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1.
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
2.
Genes Dev ; 34(3-4): 239-249, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31919193

RESUMO

Addressing the complexity of organogenesis at a system-wide level requires a complete understanding of adult cell types, their origin, and precursor relationships. The Drosophila ovary has been a model to study how coordinated stem cell units, germline, and somatic follicle stem cells maintain and renew an organ. However, lack of cell type-specific tools have limited our ability to study the origin of individual cell types and stem cell units. Here, we used a single-cell RNA sequencing approach to uncover all known cell types of the developing ovary, reveal transcriptional signatures, and identify cell type-specific markers for lineage tracing. Our study identifies a novel cell type corresponding to the elusive follicle stem cell precursors and predicts subtypes of known cell types. Altogether, we reveal a previously unanticipated complexity of the developing ovary and provide a comprehensive resource for the systematic analysis of ovary morphogenesis.


Assuntos
Drosophila/citologia , Folículo Ovariano/citologia , Células-Tronco/citologia , Animais , Drosophila/genética , Drosophila/metabolismo , Feminino , Modelos Animais , Ovário/citologia , Análise de Sequência de RNA , Análise de Célula Única , Transcrição Genética
3.
Toxicol Lett ; 319: 175-186, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31733319

RESUMO

Information on the effects of gibberellic acid (gibberellin A3, GA3) on ovarian follicle development is limited. In our present study, 21-day-old female Wistar rats were exposed to GA3 by gavage (25, 50, and 100 mg/kg body weight, once per day) for eight weeks to evaluate the influence of GA3 on ovarian follicle development. After treatment, significant (P < 0.05) increases (to 40.17 % and 44.5 %, respectively) in atretic follicle proportions and significant decreases (to 19.49 % and 17.86 %, respectively) in corpus luteum proportions were observed in the 50 and 100 mg/kg treatment groups compared to the control group. Significant (P < 0.05) increases (to 31.3 % and 42.0 %, respectively) in follicle apoptosis were observed in the 50 and 100 mg/kg treatment groups by transmission electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays. Significantly increased expression of caspase-3, caspase-8, caspase-9 and Fas was observed by real-time PCR and Western blotting. Bisulfite sequencing PCR (BSP) revealed obviously decreased total methylation percentages of the caspase-3 promoter region in the two treatment groups. Real-time quantitative PCR also showed significantly decreased mRNA expression of DNA methyltransferase (Dnmt) 3a and Dnmt3b. Further in vitro studies showed that a DNA methylation inhibitor could enhance the GA3-induced increase in the mRNA expression of caspase-3. Overall, our present study indicates that GA3 administration from weaning until sexual maturity can affect ovarian follicle development by inducing apoptosis and suggests that signaling through the Fas-mediated apoptotic pathway may be an important underlying mechanism of this apoptosis. In addition, GA3-induced aberrant DNA methylation patterns might be partly responsible for upregulation of caspase-3 gene expression.


Assuntos
Apoptose/efeitos dos fármacos , Caspase 3/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Proteína de Domínio de Morte Associada a Fas/biossíntese , Giberelinas/toxicidade , Células da Granulosa/efeitos dos fármacos , Ovário/citologia , Transdução de Sinais/efeitos dos fármacos , Animais , DNA (Citosina-5-)-Metiltransferases/biossíntese , DNA (Citosina-5-)-Metiltransferases/genética , Proteína de Domínio de Morte Associada a Fas/efeitos dos fármacos , Feminino , Folículo Ovariano/efeitos dos fármacos , Ovário/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Ratos , Ratos Wistar , Superovulação/efeitos dos fármacos
4.
Nat Cell Biol ; 21(10): 1261-1272, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31570835

RESUMO

The repression of transposons by the Piwi-interacting RNA (piRNA) pathway is essential to protect animal germ cells. In Drosophila, Panoramix enforces transcriptional silencing by binding to the target-engaged Piwi-piRNA complex, although the precise mechanisms by which this occurs remain elusive. Here, we show that Panoramix functions together with a germline-specific paralogue of a nuclear export factor, dNxf2, and its cofactor dNxt1 (p15), to suppress transposon expression. The transposon RNA-binding protein dNxf2 is required for animal fertility and Panoramix-mediated silencing. Transient tethering of dNxf2 to nascent transcripts leads to their nuclear retention. The NTF2 domain of dNxf2 competes dNxf1 (TAP) off nucleoporins, a process required for proper RNA export. Thus, dNxf2 functions in a Panoramix-dNxf2-dependent TAP/p15 silencing (Pandas) complex that counteracts the canonical RNA exporting machinery and restricts transposons to the nuclear peripheries. Our findings may have broader implications for understanding how RNA metabolism modulates heterochromatin formation.


Assuntos
Proteínas Argonauta/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Inativação Gênica , Heterocromatina/metabolismo , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas Argonauta/química , Proteínas Argonauta/metabolismo , Montagem e Desmontagem da Cromatina , Elementos de DNA Transponíveis , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Heterocromatina/ultraestrutura , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Oócitos/metabolismo , Oócitos/ultraestrutura , Ovário/citologia , Ovário/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
5.
Results Probl Cell Differ ; 68: 217-230, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31598858

RESUMO

Creophilus maxillosus (Staphylinidae, Coleoptera, Polyphaga) has a meroistic-telotrophic ovary composed of tropharium, which contains trophocytes (nurse cells) and vitellarium, which contains growing oocytes. The trophocytes are connected to the oocytes by cytoplasmic nutritive cords, which deliver nutrients to the oocytes. The formation/differentiation of the oocytes and trophocytes takes place in the pupal ovary within linear chains of sibling cells. Each chain is composed of a single oocyte connected to a linear chain of sister trophocytes. The nuclei of the oocytes contain an extrachromosomal DNA body (extra DNA body) consisting of amplified ribosomal DNA (rDNA). During oogenesis, the prospective oocyte, located at the base (posterior) of each chain, is the only cell within the chain that amplifies rDNA and retains permanent contact with the somatic pre-follicular cells. The oogonial divisions leading to the formation of the oocyte/trophocytes chain are asymmetric, and during consecutive divisions, the rDNA body always segregates basally (posteriorly) to the prospective oocyte abutted on the somatic cells. However, the segregation of rDNA is imperfect, and within each oocyte/trophocytes chain, there is a gradient of rDNA: the prospective oocyte has the highest amount of rDNA and the trophocyte that is most distant (most anterior) from the oocyte has no or the lowest amount of rDNA. In addition, the divisions within each chain are parasynchronous, with the pro-oocyte being the most mitotically advanced cell in the chain. These observations indicate the presence of a signaling gradient emanating from the somatic cells and/or oocyte; this gradient diminishes in strength with the increasing distance from its source, i.e., the oocyte/somatic cells. Because of this phenomenon, C. maxillosus is the perfect model in which to study the germ-somatic cell interactions and signaling. This chapter describes the methods for the collection and laboratory culture of C. maxillosus and the analysis of divisions and signaling in the C. maxillosus ovary.


Assuntos
Diferenciação Celular , Divisão Celular , Besouros/citologia , Modelos Animais , Oócitos/citologia , Transdução de Sinais , Animais , Feminino , Ovário/citologia
6.
Results Probl Cell Differ ; 68: 477-494, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31598868

RESUMO

The subphylum Chelicerata represents one of the oldest groups among arthropods and comprises more than a dozen orders. Representatives of particular orders differ significantly in their external morphology, reproductive biology, behavior, and structure of internal organs, e.g. of the respiratory system. However, in almost all chelicerates (excluding some mites) the female gonads show a similar architecture. In this chapter, the chelicerate-type ovary structure and the course of oogenesis are described. Structural and functional diversities of the chelicerate-type ovary in non-matrotrophic and matrotrophic arachnids are also presented.


Assuntos
Artrópodes/anatomia & histologia , Artrópodes/citologia , Oogênese , Ovário/anatomia & histologia , Ovário/citologia , Animais , Aracnídeos/anatomia & histologia , Aracnídeos/citologia , Feminino
7.
Results Probl Cell Differ ; 68: 495-513, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31598869

RESUMO

Even though tardigrades have been known since 1772, their phylogenetic position is still controversial. Tardigrades are regarded as either the sister group of arthropods, onychophorans, or onychophorans plus arthropods. Furthermore, the knowledge about their gametogenesis, especially oogenesis, is still poor and needs further analysis. The process of oogenesis has been studied solely for several eutardigradan species. Moreover, the spatial organization of the female germ-line clusters has been described for three species only. Meroistic ovaries characterize all analyzed species. In species of the Parachela, one cell per germ-cell cluster differentiates into the oocyte, while the remaining cells become the trophocytes. In Apochela several cells in the cluster differentiate into oocytes. Vitellogenesis is of a mixed type. The eggs are covered with the egg capsule that is composed of two shells: the thin vitelline envelope that adheres to the oolemma and the thick three-layered chorion. Chorion is formed as a first followed by vitelline envelope. Several features related to the oogenesis and structure of the ovary confirm the hypothesis that tardigrades are the sister group rather for arthropods than for onychophorans.


Assuntos
Oócitos/citologia , Oogênese , Ovário/anatomia & histologia , Tardígrados/anatomia & histologia , Tardígrados/fisiologia , Animais , Feminino , Ovário/citologia , Filogenia , Tardígrados/classificação
8.
Fish Shellfish Immunol ; 94: 566-579, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31550509

RESUMO

The changes in the ovarian stroma of the fish during their spawning season become it an excellent biological model for studies on cellular and vascular elements due to the intense tissue remodeling in fish occur naturally throughout this critical period. The present study aims to investigate the cellular and vascular components of the ovarian stroma of Redbelly tilapia during the spawning season by conventional, immunohistochemical stains as well as to detect the ultrastructural characteristics for each stromal component. The histological examinations revealed a series of blood vessels with special structures, include throttle artery, glomus, spirally oriented arterioles, modified arteries, and veins as well as arteriovenous anastomosis. Various types of cells were detected in the stroma include; telocytes, rodlet cells, mast cells, eosinophils, neutrophils, lymphocytes, fibroblasts, macrophages, melanocytes, adipocytes, dendritic cells, and endocrine (steroidogenic, interstitial) cells. Moreover, these stromal cells showed a broad range of staining affinity against c-kit, desmin, and s100-protein. Bundles of nerve fibers were detected between the follicles. This study exposed various cellular and vascular components with distinct functions in the ovary of Redbelly tilapia during the spawning season.


Assuntos
Ovário/fisiologia , Células Estromais/fisiologia , Tilápia/fisiologia , Animais , Feminino , Histocitoquímica/veterinária , Imuno-Histoquímica/veterinária , Microscopia Eletrônica de Transmissão/veterinária , Ovário/citologia , Ovário/ultraestrutura , Reprodução , Estações do Ano , Células Estromais/citologia , Células Estromais/ultraestrutura
9.
Nat Commun ; 10(1): 3858, 2019 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-31451685

RESUMO

The Polycomb group of proteins is required for the proper orchestration of gene expression due to its role in maintaining transcriptional silencing. It is composed of several chromatin modifying complexes, including Polycomb Repressive Complex 2 (PRC2), which deposits H3K27me2/3. Here, we report the identification of a cofactor of PRC2, EZHIP (EZH1/2 Inhibitory Protein), expressed predominantly in the gonads. EZHIP limits the enzymatic activity of PRC2 and lessens the interaction between the core complex and its accessory subunits, but does not interfere with PRC2 recruitment to chromatin. Deletion of Ezhip in mice leads to a global increase in H3K27me2/3 deposition both during spermatogenesis and at late stages of oocyte maturation. This does not affect the initial number of follicles but is associated with a reduction of follicles in aging. Our results suggest that mature oocytes Ezhip-/- might not be fully functional and indicate that fertility is strongly impaired in Ezhip-/- females. Altogether, our study uncovers EZHIP as a regulator of chromatin landscape in gametes.


Assuntos
Proteínas Oncogênicas/metabolismo , Óvulo/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Espermatozoides/metabolismo , Adulto , Animais , Linhagem Celular Tumoral , Cromatina/metabolismo , Feminino , Células HEK293 , Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/isolamento & purificação , Oogênese , Ovário/citologia , Ovário/patologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9 , Espermatogênese , Testículo/citologia , Testículo/patologia
10.
Chem Biol Interact ; 310: 108759, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31326407

RESUMO

Sustained exogenous stimuli induce oxidative stress in granulosa cells and cause cell apoptosis, thereby resulting in follicular atresia. Hyperoside is a natural flavonoid that possesses anti-oxidant activity. The present study aimed to evaluate the effect of hyperoside on hydrogen peroxide (H2O2)-induced oxidative stress and apoptosis in granulosa cells. Cell viability was measured using MTT assay. The malondialdehyde (MDA) level and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) were detected to reflect the oxidative stress. Flow cytometry was performed to measure the apoptotic rate. Western blot was carried out to determine the expression of Bcl-2, Bax, Sonic hedgehog (SHH), Gli1, and smoothened (SMO). The mRNA levels of SHH, Gli1, and SMO were analyzed using qRT-PCR. We found that hyperoside improved cell viability in H2O2-stimulated granulosa cells. The increased MDA level and decreased activities of SOD, GSH-Px, and CAT caused by H2O2 stimulation were reversed by hyperoside treatment. The apoptotic rate of H2O2-stimulated granulosa cells was reduced after treatment with hyperoside. Hyperoside treatment caused a decrease in Bax expression and an increase in Bcl-2 expression in H2O2-stimulated granulosa cells. The mRNA and protein levels of SHH, Gli1, and SMO in H2O2-stimulated granulosa cells were elevated by hyperoside treatment. Suppression of SHH pathway by cyclopamine attenuated the protective effects of hyperoside on H2O2-induced injury. In short, hyperoside protected granulosa cells from H2O2-induced cell apoptosis and oxidative stress via activation of the SHH signaling pathway.


Assuntos
Células da Granulosa/efeitos dos fármacos , Proteínas Hedgehog/metabolismo , Peróxido de Hidrogênio/efeitos adversos , Ovário/citologia , Quercetina/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Feminino , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras , Quercetina/farmacologia , Quercetina/uso terapêutico , Ratos , Transdução de Sinais
11.
Aquat Toxicol ; 214: 105240, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31319295

RESUMO

Fish has a strong resistance to microcystins (MCs), cyclic heptapeptide cyanotoxins, known as endocrine disrupting chemicals (EDCs) which are released during cyanobacterial blooms and many laboratory and field studies have found the hepatic recovery of fish from the MCs exposure. The aim of the present study was to investigate the recovery mechanisms of reproductive function of adult zebrafish (Danio rerio) from microcystin-LR (MC-LR) exposure. Therefore, adult female zebrafish were exposed to 0, 1 or 50 µg/L of MC-LR for 21days and transferred to MC free water for another 21 days to investigate the recovery. After MC-LR exposure, marked histological lesions in the gonads, decreased the percentage of mature oocytes, decreased number of spawned eggs, decreased fertilization and hatching rates were observed. MC-LR exposure increased the concentration of 17ß-estradiol (E2), testosterone (T) and vitellogenin (VTG) in female zebrafish. Some gene transcriptions of the hypothalamic-pituitary-gonad (HPG) axis significantly changed. The protein levels of 17ßhsd and cyp19a remarkably increased in the MC-LR exposure groups. However, our laboratory observation also indicates that zebrafish transferred from microcystin exposure to toxin-free water and reared for 21 days exhibited a nearly complete recovery of reproductive functions, including histological structure, increased the percentage of matured oocytes and spawned eggs, stable hormone levels, well-balanced transcriptional and translational levels. These results indicate that after MC-LR exposure, the reproductive impairments in zebrafish are also reversible likewise hepatic recovery seen by different studies in fish. Future studies should be conducted to explore a better understanding of the recovery mechanisms of fish from microcystins exposure.


Assuntos
Exposição Ambiental , Microcistinas/toxicidade , Reprodução/efeitos dos fármacos , Peixe-Zebra/fisiologia , Animais , Disruptores Endócrinos/toxicidade , Feminino , Hormônios/sangue , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Ovário/citologia , Ovário/efeitos dos fármacos , Ovário/fisiologia , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Transcrição Genética/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/sangue , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
12.
Cell Mol Life Sci ; 76(21): 4309-4317, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31300869

RESUMO

Adult stem cells have a unique capacity to renew themselves and generate differentiated cells that are needed in the body. These cells are recruited and maintained by the surrounding microenvironment, known as the stem cell niche, during organ development. Thus, the stem cell niche is required for proper tissue homeostasis, and its dysregulation is associated with tumorigenesis and tissue degeneration. The identification of niche components and the mechanisms that regulate niche establishment and maintenance, however, are just beginning to be uncovered. Germline stem cells (GSCs) of the Drosophila ovary provide an excellent model for studying the stem cell niche in vivo because of their well-characterized cell biology and the availability of genetic tools. In this review, we introduce the ovarian GSC niche, and the key signaling pathways for niche precursor segregation, niche specification, and niche extracellular environment establishment and niche maintenance that are involved in regulating niche size during development and adulthood.


Assuntos
Drosophila melanogaster , Células-Tronco de Oogônios/citologia , Nicho de Células-Tronco/genética , Animais , Diferenciação Celular/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/fisiologia , Feminino , Células Germinativas/citologia , Células Germinativas/fisiologia , Células-Tronco de Oogônios/fisiologia , Ovário/citologia , Transdução de Sinais/genética
13.
Gen Comp Endocrinol ; 282: 113216, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31278920

RESUMO

The Atlantic Bluefin Tuna (ABFT, Thunnus thynnus) is one of the most intensely exploited fisheries resources in the world. In spite of the years of studies on ABFT, basic aspects of its reproductive biology remain uncertain. To gain insight regarding the seasonal changes of the reproductive characteristics of the eastern stock of ABFT, blood and tissue samples were collected from mature specimens caught in the Mediterranean basin during the reproductive (May-June) and non-reproductive season (Oct-Nov). Histological analysis of the gonads of May-June samples indicated that there were females which were actively spawning (contained post-ovulatory follicles) and females that were not actively spawning that had previtellogenic and fully vitellogenic oocytes. In males, testis were at early or late stage of spermatogenesis during the reproductive season. In Oct-Nov, ovaries contained mostly previtellogenic oocytes as well as ß and α atretic follicles while the testis predominantly contained spermatogonia and few cysts with spermatocytes and spermatozoa. Gonadosomatic index (GSI) in females was highest among the actively spawning individuals while in males GSI was higher in early and late spermatogenic individuals compared to those that were spent. Plasma sex steroids levels varied with the reproductive season. In females, estradiol (E2), was higher in May-June while testosterone (T) and progesterone (P) did not vary. In males, E2 and T were higher in May-June while P levels were similar at the two sampling points. Circulating follicle stimulating hormone (FSH) was higher in Oct-Nov than in May-June both in males and females. Vitellogenin (VTG) was detected in plasma from both males and females during the reproductive season with levels in females significantly higher than in males. VTG was undetected in Oct-Nov samples. Since choriogenesis is an important event during follicle growth, the expression of three genes involved in vitelline envelope formation and hardening was measured and results showed significantly higher levels in ovaries in fish caught in May-June with respect to those sampled in Oct-Nov. In addition, a set of genes encoding for ion channels that are responsible for oocyte hydration and buoyancy, as well as sperm viability, were characterized at the two time points, and these were found to be more highly expressed in females during the reproductive season. Finally, the expression level of three mRNAs encoding for different lipid-binding proteins was analyzed with significantly higher levels detected in males, suggesting sex-specific expression. Our findings provide additional information on the reproductive biology of ABFT, particularly on biomarkers for the assessment of the state of maturation of the gonad, highlighting gender-specific signals and seasonal differences.


Assuntos
Reprodução/fisiologia , Estações do Ano , Atum/fisiologia , Animais , Feminino , Hormônio Foliculoestimulante/sangue , Gametogênese/genética , Regulação da Expressão Gênica , Hormônios Esteroides Gonadais/metabolismo , Masculino , Folículo Ovariano/citologia , Ovário/citologia , Ovário/metabolismo , Testículo/citologia , Testículo/metabolismo , Atum/sangue , Atum/genética , Vitelogeninas/sangue
14.
Int J Mol Sci ; 20(13)2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31288388

RESUMO

Two methods for the cryopreservation of human ovarian tissue were compared using a xenotransplantation model to establish a safe and effective cryopreservation method. Ovarian tissues were obtained from women who underwent benign ovarian surgery in the gynecology research unit of a university hospital. The tissues were transplanted into 112 ovariectomized female severe combined immunodeficient mice 4 weeks after slow freezing or vitrification cryopreservation. Tissues were retrieved 4 weeks later. Primordial follicular counts decreased after cryopreservation and xenotransplantation, and were significantly higher in the slow freezing group than in the vitrification group (p < 0.001). Immunohistochemistry and TUNEL assay showed that the Ki-67 and CD31 markers of follicular proliferation and angiogenesis were higher in the slow freezing group (p < 0.001 and p = 0.006, respectively) and DNA damage was greater in the vitrification group (p < 0.001). Western blotting showed that vitrification increased cellular apoptosis. Anti-Müllerian hormone expression was low in transplanted samples subjected to both cryopreservation techniques. Electron microscopy revealed primordial follicle deformation in the vitrification group. Slow freezing for ovarian tissue cryopreservation is superior to vitrification in terms of follicle survival and growth after xenotransplantation. These results will be useful for fertility preservation in female cancer patients.


Assuntos
Criopreservação , Congelamento , Ovário , Vitrificação , Adulto , Animais , Biomarcadores , Contagem de Células , Proliferação de Células , Sobrevivência Celular , Criopreservação/métodos , Dano ao DNA , Feminino , Preservação da Fertilidade , Imunofluorescência , Xenoenxertos , Humanos , Camundongos , Neovascularização Fisiológica , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Folículo Ovariano/ultraestrutura , Ovário/citologia , Ovário/metabolismo , Ovário/transplante , Ovário/ultraestrutura , Adulto Jovem
15.
Cells ; 8(7)2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31261822

RESUMO

Recent studies support the existence of oogonial stem cells (OSCs) in the ovarian cortex of different mammals, including women.These cells are characterized by small size, membrane expression of DEAD(Asp-Glu-Ala-Asp)-box polypeptide-4 (Ddx4), and stemness properties (such as self-renewal and clonal expansion) as well as the ability to differentiate in vitro into oocyte-like cells. However, the discovery of OSCs contrasts with the popular theory that there is a numerically defined oocyte pool for female fertility which undergoes exhaustion with menopause. Indeed, in the ovarian cortex of postmenopausal women OSCs have been detected that possess both viability and capability to differentiate into oocytes, which is similar to those observed in younger patients. The pathophysiological role of this cell population in aged women is still debated since OSCs, under appropriate stimuli, differentiate into somatic cells, and the occurrence of Ddx4+ cells in ovarian tumor samples also suggests their potential involvement in carcinogenesis. Although further investigation into these observations is needed to clarify OSC function in ovary physiology, clinical investigators and researchers studying female infertility are presently focusing on OSCs as a novel opportunity to restore ovarian reserve in both young women undergoing early ovarian failure and cancer survivors experiencing iatrogenic menopause.


Assuntos
Plasticidade Celular/fisiologia , RNA Helicases DEAD-box/metabolismo , Células-Tronco de Oogônios/fisiologia , Ovário/fisiologia , Pós-Menopausa/fisiologia , Animais , Feminino , Humanos , Ovário/citologia
16.
Theriogenology ; 138: 66-76, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31302433

RESUMO

Our goal was to develop an objective computer-assisted volumetric method of assessing vascular flow from colour Doppler ultrasound data of ovarian structures recorded by free-hand movement. We hypothesized that a vascularity index (ratio of the region of blood flood to the region of ovarian structure) obtained from the three-dimensional volumetric analysis would be more precise (less variable) than conventional two-dimensional analysis of single images in estimating the functional status of the preovulatory follicles and corpus luteum. Doppler ultrasound cineloops of water buffaloes (Bubalus bubalis; n = 22) ovaries were recorded daily from 12 h before GnRH treatment to four days after ovulation. Cineloops were processed using Fiji and Imaris software packages for segmenting the area (two-dimensional analysis) and the volume (three-dimensional analysis) occupied by the blood-flow and associated tissue to calculate the vascularity index. For volumetric measurement, all images in a cineloop were used (i.e., no a-priori selection of images) while for two-dimensional analysis, three images from the region with apparent maximum vascularity were selected. The volumetric method was verified with theoretical ellipsoidal volume of the follicle (r = 0.96 P < 0.01) or corpus luteum (r = 0.58 P = 0.02). The variability in the follicular vascularity index among animals was lower using the volumetric method than two-dimensional analysis (0.018 ±â€¯0.002 vs 0.030 ±â€¯0.005, P < 0.01), while the variability for CL vascularity was similar between methods (P = 0.23). An increase in the follicular vascularity index was detected at 12 h after GnRH treatment using both methods (two-dimensional: 0.030 ±â€¯0.008, P < 0.01; three-dimensional: 0.016 ±â€¯0.006, P < 0.02). Buffaloes that ovulated tended to have a greater increase in 3D vascularity index than non-responding buffaloes (P = 0.06); the two-dimensional method was not able to detect these changes. Using the three-dimensional method, a moderate positive correlation (r = 0.59; P = 0.02) was evident between the follicular vascularity index at 14-16 h after GnRH treatment and follicular diameter. In conclusion, an objective volumetric method for assessing relative ovarian blood flow changes was developed using Doppler ultrasound cineloops recorded by free-hand movement. The 3-dimensional method eliminates the need for a-priori selection of images and is more precise as a result of decreased technical variability.


Assuntos
Búfalos , Corpo Lúteo/irrigação sanguínea , Corpo Lúteo/diagnóstico por imagem , Folículo Ovariano/irrigação sanguínea , Folículo Ovariano/diagnóstico por imagem , Ultrassonografia Doppler em Cores , Animais , Corpo Lúteo/citologia , Sincronização do Estro/métodos , Feminino , Hormônio Liberador de Gonadotropina/farmacologia , Hemodinâmica , Imageamento Tridimensional/veterinária , Células Lúteas/citologia , Células Lúteas/ultraestrutura , Folículo Ovariano/citologia , Ovário/irrigação sanguínea , Ovário/citologia , Ovário/diagnóstico por imagem , Ovulação/fisiologia , Detecção da Ovulação/métodos , Detecção da Ovulação/veterinária , Indução da Ovulação/métodos , Indução da Ovulação/veterinária , Fluxo Sanguíneo Regional , Ultrassonografia Doppler em Cores/métodos , Ultrassonografia Doppler em Cores/veterinária
17.
Cells ; 8(7)2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31336813

RESUMO

A population of small stem cells with diameters of up to 5 µm resembling very small embryonic-like stem cells (VSELs) were sorted from human embryonic stem cell (hESC) cultures using magnetic-activated cell sorting (MACS) based on the expression of a stem-cell-related marker prominin-1 (CD133). These VSEL-like stem cells had nuclei that almost filled the whole cell volume and expressed stem-cell-related markers (CD133, SSEA-4) and markers of germinal lineage (DDX4/VASA, PRDM14). They were comparable to similar populations of small stem cells sorted from cell cultures of normal ovaries and were the predominant cells in ascites of recurrent ovarian cancer. The sorted populations of CD133+ VSEL-like stem cells were quiescent in vitro, except for ascites, and were highly activated after exposure to valproic acid and follicle-stimulating hormone (FSH), indicating a new tool to study these cells in vitro. These VSEL-like stem cells spontaneously formed clusters resembling tumour-like structures or grew into larger, oocyte-like cells and were differentiated in vitro into adipogenic, osteogenic and neural lineages after sorting. We propose the population of VSEL-like stem cells from hESC cultures as potential original embryonic stem cells, which are present in the human embryo, persist in adult human ovaries from the embryonic period of life and are involved in cancer manifestation.


Assuntos
Carcinoma Epitelial do Ovário/patologia , Células-Tronco Embrionárias Humanas , Oócitos , Neoplasias Ovarianas/patologia , Ovário , Antígeno AC133/metabolismo , Adulto , Idoso , Ascite/patologia , Diferenciação Celular , Movimento Celular , RNA Helicases DEAD-box/metabolismo , Feminino , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/patologia , Humanos , Pessoa de Meia-Idade , Oócitos/citologia , Oócitos/patologia , Ovário/citologia , Ovário/patologia , Células Tumorais Cultivadas
18.
J Ovarian Res ; 12(1): 58, 2019 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-31228949

RESUMO

BACKGROUND: In order to preserve fertility in young women with disseminated cancer, e.g. leukemia, an approach that has been suggested is to retransplant isolated small follicles within an ovarian matrix free from malignant cells and with no risk for contamination. The present study evaluates the first step to create a bioengineered ovarian construct that can act as growth-supporting tissue for isolated small follicles that are dependent on a stroma for normal follicular maturation. The present study used the intact mouse ovary to develop a mouse ovarian scaffold through various protocols of decellularization. MATERIAL AND METHODS: Potential Immunogenic DNA and intracellular components were removed from whole mouse ovaries by agitation in a 0.5% sodium dodecyl sulfate solution (Protocol 1; P1), or in a 2% sodium deoxycholate solution (P2) or by a combination of the two (P3). The remaining decelluralized ovarian extracellular matrix structure was then assessed based on the DNA- and protein content, and was further evaluated histologically by haematoxylin and eosin-, Verhoeff's van gieson- (for elastin), Masson's trichrome- (for collagens) and Alcian blue (for glycosaminoglycans) staining. We also evaluated the decellularization efficiency using the mild detergent Triton-X100 (1%). RESULTS: Sodium dodecyl sulfate efficiently removed DNA and intracellular components from the ovarian tissue but also significantly reduced the integrity of the remaining ovarian extracellular matrix. Sodium deoxycholate, a considerably milder detergent compared to sodium dodecyl sulfate, preserved the ovarian extracellular matrix better, evident by a more distinct staining for glycosaminoglycan, collagen and elastic fibres. Triton-X100 was found ineffective as a decellularization reagent for mouse ovaries in our settings. CONCLUSIONS: The sodium dodecyl sulfate generated ovarian scaffolds contained minute amounts of DNA that may be an advantage to evade a detrimental immune response following engraftment. The sodium deoxycholate generated ovarian scaffolds had higher donor DNA content, yet, retained the extracellular composition better and may therefore have improved recellularization and other downstream bioengineering applications. These two novel types of mouse ovarian scaffolds serve as promising scaffold-candidates for future ovarian bioengineering experiments.


Assuntos
Ovário/citologia , Engenharia Tecidual/métodos , Tecidos Suporte , Animais , Órgãos Bioartificiais , DNA/análise , DNA/isolamento & purificação , Ácido Desoxicólico/química , Matriz Extracelular/química , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Ovário/química , Dodecilsulfato de Sódio/química
19.
Reprod Biol Endocrinol ; 17(1): 46, 2019 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189477

RESUMO

BACKGROUND: Reproductive aging is a robust phenotype that occurs in all females and is characterized by a significant reduction in gamete quantity and quality, which can have negative consequences on both endocrine function and fertility. Age-associated differences in the oocyte, follicle, and ovary have been well-documented, but how the broader environment changes with age is less well understood. Fat is one of the largest organs in the body, and peri-gonadal adipose tissue surrounds the rodent ovary and comprises a local ovarian environment. The goal of this study was to characterize how peri-ovarian adipose tissue changes with advanced reproductive age. METHODS: We isolated peri-gonadal adipose tissue from two cohorts of CB6F1 mice: reproductively young (6-12 weeks) and reproductively old (14-17 months). A comparative histological analysis was performed to evaluate adipocyte architecture. We then extracted lipids from the tissue and performed multiple reaction monitoring (MRM)-profiling, a mass spectrometry-based method of metabolite profiling, to compare the lipid profiles of peri-gonadal adipose tissue in these age cohorts. RESULTS: We found that advanced reproductive age was associated with adipocyte hypertrophy and a corresponding decrease in the number of adipocytes per area. Of the 10 lipid classes examined, triacylglycerols (TAGs) had significantly different profiles between young and old cohorts, despite quantitative analysis revealing a decrease in the total amount of TAGs per weight of peri-gonadal adipose tissue with age. CONCLUSIONS: These findings pinpoint age-associated physiological changes in peri-gonadal adipose tissue with respect to adipocyte morphology and lipid profiles and lay the foundation for future studies to examine how these alterations may influence both adipocyte and ovarian function.


Assuntos
Tecido Adiposo/metabolismo , Envelhecimento/fisiologia , Lipídeos/análise , Ovário/metabolismo , Reprodução/fisiologia , Adipócitos/citologia , Adipócitos/metabolismo , Tecido Adiposo/citologia , Fatores Etários , Animais , Feminino , Camundongos , Oócitos/citologia , Oócitos/metabolismo , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Ovário/citologia
20.
Mater Sci Eng C Mater Biol Appl ; 102: 670-682, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31147040

RESUMO

Since there is dearth of practical ways to obtain mature follicles from cryopreserved or native ovarian tissues, especially in patients suffering from ovarian dysfunction, tissue engineering may help in restoring ovarian function and/or fertility. In the present study, the effects of sodium dodecyl sulfate (SDS) and sodium hydroxide (NaOH) on the decellularization of ovarian tissues were studied in order to ascertain their suitability in creating suitable bioscaffolds. Cells were removed from the ovarian tissues of mouse, sheep and human. The samples were distributed among three groups, viz., control (not treated), SDS and NaOH treated. Qualitative histological evaluations, quantitative assessments (nuclear contents, collagen and glycosaminoglycan), immunohistochemistry staining (for laminin, fibronectin and Collagen I), cell viability and scanning electron microscopic (SEM) assays were performed for all experimental groups. Finally, suspensions of mouse ovarian cells were injected into human NaOH treated scaffolds and subsequently auto-transplanted to ovariectomized mice. H&E and IHC staining (GDF-9) were performed on human recellularized NaOH treated scaffolds 1 month after auto-transplantation. Although histological studies and quantitative evaluations confirmed the successful decellularization and presence of key factors in ovarian scaffolds under both treatment methods, NaOH showed more interesting outcomes. Cell metabolic activity in sheep and human ovaries treated with NaOH was statistically (p < 0.05) higher than for SDS treated samples after 72 h. Moreover, spherical associations with cuboidal cells in human NaOH treated scaffolds were observed and this follicular reconstruction was also confirmed by GDF-9. NaOH was found to be more suitable than SDS for the decellularization of ovarian tissues and it supports follicular reconstruction better than SDS. This is a valuable finding in tissue engineering research and can help in the creation of appropriate ovarian bioscaffolds.


Assuntos
Ovário/citologia , Engenharia Tecidual/métodos , Adolescente , Animais , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Camundongos , Ovário/ultraestrutura , Ovinos , Tecidos Suporte/química , Adulto Jovem
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