Acrosomal exocytosis of mouse sperm progresses in a consistent direction in response to zona pellucida.
ABSTRACT 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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ABSTRACT: The spermatozoa acrosome reaction (AR) is essential for mammalian fertilization. Few methods allow visualization of AR in real time together with Ca(2+) imaging. Here, we show that FM4-64, a fluorescent dye used to follow exocytosis, reliably reports AR progression induced by ionomycin and progesterone in human spermatozoa. FM4-64 clearly delimits the spermatozoa contour and reports morphological cell changes before, during and after AR. This strategy unveiled the formation of moving tubular appendages, emerging from acrosome reacted spermatozoa, which was confirmed by scanning electronic microscopy. Alternate wavelength illumination allowed concomitant imaging of FM4-64 and Fluo-4, a Ca(2+) indicator. These AR and [Ca(2+)]i recordings revealed that the presence of spontaneous [Ca(2+)]i oscillations both spontanous as well progesterone-induced prevent AR in human spermatozoa. Notably, the progesterone induced AR is preceded by a second [Ca(2+)]i peak and ~40% of reacting spermatozoa also manifest a slow [Ca(2+)]i rise ~ 2 minutes before AR. Our findings uncover new AR features related to [Ca(2+)]i.Biology of Reproduction 08/2014; · 3.45 Impact Factor
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ABSTRACT: In recent years, the study of mammalian acrosomal exocytosis has produced some major advances that challenge the long-held, general paradigms in the field. Principally, the idea that sperm must be acrosome-intact to bind to the zona pellucida of unfertilized eggs-- largely based on in vitro fertilization studies of mouse oocytes denuded of the cumulus oophorus--has been overturned by experiments using state-of-the-art imaging of cumulus-intact oocytes and fertilization experiments where eggs were reinseminated by acrosome-reacted sperm recovered from the perivitelline space of zygotes. In light of these results, this minireview highlights a number of unresolved questions and emphasizes that there is still much work to be done in this exciting field. Future experiments using recently advanced technologies should lead to a more complete and accurate understanding of the molecular mechanisms governing the fertilization process in mammals.Biology of Reproduction 03/2014; · 3.45 Impact Factor
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ABSTRACT: The sperm acrosome reaction is an all-or-none secretion process, mainly following the principles of calcium-regulated exocytosis. However, the relationship between the formation of hundreds of fusion pores and the required mobilization of calcium from the lysosome-related acrosomal vesicle has only been partially defined. Hence, the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), known to promote efflux of calcium from lysosome-like acidic compartments, was analyzed for its ability to trigger acrosome reaction in mouse sperm. In addition, the expression of two-pore channel (TPC) proteins, which are primarily localized in lysosome-related acidic organelles and which present potential molecular targets of NAADP were examined in mammalian spermatozoa. Finally, the reproductive phenotype and acrosomal secretion rates were determined in a TPC1 deficient mouse line.Using the fluorescence properties of the NAADP antagonist trans-Ned-19 we found that NAADP binding sites are highly concentrated at the acrosomal region of spermatozoa. In addition, using a newly generated TPC1 specific antibody, we detected an acrosomal colocalization of trans-Ned-19 and TPC1. Immunogold electron microscopy studies showed a TPC1 distribution along the outer acrosomal membrane. Treatment of permeabilized mouse spermatozoa with NAADP resulted in a loss of the acrosomal vesicle. Induction of acrosomal exocytosis by NAADP reflected the functional properties of TPC proteins: (i) Acrosomal secretion responses were not detectable in response to its chemical analogue NADP, and (ii) where blocked by trans-Ned-19. This inhibitory effect was also observed upon treatment of spermatozoa with the physiological agonist Zona pellucida; (iii) testing a broad concentration range of NAADP, two narrow bell-shaped dose-response-curves were observed: one with a maximum effect in the nanomolar, the other with a peak in the low micromolar NAADP concentration range, thus suggesting at least two distinct NAADP-dependent mechanisms are involved in triggering acrosomal exocytosis. Antagonizing the 1,4,5-inositol trisphosphate receptor (IP3-R) attenuated acrosomal secretion rates of both the low and high NAADP-dependent pathway. In contrast, TPC1 null sperm selectively lost responsiveness to low micromolar NAADP concentrations. Remarkably, TPC1 null offspring of heterozygous breeding pairs were significantly under-represented. Moreover, disruption of the TPCN1 gene resulted in a significant reduction of sperm numbers compared with wild-type animals.Our study shows for the first time that the second messenger NAADP mediates acrosomal exocytosis in mouse spermatozoa without any additional extracellular calcium. Moreover, it was found that two convergent NAADP-dependent pathways with non-overlapping activation profiles for distinct NAADP concentrations drive acrosomal exocytosis. Thus, we suggest that both NAADP-induced cascades link different local NAADP concentrations with the efflux of acrosomal calcium, thereby safeguarding reliable and complete fusion of the large acrosomal vesicle. Activation of the IP3-R may serve as an additional amplification mechanism. The acrosome reaction is characterized by a basic sequence of functionally coupled events typical for calcium-regulated exocytosis. Thus, the results of the present study may position sperm as a valuable cellular model system to examine whether such calcium amplification mechanisms are also operative in other cells displaying NAADP regulated exocytosis.Molecular biology of the cell 01/2014; · 5.98 Impact Factor