Ultraestructura del ovocito maduro y en reabsorción de Spondylus limbatus G. B. Sowerby II, 1847 (= S. calcifer ) (Bivalvia: Spondylidae) / Ultrastructure of the mature and in resorption oocytes of Spondylus limbatus G. B. Sowerby II, 1847 (= S. calcifer ) (Bivalvia: Spondylidae)

Spondylus limbatus es una especie bajo protección especial en México, de la que existe poca información biológica y nada sobre estudios histológicos o de ultraestructura del ovario. El objetivo de esta investigación fue caracterizar la morfología ultraestructural de los gametos femeninos maduros y en degeneración. La gónada femenina de S. limbatus en estado de madurez presentó ovocitos postvitelogénicos de 60-70 µm de diámetro, que presentan el aspecto característico de células metabólicamente activas y altamente sintetizadoras. La membrana citoplasmática posee especializaciones destinadas a aumentar la superficie de absorción de la célula, las microvellosidades; el citoplasma presenta numerosos sistemas membranosos relacionados con la síntesis de material de reserva y secreción; y el patrón de organización nuclear altamente lobulado, y por consiguiente con una gran superficie que asegura el intercambio núcleo-citoplasma, se incorpora de forma estructural al proceso de vitelogénesis. Finalmente, se describen los cambios ultraestructurales resultantes de la lisis de los ovocitos: colapso de las membranas nuclear y citoplásmica, y presencia de células hemocíticas macrófagas.

Spondylus limbatus is a species under special protection in Mexico, of which there is little or no information in the literature of biological, histological or ultrastructural studies of the ovary. The objective of this research was to characterize the ultrastructural morphology of mature and degenerating female gametes. The female gonad of S. limbatus in mature state presented post-vitellogenic oocytes 60-70 µm in diameter, which have characteristics of metabolically active and highly synthesizing cells. The cytoplasmic membrane has specializations designed to increase the absorption surface of the cell, the microvilli; the cytoplasm presents numerous membranous systems related to synthesis of reserve and secretion material as well as the highly lobed nuclear organization pattern; a large surface that ensures core-cytoplasm exchange, is structurally incorporated into the vitellogenesis process. Finally, ultrastructural changes resulting from the lysis of the oocytes are described: collapse of nuclear and cytoplasmic membranes, and the presence of macrophage hemocytic cells.

Assessment of mouse oocytes ultrastructure following vitrification before and after in vitro maturation / Evaluación de la ultraestructura de los ovocitos de ratón después de la vitrificación antes y después de la maduración in vitro

SUMMARY: Vitrification is a physical process in which the concentrated cryoprotectant solution after exposure to extreme cold without ice crystal formation in living cells to be converted glassing state. In this study, maturation rate and ultrastructure of mouse oocytes followed by vitrification before or after in-virto maturation (IVM) were evaluated. A total of 373 germinal vesicle oocytes were obtained from ovaries and divided into three fresh IVM, IVM vitrified, vitrified IVM groups. Ten metaphase II oocytes were obtained from uterine tubes and considered as the control group. Oocytes in vitrified groups were vitrified by Cryotop using vitrification medium and kept in liquid nitrogen. The maturation media was a-MEM supplemented with rFSH + hCG. After 24-48 h of incubation, the oocytes were investigated for nuclear maturation and ultrastructural changes using transmission electron microscopy (TEM). The oocyte maturation rate in vIVM group was significantly lower than IVMv group, when the two groups were compared with vIVM had the highest maturity. The evaluation ultrastructure of the four groups showed that the number of cortical granules, microvilli and mitochondria-SER aggregates in vIVM group were lowest and the highest amongst the number of vacuoles. Zona pellucida was darker than the control group in two freeze groups vIVM and IVMv. Most similar groups to the control group were group vIVM, Group IVMv and ultimately vIVM group, respectively. According to the results, IVM procedure is more efficient when it is performed before oocyte vitrification.

RESUMEN: La vitrificación es un proceso físico en el que la solución concentrada de crioprotectores, después de la exposición al frío extremo sin formación de cristales de hielo en las células vivas, se convierte en estado de cristal. En este estudio, se evaluaron la velocidad de maduración y la ultraestructura de los ovocitos de ratón seguidos por la vitrificación antes o después de la maduración in vitro (IVM). Se obtuvieron un total de 373 ovocitos, de vesículas germinales de ovarios, y se dividieron en tres grupos de IVM vitrificados, IVM e IVM frescos. Diez ovocitos metafase II se obtuvieron a partir de tubas uterinas y se consideraron como el grupo de control. Los ovocitos en grupos vitrificados fueron vitrificados por Cryotop usando medio de vitrificación y mantenidos en nitrógeno líquido. El medio de maduración fue a-MEM suplementado con rFSH + hCG. Después de 24-48 h de incubación, fueron observados en los ovocitos la maduración nuclear y cambios ultraestructurales utilizando microscopía electrónica de transmisión (MET). La tasa de maduración de los ovocitos en el grupo vIVM fue significativamente más baja que en el grupo IVM v, cuando los dos grupos se compararon con los que tenían la mayor madurez. La evaluación de la ultraestructura de los cuatro grupos mostró que el número de gránulos corticales, microvellosidades y acúmulos de mitocondrias-SER en el grupo vIVM fue el más bajo y el más alto entre el número de vacuolas. La zona pelúcida fue más oscura en dos grupos de congelación vIVM e IVMv, que en el grupo control. La mayoría de los grupos, similares al grupo de control, fueron los grupos vIVM, IVMv y,finalmente, el grupo vIVM, respectivamente. De acuerdo con los resultados, el procedimiento de IVM es más eficiente cuando se realiza antes de la vitrificación de ovocitos.

Efecto de la vitrificación de ovocitos humanos sobre la capacidad de unión y el estado acrosomal de espermatozoides humanos / Effect of the vitrification of human oocytes on the binding capacity and acrosome status of human sperm

Conocer los aspectos moleculares que acontecen en el proceso de unión de los espermatozoides humanos a la zona pelúcida (ZP) humana es uno de los grandes retos de la biología de la Reproducción. Por otra parte conocer si el proceso de fecundación puede verse afectado por la criopreservación de los gametos femeninos sigue siendo otra cuestión debatida en la literatura. En base a esto, el objetivo principal de este trabajo fue conocer si la vitrificación ovocitaria puede alterar la interacción de los espermatozoides con el glicocáliz de la ZP y demostrar si la ZP de estos ovocitos pierde la capacidad de inducir la reacción acrosómica en los espermatozoides. Según nuestros resultados el método de vitrificación ovocitaria cerrado (S3) no altera la capacidad de unión de los espermatozoides a la zona pelúcida, ni la capacidad de ésta para inducir la reacción acrosómica.

To know the molecular aspects that occur in the process of human sperm binding to the human zona pellucida (ZP) is one of the great challenges of reproduction biology. Moreover knowing if the fertilization process may be affected by cryopreservation of female gametes is still another issue discussed in the literature. Based on this, the main objective of this study was to determine whether the oocyte vitrification may alter the interaction of sperm with the glycocalyx of ZP and show whether these oocytes lost the ability to induce the acrosome reaction in sperm. According to our results the oocyte closed vitrification method (S3) does not alter the ability of the sperm binding to the zona pellucida, and their ability to induce the acrosome reaction.

Histological description of oogenesis in chiton virgulatus (Mollusca: Polyplacophora) / Descripción histológica de la ovogénesis de chiton virgulatus (Mollusca: Polyplacophora)

This paper describes the oogenesis of Chiton virgulatus, based on histological observations under transmission and scanning electron microscopy. Three oocyte types were identified: i) previtellogenic oocytes with a mean diameter of 50±20.5 µm, surrounded by elongated follicular cells of approximately 5 µm, ii) immature vitellogenic oocytes with a mean diameter of 113±15.3 µm and small cytoplasmic projections denoting the onset of the oocyte hull development; adjacent to each projection are pores approximately 0.7 µm in diameter, and iii) mature vitellogenic oocytes with a mean diameter of 146±24.8 µm; the oocyte cytoplasmic projections grow and its apical zone becomes trident-shaped; follicular cells adopt a bulbous shape due to the growth of the elongation and can reach up to 20 µm in length. The morphology and ultrastructure of the projections of the mature vitellogenic oocyte, as well as the size of pores at their base, are specific to C. virgulatus; therefore, these features could be used in taxonomic or fertilization studies.

En el presente trabajo se describe la ovogénesis de Chiton virgulatus, utilizando histología y las técnicas de microscopía electrónica de barrido y de transmisión. Se identificaron tres tipos de ovocitos: i) ovocitos previtelogénicos con un diámetro promedio de 50±20,5 µm, rodeados por células foliculares de forma alargada y un tamaño de aproximadamente 5 µm, ii) ovocitos vitelogénicos inmaduros con un diámetro promedio de 113±15,3 µm, este tipo de ovocitos presentan pequeñas proyecciones citoplasmáticas, que indican el inicio del desarrollo del casco del ovocito. Adyacentes a cada prolongación se presentan poros con un diámetro aproximado de 0,7 µm y iii) ovocitos vitelogénicos maduros con un diámetro promedio de 146±24,8 µm, las proyecciones citoplasmáticas del casco del ovocito crecen y en su parte apical adquieren la forma de un tridente, las células foliculares, dado el crecimiento de la prolongación toman el aspecto bulboso y llegan a medir hasta 20 µm de longitud. La morfología y la ultraestructura de las proyecciones del casco del ovocito vitelogénico maduro, así como el tamaño del poro en la base de las proyecciones son particulares para C. virgulatus, dichas características podrían ser utilizadas en trabajos de taxonomía y fertilización.

Effect of leptin on in vitro nuclear maturation and apoptosis of buffalo [bubalus bubalis] oocyte

Leptin, as a 16 kDa adipokine, is a pleiotropic cytokine-like hormone that primarily secreted from adipose tissue. It also involves in the regulation of energy homeostasis, neuroendocrine function, immunity, lipid and glucose homeostasis, fatty acid oxidation, angiogenesis, puberty and reproduction. The aim of this study was to investigate the effects of in vitro addition of leptin to in vitro maturation [IVM] medium on buffalo oocyte maturation and apoptosis. In this experimental study, Ovaries from apparently normal reproductive organs of slaughtered adult buffaloes [Bubalus bubalis] with unknown breeding history were collected from Urmia Abattoir, Urmia, Iran, and were transported immediately to the laboratory in a thermos flask containing sterile normal saline with added antibiotics. Oocytes were aspirated from 2-8 mm visible follicles of the ovaries using an 18-G needle attached to a 10 ml syringe. IVM medium included tissue culture medium-199 [TCM-199], 10% fetal bovine serum [FBS], 22 microg/ml sodium pyruvate, 0.5 IU/ml ovine follicle-stimulating hormone [oFSH], 0.5 IU/ml ovine luteinizing hormone [oLH], 1 microg/ml oestradiol, 50 microg/ml gentamycin, and leptin [0 [control], 10, 50, and 100 ng/ml]. The good quality buffalo oocytes [batches of 10 oocytes] were placed in a culture plate containing six 50 microl droplets of maturation medium, covered with sterilized mineral oil, and then incubated at 38.5?C with 5% CO2 in air for 24 hours. The maturation of oocytes was evaluated under a stereomicroscope by detecting the first polar body extrusion of oocytes. FITC-Annexin V - propidium iodide [PI] staining method was used to detect oocyte apoptosis. From a total of 115 collected ovaries, 1100 oocytes were recovered among which 283 oocyte were suitable for IVM. In the groups of leptin treated with 0 [control], 10, 50 and 100 ng/ml, the percentage of oocytes maturation was 74.65, 83.81, 77.85, and 75.40%, while the percentage of oocytes apoptosis was 9.83, 9.54, 9.93, and 10.42%, respectively. Our results showed that addition of 10 ng/ml leptin to buffalo IVM medium increased oocyte maturation, significantly, as compared with that in control group. However, addition of leptin to IVM medium had no significant influence on buffalo oocyte apoptosis. Our findings suggested that addition of 10 ng/ml leptin to IVM medium of buffalo oocyte can improve oocyte nuclear maturation. Furthermore, we showed that there is no relation between in vitro addition of leptin to buffalo oocyte IVM medium and oocyte apoptosis

Análise invasiva e não invasiva do fuso meiótico de oócitos humanos obtidos de ciclos estimulados: dados preliminares / Invasive and noninvasive analysis of the meiotic spindle of human oocytes obtained from stimulate cycles: preliminary results

OBJETIVOS: Avaliar a concordância entre as técnicas de microscopia de polarização e microscopia confocal na avaliação do fuso meiótico de oócitos humanos maturados in vivo. MÉTODOS: Estudo prospectivo que avaliou oócitos com o primeiro corpúsculo polar extruído obtidos de mulheres inférteis submetidas à estimulação ovariana para realização de injeção intracitoplasmática de espermatozoide. Os oócitos com o primeiro corpúsculo polar extruído foram avaliados por meio da microscopia de polarização e, imediatamente após, foram fixados e corados para avaliação dos microtúbulos e cromatina pela microscopia confocal de alto desempenho. Foram comparadas as técnicas de microscopia de polarização e confocal, de acordo com a visualização ou não do fuso meiótico pela microscopia de polarização e a presença ou não de anomalias meióticas à análise pela microscopia confocal. Foram calculados os intervalos de confiança, o índice de Kappa e a concordância entre as metodologias, considerando a análise da microscopia de imunofluorescência como padrão-ouro para avaliação de normalidade do fuso e distribuição cromossômica oocitária. RESULTADOS: Observou-se que 72,7% dos oócitos em metáfase II com fuso celular não visível à polarização apresentaram anormalidades meióticas à análise confocal e que 55,6% dos oócitos em metáfase II com fuso celular visível à polarização apresentaram-se como oócitos anormais à análise confocal. Somente 44,4% dos oócitos com fuso celular visível à polarização apresentaram-se como normais à análise confocal. A concordância entre os métodos foi de 51,1% (Kappa: 0,11; IC95% -0,0958 - 0,319). CONCLUSÕES: A baixa concordância entre a microscopia de polarização e a confocal na avaliação do fuso meiótico oocitário sugere que a visualização do fuso meiótico de oócitos humanos em metáfase II pela microscopia de polarização tem limitado o valor preditivo de normalidade meiótica oocitária.

PURPOSE: To evaluate the concordance between polarization microscopy and confocal microscopy techniques in the evaluation of the meiotic spindle of human oocytes matured in vivo. METHODS: Prospective study that evaluated oocytes with the first polar extruded body obtained from infertile women who had undergone ovarian stimulation for intracytoplasmic sperm injection. The oocytes with the first polar extruded body were evaluated by polarization microscopy and were then immediately fixed and stained for microtubule and chromatin evaluation by high-performance confocal microscopy. We determined the correlation of polarization microscopy with confocal microscopy in the detection of meiotic oocyte anomalies, and we also evaluated the percentage of oocytes with a visible and non-visible cell spindle by polarization microscopy and with meiotic normality and abnormalities by confocal microscopy. Confidence intervals, Kappa's index and concordance between the methodologies were calculated, considering immunofluorescence microscopy analysis as the golden-standard for evaluating normal spindle and oocyte chromosome distribution. RESULTS: We observed that 72.7% of metaphase II oocytes with a nonvisible meiotic spindle by polarization microscopy showed no meiotic abnormalities by confocal analysis and 55.6% of metaphase II oocytes with a visible meiotic spindle by polarization microscopy were found to be abnormal oocytes by the confocal analysis. Only 44.4% of oocytes with a visible meiotic spindle by polarization microscopy were found to be normal by confocal analysis. Concordance between the methods was 51.1% (Kappa: 0.11; 95%CI -0.0958 - 0.319). CONCLUSIONS: The low correlation between polarization microscopy and confocal microscopy in the assessment of oocyte meiotic spindle suggests that visualization of the meiotic spindle of human oocytes at metaphase II by polarization microscopy is not a good indicator of oocyte meiotic normality.

Molecular characterization, electrophysiological and contraceptive effect of chilean latrodectus venom / Caracterización molecular, electrofisiológica y efecto anticonceptivo del veneno de latrodectus chilena

Since the 1970s, There have been studies of the venom of Latrodectus sp. spiders, in particular the latrotoxin (LTX) of Latrodectus mactans. Many of the studies were aimed at understanding the action of the venom on the muscular system. Now accepted that LTX is able to generate a calcium-permeable membrane pore and modulate the release of synaptic vesicles that activate a receptor and induce cellular changes. Interestingly, when work began with venom obtained from the Latrodectus sp present in Chile, it generated clinical indications similar to the bite of this spider in another country, with some differences in intensity. The purpose of the first studies was to understand the systemic mechanisms of this venom, and other active compounds were studied for biological interest. It was found that these molecules are capable of causing systemic effects such as changes in muscle contraction; of generating vascular relaxation and synaptic and cellular modulation; and of altering potassium conductance channels. Based on this evidence, we suggested biotechnological applications to characterize low molecular-weight compounds obtained from the Chilean Latrodectus venom and exploring the effects on the electrophysiology in oocytes and neurons, and the contraceptive effect on spermatozoa.

Desde los años 70, se han realizado estudios con el veneno de arañas Latrodectus sp, en particular la latrotoxina (LTX) de Latrodectus mactans. Muchos de estos estudios estuvieron enfocados a entender la acción del veneno sobre el sistema muscular. Hoy en día es aceptado que la LTX es capaz de generar un poro de membrana permeable a calcio y modular la liberación de vesículas sinápticas que activan un receptor e inducen cambios celulares. Interesantemente, cuando comenzamos a trabajar con el veneno obtenido de Latrodectus sp. presente en Chile, ésto generó indicaciones clínicas similares a la picadura de esta araña en otros países, con algunas diferencias en su intensidad. El propósito de estos primeros estudios fue entender los mecanismos sistémicos de este veneno y además otros compuestos activos fueron estudiados para interés biológico. Se ha encontrado que estas moléculas son capaces de causar efectos sistémicos así como cambios en la contracción muscular; generar relajación vascular y modulación sináptica y celular; y de alterar los canales de conductancia de potasio. Basados en estas evidencias, nosotros sugerimos usar aplicaciones biotecnológicas para caracterizar los compuestos de bajo peso molecular obtenidos del veneno de Latrodectus Chilena y explorar los efectos sobre la electrofisiología en ovocitos y neuronas, y el efecto anticonceptivo sobre los espermatozoides.

Estructura y la ultraestructura del ovario de Cichlasoma urophthalmus (Perciformes: Cichlidae)

Structure and ultrastructure of the ovary of Cichlasoma urophthalmus (Osteichthyes: Cichlidae). The study of the normal development, differentiation, structure and function of various components of developing follicles in the ovaries of numerous fish species have been a consistent focus of comparative reproduction. The structural and ultrastructural features of gonads from Cichlasoma urophthalmus have received scarce attention. In this work, we realized a descriptive study of female gonads of Cichlasoma urophthalmus. A total of 40 samples were collected in the Veracruz Alvarado Lagoon, Mexico in 2007-2008 period including the windy, dry and rainy seasons. Female gonads were extracted and a portion was fixed in 4% formaldehyde for treatment for routine histology hematoxylin and eosin (HE) and another part was processed for transmission electron microscopy (TEM). The gonads were fixed in 3% glutaraldehyde and 2% osmium tetroxide, followed by dehydrated in ethanol 50%, 70%, 80%, 95% and 100% for inclusion in Epon, thin sections were then prepared and were contrasted with lead citrate and uranyl acetate. The process of oocyte development can be divided into five distinct stages (formation of oocytes from oogonia, primary growth, lipid stage, vitellogenesis and maturation). In this work, we found that the primary growth stage is characterized by intense RNA synthesis and the differentiation of the vitelline envelope. Secondary growth starts with the accumulation of lipid droplets in the oocyte cytoplasm (lipid stage), which is then followed by massive uptake and processing of proteins into yolk platelets (vitellogenic stage). During the maturation stage, the lipid inclusions coalesce into a single oil droplet, and hydrolysis of the yolk platelets leads to the formation of a homogeneous mass of fluid yolk in mature eggs. In conclusion, further studies should elucidate structure and ultrastructural changes in the ovarian follicular components, in C. urophthalmus during different stages of oocyte growth. Rev. Biol. Trop. 59 (2): 743-750. Epub 2011 June 01.

Se realizó un estudio descriptivo de las gónadas femeninas de Cichlasoma urophthalmus. Las muestras fueron recolectadas en la Laguna de Alvarado Veracruz, México en el período 2007-2008 que incluyó las temporadas de Nortes, Secas y Lluvias. Se extrajeron las gónadas femeninas y una parte se fijó en formol al 4% para su tratamiento por técnica histológica de rutina hematoxilina y Eosina (H-E) y otra parte se procesó para microscopia electrónica de transmisión. Las gónadas se fijaron en glutaraldehído al 3% y OsO4 al 2%, se deshidrataron en etanol de 50 al 100% para ser incluidas en Epón. Se realizaron cortes finos y semifinos contrastados con citrato de plomo y acetato de uranilo. Los ovarios de C. urophthalmus son pareados presentan un desarrollo asincrónico con ovocitos previtelogénicos en estadio perinuclear tardío, asociados a las lamelas ovígeras y ovocitos vitelogénicos del VII estadio, éstos últimos presentan una zona radiada bien definida, con gránulos de vítelo lipídico y vesículas de vítelo proteico que se distribuyen en capas concéntricas, que rodean al núcleo. El presente estudio, permitió conocer más a fondo los cambios de la estructura y ultraestructura de los componentes de los folículos ováricos, en C. urophthalmus durante las diferentes etapas de crecimiento de los oocitos.

[ ultrastructural study of oocyte and zona pellucida in ovarian follicles of untreated and and metformin-treated diabetic rats subsequent to induction of experimental diabetes]

Diabetes is a metabolic disorder affecting the whole body systems including the female reproductive organs. Moreover, diabetes is an important cause of infertility. Metformin is commonly used to control hyperglycemia in patients with diabetes. This study was done to evaluate the ultrastructural changes of ovarian follicles in diabetic rats and their response to metformin. Thirty-six adult Sprague-Dawley female rats [170-210 g] were studied in three groups [Control, diabetic and metformin-treated rats]. In the second and third groups, diabetes was induced by injection of streptozotocin [45 mg/kg]. The rats in the third group were later treated by metformin monohydrochloride [100 mg/kg]. At the end of the experiment, rats were sacrificed and their right ovaries were observed under transmission electron microscope. Quantitative data were analyzed by student t-test in SAS software. In comparison with the control group, significant decreases in zona pellucida thickness and the mean number of microvilli were observed [respectively, P<0.01 and P<0.001] in diabetic rats. Significant decreases in zona pellucida thickness were also observed in metformin-treated rats [P<0.05] but changes in the number of microvilli were non-significant. The number of organelles in oocyte cytoplasm was higher and they were natural or natural-looking in metformin-treated rats versus the diabetic ones. Reduction in the number of mitochondria and their ballooning cristae were of the most noticeable changes in diabetic rats. Diabetes decreases the number of microvilli and oocyte organelles and diminishes zona pellucida thickness leading to structural changes in the organelles but metformin could improve the aforesaid conditions

Oocyte structure and ultrastructure in the Mexican silverside fish Chirostoma humboldtianum (Atheriniformes: Atherinopsidae)

the structural and ultrastructural features of gonads from endemic Mexican fish have received scarce attention. This study describes the histological and ultrastructural characteristics of the oocyte in Chirostoma humboldtianum. The ovary is asynchronic, and as such, most phases of oocyte development are found in the same ovary. The complete process of oogenesis was divided in five stages: oogonium and folliculogenesis, primary growth, cortical alveoli and lipid inclusions, vitellogenesis and maturation. The presence of big filaments, which appear at the end of primary growth, induces some common follicular adaptation. During primary growth, abundant ribosomes, rough endoplasmic reticulum, and mitochondria are grouped in the cytoplasm. At the end of this stage, the Z1 layer of the chorion is developed, while microvilli start to be evident as well. In the cortical alveoli and lipid droplets phase, intense PAS positive vesicles, some of them containing nucleoid material, are observed in the peripheral cytoplasm and the lipid droplets take a more central position. In vitellogenesis, the proteic yolk accumulates in a centripetal way while the chorion is completely formed. In maturation, the germinal vesicle migrates to the animal pole, meiosis is restored, and there is nuclear breakdown. The oocyte increases its size and holds some oil droplets and a big fluid mass of yolk. On the outside, filaments surround the oocyte completely. Rev. Biol. Trop. 56 (4): 1825-1835. Epub 2008 December 12.

Los aspectos estructurales y ultraestructrurales de las gónadas de peces mexicanos endémicos han sido poco estudiados. En el presente trabajo reportamos las características histológicas y ultraestructurales del ovocito de Chirostoma hulmboldtianum. El ovario es de tipo asincrónico, por ende, la mayoría de las fases del desarrollo del ovocito pueden ser encontradas en el mismo ovario. El desarrollo del ovocito fue dividido en cinco etapas: ovo-gonia y foliculogénesis, crecimiento primario del ovocito, inclusiones lipídicas y gránulos corticales, vitelogénesis y maduración. La presencia de grandes filamentos que aparecen al final de la etapa de crecimiento primario, inducen adaptaciones foliculares. Durante el crecimiento primario, en el citoplasma se encuentran abundantes ribosomas, retículo endoplásmico rugoso y agrupamientos de mitocondrias. Al final de esta etapa, inicia el desarrollo de la capa Z1 del corion, comenzando a ser evidentes las microvellosidades del ovocito. Durante la etapa de inclusiones lipídicas y gránulos corticales, vesículas PAS positivas, algunas de ellas con material nucleoide, se ubican en la periferia del ovocito, mientras que las que contienen material graso toman una posición más central en la célula. Durante la vitelogénesis se presenta una acumulación de vitelo protéico en un sentido centrípeto; durante esta etapa, el corion está completamente formado. En la maduración, la vesicular germinal migra hacia el polo animal, se reinicia la meiosis y se rompe la envoltura nuclear. El ovocito incrementa su tamaño y en el citoplasma se pueden observar algunas gotas de grasa y el vitelo se presenta como una gran masa acuosa. En el exterior, los filamentos rodean completamente al ovocito.

Oocyte structure and ultrastructure in the Mexican silverside fish Chirostoma humboldtianum (Atheriniforme: Atherinopsidae)

The structural and ultrastructural features of gonads from endemic Mexican fish have received scarce attention. This study describes the histological and ultrastructural characteristics of oocyte from Chirostoma humboldtianum. The ovary is asynchronic, and as such, most phases of oocyte development are found in the same ovary. The complete process of oogenesis was divided in five stages: oogonium and folliculogenesis, primary growth, cortical alveoli and lipid inclusions, vitellogenesis, and maturation. The presence of big filaments, which appear at the end of primary growth, induces some common follicular adaptation. During primary growth, abundant ribosomes, the rough endoplasmic reticulum, and mitochondria are grouped in the cytoplasm. At the end of this stage, the Z1 layer of the chorion is developed, while microvilli start to be evident. In the cortical alveoli and lipid droplets phase, intense PAS positive vesicles, some of them containing nucleoid material, are observed in the peripheral cytoplasm and the lipid droplets take a more central position. In vitellogenesis, the proteic yolk accumulates in a centripetal way while the chorion is completely formed. During maturation, the germinal vesicle migrates to the animal pole, meiosis is restored, and there is nuclear breakdown. The oocyte increases its size and holds some oil droplets and a big fluid mass of yolk. On the outside, filaments completely surround the oocyte. Rev. Biol. Trop. 56 (3): 1371-1380. Epub 2008 September 30.

Morfologia do primeiro corpúsculo polar e taxas de fertilização, clivagem e qualidade embrionária / First polar body morphology and fertilization rate, cleavage rate, and embryo quality

OBJETIVO: determinar a relação entre a morfologia do primeiro corpúsculo polar (CP) de oócitos humanos e as taxas de fertilização e clivagem e a qualidade embrionária em procedimentos de Injeção Intracitoplasmática de Espermatozóide (ICSI). MÉTODOS: estudo retrospectivo de 582 ciclos consecutivos de ICSI no período de julho de 2003 a julho de 2005. A morfologia do primeiro CP foi avaliada com revisão de 3.177 oócitos em metáfase II, imediatamente antes da realização da ICSI, sempre pelo mesmo observador. O CP foi classificado nas seguintes categorias: CP intacto e de tamanho normal, CP fragmentado ou CP de tamanho aumentado. Avaliamos as taxas de fertilização e de clivagem, o número e a proporção de embriões de boa qualidade em cada um dos três grupos avaliados 48 horas após a ICSI (D2). Foram considerados de boa qualidade os embriões com quatro células, sem fragmentação e com blastômeros simétricos em D2. RESULTADOS: as taxas de fertilização, clivagem e de formação de embriões de boa qualidade resultantes da inseminação de oócitos com o CP aumentado (20,7, 18,7 e 5 por cento, respectivamente) foram significativamente menores que as de oócitos com o CP intacto e de tamanho normal (70,8, 62,5 e 19 por cento, respectivamente) ou CP fragmentado (69,7, 60,5 e 17,1 por cento, respectivamente). CONCLUSÕES: observamos que a presença do primeiro CP aumentado relaciona-se com piores taxas de fertilização, clivagem e de formação de embriões de má qualidade. Entretanto, a fragmentação no primeiro CP parece não interferir nos resultados da ICSI.

PURPOSE: to determine the relationship between the morphology of the first spindle pole of human oocytes and rates of fertilization, fragmentation and embryo quality in procedures of Intracytoplasmic Sperm Injection (ICSI). METHODS: retrospective study of 582 consecutive ICSI cycles, from July 2003 to July 2005. The morphology of the first spindle pole (SP) was assessed through the analysis of 3,177 oocytes in metaphase II, immediately before the ICSI procedure, always by the same observer. SP has been classified in the following categories: normal size intact, fragmented or augmented SP. Fertilization rate and fragmentation, and the number and rate of good quality embryos in each one of the three groups studied have been evaluated, 48 hours after ICSI (D2). Embryos with four cells, without fragmentation and with symmetric blastomeres in D2 were considered as of good quality. RESULTS: rates of fertilization, fragmentation and of good quality embryo formation, resulting from oocyte insemination, with augmented SP (20.7, 16.7 and 5 percent respectively) were significantly lower than the ones from intact and normal size SP (70.8, 62.5 and 19 percent, respectively) or from fragmented SP oocytes (69.7, 60.5 and 17.1 percent, respectively). CONCLUSIONS: it has been observed that the presence of augmented first spindle pole is related to worse rates of fertilization, fragmentation and bad quality embryo formation. Nevertheless, fragmentation in the first spindle pole of the oocyte does not seem to affect ICSI results.

Structural and ultrastructural aspects of folliculogenesis in Didelphis albiventris, the South-American opossum / Aspectos estructurales y ultraestructurales de la foliculogénesis de Didelphis albiventris, la zarigüeña sudamericana

The ovarian histology, the structural and the ultrastructural characteristics of the folliculogenesis in Didelphis albiventris were described in detail. Recent studies suggest that methatherian mammals have unusual reproductive cycle but there are few informations regarding the marsupials reproductive life. Despite of the opossum folliculogenesis pattern resembles methatherian and eutherian pattern in many aspects, the analysis shows some peculiar features of the oocyte structure and ultrastructure that make available new data on the reproductive biology of marsupials.

Fueron descritas con detalles la histología ovárica, las características estructurales y ultra-estructurales de la foliculogénesis del Didelphys albiventris. Estudios recientes sugieren que mamíferos metaterios tienen un ciclo reproductivo inusual, pero existen pocas informaciones en relación a la vida reproductiva de los marsupiales. A pesar de que el modelo de foliculogénesis de la zarigüeya se parece al modelo metaterio y euterio en muchos aspectos, el análisis muestra algunos rasgos peculiares de las estructura y ultra-estructura del oocito, que colocan a disposición nuevos datos en la biología reproductiva de los marsupiales.

Vitrification of mouse oocyte using open pulled straws compared with needles.

OBJECTIVE: To compare the survival rate of mouse oocytes and fertilization rate between using open pulled straws (OPS) and needles for vitrification. MATERIAL AND METHOD: Meiosis II oocytes from female C57B/6J mice aged 7-8 weeks were collected and allocated to two groups for vitrification by using OPS or needles. Vitrified oocytes were thawed, morphological survival and fertilization rate were examined. RESULTS: There was no obvious difference between the morphological survival rates of vitrified mouse oocytes using OPS and needles (66.7% vs 64.8%). Proportions Difference 1.9% (95% CI -7.1, 10.7). The vitrified oocytes from the needle had significantly higher percentages of fertilization rate than OPS (76.8% vs 62.5%). Proportions Difference -14.3% (95% CI -24.5, -3.6). CONCLUSION: Vitrification method of mouse oocytes using needles when compared to OPS provides a similar morphological survival rate and higher fertilization rate.

Identification of the stages of ovarian maturation of the Argentine hake Merluccius hubbsi Marini, 1933 (Teleostei: Merlucciidae): advantages and disadvantages of the use of the macroscopic and microscopic scales

The Argentine hake Merluccius hubbsi is a demersal-pelagic species on which few studies have been undertaken, despite its importance for the fisheries of the South-Southeastern Brazilian region, . The species is the most important commercial fishery resource in Uruguay and Argentina, where several studies have permitted the proper monitoring of the species. The ovarian maturation of the Argentine hake is analysed in this study. A scale of maturation is presented in the light of the oocyte development and the use of macro and microscopic scales of ovarian maturation are compared. It was detected that the oocyte types and derived structures present in the ovaries are similar to those already described both for the species and for teleosts in general; group synchronous development and multiple spawning are typical. The identification of ovarian maturation based only on external morphological characteristics of the gonads presents many errors, mainly in the characterization of the stages of maturation, which makes imperative the parallel analysis of the oocyte development, undertaken through histological ovarian cross-sections.

A merluza Merluccius hubbsi é uma espécie demerso-pelágica sobre a qual, apesar de sua importância nas pescarias da Região Sudeste-Sul do Brasil, poucos estudos foram realizados. Neste aspecto, destacam-se o Uruguai e a Argentina, países onde ela é o principal recurso pesqueiro e onde a existência de inúmeros estudos tem permitido o monitoramento da espécie. Neste estudo a maturação ovariana da merluza é analisada. Através do desenvolvimento ovocitário, apresenta-se uma escala de maturação e compara-se o emprego de escalas macro e microscópicas de maturação dos ovários. Constatou-se que os tipos de ovócitos e estruturas derivadas presentes nos ovários são semelhantes aos já descritos para a espécie e para teleósteos; o desenvolvimento dos ovócitos é sincrônico em grupos e a desova é parcelada. A identificação da maturação ovariana com base apenas em características morfológicas externas das gônadas apresenta muitos erros, principalmente na caracterização do estádio em maturação, sendo imprescindível a análise paralela do desenvolvimento ovocitário, realizada através de cortes histológicos dos ovários.

Single-channel response of hamster oocytes to fertilization with homologous spermatozoa

Electrophysiological events occur early after fertilization, along with changes in intracellular Ca2+ concentration. Passive electrical parameters were determined in golden hamster oocytes by whole cell patch-clamp method. In separate experiments the effect of 4-aminopyridine on resting oocytes was tested. The single-channel patch clamp configuration was employed to assess the electrical response to fertilization with homologous sperm. Structure of oocytes submitted to patch clamp was evaluated with scanning electron microscopy and found to be preserved.Oocyte diameter was 70.2 ± 2.2 µm; their resting parameters were: membrane potential 23.8 ± 0.8 mV; total membrane specific resistance 519.1 ± 94.6 Ù.cm2, and specific capacity 0.99 ± 0.03 µF.cm-2. Total membrane current was decreased by 42 % by 4-aminopyridine.Control oocytes and oocytes exposed to sperm differed in their membrane currents in response to a voltage ramp clamping membrane potential from - 100 mV to + 100 mV. In both cases, currents were largest at the most negative potentials, but sperm-exposed oocytes had larger currents. Additionally, while in controloocytes the current was inward at negative potentials but outward at positive potentials, in the presence of spermatozoa oocytes was inward within the whole voltage range tested. This latter current may represent Ca2+ en try

Seasonal variations in the heterologous binding of viscacha spermatozoa: A scanning electron microscope study

Seasonal changes in the reproductive activity of the adult male viscacha (Lagostomus maximus maximus) were investigated during the annual reproductive cycle. Assays of heterologous in vitro binding between compatible gametes were used to evaluate the ability of viscacha spermatozoa to achieve primary binding during its annual reproductive cycle. Sperm were collected by mincing cauda epididymis in HECM-3 medium and the sperm concentration and motility were evaluated. Cumulus-free and zona-free oocytes obtained from superovulated hamsters were inseminated in vitro with capacitated sperm suspensions, incubated at 37ºC, 5% CO2 for 3 h, and then processed for studies by scanning electronic microscopy. Statistical analysis was used to compare the quantitative differences. The number of spermatozoa significantly decreases during the regression period, while sperm motility was progressive speed in both periods. During the active period elevated sperm binding to cumulus-free and zona-free oocytes was observed, while the binding during the regression period decreased drastically. In both periods, oocyte microvilli covered sperm heads and tails. These results suggest that the ability of viscacha spermatozoa to participate in gamete recognition is profoundly affected. This would likely be related to different functional stages of the spermatozoa and their epididymal microenvironment during the annual reproductive cycle of viscacha.

Oogenesis in the swamp eel Synbranchus marmoratus (Bloch, 1795) (Teleostei; Synbranchidae). Ovarian anatomy, stages of oocyte development and micropyle structure

Synbranchus marmoratus (Synbranchidae), commonly known as the swamp eel, is a protogynous diandric teleost fish widely distributed throughout South America. The purpose of this work was to study the ovarian anatomy and to describe oocyte developmental stages in the swamp eel, Synbranchus marmoratus. S. marmoratus has a unique sacular ovary. It is covered by a conspicuous muscular wall, probably involved in an egg-releasing system acting as a peristaltic-like mechanism. The internal ovarian anatomy shows a U-shaped ovarian lamella delimiting a dorsal ovarian lumen. The microscopic study shows evidence of the existence of a germinal epithelium in the inner surface of the lamella, which contains germinal cells, pre-follicular cells and epithelial cells. The complete oogenesis process is divided into four stages: oogonia, primary growth, cortical alveoli and vitellogenesis. Besides, the ovulated oocytes, and atretic structures were described. The structure of the micropyle was studied by scanning electron microscopy (MEB). Near the animal pole the vitelline envelope forms crests that fuse together becoming furrow-like structures with a slightly spiraled direction that converge into the micropyle pit where is located the micropylar canal. Although the sex reversal process of Synbranchids has been subject of many studies, this is the first complete description of the ovarian anatomy and oogenesis.

Ultrastructure of ovarian follicular epithelium of the Amazonian fish Pseudotylosurus microps (Teleostei: Belonidae): morphological and histochemical characterization of the intercellular deposits

The present paper reports the presence of great quantities of electrondense intercellular material in the follicular epithelium of P. microps. The material apparently is uptaken from circulation and enter the follicle through the intercellular spaces accumulating in the epithelial median-apical intercellular spaces and in perioocytic space. The accumulation starts in the early growth of the primary oocyte and proceed until vitellogenesis. The possible chemical nature and function of the deposits are discussed

Correlation of oocyte morphology with fertilization rate and embryo quality after intracytoplasmic sperm injection.

To determine whether the morphology of the oocyte assessed under light microscopy is related to the results of intracytoplasmic sperm injection (ICSI). 135 ICSI oocytes from 15 patients were analyzed. Transvaginal ultrasound-guided oocytes retrieval with oocyte morphology evaluation based on shape of the first polar body and perivitelline space followed by ICSI. After 48 hours, embryo quality was evaluated and compared to each pre-injected oocyte morphology. Normal fertilization was achieved in 81.5 per cent of the oocytes (110/135). Abnormal fertilization (3 pronuclei) was 1.5 per cent (2/135). Fertilization rate of oocytes with good morphology was higher than those with poor morphology, but there was no statistical significance (82.4% vs 79.5%; p > 0.05). Oocytes with good morphology were significantly fertilized to be embryos with good quality (p < 0.001). This study suggests that oocyte morphology correlates with embryo quality after ICSI.