Oocyte Chromatin Configuration Research Articles

Oocyte Competence of Prepubertal Sheep and Goat Oocytes: An Assessment of Large-Scale Chromatin Configuration and Epidermal Growth Factor Receptor Expression in Oocytes and Cumulus Cells.

The oocyte competence of prepubertal females is lower compared to that of adults, mainly because they originate from small follicles. In adult females, the germinal vesicle (GV) and epidermal growth factor receptor (EGFR) have been associated with oocyte competence. This study aimed to analyze GV chromatin configuration and EGFR expression in prepubertal goat and sheep oocytes obtained from small (<3 mm) and large (≥3 mm) follicles and compare them with those from adults. GV chromatin was classified from diffuse to condensed as GV1, GVn, and GVc for goats and NSN, SN, and SNE for sheep. EGFR was quantified in cumulus cells (CCs) by Western blotting and in oocytes by immunofluorescence. Oocytes from prepubertal large follicles and adults exhibited highly condensed chromatin in goats (71% and 69% in GVc, respectively) and sheep (59% and 75% in SNE, respectively). In both species, EGFR expression in CCs and oocytes was higher in prepubertal large follicles than in small ones. In adult females, EGFR expression in oocytes was higher than in prepubertal large follicles. In conclusion, GV configuration and EGFR expression in CCs and oocytes were higher in the large than small follicles of prepubertal females.

Chromatin Configuration in Diplotene Mouse and Human Oocytes during the Period of Transcriptional Activity Extinction.

In the oocyte nucleus, called the germinal vesicle (GV) at the prolonged diplotene stage of the meiotic prophase, chromatin undergoes a global rearrangement, which is often accompanied by the cessation of its transcriptional activity. In many mammals, including mice and humans, chromatin condenses around a special nuclear organelle called the atypical nucleolus or formerly nucleolus-like body. Chromatin configuration is an important indicator of the quality of GV oocytes and largely predicts their ability to resume meiosis and successful embryonic development. In mice, GV oocytes are traditionally divided into the NSN (non-surrounded nucleolus) and SN (surrounded nucleolus) based on the specific chromatin configuration. The NSN-SN transition is a key event in mouse oogenesis and the main prerequisite for the normal development of the embryo. As for humans, there is no single nomenclature for the chromatin configuration at the GV stage. This often leads to discrepancies and misunderstandings, the overcoming of which should expand the scope of the application of mouse oocytes as a model for developing new methods for assessing and improving the quality of human oocytes. As a first approximation and with a certain proviso, the mouse NSN/SN classification can be used for the primary characterization of human GV oocytes. The task of this review is to analyze and discuss the existing classifications of chromatin configuration in mouse and human GV oocytes with an emphasis on transcriptional activity extinction at the end of oocyte growth.

Acrylamide impairs the developmental potential of germinal vesicle oocytes by inducing mitochondrial dysfunction and autophagy/apoptosis in mice

Background: Acrylamide (ACR), an important endogenous contaminant in carbohydrate-rich foods, has been involved in various negative effects on multiple organ networks, including the reproductive system. Previous studies have reported that ACR affects oocyte quality and fertility.Purpose: This study aimed to explore the toxic effects and regulatory mechanisms of ACR on mouse germinal vesicle (GV) oocytes. Research Design: In this study, adult female mice were exposed to ACR at 10mg/kg/day/body weight through their drinking water continuously for 4weeks. Study Sample and Data Analysis: The mitochondrial function, autophagy/apoptosis, and development potential of GV oocytes were investigated. Results: The results showed that ACR reduced the oocyte diameter, sperm-binding ability, parthenogenetic activation and in vitro fertilization (IVF) rate, and development potential of pre-implantation embryos. We also found that ACR exposure disrupted chromatin configuration, mitochondrial distribution, and membrane potential (Δφm) of oocytes. Actin filament expression was significantly reduced in both the membrane and cytoplasm of mouse oocytes. Moreover, ACR exposure increased LC3-positive signals, early apoptosis rate, aberrant ATG3, ATG5, LC3, Beclin1, and mTOR mRNA expression. Conclusions: These results suggest that ACR exposure can affect the developmental potential of GV oocytes by inducing mitochondrial dysfunction, actin filament assembly, and autophagy/apoptosis.

Characterization and control of oocyte large-scale chromatin configuration in different cattle breeds

Differences in reproductive physiology between cattle breeds may help to explain distinct responses to assisted reproductive techniques and to define breed-specific protocols with improved efficiency. Germinal vesicle (GV) stage oocytes are characterized by increasing levels of chromatin compaction enclosed within the nucleus (graded from GV0 to GV3), associated with different developmental competence. The first objective of this study was to characterize chromatin configuration of GV stage oocytes recovered by OPU at random days of the estrous cycle from Nelore (Bos indicus) and Holstein (Bos taurus) cows. In Nelore 90% of the oocytes presented advanced stages of chromatin compaction associated with higher developmental competence (GV2 and GV3), while in Holstein, only 65% of the oocytes were at these stages. Then, aiming to obtain a more homogeneous population of oocytes in Holstein, we tested two synchronization protocols combining aspiration of all visible follicles at a random day (day 0), two IM injections of FSH 12 h apart on day 2, and OPU on day 4 (OPU/D4) or 5 (OPU/D5). The protocol OPU/D4 provided around 45% of the oocytes with low chromatin compaction (GV1), while the protocol OPU/D5 provided 70% of the oocytes at GV2 and 20% at GV3. Finally, we assessed the effects of a culture system known to prevent meiotic resumption on chromatin configuration of the GV2 enriched oocyte population obtained with the protocol OPU/D5. After 9 h of culture most oocytes transited from GV2 to GV3, with 90% of the oocytes at GV3 stage. This study demonstrates differences between Nelore and Holstein cows regarding patterns of chromatin configuration that may account for their different performance in IVM/IVF. In addition, it provides novel references for the design of protocols aiming to regulate oocyte quality before IVM for the optimization of IVF outcomes.

Follicular factors determining the developmental competence of porcine oocyte.

PurposeThis study aimed to examine the relationship between granulosa cells (GCs), number of follicles, and the ability of follicular fluid to support in vitro growth of oocytes.MethodsThe culture medium was supplemented with follicular fluid (FF) collected from GC‐rich ovaries and GC‐poor ovaries, and its effect on in vitro growth and quality of oocytes derived from early antral follicles (EAFs) was assessed.ResultsGC‐rich FF treatment enhanced oocyte growth and augmented changes in the chromatin configuration and lipid content of oocytes when compared to oocytes treated with GC‐poor FF. Moreover, oocytes treated with GC‐rich FF had a higher ability to progress to the blastocyst stage, than oocytes derived from large antral follicles (3‐5 mm in diameter). In addition, supplementation of the culture medium with either GC‐rich or GC‐poor FF enhanced histone acetylation in oocytes grown in vitro.ConclusionGC‐rich FF contains key factors that support in vitro oocyte growth; hence, oocytes grown in GC‐rich FF medium had high developmental competence, which was comparative to the oocytes grown in vivo.

Cilostamide affects in a concentration and exposure time-dependent manner the viability and the kinetics of in vitro maturation of caprine and bovine oocytes

This study investigated: 1) the kinetics of oocyte chromatin configuration during in vitro maturation (IVM) of caprine and bovine oocytes; and 2) the effect of in vitro pre-maturation (IVPM) with cilostamide with or without association of the follicular wall (FW) on the same parameters. In experiment I, cumulus-oocyte complexes (COCs) were cultured in vitro in a standard maturation medium for 6, 12, 18 or 30 h. For experiment II, the COCs were cultured for 30 h, either in a standard IVM medium or in IVPM containing cilostamide (10 or 20 μM) and FW alone or in combination, for 6 or 12 h before the onset of maturation. The MII rate was similar (P > .05) between 18 and 30 h of maturation, both of which were higher (P < .05) than 6 and 12 h IVM in both species (Experiment I). Contrary to caprine, all IVPM treatments presented a higher (P < .05) percentage of bovine oocytes arrested at the GV stage than the control treatment after 6 h of culture. The percentage of MII oocytes after 30 h (IVPM+IVM) of culture in bovine oocytes treated with 10 μM cilostamide associated with FW and FW alone cultured for 6 h presented MII percentages similar to the control. However, in caprine, these treatments significantly reduced the percentages of MII in relation to the control treatment (Experiment II). In conclusion, the combination of concentration-exposure time to cilostamide during IVPM delayed meiotic progression in bovine after 6 and 12 h of culture. However, overall the culture period (IVPM+IVM) influenced the oocyte chromatin configuration and kinetics in both species.

173 Size Matters: a Follicle-of-Origin Effect on the Preovulatory Epigenetic Constitution of Germinal Vesicle-Stage Porcine Oocytes

The goal of this study was to evaluate global levels of a variety of histone modifications at different lysine (K) residues on Histone 3 (H3) within the chromatin of porcine germinal vesicle (GV)-stage oocytes that were aspirated from follicles of different sizes. We hypothesised that we would see evidence of a transition from open, transcriptionally active chromatin (in oocytes from smaller, growing follicles) to more closed, transcriptionally silent chromatin associated with fully grown oocytes (aspirated from large, preovulatory follicles). Cumulus-enclosed oocytes were aspirated from small (&lt;3 mm) or large (&gt;7 mm) follicles from abattoir-derived pig ovaries. Oocytes were denuded immediately after aspiration and then immunoprobed with antibodies specific for trimethylated (me3) H3K4, H3K9me3, and H3K27me3. Background-corrected nuclear fluorescence levels for each histone mark were collected from multiple oocytes from each of at least three experimental replicates (aspiration days). Data were subjected to one-way ANOVA with a Bonferroni multiple testing correction to determine whether there were differences in fluorescence intensities in the nuclei (germinal vesicles) of oocytes from small v. large follicles. Oocytes from large follicles displayed more intense nuclear staining for all 3 histone marks: average nuclear H3K4me3 intensity was 31.4% higher (P = 0.0004), H3K9me3 was 70.3% higher (P = 0.0218), and H3K27me3 was 32.0% higher (P = 0.0231) in oocytes from large follicles. An ancillary analysis of the data revealed no effect (P &gt; 0.1) of pubertal status (i.e. whether small and large follicles were aspirated from pre- v. post-pubertal ovaries) on the intensity of nuclear fluorescence for any of the marks evaluated. In continuation, 3 oocytes from both follicle types were collected on each of 6 aspiration days (i.e. 18 individual oocytes from each follicle type), and the mRNA from these were used for an RT-qPCR experiment to detect the relative abundance of transcripts from 21 different genes coding for histone methyltransferase or demethylase enzymes in oocytes from large v. small follicles. Of the 21 genes tested, 5 genes (KDM4C, KDM4D, KDM5B, KDM5C, and SETD7) were not detectable in our individual oocyte samples, but transcripts from 6 of the 16 remaining genes (KDM6A, KMT2B, MLL3, SETD1B, SETDB1, and SUV39H2) were shown to be significantly more abundant in oocytes from large follicles (at least 2-fold greater abundance and P &lt; 0.05). Although our expectation-that histone marks (and related transcripts) would consistently reflect a globally “repressive” chromatin configuration in oocytes from large follicles and a more “open” configuration in oocytes from small follicles-turned out to be untrue, the evidence suggests that the epigenetic constitution of oocytes from small follicles may indeed vary from that of oocytes from large, preovulatory follicles, and this phenomenon warrants further investigation.

Relationship between follicular dynamics and oocyte maturation during in vitro culture as a non-invasive sign of caprine oocyte meiotic competence

The search for non-invasive signs of oocyte meiotic competence is very important for the development of in vitro follicle culture (IVFC) systems. The aims of the present study were: (1) to investigate the effect of in vitro maturation (IVM) of in vivo grown goat COCs, in group or individually, on oocyte chromatin configuration (Experiment 1), and (2) the influence of IVFC period (12 vs. 18 days) on the ability of the oocyte to resume meiosis immediately after IVFC (before in vitro maturation; IVM), or after IVM (Experiment 2). In experiment 1, in vivo grown cumulus-oocyte complexes (COCs) were submitted to IVM in groups (10 COCs/100 μL-drop) or individually (1 COC/10 μL-drop), and chromatin configuration was assessed. In experiment 2, isolated follicles were individually cultured for 12 or 18 days, and submitted to individual IVM afterwards. The following end points were evaluated: follicular growth and morphology, oocyte diameter, viability and chromatin configuration, as well as individual follicular estradiol production. Similar maturation rates were obtained between in vivo grown COCs matured individually and in groups (66.7% vs. 63.6%, respectively) (Experiment 1). Only after 18 days of IVFC, oocytes were able to grow during IVM, reaching a mean oocyte diameter of 119 μm. Also, this treatment produced the highest rate of metaphase II oocytes (46.2% out of the total number of cultured follicles). Finally, it was observed that follicles with a daily growth rate >7.1 μm/day (fast-growing) and that reached at least 600 μm in diameter, were more likely (P < 0.05) to produce oocytes capable of attaining MII. In conclusion, caprine oocytes can be individually matured in vitro, as efficiently as in groups. This result was essential to pair in vitro follicle development and in vitro oocyte maturation with specific individual follicles. Using this approach, it was possible to establish non-invasive signs for the efficiency of IVFC based on follicle daily growth rate and diameter, and oocyte diameter: follicle daily growth >7 μm, follicle diameter of at least 600 μm, and oocyte diameter ≥120 μm. In addition, 18 days seems to be the most suitable culture time for caprine early antral follicles.

An improved IVM method for cumulus-oocyte complexes from small follicles in polycystic ovary syndrome patients enhances oocyte competence and embryo yield.

Are meiotic and developmental competence of human oocytes from small (2-8 mm) antral follicles improved by applying an optimized IVM method involving a prematuration step in presence of C-Type Natriuretic Peptide (CNP) followed by a maturation step in presence of FSH and Amphiregulin (AREG)? A strategy involving prematuration culture (PMC) in the presence of CNP followed by IVM using FSH + AREG increases oocyte maturation potential leading to a higher availability of Day 3 embryos and good-quality blastocysts for single embryo transfer. IVM is a minimal-stimulation ART with reduced hormone-related side effects and risks for the patients, but the approach is not widely used because of an efficiency gap compared to conventional ART. In vitro systems that enhance synchronization of nuclear and cytoplasmic maturation before the meiotic trigger are crucial to optimize human IVM systems. However, previous PMC attempts have failed in sustaining cumulus-oocyte connections throughout the culture period, which prohibited a normal cumulus-oocyte communication and precluded an adequate response by the cumulus-oocyte complex (COC) to the meiotic trigger. A first prospective study involved sibling oocytes from a group of 15 patients with polycystic ovary syndrome (PCOS) to evaluate effects of a new IVM culture method on oocyte nuclear maturation and their downstream developmental competence. A second prospective study in an additional series of 15 women with polycystic ovaries characterized and fine-tuned the culture conditions. Fifteen women with PCOS (according to Rotterdam criteria) underwent IVM treatment after 3-5 days of highly purified human menopausal gonadotropin (HP-hMG) stimulation and no human chorionic gonadotropin (hCG) trigger before oocyte retrieval. A first study was designed with sibling oocytes to prospectively evaluate the impact of an IVM culture method: 24 h PMC with CNP + 30 h IVM with FSH and AREG, on embryo yield, in comparison to the standard (30 h) IVM clinical protocol (Group I, n = 15). A second prospective study was performed in 15 women with polycystic ovaries, to characterize and optimize the PMC conditions (Group II, n = 15). The latter study involved the evaluation of oocyte meiotic arrest, the preservation of cumulus-oocyte transzonal projections (TZPs), the patterns of oocyte chromatin configuration and cumulus cells apoptosis following the 24 and 46 h PMC. Furthermore, oocyte developmental potential following PMC (24 and 46 h) + IVM was also evaluated. The first 20 good-quality blastocysts from PMC followed by IVM were analysed by next generation sequencing to evaluate their aneuploidy rate. PMC in presence of CNP followed by IVM using FSH and AREG increased the meiotic maturation rate per COC to 70%, which is significantly higher than routine standard IVM (49%; P ≤ 0.001). Hence, with the new system the proportion of COCs yielding transferable Day 3 embryos and good-quality blastocysts increased compared to routine standard IVM (from 23 to 43%; P ≤ 0.001 and from 8 to 18%; P ≤ 0.01, respectively). CNP was able to prevent meiosis resumption for up to 46 h. After PMC, COCs had preserved cumulus-oocyte TZPs. The blastocysts obtained after PMC + IVM did not show increased aneuploidy rates as compared to blastocysts from conventional ART. The novel IVM approach in PCOS patients was tested in oocytes derived from small antral follicles which have an intrinsically low developmental potential. Validation of the system would be required for COCs from different (larger) follicular sizes, which may involve further adjustment of PMC conditions. Furthermore, considering that this is a novel strategy in human IVM treatment, its global efficiency needs to be confirmed in large prospective randomized controlled trials. The further application in infertile patients without PCOS, e.g. cancer patients, remains to be evaluated. The findings of this pilot study suggest that the efficiency gap between IVM and conventional IVF can be reduced by fine-tuning of the culture methods. This novel strategy opens new perspectives for safe and patient-friendly ART in patients with PCOS. IVM research at the Vrije Universiteit Brussel has been supported by grants from: the Institute for the Promotion of Innovation by Science and Technology in Flanders (Agentschap voor Innovatie door Wetenschap en Technologie-IWT, project 110680); the Fund for Research Flanders (Fonds Wetenschappelijk Onderzoek-Vlaanderen-FWO, project G.0343.13), the Belgian Foundation Against Cancer (HOPE project, Dossier C69). The authors have no conflicts of interest.

Toxicity effect of Auxemma oncocalyx fraction and its active principle oncocalyxone A on in vitro culture of caprine secondary follicles and in vitro oocyte maturation

Crude extract of the heartwood of Auxemma oncocalyx (A. oncocalyx) and its main component i.e., Oncocalyxone A (onco A), have elevated antioxidant and anti-tumoral activity, but studies on the action of these drugs regarding folliculogenesis are lacking. The aim of this study was to evaluate the effect of A. oncocalyx and onco A on the in vitro culture of isolated secondary follicles and on the in vitro maturation of oocytes from caprine antral follicles grown in vivo. Isolated secondary follicles were randomly distributed in six groups; the non-cultured control was immediately fixed upon isolation. The remaining follicles were cultured for 7 days in ?-MEM+ alone (control) or supplemented with DMSO, doxorrubicin, A. oncocalyx or onco A. After culture, follicles were evaluated for antrum formation, growth rate, apoptosis (TUNEL) and cellular proliferation (PCNA), as well as gene expression of Bcl2 and Bax. Additionally, cumulus oocyte complexes (COCs) were aspirated and allocated into five treatments for in vitro maturation: control, cultured only in maturation base medium (TCM 199+); or supplemented with DMSO; DXR; A. oncocalyx or onco A. After in vitro maturation, oocyte chromatin configuration and viability were assessed. After 7 days of culture, there was a reduction (P &lt; 0.05) in the percentage of morphologically intact follicles, antrum formation, growth rate and number of PCNA positive granulosa cells in DXR treatment compared to the other treatments. In the DXR treatment a higher percentage (P &lt; 0.05) of TUNEL positive follicles and higher (P &lt; 0.05) relative BAX:BCL2 mRNA ratio’s were observed. After in vitro maturation of the COCs DXR, A. oncocalyx and onco A treatments had a greater (P &lt; 0.05) percentage of abnormal oocytes and a lower (P &lt; 0.05) percentage of viable oocytes as compared with the control group. However, only DXR and onco A treatments increased (P &lt; 0.05) the percentage of alive oocytes with abnormal chromatin configuration. There were no differences in maturation rates between the control group and DXR, A. oncocalyx and onco A treatments. In conclusion, under our culture conditions, A. oncocalyx and onco A do not exhibit a toxic effect on isolated secondary follicles and on maturation rates of COCs recovered from antral follicles, however, these drugs negatively affected the COCs viability. Thus, the use of culture biotechnologies as an in vitro secondary follicle culture and in vitro oocyte maturation toxicity testing are appropriated methods to evaluate the possible effects of drugs in folliculogesis.

Differences in cumulus cell gene expression indicate the benefit of a pre-maturation step to improve in-vitro bovine embryo production.

Does the gene expression profile of cumulus cells (CC) accompanying oocytes with different degrees of chromatin compaction within the germinal vesicle (GV) reflect the oocyte's quality and response in culture during in-vitro embryo production (IVP). The transcriptomic profile of the CC is related to oocyte competence, setting the stage for the development of customized pre-maturation strategies to improve IVP. Oocytes complete the acquisition of their competence during antral follicle development. During this period, the chromatin configuration within the GV changes dynamically and is indicative of oocyte's developmental potential. The interactions between somatic and germ cells modulate chromatin morphology and function and are critical for acquisition of oocyte competence. Bovine cumulus-oocyte complexes (COC) were isolated from 0.5 to 6 mm antral follicles. Surrounding CC were separated from the oocyte and classified as GV0, GV1, GV2 and GV3 according to the degree of the oocyte's chromatin compaction. RNA extracted from CC of each group was amplified and hybridized on a bovine embryo-specific 44 K Agilent slide. The CC_GV1, CC_GV2 and CC_GV3 classes were each hybridized against the CC_GV0 class, representing an early oocyte differentiation stage with poor development competence. The data were normalized and fold changes of the differentially expressed genes were determined. Microarray data were validated using quantitative RT-PCR on selected targets. Microarray data were further analyzed through: (i) between-group analysis (BGA), which classifies the samples according to their transcriptomic profiles; (ii) cluster analysis according to the expression profile of each gene; and (iii) Ingenuity Pathway Analysis (IPA) to study gene regulation patterns and predicted functions. Furthermore, CC of each GV group were cultured and apoptotic cells were assessed after 3 h by caspase analysis. Finally, based on the analysis of CC transcriptomic profiles and the relationship between morphological features of the COC and the oocyte chromatin configuration, a customized, stage-dependent oocyte pre-maturation (pre-IVM) system was used to improve oocyte developmental potential before IVM. For this, the blastocyst rate and quality were assessed after in-vitro maturation and fertilization of pre-matured oocytes. Overall, quantitative RT-PCRresults of a subset of five selected genes were consistent with the microarray data. Clustering analysis generated 16 clusters representing the main profiles of transcription modulation. Of the 5571 significantly differentially expressed probes, the majority (25.49%) best fitted with cluster #6 (downregulation between CC_GV0 and CC_GV1 and stable low levels in successive groups). IPA identified the most relevant functions associated with each cluster. Genes included in cluster #1 were mostly related to biological processes such as 'cell cycle' and 'cell death and survival', whereas genes included in cluster #5 were mostly related to 'gene expression'. Interestingly, 'lipid metabolism' was the most significant function identified in clusters #6, #9 and #12. IPA of gene lists obtained from each contrast (i.e., CC_GV0 vs. CC_GV1; CC_GV0 vs. CC_GV2; CC_GV0 vs. CC_GV3) revealed that the main affected function in each contrast was 'cell death and survival'. Importantly, apoptosis was predicted to be inhibited in CC_GV1 and CC_GV2, but activated in CC_GV3. Caspase analysis indicated that a low percentage of CC_GV0 was prone to undergo apoptosis but apoptosis increased significantly in CC from oocytes with condensed chromatin, reaching a peak in CC_GV3 (P <0.05). Finally, the tailored oocyte pre-maturation strategy, based on morphological features of the COC and the oocyte chromatin configuration, demonstrated that pre-IVM improved the developmental capability of oocytes at early stages of differentiation (GV1-enriched COC) but was detrimental for oocytes at more advanced stages of development (GV2 and GV3-enriched COC). The data are available through the GEO series accession number GSE79886. This study was conducted with bovine samples. Whether or not the results are applicable to human oocytes requests further elucidation. Embryo transfer experiments are required to determine whether the improvement in blastocyst rates in the tailored system leads to increased live birth rates. The identification of multiple non-invasive biomarkers predictive of oocyte quality can greatly strengthen the pre-IVM approach aimed to improve IVM outcomes. These results have potentially important implications in treating human infertility and in developing breeding schemes for domestic mammals. This work was supported in part by NSERC Strategic Network EmbryoGENE, Canada and in part by CIG-Marie Curie Actions-Reintegration Grants within the EU 7FP (n. 303640, 'Pro-Ovum'). The authors declare no potential conflict of interest.

Immature Oocytes from Unprimed Juvenile Mice Become a Valuable Source for Embryo Production When Using C-Type Natriuretic Peptide as Essential Component of Culture Medium.

C-type natriuretic peptide (CNP) and its receptor natriuretic peptide receptor 2 (NPR2) play a paramount role in the maintenance of oocyte meiotic arrest in antral follicles via the regulation of the intra-oocyte levels of cyclic guanosine monophosphate and cyclic adenosine monophosphate. We investigated the potential of CNP 1) to maintain oocyte meiotic arrest during a prolonged prematuration culture and 2) to sustain acquisition of developmental competence of immature cumulus-oocyte complexes (COCs). Compact COCs were collected from small antral follicles of prepubertal unprimed mice and placed in prematuration culture under different CNP-supplemented media conditions. A preliminary analysis showed a dose-dependent effect of CNP on the maintenance of meiotic arrest. A dose of 25 nM maintained oocytes under meiotic arrest for 24 h, and this period was extended to 48 h in the presence of estradiol. Analysis of transzonal projections of COCs cultured with CNP indicated that oocyte-cumulus connections were well preserved after the prolonged prematuration culture. Furthermore, CNP medium supplemented with FSH and GDF9 promoted oocyte growth and induced a shift in oocyte chromatin configuration from a predominantly dispersed to a condensed configuration. Following in vitro maturation, oocytes cultured under CNP were capable of extruding the first polar body at a high rate (around 80%). Blastocyst formation was significantly improved when oocytes were cultured under CNP-supplemented medium containing FSH and GDF9. This study reports for the first time a prolonged prematuration culture system, with CNP as the pivotal factor, that can efficiently maintain oocytes retrieved from unprimed prepubertal mice under meiotic arrest while promoting their acquisition of developmental competence.

The relationship between apoptosis, chromatin configuration, histone modification and competence of oocytes: A study using the mouse ovary-holding stress model

The epigenetic factors causing competence differences between SN (surrounded nucleolus) and NSN (non-surrounded nucleolus) oocytes, the significance for the increased histone acetylation and methylation in SN oocytes, and whether chromatin configuration or histone modification determines oocyte competence, are unclear. This study has addressed these issues by using the ovary-holding (OH) stress models where oocyte SN configuration was uncoupled from histone modifications and developmental potential. Prepubertal mouse ovaries containing high percentages of NSN oocytes were preserved at 37 or 39 °C for 1 or 2 h before examination for oocyte chromatin configuration, developmental competence, histone modification and apoptosis. Whereas 1-h OH at 37 °C caused a moderate apoptosis with increased oocyte competence, improved histone modification and a normal NSN-to-SN transition, harsher OH conditions induced a severe apoptosis with decreased oocyte competence, impaired histone modification and a pseudo (premature) NSN-to-SN transition. Observations on Fas/FasL expression and using the gld (generalized lymphoproliferative disorder) mice harboring FasL mutations indicated that OH triggered oocyte apoptosis with activation of the Fas signaling. It was concluded that OH stress caused oocyte apoptosis with activation of the Fas/FasL system and that oocyte competence was more closely correlated with histone modification than with chromatin configuration.

An essential role for the intra-oocyte MAPK activity in the NSN-to-SN transition of germinal vesicle chromatin configuration in porcine oocytes.

The mechanisms for the transition from non-surrounded nucleolus (NSN) to surrounded nucleolus (SN) chromatin configuration during oocyte growth/maturation are unclear. By manipulating enzyme activities and measuring important molecules using small-follicle pig oocytes with a high proportion of NSN configuration and an extended germinal vesicle stage in vitro, this study has the first time up-to-date established the essential role for intra-oocyte mitogen-activated protein kinase (MAPK) in the NSN-to-SN transition. Within the oocyte in 1–2 mm follicles, a cAMP decline activates MAPK, which prevents the NSN-to-SN transition by activating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) while inhibiting histone deacetylase (HDAC). In cumulus cells of 1–2 mm follicles, a lower level of estradiol and oocyte-derived paracrine factor (ODPF) reduces natriuretic peptide receptor 2 (NPR2) while enhancing FSH and cAMP actions. FSH elevates cAMP levels, which decreases NPR2 while activating MAPK. MAPK closes the gap junctions, which, together with the NPR2 decrease, reduces cyclic guanosine monophosphate (cGMP) delivery leading to the cAMP decline within oocytes. In 3–6 mm follicles, a higher level of estradiol and ODPF and a FSH shortage initiate a reversion of the above events leading to MAPK inactivation and NSN-to-SN transition within oocytes.

Restraint Stress on Female Mice Diminishes the Developmental Potential of Oocytes

The mechanisms by which restraint stress impairs oocyte developmental potential are unclear. Factors causing differences between the developmental potential of oocytes with surrounded nucleolus (SN) and that of oocytes with nonsurrounded nucleolus (NSN) are not fully characterized. Furthermore, the relationship between increased histone acetylation and methylation and the increased developmental competence in SN oocytes is particularly worth exploring using a system where the SN configuration can be uncoupled (dissociated) from increased histone modifications. In this study, female mice were subjected to restraint for 24 or 48 h or for 23 days before being examined for oocyte chromatin configuration, histone modification, and development in vitro and in vivo. Results showed that restraint for 48 h or 23 days impaired NSN-to-SN transition, histone acetylation and methylation in SN oocytes, and oocyte developmental potential. However, whereas the percentage of stressed SN oocytes returned to normal after a 48-h postrestraint recovery, neither histone acetylation/methylation in SN oocytes nor developmental competence recovered following postrestraint recovery with equine chorionic gonadotropin (eCG) injection. Priming unstressed mice with eCG expedited oocyte histone modification to an early completion. Contrary to the levels of acetylated and methylated histones, the level of phosphorylated H3S10 increased significantly in the stressed SN oocytes. Together, the results suggest that 1) restraint stress impaired oocyte potential with disturbed histone modifications; 2) SN configuration was uncoupled from increased histone acetylation/methylation in the restraint-stressed oocytes; and 3) the developmental potential of SN oocytes is more closely correlated with epigenetic histone modification than with chromatin configuration.

Effects of reaggregated granulosa cells and oocytes derived from early antral follicles on the properties of oocytes grown in vitro.

In this study, we examined the effects of reconstructed oocyte–granulosa cell complexes (OGCs) on the development of porcine oocytes derived from early antral follicles (EAFs; 0.5–0.7 mm in diameter). When denuded oocytes were cocultured with granulosa cells derived from other EAFs, the oocytes and granulosa cells aggregated to form OGCs after 2 days of culture. After 14 days of culture, we compared cell number, oocyte diameter, and oocyte chromatin configuration in unmanipulated (natural) OGCs, reconstructed OGCs, and OGCs collected from antral follicles (AFs, 3.0–6.0 mm in diameter). The diameters of oocytes from reconstructed OGCs grown in vitro were not different from those of oocytes from natural OGCs, although they were significantly smaller than those of oocytes from antral follicle (AF) OGCs. Oocyte chromatin configuration did not differ among the 3 OGC groups, but the oocyte nuclear maturation rate was lower in the reconstructed OGCs and higher in the AF OGCs. However, when the in vitro culture period for the reconstructed OGCs was extended by 2 days, the nuclear maturation rate of oocytes from reconstructed OGCs was similar to that of oocytes from natural OGCs. In addition, blastocysts were successfully obtained from oocytes from reconstructed OGCs. In conclusion, we established an innovative culture method that allows oocytes and granulosa cells from EAFs to reaggregate as reconstructed OGCs, which yield oocytes with the ability to develop to the blastocyst stage.

Restraint stress on female mice diminishes the developmental potential of oocytes: roles of chromatin configuration and histone modification in germinal vesicle stage oocytes.

The mechanisms by which restraint stress impairs oocyte developmental potential are unclear. Factors causing differences between the developmental potential of oocytes with surrounded nucleolus (SN) and that of oocytes with nonsurrounded nucleolus (NSN) are not fully characterized. Furthermore, the relationship between increased histone acetylation and methylation and the increased developmental competence in SN oocytes is particularly worth exploring using a system where the SN configuration can be uncoupled (dissociated) from increased histone modifications. In this study, female mice were subjected to restraint for 24 or 48 h or for 23 days before being examined for oocyte chromatin configuration, histone modification, and development in vitro and in vivo. Results showed that restraint for 48 h or 23 days impaired NSN-to-SN transition, histone acetylation and methylation in SN oocytes, and oocyte developmental potential. However, whereas the percentage of stressed SN oocytes returned to normal after a 48-h postrestraint recovery, neither histone acetylation/methylation in SN oocytes nor developmental competence recovered following postrestraint recovery with equine chorionic gonadotropin (eCG) injection. Priming unstressed mice with eCG expedited oocyte histone modification to an early completion. Contrary to the levels of acetylated and methylated histones, the level of phosphorylated H3S10 increased significantly in the stressed SN oocytes. Together, the results suggest that 1) restraint stress impaired oocyte potential with disturbed histone modifications; 2) SN configuration was uncoupled from increased histone acetylation/methylation in the restraint-stressed oocytes; and 3) the developmental potential of SN oocytes is more closely correlated with epigenetic histone modification than with chromatin configuration.

Influência das fases de anestro e diestro sobre a configuração da cromatina em vesícula germinativa de oócitos caninos

In canine specie, oocyte maturation rates are low and the percentage of oocytes that remain in the stage of germinal vesicle (GV) regardless of culture conditions is high. During maturation oocyte undergoes modification and the GV chromatin remodeling manifested by changes in the configuration and positioning. The objective of this work is to evaluate the configuration and positioning of chromatin of oocytes in GV stage during anestrus and diestrus bitches. The ovaries of 33 females (20 bitches in anestrous and 13 in diestrus) were isolated, sliced and only cumulus-oocyte complexes (COCs) grade 1 were subjected to solution of 0.2% hyaluronidase for release in cumulus cells. After this process, the selected oocytes were stained and evaluated. From a total of 920 oocytes, 566 were classified as grade 1 and the stages of chromatin configuration identified as GV-1, GV-2, GV-3 and GV-4. The observed changes in chromatin configuration been characterized as a transition dispersed chromatin (GV-1, GV-2) for partially condensed (GV-3) until it reaches a fully condensed stage (GV-4). The data analyzed from the chromatin configuration showed a significant difference between the stages with a higher proportion of GV-1 and GV-2 for the anoestrus and GV-3 and GV-4 during diestrus. There is need for further studies to be able to have a proper understanding of the influence of chromatin configuration of oocytes in GV stage in resumption of meiosis and consequently in oocyte meiotic competence.

Halogenated nucleotide labeling of nascent RNAs reveals dynamic transcription in growing pig oocytes

Germ cells differentiate into oocytes in females and are arrested at the first meiotic prophase. However, during arrest, oocytes undergo a growth phase leading to a dramatic increase in size, which is under control of transcription events. In the current study, we examined the transcriptional activity of growing pig oocytes using an immunocytochemical approach. Our data showed that fluorouridine (FU), a halogenated nucleotide, can be successfully incorporated into synthesizing RNAs and detected using a specific monoclonal antibody. Using this method, we identified dynamic changes in transcriptional activity patterns in growing pig oocytes. Oocytes obtained from small follicles exhibited the highest level of transcription, while at the final phase of growth, transcription was no longer detected. These transcriptional changes were concomitant with chromatin compaction resulting in a tightly packed ring-like chromatin conformation surrounding the nucleolar structure. Also, FU incorporation appeared sensitive to the biochemical manipulation of transcription, because transcriptional inhibitors induced a decrease in signal intensity from FU labeling and transcriptional activation caused an increase in FU signal intensity. Our data collectively support that a direct link exists between chromatin configuration and transcriptional activity in pig oocytes, and support the suitability of FU for studies on transcription-related events in mammalian oocytes.

A Novel Oocyte Chromatin Configuration Classification Method: Based on the Degree of Aggregation and Spatial Distribution of Germinal Vesicle Chromatin

A Novel Oocyte Chromatin Configuration Classification Method: Based on the Degree of Aggregation and Spatial Distribution of Germinal Vesicle Chromatin