Altrenogest during early pregnancy modulates uterine glandular epithelium and endometrial growth factor expression at the time implantation in pigs

This study evaluated the effects of supplying altrenogest from day 6-12 of pregnancy on the endometrial glandular epithelium, corpora lutea (CL) morphology, and endometrial and CL gene expression. A total of 12 crossbred females (Landrace × Large White) were used. The females were assigned to 4 treatments according to a random design with a 2 × 2 factorial arrangement, with two categories (sow or gilt) and two treatments (non-treated and treated with altrenogest). On day 6 of pregnancy, animals were allocated to one of the following groups: non-treated (NT, n = 6; 3 sows and 3 gilts), and (T, n = 6; 3 sows and 3 gilts) treated daily with 20 mg of altrenogest, from day 6-12 of pregnancy. All animals were euthanized on day 13 of pregnancy. All CLs were individually weighed, and their volume were determined. The endometrial glandular density (GD), mean glandular area (MGA), and vascular density (VD) were determined by histomorphometric and immunohistochemical analyses. Endometrium samples were collected and analyzed by qRT-PCR to evaluate the abundance of transcripts for VEGF and IGF-I. Females in the T group had higher MGA (P < 0.05) compared to the NT group. There was no effect of treatment on GD or VD for both experimental groups. Sows in the T group had augmented expression of IGF-I (P < 0.05). Progestagen had no detrimental effect on CL morphology. In conclusion, altrenogest improves the uterine environment during the peri-implantation period in pigs without compromising corpora lutea development.(AU)

Pluripotent stem cells proliferation is associated with placentation in dogs

Pluripotent stem cells have been studied as source of cells for regenerative medicine and acquire or genetic diseases, as an innovative therapy. Most tissues have stem cells populations, however in few quantities or impossible to be used during adult life, which lead to scientists look for new sources. Thus, this study aimed to analyze the presence of pluripotent cells in the uterus and placenta, following up non-pregnant, pregnant (begin, middle, and final), and postpartum periods in dogs. The uteri were obtained from social castration programs for population control in Pirassununga, Sao Paulo, Brazil. It was collected 20 uteri at different stages. The samples were fixed and processed for immunohistochemical analysis of NANOG, OCT4 and SOX2 expression, knowing as pluripotent stem cells makers. Our results showed positive expression for NANOG, OCT4 and SOX2 in all stages of gestation and nonpregnant uterus; however, we highlight some quantitative different between stages. OCT4 showed more expression in non-pregnant uterus than NANOG and SOX2, and its expression increased in pregnant uterus. In pregnant uterus there was more expression of NANOG than OCT4 and SOX2. Interesting, no difference was found between these markers in the other periods. In conclusion, it was possible to identify pluripotent stem cells in all periods in dog placenta and uterus, however during the early stage of pregnancy we observed more pluripotent stem cells than in all the others periods confirming the high plasticity and regeneration capacity of the uterine tissue.

Pluripotent stem cells proliferation is associated with placentation in dogs

Pluripotent stem cells have been studied as source of cells for regenerative medicine and acquire or genetic diseases, as an innovative therapy. Most tissues have stem cells populations, however in few quantities or impossible to be used during adult life, which lead to scientists look for new sources. Thus, this study aimed to analyze the presence of pluripotent cells in the uterus and placenta, following up non-pregnant, pregnant (begin, middle, and final), and postpartum periods in dogs. The uteri were obtained from social castration programs for population control in Pirassununga, Sao Paulo, Brazil. It was collected 20 uteri at different stages. The samples were fixed and processed for immunohistochemical analysis of NANOG, OCT4 and SOX2 expression, knowing as pluripotent stem cells makers. Our results showed positive expression for NANOG, OCT4 and SOX2 in all stages of gestation and nonpregnant uterus; however, we highlight some quantitative different between stages. OCT4 showed more expression in non-pregnant uterus than NANOG and SOX2, and its expression increased in pregnant uterus. In pregnant uterus there was more expression of NANOG than OCT4 and SOX2. Interesting, no difference was found between these markers in the other periods. In conclusion, it was possible to identify pluripotent stem cells in all periods in dog placenta and uterus, however during the early stage of pregnancy we observed more pluripotent stem cells than in all the others periods confirming the high plasticity and regeneration capacity of the uterine tissue.(AU)

Endometrial prostaglandin F2α in vitro production and its modulation regarding dominant follicle position in cattle / Produção in vitro de PGF F2α endometrial e sua modulação referente ao folículo dominante em bovinos

Prostaglandin F2α (PGF2α) determines luteolysis in cattle, and the ability to manipulate its endogenous synthesis is indispensible for large-scale animal breeding. Estradiol (E2) and progesterone (P4) modulate several molecular pathways in endometrial cells, including the synthesis of PGF2α; however, its specific mechanisms are still not totally known. This study investigated the production in vitro and possible modulation of endometrial PGF2α due to a local effect of endogenous E2 in the ipsilateral uterine horn (UH) containing the dominant follicle (DF) or from P4 in ipsilateral horn containing the corpus luteum (CL). The PGF2α stimulators oxytocin (OT) and phorbol 12,13-dibutyrate (PDBu) were incubated with endometrial explants, and PGF2α content was measured. For that, cycling cows were synchronized, the development of DF and CL was examined by ultrasonography and on the seventh day of the estrous cycle, endometrial explants were collected and cultured in medium supplemented with 10-6 M PDBu or 10-6 M OT or non-supplemented. Media samples were collected immediately after treatment and 60 min later. Radioimmunoassay showed that the PGF2α content of the UH ipsilateral to the DF was 49% less than that of the contralateral UH (8.22 ± 0.95 vs. 12.24 ± 0.95 pg/mL/mg tissue, respectively; P < 0.01). However, the PGF2α levels did not differ between the UHs as a function of the CL position (9.46 ± 0.95 vs. 11 ± 0.95 pg/mL/mg; P > 0.05). The cellular stimulators promoted an increase in PGF2α synthesis (P < 0.02), and the effects differed among the animals (P < 0.04). The PGF2a production was higher in the explants treated with PDBu rather than OT (13.68 ± 1.16 vs. 10.01 ± 1.16 pg/mL/mg tissue, respectively; P < 0.05). In conclusion, PGF2α synthesis is modulated by the presence of the DF (local E2) but not the CL (local P4), and both PDBu and OT stimulated PGF2a synthesis.(AU)

A prostaglandina F2α (PGF2α) determina a luteólise em bovinos. A capacidade de manipular sua síntese endógena é indispensável para a produção animal em grande escala. O estradiol (E2) e a progesterona (P4) modulam diversas vias moleculares das células endometriais, incluindo a síntese de PGF2α; no entanto, pouco se sabe sobre seus mecanismos específicos. Este trabalho investigou a produção in vitro e a possível modulação da PGF2α endometrial devido a um efeito local do E2 endógeno no corno uterino ipsilateral ao folículo dominante (FD) ou da P4 no corno ipsilateral ao corpo lúteo (CL). Os estimuladores de PGF2α oxitocina (OT) e 12,23-dibutirato de forbol (PDBu) foram incubados com explantes endometriais, e o conteúdo de PGF2α foi mensurado. Para tal, vacas cíclicas foram sincronizadas, o desenvolvimento de FD e CL foi examinado por ultrassonografia, e no 17º dia do ciclo estral os explantes endometriais foram coletados e cultivados em meio ou suplementados com PDBu 10-6M ou 10-6M OT. As amostras de meio foram coletadas imediatamente após o tratamento e sessenta minutos depois. O radioimunoensaio mostrou que o conteúdo de PGF2α do corno ipsilateral ao FD foi 49% menor que o do corno contralateral (8,22 ± 0,95 vs. 12,24 ± 0,95 pg/mL/mg de tecido, respectivamente, P < 0,01). No entanto, os níveis de PGF2α não diferiram entre os cornos em função da posição do CL (9,46 ± 0,95 versus 11 ± 0,95 pg/mL/mg; P > 0,05). Os estimuladores celulares promoveram um aumento na síntese de PGF2α (P < 0,02), e os efeitos diferiram entre os animais (P < 0,04). A produção de PGF2α foi maior nos explantes tratados com PDBu em comparação à OT (13,68 ± 1,16 versus 10,01 ± 1,16 pg/mL/mg de tecido, respectivamente, P < 0,05). A conclusão obtida foi que a síntese de PGF2α é: modulada pela presença do FD (E2 local), mas não do CL (P4 local); e estimulada por PDBu e OT.(AU)

Endometrial prostaglandin F2α in vitro production and its modulation regarding dominant follicle position in cattle / Produção in vitro de PGF F2α endometrial e sua modulação referente ao folículo dominante em bovinos

Prostaglandin F2α (PGF2α) determines luteolysis in cattle, and the ability to manipulate its endogenous synthesis is indispensible for large-scale animal breeding. Estradiol (E2) and progesterone (P4) modulate several molecular pathways in endometrial cells, including the synthesis of PGF2α; however, its specific mechanisms are still not totally known. This study investigated the production in vitro and possible modulation of endometrial PGF2α due to a local effect of endogenous E2 in the ipsilateral uterine horn (UH) containing the dominant follicle (DF) or from P4 in ipsilateral horn containing the corpus luteum (CL). The PGF2α stimulators oxytocin (OT) and phorbol 12,13-dibutyrate (PDBu) were incubated with endometrial explants, and PGF2α content was measured. For that, cycling cows were synchronized, the development of DF and CL was examined by ultrasonography and on the seventh day of the estrous cycle, endometrial explants were collected and cultured in medium supplemented with 10-6 M PDBu or 10-6 M OT or non-supplemented. Media samples were collected immediately after treatment and 60 min later. Radioimmunoassay showed that the PGF2α content of the UH ipsilateral to the DF was 49% less than that of the contralateral UH (8.22 ± 0.95 vs. 12.24 ± 0.95 pg/mL/mg tissue, respectively; P < 0.01). However, the PGF2α levels did not differ between the UHs as a function of the CL position (9.46 ± 0.95 vs. 11 ± 0.95 pg/mL/mg; P > 0.05). The cellular stimulators promoted an increase in PGF2α synthesis (P < 0.02), and the effects differed among the animals (P < 0.04). The PGF2a production was higher in the explants treated with PDBu rather than OT (13.68 ± 1.16 vs. 10.01 ± 1.16 pg/mL/mg tissue, respectively; P < 0.05). In conclusion, PGF2α synthesis is modulated by the presence of the DF (local E2) but not the CL (local P4), and both PDBu and OT stimulated PGF2a synthesis.(AU)

A prostaglandina F2α (PGF2α) determina a luteólise em bovinos. A capacidade de manipular sua síntese endógena é indispensável para a produção animal em grande escala. O estradiol (E2) e a progesterona (P4) modulam diversas vias moleculares das células endometriais, incluindo a síntese de PGF2α; no entanto, pouco se sabe sobre seus mecanismos específicos. Este trabalho investigou a produção in vitro e a possível modulação da PGF2α endometrial devido a um efeito local do E2 endógeno no corno uterino ipsilateral ao folículo dominante (FD) ou da P4 no corno ipsilateral ao corpo lúteo (CL). Os estimuladores de PGF2α oxitocina (OT) e 12,23-dibutirato de forbol (PDBu) foram incubados com explantes endometriais, e o conteúdo de PGF2α foi mensurado. Para tal, vacas cíclicas foram sincronizadas, o desenvolvimento de FD e CL foi examinado por ultrassonografia, e no 17º dia do ciclo estral os explantes endometriais foram coletados e cultivados em meio ou suplementados com PDBu 10-6M ou 10-6M OT. As amostras de meio foram coletadas imediatamente após o tratamento e sessenta minutos depois. O radioimunoensaio mostrou que o conteúdo de PGF2α do corno ipsilateral ao FD foi 49% menor que o do corno contralateral (8,22 ± 0,95 vs. 12,24 ± 0,95 pg/mL/mg de tecido, respectivamente, P < 0,01). No entanto, os níveis de PGF2α não diferiram entre os cornos em função da posição do CL (9,46 ± 0,95 versus 11 ± 0,95 pg/mL/mg; P > 0,05). Os estimuladores celulares promoveram um aumento na síntese de PGF2α (P < 0,02), e os efeitos diferiram entre os animais (P < 0,04). A produção de PGF2α foi maior nos explantes tratados com PDBu em comparação à OT (13,68 ± 1,16 versus 10,01 ± 1,16 pg/mL/mg de tecido, respectivamente, P < 0,05). A conclusão obtida foi que a síntese de PGF2α é: modulada pela presença do FD (E2 local), mas não do CL (P4 local); e estimulada por PDBu e OT.(AU)

Células-tronco pluripotentes induzidas (células iPS) em animais domésticos e a possibilidade de geração in vitro de gametas / Induced pluripotent stem cells (iPS cells) in domestic animals and the possibility to generate gametes in vitro

Células-tronco são conhecidas pela característica de alto potencial de auto-renovação e podem ser classificadas segundo seu estágio de indiferenciação e perfil epigenético. Células-tronco embrionárias (CTEs) e células-tronco adultas são bastante estudadas e caracterizadas, principalmente nos modelos humano e em camundongos por apresentarem novas possibilidades tanto para a medicina regenerativa quanto para o entendimento do desenvolvimento inicial dos mamíferos. Contudo, a derivação e a manutenção de CTEs em espécies domésticas é desafiadora e apresenta resultados inconsistentes em relação à manutenção da pluripotência in vitro. Nesse contexto, a geração das células pluripotentes induzidas in vitro (células iPS ou iPSCs) é imprescindível para a geração de novas possibilidades na medicina veterinária regenerativa e reprodutiva devido à capacidade de diferenciação destas em uma variedade de outros tipos celulares, inclusive em animais. Assim, esta revisão apresenta a geração das células iPS em animais domésticos, e em especial, recentes estudos sobre as etapas necessárias para a possibilidade de geração de gametas funcionais in vitro, uma importante contribuição na correção de infertilidades, conservação de espécies ameaçadas de extinção e geração de indivíduos geneticamente superiores ou modificados para aplicações agropecuárias ou biomédicas.(AU)

Stem cells are widely known for their high potential for self-renewal, being classified according to their stage of undifferentiation and epigenetic profile. Embryonic stem cells (ES) and adult stem cells are already well studied and characterized, mainly in human and mouse models, once they present new possibilities both for regenerative medicine and for understanding the initial development of mammals. However, the derivation and maintenance of ES in domestic species is challenging and presents inconsistent results regarding maintenance of in vitro pluripotency. In this context, the derivation of in vitro induced pluripotent cells (iPS cells or iPSCs) enables new possibilities in regenerative and reproductive veterinary medicine due to their ability to differentiate into a variety of other cell types. Therefore, this review presents the generation of iPS cells in domestic animals, and focuses on recent studies on the steps necessary for the generation of functional gametes in vitro, an important contribution of stem cells aiming the correction of infertility, the conservation of species in risk of extinction and for the generation of genetically superior or modified organisms for agricultural and biomedical applications.(AU)

Células-tronco pluripotentes induzidas (células iPS) em animais domésticos e a possibilidade de geração in vitro de gametas / Induced pluripotent stem cells (iPS cells) in domestic animals and the possibility to generate gametes in vitro

Células-tronco são conhecidas pela característica de alto potencial de auto-renovação e podem ser classificadas segundo seu estágio de indiferenciação e perfil epigenético. Células-tronco embrionárias (CTEs) e células-tronco adultas são bastante estudadas e caracterizadas, principalmente nos modelos humano e em camundongos por apresentarem novas possibilidades tanto para a medicina regenerativa quanto para o entendimento do desenvolvimento inicial dos mamíferos. Contudo, a derivação e a manutenção de CTEs em espécies domésticas é desafiadora e apresenta resultados inconsistentes em relação à manutenção da pluripotência in vitro. Nesse contexto, a geração das células pluripotentes induzidas in vitro (células iPS ou iPSCs) é imprescindível para a geração de novas possibilidades na medicina veterinária regenerativa e reprodutiva devido à capacidade de diferenciação destas em uma variedade de outros tipos celulares, inclusive em animais. Assim, esta revisão apresenta a geração das células iPS em animais domésticos, e em especial, recentes estudos sobre as etapas necessárias para a possibilidade de geração de gametas funcionais in vitro, uma importante contribuição na correção de infertilidades, conservação de espécies ameaçadas de extinção e geração de indivíduos geneticamente superiores ou modificados para aplicações agropecuárias ou biomédicas.

Stem cells are widely known for their high potential for self-renewal, being classified according to their stage of undifferentiation and epigenetic profile. Embryonic stem cells (ES) and adult stem cells are already well studied and characterized, mainly in human and mouse models, once they present new possibilities both for regenerative medicine and for understanding the initial development of mammals. However, the derivation and maintenance of ES in domestic species is challenging and presents inconsistent results regarding maintenance of in vitro pluripotency. In this context, the derivation of in vitro induced pluripotent cells (iPS cells or iPSCs) enables new possibilities in regenerative and reproductive veterinary medicine due to their ability to differentiate into a variety of other cell types. Therefore, this review presents the generation of iPS cells in domestic animals, and focuses on recent studies on the steps necessary for the generation of functional gametes in vitro, an important contribution of stem cells aiming the correction of infertility, the conservation of species in risk of extinction and for the generation of genetically superior or modified organisms for agricultural and biomedical applications.

Achievements and perspectives in cloned and transgenic cattle production by nuclear transfer: influence of cell type, epigenetic status and new technology

Genetically modified cattle production is motivated by many factors, including recombinant protein production for therapeutic purposes, disease models and animals presenting improved production traits. Nuclear transfer (NT), combined with efficient cultivation methods, genetic modification and donor cell selection is important for transgenic cattle production. Studies have found that adult cells (such as fibroblasts and cumulus cells, among others) used as nuclear donors achieved results similar to those of fetal cells, with the advantages of easier collection and a known genotype/phenotype. However, no consensus has been reached on the influence of cell type on transgene expression levels and post-reprogramming capacity after nuclear transfer, and these factors appear to be related to epigenetic factors. The development of new strategies, such as chromatin-modifying agents (CMAs), in vitro generation of induced pluripotent cells (iPS cells) and precise genome editing techniques are being explored and may influence nuclear reprogramming success for efficiently producing genetically modified bovine clones.

Achievements and perspectives in cloned and transgenic cattle production by nuclear transfer: influence of cell type, epigenetic status and new technology

Genetically modified cattle production is motivated by many factors, including recombinant protein production for therapeutic purposes, disease models and animals presenting improved production traits. Nuclear transfer (NT), combined with efficient cultivation methods, genetic modification and donor cell selection is important for transgenic cattle production. Studies have found that adult cells (such as fibroblasts and cumulus cells, among others) used as nuclear donors achieved results similar to those of fetal cells, with the advantages of easier collection and a known genotype/phenotype. However, no consensus has been reached on the influence of cell type on transgene expression levels and post-reprogramming capacity after nuclear transfer, and these factors appear to be related to epigenetic factors. The development of new strategies, such as chromatin-modifying agents (CMAs), in vitro generation of induced pluripotent cells (iPS cells) and precise genome editing techniques are being explored and may influence nuclear reprogramming success for efficiently producing genetically modified bovine clones.(AU)

Rabbit olfactory stem cells. Isolation protocol and characterization

PURPOSE:To describe a new technique for isolation of a mesenchymal stem cells (MSCs) population from the olfactory mucosa in rabbits.METHODS:Olfactory stem cells (OSCs) were retrieved from under the cribriform plate of the Ethmoid bone. Several assays were accomplished to characterize the cell population and attest its viability in vitro. The cells were submitted to flow cytometry with the antibodies CD34, CD45, CD73, CD79, CD90 and CD105 and also they were induced to differentiate in three lineages. Functional evaluation involved analysis of in vitro growth behavior, colony forming unit like fibroblasts (CFU-f) and cryopreservation response. Further transduction with Green Fluorescent Protein (GFP) was also performed.RESULTS:The OSCs showed mesenchymal features, as positive response to CD34, CD73 and CD90 antibodies and plasticity. Additionally, these cells have high proliferated rate, and they could be cultured through many passages and kept the ability to proliferate and differentiate after cryopreservation. The positive response to the transduction signalizes the possibility of cellular tracking in vivo. This is a desirable feature in case those cells are used for pre-clinical trials.CONCLUSION:The cells harvested were mesenchymal stem cells and the technique described is therefore efficient for rabbit olfactory stem cells isolation.(AU)

Challenges and perspectives to enhance cattle production via in vitro techniques: focus on epigenetics and cell-secreted vesicles / Desafios e perspectivas na produção in vitro de embriões bovinos: enfoque epigenético e em vesículas secretadas por células

This review aim to present some clinical problems found in IVP-derived animals focusing on NT procedures and to discuss the possible role of epigenetics in such process. Also, as cell-secreted vesicles have been reported as possible regulators of important physiological reproductive processes such as folliculogenesis and fertilization, it is also presented herein a new perspective of manipulating the pre-implantation period trough effector molecules contained in such vesicles.(AU)

Nesta revisão, apresentamos alguns problemas clínicos encontrados nos animais derivados de PIV, principalmente derivados de transferência de núcleo, e discutimos o possível papel da epigenética em tais processos. Além disso, uma vez que vesículas secretadas por células têm sido descritas como possíveis reguladores de processos reprodutivos fisiológicos importantes, tais como a foliculogênese e a fertilização, estas são aqui apresentadas como uma possível nova ferramenta para a manipulação do período embrionário pré-implantacional através de moléculas efetoras, contidas em tais vesículas.(AU)

Exploring the effects of second estrus synchronization and dietary flushing on the incidence of ovarian cysts in gilts by using exogenous gonadotropins / Explorando os efeitos da sincronização do segundo estro e flushing alimentar sobre a incidência de cistos ovarianos em marrãs utilizando gonadotrofinas exógenas

Estrus stimulation by exogenous gonadotropins (EG) in association with dietary flushing is an important tool for theimprovement of gilt reproductive performance. However, there is evidence associating both flushing and EG with a disturbance in the endocrine balance that could lead to increased ovarian cysts. The aim of this study was to evalu- ate whether flushing or EG might affect the ovulation rate and the incidence of ovarian cysts. Seventy-one gilts were randomly distributed into 2x2 factorial design with four treatments: flushing and hormone (wFwH); no flushing and hormone (nFwH); flushing without hormone (wFnH); and neither flushing nor hormone (nFnH). Gilts were slaughtered for macroscopic and histopathological ovary examination approximately five days after AI. The characterization of these cysts was performed by optical microscopy in the following: follicular cysts (FC), luteinizedcysts (LC) or cystic corpora lutea (CCL). The number of ovulations did not differ between treatments. There was no interaction between the factors in any analyzed variable. The frequency of gilts with CCL and LC was not affected by flushing and EG. No difference was found in the incidence of FC, with 12.5% and 5.88% in gilts from wFwH and nFwH treatments, respectively. There were no differences in the proportion of CCL between FC and LC (9.85 vs. 4.22 and 4.22%, respectively). In conclusion, the use of exogenous gonadotropins for second estrus synchronization in gilts, either alone or in association with dietary flushing, does not increase the incidence of ovarian cysts, nor does it decrease the ovulation rate.(AU)

A estimulação do estro por gonadotrofinas exógenas (GE) associada ao flushing alimentar é uma ferramenta importante na melhoria do desempenho reprodutivo de marrãs. Contudo, há evidência da associação do flushing com GE levando ao desequilíbrio no sistema endócrino que poderia levar ao aumento de cistos ovarianos. O objetivo deste estudo foi avaliar se o flushing ou GE pode afetar a taxa de ovulação e a incidência de cistos ovarianos. Setenta e uma marrãs foramdistribuídas aleatoriamente em arranjo fatorial 2x2 com quatro tratamentos: flushing e hormônio (cFcH); sem flushing e com hormônio (sFcH); com flushing e sem hormônio (cFsH) e sem flushing e hormônio (sFsH). Marrãs foram abatidas para exame macroscópico e histopatológico dos ovários, aproximadamente cinco dias após IA. A caracterização desses cistos foi realizada por microscopia óptica: cistos foliculares (CF), cistos luteinizados (CL) ou corpos lúteos císticos(CCL). O número de ovulações não diferiu entre os tratamentos. Não houve interação entre os fatores em qualquer variável analisada. A frequência de leitoas com CCL e CL não foi afetada pelo flushing e GE. Não houve diferença na incidência de CF, com 12,5% e 5,88 % em leitoas dos tratamentos cFcH e sFcH, respectivamente. Não foram obtidas diferenças na proporção de CCL entre CF e CL (9,85 vs. 4,22 e 4,22%, respectivamente). Em conclusão, a utilização de gonadotrofinas exógenas para sincronização do segundo estro de marrãs, isoladamente ou em associação com o flushing, não aumenta a incidência de cistos ovarianos e não diminui a taxa de ovulação.(AU)

In vitro development and cell allocation after aggregation of syngeneic wild type and fluorescence-expressing bovine cloned embryos

Background: The in vitro production (IVP) of embryos by in vitro fertilization or cloning procedures has been known to cause epigenetic changes in the conceptus that in turn are associated with abnormalities in pre-and postnatal development. Handmade cloning (HMC) procedures and the culture of zona-free embryos in individual microwells provide excellent tools for studies in developmental biology, since embryo development and cell allocation patterns can be evaluated under a wide range of embryo reconstruction arrangements and in in vitro embryo culture conditions. As disturbances in embryonic cell allocation after in vitro embryo manipulations and unusual in vivo conditions during the first third of pregnancy appear to be associated with large offspring, embryo aggregation procedures may allow a compensation for epigenetic defects between aggregated embryos or even may influence more favorable cell allocation in embryonic lineages, favoring subsequent development. Thus, the aim of this study was to evaluate in vitro embryo developmental potential and the pattern of cell allocation in blastocysts developed after the aggregation of handmade cloned embryos produced using syngeneic wild type and/or transgenic somatic cells. Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then, two enucleated hemi-oocytes were paired and fused with either a wild type (WT) or a GFP-expressing (GFP) fetal skin cell at the 11th and 19th passages, respectively. Following chemical activation, reconstructed cloned embryos and zona-free parthenote embryos were in vitro-cultured in microwells, for 7 days, either individually (1 x 100%) or after the aggregation of two structures (2 x 100%) per microwell, as follows: (G1) one WT cloned embryo; (G2) two aggregated WT embryos; (G3) one GFP cloned embryo; (G4) two aggregated GFP embryos; (G5) aggregation of a WT embryo and a GFP embryo; (G6) one parthenote embryo; or (G7) two aggregated parthenote embryos. Fusion (clones), cleavage (Day 2), and blastocyst (Day 7) rates, and embryonic cell allocation were compared by the x² or Fisher tests. Total cell number (TCN) in blastocysts was analyzed by the Student's test (P < 0.05). Fusion and cleavage rates, and cell allocation were similar between groups. On a per WOW basis, development to the blastocyst stage was similar between groups, except for lower rates of development seen in G3. However, when based on number of embryos per group (one or two), blastocyst development was higher in G1 than all other groups, which were similar between one another. Cloned GFP embryos had lower in vitro development to the blastocyst stage than WT embryos, which had more TCN than parthenote or aggregated chimeric WT/GFP embryos. Aggregated GFP embryos had fewer cells than the other embryo groups. Discussion: The in vitro development of GFP cloned embryos was lower than WT embryos, with no effects on cell allocation in resulting blastocysts. Differences in blastocyst rate between groups were likely due to lower GFP-expressing cell viability, as GFP donor cells were at high population cell doublings when used for cloning. On a per embryo basis, embryo aggregation on Day 1 resulted in blastocyst development similar to non-aggregated embryos on Day 7, with no differences in cell proportion between groups. The use of GFP-expressing cells was proven a promising strategy for the study of cell allocation during embryo development, which may assist in the elucidation of mechanisms of abnormalities after in vitro embryo manipulations, leading to the development of improved protocols for the in vitro production (IVP) of bovine embryos.

Reprogramming somatic cells: pluripotency through gene induction and nuclear transfer

Background: The understanding of nuclear reprogramming pathways provides important contributions to applied and basic sciences such as the development of autologous cellular therapies for the treatment of numerous diseases, the improved efficiency of animal-based biotechnology or the generation of functional gametes in vitro. Strategies such as nuclear transfer and induced reprogramming have been used to induce somatic cells into an embryonic-like pluripotent state. Both techniques have been routinely performed worldwide, and live offspring have been successfully derived from them, resulting in a proof of efficacy of both techniques. Detailed studies on cellular and molecular mechanisms that mediate reprogramming, however, still require further investigation to develop practical applications in veterinary and human medicine. Review: Studies on cell reprogramming, differentiation and proliferation have revealed that a core of transcription factors, as for example, OCT4, SOX2 and NANOG, act together promoting cell commitment or pluripotency. Mechanisms of induced reprogramming by pluripotency-related transcription factors forced expression or nuclear transfer seems to be mediated by the same pathways observed in fertilization, eliciting nuclear remodeling and modulating gene expression. However, abnormal chromatin conformation, often leading to disrupted imprinting and atypical gene expression patterns are frequently observed on in vitro reprogramming. Strategies used to facilitate nuclear remodeling, such as chromatin modifying agents, as for example, histone deacetilases inhibitors or DNA methyltransferases; or chemicals responsible for the inhibition of development related pathways, as for example, MEK and GSK3 inhibitors, when used in the in vitro culture of cells or embryos, have proved to favors transcriptional regulation and improve reprogramming. Such alternatives are highly prone to enable the routine use of in vitro reprogramming in animal production and medical sciences, for example, by promoting the generation of functional male and female functional gametes capable of producing viable offspring. Thus, the properties, deficiencies and implications of induced reprogramming and nuclear transfer techniques in somatic cells were discussed in this review, as well as its probable outcomes. Conclusions: The combination of both reprogramming techniques - induced reprogramming and nuclear transfer, may be essential to clarify the mechanisms of gene expression that are responsible for induced pluripotency. As discussed here, the mechanisms responsible for triggering the pluripotency status of a somatic cell are probably closely related to the epigenetic changes and gene expression profiles present in early development following fertilization. We report here that the nuclear transfer of SOX2 expressing donor cells resulted in similar rates of embryo production when compared to control cells. A better understanding of the contribution of each reprogramming factor used in induced reprogramming may result in the establishment of strategies aiming to enhance in vitro reprogramming performance. Such knowledge will contribute to in vitro animal production by increasing the cloning efficiency and regenerative medicine through the derivation and adequate culture of reprogrammed embryonic stem cells.

Geração de células pluripotentes através da indução gênica e transferência de núcleo: modelo bovino de aquisição de pluripotência / Generation of pluripotent cells through gene induction and nuclear transfer: a bovine model of pluripotency

Estratégias como a transferência nuclear e a reprogramação induzida vêm sendo empregadas com o objetivo de induzir células somáticas a um estado pluripotente similar ao embrionário. O processo de reprogramação nuclear e extremamente desejável e possui importantes contribuições tanto no estudo da ciência básica como aplicada, como por exemplo, no aumento da eficiência das biotécnicas de produção animal ou na medicina, com a possibilidade de terapia celular autóloga. Uma série de estudos, porem, ainda são necessários para que tais aplicações sejam viáveis, uma vez que os mecanismos fundamentais das técnicas empregadas ainda não estão totalmente elucidados. Esta proposta teve como objetivo gerar células bovinas pluripotentes através da reprogramação direta e utilizá-las na transferência de núcleo para a produção animal visando o aumento da eficiência da reprogramação celular. Para tal, foi analisada a capacidade de indução e manutenção da pluripotência em células somáticas bovinas comparando-as com células humanas e equinas (células pluripotentes induzidas - iPSC), assim como a capacidade de desenvolvimento de embriões produzidos através da combinação das técnicas em bovinos. As células iPS derivadas neste estudo foram produzidas mediante transdução lentiviral de fatores de transcrição (OSKM) murinos, caracterizadas e utilizadas como doadoras de núcleo na clonagem. Resumidamente, oócitos bovinos obtidos de ovários provenientes de abatedouros foram maturados in vitro por 18h, enucleados e reconstruídos com células iPS (n=203 ou fibroblastos fetais bovinos (bFF, n=153), em cinco repetições. [...] O conhecimento da contribuição de cada fator utilizado na reprogramação induzida, aliado a estudos de comparação com a capacidade de desenvolvimento in vitro de organismos derivados de células reprogramadas deverá contribuir para o aumento da eficiência da clonagem e produção animal in vitro como para a medicina regenerativa.

Nuclear transfer and induced reprogramming are technologies usually used for the induction of somatic cells into an embryonic-like pluripotent status. The knowledgment of nuclear reprogramming process is highly desirable, leading to important contributions for both basic and applied sciences; for example, resulting in the increase in the efficiency of several animal biotechnologies, or else enabling autologous cellular therapy for medical purposes. However, basic studies are still needed in order to enable such applications, once the mechanisms controlling in vitro reprogramming are yet to be unraveled. This study aims to generate induced pluripotent bovine stem cells through direct reprogramming and its use in nuclear transfer in order to enhance the cellular reprogramming efficiency, For that, the potential of pluripotency induction and maintenance was analyzed in bovine somatic cells, comparing those with human and equine cells, as well as the potential of embryonic development after combining direct and nuclear reprogramming. iPS cells derived in this study were produced trought lentivirus transduction of mouse transcription factors (OSKM), further characterized and used as nuclei donors for cloning. In summary, bovine oocytes were obtained from slaughterhouse ovaries, in vitro matured for 18h, enucleated and reconstructed with iPS cells (n=203) or fetal fibroblasts (bFF, n=153), in five replicates. Embryos were reconstructed, chemically activated with ionomycin and 6-DMAP and cultured in vitro until blastocyst stage. [...] The knowledge of each reprogramming factor influence on in vitro reprogramming, together with comparison studies on in vitro developmental potential of organisms derived from reprogrammed cells should help enhancing not only the cloning efficiency and in vitro animal production, but also the regenerative medicine.

Geração de células pluripotentes através da indução gênica e transferência de núcleo: modelo bovino de aquisição de pluripotência / Generation of pluripotent cells through gene induction and nuclear transfer: a bovine model of pluripotency

Estratégias como a transferência nuclear e a reprogramação induzida vêm sendo empregadas com o objetivo de induzir células somáticas a um estado pluripotente similar ao embrionário. O processo de reprogramação nuclear e extremamente desejável e possui importantes contribuições tanto no estudo da ciência básica como aplicada, como por exemplo, no aumento da eficiência das biotécnicas de produção animal ou na medicina, com a possibilidade de terapia celular autóloga. Uma série de estudos, porem, ainda são necessários para que tais aplicações sejam viáveis, uma vez que os mecanismos fundamentais das técnicas empregadas ainda não estão totalmente elucidados. Esta proposta teve como objetivo gerar células bovinas pluripotentes através da reprogramação direta e utilizá-las na transferência de núcleo para a produção animal visando o aumento da eficiência da reprogramação celular. Para tal, foi analisada a capacidade de indução e manutenção da pluripotência em células somáticas bovinas comparando-as com células humanas e equinas (células pluripotentes induzidas - iPSC), assim como a capacidade de desenvolvimento de embriões produzidos através da combinação das técnicas em bovinos. As células iPS derivadas neste estudo foram produzidas mediante transdução lentiviral de fatores de transcrição (OSKM) murinos, caracterizadas e utilizadas como doadoras de núcleo na clonagem. Resumidamente, oócitos bovinos obtidos de ovários provenientes de abatedouros foram maturados in vitro por 18h, enucleados e reconstruídos com células iPS (n=203 ou fibroblastos fetais bovinos (bFF, n=153), em cinco repetições. [...] O conhecimento da contribuição de cada fator utilizado na reprogramação induzida, aliado a estudos de comparação com a capacidade de desenvolvimento in vitro de organismos derivados de células reprogramadas deverá contribuir para o aumento da eficiência da clonagem e produção animal in vitro como para a medicina regenerativa. (AU)

Nuclear transfer and induced reprogramming are technologies usually used for the induction of somatic cells into an embryonic-like pluripotent status. The knowledgment of nuclear reprogramming process is highly desirable, leading to important contributions for both basic and applied sciences; for example, resulting in the increase in the efficiency of several animal biotechnologies, or else enabling autologous cellular therapy for medical purposes. However, basic studies are still needed in order to enable such applications, once the mechanisms controlling in vitro reprogramming are yet to be unraveled. This study aims to generate induced pluripotent bovine stem cells through direct reprogramming and its use in nuclear transfer in order to enhance the cellular reprogramming efficiency, For that, the potential of pluripotency induction and maintenance was analyzed in bovine somatic cells, comparing those with human and equine cells, as well as the potential of embryonic development after combining direct and nuclear reprogramming. iPS cells derived in this study were produced trought lentivirus transduction of mouse transcription factors (OSKM), further characterized and used as nuclei donors for cloning. In summary, bovine oocytes were obtained from slaughterhouse ovaries, in vitro matured for 18h, enucleated and reconstructed with iPS cells (n=203) or fetal fibroblasts (bFF, n=153), in five replicates. Embryos were reconstructed, chemically activated with ionomycin and 6-DMAP and cultured in vitro until blastocyst stage. [...] The knowledge of each reprogramming factor influence on in vitro reprogramming, together with comparison studies on in vitro developmental potential of organisms derived from reprogrammed cells should help enhancing not only the cloning efficiency and in vitro animal production, but also the regenerative medicine. (AU)

Produção de animais transgênicos por transferência nuclear como modelo de estudo biológico / Production of transgenic bovine by nuclear transfer: model for biological studies

A produção de animais transgênicos possui aplicações que envolvem desde a pesquisa básica à produção agropecuária. O recente progresso na clonagem animal por transferência nuclear (TN) possibilitou a produção de animais transgênicos utilizando linhagens de células doadoras de núcleo previamente modificadas geneticamente. A possibilidade de manipulação genética, estudo da expressão gênica e adequada seleção da célula doadora de núcleo na TN não somente pode garantir a presença da construção gênica em toda a prole, como também pode evitar a produção de animais portadores de modificações indesejáveis resultantes da inserção do inserto em regiões codificantes do genoma, em decorrência da inserção aleatória das técnicas de transferência gênica mais comuns. Este trabalho teve como objetivo geral produzir animais transgênicos a partir de transferência nuclear utilizando como células doadoras de núcleo fibroblastos modificados geneticamente por transdução lentiviral. Objetivos específicos foram a produção e caracterização de linhagens de fibroblasto fetal portadores do gene da Proteína Fluorescente Verde (eGFP) quanto à seleção da expressão do transgene, passagem celular e posição da inserção do transgene e sua utilização na técnica de transferência de núcleos para a análise da competência de desenvolvimento a blastocisto e estabelecimento de gestações. Para tal, fibroblastos fetais bovinos foram transduzidos pelo sistema lentiviral. Células expressando o gene da eGFP foram selecionadas por citometria de fluxo e utilizadas como doadoras de núcleo na TN. Foram analisados o efeito do período de cultivo dos fibroblastos, assim como o efeito da reclonagem na competência de desenvolvimento a blastocisto e estabelecimento de gestações. O cultivo celular submetido à reclonagem foi analisado quanto à posição de inserção do transgene, sendo constatado nos fetos produzidos neste experimento a presença de uma inserção única em região não transcrita do cromossomo 14. Não houve efeito neste experimento do tempo de cultivo na competência de desenvolvimento a blastocisto, mas houve efeito benéfico da reclonagem celular. Além disso, foram obtidos 4 fetos, sendo 3 transgênicos, dos embriões transferidos provenientes das TNs que utilizaram células transgênicas de inserção aleatória em baixas e altas passagens e 6 fetos dos 37 embriões transferidos provenientes das TN que utilizaram células reclonadas, sendo todos transgênicos. Conclui-se que a produção de células transgênicas mediante mecanismo de transdução lentiviral, pode resultar, após TNCS, em embriões geneticamente idênticos à células doadora capazes de sustentar o desenvolvimento in vitro a blastocisto e o estabelecimento de gestações. Finalmente, são discutidos fatores ligados ao processo de seleção e reclonagem que aumentam a eficiência da produção de gestações por TNCS

Genetically modified animals have numerous applications ranging from basic research to agriculture production. Recent progress in animal cloning by nuclear transfer (NT) has made possible the production of transgenic animals using previously genetically modified cell lineages. The possibility of genetic manipulation, gene expression studies and adequate selection of the nuclei donor cell for NT not only can guarantee the presence of the gene construction in the offspring, but also can avoid the production of animals that carries undesirable characteristics, often as a result of the random insertion of transgenes in transcripted areas of the genome. General objective of this study was to produce transgenic animals by nuclear transfer using lentivirus-genetically modified nuclei donor fibroblasts. Specifically, objectives were the production and characterization of fetal fibroblasts lineages expressing eGFP (enhanced Green Fluorescent Protein, eGFP) gene in different cell passages regarding transgene insertion position and its use for the nuclear transfer procedure. The potential of blastocyst development and pregnancy establishment were analyzed in embryos reconstructed with late and early passages and with clonal or random insertion of transgenes (recloning). For that, bovine fetal fibroblasts were transduced with lentiviruses. eGFP expressing cells were selected by flow citometry sorting and used as nuclei donor cells for NT. Transgene integration site of the cell culture submitted to recloning was analised. It was observed that an unique insertion in a non-transcribed área of chromossome 14 was present in the fetuses recovered in this recloning experiment. No effect of culture time on development of blastocysts was observed, however, there was a beneficial effect of the cell recloning Besides, 4 fetuses (3 of them were transgenic) were obtained when 64 embryos reconstructed with random transgene position cells in late and early passages were transferred and 6 fetuses were obtained when 37 embryos reconstructed with recloned cells were transferred (all of them were transgenic). In conclusion, lentivirus transduction was able to produce transgenic cells with a stable expression of the transgene. These cells, when used for SCNT, can be reprogrammed and genetically identical embryos able to sustain in vitro culture, pregnancy establishment and recognition. Finally, recloning and cell selection procedures are discussed as a possible approach to increase pregnancy efficiency after TNCS