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Supplemented tissue culture medium 199 is a better medium for in vitro maturation of oocytes from women with polycystic ovary syndrome women than human tubal fluid

ARAUJO, Carlos Henrique Medeiros de; NOGUEIRA, Daniela; ARAUJO, Maria Cristina Picinato Medeiros de; MARTINS, Wellington de Paula; FERRIANI, Rui Alberto; REIS, Rosana Maria dos
Fonte: ELSEVIER SCIENCE INC Publicador: ELSEVIER SCIENCE INC
Tipo: Artigo de Revista Científica
Português
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Objective: To compare oocyte maturation, fertilization and cleavage rates, and embryonic developmental quality after culture of human immature oocytes from polycystic ovary syndrome (PCOS) patients in human tubal fluid (HTF) or tissue culture medium (TCM) 199. Design: Prospective, randomized, controlled trial. Setting: University hospital. Patient(s): Thirteen women undergoing 23 in vitro maturation cycles, from whom 119 oocytes were retrieved. Intervention(s): Cumulus-enclosed germinal vesicle-stage oocytes matured in TCM-199-supplemented or HTF-supplemented media. Main Outcome Measure(s): Oocyte maturation and fertilization rates, embryonic developmental quality. Result(s): Significant differences were observed between TCM 199 and HTF regarding maturation rate (82% vs. 56.9%), fertilization rate (70% vs. 39.4%), and embryo quality (81.3% vs. 41.7%). Conclusion(S): Human tubal fluid medium, although widely used for embryo fertilization and maintenance in IVF techniques, is not,in appropriate medium for the maturation of oocytes obtained from PCOS patients in nonstimulated cycles. (Fertil Steril(R) 2009;91:509-13. (C)2009 by American Society for Reproductive Medicine.)

Efeito da melatonina sobre a maturação dos ovócitos em sistema tradicional de produção in vitro de embriões bovinos; Effect of melatonin on oocyte maturation in traditional system of bovine embryo in vitro production

Takada, Luciana
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 01/07/2008 Português
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Este trabalho teve objetivou-se avaliar o efeito da melatonina adicionada ao meio de maturação (meio B199) sobre a maturação nuclear, a distribuição dos grânulos corticais e dos microtúbulos, a produção in vitro de embriões e sobre a apoptose das células do cumulus (CC) e dos embriões. Os complexos cumulus-ovócitos (COCs), aspirados de folículos ovarianos obtidos de ovários de vacas oriundas de abatedouros, foram cultivados por 24 h, conforme os tratamentos: 1) Grupo Controle: meio B199 acrescidos de 0,5 µg/mL de FSH e 5,0 µg/mL de LH; 2) Grupo MEL: meio B199 com 1 µg/mL de melatonina; 3) Grupo MEL/FSH/LH: meio B199 com 1 µg/mL de melatonina e 0,5 µg/mL de FSH e 5,0 µg/mL de LH. Após a maturação in vitro (MIV), os COCs de todos os grupos foram submetidos à fecundação in vitro (FIV) e desenvolvimento embrionário in vitro. A condição de cultivo em todas as etapas foi microgotas, sob óleo de silicone, com atmosfera de 5% de CO2 em ar, a 38,5ºC. Foram avaliadas as taxas de clivagem e de embriões produzidos no dia 7 (D-7) pós-inseminação in vitro. Após a MIV, os ovócitos de todos os grupos também foram corados com Hoechst 33342, com anticorpo monoclonal anti-tubulina conjugada com FITC ou cultivados com aglutinina Lens Culinaris conjugada a FITC para avaliação da taxa de maturação nuclear (progressão da meiose)...

Ovarian morphometric characterization and in vitro maturation of oocytes obtained from buffalo (Bubalus bubalis) ovaries - partial results

Leal, L. S.; Oba, E.; Fernandes, C. B.; Moya, C. F.; Martins, L. R.; Martin, I.; Landim-Alvarenga, F. C.
Fonte: Pagepress Publ Publicador: Pagepress Publ
Tipo: Artigo de Revista Científica Formato: 804-806
Português
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Buffalo ovaries were collected from a slaughterhouse (Frigol, Brazil) and transported to the laboratory in saline solution at 36 degrees C. The ovaries were dissected to realize the evaluations (weight, length, width and height of the ovary; corpus luteum and dominant follicle diameters). The Cumulus-oocyte complexes (COCs) were recovered by aspiration of 2-8 mm follicles. Selected COCs were matured in TCM 199 supplemented with 10% fetal bovine serum, sodium pyruvate, LH, FSH, estradiol and gentamicin. In vitro maturation was carried out at 38.5 degrees C for 22-24 h and 34-36 h. For the evaluation of the nuclear maturation the oocytes were placed in TCM 199 medium added with type v hialuronidase where the granulosa cells were extracted. The denuded oocytes were transferred to 10 mu l of Hoescht 33342 and the chromosomic configuration was evaluated. The oocytes were classified according to meiosis stage in: Germinal Vesicle, Germinal Vesicle Breakdown, Metaphase I, Metaphase II and Degenerated. The means of weight, length, width and height of the ovary were 3.83 g, 2.27 cm, 1.08 cm and 1.56 cm, respectively. The means of corpus luteum and dominant follicle diameters were 1.40 cm and 7.77 mm. The proportion of oocytes that reached metaphase II stage was: 36.68%.

THE EFFECT OF SERUM ON IN-VITRO MATURATION, IN-VITRO FERTILIZATION AND STEROIDOGENESIS OF BOVINE OOCYTES COCULTURED WITH GRANULOSA-CELLS

Mingoti, G. Z.; Garcia, J. M.; Rosaesilva, AAM
Fonte: Associação Brasileira de Divulgação Científica (ABRADIC) Publicador: Associação Brasileira de Divulgação Científica (ABRADIC)
Tipo: Artigo de Revista Científica Formato: 213-217
Português
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The influence of fetal calf serum alone (FCS) or associated with proestrous (FCS+PCS), estrous (FCS+ECS) or metaestrous (FCS+MCS) cow serum added to the culture medium and of the steroids produced by co-cultured granulosa cells were evaluated in terms of the in vitro maturation (TVM) and fertilization (IVF) of bovine oocytes. Supplementation of the medium with FCS+ECS and FCS+MCS resulted in higher proportions of oocytes that reached metaphase II (96.0% and 93.3%, respectively) and in higher proportions of embryos that reached the four- and eight-cell/morula stages (51.9% and 65.6%, respectively), whereas the supplementation with FCS and FCS+PCS resulted in only 79.2% and 67.5%, respectively, of matured oocytes and 26.7% and 34.3%, respectively, of cleaved embryos. These findings show that the best IVM and IVF were obtained at lower concentrations of estradiol produced by co-cultured granulosa cells (supplementation with FCS+ECS: 10.3 ng/ml and FCS+MCS: 2.1 ng/ml), whereas the worst-results in IVM and IVF occurred at higher concentrations of estradiol that were obtained with FCS (33.1 ng/ml) and FCS+PCS (19.9 ng/ml) supplementation. These data suggest an inhibitory effect of estradiol on resumption of oocyte meiosis in vitro.

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

Cruz, Maria Helena Coelho
Fonte: Universidade Estadual Paulista (UNESP) Publicador: Universidade Estadual Paulista (UNESP)
Tipo: Dissertação de Mestrado Formato: xvi, 73 f. : il.
Português
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Pós-graduação em Medicina Veterinária - FCAV; Este estudo objetivou avaliar a influência do fluido folicular (FF) e da proteína recombinante humana GDF9 adicionados ao meio de maturação sobre a qualidade de embriões bovinos produzidos in vitro. Foram realizados três experimentos utilizando o meio TCM199 como meio básico de maturação. No primeiro experimento, os oócitos foram maturados em cinco diferentes meios (0%, 25%, 50%, 75% ou 100% de FF) em quatro tempos de maturação (22, 24, 26 ou 28 h). No segundo experimento, os oócitos foram maturados nos mesmos meios utilizados no primeiro experimento e fecundados em três diferentes tempos (24, 26 ou 28 h). No terceiro experimento, foram utilizados dois meios: controle (sem FF) ou com 25% de FF e quatro níveis de suplementação (0, 25, 100 ou 200 ng/mL) com GDF9. O FF promoveu retardo da progressão meiótica, afetando a maturação nuclear e a migração de grânulos corticais para a periferia dos oócitos. O FF em altas concentrações influenciou negativamente as taxas de clivagem e de blastocisto. O GDF9, com (25%) ou sem FF, não influenciou as taxas de clivagem e de blastocisto, no entanto...

Characterisation of the methylation pattern in the intragenic CpG island of the IGF2 gene in Bos taurus indicus cumulus cells during in vitro maturation

Franco, Mauricio Machaim; Fagundes, Nadia Simarro; Michalczechen-Lacerda, Valquiria Alice; Caixeta, Ester Siqueira; Rodrigues, Fernanda de Castro; Machado, Grazieli Marinheiro; Ferreira, Allice Rodrigues; Nunes Dode, Margot Alves
Fonte: Springer Publicador: Springer
Tipo: Artigo de Revista Científica Formato: 115-120
Português
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The aim of this study was to characterise the methylation pattern in a CpG island of the IGF2 gene in cumulus cells from 1-3 mm and a parts per thousand yenaEuro parts per thousand 8.0 mm follicles and to evaluate the effects of in vitro maturation on this pattern.Genomic DNA was treatment with sodium bisulphite. Nested PCR using bisulphite-treated DNA was performed, and DNA methylation patterns have been characterised.There were no differences in the methylation pattern among groups (P > 0.05). Cells of pre-IVM and post-IVM from small follicles showed methylation levels of 78.17 +/- 14.11 % and 82.93 +/- 5.86 %, respectively, and those from large follicles showed methylation levels of 81.81 +/- 10.40 % and 79.64 +/- 13.04 %, respectively. Evaluating only the effect of in vitro maturation, cells of pre-IVM and post-IVM COCs showed methylation levels of 80.17 +/- 12.01 % and 81.19 +/- 10.15 %.In conclusion, the methylation levels of the cumulus cells of all groups were higher than that expected from the imprinted pattern of somatic cells. As the cumulus cells from the pre-IVM follicles were not subjected to any in vitro manipulation, the hypermethylated pattern that was observed may be the actual physiological methylation pattern for this particular locus in these cells. Due the importance of DNA methylation in oogenesis...

In vitro maturation of porcine oocyte: effects of lipid manipulation on fertilization, embryo development and cryo-resistance

Prates, Elsa Cristina da Graça
Fonte: Universidade de Évora Publicador: Universidade de Évora
Tipo: Tese de Doutorado
Português
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Os objectivos desta tese foram implementar a técnica de produção in vitro de embriões de suíno e investigar os efeitos da modulação lipídica durante a maturação in vitro (MIV) usando o isómero trans-10,cis-12 do ácido linoleico conjugado (t10,c12 CLA) e a forscolina. A modulação lipídica durante a MIV induziu alterações na morfologia do oócito e perfil em ácidos gordos (AG)/ plasmalogénios dos complexos cumulus-oócito. Após 44 h de suplementação com t10,c12 CLA ocorreu aclaramento citoplásmico mas a taxa de blastocistos diminuiu. A suplementação em períodos longos com forscolina poderá reduzir o crescimento do oócito, a progressão nuclear e a capacidade fertilizante, ou apenas interferir na morfologia e composição das gotas lipídicas (2 h de suplementação). Os dois suplementos em simultâneo melhoraram a taxa de maturação. Apesar das alterações na composição em AG/ plasmalogénios dos oócitos tratados, não se detetaram efeitos sobre a crio-resistência dos blastocistos; ABSTRACT: The purpose of this thesis was to implement the in vitro technique for porcine embryo production and to investigate the effects of lipid content modulation during oocyte in vitro maturation (IVM) using trans-10...

Oxygen concentration during mouse oocyte in vitro maturation affects embryo and fetal development

Banwell, K.; Lane, M.; Russell, D.; Kind, K.; Thompson, J.
Fonte: Oxford Univ Press Publicador: Oxford Univ Press
Tipo: Artigo de Revista Científica
Publicado em //2007 Português
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BACKGROUND: Little is known of how the oxygen environment in the ovarian follicle affects oocyte and embryo development, but this has an important impact on the conditions used for in vitro maturation (IVM) of oocytes. We investigated the effect of varying oxygen concentrations during IVM on subsequent pre and post-implantation development. METHODS: IVM of mouse cumulus-oocyte complexes (COCs) was performed under 2, 5, 10 or 20% O(2) (6% CO(2), balance N(2)). In vivo-matured COCs were collected post ovulation. Embryos were generated by IVF and culture. Blastocyst development, cell number and apoptosis were assessed, and fetal and placental outcomes analysed following embryo transfer at day 18 of pregnancy. RESULTS: Oxygen concentration during IVM did not affect oocyte maturation or subsequent fertilization, cleavage and blastocyst development rates. Maturation of oocytes under 2% O(2) increased blastocyst trophectoderm cell number compared with all groups and numbers at 5% were higher than 20% (both P < 0.05). Percentage of apoptotic cells was increased in blastocysts developed from 2% O(2)-matured oocytes, compared with maturation at 5% O(2) or in vivo (P < 0.05). Rates of embryo implantation and development into a viable fetus were not altered by IVM oxygen. However...

Disruption of Bidirectional Oocyte-Cumulus Paracrine Signaling During In Vitro Maturation Reduces Subsequent Mouse Oocyte Developmental Competence

Yeo, C.; Gilchrist, R.; Lane, M.
Fonte: Soc Study Reproduction Publicador: Soc Study Reproduction
Tipo: Artigo de Revista Científica
Publicado em //2009 Português
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Oocyte-cumulus cell bidirectional communication is essential for normal development of the oocyte and cumulus cells (CCs) within the follicle. We showed recently that addition of recombinant growth differentiation factor 9 (GDF9), which signals through the SMAD2/3 pathway, during mouse oocyte in vitro maturation (IVM) increased fetal viability. This study thus aimed to observe the effects of disrupting oocyte-CC bidirectional communication during IVM on oocyte developmental competence and fetal outcomes. Cumulus-oocyte complexes (COCs) from equine chorionic gonadotropin-primed prepubertal (CBA/C57BL6) mice were cultured with or without 50 mIU/ml follicle-stimulating hormone (FSH) and 10 ng/ml epidermal growth factor (EGF) or 4 μM SMAD2/3 inhibitor SB-431542. Cumulus expansion and first polar body extrusion were then assessed, or COCs were fertilized and stained to evaluate sperm entry or cultured to the blastocyst stage. Embryo development and blastocyst quality were assessed, and Day 4.5 blastocysts were transferred to pseudopregnant recipients to analyze fetal outcomes. SMAD2/3 inhibition or FSH/EGF absence during IVM resulted in decreased cumulus expansion. First polar body extrusion and sperm entry were decreased in the absence of FSH/EGF...

Simulated physiological oocyte maturation (SPOM): a novel in vitro maturation system that substantially improves embryo yield and pregnancy outcomes

Albuz, F.; Sasseville, M.; Lane, M.; Armstrong, D.; Thompson, J.; Gilchrist, R.
Fonte: Oxford Univ Press Publicador: Oxford Univ Press
Tipo: Artigo de Revista Científica
Publicado em //2010 Português
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Background: Oocyte in vitro maturation (IVM) reduces the need for gonadotrophin-induced ovarian hyperstimulation and its associated health risks but the unacceptably low conception/pregnancy rates have limited its clinical uptake.We report the development of a novel in vitro simulated physiological oocyte maturation (SPOM) system. Methods and Results: Bovine or mouse cumulus–oocyte complexes (COCs) were treated with cAMP modulators for the first 1–2 h in vitro (pre-IVM), increasing COC cAMP levels ~100-fold. To maintain oocyte cAMP levels and prevent precocious oocyte maturation, COCs were treated during IVM with an oocyte-specific phosphodiesterase inhibitor and simultaneously induced to mature with FSH. Using SPOM, the pre-IVM and IVM treatments synergized to increase bovine COC gap-junctional communication and slow meiotic progression (both P , 0.05 versus control), extending the normal IVM interval by 6 h in bovine and 4 h in mouse. FSH was required to complete maturation and this required epidermal growth factor signalling. These effects on COC had profound consequences for oocyte developmental potential. In serum-free conditions, SPOM increased bovine blastocyst yield (69 versus 27%) and improved blastocyst quality (184 versus 132 blastomeres; both P ...

Effects of differing oocyte-secreted factors during mouse in vitro maturation on subsequent embryo and fetal development

Sudiman, J.; Ritter, L.; Inglis, D.; Wang, X.; Chan, K.; Mottershead, D.; Robertson, D.; Thompson, J.; Gilchrist, R.
Fonte: Kluwer Academic/Plenum Publ Publicador: Kluwer Academic/Plenum Publ
Tipo: Artigo de Revista Científica
Publicado em //2014 Português
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Purpose We hypothesised that varying native oocyte-secreted factor (OSF) exposure or using different recombinant OSF peptides would have differential effects on post-in vitro maturation (IVM) embryo and fetal development. Methods Mouse cumulus oocyte complexes (COCs) were treated with the purified mature domain of GDF9 and/or BMP15 or were co-cultured with denuded oocytes (DOs) from 0 h or 3 h of IVM. DOs were matured for 3 h as either intact COCs+/-FSH before denuding, or as DOs + FSH. COCs were fertilised and blastocyst development was assessed on days 5 and 6, and either differentially stained for ICM numbers or vitrified/warmed embryos were transferred to recipients to assess implantation and fetal rates. Results No improvement in embryo development was observed with the addition of GDF9 and/or BMP15 to IVM. In contrast, embryos derived from COCs co-cultured with DOs had significantly improved blastocyst rates and ICM numbers compared to controls (P <0.05). The highest response was obtained when DOs were first added to COCs at 3 h of IVM, after being pre-treated (0–3 h) as COCs + FSH. Compared to control, co-culture with DOs from 3 h did not affect implantation rates but more than doubled fetal yield (21 % vs 48 %; P <0.05). GDF9 Western blot analysis was unable to detect any differences in quantity or form of GDF9 (17 and 65 kDa) in extracts of DO at 0 h or 3 h. Conclusions This study provides new knowledge on means to improve oocyte quality in vitro which has the potential to significantly aid human infertility treatment and animal embryo production technologies.; J. Sudiman & L. J. Ritter & D. K. Feil & X.Wang & K. Chan & D. G. Mottershead & D. M. Robertson & J. G. Thompson & R. B. Gilchrist

Spontaneous pregnancies after in vitro maturation treatment in two polycystic ovary syndrome patients

Braam, S.; Maas, P.; van Kamp, A.; Mol, B.; de Bruin, J.
Fonte: Expert Reviews Publicador: Expert Reviews
Tipo: Artigo de Revista Científica
Publicado em //2012 Português
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Women with polycystic ovary syndrome who need IVF are at increased risk for ovarian hyperstimulation syndrome. Immature oocyte retrieval, and subsequent oocyte in vitro maturation (IVM), is an alternative that does not bear such a risk. We report two cases of spontaneous conception in women with polycystic ovary syndrome-related anovulation, who did not conceive after an IVM treatment. In both patients, spontaneous ovulation and conception occurred within 4 months after the IVM treatment. We hypothesize that this is a beneficial side effect of the ovarian puncture, comparable to the effect of ovarian drilling.; Sanne C Braam, Pettie H Maas, Anton A van Kamp, Ben Willem J Mol and Jan Peter de Bruin

Chromosome constitution of human embryos generated after in vitro maturation including 3-isobutyl-1-methylxanthine in the oocyte collection medium.

Spits, C.; Guzman, L.; Mertzanidou, A.; Jacobs, K.; Ortega-Hrepich, C.; Gilchrist, R.B.; Thompson, J.G.; De Vos, M.; Smitz, J.; Sermon, K.
Fonte: Oxford University Press (OUP) Publicador: Oxford University Press (OUP)
Tipo: Artigo de Revista Científica
Publicado em //2014 Português
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STUDY QUESTION: Do cleavage-stage embryos obtained from oocytes matured in vitro after pre-incubation with a phosphodiesterase inhibitor (IBMX) carry more chromosomal abnormalities than those generated from oocytes matured in vivo? SUMMARY ANSWER: The rate and type of chromosomal abnormalities in normally developing cleavage-stage embryos generated with an in vitro maturation (IVM) system including pre-incubation with IBMX are not different from those observed in supernumerary embryos obtained from oocytes matured in vivo. WHAT IS KNOWN ALREADY: Very limited information is available about the chromosomal constitution of IVM embryos. Previous studies were carried out using FISH on single biopsied blastomeres or arrested whole embryos and only provided fragmentary information on chromosomal abnormalities in IVM embryos. There is no systematic study of chromosomal abnormalities in all blastomeres of human Day 3 embryos with good morphology. STUDY DESIGN, SIZE, DURATION: Between July 2012 and December 2012, 16 young (age <35 years old) egg donors underwent 18 IVM cycles for the generation of research embryos. Eighteen embryos developed to Day 3 and were analysed using array comparative genomic hybridization (aCGH). PARTICIPANTS/MATERIALS...

Optimisation of oocyte in vitro maturation using oocyte secreted factors.

Sudiman, Jaqueline
Fonte: Universidade de Adelaide Publicador: Universidade de Adelaide
Tipo: Tese de Doutorado
Publicado em //2014 Português
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During follicular development, oocyte and somatic cells communicate by producing signals such as proteins, growth factors, hormones that interact in complex harmony. The oocyte plays an active role in this communication by secreting soluble oocyte secreted factors (OSFs) including growth differentiation factor 9 (GDF9) and bone morphogenetic factor 15 (BMP15). Those two growth factors have been implicated in follicular development and are related to female fertility. In vitro matured oocytes have aberrant gene expression and altered matrix protein profiles in cumulus cells compared to their in vivo mature counterparts, which leads to a decrease in oocyte quality and embryo development post fertilisation. The first aim of this study was to determine the effect of exogenous native OSFs from denuded oocytes (DOs) on mouse in vitro maturation (IVM). In a series of experiments, native OSFs were used in various ways to observe the role of FSH, cumulus cells, and temporal effects, on production of native OSFs. Overall, co-culture of COCs with DOs improved mouse embryo development. The highest improvement in embryo development and embryo quality post co-culture of COCs with DOs was when COCs were matured in separate IVM media in the presence of FSH...

The promise of in vitro maturation in assisted reproduction and fertility preservation

Smitz, J.; Thompson, J.; Gilchrist, R.
Fonte: Thieme Medical Publ Inc Publicador: Thieme Medical Publ Inc
Tipo: Artigo de Revista Científica
Publicado em //2011 Português
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An innovative approach to in vitro maturation (IVM) for application in infertility treatment and fertility preservation is required to bring this patient-friendly treatment into routine practice. Current approaches to IVM never report more than a 10 to 15% implantation rate per embryo transferred, which is two to three times lower and early pregnancy losses are higher than in conventional in vitro fertilization/intracytoplasmic sperm injection. The cornerstone of such an innovative culture technique is the use of pharmacological compounds that allow synchronization of nuclear and cytoplasmic maturation processes within the oocyte. The rationale of a prolonged oocyte maturation period is to promote a longer interaction between the immature oocyte with adequately conditioned cumulus cells. Successful introduction of a new approach to IVM will reduce the requirement of fertility hormones and will be less invasive to the patient's daily life by reducing the need for monitoring of serum hormone levels and intravaginal ultrasound. The new IVM conditions will reduce a whole range of minor and major complications in assisted reproductive technology and finally will also reduce the total cost for treatment. The minimal invasiveness of this procedure will benefit cancer patients who want to store gonadal tissue before undergoing therapy that devastates subsequent germ-cell competence.; http://www.ncbi.nlm.nih.gov/pubmed/21207332; Smitz...

Effect of Hormonal Supplementation Periods and In Vitro Maturation Media on Developmental Competence of Pig Oocytes

LIMA, Alecssandra Sobreira de; MARQUES, Mariana Groke; NASCIMENTO, Anibal Ballarotti; ASSUMPCAO, Mayra Elena Ortiz D'Avila; VISITIN, Jose Antonio
Fonte: UNIV FED RIO GRANDE DO SUL Publicador: UNIV FED RIO GRANDE DO SUL
Tipo: Artigo de Revista Científica
Português
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Background: The oocyte ability to undergo successful fertilization, cleavage and embryonic development depends on meiotic maturation and developmental competence acquisition. In vitro maturation (IVM) protocols currently use eCG, hCG or a combination of both, the effect of these gonadotrophins during IVM and subsequent embryonic development is still controversial. Several media have been used for IVM of porcine oocytes: TCM199, Whitten's and NCSU23 have also been shown to support pig oocyte IVM. This study was designed to determine the effect of hormonal supplementation period and maturation media during in vitro maturation of pig oocytes (1) and subsequent embryonic development (2). Materials, Methods & Results: Oocytes with intact cumulus oophurus layers and homogeneous cytoplasm were collected from prebubertal gilts. IVM was subjected in NCSU23, TCM199 or Whitten's media supplemented with 10 IU/mL eCG and 10 IU/mL hCG for the first 24 or 48 h of IVM. In each replicate the oocytes were fixed every 4 h from 32 to 48 h IVM or the past 48 h after IVM, oocytes were fertilized in vitro in mTBM medium for six hours and cultured in NCSU23 medium for nine days. Cleavage, blastocyst and hatching rates were evaluated at 48 h (day 2), 168 h (day 7) and 216 h (day 9)...

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

Ling,B.; Feng,D.Q.; Zhou,Y.; Gao,T.; Wei,H.M.; Tian,Z.G.
Fonte: Associação Brasileira de Divulgação Científica Publicador: Associação Brasileira de Divulgação Científica
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/11/2008 Português
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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...

Activation of 5' adenosine monophosphate-activated protein kinase blocks cumulus cell expansion through inhibition of protein synthesis during in vitro maturation in swine

Santiquet, N.; Sasseville, M.; Laforest, M.; Guillemette, C.; Gilchrist, R.B.; Richard, F.J.
Fonte: Society for the Study of Reproduction Publicador: Society for the Study of Reproduction
Tipo: Artigo de Revista Científica
Publicado em //2014 Português
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95.74%
The serine/threonine kinase 5' adenosine monophosphate-activated protein kinase (AMPK), a heterotrimeric protein known as a metabolic switch, is involved in oocyte nuclear maturation in mice, cattle, and swine. The present study analyzed AMPK activation in cumulus cell expansion during in vitro maturation (IVM) of porcine cumulus-oocyte complexes (COC). 5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) is a well-known activator of AMPK. It inhibited oocyte meiotic resumption in COC. Moreover, cumulus cell expansion did not occur in the presence of AICAR, demonstrating its marked impact on cumulus cells. Activation of AMPK was supported by AICAR-mediated phosphorylation of alpha AMPK subunits. Furthermore, the presence of AICAR increased glucose uptake, a classical response to activation of this metabolic switch in response to depleted cellular energy levels. Neither nuclear maturation nor cumulus expansion was reversed by glucosamine, an alternative substrate in hyaluronic acid synthesis, through the hexosamine biosynthetic pathway, which ruled out possible depletion of substrates. Both increased gap junction communication and phosphodiesterase activity in COC are dependent on protein synthesis during the initial hours of IVM; however...

Influência da temperatura de transporte de ovários na maturação in vitro de oócitos caninos coletados em diferentes estágios do ciclo estral; Effect of transport temperature of ovaries on in vitro maturation of canine oocytes collected in different stages of the estrous cycle

Pereira, Leda Maria Costa; Bersano, Paulo Ricardo Oliveira; Lopes, Maria Denise
Fonte: Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia Publicador: Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia
Tipo: info:eu-repo/semantics/article; info:eu-repo/semantics/publishedVersion; ; ; ; Formato: application/pdf
Publicado em 30/06/2015 Português
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This study evaluated the influence of estrous cycle stage and transport temperature of ovaries on in vitro maturation of canine oocytes. The bitches were categorized into two groups based on stage of estrus cycle: diestrus or anestrus. One ovary of each pair collected was transported in saline solution at 4°C while the other was transported at 37°C. Thus, ovarian tissue was sliced in PBS to release cumulus oocyte complexes (COCs). A total of 345 COCs (n = 186 oocytes from ovaries of bitches in anestrus and 159 in diestrus) were cultivated in TCM 199 supplemented with HEPES, sodium pyruvate, cysteine, follicle stimulating hormone (FSH), human chorionic gonadotropin (hCG), estrogen (E2) and epidermal growth factor (EGF). After 72h of maturation, the COCs were denuded, fixed and stained to assess nuclear maturation. The Fisher test was applied to examine the differences between the groups. The significance level adopted was 0.05. The oocytes obtained from the ovaries from bitches in diestrus transported at 4°C shown increased frequency of oocytes in metaphase II stage than those maintained at 37°C (p < 0.01). Similarly, there was increased frequency of oocytes in metaphase II (11.1%) stage from the ovaries of bitches in anestrus and transported at 4°C...

Efeito do fator de crescimento epidermal (EGF) na maturação in vitro de oócitos caninos; Effect of epidermal growth factor (EGF) on in vitro maturation of canine oocyte

Pereira, Leda Maria Costa; Bersano, Paulo Ricardo de Oliveira; Lopes, Maria Denise
Fonte: Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia Publicador: Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia
Tipo: info:eu-repo/semantics/article; info:eu-repo/semantics/publishedVersion; ; ; ; Formato: application/pdf
Publicado em 23/09/2014 Português
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Este trabalho teve o objetivo de avaliar a influência do EGF na maturação in vitro (MIV) de cadelas. Realizou-se o transporte dos ovários em solução de cloreto de sódio 0,9%, que foram seccionados em solução salina fosfato tamponado (PBS) e os complexos cumulus oócito (COCs) selecionados em meio de cultura de tecidos (TCM) 199 suplementado com HEPES. Foram coletados 405 oócitos grau 1 de cadelas em anestro e diestro. Os oócitos provenientes das duas fases do ciclo estral foram submetidos a dois tratamentos: meio com adição de 10 ηg/mL do fator de crescimento epidermal (EGF) (T) e meio sem suplementação (C). Depois de 72 horas de maturação, os COCs foram desnudados, fixados e corados com HOESCHT 33342 para avaliação da maturação nuclear. Os oócitos obtidos dos ovários da fase de diestro do grupo T demonstraram maior porcentagem (18,8%) de oócitos no estágio de metáfase II (M-II), em relação ao grupo C (1,3%) (p < 0,01). Nos oócitos obtidos da fase de anestro houve diferença (p < 0,05) entre os grupos, observando-se menor parcela (4,1%) de oócitos no estágio de vesícula germinativa (VG) no grupo T, quando comparado ao grupo C (16,1%). Comparando-se as diferentes fases reprodutivas, em meios suplementados com EGF...