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1.
Mol Reprod Dev ; 87(6): 704-710, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32415813

RESUMO

Dromedary camel oocytes are unique in their capability for intrafollicular and in vitro spontaneous parthenogenetic activation (SPA) and development. This study was designed for (a) observing the incidence of SPA and development of dromedary camel oocytes retrieved from ovaries; (b) assessing intrafollicular development of dromedary camel oocytes using histological examination; (c) evaluating the abilities of dromedary camel oocytes to mature, SPA, and develop in vitro; and (d) identifying the transcript abundance of Cdx2 messenger RNA (mRNA) expression in different stages of SPA and developed camel embryos. The results revealed that 2.33% of oocytes retrieved from dromedary camel ovaries were SPA and developed to blastocyst stage. Serial sections of dromedary camel ovaries also demonstrated the presence of 1.4 SPA and parthenotes per ovary, which included from two-cell to the blastocysts with demarcated trophectoderm and inner cell mass layers. A total of 2.6% in vitro matured dromedary camel oocytes developed into morulae. The SPA and developed dromedary embryos expressed transcript abundance for Cdx2 mRNA with the highest (p < .05) at the blastocyst. The present work determines for the first time the intrafollicular oocytes from the dromedary camel display SPA, and the parthenotes can develop into blastocysts and expressing Cdx2 mRNA.


Assuntos
Camelus/fisiologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/fisiologia , Partenogênese/fisiologia , Animais , Blastocisto/fisiologia , Fator de Transcrição CDX2/genética , Fator de Transcrição CDX2/metabolismo , Células Cultivadas , Desenvolvimento Embrionário/genética , Feminino , Recuperação de Oócitos/veterinária , Oócitos/citologia , Oogênese/genética , Oogênese/fisiologia , Partenogênese/genética
2.
Anim Sci J ; 89(4): 640-647, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29318710

RESUMO

When European Union regulations restricted the use of estrogenic compounds in food-producing animals, refined hormonal protocols were no longer applicable for anovulatory cows. However, Ovsynch and its adaptations are routinely and uniformly applied to all cows regardless of ovarian function. To evaluate their efficacy on anovulatory cows, 143, 147 and 144 anovulatory cows received Ovsynch, Presynch and G6G protocols, respectively. In comparison, 150 cyclic cows were bred without using a synchronized protocol. Results showed that cows in the Presynch group had luteolysis responding to the last prostaglandin F2α (PGF2α ) injection greater than the Ovsynch group. The serous progesterone levels at the first gonadotropin-releasing hormone of Ovsych and the last PGF2α injection was greater in the G6G group than the other two hormonal treatment groups. Concentrations of Ca2+ and total protein in cervical mucus in all three hormone-treated groups before artificial insemination (AI) were significantly different from the controls. The G6G group obtained a greater pregnancy rate compared with Ovsynch and Presynch, but significantly less than the controls. For open cows in the Ovsynch group, estrus rate within 24 days after the first AI was significantly less than the controls. In conclusion, the G6G treatment resulted to better reproductive performance in anovulatory cows.


Assuntos
Anovulação/tratamento farmacológico , Bovinos/metabolismo , Bovinos/fisiologia , Estrogênios/administração & dosagem , Sincronização do Estro/métodos , Lactação/fisiologia , Animais , Anovulação/fisiopatologia , Cálcio/metabolismo , Muco do Colo Uterino/metabolismo , Dinoprosta/administração & dosagem , Estro , Feminino , Hormônio Liberador de Gonadotropina/administração & dosagem , Injeções , Inseminação Artificial/métodos , Luteólise , Gravidez , Taxa de Gravidez , Progesterona/sangue , Proteínas/metabolismo
3.
Reprod Fertil Dev ; 29(6): 1209-1216, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27165775

RESUMO

Interferon (IFN)-stimulated gene 15 (ISG15) is one of several proteins induced by conceptus-derived Type I or II IFNs in the uterus, and is implicated as an important factor in determining uterine receptivity to embryos in ruminants. But little is known about the role the ISG15 gene or gene product plays during embryo development. In the present study, both the expression profile and function of ISG15 were investigated in early bovine embryos in vitro. ISG15 mRNA was detectable in Day 0, 2, 6 and 8 bovine embryos, but IFN-τ (IFNT) mRNA only appeared from Day 6. This means that embryonic expression of ISG15 on Days 0 and 2 was not induced by embryonic IFNT. However, ISG15 mRNA expression paralleled the expression of IFNT mRNA in Day 6 and 8 embryos. ISG15-lentivirus interference plasmid (ISG15i) was injected into 2-cell embryos to knockdown ISG15 expression. This resulted in decreases in the proportion of hatching blastocysts, the diameter of blastocysts and cell number per diameter of blastocysts compared with control embryos. In addition, ISG15i inhibited IFNT, Ets2 (E26 oncogene homolog 2) mRNA and connexion 43 protein expression in Day 8 blastocysts, whereas exogenous IFNT treatment (100ngmL-1, from Day 4 to Day 8) improved ISG15 mRNA and connexion 43 protein expression. In conclusion, it appears that ISG15 is involved in early bovine embryo development and that it regulates IFNT expression in the blastocyst.


Assuntos
Blastocisto/metabolismo , Citocinas/metabolismo , Ectogênese , Regulação da Expressão Gênica no Desenvolvimento , Interferon Tipo I/metabolismo , Proteínas da Gravidez/metabolismo , Ubiquitinas/metabolismo , Regulação para Cima , Matadouros , Animais , Animais Endogâmicos , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Bovinos , Criopreservação , Citocinas/antagonistas & inibidores , Citocinas/genética , Ectogênese/efeitos dos fármacos , Feminino , Fertilização in vitro , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Técnicas de Maturação in Vitro de Oócitos , Interferon Tipo I/genética , Interferon Tipo I/farmacologia , Masculino , Proteínas da Gravidez/genética , Proteínas da Gravidez/farmacologia , Interferência de RNA , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Preservação do Sêmen , Ubiquitinas/antagonistas & inibidores , Ubiquitinas/genética , Regulação para Cima/efeitos dos fármacos
4.
Reprod Biol ; 16(4): 295-299, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27839674

RESUMO

Postpartum uterine health in dairy cows is crucial for the maintenance of good reproductive performance. In order to improve uterine health and reduce puerperal intrauterine infection, 608 Holstein cows received a weekly PGF2α protocol (3 i.m. injections of PGF2α at 7, 14 and 21 d postpartum). For comparison, 593 cows in the control group received injections of sterile saline at the same time. Uterine score at 14 d postpartum, the prevalence of endometritis at 21-27 d postpartum, and subsequent reproduction performance was evaluated. Cows in the treated group exhibited higher tonicity (P<0.05) of the uterus, with less prevalence of endometritis (10.4%, 63/608 vs. 34.6%, 205/593; P<0.001), and required shorter time to the first AI postpartum (67.5±3.4 d vs. 84.4±3.7 d, P<0.05) and to pregnancy (114.5±5.4 d vs. 131.4±5.8 d, P<0.05). In conclusion, the present study demonstrated that uterine health in Holstein cows can be promoted while puerperal infection can be suppressed by this weekly postpartum PGF2α protocol.


Assuntos
Doenças dos Bovinos/prevenção & controle , Dinoprosta/farmacologia , Endometrite/veterinária , Período Pós-Parto , Infecção Puerperal/veterinária , Útero/efeitos dos fármacos , Animais , Bovinos , Doenças dos Bovinos/diagnóstico , Dinoprosta/uso terapêutico , Endometrite/diagnóstico , Endometrite/prevenção & controle , Feminino , Infecção Puerperal/diagnóstico , Infecção Puerperal/prevenção & controle
5.
Biochem Biophys Res Commun ; 473(1): 311-316, 2016 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-27016483

RESUMO

The transcription factor nuclear factor-κB (NF-κB) plays an important role in diverse processes, including cell proliferation and differentiation, apoptosis and inflammation. However, the role of NF-κB in porcine follicle development is not clearly elucidated. In this study, we demonstrated that follicle stimulating hormone (FSH) increased the level of inhibitor of NF-κB (IκB) protein and promoted the cytoplasmic localization of p65, indicating that FSH inhibits the activation of NF-κB in porcine granulosa cells. Moreover, inhibition of NF-κB by FSH or another specific inhibitor of NF-κB, pyrrolidine dithiocarbamate (PDTC), could activate JNK signaling and enhance autophagic activity in porcine granulosa cells. Knockdown of RelA (p65) Subunit of NF-κB by RNA interference abrogated the activation of JNK signaling pathway and the increase of autophagic protein expression by FSH. Meanwhile, the functional significance of FSH or PDTC-mediated autophagy were further investigated. Our results demonstrated that the increased autophagy promoted progesterone secretion in porcine granulosa cells. Blockage of autophagy by chloroquine obviated the FSH or PDTC-induced progesterone production. Taken together, these results indicate that inhibition of NF-κB increased autophagy via JNK signaling, and promote steroidogenesis in porcine granulosa cells. Our results provide new insights into the regulation and function of autophagy in mammalian follicle development.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Autofagia , Células da Granulosa/metabolismo , MAP Quinase Quinase 4/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Animais , Apoptose , Núcleo Celular/metabolismo , Proliferação de Células , Citoplasma/metabolismo , Feminino , Hormônio Foliculoestimulante/metabolismo , Marcação In Situ das Extremidades Cortadas , Microscopia de Fluorescência , NF-kappa B/antagonistas & inibidores , Folículo Ovariano/metabolismo , Progesterona/química , Pirrolidinas/química , Suínos , Tiocarbamatos/química , Fator de Transcrição RelA/metabolismo
6.
Fertil Steril ; 105(5): 1351-1359.e4, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26812245

RESUMO

OBJECTIVE: To determine whether the levels of CBP/p300 interacting transactivator with ED-rich tail 2 (Cited2) protein in cumulus cells (CCs) derived from patients undergoing IVF related to infertility factors, embryo quality, and clinical outcomes in one IVF cycle. DESIGN: Retrospective analysis of human CCs. SETTING: Public hospital and university. PATIENT(S): A total of 103 (conventional) IVF patients and 32 intracytoplasmic sperm injection patients. INTERVENTION(S): All CCs from each patient's oocytes were considered as one sample. The patients were divided into two groups according to whether the Cited2/ß-actin levels in their CCs were above or below the mean level detected for all patients. MAIN OUTCOME MEASURE(S): Embryo quality and clinical outcomes of IVF patients. RESULT(S): The oocytes derived from the group of patients whose CCs showed lower Cited2 levels displayed higher fertilization, transferable embryo, and implantation rates. Moreover, the patients in this group were more likely to have a successful pregnancy outcome. Among different infertility factors, a total of 78.6% of patients with polycystic ovary syndrome had a higher Cited2 level in CCs. Additionally, patients with a lower basal FSH level belonged to the higher Cited2 levels group. The expression of two genes (phosphoenolpyruvate carboxykinase 1 [PCK1] and progesterone receptor [PR]) and the glucose content in CCs were also markedly increased in CCs derived from patients with higher Cited2 levels. CONCLUSION(S): The present findings imply that Cited2 level in CCs is associated with polycystic ovary syndrome, embryo quality, and pregnancy outcome of IVF patients.


Assuntos
Células do Cúmulo/metabolismo , Implantação do Embrião/fisiologia , Fertilização in vitro/métodos , Resultado da Gravidez , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Adulto , Biomarcadores/metabolismo , Transferência Embrionária/métodos , Transferência Embrionária/tendências , Feminino , Fertilização in vitro/tendências , Humanos , Gravidez , Estudos Retrospectivos
7.
Theriogenology ; 84(5): 811-7, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26100237

RESUMO

Progesterone is an important steroid hormone in the regulation of the bovine estrous cycle. The steroidogenic acute regulatory protein (StAR) is an indispensable component for transporting cholesterol to the inner mitochondrial membrane, which is one of the rate-limiting steps for progesterone synthesis. Low-density lipoprotein (LDL) supplies cholesterol precursors for progesterone formation, and the lysosomal degradation pathway of LDL is essential for progesterone biosynthesis in granulosa cells after ovulation. However, it is currently unknown how LDL and lysosomes coordinate the expression of the StAR gene and progesterone production in bovine granulosa cells. Here, we investigated the role of lysosomes in LDL-treated bovine granulosa cells. Our results reported that LDL induced expression of StAR messenger RNA and protein as well as expression of cholesterol side-chain cleavage cytochrome P-450 (CYP11A1) messenger RNA and progesterone production in cultured bovine granulosa cells. The number of lysosomes in the granulosa cells was also significantly increased by LDL; whereas the lysosomal inhibitor, chloroquine, strikingly abolished these LDL-induced effects. Our results indicate that LDL promotes StAR expression, synthesis of progesterone, and formation of lysosomes in bovine granulosa cells, and lysosomes participate in the process by releasing free cholesterol from hydrolyzed LDL.


Assuntos
Bovinos/metabolismo , Células da Granulosa/metabolismo , Lipoproteínas LDL/fisiologia , Lisossomos/fisiologia , Fosfoproteínas/metabolismo , Progesterona/biossíntese , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Lipoproteínas LDL/farmacologia , Fosfoproteínas/genética
8.
Theriogenology ; 84(2): 184-92, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-25881989

RESUMO

No successful method exists to maintain the three-dimensional architecture of hatched embryos in vitro. Alginate, a linear polysaccharide derived from brown algae, has characteristics that make it an ideal material as a three-dimensional (3D) extracellular matrix for in vitro cell, tissue, or embryo culture. In this study, alginate hydrogel was used for IVC of posthatched bovine embryos to observe their development under the 3D system. In vitro-fertilized and parthenogenetically activated posthatched bovine blastocysts were cultured in an alginate encapsulation culture system (AECS), an alginate overlay culture system (AOCS), or control culture system. After 18 days of culture, the survival rate of embryos cultured in AECS was higher than that in the control group (P < 0.05), and the embryos were expanded and elongated in AECS with the maximal length of 1.125 mm. When the AECS shrinking embryos were taken out of the alginate beads on Day 18 and cultured in the normal culture system, 9.09% of them attached to the bottoms of the plastic wells and grew rapidly, with the largest area of an attached embryo being 66.00 mm(2) on Day 32. The embryos cultured in AOCS developed monovesicular or multivesicular morphologies. Total cell number of the embryos cultured in AECS on Day 19 was significantly higher than that of embryos on Day 8. Additionally, AECS and AOCS supported differentiation of the embryonic cells. Binuclear cells were visible in Day-26 adherent embryos, and the messenger RNA expression patterns of Cdx2 and Oct4 in AOCS-cultured embryos were similar to those in vivo embryos, whereas IFNT and ISG15 messenger RNA were still expressed in Day-26 and Day-32 prolong-cultured embryos. In conclusion, AECS and AOCS did support cell proliferation, elongation, and differentiation of hatched bovine embryos during prolonged IVC. The culture system will be useful to further investigate the molecular mechanisms controlling ruminant embryo elongation and implantation.


Assuntos
Alginatos , Bovinos/embriologia , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário , Animais , Blastocisto/citologia , Blastocisto/fisiologia , Diferenciação Celular , Proliferação de Células , Técnicas de Cultura Embrionária/instrumentação , Técnicas de Cultura Embrionária/métodos , Implantação do Embrião/fisiologia , Feminino , Fertilização in vitro/veterinária , Expressão Gênica , Ácido Glucurônico , Ácidos Hexurônicos , Hidrogéis , Morfogênese
9.
Theriogenology ; 83(9): 1493-501, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25784452

RESUMO

Oocyte aging due to delayed fertilization is associated with declining quality and developmental potential. Intracellular calcium (Ca(2+)) concentration ([Ca(2+)]i) regulates oocyte growth, maturation, and fertilization and has also been implicated in aging. Using bovine oocytes, we tested the hypothesis that oocyte aging could be delayed by reducing [Ca(2+)]ivia blocking the influx of extracellular Ca(2+) or chelating ooplasmic free Ca(2+). After IVM, cumulus-oocyte complexes or denuded oocytes were cultured in medium supplemented with 1-octanol, phorbol 12-myristate 13-acetate, or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis-acetoxymethyl ester (BAPTA-AM) to manipulate [Ca(2+)]i. Addition of 1-mM 1-octanol increased blastocyst development rates in the cumulus-oocyte complexes aged for 6 hours by IVF and for 6, 12, and 24 hours by parthenoactivation, and this effect was independent of the presence of cumulus cells. The intracellular levels of ATP, Glutathione, and Glutathione disulfide were not affected by 1-octanol, but [Ca(2+)]i was significantly decreased. When oocytes were cultured in Ca(2+)-free medium for 12 hours, the blastocyst development rate was greater and the beneficial effects of 1-octanol on oocyte aging were abolished. However, when the medium was supplemented with phorbol 12-myristate 13-acetate, [Ca(2+)]i increased and the blastocyst development rate decreased. Moreover, BAPTA-AM reduced [Ca(2+)]i and increased blastocyst development rates after IVF or parthenoactivation. We conclude that the age-associated developmental potency decline was delayed by blocking the influx of extracellular Ca(2+) or reducing ooplasmic free Ca(2+). 1-Octanol, BAPTA-AM, or Ca(2+)-free medium could be used to lengthen the fertilization windows of aged bovine oocytes.


Assuntos
Cálcio/metabolismo , Bovinos/fisiologia , Oócitos/efeitos dos fármacos , 1-Octanol/farmacologia , Fatores Etários , Animais , Cálcio/química , Senescência Celular , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Desenvolvimento Embrionário/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Oócitos/fisiologia , Acetato de Tetradecanoilforbol/farmacologia
10.
J Dairy Sci ; 97(11): 6917-25, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25242422

RESUMO

Interferon-τ (IFNT), produced in ruminants by embryonic trophoblastic cells before implantation, is involved in the maternal recognition of pregnancy. It is a pleiotropic molecule that alters the synthesis of endometrial proteins and inhibits the proliferation of some cells. The present study investigated the effects of recombinant bovine IFNT on the development of early-stage bovine embryos and the molecular mechanism underlying this effect. This study demonstrated that expression of mRNA encoding type I IFN receptor subunits was detectable from d 4 to 8 in in vitro fertilized (IVF) bovine embryos. A considerable number of IVF (n = 1,941) and parthenogenetic activated (n = 1,552) bovine embryos demonstrated that supplementing the culture medium with IFNT (100 ng/mL) produced a greater percentage of blastocysts, and the total cell number within the resulting blastocysts was higher. In addition, IFNT upregulated the expression levels of both mRNA and protein for connexin 43 (GJA1) and E-cadherin (CDH1) and expression levels for granulocyte-macrophage colony-stimulating factor and insulin-like growth factor 2 mRNA but not for their proteins in d 8 embryos. However, IFNT inhibited mRNA expression for leukemia inhibitory factor (LIF), LIF receptor α, and the sodium/potassium-transporting ATPase subunit ß-1. We concluded that IFNT promoted the development of bovine embryos by upregulating the expression of GJA1 and CDH1. Thus, supplementing embryo cultures or transfer medium with IFNT may stimulate embryo development and improve embryo transfer efficiency.


Assuntos
Caderinas/metabolismo , Bovinos/embriologia , Conexina 43/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Interferon Tipo I/farmacologia , Proteínas da Gravidez/farmacologia , Animais , Blastocisto/metabolismo , Bovinos/fisiologia , Transferência Embrionária/veterinária , Feminino , Fertilização in vitro/veterinária , Gravidez , RNA Mensageiro/metabolismo , Proteínas Recombinantes/farmacologia , Regulação para Cima
11.
Biol Reprod ; 89(5): 124, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24089204

RESUMO

Accumulating evidence indicates that cellular and molecular abnormalities occur during oocyte aging, including fragmentation, increases in intracellular reactive oxygen species (ROS), and abnormal Ca(2+) oscillations. The objective of the present study was to characterize the relationships between intracellular ROS, Ca(2+) homeostasis of endoplasmic reticulum (ER), and fragmentation in aged porcine MII oocytes. Prolonged culture (36 h) of porcine oocytes resulted in elevated intracellular ROS level, impaired ER Ca(2+) homeostasis (i.e., Ca(2+) storage, Ca(2+) rising patterns after electroactivation, and the cluster distribution of ER), and increased fragmentation rates. However, when the porcine oocytes were treated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester), an intracellular Ca(2+) chelator, the fragmentation was significantly inhibited during in vitro aging. In order to pursue the underlying mechanisms, H2O2 and cycloheximide (CHX) were used to artificially increase or inhibit, respectively, the intracellular ROS levels in aged porcine oocytes during in vitro culture. The results demonstrated that incubation of porcine MII oocytes with H2O2 damaged the ER clusters and the Ca(2+) regulation of ER, leading to a high proportion of fragmented oocytes. In contrast, CHX, an intracellular inhibitor of ROS generation, prevented both increase of ROS level and damage of the ER Ca(2+) homeostasis in porcine oocytes during aging, resulting in low fragmentation rate. We conclude that the increased intracellular ROS damaged the ER clusters and ER Ca(2+) homeostasis, resulting in a disorder in ooplasmic free Ca(2+), which caused the fragmentations seen in porcine MII oocytes during aging.


Assuntos
Cálcio/metabolismo , Senescência Celular/fisiologia , Fragmentação do DNA/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Oócitos/efeitos dos fármacos , Espécies Reativas de Oxigênio/efeitos adversos , Suínos/fisiologia , Animais , Células Cultivadas , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Feminino , Homeostase/efeitos dos fármacos , Oócitos/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Suínos/metabolismo
12.
Biol Reprod ; 89(3): 56, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23843241

RESUMO

Oocyte-secreted factors (OSFs) maintain the low incidence of cumulus cell apoptosis. In this report, we described that the presence of oocytes suppressed the expression of proapoptotic protein BCL-2-interacting mediator of cell death-extra long (BIMEL) in porcine cumulus cells. Atretic (terminal deoxynucleotidyl transferase dUTP nick end labeling-positive) cumulus cells strongly expressed BIMEL protein. The healthy cumulus- oocyte complex exhibited a low BIMEL expression in cumulus cell while the removal of oocyte led to an about 2.5-fold (P < 0.5) increased expression in oocytectomized complex (OOX). Coculturing OOXs with denuded oocytes decreased BIMEL expression to the normal level. The similar expression pattern could also be achieved in OOXs treated with exogenous recombinant mouse growth differentiation factor 9 (GDF9), a well-characterized OSF. This inhibitory action of GDF9 was prevented by the addition of a phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Luciferase assay further demonstrated that BIM gene expression was forkhead box O3a (FOXO3a)-dependent because mutation of FOXO3a-binding site on the BIM promoter inhibited luciferase activities. Moreover, the activity of BIM promoter encompassing the FOXO3a-binding site could be regulated by GDF9. Additionally, we found that GDF9 elevated the levels of phosphorylated AKT and FOXO3a, and this process was independent of the SMAD signal pathway. Taken together, we concluded that OSFs, particularly GDF9, maintained the low level of BIMEL expression in cumulus cell through activation of the PI3K/FOXO3a pathway.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Células do Cúmulo/efeitos dos fármacos , Fator 9 de Diferenciação de Crescimento/fisiologia , Proteínas de Membrana/genética , Oócitos/metabolismo , Proteínas Proto-Oncogênicas/genética , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Células Cultivadas , Células do Cúmulo/fisiologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Feminino , Fatores de Transcrição Forkhead/metabolismo , Fator 9 de Diferenciação de Crescimento/farmacologia , Masculino , Proteínas de Membrana/metabolismo , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Suínos
13.
Microbiol Immunol ; 56(3): 163-70, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22308980

RESUMO

Interferon stimulated gene 15 (ISG15), an ubiquitin cross-reactive protein, can conjugate to target proteins. Unlike ubiquitination, protein modification by ISG15 does not target protein for degradation, but enhances the cellular response to interferon (IFN), which plays a key role in antiviral responses. In this study, Western blot and/or immunocytochemistry were performed to explore the ISG15 expression patterns in explants of bovine endometrium, mammary gland and kidney, as well as Madin-Darby bovine kidney (MDBK), endometrial and mammary cells stimulated by IFN-α, -ß, and -τ. Western blot indicated that there are differential minimum antiviral units among recombinant bovine interferon-α (rbIFN-α, 10(2) IU/mL), rbIFN-ß (10(3) IU/mL) and rbIFN-τ (10(4) IU/mL) in regard to stimulating saturation expression of free and ISG15-conjugated proteins by MDBK cells and endometrial and mammary explants. These results were further confirmed through immunocytochemical analysis of MDBK, endometrial and mammary cells. For the first time it has been shown that the expression pattern of ISG15-conjugated proteins occurs in a tissue-specific manner. Furthermore, the present findings provide the first evidence of 10- to 100-fold differences in minimum antiviral units of rbIFN-α, rbIFN-ß, and rbIFN-τ in regard to stimulating saturation expression of ISG15.


Assuntos
Citocinas/biossíntese , Interferons/imunologia , Ubiquitinas/biossíntese , Animais , Western Blotting , Bovinos , Células Cultivadas , Endométrio/citologia , Feminino , Perfilação da Expressão Gênica , Imuno-Histoquímica , Rim/citologia , Glândulas Mamárias Animais/citologia , Técnicas de Cultura de Órgãos , Proteínas Recombinantes/imunologia
14.
J Biol Chem ; 287(13): 10166-10177, 2012 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-22235114

RESUMO

The pro-apoptotic protein Bim (B-cell lymphoma-2 (Bcl-2)-interacting modulator of cell death) has recently been identified and shown to promote cell death in response to several stimuli. In this report, we investigated the role of Bim in porcine follicular atresia. Initially, Bim cDNA was cloned and characterized from porcine ovarian tissue. Porcine Bim had three alternative splicing variants (Bim-extra long, Bim-long, and Bim-short), all containing the consensus Bcl-2 homology 3 domain. We then found the Bim-extra long (Bim(EL)) protein, the most abundant isoform of Bim, was strongly expressed and co-localized with apoptotic (TUNEL-positive) granulosa cells from porcine atretic follicles. Furthermore, overexpression of Bim(EL) triggered apoptosis in granulosa cells. In primary granulosa cell cultures under basal conditions, we observed that Bim(EL) expression was dampened by treatment with follicle-stimulating hormone (FSH). The role of the PI3K/Akt pathway in the regulation of repression was clarified by the use of the PI3K inhibitor, LY294002, and by transfection with Akt siRNA. Forkhead Box Protein O3a (FoxO3a), a well defined transcriptional activator of Bim, was phosphorylated at Ser-253 and inactivated after FSH stimulation. Also, FSH abolished FoxO3a nuclear accumulation in response to LY294002. Finally, chromatin immunoprecipitation assays demonstrated that FoxO3a directly bound and activated the bim promoter. Taken together, we conclude that Bim(EL) induces porcine granulosa cell apoptosis during follicular atresia, and its expression is regulated by FSH via the PI3K/Akt/FoxO3a pathway.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Hormônio Foliculoestimulante/farmacologia , Células da Granulosa/metabolismo , Hormônios/farmacologia , Proteínas de Membrana/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Processamento Alternativo/efeitos dos fármacos , Processamento Alternativo/fisiologia , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Proteína 11 Semelhante a Bcl-2 , Células Cultivadas , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Fatores de Transcrição Forkhead/metabolismo , Células da Granulosa/citologia , Humanos , Camundongos , Dados de Sequência Molecular , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/farmacologia , Suínos
15.
Theriogenology ; 76(8): 1487-95, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21835451

RESUMO

Whether cumulus cells (CCs) contribute to oocyte aging remains controversial; in that regard, little is known about biochemical processes of gene expression in CCs surrounding aged oocytes. The objective was to elucidate contributions of CCs to porcine oocyte aging and degeneration, apoptosis and BIM expression in CCs during oocyte aging in vitro. When culture of cumulus oocyte complexes (COCs) was prolonged (68 h, which resulted in 24 h of aging), the rate of blastocyst formation following electro-activation was lower than that of oocytes aged without CCs (2.6 ± 0.1 vs 13.5 ± 1.3%, mean ± SEM; P < 0.05). In addition, the presence of CCs significantly accelerated spontaneous fragmentation of oocytes following prolonged (92 h) culture. Apoptotic CCs were present in COCs cultured for 68 h, and the abundance of Bim mRNA in CCs progressively increased after 56 h of culture (P < 0.05). Based on immunofluorescence, BIM protein expression was up-regulated in CCs surrounding aged oocytes; furthermore, quantification (Western blot) of BIM(EL) protein progressively increased after 56 h of culture. Lastly, in a series of experiments to elucidate the signal pathway, blocking gap junctions (with 1-octanol) during aging did not eliminate the effect of CCs on accelerating oocyte aging, but prolonged co-culture of denuded oocytes with COCs after in vitro maturation reduced blastocyst rate relative to culture of denuded oocytes aged alone (4.15 ± 0.1 vs 11.0 ± 0.7%, P < 0.05). We concluded that apoptotic CCs, in which BIM(EL) up-regulation was involved, accelerated oocyte aging and degeneration in vitro via a paracrine action.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Células do Cúmulo/fisiologia , Proteínas de Membrana/metabolismo , Oócitos/fisiologia , Comunicação Parácrina/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Regulação da Expressão Gênica/fisiologia , Proteínas de Membrana/genética , Oócitos/citologia , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Suínos , Fatores de Tempo
16.
Theriogenology ; 76(7): 1207-14, 2011 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-21820723

RESUMO

Dromedary camel oocytes have the ability to spontaneous parthenogenetic activation and development in vivo and in vitro. The present study was conducted to investigate changes in mitochondrial distribution, adenosine triphosphate (ATP), and glutathione (GSH) contents and [Ca(2+)] oscillation during in vitro maturation and spontaneous parthenogentic activation of dromedary camel oocytes. Dromedary camel cumulus-oocyte complexes (COCs) were matured in TCM199 medium supplemented with 10% FCS + 10 µg/mL FSH + 10 IU hCG + 10 IU eCG + 10 ng/mL EGF and 50 µg/mL gentamycine. Maturation was performed at 38.5 °C under 5% CO(2) in humidified air for 40 h. After maturation and removal of cumulus cells, oocytes were classified into: immature cultured (Group 1); metaphase II (M II, Group 2); and spontaneously parthenogenetically activated (with 2 polar bodies, Group 3); cleaved embryos (Group 4); and immature oocytes served as a control (Group 5). Cytoplasmic mitochondrial distribution, ATP-GSH contents, calcium [Ca(2+)] oscillation were determined. Results indicated that M II and spontaneously parthenogenetically activated oocytes represent 37.53% and 32.67% of the cultured oocytes, respectively, and 3.3% cleaved and developed to 2-16-cell stage embryos. Mitochondrial distribution, ATP-GSH contents and [Ca(2+)] oscillation were significantly (P < 0.01) differ between immature and matured dromedary camel oocytes. Mitochondrial distribution showed clustering form in matured oocytes without polar body. High polarized mitochondrial distribution (HPM) was detected in M II and spontaneously parthenogenetically activated oocytes, and the intensity of MitoTracker Red was higher in spontaneously parthenogenetically activated than M II. ATP-GSH contents and the duration of [Ca(2+)] oscillation were significantly (P < 0.01) higher in spontaneously parthenogenetically activated than M II oocytes or that matured without polar body. In conclusion, the higher incidence of spontaneously parthenogenetically activated in vitro matured dromedary camel oocytes could be attributed to the high polarized mitochondrial distribution associated with significantly higher ATP-GSH contents and duration of [Ca(2+)] oscillation.


Assuntos
Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Camelus , Glutationa/metabolismo , Técnicas de Maturação in Vitro de Oócitos/veterinária , Mitocôndrias/metabolismo , Oócitos/metabolismo , Animais , Polaridade Celular , Feminino , Mitocôndrias/ultraestrutura , Oócitos/ultraestrutura
17.
Theriogenology ; 75(4): 638-46, 2011 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-21074839

RESUMO

Deterioration in the quality of mammalian mature oocytes during metaphase-II (M-II) arrest is called "oocyte aging". Although histone acetylation may affect the progression of aging in murine oocytes, the mechanism is unknown. The objective was to determine the role of ooplasmic reactive oxygen species (ROS) in acetylation of histone H4 at lysine 12 (acH4K12) in porcine aged oocytes in vitro. Based on immunostaining with a specific antibody, acetylation of H4K12 in porcine oocytes increased during in vitro aging, which coincided with changing patterns of ooplasmic ROS content. Furthermore, both hydrogen peroxide (H(2)O(2)), and the mitochondrial membrane potential disrupter, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), which can moderately elevate oocyte ROS content, significantly increased acetylation levels of H4K12 in porcine oocytes. It was noteworthy that acetylation in the CCCP group was decreased when ROS was counteracted by cysteine, a common antioxidant. In addition, the intracellular mRNA abundance of acetyltransferase gene HAT1 in aged and H(2)O(2) treated oocytes was higher than in M-II phase oocytes, suggesting that HAT1 was involved in this reaction. After parthenogenetic activation, a lower proportion of oocytes developed to the blastocyst stage after CCCP or H(2)O(2) treatment when compared with M-II phase oocytes (20 and 0% for CCCP and H(2)O(2) groups, respectively, versus 42% for the M-II group, P < 0.05). In conclusion, elevated levels of H4K12 acetylation were attributed to increased ooplasmic ROS content during porcine oocyte aging in vitro.


Assuntos
Histonas/metabolismo , Oócitos/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/metabolismo , Suínos/genética , Acetilação/efeitos dos fármacos , Animais , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Técnicas de Cultura de Células , Cisteína/farmacologia , Desenvolvimento Embrionário/efeitos dos fármacos , Epigenômica , Feminino , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Peróxido de Hidrogênio/farmacologia , Oócitos/efeitos dos fármacos
18.
Endocr Res ; 35(1): 17-23, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20136515

RESUMO

Melatonin is capable of improving the developmental capacity of ovine, porcine and bovine embryos in vitro. However, whether melatonin possesses similar benefits to the in vitro mouse embryonic development has yet to be determined. In this study, we assessed the effects of various concentrations of melatonin (10-13 to 10-3 M) on the in-vitro development of mouse embryos cultured in HTF medium for 96 hr; embryos cultured without melatonin were used as control. The in vitro development of mouse two-cell embryos significantly benefited from treatment with melatonin in a concentration-dependent manner. The effects of melatonin on the rates of blastocyst formation, hatching/hatched blastocysts and cell number per blastocyst were bi-phasic; all significantly increased by melatonin at 10-13 to 10-5 M and decreased by melatonin at 10-3 M. Maximal benefit of melatonin on in vitro mouse 2-cell embryo development was achieved at a concentration of 10-9 M. In comparison to control, 10-9 M melatonin increased blastocyst formation rate from 48.08 +/- 5.25% to 82.08 +/- 2.34% (p < 0.05), hatched blastocyst rate from 25.65 +/- 11.79% to 66.47 +/- 4.94% (p < 0.05), and cell number per blastocyst 62.71 +/- 5.97 to 77.91 +/- 10.63 (p < 0.05). Thus, our datas demonstrated firstly that melatonin has beneficial effects on the in vitro development of 2-cell mouse embryos cultured in HTF medium.


Assuntos
Meios de Cultura , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário/efeitos dos fármacos , Melatonina/farmacologia , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/fisiologia , Relação Dose-Resposta a Droga , Feminino , Melatonina/administração & dosagem , Camundongos , Microscopia de Fluorescência , Gravidez , Zigoto
19.
J Pineal Res ; 47(4): 318-23, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19817971

RESUMO

This study focused on the effect of melatonin on in vitro maturation of porcine oocytes and their parthenogenetic embryonic development. Melatonin was measured in porcine follicular fluid of follicles of different sizes in the same ovary. Melatonin exists in follicular fluid, and the concentration is approximately 10(-11) m. Its concentration decreased as the diameter of follicle increased, which suggests an effect of melatonin on oocyte maturation. Therefore, immature oocytes were cultured in vitro in maturation medium supplemented with melatonin (10(-11), 10(-9), 10(-7), 10(-5) and 10(-3) m) or without melatonin. The oocytes at maturation stage were collected and activated. The parthenogenetic embryos were cultured and observed in medium supplemented with or without melatonin. Fresh immature oocytes without melatonin treatment were used as control. When only maturation medium was supplemented with 10(-9) m melatonin, the cleavage rate, blastocyst rate and the cell number of blastocyst (70 +/- 4.5%, 28 +/- 2.4% and 50 +/- 6.5%) were significantly higher (P < 0.05) than that of controls; when only culture medium was supplemented with melatonin, the highest cleavage rate, blastocyst rate and the cell number of blastocyst was observed at 10(-7) m melatonin, which were significantly higher than that of controls (P < 0.05). The best results (cleavage rates 79 +/- 8.4%, blastocyst rates 35 +/- 6.7%) were obtained when both the maturation and culture medium were supplemented with 10(-9) m melatonin respectively (P < 0.05). In conclusion, exogenous melatonin at the proper concentration may improve the in vitro maturation of porcine oocytes and their parthenogenetic embryonic development. Further research is needed to identify the effect of melatonin on in vitro and in vivo oocyte maturation and embryo development in porcine.


Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Líquido Folicular/metabolismo , Melatonina/metabolismo , Melatonina/farmacologia , Oócitos/citologia , Oócitos/efeitos dos fármacos , Animais , Blastocisto/efeitos dos fármacos , Células Cultivadas , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Suínos
20.
Cryo Letters ; 30(2): 112-8, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19448860

RESUMO

Farmed blue fox was used as a model to develop cryopreservation protocol for nondomestic canine species. We report here the developmental potential of farmed blue fox oocytes after vitrification with a two-step OPS method. Oocytes were collected and pre-cultured for 0, 24, 48, 72 hours respectively before cryopreservation. Vitrification of oocytes was achieved by a 30 sec treatment in 10% ethylene glycol (EG) or 10% EG + 10% dimethyl sulfoxide (DMSO) at 25 degree C followed by a 25 sec equilibration in EFS30 (30% (v/v) EG +21% (w/v) Ficoll +0.35M sucrose) or EDFS30 (15% (v/v) EG +15% (v/v) DMSO +21% (w/v) Ficoll +0.35M sucrose), before plunging into liquid nitrogen. The survival of oocytes after vitrification was assessed morphologically immediately after warming, and cultured for in vitro maturation. For comparison, control oocytes were cultured for in vitro maturation for 96 hours. The best result was obtained when oocytes were pre-cultured for 72 hours, first exposed to 10 percent EG + 10% DMSO and vitrified in EDFS30. The survival percentage of oocytes under these conditions was not significantly different (P > 0.05) from that of the control.


Assuntos
Criopreservação/veterinária , Dimetil Sulfóxido , Etilenoglicol , Raposas/fisiologia , Oócitos/fisiologia , Animais , Sobrevivência Celular , Células Cultivadas , Criopreservação/métodos , Feminino , Oócitos/citologia
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