Sperm mediated gene transfer in pig: Selection of donor boars and optimization of DNA uptake

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.     

Sperm nuclear transfer and transgenic production in the fish medaka

Sperm nuclear transfer or intracytoplasmic sperm injection (ICSI) is a powerful assisted reproductive technology (ART) for treating human male infertility. Controversial reports of increased birth defects have raised concerns about the ART's safety. The cause for birth defects, however, has remained elusive for analysis in human because of the sample size, male infertility genetics, physiological heterogeneity and associated procedures such as embryo manipulations. Animal models are required to evaluate factors leading to the increased birth defects. Here we report the establishment of medakafish model for ICSI and transgenic production. This small laboratory fish has high fecundity and easy embryology. We show that ICSI produced a 5% high percentage of fertile animals that exhibited both paternal and maternal contribution as evidenced by the pigmentation marker. Furthermore, when sperm were pre-incubated with a plasmid ubiquitously expressing RFP and subjected to ICSI, 50% of sperm nuclear transplants showed germline transmission. We conclude that medaka is an excellent model for ICSI to evaluate birth defects and that sperm nuclear transfer can mediate stable gene transfer at high efficiency. Although more demanding for experimentation, sperm-mediated transgenesis should be particularly applicable for aquaculture species with a lengthy generation time and/or a large adult body size.     

Multi-transgenic pigs expressing three fluorescent proteins produced with high efficiency by sperm mediated gene transfer

Multi-gene transgenic pigs would be of benefit for large animal models in medical, agricultural, and pharmaceutical applications; in particular for xenotransplantation, where extensive genetic manipulation of donor pigs is required to make them suitable for organ grafting to humans. We used the sperm mediated gene transfer (SMGT) method to produce with high efficiency multi-gene transgenic pigs using three genes coding for fluorescent proteins: enhanced blue (EBFP), green (EGFP), and red (DsRed2). All three fluorescent proteins were expressed in 171 out of 195 normally developed morula/blastocysts examined at day 6 post insemination (88%). Genomic DNA of 18 piglets born from two litters was screened by PCR, showing that all piglets were transgenic with at least one gene, 7/18 piglets were triple transgenic, 7/18 double transgenic, and 4/18 single transgenic. Fluorescence in situ hybridization (FISH) analysis revealed multiple sites of integration of the transgenes. RNA and protein expression was found in muscle, heart, liver, hair, and peripheral blood mononuclear cells (PBMCs). These results show that SMGT is an effective method for introducing multiple genes into pigs as shown by the simultaneous expression of three fluorescent proteins.     

Sperm head abnormalities are more frequent in songbirds with more helical sperm: A possible trade‐off in sperm evolution

Sperm morphology varies enormously across the animal kingdom. Whilst knowledge of the factors that drive the evolution of interspecific variation in sperm morphology is accumulating, we currently have little understanding of factors that may constrain evolutionary change in sperm traits. We investigated whether susceptibility to sperm abnormalities could represent such a constraint in songbirds, a group characterized by a distinctive helical sperm head shape. Specifically, using 36 songbird species and data from light and scanning electron microscopy, we examined among‐species correlations between the occurrence of sperm head abnormalities and sperm morphology, as well as the correlation between sperm head abnormalities and two indicators of sperm competition. We found that species with more helically shaped sperm heads (i.e., a wider helical membrane and more pronounced cell waveform) had a higher percentage of abnormal sperm heads than species with less helical sperm (i.e., relatively straight sperm) and that sperm head traits were better predictors of head abnormalities than total sperm length. In contrast, there was no correlation between sperm abnormalities and the level of sperm competition. Given that songbird species with more pronounced helical sperm have higher average sperm swimming speed, our results suggest an evolutionary trade‐off between sperm performance and the structural integrity of the sperm head. As such, susceptibility to morphological abnormalities may constrain the evolution of helical sperm morphology in songbirds.     

精子结合外源DNA的特征及影响因素

外源DNA与精子相互作用后的定位及内化率是精子载体法制备转基因动物的关键环节。实验以标记的DNA片段为示踪材料,就精子与外源DNA相互作用的基本特征及影响因素进行了研究。结果表明：山羊精子可自发性结合外源DNA,外源DNA最初结合于顶体后区质膜外表面,随后部分内化进入细胞内。精子对外源DNA的结合和内化能力随供体的不同而差异明显,在实验所检查的35只公羊中,结合率（DNaseⅠ消化前）波动于4.6% ~ 62.4%,内化率（DNaseⅠ消化后）波动于2.1% ~ 53.8%,个体间差异显著（P<0.01）。对于同一供体的精子而言,阻止DNA结合的最主要因素是精浆,与射出的原精液相比,洗涤后精子的结合率和内化率分别提高了3倍和5倍;其次精子获能也将导致结合率和内化率降低（P<0.01）。死精子不能完成外源DNA的内化过程,但反复冻融导致质膜破裂的死精子具有更高的结合率,而且阳性率与动物个体无关。上述结果提示,筛选合适的精子供体,采用优化的转染处理方法是提高精子载体方法效率的前提和保证。     

Sperm morphology and sperm velocity in passerine birds

Sperm velocity is one of the main determinants of the outcome of sperm competition. Since sperm vary considerably in their morphology between and within species, it seems likely that sperm morphology is associated with sperm velocity. Theory predicts that sperm velocity may be increased by enlarged midpiece (energetic component) or flagellum length (kinetic component), or by particular ratios between sperm components, such as between flagellum length and head size. However, such associations have rarely been found in empirical studies. In a comparative framework in passerine birds, we tested these theoretical predictions both across a wide range of species and within a single family, the New World blackbirds (Icteridae). In both study groups, sperm velocity was influenced by sperm morphology in the predicted direction. Consistent with theoretical models, these results show that selection on sperm morphology and velocity are likely to be concomitant evolutionary forces.     

Sperm transfer during mating, movement of sperm in the female reproductive tract, and sperm precedence in the common cutworm Spodoptera litura

Abstract. Mating behaviour, sperm transfer and sperm precedence were studied in the moth Spodoptera litura (Fabr.) (Lepidoptera: Noctuidae). There existed a rhythmic, diel pattern of mating behaviour of this moth during the scotophase, presumably set with respect to an endogenous activity rhythm. Approximately 30 min after copulation had started, the formation of the corpus of the spermatophore began in the bursa copulatrix of the female moth, but full inflation of the corpus was not completed until 45–60 min after mating had started. The mature spermatophore contained about 350 eupyrene sperm bundles and a large number of individual (loose) apyrene spermatozoa. The mating status and the age of the male insect influenced the number of sperm transferred to the female within the spermatophore, and also affected the consequent fertility. There was no evidence of sperm reflux within the male tract. Within the female, dissociation of eupyrene sperm bundles was evident within the spermatophore less than 15 min after the completion of mating. Spermatozoa began to move from the bursa (in which the spermatophore is lodged) into the spermatheca 30–45 min after the end of the copulation, and the quantity of sperm in the spermatheca reached a plateau at 90 min after mating. Apyrene sperm reached the spermatheca first, followed by eupyrene sperm. Examination of total (apyrene plus eupyrene) sperm in the female tract showed that 86% of mated females received an apparently normal amount of total sperm from the male. Examination of eupyrene sperm alone showed that 81% of matings resulted in an apparently normal transfer of eupyrene sperm. A small proportion (approximately 8%) of the matings, however, were identified as transferring a clearly subnormal quantity of eupyrene sperm to the spermatheca. The eggs produced as a result of such pairings displayed much reduced fertility (about 43%) compared to those from matings confirmed to have transferred normal quantities of sperm, which showed about 92% fertility. This shows that the availability of eupyrene sperm in the spermatheca may be an important constraint on fertility in normal populations of insects. In the laboratory, S. litura females exhibited multiple matings. Of the females, 93% mated, and the mean frequency of mating was 1.69. Mating with a fertile male led to the oviposition of an increased number of eggs. This effect continued even when the female subsequently mated with an infertile male. Displacement of sperm from previous matings is known to be an important factor in the evolution of multiple mating strategies. Our results on sperm utilization by S. litura indicated that after a second mating, the sperm utilized for subsequent fertilization were almost exclusively from the last mating with little mixing. The proportion of eggs fertilized by sperm from the second mating (P₂) was calculated as 0.95, indicating almost complete sperm precedence from the last mating.     

Ras-related proteins (Rab) play significant roles in sperm motility and capacitation status

Rab proteins are widely known for their involvement in establishing Golgi apparatus and controlling Golgi trafficking in eukaryotic cells. Specifically, Rab proteins play significant roles in acrosome formation and exocytosis. Furthermore, mechanisms involved in the regulation of Rab proteins during capacitation have been identified. However, there has been no direct evaluation to assess the correlation between Rab proteins and sperm function. Consequently, this study was designed to analyze the correlation between Rab proteins and sperm functions. Individually, we analyzed the sperm motility patterns, motion kinematics, capacitation status, and Rab protein expression levels of sperm samples from 31 boars before and after capacitation. As a result, we discovered that Rab3A, Rab5, Rab11, Rab14, and Rab27A correlated with various sperm motility patterns, motion kinematics before capacitation. Rab3A, Rab5, Rab11, Rab14, and Rab34 correlated with various sperm motility patterns, motion kinematics after capacitation. Moreover, Rab4 and Rab34 were associated with capacitation status before capacitation, and Rab3A, 25, and 27A correlated with capacitation status after capacitation. This is the first study to analyze the correlation between Rab proteins and sperm functions. Collectively, our results indicate that specific sperm motility and kinematics, as well as the structural condition of the sperm head and capacitation status, regulate individual Rab protein. Therefore, we expect that the current findings will be used to identify the etiology of idiopathic male infertility patients and to diagnose male fertility and that Rab proteins will be employed as biomarkers to predict and analyze male fertility.     

Electro‐mediated gene transfer and expression are controlled by the life‐time of DNA/membrane complex formation

Background

Electroporation is a physical method used to transfer molecules into cells and tissues. Clinical applications have been developed for antitumor drug delivery. Clinical trials of gene electrotransfer are under investigation. However, knowledge about how DNA enters cells is not complete. By contrast to small molecules that have direct access to the cytoplasm, DNA forms a long lived complex with the plasma membrane and is transferred into the cytoplasm with a considerable delay.

Methods

To increase our understanding of the key step of DNA/membrane complex formation, we investigated the dependence of DNA/membrane interaction and gene expression on electric pulse polarity and repetition frequency.

Results

We observed that both are affected by reversing the polarity and by increasing the repetition frequency of pulses. The results obtained in the present study reveal the existence of two classes of DNA/membrane interaction: (i) a metastable DNA/membrane complex from which DNA can leave and return to external medium and (ii) a stable DNA/membrane complex, where DNA cannot be removed, even by applying electric pulses of reversed polarity. Only DNA belonging to the second class leads to effective gene expression.

Conclusions

The life‐time of DNA/membrane complex formation is of the order of 1 s and has to be taken into account to improve protocols of electro‐mediated gene delivery. Copyright © 2009 John Wiley & Sons, Ltd.     

Age-related sperm transfer and sperm competitive ability in the male hide beetle

The influence of male age on reproductive success after a singlemating has been explored widely; however, few studies have investigatedwhether quantitative or qualitative differences in male spermare responsible for the observed patterns. Moreover, the roleof male age on sperm competitive ability has been largely ignored.We examined the importance of male age on the probability andamount of sperm transferred during a single mating and exploredwhether sperm competitive ability varies with male age in thehide beetle Dermestes maculatus, a species where sperm viabilitydoes not vary with male age. We also investigated whether spermtransfer rates varied with female age. We found that the probabilityof sperm transfer and the amount of sperm transferred variedwith male, but not female, age. All males performed behaviorallysuccessful copulations, but intermediate-age and old males weremore likely to transfer sperm successfully and also transferreda greater quantity of sperm than young males. Old males wereless likely to transfer sperm than intermediate-age males, butif they did transfer sperm successfully, they transferred comparableamounts. Sperm competitive ability varied with male age andreflected the quantity of sperm transferred. On average, intermediate-agemales achieved greater fertilization success when competingagainst young or old males than when competing against otherintermediate-age males. Old males were poor competitors againstintermediate-age males, but they achieved significantly higherrates of fertilization when competing against young males. Ourfindings suggest that quantitative differences in the amountof sperm transferred determine male success in sperm competitionin the hide beetle.     

Production of inbred and hybrid transgenic mice carrying large (> 200 kb) foreign DNA fragments by intracytoplasmic sperm injection

We have developed a mouse transgenesis technique that facilitates the insertion of large (approximately 200 kilo base pairs) DNA fragments into host genomes of both inbred and hybrid mice. Six inbred and three hybrid transgenic mice carrying a single bacterial artificial chromosome (BAC) clone with genes located in the Down syndrome critical region of human chromosome 21 were produced using this technology.     

No evidence of trade‐offs in the evolution of sperm numbers and sperm size in mammals

Post‐copulatory sexual selection, in the form sperm competition, has influenced the evolution of several male reproductive traits. However, theory predicts that sperm competition would lead to trade‐offs between numbers and size of spermatozoa because increased costs per cell would result in a reduction of sperm number if both traits share the same energetic budget. Theoretical models have proposed that, in large animals, increased sperm size would have minimal fitness advantage compared with increased sperm numbers. Thus, sperm numbers would evolve more rapidly than sperm size under sperm competition pressure. We tested in mammals whether sperm competition maximizes sperm numbers and size, and whether there is a trade‐off between these traits. Our results showed that sperm competition maximizes sperm numbers in eutherian and metatherian mammals. There was no evidence of a trade‐off between sperm numbers and sperm size in any of the two mammalian clades as we did not observe any significant relationship between sperm numbers and sperm size once the effect of sperm competition was taken into account. Maximization of both numbers and size in mammals may occur because each trait is crucial at different stages in sperm's life; for example size‐determined sperm velocity is a key determinant of fertilization success. In addition, numbers and size may also be influenced by diverse energetic budgets required at different stages of sperm formation.     

Efficient and simple production of transgenic mice and rabbits using the new DMSO-sperm mediated exogenous DNA transfer method

A high efficient and simple transgenic technology on mice and rabbits to transfect spermatozoa with exogenous DNA/DMSO complex to obtain transgenic offspring, which is namely called DMSO-sperm mediated gene transfer (SMGT). Mouse sperm could be either directly transfected via injection into testis or cultured in vitro with the plasmed DNA containing the enhanced green fluorescent protein (EGFP) that could be expressed in the embryos and offspring. Then, 36 living transgenic rabbits were produced using the same technology, and the transgenic ratio of 56.3% was detected using PCR and Southern blot. As the controls, the transgenic ratios of 39.6% and 47.8% have also been tested using the liposomes mediated technology of Tfx-50 Reagent or Lipefectamin-2000, respectively. The results show that the female transgenic rabbits, as the mammary gland bioreactor models, could express the human tissue plasminogen activator mutant (htPAm) in their mammary cells when they are adult.     

Adenovirus‐mediated introduction of DNA into pig sperm and offspring

The ability of adenoviral vectors to transfer DNA into boar spermatozoa and to offspring was tested. Exposure of spermatozoa to adenovirus bearing the E. coli lacZ gene resulted in the transfer of the gene to the head of the spermatozoa. Treatment did not affect either viability or acrosomal integrity of boar sperm. Of the 2‐ to 8‐cell embryos obtained after in vitro fertilization with adenovirus‐exposed sperm, 21.7% expressed the LacZ product. Four out of 56 piglets (about 7%) obtained after artificial insemination with adenovirus‐exposed spermatozoa were positive in PCR analyses, even though none of the piglets showed the LacZ gene after southern blot analysis. RT‐PCR analysis performed in tissues from two positive stillborn piglets showed the presence of the LacZ mRNA in all of the tissues tested. The offspring obtained after mating two positive animals did not show LacZ gene presence. Our results indicate that adenovirus could be a feasible mechanism for the delivery of DNA into spermatozoa, even though the transfer of the transgene may be limited to the first generation. Mol. Reprod. Dev. 53:149–158, 1999. © 1999 Wiley‐Liss, Inc.     

Sperm structure and sperm transfer in Pseudopythina subsinuata (Bivalvia; Galeommatoidea)

Males and females of the commensal protandric bivalve Pseudopythina subsinuata have paired seminal receptacles, the interior of which contains many slender elongate cells. The testis produces small euspermatozoa and comparatively few and much larger paraspermatozoa. The ?16-μm-long and 3-5-μm-thick paraspermatozoa have a terminally placed irregularly cork-screw-shaped acrosome and a bundle of ca. 16 flagella emerging from behind the nucleus. The role of the paraspermatozoa is obscure. Euspermatozoa are transferred to the seminal receptacles of the females and attach with the tip of the acrosome to the elongate cells. Most females contain one to three “sperm trees”, structures consisting of a short stem and numerous branches. They are firmly implanted in the abfrontal part of the gill filament and protrude into the posterior part of the suprabranchial (brooding) chamber. Implantation of the trees causes the gill tissue to swell around the stem and some of the nearest filaments to coalesce. All branches are densely coated with euspermatozoa that are attached by means of their acrosomes. It is conjectured that the syncytial and multinucleate trees arise from seminal receptacle cells that detach from the receptacle and thereupon fuse. A similar process is known in the allied P. tsurumaru, but the resulting structure (“sperm-carrying body”) is not attached to the gills.     

Sperm competition and sperm precedence in the dragonfly Nanophya pygmaea

Abstract. The libellulid dragonfly, Nanophya pygmaea Rambur, has an average ejaculate volume of 0.16 mm³. During successive copulations the volume of sperm stored in the female's sperm storage organs increases in steps equivalent to this volume, suggesting that the sperm competition mechanism in this species is sperm repositioning, i.e. adding an ejaculate to what is already present in the female's sperm storage organ. By using sterile/normal males in double matings with females we have shown that this mechanism results in last male sperm precedence (P²= 0.979).     

Remating, sperm transfer, and sperm displacement in the cactophilic species Drosophila buzzatii Patterson & Wheeler (Diptera: Drosophilidae)

The mating system of Drosophila buzzatii is characterized by short copulation duration, frequent remating in both males and females, and male ejaculate partitioning. Additional features of the system are strong sperm displacement and a high frequency of sterile matings. Remating frequencies and the effects of remating on various mating parameters were studied. In order to characterize variation, five isofemale lines from geographically distant localities in Australia (three localities), Brazil and the Canary Islands were used. Mating parameters studied were: premating time, copulation duration, interval between successive matings, and progeny number as a measure of sperm transfer. Variation for sperm displacement was studied in crosses between laboratory stocks and a number of isofemale lines from Australia. There were significant between‐line differences in female remating frequencies, premating time, copulation duration, interval between successive matings, and progeny numbers, indicating genetic variation for these traits. Females from the five lines mated on average 1.6 to 3.1 times in 4 h, with a maximum of eight matings for one female. The males were given a maximum of ten virgin females in sequence and more than one‐third of the males mated all ten females in the 2 h observation period. Copulation duration decreased and interval between matings increased with copulation number in multiply mated males. Mean copulation duration was c. 2 min. Sperm transfer, measured as the average number of progeny from a single mating, was low (c. 25) and multiply mated females gave more progeny than single mated females, although with much lower progeny numbers than observed in wild‐caught non‐virgin females. A surprisingly high proportion of observed matings gave no progeny, i.e. they were sterile matings. Sperm displacement was strong in most crosses and remained strong in multiply mated females. The results are discussed in relation to the evolution of mating patterns in Drosophila.