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

AbstractWe 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

Lactation and neonatal nutrition: defining and refining the critical questions.

This paper resulted from a conference entitled "Lactation and Milk: Defining and refining the critical questions" held at the University of Colorado School of Medicine from January 18-20, 2012. The mission of the conference was to identify unresolved questions and set future goals for research into human milk composition, mammary development and lactation. We first outline the unanswered questions regarding the composition of human milk (Section I) and the mechanisms by which milk components affect neonatal development, growth and health and recommend models for future research. Emerging questions about how milk components affect cognitive development and behavioral phenotype of the offspring are presented in Section II. In Section III we outline the important unanswered questions about regulation of mammary gland development, the heritability of defects, the effects of maternal nutrition, disease, metabolic status, and therapeutic drugs upon the subsequent lactation. Questions surrounding breastfeeding practice are also highlighted. In Section IV we describe the specific nutritional challenges faced by three different populations, namely preterm infants, infants born to obese mothers who may or may not have gestational diabetes, and infants born to undernourished mothers. The recognition that multidisciplinary training is critical to advancing the field led us to formulate specific training recommendations in Section V. Our recommendations for research emphasis are summarized in Section VI. In sum, we present a roadmap for multidisciplinary research into all aspects of human lactation, milk and its role in infant nutrition for the next decade and beyond

Mouse oviduct-specific glycoprotein is an egg-associated ZP3-independent sperm-adhesion ligand

Mouse sperm-egg binding requires a multiplicity of receptor-ligand interactions, including an oviduct-derived, high molecular weight, wheat germ agglutinin (WGA)-binding glycoprotein that associates with the egg coat at ovulation. Herein, we report the purification and identification of this sperm-binding ligand. WGA-binding, high molecular weight glycoproteins isolated from hormonally primed mouse oviduct lysates competitively inhibit sperm-egg binding in vitro. Within this heterogeneous glycoprotein preparation, a distinct 220 kDa protein selectively binds to sperm surfaces, and was identified by sequence analysis as oviduct-specific glycoprotein (OGP). The sperm-binding activity of OGP was confirmed by the loss of sperm-binding following immunodepletion of OGP from oviduct lysates, and by the ability of both immunoprecipitated OGP and natively purified OGP to competitively inhibit sperm-egg binding. As expected, OGP is expressed by the secretory cells of the fimbriae and infundibulum; however, in contrast to previous reports, OGP is also associated with both the zona pellucida and the perivitelline space of mouse oocytes. Western blot analysis and lectin affinity chromatography demonstrate that whereas the bulk of OGP remains soluble in the ampullar fluid, distinct glycoforms associate with the cumulus matrix, zona pellucida and perivitelline space. The sperm-binding activity of OGP is carbohydrate-dependent and restricted to a relatively minor peanut agglutinin (PNA)-binding glycoform that preferentially associates with the sperm surface, zona pellucida and perivitelline space, relative to other more abundant glycoforms. Finally, pretreatment of two-cell embryos, which do not normally bind sperm, with PNA-binding OGP stimulates sperm binding

A novel role for SED1 (MFG-E8) in maintaining the integrity of the epididymal epithelium

The epididymis is a highly convoluted tubule that connects the testis with the vas deferens, and in which mammalian sperm acquire the ability to fertilize eggs. The most proximal portion of the epididymis, or initial segment, secretes numerous factors that are critical for sperm maturation and storage. One such factor is SED1 (also known as MFG-E8) a bi-motif protein composed of two N-terminal EGF domains, the second of which contains an RGD motif, and two C-terminal discoidin domains (also known as F5/8 type C domains). Previous studies have reported that SED1 is secreted into the epididymal lumen, where it coats sperm and later facilitates sperm-egg binding. Herein, we report that SED1-null males also harbor unexpected epididymal pathologies, including detached epithelia and spermatic granulomas. We therefore examined whether SED1 has a tissue-intrinsic role in the epididymis, in addition to its role in sperm-egg adhesion. Improved fixation protocols revealed that SED1 is found in the basolateral domains of epididymal epithelial cells in vivo, and similarly, SED1 is secreted both apically and basally from polarized epididymal cells in vitro. The basolateral distribution of SED1 suggests that it may play a novel role in epididymal cell adhesion. Consistent with this, in vitro assays showed that SED1 supports epididymal cell adhesion via RGD binding to αV integrin receptors on epididymal epithelial cells. Finally, epididymal cells from SED1-null males showed reduced adhesion in vitro, a phenotype that can be rescued with exogenous SED1. These results suggest that SED1 facilitates epididymal cell adhesion, and that its loss leads to breakdown of the epididymal epithelium and consequent development of spermatic granulomas