Evaluation of DNase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of the Brazilian flounder Paralichthys orbignyanus

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.     

Transgene transmission in chickens by sperm-mediated gene transfer after seminal plasma removal and exogenous DNA treated with dimethylsulfoxide or N,N-dimethylacetamide

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.     

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。     

The negative effects of exogenous DNA binding on porcine spermatozoa are caused by removal of seminal fluid

Sperm-mediated gene transfer (SMGT) might become the most efficient and cost effective technique to generate transgenic animals, which will significantly increase their application in biomedical research and in commercial production. Despite some successes, the technique has remained controversial for almost 20 years and despite number of studies the reasons for poor reproducibility of this promising technology has not been understood. We suggest that the reason for poor reproducibility is the presence of natural defences against exogenous DNA invasion acting in spermatozoa or in embryo. Based on previous reports we have investigated the effect of foreign DNA binding on spermatozoa by monitoring motility, viability and genomic DNA damage. Evaluation of DNA binding in sperm collected from 16 boars demonstrated that 28-45% of the added pEGFP plasmid was bound to spermatozoa with 9-32% being internalized in sperm nucleus. In agreement with previous reports, our results demonstrated that the pEGFP-treated sperm show an average a 2-fold decrease in motility (p<0.05), 5-fold decrease in progressive motility (p<0.05), and 1.4-fold increase in number of sperm with highly damaged DNA (p<0.05) as detected by Comet assay. In contrast with previous reports, we demonstrate that all such changes were associated with the removal of seminal plasma during the washing step and not with foreign DNA binding per se. We suggest that poor reproducibility of SMGT most likely result from selection against DNA-loaded sperm at later stages of fertilization.     

Transgene transmission in South American catfish (<Emphasis Type="Italic">Rhamdia quelen</Emphasis>) larvae by sperm-mediated gene transfer

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.     

Association of rabbit sperm cells with exogenous DNA

The ability of rabbit spermatozoa to bind exogenous DNA during sperm-mediated gene transfer (SMGT) was tested in our study. Fresh collected semen, or fully capacitated sperm cells, was co-cultured with plasmid DNA labeled with tetramethyrodamine-6-dUTP. Fluorescent spermatozoa were counted before and after DNaseI treatment. Results showed that fluorescent-labeled plasmid DNA could be taken up by capacitated rabbit sperm cells. 66% spermatozoa carried exogenous DNA in the presence of lipofectin. Bovine serum albumin could block this process effectively. Associated DNA was mainly located in the posterior area of the sperm head. In order to verify whether exogenous DNA was carried into the embryo and expressed in the offspring, further SMGT experiments were carried out using the pHM-CR plasmid which contains LacZ and Neomycin genes. beta-galactosidase was expressed in different stages of embryo development and in the tissues of young rabbit as detected by using X-gal staining. Large portion of embryos survived under the selection pressure in G418 containing medium, after SMGT. Transgene integration was further verified by PCR analysis. These results confirmed the ability of rabbit sperm cells to carry transgene into the embryo during in vitro fertilization.     

Spontaneous uptake of exogenous DNA by goat spermatozoa and selection of donor bucks for sperm-mediated gene transfer

Sperm-mediated gene transfer (SMGT) has been long heralded as a faster and cheaper alternative to more commonly used methods of producing transgenic animals. In this study, the capra semen ejaculates were pooled together and then incubated in vitro with DIG-labeled DNA. The binding and internalizing rates were observed by the in situ hybridization methods. We also compared the standard sperm parameters and the efficiencies of interaction with exogenous DNA of 60 individuals to select donor bucks for SMGT. It was showed that labeled exogenous DNA was detected in different localizations in spermatozoa but genuine DNA uptake, in contrast to mere binding, seems to be limited to the postacrosomal region. The removal of seminal plasma increased significantly (P < 0.01) the capability in picking up exogenous DNA. Use of frozen-thawed semen (without cryoprotectant agents) and Triton X-100 treatment also increased significantly (P < 0.01) the DNA-binding capacity, but reduced the sperm viability. The binding rates (the proportion of labeled-DNA positive spermatozoa to all the spermatozoa counted) of 60 buck individuals were in the range of 3.08–73.39%, and the internalizing rates (the proportion of DNaseI-treated labeled-DNA positive spermatozoa to all the spermatozoa counted) were 4.83–70.00%. About 8.34% (5/60) bucks showed high binding, but low internalizing ability. Chi-square test showed that there was significant difference among the breeds (x ² = 26.515, P < 0.01). Eight individual bucks that demonstrated high DNA uptake were selected for SMGT. It was demonstrated that the goat spermatozoa was capable of spontaneous uptake of exogenous DNA. Seminal fluid inhibits DNA uptake and that membrane disruption increases DNA binding but greatly diminishes uptake.     

Exogenous DNA uptake by bovine spermatozoa does not induce DNA fragmentation

Sperm-mediated gene transfer (SMGT) is a fast and low-cost method used to produce transgenic animals. The objective of this study was to evaluate the effects of the concentration of exogenous DNA and the duration of incubation on DNA uptake by bovine spermatozoa and subsequently the integrity of sperm DNA and sperm apoptosis. Spermatozoa (5 × 10⁶ cells/mL) were incubated with 100, 300, or 500 ng of exogenous DNA (pEYFP-Nuc plasmid) for 60 or 120 min at 39 °C. The amount of exogenous DNA associated with spermatozoa was quantified by real-time PCR, and the percentages of DNA fragmentation in spermatozoa were evaluated using SCSA and a TUNEL assay, coupled with flow cytometry. Uptake of exogenous DNA increased significantly as incubation increased from 60 to 120 min (0.0091 and 0.028 ng, respectively), but only when the highest exogenous DNA concentration (500 ng) was used (P < 0.05). Based on SCSA and TUNEL assays, there was no effect of exogenous DNA uptake or incubation period on sperm DNA integrity. In conclusion, exogenous DNA uptake by bovine spermatozoa was increased with the highest exogenous DNA concentration and longest incubation period, but fragmentation of endogenous DNA was apparently not induced.     

On the integrity of sperm DNA

Ejaculated rabbit spermatozoa washed with buffer prior to decondensation by Triton X-100 and dithiothreitol were good templates for DNA synthesis by Escherichia coli DNA polymerase. This result agrees with the observations of Zirkin and Chang [1977], and implies that the sperm DNA is nicked. Template activity, however, was reduced if spermatozoa were extensively washed before decondensation, and if DNase inhibitors EDTA or Na₂SO₄ were present during decondensation. Template activity was also low if decondensation was induced with DNase inhibitors thioglycollic acid, Na₂SO₃ or sodium dodecylsulphate and dithiothreitol instead of with Triton X-100 and dithiothreitol. Calf thymus DNA was completely degraded when incubated with rabbit seminal plasma or buffer-washed spermatozoa, but much less degradation was observed if EDTA, Na₂SO₄, thioglycollic acid, Na₂SO₃ or sodium dodecylsulphate were also present, or if spermatozoa were extensively washed with buffer. Centrifugation of spermatozoa through 2.05 M sucrose completely removed contaminating DNase, and such spermatozoa were inactive as DNA templates after decondensation. The DNA template activity of swollen rabbit sperm nuclei thus parallels the activity of a contaminating seminal plasma DNase. This suggest that the nicks in sperm DNA enabling it to act as a template for DNA synthesis were generated by the DNase during decondensation and thus are not a natural structural feature of the DNA. The presence of breaks in the DNA of decondensed buffer-washed spermatozoa (DNase contaminated) was confirmed by their incorporation of phosphate from [γ^?32 P] ATP in the presence of the enzyme polynucleotide kinase. These spermatozoa were found to contain as few as two breaks/mole of DNA, but sucrose-washed spermatozoa (DNase free) were free of breaks. The possible use of this enzymic procedure for the assessment of sperm genome damage and the evaluation of the quality of a sperm population are discussed.     

精子因素对精子载体法制备转基因山羊的影响

精子具有主动结合、转运、整合外源DNA的能力,并在受精时导入卵母细胞,获得转基因动物.精子介导基因转移(sperm-mediated gene transfer,SMGT)是目前获得转基因动物简单而高效的方法之一.精子因素是影响SMGT方法生产转基因动物的重要方面.本论文结合我们的研究针对转染用山羊(Capra hircus)精液的来源、精子质膜完整性、精液品质及发育阶段等精子因素影响精子结合外源DNA和SMGT方法生产转基因山羊的效率进行了论述,并从这些影响因素入手,提出了筛选精子供体、保持精液品质、调控质膜等措施,提高精子转染外源DNA能力和生产转基因动物的效率.     

Regulation of foreign DNA uptake by mouse spermatozoa

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.     

Seminal DNase frees spermatozoa entangled in neutrophil extracellular traps

Insemination always stimulates neutrophil migration into the female reproductive tract (FRT), which eliminates excess spermatozoa and bacterial contaminants introduced by the breeding process. However, the presence of neutrophils in the FRT at the time of semen deposition has been shown to result in sperm-neutrophil binding that reduces motility and fertility. Although the binding and trapping mechanism has not been determined, seminal plasma (SP) was found to include a protein factor or factors that reduced sperm-neutrophil binding and improved fertility of sperm inseminated in the presence of neutrophils. Although DNase has been shown to be present in the SP of different species and has been associated with improved fertility in bulls, the mechanism(s) explaining this association and the paradox of DNA-packed cells being associated with DNase have remained unresolved. We demonstrate that sperm-activated neutrophils extrude their DNA, which in turn traps sperm cells and hinders their motility (and ultimately may hinder sperm transport to the fertilization site). DNase activity present in the SP digests the extruded DNA and frees entangled spermatozoa, which in turn may allow more spermatozoa to reach the oviduct, and explains at least one mechanism by which SP increases the rate of fertility. The ability of SP proteins to suppress neutrophil activation in the presence of spermatozoa did not render neutrophils incapable of combating bacteria, demonstrating that SP proteins are highly selective for suppressing neutrophils activated by spermatozoa, but not by bacteria.     

Seminal plasma reduces exogenous oxidative damage to human sperm, determined by the measurement of DNA strand breaks and lipid peroxidation

Exposure of spermatozoa to reactive oxygen species (ROS) has been associated with cellular injury, that includes DNA damage and lipid peroxidation. In addition, sperm preparation techniques such as centrifugation, commonly used prior to in vitro fertilization and scientific studies, are associated with the generation of ROS and an increase in the level of DNA damage. The preservation, therefore, of sperm in vitro that might decrease the potential for oxidative DNA damage to arise and allow for an improvement in semen quality used for artificial insemination, is of importance. Seminal plasma is a rich source of antioxidants, which, potentially, safeguards sperm from oxidative attack during storage and once ejaculated. We have investigated the protection of human spermatozoa from ROS afforded by seminal plasma. Sperm were exposed to exogenous ROS by incubating the cells with hydrogen peroxide in the presence of ferrous sulfate and ADP. Aliquots of seminal plasma were added to the incubation mixture in differing amounts, and the generation of DNA strand breaks and thiobarbituric acid reactive species (TBARS), indicative of lipid peroxidation, determined. Incubation of sperm with exogenous ROS resulted in a significant generation of DNA strand breaks and lipid peroxidation compared to basal levels of damage (P<0.05). Addition of seminal plasma to the incubation media produced a significant decrease in DNA strand breaks and TBARS (P<0. 05), when the amount of plasma added exceeded 60% v/v. The results indicate that spermatozoal oxidative damage induced by exogenous ROS, specifically DNA damage and lipid peroxidation, is reduced by the presence of seminal plasma.     

Effect of exogenous DNA on bovine sperm functionality using the sperm mediated gene transfer (SMGT) technique

Sperm mediated gene transfer (SMGT) could provide the opportunity to carry out transgenesis on a mass scale using spermatozoa as vectors for exogenous DNA. However, the efficiency of sperm‐mediated DNA transfer is still questionable, and the mode of transmission to the egg has not yet been well understood. Our aim was to investigate the capacity of bovine spermatozoa to carry exogenous DNA and its relationship to sperm functionality. We studied these parameters using flow cytometry to measure viability (necrosis and apoptosis) and capacitation status, computer‐assisted semen analysis (CASA) to measure motility parameters and in vitro fertilization (IVF) to assess fertilizing capacity. Furthermore, we studied the effect of capacitation status on interaction with exogenous DNA, and the role of heparin supplementation in this process. Bull spermatozoa showed a high capacity to bind DNA quickly and reached a maximum after 30 min, with approximately half of the DNA‐bound spermatozoa being viable. Incubation with exogenous DNA induced a decrease in sperm viability and motility and increased the proportion of apoptotic cells, but did not affect the cleavage rate in IVF assay. Heparin increased high‐lipid disorder and the number of sperm with DNA bound (viable and dead). In conclusion, this study shows that live spermatozoa can bind exogenous DNA with a slight negative effect in some parameters of sperm function that in our opinion, would not drastically compromise fertility. Mol. Reprod. Dev. 77: 687–698, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Effect of seminal plasma on the motility of epididymal and ejaculated spermatozoa of the ram and bull during the cryopreservation process

Experiments were conducted to investigate the effect of seminal plasma on sperm motility during the cryopreservation process. Ejaculated and epididymal spermatozoa from the ram and the bull were washed by centrifugation and resuspended in either seminal plasma or a modified Tyrode's medium (TALP) prior to dilution in medium suitable for cryopreservation. Resuspension of washed ejaculated ram spermatozoa in seminal plasma resulted in higher percentages of motile spermatozoa than resuspension in TALP after the spermatozoa were cooled to 5 degrees C (52 vs 35%), and after thawing (14 vs 9%), respectively. Resuspension of epididymal ram spermatozoa in seminal plasma had no beneficial effect in maintaining sperm motility after cooling (78 vs 73%); however, seminal plasma was beneficial to epididymal ram spermatozoa after thawing (34 vs 3%), respectively. Resuspension of washed ejaculated bull spermatozoa in either seminal plasma or TALP had no effect on the percentage of motile spermatozoa after cooling to 5 degrees C (73 vs 75%) or after thawing (60 vs 60%), respectively. In addition, seminal plasma had no beneficial effect on the percentage of motile epididymal bull spermatozoa when compared with that of TALP-treated spermatozoa after cooling (75 vs 72%) or after thawing (66 vs 63%), respectively. Seminal plasma from different sires (ram and bull) affected epididymal sperm motility. The ability of sperm cells to withstand damage during cryopreservation, however, appears to reside in the sperm cells themselves, probably due to sperm cell composition.     

DNA uptake in swine sperm: Effect of plasmid topology and methyl‐beta‐cyclodextrin‐mediated cholesterol depletion

Sperm‐mediated gene transfer (SMGT), the ability of sperm cells to spontaneously incorporate exogenous DNA and to deliver it to oocytes during fertilization, has been proposed as an easy and efficient method for producing transgenic animals. SMGT is still undergoing development and optimization to improve the uptake efficiency of foreign DNA by sperm cells, which is a preliminary, yet critical, step for successful SMGT. Towards this aim, we developed a quantitative, real‐time PCR‐based assay to assess the absolute number of exogenous plasmids internalized into the spermatozoon. Using this technique, we found that the circular form of the DNA is more efficiently taken up than the linearized form. We also found that DNA internalization into the nucleus of porcine sperm cells is better under specific methyl‐β‐cyclodextrin (MCD)‐treated conditions, where the plasma membrane properties were altered without significantly compromising sperm physiology. These results provide the first evidence that membrane cholesterol depletion by MCD might represent a novel strategy for enhancing the ability of sperm to take up heterologous DNA. Mol. Reprod. Dev. 79: 853–860, 2012. © 2012 Wiley Periodicals, Inc.     

The associations among semen quality,oxidative DNA damage in human spermatozoa and concentrations of cadmium,lead and selenium in seminal plasma

To explore the associations among semen quality, oxidative DNA damage in human spermatozoa and concentrations of cadmium, lead and selenium in seminal plasma, 56 non-smoking subjects were asked to collect semen by masturbation into a sterile wide-mouth metal-free plastic container after 3 days of abstinence. The conventional semen parameters were analysed. The concentrations of Cd, Pb and Se in seminal plasma were detected using atomic absorption spectrophotometer. 8-OHdG levels in sperm DNA were measured using HPLC-EC. The results showed that the geometric mean concentrations of Cd, Pb and Se were 0.78, 7.8 and 51.4 microg/l, respectively. The geometric mean of 8-OHdG/10(6) dG was 51.4 (95% CI: 21.5-123.0). A significant inverse correlation exists between Cd and sperm density (r=-0.28, P<0.05), and between Cd and sperm number per ejaculum (r=-0.27, P<0.05). In contrast, there was a significantly positive correlation between Se and sperm density (r=0.50, P<0.01), between Se and sperm number (r=0.49, P<0.01), between Se and sperm motility (r=0.40, P<0.01), and between Se and sperm viability (r=0.38, P<0.01). No statistically significant correlation was observed between Pb and semen quality. A significant inverse correlation was observed between 8-OHdG and sperm density (r=-0.34, P<0.01), between 8-OHdG and sperm number per ejaculum (r=-0.30, P<0.01), and 8-OHdG and sperm viability (r=-0.24, P<0.05). 8-OHdG was significantly correlated with Cd in seminal plasma (r=0.55, P<0.01). A significant but weak positive correlation was found between 8-OHdG and Pb concentration in seminal plasma (r=0.28, P<0.05). In contract, a significant inverse correlation was observed between 8-OHdG and Se concentration in seminal plasma (r=-0.40, P<0.01). The results indicate that Cd in seminal plasma could affect semen quality and oxidative DNA damage in human spermatozoa. Se could protect against oxidative DNA damage in human sperm cells. Pb did not appear to have any association with the semen quality when concentration of Pb in seminal plasma was below 10 microg/l.     

Regulation of acrosomal exocytosis. II. The zona pellucida-induced acrosome reaction of bovine spermatozoa is controlled by extrinsic positive regulatory elements

The effects of accessory sex gland secretions on the zona pellucida-induced acrosome reaction of bovine spermatozoa were investigated. Soluble extracts of zonae pellucidae initiated exocytosis in ejaculated spermatozoa. This process had an ED50 of 20 ng/microliter zona pellucida protein and saturated at 50 ng/microliter (Florman and First, 1988. Dev. Biol. 128, 453-463). In epididymal sperm this dose-response relationship was shifted toward greater agonist concentrations by at least a factor of 10(3). Reconstitution of high potency agonist response was achieved in vitro by incubation of epididymal sperm with bovine seminal plasma. Reconstitution was dependent on the seminal plasma protein concentration. The ED50 of this process was 62 micrograms protein/10(8) sperm and saturation was observed with 124 micrograms protein/10(8) sperm. Agonist responses in reconstituted epididymal sperm and in ejaculated sperm were indistinguishable with regard to dependence on the zona pellucida protein concentration and the kinetics of induced acrosome reactions. Kinetic studies suggest that reconstitution is due to adsorption of regulatory factors from seminal plasma. In addition to the positive regulatory elements responsible for reconstituting activity, seminal plasma also contains negative regulatory elements which inhibit agonist response. These negative factors are inactivated during sperm capacitation, permitting the expression of positive regulators. Acting together, these regulatory elements could coordinate high affinity agonist response with the availability of eggs in vivo.     

Foreign DNA introduced into the vas deferens is gained by mammalian spermatozoa

The uptake of exogenous DNA by mouse and rat spermatozoa was analyzed using in vitro and in vivo methods. Two DNA constructs were used, one containing the Growth hormone (GH) gene and the other the c-myc oncogene linked to the αA-crystallin promoter (CPV-1 plasmid). For the in vitro approach, washed epididymal spermatozoa were incubated for 2 hr in the presence of linearized DNA. For in vivo experiments, DNA was injected into the proximal region of the vas deferens, and spermatozoa were recovered 6 hr later. In situ hybridization employing fluorescent markers and electron microscopy were used to localize the exogenous genes in spermatozoa. The precise localization of the foreign DNA in spermatozoa was visualized by tridimensional reconstructions using a confocal laser microscopy. Uptake of exogenous DNA occurred in 60–70% of the spermatozoa after in vitro or in vivo treatments. A positive signal was detected in the sperm nucleus and was not affected by DNase treatments. Incorporation of exogenous DNA was also evaluated by slot blot and PCR techniques using the DNA isolated from the sperm nuclei and the corresponding labelled probes. Comparison of a nucleotide sequence between the DNA isolated from in vivo treated spermatozoa and CPV-1 plasmid showed a 98.6% identity. These results show the in vivo capacity of spermatozoa to incorporate exogenous DNA, the ability of this DNA to reach the nucleus, and also demonstrate that epididymal and vas deferens secretions do not block these capacities. Mol. Reprod. Dev. 51:42–52, 1998. © 1998 Wiley-Liss, Inc.