Vitrificación de ovocitos bovinos parcialmente madurados de la raza frisón rojo chileno / Vitrification of partially matured bovine oocytes of the Chilean red friesian breed

Se evaluó la tasa de maduración in vitro post descongelación de ovocitos bovinos, de la raza Frisón Rojo Chileno, parcialmente madurados y vitrificados. Complejos Cúmulus-ovocito fueron obtenidos por aspiración folicular, clasificados morfológicamente y aleatoriamente cultivados in vitro en TCM199 (10 % Suero Fetal Bovino (SFB), 50 mg/mL gentamicina, 0,2 mM piruvato de sodio, 0,08 µg/mL FSH, 1 µg/mL LH y 1 µg/mL estradiol) en los grupos: a) control (n= 137), madurados por 24 h a 38,5 C, 5 % CO2 y 99 % humedad y, b) tratamiento (n= 156), madurados por 6 h, parcialmente denudados, e incubados hasta completar 20 h, para luego ser vitrificados por el método Open Pulled Straws (OPS). Los ovocitos fueron expuestos a la solución de vitrificación uno (SV1) (Buffer Fosfato Salino (PBS), 10 % Etilenglicol (EG), 10% DMSO) por 30 s, posteriormente traspasados a la SV2 (PBS, 20 % EG, 20 % DMSO) por 25 s. Inmediatamente los ovocitos fueron cargados en pajuelas francesas estiradas (OPS) y sumergidos en nitrógeno líquido. Las ovocitos fueron descongelados introduciéndolos en una secuencia de soluciones con concentraciones decrecientes de sucrosa (0,3; 0,15 y 0 M respectivamente). Finalmente, los ovocitos continuaron con la maduración por 4 h adicionales. Posterior al periodo de maduración, los ovocitos de ambos grupos fueron fijados, teñidos y evaluados. Las proporciones de ovocitos en Metafase I (MI), Metafase II (MII) y degenerados fueron comparadas mediante el test de Chi cuadrado. La vitrificación aumentó (p 0,05) el porcentaje de pérdida y de ovocitos dañados en comparación al control. Además, aumentó (p 0,05) la tasa de ovocitos en MI y el número de ovocitos degenerados, y redujo el porcentaje de ovocitos MII, en comparación al control. Por tanto, la vitrificación por el método Open Pulled Straw de ovocitos parcialmente madurados in vitro es una alternativa viable para la conservación de material genético de hembras Frisón Rojo Chileno.

Post thawing in vitro maturation rate was evaluated for partially matured vitrified oocytes from Chilean Red Friesian cattle. Cumulus-Oocytes Complexes were obtained by follicular aspiration, classified by morphology and randomly in vitro matured in TCM199 (10 % Bovine Fetal Serum (BFS), 50 mg/mL gentamicine, 0.2 mM sodium piruvate, 0.08 µg/ml FSH, 1 µg/mL LH and 1 µg/mL estradiol) in the following groups: a) control (n= 137), matured for 24 h at 38.5 C, 5 % CO2 y 99 % humidity, and b) treatment (n= 156), matured for 6 h, partially denuded, and incubated until completion of 20 h. Then, oocytes were vitrified by the Open Pulled Straws (OPS) method. Oocytes were exposed to vitrification solution one (VS1) (Phosphate Buffered Saline (PBS), 10 % Ethylen glycol (EG), 10 % DMSO) for 30 s, then they were exposed to VS2 (PBS, 20% EG, 20% DMSO) for 25 s. Afterwards, oocytes were loaded into open pulled straws and submerged into liquid nitrogen. Oocytes were thawed by exposure to sequential solutions with decreasing concentrations of sucrose (0.3; 0.15 y 0 M respectively). Finally, oocytes continued the in vitro maturation for 4 additional hours. After completion of maturation period oocytes from both groups were fixated, stained and evaluated. The proportion of lost and damaged, MI, MII, and degenerate oocytes were compared between groups by Chi square test. Vitrification procedure increased (p 0.05) the percentage of oocytes lost and damaged when compared to control group. Additionally, vitrification increased (p 0.05) the proportion of MI and degenerated oocytes, and decreased the proportion of MII oocytes. Therefore, vitrification by the OPS method of partially matured bovine oocytes is a reliable alternative for the conservation of germinal cells from Chilean Red Friesian females.

Limitações e controvérsias na determinação do valor preditivo de critérios de morfologia oocitária e embrionária / Limitations and controversies in determining the predictive value of oocyte and embryo morphology criteria

Com o objetivo de aumentar as taxas de sucesso das pacientes que são submetidas a técnicas de reprodução humana assistida (RHA), numerosos estudos apresentam como foco a identificação do embrião com maior potencial de implantação. Apesar dos avanços tecnológicos significativos da Medicina Reprodutiva baseados no advento da era da genômica, proteômica e metabolômica (OMICS), técnicas rotineiramente aplicáveis ainda não estão disponíveis. Dessa forma, laboratórios de fertilização in vitro(FIV) de todo o mundo selecionam para transferência embriões humanos cultivados in vitrobaseados em parâmetros morfológicos avaliados em microscopia de luz. Diversos parâmetros morfológicos podem ser avaliados desde o estágio pronuclear até o estágio de blastocisto para embriões humanos cultivados in vitro. De modo geral, independentemente do dia da transferência, tais critérios parecem apresentar valor preditivo de viabilidade embrionária quando avaliados individualmente ou coletivamente. No entanto, a subjetividade da avaliação morfológica, bem como a ampla diversidade de sistemas de classificação embrionária aplicados por diferentes clínicas, implica em resultados contraditórios, tornando extremamente difícil a implementação de um consenso do valor preditivo dos diferentes parâmetros morfológicos avaliados. A otimização da seleção embrionária representa um grande potencial de aumento das taxas de sucesso do tratamento, além de possibilitar a realização da transferência de um número reduzido de embriões, minimizando os riscos derivados de estações múltiplas.

In order to increase the success rate of in vitrofertilization cycles, several studies have focused on the identification of the embryo with higher implantation potential. Despite recent advances in the reproductive medicine, based on the OMICs technology, routinely applicable methodologies are still needed. Thus, in most fertilization centers embryo selection for transfer is still based on morphological parameters evaluated under light microscopy. Several morphological parameters may be evaluated, ranging from the pronuclear to blastocyst stage. In general, despite the day of transfer, some criteria are suggested to present a predictive value for embryo viability when analyzed independently or combined. However, the subjectivity of morphological evaluation, as well as the wide diversity of embryo classification systems used by different fertilization centers shows contrasting results, making the implementation of a consensus regarding different morphological criteria and their predictive value a difficult task. The optimization of embryo selection represents a large potential to increase treatment success rates, allowing the transfer of a reduced number of embryos and inimizing the risks of multiple pregnancy.

A spatial model showing differences between juxtacrine and paracrine mutual oocyte-granulosa cells interactions.

The bidirectional communication between oocytes and granulosa cells are mediated by several factors via a local feedback loop(s). The current model was carried out to study the spatial mutual interaction of porcine denuded oocytes and granulosa cells either in direct contact (juxtacrine) or paracrine co-culture using transwell system. Transwell 0.4 µm polyester membrane inserts were used to permit oocytes-granulosa cells paracrine communication with a distance of 2 mm between them in co-culture. Oocytes were cultured with granulosa cells in a defined basic maturation medium for 44 h. In results, oocyte secreted factors (OSFs; GDF9 and BMP15) temporal expression showed progressive decrement by the end of culture in case of direct contact with granulosa cells while it was increased progressively in the paracrine co-culture groups. However, oocytes that were cultured in direct contact showed a significant increase in blastocyst development after parthenogenetic activation than the paracrine co-cultured ones (20% vs. 11.5%, respectively). By the end of culture, granulosa cell count in direct contact showed a significant decrease than the indirect co-culture group (1.2 × 105 cell/mL vs. 2.1 × 105 cell/mL, respectively). Steroids (P4 and E2) and steriodogenesis enzymes mRNA levels showed significant temporal alterations either after 22 h and 44 h of IVM in both juxtacrine and paracrine co-culture systems (P ≤ 0.05). CX43 was much more highly expressed in the granulosa of the direct contact group than the indirect co-culture group. These results indicate the difference in mutual communication between oocytes and granulosa cells that were cocultured either in direct contact (juxtacrine) or with a short distance (paracrine) and propose a new paradigm to study different ovarian follicular cells interaction.

A Correlative Study of Oocytes Morphology with Fertilization, Cleavage, Embryo Quality and Implantation Rates after Intra Cytoplasmic Sperm Injection.

ntroduction: Non-invasive selection of developmentally human oocytes may increase the overall effi ciency of human assisted reproduction. Morphologic abnormalities in the oocyte are relevant for determining its developmental fate. Th e objective is to evaluate the infl uence of MII oocyte morphology on intra cytoplasmic sperm injection (ICSI) outcomes. Material and Methods: 132 patients undergoing ICSI cycles and having female factors of infertility and unexplained infertility. Couples having male factors of infertility were excluded. A total of 1200 oocytes were retrieved from 132 ICSI cycles, of which 1056 MII oocytes were evaluated. Th e criteria for morphological evaluations were: (i) Normal MII oocytes showing clear cytoplasm with uniform texture and homogenous fi ne granularity, a round or ovoid fi rst polar body with a smooth surface, and perivitelline space of normal size. (ii) MII oocytes with extra cytoplasmic abnormalities (fi rst polar body and perivitelline space abnormalities). (iii) MII oocytes with cytoplasmic abnormalities (dark cytoplasm, granular cytoplasm, inclusion body and presents of vacuoles). (iv) MII oocytes with combined abnormalities. Result: From 1056 MII oocytes, 180 (17.04%) had normal morphology while 876 (82.95%) had at least one demonstrable morphological abnormality. Cytoplasmic abnormalities were observed in 516 (58.9%) of the oocytes. Extra cytoplasmic abnormalities were observed in 104 (11.87%) while combined abnormalities were responsible for the remaining 256 (29.22%). Th ere were no signifi cant diff erences in fertilization, cleavage, and embryo quality between the groups but there was a highly signifi cant diff erence in implantation rate which was higher in the group of normal oocytes morphology than abnormal oocytes morphology, oocytes with cytoplasmic, extracytoplasmic and combined abnormality 11.11%, 7.33%, 9.03%, 2.3%, and 4.34% respectively. Conclusion: MII oocyte morphology did not aff ect fertilization, cleavage, and embryo quality, but aff ecting implantation rate.

Signaling Pathways in Insulin- and IGF-I Mediated Oocyte Maturation in Lower Vertebrates.

The endocrine control of oocyte maturation in fish and amphibians has proved to be a valuable model for investigating the rapid and non-genomic steroid actions at the cell surface. Considerable progress has made over the last decade in elucidating signaling pathways in steroid-induced oocyte maturation. In addition to steroids, various growth factors have also been reported to be involved in this process and progress being made to elucidate their mechanism of actions. Exposure of fully-grown oocytes to steroids or growth factors (insulin/IGFs) initiates various signaling cascade, leading to formation and activation of maturation-promoting factor (MPF), a key enzyme that catalyzes entry into M-phase of meiosis I and II. Whereas the function of MPF in promoting oocyte maturation is ubiquitous, there are differences in signaling pathways between steroids- and growth factors-induced oocyte maturation in amphibian and fish. Here, we have reviewed the recent advances on the signaling pathways in insulin- and IGF-I-induced oocyte maturation in these two groups of non-mammalian vertebrates. New findings demonstrating the involvement of PI3 kinase and MAP kinase in induction of oocyte maturation by insulin and IGF-I are presented.

Morphological indicators of initial reproductive commitment in Mustelus schmitti (Springer 1939) (Chondrichthyes, Triakidae): folliculogenesis and ovarian structure over the life cycle / Indicadores morfológicos de compromisso reprodutivo inicial em Mustelus schmitti (Springer 1939) (Chondrichthyes, Triakidae): folliculogenesis e estrutura ovariana ao longo do ciclo de vida

This work provides information about the sexual commitment and the folliculogenesis of the gatuzo, Mustelus schmitti. A total of 112 females of all maturity stages were fished in the Bahía Blanca estuary, between 2009 and 2010. The oogonia were present throughout the life cycle of the animals. The folliculogenesis follows a pattern similar to other elasmobranchs. The granulosa layer keeps monolayered throughout the folliculogenesis, but with two cell types in the vitellogenic follicle. The zona pellucida forms in the primordial follicles. The thecal system shows a connective inner layer and a glandular outer sheath. The microscopic beginning of the sexual commitment, indicated by the vitello hoarding, takes place in follicles from 500 micrometres, while the macroscopic evidence appears in follicles of 2500-3000 micrometres. The results presented in this study suggest that the fishery pressure may affect a susceptible range of sizes of the species, not previously considered and provides a biological framework for the development of fisheries policy.

Este trabalho provê informações sobre o compromisso sexual e da foliculogênese do gatuzo, Mustelus schmitti. Um total de 112 fêmeas de todas as fases de maturidade foram pescados no estuário Bahía Blanca, entre 2009 e 2010. O oogônias foi presentes durante todo do ciclo de vida dos animais. A foliculogênese segue um padrão semelhante a outros elasmobrânquios. A capa granulosa mantém-se simples durante toda a foliculogénese, mas com dois tipos de células no folículo vitelogênico. A zona pelúcida forma-se nos folículos primordiais. O sistema mostra uma capa tecal interior de tecido conjuntivo e uma bainha exterior glandular. O início microscópico do compromisso sexual, indicado pela acumulação do vitello, realiza-se em folículos de 500 micrómetros, enquanto que a evidência macroscópica aparece em folículos de 2500-3000 micrómetros. Os resultados apresentados neste estudo sugerem que a pressão da pesca pode afetar um amplo intervalo de tamanho das espécies não considerado anteriormente, e fornece uma base biológica para o desenvolvimento de política comum da pesca.

Impact of the endometrioma on ovarian response and pregnancy rate in in vitro fertilization cycles

Our objective was to evaluate the effect of ovarian endometrioma on ovarian stimulation outcomes in in vitro fertilization cycles [IVF]. In this prospective cohort study, we followed 103 patients who underwent intra-cytoplasmic sperm injection [ICSI] procedures over a 24-months period. The study group consisted of 47 infertile women with either unilateral or bilateral ovarian endometrial cysts of less than 3 cm. The control group consisting of 57 patients with mild male factor infertility was candidate for ICSI treatment during the same time period as the study groups. Both groups were compared for number of oocytes retrieved, grades of oocytes, as well as embryo quantity and quality. Our results showed similar follicle numbers, good embryo grades [A or B] and pregnancy rates in the compared groups. However, patients with endometrioma had higher gonadotropin consumption than the control group. The mean number of retrieved oocytes in patients with endometrioma was significantly lower than control group [6.6 +/- 3.74 vs. 10.4 +/- 5.25] [p<0.001]. In addition, patients with endometrioma had significantly lower numbers of metaphase II [MII] oocytes [5 +/- 3.21] than controls [8.2 +/- 5.4] [p<0.001]. In patients with unilateral endometrioma, there were no significant differences in main outcome measures between normal and involved ovaries in the patients with endometrioma. Patients with ovarian endometrioma had poor outcome. They showed poor ovarian response with lower total numbers of retrieved oocytes and lower MII oocytes during the stimulation phase; however, it does not affect the total number of embryos transferred per patient, quality of embryos, and pregnancy rate per patient

Assessment of ovarian tissues autografted to various body sites followed by IVM in mouse

Ovarian tissue transplantation is emerging technologies for fertility preservation. In addition, in vitro maturation [IVM] of oocytes retrieved from ovarian tissues may overcome the fertility defects in certain cases. The aim was to evaluate the best site for ovarian tissue transplantation in mice. Also, feasibility of IVM of oocytes retrieved from auto grafted ovarian tissues was freshly assessed. Hemi-ovaries from 6 weeks old mice were auto grafted into kidney capsule [K] versus the back muscle [B] and leg muscle [L] in a mouse auto graft model which was stimulated with gonadotrophins. Then ovarian grafts were recovered and processed histologically for follicle assessment compared with control, also the ability of oocytes to mature with IVM was studied 14 days after transplantation. Total follicle count was significantly higher in K-graft [3.5 +/- 3.17] and the antral follicles were only observed in K-site model. The number of retrieved immature oocytes as well as successful IVM in K-grafts was significantly higher than other groups [p=0.008, p=0.016]. The kidney capsule is a promising site for ovarian tissue auto graft in mice. This resulted in better follicular survival and IVM outcomes

Anti-Mullerian Hormone Levels in the Follicular Fluid of the Preovulatory Follicle: A Predictor for Oocyte Fertilization and Quality of Embryo

This prospective study investigated the relationship between anti-Mullerian hormone (AMH) level in the follicular fluid (FF) and the quality of the oocyte and embryo. A total of 65 FF samples from 54 women were included in this study. FF was collected from the largest preovulatory follicle sized> or =20 mm of mean diameter from each ovary. Samples were divided into 3 groups according to the FF AMH levels: below the 33th percentile (low group, FF AMH3.6 ng/mL, n=22). The quality of the ensuing oocytes and embryos was evaluated by fertilization rate and embryo score. FF AMH levels correlated positively with the matched embryo score on day 3 after fertilization (r=0.331, P=0.015). The normal fertilization rate was significantly lower in the low group than in the intermediate group (61.9% vs. 95.5% vs. 77.3%, respectively, P=0.028). Our results suggest that the FF AMH level could be a predictor of the ensuing oocyte and embryo quality.

Produção de oócitos em mamíferos adultos: o camundongo como modelo / Oocyte production in adult mammals: mice as the model

Objetivo: identificar produção de oócitos em mamíferos adultos com o uso de camundongo como modelo experimental. Método: empregamos a técnica de imuno-histoquímica em cortes de ovários de camundongos Balb-c (45 dias de idade) com o uso de anticorpo específico para marcação de células germinativas. Como controles positivos da reação, usamos cortes de testículos de camundongos. Resultados: as células germinativas (espermatogônias, espermatócitos e espermátides) dos controles positivos sofreram marcação, enquanto células não pertencentes a essa linhagem (células de Leydig e de Sertoli) mostraram negatividade de reação; nos cortes ovarianos observou-se marcação de oócitos de folículos em diferentes estágios de maturação, mas houve também marcação de células não englobadas pela estrutura folicular. Conclusões: os achados sugerem que durante a puberdade ovários de camundongos fêmeas contêm células da linhagem germinativa em estágios anteriores à formação folicular, o que corrobora estudos anteriores; o trabalho é pioneiro no Brasil e progredirá para a completa caracterização de células com potencial oogênico em outras espécies de mamíferos. Resultados positivos poderão alterar o entendimento da biologia reprodutiva e abrir novas portas para o tratamento de infertilidade.

Objective: to identify oocyte production in adult mammals using the mouse as the experimental model. Method: we used the immunohistochemistry technique on ovary sections of Balb-c mice (45 days old), with antibody that labels germline cells specifically. We used sections of mice’s testes as positive reaction controls.Results: in testes samples, germ cells (spermatogonia, spermatocytes and spermatids) were stained, while cells not belonging to germ lineage (Leydig and Sertoli cells) showed negativereaction; in ovarian samples, oocytes from follicles in different stages of maturation werestained, but the reaction was also positive for cells not enclosed by the follicular structure. Conclusions: the findings suggest that, during puberty, female mice ovaries contain germline cells in earlier stages before follicular formation, as was found in previous studies. Thework, pioneering in Brazil, must progress to a complete characterization of these cells (with oogenesis potential) in mice and in other mammal species. Positive results may change the understanding of the reproductive biology and open new possibilities for infertility treatment.

Maturação in vitro de oócitos bovinos em meios suplementados com quercetina e seu efeito sobre o desenvolvimento embrionário / In vitro maturation of bovine oocytes in medium supplemented with quercetin, and its effect on embryonic development

A quercetina é um flavonoide, amplamente encontrada em frutas, vegetais, grãos, flores, com elevada concentração no vinho tinto, e tem sido caracterizada funcionalmente pela atividade antioxidante. Para avaliação da maturação nuclear e do desenvolvimento embrionário bovino, os oócitos foram maturados por 22h na presença de quercetina (0,4, 2, 10 e 50µM), cisteamina (100µM) e na ausência dos antioxidantes. Os oócitos maturados foram corados com Hoechst para avaliação da maturação in vitro. Para avaliação do desenvolvimento embrionário, os oócitos foram fertilizados e cultivados in vitro, as taxas de desenvolvimento embrionário foram determinadas no sétimo dia de cultivo e o percentual de eclosão e o número de células dos embriões no oitavo dia. Os níveis de glutationa (GSH) dos oócitos foram mensurados por emissão de fluorescência com CMF2HC. A porcentagem de maturação nuclear (±89%) não diferiu entre os grupos. O desenvolvimento embrionário variou entre os tratamentos, o percentual de blastocisto foi superior (P<0,05) nos grupos tratados com 0,4, 2, 10 e 50∝M de quercetina (56,9, 59,5, 53,6 e 49,6%, respectivamente) e com 100∝M de cisteamina (50,4%) em relação ao grupo controle (42,3%). Na comparação entre os dois antioxidantes, a quercetina (0,4 e 2µM) foi superior na produção de embriões (56,9 e 59,5%, respectivamente) em comparação com cisteamina (50,4%). As taxas de embriões eclodidos foram similares (P>0,05) entre os grupos (±63,0%). O número médio de células dos embriões também foi similar entre os grupos (±233). Os níveis intracelulares de GSH foram superiores nos oócitos maturados com cisteamina, mas similares entre os oócitos tratados com quercetina e o controle. A suplementação da maturação in vitro com antioxidantes melhora as taxas de blastocistos. A quercetina foi superior à cisteamina, que, por sua vez, foi superior ao controle. Mas os níveis de GSH foram superiores somente nos oócitos tratados com cisteamina.

Quercetin is a flavonoid widely found in fruit, vegetables, grains and flowers, with a high concentration in red wine, and has been functionally characterized by its antioxidant activity. For assessment of nuclear maturation and bovine embryo, oocytes were matured for 22h in the presence of quercetin (0.4, 2, 10 and 50µM), cysteamine (100µM) and in the absence of antioxidants. The matured oocytes were stained with Hoechst to evaluate the in vitro maturation. To assess embryonic development, oocytes were fertilized and cultured in vitro and rates of embryo development were obtained in the seventh day of culture and the percentage of hatching and the number of cells on eighth day embryos. The levels of glutathione (GSH) of the oocytes were measured by fluorescence emission with CMF2HC. The percentage of nuclear maturation (±89%) did not differ between groups. Embryonic development varied between treatments, the percentage of blastocyst was higher (P<0.05) in the groups treated with 0.4, 2, 10 and 50∝M of quercetin (56.9, 59.5, 53.6 and 49.6%, respectively) and 100 ∝M cysteamine (50.4%) compared to the control group (42.3%). Comparing the two antioxidants, quercetin (0.4 to 2µM) was superior in embryo production (56.9 and 59.5% respectively) compared with cysteamine (50.4%). The rates of hatched embryos were similar (P>0.05) between groups (±63.0%). The average number of embryo cells was also similar in both groups (±233). The intracellular GSH levels were higher in oocytes matured with cysteamine, but similar between the oocytes treated with quercetin and control. The supplementation of matured in vitro with antioxidants improves blastocyst rates. Quercetin was greater than cysteamine, which in turn was superior to the control. However, GSH levels were higher in oocytes treated only with cysteamine.

Induced spawning and reproductive variables of the catfish Lophiosilurus alexandri Steindachner, 1876 (siluriformes: pseudopimelodidae)

Lophiosilurus alexandri is an endemic fish from the São Francisco River basin, Brazil. The aim of this study was to induce L. alexandri to spawn and to obtain data on several reproductive variables for this species. For induced spawning, adults were submitted to Cyprinus carpio pituitary homogenate (CPH). Nine of the 12 females (75%) responded positively to the treatment. The stripping of oocytes was performed 8.4 h after the second dose of CPH with the water temperature maintained at 26ºC. The number of stripped oocytes per gram of ova was 74 ± 5 oocytes g-1, and the mean oocyte diameter was 3.1 ± 0.2 and 3.6 ± 0.2 mm, before and after hydration, respectively. The oocytes were opaque, yellowish, demersal, highly adhesive, and covered by a gelatinous coat. The total fecundity was 4,534 ± 671 oocytes, and the fertilization rate was 59%. The initial and final fertilities were 2,631 ± 740 and 1,542 ± 416 embryos, respectively. Larval hatching occurred up to 56 h after fertilization, and the larvae had a total length of 8.4 ± 0.1 mm. This work provides important biological information for L. alexandri that can be used for management and conservation of this species.

Lophiosilurus alexandri é um peixe endêmico da bacia do rio São Francisco, Brasil. O objetivo do trabalho foi induzir L. alexandri à desova e obter dados sobre várias variáveis reprodutivas para esta espécie. Para desova induzida, adultos foram submetidos ao homogeneizado de hipófise de Cyprinus carpio (HHC). Nove das 12 fêmeas (75%) responderam positivamente ao tratamento. A extrusão dos ovócitos aconteceu 8,4 h após a segunda dose de HHC com a temperatura da água mantida a 26ºC. O número de ovócitos liberados por grama de ova foi de 74 ± 5 ovócitos g-1 e a média do diâmetro ovocitário foi de 3,1 ± 0,2 e 3,6 ± 0,2 mm, antes e depois da hidratação, respectivamente. Os ovócitos foram opacos, amarelo-castanho, demersais, altamente adesivos e revestidos por capa gelatinosa. A fecundidade total apresentou 4.534 ± 671 ovócitos e a taxa de fertilização foi de 59%. As fertilidades inicial e final foram de 2.631 ± 740 e 1.542 ± 416 embriões, respectivamente. A eclosão das larvas aconteceu até 56 h após a fertilização e as larvas tiveram comprimento total de 8,4 ± 0,1 mm. Este trabalho fornece informações biológicas importantes para L. alexandri, que podem ser utilizadas para o manejo e conservação desta espécie.

Dynamics of ovarian maturation during the reproductive cycle of Metynnis maculatus, a reservoir invasive fish species (Teleostei: Characiformes)

In this study, we evaluated the dynamics of ovarian maturation and the spawning processes during the reproductive cycle of Metynnis maculatus. Adult females (n = 36) were collected bimonthly between April 2010 and March 2011. The mean gonadosomatic index (GSI) was determined, ovarian and blood samples were submitted for morphometric evaluation and the steroid plasma concentration was determined by ELISA. This species demonstrated asynchronous ovarian development with multiple spawns. This study revealed that, although defined as a multiple spawning species, the ovaries of M. maculatus have a pattern of development with a predominance of vitellogenesis between April and August and have an intensification in spawning in September; in October, a drop in the mean GSI values occurred, and the highest frequencies of post-ovulatory follicles (POFs) were observed. We observed a positive correlation between the POF and the levels of 17α-hydroxyprogesterone. Metynnis maculatus has the potential to be used as a source of pituitary tissue for the preparation of crude extracts for hormonal induction; the theoretical period for use is from September to December, but specific studies to determine the feasibility of this approach must be conducted.

Neste estudo, avaliamos a dinâmica da maturação ovariana a desova durante o ciclo reprodutivo de Metynnis maculatus. Fêmeas adultas (n = 36) foram coletadas bimestralmente entre abril de 2010 e março de 2011. O índice gonadossomático (IGS) foi calculado e amostras de ovário e de sangue foram submetidas à avaliação morfométrica e das concentrações plasmáticas dos esteroides por ELISA, respectivamente. A espécie apresenta desenvolvimento ovariano assincrônico, com múltiplas desovas. Neste estudo revelamos que mesmo sendo de desova parcelada, os ovários do M. maculatus mostraram um padrão de desenvolvimento com predomínio de atividade vitelogênica entre abril a agosto e intensificação da desova em setembro. Em outubro houve uma diminuição nos valores médios de IGS, bem como registramos as maiores frequências de folículos pós-ovulatórios (FPOs). Observamos uma correlação positiva entre a frequência de FPOs e a concentração plasmática de 17 α-OHP. O M. maculatus tem potencial para ser usado como fonte para uso de hipófise para preparo de extrato bruto para indução hormonal, sendo o período teórico para coleta de hipófises de setembro a outubro, mas estudos específicos para esta finalidade ainda precisam ser desenvolvidos.

Quercetin improves the in vitro development of porcine oocytes by decreasing reactive oxygen species levels

Quercetin is a plant-derived flavonoid found in fruits or vegetables that has antioxidant properties and acts as a free radical scavenger. We investigated the effects of quercetin on porcine oocyte nuclear maturation and embryonic development after parthenogenetic activation. We then evaluated the antioxidant activities of quercetin by measuring reactive oxygen species (ROS) levels in matured oocytes. Immature oocytes were untreated or treated with 1, 10, and 50 microg/mL quercetin during in vitro maturation (IVM). Quercetin treatment did not improve oocyte nuclear maturation, but significantly higher blastocyst rates (p < 0.05) of parthenogenetically activated oocytes were achieved when the IVM medium was supplemented with an adequate concentration of quercetin (1 microg/mL). However, cleavage rates and blastocyst cell numbers were not affected. Oocytes treated with 1 or 10 microg/mL quercetin had significantly lower (p < 0.05) levels of ROS than the control and group treated with the highest concentration of quercetin (50 microg/mL). Moreover, this highest concentration was detrimental to oocyte nuclear maturation and blastocyst formation. Based on our findings, we concluded that exogenous quercetin reduces ROS levels during oocyte maturation and is beneficial for subsequent embryo development.

Quercetin improves the in vitro development of porcine oocytes by decreasing reactive oxygen species levels

Quercetin is a plant-derived flavonoid found in fruits or vegetables that has antioxidant properties and acts as a free radical scavenger. We investigated the effects of quercetin on porcine oocyte nuclear maturation and embryonic development after parthenogenetic activation. We then evaluated the antioxidant activities of quercetin by measuring reactive oxygen species (ROS) levels in matured oocytes. Immature oocytes were untreated or treated with 1, 10, and 50 microg/mL quercetin during in vitro maturation (IVM). Quercetin treatment did not improve oocyte nuclear maturation, but significantly higher blastocyst rates (p < 0.05) of parthenogenetically activated oocytes were achieved when the IVM medium was supplemented with an adequate concentration of quercetin (1 microg/mL). However, cleavage rates and blastocyst cell numbers were not affected. Oocytes treated with 1 or 10 microg/mL quercetin had significantly lower (p < 0.05) levels of ROS than the control and group treated with the highest concentration of quercetin (50 microg/mL). Moreover, this highest concentration was detrimental to oocyte nuclear maturation and blastocyst formation. Based on our findings, we concluded that exogenous quercetin reduces ROS levels during oocyte maturation and is beneficial for subsequent embryo development.

Oócitos aparentemente maduros injetados em telófase I apresentam piores resultados de reprodução assistida / Apparently matured oocytes injected in telophase I have worse outcomes from assisted reproduction

OBJETIVO: Avaliar o estágio de maturação nuclear de oócitos com o primeiro corpúsculo polar (CP) visível de pacientes inférteis submetidas à estimulação ovariana para injeção intracitoplasmática de espermatozoide (ICSI) e comparar os resultados da injeção intracitoplasmática de espermatozoide entre os oócitos em telófase I (TI) e metáfase II (MII), e entre aqueles em metáfase II com e sem fuso celular visível. MÉTODOS: Estudo prospectivo que incluiu 106 pacientes inférteis submetidas à injeção intracitoplasmática de espermatozoide. Foram incluídas pacientes com idade menor ou igual a 38 anos, hormônio folículo estimulante (FSH) basal menor que 10 mIU/mL e índice de massa corpórea (IMC) menor que 30 kg/m². Foram excluídas pacientes com doenças sistêmicas, com qualquer infecção ativa, tabagistas ou que fizeram uso de medicações hormonais e anti-inflamatórias hormonais e não hormonais nos últimos dois meses, previamente à programação para o procedimento de reprodução assistida. Os oócitos com extrusão do primeiro corpúsculo polar foram avaliados pela microscopia de polarização, imediatamente antes da realização da injeção intracitoplasmática de espermatozoide, e caracterizados quanto ao estágio de maturação nuclear (telófase I ou metáfase II). Os oócitos em metáfase II foram avaliados de acordo com a presença ou não do fuso meiótico. Foram analisadas as taxas de fertilização, clivagem e o número de embriões de boa qualidade no segundo dia (D2) de desenvolvimento. Os dados foram analisados comparativamente através do teste exato de Fisher. Em todas as análises foi considerado o nível de significância de 5% (p<0,05). RESULTADOS: O fuso meiótico de 516 oócitos foi visualizado através da microscopia de polarização. Dezessete dos 516 oócitos avaliados estavam em telófase I (3,3%) e 499 (96,7%) em metáfase II. Os oócitos injetados em telófase I apresentaram taxas de fertilização significativamente menores do que os injetados em metáfase II (53 e 78%, respectivamente) e não produziram nenhum embrião de boa qualidade no segundo dia. Comparando-se os oócitos com e sem fuso celular visível, não foi observada diferença significativa nos resultados de injeção intracitoplasmática de espermatozoide. CONCLUSÕES: Oócitos injetados em telófase I apresentaram menores taxas de fertilização quando comparados aos em metáfase II. É possível que a análise do estágio de maturação nuclear oocitária, por meio da microscopia de polarização, possa ser utilizada como fator de predição das taxas de fertilização pós-injeção intracitoplasmática de espermatozoide.

PURPOSE: To evaluate the nuclear maturation stage and the presence of meiotic spindles of in vivo matured oocytes from infertile women undergoing stimulated cycles for intracytoplasmic sperm injection (ICSI) and compare intracytoplasmic sperm injection outcomes between oocytes in telophase I (TI) and metaphase II (MII), and the ones with and without visible meiotic spindle. METHODS: A prospective and controlled study with 106 infertile patients who underwent ovarian stimulation for intracytoplasmic sperm injection purposes. Patients aged 38 years or less, with basal follicle stimulating hormone (FSH) less than 10 mIU/mL and body mass index (BMI) less than 30 kg/m². Were included patients presenting any systemic diseases, any active infection, smokers or patients who had been using hormonal medications and hormonal and nonhormonal anti-inflammatory drugs for the past two months prior to the assisted reproduction procedure were excluded. The oocytes with the first polar body extruded (in vivo matured oocytes) were imaged by polarization microscopy immediately before intracytoplasmic sperm injection and characterized according to nuclear maturation stage (telophase I and metaphase II) and to the presence of a meiotic spindle. We analyzed the fertilization rates, cleavage, number of good quality embryos on the second day (D2) from oocytes on telophase I versus those in metaphase II, and metaphase II visible spindle versus non-visible ones. Data were analyzed comparatively by Fisher's exact test. The level of significance was set at 5% in all analyses (p<0.05). RESULTS: The meiotic spindles of 516 oocytes were imaged using polarization microscopy. From the 516 oocytes analyzed, seventeen were in telophase I (3.3%) and 499 (96.7%) in metaphase II. The oocytes injected in telophase I had significantly lower fertilization rates than those injected in metaphase II (53 and 78%, respectively) and produced no good quality embryos on day 2. When the oocytes with and without a visible meiotic spindle were compared, there was no significant difference in the intracytoplasmic sperm injection results. CONCLUSIONS: Oocytes injected in telophase I showed lower fertilization rates when compared to those in metaphase II. It is possible that the analysis of oocyte nuclear maturation by polarization microscopy can be used as a predictor of fertilization after intracytoplasmic sperm injection.

Oocyte Maturity in Relation to Woman's Age in In Vitro Fertilization Cycles Stimulated by Single Regimen

PURPOSE: During stimulated in vitro fertilization (IVF) cycle, up to 30% of the recovered oocytes are immature ones which have poor fertilization capacity; however, the precise influencing factors are largely unknown. Here, we analyzed the association of oocyte immaturity with woman's age in IVF cycles stimulated by single regimen. MATERIALS AND METHODS: A total of one-hundred ninety five IVF cycles stimulated by recombinant FSH and GnRH antagonist protocol between 2003 and 2009 were analyzed retrospectively. The mean age of women was 34.2+/-4.0 (26-45 years). After triggering by exogenous hCG, an ultrasound-guided retrieval of oocytes was performed 35-36 hours later. All clinical data were stratified by woman's age; group I: or =41 (n=19). RESULTS: The total retrieved oocytes, as well as immature oocytes, were significantly lower in group IV, however, the mean % of immature oocytes was significantly higher in group IV than other age groups. Oocyte immaturity tended to decrease as increasing age in women aged 40 years or less. CONCLUSION: In stimulated IVF cycle, much higher oocyte immaturity was noted in women aged 41 years or more.

Application of Sperm Selection Using Hyaluronic Acid Binding in Intracytoplasmic Sperm Injection Cycles: A Sibling Oocyte Study

The purpose of this study was to investigate whether sperm selection by hyaluronic acid (HA) binding could improve fertilization rate and embryo quality in intracytoplasmic sperm injection (ICSI) cycles. Two hundred nineteen oocytes obtained from eighteen women were injected with either HA-bound (n = 107) or conventionally selected spermatozoa (n = 112) in a randomized way. All of the participants were infertile couples who had normal sperm parameters but low fertilization rate in previous in vitro fertilization (IVF) cycle (n = 5) or experienced multiple IVF failures (n = 13). Lower fertilization (75.7% vs 83.0%) and cleavage rate on day 2 (72.9% vs 83.0%) was observed in oocytes injected with HA-bound spermatozoa than the conventional group, but the difference was not significant. Significantly lower cleavage rate was observed on day 3 in HA group (56.0% vs 69.6%, P = 0.038). Blastocyst formation rate and the number of transferred embryos were similar in both groups. In multiple IVF failure patients, significantly reduced fertilization rate (71.8% vs 85.3%, P = 0.046) and cleavage rate on day 2 (70.4% vs 85.3%, P = 0.029) and day 3 (53.5% vs 77.3%, P = 0.002) were noticed in HA group. Five women achieved pregnancy continuing more than 12 weeks after transfer (27.8%). Success of ICSI was not related with the number of embryos fertilized by HA-bound spermatozoa. Application of ICSI by sperm selection using HA binding is not helpful in couples with repeated poor fertilization or implantation despite normal sperm parameters.

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.