Investigation of assisted fertilization and biology of reproduction by sperm microinjection

Recent success in assisted fertilization mainly depended on the development of sperm microinjection methods: intracytoplasmic sperm injection and subzonal insemination. Some basic mechanisms that under-lie fertilization were revealed by using intracytoplasmic sperm injection. In respect to this, problems of fertility, oocyte activation, formation of pronuclei and practical aspects of intracytoplasmic sperm injection are discussed.     

Catabolite inactivation of the maltose transporter in nitrogen-starved yeast could be due to the stimulation of general protein turnover

Addition of glucose to Saccharomyces cerevisiae inactivates the maltose transporter. The general consensus is that this inactivation, called catabolite inactivation, is one of the control mechanisms developed by this organism to use glucose preferentially whenever it is available. Using nitrogen-starved cells (resting cells), it has been shown that glucose triggers endocytosis and degradation of the transporter in the vacuole. We now show that maltose itself triggers inactivation and degradation of its own transporter as efficiently as glucose. This fact, and the observation that glucose inactivates a variety of plasma membrane proteins including glucose transporters themselves, suggests that catabolite inactivation of the maltose transporter in nitrogen-starved cells is not a control mechanism specifically directed to ensure a preferential use of glucose. It is proposed that, in this metabolic condition, inactivation of the maltose transporter might be due to the stimulation of the general protein turnover that follows nitrogen starvation.     

On the Importance of Hysteresis in Numerical Modeling of Surfactant-Induced Unsaturated Flow

It is known that surfactants can induce flow in unsaturated porous media due to the dependence of capillary pressure on surface tension. A commonly observed feature in systems with surfactant-induced flow is a transient wetting/drying/wetting sequence associated with the propagation of a surfactant solute front under monotonic flow conditions. Previous efforts to model surfactant-induced flow in relatively complex (e.g., two-dimensional systems) have not successfully incorporated hysteretic moisture retention properties. In this research, hysteretic, two-dimensional simulations of surfactant-induced flow were performed to assess the potential importance of considering hysteresis in such simulations. Hysteretic simulation results were compared to experimental data and to non-hysteretic simulations. The results suggest that the inclusion of hysteresis in numerical simulations can improve the match between simulated and experimental results in systems with surfactant-induced unsaturated flow. Furthermore, the inclusion of hysteresis in numerical simulations played a significant role in predicting the distribution of the contaminant and correct pressure head/moisture condition at the end of the experiment.     

Translocation - A key factor limiting the efficiency of nitrogen fixation in legume nodules

A hypothesis is presented that the availability of water for export of nitrogenous products from legume nodules is a major factor limiting the efficiency of symbiotic nitrogen fixation. Water for export of solutes in the xylem probably depends largely on the import of water and reduced carbon in the phloeum, and one function of respiration may be to dispose of reduced carbon in order to increase the supply of water. A second hypothesis presented is that control of gas diffusion in soybean nodules is largely restricted to the cortex nearby the vascular bundles, thus making possible the linkage of solute balances in xylem and phloem with resistance to diffusion. These concepts are used in a re-examination of literature on manipulations of nodules and nodulated plants such as lowering of light levels, water stress, defoliation, stem girdling, and alteration of oxygen supply. The concept of translocation as a major factor limiting efficiency of symbiotic fixation is consistent with the failure of superior rhizobial isolates to improve N input significantly, and this limitation could also prevent exploitation of superior bacterial symbionts in the future     

Differential Localisation of PARP-1 N-Terminal Fragment in PARP-1+/+ and PARP-1?/? Murine Cells

Human PARP family consists of 17 members of which PARP-1 is a prominent member and plays a key role in DNA repair pathways. It has an N-terminal DNA-binding domain (DBD) encompassing the nuclear localisation signal (NLS), central automodification domain and C-terminal catalytic domain. PARP-1 accounts for majority of poly-(ADP-ribose) polymer synthesis that upon binding to numerous proteins including PARP itself modulates their activity. Reduced PARP-1 activity in ageing human samples and its deficiency leading to telomere shortening has been reported. Hence for cell survival, maintenance of genomic integrity and longevity presence of intact PARP-1 in the nucleus is paramount. Although localisation of full-length and truncated PARP-1 in PARP-1 proficient cells is well documented, subcellular distribution of PARP-1 fragments in the absence of endogenous PARP-1 is not known. Here we report the differential localisation of PARP-1 N-terminal fragment encompassing NLS in PARP-1^+/+ and PARP-1^−/− mouse embryo fibroblasts by live imaging of cells transiently expressing EGFP tagged fragment. In PARP-1^+/+ cells the fragment localises to the nuclei presenting a granular pattern. Furthermore, it is densely packaged in the midsections of the nucleus. In contrast, the fragment localises exclusively to the cytoplasm in PARP-1^−/− cells. Flourescence intensity analysis further confirmed this observation indicating that the N-terminal fragment requires endogenous PARP-1 for its nuclear transport. Our study illustrates the trafficking role of PARP-1 independently of its enzymatic activity and highlights the possibility that full-length PARP-1 may play a key role in the nuclear transport of its siblings and other molecules.     

Dynamic resource allocation between pre- and postcopulatory episodes of sexual selection determines competitive fertilization success

In polyandrous mating systems, male reproductive success depends on both mate-acquisition traits (precopulatory) and sperm competitive abilities (postcopulatory). Empirical data on the interaction between these traits are inconsistent; revealing positive, negative or no relationships. It is generally expected that the investment in pre- and postcopulatory traits is mediated by environmental conditions. To test how dietary resource availability affects sexual ornamentation, sperm quality and their interrelationship in three-spined sticklebacks (Gasterosteus aculeatus), full-sibling groups were raised under three conditions differing in food quantity and/or quality (i.e. carotenoid content): (i) high-quantity/high-quality, (ii) high-quantity/low-quality or (iii) low-quantity/low-quality. After 1 year of feeding, food-restricted males developed a more intense breeding coloration and faster sperm compared with their well-fed brothers, indicating that they allocated relatively more in pre- and postcopulatory traits. Moreover, they outcompeted their well-fed, carotenoid-supplemented brothers in sperm competition trials with equal numbers of competing sperm, suggesting that food-restricted males maximize their present reproductive success. This may result in reduced future reproductive opportunities as food-restricted males suffered from a higher mortality, had an overall reduced body size, and sperm number available for fertilization. In accordance with theory, a trade-off between the investment in pre- and postcopulatory traits was observed in food-restricted males, whereas well-fed males were able to allocate to both traits resulting in a significantly positive relationship.     

Interaction between atmospheric and pedospheric nitrogen nutrition in spruce (Picea abies L. Karst) seedlings

The effect of NO₂ fumigation on root N uptake and metabolism was investigated in 3-month-old spruce (Picea abics L. Karst) seedlings. In a first experiment, the contribution of NO₂ to the plant N budget was measured during a 48 h fumigation with 100mm³m^?3 NO₂. Plants were pre-treated with various nutrient solutions containing NO₂ and NH₄⁺, NO₃^? only or no nitrogen source for 1 week prior to the beginning of fumigation. Absence of NH₄⁺ in the solution for 6d led to an increased capacity for NO₃^? uptake, whereas the absence of both ions caused a decrease in the plant N concentration, with no change in NO₃^? uptake. In fumigated plants, NO₂ uptake accounted for 20–40% of NO₃^? uptake. Root NO₃^? uptake in plants supplied with NH₄⁺plus NO₃^? solutions was decreased by NO₂ fumigation, whereas it was not significantly altered in the other treatments. In a second experiment, spruce seedlings were grown on a solution containing both NO₂ and NH₄⁺ and were fumigated or not with 100mm³m^?3 NO₂ for 7 weeks. Fumigated plants accumulated less dry matter, especially in the roots. Fluxes of the two N species were estimated from their accumulations in shoots and roots, xylem exudate analysis and ¹⁵N labelling. Root NH₄⁺ uptake was approximately three times higher than NO₃^? uptake. Nitrogen dioxide uptake represented 10–15% of the total N budget of the plants. In control plants, N assimilation occurred mainly in the roots and organic nitrogen was the main form of N transported to the shoot. Phloem transport of organic nitrogen accounted for 17% of its xylem transport. In fumigated plants, neither NO₃^? nor NH₄⁺ accumulated in the shoot, showing that all the absorbed NO₂ was assimilated. Root NO₃^? reduction was reduced whereas organic nitrogen transport in the phloem increased by a factor of 3 in NO₂-fimugated as compared with control plants. The significance of the results for the regulation of whole-plant N utilization is discussed.     

Iron uptake in Pseudomonas aeruginosa mediated by N-(2,3-dihydroxybenzoyl)-L-serine and 2,3-dihydroxybenzoic acid

Abstract Pseudomonas aeruginosa is known to have an inducible uptake system for the enterobacterial siderophore enterobactin. In this work we have examined iron transport mediated by the biosynthetic precursor 2,3-dihydroxybenzoic acid and N -(2,3-dihydroxybenzoyl)- l -serine, a breakdown product of enterobactin. Iron complexed with 2,3-dihydroxybenzoyl-L-serine was transported into P. aeruginosa IA1 via a transport system which is energy-dependent and iron-repressible. The rate of transport was not altered by growing the cells in the presence of either pyoverdin or pyochelin, which have been shown previously to induce transport via that system. Growth of the cells in the presence of enterobactin did cause an increase in the rate of transport, indicating that the complex can be transported by the inducible enterobactin uptake system, but also that a separate system must exist. In contrast, transport of iron complexed with 2,3-dihydroxybenzoic acid was neither iron-repressible nor strongly energy-dependent, from which we conclude that there must be a novel mode of transport not characteristic of iron-siderophore transport systems.     

Carboxyl-terminal Truncations of ClC-Kb Abolish Channel Activation by Barttin Via Modified Common Gating and Trafficking

ClC-K chloride channels are crucial for auditory transduction and urine concentration. Mutations in CLCNKB, the gene encoding the renal chloride channel hClC-Kb, cause Bartter syndrome type III, a human genetic condition characterized by polyuria, hypokalemia, and alkalosis. In recent years, several Bartter syndrome-associated mutations have been described that result in truncations of the intracellular carboxyl terminus of hClC-Kb. We here used a combination of whole-cell patch clamp, confocal imaging, co-immunoprecipitation, and surface biotinylation to study the functional consequences of a frequent CLCNKB mutation that creates a premature stop codon at Trp-610. We found that W610X leaves the association of hClC-Kb and the accessory subunit barttin unaffected, but impairs its regulation by barttin. W610X attenuates hClC-Kb surface membrane insertion. Moreover, W610X results in hClC-Kb channel opening in the absence of barttin and prevents further barttin-mediated activation. To describe how the carboxyl terminus modifies the regulation by barttin we used V166E rClC-K1. V166E rClC-K1 is active without barttin and exhibits prominent, barttin-regulated voltage-dependent gating. Electrophysiological characterization of truncated V166E rClC-K1 demonstrated that the distal carboxyl terminus is necessary for slow cooperative gating. Since barttin modifies this particular gating process, channels lacking the distal carboxyl-terminal domain are no longer regulated by the accessory subunit. Our results demonstrate that the carboxyl terminus of hClC-Kb is not part of the binding site for barttin, but functionally modifies the interplay with barttin. The loss-of-activation of truncated hClC-Kb channels in heterologous expression systems fully explains the reduced basolateral chloride conductance in affected kidneys and the clinical symptoms of Bartter syndrome patients.