Characterization of the main steps in first shell formation in Mytilus galloprovincialis: possible role of tyrosinase

Bivalve biomineralization is a highly complex and organized process, involving several molecular components identified in adults and larval stages. However, information is still scarce on the ontogeny of the organic matrix before calcification occurs. In this work, first shell formation was investigated in the mussel Mytilus galloprovincialis. The time course of organic matrix and CaCO3 deposition were followed at close times post fertilization (24, 26, 29, 32, 48 h) by calcofluor and calcein staining, respectively. Both components showed an exponential trend in growth, with a delay between organic matrix and CaCO3 deposition. mRNA levels of genes involved in matrix deposition (chitin synthase; tyrosinase- TYR) and calcification (carbonic anhydrase; extrapallial protein) were quantified by qPCR at 24 and 48 hours post fertilization (hpf) with respect to eggs. All transcripts were upregulated across early development, with TYR showing highest mRNA levels from 24 hpf. TYR transcripts were closely associated with matrix deposition as shown by in situ hybridization. The involvement of tyrosinase activity was supported by data obtained with the enzyme inhibitor N-phenylthiourea. Our results underline the pivotal role of shell matrix in driving first CaCO3 deposition and the importance of tyrosinase in the formation of the first shell in M. galloprovincialis

Adult Body Weight Is Programmed by a Redox-Regulated and Energy-Dependent Process during the Pronuclear Stage in Mouse

In mammals fertilization triggers a series of Ca2+ oscillations that not only are essential for events of egg activation but also stimulate oxidative phosphorylation. Little is known, however, about the relationship between quantitative changes in egg metabolism and specific long-term effects in offspring. This study assessed whether post-natal growth is modulated by early transient changes in NAD(P)H and FAD2+ in zygotes. We report that experimentally manipulating the redox potential of fertilized eggs during the pronuclear (PN) stage affects post-natal body weight. Exogenous pyruvate induces NAD(P)H oxidation and stimulates mitochondrial activity with resulting offspring that are persistently and significantly smaller than controls. Exogenous lactate stimulates NAD+ reduction and impairs mitochondrial activity, and produces offspring that are smaller than controls at weaning but catch up after weaning. Cytosolic alkalization increases NAD(P)+ reduction and offspring of normal birth-weight become significantly and persistently larger than controls. These results constitute the first report that post-natal growth rate is ultimately linked to modulation of NAD(P)H and FAD2+ concentration as early as the PN stage

Automatic Hotspots Detection for Intracellular Calcium Analysis in Fluorescence Microscopic Videos

In recent years, life-cell imaging techniques and their software applications have become powerful tools to investigate complex biological mechanisms such as calcium signalling. In this paper, we propose an automated framework to detect areas inside cells that show changes in their calcium concentration i.e. the regions of interests or hotspots, based on videos taken after loading living mouse cardiomyocytes with fluorescent calcium reporter dyes. The proposed system allows an objective and efficient analysis through the following four key stages: (1) Pre-processing to enhance video quality, (2) First level segmentation to detect candidate hotspots based on adaptive thresholding on the frame level, (3) Second-level segmentation to fuse and identify the best hotspots from the entire video by proposing the concept of calcium fluorescence hit-ratio, and (4) Extraction of the changes of calcium fluorescence over time per hotspot. From the extracted signals, different measurements are calculated such as maximum peak amplitude, area under the curve, peak frequency, and inter-spike interval of calcium changes. The system was tested using calcium imaging data collected from Heart muscle cells. The paper argues that the automated proposal offers biologists a tool to speed up the processing time and mitigate the consequences of inter-intra observer variability

Mitochondrial Physiology and Gene Expression Analyses Reveal Metabolic and Translational Dysregulation in Oocyte-Induced Somatic Nuclear Reprogramming

While reprogramming a foreign nucleus after somatic cell nuclear transfer (SCNT), the enucleated oocyte (ooplasm) must signal that biomass and cellular requirements changed compared to the nucleus donor cell. Using cells expressing nuclear-encoded but mitochondria-targeted EGFP, a strategy was developed to directly distinguish maternal and embryonic products, testing ooplasm demands on transcriptional and post-transcriptional activity during reprogramming. Specifically, we compared transcript and protein levels for EGFP and other products in pre-implantation SCNT embryos, side-by-side to fertilized controls (embryos produced from the same oocyte pool, by intracytoplasmic injection of sperm containing the EGFP transgene). We observed that while EGFP transcript abundance is not different, protein levels are significantly lower in SCNT compared to fertilized blastocysts. This was not observed for Gapdh and Actb, whose protein reflected mRNA. This transcript-protein relationship indicates that the somatic nucleus can keep up with ooplasm transcript demands, whilst transcription and translation mismatch occurs after SCNT for certain mRNAs. We further detected metabolic disturbances after SCNT, suggesting a place among forces regulating post-transcriptional changes during reprogramming. Our observations ascribe oocyte-induced reprogramming with previously unsuspected regulatory dimensions, in that presence of functional proteins may no longer be inferred from mRNA, but rather depend on post-transcriptional regulation possibly modulated through metabolism

Release of sICAM-1 in Oocytes and In Vitro Fertilized Human Embryos

Background: During the last years, several studies have reported the significant relationship between the production of soluble HLA-G molecules (sHLA-G) by 48–72 hours early embryos and an increased implantation rate in IVF protocols. As consequence, the detection of HLA-G modulation was suggested as a marker to identify the best embryos to be transferred. On the opposite, no suitable markers are available for the oocyte selection. Methodology/Principal Findings: The major finding of the present paper is that the release of ICAM-1 might be predictive of oocyte maturation. The results obtained are confirmed using three independent methodologies, such as ELISA, Bio-Plex assay and Western blotting. The sICAM-1 release is very high in immature oocytes, decrease in mature oocytes and become even lower in in vitro fertilized embryos. No significant differences were observed in the levels of sICAM-1 release between immature oocytes with different morphological characteristics. On the contrary, when the mature oocytes were subdivided accordingly to morphological criteria, the mean sICAM-I levels in grade 1 oocytes were significantly decreased when compared to grade 2 and 3 oocytes. Conclusions/Significance: The reduction of the number of fertilized oocytes and transferred embryos represents the main target of assisted reproductive medicine. We propose sICAM-1 as a biochemical marker for oocyte maturation and grading

Parental and Grandparental Ages in the Autistic Spectrum Disorders: A Birth Cohort Study

Background: A number of studies have assessed ages of parents of children with autistic spectrum disorders (ASD), and reported both maternal and paternal age effects. Here we assess relationships with grandparental ages. Methods and Findings: We compared the parental and grandparental ages of children in the population-based Avon Longitudinal Study of Parents and Children (ALSPAC), according to their scores in regard to 4 autistic trait measures and whether they had been given a diagnosis of ASD. Mean maternal and paternal ages of ASD cases were raised, but this appears to be secondary to a maternal grandmother age effect (P = 0.006): OR = 1.66[95%CI 1.16, 2.37] for each 10-year increase in the grandmother’s age at the birth of the mother. Trait measures also revealed an association between the maternal grandmother’s age and the major autistic trait–the Coherence Scale (regression coefficient b = 0.142, [95%CI = 0.057, 0.228]P = 0.001). After allowing for confounders the effect size increased to b = 0.217[95%CI 0.125, 0.308](P,0.001) for each 10 year increase in age. Conclusions: Although the relationship between maternal grandmother’s age and ASD and a major autistic trait was unexpected, there is some biological plausibility, for the maternal side at least, given that the timing of female meiosis I permits direct effects on the grandchild’s genome during the grandmother’s pregnancy. An alternative explanation is the meiotic mismatch methylation (3 M) hypothesis, presented here for the first time. Nevertheless the findings should b

Age-Associated Metabolic and Morphologic Changes in Mitochondria of Individual Mouse and Hamster Oocytes

Background: In human oocytes, as in other mammalian ova, there is a significant variation in the pregnancy potential, with approximately 20% of oocyte-sperm meetings resulting in pregnancies. This frequency of successful fertilization decreases as the oocytes age. This low proportion of fruitful couplings appears to be influenced by changes in mitochondrial structure and function. In this study, we have examined mitochondrial biogenesis in both hamster (Mesocricetus auratus) and mouse (Mus musculus) ova as models for understanding the effects of aging on mitochondrial structure and energy production within the mammalian oocyte. Methodology/Principal Findings: Individual metaphase II oocytes from a total of 25 young and old mice and hamsters were collected from ovarian follicles after hormone stimulation and prepared for biochemical or structural analysis. Adenosine triphosphate levels and mitochondrial DNA number were determined within individual oocytes from young and old animals. In aged hamsters, oocyte adenosine triphosphate levels and mitochondrial DNA molecules were reduced 35.4% and 51.8%, respectively. Reductions of 38.4% and 44% in adenosine triphosphate and mitochondrial genomes, respectively, were also seen in aged mouse oocytes. Transmission electron microscopic (TEM) analysis showed that aged rodent oocytes had significant alterations in mitochondrial and cytoplasmic lamellae structure. Conclusions/Significance: In both mice and hamsters, decreased adenosine triphosphate in aged oocytes is correlated with a similar decrease in mtDNA molecules and number of mitochondria. Mitochondria in mice and hamsters undergo significant morphological change with aging including mitochondrial vacuolization, cristae alterations, and changes in cytoplasmic lamellae

Elevated Non-Esterified Fatty Acid Concentrations during Bovine Oocyte Maturation Compromise Early Embryo Physiology

Elevated concentrations of serum non-esterified fatty acids (NEFA), associated with maternal disorders such as obesity and type II diabetes, alter the ovarian follicular micro-environment and have been associated with subfertility arising from reduced oocyte developmental competence. We have asked whether elevated NEFA concentrations during oocyte maturation affect the development and physiology of zygotes formed from such oocytes, using the cow as a model. The zygotes were grown to blastocysts, which were evaluated for their quality in terms of cell number, apoptosis, expression of key genes, amino acid turnover and oxidative metabolism. Oocyte maturation under elevated NEFA concentrations resulted in blastocysts with significantly lower cell number, increased apoptotic cell ratio and altered mRNA abundance of DNMT3A, IGF2R and SLC2A1. In addition, the blastocysts displayed reduced oxygen, pyruvate and glucose consumption, up-regulated lactate consumption and higher amino acid metabolism. These data indicate that exposure of maturing oocytes to elevated NEFA concentrations has a negative impact on fertility not only through a reduction in oocyte developmental capacity but through compromised early embryo quality, viability and metabolism

Gene Expression Rhythms in the Mussel Mytilus galloprovincialis (Lam.) across an Annual Cycle

Seasonal environmental changes may affect the physiology of Mytilus galloprovincialis (Lam.), an intertidal filter-feeder bivalve occurring commonly in Mediterranean and Atlantic coastal areas. We investigated seasonal variations in relative transcript abundance of the digestive gland and the mantle (gonads) of males and females. To identify gene expression trends – in terms of relative mRNA abundance- we used a medium-density cDNA microarray (1.7 K probes) in dual-color competitive hybridization analyses. Hierarchical clustering of digestive gland microarray data showed two main branches, distinguishing profiles associated with the “hot” months (May–August) from the other months. Genes involved in chitin metabolism, associated with mussel nutrition and digestion showed higher mRNA levels during summer. Moreover, we found different gene transcriptomic patterns in the digestive glands of males when compared to females, during the four stages of mussel gonadal development. Microarray data from gonadal transcripts also displayed clear patterns during the different developmental phases respect to the resting period (stage I) with peak relative mRNA abundance at the ripe phase (stage III) for both sexes. These data showed a clear temporal pattern in transcriptomic profiles of mussels sampled over an annual cycle. Physiological response to thermal variation, food availability, and reproductive status across months may contribute to variation in relative mRNA abundance