site of ER and strongly suggest that, whichever ring is hydroxylated, the orientation of the TPE in the steroid binding site is always the same. A hydroxyl group in a given position always interacts with the same location within the site.

Mono-hydroxylation of the highly hydrophobic non-substituted TPE skeleton led to a large increase in relative binding affinity for ER which could be explained by a dual mechanism whereby the interaction specific to the hydroxyl is accompanied by a temperature- or time-dependent binding process that is not related to the hydroxylation position.

After neonatal administration of supraphysiological doses of oestradiol, the concentration of tissue proteins, in adult mice, was significantly reduced by 39, 45 and 56% in epididymis, vas deferens and seminal vesicle respectively. The protein profiles showed persistent alterations. In epididymis, 4 protein bands were differentially increased (14.4, 43 and 67 kDa) or reduced (24 kDa) in oestrogenized males. In vas deferens, 4 proteins were increased (14.4, 49,67 and 76 kDa) and one (34 kDa) virtually absent. In seminal vesicle, about 20 proteins of varying molecular weights (12–140 kDa) were differentially increased or decreased. Testosterone substitution, at adulthood, was unable to reverse these effects. Treatments with oestradiol during adult life induced persistent alterations in the protein profiles of the 3 organs but, in contrast to neonatal treatment, these alterations could be reversed by androgen therapy. A cDNA library has been constructed with RNA prepared from adult seminal vesicle and screened by differential hybridization. Neonatal oestrogenization strongly reduced the abundance of some mRNA species. Eleven recombinants containing putative oestrogen-sensitive sequences were isolated. Two of them, having an insert of about 500 base pairs, were used for dot-blot hybridization. Results showed that the two clones contained sequences which were differently regulated by androgens.

Ovarian cells of pregnant rats were cultured with synthetic progestins (R5020, R2323), dexamethasone and RU486. Progesterone and 20α-hydroxy-pregn-4-en-3-one (20α-dihydroprogesterone) in the medium were measured by specific radioimmunoassay. Both R5020 and R2323 increased concentrations of these intrinsic progestins. RU486 decreased concentrations of progesterone, however, the addition of R5020 or R2323 counteracted this action.

Immature hypophysectomized rats treated with pregnant mare serum gonadotropin (PMS) and human chorionic gonadotropin (hCG) were administered with RU486; the serum levels of progesterone and 20α-dihydroprogesterone tended to decrease.

R5020 and R2323 inhibited the effect of 3β -hydroxy steroid dehydrogenase (3β-HSD), whereas RU486 did not. Inhibition of the cholesterol side chain cleavage enzyme (CSCC) by RU486 was more marked than that by R5020 or R2323.

These results show that RU486 decreases progesterone synthesis in cultured ovarian cells. A part of the mechanism may involve an inhibition of CSCC.

Methandrostenolone dose (amount and duration) and methods of isolation from urine can influence the identification and quantitation of methandrostenolone metabolites. Long-term use of methandrostenolone at high dosages led to the appearance of unmetabolized drug in the urine and contributed to the identification of a previously unreported metabolite, 3β,6§,17β-trihydroxy-17α-methyl-5§-1-androstene. Exposure of methandrostenolone in vitro to acid conditions induced a retropinacol rearrangement in the D-ring of the methandrostenolone molecule, causing the formation of 18-nor-17,17-dimethyl-1,4,13(14)-androstatrien-3-one in large amounts. The same acidic conditions led to the addition of a hydroxyl at the 6 position of the B-ring of either the retropinacol rearrangement products or native methandrostenolone resulting in the formation of 6β-hydroxy-18-nor-17, 17-dimethyl-1,4,13(14)-androstatrien-3-one,6α-hydroxy-18-nor-17,17-dimethyl-1,4,13(14)-androstatrien, 6β-17α-methyl-1,4-androstadien-3-one and 6α,17β-dihydroxy-17α-methyl-1,4-androstadien-3-one. Hydroxylation of native methandrostenolone at the 6 position also occurs endogenously. However, no evidence of an endogenous retropinacol rearrangement was found. Silylating agents alone can induce the formation of small amounts of 6β,17β-dihydroxy-17α-methyl-1,4-androstadien-3-one. Discrepancies between previously published reports on methandrostenolone metabolism in man are discussed and compared with an animal model.

Granulosa cells of the ovarian follicle are unique in that they both synthesize steroid hormones and respond to exogenously-administered steroids. Isolated granulosa cells from ovaries of gonadotropin-primed rats were incubated in the presence of [³H]testosterone, which the cells convert to [³H]estradiol. Nuclear extracts of these cells were analyzed by high-performance liquid chromatography in a system of 40% acetonitrile. When cells were incubated in the presence of [³H]testosterone alone, a significant portion of the radioactivity present in nuclei co-eluted with authentic estradiol. The nuclear binding was considered to be specific, since 50–75% of total binding was suppressed when the incubation medium contained excess unlabeled estrogen. Moreover, when an antibody to estradiol was included in the medium, specific nuclear uptake of [³H]estradiol was not abolished, but rather was increased. Granulosa cells may, therefore, directly utilize endogenously-produced estradiol, a mechanism which may play a role in the regulation of ovarian cells.

The effects of the antiprogestins (APs) ZK 98.299, ZK 98.734 and RU 486 on GnRH-stimulated LH secretion and their antagonistic activity on progesterone (P) actions were investigated in cultured pituitary cells from adult female Wistar rats. P (100 nM) was able to exert a facilitatory effect on GnRH (1 nM)-induced LH secretion after short-term (4h) treatment of estradiol-primed (1 nM, 48 h) rat pituitary cells. When the APs (10 pM–10 μM) were introduced during the 4 h incubation period with P the facilitatory effect of P was totally abolished at concentrations > 10 nM (ZK 98.299, ZK 98.734) and > 1 nM (RU 486). Also the APs were shown to block the inhibitory action of P which occurs after long-term incubation of pituitary cells with this steroid. However at concentrations > 10 nM (ZK 98.734, RU 486) and > 100 nM (ZK 98.299) this antagonistic action of the APs was lost. To evaluate whether the APs have direct effects on GnRH-induced LH secretion in the absence of exogenous P pituitary cells cultivated for 48 h with or without 1 nM estradiol were incubated for 4 or 24 h with increasing concentrations of the APs (10 pM–10 μM). Four hour treatment of nonestradiol-primed cells with ZK 98.299 or ZK 98.734 was without any effect on the LH response to a 1 nM GnRH-stimulus. Only the highest concentration of RU 486 (10 μM) reduced the LH response. Twenty-four hour treatment of the cultures with the APs led to enhancement of GnRH-stimulated LH secretion by up to 113, 37 and 33% for ZK 98.734, ZK 98.299 and RU 486, respectively. When estradiol-primed cells were used for the same experiments we observed exclusively inhibitory effects on GnRH-induced LH secretion after 4 and 24 h treatment periods.

It is concluded that these new APs are potent inhibitors of P-actions, but also per se they induce diverse effects on GnRH-stimulated LH secretion in cultured rat pituitary cells which have to be taken into account.