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.

In vitro microtubule assembly on centrioles from mammalian spermatids.

In the present study we have shown that the centriolar structures, which form the neck region of the spermatid tail, can act as microtubule-organizing centers.

Nucleolar fibrillar centers in mouse spermatid nucleoli.

The nucleoli of young spermatids of mice are described. They exhibit a very special shape resembling a "padlock" in which three different areas can be distinguished: (a) a compact zone corresponding to the fibrillar component, (b) the granular component and (c) a fibrillar center of low density. Fibrillar and granular components usually appear segregated. This nucleolus has been reconstructed based on serial sectioning. When the silver impregnation technique is employed, both fibrillar and granular components show a positive reaction, although the fibrillar center is free of granules. The morphology of the fibrillar center seems to be similar to that reported in other cells. The possibility that these fibrillar centers correspond to the nucleolar organizer is discussed.

Tissue-specific and hormonally regulated expression of the puromycin N-acetyltransferase-encoding gene under control of the rabbit uteroglobin promoter in transgenic mice.

Transgenic mice bearing two fragments of the rabbit uteroglobin 5'-flanking region fused to the new reporter gene (pac) encoding puromycin N-acetyltransferase (PAC) showed a different pattern of expression. Transgenic lines (C0.4) harboring a 404-bp fragment (-396/+8) had a uterus-specific expression slightly inducible by estrogen, lacking detectable expression in other tissues where the uteroglobin-encoding gene is naturally expressed in rabbit. Transgenic lines (C3.2) bearing a longer fragment of 3.2-kb (-3254/+8) showed hormonally regulated expression in the uterus and the male genital tract, and detectable expression in the lung. In addition, the nonstimulated uterine expression of the transgene was higher in C0.4 lines than in C3.2 lines. It could be concluded that all sequences required for uterus-specific expression should be present within the 404-bp fragment, and that other upstream (-396) sequences are responsible for expression in the lung and male genital tract, as well as for a possible down modulation of expression in the uterus.

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.

Epididymal fluid macromolecules do not act as auto- or alloantigens.

Because epididymal secretory glycoproteins form important functional associations with maturing spermatozoa, the possibility has been explored that epididymal antigens might be useful in contraceptive vaccine development. Male and female rabbits, hamsters, and rats were immunized for several weeks with epididymal fluid, initially either with complete Freund's adjuvant, or conjugated with glutaraldehyde. Some female rabbits were also immunized using albumin-free rabbit epididymal fluid. The sera of all the immunized animals were then examined for autogenous, allogeneic, or [in guinea pigs also] xenogeneic immune responses, using immunodiffusion and ELISA techniques. In each instance, xenogeneic immunization produced a marked antibody response. However, no animals developed any detectable antibody reactive with epididymal fluids following auto- or alloimmunization (ie with fluid from the same species), and they remained fertile even when immunized males were mated with immunized females. Furthermore, when female rabbits were immunized with ejaculated rabbit spermatozoa, they produced antibodies reactive only with the sperm homogenate, but not with any epididymal fluid component. These results indicate that macromolecules secreted by the epididymis, including those that associate with spermatozoa, do not act as auto- or alloantigens and, at present, would seem to have no immediate promise for contraceptive vaccine development in males or females.

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.

Nucleolar changes induced by isoproterenol in mouse acinar parotid cells.

This paper deals with the analysis of the nucleolar morphology and distribution of the nucleolar component (i.e. fibrils and granules) in normal and isoproterenol-treated parotid acinar cells of mice. Normally nucleoli present both components intermingled, but at 2 h treatment a considerable decrease of the fibrillar area is detected by a silver staining method. Normal nucleolar characteristics are recovered after eight hours treatment. In hypertrophic cells nucleolar size is considerably increased, but the ratio total nucleolar area/fibrillar area is similar to control cells as shown by a stereometrical computerized analysis. Rounded nucleolar bodies occur in these nuclei which do not seem to be dependent on the modifications induced by the drug.

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.

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.

Spermatozoon maturation in vertebrates with internal fertilization.

Sperm maturation occurs in the mammalian spermatozoon during its passage through the epididymis. Maturation comprises a series of morpho-physiological changes, which includes the acquisition of the fertilizing capacity in the gamete. This maturative process has been particularly studied in mammals, but different data reveal that birds, reptiles and some kind of fish have a similar characteristic. Anatomical and histological analyses of mammalian epididymis and of Wolffian ducts of some birds and reptiles show the predominance of a secretory cell system. Proteins secreted by the male ducts seem to be an important factor involved in the acquisition of motility, as well as in the changes in the molecular organization of the plasma membrane. Changes occurring in the plasma membrane of the mammalian spermatozoon are related to the acquisition of foreign proteins (of epididymal origin). Some of these membrane changes seem to be connected with the capacitation phenomena and also with gamete interaction during fertilization. The use of antibodies against Wolffian duct proteins has shown that spermatozoa birds and reptiles also acquire proteins during their passage through the male duct. Nevertheless, in birds, and probably in reptiles, capacitation is not a pre-requisite for fertilization and some testicular spermatozoa are able to fertilize the egg. Then, what is the real significance of the membrane maturative changes in these subtherian vertebrates? Proteins acquired during maturation in such species must have different functions from those in mammals, to support spermatozoon survival and/or transport in the female tract, where spermatozoa are stored for a long time. Surface changes in mammals would possibly have similar roles when the gametes are in the female tract.

Spermiogenesis and spermatozoa in mammals.

This is a summarizing review about some aspects of the morphogenesis and characteristics of mammalian spermatozoon. The morphology, biogenesis and function of the different spermatozoon structures are analysed, as well as different aspects, like immunology and endocrine control. Comparative morphology, fate and function of the male gamete during its passage through the epididymis and female tract are commented. The fact, that the phenomenon of spermiogenesis continues with the maturative changes in the epididymis, and that modifications occur in the female genital tract is put forward; this idea seems to be basic for understanding the role of many spermatozoon structures, as well as many biochemical and molecular changes that take place in the gamete. On the other hand, all these changes (spermiogenesis, maturation, capacitation and acrosomal reaction in the female tract), as well as the interaction sperm/egg are sequentially dependent, and are focussed on the sperming capacity of reaching and fertilizing the egg. Many gaps exist in regard to the functional role of different spermatozoon structures, and three important questions are emphasized with a view to solving these problems: a) The importance of the comparative analysis of spermatozoon and egg structures in different mammalian species. b) The necessity to develop ample studies using in vivo situations, particularly in reference to the changes occurred in the female tract. In this regard, the majority of studies have been developed in the in vitro situation, which is very different from the natural environment. c) The importance of using and compiling the different data (morphological, biochemical, molecular, etc.) about spermiogenesis, spermatozoon and fertilization. In the future, the use of new technologies appears as a promising idea to clarify doubts about spermatozoa biogenesis and function in mammals.

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.

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.

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.

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.