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.
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.
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.
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.
A miniature system for two‐dimensional polyacrylamide gel electrophoresis is presented. Discontinuous sodium dodecyl sulfate gel electrophoresis in micro‐slab gels following high resolution micro‐electrophoresis in cylindrical continuous polyacrylamide gradient gels separates macromolecules after an initial size fractionation into their subunits. The construction of a glass cuvette for micro‐slab gels and of a suitable electrophoresis tank is described in detail. Furthermore, procedures for silver staining and fluorography initially developed for gels of normal size were adapted to micro‐slab gels. Crude tissue extracts of Funaria hygrometrica L. Sibth. were analyzed without any further purfication. Twenty samples can be processed in about eight hours.
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