Lipsome-mediated uptake of exogenous DNA by Sahiwal cattle spermatozoa

Aim: To investigate the influence of lipofection treatment and exogenous DNA uptake on the quality of sahiwal cattle spermatozoa. Materials and Methods: Semen collected from sahiwal bulls (n=7) were evaluated separately for color, volume, mass activity, concentration, motility and viability using standard procedures. Pooled sperm samples from selected bulls (n=3) were transfected with a model gene construct enhanced green fluorescent protein (p-EGFP) via lipofection method and confirmed the genome integration by PCR technique. Furthermore the effect of transfection on spermatozoa was assessed based on apotosis, viability and motility. Results: In the current investigation sahiwal bulls were selected based on their breeding records and better semen characteristics. Although the transfected sperm samples failed to show florescence under fluorescence microscope, PCR studies confirmed the successful uptake of the p-EGFP gene in to the host sperm cell genome. Moreover transfected samples showed a significant reduction in the viability and motility without causing any DNA damage induced apoptosis as demonstrated by DNA Ladder assay.


Introduction
mammalian spermatozoa and subsequently carried into an ovum during the process of fertilization.This Liposome mediated DNA delivery method i.e. finding and its implications were ignored for around lipofection is a technique used for introducing nucleic twenty years and rediscovered after the report by acids into cells and embryos.Liposomes are small Lavitrano et al, in 1989 that mouse sperm cell can act vesicles consisting of membrane like lipid layers that as a vector for transferring foreign gene to the next can actually protect foreign DNA from digestion of generation [3].Similarly it has been reported in Sea proteases and DNases.Cationic liposomes are capable urchin sperm cells.[4].After this many success reports of spontaneously interacting with DNA molecules, of in-vitro uptake of DNA constructs by animal sperm giving rise to lipid-DNA complexes [1].
cells have been presented [3,5-10].Sperm cells constitute the male genetic material Green-fluorescent protein (GFP), is one of the with haploid number of chromosomes, having hottest new biological tool responsible for the capability of maintaining constancy of chromosome stunning bioluminescence of the Pacific Northwest number in a species through fertilization.Sperm cells Jellyfish, Aequorea Victoria [11].In order to from echinoderm to man under certain conditions can overcome the slow rate of flourescence acquisition in take up foreign genes and integrate into its genome.wild type GFP, a mutant variety was introduced known The capacity of sperm cells to capture foreign DNA as Enhanced GFP (EGFP) and it is commonly used as a had been reported by Brackett et al. (1971) where, they transfection reporter.EGFP permits the use of observed that Simian virus 40 (SV40) adsorbs on fluorescence-based technologies not only qualitative, surface of rabbit spermatozoa, but does not penetrate but quantitative analysis of fluorescence intensity the cells [2].This report provided the first evidence [12].that a heterologous genome can be incorporated into

Lipsome-mediated uptake of exogenous DNA by Sahiwal cattle spermatozoa
The sperm mediated gene transfer (SMGT) is a Breeding Complex, NDRI, Karnal.The animals were novel method for the generation of transgenic animals, housed in bull pen and maintained on grass (hay) where sperm is used as a vector for transferring the supplemented with concentrate, minerals and gene of interest to the zygote.Success is dependant on vitamins through out the experiment.the sperm's ability to bind and internalize exogenous Collection of semen: Semen was collected from the DNA and to transfer it into ovum at fertilization [3,7].selected sahiwal bulls twice a week for a month using This method offers a powerful tool in the field of sterile artificial vagina (CMV France) maintaining animal transgenesis and biotechnology.This internal temperature of 42-44°C.Sterile and hygienic technique has a widespread application to all species conditions were maintained during collection. in which reproduction is mediated by gametes, but is Collection of semen was according to the routine refractory to microinjection technique, for example in practices followed at Artificial Breeding Complex, fish, insects, amphibians, swine, and cattle [13,14].NDRI, Karnal.

Sahiwal cattle is one of the well established disease
Semen evaluation: Semen collected from selected and heat resistant breed of tropical and sub tropical sahiwal bulls were evaluated separately for color, areas thereby serves as a model for determining the volume, mass activity, concentration, motility and potential role of indigenous cattle in contributing to the economic and efficient milk and meat supply under viability using standard procedures.the prevailing tropical conditions [15].Despite of its Mass activity was assessed by visual analysis utmost importance and unique characteristics to date using an arbitrary scale ranging from + 1to +4 based on there are no reports were presented regarding the the whirling movement of the semen sample drops on genetic manipulation of sahiwal cattle spermatozoa.
a warm stage at 37 °C under light microscope at 10X In the current study sperm cells from the selected magnification.Sperm concentration and viability sahiwal bulls were lipofected with a model gene were calculated by eosin-nigrosine staining procedure.pEGFP and evaluated its efficacy with respect to Enumeration of diluted sample on haemocytometer viability, motility, apoptosis and gene expression ,which was determined by counting the sperm cells in studies.It is a preliminary work but definitely paves the central chamber of Neubauer's haemocytometer way for further studies to find widespread application under light microscope at 100x magnification and the in gene transfer for welfare of livestock species.number of total spermatozoa in four corner and one central square of RBC chamber were counted separately and

Materials and Methods
concentration was calculated as per formula given

Experimental design
below.Number of spermatozoa (millions /ml) = 50N x 3 Experiment 1: Two step procedure for the selection DF x 10 , where N = no. of spermatozoa in five squares of Sahiwal bulls for the transfection experiment.and DF = dilution factor.Number of viable sperms that Experiment 2: Based on the results obtained from did not take up stain were considered as live and those experiment 1, three animals were selected for semen stained pink were considered as dead.Percentage of collection and for further transfection studies.Transfected viable sperms= [Total no. of sperms -stained sperms cells were evaluated for efficiency with respect to (in 10 fields)] /total no. of sperms X 100.

apoptosis, viability, motility and corresponding gene
Transfection of spermatozoa: Based on the expression.
evaluation of semen characterstics, three sahiwal bulls Selection and Management of Bulls: In the present were selected and semen collected from these animals investigation six sahiwal cattle bulls, with an average was pooled, diluted, processed and subjected for age of 22-30 months were selected based on their body further transfection studies.Fresh semen samples weight and activity index from the animal herd were transfected with linearized pEGFP using cationic maintained at Artificial breeding complex, National liposomes (DNAfectin) as mediator.

Dairy Research Institute, Karnal. All animal procedures
Transgene construct: pEGFP-C1 (Fig: 1) encodes a were approved under the guidelines of the institutional red-shifted variant of wild-type GFP, which has been animal ethics committee, NDRI, India.The selected optimized for brighter fluorescence and higher bulls were of high activity score (above +2.5) [16] and expression in mammalian cells (Excitation body weight ranging from 400-500kg, in addition maximum=488nm; emission maximum=507nm.).these bulls were screened against genital infections The vector contains an SV-40 origin for replication in also.The selected sahiwal bulls were maintained as mammalian cells expressing the SV-40 T antigen.A per the standard practices followed at the Artificial SV-40 early promoter for neomycin resistance cassette The neat semen sample was diluted with egg and bacterial promoter for kanamycin resistance yolk citrate diluent (EYC) at a ratio of 1:10.The 2+ 2+ cassette are present in back bones of this vector.Vector diluted semen was again diluted with Ca , Mg free DNA was linearized by digesting with restriction PBS, so that the sperm numbers in the samples were enzyme:Mlu I.This linearized vector was purchased adjusted to four millions.The samples were washed from Chromus Co. Bangalore.(Genebank accession twice with Ca , Mg free PBS by centrifugation at no.# U55763).1000 rpm for 3 min.The supernatant was discarded and the pellet obtained was resuspended in 500µl of Preparation of sperm samples: The transfection of Opti-MEM-I medium, a commercial medium that is spermatozoa with pEGFP-DNAfectin complex with specially used for lipid mediated transfections.Optirespect to their corresponding concentrations and MEM-I is a modification of eagle's minimal essential incubation time were already optimized in our lab.Out medium, buffered with HEPES and sodium bicarbonate of different combinations, 4 million spermatozoa, and supplemented with hypoxanthine, thymidine, 2hrs of incubation time and 1.5µg of pEGFPsodium pyruvate, L-glutamine, trace minerals and DNAfectin complex were found to be optimum for growth factors.liposome mediated transfection in bull spermatozoa ( Data not shown).complex: DNAfectin (0.50µg) and pEGFP (1.5µg) sequences for EGFP; were mixed in 125 µl of Opti-MEM-I medium taken in (F-primer:5'ATGGTGAGCAAGGGGCGAGGAG CT a cryovial, by gentle movement and incubated at room R-primer:5' GTACCGTCGACTGCAGAATTCGAA temperature for 30 min to allow the formation of lipid GCT) DNA complexes.

Preparation of plasmid (pEGFP) -DNAfectin
The thermal cycling conditions in this study 0 were initially at 94 C for 4 min denaturation, followed Transfection of spermatozoa with pEGFP-DNA 0 0 0 by 30 cycles of 94 C for 30 sec, 56 C for 30 sec, 72 C fectin complex : A six well plate (sterile TC grade, for 1min respectively.Final extension step was Orange scientific, USA) was used for transfection 0 performed at 72 C for 15 min.The amplified products trials.Trials were performed for both fresh and were resolved on 1.8% agarose gel electrophoresis, cryopreserved semen samples separately.The wells stained with ethidium bromide, and photographed were seeded with sperm suspension in Opti-MEM-I 6 under ultraviolet illumination (Alpha Imagetech, USA).medium at a rate of 4x10 cells in duplicates .The pEGFP -DNAfectin complex prepared in Opti-MEM-Results I medium was added to each well.Controls were also From the herd maintained in the NDRI cattle similarly processed without pEGFP-DNA fectin farm, based on the average activity index (2-3) and complex.The plates were incubated for 45 minute at 0 body weight (300-400kg), six donor Sahiwal bulls 37 C in a CO incubator under controlled conditions.

were shortlisted (Table-1). This was also confirmed
The transfected samples at the end of incubation from farm breeding records.In the present study the period were treated with DNase (Promega.USA) for 0 semen was evaluated from these selected animals for 30 min at 37 C and terminated its action by co-0 different parameter like color, volume, mass activity, incubating with stopping buffer at 65 C for further 10 motility, viability, concentration and semen samples min.After the end of the specific incubation period from three bulls were utilized for further transfection the above samples were observed for fluorescence studies (SW-1719, SW-1681, SW-1772).Data of under fluorescent microscope using FITC filter.
various semen parameters of the selected Sahiwal Evaluating the effect of transfection on sperm bulls were displayed in Table-2.As spermatozoa are cells: Transfected sperm cells of either group were very sensitive to thermal shocks, all evaluation steps evaluated in terms of viability, number of motile were done at 37°C on a thermostat stage.spermatozoa, apoptosis, and pEGFP gene uptake parameters.After the specific incubation period the million spermatozoa by standard salting out protocol using DTT and Proteinase K [18].Purity of the The semen samples incubated with p EGFP gene genomic DNA was checked by measuring the for a period of 2 h were checked for motility and absorbances at 260 nm and 280 nm respectively.1µg viability parameters along with respective controls. of extracted DNA was subjected to electrophoresis on The percentage of motile and viable sperms 0.8% agarose gel, using 1kb ladder as marker.
significantly (P<0.05)decreased after subjeced to transfection with p EGFP gene-DNA fectin complex, Genomic DNA extraction and PCR for evaluating uptake of p EGFP: Genomic DNA was extracted (Table : 3).In addition the transfected sperm samples from control and transfected sperm samples after lysis when observed under fluorescence microscope found negative for green fluorescence.This could be due to [16] using DNA extraction kit according to the less active transcription and translation machinery manufacturer's instructions.The extracted genomic in sperm cells.The purity of extracted DNA samples as DNA was subjected to quality check by taking the ratio analyzed by taking the ratio was observed to range of the absorbances at 260 and 280 nm.Genomic DNA between 1.8-1.9.Apoptosis in sperm samples due to (4 µl. for each sample) was amplified by polymerase DNA fragmentation was evaluated by DNA ladder chain reaction (PCR) using the specific primer Lipsome-mediated uptake of exogenous DNA by Sahiwal cattle spermatozoa assay on 0.8% agarose gel, Apoptosis was not Liposomes have been used to introduce a variety observed in any of the samples before and after of molecules into living cells [8,9,10], but their utility as a carrier for exogenous DNA in transgenic transfection (Fig. 2).Furthermore the gene uptake via experiment was not explored until a later stage.lipofection was evaluated by PCR amplification of the Lipofection is one of the most sought methods for pEGFP gene in the transfected sperm samples.An transfection of DNA molecules into spermatozoa .The 885bp product was observed on 1.8% agarose gel, efficiency of sperm-mediated gene transfer (SMGT) demonstrating the successful uptake of the pEGFP via lipofection appears to be much higher than that gene to the host sperm by lipofection technique (Fig- 3).
obtained with other transfection methods and rates as Discussion high as 88% have been obtained in some liposomemediated uptake of exogenous DNA by spermatozoa Transfection is a tool for the introduction of and applications in SMGT studies [21], compared to foreign DNA into eukaryotic or prokaryotic cells and it only 1-8% of transgenic offspring after microinjection is one of the important steps in transgenesis.
techniques [22].The major benefits of this technique Transgenic technology is of particular relevance in the are the high efficiency, low cost, and ease of use rapid genetic modification of farm animal species as it compared with that of other transfection methods [23].

facilitates augmentation of production characteristics
The use of transgenic sperm has the advantage over like growth, development, disease resistance, reprosomatic cell nuclear transfer for producing transgenic duction, lactation performance, feed efficiency, immune animals as it is a simple procedure and there is no response and fibre production [19].For example, trauma to the oocytes [22].Additionally, sperm transgenic cattle expressing a mammary specific mediated gene transfer has resulted in germ-line transgene encoding lysostaphin are resistant to transmission of transgenes by incorporation into F1 mastitis due to Staphylococcus aureus, and this and later progeny [24].Therefore, SMGT offers potential finding leads the way for targeting specific infections advantages over other methods for inducing transgenesis through the use of transgenic technology [20].For the in domestic livestock as been successfully reported present study sahiwal donor bulls were selected based on their semen qualities and thereby demon-strated [6,25].that good quality sperm cells can be successfully DNA uptake is directly correlated with semen transfected with pEGFP via lipofection without quality.Lavitrano et al., (2003)  an independent measure of sperm quality apart from 5. considered as an alternative method for the production 10.Wang, H.J., Lin, A.X., and Chen, Y.F.(2003) of genetically modified superior quality farm animals.
Figure-1.pEGFP-C1 transgene construct used as model gene for transfection studies in sperm samples.

Figure- 2 .
Figure-2.Apoptosis assay of sperm samples before and after transfection M-100bp DNA ladder, NF-Non Transfected samples and TF-Transfected samples.
Rottmann, O., Antes, R., Hofer, P., Sommer, B., and other sperm parameters.In the present study we found Wanner, G. (1996) Liposome-mediated gene transfer that transfection of sperm cells by lipofection method via sperm cells: High transfer efficiencyand does not cause any DNA damage induced apoptosis as persistence of transgenes by use of liposomes and sperm cells and a murine amplification element.J. demonstrated by the DNA ladder assay.Anim.Breed Genet.113: 401-411.Conclusion 6. Shemesh, M., Gurevich, M., Harel-Markowitz, E., Benvenisti, L., Shore, L.S., and Stram, Y. (2000) Gene To conclude in the present study, we demonstrated integration into bovine sperm genome and its that Sahiwal cattle sperm cells can be successfully expression in transgenic offspring.Mol Reprod Devel.transfected with pEGFP gene of interest via lipofection 56:306-308.without causing DNA damage.In this study, the 7. Maione, B., Pittoggi, C., Achene, L., Lorenzini, R., and Spadafora, C. (1997) Activation of endogenous motility and viability of sperm cells had significantly nucleases in mature sperm cells upon interaction with reduced post transfection.It will be an inexpensive exogenous DNA.DNA Cell Biol.16(9):1087-97.technique for transfer of genes.Keeping in view of 8. Yang, C.C., Chang, H.S., Lin, C.J., Hsu, C.C., and enhancing the socio-economic conditions of farming Cheung, J.I. (2004) Cock spermatozoa serve as the community and to bridge the gap of supply and gene vector for generation of transgenic chicken demand of animal protein in the developing countries, (Gallus gallus).Asian Austral J Anim Sci.17: traits of commercial value such as milk yield and feed 885-891.9. Yonezawa, T., Furahata, Y., Hirabayashi, K., Suzuki, efficiency needs genetic improvement in indigenous M., and Yamanouchi, K. (2002) Protamine-derived breeds like Sahiwal cattle.Considering this research synthetic peptide enhances the efficiency ofspermoutcome along with the extensive availability of mediated gene transfer using liposome-peptide-DNA livestock semen, sperm mediated gene transfer can be complex.J Reprod Devel.48:281-286.

Table - 2
. Evaluation of semen parameters of the selected sahiwal bulls.-Selected animals for further transfection experiments. *

Table - 3
. Ev26]ation motility and viability of the transfected sperm samples.Values with different superscripts in the same column are significantly different (P<0•05) x,y, Values with different superscripts in the same column are significantly different (P<0•05)SMGT technique to be effective, quality of the semen Acknowledgements sample is primarily dependent on viability and motility Authors are thankful to the Department of of spermatozoa in the samples[24,26].The semen Biotechnology, Ministry of Science and Technology, samples should pass the evaluation tests for selection Government of India for the financial assistance.as a good vector for exogenous DNA, for example volume, concentration, presence of abnormal sperm, motility etc. Competing interest The ability of the sperm cells to internalize exogenous Authors declares that they have no competing interest.DNA is directly correlated with the progressive motility of spermatozoa in the samples[24]. a,b