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1.
Anim Reprod Sci ; 261: 107407, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38217925

RESUMO

The present study aims to establish the morphological, morphometric, and immunostaining patterns of the steroidogenic enzymes 17ß-HSD and 5α-reductase and androgen receptors (AR) during the prenatal development of the male gonad and epididymis of Cavia porcellus. Fetuses at 22, 25, 30, 40, 45, 50, and 60 days of gestation (DG) were used. Specimens were dissected and subjected to macroscopic, histological, histomorphometric, and immunohistochemical analyses. Genital and scrotal protrusions were identified in 22 DG embryos. Gonocytes were identified at 25 DG and the formation of primary testicular cords was observed at 30 DG. Through anatomical evaluation, we observed differentiation of the epididymis into the head, body, and tail at 45 DG. During development, there is a progressive decrease in the diameters of the testicular cords and epididymal ducts. 17ß-HSD enzyme immunostaining was observed in Leydig cells at all ages, while 5α-reductase was observed in Leydig cell cytoplasm and gonocytes at 40, 50, and 60 DG. AR shows gonocyte labeling at 30 DG. Thus, from the second trimester of pregnancy, it is possible to observe patterns of anatomical development, such as genital and scrotal prominence (22 DG), the appearance of gonocytes in the testicular cords at 25 DG, and the beginning of the organization of primary testicular cords at 30 DG, suggesting sexual differentiation. The 17ß-HSD, 5α-reductase, and ARs play an essential role in sexual development and differentiation, presenting immunostaining at different reproductive process times.


Assuntos
Epididimo , Testículo , Gravidez , Feminino , Cobaias , Masculino , Animais , Células Intersticiais do Testículo , Oxirredutases , Receptores Androgênicos
2.
Theriogenology ; 211: 151-160, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37639997

RESUMO

This study aims to define the best method (slow freezing or vitrification) and fragment size (1, 5, or 9 mm³) for prepubertal goat testis cryopreservation, as well as to evaluate testicular morphological integrity after cryopreservation and in vitro culture (IVC). Initially (experiment I), 1, 5, or 9 mm³ testis fragments were cryopreserved by slow freezing using a Mr. Frosty container with 20% Dimethylsulfoxide (DMSO) or vitrified using the Ovarian Tissue Cryosystem (OTC) device, (Equilibration solution - ES: 10% DMSO and 10% ethylene glycol - EG; Vitrification solution - VS: 20% DMSO and 20% EG) and then subjected to morphological analysis, type I and III collagen quantification and gene expression (Oct4, C-kit, Bax, and Bcl-2). Subsequently, (experiment II), fresh or cryopreserved by slow freezing testis fragments were cultured in vitro and submitted to morphological analysis by scanning electron microscopy. The data from the experiment I revealed fewer morphological alterations in 1 and 5 mm³ fragments after vitrification and slow freezing, respectively. The percentage of type I collagen fibers in 5 and 9 mm³ frozen was higher than in fresh or vitrified fragments. For type III collagen, fresh or frozen fragments of 1 and 5 mm3 showed a higher percentage than fragments of 9 mm3. Gene expression for Oct4 and C-kit after slow freezing or vitrification in the 5 mm3 fragments was lower than that observed in the fresh fragments. The Bax:Bcl-2 ratio in the 1 and 9 mm³ fragments was lower than in the 5 mm³ fragments for fresh fragments or after freezing. In experiment II, fragments cultured in vitro, previously frozen or not, showed more morphological alterations than fresh or frozen fragments. We concluded that slow freezing of 5 mm³ fragments was the best protocol for cryopreserving prepubertal goat testis and although the results of IVC are encouraging, it still needs improvement to restore testicular function after cryopreservation.


Assuntos
Dimetil Sulfóxido , Cabras , Animais , Masculino , Proteína X Associada a bcl-2 , Criopreservação/veterinária , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas c-kit
3.
Zygote ; 30(3): 419-422, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34689852

RESUMO

The aim of this study was to evaluate the effect of 1 µmol/l zearalenone (ZEN) and 1 µmol/l matairesinol (MAT), alone or in combination, on the morphology of in vitro-cultured ovarian preantral follicles. Ovaries from four adult sheep were collected at a local slaughterhouse and fragmented, and the ovarian pieces were submitted to in vitro culture for 3 days in the presence or absence of the test compounds. The morphology of primordial and primary follicles was impaired by ZEN. The plant lignan MAT alone did not maintain the morphology of the ovarian follicles; its combination with ZEN counteracted the negative effects observed when follicles were cultured in the presence of the mycotoxin alone. However, MAT was not able to promote the in vitro development of the ovarian follicles.


Assuntos
Lignanas , Zearalenona , Animais , Feminino , Furanos , Lignanas/farmacologia , Folículo Ovariano , Ovário , Ovinos , Zearalenona/toxicidade
4.
Zygote ; 30(1): 144-147, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33988116

RESUMO

Ovine ovarian fragments (3 × 3 × 1 mm) were fixed in neutral buffered formalin (NBF), Carnoy's solution (CAR), Davidson's solution (DAV), or paraformaldehyde (PFA) for 12 h or 24 h. After this fixation time, each fragment was prepared for histological analysis. Although fixative and fixation period did not affect follicular and stromal cells density, the percentages of morphologically normal primordial and primary follicles was affected by the fixative type and period of fixation. Paraformaldehyde was not indicated as a fixative for ovarian fragments. Formalin was a suitable fixative only when the period of fixation was 12 h, while Carnoy was efficient after a fixation period of 24 h. In conclusion, the most indicated fixative for the morphological evaluation of ovarian preantral follicles was DAV, regardless of the fixation period, that is 12 or 24 h.


Assuntos
Folículo Ovariano , Ovário , Animais , Feminino , Fixadores/farmacologia , Ovinos , Fixação de Tecidos
5.
Theriogenology ; 174: 124-130, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34428678

RESUMO

The aim of this study was to evaluate the effect of 1 µmol/L zearalenone (ZEN) and 1 µmol/L enterolactone (ENL), alone or in combination, on the survival and morphology of in vitro cultured ovarian preantral follicles. Ovaries from 10 sheep were collected at a local abattoir and fragmented, and the ovarian pieces were submitted to in vitro culture for 3 days in the presence or absence of the test compounds. The morphology of primordial and primary follicles was impaired by ZEN, whereas that of cultured secondary follicles was improved by ENL. However, the combination of ENL with ZEN impaired the quality of primary and secondary follicles. Both ZEN and ENL induced apoptosis, but only ZEN was responsible for oocyte autophagy. None of these xenoestrogens affected endoplasmic reticulum stress as observed by the unaltered expression of ERP29. Differently from ZEN, ENL increased the expression of the efflux transporter ABCG2. In conclusion, although ENL can counteract the negative effects of ZEN on primordial and primary follicles, this positive effect is not similar to that observed in ovarian tissue cultures in the presence of ENL alone.


Assuntos
Zearalenona , 4-Butirolactona/análogos & derivados , Animais , Feminino , Lignanas , Oócitos , Folículo Ovariano , Ovário , Ovinos , Zearalenona/toxicidade
6.
Theriogenology ; 111: 69-77, 2018 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-29428847

RESUMO

We aimed to evaluate the effect of two vitrification methods on the morphology and functionality of vitrified feline preantral follicles. Feline ovarian tissue was vitrified with EG + trehalose combined or not with dimethyl sulphoxide (DMSO), using two different techniques (open or closed systems). Morphology, developmental capacity and mRNA expression of markers for follicle survival and quality were assessed before and after in vitro culture (IVC). Both vitrification and culture media were serum-free. Vitrification of feline ovarian tissue from five adult domestic cats was performed with EG + trehalose combined or not with DMSO. Two systems were used: the open system solid-surface vitrification (SSV) and the closed system ovarian tissue cryosystem (OTC). Histological analysis of follicle integrity showed that the percentages of normal follicles in previously vitrified ovarian fragments decreased after 7 days of in vitro culture (IVC), independently of the protocol used. Although follicular activation was observed by Ki-67 labelling, this was accompanied by extensive follicular degeneration as detected by a 3-4-fold decrease in follicular density. Remarkable follicle activation was observed in the ovarian tissue vitrified using OTC and subjected to IVC, probably due to a higher rate of degeneration of developing follicles. Even with such follicular loss, the results are promising for the combination of EG + DMSO + trehalose in a serum-free medium when applying the SSV method, with this approach resulting in the highest rates of normal developing follicles (19%) after 7 days IVC, together with granulosa cells proliferating at the same rate observed in fresh tissue.


Assuntos
Gatos , Criopreservação/veterinária , Ovário/fisiologia , Vitrificação , Animais , Apoptose , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Ecossistema , Etilenoglicol/farmacologia , Feminino
7.
Cell Tissue Res ; 355(2): 471-80, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24362491

RESUMO

Ovarian fragments were exposed to 0.5 M sucrose and 1 M ethylene glycol (freezing solution; FS) with or without selenium or Trolox. Histological and ultrastructural analyses showed that the percentages of normal follicles in control tissue and in tissue after exposure to FS + 50 µM Trolox were similar. Trolox prevented endoplasmic reticulum (ER)-related vacuolization, which is commonly observed in oocytes and stromal tissue after exposure to FS. From the evaluated stress markers, superoxide dismutase 1 (SOD1) was up-regulated in ovarian tissue exposed to FS + 10 ng/ml selenium. Ovarian fragments were subsequently frozen-thawed in the presence of FS with or without 50 µM Trolox, followed by in vitro culture (IVC). Antioxidant capacity in ovarian fragments decreased after freeze-thawing in Trolox-free FS compared with FS + 50 µM Trolox. Although freezing itself minimized the percentage of viable follicles in each solution, Trolox supplementation resulted in higher rates of viable follicles (67 %), even after IVC (61 %). Furthermore, stress markers SOD1 and ERp29 were up-regulated in ovarian tissue frozen-thawed in Trolox-free medium. Relative mRNA expression of growth factors markers was evaluated after freeze-thawing followed by IVC. BMP4, BMP5, CTGF, GDF9 and KL were down-regulated independently of the presence of Trolox in FS but down-regulation was less pronounced in the presence of Trolox. Thus, medium supplementation with 50 µM Trolox prevents ER stress and, consequently, protects ovarian tissue from ER-derived cytoplasmic vacuolization. ERp29 but not ERp60, appears to be a key marker linking stress caused by freezing-thawing and cell vacuolization.


Assuntos
Cebus/metabolismo , Cromanos/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Congelamento , Ovário/efeitos dos fármacos , Ovário/patologia , Vitamina E/análogos & derivados , Animais , Calreticulina/metabolismo , Crioprotetores/farmacologia , Feminino , Proteínas de Choque Térmico HSP70/metabolismo , Ovário/metabolismo , Ovário/ultraestrutura , Superóxido Dismutase/metabolismo
8.
Zygote ; 21(2): 167-71, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-22475447

RESUMO

There is no tradition in studies reporting the effect of exposure to cryoprotectants or simply hypoxia and hypothermia on gene expression in the ovarian tissue and there has been only one study on reference or target genes quantification, and comparisons of normoxic with hypoxic, hypothermic and toxic conditions. Our aim in the present study was to investigate the stability of three reference genes in the ovarian tissue of capuchin monkeys (Cebus apella). To this end, fresh and cryoprotectant-exposed ovarian biopsies were used. Both fresh and exposed ovarian tissues were subjected to total RNA extraction and synthesis of cDNA. cDNA was amplified by real-time polymerase chain reaction (PCR), and GeNorm, BestKeeper and NormFinder software were used to evaluate the stability of glyceraldehyde-2-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and TATA-binding protein (TBP). Results demonstrated that, in the ovarian tissue from capuchin monkeys, HPRT1 and TBP were the most suitable reference genes and thus could be used as parameters to normalize data in future studies. In contrast, GAPDH appeared as the least stable gene among the tested reference genes. In conclusion, HPRT1 and TBP were the most stable reference genes in fresh and cryoprotectant-exposed ovarian tissue from capuchin monkeys.


Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/genética , Hipoxantina Fosforribosiltransferase/genética , Ovário/efeitos dos fármacos , Padrões de Referência , Proteína de Ligação a TATA-Box/genética , Animais , Cebus , Crioprotetores/farmacologia , Feminino , Hipotermia , Ovário/citologia , Ovário/metabolismo , Oxigênio/farmacologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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