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PubMed2003

Fras1 deficiency results in cryptophthalmos, renal agenesis and blebbed phenotype in mice.

Loss of tight association between epidermis and dermis underlies several blistering disorders and is frequently caused by impaired function of extracellular matrix (ECM) proteins. Here we describe a new protein in mouse, Fras1, that is specifically detected in a linear fashion underlying the epidermis and the basal surface of other epithelia in embryos. Loss of Fras1 function results in the formation of subepidermal hemorrhagic blisters as well as unilateral or bilateral renal agenesis during mouse embryogenesis. Postnatally, homozygous Fras1 mutants have fusion of the eyelids and digits and unilateral renal agenesis or dysplasia. The defects observed in Fras1-/- mice phenocopy those of the existing bl (blebbed) mouse mutants, which have been considered a model for the human genetic disorder Fraser syndrome. We show that bl/bl homozygous embryos are devoid of Fras1 protein, consistent with the finding that Fras1 is mutated in these mice. In sum, our data suggest that perturbations in the composition of the extracellular space underlying epithelia could account for the onset of the blebbed phenotype in mouse and Fraser syndrome manifestation in human.

Animals↗
PubMed1983

The inter-ovarian distribution of twin ovulations and embryo survival in the bovine.

Data in the literature on twin ovulations and twin pregnancies were used to examine the distribution (unilateral vs bilateral) of twin ovulations and associated effects on embryo survival. Unilateral twin ovulations averaged 52% but there was significant heterogeneity among sources. Twenty-five percent of subjects which became pregnant following twin ovulations had unilateral twin ovulations. This suggests that fertilisation failure or embryo mortality, or both, are greater for unilateral twin ovulations. Using data on pregnant cows, the probability of embryo loss increased by an estimated 0.22 for unilateral twin ovulations compared with bilateral twin ovulations or single ovulations. Using the estimates obtained, the expected litter size was calculated for a range of ovulation rates with varying proportions of unilateral twin ovulations and different levels of embryo mortality. Litter size was not very sensitive to the distribution of twin ovulations.

Journal Article↗
PubMed2004

Use of assisted reproductive technologies in the propagation of rhesus macaque offspring.

The assisted reproductive technologies (ARTs) as tailored to the production of rhesus monkeys at the Oregon National Primate Research Center (ONPRC) are described. Efficient fertilization of mature oocytes recovered by aspiration from females subjected to follicular stimulation was achieved with fresh or frozen sperm by intracytoplasmic sperm injection (ICSI). Embryo development to the early cleavage stage occurred at high frequency. Cryopreserved embryos showed high postthaw survival and were also transferred in efforts to establish pregnancies. Three methods of transfer were evaluated, two involving embryo placement into the oviduct, laparoscopy and minilaparotomy, and a nonsurgical, transcervical approach that resulted in uterine deposition. Early cleaving embryos (Days 1-4) were transferred into the oviducts of synchronized recipients with optimal results and pregnancy rates of up to 36%. Pregnancy rates were similar when two fresh or frozen embryos were transferred (28- 30%), although more than two embryos had to be thawed to compensate for embryo loss during freeze-thawing. Normal gestational lengths, birth weights, and growth curves were seen with ART-produced infants compared with infants produced by natural mating in the timed mated breeding (TMB) colony at the ONPRC. In 72 singleton pregnancies established following the transfer of ART-produced embryos, the live-birth rate, at 87.5%, was statistically identical to that for the TMB colony. Further development of the ARTs should result in increasing use of these techniques to augment conventional approaches to propagating monkeys, especially those of defined genotypes.

Animals↗
PubMed2005

Fertility in South Australian commercial Merino flocks: sources of reproductive wastage.

To identify reasons for low fertility in the Merino in South Australia, we defined the extent and sources of reproductive wastage in flocks of maiden (n=14) and mature age (n=54) Merino ewes managed on 43 properties over 4 year. In a second study, reproductive wastage was examined in a very high ovulating flock of mature age South Australian Merino ewes heterozygous for the FecB gene, mated to lamb in either the autumn or spring. Losses in the latter flock were examined in less detail than the large-scale study, but allowed wastage to be partitioned between pre- and peri/post-natal sources. In the first experiment, reproductive wastage between mating and weaning was estimated at 59.7 potential lambs or ova lost per 100 ewes exposed to rams, representing 42.4% of ova shed. The main source of wastage occurred at lambing (55.6% of total loss), with death of twins a major contributor (35.3% of total loss). Other important sources of wastage were from partial failure of multiple ovulation (PFMO; 20.4% of total loss) and from ewes mating but not lambing (13.2% of total loss). Reproductive wastage did not vary with either age of ewe (maiden versus mature) or season of mating (October-December versus January-March). Mean ovulation rate, estimated as 141 per 100 ewes ovulating (range 100-200), varied with age of ewe (129 versus 144; maiden versus mature) but not with season of mating or year (1990-1991). Values for fertility, fecundity and lamb survival were 90, 127 and 73%, respectively. Fecundity of maiden ewes was lower than that of mature ewes (116 versus 130). Survival of single and twin lambs between birth and tail docking was 83 and 56%, respectively. Net reproductive efficiency, or lambs weaned per 100 ewes exposed to rams, averaged 81 (range 31-122). In the second experiment, mean ovulation rate of FecB Merino ewes was 316 per 100 ewes ovulating, with 242 ova (potential lambs) lost between ovulation and tail docking. This loss was equally shared between pre- and peri/post-natal sources (123 versus 119), and demonstrates severe limitation of the Merino to successfully bear and rear multiple litters. We concluded that future research in commercial Merino flocks be focused on lamb mortality, particularly of twins, and on PFMO, the major source of embryo loss.

Aging↗
PubMed1998

Anti-phospholipid syndrome: from patient's bedside to experimental animal models and back to the patient's bedside.

The availability of animal models of the APS has provided a lot of experimental data which might be considered in trying to unravel several questions concerning this complicated disease. The main clinical manifestations associated with this disorder are repeated pregnancy loss, thrombocytopenia and thrombotic events. Other manifestations have been reported in relation to APS. However, the association with anti-phospholipid antibodies (aPL) are still uncertain. In the APS murine models presented here, both the Lupus-prone mice and the naive mice with induced APS, fetal resorption (parallels to embryo loss) and reduced fecundity rate were prominent features strongly associated with pathogenic aCL antibodies, making these models appropriate for investigating the human disease. Utilizing these models for APS have enabled to show the pathogenicity of aPL in pregnancy loss, neurological and behavioral changes, renal involvement and thrombus formation. Antiphospholipid antibodies from patients with APS, as well as natural aCL antibodies exerted pathogenic effects in naive mice, and in an in vivo thrombosis model. Several therapeutic modalities were found promising for application in the clinics. These include the antithrombotic and anticoagulant treatments using aspirin or LMWH, IL-3, or immunomodulation by high dose IVIG, specific anti-idiotypic or anti-CD4 antibodies, cyprofloxacin or bromocriptin administration.

Animals↗
PubMed1993

Embryonic losses after in vitro fertilization and embryo transfer.

Early loss of embryos is a major problem after in vitro fertilization (IVF) and embryo transfer (ET). Because implantation of the embryo is a precondition for an increasing serum beta-hCG level, the embryonic development cannot be evaluated during the pre- and peri-implantation period. Early pregnancy factor (EPF) is a marker of the development of the embryo before and during implantation. Therefore serum samples of 82 patients were screened for EPF and beta-hCG after IVF/ET. 63% (52/82) of all patients were positive for EPF, 21% (17/82) showed elevated serum beta-hCG levels. Clinical pregnancies occurred in 12 patients (15%) resulting in nine deliveries. After difficult ET EPF turned negative more frequently in the peri-implantation period compared to transfers without problems. Emphasis should be directed towards optimizing the placement of the embryos and towards minimizing the trauma of the uterus.

Abortion, Spontaneous↗
PubMed1997

Reproductive efficiency in mink (Mustela vison) treated with the pesticides lindane, carbofuran and pentachlorophenol.

Mink are carnivores of agroforestry fringe habitats and are exposed to pesticides that biomagnify within the food chain. Some pesticides are thought to disrupt reproductive and endocrine functions. In Expt 1, four groups of mink (n = 10) were fed either a control diet, or diets treated with lindane (1 mg kg-1 day-1), carbofuran (0.05 mg kg-1 day-1) or pentachlorophenol (1 mg kg-1 day-1) from before breeding until weaning. Mink were mated twice, at 7-8 day intervals. The treatments had no effect on the proportion of mink accepting the first mating; however, lindane and pentachlorophenol caused a decrease in the percentage of females accepting the second mating. Lindane and pentachlorophenol caused a decrease in whelping rate, although litter size was not affected. Carbofuran had no effect on fertility. Mink that mated only once had a lower whelping rate than mink that mated twice; therefore, it could not be determined whether the decreased whelping rates were due to the lack of a second mating or to increased embryo loss. In Expt 2, two groups of mink (n = 15) were fed a control diet or a diet treated with lindane (1 mg kg-1 day-1) from before mating until weaning. Mink were mated twice on two consecutive days. Lindane did not affect mating response at either mating. Whelping rate, but not implantation rate, was decreased by the lindane treatment. The proportion of embryos lost after implantation (implantation scars not represented by kits at whelping) was increased by the lindane treatment. In conclusion, both lindane and pentachlorophenol decreased fertility in mink, and the lindane effect was primarily a result of embryo mortality after implantation.

Animals↗
PubMed2005

Epigenetic programming in the preimplantation rat embryo is disrupted by chronic paternal cyclophosphamide exposure.

Preconceptional paternal exposure to cyclophosphamide, a widely used anticancer agent, leads to increases in embryo loss, malformations, and behavioral deficits in offspring; these abnormalities are transmissible to subsequent generations [Auroux, M., Dulioust, E., Selva, J. & Rince, P. (1990) Mutat. Res. 229, 189-200]. Little information exists on the mechanisms underlying this male-mediated developmental toxicity. We assessed the impact of paternal cyclophosphamide exposure on the dynamic regulation of histone H4 acetylation at lysine 5 and DNA methylation in preimplantation rat embryos. Zygotes sired by drug-treated males displayed advanced developmental progression, increased pronuclear areas, and disruption of the epigenetic programming of both parental genomes. Early postfertilization zygotic pronuclei were hyperacetylated; by mid-zygotic development, male pronuclei were dramatically hypomethylated, whereas female pronuclei were hypermethylated. Micronuclei were substantially elevated, and histone H4 acetylation at lysine 5 localization to the nuclear periphery was disrupted in two-cell embryos fertilized by cyclophosphamide-exposed spermatozoa. This finding demonstrates that paternal exposure to this drug induces aberrant epigenetic programming in early embryos. We hypothesize that disturbances in epigenetic programming contribute to heritable instabilities later in development, emphasizing the importance of epigenetic risk assessment after chemotherapy.

Acetylation↗
PubMed1999

Positive outcome after preimplantation diagnosis of aneuploidy in human embryos.

usromosomal abnormalities are responsible for a great deal of embryo wastage, which is reflected, at least partially, in decreased implantation and increased miscarriage in older women. To address this problem the transfer of only chromosomally normal embryos previously selected by preimplantation genetic diagnosis (PGD) has been proposed. We designed a multi-centre in-vitro fertilization (IVF) study to compare controls and a test group that underwent embryo biopsy and PGD for aneuploidy. Patients were matched retrospectively, but blindly, for average maternal age, number of previous IVF cycles, duration of stimulation, oestradiol concentrations on day +1, and average mature follicles. All these parameters were similar in test and control groups. Only embryos classified as normal for those chromosomes were transferred after PGD. The results showed that the rates of fetal heart beat (FHB)/embryo transferred between the control and test groups were similar. However, spontaneous abortions, measured as FHB aborted/FHB detected, decreased after PGD (P < 0.05), and ongoing pregnancies and delivered babies increased (P < 0.05) in the PGD group of patients. Two conclusions were obtained: (i) PGD of aneuploidy reduced embryo loss after implantation; (ii) implantation rates were not significantly improved, but the proportion of ongoing and delivered babies was increased.

Aneuploidy↗
PubMed1994

Optimization of superovulation in the reproductively mature mouse.

Optimum gonadotropin doses and chronology were established for the induction of superovulation in sexually mature hybrid mice (BALB/cBy x C57BL/6By). A regime of 12 IU pregnant mares' serum gonadotropin (PMSG), followed 48 hr later by 20 IU human chorionic gonadotropin (hCG) administered 1 hr before the midpoint of the light cycle (1200), gave the maximum ovulatory response. There was no evidence that endogenous luteinizing hormone influenced the superovulation response to exogenous gonadotropins. Fewer than 50% of zygotes reached the blastocyst stage (90-93 hr post hCG), with the greatest rate of loss at the two- to four-cell stage. Litter size following superovulation was 19.6 +/- 0.9. There was no significant difference between the number of blastocysts observed and litter size. Similarly, counts of mature follicles in ovaries prior to hCG stimulation were not significantly greater than the number of secondary oocytes that subsequently ovulated. These data indicate that standard superovulation protocols may require fine-tuning to maximize productivity and confirm that embryo loss is greatest between the first cleavage division and blastocyst formation.

Analysis of Variance↗
PubMed1996

Retinoic acid induces stage-specific repatterning of the rostral central nervous system.

We had previously reported that in gastrulating mouse embryos retinoic acid (RA) induces morphological as well molecular alterations strictly depending on the time of administration. In particular, embryos treated with RA at the mid-late streak stage share reduction of the rostral central nervous system (CNS) and increase of the hindbrain. In the same embryos, loss of the forebrain-expressed genes, such as Emx1, Emx2, and Dlx1, and rostral ectopic expression of the Hoxb-1 gene suggest an antero-posterior (A/P) ordered repatterning of the fore-, mid-, and hindbrain regions. Several genes, such as Pax-2, Wnt-1, En-2, and En-1, are involved in the establishment of midbrain and rostral hindbrain regional identities and boundaries. We report that these genes become coordinately anteriorized only in embryos treated with RA at the late streak stage. Moreover, in the hindbrain of the same embryos, at 8.5 days post coitum (dpc), Wnt-1 and Pax-2 are rostrally induced all along the neural plate. Considering that forebrain markers are repressed in embryos treated with RA at the same time, these findings strongly support the idea that RA administration at the late streak stage induces an ordered repatterning of the rostral CNS, possibly altering the A/P nature of mesendodermal inductive signals.

Animals↗
PubMed2004

Allelic loss underlies type 2 segmental Hailey-Hailey disease, providing molecular confirmation of a novel genetic concept.

Hailey-Hailey disease (HHD) is an autosomal dominant trait characterized by erythematous and oozing skin lesions preponderantly involving the body folds. In the present unusual case, however, unilateral segmental areas along the lines of Blaschko showing a rather severe involvement were superimposed on the ordinary symmetrical phenotype. Based on this observation and similar forms of mosaicism as reported in other autosomal dominant skin disorders, we postulated that in such cases, 2 different types of segmental involvement can be distinguished. Accordingly, the linear lesions as noted in the present case would exemplify type 2 segmental HHD. In the heterozygous embryo, loss of heterozygosity occurring at an early developmental stage would have given rise to pronounced linear lesions reflecting homozygosity or hemizygosity for the mutation. By analyzing DNA and RNA derived from blood and skin samples as well as keratinocytes of the index patient with various molecular techniques including RT-PCR, real-time PCR, and microsatellite analysis, we found a consistent loss of the paternal wild-type allele in more severely affected segmental skin regions, confirming this hypothesis for the first time, to our knowledge, at the molecular and cellular level.

Alleles↗
PubMedDate not supplied

Effect of ginger tea on the fetal development of Sprague-Dawley rats.

This study investigated the effect of ginger, a common morning sickness remedy, on fetal development. Pregnant Sprague-Dawley rats were administered, from gestation day 6 to 15, 20 g/liter or 50 g/liter ginger tea via their drinking water and then sacrificed at day 20. No maternal toxicity was observed, however embryonic loss in the treatment groups was double that of the controls (P<0.05). No gross morphologic malformations were seen in the treated fetuses. Fetuses exposed to ginger tea were found to be significantly heavier than controls, an effect that was greater in female fetuses and was not correlated with increased placental size. Treated fetuses also had more advanced skeletal development as determined by measurement of sternal and metacarpal ossification centers. The results of this study suggest that in utero exposure to ginger tea results in increased early embryo loss with increased growth in surviving fetuses.

Abnormalities, Drug-Induced↗
PubMed2003

TNF-alpha protects embryos exposed to developmental toxicants.

BACKGROUND: Tumor necrosis factor alpha (TNF-alpha) has been implicated in mediating post-implantation embryo loss or the embryonic maldevelopment induced by development toxicants or maternal metabolic imbalances. In order to clarify the role of TNF-alpha further, a comparative study was performed in TNF-alpha, knockout and TNF-alpha, positive mice, exposed to a reference teratogen, cyclophosphamide (CP). METHODS: Cyclophosphamide was injected on day 12 of pregnancy and 18-day fetuses were examined for external structural anomalies. Apoptosis and cell proliferation were measured by TdT-mediated biotin-dUTP nick-end labeling and 5'-bromo-2'-deoxyuridine incorporation, respectively, in the brain (an organ, sensitive to the teratogen) of embryos 24 hr after CP injection. NF-kappaB DNA-binding activity by electrophoretic mobility shift assay (EMSA) and the expression of Re1lA (an NF-kappaB subunit) and I(kappa)B(alpha) proteins by Western blot analysis were assessed in the brain of embryos tested 24 and 48 hr after CP treatment. RESULTS: Surprisingly, the proportion of fetuses with craniofacial, trunk and severe limb reduction anomalies were significantly higher in TNF-alpha -/- females, than in TNF-alpha,+/+ mice. Excessive apoptosis and suppression of cell proliferation was found in the brain, and they were more prominent in TNF-alpha -/- than TNF-alpha +/+ embryos, when examined 24 hr after CP injection. Finally, CP-induced suppression of NF-kappaB DNA-binding activity was found to be enhanced in the brain of TNF-alpha -/- embryos, and the restoration of NF-kappaB DNA-binding activity was compromised. CONCLUSION: This work demonstrates for the first time that TNF-alpha may act as a protector of embryos exposed to teratogenic stress. One possible mechanism may be restoration of NF-kappaB activity in embryonic cells surviving the teratogenic insult.

Animals↗
PubMed1996

Offspring sex-ratio and reproductive performance in heterogametic females of the South American field mouse Akodon azarae. Reproduction in heterogametic Akodon azarae females.

We have compared the breeding performance of homogametic (XX) and heterogametic (XY*) females of the South American sigmodontine rodent Akodon azarae under laboratory conditions. XY* females showed an enhanced reproductive performance when compared with normal, XX, females. The XY* females had a longer reproductive lifespan. They started to reproduce early, had more frequent litters, and stopped reproduction later than XX females. Their progeny showed a biased 1:2 male:female sex ratio which may be explained by the early loss of YY* zygotes after fertilization. However, litter size at birth was similar both in XY* and XX females, and no difference in ovulation rate was detected between them. This indicates that an "automatic" rather than an "evolved" reproductive compensation mechanism may be acting in heterogametic females. A separate study has shown that self-synapsis of both the X and Y* chromosomes takes place during meiosis, allowing the oocytes to escape from functional deterioration. It is suggested that self-synapsis and "automatic" reproductive compensation account for the preservation of fertility in heterogametic females in spite of the heteromorphic sex chromosomes and the early embryo loss they experience. However, these mechanisms do not account for the enhancement of reproductive lifespan. The possibility that an intrauterine position phenomenon is acting in A. azarae is discussed.

Animals↗
PubMedDate not supplied

Reproductive failure due to spontaneous abortion and recurrent miscarriage.

The epidemiology, aetiology, diagnosis and clinical management of spontaneous and recurrent abortion and of the failure of embryo implantation are discussed in a retrospective overview of the major studies conducted since 1975 identified through a Medline search. Infertile women who experienced spontaneous single (32%) and recurrent (0.5%) abortion as well as those who became pregnant after induction of ovulation with gonadotrophins (abortion rate 17-31%) and those who underwent assisted fertilization programmes (abortion rate 18-34%) are considered. Causes and treatments are here reported. Medical treatments for immunologically mediated abortion (IMA) are based on prednisolone, heparin, aspirin and intravenous immunoglobulin. Efficacy of the medical treatment of patients with a history of IMA has yet to be completely demonstrated. Genetic disorders are possible causes of both failure in implantation and early abortion; this cause is more prominent with advanced age and currently cannot be treated. Endocrine factors may also be responsible for miscarriage, and correction of hormone abnormalities is discussed. Infections, endometriosis and psychological factors are other possible important causes of embryo loss without specific widely accepted treatments. Prominent areas of research are the identification of genetic preimplantation abnormalities, and pharmacological intervention for abnormal spontaneous uterine contractility. The data here reported are encouraging, but the efficacy of different treatments is still not convincing. The information available is sufficient to develop new diagnostic and therapeutic tools to evaluate their efficacy in reducing spontaneous abortion at an early stage.

Abortion, Habitual↗
PubMed2005

Drosophila activating transcription factor-2 is involved in stress response via activation by p38, but not c-Jun NH(2)-terminal kinase.

Activating transcription factor (ATF)-2 is a member of the ATF/cAMP response element-binding protein family of transcription factors, and its trans-activating capacity is enhanced by stress-activated protein kinases such as c-Jun NH(2)-terminal kinase (JNK) and p38. However, little is known about the in vivo roles played by ATF-2. Here, we identified the Drosophila homologue of ATF-2 (dATF-2) consisting of 381 amino acids. In response to UV irradiation and osmotic stress, Drosophila p38 (dp38), but not JNK, phosphorylates dATF-2 and enhances dATF-2-dependent transcription. Consistent with this, injection of dATF-2 double-stranded RNA (dsRNA) into embryos did not induce the dorsal closure defects that are commonly observed in the Drosophila JNK mutant. Furthermore, expression of the dominant-negative dp38 enhanced the aberrant wing phenotype caused by expression of a dominant-negative dATF-2. Similar genetic interactions between dATF-2 and the dMEKK1-dp38 signaling pathway also were observed in the osmotic stress-induced lethality of embryos. Loss of dATF-2 in Drosophila S2 cells by using dsRNA abrogated the induction of 40% of the osmotic stress-induced genes, including multiple immune response-related genes. This indicates that dATF-2 is a major transcriptional factor in stress-induced transcription. Thus, dATF-2 is critical for the p38-mediated stress response.

Amino Acid Sequence↗
PubMed2000

A study of the inheritance of a bleeding disorder in Simmental cattle.

A study was designed to determine the inheritance pattern of a blood platelet aggregation disorder in Simmental cattle utilizing embryo transfer technology. A Simmental donor cow that had previously produced a calf with the platelet aggregation disorder was superovulated and mated to a bull that had also produced affected offspring. Twenty-seven calves were produced from the 63 (42.9%) embryos transferred. This somewhat lower than expected pregnancy rate is suggestive of an increased rate of embryo loss. Twenty-three of 25 (92%) calves had normal platelet aggregation patterns and 2 failed to show any evidence of platelet aggregation. Data are suggestive that inheritance is not simple Mendelian recessive. A more likely scenario is that the defect is the result of the inheritance of at least 2 genes, which is also consistent with the sporadic incidence reported in the population at large.

Animals↗