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Vitro Maturation (vitro + maturation)

Distribution by Scientific Domains

Life Sciences	51%
Medical Sciences	48%

Selected Abstracts

Effects of Gonadotropins on In Vitro Maturation and of Electrical Stimulation on Parthenogenesis of Canine Oocytes

REPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2010
BS Kim
Contents The objective of this study was to determine the effects of gonadotropins on in vitro maturation (IVM) and electrical stimulation on the parthenogenesis of canine oocytes. In experiment I, cumulus oocyte complexes were collected from ovaries at a random phase of the oestrus cycle and cultured on maturation medium treated with hCG or eCG for 48 or 72 h. There were no significant differences in the effects on the metaphase II (MII) rate between the hCG and eCG treatment groups over 48 h (5.4% vs 5.5%). The MII rate in the co-treatment group of hCG and eCG for 48 h was higher than in each hormone treated group (15.5%, p < 0.05). In experiment 2, the parthenogenetic effect on oocyte development, at various electrical field strengths (1.0, 1.5, 2.0 kV/cm DC) for 60 or 80 ,s with a single DC pulse after IVM on the co-treatment of hCG and eCG, was examined. The rate of pronuclear formation (37.1%) in electrical activation at 1.5 kV/60 ,s without cytochalasin B (CB) was higher than that of oocytes activated in the other groups (p < 0.05). However, we did not observe the cleavage stages. Also, CB did not influence parthenogenesis of canine oocytes. The results showed that the pronucleus formation rate, indicative of the parthenogenesis start point, could be increased by electrical stimulation. Therefore, these results can provide important data for the parthenogenesis of canine oocytes and suggest the probability of parthenogenesis in canines. [source]

Transcriptional Analysis of Buffalo (Bubalus bubalis) Oocytes During In Vitro Maturation Using Bovine cDNA Microarray

REPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2010
OM Kandil
Contents The need for improving in vitro production of buffalo embryos necessitates a better understanding of the molecular mechanisms regulating early development including oocyte maturation. Here, we used bovine cDNA microarray platform to investigate mRNA abundance of buffalo oocytes before and after in vitro maturation. For this, a total of six pools each contains 50 immature or in vitro matured buffalo oocytes were used for mRNA isolation and subsequent cDNA synthesis. The BlueChip bovine cDNA microarray (with approximately 2000 clones) was used to analyse gene expression profiles between immature and matured oocytes. Statistical analysis of microarray data revealed a total of 104 transcripts to be differentially expressed between the two oocyte groups. Among these, transcription factors (ZFP91), M-phase mitotic cell cycle (MPHOSPH9), growth factor (BMP15) and DNA binding (HMGN2) were found to be up-regulated in immature oocytes. Similarly, matured oocytes were found to be enriched with genes involved in cytoskeleton (ACTB), hydrogen ion transporting (ATP6V1C2) and structural constituent of ribosome (RPS27A). Quantitative real-time polymerase chain reaction validated the expression profile of some selected transcripts during array analysis. In conclusion, to our knowledge, this is the first large-scale expression study to identify candidate genes differentially abundant and with potential role during buffalo oocyte maturation. [source]

Elevated Histone H1 (MPF) and Mitogen-activated Protein Kinase Activities in Pig Oocytes Following In Vitro Maturation do not Indicate Cytoplasmic Maturation

REPRODUCTION IN DOMESTIC ANIMALS, Issue 2 2009
MA Setiadi
Contents Effects of different media (TCM 199 + BSA, TCM 199 + FCS, TCM 199 + NBCS, Whitten's medium + BSA) supplemented with estradiol-17, and two isolated and everted follicle shells on MPF and MAP kinase activities and the sensitivity to parthenogenetic activation of pig oocytes were examined at the end of culture (48 h). Elevated (P < 0.05) activities of MAP kinase were recorded in metaphase II oocytes following culture in Whitten's medium, whereas MPF levels were lowest (P < 0.05) in MII oocytes matured in TCM 199 supplemented with BSA. Oocytes matured in TCM 199 based media showed higher (P < 0.05) activation rates when compared to oocytes incubated in Whitten's medium. Whitten's medium supplemented with different protein sources (amino acids, FCS, BSA) was used to study the effects of different exposure periods to eCG/hCG stimulation on MPF and MAP kinase activities and in vivo fertilisability following culture for 48 h. MPF and MAP kinase activities were significantly increased by eCG/hCG stimulation of COCs during maturation. Further, the continuous presence of eCG/hCG during culture (48 h) significantly increased the levels of both kinases in comparison to stimulation by gonadotrophins alone during the first 24 h of incubation. In vivo fertilisation of oocytes matured in Whitten's medium supplemented with eCG/hCG for 24 or 48 h led to a significant retardation of early embryonic development compared to ovulated oocytes. In conclusion, media composition and gonadotrophin stimulation affect MPF/MAP kinase activities and the susceptibility to parthenogenetic activation of IVM oocytes. However, elevated kinase levels in pig oocytes following culture do not indicate complete cytoplasmic maturation. [source]

Chromatin Configurations in the Ferret Germinal Vesicle that Reflect Developmental Competence for In Vitro Maturation

REPRODUCTION IN DOMESTIC ANIMALS, Issue 2 2009
X Sun
Contents In several mammalian species, the configuration of germinal vesicle (GV) chromatin correlates with the developmental competence of oocytes. Yet, no study has been published on the configuration of GV chromatin in ferret, nor is it known whether a specific configuration predicts meiotic competence in this species, in spite of the potential importance of ferret cloning to the study of human disease and to species conservation efforts. Here, we report on an analysis of the chromatin configuration in ferret GV oocytes and on how they correlate with meiotic development. Three distinct configurations were identified based on the degree of chromatin condensation: (1) fibrillar chromatin (FC), featuring strands of intertwined chromatin occupying most of the visible GV region; (2) intermediate condensed chromatin (ICC), characterized by dense, irregular chromatin masses throughout the GV; and (3) condensed chromatin (CC), which is highly compact and centered around the nucleolus. We also found that chromatin configuration was related to the extent of association with cumulus cells in cumulus,oocyte complexes; CC-configured oocytes were most often surrounded by a compact cumulus layer and also a compact corona but FC-configured oocytes were associated with neither. In addition, increasing chromatin condensation corresponded to an increase in oocyte diameter. Finally, following in vitro culture, significantly more CC-configured oocytes underwent maturation to meiotic metaphase II than did FC- or ICC-configured oocytes. We conclude that, in ferret, chromatin condensation is related to the sequential achievement of meiotic competencies during oocyte growth and differentiation, and thus can be used as a predictor of competence. [source]

Development of a Competitive Quantitative PCR Strategy for Evaluating the Expression Stability of 18s rRNA during In Vitro Maturation of Buffalo (Bubalus bubalis) Follicular Oocytes

REPRODUCTION IN DOMESTIC ANIMALS, Issue 2 2007
APS Aswal
Contents The present work describes the development of a quantitative competitive PCR strategy for quantifying the relative abundance of 18s rRNA transcripts in buffalo oocytes during in vitro maturation (IVM). As a method, the competitive PCR overcomes some of the shortcomings of conventional reverse transcriptase polymerase chain reaction (RT-PCR) procedure making it a more authentic quantitative method. A composite primer based approach was used to generate the competitor cDNA to be used as external control. Validity of the method for its efficiency was demonstrated by quantitative analysis of the competition parameters. Using this method the relative abundance of buffalo oocyte 18s rRNA transcript over the period of IVM was found to vary within a narrow range of 0.93,1.06 folds which establishes the accuracy of the method and reflects the stability of its expression during IVM. This qualifies the use of this house keeping gene as a valid internal control in studies investigating the gene expression pattern in buffalo oocytes. The competitive PCR approach described in this study could be used for quantification of other transcripts from a limited number of oocytes where a conventional RT-PCR method is either difficult to use or multiplexing it with highly abundant house keeping genes is apparently problematic. [source]

Effect of Alpha-Tocopherol and Ascorbic Acid on Bovine Oocyte in Vitro Maturation

REPRODUCTION IN DOMESTIC ANIMALS, Issue 2 2005
G Dalvit
Contents In vitro culture results in higher oxygen concentrations than in vivo environments, leading to an increased level of reactive oxygen species (ROS) that cause lipid peroxidation of cellular membranes. Alpha-tocopherol (active form of vitamin E) is an antioxidant that protects mammalian cells against lipid peroxidation, which is regenerated by ascorbic acid. The aim of this study was to determine the effect of the addition of alpha-tocopherol and/or ascorbic acid to the maturation medium on bovine oocyte in vitro maturation (IVM) and subsequently on in vitro fertilization (IVF) and embryo development. Cumulus,oocyte complexes (COCs) were matured in Medium 199 (control), and with the addition of alpha-tocopherol and/or ascorbic acid. The concentration of alpha-tocopherol in COCs was determined by high-performance liquid chromatography (HPLC). IVF and in vitro culture (IVC) were carried out in modified synthetic oviductal fluid (mSOF). The quantity of alpha-tocopherol naturally present in COCs diminished by half during IVM (p < 0.05), although in the presence of ascorbic acid it remained constant. A greater amount of alpha-tocopherol was detected in COCs matured in medium supplemented with this antioxidant (p < 0.05), but the addition of alpha-tocopherol plus ascorbic acid maintained higher levels of alpha-tocopherol (p < 0.05). Significant differences were not observed in the percentages of nuclear maturation and fertilization among different treatments. The presence of alpha-tocopherol or ascorbic acid in the maturation medium failed to modify the percentage of blastocysts obtained, unlike the addition of both antioxidants when a significant decrease was observed (p < 0.05). Absorbic acid maintained the antioxidant capacity of the alpha-tocopherol incorporated to COC membranes during IVM. The active form of vitamin E during maturation impaired the acquisition of oocyte developmental competence. [source]

Comparison of In Vitro Maturation, In Vitro Fertilization, Metabolism and Ultrastructure of Oocytes from Prepubertal and Adult Pigs

REPRODUCTION IN DOMESTIC ANIMALS, Issue 3-4 2000
JK O'Brien
Contents In vitro maturation and fertilization, metabolism and ultrastructure were examined for oocytes from prepubertal and adult pigs. The proportion of oocytes undergoing in vitro maturation was lower for oocytes derived from prepubertal (piglets aged 8,10 weeks; 59.8% and gilts aged 5,6 months; 79.2%) than for those from adult ( ows aged 4,6 years; 92.2%) pigs (p < 0.05). A higher proportion of oocytes from piglets (22.8%) degenerated during in vitro maturation than from gilts (4.8%) and adult pigs (4.7%; p < 0.05). There was an increased incidence of polyspermic fertilization in prepubertal (piglet; 52.2%, gilt; 23.8%) compared with adult (7.1%) oocytes (p < 0.05). There were no differences in the metabolism of glucose, glutamine or pyruvate between oocytes from prepubertal and adult pigs. The metabolism of glutamine by prepubertal and adult oocytes matured in vitro was significantly higher than that by prepubertal oocytes matured in vivo (p < 0.05). Oocyte size and ultrastructure was also similar for oocytes from prepubertal and adult pigs. These data show that prepubertal and adult pig oocytes have a different in vitro developmental capacity, but similar metabolic activity and ultrastructure, under the conditions investigated. [source]

Improving in vitro Maturation of Oocytes in the Human Taking Lessons from Experiences in Animal Species

REPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2001
J Smitz
One to three per cent of infertile women develop severe ovarian hyperstimulation syndrome after superovulation for assisted reproduction treatment (ART). This severe complication can be avoided when oocytes are obtained at an immature stage (germinal vesicle stage) out of small or medium-sized follicles. This hypothesis has been tested in several infertile women, but clinical pregnancies are disappointlingly low. This new approach in ART is still at an experimental phase and this treatment has still to be improved before routine clinical application. Experimental work in animals and humans suggest a beneficial effect in providing a short preliminary pretreatment with follicle-stimulating hormone to select for a developing cohort of follicles. The aspiration of oocyte cumulus complexes is carried out with a short needle applying reduced aspiration pressure. A crucial point is to provide the appropriate culture environment for the immature oocytes. An optimal cumulus-enclosed human oocyte culture system needs to be defined. The composition of the culture medium could be suggested by in vitro work carried out in animal models. As developmental competence is established during the latest phases of oocyte growth and is dependent on the storage of RNA, a prolonged in vitro maturation period (before inducing nuclear maturation) could provide the necessary transcriptional and translational changes. The conditions to achieve this improved cytoplasmic maturation by prolonging the in vitro culture remain to be defined. More objective noninvasive parameters for oocyte maturity are also needed to pursue research in this field. [source]

Protein kinase C, is differentially activated during neonatal and adult erythropoiesis and favors expression of a reporter gene under the control of the A, globin-promoter in cellular models of hemoglobin switching

JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2007
Angela Di Baldassarre
Abstract PKC, was found to be expressed (mRNA and protein) throughout the in vitro maturation of primary human erythroblasts but its activity (phosphorylation levels and nuclear localization) was consistently higher in cells derived from human neonatal rather than adult blood. Since the ,/,,+,, globin expression ratio represented the major difference between neonatal and adult erythroblasts (58,±,12 vs. 7,±,3, respectively), we tested the hypothesis that PKC, might affect ,-globin expression by measuring the levels of A,- or ,-promoter-driven reporter activity in erythroid cells stably (GM979) or transiently (K562, primary adult and neonatal erythroblasts) transfected with a dual µLCR,prRlucA,prFluc reporter in the presence of transient expression of either the constitutively active (sPKC,) or catalytically inactive (iPKC,) PKC,. As further control, GM979 cells were incubated with the PKC inhibitor rottlerin (30 µM). In all the cells analyzed, sPKC, significantly increased (by two- to sixfold) the levels of luciferase activity driven by the A,-promoter and the A,-F/(A,-F,+,2,-R) expression ratio. In GM979 cells, rottlerin inhibited (by 50%) the A,-driven luciferase activity and the A,-F/(A,-F,+,2,-R) expression ratio. These results suggest that different PKC isoforms may exert ontogenetic-specific functions in erythropoiesis and that modulation of PKC, might affect the activity of A,-promoter-driven reporters. J. Cell. Biochem. 101: 411,424, 2007. © 2007 Wiley-Liss, Inc. [source]

Recent Discoveries on the Control of Gonadotrophin-Releasing Hormone Neurones in Nonhuman Primates

JOURNAL OF NEUROENDOCRINOLOGY, Issue 7 2010
E. Terasawa
Since Ernst Knobil proposed the concept of the gonadotrophin-releasing hormone (GnRH) pulse-generator in the monkey hypothalamus three decades ago, we have made significant progress in this research area with cellular and molecular approaches. First, an increase in pulsatile GnRH release triggers the onset of puberty. However, the question of what triggers the pubertal increase in GnRH is still unclear. GnRH neurones are already mature before puberty but GnRH release is suppressed by a tonic GABA inhibition. Our recent work indicates that blocking endogenous GABA inhibition with the GABAA receptor blocker, bicuculline, dramatically increases kisspeptin release, which plays an important role in the pubertal increase in GnRH release. Thus, an interplay between the GABA, kisspeptin, and GnRH neuronal systems appears to trigger puberty. Second, cultured GnRH neurones derived from the olfactory placode of monkey embryos exhibit synchronised intracellular calcium, [Ca2+]i, oscillations and release GnRH in pulses at approximately 60-min intervals after 14 days in vitro (div). During the first 14 div, GnRH neurones undergo maturational changes from no [Ca2+]i oscillations and little GnRH release to the fully functional state. Recent work also shows GnRH mRNA expression increases during in vitro maturation. This mRNA increase coincides with significant demethylation of a CpG island in the GnRH 5,-promoter region. This suggests that epigenetic differentiation occurs during GnRH neuronal maturation. Third, oestradiol causes rapid, direct, excitatory action in GnRH neurones and this action of oestradiol appears to be mediated through a membrane receptor, such as G-protein coupled receptor 30. [source]

N-methyl-D-aspartate-stimulated ERK1/2 signaling and the transcriptional up-regulation of plasticity-related genes are developmentally regulated following in vitro neuronal maturation

JOURNAL OF NEUROSCIENCE RESEARCH, Issue 12 2009
Xianju Zhou
Abstract The general features of neuroplasticity are developmentally regulated. Although it has been hypothesized that the loss of plasticity in mature neurons may be due to synaptic saturation and functional reduction of N-methyl-D-aspartate receptors (NMDAR), the molecular mechanisms remain largely unknown. We examined the effects of NMDAR activation and KCl-mediated membrane depolarization on ERK1/2 signaling following in vitro maturation of cultured cortical neurons. Although NMDA stimulated a robust increase in intracellular calcium at both DIV (day in vitro) 3 and 14, the activation of ERK1/2 and cAMP responsive element-binding protein (CREB) was impaired at DIV 14. Specifically, the phosphorylation of ERK1/2 was stimulated by both NMDA and KCl at DIV 3. However, at DIV 14, NMDA- but not KCl-stimulated ERK1/2 and CREB phosphorylation was significantly diminished. Consistently, the NMDA-induced transcription of ERK/CREB-regulated genes Bdnf exon 4, Arc, and zif268 was significantly attenuated at DIV 14. Moreover, in comparison with 3 DIV neurons, the phosphorylated-ERK1/2 in 14 DIV neurons displayed a tremendous increase following maturation and was more susceptible to dephosphorylation. Blocking calcium channels by nifedipine or NMDAR by APV caused a more dramatic ERK dephosphorylation in 14 DIV neurons. We further demonstrate that the loss of plasticity-related signaling is unrelated to NMDA-induced cell death of the 14 DIV neurons. Taken together, these results suggest that the attenuation of certain aspects of neuroplasticity following maturation may be due to the reduction of NMDAR-mediated gene transcription and a saturation of ERK1/2 activity. © 2009 Wiley-Liss, Inc. [source]

Behaviors of ATP-dependent chromatin remodeling factors during maturation of bovine oocytes in vitro

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2010
Gabbine Wee
The mammalian oocyte undergoes dynamic changes in chromatin structure to reach complete maturation. However, little known is about behaviors of ATP-dependent chromatin remodeling factors (ACRFs) during meiosis. Here, we found that respective ACRFs may differently behave in the process of oocyte maturation in the bovine. All ACRFs interacted with oocytic chromatin at the germinal vesicle (GV) stage. Mi-2 and hSNF2H disappeared from GV-chromatin within 1,hr of in vitro culture whereas Brg-1 and BAF-170 were retained throughout germinal vesicle break down (GVBD). Brg-1 was localized on the condensed chromatin outside, whereas BAF-170 was entirely excluded from condensed chromatin. Thereafter, Brg-1 and BAF-170 interacted with metaphase I and metaphase II chromosomes. These results imply that Mi-2 and hSNF2H may initiate the meiotic resumption, and Brg-1 and BAF-170 may support chromatin condensation during meiosis. In addition, DNA methylation and methylation of histone H3 at lysine 9 (H3K9) seem to be constantly retained in the oocyte chromatin throughout in vitro maturation. Inhibition of ACRF activity by treatment with the inhibitor apyrase led to retarded chromatin remodeling in bovine oocytes, thereby resulting in poor development of fertilized embryos. Therefore, these results indicate that precise behaviors of ACRFs during meiosis are critical for nuclear maturation and subsequent embryonic development in the bovine. Mol. Reprod. Dev. 77: 126,135, 2010. © 2009 Wiley-Liss, Inc. [source]

Molecular characterization and polyadenylation-regulated expression of cyclin B1 and Cdc2 in porcine oocytes and early parthenotes

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 1 2010
Ding-Xiao Zhang
Meiotic maturation of mammalian oocytes is controlled by the maturation/M-phase promotion factor (MPF), a complex of Cdc2 kinase and cyclin B protein. To better understand the molecular mechanism of oocyte maturation, we characterized porcine cyclin B1 and Cdc2 genes, both of which are widely expressed in pig tissues. We further analyzed their expression profiles during in vitro maturation of pig oocyte and early embryonic development at both the mRNA and protein level. Two isoforms of cyclin B1, comprising the same open reading frame but differing in 3,-UTR length, were identified. Cyclin B1 transcripts was up-regulated after 30,hr of maturation, while Cdc2 mRNA levels were unchanged during maturation except for a sharp decline at 44,hr. Cyclin B1 protein synthesis increased with oocyte maturation. Cdc2 protein expression was relatively low during 0,18,hr, followed by a higher level of expression up to 44,hr of maturation. Poly(A)-test PCR clearly revealed that both cyclin B1 isoforms underwent cytoplasmic polyadenylation starting around 18,24,hr during maturation, while a substantial de-adenylation and degradation of Cdc2 isoforms were observed in metaphase II oocytes and during embryo development after parthenogenetic activation. Porcine MII oocytes derived from small follicles (,3,mm) and bad quality 2-cell parthenotes showed lower developmental competence and lower levels of cyclin B1 protein, and Cdc2 mRNA or both gene mRNAs, respectively, compared to their control counterparts. These results suggested that cyclin B1 was regulated posttranscriptionally by cytoplasmic polyadenylation during porcine oocyte maturation. Further, the decreased expression of maternal cyclin B1 and Cdc2 at the mRNA or protein level in developmentally incompetent oocytes and embryos was responsible for, at least in part, a profound defect in further embryonic development. Mol. Reprod. Dev. 77: 38,50, 2010. © 2009 Wiley-Liss, Inc. [source]

Factors affecting the in vitro action of cumulus cells on the maturing mouse oocytes

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 1 2008
Li Ge
Abstract The removal of cumulus cells (CCs) from oocytes at the germinal vesicle (GV) stage still represents a major limitation in such embryo techniques as GV transfer, somatic cell haploidization, and oocyte cryopreservation. However, no efficient in vitro maturation (IVM) system for CC-denuded oocytes (DOs) has been established in mammalian species. Although follicular cells are considered to play an important role in oocyte maturation, the specific role and mechanisms of action of different cell types are poorly understood. Reports on whether junctional association between CCs and the oocyte is essential for the beneficial effect of CC co-culture on oocyte maturation are in conflict. Our objective was to try to address these issues using the mouse oocyte model. The results indicated that while co-culture with the CC monolayer could only partially restore the developmental potential of DOs without corona cells, it restored the competence of corona-enclosed DOs completely. Culture in medium conditioned with CC monolayer also promoted maturation of DOs. However, co-culture with the monolayer of mural granulosa cells had no effect. The efficiency of CC co-culture was affected by various factors such as density and age of the CCs, the presence of gonadotropin in the maturation medium and the duration for in vivo (IVO) gonadotropin priming. It is concluded that mouse CCs produce a diffusible factor(s) that support DO maturation in a CC-oocyte junctional communication dependent manner. The data will contribute to our understanding the mechanisms by which CCs promote oocyte maturation and to the establishment of an efficient DO IVM system. Mol. Reprod. Dev. 75: 136,142, 2008. © 2007 Wiley-Liss, Inc. [source]

Effects of thiol compounds on in vitro maturation of canine oocytes collected from different reproductive stages

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 9 2007
Mohammad Shamim Hossein
Abstract Various thiol compounds are known to improve cytoplasmic and/or nuclear maturation of oocytes in vitro. The present study examined the effects of two thiol compounds, cysteine (0.1, 0.5, and 1.0 mM) and cysteamine (50, 100, and 200 µM), on cytoplasmic and nuclear maturation of canine oocytes. Oocytes collected from different reproductive stages were cultured in TCM-199 supplemented with 10% fetal bovine serum, 2.2 mg/ml sodium carbonate, 2.0 µg/ml estrogen, 0.5 µg/ml FSH, 0.03 IU/ml hCG, and 1% penicillin,streptomycin solution for 72 h. Data were analyzed by two-way ANOVA after arcscine transformation and protected by Bonferroni post hoc test. The effects of cysteine and cysteamine on canine IVM were varied depending on the reproductive stage of oocyte donor bitches. In the follicular stage, significantly more oocytes reached the metaphase II (M II) stage when cultured with 0.5 or 1.0 mM cysteine (16.7% and 16.9%, respectively) compared to the control (6.2%). In the follicular stage, cysteamine increased oocyte maturation rate upto the M II stage (15.1% to 17.0%) compared to the control (4.4%). Both the 0.5 mM cysteine and 100 µM cysteamine, alone or together, increased the intracellular GSH level of canine oocytes compared to the control. Irrespective of reproductive stage, no further beneficial effects on nuclear or cytoplasmic maturation were observed when 0.5 mM cysteine and 100 µM cysteamine were supplemented together. In conclusion, addition of 0.5 mM cysteine and 100 µM cysteamine to the maturation medium improved IVM of canine oocytes. Mol. Reprod. Dev. 74: 1213,1220, 2007. © 2007 Wiley-Liss, Inc. [source]

Reduced oxygen concentration improves the developmental competence of mouse oocytes following in vitro maturation

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 7 2007
Kimberly A. Preis
Abstract Reduced atmospheric oxygen concentration is beneficial to embryo development; however, optimal oxygen concentration for oocyte maturation remains undetermined. Likewise, there is no consensus of appropriate medium supplementation during maturation. The objective of this study was to determine whether oxygen tension (20% or 5% O2) and epidermal growth factor (EGF) affect oocyte metabolism and subsequent embryo development. Cumulus-oocyte complexes (COCs) were collected from 28-day-old equine chorionic gonadotropin (eCG) primed or unprimed F1 (C57BL/6xCBA) mice. COCs were matured in defined medium in one of four groups: 20% O2, 20% O2,+,EGF, 5% O2, 5% O2,+,EGF. In vivo matured COCs were also collected for analysis. COCs from unprimed mice, matured in 5% O2,±,EGF or 20% O2,+,EGF had higher metabolic rates than COCs matured in 20% O2 (P,<,0.05). COCs from primed mice had higher metabolic rates when matured in the presence of EGF, regardless of oxygen tension (P,<,0.01). Oxygen uptake and mitochondrial membrane potential were higher for in vivo matured oocytes and oocytes matured under 5% O2 compared to oocytes matured under 20% O2 (P,<,0.05). Blastocyst formation was not different between maturation groups (primed or unprimed); however, embryo cell numbers were 20,45% significantly higher when COCs were matured at 5% O2 (P,<,0.05). Results suggest that oocytes matured in physiological concentrations of oxygen have improved development and metabolic activity, more closely resembling in vivo maturation. These findings have implications for oocyte maturation in both clinical and research laboratories. Mol. Reprod. Dev. 74: 893,903, 2007. © 2006 Wiley-Liss, Inc. [source]

Mitochondrial organization in prepubertal goat oocytes during in vitro maturation and fertilization

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 5 2006
Esther Velilla
Abstract The aim of this study was to evaluate mitochondrial distribution during in vitro maturation (at 0, 15, 20, and 27 hr of IVM) and fertilization of prepubertal goat oocytes compared to mitochondrial distribution of ovulated and in vitro fertilized oocytes from adult goats. Oocytes from prepubertal goats were recovered from a slaughterhouse and were matured in M199 with hormones and serum for 27 hr. Ovulated oocytes were collected from gonadotrophin-treated Murciana goats. Frozen-thawed spermatozoa were selected by centrifugation in Percoll gradient and were capacitated in DMH with 20% steer serum for 1 hr. Ovulated and IVM-oocytes were inseminated in DMH medium with steer serum and calcium lactate for 20 hr. Oocytes and presumptive zygotes were stained with Mitotraker Green FM and observed under a confocal laser scanning microscope. Ultrastructural morphology of oocytes and presumptive zygotes were analyzed by transmission electron microscopy (TEM). Prepubertal goat oocytes at germinal vesicle stage (GV) presented mitochondria localized in the cortical and perinuclear region. IVM-oocytes at metaphase II presented mitochondria peripheral polarized to the region opposite were the metaphase spindle is positioned and within the polar body. Ovulated oocytes presented peripheral mitochondria distribution and mitochondrial aggregation around the MII spindle. At 20 hr post-insemination, mitochondria were distributed around the two synchronous pronuclei (2PN rpar; in zygotes ovulated oocytes whereas in prepubertal 2PN-zygotes mitochondria presented a peripheral polarized distribution. Images by TEM detected that immature prepubertal goat oocytes that are less electrodense and present fewer cristae than in vitro matured prepubertal goat oocytes; these are characterized by being associated to swollen vesicles. Mol. Reprod. Dev. 73: 617,626, 2006 © 2006 Wiley-Liss, Inc. [source]

EGF-induced EGF-receptor and MAP kinase phosphorylation in goat cumulus cells during in vitro maturation

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2005
Laurence Gall
Abstract EGF has been shown to influence meiotic maturation and development competence of oocyte in various mammalian species. We previously reported, in goat, that the EGF receptor (EGF-R) was present both on cumulus cells and oocytes. Here, EGF-induced signaling was investigated during the in vitro maturation process in goat cumulus,oocyte complexes (COCs). Cumulus cells and oocytes were subjected to Western immunoblotting analysis using anti-MAP kinase, anti-phosphotyrosine, anti-phospho MAP kinase, and anti-phospho EGF-R antibodies. We demonstrated that treatment with EGF during the in vitro maturation process induced rapid tyrosine phosphorylation of EGF-R in a time and concentration dependent manner in cumulus cells. A similar pattern of activation by phosphorylation was observed for MAP kinase upon EGF stimulation. AG 1478, an inhibitor of the EGF kinase, suppressed EGF-stimulated phosphorylation of EGF-R and also affected the MAP kinase activation. Treatment with the MEK inhibitor PD 98059 abolished EGF-induced MAP kinase activation. We did not observe oocyte EGF-R phosphorylation in our experiments during the in vitro maturation process. Our data indicate, in goat cumulus cells, that activation of EGF-R by EGF triggers signaling through the MAP kinase pathway during in vitro maturation. This supports the hypothesis that the major site of action for EGF, that regulates oocyte maturation, is the cumulus cell. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc. [source]

Expression of peroxiredoxins in bovine oocytes and embryos produced in vitro

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 3 2004
Gregory Leyens
Abstract Peroxiredoxins (PRDXs) form a family of peroxidases involved in antioxidant protection and cell signaling. Due to their peroxide reductase activity, these enzymes might be involved in fine-tuning peroxide levels in embryos during in vitro production. In this study, RT-PCR was used to examine the expression of the six PRDX isoforms (PRDX1 to PRDX6) in bovine oocytes and embryos. PRDXs were detected in oocytes both before and after in vitro maturation. Besides, PRDX6 was up-regulated after maturation. Single embryos were analyzed from the two-cell to the blastocyst stages. PRDX1 and PRDX5 transcripts were detected throughout development. PRDX2, PRDX3, and PRDX6 were not expressed around the 9- to 16-cell stage. PRDX4 transcripts were weakly detected in pools of embryos from the 9- to 16-cell stage onwards. In situ immunodetection of PRDX5, which was previously reported to exhibit the widest subcellular distribution among PRDXs in adult mammalian cells, showed a mitochondrial distribution pattern in the bovine embryo. Finally, the potential modulation by oxidative stress of PRDX expression around the major embryonic genome activation was evaluated by culturing embryos under 20% O2 instead of 5%. No significant difference in the pattern of PRDX expression was observed under 20% O2. In conclusion, our data show for the first time that PRDXs are expressed in mammalian oocytes and early embryos. Moreover, the bovine transcripts exhibit various patterns of expression that might be related to the potential role of PRDXs in oocyte maturation and embryo development. Mol. Reprod. Dev. 69: 243,251, 2004. © 2004 Wiley-Liss, Inc. [source]

Effect of macromolecule supplementation during in vitro maturation of goat oocytes on developmental potential

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 3 2004
J.R. Herrick
Abstract In vitro maturation (IVM) of goat oocytes with serum-supplemented media results in oocytes with reduced developmental potential. The objective of this study was to develop a defined medium for IVM of goat oocytes that better supports subsequent embryonic development. Cumulus oocyte complexes (COC) were matured for 18,20 hr in: Experiment (1), tissue culture medium 199 (TCM199) with 10% (v/v) goat serum or modified synthetic oviduct fluid maturation medium (mSOFmat) with 2.5, 8.0, or 20.0 mg/ml bovine serum albumin (BSA); Experiment (2), mSOFmat with 4.0, 8.0, 12.0, or 16.0 mg/ml BSA; or Experiment (3), 1.0 mg/ml polyvinyl alcohol (PVA; control), 4.0 mg/ml BSA, 0.5 mg/ml hyaluronate plus 0.5 mM citrate, or hyaluronate, citrate, and BSA. Mature COC were coincubated for 20,22 hr with 12,15,×,106 sperm/ml in modified Brackett and Oliphant (mBO) medium. Embryos were cultured for a total of 7 days in G1/2, and evaluated for cleavage, and blastocyst development, hatching, and total cell numbers. In the first experiment, more (P,<,0.05) blastocysts developed per cleaved embryo following maturation in mSOFmat with 2.5 or 8.0 mg/ml BSA than with 20.0 mg/ml BSA or TCM199 with 10% goat serum. The various concentrations of BSA used in the second experiment did not affect (P,>,0.05) any of the developmental endpoints examined. In the third experiment, developmental potential of oocytes matured with PVA or hyaluronate with citrate was not different (P,>,0.05) from oocytes matured in the presence of BSA. These results demonstrate that developmentally competent goat oocytes can be matured under defined conditions. Mol. Reprod. Dev. 69: 338,346, 2004. © 2004 Wiley-Liss, Inc. [source]

Hypoxanthine (HX) inhibition of in vitro meiotic resumption in goat oocytes

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 3 2003
Suofeng Ma
Abstract To improve in vitro maturation and to understand the mechanism for meiotic resumption of oocytes, meiotic progression, and its control by hypoxanthine (HX) were studied in goat oocytes. Ovaries were obtained from a local abattoir, and cumulus,oocyte complexes (COCs) and follicular fluid were collected from follicles of different surface diameters (SDs). The meiotic competence and progression of oocytes were observed, and the concentration of HX in the follicular fluid and culture media was measured by high-performance liquid chromatography (HPLC). Full meiotic competence of goat oocytes was acquired in follicles of ,1.5 mm in SD with 90% of the oocytes developing to metaphase II (MII) stage after 24 hr in culture. The HX concentration in follicular fluid decreased with follicle development, from the highest level of 1.16 mM in ,0.5 mm follicles to the lowest level of 0.45 mM in ,5 mm follicles. HX inhibited meiotic resumption of goat oocytes in a concentration-related manner but this inhibitory effect declined gradually. When we renewed the medium at 4 hr of HX-199 (TCM-199 supplemented with 4 mM HX) culture, the percentage of oocytes with intact germinal vesicle (GV) did not increase but decreased significantly instead. HPLC measurement of HX in the HX-199 culture drops indicated that the HX concentration declined from 0 hr to 4 hr of culture and after medium renewal at 4 hr of culture. By adding dibutyryl cAMP (db-cAMP) at medium renewal, we found that db-cAMP held up the decline of GV percentages. Together, these results were consistent with the possibility that the decline of HX inhibitory effect was not due to HX depletion but rather due to the negative feedback of the metabolites on its further uptake by oocytes. Goat oocytes were capable of normal nuclear maturation and activation after temporal arrest by HX, but prolonged exposure to HX induced spontaneous activation. Mol. Reprod. Dev. 66: 306,313, 2003. © 2003 Wiley-Liss, Inc. [source]

Effects of meiosis-inhibiting agents and equine chorionic gonadotropin on nuclear maturation of canine oocytes

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2003
N. Songsasen
Abstract Experiments were conducted to determine the effects of meiosis-inhibiting-agents and gonadotropins on nuclear maturation of canine oocytes. The culture medium was TCM199,+,10 ng/ml epidermal growth factor supplemented with 25 ,M ,-mercaptoethanol, 0.25 mM pyruvate, and 1.0 mM L-glutamine (Basal TCM). Initially, oocytes were cultured in Basal TCM alone or in Basal TCM,+,dibutylryl cyclic adenosine monophosphate (0.5, 1, 5, or 10 mM dbcAMP) for 24 hr. Dibutylryl cAMP inhibited resumption of meiosis in a dose-dependent manner; 60% of oocytes remained at the germinal vesicle (GV) stage after being cultured for 24 hr in 5 mM dbcAMP. The meiosis-inhibitory effect of dbcAMP appeared to be reversible, as the oocytes resumed meiosis and completed nuclear maturation after being cultured for an additional 48 hr in its absence. Oocytes were then cultured in Basal TCM alone or in Basal TCM,+,roscovitine (12.5, 25, or 50 ,M) for 24 hr. Although ,60% of oocytes cultured in 25 ,M roscovitine remained at the GV stage, this percentage was not significantly different from the 48% that also remained at the GV stage when cultured in its absence. Oocytes were cultured in Basal TCM,+ 25 ,M roscovitine for 17 hr, exposed briefly to equine chorionic gonadotropin (eCG), and then cultured in Basal TCM for 48 hr. Short exposure of oocytes to eCG was beneficial, as it significantly increased the proportion of oocytes developing beyond germinal vesicle breakdown (P,<,0.05) with ,20,30% of these were metaphase I (MI) oocytes. Study of the kinetics of nuclear maturation demonstrated that large numbers of oocytes remained at MI even after being cultured for 52 hr following brief exposure to eCG. This study showed that in vitro maturation of canine oocytes can be somewhat improved by short exposure of oocytes to eCG. However, further studies are still required to derive effective methods to mature canine oocytes in vitro. Mol. Reprod. Dev. 65: 435,445, 2003. © 2003 Wiley-Liss, Inc. [source]

Synergistic Effect of Porcine Follicular Fluid and Dibutyryl Cyclic Adenosine Monophosphate on Development of Parthenogenetically Activated Oocytes from Pre-Pubertal Gilts

REPRODUCTION IN DOMESTIC ANIMALS, Issue 5 2010
AB Nascimento
Contents This study investigated the effect of porcine follicular fluid (PFF) and dibutyryl cyclic adenosine monophosphate (dbcAMP) during in vitro maturation (IVM) of porcine oocytes on meiotic maturation, fertilization and embryo development, and compared the effect of supplementing the embryo culture media with PFF or foetal bovine serum (FBS) on embryo development. Oocytes from pre-pubertal gilts were IVM for 44 h, and parthenogenetically activated or in vitro -fertilized. Embryos were cultured in porcine zygote medium (PZM3) for 7 days. Cleavage and blastocyst rates were evaluated at 48 h and 7 days of culture. The supplementation of the IVM medium with 25% PFF and 1 mm dbcAMP for the first 22 h resulted in more (p < 0.05) embryos developing to the blastocyst stage as compared with the inclusion of dbcAMP alone. The dbcAMP + PFF combination increased (p < 0.05) the average number of nuclei per blastocyst as compared with either of these components alone or in its absence. A synergistic effect of dbcAMP + PFF during IVM was also reflected in the capacity of oocytes to regulate sperm penetration and prevent polyspermy, as twice as many oocytes from the control group were penetrated by more than one sperm as compared with those matured in the presence of both dbcAMP and PFF. The supplementation of PZM3 with 10% FBS from days 5 to 7 of culture significantly improved the total cell quantity in embryos derived either from control or dbcAMP + PFF matured oocytes. There was no effect on the total cell quantity when FBS was replaced by the same concentration of PFF. These studies showed that dbcAMP, PFF and FBS can improve both the quantity (57.3% vs 41.5%) and quality (74.8 vs 33.3 nuclei) of porcine blastocysts derived from oocytes recovered of pre-pubertal gilts. [source]

In Vitro Production of Equine Embryos: State of the Art

REPRODUCTION IN DOMESTIC ANIMALS, Issue 2010
K Hinrichs
Contents In vitro embryo production is possible in the horse both clinically and for research applications. Oocytes may be collected from excised ovaries post-mortem, or from either immature follicles or stimulated pre-ovulatory follicles in the live mare. In vitro maturation of immature oocytes typically yields approximately 60% mature oocytes. As standard in vitro fertilization is not yet repeatable in the horse, fertilization is performed by intracytoplasmic sperm injection. Embryo culture requires medium with high glucose, at least during blastocyst development, and rates of blastocyst development similar to those for cattle (25% to 35%) may be obtained. Pregnancy rates after transfer of in vitro -produced blastocysts are similar to those for embryos recovered ex vivo. [source]

Effects of Gonadotropins on In Vitro Maturation and of Electrical Stimulation on Parthenogenesis of Canine Oocytes

Transcriptional Analysis of Buffalo (Bubalus bubalis) Oocytes During In Vitro Maturation Using Bovine cDNA Microarray

Cryotolerance of Bovine Blastocysts is Affected by Oocyte Maturation in Media Containing Palmitic or Stearic Acid

REPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2009
MA Shehab-El-Deen
Contents In this study, non-esterified fatty acids (NEFAs) were added during in vitro maturation at concentrations measured previously in follicular fluid (FF) of high-producing dairy cows in a negative energy status to evaluate their subsequent effect on the embryos cryotolerance. Oocytes were matured for 24 h in serum-free media with or without (negative control) the addition of NEFAs dissolved in ethanol or ethanol alone (positive control). Matured oocytes were fertilized and cultured for 7 days in synthetic oviduct fluid medium supplemented with 5% FCS. Embryos that had at least reached the blastocyst stage were vitrified by open pulled straw (OPS) vitrification. Addition of palmitic (C16 : 0) or stearic acid (C18 : 0) during oocyte maturation had significant negative effects on embryo cryotolerance, whereas ethanol or oleic acid (C18 : 1) had no effect. These in vitro results suggest that high NEFA concentrations in FF during a period of negative energy balance in high-yielding dairy cows can have carry-over effects on embryo quality. [source]

Development of a Competitive Quantitative PCR Strategy for Evaluating the Expression Stability of 18s rRNA during In Vitro Maturation of Buffalo (Bubalus bubalis) Follicular Oocytes

Effect of Alpha-Tocopherol and Ascorbic Acid on Bovine Oocyte in Vitro Maturation

Cumulus,Oocyte Communications in the Horse: Role of the Breeding Season and of the Maturation Medium

REPRODUCTION IN DOMESTIC ANIMALS, Issue 2 2004
S Colleoni
Contents Horse is a seasonal breeder and information on oocyte quality outside the breeding season is very limited. Ovaries obtained at the slaughterhouse are a convenient but often limited source of oocytes in this species. As the low quantity of ovaries leads to an intensive use of all available material, it would be useful to know whether ovaries collected during the non-breeding season are suitable for in vitro maturation (IVM). In an attempt to characterize the effect of season on oocyte quality, we investigated the permeability of the gap junctions (GJ) present between cumulus cells and oocytes because of their important role in oocyte growth and maturation. We also compared the effect of supplementing the maturation medium with bovine serum albumin (BSA) or oestrus mare serum (EMS). A total of 645 oocytes isolated from 158 and 154 ovaries collected during the breeding and the non-breeding season, respectively, were used in this study. Oocytes were matured for 30 h in TCM 199 supplemented either with 10% EMS or with 4 mg/ml BSA. The presence of permeable GJs between cumulus cells and oocytes was investigated with the injection of a 3% solution of the fluorescent dye Lucifer yellow into the ooplasm. No differences in efficiency of oocyte retrieval or oocyte meiotic competence were detected between oocytes collected during the breeding and non-breeding season. The vast majority (90%) of the oocytes collected during the breeding season had fully functional communications with their surrounding cumulus cells but such communications were completely interrupted in 55.3% of the oocytes collected during the non-breeding season. During the non-breeding season, the proportion of oocytes whose communications with cumulus cells were classified as closed or intermediate at the end of maturation was lower in the group matured with BSA than with EMS (71.4 vs 97.7, p < 0.05). The same trend, although not statistically significant, was observed during the breeding season also. The presence of BSA caused an incomplete cumulus expansion during both seasons. Our data indicate that oocytes collected during the non-breeding season do not show any meiotic deficiency but lack active communication with the surrounding cumulus cells at the time of their isolation from the ovary. No data are available at present for determining the consequences on the developmental competence even if data from other species suggest that this is likely. [source]