Silvia A. Belmonte

Acrosomal exocytosis, a special type of regulated secretion

The acrosome is a single secretory granule present in the head of mammalian ‐ and other animal groups ‐ sperm. Secretion of this granule is an absolute requirement for physiological fertilization. Acrosome exocytosis is a synchronized and tightly regulated all‐or‐nothing process, with no recycling of membranes. In the last few years, it has been shown that acrosomal exocytosis is mediated by a molecular mechanism that is homologous to that reported in the secretion of neuroendocrinal cells. Moreover, because of its particular characteristics, acrosomal exocytosis is a unique mammalian model for the study of the different steps of the membrane fusion cascade. Combining results in intact and permeabilized sperm, the following sequence of events has been proposed. In resting sperm, SNARE proteins are locked in inactive cis complexes. Sperm activation causes a calcium increase in the cytoplasm that promotes the production of cAMP and activates Rab3A. Afterwards, NSF and αSNAP disassemble cis complexes and the free SNAREs are then able to reassemble in loose trans complexes. Membrane fusion is arrested at this stage until calcium is released from inside the acrosome by inositol 1,4,5‐trisphosphate‐sensitive calcium channels to trigger the final steps of membrane fusion, which require fully assembled trans SNARE complexes and the calcium sensor synaptotagmin. This working model is still incomplete and tentative. Its improvement will be important to share light on this and other processes of regulated exocytosis. Moreover, it will bring new perspectives into the field of sperm‐related fertility and sterility. IUBMB Life, 59: 286‐292, 2007

The sesquiterpene lactone dehydroleucodine (DhL) affects the growth of cultured epimastigotes of Trypanosoma cruzi

Here,we report an inhibitory effect of a sesquiterpene lactone dehydroleucodine (DhL) on the growth of Trypanosoma cruzi in culture. At concentrations of the drug between 5 and 10 µg/ml in the medium,the parasites remained alive for at least 4 days. Higher concentrations of DhL were lethal for the parasites within a few hours. The effect of DhL is irreversible. Morphological changes induced by DhL were also observed in the parasites. The effect of DhL was blocked by the presence of reducing substrates such as glutathione or dithiothreitol, but these agents were not able to reverse the effect of DhL if added 2 days after the start of drug exposure.

Membrane-permeant Rab3A triggers acrosomal exocytosis in living human sperm.

The acrosome reaction is a regulated Ca2+‐dependent secretion event required for sperm‐egg interaction. Previous studies indicate that the process requires Rab3‐dependent tethering of membranes, SNARE complex assembly, and Ca2+‐mediated activation of synaptotagmin. Sperm are transcriptionally and translationally inactive; hence, most studies of the exocytosis mechanism are limited to membrane‐per‐meant reagents. The effect of proteins involved in exocytosis has been assessed only in permeabilized cells. Polyarginine peptides are a powerful tool for delivering macromolecules to cells. Most reports indicate that membrane translocation of arginine‐containing proteins requires endocytosis; therefore, this strategy might not be useful in sperm. However, our results indicate that GST and Rab3A, when fused with an arginine‐rich peptide, were able to translocate into sperm. Moreover, membrane‐permeant Rab3A initiated exocytosis when prenylated and activated with GTP. We show here that a key event after the cytoplas‐mic Ca2+ increase caused by progesterone is the activation of Rab3A. When active Rab3A is introduced into sperm, Ca2+ in the extracellular medium and in the cytoplasm is dispensable. However, a Ca2+ efflux from inside the acrosome is still required to achieve exocy‐tosis. In conclusion, arginine‐containing proteins can penetrate the sperm plasma membrane and thus are valuable tools to study sperm physiology in intact cells.— Lopez, C. I., Belmonte, S. A., De Blas, G. A., Mayorga, L. S. Membrane‐permeant Rab3A triggers acrosomal exocytosis in living human sperm. FASEB J. 21, 4121–4130 (2007)

Sphingosine 1-Phosphate and Sphingosine Kinase Are Involved in a Novel Signaling Pathway Leading to Acrosomal Exocytosis

Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. Sphingosine 1-phosphate is a bioactive sphingolipid that regulates crucial physiological processes. Here we report that this lipid triggers acrosomal exocytosis in human sperm by a mechanism involving a Gi-coupled receptor. Real-time imaging showed a remarkable increase of cytosolic calcium upon activation with sphingosine 1-phosphate and pharmacological experiments indicate that the process requires extracellular calcium influx through voltage and store-operated calcium channels and efflux from intracellular stores through inositol 1,4,5-trisphosphate-sensitive calcium channels. Sphingosine 1-phosphate-induced exocytosis requires phospholipase C and protein kinase C activation. We investigated possible sources of the lipid. Western blot indicates that sphingosine kinase 1 is present in spermatozoa. Indirect immunofluorescence showed that phorbol ester, a potent protein kinase C activator that can also trigger acrosomal exocytosis, redistributes sphingosine kinase 1 to the acrosomal region. Functional assays showed that phorbol ester-induced exocytosis depends on the activation of sphingosine kinase 1. Furthermore, incorporation of 32P to sphingosine demonstrates that cells treated with the phorbol ester increase their sphingosine kinase activity that yields sphingosine 1-phosphate. We present here the first evidence indicating that human spermatozoa produce sphingosine 1-phosphate when challenged with an exocytic stimulus. These observations point to a new role of sphingosine 1-phosphate in a signaling cascade that facilitates acrosome reaction providing some clues about novel lipid molecules involved in exocytosis.

Diacylglycerol stimulates acrosomal exocytosis by feeding into a PKC- and PLD1-dependent positive loop that continuously supplies phosphatidylinositol 4,5-bisphosphate.

Acrosomal exocytosis involves a massive fusion between the outer acrosomal and the plasma membranes of the spermatozoon triggered by stimuli that open calcium channels at the plasma membrane. Diacylglycerol has been implicated in the activation of these calcium channels. Here we report that this lipid promotes the efflux of intraacrosomal calcium and triggers exocytosis in permeabilized human sperm, implying that diacylglycerol activates events downstream of the opening of plasma membrane channels. Furthermore, we show that calcium and diacylglycerol converge in a signaling pathway leading to the production of phosphatidylinositol 4,5-bisphosphate (PIP(2)). Addition of diacylglycerol promotes the PKC-dependent activation of PLD1. Rescue experiments adding phosphatidic acid or PIP(2) and direct measurement of lipid production suggest that both PKC and PLD1 promote PIP(2) synthesis. Inhibition of different steps of the pathway was reverted by adenophostin, an agonist of IP(3)-sensitive calcium channels, indicating that PIP(2) is necessary to keep these channels opened. However, phosphatidic acid, PIP(2), or adenophostin could not trigger exocytosis by themselves, indicating that diacylglycerol must also activate another factor. We found that diacylglycerol and phorbol ester stimulate the accumulation of the GTP-bound form of Rab3A. Together our results indicate that diacylglycerol promotes acrosomal exocytosis by i) maintaining high levels of IP(3) - an effect that depends on a positive feedback loop leading to the production of PIP(2) - and ii) stimulating the activation of Rab3A, which in turn initiates a cascade of protein interactions leading to the assembly of SNARE complexes and membrane fusion.

COMPARTMENTALIZATION OF LYSOSOMAL ENZYMES IN CAUDA EPIDIDYMIS OF NORMAL AND CASTRATED RATS

The mammalian epididymis is an organ particularly rich in acid hydrolases, consistent with a developed lysosomal apparatus. However, some of these enzymes could also play a role in an extracellular environment, since they are actively secreted by the epithelium. In this study the authors measured the activity of five acid hydrolases distributed between the epithelium, fluid, small vesicles, and spermatozoa of the rat cauda epididymis in adult rats, and compared with that distribution under conditions of deprivation of luminal testosterone and testicular compounds (hemicastration). Lysosomal enzymes are differently compartmentalized in rat cauda epididymis. Most of g -galactosidase ( g -GAL) and aryl sulfatase (~70%) were found in soluble form within the fluid. Some 60% of N -acetyl- g -D-glucosaminidase ( g -NAG) and f -mannosidase ( f -MAN) become transiently bound to sperm, and g -glucuronidase ( g -GLU) was mostly concentrated in the epithelium. After remotion of testis this distribution changed, as the retention of f -MAN, g -GAL, g -GLU, and g -NAG by the epididiymal tissue increased. The increase of g -GLU followed an increase of synthesis of the enzyme. The distribution of enzymes in the epididymis from the contralateral side was similar to that in normal rats. The different roles for each enzyme in the epididymis are discussed.

Changes in the content of rat epididymal fluid induced by prolonged treatment with tamoxifen.

Summary Prolonged treatment with tamoxifen induces changes in the male reproductive tract in rats. In this study changes in the protein content of the rat epididymal fluid as a consequence of prolonged treatment with tamoxifen are reported. Among five lysosomal enzymes measured in the epididymal fluid, α‐mannosidase (α‐MAN) significantly diminished, but other enzymes did not. Electrophoretic analysis of fluids showed that proteins of estimated molecular weight 25, 60, 80–85 and 180 kDa decreased in the treated rats. We also detected an increase in the binding of β‐galactosidase (β‐GAL) to caudal spermatozoa in treated rats. These changes may be related in part to the loss of fertilizing capacity of spermatozoa after tamoxifen treatment.

Changes in Distribution of Phosphomannosyl Receptors During Maturation of Rat Spermatozoa

Abstract The aim of the present work was to study the distribution of the cation-independent (CI) and cation-dependent (CD) mannose-6-phosphate receptors (MPRs) in spermatozoa obtained from either rete testis or three regions of rat epididymis. We observed that both receptors underwent changes in distribution as spermatozoa passed from rete testis to cauda epididymis. CI-MPR was concentrated in the dorsal region of the head in rete testis sperm and that this labeling extended to the equatorial segment of epididymal spermatozoa. CD-MPR, however, changed from a dorsal distribution in rete testis, caput, and corpus to a double labeling on the dorsal and ventral regions in cauda spermatozoa. The percentages of spermatozoa that showed staining for either CI-MPR or CD-MPR increased from rete testis to epididymis. The observed changes were probably the result of a redistribution during transit rather than an unmasking of receptors. The fluorescence corresponding to CD-MPR and CI-MPR on the dorsal region disappeared when caudal spermatozoa underwent the acrosomal reaction. Receptors were localized on the plasmalemma of spermatozoa, as observed by immunoelectron microscopy. Changes in distribution may be related to a maturation process, which suggests new roles for the phosphomannosyl receptors.

The Molecules of Sperm Exocytosis

Exocytosis is a fundamental process used by eukaryotic cells to release biological compounds and to insert lipids and proteins in the plasma membrane. Specialized secretory cells undergo regulated exocytosis in response to physiological signals. Sperm exocytosis or acrosome reaction (AR) is essentially a regulated secretion with special characteristics. We will focus here on some of these unique features, covering the topology, kinetics, and molecular mechanisms that prepare, drive, and regulate membrane fusion during the AR. Last, we will compare acrosomal release with exocytosis in other model systems.

Expression and binding properties of the two mannose-6-phosphate receptors differ during perinatal development in rat liver.

Mammalian tissues express both cation-dependent (CD-MPR) and cation-independent (CI-MPR) mannose-6-phosphate receptors, which mediate the targeting of acid hydrolases to lysosomes. The coexistence of the two receptors in all cell types and tissues is still poorly understood. To determine whether these receptors might play a role in maturation, we studied their expression and binding properties in rat liver during perinatal development. CI-MPR expression decreases progressively from 18-day fetuses to adults, whereas the CD-MPR showed a transient decrease in newborn and at the 5th day after birth. Immunostaining of the tissues showed that both receptors localize to hepatocytes at all the ages and, additionally, the CD-MPR was reactive in megakaryocytes at early stages. Binding assays showed differences in the B(max) and K(D) values between the ages studied. These results demonstrate that both receptors change differentially during perinatal development, suggesting that they play distinct roles during organ maturation.