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Reproductive success is dependent on gamete integrity, and oxidative stress alters male nuclei, meaning that no DNA repair is possible due to chromatin compaction. The composition of sperm makes it highly sensitive to reactive oxygen species (ROS) but, at the same time, ROS are needed for sperm physiology. Over the past 30 years, much attention has been paid to the consequences of oxidative stress on sperm properties and the protective effects of antioxidant formulations to help fertility. Spermatozoa also carry epigenetic marks, critical for embryo development and the health of offspring. As DNA oxidative damage may disturb the sperm epigenome, we used an established mouse model of post-testicular sperm DNA oxidation to investigate sperm DNA methylation and hydroxymethylation. We also analyzed the potential corrective effect of oral antioxidant supplementation, proven to reduce sperm DNA oxidative damage, on sperm DNA methyl/hydroxymethyl marks. We show that sperm DNA oxidation is associated with a significant increase in overall hydroxymethylation. Oral antioxidant supplementation led to unexpected mild epigenetic changes. Antioxidant supplementation should not be proposed without proper clinical evaluation as it may alter sperm epigenetic marks, leading to a risk of paternally inherited epigenetic alterations.

Hepatoblastomas (HB) display heterogeneous cellular phenotypes that influence the clinical outcome, but the underlying mechanisms are poorly understood. Here, we use a single-cell multiomic strategy to unravel the molecular determinants of this plasticity. We identify a continuum of HB cell states between hepatocytic (scH), liver progenitor (scLP) and mesenchymal (scM) differentiation poles, with an intermediate scH/LP population bordering scLP and scH areas in spatial transcriptomics. Chromatin accessibility landscapes reveal the gene regulatory networks of each differentiation pole, and the sequence of transcription factor activations underlying cell state transitions. Single-cell mapping of somatic alterations reveals the clonal architecture of each tumor, showing that each genetic subclone displays its own range of cellular plasticity across differentiation states. The most scLP subclones, overexpressing stem cell and DNA repair genes, proliferate faster after neo-adjuvant chemotherapy. These results highlight how the interplay of clonal evolution and epigenetic plasticity shapes the potential of HB subclones to respond to chemotherapy., (© 2024. The Author(s).)

Background: Genomic analysis of circulating tumor cells (CTCs) could provide a unique and accessible representation of tumor diversity but remains hindered by technical challenges associated with CTC rarity and heterogeneity., Objective: To evaluate CTCs as surrogate samples for genomic analyses in metastatic castration-resistant prostate cancer (mCRPC)., Design, Setting, and Participants: Three isolation strategies (filter laser-capture microdissection, self-seeding microwell chips, and fluorescence-activated cell sorting) were developed to capture CTCs with various epithelial and mesenchymal phenotypes and isolate them at the single-cell level. Whole-genome amplification (WGA) and WGA quality control were performed on 179 CTC samples, matched metastasis biopsies, and negative controls from 11 patients. All patients but one were pretreated with enzalutamide or abiraterone. Whole-exome sequencing (WES) of 34 CTC samples, metastasis biopsies, and negative controls were performed for seven patients., Outcome Measurements and Statistical Analysis: WES of CTCs was rigorously qualified in terms of percentage coverage at 10× depth, allelic dropout, and uncovered regions. Shared somatic mutations between CTCs and matched metastasis biopsies were identified. A customized approach based on determination of mutation rates for CTC samples was developed for identification of CTC-exclusive mutations., Results and Limitations: Shared mutations were mostly detected in epithelial CTCs and were recurrent. For two patients for whom a deeper analysis was performed, a few CTCs were sufficient to represent half to one-third of the mutations in the matched metastasis biopsy. CTC-exclusive mutations were identified in both epithelial and nonepithelial CTCs and affected cytoskeleton, invasion, DNA repair, and cancer-driver genes. Some 41% of CTC-exclusive mutations had a predicted deleterious impact on protein function. Phylogenic relationships between CTCs with distinct phenotypes were evidenced., Conclusions: CTCs can provide unique insight into metastasis mutational diversity and reveal undiagnosed genomic aberrations in matched metastasis biopsies., Patient Summary: Our results demonstrate the clinical potential of circulating tumor cells to provide insight into metastatic events that could be critical to target using precision medicine., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

MiR-31-3p expression has been shown to be a predictive biomarker for response to anti-epithelial growth factor receptor therapy in patients with RAS wild-type metastatic colorectal cancer (mCRC). To aid in the quantification of miR-31-3p expression in formalin-fixed paraffin-embedded (FFPE) primary tumor samples from patients with mCRC, a reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay was developed and validated. Assay development included the identification of a microRNA reference standard and the determination of an appropriate relative quantification cutoff for differentiating low versus high miR-31-3p expression. Sample specimens for the validation studies included both FFPE slides and shavings. Polymerase chain reaction (PCR) efficiency and linearity, analytical sensitivity and specificity, assay robustness, reproducibility, and accuracy were demonstrated across a number of test conditions and differing quantitative PCR platforms. The data from this study provide evidence as to the feasibility of quantifying the expression of miR-31-3p from FFPE tumor tissue using a standardized RT-qPCR assay., Competing Interests: Declaration of Conflicting Interests:The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: The experiments conducted for the data reported were performed by either IntegraGen SA or Laboratorie CERBA SA. IntegraGen SA completed the statistical analyses for the data reported. L.R., S.M-L., C.M., E.L., K.F., C.V., V.D., and YG-M are employees of IntegraGen SA. F.R. and R.T. were employees of IntegraGen SA when these studies were conducted. A-H.G., J-M.C., and R.B.A. are employees of Laboratoire CERBA SA. C.D. is a paid consultant for IntegraGen SA. L.Y. is an employee of IntegraGen, Inc.

Tumor mutation load (TML) has been proposed as a biomarker of patient response to immunotherapy in several studies. TML is usually determined by tumor biopsy DNA (tDNA) whole exome sequencing (WES), therefore TML evaluation is limited by informative biopsy availability. Circulating cell free DNA (cfDNA) provided by liquid biopsy is a surrogate specimen to biopsy for molecular profiling. Nevertheless performing WES on DNA from plasma is technically challenging and the ability to determine tumor mutation load from liquid biopsies remains to be demonstrated. In the current study, WES was performed on cfDNA from 32 metastatic patients of various cancer types included into MOSCATO 01 (NCT01566019) and/or MATCHR (NCT02517892) molecular triage trials. Results from targeted gene sequencing (TGS) and WES performed on cfDNA were compared to results from tumor tissue biopsy. In cfDNA samples, WES mutation detection sensitivity was 92% compared to targeted sequencing (TGS). When comparing cfDNA-WES to tDNA-WES, mutation detection sensitivity was 53%, consistent with previously published prospective study comparing cfDNA-TGS to tDNA-TGS. For samples in which presence of tumor DNA was confirmed in cfDNA, tumor mutation load from liquid biopsy was correlated with tumor biopsy. Taken together, this study demonstrated that liquid biopsy may be applied to determine tumor mutation load. Qualification of liquid biopsy for interpretation is a crucial point to use cfDNA for mutational load estimation.

Usher syndrome (USH), the most prevalent cause of hereditary deafness-blindness, is an autosomal recessive and genetically heterogeneous disorder. Three clinical subtypes (USH1-3) are distinguishable based on the severity of the sensorineural hearing impairment, the presence or absence of vestibular dysfunction, and the age of onset of the retinitis pigmentosa. A total of 10 causal genes, 6 for USH1, 3 for USH2, and 1 for USH3, and an USH2 modifier gene, have been identified. A robust molecular diagnosis is required not only to improve genetic counseling, but also to advance gene therapy in USH patients. Here, we present an improved diagnostic strategy that is both cost- and time-effective. It relies on the sequential use of three different techniques to analyze selected genomic regions: targeted exome sequencing, comparative genome hybridization, and quantitative exon amplification. We screened a large cohort of 427 patients (139 USH1, 282 USH2, and six of undefined clinical subtype) from various European medical centers for mutations in all USH genes and the modifier gene. We identified a total of 421 different sequence variants predicted to be pathogenic, about half of which had not been previously reported. Remarkably, we detected large genomic rearrangements, most of which were novel and unique, in 9% of the patients. Thus, our strategy led to the identification of biallelic and monoallelic mutations in 92.7% and 5.8% of the USH patients, respectively. With an overall 98.5% mutation characterization rate, the diagnosis efficiency was substantially improved compared with previously reported methods.

Background: Data generated by next-generation sequencing technologies have a pivotal role in precision medicine. These high-throughput techniques are preferentially performed on fresh tissue, but there is an increasing need for protocols adapted to materials derived from formalin-fixed, paraffin-embedded tissue and cytology specimens., Methods: The aim of this work was to show that cytological material collected from archival smears processed for routine diagnoses could be used for massively parallel sequencing and array-based genomic analysis for further studies., Results: As a proof of concept, data obtained from May-Grünwald Giemsa- and Diff-Quik-stained archival smears were shown to be in keeping with those obtained from matched frozen controls., Conclusions: The quality of DNA extracted from routinely processed smears is compatible with the multitargeted sequencing of a large series of genes of interest with methods such as array-based genomic analysis and whole-exome sequencing., (© 2015 American Cancer Society.)

Study Question: What part do mitochondria play in cases of diminished ovarian reserve (DOR)?, Summary Answer: Mitochondrial biogenesis in cumulus cells may be linked with impaired oocyte competence in patients with DOR., What Is Known Already: DOR, one of the causes of infertility even in young women, is characterized by the depletion of the ovarian pool associated with a decline in oocyte competence. Mitochondria, which play a role in oocyte quality, could be involved in the pathogenesis of DOR. The study of cumulus cells offers an interesting non-invasive approach for evaluating oocyte quality and the metabolic processes on which it depends. If mitochondrial dysfunction is involved in DOR, it is likely to have an impact on the functioning of cumulus cells., Study Design, Size, Duration: This is an observational study of 74 immature oocyte-cumulus complexes retrieved from 47 women undergoing in vitro fertilization with intracytoplasmic sperm injection at the University Hospital of Angers, France, from March 2013 to March 2014. The women were divided into two groups: one group included 26 women with DOR, and the other, which included 21 women with a normal ovarian reserve (NOR), served as a control group., Participants/materials, Settings, Methods: The oocyte mitochondrial content and the average mitochondrial content of the cumulus cells were assessed by mitochondrial (mt)DNA quantification using a quantitative real-time PCR technique. Microfluidic-based quantitative RT-PCR assays were used to quantify the expression of 13 genes involved in mitochondrial functions such as apoptosis and antioxidant activity or in mitochondrial biogenesis. We used orthogonal partial least-squares discriminant analysis (OPLS-DA) to distinguish between the DOR group and the NOR group of patients, and an OPLS model to predict the value of the oocyte mtDNA content that could be used as a critical marker of oocyte quality., Main Results and the Role of Chance: The OPLS-DA model showed a good predictive capability (Q2 = 0.543). Using the variable importance in projection (VIP) metric we found three mitochondrial variables distinguishing the DOR group from the NOR group of patients, i.e. the oocyte mtDNA content (VIP = 0.92), the cumulus cell mtDNA content (VIP = 0.95) and the expression in cumulus cells of peroxisome proliferator-activated receptor γ coactivator 1 alpha (PPARGC-1A) (VIP = 1.10), all of which were lower in the DOR group than in the NOR group of patients. The OPLS model was able to satisfactorily predict the oocyte mtDNA content in only the NOR group of patients (Q2 = 0.506). We found four new variables positively linked to the oocyte mitochondrial mass, i.e. the cumulus cell mtDNA content (VIP = 1.19), and the expression in cumulus cells of three factors of mitochondrial biogenesis: polymerase gamma (POLG) (VIP = 2.13), optic atrophy 1 (OPA1) (VIP = 1.89) and the transcription factor associated with mitochondria (TFAM) (VIP = 1.32)., Limitations, Reasons of Caution: This is a descriptive study. Because of ethical concerns in human clinical practice, this study has been performed only on immature oocytes and corresponding cumulus cells, which are usually discarded during in vitro fertilization procedures., Wider Implications of the Findings: Cumulus cells may govern mitochondrial biogenesis, creating an adequate oocyte mitochondrial pool to promote embryonic development. The alteration of this process in patients with DOR may account for the impairment of oocyte quality. This suggests that some mitochondrial characteristics of cumulus cells may serve as indicators of oocyte competence and that oocyte quality may be improved by products enhancing mitochondrial biogenesis., Study Funding/competing Interests: This work was supported by a grant from the University Hospital of Angers, France: 'Appel d'offre interne à la recherche 2014'., Trial Registration Number: N/A., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Idiopathic scoliosis (IS) is a spine deformity that affects approximately 3% of the population. The underlying causes of IS are not well understood, although there is clear evidence that there is a genetic component to the disease. Genetic mapping studies suggest high genetic heterogeneity, but no IS disease-causing gene has yet been identified. Here, genetic linkage analyses combined with exome sequencing identified a rare missense variant (p.A446T) in the centriolar protein gene POC5 that cosegregated with the disease in a large family with multiple members affected with IS. Subsequently, the p.A446T variant was found in an additional set of families with IS and in an additional 3 cases of IS. Moreover, POC5 variant p.A455P was present and linked to IS in one family and another rare POC5 variant (p.A429V) was identified in an additional 5 cases of IS. In a zebrafish model, expression of any of the 3 human IS-associated POC5 variant mRNAs resulted in spine deformity, without affecting other skeletal structures. Together, these findings indicate that mutations in the POC5 gene contribute to the occurrence of IS.

Paragangliomas are neuroendocrine tumors frequently associated with mutations in RET, NF1, VHL, and succinate dehydrogenase (SDHx) genes. Methylome analysis of a large paraganglioma cohort identified three stable clusters, associated with distinct clinical features and mutational status. SDHx-related tumors displayed a hypermethylator phenotype, associated with downregulation of key genes involved in neuroendocrine differentiation. Succinate accumulation in SDH-deficient mouse chromaffin cells led to DNA hypermethylation by inhibition of 2-OG-dependent histone and DNA demethylases and established a migratory phenotype reversed by decitabine treatment. Epigenetic silencing was particularly severe in SDHB-mutated tumors, potentially explaining their malignancy. Finally, inactivating FH mutations were identified in the only hypermethylated tumor without SDHx mutations. These findings emphasize the interplay between the Krebs cycle, epigenomic changes, and cancer., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Azadinium spinosum (Elbrächter and Tillmann), a small marine dinoflagellate, has been recently described as a de novo producer of azaspiracid-1 and -2 (AZA1 and -2) diarrhoeic toxins. A culture of A. spinosum was established in our laboratory and optimised for pilot-scale production of this organism, to evaluate and understand AZA1 and -2 accumulation and biotransformation in blue mussels (Mytilus edulis) fed with A. spinosum. Adult mussels were continuously exposed to A. spinosum over 1 week in 160 L cylindrical conical tanks. Three different diets were tested for contamination: 5000, 10 000 cells mL(-1) of A. spinosum and a mixture of 5000 cells mL(-1) of A. spinosum with 5000 cells mL(-1) of Isochrysis aff. galbana (T-Iso, CCAP 927/14). During the subsequent period of detoxification (2 weeks), contaminated mussels were continuously fed with 5000 cells mL(-1) of T-Iso. Kinetics of accumulation, detoxification and biotransformation were evaluated, as well as the toxin distribution and the effect of A. spinosum on mussel digestive gland tubules. M. edulis fed on A. spinosum in the three tested conditions; this finding confirmed our recent experiments feeding A. spinosum to mussels. The original algal toxins AZA1 and -2, as well as mussel metabolites AZA3 to 12, -17, -19, -21 and -23 were found during these trials. After as little as 6 h, azaspiracid contents in mussels reached the EU regulatory limit, and metabolites were observed in all conditions at approximately 25% of the total AZA content. This fraction exceeded 50% after 24 h, and continued to increase until the end of the study. AZA17 and -19 were found to be the main metabolites, with AZA17 concentrations estimated in the same order of magnitude as that of the main algal toxin, AZA1., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objectives: Diminished ovarian reserve (DOR) is one of the causes of infertility. In this prospective study, gene expression profiling (GEP) of corona radiata cells (CRC) was performed to identify genes deregulated in DOR patients., Patients and Methods: Microarray-based GEP of CRC isolated from eight women undergoing IVF was performed to identify genes differentially expressed between patients with normal ovarian reserve and DOR patients. Microfluidic-based quantitative RT-PCR assay were used to validate selected transcripts on 40 independent patients., Results: Forty-eight transcripts were differentially expressed, including CXXC5 and FOXC1 down regulated in DOR, as well as CTGF, FSTL3, PTGS2 and SOCS2 up regulated in DOR. According to these transcripts, two DOR patients'subgroups (DOR Gr1 and Gr2) were identified. In DOR Gr2 patients, CITED2, CTGF, GAS-1, IRS2, PTGS2, SOCS2, VCAN were expressed at significantly higher levels, and CXXC5, FOXC1, GBP2 and ZMIZ1 at significantly lower level. Eleven of those genes are transcriptional targets of Estrogens and higher baseline oestradiol levels were observed in DOR Gr2 patients (P<0.006)., Discussion and Conclusion: Twelve genes deregulated in CRC of DOR patients were identified, which could be involved in DOR pathogenesis. The distinction of a particular subgroup of DOR patients suggests the possibility of deregulation of estrogen response genes., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Background: Diminished ovarian reserve (DOR) is one of the causes of infertility in young women. In this prospective study, gene expression profiling (GEP) of corona radiata cells (CRC) was performed to identify genes deregulated in DOR patients., Methods: Microarray-based GEP of CRC isolated from eight women undergoing IVF was performed to identify genes differentially expressed between patients with normal ovarian reserve and DOR patients. Microfluidic-based quantitative RT-PCR assays were used to validate selected transcripts on 40 independent patients. A principal component analysis was used to identify more homogeneous subgroups of DOR patients. In silico analyses focusing on cis-regulation were performed to refine the interactions between patient's biological characteristics and their GEP., Results: Forty-eight transcripts were differentially expressed, including CXXC finger protein 5 (CXXC5), forkhead box C1 (FOXC1) (down-regulated in DOR) as well as connective tissue growth factor (CTGF), follistatin-like 3 (FSTL3), prostaglandin-endoperoxide synthase 2 (PTGS2) and suppressor of cytokine signaling 2 (SOCS2) (up-regulated in DOR). According to these transcripts, two DOR patients' subgroups (DOR Gr1 and Gr2) were identified. In DOR Gr2 patients, C-terminal domain 2 (CITED2), CTGF, growth arrest-specific 1 (GAS1), insulin receptor substrate 2 (IRS2), PTGS2, SOCS2 and Versican (VCAN) were expressed at significantly higher levels and CXXC5, FOXC1, guanylate-binding protein 2 (GBP2) and zinc finger MIZ-domain containing 1 (ZMIZ1) at significantly lower levels. Higher baseline estradiol (E(2)) levels were observed in DOR Gr2 patients (P < 0.006). The in silico analyses suggested that all 11 genes differentially expressed between DOR Gr1 and DOR Gr2 subgroups could be transcriptional targets of estrogen., Conclusions: Despite small sample size limitations, 12 genes deregulated in the CRC of DOR patients were identified, which could be involved in DOR pathogenesis. A DOR patient's subgroup with high baseline E(2) levels and deregulated estrogen-responsive genes was also identified.

The spliceosome, a ribonucleoprotein complex that includes proteins and small nuclear RNAs (snRNAs), catalyzes RNA splicing through intron excision and exon ligation to produce mature messenger RNAs, which, in turn serve as templates for protein translation. We identified four point mutations in the U4atac snRNA component of the minor spliceosome in patients with brain and bone malformations and unexplained postnatal death [microcephalic osteodysplastic primordial dwarfism type 1 (MOPD 1) or Taybi-Linder syndrome (TALS); Mendelian Inheritance in Man ID no. 210710]. Expression of a subgroup of genes, possibly linked to the disease phenotype, and minor intron splicing were affected in cell lines derived from TALS patients. Our findings demonstrate a crucial role of the minor spliceosome component U4atac snRNA in early human development and postnatal survival.

Background: The monogenic disease osteogenesis imperfecta (OI) is due to single mutations in either of the collagen genes ColA1 or ColA2, but within the same family a given mutation is accompanied by a wide range of disease severity. Although this phenotypic variability implies the existence of modifier gene variants, genome wide scanning of DNA from OI patients has not been reported. Promising genome wide marker-independent physical methods for identifying disease-related loci have lacked robustness for widespread applicability. Therefore we sought to improve these methods and demonstrate their performance to identify known and novel loci relevant to OI., Results: We have improved methods for enriching regions of identity-by-descent (IBD) shared between related, afflicted individuals. The extent of enrichment exceeds 10- to 50-fold for some loci. The efficiency of the new process is shown by confirmation of the identification of the Col1A2 locus in osteogenesis imperfecta patients from Amish families. Moreover the analysis revealed additional candidate linkage loci that may harbour modifier genes for OI; a locus on chromosome 1q includes COX-2, a gene implicated in osteogenesis., Conclusion: Technology for physical enrichment of IBD loci is now robust and applicable for finding genes for monogenic diseases and genes for complex diseases. The data support the further investigation of genetic loci other than collagen gene loci to identify genes affecting the clinical expression of osteogenesis imperfecta. The discrimination of IBD mapping will be enhanced when the IBD enrichment procedure is coupled with deep resequencing.

Autism is a developmental disorder characterized by impairments in social interaction and communication associated with repetitive patterns of interest or behavior. Autism is highly influenced by genetic factors. Genome-wide linkage and candidate gene association approaches have been used to try and identify autism genes. A few loci have repeatedly been reported linked to autism. Several groups reported evidence for linkage to a region on chromosome 16p. We have applied a direct physical identity-by-descent (IBD) mapping approach to perform a high-density (0.85 megabases) genome-wide linkage scan in 116 families from the AGRE collection. Our results confirm linkage to a region on chromosome 16p with autism. High-resolution single-nucleotide polymorphism (SNP) genotyping and analysis of this region show that haplotypes in the protein kinase c-beta gene are strongly associated with autism. An independent replication of the association in a second set of 167 trio families with autism confirmed our initial findings. Overall, our data provide evidence that the PRKCB1 gene on chromosome 16p may be involved in the etiology of autism.

In this study, we analyze for the first time endoplasmic reticulum (ER) dynamics and organization during oogenesis and embryonic divisions of Drosophila melanogaster using a Protein Disulfide Isomerase (PDI) GFP chimera protein. An accumulation of ER material into the oocyte takes place during the early steps of oogenesis. The compact organization of ER structures undergoes a transition to an expanded reticular network at fertilization. At the syncytial stage, this network connects to the nuclear envelope as each nucleus divides. Time-lapse confocal microscopy on PDI transgenic embryos allowed us to characterize a rapid redistribution of the ER during the mitotic phases. The ER network is massively recruited to the spindle poles in prophase. During metaphase most of the ER remains concentrated at the spindle poles and shortly thereafter forms several layers of membranes along the ruptured nuclear envelope. Later, during telophase an accumulation of ER material occurs at the spindle equator. We also analyzed the subcellular organization of the ER network at the ultrastructural level, allowing us to corroborate the results from confocal microscopy studies. This dynamic redistribution of ER suggests an unexpected regulatory function for this organelle during mitosis., (Copyright 2003 Wiley-Liss, Inc.)

The presence of glutamylated tubulin, a widespread posttranslational modification of alpha- and beta-tubulin, has been investigated in Drosophila melanogaster using the specific monoclonal antibody GT335. We show here that this modification is strongly detected in brain and testis whereas other tissues analyzed did not appear to contain any glutamylated isoforms. Neuronal microtubules are glutamylated on alpha-tubulin only whereas sperm flagella showed a strong modification of both alpha- and beta-tubulin. These results argue for an essential role for glutamylation in differentiation processes that require microtubule stabilization.

We have investigated the centrosome cycle in Drosophila syncytial embryos at the ultrastructural level by using a transmission electron microscope equipped with an electron energy filtering device (Omega filter). This new technique allows the study of uncontrasted thick sections with a high resolution. We have been able to characterize two classes of filamentous structures in the centrosomal apparatus that were not detectable on ultrathin sections. These new filamentous structures are: 1) a very orderly lattice that connects the two centrioles during mitosis; and 2) a fibrogranular connection between the centrosome and the nuclear envelope. The intercentriolar linkage could be involved in the precise timing of separation of the centrioles during late anaphase. The centrosome-nuclear envelope connection probably prevents the loss of centrosomes in this syncytial environment, and ensures the proper migration of the centrosomes along the surface of the nucleus. This connection may also couple the nuclei to the cytoskeleton, thus allowing their migration and their anchorage to the cortex at the blastoderm stage. This thick section analysis has also allowed us to precisely reconstitute the centrosome cycle. From their separation at telophase and throughout most of interphase, centrosomes are composed of a single centriole. We conclude that in the early Drosophila embryo there is an unusual delay between the separation of the parent centrioles and their duplication. This leaves a surprisingly short time to assemble a daughter centriole.

The gypsy element of Drosophila melanogaster is the first retrovirus identified in invertebrates. Its transposition is controlled by a host gene called flamenco (flam): restrictive alleles of this gene maintain the retrovirus in a repressed state while permissive alleles allow high levels of transposition. To develop a cell system to study the gypsy element, we established four independent cell lines derived from the Drosophila strain SS, which contains a permissive allele of flamenco, and which is devoid of transposing copies of gypsy. The ultrastructural analysis of three SS cell lines revealed some remarkable characteristics, such as many nuclear virus-like particles, cytoplasmic dense particles, and massive cisternae filled with a fibrous material of unknown origin. Gypsy intragenomic distribution has been compared between the three cell lines and the original SS fly strain, and revealed in two of the cell lines an increase in copy number of a restriction fragment usually present in active gypsy elements. This multiplication seems to have occurred during the passage to the cell culture. Availability of SS cell lines should assist studies of gypsy transposition and infectivity and might be useful to produce high amounts of gypsy viral particles. These new lines already allowed us to show that the Envelope-like products of gypsy can be expressed as membrane proteins.

The general architecture of the mitotic apparatus was studied at the ultrastructural level in Drosophila cultured cells. Its two main characteristics are a very polarized spindle and a strong compartmentalization, ensured by large remnants of the nuclear envelope. Such compartmentalization has previously been reported for the rapid syncytial divisions of the early embryo; a similar finding in these cells with a long cycle strongly suggests that this organization constitutes a general mechanism for mitosis in Drosophila. We followed the modifications of these structures after a heat shock of 20, 50 or 120 min at 37 degrees C. Contrary to interphase cells, mitotic cells appear very sensitive to hyperthermia. This stress treatment induced a disruption of the mitotic spindle, a reappearance and an extension of the Golgi apparatus, an inactivation of microtubule nucleation and a disorganization of the centrosome. This organelle seems the first to be affected by the heat shock response. The centrosome is not only inactivated, but also is structurally affected. During the recovery phase after heat stress, the mitotic cells presented a remarkable ring-shaped accumulation of electron-dense material around the centrioles. We conclude that in Drosophila cells the mitotic phase, and more specifically the centrosome, are targets of the stress response.

Previous observations have shown that the heat shock response affects the centrosome function. We compared the ultrastructural organization of the centrosome in control (23 degrees C) and heat-shocked (37 degrees C, 50 min) interphase Drosophila cells to detect the nature of the lesions that could alter this organelle. The centrosome apparatus showed only minor modifications after the stress and the architecture of the centrioles appeared unaffected. The main difference concerned the organization of pericentriolar material which appeared more condensed and clotted. In extreme cases this material seemed to collapse on the centrioles. Recent reports proposed that Ca2+ concentrations could modify the distribution of pericentriolar material. In this study, we measured the changes in total and bound calcium in control or heat-shocked cell samples. The hyperthermia stress induced an increase of about 80% in global calcium. However, there was a decrease of about 50% in bound calcium. A heat shock stress seemed therefore to promote a change from the bound to the free state for a noticeable proportion of the element. As a preliminary hypothesis, these changes in the chemical state of calcium could be related to alterations in the pericentriolar material and thus with the functional inactivation of the centrosome. This view is also supported by calcium analysis on early Drosophila embryos. Contrary to cultured cells, Drosophila embryos did not present a stress inactivation of centrosomes. Equally, a heat shock did not disturb the bound calcium level in embryos.

Variations of the testicular permeability are demonstrated in Locusta migratoria by using tracers such as iron-containing particles of protein. The testicular region containing spermatids is never penetrated by the tracers in contrast to the apical region containing gonia and young spermatocytes. Permeability of this compartment shows variations during the intermoult.

Heterocellular gap junctions were demonstrated in germ cysts of the moth Anagasta Küehniella (Lepidoptera). They conjoin peripheral germ cells of a cyst and cells of their envelope. Their morphology differs according to the developmental stage of the germ cell involved. While gap junctional profiles are flat in cysts of gonia, in cysts of early spermatocytes they appear as button-like structures, the germ cell indenting the corresponding cyst cell. In cysts of late spermatocytes and of young spermatids, they are very numerous and often located at the extremity of conical protrusions of the germ cell. On the germ cell side, cytoplasmic microfilaments are associated with the junctional differentiation. Gap junctions are observed as being pinched off from the surface of the spermatids and, correspondingly, gap vesicles are found in the cyst cells. This, together with the fact that gap junctions are not found at later stages of development, suggests that internalization of the gap junctions might take place before elongations of the spermatids. The potential importance of these germ-somatic cell gap junctions is evaluated in light of recent physiological findings obtained by other authors on the oocyte-cumulus system and also in relation with some particularities in the development of the male germ cells in Lepidoptera.

By using horseradish peroxydase (HRP) as a tracer, it is shown that the gonial region of the locust testis is an "open" compartment which is almost always freely penetrated by the tracer. During the last larval instar, however, the penetration of HRP decreases and ceases at the time when high levels of ecdysteroids are detected in the haemolymph by radioimmunoassay. A cause and effect relationship between tracer uptake and hormonal level could not be demonstrated by the experiments carried out up to now. From ultrastructural observations of the testis, it is concluded that the temporary isolation of the gonial compartment is not based upon any morphological structure which could act as a barrier. Penetration of the macromolecule is considered as the expression of an active uptake by the testis and the short period of nonpenetrability as a state of inertia whose significance remains to be discovered.