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PubMed1990

Comparison of persistence of seven bovine viruses on bovine embryos following in vitro exposure.

The ability of seven cytopathic strains of bovine viruses to adhere to the zona pellucida of six-to-eight day-old bovine embryos were compared. Embryos were exposed to virus by placing them either in virus suspensions or by culturing them on infected bovine turbinate cultures for 18-24 h. After exposure to bovine virus diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBV), bluetongue virus (BTV), pseudorabies virus (PRV), vesicular stomatitis virus (VSV), parainfluenza 3 virus (PI3), or bovine enterovirus virus (BEV), the embryos were tested for virus by culture in bovine turbinate cells and by morphological examination using electron microscopy (EM). A special technique to minimize loss of embryos processed for EM was developed. More embryos had viral particles on the surface of the zona pellucida after exposure to 18-24 hour infected cell cultures than did embryos exposed to viral culture suspensions. The most dramatic finding was that BTV adhered in large numbers to the surface of the zona pellucida of exposed embryos. IBRV, PRV, and VSV comprised an intermediate group, with virions occasionally detected on the surface of exposed embryos after 5 washes. Therefore, extensive washing is required. The PI3 and BEV were easily removed from embryo-exposed virus by washing. BVD was difficult to identify morphologically, making assessment by EM unreliable. There was no evidence that any one of the seven viruses penetrated the intact zona pellucida. Using a micromanipulator, 42 embryos were also directly inoculated through the zona pellucida with +/- 50 picoliters of virus inoculum or medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

Alternative splicing and imprinting control of the Meg3/Gtl2-Dlk1 locus in mouse embryos.

The distal part of the mouse Chr 12 contains a cluster of reciprocally imprinted genes. Recently we found a grandparental origin-dependent, transmission-ratio distortion (TRD) in this region. The TRD resulted from postimplantation loss of embryos that inherited the distal Chr 12 alleles from the maternal grandfather. These data suggested that imprinting of one or more genes in this region was not uniformly well established or maintained in all the embryos. To elucidate the mechanism underlying such a variation, we examined the expression of two genes from the distal Chr 12 imprinted region, the maternally expressed gene 3/gene-trap locus 2 ( Meg3/ Gtl2), and the delta-like homolog 1 ( Dlk1) gene. We demonstrated that the Meg3/ Gtl2 gene had two major mRNA forms. One form, Meg3-proximal ( Meg3p), contained exons 1-3. The second form, Meg3-distal ( Meg3d) did not contain exons 1-3 and was present in oocytes and in 1- and 2-cell embryos. We observed cross-dependent and splice form-specific relaxation of imprinting of the Dlk1 and Meg3d, but not Meg3p. Expression patterns of Dlk1 and Meg3/ Gtl2 in embryos from crosses between different mouse strains suggest that 1). imprinting of the Dlk1 and Meg3/ Gtl2 genes is not strictly coordi- nated; 2). parental origin-dependent expression of these genes is under control of a strain-specific, cis-acting modifier located in a 1.5-Mb region that includes the Meg3/ Gtl2-Dlk1 locus. Biallelic expression of Dlk1 and Meg3d did not affect embryo viability and, therefore, cannot be responsible for the lethal phenotypes in UPD12 embryos or for the transmission-ratio distortion.

Alternative Splicing↗
PubMed1997

Transcription of genes for conglutin gamma and a leginsulin-like protein in narrow-leafed lupin.

The expression of genes encoding conglutin gamma and a leginsulin-like protein has been examined in narrow-leafed lupin, Lupinus angustifolius L. Conglutin gamma is a homologue of basic 7S globulin (Bg), the insulin and leginsulin binding protein from soybean. Accumulation of conglutin gamma mRNA, as assessed by northern assays and reverse-transcription PCR, was tightly regulated both spatially and temporally in lupin plants and was detected almost exclusively in developing seeds. Similar tissue and temporal specificity was demonstrated when 1.8 kb of the promoter region from the conglutin gamma gene was used to drive the expression of a beta-glucuronidase reporter gene in transgenic plants. In stably transformed tobacco the conglutin gamma promoter produced strong, temporally regulated and seed-specific expression of the reporter gene which was localised to the embryo tissues and to a layer of cells adjacent to the seed coat. A truncated 0.29 kb promoter fragment produced much reduced levels of expression and a loss of embryo specificity. Leginsulin-like mRNA was similarly detected in lupins only in developing seeds. The leginsulin-like gene detected in L. angustifolius showed 96% sequence identity to leginsulin from soybean within the 280 bp region amplified from lupin by PCR. The results demonstrate that both components of a Bg-leginsulin putative signal transduction pathway are present in the seeds of lupin.

Albumins↗
PubMedDate not supplied

[Nonsurgical embryo transplantation in mice].

Nonsurgical transplantation of the mouse embryos at the blastocyst stage into the uterus was performed using a device of original construction. The use of this device made it possible to exclude the loss of embryos, to reduce the amount of culture medium introduced together with the embryos, to lessen the damage of genital tract and ensured guaranteed "ejection" of embryos into the uterus. The females of the third day of pregnancy were used of recipients. Transplantation was performed between the mice of strains CBA/Lac, C57BL/6 and tetrahybrid CBWA. The level of newborn attained 65% of the number of transplanted blastocysts.

Animals↗
PubMedDate not supplied

Suppression of decidual cell response induced by dibutyltin dichloride in pseudopregnant rats: as a cause of early embryonic loss.

In our previous study, dibutyltin dichloride (DBTCl) caused preimplantation embryonic loss and postimplantation embryonic loss in rats following administration at 7.6 mg/kg and above on Days 0-3 and at 3.8 mg/kg and above on Days 4-7 of pregnancy, respectively. This study was designed to assess the effects of DBTCl on uterine function as a cause of early embryonic loss using pseudopregnant rats. DBTCl was given orally to pseudopregnant rats at 3.8, 7.6 or 15.2 mg/kg on pseudopregnant day (PPD) 0-3 or on PPD 4-7. The decidual cell response was induced by bilateral uterine scratch on PPD 4. The uterine weight on PPD 9 served as an index of uterine decidualization. Uterine weight and serum progesterone levels on PPD 9 were significantly decreased after administration of DBTCl at 7.6 mg/kg and above on PPD 0-3 and PPD 4-7. DBTCl had no effect on the serum estradiol levels and number of corpora lutea. Administration of progesterone reversed the suppression of uterine decidualization in rats given DBTCl on PPD 0-3. It can be concluded that DBTCl suppresses the uterine decidual cell response and decreases progesterone levels, and these effects are responsible for early embryonic loss due to DBTCl exposure.

Animals↗
PubMed2000

Sex-of-offspring-specific transmission ratio distortion on mouse chromosome X.

During our study of the DDK syndrome, we observed sex ratio distortion in favor of males among the offspring of F(1) backcrosses between the C57BL/6 and DDK strains. We also observed significant and reproducible transmission ratio distortion in favor of the inheritance of DDK alleles at loci on chromosome X among female offspring but not among male offspring in (C57BL/6 x DDK)F(1) x C57BL/6 and (C57BL/6-Pgk1(a) x DDK)F(1) x C57BL/6 backcrosses. The observed transmission ratio distortion is maximum at DXMit210 in the central region of chromosome X and decreases progressively at proximal and distal loci, in a manner consistent with the predictions of a single distorted locus model. DXMit210 is closely linked to two distortion-controlling loci (Dcsx1 and Dcsx2) described previously in interspecific backcrosses. Our analysis suggests that the female-offspring-specific transmission ratio distortion we observe is likely to be the result of the death of embryos of particular genotypic combinations. In addition, we confirm the previous suggestion that the transmission ratio distortion observed on chromosome X in interspecific backcrosses is also the result of loss of embryos.

Animals↗
PubMed1995

Factors affecting cell viability during bisection of bovine embryos.

The aim of this study was to investigate the effects of temperature, cytochalasin B, sucrose, cell number and developmental stage of embryos on cell loss and cell lysis during embryo splitting. Day-7 morulae and blastocysts were bisected using a metal blade. In Experiment 1, splitting of embryos in control medium (PBS + 10% fetal calf serum) was compared with splitting in the presence of 7.5 mul/ml cytochalasin B. In Experiment 2, the control medium was compared with medium supplemented with 200 mM sucrose. In Experiment 3, the control medium was compared with medium supplemented with sucrose and cytochalasin B. Cell viability was measured by staining nuclei of embryos with Hoechst 33258 and propidium iodide. Cells with nuclei exhibiting pink fluorescence were considered lysed, while blue fluorescence was considered an indication of viable cells. Cells disaggregated during splitting were classified as extruded cells. An effect of the developmental stage was observed in the pooled data from the control groups of the 3 experiments, with a higher proportion of viable cells in bisected morulae compared with bisected blastocysts (77.6 vs 70.0%; P = 0.003). However, as there was no effect of cell number (P = 0.85), the influence of the developmental stage can be contributed to morphological changes rather than to increase of cells associated with this change. In Experiment 1, the cytochalasin B-treated embryos contained a higher percentage of viable cells than the control embryos after removal of the developmental stage effect (P < 0.01). In Experiment 2, no effect on sucrose could be observed. In Experiment 3, the combined use of sucrose and cytochalasin B tended to increase the proportion of cells surviving bisection, but this difference was not significant. In Experiment 1, there was a correlation between viable cells and temperature during splitting (r = 0.42, P = 0.05; temperature range 8.1 degrees C to 15.6 degrees C). No correlation was found in any other group in any of the experiments, nor in the pooled data from the control groups in the 3 experiments.

Journal Article↗
PubMed1994

Comparative study of the survival rate of frozen-thawed embryos with perforated zonae from microsurgical fertilization and frozen-thawed embryos with intact zonae.

OBJECTIVE: To compare the survival rate of frozen-thawed embryos with perforated zonae from microsurgical fertilization (subzonal insemination) (SUZI) with that of embryos with intact zonae. DESIGN: Thirty-eight embryos resulting from microsurgical fertilization by SUZI were cryopreserved in 16 patient cycles. Within the same period, 140 zonae-intact embryos from 46 patient cycles were cryopreserved. The survival rate of the SUZI embryos was compared with the zonae-intact embryos after thawing. Clinical pregnancies were compared after the transfer of the thawed embryos. RESULTS: The total survival rates were 94.7% and 89.3% for thawed SUZI embryos and zonae-intact embryos, respectively. Within each type of embryo, total survival rates were similar irrespective of the age of the embryos at freezing. The blastomere loss per thawed embryo was the same for zonae-intact and SUZI embryos. One clinical pregnancy was obtained in patients who received thawed SUZI embryos and nine in patients who received embryos with intact zonae. CONCLUSIONS: It is concluded that SUZI embryos can be cryopreserved and thawed with the same degree of confidence as zonae-intact embryos. The low implantation rate of thawed SUZI embryos requires confirmation in a larger clinical series.

Cryopreservation↗
PubMed2004

Antiphosphatidylserine antibodies affect rat yolk sacs in culture: a mechanism for fetal loss in antiphospholipid syndrome.

PROBLEM: A variety of reproductive impairments have been reported in the context of the antiphospholipid syndrome (APS). APS is associated with the presence of antibodies to negatively charged phospholipids that may affect the outcome of pregnancy. METHOD OF STUDY: Rat embryos were cultured within their yolk sacs. The effects of two antiphosphatidylserine monoclonal aPS antibodies (HL5B, RR7F) regarding their influence on growth and apoptotic events of the yolk sacs, as well as on growth and the morphology of the embryos, were studied. RESULTS: Exposure of rat embryos and their yolk sacs to aPS inhibited yolk sac growth. Moreover, increased number of apoptotic events of giant cells in the aPS-exposed ectoplacental cone was found in comparison with control IgG-exposed giant cells (P < 0.05). No significant damage was observed in the embryos. CONCLUSIONS: The results suggest that aPS affect growth and apoptosis of rat ectoplacental cone.

Animals↗
PubMed2004

No association of C677T methylenetetrahydrofolate reductase and an endothelial nitric oxide synthase polymorphism with recurrent pregnancy loss.

PROBLEM: It is controversial whether polymorphisms of methylenetetrahydrofolate reductase (MTHFR) and the endothelial nitric oxide synthase (eNOS) are associated with recurrent pregnancy loss. METHOD OF STUDY: We studied the frequency of the C677T polymorphism of MTHFR and a eNOS gene polymorphism, as well as the plasma levels of homocysteine and NO, in 85 cases with a history of two or more unexplained embryonal losses, 40 patients suffering fetal loss and 76 controls. RESULTS: The frequency of the MTHFR gene T allele, which has been reported to be associated with miscarriages, in patients suffering fetal loss was rather significantly lower than in controls whereas there was no difference in the frequency of the eNOS gene A allele. There were no differences in the plasma homocysteine levels among the three groups. However, the NO concentrations in the embryonal loss and fetal loss groups were significantly higher than that in controls. CONCLUSION: We conclude that the NO concentration but not the polymorphism of MTHFR and eNOS gene and hyperhomocysteinemia are associated with recurrent pregnancy loss in Japanese.

Abortion, Habitual↗
PubMed1979

A survey of early pregnancy losses in West German thoroughbred mares.

Embryonic resorption is reported as the main reason for early pregnancy losses in the West German Thoroughbred mare. An analysis of breeding records for the last 11 years showed a resorption rat of 8.4 per cent. Evidence was obtained that these early embryonic losses were associated with too early conception after foaling and with the state of lactation.

Animals↗
PubMed1992

Early foetal death in the mare. Histological, bacteriological and cytological findings in the endometrium.

Fifteen mares which had each lost their pregnancies 2 to 8 times during the preceding 6 years were used in a study comprising 1 or 2 breeding seasons for each mare. During the research period all 15 mares conceived and 10 of the mares had normal pregnancies in the 1st experimental year. Five mares resorbed/aborted once or twice during the 1st year. This was followed by a pregnancy that terminated in a live foal. Histopathological examinations of uterine biopsies showed a wide range of histopathological conditions, from absence of changes excessive for the mare's age and parity to the most severe changes of the endometrium. Most likely, endometritis, acute as well as chronic degenerative endometritis, is the most common cause of early foetal death in the mare. Good breeding management is essential and was often enough to make the mares produce a foal. Additional treatment was needed in some of the mares and was based on the course of the conceptus loss and the condition of the endometrium, as determined by clinical, histological, bacteriological and cytological examinations.

Abortion, Habitual↗
PubMed1994

Straw thermal stabilizer for embryo cryopreservation.

Embryo cryopreservation procedures have been highly developed to support in vitro fertilization techniques, but the clinical results have not met initial expectations. Many variables influence the outcome of the embryo cryopreservation procedure. Exposure to classical freezing media does not affect further embryo development. Therefore, the first crucial step seems to be embryo thermal behavior during the freezing/thawing procedure. With the aim of avoiding thermal oscillations to the embryos due to liquid nitrogen injection into the freezing chamber, we have developed a straw thermal stabilizer (STS) cryopreservation accessory that when adapted to a programmable biological freezer offers a more exact control of the straws temperature and also a greater reproducibility of the freezing process. The STS incorporates a seeding mechanism controlled by specially designed software. For the biological assays, 2-cell mouse embryos were cooled until seeding at -7 degrees C or frozen to -120 degrees C using 1.5 M 1,2-propanediol and 0.1 M sucrose. As control groups, embryos were either untreated or were exposed to cryoprotectants, developing to blastocyst at rates of 90.8 and 88.4%, respectively. The embryos cooled until seedling or subjected to the complete freezing procedure developed into blastocysts at rates of 95.7 and 82.8%, respectively, disregarding the effect of the cryoprotectant exposure. These results show a substantial improvement in cryopreservation with no loss in embryo development due to the seeding procedure.

Animals↗
PubMed2002

Transcription factor AP-2 is an essential and direct regulator of epidermal development in Xenopus.

Expression of the Xenopus homolog of the mammalian transcription factor AP-2alpha (XAP-2) is activated throughout the animal hemisphere shortly after the midblastula transition, and becomes restricted to prospective epidermis by the end of gastrulation, under the control of BMP signal modulation. Elevated expression in the future neural crest region begins at this time. Ectopic expression of XAP-2 can restore transcription of epidermal genes in neuralized ectoderm, both in ectodermal explants and in the intact embryo. Likewise, loss of XAP-2 function, accomplished by injection of antisense oligonucleotides or by overexpression of antimorphic XAP-2 derivatives, leads to loss of epidermal and gain of neural gene expression. These treatments also result in gastrulation failure. Thus, AP-2 is a critical regulator of ectodermal determination that is required for normal epidermal development and morphogenesis in the frog embryo.

Animals↗
PubMed2003

Serum hormone characterization and exogeneous hormone rescue of bromodichloromethane-induced pregnancy loss in the F344 rat.

Previously, we demonstrated that bromodichloromethane (BDCM), a drinking water disinfection by-product, causes pregnancy loss in F344 rats when given on gestational days (GD) 6-10, encompassing the luteinizing hormone (LH)-dependent period of pregnancy (GD 7-10). Pregnancy loss, i.e., full-litter resorption, was associated with reduced serum progesterone levels; however, we were unable to identify an effect on serum LH. Here, we reevaluated serum LH levels using the more sensitive technique, DELFIA(R). We further sought to better define the temporal pattern of endocrine disruption caused by BDCM during pregnancy with more frequent sampling. Lastly, we attempted to prevent BDCM-induced pregnancy loss using exogenous progesterone or human chorionic gonadotropin (hCG), an LH-agonist. BDCM, in 10% Alkamuls(R), was dosed at 75 mg/kg/day by gavage to F344 rats on GD 6-10 (plug day = GD 0). BDCM-induced pregnancy loss was associated with marked reductions in serum progesterone and LH on GD 10. The decrease in serum LH consistently preceded the decrease in progesterone. In the hormone replacement studies, BDCM and progesterone were administered on GD 6-10, hCG on GD 8-10. BDCM was delivered at 100 mg/kg/day, progesterone at 10 mg/kg twice daily, and hCG at 0.5 IU/0.2 ml/rat. Both progesterone and hCG prevented BDCM-induced pregnancy loss. Thus, BDCM-induced pregnancy loss was associated with marked GD-10 reductions in serum LH and corresponding decreases in progesterone. Furthermore, coadministration of an LH agonist prevented pregnancy loss, supporting the hypothesis that BDCM-induced pregnancy loss in the rat occurs via an LH-mediated mode of action.

Administration, Oral↗
PubMed2005

Role of nucleophosmin in embryonic development and tumorigenesis.

Nucleophosmin (also known as NPM, B23, NO38) is a nucleolar protein directly implicated in cancer pathogenesis, as the NPM1 gene is found mutated and rearranged in a number of haematological disorders. Furthermore, the region of chromosome 5 to which NPM1 maps is deleted in a proportion of de novo human myelodysplastic syndromes (MDS), and loss of chromosome 5 is extremely frequent in therapy-related MDS. NPM is a multifunctional protein, and its role in oncogenesis is controversial as NPM has been attributed with both oncogenic and tumour suppressive functions. To study the function of Npm in vivo, we generated a hypomorphic Npm1 mutant series (Npm1+/- < Npm1(hy/hy) < Npm1-/-) in mouse. Here we report that Npm is essential for embryonic development and the maintenance of genomic stability. Npm1-/- and Npm1(hy/hy) mutants have aberrant organogenesis and die between embryonic day E11.5 and E16.5 owing to severe anaemia resulting from defects in primitive haematopoiesis. We show that Npm1 inactivation leads to unrestricted centrosome duplication and genomic instability. We demonstrate that Npm is haploinsufficient in the control of genetic stability and that Npm1 heterozygosity accelerates oncogenesis both in vitro and in vivo. Notably, Npm1+/- mice develop a haematological syndrome with features of human MDS. Our findings uncover an essential developmental role for Npm and implicate its functional loss in tumorigenesis and MDS pathogenesis.

Animals↗
PubMed1994

Sonography in early pregnancy failure.

Early pregnancy can be best divided into embryonic and fetal periods (dividing line 70 days after last menstrual period), which more naturally reflect developmental changes, morphologic appearance, loss rates, and concerns about teratogens. Newer endovaginal probes and a better understanding of anatomic landmarks and expected growth rates can improve clinical management and patient counseling.

Abortion, Spontaneous↗
PubMed2005

Xenopus aristaless-related homeobox (xARX) gene product functions as both a transcriptional activator and repressor in forebrain development.

Mutations in the aristaless-related homeobox (ARX) gene have been found in patients with a variety of X-linked mental retardation syndromes with forebrain abnormalities, including lissencephaly. Arx is expressed in the developing mouse, Xenopus, and zebrafish forebrain. We have used whole-mount in situ hybridization, overexpression, and loss-of-function studies to investigate the involvement of xArx in Xenopus brain development. We verified that xArx is expressed in the prospective diencephalon, as the forebrain is patterned and specified during neural plate stages. Expression spreads into the ventral and medial telencephalon as development proceeds through neural tube and tadpole stages. Overexpression of xArx resulted in morphological abnormalities in forebrain development, including loss of rostral midline structures, syn- or anophthalmia, dorsal displacement of the nasal organ, and ventral neural tube hyperplasia. Additionally, there is a delay in expression of many molecular markers of brain and retinal development. However, expression of some markers, dlx5 and wnt8b, was enhanced in xArx-injected embryos. Loss-of-function experiments indicated that xArx was necessary for normal forebrain development. Expansion of wnt8b expression depended on xArx function as a transcriptional repressor, whereas ectopic expression of dlx5, accompanied by development of ectopic otic structures, depended on function of Arx as a transcriptional activator. These results suggest that Arx acts as a bifunctional transcriptional regulator in brain development.

Animals↗