Mohamed Fathi

Caffeine supplementation during IVM improves frequencies of nuclear maturation and preimplantation development of dromedary camel oocytes following IVF

Caffeine supplementation during oocyte IVM has been reported to improve preimplantation embryo development and the quality of in vitro-produced blastocysts in a range of species; but no studies have been done in camels. The present study investigated the effect of caffeine supplementation during dromedary camel oocyte IVM on nuclear maturation rates, IVF events, and subsequent preimplantation development. Cumulus-oocyte complexes obtained at slaughter were matured in vitro in caffeine supplemented medium either for 30 hours (caffeine 30 hours) or in the medium without caffeine supplement for 24 hours and then transferred to freshly prepared IVM medium supplemented with 10 mM caffeine for another 6 hours (caffeine 6 hours). Cumulus-oocyte complexes matured for 30 hours in the medium without caffeine supplement were used as a control. Matured oocytes were fertilized in vitro by epididymal spermatozoa of mature male camels collected from a local slaughterhouse. Eighteen hours after insemination, presumptive zygotes were cultured in modified KSOMaa medium for 7 days. Maturation and fertilization rates were significantly higher in the caffeine 6-hour group compared with the control group (P < 0.05), whereas IVM of oocytes in caffeine-supplemented medium for 30 hours did not affect these parameters (P > 0.05). Interestingly, IVM of oocytes in caffeine supplemented medium for 6 hours significantly (P < 0.05) increased the frequencies of blastocyst development by more than two-fold when compared with control (27.78% vs. 11.76%). In conclusion, culturing dromedary camel oocytes in maturation medium without caffeine for 24 hours and then in the medium supplemented with 10 mM caffeine for 6 hours during 30-hour IVM can significantly improve frequencies of nuclear maturation, fertilization rate, and subsequent preimplantation development.

Chalcones incorporated pyrazole ring inhibit proliferation, cell cycle progression, angiogenesis and induce apoptosis of MCF7 cell line.

A Series of chalcone derivatives containing pyrazole ring was prepared and their cytotoxicity against different human cell lines, including breast (MCF-7), colon (HCT-116) liver (HEPG2) cell lines, as well as normal melanocyte HFB4 was evaluated. Two of these chalcone derivatives with different IC50 and chemical configuration were chosen for molecular studies in detail with MCF-7 cells. Our data indicated that the two compounds prohibit proliferation, angiogenesis, cell cycle progression and induce apoptosis of breast cancer cells. This inhibition is mediated by up regulation of tumor suppressor p53 associated with arrest in S-G2/M of cell cycle. This work provides a confirmation of antitumor activity of the novel chalcones and assists the development of new agents for cancer treatment.

Effect of tranquilizers on the total acetylcholine content and acetylcholinesterase activity in the brain tissue of Arvicanthis niloticus

The effect of reserpine and meprobamate on the total acetylcholine content and acetylcholinesterase activity in the brain tissue of the kusu rat, Arvicanthis niloticus, was studied. The total acetylcholine content and acetylcholinesterase activity were determined 1 hr after i.p. injection of different doses of reserpine (0.25, 0.5 and 1 mg/ml/100 g body wt) and meprobamate (6.25, 12.5 and 25 mg/ml/100 g body wt). The effect of different time intervals (1, 10, 30 min, 1, 2.5, 5, 8, 12, 24 and 48 hr) on the total acetylcholine content and acetylcholinesterase activity was investigated after i.p. injection of 0.5 mg of reserpine and 12.5 mg of meprobamate/ml/100 g body wt. Both reserpine and meprobamate caused an increase in the total ACh content in the brain tissue of Arvicanthis niloticus which was suggested to be due to a decrease in the release of ACh, since both reserpine and meprobamate inhibited AChE activity after some tested periods. The effect of meprobamate was observed to be stronger than that of reserpine.

Production of blastocysts following in vitro maturation and fertilization of dromedary camel oocytes vitrified at the germinal vesicle stage

Cryopreservation of oocytes would serve as an alternative to overcome the limited availability of dromedary camel oocytes and facilitate improvements in IVP techniques in this species. Our goal was to develop a protocol for the vitrification of camel oocytes at the germinal vesicle (GV) stage using different cryoprotectant combinations: 20% EG and 20% DMSO (VS1), 25% EG plus 25% DMSO (VS2) or 25% EG and 25% glycerol (VS3) and various cryo-carriers; straws or open pulled-straw (OPS) or solid surface vitrification (SSV); and Cryotop. Viable oocytes were cultured in vitro for 30 h. Matured oocytes were fertilized with epididymal spermatozoa and then cultured in vitro in modified KSOMaa medium for 7 days. Survival and nuclear maturation rates were significantly lower (P ≤ 0.05) in oocytes exposed to VS3 (44.8% and 34.0%, respectively) than those exposed to VS1 (68.2% and 48.0%, respectively) and VS2 (79.3% and 56.9%, respectively). Although recovery rates were significantly lower (P ≤ 0.05) in SSV and Cryotop vitrified oocytes (66.9% to 71.1%) than those vitrified by straws with VS1 or VS2 solutions (86.3% to 91.0%), survival rates were higher in the SSV and Cryotop groups (90.7% to 94.8%) than in the straw and OPS groups (68.2% to 86.5%). Among vitrified groups, maturation and fertilization rates were the highest in the Cryotop-VS2 group (51.8% and 39.2%, respectively). These values were comparable to those seen in the controls (59.2% and 44.6%, respectively). Cleavage (22.5% to 27.9%), morula (13.2% to 14.5%), and blastocyst (6.4% to 8.5%) rates were significantly higher (P ≤ 0.05) in SSV and Cryotop groups than in straws. No significant differences were observed in these parameters between the Cryotop and control groups. We report for the first time that dromedary oocytes vitrified at the GV-stage have the ability to be matured, fertilized and subsequently develop in vitro to produce blastocysts at frequencies comparable to those obtained using fresh oocytes.

Developmental competence of Dromedary camel (Camelus dromedarius) oocytes selected using brilliant cresyl blue staining - ERRATUM

The objectives of the present studies were to investigate the developmental capacity of dromedary camel oocytes selected by brilliant cresyl blue (BCB) staining and to investigate the expression of select transcripts in germinal vesicle (GV) stage oocytes. These transcripts included BMP15 and GDF9 as important transcripts for folliculogenesis and oocyte development, Zar1 and Mater as maternal transcripts required for embryonic development, Cyclin B1 and CDK1 as cell cycle regulators and Oct4 and STAT3 as transcription factors. Dromedary camel oocytes were retrieved from ovaries collected at a local slaughterhouse. After exposure to BCB staining, cumulus-oocyte complexes (COCs) from BCB+, BCB- and control (selected based on morphological criteria) groups were subjected to in vitro maturation, in vitro fertilization and in vitro culture. For gene expression studies, after BCB staining cumulus cells were stripped off and the completely denuded GV stage oocytes were used for RT-PCR analysis of selected transcripts. BCB+ oocytes showed higher maturation, and fertilization rates compared with BCB- and control groups. Indices of early embryonic development, namely, cleavage at 48 hours post insemination (hpi), and development to morula at day 5 and day 7 blastocyst rates were also significantly higher in the BCB+ group. RT-PCR revealed a higher expression of BMP15, GDF9, Zar1, Mater, Cyclin B1, CDK1, OCT4 and STAT3 in good quality oocytes that stained positively for BCB (BCB+). Collectively, results provide novel information about the use of BCB screening for selecting good quality oocytes to improve in vitro embryo production in the dromedary camel.

Effect of barbitone sodium and thiopental sodium on brain dopamine, noradrenaline, serotonin and 5-hydroxyindoleacetic acid content in Arvicanthis niloticus

The quantitative estimation of total dopamine (DA), noradrenaline (NE), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) content in the whole brain tissue of normal Nile grass rat, Arvicanthis niloticus, gives and average of 631 +/- 12 ng DA/g, 366 +/- 12 ng NE/g, 617 +/- 15 ng 5-HT/g and 431 +/- 10 ng 5-HIAA/g fresh brain tissue. The effect of barbitone sodium and thiopental sodium on the total DA, NE, 5-HT and 5-HIAA content in the brain tissue of the Nile grass rat, Arvicanthis niloticus, was studied. The total DA, NE, 5-HT and 5-HIAA contents were determined 5 hr after i.p. injection of different doses of barbitone sodium (20, 40 and 80 mg/ml/100 g body wt) and thiopental sodium (5, 10 and 20 mg/ml/100 g body wt). The effect of different time intervals (1, 10, 30 min, 1, 2.5, 5, 8, 16, 24 and 48 hr) on the total brain DA, NE, 5-HT and 5-HIAA content was investigated after i.p. injection of 40 mg of barbitone sodium and 10 mg of thiopental sodium/ml/100 g body wt. Both barbitone sodium and thiopental sodium caused an increase in DA, NE and 5-HT content and a decrease in 5-HIAA content in the brain tissue of Arvicanthis niloticus. The increase in the whole brain contents of DA, NE and 5-HT after the administration of barbitone sodium and thiopental sodium may be due either to inhibition of transmitter release by an action at the monoamine nerve terminal or to effects causing a decrease in nerve impulse flow. On the other hand, the decrease in 5-HIAA may be due to the decrease in the turnover of 5-HT.

Effect of thiopental sodium and barbitone sodium on the total acetylcholine content and acetylcholinesterase activity in the brain tissue of Arvicanthis niloticus

The total ACh content and AChE activity were determined 1 hr after the i.p. injection of different doses of thiopental sodium (5, 10 and 20 mg/ml/100 g body wt) and barbitone sodium (20, 40 and 80 mg/ml/100 g body wt). The effect of different time intervals (1 min, 10 min, 30 min, 1 hr, 2.5 hr, 5 hr, 8 hr, 12 hr, 24 hr and 48 hr) on the total ACh content and AChE activity was investigated after i.p. injection of 10 mg thiopental sodium and 40 mg barbitone sodium/ml/100 g body wt. Both thiopental sodium and barbitone sodium increased the total ACh content in the brain tissue of Arvicanthis niloticus. Both drugs inhibited the brain AChE activity. It is thought that the increase in the total ACh content in the brain tissue of Arvicanthis niloticus may be due to a decrease in the release of ACh from the neuronal tissue and a decrease in AChE activity.