Microsurgical cloning techniques for zygotes: a long-term dispute.

Zygotes (fertilized oocytes) have been considerably neglected as suitable recipients for cell nuclear transfer in cloning. Recently, it has been reported by Polish researchers that microsurgical methods using very thin pipettes for pronuclear removal from mouse zygotes seem to be crucial for successful cloning of adult mice. It was postulated that due to this technique both pronuclei are broken, leaving behind some nuclear components in the oocyte's cytoplasm. Release of pronuclear factors into the cytoplasm turns enucleated zygotes into suitable nuclear recipients for cloning.

Calgranulins in cystic fluid and serum from patients with ovarian carcinomas.

Ovarian cancer remains still associated with poor prognosis because it is diagnosed predominantly at advanced stages. Ovarian-specific tumor markers do not yet exist for early detection of the disease. At the search of diagnostic markers for ovarian cancer, proteomic-based approaches have focused on novel investigations of neoplastic processes in tumor patients. Cystic fluids of malignant and benign ovarian tumors and serum from the corresponding patients were collected and processed for two-dimensional gel electrophoresis. Proteins were visualized on the gels by silver staining. At the low molecular mass level between 10 and 20 kDa, selected protein spots were additionally processed for nanospray mass spectrometry and partial amino acid sequencing. For protein identification, the sequencing results were compared with computer information from a protein data bank. Protein patterns from cystic fluids of ovarian carcinomas differed significantly from those of benign cysts and revealed additional polypeptides at low molecular mass level between 10 and 20 kDa. Protein patterns from serum of patients with malignant ovarian tumors also contained additional polypeptides between 10 and 20 kDa that were not detected in serum from patients with benign cysts. The additional proteins in serum were present in similar electrophoretic positions compared with those found in the cystic fluid of the corresponding ovarian carcinomas. Protein spots in the range of 10-20 kDa were selected for partial amino acid sequencing. Two protein spots were identified as calgranulin A and three spots as calgranulin B. Either both proteins or only calgranulin A or B were present in cystic fluid from ovarian carcinomas and serum of the corresponding patients. These two proteins were absent or not detectable in fluid from benign ovarian cysts and in serum from those patients. Our investigations concerning protein patterns in cystic fluid of malignant and benign ovarian tumors provide new information about alterations in protein synthesis linked to neoplastic events of the ovary. With the proteomic strategy, new tumor markers are characterized and may serve for diagnostic purposes of patients with ovarian cancer.

Different proteome pattern of epidermal growth factor receptor-positive colorectal cancer cell lines that are responsive and nonresponsive to C225 antibody treatment.

The monoclonal antibody C225 directed against the epidermal growth factor receptor (EGFR) blocks downstream mitogenic signaling and is effective in patients with advanced colorectal cancer. Clinical data, however, suggest the presence of primary and secondary resistance mechanisms that are hardly understood. To define proteins involved in EGFR-triggered growth regulation and potential resistance mechanisms, we characterized the proteome profile of two colorectal cancer cell lines with a high expression of functional EGFR but a different response to treatment with C225. In Caco-2 and HRT-18, a complete saturation of EGFR was achieved after incubation with C225; whereas Caco-2 showed inhibition of proliferation, growth of HRT-18 was not suppressed. Using two-dimensional electrophoresis and subsequent mass spectrometry, we identified 14 proteins differentially expressed in both cell lines. All proteins are involved in metabolic pathways and malignant growth. Expression of enzymes such as ubiquitin carboxyl-terminal hydrolase isozyme 1, glutathione S-transferase P, and chloride intracellular channel protein 1 does not seem to interfere with the antiproliferative effect of anti-EGFR antibody. On the other hand, expression of proteins such as fatty acid binding protein and heat shock protein 27 might constitute strong antiapoptotic effects contributing to the nonresponse of HRT-18 to C225 treatment. Proteome-based investigations can help us better understand the complex protein interactions involved in EGFR signaling and its blockage by therapeutic monoclonal antibodies.

Heterotopic pregnancy: report of three cases.

OBJECTIVE: Heterotopic pregnancy, defined as concomitant intrauterine and ectopic pregnancy, is a rare event. Assisted reproductive technologies have led to an increase in the number of heterotopic pregnancies. MATERIALS AND METHOD: From 1997 to 1999 three cases of heterotopic pregnancies were referred to the gynecological unit of the hospital of St. Pölten. The condition was diagnosed at 7, 8 and 12 weeks of gestation, respectively. Two patients had undergone assisted reproductive technologies and former pelvic surgery for tubal pregnancy. Two patients had intrauterine singleton pregnancies and one patient had an intrauterine twin pregnancy. In all cases the ectopic site presented as a ruptured tubal pregnancy and the treatment consisted of laparoscopic salpingectomy. RESULTS: In all patients the postoperative course was uneventful and the intrauterine pregnancy progressed. In one case recurrent preterm labor led to vaginal delivery at 33 weeks of pregnancy. In the case of twin pregnancy, a planned cesarean section was performed at 37 weeks of gestation. One patient delivered at term. All newborns were healthy. CONCLUSION: Knowledge of heterotopic pregnancy and understanding the epidemiological risk factors underlying this condition are important for early diagnosis with the aim of improving therapy and clinical outcome.

Human embryo twinning with applications in reproductive medicine.

OBJECTIVE: To assess the efficacy of human embryo twinning by blastomere biopsy at different early embryonic stages (splitting efficiency) and to determine the in vitro developmental capacity of twinned human embryos (developmental efficiency). DESIGN: Randomized comparative study. SETTING: Private IVF centers. PATIENT(S): Couples undergoing IVF donating triploid embryos. INTERVENTION(S): Embryos at the 2- to 5- and 6- to 8-cell stage were split into twin embryos. Half the number of blastomeres from donor embryos were removed and inserted into recipient empty zonae pellucidae. After embryo splitting, donor and recipient embryos were cultured in vitro. MAIN OUTCOME MEASURE(S): Development of twinned embryos to the blastocyst stage. RESULT(S): The number of developing embryos obtained after splitting could be increased in comparison with the number of embryos available before splitting at the 6- to 8-cell stage but not at the 2- to 5-cell stage (splitting efficiency). Splitting of 6- to 8-cell embryos yielded superior rates of twin embryos developing to blastocysts (developmental efficiency). Twinning success was related to the superior morphological quality of embryos used for splitting. CONCLUSION(S): This is the first report on twinned human embryos developing to blastocysts. This study exhibits the potential for novel applications in human assisted reproduction.

E2F3a is critically involved in epidermal growth factor receptor-directed proliferation in ovarian cancer.

We describe for the first time a new integral molecular pathway, linking transcription factor E2F3a to epidermal growth factor receptor (EGFR) activation in ovarian cancer cells. Investigations on the role of E2F family members in EGFR-mediated mitogenic signaling revealed that E2F3a was selectively upregulated following EGFR activation, whereas all other E2F family members remained unaffected. In contrast, EGF treatment of healthy ovarian surface epithelial and mesothelial cells yielded a selective upregulation of proliferation-promoting E2F1 and E2F2 without influencing E2F3a expression. In ovarian cancer cell lines, the extent of EGF-induced proliferative stimulus was closely related to the magnitude of E2F3a increase, and proliferation inhibition by E2F3a knockdown was not overcome by EGF exposure. Furthermore, the EGFR-E2F3a axis was found to be signal transducer and activator of transcription 1/3 dependent and the ratio of IFN-regulatory factor (IRF)-1 to IRF-2 was shown to be determinative for E2F3a control. In a pilot study on 32 primary ovarian cancer specimens, a highly significant correlation between activated EGFR and E2F3a expression was disclosed. This new integral pathway in the EGFR-driven mitogenic cell response, which through its key player E2F3a was found to be essential in triggering proliferation in ovarian cancer cells, provides new insights into EGFR signaling and could represent the basis for appealing new therapeutic approaches in ovarian cancer.

Tissue perfusion-controlled guided biopsies are essential for the outcome of testicular sperm extraction.

OBJECTIVE: To determine if there are areas of major and minor perfusion in a single testicle, and if the quality and quantity of sperm are correlated with the level of perfusion, we collected testicular tissue from areas with different levels of perfusion. DESIGN: Controlled clinical study. SETTING: Consecutive patients with azoospermia. PATIENT(S): Patients with azoospermia undergoing testicular sperm extraction (TESE) biopsy for the retrieval of sperm to be used in an assisted reproduction program. INTERVENTION(S): Perfusion mapping was performed with the use of color Doppler ultrasound. Areas with different levels of perfusion were marked with needles. After incision with radiofrequency cutting, the exposed tissue was examined with a laser Doppler flowmeter, and biopsies were taken for TESE and histology. Sperm were analyzed using World Health Organization criteria, and prepared for intracytoplasmic sperm injection (ICSI). MAIN OUTCOME MEASURE(S): Correlation of sperm quality and quantity in testicular-tissue biopsies, with tissue-perfusion units (TPU) measured by laser Doppler flowmeter. RESULT(S): From 40 biopsies taken from 20 testicles of 12 patients, tissue was analyzed for sperm quality and quantity. Sperm quality was highest in areas of high tissue perfusion. In areas of 70 TPU, 72.3% progressive sperm were detected, whereas in areas of 10 TPU, only 13.3% progressive sperm and elevated numbers of precursor cells could be observed. The number of motile sperm isolated from tissue samples correlated well with the intensity of tissue perfusion. CONCLUSION(S): We have shown for the first time that in patients suffering from azoospermia, sperm quality and quantity depend on tissue perfusion within the testicle.

In vitro blastocyst development from serially split mouse embryos and future implications for human assisted reproductive technologies.

OBJECTIVE: To assess the efficacy of serial splitting of mouse embryos with respect to blastocyst development. DESIGN: Prospective study. SETTING: Commercial research facility. ANIMAL(S): Commercially available mouse embryos from B6C3F-1 x B6D2F-1. INTERVENTION(S): One, two, and three blastomeres were biopsied from two-, four-, and six-cell embryos, respectively, and were inserted into empty zona pellucida recipients (first split). These embryos were cultured to reach their original cell number status and then were split again (second split). Once these embryos regained their original cell status, they were split yet again (third split). MAIN OUTCOME MEASURE(S): Blastocyst development of embryos split serially at the two-, four-, and six-cell stages. RESULT(S): The blastocyst development rate for two-, four-, and six-cell embryos subjected to a first split was 74.3%, 75.0%, and 66.6%, respectively, as compared with 71.8%, 62.6%, and 48.4% (second split) and 48.4%, 38.1%, and 10.6% (third split). CONCLUSION(S): First and second splitting of cleavage-stage embryos has yielded high efficiency rates for blastocyst development when compared with the third splitting, which did not provide any beneficial advantage for further embryo splitting and multiplication. This is the first study reporting on three serial embryo splittings in a mammalian species. Embryo splitting may have significant impact and applications in human assisted reproductive technology.

Evaluation of the embryonic preimplantation potential of human adult somatic cells via an embryo interspecies bioassay using bovine oocytes.

OBJECTIVE: To examine the embryonic preimplantation potential of human adult somatic cells by creating interspecies embryos via somatic cell nuclear transfer (SCNT) using bovine oocytes. DESIGN: Prospective study. SETTING: Research facility of Reprogen. PATIENT(S): Infertile couples. INTERVENTION(S): Enucleated bovine oocytes were fused via SCNT with either human granulosa (HG) or fibroblast (HF) cells and cultured in vitro. Polymerase chain reaction (PCR) and DNA analysis were performed on the interspecies embryos. Parthenogenetically activated embryos served as controls. MAIN OUTCOME MEASURE(S): Embryonic preimplantation development after interspecies SCNT. RESULT(S): From enucleated bovine oocytes fused with HG cells (n = 48) and HF cells (n = 75), 15 HG- and 22 HF-derived embryos developed, some of which progressed to blastocysts (31.3% vs. 29.3%, respectively). The PCR and DNA analysis showed that the interspecies embryos contained human genomic DNA specific for the individual DNA profile of the HG or HF donor cells used for SCNT. In addition, both bovine- and human-specific mitochondrial DNA was detectable in the interspecies embryos up to the blastocyst stage. Parthenogenetic development was 46.8% and 64.9% for the HG and HF series, respectively. The SCNT efficiency index, defined as the ratio of SCNT and parthenogenetic success rate, was 66.8% for HG cells and 45.5% for HF cells. CONCLUSION(S): This interspecies bioassay can be utilized to determine and assess the embryonic preimplantation potential of different types of human adult somatic cells.

The potentialities of transplanted early gastrula nuclei ofDrosophila melanogaster. Production of their imago descendants by germ-line transplantation.

Wild-type nuclei, taken out of cells from five regions of early gastrula embryos, were implanted singly into unfertilizedy w sn 3lz50e eggs ofDrosophila melanogaster. The different types of nuclei initiated development with nearly equal frequencies of about 60%. 2.9% of the 1073 nuclear transfers developed as far as one of the three larval instars, and one reached the pupal stage.All individuals showed stage-specific patterns of defect. Most of these abnormalities were probably due to some inevitable damage caused by the implantation procedure such as disarrangement of the internal egg morphology and loss of peripheral egg substance. The proportions of individuals arrested at different embryonic and larval stages were similar for the five nuclear groups.Fertile imagos, descendants of all five types of donor nuclei, were produced via germ-line mosaics in two ways: (1) Pole cells of nuclear-transplant blastoderm stages were implanted into the pole cell region of host blastoderm eggs. (2) Gonads were taken from nuclear-transplant larvae and implanted into host larvae. In both cases gametes developed from the transplants as could be recognized from the genotypes of their progeny. By means of suitable crosses it was possible to get clones of flies whose large chromosomes were descended from the chromosomes of only one transplanted nucleus, that is, each clone was the descendant of one somatic nucleus. The data presented show that the nuclei remain omnipotent until the early gastrula stage.

Developmental potencies of nuclei from cleavage, preblastoderm, and syncytial blastoderm transplanted into unfertilized eggs ofDrosophila melanogaster.

Wild-type nuclei from eggs ofDrosophila melanogaster at various developmental stages and from different regions of the egg-cleavage nuclei, pole nuclei from preblastoderm, and lateral nuclei from syncytial blastoderm-were singly implanted into unfertilizedy w sn 3 lz 50e eggs to determine their developmental potencies.All three types of transplanted nuclei were almost equally effective in initiating development of unfertilized eggs. Development was arrested in one of five critieal embryonic stages or in one of the three larval instars. The frequency of individuals reaching a distinct stage was approximately the same for all three types of donor nuclei. The stage-specific pattern of defects was independent of the type of nucleus transplanted.The deviations from normal development were broadly similar to those seen in controls developing from fertilized eggs which had only been punctured or into which cytoplasm had been injected. Many defective embryos also occurred in these control experiments. These and other observations indicate that a large proportion of irregularly developed individuals found after nuclear transfer can be ascribed to loss of egg material, disturbances in the internal organization of the egg during nuclear implantation, and the difficulty the implanted nucleus has in adjusting to the autonomous processes within the egg, such as the formation and migration of cytoplasmic islands.Some of the defective embryos and larvae originating from nuclear transfer were implanted into adult hosts. After culture for 14 days the early embryonic stages had formed several larval structures, and the late embryonic and larval stages had developed all larval organs. The proliferated imaginal primordia of thesein vivo cultured embryos and larvae, as well as the imaginal disks of the third instar larva, were then implanted into larval hosts with which they passed through metamorphosis and differentiated into imaginal structures. All three types of donor nuclei were capable of producing all adult structures derivedin situ from imaginal disks. The phenotype of these structures waswild-type, thus demonstrating their origin from the transplanted nuclei.The problem as to why not all transplanted nuclei initiated development, and why development after nuclear transplantation was arrested at the third larval instar, at the latest, is discussed.

Biotechnology in reproductive medicine.

In this review I am summarizing the past and current progress in the field of pharmaceutical, diagnostic, therapeutic, and reproductive cloning in mammals. Several human gene products can be pharmaceutically explored in transgenic farm animals and employed for medical applications. Preimplantation genetic diagnosis (PGD) is utilizing modern molecular cloning techniques to detect genetic and chromosomal aberrations in early embryos originating from patients with inborn errors at risk for hereditary diseases or age-related risk for abnormal karyotype. Stem cell engineering from early human embryos is creating new and promising but also controversial applications for therapeutic and regenerative medicine. Potential risk factors for reproductive cloning are presented and discussed in the context of possible developmental malformations, frequently observed after embryo culture and cloning in farm animals. Future extension of biotechnology to human reproductive cloning is currently under worldwide dispute.

Nuclear nonhistone proteins in mouse teratocarcinomas : I. Cell lineage specificity.

The nonhistone protein pattern of four murine teratocarcinomas with different capacities for differentiation were compared: a multidifferentiated teratocarcinoma OTT2289, a nondifferentiated teratocarcinoma OTT2158, a teratocarcinoma-derived rhabdomyosarcoma TDR114, and a teratocarcinoma-derived neuroblastoma TDN2151. Their nonhistone proteins (NHP) were separated by differential salt extraction and hydroxyapatite chromatography into three fractions, NHP-I, NHP-II and NHP-III. Comparison of the NHP fractions by twodimensional gel electrophoresis in combination with a sensitive silver staining method reveals that there are several tumour line specific proteins in each NHP fraction. We suggest that specific NHP, which can be used as biochemical markers for each of the four investigated tumour lines, may be involved in cell lineage specific control of gene expression.

Protein synthesis in enuleated fertilized and unfertilized mouse eggs.

Fertilized and unfertilized C57BL/6J eggs were microsurgically enucleated and then analyzed for their capacity to synthesize proteins using 2-dimensional polyacrylamide gel electrophoresis. In both types of enucleated eggs (cytoplasts), protein synthesis continued and was still detected up to three days in culture. Shortly after enucleation, the pattern of polypeptides remained similar to the respective non-operated control eggs but it later became gradually reduced in intensity and complexity. After two days of culture the appearance of some new proteins typical for 2-cell embryos was observed in enucleated fertilized eggs only. Our findings suggest that maternal mRNA stored during oogenesis is utilized during the preimplantation period.

Protein synthesis in mouse embryos with experimentally produced asynchrony between chromosome replication and cell division.

The cleavage of fertilized mouse eggs was prevented during cytochalasin B incubation and consequently these eggs became tetraploid the following day during in vitro culture. When the eggs were cultured further in normal medium, they cleaved and gave rise to tetraploid blastocysts. Protein synthesis was analysed in these embryos at different developmental stages using two-dimensional polyacrylamide gel electrophoresis. The protein synthesis pattern of one-cell tetraploid eggs was intermediate between those of normal one- and two-cell embryos. Tetraploid two-cell embryos expressed protein sets equivalent to those of untreated four-cell embryos, and tetraploid four-cell embryos synthesized proteins similar to those of four- to eight-cell controls. At subsequent pre-implantation stages the asynchrony was no longer detectable. When fertilized eggs were cultured continuously in the presence of cytochalasin B, they became tetraploid, octoploid and more and more polyploid without cleavage occurring. The protein synthesis patterns expressed by these one-cell polyploid eggs did not resemble that of normal fertilized eggs, but were similar to those of cleaving control embryos and blastocysts of equivalent age and nuclear division. These results strongly suggest that in early mouse embryos stage-specific translation is temporally correlated with chromosome replication (karyokinesis) and independent of cell division (cytokinesis) or cell interaction.

Expression of specific genes in early mouse embryos blocked by cytochalasin.

Mouse embryos at the two cell stage derived from C57BL/6 × C3H/Aa F1-females heterozygous at the X-linked phosphoglycerate kinase locus (Pgk-1) were cultured continuously in the presence of cytochalasin B or D. Further cleavage of the two cell embryos was thus prevented and the embryos became polyploid during culture. The onset of expression of the maternally inherited Pgk-1 gene and of the paternally inherited glucosephosphate isomerase (Gpi-1) gene was determined in these polyploid embryos by cellulose acetate gel electrophoresis of single embryos. In contrast to euploid preimplantation embryos developing normally in utero or in culture without cytochalasins, expression of maternal Pgk-1 was never observed at days 4 and 5 of gestation in polyploid two cell embryos, showing that the Pgk-1 allele on the maternally inherited X chromosome is not activated independently of cytokinesis and morphogenesis. Expression of paternally derived Gpi-1, however, occurred in cleavage blocked embryos von day 5 of development. This may indicate that the activation of two genes which are both expressed during preimplantation development and which both code for glycolytic enzymes, is initiated by different signals.