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

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

Generation of developmentally competent oocytes and fertile mice from parthenogenetic embryonic stem cells

Parthenogenetic embryos, created by activation and diploidization of oocytes, arrest at mid-gestation for defective paternal imprints, which impair placental development. Also, viable offspring has not been obtained without genetic manipulation from parthenogenetic embryonic stem cells (pESCs) derived from parthenogenetic embryos, presumably attributable to their aberrant imprinting. We show that an unlimited number of oocytes can be derived from pESCs and produce healthy offspring. Moreover, normal expression of imprinted genes is found in the germ cells and the mice. pESCs exhibited imprinting consistent with exclusively maternal lineage, and higher X-chromosome activation compared to female ESCs derived from the same mouse genetic background. pESCs differentiated into primordial germ cell-like cells (PGCLCs) and formed oocytes following in vivo transplantation into kidney capsule that produced fertile pups and reconstituted ovarian endocrine function. The transcriptome and methylation of imprinted and X-linked genes in pESC-PGCLCs closely resembled those of in vivo produced PGCs, consistent with efficient reprogramming of methylation and genomic imprinting. These results demonstrate that amplification of germ cells through parthenogenesis faithfully maintains maternal imprinting, offering a promising route for deriving functional oocytes and having potential in rebuilding ovarian endocrine function.

Insights into epigenetic patterns in mammalian early embryos

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

Involvement of K+ATP and Ca2+ channels in hydrogen sulfide-suppressed ageing of porcine oocytes

BACKGROUND: Hydrogen sulfide has been shown to improve the quality of oocytes destined for in vitro fertilization. Although hydrogen sulfide is capable of modulating ion channel activity in somatic cells, the role of hydrogen sulfide in gametes and embryos remains unknown. Our observations confirmed the hypothesis that the KATP and L-type Ca2+ ion channels play roles in porcine oocyte ageing and revealed a plausible contribution of hydrogen sulfide to the modulation of ion channel activity. RESULTS: We confirmed the benefits of the activation and suppression of the KATP and L-type Ca2+ ion channels, respectively, for the preservation of oocyte quality. CONCLUSIONS: Our experiments identified hydrogen sulfide as promoting the desired ion channel activity, with the capacity to protect porcine oocytes against cell death. Further experiments are needed to determine the exact mechanism of hydrogen sulfide in gametes and embryos.

Missing and overexpressing proteins in domestic cat oocytes following vitrification and in vitro maturation as revealed by proteomic analysis

BACKGROUND: The domestic cat serves as an animal model for assisted reproductive studies of endangered felid species. To date, there are no data on the protein alterations following cryopreservation of oocytes in felid family. METHODS: Immature (germinal vesicle) domestic cat oocytes were vitrified in the vitrification solution containing 35% ethylene glycol, 20% DMSO and 0.5 mM sucrose. The vitrified-warmed oocytes were matured (metaphase II) in vitro and subjected to proteomic analysis using 1DE SDS-PAGE prefractionation combined with LC-MS/MS. RESULTS: A total of 1712 proteins were identified in in vitro matured oocytes. Of the 1712 proteins, 1454 proteins were found in both groups, whereas, 258 proteins were differentially expressed between control and vitrified-warmed groups. In vitrified-warmed oocytes, the missing proteins were membrane and nuclear proteins; whereas, apoptosis and DNA repair proteins were overrepresented. CONCLUSIONS: The identified missing and overexpressed proteins in vitrified-warmed oocytes represent potential markers of cryoinjuries and the developmental pathways of oocytes. The findings of differential expressed proteins may contribute to effective ways of proteome analysis of oocyte/embryo quality in order to assess safety of cryopreservation in felid species.

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.

Growth differentiation factor 9 and bone morphogenetic protein 15 expression in previtellogenic oocytes and during early embryonic development of Yellow-tail Kingfish Seriola lalandi

BACKGROUND: During fish oocyte maturation, specific molecules are expressed and accumulated within oocyte until fertilization and embryo development. Special attention have been paid in members of the transforming growth factor (TGF-ß) superfamily; growth differentiation factor 9 (GDF9/gdf9) and bone morphogenetic protein 15 (BMP15/bmp15), which exert regulatory functions during oocyte maturation and follicle development. However, little attention has been paid to the involvement of these molecules during embryogenesis considering its importance for the formation of a good quality egg and subsequent embryo survival. The purpose of this study was to analyze the expression of gdf9 andbmp15 in previtellogenic oocytes and during early embryonic development in Seriola lalandi, a pelagic fish with increasing prospect for its aquaculture development, which however, show high mortality at embryo and larval stages. RESULTS: Through RT-qPCR it was found that gdf9 expression was higher in previtellogenic oocytes decreasing after ovulation. This expression profile agrees with its participation in early stages of the follicular development. The transcripts for bmp15 also showed the highest levels in previtellogenic oocytes, however this expression was lower than obtained with gdf9. Conversely, in recently spawned oocytes mRNA bmp15 levels were highest than observed to gdf9. This, is consequent with the main role proposed for this growth factor at the final fish oocyte maturation: avoid the ovulation of an immature oocyte. During embryo development, low levels of mRNA were detected to gdf9, with an increase in 48 H post-fertilization embryos. The bmp15 expression did not change throughout development and was higher than gdf9 at 16 cells, blastula and appearance embryos stages. CONCLUSIONS: Both (gdf9 and bmp15) expression profiles in previtellogenic oocytes and newly spawned eggs are consistent with the described functions for these growth factors in vertebrate ovarian physiology in early and late stages of the follicular development. So, these genes could be considered as quality biomarkers at these stages. However, further studies of these proteins throughout folliculogenesis, are necessaries to fully understand their functions during the oocyte formation. In addition, the persistent expression of these growth factors during development, allows us to speculate possible roles in embryonic processes, which must also be addressed.

A simplified one-step nuclear transfer procedure alters the gene expression patterns and developmental potential of cloned porcine embryos

Various somatic cell nuclear transfer (SCNT) techniques for mammalian species have been developed to adjust species-specific procedures to oocyte-associated differences among species. Species-specific SCNT protocols may result in different expression levels of developmentally important genes that may affect embryonic development and pregnancy. In the present study, porcine oocytes were treated with demecolcine that facilitated enucleation with protruding genetic material. Enucleation and donor cell injection were performed either simultaneously with a single pipette (simplified one-step SCNT; SONT) or separately with different pipettes (conventional two-step SCNT; CTNT) as the control procedure. After blastocysts from both groups were cultured in vitro, the expression levels of developmentally important genes (OCT4, NANOG, EOMES, CDX2, GLUT-1, PolyA, and HSP70) were analyzed by real-time quantitative polymerase chain reaction. Both the developmental rate according to blastocyst stage as well as the expression levels CDX2, EOMES, and HSP70 were elevated with SONT compared to CTNT. The genes with elevated expression are known to influence trophectoderm formation and heat stress-induced arrest. These results showed that our SONT technique improved the development of SCNT porcine embryos, and increased the expression of genes that are important for placental formation and stress-induced arrest.

Interaction of lipophorin with Rhodnius prolixus oocytes: biochemical properties and the importance of blood feeding

Lipophorin (Lp) is the main haemolymphatic lipoprotein in insects and transports lipids between different organs. In adult females, lipophorin delivers lipids to growing oocytes. In this study, the interaction of this lipoprotein with the ovaries of Rhodnius prolixus was characterised using an oocyte membrane preparation and purified radiolabelled Lp (125I-Lp). Lp-specific binding to the oocyte membrane reached equilibrium after 40-60 min and when 125I-Lp was incubated with increasing amounts of membrane protein, corresponding increases in Lp binding were observed. The specific binding of Lp to the membrane preparation was a saturable process, with a Kdof 7.1 ± 0.9 x 10-8M and a maximal binding capacity of 430 ± 40 ng 125I-Lp/µg of membrane protein. The binding was calcium independent and pH sensitive, reaching its maximum at pH 5.2-5.7. Suramin inhibited the binding interaction between Lp and the oocyte membranes, which was completely abolished at 0.5 mM suramin. The oocyte membrane preparation from R. prolixus also showed binding to Lp from Manduca sexta. When Lp was fluorescently labelled and injected into vitellogenic females, the level of Lp-oocyte binding was much higher in females that were fed whole blood than in those fed blood plasma.

Oocyte aspiration and in vitro embryo production in Jersey cows with selenium-supplemented diet / Aspiração de oócitos e produção de embriões in vitro de vacas Jersey suplementadas com selênio na dieta

The effects of selenium (Se) in Jersey cows' diet on the aspiration of oocytes and production of embryos in vitro were studied. Groups with five Jersey cows received 3.2mg or 9.6mg Se daily, provided in the feed concentrate. Six follicular aspirations were carried out every 15 days, using only the last 5. The oocytes were classified, and standard procedures were carried out for maturation, fertilization and cultivation. The total number of oocytes (35.11±2.65 vs 23.10±2.16) and degree 1 oocytes (11.61±2.65 vs 4.75±0.97) were higher in the group that received 9.6mg Se and the quantity of naked oocytes (3.23±0.87 vs 6.22±1.18) was lower in this group. The aspirated oocytes from the cows treated with 9.6mg Se/day resulted in higher (P<0.05) embryo production 21.98±2.37 vs 13.12±1.59). No difference was observed in serum Se concentration between the two groups. It is recommended that the daily diet be supplemented with 100g mineral salt containing 9.6mg Se, since this rate rendered a larger production of oocytes, higher quantity of degree 1 oocytes and greater production of embryos in the process of in vitro fertilization.

Avaliou-se o efeito do selênio (Se) adicional na dieta de vacas Jersey na aspiração de oócitos e produção de embriões in vitro. Dez vacas Jerseys receberam 3,2mg de Se por dia ou 9,6mg, vinculado ao concentrado. Realizaram-se seis aspirações foliculares, com intervalo médio de 15 dias, aproveitando as cinco últimas. Os oócitos foram classificados e realizaram-se os procedimentos padrões de maturação, fertilização e cultivo in vitro. O total de oócitos, 35,11±2,65 vs 23,10±2,16, e oócitos de qualidade 1, 11,61±1,58 vs 4,75±0,97, foram mais elevados no grupo que recebeu 9,6mg de Se e a quantidade de oócitos desnudos mais baixa, 3,21±0,87 vs 6,22±1,18. A produção de embriões foi maior no grupo tratado com 9,6mg de Se/vaca/dia, 21,98±2,37 vs 13,12±1,59. Não se observou diferença na concentração de Se no soro entre os dois grupos. Conclui-se que é possível recomendar o fornecimento de 100g de sal mineral, contendo 9,6mg de Se, adicionado à dieta, pois resultou em maior produção de oócitos, maior quantidade de oócitos de grau 1 e maior produção de embriões no processo de fecundação in vitro.

In vitro oocyte transport through the oviduct of buffalo and crossbred beef cows / Transporte de oócitos in vitro através do oviduto de búfalas e vacas de corte cruzadas

The present study was conducted to verify if the elevation of plasma concentrations of estradiol during superovulatory treatments affects the oocyte transport in buffalo females, as well as if the inferior quality of buffalo oocytes and/or some functional difference on the oviduct of these animals is responsible for the low embryo recovery rate in superovulated buffaloes when compared to cows subjected to the same treatment. Oviducts of 10 buffaloes and 15 of cows, treated to induce a single ovulation were used. The oviducts were placed on Petri dishes and received the following treatments: 5 buffalo oocytes with no E2 (G-BufBuf and G-BovBuf), 5 bovine oocytes with no E2 (G-BufBov and G-BovBov), 5 buffalo oocytes with E2 (G-BufE2Buf and G-BovE2Buf) and 5 bovine oocytes with E2 (G-BufE2Bov and G-BovE2Bov; factorial 2x2x2). Oocytes were incubated for 24h. Subsequently, oviducts were washed and oocytes were recovered and counted. Since no interactions were found between E2 treatment, oviducts and oocytes species, main effects were analyzed separately. Recovery rate and number of oocytes was higher on cattle compared to buffaloes (35.0+8.6% and 1.4+0.3 vs. 10.0±4.6% and 0.5±0.2, respectively; p<0.05); no effect of E2 treatment was observed on recovery rate and number of oocytes (29.8±9.0% and 1.3±0.4 vs. 16.9±6.1% and 0.7±0.2, respectively; p>0.05); the number of buffaloes and bovine oocytes recovered were similar (1.4±0.4 and 0.6±0.2, respectively; p>0.05). Oocytes recovery rate showed a trend (P=0.07) to be higher when buffalo oocytes were implanted when compared to bovine oocytes (35.2±9.2% vs. 12.9±5.4%). Present results suggest that oocyte transport by the oviduct of buffaloes and bovine was not dependent on oocytes species or E2 supplementation to the culture medium.

O presente estudo foi realizado para verificar se a elevação das concentrações plasmáticas de estradiol durante os tratamentos superovulatórios afeta o transporte dos oócitos em fêmeas bubalinas, bem como se a qualidade inferior dos oócitos de búfalos e/ou alguma diferença funcional no oviduto destes animais é responsável pela baixa taxa de recuperação de embriões em búfalas superovuladas quando comparadas a vacas submetidas ao mesmo tratamento. Foram utilizados 10 ovidutos de búfalas e 15 de vacas, tratadas para a indução de ovulação única. Os ovidutos foram colocados em placas de Petri e receberam os seguintes tratamentos: sem E2 e inseridos com 5 oócitos de búfalas (G-BufBuf e G-BovBuf); sem E2 e com 5 oócitos de vacas (G-BufBov e G-BovBov); com E2 e com 5 oócitos de búfalas (G-BufE2Buf e G-BovE2Buf); e com E2 e com 5 oócitos de vacas (G-BufE2Bov e G-BovE2Bov; fatorial 2x2x2). Posteriormente, foram incubados por 24h e, após esse período, foram lavados para a recuperação e contagem dos oócitos. Como não foi verificado efeito de interação, foram analisados os efeitos principais. O número e a taxa de recuperação de oócitos foi maior em ovidutos de vacas que de búfalas (1,4±0,3/35,0±8,6% vs. 0,5±0,2/10,0±4,6%; P<0,05). Foi verificado que o tratamento com ou sem E2 não interferiu no número e na taxa de recuperação de oócitos (1,3±0,4/29,8±9,0% vs. 0,7±0,2/16,9±6,1%; P>0,05). Não foi verificada diferença no número de oócitos de búfalas ou de vacas recuperados (1,4±0,4 e 0,6±0,2; P>0,05). Observou-se também que houve tendência (P=0,07) de maior taxa de recuperação de oócitos de búfalas que de vacas (35,2±9,2% vs. 12,9±5,4%). Os dados são indicativos de que o transporte de oócitos pelo oviduto de búfalas e de vacas independe da espécie do oócito e não é influenciado pelo E2.

Characterization of the cGMP-dependent protein kinase SmcGK1 of Schistosoma mansoni

Schistosomes are trematode parasites and of worldwide medical importance for humans and animals. Growth and development of these parasites require a specific host environment, but also permanent communication processes between the two genders. Accumulating molecular evidence indicates that the responsible interactions are mediated by signal transduction processes. Conserved signaling molecules were identified, and first approaches made for their characterization. However, no representative of the conserved family of cGMP-dependent protein kinases (cGKs) has been described in this parasite yet. Within the Schistosoma mansoni genome data-set we identified cGK homologs, of which one was investigated in more detail in this study. We present the cloning of SmcGK1, whose sequence shows homology to cGKs of higher eukaryotes. SmcGK1 was found to be gender-independently transcribed in adult schistosomes. The occurrence of SmcGK1 sense and antisense transcripts suggests that the expression of this gene is controlled at the post-transcriptional level. In situ hybridization experiments demonstrated a gonad-preferential expression profile in both genders indicating a role of SmcGK1, at least during sexual development of schistosomes. Using a cGK-specific inhibitor to treat adult schistosomes in vitro finally resulted in a multifaceted phenotype including slow motion, oocyte congestion, and reduced egg production.

Esquistossomos são parasitas trematodos de importância médica em todo o mundo para o homem e os animais. O crescimento e o desenvolvimento destes parasitas requerem um ambiente específico do hospedeiro, mas também um processo de comunicação permanente entre parasitas dos dois sexos. Evidência molecular tem se acumulado e indica que as interações são mediadas por processos de transdução de sinal. Moléculas sinalizadoras conservadas foram identificadas, e as primeiras abordagens têm sido feitas para sua caracterização. Contudo, não foi ainda descrito nenhum representante da família conservada das proteína-quinases dependentes de cGMP (cGKs) neste parasita. Analisando o genoma do Schistosoma mansoni nós identificamos homólogos de cGK, dos quais um foi investigado em mais detalhe no presente estudo. Aqui apresentamos a clonagem do gene SmcGK1, cuja sequência mostra homologia com cGKs de eucariotos superiores. Smc- GK1 foi detectada como sendo transcrita de forma gêneroindependente em esquistossomos adultos. A ocorrência de transcritos de SmcGK1 senso e antisenso sugere que a expressão deste gene é controlada em nível pos-transcricional. Experimentos de hibridização in situ demonstraram uma expressão preferencial nas gônadas em ambos os gêneros, indicando um papel para SmcGK1, pelo menos durante o desenvolvimento de esquistossomos. Usando um inibidor específico de cGK para tratamento de esquistossomos adultos in vitro finalmente resultou em um fenótipo multifacetado, incluindo movimentos lentos, congestão dos oócitos, e redução da produção de ovos.

Bovine parthenogenotes produced by inhibition of first or second polar bodies emission

Parthenogenetic embryos are an ethically acceptable alternative for the derivation of human embryonic stem cells. In this work, we propose a new strategy to produce bovine parthenogenetic embryos inhibiting the emission of the first polar body during in vitro maturation, and allowing the extrusion of the second polar body during oocyte activation. Cytochalasin B, an inhibitor of actin microfilaments, was employed during in vitro maturation to inhibit first polar body emission or during parthenogenetic activation to block second polar body emission. Only one polar body was inhibited in each strategy in order to keep the diploid chromosome set. In experiment 1, the effect of cytochalasin B on in vitro maturation of bovine oocytes was evaluated. Most oocytes (77%) were arrested at a meiotic stage characterized by the presence of a large internal metaphase plate and absence of polar body. In experiment 2, development of embryos exposed to cytochalasin B during in vitro maturation (CytoB-IVM) or during activation (CytoB-ACT) was compared. Developmental rates did not differ between diploidization strategies, even when three agents were employed to induce activation. Both groups, CytoB-IVM and CytoB-ACT, tended to maintain diploidy. CytoB-IVM parthenogenesis could help to obtain embryos with a higher degree of homology to the oocyte donor.

Influência da interação entre o oócito e as células da granulosa nos resultados dos procedimentos de reprodução assistida / The influence of interaction between oocyte and granulosa cells on the results of procedures in assisted reproduction

A interação oócito-células da granulosa in vivo e sua influência na qualidade oocitária e embrionária tem sido alvo de inúmeros estudos, mas muitas questões ainda necessitam ser esclarecidas. O objetivo deste trabalho foi revisar a importância dessa comunicação, estabelecendo uma relação com a questão da maturação in vitro de oócitos imaturos humanos aplicando esses conhecimentos para definir possíveis marcadores moleculares que poderiam melhorar a seleção de oócitos e, consequentemente, selecionar embriões de boa qualidade para posterior transferência e sucesso de gravidez de pacientes submetidas ao tratamento da infertilidade conjugal. As células da granulosa têm um importante papel na maturação oocitária in vitro e os benefícios da presença dessas células durante essa etapa podem ser atribuídos à formação de um microambiente favorável (bioquímico e metabólico) ao redor do oócito. Foram identificados nesta revisão vários marcadores em potencial nas células do cumulus de oócitos competentes, incluindo vários genes que poderiam ser usados como preditores da competência oocitária, o que pode contribuir para a formulação de critérios mais objetivos e confiáveis para a seleção de oócitos e embriões, e consequente aprimoramento e otimização das técnicas em reprodução humana assistida que são aplicadas nos procedimentos clínicos atuais de fertilização in vitro.

The interaction of oocyte-granulosa cells in vivo and in vitro and its influence on oocyte and embryo quality has been the subject of numerous studies, but many issues still need to be clarified. The objective of this study was to promote a review about the importance of this communication establishing a connection with the issue of in vitro maturation of immature human oocytes by applying this knowledge to define potential molecular markers that could improve the selection of oocytes and consequently select good quality embryos for later transfer and success of pregnancy in patients undergoing treatment of infertility. The granulosa cells also have an important role in oocyte maturation in vitro and the venefits from the presence of these cells during this process can be atributed to the formation of a favorable micro-environment (biochemical and metabolic) around the oocyte. In this review, we identified several potential markers in the cumulus cells of competent oocytes, including several genes that could be used as predictors of oocyte competence, which contributes for more objective and reliable criteria for the selection of oocytes and embryos, thus improving and optimizing techniques in assisted human reproduction that are applied in current clinical in vitro fertilization.

Selección de ovocitos competentes, utilizando el factor de crecimiento nervioso (NFG) y el factor de crecimiento vascular endotelial (VEGF), con el fin de aumentar las posibilidades de embarazo en pacientes que se encuentran en tratamientos de fecundación in vitro: [revisión] / Competent oocytes selection, using nerve growth factor (NGF) and vascular endothelial growth factors (VEGF), with the purpose of to increase the possibilities of pregnancy in patients who are in treatments of fertilization in vitro: [review]

The attention from embryologist toward the assessment of oocyte quality in human in vitro fertilization (IVF) is increasing every day. Oocyte selection and the identification of the best oocytes, in fact, would help to limit embryo overproduction and to improve the results of oocyte cryostorage programs. Multiple methods have been proposed; but a good correlation between specific biochemical characteristics and measurable oocyte quality-linked, embryo-related variables has not been established to date. In IVF programs that include oocyte selection, levels of vascular endothelial growth factor (VEGF) and neural growth factor (NGF) could be used as an index to exclude oocytes that developed in a hypoxic follicle. The presence of both ovarian angiogenic factors provides an ideal environment to maintain the cyclical changes in vascular density that occurs during follicular development. NGF would act within a short time-frame to replenish the supply of VEGF required for the vascularization of growing preovulatory follicles and perhaps newly formed corpora lutea. Levels of these angiogenic factors can predict the necessary vascular changes within the follicles and the competence of the oocytes. A hypoxic status at this level could be responsible for the high intrafollicular and plasma concentrations of VEGF and for the low quality of the oocytes.

Purification, partial characterization and role in lipid transport to developing oocytes of a novel lipophorin from the cowpea weevil, Callosobruchus maculatus

Lipid transport in arthropods is achieved by highly specialized lipoproteins, which resemble those described in vertebrate blood. Here we describe purification and characterization of the lipid-apolipoprotein complex, lipophorin (Lp), from adults and larvae of the cowpea weevil Callosobruchus maculatus. We also describe the Lp-mediated lipid transfer to developing oocytes. Lps were isolated from homogenates of C. maculatus larvae and adults by potassio bromide gradient and characterized with respect to physicochemical properties and lipid content. The weevil Lp (465 kDa) and larval Lp (585 kDa), with hydrated densities of 1.22 and 1.14 g/mL, contained 34 and 56 percent lipids and 9 and 7 percent carbohydrates, respectively. In both Lps, mannose was the predominant monosaccharide detected by paper chromatography. SDS-PAGE revealed two apolipoproteins in each Lp with molecular masses of 225 kDa (apolipoprotein-I) and 79 kDa (apolipoprotein-II). The lipids were extracted and analyzed by thin-layer chromatography. The major phospholipids found were phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine in adult Lp, and phosphatidylcholine, phosphatidylethanolamine and sphingomyelin in larval Lp. Hydrocarbons, fatty acids and triacylglycerol were the major neutral lipids found in both Lps. Lps labeled in the protein moiety with radioactive iodine (125I-iodine) or in the lipid moiety with fluorescent lipids revealed direct evidence of endocytic uptake of Lps in live oocytes of C. maculatus.

Relationship between pronuclear scoring and embryo quality and implantation potential in IVF-ET / 华中科技大学学报（医学）（英德文版）

To assess the relationship between pronuclear scoring and day-3 embryo quality and pregnancy outcome and to determine the clinical value of pronuclear stage scoring system in human in vitro fertilization-embryo transfer (IVF-ET) program, a pronuclear scoring system was used to score zygotes 16-20 h after insemination during conventional IVF or intracytoplasmic sperm injection (ICSI). The embryos were classified into groups Z1, Z2, Z3 and Z4. Comparisons were made of the rates of arrested embryos and excellent embryos on day 3. Comparisons of pregnancy outcome were made only in those patients in whom cohorts of similarly Z-scored embryos were transferred. The results showed that there were less arrested embryos and more excellent embryos on day 3 in groups Z1 and Z2 than those in group Z3 and Z4. More embryos arrested and less excellent embryos developed in group Z4 than group Z3. The clinical pregnancy rates resulting from the transfer of single pronuclear score homologous embryo types were similar among groups Z1, Z2 and Z3. Implantation rates of group Z1 were higher (P<0.05) than that of group Z3. These findings suggests that pronuclear scoring can predict developmental ability on day 3 and implantation potential. A evaluation that combines the Z-score and day 3 embryo morphology is useful in the determination of the most viable embryos and the number of embryos for transfer.

Co-localization and interaction of human organic anion transporter 4 with caveolin-1 in primary cultured human placental trophoblasts

The human organic anion transporter 4 (hOAT4) has been identified as the fourth isoform of OAT family. hOAT4 contributes to move several negatively charged organic compounds between cells and their extracellular milieu. The functional characteristics and regulatory mechanisms of hOAT4 remain to be elucidated. It is well known that caveolin plays a role in modulating proteins having some biological functions. To address this issue, we investigated the co-localization and interaction between hOAT4 and caveolin-1. hOAT4 and caveolin-1 (mRNA and protein expression) were observed in cultured human placental trophoblasts isolated from placenta. The confocal microscopy of immuno-cytochemistry using primary cultured human trophoblasts showed hOAT4 and caveolin-1 were co-localized at the plasma membrane of the cell. This finding was confirmed by Western blot analysis using isolated caveolae-enriched membrane fractions and immune-precipitates from the trophoblasts. When synthesized cRNA of hOAT4 along with scrambled- or antisense-oligodeoxynucleotide (ODN) of Xenopus caveolin-1 were co-injected to Xenopus oocytes, the [3H]estrone sulfate uptake was significantly decreased by the co-injection of antisense ODN but not by scrambled ODN. These findings suggest that hOAT4 and caveolin-1 share a cellular expression in the plasma membrane and caveolin-1 up-regulates the organic anionic compound uptake by hOAT4 under the normal physiological condition.

Cloning and analysis of IFRG (interferon responsive gene) in rabbit oocytes and preimplantation embryos

Although there is more evidence that shows that IFNs (interferons) plays a very important role in the early development of the embryo, the mechanism of IFNs is still unclear. Our study showed that IFRG is expressed from oocytes- through to the preimplantation embryo in rabbits. This finding provides some clues for better understanding the role of IFNs in the development of the embryo. The full length of rabbit IFRG cDNA (Accession No. AJ584672), with a 2794bp encoding 131 amino acid sequence, was cloned IFRG expression can be detected in 8 different tissues: ovary, heart, lung, liver, kidney, spleen, cerebra, and the 18-day whole-body embryo. Whole-mount in situ hybridization showed that IFRG was highly expressed in the inner-cell mass of rabbit blastula. IFRG may play an important role in embryo development and tissue differentiation.

Oocyte activation and preimplantation development of bovine embryos obtained by specific inhibition of cyclin-dependent kinases

The efficiency of bohemine and roscovitine in combination with ionomycin on parthenogenetic activation and initial embryonic development of bovine oocytes was studied. Two experiments were performed: in the first, different concentrations (0, 50, 75 or 100æM) and different exposure periods (2, 4 or 6 hours) to bohemine or roscovitine were tested for activation rates of in vitro matured (IVM) bovine oocytes, which were pre-exposed to ionomycin. The best treatments, 75µM bohemine and 50µM roscovitine, both for 6h, were used in the second experiment, in which IVM bovine oocytes were exposed to ionomycin, followed or not by bohemine or roscovitine treatment, and evaluated for nuclear status, activation rate and blastocyst development were assessed. The combined treatments (ionomycin + cyclin-dependent kinases inhibitors - CDKIs) showed better results for activation rates (77.3 percent) and initial embryonic development (35.2 percent) than the single ionomycin treatment (69.4 percent for activation and 21.9 percent for development); and also lead to a more uniform activation (nearly 90 percent single pronucleus development). The results showed that CDKIs improve the effects of ionomycin on parthenogenetic activation and blastocyst development in bovine oocytes and could help to achieve more efficient activation protocols, increasing the developmental competence of embryos obtained by reproductive biotechniques.

Realizaram-se dois experimentos para avaliar a eficiência da bohemina e roscovitina associadas à ionomicina para ativação partenogenética e desenvolvimento embrionário inicial de bovinos. No primeiro, foram testadas diferentes concentrações (0, 50, 75 ou 100æM) e diferentes tempos de exposição (2, 4 ou 6 horas) à bohemina ou à roscovitina na ativação de oócitos bovinos maturados in vitro (MIV) pré-expostos à ionomicina. Os melhores tratamentos, bohemina 75µM e roscovitina 50µM, ambos por seis horas, foram utilizados no segundo experimento, no qual oócitos bovinos MIV foram expostos à ionomicina seguido ou não pelo tratamento com inibidores específicos das quinases dependentes de ciclina (CDKI), e avaliados quanto à configuração nuclear, taxa de ativação e desenvolvimento até blastocisto. Os tratamentos combinados (ionomicina+CDKI) apresentaram melhor taxa de ativação (77,3 por cento) e desenvolvimento embrionário inicial (35,2 por cento) do que a ionomicina sozinha (69,4 por cento e 21,9 por cento, respectivamente), e também promoveram ativação mais uniforme (aproximadamente 90 por cento de formação de um pronúcleo). Estes resultados demonstram que os CDKIs potencializam o efeito da ionomicina na ativação e desenvolvimento embrionário inicial e podem auxiliar na obtenção de protocolos de ativação mais eficientes, aumentando a capacidade de desenvolvimento de embriões produzidos por meio de biotécnicas reprodutivas.