In-vivo gene transfer induces transgene expression in cells and secretions of the mouse cauda epididymis.

Mouse cauda epididymis were in-vivo transfected using the lipid FuGENE 6 as gene vector. Two gene constructions were employed: the p-GeneGRIP which codifies for the Green Fluorescent Protein (GFP) and the pSEAP-control that expresses an alkaline phosphatase as a secretion. Transfection was detected by fluorescence and appeared in the nucleus and cytoplasm of epithelial cells. Transfection was observed in 39.70% of cells after 2 days and in 31.77% after 7 days, and then diminished progressively. Moreover, the presence of the transgene in the DNA isolated from treated epididymides was observed by polymerase chain reaction. GFP gene expression appeared in large areas of the cauda epididymis and it was observed exclusively in the cytoplasm of epithelial cells. GFP gene expression occurred during 2 weeks after gene injection and occupied 32.24, 29.98 and 22.37% of the area of the tubules when analyzed 2, 7 and 15 days after gene injection. The cauda was also analyzed in toto and showed similar results. The use of the pSEAP-control gene showed that cauda epididymis secretions can also be modified by the transfection procedure. A significant increase of alkaline phosphatase activity appeared in the epididymal fluids 7 days after gene injection. These results indicate that transfection procedures could be an important tool in the future to study epididymal physiology or to change the fertilizing ability of spermatozoa.

Age-induced apoptosis in the male genital tract of the mouse.

We have examined the effects of ageing on the increase in apoptotic cells numbers in the male genital tract of the house mouse (Mus musculus). We have found that not all organs have the same response. There is an induction of apoptosis in both the epididymis and ventral prostate. However, seminal vesicles and other prostatic lobes remain unaffected. Apoptosis was assessed by several methods: TUNEL, detection of the active fragment of caspase-3 and the pattern of DNA fragmentation on agarose gels. This increase in apoptosis is related to the fall in testosterone levels, although there is only a partial decrease in androgen receptor (AR). AR is still present in all tissues and only moderately reduced in the epididymis and ventral prostate. A more intense increase of lipofuscin granules, which may be indicative of oxidative stress, occurred in these tissues. Finally, testosterone supplementation reverses the changes (both in apoptosis and lipofuscin content in the tissue), suggesting a role of androgens in these processes.

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.

Phosphatidylinositol-3 kinase acts in parallel to the ERK MAP kinase in the FGF pathway during Xenopus mesoderm induction.

Phosphoinositide 3-kinases (PI3Ks) are lipid kinases that can phosphorylate phosphaditylinositides leading to the cell type-specific regulation of intracellular protein kinases. PI3Ks are involved in a wide variety of cellular events including mitogenic signalling, regulation of growth and survival, vesicular trafficking, and control of the cytoskeleton. Some of these enzymes also act downstream of receptor tyrosine kinases or G-protein-coupled receptors. Using two strategies to inhibit PI3K signalling in embryos, we have analysed the role of PI3Ks during early Xenopus development. We find that a class 1A PI3K catalytic activity is required for the definition of trunk mesoderm during the blastula stages, but is less important for endoderm and prechordal plate mesoderm induction or for organiser formation. It is required in the FGF signalling pathway downstream of Ras and in parallel to the extracellular signal-regulated kinase (ERK) MAP kinases. In addition, our results show that ERKs and PI3Ks can synergise to convert ectoderm into mesoderm. These data provide the first evidence that class 1 PI3Ks are required for a specific set of patterning events in vertebrate embryos. Furthermore, they bring new insight into the FGF signalling cascade in Xenopus.

Transfection of mouse eggs and embryos using DNA combined to cationic liposomes.

We have used plasmid DNA in combination with cationic liposomes to transfect mouse eggs and embryos. The plasmid was rhodamine labeled, which allowed a direct visualization of the DNA uptake by the cells. Immature eggs, collected from the ovaries, were easily transfected, but once the egg was ovulated the zona pellucida (ZP) acted as a barrier and prevented transfection. Permeabilization or removal of the ZP was therefore a requirement to allow transfection. Transfected eggs were capable of being fertilized in vitro giving raise to embryos that expressed the recombinant protein. Morulae and blastocysts were also transfected when the ZP was permeabilized, but the efficiency of transfection decreased and in some cases not all the blastomeres incorporated the plasmid. Pronuclear embryos were cultured and showed expression of the transgene from the 2-cell stage. This indicates that liposome-transfection of oocytes or pronuclear embryos could be a simple and suitable method to introduce foreign genes in embryos and perhaps could be also useful to generate transgenic animals.

CD52 mRNA is modulated by androgens and temperature in epididymal cell cultures.

Cultured rat epididymal tissue explants formed >90% pure, adherent growing epithelial cell monolayers. Despite their flattened and apparently androgen receptor-negative phenotype, these cells for a short period kept characteristics of the epididymal duct epithelium, i.e., expression of the tissue-specific marker CD52 and responsiveness of its mRNA toward temperature elevation and androgen withdrawal. When cells were grown on permeable supports at 33 degrees C, androgen supplementation or withdrawal specifically modulated the levels as well as the length of the CD52 mRNA. Elevation of the culture temperature to a quasi abdominal milieu of 37 degrees C selectively reduced the CD52 mRNA levels under all culture conditions. This reduction was not affected by the presence of androgens and was not accompanied by changes in length, suggesting that the modulation of CD52 mRNA in epididymal cells by androgens and by temperature is synergic, but may involve different molecular mechanisms. CD52 mRNA levels, however, were not stable in the primary cultures but decreased rapidly to undetectable levels after 4-5 days at all culture conditions. GAPDH mRNA levels, on the other hand, were stable throughout the culture period.

Effect of antibodies against seminal vesicle secretion on fertility in the rat.

Fertile female Wistar rats were immunised against rat and mouse seminal vesicle secretion (SVS) to test the production of allo-antibodies and the effect of the antibodies elicited on fertility. Twenty-six per cent of the rat and mouse SVS-immunised females were infertile after the treatment. The sera were titrated by ELISA and used in Western blots to detect the proteins recognised. Although neither the antibody titres nor the proteins recognised by the sera showed a close relation with the degree of fertility, in all females the highest antibody titre in the fluids from the genital tract was found in the oviductal fluid and during the night of oestrus. This fact suggested that the site of action of the antibody could be the oviduct. Similar results were obtained using mouse SVS as immunogen--a fact that can be related to the antigenic similarity between the SVS of the two species. The antibodies react with the spermatozoa but not with eggs or embryos. Analyses performed on embryos collected from sterile females showed that there was a delay in fertilisation and normal embryogenesis. Our results suggest that SVS proteins are antigenic and that these antigens are bound to the spermatozoa and could take part in early pre-fertilisation events such as capacitation or sperm transport.

Binding of seminal vesicle proteins to the plasma membrane of rat spermatozoa in vivo and in vitro.

The association of seminal vesicle (SV) proteins with rat spermatozoa has been studied in vivo and in vitro. SV proteins bind to the sperm plasma membrane after ejaculation but are removed progressively from the sperm plasma membrane in the female genital tract. Although some of these remain bound to spermatozoa when they reach the oviducts, they do not seem to be present at the time of fertilization. This could indicate a putative role for these SV proteins in pre-fertilization events. In addition, the binding of SV antigens was studied in vitro. It was observed that the ability to bind SV proteins is gained by the spermatozoa during epididymal maturation, and is first detectable in spermatozoa collected from the cauda epididymis. On the other hand, the binding is regulated by other proteins present in the ejaculate which are secreted by the coagulating glands. Experiments also showed that mouse spermatozoa are able to bind rat SV proteins, indicating that the binding is not a highly species-specific phenomenon.

Treatment of human spermatozoa with seminal plasma inhibits protein tyrosine phosphorylation.

It has long been known that seminal plasma contains factors that influence the fertilizing capacity of spermatozoa in many different ways. However, little is understood of the biochemical cascades triggered when spermatozoa and seminal plasma interact. In this study, we examined how incubation with seminal plasma affected protein tyrosine phosphorylation in human spermatozoa. Increased protein tyrosine phosphorylation is a hallmark of sperm capacitation in several mammalian species, including human. Seminal plasma blocks protein tyrosine phosphorylation when added to washed, non-capacitated spermatozoa. Removal of seminal plasma and incubation in capacitating medium led to partial recovery of the tyrosine phosphorylation cascade. Addition of seminal plasma to a suspension of spermatozoa previously incubated for 5 h under capacitating conditions decreased the level of tyrosine phosphorylation on all proteins in a dose-dependent manner. In this case, the phosphotyrosine signal did not increase upon removal of seminal plasma followed by overnight incubation in fresh capacitating media, indicating that removal of seminal plasma was necessary but not sufficient for protein tyrosine phosphorylation to occur. These results indicate that human seminal plasma contains factors that influence the tyrosine phosphorylation status of human spermatozoa.

Molecules involved in mammalian sperm-egg interaction.

To achieve fertilization, sperm and egg are equipped with specific molecules which mediate the steps of gamete interaction. In mammals, the first interaction between sperm and egg occurs at an egg-specific extracellular matrix, the zona pellucida (zp). The three glycoproteins, ZP1, ZP2, and ZP3, that comprise the zp have been characterized from many species and assigned different roles in gamete interaction. A large number of candidate-binding partners for the zp proteins have been described; a subset of these have been characterized structurally and functionally. Galactosyltransferase, sp56, zona receptor kinase, and spermadhesins are thought to participate in the primary binding between sperm and zp and may initiate the exocytotic release of hydrolytic enzymes in the sperm head, the acrosome reaction. Digestion of the zp by these enzymes enables sperm to traverse the zp, at which time the proteins PH20, proacrosin, sp38, and Sp17 are thought to participate in secondary binding between the acrosome-reacted sperm and zp. Once through the zp, sperm and egg plasma membranes meet and fuse in a process reported to involve the egg integrin alpha 6 beta 1 and the sperm proteins DE and fertilin. These molecules and the processes involved in gamete interaction are reviewed in this chapter within a physiological context.

Regulation of mouse epididymal epithelium in vitro by androgens, temperature and fibroblasts.

The epididymal epithelium provides the microenvironment for sperm maturation. However, the molecular basis of epididymal function is still poorly understood because of the limitations of in vivo systems. For this reason, we have developed an in vitro culture system for mouse epididymal epithelial cells. Cells were purified by enzymatic digestion and centrifugation through a Percoll gradient, and plated on inserts coated with a replacement basement membrane. Cultured cells maintained ultrastructural and immunocytochemical features of epithelia, but did not retain the androgen responsiveness of epididymal cells (as judged by androgen receptor detection and secretion of specific markers) unless cocultured with fibroblasts. The androgen receptor was detected in the nuclei of epididymal epithelial cells only when grown with epididymal fibroblasts in the subjacent chamber. Moreover, specific epididymal secretory proteins were secreted only when epithelial cells were cultured in the presence of both androgens and fibroblasts at 32 degrees C. These results highlight the importance of cell-cell interaction, as well as temperature regulation in the physiology of the epididymis. They also establish the existence of two independent pathways in the differentiation of these cells. The first, leading to the expression of epithelial characteristics, is fibroblast-independent, whereas the second, conferring tissue-specific features, depends upon coculture with fibroblasts.

Fate and distribution of seminal plasma proteins in the genital tract of the female rat after natural mating.

This paper describes the distribution and fate of seminal plasma proteins in the rat female genital tract after insemination, using immunological detection in tissue sections and in fluids collected from different regions. The localization of seminal plasma proteins in the uterus and the vagina correlated with that of spermatozoa, suggesting that passive transport mechanisms operate in these regions. No seminal plasma proteins were detected in the oviduct, indicating that their presence is probably restricted to the uterine environment. Possible mechanisms for eliminating seminal plasma molecules after copulation include leakage from the uterus after relaxation of the cervical muscles and endocytosis by the endometrial cells. Large amounts of both vesicular and coagulating gland proteins were detected in the vagina of females at the time of cervical relaxation, indicating that the first mechanism of leakage from the uterus after cervical relaxation operates. Immunocytochemical procedures were used and seminal vesicle antigens were detected inside uterine epithelial cells, which indicates that endocytosis is also a mechanism for elimination of these molecules after copulation. Western blot results suggest proteolytic cleavage as a third mechanism. However, coagulating gland antigens are neither endocytosed nor cleaved, and their elimination takes place only by backflow to the vagina. The seminal plasma distribution in experimental situations in which sperm transport is altered was also studied. The implications of our findings for mechanisms of sperm transport in the female are discussed.

Photoperiod-induced changes in the proteins secreted by the male genital tract of the rodent Octodon degus.

The proteins secreted by the male genital tract were analyzed in the seasonally breeding rodent Octodon degus. The protein patterns from the fluids collected from sexually active animals were compared with those from animals in resting period, with others which were previously castrated, and with castrated animals which received testosterone replacement treatment. Fluids from cauda epididymides (CE), seminal vesicles (SV) and prostate glands (PG) were collected, and analyzed by polyacrylamide gel electrophoresis followed by different staining methods and densitometry. Modifications were detected in the protein patterns of resting or castrated animals. In CE fluid, the decrease of one protein band (45 Kda) and the uprising of another (210 Kda) were recognized after castration. In animals during resting period the changes were not as marked as in castrated animals. SV secretion demonstrated a similar response to resting phase and castration, because Protein SVS I (200 Kda) decreased or were not observed when these conditions occurred. PG fluid proteins were also modified after castration. In general, the more severe changes in the protein spectrum were induced by castration, despite radioimmunoassay showing that testosterone fall is even higher in resting period animals than in those castrated. Testosterone replacement resulted in recovery of a protein profile which is very similar to that of sexually active males. Results suggest that the androgenic control of male tract secretions would be rather different in this seasonal hystrichomorph when compared to the regulation system described for myomorph rodents.

Interaction of a tyrosine kinase from human sperm with the zona pellucida at fertilization.

A 95-kilodalton mouse sperm protein with characteristics of a protein tyrosine kinase has been identified as a receptor for ZP3, a glycoprotein in the egg's extracellular matrix. The structure of the human homolog was determined by screening an expression library from human testis; a testis-specific complementary DNA was isolated that encodes a protein similar to receptor tyrosine kinases and appears to be expressed only in testicular germ cells. Antibodies against a synthetic peptide from the intracellular domain recognized a 95-kilodalton human sperm protein that contains phosphotyrosine; human ZP3 stimulates the kinase activity of this sperm protein. Synthetic peptides corresponding to regions of the predicted extracellular domain inhibited sperm binding to human zona pellucida. Availability of the primary sequence of a receptor for ZP3 provides a rational starting point for sperm-targeted contraceptive development.

Structure of the vaginal plugs generated by normal rats and by rats with partially removed seminal vesicles.

Normal male rats generate vaginal plugs that appear to be firmly apposed to the vagino-cervical junction and permit a large number of spermatozoa to reach the uterus. A few spermatozoa form entangled masses inside these plugs, as revealed by light microscopy. Males in which the seminal vesicles have been partially removed produce plugs that are smaller and softer than those generated by normal males, and the plugs display a cup-like structure at the proximal end. The cup-like structure is completely filled with spermatozoa that exhibit a characteristic arrangement in relation to the plug material. In this situation, the number of spermatozoa that reach the uterus is very much reduced. Experiments were also carried out to explore the restoration of sperm transport by addition of a vaginal plug. Such experiments involved successive matings of individual females with a seminal vesicle-deprived male and with a vasectomized male (which generated the plug) and also the intravaginal injection of seminal vesicle secretions after mating with a seminal vesicle-deprived male. In none of the experimental situations was transport of spermatozoa to the uterus restored, and the plug consisted of a large quantity of trapped spermatozoa inside a mass of coagulated proteins. The results suggest that the structure of the plug depends on the amount of seminal vesicle secretion present in the ejaculate and that the vaginal plug must be formed immediately after deposition of the sperm if spermatozoa are to reach the uterus.

Role of fluid from seminal vesicles and coagulating glands in sperm transport into the uterus and fertility in rats.

The relationship between the quantity of seminal vesicle secretion in the ejaculate, the percentage of spermatozoa reaching the uterus and fertility was studied in rats. Different portions of seminal vesicles were removed from male rats; 15 min after coitus (day 0), the numbers of spermatozoa in the uterus and vagina were counted and the vaginal plug characteristics were noted. Fertility was evaluated by the number of fetuses on day 14. A gradual decrease in the percentage of spermatozoa in the uterus was positively related to the reduction in seminal vesicle secretion, estimated by plug weight. This decline was not caused by a delay in sperm transport to the uterine lumen and the results suggested that the spermatozoa that fail to enter the uterus in the first minutes after coitus never enter. The vaginal plug weight, which is related to the seminal vesicle weight, and the position of the plug, which must be firmly lodged into the cervical opening, seem to be the most important conditions for promoting the rapid passage of spermatozoa into the uterus. When the seminal vesicles were partially removed, the plug was not tightly lodged and formed a 'cup' filled with spermatozoa. The number of fetuses did not show a close correlation with the quantity of seminal vesicle secretion. Studies of males in which the seminal vesicles had been removed indicated that a normal number of fetuses can be obtained despite low numbers of spermatozoa reaching the uterus. Ablation of the coagulating glands showed that, when there is no vaginal plug, no spermatozoa reach the uterus and fertility is suppressed. Nevertheless, the complete removal of coagulating glands is difficult; when small portions of these glands remain, the vaginal plug is formed and then fertility is achieved.

Electrophoretic pattern of rodent seminal vesicle proteins as revealed by silver staining.

Electrophoresis of seminal vesicle secretions (SVS) from several rodents showed a very simple pattern composed of 3-5 main protein bands when an anionic dye (Coomassie brilliant blue) was used. However, use of a silver staining method showed a more complex protein spectrum, and several minor components of 12-90 kD, were clearly revealed. Western blotting using antibodies to SVS demonstrated that these minor protein components were not serum contaminants. Rat, mouse and hamster SVS shared antigenic determinants which were not related to rabbit SVS.

Effects of melatonin on gonadal steroids and glucose plasma levels in frogs (Rana perezi and Rana temporaria).

The effect of melatonin treatment on the Gonosomatic Index (GSI), ovarian germinal epithelium, plasma estradiol and testosterone levels was studied in Rana perezi females in December. No significant changes were observed in GSI, estradiol, and testosterone levels in melatonin treated animals when compared with saline injected controls, but the percentage of previtellogenic follicles decreased in animals treated with melatonin (100 micrograms). The effect of melatonin treatment on glucose level was studied in Rana perezi females in December and Rana temporaria males in February. In Rana perezi no significant differences were observed between melatonin treated and control animals; however, significant reductions by melatonin treatment were obtained in Rana temporaria. The possibility that the inhibitory effect of melatonin can be observed only when gonadal function and metabolism are stimulated by temperature is discussed.

Comparative analysis of the neck region in spermatids and spermatozoa of some orthopteran insects.

The ultrastructure of the neck region and the participation of noncovalent and disulphide bonds in the head-tail attachment were analyzed in spermatids and spermatozoa of some orthopteran species from the families Tettigoniidae and Acrididae. This study combined conventional electron microscopy with cytochemical procedures to detect acidic proteins and lysine-rich basic proteins, and with treatments using the disruptive agents sodium dodecyl sulfate (SDS) and dithiothreitol (DTT). The organization of the neck region differs in spermatozoa among species in the families analyzed. In Acrididae the neck consists of the beginning of the axoneme attached to the nuclear envelope and surrounded by the centriolar adjunct material. Tettigoniidae species possess a complex organization in the form of a laminar "connective piece," which shows a high content of lysine-rich and acidic proteins. Experiments using SDS and DTT demonstrate that in Acrididae the head-tail connection is strong, with both noncovalent and disulphide bonds important in maintenance of the attachment. In Tettigoniidae, however, despite the presence of a morphologically well organized connective piece, the head-tail attachment is biochemically labile in comparison to that of Acrididae. The relationships between the morphology of these neck structures and their stability mediated by noncovalent and/or disulphide bonds are discussed.