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1.
J Morphol ; 281(1): 47-54, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31710388

RESUMO

Representatives of the highly specialized earwig family Hemimeridae are epizoic and viviparous. Their embryos develop inside terminal ovarian follicles (termed also embryonic follicles) and rely solely on nutrients transferred from mother tissues. In this report, we present results of ultrastructural and histochemical studies of the initial stage of Hemimerus talpoides development. Our results show that the follicular cells surrounding fully grown oocyte of Hemimerus do not degenerate after initiation of embryogenesis, but transform and gradually form the wall of the incubation chamber in which the embryo develops. We also show that amniotic cells of the early embryo remain in direct contact with transformed follicular cells. In the region of contact, short outgrowths of the amniotic cells associate with irregular surface specializations of the transformed follicular cells. We suggest that extended "postfertilization" activity of hemimerid follicular cells represents an adaptation to viviparity and matrotrophy in this insect lineage.


Assuntos
Desenvolvimento Embrionário , Epitélio/embriologia , Insetos/embriologia , Morfogênese , Folículo Ovariano/crescimento & desenvolvimento , Viviparidade não Mamífera , Animais , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/ultraestrutura , Epitélio/ultraestrutura , Feminino , Insetos/ultraestrutura , Folículo Ovariano/ultraestrutura
2.
Mol Hum Reprod ; 25(11): 706-716, 2019 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-31588500

RESUMO

The reproductive lifespan of a woman is determined by the gradual recruitment of quiescent follicles into the growing pool. In humans, ovarian tissue removal from its in vivo environment induces spontaneous activation of resting follicles. Similarly, pharmacological activation of the PI3K/Akt pathway leads to accelerated follicle recruitment, but has been associated with follicular damage. Recent findings demonstrate that everolimus (EVE), an mTORC1 inhibitor, limits primordial follicle activation. However, its potential benefit regarding growing follicle integrity remains unexplored. Ovarian cortical fragments were exposed to ± EVE for 24 h and cultured for an additional 5 days. After 0, 1 and 6 days of culture, fragments were either processed for ultrastructural analysis or subjected to follicular isolation for gene expression and immunofluorescence assessments. Data from transmission electron microscopy showed that growing follicles displayed similar ultrastructural features irrespective of the conditions and maintained close contacts between germinal and stromal compartments. Establishment of intra-follicular communication was confirmed by detection of a gap junction component, Cx43, in both groups throughout culture, whereas transzonal projections, which physically link granulosa cells to oocyte, formed later in EVE-treated follicles. Importantly, levels of GJA1 mRNA, encoding for the Cx43 protein, significantly increased from Day 0 to Day 1 in the EVE group, but not in the control group. Given that EVE-treated follicles were smaller than controls, these findings suggest that EVE might facilitate the establishment of appropriate intercellular communications without impairing follicle ultrastructure. Therefore, mTORC1 inhibitors might represent an attractive tool to delay the culture-induced primordial follicle activation while maintaining follicles in a functionally integrated state.


Assuntos
Comunicação Celular/fisiologia , Conexina 43/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/ultraestrutura , Adulto , Conexina 43/genética , Criopreservação , Everolimo/farmacologia , Feminino , Células da Granulosa/metabolismo , Humanos , Oócitos/metabolismo , Técnicas de Cultura de Órgãos , RNA Mensageiro/genética
3.
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
4.
Sci Rep ; 9(1): 1262, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718581

RESUMO

Each mammalian oocyte is nurtured by its own multi-cellular structure, the ovarian follicle. We used new methods for serial section electron microscopy to examine entire cumulus and mural granulosa cells and their projections in mouse antral ovarian follicles. Transzonal projections (TZPs) are thin cytoplasmic projections that connect cumulus cells to the oocyte and are crucial for normal oocyte development. We studied these projections in detail and found that most TZPs do not reach the oocyte, and that they often branch and make gap junctions with each other. Furthermore, the TZPs that connect to the oocyte are usually contacted on their shaft by oocyte microvilli. Mural granulosa cells were found to possess randomly oriented cytoplasmic projections that are strikingly similar to the free-ended TZPs. We propose that granulosa cells use cytoplasmic projections to search for the oocyte, and cumulus cell differentiation results from a contact-mediated paracrine interaction with the oocyte.


Assuntos
Extensões da Superfície Celular/ultraestrutura , Citoplasma/ultraestrutura , Camundongos , Folículo Ovariano/ultraestrutura , Animais , Comunicação Celular , Células do Cúmulo/citologia , Células do Cúmulo/ultraestrutura , Feminino , Junções Comunicantes/ultraestrutura , Células da Granulosa/citologia , Células da Granulosa/ultraestrutura , Camundongos/anatomia & histologia , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Microvilosidades/ultraestrutura , Oócitos/citologia , Folículo Ovariano/citologia , Pseudópodes/ultraestrutura
5.
Sci Rep ; 9(1): 1150, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718584

RESUMO

Recently, the interest of the scientific community is focused on the application of tissue engineering approach for the fertility restoration. In this paper innovative patterned electrospun fibrous scaffolds were fabricated and used as 3D system for porcine follicles culture. The obtained scaffolds demonstrated to be a suitable support which did not alter or interfere with the typical spherical follicles morphology. The fibrillar structure of the scaffolds mimics the morphology of the healthy native tissue. The use of porcine follicles implied many advantages respect to the use of mouse model. Relevant results showed that more than the scaffold pattern and struts dimension, the selection of proper biomaterials improve the follicles adhesion and development.


Assuntos
Materiais Biocompatíveis/química , Nanofibras/química , Folículo Ovariano/crescimento & desenvolvimento , Engenharia Tecidual/métodos , Tecidos Suporte/química , Animais , Estudos de Viabilidade , Feminino , Folículo Ovariano/ultraestrutura , Porosidade , Suínos
6.
J Morphol ; 280(3): 381-394, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30671996

RESUMO

The structure of the granulosa in reptilian sauropsids varies between groups. We investigated the follicle development in the desert lizard Scincus mitranus. In the germinal bed, oogonia, and primary oocytes were identified and found to be interspersed between the epithelial cells. Previtellogenesis was divided into three stages: early, transitional, and late previtellogenic stages. During the early previtellogenic stage (diplotene), the oocyte is invested by small epithelia cells that formed a complete single layer, which may be considered as a young follicle. The transitional previtellogenic stage was marked by proliferation and differentiation of the granulosa layer from a homogenous layer consisting of only small cells to a heterogeneous layer containing three cell types: small, intermediate, and large cells. The late previtellogenic stage was marked by high-synthetic activity of large cells and the initiation of cytoplasmic bridges between large granulosa cells and the oocyte. Small cells were the only type of granulosa cells that underwent division. Thus, these cells may be stem cells for the granulosa cell population and may develop into intermediate and subsequently large cells. The intermediate cells may be precursors of large cells, as suggested by their ultrastructure. The ultrastructure of the large granulosa was indicative of their high synthetic activity. Histochemical analysis indicated the presence of cholesterol and phospholipids in the cytoplasm of large cells, the zona pellucida, among the microvilli, in the bridges region, and in the cortical region of the oocyte cytoplasm. These materials may be transferred from large cells into the oocyte through cytoplasmic bridges and provide nutritive function to large cells rather than functioning in steroidogenesis or vitellogenesis.


Assuntos
Lagartos/anatomia & histologia , Lagartos/fisiologia , Oogênese/fisiologia , Folículo Ovariano/ultraestrutura , Animais , Feminino
7.
Res Vet Sci ; 120: 33-40, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30195149

RESUMO

This study evaluated the effects of frutalin (0.6, 6.0 or 60.0 µg/mL) and doxorubicin (0.3 µg/mL) on survival, growth and ultrastructure of in-vitro cultured goat secondary follicles. The effects of these substances on the levels of mRNA for Casp3, Casp6, Bax, and Bcl2 were also investigated. Results showed that, after 6 days of culture, frutalin or doxorubicin reduced the percentage of normal follicles (P < 0.05), but doxorubicin had higher toxicity than frutalin. Except for follicles cultured with 0.6 µg/mL frutalin, follicular growth rate was reduced after culture with doxorubicin or frutalin (P < 0.05). The presence doxorubicin or 60.0 µg/mL frutalin increased the levels of mRNA for Casp3, Casp6, Bax, and Bcl2 (P < 0.05). Higher mRNA levels for Casp3, Casp6 and Bax were found in follicles cultured with doxorubicin, but higher levels of Bcl2 mRNA were found in follicles cultured with frutalin (P < 0.05). In conclusion, frutalin has lower toxic effects than doxorubicin on secondary follicles cultured in vitro.


Assuntos
Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Galectinas/farmacologia , Cabras , Folículo Ovariano/efeitos dos fármacos , Animais , Antineoplásicos/administração & dosagem , Relação Dose-Resposta a Droga , Feminino , Galectinas/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Folículo Ovariano/ultraestrutura , RNA Mensageiro/genética , Técnicas de Cultura de Tecidos
8.
J Ovarian Res ; 11(1): 55, 2018 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-29958542

RESUMO

BACKGROUND: PCOS is a reproductive hormonal abnormality and a metabolic disorder. It is frequently associated with insulin resistance, hyperandrogenism, chronic inflammation, and oxidative stress. We aim to investigate the potential therapeutic effects of combined therapy of resveratrol and metformin on polycystic ovaries via SIRT1 and AMPK activation. METHODS: Wistar albino rats were divided into control and experimental (PCOS) groups. DHEA-induced PCOS rats were given resveratrol (20 mg/kg/day), metformin (300 mg/kg/day) and combined therapy. At the end of the experiment, the body and ovarian weight of rats were measured and blood samples were analyzed for FSH, LH, testosterone, AMH, TNF-α and MDA levels. Histopathological evaluation of ovaries were carried out by light and electron microscopy. SIRT1 and AMPK immunreactivity and TUNEL assay were scored. Data were statistically analyzed by SPSS programme. RESULTS: Metformin and combined treatment groups reduced the body and ovary weights compared to the PCOS group. Serum testosterone levels were significantly higher in the PCOS group than in the control group and this was reduced when PCOS was treated with all but especially resveratrol. All the treatment groups decreased LH, LH/FSH, TNF-α and tissue AMH levels which were induced in the PCOS group, whereas metformin was unable to improve the increased MDA and plasma AMH levels. Treatment with resveratrol and/or metformin ameliorated the elevated number of secondary and atretic follicles and the decreased number of Graafian follicles in the PCOS group, which indicates the effect of the treatments on the maintenance of folliculogenesis. Light and electron microscopic findings supported the analysis of follicular count. Increased number of TUNEL (+) granulosa cells in the PCOS group were reduced significantly in the treatment groups. Resveratrol and metformin increased SIRT1 and AMPK immunreactivity, respectively, compared to the PCOS group. CONCLUSIONS: The results suggest that combined therapy of metformin and resveratrol may improve the weight gain, hormone profile and ovarian follicular cell architecture by inducing antioxidant and antiinflammatory systems via SIRT1 and AMPK activation in PCOS.


Assuntos
Metformina/administração & dosagem , Metformina/farmacologia , Reserva Ovariana/efeitos dos fármacos , Ovário , Síndrome do Ovário Policístico/tratamento farmacológico , Estilbenos/administração & dosagem , Estilbenos/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Apoptose/efeitos dos fármacos , Contagem de Células , Desidroepiandrosterona/efeitos adversos , Modelos Animais de Doenças , Quimioterapia Combinada , Feminino , Hormônios/sangue , Hormônios/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/ultraestrutura , Ovário/efeitos dos fármacos , Ovário/patologia , Síndrome do Ovário Policístico/induzido quimicamente , Síndrome do Ovário Policístico/metabolismo , Síndrome do Ovário Policístico/patologia , Ratos , Ratos Wistar , Resveratrol , Sirtuína 1/metabolismo , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/metabolismo , Vagina/patologia
9.
Toxicol Mech Methods ; 28(9): 653-659, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29968488

RESUMO

BACKGROUND: Ovarian failure is diagnosed by ovarian destruction and reducing sex hormonal levels. Platelet-rich plasma (PRP) contains several growth factors that induce tissue repair and may induce folliculogenesis. OBJECTIVE: This study evaluates the effect of PRP on ovarian structures and function in cyclophosphamide (Cy)-induced ovarian failure in female rats by a stereological method. METHODS: Thirty-two adult female rats were divided into four groups (Control, Cy, Cy + PRP, and PRP). Female infertility was induced by Cy (75 mg/kg, single dose, intraperitoneally). Animals were treated by PRP which was obtained from age-matched male rats (200 µL, single dose, intraperitoneally). Blood samples were collected for measurement of hormones. The animals were dissected and the right ovaries were removed, fixed, sectioned, and stained by H&E. Stereological methods were used to estimate cortex and medulla volume, and the number and diameter of follicles, follicular cell, and oocyte using light microscopy. RESULTS: Cyclophosphamide had the maximum effect in decreasing on cortex volume, the pre-antral follicles number, a diameter of follicular cells and oocyte diameter in the antral follicle and the reduction of estradiol and progesterone levels compared with the control group. PRP had a dominant positive effect on the ovarian cortex volume, pre-antral follicles number and antral follicle diameter relative to the control group. PRP also decreased oocyte diameter in pre-antral follicle in infertile animals (p < 0.001). CONCLUSION: It seems that PRP has a protective effect on ovarian failure in the infertile female rat model.


Assuntos
Ciclofosfamida/toxicidade , Oócitos/efeitos dos fármacos , Doenças Ovarianas/induzido quimicamente , Folículo Ovariano/efeitos dos fármacos , Plasma Rico em Plaquetas , Animais , Estradiol/sangue , Feminino , Oócitos/ultraestrutura , Doenças Ovarianas/sangue , Doenças Ovarianas/patologia , Folículo Ovariano/ultraestrutura , Progesterona/sangue , Ratos Sprague-Dawley , Reprodução/efeitos dos fármacos
10.
Cryobiology ; 83: 9-14, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29981301

RESUMO

Ovarian tissue cryopreservation is a promising technique for fertility maintenance. The aim of this study was to compare the morphology of domestic cat ovarian follicles after tissue cryopreservation with ethylene glycol (EG) and dimethyl sulfoxide (Me2SO). Ovaries from healthy adult cats undergoing elective ovariohysterectomy were used. Eight fragments were obtained from each pair of ovaries: two were used as fresh controls; three were submitted to fresh perfusion toxicity test and perfused with M199, 10% fetal calf serum and 0.4% sucrose containing Me2SO 1.5 M, EG 1.5 M or Me2SO 0.75 M + EG 0.75 M; and the remaining three fragments were perfused as described and submitted to slow freezing. After 45 days of cryopreservation, the samples were thawed, fixed and processed for light and transmission electron microscopy (TEM). The percentages of morphologically normal follicles identified by light microscopy were higher in the control group (94.45%) in comparison to the frozen groups (80.56% with EG, 78.7% with Me2SO and 75.87% with EG + Me2SO). The fresh perfused tissue showed no statistical difference compared to control or frozen samples. The TEM analysis showed less damage in the ultrastructure of follicles from the Me2SO group in comparison with the EG and Me2SO + EG groups. According to the morphological analysis, 1.5 M Me2SO is the best cryoprotectant for cryopreservation of domestic cat ovarian tissue regarding the morphology of preantral follicles after thawing. Further studies regarding the viability of these follicles should be performed.


Assuntos
Criopreservação/veterinária , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Etilenoglicol/farmacologia , Folículo Ovariano/ultraestrutura , Animais , Gatos , Feminino , Congelamento , Microscopia Eletrônica de Transmissão
11.
J Fish Biol ; 93(2): 424-427, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29951997

RESUMO

The presence of follicular cellular processes (FCP) that cross the zona pellucida, has been recorded in the ovarian follicles of Callorhinchus callorhynchus. This constitutes the first report describing the presence of these structures in a species of the Holocephali. Considering that FCPs have only previously been reported in the Selachii, these findings suggest that FCPs could have been lost by the Batoidea after their divergence, around 280 M B.P.


Assuntos
Peixes/anatomia & histologia , Folículo Ovariano/ultraestrutura , Zona Pelúcida/ultraestrutura , Animais , Feminino , Ovário
12.
Protoplasma ; 255(6): 1703-1712, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29756169

RESUMO

Vitellogenin receptor (VgR) is a low-density lipoprotein receptor responsible for the mediated endocytosis of vitellogenin (Vg) during egg formation in insects. The maturing oocyte is enveloped by a follicular epithelium, which has large intercellular spaces during Vg accumulation (patency). However, Vg has been reported in the cytoplasm of follicular cells, indicating that there may be a transcellular route for its transport. This study verified the presence of VgR in the follicular cells of the ovaries of the honeybee Apis mellifera and the wasp Polistes simillimus in order to evaluate if Vg is transported via transcytosis in these insects. Antibodies specific for vitellogenin receptor (anti-VgR), vitellogenin (anti-Vg), and clathrin (anti-Clt) were used for immunolocalization. The results showed the presence of VgR on the apical and basal plasma membranes of follicular cells of the vitellogenic follicles in both species, indicating that VgR may have been transported from the basal to the apical cell domain, followed by its release into the perivitelline space, evidenced by the presence of apical plasma membrane projections containing VgR. Co-localization proved that Vg bind to VgR and that the transport of this protein is mediated by clathrin. These data suggest that, in these social insects, Vg is transported via clathrin-mediated VgR transcytosis in follicular cells.


Assuntos
Abelhas/citologia , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Transcitose , Vitelogeninas/metabolismo , Vespas/citologia , Animais , Proteínas do Ovo/metabolismo , Feminino , Proteínas de Membrana/metabolismo , Folículo Ovariano/ultraestrutura , Receptores de Superfície Celular/metabolismo
13.
Reprod Biomed Online ; 37(2): 153-162, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29802069

RESUMO

RESEARCH QUESTION: Does synchrotron X-ray fluorescence (XRF) provide novel chemical information for the evaluation of human ovarian tissue cryopreservation protocols? DESIGN: Tissues from five patients undergoing laparoscopic surgery for benign gynaecological conditions were fixed for microscopic analysis either immediately or after cryopreservation. After fixation, fresh and slowly frozen samples were selected by light microscopy and transmission electron microscopy, and subsequently analysed with synchrotron XRF microscopy at different incident energies. RESULTS: The distributions of elements detected at 7.3 keV (S, P, K, Cl, Fe, and Os) and 1.5 keV (Na and Mg) were related to the changes revealed by light microscopy and transmission electron microscopy analyses. The light elements showed highly informative findings. The S distribution was found to be an indicator of extracellular component changes in the stromal tissues of the freeze-stored samples, further revealed by the transmission electron microscopy analyses. Low-quality follicles, frequent in the freeze-thawed tissues, showed a high Na level in the ooplasm. On the contrary, good-quality follicles were detected by a homogeneous Cl distribution. The occurrence of vacuolated follicles increased after cryopreservation, and the XRF analyses showed that the vacuolar structures contained mainly Cl and Na. CONCLUSIONS: The study demonstrates that elemental imaging techniques, particularly revealing the distribution of light elements, could be useful in establishing new cryopreservation protocols.


Assuntos
Criopreservação/métodos , Preservação de Órgãos/métodos , Ovário/ultraestrutura , Feminino , Humanos , Microscopia Eletrônica de Transmissão , Folículo Ovariano/ultraestrutura
14.
Biol Reprod ; 99(3): 590-599, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29659700

RESUMO

The extracellular matrix (ECM) is a group of molecules that offer structural and biochemical support to cells and interact with them to regulate their function. Also, growth factors (GFs) stored in the ECM can be locally released during ECM remodeling. Here, we hypothesize that the balance between ECM components and remodelers is regulated according to the ovarian steroid milieu to which the oviduct is exposed during the periovulatory period. Follicular growth was manipulated to generate cows that ovulated small follicles (SF-small corpus luteum [SCL]; n = 20) or large follicles (LF-large corpus luteum [LCL]; n = 21) and possess corresponding Estradiol (E2) and Progesterone (P4) plasmatic concentrations. Ampulla and isthmus samples were collected on day 4 (day 0 = ovulation induction) and immediately frozen or fixed. The transcriptional profile (n = 3/group) was evaluated by RNA sequencing. MMP Antibody Array was used to quantify ECM remodelers' protein abundance and immunohistochemistry to quantify type I collagen. Transcriptome analysis revealed the over-representation of ECM organization and remodeling pathways in the LF-LCL group. Transcription of ECM components (collagens), remodelers (ADAMs and MMPs), and related GFs were upregulated in LF-LCL. Protein intensities for MMP3, MMP8, MMP9, MMP13, and TIMP4 were greater for the LF-LCL group. Type I collagen content in the mucosa was greater in SF-SCL group. In conclusion, that the earlier and more intense exposure to E2 and P4 during the periovulatory period in LF-LCL animals stimulates ECM remodeling. We speculate that differential ECM regulation may contribute to oviductal receptivity to the embryo.


Assuntos
Matriz Extracelular/fisiologia , Hormônios Esteroides Gonadais/fisiologia , Oviductos/fisiologia , Proteínas ADAM/metabolismo , Animais , Bovinos , Colágeno Tipo I/biossíntese , Colágeno Tipo I/genética , Biologia Computacional , Estradiol/sangue , Matriz Extracelular/ultraestrutura , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Metaloproteinases da Matriz/metabolismo , Folículo Ovariano/fisiologia , Folículo Ovariano/ultraestrutura , Oviductos/ultraestrutura , Ovulação/fisiologia , Gravidez , Progesterona/sangue
15.
Cryobiology ; 81: 34-42, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29481782

RESUMO

The aim of this study was to define the population, morphological and ultrastructural characteristics of bitch preantral follicles (PAFs) and to compare the effects on the morphology of PAF of two cryopreservation techniques - slow freezing (SF) and vitrification (V) - of bitches' ovarian tissue. The average population (number per ovary) of PAFs was 48,541 ±â€¯18,366, where 94.25% were primordial (45,145 ±â€¯16,076). The average diameter of the primordial follicles was 27.5 ±â€¯4.2 µm. The overall percentage of morphologically normal PAFs was 93.66 ±â€¯6.81% for the control group, 86.16 ±â€¯11.05% after SF and 68.14 ±â€¯12.75% after V. The percentage of normal primordial follicles was 96.69 ±â€¯4.72% in control, 89.51 ±â€¯10.39% in SF and 75.32 ±â€¯9.23% in V. There was no significant difference in the overall percentage of normal PAFs among SF and the control. However, slow frozen follicles presented ultrastructural damage, while vitrified primordial and primary follicles were well preserved. In conclusion, although slow freezing seemed to be a good preserving method, vitrification was more effective than slow freezing in preserving the ultrastructure of primordial and primary follicles of bitches.


Assuntos
Criopreservação/veterinária , Cães , Folículo Ovariano/ultraestrutura , Vitrificação , Animais , Crioprotetores/farmacologia , Feminino , Congelamento , Folículo Ovariano/efeitos dos fármacos
16.
Cell Tissue Bank ; 19(1): 123-132, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28365880

RESUMO

Most protocols to cryopreserve ovarian tissue utilize the permeable cryoprotective agents (CPAs) in 1.5 M concentration. However the issues related to the ability to use higher concentrations of CPAs have remained open. The research aim was to assess the efficiency of media containing osmotically active sugars (sucrose, mannitol) at stepwise adding/removing of 3 M dimethylsulphoxide (DMSO) and propanediol (PROH) on ovarian tissue integrity. After the CPAs adding/removing the ovarian tissue injury was histologically examined, as well as the oocyte volume in tissue structure was assessed. It has been found, that after adding/removing of PROH and DMSO solutions the maximum amount of normal follicles made 67-93% when using Dulbecco's Modified Eagle Medium (DMEM) with 200 mM sucrose. Assessment of tissue damage after adding/removing of CPAs has demonstrated that the percentage of normal follicles was 83-87% using DMSO in presence of both sucrose and mannitol as the dilution media components. While removing PROH the level of follicles preservation was 2.5× higher when using mannitol compared with sucrose. Our results indicate that the ovarian tissue injury was minimal during adding 3 M CPAs using DMEM, containing sucrose and following application of mannitol at removing both DMSO and PROH.


Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Manitol/farmacologia , Folículo Ovariano/citologia , Folículo Ovariano/efeitos dos fármacos , Animais , Dimetil Sulfóxido/metabolismo , Dimetil Sulfóxido/farmacologia , Feminino , Pressão Osmótica/efeitos dos fármacos , Folículo Ovariano/metabolismo , Folículo Ovariano/ultraestrutura , Propilenoglicóis/metabolismo , Propilenoglicóis/farmacologia , Ratos Wistar
17.
Histol Histopathol ; 33(1): 41-53, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28197987

RESUMO

Frutalin is a galactose-binding lectin that has an irreversible cytotoxic effect on HeLa cervical cancer cells, by inducing apoptosis and inhibiting cell proliferation. It was previously shown that after in vitro incubation, frutalin is internalized into HeLa cells nucleus, which indicates that frutalin apoptosis-inducing activity might be linked with its nuclear localization. Considering that drugs commonly used for cancer treatment have a deleterious effect on germ cells, the aim of this study was to evaluate the effect of frutalin on the activation, survival, ultrastructure and gene expression in follicles cultured within ovarian tissue. Goat ovarian fragments were cultured for 6 days in α-MEM⁺ alone or supplemented with frutalin (1, 10, 50, 100 or 200 µg/ml). Non-culturad and cultured tissues were processed for histological and ultrastructural analysis and they were also stored to evaluate the expression of anti- and pro-apoptotic genes by quantitative polymerase chain reaction (qPCR). The results showed that the frutalin, at all concentrations tested, reduced follicular survival when compared with control medium. Higher concentrations of frutalin (50, 100 or 200 µg/ml) also reduced follicular survival when compared with those tissues cultured with 1 or 10 µg/ml of frutalin. The ultrastructural analysis showed that atretic cultured follicles had retracted oocytes and a large number of vacuoles spread throughout the cytoplasm. In addition, signs of damage of mitochondrial membranes and cristae were observed. Moreover, although a dose-response effect on gene expression has not been observed, when compared with tissues culture in control medium, the presence of frutalin increased in mRNA expression pro-apoptotic genes. In conclusion, frutalin reduces follicular survival at all concentrations tested, its effects being more pronounced when high concentrations of this lectin (50, 100 and 200 µg/ml) are used. Gene expression profile and ultrastrutural features of cultured follicles suggest that follicular death in goat ovarian tissue cultured in presence of frutalin occurs via necrosis.


Assuntos
Antineoplásicos Fitogênicos/toxicidade , Galectinas/toxicidade , Folículo Ovariano/efeitos dos fármacos , Ovário/efeitos dos fármacos , Animais , Morte Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica , Cabras , Necrose , Folículo Ovariano/metabolismo , Folículo Ovariano/ultraestrutura , Ovário/metabolismo , Ovário/ultraestrutura , Fatores de Tempo , Técnicas de Cultura de Tecidos
18.
J Assist Reprod Genet ; 35(1): 41-48, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29236205

RESUMO

PURPOSE: The aim of this study is to optimize fibrin matrix composition in order to mimic human ovarian tissue architecture for human ovarian follicle encapsulation and grafting. METHODS: Ultrastructure of fresh human ovarian cortex in age-related women (n = 3) and different fibrin formulations (F12.5/T1, F30/T50, F50/T50, F75/T75), rheology of fibrin matrices and histology of isolated and encapsulated human ovarian follicles in these matrices. RESULTS: Fresh human ovarian cortex showed a highly fibrous and structurally inhomogeneous architecture in three age-related patients, but the mean ± SD of fiber thickness (61.3 to 72.4 nm) was comparable between patients. When the fiber thickness of four different fibrin formulations was compared with human ovarian cortex, F50/T50 and F75/T75 showed similar fiber diameters to native tissue, while F12.5/T1 was significantly different (p value < 0.01). In addition, increased concentrations of fibrin exhibited enhanced storage modulus with F50/T50, resembling physiological ovarian rigidity. Excluding F12.5/T1 from further analysis, only three remaining fibrin matrices (F30/T50, F50/T50, F75/T75) were histologically investigated. For this, frozen-thawed fragments of human ovarian tissue collected from 22 patients were used to isolate ovarian follicles and encapsulate them in the three fibrin formulations. All three yielded similar follicle recovery and loss rates soon after encapsulation. Therefore, based on fiber thickness, porosity, and rigidity, we selected F50/T50 as the fibrin formulation that best mimics native tissue. CONCLUSIONS: Of all the different fibrin matrix concentrations tested, F50/T50 emerged as the combination of choice in terms of ultrastructure and rigidity, most closely resembling human ovarian cortex.


Assuntos
Órgãos Artificiais , Fibrina/química , Ovário , Materiais Biomiméticos/química , Composição de Medicamentos , Elasticidade , Feminino , Dureza , Humanos , Fenômenos Mecânicos , Folículo Ovariano/transplante , Folículo Ovariano/ultraestrutura , Ovário/química , Ovário/citologia , Ovário/ultraestrutura
19.
Anim Reprod Sci ; 187: 193-202, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29126831

RESUMO

This study aimed to characterize the ovarian follicular population and to determine its correlations with the age and nutritional status of donkeys of the Northeastern Brazilian breed. A total of 10 females with a mean age of 5.1±3.2years were submitted to ovariectomy by videolaparoscopy to obtain the ovaries. In the laboratory, the ovaries were sectioned into 6-12 fragments of approximately 7mm in diameter, which were fixed in Carnoy, dehydrated in increasing concentrations of alcohol (85%, 95% and absolute), clarified using xylol and embedded in blocks of histological paraffin. The blocks were cut in sections of 7µm and each 120th section was mounted on a slide for observation using optical microscopy. The follicle counting and identification allowed the characterization of the population of the preantral follicles. A total of 21.135±10.821 preantral follicles was counted, of which, 91.3% were primordial, 8.3% were primary follicles and 0.4% were secondary follicles. There were no differences between the two ovaries of each animal regarding the follicular population (P>0.05). There was a rate of 9.77% degenerated follicles. Values of 0.99% follicles containing two oocytes were also identified and classified as multi-oocyte follicles, always of the primordial category. The thickness of the granulosa cell layer was 1.85µm±1.39, 3.56µm±2.08 and 21.85µm±17.27, for primordial, primary and secondary follicles, respectively. There was a significant inverse correlation (r=-0.66; P<0.001) between the age of the animals and the population of ovarian follicles. A negative influence of the weight and body score on the ovarian follicular population was also observed, when donkeys had very little or a great amount of body condition. This is the first study to describe the morphometric characteristics and estimation of the population of preantral ovarian follicles in Northeastern Brazilian donkey, showing that number of preantral follicles decreased with increasing age of the animals and this finding may be affected by nutritional status of the animals.


Assuntos
Equidae/crescimento & desenvolvimento , Microscopia/métodos , Estado Nutricional , Folículo Ovariano/citologia , Folículo Ovariano/fisiologia , Fatores Etários , Animais , Brasil , Equidae/metabolismo , Feminino , Oócitos/citologia , Oócitos/fisiologia , Oócitos/ultraestrutura , Folículo Ovariano/ultraestrutura
20.
Biol Reprod ; 97(3): 426-437, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-29025092

RESUMO

Hyperthermia or heat stress (HS) occurs when heat dissipation mechanisms are overwhelmed by external and internal heat production. Hyperthermia negatively affects reproduction and potentially compromises oocyte integrity and reduces developmental competence of ensuing embryos. Autophagy is the process by which cells recycle energy through the reutilization of cellular components and is activated by a variety of stressors. Study objectives were to characterize autophagy-related proteins in the ovary following cyclical HS during the follicular phase. Twelve gilts were synchronized and subjected to cyclical HS (n = 6) or thermal neutral (n = 6) conditions for 5 days during the follicular phase. Ovarian protein abundance of Beclin 1 and microtubule associated protein light chain 3 beta II were each elevated as a result of HS (P = 0.001 and 0.003, respectively). The abundance of the autophagy related (ATG)12-ATG5 complex was decreased as a result of HS (P = 0.002). Regulation of autophagy and apoptosis occurs in tight coordination, and B-cell lymphoma (BCL)2 and BCL2L1 are involved in regulating both processes. BCL2L1 protein abundance, as detected via immunofluorescence, was increased in both the oocyte (∼1.6-fold; P < 0.01) and granulosa cells of primary follicles (∼1.4-fold P < 0.05) of HS ovaries. These results suggest that ovarian autophagy induction occurs in response to HS during the follicular phase, and that HS increases anti-apoptotic signaling in oocytes and early follicles. These data contribute to the biological understanding of how HS acts as an environmental stress to affect follicular development and negatively impact reproduction.


Assuntos
Autofagia , Transtornos de Estresse por Calor/patologia , Folículo Ovariano/patologia , Ovário/patologia , Animais , Apoptose/genética , Feminino , Febre/patologia , Genes bcl-2/genética , Células da Granulosa/metabolismo , Temperatura Alta , Infertilidade Feminina/etiologia , Infertilidade Feminina/fisiopatologia , Folículo Ovariano/ultraestrutura , Ovário/ultraestrutura , Gravidez , Transdução de Sinais/genética , Sus scrofa , Suínos , Vacúolos/ultraestrutura , Proteína bcl-X/genética
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