Concepts to Reveal Parvovirus–Nucleus Interactions

Parvoviruses are small single-stranded (ss) DNA viruses, which replicate in the nucleoplasm and affect both the structure and function of the nucleus. The nuclear stage of the parvovirus life cycle starts at the nuclear entry of incoming capsids and culminates in the successful passage of progeny capsids out of the nucleus. In this review, we will present past, current, and future microscopy and biochemical techniques and demonstrate their potential in revealing the dynamics and molecular interactions in the intranuclear processes of parvovirus infection. In particular, a number of advanced techniques will be presented for the detection of infection-induced changes, such as DNA modification and damage, as well as protein–chromatin interactions.

On Wars

Representatives of the states of the world must find other means of conflict handling than warfare. The increasing tension between open and closed societies calls for new mechanisms of intergovernmental interaction. Intervention in Burma is necessary to hinder a new example of the meaninglessness of warfare. The war in Palestine fits perfectly the concept of a senseless warfare. The wisdom of classical theorists is no longer relevant in the nuclear stage of mankind.

Development of female hametophytis and embryogenesis of introduced species Picea A. Dietr. in the Taiga Zone (Karelia)

The aim of the research was to establish features in macrosporogenesis, macrogametogenesis, and embryogenesis in Picea species (native species P. abies and introduced species P. canadensis and P. pungens) in the Botanical Garden of Petrozavodsk State University (middle taiga subzone). To study the development of the generative sphere, shoots with generative buds and strobiles from the southwestern side of the crown were cut from 10 trees and fixed in a mixture of alcohol and glycerin. The development of the female gametophyte and the embryo was studied with constant preparations. Preparations for anatomical examination of the kidneys were prepared according to standard methods. In this case, double fixation in glutaraldehyde and osmium was used, wiring through alcohol and acetone, and encapsulation in epoxy resin. Sections with a thickness of about 1 μm were prepared on an ultramicrotome and stained in a 1% aqueous solution of methylene blue. Studies have established that in mid-May, the female gametophyte is still at a free stage, and by the beginning of June it already has a cellular structure. Embryogenesis begins in late July and ends in the second decade of August. In introduced species, various deviations are observed in the development of the female generative sphere and during embryogenesis. The main ones are the degeneration of the female gametophyte at the free-nuclear stage and the destruction of archegonia at the stage of proembryogenesis. For this reason, from 37 to 51% of the seeds of introducers are devoid of the embryo. The largest percentage of empty seeds and the most recent stages of the passage of the main stages of morphogenesis are observed in P. pungens. This indicates the least degree of adaptability of the generative sphere of this species of introducer to new conditions.

The Nuclear Localization of the DnaJ-Like Zinc Finger Domain-Containing Protein EDA3 Affects Seed Development in Arabidopsis thaliana

The DnaJ-like zinc finger domain-containing proteins are involved in different aspects of plastid function and development. Some of these proteins were recently reported to have dual subcellular localization in the nucleus and plastids. One member of this family, PSA2 (AT2G34860), was found to localize to the thylakoid lumen and regulate the assembly of photosystem I (PSI). However, PSA2 was also annotated as Embryo sac Development Arrest 3 (EDA3) from the observation that its embryo sac development was arrested at the two-nuclear stage. In this study, we characterized the eda3 mutant, and demonstrated that, as compared with the wild-type (WT) plants, the mutant has shorter siliques, fewer siliques per plant, and fewer seeds per silique. Both aborted and undeveloped ovules were observed in siliques of the mutant. By immunoblot analysis, we found that, different from the chloroplast localization in mature leaves, EDA3 localizes in the nucleus in seeds. A nuclear localization signal was identified from the deduced amino acid sequence of EDA3, and also proved to be sufficient for directing its fusion peptide into the nucleus.

Phosphatidylinositol 5 Phosphate (PI5P): From Behind the Scenes to the Front (Nuclear) Stage

Phosphatidylinositol (PI)-related signaling plays a pivotal role in many cellular aspects, including survival, cell proliferation, differentiation, DNA damage, and trafficking. PI is the core of a network of proteins represented by kinases, phosphatases, and lipases which are able to add, remove or hydrolyze PI, leading to different phosphoinositide products. Among the seven known phosphoinositides, phosphatidylinositol 5 phosphate (PI5P) was the last to be discovered. PI5P presence in cells is very low compared to other PIs. However, much evidence collected throughout the years has described the role of this mono-phosphoinositide in cell cycles, stress response, T-cell activation, and chromatin remodeling. Interestingly, PI5P has been found in different cellular compartments, including the nucleus. Here, we will review the nuclear role of PI5P, describing how it is synthesized and regulated, and how changes in the levels of this rare phosphoinositide can lead to different nuclear outputs.

Optimal doses of EGF and GDNF act as biological response modifiers to improve porcine oocyte maturation and quality

SummaryIt is well documented that both epidermal growth factor (EGF) and glial cell line-derived neurotrophic factor (GDNF) are critical for porcine oocyte maturation, however, little information is known about their mechanism of action in vitro. To gain insight into the mechanisms of action of the optimum doses of EGF and GDNF on porcine oocyte maturation, porcine cumulus–oocyte complexes (COCs) were matured in defined porcine oocyte medium supplemented with EGF, GDNF or a combination of both at varying concentrations (0–100 ng/ml) for 44 h. Nuclear and cytoplasmic maturation were determined in terms of nuclear stage after DNA staining with Hoechst and cortical granule distribution after lectin labeling, respectively. Mature oocytes were subsequently collected for gene expression analysis or subjected to in vitro fertilization and cultured for 7 days. The results showed that EGF and/or GDNF, when administered in a certain dose (50 ng/μl) to the maturation medium, not only effectively improved the synchronization of nuclear and cytoplasmic maturation processes within the oocyte, but enhanced expression of their corresponding receptors in mature oocytes (P < 0.05). Moreover, supplementation with an optimal combination of EGF + GDNF resulted in elevation of TFAM transcripts as well as a decrease of caspase-3 transcripts compared with the other studied groups (P < 0.05). Collectively, our results indicate that treatment of porcine oocytes with specific-dose combinations of EGF and GDNF stimulates oocyte quality and competence by transcriptional modulation of genes involved in oocyte survival and competence.

171 PRE-IN VITRO MATURATION OF PORCINE OOCYTES USING PITUITARY ADENYLATE CYCLASE-ACTIVATING PEPTIDE: EFFECTS ON MEIOSIS PROGRESSION AND DEVELOPMENTAL COMPETENCE

Porcine immature oocytes derived from small follicle (SF, ≤3 mm in diameter) are able to resume meiosis, but few oocytes are capable to progress to the metaphase 2 stage. To improve capacitation of oocytes derived from SF and inhibit spontaneous maturation, a pre-in vitro maturation (IVM) system was applied to in vitro culturing of cumulus–oocyte complexes (COC). Pituitary adenylate cyclase-activating peptides (PACAP), increasing cellular cyclic adenosine 3′5′-monophosphate, have pleiotropous actions and multiple functions throughout the body as a neuropeptide. Recently, studies have described the role PACAP play in fertility and reproduction, including follicular development, antiapoptotic effects, and implantation. The purpose of this study is to improve the developmental competence of oocytes derived from SF by exogenous addition of PACAP on pre-IVM. In the conventional IVM group, COC obtained from follicles ≤3 mm in diameter (SF group) and 3 to 6 mm in diameter (medium follicles; MF group) were subjected to IVM for 42 h. In the pre-IVM group, COC obtained from SF were matured with nontreatment [pre-SF(-)PACAP group] and 1μM PACAP [pre-SF(+)PACAP group] for 18 h pre-IVM and were immediately subjected to IVM for 42 h. We examined nuclear stage assessment, intracellular reduced glutathione and reactive oxygen species levels, and embryo developmental competence by parthenogenesis and IVF. After IVM, the result of the nuclear stage assessment of groups showed that the pre-SF(+)PACAP group had the highest metaphase 2 rate in the groups (P < 0.05). Reduced glutathione levels in MF and pre-SF(+)PACAP groups showed significantly higher levels than that of the other groups (P < 0.05). After parthenogenesis, the cleavage rates were significantly (P < 0.05) higher than the others in pre-SF(+)PACAP group. In the IVF experiment, the embryo cleavage rate was significantly higher in that of MF and pre-SF(+)PACAP groups compared with that of SF and pre-SF(-)PACAP groups (P < 0.05). Moreover, no significant differences were found in the cleavage rate between MF and pre-SF(+)PACAP groups. In all groups derived from SF, the pre-SF(+)PACAP group rate of blastocyst formation and total cell number of blastocysts was significantly higher than that of the other groups (P < 0.05). These results indicated that pre-IVM system using PACAP is able to improve meiotic maturation and developmental competence although the oocytes were derived from SF.

254 IN VITRO BLASTOCYST PRODUCTION FROM PREPUBERTAL GOAT OOCYTES ACCORDING TO SEASON

In our laboratory, we have been working for more than 20 years on in vitro embryo production from prepubertal goat oocytes. We have observed significant differences on blastocyst production according to the season of year. The present study is aimed to asses these differences in summer, autumn, winter, and spring. Oocytes with more than 3 compact cumulus layers (cumulus-oocyte complex, COC) and homogeneous cytoplasm were obtained after slicing prepubertal goat ovaries (45-day-old Murciano-Granadina rearing animals) recovered from slaughterhouse. Groups of 30 COC were matured in 100-µL drops of TCM199 medium (10% FBS) for 24 h with 5% CO2 and humidified atmosphere. Fresh buck semen (pool of 3 males) was selected by swim-up and capacitated with 0.05 mg mL–1 of heparin for 45 min. The IVF was performed by transferring 20 oocytes to 100-μL drops of Tyrode's medium with 4 × 106 capacitated sperm with 5% CO2 and humidity. After 17 h of IVF, a sample of oocytes was fixed in ethanol:acetic (3 : 1) for staining (1% lacmoid) to assess nuclear stage (Table 1). At 20 h post-insemination (pi), groups of 10 presumptive zygotes were transferred to 10-μL drops of SOF medium (10% FBS) under paraffin oil in a humidified atmosphere with 90% N2 for 8 days. Cleavage rate was evaluated at 48 h pi, and blastocyst rate was recorded at Day 8 pi (Table 1). Table 1 shows a decrease in normal pronuclear formation during winter (43%) and spring (47%). However, winter (16%) and spring (16%) produced significantly more blastocysts than summer (10%) and autumn (5%). Similar results were found by Sousa et al. (2014 Theriogenology 82, 1149–1162) in adult goats producing significantly higher blastocysts in the anoestrus season. We hypothesise that prepubertal goat oocytes in winter and spring may have a better cytoplasmic quality, producing more blastocyst even though the penetration rate is significantly lower than in autumn. In conclusion, under in vitro conditions and using fresh semen, the rate of penetration, cleavage, and blastocysts in prepubertal goats is influenced by season. Further studies in oocyte and sperm quality should be done to clarify this result. Table 1.Nuclear stage of prepubertal goat oocytes according to season (3 replicates; top) and in vitro blastocyst production of prepubertal goat oocytes according to season at Day 8 postinsemination (7 replicates; bottom)1

Female gametophyte development and embryogenesis in Taxus baccata L.

Crassinucellate ovules are initiated in <em>Taxus</em>, directly from the shoot apex. The rudimentary pollen chamber is formed in the nucellus. A linear tetrad of megaspores with a functional chalazal megaspore is formed. A free-nuclear stage is charac-teristic at the beginning of megagametophyte development. Archegonia without ventral canal cell are solitary or in complexes. The embryo has a very long suspensor even after maturation. Two types of polyembryony have been revealed: i) embryogenic redifferentiation of suspensor cells and ii) cleavage of embryonic region in the early embryo. In the northern temperate climate of St. Petersburg one month delay in development of reproductive structures has been noted.

223 CRYOPRESERVATION OF IMMATURE BOVINE CUMULUS–OOCYTE COMPLEXES BY SLOW RATE FREEZING AND VITRIFICATION

Cryopreservation of mature oocytes can result in damage to the metaphase spindle due to the temperature sensitivity of microfilaments and microtubules. Cryopreservation of immature oocytes may circumvent this problem because these structures have not formed yet and the genetic material is enclosed within a nuclear envelope. Because intact oocyte cumulus-oocyte complexes (COC) are essential for normal maturation, we chose to cryopreserve immature intact COC. The aim of this study was to determine if immature COC cryopreserved by slow rate freezing or vitrification would resume meiosis upon thawing. In 2 separate experiments, immature COC (n = 102 and 79) were collected from cross-bred cattle by transvaginal ultrasound-guided aspiration and divided into 2 groups. For both experiments, the first group (n = 64 and 40) was placed directly into maturation medium (TCM 199 supplemented with 10% fetal bovine serum, 0.2 mM sodium pyruvate, 2 mM glutamine, and 5 μg mL–1 of FSH) and cultured for 22 h under 5% CO2 in air atmosphere at 39°C. In experiment 1, COC (n = 38) in the second group were cryopreserved by a slow rate freezing protocol. The COC were equilibrated for 5 min in 1.5 M ethylene glycol (EG), and 5 COC were loaded into 0.25-mL straws and placed into the cooling chamber of a Freeze Control unit at –6°C. After 5 min, straws were seeded, then cooled at 0.5°C per min to –35°C before plunging into LN. After storage in LN for 5 days, straws were removed from LN, thawed by placing straws in a 35°C water bath, and COC put into maturation as described above. In experiment 2, COC (n = 39) in the second group were cryopreserved by vitrification using a 3-step procedure. The COC were equilibrated in solutions consisting of 10% glycerol then 10% glycerol and 20% EG in PBS for 5 min each. The COC were then placed in a solution of 24% glycerol and 26% EG, and 1 to 3 COC were placed onto a cryotop device in minimal medium and plunged into LN within 45 s. After storage in LN for 2 days, COC were thawed by placing the cryotop device directly into a warmed dilution solution consisting of 0.5 M galactose in PBS. After 5 min in the dilution solution, COC were put into maturation as described above. For all groups, after 22 h of maturation cumulus cells were stripped from the oocytes by vortexing and oocytes were placed on slides for fixation in methanol acetic acid and stained with 1% orcein to determine the nuclear stage. In experiment 1, oocytes cryopreserved by slow rate freezing matured to MII at the same rate as control oocytes (45 v. 55%, P = 0.44). In experiment 2, oocytes cryopreserved by vitrification matured to MII at a lower rate than controls (49 v. 79%, P = 0.01). These results show that cryopreservation of immature intact COC is a viable alternative to cryopreservation of mature oocytes. Further studies are needed to optimize either slow rate freezing or vitrification of intact COC and determine the developmental competence of cryopreserved oocytes following fertilization.