The incorporation of macromolecules into the germ cells of male mice via electroporation and dimethyl sulfoxide

Electroporation (incorporation of macromolecules into the living cells by means of electric pulses) provides inclusion of plasmid 14C-DNA into immature cells of spermatogenic epithelium. The highest level of foreign DNA incorporation into spermatocytes and spermatids has been induced by 8kV electric pulses applied 3 times with 20 sec intervals. Meanwhile, mature sperms are found to be exclusively resistant to exogenous DNA irrespective of the voltage level, the number of pulses and Ca++ uptake (contents). Incubation of mature sperms for two hours in the medium with Ca++ (10 mM) and dimethylsulfoxide--(DMSO, 33%) provides highly reliable incorporation of plasmid 14C-DNA into sperm heads. The sperm cells with foreign DNA incorporated by means of Ca++ and DMSO treatment still remain alive and mobile. The possibilities of mature sperms loaded with foreign DNA for the creation of transgenic mammals are discussed.     

Immunocytochemical localization of actin in the sperm head of Talpa europaea (Insectivora)

Actin in the sperm head of Talpa europaea was observed by immunofluorescence and immunoelectron microscopy. The indirect immunofluorescence technique, using both anti-actin and DNase anti-DNase methods, showed a shining fluorescent band around the sperm head in some spermatozoa, whereas in others the fluorescence was found in the postacrosomal region. Since no labeling was detected in sperms treated with NBD-phallacidin, it is likely that mature mole sperms contain G-actin but not F-actin. The results of electron microscopy indicated the deposition of the anti-actin antibodies in two places in mole spermatozoa: the postacrosomal region and the nuclear segment of the acrosome. In the first case, the actin was localized in the space between the outer surface of the postacrosomal sheath and the plasma membrane; in the second one, the actin was localized in the space between the outer acrosomal membrane and the plasma membrane. The significance of the presence of actin and its role(s) during fertilization are discussed.     

Identification and distribution of actin in spermatogenic cells and spermatozoa of the rabbit

Using a monoclonal antibody as a highly specific probe and a seminal particle-free fraction of rabbit ejaculated spermatozoa, actin has been localized in the postacrosomal region of mature rabbit spermatozoa. The sperm actin has been extracted and identified on two-dimensional PAGE immunoblots as a single spot of pI = 5.45 and Mr = 43,000. Rabbit sperm actin is present in a nonfilamentous form and is not removed by removing the plasma membrane. Unlike mature spermatozoa, however, filamentous actin is present in spermatogenic cells, as determined by rhodamine phalloidin staining. Starting as diffusely distributed in spermatocytes, actin accumulates in the subacrosomal space and appears as a band in conjunction with the developing acrosome. This band lengthens throughout the spermatid stage and becomes continuous with the postacrosomal region staining in testicular spermatozoa. Actin may therefore function during spermatogenesis to both shape the acrosome to the nucleus and to anchor inner acrosomal membrane proteins.     

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.     

The transfer of foreign DNA by rabbit spermatozoa into hamster eggs

Capacitated rabbit sperm was incubated with pCMVlacZ plasmid and then used to fertilize hamster oocytes liberated from zona pellucida. After treatment with DNase I, these oocytes were examined for the presence of exogenous DNA by PCR. DNA of pCMVlacZ was not found in oocytes after simple incubation with male gametes. The presence of DNA was recorded in the experiments where the sperms were treated with DMSO and subjected to heat shock prior to fertilization.     

Actin polymerization in the equatorial and postacrosomal regions of guinea pig spermatozoa during the acrosome reaction is regulated by G proteins

The acrosome reaction (AR) is an exocytotic process of spermatozoa, and an absolute requirement for fertilization. During AR, actin polymerization is necessary in the equatorial and postacrosomal regions of guinea pig sperm for spermatozoa incorporation deep into the egg cytoplasm, but not for plasma membrane (PM) fusion nor the early steps of egg activation. To identify the mechanisms involved in this sperm actin polymerization, we searched for the protein members, known to be involved in a highly conserved model, that may apply to any cellular process in which de novo actin polymerization occurs from G protein activation. WASP, Arp 2/3, profilins I and II, and Cdc42, RhoA and RhoB GTPases were localized by indirect immunofluorescence (IIF) in guinea pig spermatozoa and their presence corroborated by Western blotting. WASP and profilin II were translocated to the postacrosomal region (Arp2/3 already were there) in long-term capacitated and acrosome-reacted spermatozoa, at the same time as actin polymerization occurred. These events were inhibited by GDP-beta-S and promoted by lysophosphatidic acid (LPA) and GTP-gamma-S, a small GTPase inhibitor and two activators, respectively. By immunoprecipitation, Cdc42-WASp association was identified in capacitated but not in noncapacitated gametes. Polymerized actin in the postacrosomal region is apparently anchored both to the postacrosomal perinuclear theca region and the overlying PM. Results suggest that GTPases are involved in sperm actin polymerization, in the postacrosomal region and the mechanism for polymerization might fit a previously proposed model (Mullins, 2000: Curr Opin Cell Biol 12:91-96).     

Uptake of exogenous DNA by mammalian spermatozoa: specific localization of DNA on sperm heads.

When mouse spermatozoa were briefly exposed in culture to radioactively labelled DNA (pSV2CAT plasmid), radioactivity could be detected by high-resolution autoradiography on the surface and within the nucleus of the spermatozoa. Spermatozoa from other mammalian species (boar, bull, man) could also bind foreign DNA. With the exclusion of human spermatozoa, which in most experiments showed very low labelling values, labelling percentages (evaluated by light microscope autoradiography) ranged between 39 and 78%. In all four species the DNA-binding ability was mainly confined to a specific region of the sperm head (equatorial segment and postacrosomal region), and the sperm-DNA association kinetics were rapid (maximum values were reached within 20-40 min). The data also indicate that factor(s) in seminal plasma might protect spermatozoa from accidental transfection by foreign DNA that may be present in the genital tracts from bacterial or viral sources.     

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。     

A monoclonal antibody, MN13, that recognizes specifically a novel substance between the postacrosomal sheath and the overlying plasma membrane in the mammalian sperm head

Monoclonal antibody MN13 raised against mouse spermatozoa specifically recognizes the postacrosomal region of the sperm head in several mammalian species. Colloidal gold-immunoelectron microscopy of demembranated mouse spermatozoa indicated that the antigen is associated with the outer layer of the periodic substructure apparently linking the postacrosomal sheath to the overlying plasma membrane. The antigen recognized by MN13 may contribute to the intimate association of the postacrosomal sheath with the overlying plasma membrane.     

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.     

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.     

Use of liposomes for the association of foreign genetic material to spermatozoa]

The binding of liposomes of different composition with sperm cells and their effect on cell behaviour were studied. Experiments have demonstrated, that liposomes, containing positively charged phospholipids, had maximal degree of association with sperm cells. Besides, liposomes seem to be nontoxic for sperms and do not change the functional activity of the latter. Theoretically, sperm cells may serve as a natural vector for transfer of liposome-encapsulated DNA into the ovary cells of animals for obtaining transgenic animals.     

Distribution of filipin-sterol complexes in the plasma membrane of stallion spermatozoa during the epididymal maturation process

The presence and distribution of cholesterol in mature and immature epididymal spermatozoa was analyzed using filipin as a cytochemical tool in freeze-fracture replicas and thin section preparations. The polyenic-antibiotic filipin formed complexes with 3, beta -OH sterols, producing characteristic protrusions, or pits, that were heterogeneously distributed in the plasma membrane of stallion spermatozoa, revealing a specific organization in a functionally specialized area of the gamete. The acrosomal region of the sperm head presented a significantly higher density of filipin sterol complexes than the postacrosomal region, which was usually free of these complexes. The plasma membrane of the flagellum also showed filipin sterol complexes randomly distributed in freeze-fracture replicas. The strong filipin labeling observed in the membrane of spermatozoa obtained from the caput region of the epididymis decreased significantly during epididymal passage. The significance of these changes is not completely understood, but they might contribute to establishing the molecular organization necessary for sperm transit and storage in the epididymis as well as to development of motile spermatozoa that are able to fertilize the oocyte and induce normal embryonic development.     

Comparative scanning electron microscope study of boar,bull and ram spermatozoa

The comparative ultrastructure of ejaculated boar, bull and ram spermatozoa is studied by scanning electron microscopy. After washing, the spermatozoa are fixed in glutaraldehyde or im picric acid-formaldehyde-glutaraldehyde mixture. Samples are prepared either by critical point drying (Freon) on Millipore filters or by air drying on glass cover slips. In all the species studied, three regions may be distinguished in the paddle-shaped head of the sperm: an anterior segment (surrounded by the marginal thickening) and an equatorial segment constituting together the acrosome, and the postacrosomal region. Most of the feature of the postacrosomal lamina described in transmission electron microscopy are visible through the plasma membrane, particularly after air drying. The surface morphology of the neck and of the different segments of the flagellum is also evident. Some species differences are encountered, e.g. rough surface of acrosome and absence of serrations in postacrosomal lamina of boar spermatozoa only. The techniques employed result in good general morphology and fine resolution of surface detail of the sperm samples; they also permit analysis of spermatozoa treated by freezing or submitted to acrosomal extraction.     

Prolactin signaling through the short isoform of the mouse prolactin receptor regulates DNA binding of specific transcription factors, often with opposite effects in different reproductive issues

Background  

Externalization of phosphatidylserine (EPS) occurs in apoptotic-like spermatozoa and could be used to remove them from sperm preparations to enhance sperm quality for assisted medical procreation. We first characterized EPS in sperms from infertile patients in terms of frequency of EPS spermatozoa as well as localization of phosphatidylserine (PS) on spermatozoa. Subsequently, we determined the impact of depleting EPS spermatozoa on sperm quality.     

Properties and function of caltrin, the calcium-transport inhibitor of bull seminal plasma

Indirect immunofluorescence studies with polyclonal antibodies show that caltrin binds to the plasma membrane over the acrosome and principal tail regions of bovine spermatozoa but not to the postacrosomal area or the midpiece. Calcium influx into bovine epididymal spermatozoa maintained in a simple salt medium containing DL-beta-hydroxybutyrate is prevented by caltrin freshly prepared from bovine seminal plasma through a procedure employing only gel permeation columns. Older preparations, on the other hand, enhance calcium uptake into these cells. Caltrin freshly prepared through a purification scheme that includes a cation exchanger only induces enhancement of calcium uptake into bovine epididymal spermatozoa maintained under identical conditions. It is postulated that early during sperm transit through the female reproductive tract, caltrin bound to the sperm plasma membrane protects the sperm cells from calcium influx. As the cells enter the oviduct where meeting with the egg could take place, factors present in the surrounding milieu may cause caltrin to change from an inhibitor to an enhancer of calcium uptake. The acrosome reaction and possibly hyperactivation, two components of capacitation that require calcium influx as an initial event, then take place.     

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.     

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.     

Diffusion barriers in ram and boar sperm plasma membranes: directionality of lipid diffusion across the posterior ring

The plasma membrane of mammalian spermatozoa, like that of other differentiated cells, is compartmentalized into discrete regions or domains that are biochemically and functionally distinct from one another. Physical structures within the membrane, such as the posterior ring at the juncture of the sperm head and tail, have long been thought to act as diffusion barriers to help segregate important molecules required for fertilization within specific domains and to regulate migration of molecules between domains. In this investigation, we used a quantitative photobleaching technique (video-FRAP) to assess the efficacy of the posterior ring as a barrier to exchange of lipids between the postacrosomal and midpiece plasma membranes. A lipid reporter probe (1,1'-diduodecyl-3,3,3', 3'-tetramethylindocarbocyanine; DiIC(12)) was incorporated into the plasma membrane of live ram and boar spermatozoa, and the directionality of its diffusion across the posterior ring was measured by line-profile analysis. Results showed that DiIC(12) was able to traverse the posterior ring from the direction of the postacrosomal plasma membrane and to diffuse onto the midpiece plasma membrane. These results suggest that the posterior ring is not an immutable barrier to lipid exchange in mature spermatozoa and that there are other mechanisms for maintaining in-plane lipid asymmetry, such as differential phase behavior and interaction with the submembranous cytoskeleton.     

Study of the transfer of foreign genes into mussel Mytilus Galloprovincialis Lam. eggs by spermatozoa

The possibility of transferring exogenous DNA into eggs by mussel Mytilus galloprovincialis Lam. sperms both with the use of certain methods of transfection and without them was studied. The efficacy of egg fertilization by sperms treated with foreign DNA and the development of larvae at early stages of embryogenesis were evaluated. Negative effects of the contact between mussel sperms and exogenous DNA on fertilization and subsequent development were noted. The proportion of developing larvae decreased with increasing DNA concentration and sperm exposure. Transfer of plasmids pCMVlacZ and pMTbGH into eggs was observed in group crosses. With the use of PCR, foreign DNA sequences were found in the larvae at the stage of veliger 48 h after fertilization. An intense signal was recorded after sperm electroporation in 10% DMSO.