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Öğe The Growth Promoting Effects of bFGF, PD-ECGF and Vegf on Cultured Postimplantation Rat Embryos Deprived of Serum Fractions(Wıley, 2000) Ulger, Harun; Karabulut, Ahmet K.; Pratten, Margaret K.Serum components in which embryos are cultured in vitro are very important for normal embryonic development. In this study, rat serum was fractionated using Macrosep filters to study the effect of a single growth factor. The fractionated serum, both that containing only material greater than 30 kDa molecular weight (> 30 kDa) and that from which material between 30 kDa and 50 kDa had been removed (< 30 kDa + > 50 kDa), caused significant embryonic growth retardation. Addition of different concentrations of basic fibroblast growth factor (bFGF, 18 kDa), vascular endothelial growth factor (VEGF, 45 kDa) and platelet-derived endothelial growth factor (PD-ECGF, 45 kDa), to fractionated serum (bFGF to > 30 kDa serum and VEGF or PD-ECGF to < 30 kDa + > 50 kDa serum) partially restored embryonic growth and development according to a morphological scoring system and protein assay. This restoration was clear by all criteria, as well as in yolk sac vascularisation and heart development. The growth promoting effects of all 3 factors were significant but did not reach the level seen in embryos grown in whole rat serum. The effect of these growth factors was also investigated on anembryonic yolk sac development using a concentration for which maximum whole embryonic growth was seen (128 ng/ml bFGF, 1.6 ng/ml VEGF and 4 ng/ml PD-ECGF), and significant anembryonic yolk sac development was found. These findings suggest that the angiogenic factors may have a growth promoting effect on total embryonic development and vascularisation.Öğe Growth Promoting Effects of Human Placental Lactogen During Early Organogenesis: A Link to Insulin-Like Growth Factors(Wıley, 2001) Karabulut, Ahmet Kağan; Layfield, Robert; Pratten, Margaret K.Many maternally derived factors may be involved in the regulation of embryonic growth but the control mechanisms involved are poorly understood. Human placental lactogen (hPL) has been implicated in playing a role in the control of embryonic growth. Several investigators suggested that there may be a possible link between the effects of this hormone and insulin-like growth factors (IGFs). In order to determine the growth promoting potential of hPL and involvement of IGFs in the mechanism of action of the hormone, 9.5 d rat embryos were cultured in vitro for 48 h in depleted serum in the presence and absence of hPL with additional IGF antisera. The growth supporting capacity of the serum was reduced by removal of low molecular weight molecules by prolonged filtration of the serum using filters with a molecular weight exclusion of 30 kDa. Addition of hPL (3.2-25.6 ng/ml) to depleted serum significantly improved embryonic growth and development, suggesting that the developing embryo may utilise hPL. The presence of antisera against hPL, IGF-I and -II abolished the hPL-induced increase in the development in all parameters suggesting that there may be a possible link between the IGFs and the effects of hPL on rat embryonic development and this hormone may achieve its growth promoting effects via IGFs.Öğe Isolation and culture of endothelial cells from embryonic rat yolk sac(SCIENDO, 2017) Ulger, Harun; Karabulut, Ahmet K.; Pratten, Margaret K.Yolk sac blood islands are the first morphologic evidence of hematopoietic development during mammalian embryogenesis, and visseral yolk sac mesoderm gives rise to the first embryonic blood cells within a rich endothelial network. Present study reports the isolation and culture of endothelial cells from 11.5 days old embryonic rat yolk sac. The embryos were dissected from 11.5 days pregnant Wistar rat (Rattus norvegicus) and the external yolk sac membrane and embryos were removed under aseptic condition. After washing three times with Calcium-Magnesium free Hank's balanced salt solution (CMF-HBSS), the tissue was minced, and fragments were incubated in CMF-HBSS containing 2mg/ml Trypsin, 100mg/ml collagenase I and 40mg/ml DNAse at 37 degrees C until the tissue was completely dispersed. The digestion effect was then neutralized by fetal bovine serum at 1:3 (v/v). The cell suspension was centrifuged at 1000 rpm for 10 min., the supernatants were discarded and the cell pellets resuspended in Dulbecco modified Eagle medium containing 15% fetal bovine serum, 1.25mg/ml amphotericin B, 25mg/ml gentamycin sulphate and 100mg/ml endothelial cell growth supplement. The resuspended cells were plated in two diverse 25cm(2) culture flasks for overnight differential adherence at 37 degrees C. The non-adherent cells were removed by gentle aspiration and adherent cells refed with fresh medium. The cells were transferred using 1ml of 0.2% Trypsin when cultures reached near-confluence. The cultured yolk sac endothelial cells had characteristic cobblestone appearence and positive immunofluorescent staining for von Willebrand Factor (vWF). Weibel-Palade bodies, the major ultrastructural marker for endothelium, were also detected in cultured cells by electron microscopy.Öğe Visualisation of the uptake of prolactin ( PRL ) in rat embryonic tissues(1999) Karabulut, A. Kağan; Ülger, Harun; Pratten, Margaret K.Objective: Evidences implicate roles for prolactin (PRL) in the regulation of embryonic growth. To clarify the roles of PRL in rat embryogenesis we examined the uptake and expression of the hormone in embryonic tissues. Methods: Nine and a half day postimplantation rat embryos were cultured in vitro for 44h in rat serum and serum depleted of low molecular weight molecules (retenate). The embryos were transferred to M199 for the last 4h, and 12.8 ng/ml rat PRL was added to culture medium for different times (4h - 15 min) and/or different temperatures (37ºC and 4ºC). As a control tissue, pituitary glands from 11.5 and 18.5d pregnant rats were used. Embryos and tissues were then examined by an indirect immunofluorescence protocol. Results: The pituitary glands showed positive immunoreactivity for anti-PRL antibody whilst there was no stain in the control brain tissue. Immunoreactivity was observed in embryos grown in rat serum, and intensity was much greater in the presence of additional rat PRL, whilst there was no immunoreactivity detected in those grown in retenate only. Shorter incubations and incubations at 37ºC caused a greater immunoreactivity for PRL, suggesting that this is an active and temperature dependent metabolic process. Conclusion: These results show the uptake and distribution of PRL by the yolk sac and embryonic tissues which might be interpreted for the presence of PRL receptors.
