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1.
J Vis Exp ; (161)2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32716390

RESUMO

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that during the reproductive life span only about 1% of the oocytes in an ovary mature and ovulate, several techniques have been developed to increase the exploitation of the ovarian reserve to the growing population of non-ovulatory follicles. Such technologies have allowed interventions of fertility preservation, selection programs in livestock, and conservation of endangered species. However, the vast potential of the ovarian reserve is still largely unexploited. In cows, for instance, some attempts have been made to support in vitro culture of oocytes at specific developmental stages, but efficient and reliable protocols have not yet been developed. Here we describe a culture system that reproduce the physiological conditions of the corresponding follicular stage, defined to develop in vitro growing oocytes collected from bovine early antral follicles to the fully-grown stage, corresponding to the medium antral follicle in vivo. A combination of hormones and a phosphodiesterase 3 inhibitor was used to prevent untimely meiotic resumption and to guide oocyte's differentiation.


Assuntos
Técnicas de Cultura de Células/métodos , Oócitos/fisiologia , Reserva Ovariana/fisiologia , Reprodução/fisiologia , Animais , Bovinos , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Feminino , Oogênese/fisiologia , Folículo Ovariano/fisiologia , Ovário/citologia , Ovário/fisiologia
2.
Gene ; 758: 144955, 2020 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-32683076

RESUMO

Cyclin B functions as a regulatory protein through association with its catalytic partner Cdc2 kinase forming M-phase promoting factor (MPF), which plays a central role in the meiotic maturation of oocyte. To gain insight into the molecular events, we here cloned a cyclin B cDNA from the ovary of the prawn Macrobrachium rosenbergii and compared its spatial-temporal expression patterns during oocyte maturation with those of crab Eriocheir sinensis. The prawn cyclin B cDNA encodes a 398 amino acid protein with predicted molecular weight of 45.16 kDa. Immunodetection of cyclin B protein by Western blot showed that a target band of approximately 53 kDa protein in the prawn ovaries at both late vitellogenesis (lVt) and germinal vesicle breakdown (GVBD) stages, whereas a 41 kDa band was present in the crab ovaries. Cyclin B protein expression changes indicating that the newly synthesis of cyclin B proteins could be required for GVBD in both prawn and crab. Immunohistochemical analysis revealed that both the prawn and crab cyclin B proteins, were localized in the ooplasm of previtellogenic oocytes, then relocated into germinal vesicle at vitellogenesis stage and localized on meiotic spindle at M phase. These similar behaviors suggested that the prawn and the crab cyclin B proteins associated with Cdc2 kinase have conserved roles in inducing GVBD and regulating the formation of meiotic spindle. The similar expression patterns of the cyclin B proteins during oocyte maturation implicated that the molecular mechanisms for MPF activation could be identical between the prawn and the crab.


Assuntos
Braquiúros/embriologia , Ciclina B/metabolismo , Oócitos/crescimento & desenvolvimento , Oogênese/fisiologia , Palaemonidae/embriologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteína Quinase CDC2/metabolismo , Clonagem Molecular , Ciclina B/genética , Feminino , Regulação da Expressão Gênica/genética , Oogênese/genética , Ovário/metabolismo , RNA Mensageiro/genética , Fuso Acromático/metabolismo , Vitelogênese/fisiologia
3.
PLoS One ; 15(6): e0235043, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32589675

RESUMO

Captive breeding has been explored in Chinese sturgeon (Acipenser sinensis) for species protection. However, gonad development from stage II to IV of cultured female broodstocks is a handicap. This study aimed to explore the physiological and metabolic changes during the ovary development from stage II to IV of female Chinese sturgeon and the related energy regulatory mechanism, which may be helpful to address the developmental obstacle. The results showed that the oocyte volume increased and the muscle lipid content decreased with the ovary development. Ovarian RNA levels of most genes related to lipid and amino acid metabolism were higher in stage II and III than in stage IV. Serum contents of differential metabolites in arginine, cysteine, methionine, purine, tyrosine, lysine, valine, leucine and isoleucine metabolism pathways peaked at stage III, while the contents of sarcosine, alanine and histidine, as well as most oxylipins derived from fatty acids peaked at stage IV. These results indicated the more active amino acids, lipid metabolism, and energy dynamics of fish body in response to the high energy input of ovary developing from stage II to III, and the importance of alanine, histidine, taurine, folate and oxylipins for fish with ovary at stage IV.


Assuntos
Aminoácidos/metabolismo , Ácidos Graxos/metabolismo , Peixes/fisiologia , Metabolômica/métodos , Oogênese/fisiologia , Ovário/metabolismo , Animais , China , Espécies em Perigo de Extinção , Feminino , Expressão Gênica/fisiologia
4.
Front Biosci (Schol Ed) ; 12: 116-136, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32114451

RESUMO

Oocyte quality influences early embryonic survival, establishment and maintenance of pregnancy, fetal development and adult diseases. The developmental competence of oocytes is acquired gradually and increases with follicular development. The ability of an oocyte to develop into an embryo depends on, having enough specific information in the form of mRNA or proteins. If this information is insufficient, defects in nuclear or cytoplasmic maturation, or in both processes, may arise and thus affect the in vitro development of fertilized oocytes. The greater developmental competence of oocytes aspirated from larger follicles is reported as compared with smaller follicles. Oocyte developmental competence is greatly correlated with the morphology of the cumulus oocyte complexes (COCs). Apart from morphological or biochemical markers, molecular markers have also been investigated. Until now, no specific markers of oocyte developmental competence could be described for the oocyte developmental competence. To, utilize female germplasm to its maximum, there is a need to enhance developmental competence of lesser competent oocytes derived from the follicles which are not fully grown. The oocyte pre-maturation and maturation conditions affect gene expression not only in the oocyte but till the blastocyst stages too. Strategies have been discussed in this review would be useful to enhance the developmental competence of oocytes.


Assuntos
Oócitos/fisiologia , Animais , Blastocisto/citologia , Blastocisto/fisiologia , Células do Cúmulo/citologia , Células do Cúmulo/fisiologia , Desenvolvimento Embrionário/fisiologia , Feminino , Humanos , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Oogênese/fisiologia , Gravidez
5.
Nat Commun ; 11(1): 1399, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170089

RESUMO

Deleted in azoospermia-like (DAZL) is an RNA-binding protein critical for gamete development. In full-grown oocytes, the DAZL protein increases 4-fold during reentry into the meiotic cell cycle. Here, we have investigated the functional significance of this accumulation at a genome-wide level. Depletion of DAZL causes a block in maturation and widespread disruption in the pattern of ribosome loading on maternal transcripts. In addition to decreased translation, DAZL depletion also causes translational activation of a distinct subset of mRNAs both in quiescent and maturing oocytes, a function recapitulated with YFP-3'UTR reporters. DAZL binds to mRNAs whose translation is both repressed and activated during maturation. Injection of recombinant DAZL protein in DAZL-depleted oocytes rescues the translation and maturation to MII. Mutagenesis of putative DAZL-binding sites in these mRNAs mimics the effect of DAZL depletion. These findings demonstrate that DAZL regulates translation of maternal mRNAs, functioning both as the translational repressor and activator during oocyte maturation.


Assuntos
Oócitos/metabolismo , Oogênese/genética , Oogênese/fisiologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos/embriologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Proteínas da Gravidez/metabolismo , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma , Fatores de Poliadenilação e Clivagem de mRNA
6.
PLoS One ; 15(2): e0229391, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32092110

RESUMO

Our previous work documented significant advancements in steroid-induced progression of oogenesis, demonstrating that co-treatment of female eels with 11-ketotestosterone (11KT) and estradiol-17ß (E2) successfully induced uptake of vitellogenin by oocytes. Here we evaluate the effects of this steroid co-treatment on subsequent time to ovulation and egg quality in shortfinned eels artificially matured by hypophysation. Co-treatment with 11KT (1 mg) and E2 (0.2 or 2 mg) significantly reduced time to ovulation and therefore, the amount of pituitary homogenate required, without any detrimental effects on gonadosomatic index, oocyte diameter or the total weight of stripped eggs. E2 treatment resulted in promising increases in fertilization rates. These indicators suggest that co-treatment with 11KT and E2 holds promise for future artificial maturation practices in terms of minimising fish handling and stress, and of reducing the need for expensive pituitary preparations.


Assuntos
Anguilla , Estradiol/farmacologia , Oogênese/efeitos dos fármacos , Indução da Ovulação , Testosterona/análogos & derivados , Anguilla/fisiologia , Animais , Feminino , Fertilidade/efeitos dos fármacos , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Oogênese/fisiologia , Ovário/citologia , Ovário/efeitos dos fármacos , Indução da Ovulação/métodos , Indução da Ovulação/veterinária , Testosterona/farmacologia
7.
Development ; 147(4)2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32054660

RESUMO

La-related protein 6 (Larp6) is a conserved RNA-binding protein found across eukaryotes that has been suggested to regulate collagen biogenesis, muscle development, ciliogenesis, and various aspects of cell proliferation and migration. Zebrafish have two Larp6 family genes: larp6a and larp6b Viable and fertile single and double homozygous larp6a and larp6b zygotic mutants revealed no defects in muscle structure, and were indistinguishable from heterozygous or wild-type siblings. However, larp6a mutant females produced eggs with chorions that failed to elevate fully and were fragile. Eggs from larp6b single mutant females showed minor chorion defects, but chorions from eggs laid by larp6a;larp6b double mutant females were more defective than those from larp6a single mutants. Electron microscopy revealed defective chorionogenesis during oocyte development. Despite this, maternal zygotic single and double mutants were viable and fertile. Mass spectrometry analysis provided a description of chorion protein composition and revealed significant reductions in a subset of zona pellucida and lectin-type proteins between wild-type and mutant chorions that paralleled the severity of the phenotype. We conclude that Larp6 proteins are required for normal oocyte development, chorion formation and egg activation.


Assuntos
Autoantígenos/genética , Autoantígenos/fisiologia , Córion/fisiologia , Oócitos/fisiologia , Ribonucleoproteínas/genética , Ribonucleoproteínas/fisiologia , Animais , Movimento Celular , Proliferação de Células , Colágeno/fisiologia , Proteínas do Ovo/fisiologia , Feminino , Edição de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Genótipo , Heterozigoto , Homozigoto , Lectinas/fisiologia , Masculino , Mutação , Oócitos/citologia , Oogênese/fisiologia , Fenótipo , Peixe-Zebra , Zona Pelúcida/fisiologia
8.
Gen Comp Endocrinol ; 290: 113398, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31981692

RESUMO

Pentraxins (PTX), belong to an evolutionarily conserved family, containing a PTX protein domain, having role in acute immunological responses and fertility in higher vertebrates. However, information regarding the action of ptx on reproduction is extremely limited in fish. To study this, ptx cDNA was cloned for downstream analysis. Tissue distribution and ontogeny expression analysis indicated the prevalence of ptx in ovary. Varied phase-wise expression during carp ovarian cycle and elevated ptx expression after human chorionic gonadotropin induction, in vitro and in vivo, indicated probable regulation of gonadotropin. In situ hybridization and immunohistochemistry revealed the presence of ptx transcript and protein in the follicular layer of stage-III/IV oocytes indicating a role in ovarian growth. To assess the functional significance of ptx, transient silencing was performed using follicular primary cell culture, in vitro and in common carp, in vivo, through ovary-targeted injection of PEI-siRNA. Transient silencing of ptx-siRNA reduced the expression of various genes/factors related to oogenesis such as transcription factors, several steroidogenic enzymes, and esrs genes. These alterations in expression suggested a plausible role for ptx in ovarian steroidogenesis either, directly or indirectly, which is evident from the changes in the serum estradiol-17ß (E2) and 17α,20ß-dihydroxyprogesterone levels. Furthermore, downregulation of aromatase activity was also noticed after transient silencing. Increased ptx expression after E2 induced sex reversal to juvenile carp showed the correlative role of ptx during ovarian differentiation and development. Taken together, these findings suggest that ptx exerts an important role during ovarian growth, maturation and/or recrudescence of common carp.


Assuntos
Carpas , Oogênese/fisiologia , Ovário/metabolismo , Animais , Diferenciação Celular , Feminino
9.
Cryobiology ; 92: 161-167, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31917962

RESUMO

The objective was to evaluate the developmental competence of immature and matured ovine oocytes after removing, maintaining or adding cumulus cells (CC) associated to vitrification by Cryotop method. Three experiments were performed involving 3,144 oocytes. In Experiment 1, CC were removed from immature, matured or fertilized oocytes subjected to in vitro embryo production. In Experiment 2, oocytes were vitrified either in MI or MII stage with or without CC, while a control group with CC remained unvitrified. In Experiment 3, oocytes partially denuded from CC were vitrified either in MI or MII stage, and a co-culture of fresh CC was added or not soon after warming to complete in vitro maturation (IVM) and in vitro fertilization (IVF), or IVF, respectively, while a control group remained unvitrified. In Experiment 1, the cleavage rate, development rate on Day 6 and blastocyst rate on Day 8 were improved when CC were maintained until the end of IVF (P < 0.05). In Experiment 2, vitrification of oocytes with enclosed CC showed a tendency to increase cleavage (P = 0.06) and improved blastocyst rate (P < 0.05). In Experiment 3, adding CC as co-culture after vitrification-warming tended to improve cleavage rate (P = 0.06) and increased hatching rate (P < 0.05). Regarding oocyte stage, vitrification of in vitro matured oocytes resulted in greater developmental competence than immature stages (P < 0.05). In conclusion, CC seems to have a relevant role for in vitro embryo development in either fresh or vitrified oocytes.


Assuntos
Criopreservação/métodos , Células do Cúmulo/citologia , Desenvolvimento Embrionário/fisiologia , Oócitos/citologia , Oogênese/fisiologia , Vitrificação , Animais , Blastocisto/citologia , Técnicas de Cocultura , Feminino , Fertilização In Vitro/métodos , Técnicas de Maturação in Vitro de Oócitos , Ovinos
10.
Artigo em Inglês | MEDLINE | ID: mdl-31629811

RESUMO

Prohibitin (PHB) is an evolutionarily conserved multifunctional protein with ubiquitous expression. In this study, we cloned the PHB gene from the testis of the swimming crab Portunus trituberculatus (PtPHB) and analyzed the deduced amino acid sequence. The expression level of phb mRNA in larvae was analyzed using qRT-PCR. The expression level of phb mRNA and PHB protein in different tissues were analyzed using qRT-PCR and Western blot respectively. Enzyme-linked immunosorbent assay analyses of the PHB protein were conducted with the testis and ovaries from P. trituberculatus specimens at different developmental stages. PHB was localized with mitochondria and ubiquitin in the testis and ovaries. The PtPHB gene was found to contain an open reading frame of 825 bp, encoding a predicted peptide with 275 amino acids, sharing between 65.9% and 96.7% similarity with that of other species. The qRT-PCR and Western blot results showed that the phb gene and PHB protein both expressed less in the testis and ovary than in other tissues, and the phb gene presented the lowest expression in the Z1 stage. Furthermore, the phb gene and PHB protein expression were different in the testis and ovaries at different developmental stages. PHB was mainly found to be co-localized with mitochondria and ubiquitin in cytoplasm and acrosome complex during spermatogenesis and in follicular cells during oogenesis. Interestingly, PHB-mitochondria signals and ubiquitin signal were also found in oocytes. These results indicated that PHB might play important roles during spermatogenesis and oogenesis by regulating mitochondrial activities.


Assuntos
Proteínas de Artrópodes/metabolismo , Braquiúros/metabolismo , Oogênese/fisiologia , Ovário/metabolismo , Proteínas Repressoras/metabolismo , Espermatogênese/fisiologia , Testículo/metabolismo , Animais , Feminino , Masculino , Mitocôndrias/metabolismo
11.
Theriogenology ; 142: 320-327, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31711691

RESUMO

To optimize the protocols for assisted reproductive techniques (ARTs) in collared peccary (Pecari tajacu Linnaeus, 1758), we evaluated various conditions for oocyte in vitro maturation (IVM) and chemical activation. Initially, we assessed the IVM rates, cumulus-oocyte complex (COC) quality, and oocyte morphometry in the absence or presence of epidermal growth factor (EGF). There was no difference between the COCs matured in absence or presence of EGF for the expansion of cumulus cells (97.6% ±â€¯1.2 vs. 100% ±â€¯0.0), presence of first polar body (65.9% ±â€¯1.2 vs. 70.5% ±â€¯1.8), nuclear status in second metaphase (62.5% ±â€¯11.6 vs. 68.4% ±â€¯4.9), cytoplasmic maturation (100.0% ±â€¯0.7 vs. 75.0% ±â€¯0.7), reactive oxygen species levels (0.5 ±â€¯0.2 vs. 0.3 ±â€¯0.1), and mitochondrial membrane potential (1.1 ±â€¯0.2 vs. 1.1 ± 0.1). However, the zona pellucida thickness of matured COCs was reduced in the presence of EGF. Thus, the EGF group was used for further experiments. The oocytes were artificially activated with ionomycin and four secondary activator combinations [6-dimethylaminopurine (6D), 6D and cytochalasin B (6D + CB), cycloheximide (CHX), and CHX and CB (CHX + CB)]. The effect of immature COCs based on cumulus cell layers and cytoplasm homogeneity (GI and GII or GIII COCs) on embryonic development and quality was evaluated. There was no difference in the cleavage rates among the groups of secondary activators. The cleavage rates of embryos derived from GI/GII and GIII COCs were greater than 72.2% and 25.0%, respectively. Moreover, treatment with CHX showed a reduction in the cleavage rate of embryos derived from GIII COCs when compared to the cleavage rate of embryos derived from GI/GII COCs (P < 0.05). Nevertheless, higher rates of blastocyst/total GI and GII COCs were observed in the 6D group (27.6% ± 0.3) compared to CHX group (6.9% ± 0.3). Additionally, only 6D treatment resulted in the production of embryos derived from GIII COCs (25.0% ± 0.2). The percentage of the ICM/total cell ratio was also greater in blastocysts derived from 6D (42.5% ± 19.0), 6D + CB (37.9% ± 21.9), and CHX + CB (43.8% ± 19.6) groups when compared to CHX (3.6% ± 0.1) group. Thus, the combination of ionomycin and 6D could produce collared peccary embryos by activation of both GI/GII COCs and GIII COCs. These optimized IVM conditions using EGF and chemical activation using ionomycin and 6D in collared peccaries form the first steps for establishing ARTs to conserve this species.


Assuntos
Adenina/análogos & derivados , Artiodáctilos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Ionomicina/farmacologia , Oócitos/efeitos dos fármacos , Partenogênese/efeitos dos fármacos , Adenina/farmacologia , Animais , Artiodáctilos/embriologia , Células Cultivadas , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/fisiologia , Desenvolvimento Embrionário/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Feminino , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Transferência Nuclear/veterinária , Oócitos/citologia , Oócitos/fisiologia , Oogênese/efeitos dos fármacos , Oogênese/fisiologia , Partenogênese/fisiologia
12.
Elife ; 82019 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-31755866

RESUMO

Human oocytes frequently generate aneuploid embryos that subsequently miscarry. In contrast, Drosophila oocytes from outbred laboratory stocks develop fully regardless of maternal age. Since mature Drosophila oocytes are not extensively stored in the ovary under laboratory conditions like they are in the wild, we developed a system to investigate how storage affects oocyte quality. The developmental capacity of stored mature Drosophila oocytes decays in a precise manner over 14 days at 25°C. These oocytes are transcriptionally inactive and persist using ongoing translation of stored mRNAs. Ribosome profiling revealed a progressive 2.3-fold decline in average translational efficiency during storage that correlates with oocyte functional decay. Although normal bipolar meiotic spindles predominate during the first week, oocytes stored for longer periods increasingly show tripolar, monopolar and other spindle defects, and give rise to embryos that fail to develop due to aneuploidy. Thus, meiotic chromosome segregation in mature Drosophila oocytes is uniquely sensitive to prolonged storage. Our work suggests the chromosome instability of human embryos could be mitigated by reducing the period of time mature human oocytes are stored in the ovary prior to ovulation.


Assuntos
Drosophila/fisiologia , Oócitos/fisiologia , Oogênese/fisiologia , Ovário/fisiologia , Fuso Acromático/fisiologia , Envelhecimento , Aneuploidia , Animais , Segregação de Cromossomos , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , RNA Mensageiro/metabolismo , Ribossomos , Temperatura
13.
Elife ; 82019 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-31650955

RESUMO

The TORC1 regulator GATOR1/SEACIT controls meiotic entry and early meiotic events in yeast. However, how metabolic pathways influence meiotic progression in metazoans remains poorly understood. Here we examine the role of the TORC1 regulators GATOR1 and GATOR2 in the response to meiotic double-stranded breaks (DSB) during Drosophila oogenesis. We find that in mutants of the GATOR2 component mio, meiotic DSBs trigger the constitutive downregulation of TORC1 activity and a permanent arrest in oocyte growth. Conversely, in GATOR1 mutants, high TORC1 activity results in the delayed repair of meiotic DSBs and the hyperactivation of p53. Unexpectedly, we found that GATOR1 inhibits retrotransposon expression in the presence of meiotic DSBs in a pathway that functions in parallel to p53. Thus, our studies have revealed a link between oocyte metabolism, the repair of meiotic DSBs and retrotransposon expression.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Drosophila/metabolismo , Drosophila/fisiologia , Meiose , Complexos Multiproteicos/metabolismo , Oogênese/fisiologia , Animais , Regulação da Expressão Gênica , Mapas de Interação de Proteínas
14.
Cryobiology ; 90: 96-99, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31550455

RESUMO

The aim of this study was to determine pore size of nylon mesh (NM) device suitable for cryosurvival of bovine mature oocytes and to apply the device to vitrification of large quantities of the oocytes. Ten to twelve oocytes were loaded onto an NM device (a square opening 37-, 57- or 77-µm on a side length). After removal of the excess volume of vitrification solution by paper absorption, the oocytes were vitrified-warmed, fertilized and cultured in vitro. Oocyte recovery and morphological survival were comparable among the three groups. However, blastocyst yield in the 37-µm group (39%) was higher than that in the 77-µm group (28%), and the yield in the 57-µm group (31%) was the intermediate. The 37-µm NM device was applicable for increased oocyte number >40 (blastocyst yield, 33%). These results suggest that 37-µm-pore sized NM can serve as cryodevice to vitrify large quantities of in vitro-matured bovine oocytes.


Assuntos
Blastocisto/citologia , Criopreservação/métodos , Oócitos/citologia , Telas Cirúrgicas , Animais , Blastocisto/fisiologia , Bovinos , Sobrevivência Celular/fisiologia , Feminino , Fertilização In Vitro , Técnicas de Maturação in Vitro de Oócitos/métodos , Nylons , Oócitos/fisiologia , Oogênese/fisiologia , Vitrificação
15.
Anim Reprod Sci ; 209: 106173, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31514919

RESUMO

The reproduction of Iheringichthys labrosus (Lütken, 1874) from the Turvo River, Brazil, was studied using anatomical, biometric, histological, and ultrastructural techniques. Between April 2014 and March 2015, a total of 278 males and 512 females were captured bimonthly. The testes of Iheringichthys labrosus are fringed and possess a cranial spermatogenic region and an exclusively secretory caudal region. Histologically, the cranial region is composed of seminiferous tubules with spermatogenesis being completed in cysts. The spermatozoa are of the primitive type with a spherical head and have a rudimentary intermediate piece and a long tail with an axonemic arrangement of 9 + 2. The caudal region does not form an individualized gland, and cells in this testis area have characteristics of protein secretion. A variable density electron-dense secretion accumulates in the cisternae of the rough endoplasmic reticulum in the lumen of the seminiferous tubules and in the testicular ducts during maturation. The cortical alveoli are discontinuous, and the zona pellucida consists of three layers crossed by pore canals, and the follicular cells are squamous in the early stages of oogenesis and cuboidal in advanced stages. The gonadosomatic index was associated with the maturation of the gonads while the condition factor indicated that the fish feed less and utilize adipose reserves during the reproductive period. Males and females reproductively functional throughout the year with spawning being partial or multiple, similar to that reported in studies of the species in lentic environments.


Assuntos
Peixes-Gato/fisiologia , Gônadas/anatomia & histologia , Gônadas/citologia , Reprodução/fisiologia , Animais , Peixes-Gato/anatomia & histologia , Tamanho Celular , Feminino , Gônadas/ultraestrutura , Masculino , Oogênese/fisiologia , Tamanho do Órgão , Ovário/anatomia & histologia , Ovário/citologia , Ovário/ultraestrutura , Estações do Ano , Espermatogênese/fisiologia , Espermatozoides/citologia , Espermatozoides/ultraestrutura , Temperatura , Testículo/citologia , Testículo/ultraestrutura
16.
Anim Reprod Sci ; 209: 106137, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31514927

RESUMO

To evaluate follicular dynamics, there was assessment of superovulatory response and in vivo embryo production in ewes treated with relatively smaller doses of exogenous pFSH than typically used in combination with a dose of eCG at the beginning of the gonadotropin treatment period. Santa Inês ewes (n = 24) were randomly divided into three groups, based on mg dose of pFSH administered: G200 (n = 8), G133 (n = 8) and G100 (n = 8) in eight decreasing doses at 12 -h intervals. All ewes were treated with 300 IU of eCG concomitantly starting with first pFSH administration. Ovulatory follicular dynamics and follicular wall vascularization (FWV) were evaluated using a B-mode and color Doppler ultrasonic machine, respectively. Superovulatory response and embryo production were evaluated 6 days after estrous detection. In the G200 group, the preovulatory follicle size (PFS) were less (P <  0.05), ovulation time later (P <  0.05), and PFS rate greater (P <  0.05); while in the G100 group ovulation rate, and number and percentage of unfertilized eggs were greater (P <  0.05) than in the G133 group (P <  0.05). Number and percentage of viable embryos were greater in the G200 and G100 compared to G133 group (P <  0.05). The dose of 100 mg of FSH was as efficacious as the traditional dose of 200 mg, in combination with a dose of eCG, for superovulatory response and viable embryo production but there was a greater percentage of unfertilized eggs with this treatment.


Assuntos
Hormônio Foliculoestimulante/administração & dosagem , Inseminação Artificial , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/fisiologia , Indução da Ovulação , Ovinos , Animais , Brasil , Tamanho Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Feminino , Inseminação Artificial/métodos , Inseminação Artificial/veterinária , Oogênese/efeitos dos fármacos , Oogênese/fisiologia , Folículo Ovariano/irrigação sanguínea , Ovulação/efeitos dos fármacos , Indução da Ovulação/métodos , Indução da Ovulação/veterinária , Gravidez , Ovinos/embriologia , Superovulação/fisiologia , Clima Tropical
17.
Mol Biol Cell ; 30(19): 2490-2502, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31390285

RESUMO

Collective cell migration is emerging as a major driver of embryonic development, organogenesis, tissue homeostasis, and tumor dissemination. In contrast to individually migrating cells, collectively migrating cells maintain cell-cell adhesions and coordinate direction-sensing as they move. While nonmuscle myosin II has been studied extensively in the context of cells migrating individually in vitro, its roles in cells migrating collectively in three-dimensional, native environments are not fully understood. Here we use genetics, Airyscan microscopy, live imaging, optogenetics, and Förster resonance energy transfer to probe the localization, dynamics, and functions of myosin II in migrating border cells of the Drosophila ovary. We find that myosin accumulates transiently at the base of protrusions, where it functions to retract them. E-cadherin and myosin colocalize at border cell-border cell contacts and cooperate to transmit directional information. A phosphomimetic form of myosin is sufficient to convert border cells to a round morphology and blebbing migration mode. Together these studies demonstrate that distinct and dynamic pools of myosin II regulate protrusion dynamics within and between collectively migrating cells and suggest a new model for the role of protrusions in collective direction sensing in vivo.


Assuntos
Movimento Celular/fisiologia , Miosina Tipo II/metabolismo , Ovário/metabolismo , Actomiosina/metabolismo , Animais , Adesão Celular , Polaridade Celular/fisiologia , Proteínas do Citoesqueleto , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Células Epiteliais/metabolismo , Feminino , Miosina Tipo II/fisiologia , Miosinas/metabolismo , Miosinas/fisiologia , Oogênese/fisiologia
18.
BMC Dev Biol ; 19(1): 14, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31277577

RESUMO

BACKGROUND: Insulin/insulin-like growth peptide signaling (IIS) down-regulates hemolymph sugar level and facilitates larval growth in the soybean pod borer, Maruca vitrata. The objective of this study is to determine whether IIS of M. vitrata can mediate ovarian development of adult females. RESULTS: A pair of ovaries consists of 8 ovarioles, each of which is separated into distal germarium and proximal vitellarium in M. vitrata. In the germarium, oocyte development occurred with active mitotic activity which was visible by incorporating bromodeoxyribose uridine. Previtellogenic development and subsequent vitellogenesis began soon after adult emergence. They continued with increase of female age. Oocyte development was facilitated by up-regulation of vitellogenin (Vg) and Vg receptor (VgR) gene expression. Larval diets significantly influenced on ovarian development of M. vitrata because oocyte development varied with pupal size derived from larvae treated with different nutritional diets. Its ovarian development was dependent on endocrine signal(s) from the head because decapitation soon after adult emergence prevented oogenesis and subsequent vitellogenesis along with marked reduction of Vg and VgR expression. Topical application of juvenile hormone (JH) significantly recovered its ovarian development whereas farnesoic acid (a precursor of JH biosynthesis) or 20-hydroxyecdysone treatment did not. JH stimulated vitellogenesis and choriogenesis, but not previtellogenic development. In contrast, insulin injection to decapitated females stimulated oocyte differentiation and vitellogenesis along with increase of Vg and VgR expression. To further analyze the effect of insulin on ovarian development, expression of four IIS components (InR, FOXO, Akt, and TOR) genes was manipulated by RNA interference. Hemocoelic injection of gene-specific double stranded RNAs significantly reduced their target gene mRNA levels and interfered with ovarian development. An addition of insulin to JH treatment against decapitated females enhanced the gonadotropic effect of JH by stimulating oogenesis. CONCLUSIONS: IIS plays crucial role in mediating previtellogenic development of M. vitrata in response to nutrient signal. It also enhances the gonadotropic effect of JH II on vitellogenesis.


Assuntos
Fator de Crescimento Insulin-Like I/metabolismo , Insulina/metabolismo , Hormônios Juvenis/metabolismo , Ovário/crescimento & desenvolvimento , Vitelogênese/fisiologia , Animais , Ecdisterona/farmacologia , Proteínas do Ovo/genética , Proteínas do Ovo/metabolismo , Ácidos Graxos Insaturados/farmacologia , Feminino , Proteína Forkhead Box O1/genética , Mariposas , Oogênese/fisiologia , Proteínas Proto-Oncogênicas c-akt/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores de Somatomedina/genética , Serina-Treonina Quinases TOR/genética , Vitelogeninas/genética , Vitelogeninas/metabolismo
19.
In Vitro Cell Dev Biol Anim ; 55(7): 548-558, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31313007

RESUMO

Recently, the mean maternal age at first birth has been continuing to increase. The decline in the age-related fertility is due to the reduction in the number and the quality of the oocyte. An elevation in intra-ovarian reactive oxygen species (ROS) is correlated with the increase in maternal age, and the oxidative stress is involved in the decline in oocyte quality. Although ß-carotene, a very effective quencher of ROS, has been found to have the beneficial contribution to the ovarian development and steroidogenesis, it is unknown the effect of ß-carotene on the oocyte development especially oocyte maturation. This investigation aimed to explore the beneficial contribution of ß-carotene on oocyte maturation under oxidative stress and the underlying mechanism. We found that the oxidative stress induced by ROS reagent Rosup inhibited oocyte development/maturation and parthenogenetic activation which could be dramatically rescued by ß-carotene (57.1 ± 4.7% vs 78.9 ± 3.8%; p < 0.05) in vitro. The underlying mechanisms include that ß-carotene not only reduces ROS formation and cell apoptosis, but also it can restore actin expression, cortical granule-free domain (CGFD) formation, mitochondria homogeneous distribution, and nuclear maturation. The data suggest that ß-carotene acts as a potential antioxidant in the oocyte. Therefore, the findings from this investigation provide the fundamental 7knowledge for using ß-carotene as an antioxidant to improve the oocyte quality and even the ovarian function.


Assuntos
Antioxidantes/farmacologia , Oócitos/citologia , Oogênese/fisiologia , Estresse Oxidativo/efeitos dos fármacos , beta Caroteno/farmacologia , Actinas/biossíntese , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Feminino , Idade Materna , Camundongos , Mitocôndrias/fisiologia , Espécies Reativas de Oxigênio/metabolismo
20.
Dev Biol ; 454(2): 97-107, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31251895

RESUMO

Dietary proteins are crucial for oogenesis. The Target of Rapamycin (TOR) is a major nutrient sensor controlling organismal growth and fertility, but the downstream effectors of TOR signaling remain largely uncharacterized. We previously identified Drosophila Spargel/dPGC-1 as a terminal effector of the TOR-TSC pathway, and now report that Spargel connects nutrition to oogenesis. We found that Spargel is expressed predominantly in the ovaries of adult flies, and germline spargel knockdown inhibits cyst growth, ultimately leading to egg chamber degeneration and female sterility. In situ staining demonstrated nuclear localization of Spargel in the nurse cells and follicle cells of the ovariole. Furthermore, Spargel/dPGC-1 expression is influenced by dietary yeast concentration and TOR signaling, suggesting Spargel/dPGC-1 might transmit nutrient-mediated signals into ovarian growth. We propose that potentiating Spargel/dPGC-1 expression in the ovary is instrumental in nutrient-mediated regulation of oogenesis.


Assuntos
Proteínas de Drosophila/metabolismo , Oogênese/fisiologia , Ovário/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Animais , Proteínas na Dieta/metabolismo , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/metabolismo , Feminino , Células Germinativas/metabolismo , Nutrientes , Folículo Ovariano/metabolismo , Ovário/crescimento & desenvolvimento , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fator B de Elongação Transcricional Positiva/fisiologia , Transdução de Sinais , Sirolimo/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/fisiologia
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