Forward Genetics Identifies a Requirement for the Izumo-like Immunoglobulin Superfamily spe-45 Gene in Caenorhabditis elegans Fertilization

Fertilization is a conserved process in all sexually reproducing organisms whereby sperm bind and fuse with oocytes. Despite the importance of sperm-oocyte interactions in fertilization, the molecular underpinnings of this process are still not well understood. The only cognate ligand-receptor pair identified in the context of fertilization is sperm-surface Izumo and egg-surface Juno in the mouse [1]. Here we describe a genetic screening strategy to isolate fertilization mutants in Caenorhabditis elegans in order to generate a more complete inventory of molecules required for gamete interactions. From this screening strategy, we identified, cloned, and characterized spe-45, a gene that encodes an Izumo-like immunoglobulin superfamily protein. Mammalian Izumo is required for male fertility and has the same basic mutant phenotype as spe-45. Worms lacking spe-45 function produce morphologically normal and motile sperm that cannot fuse with oocytes despite direct contact in the reproductive tract. The power of this screen to identify proteins with ancient sperm functions suggests that characterization of additional mutants from our screen may reveal other deeply conserved components in fertility pathways and complement studies in other organisms

EXAMINING CALIFORNIA’S ASSEMBLY BILL 1629 AND THE LONG-TERM CARE REIMBURSEMENT ACT: DID NURSING HOME NURSE STAFFING CHANGE?

California’s elderly population over age 85 is estimated to grow 361% by the year 2050. Many of these elders are frail and highly dependent on caregivers making them more likely to need nursing home care. A 1998 United States Government Accountability Office report identified poor quality of care in California nursing homes. This report spurred multiple Assembly Bills in California designed to increase nursing home nurse staffing, change the state’s Medi-Cal reimbursement methodology, or both. The legislation culminated in Assembly Bill (AB) 1629, signed into law in September 2004, which included the Long-Term Care Reimbursement Act. This legislation changed the state’s Medi-Cal reimbursement from a prospective, flat rate to a prospective, cost-based methodology and was designed in part to increase nursing home nurse staffing. It is estimated that this methodology change moved California from the bottom 10% of Medicaid nursing home reimbursement rates nationwide to the top 25%. This study analyzed the effect of AB 1629 on a panel of 567 free-standing nursing homes that were in continuous operation between the years 2002 – 2007. Resource Dependence Theory was used to construct the conceptual framework. Ordinary least squares (OLS) and first differencing with instrumental variable estimation procedures were used to test five hypotheses concerning Medi-Cal resource dependence, bed size, competition (including assisted living facilities and home health agencies), resource munificence, and slack resources. Both a 15 and 25 mile fixed radius were used as alternative market definitions instead of counties. The OLS results supported that case-mix adjusted licensed vocational nurse (LVN) and total nurse staffing hours per resident day increased overall. Nursing homes with the highest Medi-Cal dependence increased only increased NA staffing more than nursing homes with the lowest Medi-Cal dependence post AB 1629. The fixed effects with instrumental variable estimation procedure provided marginal support that nursing homes with more home health agency competition, in a 15 mile market, had higher LVN staffing. This estimation procedure also supported that nursing homes with more slack resources (post AB 1629) increased nurse aide and total nurse staffing while nursing homes located in markets with a greater percentage of residents over the age of 85 had more nurse aide staffing

Control of PNG kinase, a key regulator of mRNA translation, is coupled to meiosis completion at egg activation

The oocyte-to-embryo transition involves extensive changes in mRNA translation, regulated in Drosophila by the PNG kinase complex whose activity we show here to be under precise developmental control. Despite presence of the catalytic PNG subunit and the PLU and GNU activating subunits in the mature oocyte, GNU is phosphorylated at Cyclin B/CDK1sites and unable to bind PNG and PLU. In vitro phosphorylation of GNU by CyclinB/CDK1 blocks activation of PNG. Meiotic completion promotes GNU dephosphorylation and PNG kinase activation to regulate translation. The critical regulatory effect of phosphorylation is shown by replacement in the oocyte with a phosphorylation-resistant form of GNU, which promotes PNG-GNU complex formation, elevation of Cyclin B, and meiotic defects consistent with premature PNG activation. After PNG activation GNU is destabilized, thus inactivating PNG. This short-lived burst in kinase activity links development with maternal mRNA translation and ensures irreversibility of the oocyteto- embryo transition.National Institutes of Health (U.S.) (GM39341)National Institutes of Health (U.S.) (GM118098

Divergence in transcriptional and regulatory responses to mating in male and female fruitflies

Mating induces extensive physiological, biochemical and behavioural changes in female animals of many taxa. In contrast, the overall phenotypic and transcriptomic consequences of mating for males, hence how they might differ from those of females, are poorly described. Post mating responses in each sex are rapidly initiated, predicting the existence of regulatory mechanisms in addition to transcriptional responses involving de novo gene expression. That post mating responses appear different for each sex also predicts that the genome-wide signatures of mating should show evidence of sex-specific specialisation. In this study, we used high resolution RNA sequencing to provide the first direct comparisons of the transcriptomic responses of male and female Drosophila to mating, and the first comparison of mating-responsive miRNAs in both sexes in any species. As predicted, the results revealed the existence of sex- and body part-specific mRNA and miRNA expression profiles. More genes were differentially expressed in the female head-thorax than the abdomen following mating, whereas the opposite was true in males. Indeed, the transcriptional profile of male head-thorax tissue was largely unaffected by mating, and no differentially expressed genes were detected at the most stringent significance threshold. A subset of ribosomal genes in females were differentially expressed in both body parts, but in opposite directions, consistent with the existence of body part-specific resource allocation switching. Novel, mating-responsive miRNAs in each sex were also identified, and a miRNA-mRNA interactions analysis revealed putative targets among mating-responsive genes. We show that the structure of genome-wide responses by each sex to mating is strongly divergent, and provide new insights into how shared genomes can achieve characteristic distinctiveness

De novo assembly of a transcriptome from the eggs and early embryos of Astropecten aranciacus

Starfish have been instrumental in many fields of biological and ecological research. Oocytes of Astropecten aranciacus, a common species native to the Mediterranean Sea and the East Atlantic, have long been used as an experimental model to study meiotic maturation, fertilization, intracellular Ca2+ signaling, and cell cycle controls. However, investigation of the underlying molecular mechanisms has often been hampered by the overall lack of DNA or protein sequences for the species. In this study, we have assembled a transcriptome for this species from the oocytes, eggs, zygotes, and early embryos, which are known to have the highest RNA sequence complexity. Annotation of the transcriptome identified over 32,000 transcripts including the ones that encode 13 distinct cyclins and as many cyclin-dependent kinases (CDK), as well as the expected components of intracellular Ca2+ signaling toolkit. Although the mRNAs of cyclin and CDK families did not undergo significant abundance changes through the stages from oocyte to early embryo, as judged by real-time PCR, the transcript encoding Mos, a negative regulator of mitotic cell cycle, was drastically reduced during the period of rapid cleavages. Molecular phylogenetic analysis using the homologous amino acid sequences of cytochrome oxidase subunit I from A. aranciacus and 30 other starfish species indicated that Paxillosida, to which A. aranciacus belongs, is not likely to be the most basal order in Asteroidea. Taken together, the first transcriptome we assembled in this species is expected to enable us to perform comparative studies and to design gene-specific molecular tools with which to tackle long-standing biological questions

Nature And Regulation Of Protein Phosphorylation Changes During Egg Activation In Drosophila Melanogaster

Mature oocytes are held in a developmentally-quiescent, arrested state. For development to occur, these oocytes must transition to a new cellular state that can support the processes of embryogenesis. This transition is achieved by the events of egg activation. My studies focused on protein phosphorylation changes that take place during egg activation in Drosophila. Because there is little or no transcription at this time, egg activation is directed by maternal mRNAs and proteins regulated through posttranscriptional and post-translational mechanisms. Phosphorylation is an abundant post-translational modification with a wide array of regulatory effects. In addition, phosphorylation regulators such as CaMKII and calcineurin are required for egg activation in a variety of organisms. We hypothesize that simultaneously changing the phosphorylation states of a large number of proteins is a key contributor to the cellular changes that encompass the oocyte-to-embryo transition. I applied two different proteomic methods, IMAC and 2D-gel electrophoresis, to identify the proteins that change in phosphorylation state between mature oocytes and unfertilized, activated eggs. This led to the identification of 311 proteins that are phospho-modulated during egg activation. I used RNAi to knock down the genes that encode some of these proteins, testing a total of 71 genes for effects on female fertility. I identified multiple candidates for future study including, mrityu, which is required for progression through the early rounds of embryonic mitosis. I also used the phosphorylation changes of two proteins identified from the proteomics experiments, Spindly and Vap-33-1, as "molecular markers" to examine how the egg activation genes sarah, cortex, and prage relate to the phosphorylation changes that take place at egg activation. I showed that all three genes are upstream of Spindly dephosphorylation, but only sarah and cortex are upstream of Vap-33-1 phosphorylation. These data, along with previous findings in the lab, suggest that sarah and cortex act in a common pathway. Overall, my studies have contributed to our understanding of the roles of protein phosphorylation during egg activation. My results show that phosphorylation is an important area of study if we are to discover the proteins and pathways that regulate the oocyte-to-embryo transition

Maternal Proteins That Are Phosphoregulated upon Egg Activation Include Crucial Factors for Oogenesis, Egg Activation and Embryogenesis in Drosophila melanogaster

Egg activation is essential for the successful transition from a mature oocyte to a developmentally competent egg. It consists of a series of events including the resumption and completion of meiosis, initiation of translation of some maternal mRNAs and destruction of others, and changes to the vitelline envelope. This major change of cell state is accompanied by large scale alteration in the oocyte’s phosphoproteome. We hypothesize that the cohort of proteins that are subject to phosphoregulation during egg activation are functionally important for processes before, during, or soon after this transition, potentially uniquely or as proteins carrying out essential cellular functions like those they do in other (somatic) cells. In this study, we used germline-specific RNAi to examine the function of 189 maternal proteins that are phosphoregulated during egg activation in Drosophila melanogaster. We identified 53 genes whose knockdown reduced or abolished egg production and caused a range of defects in ovarian morphology, as well as 51 genes whose knockdown led to significant impairment or abolishment of the egg hatchability. We observed different stages of developmental arrest in the embryos and various defects in spindle morphology and aberrant centrosome activities in the early arrested embryos. Our results, validated by the detection of multiple genes with previously-documented maternal effect phenotypes among the proteins we tested, revealed 15 genes with newly discovered roles in egg activation and early embryogenesis in Drosophila. Given that protein phosphoregulation is a conserved characteristic of this developmental transition, we suggest that the phosphoregulated proteins may provide a rich pool of candidates for the identification of important players in the egg-to-embryo transition