O papel da gota citoplasmática na funcionalidade de espermatozoides em mamíferos: uma revisão atualizada / The role of cytoplasmic droplet over sperm functionality in mammals: an updated review

A gota citoplasmática é uma protuberância de citoplasma geralmente posicionada na peça intermediária dos espermatozoides. É considerada uma organela transitória, uma vez que migra pelo espermatozoide durante o trânsito epididimário, quando é removida dessas células na maioria dos mamíferos. Dúvidas sobre se a presença desta gota está relacionada de forma positiva ou negativa com a função e fertilidade espermática são frequentes, já que sua presença no ejaculado pode afetar a fertilidade espermática e o uso do sêmen em biotécnicas da reprodução. Porém, mesmo não sendo totalmente compreendida a nível molecular e funcional, sabe-se que a gota citoplasmática é necessária ao espermatozoide durante seu processo de maturação no epidídimo. Por isso, o objetivo desta revisão foi abordar pontos sobre a fisiologia e funções das gotas citoplasmáticas, bem como relacionar sua presença com a maturação e fertilidade dos espermatozoides.(AU)

The cytoplasmic droplet is a protuberance of cytoplasm usually positioned in the sperm midpiece. It is considered a transient organelle that migrates through the sperm cell during its epididymal transit when it is finally removed from the cell in most mammals. Doubts over whether the presence of cytoplasmic droplets is positively or negatively related to sperm functions and fertility are common, as its presence in the ejaculate may affect sperm fertility and the use of semen in assisted reproductive technology. Although the understanding of cytoplasmic droplets function is not fully clarified at molecular and functional levels, it is known that cytoplasmic droplet is necessary for spermatozoa, especially during their maturation in the epididymal duct. Therefore, the aim of this review was to discuss aspects of the physiology and function of cytoplasmic droplets, as well as to relate their presence to sperm maturation and their consequent fertility.(AU)

O papel da gota citoplasmática na funcionalidade de espermatozoides em mamíferos: uma revisão atualizada / The role of cytoplasmic droplet over sperm functionality in mammals: an updated review

A gota citoplasmática é uma protuberância de citoplasma geralmente posicionada na peça intermediária dos espermatozoides. É considerada uma organela transitória, uma vez que migra pelo espermatozoide durante o trânsito epididimário, quando é removida dessas células na maioria dos mamíferos. Dúvidas sobre se a presença desta gota está relacionada de forma positiva ou negativa com a função e fertilidade espermática são frequentes, já que sua presença no ejaculado pode afetar a fertilidade espermática e o uso do sêmen em biotécnicas da reprodução. Porém, mesmo não sendo totalmente compreendida a nível molecular e funcional, sabe-se que a gota citoplasmática é necessária ao espermatozoide durante seu processo de maturação no epidídimo. Por isso, o objetivo desta revisão foi abordar pontos sobre a fisiologia e funções das gotas citoplasmáticas, bem como relacionar sua presença com a maturação e fertilidade dos espermatozoides.

The cytoplasmic droplet is a protuberance of cytoplasm usually positioned in the sperm midpiece. It is considered a transient organelle that migrates through the sperm cell during its epididymal transit when it is finally removed from the cell in most mammals. Doubts over whether the presence of cytoplasmic droplets is positively or negatively related to sperm functions and fertility are common, as its presence in the ejaculate may affect sperm fertility and the use of semen in assisted reproductive technology. Although the understanding of cytoplasmic droplets function is not fully clarified at molecular and functional levels, it is known that cytoplasmic droplet is necessary for spermatozoa, especially during their maturation in the epididymal duct. Therefore, the aim of this review was to discuss aspects of the physiology and function of cytoplasmic droplets, as well as to relate their presence to sperm maturation and their consequent fertility.

Influence of glycerol addition on the quality of cat epididymal sperm during the freeze-thaw process / Influência da adição do glicerol sobre a qualidade dos espermatozoides epididimários de gato durante as etapas da congelação-descongelação

Cryopreservation of epididymal sperm is a useful tool for preserving the genetic potential of valuable animal specimens. The domestic cat is used as a model to study and develop cryogenics for other felines. However, regulation of the entire cryopreservation process is essential for the success of this biotechnology. Thus, our aim was to evaluate the effects of glycerol equilibration time and freezethaw stages on the quality of epididymal sperm obtained from domestic cats. Epididymal sperm were recovered with TRIS and immediately evaluated for total motility (TM), vigor, viability, membrane functionality (HOST), and morphology. Then, TRIS-20% egg yolk was added to the samples, which were equally divided into two 1.5 mL tubes and refrigerated at 4 ºC for 1 hour. Subsequently, glycerol was added at a final concentration of 5%. The samples were incubated with glycerol (equilibration time) for either 5 or 10 minutes (groups G5 and G10, respectively) and then frozen. Thawing occurred at 37 ºC for 30 seconds. The samples were evaluated at all stages. A reduction in TM was observed only after thawing; however, it was higher in G5 (39.00 ± 4.07%) than in G10 (18.50 ± 4.54%). Vigor declined in both groups after thawing; however, they did not differ from each other. Sperm viability was maintained in G5 after glycerolization (53.60 ± 2.59%); in G10, sperm viability decreased in the glycerolized sample (48.80 ± 2.93%) when compared to that in the fresh sample (59.90 ± 1.74%). Post-thaw viability of G5 (33.80 ± 1.89%) was higher than that of G10 (18.80 ± 3.01%). In the HOST, a decrease in viability was only observed after thawing, with no difference between the groups (41.50 ± 2.84% for G5 and 40.20 ± 3.49% for G10). With regard to sperm morphology, normal sperm decreased while sperm with post-thaw secondary defects increased in both groups. In conclusion, a shorter equilibration time for glycerolization preserves epididymal sperm quality better and the freeze-thaw process is the most critical stage of thawing.(AU)

A criopreservação dos espermatozoides epididimários é uma ferramenta útil para preservar o potencial genético de um animal valioso. Além disso, o gato doméstico é modelo eleito para o estudo e desenvolvimento da criogenia para os demais felinos. Contudo, para o sucesso dessa biotécnica é essencial o controle de todo o processo de criopreservação. Assim, objetivou-se avaliar o efeito do tempo de equilíbrio da glicerolização e das etapas da congelação-descongelação sobre a qualidade dos espermatozoides epididimários de gato doméstico. Para tanto, espermatozoides epididimários foram recuperados com TRIS e imediatamente avaliados quanto à motilidade total (MT), vigor, viabilidade, funcionalidade de membrana (HOST) e morfologia. Em seguida, as amostras foram adicionadas de TRIS-gema a 20%, fracionadas igualmente em dois tubos de 1,5 mL, refrigeradas a 4 ºC por 1 hora e, posteriormente, adicionadas de glicerol na concentração final de 5%. As amostras foram incubadas com glicerol (tempo de equilíbrio) por 5 ou 10 minutos (grupos G5 e G10, respectivamente) e depois congeladas. A descongelação ocorreu a 37 ºC por 30 segundos. As amostras foram avaliadas em todas as etapas. Uma redução na MT foi observada apenas na pós-descongelação, no entanto G5 (39,00 ± 4,07%) foi superior ao G10 (18,50 ± 4,54%). O vigor declinou pós-descongelação em ambos os grupos; contudo, não diferiram entre si. A viabilidade espermática foi mantida no G5 pós-glicerolização (53,60 ± 2,59%), diferentemente do observado em G10, em que a amostra glicerolizada (48,80 ± 2,93%) reduziu em relação à fresca (59,90 ± 1,74%). A viabilidade pós-descongelação de G5 (33,80 ± 1,89%) foi superior à de G10 (18,80 ± 3,01%). No HOST, uma redução da viabilidade só foi observada pósdescongelação, não havendo diferença entre os grupos (41,50 ± 2,84% para G5 e 40,20 ± 3,49% para G10). Em relação à morfologia espermática, os espermatozoides normais diminuíram, enquanto os espermatozoides com defeitos secundários pós-descongelação aumentaram em ambos os grupos. Conclui-se que um menor tempo de equilíbrio para a glicerolização preserva melhor a qualidade dos espermatozoides epididimários e a etapa mais crítica do processo de congelação-descongelação é a descongelação.(AU)

Cathepsin L Inhibitors with Activity against the Liver Fluke Identified From a Focus Library of Quinoxaline 1,4-di-N-Oxide Derivatives.

Infections caused by Fasciola species are widely distributed in cattle and sheep causing significant economic losses, and are emerging as human zoonosis with increasing reports of human cases, especially in children in endemic areas. The current treatment is chemotherapeutic, triclabendazole being the drug of preference since it is active against all parasite stages. Due to the emergence of resistance in several countries, the discovery of new chemical entities with fasciolicidal activity is urgently needed. In our continuous search for new fasciolicide compounds, we identified and characterized six quinoxaline 1,4-di-N-oxide derivatives from our in-house library. We selected them from a screening of novel inhibitors against FhCL1 and FhCL3 proteases, two essential enzymes secreted by juvenile and adult flukes. We report compounds C7, C17, C18, C19, C23, and C24 with an IC50 of less than 10 µM in at least one cathepsin. We studied their binding kinetics in vitro and their enzyme-ligand interactions in silico by molecular docking and molecular dynamic (MD) simulations. These compounds readily kill newly excysted juveniles in vitro and have low cytotoxicity in a Hep-G2 cell line and bovine spermatozoa. Our findings are valuable for the development of new chemotherapeutic approaches against fascioliasis, and other pathologies involving cysteine proteases.

Evolução da utilização de espermatozoides sexados / Evolution of sexed spermatozoa utilization

A sexagem de espermatozoides tem relevante aplicação na produção animal. Várias publicações demonstram a eficiência do processo de sexagem por citometria de fluxo que separa os espermatozoides X e Y em função da mensuração do conteúdo de DNA. Importantes modificações no processo de sexagem por citometria de fluxo, tais como pressão, intensidade do laser, diluidores tem levado à diminuição dos danos nos espermatozoides e aumentado a viabilidade dos mesmos.

Spermatozoa sexing has an important application for livestock production. Results have been published worldwide that demonstrate effectiveness of the flow cytometer sexing process based on sorting sperm with differential DNA content as the X and Y sperm marker. Major improvements in the sexing sorting process as pressure, laser power, extenders had led to the decrease of sperm damage and increase of sperm viability

Evolução da utilização de espermatozoides sexados / Evolution of sexed spermatozoa utilization

A sexagem de espermatozoides tem relevante aplicação na produção animal. Várias publicações demonstram a eficiência do processo de sexagem por citometria de fluxo que separa os espermatozoides X e Y em função da mensuração do conteúdo de DNA. Importantes modificações no processo de sexagem por citometria de fluxo, tais como pressão, intensidade do laser, diluidores tem levado à diminuição dos danos nos espermatozoides e aumentado a viabilidade dos mesmos.(AU)

Spermatozoa sexing has an important application for livestock production. Results have been published worldwide that demonstrate effectiveness of the flow cytometer sexing process based on sorting sperm with differential DNA content as the X and Y sperm marker. Major improvements in the sexing sorting process as pressure, laser power, extenders had led to the decrease of sperm damage and increase of sperm viability(AU)

Glycerol-3-phosphate acyltransferase 2 is essential for normal spermatogenesis.

Glycerol-3-phosphate acyltransferases (GPATs) catalyze the first and rate-limiting step in the de novo glycerolipid synthesis. The GPAT2 isoform differs from the other isoforms because its expression is restricted to male germ cells and cancer cells. It has been recently reported that GPAT2 expression in mouse testis fluctuates during sexual maturation and that it is regulated by epigenetic mechanisms in combination with vitamin A derivatives. Despite progress made in this field, information about GPAT2 role in the developing male germ cells remains unclear. The aim of the present study was to confirm the hypothesis that GPAT2 is required for the normal physiology of testes and male germ cell maturation. The gene was silenced in vivo by inoculating lentiviral particles carrying the sequence of a short-hairpin RNA targeting Gpat2 mRNA into mouse testis. Histological and gene expression analysis showed impaired spermatogenesis and arrest at the pachytene stage. Defects in reproductive fitness were also observed, and the analysis of apoptosis-related gene expression demonstrated the activation of apoptosis in Gpat2-silenced germ cells. These findings indicate that GPAT2 protein is necessary for the normal development of male gonocytes, and that its absence triggers apoptotic mechanisms, thereby decreasing the number of dividing germ cells.

Participation of phosphofructokinase, malate dehydrogenase and isocitrate dehydrogenase in capacitation and acrosome reaction of boar spermatozoa.

The aim of this work was to determine the enzymatic activity of phosphofructokinase (PFK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in boar spermatozoa and study their participation in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction. Enzymatic activity of these enzymes was determined spectrophotometrically in extracts of boar spermatozoa. Sperm suspensions were incubated in the presence of bicarbonate (40 mM), a well-known capacitation inducer, or follicular fluid (30%), as an acrosome reaction inducer, and different concentrations of oxoglutarate, oxalomalate and hydroxymalonate, inhibitors of PFK, IDH and MDH, respectively. Capacitation percentages were determined by the fluorescence technique of chlortetracycline (CTC), and true acrosome reaction was determined by trypan blue and differential-interferential contrast, optical microscopy. The activity of PFK in boar spermatozoa enzymatic extracts was 1.70 ± 0.19 U/1010 spermatozoa, the activity of NAD- and NADP-dependent IDH was 0.111 ± 0.005 U/1010 and 2.22 ± 0.14 U/1010 spermatozoa, respectively, and the activity of MDH was 4.24 ± 0.38 U/1010 spermatozoa. The addition of the specific inhibitors of these enzymes prevented sperm capacitation and decreased sperm motility during capacitation and inhibited the acrosome reaction (AR), without affecting the sperm motility during this process. Our results demonstrate the participation of PFK, IDH and MDH in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction in boar spermatozoa, contributing to elucidate the mechanisms that produce energy necessary for these processes in porcine spermatozoa.

Protein kinase C activity in boar sperm.

Male germ cells undergo different processes within the female reproductive tract to successfully fertilize the oocyte. These processes are triggered by different extracellular stimuli leading to activation of protein phosphorylation. Protein kinase C (PKC) is a key regulatory enzyme in signal transduction mechanisms involved in many cellular processes. Studies in boar sperm demonstrated a role for PKC in the intracellular signaling involved in motility and cellular volume regulation. Experiments using phorbol 12-myristate 13-acetate (PMA) showed increases in the Serine/Threonine phosphorylation of substrates downstream of PKC in boar sperm. In order to gain knowledge about those cellular processes regulated by PKC, we evaluate the effects of PMA on boar sperm motility, lipid organization of plasma membrane, integrity of acrosome membrane and sperm agglutination. Also, we investigate the crosstalk between PKA and PKC intracellular pathways in spermatozoa from this species. The results presented here reveal a participation of PKC in sperm motility regulation and membrane fluidity changes, which is probably associated to acrosome reaction and to agglutination. Also, we show the existence of a hierarchy in the kinases pathway. Previous works on boar sperm suggest a pathway in which PKA is positioned upstream to PKC and this new results support such model.

Efeito da Insulina-N sobre a Integridade das Membranas Plasmáticas em Sêmen Fresco de Ovino, utilizado Gema de Ovo de Codorna / Insulin- N Effects on Plasma Membranes Integrity in Ovine Fresh Semen, using Quail Egg of Yolk

This study aimed to evaluate the effect of insulin on the membrane integrity evaluated by the eosin testand nigrosine in ovine semen. Seven sheep were used, harvest were made with the aid of eletroejaculator. Theyolk-citrate extender was used for dilution of the ejaculate (25x106 sperm / mL) using quail's egg yolk. Eachejaculate was divided into four equal fractions, corresponding to treatments: control group (Gcont) using onlydilutive citrat gem; Group 50 (G50) plus dilutive 50UI/ml insulin type N; Group 100 (G100) plus dilutive 100IU/Ml Insulin type N; Group 200 (G200) plus dilutive 200 IU/Ml Insulin type N. After dilution, the semenremained in water - bath at 45°C to perform the eosin test and nigrosine for evaluation by time (0, 20, 40 and 60minutes) for membrane integrity. Thus, the use of insulin does not preserve the integrity of the membranesduring the incubation period of 45C, with a gradual decrease due to stress caused by time and temperature atwhich the semen was subjected.

Efeito da Insulina-N sobre a Integridade das Membranas Plasmáticas em Sêmen Fresco de Ovino, utilizado Gema de Ovo de Codorna / Insulin- N Effects on Plasma Membranes Integrity in Ovine Fresh Semen, using Quail Egg of Yolk

This study aimed to evaluate the effect of insulin on the membrane integrity evaluated by the eosin testand nigrosine in ovine semen. Seven sheep were used, harvest were made with the aid of eletroejaculator. Theyolk-citrate extender was used for dilution of the ejaculate (25x106 sperm / mL) using quail's egg yolk. Eachejaculate was divided into four equal fractions, corresponding to treatments: control group (Gcont) using onlydilutive citrat gem; Group 50 (G50) plus dilutive 50UI/ml insulin type N; Group 100 (G100) plus dilutive 100IU/Ml Insulin type N; Group 200 (G200) plus dilutive 200 IU/Ml Insulin type N. After dilution, the semenremained in water - bath at 45°C to perform the eosin test and nigrosine for evaluation by time (0, 20, 40 and 60minutes) for membrane integrity. Thus, the use of insulin does not preserve the integrity of the membranesduring the incubation period of 45C, with a gradual decrease due to stress caused by time and temperature atwhich the semen was subjected.(AU)

Cadmium inhibits motility, activities of plasma membrane Ca(2+)-ATPase and axonemal dynein-ATPase of human spermatozoa.

Cd(2+) has been associated with decreased sperm motility in individuals exposed to this element, such as smokers. Among other factors, this lowered motility could be the result of inhibition exerted by Cd(2+) on the activity of the sperm ATPases associated with sperm motility. In this study, we evaluated the plasma membrane Ca(2+)-ATPase and the axonemal dynein-ATPase activities as well as sperm motility, in the presence of different free Cd(2+) concentrations in the assay media. It was found that spermatozoa incubated for 5 h in a medium containing 25 nm free Cd(2+) showed a significant inhibition of progressive motility, reaching values even lower at higher Cd(2+) concentrations. In addition, it was found that the activity of the plasma membrane Ca(2+)-ATPase reached maximal inhibition at 50 nm free Cd(2+), with a K50% inhibition of 18.3 nm free Cd(2+). The dynein-ATPase activity was maximally inhibited by 25 nm free Cd(2+) in the assay medium, with a K50% inhibition of 11.3 nm Cd(2+). Our results indicate that the decreased activity of the sperm ATPases might have a critical importance in the biochemical mechanisms underlying the decreased sperm motility of individuals exposed to Cd(2+).

Progesterone causes metabolic changes involving aminotransferases and creatine kinase in cryopreserved bovine spermatozoa.

Progesterone (P4) is capable of inducing acrosome reaction in many species. The objective of this study was to determine the activity of enzymes involved in metabolism that contribute to the redox state and supply energy for acrosome reaction in cryopreserved bull spermatozoa. To accomplish this aim, acrosome reaction was induced by P4 in capacitated and non-capacitated samples. Alanine and aspartate aminotransferases (ALT, AST) and creatine kinase (CK) activities were measured spectrophotometrically at 340 nm after acrosome reaction with P4. Oxygen consumption was measured polarographically. ALT and AST activities increased by the addition of P4 capacitated and non-capacitated samples. P4 addition provoked an increase in CK activity in non-capacitated spermatozoa compared to heparin capacitated spermatozoa with or without P4 addition. P4 increased oxygen consumption, the percentage of acrosome reacted spermatozoa as well as the absence of acrosome integrity in both capacitated and non-capacitated bovine spermatozoa, but oxygen consumption in P4 samples was significantly lower than in heparin capacitated spermatozoa (P<0.05). Acrosome reaction induction by P4 required different creatine kinase activity with the same oxygen consumption and transaminases level to maintain oxidative metabolism and redox state through reducing equivalents transfer between cytosolic and mitochondrial compartment. In conclusion, P4 induces a lower oxidative metabolism during acrosome reaction in bovine cryopreserved spermatozoa, compared to heparin induced capacitation process.

Organic selenium supplementation increases PHGPx but does not improve viability in chilled boar semen.

This study evaluated the effects of dietary organic selenium (Se) on viability of chilled boar semen. Twelve boars were divided into three groups: control (CON), 0.3 mg kg(-1) sodium selenite; inorganic (INO), 0.5 mg kg(-1) sodium selenite and organic (ORG), 0.5 mg kg(-1) Se yeast. The experiment was conducted within 10 weeks, and analysis was performed fortnightly, in storage semen by 72 h. No effect was observed on motility; however, straightness and linearity percentages were higher (P < 0.05) in the animals receiving CON diet compared with INO group. Percentages of cells with both plasma and acrosomal intact membranes, lipidic membrane peroxidation and mitochondrial membrane potential were similar on all treatments. Animals receiving CON diet presented higher (P < 0.05) values of ATP when compared with INO group. The PHGPx was higher (P < 0.05) in animals that received ORG in comparison with INO group. In conclusion, organic selenium supplementation increases PHGPx but does not improve chilled semen viability in 72 h.

Central role of soluble adenylyl cyclase and cAMP in sperm physiology.

Cyclic adenosine 3',5'-monophosphate (cAMP), the first second messenger to be described, plays a central role in cell signaling in a wide variety of cell types. Over the last decades, a wide body of literature addressed the different roles of cAMP in cell physiology, mainly in response to neurotransmitters and hormones. cAMP is synthesized by a wide variety of adenylyl cyclases that can generally be grouped in two types: transmembrane adenylyl cyclase and soluble adenylyl cyclases. In particular, several aspects of sperm physiology are regulated by cAMP produced by a single atypical adenylyl cyclase (Adcy10, aka sAC, SACY). The signature that identifies sAC among other ACs, is their direct stimulation by bicarbonate. The essential nature of cAMP in sperm function has been demonstrated using gain of function as well as loss of function approaches. This review unifies state of the art knowledge of the role of cAMP and those enzymes involved in cAMP signaling pathways required for the acquisition of fertilizing capacity of mammalian sperm. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease.

Calcineurin regulates progressive motility activation of Rhinella (Bufo) arenarum sperm through dephosphorylation of PKC substrates.

Animals with external fertilization, as amphibians, store their sperm in a quiescent state in the testis. When spermatozoa are released into natural fertilization media, the hypotonic shock triggers activation of sperm motility. Rhinella (Bufo) arenarum sperm are immotile in artificial seminal plasma (ASP, resembling testicular plasma tonicity) but acquire in situ flagellar beating upon dilution. However, if components from the egg shelly coat are added to this medium, motility shifts to a progressive pattern. Recently, we have shown that the signal transduction pathway required for in situ motility activation involves a rise in intracellular cAMP through a transmembrane adenylyl cyclase and activation of PKA, mostly in the midpiece and in the sperm head. In this report, we demonstrate that activation of calcineurin (aka PP2B and PPP3) is required for the shift from in situ to progressive sperm motility. The effect of calcineurin is manifested by dephosphorylation of PKC substrates, and can be promoted by intracellular calcium rise by Ca(2+) ionophore. Both phosphorylated PKC substrates and calcineurin localized to the flagella, indicating a clear differentiation between compartmentalization of PKA and calcineurin pathways. Moreover, no crosstalk is observed between these signaling events, even though both pathways are required for progressive motility acquisition as discussed.

Protein phosphatases decrease their activity during capacitation: a new requirement for this event.

There are few reports on the role of protein phosphatases during capacitation. Here, we report on the role of PP2B, PP1, and PP2A during human sperm capacitation. Motile sperm were resuspended in non-capacitating medium (NCM, Tyrode's medium, albumin- and bicarbonate-free) or in reconstituted medium (RCM, NCM plus 2.6% albumin/25 mM bicarbonate). The presence of the phosphatases was evaluated by western blotting and the subcellular localization by indirect immunofluorescence. The function of these phosphatases was analyzed by incubating the sperm with specific inhibitors: okadaic acid, I2, endothall, and deltamethrin. Different aliquots were incubated in the following media: 1) NCM; 2) NCM plus inhibitors; 3) RCM; and 4) RCM plus inhibitors. The percent capacitated sperm and phosphatase activities were evaluated using the chlortetracycline assay and a phosphatase assay kit, respectively. The results confirm the presence of PP2B and PP1 in human sperm. We also report the presence of PP2A, specifically, the catalytic subunit and the regulatory subunits PR65 and B. PP2B and PP2A were present in the tail, neck, and postacrosomal region, and PP1 was present in the postacrosomal region, neck, middle, and principal piece of human sperm. Treatment with phosphatase inhibitors rapidly (≤1 min) increased the percent of sperm depicting the pattern B, reaching a maximum of ∼40% that was maintained throughout incubation; after 3 h, the percent of capacitated sperm was similar to that of the control. The enzymatic activity of the phosphatases decreased during capacitation without changes in their expression. The pattern of phosphorylation on threonine residues showed a sharp increase upon treatment with the inhibitors. In conclusion, human sperm express PP1, PP2B, and PP2A, and the activity of these phosphatases decreases during capacitation. This decline in phosphatase activities and the subsequent increase in threonine phosphorylation may be an important requirement for the success of sperm capacitation.

Functional human sperm capacitation requires both bicarbonate-dependent PKA activation and down-regulation of Ser/Thr phosphatases by Src family kinases.

In all mammalian species studied so far, sperm capacitation correlates with an increase in protein tyrosine (Tyr) phosphorylation mediated by a bicarbonate-dependent cAMP/protein kinase A (PKA) pathway. Recent studies in mice revealed, however, that a Src family kinase (SFK)-induced inactivation of serine/threonine (Ser/Thr) phosphatases is also involved in the signaling pathways leading to Tyr phosphorylation. In view of these observations and with the aim of getting a better understanding of the signaling pathways involved in human sperm capacitation, in the present work we investigated the involvement of both the cAMP/PKA and SFK/phosphatase pathways in relation to the capacitation state of the cells. For this purpose, different signaling events and sperm functional parameters were analyzed as a function of capacitation time. Results revealed a very early bicarbonate-dependent activation of PKA indicated by the rapid (1 min) increase in both phospho-PKA substrates and cAMP levels (P < 0.05). However, a complete pattern of Tyr phosphorylation was detected only after 6-h incubation at which time sperm exhibited the ability to undergo the acrosome reaction (AR) and to penetrate zona-free hamster oocytes. Sperm capacitated in the presence of the SFK inhibitor SKI606 showed a decrease in both PKA substrate and Tyr phosphorylation levels, which was overcome by exposure of sperm to the Ser/Thr phosphatase inhibitor okadaic acid (OA). However, OA was unable to induce phosphorylation when sperm were incubated under PKA-inhibitory conditions (i.e. in the absence of bicarbonate or in the presence of PKA inhibitor). Moreover, the increase in PKA activity by exposure to a cAMP analog and a phosphodiesterase inhibitor did not overcome the inhibition produced by SKI606. Whereas the presence of SKI606 during capacitation produced a negative effect (P < 0.05) on sperm motility, progesterone-induced AR and fertilizing ability, none of these inhibitions were observed when sperm were exposed to SKI606 and OA. Interestingly, different concentrations of inhibitors were required to modulate human and mouse capacitation revealing the species specificity of the molecular mechanisms underlying this process. In conclusion, our results describe for the first time the involvement of both PKA activation and Ser/Thr phosphatase down-regulation in functional human sperm capacitation and provide convincing evidence that early PKA-dependent phosphorylation is the convergent regulatory point between these two signaling pathways.

Período de permanência de espermatozoides em glândulas hospedeiras de espermatozoides e glândulas infundibulares em codorna de corte / Stay period of sperm in sperm-storage glands and in infundibular glands in quails

Para determinar o tempo de permanência de espermatozoides nas glândulas hospedeiras de espermatozoides (GHEs) e nas glândulas infundibulares (GIs) de codorna de corte (Coturnix coturnix coturnix), foram utilizados 12 machos e 66 fêmeas, totalizando 78 codornas em fase reprodutiva. As fêmeas foram distribuídas em 11 grupos e acasaladas por 24 horas em gaiolas individuais. Os machos, utilizados de modo intercalado, foram separados do contato com as fêmeas e colocados em descanso. As aves do grupo-controle (G0 - seis fêmeas) foram abatidas no início do experimento, enquanto as 60 fêmeas acasaladas foram distribuídas em 10 grupos (G1 a G10, com seis fêmeas cada) e abatidas a cada período de 24 horas, de forma sequencial. Fragmentos foram obtidos da região uterovaginal e do infundíbulo e submetidos às análises histológica, histoquímica e histométrica com técnicas de rotina. Os resultados morfométricos mostraram que 46% das GHEs continham espermatozoides em seu lume no primeiro dia após o acasalamento, diminuindo gradativamente nos dias posteriores chegando a 3% no quinto dia. Nesse período, os espermatozoides ascendem em direção às GIs, onde permanecem viáveis e férteis por, pelo menos, 96 horas após deixarem as GHEs, possibilitando a postura de ovos férteis por 10 dias, em média, após o acasalamento.

Sperm-Storage Tubules (SSPs) and Infundibular Tubules (ITs) are the structures responsible for sperm storage in the oviduct of birds, snakes, alligators and turtles after mating. Aiming to determine length of stay of sperm-storage tubules (SSPs) and infundibular tubules (ITs) cutting quail, Coturnix coturnix coturnix, we used 12 males and 66 females, totaling 79 quails in the reproductive phase. The females were allocated into 11 groups and mated for 24 hours in individual cages. The males used were merged and separated from contact with females and placed at rest. The poultry of the control-group (G0 six females) was slaughtered at the beginning of the experiment, the 60 previously mated females were allocated into 10 groups (G1 to G10, with six females each) and were slaughtered sequentially. On the 10th day, the last group (G10) was shot. The fragments obtained from the utero-vaginal region and the infundibulum of each female underwent histological techniques, immunohistochemistry and morphometry routine. The morphometric results showed that GHEs had 46% of the sperm in his heat on day 1 after mating, decreasing gradually in the after days reaching 3% on day 5. At this time they increase toward the infundibular tubules, where they remain viable and fertile for at least another 96 hours (4 days) after leaving the SSPs, allowing these birds to lay fertile eggs for 10 days on average after mating.

Activation of proacrosin accompanies upregulation of sp32 protein tyrosine phosphorylation in pig sperm.

This study investigated the relationship between acrosin activation and pig sperm proacrosin binding protein (sp32) phosphorylation levels. Differently processed pig spermatozoa (fresh semen sperm, capacitation sperm, acrosome reaction sperm, capacitation-like sperm, and thawed sperm) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis. The fresh semen and capacitation sperm groups both produced proacrosin protein bands of 55 kDa; however, the result of the fresh semen sperm group was clearer than that of the capacitation sperm group. The thawed sperm group showed a shallow strip at 55 kDa. The capacitation and acrosome reaction sperm groups produced obvious proacrosin protein bands at 35 kDa, and the strips of the capacitation sperm group were again clearer. A faint band was visible at 32 kDa in the acrosome reaction sperm group. The capacitation, thawed, and acrosome reaction sperm groups showed significant strips in sp32, and the bands of the acrosome reaction sperm group were shallower than those of the 2 other groups. The capacitation and thawed sperm groups produced significant strips at 40 kDa, and the capacitation sperm group produced an additional strip at 55 kDa. In conclusion, sp32 phosphorylation levels can promote proacrosin activation into the active acrosin.