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Somatic cell nuclear transfer (SCNT) is a powerful tool to understand the mechanisms driving the cellular reprogramming, as well as addressing fundamental questions on developmental biology. Hundreds of animals have already been generated through this technology, however its large-scale use is still hampered by the low efficiency and disrupted phenotypes reported in SCNT derived newborns. Therefore, this review aims to expose some of the consequences often seen in cloned animals, as well as to discuss and propose some possibilities and perspectives to avoid epigenetic failures during reprogramming.

Developing embryos are susceptible to fluctuations in the nutrients and metabolites concentrations within the reproductive tract, which can lead to alterations in their developmental trajectory. Ketotic dairy cows have diminished fertility, and elevated levels of the ketone body beta-hydroxybutyrate (BHB) have been associated with poor embryonic development. We used an in vitro model based on either in vitro fertilization (IVF) or parthenogenesis to investigate the effects of BHB on the preimplantation bovine embryo development, epigenome, and transcriptome. Embryo culture medium was supplemented with BHB at a similar concentration to that present in the blood of cows suffering with severe ketosis, followed by analysis of blastocysts formation rate, diameter, total number of cells, levels of H3K9 beta-hydroxybutyrylation (H3K9bhb), apoptosis, and transcriptional alterations. As a result, we observed that BHB reduced the blastocysts rates, the diameter and the total number of cells in both parthenotes and IVF embryos. Exposure to BHB for either 3 or 7 days greatly increased the H3K9bhb levels in parthenotes at the 8-cells and blastocyst stages, and affected the expression of HDAC1, TET1, DNMT1, KDM6B, NANOG, and MTHFD2 genes. Additionally, culture of IVF embryos with BHB for 7 days dramatically increased H3K9bhb and reduced NANOG in blastocysts. RNA-seq analysis of IVF blastocysts revealed that BHB modulated the expression of 118 genes, which were involved with biological processes such as embryonic development, implantation, reproduction, proliferation, and metabolism. These findings provided valuable insights into the mechanisms through which BHB disrupts preimplantation embryonic development and affects the fertility in dairy cows., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background and Aims: Ovum pick-up (OPU) is an intrinsic step of in vitro fertilization procedures. Nevertheless, it can cause ovarian lesions and compromise female fertility in bovines. Recently, we have shown that intraovarian injection of adipose-derived mesenchymal stromal cells (AD-MSCs) effectively preserves ovarian function in bovines. Given that MSC-derived extracellular vesicles (MSC-EVs) have been shown to recapitulate several therapeutic effects attributed to AD-MSCs and that they present logistic and regulatory advantages compared to AD-MSCs, we tested whether MSC-EVs would also be useful to treat OPU-induced lesions., Methods: MSC-EVs were isolated from the secretome of bovine AD-MSCs, using ultrafiltration (UF) and ultracentrifugation methods. The MSC-EVs were characterized according to concentration and mean particle size, morphology, protein concentration and EV markers, miRNA, mRNA, long noncoding RNA profile, total RNA yield and potential for induction of the proliferation and migration of bovine ovarian stromal cells. We then investigated whether intraovarian injection of MSC-EVs obtained by UF would reduce the negative effects of acute OPU-induced ovarian lesions in bovines. To do so, 20 animals were divided into 4 experimental groups (n = 5), submitted to 4 OPU cycles and different experimental treatments including vehicle only (G1), MSC-EVs produced by 7.5 × 10 6 AD-MSCs (G2), MSC-EVs produced by 2.5 × 10 6 AD-MSCs (G3) or 3 doses of MSC-EVs produced by 2.5 × 10 6 AD-MSCs, injected after OPU sessions 1, 2 and 3 (G4)., Results: Characterization of the MSC-EVs revealed that the size of the particles was similar in the different isolation methods; however, the UF method generated a greater MSC-EV yield. MSC-EVs processed by both methods demonstrated a similar ability to promote cell migration and proliferation in ovarian stromal cells. Considering the higher yield and lower complexity of the UF method, UF-MSC-EVs were used in the in vivo experiment. We evaluated three therapeutic regimens for cows subjected to OPU, noting that the group treated with three MSC-EV injections (G4) maintained oocyte production and increased in vitro embryo production, compared to G1, which presented compromised embryo production following the OPU-induced lesions., Conclusions: MSC-EVs have beneficial effects both on the migration and proliferation of ovarian stromal cells and on the fertility of bovines with follicular puncture injury in vivo., Competing Interests: Declaration of competing interest Authors MP and PM declare competing financial interests as owners of the company Bio. All other authors declare that they have no conflicting interests., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

This study explored the migration of follicular fluid (FF)-derived extracellular vesicles (EVs) of the uterine environment to the bloodstream and their interaction with neutrophils in vivo and in vitro . For the in vivo experiment, six Nellore heifers ( Bos indicus ) received an intrauterine infusion seven days after ovulation with 1X PBS only (sham group; n=1), 1X PBS stained with lipophilic dye PKH26 (control group; n=2), or FF-derived EVs stained with PKH26 (treated group; n=3). Plasma was collected at 0, 10, 30, 60-, 180-, 360-, 720-, and 1440-min post-infusion to obtained EVs for analysis by nano flow cytometry. Labeled EVs were present in the bloodstream at 30- and 60-min post-infusion in the treatment group. Additionally, plasma derived-EVs from all groups were positive for Calcein-AM, Alix, Syntenin, and Calnexin, which confirm the presence of EVs. The second experiment utilized the plasma-derived EVs from the heifers from 30 and 60 min timepoints to evaluate if neutrophils can uptake EVs in vitro . As results, it was possible to observe the presence of labeled EVs in neutrophils treated with plasma derived-EVs from the treatment group. In summary, our results suggest that labeled EVs can migrate from the uterine environment rapidly and interact with circulating immune cells in bovine., Competing Interests: Conflicts of interest: The authors have no conflict of interest to declare., (Copyright © The Author(s).)

Multiple sclerosis is a chronic inflammatory disease of the central nervous system characterized by autoimmune destruction of the myelin sheath, leading to irreversible and progressive functional deficits in patients. Pre-clinical studies involving the use of neural stem cells (NSCs) have already demonstrated their potential in neuronal regeneration and remyelination. However, the exclusive application of cell therapy has not proved sufficient to achieve satisfactory therapeutic levels. Recognizing these limitations, there is a need to combine cell therapy with other adjuvant protocols. In this context, extracellular vesicles (EVs) can contribute to intercellular communication, stimulating the production of proteins and lipids associated with remyelination and providing trophic support to axons. This study aimed to evaluate the therapeutic efficacy of the combination of NSCs and EVs derived from oligodendrocyte precursor cells (OPCs) in an animal model of multiple sclerosis. OPCs were differentiated from NSCs and had their identity confirmed by gene expression analysis and immunocytochemistry. Exosomes were isolated by differential ultracentrifugation and characterized by Western, transmission electron microscopy and nanoparticle tracking analysis. Experimental therapy of C57BL/6 mice induced with experimental autoimmune encephalomyelitis (EAE) were grouped in control, treated with NSCs, treated with OPC-derived EVs and treated with a combination of both. The treatments were evaluated clinically using scores and body weight, microscopically using immunohistochemistry and immunological profile by flow cytometry. The animals showed significant clinical improvement and weight gain with the treatments. However, only the treatments involving EVs led to immune modulation, changing the profile from Th1 to Th2 lymphocytes. Fifteen days after treatment revealed a reduction in reactive microgliosis and astrogliosis in the groups treated with EVs. However, there was no reduction in demyelination. The results indicate the potential therapeutic use of OPC-derived EVs to attenuate inflammation and promote recovery in EAE, especially when combined with cell therapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Santos, Ortiz-Peñuela, de Paula Filho, Tomiyama, Coser, da Silveira, Martins, Ciena, de Oliveira and Ambrósio.)

During oocyte meiosis resumption, a coordinated program of transcript translation and decay machinery promotes a remodeling of mRNA stores, which determines the success of the acquisition of competence and early embryo development. We investigated levels of two genes related to mRNA translation ( CPEB1 and CPEB4 ) and two related to mRNA degradation ( CNOT7 and ZFP36L2 ) machinery and found ZFP36L2 downregulated in in vitro -matured bovine oocytes compared to in vivo counterparts. Thereafter, we tested the effects of a pre-IVM step with NPPC and a modified IVM with AREG on the modulation of members of mRNA translation and degradation pathways in cumulus cells and oocytes. Our data showed a massive upregulation of genes associated with translational and decay processes in cumulus cells, promoted by NPPC and AREG supplementation, up to 9h of IVM. The oocytes were less affected by NPPC and AREG, and even though ZFP36L2 transcript and protein levels were downregulated at 9 and 19h of IVM, only one ( KDM4C ) from the ten target genes evaluated was differently expressed in these treatments. These data suggest that cumulus cells are more prone to respond to NPPC and AREG supplementation in vitro , regarding translational and mRNA decay programs. Given the important nursing role of these cells, further studies could contribute to a better understanding of the impact of these modulators in maternal mRNA modulation and improve IVM outcomes., Competing Interests: Conflicts of interest: The authors have no conflict of interest to declare., (Copyright © The Author(s).)

Worldwide meat consumption and production have nearly quintupled in the last 60 years. In this context, research and the application of new technologies related to animal reproduction have evolved in an accelerated way. The objective of the present study was to apply nanoemulsions (NEs) as carriers of lipids to feed bovine embryos in culture media and verify their impact on the development of embryos produced in vitro . The NEs were characterized by particle size, polydispersity, size distribution, physical stability, morphology using atomic force microscopy (AFM), surface tension, density, pH, and rheological behavior. The NEs were prepared by the emulsification/evaporation technique. A central composite rotatable design (CCRD) was used to optimize the NE fabrication parameters. The three optimized formulations used in the embryo application showed an emulsion stability index (ESI) between 0.046 and 0.086, which reflects high stability. The mean droplet diameter analyzed by laser diffraction was approximately 70-80 nm, suggesting a possible transit across the embryonic zona pellucida with pores of an average 90 nm in diameter. AFM images clearly confirm the morphology of spherical droplets with a mean droplet diameter of less than 100 nm. The optimized formulations added during the higher embryonic genome activation phase in bovine embryos enhanced early embryonic development.

While cryopreservation of cauda epididymal sperm (SpCau) allows the preservation of post-mortem bulls' gametes, the process triggers sperm damage. Although improving post-thaw sperm quality, using egg yolk extenders (EY) raises biosafety concerns which forces the use of EY-free extenders (EYFE). Since EYFE are less efficient in preserving post-thaw sperm quality, a strategy for ejaculated sperm (SpEj) frozen with EYFE is to add an Equilibrium Time (ET) step period to the cryopreservation process. However, the ET effect on the quality of SpCau cryopreserved in EYFE remains unknown. Distinct from SpEJ, SpCau physiologically displays cytoplasmic droplets (CDs) in the flagellum that may benefit cell exchange during ET. We hypothesized that using ET in SpCau cryopreserved with EYFE impacts sperm morphofunctional features, CD area, and in vitro fertility ability. Extender nanoparticles were also assessed. Following collection from the cauda epididymis of six Nellore bulls by retrograde flow, SpCau were cryopreserved in EYFE BoviFree® (Minitube, Germany) using three ET protocols: ET0 (no-ET); ET2.5 (2.5 h-ET); and ET5 (5 h-ET). SpCau from ET2.5 and ET5 showed a higher (P ≤ 0.05) percentage of motility and integrity of plasma and acrosome membranes and a smaller (P ≤ 0.05) distal CD area. There are no differences in sperm abnormalities, oxidative stress, capacitation-like events, and in vitro fertility ability. However, a better sperm recovery was found after Percoll® selection for ET2.5 and ET5. Interestingly, the number of nanoparticles in the extender decreased in post-thawed samples. In conclusion, an ET of 2.5 or 5 h is required for an efficient SpCau cryopreservation using an EYFE., Competing Interests: Declaration of competing interest The authors affirm that they have no competing or conflicts of interest., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

We tested the hypothesis that the biosensor capability of the endometrium is mediated in part, by the effect of different cargo contained in the extracellular vesicles secreted by the conceptus during the peri-implantation period of pregnancy. We transferred Bos taurus taurus embryos of different origin, in vivo (high developmental potential (IV)), in vitro (intermediate developmental potential (IVF)), or cloned (low developmental potential (NT)), into Bos taurus indicus recipients. Extracellular vesicles (EVs) recovered from Day 16 conceptus-conditioned medium were characterized and their microRNA (miRNA) cargo sequenced alongside RNA sequencing of their respective endometria. There were substantial differences in the endometrial response to in vivo versus in vitro and in vivo versus cloned conceptuses (1153 and 334DEGs respectively) with limited differences between in vitro Vs cloned conceptuses (36 DEGs). The miRNA cargo contained in conceptus-derived EVs was similar between all three groups (426 miRNA in common). Only 8 miRNAs were different between in vivo and cloned conceptuses, while only 6 miRNAs were different between in vivo and in vitro-derived conceptuses. Treatment of endometrial epithelial cells with mimic or inhibitors for miR-128 and miR-1298 changed the proteomic content of target cells (96 and 85, respectively) of which mRNAs are altered in the endometrium in vivo (PLXDC2, COPG1, HSPA12A, MCM5, TBL1XR1, and TTF). In conclusion, we have determined that the biosensor capability of the endometrium is mediated in part, by its response to different EVs miRNA cargo produced by the conceptus during the peri-implantation period of pregnancy., (© 2024 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

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., Competing Interests: Conflicts of interest: The authors have no conflict of interest to declare.

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., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Ferronato et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Bastos et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., Competing Interests: Conflicts of interest: The authors have no conflict of interest to declare.

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., (© 2022. The Author(s).)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Marchi, Vendramini, Perini, Zafalon, Amaral, Ochamotto, Da Silveira, Dagli and Brunetto.)

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., (© 2022. The Author(s).)

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., Competing Interests: The authors declare no conflict of interest.

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.

Besides their canonical roles as energy sources, short-chain fatty acids act as metabolic regulators of gene expression through histone posttranslational modifications. Ketone body β-hydroxybutyrate (BHB) causes a novel epigenetic modification, histone lysine β-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., (© 2022 Wiley Periodicals LLC.)

Extracellular vesicles (EVs) found in various biological fluids and particularly in reproductive fluids, have gained considerable attention for their possible role in cell- to- cell communication. Among, the different bioactive molecules cargos of EVs, MicroRNAs (miRNAs) are emerging as promising diagnostic biomarkers with high clinical potential. Aiming to understand the roles of EVs in bovine reproductive tract, we intended to characterize and profile the EVs of oviduct and uterine fluids (OF-EVs, UF-EVs) and their miRNA across the estrous cycle. Nanoparticle tracking analysis and transmission electron microscopy confirmed the existence of small EV population in OF and UF at all stages, (size between 30 and 200 nm; concentration: 3.4 × 10 10  EVs/ml and 6.0 × 10 10  EVs/ml for OF and UF, respectively, regardless of stage). The identification of EV markers (CD9, HSP70, and ALIX proteins) was confirmed by western blot. The miRNA analysis revealed the abundance of 310 and 351 miRNAs in OF-EVs and UF-EVs, respectively. Nine miRNAs were differentially abundant in OF-EVs between stages of the cycle, eight of them displayed a progressive increase from S1 to S4 (p < .05). In UF-EVs, a total of 14 miRNAs were differentially abundant between stages. Greater differences were observed between stage 1 (S1) and stage 3 (S3), with 11 miRNAs enriched in S3 compared to S1. Functional enrichment analysis revealed the involvement of these miRNAs in relevant pathways such as cell signaling, intercellular junctions, and reproductive functions that may be implicated in oviduct and uterus modulation across the cycle, but also in their preparation for embryo/conceptus presence and development., (© 2021 Federation of American Societies for Experimental Biology.)

Cell communication within the ovarian follicle is crucial during folliculogenesis to assure an ideal environment for the oocyte to achieve full developmental competence. Intercellular communication is facilitated by the presence of follicular fluid, which mediates the transfer of signaling molecules. Recently, extracellular vesicles (exosomes and microvesicles) containing mRNAs, miRNAs and proteins were described in mammalian follicular fluid. Besides these molecules, extracellular vesicles (EVs) can mediate the transfer of lipids that can act as signal transducers activating second messengers and modulating intracellular pathways. Our goal was to determine the lipid profile of exosomes (small extracellular vesicles) and microvesicles (large extracellular vesicles) from bovine ovarian follicles containing oocytes with different developmental capabilities to verify potential relationships to competence. Using mass spectrometry, we examined the lipid content of EVs present in the follicular fluid of follicles enclosing oocytes that were either unable to cleave (NCLEAVE), arrested at cleavage stage (CLEAVE), or developed to the blastocyst stage (BLAST) after parthenogenetic activation. Although most of the 514 lipids identified in the follicular fluid EVs were common among all groups, 10 exosome-derived lipids and 15 microvesicle-derived lipids were present exclusively in the BLAST group, suggesting a potential relationship with developmental competence. Therefore, our data indicate that the EVs present in follicular fluid of antral follicles of similar morphology contain lipids that may be used as biomarkers associated with the developmental capability of the oocyte to develop to the blastocyst stage., Competing Interests: Declaration of competing interest The all authors declare that they have no competing interests. SCIEX of Brazil employs Helio A Martins-Júnior and he declares no conflict of interests or competing interests and financial disclosure that declare the affiliations to this company or marketed products related to this article., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In vivo- and in vitro-produced bovine embryos have different metabolic profiles and differences in gene transcription patterns. These embryos also have a distinct ability to establish and sustain early pregnancies. Small extracellular vesicles (sEVs) are secreted by embryos and carry bioactive molecules, such as miRNAs. We hypothesize that in vivo or in vitro-produced bovine hatched blastocysts on Day 9 and the sEVs secreted by them have different miRNA profiles. To address this hypothesis, embryos of both groups were placed in in vitro culture on Day 7. After 48 h, hatched embryos and hatched embryo-conditioned media (eCM) of both groups were collected. A total of 210 miRNAs were detected in embryos of both groups, of these 6 miRNAs were downregulated, while 7 miRNAs were upregulated in vitro group when compared to in vivo group. sEVs were isolated from eCM to determine miRNA profile. A total of 106 miRNAs were detected in both groups, including 14 miRNAs upregulated in sEVs from in vivo-eCM, and 2 miRNAs upregulated in sEVs from in vitro-eCM. These miRNAs express in embryos and sEVs secreted by them regulate early embryonic developmental and endometrial pathways, which can modify embryo-maternal communication during early pregnancy and consequently affect pregnancy establishment., (© 2021 Wiley Periodicals LLC.)

This review focuses on the innate immune events modulated by conceptus signaling during early pregnancy in ruminants. Interferon-tau (IFN-τ) plays a role in the recognition of pregnancy in ruminants, which involves more than the inhibition of luteolytic pulses of PGF2α to maintain corpus luteum function. For successful pregnancy establishment, the allogenic conceptus needs to prevent rejection by the female. Therefore, IFN-τ exerts paracrine and endocrine actions to regulate the innate immune system and prevent conceptus rejection. Additionally, other immune regulators work in parallel with IFN-τ, such as the pattern recognition receptors (PRR). These receptors are activated during viral and bacterial infections and in early pregnancy, but it remains unknown whether PPR expression and function are controlled by IFN-τ. Therefore, this review focuses on the main components of the innate immune response that are involved with early pregnancy and their importance to avoid conceptus rejection., Competing Interests: Conflict of interest: The authors have no conflict of interest to declare., (Copyright © The Author(s).)

Early embryonic development occurs in the oviduct, where an ideal microenvironment is provided by the epithelial cells and by the oviductal fluid produced by these cells. The oviductal fluid contains small extracellular vesicles (sEVs), which through their contents, including microRNAs (miRNAs), can ensure proper cell communication between the mother and the embryo. However, little is known about the modulation of miRNAs within oviductal epithelial cells (OECs) and sEVs from the oviductal fluid in pregnant cows. In this study, we evaluate the miRNAs profile in sEVs from the oviductal flushing (OF-sEVs) and OECs from pregnant cows compared to non-pregnant, at 120 h after ovulation induction. In OF-sEVs, eight miRNAs (bta-miR-126-5p, bta-miR-129, bta-miR-140, bta-miR-188, bta-miR-219, bta-miR-345-3p, bta-miR-4523, and bta-miR-760-3p) were up-regulated in pregnant and one miRNA (bta-miR-331-5p) was up-regulated in non-pregnant cows. In OECs, six miRNAs (bta-miR-133b, bta-miR-205, bta-miR-584, bta-miR-551a, bta-miR-1193, and bta-miR-1225-3p) were up-regulated in non-pregnant and none was up-regulated in pregnant cows. Our results suggest that embryonic maternal communication mediated by sEVs initiates in the oviduct, and the passage of gametes and the embryo presence modulate miRNAs contents of sEVs and OECs. Furthermore, we demonstrated the transcriptional levels modulation of selected genes in OECs in pregnant cows. Therefore, the embryonic-maternal crosstalk potentially begins during early embryonic development in the oviduct through the modulation of miRNAs in OECs and sEVs in pregnant cows., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mazzarella, Bastos, Bridi, del Collado, Andrade, Pinzon, Prado, Silva, Meirelles, Pugliesi, Perecin and da Silveira.)

Scrotal heat stress affects spermatogenesis and impairs male fertility by increasing sperm morphological abnormalities, oxidative stress and DNA fragmentation. While sperm morpho-functional changes triggered by scrotal heat stress are well described, sperm molecular alterations remain unknown. Recently, spermatozoa were described as accumulating miRNAs during the last steps of spermatogenesis and through epididymis transit, mainly by communication with small extracellular vesicles (sEVs). Herein, the aim was to investigate the impact of scrotal heat stress in miRNAs profile of sperm, as well as, seminal plasma sEVs. Six Nelore bulls (Bos indicus) were divided into two groups: Control (CON; n = 3) and Scrotal Heat Stress (SHS; n = 3; scrotal heat stressed during 96 h by scrotal bags). The day that the scrotal bags were removed from SHS group was considered as D0 (Day zero). Seminal plasma sEVs were isolated from semen samples collected seven days after heat stress (D+7) to evaluate sEVs diameter, concentration, and 380 miRNA levels. Sperm morpho-functional features and profile of 380 miRNAs were evaluated from semen collected 21 days after heat stress (D+21). As a control, sEVs and sperm were analyzed seven days before heat stress (D-7). Only semen parameters that were not significantly different (P > 0.05) among bulls on D-7 were addressed on D+7 and D+21. While no alterations in diameter and concentration were detected in sEVs on D+7 between CON and SHS groups, three sEVs-miRNAs (miR-23b-5p, -489 and -1248) were down-regulated in SHS bulls compared to CON on D+7; other three (miR-126-5p, -656 and -1307) displayed a tendency (0.05 < P < 0.10) to be altered. Sperm oxidative stress was higher, and the level of 21 sperm miRNAs was altered (18 down-, 3 up-regulated) in SHS bulls compared to CON on D+21. Functional analysis indicated that target genes involved in transcription activation, as well as cell proliferation and differentiation were related to the 18 down-regulated sperm miRNAs (miR-9-5p, -15a, -18a, -20b, -30a-5p, -30b-5p, -30d, -30e-5p -34b, -34c, -106b, -126-5p, -146a, -191, -192, -200b, -335 and -449a). Thus, the scrotal heat stress probably impacted testicular and epididymis functions by reducing the levels of a substantial proportion of sEVs and sperm miRNAs. Our findings suggest that miR-126-5p was possibly trafficked between sEVs and sperm and provide new insights on the mechanism by which sperm acquire miRNAs in the last stages of spermatogenesis and sperm maturation in cattle., Competing Interests: Declaration of competing interest The authors affirm that they have no competing or conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) are key regulators of gene expression, potentially affecting several biological processes, whose function can be altered by sequence variation. Hence, the integration of single nucleotide polymorphisms (SNP) and miRNAs can explain individual differences in economic traits. To provide new insights into the effects of SNPs on miRNAs and their related target genes, we carried out a multi-omic analysis to identify SNPs in miRNA mature sequences (miR-SNPs) associated with fatty acid (FA) composition in the Nelore cattle. As a result, we identified 3 miR-SNPs in different miRNAs (bta-miR-2419-3p, bta-miR-193a-2, and bta-miR-1291) significantly associated with FA traits ( p -value < 0.02, Bonferroni corrected). Among these, the rs110817643C>T, located in the seed sequence of the bta-miR-1291, was associated with different ω6 FAs, polyunsaturated FA, and polyunsaturated:saturated FA ratios. Concerning the other two miR-SNPs, the rs43400521T>C (located in the bta-miR-2419-3p) was associated with C12:0 and C18:1 cis-11 FA, whereas the rs516857374A>G (located in the bta-miR-193a-2) was associated with C18:3 ω6 and ratio of ω6/ω3 traits. Additionally, to identify potential biomarkers for FA composition, we described target genes affected by these miR-SNPs at the mRNA or protein level. Our multi-omics analysis outlines the effects of genetic polymorphism on miRNA, and it highlights miR-SNPs and target candidate genes that control beef fatty acid composition.

Extracellular vesicles (EVs) are vesicles released by cells, which due to their cargo and cell membrane proteins induce changes in the recipient cells. These vesicles can be a novel option to induce stem cell differentiation. Here we described a method to induce mesenchymal stem cell differentiation (MSC) into neuron-like cells using small EVs from neurons. First, we will describe a method based on neurons to induce adipocyte derived stem cells differentiation, a type of MSC, by coculturing both using inserts. Secondly, we will describe a follow-up method by using only isolated neuron-derived small EVs to directly induce ADSC differentiation in neuron-like cells. Importantly, in both methods it is possible to avoid the direct cell-to-cell contact, thus allowing for the study of soluble factors role during stem cell differentiation.

Intercellular communication can be carried out by circulating systemic and/or locally released extracellular vesicles (EVs), produced by nearly every cell type and tissue, and are involved in physiological and pathological processes. In recent years, EVs have been identified in reproductive tissues, such as oviduct and uterus, and have been shown to be related to several events important for reproductive success. The understanding of their functions in reproduction has important implications for assisted reproductive technologies, for the treatment of infertility in humans and improvement of reproduction efficiency in animals. To study such EVs, it is necessary to isolate and concentrate them from fluid samples, which in the case of reproductive tissues, are usually of limited volume. Several methods for EV isolation are available such as chromatography, ultracentrifugation, polymer-based precipitation, and immunoaffinity.Outcomes can be variable in terms of the amount and quality of isolated EVs, due to the type of isolation method. The choice of method, or a different combination of methods, may depend on the type of sample and scientific question to be addressed in a given study. In this chapter, we describe a method for isolation of EVs from bovine oviductal and uterine fluids for use in functional studies. The method combines size exclusion chromatography and ultracentrifugation. We also describe the different protocols for characterization of isolated EVs (transmission electron microscopy, nanoparticle tracking analysis, and western blot), as well as the isolation of RNA content in EVs, and their miRNAs profiling for functional studies.

We evaluated the effect of the antral follicle count (AFC) on ovarian follicular dynamics, pregnancy rates, progesterone concentrations, and transcriptional patterns of genes in Nelore cattle (Bos taurus indicus) after a timed artificial insemination (TAI) programme. Cows were separated based on the AFC, and those with a high AFC showed a larger (P < 0.0001) ovarian diameter and area than those with a very low AFC. Females with a very low AFC exhibited a larger (P < 0.01) diameter of the dominant follicle at TAI (13.6 ± 0.3 vs. 12.2 ± 0.4 mm) and a tendency (P = 0.06) to have different serum progesterone concentrations (2.9 ± 0.3 vs. 2.1 ± 0.3 ng/mL; on day 18, considering day 0 as the beginning of the synchronization protocol) than those with a high AFC. The pregnancy rate was higher (P ≤ 0.05) in animals with a very low (57.9%) and low (53.1%) AFC than in those with a high AFC (45.2%). The expression of genes related to intercellular communication, meiotic control, epigenetic modulation, cell division, follicular growth, cell maintenance, steroidogenesis and cellular stress response was assessed on day 5. In females with a low AFC, 8 and 21 genes in oocytes and cumulus cells, respectively, were upregulated (P < 0.05), while 3 and 6 genes in oocytes and cumulus cells, respectively, were downregulated. The results described here will help elucidate the differences in ovarian physiology and the reproductive success of Bos indicus females with a low or high AFC.

C-type natriuretic peptide (CNP) and its natriuretic peptide receptors subtype 2 (NPR2) are essential for the maintenance of oocyte meiotic arrest in different species. Extracellular vesicles (EVs) in bovine follicular fluid (FF) are important for cell communication within the ovarian follicle. This study investigated the involvement of EVs from FF of bovine ovarian follicles in the CNP-NPR2 system, first by analyzing the presence of CNP in the EV contents, followed by addition of EVs to in-vitro maturation (IVM) medium, to evaluate the effect on maintenance of oocyte meiosis arrest and improvements in in-vitro embryo production. As expected, CNP was observed in FF and granulosa cells from the ovarian follicles. To the best of our knowledge, this is the first time that CNP has been found in the EV contents. To evaluate the possible effect of EVs on the progression of oocyte meiosis, the IVM was performed under three conditions: CNP and EV supplementation and control condition. Both the CNP and EV treatments inhibited meiosis resumption in the oocyte within 9 h of IVM. CNP treatment increased cGMP levels in cumulus cells within 6 h of IVM compared to the control group, but the EV treatment did not. In contrast, the relative mRNA abundance of adenylate cyclase 3 and 9 (ADCY3 and ADCY9) was upregulated in oocytes after 6 h of IVM under EV treatment compared to the control group, but not under CNP treatment. Last, these treatments in the IVM medium had no significant effect on the in-vitro embryo production. In conclusion, we demonstrated the presence of endogenous CNP in bovine reproductive structures, especially in the EVs from the FF of antral follicles. The presence of CNP in the EVs suggests an important involvement of this cell-communication system in the CNP-NPR2 system. Therefore, we indeed observed that the EVs from FF can modulate the arrest of oocyte meiosis, acting similarly to the CNP-NPR2 system to block the oocyte in the GV state. However, the mechanism of each system might be different; the CNP-NPR2 system seems to be involved in modulating the cGMP levels, while the contents of EVs might be involved in modulating the cAMP levels., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Preterm labor (PTL) and Preterm Premature Rupture of Membranes (PPROM) impose substantial morbimortality on mothers and newborns. Exosomes act in intercellular communication carrying molecules involved in physiopathological processes. Little is known about exosomal proteins in prematurity. Our aim was to evaluate the protein expression of hemopexin, C1 inhibitor (C1INH) and alpha-2-macroglobulin (A2M) from circulating exosomes of women with PTL and PPROM. Plasma was obtained from PTL, PPROM, Term in labor and Term out of labor (T) patients, exosomes were isolated by ultracentrifugation, then lysed and the proteins quantified. Western Blot (WB) and Nanoparticle Tracking Analysis (NTA) were performed. Data were compared by Kruskal-Wallis, unpaired T-test and one-way ANOVA. WB and NTA confirmed exosome isolation (concentration: 4.3 × 10 10  particles/ml ± 1.9 × 10 10 ). There was no difference regarding hemopexin or C1INH expression between the groups. For A2M, the fold change was significantly higher on preterm groups when compared to term groups (1.07 ± 0.30 vs. 0.42 ± 0.17, p < 0.0001). Higher levels of A2M in circulating exosomes are linked to preterm pregnancies. sEV are strong candidates to intermediate maternal-fetal communication, carrying preterm labor-related immunomodulatory proteins.

Orchestrated events, including extensive changes in epigenetic marks, allow a somatic nucleus to become totipotent after transfer into an oocyte, a process termed nuclear reprogramming. Recently, several strategies have been applied in order to improve reprogramming efficiency, mainly focused on removing repressive epigenetic marks such as histone methylation from the somatic nucleus. Herein we used the specific and non-toxic chemical probe UNC0638 to inhibit the catalytic activity of the histone methyltransferases EHMT1 and EHMT2. Either the donor cell (before reconstruction) or the early embryo was exposed to the probe to assess its effect on developmental rates and epigenetic marks. First, we showed that the treatment of bovine fibroblasts with UNC0638 did mitigate the levels of H3K9me2. Moreover, H3K9me2 levels were decreased in cloned embryos regardless of treating either donor cells or early embryos with UNC0638. Additional epigenetic marks such as H3K9me3, 5mC, and 5hmC were also affected by the UNC0638 treatment. Therefore, the use of UNC0638 did diminish the levels of H3K9me2 and H3K9me3 in SCNT-derived blastocysts, but this was unable to improve their preimplantation development. These results indicate that the specific reduction of H3K9me2 by inhibiting EHMT1/2 during nuclear reprogramming impacts the levels of H3K9me3, 5mC, and 5hmC in preimplantation bovine embryos.

This study characterised the expression of interferon (IFN)-τ-stimulated genes (ISGs) and Type I IFN receptors in circulating polymorphonuclear cells (PMNs) of beef heifers and compared it with expression in peripheral blood mononuclear cells (PBMCs) up to Day 20 of gestation. Nelore heifers (n=26) were subjected to fixed-time AI (FTAI) on Day 0. PMNs and PBMCs were isolated on Days 0, 10, 14, 16, 18 and 20 after FTAI. The abundance of target transcripts (ubiquitin-like protein (ISG15), 2'-5'-oligoadenylate synthetase 1 (OAS1), myxovirus resistance 1 (MX1), myxovirus resistance 2 (MX2), IFN receptor I (IFNAR1) and IFN receptor 2 (IFNAR2)) was determined using real-time quantitative polymerase chain reaction and compared between pregnant (n=8) and non-pregnant (n=9) females. In both PBMCs and PMNs, ISG15 and OAS1 expression was greater in pregnant than non-pregnant heifers on Days 18 and 20. There were no significant differences in the expression of ISGs between PBMCs and PMNs. A time effect on expression was found for IFNAR1 in PBMCs and IFNAR2 in PMNs, with decreased expression of both genes on Days 18 and 20. When the expression of these genes was compared between cell types only in pregnant heifers, IFNAR2 expression in PMNs had an earlier decrease when compared to its expression in PBMCs, starting from Day 18. In conclusion, PMNs do not respond earlier to the conceptus stimulus, and ISG15 and OAS1 expression in both PMNs and PBMCs can be used as a suitable marker for pregnancy diagnosis on Days 18 and 20. In addition, gestational status did not affect IFNAR1 and IFNAR2 expression, but IFNAR2 showed a distinct response between PMNs and PBMCs of pregnant heifers.

Intercellular communication is an essential mechanism for development and maintenance of multicellular organisms. Extracellular vesicles (EVs) were recently described as new players in the intercellular communication. EVs are double-membrane vesicles secreted by cells and are classified according to their biosynthesis, protein markers and morphology. These extracellular vesicles contain bioactive materials such as miRNA, mRNA, protein and lipids. These characteristics permit their involvement in different biological processes. Reproductive physiology is complex and involves constant communication between cells. Different laboratories have described the presence of EVs secreted by ovarian follicular cells, oviductal cells, in vitro produced embryos and by the endometrium, suggesting that EVs are involved in the development of gametes and embryos, in animals and humans. Therefore, is important to understand physiological mechanisms and contributions of EVs in female reproduction in order to develop new tools to improve in vivo reproductive events and assisted reproductive techniques (ARTs). This review will provide the current knowledge related to EVs in female reproductive tissues and their role in ARTs., (Copyright © The Author(s). Published by CBRA.)

Extracellular vesicles (EVs) are nanoparticles secreted by ovarian follicle cells. Extracellular vesicles are an important form of intercellular communication, since they carry bioactive contents, such as microRNAs (miRNAs), mRNAs, and proteins. MicroRNAs are small noncoding RNA capable of modulating mRNA translation. Thus, EVs can play a role in follicle and oocyte development. However, it is not clear if EV contents vary with the estrous cycle stage. The aim of this study was to investigate the bovine miRNA content in EVs obtained from follicles at different estrous cycle stages, which are associated with different progesterone (P4) levels in the follicular fluid (FF). We collected FF from 3 to 6 mm follicles and evaluated the miRNA profile of the EVs and their effects on cumulus-oocyte complexes during in vitro maturation. We observed that EVs from low P4 group have a higher abundance of miRNAs predicted to modulate pathways, such as MAPK, RNA transport, Hippo, Cell cycle, FoxO, oocyte meiosis, and TGF-beta. Additionally, EVs were taken up by cumulus cells and, thus, affected the RNA global profile 9 h after EV supplementation. Cumulus cells supplemented with EVs from low P4 presented upregulated genes that could modulate biological processes, such as oocyte development, immune responses, and Notch signaling compared with genes of cumulus cells in the EV free media or with EVs from high P4 follicles. In conclusion, our results demonstrate that EV miRNA contents are distinct in follicles exposed to different estrous cycle stage. Supplementation with EVs impacts gene expression and biological processes in cumulus cells., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Follicles are composed of different interdependent cell types including oocytes, cumulus, granulosa, and theca cells. Follicular cells and oocytes exchange signaling molecules from the beginning of the development of the primordial follicles until the moment of ovulation. The follicular structure transforms during folliculogenesis; barriers form between the germ and the somatic follicular cells, and between the somatic follicular cells. As such, communication systems need to adapt to maintain the exchange of signaling molecules. Two critical barriers are established at different stages of development: the zona pellucida, separating the oocyte and the cumulus cells limiting the communication through specific connections, and the antrum, separating subpopulations of follicular cells. In both situations, communication is maintained either by the development of specialized connections as transzonal projections or by paracrine signaling and trafficking of extracellular vesicles through the follicular fluid. The bidirectional communication between the oocytes and the follicle cells is vital for driving folliculogenesis and oogenesis. These communication systems are associated with essential functions related to follicular development, oocyte competence, and embryonic quality. Here, we discuss the formation of the zona pellucida and antrum during folliculogenesis, and their importance in follicle and oocyte development. Moreover, this review discusses the current knowledge on the cellular mechanisms such as the movement of molecules via transzonal projections, and the exchange of extracellular vesicles by follicular cells to overcome these barriers to support female gamete development. Finally, we highlight the undiscovered aspects related to intrafollicular communication among the germ and somatic cells, and between the somatic follicular cells and give our perspective on manipulating the above-mentioned cellular communication to improve reproductive technologies., Competing Interests: Conflict of interest
 The authors declare that they have no competing interests., (Copyright © The Author(s). Published by CBRA.)

Potential mechanisms involved in neural differentiation of adipocyte derived stem cells (ADSCs) are still unclear. In the present study, extracellular vesicles (EVs) were tested as a potential mechanism involved in the neuronal differentiation of stem cells. In order to address this, ADSCs and neurons (BRC) were established in primary culture and co-culture at three timepoints. Furthermore, we evaluated protein and transcript levels of differentiated ADSCs from the same timepoints, to confirm phenotype change to neuronal linage. Importantly, neuron-derived EVs cargo and EVs originated from co-culture were analyzed and tested in terms of function, such as gene expression and microRNA levels related to the adult neurogenesis process. Ideal neuron-like cells were identified and, therefore, we speculated the in vivo function of these cells in acute sciatic nerve injury. Overall, our data demonstrated that ADSCs in indirect contact with neurons differentiated into neuron-like cells. Neuron-derived EVs appear to play an important role in this process carrying SNAP25, miR-132 and miR-9. Additionally, in vivo neuron-like cells helped in microenvironment modulation probably preventing peripheral nerve injury degeneration. Consequently, our findings provide new insight of future methods of ADSC induction into neuronal linage to be applied in peripheral nerve (PN) injury.

The biotechnology for in vitro embryo production is becoming increasingly popular, being applied to humans and domestic animals. Embryo development can be achieved with either 20% or 5% oxygen tension. The extracellular vesicles (EVs) are secreted by different cell types and carry bioactive materials. Our objective was to determine the secretion pattern and micro RNA (miRNA) contents of EVs released in the bovine embryo culture environment-embryo and cumulus cell monolayer-on Days 3 and 7 of in vitro culture under two different oxygen tensions: High (20%) and low (5%). The EVs were isolated from the medium and analyzed to determine size, concentration, and miRNA levels. EVs concentration in low oxygen tension increased on Day 3 and decreased on Day 7. Additionally, altered EV miRNAs derived from the embryo-cumulus culture medium were predicted to regulate survival and proliferation-related pathways on Days 3 and 7. Moreover, miR-210 levels decreased in EVs isolated from the culture medium under high oxygen tension suggesting that this miRNA can be used as a marker for normoxia since it is associated with low oxygen tension. In summary, this study provides knowledge of the oxygen tension effects on EVs release and content, and potentially, on cell-to-cell communication during in vitro bovine embryo production., (© 2019 Wiley Periodicals, Inc.)

Semen fertilizing potential is dependent upon the morphological, functional and molecular attributes of sperm. Sperm microRNAs (miRNAs) were recently shown to hold promise regarding their association with different fertility phenotypes. However, their role in fertility regulation remains to be determined. We postulated that sperm miRNAs might regulate early embryonic development. From this perspective, sperm quality and 380 sperm miRNAs were investigated in frozen-thawed semen from high (HF; 54.3 ± 1.0% pregnancy rate) and low (LF; 41.5 ± 2.3%) fertility bulls. Out of nine miRNAs that showed different levels in sperm cells, miR-216b was present at lower levels in HF sperm cells and zygotes. Among miR-216b target genes (K-RAS, BECN1 and JUN), K-RAS, related to cell proliferation, revealed a higher level in HF two-cell embryos. First cleavage rate, blastocyst cell number and division number were also higher in HF. In addition, by using a model based on polyspermy embryos, we demonstrated an increase in miR-216b levels in zygotes associated with sperm cell entry. Our results shed light on a possible mechanism of paternal contribution involving sperm-borne miR-216b that modulates levels of miR-216b in zygotes and K-RAS in two-cell embryos. This modulation might regulate early development by interfering with the first cleavage and blastocyst quality.

Introduction: Pluripotent stem cells are believed to have greater clinical potential than mesenchymal stem cells due to their ability to differentiate into almost any cell type of an organism, and since 2006, the generation of patient-specific induced pluripotent stem cells (iPSCs) has become possible in multiple species., Objectives: We hypothesize that different cell types respond differently to the reprogramming process; thus, the goals of this study were to isolate and characterize equine adult and fetal cells and induce these cells to pluripotency for future regenerative and translational purposes., Methods: Adult equine fibroblasts (eFibros) and mesenchymal cells derived from the bone marrow (eBMmsc), adipose tissue (eADmsc), and umbilical cord tissue (eUCmsc) were isolated, their multipotency was characterized, and the cells were induced in vitro into pluripotency (eiPSCs). eiPSCs were generated through a lentiviral system using the factors OCT4, SOX2, c-MYC, and KLF4. The morphology and in vitro pluripotency maintenance potential (alkaline phosphatase detection, embryoid body formation, in vitro spontaneous differentiation, and expression of pluripotency markers) of the eiPSCs were characterized. Additionally, a miRNA profile analysis of the mesenchymal and eiPSCs was performed., Results: Multipotent cells were successfully isolated, but the eBMmsc failed to generate eiPSCs. The eADmsc-, eUCmsc-, and eFibros-derived iPSCs were positive for alkaline phosphatase, OCT4 and NANOG, were exclusively dependent on bFGF, and formed embryoid bodies. The miRNA profile revealed a segregated pattern between the eiPSCs and multipotent controls: the levels of miR-302/367 and the miR-92 family were increased in the eiPSCs, while the levels of miR-23, miR-27, and miR-30, as well as the let-7 family were increased in the nonpluripotent cells., Conclusions: We were able to generate bFGF-dependent iPSCs from eADmsc, eUCmsc, and eFibros with human OSKM, and the miRNA profile revealed that clonal lines may respond differently to the reprogramming process.