JOURNAL OF FOOD BIOCHEMISTRY, Issue 6 2007
KRISNA RUNGRUANGSAK-TORRISSEN
ABSTRACT Maturing Atlantic mackerel with and without artificial feeding, kept in sea pens (September to May), showed differences in digestive efficiency (protease activity ratio of trypsin to chymotrypsin), muscle growth (concentrations of RNA, protein, RNA/protein ratio and free amino acids [FAA]) and oocyte quality (trypsin-like specific activity, and concentrations of RNA, RNA/protein ratio and FAA). The artificially fed mackerel had higher body weights (1.7 times) but with less white muscle protein concentration (0.5 time), compared to the control group. Both groups showed higher levels of capacity for protein synthesis in the oocytes than in the white muscle, but it was about two times higher in the artificially fed fish whereas about four times higher in the control group. This indicated that, during maturation, development of oocytes and muscle for growth occurred concurrently in higher growth mackerel, while development of oocytes dominated in slower growth fish. A higher trypsin-like specific activity with higher FAA levels in the oocytes from females fed with an artificial diet, compared to the control group, suggested differences in development and quality between the gametes of the fish with different feedings. PRACTICAL APPLICATIONS The work illustrates differences in digestive efficiency and the quality of growth performance (growth and protein metabolism in muscle and oocytes) in fish with different feedings. The use of various methods for evaluating digestive efficiency and the quality of fish growth performance could provide reasonable information for some important biological differences between fish groups, especially when the number of samples are low. It is more advantageous to apply different methods simultaneously than using growth parameter alone in order to study for precise evaluation of the quality of fish growth performance. The methods are very practical for studying food utilization and growth quality of fish in different environmental conditions and with different behaviors in aquaculture as well as in natural ecosystem where food consumption rate and feeding regime cannot be under control. [source]

Postendocytic Provitellin Processing in the Growing Oocyte of the Short Horned Grasshopper, Oxyajaponicajaponica (Orthoptera: Acrididae)

ENTOMOLOGICAL RESEARCH, Issue 1 2004
Sae Youll CHO
ABSTRACT Polyclonal antibodies made against 86 kDa (86 k), 80 kDa (80 k) and 54 kDa (54 k) vitellins of Oxya japonica japonica are used for Western blotting. Anti-80k vitellin antibody is cross-reacted with a 95 kDa (95 k) vitellin. While 95 k vitellin is present both in the female hemolymph and in the oocyte, 80 k vitellin is detected only in the oocyte and the laid egg. In the growing oocytes, as 95 k vitellin is faded out gradually, 80 k vitellin is accumulated increasingly, indicating postendocytic processing of 95 k vitellin brings 80 k vitellin. Further conforming the hypothesis, partial digestion of 95 k vitellin with pepsin and ,-chymotrypsin makes several protein bands of molecular weight around 80 kDa. Thus, the 95k vitellin may have a cleavage site (s) to produce 80 k vitellin which forms fairly stable tertiary structure. In the reduced condition (20 mM glutathion), both 95 k and 80 k vitellins were digested throughly by endogenous proteinase at pH 4. Both 86 k and 54 k vitellins, respectively, show no apparent molecular weight changes in the growing oocyte and in the hemolymph. [source]

Follicular, Oocyte and Embryo Features Related to Metabolic Status in Primiparous Lactating does Fed with High-Fibre Rearing Diets

REPRODUCTION IN DOMESTIC ANIMALS, Issue 5 2010
M Arias-Álvarez
Contents Fertility of primiparous lactating does in the early postpartum (pp) period is very low mainly due to pronounced deficient energy intake, influencing oocyte and embryo developmental competence. The hypothesis used in this work was that high-lignin fibre diet supplied during the rearing period could increase feed intake and, consequently, improve the reproductive physiology and metabolic status of primiparous does in the early pp period. Diets with high-lignin [HL: 15.8% dry matter (DM)] or standard-lignin content (SL: 4.9% DM) were supplied until parturition time. No diet effects in serum oestradiol, progesterone concentrations and follicle categories were found in the histological study. Metaphase II rate of in vitro -matured oocytes was significantly higher in the SL vs the HL group (p < 0.001). Cytoplasmically degenerated oocytes (in terms of abnormal distribution of cortical granules) and follicular atresia rate were significantly lower in the SL group than in the HL group (p < 0.05 and p < 0.005 respectively). In addition, HL-fed does showed lower number of viable embryos and higher rate of retarded in vivo -recovered embryos compared with the SL group (p < 0.05). Neither in vitro embryo development of viable embryos nor conception rate was significantly different between groups. Feed intake increased during the first pregnancy in the HL group (p < 0.05), but not during early lactation. Serum protein, non-esterified fatty acid and leptin concentrations, as well as estimated body composition were similar in does fed with both diets. In conclusion, the enhancement of reproductive management by using highly lignified products in rearing diets does not seem to report physiological reproductive benefits affecting oocyte maturation rate and embryo viability in primiparous lactating does. [source]

Folliculogenesis and Morphometry of Oocyte and Follicle Growth in the Feline Ovary

REPRODUCTION IN DOMESTIC ANIMALS, Issue 2 2009
K Reynaud
Contents This study was designed to describe, both quantitatively (morphometry) and qualitatively (histological differentiation), follicle and oocyte growth in the feline ovary. The ovaries of 43 cats were collected and processed for histology. The diameters of 832 follicle/oocyte pairs were measured, with and without zona pellucida (ZP), and a special emphasis was placed on the study of early folliculogenesis. Primordial, primary, secondary, pre-antral and early antral follicles were measured at 44.3, 86.2, 126.0, 155.6 and 223.8 ,m in diameter respectively. A biphasic pattern of follicle and oocyte growth was observed. Before antrum formation, follicle (x) and oocyte (y) size were positively and linearly correlated (y = 0.500x + 20.01, r2 = 0.89). Antrum formation occurred when the follicle reached 160,200 ,m in diameter (when oocyte was at 102 ,m). After antrum formation, a decoupling was observed, a minimal increase in oocyte size contrasting with a significant follicle development (y = 0.001x + 114.39, r2 = 0.01). The pre-ovulatory follicle diameter was approximately 3500 ,m and the maximal oocyte diameter was 115 ,m. The ZP, absent in primordial and primary follicles, appeared at the secondary stage and reached almost 6 ,m at the pre-ovulatory stage. These results suggest that (i) in feline ovary, follicle and oocyte growth pattern is similar to that observed in other mammals; (ii) the antrum forms in 160,200 ,m follicles, which represents 5% of the pre-ovulatory diameter and (iii) the oocyte had achieved more than 90% of its maximal growth at the stage of antrum formation. [source]

Effect of Alpha-Tocopherol and Ascorbic Acid on Bovine Oocyte in Vitro Maturation

REPRODUCTION IN DOMESTIC ANIMALS, Issue 2 2005
G Dalvit
Contents In vitro culture results in higher oxygen concentrations than in vivo environments, leading to an increased level of reactive oxygen species (ROS) that cause lipid peroxidation of cellular membranes. Alpha-tocopherol (active form of vitamin E) is an antioxidant that protects mammalian cells against lipid peroxidation, which is regenerated by ascorbic acid. The aim of this study was to determine the effect of the addition of alpha-tocopherol and/or ascorbic acid to the maturation medium on bovine oocyte in vitro maturation (IVM) and subsequently on in vitro fertilization (IVF) and embryo development. Cumulus,oocyte complexes (COCs) were matured in Medium 199 (control), and with the addition of alpha-tocopherol and/or ascorbic acid. The concentration of alpha-tocopherol in COCs was determined by high-performance liquid chromatography (HPLC). IVF and in vitro culture (IVC) were carried out in modified synthetic oviductal fluid (mSOF). The quantity of alpha-tocopherol naturally present in COCs diminished by half during IVM (p < 0.05), although in the presence of ascorbic acid it remained constant. A greater amount of alpha-tocopherol was detected in COCs matured in medium supplemented with this antioxidant (p < 0.05), but the addition of alpha-tocopherol plus ascorbic acid maintained higher levels of alpha-tocopherol (p < 0.05). Significant differences were not observed in the percentages of nuclear maturation and fertilization among different treatments. The presence of alpha-tocopherol or ascorbic acid in the maturation medium failed to modify the percentage of blastocysts obtained, unlike the addition of both antioxidants when a significant decrease was observed (p < 0.05). Absorbic acid maintained the antioxidant capacity of the alpha-tocopherol incorporated to COC membranes during IVM. The active form of vitamin E during maturation impaired the acquisition of oocyte developmental competence. [source]

Light and Transmission Electron Microscopy of Immature Camelus Dromedarius Oocyte

ANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 4 2004
H. Nili
Summary In order to provide a consistent system for laboratory production of embryos, the characteristics of immature camel oocyte must first be described. The objective of this study was to define ultrastructural features of immature camel oocyte. Ovaries were obtained from camels at a local abattoir, and then transported to the laboratory within 2 h. Camelus cumulus oocyte complexes (COCs) were aspirated from 2,6 mm follicles using a 22-gauge needle. Excellent and good quality COCs were selected and prepared for transmission electron microscopy study using a cavity slide. The fine structure of camel oocyte is morphologically similar to that of other mammalian oocytes. However, some minor differences exist between COC of camel and other mammalian species. Different size and shape of membrane-bound vesicles, lipid droplet, mitochondria and cortical granules were distributed throughout the ooplasm. Discrete or in association with endoplasmic reticulum, Golgi complexes were observed in the periphery of the oocytes. The majority of the oocytes were in the germinal vesicle stage. [source]

Role of sperm ,v,3 integrin in mouse fertilization

DEVELOPMENTAL DYNAMICS, Issue 3 2010
Céline Chalas Boissonnas
Abstract Oocyte integrins have been described as essential for fertilization. But this concept has been challenged by deletion experiments. Recently, we have shown that sperm integrin ,6,1 plays a determinant role in mouse gamete interaction. In this study, we demonstrate the presence of ,v,3 integrin by Western blot and immunofluorescence on the sperm membrane. Oocytes and/or sperm preincubations with anti-,v or anti-,3 antibodies were performed before in vitro fertilization on cumulus-intact and zona-free egg assays. We observed inhibitory effects on the fusion process mostly by means of sperm function. An antibody directed against vitronectin inhibited gametes fusion, whereas the presence of exogenous vitronectin increased its efficiency. We suggest that vitronectin (on multimeric forms) can play a first nonspecific link corresponding to loosely bound spermatozoa to oocyte and that this link could be mediated by means of oocyte proteoglycans or integrins, and sperm ,v,3 integrin. Developmental Dynamics 239:773,783, 2010. © 2010 Wiley-Liss, Inc. [source]

Divergent roles of the DEAD-box protein BS-PL10, the urochordate homologue of human DDX3 and DDX3Y proteins, in colony astogeny and ontogeny

DEVELOPMENTAL DYNAMICS, Issue 6 2006
Amalia Rosner
Abstract Proteins of the highly conserved PL-10 (Ded1P) subfamily of DEAD-box family, participate in a wide variety of biological functions. However, the entire spectrum of their functions in both vertebrates and invertebrates is still unknown. Here, we isolated the Botryllus schlosseri (Urochordata) homologue, BS-PL10, revealing its distributions and functions in ontogeny and colony astogeny. In botryllid ascidians, the colony grows by increasing the number of modular units (each called a zooid) through a whole colony synchronized and weekly cyclical astogenic budding process (blastogenesis). At the level of the colony, both BS-PL10 mRNA and its protein (78 kDa) fluctuate in a weekly pattern that corresponds with the animal's blastogenic cycle, increasing from blastogenic stage A to blastogenic stage D. At the organ/module level, a sharp decline is revealed. Primary and secondary developing buds express high levels of BS-PL10 mRNA and protein at all blastogeneic stages. These levels are reduced four to nine times in the new set of functional zooids. This portrait of colony astogeny differed from its ontogeny. Oocytes and sperm cells express high levels of BS-PL10 protein only at early stages of development. Young embryos reveal background levels with increased expressions in some organs at more developed stages. Results reveal that higher levels of BS-PL10 mRNA and protein are characteristic to multipotent soma and germ cells, but patterns deviate between two populations of differentiating stem cells, the stem cells involved in weekly blastogenesis and stem cells involved in embryogenesis. Two types of experimental manipulations, zooidectomy and siRNA assays, have confirmed the importance of BS-PL10 for cell differentiation and organogenesis. BS-PL10 (phylogenetically matching the animal's position in the evolutionary tree), is the only member of this subfamily in B. schlosseri, featuring a wide range of biological activities, some of which represent pivotal roles. The surprising weekly cyclical expression and the participation in cell differentiation posit this molecule as a model system for studying PL10 protein subfamily. Developmental Dynamics 235:1508,1521, 2006. © 2006 Wiley-Liss, Inc. [source]

Post-ingestive effects of nectar alkaloids depend on dominance status of bumblebees

ECOLOGICAL ENTOMOLOGY, Issue 4 2009
JESSAMYN S. MANSON
Abstract 1.,Secondary metabolites have acute or chronic post-ingestive effects on animals, ranging from death to growth inhibition to reduced nutrient assimilation. 2.,Although characterised as toxic, the nectar of Gelsemium sempervirens is not lethal to pollinators, even when the concentration of the nectar alkaloid gelsemine is very high. However, little is known about the sublethal costs of nectar alkaloids. 3.,Using a microcolony assay and paired worker bumblebees, the present study measured the effects of artificial nectar containing gelsemine on oocyte development. Oocytes are a sensitive indicator of protein utilisation and general metabolic processes. We also calculated carbohydrate concentrations in the haemolymph to examine energetic costs of gelsemine consumption. 4.,High concentrations of gelsemine significantly reduced mean oocyte width in subordinate bees, while dominant bees showed only a trend towards oocyte inhibition. Gelsemine consumption did not reduce carbohydrate concentrations in haemolymph. 5.,The cost of ingesting gelsemine may be due to direct toxicity of alkaloids or may be an expense associated with detoxifying gelsemine. Detoxification of alkaloids can require reallocation of resources away from essential metabolic functions like reproduction. The risks associated with nectar alkaloid consumption are tied to both the social and nutritional status of the bee. [source]

Embryo development of Corticium candelabrum (Demospongiae: Homosclerophorida)

INVERTEBRATE BIOLOGY, Issue 3 2007
Sonia De Caralt
Abstract. Corticium candelabrum is a homosclerophorid sponge widespread along the rocky Mediterranean sublittoral. Scanning and transmission electron microscopy were used to describe the gametes and larval development. The species is hermaphroditic. Oocytes and spermatocytes are clearly differentiated in April. Embryos develop from June to July when the larvae are released spontaneously. Spermatic cysts originate from choanocyte chambers and spermatogonia from choanocytes by choanocyte mitosis. Oocytes have a nucleolate nucleus and a cytoplasm filled with yolk granules and some lipids. Embryos are surrounded by firmly interlaced follicular cells from the parental tissue. A thin collagen layer lies below the follicular cells. The blastocoel is formed by migration of blastomeres to the morula periphery. Collagen is spread through the whole blastocoel in the embryo, but is organized in a dense layer (basal lamina) separating cells from the blastocoel in the larva. The larva is a typical cinctoblastula. The pseudostratified larval epithelium is formed by ciliated cells. The basal zone of the ciliated cells contains lipid inclusions and some yolk granules; the intermediate zone is occupied by the nucleus; and the apical zone contains abundant electron-lucent vesicles and gives rise to cilia with a single cross-striated rootlet. Numerous paracrystalline structures are contained in vacuoles within both apical and basal zones of the ciliated cells. Several slightly differentiated cell types are present in different parts of the larva. Most cells are ciliated, and show ultrastructural particularities depending on their location in the larvae (antero-lateral, intermediate, and posterior regions). A few smaller cells are non-ciliated. Several features of the C. candelabrum larva seem to support the previously proposed paraphyletic position of homoscleromorphs with respect to the other demosponges. [source]

Sexual reproduction in the tropical corallimorpharian Rhodactis rhodostoma

INVERTEBRATE BIOLOGY, Issue 4 2000
Nanette E. Chadwick-Furman
Abstract. Polyps of the tropical corallimorpharian Rhodactis rhodostoma segregate sexes between center and edge positions within aggregations produced by clonal replication. On a reef flat at Eilat, northern Red Sea, infertile polyps and males occur mainly along the edges of clonal aggregations, while females mostly occupy central positions within each aggregation. In addition, on the inner to middle reef flat where polyps of this species are abundant, aggregations consist mostly of females. On the outer reef flat, where polyps are rare, a sampled aggregation consisted mostly of males and infertile polyps. Male polyps are significantly smaller than females, and the smallest polyps are infertile. Fecundity increases significantly with polyp size in females, but testis size and number do not vary with body size in males. Oocytes are present in polyps during most of the year and gradually increase in size until annual spawning in June-July during the period of maximum day length. Testes do not vary significantly in size during the year and remain a small proportion of body mass (>8%). In contrast, females invest up to 30% of their body mass into gonads during the months immediately before spawning. The annual spawning of gametes coincides with a temporary drop in the frequency of clonal replication by polyps. We estimate that each female polyp of R. rhodostoma may release up to 3000 large eggs (500 ,m in maximum diameter) each summer. The high investment of this corallimorpharian in sexual production of planktonic propagules may allow rapid dispersal to reef habitats distant from parent populations. [source]

Ultrastructure of the ovary and oogenesis in six species of patellid limpets (Gastropoda: Patellogastropoda) from South Africa

INVERTEBRATE BIOLOGY, Issue 3 2000
Alan N. Hodgson
Abstract. The ultrastructural features of the ovary and oogenesis have been described in 6 species of patellid limpets from South Africa. The ovary is a complex organ that is divided radially into numerous compartments or lacunae by plate-like, blind-ended, hollow trabeculae that extend from the outer wall of the ovary to its central lumen. Trabeculae are composed of outer epithelial cells, intermittent smooth muscle bands, and extensive connective tissue. Oocytes arise within the walls of the trabeculae and progressively bulge outward into the ovarian lumen during growth while partially surrounded by squamous follicle cells. During early vitellogenesis, the follicle cells lift from the surface of the underlying oocytes and microvilli appear in the perivitelline space. Follicle cells restrict their contact with the oocytes to digitate foot processes that form desmosomes with the oolamina. When vitellogenesis is initiated, the trabecular epithelial cells hypertrophy and become proteosynthetically active. Yolk synthesis involves the direct incorporation of extraoocytic precursors from the lumen of the trabeculae (hemocoel) into yolk granules via receptor-mediated endocytosis. Lipid droplets arise de novo and Golgi complexes synthesize cortical granules that form a thin band beneath the oolamina. A fibrous jelly coat forms between the vitelline envelope and the overlying follicle cells in all species. [source]

The zona pellucida of the koala (Phascolarctos cinereus): its morphogenesis and thickness

JOURNAL OF ANATOMY, Issue 3 2006
Jamie A. Chapman
Abstract In this study the ultrastructural organization of the koala oocyte and the thickness of the surrounding extracellular coat, the zona pellucida, has been determined to ascertain whether there is coevolution of the morphology of the female gamete with that of the highly divergent male gamete that is found in this marsupial species. Ovaries from several adult koalas were obtained and prepared for transmission electron microscopy. Oocytes in large tertiary follicles were somewhat smaller than those of most other marsupials, although their ultrastructural organization appeared similar and included many yolk vesicles. The zona pellucida surrounding the oocytes in tertiary follicles was approximately 8 µm thick and thus is of similar thickness to that of some eutherian mammals but at least twice as thick as that of most marsupial species so far studied. The results indicate that the koala oocyte is unusually small for a marsupial species whereas the zona pellucida is, by contrast, much thicker. How this relates to sperm,egg interaction at the time of fertilization has yet to be determined. [source]

Generation of pluripotent stem cells from eggs of aging mice

AGING CELL, Issue 2 2010
Junjiu Huang
Summary Oocytes can reprogram genomes to form embryonic stem (ES) cells. Although ES cells largely escape senescence, oocytes themselves do senesce in the ovaries of most mammals. It remains to be determined whether ES cells can be established using eggs from old females, which exhibit reproductive senescence. We attempted to produce pluripotent stem cell lines from artificial activation of eggs (also called pES) from reproductive aged mice, to determine whether maternal aging affects pES cell production and pluripotency. We show that pES cell lines were generated with high efficiency from reproductive aged (old) mice, although parthenogenetic embryos from these mice produced fewer ES clones by initial two passages. Further, pES cell lines generated from old mice showed telomere length, expression of pluripotency molecular markers (Oct4, Nanog, SSEA1), alkaline phosphatase activity, teratoma formation and chimera production similar to young mice. Notably, DNA damage was reduced in pES cells from old mice compared to their progenitor parthenogenetic blastocysts, and did not differ from that of pES cells from young mice. Also, global gene expression differed only minimally between pES cells from young and old mice, in contrast to marked differences in gene expression in eggs from young and old mice. These data demonstrate that eggs from old mice can generate pluripotent stem cells, and suggest that the isolation and in vitro culture of ES cells must select cells with high levels of DNA and telomere integrity, and/or with capacity to repair DNA and telomeres. [source]

The effect of elevated oocyte triiodothyronine content on development of rainbow trout embryos and expression of mRNA encoding for thyroid hormone receptors

JOURNAL OF FISH BIOLOGY, Issue 1 2004
J. C. Raine
The ability of developing rainbow trout Oncorhynchus mykiss embryos to compensate for elevated oocyte triiodothyronine (T3) content and whether elevation of oocyte T3 content within a physiologically meaningful range affects growth rates of the embryo or the expression of genes encoding for thyroid hormone receptors ,(TR,) and ,(TR,) were examined. Oocytes were immersed in ovarian fluid alone (control) or T3 -enriched ovarian fluid prior to fertilization and water hardening, to induce a dose-dependant increase in oocyte T3 content of c. 3 (control), c. 30 (LT3) or c. 110 ng egg,1(HT3). To examine the interaction of embryo somatic growth with altered thyroid state more effectively, the embryos were reared at two ambient temperatures (8·5 and 5·5°C ) to induce different growth rates. A significant decline in whole embryo T3 content was measured in the T3 -treatment groups reared at both water temperatures by 3 weeks post-fertilization (dpf), and may have reflected the action of outer ring monodeiodinase, which was present in microsomes prepared from embryos 23 dpf. Whole embryo T3 levels in the HT3 group, however, remained higher than controls until phase 2 of development [the onset of endogenous thyroid hormone (TH) release]. This suggested that the embryos exerted some control over their response to exogenous TH, but that there was a limit to the level of control exerted by the embryonic tissues. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed the presence of mRNA encoding for the two TR isoforms as early as 26 dpf, and quantitative real-time RT-PCR (qPCR) was used to examine the effect of elevated oocyte T3 content on the expression of these TR genes in embryos raised at 8·5 and 5·5° C, and sampled at similar developmental stages prior to the onset of embryonic TH synthesis, to ensure that the oocyte T3 was the only source of TH exposure to the embryo. There was a suppression of the TR, gene expression in the control 5·5° C group relative to the control 8·5° C group. In addition, both TR, and TR, mRNA accumulation was lower, relative to the controls, in the LT3 treatment group reared at 8·5° C suggesting a suppressive effect of the lower level of T3 treatment on the TR gene expression. Conversely, there were no differences from controls in the HT3 treatment group, possibly indicating that this level of exposure overrides the down-regulating capacity of the embryo. Similar patterns were seen for TR, and TR, mRNA accumulation in embryos reared at 5·5° C, but because of the temperature suppressed level of TR, mRNA in the controls, significant affects of the LT3 treatment were only found for TR,. There were no measurable effects of T3 treatment on oocyte fertility or embryo somatic growth for either temperature treatment group, nor was somatic growth hormone content (measured only in the 8·5° C treatment group) apparently related to in ovo T3 levels. The results suggest that altered in ovo T3 levels, within the ranges used here, do not induce marked affects on embryo development, probably because of the ability of the embryo to maintain the integrity of its TH milieu. [source]

Gonad development and evidence of protogyny in the red-throat emperor on the Great Barrier Reef

JOURNAL OF FISH BIOLOGY, Issue 2 2003
K. Bean
The gonad development in the red-throat emperor Lethrinus miniatus is described and the first detailed evidence for protogyny in this species provided. The identification of transitional individuals, bimodal sex-specific size-frequency distributions and female biased sex ratios suggest that L. miniatus is most likely a protogynous hermaphrodite. Transitional L. miniatus gonads were characterized by the concurrent degeneration of all oocytes and the proliferation of spermatocysts near the edge of the lamellae, an increase in blood vessels along strands of stromal tissue within the lamellae and the formation of multiple sperm sinuses. The sites of oocyte degeneration and proliferation of spermatocysts were spatially segregated. An increase in blood vessels along strands of stromal tissue within the lamellae of transitional phase gonads is likely to assist in the breakdown of oocytes and the proliferation of spermatocysts. Most mature resting females containing spermatocysts occurred within the transitional size-frequency distribution, suggesting that the presence of spermatocysts in these females may be an early sign of sex change. Oocytes within female gonads were interrupted by filamentous strands of stromal tissue within the lamellae. The testis contained a remanent ovarian lumen but no residual oocytes. Three characteristics of transitional L. miniatus gonads were found to be unusual and described for few other species of coral reef fishes. These included the absence of oocytes within testes, increased numbers of blood vessels, and the presence of strands of stromal tissue within the lamellae. [source]

Altering the Relative Abundance of GABAA Receptor Subunits Changes GABA- and Ethanol-Responses in Xenopus Oocytes

ALCOHOLISM, Issue 6 2009
Joyce H. Hurley
Background:, Variations in GABRA2 and GABRG3, genes encoding the ,2 and ,3 subunits of the pentameric GABAA receptor, are associated with the risk of developing alcoholism in adults, conduct disorder at younger ages, and with differences in electroencephalographic power in the , frequency range. The SNPs associated with alcoholism did not alter the coding of these genes, and extensive DNA sequencing of GABRA2 did not find coding changes in the high-risk haplotypes. Therefore, we hypothesize that the associations arise from differences in gene expression. Methods:, Here we report studies in Xenopus oocytes to examine the functional effects of altering the relative abundance of these 2 receptor subunits on GABA current and response to ethanol, as a model of potential effects of regulatory differences. Results:, When human ,2,2,3 subunits are co-expressed, increasing the amount of the ,2 subunit mRNA increased GABA current; in contrast, increasing the amount of the ,3 subunit decreased GABA currents. Acute ethanol treatment of oocytes injected with a 1:1:1 or 2:2:1 ratio of ,2:,2:,3 subunit mRNAs resulted in significant potentiation of GABA currents, whereas ethanol inhibited GABA currents in cells injected with a 6:2:1 ratio. Overnight treatment with ethanol significantly reduced GABA currents in a manner dependent on the ratio of subunits. Conclusions:, These studies demonstrate that changes in relative expression of GABAA receptor subunits alter the response of the resulting channels to GABA and to ethanol. [source]

Inhibition of the Activity of Excitatory Amino Acid Transporter 4 Expressed in Xenopus Oocytes After Chronic Exposure to Ethanol

ALCOHOLISM, Issue 7 2008
Seung-Yeon Yoo
Background:, The extracellular glutamate concentration is tightly controlled by excitatory amino acid transporters (EAATs). EAAT4 is the predominant EAAT in the cerebellar Purkinje cells. Purkinje cells play a critical role in motor coordination and may be an important target for ethanol to cause motor impairments. We designed this study to determine the effects of chronic ethanol exposure on the activity of EAAT4 and evaluate the involvement of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) in these effects. Methods:, EAAT4 was expressed in Xenopus oocytes following injection of EAAT4 mRNA. Oocytes were incubated with ethanol-containing solution for 24 to 96 hours. Membrane currents induced by l -aspartate were recorded using 2-electrode voltage clamps. Responses were quantified by integration of the current trace and reported in microCoulombs (,C). Results:, Ethanol dose- and time-dependently reduced EAAT4 activity. EAAT4 activity after a 96-hour exposure was significantly decreased compared to the control values at all concentrations tested (10 to 100 mM). Ethanol (50 mM) significantly decreased the Vmax (2.2 ± 0.2 ,C for control vs. 1.6 ± 0.2 ,C for ethanol, n = 18, p < 0.05) of EAAT4 for l -aspartate. Preincubation of ethanol-treated (50 mM for 96 hours) oocytes with phorbol-12-myrisate-13-acetate (100 nM for 10 minutes) abolished the ethanol-induced decrease in EAAT4 activity. While staurosporine (2 ,M for 1 hour) or chelerythrine (100 ,M for 1 hour) significantly decreased EAAT4 activity, no difference was observed in EAAT4 activity among the staurosporine, ethanol, or ethanol plus staurosporine groups. Similarly, EAAT4 activity did not differ among the chelerythrine, ethanol, or ethanol plus chelerythrine groups. Pretreatment of the oocytes with wortmannin (1 ,M for 1 hour) also significantly decreased EAAT4 activity. However, no difference was observed in the wortmannin, ethanol, or ethanol plus wortmannin groups. Conclusions:, The results of this study suggest that chronic ethanol exposure decreases EAAT4 activity and that PKC and PI3K may be involved in these effects. These effects of ethanol on EAAT4 may cause an increase in peri-Purkinje cellular glutamate concentration, and may be involved in cerebellar dysfunction and motor impairment after chronic ethanol ingestion. [source]

Ethanol Potentiation of Glycine Receptors Expressed in Xenopus Oocytes Antagonized by Increased Atmospheric Pressure

ALCOHOLISM, Issue 5 2003
Daryl L. Davies
Background: Behavioral and biochemical studies indicate that exposure to 12 times normal atmospheric pressure (12 ATA) of helium-oxygen gas (heliox) is a direct, selective ethanol antagonist. The current study begins to test the hypothesis that ethanol acts by a common mechanism on ligand-gated ion channels by expanding previous hyperbaric investigations on ,-aminobutyric acid type A (GABAA) receptors (GABAARs) at the biochemical level to ,1glycine (GlyRs) expressed in Xenopus oocytes. Methods: Oocytes expressing wild-type ,1 homomeric GlyRs were voltage-clamped (,70 mV) and tested in the presence of glycine (EC2) ± ethanol (50,200 mM) under 1 ATA control and 3 to 12 ATA heliox conditions. Glycine concentration response curves, strychnine/glycine interactions, and zinc (Zn2+) modulation of GlyR function was also tested. Results: Pressure reversibly antagonized the action of ethanol. The degree of antagonism increased as pressure increased. Pressure did not significantly alter the effects of glycine, strychnine, or Zn2+, indicating that ethanol antagonism by pressure cannot be attributed to alterations by pressure of normal GlyR function. The antagonism did not reflect tolerance to ethanol, receptor desensitization, or receptor rundown. Conclusion: This is the first use of hyperbarics to investigate the mechanism of action of ethanol in recombinant receptors. The findings indicate that pressure directly and selectively antagonizes ethanol potentiation of ,1GlyR function in a reversible and concentration- and pressure-dependent manner. The sensitivity of ethanol potentiation of GlyR function to pressure antagonism indicates that ethanol acts by a common, pressure-antagonism,sensitive mechanism in GlyRs and GABAARs. The findings also support the hypothesis that ethanol potentiation of GlyR function plays a role in mediating the sedative-hypnotic effects of ethanol. [source]

Production of cloned dogs by decreasing the interval between fusion and activation during somatic cell nuclear transfer

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 5 2009
Sue Kim
To improve the efficiency of somatic cell nuclear transfer (SCNT) in dogs, we evaluated whether or not the interval between fusion and activation affects the success rate of SCNT. Oocytes retrieved from outbred dogs were reconstructed with adult somatic cells from a male or female Golden Retriever. In total, 151 and 225 reconstructed oocytes were transferred to 9 and 14 naturally synchronized surrogates for male and female donor cells, respectively. Chromosomal morphology was evaluated in 12 oocytes held for an interval of 2 hr between fusion and activation and 14 oocytes held for an interval of 4 hr. Three hundred seventy-six and 288 embryos were transferred to 23 and 16 surrogates for the 2 and 4 hr interval groups, respectively. Both the male (two pregnant surrogates gave birth to three puppies) and female (one pregnant surrogate gave birth to one puppy) donor cells gave birth to live puppies (P,>,0.05). In the 2 hr group, significantly more reconstructed oocytes showed condensed, metaphase-like chromosomes compared to the 4 hr group (P,<,0.05). A significantly higher pregnancy rate and a greater number of live born puppies were observed in the 2 hr group (13.0% and 1.1%, respectively) compared to the 4 hr group (0%) (P,<,0.05). In total, three surrogate dogs carried pregnancies to term and four puppies were born. These results demonstrate that decreasing the interval between fusion and activation increases the success rate of clone production and pregnancy. These results may increase the overall efficiency of SCNT in the canine family. Mol. Reprod. Dev. 76: 483,489, 2009. © 2008 Wiley-Liss, Inc. [source]

Ultrastructure of bovine oocytes exposed to Taxol prior to OPS vitrification

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 8 2008
Roser Morató
Abstract Our objective was to document potential subcellular consequences of treatment with the microtubule stabilizer Taxol with or without subsequent vitrification of cow and calf oocytes by the open pulled straw (OPS) method. Oocytes were divided into four experimental groups for cows and four groups for calves: (1) a control group fixed immediately after maturation; (2) an OPS group cryopreserved by conventional OPS; (3) a Taxol/CPA group exposed to 1 ,M Taxol and cryoprotective agents (CPAs); and (4) a Taxol/OPS group vitrified by OPS including 1 ,M Taxol to the vitrification solution. All oocytes were processed for light and transmission electron microscopy. The main injuries were observed on the metaphase plate and the spindle. In control oocytes, the metaphase appeared as condensed chromosomes arranged in a well-organized metaphase plate and the spindle showed well organized microtubules in both cow and calf oocytes. However, in cow OPS oocytes, the metaphase plate was disorganized into scattered chromosomes or the chromosomes were condensed into a single block of chromatin. In addition, microtubules were not organized as typical spindles. In contrast, cow Taxol/OPS oocytes as well as both cow and calf Taxol/CPAs oocytes showed well-organized metaphase plates and normal spindle morphology. All calf OPS and calf Taxol/OPS oocytes displayed a single block of chromatin and no microtubules could be observed around the chromosomes. In conclusion, treatment with 1 µM Taxol before and during vitrification did not induce adverse changes in the oocyte cytoplasm or metaphase spindles in adult bovine oocytes, but stabilized the metaphase and spindle morphology. Mol. Reprod. Dev. 75: 1318,1326, 2008. © 2008 Wiley-Liss, Inc. [source]

Effects of thiol compounds on in vitro maturation of canine oocytes collected from different reproductive stages

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 9 2007
Mohammad Shamim Hossein
Abstract Various thiol compounds are known to improve cytoplasmic and/or nuclear maturation of oocytes in vitro. The present study examined the effects of two thiol compounds, cysteine (0.1, 0.5, and 1.0 mM) and cysteamine (50, 100, and 200 µM), on cytoplasmic and nuclear maturation of canine oocytes. Oocytes collected from different reproductive stages were cultured in TCM-199 supplemented with 10% fetal bovine serum, 2.2 mg/ml sodium carbonate, 2.0 µg/ml estrogen, 0.5 µg/ml FSH, 0.03 IU/ml hCG, and 1% penicillin,streptomycin solution for 72 h. Data were analyzed by two-way ANOVA after arcscine transformation and protected by Bonferroni post hoc test. The effects of cysteine and cysteamine on canine IVM were varied depending on the reproductive stage of oocyte donor bitches. In the follicular stage, significantly more oocytes reached the metaphase II (M II) stage when cultured with 0.5 or 1.0 mM cysteine (16.7% and 16.9%, respectively) compared to the control (6.2%). In the follicular stage, cysteamine increased oocyte maturation rate upto the M II stage (15.1% to 17.0%) compared to the control (4.4%). Both the 0.5 mM cysteine and 100 µM cysteamine, alone or together, increased the intracellular GSH level of canine oocytes compared to the control. Irrespective of reproductive stage, no further beneficial effects on nuclear or cytoplasmic maturation were observed when 0.5 mM cysteine and 100 µM cysteamine were supplemented together. In conclusion, addition of 0.5 mM cysteine and 100 µM cysteamine to the maturation medium improved IVM of canine oocytes. Mol. Reprod. Dev. 74: 1213,1220, 2007. © 2007 Wiley-Liss, Inc. [source]

Effects of delayed excision of oviducts/ovaries on mouse oocytes and embryos

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2007
De-Qiang Miao
Abstract To achieve the best and reproducible results of experiments, effects of delayed excision of oviducts/ovaries on mouse ovarian/ovulated oocytes and embryos have been studied. Oviducts/ovaries were excised at different times after death of mice and effects of the postmortem interval on ovarian/ovulated oocytes and embryos were analyzed. When oviduct excision was delayed 10 min, many ovulated oocytes lysed or underwent in vitro spontaneous activation, and this postmortem effect aggravated with the extension of postmortem interval and oocyte aging. Oocytes from different mouse strains responded differently to delayed oviduct removal. Delayed oviduct excision did not cause lysis of zygotes or embryos but compromised their developmental potential. When ovaries were excised at 30 min after death, percentages of atretic follicles increased while blastocyst cell number declined significantly after oocyte maturation in vitro. Preservation of oviducts in vitro, in intact or opened abdomen at different temperatures and histological analysis of oviducts from different treatments suggested that toxic substance(s) were secreted from the dying oviducts which induced oocyte lysis and spontaneous activation and both this effect itself and the sensitivity of oocytes to this effect was temperature dependent. It is concluded that a short delay of oviduct/ovary removal had marked detrimental effects on oocytes and embryos. This must be taken into account in experiments using oocytes or embryos from slaughtered animals. The data may also be important for estimation of the time of death in forensic medicine and for rescue of oocytes from deceased valuable or endangered mammals. Mol. Reprod. Dev. 74: 468,477, 2007. © 2006 Wiley-Liss, Inc. [source]

Changes of maternal transcripts in oocytes from persistent follicles in cattle

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 3 2007
Brandon M. Lingenfelter
Abstract A high incidence of early embryonic loss is associated with prolonged dominance of follicles. The objective of the present experiment was to determine if persistence of a follicle resulted in alterations in mRNA expression of important genes in the oocyte. Cows were assigned to four groups: growing follicles on day 6 (G0h) or day 8 (G48h) and persistent follicles on day 13 (P0h) or day 15 (P48h) of the estrous cycle (estrus,=,day 0). All cows were super-stimulated on day 1,4. Cows in G48h, P0h, and P48h groups received 25 mg prostaglandin (PG) F2, on day 6. Cows in P0h and P48h groups received progesterone from CIDR-B devices on day 5 through 13. Ovaries of cows in G0h, G48h, P0h, and P48h groups were removed on day 6, 8, 13, and 15, respectively. Oocytes were aspirated immediately after colpotomy and denuded of cumulus cells. Quantitative real-time PCR was used to measure the mRNA abundances of 10 selected genes important for early embryogenesis in oocytes obtained from growing and persistent follicles. Relative abundances of MSY2, PARN, and YY1 mRNA (P,<,0.05) were significantly lower in oocytes from persistent than from growing follicles. Oocytes from persistent follicles, however, had greater abundances of PAP and eIF-4E transcripts (P,<,0.05). The data indicate that persistence of a follicle leads to altered abundances of mRNA for genes important for regulation of transcription and protein translation in the oocyte, which could compromise development of early embryos in cows that ovulate a persistent follicle. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc. [source]

Mitochondrial organization in prepubertal goat oocytes during in vitro maturation and fertilization

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 5 2006
Esther Velilla
Abstract The aim of this study was to evaluate mitochondrial distribution during in vitro maturation (at 0, 15, 20, and 27 hr of IVM) and fertilization of prepubertal goat oocytes compared to mitochondrial distribution of ovulated and in vitro fertilized oocytes from adult goats. Oocytes from prepubertal goats were recovered from a slaughterhouse and were matured in M199 with hormones and serum for 27 hr. Ovulated oocytes were collected from gonadotrophin-treated Murciana goats. Frozen-thawed spermatozoa were selected by centrifugation in Percoll gradient and were capacitated in DMH with 20% steer serum for 1 hr. Ovulated and IVM-oocytes were inseminated in DMH medium with steer serum and calcium lactate for 20 hr. Oocytes and presumptive zygotes were stained with Mitotraker Green FM and observed under a confocal laser scanning microscope. Ultrastructural morphology of oocytes and presumptive zygotes were analyzed by transmission electron microscopy (TEM). Prepubertal goat oocytes at germinal vesicle stage (GV) presented mitochondria localized in the cortical and perinuclear region. IVM-oocytes at metaphase II presented mitochondria peripheral polarized to the region opposite were the metaphase spindle is positioned and within the polar body. Ovulated oocytes presented peripheral mitochondria distribution and mitochondrial aggregation around the MII spindle. At 20 hr post-insemination, mitochondria were distributed around the two synchronous pronuclei (2PN rpar; in zygotes ovulated oocytes whereas in prepubertal 2PN-zygotes mitochondria presented a peripheral polarized distribution. Images by TEM detected that immature prepubertal goat oocytes that are less electrodense and present fewer cristae than in vitro matured prepubertal goat oocytes; these are characterized by being associated to swollen vesicles. Mol. Reprod. Dev. 73: 617,626, 2006 © 2006 Wiley-Liss, Inc. [source]

Protein synthesis and mRNA storage in cattle oocytes maintained under meiotic block by roscovitine inhibition of MPF activity

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2004
Céline Vigneron
Abstract Roscovitine, a specific inhibitor of MPF kinase activity, has been shown to block efficiently and reversibly the meiotic resumption of oocytes from different species, including cattle. In view to verify that oocytes maintain germinal vesicle like molecular activities under roscovitine treatment, we compared in the present study the M-phase Promoting Factor (MPF) and Mitogen Activated Protein (MAP) kinase activities; protein synthesis and phosphorylation patterns in oocytes and cumulus cells; and CDK1 and Cyclin B messengers storage under control culture and under roscovitine inhibition. We observed that roscovitine induced a full and reversible inhibition of MPF kinase activity and of the activating phosphorylation of both ERK1/2 MAPK. During in vivo maturation, there was a highly significant increase in the relative mRNA level of both cyclin B1 and CDK1 whereas during in vitro culture, the relative amount of CDK1 messenger was reduced. These messengers may be used as markers for the optimization of in vitro maturation treatment. Roscovitine reversibly prevented this drop in relative quantities of CDK1 messenger. Oocytes cultured in the presence of roscovitine maintained a GV like profile of protein synthesis except that two proteins of 48 and 64 kDa specific of matured oocytes also appeared under roscovitine treatment. However, roscovitine did not prevent most of the modifications of protein phosphorylation pattern observed during maturation. In conclusion, results of this study revealed that the use of roscovitine did not prevent all the events related to maturation of bovine oocytes. Mol. Reprod. Dev. 69: 457,465, 2004. © 2004 Wiley-Liss, Inc. [source]

Effects of meiosis-inhibiting agents and equine chorionic gonadotropin on nuclear maturation of canine oocytes

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2003
N. Songsasen
Abstract Experiments were conducted to determine the effects of meiosis-inhibiting-agents and gonadotropins on nuclear maturation of canine oocytes. The culture medium was TCM199,+,10 ng/ml epidermal growth factor supplemented with 25 ,M ,-mercaptoethanol, 0.25 mM pyruvate, and 1.0 mM L-glutamine (Basal TCM). Initially, oocytes were cultured in Basal TCM alone or in Basal TCM,+,dibutylryl cyclic adenosine monophosphate (0.5, 1, 5, or 10 mM dbcAMP) for 24 hr. Dibutylryl cAMP inhibited resumption of meiosis in a dose-dependent manner; 60% of oocytes remained at the germinal vesicle (GV) stage after being cultured for 24 hr in 5 mM dbcAMP. The meiosis-inhibitory effect of dbcAMP appeared to be reversible, as the oocytes resumed meiosis and completed nuclear maturation after being cultured for an additional 48 hr in its absence. Oocytes were then cultured in Basal TCM alone or in Basal TCM,+,roscovitine (12.5, 25, or 50 ,M) for 24 hr. Although ,60% of oocytes cultured in 25 ,M roscovitine remained at the GV stage, this percentage was not significantly different from the 48% that also remained at the GV stage when cultured in its absence. Oocytes were cultured in Basal TCM,+ 25 ,M roscovitine for 17 hr, exposed briefly to equine chorionic gonadotropin (eCG), and then cultured in Basal TCM for 48 hr. Short exposure of oocytes to eCG was beneficial, as it significantly increased the proportion of oocytes developing beyond germinal vesicle breakdown (P,<,0.05) with ,20,30% of these were metaphase I (MI) oocytes. Study of the kinetics of nuclear maturation demonstrated that large numbers of oocytes remained at MI even after being cultured for 52 hr following brief exposure to eCG. This study showed that in vitro maturation of canine oocytes can be somewhat improved by short exposure of oocytes to eCG. However, further studies are still required to derive effective methods to mature canine oocytes in vitro. Mol. Reprod. Dev. 65: 435,445, 2003. © 2003 Wiley-Liss, Inc. [source]

Glutathione and adenosine triphosphate content of in vivo and in vitro matured porcine oocytes

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2003
A.M. Brad
Abstract Glutathione (GSH) content in mature porcine oocytes is correlated with subsequent fertilization and developmental success. Adenosine triphosphate (ATP) is an important energy source for maintaining cellular activities and protein synthesis. The objective of this study was to compare GSH and ATP concentrations of in vivo and in vitro matured porcine oocytes. Ovulated, in vivo matured oocytes were frozen at ,80°C in groups of 10,20 (GSH) or 5,10 (ATP). In vitro oocytes were matured in either tissue culture medium-199 (TCM199) supplemented with polyvinyl alcohol (PVA) or hyaluronic acid (MAP5), or North Carolina State University-23 (NCSU23) supplemented with porcine follicular fluid (pFF) and frozen as described, or fertilized and cultured. GSH content was determined by the dithionitrobenzoic acid,glutathione disulfide (DTNB,GSSG) reductase recycling assay. ATP content was determined by using the Bioluminescent Somatic Cell Assay Kit. Oocytes matured in vitro in defined TCM199 with PVA or hyaluronic acid, or NCSU23 with pFF had significantly lower concentrations (P,<,0.05) of GSH (n,=,207, 9.82,±,0.71 pmol/oocyte; n,=,104, 9.73,±, 0.81 pmol/oocyte; n,=,108, 7.89,±,0.66 pmol/oocyte, respectively) compared to in vivo matured oocytes (n,=,217, 36.26,±,11.00 pmol/oocyte). Concentrations of ATP were not different between treatments (in vivo, n,=,70, 0.97,±,0.07 pmol/oocyte; TCM,PVA, n,=,117, 0.81,±,0.13 pmol/oocyte; TCM,MAP, n,=,107, 1.02,±,0.18 pmol/oocyte; NCSU,pFF, n,=,134, 0.71,±,0.08 pmol/oocyte). Intracellular ATP content does not appear to be related to developmental potential in porcine oocytes. Low intracellular GSH may be responsible, in part, for lower developmental competence observed in in vitro matured porcine oocytes. Mol. Reprod. Dev. 64: 492,498, 2003. © 2003 Wiley-Liss, Inc. [source]

Synergistic Effect of Porcine Follicular Fluid and Dibutyryl Cyclic Adenosine Monophosphate on Development of Parthenogenetically Activated Oocytes from Pre-Pubertal Gilts

REPRODUCTION IN DOMESTIC ANIMALS, Issue 5 2010
AB Nascimento
Contents This study investigated the effect of porcine follicular fluid (PFF) and dibutyryl cyclic adenosine monophosphate (dbcAMP) during in vitro maturation (IVM) of porcine oocytes on meiotic maturation, fertilization and embryo development, and compared the effect of supplementing the embryo culture media with PFF or foetal bovine serum (FBS) on embryo development. Oocytes from pre-pubertal gilts were IVM for 44 h, and parthenogenetically activated or in vitro -fertilized. Embryos were cultured in porcine zygote medium (PZM3) for 7 days. Cleavage and blastocyst rates were evaluated at 48 h and 7 days of culture. The supplementation of the IVM medium with 25% PFF and 1 mm dbcAMP for the first 22 h resulted in more (p < 0.05) embryos developing to the blastocyst stage as compared with the inclusion of dbcAMP alone. The dbcAMP + PFF combination increased (p < 0.05) the average number of nuclei per blastocyst as compared with either of these components alone or in its absence. A synergistic effect of dbcAMP + PFF during IVM was also reflected in the capacity of oocytes to regulate sperm penetration and prevent polyspermy, as twice as many oocytes from the control group were penetrated by more than one sperm as compared with those matured in the presence of both dbcAMP and PFF. The supplementation of PZM3 with 10% FBS from days 5 to 7 of culture significantly improved the total cell quantity in embryos derived either from control or dbcAMP + PFF matured oocytes. There was no effect on the total cell quantity when FBS was replaced by the same concentration of PFF. These studies showed that dbcAMP, PFF and FBS can improve both the quantity (57.3% vs 41.5%) and quality (74.8 vs 33.3 nuclei) of porcine blastocysts derived from oocytes recovered of pre-pubertal gilts. [source]

Expression Pattern of Apoptotic Genes in Vitrified-Thawed Bovine Oocytes

REPRODUCTION IN DOMESTIC ANIMALS, Issue 5 2010
VM Anchamparuthy
Contents This study describes a method for quantification of transcripts from low numbers of bovine oocytes using real time RT-PCR. The objective was to evaluate the expression pattern of apoptotic genes (Fas, FasL, Bax and Bcl-2) in vitrified-thawed oocytes. Oocytes were evaluated at germinal vesicle stage; at 15 h of maturation; after vitrification and warming at 15 h of maturation and at 9 h of additional maturation. All transcripts showed an increase in at least 1.2-fold change post-vitrification warming, but the levels tended to decrease at 9 h of maturation post-vitrification warming. Transcript abundance for Fas mRNA was 1.4-fold for oocytes after vitrification and warming. The level of Fas mRNA upon maturation was 0.8-fold. The increase in the abundance of FasL mRNA was 2.1, while it was 0.5-fold relative to control. Vitrification resulted in 1.5-fold change in Bax mRNA expression in oocytes. After 9 h of maturation post-vitrification warming, the level for Bax mRNA was 0.6-fold. The mRNA for Bcl-2 was nearly the same after vitrification and warming. The abundance of mRNA for Bcl-2 was 1.2-fold in vitrified oocytes and fell (p = 0.05) to 0.5 at 9 h of maturation post-vitrification and warming. The up-regulation of apoptotic genes in vitrified oocytes may be an early indicator of reduced developmental competence following vitrification. Yet, results from terminal deoxynucleotidyl transferase dUTP nick end labelling and caspase assays did not support the evidence of apoptosis in embryos derived from large numbers of vitrified oocytes. [source]