Extracellular vesicles secreted by cumulus cells contain microRNAs that are potential regulatory factors of mouse oocyte developmental competence.

The role of cumulus cells (CCs) in the acquisition of oocyte developmental competence is not yet fully understood. In a previous study, we matured cumulus-denuded fully-grown mouse oocytes to metaphase II (MII) on a feeder layer of CCs (FL-CCs) isolated from developmentally competent (FL-SN-CCs) or incompetent (FL-NSN-CCs) SN (surrounded nucleolus) or NSN (not surrounding nucleolus) oocytes, respectively. We observed that oocytes cultured on the former could develop into blastocysts, while those matured on the latter arrested at the 2-cell stage. To investigate the CC factors contributing to oocyte developmental competence, here we focused on the CCs' release into the medium of extracellular vesicles (EVs) and on their miRNA content. We found that, during the 15-h transition to MII, both FL-SN-CCs and FL-NSN-CCs release EVs that can be detected, by confocal microscopy, inside the zona pellucida (ZP) or the ooplasm. The majority of EVs are <200 nm in size, which is compatible with their ability to cross the ZP. Next-generation sequencing of the miRNome of FL-SN-CC versus FL-NSN-CC EVs highlighted 74 differentially expressed miRNAs, with 43 up- and 31 down-regulated. Although most of these miRNAs do not have known roles in the ovary, in silico functional analysis showed that seven of these miRNAs regulate 71 target genes with specific roles in meiosis resumption (N = 24), follicle growth (N = 23), fertilization (N = 1), and the acquisition of oocyte developmental competence (N = 23). Overall, our results indicate CC EVs as emerging candidates of the CC-to-oocyte communication axis and uncover a group of miRNAs as potential regulatory factors.

Oocyte vitrification for fertility preservation is an evolving practice requiring a new mindset: societal, technical, clinical, and basic science-driven evolutions.

Infertility is a condition with profound social implications. Indeed, it is not surprising that evolutions in both medicine and society affect the way in vitro fertilization is practiced. The keywords in modern medicine are the four principles, which implicitly involve a constant update of our knowledge and our technologies to fulfill the "prediction" and "personalization" tasks, and a continuous reshaping of our mindset in view of all relevant societal changes to fulfill the "prevention" and "participation" tasks. A worldwide aging population whose life priorities are changing requires that we invest in fertility education, spreading actionable information to allow women and men to make meaningful reproductive choices. Fertility preservation for both medical and nonmedical reasons is still very much overlooked in many countries worldwide, demanding a comprehensive update of our approach, starting from academia and in vitro fertilization laboratories, passing through medical offices, and reaching out to social media. Reproduction medicine should evolve from being a clinical practice to treat a condition to being a holistic approach to guarantee patients' reproductive health and well-being. Oocyte vitrification for fertility preservation is the perfect use case for this transition. This tool is acquiring a new identity to comply with novel indications and social needs, persisting technical challenges, brand-new clinical technologies, and novel revolutions coming from academia. This "views and reviews" piece aims at outlining the advancement of oocyte vitrification from all these tightly connected perspectives.

MALDI mass spectrometry imaging shows a gradual change in the proteome landscape during mouse ovarian folliculogenesis.

Our knowledge regarding the role proteins play in the mutual relationship among oocytes, surrounding follicle cells, stroma, and the vascular network inside the ovary is still poor and obtaining insights into this context would significantly aid our understanding of folliculogenesis. Here, we describe a spatial proteomics approach to characterize the proteome of individual follicles at different growth stages in a whole prepubertal 25-day-old mouse ovary. A total of 401 proteins were identified by nano-scale liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS/MS), 69 with a known function in ovary biology, as demonstrated by earlier proteomics studies. Enrichment analysis highlighted significant KEGG and Reactome pathways, with apoptosis, developmental biology, PI3K-Akt, epigenetic regulation of gene expression, and extracellular matrix organization being well represented. Then, correlating these data with the spatial information provided by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) on 276 follicles enabled the protein profiles of single follicle types to be mapped within their native context, highlighting 94 proteins that were detected throughout the secondary to the pre-ovulatory transition. Statistical analyses identified a group of 37 proteins that showed a gradual quantitative change during follicle differentiation, comprising 10 with a known role in follicle growth (NUMA1, TPM2), oocyte germinal vesicle-to-metaphase II transition (SFPQ, ACTBL, MARCS, NUCL), ovulation (GELS, CO1A2), and preimplantation development (TIF1B, KHDC3). The proteome landscape identified includes molecules of known function in the ovary, but also those whose specific role is emerging. Altogether, this work demonstrates the utility of performing spatial proteomics in the context of the ovary and offers sound bases for more in-depth investigations that aim to further unravel its spatial proteome.

Biomechanical forces and signals operating in the ovary during folliculogenesis and their dysregulation: implications for fertility.

BACKGROUND: Folliculogenesis occurs in the highly dynamic environment of the ovary. Follicle cyclic recruitment, neo-angiogenesis, spatial displacement, follicle atresia and ovulation stand out as major events resulting from the interplay between mechanical forces and molecular signals. Morphological and functional changes to the growing follicle and to the surrounding tissue are required to produce oocytes capable of supporting preimplantation development to the blastocyst stage. OBJECTIVE AND RATIONALE: This review will summarize the ovarian morphological and functional context that contributes to follicle recruitment, growth and ovulation, as well as to the acquisition of oocyte developmental competence. We will describe the changes occurring during folliculogenesis to the ovarian extracellular matrix (ECM) and to the vasculature, their influence on the mechanical properties of the ovarian tissue, and, in turn, their influence on the regulation of signal transduction. Also, we will outline how their dysregulation might be associated with pathologies such as polycystic ovary syndrome (PCOS), endometriosis or premature ovarian insufficiency (POI). Finally, for each of these three pathologies, we will highlight therapeutic strategies attempting to correct the altered biomechanical context in order to restore fertility. SEARCH METHODS: For each area discussed, a systematic bibliographical search was performed, without temporal limits, using PubMed Central, Web of Science and Scopus search engines employing the keywords extracellular matrix, mechanobiology, biomechanics, vasculature, angiogenesis or signalling pathway in combination with: ovary, oogenesis, oocyte, folliculogenesis, ovarian follicle, theca, granulosa, cumulus, follicular fluid, corpus luteum, meiosis, oocyte developmental competence, preimplantation, polycystic ovary syndrome, premature ovarian insufficiency or endometriosis. OUTCOMES: Through search engines queries, we yielded a total of 37 368 papers that were further selected based on our focus on mammals and, specifically, on rodents, bovine, equine, ovine, primates and human, and also were trimmed around each specific topic of the review. After the elimination of duplicates, this selection process resulted in 628 papers, of which 287 were cited in the manuscript. Among these, 89.2% were published in the past 22 years, while the remaining 8.0%, 2.4% or 0.3% were published during the 1990s, 1980s or before, respectively. During folliculogenesis, changes occur to the ovarian ECM composition and organization that, together with vasculature modelling around the growing follicle, are aimed to sustain its recruitment and growth, and the maturation of the enclosed oocyte. These events define the scenario in which mechanical forces are key to the regulation of cascades of molecular signals. Alterations to this context determine impaired folliculogenesis and decreased oocyte developmental potential, as observed in pathological conditions which are causes of infertility, such as PCOS, endometriosis or POI. WIDER IMPLICATIONS: The knowledge of these mechanisms and the rules that govern them lay a sound basis to explain how follicles recruitment and growth are modulated, and stimulate insights to develop, in clinical practice, strategies to improve follicular recruitment and oocyte competence, particularly for pathologies like PCOS, endometriosis and POI.

Maternal effect factors that contribute to oocytes developmental competence: an update.

Oocyte developmental competence is defined as the capacity of the female gamete to be fertilized and sustain development to the blastocyst stage. Epigenetic reprogramming, a correct cell division pattern, and an efficient DNA damage response are all critical events that, before embryonic genome activation, are governed by maternally inherited factors such as maternal-effect gene (MEG) products. Although these molecules are stored inside the oocyte until ovulation and exert their main role during fertilization and preimplantation development, some of them are already functioning during folliculogenesis and oocyte meiosis resumption. This mini review summarizes the crucial roles played by MEGs during oocyte maturation, fertilization, and preimplantation development with a direct/indirect effect on the acquisition or maintenance of oocyte competence. Our aim is to inspire future research on a topic with potential clinical perspectives for the prediction and treatment of female infertility.

Three-dimensional imaging and reconstruction of the whole ovary and testis: a new frontier for the reproductive scientist.

The 3D functional reconstruction of a whole organ or organism down to the single cell level and to the subcellular components and molecules is a major future scientific challenge. The recent convergence of advanced imaging techniques with an impressively increased computing power allowed early attempts to translate and combine 2D images and functional data to obtain in-silico organ 3D models. This review first describes the experimental pipeline required for organ 3D reconstruction: from the collection of 2D serial images obtained with light, confocal, light-sheet microscopy or tomography, followed by their registration, segmentation and subsequent 3D rendering. Then, we summarise the results of investigations performed so far by applying these 3D image analyses to the study of the female and male mammalian gonads. These studies highlight the importance of working towards a 3D in-silico model of the ovary and testis as a tool to gain insights into their biology during the phases of differentiation or adulthood, in normal or pathological conditions. Furthermore, the use of 3D imaging approaches opens to key technical improvements, ranging from image acquisition to optimisation and development of new processing tools, and unfolds novel possibilities for multidisciplinary research.

Cytoplasmic movements of the early human embryo: imaging and artificial intelligence to predict blastocyst development.

RESEARCH QUESTION: Can artificial intelligence and advanced image analysis extract and harness novel information derived from cytoplasmic movements of the early human embryo to predict development to blastocyst? DESIGN: In a proof-of-principle study, 230 human preimplantation embryos were retrospectively assessed using an artificial neural network. After intracytoplasmic sperm injection, embryos underwent time-lapse monitoring for 44 h. For comparison, standard embryo assessment of each embryo by a single embryologist was carried out to predict development to blastocyst stage based on a single picture frame taken at 42 h of development. In the experimental approach, in embryos that developed to blastocyst or destined to arrest, cytoplasm movement velocity was recorded by time-lapse monitoring during the first 44 h of culture and analysed with a Particle Image Velocimetry algorithm to extract quantitative information. Three main artificial intelligence approaches, the k-Nearest Neighbour, the Long-Short Term Memory Neural Network and the hybrid ensemble classifier were used to classify the embryos. RESULTS: Blind operator assessment classified each embryo in terms of ability to develop to blastocyst, with 75.4% accuracy, 76.5% sensitivity, 74.3% specificity, 74.3% precision and 75.4% F1 score. Integration of results from artificial intelligence models with the blind operator classification, resulted in 82.6% accuracy, 79.4% sensitivity, 85.7% specificity, 84.4% precision and 81.8% F1 score. CONCLUSIONS: The present study suggests the possibility of predicting human blastocyst development at early cleavage stages by detection of cytoplasm movement velocity and artificial intelligence analysis. This indicates the importance of the dynamics of the cytoplasm as a novel and valuable source of data to assess embryo viability.

Three-Dimensional Micro-Computed Tomography of the Adult Mouse Ovary.

In the mouse ovary, folliculogenesis proceeds through eight main growth stages, from small primordial type 1 (T1) to fully grown antral T8 follicles. Most of our understanding of this process was obtained with approaches that disrupted the ovary three-dimensional (3D) integrity. Micro-Computed Tomography (microCT) allows the maintenance of the organ structure and a true in-silico 3D reconstruction, with cubic voxels and isotropic resolution, giving a precise spatial mapping of its functional units. Here, we developed a robust method that, by combining an optimized contrast procedure with microCT imaging of the tiny adult mouse ovary, allowed 3D mapping and counting of follicles, from pre-antral secondary T4 (53.2 ± 12.7 µm in diameter) to antral T8 (321.0 ± 21.3 µm) and corpora lutea, together with the major vasculature branches. Primordial and primary follicles (T1-T3) could not be observed. Our procedure highlighted, with unprecedent details, the main functional compartments of the growing follicle: granulosa, antrum, cumulus cells, zona pellucida, and oocyte with its nucleus. The results describe a homogeneous distribution of all follicle types between the ovary dorsal and ventral regions. Also, they show that each of the eight sectors, virtually segmented along the dorsal-ventral axis, houses an equal number of each follicle type. Altogether, these data suggest that follicle recruitment is homogeneously distributed all-over the ovarian surface. This topographic reconstruction builds sound bases for modeling follicles position and, prospectively, could contribute to our understanding of folliculogenesis dynamics, not only under normal conditions, but, importantly, during aging, in the presence of pathologies or after hormones or drugs administration.

Bacillus subtilis as a host for mosquitocidal toxins production.

Aedes albopictus transmits several arboviral infections. In the absence of vaccines, control of mosquito populations is the only strategy to prevent vector-borne diseases. As part of the search for novel, biological and environmentally friendly strategies for vector control, the isolation of new bacterial species with mosquitocidal activity represents a promising approach. However, new bacterial isolates may be difficult to grow and genetically manipulate. To overcome these limits, here we set up a system allowing the expression of mosquitocidal bacterial toxins in the well-known genetic background of Bacillus subtilis. As a proof of this concept, the ability of B. subtilis to express individual or combinations of toxins of Bacillus thuringiensis israelensis (Bti) was studied. Different expression systems in which toxin gene expression was driven by IPTG-inducible, auto-inducible or toxin gene-specific promoters were developed. The larvicidal activity of the resulting B. subtilis strains against second-instar Ae. albopictus larvae allowed studying the activity of individual toxins or the synergistic interaction among Cry and Cyt toxins. The expression systems here presented lay the foundation for a better improved system to be used in the future to characterize the larvicidal activity of toxin genes from new environmental isolates.