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1.
Theriogenology ; 141: 91-97, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31521883

RESUMO

Autophagy is a critical process in early mammalian embryogenesis. Mammalian target of rapamycin (mTOR) inhibitors are major regulators of autophagy. However, mTOR plays a vital role in major signaling pathways controlling cell growth and metabolism; thus, more secure autophagy activation methods should be considered. The present study investigated the effects of supplementary trehalose, a novel mTOR-independent autophagy enhancer, on oocyte maturation and embryonic development after parthenogenetic activation (PA). Trehalose treatment during in vitro maturation (IVM) did not affect the nuclear maturation rates of oocytes. Oocytes treated with 25 mM trehalose during IVM had a significantly higher (P < 0.05) blastocyst formation rate (64.2%) after PA compared to that in control oocytes (52.0%). Blastocyst quality was also improved in the trehalose-treated group. The total cell numbers for blastocyst formation and expanded blastocyst formation were significantly increased in the trehalose-treated group (52.2% and 27.7%, respectively) compared to those in the control group (36.9% and 11.0%, respectively). Trehalose treatment led to the increased expression of LC3, an autophagy marker, in metaphase II oocytes and 4-cell stage embryos. Gene expression analysis revealed that the expression of several autophagy related genes (LAMP2, pATG5, and LC3) increased, while the Bax/Bcl2 ratio and pro-apoptotic Bak transcript levels were decreased in the trehalose-treated group. In conclusion, these results indicate that treatment with trehalose during IVM improved the developmental potential of porcine embryos by down-regulation of pro-apoptotic genes and up-regulation of autophagy-related genes and marker. Trehalose may be useful for the large-scale production of high-quality porcine blastocysts in vitro.

2.
Reprod Domest Anim ; 54(11): 1449-1458, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31381179

RESUMO

The objectives of this study were to investigate the effects of polyvinyl alcohol (PVA) as a chemically defined compound in egg yolk (EY)-free extender by determining the appropriate concentration of PVA and the effect of pH adjustment in EY-free PVA extenders on dog spermatozoa. Spermatozoa (1 × 108  cells/ml) were frozen with EY-free extenders supplemented with 0 (control), 0.025, 0.05, 0.1, 0.2 or 0.3 g/100 ml PVA. Sperm progressive motility (PM) was assessed immediately after thawing (IAT) and post-thaw incubation (PTI), while viability, acrosome integrity and reactive oxygen species (ROS) levels were evaluated after PTI. Additionally, spermatozoa were frozen using EY-free PVA extenders before pH adjustment (6.45) and after adjustment of pH (6.85). Viability, PM, ROS and gene expression (BCL2 and SMCP) were assessed. Supplementation with 0.05 g/100 ml or more PVA significantly increased PM compared to the control group in the IAT and PTI. Post-thaw incubation significantly increased sperm motility in all groups. The acrosome integrity in all PVA groups was higher (p < .05) than the control without an effect on ROS and viability. Adjustment of the pH to 6.85 improved (p < .05) sperm PM compared to the non-adjusted groups without affecting viability, ROS or expression of BCL2 and SMCP. We suggest that PVA supplementation in EY-free Tris extenders can effectively protect dog spermatozoa during freezing and can maintain higher motility and acrosome integrity. Adjustment of pH in EY-free PVA extenders can improve post-thaw sperm motility. Therefore, PVA can be used as a compound in EY-free extender for the cryopreservation of dog spermatozoa.

3.
Biol Reprod ; 101(1): 63-75, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31004472

RESUMO

Growth differentiation factor 8 (GDF8), also known as myostatin, is a member of the transforming growth factor-ß (TGF-ß) family and has been identified as a strong physiological regulator of muscle differentiation. Recently, the functional role of GDF8 in reproductive organs has received increased interest following its detection in the human placenta and uterus. To investigate the effects of GDF8 during porcine oocyte in vitro maturation (IVM), we assessed the quality of matured oocytes. Furthermore, we investigated the specific gene transcription and protein activation levels in oocytes and cumulus cells after IVM and subsequent embryonic development after in vitro fertilization and parthenogenetic activation. Prior to these experiments, the concentration of GDF8 in porcine follicular fluid was determined. During the entire IVM period, 1.3 ng/mL GDF8 and its signaling inhibitor SB431542 (SB) at 5 µM were added as control, SB, SB + GDF8, and GDF8 groups, respectively. Our results demonstrate that supplementation with GDF8 during porcine oocyte IVM enhanced both meiotic and cytoplasmic maturation, with altered transcriptional patterns, via activation of Sma- and Mad-related protein 2/3 (SMAD2/3). Using the pharmacological inhibitor SB431542, we demonstrated that inhibition of GDF8-induced Smad2/3 signaling reduces matured oocyte quality. In conclusion, for the first time, we demonstrated paracrine factor GDF8 in porcine follicular fluid in vivo. Furthermore, we showed that GDF8 supplementation improved mature oocyte quality by regulating p38 mitogen-activated protein kinase phosphorylation and intracellular glutathione and reactive oxygen species levels during porcine IVM.

4.
Theriogenology ; 129: 146-153, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30851478

RESUMO

The success of in vitro embryo production demonstrates that the oviduct can be bypassed during early embryonic development. Using an ex vivo model of porcine uterus is one of the strategies used to investigate fertilization within the oviductal environment. In this study, in vitro-matured porcine oocytes (MII) were fertilized with 7.5 × 107, 15 × 107, or 30 × 107 sperm cells for 20 min in the oviduct of a porcine uterine ex vivo model. MII oocytes used for in vitro fertilization (IVF) served as control 1; those cultured in the oviduct of the ex vivo model for 20 min before IVF served as control 2. In present study, the penetration rate, polyspermy, and fertilization efficiency, and accumulated reactive oxygen species (ROS) levels in the treatment groups were significantly decreased compared to those in the control 1 group. During embryonic development, the cleavage rates in the treatment groups were significantly lower than those in the control groups. The cleavage rate in the 30 × 107 sperm cell-treated group was higher than that in the 7.5 × 107 sperm cell-treated group. The blastocyst formation rate in control 1 and 2, and 30 × 107 sperm cell-treated groups increased compared to that in the 7.5 and 15 × 107 sperm cell-treated groups. PCNA, HSP70.2, and GLUT1 were upregulated in the treatment groups and POU5F1, BAX, GPX1 were upregulated in the treatment and control 2 groups, compared to the control 1 group. These results suggest that an ex vivo model may decrease the penetration rate and fertilization efficiency by increasing the accumulated ROS levels and inducing the expression of apoptosis- and stress-related genes. However, the model improved the monospermy rate and expression of embryo developmental competence genes. This is the first study that evaluates the effect of an ex vivo model of porcine uterus on fertilization parameters, and the development of porcine embryos.


Assuntos
Blastocisto/fisiologia , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário , Fertilização , Suínos , Animais , Técnicas de Cultura Embrionária/métodos , Feminino , Fertilização In Vitro/métodos , Fertilização In Vitro/veterinária , Glutationa/metabolismo , Técnicas de Maturação in Vitro de Oócitos/veterinária , Espécies Reativas de Oxigênio/metabolismo , Transcriptoma , Útero
5.
Theriogenology ; 129: 70-76, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30825707

RESUMO

Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-ß family and a physiological regulator. According to recent studies, GDF8 can be detected in follicular fluid and the uterus, suggesting that GDF8 may affect preimplantation embryonic development and act in a paracrine manner to improve the success of late-blastocyst implantation in vivo. We investigated the effect of GDF8 supplementation during in vitro culture (IVC) of porcine embryos derived from in vitro fertilization (IVF) and parthenogenetic activation (PA) on cleavage, blastocyst formation rate, and total cell number and analysed gene transcription levels and cell linage specification in the resulting blastocysts. First, the concentration of GDF8 in porcine oviductal fluid was determined to be 139.8 pg/mL. Then, 0, 0.2, 2, or 20 ng/mL GDF8 was added to embryos throughout the entire IVC period. Our results showed that supplementation with GDF8 during porcine preimplantation embryo IVC enhanced blastocyst formation and total cell number and altered the transcriptional patterns of genes that regulate pluripotency and cavitation. Furthermore, using differential immunostaining, we demonstrated that supplementation with GDF8 enhanced the expression of the genuine inner cell mass (ICM) marker SOX2 and the ICM/trophectoderm ratio, improving IVF blastocyst quality. In conclusion, for the first time, we demonstrated the presence of the in vivo oviductal factor GDF8 in oviductal fluid. Furthermore, we found that GDF8 supplementation at 0.2 ng/mL increased the blastocyst total cell number and ICM/trophectoderm ratio by inducing the transcription of genes involved in developmental competence and the expression of genuine ICM marker SOX2 during porcine IVF embryo development in vitro.


Assuntos
Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , Miostatina/farmacologia , Suínos/embriologia , Animais , Biomarcadores/metabolismo , Linhagem da Célula , Técnicas de Cultura Embrionária/métodos , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Maturação in Vitro de Oócitos/veterinária , Suínos/metabolismo
6.
Theriogenology ; 108: 46-55, 2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-29197292

RESUMO

Inhibitor of DNA binding (ID) proteins, ID1, ID2, ID3, and ID4 are transcriptional regulators that have a helix-loop-helix (HLH) domain but not a basic DNA binding domain. ID proteins inhibit the functions of basic HLH transcription factors and regulate cell proliferation and differentiation. However, the expression and function of ID1, ID2, ID3, and ID4 at the maternal-conceptus interface are not fully understood in pigs. Therefore, we determined the expressions of ID1, ID2, ID3, and ID4 in porcine endometrium, conceptus, and chorioallantoic tissues. ID1, ID2, ID3, and ID4 mRNAs were expressed in the endometrium, with lower levels of ID1, ID2, and ID4 on Day 12 of pregnancy than during the estrous cycle. ID1, ID2, ID3, and ID4 mRNAs were also detected in conceptus and chorioallantoic tissues during pregnancy. ID2 protein was mainly localized to luminal epithelia and weakly to vascular smooth muscle cells in the endometrium and conceptus trophectoderm. Increasing doses of interleukin-1ß decreased levels of ID2 mRNA, while estradiol-17ß increased levels of ID3 mRNA in endometrial explants. The expressions of ID2 and ID4 mRNAs were higher in endometria from gilts with somatic cell nuclear transfer-derived conceptuses compared with endometria from gilts carrying conceptuses derived from natural mating on Day 12. These results indicate that the expressions of ID family genes are dynamically regulated at the maternal-conceptus interface, suggesting that ID proteins may play critical roles in the regulation of endometrial epithelial cell function and conceptus development to support the establishment and maintenance of pregnancy in pigs.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Troca Materno-Fetal , Suínos/fisiologia , Fatores de Transcrição/metabolismo , Animais , Clonagem de Organismos , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos , Ciclo Estral , Feminino , Regulação da Expressão Gênica , Técnicas de Transferência Nuclear/veterinária , Placenta , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/genética , Útero/metabolismo
7.
J Reprod Dev ; 62(6): 635-638, 2016 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-27488694

RESUMO

Zinc supplementation (0.8 µg/ml) in in vitro maturation (IVM) medium significantly enhances oocyte quality. In this study, we compared the development of somatic cell nuclear transfer (SCNT) embryos produced from conventional IVM (control) and zinc-supplemented IVM oocytes. A total of 1206 and 890 SCNT embryos were produced using control and zinc-supplemented oocytes, respectively, and then were transferred to 11 and 8 recipients, respectively. Five control recipients and three zinc-supplemented recipients became pregnant. Two live piglets and eight mummies were born from two control recipients, and ten live piglets and six stillborn piglets were born from three zinc-supplemented recipients. The production efficiency significantly increased in the zinc-supplemented group (0.33% vs. 3.02%). This report suggests that zinc supplementation in IVM medium improved the production efficiency of cloned pigs.


Assuntos
Clonagem de Organismos/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Zinco/administração & dosagem , Animais , Clonagem de Organismos/métodos , Feminino , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Transferência Nuclear , Gravidez , Resultado da Gravidez , Suínos
8.
PLoS One ; 11(7): e0160289, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27472781

RESUMO

Pigs provide outstanding models of human genetic diseases due to their striking similarities with human anatomy, physiology and genetics. Although transgenic pigs have been produced using genetically modified somatic cells and nuclear transfer (SCNT), the cloning efficiency was extremely low. Here, we report an improved method to produce diploid cloned embryos from porcine induced pluripotent stem cells (piPSCs), which were synchronized to the G2/M stage using a double blocking method with aphidicolin and nocodazole. The efficiency of this synchronization method on our piPSC lines was first tested. Then, we modified our traditional SCNT protocol to find a workable protocol. In particular, the removal of a 6DMAP treatment post-activation enhanced the extrusion rate of pseudo-second-polar bodies (p2PB) (81.3% vs. 15.8%, based on peak time, 4hpa). Moreover, an immediate activation method yielded significantly more blastocysts than delayed activation (31.3% vs. 16.0%, based on fused embryos). The immunofluorescent results confirmed the effect of the 6DMAP treatment removal, showing remarkable p2PB extrusion during a series of nuclear transfer procedures. The reconstructed embryos from metaphase piPSCs with our modified protocol demonstrated normal morphology at 2-cell, 4-cell and blastocyst stages and a high rate of normal karyotype. This study demonstrated a new and efficient way to produce viable cloned embryos from piPSCs when synchronized to the G2/M phase of the cell cycle, which may lead to opportunities to produce cloned pigs from piPSCs more efficiently.


Assuntos
Clonagem de Organismos , Diploide , Embrião de Mamíferos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Metáfase , Suínos/embriologia , Animais , Blastocisto , Células Cultivadas , Camundongos
9.
J Reprod Dev ; 62(4): 345-50, 2016 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-27064112

RESUMO

Interspecies somatic cell nuclear transfer (iSCNT) can be a solution for preservation of endangered species that have limited oocytes. It has been reported that blastocyst production by iSCNT is successful even if the genetic distances between donors and recipients are large. In particular, domestic pig oocytes can support the development of canine to porcine iSCNT embryos. Therefore, we examined whether porcine oocytes may be suitable recipient oocytes for Korean raccoon dog iSCNT. We investigated the effects of trichostatin A (TSA) treatment on iSCNT embryo developmental patterns and nucleolus formation. Enucleated porcine oocytes were fused with raccoon dog fibroblasts by electrofusion and cleavage, and blastocyst development and nucleolus formation were evaluated. To our knowledge, this study is the first in which raccoon dog iSCNT was performed using porcine oocytes; we found that 68.5% of 158 iSCNT embryos had the ability to cleave. However, these iSCNT embryos did not develop past the 4-cell stage. Treatment with TSA did not affect iSCNT embryonic development; moreover, the nuclei failed to form nucleoli at 48 and 72 h post-activation (hpa). In contrast, pig SCNT embryos of the control group showed 18.8% and 87.9% nucleolus formation at 48 and 72 hpa, respectively. Our results demonstrated that porcine cytoplasts efficiently supported the development of raccoon dog iSCNT embryos to the 4-cell stage, the stage of porcine embryonic genome activation (EGA); however, these embryos failed to reach the blastocyst stage and showed defects in nucleolus formation.


Assuntos
Nucléolo Celular/metabolismo , Clonagem de Organismos/veterinária , Desenvolvimento Embrionário/fisiologia , Técnicas de Transferência Nuclear/veterinária , Oócitos/citologia , Animais , Feminino , Gravidez , Cães Guaxinins , Suínos
10.
J Reprod Dev ; 62(2): 177-85, 2016 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-26821870

RESUMO

The ultrastructure of porcine putative embryonic stem cells and porcine fetal fibroblasts (PFFs) was analyzed by transmission electron microscopy. The aim of this study was to compare the features of organelles in in vitro fertilization (IVF) derived porcine embryonic stem cells (IVF-pESCs) and somatic cell nuclear transfer (SCNT) derived pESCs (SCNT-pESCs). Also, the features of organelles in high-passage IVF-pESCs were compared with those in low-passage cells. The ultrastructure of PFFs showed rare microvilli on the cell surfaces, polygonal or irregular nuclei with one to two reticular-shaped nucleoli and euchromatin, low cytoplasm-to-nucleus ratios, rare ribosomes, rare rough endoplasmic reticulum, elongated mitochondria, rich lysosomes and rich phagocytic vacuoles. IVF-pESCs showed rare microvilli on the cell surfaces, round or irregular nuclei with one to two reticular-shaped nucleoli and euchromatin, low cytoplasm-to-nucleus ratios, rich ribosomes, long stacks of rough endoplasmic reticulum, elongated mitochondria, rare lysosomes and rare autophagic vacuoles. By contrast, SCNT-pESCs showed rich microvilli with various lengths and frequencies on the cell surfaces, polygonal nuclei with one reticular shaped nucleoli and heterochromatin, high cytoplasm-to-nucleus ratios, rare ribosomes, rare rough endoplasmic reticulum, round mitochondria, rich lysosomes and rich phagocytic vacuoles with clear intercellular junctions. Furthermore, high-passage IVF-pESCs showed irregularly shaped colonies, pyknosis and numerous lysosomes associated with autophagic vacuoles showing signs of apoptosis. In conclusion, this study confirms that the ultrastructural characteristics of pESCs differ depending on their origin. These ultrastructural characteristics might be useful in biomedical research using pESCs, leading to new insights regarding regenerative medicine and tissue repair.


Assuntos
Células-Tronco Embrionárias/ultraestrutura , Fertilização In Vitro/métodos , Técnicas de Transferência Nuclear , Animais , Apoptose , Blastocisto/citologia , Linhagem Celular , Núcleo Celular/ultraestrutura , Técnicas de Cocultura , Citoplasma/ultraestrutura , Células-Tronco Embrionárias/citologia , Retículo Endoplasmático Rugoso/ultraestrutura , Fibroblastos/ultraestrutura , Camundongos , Camundongos Endogâmicos ICR , Microscopia Eletrônica de Transmissão , Microvilosidades/ultraestrutura , Mitocôndrias/ultraestrutura , Fagocitose , Suínos
11.
Theriogenology ; 85(4): 601-16, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26542134

RESUMO

The establishment of porcine embryonic stem cells (ESCs) would have great impact in biomedical studies and preclinical trials through their use in genetic engineering. However, authentic porcine ESCs have not been established until now. In this study, a total of seven putative ESC lines were derived from porcine embryos of various origins, including in vitro fertilization, parthenogenetic activation, and, in particular, induced pluripotent stem (iPS) nuclear transfer (NT) from a donor cell with induced pluripotent stem cells (iPSCs). To characterize these cell lines, several assays including an assessment of intensive alkaline phosphatase activity, karyotyping, embryoid body formation, expression analysis of the pluripotency-associated markers, and the three germ layerassociated markers were performed. Based on quantitative polymerase chain reaction, the expression levels of REX1 and FGFR2 in iPS-NT lines were higher than those of cells of other origins. Additionally, only iPS-NT lines showed multiple aberrant patterns of nuclear foci elucidated by immunofluorescence staining of H3K27me3 as a marker of the state of X chromosome inactivation and a less mature form of mitochondria like naive ESCs, by transmission electron microscopy. Together, these data suggested that established putative porcine ESC lines generally exhibited a primed pluripotent state, like human ESCs. However, iPS-NT lines have especially unique characteristics distinct from other origins because they have more epigenetic instability and naive-like mitochondrial morphology than other putative ESC lines. This is the first study to establish and characterize the iPSC-derived putative ESC lines and compare them with other lines derived from different origins in pigs.


Assuntos
Blastocisto/citologia , Clonagem de Organismos , Células-Tronco Embrionárias/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Suínos/embriologia , Animais , Blastocisto/fisiologia , Proliferação de Células , Células Alimentadoras , Regulação da Expressão Gênica no Desenvolvimento , Cariotipagem , Camundongos , Microscopia Eletrônica de Transmissão , Mitocôndrias/fisiologia , Partenogênese
12.
J Reprod Dev ; 61(6): 549-57, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26370787

RESUMO

Ganglioside is an acidic glycosphingolipid with sialic acids residues. This study was performed to investigate the effect and mechanism of ganglioside GT1b in porcine oocytes in the process of in vitro maturation (IVM) and preimplantation development. Metaphase II (MII) rates were significantly (P < 0.05) different between the control group and the 5 nM GT1b treatment group. Intracellular glutathione (GSH) levels in oocytes matured with 5 nM and 20 nM and GT1b decreased significantly (P < 0.05). The 10 nM group showed a significant (P < 0.05) decrease in intracellular reactive oxygen species (ROS) levels compared with the control group. Subsequently, the level of intracellular Ca(2+) in oocytes treated with different concentrations of GT1b was measured. Intracellular Ca(2+) was significantly (P < 0.05) increased with a higher concentration of GT1b in a dose-dependent manner. Real-time PCR was performed and showed that the expression of bradykinin 2 receptor (B2R) and calcium/calmodulin-dependent protein kinase II delta (CaMKIIδ) in cumulus cells was significantly (P < 0.05) decreased in the 20 nM GT1b treatment group. Treatment with 5 nM GT1b significantly (P < 0.05) decreased the expression of CaMKIIδ. In oocytes, treatment with 5 nM GT1b significantly (P < 0.05) decreased CaMKIIγ and POU5F1 (POU domain, class 5, transcription factor 1). However, treatment with 20 nM GT1b significantly (P < 0.05) increased the expression of POU5F1. Finally, embryonic developmental data showed no significant differences in the two experiments (parthenogenesis and in vitro fertilization). In conclusion, the results of the present study indicated that GT1b plays an important role in increasing the nuclear maturation rate and decreasing the intracellular ROS levels during IVM. However, GT1b inhibited maturation of the cytoplasm by maintaining intracellular Ca(2+) in the process of oocyte maturation regardless of the cell cycle stage. Therefore, GT1b is thought to act on another mechanism that controls intracellular Ca(2+).


Assuntos
Desenvolvimento Embrionário/fisiologia , Gangliosídeos/fisiologia , Técnicas de Maturação in Vitro de Oócitos , Suínos/fisiologia , Animais , Cálcio/análise , Feminino , Fertilização In Vitro , Glutationa/análise , RNA Mensageiro/análise , Espécies Reativas de Oxigênio , Reação em Cadeia da Polimerase em Tempo Real , Receptor B2 da Bradicinina/análise
13.
Mol Med Rep ; 12(4): 5973-82, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26238161

RESUMO

The present study investigated the effects of zinc deficiency during in vitro maturation (IVM) of porcine oocytes. Zinc deficiency was induced by administering the membrane­permeable zinc chelator N,N,N',N'­tetrakis­(2­pyridylmethyl)­ethylendiamine (TPEN). First, the effects of zinc deficiency during IVM on a TPEN­treated group and a TPEN+zinc-treated group compared with a control group were assessed. The oocyte maturation rates and subsequent embryonic developmental competence of the TPEN+zinc­treated oocytes were similar to those of the control oocytes (metaphase II [MII] rate, 93.0 and 92.7%, respectively, and blastocyst [BL] formation rate, 42.0 and 40.0%, respectively). These results were significantly different from those obtained for the TPEN­treated oocytes (MII rate, 0.61%; BL formation rate, 0%). Although the TPEN­treated oocytes were arrested at metaphase I (MI), the distribution of microtubules was normal. However, microfilament formation was abnormal in the TPEN­treated oocytes. Furthermore, the effect of a temporary zinc deficiency during IVM on oocyte maturation and subsequent embryonic development was assessed. TPEN (10 µM) was added to the IVM medium for 0, 7, 15 or 22 h. The 0 h­treated oocytes showed an 83.9% MII rate, while the 7 h­treated oocytes had a significantly lower MII rate (44.8%). Most of the 15- and 22 h­treated oocytes were arrested at MI (MI rate: 98.0 and 97.2%, respectively; MII rate, 0% in both groups). Reductions in the BL formation were dependent on the TPEN treatment duration (29.3, 9.2, 0, and 0% after 0, 7, 15 and 22 h, respectively). In conclusion, zinc is an essential element for successful oocyte maturation and embryonic development in pigs. Zinc deficiency caused a meiotic block and had lasting effects on early embryonic development.


Assuntos
Oócitos/efeitos dos fármacos , Zinco/deficiência , Animais , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Células Cultivadas , Desenvolvimento Embrionário/efeitos dos fármacos , Etilenodiaminas/efeitos adversos , Glutationa/metabolismo , Metáfase , Oócitos/citologia , Espécies Reativas de Oxigênio/metabolismo , Suínos
14.
Theriogenology ; 84(7): 1075-87, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26194698

RESUMO

Granulocyte colony-stimulating factor (G-CSF) is required for proliferation, differentiation, and survival of cells. It is also a biomarker of human oocyte developmental competence for embryo implantation. In humans, the G-CSF concentration peaks during the ovulatory phase of the ovarian cycle. In this study, the expressions of G-CSF and its receptor were analyzed by polymerase chain reaction in granulosa cells (GCs), CL, cumulus cells (CCs), and oocytes. Cumulus-oocyte complexes were aspirated from antral follicles of 1 to 3 mm (small follicles) and 4 to 6 mm (medium follicles). Cumulus-oocyte complexes from two kinds of follicles were matured in protein-free maturation medium supplemented with various concentrations of G-CSF (0, 10, and 100 ng/mL). By real-time polymerase chain reaction, the expressions of G-CSF and its receptor were detected in GCs, CL, CCs, and oocytes. Interestingly, the G-CSF transcript levels were significantly lower in oocytes than in the other cell types, whereas the G-CSF receptor transcript levels in oocytes were similar to those in GCs. After 44 hours of IVM, no differences in the rate of nuclear maturation were detected; however, the intracellular reactive oxygen species levels in oocytes from both groups of follicles matured with 10 ng/mL of human recombinant G-CSF (hrG-CSF) groups were significantly lower (P < 0.05). After parthenogenetic activation, the cleavage rates were significantly (P < 0.05) higher in 100 ng/mL hrG-CSF-treated small (63.3%) follicles than in 0, 10 ng/mL hrG-CSF-treated small (38.6% and 49.0%, respectively) follicles and 0 ng/mL hrG-CSF-treated medium (52.1%) follicles, and the cleavage rates were significantly (P < 0.05) higher in 10 ng/mL hrG-CSF-treated medium (76.3%) follicles than in all other groups. The blastocyst formation rates were significantly (P < 0.05) higher in 100 ng/mL hrG-CSF-treated small (31.2%) follicles than in 0 and 10 ng/mL hrG-CSF small (10.4% and 15.6%, respectively) follicles, and the 10 ng/mL hrG-CSF medium (45.7%) follicle was significantly (P < 0.05) higher than in all other groups. The total cell number in blastocysts from the 10 ng/mL hrG-CSF medium (106.5) follicles was significantly (P < 0.05) increased compared to 0, 10, 100 ng/mL hrG-CSF small (55.0, 73.7 and 59.5, respectively) follicles and 0, 100 ng/mL hrG-CSF-treated medium (82.5 and 93.5, respectively) follicles. After IVF, the blastocysts stage was significantly (P < 0.05) increased in 10 ng/mL hrG-CSF-treated medium (36.4%) follicles. Fertilization efficiency was significantly high in 100 ng/mL of small (29.1%) and 10 ng/mL of medium (44.0%) follicles. We also examined the Bcl2 and ERK2 transcript levels and found that they were significantly higher in the small and medium follicle treatment groups. In conclusion, these results indicate that hrG-CSF improve the viability of porcine embryos.


Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Suínos , Animais , Blastocisto/fisiologia , Células do Cúmulo/química , Feminino , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos/análise , Fator Estimulador de Colônias de Granulócitos/genética , Células da Granulosa/química , Humanos , Oócitos/química , RNA Mensageiro/análise , Espécies Reativas de Oxigênio/análise , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Fator Estimulador de Colônias de Granulócitos/análise , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Proteínas Recombinantes
15.
Theriogenology ; 84(4): 531-7, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26047708

RESUMO

This study aimed to investigate the effect of zinc on in vitro development of porcine embryos. We evaluated the effects of zinc on blastocysts formation and investigated gene expression at zinc-deficient and supplemented conditions. Zinc-deficient in vitro condition was induced by 10-µM N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylendiamine (TPEN) (zinc chelator) treatment during IVC. On parthenogenetic activated embryos, this treatment significantly decreased cleavage rate and blastocyst formation compared with the control (0.0% and 0.0% vs. 69.0% and 36.0%, respectively). And time effect of the zinc deficiency exposure is observed. Blastocyst formation rate was significantly decreased as zinc-deficient time increases (54.1%, 31.0%, 9.0%, and 1.2% for zinc deficiency during 0, 3, 5, and 7 hours). However, zinc supplementation during IVC supported in vitro embryonic development. On parthenogenetic activated embryos, supplementation of 0.8 µg/mL of zinc during IVC significantly increased blastocyst formation compared with other groups (43.9%, 57.8%, 67.1%, 51.4%, and 52.6% for zinc supplementation of 0, 0.4, 0.8, 1.2, and 1.6 µg/mL). In vitro-fertilized (IVF) embryos showed similar results. The blastocyst formation rate was significantly higher in the 0.8 µg/mL of zinc-supplemented group than in the other groups (21.3%, 24.1%, 36.1%, 25.9%, and 25.2% for zinc supplementation of 0, 0.4, 0.8, 1.2, and 1.6 µg/mL). PCNA, POU5F1, and Bcl2 messenger RNA expressions were unregulated in IVF-derived blastocysts in the 0.8 µg/mL of zinc-supplemented group compared with the control. These results suggest that zinc is required for embryonic development, and supplementation with adequate zinc concentrations during IVC improved the viability of porcine embryos, possibly by increasing PCNA, POU5F1, and Bcl2 gene expression of embryos.


Assuntos
Técnicas de Cultura Embrionária/veterinária , Suínos/embriologia , Zinco/farmacologia , Animais , Meios de Cultura/química , Etilenodiaminas/toxicidade , Regulação da Expressão Gênica no Desenvolvimento , Partenogênese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
16.
Theriogenology ; 84(2): 226-36, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-25913277

RESUMO

Carboxyethylgermanium sesquioxide (Ge-132) is an organogermanium compound known to exert biological activities, such as antioxidant and anticancer effects. In this study, we investigated the effect of Ge-132 on the IVM of porcine oocytes via analysis of nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, and subsequent embryonic development after parthenogenetic activation (PA) and IVF. After 40 hours of IVM, no significant difference in nuclear maturation was observed in the 100, 200, and 400 µg/mL of Ge-132 treatment groups (89.9%, 91.3%, and 90.4%, respectively) compared with the control group (89.0%). However, intracellular GSH levels in oocytes treated with 200 µg/mL of Ge-132 increased significantly (P < 0.05), and the 200 and 400 µg/mL of Ge-132 treatment groups exhibited a significant (P < 0.05) decrease in intracellular ROS levels compared with the control group. Oocytes matured with 200 and 400 µg/mL of Ge-132 during IVM displayed significantly higher cleavage rates (78.7% and 82.7% vs. 67.5%, respectively), and the 200 µg/mL of Ge-132 treatment group displayed higher blastocyst formation rates and greater total cell numbers after PA (59.5% and 67.8 vs. 38.2% and 55.3, respectively) than the control group. Furthermore, oocytes matured with 200 µg/mL of Ge-132 during IVM failed to display significantly higher blastocyst formation rates (31.6% vs. 36.7%) but exhibited greater total cell numbers after IVF (71.5 vs. 101.3, respectively) than the control group. We also found that the Ge-132-treated oocytes showed significantly higher messenger RNA (mRNA) expression levels of the oxidative-related gene Nrf-2 and lower mRNA expression levels of the proapoptotic gene Bax than the control group (P < 0.05). In conclusion, our results suggest that treatment with Ge-132 during IVM improves the developmental potential of PA and IVF porcine embryos by increasing the intracellular GSH levels, thereby decreasing the intracellular ROS levels and reducing oxidative stress-induced apoptosis, thereby regulating the mRNA expression of oocytes during oocyte maturation.


Assuntos
Antioxidantes/farmacologia , Desenvolvimento Embrionário/efeitos dos fármacos , Fertilização In Vitro/veterinária , Técnicas de Maturação in Vitro de Oócitos/métodos , Compostos Organometálicos/farmacologia , Sus scrofa/fisiologia , Animais , Apoptose/genética , Blastocisto/citologia , Contagem de Células , Núcleo Celular/fisiologia , Meios de Cultura , Células do Cúmulo/metabolismo , Citoplasma/fisiologia , Reparo do DNA/genética , Técnicas de Cultura Embrionária/veterinária , Feminino , Expressão Gênica/efeitos dos fármacos , Glutationa/análise , Oócitos/química , Oócitos/metabolismo , Oócitos/ultraestrutura , Partenogênese , Espécies Reativas de Oxigênio/análise , Sus scrofa/embriologia
17.
Mol Med Rep ; 12(2): 2140-8, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25892608

RESUMO

Porcine embryonic stem cells (pESCs) have great potential for application in translational biomedical research, including xenotransplantation and disease models. Obtaining high-quality blastocysts is the most important factor in the isolation and colonization of primary ESCs and the establishment of ESC lines. In pigs, in vitro-derived blastocysts have a limited cell number compared to in vivo-derived blastocysts and show an indefinite inner cell mass, which may result in failure to establish pESC lines. In the present study, the effects of resveratrol (RES), granulocyte-macrophage colony stimulating factor (GM-CSF) and ß-mercaptoethanol (ß-ME) on the quality of blastocysts and the efficiency of colony derivation were investigated for the establishment of ESCs. A novel culturing system was developed in which 2 µM RES was added to the oocyte in vitro maturation (IVM) medium, and 10 ng/ml pGM-CSF and 10 µM ß-ME were added to embryo in vitro culture (IVC) medium. This novel system showed significantly more parthenogenetic activation (PA) blastocysts (54.5 ± 1.8% vs. 43.4 ± 1.2%; P<0.05) and in vitro fertilization (IVF) blastocysts (36.9 ± 3.3% vs. 26.2 ± 2.9%; P<0.06) at day seven as compared with that in the control system. The PA and IVF blastocysts from the novel system showed a significantly greater hatching rate (P<0.05) and greater cell numbers (55.1 ± 2.0 vs. 45.6 ± 2.0; P<0.05 and 78.9 ± 6.8 vs. 58.5 ± 7.2; P<0.06, for PA and IVF, respectively) at day seven compared to that in the control system. After seeding on feeder cells, the PA blastocysts produced by the novel system showed a significantly increased rate of attachment (28.8 ± 3.9% vs. 17.2 ± 2.4%; P<0.062). Finally, two putative pESC lines from PA embryos produced by the novel system and one by the control system were established. In conclusion, the novel system improved blastocyst quality and increased the derivation efficiency of putative pESC lines from porcine PA and IVF embryos produced in vitro.


Assuntos
Blastocisto/citologia , Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias/citologia , Suínos/embriologia , Animais , Blastocisto/metabolismo , Proliferação de Células , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Feminino , Fertilização In Vitro , Perfilação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Masculino , Mercaptoetanol/metabolismo , Camundongos , Partenogênese , Resveratrol , Estilbenos/metabolismo
18.
Theriogenology ; 83(2): 294-305, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25442018

RESUMO

In the process of IVM, cumulus-oocyte complexes (COCs) separate from the follicular microenvironment, leading to the loss of endocrine interactions between follicular mural somatic cells and COCs. To restore the microenvironment, a coculture system was established using cumulus-derived somatic cells (CSCs) for IVM. The CSCs were cultured in Dulbecco's modified Eagle's medium for 48 hours with varying numbers of CSCs (0.0, 2.5 × 10(4), 5.0 × 10(4), and 10.0 × 10(4)) and then cultured in tissue culture medium 199 (TCM 199) for 4 hours before adding the oocytes. Cumulus-oocyte complexes from 3- to 6-mm follicles were matured in 500 µL of TCM 199 with eCG and hCG for 22 hours and then cultured in TCM 199 without hormones for 22 hours. After IVM, the group with 2.5 × 10(4) CSCs showed a significant increase in intracellular glutathione levels compared with the control group. In the evaluation of sperm penetration, efficient fertilization was increased in the groups with 2.5 × 10(4) and 5.0 × 10(4) CSCs compared with controls (44.9 and 46.5 vs. 32.1, respectively). The mRNA expression pattern analysis in matured COCs showed a significant upregulation of PCNA, COX-2, Has2, Ptx3, and Nrf2 in the 2.5 × 10(4) CSC group compared with controls. During COC maturation at 0, 11, 22, 33, and 44 hours, the 2.5 × 10(4) and 5.0 × 10(4) CSC groups showed a significantly altered mRNA expression of BMP15 and GDF9. The developmental competence of the matured oocytes in all groups was evaluated after IVF and parthenogenetic activation (PA). After IVF, the 2.5 × 10(4) CSC group showed significantly higher cleavage, blastocyst formation rate, and total cell numbers compared with controls (60.0%, 35.7%, and 127.3 vs. 43.2%, 21.1%, and 89.3, respectively). After PA, the 2.5 × 10(4) CSC group had significantly higher blastocyst formation rate and total cell number than the control group (52.0% and 120.4 vs. 35.4% and 90.9, respectively). In conclusion, these results suggest that the presence of a population of 2.5 × 10(4) CSCs during IVM synergistically improved the developmental potential of IVF- and PA-derived porcine embryos by increasing the intracellular glutathione level via changing of a specific gene expression pattern during oocyte maturation.


Assuntos
Técnicas de Cocultura , Células do Cúmulo/fisiologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/fisiologia , Sus scrofa , Animais , Blastocisto/fisiologia , Desenvolvimento Embrionário , Feminino , Fertilização In Vitro/veterinária , Expressão Gênica , Glutationa/análise , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/química , Partenogênese , RNA Mensageiro/análise , Espécies Reativas de Oxigênio/análise
19.
Theriogenology ; 82(6): 866-74, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25091527

RESUMO

We investigated the effects of zinc supplementation during the IVM of porcine oocytes. Nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, subsequent embryonic development, and gene expression were evaluated. Zinc concentrations in porcine plasma and follicular fluid were 0.82 and 0.84 µg/mL, respectively. Zinc was not detected in IVM medium. After treatment with various zinc concentrations (0.0, 0.4, 0.8, 1.2, and 1.6 µg/mL), no significant difference in IVM was observed among groups (85.7%, 88.7%, 90.4%, 90.3%, and 87.2%, respectively). The effects of different zinc concentrations on porcine oocyte intracellular GSH and ROS levels were examined in mature oocytes. Intracellular GSH levels were significantly higher in the 0.8-, 1.2-, and 1.6-µg/mL groups than in the control (P < 0.05). Intracellular ROS levels of oocytes matured with 0.8, 1.2, and 1.6 µg/mL were reduced significantly (P < 0.05) compared with the control and 0.4-µg/mL groups. The developmental competence of oocytes matured with different zinc concentrations was evaluated after parthenogenetic activation (PA) and in vitro fertilization (IVF). Oocytes treated with zinc during IVM showed no significant difference in cleavage rate after PA. Oocytes treated with 0.8 and 1.2 µg/mL zinc during IVM had significantly higher blastocyst formation rates after PA (41.5% and 41.1%, respectively) than the control (27.2%). IVF embryos showed similar results. The blastocyst formation rate was significantly higher (28.2%) in the 0.8-µg/mL group. TNFAIP2 and Bax were decreased in zinc-treated cumulus cells. Increased POU5F1 and decreased Bax transcript levels were observed in zinc-treated oocytes. POU5F1 and Bcl-2 transcript levels were significantly higher in zinc-treated IVF blastocysts. These results indicate that treatment with adequate zinc concentrations during IVM improved the developmental potential of porcine embryos by regulating the intracellular GSH concentration, the ROS level, and transcription factor expression.


Assuntos
Blastocisto/fisiologia , Desenvolvimento Embrionário/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/efeitos dos fármacos , Animais , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glutationa/metabolismo , Partenogênese , Espécies Reativas de Oxigênio
20.
Theriogenology ; 81(7): 974-81, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24576710

RESUMO

The objective of this study was to examine the developmental competence of IVM pig oocytes in relation to the pattern of morphologic changes after exposure to hyperosmotic medium to select oocytes of a higher quality. IVM oocytes were treated with a hyperosmotic (593 mOsm) medium containing NaCl, sorbitol, or sucrose. Oocytes that shrunk spherically (SSP oocytes) or in irregular shapes (SIR oocytes) were collected separately, and washed for 15 minutes in an isotonic (297 mOsm) medium for recovery. Irrespective of the chemicals used, hyperosmotic treatment of oocytes for 1 hour or 15 minutes did not alter embryonic development after parthenogenesis (PA) and SCNT. A significantly higher proportion of SSP oocytes developed to the blastocyst stage (34.0%) compared with SIR oocytes (15.8%) after PA. The intracellular glutathione content was significantly higher in SSP oocytes than in SIR oocytes. Conversely, the reactive oxygen species level was significantly higher in SIR oocytes than in SSP oocytes. The maturation promoting factor level as measured by p34(cdc2) kinase activity was not influenced by hyperosmotic treatment itself but was 1.3-fold higher (P < 0.05) in SSP oocytes than in SIR oocytes. When IVM oocytes were divided into two groups according to their diameters (large and small), and treated separately in hyperosmotic medium, significantly more SSP oocytes (71.4%) were found in the large oocytes than in the small oocytes (51.4%). Moreover, the proportion of metaphase II oocytes was significantly higher in SSP oocytes than in SIR oocytes in both groups (98.5% vs. 73.1% in large oocytes, and 92.2% vs. 48.0% in small oocytes). After SCNT, a significantly higher proportion of SSP oocytes displayed blastocyst formation (36.4%) than untreated (29.0%) and SIR oocytes (22.1%). Our results demonstrated that SSP oocytes were of a higher quality than SIR oocytes, which was shown by higher intracellular glutathione and maturation promoting factor levels, lower reactive oxygen species levels, and improved embryonic development to the blastocyst stage after PA and SCNT.


Assuntos
Técnicas de Maturação in Vitro de Oócitos/veterinária , Técnicas de Transferência Nuclear/veterinária , Oócitos/citologia , Pressão Osmótica/fisiologia , Suínos/fisiologia , Animais , Clonagem de Organismos/veterinária , Técnicas de Cultura Embrionária/veterinária , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário , Feminino , Oócitos/fisiologia
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