Repositório RCAAP

Nectaries in leaves of Gentianaceae have been poorly studied. The present study aims to describe the distribution, anatomy, and ecological aspects of extrafloral nectaries (EFNs) of three Calolisianthusspecies and in particular the ultrastructure of EFNs in Calolisianthus speciosus during leaf development, discussing its unusual structure. Leaves of Calolisianthus species were fixed and processed by the usual methods for studies using light, scanning microscopy and transmission electron microscopy (TEM). Ion chromatography was used to analyze the nectar exudates of C. speciosus. The distribution patterns of nectar secretion units were analysed by ANOVA and t-tests. Two EFNs that can be seen macroscopically were observed at the bases of C. speciosus and C. pendulus leaves. Such large nectaries are absent there in C. amplissimus. Another similarly large EFN is observed at the apex of each leaf in all species. The EFNs at the base of the young leaves in C. speciosus are visited by ants during the rainy season. EFNs are formed by several nectar secretory units (nectarioles) that are present throughout the leaves. Each nectariole is formed by rosette cells with a central channel from which the nectar is released. Channels of old C. speciosus and C. pendulus EFNs were obstructed by fungi. TEM of EFNs in young leaves showed cytoplasms with secretion, small vacuoles, mitochondria, cell wall ingrowth, and plasmodesmata. TEM of EFNs in old leaves demonstrated dictyosomes, plastids, mitochondria, segments of endoplasmatic reticulum, and lipid droplets. The nectar contains sucrose, glucose and fructose.

Avaliou-se o efeito da adição de diferentes tipos e concentrações de soro sobre o desenvolvimento e a sobrevivência de folículos ovarianos pré-antrais (FOPA) caprinos in vitro. Além disso, verificou-se a relação entre as concentrações de nitrito presentes no meio de cultivo e a viabilidade folicular. Cada par ovariano foi dividido em 29 fragmentos, sendo um destinado ao controle. Os fragmentos foram cultivados por um ou sete dias em meio essencial mínimo suplementado (MEM+) ou MEM+ com diferentes concentrações (10 ou 20%) de soro fetal bovino (SFB), soro de cabra em estro (SCE) ou soro de cabra em diestro (SCD). Na análise morfológica após sete dias, apenas o tratamento com 10% de SFB apresentou percentual de FOPA normais similar ao MEM+ (P>0,05). A análise ultra-estrutural dos folículos cultivados por sete dias com MEM+ ou MEM+ com 10% de SFB mostrou danos oocitários, porém células da granulosa normais. A análise do meio de cultivo revelou correlação positiva entre a viabilidade folicular e a produção de nitrito. A suplementação com soro não melhorou a viabilidade de FOPA e a concentração de nitrito no meio de cultivo funcionou como um indicador da viabilidade das células da granulosa de FOPA caprinos cultivados in vitro. ______________________________________________________________________________________________________________ ABSTRACT

The objective was to develop an efficient protocol for cryopreservation of agouti (Dasyprocta aguti) ovarian tissue. Agouti ovarian fragments were placed, for 10 min, in a solution containing MEM and fetal bovine serum plus 1.5 M dimethyl sulfoxide (DMSO), ethylene glycol (EG) or propanediol (PROH); some of those fragments were subsequently cryopreserved in a programmable freezer. After exposure and/or thawing, all samples were fixed in Carnoy prior to histological analysis. To evaluate ultrastructure, follicles from the control and all cryopreserved treatments were fixed in Karnovsky and processed for transmission electron microscopy. After exposure and freezing, there was a significant decrease in the percentage of morphologically normal preantral follicles in all treatments when compared to the control (92.67 ± 2.79, mean ± SD). However, there were no significant difference when the exposure and freezing procedures were compared using the same cryoprotectant. Moreover, there was no significant difference among cryoprotectants at the time of exposure (DMSO: 64.7 ± 3.8; EG: 70.7 ± 11.2, PROH: 63.3 ± 8.5) or after freezing (DMSO: 60.6 ± 3.6, EG: 64.0 ± 11.9; PROH: 62.0 ± 6.9). However, only follicles frozen with PROH had normal ultrastructure. In conclusion, preantral follicles enclosed in agouti ovarian tissue were successfully cryopreserved using 1.5 M PROH, with satisfactory maintenance of follicle morphology and ultrastructure.

The morphological aspects of male reproductive tract, spermiogenesis and spermatozoa are typical for each species and reflect its evolution, establishing a unique source of characters, which has been used to help solve phylogenetic problems. In Hypanthidium foveolatum the reproductive tract is composed of the testes comprising 28 testicular tubules, deferent ducts, seminal vesicles, accessory glands and an ejaculatory duct. The differentiation of spermatids occurs within cysts of up to 128 germ line cells each one. During the early spermatid phase, the nucleus resembles that of somatic cells. There follows a gradual chromatin condensation with an increase in nuclear electron density. In the spermatozoon, the nucleus contains heterogeneous chromatin with a loose appearance. The acrosome, shaped with the active participation of the Golgi complex, shows an electron-dense perforatorium involved by four electron-lucent acrosomal vesicle projections. The sperm tail presents an axoneme with a 9 + 9 + 2 microtubule pattern and two mitochondrial derivatives, which appear with different sizes. A dense crystalloid is formed initially in the mitochondrial matrix of the large derivative. The mitochondrial derivatives’ differentiation occurs concomitantly with an axoneme outgrowth. The centriolar adjunct is observed near the axoneme, anterior to the smaller mithocondrial derivative and exhibits an approximately triangular shape in cross-sections. Microtubules were observed around the head region and flagellar components during spermiogenesis.

The main objective was to compare the efficiency of vitrification techniques and solutions on the preservation of morphology, ultrastructure and viability of sheep preantral follicles enclosed in ovarian tissue fragments. The fragments were cryopreserved by using macrotube vitrification (MTV), solid-surface vitrification (SSV) or conventional vitrification (CV). These techniques were combined with one of the six solutions containing 6 M ethylene glycol (EG) and with or without sucrose (SUC) (0.25 or 0.50 M) and with or without fetal calf serum (FCS) (10%). After one week, samples were warmed and histological analysis was performed, showing that the percentage of normal follicles after CV (66.20 ± 8.87%) using a solution containing 6 M EG, 0.25 M SUC and 10% FCS (vitrification solution 4 – VS4) was similar to fresh control (79.40 ± 7.83%), MTV (53.40 ± 10.60%) and SSV (56.75 ± 15.33%), all of them with the same vitrification solution (P < 0.05). For follicular viability evaluation, ovarian fragments were vitrified as described above. After warming, follicles were assessed by trypan blue dye. Controversially, the highest percentage of viable follicles was observed in MTV (97.06%) and was similar to fresh control (92.62%) (P < 0.05), but was significantly different from SSV (81.08%) and CV (83.81%) (P < 0.05). These results were validated by transmission electron microscopy that showed normal follicles observed in MTV and in fresh control. In addition, to verify the MTV with VS4 (a combination of the best technique plus the best solution), follicle viability was evaluated after 48 h in vitro culture. The viability assay was performed by fluorescence microscopy (calcein-AM and ethidium homodimer-1) analysis as follows: follicles isolated from fresh tissue were forthwith analyzed or underwent 48 h in vitro culture before analysis, whereas others fragments were vitrified/warmed and immediately analyzed or underwent 48 h in vitro culture before analysis. These results showed that, although follicular viability after MTV/VS4 (65%) was reduced when compared to the non-vitrified follicles at day 0 (100%), follicular viability after MTV/VS4 at day 2 (36.5%) was similar to follicles vitrified at day 0 (65%) and similar to non-vitrified follicles at day 2 (62.5%) (P > 0.05). As the decrease of viability in non-vitrified follicles at day 2 was similar to the decrease of MTV/VS4 in the same time, follicle viability at day 2 is not affected by MTV/VS4. In conclusion, using the experimental conditions of the present study, an efficient solution (VS4: 6 M EG, 0.25 M SUC and 10% FCS) and technique (MTV) were successfully used to vitrify ovine ovarian tissue.

In the very late phase of baculovirus infection, virions are occluded in a crystalline matrix called polyhedra, which is mainly composed of polyhedrin. This protein is highly conserved among baculoviruses and changes in its amino acid sequence may lead to mutant polyhedra. During the purification of an AcMNPV recombinant virus, a mutant virus was isolated. Structural and ultrastrutural analysis by light and transmission electron microscopy (TEM) of insect cells infected with this mutant virus did not show polyhedra formation and differed from the wild-type infection by the presence of a proteinaceous mass dispersed in the cytoplasm and nucleus of the infected cells, which was confirmed by immunogold labelling to be polyhedrin. The polyhedrin gene was amplified by PCR and sequenced. The only change observed was the substitution of a G to a T at the nucleotide +352, which resulted in a Val to Phe change. A recombinant virus was constructed by transferring the mutant gene into a polyhedrin negative virus. The phenotype of this recombinant virus was the same as the mutant one, confirming that this single mutation alone was responsible for the mutant phenotype.

The maintenance of follicle quality during the transportation of ovaries is essential for the successful cryopreservation and in vitro development of preantral follicles. The objective of this study was to evaluate the effect of cooling ovarian tissue on the conservation of zebu cow preantral follicles. Ovarian pieces were immersed in saline or coconut water (CW) solutions and maintained at 4 or 20 °C for 6, 12, or 18 h. Preantral follicles were evaluated by histology and transmission electron microscopy. Storage of ovarian pieces at 20 °C for 12 or 18 h significantly reduced the percentage of morphologically normal follicles compared to controls. In contrast, conservation at 4 °C for up to 18 h and at 20 °C for up to 6 h kept the percentage of normal follicles similar to controls. However, the type of solution that the ovaries were immersed in had little effect on the results. Decreased cellular metabolism probably accounted for better preservation of preantral follicles at 4 °C. In conclusion, zebu cow ovaries were successfully stored at 4 °C for up to 18 h with no morphological damage to preantral follicles. However, at 20 °C, ovaries could only be stored for 6 h.

The present study was undertaken to elucidate some aspects about the nature of the spermatozoon ultrastructure of Crotallus durissus using cytochemical methods. We also provide for the first time the ultrastructural description of this species spermatozoon. Cytochemical studies of spermatozoa have not been performed so far in the Serpentes, and species spermatozoon may prove helpful to better understand the reproductive biology of this group. Besides the synapomorphies of the Squamata and Serpentes, the C. durissus spermatozoon possess the following: circular acrosome tip; rounded perforatorium tip with a stopper-like basal modification; bilateral stratified laminar structures; central electron-dense structure within the proximal centriole; fibrous sheath extending until the level of the second mitochondrial ring; rounded mitochondria in cross-section, but with variable shape and organization in longitudinal and oblique sections, respectively; linear annulus; developed multilaminar membranes in the nuclear region and the midpiece. The formation of membrane filipin–sterol complexes occur sparsely along the head region, specially around the nucleus; the complexes were also present in the midpiece membrane and scarcely lining the flagellum. The complexes were present in the different layers of the multilaminar membranes. The ethanol-phosphotungstic acid (E-PTA) treatment releaved the presence of basic proteins in acrosome vesicle, pericentriolar material, peripheral fibers of the axoneme and fibrous sheath. The tannic acid technique revealed the microtubules of the centrioles and the axoneme; the extracellular tubules encircling the spermatozoa and those spread in the epididymal lumen were also observed. However, the immunocytochemistry assay using antibodies against alpha-tubulin and beta-tubulin, the primary microtubule monomers, does not support the existence of composition similarity between these tubular structures, since the extracellular tubules were not labeled by the antibodies. The results obtained in this work demonstrate that the utilization of electron microscopic techniques may provide relevant information to the study of ophidian reproductive biology, particularly in analyses concerning spermatozoal ultrastructure.

This study aims to investigate the effects of follicle stimulating hormone (FSH) and fibroblast growth factor-2 (FGF-2) on the survival and growth of caprine preantral follicles. Ovarian tissues were cultured for 1, 7, 14, 21 or 28 days in medium supplemented with FSH (FSH-2d or FSH-7d, i.e., with replacement of the culture medium every 2 or 7 days, respectively) or FSH + FGF-2 (replacement of the medium every 2 days). Non-cultured (control) and cultured ovarian fragments were processed for histological and ultrastructural analysis. After 28 days of culture, the media supplemented with FSH-2d was the most effective in maintaining the percentage of normal follicles and in promoting follicular growth. Furthermore, both treatments with FSH increased the percentage of the primary follicles. However, ultrastructural studies did not confirm follicular integrity from 14 days of culture onward. In conclusion, culturing tissue for up to 7 days in medium containing FSH alone or combined with FGF-2 maintains caprine preantral follicle integrity and promotes their growth in vitro.

Tese (doutorado)—Universidade de Brasília, Faculdade de Ciências da Saúde, 2006.

Os efeitos do α-tocoferol e da ternatina sobre morfologia, ativação e crescimento de folículos pré-antrais caprinos cultivados in vitro, por um ou cinco dias, foram avaliados. Os fragmentos ovarianos foram imediatamente fixados (controle não-cultivado) ou cultivados in vitro, por um ou cinco dias, em Meio Essencial Mínimo (MEM) com ou sem suplementação com α-tocoferol ou ternatina nas concentrações de 5, 10 ou 15M, formando os tratamentos MEM, TOC5, TOC10, TOC 15, TER5, TER10, TER15. O percentual de folículos pré-antrais normais no controle não-cultivado foi de 73,2%, depois de cinco dias de cultivo, houve redução desse percentual em todos os tratamentos, quando comparados com o controle não-cultivado (P<0,05). O cultivo por cinco dias aumentou a ativação folicular em todos os tratamentos (P<0,05). A análise ultra-estrutural não mostrou folículos pré-antrais íntegros após cinco dias de cultivo em meio contendo antioxidantes. Concluiu-se que o α -tocoferol e a ternatina podem promover a ativação folicular, no entanto a adição desses antioxidantes nas concentrações testadas reduziu a viabilidade folicular após o cultivo in vitro. ______________________________________________________________________________________________________________ ABSTRACT

The sperm of Microstigmus arlei and Microstigmus nigrophthalmus are twisted in a spiral and consist of two regions: the head, formed by an acrosome and a nucleus, and the flagellum, formed by two asymmetric mitochondrial derivatives, a long centriolar adjunct, an axoneme (9 + 9 + 2) and two accessory bodies. The head shows a characteristic morphology. The acrosome is very long and is basically made up of a paracrystalline structure. In the central head region, the acrosome is inserted into the nucleus, which is observed coiling laterally around the paracrystalline structure. In the subsequent part of the spermatozoon the nucleus appears round in transverse sections, and over some length it is still penetrated by the acrosome until shortly distal to the flagellar insertion. At this point the nucleus forms an inverted cone-shaped projection. These morphological characteristics of acrosome and nucleus of the Microstigmus wasp have not been previously described in Apoidea and are useful for phylogenetic evaluation of this superfamily.

The three Pegoscapus species present the same internal reproductive tract features comprising testes with a single testicular tubule, seminal vesicles, vasa deferentia, accessory glands and an ejaculatory duct. The seminal vesicle shows two morphologically distinct portions although they do not resemble the separate chambers found in other Chalcidoidea. The anterior portion of the seminal vesicle shows a prominent epithelium and stores the mature spermatozoa, while the posterior region is formed by a thicker muscular sheath that participates on ejaculation. The sexual maturation in Pegoscapus is achieved at emergence, when the testicular degeneration occurs. The spermatozoa of Pegoscapus reveal a basic structure similar to that of other Chalcidoidea. In Pegoscapus sp1. and Pegoscapus sp2. they present the same features, whereas Pegoscapus tonduzi comprises some different characteristics. It measures approximately 160 μm in Pegoscapus sp1. and Pegoscapus sp2., while in P. tonduzi the spermatozoa measure about 360 μm. The extracellular sheath thickness is another difference among the species. While Pegoscapus sp1. and Pegoscapus sp2. show a thick extracellular sheath, in P. tonduzi this sheath is very thin resulting in a large space intervening between the extracellular sheath and the nucleus. Despite these differences, the three species analyzed share some characteristics that allow the establishment of an identity to the spermatozoon of the genus Pegoscapus: the seminal vesicle not divided in chambers; the absence of acrosomal structures in the spermatozoa; the length of the extracellular sheath; the central microtubules being the firsts to terminate in the sequence of microtubular cutoff at the final axonemal portion.

Tese (doutorado)—Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, Programa de Pós-Graduação em Agronomia, 2014.

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Educação, Programa de Pós-Graduação, Mestrado em Educação, 2014.

Dissertação (mestrado)—Universidade de Brasília, Instituto de Geociências, 2014.

Dissertação (mestrado) - Universidade de Brasília, Faculdade de Tecnologia, 2013.

Dissertação (mestrado)—Universidade de Brasília, Instituto de Relações Internacionais, 2014.

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciência da Saúde, 2014.

Tese (doutorado)—Universidade de Brasília, Faculdade de Tecnologia, Departamento de Engenharia Civil e Ambiental, 2008.