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The impact of exogenous DNA on the structure of sperm of olive flounder (Paralichthys olivaceus)
Abstract
Sperm-mediated gene transfer (SMGT) is a promising transgenic technology that relies on the capability of sperm to internalize exogenous DNA. In marine fish, however, the interaction between sperm and exogenous DNA appears to be deficient. Here, we demonstrated significant DNase activity in the seminal plasma of the olive flounder. When incubated with naked-DNA, the spermatozoa lost their structural integrity, including the head, mitochondria and flagellum, in an incubation time-dependent manner. However, internalization of a liposome-DNA complex resulted in the structural integrity of the spermatozoa being maintained, even when using incubation times of up to 50 min. We concluded that in the olive flounder, SMGT is possible by integrating liposome-DNA complexes, rather than naked-DNA alone, into the sperm. In brief, removal of the seminal plasma and packaging the exogenous DNA were necessary for successful SMGT in the olive flounder.
... In fish, the electroporation method to directly introduce foreign DNA into the embryo was firstly used in medaka (Oryzias latipes) in 1990 (Inoue et al. 1990). The most electroporation studies were carried out by sperm-mediated gene transfer (SMGT) to introduce foreign genes into the unfertilized eggs (Khoo et al. 1992), but the transgenic efficiency of SMGT was almost not successful in marine fish because the interaction between the sperm and the exogenous DNA was not fully understood (Xin et al. 2014). In the meanwhile, the traditional electroporation operation could cause great damage to the embryo, and lead to low efficiency and survival rate (Tsai and Tseng 1994), so this technology is not widely used. ...
... Flounder eggs, like many marine fish eggs, have hard and elastic chorion, and a high internal pressure, which make the microinjection be not carried out easily. Besides, previous study showed that the seminal plasma of the flounder exhibited obvious DNase activity so exogenous DNA couldn't be transferred into embryos through SMGT which limited the adhibition (Xin et al. 2014). So, it is necessary to find a simple and valid method to introduce foreign substances into eggs. ...
As a new breeding technology, genome editing becomes a powerful tool owing to its high efficiency of gene targeting. In CRISPR/Cas9 system, how to efficiently transfer gRNA and Cas9 mRNA into embryos is an important step. Though microinjection is the most common method for operating on fish embryos, it is not easy to inject the RNA into pelagic and telolecithal eggs with hard egg chorion, such as the olive flounder (Paralichthys olivaceus) eggs. Therefore, an efficient and simple technology is urgently needed for this kind of study. In the present study, we used the electroporation method to introduce foreign gene into the flounder eggs. The results showed that the proper electroporation condition was 3 pulses for 1 millisecond (ms), 50 ms interval, at 25 V with high survival rate. Under this condition, the effect of CRISPR/Cas9 system on genome editing by using two different genes, myomaker and gonadal soma derived factor (gsdf) was investigated. Around 12% and 7% of the electroporated embryos for myomaker and gsdf hatched, respectively. The mutation sites including insert and deletion mutations at the candidate sites were visible for both targeted genes in the hatched larvae. The checked frame-shift and start codon deletion mutations would lead to complete destruction of these genes’ structure. Above results implied that CRISPR/Cas9 system could work well in marine fish with pelagic eggs by using electroporation, and genome editing could be achieved on a large scale which may be useful for study of gene function in marine fish.
... Se conoce como ultraestructura espermática a la morfología estructural de los espermatozoides, que es obtenida a través de Microscopía Electrónica de Barrido (MEB) y de Transmisión (TEM) (Rodao et al., 2015;Medina-Robles, 2020). Además, el análisis por MEB y TEM provee infor- mación detallada de la ultraestructura externa y subcelular espermática, lo que permite dilucidar la morfología normal de los gametos, esta información es de mucho valor para desarrollar protocolos de crioconservación y para hacer seguimiento y evaluación del potencial daño celular, causados por la exposición a algún agente nocivo, previo a procesos de fertilización (Xin et al., 2014). Los espermatozoides de los peces se clasifican según su forma de fertilización: en aquaespermatozoides, cuando la fertilización es externa y en introespermatozoides si este proceso se da internamente. ...
La acuicultura a nivel mundial cada vez toma más relevancia y se ha convertido en una fuente de alimento e ingresos económicos de muchas comunidades alrededor del mundo. Sin embargo, actividades como la sobreexplotación del recurso hídrico y pesquero, el aumento de la población humana, la contaminación ambiental, el cambio climático, entre otros factores, están poniendo en riesgo no solo la seguridad alimentaria del planeta, sino también la biodiversidad de organismos, entre ellos los peces. Una de las herramientas para salvaguardar los recursos biológicos acuáticos, es la crioconservación, que en peces se enfoca principalmente en la conservación de semen en nitrógeno líquido y esta, pesar de ser una técnica muy útil, es susceptible a mejoramiento, ya que se ha reportado que las bajas temperaturas usadas en la crioconservación pueden causar efectos negativos en las células espermáticas, debido a esto es necesario establecer protocolos especie específicos para sacarle todo el potencial a esta forma de conservación. Por lo tanto, el objetivo de esta revisión es recopilar información sobre los principales parámetros de calidad espermática que se ven afectados en el momento de crioconservar el semen, haciendo énfasis en las especies dulceacuícolas.
... Hence, the role of seminal plasma miRNAs may be restricted to maintaining a conducive environment for spermatozoa before the spermiation. Seminal plasma contains inhibitory factors that prevent the internalization of foreign nucleic acids [52][53][54][55]. It has been demonstrated that DNA-binding proteins at the sperm cell surface allow internalization of exogenous DNA in the absence of inhibitory factors in the seminal plasma [56]. ...
The origin and contribution of seminal plasma RNAs into the whole semen RNA repertoire are poorly known, frequently being overlooked or neglected. In this study, we used high-throughput sequencing and RT-qPCR to profile microRNA (miRNA) constituents in the whole semen, as well as in fractionated spermatozoa and seminal plasma of Atlantic salmon (Salmo salar). We found 85 differentially accumulated miRNAs between spermatozoa and the seminal plasma. We identified a number of seminal plasma-enriched and spermatozoa-enriched miRNAs. We localized the expression of some miRNAs in juvenile and mature testes. Two abundant miRNAs, miR-92a-3p and miR-202-5p, localized to both spermatogonia and somatic supporting cells in immature testis, and they were also highly abundant in somatic cells in mature testis. miR-15c-5p, miR-30d-5p, miR-93a-5p, and miR-730-5p were detected only in mature testis. miRs 92a-3p, 202-5p, 15c-5p, and 30d-5p were also detected in a juvenile ovary. The RT-qPCR experiment demonstrated lack of correlation in miRNA transcript levels in seminal plasma versus blood plasma. Our results indicate that salmon semen is rich in miRNAs, which are present in both spermatozoa and seminal plasma. Testicular-supporting somatic cells are likely the source of seminal plasma enrichment, whereas blood plasma is unlikely to contribute to the seminal plasma miRNA repertoire.
... Hence, the role of seminal plasma miRNAs may be restricted to maintaining conducive environment for spermatozoa before the spermiation. Seminal plasma contains inhibitory factors that prevent the internalization of foreign nucleic acids [53][54][55][56]. It has been demonstrated that DNA-binding proteins at sperm cell surface allow internalization of exogenous DNA in the absence of inhibitory factors in the seminal plasma [ 57 ]. ...
Background The origin and contribution of seminal plasma RNAs into the whole semen RNA repertoire are poorly known, frequently being overlooked or neglected. Virtually nothing is known about seminal plasma RNAs in fish, including small RNAs, which have regulatory functions in gonadal development. Results In this study, we profiled microRNA (miRNA) constituents in the whole semen, as well as in fractionated spermatozoa and seminal plasma of Atlantic salmon (Salmo salar). Among 306 conserved miRNAs, 85 were differentially accumulated (>2 log-fold change and p-value
... Such reduction in the efficiency of SMGT can be overcome by lipofection. The use of liposomes to introduce exogenous DNA into spermatozoa has been widely demonstrated (Ball et al., 2008;Campos et al., 2011a;Xin et al., 2014), but the reports of its application in bovine SMGT using sexsorted cells are scarce. ...
Bovine sex-sorted sperm have been commercialized and successfully used for the production of transgenic embryos of the desired sex through the sperm-mediated gene transfer (SMGT) technique. However, sex-sorted sperm show a reduced ability to internalize exogenous DNA. The interaction between sperm cells and the exogenous DNA has been reported in other species to be a CD4-like molecule-dependent process. The flow cytometry-based sex-sorting process subjects the spermatozoa to different stresses causing changes in the cell membrane. The aim of this study was to elucidate the relationship between the redistribution of CD4-like molecules and binding of exogenous DNA to sex-sorted bovine sperm. In the first set of experiments, the membrane phospholipid disorder and the redistribution of the CD4 were evaluated. The second set of experiments was conducted to investigate the effect of CD4 redistribution on the mechanism of binding of exogenous DNA to sperm cells and the efficiency of lipofection in sex-sorted bovine sperm. Sex-sorting procedure increased the membrane phospholipid disorder and induced the redistribution of CD4-like molecules. Both X-sorted and Y-sorted sperm had decreased DNA bound to membrane in comparison with the unsorted sperm; however, the binding of the exogenous DNA was significantly increased with the addition of liposomes. Moreover, we demonstrated that the number of sperm-bound exogenous DNA was decreased when these cells were preincubated with anti-bovine CD4 monoclonal antibody, supporting our hypothesis that CD4-like molecules indeed play a crucial role in the process of exogenous DNA/bovine sperm cells interaction.
Las observaciones construidas en el proyecto de investigación que da origen a este producto de investigación, se hicieron a partir de la discusión de sistemas sociales de Niklas Luhmann (teoría de sistemas de tercera generación o teoría de la autoproducción con su concepto medular de autopoiesis). Este capítulo recoge aportes del libro “Interjuego de sueños y realidades en relatos de empresarios”, de los investigadores Vallejo-Correa, López-Isaza y Cubillos-Arcila (2019), quienes requirieron en el tratamiento de los discursos de 7 empresarios grabados en las entrevistas, en su proceso de transcripción, procesamiento y codificación, del software NVivo, versión 11.4.3 (2084) para Mac OSX.
En la fase uno, a partir de una distinción de nueve categorías de nodos o variables, distinguidas como:
“conflicto”, “decisiones”, “elementos portadores de confianza”, “elementos que dan sentido a las decisiones”, “estados”, “formas de desarrollo empresarial”, “premisas de decisión”, “presente” y “sucesos”,
que ejercieron como agrupadoras de elementos que fungen contingentemente como premisas de decisión, emergieron cuarenta y cuatro premisas, las cuales cotematizan la selección de su relación con otras que serán también premisas de decisión, al ser decisiones que fungirán como tales, otorgando sentidos comunicativos, con pretensiones de unidad de sentido, a las formas de desarrollo empresarial de los empresarios entrevistados.
En la fase dos, a partir de las cuarenta y cuatro premisas de decisión y observando los nodos comparados por número de referencias de codificaciones, se obtuvieron los valores de fuerza de prominencia de acuerdo al número de referencias codificadas. En la tercera fase se describió cómo las decisiones que toman los empresarios entrevistados, y desde una perspectiva de complejización sistémica luhmanniana, tienen en su origen una doble causalidad sistémica, al emerger tanto del sistema psíquico como en el seno de la sociedad, más concretamente en el de los sistemas sociales organizacionales empresariales, al ser las decisiones comunicaciones realizadas, que al serlo constituyen al sistema social sociedad. Vale recordar que ambos sistemas: psíquico y sociedad se encuentran acoplados estructuralmente, es decir se suponen mutuamente. En esta fase se distinguieron las siguientes pautas de pautas (metapautas) u observaciones de segundo orden:
“Construcción de futuro”, “Desafiantes-tenacidad-persistencia-insistencia” o “Irritación permanente como constitutiva de estado de confort”.
La “construcción de futuro” emergió a partir del lenguaje “conflicto”, que a su vez surge en la relación ensayo error, significando para quien lo sabe ver una oportunidad de cambio y aprendizaje, pero también un equivalente funcional para el lenguaje “construcción”. El conflicto, como irritación o perturbación, funge como carga para las decisiones, con su propiedad de movilizar y estimular las operaciones autopoiéticas de los sistemas psíquicos (pensamientos que se traducen en decisiones) y sociales (comunicaciones como decisiones).
Se enaltece la capacidad de persistencia, insistencia y tenacidad que subyace en la personalidad de cada uno de estos empresarios. Es indudable que sin esta cualidad no podrían llegar a alcanzar sus metas. Por otro lado persiguen objetivos con ahínco, buscan caminos distintos apuntando a la eficacia, así mismo desarrollan agudeza dada por la experiencia, para dectectar oportunidades. En este tránsito ganan confianza en sí mismos, intuyen el valor de la paciencia y lo combinan con renuncias temporales, con el fin de conseguir sus propósitos.
Mitochondria are cellular organelles with crucial roles in ATP synthesis, metabolic integration, reactive oxygen species (ROS) synthesis and management, the regulation of apoptosis (namely via the intrinsic pathway), among many others. Additionally, mitochondria in different organs or cell types may have distinct properties that can decisively influence functional analysis. In terms of the importance of mitochondria in mammalian reproduction, and although there are species-specific differences, these aspects involve both energetic considerations for gametogenesis and fertilization, control of apoptosis to ensure the proper production of viable gametes, and ROS signaling, as well as other emerging aspects. Crucially, mitochondria are the starting point for steroid hormone biosynthesis, given that the conversion of cholesterol to pregnenolone (a common precursor for all steroid hormones) takes place via the activity of the cytochrome P450 side-chain cleavage enzyme (P450scc) on the inner mitochondrial membrane. Furthermore, mitochondrial activity in reproduction has to be considered in accordance with the very distinct strategies for gamete production in the male and female. These include distinct gonad morpho-physiologies, different types of steroids that are more prevalent (testosterone, estrogens, progesterone), and, importantly, the very particular timings of gametogenesis. While spermatogenesis is complete and continuous since puberty, producing a seemingly inexhaustible pool of gametes in a fixed environment; oogenesis involves the episodic production of very few gametes in an environment that changes cyclically. These aspects have always to be taken into account when considering the roles of any common element in mammalian reproduction.
The domesticated silkworm Bombyx mori L. has important roles in basic biological research and applied science. To explore the practical use of transgenic technology in agricultural silkworm varieties, we fused the neomycin-resistance gene (Neo(R)) and the green fluorescent protein gene (gfp) into the piggyBac-based transposon vector and transduced it into silkworms by sperm-mediated gene transfer (SMGT). Fluorescence observation indicated the positive rate of G0 egg-batches is 72.7%. After screening against the antibiotic G418, development of individual larvae in the same brood showed significant size differences. PCR detection indicated the existence of gfp and Neo(R) and confirmed the positive rate of transgenesis is 0.47%. Southern blotting confirmed the presence of the exogenous genes in the genome of G7 larvae. These results show that our strategy is practicable and markedly improves the efficiency of SMGT.
Although graphic strategies, such as graphic organizers and knowledge maps, have proved helpful for text learning, certain important application issues such as surface processing and cognitive overload have yet to be resolved. The authors tested the learning effects of a concept-mapping strategy. They designed 3 concept-mapping approaches-map correction, scaffold fading, and map generation-to determine their effects on students' text comprehension and summarization abilities. The experimental results from 126 fifth graders showed that the map-correction method enhanced text comprehension and summarization abilities and that the scaffold-fading method facilitated summarization ability.
Transgenic animals have been successfully produced by mass gene transfer techniques such as sperm-mediated gene transfer (SMGT). The aim of this work was to demonstrate transgene transmission by SMGT in chickens using dimethylsulfoxide (DMSO) or N,N-dimethylacetamide (DMAc) as transfectants after seminal plasma removal to prevent DNase activity. Sperm samples were prepared by repetitive washes, and after each wash sperm motility, seminal plasma proteins, exogenous DNA integrity and its uptake by spermatozoa were evaluated. Laying hens were inseminated using spermatozoa transfected with pEGFP-N1 vector in the presence of DMSO or DMAc. Transgene transmission in newborn chicks was evaluated by in vivo enhanced green fluorescent protein (EGFP) expression, RT-PCR and PCR analysis. DNA internalization was limited to sperm samples washed twice. The presence of DMSO or DMAc during transfection had no effect on fertilization or hatching rates. PCR analysis detected the presence of EGFP DNA in 38% of newborn chicks from the DMSO group and 19% from the DMAc group. EGFP mRNA was detected in 21% of newborn chicks from the DMSO group, as against 8.5% from the DMAc group. However, in vivo expression of EGFP was only observed in a single animal from the DMSO group. Our data revealed that the plasmid DNA-DMSO combination coupled with sperm washes can be an efficient method for transfection in chickens.
Mitochondria and its role in male reproduction has remained an enigma since long. Similarly, etiology of male infertility in a large percentage of individuals, mainly primary infertility, has evaded concrete conclusions. Oxidative metabolism, energy production and free radical generation are the principal biological reactions occurring inside mitochondria. In addition to the above, mitochondria participates in an important process of apoptosis. Mitochondrial causes of infertility have triggered interest because of its presence in the tail of sperm and immense need of energy for sperm motility. Several studies on mitochondria have strongly suggested its role in fertility, some of which support mitochondrial role presenting numerous hypotheses, whereas others deny its very existence as a causative factor. We have, in the present review of existing literature, covered the role of mitochondria right from spermatogenesis to male infertility.
The silver catfish (Rhamdia quelen) is an endemic American fish species. The sperm of each species has its own peculiarities and biological characteristics, which influence the success of mass DNA transfer methods. Our objective in this study was to evaluate different sperm-mediated gene transfer (SMGT) methods to obtain transgenic silver catfish. Different treatments for the incorporation of a foreign pEGFP plasmid group were used: (1) dehydrated/rehydrated (DR), (2) dehydrated/rehydrated/electroporated (DRE), (3) electroporated (E), (4) incubated with seminal plasma (INC); and (5) incubated in the absence of seminal plasma (INCSP). Sperm motility, time of activity duration (TAD), fertilization rate (FR), hatching rate (HR) and sperm morphology were also evaluated. The polymerase chain reaction (PCR) positivity rates for the presence of the transgene were: DRE 60%; DR 40%; E 25%; INC 5% and INCSP 25%. The rates of embryo EGFP expression were: DRE 63%; DR 44%; E 34%; INC 8% and INCSP 38%. The fertilization rate in the control and DRE treatments groups were higher than in the DR group, but the E,INC and INCSP treatment groups had the lowest rate. The hatching rates of the DRE, DR and control groups were higher than in the INCSP, INC and E treatment groups (P>0.05). There were no differences among the DRE and DR, E and DR, E and INCSP groups in expression and PCR positivity rates of enhanced green fluorescent protein (EGFP) in embryos. Scanning electron microscopy also did not show any change in sperm morphology among treatment groups. To the best of our knowledge, this is the first report on transgene transmission of exogenous DNA into silver catfish larvae through SMGT technology.
Epididymal sperm cells, incubated with plasmid DNA, showed a spontaneous tendency to interact with the exogenous nucleic acid. We have investigated the molecular basis of such interaction. Exogenous DNA is taken up by sperm cells over a 15- to 20-min period and is specifically localized on the nuclear area of the sperm head. DNA was reversibly bound to spermatozoa since it can be competed out by excess of cold competitor DNA or by other polyanions as heparin and dextran sulphate. By contrast, poly-L-lysine, a polycation, favours the uptake. DNA molecules of large size (7 kb) were preferentially taken up as compared to smaller ones (150-750 bp). Acidic proteins were also taken up and concentrated, as for DNA, at the nuclear level. These data strongly suggested that ionic interactions may occur between foreign molecules and a substrate located in the sperm head. On the basis of Southwestern analysis, a sperm head protein(s) of 30-35 KD is identified as potential substrate for exogenous DNA binding. Moreover, we have found that seminal plasma contains factor(s) which abolish sperm permeability, exerting a powerful inhibitor effect on DNA uptake. The presence of a specific binding protein for the DNA and of a factor inhibiting such interaction support the existence of a mechanism controlling, through specific factors, the sperm-DNA interaction.
Mature mouse sperm cells incubated in an isotonic buffer with cloned DNA capture DNA molecules over a 15 min period. Spermatozoa incubated with pSV2CAT plasmid in either circular or linear form were used to fertilize mouse eggs in vitro. Sequences complementary to pSV2CAT were identified in approximately 30% of 250 progeny by Southern blotting. A genomic library was constructed from the DNA of a positive mouse. Three positive clones were identified and two adjacent HincII restriction fragments of 240 and 370 bp showed identical sequences to the corresponding fragments of the pSV2CAT plasmid. F1 progeny showed paternal and maternal transmission of the transgenes from founders. CAT gene expression was detected on tissues of adult F1 individuals, preferentially on tails and muscle. We conclude that transgenic mice can be obtained using sperm cells as foreign DNA vectors.
Mature sperm cells have the spontaneous capability of taking up exogenous DNA. Potential substrates for the interaction of the DNA with the sperm heads are specific classes of DNA-binding proteins. In the present work three major classes of DNA-binding proteins were identified by Southwestern analysis of sperm head protein extracts: a first class of about 50 kDa in molecular weight, a second one of 30-35 kDa, and finally a third one below 20 kDa. The latter group most probably contains sperm protamines. Our attention was particularly focused on the 30- to 35-kDa proteins as a substrate for DNA binding, as they represented the only group whose electrophoretic mobility was conserved among mammalian species. In addition they were the only class of DNA-binding proteins accessible to exogenous DNA in intact sperm cells. The purified 30- to 35-kDa proteins interacted in vitro with exogenous DNA and generated discrete protein/DNA complexes as determined by band shift assay. A factor blocking the binding of exogenous DNA to sperm cells was also identified in the seminal fluid of mammals and in echinoid spermatoza. The factor also exerted a powerful inhibitory effect on DNA uptake in sperm cells of heterologous species. The 30- to 35-kDa DNA-binding proteins appeared to be the specific target through which the inhibition was mediated. In the presence of the inhibitory factor, the 30- to 35-kDa lost the ability to bind exogenous DNA. Thus, the interaction of exogenous DNA with sperm cells does not appear to be a casual event but, on the contrary, relies on a molecular mechanism based on the cooperation of specific protein factors.
Mature sperm cells, either of epididymal origin or ejaculated and depleted of seminal fluid, are spontaneously able to bind exogenous DNA molecules which are subsequently internalized into sperm nuclei. Southern blot analysis showed that the internalized DNA was specifically cleaved by sperm endonucleases and showed typical fragmentation patterns of localized hypersensitivity. Nucleases were activated in response to the internalization of exogenous DNA by sperm cells and their activity increased with the DNA concentration. Nuclease activation was efficient in epididymal sperm cells, while being drastically reduced in ejaculated washed spermatozoa. Nucleases were Ca++ dependent, and were, respectively, inhibited and activated by preincubating sperm cells with Aurintricarboxylic Acid (ATA) and Ca++ Ionophore A23187, which are known to, respectively, inhibit and activate apoptosis in somatic cells. Moreover, nuclease activation also caused a partial degradation of the sperm endogenous chromosomal DNA; cleaved DNA fragments were released from the sperm cells to the medium. Taken together, these results suggest that a metabolically active process similar to apoptosis is triggered in the nuclei of mature sperm cells upon interaction with exogenous DNA.
Sperm-mediated DNA transfer to offspring has the potential to markedly simplify the generation of transgenic animals, but the efficiency in mice has been controversial. To determine the basis of the variability of the procedure in mice, we undertook a large, collaborative study of sperm-mediated DNA transfer to mouse eggs in well-established laboratory conditions for in vitro fertilization and offspring development following embryo transfer. Sperm were incubated with plasmid DNA during the capacitation period and then added to freshly ovulated mouse oocytes for fertilization; cleaved embryos were then transferred to the oviducts of pseudopregnant recipients for gestation. From a total of 75 experiments, 13 produced 130 transgenic offspring, amounting to 7.4% of total fetuses. In five experiments, more than 85% of offspring were transgenic, but the factors leading to this high success rate were not discovered. Clustering of such a low frequency event could account for the disparate reports of transgenic success with sperm-mediated DNA transfer to mouse offspring. Discovering the factors important to success would not only allow this simplified approach to become an important tool in the generation of transgenic mice, but could also lead to important insights into natural protective mechanisms against sperm-mediated transfer of foreign DNA.
Transgenic animals have become valuable tools for both research and applied purposes. The current method of gene transfer, microinjection, which is widely used in transgenic mouse production, has only had limited success in producing transgenic animals of larger or higher species. Here, we report a linker based sperm-mediated gene transfer method (LB-SMGT) that greatly improves the production efficiency of large transgenic animals.
The linker protein, a monoclonal antibody (mAb C), is reactive to a surface antigen on sperm of all tested species including pig, mouse, chicken, cow, goat, sheep, and human. mAb C is a basic protein that binds to DNA through ionic interaction allowing exogenous DNA to be linked specifically to sperm. After fertilization of the egg, the DNA is shown to be successfully integrated into the genome of viable pig and mouse offspring with germ-line transfer to the F1 generation at a highly efficient rate: 37.5% of pigs and 33% of mice. The integration is demonstrated again by FISH analysis and F2 transmission in pigs. Furthermore, expression of the transgene is demonstrated in 61% (35/57) of transgenic pigs (F0 generation).
Our data suggests that LB-SMGT could be used to generate transgenic animals efficiently in many different species.
Background
Nausea and vomiting in pregnancy (NVP) is a multifaceted condition that affects more than half of pregnant women and can range in severity from mild nausea to severe dehydration. Presently physicians evaluate mostly physical symptoms of NVP in trying to assess the severity of the condition. The objective of this study was to investigate how factors, other than the physical morbidity of nausea and vomiting, influence self-perception of NVP by affected women.
Methods
Five hundred women with NVP calling a 1–800 NVP Healthline were asked to rate their NVP severity and report their nausea duration and number of vomiting/retching episodes.
Results
Nausea and vomiting/retching correlated significantly but very poorly with self-assessment of NVP severity. There was also a correlation between nausea duration and vomiting/retching frequency however the correlations were weak and overall physical symptoms could only explain 14% of the variability of women's feelings and perceptions through multivariate analysis.
Conclusions
Physical symptoms weakly correlate with self-assessment of NVP severity. Other aspects of this condition, most probably psychosocial, influence women's perception of NVP severity.
Intracytoplasmic sperm injection (ICSI)-mediated gene transfer has recently been shown to be an effective technique for producing transgenic pigs; however, the types of sperm pretreatment having the most beneficial effects on post-ICSI embryogenesis or transgenic efficiency have not been clarified. In the present study, we performed ICSI-mediated gene transfer using pig sperm subjected to various pretreatments and determined the developmental potential of sperm-injected oocytes and introduction efficiency of exogenous DNA. Embryos were then transferred to recipient pigs to confirm gene transfer efficiency during the fetal period. When ICSI was performed using unfrozen sperm heads with tails removed by piezo-pulse, the rates of blastocyst formation (14.2%, 17/120) and transgene (EGFP) expression (11.8%, 2/17) were both low. When unfrozen sperm heads were used that were removed by sonication, EGFP expression efficiency (11/21, 52.4%) improved significantly (P<0.05). Pretreatment of unfrozen sperm with a surfactant or acrosomal reaction did not further improve the rates of blastocyst formation and EGFP expression. However, use of the heads of sperm frozen-thawed with or without a cryoprotective agent resulted in rates of blastocyst formation and EGFP expression that tended to be generally high (23.0%, 14/61-33.8%, 26/77 and 42.9%, 6/14-66.7%, 10/15). A total of 219 in vitro matured oocytes were fertilized by ICSI-mediated gene transfer using the heads of frozen-thawed sperm and then transferred into two recipient pigs. Seven fetuses were obtained, and EGFP expression and integration of the transgene (10-30 copies) were confirmed in two of the seven fetuses. Use of unfrozen sperm thus confers no advantages on ICSI-mediated gene transfer, and although further investigations are needed, frozen-thawed sperm heads appear to be useful in ICSI-mediated gene transfer.
Coinjection of unfertilized mouse oocytes with sperm heads and exogenous DNA encoding either a green fluorescent protein (GFP) or β-galactosidase reporter produced 64 to 94 percent transgene-expressing embryos, reflecting DNA–sperm head association before coinjection. Nonselective transfer to surrogate mothers of embryos in the GFP series generated about 20 percent offspring expressing the integrated transgene. These data indicate that exogenous DNA can reproducibly be delivered into an oocyte by microinjected spermatozoa and suggest an adaptable method of transgenesis.
Paracentrotus lividusStrongylocentrotus purpuratus and Arbacia lixula sperm is capable of incorporating homologous as well as heterologous macromolecules and vehiculating them into eggs of the same species. If Strongylocentrotus purpuratus eggs are fertilized with sperm previously incubated with pSV2-cat or pRSV-cat plasmids, chloramphenicol acetyl transferase (CAT) activity is found in the embryos at the swimming blastula stage, showing that the plasmids bearing the CAT gene are vehiculated by the sperm into the eggs and the bacterial gene is expressed in the embryos.
Mature female zebrafish (Brachydanio rerio) eggs were in-vitro fertilized with zebrafish sperm cells incubated with pUSVCAT plasmid in either circular or linearized form. Analysis of adult pectoral fins showed that 23.33% (21 out of 89) and 37.5% (3 out of 8) of the treated founder fish (P), fertilized with sperms incubated with circular and linearized plasmid DNA, respectively, showed positive hybridization with whole pUSVCAT plasmid32P radiolabelled probes. When crossbred to nont-ransgenic fish, these putative founder transgenics (P) produced transgenic F1 progenies which in turn produced transgenic F2 fry when crossbred with non-transgenics. No expression of the CAT has been detected so far. Sperm-mediated transfer of foreign genes into the zebrafish is therefore possible; however, the nature of the incorporation is not known at the moment. It would appear from the results obtained that the introduced foreign DNA exists extrachromosomally. Although it has been shown that sperms can be used to insert genes into the fish, methods to integrate the introduced gene into the fish genome need to be explored further.
The objectives were to investigate whether: 1) nanotransfectants are more effective than other common transfection methods for SMGT; 2) NanoSMGT is able to transmit exogenous DNA molecules to bovine embryos; and 3) halloysite clay nanotubes (HCNs) can be used as a transfection reagent to improve transgene transmission. Four transfection systems were used: naked DNA (without transfectant), lipofection, nanopolymer, and halloysite clay nanotubes. Plasmid uptake by sperm and its transfer to embryos were quantified by conventional and real-time PCR, as well as EGFP expression by fluorescence microscopy. Furthermore, sperm motility and viability, and embryo development were investigated. Mean number of plasmids taken up was affected (P < 0.05) by transfection procedure, with the nanopolymer being the most effective transfectant (∼ 153 plasmids per spermatozoon). None of the treatments affected sperm motility or viability. The mean number of plasmids transmitted to four-cell stage embryos was higher (P < 0.05) in nanopolymer and HCNs than liposomes and naked DNA groups. The number of embryos carrying the transgene increased from 8-10% using naked DNA or liposomes to 40-45% using nanopolymer or HCN as transfectants (P < 0.05). There were no significant differences among transfection procedures regarding blastocyst formation rate of resulting embryos. However, no EGFP-expressing embryo was identified in any treatment. Therefore, nanotransfectants improved transgene transmission in bovine embryos without deleterious effects on embryo development. To our knowledge, this was the first time that bovine embryos carrying a transgene were produced by NanoSMGT.
Sperm from different species shows biological differences, determining the success or failure of the sperm-mediated gene transfer (SMGT) technique. There is evidence that exogenous DNA uptake by the spermatozoa is a species-specific and highly regulated phenomenon. Problems involving SMGT procedures might be related to activation of defenses in spermatozoa and in seminal plasma such as DNase enzymes. The objective in the present study was to transfect South American catfish spermatozoa after seminal plasma removal. Seminal plasma had a strong DNase activity that is reduced after sperm washes in isosmotic solution, in which Western blot analysis demonstrated a reduction in the DNase content after washes and Southern blot evaluations show the presence of plasmid after sperm washes. The seminal plasma DNase digests exogenous DNA in a few minutes and has an optimal activity at 43°C. Also, EDTA at 30 mM concentration inhibits the DNase activity. Using PCR the pEGFP vector was internalized by sperm cells even at lesser concentrations (5-40 ng/10(6) spermatozoa) without motility loss after seminal plasma removal. Conversely, using greater pEGFP concentrations (100 ng/10(6) spermatozoa), there were no motile cells, suggesting toxicity of exogenous DNA for sperm cells. These results are interpreted to provide information that can improve the protocol for generation of transgenic South American catfish.
Sperm mediated gene transfer (SMGT) has been successfully used in mammals, amphibians, birds, and some invertebrates. In fish, this methodology has failed or had poor efficiency for the production of transgenic specimens, presumably because the processes regulating the interaction between spermatozoa and exogenous DNA are not well understood. Therefore, the objective was to develop a SMGT protocol for the Brazilian flounder Paralichthys orbignyanus, with an emphasis on the role of seminal plasma DNase on exogenous DNA uptake by fish spermatozoa. In this study, there was strong DNase activity in the seminal plasma of P. orbignyanus; however, this DNase activity was decreased or eliminated by washing the spermatozoa with solutions containing EDTA (DNase activity was completely inhibited by 40 mM EDTA). Three washing solutions were tested, all of which maintained sperm quality. Moreover, it was determined that the no more than 50 ng of exogenous DNA/10(6) cells should be used for SMGT in fish. Finally, it was demonstrated that fish spermatozoa were capable of spontaneous uptake of exogenous DNA after elimination of DNase activity; this was confirmed by exogenous DNA amplification (PCR using sperm genomic DNA as a template) after DNase I treatment. We concluded that whereas DNase activity was an important obstacle for exogenous DNA uptake by fish spermatozoa; controlling this activity improved the efficiency of SMGT in fish.
This study was conducted to evaluate the effects of an electric pulse (electroporation/electropermeabilization) on the binding of foreign DNA molecules to porcine spermatozoa. We previously examined various parameters involved in the association of foreign DNA with sperm after a simple incubation procedure and now report the effects of electroporation on this association. Using end-labeled and random primer labeled lambda HindIII DNA fragments (23-0.125 kb), it was demonstrated that the DNA fragments interacted with the sperm after electroporation. These samples were then centrifuged and washed extensively to establish if any of the labeled DNA was associated with the spermatozoa. It was determined that approximately 10(8) molecules of DNA were associated with 1.5 x 10(7) mL-1 motile spermatozoa after five medium washes. After each wash, samples were withdrawn for gel analysis and scintillation counting. Gel analysis followed by autoradiography revealed the distinctive band pattern of lambda HindIII DNA associating with sperm. In situ visualization studies with biotin-labeled DNA revealed that approximately 75% of motile sperm carried DNA bound to the post-acrosomal region. However, the intensity of the binding varied, with some sperm being more strongly stained than others. Using [3H]dCTP-labeled DNA followed by light microscope autoradiography, approximately 70% of the sperm were strongly stained in the post-acrosomal region. There was a 5-10% increase in the amount of DNA bound by sperm when the samples were electroporated.
Simian virus 40 (SV40) adsorbs on rabbit spermatozoa but does not penetrate the cells, as indicated by the absence of radioactive material seen on autoradiography of spermatozoa exposed to [(3)H]thymidine-labeled SV40. In contrast, after exposure of spermatozoa to labeled SV40 DNA, radioactive material was found in the postacrosomal area of the spermatozoa. Furthermore, when spermatozoa exposed to SV40 DNA were fused with cells of the CV-1 line of African green monkey kidney cells, infectious SV40 was isolated. After uterine insemination of rabbits with spermatozoa infected with SV40 DNA, both unfertilized and one- and two-celled fertilized ova were obtained. When the fertilized ova were cocultivated with CV-1 cells, infectious virus was recovered. In contrast, CV-1 cells exposed to the unfertilized ova or to zonae pellucidae or polar bodies from the fertilized ova did not show a cytopathic effect. This report provides the first evidence that a heterologous genome can be incorporated into a mammalian spermatozoon and subsequently carried into an ovum during the process of fertilization.
Mature Xenopus laevis spermatozoa are capable of binding plasmid pAPrC carrying the complete Rous sarcoma virus (RSV) DNA. Each sperm cell associates, on an average, with 70-160 molecules of the plasmid DNA in a DNase resistant form, if the spermatozoa were exposed to the DNA at a concentration of 1.0-1.4 micrograms/10(7) sperm cells. Fertilization with pAPrC-treated spermatozoa induced developmental malformations in 25-30% of embryos. Immunohistochemical analysis of tissue sections from defective animals revealed aberrations in myotomal structures, and increased expression of pp60src protein in myoblasts, neuronal tube, and epidermis. The presence of characteristic v-src and RSV-long terminal repeat (LTR) sequences in X. laevis DNA was detected by PCR analysis. Embryonic RNA hybridized with a src-specific and an RSV-LTR specific probes indicating expression of the viral DNA. Plasmid DNAs without the v-src gene (pATV9) or completely free of any RSV sequences (pBR322) did not induce any changes in embryonic development. Our results provide evidence that the pBR322-cloned DNA form of the RSV genome associates with frog sperm cells in a DNase-resistant manner suggesting internalization and may be subsequently carried into eggs during the process of artificial fertilization. Correlation between the defective morphogenesis of X. laevis and increased expression of the src gene as well as an interference of RSV DNA with the developmental programs of frog embryos are discussed.
Coinjection of unfertilized mouse oocytes with sperm heads and exogenous DNA encoding either a green fluorescent protein (GFP) or beta-galactosidase reporter produced 64 to 94 percent transgene-expressing embryos, reflecting DNA-sperm head association before coinjection. Nonselective transfer to surrogate mothers of embryos in the GFP series generated about 20 percent offspring expressing the integrated transgene. These data indicate that exogenous DNA can reproducibly be delivered into an oocyte by microinjected spermatozoa and suggest an adaptable method of transgenesis.
We have studied some features of DNA uptake in both mature and immature mammalian spermatozoa. Mature sperm collected from the cauda epididymis are able to incorporate foreign DNA in a buffer containing only salts and calcium. Immature spermatozoa, however, are unable to bind DNA. This seems to be caused by the lack of a functional receptor in the sperm membrane since once this membrane is disrupted by sonication, DNA can be detected in the postacrosome region of the sperm nucleus, matching the distribution of the mature spermatozoa. Comparison between the DNA binding proteins of mature and immature spermatozoa allowed us to identify two bands that could be part of the putative membrane receptor for the DNA. On the other hand, DNA uptake in mature sperm is prevented by the seminal plasma. We have identified two components of the seminal plasma, a calcium-dependent DNase present in the seminal vesicle fluid and several DNA binding proteins secreted by the ventral prostate, that could account for the inhibitory activity. Taken as a whole, our results indicate that DNA uptake by the mammalian spermatozoa is a very specific and highly regulated phenomenon.
The objective of this study was to evaluate in vitro and in vivo development of porcine in vitro matured (IVM) porcine oocytes fertilised by intracytoplasmic sperm injection (ICSI) and the possibility of producing transgenic embryos and offspring with this procedure. Activated ICSI oocytes had a higher pronuclear formation than non-activated ICSI oocytes (mean 64.8+/-17.3% vs 28.5+/-3.4%, p<0.05). When the zygotes with two pronuclei were cultured to day 2, there was no difference (p<0.05) in the cleavage rate (mean 60.0+/-7.0% vs 63.3+/-12.7%) between the two groups. The blastocyst rate in the activation group was significantly higher than that in the non-activation group (mean 30.0+/-11.6% vs 4.6+/-4.2%, p<0.05). After injection of the sperm transfected with DNA/liposome complex, destabilised enhanced green fluorescent protein (d2EGFP) expression was not observed on day 2 in either cleaved or uncleaved embryos. But from day 3, some of the embryos at the 2-cell to 4-cell stage started to express d2EGFP. On day 7, about 30% of cleaved embryos, which were in the range of 2-cell to blastocyst stage, expressed d2EGFP. However, for the IVF oocytes inseminated with sperm transfected with DNA/liposome complex, and for oocytes injected with sperm transfected with DNA/liposome complex, and for oocytes injected with DNA/liposome complex following insemination with sperm not treated with DNA/liposome complex, none of the embryos expressed d2EGFP. Sixteen day 4 ICSI embryos derived from sperm not treated with DNA/liposome complex were transferred into a day 3 recipient. One recipient delivered a female piglet with normal birthweight. After transfer of the ICSI embryos derived from sperm transfected with DNA/liposome complex, none of the four recipients maintained pregnancy.
Transgenic animals are produced primarily by microinjecting exogenous DNA into the male pronuclei of a zygote. Microinjection is successful in mice but not efficient in farm animals, limiting its general utility. We have pursued an alternative technology for producing transgenic animals: Sperm Mediated Gene Transfer (SMGT). Based on our finding that sperm cells bind and internalize exogenous DNA, we used sperm as a vector for transmitting, not only their own DNA, but also, the exogenously-introduced gene of interest to the zygote. SMGT is highly efficient (up to greater than 80%) and relatively inexpensive; it can be used in species refractory to microinjection, whenever reproduction is mediated by gametes. In this report, we describe the procedure for selection of sperm donors and optimization of DNA uptake that are the key steps for the successful outcome of SMGT. We found that the nominal parameters that boar sperm should possess to serve as a good vector for exogenous DNA are the quality of semen based on standard parameters used in conventional animal breeding programs (volume, concentration, presence of abnormal sperm cells, motility at time of collection, and high progressive motility after 2 hr) and the ability of the sperm cells to take up and internalize exogenous DNA. The results described provide significant advances in SMGT technology applied to pigs, so that transgenic pigs can be efficiently obtained. Mol.
We have been interested in developing convenient mass gene transfer methods for producing strains of silver sea bream (Sparus sarba) with superior genetic traits for aquaculture. A transgene construct carrying rainbow trout growth hormone (rtGH) complementary DNA driven by a common carp b-actin promoter was introduced into silver sea bream by electroporating the sperm with the rtGH transgene and using the treated sperm to fertilize eggs stripped from mature females. The presence of the GH transgene in presumptive transgenic individuals was detected by polymerase chain reaction (PCR) analysis. Between 56% and 70% of the animals carried the GH transgene. We refer to this method as sperm-mediated gene transfer (SMGT). Since the handling stress of stripping gametes from female sliver sea bream brood fish could cause severe mortality, an alternative gene transfer method would be highly desirable. We developed a liposome-based method to transfer the GH transgene into the fish. This method, referred as testis-mediated gene transfer (TMGT), involves injecting the liposome-transgene mixture into the gonads of male sea bream at least 48 hours before spawning. The males were mated to reproductively active females, and fertilized eggs were collected for further incubation. Between 59% and 76% of the hatched fry were found by PCR analysis to carry the rtGH transgene. The efficiency of gene transfer was improved more than 80% by injecting multiple doses of the liposome-transgene mixture into the gonads of treated males. Results of Southern blot analysis of DNA isolated from PCR-positive animals showed that the transgene was integrated into the host genome and could be transmitted to its offspring. The rtGH transgene was expressed in many of the rtGH-transgenic fish. Several P1 GH-transgenic silver sea bream exhibited significant growth enhancement compared with nontransgenic controls. Our studies showed that faster-growing silver sea bream could be produced by a variety of mass gene transfer technologies. These gene transfer technologies would be of great value to aquaculture.
Sperm-mediated DNA transfer can be used to transfer exogenous DNA into the oocyte for the production of transgenic animals. In spite of controversy in the literature, sperm-mediated DNA transfer is a simple and quick technique that can be used in routine breeding programs (AI, embryo transfer and IVF). The main objective of this study was to determine the factors affecting the spontaneous uptake of exogenous DNA by bull spermatozoa. For this purpose, fresh and frozen spermatozoa (0.25 x 10(6)), from the same ejaculate from each of four bulls were co-incubated with fluorescent-labeled green fluorescent protein (GFP) and chloremphenicol acetyltransferase (CAT) plasmids at 37 degrees C for 30 min. Neither bull nor plasmid significantly affected the uptake of exogenous DNA. However, transfection efficiency was higher in frozen-thawed versus fresh spermatozoa (P<0.001). Regardless of whether transfected spermatozoa were alive or dead, all transfected spermatozoa were immotile. It can be concluded that a population of spermatozoa is present in bull semen which has the ability to uptake exogenous DNA spontaneously. There is tremendous scope to improve transfection efficiency of spermatozoa while maintaining motility; this needs to be achieved in order to more easily use this technique in transgenesis. However, live-transfected bull spermatozoa clearly can incorporate exogenous DNA and should be usable in intracytoplasmic sperm injection protocols.
Since 1989, a new method for the production of transgenic animals has been available, namely sperm-mediated gene transfer (SMGT), based on the intrinsic ability of sperm cells to bind and internalise exogenous DNA molecules and to transfer them into the oocyte at fertilisation. We first described the SMGT procedure in a small animal model, with high efficiency reported in the mouse. In addition, we successfully adapted and optimised the technique for use in large animals; it was, in fact, highly efficient in the generation of human decay accelerating factor transgenic pig lines, as well as multigene transgenic pigs in which three different reporter genes, namely enhanced green fluorescent protein, enhanced blue fluorescent protein and red fluorescent protein, were introduced. The major benefits of the SMGT technique were found to be its high efficiency, low cost and ease of use compared with other methods. Furthermore, SMGT does not require embryo handling or expensive equipment. Sperm-mediated gene transfer could also be used to generate multigene transgenic pigs that would be of benefit as large animal models for medical research, for agricultural and pharmaceutical applications and, in particular, for xenotransplantation, which requires extensive genetic manipulation of donor pigs to make them suitable for grafting to humans.
In this study, we compared the transfection effectiveness of liposomes with the new transfection reagent FuGene 6 in bovine sperm mediated gene transfer (SMGT). Furthermore, we examined whether plasmid architecture affects overall efficiency by comparing two plasmids, one of them bearing an additional murine nontranscribed spacer (nts) insert (CMV-INF-tau-IRES-EGFP versus CMV-INF-tau-IRES-EGFP-nts). To accomplish that, we quantified plasmid binding and uptake to spermatozoon and transfer and expression of foreign DNA into embryos by real time PCR. More plasmids bound to spermatozoa when treated with FuGene 6 than with liposome treatment (p<0.05) reaching highest counts in plasmids bearing the nts sequence (p<0.05). Mean number of plasmids taken up was significantly (p<0.05) affected by transfection strategy (1-3 versus 15-81 versus 120-162) with plasmids bearing the nts sequence being 2-8 fold more effective (p<0.05). Culture of SMGT derived embryos up to day 9 did not result in any difference in terms of cleavage rate (64.2-84.2%) and development to blastocyst stage (18.8-26.3%) between different groups. Insert of the nts fragment significantly (p<0.05) affected mean number of transmitted plasmids to 4-cell stage embryos (44 versus 7) and relative INF-tau mRNA expression level in day 9 blastocysts (7-8 fold). However, only six blastocysts (3.6%) exhibited green fluorescence indicating low EGFP protein production. In conclusion, we were able to show effectiveness of sperm mediated gene transfer is significantly affected by choice of transfection reagent and by plasmid architecture.
- N Xin
N. Xin et al. / Animal Reproduction Science 149 (2014) 305–310