Functional studies of p.R132C, p.R149C, p.M283V, p.E431K, and a novel c.652-2A>G mutations of the CYP21A2 gene.

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is the most frequent inborn error of metabolism and accounts for 90-95% of CAH cases. In the present work, we analyzed the functional consequence of four novel previously reported point CYP21A2 mutations -p.R132C, p.R149C, p.M283V, p.E431K- found in Argentinean 21-hydroxylase deficient patients. In addition, we report an acceptor splice site novel point mutation, c.652-2A>G, found in a classical patient in compound heterozygosity with the rare p.R483Q mutation. We performed bioinformatic and functional assays to evaluate the biological implication of the novel mutation. Our analyses revealed that the residual enzymatic activity of the isolated mutants coding for CYP21A2 aminoacidic substitutions was reduced to a lesser than 50% of the wild type with both progesterone and 17-OH progesterone as substrates. Accordingly, all the variants would predict mild non-classical alleles. In one non-classical patient, the p.E431K mutation was found in cis with the p.D322G one. The highest decrease in enzyme activity was obtained when both mutations were assayed in the same construction, with a residual activity most likely related to the simple virilizing form of the disease. For the c.652-2A>G mutation, bioinformatic tools predicted the putative use of two different cryptic splicing sites. Nevertheless, functional analyses revealed the use of only one cryptic splice acceptor site located within exon 6, leading to the appearance of an mRNA with a 16 nt deletion. A severe allele is strongly suggested due to the presence of a premature stop codon in the protein only 12 nt downstream.

Characterization and seasonal changes in LHß and FSHß mRNA of Rhinella arenarum (Amphibia, Anura).

In anurans, two types of gonadotropins were described in several species of Ranidae and Pipidae families but only in one of the Bufonidae family. Rhinella arenarum is a bufonid that have the lowest concentration of plasma androgens during the breeding. The objective of this paper was to characterize the cDNA sequence of ß subunit of LH and FSH from toad pituitary and study seasonal variation in gonadotropins mRNA using quantitative real-time RT-PCR. The LHß cDNA is a 636 bp sequence containing an open reading frame (ORF), 45 bp of 5'-untranslated region (UTR) and 174 bp of 3'-UTR. The ORF encodes for a signal peptide of 26 amino acids and a mature protein of 113 amino acids with one N-glycosylation site at the 34th position. The FSHß cDNA sequence is a 535 bp fragment containing an ORF, 8 bp of 5'-UTR and 152 bp of 3'-UTR. The ORF encodes for a signal peptide of 20 amino acids and a mature protein of 104 amino acids with two N-glycosylation sites at 25th and 42nd positions. Multiple alignments of aminoacid deduced sequences of LHß and FSHß (teleosts, amphibians, birds, mammals) showed that all the tetrapods studied conserve 12 cysteins and one (LH) or two (FSH) N-Glycosylation sites. LHß is closer to teleosts than to mammals and birds while FSHß is closer to mammals. The analysis of seasonal changes in LHß and FSHß mRNA indicates that transcript levels have seasonal variations and that the profile of androgens is opposite to that of the gonadotropins mRNA.

Mechanism of hCG-induced spermiation in the toad Rhinella arenarum (Amphibia, Anura).

In Rhinella arenarum spermiation occurs as a consequence of LH/FSH increase during the amplexus or by a single dose of hCG, among other gonadotropins. The present study employs an in vitro system to study the mechanism of action of hCG in the spermiation of R. arenarum. Testicular fragments were incubated for 2h at 28°C in the presence or absence of 20IU hCG with or without different PKA/PKC inhibitors and activators as well as ouabain and amiloride as Na(+)/K(+) ATPase and transcellular Na(+) transport inhibitors, respectively. Ouabain did not induce spermiation in absence of hCG and inhibited hCG-induced spermiation in a dose-dependent manner, reaching 90% inhibition with the higher concentration. In contrast, amiloride neither affected spermiation nor steroidogenesis. Activation of PKA with 8Br-cAMP induced spermiation in the absence of hCG while its inhibition with H89 blocked hCG action. On the other hand, PKC inhibition with Bi or STP did not affect hCG-induced spermiation although PKC activation significantly decreased hCG-dependent sperm release. These results suggest that PKC inhibits spermiation but also that the inhibition exerted by the kinase could be blocked by hCG. Taken together, these observations could indicate that PKA is involved in the mechanism of the gonadotropin action, mechanism also requiring the activation of a non-pumping Na(+)/K(+) ATPase pathway.

New lead compounds in the search for pure antiglucocorticoids and the dissociation of antiglucocorticoid effects.

Antiglucocorticoids that act as antagonists at the glucocorticoid receptor (GR) level may be used to block or modulate the undesirable effects of glucocorticoid excess (from endogenous or exogenous origin). RU486 developed in the early 80s, is an antiglucocorticoid but also a potent antiprogestin and abortifacient, nevertheless it still remains as the only GR antagonist drug in the market. Further on, in view of the variety of physiological processes in which glucocorticoids are involved, selective antiglucocorticoids that can block only some of these processes (eventually with tissue specificity) would be highly desirable. The bridged pregnane 21-hydroxy-6,19-epoxyprogesterone, was developed as an alternative lead being an antagonist of the GR with no affinity for mineralocorticoid and progesterone receptors. Antagonistic activity was evidenced by partial blocking of dexamethasone induction of tyrosine aminotransferase (TAT) and thymocyte apoptosis. Replacement of the oxygen bridge by a sulfur bridge gave a less bent, more flexible molecule. 21-Hydroxy-6,19-epithioprogesterone exhibited improved antiapoptotic activity on thymocytes but was not effective blocking TAT induction. This selectivity was improved further by oxidation to the sulfone. The sulfone but not the reduced compound also reverted the dexamethasone-mediated inhibition of NFkappaB activity in HeLa cells. Blocking of the apoptotic effect of TNFalpha by dexamethasone in the L929 cell line (mouse fibroblasts), was only reverted partially by the sulfone which exhibited a mild agonistic/antagonistic activity in this assay. None of these compounds showed antiprogestin activity. Similar overall molecular shapes but more lipophylic and with higher metabolic stability were obtained by introduction of a methylene bridge (6,19-methanoprogesterone) or by a direct bond between C-6 and C-19 (6,19-cycloprogesterone and its 21-hydroxy derivative). The latter highly bent steroids showed affinity for the GR. Recently we performed molecular dynamics simulations of GR-ligand complexes to investigate the molecular basis of the passive antagonism exhibited by 21-hydroxy-6,19-epoxyprogesterone. On the basis of our findings, we proposed that the passive antagonist mode of action of this antiglucocorticoid analog resides, at least in part, in the incapacity of GR-21-hydroxy-6,19-epoxyprogesterone complex to dimerize, making the complex unable to activate gene transcription.

Relationship between steroidogenesis and spermiation in Rana catesbeiana and Leptodactylus ocellatus.

The present study employs an in vitro system to analyse the role of steroid hormones in hCG-induced spermiation in two species of anuran amphibian: Rana catesbeiana and Leptodactylus ocellatus. In vitro spermiation was induced with 10 IU hCG and the effect of different steroid-biosynthesis inhibitors was analysed. Cyanoketone (10(-5)M), an inhibitor of 3-oxo-4-ene steroid biosynthesis, did not block hCG-inducing activity even when biosynthesis of androgen was significantly reduced. These results clearly showed that, in both species, spermiation-inducing action of hCG does not depend on the biosynthesis of 3-oxo-4-ene steroids. Moreover, when combined inhibitors, aminoglutethimide (10(-5)M) plus cyanoketone (10(-5)M), were employed, spermiation evoked by hCG was not modified while hCG-induced androgen secretion significantly decreased. Additionally, none of the steroids used, progesterone, 17, 20 alpha-dihydroxy-4-pregnen-3-one, testosterone and 5 alpha-dihydrotestosterone, were able to induce spermiation in the absence of hCG, confirming that steroids are not involved in that process. In conclusion, as previously described in Bufo arenarum, in L. ocellatus and R. catesbeiana hCG-induced spermiation does not depend on steroid biosynthesis.

Effects of mGnRH on testicular steroidogenesis in the toad Bufo arenarum.

GnRH controls vertebrate reproduction in several ways. This hormone not only affects the secretion of gonadotropins from the pituitary gland but also has a direct influence on several gonadal functions such as steroidogenesis, spermatogenesis, and spermiation. In the present paper we have studied the in vitro effects of GnRH on the testicular steroidogenesis of Bufo arenarum to ascertain the role of this peptide in the control of the steroidogenic pathway previously described in this species. It was found that GnRH is able to reduce basal as well as hCG-stimulated testosterone release, having an inhibitory effect on P450(c17) activity. Thus, GnRH could be involved in the mechanism that regulates the metabolic change in the testicular steroidogenesis. Additionally, testicular GnRH binding site has been characterised, showing a K(d) of 34 nM and a maximum binding of 4.7 pmol/mg protein.