Estrogens: a new player in spermatogenesis.

The mammalian testis serves two main functions: production of spermatozoa and synthesis of steroids; among them, estrogens are the end products obtained from the irreversible transformation of androgens by aromatase. The aromatase is encoded by a single gene (cyp19) in humans which contains 18 exons, 9 of them being translated. In rat the aromatase activity is mainly located in Sertoli cells of immature animals and then in Leydig cells of adults. Moreover rat germ cells represent an additional source of estrogens: the amount of P450arom transcript is 3-fold higher in pachytene spermatocytes (PS) compared to gonocytes or round spermatids (RS); conversely, aromatase activity is more intense in haploid cells. Male germ cells of mice, bank vole, bear and monkey express also aromatase. In man besides Leydig cells, we have shown the presence of a biologically active aromatase and of estrogen receptors in ejaculated spermatozoa and in immature germ cells. Concerning aromatase, a 30% decrease of the amount of mRNA is observed in immotile compared to motile sperm fraction from the same sample; moreover the aromatase activity is also diminished of 34%. In asthenoteratozoospermic and teratozoospermic patients the aromatase gene expression is decreased by 67 and 52%, respectively when compared to normospermic controls. Statistical analyses between the sperm morphology and the aromatase/GAPDH ratio have revealed a high degree of correlation (r=-0.64) between the ratio and the percentage of abnormal spermatozoa (especially microcephaly and acrosmome malformations). Alterations of sperm number and motility have been described in men genetically deficient in aromatase, which together with our data, suggest a likely role for aromatase/estrogens in the acquisition of sperm motility. Therefore besides gonadotrophins and testosterone, estrogens produced locally should be considered as a physiologically relevant hormone involved in the regulation of spermatogenesis and spermiogenesis

Aromatase expression and role of estrogens in male gonad : a review

The ability of the testis to convert irreversibly androgens into estrogens is related to the presence of a microsomal enzymatic complex named aromatase, which is composed of a specific glycoprotein, the cytochrome P450 aromatase (P450arom) and an ubiquitous reductase. The aromatase gene is unique in humans and contained 18 exons, 9 of them being translated. In the rat testis we have immunolocalized the P450arom not only in Leydig cells but also in germ cells and especially in elongated spermatids. Related to the stage of germ cell maturation, we have shown that the level of P450arom mRNA transcripts decreases, it is much more abundant in pachytene spermatocytes and round spermatids than in mature germ cells whereas the aromatase activity is 2–4 fold greater in spermatozoa when compared to the younger germ cells. Using a highly specific quantitative competitive RT-PCR method we have evidenced that several factors direct the expression of the aromatase gene in Leydig cells, Sertoli cells, pachytene spermatocytes and round spermatids, and it is obvious that promoter PII is the main one but other promoters could be concerned. In the bank-vole testis we have observed a positive correlation between a fully developed spermatogenesis and a strong immunoreactivity for both P450arom and estrogen receptor β not only in Sertoli cells but also in pachytene spermatocytes and round spermatids. Our recent data obtained from ejaculated human spermatozoa demonstrate the presence of aromatase both in terms of mRNA and protein, and in addition, we suggest that aromatase could be involved in the acquisition of sperm motility. Indeed in men the congenital aromatase deficiency is associated with severe bone maturation problems and sterility. Together with the widespread distribution of estrogen receptors in testicular cells these data clearly show that estrogens play a physiological role in the regulation of spermatogenesis in mammals

The Collagen V Homotrimer [α1(V)]3 Production Is Unexpectedly Favored over the Heterotrimer [α1(V)]2α2(V) in Recombinant Expression Systems

Collagen V, a fibrillar collagen with important functions in tissues, assembles into distinct chain associations. The most abundant and ubiquitous molecular form is the heterotrimer [α1(V)]2α2(V). In the attempt to produce high levels of recombinant collagen V heterotrimer for biomedical device uses, and to identify key factors that drive heterotrimeric chain association, several cell expression systems (yeast, insect, and mammalian cells) have been assayed by cotransfecting the human proα1(V) and proα2(V) chain cDNAs. Suprisingly, in all recombinant expression systems, the formation of [α1(V)]3 homotrimers was considerably favored over the heterotrimer. In addition, pepsin-sensitive proα2(V) chains were found in HEK-293 cell media indicating that these cells lack quality control proteins preventing collagen monomer secretion. Additional transfection with Hsp47 cDNA, encoding the collagen-specific chaperone Hsp47, did not increase heterotrimer production. Double immunofluorescence with antibodies against collagen V α-chains showed that, contrary to fibroblasts, collagen V α-chains did not colocalized intracellularly in transfected cells. Monensin treatment had no effect on the heterotrimer production. The heterotrimer production seems to require specific machinery proteins, which are not endogenously expressed in the expression systems. The different constructs and transfected cells we have generated represent useful tools to further investigate the mechanisms of collagen trimer assembly

Evaluation of branched GDGTs and leaf wax n-alkane δ2H as (paleo) environmental proxies in East Africa

The role of mountain evolution on local climate is poorly understood and potentially underestimated in climate models. One prominent example is East Africa, which underwent major geodynamic changes with the onset of the East African Rift System (EARS) more than 250 Myr ago. This study explores, at the regional East African scale, a molecular approach for terrestrially-based paleo-climatic reconstructions that takes into account both changes in temperature and in altitude, potentially leading to an improved concept in paleo-climatic reconstructions. Using surface soils collected along pronounced altitudinal gradients in Mt. Rungwe (n=40; Southwest Tanzania) and Mt. Kenya (n=20; Central Kenya), we investigate the combination of 2 terrestrial proxies, leaf wax n-alkane δ2H (δ2Hwax) and branched glycerol dialkyl glycerol tetraether (br GDGT) membrane lipids, as (paleo) elevation and (paleo) temperature proxies, respectively. At the mountain scale, a weak link between δ2Hwax and altitude (R2 = 0.33) is observed at Mt. Kenya, but no relationship is observed at Mt. Rungwe. It is likely that additional parameters, such as decreasing relative humidity (RH) or vegetation changes with altitude, are outcompeting the expected 2H-depletion trend along Mt. Rungwe. In contrast, br GDGT-derived absolute mean annual air temperature (MAAT) and temperature lapse rate (0.65 °C/100 m) for both mountains are in good agreement with direct field measurements, further supporting the robustness of this molecular proxy for (paleo) temperature reconstructions. At the regional scale, estimated and observed δ2H data in precipitation along 3 mountains in East Africa (Mts. Rungwe, Kenya and Kilimanjaro) highlight a strong spatial heterogeneity, preventing the establishment of a regional based calibration of δ2Hwax for paeloaltitudinal reconstructions. Different from that, an improved regional soil calibration is developed between br GDGT distribution and MAAT by combining the data from this study (Mts. Rungwe and Kenya) with previous results from East African surface soils along Mts. Kilimanjaro (Tanzania) and Rwenzori (Uganda). This new regional calibration, based on 105 samples, improves both the R2 (0.77) and RMSE (root mean square error; 2.4 °C) of br GDGT-derived MAAT over the global soil calibrations previously established (R2 = 0.56; RMSE = 4.2 °C) and leads to more accurate (paleo) temperature reconstructions in the region

La protéine eFF-1DELTA et le développement précoce de l'oursin Sphaerechinus granularis

Protein synthesis is composed of three phases: initiation, elongation and termination. The EFF-1 factor involved in the elongation phase of protein synthesis. It consists of two elements: a protein G: EFF-1A and a complex exchange GDP / GTP: eEF-1B. The composition of the EFF-1B complex is different in different species eEF-1ßy or eEF-1ßyδ associated with the valyl-tRNA synthetase in animals and eEF-1ßß'y in plants.We cloned and sequenced two cDNAs of 2.0 and 2.7 kb encoding eEF-1δ in the sea urchin Sphaerechinus granularis. They encode a 35 kDa protein identified to eEF-1δ on three criteria: the presence of a leucine zipper domain in the N-terminal portion, an area of exchange of GDP / GTP in the C-terminal portion and its integration into a heterologous eEF-1 complex.The two cDNAs are identical in sequences 5 'non-coding and coding, they differ in the length of their 3' UTR region. Their expression is regulated during development. The expression of mRNA encoding eEF-1δ is decoupled from that of his eEF-1A partner during early development.Phylogenetic analysis of sequences of protein-1ß kind eEF / ß 'and eEF-1δ showed that eEF-1δ and ß' were paralogous genes and eEF-1ß was from the fusion of a C-terminal domain of an ancestral gene ß leucine zipper domain with.We used the eEF-1δ recombinant protein to study the regulation (s) activity (s) eEF-1δ kinase (s) during early development. We showed a transient activity eEF-1δ kinase activity peaking 24 to 30 hours after fertilization. The eEF-1δ kinase was identified in the CK2 kinase phosphorylating nucleotide exchange factors: eEF-1ß and eEF-1δ. Inhibitors affect the CK2-gastrula blastula transition, suggesting a role of the kinase in early development.The regulation of the expression of eEF-1δ during development as well as phosphorylation of the protein by several protein kinases (PKA, PKC, CDK1 / cyclin B, multipotential S6 kinase, CK2, U (L) 13) suggest a role of signal integrator for the regulation of the activity of the eEF-1B complex whose eEF-1δ is a component in animals.La synthèse protéique est composée de trois phases : l'initiation, l'élongation et la terminaison. Le facteur eFF-1 intervient dans la phase d'élongation de la synthèse protéique. Il est constitué de deux éléments : d'une protéine G : eFF-1A et d'un complexe d'échange de GDP/GTP : eEF-1B. La composition du complexe eFF-1B est différente selon les espèces : eEF-1ßy ou eEF-1ßyδ associé à la valyl-ARNt synthétase chez les animaux et eEF-1ßß'y chez les végétaux. Nous avons cloné et séquencé deux ADNc de 2,0 et 2,7 kb codant pour eEF-1δ chez l'oursin Sphaerechinus granularis. Ils codent pour une protéine de 35 kDa identifiée à eEF-1δ sur trois critères : la présence d'un domaine leucine zipper dans la partie N-terminale, d'un domaine d'échange de GDP/GTP dans la partie C-terminale et son intégration dans un complexe eEF-1 hétérologue. Les deux ADNc sont identiques dans leurs séquences 5' non codante et codante, ils diffèrent par la longueur de leur région 3'UTR. Leur expression est régulée au cours du développement. L'expression des ARNm codant pour eEF-1δ est découplée de celle de son partenaire eEF-1A au cours du développement précoce.L'analyse phylogénétique des séquences des protéines de type eEF-1ß/ß' et eEF-1δ a montré que eEF-1δ et ß' étaient des gènes paralogues et que eEF-1ß provenait de la fusion d'un domaine C-terminal d'un gène ancestral ß avec un domaine leucine zipper.Nous avons utilisé la protéine recombinante eEF-1δ pour étudier la régulation de(s) activité(s) eEF-1δ kinase(s) au cours du développement précoce. Nous avons montré une activité transitoire eEF-1δ kinase atteignant son maximum d'activité 24 à 30h après la fécondation. La eEF-1δ kinase a été identifiée à la CK2, kinase phosphorylant les facteurs d'échange de nucléotides : eEF-1ß et eEF-1δ. Les inhibiteurs de la CK2 affectent la transition blastula-gastrula, suggérant un rôle de la kinase dans le développement précoce. La régulation de l'expression de eEF-1δ au cours du développement ainsi que la phosphorylation de la protéine par plusieurs protéines kinases (PKA, PKC, CDK1/cycline B, S6 kinase multipotentielle, CK2, U (L)13) laissent présager un rôle d'intégrateur de signaux pour la régulation de l'activité du complexe eEF-1B dont eEF-1δ est un constituant chez les animaux

Dosage simultané de 25 hormones stéroïdiennes par UHPLC-MS/MS

International audienceLes stéroïdes jouent un rôle essentiel dans la régulation de l'eau, des sels, du métabolisme, dans la réponse au stress ainsi que dans l'initiation et le maintien de la différentiation sexuelle et de la reproduction (Schiffer et al. 2019). Considérés comme des messagers biologiques, ces composés ont une grande importance dans le domaine de la recherche clinique (Guo et al. 2006). Une étude en endocrinologie a démontré que le métabolome stéroïdien pouvait être altéré durant certaines pathologies telles que le cancer (Storbeck et al. 2019), démontrant ainsi que l'analyse fiable et simultanée d'un large panel de stéroïdes pourrait être un outil puissant dans le cadre de diagnostics cliniques.Dosage simultané de 25 hormones stéroïdiennes par UHPLC-MS/MSAdeline Clergé a,d,Christelle Delalande - Lecapitaine a, Raphaël Delépée b,c,da. Laboratoire OeReCa, EA 2608, Caenb. INSERM U1086 ANTICIPE (Interdisciplinary Research Unit for Cancers prevention and Treatment) – Axis BioTICLA(Biology and Innovative Therapeutics for Ovarian Carcinomas), Caenc. Comprehensive Cancer Center François Baclesse, UNICANCER, Caend. PRISMM, Plateforme de Recherche et d’Innovation en Spectrométrie de Masse et Métabolomique, ICORE, CaenContact : [email protected] stéroïdes jouent un rôle essentiel dans la régulation de l’eau, des sels, du métabolisme, dans la réponseau stress ainsi que dans l’initiation et le maintien de la différentiation sexuelle et de la reproduction (Schifferet al. 2019). Considérés comme des messagers biologiques, ces composés ont une grande importance dansle domaine de la recherche clinique (Guo et al. 2006). Une étude en endocrinologie a démontré que lemétabolome stéroïdien pouvait être altéré durant certaines pathologies telles que le cancer (Storbeck et al.2019), démontrant ainsi que l’analyse fiable et simultanée d’un large panel de stéroïdes pourrait être un outilpuissant dans le cadre de diagnostics cliniques.La plupart des méthodes d’analyses des hormones stéroïdiennes sont basée sur l’utilisation de l’immunofluorescence, méthode rapide et simple d’utilisation. Cependant, la fiabilité de cette technique pour l’analyse des stéroïdes a rapidement été remise en question en raison du manque de spécificité, des effets matrice et de la nécessité de réaliser un test par composé (Rauh 2010). La chromatographie liquide couplée à la spectrométrie de masse en tandem (LC-MS/MS) est progressivement venue remplacer l’immunofluorescence dans les laboratoires d’analyse clinique en raison de ses faibles volumes d’échantillons, ses analyses rapides, son automatisation possible et sa faculté de pouvoir étudier plusieurs analytes en simultané.Différentes études portent sur l’analyse de quelques stéroïdes ciblés, en lien avec une pathologie (Antonelli et al. 2015; Peitzsch et al. 2015). Dans notre étude, 25 composés ont été sélectionnés (incluant des progestagènes, des minéralocorticoïdes, des glucocorticoïdes, des androgènes et des œstrogènes). Le but est de mettre au point une méthode de dosage afin d’obtenir un profil stéroïdien complet malgré les très faibles concentrations de certains composés (tels que les œstrogènes). Cette méthode pourrait permettre d’évaluer l’impact de ces composés sur différents tissus et fonctions d’organes et d’améliorer, accélérer et automatiserles processus de diagnostic

Estrogens: new players in spermatogenesis.

International audienceAromatase that irreversibly transforms androgens into estrogens is present in the smooth endoplasmic reticulum of nearly all cell types in the mammalian testis. In rodents, all testicular cells except for myoid cells express aromatase activity. We have demonstrated the presence of the functional aromatase (transcript or protein, and biological activity) in adult rat germ cells including pachytene spermatocytes and round spermatids. We have also demonstrated estrogen output from these cells equivalent to that of Leydig cells. Unlike androgen receptors, which are localized mainly in testicular somatic cells, estrogen receptors are present in both somatic and germ cells in the testis. Moreover, we have recently described the rapid membrane effects of estrogens (via G protein-coupled receptor [GPER]) in purified rat germ cells. On the basis of various experimental models, in vitro studies and clinical data, it can be concluded that estrogens play an essential role in male reproduction, specifically in the development of spermatozoa

Mammalian sperm quality and aromatase expression

SynthèseInternational audienceIn most mammalian species the aromatase is encoded by a single gene (cyp19), which contains 18 exons, 9 of them being translated. In adult rats, together with Leydig cells germ cells represent an additional source of estrogens. The amount of P450arom transcript is threefold higher in pachytene spermatocytes compared to younger cells (spermatogonia-preleptotene spermatocyte) or round spermatids; conversely, aromatase activity is more intense in haploid cells. In man besides Leydig cells, we have shown the presence of a biologically active aromatase and of estrogen receptors (ER alpha and ER beta) in immature germ cells and ejaculated spermatozoa. Concerning aromatase, a 30% decrease of the amount of mRNA is observed in immotile compared to motile sperm fraction from the same sample; moreover, the aromatase activity is diminished. We have amplified aromatase mRNA by RT-real time PCR in spermatozoa from asthenospermic, teratospermic, and asthenoteratospermic men and recorded respectively 44, 52, and 67% decreases of the amount of transcripts as compared to controls. Statistical analyses between the sperm morphology and the aromatase/GAPDH ratio have revealed a high degree of correlation (r = -0.64) with the percentage of abnormal spermatozoa (especially microcephaly and acrosome malformations). Alterations of sperm number and motility have been described in men genetically deficient in aromatase, which together with our data, suggest a likely role for aromatase/estrogens in the acquisition of sperm motility. Therefore besides gonadotrophins and testosterone, estrogens produced locally should be considered as a physiologically relevant hormone involved in the regulation of mammalian spermatogenesis

Estrogens: new players in spermatogenesis.

International audienceAromatase that irreversibly transforms androgens into estrogens is present in the smooth endoplasmic reticulum of nearly all cell types in the mammalian testis. In rodents, all testicular cells except for myoid cells express aromatase activity. We have demonstrated the presence of the functional aromatase (transcript or protein, and biological activity) in adult rat germ cells including pachytene spermatocytes and round spermatids. We have also demonstrated estrogen output from these cells equivalent to that of Leydig cells. Unlike androgen receptors, which are localized mainly in testicular somatic cells, estrogen receptors are present in both somatic and germ cells in the testis. Moreover, we have recently described the rapid membrane effects of estrogens (via G protein-coupled receptor [GPER]) in purified rat germ cells. On the basis of various experimental models, in vitro studies and clinical data, it can be concluded that estrogens play an essential role in male reproduction, specifically in the development of spermatozoa

Estrogens in male germ cells

The biosynthesis of steroids and the production of spermatozoa are two major functions of the mammalian testis which are tightly controlled by gonadotropins and numerous locally produced factors. Among these are the estrogens that are produced within the seminiferous epithelium via the irreversible transformation of androgens (C19) into estrogens (C18) by aromatase. We have recently reported that male germ cells are the new source of estrogens in the testis. For instance, estrogen receptors (ER) are found mainly in spermatids that give rise to spermatozoa. Moreover, it is noteworthy that GPR 30 (a transmembrane ER) induces rapid responses after estradiol binding, which, in turn, modulates cyclins and proapoptotic factors (e.g., BAX) to affect germ cell cycle progression and apoptosis. In summary, at least in the animal species that were studied thus far, germ cells are the major source and the target of estrogens, affecting normal male gonadal development and spermatogenesis, in particular spermiogenesis. These findings have also shed new light on the possible adverse effects of endocrine disruptors having estrogenic activities that can cause abnormal development of the male genital tract