Effects of Hormonal Supplementation on Nuclear Maturation and Cortical Granules Distribution of Canine Oocytes During Various Reproductive Stages

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

Effect of conditioned medium of mesenchymal stem cells on the in vitro maturation and subsequent development of mouse oocyte

Mesenchymal stem cells (MSCs) secrete a variety of cytokines and growth factors in addition to self-renewal and multiple forms of differentiation. Some of these secreted bioactive factors could improve meiotic maturation in vitro and subsequent embryo developmental potential. The aim of the present study was to determine whether in vitro maturation (IVM) of mouse oocyte with or without cumulus cells could be improved by contact with conditioned medium (CM) of MSCs as well as the efficiency of CM to support follicular growth and oocyte maturation in the ovarian organ of mice cultured on soft agar. The developmental potential of matured oocyte was assessed by blastocyst formation after in vitro fertilization (IVF). Germinal vesicle stage oocytes with or without cumulus cells were subjected to IVM in either CM, Dulbecco's modified Eagle's medium (DMEM), α-minimum essential medium (α-MEM) or human tubal fluid (HTF). Approximately 120 oocytes were studied for each medium. CM produced a higher maturation rate (91.2%) than DMEM (54.7%), α-MEM (63.5%) and HTF (27.1%). Moreover, CM improved embryo development to blastocyst stage significantly more than DMEM and HTF (85 vs 7% and 41.7%, respectively) but there was no significant difference compared with α-MEM (85 vs 80.3%). The behavior of cortical granules of IVM oocytes cultured in CM revealed cytoplasmic maturation. Moreover, CM also supported preantral follicles growth well in organotypic culture on soft agar resulting in the maturation of 60% of them to developmentally competent oocytes. The production of estrogen progressively increased approximately 1-fold every other day during organ culture, while a dramatic 10-fold increase in progesterone was observed 17 h after human chorionic gonadotropin stimulus at the end of culture. Thus, CM is an effective medium for preantral follicle growth, oocyte maturation, and sequential embryo development.

Cytoplasmic maturation of bovine oocytes: Structural and biochemical modifications and acquisition of developmental competence

Oocyte maturation is a long process during which oocytes acquire their intrinsic ability to support the subsequent stages of development in a stepwise manner, ultimately reaching activation of the embryonic genome. This process involves complex and distinct, although linked, events of nuclear and cytoplasmic maturation. Nuclear maturation mainly involves chromosomal segregation, whereas cytoplasmic maturation involves organelle reorganization and storage of mRNAs, proteins and transcription factors that act in the overall maturation process, fertilization and early embryogenesis. Thus, for didactic purposes, we subdivided cytoplasmic maturation into: (1) organelle redistribution, (2) cytoskeleton dynamics, and (3) molecular maturation. Ultrastructural analysis has shown that mitochondria, ribosomes, endoplasmic reticulum, cortical granules and the Golgi complex assume different positions during the transition from the germinal vesicle stage to metaphase II. The cytoskeletal microfilaments and microtubules present in the cytoplasm promote these movements and act on chromosome segregation. Molecular maturation consists of transcription, storage and processing of maternal mRNA, which is stored in a stable, inactive form until translational recruitment. Polyadenylation is the main mechanism that initiates protein translation and consists of the addition of adenosine residues to the 3` terminal portion of mRNA. Cell cycle regulators, proteins, cytoplasmic maturation markers and components of the enzymatic antioxidant system are mainly transcribed during this stage. Thus, the objective of this review is to focus on the cytoplasmic maturation process by analyzing the modifications in this compartment during the acquisition of meiotic competence for development. (c) 2009 Elsevier Inc. All rights reserved.

Ooplast-mediated developmental rescue of bovine oocytes exposed to ethidium bromide

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

Isolamento e caracterização ultraestrutural de folículos pré-antrais de vacas da raça Nelore (Bos taurus indicus)

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

Evaluation of zona pellucida birefringence intensity during in vitro maturation of oocytes from stimulated cycles

Background: This study evaluated whether there is a relationship between the zona pellucida birefringence (ZP-BF) intensity and the nuclear (NM) and cytoplasmic (CM) in vitro maturation of human oocytes from stimulated cycles.Results: The ZP-BF was evaluated under an inverted microscope with a polarizing optical system and was scored as high/positive (when the ZP image presented a uniform and intense birefringence) or low/negative (when the image presented moderate and heterogeneous birefringence). CM was analyzed by evaluating the distribution of cortical granules (CGs) throughout the ooplasm by immunofluorescence staining. CM was classified as: complete, when CG was localized in the periphery; incomplete, when oocytes presented a cluster of CGs in the center; or in transition, when oocytes had both in clusters throughout cytoplasm and distributed in a layer in the cytoplasm periphery Nuclear maturation: From a total of 83 germinal vesicle (GV) stage oocytes, 58 of oocytes (69.9%) reached NM at the metaphase II stage. From these 58 oocytes matured in vitro, the high/positively scoring ZP-BF was presented in 82.7% of oocytes at the GV stage, in 75.8% of oocytes when at the metaphase I, and in 82.7% when oocytes reached MII. No relationship was observed between NM and ZP-BF positive/negative scores (P = 0.55). These variables had a low Pearson's correlation coefficient (r = 0.081). Cytoplasmic maturation: A total of 85 in vitro-matured MII oocytes were fixed for CM evaluation. Forty-nine oocytes of them (57.6%) showed the complete CM, 30 (61.2%) presented a high/positively scoring ZP-BF and 19 (38.8%) had a low/negatively scoring ZP-BF. From 36 oocytes (42.3%) with incomplete CM, 18 (50%) presented a high/positively scoring ZPBF and 18 (50%) had a low/negatively scoring ZP-BF. No relationship was observed between CM and ZP-BF positive/negative scores (P = 0.42). These variables had a low Pearson's correlation coefficient (r = 0.11).Conclusions: The current study demonstrated an absence of relationship between ZP-BF high/positive or low/negative score and nuclear and cytoplasmic in vitro maturation of oocytes from stimulation cycles.

Collection Rates and Morphology of Equine Oocytes Obtained from Immature Follicles after Treatment with Equine Pituitary Extract (EPE) and Human Chorionic Gonadotropin

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Use of strontium for bovine oocyte activation

Strontium efficiently activates mouse oocytes, however, there is limited information on its use in cattle. Thus, the objective of this study was to establish a suitable protocol for activating bovine oocyte with strontium. For pronuclear development, the absence of calcium and magnesium in the activation medium (TALP) with 10 and 50mM strontium (34.4 and 53.1%, respectively) was superior to the complete TALP (6.5 and 19.4%, respectively). In all activation media, better results were observed with 25 and 50 mM strontium (21.9-53.1 and 19.4-53.1%, respectively). Incubation for 4 h promoted similar results in all strontium concentrations. However, strontium at 15, 20, and 25 mM for 6 and 8 h (40.7, 46.7, and 48.3%, and 29.3, 48.3, and 40.7%, respectively) were superior to control (15.5 and 10%, respectively). After in vitro maturation for 26 h, strontium (S; 20 mM in Ca2+ and Mg2+-free TALP for 6 h), ionomycin + strontium (IS), and strontium + ionomycin (SI) (60, 63.3, and 65%, respectively) were similar in pronuclear development and superior to ionomycin (I; 5 mu M for 5 min; 36.7%). In treatments S and I, only 1 PN zygotes were observed. In treatment S, most of them had 1 and 2 PB (35.7 and 60.7%, respectively), and in treatment I, 0, 1, and 2 PB (14.3, 57.1, and 28.6%, respectively). Most of the zygotes in treatment IS and SI were 1 PN 2 PB (77.4 and 61.6%, respectively). The number of oocytes with clusters of cortical granules was similar in all treated groups (11-29%). Cortical granule exocytosis in treatment IS (68%) was similar to S (54%) and superior to 1, SI, and control (27, 45, and 5.0%, respectively). Cleavage and blastocyst rates were similar for S, I, IS, and SI treatments (61.7-76.7, and 8.3-13.3%, respectively) and the same was observed for ICM, TE, and total cell number, and ICM/total cell ratio (22-25, 64-69, and 86-95, and 0.26-0.27). In conclusion, strontium may be efficiently applied for bovine oocyte activation at 20 mM in Ca2+-and Mg2+-free TALP medium for 6 h.

Effect of roscovitine and cycloheximide on ultrastructure of sheep oocytes

It is believed the temporary meiosis arrest with roscovitine or cycloheximide may improve the in vitro developmental competence of oocytes in different animal species. However, little is known about the effects of these inhibitors on ultrastructure of ovines cumulus-oocyte complexes (COCs). The aim of this study was to evaluate the progression of cytoplasmic maturation and the ultrastructural changes in sheep COCs exposed to roscovitine or cycloheximide, at acceptable concentrations. COCs were in vitro cultured for 24. h in maturation medium (control group) containing 100 μM roscovitine or 1 μg/mL cycloheximide (treatment groups). After this time, some COCs were cultured for further 22. h in inhibitor-free medium. The ultrastructure organization of COCs was evaluated by transmission electron microscopy before (immature group) and after in vitro culture for 24 and 46. h. As expected, signs of immaturity and maturity were observed in immature and control groups, respectively. In treatment with roscovitine, there were cumulus cells degeneration, swelling of mitochondrias, few cortical granules and many vesicles with electron-dense material. However, in cycloheximide treatment there were not signs of degeneration or cellular senescence. Metabolic units and mitochondrial pleomorphism were found in all experimental groups. These evidences demonstrate that roscovitine promoted irreversible ultrastructural changes while cycloheximide did not affect the cytoplasmic maturation. However, the implications on embryo development are still unclear. © 2012 Elsevier B.V.

Influência do fluido folicular e da proteína recombinante humana GDF9 no meio de maturação sobre a qualidade de embriões bovinos produzidos in vitro

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeitos da demecolcina na cinética de maturação, microtúbulos e na enucleação química de oócitos bovinos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

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

Estímulo da síntese da glutationa na maturação in vitro de oócitos bovinos e sua influência no desenvolvimento embrionário

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)

Vitrificação de ovócitos e embriões bovinos utilizando-se etilenoglicol, dimetilsulfóxido e dimetilformamida como agentes crioprotetores

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