Evaluation of polyhydroxyalkanoate (PHA) synthesis by Pichia sp. TSLS24 yeast isolated in Vietnam.

Following the rising concern on environmental issues caused by conventional fossil-based plastics and depleting crude oil resources, polyhydroxyalkanoates (PHAs) are of great interest by scientists and biodegradable polymer market due to their outstanding properties which include high biodegradability in various conditions and processing flexibility. Many polyhydroxyalkanoate-synthesizing microorganisms, including normal and halophilic bacteria, as well as algae, have been investigated for their performance in polyhydroxyalkanoate production. However, to the best of our knowledge, there is still limited studies on PHAs-producing marine yeast. In the present study, a halophilic yeast strain isolated from Spratly Island in Vietnam were investigated for its potential in polyhydroxyalkanoate biosynthesis by growing the yeast in Zobell marine agar medium (ZMA) containing Nile red dye. The strain was identified by 26S rDNA analysis as Pichia kudriavzevii TSLS24 and registered at Genbank database under code OL757724. The amount of polyhydroxyalkanoates synthesized was quantified by measuring the intracellular materials (predicted as poly(3-hydroxybutyrate) -PHB) by gravimetric method and subsequently confirmed by Fourier transform infrared (FTIR) spectroscopic and nuclear magnetic resonance (NMR) spectroscopic analyses. Under optimal growth conditions of 35 °C and pH 7 with supplementation of glucose and yeast extract at 20 and 10 gL-1, the isolated strain achieved poly(3-hydroxybutyrate) content and concentration of 43.4% and 1.8 gL-1 after 7 days of cultivation. The poly(3-hydroxybutyrate) produced demonstrated excellent biodegradability with degradation rate of 28% after 28 days of incubation in sea water.

Excess polyspermy reduces the ability of porcine oocytes to promote male pronuclear formation after in vitro fertilization.

Male pronucleus (MPN) formation is a very important physiological event during fertilization, which affects in vitro production of transferrable embryos. The aim of this study was to find out the correlation between the number of penetrated sperm and the occurrence of failure of MPN formation in porcine oocytes. In vitro matured porcine oocytes were fertilized in vitro with frozen epididymal sperm. Two different frozen sperm lots were tested in this study, which were different in terms of polyspermy rates. The numbers and the status of penetrated sperm in oocytes were evaluated 10 h after insemination. Under high polyspermy condition, the polyspermy rate was 83.5% with an average mean of 3.5 sperms per penetrated oocyte, whereas the percentage of polyspermy was 65.5% with an average mean of 2.4 sperms per penetrated oocyte under moderate polyspermic condition. Correlation analysis revealed a negative correlation between the number of penetrated sperm and their MPN formation percentage both in the sperm lot of high polyspermy (R = -0.560, p < 0.05) and in the sperm lot of moderate polyspermy (R = -0.405, p < 0.05) which suggests that penetration of excessive spermatozoa disables the oocyte cytoplasm to promote MPN formation.

Pluripotency-associated genes reposition during early embryonic developmental stages in pigs.

We examined the allelic expression and positioning of two pluripotency-associated genes, OCT4 and SOX2, and two housekeeping genes, ACTB and TUBA, in 4- and 8-cell porcine embryos utilizing RNA and DNA fluorescence in situ hybridization (FISH) in single blastomeres. The proportion of blastomeres expressing SOX2 bi-allelically increased from 45% at the 4-cell stage to 60% at the 8-cell stage. Moreover, in 8-cell embryos, SOX2 was expressed bi-allelically in significantly more blastomeres than was the case for OCT4, and this was associated with a tendency for SOX2 alleles to move toward the nuclear interior during 4- to 8-cell transition. However, the radial location of OCT4 alleles did not change significantly during this transition. The locations of active and inactive alleles based on DNA and RNA FISH signals were also calculated. Inactive OCT4 alleles were located in very close proximity to the nuclear membrane, whereas active OCT4 alleles were more centrally disposed in the nucleus. Nevertheless, the nuclear location of active and inactive SOX2 alleles did not change in either 4- or 8-cell blastomeres. Our RNA and DNA FISH data provide novel information on the allelic expression patterns and positioning of pluripotency-associated genes, OCT4 and SOX2, during embryonic genome activation in pigs.

Selection based on morphological features of porcine embryos produced by in vitro fertilization: Timing of early cleavages and the effect of polyspermy.

The aim of this study was to examine whether a morphological approach is efficient for selecting high-quality porcine embryos produced by in vitro fertilization (IVF) under high polyspermy conditions. Frozen-thawed Meishan epididymal spermatozoa showing moderate and high polyspermy were subjected to IVF (1 × 105  sperms/ml). Under conditions of moderate polyspermy, 4-cell embryos selected at 48 hr after IVF (single selection) and 8-cell embryos selected at 79 hr after IVF from the collected 4-cell embryos (double selection) showed high developmental competence. Likewise, 4- and 8-cell embryos produced by IVF under high polyspermy conditions also showed high competence for development to blastocysts. However, blastocysts derived from high polyspermy conditions had significantly fewer cells than those produced under moderate polyspermy conditions. Furthermore, the frequency of nuclear and chromosomal abnormalities in 4- and 8-cell embryos produced under conditions of high polyspermy was significantly (p < .05) higher in comparison to moderate polyspermy conditions. These findings suggest that although high polyspermy affects the frequency of nuclear and chromosomal anomalies in porcine IVF embryos, subsequent selection based on morphological features of 4- and 8-cell embryos even under high polyspermy conditions, could be an alternative option for selecting porcine IVF embryos with high development ability.

Cryopreservation of immature oocytes of the indigeneous Vietnamese Ban Pig.

We report the cryopreservation of oocytes from Ban miniature pigs which are endemic in Vietnam. Immature cumulus-oocyte complexes were collected from antral follicles of 7-8 mo old female cyclic Ban pigs and vitrified in micro-drops. Oocyte morphology, lipid content, post-warming survival, nuclear maturation, and embryo development were compared to those of oocytes from commercially slaughtered Landrace × Large white hybrid pigs. The size of oocytes in the two breeds was similar. However, significantly lower amounts of intracellular lipid were detected in Ban oocytes. There was no difference (p > 0.05) between Ban and Landrace × Large white oocytes in percentages of post-warming survival (93.1 ± 3.4% vs. 70.7 ± 16.7%, respectively) and nuclear maturation after in vitro maturation (80.4 ± 5.1% vs. 90.0 ± 1.3% respectively). Similarly, cleavage (30.8 ± 7.8% vs. 10.3 ± 6.1%, respectively) and blastocyst development rates (9.4 ± 5.0% vs. 0.79 ± 0.79, respectively) were not different (p > 0.05) between vitrified Ban and Landrace × Large white oocytes after in vitro fertilization and embryo culture. In conclusion, high survival and maturation rates were achieved after vitrification of immature Ban oocytes and their cryo-tolerance was similar to that of Landrace × Large white oocytes, despite the difference in lipid content. We succeeded to generate reasonable rates of blastocysts from vitrified Ban oocytes by in vitro fertilization.

Sucrose assists selection of high-quality oocytes in pigs.

We investigated whether high-quality in vitro matured (IVM) oocytes can be distinguished from poor ones based on the morphological changes after treatment with hyperosmotic medium containing 0.2 mol/L sucrose in pigs. We hypothesize that IVM oocytes maintaining round shape have higher quality than mis-shapened oocytes following dehydration. Oocyte quality was verified by determining embryonic developmental competence using in vitro fertilization, nuclear transfer and parthenogenetic activation. In all cases, the round oocytes had greater (p < .05) developmental competence than that of mis-shapened oocytes in terms of blastocyst rate and total cell number in blastocysts obtained after 6 days of in vitro culture. We also confirm that round aged oocytes are higher in quality than mis-shapened aged oocytes. In an attempt to find out why high-quality oocytes maintain a round shape whereas poorer oocytes become mis-shapened following sucrose treatment, we examined the arrangement of actin microfilaments and microtubules. Abnormal organization of these cytoskeletal components was higher (p < .05) in mis-shapened oocytes compared to round oocytes after 52 hr of IVM. In conclusion, sucrose treatment helps selection of high-quality oocytes, including aged oocytes, in pigs. Abnormal cytoskeleton arrangements partly explain for low developmental competence of mis-shapened oocytes.

Expression of DNA repair genes in porcine oocytes before and after fertilization by ICSI using freeze-dried sperm.

Boar sperm freeze-dried with trehalose showed a protective effect against sperm DNA fragmentation. However, normal fertilization and embryonic development were not improved. Damaged sperm may activate maternal DNA repair genes when injected into oocytes. Therefore, we investigated the expression profile of some DNA repair genes in porcine oocytes after intra-cytoplasmic sperm injection. First, the expression levels of MGMT, UDG, XPC, MSH2, XRCC6 and RAD51 genes that are concerned with different types of DNA repair were examined in in vitro mature (IVM) oocytes injected with ejaculated sperm, or freeze-dried sperm with or without trehalose. Quantitative reverse transcription polymerase chain reaction revealed that expression of six DNA repair genes in the oocytes at 4 h after injection did not differ among the four groups. Next, we investigated the gene expression levels of these genes at different stages of maturation. The relative expression levels of UDG and XPC were significantly up-regulated in mature oocytes compared with earlier stages. Furthermore, there was an increased tendency in relative expression of MSH2 and RAD51. These results suggested two possible mechanisms that messenger RNA of DNA repair genes are either accumulated during IVM to be ready for fertilization or increased expression levels of DNA repair genes in oocytes caused by suboptimal IVM conditions.

Development of large and small blastomeres from 2-cell embryos produced in vitro in pigs.

In the present study, we examined the development to blastocysts of large and small blastomeres from unevenly cleaved 2-cell embryos (uneven 2-cell embryos) in pigs. Proportion of blastocysts derived from large blastomeres (52.8 ± 6.4%) was significantly higher (P<0.05) compared with small ones (32.1 ± 4.6%). However, there were no differences in total cell number, inner cell mass (ICM) cell number and ICM/total cells ratio between them. Of 53 sister blastomere pairs in the same embryos examined there were 12 pairs (22.6%) in which both blastomeres developed to blastocysts, 16 pairs (30.2%) in which only large blastomeres developed to blastocysts, and five pairs (9.4%) in which only small blastomeres developed to blastocysts. Relative total amount of active mitochondria in small blastomeres were lower (P<0.05) than that of large blastomeres and blastomeres from evenly cleaved 2-cell embryos. However, there was no difference in relative density of active mitochondria in these three types of blastomeres. In conclusion, blastocysts derived from small and large blastomeres in uneven 2-cell embryos had comparable quality in terms of cell number, ICM number, ICM/total cell ratio and distribution of active mitochondria. The results suggest that these blastomeres may contribute multiple offspring production in pigs.

Fertilization ability of porcine oocytes reconstructed from ooplasmic fragments produced and characterized after serial centrifugations.

Mitochondria are reported to be critical in in vitro maturation of oocytes and subsequent embryo development after fertilization, but their contribution for fertilization has not been investigated in detail. In the present study, we investigate the contribution of mitochondria to fertilization using reconstructed porcine oocytes by fusion of ooplasmic fragments produced by serial centrifugations (centri-fusion). Firstly, we evaluated the characteristics of ooplasmic fragments. Three types of fragments were obtained by centrifugation of porcine oocytes matured in vitro for 46 h: brownish (B), transparent (T) and large (L) fragments containing both B and T parts in a fragment. The production efficiencies of these types of fragments were 71.7, 91.0 and 17.8 fragments/100 oocytes, respectively. In experiments, L fragments were excluded because they contained both brownish and transparent components that were apparently intermediate between B and T fragments. Observations by confocal microscopy after staining with MitoTracker Red CMXRos® and transmission electron microscopy revealed highly condensed active mitochondria in B fragments in contrast to T fragments that contained only sparse organelles. We reconstructed oocytes by fusion of a karyoplast and two cytoplasts from B and T fragments (B and T oocytes, respectively). The B oocytes showed higher sperm penetration (95.8%) and male pronuclear formation rates (94.2%) by in vitro fertilization than T oocytes (66.7% and 50.0%, respectively). These results suggest that the active mitochondria in oocytes may be related to their ability for fertilization.

Naloxone increases maturation rate and ratio of inner cell mass to total cells in blastocysts in pigs.

The purposes of the present study were to examine the effect of naloxone, a mu-opioid receptor (MOR) antagonist, on porcine oocyte maturation and embryo development. MOR gene was expressed in germinal vesicle (GV) and metaphase II (M-II) porcine oocytes, one-, four-cell stage embryos and blastocysts. In blastocysts, MOR gene was mainly expressed in inner cell mass (ICM) cells. Supplementation of 10(-8) mol/L naloxone in in vitro maturation (IVM) medium increased the maturation rate (P < 0.05). However, 10(-4) mol/L naloxone reduced the maturation rate (P < 0.05) compared with the control. The presence of naloxone during IVM had no effects on fertilization status and subsequent embryonic development after in vitro culture (IVC). The addition of 10(-3) mol/L dibutyryl cyclic adenosine monophosphate (dbcAMP), and 10(-8 ) mol/L naloxone together into IVM medium increased nuclear maturation (P < 0.05) compared with the addition of either dbcAMP or naloxone alone. Supplementation with naloxone in IVC medium did not improve embryonic development. However, at the concentrations of 10(-6) mol/L and 10(-8) mol/L, naloxone increased the ratio of ICM to total cells in blastocysts (P < 0.05). In conclusion, at low concentration, naloxone increases maturation rate and the ratio of ICM to total cells in blastocysts. Naloxone and cAMP have a synergistic effect on oocyte maturation.

Improvement of porcine oocytes with low developmental ability after fusion of cytoplasmic fragments prepared by serial centrifugation.

We have shown in pigs that oocytes denuded of cumulus cells at 24 h of in vitro maturation culture and subsequently matured for a total of 46 h (DO24 oocytes) have lower cytoplasmic maturity than those matured with cumulus cells for 46 h and then denuded (DO46 oocytes). In the present study, DO24 zona-free oocytes were fused with one (1C) or two (2C) cytoplasmic fragments produced by serial centrifugation ("centri-fusion") of DO46 oocytes (DO24+1C and DO24+2C oocytes, respectively). Groups of (1) DO46 (a control), (2) DO24, (3) DO24+1C and (4) DO24+2C oocytes were partheno-activated by an electrical pulse or fertilized in vitro and subsequently cultured for 6 days. In the fused groups, female pronucleus (FPN) formation rates were higher than that in the DO24 group after parthenogenetic activation (PA); however, the blastocyst rates were intermediate between those of the control and DO24 groups. After in vitro fertilization, the male pronucleus (MPN) formation rates in the fused groups were similar to that in the control group and higher than that in the DO24 group; the normal fertilization rate in the DO24+2C group was higher than that in the DO24 group and similar to that in the control group, resulting in significantly higher blastocyst rates in the DO24+2C and control groups than that in the DO24 group. These results suggest that centri-fusion using ooplasm from fully matured DO46 oocytes can offer a potentially novel approach for restoration of cytoplasmic maturity to oocytes with low developmental ability and subsequent improvement of fertilization and developmental competence.

Evaluation of developmental competence of in vitro-produced porcine embryos based on the timing, pattern and evenness of the first cleavage and onset of the second cleavage.

The following selection markers for in vitro-produced porcine embryos were investigated: the timing, pattern and evenness of the first cleavage and the timing of the second cleavage. The embryos that cleaved by 30 h post-insemination (hpi) developed to blastocysts at a significantly higher rate (60.9%) and with a significantly higher cell number (33.6 cells) than those of embryos cleaved by 36 hpi (26.4% and 23.6 cells, respectively, P<0.05). Blastocyst proportions derived from 2- and 3-cell embryos cleaved by 30 hpi (68.2 and 65.3%, respectively) were significantly higher than those of 4- and >4-cell embryos (46.3 and 42.6%, respectively, P<0.05). The cell number per blastocyst generated from 2-cell embryos was significantly greater (37.3 cells) than those from 3-, 4- and >4-cell embryos (23.6-27.8 cells, P<0.05). Among embryos cleaved by 30 hpi, the blastocysts derived from evenly cleaved embryos (40.6 cells) were of significantly better quality than those derived from unevenly cleaved embryos (33.2 cells, P<0.05), although their blastocyst rates did not differ. The evenly cleaved embryos that underwent subsequent cleavage within 18 h had significantly higher blastocyst rates (72.7-81.0%) and quality (36.2-40.9 cells) than those without subsequent cleavage (48.3% and 22.5 cells, respectively, P<0.05) during the same period. In conclusion, the timing, pattern and evenness of the first cleavage and the timing of the second cleavage affected the developmental competence and quality of in vitro-produced porcine embryos.

Effects of cysteine during in vitro maturation of porcine oocytes under low oxygen tension on their subsequent in vitro fertilization and development.

In this study, we evaluated the effect of different concentrations of cysteine in in vitro maturation (IVM) medium during IVM under low oxygen tension (5% O(2)) of porcine oocytes on the intracellular content of glutathione (GSH) and subsequent in vitro fertilization (IVF) and development. Cumulus oocyte complexes (COCs) were collected from ovaries obtained at a local slaughterhouse, cultured in IVM medium supplemented with 0 (control), 0.05, 0.1, 0.2 or 0.6 mM cysteine for 44-46 h, fertilized in vitro and subsequently cultured for 6 days in total. The GSH content of the IVM oocytes exposed to 0, 0.05, 0.1, 0.2 or 0.6 mM cysteine increased significantly (P<0.05) as the concentration of cysteine increased (12.2, 14.0, 15.1, 16.4 and 16.4 pmol/oocyte, respectively). However, the rates of oocyte maturation, sperm penetration, male pronuclear formation, monospermy and even cleavage on Day 2 (the day of IVF was defined as Day 0) and blastocyst formation on Day 6 did not differ among the groups. Moreover, the cell numbers of blastomeres in blastocysts were uniform among the groups. These results indicate that supplementation with 0.05-0.6 mM cysteine during IVM under 5% O(2) tension significantly increased the intracellular GSH contents of IVM oocytes; however, it had no promoting effects on nuclear maturation, fertilization, male pronucleus formation and subsequent embryonic development to the blastocyst stage.