Essential role of Mg2+ in mouse preimplantation embryo development revealed by TRPM7 chanzyme-deficient gametes.

TRPM7 (transient receptor potential cation channel subfamily M member 7) is a chanzyme with channel and kinase domains essential for embryo development. Using gamete-specific Trpm7-null lines, we report that TRPM7-mediated Mg2+ influx is indispensable for reaching the blastocyst stage. TRPM7 is expressed dynamically from gametes to blastocysts; displays stage-specific localization on the plasma membrane, cytoplasm, and nucleus; and undergoes cleavage that produces C-terminal kinase fragments. TRPM7 underpins Mg2+ homeostasis, and excess Mg2+ but not Zn2+ or Ca2+ overcomes the arrest of Trpm7-null embryos; expressing Trpm7 mRNA restores development, but mutant versions fail or are partially rescued. Transcriptomic analyses of Trpm7-null embryos reveal an abundance of oxidative stress-pathway genes, confirmed by mitochondrial dysfunction, and a reduction in transcription factor networks essential for proliferation; Mg2+ supplementation corrects these defects. Hence, TRPM7 underpins Mg2+ homeostasis in preimplantation embryos, prevents oxidative stress, and promotes gene expression patterns necessary for developmental progression and cell-lineage specification.

Knockin' on Egg's Door: Maternal Control of Egg Activation That Influences Cortical Granule Exocytosis in Animal Species.

Fertilization by multiple sperm leads to lethal chromosomal number abnormalities, failed embryo development, and miscarriage. In some vertebrate and invertebrate eggs, the so-called cortical reaction contributes to their activation and prevents polyspermy during fertilization. This process involves biogenesis, redistribution, and subsequent accumulation of cortical granules (CGs) at the female gamete cortex during oogenesis. CGs are oocyte- and egg-specific secretory vesicles whose content is discharged during fertilization to block polyspermy. Here, we summarize the molecular mechanisms controlling critical aspects of CG biology prior to and after the gametes interaction. This allows to block polyspermy and provide protection to the developing embryo. We also examine how CGs form and are spatially redistributed during oogenesis. During egg activation, CG exocytosis (CGE) and content release are triggered by increases in intracellular calcium and relies on the function of maternally-loaded proteins. We also discuss how mutations in these factors impact CG dynamics, providing unprecedented models to investigate the genetic program executing fertilization. We further explore the phylogenetic distribution of maternal proteins and signaling pathways contributing to CGE and egg activation. We conclude that many important biological questions and genotype-phenotype relationships during fertilization remain unresolved, and therefore, novel molecular players of CG biology need to be discovered. Future functional and image-based studies are expected to elucidate the identity of genetic candidates and components of the molecular machinery involved in the egg activation. This, will open new therapeutic avenues for treating infertility in humans.

Deletion of TRPV3 and CaV3.2 T-type channels in mice undermines fertility and Ca2+ homeostasis in oocytes and eggs.

Ca2+ influx during oocyte maturation and after sperm entry is necessary to fill the internal Ca2+ stores and for complete egg activation. We knocked out the transient receptor potential vanilloid member 3 (TRPV3) and the T-type channel, CaV3.2, to determine their necessity for maintaining these functions in mammalian oocytes/eggs. Double-knockout (dKO) females were subfertile, their oocytes and eggs showed reduced internal Ca2+ stores, and, following sperm entry or Plcz (also known as Plcz1) cRNA injection, fewer dKO eggs displayed Ca2+ responses compared to wild-type eggs, which were also of lower frequency. These parameters were rescued and/or enhanced by removing extracellular Mg2+, suggesting that the residual Ca2+ influx could be mediated by the TRPM7 channel, consistent with the termination of divalent-cation oscillations in dKO eggs by a TRPM7 inhibitor. In total, we demonstrated that TRPV3 and CaV3.2 mediate the complete filling of the Ca2+ stores in mouse oocytes and eggs. We also showed that they are required for initiating and maintaining regularly spaced-out oscillations, suggesting that Ca2+ influx through PM ion channels dictates the periodicity and persistence of Ca2+ oscillations during mammalian fertilization.

RGD-binding integrins and TGF-ß in SARS-CoV-2 infections - novel targets to treat COVID-19 patients?

The new coronavirus SARS-CoV-2 is a global pandemic and a severe public health crisis. SARS-CoV-2 is highly contagious and shows high mortality rates, especially in elderly and patients with pre-existing medical conditions. At the current stage, no effective drugs are available to treat these patients. In this review, we analyse the rationale of targeting RGD-binding integrins to potentially inhibit viral cell infection and to block TGF-ß activation, which is involved in the severity of several human pathologies, including the complications of severe COVID-19 cases. Furthermore, we demonstrate the correlation between ACE2 and TGF-ß expression and the possible consequences for severe COVID-19 infections. Finally, we list approved drugs or drugs in clinical trials for other diseases that also target the RGD-binding integrins or TGF-ß. These drugs have already shown a good safety profile and, therefore, can be faster brought into a trial to treat COVID-19 patients.

TRPM8 Channel Promotes the Osteogenic Differentiation in Human Bone Marrow Mesenchymal Stem Cells.

Various families of ion channels have been characterized in mesenchymal stem cells (MSCs), including some members of transient receptor potential (TRP) channels family. TRP channels are involved in critical cellular processes as differentiation and cell proliferation. Here, we analyzed the expression of TRPM8 channel in human bone marrow MSCs (hBM-MSCs), and its relation with osteogenic differentiation. Patch-clamp recordings showed that hBM-MSCs expressed outwardly rectifying currents which were increased by exposure to 500 µM menthol and were partially inhibited by 10 µM of BCTC, a TRPM8 channels antagonist. Additionally, we have found the expression of TRPM8 by RT-PCR and western blot. We also explored the TRPM8 localization in hBM-MSCs by immunofluorescence using confocal microscopy. Remarkably, hBM-MSCs treatment with 100 µM of menthol or 10 µM of icilin, TRPM8 agonists, increases osteogenic differentiation. Conversely, 20 µM of BCTC, induced a decrease of osteogenic differentiation. These results suggest that TRPM8 channels are functionally active in hBM-MSCs and have a role in cell differentiation.

Hypoxic Regulation of the Large-Conductance, Calcium and Voltage-Activated Potassium Channel, BK.

Hypoxia is a condition characterized by a reduction of cellular oxygen levels derived from alterations in oxygen balance. Hypoxic events trigger changes in cell-signaling cascades, oxidative stress, activation of pro-inflammatory molecules, and growth factors, influencing the activity of various ion channel families and leading to diverse cardiovascular diseases such as myocardial infarction, ischemic stroke, and hypertension. The large-conductance, calcium and voltage-activated potassium channel (BK) has a central role in the mechanism of oxygen (O2) sensing and its activity has been related to the hypoxic response. BK channels are ubiquitously expressed, and they are composed by the pore-forming α subunit and the regulatory subunits ß (ß1-ß4), γ (γ1-γ4), and LINGO1. The modification of biophysical properties of BK channels by ß subunits underly a myriad of physiological function of these proteins. Hypoxia induces tissue-specific modifications of BK channel α and ß subunits expression. Moreover, hypoxia modifies channel activation kinetics and voltage and/or calcium dependence. The reported effects on the BK channel properties are associated with events such as the increase of reactive oxygen species (ROS) production, increases of intracellular Calcium ([Ca2+]i), the regulation by Hypoxia-inducible factor 1α (HIF-1α), and the interaction with hemeproteins. Bronchial asthma, chronic obstructive pulmonary diseases (COPD), and obstructive sleep apnea (OSA), among others, can provoke hypoxia. Untreated OSA patients showed a decrease in BK-ß1 subunit mRNA levels and high arterial tension. Treatment with continuous positive airway pressure (CPAP) upregulated ß1 subunit mRNA level, decreased arterial pressures, and improved endothelial function coupled with a reduction in morbidity and mortality associated with OSA. These reports suggest that the BK channel has a role in the response involved in hypoxia-associated hypertension derived from OSA. Thus, this review aims to describe the mechanisms involved in the BK channel activation after a hypoxic stimulus and their relationship with disorders like OSA. A deep understanding of the molecular mechanism involved in hypoxic response may help in the therapeutic approaches to treat the pathological processes associated with diseases involving cellular hypoxia.

Divalent cation influx and calcium homeostasis in germinal vesicle mouse oocytes.

Prior to maturation, mouse oocytes are arrested at the germinal vesicle (GV) stage during which they experience constitutive calcium (Ca2+) influx and spontaneous Ca2+ oscillations. The oscillations cease during maturation but Ca2+ influx continues, as the oocytes' internal stores attain maximal content at the culmination of maturation, the metaphase II stage. The identity of the channel(s) that underlie this Ca2+ influx has not been completely determined. GV and matured oocytes are known to express three Ca2+ channels, CaV3.2, TRPV3 and TRPM7, but females null for each of these channels are fertile and their oocytes display minor modifications in Ca2+ homeostasis, suggesting a complex regulation of Ca2+ influx. To define the contribution of these channels at the GV stage, we used different divalent cations, pharmacological inhibitors and genetic models. We found that the three channels are active at this stage. CaV3.2 and TRPM7 channels contributed the majority of Ca2+ influx, as inhibitors and oocytes from homologous knockout (KO) lines showed severely reduced Ca2+ entry. Sr2+ influx was promoted by CaV3.2 channels, as Sr2+ oscillations were negligible in CaV3.2-KO oocytes but robust in control and Trpv3-KO GV oocytes. Mn2+ entry relied on expression of CaV3.2 and TRPM7 channels, but Ni2+ entry depended on the latter. CaV3.2 and TRPV3 channels combined to fill the Ca2+ stores, although CaV3.2 was the most impactful. Studies with pharmacological inhibitors effectively blocked the influx of divalent cations, but displayed off-target effects, and occasionally agonist-like properties. In conclusion, GV oocytes express channels mediating Ca2+ and other divalent cation influx that are pivotal for fertilization and early development. These channels may serve as targets for intervention to improve the success of assisted reproductive technologies.

TRPM7 and CaV3.2 channels mediate Ca2+ influx required for egg activation at fertilization.

The success of mammalian development following fertilization depends on a series of transient increases in egg cytoplasmic Ca2+, referred to as Ca2+ oscillations. Maintenance of these oscillations requires Ca2+ influx across the plasma membrane, which is mediated in part by T-type, CaV3.2 channels. Here we show using genetic mouse models that TRPM7 channels are required to support this Ca2+ influx. Eggs lacking both TRPM7 and CaV3.2 stop oscillating prematurely, indicating that together they are responsible for the majority of Ca2+ influx immediately following fertilization. Fertilized eggs lacking both channels also frequently display delayed resumption of Ca2+ oscillations, which appears to require sperm-egg fusion. TRPM7 and CaV3.2 channels almost completely account for Ca2+ influx observed following store depletion, a process previously attributed to canonical store-operated Ca2+ entry mediated by STIM/ORAI interactions. TRPM7 serves as a membrane sensor of extracellular Mg2+ and Ca2+ concentrations and mediates the effects of these ions on Ca2+ oscillation frequency. When bred to wild-type males, female mice carrying eggs lacking TRPM7 and CaV3.2 are subfertile, and their offspring have increased variance in postnatal weight. These in vivo findings confirm previous observations linking in vitro experimental alterations in Ca2+ oscillatory patterns with developmental potential and offspring growth. The identification of TRPM7 and CaV3.2 as key mediators of Ca2+ influx following fertilization provides a mechanistic basis for the rational design of culture media that optimize developmental potential in research animals, domestic animals, and humans.

Ion Channel Function During Oocyte Maturation and Fertilization.

The proper maturation of both male and female gametes is essential for supporting fertilization and the early embryonic divisions. In the ovary, immature fully-grown oocytes that are arrested in prophase I of meiosis I are not able to support fertilization. Acquiring fertilization competence requires resumption of meiosis which encompasses the remodeling of multiple signaling pathways and the reorganization of cellular organelles. Collectively, this differentiation endows the egg with the ability to activate at fertilization and to promote the egg-to-embryo transition. Oocyte maturation is associated with changes in the electrical properties of the plasma membrane and alterations in the function and distribution of ion channels. Therefore, variations on the pattern of expression, distribution, and function of ion channels and transporters during oocyte maturation are fundamental to reproductive success. Ion channels and transporters are important in regulating membrane potential, but also in the case of calcium (Ca2+), they play a critical role in modulating intracellular signaling pathways. In the context of fertilization, Ca2+ has been shown to be the universal activator of development at fertilization, playing a central role in early events associated with egg activation and the egg-to-embryo transition. These early events include the block of polyspermy, the completion of meiosis and the transition to the embryonic mitotic divisions. In this review, we discuss the role of ion channels during oocyte maturation, fertilization and early embryonic development. We will describe how ion channel studies in Xenopus oocytes, an extensively studied model of oocyte maturation, translate into a greater understanding of the role of ion channels in mammalian oocyte physiology.

Small and Intermediate Calcium-Activated Potassium Channel Openers Improve Rat Endothelial and Erectile Function.

Modulation of endothelial calcium-activated potassium (KCa) channels has been proposed as an approach to restore endothelial function. The present study investigated whether novel openers of KCa channels with small (KCa2.x) and intermediate (KCa3.1) conductance, NS309 and NS4591, improve endothelium-dependent relaxation and erectile function. Rat corpus cavernosum (CC) strips were mounted for isometric tension recording and processed for immunoblotting. Mean arterial pressure (MAP), intracavernosal pressure (ICP), and electrocardiographic (ECG) measurements were conducted in anesthetized rats. Immunoblotting revealed the presence of KCa2.3 and large KCa conductance (KCa1.1) channels in the corpus cavernosum. NS309 and NS4591 increased current in CC endothelial cells in whole cell patch clamp experiments. Relaxation induced by NS309 (<1 µM) was inhibited by endothelial cell removal and high extracellular potassium. An inhibitor of nitric oxide (NO) synthase, and blockers of KCa2.x and KCa1.1 channels, apamin and iberiotoxin also inhibited NS309 relaxation. Incubation with NS309 (0.5 µM) markedly enhanced acetylcholine relaxation. Basal erectile function (ICP/MAP) increased during administration of NS309. Increases in ICP/MAP after cavernous nerve stimulation with NS309 were unchanged, whereas NS4591 significantly improved erectile function. Administration of NS309 and NS4591 caused small changes in the electrocardiogram, but neither arrhythmic events nor prolongation of the QTc interval were observed. The present study suggests that openers of KCa2.x and KCa3.1 channels improve endothelial and erectile function. The effects of NS309 and NS4591 on heart rate and ECG are small, but will require additional safety studies before evaluating whether activation of KCa2.3 channels has a potential for treatment of erectile dysfunction.

Conotoxins as Tools to Understand the Physiological Function of Voltage-Gated Calcium (CaV) Channels.

Voltage-gated calcium (CaV) channels are widely expressed and are essential for the completion of multiple physiological processes. Close regulation of their activity by specific inhibitors and agonists become fundamental to understand their role in cellular homeostasis as well as in human tissues and organs. CaV channels are divided into two groups depending on the membrane potential required to activate them: High-voltage activated (HVA, CaV1.1-1.4; CaV2.1-2.3) and Low-voltage activated (LVA, CaV3.1-3.3). HVA channels are highly expressed in brain (neurons), heart, and adrenal medulla (chromaffin cells), among others, and are also classified into subtypes which can be distinguished using pharmacological approaches. Cone snails are marine gastropods that capture their prey by injecting venom, "conopeptides", which cause paralysis in a few seconds. A subset of conopeptides called conotoxins are relatively small polypeptides, rich in disulfide bonds, that target ion channels, transporters and receptors localized at the neuromuscular system of the animal target. In this review, we describe the structure and properties of conotoxins that selectively block HVA calcium channels. We compare their potency on several HVA channel subtypes, emphasizing neuronal calcium channels. Lastly, we analyze recent advances in the therapeutic use of conotoxins for medical treatments.

TRPM7-like channels are functionally expressed in oocytes and modulate post-fertilization embryo development in mouse.

The Transient Receptor Potential (TRP) channels are a family of cationic ion channels widely distributed in mammalian tissues. In general, the global genetic disruption of individual TRP channels result in phenotypes associated with impairment of a particular tissue and/or organ function. An exception is the genetic ablation of the TRP channel TRPM7, which results in early embryonic lethality. Nevertheless, the function of TRPM7 in oocytes, eggs and pre-implantation embryos remains unknown. Here, we described an outward rectifying non-selective current mediated by a TRP ion channel in immature oocytes (germinal vesicle stage), matured oocytes (metaphase II eggs) and 2-cell stage embryos. The current is activated by specific agonists and inhibited by distinct blockers consistent with the functional expression of TRPM7 channels. We demonstrated that the TRPM7-like channels are homo-tetramers and their activation mediates calcium influx in oocytes and eggs, which is fundamental to support fertilization and egg activation. Lastly, we showed that pharmacological inhibition of the channel function delays pre-implantation embryo development and reduces progression to the blastocyst stage. Our data demonstrate functional expression of TRPM7-like channels in mouse oocytes, eggs and embryos that may play an essential role in the initiation of embryo development.

TRPV3 channels mediate Ca²âº influx induced by 2-APB in mouse eggs.

Fertilization in mammals is initiated when a sperm fuses with a mature MII oocyte, also known as egg, and triggers a plethora of finely controlled processes identified as egg activation. The completion of all events of egg activation is driven by and depends on a series of repetitive calcium (Ca(2+)) increases (Ca(2+) oscillations), which rely on Ca(2+) influx from the extracellular media. Ca(2+) channels on the egg plasma membrane (PM) are thought to mediate this influx. The TRP Ca(2+) channel TRPV3 is differentially expressed during oocyte maturation, being most active at the MII stage. Specific stimulation of TRPV3 channels promotes Ca(2+) influx sufficient to induce egg activation and parthenogenesis. Here, we explore the function and distribution dynamics of the TRPV3 channel protein during maturation. Using dsRNA, TrpV3 overexpression, and inhibitors of protein synthesis, we modified the expression levels of the channel and showed that the TRPV3 protein is synthesized and translocated to the PM during maturation. We demonstrated that 2-APB at the concentrations used here to promote Ca(2+) influx in eggs, specifically and reversibly targets TRPV3 channels without blocking IP3R1. Finally, we found that the activity of TRPV3 channels is dependent upon an intact actin cytoskeleton, suggesting an actin-based regulation of its expression and/or function on the PM. Collectively, our results show TRPV3 is a target of 2-APB in eggs, a condition that can be used to induce parthenogenesis. The need of an intact actin cytoskeleton for the function of TRPV3 channels in oocytes is a novel finding and suggests the rearrangements of actin that occur during maturation could regulate both the presence on the PM and/or the function of TRPV3 and of other Ca(2+) channels involved in oocyte maturation and fertilization.

TRPV3 channels mediate strontium-induced mouse-egg activation.

In mammals, calcium influx is required for oocyte maturation and egg activation. The molecular identities of the calcium-permeant channels that underlie the initiation of embryonic development are not established. Here, we describe a transient receptor potential (TRP) ion channel current activated by TRP agonists that is absent in TrpV3(-/-) eggs. TRPV3 current is differentially expressed during oocyte maturation, reaching a peak of maximum density and activity at metaphase of meiosis II (MII), the stage of fertilization. Selective activation of TRPV3 channels provokes egg activation by mediating massive calcium entry. Widely used to activate eggs, strontium application is known to yield normal offspring in combination with somatic cell nuclear transfer. We show that TRPV3 is required for strontium influx, because TrpV3(-/-) eggs failed to conduct Sr(2+) or undergo strontium-induced activation. We propose that TRPV3 is a major mediator of calcium influx in mouse eggs and is a putative target for artificial egg activation.

An aqueous H+ permeation pathway in the voltage-gated proton channel Hv1.

Hv1 voltage-gated proton channels mediate rapid and selective transmembrane H(+) flux and are gated by both voltage and pH gradients. Selective H(+) transfer in membrane proteins is commonly achieved by Grotthuss proton 'hopping' in chains of ionizable amino acid side chains and intraprotein water molecules. To identify whether ionizable residues are required for proton permeation in Hv1, we neutralized candidate residues and measured expressed voltage-gated H(+) currents. Unexpectedly, charge neutralization was insufficient to abrogate either the Hv1 conductance or coupling of pH gradient and voltage-dependent activation. Molecular dynamics simulations revealed water molecules in the central crevice of Hv1 model structures but not in homologous voltage-sensor domain (VSD) structures. Our results indicate that Hv1 most likely forms an internal water wire for selective proton transfer and that interactions between water molecules and S4 arginines may underlie coupling between voltage- and pH-gradient sensing.

[Factors associated with access to health services prior to pregnancy by pregnant adolescents]. / Fatores associados ao acesso anterior à gestação a serviços de saúde por adolescentes gestantes.

OBJECTIVE: To assess determinants of access to primary care services prior to pregnancy by pregnant adolescents. METHODS: Theory-based cross-sectional study conducted to examine access to heath services at five dimensions: geographic, economic, administrative, psychosocial and information. There were included in the study 200 first-time pregnant adolescents (aged 10 to 19 years) who attended a primary care unit in the municipality of Indaiatuba, Southeastern Brazil, in 2003. Data was collected using a semi-structured questionnaire applied in face-to-face interviews conducted just before their first pre-natal care visit. Data analyses were performed through Person's chi-square or Fisher's exact test, and by a multivariate logistic regression model including all five dimensions of access. RESULTS: More than half (63.7%) of the adolescents utilized some gynecological care service. Information (43.8%) or psychosocial feelings of embarrassment or fear (37.0%) barriers were the most frequent reasons given for not having sought care before. The main barrier to health service access was related to the psychosocial dimension, reported by 77.0% of the adolescents studied. CONCLUSIONS: Among all barriers to health service access, the most important were those related to the psychosocial dimension. There is a need for new strategies to facilitate access to health services by adolescents, including actions to reduce gender-based barriers taking into consideration sociodemographic characteristics of this population and the relationship they have with their partners.

Fatores associados ao acesso anterior à gestação a serviços de saúde por adolescentes gestantes / Factores asociados con el acceso anterior a la gestación a los servicios de salud por adolescentes gestantes / Factors associated with access to health services prior to pregnancy by pregnant adolescents

OBJETIVO: Analisar os fatores determinantes do acesso de adolescentes gestantes a serviços de atenção primária à saúde, anterior à ocorrência da gestação. MÉTODOS: Estudo transversal baseado em referencial teórico. O acesso a serviços foi analisado em cinco dimensões: geográfico, econômico, administrativo, psicossocial e de informação. Participaram 200 adolescentes primigestas (10 a 19 anos) atendidas em uma unidade básica de saúde do município de Indaiatuba (SP), em 2003. Um questionário com perguntas abertas e fechadas referentes ao acesso ao último serviço de saúde utilizado, anterior à gestação, foi aplicado às participantes no momento de sua primeira consulta de pré-natal. Os dados foram analisados por meio do teste de qui-quadrado de Pearson ou exato de Fisher e por regressão logística múltipla, considerando as cinco dimensões de acesso. RESULTADOS: Mais da metade (63,7 por cento) das adolescentes utilizou algum serviço de saúde para consulta ginecológica...

OBJECTIVE: To assess determinants of access to primary care services prior to pregnancy by pregnant adolescents. METHODS: Theory-based cross-sectional study conducted to examine access to heath services at five dimensions: geographic, economic, administrative, psychosocial and information. There were included in the study 200 first-time pregnant adolescents (aged 10 to 19 years) who attended a primary care unit in the municipality of Indaiatuba, Southeastern Brazil, in 2003. Data was collected using a semi-structured questionnaire applied in face-to-face interviews conducted just before their first pre-natal care visit. Data analyses were performed through Person's chi-square or Fisher's exact test, and by a multivariate logistic regression model including all five dimensions of access. RESULTS: More than half (63.7 percent) of the adolescents utilized some gynecological care service. Information (43.8 percent) or psychosocial feelings of embarrassment or fear (37.0 percent)...

OBJETIVO: Analizar los factores determinantes del acceso de adolescentes gestantes a los servicios de atención primaria a la salud, anterior a la ocurrencia de la gestación. MÉTODOS: Se efectuó estudio transversal basado en referencial teórico. El acceso a servicios fue analizado en cinco dimensiones: geográfico, económico, administrativo, psicosocial y de información. Participaron 200 adolescentes primigestas (10 a 19 años) atendidas en una unidad básica de salud del municipio de Indaiatuba (Sureste de Brasil), en 2003. Se aplicó a las participantes en el momento de su primera consulta pre-natal un cuestionario con preguntas abiertas y cerradas referentes al acceso al último servicio de salud utilizado, anterior a la gestación. Los datos fueron analizados por medio de la prueba de Chi cuadrado de Pearson o exacto de Fischer y por regresión logística múltiple, considerando las cinco dimensiones del acceso...

[Knowledge of pregnant adolescents about reproductive anatomy and physiology in a municipality of Southern Brazil]. / Conhecimento de adolescentes grávidas sobre anatomia e fisiologia da reprodução.

OBJECTIVES: To study knowledge of some aspects of the female reproductive anatomy and physiology and their association with socio-demographic and reproductive "choices" of pregnant adolescents. METHODS: A cross-sectional study was performed with 200 first time pregnant adolescents who attended a public women's health clinic in the municipality of Indaiatuba, Sao Paulo, Brazil. During their first prenatal care visit, face-to-face interviews were conducted using a structured questionnaire and a three-dimensional handmade female model. Bivariate data analyses were performed using Pearson's Chi-square or Fisher's Exact test. Data were also analyzed using multivariate logistic regression models to test for associations of indicators of knowledge of female reproductive anatomy, physiology of female reproductive organs and physiology of reproduction with socio-demographic characteristics and reproductive "choices". RESULTS: The majority had little knowledge of anatomy (55.5%), with external organs more easily identified and placed than the internal; of physiology of reproductive organs (61.0%), and of physiology of reproduction (76.5%). Associations were found between knowledge and age of partner, couple difference of age, maintenance of the relationship with partner after pregnancy, religious affiliation, and level of education. No association was found between indicators of knowledge with use of contraceptives at first intercourse and with intention of having the baby at that time. CONCLUSION: This study addressed the complexity of the relationship between knowledge of reproductive anatomy and physiology and the theme of adolescent pregnancy, and emphasized the need for more contextualized approaches of programmatic contents on sexual education, in view of the intention to reduce early pregnancy.

Conhecimento de adolescentes grávidas sobre anatomia e fisiologia da reprodução / Knowledge of pregnant adolescents about reproductive anatomy and physiology in a municipality of Southern Brazil

OBJETIVOS: Verificar o conhecimento sobre alguns aspectos do aparelho genital feminino, da fisiologia da reprodução e sua associação com características sociodemográficas e "escolhas" reprodutivas em adolescentes gestantes. MÉTODOS: Realizou-se um estudo de corte transversal com 200 adolescentes primigestas, durante a primeira consulta de pré-natal no Ambulatório da Mulher de Indaiatuba, São Paulo. Foram feitas entrevistas face a face, com questionário estruturado e um modelo feminino tridimensional confeccionado artesanalmente para coleta dos dados. Os dados foram analisados utilizando-se os testes Qui-quadrado de Pearson, ou Exato de Fisher, e por regressões logísticas múltiplas, para verificar associações entre indicadores do conhecimento sobre anatomia dos órgãos genitais femininos, fisiologia dos órgãos e da reprodução com características sociodemográficas e "escolhas" reprodutivas. RESULTADOS: A maioria tinha conhecimento insatisfatório sobre anatomia (55,5 por cento), com os órgãos externos sendo identificados com maior facilidade e melhor localizados do que os internos; fisiologia dos órgãos (61 por cento); e aspectos fisiológicos da reprodução (76,5 por cento). Algumas associações significativas foram estabelecidas entre o conhecimento e a idade dos parceiros, diferença de idade do casal, manutenção do vinculo após ocorrência da gravidez, filiação religiosa e escolaridade da adolescente. Não houve associação entre os indicadores de conhecimento estudados com a utilização de método anticoncepcional na primeira relação sexual e a intenção de ter um filho naquele momento. CONCLUSÃO: Este estudo faz emergir a complexidade da relação entre o conhecimento sobre anatomia e fisiologia reprodutivas e a temática da gravidez na adolescência, evidenciando a necessidade de abordagens mais contextualizadas dos conteúdos de programas de educação sexual, quando seu foco for a redução da gravidez precoce.

OBJECTIVES: To study knowledge of some aspects of the female reproductive anatomy and physiology and their association with socio-demographic and reproductive "choices" of pregnant adolescents. METHODS: A cross-sectional study was performed with 200 first time pregnant adolescents who attended a public women's health clinic in the municipality of Indaiatuba, Sao Paulo, Brazil. During their first prenatal care visit, face-to-face interviews were conducted using a structured questionnaire and a three-dimensional handmade female model. Bivariate data analyses were performed using Person's Chi-square or Fisher Exact test. Data were also analyzed using multivariate logistic regression models to test for associations of indicators of knowledge of female reproductive anatomy, physiology of female reproductive organs and physiology of reproduction with socio-demographic characteristics and reproductive "choices". RESULTS: The majority had little knowledge of anatomy (55.5 percent), with external organs more easily identified and placed than the internal; of physiology of reproductive organs (61.0 percent), and of physiology of reproduction (76.5 percent). Associations were found between knowledge and age of partner, couple difference of age, maintenance of the relationship with partner after pregnancy, religious affiliation, and level of education. No association was found between indicators of knowledge with use of contraceptives at first intercourse and with intention of having the baby at that time. CONCLUSION: This study addressed the complexity of the relationship between knowledge of reproductive anatomy and physiology and the theme of adolescent pregnancy, and emphasized the need for more contextualized approaches of programmatic contents on sexual education, in view of the intention to reduce early pregnancy.

Intrinsic electrostatic potential in the BK channel pore: role in determining single channel conductance and block.

The internal vestibule of large-conductance Ca(2+) voltage-activated K(+) (BK) channels contains a ring of eight negative charges not present in K(+) channels of lower conductance (Glu386 and Glu389 in hSlo) that modulates channel conductance through an electrostatic mechanism (Brelidze, T.I., X. Niu, and K.L. Magleby. 2003. Proc. Natl. Acad. Sci. USA. 100:9017-9022). In BK channels there are also two acidic amino acid residues in an extracellular loop (Asp326 and Glu329 in hSlo). To determine the electrostatic influence of these charges on channel conductance, we expressed wild-type BK channels and mutants E386N/E389N, D326N, E329Q, and D326N/E329Q channels on Xenopus laevis oocytes, and measured the expressed currents under patch clamp. Contribution of E329 to the conductance is negligible and single channel conductance of D326N/E329Q channels measured at 0 mV in symmetrical 110 mM K(+) was 18% lower than the control. Current-voltage curves displayed weak outward rectification for D326N and the double mutant. The conductance differences between the mutants and wild-type BK were caused by an electrostatic effect since they were enhanced at low K(+) (30 mM) and vanished at high K(+) (1 M K(+)). We determine the electrostatic potential change, Deltaphi, caused by the charge neutralization using TEA(+) block for the extracellular charges and Ba(2+) for intracellular charges. We measured 13 +/- 2 mV for Deltaphi at the TEA(+) site when turning off the extracellular charges, and 17 +/- 2 mV for the Deltaphi at the Ba(2+) site when the intracellular charges were turned off. To understand the electrostatic effect of charge neutralizations, we determined Deltaphi using a BK channel molecular model embedded in a lipid bilayer and solving the Poisson-Boltzmann equation. The model explains the experimental results adequately and, in particular, gives an economical explanation to the differential effect on the conductance of the neutralization of charges D326 and E329.