Michael A. Ensslin

Identification of Mouse Sperm SED1, a Bimotif EGF Repeat and Discoidin-Domain Protein Involved in Sperm-Egg Binding

We report the identification of SED1, a protein required for mouse sperm binding to the egg zona pellucida. SED1 is homologous to a small group of secreted cell-matrix adhesive proteins that contain Notch-like EGF repeats and discoidin/F5/8 type C domains. SED1 is expressed in spermatogenic cells and is secreted by the initial segment of the caput epididymis, resulting in SED1 localization on the sperm plasma membrane overlying the acrosome. SED1 binds specifically to the zona pellucida of unfertilized oocytes, but not to the zona of fertilized eggs. Recombinant SED1 and anti-SED1 antibodies competitively inhibit sperm-egg binding, as do truncated SED1 proteins containing a discoidin/C domain. SED1 null males are subfertile and their sperm are unable to bind to the egg coat in vitro. These studies illustrate that Notch-like EGF and discoidin/C domains, protein motifs that facilitate a variety of cellular interactions, participate in gamete recognition as well.

Milk fat globule–EGF factor 8/lactadherin plays a crucial role in maintenance and repair of murine intestinal epithelium

Milk fat globule-EGF factor 8 (MFG-E8)/lactadherin participates in several cell surface-mediated regulatory events. Although its mRNA is present in the gut, the physiological roles of MFG-E8 in the intestinal mucosa have not been explored. Here we show that MFG-E8 was expressed in intestinal lamina propria macrophages from mice. Using a wound-healing assay, MFG-E8 was shown to promote the migration of intestinal epithelial cells through a PKCepsilon-dependent mechanism. MFG-E8 bound to phosphatidylserine and triggered reorientation of the actin cytoskeleton in intestinal epithelial cells at the wound edge. Depleting MFG-E8 in mice by administration of anti-MFG-E8 antibody or targeted deletion of the MFG-E8 gene resulted in a slowing of enterocyte migration along the crypt-villus axis and focal mucosal injury. Moreover, in septic mice, intestinal MFG-E8 expression was downregulated, which correlated with intestinal injury, interrupted enterocyte migration, and impaired restitution. Treatment with recombinant MFG-E8 restored enterocyte migration, whereas deletion of MFG-E8 impeded mucosal healing in mice with sepsis. These results suggest that a decrease in intestinal MFG-E8 impairs intestinal mucosal repair in sepsis. Together, our data indicate that MFG-E8 plays an important role in the maintenance of intestinal epithelial homeostasis and the promotion of mucosal healing and suggest that recombinant MFG-E8 may be beneficial for the treatment of bowel injuries.

SED1/MFG-E8: a bi-motif protein that orchestrates diverse cellular interactions

MFG‐E8 was initially identified as a principle component of the Milk Fat Globule, a membrane‐encased collection of proteins and triglycerides that bud from the apical surface of mammary epithelia during lactation. It has since been independently identified in many species and by many investigators and given a variety of names, including p47, lactadherin, rAGS, PAS6/7, and BA‐46. The acronym SED1 was proposed to bring cohesion to this nomenclature based upon it being a Secreted protein that contains two distinct functional domains: an N‐terminal domain with two EGF‐repeats, the second of which has an integrin‐binding RGD motif, and a C‐terminal domain with two Discoidin/F5/8C domains that bind to anionic phospholipids and/or extracellular matrices. SED1/MFG‐E8 is now known to participate in a wide variety of cellular interactions, including phagocytosis of apoptotic lymphocytes and other apoptotic cells, adhesion between sperm and the egg coat, repair of intestinal mucosa, mammary gland branching morphogenesis, angiogenesis, among others. This article will explore the various roles proposed for SED1/MFG‐E8, as well as its provocative therapeutic potential. J. Cell. Biochem. 106: 957–966, 2009.

Composition and significance of detergent resistant membranes in mouse spermatozoa

Mammalian spermatozoa acquire the ability to fertilize an oocyte as they ascend the female reproductive tract. This process is characterized by a complex cascade of biophysical and biochemical changes collectively know as “capacitation.” The attainment of a capacitated state is accompanied by a dramatic reorganization of the surface architecture to render spermatozoa competent to recognize the oocyte and initiate fertilization. Emerging evidence indicates that this process is facilitated by molecular chaperone‐mediated assembly of a multimeric receptor complex on the sperm surface. However, the mechanisms responsible for gathering key recognition molecules within this putative complex have yet to be defined. In this study, we provide the first evidence that chaperones partition into detergent resistant membrane fractions (DRMs) within capacitated mouse spermatozoa and co‐localize in membrane microdomains enriched with the lipid raft marker, GM1 ganglioside. During capacitation, these microdomains coalesce within the apical region of the sperm head, a location compatible with a role in sperm–zona pellucida interaction. Significantly, DRMs isolated from spermatozoa possessed the ability to selectively bind to the zona pellucida of unfertilized, but not fertilized, mouse oocytes. A comprehensive proteomic analysis of the DRM fractions identified a total of 100 proteins, a number of which have previously been implicated in sperm–oocyte interaction. Collectively, these data provide compelling evidence that mouse spermatozoa possess membrane microdomains that provide a platform for the assembly of key recognition molecules on the sperm surface and thus present an important mechanistic insight into the fundamental cell biological process of sperm–oocyte interaction. J. Cell. Physiol. 218: 122–134, 2009.

Galactosyltransferase Function during Mammalian Fertilization

Gamete recognition has been studied extensively in the mouse. In this system, it is generally believed that sperm bind to a class of O-linked oligosaccharides on the zona pellucida glycoprotein, ZP3. The best characterized sperm receptor for ZP3 is β1,4-galactosyltransferase (GalT), which functions in a lectin-like capacity by binding to N-terminal N-acetylglucosamine residues on ZP3 oligosaccharides. Multivalent oligosaccharides on ZP3, as well as synthetic polymers terminating in N-acetylglucosamine aggregate GalT, leading to activation of a heterotrimeric G protein cascade and culminating in the acrosome reaction. Following fertilization, cortical granules release N-acetylglucosaminidase, which removes the binding site for sperm GalT and facilitates the zona block to polyspermic binding. Genetic manipulation of GalT expression has confirmed its function as a ZP3 receptor. Overexpressing GalT on sperm leads to increased binding of ZP3, increased G protein activation, and precocious acrosome reactions. In contrast, sperm from mice made null for GalT by homologous recombination are refractory to ZP3, in that they are unable to bind soluble ZP3 and fail to undergo the acrosome reaction in response to zona glycoproteins. Surprisingly, GalT null sperm still bind to the zona and achieve low rates of fertilization in vitro. This then suggests that sperm-egg binding involves receptor-ligand interactions independent of GalT and ZP3. The current model suggests that GalT functions as the ZP3 receptor that is responsible for inducing the acrosome reaction, whereas initial sperm-zona binding is dictated by other sperm surface receptors. Consistent with this, at least three other zona pellucida monosaccharides have been implicated in sperm binding, and novel sperm surface glycoproteins have been suggested to function in gamete binding. A large scaffolding protein has been identified that associates with the GalT cytoplasmic domain and may be responsible for orchestrating its signal transduction capacities that lead to the acrosome reaction.

Identification of novel gamete receptors that mediate sperm adhesion to the egg coat.

Mammalian fertilization is initiated by the species-specific binding of sperm to the zona pellucida, or egg coat. Earlier studies suggested that sperm-egg adhesion in mouse is mediated by the binding of beta1,4-galactosyltransferase-I (GalT) on the sperm surface to specific glycoside ligands on the egg coat glycoprotein, ZP3. Binding of multiple ZP3 oligosaccharides induces GalT aggregation, triggering a pertussis toxin-sensitive G-protein cascade leading to induction of the acrosome reaction. Consistent with this, sperm bearing targeted deletions in GalT are unable to bind ZP3 nor undergo ZP3-dependent acrosomal exocytosis; however, GalT-null sperm are still able to bind to the egg coat. This indicates that sperm-egg binding requires at least two independent binding mechanisms: a GalT-ZP3-independent event that mediates initial adhesion, followed by a GalT-ZP3 interaction that facilitates acrosomal exocytosis. During the past few years, novel GalT-ZP3-independent gamete receptors have been identified that appear to participate in initial gamete adhesion. On such receptor is SED1, an EGF repeat and discoidin domain protein that coats sperm as they traverse through the epididymis, and which is required for sperm to bind the egg coat. Similarly, a novel egg coat ligand is present on ovulated oocytes, but not on ovarian eggs, and which also appears to function in initial sperm binding. The identification of novel gamete receptors that are required for sperm-egg binding opens up new avenues for the development of specific contraceptive strategies.

The EGF repeat and discoidin domain protein, SED1/MFG-E8, is required for mammary gland branching morphogenesis

SED1, also known as MFG-E8, is a secreted protein composed of two EGF repeats (the second of which contains an RGD motif) and two discoidin/Factor V/VIII C domains. SED1 is expressed by a wide range of cell types, where it participates in diverse cellular interactions, such as sperm binding to the egg coat and macrophage recognition of apoptotic lymphocytes. Although SED1 was originally identified as a milk protein, its function in the mammary gland remains unclear; suggested functions include inhibition of viral infection and clearance of apoptotic cells during mammary gland involution. We report here that SED1 has an unexpected obligatory role during mammary gland development. Unlike that seen in WT glands, SED1-null glands show severely reduced branching from epithelial ducts and from terminal end buds, which are thin and poorly developed. SED1 is expressed by both luminal and myoepithelial cells in the developing epithelial duct, and binds to αv integrin receptors on myoepithelial cells leading to MAPK activation and cell proliferation. The absence of SED1 leads to greatly reduced levels of activated MAPK and a concomitant reduction in cell proliferation and branching throughout the epithelial tree. These results suggest that SED1 contributes, at least partly, to the intercellular signaling between luminal and myoepithelial cells that is required for branching morphogenesis.

PIKE-A is required for prolactin-mediated STAT5a activation in mammary gland development

PI 3‐kinase enhancer A (PIKE‐A) is critical for the activation of Akt signalling, and has an essential function in promoting cancer cell survival. However, its physiological functions are poorly understood. Here, we show that PIKE‐A directly associates with both signal transducer and activator of transcription 5a (STAT5a) and prolactin (PRL) receptor, which is essential for PRL‐provoked STAT5a activation and the subsequent gene transcription. Depletion of PIKE‐A in HC11 epithelial cells diminished PRL‐induced STAT5 activation and cyclin D1 expression, resulting in profoundly impaired cell proliferation in vitro. To confirm the function of PIKE‐A in PRL signalling in vivo, we generated PIKE knockout (PIKE−/−) mice. PIKE−/− mice displayed a severe lactation defect that was characterized by enhanced apoptosis and impaired proliferation of mammary epithelial cells. At parturition, STAT5 activation and cyclin D1 expression were substantially reduced in the mammary epithelium of PIKE−/− mice. The defective mammary gland development in PIKE−/− mice was rescued by overexpression of a mammary‐specific cyclin D1 transgene. These data establish a critical function for PIKE‐A in mediating PRL functions.

Loss of SED1/MFG-E8 results in altered luminal physiology in the epididymis

SED1/MFG‐E8, herein referred to as SED1, is a bimotif adhesive protein with ascribed functions in a range of cell–cell interactions, including sperm‐egg binding. In the male reproductive tract, SED1 is secreted by the initial segment of the epididymis, where it coats sperm and subsequently facilitates binding to the egg zona pellucida. We have recently reported that SED1‐null epididymides show an unexpected incidence of spermatic granulomas, reflecting breakdown of the epithelium and a consequent autoimmune response against sperm antigens. However, spermatic granulomas are most often manifest in the distal segments of the epididymis, whereas the bulk of SED1 is expressed in the proximal epididymis. In some models, the presence of granulomas in the distal epididymis is associated with an underlying defect in the maintenance of luminal fluid homeostasis. Herein, we report that SED1‐null epididymal fluid is both hypo‐osmotic and alkaline, relative to wildtype epididymal fluid. Furthermore, the SED1‐null epididymal epithelium exhibits various hallmarks of disrupted fluid reabsorption and pH regulation, including altered morphology of clear cells, increased intracellular vesicles, and apical distribution of VATPase. Results indicate that the SED1‐null epididymal pathologies are not the secondary consequences of defective testes or efferent ducts or of improper epididymal differentiation, unlike that seen in other epididymal models. The expression and distribution of various ion exchangers, channels, and enzymes that mediate fluid transport and pH regulation are examined in wildtype and SED1‐null epididymides, and models to account for how SED1 functions in luminal fluid dynamics are discussed. Mol. Reprod. Dev. 77: 550–563, 2010.