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Benzyl butyl phthalate (BBP), a plasticizer found in a wide range of consumer products including vinyl flooring, carpet backing, food packaging, personal care products, and children's toys, is an endocrine-disrupting chemical linked to impaired reproduction and development in humans. Despite evidence that BBP exposure perturbs the integrity of male and female gametes, its direct effect on early meiotic events is understudied. Here, using the nematode Caenorhabditis elegans, we show that BBP exposure elicits a non-monotonic dose response on the rate of X-chromosome nondisjunction measured using a high-throughput screening platform. From among the range of doses tested (1, 10, 100 and 500 μM BBP), we found that 10 μM BBP elicited the strongest effect on the germline, resulting in increased germ cell apoptosis and chromosome organization defects. Mass spectrometry analysis shows that C. elegans efficiently metabolizes BBP into its primary metabolites, monobutyl phthalate (MBP) and monobenzyl phthalate (MBzP), and that the levels of BBP, MBP, and MBzP detected in the worm are within the range detected in human biological samples. Exposure to 10 μM BBP leads to germlines with enlarged mitotic nuclei, altered meiotic progression, activation of a p53/CEP-1-dependent DNA damage checkpoint, increased double-strand break levels throughout the germline, chromosome morphology defects in oocytes at diakinesis, and increased oxidative stress. RNA sequencing analysis indicates that BBP exposure results in the altered expression of genes involved in xenobiotic metabolic processes, extracellular matrix organization, oocyte morphogenesis, meiotic cell cycle, and oxidoreduction. Taken together, we propose that C. elegans exposure to BBP leads to increased oxidative stress and double-strand break formation, thereby compromising germline genomic integrity and chromosome segregation. Author summary: Endocrine disrupting chemicals (EDCs) have been linked to various health problems including effects on reproductive health. Benzyl butyl phthalate (BBP) is a commonly used plasticizer found in many consumer products that has been shown to act as an EDC and affect human reproduction and development. However, how it might impact the germline and affect the specialized cell division program of meiosis, which results in the formation of haploid gametes (i.e. eggs and sperm), remained unclear. Here, using the nematode C. elegans, we show that BBP exposure at levels within the range of what is detected in humans impairs accurate chromosome segregation, which can result in aneuploidy. Similar to mammals, BBP exposure in C. elegans results in a non-monotonic dose response, whereby lower doses result in the strongest effects, and BBP is efficiently metabolized, underscoring the use of this model system for gaining mechanistic insights into how this EDC impacts the germline. BBP results in increased levels of DNA double-strand breaks (DSBs), activation of a DNA damage checkpoint, and altered chromosome morphology in exposed germlines. We propose that BBP exposure alters gene expression and results in increased oxidative stress, resulting in increased DSBs, thereby compromising chromosome morphology and accurate segregation. [ABSTRACT FROM AUTHOR]

Mitosis is an important process in the cell cycle required for cells to divide. Never in mitosis (NIMA)-like kinases (NEKs) are regulators of mitotic functions in diverse organisms. Plasmodium spp., the causative agent of malaria is a divergent unicellular haploid eukaryote with some unusual features in terms of its mitotic and nuclear division cycle that presumably facilitate proliferation in varied environments. For example, during the sexual stage of male gametogenesis that occurs within the mosquito host, an atypical rapid closed endomitosis is observed. Three rounds of genome replication from 1N to 8N and successive cycles of multiple spindle formation and chromosome segregation occur within 8 min followed by karyokinesis to generate haploid gametes. Our previous Plasmodium berghei kinome screen identified 4 Nek genes, of which 2, NEK2 and NEK4, are required for meiosis. NEK1 is likely to be essential for mitosis in asexual blood stage schizogony in the vertebrate host, but its function during male gametogenesis is unknown. Here, we study NEK1 location and function, using live cell imaging, ultrastructure expansion microscopy (U-ExM), and electron microscopy, together with conditional gene knockdown and proteomic approaches. We report spatiotemporal NEK1 location in real-time, coordinated with microtubule organising centre (MTOC) dynamics during the unusual mitoses at various stages of the Plasmodium spp. life cycle. Knockdown studies reveal NEK1 to be an essential component of the MTOC in male cell differentiation, associated with rapid mitosis, spindle formation, and kinetochore attachment. These data suggest that P. berghei NEK1 kinase is an important component of MTOC organisation and essential regulator of chromosome segregation during male gamete formation. [ABSTRACT FROM AUTHOR]

Molecular dissection of meiotic recombination in mammals, combined with population-genetic and comparative studies, have revealed a complex evolutionary dynamic characterized by short-lived recombination hotspots. Hotspots are chromosome positions containing DNA sequences where the protein PRDM9 can bind and cause crossing-over. To explain these fast evolutionary dynamic, a so-called intra-genomic Red Queen model has been proposed, based on the interplay between two antagonistic forces: biased gene conversion, mediated by double-strand breaks, resulting in hotspot extinction (the hotspot conversion paradox), followed by positive selection favoring mutant PRDM9 alleles recognizing new sequence motifs. Although this model predicts many empirical observations, the exact causes of the positive selection acting on new PRDM9 alleles is still not well understood. In this direction, experiment on mouse hybrids have suggested that, in addition to targeting double strand breaks, PRDM9 has another role during meiosis. Specifically, PRDM9 symmetric binding (simultaneous binding at the same site on both homologues) would facilitate homology search and, as a result, the pairing of the homologues. Although discovered in hybrids, this second function of PRDM9 could also be involved in the evolutionary dynamic observed within populations. To address this point, here, we present a theoretical model of the evolutionary dynamic of meiotic recombination integrating current knowledge about the molecular function of PRDM9. Our modeling work gives important insights into the selective forces driving the turnover of recombination hotspots. Specifically, the reduced symmetrical binding of PRDM9 caused by the loss of high affinity binding sites induces a net positive selection eliciting new PRDM9 alleles recognizing new targets. The model also offers new insights about the influence of the gene dosage of PRDM9, which can paradoxically result in negative selection on new PRDM9 alleles entering the population, driving their eviction and thus reducing standing variation at this locus. Author summary: Meiosis is an important step in the eukaryotic life cycle, leading to the formation of gametes and implementing genetic mixing by recombination of paternal and maternal genomes. A key step of meiosis is the pairing of homologous chromosomes, which is required in order to distribute them evenly into the gametes. Chromosome pairing will also determine the exact position at which paternal and maternal chromosomes will exchange material. Research on the molecular basis of meiosis has revealed the role of a key gene, PRDM9. The protein encoded by PRDM9 binds to specific DNA sequences, by which it determines the location of recombination points. Symmetric binding of the protein (at the same position on the homologous chromosomes) also facilitates chromosome pairing. This molecular mechanism, however, has paradoxical consequences, among which the local destruction of the DNA sequences recognized by PRDM9, leading to their rapid loss at the level of the population over a short evolutionary time. In order to better understand why recombination is maintained over time despite this process, we have developed a simulation program implementing a model taking into account these molecular mechanisms. Our model makes realistic predictions about recombination evolution and confirms the important role played by PRDM9 during meiosis. [ABSTRACT FROM AUTHOR]

During meiosis, pairing of homologous chromosomes (homologs) ensures the formation of haploid gametes from diploid precursor cells, a prerequisite for sexual reproduction. Pairing during meiotic prophase I facilitates crossover recombination and homolog segregation during the ensuing reductional cell division. Mechanisms that ensure stable homolog alignment in the presence of an excess of non-homologous chromosomes have remained elusive, but rapid chromosome movements during prophase I appear to play a role in the process. Apart from homolog attraction, provided by early intermediates of homologous recombination, dissociation of non-homologous associations also appears to contribute to homolog pairing, as suggested by the detection of stable non-homologous chromosome associations in pairing-defective mutants. Here, we have developed an agent-based model for homolog pairing derived from the dynamics of a naturally occurring chromosome ensemble. The model simulates unidirectional chromosome movements, as well as collision dynamics determined by attractive and repulsive forces arising from close-range physical interactions. Chromosome number and size as well as movement velocity and repulsive forces are identified as key factors in the kinetics and efficiency of homologous pairing in addition to homolog attraction. Dissociation of interactions between non-homologous chromosomes may contribute to pairing by crowding homologs into a limited nuclear area thus creating preconditions for close-range homolog attraction. Incorporating natural chromosome lengths, the model accurately recapitulates efficiency and kinetics of homolog pairing observed for wild-type and mutant meiosis in budding yeast, and can be adapted to nuclear dimensions and chromosome sets of other organisms. Author summary: Pairing of homologous chromosomes is a key feature of multiple cellular processes including gene expression control, chromosome break repair, and chromosome segregation. Homolog pairing during meiosis is shared among all sexually reproducing eukaryotes. Mechanistic determinants of homology-specific chromosome alignment are presently unknown. We have developed an agent-based model where contributions of the entire chromosome set to the pairing process is taken into account, comprising both homologous and non-homologous chromosomal encounters. Predictions from the model are readily compared to experimental data from budding yeast, parameters can be adjusted to other cellular systems and predictions can be tested via experimental manipulation of the relevant chromosomal features. [ABSTRACT FROM AUTHOR]

Egg activation, representing the critical oocyte-to-embryo transition, provokes meiosis completion, modification of the vitelline membrane to prevent polyspermy, and translation of maternally provided mRNAs. This transition is triggered by a calcium signal induced by spermatozoon fertilization in most animal species, but not in insects. In Drosophila melanogaster, mature oocytes remain arrested at metaphase-I of meiosis and the calcium-dependent activation occurs while the oocyte moves through the genital tract. Here, we discovered that the oenocytes of fruitfly females are required for egg activation. Oenocytes, cells specialized in lipid-metabolism, are located beneath the abdominal cuticle. In adult flies, they synthesize the fatty acids (FAs) that are the precursors of cuticular hydrocarbons (CHCs), including pheromones. The oenocyte-targeted knockdown of a set of FA-anabolic enzymes, involved in very-long-chain fatty acid (VLCFA) synthesis, leads to a defect in egg activation. Given that some but not all of the identified enzymes are required for CHC/pheromone biogenesis, this putative VLCFA-dependent remote control may rely on an as-yet unidentified CHC or may function in parallel to CHC biogenesis. Additionally, we discovered that the most posterior ventral oenocyte cluster is in close proximity to the uterus. Since oocytes dissected from females deficient in this FA-anabolic pathway can be activated in vitro, this regulatory loop likely operates upstream of the calcium trigger. To our knowledge, our findings provide the first evidence that a physiological extra-genital signal remotely controls egg activation. Moreover, our study highlights a potential metabolic link between pheromone-mediated partner recognition and egg activation. Author summary: Efficient sexual reproduction requires gamete formation and maturation, partner mating, fecundation and the triggering of embryonic development. Deciphering the underlying physiological regulatory processes represents a major scientific challenge, crucial both for medical issues and agricultural pest control. A pivotal step in the reproductive process, known as egg activation, is the oocyte-to-embryo transition. In a wide range of metazoan species, this activation is provoked by the entry of the spermatozoon into the oocyte. Conversely, in insects, egg activation is initiated as the oocyte moves through the female genital tract, operating independently of spermatozoon entry. Here, we discovered that egg activation in Drosophila depends on a set of anabolic enzymes required for very-long-chain fatty acid (VLCFA) synthesis in the oenocytes. The oenocytes, abdominal cells specialized in lipid metabolism, are necessary for the biogenesis of cuticular hydrocarbons and pheromones. To our knowledge, our findings provide the first evidence that a non-genital physiological signal remotely governs egg activation. Given that pheromone-deficient Drosophila females attract males regardless of their species, we propose that pheromone biogenesis and this VLCFA-dependent remote signal might have co-evolved to prevent the development of hybrid individuals. [ABSTRACT FROM AUTHOR]

The meiotic recombination checkpoint reinforces the order of events during meiotic prophase I, ensuring the accurate distribution of chromosomes to the gametes. The AAA+ ATPase Pch2 remodels the Hop1 axial protein enabling adequate levels of Hop1-T318 phosphorylation to support the ensuing checkpoint response. While these events are localized at chromosome axes, the checkpoint activating function of Pch2 relies on its cytoplasmic population. In contrast, forced nuclear accumulation of Pch2 leads to checkpoint inactivation. Here, we reveal the mechanism by which Pch2 travels from the cell nucleus to the cytoplasm to maintain Pch2 cellular homeostasis. Leptomycin B treatment provokes the nuclear accumulation of Pch2, indicating that its nucleocytoplasmic transport is mediated by the Crm1 exportin recognizing proteins containing Nuclear Export Signals (NESs). Consistently, leptomycin B leads to checkpoint inactivation and impaired Hop1 axial localization. Pch2 nucleocytoplasmic traffic is independent of its association with Zip1 and Orc1. We also identify a functional NES in the non-catalytic N-terminal domain of Pch2 that is required for its nucleocytoplasmic trafficking and proper checkpoint activity. In sum, we unveil another layer of control of Pch2 function during meiosis involving nuclear export via the exportin pathway that is crucial to maintain the critical balance of Pch2 distribution among different cellular compartments. Author summary: Meiosis is a specialized cell division essential for sexual reproduction because it is responsible for halving the number of chromosomes during gametogenesis. Meiosis involves a series of events that culminate in the interaction between maternal and paternal chromosomes, and the exchange of genetic material by recombination. These processes not only generate genetic diversity, but also, and more important, are essential to promote proper distribution of chromosomes to the gametes. Meiotic cells possess surveillance mechanisms, called checkpoints, that reinforce the proper order of events during the meiotic cell cycle, ensuring the accurate segregation of chromosomes and preventing the formation of aneuploid gametes. The Pch2 protein is an evolutionarily-conserved ATPase that functions in the yeast meiotic recombination checkpoint. Pch2 localizes in the nucleolus, chromosomes, and the cytoplasm, performing distinct and, in some cases, opposite functions in these different subcellular compartments. In this work, we uncover the mechanism that Pch2 uses to travel from the nucleus to the cytoplasm; in particular, the Crm1 exportin pathway. We also identify and characterize a nuclear export signal in the non-catalytic domain of Pch2 required for its nucleocytoplasmic trafficking. Thus, our work contributes to understanding how the critical balance of Pch2 subcellular distribution is achieved to support faithful meiotic completion. [ABSTRACT FROM AUTHOR]

From insects to mammals, oocytes and sperm develop within germline cysts comprising cells connected by intercellular bridges (ICBs). In numerous insects, formation of the cyst is accompanied by growth of the fusome—a membranous organelle that permeates the cyst. Fusome composition and function are best understood in Drosophila melanogaster: during oogenesis, the fusome dictates cyst topology and size and facilitates oocyte selection, while during spermatogenesis, the fusome synchronizes the cyst's response to DNA damage. Despite its distinct and sex-specific roles during insect gametogenesis, elucidating fusome growth and inheritance in females and its structure and connectivity in males has remained challenging. Here, we take advantage of advances in three-dimensional (3D) confocal microscopy and computational image processing tools to reconstruct the topology, growth, and distribution of the fusome in both sexes. In females, our experimental findings inform a theoretical model for fusome assembly and inheritance and suggest that oocyte selection proceeds through an 'equivalency with a bias' mechanism. In males, we find that cell divisions can deviate from the maximally branched pattern observed in females, leading to greater topological variability. Our work consolidates existing disjointed experimental observations and contributes a readily generalizable computational approach for quantitative studies of gametogenesis within and across species. Author summary: The ubiquity of germline cysts across animals and accelerating advances in microscopy call for quantitative and highly resolved studies of their developmental dynamics. Here we use Drosophila melanogaster gametogenesis as a model system, alongside a supervised learning algorithm to study a shared organelle that arises during sperm and oocyte development—the fusome. The fusome is a highly specialized membranous organelle that permeates the cyst in both sexes. Our three-dimensional (3D) reconstructions of the fusome and quantitative measurements at successive stages of cyst development during oogenesis shed light on the evolution of cell fate asymmetry within the germline cyst in females, where the cyst gives rise to a single oocyte. In males, where each cell of the cyst goes on to form sperm, the fusome fragments and exhibits topologies that deviate from the stereotypic maximally branched topology found in females. Our findings can be interpreted in the context of the divergent outcomes of gametogenesis in both sexes and highlight the centrality of quantitative measurements in evaluating hypotheses in biological sciences. [ABSTRACT FROM AUTHOR]

Chromosome movements and licensing of synapsis must be tightly regulated during early meiosis to ensure accurate chromosome segregation and avoid aneuploidy, although how these steps are coordinated is not fully understood. Here we show that GRAS-1, the worm homolog of mammalian GRASP/Tamalin and CYTIP, coordinates early meiotic events with cytoskeletal forces outside the nucleus. GRAS-1 localizes close to the nuclear envelope (NE) in early prophase I and interacts with NE and cytoskeleton proteins. Delayed homologous chromosome pairing, synaptonemal complex (SC) assembly, and DNA double-strand break repair progression are partially rescued by the expression of human CYTIP in gras-1 mutants, supporting functional conservation. However, Tamalin, Cytip double knockout mice do not exhibit obvious fertility or meiotic defects, suggesting evolutionary differences between mammals. gras-1 mutants show accelerated chromosome movement during early prophase I, implicating GRAS-1 in regulating chromosome dynamics. GRAS-1-mediated regulation of chromosome movement is DHC-1-dependent, placing it acting within the LINC-controlled pathway, and depends on GRAS-1 phosphorylation at a C-terminal S/T cluster. We propose that GRAS-1 coordinates the early steps of homology search and licensing of SC assembly by regulating the pace of chromosome movement in early prophase I. Author summary: Successful sexual reproduction depends on the formation of gametes (i.e., eggs and sperm) carrying a correct number of chromosomes. This involves a series of well-choreographed steps during meiosis, the specialized cell division program resulting in the formation of eggs and sperm. Some of these steps include pairing between homologous chromosomes, assembly of a scaffold (the synaptonemal complex) between homologs, and inter-homolog recombination. These steps must be properly coordinated to ensure normal meiotic progression. Here, we show that the GRAS-1 protein in the nematode C. elegans, which shares sequence conservation with the mammalian proteins GRASP/Tamalin and CYTIP, contributes to normal meiotic progression by coordinating early events in meiosis. GRAS-1 is required for the normal speed of chromosome movements early in prophase I in a manner dependent of its phosphorylation and on dynein heavy chain. Delayed homologous chromosome pairing, synaptonemal complex assembly, and DNA double-strand break repair progression are partially rescued by the expression of human CYTIP in gras-1 mutants, supporting functional conservation. We identified a new factor coordinating the early steps of pairing and synapsis by regulating the pace of chromosome movement in early prophase I. [ABSTRACT FROM AUTHOR]

Meiotic recombination is a driving force for genome evolution, deeply characterized in a few model species, notably in the budding yeast Saccharomyces cerevisiae. Interestingly, Zip2, Zip3, Zip4, Spo16, Msh4, and Msh5, members of the so-called ZMM pathway that implements the interfering meiotic crossover pathway in S. cerevisiae, have been lost in Lachancea yeast species after the divergence of Lachancea kluyveri from the rest of the clade. In this context, after investigating meiosis in L. kluyveri, we determined the meiotic recombination landscape of Lachancea waltii. Attempts to generate diploid strains with fully hybrid genomes invariably resulted in strains with frequent whole-chromosome aneuploidy and multiple extended regions of loss of heterozygosity (LOH), which mechanistic origin is so far unclear. Despite the lack of multiple ZMM pro-crossover factors in L. waltii, numbers of crossovers and noncrossovers per meiosis were higher than in L. kluyveri but lower than in S. cerevisiae, for comparable genome sizes. Similar to L. kluyveri but opposite to S. cerevisiae, L. waltii exhibits an elevated frequency of zero-crossover bivalents. Lengths of gene conversion tracts for both crossovers and non-crossovers in L. waltii were comparable to those observed in S. cerevisiae and shorter than in L. kluyveri despite the lack of Mlh2, a factor limiting conversion tract size in S. cerevisiae. L. waltii recombination hotspots were not shared with either S. cerevisiae or L. kluyveri, showing that meiotic recombination hotspots can evolve at a rather limited evolutionary scale within budding yeasts. Finally, L. waltii crossover interference was reduced relative to S. cerevisiae, with interference being detected only in the 25 kb distance range. Detection of positive inference only at short distance scales in the absence of multiple ZMM factors required for interference-sensitive crossovers in other systems likely reflects interference between early recombination precursors such as DSBs. Author summary: Studying non-model species is relevant to understand better biological processes by shedding light on their evolutionary variations. Here we chose the non-model budding yeast Lachancea waltii to study meiotic recombination. In sexually reproducing, meiotic recombination is essential for gamete formation, and it shuffles parental genetic combinations notably by crossovers that cluster in hotspots at the population level. We found remarkable variations compared to both the canonical Saccharomyces cerevisiae model, also known as the baker's yeast, and another close relative Lachancea kluyveri. L. waltii meiotic chromosomes are frequently devoid of crossover, suggesting the existence of an alternative mechanism that efficiently ensures gamete formation. In addition, in line with the L. waltii specific loss of several genes controlling interference between meiotic crossovers, a process promoting even crossovers spacing, we found only residual crossover interference in L. waltii. This residual crossover interference is likely the result of the modest interference existing between recombination precursors that is often disregarded. Finally, while crossover hotspots were found to be remarkably stable across the Saccharomyces species, we found here that they are not conserved between the Lachancea and the Saccharomyces clades. This shows that crossover hotspots can evolve at a rather limited evolutionary scale within budding yeasts. [ABSTRACT FROM AUTHOR]

Gametogenesis requires coordinated signaling between germ cells and somatic cells. We previously showed that Gap junction (GJ)-mediated soma-germline communication is essential for fly spermatogenesis. Specifically, the GJ protein Innexin4/Zero population growth (Zpg) is necessary for somatic and germline stem cell maintenance and differentiation. It remains unknown how GJ-mediated signals regulate spermatogenesis or whether the function of these signals is restricted to the earliest stages of spermatogenesis. Here we carried out comprehensive structure/function analysis of Zpg using insights obtained from the protein structure of innexins to design mutations aimed at selectively perturbing different regulatory regions as well as the channel pore of Zpg. We identify the roles of various regulatory sites in Zpg in the assembly and maintenance of GJs at the plasma membrane. Moreover, mutations designed to selectively disrupt, based on size and charge, the passage of cargos through the Zpg channel pore, blocked different stages of spermatogenesis. Mutations were identified that progressed through early germline and soma development, but exhibited defects in entry to meiosis or sperm individualisation, resulting in reduced fertility or sterility. Our work shows that specific signals that pass through GJs regulate the transition between different stages of gametogenesis. Author summary: Gap-junctions allow neighboring cells to communicate by connecting their cytoplasm. Gap-junctions play an essential role during sperm development by facilitating communication between the two cell types found in the testes, the germline which produces sperm, and the soma, which provides an essential supportive environment to the germline. We sought to better understand the ways in which gap-junctions help germline and somatic cells to communicate. We introduced nearly twenty different mutations into a gap-junction gene that connects the soma and germline in the fly testes. These mutations were chosen based on bioinformatics and analysis of the predicted structure of the gap-junction protein. We replaced the normal version of the gap-junction with the mutated versions in flies, and analysed how sperm development was affected. Based on this analysis we identified key parts of the protein that were required for the assembly and maintenance of the gap-junctions. Moreover, mutations designed to selectively disrupt the passage of specific materials through the gap-junction blocked different stages of sperm development. Mutations were identified that progressed through early sperm development, but exhibited defects in later stages, resulting in sterility. Our work shows that specific signals that pass-through gap-junctions regulate the transition between different stages of sperm development. [ABSTRACT FROM AUTHOR]

During meiosis, DNA double-strand breaks (DSBs) occur throughout the genome, a subset of which are repaired to form reciprocal crossovers between chromosomes. Crossovers are essential to ensure balanced chromosome segregation and to create new combinations of genetic variation. Meiotic DSBs are formed by a topoisomerase-VI-like complex, containing catalytic (e.g. SPO11) proteins and auxiliary (e.g. PRD3) proteins. Meiotic DSBs are formed in chromatin loops tethered to a linear chromosome axis, but the interrelationship between DSB-promoting factors and the axis is not fully understood. Here, we study the localisation of SPO11-1 and PRD3 during meiosis, and investigate their respective functions in relation to the chromosome axis. Using immunocytogenetics, we observed that the localisation of SPO11-1 overlaps relatively weakly with the chromosome axis and RAD51, a marker of meiotic DSBs, and that SPO11-1 recruitment to chromatin is genetically independent of the axis. In contrast, PRD3 localisation correlates more strongly with RAD51 and the chromosome axis. This indicates that PRD3 likely forms a functional link between SPO11-1 and the chromosome axis to promote meiotic DSB formation. We also uncovered a new function of SPO11-1 in the nucleation of the synaptonemal complex protein ZYP1. We demonstrate that chromosome co-alignment associated with ZYP1 deposition can occur in the absence of DSBs, and is dependent on SPO11-1, but not PRD3. Lastly, we show that the progression of meiosis is influenced by the presence of aberrant chromosomal connections, but not by the absence of DSBs or synapsis. Altogether, our study provides mechanistic insights into the control of meiotic DSB formation and reveals diverse functional interactions between SPO11-1, PRD3 and the chromosome axis. Author summary: Most eukaryotes rely on the formation of gametes with half the number of chromosomes for sexual reproduction. Meiosis is a specialised type of cell division essential for the transition between a diploid and a haploid stage during gametogenesis. In early meiosis, programmed-DNA double strand breaks (DSBs) occur across the genome. These DSBs are processed by a set of proteins and the broken ends are repaired using the genetic information from the homologous chromosomes. These reciprocal exchanges of information between two chromosomes are called crossovers. Crossovers physical link chromosomes in pair which is essential to ensure their correct segregation during the two rounds of meiotic division. Crossovers are also essential for the creation of genetic diversity as they break genetic linkages to form novel allelic blocks. The formation of DSBs is not completely understood in plants. Here we studied the function of SPO11-1 and PRD3, two proteins involved in the formation of DSBs in Arabidopsis. We discovered functional differences in their respective mode of recruitment to the chromosomes, their interactions with proteins forming the chromosome core and their roles in chromosome co-alignment. These indicate that, although the SPO11-1 and PRD3 share a role in the formation of DSBs, the two proteins have additional and distinct roles beside DSB formation. [ABSTRACT FROM AUTHOR]

An intriguing outcome of hybridisation is the emergence of clonally and hemiclonally reproducing hybrids, that can sustain, reproduce, and lead to the emergence of polyploid forms. However, the maintenance of diploid and polyploid hybrid complexes in natural populations remains unresolved. We selected water frogs from the Pelophylax esculentus complex to study how diploid and triploid hybrids, which reproduce hemiclonally via hybridogenesis, are maintained in natural populations. During gametogenesis in diploid hybrids, one of the parental genomes is eliminated, and the remaining genome is endoreplicated. In triploid hybrids, the single-copy genome is typically eliminated, while genome endoreplication does not occur. To investigate how diploid and triploid hybrid frogs reproduce in populations without parental species, we crossed these hybrid animals from two separate pure hybrid populations located in Poland. Using cytogenetic analysis of tadpoles that emerged from the crosses, we established which gametes were produced by parental hybrids. The majority of hybrid females and hybrid males produced one type of gamete with the P. ridibundus genome. However, in both studied populations, approximately half of the diploid and triploid hybrids simultaneously produced gametes with different genome compositions and ploidy levels, specifically, the P. ridibundus and P. lessonae genomes, as well as diploid gametes with genomes of both parental species. Triploid hybrid males and females mostly produced haploid gametes with the P. lessonae genome; however, gametes with the P. ridibundus genome have also been observed. These results suggest that not all hybrids follow the classical hybridogenetic reproduction program and reveal a significant level of alterations in the gametogenesis pathways. In addition, we found a variable survival rate of particular progeny genotypes when we crossed hybrid females with different males suggesting the important role of postzygotic barriers on the maintenance of pure hybrid systems. We suggest that the observed variability in produced gametes and the different survival rate of the progeny with certain genotypes is crucial for the existence of pure hybrid systems. [ABSTRACT FROM AUTHOR]

The number and location of crossovers across genomes are highly regulated during meiosis, yet the key components controlling them are fast evolving, hindering our understanding of the mechanistic causes and evolutionary consequences of changes in crossover rates. Drosophila melanogaster has been a model species to study meiosis for more than a century, with an available high-resolution crossover map that is, nonetheless, missing for closely related species, thus preventing evolutionary context. Here, we applied a novel and highly efficient approach to generate whole-genome high-resolution crossover maps in D. yakuba to tackle multiple questions that benefit from being addressed collectively within an appropriate phylogenetic framework, in our case the D. melanogaster species subgroup. The genotyping of more than 1,600 individual meiotic events allowed us to identify several key distinct properties relative to D. melanogaster. We show that D. yakuba, in addition to higher crossover rates than D. melanogaster, has a stronger centromere effect and crossover assurance than any Drosophila species analyzed to date. We also report the presence of an active crossover-associated meiotic drive mechanism for the X chromosome that results in the preferential inclusion in oocytes of chromatids with crossovers. Our evolutionary and genomic analyses suggest that the genome-wide landscape of crossover rates in D. yakuba has been fairly stable and captures a significant signal of the ancestral crossover landscape for the whole D. melanogaster subgroup, even informative for the D. melanogaster lineage. Contemporary crossover rates in D. melanogaster, on the other hand, do not recapitulate ancestral crossovers landscapes. As a result, the temporal stability of crossover landscapes observed in D. yakuba makes this species an ideal system for applying population genetic models of selection and linkage, given that these models assume temporal constancy in linkage effects. Our studies emphasize the importance of generating multiple high-resolution crossover rate maps within a coherent phylogenetic context to broaden our understanding of crossover control during meiosis and to improve studies on the evolutionary consequences of variable crossover rates across genomes and time. Author summary: Meiotic recombination is a fundamental cellular process required for the formation of gametes in most eukaryotic organisms. Recombination also plays a fundamental role in evolution, increasing rates of adaptation. Paradoxically for such an essential process, key components evolve fast, and model organisms differ vastly in number of recombination events and distribution across chromosomes. This variation has limited our understanding of how recombination is controlled or why it can vary according to environmental cues. D. melanogaster has been a model species to study meiosis and recombination for more than a century and, in this study, we applied a new and highly efficient whole-genome genotyping scheme to identify recombination properties for a closely related species, D. yakuba, thus providing an informative counterpoint and needed phylogenetic context. Our results describe important differences relative to D. melanogaster, including the first description of a mechanism favoring the segregation of recombinant chromatids to the functional egg pole under benign conditions. We argue that such a mechanism may also be active in other species. Moreover, our studies suggest that D. yakuba has maintained its recombination landscape for a longer time than D. melanogaster, indicating that the former species may be more adequate for evolutionary analyses. Combined, we show the importance of tackling the current siloed approach that focuses on model species with a more comprehensive analysis that requires the inclusion of closely related species when studying the causes and consequences of recombination variation. [ABSTRACT FROM AUTHOR]

Early diverging lineages such as trypanosomes can provide clues to the evolution of sexual reproduction in eukaryotes. In Trypanosoma brucei, the pathogen that causes Human African Trypanosomiasis, sexual reproduction occurs in the salivary glands of the insect host, but analysis of the molecular signatures that define these sexual forms is complicated because they mingle with more numerous, mitotically-dividing developmental stages. We used single-cell RNA-sequencing (scRNAseq) to profile 388 individual trypanosomes from midgut, proventriculus, and salivary glands of infected tsetse flies allowing us to identify tissue-specific cell types. Further investigation of salivary gland parasite transcriptomes revealed fine-scale changes in gene expression over a developmental progression from putative sexual forms through metacyclics expressing variant surface glycoprotein genes. The cluster of cells potentially containing sexual forms was characterized by high level transcription of the gamete fusion protein HAP2, together with an array of surface proteins and several genes of unknown function. We linked these expression patterns to distinct morphological forms using immunofluorescence assays and reporter gene expression to demonstrate that the kinetoplastid-conserved gene Tb927.10.12080 is exclusively expressed at high levels by meiotic intermediates and gametes. Further experiments are required to establish whether this protein, currently of unknown function, plays a role in gamete formation and/or fusion. Author summary: African Trypanosomes are single-celled protozoan parasites that cause disease in humans and livestock. They have a complex life cycle that spans a mammalian and tsetse fly host. Within the tsetse fly, the parasite first travels into the midgut when the fly takes up an infectious blood meal. As it develops it moves into the proventriculus followed by the salivary glands taking on distinct morphological forms in each of these tissues. In the salivary glands, the parasite can undergo non-obligatory sexual reproduction via meiosis and the production of gametes. However, the biological processes that underly this sexual developmental and the molecular signatures that define these morphological forms remain elusive because they are found within heterogeneous populations that also contain mitotically dividing forms. Here we have used single-cell RNAseq to profile the transcriptomes of parasites across development in the tsetse with a focus on identifying the patterns of expression that define these sexual stages. We showed that the sexual forms have a unique transcriptional profile and we connect these expression patterns to specific morphological stages of sexual development using a fluorescent reporter. This allowed us to identify a new gene that may be involved in reproduction. Elucidating the mechanisms underlying sexual reproduction and genetic exchange is fundamental to understanding the evolution of key traits such as virulence and drug resistance. [ABSTRACT FROM AUTHOR]

Formation of gametes in the malaria parasite occurs in the midgut of the mosquito and is critical to onward parasite transmission. Transformation of the male gametocyte into microgametes, called microgametogenesis, is an explosive cellular event and one of the fastest eukaryotic DNA replication events known. The transformation of one microgametocyte into eight flagellated microgametes requires reorganisation of the parasite cytoskeleton, replication of the 22.9 Mb genome, axoneme formation and host erythrocyte egress, all of which occur simultaneously in <20 minutes. Whilst high-resolution imaging has been a powerful tool for defining stages of microgametogenesis, it has largely been limited to fixed parasite samples, given the speed of the process and parasite photosensitivity. Here, we have developed a live-cell fluorescence imaging workflow that captures the entirety of microgametogenesis. Using the most virulent human malaria parasite, Plasmodium falciparum, our live-cell approach captured early microgametogenesis with three-dimensional imaging through time (4D imaging) and microgamete release with two-dimensional (2D) fluorescence microscopy. To minimise the phototoxic impact to parasites, acquisition was alternated between 4D fluorescence, brightfield and 2D fluorescence microscopy. Combining live-cell dyes specific for DNA, tubulin and the host erythrocyte membrane, 4D and 2D imaging together enables definition of the positioning of newly replicated and segregated DNA. This combined approach also shows the microtubular cytoskeleton, location of newly formed basal bodies, elongation of axonemes and morphological changes to the erythrocyte membrane, the latter including potential echinocytosis of the erythrocyte membrane prior to microgamete egress. Extending the utility of this approach, the phenotypic effects of known transmission-blocking inhibitors on microgametogenesis were confirmed. Additionally, the effects of bortezomib, an untested proteasomal inhibitor, revealed a clear block of DNA replication, full axoneme nucleation and elongation. Thus, as well as defining a framework for broadly investigating microgametogenesis, these data demonstrate the utility of using live imaging to validate potential targets for transmission-blocking antimalarial drug development. Author summary: Upon transmission of Plasmodium, the causative agents of malaria, male and female gametes form in the mosquito midgut in a remarkably rapid and complex cellular transformation. Microgametogenesis, or male gamete formation, describes the formation of eight haploid microgametes from a single gametocyte in only 15 minutes. Simultaneous egress from the host erythrocyte, substantial cytoskeletal rearrangement and DNA replication occurs during this characteristically explosive transformation. Whilst microgametogenesis has been extensively studied using various microscopy techniques, efforts to date have been limited by the need for complex transgenic parasite generation and fixed-parasite protocols. Here, we have developed a live-cell fluorescence imaging approach to study microgametogenesis, overcoming both the laborious staining steps and spatiotemporal limitations of existing approaches. Crucially, our newly devised workflow utilises widefield fluorescence microscopy, commercially available stains and an open-access analysis software and is hence accessible to the wider malaria research community. We demonstrate the ability to retrieve and quantify volumetric data from our live-cell approach and highlight the applicability of our live-cell approach to transmission-blocking drug discovery. Using our workflow, we have elucidated the cellular phenotypes of both existing and unknown inhibitors of microgametogenesis. Using this approach, we find the proteasome to be a potential microgametogenesis-blocking cellular target. [ABSTRACT FROM AUTHOR]

Meiosis is a complex process involving the expression and interaction of numerous genes in a series of highly orchestrated molecular events. Fam9b localized in Xp22.3 has been found to be expressed in testes. However, FAM9B expression, localization, and its role in meiosis have not been previously reported. In this study, FAM9B expression was evaluated in the human testes and ovaries by RT-PCR, qPCR, and western blotting. FAM9B was found in the nuclei of primary spermatocytes in testes and specifically localized in the synaptonemal complex (SC) region of spermatocytes. FAM9B was also evident in the follicle cell nuclei and diffusely dispersed in the granular cell cytoplasm. FAM9B was partly co-localized with SYCP3, which is essential for both formation and maintenance of lateral SC elements. In addition, FAM9B had a similar distribution pattern and co-localization as γH2AX, which is a novel biomarker for DNA double-strand breaks during meiosis. All results indicate that FAM9B is a novel meiosis-associated protein that is co-localized with SYCP3 and γH2AX and may play an important role in SC formation and DNA recombination during meiosis. These findings offer a new perspective for understanding the molecular mechanisms involved in meiosis of human gametogenesis. [ABSTRACT FROM AUTHOR]

During meiosis, defects in critical events trigger checkpoint activation and restrict cell cycle progression. The budding yeast Pch2 AAA+ ATPase orchestrates the checkpoint response launched by synapsis deficiency; deletion of PCH2 or mutation of the ATPase catalytic sites suppress the meiotic block of the zip1Δ mutant lacking the central region of the synaptonemal complex. Pch2 action enables adequate levels of phosphorylation of the Hop1 axial component at threonine 318, which in turn promotes activation of the Mek1 effector kinase and the ensuing checkpoint response. In zip1Δ chromosomes, Pch2 is exclusively associated to the rDNA region, but this nucleolar fraction is not required for checkpoint activation, implying that another yet uncharacterized Pch2 population must be responsible for this function. Here, we have artificially redirected Pch2 to different subcellular compartments by adding ectopic Nuclear Export (NES) or Nuclear Localization (NLS) sequences, or by trapping Pch2 in an immobile extranuclear domain, and we have evaluated the effect on Hop1 chromosomal distribution and checkpoint activity. We have also deciphered the spatial and functional impact of Pch2 regulators including Orc1, Dot1 and Nup2. We conclude that the cytoplasmic pool of Pch2 is sufficient to support the meiotic recombination checkpoint involving the subsequent Hop1-Mek1 activation on chromosomes, whereas the nuclear accumulation of Pch2 has pathological consequences. We propose that cytoplasmic Pch2 provokes a conformational change in Hop1 that poises it for its chromosomal incorporation and phosphorylation. Our discoveries shed light into the intricate regulatory network controlling the accurate balance of Pch2 distribution among different cellular compartments, which is essential for proper meiotic outcomes. Author summary: During gametogenesis, the number of chromosomes is reduced by half and it returns to the normal ploidy when the two gametes fuse during fertilization. Meiosis lies at the heart of gametogenesis because it is the specialized cell division making possible the reduction in ploidy. The fidelity in this process is essential to maintain the chromosome complement characteristic of the species and to avoid aneuploidies. Meiotic cells possess an intricate surveillance network that monitors crucial meiotic events. In response to defects in synapsis and recombination, the meiotic recombination checkpoint blocks meiotic cell cycle progression, thus avoiding aberrant chromosome segregation and formation of defective gametes. The AAA+ ATPase Pch2 is an essential component of the checkpoint response triggered by the recombination defects occurring in the zip1Δ mutant lacking the central region of the synaptonemal complex. Pch2 supports proper chromosomal localization and phosphorylation of the Hop1 axial component required for the ensuing checkpoint response. We reveal here the biological relevance of a cytoplasmic population of Pch2 that is necessary for meiotic events occurring on chromosomes. Using a variety of strategies, we demonstrate that the checkpoint activating function of Pch2 takes place outside the nucleus, whereas the nuclear accumulation of Pch2 has deleterious consequences. Our work highlights the importance of nucleocytoplasmic communication for a balanced distribution of Pch2 among different subcellular compartments and how it impinges on Hop1 dynamics, which is crucial for proper completion of the meiotic program. [ABSTRACT FROM AUTHOR]

During meiosis I, ring-shaped cohesin complexes play important roles in aiding the proper segregation of homologous chromosomes. RAD21L is a meiosis-specific vertebrate cohesin that is required for spermatogenesis in mice but is dispensable for oogenesis in young animals. The role of this cohesin in other vertebrate models has not been explored. Here, we tested if the zebrafish homolog Rad21l1 is required for meiotic chromosome dynamics during spermatogenesis and oogenesis. We found that Rad21l1 localizes to unsynapsed chromosome axes. It is also found between the axes of the mature tripartite synaptonemal complex (SC) in both sexes. We knocked out rad21l1 and found that nearly all rad21l1-/- mutants develop as fertile males, suggesting that the mutation causes a defect in juvenile oogenesis, since insufficient oocyte production triggers female to male sex reversal in zebrafish. Sex reversal was partially suppressed by mutation of the checkpoint gene tp53, suggesting that the rad21l1 mutation activates Tp53-mediated apoptosis or arrest in females. This response, however, is not linked to a defect in repairing Spo11-induced double-strand breaks since deletion of spo11 does not suppress the sex reversal phenotype. Compared to tp53 single mutant controls, rad21l1-/-tp53-/- double mutant females produce poor quality eggs that often die or develop into malformed embryos. Overall, these results indicate that the absence of rad21l1-/- females is due to a checkpoint-mediated response and highlight a role for a meiotic-specific cohesin subunit in oogenesis but not spermatogenesis. Author summary: Cohesins are multi-protein, ring-shaped complexes that tether DNA duplexes to one another and are important for nuclear organization of DNA and proper chromosome segregation during cell division. Specialized cohesin subunits play important roles in meiosis, a cell division required to produce gametes. Segregation errors in meiosis can lead to the formation of gametes with the incorrect number of chromosomes (aneuploidy), a major cause of birth defects and pregnancy loss in humans. Here we assess the role of Rad21l1, a cohesin subunit specific to vertebrate animals, by knocking out the gene in zebrafish and examining how oogenesis and spermatogenesis are affected. While males produce normal sperm in the absence of Rad21l1, oocyte production is severely compromised. Oocytes develop partway but then undergo cell death. Deleting tp53, a gene involved in sensing cellular stress, prevents cell death and allows oogenesis to be completed, though most of the resulting eggs are severely poor quality. Thus, our work shows that zebrafish oogenesis requires a special cohesin subunit, without which oocytes die. In contrast, spermatogenesis does not require this subunit. [ABSTRACT FROM AUTHOR]

Meiotic crossovers (COs) are important for reshuffling genetic information between homologous chromosomes and they are essential for their correct segregation. COs are unevenly distributed along chromosomes and the underlying mechanisms controlling CO localization are not well understood. We previously showed that meiotic COs are mis-localized in the absence of AXR1, an enzyme involved in the neddylation/rubylation protein modification pathway in Arabidopsis thaliana. Here, we report that in axr1-/-, male meiocytes show a strong defect in chromosome pairing whereas the formation of the telomere bouquet is not affected. COs are also redistributed towards subtelomeric chromosomal ends where they frequently form clusters, in contrast to large central regions depleted in recombination. The CO suppressed regions correlate with DNA hypermethylation of transposable elements (TEs) in the CHH context in axr1-/- meiocytes. Through examining somatic methylomes, we found axr1-/- affects DNA methylation in a plant, causing hypermethylation in all sequence contexts (CG, CHG and CHH) in TEs. Impairment of the main pathways involved in DNA methylation is epistatic over axr1-/- for DNA methylation in somatic cells but does not restore regular chromosome segregation during meiosis. Collectively, our findings reveal that the neddylation pathway not only regulates hormonal perception and CO distribution but is also, directly or indirectly, a major limiting pathway of TE DNA methylation in somatic cells. Author summary: In sexually reproducing organisms, each parent transmits one and only one copy of each chromosome to their progeny via their packaging in haploid gametes. To ensure the proper transmission of the chromosomes, pairs of homologous chromosomes must associate and exchange genetic information (also called reciprocal recombination) during a special division called meiosis that lead to the formation of the gametes. The recombination process is highly controlled in terms of number and localization of the events along the chromosomes. Disruption of this control may cause an inappropriate transmission of the chromosomes in the gametes leading to abnormal chromosome numbers in the offspring which is usually deleterious. In the plant Arabidopis thaliana, we show that when the pathway modifying proteins through ubiquitination/neddylation is impaired, the number of reciprocal recombination events is maintained but they are delocalized toward the ends of the chromosomes and some chromosomes do not exchange material. We also detected changes of patterns for DNA methylation, an epigenetic modification localised on DNA cytosines. Furthermore, we demonstrate that the methylation of cytosines is not causal to the localization change of meiotic recombination events. [ABSTRACT FROM AUTHOR]

Polyploidy has been an influential force in plant evolution, playing a crucial role in diversification. Differences in polyploid and diploid distributions have been long noted, with polyploid taxa especially abundant in harsh environments. These plants have higher photosynthetic rates and/or higher tolerance to water deficits. Moreover, there is data pointing to an increase in the rate of unreduced gamete formation by plants under conditions of stress. Accordingly, a higher frequency of polyploid individuals would be expected in populations living under extreme environments, a phenomenon that may be relevant when considering the origin of allopolyploid species. Hybridization between distinct autopolyploids is known to produce allopolyploids and hence, a high frequency of compatible autopolyploids in an area could enhance the formation of stable populations of the corresponding allopolyploid hybrid. Here we consider the allopolyploid species Gypsophila bermejoi G. López and its parental taxa G. struthium L. subsp. struthium and G. tomentosa L. We have used Species Distribution Models to locate areas with low bioclimatic suitability for both parental taxa during the Last Glacial Maximum (LGM), hypothesizing that the rate of tetraploid hybrid formation would be higher than expected where low suitability areas of both parental species overlap. We selected those areas taking into account the strict gypsophyllic nature of these taxa. There is data pointing to a post-glacial origin of the current G. bermejoi populations and according to our hypothesis, such locations could be centers for hybrid tetraploid formation or potential cradles of this species. Indeed, potential Mid-Holocene cradles were also identified in this manner. The evolution of bioclimatic suitability in both LGM and Mid-Holocene cradles was studied to assess the possible survival of the hybrids, and the current distribution of G. bermejoi proved to be consistent with our hypothesis. [ABSTRACT FROM AUTHOR]

A set of sex chromosomes is required for gametogenesis in both males and females, as represented by sex chromosome disorders causing agametic phenotypes. Although studies using model animals have investigated the functional requirement of sex chromosomes, involvement of these chromosomes in gametogenesis remains elusive. Here, we elicit a germ cell-intrinsic effect of sex chromosomes on oogenesis, using a novel culture system in which oocytes were induced from embryonic stem cells (ESCs) harboring XX, XO or XY. In the culture system, oogenesis using XO and XY ESCs was severely disturbed, with XY ESCs being more strongly affected. The culture system revealed multiple defects in the oogenesis of XO and XY ESCs, such as delayed meiotic entry and progression, and mispairing of the homologous chromosomes. Interestingly, Eif2s3y, a Y-linked gene that promotes proliferation of spermatogonia, had an inhibitory effect on oogenesis. This led us to the concept that male and female gametogenesis appear to be in mutual conflict at an early stage. This study provides a deeper understanding of oogenesis under a sex-reversal condition. Author summary: Oogenesis is a highly sex-dependent differentiation process that commences at the mid-gestation stage. Oogenesis is severely disturbed in XO and XY females, although the mechanisms underlying the disturbance are still elusive. In this study, we subjected XO and XY ES cells to an in vitro culture system, in which female (XX) ES cells differentiate into functional oocytes. Our results showed that oogenesis from XO and XY ES cells was severely disturbed due to defective processes such as delayed meiotic initiation/progression, aberrant gene expression and mispairing of homologous chromosomes. In addition, our results demonstrate that a Y-linked gene, Eif2s3y that promotes spermatogenesis, has conversely a negative impact on oogenesis. This study provides a culture system that serves as a useful model of sex reversal and contributes the concept of a substantial germ cell-intrinsic conflict between male and female gametogenesis. Further study using the culture system will reveal molecular insights into infertility with sex chromosome disorder(s). [ABSTRACT FROM AUTHOR]

Autologous transplantation of spermatogonial stem cells is a promising new avenue to restore fertility in infertile recipients. Expansion of the initial spermatogonial stem cell pool through cell culturing is a necessary step to obtain enough cells for effective repopulation of the testis after transplantation. Since in vitro propagation can lead to (epi-)genetic mutations and possibly malignant transformation of the starting cell population, we set out to investigate genome-wide DNA methylation status in uncultured and cultured primary testicular ITGA6+ sorted cells and compare them with germ cell tumor samples of the seminoma subtype. Seminomas displayed a severely global hypomethylated profile, including loss of genomic imprinting, which we did not detect in cultured primary testicular ITGA6+ cells. Differential methylation analysis revealed altered regulation of gamete formation and meiotic processes in cultured primary testicular ITGA6+ cells but not in seminomas. The pivotal POU5F1 marker was hypomethylated in seminomas but not in uncultured or cultured primary testicular ITGA6+ cells, which is reflected in the POU5F1 mRNA expression levels. Lastly, seminomas displayed a number of characteristic copy number variations that were not detectable in primary testicular ITGA6+ cells, either before or after culture. Together, the data show a distinct DNA methylation patterns in cultured primary testicular ITGA6+ cells that does not resemble the pattern found in seminomas, but also highlight the need for more sensitive methods to fully exclude the presence of malignant cells after culture and to further study the epigenetic events that take place during in vitro culture. [ABSTRACT FROM AUTHOR]

Many closely related species are capable of mating to produce hybrid offspring, which are usually sterile. Nevertheless, altering the gametogenesis of hybrid offspring can rescue hybrids from sterility by enabling asexual reproduction. Hybridogenesis is one of the most complicated asexual reproductive modes, and it includes drastic genome reorganization only in the germline; this is achieved through elimination of one parental genome and duplication of the remaining one to restore diploid chromosomal set and overcome blocks in meiotic progression. We investigated a model of hybridogenesis, namely, water frogs from the Pelophylax esculentus complex, for the emergence of asexual reproduction. Further, we assessed the impact of its asexual reproduction on the maintenance of interspecies hybrids from two populations on the western edge of the P. esculentus range, in which hybrids coexist with either both parental species or with only one parental species. After analysing tadpole karyotypes, we conclude that in both studied populations, the majority of diploid hybrid males produced haploid gametes with the P. ridibundus genome after elimination of the P. lessonae genome. Hybrid females exhibited problems with genome elimination and duplication; they usually produced oocytes with univalents, but there were observations of individual oocytes with 13 bivalents and even 26 bivalents. In some hybrid tadpoles, especially F1 crosses, we observed failed germ cell development, while in tadpoles from backcrosses, germ cells were normally distributed and contained micronuclei. By identifying chromosomes present in micronuclei, we estimated that the majority of tadpoles from all crosses were able to selectively eliminate the P. lessonae chromosomes. According to our results, hybridogenesis in hybrids can appear both from crosses of parental species and crosses between sexual species with hybrid individuals. The ability to eliminate a genome and perform endoreplication to ensure gamete formation differed between male and female hybrids from the studied populations. Some diploid hybrid females can rarely produce not only haploid gametes but also diploid gametes, which is a crucial step in the formation of triploid hybrids. [ABSTRACT FROM AUTHOR]

Animal development requires the execution of specific transcriptional programs in different sets of cells to build tissues and functional organs. Transcripts are exported from the nucleus to the cytoplasm where they are translated into proteins that, ultimately, carry out the cellular functions. Here we show that in Caenorhabditis elegans, reduction of mRNA export strongly affects epithelial morphogenesis and germline proliferation while other tissues remain relatively unaffected. Epithelialization and gamete formation demand a large number of transcripts in the cytoplasm for the duration of these processes. In addition, our findings highlight the existence of a regulatory feedback mechanism that activates gene expression in response to low levels of cytoplasmic mRNA. We expand the genetic characterization of nuclear export factor NXF-1 to other members of the mRNA export pathway to model mRNA export and recycling of NXF-1 back to the nucleus. Our model explains how mutations in genes involved in general processes, such as mRNA export, may result in tissue-specific developmental phenotypes. [ABSTRACT FROM AUTHOR]

In haplodiplontic lineages, sexual reproduction occurs in haploid parents without meiosis. Although widespread in multicellular lineages such as brown algae (Phaeophyceae), haplodiplontic gametogenesis has been little studied at the molecular level. We addressed this by generating an annotated reference transcriptome for the gametophytic phase of the sugar kelp, Saccharina latissima. Transcriptional profiles of microscopic male and female gametophytes were analysed at four time points during the transition from vegetative growth to gametogenesis. Gametogenic signals resulting from a switch in culture irradiance from red to white light activated a core set of genes in a sex-independent manner, involving rapid activation of ribosome biogenesis, transcription and translation related pathways, with several acting at the post-transcriptional or post-translational level. Additional genes regulating nutrient acquisition and key carbohydrate-energy pathways were also identified. Candidate sex-biased genes under gametogenic conditions had potentially key roles in controlling female- and male-specific gametogenesis. Among these were several sex-biased or -specific E3 ubiquitin-protein ligases that may have important regulatory roles. Females specifically expressed several genes that coordinate gene expression and/or protein degradation, and the synthesis of inositol-containing compounds. Other female-biased genes supported parallels with oogenesis in divergent multicellular lineages, in particular reactive oxygen signalling via an NADPH-oxidase. Males specifically expressed the hypothesised brown algal sex-determining factor. Male-biased expression mainly involved upregulation of genes that control mitotic cell proliferation and spermatogenesis in other systems, as well as multiple flagella-related genes. Our data and results enhance genome-level understanding of gametogenesis in this ecologically and economically important multicellular lineage. [ABSTRACT FROM AUTHOR]

Primnoa pacifica is the most ecologically important coral species in the North Pacific Ocean and provides important habitat for commercially important fish and invertebrates. Ocean acidification (OA) is more rapidly increasing in high-latitude seas because anthropogenic CO2 uptake is greater in these regions. This is due to the solubility of CO2 in cold water and the reduced buffering capacity and low alkalinity of colder waters. Primnoa pacifica colonies were cultured for six to nine months in either pH 7.55 (predicted Year 2100 pH levels) or pH 7.75 (Control). Oocyte development and fecundity in females, and spermatocyst stages in males were measured to assess the effects of pH on gametogenesis. Oocyte diameters were 13.6% smaller and fecundities were 30.9% lower in the Year 2100 samples. A higher proportion of vitellogenic oocytes (65%) were also reabsorbed (oosorption) in the Year 2100 treatment. Lower pH appeared to advance the process of spermatogenesis with a higher percentage of later stage sperm compared to Control. There was a laboratory effect observed in all measurement types, however this only significantly affected the analyses of spermatogenesis. Based on the negative effect of acidification on oogenesis and increased rate of oosorption, successful spawning could be unlikely in an acidified ocean. If female gametes were spawned, they are likely to be insufficiently equipped to develop normally, based on the decreased overall size and therefore subsequent limited amount of lipids necessary for successful larval development. [ABSTRACT FROM AUTHOR]

RWP-RKs represent a small family of transcription factors (TFs) that are unique to plants and function particularly under conditions of nitrogen starvation. These RWP-RKs have been classified in two sub-families, NLPs (NIN-like proteins) and RKDs (RWP-RK domain proteins). NLPs regulate tissue-specific expression of genes involved in nitrogen use efficiency (NUE) and RKDs regulate expression of genes involved in gametogenesis/embryogenesis. During the present study, using in silico approach, 37 wheat RWP-RK genes were identified, which included 18 TaNLPs (2865 to 7340 bp with 4/5 exons), distributed on 15 chromosomes from 5 homoeologous groups (with two genes each on 4B,4D and 5A) and 19 TaRKDs (1064 to 5768 bp with 1 to 6 exons) distributed on 12 chromosomes from 4 homoeologous groups (except groups 1, 4 and 5); 2–3 splice variants were also available in 9 of the 37 genes. Sixteen (16) of these genes also carried 24 SSRs (simple sequence repeats), while 11 genes had targets for 13 different miRNAs. At the protein level, MD simulation analysis suggested their interaction with nitrate-ions. Significant differences were observed in the expression of only two (TaNLP1 and TaNLP2) of the nine representative genes that were used for in silico expression analysis under varying levels of N at post-anthesis stage (data for other genes was not available for in silico expression analysis). Differences in expression were also observed during qRT-PCR, when expression of four representative genes (TaNLP2, TaNLP7, TaRKD6 and TaRKD9) was examined in roots and shoots of seedlings (under different conditions of N supply) in two contrasting genotypes which differed in NUE (C306 with low NUE and HUW468 with high NUE). These four genes for qRT-PCR were selected on the basis of previous literature, level of homology and the level of expression (in silico study). In particular, the TaNLP7 gene showed significant up-regulation in the roots and shoots of HUW468 (with higher NUE) during N-starvation; this gene has already been characterized in Arabidopsis and tobacco, and is known to be involved in nitrate-signal transduction pathway. [ABSTRACT FROM AUTHOR]

Meiotic drivers are selfish alleles that subvert gametogenesis to increase their transmission into progeny. Drivers impose a fitness cost, putting pressure on the genome to evolve suppressors. Here we investigate the wtf gene family from Schizosaccharomyces pombe, previously shown to contain meiotic drivers in wild isolates. We discovered that wtf13 found in lab stocks is a meiotic driver. wtf13 kills spores that do not inherit it by generating both a diffusible poison and a spore-specific antidote. Additionally, we demonstrate that wtf13 is suppressed by another wtf gene, wtf18-2, that arose spontaneously in the lab and makes only an antidote. Wtf18-2 does not act indiscriminately to prevent spore destruction. Instead, it rescues only the spores that inherit wtf18-2. In this way, wtf18-2 selfishly gains a transmission advantage of its own while dampening the drive of wtf13. This establishes a novel paradigm for meiotic drive suppressors and provides insight into the mechanisms and evolution of drive systems. [ABSTRACT FROM AUTHOR]

Restoring and conserving coastal resilience faces increasing challenges under current climate change predictions. Oyster restoration, in particular, faces threats from alterations in precipitation, warming water temperatures, and urbanization of coastlines that dramatically change salinity patterns, foster the proliferation and spread disease, and disrupt habitat connectivity, respectively. New York City (NYC) coastal waters, once home to a booming oyster fishery for eastern oysters (Crassostrea virginica), are now nearly devoid of live oyster reefs. Oyster restoration in urban estuaries is motivated by the synergistic ecosystem benefits this native keystone species can deliver. Recent surveys have documented substantial remnant populations of adult oysters in the upper low salinity zone of the Hudson/Raritan Estuary (HRE) near Tarrytown, NY. This study assessed fitness-related performance across the HRE salinity gradient to evaluate habitat suitability on an estuarine scale. Oysters were hatchery-produced from wild, moderate-salinity broodstock, then outplanted for measurement of growth, survival, reproduction and disease prevalence over two years. Survival was generally higher in the lower salinity river sites and in the higher salinity Jamaica Bay sites relative to mesohaline NYC harbor sites. Growth rate was highest in Jamaica Bay and had high variation among other sites. Surprisingly, the highest proportion of individuals with sex-differentiated gametes and the highest average gonad maturation index was found at a low salinity site. Consistent with the advanced gametogenesis measured in experimental animals at low salinity, annual wild recruitment was documented near the low salinity remnant population in each of five monitored years. These results suggest that the remnant HRE oyster population is a robust, self-sustaining population that can be leveraged to support restoration of subpopulations in other parts of the estuary, but further research is required to determine if the mesohaline and near-ocean reaches of the HRE can support the full oyster life cycle. [ABSTRACT FROM AUTHOR]

Breast cancer susceptibility gene 1 (BRCA1) and binding partner BRCA1-associated RING domain protein 1 (BARD1) form an essential E3 ubiquitin ligase important for DNA damage repair and homologous recombination. The Caenorhabditis elegans orthologs, BRC-1 and BRD-1, also function in DNA damage repair, homologous recombination, as well as in meiosis. Using functional GFP fusions we show that in mitotically-dividing germ cells BRC-1 and BRD-1 are nucleoplasmic with enrichment at foci that partially overlap with the recombinase RAD-51. Co-localization with RAD-51 is enhanced under replication stress. As cells enter meiosis, BRC-1-BRD-1 remains nucleoplasmic and in foci, and beginning in mid-pachytene the complex co-localizes with the synaptonemal complex. Following establishment of the single asymmetrically positioned crossover on each chromosome pair, BRC-1-BRD-1 concentrates to the short arm of the bivalent. Localization dependencies reveal that BRC-1 and BRD-1 are interdependent and the complex fails to properly localize in both meiotic recombination and chromosome synapsis mutants. Consistent with a role for BRC-1-BRD-1 in meiotic recombination in the context of the synaptonemal complex, inactivation of BRC-1 or BRD-1 enhances the embryonic lethality of mutants defective in chromosome synapsis. Our data suggest that under meiotic dysfunction, BRC-1-BRD-1 stabilizes the RAD-51 filament and alters the recombination landscape; these two functions can be genetically separated from BRC-1-BRD-1’s role in the DNA damage response. Together, we propose that BRC-1-BRD-1 serves a checkpoint function at the synaptonemal complex where it monitors and modulates meiotic recombination. [ABSTRACT FROM AUTHOR]

Reproductive function is under the control of the neurohormone GnRH, which activates a G-protein-coupled receptor (GnRHR) expressed in pituitary gonadotrope cells. GnRHR activates a complex signaling network to regulate synthesis and secretion of the two gonadotropin hormones, luteinizing hormone and follicle-stimulating hormone, both regulating gametogenesis and steroidogenesis in gonads. Recently, in an attempt to identify the mechanisms underlying GnRHR signaling plasticity, we identified the first interacting partner of GnRHR, the proto-oncogene SET. We showed that SET binds to intracellular domains of GnRHR to enhance its coupling to cAMP pathway in αT3-1 gonadotrope cells. Here, we demonstrate that SET protein is rapidly regulated by GnRH, which increases SET phosphorylation state and decreases dose-dependently SET protein level. Our results highlight a post-translational regulation of SET protein involving the proteasome pathway. We determined that SET phosphorylation upon GnRH stimulation is mediated by PKC and that PKC mediates GnRH-induced SET down-regulation. Phosphorylation on serine 9 targets SET for degradation into the proteasome. Furthermore, a non-phosphorylatable SET mutant on serine 9 is resistant to GnRH-induced down-regulation. Altogether, these data suggest that GnRH-induced SET phosphorylation on serine 9 mediates SET protein down-regulation through the proteasome pathway. Noteworthy, SET down-regulation was also observed in response to pulsatile GnRH stimulation in LβT2 gonadotrope cells as well as in vivo in prepubertal female mice supporting its physiological relevance. In conclusion, this study highlights a regulation of SET protein by the neurohormone GnRH and identifies some of the mechanisms involved. [ABSTRACT FROM AUTHOR]

Germ cells give rise to eggs or sperm. However, recent analyses in medaka (Oryzias latipes) showed that germ cells are also important for feminization of gonads, although this novel role of germ cells has not been characterized in detail. Here, we show that the feminizing effect is inherent to germ cells and is not affected by gametogenic stages or the sexual fate of germ cells. Three medaka mutants were generated to demonstrate this effect: figlα mutants, in which follicle formation is disrupted; meioC mutants, in which germ cells are unable to commit to gametogenesis and meiosis; and dazl mutants, in which germ cells do not develop into gonocytes. All these different stages of germ cells in XX mutants have an ability to feminize the gonads, resulting in the formation of gonads with ovarian structures. In addition to normal ovarian development, we also suggest that the increased number of gonocytes is sufficient for male to female sex reversal in XY medaka. These results may genetically demonstrate that the mechanism underlying the feminizing effect of germ cells is activated before the sexual fate decision of germ cells and meiosis, probably by the time of gonocyte formation in medaka. Author summary: Germ cells are the only cells that can transfer genetic materials to the next generation via the sperm or egg. However, recent analyses in teleosts revealed another essential role of germ cells: feminizing the gonads. In our study, medaka mutants in which gametogenesis was blocked at specific stages provides the novel view that the feminizing effect of germ cells occurs in parallel with other reproductive elements, such as meiosis, the sexual fate decision of germ cells, and gametogenesis. Germ cells in medaka may have a potential to feminize gonads at the moment they have developed. [ABSTRACT FROM AUTHOR]

The 24-nucleotides (nt) phased secondary small interfering RNA (phasiRNA) is a unique class of plant small RNAs abundantly expressed in monocot anthers at early meiosis. Previously, 44 intergenic regions were identified as the loci for longer precursor RNAs of 24-nt phasiRNAs (24-PHASs) in the rice genome. However, the regulatory mechanism that determines spatiotemporal expression of these RNAs has remained elusive. ETERNAL TAPETUM1 (EAT1) is a basic-helix-loop-helix (bHLH) transcription factor indispensable for induction of programmed cell death (PCD) in postmeiotic anther tapetum, the somatic nursery for pollen production. In this study, EAT1-dependent non-cell-autonomous regulation of male meiosis was evidenced from microscopic observation of the eat1 mutant, in which meiosis with aberrantly decondensed chromosomes was retarded but accomplished somehow, eventually resulting in abortive microspores due to an aberrant tapetal PCD. EAT1 protein accumulated in tapetal-cell nuclei at early meiosis and postmeiotic microspore stages. Meiotic EAT1 promoted transcription of 24-PHAS RNAs at 101 loci, and importantly, also activated DICER-LIKE5 (DCL5, previous DCL3b in rice) mRNA transcription that is required for processing of double-stranded 24-PHASs into 24-nt lengths. From the results of the chromatin-immunoprecipitation and transient expression analyses, another tapetum-expressing bHLH protein, TDR INTERACTING PROTEIN2 (TIP2), was suggested to be involved in meiotic small-RNA biogenesis. The transient assay also demonstrated that UNDEVELOPED TAPETUM1 (UDT1)/bHLH164 is a potential interacting partner of both EAT1 and TIP2 during early meiosis. This study indicates that EAT1 is one of key regulators triggering meiotic phasiRNA biogenesis in anther tapetum, and that other bHLH proteins, TIP2 and UDT1, also play some important roles in this process. Spatiotemporal expression control of these bHLH proteins is a clue to orchestrate precise meiosis progression and subsequent pollen production non-cell-autonomously. [ABSTRACT FROM AUTHOR]

Bacterial morphology is a complex trait that is highly sensitive to changes in the environment. For heterotrophic organisms, such as Escherichia coli, increases in nutrient levels are frequently accompanied by several-fold increases in both size and growth rate. Despite the dramatic nature of these changes, how alterations in nutrient availability translate into changes in growth and morphology remains a largely open question. To understand the signaling networks coupling nutrient availability with size and shape, we examined the impact of deletions in the entirety of non-essential central carbon metabolic genes on E. coli growth rate and cell size. Our data reveal the presence of multiple metabolic nodes that play important yet distinctive roles in dictating biosynthetic capacity and shaping cell morphology. Specifically, perturbations of acetyl-CoA metabolism impact cell size and division through changes in fatty acid synthesis. Additionally, we identify a genetic pathway linking glucose levels to cell width through the signaling molecule cyclic-AMP. Together our findings highlight a surprising diversity of factors and mechanisms contributing to growth potential and cell morphology, providing a foundation for further studies. [ABSTRACT FROM AUTHOR]

In this work, we study if ploidy (i.e. number of copies of chromosomes) in the oyster Crassostrea gigas may introduce differences in behavior and in its synchronization by the annual photoperiod. To answer to the question about the effect of the seasonal course of the photoperiod on the behavior of C. gigas according to its ploidy, we quantified valve activity by HFNI valvometry in situ for 1 year in both diploid and triploid oysters. Chronobiological analyses of daily, tidal and lunar rhythms were performed according the annual change of the photoperiod. In parallel, growth and gametogenesis status were measured and spawning events were detected by valvometry. The results showed that triploids had reduced gametogenesis, without spawning events, and approximately three times more growth than diploids. These differences in physiological efforts could explain the result that photoperiod (daylength and/or direction of daylength) differentially drives and modulates seasonal behavior of diploid and triploid oysters. Most differences were observed during long days (spring and summer), where triploids showed longer valve opening duration but lower opening amplitude, stronger daily rhythm and weaker tidal rhythm. During this period, diploids did major gametogenesis and spawning whereas triploids did maximal growth. Differences were also observed in terms of moonlight rhythmicity and neap-spring tidal cycle rhythmicity. We suggest that the seasonal change of photoperiod differentially synchronizes oyster behavior and biological rhythms according to physiological needs based on ploidy. [ABSTRACT FROM AUTHOR]

The methylation of cytosine at CG sites in the mammalian genome is dynamically reprogrammed during gametogenesis and preimplantation development. It was previously shown that oocyte-derived DNMT1 (a maintenance methyltransferase) is essential for maintaining and propagating CG methylation at imprinting control regions in preimplantation embryos. In mammalian somatic cells, hemimethylated-CG-binding protein UHRF1 plays a critical role in maintaining CG methylation by recruiting DNMT1 to hemimethylated CG sites. However, the role of UHRF1 in oogenesis and preimplantation development is unknown. In the present study, we show that UHRF1 is mainly, but not exclusively, localized in the cytoplasm of oocytes and preimplantation embryos. However, smaller amounts of UHRF1 existed in the nucleus, consistent with the expected role in DNA methylation. We then generated oocyte-specific Uhrf1 knockout (KO) mice and found that, although oogenesis was itself unaffected, a large proportion of the embryos derived from the KO oocytes died before reaching the blastocyst stage (a maternal effect). Whole genome bisulfite sequencing revealed that blastocysts derived from KO oocytes have a greatly reduced level of CG methylation, suggesting that maternal UHRF1 is essential for maintaining CG methylation, particularly at the imprinting control regions, in preimplantation embryos. Surprisingly, UHRF1 was also found to contribute to de novo CG and non-CG methylation during oocyte growth: in Uhrf1 KO oocytes, transcriptionally-inactive regions gained less methylation, while actively transcribed regions, including the imprinting control regions, were unaffected or only slightly affected. We also found that de novo methylation was defective during the late stage of oocyte growth. To the best of our knowledge, this is the first study to demonstrate the role of UHRF1 in de novo DNA methylation in vivo. Our study reveals multiple functions of UHRF1 during the global epigenetic reprogramming of oocytes and early embryos. [ABSTRACT FROM AUTHOR]

Two types of natural hybrids were discovered in populations of three Hexagrammos species (Teleostei: Hexagrammidae) distributed off the southern coast of Hokkaido in the North Pacific Ocean. Both hybrids reproduce by hybridogenesis, in which the maternal haploid genome is transmitted to offspring without recombination and the paternal haploid genome is eliminated during gametogenesis. While natural hybrids are unisexual and reproduce hemiclonally by backcrossing with the paternal species (BC-P), artificial F1-hybrids between the pure species produce recombinant gametes. Thus, despite having the same genome composition, the natural hybrids and the F1-hybrids are not genetically identical. Here, to clarify the differences between both hybrids, we examined the karyotypes of the three Hexagrammos species, their natural hybrids, the artificial F1-hybrids, and several backcrosses. Artificial F1-hybrids have karyotypes and chromosome numbers that are intermediate between those of the parental species. Conversely, the natural hybrids differed from F1-hybrids by having several large metacentric chromosomes and microchromosomes. Since the entire maternal haploid genome is inherited by the natural hybrids, maternal backcrosses (BC-M) between natural hybrids and males of the maternal species (H. octogrammus; Hoc) have a hemiclonal Hoc genome with large chromosomes from the mother and a normal Hoc genome from the father. However, the large chromosomes disappear in offspring of BC-M, probably due to fissuring during gametogenesis. Similarly, microsatellite DNA analysis revealed that chromosomes of BC-M undergo recombination. These findings suggest that genetic factors associated with hemiclonal reproduction may be located on the large metacentric chromosomes of natural hybrids. [ABSTRACT FROM AUTHOR]

Sporadic outbreaks of the coral-eating crown-of-thorns starfish are likely to be due, at least in part, to spatial and temporal variation in reproductive and settlement success. For gonochoric and broadcast spawning species such as crown-of-thorns starfish, spawning synchrony is fundamental for achieving high rates of fertilization. Highly synchronized gamete release within and among distinct populations is typically the result of the entrainment of neurohormonal endogenous rhythms by cues from the environment. In this study, we conducted multiple spawning assays to test the effects of temperature change, reduced salinity and nutrient enrichment of seawater, phytoplankton, gametes (sperm and eggs), and the combined effect of sperm and phytoplankton on the likelihood of spawning in male and female crown-of-thorns starfish. We also investigated sex-specific responses to each of these potential spawning cues. We found that (1) abrupt temperature change (an increase of 4°C) induced spawning in males, but less so in females; (2) males often spawned in response to the presence of phytoplankton, but none of the females spawned in response to these cues; (3) the presence of sperm in the water column induced males and females to spawn, although additive and synergistic effects of sperm and phytoplankton were not significant; and (4) males are more sensitive to the spawning cues tested and most likely spawn prior to females. We propose that environmental cues act as spawning ‘inducers’ by causing the release of hormones (gonad stimulating substance) in sensitive males, while biological cues (pheromones) from released sperm, in turn, act as spawning ‘synchronizers’ by triggering a hormonal cascade resulting in gamete shedding by conspecifics. Given the immediate temporal linkage between the timing of spawning and fertilization events, variability in the extent and synchronicity of gamete release will significantly influence reproductive success and may account for fluctuations in the abundance of crown-of-thorns starfish. [ABSTRACT FROM AUTHOR]

Global environmental change, in marine ecosystems, is associated with concurrent shifts in water temperature, circulation, stratification, and nutrient input, with potentially wide-ranging biological effects. Variations in seawater temperature might alter physiological functioning, reproductive efficiency, and demographic traits of marine organisms, leading to shifts in population size and abundance. Differences in temperature tolerances between organisms can identify individual and ecological characteristics, which make corals able to persist and adapt in a climate change context. Here we investigated the possible effect of temperature on the reproductive output of the solitary non-zooxanthellate temperate coral Leptopsammia pruvoti, along an 8° latitudinal gradient. Samples have been collected in six populations along the gradient and each polyp was examined using histological and cyto-histometric analyses. We coupled our results with previous studies on the growth, demography, and calcification of L. pruvoti along the same temperature gradient, and compared them with those of another sympatric zooxanthellate coral Balanophyllia europaea to understand which trophic strategy makes the coral more tolerant to increasing temperature. The non-zooxanthellate species seemed to be quite tolerant to temperature increases, probably due to the lack of the symbiosis with zooxanthellae. To our knowledge, this is the first field investigation of the relationship between reproductive output and temperature increase of a temperate asymbiotic coral, providing novel insights into the poorly studied non-zooxanthellate scleractinians. [ABSTRACT FROM AUTHOR]

The greater amberjack Seriola dumerili is a large teleost fish with rapid growth and excellent flesh quality, whose domestication represents an ambitious challenge for aquaculture. The occurrence of reproductive dysfunctions in greater amberjack reared in captivity was investigated by comparing reproductive development of wild and captive-reared individuals. Wild and captive-reared breeders were sampled in the Mediterranean Sea during three different phases of the reproductive cycle: early gametogenesis (EARLY, late April-early May), advanced gametogenesis (ADVANCED, late May-early June) and spawning (SPAWNING, late June-July). Fish reproductive state was evaluated using the gonado-somatic index (GSI), histological analysis of the gonads and determination of sex steroid levels in the plasma, and correlated with leptin expression in the liver and gonad biochemical composition. The GSI and sex steroid levels were lower in captive-reared than in wild fish. During the ADVANCED period, when the wild greater amberjack breeders were already in spawning condition, ovaries of captive-reared breeders showed extensive atresia of late vitellogenic oocytes and spermatogenic activity ceased in the testes of half of the examined males. During the SPAWNING period, all captive-reared fish had regressed gonads, while wild breeders still displayed reproductive activity. Liver leptin expression and gonad proximate composition of wild and captive greater amberjack were similar. However, the gonads of captive-reared fish showed different total polar lipid contents, as well as specific lipid classes and fatty acid profiles with respect to wild individuals. This study underlines the need for an improvement in rearing technology for this species, which should include minimum handling during the reproductive season and the formulation of a specific diet to overcome the observed gonadal decrements of phospholipids, DHA (22:6n-3) and ARA (20:4n-6), compared to wild breeders. [ABSTRACT FROM AUTHOR]

The origin of the germline–soma distinction is a fundamental unsolved question. Plants and basal metazoans do not have a germline but generate gametes from pluripotent stem cells in somatic tissues (somatic gametogenesis). In contrast, most bilaterians sequester a dedicated germline early in development. We develop an evolutionary model which shows that selection for mitochondrial quality drives germline evolution. In organisms with low mitochondrial replication error rates, segregation of mutations over multiple cell divisions generates variation, allowing selection to optimize gamete quality through somatic gametogenesis. Higher mutation rates promote early germline sequestration. We also consider how oogamy (a large female gamete packed with mitochondria) alters selection on the germline. Oogamy is beneficial as it reduces mitochondrial segregation in early development, improving adult fitness by restricting variation between tissues. But it also limits variation between early-sequestered oocytes, undermining gamete quality. Oocyte variation is restored through proliferation of germline cells, producing more germ cells than strictly needed, explaining the random culling (atresia) of precursor cells in bilaterians. Unlike other models of germline evolution, selection for mitochondrial quality can explain the stability of somatic gametogenesis in plants and basal metazoans, the evolution of oogamy in all plants and animals with tissue differentiation, and the mutational forces driving early germline sequestration in active bilaterians. The origins of predation in motile bilaterians in the Cambrian explosion is likely to have increased rates of tissue turnover and mitochondrial replication errors, in turn driving germline evolution and the emergence of complex developmental processes. [ABSTRACT FROM AUTHOR]

The pennatulid Malacobelemnon daytoni is one of the dominant species in Potter Cove, Antarctica. Its abundance and range of distribution have increased in recent years probably related to climate change mediated alterations of environmental factors. This work is the second part of a study dealing on the reproductive ecology of Malacobelemnon daytoni, and aims to assess its reproductive seasonality over a two-year period. Sampling was carried out every month during 2009–2010 and samples were examined by histological analysis. Gametogenesis exhibited a seasonal pattern evidenced by the maturity stage index (MSI) and the number of mature oocytes and cysts throughout the year. Immature oocytes and spermatocytes were present year-round, but maturation was seasonal and it seems that more than one spawning per year was possible. These spawnings could be more linked with suspended particulate matter (SPM) (probably available via resuspension events) than with primary production pulses. This idea reinforces the hypothesis that winter time is not so stressful, in energy terms, in Potter Cove, which seems to depend on energy sources other than local phytoplankton production. There was not a strong inter-annual variability between the reproductive characteristics analyzed in 2009 and 2010; the only variable different was the size of oocytes (higher in 2009), suggesting different energy availability in each year, related with a higher concentration of SPM in 2009 (although it was not significant). Malacobelemnon daytoni could be the first reported Antarctic suspension feeder species that presents a reproductive cycle with more than a spawning event per year. This strategy would help to explain the success of this species in the Potter Cove ecosystem and in high ice-impacted areas. [ABSTRACT FROM AUTHOR]

Induction of sexual reproduction in the facultatively sexual Chlamydomonas reinhardtii is cued by depletion of nitrogen. We explore the capacity for indirect monitoring of population variation in the gametogenic process using flow cytometry. We describe a high-throughput method capable of identifying fluorescence, ploidy and scatter profiles that track vegetative cells entering and undergoing gametogenesis. We demonstrate for the first time, that very early and late growth phases reduce the capacity to distinguish putative gametes from vegetative cells based on scatter and fluorescence profiles, and that early/mid-logarithmic cultures show the optimal distinction between vegetative cells and gamete scatter profiles. We argue that early/mid logarithmic cultures are valuable in such high throughput comparative approaches when investigating optimisation or quantification of gametogenesis based on scatter and fluorescence profiles. This approach provides new insights into the impact of culture conditions on gametogenesis, while documenting novel scatter and fluorescence profile shifts which typify the process. This method has potential applications to; enabling quick high-throughput monitoring, uses in increasing efficiency in the quantification of gametogenesis, as a method of comparing the switch between vegetative and gametic states across treatments, and as criteria for enrichment of gametic phenotypes in cell sorting assays. [ABSTRACT FROM AUTHOR]

Venus kinase receptors (VKRs) are invertebrate receptor tyrosine kinases (RTKs) formed by an extracellular Venus Fly Trap (VFT) ligand binding domain associated via a transmembrane domain with an intracellular tyrosine kinase (TK) domain. Schistosoma mansoni VKRs, SmVKR1 and SmVKR2, are both implicated in reproductive activities of the parasite. In this work, we show that the SH2 domain-containing protein SmShb is a partner of the phosphorylated form of SmVKR1. Expression of these proteins in Xenopus oocytes allowed us to demonstrate that the SH2 domain of SmShb interacts with the phosphotyrosine residue (pY979) located in the juxtamembrane region of SmVKR1. This interaction leads to phosphorylation of SmShb on tyrosines and promotes SmVKR1 signaling towards the JNK pathway. SmShb transcripts are expressed in all parasite stages and they were found in ovary and testes of adult worms, suggesting a possible colocalization of SmShb and SmVKR1 proteins. Silencing of SmShb in adult S. mansoni resulted in an accumulation of mature sperm in testes, indicating a possible role of SmShb in gametogenesis. [ABSTRACT FROM AUTHOR]

Global DNA de-methylation is thought to occur only during pre-implantation and gametogenesis in mammals. Scalable, cell-wide de-methylation has not been demonstrated beyond totipotent stages. Here, we observed a large scale de-methylation and subsequent re-methylation (CDR) (including 5-methylcytosine (5mC) and 5-hydroxylmethylcytosine (5hmC)) in post-mitotic cerebellar Purkinje cells (PC) through the course of normal development. Through single cell immuno-identification and cell-specific quantitative methylation assays, we demonstrate that the CDR event is an intrinsically scheduled program, occurring in nearly every PC. Meanwhile, cerebellar granule cells and basket interneurons adopt their own DNA methylation program, independent of PCs. DNA de-methylation was further demonstrated at the gene level, on genes pertinent to PC development. The PC, being one of the largest neurons in the brain, may showcase an amplified epigenetic cycle which may mediate stage transformation including cell cycle arrest, vast axonal-dendritic growth, and synaptogenesis at the onset of neuronal specificity. This discovery is a key step toward better understanding the breadth and role of DNA methylation and de-methylation during neural ontology. [ABSTRACT FROM AUTHOR]

Planar morphogenesis, a distinct feature of multicellular organisms, is crucial for the development of ovule, progenitor of seeds. Both receptor-like kinases (RLKs) such as STRUBBELIG (SUB) and auxin gradient mediated by PIN-FORMED1 (PIN1) play instructive roles in this process. Fine-tuned intercellular communications between different cell layers during ovule development demands dynamic membrane distribution of these cell-surface proteins, presumably through vesicle-mediated sorting. However, the way it’s achieved and the trafficking routes involved are obscure. We report that HAPLESS13 (HAP13)-mediated trafficking of SUB is critical for ovule development. HAP13 encodes the μ subunit of adaptor protein 1 (AP1) that mediates protein sorting at the trans-Golgi network/early endosome (TGN/EE). The HAP13 mutant, hap13-1, is defective in outer integument growth, resulting in exposed nucellus accompanied with impaired pollen tube guidance and reception. SUB is mis-targeted in hap13-1. However, unlike that of PIN2, the distribution of PIN1 is independent of HAP13. Genetic interference of exocytic trafficking at the TGN/EE by specifically downregulating HAP13 phenocopied the defects of hap13-1 in SUB targeting and ovule development, supporting a key role of sporophytically expressed SUB in instructing female gametogenesis. [ABSTRACT FROM AUTHOR]

Gnrh is the major neuropeptide regulator of vertebrate reproduction, triggering a cascade of events in the pituitary-gonadal axis that result in reproductive competence. Previous research in mice and humans has demonstrated that Gnrh/GNRH null mutations result in hypogonadotropic hypogonadism and infertility. The goal of this study was to eliminate gnrh3 (the hypophysiotropic Gnrh form) function in zebrafish (Danio rerio) to determine how ontogeny and reproductive performance are affected, as well as factors downstream of Gnrh3 along the reproductive axis. Using the TALEN technology, we developed a gnrh3-/- zebrafish line that harbors a 62 bp deletion in the gnrh3 gene. Our gnrh3-/- zebrafish line represents the first targeted and heritable mutation of a Gnrh isoform in any organism. Using immunohistochemistry, we verified that gnrh3-/- fish do not possess Gnrh3 peptide in any regions of the brain. However, other than changes in mRNA levels of pituitary gonadotropin genes (fshb, lhb, and cga) during early development, which are corrected by adulthood, there were no changes in ontogeny and reproduction in gnrh3-/- fish. The gnrh3-/- zebrafish are fertile, displaying normal gametogenesis and reproductive performance in males and females. Together with our previous results that Gnrh3 cell ablation causes infertility, these results indicate that a compensatory mechanism is being activated, which is probably primed early on upon Gnrh3 neuron differentiation and possibly confined to Gnrh3 neurons. Potential compensation factors and sensitive windows of time for compensation during development and puberty should be explored. [ABSTRACT FROM AUTHOR]

Mating and gametogenesis are two essential components of animal reproduction. Gametogenesis must be modulated by the need for gametes, yet little is known of how mating, a process that utilizes gametes, may modulate the process of gametogenesis. Here, we report that mating stimulates female germline stem cell (GSC) proliferation in Drosophila melanogaster. Mating-induced increase in GSC number is not simply owing to the indirect effect of emission of stored eggs, but rather is stimulated by a male-derived Sex Peptide (SP) and its receptor SPR, the components of a canonical neuronal pathway that induces a post-mating behavioral switch in females. We show that ecdysteroid, the major insect steroid hormone, regulates mating-induced GSC proliferation independently of insulin signaling. Ovarian ecdysteroid level increases after mating and transmits its signal directly through the ecdysone receptor expressed in the ovarian niche to increase the number of GSCs. Impairment of ovarian ecdysteroid biosynthesis disrupts mating-induced increase in GSCs as well as egg production. Importantly, feeding of ecdysteroid rescues the decrease in GSC number caused by impairment of neuronal SP signaling. Our study illustrates how female GSC activity is coordinately regulated by the neuroendocrine system to sustain reproductive success in response to mating. [ABSTRACT FROM AUTHOR]

The article discusses the genetic relation of recombination and speciation. It refers to the crucial role of meiosis in speciation which is also a fundamental part of gametogenesis. It notes the incompatibility between Prdm9 and Hstx2 that could inhibit the repair of double strand breaks and disrupt synapsis of chromosomes and meiotic arrest.

Cell fate choices are tightly controlled by the interplay between intrinsic and extrinsic signals, and gene regulatory networks. In Saccharomyces cerevisiae, the decision to enter into gametogenesis or sporulation is dictated by mating type and nutrient availability. These signals regulate the expression of the master regulator of gametogenesis, IME1. Here we describe how nutrients control IME1 expression. We find that protein kinase A (PKA) and target of rapamycin complex I (TORC1) signalling mediate nutrient regulation of IME1 expression. Inhibiting both pathways is sufficient to induce IME1 expression and complete sporulation in nutrient-rich conditions. Our ability to induce sporulation under nutrient rich conditions allowed us to show that respiration and fermentation are interchangeable energy sources for IME1 transcription. Furthermore, we find that TORC1 can both promote and inhibit gametogenesis. Down-regulation of TORC1 is required to activate IME1. However, complete inactivation of TORC1 inhibits IME1 induction, indicating that an intermediate level of TORC1 signalling is required for entry into sporulation. Finally, we show that the transcriptional repressor Tup1 binds and represses the IME1 promoter when nutrients are ample, but is released from the IME1 promoter when both PKA and TORC1 are inhibited. Collectively our data demonstrate that nutrient control of entry into sporulation is mediated by a combination of energy availability, TORC1 and PKA activities that converge on the IME1 promoter. [ABSTRACT FROM AUTHOR]