Possible role of 5'AMP-activated protein kinase in the metformin-mediated arrest of bovine oocytes at the germinal vesicle stage during in vitro maturation

The 5'AMP-activated protein kinase (AMPK) activation is involved in the meiotic maturation of oocytes in the ovaries of mice and pigs. However, its effects on the oocyte appear to be species-specific. We investigated the patterns of AMPK and mitogen-activated protein kinases (MAPK3/1) phosphorylation during bovine in vitro maturation (IVM) and the effects of metformin, an AMPK activator, on oocyte maturation in cumulus-oocyte complexes (COCs) and denuded bovine oocytes (DOs). In bovine COCs, PRKAA Thr172 phosphorylation decreased, whereas MAPK3/1 phosphorylation increased in both oocytes and cumulus cells during IVM. Metformin (5 and 10 mM) arrested oocytes at the GV stage in COCs but not in DOs. In COCs, this arrest was associated with the inhibition of cumulus cell expansion, an increase in PRKAA Thr172 phosphorylation, and a decrease in MAPK3/1 phosphorylation in both oocytes and cumulus cells. However, the addition of compound C (10 muM), an inhibitor of AMPK, accelerated the initiation of the GV breakdown (GVBD) process without any alteration of MAPK3/1 phosphorylation in oocytes from bovine COCs. Metformin decreased AURKA and CCNB1 protein levels in oocytes. Moreover, after 1 h of IVM, metformin decreased RPS6 phosphorylation and increased EEF2 phosphorylation, suggesting that protein synthesis rates were lower in oocytes from metformin-treated COCs. Most oocytes were arrested after the GVBD stage following the treatment of COCs with the MEK inhibitor, U0126 (100 micromoles). Thus, in bovine COCs, metformin blocks meiotic progression at the GV stage, activates PRKAA, and inhibits MAPK3/1 phosphorylation in both the oocytes and cumulus cells during IVM. Moreover, cumulus cells were essential for the effects of metformin on bovine oocyte maturation, whereas MAPK3/1 phosphorylation was not.     

Morphological and biochemical analysis of immature ovine oocytes vitrified with or without cumulus cells

The cryopreservation of oocytes is an open problem as a result of their structural sensitivity to the freezing process. This study examined (i) the survival and meiotic competence of ovine oocytes vitrified at the GV stage with or without cumulus cells; (ii) the viability and functional status of cumulus cells after cryopreservation; (iii) the effect of cytochalasin B treatment before vitrification; (iv) chromatin and spindle organization; (v) the maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activity of vitrified oocytes after in vitro maturation. Sheep oocytes were vitrified at different times during in vitro maturation (0, 2, and 6 h) with (COCs) or without cumulus cells (DOs). After warming and in vitro maturation, oocytes denuded at 0 h culture showed a significantly higher survival and meiotic maturation rate compared to the other groups. Hoechst 33342/propidium iodide double staining of COCs and microinjection of Lucifer Yellow revealed extensive cumulus cell membrane damage and reduced oocyte-cumulus cell communications after vitrification. Cytochalasin B treatment of COCs before vitrification exerted a negative effect on oocyte survival. After in vitro maturation, the number of vitrified oocytes with abnormal spindle and chromatin configuration was significantly higher compared to control oocytes, independently of the presence or absence of cumulus cells. The removal of cumulus cells combined with vitrification significantly decreased the MPF and MAPK levels. This study provides evidence that the removal of cumulus cells before vitrification enhances oocyte survival and meiotic competence, while impairing the activity of important proteins that could affect the developmental competence of oocytes.     

Parthenogenic Blastocysts Derived from Cumulus-Free In Vitro Matured Human Oocytes

Background

Approximately 20% of oocytes are classified as immature and discarded following intracytoplasmic sperm injection (ICSI) procedures. These oocytes are obtained from gonadotropin-stimulated patients, and are routinely removed from the cumulus cells which normally would mature the oocytes. Given the ready access to these human oocytes, they represent a potential resource for both clinical and basic science application. However culture conditions for the maturation of cumulus-free oocytes have not been optimized. We aimed to improve maturation conditions for cumulus-free oocytes via culture with ovarian paracrine/autocrine factors identified by single cell analysis.

Methodology/Principal Finding

Immature human oocytes were matured in vitro via supplementation with ovarian paracrine/autocrine factors that were selected based on expression of ligands in the cumulus cells and their corresponding receptors in oocytes. Matured oocytes were artificially activated to assess developmental competence. Gene expression profiles of parthenotes were compared to IVF/ICSI embryos at morula and blastocyst stages. Following incubation in medium supplemented with ovarian factors (BDNF, IGF-I, estradiol, GDNF, FGF2 and leptin), a greater percentage of oocytes demonstrated nuclear maturation and subsequently, underwent parthenogenesis relative to control. Similarly, cytoplasmic maturation was also improved as indicated by development to blastocyst stage. Parthenogenic blastocysts exhibited mRNA expression profiles similar to those of blastocysts obtained after IVF/ICSI with the exception for MKLP2 and PEG1.

Conclusions/Significance

Human cumulus-free oocytes from hormone-stimulated cycles are capable of developing to blastocysts when cultured with ovarian factor supplementation. Our improved IVM culture conditions may be used for obtaining mature oocytes for clinical purposes and/or for derivation of embryonic stem cells following parthenogenesis or nuclear transfer.     

Large-scale chromatin remodeling in germinal vesicle bovine oocytes: interplay with gap junction functionality and developmental competence

In mammals, oocyte acquires a series of competencies sequentially during folliculogenesis that play critical roles at fertilization and early stages of embryonic development. In mouse, chromatin in germinal vesicle (GV) undergoes dynamic changes during oocyte growth and its progressive condensation has been related to the achievement of developmental potential. Cumulus cells are essential for the acquisition of meiotic competence and play a role in chromatin remodeling during oocyte growth. This study is aimed to characterize the chromatin configuration of growing and fully grown bovine oocytes, the status of communications between oocyte and cumulus cells and oocyte developmental potential. Following nuclear staining, we identified four discrete stages of GV, characterized by an increase of chromatin condensation. GV0 stage represented 82% of growing oocytes and it was absent in fully grown oocytes. GV1, GV2, and GV3 represented, respectively, 24, 31, and 45% of fully grown oocytes. Our data indicated a moderate but significant increase in oocyte diameter between GV0 and GV3 stage. By dye coupling assay the 98% of GV0 oocytes showed fully open communications while the number of oocytes with functionally closed communications with cumulus cells was significantly higher in GV3 group than GV1 and GV2. However, GV0 oocytes were unable to progress through metaphase II while GV2 and GV3 showed the highest developmental capability. We conclude that in bovine, the progressive chromatin condensation is related to the sequential achievement of meiotic and embryonic developmental competencies during oocyte growth and differentiation. Moreover, gap-junction-mediated communications between oocyte and cumulus cells could be implicated in modulating the chromatin remodeling process.     

Glucosamine supplementation during in vitro maturation inhibits subsequent embryo development: possible role of the hexosamine pathway as a regulator of developmental competence

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

The influence of cumulus-oocyte complex morphology and meiotic inhibitors on the kinetics of nuclear maturation in cattle

This study evaluated whether pre-established morphological classes of bovine cumulus oocyte complex (COCs) differ in their kinetics of meiosis resumption after 4 h of incubation and whether the timing of COCs resumption of meiosis differed after a period of maintained meiotic arrest. Bovine COCs were aspirated from 2- to 5- mm follicles and classified according to the state of their cumulus cells and cytoplasm (Classes 1 to 3). Groups of 15 to 20 COCs were fixed at 0 h or after an incubation period of 4 h. In addition, COCs from Class 1 were first incubated for 4 h on a theca cell monolayer or in the presence of 2 microg/mL of cycloheximide, rinsed and then incubated in cycloheximide and theca cell-free medium for another 4 h. Oocytes then were fixed and evaluated for state of nuclear maturation. Results show that at 0 h, COCs from Class 3 have fewer oocytes at the GV stage than COCs from Class 1 and Class 2 (respectively 69.3+/-3.2 vs 88.8+/-3.4% and 86.9% GV+/-4.3% SEM; P < 0.05). After 4 h of incubation, all COCs classes show a significant decrease in the number of COCs at the GV stage. The COCs maintained in meiotic arrest and then incubated for 4 h resume meiosis faster than COCs incubated in cycloheximide and theca cell-free medium (19.4+/-2.5, 33.3+/-7.3 and 59.9+/-6.5% GV SEM, respectively). The COCs of Class 3 have fewer oocytes at the GV stage at the beginning of incubation than all other classes. The number of COCs at the GV stage after 4 h of incubation in cycloheximide and theca cell-free medium is not significantly different than those COCs incubated in the presence of theca cell monolayers for 24 h (58.8+/-6.5 vs. 56.4+/-6.4% SEM; respectively). Our results indicate that the ability of theca cells to maintain oocytes at the GV stage could be limited to those oocytes that were not committed or primed in vivo to resume maturation as indicated by their faster maturation kinetics.     

Effects of cumulus cell density during in vitro maturation of the developmental competence of bovine oocytes

To determine the role of cumulus cells in oocyte maturation, we carried out an investigation on the effects of addition of cumulus cells to the maturation medium on the developmental competence of corona-enclosed oocytes and oocytes denuded from their somatic cells. The addition of cumulus cell (1.6 x 10(6) cells/mL) improved the development of bovine corona-enclosed oocytes, however, addition of a similar number of cumulus cells as cumulus-oocyte-complexes (COCs, cumulus cell density: 4.2 x 10(6) cells/mL) had no effect on the development of oocytes denuded from their somatic cells. To determine if corona-enclosed oocytes can obtain developmental competence without the addition of extra cumulus cells, the effects of cell density during in vitro maturation on the developmental competence were studied. A density of 1.6 to 3.2 x 10(6) cumulus cells/mL was the most effective for in vitro maturation of oocytes with intact gap junctions. The effects of the medium conditioned by COCs on the developmental competence of oocytes was also examined. It was demonstrated that COC-conditioned medium improved the development of bovine oocytes to the blastocyst stage. These data suggest that the developmental competence of bovine oocytes surrounded with corona cells is supported in a cell density-dependent manner in the maturation medium. In addition, the data indicate that cumulus cells benefit bovine oocyte development either by secreting soluble factors which induce developmental competence or by removing an embryo development-suppressive component from the medium.     

Release of sICAM-1 in Oocytes and In Vitro Fertilized Human Embryos

Background

During the last years, several studies have reported the significant relationship between the production of soluble HLA-G molecules (sHLA-G) by 48–72 hours early embryos and an increased implantation rate in IVF protocols. As consequence, the detection of HLA-G modulation was suggested as a marker to identify the best embryos to be transferred. On the opposite, no suitable markers are available for the oocyte selection.

Methodology/Principal Findings

The major finding of the present paper is that the release of ICAM-1 might be predictive of oocyte maturation. The results obtained are confirmed using three independent methodologies, such as ELISA, Bio-Plex assay and Western blotting. The sICAM-1 release is very high in immature oocytes, decrease in mature oocytes and become even lower in in vitro fertilized embryos. No significant differences were observed in the levels of sICAM-1 release between immature oocytes with different morphological characteristics. On the contrary, when the mature oocytes were subdivided accordingly to morphological criteria, the mean sICAM-I levels in grade 1 oocytes were significantly decreased when compared to grade 2 and 3 oocytes.

Conclusions/Significance

The reduction of the number of fertilized oocytes and transferred embryos represents the main target of assisted reproductive medicine. We propose sICAM-1 as a biochemical marker for oocyte maturation and grading, with a possible interesting rebound in assisted reproduction techniques.     

Development of in vitro embryo production systems for red deer (Cervus elaphus). Part 2. The timing of in vitro nuclear oocyte maturation

The time course of in vitro red deer nuclear oocyte maturation was determined. Ovaries were obtained at slaughter and oocytes were aspirated from follicles greater than 2mm in diameter. Oocytes with compact cumulus cells were matured in 50 microl microdrops (10 per drop) under mineral oil containing TCM 199 supplemented with 0.33 mM pyruvate, 10 microg LH and FSH, 1 microg oestradiol and 10% foetal bovine serum. Oocytes were matured at 39 degrees C and 5% CO(2) in air. At 3h intervals (0-27 h) oocytes were removed from incubation, cumulus expansion scored and removed, and fixed oocytes in ethanol:acetic acid (3:1) for 48 h. Oocytes were stained with lacmoid (1%) and nuclear maturation assessed. Oocytes were arrested in the germinal vesicle (GV) stage at aspiration and up to 6h of incubation. The nuclear membrane began to disperse after 6h and by 10.6+/-0.6h of incubation 75% of the oocytes exhibited germinal vesicle breakdown (GVBD). The mean time for 50% of the oocytes to reach metaphase one (MI) and metaphase two (MII) was 11.7+/-0.4 and 24.8+/-0.9h, respectively. Cumulus oophorus were tightly compacted at aspiration and did not begin expansion until 12h of culture. Full expansion was complete by 18 h of culture. Corona radiata cells did not begin expansion until 15 h and were fully expanded by 24h. Results indicate that in vitro red deer oocyte maturation follows a similar time course of nuclear maturation as reported for bovine and ovine oocytes.     

Proteomic analysis of human follicular fluid from fertile women

Background

Follicular fluid is a unique biological fluid in which the critical events of oocyte and follicular maturation and somatic cell-germ cell communication occur. Because of the intimate proximity of follicular fluid to the maturing oocyte, this fluid provides a unique window into the processes occurring during follicular maturation. A thorough identification of the specific components within follicular fluid may provide a better understanding of intrafollicular signaling, as well as reveal potential biomarkers of oocyte health for women undergoing assisted reproductive treatment. In this study, we used high and low pH HPLC peptide separations followed by mass spectrometry to perform a comprehensive proteomic analysis of human follicular fluid from healthy ovum donors. Next, using samples from a second set of patients, an isobaric mass tagging strategy for quantitative analysis was used to identify proteins with altered abundances after hCG treatment.

Results

A total of 742 follicular fluid proteins were identified in healthy ovum donors, including 413 that have not been previously reported. The proteins belong to diverse functional groups including insulin growth factor and insulin growth factor binding protein families, growth factor and related proteins, receptor signaling, defense/immunity, anti-apoptotic proteins, matrix metalloprotease related proteins, and complement activity. In a quantitative analysis, follicular fluid samples from age-matched women undergoing in vitro fertilization oocyte retrieval were compared and 17 follicular fluid proteins were found at significantly altered levels (p < 0.05) between pre-hCG and post-hCG samples. These proteins belong to a variety of functional processes, including protease inhibition, inflammation, and cell adhesion.

Conclusions

This database of FF proteins significantly extends the known protein components present during the peri-ovulatory period and provides a useful basis for future studies comparing follicular fluid proteomes in various fertility, disease, and environmental exposure conditions. We identified 17 differentially expressed proteins after hCG treatment and together these data showed the feasibility for defining biomarkers that illuminate how the ovarian follicle microenvironment is altered in various infertility-related conditions.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9077-6) contains supplementary material, which is available to authorized users.     

Changes in germinal vesicle (GV) chromatin configurations during growth and maturation of porcine oocytes

Changes in germinal vesicle (GV) chromatin configurations during growth and maturation of porcine oocytes were studied using a new method that allows a clearer visualization of both nucleolus and chromatin after Hoechst staining. The GV chromatin of porcine oocytes was classified into five configurations, based on the degree of chromatin condensation, and on nucleolus and nuclear membrane disappearance. While the GV1 to 4 configurations were similar to those reported by previous studies, the GV0 configuration was distinct by the diffuse, filamentous pattern of chromatin in the whole nuclear area. Most of the oocytes were at the GV0 stage in the <1 and 1-1.9 mm follicles, but the GV0 pattern disappeared completely in the 2-2.9 and 3-6 mm follicles. As follicles grew, the number of oocytes with GV1 configurations increased and reached a maximum in the preovulatory follicles 4 hr post-hCG injection. During maturation in vivo, the number of GV1 oocytes decreased while oocytes undergoing GVBD increased. The percentage of oocytes with GV3 and GV4 configurations was constant during oocyte growth except at the 2-2.9 mm follicle stage, but these configurations disappeared completely after hCG injection. On the contrary, the in vitro maturing oocytes showed a large proportion of GV3 and GV4 configurations. There was no significant difference in distribution of chromatin configurations between the nonatretic and atretic follicles, and between oocytes with more than two layers of cumulus cells and those with less than one layer or no cumulus cells. Overall, our results suggested that (i) the GV0 configuration in porcine oocytes corresponded to the "nonsurrounded nucleolus" pattern in mice and other species; (ii) all the oocytes were synchronized at the GV1 stage before GVBD and this pattern might, therefore, represent a nonatretic state; (iii) the GV3 and GV4 configurations might represent stages toward atresia, or transient events prior to GVBD that could be switched toward either ovulation or atresia, depending upon circumstances; (iv) the in vitro systems currently used were not favorable for oocytes to switch toward ovulation (or final maturation); (v) the number of cumulus cells was not correlated with the chromatin configuration of oocytes, indicating that the beneficial effect of cumulus cells on oocyte maturation and development may simply be attributed to their presence during in vitro culture.     

HCO3 ?/Cl? Exchange Inactivation and Reactivation during Mouse Oocyte Meiosis Correlates with MEK/MAPK-Regulated Ae2 Plasma Membrane Localization

Background

Germinal Vesicle (GV) stage mouse oocytes in first meiotic prophase exhibit highly active HCO₃ ⁻/Cl⁻ exchange—a class of transport nearly ubiquitously involved in regulation of intracellular pH and cell volume. During meiosis, however, oocyte HCO₃ ⁻/Cl⁻ exchange becomes inactivated during first metaphase (MI), remains inactive in second metaphase (MII), and is reactivated only after egg activation. Previous work using pharmacological manipulations had indicated that activity of the MEK/MAPK signaling pathway was negatively correlated with HCO₃ ⁻/Cl⁻ exchange activity during meiosis. However, the mechanism by which the exchanger is inactivated during meiotic progression had not been determined, nor had the role of MEK/MAPK been directly established.

Methodology/Principal Findings

Expression of a constitutively active form of MEK (MAP kinase kinase), which prevented the normal downregulation of MAPK after egg activation, also prevented reactivation of HCO₃ ⁻/Cl⁻ exchange. Conversely, suppression of endogenous MAPK activity with dominant negative MEK activated the normally quiescent HCO₃ ⁻/Cl⁻ exchange in mature MII eggs. A GFP-tagged form of the HCO₃ ⁻/Cl⁻ exchanger isoform Ae2 (Slc4a2) was strongly expressed at the GV oocyte plasma membrane, but membrane localization decreased markedly during meiotic progression. A similar pattern for endogenous Ae2 was confirmed by immunocytochemistry. The loss of membrane-localized Ae2 appeared selective, since membrane localization of a GFP-tagged human dopamine D1 receptor did not change during meiotic maturation.

Conclusions

Direct manipulation of MAPK activity indicated that GFP-tagged Ae2 localization depended upon MAPK activity. Inactivation of HCO₃ ⁻/Cl⁻ exchange during the meiotic cell cycle may therefore reflect the loss of Ae2 from the oocyte plasma membrane, downstream of MEK/MAPK signaling. This identifies a novel role for MEK/MAPK-mediated cytostatic factor (CSF) activity during meiosis in membrane protein trafficking in mouse oocytes, and shows for the first time that selective retrieval of membrane proteins is a feature of meiosis in mammalian oocytes.     

Apoptosis in cumulus cells, but not in oocytes, may influence bovine embryonic developmental competence

Aim of our study was to clarify if the occurrence of apoptosis in oocytes and cumulus cells is correlated to bovine oocyte developmental competence. The cumulus-oocyte complexes (COCs) were selected according to cumulus status: G1 with more than five layers of compact cumulus cells, G2 with one to five layers of compact cumulus cells and G3 with expanded cumulus cells. The degree of apoptosis in cumulus cells and oocytes measured by caspase staining and TUNEL assay before and after maturation, and 24 h post-insemination was compared to the cleavage, blastocyst formation and hatching rates of each group. Highest cleavage, blastocyst and hatching rates were found in cumulus-oocyte complexes with more than five layers of compact cumulus cells, but no apoptosis was detected in immature or in vitro matured oocytes, regardless of the cumulus status. Many cumulus cells contained active caspases before maturation, but caspase activity declined dramatically after maturation. TUNEL positive cells were rarely observed in each cumulus-oocyte complex upon oocyte recovery, but a huge increase of them was seen after in vitro maturation. Significantly more TUNEL and caspase positive cells were found in G2 cumulus-oocyte complexes. Our results suggest that: (i) oocyte apoptosis does not account for the inferior oocyte quality of G2 and G3; (ii) apoptosis occurs in cumulus cells regardless of the number and compactness of cumulus cells; and (iii) the degree of apoptosis in the compact cumulus-oocyte complexes (G1 and G2) is negatively correlated to the developmental competence of oocyte.     

Towards a Rigorous Assessment of Systems Biology Models: The DREAM3 Challenges

Background

Systems biology has embraced computational modeling in response to the quantitative nature and increasing scale of contemporary data sets. The onslaught of data is accelerating as molecular profiling technology evolves. The Dialogue for Reverse Engineering Assessments and Methods (DREAM) is a community effort to catalyze discussion about the design, application, and assessment of systems biology models through annual reverse-engineering challenges.

Methodology and Principal Findings

We describe our assessments of the four challenges associated with the third DREAM conference which came to be known as the DREAM3 challenges: signaling cascade identification, signaling response prediction, gene expression prediction, and the DREAM3 in silico network challenge. The challenges, based on anonymized data sets, tested participants in network inference and prediction of measurements. Forty teams submitted 413 predicted networks and measurement test sets. Overall, a handful of best-performer teams were identified, while a majority of teams made predictions that were equivalent to random. Counterintuitively, combining the predictions of multiple teams (including the weaker teams) can in some cases improve predictive power beyond that of any single method.

Conclusions

DREAM provides valuable feedback to practitioners of systems biology modeling. Lessons learned from the predictions of the community provide much-needed context for interpreting claims of efficacy of algorithms described in the scientific literature.     

A Quantitative Image Cytometry Technique for Time Series or Population Analyses of Signaling Networks

Background

Modeling of cellular functions on the basis of experimental observation is increasingly common in the field of cellular signaling. However, such modeling requires a large amount of quantitative data of signaling events with high spatio-temporal resolution. A novel technique which allows us to obtain such data is needed for systems biology of cellular signaling.

Methodology/Principal Findings

We developed a fully automatable assay technique, termed quantitative image cytometry (QIC), which integrates a quantitative immunostaining technique and a high precision image-processing algorithm for cell identification. With the aid of an automated sample preparation system, this device can quantify protein expression, phosphorylation and localization with subcellular resolution at one-minute intervals. The signaling activities quantified by the assay system showed good correlation with, as well as comparable reproducibility to, western blot analysis. Taking advantage of the high spatio-temporal resolution, we investigated the signaling dynamics of the ERK pathway in PC12 cells.

Conclusions/Significance

The QIC technique appears as a highly quantitative and versatile technique, which can be a convenient replacement for the most conventional techniques including western blot, flow cytometry and live cell imaging. Thus, the QIC technique can be a powerful tool for investigating the systems biology of cellular signaling.