Characterization of polygalacturonases produced by the endophytic fungus Penicillium brevicompactum in solid state fermentation - SSF

Polygalacturonases belong to the family of pectinases, enzymes that are in high demand in industry because of their many different applications. This study therefore sought to examine the production of polygalacturonases using an endophytic fungus, Penicilium brevicompactum, isolated from Baccharis dracunculifolia D.C. (Asteraceae) through semi solid fermentation using orange peels and citric pectin 2% as base substrate, supplemented with different carbon sources. After the fermentation process, the enzyme was characterized. The results showed that the micro-organism was able to use a wide range of carbon sources, but with polygalacturonase activity varying with each source. The highest yield, however, was achieved after 30 hours of incubation in the presence of 4% of galactose and 2% of pectin. Studies on the characterization of the polygalacturonase revealed that the optimal temperature of this enzyme is 72°C and that it maintains 60 and 15% of its maximum activity when incubated for 2 hours at 40 and 90°C, respectively. The optimal pH for the activity of the enzyme was 4.6. The enzyme retained 65 and 30% of its maximum activity when incubated at pH 3.5 and 9.5, respectively, for 24 hours at ambient temperature. The enzyme activity was stimulated by Mg2+ ions. On the other hand, it was inhibited by the ions Cs+2, Hg+2, Li+2 and Sr+2. The ions Zn+2 and Cu+2 inhibited it by 94% and 69%, respectively

Localization of DNA methyltransferase-1 during oocyte differentiation, in vitro maturation and early embryonic development in cow

DNA methyltransferase-1 (Dnmt1) is involved in the maintenance of DNA methylation patterns and is crucial for normal mammalian development. The aim of the present study was to assess the localization of Dnmt1 in cow, during the latest phases of oocyte differentiation and during the early stages of segmentation. Dnmt1 expression and localization were assessed in oocytes according to the chromatin configuration, which in turn provides an important epigenetic mechanism for the control of global gene expression and represents a morphological marker of oocyte differentiation. We found that the initial chromatin condensation was accompanied by a slight increase in the level of global DNA methylation, as assessed by 5-methyl-cytosine immunostaining followed by laser scanning confocal microscopy analysis (LSCM). RT-PCR confirmed the presence of Dnmt1 transcripts throughout this phase of oocyte differentiation. Analogously, Dnmt1 immunodetection and LSCM indicated that the protein was always present and localized in the cytoplasm, regardless the chromatin configuration and the level of global DNA methylation. Moreover, our data indicate that while Dnmt1 is retained in the cytoplasm in metaphase II stage oocytes and zygotes, it enters the nuclei of 8-16 cell stage embryos. As suggested in mouse, the functional meaning of the presence of Dnmt1 in the bovine embryo nuclei could be the maintainement of the methylation pattern of imprinted genes. In conclusion, the present work provides useful elements for the study of Dnmt1 function during the late stage of oocyte differentiation, maturation and early embryonic development in mammals

Reductions in the number of mid-sized antral follicles are associated with markers of premature ovarian senescence in dairy cows

High-producing dairy cows are subfertile; however, the mechanisms responsible for the decreased fertility are unknown. The aim of the present study was to test the hypothesis that culled dairy cows (4\u20138 years old) characterised by \u2018Lo\u2019 ovaries (i.e. those with <10 mid-antral follicles) are affected by premature ovarian senescence. Cows in which both ovaries were \u2018Lo\u2019 ovaries represented 5% of the total population analysed, and exhibited reduced ovarian size (P < 0.001) and increased perifollicular stroma (P < 0.05) compared with age-matched controls (i.e. cows in which both ovaries had >10 mid-antral follicles; \u2018Hi\u2019 ovaries). The total number of follicles, including healthy and atretic primordial, primary, secondary and small antral follicles, was lower in Lo ovaries (P < 0.01). Interestingly, the primordial follicle population in Lo ovaries was lower (P < 0.05) than in the control. Finally, the follicular fluid of mid-antral follicles from Lo ovaries had reduced oestradiol and anti-M\ufcllerian hormone levels (P < 0.05), but increased progesterone concentrations (P < 0.05). Together, these data account for the reduced fertility of cows with Lo ovaries and are in agreement with previous observations that oocytes isolated from Lo ovaries have reduced embryonic developmental competence. Cows with a specific Lo ovary condition may represent a suitable model to address the causes of low fertility in high-yielding dairy cows, as well as the condition of premature ovarian aging in single-ovulating species

Expression of progesterone receptor membrane component-1 in bovine reproductive system during estrous cycle

Several reports suggest the participation of progesterone receptor membrane component 1 (PGRMC1) in progesterone signaling in the reproductive system. This study aimed at investigating the presence and localization of PGRMC1 in bovine ovary, oviduct and uterus, during the follicular and luteal phases of the estrous cycle. In the ovary, PGRMC1 has been detected in surface germinal epithelium, granulosa cells, theca cells and in the germinal vesicle of the oocytes at all stages of folliculogenesis. In the corpus luteum the expression of PGRMC1 was influenced by the stage of the estrous cycle. In the oviducts and in the uterus horns, PGRMC1 was immunolocalized in the luminal epithelium, in the muscle layer cells and in the endothelial cells. In the uterus, PGRMC1 was intensely localized also in the glandular endometrium. However, in the oviducts and in the uterus horns, the localization of PGRMC1 was independent on the stage of the estrous cycle and on whether evaluating the ipsilateral or the contralateral organ. In conclusion, the present immunohistochemical study showed that PGRMC1 is located in various compartments of the bovine female reproductive organs. With the exception of the corpora lutea, PGRMC1 localization showed similar pattern during different stages of the estrous cycle

Characterization of alpha-Amylase Produced by the Endophytic Strain of Penicillium digitatum in Solid State Fermentation (SSF) and Submerged Fermentation (SmF)

Α-Amylases are enzymes responsible for breaking the α-1.4 bond in polysaccharides with three or more glucose units, occupying the second place in the most widely employed enzymes in industry in the world. The objective of this study was to compare the yields of α-amylase produced by the endophytic fungus, Penicillium digitatum, strain D1-FB, isolated from Baccharis dracunculifolia D.C. (Asteraceae), through the solid state fermentation (SSM) and submerged fermentation (SmF) processes, in addition to characterizing the produced enzyme. The two fermentations were conducted for 120 hours, taking samples every 24 hours to obtain the peaks of production. The enzymes were characterized according to their optimal pH and temperature for performance and stability regarding the incubation in the presence of ions, variations in pH and temperature. The maximum yield of the enzyme was observed with SSF, using rice bran as substrate after 72 hours of fermentation, with 1,625 U/mL. The α-amylase had an optimal pH at 6.5 and optimal temperature at 45°C. All the ions resulted in a decrease in the activity of α-amylase in the concentration of 5mM. The enzyme proved to be quite stable in a pH range of 6.0 to 7.5 and up to the temperature of 37°C, but it presented great drops in activity with temperatures above 45°C and in the presence of ions at the concentration of 5 mM

Role of gap junction-mediated communications in regulating large-scale chromatin configuration remodeling and embryonic developmental competence acquisition in fully grown bovine oocyte

PURPOSE: This study was aimed to test the hypothesis that gap junction mediated communications (GJC) are required to allow the progressive chromatin configuration remodeling (from GV1 to GV3) process to occur in fully grown oocytes in order to gain the final step of developmental competence acquisition, and that a premature disruption of GJC can alter this process. METHODS: Bovine cumulus-oocytes complexes collected from medium antral follicles were cultured for 2, 4, 6 and 8 h in the presence of 10-4 IU/ml of r-hFSH and with 2 mM of the non-selective PDE inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) to prevent meiotic resumption. GJC functionality and chromatin configuration were monitored during the culture period. After meiotic arrest, the developmental capability of oocytes was assessed after IVM and IVF. RESULTS: IBMX was effective in significantly sustaining GJC up to 6 h and maintaining meiotic arrest, when compared to control group. Moreover, the percentage of oocytes with less condensed chromatin (GV1) decreased within 4 h of culture, while the proportion of GV2 oocytes gradually increased up to 6 h. Interestingly, a decline in the proportion of GV2 oocytes and an increase in the proportion of GV3 oocytes were observed after 6 h of culture, when the major drop of GJC occurred. On the contrary, when GJC were uncoupled by adding 3 mM of 1-heptanol or through cumulus cells removal, chromatin condensation occurred rapidly throughout the culture period, more promptly in denuded oocytes. Moreover, the maintenance of GJC during meiotic arrest was accompanied by a significant increase of developmental competence compared to the control, as indicated by a higher percentage of hatched blastocysts and blastocyst cell number. CONCLUSIONS: Altogether, our data indicate that both paracrine and junctional mechanisms are involved in modulating large-scale chromatin structure during the final phase of oocyte differentiation

The application of stem cells from different tissues to cartilage repair

The degeneration of articular cartilage represents an ongoing challenge at the clinical and basic level. Tissue engineering and regenerative medicine using stem/progenitor cells have emerged as valid alternatives to classical reparative techniques. This review offers a brief introduction and overview of the field, highlighting a number of tissue sources for stem/progenitor cell populations. Emphasis is given to recent developments in both clinical and basic sciences. The relative strengths and weaknesses of each tissue type are discussed

The endothelial nitric oxide synthase/nitric oxide system is involved in the defective quality of bovine oocytes from low mid-antral follicle count ovaries

In a previous survey concerning cows of reproductive age, we demonstrated that oocytes isolated from ovaries with 10 medium antral follicles (High ovaries, Hi). The aim of the present study was to evaluate whether a defective endothelial nitric oxide synthase/nitric oxide (eNOS/NO) system and vasculature in healthy medium antral follicles is likely to reduce oocyte competence from Lo ovaries. Thus, experiments were conducted to: (1) immunolocalize eNOS protein during folliculogenesis; (2) quantify eNOS protein/vasculature in the follicle wall; and (3) verify if NO donor, S-nitroso acetyl penicillamine (SNAP) administration during in vitro maturation affects developmental competence of oocytes isolated from Lo ovaries. Endothelial-NOS protein was detected in granulosa and theca cells, as well as in blood vessels from primordial to antral follicles. Quantitative analysis indicated that in medium antral follicles from Lo ovaries, eNOS protein expression and vasculature were reduced (P < 0.05). The addition of SNAP improved blastocyst and hatching rates of oocytes from Lo ovaries, promoting a percentage similar to oocytes from Hi ovaries, and reduced the percentage of apoptotic nuclei in in vitro-produced blastocysts (P < 0.05). Results from our study suggest that in bovine ovaries with low mid antral follicle number, a defective eNOS/NO system is related to a reduced follicle vasculature and may impact oocyte quality, thus inducing a premature decline of fertility

Morphological markers to select populations of oocytes with different cultural needs for dedicated pre-maturation protocols

Oocyte’s chromatin gradually becomes more compacted during the final stage of oocyte development and the level of chromatin compaction is considered a marker of oocyte differentiation [Luciano et al, 2014]. Moreover, several studies demonstrate that in vitro pre-maturation treatments (Pre-IVM), aimed to improve the developmental capability of immature oocytes, might behave differently depending on the oocyte metabolic status, when it is isolated from follicle [Luciano et al., 2011]. This study aims at identifying correlations between cumulus-oocyte complex (COC) morphology and oocyte chromatin configuration and secondly at testing the hypothesis that only fully grown oocytes at earlier stages of differentiation with loosely compacted chromatin  (GV1) can benefit from Pre-IVM treatment.   COCs were collected from bovine 2-6mm ovarian follicles, and further divided in three groups according to their morphology (Class-1, 2 and 3) as previously described [Blondin & Sirard, 1995]. Analysis of chromatin configuration revealed that only Class-1 COC was enriched in GV1 oocyte, while Class-2 and 3 presented a similar distribution of GV1, GV2 and GV3 oocytes, where GV2 and 3 oocytes are characterized by increased chromatin compaction [Lodde et al., 2007]. Then COCs were divided into two groups, one containing Class-1 COCs and the other containing Class-2 and 3 COCs and subjected to pre-IVM for 6 hours in presence of cilostamide and 10-4 UI/ml rhFSH. Finally, COCs underwent standard in vitro maturation (IVM) for 22 hours, in vitro fertilization and embryo culture. Blastocyst rate and embryos cell number were assessed at day 7. Pre-IVM positively affected developmental competences of Class-1, while in Classes 2 and 3 Pre-IVM had detrimental effects.In conclusion COCs morphology could be used as a non-invasive approach to select population of oocyte with different cultural needs. These data could be useful in setting-up dedicated IVM protocols considering specific genes and pathways to improve IVP efficiency

Hypoxia as a stimulus upon neonatal swinemeniscus cells: highway to phenotypic maturation of meniscal fibro-chondrocytes?

Menisci are essential structures in the knee joint where they cover fundamental biomechanical and protective roles (1-3). Menisci are characterized by a peculiar structure that, on one hand, allow them to perform their particular role in the stifle joint, but simultaneously make them a very challenging structure to deal with (2). Immature menisci are featured by numerously elongated cells (fibrocytes-like) in a disorganized matrix composed almost completely of collagen type I and few glycosaminoglycans (GAGs) and have a rich vascularization, on the other hand, mature and functional menisci are characterized by few round-shaped cells,a matrix rich of well ordinated collagen fibres (above all collagen type II) and GAGs, and preserve vascularization only in the outer zone (aka red zone) (1). Great interest, in both human and veterinary medicines, is reserved to the treatment of the injuries of the inner and avascular zone (aka white zone) of the meniscus: until now, there are no perfect solutions for the regeneration or the replacement of this tissue once injured (3). This work is focused on the utilization of an environmental factor like hypoxia in meniscal tissue culture, in order to evaluate if it could be utilized to improve meniscal culture with a view to tissue engineering. Ninety menisci from neonatal pigs (day 0) were harvested and cultured under two different atmospheric conditions (hypoxia with 1% O2 and normoxia) until 14 days. Samples were analysed at 0, 7 and 14 days through histochemical (Safranin-O staining), immunofluorescence and RT-PCR (Sox-9, Hif-1a, Hif-2, Collagen I and II, both methods) and biochemical (DNA, GAGs, DNA/GAGs ratio) techniques to record any possible differences in maturation of meniscal cells. Safranin-O staining allowed to show an increment in matrix deposition and round-shape \u201cfibro-chondrocytic\u201d cells quantity of hypoxia-cultured menisci respect to controls under normal atmospheric conditions. The same maturation shifting was observed by means of immunofluorescence and RT-PCR analysis, characterized by an increment of Sox-9 and collagen II, moving from day zero to 14-days under hypoxic environment, and by biochemical analysis,with an increment of DNA/GAGs ratio typical of mature meniscal tissue (characterized by few cells and much GAGs). This study shows that hypoxia can be considered as a booster to achieve meniscal cells maturation and opens considerably opportunities in the field of meniscus tissue engineering. References 1. Dai Z, et al. J Orthop Res 2013 ;31:1514-9, 2. Fox AJS, et al. Clin Anat 2015 ;28:269-87 3. Sosio C, et al. Tissue Eng Part A 2015 ;21:3-4