1,720,966 research outputs found
Towards the optimization of targeted systems for the in vitro culture of isolated bovine primordial follicles
No full textTo date, the production of live offspring through in vitro follicle culture from the ovarian reserve has only been achieved in mice, providing the proof-of-principle of the potential value of primordial follicles (PMF) as a source of fully grown oocytes. The ability to grow undifferentiated oocytes in vitro from PMF would increase the supply of fully grown oocytes destined for downstream applications in the livestock industry and fertility preservation programs. However, in large mammals, such as bovine, in vitro follicle culture systems to produce mature oocytes from PMF are still experimental due to high follicle mortality following isolation from the surrounding tissue. As evidenced in humans, isolated PMF undergo cell death after a short period in culture. While understanding the cause of follicle death is necessary to inhibit the induction of programmed cell death (PCD), it is also essential to elucidate the mechanisms responsible for activating PMF and steering them towards the next stage of development, i.e., primary follicles (PF). Mechanistically resolving both sides of the story - PMF death and development - would then provide insights into key factors responsible for deciding the fate of the PMF in culture. Condensation of this genetic repertoire would then optimize the current base culture system and improve the development of isolated bovine PMF to PF entirely in vitro. Heifer ovaries were collected from the abattoir and transported on ice to the laboratory in saline. PMF and PF were mechanically isolated, and PMF were cultured in a defined system. Follicle viability of freshly isolated (PMF T0) and PMF cultured for 16 (PMF T16) and 24 hours (PMF T24) were assessed. PMF T0, PMF T16, and PF T0 were subjected to bulk RNA sequencing, generating 50bp paired-end reads. Raw data were trimmed with TrimGalore to remove artificial constructs and low-quality bases. Trimmed data were mapped to the Bos taurus ARS-UCD1.3 transcriptome, and reads were quantified with Salmon. Differentially expressed genes (DEGs) between PMF T0 and PMF T16 were then obtained with DESeq2 (Wald’s Test). A chi-square test was performed to determine an association of the DEGs with PCD genes. DESeq2 (Likelihood Ratio Test) was further employed to perform an inter-follicle (PMF T0-PMF T16-PF T0) time course gene expression analysis followed by hierarchical clustering of significant genes (IFGEA) with the DEGreport package in R. We observed a significant reduction in PMF viability after 16 hours of culture, while no significant differences were observed between 16 and 24 hours (p<0.0001 and p=0.9753, respectively, two-way ANOVA followed by Tukey's test). Furthermore, we report the transcriptome profiles of freshly isolated bovine PMF and PF and 16-hour cultured PMF for the first time. PCA showed clear clustering of all the samples. 1949DEGs (adjusted p-value<0.1) were identified, which were determined to be associated with genes involved in ferroptosis and autophagy (chi-square test, p-value<0.05). IFGEA yielded 4 clusters of 1173, 1570, 162, and 22 genes (padj<0.05) with similar expression patterns in clusters 1-4, respectively. Our transcriptome analyses indicate that ferroptosis and autophagy are the elected PCD mechanisms bringing about PMF death in vitro and can be inhibited to nurture current culture systems. Based on comparison with PF, systems may be further optimized to allow the holistic development of isolated PMF entirely in vitro in the future. Work funded by H2020 MSCA-ITN-ETN n.860960 (EUROVA) and RL PSR2014-2020 No.202102146691 (R-INNOVA)
044 Gene expression profiling using next generation sequencing of primordial, primary and secondary bovine follicles
Get PDFDevelopment of an efficient and effective protocol for the isolation and culture of bovine primordial follicles
No full textBACKGROUND-AIM The ability to grow undifferentiated oocytes in vitro from primordial follicles (PMF) would increase the supply of fully grown oocytes, to be destined to downstream applications in the livestock industry and fertility preservation programs. To date, the production of living offspring using in vitro development of oocytes from the PMF reserve has only been achieved in mice, providing the proof of principle of the potential value of follicle culture as a source of fully grown oocytes. However, culture systems to produce mature oocytes from PMF are still experimental. One of the main limiting factor in PMFs in vitro culture’s efficiency is follicle death occurring shortly after isolation from surrounding tissue. Therefore, we hypothesize that counteracting the cell-death signaling network(s) triggered upon isolation from the surrounding ovarian cortex should improve the outcome of PMF in vitro culture. To pursue this objective, we started by developing a reliable and efficient protocol for the isolation of viable PMF from the bovine ovarian cortex. METHODS Fragments of ovarian cortex of about 2 cm2 and 1 mm thick removed from slaughter-derived heifers (14-22 months) and adult cows (48-60 months) were chopped up into small fragments with a blade, dispersed in manipulation medium and homogenized. The resultant homogenate was then passed through a serial sieves system up to the lower limit of 30 μm. In the first set of experiments, homogenization speed and time were optimized to reduce follicle damage and foam formation. The second set of experiments was conducted to evaluate the viability after 16 hrs of culture by live/death staining and TUNEL and Caspases 3/7 assay. RESULTS
The obtained results indicate that the number of follicles isolated from 2 cm2 and 1 mm thick ovarian cortex is inversely related to the age (mean of 100 vs. 40 from heifers and adult cows, respectively). Moreover, PMFs revealed a 30% decline in viability after culture compared to the freshly isolated ones. Finally, live/death staining showed two different patterns, suggestive of different modes of PMF cell death. CONCLUSIONS Our protocol can allow isolation of a high number of PMF per animal, higher than previously reported, and poses the basis for a morphological and functional characterization of isolated PMFs
Follicle-stroma interplay to unravel the mechanisms involved in bovine early folliculogenesis
No full textExploiting primordial follicle reserve to improve assisted reproductive technologies is still ineffective. So far, no defined culture system has been able to replicate the folliculogenesis in vitro due to the lack of knowledge of the mechanisms involved. During folliculogenesis, diverse and unique pathways control the fate of primordial follicles, from remaining dormant to undergoing cell death or activation. Morphological and functional changes occur within the follicles and interact with the ovarian stromal environment, particularly the ovarian cortex. The ovarian cortex surrounds the preantral follicles and mediates dialogue through paracrine factors. It is reported that the ovarian cortical matrix can influence the action of local paracrine biochemical signaling pathways to sustain the development of primordial, primary, and secondary follicles.
The present study aims to delineate the signaling networks involved in preantral follicle differentiation and the role of the ovarian cortex in guiding early folliculogenesis from the primordial to the secondary follicle stage. A bovine follicle dataset was generated in-house by mechanically isolating pools of primordial (n=3), primary (n=3), and secondary (n=3) follicles and subjecting them to 50bp paired-end bulk RNA sequencing on Illumina NextSeq2000. Sequenced data were trimmed with TrimGalore, mapped to the bovine transcriptome assembly ARS UCD 1.3, and quantified with Salmon. Differential gene expression analyses were performed with DESeq2 on R. Functional enrichment analyses were then conducted to identify overrepresented pathways. The transcriptome analysis of primordial, primary, and secondary follicles reveals an evident clustering of the samples via principal component analysis. Pairwise comparisons of the subsequent follicle stages, i.e., primordial-primary and primary-secondary, identified 1083 and 4596 significant differentially expressed genes (padj<0.1), respectively. Collectively, overrepresentation analyses of the differentially expressed genes revealed signaling pathways, such as PI3K-Akt, Wnt, mTOR, and cAMP, guiding the transitional phases. To explore preantral follicle gene expression dynamics, we simultaneously compared all three follicle categories using the Likelihood Ratio Test of R(DESeq2). Significant genes (padj<0.1) were subjected to hierarchical clustering. Four clusters were generated that contained 2030, 1527, 536, and 147 genes in clusters 1-4, respectively, showing distinct expression trends.
To better understand the interplay between preantral follicles and the ovarian cortex, a meta-analysis was then performed using the publicly available data (GSE147176), downloaded from GEO, processed and analyzed with the same pipeline described above. The metanalysis between the follicles and the ovarian cortex transcriptomic profiles shortlisted 2479 differentially expressed genes (padj<0.01) with substantial differences in expression levels (log2FoldChange <-4 and >4). Our preliminary data report key overrepresented pathways between follicles and cortex, such as focal adhesion. Further studies are needed to narrow down key regulators involved in follicle-stroma communication. However, the definition of pathways involved in follicle-cortex crosstalk may translate into effective systems for supporting early folliculogenesis in vitro. These analyses uncover the networks governing the development of follicles, from the primordial to the secondary stage, also by including the signal transduction from the ovarian cortical matrix. Indeed, understanding the support provided by the ovarian cortex during early folliculogenesis is essential for reconstructing 3D culture systems to sustain the growth of follicles in vitro. The in vitro development of preantral follicles can represent a significant achievement in mammalian fertility preservation plans to maximize the exploitation of the ovarian reserve, adding a step to assisted reproductive technologies.
Funded by MUR PRIN2020, No. 20209L8BN4 (InfinitEGG). NM funded by PON MUR 2014-2020
043 A transcriptomic approach towards the improvement of physiological systems for the in vitro culture of isolated bovine primordial follicles
Get PDF
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
- …
