Maturação nuclear de ovócitos de cadelas em estro e anestro submetidos à maturação in vitro

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Influência da sazonalidade e da condição ovariana sobre a produção embrionária in vitro de gato doméstico (Felis catus, Linnaeus. 1758)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeitos de diferentes inibidores da meiose sobre a maturação in vitro de oócitos bovinos e subsequente tolerância dos embriões a vitrificação

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Oogenesis in Laetacara araguaiae (Ottoni and Costa, 2009) (Labriformes: Cichlidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influencia das Fases de Anestro e Diestro sobre a Configuração da Cromatina em Vesícula Germinativa do Oócitos Caninos.

Fundação de Apoio à Pesquisa do Estado de São Paulo (FAPESP)

Efeito do fator de crescimento epidermal (EGF) na maturação in vitro de oócitos caninos

Fundação de Apoio à Pesquisa do Estado de São Paulo (FAPESP)

Heat stress induced alteration in bovine oocytes: functional and cellular aspects

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Gene Expression in Bovine Oocytes and Cumulus Cells After Meiotic Inhibition with the Cyclin-Dependent Kinase Inhibitor Butyrolactone I

Contents The aim of this study was to determine the effect of temporary inhibition of meiosis using the cyclin-dependent kinase inhibitor butyrolactone I (BLI) on gene expression in bovine oocytes and cumulus cells. Immature bovine cumulusoocyte complexes (COCs) were assigned to groups: (i) Control COCs collected immediately after recovery from the ovary or (ii) after in vitro maturation (IVM) for 24 h, (iii) Inhibited COCs collected 24 h after incubation with 100 mu m BLI or (iv) after meiotic inhibition for 24 h followed by IVM for a further 22 h. For mRNA relative abundance analysis, pools of 10 denuded oocytes and respective cumulus cells were collected. Transcripts related to cell cycle regulation and oocyte competence were evaluated in oocytes and cumulus cells by quantitative real-time PCR (qPCR). Most of the examined transcripts were downregulated (p < 0.05) after IVM in control and inhibited oocytes (19 of 35). Nine transcripts remained stable (p > 0.05) after IVM in control oocytes; only INHBA did not show this pattern in inhibited oocytes. Seven genes were upregulated after IVM in control oocytes (p < 0.05), and only PLAT, RBP1 and INHBB were not upregulated in inhibited oocytes after IVM. In cumulus cells, six genes were upregulated (p < 0.05) after IVM and eight were downregulated (p < 0.05). Cells from inhibited oocytes showed the same pattern of expression regarding maturation profile, but were affected by the temporary meiosis inhibition of the oocyte when the same maturation stages were compared between inhibited and control groups. In conclusion, changes in transcript abundance in oocytes and cumulus cells during maturation in vitro were mostly mirrored after meiotic inhibition followed by maturation.

Protein diets promote the maturation of oocytes and spawning of Piaractus mesopotamicus kept in cages

The objective of this study was to evaluate the effects of balanced diets on the maturation of oocytes and the reproductive performance of P. mesopotamicus in cages. A completely random design with 224 fish in 16 cages measuring 5 m(3) was employed for this purpose. The treatments consisted of diets containing 18, 24, 30, and 36% crude protein (CP) provided ad libitum. The external and internal morphological characteristics of the specimens were examined, as well as: the position of the germinal vesicle, the distribution of oocyte diameters, the fertilization and hatching rates, the number of oocytes released, the total number of oocytes, the remaining weight and total weight of the ovaries, the gonadosomatic index, the condition factor (K), and the histology of the oocytes and ovaries post-spawning and during ovarian regression. The diameters of the oocytes collected before the first hormonal application displayed a unimodal distribution for the lowest protein content and a polymodal distribution for the other treatments. A similar situation was seen during spawning. The lowest fertilization and hatching rates were found as a consequence of the treatment with 30% CP (P < 0.05). The greatest hatching rate occurred in the females fed 18% CP. The greatest total oocyte weight was found in the specimens that received between 30 and 36% CP. The lowest K index was found in the females fed 36% CP. In conclusion, a diet containing 18% CP satisfies the reproductive requirements of females adapted to this system.

Identification and characterization of the vegetally localized Hermes mRNA

One way developing embryos regulate the expression of their genes is by localizing mRNAs to specific subcellular regions. In the oocyte of the frog, Xenopus laevis, many RNAs are localized specifically to the animal or the vegetal halves of the oocyte. The localization of these RNAs contributes to the primary polarity of the oocyte, the asymmetry that is the basis for patterning and lineage specification in the embryo. I have screened a cDNA library for clones containing the Xlsirt repeat, an element known to target RNAs to the vegetal cortex of the oocyte. I have identified seventeen cDNA clones that contain this element. One of these cDNAs encodes the RNA binding protein Hermes. The Hermes mRNA is localized to the vegetal cortex of the oocyte. Additionally, Hermes protein is also vegetally localized in the oocyte and is found in subcellular structures known to contain localized mRNAs. This suggests that Hermes might interact with localized RNAs. While Hermes protein is present in oocytes, it disappears at germinal vesicle breakdown during maturation. We therefore believe that the time period during which Hermes functions is during oogenesis or maturation prior to the time of Hermes degradation. To determine Hermes function, an antisense depletion strategy was used that involved injecting morpholino oligos (HE-MO) into oocytes. Injection of these morpholinos causes the level of Hennes protein to drop prematurely during maturation. Embryos produced from these oocytes exhibit cleavage defects that are most prevalent in the vegetal blastomeres. The phenotype can be partially rescued by injection of a heterologous Hermes mRNA and is therefore specific to Hermes. The Hermes expression and depletion results are consistent with a model in which Hermes interacts with one or more vegetally localized mRNAs in the oocyte and during the early stages of maturation. The interaction is required for cleavage of the vegetal blastomeres. Therefore, it is likely that at least one mRNA that interacts with Hermes is a cell cycle regulator. ^

Assembly of the Nuclear Transcription and Processing Machinery: Cajal Bodies (Coiled Bodies) and Transcriptosomes

We have examined the distribution of RNA transcription and processing factors in the amphibian oocyte nucleus or germinal vesicle. RNA polymerase I (pol I), pol II, and pol III occur in the Cajal bodies (coiled bodies) along with various components required for transcription and processing of the three classes of nuclear transcripts: mRNA, rRNA, and pol III transcripts. Among these components are transcription factor IIF (TFIIF), TFIIS, splicing factors, the U7 small nuclear ribonucleoprotein particle, the stem–loop binding protein, SR proteins, cleavage and polyadenylation factors, small nucleolar RNAs, nucleolar proteins that are probably involved in pre-rRNA processing, and TFIIIA. Earlier studies and data presented here show that several of these components are first targeted to Cajal bodies when injected into the oocyte and only subsequently appear in the chromosomes or nucleoli, where transcription itself occurs. We suggest that pol I, pol II, and pol III transcription and processing components are preassembled in Cajal bodies before transport to the chromosomes and nucleoli. Most components of the pol II transcription and processing pathway that occur in Cajal bodies are also found in the many hundreds of B-snurposomes in the germinal vesicle. Electron microscopic images show that B-snurposomes consist primarily, if not exclusively, of 20- to 30-nm particles, which closely resemble the interchromatin granules described from sections of somatic nuclei. We suggest the name pol II transcriptosome for these particles to emphasize their content of factors involved in synthesis and processing of mRNA transcripts. We present a model in which pol I, pol II, and pol III transcriptosomes are assembled in the Cajal bodies before export to the nucleolus (pol I), to the B-snurposomes and eventually to the chromosomes (pol II), and directly to the chromosomes (pol III). The key feature of this model is the preassembly of the transcription and processing machinery into unitary particles. An analogy can be made between ribosomes and transcriptosomes, ribosomes being unitary particles involved in translation and transcriptosomes being unitary particles for transcription and processing of RNA.

Mos/mitogen-activated protein kinase can induce early meiotic phenotypes in the absence of maturation-promoting factor: a novel system for analyzing spindle formation during meiosis I.

Mitogen-activated protein kinase (MAPK) is selectively activated by injecting either mos or MAPK kinase (mek) RNA into immature mouse oocytes maintained in the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). IBMX arrests oocyte maturation, but Mos (or MEK) overexpression overrides this block. Under these conditions, meiosis I is significantly prolonged, and MAPK becomes fully activated in the absence of p34cdc2 kinase or maturation-promoting factor. In these oocytes, large openings form in the germinal vesicle adjacent to condensing chromatin, and microtubule arrays, which stain for both MAPK and centrosomal proteins, nucleate from these regions. Maturation-promoting factor activation occurs later, concomitant with germinal vesicle breakdown, the contraction of the microtubule arrays into a precursor of the spindle, and the redistribution of the centrosomal proteins into the newly forming spindle poles. These studies define important new functions for the Mos/MAPK cascade in mouse oocyte maturation and, under these conditions, reveal novel detail of the early stages of oocyte meiosis I.

The environmental control of reproduction in the female dace, Leuciscus leuciscus

An investigation was made into the nature and control of the annual reproductive cycle of the dace, Leuciscus leuciscus. It includes 1) a study of the natural reproductive cycle, 2) the use of Carp Pituitary Extract (CPE) to induce final maturation and ovulation in captive fish, 3) the effect of artificial light treatments on ovarian development and 4) the measurement of serum melatonin levels under different photoperiod regimes. Ovarian development was monitored by endocrinological data, notably serum cycles of 17-oestradiol (E2), testosterone (T), and calcium (as an index of vitellogenin), oocyte diameter, the gonadosomatic index and histological studies of the ovary. Under natural conditions, ovarian development can broadly be divided into 4 stages: 1) oogenesis which occurs immediately after spawning; 2) a primary growth phase (previtellogenic growth) prevalent between spawning and June; 3) a secondary growth phase (yolk vesicle plus vitellogenic growth) occurring between June and December and 4) final maturation and ovulation which occurs in mid-March. During the annual ovarian cycle, the sex steroids E2 and T showed two clear elevations. The first occurred initially in April followed by a rise in serum calcium levels. This subsequently initiated the appearance of yolk granules in the oocytes in June. The second rise occurred in September and levels were maintained until December, after which there was a decline in serum E2 levels. It is proposed that in the dace, high serum E2 levels between September and December were required to maintain vitellogenin production and therefore its uptake into the developing oocytes which occurred during this time, albeit at a slower rate than in the summer months. After December, prior to final maturation, whereas serum E2 and calcium levels declined, serum T levels remained elevated. In captivity, final maturation beyond the germinal vesicle migration stage failed to occur suggesting that the stimuli required for these events were absent. However ovulation could be induced by a single injection of CPE, which induced ovulation between 6 and 14 hours after treatment. Endocrine events associated with the artificial induction of spawning included a rise in serum levels of E2, T and the maturation inducing steroid 1720-dihydroxy progesterone. Photoperiodic manipulation demonstrated that whereas short or increasing daylengths were stimulatory to ovarian development, long days delayed development. Changes from long to short and constant short daylengths early in the reproductive cycle advanced maturation (up to 5 months), suggesting that the stimulus for ovarian development and maturation was a short day. However, experiments conducted later in the reproductive cycle demonstrated that only a simulated ambient photoperiod could induce final maturation. It is proposed therefore that under natural conditions the environmental stimulus for ovarian development and final maturation are short and increasing daylengths respectively. Further support that photoperiod is the dominant timing cue in this species was provided by the pattern of serum melatonin levels. Under different photoperiod treatments, serum melatonin, which is believed to be the chemical transducer of photoperiodic information (similar to other photoperiodic species) was elevated for the duration of the dark phase, indicating that the dace at least has the ability to `measure' changes in daylength.

Kisspeptina: efeito na maturação in vitro de ovócitos bovinos

This study aimed to evaluate different concentrations of kisspeptin, as well as the interaction of kisspeptin and FSH/LH in vitro maturation and oocyte competence in cattle. In Experiment 1 was determined the minimum concentration of Kisspeptin (Kp) to be used, and in Experiment 2 was evaluated its interection with FSH and LH. The oocytes were collected in a commercial slaughterhouse and only Grade I oocytes were utilized. The oocytes were cultured in TCM-199 medium with bicarbonate plus 10% FBS, sodium pyruvate (22μg/mL), amikacin (83mg/mL), FSH (0.5μg/mL), with different concentrations of Kp, the treatments were: FSH + 0M Kp-10; FSH + 10-7M Kp-10, FSH + 10-6M Kp-10; FSH + 10-5M Kp-10. In Experiment 2, was used better concentration of Kp found in Experiment 1, the following treatments: no hormones; FSH; FSH + Kp-10; FSH + LH; FSH, LH + Kp-10; Kp-10. The oocyte competence was determined by nuclear maturation, mitochondrial distribution, MitoTracker® Orange CMTMRos fluorescence intensity and DCF. The evaluation of nuclear maturation was made after 24 hours incubation and the oocytes were stained with DAPI to determine the nuclear stage (Germinal Vesicle-GV, Metaphase I-MI and Metaphase II-MII).The mitochondrial distribution was classified as peripheral/semiperipheral and diffuse in clusters/granules, evaluated after stained with the MitoTracker® Orange CMTMRos, and was also identified the intensity of it. To determine the intensity of ROS oocytes were stained with DCF. The statistical analysis was performed by SAS GLIMMIX PROC. In Experiment 1 oocytes matured only with the FSH reached a smaller nuclear maturation when compared to those who were matured with Kisspeptin at different concentrations (FSH:13/33; FSH + 10-7M Kp-10: 28/35; FSH + 10-6M Kp-10:30/34; FSH + 10-5M Kp-10:28/32; P=0,0001). There was no statistical difference in mitochondrial distribution between treatments (P>0.05). The fluorescence intensity of MitoTracker did not differ among treatments (P>0.05). The DCF fluorescence intensity was lower when the concentration of Kp was increased in the medium (FSH:12177726,1; FSH + 10-7M Kp-10:10945982,83; FSH + 10-6M Kp-10:9820536,53; FSH + 10-5M Kp-10:9147016,38; P<0,0001). Based in the Experiment 1 results, the concentration of Kp was determined in 10-7M. In Experiment 2 the mitochondrial distribution was different between treatments, because oocytes matured only with Kp or FSH+LH, reached a oocyte competence greater than those maturated with FSH only or without hormone addition (no hormones:66,66%; FSH:66,66%; FSH + Kp-10:75,86%; FSH + LH:91,17%; FSH, LH + Kp-10:82,85%; Kp-10:91,17%; P<0,05). The no hormones resulted in a lower nuclear maturation than the other treatments (no hormones: 5/18; FSH:18/32; FSH + Kp-10:22/29; FSH + LH:26/33; FSH, LH + Kp-10:26/34; Kp-10:25/34; P=0,0094). The fluorescence intensity of probes MitoTracker and DCF was lower when Kp was added to the maturation medium (no hormones:1228363/540069; FSH:2307984/1395751; FSH + Kp-10:1941890/1114948; FSH + LH:2502145/1722376; FSH, LH + Kp-10:2286173/1467782; Kp-10:1859411/979325 P<0,0001). So this is the first study that shows that Kisspeptin stimulates oocyte maturation without the presence of gonadotropins in the maturation medium.

Avaliação da crioinjúria em ovócitos imaturos e maturados in vitro e produção de embriões a partir de ovócitos bovinos vitrificados em Open Pulled Straw (OPS) e Cryotop

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciências Médicas, Programa de Pós-Graduação em Ciências Médicas, 2012.