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1.
J Vis Exp ; (169)2021 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33843932

RESUMO

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, chemo- and radiation-therapies, as well as ovarian surgeries. In unmarried women with severe ovarian dysfunction, fertility preservation is important for future pregnancies. Although oocyte cryopreservation is an established method for fertility preservation, these patients could only preserve a limited number of oocytes even after ovarian hyperstimulation, leading to repeated stimulations to ensure sufficient oocytes to guarantee future pregnancy. To solve this issue, we have recently developed a drug-free in vitro activation (IVA) procedure, which enable us to stimulate early stages of ovarian follicles to develop to the preantral follicle stage. These preantral follicles can respond to the unique protocol of gonadotropin stimulation, resulting in increased number of retrieved oocytes per ovarian stimulation for cryopreservation. The drug-free IVA comprised from the surgical approach and ovarian stimulation. We removed a part of cortex from one or both ovaries from patients under laparoscopic surgery. The ovarian cortical tissues were cut into small cubes to disrupt the Hippo signaling pathway and stimulate the development of early stage follicles. These cubes were grafted orthotropically into remaining ovaries as well as beneath the serosa of both Fallopian tubes. We have already published the surgical procedure of the drug-free IVA and the protocol of subsequent ovarian stimulation, but herein we present the details of laboratory methods required for drug-free IVA.


Assuntos
Criopreservação/métodos , Preservação da Fertilidade/métodos , Infertilidade Feminina/terapia , Oócitos/transplante , Ovário/transplante , Insuficiência Ovariana Primária/terapia , Feminino , Humanos , Oócitos/citologia , Oócitos/fisiologia , Ovário/citologia , Ovário/fisiologia , Indução da Ovulação , Gravidez
2.
Methods Mol Biol ; 2273: 75-84, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33604845

RESUMO

The technological revolution in reproductive biology that started with artificial insemination procedures and embryo transfer led to the development of assisted reproduction techniques such as in vitro fertilization or even cloning of domestic animals by nuclear transfer from somatic cells. Currently, procedures of isolated immature ovarian follicles in vitro culture are becoming the prominent technology aimed to preserve or restore fertility especially of young oncological patients or those at risk of premature ovarian failure.Here, we describe a protocol that can be applied for in vitro growth of porcine, preantral ovarian follicles in three-dimensional (3D) culture conditions. After enzymatic isolation from the ovarian cortex, preantral follicles are suspended in a drop of medium and enclosed with fluorinated ethylene propylene (FEP) powder particles (microbioreactors). Such microbioreactors maintain the 3D structure of the follicles during the whole process of in vitro growth what is crucial to ensure proper folliculogenesis progression and their ability to survive.


Assuntos
Técnicas de Cultura de Células/métodos , Fertilização In Vitro/métodos , Folículo Ovariano/crescimento & desenvolvimento , Animais , Meios de Cultura/química , Transferência Embrionária/métodos , Feminino , Humanos , Oócitos/citologia , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Ovário/citologia , Suínos
3.
J Vis Exp ; (167)2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33522510

RESUMO

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors from somatic cells and cumulus cell-oocyte communication. Little is known about the ovarian microenvironment and how the cytokines and steroids produced in the surrounding milieu affect follicle progression or arrest. In vitro culture of ovarian cortex enables follicles to develop in a normalized environment that remains supported by adjacent stroma. Our objective was to determine the effect of nutritional Stair-Step diet on the ovarian microenvironment (follicle development, steroid, and cytokine production) through in vitro culture of bovine ovarian cortex. To accomplish this, ovarian cortical pieces were removed from heifers undergoing two different nutritionally developed schemes prior to puberty: Control (traditional nutrition development) and Stair-Step (feeding and restriction during development) that were cut into approximately 0.5-1 mm3 pieces. These pieces were subsequently passed through a series of washes and positioned on a tissue culture insert that is set into a well containing Waymouth's culture medium. Ovarian cortex was cultured for 7 days with daily culture media changes. Histological sectioning was performed to determine follicle stage changes before and after the culture to determine effects of nutrition and impact of culture without additional treatment. Cortex culture medium was pooled over days to measure steroids, steroid metabolites, and cytokines. There were tendencies for increased steroid hormones in ovarian microenvironment that allowed for follicle progression in the Stair-Step versus Control ovarian cortex cultures. The ovarian cortex culture technique allows for a better understanding of the ovarian microenvironment, and how alterations in endocrine secretion may affect follicle progression and growth from both in vivo and in vitro treatments. This culture method may also prove beneficial for testing potential therapeutics that may improve follicle progression in women to promote fertility.


Assuntos
Ovário/fisiologia , Técnicas de Cultura de Tecidos/métodos , Animais , Bovinos , Quimiocinas/metabolismo , Meios de Cultura , Feminino , Imageamento Tridimensional , Metaboloma , Oócitos/citologia , Folículo Ovariano/citologia , Ovário/citologia , Coloração e Rotulagem , Esteroides/metabolismo
4.
Dev Biol ; 469: 111-124, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33141038

RESUMO

Although somatic cells play an integral role in animal gametogenesis, their organization and function are usually poorly characterized, especially in non-model systems. One such example is a peculiar cell found in leech ovaries - the apical cell (AC). A single AC can be found at the apical tip of each ovary cord, the functional unit of leech ovaries, where it is surrounded by other somatic and germline cells. The AC is easily distinguished due to its enormous size and its numerous long cytoplasmic projections that penetrate the space between neighboring cells. It is also characterized by a prominent accumulation of mitochondria, Golgi complexes and electron-dense vesicles. ACs are also enriched in cytoskeleton, mainly in form of intermediate filaments. Additionally, the AC is connected to neighboring cells via junctions that structurally resemble hemidesmosomes. In spite of numerous descriptive data about the AC, its functions remain poorly understood. Its suggested functions include a role in forming skeleton for the germline cells, and a role in defining a niche for germline stem cells. The latter is more speculative, since germline stem cells have not been identified in leech ovaries. Somatic cells with similar morphological properties to those of the AC have been found in gonads of nematodes - the distal tip cell - and in insects - Verson's cell, hub cells and cap cells. In the present article we summarize information about the AC structure and its putative functions. AC is compared with other well-described somatic cells with potentially similar roles in gametogenesis.


Assuntos
Sanguessugas/citologia , Ovário/citologia , Animais , Núcleo Celular/ultraestrutura , Citoplasma/ultraestrutura , Feminino , Oogênese , Ovário/fisiologia , Ovário/ultraestrutura , Nicho de Células-Tronco
5.
J Fish Dis ; 44(1): 119-122, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33068031

RESUMO

The channel catfish (Ictalurus punctatus, Rafinesque) ovary (CCO) cell line is the standard cell line used for channel catfish diagnostics. Next-gen sequencing studies of a virus cultured in the CCO cells revealed mitochondrial sequences matching those of brown bullhead (Ameiurus nebulosus, Lesueur). Therefore, we systematically performed partial cytochrome oxidase 1 gene sequencing of several sources of the CCO cell line and all matched the brown bullhead and not the channel catfish.


Assuntos
Linhagem Celular , Complexo IV da Cadeia de Transporte de Elétrons/genética , Proteínas de Peixes/genética , Ictaluridae/genética , Ovário/citologia , Animais , Feminino , Análise de Sequência de DNA
6.
Methods Mol Biol ; 2180: 317-330, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797418

RESUMO

Cryoprotectants are essential to prevent ice formation during tissue cryopreservation procedures. However, the control of their concentration and spatial distribution in the tissue is necessary to avoid toxicity and other damages associated with the cryopreservation procedures, especially for bulky samples such as tissues and organs. X-ray computed tomography measures the attenuation of an X-ray beam when it passes through a substance, depending on the material properties of the samples. The high electronic density of the sulfur atom of the dimethyl sulfoxide makes it an excellent cryoprotectant to be assessed by X-ray CT, and its concentration is proportional to the X-ray attenuation either at room or cryogenic temperatures. In addition, this imaging technique also allows to detect the formation of ice and eventual fractures within tissues during the cooling and warming processes. Therefore, X-ray CT technology is an excellent tool to assess and develop new cryopreservation procedures for tissues and organs.


Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Processamento de Imagem Assistida por Computador/métodos , Ovário/citologia , Tomografia Computadorizada por Raios X/métodos , Feminino , Humanos , Ovário/diagnóstico por imagem , Ovário/efeitos dos fármacos , Transição de Fase , Manejo de Espécimes
7.
Methods Mol Biol ; 2180: 469-483, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797428

RESUMO

Genetic modifications in combination with highly sophisticated assisted reproductive technologies such as in vitro oocyte maturation and development, in vitro fertilization, intracytoplasmic sperm injection, and in vitro embryo culture have opened many research avenues and treatment options for both animals and humans. The number of genetically modified (GM) rodent strains increased considerably during the last several decades, and their numbers are expected to increase due to efficient gene editing technologies including the CRISPR/Cas9. Rodent ovarian tissues (OT) cryopreservation and transplantation procedures have several applications in biomedical field: they provide a fertility restoration option for GM rodent strains in some circumstances. They also serve as models to investigate OT cryopreservation as potential alternatives for human infertility patients as well as other domestic and wildlife species for the development of improved cryopreservation and subsequent transplantation strategies. The modeling studies enable determining effective cryoprotective agents (CPA), CPA and water permeability kinetics, and cooling and warming rates during the development of OT cryopreservation procedures. Furthermore, rodent models are extremely useful for determining post-thaw OT graft sites as well as potential medical interventions in an effort to expedite angiogenesis and inhibit inflammatory/immune response, OT longevity, and follicular integrity. Here we describe methodologies for rodent OT cryopreservation and potential transplantation sites for frozen-thawed rat and mouse OT.


Assuntos
Bancos de Espécimes Biológicos , Pesquisa Biomédica , Criopreservação/métodos , Crioprotetores/farmacologia , Genoma , Ovário/citologia , Animais , Feminino , Camundongos , Ovário/efeitos dos fármacos , Ratos
8.
Methods Mol Biol ; 2180: 485-499, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797429

RESUMO

Cryopreserved ovarian cortex tissue can be used to improve or restore female fertility. It can be used for cancer patients to restore fertility after chemotherapy treatment or for social reasons for women who want to postpone their pregnancy wish. In order to preserve ovarian tissue viability in these cases, the tissue needs to be stored by cryopreservation. In this chapter we describe the entire process chain needed to prepare, transport, and cryopreserve human ovarian cortex tissues as well as to subsequently thaw and implant it.


Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Preservação da Fertilidade/métodos , Ovário/citologia , Feminino , Humanos , Ovário/efeitos dos fármacos , Gravidez
9.
PLoS Biol ; 18(12): e3001025, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33351795

RESUMO

Primordial follicle assembly in the mouse occurs during perinatal ages and largely determines the ovarian reserve that will be available to support the reproductive life span. The development of primordial follicles is controlled by a complex network of interactions between oocytes and ovarian somatic cells that remain poorly understood. In the present research, using single-cell RNA sequencing performed over a time series on murine ovaries, coupled with several bioinformatics analyses, the complete dynamic genetic programs of germ and granulosa cells from E16.5 to postnatal day (PD) 3 were reported. Along with confirming the previously reported expression of genes by germ cells and granulosa cells, our analyses identified 5 distinct cell clusters associated with germ cells and 6 with granulosa cells. Consequently, several new genes expressed at significant levels at each investigated stage were assigned. By building single-cell pseudotemporal trajectories, 3 states and 1 branch point of fate transition for the germ cells were revealed, as well as for the granulosa cells. Moreover, Gene Ontology (GO) term enrichment enabled identification of the biological process most represented in germ cells and granulosa cells or common to both cell types at each specific stage, and the interactions of germ cells and granulosa cells basing on known and novel pathway were presented. Finally, by using single-cell regulatory network inference and clustering (SCENIC) algorithm, we were able to establish a network of regulons that can be postulated as likely candidates for sustaining germ cell-specific transcription programs throughout the period of investigation. Above all, this study provides the whole transcriptome landscape of ovarian cells and unearths new insights during primordial follicle assembly in mice.


Assuntos
Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/metabolismo , Ovário/metabolismo , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Células Germinativas , Células da Granulosa/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/metabolismo , Folículo Ovariano/fisiologia , Ovário/citologia , Gravidez , Análise de Célula Única/métodos , Transcriptoma/genética
10.
Anim Sci J ; 91(1): e13465, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33222358

RESUMO

The present study was conducted to investigate the effect of cold storage time on apoptosis of cumulus cells (CCs) from porcine ovaries, and to compare the sensitivity of four apoptosis-detection methods. Porcine ovaries were stored in physiological saline solution at 4°C for 0, 7, 24 and 48 hr, and then cumulus cells or granulosa cells (GCs) in antral follicles were retrieved to detect cell apoptosis. Cumulus cells isolated from stored ovaries for 24 hr presented obvious apoptosis using terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP nick end-labeling (TUNEL) assay. A typical DNA ladder pattern of apoptosis was observed in GCs 24 hr post storage treatment. The mean Olive Tail Moment of CCs was significantly increased after 24 hr using comet assay; however, the mean tail migration and mean tail DNA increased gradually after 7 hr of storage. In addition, annexin V/PI staining assay showed an obvious increase in apoptotic CCs (Annexin V positive, PI negative) 7 hr after treatment, and the apoptotic rate reached to a peak at 24 hr followed by a decline after 48 hr of storage to the level at 7 hr. In conclusion, cold storage of porcine ovary in physiological saline solution induced a time-dependent increase in apoptosis of cumulus cells, and annexin V/PI staining combined with comet assay provided a sensitive and reliable method to detect early damages in cumulus cells induced by cold storage of ovary.


Assuntos
Apoptose , Temperatura Baixa/efeitos adversos , Células do Cúmulo/patologia , Preservação de Órgãos/efeitos adversos , Preservação de Órgãos/métodos , Folículo Ovariano/citologia , Ovário , Animais , Separação Celular , Células Cultivadas , Células do Cúmulo/fisiologia , DNA , Feminino , Marcação In Situ das Extremidades Cortadas/métodos , Ovário/citologia , Suínos , Fatores de Tempo
11.
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
12.
Fertil Steril ; 114(1): 33-43, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32622411

RESUMO

OBJECTIVE: To identify cell types in the male and female reproductive systems at risk for SARS-CoV-2 infection because of the expression of host genes and proteins used by the virus for cell entry. DESIGN: Descriptive analysis of transcriptomic and proteomic data. SETTING: Academic research department and clinical diagnostic laboratory. PATIENT(S): Not applicable (focus was on previously generated gene and protein expression data). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Identification of cell types coexpressing the key angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) genes and proteins as well as other candidates potentially involved in SARS-CoV-2 cell entry. RESULT(S): On the basis of single-cell RNA sequencing data, coexpression of ACE2 and TMPRSS2 was not detected in testicular cells, including sperm. A subpopulation of oocytes in nonhuman primate ovarian tissue was found to express ACE2 and TMPRSS2, but coexpression was not observed in ovarian somatic cells. RNA expression of TMPRSS2 in 18 samples of human cumulus cells was shown to be low or absent. There was general agreement between publicly available bulk RNA and protein datasets in terms of ACE2 and TMPRSS2 expression patterns in testis, ovary, endometrial, and placental cells. CONCLUSION(S): These analyses suggest that SARS-CoV-2 infection is unlikely to have long-term effects on male and female reproductive function. Although the results cannot be considered definitive, they imply that procedures in which oocytes are collected and fertilized in vitro are associated with very little risk of viral transmission from gametes to embryos and may indeed have the potential to minimize exposure of susceptible reproductive cell types to infection in comparison with natural conception.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Fertilidade/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , Pneumonia Viral/metabolismo , Reprodução/fisiologia , Internalização do Vírus , Adolescente , Adulto , Animais , Betacoronavirus/genética , Linhagem Celular , Infecções por Coronavirus/genética , Feminino , Humanos , Macaca fascicularis , Masculino , Ovário/citologia , Ovário/metabolismo , Ovário/virologia , Pandemias , Peptidil Dipeptidase A/biossíntese , Peptidil Dipeptidase A/genética , Pneumonia Viral/genética , Gravidez , Proteômica/métodos , Serina Endopeptidases/biossíntese , Serina Endopeptidases/genética , Testículo/citologia , Testículo/metabolismo , Testículo/virologia , Transcriptoma/fisiologia , Adulto Jovem
13.
Nat Commun ; 11(1): 3147, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561720

RESUMO

Transposons are known to participate in tissue aging, but their effects on aged stem cells remain unclear. Here, we report that in the Drosophila ovarian germline stem cell (GSC) niche, aging-related reductions in expression of Piwi (a transposon silencer) derepress retrotransposons and cause GSC loss. Suppression of Piwi expression in the young niche mimics the aged niche, causing retrotransposon depression and coincident activation of Toll-mediated signaling, which promotes Glycogen synthase kinase 3 activity to degrade ß-catenin. Disruption of ß-catenin-E-cadherin-mediated GSC anchorage then results in GSC loss. Knocking down gypsy (a highly active retrotransposon) or toll, or inhibiting reverse transcription in the piwi-deficient niche, suppresses GSK3 activity and ß-catenin degradation, restoring GSC-niche attachment. This retrotransposon-mediated impairment of aged stem cell maintenance may have relevance in many tissues, and could represent a viable therapeutic target for aging-related tissue degeneration.


Assuntos
Proteínas Argonauta/metabolismo , Senescência Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Células Germinativas/metabolismo , Animais , Proteínas Argonauta/genética , Caderinas/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Inativação Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Ovário/citologia , Ovário/metabolismo , Retroelementos/genética , Transdução de Sinais , Nicho de Células-Tronco/fisiologia , Células-Tronco/metabolismo , Receptores Toll-Like/metabolismo , beta Catenina/metabolismo
14.
PLoS One ; 15(6): e0235140, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32574203

RESUMO

BACKGROUND: Due to improved treatment, there is an increasing focus on the reproductive potential of survivors of childhood cancer. Cytotoxic chemotherapy accelerates the decline in the number of primordial follicles within the mammalian ovary at all ages, but effects on the developmental potential of remaining oocytes following prepubertal cancer treatment are unclear. OBJECTIVES: To investigate whether cyclophosphamide (CY) exposure in the prepubertal period in female mice influences ovarian function and the functional competence of oocytes in adulthood. METHODS: This study used Swiss albino mice as the experimental model. Female mice were treated with 200 mg/kg CY on either postnatal day 14 (CY14), 21 (CY21) or 28 (CY28) i.e at a prepubertal and 2 young postpubertal ages. At 14 weeks of life, ovarian function, functional competence of oocytes, and embryo quality were assessed. RESULTS: The number of primordial follicles decreased significantly in CY14 and CY21 groups compared to control (p < 0.01). The number of oocytes from superovulated was 8.5 ± 1.4, 24.1 ± 2.9 and 26.8 ± 2.1 in CY14, CY21 and CY28 respectively which was significantly lower than control (50.2 ± 3.2; p < 0.001). In vitro culture of CY14 embryos demonstrated only 55.4% blastocyst formation (p < 0.0001) and reduced ability of inner cell mass (ICM) to proliferate in vitro (p < 0.05) at 120 and 216 h post insemination respectively. On the other hand, ICM proliferation was unaltered in 2 young postpubertal ages. CONCLUSION: Our results indicate long-term effects on the developmental competence of oocytes exposed to CY in early but not adult life. These data provide a mechanism whereby long-term fertility can be impaired after chemotherapy exposure, despite the continuing presence of follicles within the ovary, and support the need for fertility preservation in prepubertal girls before alkylating agent exposure.


Assuntos
Blastocisto/efeitos dos fármacos , Ciclofosfamida/farmacologia , Desenvolvimento Embrionário/efeitos dos fármacos , Reserva Ovariana/efeitos dos fármacos , Maturidade Sexual/fisiologia , Animais , Hormônio Antimülleriano/sangue , Antineoplásicos Alquilantes/farmacologia , Blastocisto/citologia , Blastocisto/fisiologia , Peso Corporal/efeitos dos fármacos , Peso Corporal/fisiologia , Proliferação de Células/efeitos dos fármacos , Ciclofosfamida/administração & dosagem , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário/fisiologia , Feminino , Fertilidade/efeitos dos fármacos , Fertilidade/fisiologia , Camundongos , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Tamanho do Órgão/efeitos dos fármacos , Reserva Ovariana/fisiologia , Ovário/anatomia & histologia , Ovário/citologia , Ovário/efeitos dos fármacos , Fatores de Tempo
15.
Sci Data ; 7(1): 165, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32471976

RESUMO

The North Sea plaice, Pleuronectes platessa (Linnaeus, 1758), is a commonly studied commercial flatfish with poorly known ovarian histology. The following dataset is a collection of female plaice gonad images and their corresponding histological slides, collected during a complete season of the plaice's reproduction cycle. Stereology was used to determine the percentage of different structures found throughout the ovaries. Inter-agent calibrations were accomplished in order to harmonize the stereological readings, and were based on a comprehensive reading protocol and histological lexicon that were specifically written for the plaice's ovaries. The distribution and homogeneity of the different cell types found throughout the ovaries were also evaluated. This dataset can be used to automate the stereological reading process (through statistical learning methods for example) or to objectively determine the plaice's maturity phase, and link that information to either macroscopic measurements or through image analysis of the full ovaries.


Assuntos
Linguado , Imageamento Tridimensional , Ovário/anatomia & histologia , Animais , Feminino , Histologia , Ovário/citologia
16.
PLoS One ; 15(5): e0233885, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32470029

RESUMO

In the Danio species, interspecific hybridization has been conducted in several combinations. Among them, only the hybrid between a zebrafish (D. rerio) female and a spotted danio (D. nigrofasciatus) male was reported to be fertile. However, beyond these investigations, by means of reproductive biology, gametes of the hybrid have also not been investigated genetically. For this study, we induced a hybrid of the D. rerio female and D. nigrofasciatus male in order to study its developmental capacity, reproductive performance and gametic characteristics. Its hybrid nature was genetically verified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the rhodopsin gene. Almost all the hybrids (36/37) were males, and only one was female. Developing oocytes were observed in the hybrid female, but ovulated eggs have not been obtained thus far. Microscopic observation revealed various head sizes of sperm in the hybrid males. Flow cytometry showed that the hybrid males generated aneuploid sperm with various ploidy levels up to diploidy. In backcrosses between D. rerio females and hybrid males, fertilization rates were significantly lower than the control D. rerio, and most resultant progeny with abnormal appearance exhibited various kinds of aneuploidies ranging from haploidy to triploidy, but only one viable progeny, which survived more than four months, was triploid. This suggested the contribution of fertile diploid sperm of the hybrid male to successful fertilization and development.


Assuntos
Aneuploidia , Fertilização/fisiologia , Hibridização Genética , Espermatozoides/fisiologia , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Animais , Cruzamentos Genéticos , DNA/genética , Feminino , Masculino , Ovário/citologia , Ploidias , Rodopsina/genética , Razão de Masculinidade , Espermatozoides/citologia
17.
PLoS Biol ; 18(4): e3000538, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32339165

RESUMO

Oogenesis is a complex developmental process that involves spatiotemporally regulated coordination between the germline and supporting, somatic cell populations. This process has been modeled extensively using the Drosophila ovary. Although different ovarian cell types have been identified through traditional means, the large-scale expression profiles underlying each cell type remain unknown. Using single-cell RNA sequencing technology, we have built a transcriptomic data set for the adult Drosophila ovary and connected tissues. Using this data set, we identified the transcriptional trajectory of the entire follicle-cell population over the course of their development from stem cells to the oogenesis-to-ovulation transition. We further identify expression patterns during essential developmental events that take place in somatic and germline cell types such as differentiation, cell-cycle switching, migration, symmetry breaking, nurse-cell engulfment, egg-shell formation, and corpus luteum signaling. Extensive experimental validation of unique expression patterns in both ovarian and nearby, nonovarian cells also led to the identification of many new cell type-and stage-specific markers. The inclusion of several nearby tissue types in this data set also led to our identification of functional convergence in expression between distantly related cell types such as the immune-related genes that were similarly expressed in immune cells (hemocytes) and ovarian somatic cells (stretched cells) during their brief phagocytic role in nurse-cell engulfment. Taken together, these findings provide new insight into the temporal regulation of genes in a cell-type specific manner during oogenesis and begin to reveal the relatedness in expression between cell and tissues types.


Assuntos
Drosophila melanogaster/citologia , Oogênese/genética , Ovário/citologia , Animais , Animais Geneticamente Modificados , Diferenciação Celular/genética , Linhagem da Célula , Drosophila melanogaster/genética , Feminino , Perfilação da Expressão Gênica , Marcadores Genéticos , Hemócitos/citologia , Hemócitos/fisiologia , Mitose/genética , Folículo Ovariano/citologia , Ovário/fisiologia , Ovulação/genética , Análise de Sequência de RNA , Análise de Célula Única/métodos
18.
In Vivo ; 34(2): 533-541, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32111751

RESUMO

BACKGROUND/AIM: Transportation of ovarian cortex prior to freezing is used clinically; however, basic investigations of ovarian storage are limited and the question remains what temperature is optimal for transport over long distances and time periods. The aim of this study was to evaluate the rate of follicular loss over various time periods under two different temperatures and assess whether ovarian follicle viability is affected following cryopreservation and thawing subsequent to the transportation of ovarian tissue. MATERIALS AND METHODS: Pig ovaries were transported at 4°C (n=10) or at 38°C (n=10) prior to cryopreservation. At 0, 4, 12 and 24 h tissues were fixed for histological examination and a LIVE/DEAD Assay. At the same time-points ovarian tissues were cryopreserved and analysed after thawing. RESULTS: Histological evaluation and LIVE/DEAD Assay of freshly transported ovarian tissue showed significantly better follicle survival at 4°C during transportation duration. In cryopreserved ovarian tissues the LIVE/DEAD Assay showed a significant difference in the number of intact and dead follicles at 24 h in favor of 4°C (p<0.05). CONCLUSION: Ovarian tissue transportation should be kept at a minimum to prevent potential damage.


Assuntos
Sobrevivência Celular , Folículo Ovariano/fisiologia , Ovário/fisiologia , Temperatura , Animais , Biomarcadores , Temperatura Corporal , Criopreservação , Feminino , Preservação da Fertilidade , Humanos , Imuno-Histoquímica , Folículo Ovariano/citologia , Ovário/citologia , Suínos
19.
Nat Commun ; 11(1): 1147, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32123174

RESUMO

The human ovary orchestrates sex hormone production and undergoes monthly structural changes to release mature oocytes. The outer lining of the ovary (cortex) has a key role in defining fertility in women as it harbors the ovarian reserve. It has been postulated that putative oogonial stem cells exist in the ovarian cortex and that these can be captured by DDX4 antibody isolation. Here, we report single-cell transcriptomes and cell surface antigen profiles of over 24,000 cells from high quality ovarian cortex samples from 21 patients. Our data identify transcriptional profiles of six main cell types; oocytes, granulosa cells, immune cells, endothelial cells, perivascular cells, and stromal cells. Cells captured by DDX4 antibody are perivascular cells, not oogonial stem cells. Our data do not support the existence of germline stem cells in adult human ovaries, thereby reinforcing the dogma of a limited ovarian reserve.


Assuntos
Células-Tronco de Oogônios , Ovário/citologia , Análise de Célula Única/métodos , Adulto , Biomarcadores/metabolismo , Células Cultivadas , RNA Helicases DEAD-box/imunologia , RNA Helicases DEAD-box/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Procedimentos de Readequação Sexual , Transcriptoma
20.
J Morphol ; 281(4-5): 491-499, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32198946

RESUMO

Arapaima gigas is a giant air-breathing and bony tongue fish from the Amazon basin and a promising species for aquaculture. A. gigas farming industry is still not established because of the lack of information on its reproductive physiology. Reproduction in captivity cannot be manipulated or stimulated, and the identification of males and females in a broodstock is not easy. We aimed to reveal the morphological sex differentiation of pirarucu as studies involving gonad development are essential to understanding the reproductive physiology of any species. We performed histological analysis on the whole body and extracted the gonads of 150 juveniles. The first sign of ovary differentiation is the sex-specific rearrangement of the germ cells. In 9 cm total length females, the germ cells group into nests and are restricted to the lateral face of the gonad, in close contact with the abdomen wall. With further development, this region invaginates and that later develops into ovigerous lamellae. Meiosis starts soon after ovary differentiation. In males, the germ cells are scattered along the elongated differentiating testis at first, and later become more restricted to the central region where the spermatogonial cysts start to develop. Somatic and germ cells are jointly involved in the cellular reorganization during gonadal differentiation, specifically when the germ cells begin to establish new associations during the development of both the germinal epithelium and stroma. RESEARCH HIGHLIGHTS: In Arapaima gigas, the ovary differentiation occurs in 9 cm TL females and it is marked by the rearrangement of germ and somatic cells; and the germ cells entering meiosis with no formation of ovarian cavity; testis differentiation occurs later and meiosis does not start in males smaller than 80 cm TL.


Assuntos
Peixes/anatomia & histologia , Gônadas/anatomia & histologia , Diferenciação Sexual , Animais , Diferenciação Celular , Feminino , Peixes/crescimento & desenvolvimento , Peixes/fisiologia , Masculino , Ovário/anatomia & histologia , Ovário/citologia , Testículo/anatomia & histologia , Testículo/crescimento & desenvolvimento , Vitelogênese
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