Extracellular vesicles-coupled miRNAs from oviduct and uterus modulate signaling pathways related to lipid metabolism and bovine early embryo development.

BACKGROUND: Extracellular vesicles (EVs) present in oviductal (OF) and uterine fluid (UF) have been shown to enhance bovine embryo quality during in vitro culture by reducing lipid contents and modulating lipid metabolism-related genes (LMGs), while also influencing cell proliferation, suggesting their involvement on the regulation of different biological pathways. The regulation of signaling pathways related to cell differentiation, proliferation, and metabolism is crucial for early embryo development and can determine the success or failure of the pregnancy. Bioactive molecules within EVs in maternal reproductive fluids, such as microRNAs (miRNAs), may contribute to this regulatory process as they modulate gene expression through post-transcriptional mechanisms. RESULTS: From the 20 differentially expressed miRNAs, 19 up-regulated in UF-EVs (bta-miR-134, bta-miR-151-3p, bta-miR-155, bta-miR-188, bta-miR-181b, bta-miR-181d, bta-miR-224, bta-miR-23b-3p, bta-miR-24-3p, bta-miR-27a-3p, bta-miR-29a, bta-miR-324, bta-miR-326, bta-miR-345-3p, bta-miR-410, bta-miR-652, bta-miR-677, bta-miR-873 and bta-miR-708) and one (bta-miR-148b) in OF-EVs. These miRNAs were predicted to modulate several pathways such as Wnt, Hippo, MAPK, and lipid metabolism and degradation. Differences in miRNAs found in OF-EVs from the early luteal phase and UF-EVs from mid-luteal phase may reflect different environments to meet the changing needs of the embryo. Additionally, miRNAs may be involved, particularly in the uterus, in the regulation of embryo lipid metabolism, immune system, and implantation. This study evaluated miRNA cargo in OF-EVs from the early luteal phase and UF-EVs from the mid-luteal phase, coinciding with embryo transit within oviduct and uterus in vivo, and its possible influence on LMGs and signaling pathways crucial for early embryo development. A total of 333 miRNAs were detected, with 11 exclusive to OF, 59 to UF, and 263 were common between both groups. CONCLUSIONS: Our study suggests that miRNAs within OF- and UF-EVs could modulate bovine embryo development and quality, providing insights into the intricate maternal-embryonic communication that might be involved in modulating lipid metabolism, immune response, and implantation during early pregnancy.

High-content analysis identified synergistic drug interactions between INK128, an mTOR inhibitor, and HDAC inhibitors in a non-small cell lung cancer cell line.

BACKGROUND: The development of drug resistance is a major cause of cancer therapy failures. To inhibit drug resistance, multiple drugs are often treated together as a combinatorial therapy. In particular, synergistic drug combinations, which kill cancer cells at a lower concentration, guarantee a better prognosis and fewer side effects in cancer patients. Many studies have sought out synergistic combinations by small-scale function-based targeted growth assays or large-scale nontargeted growth assays, but their discoveries are always challenging due to technical problems such as a large number of possible test combinations. METHODS: To address this issue, we carried out a medium-scale optical drug synergy screening in a non-small cell lung cancer cell line and further investigated individual drug interactions in combination drug responses by high-content image analysis. Optical high-content analysis of cellular responses has recently attracted much interest in the field of drug discovery, functional genomics, and toxicology. Here, we adopted a similar approach to study combinatorial drug responses. RESULTS: By examining all possible combinations of 12 drug compounds in 6 different drug classes, such as mTOR inhibitors, HDAC inhibitors, HSP90 inhibitors, MT inhibitors, DNA inhibitors, and proteasome inhibitors, we successfully identified synergism between INK128, an mTOR inhibitor, and HDAC inhibitors, which has also been reported elsewhere. Our high-content analysis further showed that HDAC inhibitors, HSP90 inhibitors, and proteasome inhibitors played a dominant role in combinatorial drug responses when they were mixed with MT inhibitors, DNA inhibitors, or mTOR inhibitors, suggesting that recessive drugs could be less prioritized as components of multidrug cocktails. CONCLUSIONS: In conclusion, our optical drug screening platform efficiently identified synergistic drug combinations in a non-small cell lung cancer cell line, and our high-content analysis further revealed how individual drugs in the drug mix interact with each other to generate combinatorial drug response.

3D culture applied to reproduction in females: possibilities and perspectives.

In vitro cell culture is a well-established technique present in numerous laboratories in diverse areas. In reproduction, gametes, embryos, and reproductive tissues, such as the ovary and endometrium, can be cultured. These cultures are essential for embryo development studies, understanding signaling pathways, developing drugs for reproductive diseases, and in vitro embryo production (IVP). Although many culture systems are successful, they still have limitations to overcome. Three-dimensional (3D) culture systems can be close to physiological conditions, allowing greater interaction between cells and cells with the surrounding environment, maintenance of the cells' natural morphology, and expression of genes and proteins such as in vivo. Additionally, three-dimensional culture systems can stimulated extracellular matrix generating responses due to the mechanical force produced. Different techniques can be used to perform 3D culture systems, such as hydrogel matrix, hanging drop, low attachment surface, scaffold, levitation, liquid marble, and 3D printing. These systems demonstrate satisfactory results in follicle culture, allowing the culture from the pre-antral to antral phase, maintaining the follicular morphology, and increasing the development rates of embryos. Here, we review some of the different techniques of 3D culture systems and their applications to the culture of follicles and embryos, bringing new possibilities to the future of assisted reproduction.

Corpus luteum presence in the bovine ovary increase intrafollicular progesterone concentration: consequences in follicular cells gene expression and follicular fluid small extracellular vesicles miRNA contents.

BACKGROUND: It is well described that circulating progesterone (P4) plays a key role in several reproductive events such as oocyte maturation. However, during diestrus, when circulating P4 is at the highest concentrations, little is known about its local impact on the follicular cells such as intrafollicular P4 concentration due to corpus luteum (CL) presence within the same ovary. Based on that, our hypothesis is that the CL presence in the ovary during diestrus alters intrafollicular P4 concentrations, oocyte competence acquisition, follicular cells gene expression, and small extracellular vesicles (sEVs) miRNAs contents. RESULTS: P4 hormonal analysis revealed that ipsilateral to the CL follicular fluid (iFF) presented higher P4 concentration compared to contralateral follicular fluid (cFF). Furthermore, oocyte maturation and miRNA biogenesis pathways transcripts (ADAMTS-1 and AGO2, respectively) were increased in cumulus and granulosa cells of iFF, respectively. Nevertheless, a RT-PCR screening of 382 miRNAs showed that three miRNAs were upregulated and two exclusively expressed in sEVs from iFF and are predicted to regulate cell communication pathways. Similarly, seven miRNAs were higher and two exclusively expressed from cFF sEVs and are predicted to modulate proliferation signaling pathways. CONCLUSION: In conclusion, intrafollicular P4 concentration is influenced by the presence of the CL and modulates biological processes related to follicular cell development and oocyte competence, which may influence the oocyte quality. Altogether, these results are crucial to improve our knowledge about the follicular microenvironment involved in oocyte competence acquisition.

Laterality influence on gene expression of DNA damage repair in colorectal cancer.

Colorectal carcinoma (CRC) is the third most common malignancy worldwide, and second in number of deaths in the world. The molecular pathogenesis of CRC is heterogeneous and can affect several genes. Moreover, genomic instability is recognized as an important part of CRC carcinogenesis and is tightly connected to DNA damage response. DNA damage repair (DDR) pathways are intrinsically associated with cancer development and establishment. Traditionally, CRC is considered as one coherent disease, however, new evidence shows that left and right-sided CRC present differences observed in clinical settings, as well as in pre-clinical studies. Therefore, this study aimed to investigate the impact of DDR transcriptional profiles on survival in different sublocations of the colon and rectum using Cox regression, survival analysis and differential gene expression. Right side colon (RSC) has DDR genes' expression associated only with higher risk of death, while left side colon (LSC) and Rectum have most genes' expression associated with lower risk. The pattern is the same with survival analysis. All significant DDR genes had lower expression associated with better survival in RSC, as opposed to LSC and Rectum. Our results demonstrate that RSC is distinctively different from LSC and Rectum. LSC and Rectum have similar DDR expression profiles.

Contribution of lipids to the organelle differential profile of in vitro-produced bovine embryos.

Each living organism is unique because of the lipid identity of its organelles. The diverse distribution of these molecules also contributes to the role of each organelle in cellular activity. The lipid profiles of whole embryos are well documented in the literature. However, this approach can often lead to the loss of relevant information at the subcellular and consequently, metabolic levels, hindering a deeper understanding of key physiological processes during preimplantation development. Therefore, we aimed to characterize four organelles in vitro-produced bovine embryos: lipid droplets (LD), endoplasmic reticulum (ER), mitochondria (MIT), and nuclear membrane (NUC), and evaluate the contribution of the lipid species to each organelle evaluated. Expanded blastocysts were subjected to cell organelle isolation. Thereafter, lipid extraction from cell organelles and lipid analysis using the Multiple Reaction Monitoring (MRM) profiling method were performed. The LD and ER displayed a greater number of lipids (Phosphatidylcholine - PC, Ceramide - Cer, and Sphingomielin - SM) with high signal-to-noise intensities. This result is due to the high rate of biosynthesis, lipid distribution, and ability to store and recycle lipid species of these organelles. The NUC had a more distinct lipid profile than the other three organelles, with high relative intensities of PC, SM, and triacylglycerols (TG), which is consistent with its high nuclear activity. MIT had an intermediate profile that was close to that of LD and ER, which aligns with its autonomous metabolism for some classes of phospholipids (PL). Our study revealed the lipid composition of each organelle studied, and the roles of these lipids could be associated with the characteristic organellar activity. Our findings highlight the lipid species and classes that are relevant for the homeostasis and function of each associated organelle and provide tentative biomarkers for the determination of in vitro embryonic development and quality.

Bovine in vitro oocyte maturation and embryo culture in liquid marbles 3D culture system.

Despite the advances in in vitro embryo production (IVP) over the years, the technique still has limitations that need to be overcome. In cell cultures, it is already well established that three-dimensional culture techniques are more physiological and similar to the in vivo development. Liquid marble (LM) is a three-dimensional system based on the use of a hydrophobic substance to create in vitro microbioreactors. Thus, we hypothesized that the LM system improves bovine in vitro oocyte maturation and embryo culture. In experiment I, bovine cumulus-oocyte complexes (COCs) were placed for in vitro maturation for 22h in two different groups: control (conventional 2D culture) and LM (three-dimensional culture). We found that oocyte nuclear maturation was not altered by the LM system, however it was observed a decrease in expression of genes important in the oocyte maturation process in cumulus cells of LM group (BCL2, EIF4E, and GAPDH). In experiment II, the COCs were conventionally matured and fertilized, and for culture, they were divided into LM or control groups. There was a decrease in blastocyst rate and cell counting, a down-regulation of miR-615 expression, and an increase in the DNA global methylation and hydroxymethylation in embryos of LM group. Therefore, for the bovine in vitro embryo production, this specific three-dimensional system did not present the advantages that we expected, but demonstrated that the embryos changed their development and epigenetics according to the culture system.

Circulating Extracellular Vesicles microRNAs Are Altered in Women Undergoing Preterm Birth.

Preterm labor (PTL) and preterm premature rupture of membranes (PPROM) lead to high perinatal morbidity/mortality rates worldwide. Small extracellular vesicles (sEV) act in cell communication and contain microRNAs that may contribute to the pathogenesis of these complications. We aimed to compare the expression, in sEV from peripheral blood, of miRNAs between term and preterm pregnancies. This cross-sectional study included women who underwent PTL, PPROM, and term pregnancies, examined at the Botucatu Medical School Hospital, SP, Brazil. sEV were isolated from plasma. Western blot used to detect exosomal protein CD63 and nanoparticle tracking analysis were performed. The expression of 800 miRNAs was assessed by the nCounter Humanv3 miRNA Assay (NanoString). The miRNA expression and relative risk were determined. Samples from 31 women-15 preterm and 16 term-were included. miR-612 expression was increased in the preterm groups. miR-612 has been shown to increase apoptosis in tumor cells and to regulate the nuclear factor κB inflammatory pathway, processes involved in PTL/PPROM pathogenesis. miR-1253, miR-1283, miR378e, and miR-579-3p, all associated with cellular senescence, were downregulated in PPROM compared with term pregnancies. We conclude that miRNAs from circulating sEV are differentially expressed between term and preterm pregnancies and modulate genes in pathways that are relevant to PTL/PPROM pathogenesis.

High body energy reserve influences extracellular vesicles miRNA contents within the ovarian follicle.

Aiming to evaluate the effects of increased body energy reserve (BER) in Nellore cows' reproductive efficiency, cows were fed with different nutritional plans to obtain animals with high BER (HBER; Ad libitum diet) and moderate BER (MBER: cows fed 70% of HBER group ingestion). To evaluate the BER, cows were weekly weighted and evaluated for subcutaneous fat thickness and insulin serum concentration along the experimental period. At the end of the experimental period, animals were submitted to estrous synchronization and artificial insemination. Animals were slaughtered approximately 120 h after ovulation induction and the reproductive tracts were collected for embryo recovery and samples collection. Cumulus-oocyte-complexes (COC) and follicular fluid were collected from 3-6 mm in diameter ovarian follicles to perform miRNA analysis of cumulus cells (CC) and extracellular vesicles from follicular fluid (EV FF). As expected, differences were observed among MBER and HBER groups for body weight, fat thickness, and insulin serum concentration. HBER animals showed lower ovulation and embryo recovery rates compared to MBER animals. Different miRNAs were found among CC and EV FF within groups, suggesting that the BER may influence follicular communication. This suggests that small follicles (3-6 mm diameter) are already under BER effects, which may be greater on later stages of follicular development.

Adipose and amnion-derived mesenchymal stem cells: Extracellular vesicles characterization and implication for reproductive biotechnology.

The stem cell-based research for reproductive biotechnology has been widely studied and shows promise for repairing defective tissue or degenerated cells to treat different diseases. The adipose tissue and amniotic membrane have awakened great interest in regenerative medicine and arises as a promising source of mesenchymal stem cells. Both types, adipose and amniotic derived mesenchymal stem cells (AMSCs) are multipotent cells with an enhanced ability to differentiate into multiple lineages.. We aimed to evaluate the effect of basal supplementation of exosomes in cell cultures with canine amniotic mesenchymal stem cells (MSCs). Mesenchymal stem cells derived from canine amniotic and adipose tissue were isolated and cultured performing cell passages until 80-90% confluence was reached. The growth curve was determined and peak cell growth was observed in the second passage. The cells were then characterized and differentiated into adipogenic, chondrogenic and osteogenic lineages. Extracellular vesicles from amnion were isolated using an ultracentrifugation protocol and characterized by nanosight analysis. To evaluate their ability to improve cellular viability in naturally inefficient passages, exosomes were co-cultures to the MSC cells. The results showed a 15-20% increase in the expansion rate of cultures supplemented with vesicles extracted in the first and second passages when compared to the control group. Statistical analysis using the Dunnett test (p ≤ 0.05) corroborated this result, showing a positive correlation between supplementation and expansion rate. These results indicate not only the importance of exosomes in the cell communication process but also the feasibility of the culture supplementation protocol for therapeutic purposes. The potential of the AMSCs for reproductive biotechnology is undoubted, however, their application to repair reproductive disorders and the involved mechanisms remain elusive. The strategies to enable the Adipose Stem Cells and AMSCs application in reproductive biotechnology and optimize their use for tissue regeneration open new venues using exosomes interactions.

MicroRNA Profiling Using a PCR-Based Method.

MicroRNAs (miRNAs) are small non-coding RNA molecules involved in the post-transcriptional regulation of specific mRNA targets, thus possibly controlling many biological processes. The miRNA profiling analysis can contribute to understanding several signaling pathways, as biomarkers for molecular diagnostic, as well as potential to be used as therapeutic targets. The miRNAs expression can be analyzed by quantitative reverse transcription PCR (RT-qPCR), microarrays, and RNA sequencing. The RT-qPCR method is sensitive and specific and has a lower cost when compared to other techniques as microarrays and RNA sequencing. Therefore, the protocol presented in this chapter describes step by step all the details to perform miRNA analysis using primer-based RT-qPCR.

Micro-vibration results in vitro-derived bovine blastocysts with greater cryotolerance, epigenetic abnormalities, and a massive transcriptional change.

Much effort has been employed to improve the quality of embryos obtained by in vitro production (IVP) given the relevance of this technology to current livestock systems. In this context, dynamic IVP systems have proved beneficial to the embryo once they mimic fluid flows and mechanical forces resulting from the movement of ciliated cells and muscle contraction in the reproductive tract. In the present study, we sought to confirm these initial findings as well as assess potential molecular consequences to the embryo by applying micro-vibration (45 Hz for 5 s once per 60 min) during both oocyte maturation and embryo culture in cattle. As a result, micro-vibration led to lower incidence of apoptosis in blastocysts following vitrification-thawing. Further analyses revealed epigenetic and transcriptional changes in blastocysts derived from the micro-vibration treatment, with a total of 502 differentially expressed genes. Enrichment analyses linked differentially expressed genes to 'Oxidative phosphorylation', 'Cytokine-cytokine receptor interaction', and 'Signaling pathways regulating pluripotency of stem cells'. Yet, a meta-analysis indicated that the transcriptional changes induced by micro-vibration were not toward that of in vivo-derived embryos. In conclusion, micro-vibration increases the cryoresistance of bovine embryos, but caution should be taken given the unclear consequences of epigenetic and transcriptional abnormalities induced by the treatment.

Secretion pattern of canine amniotic stem cells derived extracellular vesicles.

Extracellular vesicles (EVs) derived from stem cells (SCs) have regenerative potential and the possibility of being used in treating chronic diseases. EVs present lower risk of tumorigenicity and easily to isolation and storage. Therefore, this research aims to compare the morphological characteristics of the EVs (up to 150nm) derived from stem cells obtained from canine amniotic membranes in different passages during the in vitro culture. For this, cells from the amniotic membranes were isolated, cultured, and characterized. In order to answer our aim, the number of cells was normalized at each passage to generate conditioned media for EVs separation. The cells were differentiated into adipogenic, chondrogenic, and osteogenic tissue, to characterize these cells as mesenchymal stem cells (MSC). Moreover, flow cytometry analysis was performed and showed that the MSC were positive for CD90, CD105 and negative for CD34, CD45, mesenchymal and hematopoietic markers, respectively. For EVs analysis, MSC in different passages (P0-P2) were culture until 80% of confluence, then the medium was replaced by EVs depleted medium. After 48h, culture medium was collected and centrifuged to separate EVs, followed by nanoparticle tracking analysis. The EVs were also characterized by western blot and transmission electron microscopy (TEM). EVs were positive for Alix and negative for Cytochrome C as well as presented the traditional cup-shape by transmission electronic microscopy. Our results demonstrated that the concentration in the different passages was increased in P0 compared to P1 and P2 (p<0.05). No differences were found in EVs size (P0=132nm, P1=130nm and P2=120nm). Together, these results demonstrate that P0 of MSC is enriched of EVs when compared to later passages, suggesting that this passage would be the best to be applied in pre-clinical tests. Despite that, more studies are necessary to identify the EVs content and how the cells will respond to treatment with them.

Biomimetic Nanovesicles-Sources, Design, Production Methods, and Applications.

Despite all the progress in the field of liposomes and nanoparticles for applications as drug and gene delivery systems, the specific targeting and immune system escape capabilities of these systems are still limited. Biomimetic nanovesicles emerged as a strategy to overcome these and other limitations associated with synthetic carriers, such as short circulation time, cytotoxicity, and difficulty in crossing biological barriers, since many of the desirable abilities of drug delivery systems are innate characteristics of biological vesicles. Thus, the question arises: would biomimetic nanovesicles be responsible for addressing these advances? It is currently known that biomimetic nanovesicles (BNV) can combine the intrinsic advantages of natural materials with the well-known production methods and controllability of synthetic systems. Besides, the development of the biotechnology and nanotechnology fields has provided a better understanding of the functionalities of biological vesicles and the means for the design and production of biomimetic nanovesicles (BNV). Based on this, this work will focus on tracking the main research on biomimetic nanovesicles (BNV) applied as drug and gene delivery systems, and for vaccines applications. In addition, it will describe the different sources of natural vesicles, the technical perspectives on obtaining them, and the possibility of their hybridization with synthetic liposomes.

Extracellular vesicles from oviductal and uterine fluids supplementation in sequential in vitro culture improves bovine embryo quality.

BACKGROUND: In vitro production of bovine embryos is a well-established technology, but the in vitro culture (IVC) system still warrants improvements, especially regarding embryo quality. This study aimed to evaluate the effect of extracellular vesicles (EVs) isolated from oviductal (OF) and uterine fluid (UF) in sequential IVC on the development and quality of bovine embryos. Zygotes were cultured in SOF supplemented with either BSA or EVs-depleted fetal calf serum (dFCS) in the presence (BSA-EV and dFCS-EV) or absence of EVs from OF (D1 to D4) and UF (D5 to D8), mimicking in vivo conditions. EVs from oviducts (early luteal phase) and uterine horns (mid-luteal phase) from slaughtered heifers were isolated by size exclusion chromatography. Blastocyst rate was recorded on days 7-8 and their quality was assessed based on lipid contents, mitochondrial activity and total cell numbers, as well as survival rate after vitrification. Relative mRNA abundance for lipid metabolism-related transcripts and levels of phosphorylated hormone-sensitive lipase (pHSL) proteins were also determined. Additionally, the expression levels of 383 miRNA in OF- and UF-EVs were assessed by qRT-PCR. RESULTS: Blastocyst yield was lower (P < 0.05) in BSA treatments compared with dFCS treatments. Survival rates after vitrification/warming were improved in dFCS-EVs (P < 0.05). EVs increased (P < 0.05) blastocysts total cell number in dFCS-EV and BSA-EV compared with respective controls (dFCS and BSA), while lipid content was decreased in dFCS-EV (P < 0.05) and mitochondrial activity did not change (P > 0.05). Lipid metabolism transcripts were affected by EVs and showed interaction with type of protein source in medium (PPARGC1B, LDLR, CD36, FASN and PNPLA2, P < 0.05). Levels of pHSL were lower in dFCS (P < 0.05). Twenty miRNA were differentially expressed between OF- and UF-EVs and only bta-miR-148b was increased in OF-EVs (P < 0.05). CONCLUSIONS: Mimicking physiological conditions using EVs from OF and UF in sequential IVC does not affect embryo development but improves blastocyst quality regarding survival rate after vitrification/warming, total cell number, lipid content, and relative changes in expression of lipid metabolism transcripts and lipase activation. Finally, EVs miRNA contents may contribute to the observed effects.

Obesity, inflammation, and cancer in dogs: Review and perspectives.

Obesity is the most common nutritional disease in dogs, and its prevalence has increased in recent decades. Several countries have demonstrated a prevalence of obesity in dogs similar to that observed in humans. Chronic low-grade inflammation is a prominent basis used to explain how obesity results in numerous negative health consequences. This is well known and understood, and recent studies have pointed to the association between obesity and predisposition to specific types of cancers and their complications. Such elucidations are important because, like obesity, the prevalence of cancer in dogs has increased in recent decades, establishing cancer as a significant cause of death for these animals. In the same way, intensive advances in technology in the field of human and veterinary medicine (which even proposes the use of animal models) have optimized existing therapeutic methods, led to the development of innovative treatments, and shortened the time to diagnosis of cancer. Despite the great challenges, this review aims to highlight the evidence obtained to date on the association between obesity, inflammation, and cancer in dogs, and the possible pathophysiological mechanisms that link obesity and carcinogenesis. The potential to control cancer in animals using existing knowledge is also presented.

Evaluation of circulating extracellular vesicles and miRNA in neutered and obese female dogs.

Adipose tissue is a metabolic and endocrine organ, and its adipocytes can synthesize and secrete extracellular vesicles (EVs), thus allowing intercellular communication. EVs are nanoparticles that transport lipids, proteins, metabolites, and nucleic acids (mRNA and microRNAs). MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression. miR-132, miR-26b, and miR-155 are associated with obesity, lipid metabolism and adipogenesis. The aim of this study was to evaluate the enriched EVs fraction containing miRNAs (miR-132, miR-26b, and miR-155) in serum from obese female dogs. Thirty-two neutered females in good general condition were recruited, including 21 obese and 11 healthy controls. The initial evaluation of the females included a general physical examination and laboratory tests. Small EVs (sEVs) were isolated from whole blood by serial centrifugation and ultracentrifugation, and nanoparticle analysis was used to determine the size and concentration of serum sEVs. miRNAs were extracted from sEVs enriched fraction and analyzed by real-time polymerase chain reaction. Obese female dogs with hypertriglyceridemia showed an increase in the sEVs concentration and in the expression of miR-132 and miR-26b in sEVs enriched fraction. No changes were observed in the group of obese female dogs with normal serum biochemical profile and in relation to miR-155 expression. These results suggest that obese female dogs with hypertriglyceridemia may present alterations in sEVs and in the expression of miRNAs related to lipid metabolism and adipogenesis.

Cellular responses and microRNA profiling in bovine spermatozoa under heat shock.

In brief: Elevated temperatures disturbed sperm physiology. Bovine sperm cells exposed to heat shock led to diminished mitochondrial activity, fertilizing ability, increased oxidative stress and caspase activity concomitant with a delay in embryonic developmental kinetics and modulation of sperm-borne microRNAsmiRNAs. Abstract: Sperm function is susceptible to adverse environmental conditions. It has been demonstrated that in vivo and in vitro exposure of bovine sperm to elevated temperature reduces sperm motility and fertilizing potential. However, the cascade of functional, cellular, and molecular events triggered by elevated temperature in the mature sperm cell remains not fully understood. Therefore, the aim of this study was to determine the effect of heat shock on mature sperm cells. Frozen-thawed Holstein sperm were evaluated immediately after Percoll purification (0 h non-incubation control) or after incubation at 35, 38.5, and 41°C for 4 h. Heat shock reduced sperm motility after 3-4 h at 41°C while mitochondrial activity was reduced by 38.5 and 41°C when compared to the control. Heat shock also increased sperm reactive oxygen species production and caspase activity. Heat-shocked sperm had lower fertilizing ability, which led to diminished cleavage and blastocyst rates. Preimplantation embryo developmental kinetics was also slowed and reduced by sperm heat shock. The microRNA (miR) profiling identified >300 miRs in bovine sperm. Among these, three and seven miRs were exclusively identified in sperm cells exposed to 35 and 41°C, respectively. Moreover, miR-181d was enriched in sperm cells exposed to higher temperatures. Hence, elevated temperature altered the physiology of mature sperm cells by perturbing cellular processes and the miR profile, which collectively led to lower fertilizing ability and preimplantation development.

Characterization of histone lysine ß-hydroxybutyrylation in bovine tissues, cells, and cumulus-oocyte complexes.

Besides their canonical roles as energy sources, short-chain fatty acids act as metabolic regulators of gene expression through histone posttranslational modifications. Ketone body ß-hydroxybutyrate (BHB) causes a novel epigenetic modification, histone lysine ß-hydroxybutyrylation (Kbhb), which is associated with genes upregulated in starvation-responsive metabolic pathways. Dairy cows increase BHB in early lactation, and the effects of this increase on cellular epigenomes are unknown. We searched for and identified that Kbhb is present in bovine tissues in vivo and confirmed that this epigenetic mark is responsive to BHB in bovine and human fibroblasts cultured in vitro in a dose-dependent manner. Maturation of cumulus-oocyte complexes with high concentrations of BHB did not affect the competence to complete meiotic maturation or to develop until the blastocyst stage. BHB treatment strongly induced H3K9bhb in cumulus cells, but faintly in oocytes. RNA-seq analysis in cumulus cells indicated that BHB treatment altered the expression of 345 genes. The downregulated genes were mainly involved in glycolysis and ribosome assembly pathways, while the upregulated genes were involved in mitochondrial metabolism and oocyte development. The genes and pathways altered by BHB will provide entry points to carry out functional experiments aiming to mitigate metabolic disorders and improve fertility in cattle.