Biomedical subjects

P Esponda

Publications and source records attributed to P Esponda.

At least 19 recordsLinked to original sources

PubMed2004

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.

Aging↗

PubMed2004

Postovulatory ageing induces structural changes in the mouse zona pellucida.

The structure of the zona pellucida (ZP) was analyzed in mouse oocytes collected soon after ovulation and in others retrieved 20 h after. The conventional methods for electron microscopy and the alcoholic PTA staining procedure which preferentially contrasts lysine-rich proteins were employed. The ZP of aged oocytes showed several structural changes which were particularly observed after using the PTA procedure. In 82.14% of the aged oocytes the ZP appeared clearly composed of two different regions: an inner dense and an outer of low density. The ZP showed a fibrillar banded structure with a parallel arrangement of fibrillar threads in both the outer and inner regions. The in vitro fertilization analysis showed that only 16.85% of the aged gametes attained the two cell embryo stage in comparision to 66.93% shown by the freshly ovulated eggs. The non-fertilized oocytes showed that no sperm penetration through the ZP occurred.

Aging↗

PubMed2001

The Drosophila melanogaster X-linked mfs(1)6E locus is required for production of normal seminal fluid by the male accessory glands.

The Drosophila male accessory glands (paragonias) are two male-specific organs that produce seminal fluid, a secretion involved in sperm storage and subsequent sperm utilization by the female. This paper reports the first X-linked locus, male-female-sterile in region 6E [mfs(1)6E], required for the production of normal seminal fluid. Mutant males produce motile spermatozoa, which are transferred to females during mating, but which are not stored. Sterility of these males is mainly due to severe affected transfer of seminal fluid to females during mating. In addition, the mutant seminal fluid seems defective in triggering the behavioral (reduced receptivity to further mating) and physiological (increased egg-laying) changes normally observed in mated females. Mutant male accessory glands show notable abnormalities, connected with glandular secretion as well as qualitative and quantitative differences in their protein content.

Animals↗

PubMed2001

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.

Animals↗

PubMed2000

Generation of genetically modified mice by spermatozoa transfection in vivo: preliminary results.

Mouse vas deferens were injected with a plasmid DNA encoding the GFP (Green Fluorescent Protein). The night after injection males were mated with normal oestrus females, and the offspring were analyzed. From 53 newborns, 4 were found positive by PCR for the GFP gene. In these positive animals, some tissues showed expression for GFP as evidenced by a strong green cytoplasmic fluorescence. GFP expression was particularly patent in the liver (hepatocytes), kidney (renal corpuscle and tubules), abdominal wall, and lung. These preliminary results indicate the possibility to use this method as a simple alternative procedure to create transgenic animals, and it could be especially helpful in species in which the microinjection procedure is not feasible.

Animals↗

PubMed2000

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.

Animals↗

PubMed2000

In-vivo transfection of the female reproductive tract epithelium.

Mouse female genital tract was transfected in vivo using the ss-galactosidase reporter gene. To transfect the female tract, DNA/liposome complexes were injected through the infundibulum of the oviducts of adult, immature, and pseudopregnant females. Females which were in different stages of the ovarian cycle were also employed. Transfection was analysed using histochemical, immunological and molecular (Southern blotting, polymerase chain reaction and gene sequencing) procedures. The lower region of the uterine glands and the oviduct epithelium in the isthmus and juncture regions were the most conspicuous transfected areas. The greatest numbers of transfected cells were 6% in the oviduct and 9% in the uterus, meanwhile the duration of expression reached a maximum of 7 days in the oviduct and 14 days in the uterus. The hormonal stage of the genital tract epithelium directly affected transfection, as the highest number of successful transfections occurred during the meta-oestrus and pseudopregnancy stages.

Age Factors↗

PubMed1999

Structure, cytoskeleton, and development of the acrosome of Platycleis albopunctata (Orthoptera: Tettigoniidae).

The acrosome of Platycleis albopunctata (Orthoptera: Tettigoniidae) is relatively large and complex, consisting of an apical vesicle and two large wing-like extensions that give the spermatozoon the shape of an arrow. The wings have actin microfilaments and microtubules and are covered with a noticeable extracellular material. Actin filaments are present in the acrosome when it first appears in spermatid stages. The acrosome and the acrosomal attachment to the nucleus are more resistant than other structures to the reducing agents DTT and SDS. At the end of spermiogenesis, groups of spermatozoa juxtapose their sperm heads and become joined to form a spermatodesm encircled by an amorphous material. Treatment with the ionophore A23187 rapidly disrupted acrosomes of the free gametes, but acrosomes from spermatozoa contained in the spermatodesm were not disassembled. Packaging of sperm in a spermatodesm appears to protect the acrosome.

Acrosome↗

PubMed1999

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.

Animals↗

PubMed1998

Acrosomal status of mouse spermatozoa in the oviductal isthmus.

The principal purpose of this study was to establish the acrosomal status of mouse spermatozoa stored in the isthmus of the oviduct after natural mating. Scanning electron microscopy of oviducts fixed about 6 hr before the estimated ovulation time showed numerous acrosome-intact spermatozoa attached to the mucosal surface of the oviduct, or trapped in the oviductal crypts. Nevertheless, an unambiguous assessment of the state of the acrosome requires transmission electron microscopy. Using this method, it was observed that the acrosome was intact in the 81% of spermatozoa attached to the mucosal surface but in only 31% of spermatozoa that were free in the lumen. Most of the free spermatozoa showed swelling or disruption of the acrosome. This result might indicate that the in vivo spermatozoon-oviductal mucosa interaction maintains the acrosome intact. Alternatively, it could mean that only ejaculated spermatozoa with a normal acrosome can establish such a mucosal relationship.

Acrosome↗

PubMed1998

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 alphaA-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.

Animals↗

PubMed1998

In vivo gene transfer to the mouse oviduct epithelium.

OBJECTIVE: To transfect the mouse oviduct in vivo. DESIGN: Prospective study. SETTING: Research laboratory. ANIMAL(S): Sixteen female Swiss albino mice. INTERVENTION(S): The left oviduct of 10 female mice was instilled with a liposome DNA solution. In addition, 2 control mice received liposome solution, 2 received phosphate-buffered saline solution, and 2 received no injection. MAIN OUTCOME MEASURE(S): The expression of the gene transfected (beta-galactosidase) was detected in the oviduct epithelium with the use of a routine histochemical analysis. RESULT(S): The 90% of the female mice that were transfected with liposome/beta-Gal complexes expressed the gene in the oviduct mucosa. The controls did not show beta-Gal expression. CONCLUSION(S): Cationic liposome/DNA complexes can be used for in vivo transfection of the mouse fallopian tubes. The foreign gene expression occurs in clusters of cells located along the mucosa of the isthmus and juncture regions.

Animals↗

PubMed1998

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.

Animals↗

PubMed1997

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.

Animals↗

PubMed1996

Immunocytochemical localisation of the nucleolar protein fibrillarin and RNA polymerase I during mouse early embryogenesis.

We have employed immunocytochemical procedures to localise the nucleolar protein fibrillarin and the enzyme RNA polymerase I in the numerous dense fibrillar bodies (nucleolar precursor bodies) which appear in the nuclei of mammalian early embryos. The aim of this study was to search for relationships between the localisation of these proteins, the changes in the structure of the nucleolar precursor bodies and the resumption of rRNA gene transcription during mouse early embryogenesis. Three human autoimmune sera which recognised fibrillarin and a rabbit antiserum created against RNA polymerase I were employed for fluorescence and electron microscopic immunocytochemical assays. A statistical analysis was also applied. Immunocytochemistry revealed that fibrillarin and RNA polymerase I showed the same localisation in the nucleolar precursor bodies. These proteins were immunolocalised only from the late 2-cell stage onward. Fibrillarin was initially detected at the periphery of the nucleolar precursor bodies and the labelling gradually increased until the morula and blastocyst stages, where normally active nucleoli are found. The pattern of increase of fibrillarin during early embryogenesis shows a parallelism with the rise in rRNA gene transcription occurring during these embryonic stages, and a possible correlation between these two phenomena is suggested. Results demonstrated that nucleolar precursor bodies differ in their biochemical composition from the nucleolus and also from the prenucleolar bodies which appear during mitosis. When anti-fibrillarin antibodies were microinjected into the male pronucleus of mouse embryos to analyse the functions of fibrillarin during early development, they partially blocked the early development of mouse embryos and only 23.8% of injected embryos reach the blastocyst stage.

Animals↗

PubMed1995

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.

Animals↗

PubMed1993

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.

Animals↗

PubMed1993

Temperature and androgens regulate the biosynthesis of secretory proteins from rabbit cauda epididymidis.

We have compared the biosynthesis of secretory proteins in rabbit cauda epididymidis maintained for 15 days at abdominal temperature with that of the scrotal cauda. Explants from both situations were incubated in vitro in the presence of [35S] methionine, and the labelled proteins released into the incubation medium were analyzed by polyacrylamide gel electrophoresis. Body temperature specifically inhibited the synthesis of at least two polypeptides of 43 kDa and 21 kDa (designated EP21), whereas the synthesis of polypeptides of 80, 39, 31, and 24 kDa was increased. These changes resembled those produced by castration, but androgen treatment was not able to reverse the effect of body temperature. To confirm these observations, poly(A)+ RNA from the scrotal and the abdominal cauda respectively, was translated in vitro and the synthesized products were immunoprecipitated with an antibody against EP21 polypeptide. Both castration and body temperature strongly decreased the concentration of EP21 mRNA. In vivo testosterone administration restored the content of EP21 mRNA in cauda from castrated animals, but not in cauda maintained at body temperature. The changes observed might be related to the adverse effect of body temperature on sperm storage in the cauda epididymidis.

Animals↗