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Protein Cell 456 Tility Protein Cell 2013, 4(6): 456–466 DOI 10.1007/s13238-013-3019-8 Protein & Cell RESEARCH ARTICLE Cytosolic Ca2+ as a multifunctional modulator is required for spermiogenesis in Ascaris suum Yunlong Shang1,2, Lianwan Chen1, Zhiyu Liu1,2, Xia Wang1, Xuan Ma1, Long Miao1 1 Laboratory of Noncoding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 2 University of Chinese Academy of Sciences, Beijing 100049, China Correspondence: [email protected] Received March 6, 2013 Accepted April 7, 2013 Cell & ABSTRACT tial for many biological processes such as embryogenesis, immune surveillance and wound healing. Typically, actin and The dynamic polar polymers actin fi laments and microtu- microtubule cytoskeletons are employed to establish and bules are usually employed to provide the structural ba- maintain cell polarity (Li and Gundersen, 2008). Spermiogen- sis for establishing cell polarity in most eukaryotic cells. esis (sperm activation), in which round sessile spermatids dif- Protein Radially round and immotile spermatids from nematodes ferentiate into asymmetric motile spermatozoa, is a symmetry- contain almost no actin or tubulin, but still have the abil- breaking process. Dynamic and pronounced morphological ity to break symmetry to extend a pseudopod and initiate changes occur in the radially symmetrical spermatids during the acquisition of motility powered by the dynamics of the process of mammalian sperm activation, including the cytoskeleton composed of major sperm protein (MSP) formation of an elongated nucleus with condensed chromatin during spermiogenesis (sperm activation). However, covered by a well-shaped acrosome in the head and a long the signal transduction mechanism of nematode sperm fl agellum. Cytoskeletal networks composed of actin fi laments, activation and motility acquisition remains poorly under- intermediate fi laments and microtubules are required for this 2+ stood. Here we show that Ca oscillations induced by the morphological transformation during spermiogenesis (Sperry, 2+ 2+ Ca release from intracellular Ca store through inositol 2012). Remarkably, this acquisition of function occurs while (1,4,5)-trisphosphate receptor are required for Ascaris these cells are transcriptionally and translationally silent and is 2+ suum sperm activation. The chelation of cytosolic Ca therefore highly dependent on posttranslational modifi cations suppresses the generation of a functional pseudopod, to their existing protein components. In addition, intracellular and this suppression can be relieved by introducing ex- Ca2+ and Ca2+-dependent proteolysis have also been implicat- 2+ 2+ ogenous Ca into sperm cells. Ca promotes MSP-based ed in mammalian spermiogenesis (Berrios et al., 1998; Ben- sperm motility by increasing mitochondrial membrane Aharon et al., 2005). potential and thus the energy supply required for MSP Nematode sperm also require a functional maturation 2+ cytoskeleton assembly. On the other hand, Ca promotes process, in which round immotile spermatids transform into 2+ MSP disassembly by activating Ca /calmodulin-depend- asymmetrical crawling spermatozoa, to achieve fertilizing ent serine/threonine protein phosphatase calcineurin. In competence in the female reproductive tract (Ma et al., 2012). 2+ addition, Ca /camodulin activity is required for the fusion Upon activation, sperm extend a single pseudopod for migra- of sperm-specifi c membranous organelle with the plasma tion, instead of the beating flagellum found in mammalian membrane, a regulated exocytosis required for sperm mo- spermatozoa. In nematode Ascaris suum (Ascaris hereafter), 2+ tility. Thus, Ca plays multifunctional roles during sperm vas deferens extract (VDE) has the capacity to trigger sperm activation in Ascaris suum. activation (Abbas and Foor, 1978). Our previous studies dem- onstrate that a trypsin-like serine protease As_TRY-5 purifi ed 2+ KEYWORDS spermiogenesis, Ca , major sperm protein, from VDE was identifi ed as the sperm activator (Zhao et al., Ascaris suum 2012). Its homolog in C. elegans was identifi ed as the male sperm activator by genetic approaches (Smith and Stanfi eld, INTRODUCTION 2011). The establishment and maintenance of cell polarity is essen- Nematode sperm possess neither actin nor tubulin; instead, 456 | June 2013 | Volume 4 | Issue 6 © Higher Education Press and Springer-Verlag Berlin Heidelberg 2013 Cytosolic Ca2+ modulates nematode spermiogenesis. RESEARCH ARTICLE their activation and amoeboid migration depend on controlled upon activation are required for sperm motility and male fertility assembly/disassembly of the major sperm protein (MSP) cy- (L’Hernault, 2009). C. elegans spermatids from the MO fusion- toskeleton (Roberts and Stewart, 2000). During sperm activa- defective mutant fer-1 extend pseudopods in response to the tion, the sperm specifi c membranous organelle (MO) derived artificial activator (Washington and Ward, 2006), indicating from e ndoplasmic reticulum/Golgi apparatus fuses with the that MO fusion and pseudopod extension are two separate plasma membrane (PM), leaving a permanent invagination on events during sperm activation. Pseudopod extension can be the cell surface and resulting in the exocytosis and transloca- visualized under light microscopy and the fused MOs can be tion of MOs components (Washington and Ward, 2006; Zhao detected as fl uorescent puncta of FM1–43 formed at the rear et al., 2012). In fl agellated sperm, Ca2+ modulates nearly every edge of the cell body (Washington and Ward, 2006; Zhao et step of sperm maturation and fertilization including sperm al., 2012). Our ex vivo time-lapse imaging showed that pseu- capacitation, hyperactivation, chemotaxis, acrosome reaction dopod protrusion precedes MO fusion (Fig. 1E and Movie S1). and sperm-egg recognition (Breitbart, 2002; Kirichok et al., The following analyses dissect the roles of Ca2+ in pseudopod 2006; Kaupp et al., 2008; Teves et al., 2009). However, the role extension and MO fusion. of Ca2+ in nematode sperm activation was seldom reported. Previously, Ca2+ was implicated in the regulation of C. elegans Ca2+ oscillations are required for sperm activation and are sperm activation (Shakes and Ward, 1989; Washington and regulated by IP3R and PLC Ward, 2006). However, the underlying mechanisms remain to The rise of cytoplasmic Ca2+ levels during sperm activation be elucidated. Here we show that cytosolic Ca2+ oscillations might be caused by the infl ux of extracellular Ca2+ or the re- regulated by phospholipase C (PLC) and i nositol (1,4,5)-tris- lease of Ca2+ from intracellular store. Nematode spermatids Cell phosphate receptor (IP3R) synchronize with sperm activation in 2+ can be activated in Ca -free medium (Movie S2) (Ward et & Ascaris. Ca2+ promotes MSP-based sperm motility by increas- al., 1983; Washington and Ward, 2006), indicating that the ing mitochondrial membrane potential and thus the energy [Ca2+] increase might be caused by Ca2+ release from intra- production required for MSP cytoskeleton assembly, and by i cellular store. Ca2+ oscillations are primarily regulated by IP R modulating the activity of Ca2+/calmodulin-dependent serine/ 3 (Berridge, 2007), which can be activated by inositol (1,4,5)-tri- threonine protein phosphatase calcineurin (CaN) for inhibiting sphosphate (IP ) generated through cleavage of phosphati- Protein MSP assembly and promoting MSP disassembly. In addition, 3 dylinositol 4,5-bisphosphate (PIP ) by PLC in a variety of cell we show that Ca2+/calmodulin activity is required for the sperm 2 types (Berridge, 2007). To investigate whether the IP /Ca2+ exocytosis, which is necessary for functional spermatozoa mi- 3 signaling cascade is required for sperm pseudopod exten- gration. Thus, Ca2+ plays multifunctional roles in Ascaris sperm sion and MO fusion, we treated spermatids with U73122, a activation. specifi c PLC inhibitor (Gulbransen et al., 2012) or with 2-APB, a cell-permeable IP R inhibitor (Estrada et al., 2001), and RESULTS 3 found that both U73122 (100 μmol/L) and 2-APB (200 μmol/L) Cytosolic Ca2+ oscillations synchronize with pseudopod blocked VDE-induced sperm activation. These drugs inhibited extension during sperm activation both pseudopod formation and MO fusion, whereas the inac- tive analog of U73122, U73343, had no inhibitory effect on To elucidate the molecular mechanism underlying Ca2+ modu- MO fusion and much less infl uence on pseudopod formation lation of the MSP-based cytoskeletal dynamics during nema- (Fig. 2A). Pseudopod extension was inhibited for ~85% and tode sperm activation, Ascaris sperm were employed as they ~80% of the sperm treated with U73122 and 2-APB, respec- have the following advantages: (i) Ascaris spermatids and their tively (Fig. 2B). Consistent with the FM1-43 staining assay endogenous activator VDE (Zhao et al., 2012) can be obtained (Fig. 2A, bottom panels), immunoblot results also showed that in large quantities; (ii) sperm activation can be studied ex vivo; U73122 and 2-APB inhibited VDE-triggered secretion of As_ (iii) the motile apparatus of Ascaris sperm can be reconstituted SRP-1 (Fig. 2C and 2D), which was previously identifi ed as an in vitro (Italiano et al., 1996; Miao et al., 2003). To investigate MO component (Zhao et al., 2012). the roles of Ca2+ in sperm activation, we labeled cytosolic Ca2+ To validate the inhibitory effect of 2-APB on IP R, the with Fluo 4-AM, a cell-permeable indicator, and monitored in- 3 [Ca2+] dynamics of 2-APB-treated cells were examined. Time- tensity dynamics of Fluo 4 fl uorescence during
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