Biosensor capability of the endometrium is mediated in part, by altered miRNA cargo from conceptus-derived extracellular vesicles.

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.

Small extracellular vesicles derived from the crosstalk between early embryos and the endometrium potentially mediate corpus luteum function.

The first interactions among the embryo, endometrium, and corpus luteum (CL) are essential for pregnancy success. Small extracellular vesicles (sEVs) are part of these interactions. We previously demonstrated that sEVs from in vivo- or in vitro-produced bovine embryos contain different miRNA cargos. Herein we show: 1) the presence and origin (in vivo or in vitro) of the blastocyst differentially reprograms endometrial transcriptional profiles; 2) the endometrial explant (EE) cultured with in vivo or in vitro embryos release sEVs with different miRNA contents, and; 3) the luteal explant (CLE) exposed to these sEVs have distinct mRNA and miRNA profiles. To elucidate this, the EE were cultured in the presence or absence of a single Day-7 in vivo (EE-AI) or in vitro (EE-IVF) embryo. After of culture we found, in the EE, 45 and 211 differentially expressed genes (DEGs) associated with embryo presence and origin, respectively. SEVs were recovered from the conditioned media (CM) in which EE and embryos were co-cultured. Four miRNAs were differentially expressed between sEVs from CM-EE-AI and CM-EE-IVF. Luteal explants exposed in culture to these sEVs showed 1360 transcripts, and fifteen miRNAs differentially expressed. The DEGs associated with embryo presence and origin, modulating cells' proliferation, and survival. These results demonstrate that in vivo- or in vitro-produced bovine embryos induce molecular alterations in the endometrium; and that the embryo and endometrium release sEVs capable of modifying the mRNA and miRNA profile in the CL. Therefore, the sEVs-mediated embryo-endometrium-CL interactions possibly regulate the CL viability to ensure pregnancy success.

Extracellular vesicles derived from bovine adipose-derived mesenchymal stromal cells enhance in vitro embryo production from lesioned ovaries.

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 × 106 AD-MSCs (G2), MSC-EVs produced by 2.5 × 106 AD-MSCs (G3) or 3 doses of MSC-EVs produced by 2.5 × 106 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.

Small extracellular vesicles derived from in vivo- or in vitro-produced bovine blastocysts have different miRNAs profiles-Implications for embryo-maternal recognition.

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.

Estrous cycle impacts microRNA content in extracellular vesicles that modulate bovine cumulus cell transcripts during in vitro maturation†.

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.

Extracellular Vesicles Mediated Early Embryo-Maternal Interactions.

Embryo-maternal crosstalk is an important event that involves many biological processes, which must occur perfectly for pregnancy success. This complex communication starts from the zygote stage within the oviduct and continues in the uterus up to the end of pregnancy. Small extracellular vesicles (EVs) are part of this communication and carry bioactive molecules such as proteins, lipids, mRNA, and miRNA. Small EVs are present in the oviductal and uterine fluid and have important functions during fertilization and early embryonic development. Embryonic cells are able to uptake oviductal and endometrium-derived small EVs. Conversely, embryo-derived EVs might modulate oviductal and uterine function. In this review, our aim is to demonstrate the role of extracellular vesicles modulating embryo-maternal interactions during early pregnancy.

1α, 25-Dihydroxyvitamin D3 alters ectonucleotidase expression and activity in human cutaneous melanoma cells.

PURPOSE: We hypothesized that vitamin D decreases rates of adenosine formation in human cutaneous melanoma cells through the inhibition of extracellular adenosine 5'-triphosphate breakdown, thereby affecting tumor cell viability. Therefore, the objective of this study was to explore the mechanisms of action of 1α, 25-dihydroxyvitamin D3 (1,25(OH)2 D3) on the activity and expression of ectonucleotidases in cutaneous melanoma cells. METHODS: A human melanoma cell line, SK-Mel-28, was treated with 1 to 50 nM of the active vitamin D metabolite (1,25(OH)2 D3) over 24 hours, followed by determination of NTPDase1/CD39 and ecto-5'-nucleotidase/CD73 activity and expression rates of the purinergic system-related NTPDASE1, NT5E and adenosine deaminase and vitamin D receptor. An 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay was used to evaluate cellular viability. RESULTS: 1,25(OH)2 D3 decreased adenosine monophosphate hydrolysis via ecto-5'-nucleotidase/CD73 and expression of CD73, but did not change NTPDase1/CD39 activity; it increased the CD39 expression. We also observed an increase of cell viability at 1 nM, but this viability decreased as the concentrations of vitamin D active metabolite increased to 50 nM. There were no differences in gene expression levels. CONCLUSION: To the best of our knowledge, we showed for the first time a mechanism of control of adenosine production via modulation of the purinergic system in cutaneous melanoma cells treated with the active metabolite of vitamin D. This study provides original information regarding mechanisms, in which vitamin D plays a key role in preventing tumor progression in human melanoma cells.

Inhibition of RNA synthesis during Scriptaid exposure enhances gene reprogramming in SCNT embryos.

Insufficient epigenetic reprogramming is incompatible with normal development of embryos produced by somatic cell nuclear transfer (SCNT), but treatment with histone deacetylases inhibitors (HDACi) enhances development of SCNT embryos. However, the mechanisms underpinning HDACi benefits in SCNT embryos remain largely uncharacterized. We hypothesized that, in addition to enhancing reprogramming, HDACi treatment may promote expression of genes not required for early development of SCNT embryos. To test this hypothesis, RNA synthesis was inhibited by treating bovine SCNT embryos with 5,6-dichlorobenzimidazole 1-ß-D-ribofuranoside (DBR), which were concomitantly treated or not with Scriptaid (Scrip; an HDACi). Development to the blastocyst stage was significantly increased by treatment with Scrip alone (26.6%) or associated with DRB (28.6%) compared to Control (17.9%). The total number of nuclei was significantly improved only in embryos that were treated with both Scrip + DRB. Nuclear decondensation after SCNT was significantly increased by DRB treatment either alone or associated with Scrip. The relative mRNA expression, evaluated during the embryo genome activation (EGA) transition, revealed that some KDMs (KDM1A, KDM3A, KDM4C and KDM6A) and DNMT1 where prematurely expressed in Scrip-treated embryos. However, treatment with Scrip + DRB inhibited early mRNA expression of those genes, as well as several other KDMs (KDM4A, KDM4B, KDM5A, KDM5B, KDM5C and KDM7A) compared to embryos treated with Scrip alone. These findings revealed that HDACi improved development in SCNT embryos compared to Control, but altered the expression of genes involved in epigenetic regulation and did not improve embryo quality. Inhibition of RNA synthesis during HDACi treatment enhanced nuclear chromatin decondensation, modulated gene expression and improved SCNT embryo quality.

Histone 3 lysine 4, 9, and 27 demethylases expression profile in fertilized and cloned bovine and porcine embryos.

Epigenetic modifications in the C-terminal domain of histones coordinate important events during early development including embryo genome activation (EGA) and cell differentiation. In this study, the mRNA expression profile of the main lysine demethylases (KDMs) acting on the lysine 4 (H3K4), 9 (H3K9), and 27 (H3K27) of the histone H3 was determined at pre-, during and post-EGA stages of bovine and porcine embryos produced by in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). In IVF embryos, mRNA abundance of most KDMs revealed a bell-shaped profile with peak expression around the EGA period, i.e. Day 3 for porcine (KDM2B, KDM5B, KDM5C, KDM4B, KDM4C, KDM6A, KDM6B, and KDM7A), and Day 4 for bovine (KDM1A, KDM5A, KDM5B, KDM5C, KDM3A, KDM4A, KDM4C, and KDM7A). The mRNA profile of KDM1A, KDM2B, KDM3A, KDM3B, KDM6A, and KDM6B differed between porcine and bovine IVF embryos. Several differences were also observed between SCNT and IVF, which includes a precocious peak in the mRNA expression of KDM1A, KDM3A, KDM4C, KDM5A, KDM5B, KDM5C, KDM6A, and KDM7A in bovine SCNT embryos; absence of mRNA peak for KDM4B, KDM4C, and KDM6A in porcine SCNT embryos; and early decreasing in KDM5B and KDM5C mRNA in porcine SCNT embryos. Based on the mRNA profile, this study has identified several KDMs that are likely involved in the regulation of the EGA transition, KDMs that may have a species-specific role in bovine and porcine embryos, and KDMs that are improperly expressed during cell reprogramming in SCNT embryos.

Enhancing Bovine Embryo Development In Vitro Using Oil-in-Water Nanoemulsions as Specific Carriers for Essential Lipids.

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.

Labeled extracellular vesicles can be found in the blood plasma shortly after intrauterine infusion in bovine.

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.

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.

Corpus luteum proximity alters molecular signature of the small extracellular vesicles and cumulus cells in the bovine ovarian follicle environment.

Progesterone (P4) is predicted to act as a negative regulatory hormone for oocyte maturation events; however, its local effects during follicular development remain poorly understood in bovine. The complex process of oocyte meiosis progression is dependent on cellular communication among follicular cells. Besides, the breakdown of this communication, mainly between cumulus cells (CC) and oocyte, through the retraction of cumulus projections connecting these cells can impact oocyte maturation. In our study, we observed that follicles from the ovary ipsilateral to the corpus luteum (CL) containing high intrafollicular P4 concentrations enhance the abundance of proteins detected in follicular-derived small extracellular vesicles (sEVs) predicted to be involved in the retraction of membrane projections based on actin filaments, such as transzonal projections (TZPs). Conversely, we found that follicles from the ovary contralateral to the CL, which contained low intrafollicular P4 concentrations, had a high detection of proteins predicted to regulate the maintenance of TZPs. We also performed RNAseq analysis which demonstrated that 177 genes were differentially expressed in CC under the different P4 environments. Bioinformatic analysis points to changes associated to cell metabolism in cells from follicles ipsilateral to the CL in comparison to genes involved in cell communication in CC from follicles contralateral to the CL. Our functional analysis experiment confirmed that supplementation of cumulus-oocyte complexes during in vitro maturation with P4 at concentration similar to ipsilateral follicles reduces the number of TZPs. In summary, our study underscores a direct association between P4 concentration and cumulus-oocyte interaction, with potential consequences for the acquisition of oocyte competence.

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.

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.

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.

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.

Acquisition and maintenance of pluripotency are influenced by fibroblast growth factor, leukemia inhibitory factor, and 2i in bovine-induced pluripotent stem cells.

Several opportunities for embryo development, stem cell maintenance, cell fate, and differentiation have emerged using induced pluripotent stem cells (iPSCs). However, the difficulty in comparing bovine iPSCs (biPSCs) with embryonic stem cells (ESCs) was a challenge for many years. Here, we reprogrammed fetal fibroblasts by transient expression of the four transcription factors (Oct4, Sox2, Klf4, and c-Myc, collectively termed "OSKM" factors) and cultured in iPSC medium, supplemented with bFGF, bFGF2i, leukemia inhibitory factor (LIF), or LIF2i, and then compared these biPSC lines with bESC to evaluate the pluripotent state. biPSC lines were generated in all experimental groups. Particularly, reprogrammed cells treated with bFGF were more efficient in promoting the acquisition of pluripotency. However, LIF2i treatment did not promote continuous self-renewal. biPSCs (line 2) labeled with GFP were injected into early embryos (day 4.5) to assess the potential to contribute to chimeric blastocysts. The biPSC lines show a pluripotency state and are differentiated into three embryonic layers. Moreover, biPSCs and bESCs labeled with GFP were able to contribute to chimeric blastocysts. Additionally, biPSCs have shown promising potential for contributing to chimeric blastocysts and for future studies.

Changes in Oviductal Cells and Small Extracellular Vesicles miRNAs in Pregnant Cows.

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.

Reproductive seasonality influences oocyte retrieval and embryonic competence but not uterine receptivity in buffaloes.

Since buffaloes are a seasonal, polyestrous species, optimizing reproduction during the non-breeding season is a key factor in increasing the reproductive and productive efficiency of herds. Ovum pick-up associated with in vitro embryo production and embryo cryopreservation is an alternative to reduce seasonal impacts. We studied the effects of seasonality in buffalo oocyte donors and embryo recipients during the favorable and non-favorable breeding seasons. Donors were evaluated for oocyte recovery and blastocyst production rate as dFBS (donors in favorable breeding season) or dNBS (donors in non-favorable breeding season). Embryos produced from dFBS or dNBS were cryopreserved by vitrification or the slow-freeze method for direct transfer and transferred to recipients in the favorable (rFBS) or non-favorable breeding season (rNBS). The heifers or cows were subjected to a fixed-time embryo transfer protocol and conception rates were determined on day 30 and on day 60. The oocyte recovery was lower in dFBS than in dNBS (7.6 vs. 10.0 oocyte/OPU, p = 0.0262); while no difference was found comparing blastocyst production rate (23.7% vs. 30.9% of blastocysts, respectively). Embryos from dFBS resulted in greater (p = 0.0013) conception rates on day 30 compared to dNBS (46.5% vs. 22.4%, respectively), despite the breeding season. The rFBS and rNBS treatments had similar (p = 0.6714) conception rates on day 30 (38.0% vs. 33.0%, respectively), indicating similar uterine receptivity. However, heifers on FBS had higher (p = 0.0003) conception rates on day 30 than cows (73.9% vs. 13.3%, respectively) when receiving embryos from dFBS. Vitrification and direct transfer had similar (p = 0.1698) conception rates on day 30 (30.4% vs. 41.4%, respectively). In conclusion, in vitro-produced embryos derived from dFBS were more competent in establishing pregnancy than dNBS counterparts, independent of recipients' reproductive seasonality. Heifers achieved better conception rates than cows during the favorable breeding season when the embryo came from dFBS. Cryopreserved in vitro produced embryos represent a reliable alternative to reduce seasonal variations in buffalo reproduction. The data elucidate the seasonal effects on embryo competence and on recipients' uterine receptivity, affording new strategies to implement ovum pick-up associated with in vitro embryo production programs in buffalo herds.