Avaliação da maturação nuclear in vitro de oócitos de gatas domésticas (Felis catus) pré-púberes e púberes

O objetivo do trabalho foi avaliar a taxa de maturação nuclear in vitro de oócitos provenientes de gatas doméstica púbere e pré-púbere. Foram utilizadas 15 fêmeas felinas, 10 púberes e 5 pré-púberes; sendo os oócitos obtidos por aspiração quantificados e classificados. Os oócitos classificados como excelentes e regulares foram reunidos em grupos de 10, em meio de cultura, recobertos em óleo mineral em Placas de Petri siliconizadas e descartáveis. Após permanência em estufa, a 38°C e 5% de CO2 por 48 horas, os oócitos foram submetidos a duas lavagens com solução de hialuronidase a 0,4%, fixados em metanol/acido acético e corados com orceína acética. A avaliação da configuração cromossômica de oócitos maturados in vitro resultou em 44,68% das células em metáfase II no grupo das fêmeas púberes e 25,32% no grupo das doadoras pré-púberes, indicando que a puberdade influencia a capacidade dos oócitos se desenvolverem in vitro.

Efeito das células do cumulus e cisteamina durante o cultivo de maturação in vitro de oócitos bovinos sobre a maturação nuclear e aquisição da competência para desenvolvimento embrionário

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

Melatonin in maturation media fails to improve oocyte maturation, embryo development rates and DNA damage of bovine embryos

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

Selective Ablation of the Androgen Receptor in Mouse Sertoli Cells Affects Sertoli Cell Maturation, Barrier Formation and Cytoskeletal Development

The observation that mice with a selective ablation of the androgen receptor (AR) in Sertoli cells (SC) (SCARKO mice) display a complete block in meiosis supports the contention that SC play a pivotal role in the control of germ cell development by androgens. To delineate the physiological and molecular mechanism responsible for this control, we compared tubular development in pubertal SCARKO mice and littermate controls. Particular attention was paid to differences in SC maturation, SC barrier formation and cytoskeletal organization and to the molecular mediators potentially involved. Functional analysis of SC barrier development by hypertonic perfusion and lanthanum permeation techniques and immunohistochemical analysis of junction formation showed that SCARKO mice still attempt to produce a barrier separating basal and adluminal compartment but that barrier formation is delayed and defective. Defective barrier formation was accompanied by disturbances in SC nuclear maturation (immature shape, absence of prominent, tripartite nucleoli) and SC polarization (aberrant positioning of SC nuclei and cytoskeletal elements such as vimentin). Quantitative RT-PCR was used to study the transcript levels of genes potentially related to the described phenomena between day 8 and 35. Differences in the expression of SC genes known to play a role in junction formation could be shown from day 8 for Cldn11, from day 15 for Cldn3 and Espn, from day 20 for Cdh2 and Jam3 and from day 35 for ZO-1. Marked differences were also noted in the transcript levels of several genes that are also related to cell adhesion and cytoskeletal dynamics but that have not yet been studied in SC (Actn3, Ank3, Anxa9, Scin, Emb, Mpzl2). It is concluded that absence of a functional AR in SC impedes the remodeling of testicular tubules expected at the onset of spermatogenesis and interferes with the creation of the specific environment needed for germ cell development.

Inibição da maturação nuclear pela butirolactona I durante o transporte de oócitos bovinos destinados à produção in vitro de embrões (PIV)

Pós-graduação em Medicina Veterinária - FCAV

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

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Imunolocalização do fator de crescimento semelhante à insulina (IGF-I) e do fator de crescimento epidermal (EGF) em ovários caninos e seus efeitos sobre a maturação in vitro de oócitos: avaliação nuclear

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Efeito do fator de crescimento IGF-I sobre a maturação in vitro de oócitos caninos (Canis familiaris): avaliação da maturação nuclear e citoplasmática

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

Maturação nuclear e citoplasmática de oócitos de cadelas colhidos em diferentes fases do ciclo estral e cultivados in vitro em meios sequenciais com hormônios e espermatozóides

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

Avaliação dos efeitos da inibição da maturação nuclear e de antioxidantes sobre a maturação oocitária, fecundação e desenvolvimento embrionário bovino in vitro

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

Contribuição para a clonagem em eqüinos por meio de tranferência nuclear

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

In vitro Maturation of Oocytes from Santa Ines Ewes Subjected to Consecutive Sessions of Follicular Aspiration by Laparoscopy

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

Control of oocyte maturation

Oocyte maturation is a complex process involving nuclear and cytoplasmic maturation. The nuclear maturation is a chromosomal segregation and the cytoplasmic maturation involves the reorganization of the cytoplasmic organelles, mRNA transcription and storage of proteins to be used during fertilization and early embryo development. The mechanism of oocyte maturation in vivo and in vitro still are not totally understood. However it is generally accepted that the second messenger cyclic adenosine monophosphate (cAMP) plays a critical role in the maintenance of meiotic blockage of mammalian oocytes. A relative increase in the level of cAMP within the oocyte is essential for maintaining meiosis block, while a decrease in cAMP oocyte concentration allows the resumption of meiosis. The oocyte cAMP concentration is regulated by a balance of two types of enzymes: adenylate cyclase (AC) and phosphodiesterases (PDEs), which are responsible for the synthesis and degradation of cAMP, respectively. After being synthesized by AC in cumulus cells, cAMP are transferred to the oocyte through gap junctions. Thus, specific subtypes PDEs are able to inhibit or attenuate the spontaneous meiotic maturation of oocytes with PDE4 primarily involved in the metabolism of cAMP in granulosa cells and PDE3 in the oocyte. Although the immature oocytes can resume meiosis in vitro, after being removed from antral follicles, cytoplasmic maturation seems to occur asynchronously with nuclear maturation. Therefore, knowledge of the oocyte maturation process is fundamental for the development of methodologies to increase the success of in vitro embryo production and to develop treatments for various forms of infertility. This review will present current knowledge about the maintenance of the oocyte in prophase arrest, and the resumption of meiosis during oocyte maturation, focusing mainly on the changes that take place in the oocyte.

Glucosamine supplementation during in vitro maturation inhibits subsequent embryo development: Possible role of the of developmental competence

Glucose concentration during cumulus-oocyte complex (COC) maturation influences several functions, including progression of oocyte meiosis, oocyte developmental competence, and cumulus mucification. Glucosamine (GlcN) is an alternative hexose substrate, specifically metabolized through the hexosamine biosynthesis pathway, which provides the intermediates for extracellular matrix formation during cumulus cell mucification. The aim of this study was to determine the influence of GlcN on meiotic progression and oocyte developmental competence following in vitro maturation (IVM). The presence of GlcN during bovine IVM did not affect the completion of nuclear maturation and early cleavage, but severely perturbed blastocyst development. This effect was subsequently shown to be dose-dependent and was also observed for porcine oocytes matured in vitro. Hexosamine biosynthesis upregulation using GlcN supplementation is well known to increase O-linked glycosylation of many intracellular signaling molecules, the best-characterized being the phosphoinositol-3-kinase (PI3K) signaling pathway. We observed extensive O-linked glycosylation in bovine cumulus cells, but not oocytes, following IVM in either the presence or the absence of GlcN. Inhibition of O-linked glycosylation significantly reversed the effect of GlcN-induced reduction in developmental competence, but inhibition of PI3K signaling had no effect. Our data are the first to link hexosamine biosynthesis, involved in cumulus cell mucification, to oocyte developmental competence during in vitro maturation.

Bloqueio meiótico como alternativa para aumentar a produção de embriões clone por transferência nuclear

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, 2016.