Adela Mújica

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.

Summary: 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 aXenopus laevisegg extract. All antagonists inhibited both the heparin- and theX. laevisegg extract-mediated nuclear decondensation. Heparin-mediated decondensation was faster and led to loss of nuclei. TheX. laevisegg 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.

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. Mol. Reprod. Dev.

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‐β‐S and promoted by lysophosphatidic acid (LPA) and GTP‐γ‐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). Mol. Reprod. Dev. 70: 198–210, 2005.

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. Cell Motil. Cytoskeleton 56:94–108, 2003.

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. Microsc. Res. Tech. 61:76–87, 2003.

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 μM) 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. Mol. Reprod. Dev. 63: 518–528, 2002.

The sp50 protein localized at the acrosomal region of guinea pig sperm was suggested to participate in acrosome exocytosis, the acrosome reaction (AR). On the other hand, the cortical reaction (CR), also an exocytotic event, occurs during egg activation. The aim of the present work was to identify sp50 and also to define if sp50 is present in hamster eggs, as well as its location before and after CR. Sp50 was identified as calreticulin (CRT), based on: (a) its NH₂‐terminal amino acid (25 aa) sequence, (b) a cross‐recognition of pure sp50 and pure CRT with anti‐CRT (from Santa Cruz, anti‐CRTsc), and anti‐sp50 (anti‐sp50/CRT) antibodies, respectively, and (c) that both antibodies revealed a 50 kDa protein in a Brij sperm extract. On the other hand, CRT presence in eggs was positively determined by Western blotting (Wb) using anti‐sp50/CRT antibody which recognized a 60 kDa protein in the egg extract, and by indirect immunofluorescence (IIF), CRT was located in the cortical granules (CG). It was defined by a granular pattern and co‐localization with mannose, a specific carbohydrate of the CG. Additionally, a decrease in CRT concentration occurred in eggs after their activation and, in parallel, the protein was revealed in the egg's incubation medium. In activated eggs with zona pellucida (ZP), CRT remains as a halo in the perivitelline space and around the polar body. From these results we suggest that: (1) CRT is present in the CG of non‐activated hamster eggs, (2) CRT is exocytosed during the CR, in response to egg activation, and (3) CRT might participate in the block to polyspermy, together with other CG components. Mol. Reprod. Dev. 60:405‐413,

The acrosome reaction (AR) is a regulated exocytotic process. In several cell types, an actin network situated under the plasma membrane (PM) acts as a physical barrier to prevent this exocytosis. In seeking a function for a cortical skeleton in guinea pig spermatozoa, the PM and the outer acrosomal membrane (OAM) were investigated for the presence of F-actin and spectrin, proteins generally found in cell cortical skeletons. Both membrane types were visualized in whole-mount preparations by electron microscopy. PM proteins gave positive reaction to the Na⁺,K⁺-ATPase antibody and the OAM proteins did not react to the antibody. Furthermore, a Triton X-100-resistant skeleton was obtained from both membrane types. Using gold immunoelectron microscopy, F-actin was visualized in the PM and in the OAM skeletons, while spectrin was only detected in the PM skeleton. The presence of an F-actin cortical skeleton in the sperm PM suggests that F-actin may be involved in the AR. The significantly higher number of AR elicited by cytochalasin D (Cyt-D) treatment(P < 0.005) and data showing a significant (P > 0.03) decrease in F-actin relative concentration in capacitating spermatozoa, agree with this suggestion. Furthermore, the proposal is strengthened by the fact that stabilization of F-actin by phalloidin (Ph) significantly (P > 0.01) diminished AR induced by Ca²⁺ in a streptolysin O (SLO)-permeabilized sperm model. Cell Motil. Cytoskeleton 46:43–58, 2000