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Actin cytoskeleton and sperm function
Accepted Manuscript Actin cytoskeleton and sperm function Haim Breitbart, Maya Finkelstein PII:
S0006-291X(17)32164-2
DOI:
10.1016/j.bbrc.2017.11.001
Reference:
YBBRC 38793
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 6 September 2017 Accepted Date: 1 November 2017
Please cite this article as: H. Breitbart, M. Finkelstein, Actin cytoskeleton and sperm function, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.11.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Actin Cytoskeleton and Sperm Function Haim Breitbart* and Maya Finkelstein** *The Mina & Everard Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel
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Haim Breitbart The Mina & Everard Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel E-mail: [email protected]
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Corresponding Author
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**IVF Lab. Wolfson Medical Center, Holon, Israel
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ACCEPTED MANUSCRIPT Abstract For the acquisition of the ability to fertilize the egg, mammalian spermatozoa should undergo a series of biochemical transformations in the female reproductive tract, collectively called capacitation. The capacitated sperm can undergo the acrosomal exocytosis process near or on the oocyte, which allows the spermatozoon to penetrate and fertilize it. One of the main
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processes in capacitation involves dynamic cytoskeletal remodeling particularly of actin. Actin polymerization occurs during sperm capacitation and the produced F-actin should be depolymerized prior to the acrosomal exocytosis. In the present review, we describe the mechanisms that regulate F-actin formation during sperm capacitation and the F-actin
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dispersion prior to the acrosomal exocytosis. During sperm capacitation, the actin severing proteins gelsolin and cofilin are inactive and they undergo activation prior to the acrosomal
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exocytosis.
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Keywords: spermatozoa; capacitation; acrosomal exocytosis; actin; gelsolin; cofilin.
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ACCEPTED MANUSCRIPT Introduction Sperm cells undergo a number of physical and biochemical changes when presented to the female reproductive tract collectively called capacitation. These changes prepare the sperm cell for its main assignment – fusion and fertilization of the oocyte. Only capacitated sperm can undergo the acrosomal exocytosis process near or on the oocyte, which allows the
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spermatozoon to penetrate and fertilize it. One of the main processes in capacitation involves dynamic cytoskeletal remodeling particularly of actin. Actin is a well-known cytoskeletal protein, but recently in a number of papers, an additional function as a secondary messenger in signal transmission was described [1]. Actin polymerization is a process in which units of
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globular actin (G-actin) connect one another to create filamentous Actin (F-actin) under the regulation of accessory proteins [2]. A wide range of accessory proteins regulates the assembly and disassembly of F-actin as well as organizing it into a distinct complex network
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for different cellular functions [3; 4]. Actin location in the sperm head, equatorial, postacrosomal regions and in the tail of the spermatozoa indicates its crucial involvement in processes such as capacitation, acrosomal exocytosis and sperm motility [5-11]. Actin polymerization occurs during capacitation, whereas prior to the acrosomal exocytosis, F-actin must undergo depolymerization [12]. This actin filament formation is necessary for
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capacitation in bull, ram, mouse, and human spermatozoa [12-17]. We previously showed that, actin polymerization increases in the head of human spermatozoa during capacitation [18]. This increase is considered to create a physical barrier between the outer acrosomal membrane and the overlying plasma membrane, which prevents spontaneous acrosomal
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exocytosis and allows the exocytosis to occur only in the oocyte surrounding. We showed recently that actin polymerization should occur during capacitation to prevent spontaneous
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acrosomal exocytosis [19]. In addition to the increase of actin filaments in the head, actin filament elevation in the flagellum during capacitation is essential for the development of hyperactivated motility (HAM) [20]. Indeed, a low abundance of F-actin in human spermatozoa inhibits motility [18]. Epididymal mouse and ejaculated human sperm contain a curtain amount of F-actin which is important for the development of progressive motility and the further increase in F-actin levels during capacitation is important for the development of hyper-activated motility (HAM) [20]. Changes in sperm swimming pattern during the capacitation process was described in human and mouse [21-23]. During capacitation, sperm change their motility pattern from progressive to HAM [24; 25]. HAM is a movement pattern characterized by asymmetrical flagellar beating observed in spermatozoa at the site and time of fertilization in mammals [21-23], and is critical to fertilization success [26; 27]. 3
ACCEPTED MANUSCRIPT Hyperactivated motility may have a role in spermatozoa penetration of cumulus cells and zona-pellucida during fertilization [28]. Actin and related protein in sperm The increase in F-actin during capacitation depends upon activation and/or inhibition of a number of proteins. Activation of PLD and CaMKII on the one hand, and inactivation of
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actin severing proteins, such as, gelsolin and cofilin on the other hand, leads to filament elongation during sperm capacitation.
It is known that phosphatidylinositol 4,5-bisphosphate(PIP2) is a cofactor for PLD activation in many cell types [29-33]. PIP2 comprises only 1% of plasma membrane
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phospholipids; however, its extraordinary versatility puts it in the center of plasma membrane dynamics governing cell motility, adhesion, endo and exocytosis [34; 35]. PIP2 serves as an
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effector of several proteins such as MARCKS, gelsolin, PLD and PI3K. These proteins are present in spermatozoa and are involved in the regulation of sperm capacitation and/or the acrosomal exocytosis [36; 37]. We also showed that PIP2 and gelsolin are involved in regulating sperm motility and the development of hyperactivated motility [18]. Sperm total motility and hyperactivated motility are mediated by PLD-dependent actin polymerization [20]. Reduction of PIP2 synthesis inhibited actin polymerization and motility,
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and increasing PIP2 synthesis enhanced these activities. Furthermore, sperm demonstrating low motility contained low levels of PIP2 and F-actin. During capacitation, there was an increase in PIP2 and F-actin levels in the sperm head and a decrease in the tail [18]. Moreover, the localization of gelsolin in the sperm cell influenced the sperm motility. In
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sperm with high progressive motility, gelsolin was mainly localized to the sperm head before capacitation, whereas in low motility sperm, most of the gelsolin was localized to the tail
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before capacitation and translocated to the head during capacitation [18]. F-actin also increases in the sperm tail during capacitation, which is important for the development of hyper-activated motility [20]. Thus, it is likely that there are two reasons for the translocation of gelsolin from the tail to the head. First gelsolin is required in the head to depolymerize F-actin to allow the occurrence of the acrosomal exocytosis and second the exclusion of gelsolin from the tail prevents F-actin depolymerization in the tail allowing the development of hyper-activated motility. F-actin formation during sperm capacitation is also controlled by Rho GTPases [12; 38]. The small GTPases Rho A, Rho B, Rac1 and Cdc42 are present in the head and tail of several mammalian spermatozoa [39]. These small GTPases can interact with Wasp, which involves in actin nucleation together with Arp2/3 and profilins [40]. Wasp,
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ACCEPTED MANUSCRIPT profilin, Arp2/3 and cdc42 are localized to the head and tail of sperm cells [10]. It was shown that Wasp, RhoA, RhoB and Cdc42 are involved in actin remodeling in sperm capacitation and the acrosomal exocytosis processes [38]. Gelsolin and cofilin activities are inhibited during capacitation: The presence of actin severing proteins such as gelsolin and cofilin in mammalian sperm
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suggests that the assembly and disassembly of F-actin are well-controlled events. Gelsolin severs assembled actin filaments, and caps the fast growing plus end of free or newly severed filaments in response to Ca2+, and is inhibited by binding to PIP2 and by phosphorylation on tyr-438. We showed that gelsolin must be inhibited during capacitation for actin
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polymerization to occur [41]. Gelsolin can be inhibited by its Src-dependent phosphorylation on tyr-438 and/or its binding to PIP2, two processes that occur during sperm capacitation. In
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human sperm, Src is involved in regulating capacitation, Ca2+ fluxes, protein tyrosine phosphorylation and the acrosomal exocytosis [42-44]. The binding of gelsolin to PIP2 promotes its tyrosine phosphorylation by Src, which keeps gelsolin in an inactive form, allowing F-actin formation during capacitation [41]. Increase in PIP2 in the sperm head during capacitation occurs simultaneously to the increase in F-actin and gelsolin in the sperm head. Moreover, gelsolin phosphorylation on Tyr438 is enhanced during sperm capacitation
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mainly towards the mid and end of the capacitation process [18]. Inhibition of PIP2 synthesis prevented the translocation of gelsolin to the head while enhancing PIP2 synthesis significantly increased it. Furthermore, tyr-438 phosphorylation/ inhibition of gelsolin is reduced when PIP2 levels are decreased and vice versa [18].
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Previous studies suggested that Src is not directly involved in protein tyrosine phosphorylation during sperm capacitation, but rather, inhibits protein phosphatase resulting
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in an increase in tyrosine phosphorylation of proteins [45]. It was shown elsewhere that capacitation is regulated by activation of PKA, which activates Src leading to inactivation of Ser/Thr phosphatase [43; 46] (Fig.1). We recently showed that activation of Src by PKA, inhibits the Ser/Thr phosphatase PP1 resulting in CaMKII activation leading to activation of Pyk2 which phosphorylates PI3K on tyrosine-845 [47] (See Fig.1). Src was found in the sperm tail and head and is localized to the membrane fraction [46], similar to gelsolin localization and consistent with our assumption about its inactivating function. These assumptions were verified when Src-dependent phosphorylation of gelsolin on tyr-438 during sperm capacitation was found [18]. In addition, we found that activation of PKA/Src cause Factin formation which was inhibited by inhibiting Src activity [41], suggesting that activation
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ACCEPTED MANUSCRIPT of Src causes gelsolin inhibition and an increase in F-actin. In addition, PBP10-induced F-actin depolymerization is inhibited by activating Src or by inhibition of tyrosinephosphatase, suggesting that although gelsolin is released from its binding to PIP2, it is still highly phosphorylated and inactive. PBP10 is a peptide which contains the gelsolin binding domain to PIP2, competes with gelsolin to bind to PIP2 resulting in the release of gelsolin
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from PIP2. The levels of Tyr-p-gelsolin are enhanced by elevating the cellular levels of PIP2 and vice versa [18], suggesting that binding of gelsolin to PIP2 increases its phosphorylation. These findings is further supported by showing a decrease in Tyr-p-gelsolin by releasing gelsolin from PIP2 by activation of phospholipase C (PLC) which hydrolyze PIP2 or by using
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PBP10. These findings also suggest that free Tyr-p-gelsolin is more sensitive to Tyr-
phosphatase activity compared to the PIP2-bound p-gelsolin. Similarly, activation of sperm EGFR caused PLC-dependent dephosphorylation of p-gelsolin, after 1h of incubation under
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capacitation conditions [18]. These results confirm our hypothesis that gelsolin is inhibited during capacitation due to its binding to PIP2 and its Tyr-phosphorylation by Src. Cofilin is an additional actin severing protein present in sperm cells that undergo phosphorylation/inactivation on serine 3 by Lim kinases (LIMK) and by Tes kinases (TESK) [48]. LIMK can be activated by several pathways, including one through the
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Rho/ROCK/LIMK cascade [49-51]. Another way of activating LIMK is its dimerization and transphosphorylation by Hsp90 [52]. It is possible that cofilin phosphorylation is modulated by PKA activation, but there are conflicting reports about its impact. It was shown that PKA inhibits ROCK phosphorylation and activation by phosphorylation/inhibition of RhoA on
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serine-188 [53]. Other works support this idea by showing that elevated cAMP levels may indirectly lead to cofilin dephosphorylation [54; 55]. In contrast, another study showed that
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LIMK is directly activated by PKA through phosphorylation on serine-323 and -596 [56]. Another pathway of cofilin regulation is phosphorylation on tyrosine 68 by v-Src, leading to cofilin ubiquitination and degradation by the proteasome [57]. Supporting that, we found that inhibition of PKA leads to cofilin dephosphorylation [58]. In a recent study, we demonstrated that like gelsolin, activation of sperm cofilin leads to actin depolymerization, inhibition of hyper-activated motility and the induction of the acrosomal exocytosis [58]. However, the kinetics of inactivation during capacitation is different between cofilin and gelsolin. Cofilin is highly phosphorylated/inactivated at the beginning of the capacitation process, whereas gelsolin shows high phosphorylation/inactivation towards the mid-end of the capacitation process. In addition, the
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ACCEPTED MANUSCRIPT level of PIP2 does not affect cofilin phosphorylation, as it does in the case of gelsolin [18; 58]. Despite the different regulation of the two proteins, the role of cofilin appears similar to that of gelsolin, and its activation leads to actin depolymerization, inhibition of sperm motility and induction of the acrosomal exocytosis. Moreover, like gelsolin, cofilin
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translocates from the tail to the head during capacitation. Gelsolin and cofilin play a similar role in F-actin depolymerization prior to the acrosomal exocytosis but their pattern of phosphorylation/inactivation during the capacitation process is different.
Thus, for the sperm to achieve high levels of F-actin along the capacitation process both
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gelsolin and cofilin must be inactive at different times during the capacitation period and both are activated prior to the acrosomal exocytosis. It is important to mention that for its activity gelsolin needs the actin-depolymerizing-factor (ADF)/cofilin to depolymerize F-actin [59].
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In conclusion, our data suggest the following model (see Fig.1): The relatively small increase in [Ca2+]i during capacitation leads to conformational changes in gelsolin revealing the F-actin binding site. This change and the increase in F-actin and PIP2 in the sperm head, result in gelsolin translocation to the head. Nevertheless, the elevation of PIP2 levels and PKA/Src activation, maintain gelsolin in a phosphorylated/inactivated state and actin
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polymerization occurs. Activation of Src during capacitation lead to protein-phosphatase1γ2 (PP1γ2) inhibition resulting in activation of the cascade CaMKII-Pyk2-PI3K that mediates actin polymerization [19; 47; 60] as described in Figure 1 here. The increase in F-actin in the tail leads to the development of hyper-activated motility as part of the capacitation process.
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The model for cofilin phosphorylation/inactivation during capacitation is seen in Figure 1. HCO3- activates the soluble-adenylyl cyclase (sAC) to produce cAMP, which activates PKA
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leading to LIMK activation and cofilin phosphorylation. It is possible that the phosphatase SSH1L which dephosphorylates p-cofilin is inhibited during capacitation by CaMKII, keeping the cofilin in its phosphorylated/inactivated form. This suggestion should be confirmed in the future.
Activation of gelsolin and cofilin prior to acrosomal exocytosis: Gelsolin activation is regulated by calcium ions, phosphoinositides [61; 62] and by Srcdependent phosphorylation on tyr-438 [63]. Low concentration of calcium ions cause conformational changes in the C- terminus of gelsolin, which exposed its F-actin binding site and higher calcium caused a second conformational change exposing the catalytic site [62]. In human sperm, activation of gelsolin by enhancing intracellular calcium concentration or by
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ACCEPTED MANUSCRIPT using the peptide PBP10 causes fast depolymerization of F-actin and induction of the acrosomal exocytosis in capacitated sperm [41]. In Sertoli cells, the hydrolysis of PIP2 by PLC resulted in the release the bound gelsolin and its activation [64]. Releasing of gelsolin from binding to PIP2 due to its hydrolysis by phospholipase C (PLC) induced reverse translocation of gelsolin from the head to the tail [41]. It is well known that there is a high
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increase in intracellular Ca2+ concentrations as a result of the interaction between capacitated sperm and the egg zona pellucida [65]. This increase in calcium is essential for the
occurrence of the acrosomal exocytosis which is known to be mediated by PLC activity [60]. Interestingly, activation of gelsolin by PBP10 in capacitated sperm, which induces PLCindependent acrosomal exocytosis, occurs under conditions by which intracellular Ca2+
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concentration is relatively low, further indicating that the increase in intracellular Ca2+ is essential for the activation of PLC leading to gelsolin activation and F-actin dispersion an
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essential step for acrosomal exocytosis to occur. Activation of cofilin by 17allylaminogelanamycin (17-AAG) resulted in F-actin breakdown, inhibition of hyperactivated motility and significant increase in acrosomal exocytosis [58]. 17-AAG inhibits Hsp90/LIMK and cofilin phosphorylation resulting in p-cofilin dephosphorylation/activation. Moreover, activation of cofilin during the entire capacitation period prevents the cells from
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undergoing proper capacitation. Thus, cofilin regulates sperm capacitation by controlling the cellular F-actin levels. This conclusion is further supported by the translocation of cofilin from the tail to the head during capacitation [58]. The decrease of cofilin in the tail during the capacitation allows the retention of a high level of F-actin in the tail, which is important for
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the development of hyperactivated motility, while its increase in the head is important for Factin breakdown prior to the acrosomal exocytosis. It should be mentioned that inhibition of
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Hsp90 by 17 AAG results in Akt inactivation [66; 67], and that Akt is related to the regulation of human sperm motility and hyper-activated motility [68]; thus, it is possible that 17-AAG also inhibits sperm motility via this mechanism. It is well accepted that PKA is a key enzyme activated at the beginning of the capacitation process [69]. As mentioned above phosphorylation/inactivation of cofilin in sperm is a PKA dependent process [58]. We could not see any change in cofilin phosphorylation level by adding the PKA activator 8Br-cAMP to the cells [58]. In previous work activation of PKA by adding 8Br-cAMP to human sperm demonstrated activation of Src to phosphorylate/ inactivate gelsolin [41]. Also, 8Br-cAMP enhances F-actin formation which is inhibited by inhibition of Src [41] or by inhibition of PKA [70]. We also showed that inhibition of PKA caused almost complete inhibition of human sperm hyperactivated motility [71], and 8
ACCEPTED MANUSCRIPT inhibition of F-actin formation inhibits mouse and human sperm motility [20]. Thus, 8BrcAMP stimulates F-actin formation due to the enhanced phosphorylation/inactivation of gelsolin [41], but it does not influence cofilin phosphorylation at the beginning of the capacitation process probably because the intracellular concentration of cAMP is already high at the beginning of the capacitation process [72]. Elevation of intracellular Ca2+
this dephosphorylation is PLC-independent. In contrast, gelsolin
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significantly reduced p-cofilin which was not reversed by inhibiting PLC [58], indicating that
dephosphorylation/activation depends on PLC activity [41]. It has been shown elsewhere that Ca2+-induced cofilin dephosphorylation is mediated by calcineurin-dependent
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dephosphorylation activated by SSH1L [73]. CaMKII negatively regulates SSH1L activity by phosphorylation/activation of LIMK1, which regulates the subcellular localization of SSH1L [74]. Inhibition of sperm CaMKII during incubation under capacitation conditions prevents
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F-actin formation resulting in an increase in spontaneous acrosomal exocytosis [19]. We suggest that inhibition of CaMKII would allow SSH1L activation and p-cofilin dephosphorylation/activation which will prevent F-actin formation resulting in an increase in spontaneous acrosomal exocytosis. It has been shown elsewhere that PI3K mediates the activation of SSH1L [75]. We showed elsewhere that towards the end of sperm capacitation
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CaMKII undergoes dephosphorylation/inactivation whereas PI3K is activated [47]. Thus, it is possible that inactivation of CaMKII and activation of PI3K towards the end of the capacitation together with the increase in [Ca2+]i activates SSH1L which dephosphorylates/activates cofilin to disperses F-actin allowing the acrosomal exocytosis to
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take place.
To summarize this point we suggest the following: activation of CaMKII during
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capacitation leads to SSH1L inhibition and F-actin formation, whereas prior to the acrosomal exocytosis, inactivation of CaMKII and activation of PI3K leads to SSH1L activation resulting in dephosphorylation/activation of cofilin, leading to F-actin breakdown and the occurrence of the acrosomal exocytosis. We suggest a model for the effects of gelsolin and cofilin on actin during capacitation (Fig. 1). The two proteins affect F-actin levels at different times in the capacitation process: inhibition of cofilin maintains relatively high levels of F-actin at the early stages of capacitation, whereas gelsolin inhibition enables high F-actin in the late steps of the capacitation process. The fact that cofilin phosphorylation/inactivation is independent of PIP2, whereas gelsolin phosphorylation/inactivation does, further support the suggested time course for these phosphorylation. The levels of PIP2 are relatively low at the beginning of the 9
ACCEPTED MANUSCRIPT capacitation process [18], therefore it is not expected that PIP2 will regulate cofilin activity. In addition, PIP2 levels are enhanced in the sperm head during capacitation, leading to phosphorylation/inactivation of gelsolin in the head allowing F-actin increase in the sperm head [18]. Although both gelsolin and cofilin translocate from the tail to the head during capacitation, gelsolin phosphorylation/inactivation, but not cofilin phosphorylation is
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regulated by PIP2 in the head; however, it is not clear how cofilin phosphorylation is regulated. The fact that translocation of cofilin from the tail to the head depends upon its phosphorylation suggests that cofilin is phosphorylated in the tail. The phosphorylation
dependency of cofilin translocation is supported by other studies. It was shown in NIH3T3
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cells that subcellular localization of cofilin depends on the phosphorylation state of Ser-3, in which non-phosphorylated cofilin accumulates within the nuclei [76]. However, in HL-60 cells, cofilin translocate to the plasma membrane concomitantly to activation-induced cofilin
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dephosphorylation [77]. Tyrosine phosphorylation of gelsolin is mediated by PKA/Src activities and serine-phosphorylation of cofilin is also mediated by PKA which probably phosphorylates/activates LIMK [56], which phosphorylates/inactivates cofilin [50]. In summary, we suggest that both cofilin and gelsolin are essential factors that regulate sperm capacitation and the acrosomal exocytosis by modulating actin. The relationships
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between activation/inactivation of cofilin and gelsolin suggest that inhibition of cofilin is important for allowing F-actin formation at the beginning of the capacitation process, whereas inhibition of gelsolin is important later on during the capacitation process. Moreover, the different regulation of the actin-severing proteins, in which gelsolin activity is regulated
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by PIP2 and PLC whereas cofilin is not, and the fact that gelsolin activation but not cofilin is calcium-regulated [78], suggest a better and safer way to control actin dynamics during sperm
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capacitation. The need for two actin-severing proteins, or perhaps more, and at least two pathways, PLD and CaMKII for regulation actin remodeling emphasize the importance of this process for achieving successful fertilization.
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ACCEPTED MANUSCRIPT Figure Legend Figure 1: A model describing the biochemical cascade in sperm capacitation: Intracellular HCO3− activates sAC to generate cAMP leading to PKA activation and cholesterol efflux from the sperm plasma membrane which further stimulate the HCO3− /sAC/cAMP/PKA cascade. The relatively small increase in [Ca2+]i during capacitation leads
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to conformational changes in gelsolin revealing the F-actin binding site. This change and the increase in F-actin and PIP2 in the sperm head, result in gelsolin translocation to the head. PKA activates Src to phosphorylate/inactivate PIP2-bound gelsolin. The elevation of PIP2 levels and PKA/Src activation, maintain gelsolin in a phosphorylated/inactivated state and
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actin polymerization occurs. Activation of Src during capacitation lead to protein-
phosphatase1γ2 (PP1γ2) inhibition resulting in activation of the cascade CaMKII-Pyk2-PI3K which mediates actin polemerization. PIP2 is a cofactor for PLD activation by PKCα, leading
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to phosphatidylcholine hydrolysis and production of phosphatidic acid (PA) which mediates the conversion of G-actin to F-actin. Cofilin is also highly phosphorylated/inactivated at the beginning of the capacitation process through LIMK activation and inhibition of PKA leads to cofilin dephosphorylation. LIMK is directly activated by PKA through phosphorylation on serine-323 and -596. It is possible that the phosphatase SSH1L which dephosphorylates p-
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cofilin is inhibited during capacitation by CaMKII, keeping the cofilin in its
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phosphorylated/inactivated form.
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Highlights Actin polymerization occurs during sperm capacitation
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F-actin dispersion occurs prior to the acrosomal exocytosis
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Gelsolin and cofilin are inactive during sperm capacitation.
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Gelsolin and cofilin are activated prior to the acrosomal exocytosis.
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Conflict of Interest We have no conflict of interest
S0006-291X(17)32164-2
DOI:
10.1016/j.bbrc.2017.11.001
Reference:
YBBRC 38793
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 6 September 2017 Accepted Date: 1 November 2017
Please cite this article as: H. Breitbart, M. Finkelstein, Actin cytoskeleton and sperm function, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.11.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Actin Cytoskeleton and Sperm Function Haim Breitbart* and Maya Finkelstein** *The Mina & Everard Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel
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Haim Breitbart The Mina & Everard Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel E-mail: [email protected]
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Corresponding Author
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**IVF Lab. Wolfson Medical Center, Holon, Israel
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ACCEPTED MANUSCRIPT Abstract For the acquisition of the ability to fertilize the egg, mammalian spermatozoa should undergo a series of biochemical transformations in the female reproductive tract, collectively called capacitation. The capacitated sperm can undergo the acrosomal exocytosis process near or on the oocyte, which allows the spermatozoon to penetrate and fertilize it. One of the main
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processes in capacitation involves dynamic cytoskeletal remodeling particularly of actin. Actin polymerization occurs during sperm capacitation and the produced F-actin should be depolymerized prior to the acrosomal exocytosis. In the present review, we describe the mechanisms that regulate F-actin formation during sperm capacitation and the F-actin
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dispersion prior to the acrosomal exocytosis. During sperm capacitation, the actin severing proteins gelsolin and cofilin are inactive and they undergo activation prior to the acrosomal
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exocytosis.
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Keywords: spermatozoa; capacitation; acrosomal exocytosis; actin; gelsolin; cofilin.
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ACCEPTED MANUSCRIPT Introduction Sperm cells undergo a number of physical and biochemical changes when presented to the female reproductive tract collectively called capacitation. These changes prepare the sperm cell for its main assignment – fusion and fertilization of the oocyte. Only capacitated sperm can undergo the acrosomal exocytosis process near or on the oocyte, which allows the
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spermatozoon to penetrate and fertilize it. One of the main processes in capacitation involves dynamic cytoskeletal remodeling particularly of actin. Actin is a well-known cytoskeletal protein, but recently in a number of papers, an additional function as a secondary messenger in signal transmission was described [1]. Actin polymerization is a process in which units of
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globular actin (G-actin) connect one another to create filamentous Actin (F-actin) under the regulation of accessory proteins [2]. A wide range of accessory proteins regulates the assembly and disassembly of F-actin as well as organizing it into a distinct complex network
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for different cellular functions [3; 4]. Actin location in the sperm head, equatorial, postacrosomal regions and in the tail of the spermatozoa indicates its crucial involvement in processes such as capacitation, acrosomal exocytosis and sperm motility [5-11]. Actin polymerization occurs during capacitation, whereas prior to the acrosomal exocytosis, F-actin must undergo depolymerization [12]. This actin filament formation is necessary for
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capacitation in bull, ram, mouse, and human spermatozoa [12-17]. We previously showed that, actin polymerization increases in the head of human spermatozoa during capacitation [18]. This increase is considered to create a physical barrier between the outer acrosomal membrane and the overlying plasma membrane, which prevents spontaneous acrosomal
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exocytosis and allows the exocytosis to occur only in the oocyte surrounding. We showed recently that actin polymerization should occur during capacitation to prevent spontaneous
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acrosomal exocytosis [19]. In addition to the increase of actin filaments in the head, actin filament elevation in the flagellum during capacitation is essential for the development of hyperactivated motility (HAM) [20]. Indeed, a low abundance of F-actin in human spermatozoa inhibits motility [18]. Epididymal mouse and ejaculated human sperm contain a curtain amount of F-actin which is important for the development of progressive motility and the further increase in F-actin levels during capacitation is important for the development of hyper-activated motility (HAM) [20]. Changes in sperm swimming pattern during the capacitation process was described in human and mouse [21-23]. During capacitation, sperm change their motility pattern from progressive to HAM [24; 25]. HAM is a movement pattern characterized by asymmetrical flagellar beating observed in spermatozoa at the site and time of fertilization in mammals [21-23], and is critical to fertilization success [26; 27]. 3
ACCEPTED MANUSCRIPT Hyperactivated motility may have a role in spermatozoa penetration of cumulus cells and zona-pellucida during fertilization [28]. Actin and related protein in sperm The increase in F-actin during capacitation depends upon activation and/or inhibition of a number of proteins. Activation of PLD and CaMKII on the one hand, and inactivation of
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actin severing proteins, such as, gelsolin and cofilin on the other hand, leads to filament elongation during sperm capacitation.
It is known that phosphatidylinositol 4,5-bisphosphate(PIP2) is a cofactor for PLD activation in many cell types [29-33]. PIP2 comprises only 1% of plasma membrane
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phospholipids; however, its extraordinary versatility puts it in the center of plasma membrane dynamics governing cell motility, adhesion, endo and exocytosis [34; 35]. PIP2 serves as an
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effector of several proteins such as MARCKS, gelsolin, PLD and PI3K. These proteins are present in spermatozoa and are involved in the regulation of sperm capacitation and/or the acrosomal exocytosis [36; 37]. We also showed that PIP2 and gelsolin are involved in regulating sperm motility and the development of hyperactivated motility [18]. Sperm total motility and hyperactivated motility are mediated by PLD-dependent actin polymerization [20]. Reduction of PIP2 synthesis inhibited actin polymerization and motility,
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and increasing PIP2 synthesis enhanced these activities. Furthermore, sperm demonstrating low motility contained low levels of PIP2 and F-actin. During capacitation, there was an increase in PIP2 and F-actin levels in the sperm head and a decrease in the tail [18]. Moreover, the localization of gelsolin in the sperm cell influenced the sperm motility. In
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sperm with high progressive motility, gelsolin was mainly localized to the sperm head before capacitation, whereas in low motility sperm, most of the gelsolin was localized to the tail
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before capacitation and translocated to the head during capacitation [18]. F-actin also increases in the sperm tail during capacitation, which is important for the development of hyper-activated motility [20]. Thus, it is likely that there are two reasons for the translocation of gelsolin from the tail to the head. First gelsolin is required in the head to depolymerize F-actin to allow the occurrence of the acrosomal exocytosis and second the exclusion of gelsolin from the tail prevents F-actin depolymerization in the tail allowing the development of hyper-activated motility. F-actin formation during sperm capacitation is also controlled by Rho GTPases [12; 38]. The small GTPases Rho A, Rho B, Rac1 and Cdc42 are present in the head and tail of several mammalian spermatozoa [39]. These small GTPases can interact with Wasp, which involves in actin nucleation together with Arp2/3 and profilins [40]. Wasp,
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ACCEPTED MANUSCRIPT profilin, Arp2/3 and cdc42 are localized to the head and tail of sperm cells [10]. It was shown that Wasp, RhoA, RhoB and Cdc42 are involved in actin remodeling in sperm capacitation and the acrosomal exocytosis processes [38]. Gelsolin and cofilin activities are inhibited during capacitation: The presence of actin severing proteins such as gelsolin and cofilin in mammalian sperm
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suggests that the assembly and disassembly of F-actin are well-controlled events. Gelsolin severs assembled actin filaments, and caps the fast growing plus end of free or newly severed filaments in response to Ca2+, and is inhibited by binding to PIP2 and by phosphorylation on tyr-438. We showed that gelsolin must be inhibited during capacitation for actin
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polymerization to occur [41]. Gelsolin can be inhibited by its Src-dependent phosphorylation on tyr-438 and/or its binding to PIP2, two processes that occur during sperm capacitation. In
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human sperm, Src is involved in regulating capacitation, Ca2+ fluxes, protein tyrosine phosphorylation and the acrosomal exocytosis [42-44]. The binding of gelsolin to PIP2 promotes its tyrosine phosphorylation by Src, which keeps gelsolin in an inactive form, allowing F-actin formation during capacitation [41]. Increase in PIP2 in the sperm head during capacitation occurs simultaneously to the increase in F-actin and gelsolin in the sperm head. Moreover, gelsolin phosphorylation on Tyr438 is enhanced during sperm capacitation
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mainly towards the mid and end of the capacitation process [18]. Inhibition of PIP2 synthesis prevented the translocation of gelsolin to the head while enhancing PIP2 synthesis significantly increased it. Furthermore, tyr-438 phosphorylation/ inhibition of gelsolin is reduced when PIP2 levels are decreased and vice versa [18].
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Previous studies suggested that Src is not directly involved in protein tyrosine phosphorylation during sperm capacitation, but rather, inhibits protein phosphatase resulting
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in an increase in tyrosine phosphorylation of proteins [45]. It was shown elsewhere that capacitation is regulated by activation of PKA, which activates Src leading to inactivation of Ser/Thr phosphatase [43; 46] (Fig.1). We recently showed that activation of Src by PKA, inhibits the Ser/Thr phosphatase PP1 resulting in CaMKII activation leading to activation of Pyk2 which phosphorylates PI3K on tyrosine-845 [47] (See Fig.1). Src was found in the sperm tail and head and is localized to the membrane fraction [46], similar to gelsolin localization and consistent with our assumption about its inactivating function. These assumptions were verified when Src-dependent phosphorylation of gelsolin on tyr-438 during sperm capacitation was found [18]. In addition, we found that activation of PKA/Src cause Factin formation which was inhibited by inhibiting Src activity [41], suggesting that activation
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ACCEPTED MANUSCRIPT of Src causes gelsolin inhibition and an increase in F-actin. In addition, PBP10-induced F-actin depolymerization is inhibited by activating Src or by inhibition of tyrosinephosphatase, suggesting that although gelsolin is released from its binding to PIP2, it is still highly phosphorylated and inactive. PBP10 is a peptide which contains the gelsolin binding domain to PIP2, competes with gelsolin to bind to PIP2 resulting in the release of gelsolin
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from PIP2. The levels of Tyr-p-gelsolin are enhanced by elevating the cellular levels of PIP2 and vice versa [18], suggesting that binding of gelsolin to PIP2 increases its phosphorylation. These findings is further supported by showing a decrease in Tyr-p-gelsolin by releasing gelsolin from PIP2 by activation of phospholipase C (PLC) which hydrolyze PIP2 or by using
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PBP10. These findings also suggest that free Tyr-p-gelsolin is more sensitive to Tyr-
phosphatase activity compared to the PIP2-bound p-gelsolin. Similarly, activation of sperm EGFR caused PLC-dependent dephosphorylation of p-gelsolin, after 1h of incubation under
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capacitation conditions [18]. These results confirm our hypothesis that gelsolin is inhibited during capacitation due to its binding to PIP2 and its Tyr-phosphorylation by Src. Cofilin is an additional actin severing protein present in sperm cells that undergo phosphorylation/inactivation on serine 3 by Lim kinases (LIMK) and by Tes kinases (TESK) [48]. LIMK can be activated by several pathways, including one through the
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Rho/ROCK/LIMK cascade [49-51]. Another way of activating LIMK is its dimerization and transphosphorylation by Hsp90 [52]. It is possible that cofilin phosphorylation is modulated by PKA activation, but there are conflicting reports about its impact. It was shown that PKA inhibits ROCK phosphorylation and activation by phosphorylation/inhibition of RhoA on
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serine-188 [53]. Other works support this idea by showing that elevated cAMP levels may indirectly lead to cofilin dephosphorylation [54; 55]. In contrast, another study showed that
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LIMK is directly activated by PKA through phosphorylation on serine-323 and -596 [56]. Another pathway of cofilin regulation is phosphorylation on tyrosine 68 by v-Src, leading to cofilin ubiquitination and degradation by the proteasome [57]. Supporting that, we found that inhibition of PKA leads to cofilin dephosphorylation [58]. In a recent study, we demonstrated that like gelsolin, activation of sperm cofilin leads to actin depolymerization, inhibition of hyper-activated motility and the induction of the acrosomal exocytosis [58]. However, the kinetics of inactivation during capacitation is different between cofilin and gelsolin. Cofilin is highly phosphorylated/inactivated at the beginning of the capacitation process, whereas gelsolin shows high phosphorylation/inactivation towards the mid-end of the capacitation process. In addition, the
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ACCEPTED MANUSCRIPT level of PIP2 does not affect cofilin phosphorylation, as it does in the case of gelsolin [18; 58]. Despite the different regulation of the two proteins, the role of cofilin appears similar to that of gelsolin, and its activation leads to actin depolymerization, inhibition of sperm motility and induction of the acrosomal exocytosis. Moreover, like gelsolin, cofilin
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translocates from the tail to the head during capacitation. Gelsolin and cofilin play a similar role in F-actin depolymerization prior to the acrosomal exocytosis but their pattern of phosphorylation/inactivation during the capacitation process is different.
Thus, for the sperm to achieve high levels of F-actin along the capacitation process both
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gelsolin and cofilin must be inactive at different times during the capacitation period and both are activated prior to the acrosomal exocytosis. It is important to mention that for its activity gelsolin needs the actin-depolymerizing-factor (ADF)/cofilin to depolymerize F-actin [59].
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In conclusion, our data suggest the following model (see Fig.1): The relatively small increase in [Ca2+]i during capacitation leads to conformational changes in gelsolin revealing the F-actin binding site. This change and the increase in F-actin and PIP2 in the sperm head, result in gelsolin translocation to the head. Nevertheless, the elevation of PIP2 levels and PKA/Src activation, maintain gelsolin in a phosphorylated/inactivated state and actin
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polymerization occurs. Activation of Src during capacitation lead to protein-phosphatase1γ2 (PP1γ2) inhibition resulting in activation of the cascade CaMKII-Pyk2-PI3K that mediates actin polymerization [19; 47; 60] as described in Figure 1 here. The increase in F-actin in the tail leads to the development of hyper-activated motility as part of the capacitation process.
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The model for cofilin phosphorylation/inactivation during capacitation is seen in Figure 1. HCO3- activates the soluble-adenylyl cyclase (sAC) to produce cAMP, which activates PKA
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leading to LIMK activation and cofilin phosphorylation. It is possible that the phosphatase SSH1L which dephosphorylates p-cofilin is inhibited during capacitation by CaMKII, keeping the cofilin in its phosphorylated/inactivated form. This suggestion should be confirmed in the future.
Activation of gelsolin and cofilin prior to acrosomal exocytosis: Gelsolin activation is regulated by calcium ions, phosphoinositides [61; 62] and by Srcdependent phosphorylation on tyr-438 [63]. Low concentration of calcium ions cause conformational changes in the C- terminus of gelsolin, which exposed its F-actin binding site and higher calcium caused a second conformational change exposing the catalytic site [62]. In human sperm, activation of gelsolin by enhancing intracellular calcium concentration or by
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ACCEPTED MANUSCRIPT using the peptide PBP10 causes fast depolymerization of F-actin and induction of the acrosomal exocytosis in capacitated sperm [41]. In Sertoli cells, the hydrolysis of PIP2 by PLC resulted in the release the bound gelsolin and its activation [64]. Releasing of gelsolin from binding to PIP2 due to its hydrolysis by phospholipase C (PLC) induced reverse translocation of gelsolin from the head to the tail [41]. It is well known that there is a high
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increase in intracellular Ca2+ concentrations as a result of the interaction between capacitated sperm and the egg zona pellucida [65]. This increase in calcium is essential for the
occurrence of the acrosomal exocytosis which is known to be mediated by PLC activity [60]. Interestingly, activation of gelsolin by PBP10 in capacitated sperm, which induces PLCindependent acrosomal exocytosis, occurs under conditions by which intracellular Ca2+
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concentration is relatively low, further indicating that the increase in intracellular Ca2+ is essential for the activation of PLC leading to gelsolin activation and F-actin dispersion an
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essential step for acrosomal exocytosis to occur. Activation of cofilin by 17allylaminogelanamycin (17-AAG) resulted in F-actin breakdown, inhibition of hyperactivated motility and significant increase in acrosomal exocytosis [58]. 17-AAG inhibits Hsp90/LIMK and cofilin phosphorylation resulting in p-cofilin dephosphorylation/activation. Moreover, activation of cofilin during the entire capacitation period prevents the cells from
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undergoing proper capacitation. Thus, cofilin regulates sperm capacitation by controlling the cellular F-actin levels. This conclusion is further supported by the translocation of cofilin from the tail to the head during capacitation [58]. The decrease of cofilin in the tail during the capacitation allows the retention of a high level of F-actin in the tail, which is important for
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the development of hyperactivated motility, while its increase in the head is important for Factin breakdown prior to the acrosomal exocytosis. It should be mentioned that inhibition of
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Hsp90 by 17 AAG results in Akt inactivation [66; 67], and that Akt is related to the regulation of human sperm motility and hyper-activated motility [68]; thus, it is possible that 17-AAG also inhibits sperm motility via this mechanism. It is well accepted that PKA is a key enzyme activated at the beginning of the capacitation process [69]. As mentioned above phosphorylation/inactivation of cofilin in sperm is a PKA dependent process [58]. We could not see any change in cofilin phosphorylation level by adding the PKA activator 8Br-cAMP to the cells [58]. In previous work activation of PKA by adding 8Br-cAMP to human sperm demonstrated activation of Src to phosphorylate/ inactivate gelsolin [41]. Also, 8Br-cAMP enhances F-actin formation which is inhibited by inhibition of Src [41] or by inhibition of PKA [70]. We also showed that inhibition of PKA caused almost complete inhibition of human sperm hyperactivated motility [71], and 8
ACCEPTED MANUSCRIPT inhibition of F-actin formation inhibits mouse and human sperm motility [20]. Thus, 8BrcAMP stimulates F-actin formation due to the enhanced phosphorylation/inactivation of gelsolin [41], but it does not influence cofilin phosphorylation at the beginning of the capacitation process probably because the intracellular concentration of cAMP is already high at the beginning of the capacitation process [72]. Elevation of intracellular Ca2+
this dephosphorylation is PLC-independent. In contrast, gelsolin
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significantly reduced p-cofilin which was not reversed by inhibiting PLC [58], indicating that
dephosphorylation/activation depends on PLC activity [41]. It has been shown elsewhere that Ca2+-induced cofilin dephosphorylation is mediated by calcineurin-dependent
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dephosphorylation activated by SSH1L [73]. CaMKII negatively regulates SSH1L activity by phosphorylation/activation of LIMK1, which regulates the subcellular localization of SSH1L [74]. Inhibition of sperm CaMKII during incubation under capacitation conditions prevents
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F-actin formation resulting in an increase in spontaneous acrosomal exocytosis [19]. We suggest that inhibition of CaMKII would allow SSH1L activation and p-cofilin dephosphorylation/activation which will prevent F-actin formation resulting in an increase in spontaneous acrosomal exocytosis. It has been shown elsewhere that PI3K mediates the activation of SSH1L [75]. We showed elsewhere that towards the end of sperm capacitation
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CaMKII undergoes dephosphorylation/inactivation whereas PI3K is activated [47]. Thus, it is possible that inactivation of CaMKII and activation of PI3K towards the end of the capacitation together with the increase in [Ca2+]i activates SSH1L which dephosphorylates/activates cofilin to disperses F-actin allowing the acrosomal exocytosis to
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take place.
To summarize this point we suggest the following: activation of CaMKII during
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capacitation leads to SSH1L inhibition and F-actin formation, whereas prior to the acrosomal exocytosis, inactivation of CaMKII and activation of PI3K leads to SSH1L activation resulting in dephosphorylation/activation of cofilin, leading to F-actin breakdown and the occurrence of the acrosomal exocytosis. We suggest a model for the effects of gelsolin and cofilin on actin during capacitation (Fig. 1). The two proteins affect F-actin levels at different times in the capacitation process: inhibition of cofilin maintains relatively high levels of F-actin at the early stages of capacitation, whereas gelsolin inhibition enables high F-actin in the late steps of the capacitation process. The fact that cofilin phosphorylation/inactivation is independent of PIP2, whereas gelsolin phosphorylation/inactivation does, further support the suggested time course for these phosphorylation. The levels of PIP2 are relatively low at the beginning of the 9
ACCEPTED MANUSCRIPT capacitation process [18], therefore it is not expected that PIP2 will regulate cofilin activity. In addition, PIP2 levels are enhanced in the sperm head during capacitation, leading to phosphorylation/inactivation of gelsolin in the head allowing F-actin increase in the sperm head [18]. Although both gelsolin and cofilin translocate from the tail to the head during capacitation, gelsolin phosphorylation/inactivation, but not cofilin phosphorylation is
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regulated by PIP2 in the head; however, it is not clear how cofilin phosphorylation is regulated. The fact that translocation of cofilin from the tail to the head depends upon its phosphorylation suggests that cofilin is phosphorylated in the tail. The phosphorylation
dependency of cofilin translocation is supported by other studies. It was shown in NIH3T3
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cells that subcellular localization of cofilin depends on the phosphorylation state of Ser-3, in which non-phosphorylated cofilin accumulates within the nuclei [76]. However, in HL-60 cells, cofilin translocate to the plasma membrane concomitantly to activation-induced cofilin
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dephosphorylation [77]. Tyrosine phosphorylation of gelsolin is mediated by PKA/Src activities and serine-phosphorylation of cofilin is also mediated by PKA which probably phosphorylates/activates LIMK [56], which phosphorylates/inactivates cofilin [50]. In summary, we suggest that both cofilin and gelsolin are essential factors that regulate sperm capacitation and the acrosomal exocytosis by modulating actin. The relationships
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between activation/inactivation of cofilin and gelsolin suggest that inhibition of cofilin is important for allowing F-actin formation at the beginning of the capacitation process, whereas inhibition of gelsolin is important later on during the capacitation process. Moreover, the different regulation of the actin-severing proteins, in which gelsolin activity is regulated
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by PIP2 and PLC whereas cofilin is not, and the fact that gelsolin activation but not cofilin is calcium-regulated [78], suggest a better and safer way to control actin dynamics during sperm
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capacitation. The need for two actin-severing proteins, or perhaps more, and at least two pathways, PLD and CaMKII for regulation actin remodeling emphasize the importance of this process for achieving successful fertilization.
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ACCEPTED MANUSCRIPT Figure Legend Figure 1: A model describing the biochemical cascade in sperm capacitation: Intracellular HCO3− activates sAC to generate cAMP leading to PKA activation and cholesterol efflux from the sperm plasma membrane which further stimulate the HCO3− /sAC/cAMP/PKA cascade. The relatively small increase in [Ca2+]i during capacitation leads
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to conformational changes in gelsolin revealing the F-actin binding site. This change and the increase in F-actin and PIP2 in the sperm head, result in gelsolin translocation to the head. PKA activates Src to phosphorylate/inactivate PIP2-bound gelsolin. The elevation of PIP2 levels and PKA/Src activation, maintain gelsolin in a phosphorylated/inactivated state and
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actin polymerization occurs. Activation of Src during capacitation lead to protein-
phosphatase1γ2 (PP1γ2) inhibition resulting in activation of the cascade CaMKII-Pyk2-PI3K which mediates actin polemerization. PIP2 is a cofactor for PLD activation by PKCα, leading
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to phosphatidylcholine hydrolysis and production of phosphatidic acid (PA) which mediates the conversion of G-actin to F-actin. Cofilin is also highly phosphorylated/inactivated at the beginning of the capacitation process through LIMK activation and inhibition of PKA leads to cofilin dephosphorylation. LIMK is directly activated by PKA through phosphorylation on serine-323 and -596. It is possible that the phosphatase SSH1L which dephosphorylates p-
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cofilin is inhibited during capacitation by CaMKII, keeping the cofilin in its
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Highlights Actin polymerization occurs during sperm capacitation
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F-actin dispersion occurs prior to the acrosomal exocytosis
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Gelsolin and cofilin are inactive during sperm capacitation.
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Gelsolin and cofilin are activated prior to the acrosomal exocytosis.
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Conflict of Interest We have no conflict of interest