Single embryo and oocyte lipid fingerprinting by mass spectrometry

Methods used for lipid analysis in embryos and oocytes usually involve selective lipid extraction from a pool of many samples followed by chemical manipulation, separation and characterization of individual components by chromatographic techniques. Herein we report direct analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of single and intact embryos or oocytes from various species. Biological samples were simply moisturized with the matrix solution and characteristic lipid ( represented by phosphatidylcholines, sphingomyelins and triacylglycerols) profiles were obtained via MALDI-MS. As representative examples, human, bovine, sheep and fish oocytes, as well as bovine and insect embryos were analyzed. MALDI-MS is shown to be capable of providing characteristic lipid profiles of gametes and embryos and also to respond to modifications due to developmental stages and in vitro culture conditions of bovine embryos. Investigation in developmental biology of the biological roles of structural and reserve lipids in embryos and oocytes should therefore benefit from these rapid MALDI-MS profiles from single and intact species.-Ferreira, C. R., S. A. Saraiva, R. R. Catharino, J. S. Garcia, F. C. Gozzo, G. B. Sanvido, L. F. A. Santos, E. G. Lo Turco, J. H. F. Pontes, A. C. Basso, R. P. Bertolla, R. Sartori, M. M. Guardieiro, F. Perecin, F. V. Meirelles, J. R. Sangalli, and M. N. Eberlin. Single embryo and oocyte lipid fingerprinting by mass spectrometry. J. Lipid Res. 2010. 51: 1218-1227.

Influence of nitric oxide during maturation on bovine oocyte meiosis and embryo development in vitro

The effect of s-nitroso-N-acetyl-1,1-penicillamine (SNAP, a nitric oxide donor) during in vitro maturation (IVM) on nuclear maturation and embryo development was investigated. The effect of increasing nitric oxide (NO) during prematuration or maturation, or both, on embryo development was also assessed. 10(-3) M SNAP nearly blocked oocytes reaching metaphase II (MII) (7%, P < 0.05) while 10(-5) M SNAP showed intermediate proportions (55%). For 10(-7) M SNAP and controls (without SNAP), MII percentages were similar (72% for both, P > 0.05), but superior to the other treatment groups (P < 0.05). Blastocyst development, however, was not affected (38% for all treatments, P < 0.05). TUNEL-positive cells in hatched blastocysts (Day 9) increased when IVM included 10(-5) M SNAP (8 v. 3 to 4 cells in the other treatments, P > 0.05), without affecting total cell numbers (240 to 291 cells, P > 0.05). When oocytes were prematured followed by IVM with or without 10(-7) M SNAP, during either culture period or both, blastocyst development was similar (26 to 40%, P > 0.05). When SNAP was included during both prematuration and IVM, the proportion of Day 9 hatched embryos increased (28% v. 14 to 19% in the other treatments, P < 0.05). Apoptotic cells, however, increased when SNAP was included (6 to 10 cells) in comparison to prematuration and maturation without SNAP (3 cells, P < 0.05). NO may be involved in meiotic progression and apoptosis during embryo development.

Embryo Mitochondrial DNA Depletion Is Reversed During Early Embryogenesis in Cattle

The extensive replication of mitochondria during oogenesis and the wide variability in mitochondrial DNA ( mtDNA) copy numbers present in fully grown oocytes indicate that mtDNA amount may play an important role during early embryogenesis. Using bovine oocytes derived from follicles of different sizes to study the influence of mtDNA content on development, we showed that oocytes obtained from small follicles, known to be less competent in developing into blastocysts, contain less mtDNA than those originating from larger follicles. However, because of the high variability in copy number, a more accurate approach was examined in which parthenogenetic one-cell embryos were biopsied to measure their mtDNA content and then cultured to assess development capacity. Contrasting with previous findings, mtDNA copy number in biopsies was not different between competent and incompetent embryos, indicating that mtDNA content is not related to early developmental competence. To further examine the importance of mtDNA on development, one-cell embryos were partially depleted of their mtDNA (64% +/- 4.1% less) by centrifugation followed by the removal of the mitochondrial-enriched cytoplasmic fraction. Surprisingly, depleted embryos developed normally into blastocysts, which contained mtDNA copy numbers similar to nonmanipulated controls. Development in depleted embryos was accompanied by an increase in the expression of genes (TFAM and NRF1) controlling mtDNA replication and transcription, indicating an intrinsic ability to restore the content of mtDNA at the blastocyst stage. Therefore, we concluded that competent bovine embryos are able to regulate their mtDNA content at the blastocyst stage regardless of the copy numbers accumulated during oogenesis.

Functional analysis of a novel missense NBC1 mutation and of other mutations causing proximal renal tubular acidosis

Mutations in the Na+-HCO3- cotransporter NBC1 cause severe proximal tubular acidosis (pRTA) associated with ocular abnormalities. Recent studies have suggested that at least some NBC1 mutants show abnormal trafficking in the polarized cells. This study identified a new homozygous NBC1 mutation (G486R) in a patient with severe pRTA. Functional analysis in Xenopus oocytes failed to detect the G486R activity due to poor surface expression. In ECV304 cells, however, G486R showed the efficient membrane expression, and its transport activity corresponded to approximately 50% of wild-type (WT) activity. In Madin-Darby canine kidney (MDCK) cells, G486R was predominantly expressed in the basolateral membrane domain as observed for WT. Among the previously identified NBC1 mutants that showed poor surface expression in oocytes, T485S showed the predominant basolateral expression in MDCK cells. On the other hand, L522P was exclusively retained in the cytoplasm in ECV304 and MDCK cells, and functional analysis in ECV304 cells failed to detect its transport activity. These results indicate that G486R, like T485S, is a partial loss of function mutation without major trafficking abnormalities, while L522P causes the clinical phenotypes mainly through its inability to reach the plasma membranes. Multiple experimental approaches would be required to elucidate potential disease mechanism by NBC1 mutations.

Reproductive cycle of Ophionereis reticulata (Ophiuroidea, Echinodermata) on the southeast coast of Brazil

The reproductive cycle of Ophionereis reticulata, a common sediment-rocky shore-interface ophiuroid, was examined monthly from January 2002 to January 2003 at Praia Grande beach (Sao Sesbatiao, state of Sao Paulo, Brazil). Mature individuals were found from January 2002 to April 2002 and from November 2002 to January 2003. Spawning was regcorded from January 2002 to March 2002 and from November 2002 to January 2003. Mature sperm is still present in April which could be involved in the fertilization of the last oocytes spawned in March and April. November and December marked the final point in the maturation process, with a high concentration of yolk and lipid nutrients in the oocytes. Ophionereis reticulata showed, for the period analyzed, a single spawning period, during spring and summer.

Cloning and characterization of a zebrafish homologue of human AQP1: a bifunctional water and gas channel

Chen LM, Zhao J, Musa-Aziz R, Pelletier MF, Drummond IA, Boron WF. Cloning and characterization of a zebrafish homologue of human AQP1: a bifunctional water and gas channel. Am J Physiol Regul Integr Comp Physiol 299: R1163-R1174, 2010. First published August 25, 2010; doi:10.1152/ajpregu.00319.2010.-The mammalian aquaporins AQP1, AQP4, and AQP5 have been shown to function not only as water channels but also as gas channels. Zebrafish have two genes encoding an AQP1 homologue, aqp1a and aqp1b. In the present study, we cloned the cDNA that encodes the zebrafish protein Aqp1a from the 72-h postfertilization (hpf) embryo of Danio rerio, as well as from the swim bladder of the adult. The deduced amino-acid sequence of aqp1a consists of 260 amino acids and is 59% identical to human AQP1. By analyzing the genomic DNA sequence, we identified four exons in the aqp1a gene. By in situ hybridization, aqp1a is expressed transiently in the developing vasculature and in erythrocytes from 16 to 48 h of development. Later, at 72 hpf, aqp1a is expressed in dermal ionocytes and in the swim bladder. Western blot analysis of adult tissues reveals that Aqp1a is most highly expressed in the eye and swim bladder. Xenopus oocytes expressing aqp1a have a channel-dependent (*) osmotic water permeability (P(f)*) that is indistinguishable from that of human AQP1. On the basis of the magnitude of the transient change in surface pH (Delta pHS) that were recorded as the oocytes were exposed to either CO(2) or NH(3), we conclude that zebrafish Aqp1a is permeable to both CO(2) and NH(3). The ratio (Delta pHS*)CO2/P(f)* is about half that of human AQP1, and the ratio (Delta pHS*)NH3/P(f)* is about one-quarter that of human AQP1. Thus, compared with human AQP1, zebrafish Aqp1a has about twice the selectivity for CO(2) over NH(3).

Patterns of oocyte development in natural habitat and captive Salminus hilarii Valenciennes, 1850 (Teleostei: Characidae)

Fecundity and oocyte development in Salminus hilarii female brood stock were analyzed with the aim of investigating the impact of migration impediment on oogenesis. Histological analyses of the ovaries were performed in adult females caught in two different environments-the TietA(a) River (natural) and captivity-and the gonadossomatic index, oocyte diameter and fecundity determined. Five germ cell development stages (oogonium, perinucleolar, cortical alveoli, vitellogenic, ripe) and two other structures (postovulatory follicles and atretic oocytes) were observed in females caught in the river. Captive animals lacked the ripe oocytes and postovulatory follicles and had a relatively higher number of atretic oocytes. Females in captivity are known to produce larger oocytes, and they release fewer eggs in each spawn (absolute fecundity) when compared with animals that are able to migrate. Our results suggest that the TietA(a) River is undergoing alterations which are being reflected in the reproductive performance of S. hilarii, mainly due to the presence of atretic oocytes in females caught in the river. The lack of postovulatory follicles and ripe oocytes in captive animals reveals that migratory impediment negatively impacts final oocyte maturation. However, the stage of maturation reached is adequate for ovulation induction with hormone manipulation.

Three-dimensional Reconstruction of Ovaries of Leaf-cutting Ant (Atta sexdens rubropilosa) Queens (Hymenoptera: Formicidae)

In this study, the ovary morphology of newly emerged ant queens of Atta sexdens rubropilosa was studied in whole mount preparations by confocal microscopy. The ovaries are composed of approximately 40 ovarioles, showing non-synchronic oocyte maturation. The terminal filament with clusters of undifferentiated cells was found at the distal end of the ovarioles. Next to this region is the germarium, composed of several elongated cystocytes interconnected by cytoplasmic bridges. The nurse cells (23-28 cells) result from asymmetric mitosis. Cytoskeleton analysis showed F-actin concentrated at the muscle cells of the external tunica and in fusomes inside the ovarioles. Microtubules were concentrated around the nuclei of the nurse and follicular cells. In contrast, the oocytes and the external tunica showed faint staining for tubulin.

Transcriptome and gene expression profile of ovarian follicle tissue of the triatomine bug Rhodnius prolixus

Insect oocytes grow in close association with the ovarian follicular epithelium (OFE), which escorts the oocyte during oogenesis and is responsible for synthesis and secretion of the eggshell. We describe a transcriptome of OFE of the triatomine bug Rhodnius prolixus, a vector of Chagas disease, to increase our knowledge of the role of FE in egg development. Random clones were sequenced from a cDNA library of different stages of follicle development. The transcriptome showed high commitment to transcription, protein synthesis, and secretion. The most abundant cDNA was a secreted (S) small, proline-rich protein with maximal expression in the vitellogenic follicle, suggesting a role in oocyte maturation. We also found Rp45, a chorion protein already described, and a putative chitin-associated cuticle protein that was an eggshell component candidate. Six transcripts coding for proteins related to the unfolded-protein response (UPR) by were chosen and their expression analyzed. Surprisingly, transcripts related to UPR showed higher expression during early stages of development and downregulation during late stages, when transcripts coding for S proteins participating in chorion formation were highly expressed. Several transcripts with potential roles in oogenesis and embryo development are also discussed. We propose that intense protein synthesis at the FE results in reticulum stress (RS) and that lowering expression of a set of genes related to cell survival should lead to degeneration of follicular cells at oocyte maturation. This paradoxical suppression of UPR suggests that ovarian follicles may represent an interesting model for studying control of RS and cell survival in professional S cell types. (C) 2011 Elsevier Ltd. All rights reserved.

Effects of Plasmodium gallinaceum on hemolymph physiology of Aedes aegypti during parasite development

Insect disease vectors show diminished fecundity when infected with Plasmodium. This phenomenon has already been demonstrated in laboratory models such as Aedes aegypti, Anopheles gambiae and Anopheles stephensi. This study demonstrates several changes in physiological processes of A. aegypti occurring upon infection with Plasmodium gallinaceum, such as reduced ecdysteroid levels in hemolymph as well as altered expression patterns for genes involved in vitellogenesis, lipid transport and immune response. Furthermore, we could show that P. gallinaceum infected A. aegypti presented a reduction in reproductive fitness, accompanied by an activated innate immune response and increase in lipophorin expression, with the latter possibly representing a nutritional resource for Plasmodium sporozoites. (C) 2010 Elsevier Ltd. All rights reserved.

Antimicrobial activity in the tick Rhipicephalus (Boophilus) microplus eggs: Cellular localization and temporal expression of microplusin during oogenesis and embryogenesis

Arthropods display different mechanisms to protect themselves against infections, among which antimicrobial peptides (AMPs) play an important role, acting directly against invader pathogens. We have detected several factors with inhibitory activity against Candida albicans and Micrococcus luteus on the surface and in homogenate of eggs of the tick Rhipicephalus (Boophilus) microplus. One of the anti-M. luteus factors of the egg homogenate was isolated to homogeneity. Analysis by electrospray mass spectrometry (ESI-MS) revealed that it corresponds to microplusin, an AMP previously isolated from the cell-free hemolymph of X (B.) microplus. Reverse transcription (RT) quantitative polymerase chain reactions (qPCR) showed that the levels of microplusin mRNA gradually increase along ovary development, reaching an impressive highest value three days after the adult females have dropped from the calf and start oviposition. Interestingly, the level of microplusin mRNA is very low in recently laid eggs. An enhance of microplusin gene expression in eggs is observed only nine days after the onset of oviposition, achieving the highest level just before the larva hatching, when the level of expression decreases once again. Fluorescence microscopy analysis using an anti-microplusin serum revealed that microplusin is present among yolk granules of oocytes as well as in the connecting tube of ovaries. These results, together to our previous data. suggest that microplusin may be involved not only in protection of adult female hemocele, but also in protection of the female reproductive tract and embryos, what points this AMP as a considerable target for development of new methods to control R. (B.) microplus as well as the vector-borne pathogens. (c) 2009 Elsevier Ltd. All rights reserved.

Structural-electronic aspects related to the near-infrared light emission of Fe-doped silicon films

The need of efficient (fast and low consumption) optoelectronic devices has always been the driving force behind the investigation of materials with new or improved properties. To be commercially attractive, however, these materials should be compatible with our current micro-electronics industry and/or telecommunications system. Silicon-based compounds, with their matured processing technology and natural abundance, partially comply with such requirements-as long as they emit light. Motivated by these issues, this work reports on the optical properties of amorphous Si films doped with Fe. The films were prepared by sputtering a Si+Fe target and were investigated by different spectroscopic techniques. According to the experimental results, both the Fe concentration and the thermal annealing of the samples induce changes in their atomic structure and optical-electronic properties. In fact, after thermal annealing at similar to 750 degrees C, the samples partially crystallize with the development of Si and/or beta-FeSi(2) crystallites. In such a case, certain samples present light emission at similar to 1500 nm that depends on the presence of beta-FeSi(2) crystallites and is very sensitive to the annealing conditions. The most likely reasons for the light emission (or absence of it) in the considered Fe-doped Si samples are presented and discussed in view of their main structural-electronic characteristics. (C) 2011 Elsevier Ltd. All rights reserved.

Utilização de práticas de gerenciamento de projetos aplicadas em projetos de responsabilidade social corporativa: um estudo de caso em uma empresa brasileira

O estudo desenvolvido tem como objetivo apresentar uma visão geral da relevância e das relações entre os temas responsabilidade social e gerenciamento de projetos. Apresentando a justificativa para a necessidade de se segregar os dois temas na gestão dos projetos de responsabilidade social, o estudo aborda não só a questão da implantação da responsabilidade social nas empresas, como também busca medir o nível de utilização de práticas em gerenciamento nos projetos de responsabilidade social em uma empresa brasileira. O trabalho apresenta os principais conceitos que abordam o tema Responsabilidade Social Corporativa, bem como, os conceitos que tratam o tema Gerenciamento de Projetos. Como o tema Responsabilidade Social Corporativa deixou de ser para as empresas apenas uma questão de filantropia, mas de gestão organizacional e estratégica, o presente trabalho direciona para o levantamento da relevância do tema e da necessidade de se aplicar conceitos e práticas em gerenciamento de projetos de responsabilidade social. A questão explorada no estudo de caso desenvolvido é quanto a empresa estudada utiliza das práticas e metodologias de gerenciamento de projetos aplicadas aos projetos de responsabilidade social. Visando avaliar o cenário das organizações brasileiras, o trabalho abordou um estudo de caso de uma organização brasileira que desenvolve diversos projetos em responsabilidade social e tem ações de responsabilidade social definidas em sua estratégia organizacional. O resultado obtido no estudo de caso demonstrou que embora a empresa tenha iniciado o processo de transferência de conhecimento e padronização de práticas de gerenciamento de projetos, a percepção dos gestores e o resultado da pesquisa refletiram que essas práticas devem ser amadurecidas no ambiente de projetos corporativos.

Social entrepreneurship practices and social change In Brazil: a qualitative study in three non-governmental organizations

Social Entrepreneurship (SE) has attracted growing interest from a wide variety of actors over the last 30 years, especially due to a general agreement that it could be an important tool for tackling many of the world’s social ills. In the academic sphere, this growing interest did not translate into a matured field of study. Quite the opposite, a quick look at this literature makes it evident that: SE has been consistently subjected to numerous theoretical discussions and disagreements, especially over the definition of the concept of SE which is often based on a taken-for-granted notion of social change; it has been more systematically investigated in restricted contexts, often leaving aside so called developing/emerging countries like Brazil and especially lacking in-depth qualitative studies; SE literature lags behind SE practices and few studies focus on how SE actually occurs in a daily and bottom-up manner. In order to address such gaps, this thesis examines how social entrepreneurship practices accomplish social change in the context of Brazil. In this investigation I conducted an inductive practice-based, qualitative/ethnographic study in three Non-Governmental Organizations (NGOs) located in different cities in the Brazilian state of São Paulo. Data collection lasted from February 2014 until March 2015 and was mainly done through participant observations and through in-depth unstructured conversations with research participants. Secondary data and documents were also collected whenever available. The participants of this study included a variety of the studied organizations’ stakeholders: two founders, volunteers, employees, donors and beneficiaries. Observation data was kept in fieldnotes, conversations were recorded whenever possible and were later transcribed. Data was analyzed through an iterative thematic analysis. Through this I identified eight recurrent themes in the data: (1) structure; (2) relationship with other organizational actors (sub-themes: relationship with state, relationship with businesses and relationship with other NGOs); (3) beliefs, spirituality and moral authority; (4) social position of participants, (5) stakeholders’ mobilization and participation; (6) feelings; (7) social purpose; and (8) social change. These findings were later discussed under the lens of practice theory, and in this discussion I argue and show that, in the context studied: (a) even though SE embraces a wide variety of different social purposes, they are intertwined with a common notion of social change based on a general understanding and aspiration for social equality; (b) this social change is accomplished in a processual and ongoing manner as stakeholders from antagonistic social groups felt compelled to and participated in SE practices. In answering the proposed research question the contributions of this thesis are: (i) the elaboration a working definition for SE based on its relationship with social change; (ii) providing in-depth empirical evidence which accounts for and explains this relationship; (iii) characterizing SE in the Brazilian context and reflecting upon its transferability to other contexts. This thesis also makes a methodological contribution, for it demonstrates how thematic analysis can be used in practice-based studies.

Gonad development and hormone titres in Loggerhead Sea Turtles (Caretta caretta) in the NE Atlantic

The study proposed to describe sexual development in pelagic stage loggerhead sea turtles Caretta caretta and compare this to hatchlings and adults. It is meant as an ontogenic approach, in order to understand reproductive development and population composition and their dynamics in the pelagic environment. The study focused on the pelagic loggerheads that are found in the waters offshore Madeira Island (Portugal) in the North-eastern Atlantic and use it as a developmental habitat. The innovating character of this work relied on the lack of any description regarding the gonad ontogenesis and reproductive development for the pelagic stage in any of the 7 existing sea turtle species, all of them in danger of extinction. Three methods were used to diagnose the sex of each juvenile individual and asses the level of reproductive development: (1) laparoscopy, (2) gonad biopsy and (3) the assessment of two sex steroids circulating levels, namely testosterone and estradiol. In order to cover all life stages and compare data obtained for the juvenile stage, hatchlings and nesting female adults were sampled at the nearest nesting rookery at Boa Vista Island in the Cape Verde Archipelago. Gonads from dead hatchlings were collected for gonad histology and blood was collected from nesting females for sex steroids assessment. Laparoscopies revealed to be a valid sexing method for the juvenile stage, since gonads are morphologically differentiated at these size classes. Moreover, laparoscopy was validated using gonad histology. Gonad histology of juveniles showed that gonads are already completely differentiated into ovaries or testes at the size classes examined, but development seems to be quiescent. Males present already developed seminiferous tubules with spermatogonia lining the interior of the seminiferous tubule. Female gonads present oocytes at different development stages, but only oocytes up to stage III were observed. The maximum oocyte diameter in each individual correlated with body size, suggesting that reproductive development is an on-going process in juvenile females. The circulating levels of both testosterone and estradiol in juveniles of both sexes were very low and consistently lower than the ones observed in the nesting females from Boa Vista Island. No bimodal distribution was found for any of the sex steroids analysed and thus circulating hormone levels were not a reliable tool for sexing juvenile individuals with a non-invasive technique. The ratio testosterone:estradiol did not show a bimodal distribution either. The levels of testosterone correlated with sea surface temperature. The fact that temperatures observed during this study were below 24ºC might have hindered a differential testosterone pattern between juvenile males and females. Sex ratios for this population were generated according to laparoscopy results and compared among years and size classes. An overall sex ratio of 2 females for each male was found, but they varied among size classes but not among years. Possible causes for the sex ratios observed are discussed. This study is a contribution to our knowledge on the pelagic stage of loggerhead turtles, namely on the population structure regarding sex ratio, which is a vital tool for implementing conservation strategies.