Morphological study on the effects of sample size on ovarian tissue vitrification.

OBJECTIVE: The present study aimed to assess the effects of ovarian cortex sample size on tissue morphological integrity after vitrification in a metal capsule. METHODS: Bovine ovarian tissue samples cut in large and small fragments (1x1x5 and 1x1x3 mm, respectively - 5 and 3 mm refer to length), vitrified in a metal capsule were fixed for histological analysis immediately after rewarming or after 48 hours culture. We assessed primordial, primary and secondary follicle morphology and stromal integrity. RESULTS: Primordial follicles showed the highest rates of normal morphology after rewarming and after 48 hours culture in both, small and large tissue fragments. Primary follicles presented a significant drop in normal morphology in large samples, after 48 hours in culture. Stromal integrity was well-preserved immediately after rewarming in small and large fragments but presented a significant drop in normal morphology in large samples, after 48 hours in culture. CONCLUSIONS: The ovarian reserve, represented by Primordial follicles, is well-preserved in small or large fragments, after vitrification and culture. However, the stromal components present better preservation after vitrification\rewarming, when tissue samples are cut in small fragments. Thus, small cortex samples should be preferred for ovarian tissue vitrification.

Interaction of fibroblasts and induced pluripotent stem cells with poly(vinyl alcohol)-based hydrogel substrates.

Induced pluripotent stem cells (iPSCs) provide a promising means of creating custom-tailored cell lines for cellular therapies. Their application in regenerative medicine, however, depends on the possibility that the maintenance and differentiation of cells and organs occur under defined conditions. One major component of stem cell culture systems is the substrate, where the cells must attach and proliferate. The present study aimed to investigate the putative cytotoxic effects of poly(vinyl alcohol) (PVA)-based matrices on the in vitro culture of mouse fetal fibroblasts. In addition, the PVA-based hydrogels were used to determine the capacity of bovine induced pluripotent stem cells (biPSCs) to adhere and proliferate on synthetic substrates. Our results show that both cell types interacted with the substrate and presented proliferation during culture. The biPSCs formed new colonies when cell suspensions were placed onto the hydrogel surface for culture. These results may represent a new characterized xeno-free clinical grade culture system to be widely applied in cell-based therapies.

Morphological and morphometric analysis of skeletal muscle between male and female young adult Colossoma macropomum (Characiformes: Serrasalmidae)

This study aimed to evaluate muscle organization in tambaqui in order to describe the muscle growth process. We analyzed the morphometric pattern of fibers from white muscle of young-adults (300 days) by smaller diameter. The organization of white muscle exhibited a typical morphological pattern found in other fish species. Heavier animals showed higher frequency of larger diameter fibers (>50 m ) and smaller animals had higher frequency of smaller diameter fibers ( 20 m ) (P =0.005). However, both animals showed the same frequency of intermediate diameter fibers (20-50 m ). Body weight showed a positive correlation with muscle diameter fiber (r=0.45), being 20-50 m the diameters that contributed the most to animal weight (P 0.0001). A weak correlation between fiber diameter and animal sex was observed (r=0.2). Females showed higher frequency of large fiber diameters (>50 m ) than males. However, there was no difference between body weight and sex (P =0.8). Our results suggest that muscle growth is by hypertrophy and hyperplasia due to a mosaic appearance from different diameters fibers, which is characteristic of large size fish species.

O objetivo deste trabalho foi avaliar a organização muscular em tambaqui, a fim de descrever o processo de crescimento muscular. Foi analisado o padrão morfométrico das fibras do músculo branco de animais com 300 dias de idade usando o método de diâmetro menor. O músculo branco apresentou uma organização morfológica padrão encontrado em peixes. Animais de maior peso apresentaram maior frequência de fibras de maior diâmetro (> 50 m ) e os animais de menor peso apresentaram maior frequência de fibras de menor diâmetro ( 20 m ) (P = 0,005). Entretanto, ambos os animais, com maior e menor peso, apresentaram frequências semelhantes de fibras de diâmetro intermediário (20-50 m ). O parâmetro peso corporal mostrou correlação positiva com o diâmetro da fibra muscular (r = 0,45), sendo as fibras de diâmetro intermediários (20-50 m ) que mais contribuíram para o peso do animal (P 0,0001). Fêmeas apresentaram maior frequência de fibras de maior diâmetro (>50 m ) que machos. Observou-se uma fraca correlação entre o diâmetro da fibra e o sexo dos animais (r = 0,2). Apesar de fraca, a correlação estimada é corroborada pela fibras de grandes diâmetros (> 50 m ) serem mais frequente nas fêmeas que nos machos. No entanto, não houve diferença entre o peso corporal dos animais aos 300 dias de idade e sexo (P = 0,8). Os resultados encontrados sugerem que o crescimento muscular ocorre por hipertrofia e hiperplasia, caracterizado pela aparência em mosaico de fibras de diferentes diâmetros, característico de peixes de grande tamanho.

Embryogenesis of Heliconius erato (Lepidoptera, Nymphalidae): a contribution to the anatomical development of an evo-devo model organism.

This study reports on the embryogenesis of Heliconius erato phyllis between blastoderm formation and the prehatching larval stage. Syncytial blastoderm formation occurred approximately 2 h after egg laying (AEL) and at about 4 h, the cellular blastoderm was formed. The germ band arose from the entire length of the blastoderm, and rapidly became compacted occupying approximately two-thirds of the egg length. At about 7 h AEL, protocephalon and protocorm differentiation occurred. Continued proliferation of the germ band was followed by penetration into the yolk mass, forming a C-shaped embryo at about 10 h. Approximately 12 h AEL, the gnathal, thoracic and abdominal segments became visible. The primordium of the mouthparts and thoracic legs formed as paired evaginations, while the prolegs formed as paired lobes. At about 30 h, the embryo reversed dorsoventrally. Approximately 32 h AEL, the protocephalon and gnathal segments fused, shifting the relative position of the rudimentary appendages in this region. At about 52 h, the embryo was U-shaped in lateral view and at approximately 56 h, the bristles began evagination from the larval cuticle. Larvae hatched at about 72 h. We found that H. erato phyllis followed an embryonic pattern consistent with long-germ embryogenesis. Thus, we believe that H. erato phyllis should be classified as a long-germ lepidopteran. The study of H. erato phyllis embryogenesis provided a structural glimpse into the morphogenetic events that occur in the Heliconius egg period. This study could help future molecular approaches to understanding the evolution of Heliconius development.

Ovarian tissue vitrification: the use of a novel metal closed system for clinical grade cryopreservation.

OBJECTIVE: To investigate whether a metal chamber is an appropriate system for vitrification of ovarian tissue under clinical grade. METHODS: Experimental study, control versus treatment. Bovine ovarian cortices cut in 1x1x1 mm fragments were vitrified using ethylene glycol and dimethyl sulfoxide inside steel cryovials, whose bases were in touch with Liquid Nitrogen (LN2). Screw caps closed the cryovials before plunging into LN2. Primordial (n=356) and primary (n=327) follicles and the stroma were analyzed after histological preparation using light microscopy. RESULTS: High rate of primordial (93%) and primary (80%) follicles presented normal morphology in the rewarmed fragments. There was not a significant difference between controls and primordial follicles morphology (P=0.1519). Significant difference was observed for the primary follicles (P=0.0097). Important to point out that stromal cells and collagen fibers presented a remarkable integrity, without major alterations in the cryopreserved tissues. CONCLUSIONS: The steel cryovial seems to be a safe means of vitrification under clinical grade conditions, with very fast cooling rates and no direct contact of the biological material with the liquid nitrogen (LN2). Ovarian reserve represented by primordial and primary follicles and stroma are very well preserved in this vitrification system.

The use of a metal container for vitrification of mouse ovaries, as a clinical grade model for human ovarian tissue cryopreservation, after different times and temperatures of transport.

PURPOSE: Cryopreservation of ovarian tissue is paramount for fertility preservation, with important clinical applications, especially for women suffering from an oncological condition. Several cryopreservation methodologies have been tried in search of better outcomes, especially in terms of primor-dial and primary follicles integrity post-cryopreservation. Vitrification has successfully been applied to ovarian tissue using different carriers for tissue exposure to the liquid nitrogen (LN2). METHODS: We developed an enclosed metal vessel, which has the advantage of a faster heat transfer, when in contact with LN2 avoiding at the same time, the direct contact with tissue. Additionally, we assessed the effect of different times and temperatures of transport between the collection of mouse ovaries and the beginning of cryopreservation, on follicular morphology after vitrification. RESULTS: Our results suggest that 37 °C and R.T. help to maintain normal primordial and primary follicle morphology for up to 4 hrs after collection and beginning of vitrification in a metal container. CONCLUSION: These data show that the metal container is an appropriate carrier for mouse ovary vitrification. The rate of morphologically normal primordial follicles up to 4 hrs.

Expression of mdr isoforms in mice during estrous cycle and under hormone stimulation

The multidrug resistance (MDR) phenotype is associated with the expression of P-glycoprotein (Pgp), coded by the multigenic mdr family. Mice present the isoforms mdr1 and mdr3, which are responsible for multidrug resistance, and mdr2, that is involved in the transport of phospholipids. mdr1 expression has more recently been associated also with the secretion of steroid hormones. This work presents an RT-PCR analysis of the expression of mdr isoforms, in several organs of mice during different phases of the estrous cycle. Additionally, females were ovariectomized, submitted to different hormone treatments, and their uterus was analyzed for the expression of mdr isoforms. The results show that in the adrenal gland and ovaries mdr1 is the main isoform during proestrus, and that progesterone or a combination of progesterone and estrogen induce the expression of all mdr isoforms in the uterus of ovariectomized females. We suggest that the functions of mdr1 and mdr3 are overlapping, that mdr3 may be the more efficient isoform in the detoxification function, and that mdr1 may be more closely related to the secretion of steroid hormones.