Effects of ascorbic acid on in vitro culture of bovine preantral follicles

Summary The objective of this study was to evaluate the effects of adding ascorbic acid to the media for in vitro culture of cattle ovarian fragments and to determine their effects on growth activation and viability of early-stage follicles. The ovarian cortex was divided into small fragments; one fragment was immediately fixed (control) and the other fragments were cultured in minimum essential medium (MEM) supplemented or not with various doses of ascorbic acid. Ovarian tissue was processed for histology, transmission electron microscopy (TEM) and immunohistochemical demonstration of proliferating cell nuclear antigen (PCNA). Compared with control fragments, the percentage of primordial follicles was reduced (p < 0.05) and the percentage of growing follicles had increased (p < 0.05) in cultured cortical fragments, independent of the tested medium or incubation time. Furthermore, compared with control tissue, culture of ovarian cortex for 8 days reduced the percentages of healthy, viable follicles (p < 0.05), but not when cultures were supplemented with 25, 50 or 100 μg/ml of ascorbic acid. Ultrastructural and immunohistochemical analysis of 8 day cultured ovarian cortical fragments, however, showed the integrity and viability of follicles only when fragments were cultured in presence of 50 μg/ml of ascorbic acid. In conclusion, this study demonstrated that addition of ascorbic acid to MEM at a concentration of 50 μg/ml not only stimulates the activation of 8 day in vitro cultured cattle primordial follicles and subsequent growth of activated follicles, but also safeguards the viability of these early-stage follicles.     

Improvement of an electrical activation protocol for porcine oocytes

Factors influencing pig oocyte activation by electrical stimulation were evaluated by their effect on the development of parthenogenetic embryos to the blastocyst stage to establish an effective activation protocol for pig nuclear transfer. This evaluation included 1) a comparison of the effect of epidermal growth factor and amino acids in maturation medium, 2) an investigation of interactions among oocyte age, applied voltage field strength, electrical pulse number, and pulse duration, and 3) a karyotype analysis of the parthenogenetic blastocysts yielded by an optimized protocol based on an in vitro system of oocyte maturation and embryo culture. In the first study, addition of amino acids in maturation medium was beneficial for the developmental competence of activated oocytes. In the second study, the developmental response of activated oocytes was dependent on interactions between oocyte age at activation and applied voltage field strength, voltage field strength and pulse number, and pulse number and duration. The formation of parthenogenetic blastocysts was optimal when activation was at 44 h of maturation using three 80-microsec consecutive pulses of 1.0 kV/cm DC. Approximately 84% of parthenogenetic blastocysts yielded by this protocol were diploid, implying a potential for further in vivo development.     

Use of Monodispersed, Fluorescently Labeled Bacteria to Estimate In Situ Protozoan Bacterivory

We have developed a procedure for preparing monodispersed, fluorescently labeled bacteria (FLB), which may be used to measure virtually instantaneous rates of protozoan bacterivory in natural waters. FLB can be prepared both from natural bacterioplankton assemblages and from clonal isolates and can be stored in frozen suspension or freeze-dried without apparent loss of fluorescence intensity. They are not toxic to protozoa and can be metabolized to support bacterivorous protozoan growth rates equal to those on the same strain of unstained, viable bacteria. In experiments comparing uptake of FLB with uptake of fluorescent latex microspheres by protozoan assemblages in a salt marsh tidal creek, we found that both pelagic oligotrichous ciliates and phagotrophic flagellates ingested FLB with a frequency 4- to 10-fold greater than they ingested the microspheres. Consequently, it appears that the use of latex microspheres leads to underestimation of protozoan bacterivory and that the FLB technique is superior for estimating instantaneous rates of in situ protozoan grazing on bacterioplankton.     

Satellite tracking reveals distinct movement patterns for Type B and Type C killer whales in the southern Ross Sea, Antarctica

During January/February 2006, we satellite-tracked two different ecotypes of killer whales (Orcinus orca) in McMurdo Sound, Ross Sea, Antarctica, using surface-mounted tags attached with sub-dermal darts. A single Type B whale (pinniped prey specialist), tracked for 27 days, traveled an average net distance of 56.8 ± 32.8 km day⁻¹, a maximum of 114 km day⁻¹, and covered an estimated area of 49,351 km². It spent several days near two large emperor penguin (Aptenodytes forsteri) colonies, a potential prey item for this form. By contrast, four Type C killer whales (fish prey specialists) tracked for 7–65 days, traveled an average net distance of 20 ± 8.3 km day⁻¹, a maximum of 56 net km day⁻¹, and covered an estimated area of only 5,223 km². These movement patterns are consistent with those of killer whale ecotypes in the eastern North Pacific where mammal-eating ‘transients’ travel widely and are less predictable in their movements, and fish-eating ‘residents’ have a more localized distribution and more predictable occurrence, at least during the summer months.     

Interferon-Induced Antiviral Mx1 GTPase Is Associated with Components of the SUMO-1 System and Promyelocytic Leukemia Protein Nuclear Bodies

Mx proteins are interferon-induced large GTPases, some of which have antiviral activity against a variety of viruses. The murine Mx1 protein accumulates in the nucleus of interferon-treated cells and is active against members of the Orthomyxoviridae family, such as the influenza viruses and Thogoto virus. The mechanism by which Mx1 exerts its antiviral action is still unclear, but an involvement of undefined nuclear factors has been postulated. Using the yeast two-hybrid system, we identified cellular proteins that interact with Mx1 protein. The Mx1 interactors were mainly nuclear proteins. They included Sp100, Daxx, and Bloom's syndrome protein (BLM), all of which are known to localize to specific subnuclear domains called promyelocytic leukemia protein nuclear bodies (PML NBs). In addition, components of the SUMO-1 protein modification system were identified as Mx1-interacting proteins, namely the small ubiquitin-like modifier SUMO-1 and SAE2, which represents subunit 2 of the SUMO-1 activating enzyme. Analysis of the subcellular localization of Mx1 and some of these interacting proteins by confocal microscopy revealed a close spatial association of Mx1 with PML NBs. This suggests a role of PML NBs and SUMO-1 in the antiviral action of Mx1 and may allow us to discover novel functions of this large GTPase.     

Intact p53-Dependent Responses in miR-34-Deficient Mice

MicroRNAs belonging to the miR-34 family have been proposed as critical modulators of the p53 pathway and potential tumor suppressors in human cancers. To formally test these hypotheses, we have generated mice carrying targeted deletion of all three members of this microRNA family. We show that complete inactivation of miR-34 function is compatible with normal development in mice. Surprisingly, p53 function appears to be intact in miR-34-deficient cells and tissues. Although loss of miR-34 expression leads to a slight increase in cellular proliferation in vitro, it does not impair p53-induced cell cycle arrest or apoptosis. Furthermore, in contrast to p53-deficient mice, miR-34-deficient animals do not display increased susceptibility to spontaneous, irradiation-induced, or c-Myc-initiated tumorigenesis. We also show that expression of members of the miR-34 family is particularly high in the testes, lungs, and brains of mice and that it is largely p53-independent in these tissues. These findings indicate that miR-34 plays a redundant function in the p53 pathway and suggest additional p53-independent functions for this family of miRNAs.