Acrosomal Exocytosis of Mouse Sperm Progresses in a Consistent Direction in Response to Zona Pellucida

Department of Obstetrics and Gynecology, Center for Research on Reproduction and Women's Health, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6160, USA.
Journal of Cellular Physiology (Impact Factor: 3.84). 09/2009; 220(3):611-20. DOI: 10.1002/jcp.21781
Source: PubMed


Sperm acrosomal exocytosis is essential for successful fertilization, and the zona pellucida (ZP) has been classically considered as the primary initiator in vivo. At present, following what is referred to as primary binding of the sperm to the ZP, the acrosome reaction paradigm posits that the outer acrosomal membrane and plasma membrane fuse at random points, releasing the contents of the acrosome. It is then assumed that the inner acrosomal membrane mediates secondary binding of the sperm to the ZP. In the present work we used a live fluorescence imaging system and mouse sperm containing enhanced green fluorescent protein (EGFP) in their acrosomes. We compared the processes of acrosomal exocytosis stimulated by the calcium ionophore ionomycin or by solubilized ZP. As monitored by the loss of EGFP from the sperm, acrosomal exocytosis driven by these two agents occurred differently. When ionomycin was used, exocytosis started randomly (no preference for the anterior, middle or posterior acrosomal regions). In contrast, following treatment with solubilized ZP, the loss of acrosomal components always started at the posterior zone of the acrosome and progressed in an anterograde direction. The exocytosis was slower when stimulated with ZP and on the order of 10 sec, which is in accordance with other reports. These results demonstrate that ZP stimulates acrosomal exocytosis in an orderly manner and suggest that a receptor-mediated event controls this process of membrane fusion and release of acrosomal components. These findings are incorporated into a model.

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Available from: George L Gerton, Mar 23, 2015
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    • "In contrast, AR induced by a more physiological stimulus like Progesterone typically starts at the anterior region of the sperm head. A similar conclusion was reached in mouse spermatozoa regarding the starting point of AR promoted by either zona pellucida or Ca 2+ ionophore[31]. Similarly to what we observed in mouse sperm, in humans, most of sperm undergoing[Ca 2+ ]i oscillations after Progesterone addition fail to undergo AR. "
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    ABSTRACT: During capacitation, sperm acquire the ability to undergo the acrosome reaction (AR), an essential step in fertilization. Progesterone produced by cumulus cells has been associated with various physiological processes in sperm, including stimulation of AR. An increase in intracellular Ca(2+) ([Ca(2+)]i) is necessary for AR to occur. In this study, we investigated the spatio-temporal correlation between the changes in ([Ca(2+)]i and AR in single mouse spermatozoa in response to Progesterone. We found that Progesterone stimulates an ([Ca(2+)]i increase in five different patterns: gradual increase, oscillatory, late transitory, immediate transitory and sustained. We also observed that the ([Ca(2+)]i increase promoted by Progesterone starts at either the flagellum or the head. We validated the use of FM4-64 as an indicator for the occurrence of the AR by simultaneously detecting its fluorescence increase and the loss of EGFP in transgenic EGFPAcr sperm. For the first time, we have simultaneously visualized the rise in ([Ca(2+)]i and the process of exocytosis in response to Progesterone and found that only a specific transitory increase in ([Ca(2+)]i originated in the sperm head promotes the initiation of AR.
    Full-text · Article · Jan 2016 · Biology of Reproduction
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    • "Men or mice carrying mutations affecting the process of acrosomal exocytosis are infertile or display some degree of subfertility (Dam et al., 2007; Kang-Decker et al., 2001; Lin et al., 2007). To penetrate the zona pellucida (ZP), the extracellular matrix surrounding the egg, mammalian sperm must undergo acrosomal exocytosis in an orderly manner (Buffone et al., 2009; Yanagimachi, 1994). In addition, only acrosome-reacted sperm are able to relocalize Izumo1, a protein essential for sperm egg-fusion, to the equatorial segment (Miranda et al., 2009). "
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    ABSTRACT: Mammalian sperm must acquire their fertilizing ability after a series of biochemical modifications in the female reproductive tract collectively called capacitation to undergo acrosomal exocytosis, a process that is essential for fertilization. Actin dynamics play a central role in controlling the process of exocytosis in somatic cells as well as in sperm from several mammalian species. In somatic cells, small GTPases of the Rho family are widely known as master regulators of actin dynamics. However, the role of these proteins in sperm has not been studied in detail. In the present work we characterized the participation of small GTPases of the Rho family in the signaling pathway that leads to actin polymerization during mouse sperm capacitation. We observed that most of the proteins of this signaling cascade and their effector proteins are expressed in mouse sperm. The activation of the signaling pathways of cAMP/PKA, RhoA/C and Rac1 are essential for LIMK1 activation by phosphorylation on Threonine 508. Serine 3 of Cofilin is phosphorylated by LIMK1 during capacitation in a transiently manner. Inhibition of LIMK1 by specific inhibitors (BMS-3) resulted in lower levels of actin polymerization during capacitation and a dramatic decrease in the percentage of sperm that undergo acrosomal exocytosis. Thus, we demonstrated for the first time that the master regulators of actin dynamics in somatic cells are present and active in mouse sperm. Combining the results of our present study with other results from the literature, we have proposed a working model regarding how LIMK1 and Cofilin control acrosomal exocytosis in mouse sperm. Copyright © 2015. Published by Elsevier Inc.
    Full-text · Article · Jul 2015 · Developmental Biology
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    • "To determine the percentage of spermatozoa with EGFP in their acrosomes, 10 ml aliquots of the sperm suspensions were placed on poly-L-lysine-coated glass slides and covered with coverslips. To detect the presence of EGFP in the acrosomes, the cells were quantified using a Nikon TE2000 inverted fluorescence microscope with fluorescence optics (excitation: 480 nm; emission: 515 nm), as previously described (Buffone et al. 2009a,b). In other experiments, AR was quantified as previously described by flow cytometry using a FACSCanto II flow cytometer (BD) (Muro et al. 2012). "
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    ABSTRACT: Mammalian spermatozoa must undergo complex physiological and morphological alterations within the female reproductive tract before they become fertilization-competent. Two important alterations are capacitation and the acrosome reaction (AR), by which spermatozoa become capable of penetrating the zona pellucida (ZP) of the oocyte. Although various biochemical stimulants have been reported to induce the AR, the true physiological inducer in vivo remains to be identified. Previously, it was reported that most fertilizing spermatozoa undergo the AR before contacting the ZP, and that only a small fraction of in vitro capacitated spermatozoa can penetrate the ZP. Therefore, it is important to identify which capacitating spermatozoa undergo the AR in response to potential AR inducers such as progesterone. Here we show that spermatozoa undergo a dynamic rearrangement of the acrosome during in vitro capacitation. This involves the rapid movement of an artificially introduced soluble component of the acrosome, enhanced green fluorescent protein (EGFP), from the acrosomal cap region to the equatorial segment of the sperm head (EQ). Spermatozoa exhibiting the EQ pattern were more sensitive to progesterone than those without it. We suggest that spermatozoa that are ready for acrosomal exocytosis can be detected by real-time EGFP imaging. This offers a promising new method for identifying where spermatozoa undergo the AR in the female reproductive tract in vivo.
    Full-text · Article · Apr 2015 · Reproduction
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