Analysis of nuclear maturation, DNA damage and repair gene expression of bovine oocyte and cumulus cells submitted to ionizing radiation'

Radiotherapy causes destruction of tumor cells, but also threatens the integrity and survival of surrounding normal cells. Then, woman submitted to irradiation for cancer treatment may present permanent ovary damage, resulting in impaired fertility. The objective of this study was to investigate the effects of therapeutic doses of ionizing radiation (IR), used for ovarian cancer treatment in humans, on bovine cumulus-oocyte complexes (COCs) as experimental model. Bovine ovaries were exposed to 0.9 Gy, 1.8 Gy, 3.6 Gy or 18.6 Gy IR, and then COCs were collected and used to evaluate: (a) oocyte nuclear maturation; (b) presence of phosphorylated H2A.X (γH2AX), as an indicator of DNA double-strand breaks (DSBs); and (c) expression of genes involved in DNA repair (TP53BP1, RAD52, ATM, XRCC6 and XRCC5) and apoptosis (BAX). The radiation doses tested in this study had no detrimental effects on nuclear maturation and did not increase γH2AX in the oocytes. However, IR treatment altered the mRNA abundance of RAD52 (RAD52 homolog, DNA repair protein) and BAX (BCL2-associated X protein). We conclude that although IR doses had no apparent effect on oocyte nuclear maturation and DNA damage, molecular pathways involved in DNA repair and apoptosis were affected by IR exposure in cumulus cells.(AU)

In search of the transcriptional blueprints of a competent oocyte

The oocyte undergoes a remarkably long andelaborated journey within the follicle before becomingfully equipped to sustain embryonic development. Itsability to support early embryonic development relieslargely on the maternal transcripts accumulated duringits growth and maturation. However, it is still not clearwhat transcriptome blueprint composes a competentoocyte. A number of extensive studies provided adetailed characterization of the mRNA molecules thatare gradually accumulated in the oocyte cytoplasm. Thedetail of our knowledge has gradually increased throughthe years also thanks to the development andimprovement of the analytical techniques. From realtimePCR analysis of single transcripts, to the wholetranscriptome approach of gene arrays and newgenereation sequencing, scientists accumulated anexponentially growing amount of new information.More recently, the discovery of non-coding RNAsrevealed a new layer of complexity in the mechanismsthat modulate gene expression at the mRNA level, infolliculogenesis and oogenesis. In particular, data areemerging on the potential role of microRNAs incontrolling ovarian function, oocyte maturation and theoocyte-somatic cell cross talk. This review will try tosummarize the vast amount of data currently available onthe mRNAs and microRNAs associated with the ovarianfunction and to find their biological significance.

In search of the transcriptional blueprints of a competent oocyte

The oocyte undergoes a remarkably long andelaborated journey within the follicle before becomingfully equipped to sustain embryonic development. Itsability to support early embryonic development relieslargely on the maternal transcripts accumulated duringits growth and maturation. However, it is still not clearwhat transcriptome blueprint composes a competentoocyte. A number of extensive studies provided adetailed characterization of the mRNA molecules thatare gradually accumulated in the oocyte cytoplasm. Thedetail of our knowledge has gradually increased throughthe years also thanks to the development andimprovement of the analytical techniques. From realtimePCR analysis of single transcripts, to the wholetranscriptome approach of gene arrays and newgenereation sequencing, scientists accumulated anexponentially growing amount of new information.More recently, the discovery of non-coding RNAsrevealed a new layer of complexity in the mechanismsthat modulate gene expression at the mRNA level, infolliculogenesis and oogenesis. In particular, data areemerging on the potential role of microRNAs incontrolling ovarian function, oocyte maturation and theoocyte-somatic cell cross talk. This review will try tosummarize the vast amount of data currently available onthe mRNAs and microRNAs associated with the ovarianfunction and to find their biological significance.(AU)

Avaliação morfológica e nuclear de oócitos bovinos imaturos, obtidos de ovários com e sem a presença de corpo lúteo / Morphological evaluation and nuclear oocytes immature, obtained from ovarian with and without the body presence luteum

A produção in vitro de embriões (PIVE) é uma biotecnologia que possibilita principalmente o rápido aumento no número de descendentes com mérito genético superior. Atualmente o Brasil é líder mundial na PIVE e grande parte desse sucesso deve-se às inúmeras pesquisas realizadas nessa área. A seleção de oócitos de boa qualidade para essa técnica é determinante para obtenção de embriões de qualidade e satisfatórios índices de prenhez. Desta forma, esse trabalho teve como objetivo avaliar oócitos, obtidos de ovários com e sem a presença de corpo lúteo (CL), de acordo com a classificação morfológica e nuclear, afim de identificar a possível influência do CL na viabilidade dos oócitos. Foram coletados ovários de abatedouro e separados em dois grupos, com e sem CL. Os folículos foram aspirados, e os oócitos obtidos foram selecionados, classificados e em seguida corados com Hoechst 33342 e avaliados sob microscópio de epifluorescência, para avaliação nuclear. Realizou-se análise descritiva de cada tratamento com relação a classificação morfológica e nuclear. Os resultados obtidos foram analisados através de testes não-paramétricos, sendo o efeito do corpo lúteo verificado por meio do teste Qui-quadrado (2) (R core Team, 2013). Concluiu-se que a presença de CL não influencia na qualidade de oócitos imaturos, do ponto de vista quantitativo, morfológico e nuclear.

The in vitro embryo production (IVP) is a biotechnology that primarily enables the rapid increase in the number of offspring with superior genetic merit . Currently, Brazil is the world leader in IVP and much of this success is due to the extensive research carried out in that area. The selection of good quality oocytes for this technique is critical to obtaining quality embryos and satisfactory pregnancy rates. Thus, this study aimed to assess oocytes obtained from ovaries with and without the presence of corpus luteum (CL), according to the morphological and nuclear classification, in order to identify the possible influence of CL on the viability of oocytes. They were collected from slaughterhouse ovaries and separated into two groups, with and without CL. Follicles were aspirated and the oocytes were selected, ranked, and then stained with Hoechst 33342 and evaluated by epifluorescence microscope for nuclear evaluation. A descriptive analysis of each treatment with respect to morphological and nuclear classification. The results were analyzed using non-parametric tests, with the effect of the corpus luteum verified using the chisquare test (χ2) (R Core Team, 2013). It was concluded that the presence of CL does not influence the quality of immature oocytes, from the quantitative and morphological nuclear standpoint.

Avaliação morfológica e nuclear de oócitos bovinos imaturos, obtidos de ovários com e sem a presença de corpo lúteo / Morphological evaluation and nuclear oocytes immature, obtained from ovarian with and without the body presence luteum

A produção in vitro de embriões (PIVE) é uma biotecnologia que possibilita principalmente o rápido aumento no número de descendentes com mérito genético superior. Atualmente o Brasil é líder mundial na PIVE e grande parte desse sucesso deve-se às inúmeras pesquisas realizadas nessa área. A seleção de oócitos de boa qualidade para essa técnica é determinante para obtenção de embriões de qualidade e satisfatórios índices de prenhez. Desta forma, esse trabalho teve como objetivo avaliar oócitos, obtidos de ovários com e sem a presença de corpo lúteo (CL), de acordo com a classificação morfológica e nuclear, afim de identificar a possível influência do CL na viabilidade dos oócitos. Foram coletados ovários de abatedouro e separados em dois grupos, com e sem CL. Os folículos foram aspirados, e os oócitos obtidos foram selecionados, classificados e em seguida corados com Hoechst 33342 e avaliados sob microscópio de epifluorescência, para avaliação nuclear. Realizou-se análise descritiva de cada tratamento com relação a classificação morfológica e nuclear. Os resultados obtidos foram analisados através de testes não-paramétricos, sendo o efeito do corpo lúteo verificado por meio do teste Qui-quadrado (2) (R core Team, 2013). Concluiu-se que a presença de CL não influencia na qualidade de oócitos imaturos, do ponto de vista quantitativo, morfológico e nuclear.(AU)

The in vitro embryo production (IVP) is a biotechnology that primarily enables the rapid increase in the number of offspring with superior genetic merit . Currently, Brazil is the world leader in IVP and much of this success is due to the extensive research carried out in that area. The selection of good quality oocytes for this technique is critical to obtaining quality embryos and satisfactory pregnancy rates. Thus, this study aimed to assess oocytes obtained from ovaries with and without the presence of corpus luteum (CL), according to the morphological and nuclear classification, in order to identify the possible influence of CL on the viability of oocytes. They were collected from slaughterhouse ovaries and separated into two groups, with and without CL. Follicles were aspirated and the oocytes were selected, ranked, and then stained with Hoechst 33342 and evaluated by epifluorescence microscope for nuclear evaluation. A descriptive analysis of each treatment with respect to morphological and nuclear classification. The results were analyzed using non-parametric tests, with the effect of the corpus luteum verified using the chisquare test (χ2) (R Core Team, 2013). It was concluded that the presence of CL does not influence the quality of immature oocytes, from the quantitative and morphological nuclear standpoint.(AU)

Survival, growth, and hormone production in vitro from preantral follicles of crossbred female goats (Saanen × Anglo-Nubian)

The present study aimed to evaluate the survival, growth, antrum formation, oocyte extrusion, and hormone production during in vitro culture of caprine preantral follicles isolated from pure-breed (Saanen) or crossbreed (F1 generation: ½ Saanen + ½ Anglo-Nubian) goats. Secondary follicles (diameter: 150-250 µm) from Saanen or crossbreed (Saanen × Anglo-Nubian) goats were isolated from the ovarian cortex by microdissection and cultured in vitro for 18 days in α-modified minimum essential medium (α-MEM+) supplemented with vascular endothelial growth factor and increasing concentrations of follicle-stimulating hormone. Every six days follicular morphology, growth, antrum formation, and follicular extrusion were evaluated. In addition, on days 2, 6, 12, and 18 of culture the medium samples were collected and stored at -20°C for further measurement of estradiol and progesterone. The follicular survival, antrum formation, and oocyte extrusion were analyzed by the Chi square test. Follicular diameter and hormone assays were compared using the Kruskal-Wallis test. Survival rates, growth, antral follicle formation, and oocyte extrusion of preantral follicles cultured in vitro were similar between the different genetic groups. The production of estradiol and progesterone indicated the maintenance of cell viability throughout the culture. In conclusion, preantral follicles from pure-breed or crossbreed goats can be used with the same efficiency for in vitro culture of isolated caprine preantral follicles.

Survival, growth, and hormone production in vitro from preantral follicles of crossbred female goats (Saanen × Anglo-Nubian)

The present study aimed to evaluate the survival, growth, antrum formation, oocyte extrusion, and hormone production during in vitro culture of caprine preantral follicles isolated from pure-breed (Saanen) or crossbreed (F1 generation: ½ Saanen + ½ Anglo-Nubian) goats. Secondary follicles (diameter: 150-250 µm) from Saanen or crossbreed (Saanen × Anglo-Nubian) goats were isolated from the ovarian cortex by microdissection and cultured in vitro for 18 days in α-modified minimum essential medium (α-MEM+) supplemented with vascular endothelial growth factor and increasing concentrations of follicle-stimulating hormone. Every six days follicular morphology, growth, antrum formation, and follicular extrusion were evaluated. In addition, on days 2, 6, 12, and 18 of culture the medium samples were collected and stored at -20°C for further measurement of estradiol and progesterone. The follicular survival, antrum formation, and oocyte extrusion were analyzed by the Chi square test. Follicular diameter and hormone assays were compared using the Kruskal-Wallis test. Survival rates, growth, antral follicle formation, and oocyte extrusion of preantral follicles cultured in vitro were similar between the different genetic groups. The production of estradiol and progesterone indicated the maintenance of cell viability throughout the culture. In conclusion, preantral follicles from pure-breed or crossbreed goats can be used with the same efficiency for in vitro culture of isolated caprine preantral follicles.(AU)

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.(AU)

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.(AU)

The effects of cyanocobalamin supplementation during the thawing of frozen boar semen on spermatozoa, in vitro fertilization, and embryonic development

The objective of this study was to assess the in vitro fertilization (IVF) of pig oocytes using frozen - thawed boar sperm in media supplemented with cyanocobalamin . Frozen semen pellets were thawed and incubated for 1 h in fe rtilization media containing cyanocobalamin (0, 0.5, 1.0 or 2.0 μ m ) and evaluated for forward progressive motility, viability , and embryo cleavage . Forward progressive motility of the 0.5 and 1 .0 μ m cyanocobalamin supplements was higher (P < 0.05) than the 0 and 2 .0 μ m cyanocobalamin supplements. Membrane v iability of sperm supplemented with 0.5 μ m cyanocobalamin was higher (P < 0.05) than all other groups. Oocytes were matured and fertilized with frozen - thawed boar semen that was previously incubated for 1 h in fertilization media containing cyanocobalamin (0 or 0.5 μ m ; 100 oocytes/treatment). Fertilization characteristics were evaluated 12 h after IVF of oocytes and embryo development was analyzed at 48 h and 144 h post - IVF. There were no significant differences between treatment groups when evaluating fertilization , polyspermic penetration or male pronucleus development. Embryos derived from oocytes fertilized with 0.5 μ m cyanocobalamin supplemented sperm had a higher percentage (P < 0.05) of cleaved embryos compared to those without cyanocobalamin supplementation at 48 h after IVF. There were no significant differences in the percent ages of embryos reaching the blastocyst stage by 144 h afte r IVF between treatment groups. The results of this study suggest that there are positive effects of 0.5 μ m cyanocobalamin supplementation during the incubation of frozen - thawed boar semen on early development of IVF derived pig embryos.

Ovarian follicle reserve: emerging concepts and applications

This paper presents new concepts in the study of folliculogenesis and describes some of the current applications to reproductive biotechnology. The importance of better understanding this issue is addressed both for basic and applied research. After a brief review of the basic conceptions of the origin, formation, and growth of follicules according to stablished concepts, some controversial points, as the postnatal production of the follicles and the role of multioocyte follicles, are discussed. The importance of the ovarian follicular reserve is considered for fertility and reproductive parameters, as well as some questiones about the presence of multioocyte follicles in adult ovaries. Finally, some future prospects are proposed.

The effects of cyanocobalamin supplementation during the thawing of frozen boar semen on spermatozoa, in vitro fertilization, and embryonic development

The objective of this study was to assess the in vitro fertilization (IVF) of pig oocytes using frozen - thawed boar sperm in media supplemented with cyanocobalamin . Frozen semen pellets were thawed and incubated for 1 h in fe rtilization media containing cyanocobalamin (0, 0.5, 1.0 or 2.0 μ m ) and evaluated for forward progressive motility, viability , and embryo cleavage . Forward progressive motility of the 0.5 and 1 .0 μ m cyanocobalamin supplements was higher (P < 0.05) than the 0 and 2 .0 μ m cyanocobalamin supplements. Membrane v iability of sperm supplemented with 0.5 μ m cyanocobalamin was higher (P < 0.05) than all other groups. Oocytes were matured and fertilized with frozen - thawed boar semen that was previously incubated for 1 h in fertilization media containing cyanocobalamin (0 or 0.5 μ m ; 100 oocytes/treatment). Fertilization characteristics were evaluated 12 h after IVF of oocytes and embryo development was analyzed at 48 h and 144 h post - IVF. There were no significant differences between treatment groups when evaluating fertilization , polyspermic penetration or male pronucleus development. Embryos derived from oocytes fertilized with 0.5 μ m cyanocobalamin supplemented sperm had a higher percentage (P < 0.05) of cleaved embryos compared to those without cyanocobalamin supplementation at 48 h after IVF. There were no significant differences in the percent ages of embryos reaching the blastocyst stage by 144 h afte r IVF between treatment groups. The results of this study suggest that there are positive effects of 0.5 μ m cyanocobalamin supplementation during the incubation of frozen - thawed boar semen on early development of IVF derived pig embryos.(AU)

Ovarian follicle reserve: emerging concepts and applications

This paper presents new concepts in the study of folliculogenesis and describes some of the current applications to reproductive biotechnology. The importance of better understanding this issue is addressed both for basic and applied research. After a brief review of the basic conceptions of the origin, formation, and growth of follicules according to stablished concepts, some controversial points, as the postnatal production of the follicles and the role of multioocyte follicles, are discussed. The importance of the ovarian follicular reserve is considered for fertility and reproductive parameters, as well as some questiones about the presence of multioocyte follicles in adult ovaries. Finally, some future prospects are proposed.(AU)

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.(AU)

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.(AU)

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.(AU)

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.(AU)

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.(AU)

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.(AU)

Padrão de metilação dos genes xist e igf2 em ovocitos de folículos antrais de vacas nelore

Modificações epigenéticas são eventos que controlam a expressão diferencial de genes, sendo a metilação do DNA um dos mais conhecidos, importante para a reprogramação epigenética durante a gametogênese. Entender como isso ocorre na ovogênese é importante para criar parâmetros para a competência ovocitária com o intuito de melhorar a produção in vitro de embriões. Nesse trabalho objetivou-se avaliar o padrão de metilação em ovócitos de duas categorias de folículos antrais: entre 1 e 3 mm e maiores que 6 mm. Para isso foram escolhidas duas ilhas CpGs, uma no éxon 1 do gene XIST – que está relacionado com a inativação do cromossomo X em fêmeas, e outra no último éxon do gene IGF2 – envolvido no desenvolvimento embrionário e placentação. Os ovócitos foram obtidos a partir de ovários de vacas Nelore provenientes de abatedouros usando o tissue chopper e sucessivas pipetagens para o isolamento dos ovócitos. A classificação dos ovócitos foi realizada de acordo com o seu diâmetro. O DNA extraído dos ovócitos foi tratado com bissulfito de sódio e os genes de interesse foram amplificados através de PCR nested. Os produtos purificados de gel de agarose 2% foram utilizados clonagem em células DH5α. Os clones foram sequenciados usando a metodologia dideoxi e analisados no programa BiQ Analyzer utilizando sequências controle do “GenBank”. Apenas sequências com mais de 90% de identidade e de conversão pelo bissulfito de sódio foram consideradas para análises. Os resultados mostraram que os ovócitos de folículos entre 1 e 3 mm e maiores que 6 mm possuem padrão de metilação iguais, tanto para o gene XIST (89,5% e 92,7%, respectivamente) quanto para o gene IGF2 (73,4% e 61,5%, respectivamente). Esses dados sugerem que, pelo menos para as regiões estudadas, o padrão de metilação já está estabelecido a partir da fase antral do folículo ovariano em ovócitos imaturos.

From cattle to sheep: a view of the difficulties and success of commercial in vitro production of sheep embryos

The in vitro production (IVP) of sheep embryos, unlike that for cattle, requires hormonal treatment with progesterone or a progestagen to improve efficiency of ovum pick - up (OPU). Another difference is that the process used to obtain oocytes is by OPU in cattle and LOPU (laparoscopic ovum pick - up) in sheep. Several factors affect oocyte recovery, including the skill of the technician, suction pressure and the aspiration system for LOPU. The LOPU technique is less invasive and can be p erformed weekly, biweekly or monthly in ewes. LOPU is an option to MOET (multiple ovulation and embryo transfer), mainly for animals of high genetic value that do not respond to the superovulatory treatment or only produce unfertilized oocytes. The embryos in the initial development stages can be transferred into the oviduct or into the cranial portion of lumen of the uterine horn, reducing the time for in vitro culture, which can be harmful to embryos. Although early results are promising, there are still many points to be improved in IVP of sheep embryos before it is used extensively in the commercial sheep industry.

Oocyte maturation in bitches

The canine species has been used as an experimental model for preservation of endangered species. Biotechnologies of reproduction, such as in vitro maturation (IVM), have been used to meet this objective. Several protocols for in vitro embryo production (IVEP) in swine and bovine species have been adapted for canids. Ho wever, the highest rate reported for in vitro maturation in canids is only 39%, which is still lower than those in other species. Therefore, current research on assisted reproduction in canids have focused on several IVM protocols, including the addition of proteins, hormones, meiosis inhibitors, growth factors and antioxidants to the maturation media and the determination of suitable timing for culture, so that variables involved in the process can be fine-tuned. This review has the main objective of describing major developments and limitations in the process of oocyte maturation in bitches.

Paracrine and autocrine factors in the differentiation of the cumulus-oocyte complex

A better understanding of the paracrine and autocrine regulatory loops within the cumulus-oocyte complex (COC) is fundamental for the improvement of in vitro maturation (IVM) outcomes in humans and domestic species. This review presents the most important local regulators identified in the COC to date with special attention to th ose secreted by the oocyte and acting on cumulus cells, as well as their roles in different processes crucial for the successful maturation of the COC. An autocrine re gulatory loop mediated by epidermal growth factor-lik e (EGF-like) peptides in cumulus cells triggers COC maturation. During COC differentiation, oocyte s ecreted factors (OSFs), particularly members of the transforming growth factor-  (TGF  ) and fibroblast growth factor (FGF) families, regulate meiotic resumption, cumulus expansion, cumulus metabolism, apoptosis and steroidogenesis.

Maternal metabolic health and oocyte quality: the role of the intrafollicular environment

Reduced oocyte and embryo quality are currently recognized as key factors in the problem of disappointing fertility in high producing dairy cows, but also in women undergoing assisted reproductive treatment. This review aims to highlight the importance of intrafollicular conditions in the subfertility problem, topical in both bovine and human research. Metabolic disturbances, like a negative energy balance (NEB) early postpartum in the bovine or obesity and type II diabetes in women, are associated with ovarian dysfunction. Changes in the growth pattern of the ovarian follicle, due to suboptimal metabolic conditions, can indirectly affect oocyte quality. Furthermore, maternal metabolic disorders (nutritionally induced, linked with NEB or caused by for example obesity) may alter the endocrine and biochemical composition of the follicular fluid, the microenvironment of the growing and maturing female gamete. The maturing oocyte is very sensitive to any perturbation in its direct environment and in vitro maturation models revealed that some of these metabolic changes have the potential to reduce the oocyte’s developmental co mpetence. Also embryo quality is significantly reduced due to maturation in adverse conditions. Well-balanced and timed oocyte metabolism and gene expression are crucial to safeguard an optimal oocyte development. In that perspective metabolic and transcriptomic parameters of the oocyte may serve to predict reproductive success rates. Importantly, there is also gr owing evidence that adverse conditions for oocyte growth and maturation may jeopardize the metabolism and health of the offspring.