Protective effect of butylated hydroxytoluene on sperm function in human spermatozoa cryopreserved by vitrification technique.

Butylhydroxytoluene (BHT), a synthetic analogue of vitamin E, shows antioxidant and antiviral properties and has been successfully used for mammalian sperm cryopreservation. In this study, BHT was included in a vitrification solution to determine its cryoprotective effect on human spermatozoa. Spermatozoa were selected by swim-up and vitrified in close sealed straw using either a combination of human tubal fluid (HTF), sucrose and BHT 1 mm (VMBHT), or only HTF and sucrose (VM). The optimal concentration of BHT was determined by the observation of preserved progressive sperm motility (PSM) after warming and detection of plasma membrane (PMI), membrane mitochondrial potential (ΔΨm) and DNA integrity. The presence of reactive oxygen species (ROS) was also detected. The PSM was significantly higher in the VMBHT group (80.86 ± 5.41%) compared with the VM group (68.9 ± 3.67%) (P < 0.05). Butylhydroxytoluene significantly preserved DNA integrity (4.0 ± 0.1% versus 6.1 ± 1.6%; P < 0.05) and reduced ROS production (5.5 ± 2.2 versus 8.6 ± 1.8%; P < 0.05). Plasma membrane and ΔΨm showed no statistical differences. One millimolar BHT effectively maintained cell function and due to its antioxidant and antiviral properties could be used in semen cryopreservation of patients with viral infections transmitted by seminal plasma.

Female sex hormones regulate the Th17 immune response to sperm and Candida albicans.

STUDY QUESTION: What role do female sex hormones play in the antisperm immune response? SUMMARY ANSWER: We found that sperm induce a Th17 immune response and that estradiol down-regulates the antisperm Th17 response by dendritic cells. WHAT IS KNOWN ALREADY: Estradiol down-regulates the immune response to several pathogens and impairs the triggering of dendritic cell maturation by microbial products. STUDY DESIGN, SIZE, DURATION: Ex vivo and in vivo murine models of vaginal infection with sperm and Candida albicans were used to study the induction of Th17 and its hormonal regulation. PARTICIPANTS/MATERIALS, SETTING, METHODS: We analyzed the induction of Th17 cytokines and T cells in splenocytes obtained from BALB/c mice challenged with sperm and C. albicans. For the in vivo vaginal infection models, we used ovariectomized mice treated with vehicle, estradiol or progesterone, and we assessed the effect of these hormones on the immune response in the lymph nodes. MAIN RESULTS AND THE ROLE OF CHANCE: Th17 cytokines and T cells were induced by sperm antigens in both ex vivo and in vivo experiments. Estrus levels of estradiol down-regulated the Th17 response to sperm and C. albicans in vivo. LIMITATIONS, REASONS FOR CAUTION: This study was conducted using murine models; whether or not the results are applicable to humans is not known. WIDER IMPLICATIONS OF THE FINDINGS: Our results describe an adaptive mechanism that reconciles immunity and reproduction and further explains why unregulated Th17 could be linked to infertility and recurrent infections. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by research grants from the Instituto de Salud Carlos III (ISCIII) (PI10/00897) and Fundación Mutua Madrileña to M.R. M.R. holds a Miguel Servet contract from the ISCIII (CP08/00228). M.A.M.-F. was supported by (ISCIII) INTRASALUD PI09/02029. We have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: Not required.

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.

Transfection of eggs in the bivalve mollusc Chamelea gallina (Bivalvia, Veneridae).

Eggs from the bivalve mollusc Chamelea gallina were transfected in vitro. The p-GeneGrip gene construction that expresses the green fluorescent protein (GFP) was employed. It was necessary to remove the jelly coat which covers the egg surface for a successful transfection, and then 44.2% of gametes appeared transfected after using naked DNA. On the other hand, cationic liposomes (Lipofectamine) and neutral lipids (GenePORTER) were employed as gene vectors. After the employ of Lipofectamine 35.6% of eggs were transfected and 41.4% after using GenePORTER. Fluorescence analysis showed that the foreign gene appeared principally located in the egg cytoplasm, but laser confocal microscopy showed that it was also present in the nucleus. Furthermore, PCR analysis demonstrated that the foreign DNA appeared in the DNA extracted from the treated eggs. This simple method for the transfection of mollusc eggs would be interesting for future biotechnological applications in species of commercial interest.

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.

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.

Plasma membrane glycoproteins during spermatogenesis and in spermatozoa of some fishes.

Plasma membrane glycoproteins were analyzed during spermatogenesis and in the spermatozoa of the teleost fish Xiphophorus maculatus and of the Elasmobranch Schroederichthys chilensis. The analysis was undertaken using the fluoresceinated lectins concanavalin A (ConA) and wheat germ agglutinin (WGA) to detect glycoproteins in the plasma membrane using confocal laser microscopy. In Xiphophorus, a species with acrosomeless spermatozoa, primary spermatocytes and rounded shaped spermatids showed that the whole cell surface was labeled by both lectins. As spermiogenesis proceeded surface glycoproteins diminished and in mature spermatozoa a discrete and non-random localized fluorescence was observed exclusively on the surface of the spermatozoon head after the employ of ConA and WGA. In the elasmobranch Schroederichthys chilensis the spermatozoon displays an acrosome as a small vesicle. After ConA and WGA labeling, the region of the plasma membrane that covers the acrosome was the only fluorescent region in the gamete. The physiological significance of plasma membrane glycoproteins is discussed regarding spermatozoon physiology.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

El síndrome de inmunodeficiencia adquirida en la unidad de terapia intensiva el Hospital Angeles del Pedregal / Acquired immunodeficiency syndrome in the intensive care unit of the hospital Angeles del Pedregal of Mexico

El presente trabajo describe retrospectivamente las características clínicas y la evolución de 24 pacientes con VIH/SIDA que requirieron manejo en la Unidad de Terapia Intensiva (UTI) del Hospital Angeles del Pedregal durante su hospitalización. En cinco pacientes (21 por ciento) el diagnóstico de infección por VIH se realizó al momento de su ingreso a la UTI. La causa más frecuente de ingreso a la UTI fue insuficiencia respiratoria que requirió ventilación mecánica en 81 por ciento de los casos. La mortalidad relacionada con el accidente que provocó ingreso a la UTI fue cercana a 80 por ciento. Sin embargo, en aquellos pacientes en los que la causa del ingreso a terapia intensiva fue una neumonía por P. carinii, la mortalidad fue de 55 por ciento. En conclusión, el uso de unidades de cuidados intensivos en el manejo de pacientes con VIH/SIDA puede ser de utilidad en algunos pacientes con SIDA que tienen insuficiencia respiratoria, particularmente cuando ésta es debida a neumonía por P. carinii. Sin embargo, en otras situaciones la utilidad del manejo en la UTI no es tan clara. Con frecuencia el diagnóstico de infección por VIH se realizó por vez primera en la UTI, pero en aquellos pacientes en los que ya se conoce su infección por VIH es indispensable que el médico tratante discuta con el paciente acerca de sus deseos de ingresar o no a una unidad de terapia intensiva durante el curso de su padecimiento, con el fin de evitar intervenciones no deseadas en aquellos pacientes que así lo indiquen

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.