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.

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.

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.

Tissue perfusion essential for spermatogenesis and outcome of testicular sperm extraction (TESE) for assisted reproduction.

PURPOSE: In order to determine if there are areas of major and minor perfusion in a single testicle and if the quality of sperm is correlated with quantity of perfusion we collected testicle tissue for TESE in accordance to the local testicle tissue perfusion. METHODS: A patient undergoing TESE underwent testicular perfusion mapping using contrast enhanced ultrasound. The exposed tissue was scanned with a Laser Doppler scanner and perfusion rates were determined measuring tissue perfusion units (TPUs). Tissue was biopsied and sperm were selected and prepared for assisted reproduction. RESULTS: The total amount of isolated sperm correlated highly with the intensity of tissue perfusion showing high number of sperm in areas with high TPUs. CONCLUSIONS: This is the first demonstration that sperm quality and quantity is depending on tissue perfusion within the testicle. To further improve infertility treatment we propose that random biopsies could be replaced by perfusion-dependent collection of testicular tissue.

[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.

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.

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.

Different protein patterns derived from follicular fluid of mature and immature human follicles.

The purpose of our study was to compare the protein patterns originating from fluids of mature and immature human follicles in order to gain further insight into their biochemical composition. A total of 10 patients were stimulated for in-vitro fertilization (IVF) using different stimulation protocols. Follicular fluids were aspirated transvaginally and analysed microscopically for the presence of oocytes. Follicular fluids were stored at -18 degrees C. Samples of 500 microliters were processed for two-dimensional gel electrophoresis. Up to 60 proteins in various groups could be detected. Seven protein spots were selected for chemical analysis by cutting them out of the gels and subjecting them to internal amino acid sequencing procedures. Our results can be summarized as follows: (i) major differences were not detected between the protein patterns from the various mature follicles of a particular patient, nor were significant differences observed in the proteins derived from follicular fluids collected from the seven patients with mature follicles; (ii) considerable differences were observed in the protein patterns derived from fluids of immature compared with mature follicles. Fluid from the three patients with immature follicles contained many fewer proteins, some of which were expressed at low levels. We conclude that the observed variations in protein composition of follicles of different developmental age reflect their physiological condition and serve as biomedical markers for follicular maturity.

Comparison of protein analysis between embryonic and extraembryonic tissues during the 11th day of gestation of the mouse.

At day 11 of gestation, embryos and their extraembryonic tissues were isolated from the uterus of Him OF1/SPF mice and incubated in Dulbecco's modified Eagle's medium (DMEM) containing L-[35S]methionine. After 4 h of incubation, the embryos were dissected to obtain the heart, liver, limb buds, and brain. The latter was fragmented into the telencephalon, mesencephalon, and myelencephalon. These organs and the extraembryonic tissues such as chorion, yolk sac, and placenta were processed for two-dimensional (2-D) gel electrophoresis. About 1000 proteins with relative molecular weights (M(r) varying from 10,000 to 200,000 and isoelectric points ranging from 4 to 10 could be detected on these gels. The protein patterns of the various organs and tissues were analyzed for organ- and cell lineage-specific protein spots. We detected subtle differences in the protein patterns of the three cerebral areas when compared to each other. In addition, we found protein spots characteristic for the entire brain. We also found several heart-specific protein spots. Distinct protein synthesis was also detected in liver and limb buds. Several groups of protein spots seem to be differentially regulated in these organs. Substantial differences between the patterns of embryonic and extraembryonic tissues were observed. In addition, several clusters of protein spots of well-defined molecular weight could be detected only in extraembryonic tissues. We propose that organ- and tissue-specific differences in protein synthesis are linked to some of the morphogenetic and functional processes during mammalian embryogenesis. Identification of particular proteins will serve as a basis to search for the corresponding genes.

Protein synthesis in murine organs during postimplantation development detected by two-dimensional gel electrophoresis.

Mouse embryos were isolated from the uterus on days 10 to 11 of gestation and incubated in Dulbecco's modified Eagle's medium (DMEM) with [35S]methionine for 4 h. Subsequently, their hearts and the brains were dissected. The brain was divided into three parts, containing the telencephalon, mesencephalon, and myelencephalon. These tissues were then processed for two-dimensional (2-D) gel electrophoresis. Protein synthesis of the isolated tissues was analyzed for organ-and cell lineage-specific patterns. We studied proteins with isoelectric points (pI) ranging from 4 to 10 and relative molecular weights (M(r)) varying from 10000 to 200000 and found several significant quantitative and qualitative differences between the tissues and the developmental stages analyzed. In particular, we were able to distinguish between protein spots that we now attribute putatively to the corresponding embryonic organs. These differences may reflect some of the organ- and cell lineage-specific changes in protein synthesis and gene expression during early mammalian differentiation.

Protein synthesis in embryonic tissues during mouse postimplantation development.

Mouse embryos of the NMRI strain between the 7th and 9th day of gestation were isolated from the uterus and dissected into the various tissue derivatives in order to investigate newly synthesized proteins during morphogenesis. The day 7 embryo was fragmented into trophoblast and ectoplacental cone, distal and proximal endoderm, extraembryonic and embryonic ectoderm. The day 8 and day 9 embryos were divided into trophoblast and placental anlage, yolk sac, amnion, and allantois, as well as cranial, central, and caudal embryonic tissue. The intact embryos were incubated in Dulbecco's minimum essential medium in the presence of 35S-methionine for 4 h, then dissected into the various fragments, and further processed for two-dimensional gel electrophoresis. Protein synthesis of the isolated tissue derivatives was analyzed and compared for the three developmental stages. Concerning the proteins with isoelectric points in the range of 4.5 to 8.0 and molecular weight ratio (M(r)) values between 20,000 and 200,000, we found several significant quantitative and qualitative differences in the various tissue fragments. In addition, we observed further quantitative and qualitative differences in protein synthesis during the postimplantation period investigated. We propose that the differences reflect some of the cell lineage- and developmental stage-specific changes in gene expression during early mammalian differentiation.

Nuclear transfer from teratocarcinoma cells into mouse oocytes and eggs.

A spontaneous ovarian teratocarcinoma was isolated from a LT/Sv mouse female and converted into an ascites tumor from which embryonal carcinoma (EC) cells were dissociated. Non-enucleated and enucleated, activated oocytes were fused with EC cells and either cultured in vitro or transferred into ligated oviducts of Swiss/A females. The nucleocytoplasmic hybrids cultured in vitro up to 22 h were examined cytologically at various time intervals. EC nuclei showed morphological remodelling in the foreign cytoplasm. EC chromosomes and female pronuclear chromosomes together formed a common metaphase. The nucleocytoplasmic hybrids developed in vivo were analyzed cytologically between the first and third day after oviduct transfer. The majority of embryos developed abnormally and, in a few instances, they had passed several cleavage divisions and reached, at best, a developmental stage resembling a premature morula. Fertilized, enucleated eggs were fused with EC cells or microinjected with EC nuclei. The resulting nucleocytoplasmic hybrids were either cultured in vitro or in vivo up to the fourth day. Enzyme tests were carried out on the nuclear transplant embryos, using electrophoretic variants of glucose phosphate isomerase (GPI) in order to distinguish between EC nuclei (GPI-A) and recipient eggs (GPI-B). The EC-specific GPI could be detected in about one third of the embryos analyzed and, in several instances, also together with the egg-specific GPI. Most of them were arrested during early cleavage divisions. Some embryos cleaved abnormally or mimicked normal embryogenesis. In a few instances, development resulted in embryos that resembled late preimplantation embryos.

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.

Sequential expression of maternally inherited phosphoglycerate kinase-1 in the early mouse embryo.

Enzyme activities of X-linked phosphoglycerate kinase (PGK-1) and autosomal glucose phosphate isomerase (GPI-1) were determined in intact mouse blastocysts and isolated inner cell masses (ICMs). Blastocysts were recovered from the uterus on day 4 of gestation and cultured overnight in vitro. ICMs were isolated by treatment with calcium ionophore A23187. On day 4, approximately 35% of the total activity of both PGK-1 and GPI-1 was located in the ICM. After overnight culture, the PGK-1 activity of the whole blastocyst nearly doubled, due to the activation of only the maternally derived gene coding for PGK-1. In the ICM, however, a pronounced decrease of PGK-1 activity was measured: only 10% of the total PGK-1 activity was measured in the ICM on day 5. In contrast to PGK-1, GPI-1 activity of the intact blastocyst remained stable from day 4 to day 5. In the ICM, the GPI-1 activity did decline, but to a lesser extent than PGK-1 activity: 20% of total GPI-1 activity was found in the ICM on day 5. These results, when compared with the data of Handyside and Hunter, suggest that the decline in GPI-1 activity in the ICM is due to a change in the ratio of trophectoderm (TE) to ICM cells. The greater reduction of PGK-1 activity in the ICM cannot, however, be explained solely by this mechanism. To explain the observed additional decrease, we postulate that Pgk-1 is not activated in the ICM prior to day 6. This implies that on day 4 maternal Pgk-1 is activated in the TE exclusively.

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.

[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.

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.