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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.
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Selection strategies are performed post-fertilization when the random combination of paternal and maternal genomes has already occurred. It would be greatly advantageous to eliminate meiotic uncertainty by selecting genetically superior gametes before fertilization. To achieve this goal, haploid embryonic cells and embryonic stem cell lineages could be derived, genotyped, and used to substitute gametes. On the paternal side, androgenetic development can be achieved by removing the maternal chromosomes from the oocyte before or after fertilization. We have shown that once developed into an embryo, haploid cells can be removed for genotyping and, if carrying the selected genome, be used to replace sperm at fertilization. A similar strategy can be used on the maternal side by activating the oocyte parthenogenetically and using some embryonic cells for genotyping while the remaining are used to produce diploid embryos by fertilization. Placed together, both androgenetic and parthenogenetic haploid cells that have been genotyped to identify optimal genomes can be used to produce offspring with predetermined genomes. Successes and problems in developing such a breeding platform to achieve this goal are described and discussed below.
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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.
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The uterine tube extracellular matrix is a key component that regulates tubal tissue physiology, and it has a region-specific structural distribution, which is directly associated to its functions. Considering this, the application of biological matrices in culture systems is an interesting strategy to develop biomimetic tubal microenvironments and enhance their complexity. However, there are no established protocols to produce tubal biological matrices that consider the organ morphophysiology for such applications. Therefore, this study aimed to establish region-specific protocols to obtain decellularized scaffolds derived from porcine infundibulum, ampulla, and isthmus to provide suitable sources of biomaterials for tissue-engineering approaches. Porcine uterine tubes were decellularized in solutions of 0.1% SDS and 0.5% Triton X-100. The decellularization efficiency was evaluated by DAPI staining and DNA quantification. We analyzed the ECM composition and structure by optical and scanning electronic microscopy, FTIR, and Raman spectroscopy. DNA and DAPI assays validated the decellularization, presenting a significative reduction in cellular content. Structural and spectroscopy analyses revealed that the produced scaffolds remained well structured and with the ECM composition preserved. YS and HEK293 cells were used to attest cytocompatibility, allowing high cell viability rates and successful interaction with the scaffolds. These results suggest that such matrices are applicable for future biotechnological approaches in the reproductive field.
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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.
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Criopreservação , Crioprotetores , Epididimo , Nanopartículas , Preservação do Sêmen , Motilidade dos Espermatozoides , Espermatozoides , Masculino , Animais , Criopreservação/métodos , Criopreservação/veterinária , Preservação do Sêmen/métodos , Preservação do Sêmen/veterinária , Crioprotetores/farmacologia , Espermatozoides/citologia , Epididimo/citologia , Bovinos , Nanopartículas/química , Gema de Ovo/química , Análise do Sêmen , CitoplasmaRESUMO
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.
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Vesículas Extracelulares , MicroRNAs , Feminino , Animais , Bovinos , Líquido Folicular/metabolismo , Progesterona/metabolismo , Folículo Ovariano/metabolismo , Ovário/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Oócitos/metabolismo , Corpo Lúteo/metabolismo , Vesículas Extracelulares/genética , Expressão GênicaRESUMO
Histone deacetylation is an important mechanism involved in human breast cancer tumorigenesis and recent veterinary oncology studies also demonstrate a similar relationship in some canine neoplasms. The use of HDAC inhibitors in vitro and in vivo has demonstrated antitumor action on several strains of human and animal cancers. The present study aims to correlate the expression of H3K9Ac, H4K12Ac, HDAC1, HDAC2 and HDAC6 in simple mammary carcinomas in dogs with clinicopathological parameters and overall survival time. To this end, 61 samples of simple breast carcinomas were analyzed by the immunohistochemistry technique with subsequent validation of the antibodies by the Western Blot technique. The expressions obtained via a semi-quantitative way were categorized by assigning scores and classified into high or low expressions according to the given score, except for HDAC6, when the marking percentage was considered and subdivided into high and low expressions using the median value. For statistical analysis, the chi-square test or Fisher exact test were used as univariate analysis and correspondence analysis as a multivariate test, in addition to the Kaplan-Meier survival analysis. In the studied samples, the highest frequencies were determined for the high expression proteins H4K12Ac (88.5%), HDAC2 (65.6%) and HDAC6 (56.7%) and the low expression proteins H3K9Ac (73.8%) and HDAC1 (54.1%). An association between the low expression of HDAC1 and the presence of lymph node metastasis (p = 0.035) was indicated by univariate analysis while the high expression of HDAC1 was associated with favorable prognostic factors, such as the absence of lymph node metastasis and low mitotic index by multivariate analysis. Also, by multivariate analysis, the low expression of HDAC6 was correlated with the low expression of Ki67, smaller tumors, and better prognosis factors as well. Protein expression was not correlated with patients' overall survival time (p > 0.05). The high expressions of HDAC2 and HDAC6 in mammary carcinomas in female dogs may be useful information for research involving therapeutic targets with iHDACs since their inhibition favors hyperacetylation and transcription of tumor suppressor genes.
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Mechanisms of cell reprogramming by pluripotency-related transcription factors or nuclear transfer seem to be mediated by similar pathways, and the study of the contribution of OCT4 and SOX2 in both processes may help elucidate the mechanisms responsible for pluripotency. Bovine fibroblasts expressing exogenous OCT4 or SOX2, or both, were analyzed regarding the expression of pluripotency factors and imprinted genes H19 and IGF2R, and used for in vitro reprogramming. The expression of the H19 gene was increased in the control sorted group, and putative iPSC-like cells were obtained when cells were not submitted to cell sorting. When sorted cells expressing OCT4, SOX2, or none (control) were used as donor cells for somatic cell nuclear transfer, fusion rates were 60.0% vs. 64.95% and 70.53% vs. 67.24% for SOX2 vs. control and OCT4 vs. control groups, respectively; cleavage rates were 66.66% vs. 81.68% and 86.47% vs. 85.18%, respectively; blastocyst rates were 33.05% vs. 44.15% and 52.06% vs. 44.78%, respectively. These results show that the production of embryos by NT resulted in similar rates of in vitro developmental competence compared to control cells regardless of different profiles of pluripotency-related gene expression presented by donor cells; however, induced reprogramming was compromised after cell sorting.
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The present study developed a review and exploration of data in public and already validated repositories. The main objective was to identify the pathways involved in ruminants' cervical dilatation, which are conserved between cattle and sheep in the follicular and luteal phases of the reproductive cycle. In cattle, 1961 genes were more differentially expressed in the follicular phase and 1560 in the luteal phase. An amount of 24 genes were considered exclusively expressed from these. A total of 18 genes were in the follicular phase and 6 genes were in the luteal phase. In sheep, 2126 genes were more differentially expressed in the follicular phase and 2469 genes were more differentially expressed in the luteal phase. Hoxb genes were identified in both species and are correlated with the PI3K/Akt pathway. PI3K/Akt was also found in both cattle and sheep, appearing prominently in the follicular and luteal phases of both species. Our analyses have pointed out that the PI3K/Akt pathway and the Hoxb genes appear in prominence in modulating mechanisms that involve estrus alterations in the cervix. PI3K/Akt appears to be an important pathway in the cervical relaxation process.
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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.
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Técnicas de Maturação in Vitro de Oócitos , Oócitos , Feminino , Animais , Bovinos , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/metabolismo , Oogênese/genética , Células do Cúmulo/metabolismo , Embrião de Mamíferos , Blastocisto , Fertilização in vitro/veterinária , Fertilização in vitro/métodos , Desenvolvimento Embrionário/fisiologiaRESUMO
Cloning by somatic cell Nuclear Transfer (SCNT) is a powerful technology capable of reprograming terminally differentiated cells to totipotency for generating whole animals or pluripotent stem cells for use in cell therapy, drug screening, and other biotechnological applications. However, the broad usage of SCNT remains limited due to its high cost and low efficiency in obtaining live and healthy offspring. In this chapter, we first briefly discuss the epigenetic constraints responsible for the low efficiency of SCNT and current attempts to overcome them. We then describe our bovine SCNT protocol for delivering live cloned calves and addressing basic questions about nuclear reprogramming. Other research groups can benefit from our basic protocol and build up on it to improve SCNT in the future. Strategies to correct or mitigate epigenetic errors (e.g., correcting imprinting loci, overexpression of demethylases, chromatin-modifying drugs) can integrate the protocol described here.
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Técnicas de Transferência Nuclear , Células-Tronco Pluripotentes , Bovinos , Animais , Técnicas de Transferência Nuclear/veterinária , Clonagem de Organismos/métodos , Biotecnologia , Clonagem MolecularRESUMO
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.
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Insulinas , MicroRNAs , Feminino , Bovinos , Animais , MicroRNAs/genética , MicroRNAs/farmacologia , Folículo Ovariano , Oócitos , Líquido Folicular , ProgesteronaRESUMO
BACKGROUND: The generation of induced pluripotent stem cells (iPSC) has been a game-changer in translational and regenerative medicine; however, their large-scale applicability is still hampered by the scarcity of accessible, safe, and reproducible protocols. The porcine model is a large biomedical model that enables translational applications, including gene editing, long term in vivo and offspring analysis; therefore, suitable for both medicine and animal production. AIM: To reprogramme in vitro into pluripotency, and herein urine-derived cells (UDCs) were isolated from porcine urine. METHODS: The UDCs were reprogrammed in vitro using human or murine octamer-binding transcription factor 4 (OCT4), SRY-box2 (SOX2), Kruppel-like factor 4 (KLF4), and C-MYC, and cultured with basic fibroblast growth factor (bFGF) supplementation. To characterize the putative porcine iPSCs three clonal lineages were submitted to immunocytochemistry for alkaline phosphatase (AP), OCT4, SOX2, NANOG, TRA1 81 and SSEA 1 detection. Endogenous transcripts related to the pluripotency (OCT4, SOX2 and NANOG) were analyzed via reverse transcription quantitative real-time polymerase chain reaction in different time points during the culture, and all three lineages formed embryoid bodies (EBs) when cultured in suspension without bFGF supplementation. RESULTS: The UDCs were isolated from swine urine samples and when at passage 2 submitted to in vitro reprogramming. Colonies of putative iPSCs were obtained only from UDCs transduced with the murine factors (mOSKM), but not from human factors (hOSKM). Three clonal lineages were isolated and further cultured for at least 28 passages, all the lineages were positive for AP detection, the OCT4, SOX2, NANOG markers, albeit the immunocytochemical analysis also revealed heterogeneous phenotypic profiles among lineages and passages for NANOG and SSEA1, similar results were observed in the abundance of the endogenous transcripts related to pluripotent state. All the clonal lineages when cultured in suspension without bFGF were able to form EBs expressing ectoderm and mesoderm layers transcripts. CONCLUSION: For the first time UDCs were isolated in the swine model and reprogrammed into a pluripotent-like state, enabling new numerous applications in both human or veterinary regenerative medicine.
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Turner syndrome (TS) is a genetic disorder in females with X Chromosome monosomy associated with highly variable clinical features, including premature primary gonadal failure leading to ovarian dysfunction and infertility. The mechanism of development of primordial germ cells (PGCs) and their connection with ovarian failure in TS is poorly understood. An in vitro model of PGCs from TS would be beneficial for investigating genetic and epigenetic factors that influence germ cell specification. Here we investigated the potential of reprogramming peripheral mononuclear blood cells from TS women (PBMCs-TS) into iPSCs following in vitro differentiation in hPGCLCs. All hiPSCs-TS lines demonstrated pluripotency state and were capable of differentiation into three embryonic layers (ectoderm, endoderm, and mesoderm). The PGCLCs-TS recapitulated the initial germline development period regarding transcripts and protein marks, including the epigenetic profile. Overall, our results highlighted the feasibility of producing in vitro models to help the understanding of the mechanisms associated with germ cell formation in TS.
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Técnicas de Cultura de Células/métodos , Células Germinativas/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Síndrome de Turner/patologia , Biomarcadores/metabolismo , Estudos de Casos e Controles , Diferenciação Celular/genética , Linhagem Celular , Reprogramação Celular/genética , Análise Citogenética , Corpos Embrioides/citologia , Epigênese Genética , Vetores Genéticos/metabolismo , Células Germinativas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Plasmídeos/genéticaRESUMO
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.
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Vesículas Extracelulares , Oogênese , Animais , Bovinos , Comunicação Celular , Feminino , Líquido Folicular , Lipídeos , OócitosRESUMO
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.
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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.
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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.
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Folículo Ovariano/fisiologia , Taxa de Gravidez , Progesterona/sangue , Transcriptoma , Animais , Bovinos , Células do Cúmulo/citologia , Feminino , Inseminação Artificial/veterinária , Oócitos/citologia , Folículo Ovariano/citologia , Ovário/citologia , Ovário/fisiologia , GravidezRESUMO
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.
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Reprogramação Celular/genética , Metilação de DNA/genética , Desenvolvimento Embrionário/genética , Histona Metiltransferases/genética , Animais , Blastocisto , Bovinos , Diferenciação Celular , Clonagem de Organismos/métodos , Transferência Embrionária/métodos , Epigênese Genética/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/genética , Técnicas de Transferência Nuclear , Oócitos/crescimento & desenvolvimento , Processamento de Proteína Pós-Traducional/genética , Quinazolinas/farmacologiaRESUMO
Gene editing in large animal models for future applications in translational medicine and food production must be deeply investigated for an increase of knowledge. The mitochondrial transcription factor A (TFAM) is a member of the HMGB subfamily that binds to mtDNA promoters. This gene maintains mtDNA, and it is essential for the initiation of mtDNA transcription. Lately, we generated a new cell line through the disruption of the TFAM gene in bovine fibroblast cells by CRISPR/Cas 9 technology. We showed that the CRISPR/Cas9 design was efficient through the generation of heterozygous mutant clones. In this context, once this gene regulates the mtDNA replication specificity, the study aimed to determine if the post-edited cells are capable of in vitro maintenance and assess if they present changes in mtDNA copies and mitochondrial membrane potential after successive passages in culture. The post-edited cells were expanded in culture, and we performed a growth curve, doubling time, cell viability, mitochondrial DNA copy number, and mitochondrial membrane potential assays. The editing process did not make cell culture unfeasible, even though cell growth rate and viability were decreased compared to control since we observed the cells grow well when cultured in a medium supplemented with uridine and pyruvate. They also exhibited a classical fibroblastoid appearance. The RT-qPCR to determine the mtDNA copy number showed a decrease in the edited clones compared to the non-edited ones (control) in different cell passages. Cell staining with Mitotracker Green and red suggests a reduction in red fluorescence in the edited cells compared to the non-edited cells. Thus, through characterization, we demonstrated that the TFAM gene is critical to mitochondrial maintenance due to its interference in the stability of the mitochondrial DNA copy number in different cell passages and membrane potential confirming the decrease in mitochondrial activity in cells edited in heterozygosis.