Dr Fleming is Director of Embryology at ORIGIO a/s, where he is primarily concerned with the education and training of both trainee and experienced embryologists (http://www.origio.com/training-lab/). Previously, the Scientific Director of Westmead Fertility Centre at Westmead Hospital and Senior Lecturer in Obstetrics and Gynaecology, he now holds an honorary position at the University of Sydney (http://sydney.edu.au/medicine/people/academics/profiles/s.fleming.php). He is an author and editor of several books and textbooks on IVF units (http://www.springer.com/gp/book/9783319293714), embryology (http://www.vividpublishing.com.au/SIRT/) and micromanipulation (http://www.cambridge.org/au/academic/subjects/medicine/obstetrics-and-gynecology-reproductive-medicine/micromanipulation-assisted-conception?format=PB).
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
... Oehninger, S., Acosta, A., Veeck, LL, Simonetti, S. and Muasher, SJ (1989) Delayed fertilizat... more ... Oehninger, S., Acosta, A., Veeck, LL, Simonetti, S. and Muasher, SJ (1989) Delayed fertilization during in vitro fertilization and embryo transfer cycles ... Jenny Hall1, Simon Fishel, Steven Green, Steven Fleming, Alison Hunter, Neil Stoddart, Ken Dowell and Simon Thornton ...
Ubiquitin is a small protein involved in many intracellular processes. We have previously shown t... more Ubiquitin is a small protein involved in many intracellular processes. We have previously shown that levels of ubiquitin change during the process of decidualisation in the human uterus at the beginning of pregnancy. Other workers have shown that the ubiquitin system may be essential for normal murine placental development. In this investigation we employed immunohistochemistry and immunoblotting techniques to study the distribution and abundance of ubiquitin and ubiquitin-protein conjugates within human placental specimens from throughout gestation. Trophoblast from two pathological conditions, ectopic pregnancy and pregnancy-induced hypertension (PIH), was also investigated. Ubiquitin was detected within both the cytoplasm and nucleus of the cytotrophoblast layer only. Both monomeric and conjugated forms of ubiquitin were detected. The relative abundance of ubiquitin did not change through gestation or in the two disorders of pregnancy studied. Ubiquitin cross-reactive protein was not detected in the tissues of interest. This is the first report to demonstrate the cell-specific localisation of ubiquitin and ubiquitin-protein conjugates in the human cytotrophoblast and provides supportive evidence that ubiquitin may be important during placental development.
We have examined the distribution of ubiquitin and the related ubiquitin cross-reactive protein (... more We have examined the distribution of ubiquitin and the related ubiquitin cross-reactive protein (UCRP) in paraffin-embedded sections of human and baboon endometrium and decidua by immunoperoxidase or immunofluorescence cytochemistry with antibodies raised against ubiquitin, UCRP, CD45, and insulin-like growth factor-binding protein-1. Anti-ubiquitin immunoreactivity was present in the nonpregnant endometrium, particularly in the glandular epithelial cells, and up-regulated in endometrial stromal cells as they decidualized at the beginning of pregnancy. Anti-UCRP immunoreactivity was absent from nonpregnant tissue but accumulated to high levels in decidual cells during pregnancy. Western blotting indicated that immunoreactivity was primarily due to the presence of ubiquitin and UCRP conjugated to other proteins, and that although levels of ubiquitin-protein conjugates do not change substantially during pregnancy, decidualization is accompanied by the appearance of conjugates of UCRP. Baboon uterine tissues demonstrated a similar distribution of the two proteins, which indicates that the baboon may be a useful model for study of the role of the ubiquitin system and UCRP in the establishment of pregnancy in humans.
Journal of assisted reproduction and genetics, 1998
Our purpose was to determine whether there is a correlation between human chorionic gonadotropin ... more Our purpose was to determine whether there is a correlation between human chorionic gonadotropin (hCG) blood levels and oocyte maturation. Three samples of blood were obtained at different times from hCG administration as follows: 12 hr, 36 hr, during oocyte recovery, and at 84 hr, when the patient comes for embryo transfer. A total of 5036 oocytes was retrieved from 404 patients prospectively recruited between April 1996 and March 1997. The percentage of metaphase-II oocytes at different blood levels ranged from 84 to 88%. The general trend does not show any significant increase in percentage of metaphase-II oocytes in association with an increasing serum hCG concentration. The results of this study suggest that at 12, 36, and 84 hr after hCG administration, levels as low as 50, 45, and 9 IU/L of hCG, respectively, are equally potent as higher levels at initiating maximal oocyte maturity.
Ovulation is associated with a rise in activin A and a decline in pro-alpha C, inhibin A and inhi... more Ovulation is associated with a rise in activin A and a decline in pro-alpha C, inhibin A and inhibin B secretion. It is believed that the actions of inhibin and activin during human chorionic gonadotrophin (HCG) stimulation are mediated by protein kinase A (PKA) and/or protein kinase C (PKC). Using an in-vitro murine prenatal follicle culture model, the effects of a PKA inhibitor, Rp-cAMP, and a PKC inhibitor, PKIM, on inhibin and activin gene expression, secretion, ovulation and oocyte maturation were studied during HCG stimulation. Both Rp-cAMP (0.1 micromol/l and 1.0 micromol/l) and PKIM (1.0 micromol/l) significantly (P < 0.001) inhibited the action of HCG by suppressing the increase in activin A secretion whilst preventing the decline in pro-alpha C, inhibin A and B. In addition, Rp-cAMP and PKIM were able to significantly (P < 0.05) reduce the rate of HCG-induced ovulation and meiotic resumption, but had no effect on the completion of oocyte maturation. Furthermore, HCG-...
In contrast to a previous report by Krussel et al., with the inclusion of larger numbers of unfer... more In contrast to a previous report by Krussel et al., with the inclusion of larger numbers of unfertilized oocytes and normal embryos and more sensitive immunofluorescence, this study shows that expression of vascular endothelial growth factor messenger ribonucleic acid can be detected from the oocyte to the blastocyst stage and that protein can be detected from the 3-cell stage to the blastocyst stage in human preimplantation embryos.
Mouse embryos secrete molecules homologous to platelet-activating factor (PAF), termed embryo-der... more Mouse embryos secrete molecules homologous to platelet-activating factor (PAF), termed embryo-derived PAF (EPAF), which act in an autocrine/paracrine fashion to stimulate embryonic development in vitro. Mouse EPAF is thought to consist predominantly of hexadecyl (C16) and octadecyl (C18) PAF-like components. Mouse pre-implantation embryos cultured with exogenous PAF from the early cleavage stages exhibit increased blastocyst cell numbers and rates of mitosis around the 8-cell stage. We investigated whether exogenous PAF could specifically stimulate embryonic cell proliferation prior to the blastocyst stage in the mouse and also compared the biological activities of the C16 and C18 PAF isoforms as follows. Embryos were cultured for either 24 h or 120 h from the 2-cell stage and their total cell numbers were determined or their development assessed in terms of their incidence of successful zona-hatching respectively. In each case, embryos were cultured in unsupplemented medium or in medium supplemented with either C16 or C18 PAF (0.5 microM). Compared with controls, culture with C16 PAF produced a significant stimulation of mean total per number per embryo and a significant increase in the incidence of successful zona-hatching, whilst culture with C18 PAF had no significant effect. We then cultured 1-cell zygotes for 48 h in unsupplemented medium or medium supplemented with either an equimolar mixture of C16 and C18 PF or with either C16 or C18 PF alone (each at 0.2 microM). Embryos were also scored for cell number at 4 h and 30 h of culture. Although no significant effect on mean cell number per embryo was seen following 4 h or 30 h of culture with a mixed C16/C18 PAF preparation, culture for 48 h with a mixed C16/C18 PAF preparation or with C16 PAF alone produced a significant increase in mean cell number per embryo compared with controls - an effect that is likely to be receptor-mediated, since culture with an equivalent concentration of C18 PAF had no significant effect compared with controls. We have demonstrated that mouse zygotes/embryos can respond in a specific manner to exogenous hexadecyl PAF in terms of increased rates of cell proliferation prior to cavitation, and must be capable of doing so at some time between the first and third, and also between the second and fourth, cell cycles. Such embryos presumably express one or more classes of functional PAF-receptor molecule during this period (i.e. as early as during the 1-, 2- or 4-cell stages). We have also demonstrated that embryonic response to exogenous PAF is significantly isoform-specific, which may reflect differences between the two isoforms either in affinity for binding to putative embryonic PAF-receptor molecules or in their ability to elicit a stimulatory response following binding. This observation calls into question the use of preparations containing a mixture of hexadecyl and octadecyl PAF isoforms, particularly in dose-response studies, in the mouse.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
... Oehninger, S., Acosta, A., Veeck, LL, Simonetti, S. and Muasher, SJ (1989) Delayed fertilizat... more ... Oehninger, S., Acosta, A., Veeck, LL, Simonetti, S. and Muasher, SJ (1989) Delayed fertilization during in vitro fertilization and embryo transfer cycles ... Jenny Hall1, Simon Fishel, Steven Green, Steven Fleming, Alison Hunter, Neil Stoddart, Ken Dowell and Simon Thornton ...
Ubiquitin is a small protein involved in many intracellular processes. We have previously shown t... more Ubiquitin is a small protein involved in many intracellular processes. We have previously shown that levels of ubiquitin change during the process of decidualisation in the human uterus at the beginning of pregnancy. Other workers have shown that the ubiquitin system may be essential for normal murine placental development. In this investigation we employed immunohistochemistry and immunoblotting techniques to study the distribution and abundance of ubiquitin and ubiquitin-protein conjugates within human placental specimens from throughout gestation. Trophoblast from two pathological conditions, ectopic pregnancy and pregnancy-induced hypertension (PIH), was also investigated. Ubiquitin was detected within both the cytoplasm and nucleus of the cytotrophoblast layer only. Both monomeric and conjugated forms of ubiquitin were detected. The relative abundance of ubiquitin did not change through gestation or in the two disorders of pregnancy studied. Ubiquitin cross-reactive protein was not detected in the tissues of interest. This is the first report to demonstrate the cell-specific localisation of ubiquitin and ubiquitin-protein conjugates in the human cytotrophoblast and provides supportive evidence that ubiquitin may be important during placental development.
We have examined the distribution of ubiquitin and the related ubiquitin cross-reactive protein (... more We have examined the distribution of ubiquitin and the related ubiquitin cross-reactive protein (UCRP) in paraffin-embedded sections of human and baboon endometrium and decidua by immunoperoxidase or immunofluorescence cytochemistry with antibodies raised against ubiquitin, UCRP, CD45, and insulin-like growth factor-binding protein-1. Anti-ubiquitin immunoreactivity was present in the nonpregnant endometrium, particularly in the glandular epithelial cells, and up-regulated in endometrial stromal cells as they decidualized at the beginning of pregnancy. Anti-UCRP immunoreactivity was absent from nonpregnant tissue but accumulated to high levels in decidual cells during pregnancy. Western blotting indicated that immunoreactivity was primarily due to the presence of ubiquitin and UCRP conjugated to other proteins, and that although levels of ubiquitin-protein conjugates do not change substantially during pregnancy, decidualization is accompanied by the appearance of conjugates of UCRP. Baboon uterine tissues demonstrated a similar distribution of the two proteins, which indicates that the baboon may be a useful model for study of the role of the ubiquitin system and UCRP in the establishment of pregnancy in humans.
Journal of assisted reproduction and genetics, 1998
Our purpose was to determine whether there is a correlation between human chorionic gonadotropin ... more Our purpose was to determine whether there is a correlation between human chorionic gonadotropin (hCG) blood levels and oocyte maturation. Three samples of blood were obtained at different times from hCG administration as follows: 12 hr, 36 hr, during oocyte recovery, and at 84 hr, when the patient comes for embryo transfer. A total of 5036 oocytes was retrieved from 404 patients prospectively recruited between April 1996 and March 1997. The percentage of metaphase-II oocytes at different blood levels ranged from 84 to 88%. The general trend does not show any significant increase in percentage of metaphase-II oocytes in association with an increasing serum hCG concentration. The results of this study suggest that at 12, 36, and 84 hr after hCG administration, levels as low as 50, 45, and 9 IU/L of hCG, respectively, are equally potent as higher levels at initiating maximal oocyte maturity.
Ovulation is associated with a rise in activin A and a decline in pro-alpha C, inhibin A and inhi... more Ovulation is associated with a rise in activin A and a decline in pro-alpha C, inhibin A and inhibin B secretion. It is believed that the actions of inhibin and activin during human chorionic gonadotrophin (HCG) stimulation are mediated by protein kinase A (PKA) and/or protein kinase C (PKC). Using an in-vitro murine prenatal follicle culture model, the effects of a PKA inhibitor, Rp-cAMP, and a PKC inhibitor, PKIM, on inhibin and activin gene expression, secretion, ovulation and oocyte maturation were studied during HCG stimulation. Both Rp-cAMP (0.1 micromol/l and 1.0 micromol/l) and PKIM (1.0 micromol/l) significantly (P < 0.001) inhibited the action of HCG by suppressing the increase in activin A secretion whilst preventing the decline in pro-alpha C, inhibin A and B. In addition, Rp-cAMP and PKIM were able to significantly (P < 0.05) reduce the rate of HCG-induced ovulation and meiotic resumption, but had no effect on the completion of oocyte maturation. Furthermore, HCG-...
In contrast to a previous report by Krussel et al., with the inclusion of larger numbers of unfer... more In contrast to a previous report by Krussel et al., with the inclusion of larger numbers of unfertilized oocytes and normal embryos and more sensitive immunofluorescence, this study shows that expression of vascular endothelial growth factor messenger ribonucleic acid can be detected from the oocyte to the blastocyst stage and that protein can be detected from the 3-cell stage to the blastocyst stage in human preimplantation embryos.
Mouse embryos secrete molecules homologous to platelet-activating factor (PAF), termed embryo-der... more Mouse embryos secrete molecules homologous to platelet-activating factor (PAF), termed embryo-derived PAF (EPAF), which act in an autocrine/paracrine fashion to stimulate embryonic development in vitro. Mouse EPAF is thought to consist predominantly of hexadecyl (C16) and octadecyl (C18) PAF-like components. Mouse pre-implantation embryos cultured with exogenous PAF from the early cleavage stages exhibit increased blastocyst cell numbers and rates of mitosis around the 8-cell stage. We investigated whether exogenous PAF could specifically stimulate embryonic cell proliferation prior to the blastocyst stage in the mouse and also compared the biological activities of the C16 and C18 PAF isoforms as follows. Embryos were cultured for either 24 h or 120 h from the 2-cell stage and their total cell numbers were determined or their development assessed in terms of their incidence of successful zona-hatching respectively. In each case, embryos were cultured in unsupplemented medium or in medium supplemented with either C16 or C18 PAF (0.5 microM). Compared with controls, culture with C16 PAF produced a significant stimulation of mean total per number per embryo and a significant increase in the incidence of successful zona-hatching, whilst culture with C18 PAF had no significant effect. We then cultured 1-cell zygotes for 48 h in unsupplemented medium or medium supplemented with either an equimolar mixture of C16 and C18 PF or with either C16 or C18 PF alone (each at 0.2 microM). Embryos were also scored for cell number at 4 h and 30 h of culture. Although no significant effect on mean cell number per embryo was seen following 4 h or 30 h of culture with a mixed C16/C18 PAF preparation, culture for 48 h with a mixed C16/C18 PAF preparation or with C16 PAF alone produced a significant increase in mean cell number per embryo compared with controls - an effect that is likely to be receptor-mediated, since culture with an equivalent concentration of C18 PAF had no significant effect compared with controls. We have demonstrated that mouse zygotes/embryos can respond in a specific manner to exogenous hexadecyl PAF in terms of increased rates of cell proliferation prior to cavitation, and must be capable of doing so at some time between the first and third, and also between the second and fourth, cell cycles. Such embryos presumably express one or more classes of functional PAF-receptor molecule during this period (i.e. as early as during the 1-, 2- or 4-cell stages). We have also demonstrated that embryonic response to exogenous PAF is significantly isoform-specific, which may reflect differences between the two isoforms either in affinity for binding to putative embryonic PAF-receptor molecules or in their ability to elicit a stimulatory response following binding. This observation calls into question the use of preparations containing a mixture of hexadecyl and octadecyl PAF isoforms, particularly in dose-response studies, in the mouse.
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