Mismatch repair proteins, meiosis, and mice: understanding the complexities of mammalian meiosis

Mammalian meiosis differs from that seen in lower eukaryotes in several respects, not least of which is the added complexity of dealing with chromosomal interactions across a much larger genome (12 MB over 16 chromosome pairs in Saccharomyces cerevisiae compared to 2500 MB over 19 autosome pairs in Mus musculus). Thus, the recombination machinery, while being highly conserved through eukaryotes, has evolved to accommodate such issues to preserve genome integrity and to ensure propagation of the species. One group of highly conserved meiotic regulators is the DNA mismatch repair protein family that, as their name implies, were first identified as proteins that act to repair DNA mismatches that arise primarily during DNA replication. Their function in ensuring chromosomal integrity has also translated into a critical role for this family in meiotic recombination in most sexually reproducing organisms. In mice, targeted deletion of certain family members results in severe consequences for meiotic progression and infertility. This review will focus on the studies involving these mutant mouse models, with occasional comparison to the function of these proteins in other organisms.     

Microspore development in Podostemaceae-Podostemoideae, with implications on the characterization of the subfamilies

Subfamilies Podostemoideae and Tristichoideae of the aquatic flowering plant family Podostemaceae are conventionally characterized by a different mode of microsporogenesis. Simultaneous meiotic division into the four microspores is found in Tristichoideae, successive meiotic division is said to be typical of Podostemoideae. In contrast, the results of the present study reveal that in subfamily Podostemoideae both modes of microsporogenesis occur. This is exemplified by the early pollen development of two neotropical species: Apinagia latifolia and Marathrum rubrum. Successive versus simultaneous meiotic cytokinesis are thus not differential characters of the two subfamilies. It is worthy to note that successive cytokinesis occurs in a family (Podostemaceae) of the Eudicots which are characterized by simultaneous cytokinesis. The occurrence of Ubisch bodies (orbicules) in several species of Apinagia and Marathrum parallels the echinate ornamentation of the pollen grains.     

Genetically determined asynapsis, spermatogenic degeneration, and infertility in men.

We report a family in which azoospermia and infertility affected two sibs whose parents were first cousins once removed. Meiotic cells of the proband, who had the chromosomal complement of a normal male (46,XY), exhibited asynapsis, defective synaptonemal complex (SC) formation, chiasma failure, and degeneration of prophase spermatocytes with asynapsis. Based on these observations, we suggest that the meiotic abnormalities and infertility in this family comprise a trait with an autosomal recessive mode of inheritance. Review of published cases of infertile men with normal chromosomal complements and disturbed meiosis suggests that genetically determined asynapsis and desynapsis similar to that established in plant and insect species also occur in humans. In humans, asynapsis appears to be inherited as an autosomal recessive. The mode of inheritance of desynapsis is not clear; X-linked recessive or autosomal dominant has been suggested in one family. Studies by us and by others reported in the literature suggest that the mode of action of genes that affect synapsis and cause a reduction in the numbers of visible chiasmata at diakinesis is dissimilar to that of the action of genes that cause defective meiotic recombination, defective repair of induced damage to DNA in somatic cells, and chromosome instability.     

WAVE2 regulates meiotic spindle stability,peripheral positioning and polar body emission in mouse oocytes

During oocyte meiotic maturation, meiotic spindles form in the central cytoplasm and then migrate to the cortex to extrude a small polar body, forming a highly polarized cell through a process involving actin and actin-related molecules. The mechanisms underlying oocyte polarization are still unclear. The Arp2/3 complex regulates oocyte polarization but it is not known whether the WASP family of proteins, a known regulator of the Arp2/3 complex, is involved in this context. In the present study, the role of WASP family member WAVE2 in mouse oocyte asymmetric division was investigated. (1) WAVE2 mRNA and protein were detected during mouse oocyte meiosis. (2) siRNA-mediated and antibody-mediated disruption of WAVE2 resulted in the failure of chromosome congression, spindle formation, spindle positioning and polar body extrusion. (3) WAVE2 regulated actin-driven chromosome migration since chromosomes were arrested in the central cytoplasm by WAVE2 RNAi in the absence of microtubules. (4) Localization of γ-tubulin and MAPK was disrupted after RNAi, confirming the effect of WAVE2 on spindle formation. (5) Actin cap and cortical granule-free domain (CGFD) formation was also disrupted, further confirming the failure of oocyte polarization. Our data suggest that WAVE2 regulates oocyte polarization by regulating meiotic spindle, peripheral positioning, probably via an actin-mediated pathway, and is involved in polar body emission during mouse oocyte meiotic maturation.     

REC, Drosophila MCM8, drives formation of meiotic crossovers

Crossovers ensure the accurate segregation of homologous chromosomes from one another during meiosis. Here, we describe the identity and function of the Drosophila melanogaster gene recombination defective (rec), which is required for most meiotic crossing over. We show that rec encodes a member of the mini-chromosome maintenance (MCM) protein family. Six MCM proteins (MCM2–7) are essential for DNA replication and are found in all eukaryotes. REC is the Drosophila ortholog of the recently identified seventh member of this family, MCM8. Our phylogenetic analysis reveals the existence of yet another family member, MCM9, and shows that MCM8 and MCM9 arose early in eukaryotic evolution, though one or both have been lost in multiple eukaryotic lineages. Drosophila has lost MCM9 but retained MCM8, represented by REC. We used genetic and molecular methods to study the function of REC in meiotic recombination. Epistasis experiments suggest that REC acts after the Rad51 ortholog SPN-A but before the endonuclease MEI-9. Although crossovers are reduced by 95% in rec mutants, the frequency of noncrossover gene conversion is significantly increased. Interestingly, gene conversion tracts in rec mutants are about half the length of tracts in wild-type flies. To account for these phenotypes, we propose that REC facilitates repair synthesis during meiotic recombination. In the absence of REC, synthesis does not proceed far enough to allow formation of an intermediate that can give rise to crossovers, and recombination proceeds via synthesis-dependent strand annealing to generate only noncrossover products.     

The spindle-associated transmembrane protein Axs identifies a new family of transmembrane proteins in eukaryotes

Axs mutations disrupt both the progression of the meiotic cell cycle and meiotic chromosome segregation in Drosophila. Axs protein co-localizes with endoplasmic reticulum components and is present within a novel structure ensheathing the meiotic spindle. We show that Axs encodes the founding member of a eukaryotic family of trans-membrane proteins.     

The Spindle-Associated Transmembrane Protein Axs Identifies a New Family of Transmembrane Proteins in Eukaryotes

Axs mutations disrupt both the progression of the meiotic cell cycle and meiotic chromosome segregation in Drosophila. Axs protein co-localizes with endoplasmic reticulum components and is present within a novel structure ensheathing the meiotic spindle. We show that Axs encodes the founding member of a eukaryotic family of trans-membrane proteins.     

Evolution of the Schlafen genes,a gene family associated with embryonic lethality,meiotic drive,immune processes and orthopoxvirus virulence

Genes of the Schlafen family, first discovered in mouse, are expressed in hematopoietic cells and are involved in immune processes. Previous results showed that they are candidate genes for two major phenomena: meiotic drive and embryonic lethality (DDK syndrome). However, these genes remain poorly understood, mostly due to the limitations imposed by their similarity, close location and the potential functional redundancy of the gene family members.     

The spindle-associated transmembrane protein Axs identifies a membranous structure ensheathing the meiotic spindle

Mutations in the aberrant X segragation (Axs) gene disrupt the segregation of achiasmate chromosomes during female meiosis in Drosophila melanogaster. We show that Axs encodes the founding member of an eukaryotic family of transmembrane proteins. Axs protein colocalizes with components of the endoplasmic reticulum and is present within a structure ensheathing the meiotic spindle. In both meiotic and mitotic cells, Axs is recruited to the microtubules of assembling spindles. We propose that Axs and the sheath represent novel mediators of meiotic spindle assembly and chromosome segregation.     

The Drosophila meiotic mutant mei-352 is an allele of klp3A and reveals a role for a kinesin-like protein in crossover distribution

The semisterile meiotic mutant mei-352 alters the distribution of meiotic exchanges without greatly affecting their total frequency. We show that the mei-352 mutation is an allele of the klp3A gene, which encodes a kinesin-like protein of the Kinesin-4 family. The semisterility observed in mei-352 females results from a known defect of klp3A oocytes in mediating pronuclear fusion. Interestingly, other klp3A alleles also exhibit defects in meiotic recombination similar to those of mei-352. Finally, we show that the Klp3A protein localizes within the oocyte nucleus during meiotic prophase, the time at which exchange distribution is established, and extensively colocalizes with DNA. The parallel of the klp3A phenotype with a meiotic defect observed for kar3 mutants in yeast suggests a role for kinesins in early meiosis and might reflect a previously suggested role for this class of kinesins in chromosome condensation.     

A family of zinc-finger proteins is required for chromosome-specific pairing and synapsis during meiosis in C. elegans

Homologous chromosome pairing and synapsis are prerequisite for accurate chromosome segregation during meiosis. Here, we show that a family of four related C2H2 zinc-finger proteins plays a central role in these events in C. elegans. These proteins are encoded within a tandem gene cluster. In addition to the X-specific HIM-8 protein, three additional paralogs collectively mediate the behavior of the five autosomes. Each chromosome relies on a specific member of the family to pair and synapse with its homolog. These "ZIM" proteins concentrate at special regions called meiotic pairing centers on the corresponding chromosomes. These sites are dispersed along the nuclear envelope during early meiotic prophase, suggesting a role analogous to the telomere-mediated meiotic bouquet in other organisms. To gain insight into the evolution of these components, we characterized homologs in C. briggsae and C. remanei, which revealed changes in copy number of this gene family within the nematode lineage.     

Critical effect of pigWee1B on the regulation of meiotic resumption in porcine immature oocytes

Porcine immature oocytes require protein synthesis for meiotic resumption, thus the importance of Cdc2 inhibitory phosphorylation in their meiotic arrest remains controversial. We examined the involvement of Cdc2 phosphorylation in the meiotic arrest of porcine oocytes with a special focus on Wee1B, an oocyte-specific Wee1 family member recently reported in mouse oocytes. We cloned a Wee1B homologue of pig by RT-PCR followed by 5’- and 3’-RACE. Overexpression of pigWee1B in porcine immature oocytes by the injection of pigWee1B mRNA almost completely blocked the germinal vesicle breakdown (GVBD) under the low cAMP concentration, which could not block their spontaneous meiotic resumption by itself. The MPF activation and cyclin B synthesis were inhibited in these oocytes. Conversely, downregulation of pigWee1B expression by the injection of specific antisense mRNA induced GVBD in the oocytes, the spontaneous meiotic resumption of which was blocked by the high concentration of cAMP (dbcAMP). In these oocytes, the MPF activity was elevated and cyclin B was accumulated. Downregulation of pigMyt1, another Wee1 family member, could not induce the GVBD under the same condition. The inhibition of tyrosine phosphatase by vanadate blocked the GVBD even in the pigWee1B-downregulated oocytes. These results suggest that the inhibitory phosphorylation of CDC2, which is catalyzed by pigWee1B, but not pigMyt1, is involved in the meiotic arrest of porcine oocytes, and that the inactivation of Wee1B in combination with the phosphatase activation induces the conversion of pre-MPF to the active MPF and starts the cyclin B synthesis, follwed by a further increase of MPF and meiotic resumption.     

Small Molecule Inhibitor of Formin Homology 2 Domains (SMIFH2) Reveals the Roles of the Formin Family of Proteins in Spindle Assembly and Asymmetric Division in Mouse Oocytes

Dynamic actin reorganization is the main driving force for spindle migration and asymmetric cell division in mammalian oocytes. It has been reported that various actin nucleators including Formin-2 are involved in the polarization of the spindle and in asymmetric cell division. In mammals, the formin family is comprised of 15 proteins. However, their individual roles in spindle migration and/or asymmetric division have not been elucidated yet. In this study, we employed a newly developed inhibitor for formin family proteins, small molecule inhibitor of formin homology 2 domains (SMIFH2), to assess the functions of the formin family in mouse oocyte maturation. Treatment with SMIFH2 during in vitro maturation of mouse oocytes inhibited maturation by decreasing cytoplasmic and cortical actin levels. In addition, treatment with SMIFH2, especially at higher concentrations (500 μM), impaired the proper formation of meiotic spindles, indicating that formins play a role in meiotic spindle formation. Knockdown of the mDia2 formins caused a similar decrease in oocyte maturation and abnormal spindle morphology, mimicking the phenotype of SMIFH2-treated cells. Collectively, these results suggested that besides Formin-2, the other proteins of the formin, including mDia family play a role in asymmetric division and meiotic spindle formation in mammalian oocytes.     

Maintenance of meiotic prophase arrest in vertebrate oocytes by a Gs protein-mediated pathway

Maintenance of meiotic prophase arrest in fully grown vertebrate oocytes depends on an elevated level of cAMP in the oocyte. To investigate how the cAMP level is regulated, we examined whether the activity of an oocyte G protein of the family that stimulates adenylyl cyclase, Gs, is required to maintain meiotic arrest. Microinjection of a dominant negative form of Gs into Xenopus and mouse oocytes, or microinjection of an antibody that inhibits the Gs G protein into zebrafish oocytes, caused meiosis to resume. Together with previous studies, these results support the conclusion that Gs-regulated generation of cAMP by the oocyte is a common mechanism for maintaining meiotic prophase arrest in vertebrate oocytes.     

Galphao/i and Galphas signaling function in parallel with the MSP/Eph receptor to control meiotic diapause in C. elegans

BACKGROUND: A conserved biological feature of sexual reproduction in animals is that oocytes arrest in meiotic prophase and resume meiosis in response to extraovarian signals. In C. elegans, sperm trigger meiotic resumption by means of the major sperm protein (MSP) signal. MSP promotes meiotic resumption by functioning as an ephrin-signaling antagonist and by counteracting inhibitory inputs from the somatic gonadal sheath cells. RESULTS: By using a genome-wide RNAi screen in a female-sterile genetic background, we identified 17 conserved genes that maintain meiotic arrest in the absence of the MSP signal. In vitro binding experiments show that MSP promotes oocyte mitogen-activated protein kinase activation and meiotic maturation in part through direct interaction with the VAB-1 Eph receptor. Four conserved proteins, including a disabled protein (DAB-1), a vav family GEF (VAV-1), a protein kinase C (PKC-1), and a STAM homolog (PQN-19), function with the VAB-1 Eph/MSP receptor in oocytes. We show that antagonistic Galphao/i and Galphas signaling pathways function in the soma to regulate meiotic maturation in parallel to the VAB-1 pathway. Galphas activity is necessary and sufficient to promote meiotic maturation, which it does in part by antagonizing inhibitory sheath/oocyte gap-junctional communication. CONCLUSIONS: Our findings show that oocyte Eph receptor and somatic cell G protein signaling pathways control meiotic diapause in C. elegans, highlighting contrasts and parallels between MSP signaling in C. elegans and luteinizing hormone signaling in mammals.