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R S Prather

Publications and source records attributed to R S Prather.

At least 91 records · Page 5Linked to original sources

Microtubule and microfilament dynamics in porcine oocytes during meiotic maturation.

Microtubule and microfilament organization in porcine oocytes during maturation in vivo and in vitro was imaged by immunocytochemistry and laser scanning confocal microscopy. At the germinal vesicle stage, microtubules were not detected in the oocyte. After germinal vesicle breakdown, a small microtubule aster was observed near the condensed chromatin. During the prometaphase stage, microtubule asters were found in association with each chromatin mass. The asters then elongated and encompassed the chromatin at the metaphase-I stage. At anaphase-I and telophase-I microtubules were detected in the meiotic spindle. Microtubules were observed only in the second meiotic spindle at the metaphase-II stage. The meiotic spindle was a symmetric, barrel-shaped structure containing anastral broad poles, located peripherally and radially oriented. Taxol, a microtubule-stabilizing agent, did not induce microtubules in oocytes at the germinal vesicle stage. After germinal vesicle breakdown, numerous cytoplasmic foci of microtubules were formed in the entire oocyte when oocytes were incubated in the presence of taxol. Microfilaments were observed as a relatively thick uniform area around the cell cortex and were also found throughout the cytoplasm of oocytes at the germinal vesicle stage. After germinal vesicle breakdown, the microfilaments were concentrated close to the female chromatin. During prometaphase, microfilaments were chromatin moved to the peripheral position. At metaphase-I, two domains, a thick and a thin microfilament area, existed in the egg cortex. Chromosomes were located in the thick microfilament domain of the cortex. In summary, these results suggest that both microtubules and microfilaments are closely involved with chromosomal dynamics after germinal vesicle breakdown and during meiotic maturation in porcine oocytes.

Actin Cytoskeleton↗

Cytoskeletal alteration in aged porcine oocytes and parthenogenesis.

The objective of this study was to examine the changes in microtubule and microfilament assembly in aged porcine oocytes and to determine their developmental pattern after parthenogenetic activation. Porcine oocytes were cultured in Whitten's medium containing 10% follicular fluid with hormonal supplements (eCG and hCG) for 22 hr and 40 hr additional culture without hormonal supplements. At 40, 50, and 60 hr of culture, the oocytes were fixed for immunocytochemistry or activated by electrical pulse. In metaphase II stage oocytes, microtubules were detected only in the meiotic spindle. Two microfilament domains existed in the egg cortex, a thick and a thin microfilament domain. In aged oocytes (50 and 60 hr of culture), the incidence of metaphase II plates observed outside of the thick microfilament domain was higher (P < 0.05) than in young oocytes (40 hr of culture). After activation, a polar body was usually emitted from the chromatin at the microfilament rich domain or two pronuclei were formed outside of the microfilament rich domain. The percentage of activated oocytes with one female pronucleus was higher (P < 0.05) in oocytes at 40 hr of maturation than at 50 and 60 hr of culture. At 24 and 30 hr after stimulation the incidence of cleavage to the 3- to 4-cell stage was higher (P < 0.05) in aged oocytes (50 and 60 hr of maturation) than that in oocytes at 40 hr of culture. These results suggested that a role of microfilaments is to retain the chromatin at the proper position in the oocyte cortex, and that aging results in a disruption of the microfilaments such that atypical development results after parthenogenetic activation.

Actin Cytoskeleton↗

Characterization of DNA synthesis during the 2-cell stage and the production of tetraploid chimeric pig embryos.

The DNA content of nuclei during the 2-cell stage as well as in presumptive tetraploid embryos was investigated. In vivo produced pig zygotes were cultured to the 2-cell stage and either monitored for cleavage to the 4-cell stage or mounted at various times post-cleavage and DNA content determined. The length of the 2-cell stage was 14.8 +/- 3.0 hr. There was a significant increase in the length of the 2-cell stage due to the time in vitro as a zygote (P < 0.001: R2 = 0.866). The DNA content increased (P < 0.05) each 2 hr postcleavage until 10 hr postcleavage. This suggested that there is a short G1 and G2 phase and a relatively long phase of DNA synthesis. Next, 2-cell stage embryos were pulsed with electricity to induce cell-to-cell fusion. Whereas only about half fused within 30 min (55%), most (96%) developed to the blastocyst stage. The DNA content of the nuclei of the embryos was consistent with them being tetraploid. A final experiment was designed to evaluate the ability of the tetraploid embryo to form a chimera with isolated inner cell mass (ICM) cells. Inner cell masses were isolated from d 6 embryos, cut into thirds, labeled with DiO (a membrane die) and injected into the perivitelline space of 4-cell-stage tetraploid embryos. Twelve of 17 formed blastocysts. In most (8/12), the ICM of the resulting blastocyst was labeled, whereas in one the only fluorescence was in the trophectoderm, and in two fluorescence was evenly distributed between the ICM and trophectoderm. These results suggest that it may be possible to create a fetus derived from ICM cells, or potentially stem cells, that has a tetraploid trophoblast.

Animals↗

gamma-Glutamyl transpeptidase of spermatozoa may decrease oocyte glutathione content at fertilization in pigs.

The presence of gamma-glutamyl transpeptidase (GGT) in boar spermatozoa and the potential role of the GGT at sperm penetration were examined using in vitro matured porcine oocytes. In the first experiment, GGT of boar spermatozoa was examined using a histochemical stain. GGT was detected in the midpiece and the acrosome regions of boar spermatozoa. In the second experiment, porcine oocytes matured in vitro were injected with approximately 40 pl of 10 mM HEPES solution alone or HEPES containing 0.5 U/ml GGT or 1 mM guanosine-5'-O-(3'-thiotriphosphate) (GTP-gamma-S; G-protein activator). When GGT was injected into oocytes, the incidence of oocytes activated (23.7 +/- 1.4%) was not different (P > 0.05) from HEPES-injected controls (24.9 +/- 1.3%) at 6 h after injection. Injected GTP-gamma-S, however, activated 76.0 +/- 5.3% of oocytes at 6 h after injection, but extrusion of the second polar body was very low (2.8 +/- 4.8%). Total content of glutathione (GSH) and glutathione disulfide (GSSG) did not differ (P > 0.05) between GTP-gamma-S injected oocytes (4.2 +/- 0.7 pmol/oocyte) and noninjected oocytes (4.0 +/- 0.1 pmol/oocyte) at 6 h after injection. However, the total content of GSH and GSSG was lower (P < 0.01) in GGT-injected oocytes (2.1 +/- 0.2 pmol/oocyte) than HEPES-injected oocytes (3.4 +/- 0.2 pmol/oocyte) at 6 h after injection. In the third experiment, in vitro matured porcine oocytes were injected with about 40 pl of 10 mM HEPES solution alone or HEPES containing 0.5 U/ml GGT and then inseminated. At 12 h after insemination, the incidence of male pronuclear formation was significantly lower in oocytes injected with GGT as compared with injected control oocytes. These results demonstrated that (1) GGT was present on the surface of spermatozoa, (2) total oocyte content of GSH and GSSG was decreased by microinjection of GGT but not by that of GTP-gamma-S, and (3) male pronuclear formation was inhibited in GGT-injected oocytes. These results suggest that sperm GGT may be a limiting factor for male pronuclear formation in polyspermic oocytes.

Animals↗

Effects of injecting calcium chloride into in vitro-matured porcine oocytes.

In vitro-matured porcine oocytes were given injections of 0.1 M CaCl2 and after 6 h evaluated for signs of early and late activation events. CaCl2 injection caused cortical granule exocytosis in 75% (3 of 4) of the oocytes tested. It also induced cell cycle resumption as monitored by the histone H1 kinase assay: the phosphorylation rate of histone H1 decreased to 36.7% of the original value. Treated oocytes completed meiosis, extruded the second polar body, and progressed to first interphase: 79.4% of them formed one or more pronuclei. The elevated intracellular Ca2+ level resulted in activation-related changes in the protein synthetic profile in 90% (9 of 10) of the oocytes. Furthermore, 14.7% (9 of 61) of the treated oocytes developed to the compact morula/early blastocyst stage after a 7-day culture in ligated porcine oviduct, and one blastocyst hatched from the zona pellucida. Control oocytes given injections of 0.1 M MgCl2 or carrier medium (10 mM Hepes) did not show the changes mentioned. The results strengthen the idea that Ca2+ is a cell messenger that plays a central part in oocyte activation; it is concluded that elevated intracellular Ca2+ level caused by a single injection of CaCl2 leads to both early and late events of porcine oocyte activation.

Animals↗

Effects of oviductal fluid on sperm penetration and cortical granule exocytosis during fertilization of pig oocytes in vitro.

The effects of oviductal fluid on sperm penetration and cortical granule exocytosis in pigs were examined. Cortical granule exocytosis in oocytes matured in vivo and in vitro was observed by staining with fluorescent-labelled lectin and laser-scanning confocal microscopy. Exocytosis of matured oocytes was classified into three categories after in vitro fertilization: complete cortical granule exocytosis and even distribution of exudate in the entire perivitelline space (type I); complete exocytosis and partial distribution of exudate (type II) and incomplete cortical granule exocytosis (type III). The incidence of oocytes with type I exocytosis was higher in oocytes matured in vivo than in those matured in vitro. The addition of oviductal fluid at a concentration of 1% or 10% to the fertilization medium decreased sperm penetration and the mean number of spermatozoa present in penetrated eggs. The distribution of cortical granule exudate was not different in the presence of 1% oviductal fluid after sperm penetration from that of control groups. When oocytes were cultured for 1.5 h in medium containing 10% or 30% oviductal fluid before insemination, the incidence of monospermy increased without a decrease in sperm penetration. Preculture of oocytes in medium containing 30% oviductal fluid increased type I cortical granule reaction and increased resistance of the zona pellucida to dissolution by 0.1% (w/v) pronase at the time of sperm penetration. These results suggest that a factor(s) from the oviductal secretion is required for the complete cortical granule reaction and in the modification of the zona pellucida.

Acrosome↗

Technical note: porcine non-surgical embryo transfer.

The cervix is a barrier for transferring porcine embryos non-surgically. A non-surgical embryo transfer instrument composed of four parts has been developed that allows the delivery of embryos into the lumen of one uterine horn. Part 1, a modified artificial insemination spirette, is used to produce a cervical lock and to assist in manipulating Part 2 through the cervix. Part 2 is made of stainless steel tubing with a unique curved tip that can be manipulated through the folds of the cervix. Part 3 is a testing bar to ensure that the tip of Part 2 reaches the bifurcation of the uterus. Part 4, a disposable tubing complex for embryo delivery, is composed of a soft plastic leader tubing, a double opened connector for embryo loading and unloading, a semi-hard plastic tubing, and a hard tubing with a water-sealable plunger. Embryos (4-cell to blastocyst stages) were surgically collected and maintained in vitro up to 5 h before the non-surgical embryo transfer. Five of 16 recipients became pregnant (31%) and farrowed an average of 6.2 +/- 3.1 pigs per litter. This easily learned non-surgical embryo transfer technique will simplify embryo transfer in the swine industry and may lead to improvements in swine breeding and scientific research.

Animals↗

In vitro development of embryos from Sinclair miniature pigs: a preliminary report.

The goal of this project was to identify conditions that result in development from the zygote or the 2-cell stage Sinclair miniature pig embryos to the blastocyst stage. Four media were selected, 2 that have been shown to result in in vitro development in domestic pigs (Hepes buffered Tyrode's medium and Whitten's medium), 1 that is compatible with similar development in the cow (CR-1), and 1 that is compatible with development in the mouse (CZB). One- and two-cell stage embryos from Sinclair miniature pigs were flushed from oviducts in Hepes buffered Tyrode's medium, allocated to 1 of the 4 media and cultured for 120 h. At the end of the culture period, embryos were morphologically scored and nuclei were counted. Morphology scores were lowest for Hepes buffered Tyrode's medium but were not different for Whitten's medium, CZB or CR-1. The highest (P < 0.07) number of nuclei was present in the oocytes cultured in Whitten's medium (21.3), with CR-1 (15.7) and CZB (16.5) not differing significantly. Similar to the morphology scores, Hepes buffered Tyrode's medium resulted in the lowest number nuclei (5.5). In a parallel experiment, domestic pig embryos were cultured in Hepes buffered Tyrode's medium versus Whitten's medium. The domestic pig embryos, while also developing better in Whitten's Medium, developed better in the Hepes buffered Tyrode's medium than did the embryos from Sinclair pigs. Thus, the Sinclair pig embryo develops best if placed in Whitten's Medium.

Journal Article↗

Differential effects of protein synthesis inhibitors on porcine oocyte activation.

This study was designed to evaluate the effects of cycloheximide and puromycin on activation and protein synthesis of porcine oocytes. When matured oocytes were electrostimulated, then cultured in the presence of cycloheximide (5 micrograms/ml) for 6 or 24 hr, 92% of oocytes were activated as indicated by pronuclear formation, vs. 2.8% for untreated oocytes, 5.3% for oocytes not electrostimulated but cultured with cycloheximide, and 60.0% for those only electrostimulated. When cultured with L-[35S]methionine in the presence of cycloheximide, puromycin (100 micrograms/ml), or no protein synthesis inhibitor for 24 hr, oocytes had mean radiolabeled incorporation rates of 36.5, 2.21, and 32.0 fmol/4 hr/oocyte, respectively. Thus, cycloheximide had little effect on protein synthesis after 24 hr of culture. A 1D-SDS PAGE showed that oocytes cultured with puromycin or cycloheximide are not activated, while electrostimulated oocytes are activated, as characterized by the conversion of a 25-kDa polypeptide to a 22-kDa polypeptide. The radiolabeling experiment was repeated, except that oocytes were cultured for 4 or 24 hr. At 4 hr, mean incorporation rates were lower in the cycloheximide group (2.34 fmol/4 hr/oocyte), but similar in the puromycin (15.7 fmol/4 hr/oocyte) and control groups (18.9 fmol/4 hr/oocyte). At 24 hr, the puromycin group 5.73 fmol/4 hr/oocyte) had a lower rate of incorporation, while the cycloheximide (22.6 fmol/4 hr/oocyte) and control (26.0 fmol/4 hr/oocyte) groups were similar. Cycloheximide was more effective earlier during culture, while puromycin was more effective later. When combined with ES, puromycin did have a higher rate (P = 0.10) of activation (87.8%) than with electrostimulation alone (73.0%).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Carry-over of mRNA during nuclear transfer in pigs.

This research was designed to evaluate the changes that might occur to protein production after nuclear transfer. Eight-cell stage pig embryo nuclei were transferred to enucleated metaphase II oocytes. Twelve hours after nuclear transfer the nuclear transfer embryos were labeled with L-[35S] methionine and subjected to 1-dimensional polyacrylamide gel electrophoresis. During normal pig embryo development, embryonic RNA is produced by the 4-cell stage and results in the production of a 51 kDa band. This 51 kDa band persists through the 8-cell and compact morula stages. Eight-cell stage blastomeres of reconstituted embryos continued the production of the 51 kDa band even after treatment with alpha-amanitin. Since alpha-amanitin should block new mRNA synthesis, the production of this 51 kDa band is likely the result of a carry-over of mRNA coding for this protein in the blastomere cytoplasm.

Amanitins↗

Parthenogenetic activation of porcine oocytes with guanosine-5'-O-(3'-thiotriphosphate).

A possible mechanism by which sperm activate oocytes may involve a G protein-mediated signal-transducing pathway. Artificial stimulation of G proteins leads to events very similar to those at fertilization in oocytes of several species. Here we report the effects of activation of a G protein with guanosine-5'-O-(3'-thiotriphosphate) (GTP-gamma-S), a hydrolysis-resistant analog of guanosine triphosphate (GTP), in porcine oocytes. Mature oocytes were injected with 1 mM GTP-gamma-S. In response to injection, oocytes underwent changes normally occurring at fertilization, i.e. cortical granule exocytosis, changes in the protein synthetic profile, pronuclear formation, and development to the blastocyst stage. Guanosine-5'-O-(2-thiodiphosphate) (GDP-beta-S; a metabolically stable analog of guanosine diphosphate [GDP] that inactivates the G protein) and the carrier medium (CM; medium in which the injected compounds were dissolved) were significantly less effective in oocyte activation. It is suggested that porcine oocytes contain the components of a G protein-mediated signaling pathway that might function in oocyte activation at fertilization.

Animals↗

Parthenogenic activation of pig oocytes by protein kinase inhibition.

Previous studies have shown that protein kinase stimulators can induce the release of the metaphase II arrest in mouse ova. The present report is about the role of protein kinase in parthenogenic activation of pig oocytes, which was studied using a broad-spectrum protein kinase inhibitor. Metaphase II oocytes were obtained via in vitro maturation. Two sources of H7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine, HCl] were tested--Sigma (H7s) and Calbiochem (H7c). Both were found to be equally effective in promoting release of the metaphase II block. Activation, release of the metaphase II arrest, and progression to the first interphase could be induced at the highest percentage with an exposure to 2.0 mM H7s for 80 min (68.1%). In another experiment, H7c resulted in 69.5% activation, while iso-H7 [1-(5-isoquinolinesulfonyl)-3-methylpiperazine, HCl] at a similar concentration and exposure duration resulted in 25.7% activation. H7s and H7c were more effective than iso-H7 in inducing the appearance of a 22-kDa protein that is associated with normal fertilization in the pig. In contrast, although pronuclei could form and the protein profiles were indicative of activation, cortical granule exocytosis did not occur, and oocytes failed to develop to the blastocyst stage after H7 treatment. In contrast to the H7-treated oocytes, electrostimulation resulted in pronuclear formation, the appearance of the 22-kDa protein, release of cortical granules, and development to the blastocyst stage. These data demonstrate that broad-spectrum inhibitors of protein kinase are unable to induce all the events associated with normal fertilization.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

Evaluation of response to hormonal therapy in prepubertal gilts of different genetic lines.

Research was conducted to determine the effect of genetic line on hormonally induced puberty. Two studies were conducted, the first to evaluate estrus response (n = 120 gilts) and the second to evaluate follicular development (n = 24 gilts). Gilts were allotted to treatments in a 2 x 2 factorial arrangement. Gilts from two genetic lines (Age at Puberty x Yorkshire [APY] and Relaxed Selection x Yorkshire [RSY] received either P.G. 600 [symbol: see text] (P) or no injection (C). The percentage in estrus within 5 d after treatment was greater (P < .05) for P than for C gilts. However, no difference was detected for the percentage of P or C gilts that had ovulated within 14 d. For the APY line, P gilts had greater (P < .05) ovulation rates than C gilts. However, for the RSY line, P and C gilts did not differ in ovulation rate. More P gilts than C gilts had follicular cysts (P < .05). At 48 h after onset of estrus, plasma progesterone concentration tended to be greater (P = .13) for P gilts than for C gilts. In Exp. 2, gilts were ovariectomized 18 h after the onset of estrus. Ova were dissected from follicles on one ovary, and follicular fluid was aspirated from the other ovary. No differences were detected for the percentages of ova in various stages of meiosis. For APY gilts, follicular fluid estradiol concentration for P gilts tended to be lower (P = .12) than that for C gilts; however, no differences were detected for RSY gilts. Progesterone concentration in follicular fluid tended (P = .14) to be greater in P gilts than in C gilts. These results indicate that the effect of genetic line on age at puberty should be considered when inducing puberty.

Aging↗

Alanine and leucine transport in unfertilized pig oocytes and early blastocysts.

Amino acid transport is facilitated by specific transporters within the plasma membrane of the cell. In mouse oocytes and cleavage-stage conceptus Na(+)-dependent L-alanine and L-leucine transport are nearly undetectable. Sodium-dependent transport via system B0,+ in the mouse conceptus increases greatly between the 8-cell and blastocyst stages. By contrast, data presented here for the pig show that L-alanine and L-leucine transport is mainly Na(+)-dependent in the oocyte; this Na(+)-dependent component of transport becomes undetectable by the blastocyst stage. The Na(+)-dependent component of transport in oocytes is inhibited by BCH (2-aminoendo-bicyclo[2.2.1] hexane-2-carboxylic acid) and L-lysine and thus could be a form of system B0,+. In both oocytes and blastocysts Na(+)-independent L-leucine transport is inhibited by BCH, which is consistent with the presence of system L. The dramatic decrease in Na(+)-dependent amino acid transport activity could occur in pig conceptuses in association with the onset of RNA synthesis during the 4-cell stage. Regardless of the precise time during development at which it occurs, however, this dramatic, developmentally regulated decrease in Na(+)-dependent alanine and leucine transport activity contrasts sharply with the large increase in Na(+)-dependent system B0,+ activity that occurs during preimplantation development of murine conceptuses. Elucidation of the molecular mechanisms by which these changes occur should contribute to an understanding of regulation of gene expression during early development.

Alanine↗

Aspartate and glutamate transport in unfertilized pig oocytes and blastocysts.

Amino acid transport is facilitated by specific transporters within the plasma membrane of the cell. Mediated Na(+)-independent transport of L-glutamate can be easily detected in mouse oocytes, but it is nearly undetectable in blastocyst-stage embryos. In contrast, the Na(+)-dependent transport of L-aspartate is not detectable in oocytes, but it is detectable in eight-cell embryos and reaches relatively high levels by the blastocyst stage. It is believed that the amino acid transporters responsible are systems x-c and X-AG, respectively. Here we report the detection of Na(+)-dependent L-aspartate transport, which increased as pig blastocysts developed, although Na(+)-dependent aspartate transport was not detected in pig oocytes. Mediated Na(+)-independent L-glutamate transport was not detected in pig oocytes, in contrast to the mouse, nor in early or hatched pig blastocysts. Thus, while the developmental regulation of system X-AG is similar in both the pig and the mouse, system x-c was not detectable in pig oocytes or blastocysts. Elucidation of the molecular mechanisms controlling amino acid transport and other gene expression in early embryos should contribute to an understanding of whether and even why some aspects of developmental regulation of gene expression may need to differ among species.

Animals↗

Cell-to-cell coupling in early-stage bovine embryos: A preliminary report.

Gap junction communication has been implicated in providing positional information within an embryo. This positional information is then used to direct the differentiation of the early embryo. To begin to gain an assessment of the cell-to-cell communication observed in the early bovine embryo, fluorescein (5%) was microinjected into single blastomeres of freshly collected embryos. Dye communication was not observed in any of the 8-to 16-cell stage embryos. Very limited dye coupling was observed in compact morula (18%) and expanded blastocysts (25%). Interestingly, none of the expanded blastocysts resulting from in vitro maturation and in vitro fertilization showed any dye coupling. The degree of coupling observed in the bovine embryo was less than that observed in compact morula mouse embryos, where almost all (95%) embryos showed dye coupling. This experimental data is discussed in context with previous electron microscopy data.

Journal Article↗

Diacylglycerol-enhanced electrical activation of porcine oocytes matured in vitro.

This study was undertaken to examine the effect of second messengers added to the electroporation medium on electric pulse-induced artificial activation of meiotic Metaphase II porcine oocytes. Six separate experiments evaluated second messengers added to electroporation medium. When added to electroporation medium, neither phospholipase C (PLC: 0 to 2.5 Units/ml), D-myo-inositol triphosphate (IP(3): 0 to 10,000 muM), nor guanosine 5'-O-(3-thiotriphosphate; GTP-gamma-S: 0 to 100 muM) had any effect (P> 0.05) on activation rates. However, addition of 1,2-dioctanoyl-sn-glycerol (DiC(8)) increased activation rates in a dose-dependent response. At a level of 1,000 muM, DiC(8) resulted in a higher activation rate (P< 0,05) than 0.0, 0.1, 1 or 10 muM of DiC(8) with a pulse, and the 1,000 and 10,000 muM of DiC(8) no-pulse control groups. Effects of DiC(8) (1,000 muM) and IP(3) (100 muM) in combination or individually were investigated. At 1,000 muM, DiC(8) caused a higher rate of activation (P< 0.05) than 100 muM IP(3), but the result was not different from DiC(8) + IP(3). In another experiment, no difference (P> 0.05) was observed between DiC(8), GTP-gamma-S and IP(3), but DiC(8) + GTP-gamma-S + IP(3) + PLC yielded a higher (P< 0.05) activation rate than PLC or the rate of the controls. No significant development (blastocyst) was observed after 5 days of culture in any of the experiments. Protein profiles of activated oocytes, determined by 1D SDS-PAGE, were characteristic of pronuclear-stage embryos. These data indicate that the addition of DiC(8) to the electroporation medium synergistically enhances the rate of activation of electrically stimulated in vitro-matured porcine oocytes.

Journal Article↗