Localization and expression of ADAM2 in the dromedary camel testis, epididymis and sperm during rutting season

ADAM2 (fertilin ) is a sperm surface protein reported in several mammalian species. However, the presence of ADAM2 in the male reproductive system and sperm of the camel is not well known. The present study was to clarify the localization and expression of ADAM2 in the dromedary camel testis, epididymis and spermatozoa during rutting season using immunohistochemistry (IHC) and the quantitative real-time polymerase chain reaction (qPCR). Tissue samples were obtained from the testis (proximal and distal) and epididymis (caput, corpus, and cauda) from eight mature male camels. Epididymal and ejaculated sperms were collected from four other fertile camels. IHC analysis clearly showed the localization of ADAM2 protein in the spermatocytes and the round and elongated spermatids of the testis, in the epithelial cells along the epididymis tract, on the posterior head of the sperm within the cauda epididymis, and on the acrosomal cap of both the epididymal and ejaculated sperm. The expression of camel ADAM2 mRNA was significantly higher (P 0.05) in the testis when compared with the epididymis. These findings may suggest an important role of ADAM2 in the fertility of male dromedary camels.(AU)

Cat epididymal semen cryopreserved with and without vitamin E: effect on sperm parameters and lipid peroxidation

The aims of this study were to investigate: 1) if the addition of α-tocopherol (vitamin E) in three concentrations (0.3, 0.6 and 0.9 mM) is able to preserve spermatozoa integrity after thawing and 2) the effect of α-tocopherol supplementation on lipid peroxidation. Fifty four domestic cats were used in this study constituting 18 pools (3 cats per pool). Each pool was submitted at four experimental groups: group 0 (control) – epididymal sperm were frozen with a commercial Botucrio® extender; group 0.3, group 0.6 and group 0.9 – the extender was supplemented with 0.3, 0.6 and 0.9 mM of α-tocopherol, respectively. Each semen sample was evaluated for motility, progressive forward motility, morphology, sperm viability (plasma membrane integrity-PMI), hypo-osmotic swelling test (HOST), before and after thawing. The evaluation of lipid peroxidation reaction by Thiobarbituric Acid Reactive Substances (TBARS) test was performed on thawed semen only. Results demonstrated that there was no significant difference between control and the three α-tocopherol groups with regards to motility and progressive motility after thawing (P > 0.05). As expected, in fresh samples viability was significantly higher than in all the cryopreserved groups in which there was no positive influence of any of the αtocopherol concentration used. Lipid peroxidation was higher in the supplemented groups 0.6 and 0.9 mM of α-tocopherol than in control and in 0.3 mM group. In conclusion, the addition of α-tocopherol to the commercial extender had no positive influence on reduction of lipid peroxidation. This topic deserves further investigations to better understand the effect of cryopreservation procedures on epididymal spermatozoa and to establish adequate strategies to counteract sperm cryodamages.

Cat epididymal semen cryopreserved with and without vitamin E: effect on sperm parameters and lipid peroxidation

The aims of this study were to investigate: 1) if the addition of α-tocopherol (vitamin E) in three concentrations (0.3, 0.6 and 0.9 mM) is able to preserve spermatozoa integrity after thawing and 2) the effect of α-tocopherol supplementation on lipid peroxidation. Fifty four domestic cats were used in this study constituting 18 pools (3 cats per pool). Each pool was submitted at four experimental groups: group 0 (control) epididymal sperm were frozen with a commercial Botucrio® extender; group 0.3, group 0.6 and group 0.9 the extender was supplemented with 0.3, 0.6 and 0.9 mM of α-tocopherol, respectively. Each semen sample was evaluated for motility, progressive forward motility, morphology, sperm viability (plasma membrane integrity-PMI), hypo-osmotic swelling test (HOST), before and after thawing. The evaluation of lipid peroxidation reaction by Thiobarbituric Acid Reactive Substances (TBARS) test was performed on thawed semen only. Results demonstrated that there was no significant difference between control and the three α-tocopherol groups with regards to motility and progressive motility after thawing (P > 0.05). As expected, in fresh samples viability was significantly higher than in all the cryopreserved groups in which there was no positive influence of any of the αtocopherol concentration used. Lipid peroxidation was higher in the supplemented groups 0.6 and 0.9 mM of α-tocopherol than in control and in 0.3 mM group. In conclusion, the addition of α-tocopherol to the commercial extender had no positive influence on reduction of lipid peroxidation. This topic deserves further investigations to better understand the effect of cryopreservation procedures on epididymal spermatozoa and to establish adequate strategies to counteract sperm cryodamages.(AU)

DHEA: el andrógeno más producido pero el menos entendido / DHEA: the androgen produced but least understood

Dehidroepiandrosterona (DHEA) y su derivada sulfatada (DHEAS) son los esteroides más abundantes en el cuerpo humano, pero aún se deconoce su mecanismo de acción y sus implicancias fisiológicas. Se le ha atribuido múltiples efectos antienvejecimiento, antiinflamatorio y antiarteriosclerótico entre otros y en EEUU se vende al público como complemento energético y para aumento del libido, sin restricción de la FDA...

Topografia de ligação de proteínas do plasma seminal à membrana de espermatozoides bovinos epididimários e ejaculados / Binding patterns of seminal plasma plasma proteins on bovine epididymal and ejaculated sperm membrane

The present study was designed to investigate the topographical distribution of seminal plasma (SP) proteins on epididymal and ejaculated bovine sperm. Using immunocytochemistry and confocal microscopy the binding patterns of bovine SP proteins BSP-A3, albumin, transferrin, prostaglandin D-synthase (PGDS) and nucleobindin in ejaculated and cauda epididymal sperm from adult bulls were evaluated. Experiments were performed using sperm from 5 males. Data showed a positive signal, only detected for anti-PGDS, in the acrosomal cap of epididymal and ejaculated sperm. In ejaculated sperm, a very weak signal for nucleobindin 2 in the midpiece and equatorial regions was detected, using the anti-rat nucleobindin. BSP-A3 was detected on all sperm regions studied, with a more evidenced signal in acrosome and midpiece. However, no binding was detected for albumin or transferrin in neither epididymal nor ejaculated sperm. In conclusion, PGDS, BSP-A3 and nucleobindin interact directly with bovine sperm, with specific topographic distribution. These findings may add to the knowledge of how these proteins modulate sperm functions, thus providing fundamental support for studies designed to evaluate how they influence sperm functions.

Investigou-se a distribuição topográfica da ligação de proteínas seminais à membrana de espermatozoides bovinos epididimários e ejaculados. Utilizando imunocitoquímica e microscopia confocal, avaliaram-se a topografia de ligação das proteínas BSP-A3, albumina, transferrina, prostaglandina D sintetase (PGDS) e nucleobindina 2 (NUC2) à membrana espermática. Os experimentos foram realizados utilizando espermatozoides de cinco touros. Os resultados mostraram que, para espermatozoides epididimários, somente detectou-se a PGDS na crista do acrossomo. Nos espermatozoides ejaculados, a PGDS ligou-se de forma mais intensa à crista acrossômica, enquanto a NUC2 apresentou sinal bastante fraco na peça intermediária e região equatorial. A BSP-A3 ligou-se a todas as regiões estudadas, de forma mais intensa na peça intermediária e acrossomo. Nenhum sinal foi detectado para albumina ou transferrina, seja em espermatozoides epididimários ou ejaculados. Concluiu-se que PGDS, BSP-A3 e NUC2 interagem diretamente com espermatozoides bovinos, e mostrou distribuição topográfica específica. Estes achados permitem melhor compreensão sobre o papel desempenhado por essas proteínas na regulação da função espermática e da fertilidade.

Binding patterns of seminal plasma plasma proteins on bovine epididymal and ejaculated sperm membrane / Topografia de ligação de proteínas do plasma seminal à membrana de espermatozoides bovinos epididimários e ejaculados

The present study was designed to investigate the topographical distribution of seminal plasma (SP) proteins on epididymal and ejaculated bovine sperm. Using immunocytochemistry and confocal microscopy the binding patterns of bovine SP proteins BSP-A3, albumin, transferrin, prostaglandin D-synthase (PGDS) and nucleobindin in ejaculated and cauda epididymal sperm from adult bulls were evaluated. Experiments were performed using sperm from 5 males. Data showed a positive signal, only detected for anti-PGDS, in the acrosomal cap of epididymal and ejaculated sperm. In ejaculated sperm, a very weak signal for nucleobindin 2 in the midpiece and equatorial regions was detected, using the anti-rat nucleobindin. BSP-A3 was detected on all sperm regions studied, with a more evidenced signal in acrosome and midpiece. However, no binding was detected for albumin or transferrin in neither epididymal nor ejaculated sperm. In conclusion, PGDS, BSP-A3 and nucleobindin interact directly with bovine sperm, with specific topographic distribution. These findings may add to the knowledge of how these proteins modulate sperm functions, thus providing fundamental support for studies designed to evaluate how they influence sperm functions.(AU)

Investigou-se a distribuição topográfica da ligação de proteínas seminais à membrana de espermatozoides bovinos epididimários e ejaculados. Utilizando imunocitoquímica e microscopia confocal, avaliaram-se a topografia de ligação das proteínas BSP-A3, albumina, transferrina, prostaglandina D sintetase (PGDS) e nucleobindina 2 (NUC2) à membrana espermática. Os experimentos foram realizados utilizando espermatozoides de cinco touros. Os resultados mostraram que, para espermatozoides epididimários, somente detectou-se a PGDS na crista do acrossomo. Nos espermatozoides ejaculados, a PGDS ligou-se de forma mais intensa à crista acrossômica, enquanto a NUC2 apresentou sinal bastante fraco na peça intermediária e região equatorial. A BSP-A3 ligou-se a todas as regiões estudadas, de forma mais intensa na peça intermediária e acrossomo. Nenhum sinal foi detectado para albumina ou transferrina, seja em espermatozoides epididimários ou ejaculados. Concluiu-se que PGDS, BSP-A3 e NUC2 interagem diretamente com espermatozoides bovinos, e mostrou distribuição topográfica específica. Estes achados permitem melhor compreensão sobre o papel desempenhado por essas proteínas na regulação da função espermática e da fertilidade.(AU)

Immunocontraceptive properties of recombinant sperm protein DE: implications for the development of novel contraceptives.

OBJECTIVE: To evaluate the immunocontraceptive properties of recombinant DE, a sperm epididymal protein involved in fertilization, via an experimental study in rats as a critical step toward the development of a human immunocontraceptive. DESIGN: In vivo study in rats. SETTING: Animal care facility of an academic research center. ANIMAL(S): Seventy-four 90-day-old Wistar male and female rats distributed into three groups. INTERVENTION(S): Animals received five injections (intramuscular and subcutaneous) of recombinant DE (recDE), native DE (nDE), or MBP (maltose-binding protein). At various times, animals were anesthetized and bled. MAIN OUTCOME MEASURE(S): Anti-DE levels and tissue specificity of sera were evaluated by enzyme-linked immunosorbent assay (ELISA) and Western blot, respectively. Fertility was analyzed by natural mating. The testes and epididymides were analyzed by histology. RESULT(S): Recombinant DE raised an immune response with the same kinetics and higher anti-DE levels than that elicited by nDE. Sera against recDE recognized epitopes of DE that were different from those recognized by anti-nDE sera but specifically reacted with DE in epididymis and sperm without cross-reacting with other tissues tested. Male and female recDE-injected animals presented a statistically significant reduction in their fertility with no evidence of pathologic effects. CONCLUSION(S): Recombinant DE is able to both elicit a specific immune response and inhibit male and female fertility, supporting the use of this sperm epididymal protein for the development of an immunocontraceptive approach.

Serum S100B protein is increased in fasting rats.

BACKGROUND: S100B is a calcium-binding protein expressed and secreted by astrocytes; serum and cerebrospinal fluid (CSF) S100B elevation has been proposed as an index of brain damage. However, other tissues are shown to produce this protein and the clinical significance of serum S100B elevation has been discussed. METHODS: We investigated the levels of serum and CSF S100B in fasting Wistar rats. Animals were divided into two groups, control and fasting for 48 h, and S100B levels in serum and CSF were determined by ELISA. S100B secretion in dissociated epididymal fat cells was investigated in the presence of epinephrine. RESULTS: We observed a significant >2-fold increase of S100B levels in serum of fasting rats, without changes in its CSF content. Moreover, we demonstrated in vitro epinephrine stimulated S100B release from fat cells. CONCLUSIONS: Present results reinforce that extracerebral sources of S100B, particularly adipocytes, contribute to its serum levels and support the idea that caution is needed when interpreting serum S100B increase as a clinical marker of brain damage.

Studies on the participation of epididymal sperm protein DE/CRISP-1 in egg activation.

Protein DE (32 kDa) associates with sperm during epididymal maturation and participates in sperm-egg fusion through its binding to complementary sites on the egg surface. In the present work we investigated the participation of DE in two mechanisms probably involved in egg activation: the ability of DE to trigger activation by its interaction with the binding sites on the egg surface (receptor model) and its ability to regulate intracellular calcium channels (sperm factor model). The incubation of eggs with DE did not promote activation parameters such as calcium oscillations or meiosis resumption. Secondly, microinjection of DE into eggs was ineffective in either eliciting calcium release or modifying oscillations induced by an activating sperm extract. Together, these results argue against the participation of DE in egg activation, restricting the activity of this protein and its egg binding sites to the sperm-egg fusion process.

Mammalian sperm-egg fusion: evidence that epididymal protein DE plays a role in mouse gamete fusion.

Rat epididymal protein DE associates with the sperm surface during epididymal maturation and is a candidate molecule for mediating gamete membrane fusion in the rat. Here, we provide evidence supporting a role for DE in mouse sperm-egg fusion. Western blot studies indicated that the antibody against rat protein DE can recognize the mouse homologue in both epididymal tissue and sperm extracts. Indirect immunofluorescence studies using this antibody localized the protein on the dorsal region of the acrosome. Experiments in which zona-free mouse eggs were coincubated with mouse capacitated sperm in the presence of DE showed a significant and concentration-dependent inhibition in the percentage of penetrated eggs, with no effect on either the percentage of oocytes with bound sperm or the number of sperm bound per egg. Immunofluorescence experiments revealed specific DE-binding sites on the fusogenic region of mouse eggs. Because mouse sperm can penetrate zona-free rat eggs, the participation of DE in this interaction was also investigated. The presence of the protein during gamete coincubation produced a significant reduction in the percentage of penetrated eggs, without affecting the binding of sperm to the oolemma. These observations support the involvement of DE in an event subsequent to sperm-egg binding and leading to fusion in both homologous (mouse-mouse) and heterologous (mouse-rat) sperm-egg interaction. The lack of disintegrin domains in DE indicates that the protein interacts with its egg-binding sites through a novel mechanism that does not involve the reported disintegrin-integrin interaction.

Relationship between the association of rat epididymal protein "DE" with spermatozoa and the behavior and function of the protein.

Rat epididymal glycoprotein DE associates with the dorsal region of the sperm head during sperm maturation, migrates to the equatorial segment (ES) with the acrosome reaction (AR), and is involved in gamete membrane fusion. In the present study we examined the association of DE with the sperm surface and the relationship of this interaction with the behavior and function of the protein. Cloning and sequencing of DE revealed a lack of hydrophobic domains and the presence of 16 cysteine residues in the molecule. Experiments in which cauda epididymal sperm were subjected to different extraction procedures indicated that while most of the protein is removable from sperm by mild ionic strength, a low amount of DE, resistant to even 2 M NaCl, can be completely extracted by agents that remove integral proteins. However, the lack of hydrophobic domains in the molecule and the failure of DE to interact with liposomes, does not support a direct insertion of the protein into the lipid bilayer. These results, and the complete extraction of the tightly bound protein by dithiothreitol, suggest that this population would correspond to a peripheral protein bound to a membrane component by strong noncovalent interactions that involve disulfide bonds. While ELISA experiments showed that no protein could be extracted by NaCl from capacitated sperm, indirect immunofluorescence studies revealed the ability of the NaCl-resistant protein to migrate to the ES. Together, these results support the existence of two populations of DE: a major, loosely bound population that is released during capacitation, and a minor strongly bound population that remains after capacitation, migrates to the ES with the AR, and thus would correspond to the one with a role in gamete fusion.

Potential contraceptive use of epididymal proteins: immunization of male rats with epididymal protein DE inhibits sperm fusion ability.

Rat epididymal protein DE associates with the sperm surface during maturation and participates in sperm-egg fusion. Immunization of male rats with DE raised specific antibodies and produced a significant reduction in the animals' fertility. The present study focused on determining the in vivo mechanism involved in fertility inhibition. Wistar males were injected with DE, and antibody levels and animal fertility were evaluated. Results revealed an association between the two parameters, since animals with absorbance values lower than 0.5 in ELISA presented high fertility rates (66%, 100%) while those with absorbance values higher than 0.5 exhibited the lowest fertility rates (0%, 33%). Histological studies showed no evidence of orchitis, epididymitis, or vasitis in DE-immunized animals. ELISA results revealed the presence of anti-DE antibodies in epididymal and vas deferential fluids. Indirect immunofluorescence and ELISA experiments indicated that these antibodies would not interfere with the synthesis or secretion of DE or with its association with the sperm surface. Finally, while epididymal sperm recovered from DE-immunized animals presented no changes in motility, viability, or ability to undergo capacitation and acrosome reaction, they exhibited a significant decrease in their ability to fuse with zona-free eggs, with no effect on their ability to bind to the oolemma. Together these results indicate that immunization of male rats with epididymal protein DE specifically interferes with the sperm fertilizing ability, supporting the use of epididymal proteins for contraceptive vaccine development.

Mammalian sperm-egg fusion: the development of rat oolemma fusibility during oogenesis involves the appearance of binding sites for sperm protein "DE".

Rat epididymal protein DE mediates gamete fusion through complementary sites localized on the egg surface. To investigate whether these egg components are involved in the development of rat oolemma fusibility, both the presence of DE-binding components and the ability of the oolemma to fuse with sperm during oogenesis were examined. Localization of DE-complementary sites by indirect immunofluorescence revealed the absence of fluorescent labeling on growing oocytes with a diameter < 50 microns, and the presence of a uniform staining over the entire surface of germinal vesicle oocytes with a diameter > 50 microns. This localization of oolemma components changed progressively to a patchy distribution during maturation. Whereas sperm incorporation was observed only in maturing oocytes, the development of the Hoechst transfer technique to evaluate membrane fusion revealed that germinal vesicle oocytes with a diameter > 50 microns were already competent to fuse with sperm. The involvement of the DE-complementary sites in the oolemma fusibility of these oocytes was confirmed by the fact that the presence of DE during gamete coincubation significantly (p < 0.001) reduced the percentage of oocytes with fused sperm. Together, these observations indicate that the acquisition of fusibility by the rat oolemma occurs during the growth period and involves the appearance of DE-binding components on the oocyte surface. This study provides novel information on the molecular mechanism by which the mammalian egg plasma membrane becomes competent to fuse with sperm during oogenesis.

Potential contraceptive use of epididymal proteins: evidence for the participation of specific antibodies against rat epididymal protein DE in male and female fertility inhibition.

Previous studies in our laboratory indicated that immunization of male and female Wistar and Lewis rats with epididymal protein DE, resulted in the development of anti-DE antibodies in over 90% of the animals, with a significant and reversible reduction of fertility. In the present study, ELISA assays performed to analyze the evolution of the immune response indicated that antibody levels in the sera of immunized animals reached a maximum at 8 weeks after the initial injection and then gradually decreased, returning to control values by the end of the sixth month. Western blot experiments demonstrated that the immune sera specifically recognized DE in epididymal sperm extracts and epididymal cytosol, while no reaction was observed with different reproductive and essential organs. The immune sera were also capable of recognizing DE on the surface of both fresh and capacitated sperm as indicated by indirect immunofluorescence experiments. Finally, the exposure of sperm to immune sera prior to uterine insemination resulted in a significant (P < 0.05) reduction in the percentage of fertilized eggs compared to controls, with no effect on sperm motility and viability, nor on their ability to undergo capacitation. Together, these results support the participation of the raised antibodies as mediators of the antifertility effect and suggest a specific interference at the sperm-egg interaction level.

Epididymal glycoprotein involved in rat sperm association.

Recently, a new head-to-head sperm association was described in the rat during epididymal transit. This association was called a rosette and a filamentous and PAS-positive material was also described joining the sperm heads. The beginning of rosette formation in the epididymis and the linking material between heads have remained unclear. Epididymides of adult rats were fixed by vascular perfusion and thin sections of the principal regions were studied by transmission electron microscopy (TEM). The first evidence of rosette formation was observed in the distal corpus. Rosettes were isolated from the distal corpus and processed for immunogold and immunofluorescence microscopy to detect an epididymal glycoprotein called DE. This glycoprotein is secreted by the corpus epididymis and appears to be involved in sperm maturation. Colloidal gold marks and fluorescence were observed in the linking material between the sperm heads. The results presented here show that rosettes begin to appear following the sites of DE secretion and permit us to postulate that DE is involved in rosette formation and constitutes another example of gamete-epididymal interaction.