Estudio Histológico en Embriones de Cobayo (Cavia porcellus) y su Utilidad como Modelo para la Comprensión del Desarrollo Embrionario Humano / Histological Study in Guinea Pig Embryos (Cavia porcellus) and its Usefulness as a Model for Understanding Human Embryonic Development

El cobayo es un modelo animal ampliamente utilizado en la investigación biomédica debido a sus similitudes biológicas con los seres humanos. El objetivo de nuestro estudio es proporcionar sustento morfológico para utilizar preparados histológicos de embriones de cobayo como modelo de estudio para comprender los procesos del desarrollo embrionario humano. Nuestros resultados muestran que los embriones de cobayo presentan características morfológicas similares a las observadas en los embriones humanos, lo que sugiere que pueden utilizarse como un modelo efectivo para estudiar el desarrollo embrionario humano. Este hallazgo tiene importantes implicancias para la investigación y la docencia utilizando este modelo animal. Se analizaron preparados histológicos de embriones de cobayo teñidos con hematoxilina eosina, adquiridos por la Universidad Autónoma de Chile. Se tomaron microfotografías de preparados histológicos de cobayo en diferentes estadios del desarrollo y se seleccionaron las mejores imágenes para la descripción de estructuras y establecer estimados de la embriogénesis. Del análisis de los preparados se desprende que órganos como esófago, médula espinal y corazón presentan similitudes anatómicas e histológicas que hacen posible compararlas con el desarrollo embrionario humano y la edad de gestación en etapas tempranas. El uso de preparados de embriones de cobayo y su análisis desde un aspecto histológico resulta ser una estrategia metodológica factible debido a las similitudes en la embriogénesis de los mamíferos y las concordancias morfológicas con el desarrollo de los órganos entre humanos y roedores. Esto permite implementar este modelo animal como una herramienta para comprender el desarrollo embrionario humano.

SUMMARY: The guinea pig is an animal model widely used in biomedical research due to its biological similarities with humans. The objective of our study is to provide morphological support to use histological preparations of guinea pig embryos as a study model to understand the processes of human embryonic development. Our results show that guinea pig embryos present morphological characteristics similar to those observed in human embryos, suggesting that they can be used as an effective model to study human embryonic development. This finding has important implications for research and teaching using this animal model. Histological preparations of guinea pig embryos stained with hematoxylin eosin, acquired by the Autonomous University of Chile, were analyzed. Photomicrographs of histological preparations of guinea pigs at different stages of development were taken and the best images were selected to describe structures and establish estimates of embryogenesis. From the analysis of the preparations it is clear that organs such as the esophagus, spinal cord and heart present anatomical and histological similarities that make it possible to compare them with human embryonic development and gestation age in early stages. The use of guinea pig embryo preparations and their analysis from a histological aspect turns out to be a feasible methodological strategy due to the similarities in mammalian embryogenesis and the morphological concordances with the development of organs between humans and rodents. This allows this animal model to be implemented as a tool to understand human embryonic development.

Ruptura prematura de membranas ovulares / Premature rupture of ovular membranes

La ruptura prematura de las membranas ovulares se define como la pérdida de la integridad del amnios y corion antes del inicio del trabajo de parto, afecta el 3 % de los embarazos, causa un tercio de los partos pretérminos, los cuales ocupan el 10,49 % de los nacimientos y es el origen de altos índices de morbimortalidad perinatal. En la actualidad, el manejo de esta patología se orienta principalmente en evitar los factores de riesgo, hacer un diagnóstico adecuado, determinar la edad gestacional en que ocurre, realizar el monitoreo exhaustivo del bienestar materno-fetal y en decidir el momento idóneo de finalización de la gestación para minimizar sus complicaciones. Debido a la compleja y lábil estructura histológica de las membranas ovulares, se ha dejado a un lado el tratamiento directo de la entidad el cual sería sellar o reparar el defecto en sí. En los últimos años, numerosos estudios y protocolos clínicos de prestigiosos centros asistenciales han servido como guía para el manejo de esta entidad, pero en muy pocos se observa una terapia destinada a la reparación de dichas membranas o en sellar tal defecto. Las evidencias científicas demuestran que la regeneración y reparación de las membranas es lenta y compleja y los tratamientos propuestos para reparar o sellar su defecto no han gozado de la aceptación científica para su aprobación, sin embargo, el uso del parche hemático transvaginal endocervical autólogo luce como una alternativa terapéutica prometedora(AU)

The premature rupture of the ovular membranes is defined as the loss of the integrity of the amnion and chorion before the on set of labor, affects 3% of pregnancies, causes athird of preterm births which occupy 10,49% of births and is the origin of high rates of perinatal morbidity and mortality. At present, the management of this pathology is mainly oriented towards avoiding risk factors, making an adequate diagnosis, determining the gestational age in which it occurs, carrying out exhaustive monitoring of maternal-fetal well-being and deciding the ideal moment to end the treatment. Pregnancy to minimizeits complications. Due to the complex and labile histological structure of the ovular membranes, the direct treatment of the entity has been set a side, which would be to seal or repairthe defect it self. In recent years, numerous studies and clinicalprotocols from prestigious health care centers have served as aguide for the management of this entity, but very few have observed a therapy aimed at repairing said membranes or sealing such a defect. Scientific evidence shows that the regeneration and repair of the membranes is slow and complex and the treatment sproposed to repair or seal their defect have not enjoyed scientific acceptance for their approval, how ever, the use of the autologous endocervical transvaginal blood patch looks like a promising therapeutic alternative(AU)

Study of ex-ovo embryonic development of Gallus gallus domesticus / Estudio inicial del desarrollo embrionario ex-ovo de Gallus gallus domesticus

SUMMARY: The domestic chicken is a species of bird that has been extensively studied in regard to its biology and as a model organism for science. The reproduction of the species is by the laying of fertilized eggs, which in a period of 21 days will develop a chick inside. Several methods have been described to develop embryos ex-ovo, allowing the observation and manipulation of the organism. This work has the propose to standardize a method that allows the development of the embryos inside the artificial incubation system, which has a low cost and is easy to make. In this work, 100 chicken eggs were used to study the effects of humidity, mineral supplementation, and the preincubation time of the egg on the incubation ex-ovo of the embryos. Embryo development was documented through the different days. Pulverized eggshell was selected as an optimal source to provide calcium, magnesium, phosphorus, and other minerals to the developing embryo. By providing 900-1200 mg of pulverized eggshell, 40 mL of the 0.001 % solution of benzalkonium chloride, and a preincubation time of approximately 56 h, the embryos were able to develop until 19 days, and even though they did not reach hatching, the incubation conditions that allowed the survival and development of embryos until late stages were achieved. Thus, due to the conditions established for calcium, humidity and preincubation time, in the present work, the chicks reached 19 days of development.

El pollo doméstico es una especie de ave que ha sido ampliamente estudiada en cuanto a su biología y como organismo modelo para la ciencia. La reproducción de la especie es por la puesta de huevos fecundados, que en un período de 21 días desarrollarán un polluelo en su interior. Se han descrito varios métodos para desarrollar embriones ex-ovo, permitiendo la observación y manipulación del organismo. Este trabajo tuvo como objetivo estandarizar un método que permita el desarrollo de los embriones dentro del sistema de incubación artificial, el cual tiene un bajo costo y es fácil de realizar. En este trabajo se utilizaron 100 huevos de gallina para estudiar los efectos de la humedad, la suplementación mineral y el tiempo de preincubación del huevo sobre la incubación ex-ovo de los embriones. El desarrollo embrionario se documentó a través de los diferentes días. Se seleccionó la cáscara de huevo pulverizada como una fuente óptima para proporcionar calcio, magnesio, fósforo y otros minerales al embrión en desarrollo. Al suministrar 900-1200 mg de cáscara de huevo pulverizada, 40 mL de la solución de cloruro de benzalconio al 0.001 % y un tiempo de preincubación de aproximadamente 56 h, los embriones lograron desarrollarse hasta los 19 días, y aunque no llegaron a eclosionar, los embriones lograron desarrollarse hasta los 19 días. Se lograron condiciones de incubación que permitieron la supervivencia y desarrollo de los embriones hasta etapas tardías. Así, debido a las condiciones establecidas de calcio, humedad y tiempo de preincubación, en el presente trabajo los pollitos alcanzaron los 19 días de desarrollo.

DYRK1-mediated phosphorylation of endocytic components is required for extracellular lumen expansion in ascidian notochord

BACKGROUND: The biological tube is a basal biology structure distributed in all multicellular animals, from worms to humans, and has diverse biological functions. Formation of tubular system is crucial for embryogenesis and adult metabolism. Ascidian Ciona notochord lumen is an excellent in vivo model for tubulogenesis. Exocytosis has been known to be essential for tubular lumen formation and expansion. The roles of endocytosis in tubular lumen expansion remain largely unclear. RESULTS: In this study, we first identified a dual specificity tyrosine-phosphorylation-regulated kinase 1 (DYRK1), the protein kinase, which was upregulated and required for ascidian notochord extracellular lumen expansion. We demonstrated that DYRK1 interacted with and phosphorylated one of the endocytic components endophilin at Ser263 that was essential for notochord lumen expansion. Moreover, through phosphoproteomic sequencing, we revealed that in addition to endophilin, the phosphorylation of other endocytic components was also regulated by DYRK1. The loss of function of DYRK1 disturbed endocytosis. Then, we demonstrated that clathrin-mediated endocytosis existed and was required for notochord lumen expansion. In the meantime, the results showed that the secretion of noto-chord cells is vigorous in the apical membrane. CONCLUSIONS: We found the co-existence of endocytosis and exocytosis activities in apical membrane during lumen formation and expansion in Ciona notochord. A novel signaling pathway is revealed that DYRK1 regulates the endocytosis by phosphorylation that is required for lumen expansion. Our finding thus indicates a dynamic balance between endocytosis and exocytosis is crucial to maintain apical membrane homeostasis that is essential for lumen growth and expansion in tubular organogenesis.

Modeling human pregastrulation development by 3D culture of blastoids generated from primed-to-naïve transitioning intermediates

Human pluripotent stem cells provide an inexhaustible model to study human embryogenesis in vitro. Recent studies have provided diverse models to generate human blastoids by self-organization of different pluripotent stem cells or somatic reprogramming intermediates. However, whether blastoids can be generated from other cell types or whether they can recapitulate postimplantation development in vitro is unknown. Here, we develop a strategy to generate human blastoids from heterogeneous intermediates with epiblast, trophectoderm, and primitive endoderm signatures of the primed-to-naïve conversion process, which resemble natural blastocysts in morphological architecture, composition of cell lineages, transcriptome, and lineage differentiation potential. In addition, these blastoids reflect many features of human peri-implantation and pregastrulation development when further cultured in an in vitro 3D culture system. In summary, our study provides an alternative strategy to generate human blastoids and offers insights into human early embryogenesis by modeling peri- and postimplantation development in vitro.

Bilineage embryo-like structure from EPS cells can produce live mice with tetraploid trophectoderm

Self-organized blastoids from extended pluripotent stem (EPS) cells possess enormous potential for investigating postimplantation embryo development and related diseases. However, the limited ability of postimplantation development of EPS-blastoids hinders its further application. In this study, single-cell transcriptomic analysis indicated that the "trophectoderm (TE)-like structure" of EPS-blastoids was primarily composed of primitive endoderm (PrE)-related cells instead of TE-related cells. We further identified PrE-like cells in EPS cell culture that contribute to the blastoid formation with TE-like structure. Inhibition of PrE cell differentiation by inhibiting MEK signaling or knockout of Gata6 in EPS cells markedly suppressed EPS-blastoid formation. Furthermore, we demonstrated that blastocyst-like structures reconstituted by combining the EPS-derived bilineage embryo-like structure (BLES) with either tetraploid embryos or tetraploid TE cells could implant normally and develop into live fetuses. In summary, our study reveals that TE improvement is critical for constructing a functional embryo using stem cells in vitro.

A Spacetime Odyssey of Neural Progenitors to Generate Neuronal Diversity / 神经科学通报·英文版

To understand how the nervous system develops from a small pool of progenitors during early embryonic development, it is fundamentally important to identify the diversity of neuronal subtypes, decode the origin of neuronal diversity, and uncover the principles governing neuronal specification across different regions. Recent single-cell analyses have systematically identified neuronal diversity at unprecedented scale and speed, leaving the deconstruction of spatiotemporal mechanisms for generating neuronal diversity an imperative and paramount challenge. In this review, we highlight three distinct strategies deployed by neural progenitors to produce diverse neuronal subtypes, including predetermined, stochastic, and cascade diversifying models, and elaborate how these strategies are implemented in distinct regions such as the neocortex, spinal cord, retina, and hypothalamus. Importantly, the identity of neural progenitors is defined by their spatial position and temporal patterning factors, and each type of progenitor cell gives rise to distinguishable cohorts of neuronal subtypes. Microenvironmental cues, spontaneous activity, and connectional pattern further reshape and diversify the fate of unspecialized neurons in particular regions. The illumination of how neuronal diversity is generated will pave the way for producing specific brain organoids to model human disease and desired neuronal subtypes for cell therapy, as well as understanding the organization of functional neural circuits and the evolution of the nervous system.

Oocyte quality assessment in marine invertebrates: a novel approach by fluorescence spectroscopy

BACKGROUND: The assessment of oocyte quality is, nowadays, a major challenge in aquaculture, oocyte cryopreservation, and environmental science. Oocyte quality is a determining factor in fertilization and embryo development; however, there is still a lack of rapid and sensitive cellular markers for its assessment. Currently, its estimation is pre-dominantly based on morphological analysis, which is subjective and does not consistently reflect the developmental competence of the oocytes. Despite several recent studies investigating molecular markers related to oocyte quality, methods currently available for their determination pose various technical challenges and limitations. In this study, we developed a novel approach based on fluorescence spectroscopy to assess different intrinsic physiological parameters that can be employed to evaluate egg quality in marine invertebrates that are widely used as animal models such as sea urchins and mussels. RESULTS: Different physiological parameters, such as viability, mitochondrial activity, intracellular ROS levels, plasma membrane lipid peroxidation, and intracellular pH, for egg quality evaluation have been successfully assessed in sea urchins and mussels by using specific fluorescent dyes and detecting the fluorescent signals in eggs through fluorescence spectroscopy. CONCLUSIONS: Based on our findings, we propose these physiological markers as useful predictors of egg quality in marine invertebrates; they can be estimated rapidly, selectively, and sensitively by employing this novel approach, which, due to the speed of analysis, the low cost, and easy use can be considered a powerful analytical tool for the egg quality assessment.

Sperm DNA damage compromises embryo development, but not oocyte fertilisation in pigs

BACKGROUND: The assessment of sperm DNA integrity has been proposed as a complementary test to conventional mammalian semen analysis. In this sense, single-strand (SSB) and double-strand (DSB) DNA breaks, the two types of sperm DNA fragmentation (SDF), have been reported to have different aetiologies and to be associated to different fertility outcomes in bovine and humans. Considering that no studies in porcine have addressed how SDF may affect sperm quality and fertility outcomes, the present work aimed to determine the impact of global DNA damage, SSB and DSB on sperm quality and in vitro fertilising ability. To this end, 24 ejaculates (one per boar) were split into three aliquots: the first was used to assess sperm quality parameters through a computer-assisted sperm analysis (CASA) system and flow cytometry; the second was used to perform in vitro fertilisation, and the third, to evaluate sperm DNA integrity using alkaline and neutral Comet assays. RESULTS: The results showed that global DNA damage negatively correlates (P 0.05). CONCLUSION: Considering all these findings, this work sets a useful model to study how SDF negatively influences fertility.

LIN28 coordinately promotes nucleolar/ribosomal functions and represses the 2C-like transcriptional program in pluripotent stem cells

LIN28 is an RNA binding protein with important roles in early embryo development, stem cell differentiation/reprogramming, tumorigenesis and metabolism. Previous studies have focused mainly on its role in the cytosol where it interacts with Let-7 microRNA precursors or mRNAs, and few have addressed LIN28's role within the nucleus. Here, we show that LIN28 displays dynamic temporal and spatial expression during murine embryo development. Maternal LIN28 expression drops upon exit from the 2-cell stage, and zygotic LIN28 protein is induced at the forming nucleolus during 4-cell to blastocyst stage development, to become dominantly expressed in the cytosol after implantation. In cultured pluripotent stem cells (PSCs), loss of LIN28 led to nucleolar stress and activation of a 2-cell/4-cell-like transcriptional program characterized by the expression of endogenous retrovirus genes. Mechanistically, LIN28 binds to small nucleolar RNAs and rRNA to maintain nucleolar integrity, and its loss leads to nucleolar phase separation defects, ribosomal stress and activation of P53 which in turn binds to and activates 2C transcription factor Dux. LIN28 also resides in a complex containing the nucleolar factor Nucleolin (NCL) and the transcriptional repressor TRIM28, and LIN28 loss leads to reduced occupancy of the NCL/TRIM28 complex on the Dux and rDNA loci, and thus de-repressed Dux and reduced rRNA expression. Lin28 knockout cells with nucleolar stress are more likely to assume a slowly cycling, translationally inert and anabolically inactive state, which is a part of previously unappreciated 2C-like transcriptional program. These findings elucidate novel roles for nucleolar LIN28 in PSCs, and a new mechanism linking 2C program and nucleolar functions in PSCs and early embryo development.

Quiste del rafe medio perineal / Median raphe cysts of the perineum

Introducción: los quistes de rafe medio (QRM) son lesiones infrecuentes del aparato genital masculino, pueden localizarse desde la parte distal del pene hasta la zona perianal. Se deben a defectos del cierre del rafe medio durante el desarrollo embrionario. Objetivo: presentar un lactante con el diagnóstico de QRM, discutir el diagnóstico, tratamiento y seguimiento. Caso clínico: niño de 1 año y 3 meses de edad con 7 lesiones quísticas de 0.5 cm cada una en el rafe medio escrotal desde el nacimiento, la ecografía de partes blandas reporta imagen sólida hipoecogénica de borde definidos y sin vascularización al doppler. Se realizó exéresis quirúrgica y la patología confirmó el diagnóstico de quiste de rafe medio perineal, con buena evolución en el seguimiento. Discusión: 75% de los casos de los QRM son asintomáticos es por ello que muchas veces su diagnóstico es tardío, además su desconocimiento produce confusión con patologías quísticas o tumorales similares. El diagnóstico es clínico, aunque la ecografía puede ayudar para excluir otras anomalías como las vasculares. El tratamiento de elección debe ser la extirpación quirúrgica para evitar episodios de sobreinfección o molestias locales derivadas de su localización y tamaño. Conclusión: debido a lo infrecuente de esta patología es importante darla a conocer. Se recomienda realizar la valoración integral del paciente pediátrico para lograr un adecuado diagnóstico, ofrecer el mejor tratamiento y brindar información adecuada a los padres.

Introduction: median raphe cysts (MRC) are uncommon lesions of the male genitalia. They can be found anywhere from the distal penis to the perianal area. They are caused by an incomplete closure of the median raphe during embryologic development. Objective: to present a case of MRC in an infant and provide a review on diagnosis, treatment and follow-up. Case report: one year and three months old boy, with seven 0.5 cm sized cystic lesions along the scrotum median raphe, noticed since birth. A soft tissue ultrasound demonstrated a well-circumscribed hypoechogenic solid image without any evidence of vascularity at Doppler ultrasound. Surgical excision was conducted and histopathology confirmed the diagnosis of a median raphe cyst of the perineum, showing good progression in follow-up. Discussion: 75% of cases of MRC are asymptomatic, thus their diagnosis is often delayed. Not knowing this condition leads to confuse MRC with other similar cysts or tumors. The diagnosis is mostly clinical, although ultrasound may help to exclude other anomalies such as vascular anomalies. Surgical excision is the treatment of choice to prevent superinfection or local discomfort due to its location and size. Conclusion: Recognition of this uncommon pathology is imperative. A comprehensive evaluation of the pediatric patient is recommended to achieve an adequate diagnosis and treatment and provide appropriate information to the parents.

Síndrome de heterotaxia: hallazgos tomográficos / Heterotaxy syndrome: tomographic findings

Resumen El objetivo de este trabajo es caracterizar los aspectos tomográficos relevantes en el síndrome de heterotaxia, mediante cuatro pacientes que ejemplifican los hallazgos más frecuentes en esta patología. Situs solitus es la disposición habitual de los órganos y vasos sanguíneos y situs inversus se refiere a la imagen en espejo del situs solitus. Cuando la disposición de los órganos es indeterminada e impredecible y no se corresponde con el situs solitus ni el situs inversus, estamos frente al situs ambiguus o síndrome de heterotaxia, espectro de anomalías poco frecuente en las relaciones de los órganos toracoabdominales. Puede acompañarse de isomerismo derecho o isomerismo izquierdo. Clasificarlo en dos subgrupos es habitualmente difícil, ya que ninguno de estos tiene hallazgos únicos y patognomónicos, sino que existe amplia superposición. Ambos son de mal pronóstico, en los casos de isomerismo izquierdo un 5-10% llegan a la edad adulta, siendo de peor pronóstico los casos de isomerismo derecho, debido a que presentan inmunodepresión secundaria a la asplenia y cardiopatías congénitas más severas. Se debe analizar cada caso de forma individualizada y detallada para establecer el diagnóstico, determinar la asociación lesional y establecer aquellos pacientes que presenten mayor riesgo de complicaciones.

Abstract The objective of this brief communication is to characterize the relevant tomographic aspects in the heterotaxy syndrome, by means of 4 patients that exemplify the most frequent findings in this pathology. Situs solitus is the usual arrangement of organs and blood vessels and situs inversus refers to the mirror image of situs solitus. When the arrangement of the organs is indeterminate and unpredictable and does not correspond to situs solitus or the situs inversus, we are facing the situs ambiguus or heterotaxy syndrome, abnormal spectrum of anomalies in the relations of the thoracoabdominal organs. It may be accompanied by right isomerism or left isomerism. Attempts to classify it into two subgroups are usually difficult since none of these has unique and pathognomonic findings, but rather there is broad overlap. Both are of poor prognosis, in the cases of left isomerism 5-10% reach adulthood, with a worse prognosis being the cases of right isomerism due to the fact that they have immunodepression secondary to asplenia and more severe congenital cardiopathies. Each case should be analyzed in an individualized and detailed manner to establish the diagnosis, determine the lesional association and establish those patients that present a higher risk of complications.

Follicular fluid from infertile women with mild endometriosis impairs in vitro bovine embryo development: potential role of oxidative stress / Fluido folicular de mulheres inférteis com endometriose leve prejudica o desenvolvimento in vitro de embriões bovinos: Potencial papel do estresse oxidativo

Abstract Objective To investigate whether follicular fluid (FF) from infertile women with mild endometriosis (ME) alters in vitro bovine embryo development, and whether the antioxidants N-acetyl-cysteine (NAC) and/or L-carnitine (LC) could prevent such damages. Methods Follicular fluid was obtained from infertile women (11 with ME and 11 control). Bovine oocytes were matured in vitro divided in: No-FF, with 1% of FF from control women (CFF) or ME women (MEFF); with 1.5mM NAC (CFF + NAC, MEFF + NAC), with 0.6mg/mL LC (CFF + LC, MEFF + LC), or both antioxidants (CFF + NAC + LC, MEFF + NAC + LC). After in vitro fertilization, in vitro embryo culture was performed for 9 days. Results A total of 883 presumptive zygotes were cultured in vitro. No differences were observed in cleavage rate (p = 0.5376) and blastocyst formation rate (p = 0.4249). However, the MEFF group (12.5%) had lower hatching rate than the No-FF (42.1%, p = 0.029) and CFF (42.9%, p = 0.036) groups. Addition of antioxidants in the group with CFF did not alter hatching rate (p ≥ 0.56), and in groups with MEFF, just NAC increased the hatching rate [(MEFF: 12.5% versus MEFF + NAC: 44.4% (p = 0.02); vs MEFF + LC: 18.8% (p = 0.79); versus MEFF + NAC + LC: 30.8% (p = 0.22)]. Conclusion Therefore, FF from infertile women with ME added to medium of in vitro maturation of bovine oocytes impairs hatching rate, and NAC prevented these damages, suggesting involvement of oxidative stress in worst of oocyte and embryo quality of women with ME.

Resumo Objetivo Investigar se o fluido folicular (FF) de mulheres inférteis com endometriose leve (ME, na sigla eminglês) altera o desenvolvimento in vitro de embriões bovinos, e se os antioxidantes N-acetil-cisteína (NAC) e/ou L-carnitina (LC) poderiam prevenir possíveis danos. Métodos O FF foi obtido de mulheres inférteis (11 com ME e 11 controles). Oócitos bovinos foram maturados in vitro divididos em: sem FF (No-FF), com 1% de FF de mulheres controle (CFF) ou mulheres comME (MEFF); com 1,5mMde NAC (CFF + NAC, MEFF + NAC), com 0,6mg/mL de LC (CFF + LC, MEFF + LC), ou ambos antioxidantes (CFF + NAC + LC, MEFF + NAC + LC). Depois da fertilização in vitro, o cultivo in vitro de embriões foi realizado por 9 dias. Resultados Um total de 883 zigotos presumidos foram cultivados in vitro. Nenhuma diferença foi observada na taxa de clivagem (p = 0,5376) e na taxa de formação de blastocistos (p = 0,4249). Entretanto, o grupo MEFF (12.5%) teve menor taxa de eclosão de blastocistos do que os grupos No-FF (42,1%, p = 0,029) e CFF (42,9%, p = 0,036). Adição de antioxidantes no grupo comCFF não alterou a taxa de eclosão (p ≥ 0.56), e nos grupos com MEFF, somente a NAC aumentou a taxa de eclosão [(MEFF: 12.5% versus MEFF + NAC: 44.4% (p = 0.02); versus MEFF + LC: 18.8% (p = 0.79); versus MEFF + NAC + LC: 30.8% (p = 0.22)]. Conclusão Portanto, o FF de mulheres inférteis com ME adicionado ao meio de maturação in vitro de oócitos bovinos prejudica a taxa de closão embrionária, e a NAC preveniu esses danos, sugerindo o envolvimento do estresse oxidativo na piora da qualidade oocitária e embrionária de mulheres com ME.

Protection of spawning habitat for potamodromous fish, an urgent need for the hydropower planning in the Andes

Because dams block migratory routes of potamodromous fish to their spawning areas, and energy generation changes natural flow seasonality, it is necessary to identify spawning areas and their conditions. This information will help in management decisions in the Magdalena River basin regarding the future hydropower development. We identified which characteristics of the tributaries to the Magdalena River are important for determining potamodromous fish spawning grounds, and we estimated the percentage of future loss of spawning areas because of dam development. Ichthyoplankton density is directly related to the floodplain area, and inversely related with channel slope. Low channel slopes offer adult fish a longer distance for their upstream migration and a longer time for embryo development during their drift downstream from the spawning areas to nursery habitats (floodplain lakes). These features could increase the migration distance of the adults, the time for initial embryo development, and, because of its relationship with nursery habitats access, the offspring survival. The potential loss of the actual spawning grounds in the river network was estimated to be nearly 70% because of new dams. Our findings will help to reduce conflicts between hydropower and ecological interests.(AU)

La construcción de hidroeléctricas puede afectar la reproducción de los peces migratorios potamódromos, ya sea porque las represas bloquean las rutas migratorias a sus áreas de desove, o porque la generación de energía cambia la estacionalidad del flujo natural. Esto hace necesario generar información sobre las áreas de desove y sus características, que permitan tomar decisiones de manejo, teniendo en cuenta el desarrollo hidroeléctrico propuesto a futuro en la cuenca del río Magdalena. Identificamos qué características de algunos afluentes del río Magdalena son importantes para los desoves y estimamos el porcentaje de pérdida futura de áreas de desoves debido al desarrollo hidroeléctrico. La densidad del ictioplancton se relacionó directamente con el área de la llanura aluvial e inversamente con la pendiente del canal. Estas características aumentan la distancia de migración de los adultos maduros, el tiempo para el desarrollo inicial del embrión y la supervivencia de la descendencia debido a la proximidad y/o conectividad con los hábitats de cría. La pérdida potencial de las zonas de desove en la red fluvial se estimó en casi el 70% debido a las nuevas presas. Nuestros hallazgos ayudarán a tomar decisiones sostenibles para reducir los conflictos entre intereses de desarrollo hidroeléctrico y ecológicos.(AU)

Surgical correction of bilateral ectopic ureter using the neoureterocystostomy technique / Correção cirúrgica de ureter ectópico bilateral utilizando a técnica de neoureterocistostomia

Ureteral ectopy is a rare disorder in the small animals' clinic. It is characterized as a congenital anomaly, resulting from the ducts differentiation failure during embryogenesis. In this scenario, the ureters present themselves outside the anatomical site, being inserted into the uterus, urethra, urinary vesicle neck, or vagina. The clinical signs are urinary incontinence and perivulvar dermatitis. Surgery is the accepted treatment to correct the anomaly. The surgical procedure is based on relocating the ectopic ureter and treating associated modifications. This report describes a case of intramural bilateral ureteral ectopy, corrected surgically through the neoureterocystostomy technique, making it possible to control the animal's urinary incontinence.(AU)

A ectopia ureteral é uma afecção de incidência rara na clínica de pequenos animais, sendo caracterizada como anomalia congênita resultante de falha na diferenciação dos ductos durante a embriogênese. Neste cenário, os ureteres se apresentam fora do seu local anatômico, sendo inseridos no útero, no colo da vesícula urinária, na uretra ou na vagina. Os sinais clínicos comumente apresentados são a incontinência urinária bem como a dermatite perivulvar. O tratamento de eleição para correção da anomalia é o procedimento cirúrgico, no qual a técnica de escolha é baseada na relocação do ureter ectópico e tratamento das alterações associadas. Neste contexto, o presente relato descreve um caso de ectopia ureteral bilateral intramural, corrigido cirurgicamente por meio da técnica neoureterocistostomia, o que possibilitou controle da incontinência urinária do paciente.(AU)

BPA toxicity during development of zebrafish embryo / Toxicidade do BPA durante o desenvolvimento de embriões de Zebrafish

Bisphenol A (BPA) is a monomer used in the production of polycarbonate, a polymer commonly found in plastics, epoxy resins and thermal papers. The presence of BPA in food, water, air and dust has been of great concern in recent years not only due to environmental and ecological issues but also because of its supposed risk to public health related to its mutagenic and carcinogenic potential. In this study we evaluated the toxicity of bisphenol A in zebrafish embryos (Danio rerio) and determined the 50% lethal concentration (LC50) of this chemical. BPA was used at concentrations ranging from 1 µM to 100 µM in E3 medium/0.5% dimethylsulfoxide (DMSO) from previously prepared stock solutions in 100% DMSO. Controls included embryos exposed only to E3 medium or supplemented with 0.5% DMSO. Camptothecin (CPT), a known inhibitor of cell proliferation was used as positive control at a concentration of 0.001 µM in E3 medium/0.5% DMSO. Adults zebrafish were placed for breeding a day before the experimental set up, then, viable embryos were collected and selected for use. Experiments were carried out in triplicates, according to specifications from Organization for Economic Cooperation and Development (OECD). One embryo/well (25 embryos per concentration) was distributed in 96 well microplates in presence or absence of the chemicals. The plates were kept in BOD incubators with a controlled temperature of 28.5 ºC and with photoperiod of 14 h light:10 h dark. After 24h, 48h, 72h and 96h exposure, the exposed embryos were evaluated according to the following parameters: mortality, coagulation, rate of heartbeat, hatching and presence of morphological abnormalities. Photography was obtained by photomicroscopy. Apoptosis was evaluated by DNA ladder assay. DNA was extracted by phenol:chloroform method and analyzed by 2% agarose gel electrophoresis. DNA fragments were visualized after ethidium bromide staining in ultraviolet transilluminator. The LC50 determined for BPA was 70 µM after 24 hours, 72 µM after 48 hours, 47 µM after 72 hours and 31 µM after 96 hours exposure. BPA induced morphological and physiological alterations such as yolk sac and pericardial edema, hatching delay or inhibition, spine deformation, decreasing in heartbeat rate and mortality. In conclusion, this study demonstrated that BPA induced marked malformations in zebrafish embryos at concentrations above 25 µM corroborating the current concerns related to the widespread presence of BPA in the air, food and water used by humans as well as in the bodily fluids and tissues.

Bisfenol A (BPA) é um monômero utilizado na produção de policarbonato, um polímero comumente encontrado em plásticos, resinas epóxi e papéis térmicos. A presença de BPA em alimentos, água, ar e poeira tem sido motivo de grande preocupação nos últimos anos, não só devido a questões ambientais e ecológicas, mas também ao suposto risco para a saúde pública relacionado ao seu potencial mutagênico e carcinogênico. Neste estudo avaliamos a toxicidade do bisfenol A em embriões de peixe-zebra (Danio rerio) e determinamos a concentração letal 50% (LC50) deste composto químico. O BPA foi usado na faixa de concentração entre 1 µM e 100µM em meio E3/0,5% de dimetilsulfóxido (DMSO), preparado a partir de soluções estoques em 100% DMSO. Os controles negativos incluíram embriões expostos apenas ao meio E3 ou suplementado com 0,5% DMSO. Camptotecina (CPT), um conhecido inibidor da proliferação celular, foi usado como controle positivo a uma concentração de 0,001 µM em meio E3/0,5% DMSO. Peixes-zebra adultos foram colocados para reprodução um dia antes da montagem experimental, em seguida, embriões viáveis foram coletados e selecionados para uso. Os experimentos foram realizados em triplicata, de acordo com as especificações da Organização para Cooperação e Desenvolvimento Econômico (OCDE). Um embrião/ poço (25 embriões por concentração) foi distribuído em microplacas de 96 poços na presença ou ausência dos compostos químicos. As placas foram mantidas em incubadoras BOD com temperatura controlada de 28,5 ºC e com fotoperíodo de 14h claro:10h escuro. Após 24h, 48h, 72h e 96h, os embriões expostos foram avaliados de acordo com os seguintes parâmetros: mortalidade, presença de coagulação, taxa do batimento cardíaco, eclosão e presença de anormalidades morfológicas. Fotografias foram obtidas por fotomicroscopia. A apoptose foi avaliada pelo ensaio de DNA ladder. O DNA foi extraído pelo método fenol:clorofórmio e analisado por eletroforese em gel de agarose a 2%. Fragmentos de DNA foram visualizadas após coloração com brometo de etídio em um transiluminador ultravioleta. A LC50 determinada para o BPA foi 70 µM após 24 horas, 72 µM após 48 horas, 47 µM após 72 horas e 31 µM após exposição por 96 horas. O BPA induziu alterações morfológicas e fisiológicas como edema de saco vitelino e edema pericárdico, atraso no tempo ou inibição da eclosão, deformação da coluna vertebral, diminuição da taxa de batimentos cardíacos e mortalidade. Em conclusão, este estudo demonstrou que o BPA induziu grande número de malformações em embriões de peixe-zebra em concentrações acima de 25 µM, corroborando as preocupações atuais relacionadas a presença generalizada do BPA no ar, alimento e água usados pelos seres humanos bem como nos fluidos e tecidos corporais.

Insights into epigenetic patterns in mammalian early embryos

Mammalian fertilization begins with the fusion of two specialized gametes, followed by major epigenetic remodeling leading to the formation of a totipotent embryo. During the development of the pre-implantation embryo, precise reprogramming progress is a prerequisite for avoiding developmental defects or embryonic lethality, but the underlying molecular mechanisms remain elusive. For the past few years, unprecedented breakthroughs have been made in mapping the regulatory network of dynamic epigenomes during mammalian early embryo development, taking advantage of multiple advances and innovations in low-input genome-wide chromatin analysis technologies. The aim of this review is to highlight the most recent progress in understanding the mechanisms of epigenetic remodeling during early embryogenesis in mammals, including DNA methylation, histone modifications, chromatin accessibility and 3D chromatin organization.

The role of histone deacetylases 1/2 in regulating murine oogenesis / 生理学报

Oogenesis is the basic reproductive process of female mammals and is essential for fertilization and embryo development. Recent studies have shown that epigenetic modifications play an important role in the regulation of mammalian reproductive processes (such as oogenesis, spermatogenesis, preimplantation embryo development and sex differentiation). Taking histone acetylation as an instance, the dynamic changes of histone acetyltransferases (HATs) and deacetylases (HDACs) are involved in the regulation of gene activation and inactivation when numerous key physiological events occur during reproduction. Thereinto, HDAC1 and HDAC2, which are highly homologous in terms of both structure and function, play a pivotal role in murine oogenesis. HDAC1 and 2 jointly regulate the global transcription and the incidence of apoptosis of growing oocytes and affect its subsequent growth and development, which reflects their compensatory function. In addition, HDAC1 and 2 also play a specific part in oogenesis respectively. It has shown that HDAC2 is more critical than HDAC1 for oocyte development, which regulates de novo DNA methylation and chromosome segregation. Reciprocally, HDAC1 is more critical than HDAC2 for preimplantation development. Deficiency of HDAC1 causes the decreased proliferation of embryonic stem cells and the smaller embryoid bodies with irregular shape. In this review, we summarized the role and the current research progress of HDAC1/2 in murine oogenesis, to provide a reference for further understanding the relationship between epigenetic modifications and reproductive regulation.

Design and experiment of online monitoring system for long-term culture of embryo / 生物医学工程学杂志

In the study of embryo development process, the morphological features at different stages are essential to evaluate developmental competence of the embryo, which can be used to optimize and improve the system for

The effect of melatonin on in vitro maturation fertilization and early embryo development of mouse oocytes and expression of HMGB1 gene in blastocysts

Antioxidants are commonly used for maturation, fertilization and early development of embryos. Melatonin as an antioxidant have been recently proven to be useful for the assisted reproductive technology. In the present study, we evaluated the roles of melatonin in the in vitro maturation, fertilization, development and also the gene expression of high mobility group box-1 (HMGB1) in the blastocysts. The immature oocytes of BDF1 mice were transferred to the media containing different doses of melatonin (10-6, 10-9, 10-12 M). The blastocysts that developed under in vitro fertilization from each group were stained to determine the cell number of embryos and analyzed to determine the expression level of HMGB1 by real-time PCR. The most effective doses of melatonin for maturation of oocytes were 10-6 and 10-12M (P<0.05). Fertilization rate, early development and the cell number of blastocysts were significantly higher in the group that treated with 10-12 M of melatonin comparing to the other groups. The HMGB1 expression decreased in groups that treated with 10-6M and 10-9M of melatonin and increased in the group that treated with 10-12 M of melatonin, but did not show a significant difference (p˃0.05). From the results, it may be concluded that the melatonin could be effective when the embryos undergo maturation, fertilization and early developmental processes. The HMGB1 expression, as a marker of early development in mice embryos, increased in the groups that treated with low doses of melatonin