Interspecific transplantation of polar plasm between Drosophila embryos.

Posterior polar plasm of the Drosophila egg has been shown to function autonomously in germ cell determination after transplantation to either the anterior or mid-ventral region of the early embryo. By means of similar transplantations, we have tested the ability of polar plasm of Drosophila immigrans to induce the formation of pole cells in a Drosophila melanogaster embryo. After the transplantation of polar plasm, "hybrid" pole cells were found in which both pole cell-specific organelles, the polar granules and nuclear body, were structurally similar to those characteristic of the transplanted cytoplasm. In order to determine whether these hybrid cells can function as germ cell precursors, these cells were transplanted to the posterior tip of genetically marked embryos. Approximately 5% of the flies obtained from embryos receiving potential pole cells produce offspring derived from the induced pole cells. This result demonstrates that polar plasm can function in interspecific species combinations and indicates that the molecular mechanisms of germ cell determination are conservative in evolution. Finally, in order to test whether there is any evidence for cytoplasmic inheritance of polar granules, embryos derived from hybrid pole cells were examined for their polar granule morphology. The fine structure of the granules conformed to that of the nucleus. Thus, no evidence was found for the cytoplasmic inheritance of these particular organelles.

Early postnatal depressive mood: associations with obstetric and psychosocial factors.

The central purpose of this investigation was to detect incidence and influencing factors on early postnatal depressive mood in a large hospital sample. By means of an interview we acquired information on sociodemographic data, physical and psychiatric anamnesis, and obstetric and psychologic variables. The Edinburgh Postnatal Depression Scale (EPDS) served to determine the depressive mood of our patients. The interview was carried out on 1250 women at two postnatal wards 5 days after delivery. According to the results of the German validation of the EPDS, where a cutoff of 9/10 indicates at least mild depressive disorder, the whole sample was divided into group A (EPDS score < or = 9; n = 996, 79.7%) and group B (EPDS score > or = 10; n = 254, 20.3%). Early postnatal depressive mood, as assessed by the EPDS, appeared with 20% of all women taking part in our investigation on the fifth postnatal day. Subjective measurements such as high childbirth burden, elevated trait anxiety, low life satisfaction and lower social class, and low birth weight of the infant seem to be of predominant relevance for early postnatal depressive mood.

[The evaluation of the second-look operation of patients with ovarian carcinoma and tubal carcinoma by means of a retrospective comparison study]. / Die Wertigkeit der Second-Look-Operation bei Patientinnen mit einem Ovarialkarzinom oder Tubenkarzinom mittels einer retrospektiven Vergleichsstudie.

INTRODUCTION: This investigation is a retrospective analysis to evaluate the influence of second-look surgery on the relapse-free and overall survival of patients with ovarian and tubal carcinomas. METHOD: For 208 patients with and without second-look operation out of 469 of the total collective, a matched analysis and a Cox regression model were established in the framework of a multivariate analysis. RESULTS: Second-look surgery in patients with ovarian cancer had no significant influence on the relapse-free and overall survival. The 10-year survival was equal in both groups: CONCLUSION: Second-look surgery cannot be justified on the basis of clinically noninvasive methods such as radiological findings with additional use of tumor markers. It should only be done in control clinical trials to evaluate new means of treatment.

Possible therapy of male infertility by reproductive cloning: one cloned human 4-cell embryo.

This study was conducted to evaluate the preimplantation embryonic potential of adult somatic cells from an infertile man using an interspecies bioassay for quality control and also to create human embryos via somatic cell nuclear transfer (SCNT). Skin tissue was biopsied from infertile man to obtain fibroblast cells. These cells were fused with both enucleated bovine oocytes obtained commercially and human oocytes obtained from his wife. SCNT-reconstructed oocytes were cultured in-vitro. Interspecies SCNT embryos were prepared for PCR and DNA analysis. From 13 SCNT-reconstructed bovine oocytes, 7 embryos developed (54%). DNA sequencing of these interspecies embryos showed the presence of human genomic DNA specific for the fibroblast cells of the man. From three SCNT-reconstructed human oocytes, one developed to the 4-cell stage and was subsequently transferred into the patient's uterus. Blood ss-hCG levels showed a negative pregnancy result. Human fibroblast cells from an infertile patient can promote embryonic development in interspecies SCNT. This is the first evidence of the creation and transfer of a human cloned embryo for reproductive purposes. Even though no pregnancy was established, human reproduction via SCNT may be possible and applicable in the future for patients with severe male or female infertility that have no other alternative options for procreating their own offspring.

In-vitro developmental potential of individual mouse blastomeres cultured with and without zona pellucida: future implications for human assisted reproduction.

This study was designed to compare the developmental potential of individual blastomeres derived from 2-, 4-, 6- and 8-cell mouse embryos cultured with and without zona pellucida (ZP). In the first series, one, three, five and seven blastomeres were biopsied from 2-, 4-, 6- and 8-cell embryos respectively, and inserted individually into empty ZP recipients, leaving the remaining blastomere within its original ZP. In the second series, the same protocol was used except that the biopsied blastomeres were cultured without ZP and compared with the remaining blastomere within its original ZP. For the first series, individual blastomeres derived from 2-, 4-, 6- and 8-cell embryos cultured with ZP showed blastocyst development of 82.4, 68.6, 44.4 and 23.1% respectively, with corresponding hatching rates of 70.6, 60.0, 25.9 and 7.7%. For the second series, individual blastomeres cultured without ZP progressed with blastocyst development of 73.3, 64.5, 35.7 and 22.7% respectively. Blastocyst multiplication was achieved most efficiently when using individual blastomeres from 4- and 6-cell embryos. This is the first report on comparative in-vitro propagation of single blastomeres derived from various cleavage stages in a mammalian species. Blastomere cloning with its multiple applications may be envisaged for human assisted reproductive technologies.

Efficient blastomere biopsy for mouse embryo splitting for future applications in human assisted reproduction.

The objective of the current study was to establish a safe, efficient biopsy procedure for embryo splitting using the mouse model for future applications in human assisted reproduction. From mouse embryos at the 2-, 4-, 6- and 8-cell stage, half the number of blastomeres were microsurgically biopsied and transferred into empty mouse zonae pellucidae. Twin embryonic development was monitored during in-vitro culture. Blastocyst developmental rate using 2-, 4-, 6-, and 8-cell splitting was 74.4, 75.0, 66.7 and 38.4 respectively, with corresponding hatching rates of 94.9, 97.5, 92.7 and 83.8%. Blastocysts from 2-, 4-, and 6-cell splitting resulted in elevated hatching rates compared with non-operated blastocysts (87.5%), due to the Tyrode-assisted hatching effect. Blastocyst morphology was superior from 2- and 4-cell splitting when compared with 6- and 8-cell splitting. Furthermore, outgrowth of twin blastocysts from 2- and 4-cell splitting showed well-developed colonies with trophoblast cells and clusters of ICM cells, whereas those obtained from 6- and 8-cell splitting frequently formed small-sized colonies. Due to the high twinning success rate obtained under the experimental conditions employed in this study, it appears that with further modifications and proper safeguards, such embryo splitting efforts could have potential applications in humans.

[Teratoma and the mammalian embryo]. / Teratom und Säugerembryo.

Teratomas are tumors that occur in the ovary or testis of mammals and are composed of a mixture of differentiated cells from various organs. Experimental investigations on teratomas have shown that a "biological therapy" of a tumor is feasible if these cells participate in normal differentiation and are able to reprogram changes in gene expression.

Cloning in reproductive medicine.

This review article summarizes the historical development of mammalian cloning, presents current advances and presumed risk factors in the field of reproductive cloning, discusses possible clinical applications of therapeutic and diagnostic cloning and outlines prospective commercial trends in pharmaceutical cloning. Predictable progress in biotechnology and stem cell engineering should prove to be advantageous for patients' health and for novel benefits in reproductive and regenerative medicine.

Experimental genetics of the mammalian embryo.

Recent progress in experimental mouse embryology has provided new approaches to the genetic manipulation of the mammalian embryo. The production of uniparental embryos enables one to compare maternal and paternal gene activity during development, to study the biological consequences of homozygosity of mutant genes, and to further elucidate the unsolved problem of X chromosome inactivation. Transplantation of nuclei from somatic cells into mouse eggs is considered the most vigorous functional test for the developmental capacity of the nuclear genome and provides a bioassay for the study of possible genomic changes during cellular differentiation. Transplantation of cloned eukaryotic genes into mouse eggs will permit the molecular and genetic analysis of their integration and regulation during development and, eventually, their germ line transmission as new heritable elements.

Normal genetically mosaic mice produced from malignant teratocarcinoma cells.

Malignant mouse teratocarcinoma (or embryonal carcinoma) cells with a normal modal chromosome number were taken from the "cores" of embryoid bodies grown only in vivo as an ascites tumor for 8 years, and were injected into blastocysts bearing many genetic markers, in order to test the developmental capacities, genetic constitution, and reversibility of malignancy of the core cells. Ninety-three live normal pre- and postnatal animals were obtained. Of 14 thus far analyzed, three were cellular genetic mosaics with substantial contributions of tumor-derived cells in many developmentally unrelated tissues, including some never seen in the solid tumors that form in transplant hosts. The tissues functioned normally and synthesized their specific products (e.g., immunoglobulins, adult hemoglobin, liver proteins) coded for by strain-type alleles at known loci. In addition, a tumor-contributed color gene, steel, not previously known to be present in the carcinoma cells, was detected from the coat phenotype. Cells derived from the carcinoma, which is of X/Y sex chromosome constitution, also contributed to the germ line and formed reproductively functional sperms, some of which transmitted the steel gene to the progeny. Thus, after almost 200 transplant generations as a highly malignant tumor, embryoid body core cells appear to be developmentally totipotent and able to express, in an orderly sequence in differentiation of somatic and germ-line tissues, many genes hitherto silent in the tumor of origin. This experimental system of "cycling" teratocarcinoma core cells through mice, in conjunction with experimental mutagenesis of those cells, may therefore provide a new and useful tool for biochemical, developmental, and genetic analyses of mammalian differentiation. The results also furnish an unequivocal example in animals of a non-mutational basis for transformation to malignancy and of reversal to normalcy. The origin of this tumor from a disorganized embryo suggests that malignancies of some other, more specialized, stem cells might arise comparably through tissue disorganization, leading to developmental aberrations of gene expression rather than changes in gene structure.

Totipotency and normal differentiation of single teratocarcinoma cells cloned by injection into blastocysts.

A definitive test for developmental totipotency of mouse malignant teratocarcinoma cells was conducted by cloning singly injected cells in genetically marked blastocysts. Totipotency was conclusively shown in an adult mosaic female whose tumor-strain cells had made substantial contributions to all of the wide range of its somatic tissues analyzed; the clonally propagated cell lineage had therefore differentiated in numerous normal directions. The test cells were from "cores" of embryoid bodies of a euploid, chromosomally male (X/Y), ascites tumor grown only in vivo by transplantation for 8 years. The capacity of cells from the same source to differentiate, in a phenotypic male, into reproductively functional sperms, has been shown in our previous experiments [(1975) Proc. Nat. Acad. Sci. USA 72, 3585-3589]. Cells from this transplant line therefore provide material suitable for projected somatic and germ-line genetic analyses of mammalian differentiation based on "cycling" of mutation-carrying tumor cells through developing embryos. In some animals obtained from single-cell injections tumor-derived cells were sporadically distributed in developmentally unrelated tissues. These cases can be accounted for by delayed and haphazard cellular integration, and by a marked degree of sustained cellular developmental flexibility in early mammalian development, irrespective of certain classical "germ-layer" designations. All mosaic mice obtained have thus far been free of teratomas. In one case, the injected stem cell contributed only to the pancreas and gave rise to a malignancy resembling pancreatic adenocarcinoma. The high modal frequency of euploidy in these individually tested cells thus tends to indicate that a near-normal chromosome complement is sufficient for total restoration of orderly gene expression in a normal embryonic environment; it may also be necessary for teratoma stem-cell proliferation to be terminated there.

Xenogeneic gene expression in chimeric mice derived from rat--mouse hybrid cells.

Thymidine kinase-deficient OTT6050 mouse teratocarcinoma cells were fused with hypoxanthine phosphoribosyltransferase-deficient Fu5AH rat hepatoma cells by means of inactivated Sendai virus. The resulting hybrid cells, which were selected in hypoxanthine/aminopterin/thymidine medium, retained almost all of the mouse chromosomes and various numbers of rat chromosomes, and showed many chromosomal rearrangements. The hybrid cells, as well as both parental lines, formed tumors after subcutaneous injection into athymic nude mice. Single rat--mouse hybrid cells from a clonally established subline were transplanted into C57BL6/J mouse blastocysts carrying many genetic markers suitable for the detection of hybrid cell-derived tissue contributions. From 144 blastocysts, each of which was injected with a hybrid cell and then surgically transferred to the uterus of a pseudopregnant foster mother, 62 adult mice developed without any visible coat mosaicism. However, three of these mice showed internal hybrid-cell participation in their livers and a limited number of organs of endomesodermal origin. A tumor classifiable as hemangio endothelioma was found in the liver, the only mosaic tissue, of one of the chimeric mice. Nine different rat-specific enzyme variants were detected in the mosaic organs. A considerable number of variations concerning the presence and quantitative activity of the foreign gene products probably resulted from chromosomal segregation, tissue-specific gene activity, or dosage compensation during differentiation in vivo. Our results demonstrate that cultured malignant rat--mouse hybrid cells differentiate normally and become functionally integrated during development. The appearacne in vivo of certain rat-specific gene products that are not found in the hybrid cells under conditions in vitro indicates differential gene expression of the introduced xenogeneic chromosomes.

Transplantation of posterior polar plasm in Drosophila. Induction of germ cells at the anterior pole of the egg.

In Drosophila melanogaster the primordial germ cells are normally formed at the posterior tip of the egg during the preblastoderm stage. In order to determine whether the posterior polar plasm is capable of inducing the formation of primordial germ cells in another region of the embryo, portions of this cytoplasm were transferred from wild-type embryos of the early cleavage stage to the anterior tip of mwh e embryos of the same age. At various times after the injection (15-200 min), embryos were fixed for histological analysis. Alternating thick and thin sections were examined for the presence of experimentally induced pole cells. In more than half of the embryos analyzed in this way, one to six cells were found containing the polar granules as well as round nuclear structures, both of which are characteristic of normal pole cells and are not present in blastoderm cells. In order to determine whether these "pole cells" function normally, i.e., develop further into germ cells, the cells induced at the anterior tip of mwh e blastoderm embryos were introduced into the posterior region of y w sn(3) hosts of the same age. The flies resulting from these embryos were mated to y w sn(3) partners. In addition to the expected y w sn(3) progeny, wild-type flies heterozygous for mwh e and, therefore, descended from the experimentally induced pole cells were found in 4% of the crosses. Such flies did not appear in the control experiments after transfer of normal anterior cells from noninjected blastoderm embryos. These results demonstrate that the posterior polar plasm can be transferred to the anterior tip of the embryo and that in this presumptive somatic region it still retains its capacity to determine the formation of the primordial germ cells.

Full-term development after transplantation of parthenogenetic embryonic nuclei into fertilized mouse eggs.

Diploid parthenogenetically activated oocytes were obtained after gonadotropin-induced ovulation of virgin females of the LT/Sv (LT) inbred mouse strain. These oocytes cleave spontaneously and develop into blastocysts which implant in the uterus but die within a few days. We examined the developmental potential of nuclei from parthenogenetic embryos after transplantation into fertilized eggs. The inner cell mass (ICM) and trophectoderm (TE) of LT parthenogenetic blastocysts were mechanically isolated and dissociated into single cells. Their nuclei were then injected into fertilized C57BL/6J eggs from which the male and female pronuclei were removed. Of 94 eggs injected with TE cell nuclei, 4 embryos developed to the morula stage; all 4 showed abnormalities and subsequently became arrested in development. Enzyme analysis of these embryos revealed that TE cell nuclei could neither independently initiate or support preimplantation development. However, of 54 eggs injected with nuclei from ICM cells, 3 morulae and 3 blastocysts developed and enzyme analyses of them confirmed that the preimplantation development of 2 embryos was supported by transplanted parthenogenetic nuclei. In another experimental series, 3 morulae and 4 blastocysts developed from 107 eggs injected with ICM nuclei and were transferred to uteri of foster mothers to ascertain their postimplantation development. Four female offspring were born and all of them showed a diploid karyotype and expressed enzyme activity of only the LT genotype. One female proved to be fertile and transmitted the parthenogenetic genome to the next generation. These results demonstrate that the nucleus from LT parthenogenetic blastocysts contains a complete genome necessary to support development of an adult mouse. Therefore, the early postimplantation death of parthenogenetic embryos does not seem to be related to an aberrant genotype but rather to undefined mechanisms associated with fertilization and normal morphogenetic processes.

Microsurgically produced homozygous-diploid uniparental mice.

Shortly after fertilization, either the male or the female pronucleus was microsurgically removed from 202 F(1) hybrid eggs derived from crosses of two inbred strains. Subsequent incubation of these haploid eggs in medium containing cytochalasin B, which inhibits cytokinesis but not nuclear division, enabled the remaining pronucleus to become diploid. After nuclear diploidization and transfer to regular culture medium, cleavage commenced normally, and a total of 135 successfully manipulated eggs continued in development and yielded 93 morulae and blastocysts. These embryos were surgically transferred to the uteri of pseudopregnant foster mothers who gave birth to seven live female offspring. Five of the females were derived from the maternal genome (gynogenesis) and the remaining two mice inherited only the paternal genes (androgenesis), depending on whether the female or male pronucleus had been retained in the egg, respectively. Homozygosity for a number of genetic loci positioned on different chromosomes and effecting the coat color phenotype and strain-specific allelic variants of several enzymes, urinary and plasma proteins, and hemoglobins could be demonstrated unequivocally in all instances. Chromosomal analysis revealed a normal diploid karyotype including two X chromosomes. Thus far, six of the seven homozygous-diploid (isogeneic) females have proved to be fertile and have given birth to progeny corresponding only to the pronuclear genotype of the mother.

Cytoskeletal characterization of a neuroectodermal cell line and embryonic mouse brain.

The cytoskeleton of a newly isolated mouse embryo brain-derived cell line and of dissected mesencephalon-rhombencephalon samples of the 10- to 11-day-old mouse embryo, constituted of a ventricular zone only, has been examined by immunocytochemistry, electrophoretic separation and Western blotting techniques. In accordance with their epithelial organization, the ventricular cells contain cytokeratins as main constituents of their cytoskeleton. Vimentin has been also revealed as early as day 10.5 of gestation. The complex cytokeratin polypeptide pattern puts this histological type of epithelia into the category of complex, rather than simple, epithelia and calls for explanations other than keratinization for the presence and functional role of the high-molecular weight, basic keratins. The cytoskeletal composition of the embryo brain-derived gliogenic cell line reflects the vimentin- and cytokeratin-positive character of the ventricular cells.

Developmental fate of a human insulin gene in a transgenic mouse.

A plasmid containing a genomic human insulin clone was microinjected into a pronucleus of the fertilised mouse egg. Eggs were subsequently transferred into oviducts of pseudopregnant Swiss/Alb females. Embryos developed to term and the DNA was extracted from different organs. Southern blotting analyses revealed 1 transgenic female out of 96 animals born after microinjection of C57BL/6 mouse eggs. A tandem integration was found at one locus within the mouse genome and molecular rearrangement was found within this locus. The structure of the entire locus was identical in DNA from all tissues. Both the human insulin gene sequences and the pBR322 sequences were found to be extensively methylated, although some sites were hypomethylated in the pancreas and liver. The transgenic female produced ten offspring, none of which retained the insulin gene sequences. Seven offspring retained some pBR322 sequences which were stably transmitted to the F2 and F3 generations. Homozygous F3 delta pBR/delta pBR animals were obtained, which showed neither visible defects nor sterility. The loss of the tandem locus in the F1 generation did not seem to be due to mosaicism, but involved excision due to recombination. Sequences close to the ends of the tandem locus were involved in this event. A mechanism implying excision during germ cell formation is discussed.

Developmental activation of phosphoglycerate mutase-2 in the testis of the mouse.

The expression of the phosphoglycerate mutase locus Pgam-2 which synthesizes the muscle-specific PGAM-B subunit was analyzed in the testis of the mouse. No PGAM-B activity was detected in testes of newborn mice, in which only the PGAM-AA isozyme was observed. PGAM-B was first observed between Day 14 and Day 16 of postnatal development. In adult males approximately 50% of total PGAM activity is contributed by the PGAM-B subunit and 50% by the PGAM-A subunit. Immunohistochemical studies show that in the testis PGAM-B is localized exclusively in germ cells. PGAM-B is detected in pachytene spermatocytes and in spermatids, but not in earlier stages of spermatogenesis. The muscle-specific PGAM isozyme was also found in testes of bull, cat, and rat, as well as in human sperm. PGAM-B might thus be useful as a marker for germ cell differentiation, along with other germ cell-specific proteins.

Chimeric mice derived from human-mouse hybrid cells.

Mouse teratocarcinoma cells from the OTT6050 ascites tumor were established in tissue culture and selected for 5-bromodeoxyuridine (BrdUrd) resistance. The embryonal carcinoma cells grew without a feeder layer, remained deficient for thymidine kinase (EC 2.7.1.75), and differentiated like the original tumor into various tissues after subcutaneous injection into 129 mice. We fused the BrdUrd-resistant mouse teratocarcinoma cells with HT1080-6TG human diploid fibrosarcoma cells deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) and selected for hybrid cells in hypoxanthine/aminopterin/thymidine medium. The resulting hybrid cells segregated human chromosomes quickly and retained one to three human chromosomes including chromosome 17 that carries the human genes for thymidine kinase and galactokinase (EC 2.7.1.6). Single hybrid cells from five independent clones containing human chromosome 17 were injected into mouse blastocysts bearing several genetic markers that affect the coat color phenotype and strain-specific enzyme variants in order to detect tissue differentiation derived from the injected cells. After the injection of single hybrid cells into a total of 103 experimental blastocysts that had been surgically transferred to pseudopregnant foster mothers, 49 mice were born and 2 of them clearly revealed coat mosaicism. In 2 of 17 mice thus far analyzed, the injected hybrid cells proved to be capable of participating substantially in development of seven different organs. However, human gene products have not yet been detected unequivocally in those tissues and weak human-specific galactokinase activity could be recovered only from two mosaic tissues. Our results demonstrate that, after in vitro culture and selection, at least some of the human-mouse hybrid cells still retain their in vivo potential to differentiate and become functionally integrated in the living organism. It now seems feasible to cycle mouse teratocarcinoma cells carrying human genetic material through mice via blastocyst injection to study human gene expression during differentiation.