Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the organ before imaging.^1,2 However, for organs that contain fluorescent proteins such as GFP and YFP, optical clearing protocols that use methanol dehydration and clear using benzyl alcohol:benzyl benzoate (BABB) while unprotected from light³ do not preserve the fluorescent signal. The protocol presented here is a novel way in which to perform whole organ optical clearing on mouse brain while preserving the fluorescence signal of YFP expressed in neurons. Altering the optical clearing protocol such that the organ is dehydrated using an ethanol graded series has been found to reduce the damage to the fluorescent proteins and preserve their fluorescent signal for multiphoton imaging.⁴ Using an optimized method of optical clearing with ethanol-based dehydration and clearing by BABB while shielded from light, we show high-resolution multiphoton images of yellow fluorescent protein (YFP) expression in the neurons of a mouse brain more than 2 mm beneath the tissue surface.     

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkA^taulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkA^taulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkA^taulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.     

Clearing,immunofluorescence, and confocal microscopy for the three-dimensional imaging of murine testes and study of testis biology

Optical clearing techniques provide unprecedented opportunities to study large tissue samples at histological resolution, eliminating the need for physical sectioning while preserving the three-dimensional structure of intact biological systems. There is significant potential for applying optical clearing to reproductive tissues. In testicular biology, for example, the study of spermatogenesis and the use of spermatogonial stem cells offer high-impact applications in fertility medicine and reproductive biotechnology. The objective of our study is to apply optical clearing, immunofluorescence, and confocal microscopy to testicular tissue in order to reconstruct its three-dimensional microstructure in intact samples. We used Triton-X/DMSO clearing in combination with refractive index matching to achieve optical transparency of fixed mouse testes. An antibody against smooth muscle actin was used to label peritubular myoid cells of seminiferous tubules while an antibody against ubiquitin C-terminal hydrolase was used to label Sertoli cells and spermatogonia in the seminiferous epithelium. Specimens were then imaged using confocal fluorescence microscopy. We were able to successfully clear testicular tissue and utilize immunofluorescent probes. Additionally, we successfully visualized the histological compartments of testicular tissue in three-dimensional reconstructions. Optical clearing combined with immunofluorescence and confocal imaging offers a powerful new method to analyze the cytoarchitecture of testicular tissue at histological resolution while maintaining the macro-scale perspective of the intact system. Considering the importance of the murine model, our developed method represents a significant contribution to the field of male reproductive biology, enabling the study of testicular function.     

Whole‐mount in situ hybridization of mouse brain to precisely locate mRNAs via fluorescence tomography

Nucleic acids of intact biological tissues are rich in biological information. Whole‐mount in situ hybridization is a powerful technique to mine the wealth of data contained in DNAs or RNAs, especially mRNAs. However, there are no simple, rapid approaches to precisely locate mRNAs in whole‐mount tissues such as intact brains. By combining the penetration procedures of iDISCO with the signal amplification approach termed hybridization chain reaction, we herein developed a method for whole‐brain in situ hybridization at cellular resolution. Based on fluorescence tomography instead of tissue clearing, this method provides a simple, rapid way to precisely locate mRNAs in the whole brain with cytoarchitectonic landmarks. As a proof of principle, we investigated the exact distribution of Cre mRNA in a Thy1‐Cre mouse brain. We found high levels of Cre mRNA in most regions of the subcortical nuclei and the brain stem but comparatively low levels in major areas of the cerebral cortex. This method may have broad applications in studies of RNA function and its relations with diseases.      

Laserspritzer: A Simple Method for Optogenetic Investigation with Subcellular Resolutions

To build a detailed circuit diagram in the brain, one needs to measure functional synaptic connections between specific types of neurons. A high-resolution circuit diagram should provide detailed information at subcellular levels such as soma, distal and basal dendrites. However, a limitation lies in the difficulty of studying long-range connections between brain areas separated by millimeters. Brain slice preparations have been widely used to help understand circuit wiring within specific brain regions. The challenge exists because long-range connections are likely to be cut in a brain slice. The optogenetic approach overcomes these limitations, as channelrhodopsin 2 (ChR2) is efficiently transported to axon terminals that can be stimulated in brain slices. Here, we developed a novel fiber optic based simple method of optogenetic stimulation: the laserspritzer approach. This method facilitates the study of both long-range and local circuits within brain slice preparations. This is a convenient and low cost approach that can be easily integrated with a slice electrophysiology setup, and repeatedly used upon initial validation. Our data with direct ChR2 mediated-current recordings demonstrates that the spatial resolution of the laserspritzer is correlated with the size of the laserspritzer, and the resolution lies within the 30 µm range for the 5 micrometer laserspritzer. Using olfactory cortical slices, we demonstrated that the laserspritzer approach can be applied to selectively activate monosynaptic perisomatic GABAergic basket synapses, or long-range intracortical glutamatergic inputs formed on different subcellular domains within the same cell (e.g. distal and proximal dendrites). We discuss significant advantages of the laserspritzer approach over the widely used collimated LED whole-field illumination method in brain slice electrophysiological research.     

Hub GABA neurons mediate gamma-frequency oscillations at ictal-like event onset in the immature hippocampus

Gamma-frequency oscillations (GFOs, >40?Hz) are a general network signature at seizure onset at all stages of development, with possible deleterious consequences in the immature brain. At early developmental stages, the simultaneous occurrence of GFOs in different brain regions suggests the existence of a long-ranging synchronizing mechanism at seizure onset. Here, we show that hippocamposeptal (HS) neurons, which are GABA long-range projection neurons, are mandatory to drive the firing of hippocampal interneurons in a high-frequency regime at the onset of epileptiform discharges in the intact, immature septohippocampal formation. The synchronized firing of interneurons in turn produces GFOs, which are abolished after the elimination of a small number of HS neurons. Because they provide the necessary fast conduit for pacing large neuronal populations and display intra- and extrahippocampal long-range projections, HS neurons appear to belong to the class of hub cells that play a crucial role in the synchronization of developing networks.     

Fem1a is a mitochondrial protein up-regulated upon ischemia-reperfusion injury

Various expression studies have shown a preferential muscle expression of the mouse Fem1a gene, but no data is available on the subcellular localization of the corresponding protein. Here, using a specific antibody, we show that Fem1a is expressed preferentially in cardiac muscle, brain and liver. Moreover, using immunofluorescence and electron microscopy, as well as biochemical assays, we demonstrate that Fem1a is localized within mitochondria of C2C12 myoblasts and cardiac muscle cells. Finally, we show that the expression of Fem1a, which is a cellular partner of the EP4 receptor for prostaglandin E₂, is increased in mouse hearts after myocardial infarction.     

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high spatial and temporal resolution. Combined with electrophysiology, it is widely used to study activity-related calcium signals in small subcellular compartments such as dendrites and dendritic spines. In addition to calcium transients, synaptic activity also induces postsynaptic sodium signals, the properties of which are only marginally understood. Here, we describe a method for combined whole-cell patch-clamp and multi-photon sodium imaging in cellular micro domains of central neurons. Furthermore, we introduce a modified procedure for ultra-violet (UV)-light-induced uncaging of glutamate, which allows reliable and focal activation of glutamate receptors in the tissue. To this end, whole-cell recordings were performed on Cornu Ammonis subdivision 1 (CA1) pyramidal neurons in acute tissue slices of the mouse hippocampus. Neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines. To establish UV-induced focal uncaging, several parameters including light intensity, volume affected by the UV uncaging beam, positioning of the beam as well as concentration of the caged compound were tested and optimized. Our results show that local perfusion with caged glutamate (MNI-Glutamate) and its focal UV-uncaging result in inward currents and sodium transients in dendrites and spines. Time course and amplitude of both inward currents and sodium signals correlate with the duration of the uncaging pulse. Furthermore, our results show that intracellular sodium signals are blocked in the presence of blockers for ionotropic glutamate receptors, demonstrating that they are mediated by sodium influx though this pathway. In summary, our method provides a reliable tool for the investigation of intracellular sodium signals induced by focal receptor activation in intact brain tissue.     

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.     

Quantitative assessment of optical clearing methods in various intact mouse organs

Various tissue optical clearing techniques have sprung up for large volume imaging. However, there are few methods showed clearing and imaging data on different organs while most of them were focused on mouse brain, and as a result, it is difficult to select the suitable method for organs in practical applications due to lack of quantitative evaluation and comprehensive comparison. Therefore, it is necessary to evaluate and compare the performances of clearing methods for different organs. In this paper, several typical optical clearing methods were applied, including 3DISCO, uDISCO, SeeDB, FRUIT, CUBIC, ScaleS and PACT to clear intact brain, heart, kidney, liver, spleen, stomach, lung, small intestine, skin and muscle. The clearing efficiency, sample deformation, fluorescence preservation and imaging depth of these methods were quantitatively evaluated. Finally, based on the systemic evaluation of various parameters described above, the appropriate clearing method for specific organ including kidney or intestine was screened out. This paper will provide important references for selection of appropriate clearing methods in related researches.      

Development of a Plastic Embedding Method for Large-Volume and Fluorescent-Protein-Expressing Tissues

Fluorescent proteins serve as important biomarkers for visualizing both subcellular organelles in living cells and structural and functional details in large-volume tissues or organs. However, current techniques for plastic embedding are limited in their ability to preserve fluorescence while remaining suitable for micro-optical sectioning tomography of large-volume samples. In this study, we quantitatively evaluated the fluorescence preservation and penetration time of several commonly used resins in a Thy1-eYFP-H transgenic whole mouse brain, including glycol methacrylate (GMA), LR White, hydroxypropyl methacrylate (HPMA) and Unicryl. We found that HMPA embedding doubled the eYFP fluorescence intensity but required long durations of incubation for whole brain penetration. GMA, Unicryl and LR White each penetrated the brain rapidly but also led to variable quenching of eYFP fluorescence. Among the fast-penetrating resins, GMA preserved fluorescence better than LR White and Unicryl. We found that we could optimize the GMA formulation by reducing the polymerization temperature, removing 4-methoxyphenol and adjusting the pH of the resin solution to be alkaline. By optimizing the GMA formulation, we increased percentage of eYFP fluorescence preservation in GMA-embedded brains nearly two-fold. These results suggest that modified GMA is suitable for embedding large-volume tissues such as whole mouse brain and provide a novel approach for visualizing brain-wide networks.     

Comprehensive Evaluation of Peripheral Nerve Regeneration in the Acute Healing Phase Using Tissue Clearing and Optical Microscopy in a Rodent Model

Peripheral nerve injury (PNI), a common injury in both the civilian and military arenas, is usually associated with high healthcare costs and with patients enduring slow recovery times, diminished quality of life, and potential long-term disability. Patients with PNI typically undergo complex interventions but the factors that govern optimal response are not fully characterized. A fundamental understanding of the cellular and tissue-level events in the immediate postoperative period is essential for improving treatment and optimizing repair. Here, we demonstrate a comprehensive imaging approach to evaluate peripheral nerve axonal regeneration in a rodent PNI model using a tissue clearing method to improve depth penetration while preserving neural architecture. Sciatic nerve transaction and end-to-end repair were performed in both wild type and thy-1 GFP rats. The nerves were harvested at time points after repair before undergoing whole mount immunofluorescence staining and tissue clearing. By increasing the optic depth penetration, tissue clearing allowed the visualization and evaluation of Wallerian degeneration and nerve regrowth throughout entire sciatic nerves with subcellular resolution. The tissue clearing protocol did not affect immunofluorescence labeling and no observable decrease in the fluorescence signal was observed. Large-area, high-resolution tissue volumes could be quantified to provide structural and connectivity information not available from current gold-standard approaches for evaluating axonal regeneration following PNI. The results are suggestive of observed behavioral recovery in vivo after neurorrhaphy, providing a method of evaluating axonal regeneration following repair that can serve as an adjunct to current standard outcomes measurements. This study demonstrates that tissue clearing following whole mount immunofluorescence staining enables the complete visualization and quantitative evaluation of axons throughout nerves in a PNI model. The methods developed in this study could advance PNI research allowing both researchers and clinicians to further understand the individual events of axonal degeneration and regeneration on a multifaceted level.     

Fragments of HdhQ150 Mutant Huntingtin Form a Soluble Oligomer Pool That Declines with Aggregate Deposition upon Aging

Cleavage of the full-length mutant huntingtin (mhtt) protein into smaller, soluble aggregation-prone mhtt fragments appears to be a key process in the neuropathophysiology of Huntington’s Disease (HD). Recent quantification studies using TR-FRET-based immunoassays showed decreasing levels of soluble mhtt correlating with an increased load of aggregated mhtt in the aging HdhQ150 mouse brain. To better characterize the nature of these changes at the level of native mhtt species, we developed a detection method that combines size exclusion chromatography (SEC) and time-resolved fluorescence resonance energy transfer (TR-FRET) that allowed us to resolve and define the formation, aggregation and temporal dynamics of native soluble mhtt species and insoluble aggregates in the brain of the HdhQ150 knock-in mouse. We found that mhtt fragments and not full-length mhtt form oligomers in the brains of one month-old mice long before disease phenotypes and mhtt aggregate histopathology occur. As the HdhQ150 mice age, brain levels of soluble full-length mhtt protein remain similar. In contrast, the soluble oligomeric pool of mhtt fragments slightly increases during the first two months before it declines between 3 and 8 months of age. This decline inversely correlates with the formation of insoluble mhtt aggregates. We also found that the pool-size of soluble mhtt oligomers is similar in age-matched heterozygous and homozygous HdhQ150 mouse brains whereas insoluble aggregate formation is greatly accelerated in the homozygous mutant brain. The capacity of the soluble mhtt oligomer pool therefore seems exhausted already in the heterozygous state and likely kept constant by changes in flux and, as a consequence, increased rate of insoluble aggregate formation. We demonstrate that our novel findings in mice translate to human HD brain but not HD patient fibroblasts.     

Tissue clearing‐based method for unobstructed three‐dimensional imaging of mouse penis with subcellular resolution

Although mice are widely used to elucidate factors contributing to penile disorders and develop treatment options, quantification of tissue changes upon intervention is either limited to minuscule tissue volume (histology) or acquired with limited spatial resolution (MRI/CT). Thus, imaging method suitable for expeditious acquisition of the entire mouse penis with subcellular resolution is described that relies on both aqueous‐ (clear, unobstructed brain imaging cocktails and computational analysis) and solvent‐based (fluorescence‐preserving capability imaging of solvent‐cleared organs) tissue optical clearing (TOC). The combined TOC approach allows to image mouse penis innervation and vasculature with unprecedented detail and, for the first time, reveals the three‐dimensional structure of murine penis fibrocartilage.     

A Rapid Optical Clearing Protocol Using 2,2′-Thiodiethanol for Microscopic Observation of Fixed Mouse Brain

Elucidation of neural circuit functions requires visualization of the fine structure of neurons in the inner regions of thick brain specimens. However, the tissue penetration depth of laser scanning microscopy is limited by light scattering and/or absorption by the tissue. Recently, several optical clearing reagents have been proposed for visualization in fixed specimens. However, they require complicated protocols or long treatment times. Here we report the effects of 2,2′-thiodiethanol (TDE) solutions as an optical clearing reagent for fixed mouse brains expressing a yellow fluorescent protein. Immersion of fixed brains in TDE solutions rapidly (within 30 min in the case of 400-µm-thick fixed brain slices) increased their transparency and enhanced the penetration depth in both confocal and two-photon microscopy. In addition, we succeeded in visualizing dendritic spines along single dendrites at deep positions in fixed thick brain slices. These results suggest that our proposed protocol using TDE solution is a rapid and useful method for optical clearing of fixed specimens expressing fluorescent proteins.     

Investigation of the proteolipid protein promoter activity during demyelination and repair

The transgenic plp-GFP mouse line expressing the green fluorescent protein (GFP) driven by the mouse myelin proteolipid protein (plp) gene promoter has been previously used to study the contribution of the plp lineage to oligodendrocyte development in the embryonic brain. Here, we show that the GFP fluorescence reflects the developmental expression of proteolipid protein during the postnatal development until adulthood in brain slices and in primary cultures of plp-GFP⁺ cells derived from postnatal animals. In the adult brain, plp-GFP-expressing cells are mature oligodendrocytes but not oligodendroglial progenitors. In the model of focal demyelination induced by lysolecithin (LPC) in the corpus callosum of adult plp-GFP animals, we observed an up-regulation of the morphogen Sonic Hedgehog (Shh) in the LPC-induced lesion but not in the control animals. Moreover, we show that the adenovirus-mediated transfer of Shh in the lesion results in the attenuation of the demyelination extent as evidenced by GFP fluorescence analysis in Shh-treated and control animals. Altogether these data show how plp-GFP fluorescence can be monitored to follow the oligodendrocyte lineage during demyelination and identify Shh morphogen as an important factor during repair.     

Finding Needles in a Haystack with Light: Resolving the Microcircuitry of the Brain with Fluorescence Microscopy

To understand the microcircuitry of the brain, the anatomical and functional connectivity among neurons must be resolved. One of the technical hurdles to achieving this goal is that the anatomical connections, or synapses, are often smaller than the diffraction limit of light and thus are difficult to resolve by conventional microscopy, while the microcircuitry of the brain is on the scale of 1 mm or larger. To date, the gold standard method for microcircuit reconstruction has been electron microscopy (EM). However, despite its rapid development, EM has clear shortcomings as a method for microcircuit reconstruction. The greatest weakness of this method is arguably its incompatibility with functional and molecular analysis. Fluorescence microscopy, on the other hand, is readily compatible with numerous physiological and molecular analyses. We believe that recent advances in various fluorescence microscopy techniques offer a new possibility for reliable synapse detection in large volumes of neural circuits. In this minireview, we summarize recent advances in fluorescence-based microcircuit reconstruction. In the same vein as these studies, we introduce our recent efforts to analyze the long-range connectivity among brain areas and the subcellular distribution of synapses of interest in relatively large volumes of cortical tissue with array tomography and superresolution microscopy.     

Subcellular localization of flavonol aglycone in hepatocytes visualized by confocal laser scanning fluorescence microscope

Flavonoids are widely distributed in the plant kingdom and show various biological activities. The bioavailability of flavonoids in biological samples has conventionally been quantified by high-performance liquid chromatography and mass spectrometry, but with these analytical techniques it is difficult to estimate the subcellular localization of flavonoids in intact cells. In this study, we attempted to examine the localization of flavonoids in cultured cells using a confocal laser scanning fluorescence microscope and mouse hepatoma Hepa-1c1c7 cells. Five flavonol aglycones showed autofluorescence in the cells under the conditions (Ex. 488 nm to Em. 515–535 nm), whereas three flavonol glycosides and eight compounds belonging to other flavonoid subclasses, i.e., flavones, flavanones, and catechins, did not. The autofluorescence of galangin and kaempferol appeared stronger in the nucleus than cytoplasm, suggesting that they are incorporated into the cells and accumulated in the nucleus. The proposed method provided evidence that flavonol aglycones are incorporated into, and accumulated in the nucleus of, hepatocytes.     

Retinofugal Projections from Melanopsin-Expressing Retinal Ganglion Cells Revealed by Intraocular Injections of Cre-Dependent Virus

To understand visual functions mediated by intrinsically photosensitive melanopsin-expressing retinal ganglion cells (mRGCs), it is important to elucidate axonal projections from these cells into the brain. Initial studies reported that melanopsin is expressed only in retinal ganglion cells within the eye. However, recent studies in Opn4-Cre mice revealed Cre-mediated marker expression in multiple brain areas. These discoveries complicate the use of melanopsin-driven genetic labeling techniques to identify retinofugal projections specifically from mRGCs. To restrict labeling to mRGCs, we developed a recombinant adeno-associated virus (AAV) carrying a Cre-dependent reporter (human placental alkaline phosphatase) that was injected into the vitreous of Opn4-Cre mouse eyes. The labeling observed in the brain of these mice was necessarily restricted specifically to retinofugal projections from mRGCs in the injected eye. We found that mRGCs innervate multiple nuclei in the basal forebrain, hypothalamus, amygdala, thalamus and midbrain. Midline structures tended to be bilaterally innervated, whereas the lateral structures received mostly contralateral innervation. As validation of our approach, we found projection patterns largely corresponded with previously published results; however, we have also identified a few novel targets. Our discovery of projections to the central amygdala suggests a possible direct neural pathway for aversive responses to light in neonates. In addition, projections to the accessory optic system suggest that mRGCs play a direct role in visual tracking, responses that were previously attributed to other classes of retinal ganglion cells. Moreover, projections to the zona incerta raise the possibility that mRGCs could regulate visceral and sensory functions. However, additional studies are needed to investigate the actual photosensitivity of mRGCs that project to the different brain areas. Also, there is a concern of "overlabeling" with very sensitive reporters that uncover low levels of expression. Light-evoked signaling from these cells must be shown to be of sufficient sensitivity to elicit physiologically relevant responses.     

Mouse gamete interactions during fertilization in vitro. Chlortetracycline as a fluorescent probe for the mouse sperm acrosome reaction

We have developed an assay for detecting the acrosome reaction in mouse sperm using chlortetracycline (CTC) as a fluorescent probe. Sperm known to be intact with nonreacted acrosomes show CTC fluorescence in the presence of Ca2+ over the anterior portion of the sperm head on the plasma membrane covering the acrosome. Sperm which have undergone the acrosome reaction do not show fluorescence on the sperm head. Mouse sperm bind to zonae pellucidae of cumulus-free eggs in vitro in a Ca2+-dependent reaction; these sperm are intact by the CTC assay. Intact sperm bind to mechanically isolated zonae under the same conditions: the egg is apparently unnecessary for this inital reaction. Sperm suspensions, in which greater than 50% of the motile population had completed the acrosome reaction, were prepared by incubation in hyperosmolal medium followed by treatment with the divalent cation ionophore, A23187. Cumulus-free eggs challenged with such sperm suspensions preferentially bind intact sperm; acrosome-reacted sperm do not bind. We conclude that the plasma membrane of the mouse sperm is responsible for recognition of the egg's zona pellucida and that the obligatory sequence of reactions leading to fusion of mouse gametes is binding of the intact sperm to the zona pellucida, followed by the acrosome reaction at the zona surface, followed in turn by sperm penetration of the zona.