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A targeted RNAi screen for genes involved in chromosome morphogenesis and nuclear organization in the Caenorhabditis elegans germline.

We have implemented a functional genomics strategy to identify genes involved in chromosome morphogenesis and nuclear organization during meiotic prophase in the Caenorhabditis elegans germline. This approach took advantage of a gene-expression survey that used DNA microarray technology to identify genes preferentially expressed in the germline. We defined a subset of 192 germline-enriched genes whose expression profiles were similar to those of previously identified meiosis genes and designed a screen to identify genes for which inhibition by RNA interference (RNAi) elicited defects in function or development of the germline. We obtained strong germline phenotypes for 27% of the genes tested, indicating that this targeted approach greatly enriched for genes that function in the germline. In addition to genes involved in key meiotic prophase events, we identified genes involved in meiotic progression, germline proliferation, and chromosome organization and/or segregation during mitotic growth.

Animals↗

mei-P22 encodes a chromosome-associated protein required for the initiation of meiotic recombination in Drosophila melanogaster.

Double-strand breaks (DSB) initiate meiotic recombination in a variety of organisms. Here we present genetic evidence that the mei-P22 gene is required for the induction of DSBs during meiotic prophase in Drosophila females. Strong mei-P22 mutations eliminate meiotic crossing over and suppress the sterility of DSB repair-defective mutants. Interestingly, crossing over in mei-P22 mutants can be restored to almost 50% of wild-type by X irradiation. In addition, an antibody-based assay was used to demonstrate that DSBs are not formed in mei-P22 mutants. This array of phenotypes is identical to that of mei-W68 mutants; mei-W68 encodes the Drosophila Spo11 homolog that is proposed to be an enzyme required for DSB formation. Consistent with a direct role in DSB formation, mei-P22 encodes a basic 35.7-kD protein, which, when examined by immunofluorescence, localizes to foci on meiotic chromosomes. MEI-P22 foci appear transiently in early meiotic prophase, which is when meiotic recombination is believed to initiate. By using an antibody to C(3)G as a marker for synaptonemal complex (SC) formation, we observed that SC is present before MEI-P22 associates with the chromosomes, thus providing direct evidence that the development of SC precedes the initiation of meiotic recombination. Similarly, we found that MEI-P22 foci did not appear in a c(3)G mutant in which SC does not form, suggesting that DSB formation is dependent on SC formation in Drosophila. We propose that MEI-P22 interacts with meiosis-specific chromosome proteins to facilitate DSB creation by MEI-W68.

Alleles↗

DNA methyltransferase is developmentally expressed in replicating and non-replicating male germ cells.

Genomic methylation patterns are established during maturation of primordial germ cells and during gametogenesis. While methylation is linked to DNA replication in somatic cells, active de novo methylation and demethylation occur in post-replicative spermatocytes during meiotic prophase (1). We have examined differentiating male germ cells for alternative forms of DNA (cytosine-5)-methyltransferase (DNA MTase) and have found a 6.2 kb DNA MTase mRNA that is present in appreciable quantities only in testis; in post-replicative pachytene spermatocytes it is the predominant form of DNA MTase mRNA. The 5.2 kb DNA MTase mRNA, characteristic of all somatic cells, was detected in isolated type A and B spermatogonia and haploid round spermatids. Immunobolt analysis detected a protein in spermatogenic cells with a relative mass of 180,000-200,000, which is close to the known size of the somatic form of mammalian DNA MTase. The demonstration of the differential developmental expression of DNA MTase in male germ cells argues for a role for testicular DNA methylation events, not only during replication in premeiotic cells, but also during meiotic prophase and postmeiotic development.

Animals↗

SCP2: a major protein component of the axial elements of synaptonemal complexes of the rat.

In the axial elements of synaptonemal complexes (SCs) of the rat, major protein components have been identified, with relative electrophoretic mobilities (M rs) of 30 000-33 000 and 190 000. Using monoclonal anti-SC antibodies, we isolated cDNA fragments which encode the 190 000 M r component of rat SCs. The translation product predicted from the nucleotide sequence of the cDNA, called SCP2 (for synaptonemal complex protein 2), is a basic protein (pI = 8.0) with a molecular mass of 173 kDa. At the C-terminus, a stretch of approximately 50 amino acid residues is predicted to be capable of forming coiled-coil structures. SCP2 contains two clusters of S/T-P motifs, which are common in DNA-binding proteins. These clusters flank the central, most basic part of the protein (pI = 9.5). Three of the S/T-P motifs are potential target sites for p34(cdc2) protein kinase. In addition, SCP2 has eight potential cAMP/cGMP-dependent protein kinase target sites. The gene encoding SCP2 is transcribed specifically in the testis, in meiotic prophase cells. At the amino acid sequence and secondary structural level, SCP2 shows some similarity to the Red1 protein, which is involved in meiotic recombination and the assembly of axial elements of SCs in yeast. We speculate that SCP2 is a DNA-binding protein involved in the structural organization of meiotic prophase chromosomes.

Amino Acid Sequence↗

Basic helix-loop-helix transcription factor Tcfl5 interacts with the Calmegin gene promoter in mouse spermatogenesis.

In mouse spermatogenesis, differentiating germ line cells initiate expression of specific genes at subsequent developmental steps. The Calmegin (Clgn) gene is first expressed in meiotic prophase, in primary spermatocytes, and encodes a protein that acts as a chaperone. To identify testis-specific transcription factors that control expression of the Clgn gene in spermatogenesis, we performed a yeast one-hybrid screening with a Clgn promoter sequence as bait DNA. This screening resulted in the identification of mouse Tcfl5 as a candidate Clgn promoter-binding protein. Tcfl5 is a member of the basic helix-loop-helix (bHLH) family of transcription factors, and mouse Tcfl5 shows 83% amino acid sequence identity with human TCFL5. Gel-shift and yeast one-hybrid experiments showed that Tcfl5 interacts with a non-canonical CACGCG site that is present in the Clgn promoter. By using northern blot, RT-PCR and in situ hybridization, mouse Tcfl5 mRNA was detected only in testis, with the highest expression level in primary spermatocytes and round spermatids. The highest level of Tcfl5 protein was found in primary spermatocytes at the diplotene stage of meiotic prophase, where the protein colocalizes with transcriptionally active chromatin.

Amino Acid Sequence↗

Cytological studies of human zygotes exhibiting developmental arrest.

Developmental arrest of 111 (approximately 5%) fertilized ova which had formed two pronuclei was observed during a 5-year period of an in-vitro fertilization programme. At least 30 zygotes demonstrated visible pronuclei at 44-66 h after insemination, and 42 zygotes fragmented. Of the 107 prepared zygotes, 97 were informative and revealed that developmental arrest occurred at different stages of the cell cycle: from interphase (n = 48), to transition interphase-prophase (n = 20), to prophase (n = 11), and to metaphase (n = 13). The latter 13 zygotes were characterized by chromosome sets as follows: haploid (n = 2), diploid (n = 6), triploid (n = 1) and tetraploid (n = 4). Another five zygotes demonstrated different numbers of metaphase chromosomes (between 10 and 40), as well as prematurely condensed chromosomes (PCC) as a result of marked asynchrony in pronuclear morphogenesis. A total of 18 zygotes exhibited asynchrony in the morphology of the two pronuclei. It is concluded that abnormal chromosome sets and pronuclear asynchrony might be causes for early developmental arrest.

Adult↗

Temporal and spatial distribution of pectin epitopes in differentiating anthers and microspores of fertile and sterile sugar beet.

We studied the possible involvement of several pectin epitopes in anther differentiation and microsporogenesis in fertile and cytoplasmically male sterile sugar beets. The spatial and temporal distribution of five structural motifs were traced with a panel of monoclonal antibodies in six stages: premeiosis, meiotic prophase, young and mature tetrads, young and expanding microspores. The composition of the walls of sporogenous cells and meiocytes differed than that in the tapetum, as evidenced by the presence of alpha-Fuc(1-->2)-beta-Gal and alpha-(1-->5)-L-Ara epitopes binding CCRC-M1 and LM6 antibodies. At meiotic prophase, the meiocyte walls were additionally marked by the appearance of poorly methyl-esterified domains of homogalacturonan and of (1-->4)-beta-Gal residues, detected by JIM5 and LM5. Some constituents of the meiocyte wall which reacted with JIM5 and JIM7 persisted on the surface of the special callose sheath during tetrad development. In newly formed primexine and exine layers of tetrads and microspores, epitopes that were bound by JIM5, JIM7 and LM5 were abundant. No differences in the deposition or relative abundance of pectins were found between fertile and sterile anthers until microspore release from the callose. Later, at the time of abortion, sterile microspores had much larger amounts of epitopes detected by JIM5 than their fertile counterparts.

Antibodies, Monoclonal↗

Expression profiles and chromosomal localization of genes controlling meiosis and follicular development in the sheep ovary.

In female sheep fetuses, two of the most crucial stages of ovarian development are prophase of meiosis I and follicle formation. In the present study, sheep ovaries collected on Days 25, 38, 49, 56, 67, 75, 94, and 120 of gestation, at birth, and in adulthood were tested by reverse transcription-polymerase chain reaction (RT-PCR) for the expression of 14 genes known to be involved in the ovarian differentiation in diverse organisms. The aim of this study was to determine 1) the expression pattern of six genes involved in germ cell development or meiosis (DMC1, SPO11, MSH4, MSH5, DAZL, and Boule) and five ovary-derived factors (OVOL1, SIAH2, DIAPH2, FOXL2, and FGF9), 2) the onset of gene expression for several members of the bone morphogenetic protein (BMP) pathway involved in follicular development (GDF9, BMP15, BMPR-IB), and 3) the chromosomal localization of seven of these genes in the sheep genome. The RT-PCR analysis revealed that the two germline-specific genes, DAZL and Boule, were expressed between 49 and 94 days postcoitum (dpc) with a similar pattern to typical meiosis genes (DMC1, MSH4, and MSH5), suggesting their possible participation in prophase of meiosis I. GDF9 and OVOL1 gene transcription started at 56 dpc and extended until birth, while BMP15 presented a more restricted window of expression between 94 dpc and birth, corresponding to the formation of first growing follicles. The homologous ovine genes for SPO11, DMC1, MSH5, DAZL, FGF9, DIAPH2, and SIAH2 were located on OAR 13q21-22, 3q35, 20q22, 19q13, 10q15, Xq44, and 1q41-42, respectively. In sheep, quantitative trait loci affecting female reproductive capacities are currently being detected. The ontology and precise mapping of ovarian genes will be useful to identify potential candidate genes that might underlie these effects.

Animals↗

Characterization of histone H2A.X expression in testis and specific labeling of germ cells at the commitment stage of meiosis with histone H2A.X promoter-enhanced green fluorescent protein transgene.

To study the complex molecular mechanisms of mammalian spermatogenesis, it would be useful to be able to isolate cells at each stage of differentiation, especially at the stage in which the cells switch from mitosis to meiosis. Currently, no useful marker proteins or gene promoters specific to this important stage are known. We report here a transgenic mouse line that under the control of the promoter for a histone variant, H2A.X, expressed an enhanced green fluorescent protein (EGFP) in cells at the stage of the mitosis-meiosis switch. Endogenous H2A.X is expressed in type A spermatogonia through meiotic prophase spermatocytes in testis and in some somatic cells. However, despite the fact that its expression was driven by the H2A.X promoter, the EGFP expressed in the transgenic mice specifically labeled only the intermediate spermatogonia stage through the meiotic prophase spermatocyte stage in transgenic mice containing the -600-base pair H2A.X promoter/EGFP construct. Type A spermatogonia and somatic cells of other organs were not labeled. This expression pattern made it possible to isolate living cells from the testis of the transgenic mice at the stage of the mitosis-meiosis switch in spermatogenesis using EGFP fluorescence.

Animals↗

Effect of phosphodiesterase type 3 inhibitor on developmental competence of immature mouse oocytes in vitro.

In vitro use of arresters of meiosis could improve cytoplasmic maturation of immature oocytes by controlling the period of prophase I. Phosphodiesterases (PDE) are responsible for the breakdown and concomitant inactivation of the cyclic nucleotides cAMP and cGMP and are implicated in the regulation of oocyte meiotic maturation. Selective inhibitors of phosphodiesterase type 3 (PDE3) prevent meiotic resumption of mammalian oocytes. This study evaluated the impact of meiosis arrest by PDE3 inhibitor, Org 9935, on developmental competence of geminal vesicle (GV)-stage oocytes from small antral follicles. Cumulus-oocyte complexes (COC), retrieved from antral follicles 24 h after eCG exposure and cultured in the presence of PDE3 inhibitor (10 microM) for an additional 24 h, remained arrested in the meiotic prophase. The GV configuration of oocytes before and after the arrest by PDE3 inhibitor was examined. After the period of meiosis arrest, a significantly increased proportion of oocytes had acquired a nucleolus surrounded by a condensed chromatin rim at the GV, which is a morphological correlate of transcriptional repression. Removal of inhibitor resulted in 90.6% +/- 8.3% of oocytes with the first polar body extruded. Fertilization was significantly improved in oocytes that had been arrested compared with oocytes collected 24 h after eCG and undergoing in vitro maturation immediately. Embryonic preimplantation and live offspring rates of arrested oocytes were higher, although not significantly, than those of nonarrested oocytes. These results suggest that a temporal block of meiosis by PDE3 inhibitor promotes developmental competence of mice oocytes retrieved from small antral follicles.

3',5'-Cyclic-AMP Phosphodiesterases↗

Role of deoxyribonucleic acid polymerase epsilon in spermatogenesis in mice.

Previous studies on DNA polymerase epsilon indicate that this enzyme is involved in replication of chromosomal DNA. In this study, we examined the expression of DNA polymerases alpha, delta, and epsilon during mouse testis development and germ cell differentiation. The steady-state levels of mRNAs encoding DNA polymerase epsilon and the recombination enzyme Rad51 remained constant during testis development, whereas the mRNA levels of DNA polymerases alpha and delta declined from birth until sexual maturity. Immunohistochemical staining methods, using a stage-specific model of the seminiferous epithelium, revealed dramatic differences between DNA polymerase alpha and epsilon distribution. As expected, DNA polymerase alpha and proliferating cell nuclear antigen showed relatively strong immunostaining in mitotically proliferating spermatogonia and even stronger staining in preleptotene cells undergoing meiotic DNA replication. The distribution of Rad51 was similar, but there was a dramatic peak in late pachytene cells. In contrast, DNA polymerase epsilon was detectable in mitotically proliferating spermatogonia but not in the early stages of meiotic prophase. However, DNA polymerase epsilon reappeared in late pachytene cells and remained through the two meiotic divisions, and was present in haploid spermatids up to the stage at which the flagellum starts developing. Overall, the results suggest that DNA polymerase epsilon functions in mitotic replication, in the completion of recombination in late pachytene cells, and in repair of DNA damage in round spermatids. In contrast, DNA polymerases alpha and delta appear to be involved in meiotic DNA synthesis, which occurs early in meiotic prophase, in addition to functioning in DNA replication in proliferating spermatogonia.

Animals↗

Regulation of translation during in vitro maturation of bovine oocytes: the role of MAP kinase, eIF4E (cap binding protein) phosphorylation, and eIF4E-BP1.

Meiotic maturation of mammalian oocytes (transition from prophase I to metaphase II) is accompanied by complex changes in the protein phosphorylation pattern. At least two major protein kinases are involved in these events; namely, cdc2 kinase and mitogen-activated protein (MAP) kinase, because the inhibition of these kinases arrest mammalian oocytes in the germinal vesicle (GV) stage. We show that during meiotic maturation of bovine oocytes, the translation initiation factor, eIF4E (the cap binding protein), gradually becomes phosphorylated. This substantial phosphorylation begins at the time of germinal vesicle breakdown (GVBD) and continues to the metaphase II stage. The onset of eIF4E phosphorylation occurs in parallel with a significant increase in overall protein synthesis. However, although eIF4E is nearly fully phosphorylated in metaphase II oocytes, protein synthesis reaches only basal levels at this stage, similar to that of prophase I oocytes, in which the factor remains unphosphorylated. We present evidence that a specific repressor of eIF4E, the binding protein 4E-BP1, is present and could be involved in preventing eIF4E function in metaphase II stage oocytes. Recently, two protein kinases, called Mnk1 and Mnk2, have been identified in somatic cells as eIF4E kinases, both of which are substrates of MAP kinase in vivo. In bovine oocytes, a specific inhibitor of cdk kinases, butyrolactone I, arrests oocytes in GV stage and prevents activation of both cdc2 and MAP kinase. Under these conditions, the phosphorylation of eIF4E is also blocked, and its function in initiation of translation is impaired. In contrast, PD 098059, a specific inhibitor of the MAP kinase activation pathway, which inhibits the MAP kinase kinase, called MEK function, leads only to a postponed GVBD, and a delay in MAP kinase and eIF4E phosphorylation. These results indicate that in bovine oocytes, 1) MAP kinase activation is only partially dependent on MEK kinase, 2) MAP kinase is involved in eIF4E phosphorylation, and 3) the abundance of fully phosphorylated eIF4E does not necessarily directly stimulate protein synthesis. A possible MEK kinase-independent pathway of MAP kinase phosphorylation and the role of 4E-BP1 in repressing translation in metaphase II oocytes are discussed.

Animals↗

STAG3, a novel gene encoding a protein involved in meiotic chromosome pairing and location of STAG3-related genes flanking the Williams-Beuren syndrome deletion.

Chromatin rearrangements in the meiotic prophase are characterized by the assembly and disassembly of synaptonemal complexes (SC), a protein structure that stabilizes the pairing of homologous chromosomes in prophase. We report the identification of human and mouse cDNA coding for stromalin 3 (STAG3), a new mammalian stromalin member of the synaptonemal complex. The stromalins are a group of highly conserved proteins, represented in several organisms from yeast to humans. Stromalins are characterized by the stromalin conservative domain (SCD), a specific motif found in all proteins of the family described to date. STAG3 is expressed specifically in testis, and immunolocalization experiments show that STAG3 is associated to the synaptonemal complex. As the protein encoded by the homologous gene (Scc3p) in Saccharomyces cerevisiae was found to be a subunit of a cohesin complex that binds chromosomes until the onset of anaphase, our data suggest that STAG3 is involved in chromosome pairing and maintenance of synaptonemal complex structure during the pachytene phase of meiosis in a cohesin-like manner. We have mapped the human STAG3 gene to the 7q22 region of chromosome 7; six human STAG3-related genes have also been mapped: two at 7q22 near the functional gene, one at 7q11.22, and three at 7q11.23, two of them flanking the breakpoints commonly associated with the Williams-Beuren syndrome (WBS) deletion. Since the WBS deletion occurs as a consequence of unequal meiotic crossing over, we suggest that STAG3 duplications predispose to germline chromosomal rearrangement within this region.

Amino Acid Sequence↗

Chromosome distribution: experiments on cell hybrids and in vitro.

Ostergren (1951) provided a simple explanation for both chromosome distribution in mitosis and chromosome segregation in meiosis, and more recently a molecular extension of his hypothesis has been possible. This report focuses on experimental tests of these ideas. Micromanipulation experiments on cell hybrids containing both meiotic and mitotic spindles demonstrate that differences in meiotic and mitotic chromosome behavior are determined by something intrinsic to the chromosome: meiotic chromosomes transferred to a mitotic spindle (or vice versa) behave just as they normally would. The molecular explanation postulates polarized growth or binding of microtubules at kinetochores. This has just been tested in vitro by McGill & Brinkley (1975) and by Telzer, Moses & Rosenbaum (1975), and their results are reviewed. In addition, a novel method for in vitro studies is described - mechanical demembranation of cells which is compatible with quite normal chromosome movement in anaphase. After addition of microtubule subunits to a demembranated prophase cell, chromosome orientation and movement toward an aster was observed for the first time in vitro. It is concluded that important aspects of chromosome distribution are probably understood at both the cellular and molecular levels, but final tests are still required. The outlook is hopeful indeed because the gaps in our knowledge are well known - the necessity of observations on prophase is a recurrent theme - and the means of filling the gaps are in hand.

Animals↗

Ribosomes, membranes and organelles during meiosis in angiosperms.

Evidence is presented from both observational and analytical techniques indicating profound changes to take place in the ribosome population during male and female meiosis in some flowering plants. During microsporogenesis these appear to involve the elimination of the major part of the ribosome complement early in the meiotic prophase, and its subsequent restoration by the disintegration in the tetrad cytoplasm of 'nucleoloids', themselves synthesized in the nucleus during late prophase. In female tissue the process is essentially similar except for differences in the restoration of the ribosome population. Immediately before the eradication of the ribosomes in both sexes, a sizeable proportion of the meiocyte cytoplasm is encapsulated by double, or multiple unit membrane profiles. Significantly this cytoplasm remains unaffected by the agents responsible for the degredation of the ribosome population. These events are also reflected in the organelle populations where cycles of dedifferentiation and redifferentiation take place. In view of evidence from other organisms, it is considered unlikely that these cycles are in any way a prerequisite of meiosis, but more a characteristic of cells undergoing major changes of phase, where a cytoplasmic 'clean-up' is required before the next stage of growth may begin.

Acid Phosphatase↗

An Eph receptor sperm-sensing control mechanism for oocyte meiotic maturation in Caenorhabditis elegans.

During sexual reproduction in most animals, oocytes arrest in meiotic prophase and resume meiosis (meiotic maturation) in response to sperm or somatic cell signals. Despite progress in delineating mitogen-activated protein kinase (MAPK) and CDK/cyclin activation pathways involved in meiotic maturation, it is less clear how these pathways are regulated at the cell surface. The Caenorhabditis elegans major sperm protein (MSP) signals oocytes, which are arrested in meiotic prophase, to resume meiosis and ovulate. We used DNA microarray data and an in situ binding assay to identify the VAB-1 Eph receptor protein-tyrosine kinase as an MSP receptor. We show that VAB-1 and a somatic gonadal sheath cell-dependent pathway, defined by the CEH-18 POU-class homeoprotein, negatively regulate meiotic maturation and MAPK activation. MSP antagonizes these inhibitory signaling circuits, in part by binding VAB-1 on oocytes and sheath cells. Our results define a sperm-sensing control mechanism that inhibits oocyte maturation, MAPK activation, and ovulation when sperm are unavailable for fertilization. MSP-domain proteins are found in diverse animal taxa, where they may regulate contact-dependent Eph receptor signaling pathways.

Animals↗

Telomere-mediated chromosome pairing during meiosis in budding yeast.

Certain haploid strains of Saccharomyces cerevisiae can undergo meiosis, but meiotic prophase progression and subsequent nuclear division are delayed if these haploids carry an extra chromosome (i. e., are disomic). Observations indicate that interactions between homologous chromosomes cause a delay in meiotic prophase, perhaps to allow time for interhomolog interactions to be completed. Analysis of meiotic mutants demonstrates that the relevant aspect of homolog recognition is independent of meiotic recombination and synaptonemal complex formation. A disome in which the extra chromosome is circular sporulates without a delay, indicating that telomeres are important for homolog recognition. Consistent with this hypothesis, fluorescent in situ hybridization demonstrates that a circular chromosome has a reduced capacity to pair with its homolog, and a telomere-associated meiotic protein (Ndj1) is required to delay sporulation in disomes. A circular dimer containing two copies of the same chromosome delays meiosis to the same extent as two linear homologs, implying that physical proximity bypasses the requirement for telomeres in homolog pairing. Analysis of a disome carrying two linear permuted chromosomes suggests that even nonhomologous chromosome ends can promote homolog pairing to a limited extent. We speculate that telomere-mediated chromosome movement and/or telomere clustering promote homolog pairing.

Chromosomes↗

Mouse MutS-like protein Msh5 is required for proper chromosome synapsis in male and female meiosis.

Members of the mammalian mismatch repair protein family of MutS and MutL homologs have been implicated in postreplicative mismatch correction and chromosome interactions during meiotic recombination. Here we demonstrate that mice carrying a disruption in MutS homolog Msh5 show a meiotic defect, leading to male and female sterility. Histological and cytological examination of prophase I stages in both sexes revealed an extended zygotene stage, characterized by impaired and aberrant chromosome synapsis, that was followed by apoptotic cell death. Thus, murine Msh5 promotes synapsis of homologous chromosomes in meiotic prophase I.

Animals↗