The APC/C activator FZR1 coordinates the timing of meiotic resumption during prophase I arrest in mammalian oocytes

FZR1, an activator of the anaphase-promoting complex/cyclosome (APC/C), is recognized for its roles in the mitotic cell cycle. To examine its meiotic function in females we generated an oocyte-specific knockout of the Fzr1 gene (Fzr1(Δ/Δ)). The total number of fully grown oocytes enclosed in cumulus complexes was 35-40% lower in oocytes from Fzr1(Δ/Δ) mice and there was a commensurate rise in denuded, meiotically advanced and/or fragmented oocytes. The ability of Fzr1(Δ/Δ) oocytes to remain prophase I/germinal vesicle (GV) arrested in vitro was also compromised, despite the addition of the phosphodiesterase milrinone. Meiotic competency of smaller diameter oocytes was also accelerated by Fzr1 loss. Cyclin B1 levels were elevated ~5-fold in Fzr1(Δ/Δ) oocytes, whereas securin and CDC25B, two other APC/C(FZR1) substrates, were unchanged. Cyclin B1 overexpression can mimic the effects of Fzr1 loss on GV arrest and here we show that cyclin B1 knockdown in Fzr1(Δ/Δ) oocytes affects the timing of meiotic resumption. Therefore, the effects of Fzr1 loss are mediated, at least in part, by raised cyclin B1. Thus, APC/C(FZR1) activity is required to repress cyclin B1 levels in oocytes during prophase I arrest in the ovary, thereby maintaining meiotic quiescence until hormonal cues trigger resumption.     

Apoptosis in mouse fetal and neonatal oocytes during meiotic prophase one

Background  

The vast majority of oocytes formed in the fetal ovary do not survive beyond birth. Possible reasons for their loss include the elimination of non-viable genetic constitutions arising through meiosis, however, the precise relationship between meiotic stages and prenatal apoptosis of oocytes remains elusive. We studied oocytes in mouse fetal and neonatal ovaries, 14.5–21 days post coitum, to examine the relationship between oocyte development and programmed cell death during meiotic prophase I.     

Heterochromatin and nucleolar organizers during first meiotic prophase in quail oocytes

The study of chromosomes in oocytes of the quail shows, at the pachytene stage, that microchromosomes are made of a euchromatic segment and a heterochromatic juxtacentromeric region. The heterochromatic regions of the microchromosomes amalgamate between themselves so as to constitute bulky chromocentres from which radiate the euchromatic segments which remain free. At late pachytene, nucleoli appear at the contact of these chromocentres. When the oocytes reach the diplotene stage, the nucleoli become quite large. They are stuck against chromocentres and establish a very close relationship with the euchromatic segments of the microchromosomes which surround or penetrate them. These observations lead one to think that the euchromatic segments of microchromosomes could be bearing nucleolar organizers. The close relations that the nucleolar organizers develop with the bulk of the nucleolus could explain its Feulgen-positive character in the quail.     

Localization of MMR proteins on meiotic chromosomes in mice indicates distinct functions during prophase I

Mammalian MutL homologues function in DNA mismatch repair (MMR) after replication errors and in meiotic recombination. Both functions are initiated by a heterodimer of MutS homologues specific to either MMR (MSH2-MSH3 or MSH2-MSH6) or crossing over (MSH4-MSH5). Mutations of three of the four MutL homologues (Mlh1, Mlh3, and Pms2) result in meiotic defects. We show herein that two distinct complexes involving MLH3 are formed during murine meiosis. The first is a stable association between MLH3 and MLH1 and is involved in promoting crossing over in conjunction with MSH4-MSH5. The second complex involves MLH3 together with MSH2-MSH3 and localizes to repetitive sequences at centromeres and the Y chromosome. This complex is up-regulated in Pms2-/- males, but not females, providing an explanation for the sexual dimorphism seen in Pms2-/- mice. The association of MLH3 with repetitive DNA sequences is coincident with MSH2-MSH3 and is decreased in Msh2-/- and Msh3-/- mice, suggesting a novel role for the MMR family in the maintenance of repeat unit integrity during mammalian meiosis.     

Localisation of the SMC loading complex Nipbl/Mau2 during mammalian meiotic prophase I

Evidence from lower eukaryotes suggests that the chromosomal associations of all the structural maintenance of chromosome (SMC) complexes, cohesin, condensin and Smc5/6, are influenced by the Nipbl/Mau2 heterodimer. Whether this function is conserved in mammals is currently not known. During mammalian meiosis, very different localisation patterns have been reported for the SMC complexes, and the localisation of Nipbl/Mau2 has just recently started to be investigated. Here, we show that Nipbl/Mau2 binds on chromosomal axes from zygotene to mid-pachytene in germ cells of both sexes. In spermatocytes, Nipbl/Mau2 then relocalises to chromocenters, whereas in oocytes it remains bound to chromosomal axes throughout prophase to dictyate arrest. The localisation pattern of Nipbl/Mau2, together with those seen for cohesin, condensin and Smc5/6 subunits, is consistent with a role as a loading factor for cohesin and condensin I, but not for Smc5/6. We also demonstrate that Nipbl/Mau2 localises next to Rad51 and γH2AX foci. NIPBL gene deficiencies are associated with the Cornelia de Lange syndrome in humans, and we find that haploinsufficiency of the orthologous mouse gene results in an altered distribution of double-strand breaks marked by γH2AX during prophase I. However, this is insufficient to result in major meiotic malfunctions, and the chromosomal associations of the synaptonemal complex proteins and the three SMC complexes appear cytologically indistinguishable in wild-type and Nipbl ^+/? spermatocytes.     

Structural damage to meiotic chromosomes impairs DNA recombination and checkpoint control in mammalian oocytes

Meiosis in human oocytes is a highly error-prone process with profound effects on germ cell and embryo development. The synaptonemal complex protein 3 (SYCP3) transiently supports the structural organization of the meiotic chromosome axis. Offspring derived from murine Sycp3^−/− females die in utero as a result of aneuploidy. We studied the nature of the proximal chromosomal defects that give rise to aneuploidy in Sycp3^−/− oocytes and how these errors evade meiotic quality control mechanisms. We show that DNA double-stranded breaks are inefficiently repaired in Sycp3^−/− oocytes, thereby generating a temporal spectrum of recombination errors. This is indicated by a strong residual γH2AX labeling retained at late meiotic stages in mutant oocytes and an increased persistence of recombination-related proteins associated with meiotic chromosomes. Although a majority of the mutant oocytes are rapidly eliminated at early postnatal development, a subset with a small number of unfinished crossovers evades the DNA damage checkpoint, resulting in the formation of aneuploid gametes.     

Oogenesis of Tilapia mossambica. I. Oogonia and meiotic prophase oocytes

Using light and electron microscopy and autoradiography, the morphology and synthesis of DNA, RNA and proteins in oogonia and early meiotic prophase oocytes in Tilaria mossabique were studied. According to dimensions and morphological features observed it is possible to distinguish between two groups of oogonia: large oogonia corresponding to type A spermatogonia of mammals, and small actively dividing oogonia, located in groups and identical to type B spermatogonia. The morphology of oogonia and of the early meiotic prophase oocytes well compares with the pattern described for other species of bony fishes. In the cytoplasm of these cells dense bodies, nuage-material, free ribosomes, large mitochondria with lamellar cristae and Golgi cisterns are available. In the oocyte nuclei at zygotene and pahytene stages 3H-thymidine incorporation was seen mainly into the nucleolus-associated chromatin. Besides, the formation of a heterochromatin cape and the synaptonemal complex was observed. Incorporation of 3H-uridine and 3H-leucine in the nuclei of these cells was very poor.     

The spatial relationship of the X and Y chromosomes during meiotic prophase in mouse spermatocytes

The ultrastructure of whole X-Y pairs has been reconstructed by serial sectioning and model building. Seven X-Y pairs were completely reconstructed and the lengths of the cores of the sex chromosomes were measured. These X-Y pairs corresponded to zygonema, early, middle and late pachynema. Special regions of the X-Y pair were reconstructed from thinner sections. — It has been shown that two cores exist in the sex pair during the cited stages, and that their lengths and morphology are rather constant in specific stages. The long core averages 8.9 in length and the short core is 3.5 long. Both cores have a common end region in which a synaptonemal complex is formed from zygonema up to midpachynema. This synaptonemal complex shortens progressively up to mid-pachynema and at late pachynema becomes obliterated. Each core has a free end touching the nuclear membrane. During mid-pachynema an anomalous synaptonemal complex is developed on most of the length of the long core. This complex is asymmetric and disappears at late pachynema. The meaning of the cores and the complexes are discussed, and the existence of a homologous region in the X-Y pair of the mouse is interpreted to be proved.     

Persistence of two Y chromosomes through meiotic prophase and metaphase I in an XYY man

Summary Studies of spermatogenesis in an XYY male, presenting at a subfertility clinic, confirm the tendency for the germ cells to lose the second Y chromosome but for some XYY cells to reach metaphase I (MI). Light microscope studies of MI revealed the presence of YY bivalents and EM studies of microspread, silver-stained pachytene stages showed 30% of the cells to have two Y chromosomes; 13 out of 16 of these showing a YY synaptonemal complex. Strikingly, the Y axes show only partial synapsis; in no case was synapsis of the long arm heterochromatic regions apparent.     

Nucleoli and chromosomes: Their relationships during the meiotic prophase of the human fetal oocyte

Summary The number of nucleoli and the relations between them and chromosomes in the human fetal oöcyte have been investigated in this study. The differences existing between first oöcytes and spermatocytes have been emphasized. These differences concern essentially the number of nucleoli, the stage during which they appear and the quantity of heterochromatin associated with the nucleoli.The latter appear very early in the oöcyte: they are already present at the beginning of the preleptotene stage. This stage is characterized by the processes of spiralization and despiralization, heretofore not described.The first prophase is characterized by the presence of abundant nucleolar material, especially in the diplotene stage. This abundance is certainly in relation with the active protein synthesis which characterizes the growth of the oöcyte. As in the spermatocyte of the pachytene stage, the majority of nucleolar chromosomes, in the oöcyte, are acrocentric. But in the latter, the quantity of heterochromatin associated with the nucleolus greatly exceeds the quantity present in the nucleolar organizers of acrocentric chromosomes, particularly during the leptotene stage. This supports the opinion that there are multiple sites for the synthesis of the various nucleolar componnents. Also discussed are the roles of heterochromatin and nucleolar organizers in nucleogenesis.

---

Zusammenfassung Die Zahl der Nucleolen sowie ihre Beziehungen zu den Chromosomen wurde an der menschlichen fetalen Oocyte untersucht. Dabei stellten sich deutliche Differenzen zu den Spermatocyten heraus. Diese Differenzen betreffen vor allem die Zahl der Nucleolen, das Stadium, in welchem sie erscheinen, und die Heterochromatinmenge, die mit den Nucleolen assoziiert ist.Diese erscheinen in der Oocyte sehr früh, schon zu Beginn des Präleptotäns. In diesem Stadium wurde erstmalig eine Spiralisation und Despiralisation beschrieben.Die erste Prophase ist durch die Anwesenheit von großen Mengen von Nucleolusmaterial charakterisiert, besonders im Diplotän. Dieser Befund steht sicher in Zusammenhang mit der aktiven Proteinsynthese während des Oocytenwachstums. Wie bei Pachytänspermatocyten sind auch in der Oocyte die meisten der mit dem Nucleolus verbundenen Chromosomen akrozentrisch. Doch übertrifft bei letzterer die Heterochromatinmenge beim Nucleolus die Menge der Nucleolusorganisatoren der akrozentrischen Chromosomen erheblich, besonders während des Leptotäns. Dadurch wird die Auffassung unterstützt, daß die Synthese verschiedener Nucleoluskomponenten an verschiedenen Stellen erfolgt. Die Bedeutung des Heterochromatins und der Nucleolusorganisatoren bei der Entstehung des Nucleolus wird diskutiert.

     

Developmental changes in DNA methylation of pollen mother cells of David lily during meiotic prophase I

Epigenetic marks in the form of DNA methylation are involved in the development of germ cells and are important in the maintenance of fertility. However, the controlling system of the on-off switch for DNA methylation largely remains unclear. In this study, the extent of cytosine methylation during the meiotic prophase I in David lily is assessed using high pressure liquid chromatography to evaluate the DNA methylation rates. Comparing the degree of DNA methylation before, during, and after synizesis, both de novo methylation and demethylation occurred. Mainly the methylation level decreased by 21.3% (from 54.8 to 33.5%) during synizesis in the pollen mother cells. The developmental timing of genome-wide DNA methylation acquisition during pollen mother cell development is clarified in this paper. The relative amounts of 5-methyl-deoxycytidine of global methylation in leaf DNA in David lily were also higher than in other species reported.     

Evidence for homologous pairing of chromosomes prior to meiotic prophase in maize

Relative positions of homologous heterochromatic regions of maize chromosomes were studied at premeiotic interphase, at tapetal mitotic interphase and at root tip mitotic interphase. In all three kinds of cell homologues were found to be situated significantly nearer to each other than to heterologues. It is concluded that some degree of homologous chromosome pairing may occur widely at anaphase or telophase (where it is easily overlooked) and that therefore, as has been previously suggested, homologues may be loosely aligned throughout premeiotic interphase in preparation for their subsequent synapsis.     

Robertsonian chromosomes and the nuclear architecture of mouse meiotic prophase spermatocytes

Background

The nuclear architecture of meiotic prophase spermatocytes is based on higher-order patterns of spatial associations among chromosomal domains from different bivalents. The meiotic nuclear architecture depends on the chromosome characteristics and consequently is prone to modification by chromosomal rearrangements. In this work, we consider Mus domesticus spermatocytes with diploid chromosome number 2n = 40, all telocentric, and investigate a possible modification of the ancestral nuclear architecture due to the emergence of derived Rb chromosomes, which may be present in the homozygous or heterozygous condition.

Results

In the 2n = 40 spermatocyte nuclei random associations mediated by pericentromeric heterochromatin among the 19 telocentric bivalents ocurr at the nuclear periphery. The observed frequency of associations among them, made distinguishable by specific probes and FISH, seems to be the same for pairs that may or may not form Rb chromosomes. In the homozygote Rb 2n = 24 spermatocytes, associations also mediated by pericentromeric heterochromatin occur mainly between the three telocentric or the eight metacentric bivalents themselves. In heterozygote Rb 2n = 32 spermatocytes all heterochromatin is localized at the nuclear periphery, yet associations are mainly observed among the three telocentric bivalents and between the asynaptic axes of the trivalents.

Conclusions

The Rb chromosomes pose sharp restrictions for interactions in the 2n = 24 and 2n = 32 spermatocytes, as compared to the ample possibilities for interactions between bivalents in the 2n = 40 spermatocytes. Undoubtedly the emergence of Rb chromosomes changes the ancestral nuclear architecture of 2n = 40 spermatocytes since they establish new types of interactions among chromosomal domains, particularly through centromeric and heterochromatic regions at the nuclear periphery among telocentric and at the nuclear center among Rb metacentric ones.     

Regulation of the meiotic prophase I to metaphase I transition in mouse spermatocytes

The meiotic prophase I to metaphase I transition (G2/MI) involves disassembly of synaptonemal complex (SC), chromatin condensation, and final compaction of morphologically distinct MI bivalent chromosomes. Control of these processes is poorly understood. The G2/MI transition was experimentally induced in mouse pachytene spermatocytes by okadaic acid (OA), and kinetic analysis revealed that disassembly of the central element of the SC occurred very rapidly after OA treatment, before histone H3 phosphorylation on Ser10. These events were followed by relocalization of SYCP3 and final condensation of bivalents. Enzymatic control of these G2/MI transition events was studied using small molecule inhibitors: butyrolactone I (BLI), an inhibitor of cyclin-dependent kinases (CDKs) and ZM447439 (ZM), an inhibitor of aurora kinases (AURKs). The formation of highly condensed MI bivalents and disassembly of the SC are regulated by both CDKs and AURKs. AURKs also mediate phosphorylation of histone H3 in meiosis. However, neither BLI nor ZM inhibited disassembly of the central element of the SC. Thus, despite evidence that the metaphase promoting factor is a universal regulator of the onset of cell division, desynapsis, the first and key step of the G2/MI transition, occurs independently of BLI-sensitive CDKs and ZM-sensitive AURKs.