Final Growth Phase Research Articles

Age-associated changes in cumulus cells and follicular fluid: the local oocyte microenvironment as a determinant of gamete quality.

The ovary is the first organ to age in humans with functional decline evident already in women in their early 30s. Reproductive aging is characterized by a decrease in oocyte quantity and quality, which is associated with an increase in infertility, spontaneous abortions, and birth defects. Reproductive aging also has implications for overall health due to decreased endocrinological output. Understanding the mechanisms underlying reproductive aging has significant societal implications as women globally are delaying childbearing and medical interventions have greatly increased the interval between menopause and total lifespan. Age-related changes inherent to the female gamete are well-characterized and include defects in chromosome and mitochondria structure, function, and regulation. More recently, it has been appreciated that the extra-follicular ovarian environment may have important direct or indirect impacts on the developing gamete, and age-dependent changes include increased fibrosis, inflammation, stiffness, and oxidative damage. The cumulus cells and follicular fluid that directly surround the oocyte during its final growth phase within the antral follicle represent additional critical local microenvironments. Here we systematically review the literature and evaluate the studies that investigated the age-related changes in cumulus cells and follicular fluid. Our findings demonstrate unique genetic, epigenetic, transcriptomic, and proteomic changes with associated metabolomic alterations, redox status imbalance, and increased apoptosis in the local oocyte microenvironment. We propose a model of how these changes interact, which may explain the rapid decline in gamete quality with age. We also review the limitations of published studies and highlight future research frontiers.

Ractopamine supplementation in the diet of pintado amazônico during the final growth phase

The objective of this unprecedented research was to evaluate performance traits, carcass yield, meat quality, and hematological parameters of pintado amazônico fish (Pseudoplatystoma reticulatum × Leiarius marmoratus) fed diets containing ractopamine (20 mg kg-1) for different periods, during the final growth phase. The following treatments were evaluated: 45 days without ractopamine supplementation (control diet); 30 days of control diet, followed by 15 days of ractopamine supplementation; 15 days of control diet, followed by 30 days of ractopamine supplementation; and 45 days of ractopamine supplementation. Performance traits, carcass yield and hematological parameters were not significantly influenced by the ractopamine supplementation periods. The pH, L* color and a* color parameters of the filet post-slaughter and the L* color of the filet post-thawing were significantly influenced by the period of supplementation with the additive in the diet. Cooking loss was significantly lower in the fillet of fish that received ractopamine. Ractopamine supplementation at the level of 20 mg kg-1 in the diet of pintado amazônico for 15, 30, or 45 days does not alter their production performance, carcass yield, or hematological parameters, but changes the qualitative traits of their filet.

Effect of regulated deficit irrigation on pomegranate fruit quality at harvest and during cold storage

The effect of regulated deficit irrigation (RDI) strategies on fruit yield and quality at harvest and during cold storage at 5 °C of Wonderful pomegranates was studied. Three irrigation strategies were tested: control strategy (C) using farm irrigation with 100% of crop evaporation (ETc), and two RDI strategies where 25% of ETc was applied during flowering and fruit set (RDI 1) and during the final phase of fruit growth and ripening (RDI 2). The RDI 1 approach reduced the pomegranate yield. Conversely, the RDI 2 allowed a similar pomegranate yield to C treatment. Fruit morphological characteristics were not affected by RDI at harvest and during cold storage. Both RDI strategies resulted in fruits with higher aril percentages. Physicochemical parameters of fruits at harvest and during cold storage were not affected by irrigation strategies, except the total soluble solids content that was higher in fruits from RDI 2 than in those from C and RDI 1. During storage, a reduction of the fruit weight and size was observed, particularly in the first 15 days of storage. The total soluble solids content and titratable acidity decrease, while pH increases with storage. At harvest and during cold storage, the total phenolic content and antioxidant activity evaluated by ABTS+ assay did not differ among irrigation treatments, whereas using a FRAP assay fruits from RDI trended to have higher antioxidant activity than those from C treatment. Total phenolic content and ABTS+ values were lower at the final of storage time than on the first day. Present results showed that RDI strategies applied in a final phase of fruit growth and ripening could be a convenient irrigation strategy for commercial Wonderful orchards in the south of Portugal, allowing improve the fruit quality with a water saving of 14.6%.

Identification of dental calcification stages as a predictor of skeletal development phase.

ABSTRACTObjective: This study aimed to establish a correlation between the stages of tooth calcification of mandibular canines and second molars with the phases of skeletal development. Methods: In a consecutive series of panoramic, cephalometric and hand-wrist radiographs of 113 individuals (60 females and 53 males) with an average age of 12.24 ± 1.81 years, the stages of mandibular canine and second molar calcification, cervical vertebrae maturation indicators (CVMI) and skeletal maturity indicators (SMI) were classified. The variables were correlated by means of the Spearman’s Rank test: chronological age, SMI, CVMI and tooth calcification stages. In order to assess whether the CVMI and tooth calcification stages were significant predictors of the SMI, an ordinal regression analysis was carried out. Results: The stages of CVMI (OR = 16.92; CI 95% = 6.45-44.39; p< 0.001) and calcification of the second molars (OR = 3.22; CI 95% = 1.50-6.92; p= 0.003) were significant predictors of SMI, however similar result was not observed for canines (OR = 0.52, CI 95% = 0.18-1.54; p= 0.239). Calcification stage E for boys, and E and F for girls corresponded to the pre-peak phase of pubertal growth. Stages G and H for boys, and F and G for girls coincided with peak of growth. In the final growth phase, the majority of second molars presented with root apex closure (stage H). Conclusion: The stages of calcification of the second molar may be considered predictors of the stage of skeletal development in the population studied.

Zinc supports transcription and improves meiotic competence of growing bovine oocytes.

In the last years, many studies focused on the understanding of the possible role of zinc in the control of mammalian oogenesis, mainly on oocyte maturation and fertilization. However, little is known about the role of zinc at earlier stages, when the growing oocyte is actively transcribing molecules that will regulate and sustain subsequent stages of oocyte and embryonic development. In this study, we used the bovine model to gain insights into the possible involvement of zinc in oocyte development. We first mined the EmbryoGENE transcriptomic dataset, which revealed that several zinc transporters and methallothionein are impacted by physiological conditions throughout the final phase of oocyte growth and differentiation. We then observed that zinc supplementation during in vitro culture of growing oocytes is beneficial to the acquisition of meiotic competence when subsequently subjected to standard in vitro maturation. Furthermore, we tested the hypothesis that zinc supplementation might support transcription in growing oocytes. This hypothesis was indirectly confirmed by the experimental evidence that the content of labile zinc in the oocyte decreases when a major drop in transcription occurs in vivo. Accordingly, we observed that zinc sequestration with a zinc chelator rapidly reduced global transcription in growing oocytes, which was reversed by zinc supplementation in the culture medium. Finally, zinc supplementation impacted the chromatin state by reducing the level of global DNA methylation, which is consistent with the increased transcription. In conclusion, our study suggests that altering zinc availability by culture-medium supplementation supports global transcription, ultimately enhancing meiotic competence.

Evidence of nutrient by genotype interaction in meat quail strains through reaction norm models

Quails raised under different nutrition levels could lead to genetic variation affecting the genetic gain due to the genotype by environment (GxE) interaction. Thus, reaction norm models were carried out to evaluate the GxE interaction of two meat quails’ strains (LF1 and LF2) raised under different tryptophan:lysine ratios for body weight and carcass traits. A total of 590 and 587 meat quails for LF1 and LF2, respectively, were raised under five different tryptophan:lysine ratios (0.15, 0.20, 0.25, 0.30 and 0.35) in final growth phase diet. The tryptophan:lysine ratios exhibited an important influence on the genetic parameters of body weight and carcass traits. Heritability and additive genetic variance estimates increased as tryptophan: lysine ratios increased, except for body weight at 28 and 35 days in LF2 strain. Lower correlation estimates and re-ranking of estimated breeding values were observed for slaughter weight, breast weight and thigh + leg yield in LF2 strain and for carcass yield in LF1 strain. It indicated a striking effect of amino acid ratios in quail carcass traits. The genetic evaluation and selection for carcass trait in quail strains raised under different tryptophan:lysine ratios should be considered in quail breeding programs.

The Effect of Slaughter Age and the Diet in the Final Growth Phase of Poulards on Productivity and Meat Quality

Abstract The aim of the studies was to evaluate the effect of the diet in the final growth phase and slaughter age of poulards on productivity, dressing percentage, carcass colour and physicochemical properties of meat. The studies were conducted on 78 hybrids obtained by crossing Ross 308 broiler roosters with Rhode Island Red (R-11) dual-purpose laying hens (♂Ross 308 × ♀R-11). Birds were assigned to 3 groups: control group (K) fed starter, grower and finisher diets without addition of dairy products, experimental group S offered a finisher diet supplemented with 4% whey, and experimental group M receiving 4% milk powder supplement in the final growth phase. Spaying procedure was carried out at 8 weeks of age. Poulards were slaughtered at 18 and 20 weeks of age. The studies showed that by using hybrids of dual-purpose hens with broiler roosters for production of poulards, rearing period could be shortened to 18 weeks, and the obtained meat had comparable or even better quality traits compared with poulards slaughtered at 20 weeks of age. Supplementation of poulard diet with 4% whey or milk powder in the final growth phase had a beneficial effect mostly on improvement of sensory properties of meat. There were not many significant differences in meat quality between poulards fed the diet supplemented with milk and whey but instead there were differences between these groups and the control group fed the diet without these additives.

Effect of elicitation and precursor feeding on accumulation of 20-hydroxyecdysone in Achyranthes aspera Linn. cell suspension cultures.

20-Hydroxyecdysone is one of the most common ecdysteroids in plants with potential therapeutic applications. In this study, cell suspension cultures of Achyranthes aspera were raised in shake flasks to investigate the production of 20-hydroxyecdysone. The quantification and characterization of 20-hydroxyecdysone in the cultures were done by High performance liquid chromatography (HPLC) and Liquid Chromatography-quadrupole time-of- flight mass spectrometry (LC-Q-TOF) analyses. For raising the suspension, calli initiated from in vitro grown leaf explants were cultured in liquid Murashige and Skoog (MS) medium augmented with combinations of 2, 4-dichlorophenoxyacetic acid (1mgL-1) and α-naphthaleneacetic acid (1mgL-1). Maximum growth index of the cell suspension was 9.9, which was achieved during 20th day of culture (final phase of exponential growth). At this stage, the biomass accumulated was 1.09±0.09g dry weight (DW) and the 20-hydroxyecdysone concentration was 0.24mgg-1 DW. Eliciting the cultures with 0.6mM Methyl jasmonate for 6days; enhanced the production of 20-hydroxyecdysone production to 0.35mgg-1 DW. By augmenting the cultures with the precursors namely cholesterol (10mgL-1) and 7-dehydrocholesterol (10mgL-1), production of 20-hydroxyecdysone was boosted to 0.31mgg-1 DW and 0.28mgg-1 DW respectively.

Expression patterns of the Stim1 protein during different stages of mouse tooth development

Objectives: Stromal interaction molecule 1 (STIM1), a calcium sensor protein, was shown to be significantly up-regulated during the maturation stage of amelogenesis. This result highlighted a potentially important role for STIM1 in calcium ion handling during the latter stages of enamel formation. Our work aimed to study in detail the expression patterns of STIM1 during all stages of mouse tooth development.Material and Methods: C57BL mice were used for all the experiments in this study. The embryos were obtained from time mated pregnant mice. Embryonic day 0 (E0) was designated as the day on which a vaginal plug was confirmed. Embryos at E12.5, E14.5, E15.5 and pups at PNO, PN4, PN8 and PN14 were used to describe different developmental stages of incisors (E12.5 – E15.5) and molars (PN0 – PN14).Results: Stim1 signals start to be detectable in the cells of the enamel organ at the beginning of the cap stage. Its expression was similar between the incisor and molar tooth germs. Odontoblasts appeared completely negative, while there was some strong expression in the middle of the dental papilla. Conclusion: In our study, we have shown that Stim1 was most highly expressed in the ameloblasts during the maturation stage. This highly expression of Stim-1 at the maturation stages of enamel development supports the contention that stored operated calcium channels are central to Ca2+ transport/extrusion mechanism during the final phase of enamel crystal growth.

Continuous measurements of SiF4 and SO2 by thermal emission spectroscopy: Insight from a 6-month survey at the Popocatépetl volcano

The processes linked with the emplacement and growth/destruction of a lava dome are of prime importance to understand the stability of such extrusions and assess the associated risks for local populations. During the last couple of decades, ground and space-based spectroscopic techniques have been developed to monitor such processes from a safe distance. Such approaches significantly improved our knowledge about the relationship between the chemical composition of the volcanic gas plumes and both the deep and shallow volcanic processes leading to the different types of explosive activity. The potential of the ground-based thermal emission Fourier Transform Infrared spectroscopy (FTIR) remained under-exploited due to the difficulty to properly handle the radiative-transfer phenomena. Despite the drawbacks in the complex analytical requirements, this method enables to continuously monitor (day and night) with a high temporal resolution (1meas/3min), relevant gas species such as SO2 and SiF4 in the volcanic plumes. Previous studies have related the temporal variations of the SiF4/SO2 ratio in volcanic plumes to the onset of vulcanian explosions. This study reports a 6-month SO2, SiF4, and SiF4/SO2 time series (from January to June 2015) of the Popocatepetl's gas plume obtained from FTIR thermal emission spectroscopic measurements. The infrared spectra were analyzed using the SFIT4 radiative transfer and inverse model, which we have adapted for this application. We obtained highly variable SiF4/SO2 ratios with a mean value of 3.6×10−4, with the highest values (around 3×10−3) measured during the final phase of a lava dome growth (February–March 2015). The rapid SiF4/SO2 variations were more carefully explored and compared for the first time with the seismic activity. A remarkable coincidence between sharp SiF4/SO2 rises and the seismic events are evidenced here.

Effects of season on placental, foetal and neonatal development in horses

Seasonal changes in metabolic rate have been shown in horses and we hypothesized that this leads to the birth of smaller foals early in the year. Mares and their foals were assigned to three groups by day of foaling within the year (e.g. 1 January = day 1): Group 1 (n = 10) day 40–65, group 2 (n = 8) day 67–92, group 3 (n = 9) day 94–121. Groups did not differ with regard to parity. In foals, height at withers and body weight were determined on days 1–5 and weekly until 12 weeks of age. Chest circumference, distances fetlock to carpus, carpus to elbow, poll to nose and crown-rump length were determined on day 5 and weekly until 12 weeks of age. Placental weight (p < 0.05) and surface (p < 0.01) were lower in mares of group 1 than in groups 2 and 3. Foal weight and length measurements increased over time (p < 0.001). Height at withers was consistently lower in group 1 than in groups 2 and 3 (p < 0.05) while foal weight did not differ among groups. Fetlock to carpus, carpus to elbow (both p < 0.01) and poll to nose length (p < 0.05) were lower in group 1 than in groups 2 and 3. Neither gestation length nor sex ratio of foals differed among groups. In conclusion, foetal size is reduced when the final growth phase coincides with the winter months. This also impacts neonatal growth during the first three months of life.

Phytoremediation of heavy metal contaminated buriganga riverbed sediment by Indian mustard and marigold plants

Locally available Indian mustard (Brassica juncea) and Marigold (Tagetes patula) plants can be used in a cost‐effective, environment‐friendly approach for treatment of landfills developed using heavy metal contaminated Buriganga riverbed sediments. In this study heavy metal uptake by Indian mustard and Marigold plants from heavy metal contaminated Buriganga riverbed sediments were assessed. Initial characterization of the Buriganga riverbed sediments showed concentrations of chromium, lead, copper, and zinc in the sediments higher than the toxicity reference values given for these heavy metals in soil for terrestrial plants, and soil invertebrate. The average background concentration of chromium, lead, copper, and zinc in the Buriganga riverbed sediments were found to be 141.5 mg/kg, 34.9 mg/kg, 38.7 mg/kg, and 287.5 mg/kg, respectively. Hence the newly developed area using such landfills poses a great threat to the surrounding flora and fauna. It was observed that both Indian mustard and Marigold plants accumulated these heavy metals in different parts of the plant from the contaminated sediments and were able to maintain a growth rate of more than 90% compared to that in noncontaminated soil. The results indicated rapid phytoextraction of most heavy metals by the Indian mustard during its final growth phase, whereas rapid phytoextraction of most heavy metals was observed in case of Marigold in its initial growth phase. Total chromium, lead, copper, and zinc uptakes (in mg/kg of plant dry weight) by Indian mustard plant in 12 weeks were 102.6, 28.9, 53, and 1861.5, respectively. The uptakes (in mg/kg of plant dry weight) of the same heavy metals by Marigold plant in 12 weeks were found to be 112.3, 104.25, 82.5, and 716.75, respectively. Marigold showed higher uptake efficiency for chromium, lead, and copper, while Indian mustard was found to be more efficient for zinc uptake. Hence both of these plants can be appropriately used as necessary for treating heavy metal contaminated landfills developed using Buriganga riverbed sediments. © 2015 American Institute of Chemical Engineers Environ Prog, 35: 117–124, 2016

Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor

BackgroundLeptospirosis is a zoonose that is increasingly endemic in built-up areas, especially where there are communities living in precarious housing with poor or non-existent sanitation infrastructure. Leptospirosis can kill, for its symptoms are easily confused with those of other diseases. As such, a rapid diagnosis is required so it can be treated effectively. A test for leptospirosis diagnosis using Leptospira Immunoglobulin-like (Lig) proteins is currently at final validation at Fiocruz.ResultsIn this work, the process for expression of LigB (131-645aa) in E. coli BL21 (DE3)Star™/pAE was evaluated. No significant difference was found for the experiments at two different pre-induction temperatures (28°C and 37°C). Then, the strain was cultivated at 37°C until IPTG addition, followed by induction at 28°C, thereby reducing the overall process time. Under this condition, expression was assessed using central composite design for two variables: cell growth at which LigB (131-645aa) was induced (absorbance at 600 nm between 0.75 and 2.0) and inducer concentration (0.1 mM to 1 mM IPTG). Both variables influenced cell growth and protein expression. Induction at the final exponential growth phase in shaking flasks with Absind = 2.0 yielded higher cell concentrations and LigB (131-645aa) productivities. IPTG concentration had a negative effect and could be ten-fold lower than the concentration commonly used in molecular biology (1 mM), while keeping expression at similar levels and inducing less damage to cell growth. The expression of LigB (131-645aa) was associated with cell growth. The induction at the end of the exponential phase using 0.1 mM IPTG at 28°C for 4 h was also performed in microbioreactors, reaching higher cell densities and 970 mg/L protein. LigB (131-645aa) was purified by nickel affinity chromatography with 91% homogeneity.ConclusionsIt was possible to assess the effects and interactions of the induction variables on the expression of soluble LigB (131-645aa) using experimental design, with a view to improving process productivity and reducing the production costs of a rapid test for leptospirosis diagnosis.

Accretion in giant planet circumplanetary discs

During the final growth phase of giant planets, accretion is thought to be controlled by a surrounding circumplanetary disk. Current astrophysical accretion disk models rely on hydromagnetic turbulence or gravitoturbulence as the source of effective viscosity within the disk. However, the magnetically-coupled accreting region in these models is so limited that the disk may not support inflow at all radii, or at the required rate. Here, we examine the conditions needed for self-consistent accretion, in which the disk is susceptible to accretion driven by magnetic fields or gravitational instability. We model the disk as a Shakura-Sunyaev $\alpha$ disk and calculate the level of ionisation, the strength of coupling between the field and disk using Ohmic, Hall and Ambipolar diffusevities for both an MRI and vertical field, and the strength of gravitational instability. We find that the standard constant-$\alpha$ disk is only coupled to the field by thermal ionisation within $30\,R_J$ with strong magnetic diffusivity prohibiting accretion through the bulk of the midplane. In light of the failure of the constant-$\alpha$ disk to produce accretion consistent with its viscosity we drop the assumption of constant-$\alpha$ and present an alternate model in which $\alpha$ varies radially according to the level magnetic turbulence or gravitoturbulence. We find that a vertical field may drive accretion across the entire disk, whereas MRI can drive accretion out to $\sim200\,R_J$, beyond which Toomre's $Q=1$ and gravitoturbulence dominates. The disks are relatively hot ($T\gtrsim800\,$K), and consequently massive ($M_{\text{disk}}\sim0.5\,M_J$).

Evaluation of treatment for camptodactyly: retrospective analysis on 40 fingers

Objectiveto retrospectively assess the results from cases treated in the hand surgery service, starting from a preestablished protocol; and to conduct a critical analysis on the results achieved, with separation of the cases into their respective subgroups. Methodstwenty-three patients and a total of 40 fingers were evaluated between January 2004 and December 2011. We correlated the altered anatomical structures found in the cases that underwent the surgical procedure and its results, with regard to both conservative and surgical treatment, emphasizing the main indications. Resultsthe results were analyzed using the Sierget method of the Mayo Clinic. Conclusionwe observed that the cases of camptodactyly of the little finger alone in the flexible form (>60°) that underwent surgical treatment uniformly presented excellent results. In the rigid forms, our observations indicated that there were benefits comprising gains of extension and correction of the deformity. However, the range of motion with active flexion in the proximal interphalangeal joint was always partial. With evolution over time, some cases presented some loss of the gain previously achieved, which corroborates the need for continual vigilance during the follow-up, with systematic use of braces until the final phase of skeletal growth.

Avaliação do tratamento da camptodactilia: análise retrospectiva de 40 dígitos

Objectiveto retrospectively assess the results from cases treated in the hand surgery service, starting from a preestablished protocol; and to conduct a critical analysis on the results achieved, with separation of the cases into their respective subgroups. Methods23 patients and a total of 40 fingers were evaluated between January 2004 and December 2011. We correlated the altered anatomical structures found in the cases that underwent the surgical procedure and its results, with regard to both conservative and surgical treatment, emphasizing the main indications. Resultsthe results were analyzed using the Sierget method of the Mayo Clinic. Conclusionwe observed that the cases of camptodactyly of the little finger alone in the flexible form (> 60°) that underwent surgical treatment uniformly presented excellent results. In the rigid forms, our observations indicated that there were benefits comprising gains of extension and correction of the deformity. However, the range of motion with active flexion in the proximal interphalangeal joint was always partial. With evolution over time, some cases presented some loss of the gain previously achieved, which corroborates the need for continual vigilance during the follow‐up, with systematic use of braces until the final phase of skeletal growth.

Genomic growth curves of an outbred pig population.

In the current post-genomic era, the genetic basis of pig growth can be understood by assessing SNP marker effects and genomic breeding values (GEBV) based on estimates of these growth curve parameters as phenotypes. Although various statistical methods, such as random regression (RR-BLUP) and Bayesian LASSO (BL), have been applied to genomic selection (GS), none of these has yet been used in a growth curve approach. In this work, we compared the accuracies of RR-BLUP and BL using empirical weight-age data from an outbred F2 (Brazilian Piau X commercial) population. The phenotypes were determined by parameter estimates using a nonlinear logistic regression model and the halothane gene was considered as a marker for evaluating the assumptions of the GS methods in relation to the genetic variation explained by each locus. BL yielded more accurate values for all of the phenotypes evaluated and was used to estimate SNP effects and GEBV vectors. The latter allowed the construction of genomic growth curves, which showed substantial genetic discrimination among animals in the final growth phase. The SNP effect estimates allowed identification of the most relevant markers for each phenotype, the positions of which were coincident with reported QTL regions for growth traits.

Effect of sustained and regulated deficit irrigation on fruit quality of pomegranate cv. ‘Mollar de Elche’ at harvest and during cold storage

The effect of several irrigation strategies on fruit quality at harvest and during cold storage at 5°C of ‘Mollar de Elche’ pomegranates (Punica granatum L.) was studied for three seasons. Irrigation treatments consisted of a control irrigated at 100% of crop evapotranspiration (ETc), a sustained deficit irrigation (SDI) where trees were irrigated at 50% of the ETc during the entire season, and three regulated deficit irrigation (RDI) treatments. In the RDI regimes, severe water restrictions (25% ETc) were applied during one of three phases: flowering and fruit set (RDIfl.-fr.set), fruit growth (RDIfr.gr) or the final phase of fruit growth and ripening (RDIripe). Results showed that after 8 or 19 weeks at 5°C plus 7 days of shelf life at 20°C, some fruit quality attributes such as the soluble solids content (SSC), anthocyanins and fruit colour were enhanced by deficit irrigation. Higher SSC and more reddish colouration in the fruit peel were noted at harvest and during cold storage in pomegranates from SDI and RDIripe. On the other hand, higher juice anthocyanins content were obtained in the RDIfr.gr. fruit. Control fruit showed greater susceptibility to physiological disorders manifested as peel pitting, blemishes and sinking, and to weight loss with respect to deficit irrigation samples. Weight loss increased with storage time and SDI and RDIripe showed lower weight loss compared to the control treatment. Deficit irrigation, depending on the phenological period when water shortage is applied, can be then used as a field practice to control fruit ripening timing, enhance pomegranate fruit composition and improve fruit postharvest performance.

Halogenated nucleotide labeling of nascent RNAs reveals dynamic transcription in growing pig oocytes

Germ cells differentiate into oocytes in females and are arrested at the first meiotic prophase. However, during arrest, oocytes undergo a growth phase leading to a dramatic increase in size, which is under control of transcription events. In the current study, we examined the transcriptional activity of growing pig oocytes using an immunocytochemical approach. Our data showed that fluorouridine (FU), a halogenated nucleotide, can be successfully incorporated into synthesizing RNAs and detected using a specific monoclonal antibody. Using this method, we identified dynamic changes in transcriptional activity patterns in growing pig oocytes. Oocytes obtained from small follicles exhibited the highest level of transcription, while at the final phase of growth, transcription was no longer detected. These transcriptional changes were concomitant with chromatin compaction resulting in a tightly packed ring-like chromatin conformation surrounding the nucleolar structure. Also, FU incorporation appeared sensitive to the biochemical manipulation of transcription, because transcriptional inhibitors induced a decrease in signal intensity from FU labeling and transcriptional activation caused an increase in FU signal intensity. Our data collectively support that a direct link exists between chromatin configuration and transcriptional activity in pig oocytes, and support the suitability of FU for studies on transcription-related events in mammalian oocytes.

RNA polymerase II transcriptional silencing in growing and fully grown germinal vesicle oocytes isolated from gonadotropin‐stimulated and non‐stimulated gilts

Global transcription silencing occurs in the oocyte during its final phase of growth. The particular mechanism of this silencing is not well understood. Here, we investigated the silencing of RNA polymerase II transcription in porcine oocytes. First, we investigated the transcriptional activity of germinal vesicle oocytes derived from stimulated and non-stimulated gilts, but no transcriptional activity was observed. Second, we focused on the fate of RNA polymerase II in growing and fully grown oocytes. Active and inactive forms of RNA polymerase II were detected in growing oocytes by immunofluorescence and Western blots. In contrast, only the inactive form of RNA polymerase II was detected in fully grown oocytes. To evaluate if the inactive form of RNA polymerase II is released from DNA, the oocytes were subsequently permeabilized and fixed in one step. After this modified fixation protocol, the immunofluorescent labeling was negative in fully grown oocytes, but remained unchanged (positive) in growing oocytes. These results indicate that the inactive form of RNA polymerase II is not bound to DNA during the oocyte growth. Finally, based on Western blot analysis of different stages of oocyte maturation, the inactive form of RNA polymerase II was detected in metaphase I but not in metaphase II. Our study confirmed the global transcription silencing of fully grown oocytes. Compared with other mammalian species (e.g., mouse), the mechanism of RNA polymerase II silencing in porcine oocytes seems to be similar, despite some differences in dynamics.