Non-surrounded Nucleolus Oocytes Research Articles

The chromatin accessibility landscape of mouse oocytes during configuration transition.

The transition of chromatin configuration in mammalian oocytes from a non-surrounded nucleolus (NSN) to a surrounded nucleolus (SN) is critical for acquiring the developmental competence. However, the genomic and epigenomic features underlying this process remain poorly understood. In the present study, we first establish the chromatin accessibility landscape of mouse oocytes from NSN to SN stage. Through the integrative analysis of multi-omics, we find that the establishment of DNA methylation in oocytes is independent of the dynamics of chromatin accessibility. In contrast, histone H3K4me3 status is closely associated with the dynamics of accessible regions during configuration transition. Furthermore, by focusing on the actively transcribed genes in NSN and SN oocytes, we discover that chromatin accessibility coupled with histone methylation (H3K4me3 and H3K27me3) participates in the transcriptional control during phase transition. In sum, our data provide a comprehensive resource for probing configuration transition in oocytes, and offer insights into the mechanisms determining chromatin dynamics and oocyte quality.

MIB2 Functions in Oocyte Meiosis by Modulating Chromatin Configuration

Chromatin configuration serves as a principal indicator of GV (germinal vesicle)-stage oocyte quality. However, the underlying mechanisms governing the chromatin configuration transition from NSN (non-surrounded nucleolus) to SN (surrounded nucleolus) remain unclear. In this study, by conducting a quantitative proteomic analysis, we identified an increased expression of the MIB2 (MIB E3 ubiquitin protein ligase 2) protein in SN oocytes. Specific depletion of MIB2 in SN oocytes not only leads to severe disruption of the meiotic apparatus and a higher incidence of aneuploidy but also adversely affects meiotic maturation and early embryo development. Notably, overexpression of MIB2 in NSN oocytes facilitates the chromatin configuration transition. Meantime, we observed that forced expression of MIB2 in NSN oocytes significantly mitigates spindle/chromosome disorganization and aneuploidy. In summary, our results suggest that chromatin configuration transition regulated by MIB2 is crucial for oocytes to acquire developmental competence.

The landscape of transcriptional profiles in human oocytes with different chromatin configurations

With increasingly used assisted reproductive technology (ART), the acquisition of high-quality oocytes and early embryos has become the focus of much attention. Studies in mice have found that the transition of chromatin conformation from non-surrounded nucleolus (NSN) to surrounded nucleolus (SN) is essential for oocyte maturation and early embryo development, and similar chromatin transition also exists in human oocytes. In this study, we collected human NSN and SN oocytes and investigated their transcriptome. The analysis of differentially expressed genes showed that epigenetic functions, cyclin-dependent kinases and transposable elements may play important roles in chromatin transition during human oocyte maturation. Our findings provide new insights into the molecular mechanism of NSN-to-SN transition of human oocyte and obtained new clues for improvement of oocyte in vitro maturation technique.

Comprehensive analysis of nonsurrounded nucleolus and surrounded nucleolus oocytes on chromatin accessibility using ATAC-seq.

Mouse germinal vesicle (GV) oocytes are divided into surrounded nucleolus (SN) and nonsurrounded nucleolus (NSN) oocytes based on chromatin morphology. NSN oocytes spontaneously transform into SN oocytes after accumulating enough maternal transcripts. SN oocytes show transcriptional silencing. When oocyte maturation is abnormal or takes place in vitro, NSNoocytes do not go through SN stage before proceeding to MII. Nontransitive oocytes show developmental retardation, a low fertilization rate, and arrest at the two-cell embryo stage in mice. Here, chromatin-binding ribonucleic acid polymerase II (RNAP II) activity, newly synthesized RNA, and chromatin accessibility in GV oocytes were examined. In SN oocytes, RNAP II did not bind to DNA, neo-RNA was not generated in nuclei, and the phosphorylation state of RNAP II did not affect the chromatin-binding activity. The number of accessible genes in SN oocytes was remarkably lower than that in NSN oocytes. The accessibility of different functional genes was also different between the two types of oocytes. Thus, low chromatin accessibility leads to transcriptional silencing in SN oocytes.

Fibrillarin-GFP Facilitates the Identification of Meiotic Competent Oocytes.

The nucleolus undergoes significant functional changes and plays important roles during mammalian oocyte meiotic maturation. Fibrillarin (FBL) is the component of nucleolar small nuclear ribonucleoprotein (snRNP) particle and localizes to the dense fibrillar component (DFC) of the nucleolus. We found that FBL–GFP displays an uneven and cloudy localization in the nucleolus of non-surrounded nucleolus (NSN) oocytes, while it distributes evenly and to a few bright dots in the surrounded nucleolus (SN) oocytes. Accordingly, NSN oocytes showed active nascent RNA transcription, while the SN group was transcriptionally quiescent. NSN geminal vesicles also contained more DNA damage marker γH2AX foci. Based on different FBL–GFP patterns in live oocytes, the ones with superior meiotic maturation potential can be identified. Global transcriptome profiling revealed a significant difference in single SN and NSN oocytes. Thus, FBL–GFP can serve as a marker for nucleolus activity, which also correlates with transcription activity and the quality of oocytes.

A new classification of the germinal vesicle chromatin configurations in pig oocytes.

Reported classifications of germinal vesicle (GV) chromatin configurations in pig oocytes were not done by uniform standards and they were not well correlated with oocyte competence. In this study, GV chromatin of pig oocytes was classified into nonsurrounded nucleolus (NSN), surrounded nucleolus (SN), partly NSN (pNSN) and SN (pSN), prematurely condensed NSN (cNSN), pNSN (cpNSN) and pSN (cpSN), and early diakinesis (ED) patterns. During in vitro maturation in 199 medium, NSN oocytes from 1 to 2 mm follicles went consecutively through pNSN, pSN, cpSN, and ED before undergoing GV breakdown, and chromatin in some SN oocytes from 3 to 6 mm follicles re-decondensed into a re-decondensation (RDC) configuration. Under unfavorable conditions such as follicle atresia, ovary handling or maturation in simple MEM medium, however, premature chromatin condensation occurred, forming cNSN, cpNSN, and cpSN patterns. While all NSN and pNSN and some pSN and RDC oocytes actively transcribed, no cNSN, cpNSN, or cpSN oocytes showed transcription. Maturation and embryo culture suggested that SN and pSN oocytes were more competent than NSN and pNSN oocytes; cpSN oocytes were more competent than cNSN/cpNSN oocytes; and only RDC oocytes could develop into blastocysts. It is concluded that the newly classified chromatin configurations are more closely correlated with oocyte competence than those reported previously.

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 oocyte-specific ELAVL2 isoform is a translational repressor ablated from meiotically competent antral oocytes

At the end of the growth phase, mouse antral follicle oocytes acquire full developmental competence. In the mouse, this event is marked by the transition from the so-called non-surrounded nucleolus (NSN) chromatin configuration into the transcriptionally quiescent surrounded nucleolus (SN) configuration, which is named after a prominent perinucleolar condensed chromatin ring. However, the SN chromatin configuration alone is not sufficient for determining the developmental competence of the SN oocyte. There are additional nuclear and cytoplamic factors involved, while a little is known about the changes occurring in the cytoplasm during the NSN/SN transition. Here, we report functional analysis of maternal ELAVL2 an AU-rich element binding protein. Elavl2 gene encodes an oocyte-specific protein isoform (denoted ELAVL2°), which acts as a translational repressor. ELAVL2° is abundant in fully grown NSN oocytes, is ablated during the NSN/SN transition and remains low during the oocyte-to-embryo transition (OET). ELAVL2° overexpression during meiotic maturation causes errors in chromosome segregation, indicating the significance of naturally reduced ELAVL2° levels in SN oocytes. On the other hand, during oocyte growth, prematurely reduced Elavl2 expression results in lower yields of fully grown and meiotically matured oocytes, suggesting that Elavl2 is necessary for proper oocyte maturation. Moreover, Elavl2 knockdown showed stimulating effects on translation in fully grown oocytes. We propose that ELAVL2 has an ambivalent role in oocytes: it functions as a pleiotropic translational repressor in efficient production of fully grown oocytes, while its disposal during the NSN/SN transition contributes to the acquisition of full developmental competence.

Maternal factors required for oocyte developmental competence in mice: Transcriptome analysis of non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) oocytes

During mouse antral follicle development, the oocyte chromatin gradually transforms from a less condensed state with no Hoechst-positive rim surrounding the nucleolus (NSN) to a fully condensed chromatin state with a Hoechst-positive rim surrounding the nucleolus (SN). Compared with SN oocytes, NSN oocytes display a higher gene transcription activity and a lower rate of meiosis resumption (G2/M transition), and they are mostly arrested at the two-cell stage after in vitro fertilization. To explore the differences between NSN and SN oocytes, and the maternal factors required for oocyte developmental competence, we compared the whole-transcriptome profiles between NSN and SN oocytes. First, we found that the NSN and SN oocytes were different in their metabolic pathways. In the phosphatidylinositol signaling pathway, the SN oocytes tend to produce diacylglycerol, whereas the NSN oocytes tend to produce phosphatidylinositol (3,4,5)-trisphosphate. For energy production, the SN oocytes and NSN oocytes differed in the gluconeogenesis and in the synthesis processes. Second, we also found that the key genes associated with oocyte meiosis and/or preimplantation embryo development were differently expressed in the NSN and SN oocytes. Our results illustrate that during the NSN-SN transition, the oocytes change their metabolic activities and accumulate maternal factors for further oocyte maturation and post-fertilization embryo development.

FTIR spectral signatures of mouse antral oocytes: Molecular markers of oocyte maturation and developmental competence

Mammalian antral oocytes with a Hoescht-positive DNA ring around the nucleolus (SN) are able to resume meiosis and to fully support the embryonic development, while oocytes with a non-surrounded nucleolus (NSN) cannot. Here, we applied FTIR microspectroscopy to characterize single SN and NSN mouse oocytes in order to try to elucidate some aspects of the mechanisms behind the different chromatin organization that impairs the full development of NSN oocyte-derived embryos. To this aim, oocytes were measured at three different stages of their maturation: just after isolation and classification as SN and NSN oocytes (time 0); after 10h of in vitro maturation, i.e. at the completion of the metaphase I (time 1); and after 20h of in vitro maturation, i.e. at the completion of the metaphase II (time 2). Significant spectral differences in the lipid (3050–2800cm−1) and protein (1700–1600cm−1) absorption regions were found between the two types of oocytes and among the different stages of maturation within the same oocyte type. Moreover, dramatic changes in nucleic acid content, concerning mainly the extent of transcription and polyadenylation, were detected in particular between 1000 and 800cm−1. The use of the multivariate principal component–linear discriminant analysis (PCA–LDA) enabled us to identify the maturation stage in which the separation between the two types of oocytes took place, finding as the most discriminating wavenumbers those associated to transcriptional activity and polyadenylation, in agreement with the visual analysis of the spectral data.

Contribution of the oocyte nucleus and cytoplasm to the determination of meiotic and developmental competence in mice

Mammals have two types of full-grown oocytes: those with germinal vesicles (GVs) in which the chromatin is condensed and surrounds the nucleolus (surrounded-nucleolus (SN)-type) and those in which the chromatin is less condensed and does not surround the nucleolus (non-surrounded-nucleolus (NSN)-type). Although SN oocytes possess higher meiotic and developmental competence than NSN oocytes, the factors underlying this difference are unknown. The GVs of murine SN and NSN oocytes were exchanged by nuclear transfer and the nucleus/cytoplasm of each reconstructed oocyte was classified as follows: SN/SN, NSN/SN, SN/NSN or NSN/NSN. After reconstruction, the meiotic maturation and preimplantation development of the oocytes were analysed. Few mature SN/NSN and NSN/NSN oocytes were observed (20-26%). In contrast, 88% of the NSN/SN oocytes matured; however, they rarely developed to the blastocyst stage after fertilization (4%), whereas most of the SN/SN oocytes matured (84%) and reached the blastocyst stage (83%). When the metaphase II (MII) plates of in vitro-matured NSN/SN oocytes were transferred into enucleated MII oocytes in which the contents of the SN-type GVs were spread into the cytoplasm, they completed full-term development. The differences in meiotic and developmental competence between SN and NSN oocytes are determined by factors in the cytoplasm and nucleus, respectively. In addition, material(s) within SN-type GVs, and not the chromatin configuration itself, is essential for full-term development.

Gene Expression and Chromatin Organization during Mouse Oocyte Growth

Mouse oocytes can be classified according to their chromatin organization and the presence [surrounded nucleolus (SN) oocytes] or absence [nonsurrounded nucleolus (NSN) oocytes] of a ring of Hoechst-positive chromatin around the nucleolus. Following fertilization only SN oocytes are able to develop beyond the two-cell stage. These studies indicate a correlation between SN and NSN chromatin organization and the developmental competence of the female gamete, which may depend on gene expression. In the present study, we have used the HSP70.1Luc transgene (murine HSP70.1 promoter + reporter gene firefly luciferase) to analyze gene expression in oocytes isolated from ovaries of 2-day- to 13-week-old females. Luciferase was assayed on oocytes after classification as SN or NSN type. Our data show that SN oocytes always exhibit a higher level of luciferase activity, demonstrating a higher gene expression in this category. Only after meiotic resumption, metaphase II oocytes derived from NSN or SN oocytes acquire the same level of transgene expression. We suggest that the limited availability of transcripts and corresponding proteins, excluded from the cytoplasm until GVBD in NSN oocytes, could explain why these oocytes have a lower ability to sustain embryonic development beyond the two-cell stage at which major zygotic transcription occurs. With this study we have furthered our knowledge of epigenetic regulation of gene expression in oogenesis.

Chromatin organization and meiotic non-disjunction in mouse oocytes

Two forms of oocytes termed SN (Surrounded Nucleolus) and NSN (Non Surrounded Nucleolus) differing for the spatial distribution of nuclear and nucleolar-associated chromatin have been described within the antral compartment of the ovary of a number of mammals. The biological significance of these two kinds of oocytes is as yet not completely clear. In previous studies we have shown that prior to ovulation, SN oocytes isolated from the antral compartment, cultured and fertilizedin vitro (IVM-IVF) have a far better meiotic and developmental competence than NSN oocytes. Following ovulation the proportion of SN antral oocytes diminishes and those SN and NSN oocytes that remain in the antral compartment are uncapable of embryonic development beyond the 2-cell stage. To further examine the correlation between chromatin distribution and meiotic competence of mouse antral oocytes, in the present study we have analyzed chromosome segregation at the time of first meiotic division in SN and NSN antral oocytes and in ovulated oocytes. SN and NSN antral oocytes were isolated before (48 hr post PMSG injection) or after (15 hr post hCG injection) ovulation from ovaries of females of increasing age, they were culturedin vitro to metaphase II, and their aneuploidy rate was examined. Comparison of data obtained before and after ovulation highlights two main points: 1) following ovulation a statistically significant increase of aneuploidy is observed in antral oocytes in most age groups. This increase is mainly attributable to SN oocytes, whereas the aneuploidy rate in the group of NSN oocytes does not significantly change before and after ovulation. 2) The aneuploidy rate of MII ovulated oocytes has a decreasing trend during female aging. These results suggest that in the ovarian dynamics, SN antral oocytes may be favoured in the selection for ovulation.

Analysis of aneuploidy rate in antral and ovulated mouse oocytes during female aging.

Two forms of oocytes termed SN (surrounded nucleolus) and NSN (nonsurrounded nucleolus) differing for the spatial distribution of nuclear and nucleolar-associated chromatin have been described within the antral compartment of the ovary of a number of mammals. The biological significance of these two kind of oocytes is as yet not completely clear. In previous studies we have shown that prior to ovulation, mouse SN oocytes isolated from the antral compartment, matured and fertilized in vitro have a far better meiotic and developmental competence than NSN oocytes. Immediately after ovulation SN and NSN oocytes remaining in the antral compartment do not develop beyond the 2-cell stage. To further examine the correlation between chromatin distribution and meiotic competence of mouse antral oocytes, in the present study we have analyzed chromosome segregation at the first meiotic division in antral (SN and NSN) and in ovulated oocytes. SN and NSN oocytes were isolated before (48 h post PMSG injection) or after (15 h post-hCG injection) ovulation from ovaries of females of increasing age, they were cultured in vitro to metaphase II, and their aneuploidy rate was examined. Comparison of data obtained before and after ovulation highlights two main points: 1. Following ovulation a statistically significant increase of aneuploidy is observed in antral oocytes in most age groups and it is attributable to SN oocytes. 2. The aneuploidy rate of ovulated oocytes does not increase during female aging. We have found a correlation between chromatin distribution, hormonal status, and the incidence of aneuploidy during the oocyte first meiotic division.

Chromatin organization during mouse oocyte growth.

We investigated the changes in the organization of oocyte nuclear chromatin and nucleolar-associated chromatin throughout folliculogenesis. Zona-free oocytes were isolated from ovaries, grouped into seven classes according to size and chromatin organization, and analyzed after staining with Hoechst 33342. We show that oocyte differentiation from the dictyate stage to the conclusion of maturation is associated with either of two chromatin configurations. Initially, all oocytes are in the NSN configuration (nonsurrounded nucleolus oocytes; characterized by a Hoechst positive-chromatin pattern of small clumps forming a network on the nuclear surface, with a nucleolus nonsurrounded by chromatin). While growing some of these NSN oocytes continue their development in the NSN configuration, whereas others shift (from class IV on) into the SN configuration (surrounded nucleolus oocytes; characterized by a threadlike chromatin organization that may partially surround the nucleolus or project towards the nuclear periphery). The percentage of SN oocytes increases both with increasing size of the oocyte (class I-III, 10-40 microns in diameter: 100% NSN vs. 0% SN; class VII 70-80 microns in diameter: 47.3% NSN vs. 52.3 SN, in 4-6-week-old females), and with aging (class VII: 94.1% NSN vs. 5.9% SN in 2-week-old females; 11.8% NSN vs. 8.2% SN in 56-week-old females). Further, we suggest as a working hypothesis that those oocytes that switch to the SN chromatin organization early in maturation may not be ovulated, even though this particular chromatin structure normally occurs just prior to ovulation.