Vitro Effects of Sildenafil on Murine Embryo Development - Dissertation Example

?2. Methodology 2 Experimental Plan 2 1 In vitro effects of sildenafil on murine embryo development In order to assess the impacts of sildenafil on embryo development, the experiment required two cell embryos that were taken from naturally mated mice and grown to blastocyst stage in an environment supplemented with sildenafil (Figure 2.1). Sildenafil was used at a concentration of 0.5µg/ml in order to compare well with the plasma concentrations in the male mice. The development process of the embryos was examined by assessing its rate and the cell number of the blastocysts after four days. Figure 2.1 In vitro effects of sildenafil on murine embryo development 2.1.2 In vivo effects of sildenafil on murine embryo development This part of the experiment was not conducted due to requirement for changes in the license. However, the in vivo effects on the development of oocyte would have been examined by injecting sildenafil on female mice for a period of four days with the following three doses: 0.5 mg/kg, 1.5mg/kg and 2.5mg/kg. Afterwards, the female mice would be mated naturally with males; then one-day old zygotes would be harvested from sacrificed females. The number of oocytes and corpora lutea recorded would be used to determine the rate of ovulation. Thereafter, the embryos would be grown to blastocysts again in a media not supplemented with sildenafil. The quality of development of the embryos would then be determined by examining their development rate, and assessing the numbers of the blastocyst cells (figure 2.2). Figure 2.2 In Vivo effects of sildenafil on murine embryo development 2.1.3 Expression of PDE5a in the mouse pre-implantation embryo The experiment was conducted by examining the presence of PDE5 mRNA in murine embryos at the successive stages of embryo development. The embryos used for study were obtained from mice that had been mated naturally. On the other hand, the blastocysts had been cultured in standard conditions. 2.2 Sildenafil Citrate purification from commercially available Viagra tablets In both the in vitro and in vivo experiments, sildenafil had been cleansed from commercially available Viagra tablets by leaving 20g sephadex G-25 overnight to swell in 100ml of distilled water. A column of 80ml was then applied with the sephadex gel and equilibrated in 100ml of distilled water at room temperature. A Viagra tablet of 38mg was then placed in 91.2ml of distilled water and slowly mixed with a magnetic stirrer at room temperature for a period of twenty minutes. It was then filtered for 20 minutes under a temperature of 4 degrees Celsius. The liquid in which sildenafil has been dissolved was then applied on the column. The column was rinsed with the Viagra solution just before it was flushed with 400ml of water to wash away any possible small molecules. This way, only sildenafil was left precipitated to resin. The column was then applied with 1% Formic acid to rinse the sildenafil off. The absorbance of the rinsed solution was then observed and according to Francis et al. (2003), the rinsed sildenafil had a sharp peak absorbance at 40ml (Figure 3.1). Figure 3.1 Elution of pure sildenafil from sephadex column After sildenafil was rinsed, the column was washed again with 160ml or distilled water to eliminate the formic acid. After the column was free of formic acid, it was then washed with 320ml of 0.2% sodium azide in order to preserve it for later use. The rinsed sildenafil was then frozen at temperatures of -80 degrees Celsius. It was then dried by freezing it in a high vacuum. This was done by first sublimating the contents for a period of 8 hours at 0.37 mbar and under a temperature of -53 degrees Celsius, then desorbing it at 0.001 mbar for 3 hours. The weight of the crystallized contents was determined by examining the rate of absorbance of sildenafil over the whole sildenafil that had been eluted, assuming that there had been a recovery rate of 60%. The eluted sildenafil was then dissolved in 0.1% formic acid in order to obtain the normal concentration of refined sildenafil. 2.3 Direct effect of Sildenafil Citrate on in vitro development of murine embryos 2.3.1 Embryo retrieval All experiment in this part were performed by licensed staff under license number 40/2914. Female mice aged between six to eight weeks were kept in a 12-hour light-dark environment. However, the mice were adequately provided with drinking water and laboratory food. Male mice were also kept in a similar environment and for the same period but in a separate area. The mice were allowed one week for adaptation after which they were mated monogamously. The following morning after mating, female mice showing signs of vaginal plug were isolated from the rest and kept in a different unit for a period of 24 hours. On the second day after mating, the female mice were sacrificed through dislocating the cervix. Embryos were then harvested from the oviduct by first dissecting the oviducts, which were then transferred to 1ml handling medium that had 5% Human Serum Albumin balanced at 37 degrees Celsius. Using a stereoscopic microscope, the oviducts were steadied using fine forceps while the infundibulum was identified. Using a 1ml syringe, a dulled two-cell flushing needle was inserted into the infundibulum and the equilibrated media gently flushed into the oviduct to eject the embryos from the uterine horns. The oviducts that had not been well flushed were cut using forceps and the embryos harvested from the tattered oviduct tissue. The harvested embryos were then transferred to fresh pre-equilibrated media and kept in an incubator under a temperature of 37 degrees Celsius without gas. 2.3.2 Embryo culture to blastocyst stage Harvested two-cell embryos were assigned to seven experimental groups randomly and grown in vitro for a period of four days until they reached the blastocyst stage. Sildenafil citrate was then dissolved in 0.1% formic acid and then introduced to the embryo culture. Three different concentrations were then obtained: 5µg/ml (S1), 0.5µg/ml (S2) and 0.05µg/ml (S3). Formic acid control solutions for the different groups were also obtained to prevent formic acid from having any possible effects on embryo development. The formic acid control groups were provided to the respective embryo groups as follows: 0.0005% (FA1), 0.00005% (FA2) and 0.000005% (FA3). Finally, a seventh group that did not contain any sildenafil or formic acid. Twenty-microliter portions of the culture media were covered with mineral oil and equilibrated for an hour in a humid environment with a carbon dioxide and oxygen volume of 5%. The harvested embryos were transferred from the equilibrated media to the supplemented medium with a maximum of five embryos per 20µl culture drop. From the humid atmosphere, the embryos were then transferred to a blastocyst media supplemented with 5% HAS and the suitable amounts of sildenafil. They were then cultured up to the fourth day in a humid atmosphere. The development stage of the embryos was recorded on each day during the culture. In addition, the total number of blastocyst cells was also recorded as a way of measuring the development capacity of the blastocysts. Figure 2.3 Experimental design of the in vitro culture of embryos in sildenafil 2.3.4 Blastocyst Cell Counting Blastocysts from all the five groups were tinted and the total number of their cells determined. Blastocysts were nurtured in a media containing 1% Triton-X 100 and 100µg/ml Propidium iodine for ten seconds. This made the outside of the cells to be permeable thus allowing PI, which is non-permeating, to penetrate the outer TE cells. The blastocysts were then counter-tinted in 20µl drops of 100% ethanol that had 25µg/ml bisbenzimide. They were then stored overnight under a temperature of 4 degrees Celsius. Afterwards, the blastocysts were moved to 20% glycerol slides and covered to make them flat. The blastocysts were then viewed under ultraviolet light using a microscope. The differential staining method was however not successful since, PI was making all cells permeable and Hoesht was weak. Therefore, only the total number of cells was determined. Thus, a simpler method was used as follows. Blastocysts were first washed four times in 1mg/ml PVP in PBS and injected overnight under temperatures of 4 degrees Celsius in 20µl drops of 3.5% paraformaldehyde under mineral oil. They were then washed four more times in 1mg/ml PVP in PBS and stored at 4 degrees Celsius in 1ng/ml PVP in PBS for further analysis. 2.4 Expression of PDE5 in one-cell, two-cell and blastocyst stage embryos 2.4.1 Snap freezing of murine tissue and embryos Females displaying vaginal plugs after natural mating were sacrificed and denuded zygotes removed from the swollen ampulla. The zygotes were then washed in G-MOPS twice, transferred to 1.5ml eppendorf in a small volume of PBS, and instantly frozen to dry ice. All embryos were then stored at a temperature of -80n degrees Celsius. 2.4.2 RNA extraction Mouse ovaries were ground into a fine powder in nitrogen liquid and transferred to 1.5ml eppendorfs. The nitrogen liquid was then allowed to evaporate without defrosting the tissue. Total RNA was then removed from the powder and stored under a temperature of -80 degrees Celsius. 2.4.3 cDNA Library construction The total cDNA was obtained using cDNA kit. Briefly, reactions were incubated for a period of 30 minutes under a temperature of 42 degrees Celsius for production of cDNA strands. Afterwards, the reactions were incubated in under a temperature of 95 degrees Celsius for 2 minutes in order to make the DNAse component in active. The cDNA components were then stored at -20 degrees Celsius. 2.4.4 Amplification of cDNA: Qualification PCR The cDNA samples were amplified using an unoriginal PCR in 20µl reactions. Optimized cycling conditions involved denaturing the samples for 5 minutes, at 94 degrees Celsius. This was then followed by 50 cycles of denaturing under the same temperatures. Once a gel was formed through the polymerase extension, 256bp fragments were extracted from the gel and then purified. 2.5 Statistical analyses Comparison of embryo development for all groups was done for each day during the culture using Chi-square test. Total cell numbers of the blastocysts were also determined in each group. They were then compared and analyzed using ANOVA. The significance level used was p Read More