Effects of bovine ovarian storage conditions and vitrification on isolated preantral follicle viability.

Lay summary: The decreasing rate of successful pregnancies, both naturally and through assisted conception, has led to innovations in the way eggs, sperm, and embryos are stored. Despite these advances, the use of assisted reproductive techniques to preserve endangered or rare species remains unexplored. Since the location where samples are collected and facilities are often far apart, we aim to address part of this challenge by comparing different methods to store and handle ovarian tissue before freezing. This may pave the way for further research in preserving endangered species, despite the challenges posed by the distance between sample collection sites and suitable facilities.

The need for environmentally realistic studies on the health effects of terrestrial microplastics.

Plastic pollution is now so widespread that microplastics are regularly detected in biological samples surveyed for their presence. Despite their pervasiveness, very little is known about the effects of microplastics on the health of terrestrial vertebrates. While emerging studies are showing that microplastics represent a potentially serious threat to animal health, data have been limited to in vivo studies on laboratory rodents that were force fed plastics. The extent to which these studies are representative of the conditions that animals and humans might actually experience in the real world is largely unknown. Here, we review 114 papers from the peer-reviewed literature in order to understand how the concentrations and types of microplastics being administered to rodents in lab studies compare to those found in terrestrial soils. From 73 in vivo lab studies, and 41 soil studies, we found that lab studies have heretofore fed rodents microplastics at concentrations that were hundreds of thousands of times greater than they would be exposed to in nature. Furthermore, health effects have been studied for only 20% of the microplastic polymers that are known to occur in soils. Plastic pollution is arguably one of the most pressing ecological and public health issues of our time, yet existing lab-based research on the health effects of terrestrial microplastics does not reflect the conditions that free-ranging vertebrates are actually experiencing. Going forward, performing more true-to-life research will be of the utmost importance to fully understand the impacts of microplastics and maintain the public's faith in the scientific process. Supplementary Information: The online version contains supplementary material available at 10.1186/s43591-023-00059-1.

Mining RNAseq data reveals dynamic metaboloepigenetic profiles in human, mouse and bovine pre-implantation embryos.

Metaboloepigenetic regulation has been reported in stem cells, germ cells, and tumor cells. Embryonic metaboloepigenetics, however, have just begun to be described. Here we analyzed RNAseq data to characterize the metaboloepigenetic profiles of human, mouse, and bovine pre-implantation embryos. In embryos, metaboloepigenetic reprogramming was species-specific, varied with the developmental stage and was disrupted with in vitro culture. Metabolic pathways and gene expressions were strongly correlated with early embryo DNA methylation and were changed with in vitro culture. Although the idea that the in vitro environment may influence development is not new, there has been little progress on improving pregnancy rates after decades using in vitro fertilization. Hence, the present data will contribute to understanding how the in vitro manipulation affects the metaboloepigenetic status of early embryos, which can be used to establish culture strategies aimed at improving the in vitro environment and, consequently, pregnancy rates and offspring health.

Oviductal Extracellular Vesicles Improve Post-Thaw Sperm Function in Red Wolves and Cheetahs.

Artificial insemination (AI) is a valuable tool for ex situ wildlife conservation, allowing the re-infusion and dissemination of genetic material, even after death of the donor. However, the application of AI to species conservation is still limited, due mainly to the poor survival of cryopreserved sperm. Recent work demonstrated that oviductal extracellular vesicles (oEVs) improved cat sperm motility and reduced premature acrosomal exocytosis. Here, we build on these findings by describing the protein content of dog and cat oEVs and investigating whether the incubation of cryopreserved red wolf and cheetah sperm with oEVs during thawing improves sperm function. Both red wolf and cheetah sperm thawed with dog and cat oEVs, respectively, had more intact acrosomes than the non-EV controls. Moreover, red wolf sperm thawed in the presence of dog oEVs better maintained sperm motility over time (>15%) though such an improvement was not observed in cheetah sperm. Our work demonstrates that dog and cat oEVs carry proteins important for sperm function and improve post-thaw motility and/or acrosome integrity of red wolf and cheetah sperm in vitro. The findings show how oEVs can be a valuable tool for improving the success of AI with cryopreserved sperm in threatened species.

Follicular extracellular vesicles enhance meiotic resumption of domestic cat vitrified oocytes.

Extracellular vesicles (EVs) contain multiple factors that regulate cell and tissue function. However, understanding of their influence on gametes, including communication with the oocyte, remains limited. In the present study, we characterized the proteome of domestic cat (Felis catus) follicular fluid EVs (ffEV). To determine the influence of follicular fluid EVs on gamete cryosurvival and the ability to undergo in vitro maturation, cat oocytes were vitrified using the Cryotop method in the presence or absence of ffEV. Vitrified oocytes were thawed with or without ffEVs, assessed for survival, in vitro cultured for 26 hours and then evaluated for viability and meiotic status. Cat ffEVs had an average size of 129.3 ± 61.7 nm (mean ± SD) and characteristic doughnut shaped circular vesicles in transmission electron microscopy. Proteomic analyses of the ffEVs identified a total of 674 protein groups out of 1,974 proteins, which were classified as being involved in regulation of oxidative phosphorylation, extracellular matrix formation, oocyte meiosis, cholesterol metabolism, glycolysis/gluconeogenesis, and MAPK, PI3K-AKT, HIPPO and calcium signaling pathways. Furthermore, several chaperone proteins associated with the responses to osmotic and thermal stresses were also identified. There were no differences in the oocyte survival among fresh and vitrified oocyte; however, the addition of ffEVs to vitrification and/or thawing media enhanced the ability of frozen-thawed oocytes to resume meiosis. In summary, this study is the first to characterize protein content of cat ffEVs and their potential roles in sustaining meiotic competence of cryopreserved oocytes.

Author Correction: A dog oviduct-on-a-chip model of serous tubal intraepithelial carcinoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A dog oviduct-on-a-chip model of serous tubal intraepithelial carcinoma.

Ovarian cancer is the fifth cause of cancer-related mortality in women, with an expected 5-year survival rate of only 47%. High-grade serous carcinoma (HGSC), an epithelial cancer phenotype, is the most common malignant ovarian cancer. It is known that the precursors of HGSC originate from secretory epithelial cells within the Fallopian tube, which first develops as serous tubal intraepithelial carcinoma (STIC). Here, we used gene editing by CRISPR-Cas9 to knock out the oncogene p53 in dog oviductal epithelia cultured in a dynamic microfluidic chip to create an in vitro model that recapitulated human STIC. Similar to human STIC, the gene-edited oviduct-on-a-chip, exhibited loss of cell polarization and had reduced ciliation, increased cell atypia and proliferation, with multilayered epithelium, increased Ki67, PAX8 and Myc and decreased PTEN and RB1 mRNA expression. This study provides a biomimetic in vitro model to study STIC progression and to identify potential biomarkers for early detection of HGSC.

3D printed mold leachates in PDMS microfluidic devices.

The introduction of poly(dimethylsiloxane) (PDMS) and soft lithography in the 90's has revolutionized the field of microfluidics by almost eliminating the need for a clean-room environment for device fabrication. More recently, 3D printing has been introduced to fabricate molds for soft lithography, the only step for which a clean-room environment is still often necessary, to further support the rapid prototyping of PDMS microfluidic devices. However, toxicity of most of the commercial 3D printing resins has been established, and little is known regarding the potential for 3D printed molds to leak components into the PDMS that would, in turn, hamper cells and/or tissues cultured in the devices. In the present study, we investigated if 3D printed molds produced by stereolithography can leach components into PDMS, and compared 3D printed molds to their more conventional SU-8 counterparts. Different leachates were detected in aqueous solutions incubated in the resulting PDMS devices prepared from widely used PDMS pre-polymer:curing agent ratios (10:1, 15:1 and 20:1), and these leachates were identified as originating from resins and catalyst substances. Next, we explored the possibility to culture cells and tissues in these PDMS devices produced from 3D printed molds and after proper device washing and conditioning. Importantly, we demonstrated that the resulting PDMS devices supported physiological cultures of HeLa cells and ovarian tissues in vitro, with superior outcomes than static conventional cultures.

Exploiting Microfluidics for Extracellular Vesicle Isolation and Characterization: Potential Use for Standardized Embryo Quality Assessment.

Recent decades have seen a growing interest in the study of extracellular vesicles (EVs), driven by their role in cellular communication, and potential as biomarkers of health and disease. Although it is known that embryos secrete EVs, studies on the importance of embryonic EVs are still very limited. This limitation is due mainly to small sample volumes, with low EV concentrations available for analysis, and to laborious, costly and time-consuming procedures for isolating and evaluating EVs. In this respect, microfluidics technologies represent a promising avenue for optimizing the isolation and characterization of embryonic EVs. Despite significant improvements in microfluidics for EV isolation and characterization, the use of EVs as markers of embryo quality has been held back by two key challenges: (1) the lack of specific biomarkers of embryo quality, and (2) the limited number of studies evaluating the content of embryonic EVs across embryos with varying developmental competence. Our core aim in this review is to identify the critical challenges of EV isolation and to provide seeds for future studies to implement the profiling of embryonic EVs as a diagnostic test for embryo selection. We first summarize the conventional methods for isolating EVs and contrast these with the most promising microfluidics methods. We then discuss current knowledge of embryonic EVs and their potential role as biomarkers of embryo quality. Finally, we identify key ways in which microfluidics technologies could allow researchers to overcome the challenges of embryonic EV isolation and be used as a fast, user-friendly tool for non-invasive embryo selection.

Potential Health and Environmental Risks of Three-Dimensional Engineered Polymers.

Polymer engineering, such as in three-dimensional (3D) printing, is rapidly gaining popularity, not only in the scientific and medical fields but also in the community in general. However, little is known about the toxicity of engineered materials. Therefore, we assessed the toxicity of 3D-printed and molded parts from five different polymers commonly used for prototyping, fabrication of organ-on-a-chip platforms, and medical devices. Toxic effects of PIC100, E-Shell200, E-Shell300, polydimethylsiloxane, and polystyrene (PS) on early bovine embryo development, on the transactivation of estrogen receptors were assessed, and possible polymer-leached components were identified by mass spectrometry. Embryo development beyond the two-cell stage was inhibited by PIC100, E-Shell200, and E-Shell300 and correlated to the released amount of diethyl phthalate and polyethylene glycol. Furthermore, all polymers (except PS) induced estrogen receptor transactivation. The released materials from PIC100 inhibited embryo cleavage across a confluent monolayer culture of oviduct epithelial cells and also inhibited oocyte maturation. These findings highlight the need for cautious use of engineered polymers for household 3D printing and bioengineering of culture and medical devices and the need for the safe disposal of used devices and associated waste.