Rabbit Embryos Research Articles

Somatic nucleus remodelling in immature and mature Rassir oocyte cytoplasm.

Successful production of cloned animals derived from somatic cells has been achieved in sheep, cattle, goats, mice, pigs, rabbits, etc. But the efficiency of nuclear transfer is very low in all species. The present study was conducted to examine somatic nucleus remodelling and developmental ability in vitro of rabbit embryos by transferring somatic cells into enucleated germinal vesicle (GV), metaphase I (MI) or metaphase II (MII) oocytes. Microtubules were organized around condensed chromosomes after the nucleus had been transferred into any of the three types of cytoplasm. A bipolar spindle was formed in enucleated MII cytoplasm. Most of the nuclei failed to form a normal spindle within GV and MI cytoplasm. Some chromosomes scattered throughout the cytoplasm and some formed a monopolar spindle. Pseudopronucleus formation was observed in all three types of cytoplasm. Reconstructed embryos with MI and MII cytoplasm could develop to blastcysts. Nuclei in GV cytoplasm could develop only to the 4-cell stage. These results suggest that (1) GV material is important for nucleus remodelling after nuclear transfer, and (2) oocyte cytoplasm has the capacity to dedifferentiate somatic cells during oocyte maturation.

Differential development of rabbit embryos derived from parthenogenesis and nuclear transfer.

Parthenogenetic development (PA) is often used as a model to investigate activation protocols for nuclear transfer (NT) embryos. The objective of this study was to compare the development, as well as the dynamics of the nuclear materials and microtubules of PA and NT embryos following similar activation treatment. Our results demonstrate that, during parthenogenesis, activation through either electrical pulses or chemical stimulation alone resulted in low cleavage rates and compromised development. A combination of two sets of electrical pulses and a 2-h-exposure to chemical activation medium (5 microg/ml cycloheximide (CHX) and 2 mM 6-dimethylaminopurine (6-DMAP) in KSOM+0.1% BSA) could effectively activate rabbit oocytes, and resulted in a 99% (n = 73) cleavage rate with greater than 60% (n = 73) developing to blastocysts at day 4. However, the same activation protocol following NT resulted in only 65-72% of oocytes cleaved (depending on donor cell type), with less than 20% developing to the blastocyst stage. The differences observed between NT and PA embryos subjected to the same activation protocol were also evident in terms of the time required for their development to the blastocyst stage, as well as the cell numbers present in blastocysts at day 6. Furthermore, laser confocal microscopy revealed that pronuclear formation in the NT embryos was delayed by comparison to that in the parthenotes. In conclusion, our study suggests that an effective protocol for parthenogenesis cannot promise a comparable outcome for NT embryos.

Methylation reprogramming and chromosomal aneuploidy in in vivo fertilized and cloned rabbit preimplantation embryos.

Active demethylation of the paternal genome but not of the maternal genome occurs in fertilized mouse, rat, pig, and bovine zygotes. To study whether this early demethylation wave is important for embryonic development, we have analyzed the global methylation patterns of both in vivo-fertilized and cloned rabbit embryos. Anti-5-methylcytosine immunofluorescence of in vivo-fertilized rabbit embryos revealed that the equally high methylation levels of the paternal and maternal genomes are largely maintained from the zygote up to the 16-cell stage. The lack of detectable methylation changes in rabbit preimplantation embryos suggests that genome-wide demethylation is not an obligatory requirement for epigenetic reprogramming. The methylation patterns of embryos derived from fibroblast and cumulus cell nuclear transfer were similar to those of in vivo-fertilized rabbit embryos. Fluorescence in situ hybridization with chromosome-specific BACs demonstrated significantly increased chromosomal aneuploidy rates in cumulus cell nuclear transfer rabbit embryos and embryos derived from nuclear transfer of rabbit fibroblasts into bovine oocytes compared with in vivo-fertilized rabbit embryos. The incidence of chromosomal abnormalities was correlated with subsequent developmental failure. We propose that postzygotic mitotic errors are one important explanation of why mammalian cloning often fails.

Effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection rabbit embryos.

This study assessed the effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection (ICSI) rabbit embryos. Oocytes were recovered from female rabbits superovulated with PMSG and hCG, and epididymal sperm were collected from a fertile male rabbit. The oocyte was positioned with the first polar body at 12 o'clock position, and a microinjection needle containing a sperm was inserted into the oocyte at 3 o'clock. Oolemma breakage was achieved by aspirating ooplasm, and the aspirated ooplasm and sperm were re-injected into the oocyte. The injected oocytes were cultured in M199 medium containing 10% fetal calf serum at 38 degrees C with 5% CO2 in air. The results showed that oocytes injected at 1 h post-collection produced a higher (p < 0.05) fertilization rate than those injected at 4 or 7 h post-collection. Blastocyst rate in the 1 h group was higher (p < 0.05) than in the 7 h group. Denuded oocytes (group A) and oocytes with cumulus cells (group B) were injected, respectively. Rates of fertilization and development of ICSI embryos were not significantly different (p > 0.05) between the two groups. Four ICSI methods were applied in this experiment. In methods 1 and 2, the needle tip was pushed across half the diameter of the oocyte, and oolemma breakage was achieved by either a single aspiration (method 1) or repeated aspiration and expulsion (method 2) of ooplasm. In methods 3 and 4, the needle tip was pushed to the oocyte periphery opposite the puncture site, and oolemma breakage was achieved by either a single aspiration (method 3) or repeated aspiration and expulsion (method 4) of ooplasm. Fertilization rate in method 2 was significantly higher (p < 0.05) than in methods 1 and 3. Blastocyst rates were not significantly different (p > 0.05) among methods 1, 3 and 4, but method 2 produced a higher (p < 0.05) blastocyst rate than method 3.

Stage-specific expression of leukaemia inhibitory factor and its receptor in rabbit pre-implantation embryo and uterine epithelium during early pregnancy.

Leukaemia inhibitory factor (LIF) has been shown to play an important role in the development and implantation of blastocysts in mice. In the current study, the reverse transcription-polymerase chain reaction (RT-PCR) was employed to examine the expression patterns of LIF and its receptor (LIFR) genes in rabbit embryos during pre-implantation development, and the uterine expression of LIF and LIFR was also evaluated by Western blotting. Transcripts for LIFR were detected within morula and blastocyst-stage embryos, while the LIF mRNA was only found in blastocysts (from early to fully expanded blastocoel cavities), indicating that embryo-derived LIF can act in an autocrine manner on the process of blastocyst formation. The expression levels of LIF and LIFR in uterine epithelium were gradually increased during pre-implantation period and reached their highest levels on days 6.5 of pregnancy, just before the time of blastocyst implantation, suggest that paracrine LIF circuit should exist between the endometrium and the early embryos, which may be involved in the embryo-maternal dialogue and important for the blastocyst implantation. The data present here show the stage-specific and dynamic expression patterns of LIF and LIFR, both in embryos and endometrium, during early pregnancy in rabbits, which indicated that LIF might play an important role in the pre-implantation development and subsequent implantation of rabbit embryos.

Developmental Rate of Rabbit Parthenogenetic Embryos Derived Using Different Activating Protocols

The present study compares development of rabbit embryos generated using different oocyte activation protocols and reconstructed with embryonic or cumulus cells as nuclear donor. In vivo matured oocytes were collected from New Zealand White rabbits at 16 h after ovulation treatment and were activated atl8 h of post-ovulation treatment. The following schemes of oocytes activation were tested: 1) single electric pulse (EP, 3.2 kV/cm, 3x20 μs, 0.3 M mannitol)+5 min culture in the presence of 5 mM lonomycin, 2) single electric pulse (EP, 3.2 kV/cm, 3x20 μs, 0.3 M mannitol)+1 h culture in the presence of 2 mM 6-DMAP, and 3) three electric pulses 30 min apart. Cleavage rate, percentage of expanded and hatched blastocysts as well as total cell number of blastomeres of parthenogenetic embryos were significantly higher using either EP+6-DMAP or 3xEP schemes, comparing with EP+lonomycin. Development rate up to hatched blastocyst stage of cloned rabbit embryos using the EP+6-DMAP for activation of nuclei were 19% for embryonic cell nuclei and 36% for cumulus cell nuclei. The best activation protocol optimalized in this study was the combined treatment EP+6-DMAP, which may be potentially used for nuclear transfer protocol.

The molecular basis for intersexuality part one: the developing testis.

Intersex disorders are the result of an adverse event which occurs at some stage along the pathway of normal sexual differentiation. We owe our understanding of this pathway largely to the pioneering intrauterine experiments carried out by Alfred Jost in wartime France during the 1940's. He showed that by removing the gonadal ridges in the early rabbit embryo before the stage of sexual differentiation both the internal and external genitalia went on to develop along female lines irrespective of the genetic sex. In a second series of experiments, he inserted a crystal of testosterone where the ridges had been and observed that the external genitalia in all cases masculinized although the Mullerian ducts persisted and went on to form fallopian tubes and a uterus. Jost concluded from these studies that in sexual differentiation female was the pathway of default but for normal masculinization to occur, the testes must secrete, in addition to testosterone, another substance which was responsible for suppression of the Mullerian ducts which he called ilihormone inhibitrice.i We now know this as Mullerian inhibiting substance or anti-Mullerian hormone (AMH), a TGF-betalike growth factor which is produced by the Sertoli cells early in testicular development. We have subsequently come to appreciate that testosterone, produced by the Leydig cells, is converted in the peripheral tissues by the enzyme 5 alpha reductase to dihydrotestosterone which is actually responsible for the masculinizing process and that the effectiveness of these hormones depends on the integrity of testosterone receptors in the target cells. Nonetheless, it is clear that the testis occupies a central role in the pathway of male sexual differentiation. The gonads in the early embryo are identical in both sexes and initially appear as swellings in the central portion of the urogenital ridges on either side of the dorsal midline. The first signs of testicular organization can be recognized at 42 days when cells in the medial portion of the mesonephros aggregate into cords of primordial Sertoli cells which come to envelope inflowing germ cells to form primordial tubules. Leydig cells migrate into the developing testicular interstitium where they come to rest as clusters between the tubules. These events are clearly genetically determined but the precise mechanisms responsible for initiating these steps in testicular development were, until recently, unknown.

Production of cloned rabbits by somatic nuclear transfer.

Cloning and Stem CellsVol. 5, No. 4 Methods PapersProduction of Cloned Rabbits by Somatic Nuclear TransferM. Challah-Jacques, P. Chesne, and J.P. RenardM. Challah-JacquesSearch for more papers by this author, P. ChesneSearch for more papers by this author, and J.P. RenardSearch for more papers by this authorPublished Online:5 Jul 2004https://doi.org/10.1089/153623003772032808AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited byTransgenesis and Genome Engineering: A Historical Review31 March 2023Current Progress and Prospects in Rabbit Cloning Wenbin Cao, Jinpeng Zhao, Pengxiang Qu, and Enqi Liu4 April 2022 | Cellular Reprogramming, Vol. 24, No. 2Historical DNA Manipulation Overview14 June 2022Control of the proportion of inner cells by asymmetric divisions and the ensuing resilience of cloned rabbit embryos18 April 2018 | Development, Vol. 145, No. 8CYTOMORPHOLOGICAL RESEARCH OOCYTE RABBIT IN THE PROCESS EMBRYOGENESIS IN VITRO1 November 2016 | Animal Breeding and Genetics, Vol. 52Nuclear Transfer in Rabbit20 September 2014Production of Transgenic RabbitsHeterochromatin reprogramming in rabbit embryos after fertilization, intra-, and inter-species SCNT correlates with preimplantation developmentREPRODUCTION, Vol. 145, No. 2Generation of rabbit pluripotent stem cell linesTheriogenology, Vol. 78, No. 8Postimplantation Development of Cloned Rabbit Embryos Reconstructed with Foetal and Adult Skin-Derived Fibroblast Cell Nuclei15 February 2012 | Annals of Animal Science, Vol. 12, No. 1Generation of SV40-transformed rabbit tracheal-epithelial-cell-derived blastocyst by somatic cell nuclear transfer12 January 2012 | Cell and Tissue Research, Vol. 347, No. 2Follicular Oocytes Better Support Development in Rabbit Cloning Than Oviductal Oocytes Li-Ying Sung, Chien-Hong Chen, Jie Xu, Tzu-An Lin, Hwa-Yun Su, Wei-Fang Chang, Chia-Chia Liu, Yun-Shao Sung, Winston T.K. Cheng, Jifeng Zhang, X. Cindy Tian, Jyh-Cherng Ju, Y. Eugene Chen, Shinn-Chih Wu, and Fuliang Du6 December 2011 | Cellular Reprogramming, Vol. 13, No. 6Eutherian mammals use diverse strategies to initiate X-chromosome inactivation during development6 April 2011 | Nature, Vol. 472, No. 7343Structural, metabolic and developmental evaluation of ovulated rabbit oocytes before and after cryopreservation by vitrification and slow freezingTheriogenology, Vol. 74, No. 5Statistical Analysis of 3D Images Detects Regular Spatial Distributions of Centromeres and Chromocenters in Animal and Plant Nuclei8 July 2010 | PLoS Computational Biology, Vol. 6, No. 7Animal transgenesis: an overview25 November 2009 | Brain Structure and Function, Vol. 214, No. 2-3Beneficial Effect of Young Oocytes for Rabbit Somatic Cell Nuclear Transfer Fuliang Du, Jie Xu, Jifeng Zhang, Shaorong Gao, Mark G. Carter, Chingli He, Li-Ying Sung, Sanjeev Chaubal, Rafael A. Fissore, X. Cindy Tian, Xiangzhong Yang, and Y. Eugene Chen11 September 2010 | Cloning and Stem Cells, Vol. 11, No. 1Live Birth of Somatic Cell-Cloned Rabbits following Trichostatin A Treatment and Cotransfer of Parthenogenetic Embryos Qinggang Meng, Zsuzsanna Polgar, Jun Liu, and Andras Dinnyes12 March 2009 | Cloning and Stem Cells, Vol. 11, No. 1Rabbit CloningStem Cells: Promises Versus Limitations Despoina M. Choumerianou, Helen Dimitriou, and Maria Kalmanti10 March 2008 | Tissue Engineering Part B: Reviews, Vol. 14, No. 1Cumulus-specific genes are transcriptionally silent following somatic cell nuclear transfer in a mouse modelJournal of Zhejiang University SCIENCE B, Vol. 8, No. 8Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation23 January 2007 | Reproduction, Vol. 133, No. 1Aberrant profile of gene expression in cloned mouse embryos derived from donor cumulus nuclei5 April 2006 | Cell and Tissue Research, Vol. 325, No. 2Factors affecting the efficiency of embryo transfer in the domestic ferret (Mustela putorius furo)Theriogenology, Vol. 66, No. 2Somatic Cell Nuclei in Cloning Volume 5Issue 4Dec 2003 To cite this article:M. Challah-Jacques, P. Chesne, and J.P. Renard.Production of Cloned Rabbits by Somatic Nuclear Transfer.Cloning and Stem Cells.Dec 2003.295-299.http://doi.org/10.1089/153623003772032808Published in Volume: 5 Issue 4: July 5, 2004PDF download

Developmental Stages in the Rabbit Embryo: Guidelines to Choose an Appropriate Experimental Model

Researchers involved in the field of congenital malformations are often forced to work on an animal model. Both accurate description of its normal development and comparative staging with human development will be mandatory. To complete the lacking medical literature, we herein provide such data for the rabbit model. Sampled rabbit embryos were staged using the Carnegie criteria, in order first to determine if they were consistent with the rabbit developmental pattern, and second to compare this pattern with the human one. Our results show a suitable comparison of rabbits and humans in early developmental stages, except for the neural growth.

Absence of embryo-fetal toxicity in rats or rabbits following oral dosing with nelfinavir

The potential for nelfinavir mesylate (VIRACEPT) to induce maternal and embryo-fetal toxicity was evaluated in rats and rabbits following oral administration. The drug was administered by gavage to rats at doses of 200, 500, or 1000 mg/kg/day on days 6–17 of gestation and to rabbits at doses of 200, 400, or 1000 mg/kg/day on days 7–20 of gestation. Dams and does were euthanized on GD20 and 29, respectively, and the offspring were weighed and examined for external, visceral, and skeletal alterations. Maximum plasma nelfinavir concentrations ( C max) in rats were comparable to C max values in humans and were 3- to 6-fold higher than the reported human trough levels, while plasma nelfinavir levels in rabbits were approximately 0.13–0.17× the human C max and 0.25–0.5× the human trough. In rats, no treatment-related maternal or embryo-fetal toxicity was observed at any dose level and the NOAEL for both maternal and fetal toxicity was considered to be 1000 mg/kg/day. Two rabbits in the 400 mg/kg/day group died prior to scheduled termination. Because no deaths occurred in the high dose group and there were no other treatment-related signs of clinical toxicity in any dose group, these deaths were considered unrelated to nelfinavir. Group mean body weight loss in rabbits was observed at 1000 mg/kg/day on gestation days 7–10. Food consumption was also reduced in this treatment group throughout the dosing period. There were no treatment-related findings in other maternal or fetal parameters. Thus, the no-observed-adverse-effect-level (NOAEL) for maternal toxicity in the rabbit was considered to be 400 mg/kg/day (based on maternal body weight loss in the high dose group), while the NOAEL for embryo-fetal toxicity in the rabbit was considered to be 1000 mg/kg/day. Thus, under the conditions of this study, nelfinavir was not considered to be toxic to the rat or rabbit conceptus.

Developmental toxicology studies of WHI-07, a novel nucleoside analogue-based dual-function microbicide, administered intravaginally to rabbits.

The zidovudine derivative, 5-bromo-6-methoxy-5,6-dihydro-3-azidothymidine-5'-(p-bromophenyl) methoxy alaninyl phosphate (WHI-07), is a dual-function spermicidal and anti-HIV agent with contraceptive and microbicidal activity. In previous subchronic and reproductive toxicity studies and a two-year carcinogenicity study, daily intravaginal application of 0.5 to 2.0% WHI-07 via a gel-microemulsion, was shown to cause no local, systemic and reproductive toxicity or increased carcinogenicity in mice. To evaluate the developmental toxicity potential of WHI-07 in a nonrodent model, subgroups of 20 superovulated NZW rabbits were artificially inseminated and exposed intravaginally to a gel-microemulsion containing 0, 0.5, 1.0, or 2.0% WHI-07 during major organogenesis [gestation days 6-18]. The dose of WHI-07 was equivalent to 1.4x10(6) to 5.7x10(6) times its anti-HIV IC50 and 1400 to 5700 times its spermicidal EC50. Throughout the duration of the experiment (GD 0-29), clinical observations, food consumption, and body weights were recorded. Reproductive and fetal parameters were evaluated following uterotomies on GD 29. Measurements included numbers of corpora lutea, pregnancy, number and distribution of implantations, resorptions, live and dead fetuses, fetal weight, sex ratio, and gross external and skeletal malformations and variations. Maternal food consumption and body weight gain were unaffected by WHI-07 treatment. Hematologic and clinical chemistry determinations on GD 19 and 29 revealed no treatment-related maternal effects. Prior studies of repeated intravaginal administration of WHI-07 gel-microemulsion revealed lack of local toxicity to rabbit vaginal mucosa. In the current study, no drug-related gross lesions were apparent at necropsy. Reproductive indices, ie, pregnancy rate, gravid uterine weights, litter size, number of corpora lutea, implantation sites, pre- and postimplantation losses, viable fetuses, resorptions, fetal body weights, and fetal sex ratio, were not affected by intravaginal exposure to WHI-07. External, and skeletal examinations of fetuses for malformations and variations did not reveal any evidence of teratogenicity in any WHI-07-treated groups. Intravaginal administration of WHI-07 at concentrations as high as 2% did not produce teratogenicity or other developmental toxicity in rabbit conceptus. These findings indicated that WHI-07 shows unique clinical potential to become the active ingredient of a new female-controlled topical microbicidal vaginal contraceptive for women who are at high risk of acquiring HIV/AIDS.

Analysis of gene expression in rabbit nuclear transfer embryos: Use of single-embryo mRNA differential display.

Lack of or abnormal expression of developmentally important genes is believed to hamper early development of the nuclear transfer (NT) embryo. To identify stage-specific genes in rabbit NT embryo development, mRNA differential display was used to compare the mRNA content of rabbit NT embryos at different developmental stages, from Metaphase II oocytes to 8-16-cell stage embryos. Thirty-four zygotic transcripts, which abruptly appeared at the 8-16-cell stage in rabbit NT embryos, were isolated; 11 of these were potential novel genes with no matches in the current databases. Of the remaining 23, 12 were matched with established sequence tags with functions uncharacterized and the other 11 were homologous to those in the European Molecular Biology Laboratory (EMBL) and GenBank databases. The differential expression of eight of the 34 amplicons were confirmed by reverse Northern blotting, and four positive clones were validated. Previous studies and present data indicated that these three genes were probably related to preimplantation rabbit embryo development.

Haemoglobin biosynthesis site in rabbit embryo erythroid cells.

Properly metabolized globin synthesis and iron uptake are indispensable for erythroid cell differentiation and maturation. Mitochondrial participation is crucial in the process of haeme synthesis for cytochromes and haemoglobin. We studied the final biosynthesis site of haemoglobin using an ultrastructural approach, with erythroid cells obtained from rabbit embryos, in order to compare these results with those of animals treated with saponine or phenylhydrazine. Our results are similar to those obtained in assays with adult mammals, birds, amphibians, reptiles and fish, after induction of haemolytic anaemia. Therefore, the treatment did not interfere with the process studied, confirming our previous findings. Immunoelectron microscopy showed no labelling of mitochondria or other cellular organelles supposedly involved in the final biosynthesis of haemoglobin molecules, suggesting instead that it occurs free in the cytoplasm immediately after the liberation of haeme from the mitochondria, by electrostatic attraction between haeme and globin chains.

Corrigendum to “Nonequilibrium cryopreservation of rabbit embryos using a modified (sealed) open pulled straw procedure” [Theriogenology 58 (2002) 1541–1552

Corrigendum to “Nonequilibrium cryopreservation of rabbit embryos using a modified (sealed) open pulled straw procedure” [Theriogenology 58 (2002) 1541–1552

Using the male gamete for assisted reproduction: past, present, and future.

Using the male gamete for assisted reproduction: past, present, and future.

Inositol transport in preimplantation rabbit embryos: effects of embryo stage, sodium, osmolality and metabolic inhibitors

The preimplantation period in the rabbit consists of a 3 day cleavage stage during which the number of cells increases with little change in embryo size, followed by a 3-4 day blastocyst stage during which the inner cell mass, the blastocoel and the trophectodermal layer are formed and the embryo grows rapidly in size and protein content. This study used [(3)H]inositol to investigate the transport of inositol, an essential component of the phosphatidylinositol signal transduction system, over the 6 days of preimplantation development by rabbit embryos. In the presence of 15 micromol inositol l(-1) in the incubation medium, there was a small linear increase in inositol uptake from 0.07 pmol per embryo per h at the one-cell stage (day 1) to 0.135 pmol at the late morula (day 3) stage. Inositol uptake increased to 0.58 pmol per embryo per h for early blastocysts (day 4) and 23.7 pmol for late blastocysts (day 6). There was a significant linear relationship between inositol uptake and blastocyst diameter and surface area. Efflux of inositol from early morulae was minimal (about 1.25% of embryo content per h), whereas efflux from mid-blastocysts (day 5) was much greater (about 15.6% of embryo content per h). Efflux of inositol from both early morulae and mid-blastocysts was increased by decreasing the osmolality of the incubation medium. Varying the osmolality had no effect on inositol uptake up to 2 h. Inositol uptake was dependent on sodium in cleavage-stage embryos but independent of sodium in blastocyst stages. In early morulae, inositol uptake was inhibited by glucose and the sodium-dependent hexose transport inhibitor, phloridzin, but not by the facilitated transport inhibitor, phloretin. Inositol uptake in early morulae was saturable; estimates of 0.227 and 0.288 pmol per morula per h for V(max) and 0.045 and 0.038 micromol l(-1) for K(m) were obtained for sodium-dependent transport in two separate experiments. All of these results are consistent with the hypothesis that transport in cleavage stages occurs via a sodium myo-inositol transporter (SMIT) protein. Uptake in blastocysts was non-saturable. Uptake into blastocysts appeared to take place by a transcellular rather than a paracellular route.

Inositol transport in preimplantation rabbit embryos: effects of embryo stage, sodium, osmolality and metabolic inhibitors

The preimplantation period in the rabbit consists of a 3 day cleavage stage during which the number of cells increases with little change in embryo size, followed by a 3-4 day blastocyst stage during which the inner cell mass, the blastocoel and the trophectodermal layer are formed and the embryo grows rapidly in size and protein content. This study used [(3)H]inositol to investigate the transport of inositol, an essential component of the phosphatidylinositol signal transduction system, over the 6 days of preimplantation development by rabbit embryos. In the presence of 15 micromol inositol l(-1) in the incubation medium, there was a small linear increase in inositol uptake from 0.07 pmol per embryo per h at the one-cell stage (day 1) to 0.135 pmol at the late morula (day 3) stage. Inositol uptake increased to 0.58 pmol per embryo per h for early blastocysts (day 4) and 23.7 pmol for late blastocysts (day 6). There was a significant linear relationship between inositol uptake and blastocyst diameter and surface area. Efflux of inositol from early morulae was minimal (about 1.25% of embryo content per h), whereas efflux from mid-blastocysts (day 5) was much greater (about 15.6% of embryo content per h). Efflux of inositol from both early morulae and mid-blastocysts was increased by decreasing the osmolality of the incubation medium. Varying the osmolality had no effect on inositol uptake up to 2 h. Inositol uptake was dependent on sodium in cleavage-stage embryos but independent of sodium in blastocyst stages. In early morulae, inositol uptake was inhibited by glucose and the sodium-dependent hexose transport inhibitor, phloridzin, but not by the facilitated transport inhibitor, phloretin. Inositol uptake in early morulae was saturable; estimates of 0.227 and 0.288 pmol per morula per h for V(max) and 0.045 and 0.038 micromol l(-1) for K(m) were obtained for sodium-dependent transport in two separate experiments. All of these results are consistent with the hypothesis that transport in cleavage stages occurs via a sodium myo-inositol transporter (SMIT) protein. Uptake in blastocysts was non-saturable. Uptake into blastocysts appeared to take place by a transcellular rather than a paracellular route.

Significance of 2-methoxypropionic acid formed from beta-propylene glycol monomethyl ether: integration of pharmacokinetic and developmental toxicity assessments in rabbits.

Commercial grade propylene glycol monomethyl ether (PGME), which is composed of > 99.5% alpha-isomer and < 0.5% beta-isomer, has been shown in several studies to have a low potential for developmental toxicity. Nonetheless, questions have been raised about potential human developmental toxicity due to beta-PGME, because it can be metabolized to 2-methoxypropionic acid (MPA), a compound bearing structural similarity to the teratogen, methoxyacetic acid (MAA). Accordingly, a series of in vivo developmental toxicity, whole embryo culture, and in vivo pharmacokinetic experiments were conducted in New Zealand White rabbits (highly sensitive to these compounds) to better understand the developmental toxicity potential of MPA and the kinetics of its formation from beta-PGME. For the in vivo developmental toxicity studies, groups of 20 inseminated rabbits were gavaged with 0, 10, 26, or 78 mg/kg/day of MPA on gestation day (GD) 7-19, followed by fetal evaluation on GD 28. Results with MPA were compared with those of rabbits similarly dosed with 0, 2.5, 7.5, or 15 mg/kg/day of MAA. Developmental toxicity no-observable-effect levels (NOEL) were approximately 10-fold higher for MPA (26 mg/kg/day) than for MAA (2.5 mg/kg/day). Also, the severity of effects caused by MPA was less than that of MAA, and unlike MAA, MPA was not selectively toxic to the fetus. This differential toxicity was also seen in whole embryo cultures of GD 9 rabbit embryos, in which there were no adverse effects of MPA (1.0, 5.0 mM) or its parent compound, beta-PGME (0.5, 2.0 mM), but severe dysmorphogenesis in 100% of embryos cultured in 5.0 mM MAA. The pharmacokinetics study showed rapid and complete conversion of beta-PGME to MPA, with a relatively long elimination half-life (33-44 h) for MPA. However, peak and AUC concentrations of MPA in blood associated with the MPA LOEL dose of 78 mg/kg/day were 1.3 mM and 52.9 mM-h/l, respectively, suggesting a relatively high threshold based on internal dosimetry. Taken together, these data indicate a negligible risk of developmental toxicity due to MPA formation from the small amounts of beta-isomer present in commercial PGME.

Polychlorinated biphenyls affect gene expression in the rabbit preimplantation embryo.

Polychlorinated biphenyls (PCBs) have been shown to be embryotoxic. The mechanism(s) of action is not clearly understood. The toxic effects could be either direct or indirect. Furthermore, PCB congeners vary in their toxic potential. They can be classified in coplanar PCBs binding to the transcription factor aryl hydrocarbon receptor (AhR), which induce subsequent changes in gene expression, and noncoplanar PCBs exhibiting AhR-independent effects. In order to investigate possible mechanisms, 5 and 6 days old preimplantation rabbit embryos were exposed in vitro to low levels of coplanar (PCB 77, 126, and 169) or noncoplanar PCBs (PCB 28, 52, 101, 118, 138, 153, and 180). The PCB effects were studied by semiquantitative RT-PCR analysis of AhR target genes (cytochrome P450 (CYP) 1A1, 1A2, UDP-glucuronosyl transferase 1, glutathione S-transferase pi1 and aldehyde dehydrogenase) and dioxin-responsive genes (IL 1beta, PAI 2, Cox 2, TGFalpha, EGF, erbB 1-4, c-fos, c-jun, HSP 90, cyclophilin 40), and by differential display (DD) RT-PCR. CYP 1B1 mRNA and AhR protein were localized by in situ hybridization and immunohistochemistry, respectively. From the AhR target genes studied only CYP 1B1, and cyclooxygenase 2 showed an increase in mRNA levels after coplanar and noncoplanar PCB. Interleukin 1beta and plasminogen activator inhibitor 2 were downregulated. CYP 1B1 mRNA showed a stage specific inducibility at day 6, but not at day 5. By DD RT-PCR we identified six new genes previously not reported to be regulated by PCBs. The mRNAs encoding the subunits 1, 2, 4, and 5 of the NADH ubiquinone oxidoreductase and beta-globin showed a decrease, whereas trichohyalin mRNA was increased after PCB exposure. Coplanar and noncoplanar PCB congeners elicited similar responses on the mRNA levels of the studied genes. Exposure to coplanar PCBs did not result in the AhR being translocated to the nucleus. Our results show that (i). PCBs induce changes in gene expression in rabbit day 5 and 6 preimplantation embryos and imply (ii). that the transcriptional changes observed were not mediated by the nuclear AhR.

Differential Influences in Sizes and Cell Cycle Stages of Donor Blastomeres on the Development of Cloned Rabbit Embryos

Experiments were conducted to evaluate the effect of blastomere diameters and cell cycle stages on the subsequent development of nuclear transplant rabbit embryos (NT-embryos) using nuclei derived from the 16- or 32-cell stage embryos. All blastomeres and NT-embryos were cultured individually in modified Ham's F-10 medium supplemented with 10% rabbit serum (RS) at 38°C and 5% CO 2 in air. The diameter of blastomeres from 16-cell stage embryos was found twice of those from 32-cell stage (51 vs 27 μm). Significant differences were observed in cleavage rates (>3 divisions) in the isolated single blastomeres (54 vs 48 for 16-cell; 28 vs 14 for 32-cell, p 3 divisions) of the NT-embryos were greater in the small nuclear donors than in the large donors (73 vs 55%, p 0.05). In conclusion, it appeared that no difference in the developmental competence between large and small isolated blastomeres was observed. When smaller 16-cell stage blastomeres were used as nuclear donor, the cleavage rate or development of NT-embryos was improved and was compromised when 32-cell stage blastomeres were used. Therefore, control nuclear stage of the donor cell at G 1 phase in preactivated nuclear recipients seemed to be beneficial for the cleavage rate of the reconstructed embryo in the 16-cell transplant, but not for subsequent morula or blastocyst development.