María J. Mazón

Follicle-Stimulating Hormone and Luteinizing Hormone Mediate the Androgenic Pathway in Leydig Cells of an Evolutionary Advanced Teleost

ABSTRACT The endocrine pathways controlling vertebrate spermatogenesis are well established in mammals where the pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) exclusively activate the FSH receptor (FSHR) in Sertoli cells and the LH/choriogonadotropin receptor (LHCGR) in Leydig cells, respectively. In some teleosts, however, it has been shown that Lh can cross-activate the Fshra ortholog, and that Leydig cells coexpress the Lhcgrba and Fshra paralogs, thus mediating the androgenic function of Fsh in the testis. Here, we investigated whether these proposed mechanisms are conserved in an evolutionary advanced pleuronectiform teleost, the Senegalese sole (Solea senegalensis). Transactivation assays using sole Fshra- and Lhcgrba-expressing cells and homologous single-chain recombinant gonadotropins (rFsh and rLh) showed that rFsh exclusively activated Fshra, whereas rLh stimulated both Lhcgrba and Fshra. The latter cross-activation of Fshra by rLh occurred with an EC50 4-fold higher than for rFsh. Both recombinant gonadotropins elicited a significant androgen release response in vitro and in vivo, which was blocked by protein kinase A (PKA) and 3beta-hydroxysteroid dehydrogenase inhibitors, suggesting that activation of steroidogenesis through the cAMP/PKA pathway is the major route for both Lh- and Fsh-stimulated androgen secretion. Combined in situ hybridization and immunocytochemistry using cell-specific molecular markers and antibodies specifically raised against sole Fshra and Lhcgrba demonstrated that both receptors are expressed in Leydig cells, whereas Sertoli cells only express Fshra. These data suggest that Fsh-mediated androgen production through the activation of cognate receptors in Leydig cells is a conserved pathway in Senegalese sole.

Functional Domain Analysis of the Yeast ABC Transporter Ycf1p by Site-directed Mutagenesis

The yeast cadmium factor (Ycf1p) is a vacuolar protein involved in resistance to Cd2+ and to exogenous glutathione S-conjugate precursors in yeast. It belongs to the superfamily of ATP binding cassette transporters, which includes the human cystic fibrosis transmembrane conductance regulator and the multidrug resistance-associated protein. To examine the functional significance of conserved amino acid residues in Ycf1p, we performed an extensive mutational analysis. Twenty-two single amino acid substitutions or deletions were generated by site-directed mutagenesis in the nucleotide binding domains, the proposed regulatory domain, and the fourth cytoplasmic loop. Mutants were analyzed phenotypically by measuring their ability to grow in the presence of Cd2+. Expression and subcellular localization of the mutant proteins were examined by immunodetection in vacuolar membranes. For functional characterization of the Ycf1p variants, the kinetic parameters of glutathione S-conjugated leukotriene C4 transport were measured. Our analysis shows that residues Ile711, Leu712, Phe713, Glu927, and Gly1413 are essential for Ycf1p expression. Five other amino acids, Gly663, Gly756, Asp777, Gly1306, and Gly1311, are critical for Ycf1p function, and two residues, Glu709 and Asp821, are unnecessary for Ycf1p biogenesis and function. We also identify several regulatory domain mutants in which Cd2+ tolerance of the mutant strain and transport activity of the protein are dissociated.

Activation of yeast plasma membrane ATPase by phorbol ester

Addition of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) to yeast cells produces a 2‐fold activation of the plasma membrane ATPase. The activation is reversible and time‐ and dose‐dependent. The activated enzyme shows an increased affinity for its substrate, ATP, and its optimum pH is shifted to a more alkaline range. These changes are similar to those observed in the reported activation by glucose. Upon incubation of yeast cells with 32Pi incorporation of radioactivity in a membrane polypeptide of 105 kDa is observed after addition of either glucose or TPA.

Administration of Follicle-Stimulating Hormone In Vivo Triggers Testicular Recrudescence of Juvenile European Sea Bass (Dicentrarchus labrax) 1

ABSTRACT Follicle-stimulating hormone (Fsh) is thought to act early in the process of spermatogenesis; however, its action in fish has not yet been clearly established. In the present work, we analyzed the effects of recombinant Fsh in sea bass (Dicentrarchus labrax) spermatogenesis according to two different approaches: direct injection of recombinant single-chain Fsh hormone (scFSH) and injection of scFSH coding sequence. Both approaches were efficient in increasing plasma Fsh at 7 and 15 days, respectively, after injection. The Fsh increment caused a significant increase in plasma 11-ketotestosterone levels and induced dramatic changes at the testicular level. Fsh-treated groups showed an increase in germ cell proliferation at Day 7, and cysts of spermatocytes and spermatids were observed at the end of the experiment. After treatment with Fsh, a suppression in amh transcripts and an increase of lhr transcripts were detected at Day 7 and Day 15, respectively, and an increment in fshr expression became evident at Day 23. These results show that Fsh initiates germ cell proliferation, triggering spermatogenesis in sea bass via androgen production and regulation of spermatogenesis-related genes.

Kinetic differences between two interconvertible forms of fructose-1,6-bisphosphatase from Saccharomyces cerevisiae.

Abstract Fructose-1,6-bisphosphatase (FbPase) can be partially inactivated in vivo by addition of glucose to a yeast suspension. Some kinetic properties of the active and inactivated enzymes have been studied in freshly prepared extracts. The ratio of activities found when the enzyme is assayed with 2 m m Mn 2+ and with 2 m m Mg 2+ is around 0.6 for active FbPase and increases three times for inactivated FbPase. Both forms of FbPase are inhibited by AMP noncompetitively with fructose-l,6-bisphosphate (F1,6P 2 ), however, active FbPase is less inhibited by AMP when Mn 2+ is present, while the opposite behavior is shown by the inactivated enzyme. Fructose-2,6-bisphosphate (F2,6P 2 ) is an inhibitor of both forms of FbPase the inhibition reached being dependent on the substrate concentration. The active form of FbPase is the most sensitive to F2,6P 2 inhibition ( K i in the range of 5 n m ). When assayed with Mg 2+ both forms of the enzyme were less inhibited by F2,6P 2 if AMP was present. In the presence of Mn 2+ AMP reinforced slightly the inhibition by F2,6P 2 . Inactivated FbPase has been tested at the concentrations of AMP, F2,6P 2 , and F1,6P 2 that are present in yeast treated with glucose. In these conditions the inhibition due to F2,6P 2 is only about 30%.

Ycf1-dependent cadmium detoxification by yeast requires phosphorylation of residues Ser908 and Thr911

Yeast cadmium factor (Ycf1), an ATP‐binding cassette (ABC) protein of the multidrug resistance protein subfamily, is a vacuolar GS‐conjugate transporter required for heavy metal and drug detoxification. There is evidence that phosphorylation may play a critical role in the function of ABC transporters from higher organisms. In this work, the possibility of Ycf1 phosphorylation was examined using site‐directed mutagenesis. We demonstrate that Ser908 and Thr911, within the regulatory domain (R domain), are functionally important for Ycf1 transport activity and likely sites for phosphorylation. Mutation of these residues to alanine severely impaired the Ycf1‐dependent cadmium detoxification capacity and transport activity, while replacement by acidic residues (mimicking phosphorylation) significantly suppressed the cadmium resistance and transport defects. Both in vitro treatment of Ycf1 with alkaline phosphatase and changes in the electrophoretic mobility of the S908A, T911A and double mutant S908A/T911A proteins supported the conclusion that Ycf1 is a phosphoprotein. The screening of the yeast kinome identified four protein kinases affecting cadmium detoxification, but none of them was involved directly in the phosphorylation of Ycf1. Our data strongly implicate Ycf1 phosphorylation as a key determinant in cadmium resistance in yeast, a significant finding given that very little is known about phosphorylation of ABC transporters in yeast.

Domain Interactions in the Yeast ATP Binding Cassette Transporter Ycf1p: Intragenic Suppressor Analysis of Mutations in the Nucleotide Binding Domains

The yeast cadmium factor (Ycf1p) is a vacuolar ATP binding cassette (ABC) transporter required for heavy metal and drug detoxification. Cluster analysis shows that Ycf1p is strongly related to the human multidrug-associated protein (MRP1) and cystic fibrosis transmembrane conductance regulator and therefore may serve as an excellent model for the study of eukaryotic ABC transporter structure and function. Identifying intramolecular interactions in these transporters may help to elucidate energy transfer mechanisms during transport. To identify regions in Ycf1p that may interact to couple ATPase activity to substrate binding and/or movement across the membrane, we sought intragenic suppressors of ycf1 mutations that affect highly conserved residues presumably involved in ATP binding and/or hydrolysis. Thirteen intragenic second-site suppressors were identified for the D777N mutation which affects the invariant Asp residue in the Walker B motif of the first nucleotide binding domain (NBD1). Two of the suppressor mutations (V543I and F565L) are located in the first transmembrane domain (TMD1), nine (A1003V, A1021T, A1021V, N1027D, Q1107R, G1207D, G1207S, S1212L, and W1225C) are found within TMD2, one (S674L) is in NBD1, and another one (R1415G) is in NBD2, indicating either physical proximity or functional interactions between NBD1 and the other three domains. The original D777N mutant protein exhibits a strong defect in the apparent affinity for ATP and V(max) of transport. The phenotypic characterization of the suppressor mutants shows that suppression does not result from restoring these alterations but rather from a change in substrate specificity. We discuss the possible involvement of Asp777 in coupling ATPase activity to substrate binding and/or transport across the membrane.

Efficient degradation of misfolded mutant Pma1 by endoplasmic reticulum‐associated degradation requires Atg19 and the Cvt/autophagy pathway

Misfolded proteins are usually arrested in the endoplasmic reticulum (ER) and degraded by the ER‐associated degradation (ERAD) machinery. Several mutant alleles of PMA1, the gene coding for the plasma membrane H+‐ATPase, render misfolded proteins that are retained in the ER and degraded by ERAD. A subset of misfolded PMA1 mutants exhibit a dominant negative effect on yeast growth since, when coexpressed with the wild‐type allele, both proteins are retained in the ER. We have used a pma1‐D378T dominant negative mutant to identify new genes involved in ERAD. A genetic screen was performed for isolation of multicopy suppressors of a GAL1‐pma1‐D378T allele. ATG19, a member of the cytoplasm to vacuole targeting (Cvt) pathway, was found to suppress the growth arrest phenotype caused by the expression of pma1‐D378T. ATG19 accelerates the degradation of pma1‐D378T thus allowing the co‐retained wild‐type Pma1 to reach the plasma membrane. ATG19 was also able to suppress other dominant lethal PMA1 mutations. The degradation of the mutant ATPase occurs in the proteasome and requires intact both ERAD and Cvt/autophagy pathways. We propose the cooperation of both pathways for an efficient degradation of misfolded Pma1.

Isolation and characterization of Ff1 and Gsdf family genes in European sea bass and identification of early gonadal markers of precocious puberty in males.

Puberty represents the transition from an immature to a mature reproductive stage. The mechanisms underlying the onset of normal or precocious puberty have not yet been elucidated. With the goal of gaining an understanding of early events that occur in the testes of precocious animals during this process, a hemigonadectomy was performed on male juvenile sea bass and expression levels of candidate mRNAs were determined through quantitative real-time RT-PCR. For this purpose, the gonadal soma-derived factors gsdf1 and gsdf2, the nuclear receptor 5 subfamily members nr5a1a (ff1b), nr5a1b (ff1d), nr5a2 (ff1a) and nr5a5 (ff1c) and the proliferating cell nuclear antigen or pcna, genes with a putative role in the beginning of spermatogenesis, were isolated and cloned. Hemigonadectomy proved to be a suitable strategy for the study of gonadal stages prior to the appearance of histological differences between precocious and non-precocious fish, as it allowed the subsequent classification of these gonads. The upregulation of the gene encoding the steroidogenic acute regulatory protein (Star) in precocious testes indicates that sex steroids could play a role in the onset of spermatogenesis in sea bass. In contrast, the downregulation observed in ff1b expression indicates that this initial surge in star expression is not the result of Ff1b transactivation, suggesting an alternative pathway for this transcriptional activation. Finally, a decrease in gsdf1 expression in precocious animals suggests that this gene may play a role in the onset of puberty, while its correlation with ff1b expression points to gsdf1 as a putative target for Ff1b-mediated transactivation.

Pitfalls in the measurement of membrane potential in yeast cells using tetraphenylphosphonium

The uptake of the lipophilic cation tetraphenylphosphonium (Ph4P+) by Saccharomyces cerevisiae was measured using yeast grown on glucose and harvested either at the logarithmic or at the stationary phase of growth. When yeast was collected at the stationary phase, Ph4P+ uptake proceeded steadily during several hours until an equilibrium was reached. When yeast was collected in the logarithmic phase of growth, a biphasic uptake was observed. The second phase of uptake began when the glucose of the incubation medium had been exhausted. From experiments in the presence of cycloheximide or chloramphenicol it is concluded that the second phase of Ph4P+ uptake is dependent on the synthesis of some protein(s) repressed by glucose but unrelated with the existence of functional mitochondria. The addition of compounds which collapse the membrane potential provokes an efflux from the yeast cells of the Ph4P+ accumulated both during the first phase and the second phase of uptake. It is concluded that accumulation of Ph4P+ in yeast cells is a complex process and that Ph4P+ cannot be used to give a quantitative measure of the yeast plasma membrane potential.