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J M Robl

Publications and source records attributed to J M Robl.

51 records · Page 3Linked to original sources
PubMed1990

Factors affecting the efficiency of nuclear transplantation in the rabbit embryo.

Procedures to improve nuclear transplantation efficiency in the rabbit were evaluated. We report the influence of recipient oocyte age on the different steps of nuclear transplantation. The effect of multiple pulses and the influence of manipulation medium and cytochalasin B in the post-fusion/activation medium on activation and development were studied. Recently ovulated oocytes were enucleated at a higher rate (60%) than aged oocytes (3%, p less than 0.005); they also fused at a higher rate (85% vs. 26%, p less than 0.001). Activation was low with freshly ovulated oocytes compared to aged oocytes (3% vs. 37%, respectively; p less than 0.005), but was increased by using multiple pulses (85% vs. 68%, p less than 0.05). Multiple pulses also improved development to blastocysts (48% vs. 5%, p less than 0.001). Incubation of oocytes in a bicarbonate-buffered medium with 10% fetal calf serum for manipulation also enhanced rates of activation (100% vs. 89%, p less than 0.05) and development of oocytes to blastocysts (77% vs. 26%, p less than 0.001). Furthermore, 7.5 micrograms/ml cytochalasin B in the post-fusion/activation medium increased activation rates (78% vs. 50%, p less than 0.05) and development to blastocysts of manipulated embryos (46% vs. 11%, p less than 0.001). When the above modifications were applied, 10% (23/230) of the total nuclear transplant embryos (8-16-cell-stage donor nuclei) or 21% (23/110) of those transferred to recipients developed to offspring, rates similar to the development of nonmanipulated control embryos (10%, 4/41, p greater than 0.1).

Aging↗
PubMed1989

Electrical activation of mouse oocytes.

Activation of the oocyte is the least efficient step in nuclear transplantation in the rabbit. We report the influence of age of oocytes, field strength, pulse duration and number, and shape of field on the rate of activation of mouse oocytes by electrical pulses. Regardless of oocyte age, activation rates were similar over a wide range of field strengths and pulse durations. Aged oocytes activated at a higher rate than recently ovulated oocytes (32 vs 3%), which lysed more frequently (13 vs 2%). Fragmentation rate was also higher among aged oocytes (42 vs 6%). The rate of activation increased with the number of pulses, from 9% with a single pulse, to 61% with six pulses. It also increased with the interval between pulses. Comparison of activation chamber geometries showed that the rate of activation was higher in a nonuniform field than in a uniform field and, for a particular field strength, varied from one electrode gap to another. These observations indicate that the rate of activation can be greatly increased by multiple electrical pulses. The activation rate also varied with consistent field strength in chambers with different electrode configurations.

Journal Article↗
PubMed1988

Comparison of heterospermic and homospermic inseminations as measures of male fertility.

This study attempted to determine a basis for the previously observed greater sensitivity of heterospermic tests when compared to homospermic tests for detecting differences in fertility between males. In theory, the results of heterospermic tests are an indication of the proportion of eggs fertilized per unit time whereas results of homospermic inseminations measure only the cumulative or final proportion of eggs fertilized. The fertilizing ability of sperm from males of CF1 and C57BL/6N strains of mice was compared homospermically using both relatively high and low concentrations of sperm and by measuring the proportion of eggs penetrated per unit of time. The fertilizing ability of sperm from these strains was also compared using heterospermic inseminations. When females were inseminated with a high concentration of sperm, males of both strains fertilized a high and indistinguishable percentage of eggs when examined after 30 hr. When females were inseminated with either a low concentration of sperm or when the proportion of eggs penetrated was measured at 5 hr, differences between strains of mice were distinguishable. Heterospermic insemination further magnified the observed difference between strains. The results of this study confirm that measuring the percentage of eggs fertilized per unit of time can enhance the magnitude of differences between males in fertility as compared to measuring only the final percentage of eggs fertilized. Measuring the percentage of eggs fertilized per unit of time does not, however, entirely account for the large differences observed between fertility of males when they are compared using heterospermic inseminations.

Animals↗
PubMed1988

Strain differences in early mouse embryo development in vitro: role of the nucleus.

Mouse embryos of many strains are blocked in development at the two-cell stage when placed in culture at the one-cell stage. If, however, they are placed in culture at the two-cell stage, they develop to blastocysts at a high rate. We investigated the transcriptional and translational systems in blocked embryos by assaying enzymatic activity of the protein product of an injected gene and found these systems to be functioning normally. The role of the nucleus was investigated by reciprocal nuclear transplants between blocked and nonblocked embryos. The results indicate that the source of the nucleus was of equal importance to that of the cytoplasm for the successful development of embryos cultured from the one-cell stage. When cultured from the two-cell stage, however, the nucleus was solely responsible for strain differences in culture rates.

Animals↗
PubMed1988

Nuclear reprogramming in nuclear transplant rabbit embryos.

The first six genetically verified nuclear transplant rabbits have been produced in this study. Individual eight-cell stage embryo blastomeres were transferred and fused with enucleated mature oocytes of which six full-term offspring were produced out of 164 manipulated eggs. The following efficiency rates were determined for the nuclear transplantation procedure: chromosomal removal from oocytes, 92%; fusion rate, 84%; activation rate, 46%; embryo transfer rate, 27%. Additional reasons for the low efficiency rate of nuclear transplant embryos may include limited development due to aging in recipient oocytes and asynchronous transfers of manipulated embryos to recipient females. The successful development to term may have been due to the ability of the mature oocyte to reprogram the eight-cell stage nuclei. The number of cells in blastocysts derived from isolated eight-cell blastomeres (18 +/- .08) was lower than that of nonmanipulated pronuclear (106 +/- 5.1) and nuclear transplant embryos derived from eight-cell stage nuclei (91 +/- 10.2) (p less than 0.001). This evidence along with the significant amount of nuclear swelling in nuclear transplant embryos and a delay in the time of blastocyst formation indicate that nuclear reprogramming had taken place in these embryos. Successful nuclear reprogramming indicates that serial transfers could result in the expanded multiplication of mammalian embryos.

Animals↗
PubMed1987

Penetration of mouse eggs at various intervals after insemination as influenced by concentration of sperm and strain of male.

This study was conducted to determine the extent to which the percentage of mouse eggs that were penetrated by sperm at the end of the period of sperm penetration was due to the proportion of eggs penetrated per unit of time and to the span of time of sperm penetration. Female mice of ICR strain were inseminated 1.5 hr after ovulation with 5 X 10(6) sperm/50 microliter from males of DBA/2N, CF1 or C57BL/6N strains to determine the effect of the male. To determine the effect of concentration of sperm ICR females were inseminated with 2, 4, 6, or 8 X 10(6) sperm/50 microliter from CF1 males. Females were killed at various intervals after insemination and the eggs were recovered and examined for evidence of penetration by a sperm. The time intervals from both insemination to the onset of egg penetration and from insemination to cessation of penetration were similar for the three strains of males. Throughout the period of penetration of eggs a constant percentage of eggs was penetrated per hour for a particular strain of male. The relative percentage penetrated per hour very closely approximated the relative percentage of eggs finally penetrated for each strain of male. The percentage of eggs penetrated per hour was linearly positively related to the concentration of sperm inseminated. The final percentage of eggs penetrated depended primarily on the rate at which the eggs were penetrated during the period of sperm penetration and not on the length of the period of egg penetration which was constant.

Analysis of Variance↗
PubMed1987

Nuclear transplantation in mouse embryos: assessment of nuclear function.

Enucleated mouse zygotes receiving eight-cell nuclei rarely develop beyond the two-cell stage, whereas enucleated two-cell embryos with eight-cell nuclei develop to blastocysts, and a few develop to midgestation. In this study the function of eight-cell nuclei in either the zygote or two-cell recipient was assessed by monitoring several nuclear-dependent events. These included methionine uptake, qualitative changes in protein synthesis, the time of blastocoele formation, and changes in nuclear volume. Although enucleated zygotes with nuclei from eight-cell embryos did not develop past the two-cell stage and had abnormally low levels of methionine uptake, they did show a normal zygote-to-two-cell transition in the types of polypeptides synthesized. Enucleated two-cell embryos with eight-cell nuclei formed blastocysts on schedule with the recipient cell stage and reached levels of methionine uptake equivalent to control embryos. The types of proteins synthesized by two-cell embryos with eight-cell nuclei indicated that the nucleus was developing partly in accord with its own developmental program. Transplanted nuclei did not enlarge to acquire the volume of the recipient cell's nucleus in either the zygote or the two-cell recipient. These results indicate that the difference in development between zygotes and two-cell embryos (each with eight-cell nuclei) may be the result of a greater overlap of function between the two-cell and eight-cell stages, rather than the extent of nuclear reprogramming. Nuclear function was not completely normal in either type of embryo, which may explain their failure to develop to term.

Animals↗
PubMed1987

Nuclear transplantation in the bovine embryo: assessment of donor nuclei and recipient oocyte.

Blastomeres from 2- to 32-cell bovine embryos were transferred to enucleated oocytes matured either in vivo or in vitro by micromanipulation and electrofusion. The percentage of donor cells fusing with the recipient oocytes was dependent on relative cell size or stage of development. Therefore, when smaller donor karyoplasts (17- to 32-cell vs. 2- to 8-cell) were transferred, the rate of fusion was significantly less (p less than 0.01). After fusion, nuclear transfer embryos were cultured either in vitro or in vivo (in a ligated ovine oviduct). Nuclear transfer embryos cultured in vitro developed to the 4- to 6-cell stage after 72 h (4-cell, 71%; 8-cell, 33%, 16-cell, 33%; p less than 0.30), whereas nuclear transfer embryos cultured in vivo developed to the morula or blastocyst stage (2- to 8-cell, 11.7%; 9- to 16-cell, 16.0%; 17- to 32-cell, 8.3%; p greater than 0.30) after 4 or 5 days. Freshly ovulated oocytes (collected 36 h after the onset of estrus), when used as recipients, resulted in morula/blastocyst-stage embryos more often than in vitro-matured oocytes or in vivo-matured oocytes collected 48 h after the onset of estrus (20% vs. 7.8% and 6.7%, respectively; p less than 0.02). After in vivo culture, nuclear transfer embryos were mounted and fixed or transferred nonsurgically to the uteri of 6- to 8-day postestrus heifers. Seven pregnancies resulted from the transfer of 19 embryos into 13 heifers; 2 heifers completed pregnancy with the birth of live calves.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Nuclear transplantation in bovine embryos.

This study was conducted to develop a method for transplanting nuclei in bovine embryos and to test the development of several stages of donor nuclei transplanted to enucleated pronuclear recipient embryos. Pronuclear embryos were centrifuged to reveal nuclei. Nuclei were removed without penetrating the plasma membrane as membrane-bound karyoplasts, and were inserted into enucleated zygotes by electrically induced cell fusion. The highest rate of fusion (79%) occurred in Zimmerman Cell Fusion medium at 100 V for 20 to 40 microseconds with the fusion membranes oriented parallel to the electrodes. The effect of nuclear transplantation on development was tested in pronuclear embryos in which nuclei were removed and reinserted and the embryos were then transferred to sheep oviducts for 5 d. Of the intact nuclear transplant embryos recovered, 5/29 (17%) developed to morulae or blastocysts compared with 11/30 (37%) of the non-manipulated embryos. Two nuclear transplant embryos were transferred to a recipient cow, and both developed to normal offspring. When nuclei from two-, four-, or eight-cell embryos were transplanted to pronuclear recipient embryos, no development was observed.

Animals↗
PubMed1986

Nuclear transplantation in mouse embryos: assessment of recipient cell stage.

Enucleated zygotes were compared with enucleated two-cell embryos as recipients for donor nuclei from eight-cell embryos. Only one or two cleavage divisions were observed when eight-cell nuclei were transplanted to enucleated zygotes. Development of enucleated two-cell embryos containing a transplanted eight-cell nucleus was appreciably better with 51% (45/89) of the embryos forming blastocysts in vitro and 42% (25/60) initiating implantation. Of these, eleven implantation sites on Day 10 of gestation were examined histologically and two contained normally developing embryos. No development was observed beyond Day 12 of gestation. These observations indicate that a major transition occurs between the zygote and two-cell stage that results in the two-cell recipient being more compatible with the eight-cell nucleus than with the zygote.

Animals↗
PubMed1985

Manipulation of gametes and embryos in the pig.

Several manipulation techniques including nuclear injections, nuclear transplantation, embryo splitting, chimaera production and sperm injection are discussed with special reference to their application in the pig. The nuclear injection technique is likely to be of greatest use for gene transfer. Gene transfer is feasible for pig embryos, but it is very inefficient. Efficiencies of various steps in the successful production of transgenic offspring as summarized from several published references are as follows: immediate survival of embryos following injection (60%), offspring produced from injected embryos (15%), offspring with an integrated foreign gene (24%) and offspring expressing the integrated foreign gene (60%). The overall efficiency is about 1%. Potential uses for nuclear transplantation are for the production of gynogenetic and androgenetic embryos and for cloning embryos. The feasibility of producing androgenetic and gynogenetic embryos will depend on whether in the pig, as in the mouse, the paternal and maternal genomes function differently. The feasibility of cloning by nuclear transplantation will depend upon the timing of developmental events in pig embryos and the development of methods for reprogramming nuclei. Pig embryos have been cloned by embryo splitting. Chimaera production and fertilization by sperm injection are likely to be feasible for pig embryos but have not yet found application.

Animals↗
PubMed1985

Effect of estrogen, testosterone, and phenobarbital on uterine weight and liver microsomal enzymes in prepuberal mice.

Prepuberal female mice were treated with estradiol benzoate (EB), testosterone (T), or phenobarbital (PB), or were untreated. At 8 days after treatment treated mice had lower uterine weights than did untreated mice. Mice treated with EB and PB also had higher levels of hepatic cytochrome P450 at this time. The uteri of mice treated with EB, T, or PB were capable of responding to exogenous EB 8 days following the treatment with an increase in weight. In a second experiment mice were treated with a range of doses of EB, T, or PB,. At the lower doses there was an inverse association of uterine weight and cytochrome P450 levels. At higher doses there was no apparent association. These data indicate that uterine weight may be influenced in part by prior treatment that also affects the level of liver enzymes.

Animals↗
PubMed1982

The effects of antisperm plasma membrane antibodies on sperm-egg binding, penetration, and fertilization in the pig.

A procedure using published surgical techniques is described for determining the effects of antiplasma membrane antibodies on sperm-egg binding, penetration, and fertilization in vivo in the domestic pig. Time of ovulation was controlled and sperm inseminated at precise times relative to ovulation. Sperm were exposed to antibodies (Fab) and then washed free of excess antibody and placed in one uterine horn near the uterotubal junction. Sperm exposed to control material were placed in the opposite horn as a control. One horn was tied with two ligatures near the body of the uterus. Eggs were collected by flushing the oviducts at prescribed times after insemination. in nine animals used for this study, antibodies to boar sperm plasma membranes completely blocked sperm-zona binding, penetration of the zona, and fertilization. In contrast, sperm were bound to the zona pellucida and penetrated eggs in every case on the control side. These results indicate that insemination in vivo is useful in screening antibodies produced against specific plasma membrane antigens to determine their role in fertilization.

Animals↗
PubMedDate not supplied

Development of a chicken Z-chromosome-specific DNA library.

We have developed a chicken (Gallus domesticus) Z-chromosome-specific DNA library in a phage vector by means of chromosome microisolation and microcloning. The chromosomal origin, specificity, and purity was evaluated by fluorescent in situ hybridization (FISH) on chicken metaphases. Heterologous chromosome painting using this Z-chromosome-specific probe on turkey (Meleagris gallopavo) metaphases identified its homologous Z-chromosome, under the same stringent conditions as that used in the chicken, indicating a high degree of Z-chromosome sequence homology among these two species. This chicken Z-chromosome library will facilitate the development of Z-chromosome-specific DNA markers that will be useful for genetic mapping in the domestic chicken and related avian species. The Z-chromosome-specific DNA probe will also be useful for studies pertaining to the sex chromosome evolution in avian species.

Animals↗