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First published online February 9, 2007
doi: 10.1242/10.1242/dev.02752

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Sperm binding to the zona pellucida is not sufficient to induce acrosome exocytosis

¹ Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA.
² Department of Cell Biology and Neuroscience, University of California, Riverside, CA 92521, USA.

View larger version (43K): [in this window] [in a new window]   Fig. 1. Persistent sperm binding to huZP2 rescue mouse embryos after fertilization. (A) Embryos were collected 1, 2, 4 and 24 hours after insemination and stained with Hoechst 33342 and Alexa 568-conjugated-soybean trypsin inhibitor (SBTI) before viewing by differential interference contrast (DIC) and confocal microscopy: (1-8) images of in vitro fertilization with Acr3-EGFP sperm (5x105) and wild-type (referred to as `normal') eggs; (9-16) same as 1-8, but after in vitro fertilization of huZP2 transgenic eggs; (17-24) same as 1-8, but after in vitro fertilization of huZP2 rescue eggs. Only the green and red channels of each image are displayed. Intact acrosomes are indicated by green (EGFP) on the anterior surface of sperm heads; reacted acrosomes are indicated by red because of Alexa 568-SBTI binding to the inner acrosomal membrane. Fertilization rates (>85%) were comparable among genotypes as judged by decondensing Hoechst-positive sperm nuclei in the egg cytoplasm (data not shown). (B) Bar graph (mean and s.e.m.) of sperm binding to normal (A, 1-8) huZP2 transgenic (A, 9-16) and huZP2 rescue (A, 17-24) embryos. Dotted lines represent linear regression of averages of number of sperm binding to normal (green), huZP2 transgenic (blue) or huZP2 rescue (red) embryos. (C) Postfertilization cleavage of ZP2 detected by immunoblot using monoclonal antibody to mouse ZP2, and normal eggs or embryos isolated 0, 1, 2, 4 and 24 hours after in vitro fertilization with normal sperm. Scale bar: 20 µm.

View larger version (86K): [in this window] [in a new window]   Fig. 2. Transmission electron photomicrographs of Acr3-EGFP sperm binding to mouse embryos. After in vitro fertilization, Acr3-EGFP sperm bound to: (A) the zona pellucida surrounding wild-type (referred to as `normal') mouse embryos (2 hours post-insemination) with an intact acrosome; (B) huZP2 transgenic embryos (12 hours post-insemination) having undergone acrosomal exocytosis; and (C) huZP2 rescue embryos (12 hours post-insemination) with an intact acrosome. IAM, inner acrosomal membrane. Scale bar: 1 µm.

View larger version (35K): [in this window] [in a new window]   Fig. 3. De novo Acr3-EGFP sperm binding to two-cell embryos. (A) Two-cell embryos derived from wild-type (referred to as `normal') (1-3), huZP2 transgenic (4-6) and huZP2 rescue (7-9) mice were incubated with 5x105 motile, capacitated Acr3-EGFP sperm for 1 hour and washed; Zp3-EGFP two-cell mouse embryos were used as controls (insets 1,4,7). Embryos were stained with Alexa 568-SBTI and fixed before imaging by DIC (1,4,7) and confocal microscopy to observe EGFP (2,5,8) or SBTI binding (3,6,9), reflecting acrosome-intact and acrosome-reacted sperm, respectively. (B) Aliquots of embryos from A were incubated for an additional 23 hours, and similar numbers of sperm bound to huZP2 transgenic (1-3, 49±8 s.e.m. per embryo) and huZP2 rescue (4-6, 73±15 s.e.m. per embryo) embryos. However, sperm binding to huZP2-transgenic-derived embryos were acrosome-reacted (97%), and those binding to huZP2-rescue-derived embryos remained acrosome-intact (94%). Scale bars: 30 µm.

View larger version (25K): [in this window] [in a new window]   Fig. 4. Mouse, but not human, ZP2 is cleaved following fertilization. Immunoblot of eggs (lanes 1,3,5) and two-cell embryos (lanes 2,4,6) from wild-type (referred to as `normal') (lanes 1 and 2), huZP2 rescue (lanes 3 and 4) and huZP2 transgenic (lanes 5-6) female mice. After SDS-PAGE run under reducing conditions (5% ß-mercaptoethanol), immunoblots were incubated with monoclonal antibodies specific to the carboxyl fragment of mouse (faux colored green) or human (faux colored red) ZP2 and visualized with a secondary antibody and chemiluminescence. Molecular masses (kDa) are indicated on the left.

View larger version (69K): [in this window] [in a new window]   Fig. 5. Reversible sperm binding to wild-type, huZP2 transgenic and huZP2 rescue eggs and embryos. Wild-type (referred to as `normal') (1,2), huZP2 transgenic (5,6) and huZP2 rescue (9,10) eggs or huZP2 transgenic (13,14) and huZP2 rescue (17,18) two-cell embryos were incubated with normal sperm for 1 hour (1,5,9) or 24 hours (13,17) and stained with Alexa 568-SBTI and Hoechst before imaging by DIC (1,5,9,13,17) and confocal microscopy (2,6,10,14,18). After a brief rinse, 5x105 motile, capacitated Acr3-EGFP sperm were added and incubated for an additional 1 hour. After washing to remove non-adherent sperm, eggs and embryos were stained with Alexa 568-SBTI before imaging by DIC (3,7,11,15,19) and confocal microscopy to observe SBTI and EGFP (4,8,12,16,20), reflecting acrosome-reacted and acrosome-intact sperm, respectively. Images were modified in Adobe Photoshop to remove nuclear staining from EGFP-positive sperm; thus, Hoechst-positive, EGFP-negative sperm (4,8,12,16,20) reflect those that were not displaced by EGFP-sperm. Insets (3,7,11,15,19), Zp3EGFP control two-cell mouse embryos from each incubation after washing.

View larger version (28K): [in this window] [in a new window]   Fig. 6. Passage through inert polycarbonate filters induces the sperm acrosome reaction. (A) Capacitated Acr3-EGFP sperm (2.1x104) were placed in the lower chamber and incubated for 30 minutes. The sperm acrosome status was assayed before (lower chamber) and after (upper chamber) penetration of individual polycarbonate filters (pore size, 1.2-12 µm); two example filters are imaged on the right. (B) Fixed sperm were stained with propidium iodide and divided into acrosome-intact and -reacted populations by FACS (left). The acrosome status of each population was confirmed by confocal microscopy (right). (C) Acr3-EGFP sperm, isolated before (lower chamber) and after (upper chamber) passage through individual filters (pore size, 1.2-12 µm), were fixed, stained with propidium iodide and scored by FACS for acrosome status. (D) Epididymal sperm were isolated and capacitated for 1 hour and an aliquot was passed over a glass bead column to remove acrosome-reacted sperm. The acrosome status of epididymal (left) and glass-bead-treated (right) sperm was assayed by FACS before (lower chamber) and after (upper chamber) passage through a 3 µm pore-size polycarbonate filter.

View larger version (14K): [in this window] [in a new window]   Fig. 7. Quantification of polycarbonate filter penetration assay. (A) Approximately 2.1x104 Acr3-EGFP sperm were assayed for penetration through polycarbonate filters. The average number of sperm (± s.e.m.) in the lower and upper chambers was determined by FACS and hand counting. The number of sperm recovered from the upper chamber was 123.3±38.1, 141.7±46.8, 1356.7±165.9, and 2165.0±188.0, after passage through filters with pore sizes 1.2, 3, 5 and 12 µm, respectively. (B) The acrosome status of Acr3-EGFP sperm was determined before (epididymal) and after (treated) passage through the glass bead column (controls) by FACS. Following the 30 minutes filter penetration assay, the percent of acrosome-reacted sperm in the lower (blue) and upper (red) chambers was determined by FACS and confirmed morphologically.

View larger version (22K): [in this window] [in a new window]   Fig. 8. Model of mechanosensory induction of sperm acrosome reaction. (A) Capacitated, motile and acrosome-intact sperm approach the inert polycarbonate filter lacking zona ligands. Depending on the size of the pore, the sperm is slowed or stopped as it seeks to penetrate the filter. The continued forward motility of the sperm transduces a mechanosensory signal that leads to increased intracellular Ca2+ and induction of the acrosome reaction. After passage through 3 µm or smaller pores, mostly acrosome-reacted sperm are observed on the far side of the filter. (B) Capacitated, motile and acrosome-intact sperm approach and bind to the zona pellucida. This binding (or the limiting size of the matrix interstices) immobilizes the sperm plasma membrane, inhibiting further progression of the sperm. However, the continued forward motility of the sperm transduces a mechanosensory signal that leads to increased intracellular Ca2+ and induction of the acrosome reaction. The residual acrosomal shroud is left behind bound to the surface of the zona pellucida matrix, and only acrosome-reacted sperm enter into the perivitelline space.

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