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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.

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.

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

Metabolic profiles, gene expression patterns, embryonic development, and the ability to establish and sustain early pregnancies are differences founded when in vivo- and in vitro- produced bovine embryos are compared. To the establishment of a successful pregnancy the perfect embryo-maternal communication is needed. Small extracellular vesicles (sEVs) are part of this crosstalk and carry bioactive molecules such as miRNAs that can act on target bovine cells within the reproductive system. Thereby, our hypothesis is that in vivo and in vitro bovine embryos secrete sEVs containing different miRNAs that can differentially modulate endometrial gene expression pattern and miRNAs profile in the corpus luteum (CL). To address this hypothesis, firstly, we investigated the miRNA content of in vivo and in vitro hatched blastocysts and in sEVs secreted by them. Day 9 in vivo or in vitro hatched blastocysts have distinct miRNA profiles. Small EVs secreted by them from day 7 up to day 9 of development contain different miRNAs. These miRNAs differently expressed are predicted to regulate pathways involved with early embryonic development and endometrial receptivity. Thereby, early embryo-maternal interactions can be modified and consequently can affect pregnancy success. Secondly, we investigated changes in the endometrial global transcriptome after the co-culture of endometrial explants with a day 7 in vivo or in vitro blastocysts. Differently expressed genes were identified in the endometrium and are associated with the embryo\'s presence or origin. Moreover, sEVs present in the conditioned media (C.M.) by these embryonic and endometrial cells contain different miRNAs, which are predicted to modulate the oxytocin signaling pathway. Finally, to understand if the communication among embryo, endometrium, and corpus luteum can be mediated by sEVs, luteal explants were treated with sEVs from C.M. by endometrial explants alone or cultured with a day 7 in vivo or in vitro blastocysts. MicroRNAs profile was modified in luteal explants treated with these sEVs, and we can observe an effect of the embryo presence and origin in these alterations. Together these results suggest that sEVs mediate early embryo-endometrium-corpus luteum communication, contributing to maintaining luteal viability and functionality. However, further experiments are needed to evaluate specific biological effects of these miRNAs carried by sEVs in the endometrium and in the corpus luteum. Moreover, the embryo origin (in vivo and in vitro) modify the interactions among embryo-endometrium-corpus luteum, suggesting strongly that these embryos have distinct needed to develop. Perfis metabólicos, padrões de expressão gênica, desenvolvimento embrionário, capacidade de estabelecer e manter gestações precoces são diferenças encontradas quando embriões bovinos produzidos in vivo e in vitro são comparados. Para que a gestação tenha sucesso é necessária uma perfeita comunicação entre o embrião e o organismo materno. Vesículas extracelulares pequenas (sEVs) fazem parte dessa comunicação e carregam moléculas bioativas, como miRNAs, que podem atuar em células-alvo dentro do sistema reprodutivo bovino. Com isso, nossa hipótese é que embriões bovinos produzidos in vivo e in vitro secretam sEVs contendo diferentes miRNAs que podem modular diferencialmente o padrão de expressão gênica endometrial e o perfil de miRNAs do corpo lúteo. Para isso, primeiramente, investigamos o conteúdo de miRNA em blastocistos eclodidos produzidos in vivo ou in vitro e, em sEVs secretadas por eles. Blastocistos eclodidos produzidos in vivo ou in vitro, no dia 9 de desenvolvimento, possuem diferentes perfis de miRNA. EVs pequenas secretadas por eles, do dia 7 até o dia 9 de desenvolvimento, contém miRNAs diferentes. Esses miRNAs diferentemente expressos são preditos por regularem vias envolvidas no desenvolvimento embrionário inicial e na receptividade endometrial. Por meio disso, as primeiras interações entre o embrião e o organismo materno podem ser modificadas, e isso pode afetar o sucesso da gestação. Em segundo lugar, investigamos as alterações no transcriptoma global do endométrio após o co-cultivo de explantes endometriais com um blastocisto produzido in vivo ou in vitro no dia 7 do desenvolvimento. Genes diferentemente expressos foram identificados no endométrio e estão associados com a presença e origem do embrião. Além disso, sEVs presentes nos meios condicionados (C.M.) por essas células embrionárias e endometriais contém diferentes miRNAs, que são preditos por modularem a via de sinalização da oxitocina. Finalmente, para compreender melhor se a comunicação entre o embrião, o endométrio e o corpo lúteo pode ser intermediada por sEVs, explantes luteais foram tratados com sEVs presentes nos C.M. por explantes endometriais cultivados sozinhos ou com um blastocisto bovino produzido in vivo ou in vitro no dia 7 do desenvolvimento. O perfil de miRNAs foi modificado nos explantes luteais tratados com essas sEVs e, nós podemos observar um efeito da presença e origem dos embriões nessas alterações. Juntos, esses resultados sugerem que sEVs intermedeiam a comunicação inicial entre o embrião, endométrio e o corpo lúteo, contribuindo para manter a viabilidade e funcionalidade luteal. Entretanto, futuros experimentos precisam ser realizados para avaliar efeitos biológicos específicos desses miRNAs carreados por essas sEVs no endométrio e no corpo lúteo. Além disso, a origem do embrião (in vivo ou in vitro) modifica as interações entre ele, o endométrio e o corpo lúteo, sugerindo fortemente que esses embriões tenham necessidades distintas para se desenvolverem.

Interferon tau (IFNT) is the pregnancy recognition signal in ruminants and is secreted by trophoblast cells. Paracrine action in the endometrium is well established by inhibiting luteolytic pulses of prostaglandin F2 alpha. Recently, endocrine action was documented in the corpus luteum, blood cell and liver. It was hypothesized that conditioned medium (CM) obtained from days 7, 9 and 12 parthenogenetic embryos alters luteal cell gene expression. The aim was to establish a bovine mixed luteal cell culture to evaluate cellular response associated to interferon stimulated genes, steroidogenesis and apoptosis. Conditioned medium was obtained from Days 7, 9 and 12 parthenogenetic (PA) embryos culture. Moreover, antiviral assay was performed on CM from Days 7, 9 and 12 to verify Type I interferon activity. Luteal cell culture was validated by steroidogenic and apoptotic genes (CYP11A1 , HSD3B1, BAX, BCL2, AKT and XIAP mRNA expression), and concentration of progesterone as endpoint. Luteal cell culture was treated with interferon alpha (IFNA) and CM from parthenogenetic embryos. Antiviral assay revealed Type I interferon activity on CM from embryos increasing on Days 9 and 12. ISG15 mRNA was greater in the mixed luteal cells culture treated with 1, 10 and 100ng/ml of interferon alpha (IFNA) and also on Days 7, 9 and 12 CM treatments. Concentration of progesterone was not altered in luteal cell culture regardless of treatments. Steroidogenic and apoptotic genes were similar among groups in luteal cell culture treated with different doses of IFNA or CM from PA embryos. In conclusion, parthenogenetic embryo-derived CM has antiviral activity, luteal cell culture respond to Type I interferon by expressing IGS15. These data indicate this model can be used for IFNT endocrine signaling studies.

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.

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.

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.

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.

Lady Serrano Mujica,1 Alessandra Bridi,1,2 Ricardo Della Méa,1 Vitor Braga Rissi,1 Naiara Guarda,3 Rafael Noal Moresco,3 Melissa Orlandin Premaor,4 Alfredo Quites Antoniazzi,1 Paulo Bayard Dias Gonçalves,1 Fabio Vasconcellos Comim1,4 1Laboratory of Biotechnology and Animal Reproduction, BioRep, Federal University of Santa Maria, Santa Maria, Rio Grande do Sul, 2Department of Veterinary Medicine, University of São Paulo, São Paulo, 3Laboratory of Clinical Biochemistry, Department of Clinical and Toxicological Analysis, 4Department of Clinical Medicine, Federal University of Santa Maria, Santa Maria, Rio Grande do Sul, Brazil Background: Several studies have described an enhanced inflammatory status and oxidative stress balance disruption in women with polycystic ovary syndrome (PCOS). However, there is scarce information about redox markers in the blood of androgenized animal models. Here, we evaluated the serum/plasma oxidative stress marker and metabolic parameter characteristics of prenatal (PreN) and postnatal (PostN) androgenized rat models of PCOS. Materials and methods: For PreN androgenization (n=8), 2.5mg of testosterone propionate was subcutaneously administered to dams at embryonic days 16, 17, and 18, whereas PostN androgenization (n=7) was accomplished by subcutaneously injecting 1.25mg of testosterone propionate to animals at PostN day 5. A unique control group (n=8) was constituted for comparison. Results: Our results indicate that PostN group rats exhibited particular modifications in the oxidative stress marker, an increased plasma ferric-reducing ability of plasma, and an increased antioxidant capacity reflected by higher albumin serum levels. PostN animals also presented increased total cholesterol and triglyceride–glucose levels, suggesting severe metabolic disarrangement. Conclusion: Study findings indicate that changes in oxidative stress could be promoted by testosterone propionate exposure after birth, which is likely associated with anovulation and/or lipid disarrangement. Keywords: animal models of PCOS, oxidative stress, prenatal, postnatal

Interferon tau (IFNT) is the pregnancy recognition signal in ruminants and is secreted by trophoblast cells. Paracrine action in the endometrium is well established by inhibiting luteolytic pulses of prostaglandin F2 alpha. Recently, endocrine action was documented in the corpus luteum, blood cell and liver. It was hypothesized that conditioned medium (CM) obtained from days 7, 9 and 12 parthenogenetic embryos alters luteal cell gene expression. The aim was to establish a bovine mixed luteal cell culture to evaluate cellular response associated to interferon stimulated genes, steroidogenesis and apoptosis. Conditioned medium was obtained from Days 7, 9 and 12 parthenogenetic (PA) embryos culture. Moreover, antiviral assay was performed on CM from Days 7, 9 and 12 to verify Type I interferon activity. Luteal cell culture was validated by steroidogenic and apoptotic genes (CYP11A1 , HSD3B1, BAX, BCL2, AKT and XIAP mRNA expression), and concentration of progesterone as endpoint. Luteal cell culture was treated with interferon alpha (IFNA) and CM from parthenogenetic embryos. Antiviral assay revealed Type I interferon activity on CM from embryos increasing on Days 9 and 12. ISG15 mRNA was greater in the mixed luteal cells culture treated with 1, 10 and 100ng/ml of interferon alpha (IFNA) and also on Days 7, 9 and 12 CM treatments. Concentration of progesterone was not altered in luteal cell culture regardless of treatments. Steroidogenic and apoptotic genes were similar among groups in luteal cell culture treated with different doses of IFNA or CM from PA embryos. In conclusion, parthenogenetic embryo-derived CM has antiviral activity, luteal cell culture respond to Type I interferon by expressing IGS15. These data indicate this model can be used for IFNT endocrine signaling studies.

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 metyltransferases 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 causes diverse modifications to the chromatin during early development, suggesting an intense epigenetic crosstalk during nuclear reprogramming.

The transforming growth factors beta (TGFβ) are local factors produced by ovarian cells which, after binding to their receptors, regulate follicular deviation and ovulation. However, their regulation and function during corpus luteum (CL) regression has been poorly investigated. The present study evaluated the mRNA regulation of some TGFβ family ligands and their receptors in the bovine CL during induced luteolysis in vivo. On day 10 of the estrous cycle, cows received an injection of prostaglandin F2α (PGF) and luteal samples were obtained from separate groups of cows (n= 4-5 cows per time-point) at 0, 2, 12, 24 or 48 h after treatment. Since TGF beta family comprises more than 30 ligands, we focused in some candidates genes such as activin receptors (ACVR-1A, -1B, -2A, -2B) AMH, AMHR2, BMPs (BMP-1, -2, -3, -4, -6 and -7), BMP receptors (BMPR-1A, -1B and -2), inhibin subunits (INH-A, -BA, -BB) and betaglycan (TGFBR3). The mRNA levels of BMP4, BMP6 and INHBA were higher at 2 h after PGF administration (P

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.

In vivo- and in vitro-produced bovine embryos have different metabolic characteristics, embryonic development, and gene transcription. Additionally, pregnancy rates at 30 days (on average 51% and 34% when using fixed-time AI and in vitro production, respectively) are different in beef cattle. Between Days 8 and 17 of the oestrous cycle, concurrent with embryo-maternal recognition, is when 40% of embryonic losses occur. These losses may occur due to altered embryo-maternal cross-talk. MicroRNA (miRNA) can be involved in this communication; however, its potentially regulated pathways in in vivo and in vitro embryos on Day 9 are unknown. Our hypothesis is that bovine embryos produced in vivo and in vitro contain different miRNA profiles, even after in vivo bovine embryo were in vitro cultured. Cows had the follicular wave synchronized and were superovulated to produce in vivo or in vitro bovine embryos. For the in vitro group, on Day −8 of the protocol, the dominant follicles were recovered by ovum pickup, and in vitro embryo production was performed to obtain embryos. For the in vivo group, on Day −8, the cows were inseminated 12 and 24 h after GnRH analogue application and on Day 7 after expected oestrus, uterine flushing was performed to obtain the embryos. Embryos from both groups were individually cultured for 48 h. Three pools (of 5 embryos each) per group were used for reverse transcription of miRNAs from total RNA using miScript II RT Kit (Qiagen). Relative levels of 383 bovine miRNAs were determined using the geometric mean of miR-99b, RNU43 snoRNA, and Hm/Ms/Rt U1 snRNA by RT-qPCR. Differences in relative levels of miRNAs were determined by Student's t-test. A total of 210 miRNAs were detected in in vivo and in vitro embryos, and 13 out of 210 were differently identified between the groups. In in vivo embryos, 6 miRNAs were up-regulated, whereas 7 miRNAs were up-regulated in in vitro embryos. TARGETSCAN software was used to identify genes predicted as modulated by each miRNA. The top 100 genes predicted were used to identify enriched pathways according to DAVID Bioinformatics Resources. The miRNAs (miR-129, miR-132, miR-155, miR-192, miR-215, and miR-377) up-regulated in in vivo embryos modulated pathways that include signaling pathways regulating pluripotency of stem cells (16 genes), TGF-β (11), hippo (10), oestrogen (8), and cell cycle (7). Moreover, miR-23a, miR-338, miR-34a, miR-491, miR-92b, miR-940, and miR-1271, which were increased in in vitro embryos, regulate PI3K-Akt (17 genes), signaling pathways regulating pluripotency of stem cells (10), oestrogen (9), toll-like receptor (9), Wnt (9), and HIF-1 (7). The results demonstrate that even after 48 h of in vitro culture, bovine embryos produced in vivo and in vitro have different miRNA profiles that modulate pathways associated with embryonic development on Day 9. Furthermore, these results suggest that bioactive molecules, such as miRNAs, can modify embryo-maternal cross-talk, depending on the environment where the embryos are produced. Funding was provided by FAPESP 2017/19681-9, 2014/22887-0, and 2018/13155-6.

The invitro cellular induction into pluripotency [induced pluripotent stem cell (iPSC) generation] has much evolved since its first report and may play a central role in veterinary regenerative medicine. In particular, the generation of invitro neural precursor cells (NPCs) enabled the creation of models for human genetic diseases, such as autism, schizophrenia, and Parkinson disease. The swine model is known to present many advantages over others, including the physiological similarities of pigs and humans. This project aimed to reprogram porcine embryonic fibroblasts (pEF) into iPSCs, and further differentiate them into NPCs. The iPSCs were generated through lentiviral transduction of OCT4, SOX2, c-MYC, and KLF4 genes and cultured with Dulbecco's modified Eagle's medium (DMEM)/F12 KO, 20% knockout serum replacement (KSR), nonessential amino acids, glutamine, β-mercaptoethanol, 10ngmL−1 basic fibroblast growth factor (bFGF), and antibiotics. Three clonal iPSCs lines with typical pluripotent morphology (high nuclear/cytoplasm ratio and well-defined colony borders) were alkaline phosphatase (AP) positive and were further analysed. All three lines [porcine (p)iPSCs lines 1, 2, and 3] expressed exogenous factors for at least 10 passages. Quantitative real-time (qRT)-PCR showed that line 1 expressed endogenous OCT4 and all three lines expressed NANOG. The pEFs were used as controls for 2−ΔΔCt analysis. piPSC line 1 was positive for OCT4, SOX2, NANOG, SSEA-1, TRA-1-60 through immunofluorescence; line 2 was negative for NANOG; and line 3 only presented SOX2 expression. piPSCs lines 2 and 3 differentiated into NPC-like cells. NPC induction was accomplished by 14 days of culture in commercial extracellular matrix (Geltrex) and neural induction media supplemented by SMAD inhibitors LDN193189 (0.2 μM) and SB431542 (10 μM), and then maintained at 20ngmL−1 of epidermal growth factor and basic fibroblast growth factor. They expressed NESTIN and GFAP by qRT-PCR and were positive for NESTIN, β-tubulin III and vimentin by immunofluorescence, confirming the NPC-like cell status. Interestingly, during all stages (pEF, iPSCs, and NPC-like cells), cells were positive to some degree for the neural markers tested by immunofluorescence. Flow cytometry analyses showed a 30% higher intensity of those markers at the NPC-like stage than the pEF and pluripotent stage. These results suggest preliminary evidence for neural differentiation. This will contribute to the use of the porcine model in future regenerative and translational medicine research. This study was funded by FAPESP (grant nos. 2015/26818-5, 2017/02159-8) and CAPES.

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-β-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.

Ethnopharmacological relevance Discaria americana (Rhamnaceae) root bark infusion have been used in traditional medicine as antipyretic, tonic, ameliorative of stomach and skin diseases and diabetes. This study was designed to investigate whether the methanolic extract of the root bark of Discaria americana (MEDa) exhibits antinociceptive effects in mice. Furthermore, it was investigated the involvement of the opioidergic system in MEDa mechanism of action as well the interactions with TRP/ASIC channels in its effect. Materials and methods The antinociceptive effect of intra-gastric gavage (i.g.) of MEDa (0.3–300 mg/kg) was evaluated in mice subjected to acute chemical (acetic-acid, formalin, glutamate, capsaicin, cinnamaldehyde, and acidified saline) or thermal (hot plate) tests of pain. The involvement of opioid system was evaluated in the formalin test. A nonspecific effect of MEDa was observed by measuring locomotor activity and exploratory behavior in open field test. Results MEDa significantly reduced the number of writhing induced by acetic acid and inhibited the nociception in the two phases of formalin. These effects were inhibited by pretreatment with naloxone. The nociception induced by hot plate and intraplantar injection of glutamate, capsaicin, cinnamaldehyde and acidified saline were significantly inhibited by MEDa. Only the dose of 300 mg/kg altered the locomotor activity. Conclusions Our results demonstrated, for the first time, that the methanolic extract of the root bark of Discaria americana presents antinociceptive effect in chemical and thermal stimuli and its analgesic properties can be due activation of the opioidergic system. These results support the use of Discaria americana in traditional medicine and demonstrate that this plant presents a therapeutic potential for the development of phytomedicines with antinociceptive profile.

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.

The objective of this study was to evaluate the effect of mastitis diagnosed by somatic cell count (SCC) on follicular growth, ovulation, oocytes and cumulus cells quality and the concentration and size of exosomes in follicular fluid of dairy cows. In the study, crossbred cows (Bos taurus-Holstein×Bos indicus-Gir) were classified for analysis as control (SCC 400.000 cells mL−1) groups. In Experiment 1 (follicular dynamics), cows (n=57: control=31; mastitis=26) received a progesterone intravaginal device (Sincrogest®, Ourofino Saude Animal, Cravinhos, Brazil) and 2mg of oestradiol benzoate (Sincrodiol®, Ourofino Saude Animal) injected IM. Eight days later (D8), the progesterone device was removed and cows received IM 500mg of cloprostenol (Sincrocio®, Ourofino Saude Animal), 1mg of oestradiol cypionate (SincroCP®, Ourofino Saude Animal) and 300IU of eCG (SicroeCG®, Ourofino Saude Animal). Ultrasound exams (Mindray 4900, probe linear de 5MHz, Shenzhen, China) were performed every 24h from removal of the progesterone-releasing intravaginal device (D8) until 48h later. Thereafter, evaluations were performed every 12h, until ovulation or up to 96h after removal of the progesterone-releasing intravaginal device. In Experiment 2 (oocyte, cumulus complexes, and follicular fluid evaluation), cows (n=26: control=13; mastitis=13) were submitted to follicular aspiration (ovum pickup) for oocyte quality and cumulus cells transcript evaluation. Transcript abundance of apoptosis markers (BCL2, BAX, PI3K, PTEN, FOXO3) was determined by real-time RT-PCR. Moreover, 7 days after the ovum pickup session, the dominant follicle was aspirated and follicular fluid samples were obtained. Exosomes were isolated from the follicular fluid by serial centrifugations, which were also performed for evaluation of particle size and concentration. Statistical analyses were performed using the SAS (SAS Institute Inc., Cary, NC, USA), and the GLIMMIX procedure was used to determine significant differences between groups. Gene expression and exosome data were submitted to the Student’s t-test. Ovulation rate [control 77.4% (24/31) and mastitis 57.7% (15/26); P=0.09] and viable oocytes rate [control 59.1% (130/220) and mastitis 41.9% (125/298); P=0.01] were higher in control animals. Additionally, there was a greater number of degenerate oocytes (control 6.7±1.2 and mastitis 13.3±5.5; P=0.001) in subclinical mastitis cows. There was greater abundance (P=0.003) of BAX cumulus cell transcripts and exosome mean (P=0.03) was smaller in subclinical mastitis cows. However, BCL2, PI3K, PTEN, nd FOXO3 cumulus cell transcripts was similar between treatments. In conclusion, ovulation rate, oocyte quality, and exosome diameter were smaller in cows with SCC >400.000 cells mL−1, demonstrating that subclinical mastitis can influence the fertility of dairy cows.

In this study, a GnRH agonist, leuprolide acetate (LA), was given as a single depot injection before 48 h of life to Wistar female rats allotted to prenatal (E16-18) and postnatal androgenization (day 5 of life) by the use of testosterone propionate, looking for reproductive endpoints. Remarkably, a single injection of LA increased the estrus cycles in the postnatal group (PostN) from 0% to 25% of the estrus cycles in the postnatal LA treated group (PostN L). LA also reduced the serum testosterone levels and cysts and atretic follicles in PostN L in contrast with rats (>100 days) from the PostN group (p = 0.04). Prenatally androgenized rats (PreN) exhibited significant modifications in the hypothalamic genes, such as Gnrh. To the best of our knowledge, this is the first study to show that blockage of the GnRH axis with leuprolide acetate depot prevented the development of typical features (anovulation, cysts, atretic follicles) in a postnatal testosterone propionate rat model of PCOS.

The aim was to evaluate the effect of subclinical mastitis by somatic cell count (SCC) on follicular dynamics, ovulation, oocyte and cumulus cell quality, exosome size and concentration in milk-producing cows. Crossbred cows (Bos taurus × Bos indicus; that is, Holstein × Gyr) were randomly allocated to control (SCC 400,000 cells mL−1) groups. In experiment 1 (follicular dynamics), cows (n = 57) were submitted to ultrasonographic evaluations every 24 h, after removal of an intravaginal progesterone device (Day 8) up to Day 10. From Day 10, ultrasound evaluations were performed every 12 h, until ovulation or until 96 h after progesterone device withdrawal, in order to follow final dominant follicle growth and ovulation. In experiment 2 (oocyte, cumulus cells, and follicular fluid evaluation), cows (n = 23) were submitted to follicular aspirations, preceded by synchronization of the emergence of the follicular wave. The levels of target genes in cumulus cells (BCL2, BAX, PI3K, PTEN, FOXO3) were evaluated by RT-qPCR. In the follicular fluid, the exosomes were isolated for evaluation of particle size. Data were analysed by the Glimmix procedure of SAS (SAS Institute Inc., Cary, NC, USA). Ovulation rate (P = 0.09) was higher in control cows [control 77.42% (24/31) and mastitis 57.69% (15/26)]. Viable oocyte rate (P = 0.01) was also higher in control cows [control 59.1% (130/220) and mastitis 41.9% (125/298)]. The dynamics of follicular growth did not differ between groups. The number of degenerate oocytes (P = 0.001) was higher in cows of the mastitis group. In the evaluation of cumulus cell gene expression, there was a higher abundance of BAX transcripts (P = 0.003) in cells of mastitis cows. Additionally, the mean and mode of exosome diameter in mastitis cows were smaller (P = 0.03 and P = 0.02, respectively). In conclusion, ovulation rate, oocyte quality, and follicular fluid exosome diameter were lower in cows with subclinical mastitis, demonstrating a link between mammary gland sanitary status and reproduction.

Adiponectin is the most abundantly produced human adipokine with anti-inflammatory, anti-oxidative, and insulin-sensitizing properties. Evidence from in vitro studies has indicated that adiponectin has a potential role in reproduction because it reduces the production of androstenedione in bovine theca cells in vitro. However, this effect on androgen production has not yet been observed in vivo. The current study evaluated the effect of adiponectin on androstenedione secretion and oxidative stress parameters in a rodent model. Seven-week-old female Balb/c mice (n = 33), previously treated with equine gonadotropin chorionic, were assigned to one of four different treatments: Group 1, control (phosphate-buffered saline); Group 2, adiponectin 0.1 μg/mL; Group 3, adiponectin 1.0 μg/mL; Group 4, adiponectin 5.0 μg/mL. After 24 h, all animals were euthanized and androstenedione levels were measured in the serum while oxidative stress markers were quantified in whole ovary tissue. Female mice treated with adiponectin exhibited a significant reduction (about 60%) in serum androstenedione levels in comparison to controls. Androstenedione levels decreased from 0.78 ± 0.4 ng/mL (mean ± SD) in controls to 0.28 ± 0.06 ng/mL after adiponectin (5 μg/mL) treatment (P = 0.01). This change in androgen secretion after 24 hours of treatment was associated with a significant reduction in the expression of CYP11A1 and STAR (but not CYP17A1). In addition, ovarian AOPP product levels, a direct product of protein oxidation, decreased significantly in adiponectin-treated mice (5 μg/mL); AOPP (mean ± SD) decreased to 4.3 ± 2.1 μmol/L in comparison with that of the controls (11.5 ± 1.7 μmol/L; P = 0.0003). Our results demonstrated for the first time that acute treatment with adiponectin reduced the levels of a direct oxidative stress marker in the ovary as well as decreased androstenedione serum levels in vivo after 24 h.

O objetivo deste estudo foi mostrar os efeitos da dieta ad libitum sobre a saúde dos animais de experimentação comparando-se os parâmetros bioquímicos em ratos alimentados com diferentes tipos de dieta e diferentes formas de alimentação. Três grupos de oito animais foram alimentados com ração padrão em quantidade controlada (30 g/dia)-grupo1, ração padrão ad libitum - grupo 2 e o grupo 3 ração hipercalórica ad libitum. Amostras de sangue foram coletadas antes e depois do experimento, para análise dos seguintes parâmetros bioquímicos: colesterol total (CT), lipoproteína de alta densidade (HDL-c), triglicérides(TG) e glicemia (G). Os resultados mostraram que os grupos 2 e 3, que receberam dieta ad libitum,apresentaram níveis mais elevados de CT, HDL-c e TG. Este estudo mostrou que a dieta padrão ad libitumaltera os parâmetros bioquímicos da mesma forma, que uma dieta hipercalórica, colocando em risco a saúde dos animais de experimentação.