Parthenogenetic activation of bovine oocytes using single and combined strontium, ionomycin and 6-dimethylaminopurine treatments

In vitro-matured (IVM) bovine oocytes were activated with single and combined treatments of strontium (S), ionomycin (1) and 6-DMAP (D). Using oocytes IVM for 26 h, we observed that activation altered cell cycle kinetics (faster progression, MIII arrest, or direct transition from MII to pronuclear stage) when compared to in vitro fertilization. The effect of oocyte age on early parthenogenesis was assessed in oocytes IVM for 22, 26 and 30 h. Better results in pronuclear development were obtained in treatments ISD (81.7%) at 22 h; D (66.7%), IS (63.3%), ID (73.3%) and ISD (76.7%) at 26 h; and D (86.7%), IS (85.0%) and ID (78.3%) at 30 h. Higher cleavage occurred on ISD (80.0%) at 22 h; ID (83.3%) and ISD (91.7%) at 26 h; and 1 (86.7%), IS (90.0%), ID (85.0%) and ISD (95.0%) at 30 h. More blastocysts were achieved in ID (25.0%) and ISD (18.3%) at 22h; and in ID at 26h (45.0%) and 30h (50.0%). We also observed that IS allowed higher haploid (77.4%) embryonic development, whilst ID was better for diploid (89.1%) development. It was concluded that association of S and D without I was not effective for blastocyst development; treatments using S were less influenced by oocyte age, but when S was associated with D there was a detrimental effect on aged oocytes; treatment ISD promoted higher activation and cleavage rates in young oocytes and ID protocol was the best for producing blastocysts.

Effect of Vitrification of Feline Ovarian Cortex on Follicular and Oocyte Quality and Competence

Cryopreservation of ovarian cortex has important implications in the preservation of fertility and biodiversity in animal species. Slow freezing of cat ovarian tissue resulted in the preservation of follicular morphology and in the follicular development after xenografting. Vitrification has been recently applied to ovarian tissues of different species, but no information is available on the effect of this method on feline ovarian cortex. Moreover, meiotic competence of fully grown oocytes isolated from cryopreserved tissue has not been reported. The aim of this study was to evaluate the effect of vitrification of feline ovarian cortex on follicular morphology and oocyte integrity, as well as meiotic competence. A total of 352 fragments (1.52 mm3) were obtained from ovarian cortical tissues: 176 were vitrified and 176 were used fresh as control. Histological evaluation of fresh and vitrified fragments showed intact follicles after cryopreservation procedures with no statistically significant destructive effect from primordial to antral follicles. After IVM, oocytes collected from vitrified ovarian fragment showed a higher proportion of gametes arrested at germinal vesicle (GV) stage compared to those isolated from fresh control tissue (33.8% vs 2.9%; p < 0.001). However, oocytes isolated from vitrified tissues were able to resume meiosis, albeit at lower rate than those collected from fresh tissues (39.8% vs 85.9%; p < 0.00001). Vitrification induced changes in the organization of cytoskeletal elements (actin microfilaments and microtubules) of oocytes, but significantly only for actin network (p < 0.001). Finally, chromatin configuration within the GV was not affected by the cryopreservation procedure. Our study demonstrated that vitrification preserves the integrity of ovarian follicles and that oocytes retrieved from cryopreserved tissue maintain the capability of resuming meiosis. To our knowledge, this has not previously been reported in the cat.

Demecolcine Effects on Microtubule Kinetics and on Chemically Assisted Enucleation of Bovine Oocytes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Invasion of epithelial mammalian cells by Paracoccidioides brasiliensis leads to cytoskeletal rearrangement and apoptosis of the host cell

Paracoccidioides brasiliensis (Pb) yeast cells can enter mammalian cells and probably manipulate the host cell environment to favor their own growth and survival. We studied the uptake of strain Pb 18 into A549 lung and Vero epithelial cells, with an emphasis on the repercussions in the cytoskeleton and the apoptosis of host cells. Cytoskeleton components of the host cells, such as actin and tubulin, were involved in the P. brasiliensis invasion process. Cytochalasin D and colchicine treatment substantially reduced invasion, indicating the functional participation of microfilaments (MFs) and microtubules (MTs) in this mechanism. Cytokeratin could also play a role in the P. brasiliensis interaction with the host. Gp43 was recognized by anti-actin and anti-cytokeratin antibodies, but not by anti-tubulin. The apoptosis induced by this fungus in infected epithelial cells was demonstrated by various techniques: TUNEL, DNA fragmentation and Bak and Bcl-2 immunocytochemical expression. DNA fragmentation was observed in infected cells but not in uninfected ones, by both TUNEL and gel electrophoresis methods. Moreover, Bcl-2 and Bak did not show any differences until 24 h after infection of cells, suggesting a competitive mechanism that allows persistence of infection. Overexpression of Bak was observed after 48 h, indicating the loss of competition between death and survival signals. In conclusion, the mechanisms of invasion of host cells, persistence within them, and the subsequent induction of apoptosis of such cells may explain the efficient dissemination of P. brasiliensis. (C) 2004 Published by Elsevier SAS.

Direct effects of colchicine on myocardial function - Studies in hypertrophied and failing spontaneously hypertensive rats

The aging spontaneously hypertensive rat (SHR) is a model in which the transition from chronic stable left ventricular hypertrophy to overt heart failure can be observed. Although the mechanisms for impaired function in hypertrophied and failing cardiac muscle from the SHR have been studied, none accounts fully for the myocardial contractile abnormalities. The cardiac cytoskeleton has been implicated as a possible cause for myocardial dysfunction. If an increase in microtubules contributes to dysfunction, then myocardial microtubule disruption by colchicine should promote an improvement in cardiac performance. We studied the active and passive properties of isolated left ventricular papillary muscles from 18- to 24-month-old SHR with evidence of heart failure (SHR-F, n=6), age-matched SHR without heart failure (SHR-NF, n=6), and age-matched normotensive Wistar-Kyoto rats (WKY, n=5). Mechanical parameters were analyzed before and up to 90 minutes after the addition of colchicine (10(-5), 10(-4), and 10(-3) mol/L). In the baseline state, active tension (AT) developed by papillary muscles from the WKY group was greater than for SHR-NF and SHR-F groups (WKY 5.69+/-1.47 g/mm(2) [mean+/-SD], SHR-NF 3.41+/-1.05, SHR-F 2.87+/-0.26; SHR-NF and SHR-F P<0.05 versus WKY rats). The passive stiffness was greater in SHR-F than in the WKY and SHR-NF groups (central segment exponential stiffness constant, K-cs: SHR-F 70+/-25, SHR-NF 44+/-17, WKY 41+/-13 [mean+/-SD]; SHR-F P<0.05 versus; SHR-NF and WKY rats). AT did not improve after 10, 20, and 30 minutes of exposure to colchicine (10(-5), 10(-4), and 10(-3) mol/L) in any group. In the SHR-F group, AT and passive stiffness did not change after 30 to 90 minutes of colchicine exposure (10(-4) mol/L). In summary, the data in this study fail to demonstrate improvement of intrinsic muscle function in SHR with heart failure after colchicine. Thus, in the SHR there is no evidence that colchicine-induced cardiac microtubular depolymerization affects the active or passive properties of hypertrophied or failing left ventricular myocardium.

Ultrastructure of the ovary of Dermatobia hominis (Diptera: cuterebridae). I. Development during the 3rd larval instar

The ultrastructure and distribution of gonial and somatic cells in the ovary of Dermatobia hominis was studied during the 3rd larval instar. In larvae weighing between 400 and 500 mg, the ovary is partially divided into basal and apical regions by oblong somatic cells that penetrate from the periphery; these cells show ovoid nucleus and cytoplasm full of microtubules. In both regions, gonial cells with regular outlines, large nucleus and low electron-density cytoplasm are scattered among the interstitial somatic cells. These later cells have small nucleus and electrodense cytoplasm. Clear somatic cells with small nucleus and cytoplasm of very low electron-density are restrict to the apical region of the gonad. Degenerating interstitial somatic cells are seen in the basal portion close to the ovary peduncle. During all this larval period the morphological features of the ovary remain almost the same. At the end of the period there is a gradual deposition of glycogen in the cytoplasm of the somatic cells, increase in the number and density of their mitochondria plus nuclear modification as membrane wrinkling and chromatin condensation in masses.

Effect of thyroid hormone T3 on Myosin-Va expression in the central nervous system

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Ultrastructure of Giardia duodenalis trophozoites group from hamster: some relevant aspects

Throphozoites of Giardia duodenalis group obtained from fragments or scratched of hamster's mucosa were examined by transission electron microscopy. The fine structure of the trophozoites are presented and comapred with those described for other animals. Some of the trophozoites present the cytoplasm full of glycogen, rough endoplasmic reticulum-like structures and homogeneous inclusions not enclosed by membranes, recognized as lipid drops, which had not been observed in Giardia from other animals. The adhesive disk is composed of a layer of microtubules, from which fibrous ribbons extend into the cytoplasm; these ribbons are linked by layer of crossbridge filaments that shows an intermediary dense band, described for the first time in this paper. The authors regard this band as the result of the cross-bridge filaments slinding in the medium region between adjacent fibrous ribbons, and suggest a contractile activity for them. The role of the adhesive disk on the trophozoite mechanism of attachment to host mucosa is also discussed.

Ultrastructural characterization of CD133(+) stem cells bound to superparamagnetic nanoparticles: possible biotechnological applications

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Action of the chemical agent fipronil on the reproductive process of semi-engorged females of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae). Ultrastructural evaluation of ovary cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ultrastructural aspects of the intercellular bridges between female bee germ cells

No ovário das abelhas as células germinativas e as células foliculares são interconectadas por pontes intercelulares mantidas abertas por reforços do citoesqueleto na membrana plasmática. As pontes entre as células germinativas têm comportamento dinâmico e provavelmente atuam na determinação do ovócito entre as células do clone formado pelas mitoses pré meióticas formando posteriormente uma via de transporte para que os produtos sintetizados pelas células nutridoras atinjam o ovócito durante sua maturação. Os elementos do citoesqueleto presentes nas pontes intercelulares das gônadas das abelhas são basicamente microfilamentos e microtúbulos, mas nas pontes entre os cistócitos pré-meióticos outro tipo de filamento (espesso de natureza não definida, associado a elementos do retículo endoplasmático) está presente, atravessando a ponte e prendendo-se através dos microfilamentos à membrana plasmática. Estes filamentos aparentemente controlam o vão da ponte. Terminada a fase de proliferação os cistócitos tomam a forma de uma roseta e um fusoma, formado pela convergência das pontes, aparece no centro desta. Nesta conformação os filamentos grossos não estão presentes. Nova mudança ocorre com a diferenciação do ovócito e das células nutridoras, com a reorientação de todas as pontes de maneira a canalizar o conteúdo das futuras células nutridoras para o ovócito.

Espermiogênese em Eupemphix nattereri (Anura, Leiuperidae): aspectos ultra-estruturais

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Infertilidade associada a defeito microtubular dos espermatozóides de jumento (Equus asinus) avaliados por microscopia eletrônica de transmissão

Descreve-se um caso de infertilidade de um jumento SRD confirmada por meio de microscopia eletrônica de transmissão (MET). O espermiograma, avaliado sob microscopia ótica, revelou baixa motilidade e alta concentração de anormalidades espermáticas do tipo gota citoplasmática proximal. O material foi avaliado por MET, observando-se um acúmulo desordenado de microtúbulos causando protusões irregulares na região do colo espermático. O último teste realizado correspondeu ao de fertilidade in vivo, utilizando-se quatro éguas portadoras de bom histórico reprodutivo, nas quais não foi possível confirmar nenhuma prenhez. Frente aos resultados obtidos, associados aos achados da MET, estabeleceu-se o diagnóstico de infertilidade associada a defeito microtubular dos espermatozóides.

Ultrastructure of the excretory duct in the silk gland of the sugarcane borer Diatraea saccharalis (Lepidoptera : Pyralidae)

The excretory duct in the silk gland of the sugarcane borer Diatraea saccharalis consists of two morphologically distinct regions, recognized by scanning and transmission electron microscopy. The thin posterior region, adjacent to the glandular region, presents a regular surface. Secretory vesicles containing either electron-dense or fibrillar cuticular-like materials are observed in their apical cytoplasm; the same cuticular materials were detected as extracellular deposits among the microvilli. The short anterior region, near the common duct, exhibits surface protrusions; there are no secretory vesicles in their apical cytoplasm. These results show that only the duct cells at the posterior region are involved in the secretion of the cuticular intima elements. Desmosome-like structures were visualized linking together adjacent microvillar membranes only in the cells of anterior duct region, with unknown function. The transition between the duct and the glandular region is abrupt; the cells of the glandular and posterior duct regions present large amounts of microtubules. Nerve fibers can be observed between the duct cells in their two regions, suggesting that control of silk secretion may occur in the excretory duct via neurotransmitter liberation. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

Mechanism of micronuclei formation in polyploidizated cells of Allium cepa exposed to trifluralin herbicide

The trifluralin is an agent that promotes a cellular damage due to its direct action on the microtubules. This action leads to a decontrol in the cellular division, bringing about polyploid cells. In this work, we show the evidences that the exceeding genetical material of theses polyploidizated cells tends to be eliminated from the nucleus in the form of micronucleus. Our analyses prove this fact, both by the presence of a number of cells carrying micronucleus, and by the evidences of the elimination of the exceeding material itself, after exposition of the Allium cepa root tips tested with several concentration of trifluralin herbicide. It was noticed that the residual concentration induced a number of polyploid cells, micronuclei and mini cells. Inferences about the implications of the elimination of genetic material from micronuclei, such as cell viability and apoptosis, are also presented. (c) 2007 Elsevier B.V. All rights reserved.