Techniques for augmentation of exogenous DNA uptake by ovine spermatozoa

Sperm mediated gene transfer can be an inexpensive and simple method in animal transgenesis; however its efficiency is poor, mainly due to the spermatozoa's lesser uptake of exogenous DNA. In the present study, the effects of lipofection and other augmentation techniques, such as sperm freezing and spermatozoa treatment with triton X100 and DMSO, on exogenous DNA uptake by sheep spermatozoa and motility of sperms with plasmid uptake were evaluated. In the first experiment, ram sperms were incubated with a complex of rhodamine labeled plasmid [p-EGFP] and Lipofectamine 2000TM. In the second, spermatozoa were treated with Triton X-100TM or DMSO or were frozen without cryoprotectant. The results indicated that there was no significant difference [P<0.05] in the transfection rates and in the uptake intensity of lipofected sperms with 300 and 600 ng of plasmid in comparison with control group, i.e. transfected without lipofectamine. Furthermore, lipofection could not improve sperm motility during true plasmid uptake. Almost all of triton X100 treated and frozen-thawed spermatozoa had absorbed foreign DNA, though all were immotile. In spermatozoa treated with 0.1% DMSO, plasmid absorption rate [69.40%] was significantly higher [P<0.05] than untreated spermatozoa [57.80%], but sperm motility was not significantly different from control group. In conclusion, lipofectamine® 2000 could neither improve transfection rate, nor support motility in transfected sperms. The methods inducing membrane disruption like, freeze-thaw and triton X100 treatment, can be used in ICSI-sperm mediated gene transfer without the need for sperm selection, provided that they cause no damage to sperm nucleus

[Transfection of ram spermatozoa with pEGFP carrying human lysozyme gene]

As we all know, sperm has the capacity to take up foreign DNA, therefore, sperm mediated gen transfer can be an inexpensive and simple method in animal transgenesis in various species. However, there is not sufficient evidence of DNA uptake by ovine spermatozoa. The purpose of the present study was to examine the uptake of human lysozyme gene contained plasmid [pEGFP-IRES-hLys] by ovin spermatozoa. In the first experiment, semen was prepared from three ram [each ram two times] by electrical method. After removal of seminal plasma, 1x106 spermatozoa were incubated with rhodamin-labled pEGFP-IRES-hLys in TCM199 for 15, 30, 60 and 120 minutes and then observed for motility, uptake percent and uptake intensity by florescent microscopy. Also after 60 minutes incubation sperms were treated by DNaseI to assay adsorption or uptake of pEGFP-IRES-hLys. In the second experiment, washed and unwashed sperms were incubated with rhodamin-labled pEGFP-IRES-hLys in TCM199 for 30 and 60 minutes to evaluate the effect of presence seminal plasma on sperm uptake and motility. The findings showed that increasing incubation time increased number/percentage of spermatozoa carrying exogenous DNA and its intensity. But this different was significant only up to 30 minutes. We found that 60.16% of the cells were bound to DNA after 120 minutes incubation. Incubation with exogenous DNA induced a slightly decrease in sperm total and progressive motility. But no post acrosom uptaked sperm was motile. After treatment with DNaseI, strong florescent emission from post acrosom indicated absorption of pEGFP-IRES-hLys by spermatozoa. Presence of seminal plasma induced a slightly decrease in percent of DNA absorbed spermatozoa and absorption intensity, but did not inhibit completely. Ram spermatozoa showed a high capacity to bind DNA quickly and reach a maximum after 30 min. However, no sperm with real uptake [post acrosomal] was motile. Incubation with lower DNA concentration and/or shorter time may be helpful

CT cavernosography: a new method for evaluating venous incompetence in impotent patients

Cavernosography is traditionally recognized as an imaging modality for evaluation of venous leakage in impotent men. We introduce CT cavernosography as a novel method for demonstrating leaking veins. Sixty-seven patients diagnosed as venous incompetence by Doppler study, and eight normal subjects were enrolled into the study. After intracavernosal injection of papaverine, normal saline was injected into the corpora cavernosa and the injection rate was gradually increased and the penis was observed to obtain the injection rate causing erection. Then contrast media was injected into the corpus cavernosum with obtained injection rate followed by CT scan of the penis and pelvis. In the first group, 63 patients [94%] had venous leakage by CT cavernosography. In this group, full erection was observed at the rate of 36 ml/min of saline injection in three patients and 24 ml/min in one patient. In 46 patients [73%], erection was induced with saline injection at the rates of 48-120 ml/min, and in 17 patients [27%] no full erection was demonstrated even at the injection rate of 120 ml/min. In the 2nd group we observed full erection after saline injection at the rates of 36 ml/min and 24 ml/min in five and three patients, respectively. Beside the capability of CT source images in precise visualization of all leaking veins without superimposition of bony structures, different techniques of two- or three- dimensional reformation enable us to demonstrate the anatomy of the penis and the leaking veins elegantly for further surgical planning

[Effect of gamma irradiation sterilization on the osteoinductive capacity of demineralized bone matrix]

Gamma irradiation has been widely used for secondary sterilization of bone allograft before clinical use to reduce the risk of infection and associated complications. The current study evaluated the effect of gamma irradiation sterilization on the osteoinductive capability of human demineralized bone matrix using a rat model. Eighteen euthymic rats received two separate implants consisting of 30 mg sterile-harvest demineralized bone matrix and 30 mg gamma irradiation-sterilized demineralized bone matrix. The demineralized bone matrix from each group was placed into two separate muscle pouches created in the paravertebral muscles of each rat. All 18 rats were euthanized after 4 weeks and each implantation site was removed with 0, 5 cm normal tissue around the implant. Histological examination was performed to determine the presence or absence of new bone, cartilage and bone marrow elements, All except one of 18 [94.4%] sterile-harvest demineralized bone matrix sites histologically contained new bone elements and five of 18 [27.7%] ethylene oxide sterilized demineralized bone matrix sites showed evidence of new bone elements, which was statistically significant [p<0.05]. The results of this study indicate that demineralized bone matrix sterilized with gamma irradiation loses its osteoinductive capacity in a manner similar to that of steamsterilized demineralized bone matrix, making it unsuitable as a method of secondary sterilization of demineralized bone matrix