Sperm-mediated gene transfer (SMGT) is a transgenic technique that transfers genes based on the ability of sperm cells to spontaneously bind to and internalize exogenous DNA and transport it into an oocyte during fertilization to produce genetically modified animals.1 Exogenous DNA refers to DNA that originates outside of the organism. Transgenic animals have been obtained using SMGT, but the efficiency of this technique is low. Low efficiency is mainly due to low uptake of exogenous DNA by the spermatozoa, reducing the chances of fertilizing the oocytes with transfected spermatozoa.2 In order to successfully produce transgenic animals by SMGT, the spermatozoa must attach the exogenous DNA into the head and these transfected spermatozoa must maintain their functionality to fertilize the oocyte.2 Genetically modified animals produced by SMGT are useful for research in biomedical, agricultural, and veterinary fields of study. SMGT could also be useful in generating animals as models for human diseases or lead to future discoveries relating to human gene therapy.
Deoxyribonucleic acid is a molecule composed of two chains that coil around each other to form a double helix carrying genetic instructions for the development, functioning, growth and reproduction of all known organisms and many viruses. DNA and ribonucleic acid (RNA) are nucleic acids; alongside proteins, lipids and complex carbohydrates (polysaccharides), nucleic acids are one of the four major types of macromolecules that are essential for all known forms of life.
Genetically modified animals are animals that have been genetically modified for a variety of purposes including producing drugs, enhancing yields, increase resistance to disease, etc. The vast majority of genetically modified animals are at the research stage with the number close to entering the market remains small.
The method for SMGT uses the sperm cell, a natural vector of genetic material, to transport exogenous DNA. The exogenous DNA molecules bind to the cell membrane of the head of the sperm cell. This binding and internalization of the DNA is not a random event. The exogenous DNA interacts with the DNA-binding proteins (DBPs) that are present on the surface of the sperm cell.3 Spermatozoa are naturally protected against the intrusion of exogenous DNA molecules by an inhibitory factor present in mammals’ seminal fluid. This factor blocks the binding of sperm cells and exogenous DNA because in the presence of the inhibitory factor, DBPs lose their ability to bind to exogenous DNA. In the absence of this inhibitory factor, DBPs on sperm cells are able to interact with DNA and can then translocate the DNA into the cell. Therefore, the seminal fluid must be removed from the sperm samples by extensive washing immediately after ejaculation.3 After the DNA is internalized, the exogenous DNA must be integrated into the genome. There are various mechanisms suggested for DNA integration, including integrating DNA at oocyte activation, at nucleus decondensation, or at the formation of the pronuclei, but all of these suggested mechanisms imply that the integration of DNA happens after the penetration of the sperm cell into the oocyte.3
Ejaculation is the discharge of semen from the male reproductory tract, usually accompanied by orgasm. It is the final stage and natural objective of male sexual stimulation, and an essential component of natural conception. In rare cases, ejaculation occurs because of prostatic disease. Ejaculation may also occur spontaneously during sleep. Anejaculation is the condition of being unable to ejaculate. Ejaculation is usually very pleasurable for men; dysejaculation is an ejaculation that is painful or uncomfortable. Retrograde ejaculation is the condition where semen travels backwards into the bladder rather than out the urethra.
Sperm-mediated gene transfer is considered controversial because despite the successes, it has not yet become established as a reliable form of genetic manipulation. Skepticism arises based on the assumption that evolutionary chaos could arise if sperm cells could act as vectors for exogenous DNA.4 Reasonable assumption tells us that because reproductive tracts contain free DNA molecules, sperm cells should be highly resistant to the risk of picking up exogenous DNA molecules. SMGT has been demonstrated experimentally and followed the assumption that nature has barriers against SMGT. These barriers are not always absolute and could explain the inconsistent experimental outcomes of SMGT.4 If there are natural barriers against SMGT, then the successes may only represent unusual cases in which the barriers failed. Two barriers have been identified; the inhibitory factor in seminal fluid that prevents binding to foreign DNA molecules and a sperm endogenous nuclease activity that is triggered upon interaction of sperm cells with foreign DNA molecules.4 These protections give reason to believe that unintentional interactions between sperm and exogenous genetic sequences is kept to a minimal. These barriers allow for protection against the threat that every fertilization event could become a potentially mutagenic one.4
Transgenic animals have been produced successfully using gene transfer techniques such as sperm-mediated gene transfer. Though this production has been successful, the efficiency of the process is low. Low efficiency of SMGT in the production of transgenic animals is mainly due to poor uptake of the exogenous DNA by the sperm cells, thus reducing the number of fertilized oocytes with transfected spermatozoa.5 From 1989 to 2004, there were over 30 claims for the production of viable transgenic animals using SMGT, but only about 25 percent of these demonstrated a transmission of the transgenes beyond the F0 generation.4 This transmission is required in order to claim usable animal transgenesis. According to previous studies, numerous animal species, including mammals, birds, insects, and fish, have been found susceptible to SMGT techniques, thus indicating that SMGT has broad applicability across a wide variety of Metazoan species.4 Currently, despite the low frequency of transmission of transgenes, the frequency of phenotype modifications and overall animal transgenesis has been as high as 80 percent in some experiments.4
The potential use of sperm-mediated gene transfer for embryo somatic gene therapy is a possibility for future research. Embryo somatic gene therapy would be advantageous because there seems to be an inverse correlation between the age of the patient and the effectiveness of gene therapy. Therefore, the possibility of gene therapy treatment before irreversible damage occurs would be ideal.4 A majority of the experiments that report successful SMGT provide evidence of post-fertilization transfer and maintenance of transgenes.6 SMGT has potential advantages of being a simple and cost-effective method of gene therapy, especially in contrast with pronuclear microinjection, another transgenic technique. Nevertheless, despite some successes and its potential utility, SMGT is not yet established as a reliable form of genetic modification.6
A spermatozoon is a motile sperm cell, or moving form of the haploid cell that is the male gamete. A spermatozoon joins an ovum to form a zygote.
Fertilisation or fertilization, also known as generative fertilisation, insemination, pollination, fecundation, syngamy and impregnation, is the fusion of gametes to initiate the development of a new individual organism or offspring. This cycle of fertilisation and development of new individuals is called sexual reproduction. During double fertilisation in angiosperms the haploid male gamete combines with two haploid polar nuclei to form a triploid primary endosperm nucleus by the process of vegetative fertilisation.
Intracytoplasmic sperm injection is an in vitro fertilization (IVF) procedure in which a single sperm cell is injected directly into the cytoplasm of an egg. This technique is used in order to prepare the gametes for the obtention of embryos that may be transferred to a maternal uterus. With this method acrosome reaction is skipped.
During fertilization, a sperm must first fuse with the plasma membrane and then penetrate the female egg cell in order to fertilize it. Fusing to the egg cell usually causes little problem, whereas penetrating through the egg's hard shell or extracellular matrix can present more of a problem to the sperm. Therefore, sperm cells go through a process known as the acrosome reaction which is the reaction that occurs in the acrosome of the sperm as it approaches the egg. The acrosome is a cap-like structure over the anterior half of the sperm's head.
Spermatogenesis is the process by which haploid spermatozoa develop from germ cells in the seminiferous tubules of the testis. This process starts with the mitotic division of the stem cells located close to the basement membrane of the tubules. These cells are called spermatogonial stem cells. The mitotic division of these produces two types of cells. Type A cells replenish the stem cells, and type B cells differentiate into spermatocytes. The primary spermatocyte divides meiotically into two secondary spermatocytes; each secondary spermatocyte divides into two equal haploid spermatids by Meiosis II. The spermatids are transformed into spermatozoa(sperm) by the process of spermiogenesis. These develop into mature spermatozoa, also known as sperm cells. Thus, the primary spermatocyte gives rise to two cells, the secondary spermatocytes, and the two secondary spermatocytes by their subdivision produce four spermatozoa and four haploid cells.
Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred: "transformation" is typically used to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells, including plant cells. In animal cells, transfection is the preferred term as transformation is also used to refer to progression to a cancerous state (carcinogenesis) in these cells. Transduction is often used to describe virus-mediated gene transfer into eukaryotic cells.
The zona pellucida is a glycoprotein layer surrounding the plasma membrane of mammalian oocytes. It is a vital constitutive part of the oocyte. The zona pellucida first appears in unilaminar primary oocytes. It is secreted by both the oocyte and the ovarian follicles. The zona pellucida is surrounded by the corona radiata. The corona is composed of cells that care for the egg when it is emitted from the ovary.
Capacitation is the penultimate step in the maturation of mammalian spermatozoa and is required to render them competent to fertilize an oocyte. This step is a biochemical event; the sperm move normally and look mature prior to capacitation. In vivo, capacitation occurs after ejaculation, when the spermatozoa leave the vagina and enter the superior female reproductive tract. The uterus aids in the steps of capacitation by secreting sterol-binding albumin, lipoproteins, and proteolytic and glycosidasic enzymes such as heparin.
Polly and Molly, two ewes, were the first mammals to have been successfully cloned from an adult somatic cell and to be transgenic animals at the same time. This is not to be confused with Dolly the Sheep, the first animal to be successfully cloned from an adult somatic cell where there was no genetic modification carried out on the adult donor nucleus. Polly and Molly, like Dolly the Sheep, were cloned at the Roslin Institute in Edinburgh, Scotland.
Sperm is the male reproductive cell. In the types of sexual reproduction known as anisogamy and its subtype oogamy, there is a marked difference in the size of the gametes with the smaller one being termed the "male" or sperm cell. A uniflagellar sperm cell that is motile is referred to as a spermatozoon, whereas a non-motile sperm cell is referred to as a spermatium. Sperm cells cannot divide and have a limited life span, but after fusion with egg cells during fertilization, a new organism begins developing, starting as a totipotent zygote. The human sperm cell is haploid, so that its 23 chromosomes can join the 23 chromosomes of the female egg to form a diploid cell. In mammals, sperm develops in the testicles, is stored in the epididymis, and released from the penis.
Human fertilization is the union of a human egg and sperm, usually occurring in the ampulla of the fallopian tube. The result of this union is the production of a zygote cell, or fertilized egg, initiating prenatal development. Scientists discovered the dynamics of human fertilization in the nineteenth century.
A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques from one organism to another. The introduction of a transgene (catransgenesis") has the potential to change the phenotype of an organism transgene describes a segment of DNA containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may either retain the ability to produce RNA or protein in the transgenic organism or alter the normal function of the transgenic organism's genetic code. In general, the DNA is incorporated into the organism's germ line. For example, in higher vertebrates this can be accomplished by injecting the foreign DNA into the nucleus of a fertilized ovum. This technique is routinely used to introduce human disease genes or other genes of interest into strains of laboratory mice to study the function or pathology involved with that particular gene.
Microinjection is the use of a glass micropipette to inject a liquid substance at a microscopic or borderline macroscopic level. The target is often a living cell but may also include intercellular space. Microinjection is a simple mechanical process usually involving an inverted microscope with a magnification power of around 200x.
Gene delivery is the process of introducing foreign genetic material, such as DNA or RNA, into host cells. Genetic material must reach the nucleus of the host cell to induce gene expression. Successful gene delivery requires the foreign genetic material to remain stable within the host cell and can either integrate into the genome or replicate independently of it. This requires foreign DNA to be synthesized as part of a vector, which is designed to enter the desired host cell and deliver the transgene to that cell's genome. Vectors utilized as the method for gene delivery can be divided into two categories, recombinant viruses and synthetic vectors.
A humster is a hybrid cell line made from hamster oocyte fertilized with human sperm. It always consists of single cells, and cannot form a multi-cellular being.
Zona pellucida sperm-binding protein 2 is a protein that in humans is encoded by the ZP2 gene.
Sperm guidance is the process by which sperm cells (spermatozoa) are directed to the oocyte (egg) for the aim of fertilization. In the case of marine invertebrates the guidance is done by chemotaxis. In the case of mammals, it appears to be done by chemotaxis, thermotaxis and rheotaxis.
Reproductive immunology refers to a field of medicine that studies interactions between the immune system and components related to the reproductive system, such as maternal immune tolerance towards the fetus, or immunological interactions across the blood-testis barrier. The concept has been used by fertility clinics to explain the fertility problems, recurrent miscarriages and pregnancy complications observed when this state of immunological tolerance is not successfully achieved. Immunological therapy is the new up and coming method for treating many cases of previously "unexplained infertility" or recurrent miscarriage.
Oocyteactivation is a series of processes that occur in the oocyte during fertilization.
Genetic engineering can be accomplished using multiple techniques. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism. The ability to genetically engineer organisms is built on years of research and discovery on how genes function and how we can manipulate them. Important advances included the discovery of restriction enzymes and DNA ligases and the development of polymerase chain reaction and sequencing.
1. Lavitrano M, Giovannoni R, Cerrito MG. 2013. Methods for sperm-mediated gene transfer. Methods Molecular Biology. 927:519-529.
2. García-Vázquez FA, Ruiz S, Grullón LA, Ondiz AD, Gutiérrez-Adán A, Gadea J. 2011. Factors affecting porcine sperm mediated gene transfer. Research in Veterinary Science. 91(3):446-53.
3. Lavitrano M, Busnelli M, Cerrito MG, Giovannoni R, Manzini S, Vargiolu A. 2006. Sperm-mediated gene transfer. Reproduction, Fertility and Development. 18:19-23.
4. Smith K, Spadafora C. 2005. Sperm-mediated gene transfer: applications and implications. BioEssays. 27(5):551-562.
5. Collares T, Campos VF, de Leon PM, Moura, Cavalcanti PV, Amaral, MG, et al. 2011. Transgene transmission in chickens by sperm-mediated gene transfer after seminal plasma removal and exogenous DNA treated with dimethylsulfoxide or N,N-dimethylacetamide. Journal of Biosciences. 36(4):613-620.
6. Smith K. 2004. Gene therapy: the potential applicability of gene transfer technology to the human germline. International Journal of Medical Sciences. 1(2):76-91.