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 μm in diameter: 100% NSN vs. 0% SN; class VII 70–80 μm 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. © 1995 Wiley-Liss, Inc.     

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.     

Meiotic competence acquisition is associated with the appearance of M-phase characteristics in growing mouse oocytes

To determine whether the acquisition of meiotic competence during the growth phase of oogenesis is associated with the appearance of M-phase characteristics, oocytes obtained from 13- to 30-day-old mice were evaluated by fluorescence microscopy with respect to chromatin and microtubule organization , in vitro maturation ability, and the distribution of M-phase phosphoproteins. Meiotically incompetent oocytes were distinguished from their competent counterparts in displaying elaborate interphase-like arrays of cytoplasmic microtubules and dispersed germinal vesicle chromatin. Meiotically competent oocytes were larger in size, exhibited condensation of chromatin around the nucleolus, and displayed a progressive diminution of cytoplasmic microtubules in conjunction with the appearance of multiple microtubule organizing centers. After 24 hr in culture, medium- to large-sized oocytes exhibiting perinucleolar chromatin condensation resume meiosis whereas smaller meiotically incompetent oocytes retain GVs with diffuse chromatin. Moreover, indirect immunofluorescence studies using the M-phase phosphoprotein specific monoclonal antibody MPM-2 indicate that the appearance of reactive cytoplasmic foci is directly correlated with nuclear changes characteristic of meiotically competent oocytes. Thus, the earliest transition to a meiotically competent state during oocyte growth in the immature mouse ovary is characterized by stage-specific and coordinated modifications of nuclear and cytoplasmic components.     

Position of the nucleus in mouse germinal vesicle–stage oocytes with different chromatin configurations

The mammalian germinal vesicle–stage (GV) oocytes are divided into two major types, NSN (non-surrounded nucleolus) and SN (surrounded nucleolus), and at least one intermediate type, pSN (partly surrounded nucleolus), based on large-scale chromatin configuration. In mice, the SN oocytes are considered to be the most meiotically competent, which explains active study of their phenotypic characteristics necessary for improvement of human reproductive technologies. One of such characteristics is the position of the GV (nucleus) relative to the center of the oocyte. However, the current data on this issue are contradictory and even completely absent for pSN oocytes. In this work, we have studied the GV position in 187 mouse GV oocytes belonging to NSN, SN, and pSN types using different approaches known from the literature. Our results suggest that (1) the most abundant in all examined types of oocytes are central GVs (43–66%) and the least abundant are peripheral GVs (12–39%); the pSN oocytes are closer to SN oocytes rather than to NSN oocytes according to the GV position; (3) the position of the nucleus in mouse GV oocytes is an ambiguous marker of large-scale chromatin configuration and, correspondingly, maturation competence of the oocyte; (4) the diversity of the GV position in NSN, SN, and pSN oocytes most likely reflects the ability of GVs to migrate; and (5) assessment of the GV position according to three variants (central, peripheral, and intermediate) is more informative as compared with two variants (central and peripheral).     

Enucleolation of porcine oocytes

Germinal vesicles (GVs) in immature mammalian oocytes contain prominent nucleoli whose role in the process of oocyte maturation is not fully understood. Here we report that the microsurgical removal of nucleoli from immature fully grown porcine oocytes permits germinal vesicle breakdown and chromosome condensation and the enucleolated oocytes mature up to the second metaphase. Interestingly, the enucleolation of growing oocytes which, although unable to mature, resulted in germinal vesicle breakdown and the formation of a cluster of condensed chromatin. These results indicate that the nucleolus in fully grown oocytes is dispensable at least for nuclear maturation. On the other hand, the results obtained in growing oocytes suggest the role of the nucleolus in the cell cycle regulation.     

Germinal vesicle configurations and patterns of polypeptide synthesis of procine oocytes from antral follicles of different size, as related to their competency for spontaneous maturation

The cytogenetic configurations of germinal vesicle (gv) chromatin were analyzed for pools of porcine oocytes harvested from small (1.0-2.0 mm), medium (3.0-5.0 mm), and large (6.0-10.0 mm) antral follicles. Groups of oocytes from these follicular classes also were examined by high-resolution, two-dimensional, polyacrylamide gel electrophoresis to compare their patterns of polypeptide synthesis. The results show a high incidence of gross and cytogenetic degeneration among oocytes from small antral follicles as compared with those from medium or lage follicles. Pools of oocytes could be separated, on the basis of gross morphology and integrity of adherent granulosa cells, into two classes: "Type A" which appeared normal, and "type B" which appeared to be atretic. Among selected "type A" oocytes a particular chromatin configuration, termed "fibrous" characterizes the gv of oocytes from small follicles; whereas a different configuration, termed "diffuse," characterizes the gv of oocytes from large follicles. The patterns of polypeptide synthesis were markedly different for samples of "type A" oocytes of the three follicular classes; and the patterns for oocytes from medium and large follicles were more similar to each other than to patterns for oocytes from slall follicles. The incidences of maturational development beyond the gv stage in vitro were similar for "type A" oocytes from the three follicular classes (i.e., 66% to 82% maturation); although "type B" oocytes underwent maturation beyond the gv at a significantly reduced incidence (i.e., 20% to 29% maturation). "Type A" oocytes from large follicles completed maturation in vitro (i.e., underwent the first meiotic division) at a significantly higher incidence (55%) than did oocytes from small (11% to 20%) or medium (16%) follicles. Our findings are consistent with the hypotheses that a high proportion of oocytes from small antral follicles are atretic, and that a developmental program controls the molecular and cytogenetic changes occurring in porcine oocytes during follicular growth. These changes appear to be highly correlated with the acquisition of competency to complete maturation in vitro, and possibly also are required for normal fertilization and embryogenesis.     

Configurations of germinal vesicle (GV) chromatin in the goat differ from those of other species

Configuration of germinal vesicle (GV) chromatin has been studied and found correlated with the developmental competence of oocytes in several mammalian species. A common feature in the configuration of GV chromatin in the species studied so far is that the diffuse chromatin (the so called "NSN" pattern) condenses into a perinucleolar ring (the so called "SN" configuration) with follicular growth. However, no study has been published on the configuration of GV chromatin in the goat. Nor is it known whether the perinucleolar ring of condensed chromatin (CC) in an oocyte represents a step toward final maturation or atresia. Changes in configurations of GV chromatin and RNA synthesis during goat oocyte growth, atresia and maturation in vivo and in vitro were investigated in this study. Based on both the size of nucleoli and the degree of chromatin condensation, the GV chromatin of goat oocytes was classified into GV1 characterized by large nucleoli and diffuse chromatin, GV2 with medium-sized nucleoli and condensed net-like (GV2n) or clumped (GV2c) chromatin, GV3 with small nucleoli and net-like (GV3n) or clumped (GV3c) chromatin, and GV4 with no nucleolus but clumped chromatin. The results showed that (i) the configurations of GV chromatin in the goat differ from those of other species in that the chromatin did not condense into a perinucleolar ring; (ii) most of the goat oocytes are synchronized at the GV3n configuration before GVBD; (iii) the GVn pattern might represent a healthy state, but the GVc an atretic state; (iv) in both goats and mice, the GC-specific (Chromomycin A3, CMA3) and the AT-specific (Hoechst 33342) fluorochromes followed the same pattern of distribution in GV chromatin; (v) the nucleolar size decreased significantly with oocyte growth and maturation in vivo and in vitro; and (vi) goat oocytes began GVBD at 8 hr and had completed it by 20 hr after onset of estrus. The peculiar configuration of GV chromatin of goat oocytes can be a useful model for studies of morphological and functional changes of different nuclear compartments during the cell cycle and cell differentiation, and the functional differentiation between GV3n and GV3c might be used for reference to the question whether the "SN" configuration in other species inclines toward ovulation or atresia.     

Chromatin behaviour under influence of puromycin and 6-DMAP at different stages of mouse oocyte maturation

Preovulatory mouse oocytes were cultured in vitro up to each subsequent stages of maturation: germinal vesicle (GV), germinal vesicle breakdown (GVBD), groups of not yet individualized bivalents, circular bivalents, late prometaphase I, metaphase I, anaphase I and telophase I. The stages were identified in living oocytes by fluorescence microscopy using Hoechst 33342 as a specific vital dye. Oocytes from each stage of development developed in vitro and ovulated metaphase II oocytes were subsequently cultured in the presence of puromycin or 6-dimethylaminopurine (6-DMAP), an inhibitor of protein phosphorylation. The effects on chromatin of these drugs were studied during and at the end of culture by fluorescence and electron microscopy. We found that puromycin and 6-DMAP stop meiosis when applied at all stages of oocyte maturation, except for metaphase II. Oocytes at this stage are activated by puromycin. Reaction of the oocytes to the two drugs is different at GV and at metaphase II. All of the other stages react to the drugs by chromatin compaction, which can be followed by chromatin decondensation to form a nucleus. Our results suggest that late prophase chromatin condensation, bivalent individualization and retention of their individuality, as well as individualization of monovalents from telophase and retention of their individuality at metaphase II, are dependent on protein phosphorylation. The events occurring between metaphase I and telophase I are independent of protein synthesis and phosphorylation. The events occurring between metaphase II and formation of the nucleus are independent of protein synthesis.by U. Scheer     

Reproduction,reproductive organs,and meiosis in the bisexual non-parthenogenetic mite Caloglyphus mycophagus,with reference to oocyte degeneration in virgins (Sarcoptiformes: Acaridae)

Meiosis is described in virgin females, inseminated females and males of the acarid mite Caloglyphus mycophagus (Megnin). The observed sex determining mechanism is an XO-type with the male having a diploid chromosome number of 15. Oogenesis in mated females is regular. Pachytene is the earliest meiotic stage which is readily identifiable. At metaphase I eight bivalents are observed. Both products of the first maturation division divide at the second maturation division. After the fusion of the pronuclei either 15 or 16 chromosomes are observed in cleaving eggs. Nurse cells are not observed during the growth period of the oocyte. Such oocytes are attached to a central structure of the ovary by a cone-shaped organelle. At this stage the nucleus appears as a germinal vesicle; a nucleolus is present and the diffuse chromatin appears to extend from the nucleolus to the nuclear membrane. Nuclear extrusion bodies can be seen adjacent to the nuclear membrane both within and outside of the nucleus. Virgin females do not oviposit. The aberrant morphology and behavior of bivalents in post diakinetic oocytes which have not been penetrated by a sperm are described. Neither chromatin nor a chorion could be demonstrated in aberrant oocytes situated in the oviduct. It is suggested that oocyte degeneration in virgins is an adaptive feature in an animal order in which parthenogenesis is the more common mode of reproduction.     

Aging changes the chromatin configuration and histone methylation of mouse oocytes at germinal vesicle stage

Aging decreases the fertility of mammalian females. In old oocytes at metaphase II stage (MII) there are alterations of the chromatin configuration and chromatin modifications such as histone acetylation. Recent data indicate that alterations of histone acetylation at MII initially arise at germinal vesicle stage (GV). Therefore, we hypothesized that the chromatin configuration and histone methylation could also change in old GV oocytes. In agreement with our hypothesis, young GV oocytes had non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) chromatin configurations, while old GV oocytes also had chromatin configurations that could not be classified as NSN or SN. Regarding histone methylation, young GV and MII oocytes showed dimethylation of lysines 4, 9, 36 and 79 in histone 3 (H3K4me2, H3K9me2, H3K36me2, H3K79me2), lysine 20 in histone H4 (H4K20me2) and trimethylation of lysine 9 in histone 3 (H3K9me3) while a significant percentage of old GV and MII oocytes lacked H3K9me3, H3K36me2, H3K79me2 and H4K20me2. The percentage of old oocytes lacking histone methylation was similar at GV and MII suggesting that alterations of histone methylation in old MII oocytes initially arise at GV. Besides, the expression of the histone methylation-related factors Cbx1 and Sirt1 was also found to change in old GV oocytes. In conclusion, our study reports changes of chromatin configuration and histone methylation in old GV oocytes, which could be very useful for further understanding of human infertility caused by aging.     

Aging changes the chromatin configuration and histone methylation of mouse oocytes at germinal vesicle stage

Aging decreases the fertility of mammalian females. In old oocytes at metaphase II stage (MII) there are alterations of the chromatin configuration and chromatin modifications such as histone acetylation. Recent data indicate that alterations of histone acetylation at MII initially arise at germinal vesicle stage (GV). Therefore, we hypothesized that the chromatin configuration and histone methylation could also change in old GV oocytes. In agreement with our hypothesis, young GV oocytes had non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) chromatin configurations, while old GV oocytes also had chromatin configurations that could not be classified as NSN or SN. Regarding histone methylation, young GV and MII oocytes showed dimethylation of lysines 4, 9, 36 and 79 in histone 3 (H3K4me2, H3K9me2, H3K36me2, H3K79me2), lysine 20 in histone H4 (H4K20me2) and trimethylation of lysine 9 in histone 3 (H3K9me3) while a significant percentage of old GV and MII oocytes lacked H3K9me3, H3K36me2, H3K79me2 and H4K20me2. The percentage of old oocytes lacking histone methylation was similar at GV and MII suggesting that alterations of histone methylation in old MII oocytes initially arise at GV. Besides, the expression of the histone methylation-related factors Cbx1 and Sirt1 was also found to change in old GV oocytes. In conclusion, our study reports changes of chromatin configuration and histone methylation in old GV oocytes, which could be very useful for further understanding of human infertility caused by aging.     

Spontaneous calcium oscillations and nuclear PLC-beta1 in human GV oocytes

Our aim was to investigate if human oocytes, like mouse oocytes, exhibit spontaneous Ca(2+) oscillations and nuclear translocation of PLC-beta1 prior to germinal vesicle breakdown (GVBD), and to correlate these events with the evolution of chromatin configuration as a landmark for the meiosis resumption kinetics. Human germinal vesicle (GV) oocytes were either loaded with Fluo-3 probe to record Ca(2+) signals or fixed for subsequent fluorescent labeling of both chromatin and PLC-beta1, and immunogold labeling of PLC-beta1. Here for the first time, we show that human oocytes at the GV-stage exhibit spontaneous Ca(2+) oscillations. Interestingly, only oocytes with a large diameter and characterized by a compact chromatin surrounding the nucleolus of the GV could reveal these kind of oscillations. We also observed a translocation of PLC-beta1 from the cytoplasm towards the nucleus during in vitro maturation of human oocytes. Spontaneous calcium oscillations and nuclear translocation of PLC-beta1 may reflect some degree of oocyte maturity. The impact of our results may be very helpful to understand and resolve many enigmatic problems usually encountered during the in vitro meiotic maturation of human GV oocytes.     

Selection of oocytes for in vitro maturation by brilliant cresyl blue staining： a study using the mouse model

Selecting oocytes that are most likely to develop is crucial for in vitro fertilization and animal cloning. Brilliant cresyl blue （BCB） staining has been used for oocyte selection in large animals, but its wider utility needs further evaluation. Mouse oocytes were divided into those stained （BCB＋） and those unstained （BCB-） according to their ooplasm BCB coloration. Chromatin configurations, cumulus cell apoptosis, cytoplasmic maturity and developmental competence were compared between the BCB＋ and BCB- oocytes. The effects of oocyte diameter, sexual maturity and gonadotropin stimulation on the competence of BCB＋ oocytes were also analyzed. In the large- and medium-size groups, BCB＋ oocytes were larger and showed more surrounded nucleoli （SN） chromatin configurations and higher frequencies of early atresia, and they also gained better cytoplasmic maturity （determined as the intracellular GSH level and pattern of mitochondrial distribution） and higher developmental potential after in vitro maturation （IVM） than the BCB-oocytes. Adult mice produced more BCB＋ oocytes with higher competence than the prepubertal mice when not primed with PMSG. PMSG priming increased both proportion and developmental potency of BCB＋ oocytes. The BCB＋ oocytes in the large-size group showed more SN chromatin configurations, better cytoplasmic maturity and higher developmental potential than their counterparts in the medium-size group. It is concluded that BCB staining can be used as an efficient method for oocyte selection, but that the competence of the BCB＋ oocytes may vary with oocyte diameter, animal sexual maturity and gonadotropin stimulation. Taken together, the series of criteria described here would allow for better choices in selecting oocytes for better development.     

A study on the different types of spermatogonia in buffalo (Bubalus bubalis).

As a first step to understanding spermatogenesis in the buffalo bull the cytological details of different types of spermatogonia were determined in adult buffalo bulls. Morphological changes in the nuclear details were used as a basis for classifying the different types of spermatogonia. The type A spermatogonia had a spherical to ovoid nucleus with finely granulated chromatin, homogeneously dispersed in the nucleoplasm and having one to two nucleoli adhering to the nuclear membrane. The type A0 spermatogonia were characterized by nuclei containing moderately stained, finely granulated chromatin and a nucleolus attached to the nuclear envelope. The A1 type spermatogonia, on the other hand, have pale stained, finely granulated chromatin with the nucleolus adhering to the nuclear membrane. The nuclei of A2 type spermatogonia resembled those of type A1, but contained coarse granular chromatin dispersed in the pale nucleoplasm. The intermediate type of spermatogonia acquired a central position of the nucleolus, but the chromatin remained coarsely granulated and non-clumped. Three classes of type B (B1-B3) spermatogonia were determined on the degree of clumping of the chromatin and the central position of the nucleolus. The type B1 cells were characterized by nuclei containing a few flakes of lightly stained chromatin and a centrally located nucleolus. The type B2 cells showed comparatively more clumping of chromatin than type B1 spermatogonia, which was dispersed at random in the pale nucleoplasm and along the nuclear envelope. The type B3 spermatogonia demonstrated chromophilic chromatin dispersed in the slightly grey nucleoplasm and adhering along the nuclear membrane. Since there seems to be a succession of events following differentiation of type A1 spermatogonia till the last type B cell differentiates into resting primary spermatocytes, may intermediate stages between the presently described classes of type A (A0-A2) and type B (B1-B3) could also be located in sections of the seminiferous tubules.