Role of Wasp and the small GTPases RhoA, RhoB, and Cdc42 during capacitation and acrosome reaction in spermatozoa of English guinea pigs.

Cytoskeleton remodeling is necessary for capacitation and the acrosome reaction in spermatozoa. F-actin is located in the acrosome and equatorial region during capacitation, but is relocated in the post-acrosomal region during the acrosome reaction in spermatozoa from bull, rat, mice, and guinea pig. Actin polymerization and relocalization are generally regulated by small GTPases that activate Wasp protein, which coordinates with Arp2/3, profilin I, and profilin II to complete cytoskeletal remodeling. This sequence of events is not completely described in spermatozoa, though. Therefore, the aim of this study was to determine if Wasp interacts with small GTPases (RhoA, RhoB, and Cdc42) and proteins (Arp2/3, profilin I, and profilin II) that co-localize with F-actin during capacitation and the acrosome reaction in English guinea pig spermatozoa obtained from the vas deferens. The spermatozoa were capacitated in calcium-free medium, incubated with an activator or an inhibitor of GTPases, and then induced to acrosome react using calcium. The distribution patterns of F-actin were compared to the patterns of Wasp and its putative interaction partners: Wasp and RhoB, but not RhoA or Cdc42, localization overlap with F-actin during capacitation and the acrosome reaction. Activation of small GTPases localized RhoB to the post-acrosomal region whereas their inhibition prevented acrosome exocytosis. Arp2/3 and profilin II appear to interact with Wasp in the post-acrosomal region and flagellum, while profilin I and Wasp could be found in the equatorial region. Thus, Wasp and F-actin distribution overlap during capacitation and acrosome reaction, and small GTPases play an important role in cytoskeleton remodeling during these processes in spermatozoa. Mol. Reprod. Dev. 83: 927-937, 2016 © 2016 Wiley Periodicals, Inc.

ADAM15 participates in fertilization through a physical interaction with acrogranin.

Mammalian fertilization is completed by direct interaction between sperm and egg. This process is primarily mediated by both adhesion and membrane-fusion proteins found on the gamete surface. ADAM1, 2, and 3 are members of the ADAMs protein family, and have been involved in sperm-egg binding. In this study, we demonstrate the proteolytic processing of ADAM15 during epididymal maturation of guinea pig spermatozoa to produce a mature form a size of 45 kDa. We find that the size of the mature ADAM15, 45 kDa, in cauda epididymal spermatozoa indicates that the pro-domain and metalloprotease domain are absent. In addition, using indirect immunofluorescence, ADAM15 was found throughout the acrosome, at the equatorial region and along the flagellum of guinea pig spermatozoa. After acrosome reaction, ADAM15 is lost from the acrosomal region and retained in the equatorial region and flagellum. In this study, we also report the first evidence of a complex between ADAM15 and acrogranin. By immunoprecipitation, we detected a protein band of 65 kDa which co-immunoprecipated together ADAM15. Analysis of the N-terminal sequence of this 65 kDa protein has revealed its identity as acrogranin. In addition, using cell-surface labeling, ADAM15 was found to be present on the cell surface. Assays of heterologous fertilization showed that the antibody against acrogranin inhibited the sperm-egg adhesion. Interestingly, ADAM15 and acrogranin were also found associated in two breast cancer cell lines. In conclusion, our results demonstrated that ADAM15 and acrogranin are present on and associated with the surface of guinea pig spermatozoa; besides both proteins may play a role during sperm-egg binding.

A glycolytic metabolon in Saccharomyces cerevisiae is stabilized by F-actin.

In the Saccharomyces cerevisiae glycolytic pathway, 11 enzymes catalyze the stepwise conversion of glucose to two molecules of ethanol plus two CO2 molecules. In the highly crowded cytoplasm, this pathway would be very inefficient if it were dependent on substrate/enzyme diffusion. Therefore, the existence of a multi-enzymatic glycolytic complex has been suggested. This complex probably uses the cytoskeleton to stabilize the interaction of the various enzymes. Here, the role of filamentous actin (F-actin) in stabilization of a putative glycolytic metabolon is reported. Experiments were performed in isolated enzyme/actin mixtures, cytoplasmic extracts and permeabilized yeast cells. Polymerization of actin was promoted using phalloidin or inhibited using cytochalasin D or latrunculin. The polymeric filamentous F-actin, but not the monomeric globular G-actin, stabilized both the interaction of isolated glycolytic pathway enzyme mixtures and the whole fermentation pathway, leading to higher fermentation activity. The associated complexes were resistant against inhibition as a result of viscosity (promoted by the disaccharide trehalose) or inactivation (using specific enzyme antibodies). In S. cerevisiae, a glycolytic metabolon appear to assemble in association with F-actin. In this complex, fermentation activity is enhanced and enzymes are partially protected against inhibition by trehalose or by antibodies.

The acrosomal matrix from guinea pig sperm contains structural proteins, suggesting the presence of an actin skeleton.

The mammalian sperm acrosome contains a large number of hydrolytic enzymes. When the acrosomal reaction and fertilization occur, these enzymes are released in an orderly fashion, suggesting that the acrosomal matrix is highly organized. It was decided to determine the identity of the structural scaffold underlying the organization of the acrosome. In permeabilized acrosomes and in the Triton X-100-extracted acrosomal matrices from guinea pig sperm, we used indirect immunofluorescence, immunogold labeling, and Western blotting to identify F-actin, spectrin, myosin, calmodulin, and gelsolin. These proteins were detected in the acrosomal matrix for the first time. In noncapacitated, intact spermatozoa the addition of the F-actin monomerizing agent cytochalasin D resulted in loss of the acrosome, suggesting that F-actin is needed to preserve an intact acrosome. Our results suggest that the acrosomal architecture is supported by a dynamic F-actin skeleton, which probably regulates the differential rate of release of the acrosomal enzymes during acrosomal reaction and fertilization.

Calpain modulates capacitation and acrosome reaction through cleavage of the spectrin cytoskeleton.

Research on fertilization in mammalian species has revealed that Ca(2+) is an important player in biochemical and physiological events enabling the sperm to penetrate the oocyte. Ca(2+) is a signal transducer that particularly mediates capacitation and acrosome reaction (AR). Before becoming fertilization competent, sperm must experience several molecular, biochemical, and physiological changes where Ca(2+) plays a pivotal role. Calpain-1 and calpain-2 are Ca(2+)-dependent proteases widely studied in mammalian sperm; they have been involved in capacitation and AR but little is known about their mechanism. In this work, we establish the association of calpastatin with calpain-1 and the changes undergone by this complex during capacitation in guinea pig sperm. We found that calpain-1 is relocated and translocated from cytoplasm to plasma membrane (PM) during capacitation, where it could cleave spectrin, one of the proteins of the PM-associated cytoskeleton, and facilitates AR. The aforementioned results were dependent on the calpastatin phosphorylation and the presence of extracellular Ca(2+). Our findings underline the contribution of the sperm cytoskeleton in the regulation of both capacitation and AR. In addition, our findings also reveal one of the mechanisms by which calpain and calcium exert its function in sperm.

The association of glycolytic enzymes from yeast confers resistance against inhibition by trehalose.

During stress, many organisms accumulate compatible solutes. These solutes must be eliminated upon return to optimal conditions as they inhibit cell metabolism and growth. In contrast, enzyme interactions optimize metabolism through mechanisms such as channeling of substrates. It was decided to test the (compatible solute) trehalose-mediated inhibition of some yeast glycolytic pathway enzymes known to associate and whether inhibition is prevented when enzymes are allowed to associate. Trehalose inhibited the isolated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hexokinase (HXK), but not aldolase (ALD) nor phosphoglycerate kinase (PGK). When these enzymes were mixed in pairs, both GAPDH and HXK were protected by either ALD or PGK acquiring the inhibition behavior of the resistant enzyme. GAPDH was not protected by HXK, albumin or lactate dehydrogenase (LDH). Also, ALD did not protect glucose 6-phosphate dehydrogenase (G6PDH), suggesting that protection is specific. In yeast cell extracts, fermentation was resistant to trehalose inhibition, suggesting all enzymes involved in the glucose-dependent production of ethanol were stabilized. It is suggested that during the yeast stress response, enzyme association protects some metabolic pathways against trehalose-mediated inhibition.

Possible participation of calmodulin in the decondensation of nuclei isolated from guinea pig spermatozoa.

The guinea pig spermatozoid nucleus contains actin, myosin, spectrin and cytokeratin. Also, it has been reported that phalloidin and/or 2,3-butanedione monoxime retard the sperm nuclear decondensation caused by heparin, suggesting a role for F-actin and myosin in nuclear stability. The presence of an F-actin/myosin dynamic system in these nuclei led us to search for proteins usually related to this system. In guinea pig sperm nuclei we detected calmodulin, F-actin, the myosin light chain and an actin-myosin complex. To define whether calmodulin participates in nuclear-dynamics, the effect of the calmodulin antagonists W5, W7 and calmidazolium was tested on the decondensation of nuclei promoted by either heparin or by a Xenopus laevis egg extract. All antagonists inhibited both the heparin- and the X. laevis egg extract-mediated nuclear decondensation. Heparin-mediated decondensation was faster and led to loss of nuclei. The X. laevis egg extract-promoted decondensation was slower and did not result in loss of the decondensed nuclei. It is suggested that in guinea pig sperm calmodulin participates in the nuclear decondensation process.

In guinea pig sperm, aldolase A forms a complex with actin, WAS, and Arp2/3 that plays a role in actin polymerization.

Glycolytic enzymes have, in addition to their role in energy production, other functions in the regulation of cellular processes. Aldolase A has been reported to be present in sperm, playing a key role in glycolysis; however, despite its reported interactions with actin and WAS, little is known about a non-glycolytic role of aldolase A in sperm. Here, we show that in guinea pig spermatozoa, aldolase A is tightly associated to cytoskeletal structures where it interacts with actin, WAS, and Arp2/3. We show that aldolase A spermatozoa treatment increases their polymerized actin levels. In addition, we show that there is a direct correlation between the levels of polymerized actin and the levels of aldolase A-actin interaction. Our results suggest that aldolase A functions as a bridge between filaments of actin and the actin-polymerizing machinery.

Presence, processing, and localization of mouse ADAM15 during sperm maturation and the role of its disintegrin domain during sperm-egg binding.

Successful fertilization requires gametes to complete several stages, beginning with maturation and transport along the male and female reproductive tracts and ending with the interaction between the sperm and the egg. This last step involves sperm-egg adhesion and membrane fusion. ADAMs (disintegrin and metalloprotease domain proteins) are a family of membrane-anchored glycoproteins that are thought to play diverse roles in cell-cell adhesion through their interaction with integrins. This study analyzes the presence, location, processing, and possible role of ADAM15 in mouse sperm. The presence of ADAM15 in mouse spermatozoa was detected by Western blotting, which revealed that ADAM15 is post-translationally processed, during epididymal sperm maturation and the acrosome reaction. The 35 kDa antigen present in the acrosome-reacted sperm is the last proteolytic product of the 110/75 kDa ADAM15 found in non-capacitated sperm. This 35 kDa protein contains the disintegrin domain. By indirect immunofluorescence, ADAM15 was identified in the acrosomal region and along the flagellum of mouse spermatozoa. In acrosome-reacted sperm, ADAM15 was lost from the acrosomal region, but remained diffusely distributed throughout the head and flagellum. Furthermore, the ADAM15 disintegrin domain (RPPTDDCDLPEF) partially inhibited fusion and almost completely inhibited sperm-oolemma adhesion. In conclusion, our data indicate that ADAM15 is present in the testis and in spermatozoa from the caput, corpus, and cauda epididymis, as well as in non-capacitated and acrosome-reacted gametes. Results also indicate that ADAM15 is processed during epididymal maturation and acrosome reaction and that it may play a role during sperm-egg binding.

In spermatozoa, Stat1 is activated during capacitation and the acrosomal reaction.

A role for sperm-specific proteins during the early embryonic development has been suggested by a number of recent studies. However, little is known about the participation of transcription factors in that stage. Here, we show that the signal transducer and activator of transcription 1 (Stat1), but not Stat4, was phosphorylated in response to capacitation and the acrosomal reaction (AR). Moreover, Stat1 phosphorylation correlated with changes in its localization: during capacitation, Stat1 moved from the cytoplasm to the theca/flagellum fraction. During AR, Stat1 phosphorylation increased again. In addition, blocking protein kinase A (PKA) and PKC during capacitation abolished both phosphorylation and migration of Stat1. Our results show tight spatio-temporal rearrangements of Stat1, suggesting that after fertilization Stat1 participates in the first rounds of transcription within the male pronucleus.

F-actin involvement in guinea pig sperm motility.

Sperm motility is a must for natural fertilization to occur. During their travel through the epididymis, mammalian spermatozoa gradually acquire the ability to move. This is accomplished through a sliding movement of the outer doublet microtubules of the axoneme which is energized by the dynein ATPase. Within its complex structure, the mammalian sperm flagellum contains F-actin and thus, we decided to test in the guinea pig sperm flagellum the role of F-actin in motility. During maturation, capacitation, and the acrosome reaction, a gradual decrease of the relative concentration of F-actin was observed. Motility increased as spermatozoa became able to fertilize. Gelsolin, phalloidin, and KI inhibited sperm motility. Gelsolin canceled sperm motility within 20 min of treatment while 0.6 M KI had immediate effects. Phalloidin diminished hyperactive sperm motility slightly. All three compounds significantly increased the relative concentration of F-actin. Latrunculins are conventional drugs that destabilize the F-actin cytoskeleton. Latrunculin A (LAT A) did not affect sperm motility; but significantly increased F-actin relative concentration. The results suggested that in guinea pig spermatozoa, randomly severing F-actin filaments inhibits flagellar motility; while end filament alteration does not. Thus, specific filament regions seem to be important for sperm motility.

In guinea pig spermatozoa, the procaine-promoted synchronous acrosome reaction results in highly fertile cells exhibiting normal F-actin distribution.

In guinea pig spermatozoa, procaine induces Ca(2+) independent hyperactivated motility suggestive of sperm capacitation. Nonetheless, in the presence of high extracellular Ca(2+), procaine increases cytoplasmic Ca(2+). We analyze the procaine effect on the acrosome reaction (AR) processes in guinea pig spermatozoa. Results indicated that: (i) in spermatozoa pre-incubated 5-30 min in MCM-PLG medium, procaine produced synchronous AR, (ii) the acrosome-reacted sperm number increased with the capacitation period before procaine treatment and with procaine concentration, (iii) acrosome reaction was blocked when Ca(2+) was omitted, (iv) plasma membrane-outer acrosomal membrane fusion started within 2 min after procaine treatment, (v) in acrosome-reacted spermatozoa, actin polymerization occurred and F-actin was located in the equatorial and post-acrosomal regions and (vi) procaine treatment resulted in highly fertile acrosome-reacted spermatozoa. This is the first report indicating that procaine promotes synchronic AR in mammalian spermatozoa. If procaine promotes premature AR of spermatozoa in vivo, it might be a factor for infertility in patients exposed to this local anesthetic.

Actin, myosin, cytokeratins and spectrin are components of the guinea pig sperm nuclear matrix.

The nuclear matrix (NM) of somatic cells is an internal nuclear framework structure, with a structural function and participation in DNA replication and transcription. The NM has been described in mouse, hamster and human spermatozoa. In this study, an NM structural component of the guinea pig sperm nucleus was obtained by removing nuclear proteins and DNA from DTT-CTAB nuclei. Removal was achieved with high ionic strength salt and microccocal nuclease treatments including a heparin treatment to cause a slight swelling of the nucleus and facilitate material extraction. Actin, myosin, cytokeratins and spectrin were detected associated to NM by indirect immunofluorescence, immunogold staining and Western blotting analysis using specific antibodies. The presence of NM in guinea pig sperm nucleus is shown for the first time and some of its components are identified. This is also the first report on cytokeratins and myosin presence in guinea pig sperm. A retarding effect of nuclear decondensation caused by heparin is induced after phalloidin and/or diacetyl-monoxime (a myosin ATPase activity inhibitor) treatment, suggesting a role for F-actin and myosin in the maintenance of nuclear stability in sperm. The actin role was supported by the decondensing effect that citochalasin D and gelsolin had on sperm nuclei.

Actin polymerization in the equatorial and postacrosomal regions of guinea pig spermatozoa during the acrosome reaction is regulated by G proteins.

The acrosome reaction (AR) is an exocytotic process of spermatozoa, and an absolute requirement for fertilization. During AR, actin polymerization is necessary in the equatorial and postacrosomal regions of guinea pig sperm for spermatozoa incorporation deep into the egg cytoplasm, but not for plasma membrane (PM) fusion nor the early steps of egg activation. To identify the mechanisms involved in this sperm actin polymerization, we searched for the protein members, known to be involved in a highly conserved model, that may apply to any cellular process in which de novo actin polymerization occurs from G protein activation. WASP, Arp 2/3, profilins I and II, and Cdc42, RhoA and RhoB GTPases were localized by indirect immunofluorescence (IIF) in guinea pig spermatozoa and their presence corroborated by Western blotting. WASP and profilin II were translocated to the postacrosomal region (Arp2/3 already were there) in long-term capacitated and acrosome-reacted spermatozoa, at the same time as actin polymerization occurred. These events were inhibited by GDP-beta-S and promoted by lysophosphatidic acid (LPA) and GTP-gamma-S, a small GTPase inhibitor and two activators, respectively. By immunoprecipitation, Cdc42-WASp association was identified in capacitated but not in noncapacitated gametes. Polymerized actin in the postacrosomal region is apparently anchored both to the postacrosomal perinuclear theca region and the overlying PM. Results suggest that GTPases are involved in sperm actin polymerization, in the postacrosomal region and the mechanism for polymerization might fit a previously proposed model (Mullins, 2000: Curr Opin Cell Biol 12:91-96).

The role of F-actin cytoskeleton-associated gelsolin in the guinea pig capacitation and acrosome reaction.

The acrosomal reaction (AR) is a regulated sperm exocytotic process that involves fusion of the plasma membrane (PM) with the outer acrosomal membrane (OAM). Our group has described F-actin cytoskeletons associated to these membranes. It has been proposed that in regulated exocytosis, a cortical cytoskeleton acts as a barrier that obstructs membrane fusion, and must be disassembled for exocytosis to occur. Actin-severing proteins from the gelsolin family have been considered to break this barrier. The present study attempted to determine if gelsolin has a function in guinea pig sperm capacitation and AR. By indirect immunofluorescence (IIF), gelsolin was detected in the apical and postacrosomal regions of the head and in the flagellum in both capacitated and non-capacitated guinea pig spermatozoa. By Western blotting, gelsolin was detected in isolated PM and OAM of non-capacitated spermatozoa. Gelsolin and actin were detected in a mixture of PM-OAM obtained by sonication, and both proteins were absent in membranes of capacitated spermatozoa. Inhibition of three different pathways of PIP2 hydrolysis during capacitation did not cancel gelsolin loss from membranes. Gelsolin was detected by Western blotting associated to membrane cytoskeletons obtained after phalloidin F-actin stabilization and Triton-X treatment; additionally, by immunoprecipitation, it was shown that gelsolin is associated with actin. By electron microscopy we observed that skeletons disassemble during capacitation, but phalloidin prevents disassembly. A three-dimensional skeleton was observed that apparently joins PM with OAM. Exogenous gelsolin stimulates AR assayed in a permeabilized spermatozoa model. Results suggest that gelsolin disassembles F-actin cytoskeletons during capacitation, promoting AR.

Perinuclear theca during spermatozoa maturation leading to fertilization.

Mammalian spermatozoa acquire the capacity to fertilize the ovum and display motility during their passage through the epididymis. At the same time, they undergo changes in metabolic patterns, enzymatic activities, ability to bind to zona pellucida surface, and electrophoretic properties and, furthermore, stabilization of some sperm structures by the establishment of disulphide linkages takes place in several sperm structures. The cytoplasmic perinuclear theca (PT) is a unique extranuclear cytoskeletal element that surrounds the nucleus, which is proposed to be a structural scaffold to the sperm nucleus. The purpose of this review is to describe PT changes related to epididymal sperm maturation. We will focus mainly on the protein components of the PT of eutherian mammalian spermatozoa and on quantitative protein changes during sperm maturation. The protein constituents of the PT have not been completely defined and most of them are different from the cytoskeletal proteins of somatic cells. However, they are proteins with cytoskeletal features. The morphologic changes reported for PT and the proposed functions of PT are discussed.

Cytochalasin-D retards sperm incorporation deep into the egg cytoplasm but not membrane fusion with the egg plasma membrane.

The fertilization process is impaired when spermatozoa are previously incubated with Cytochalasin-D (Cyt-D). Although this fact reveals the participation of polymerized actin in fertilization, the specific event obstructed by Cyt-D treatment has not been determined. To identify this event, we capacitated guinea pig spermatozoa in minimal capacitating medium with pyruvate and lactate (MCM-PL) with Cyt-D, to inseminate hamster zona pellucida (ZP)-free eggs. Cyt-D (70 microM) decreased F-actin relative concentration in capacitated spermatozoa to a larger extent than in spermatozoa incubated under control conditions. Cyt-D also cancelled the F-actin increase normally observed in acrosome-reacted cells, and decreased the number of these cells with normal F-actin localization at the equatorial zone. Insemination of eggs with Cyt-D treated spermatozoa did not change early fertilization events such as the egg cortical reaction (CR), membranes fusion, and egg F-actin new localization, but clearly retarded, by 16 hr, spermatozoa incorporation deep into the egg cytoplasm, and decondensation of egg metaphase II chromosomes. These results show that actin polymerization is necessary for spermatozoa incorporation deep into the egg cytoplasm, but not for plasma membrane fusion nor egg activation early steps.