Evolution of the sex-related locus and genomic features shared in microsporidia and fungi.

BACKGROUND: Microsporidia are obligate intracellular, eukaryotic pathogens that infect a wide range of animals from nematodes to humans, and in some cases, protists. The preponderance of evidence as to the origin of the microsporidia reveals a close relationship with the fungi, either within the kingdom or as a sister group to it. Recent phylogenetic studies and gene order analysis suggest that microsporidia share a particularly close evolutionary relationship with the zygomycetes. METHODOLOGY/PRINCIPAL FINDINGS: Here we expanded this analysis and also examined a putative sex-locus for variability between microsporidian populations. Whole genome inspection reveals a unique syntenic gene pair (RPS9-RPL21) present in the vast majority of fungi and the microsporidians but not in other eukaryotic lineages. Two other unique gene fusions (glutamyl-prolyl tRNA synthetase and ubiquitin-ribosomal subunit S30) that are present in metazoans, choanoflagellates, and filasterean opisthokonts are unfused in the fungi and microsporidians. One locus previously found to be conserved in many microsporidian genomes is similar to the sex locus of zygomycetes in gene order and architecture. Both sex-related and sex loci harbor TPT, HMG, and RNA helicase genes forming a syntenic gene cluster. We sequenced and analyzed the sex-related locus in 11 different Encephalitozoon cuniculi isolates and the sibling species E. intestinalis (3 isolates) and E. hellem (1 isolate). There was no evidence for an idiomorphic sex-related locus in this Encephalitozoon species sample. According to sequence-based phylogenetic analyses, the TPT and RNA helicase genes flanking the HMG genes are paralogous rather than orthologous between zygomycetes and microsporidians. CONCLUSION/SIGNIFICANCE: The unique genomic hallmarks between microsporidia and fungi are independent of sequence based phylogenetic comparisons and further contribute to define the borders of the fungal kingdom and support the classification of microsporidia as unusual derived fungi. And the sex/sex-related loci appear to have been subject to frequent gene conversion and translocations in microsporidia and zygomycetes.

Global identification of androgen response elements

Chromatin immunoprecipitation (ChIP) is an invaluable tool in the study of transcriptional regulation. ChIP methods require both a priori knowledge of the transcriptional regulators which are important for a given biological system and high-quality specific antibodies for these targets. The androgen receptor (AR) is known to play essential roles in male sexual development, in prostate cancer and in the function of many other AR-expressing cell types (e.g. neurons and myocytes). As a ligand-activated transcription factor the AR also represents an endogenous, inducible system to study transcriptional biology. Therefore, ChIP studies of the AR can make use of treatment contrast experiments to define its transcriptional targets. To date several studies have mapped AR binding sites using ChIP in combination with genome tiling microarrays (ChIP-chip) or direct sequencing (ChIP-seq), mainly in prostate cancer cell lines and with varying degrees of genomic coverage. These studies have provided new insights into the DNA sequences to which the AR can bind, identified AR cooperating transcription factors, mapped thousands of potential AR regulated genes and provided insights into the biological processes regulated by the AR. However, further ChIP studies will be required to fully characterise the dynamics of the AR-regulated transcriptional programme, to map the occupancy of different AR transcriptional complexes which result in different transcriptional output and to delineate the transcriptional networks downstream of the AR.

Role of plasmodium translationally repressed gene products in malaria transmission

Tese de doutoramento, Ciências Biomédicas (Microbiologia e Parasitologia), Universidade de Lisboa, Faculdade de Medicina, 2015

The role of the hypothalamic kisspeptin/GPR54 system in the control of GnRH neurons

Summary : The hypothalamus represents less than 1 % of the total volume of the brain tissue, yet it plays a crucial role in endocrine regulations. Puberty is defined as a process leading to physical, sexual and psychosocial maturation. The hypothalamus is central to this process, via the activation of GnRH neurons. Pulsatile GnRH secretion, minimal during childhood, increases with the onset of puberty. The primary function of GnRH is to regulate the growth, development and function of testes in boys and ovaries in girls, by stimulating the pituitary gland secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Several factors contribute to the timing of puberty, including sex and ethnicity, genetics, dietary intake and energy expenditure. Kisspeptins constitute a family of small peptides arising from the proteolytic cleavage of metastin, a peptide with 54 amino acids initially purified from human placenta. These kisspeptins were the subject of much attention following their discovery because of their antimetastatic properties, but it was more recently that their determining role in the reproductive function was demonstrated. It was shown that kisspeptins are ligands of a receptor, GPR54, whose natural inactivating mutation in humans, or knockout in the mouse, lead to infertility. GnRH neurons play a pivotal role in the central regulation of fertility. Kisspeptin greatly increases GnRH release and GnRH neuron firing activity, but the neurobiological mechanisms for these actions are unknown. Gprotein-coupled receptor 54, the receptor for kisspeptin, is expressed by GnRH neurons as well as other hypothalamic neurons, suggesting that both direct and indirect effects are possible. In the first part of my thesis, we investigated a possible connection between the acceleration of sexual development induced by leptin and hypothalamic metastin neurons. However, the data generated by our preliminary experiments confirmed that the commercially available antibodies are non-specific. This finding constituted a major drawback for our studies, which relied heavily upon the neuroanatomical study of the hypothalamic metastinergic pathways to elucidate their sensitivity to exogenous leptin. Therefore, we decided to postpone any further in vivo experiment until a better antibody becomes available, and focused on in vitro studies to better understand the mechanisms of action of kisspeptins in the modulation of the activity of GnRH neurons. We used two GnRH-expressing neuronal cell lines to investigate the cellular and molecular mechanisms of action of metastin in GnRH neurons. We demonstrated that kisspeptin induces an early activation of the MAP kinase intracellular signaling pathway in both cell lines, whereas the SAP/JNK or the Akt pathways were unaffected. Moreover, we found an increase in GnRH mRNA levels after 6h of metastin stimulation. Thus, we can conclude that kisspeptin regulates GnRH neurons both at the secretion and the gene expression levels. The MAPK pathway is the major pathway activated by metastin in GnRH expressing neurons. Taken together, these data provide the first mechanism of action of kisspeptin on GnRH neurons. Résumé : L'hypothalamus est une zone située au centre du cerveau, dont il représente moins de 1 du volume total. La puberté est la période de transition entre l'enfance et l'age adulte, qui s'accompagne de transformations somatiques, psychologiques, métaboliques et hormonales conduisant à la possibilité de procréer. La fonction principale de la GnRH est la régulation de la croissance, du développement et de la fonction des testicules chez les hommes, et des ovaires chez les femmes en stimulant la sécrétion de l'hormone lutéinisante (LH) et de l'hormone folliculostimulante (FSH) par la glande hypophysaire. Plusieurs facteurs contribuent au déclanchement de la puberté, y compris le sexe et l'appartenance ethnique, la génétique, l'apport alimentaire et la dépense énergétique. Les Kisspeptines constituent une famille de peptides résultant de la dissociation proteolytique de la métastine, un peptide de 54 acides aminés initialement purifié à partir de placenta humain. Ces kisspeptines ont fait l'objet de beaucoup d'attention à la suite de leur découverte en raison de leurs propriétés anti-metastatiques, et c'est plus récemment que leur rôle déterminant dans la fonction reproductive a été démontré. Les kisspeptines sont des ligands du récepteur GPR54, dont la mutation inactivatrice chez l'homme, ou le knockout chez la souris, conduisent à l'infertilité par hypogonadisme hypogonadotrope. Les neurones à GnRH jouent un rôle central dans le règlement des fonctions reproductrices et la kisspeptine stimule l'activité des neurones à GnRH et la libération de GnRH par ces neurones. Toutefois, les mécanismes neurobiologiques de ces actions ne sont pas connus. Dans la première partie de ma thèse, nous avons étudié le lien potentiel entre l'accélération du développement sexuel induite par la leptine et les neurones hypothalamiques à metastine. Les données générées dans cette première série d'expériences ont malheureusement confirmé que les anticorps anti-metastine disponibles dans le commerce sont aspécifiques. Ceci a constitué un inconvénient majeur pour nos études, qui devaient fortement s'appuyer sur l' étude neuroanatomique des neurones hypothalamiques à metastine pour évaluer leur sensibilité à la leptine exogène. Nous avons donc décidé de focaliser nos travaux sur une étude in vitro des mécanismes d'action de la kisspeptine pour moduler l'activité des neurones à GnRH. Nous avons utilisé deux lignées de cellules neuronales exprimant la GnRH pour étudier les mécanismes d'action cellulaires et moléculaires de la metastine dans des neurones. Nous avons ainsi pu démontrer que la kisspeptine induit une activation précoce de la voie f de signalisation de la MAP kinase dans les deux lignées cellulaires, alors que nous n'avons observé aucune activation de la voie de signalisation de la P13 Kinase et de la SAP/JNK. Nous avons en outre démontré une augmentation de l'expression de la GnRH par la stimulation avec la Kisspeptine. L'ensemble de ces données contribue à élucider le mécanisme d'action avec lequel la kisspeptine agit dans les neurones à GnRH, en démontrant un effet sur l'expression génique de la GnRH. Nous pouvons également conclure que la voie de la MAPK est la voie principale activée par la metastine dans les neurones exprimant la GnRH.

Régions de susceptibilité dans les remaniements du chromosome Y et mosaïcisme : facteurs de risque du développement sexuel anormal

Le développement sexuel est un processus complexe qui dépend de nombreux gènes, une mutation pouvant entraîner un développement sexuel anormal. Par ailleurs, des anomalies chromosomiques peuvent avoir des répercussions importantes sur la détermination gonadique, surtout lorsqu'il s'agit du chromosome Y puisqu'il porte le gène clé du développement sexuel masculin. Premièrement, nous avons identifié par cytogénétique moléculaire le point de cassure chez 5 patients avec une translocation X;Y et 10 patients avec un chromosome Y isodicentrique. Nous avons ainsi démontré que certaines régions sont plus à risque d'être remaniées, notamment lorsqu'elles contiennent des palindromes ou d'autres séquences répétées. Nous avons également établi une relation entre la distance séparant le centromère et le point de cassure et l'instabilité des chromosomes Y isodicentriques lors des divisions cellulaires. Deuxièmement, nous avons étudié en cytogénétique les gonades de 22 patients avec un chromosome Y normal ou remanié et présentant un développement sexuel anormal. Nous avons mis en évidence la perte du chromosome Y remanié dans une majorité de cellules gonadiques des 10 patients étudiés, expliquant leur phénotype sexuel anormal. Cependant, chez 11 des 12 patients avec un chromosome Y normal, aucun mosaïcisme expliquant clairement leur détermination gonadique anormale n'a été retrouvé. Finalement, nous avons analysé par immunohistochimie les gonades dysgénésiques de 30 patients avec une anomalie du développement sexuel et un chromosome Y normal ou remanié. Nos travaux ont montré la présence de cellules germinales immatures au sein de cordons sexuels primitifs sous forme de tissu gonadique indifférencié dans 15 gonades, dont 9 ont évolué en tumeur gonadique. Dans 13 autres gonades, ces cellules germinales immatures avaient disparues par apoptose. Dans l'ensemble, notre recherche met en évidence la susceptibilité du chromosome Y à subir des remaniements et à être instable lors des divisions cellulaires, et indique que le mosaïcisme peut avoir des répercussions sur la détermination gonadique. Nos travaux montrent également que le tissu gonadique indifférencié peut évoluer vers deux entités, une tumeur gonadique ou une bandelette suite à l'apoptose des cellules germinales, mettant en lumière la nécessité d'analyser le tissu gonadique des patients XY avec dysgénésie gonadique dont les gonades sont laissées en place.

Manifestaciones renales en pacientes con síndrome de Alagille: Reporte de casos y revisión de la literatura.

El síndrome de Alagille es una condición autosómica dominante que se define clínicamente por alteraciones de cinco sistemas principales: escasez de ductos biliares con colestasis crónica, compromiso cardíaco (estenosis pulmonar), óseo (vétebras en mariposa), oftálmico (embriotoxon posterior) y de la cara (fascies dismórficas leves pero reconocibles). La afectación renal es común en estos pacientes; sin embargo, no hace parte de los criterios que definen el síndrome. Reportamos los casos de 3 pacientes con síndrome de Alagille y compromiso renal y realizamos una revisión de la literatura para establecer la importancia de incluir la evaluación de este sistema en el diagnóstico del síndrome. Concluimos que el compromiso renal es frecuente, y por lo tanto sugerimos que en todos los casos se evalúe la posibilidad de compromiso renal tanto a nivel estructural como funcional glomerular y tubular.

Effect of two opposing changes in photoperiod upon age at first egg in layer-hybrid pullets

An experiment was designed to test the response of growing pullets to two changes in photoperiod (an increase from 8 to 14 h followed 5 weeks later by the reverse change, or a decrease from 14 to 8 h followed by an increase). The first change was made either at 35 days or at 56 days of age, to test the influence of age on the responses observed. Control groups were kept oil constant 8-h and constant 14-h photoperiods and the responses to appropriate single changes were also tested. Mean age at first egg varied from 111 days for birds given a single increment at 56 days to 166 days for pullets given an increase in photoperiod at 35 days followed by a reduction at 70 days. Responses to the single changes confirmed earlier reports that sensitivity to change in photoperiod varies with age ill a manner that is quantitatively predictable. Responses to the double changes could be explained by Postulating that the initial change altered the 'physiological age' of the bird to all extent that was also quantitatively predictable. An early increase in photoperiod advances sexual development and makes the bird more sensitive to a subsequent decrease than would be expected by reference to its chronological age. An early decrease in photoperiod delays sexual development, which can have the effect of making the bird more or less sensitive to a subsequent increase since, ill layer-strain pullets, sensitivity to an increment in photoperiod normally increases Lip to about 9 weeks of age but decreases thereafter. Mean age at first egg predicted using these concepts was very highly correlated with observed age at first egg. The results provide a rational basis for constructing a model to predict age at first egg for any combination of increases and decreases in photoperiod applied to growing pullets.

Growth and condition in Australian fur seals Arctocephalus pusillus doriferus (Carnivora : Pinnipedia)

Mass and length growth models were determined for male (n = 69) and female (n = 163) Australian fur seals (Arctocephalus pusillus doriferus) collected at a breeding colony on Seal Rocks (38˚31′S, 145˚06′E), Bass Strait, in south-east Australia, between February and November during 1970–72. Growth was best described by the logistic model in males and the von Bertalanffy model in females. Asymptotic mass and length were 229 kg and 221 cm for males, and 85 kg and 163 cm for females. In all, 95% of asymptotic mass and length were attained by 11 years and 11 years, respectively, in males compared with 9 years and 5 years, respectively, in females. Males grew in length faster than females and experienced a growth spurt in mass coinciding with the onset of puberty (4–5 years). The onset of puberty in females occurs when approximately 86% of asymptotic length is attained. The rate of growth and sexual development in Australian fur seals is similar to (if not faster than) that in the conspecific Cape fur seal (A. p. pusillus), which inhabits the nutrient-rich Benguela current. This suggests that the low marine productivity of Bass Strait may not be cause of the slow rate of recovery of the Australian fur seal population following the severe over-exploitation of the commercial sealing era. Sternal blubber depth was positively correlated in adult animals with a body condition index derived from the residuals of the mass–length relationship (males: r² = 0.38, n = 19, P < 0.001; females: r² = 0.22, n = 92, P < 0.001), confirming the validity of using such indices on otariids. Sternal blubber depth varied significantly with season in adult animals. In males it was lowest in winter and increased during spring prior to the breeding season (r² = 0.39, n = 19, P < 0.03) whereas in females it was greatest during winter (r² = 0.05, n = 122, P< 0.05).

Sibling incest within violent families : Children under 12 years seeking nurture.

This article will draw on theories of childhood and current sociological debates of shame to explore and understand the formation of sexual identity in children who experience a violent family. Children have limited resources available to them and in violent family situations may turn to their siblings for nurture. Contextualising this type of dependency within the usual processes of childhood sexual development, including exploration and experimentation, may increase the likelihood of sibling incest.

Effects of the aromatase inhibitor Fadrozole on plasma sex steroid secretion and oocyte maturation in female coho salmon (Oncorhynchus kisutch) during vitellogenesis

17β-estradiol, 17α-20β-dihydroxy-4-pregnen-3-one (17α-20β-P), and testosterone levels were measured in plasma samples obtained from vitellogenic coho salmon (Oncorhynchus kisutch) before and 32 days after injection of the aromatase inhibitor Fadrozole (AI). Plasma 17β-estradiol levels decreased significantly 6 h after injection in all AI treated fish. The higher the dose the longer the maintenance of low plasma 17β-estradiol levels. Inversely, plasma 17α-20β-P increased significantly 6 h after injection in all AI treated fish, and the higher the dose the longer the maintenance of high plasma 17α-20β-P levels. At 48 h after injection plasma testosterone levels were significantly higher in the AI treated groups. The oocyte maturation index showed that multiple injections with AI retarded oocyte development. Besides, oocyte diameter and GSI were lower in the same group, which presented high incidence of atresia of vitellogenic oocytes. The ovarian follicles and brain of the fish which received multiple injections secreted less 17β-estradiol, in vitro. These findings suggest that aromatase inhibitors such as Fadrozole may have a potential as a tool to regulate sexual development in salmon.

A microestrutura e o desenvolvimento dos órgãos genitais masculinos de preás (Galea spixii) criados em cativeiro

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Short- and long-term reproductive effects of prenatal and lactational growth restriction caused by maternal diabetes in male rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Differentiation of the XY Sex Chromosomes in the Fish Hoplias malabaricus (Characiformes, Erythrinidae): Unusual Accumulation of Repetitive Sequences on the X Chromosome

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Maternal exposure to aqueous extract of Jacaranda decurrens: Effects on reproductive system in male rats

Context: Jacaranda decurrens subsp. symmetrifoliolata Farias & Proenca (Bignoniaceae) is a species widely used for their medicinal properties. At least to our known, no study has been conducted concerning its toxicological profile after gestational and lactational exposure.Objective: The present study was carried out to evaluate the effects of J. decurrens on development of the reproductive system in male rats.Materials and methods: Pregnant rats were treated daily (gavage) with 250 or 500 mg/kg/day of aqueous extract of J. decurrens or vehicle, from day 12 of pregnancy to day 21 of lactation.Results and Discussion: Both doses of J. decurrens significantly anticipated (p < 0.05) the age of testicular descent to the scrotum, a parameter indicative of puberty initiation. Furthermore, at puberty, there was a significant reduction (p < 0.05) in testicular and epididymis weights in the offspring exposed to the higher dose of extract, without effect on sperm production and the histology of reproductive organs. on the other hand, at adulthood, the reproductive parameters analyzed did not differ among groups.Conclusions: J. decurrens, in this experimental model, interfered with the initial development of the reproductive system, but without lasting effects on sperm production in adulthood.

In utero protein restriction causes growth delay and alters sperm parameters in adult male rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)