Epididymal PMCA4 Expression Functional Impact and Mechanism of Sperm Uptake

Summary

Principal Investigator: PATRICIA ANASTASIA DELEON
Abstract: DESCRIPTION (provided by applicant): Almost devoid of cytoplasm, sperm have the vast majority of their proteins present on the plasma membrane (PM). Many of these proteins are fertility-modulating and in the absence of synthetic machinery sperm acquire them from the epididymis during their post-testicular maturation. To date, the majority of epididymally-acquired sperm proteins identified are glycosyl phosphatidylinositol-(GPI)-linked to the outer leaflet of th PM's lipid bilayer. However, it has recently been reported that transmembrane (TM) proteins are also epididymally-expressed and present on membranous vesicles that are known to transfer GPI-linked proteins to sperm. Past research in the DeLeon Lab has contributed to an understanding of the mechanisms of sperm uptake of GPI-linked epididymal proteins, and we are now poised to launch an investigation for TM proteins which, due to their folded nature, are unlikely to be acquired by the mechanisms employed by GPI- linked proteins. In this application we focus on PMCA4 (Plasma Membrane Ca2+-ATPase 4), a 10 TM protein, whose 4b isoform is the major Ca2+ efflux pump in murine sperm where its absence leads to loss of motility and to infertility. Exciting Preliminary results indicate murine epididymal expression of the mRNA for the 4b as well as the 4a isoform which is more efficient in clearing Ca2+ and maintaining homeostasis. We also show that PMCA4a is present in caudal epididymal luminal fluid (ELF) and interestingly, more abundant in caudal than caput sperm. Our central hypothesis is that PMCA4 is expressed in the epididymis and is secreted in the epididymal luminal fluid (ELF) where it is acquired on the sperm surface for their maturation and function. Using Pmca4 null and wild-type (WT) mice, our Specific Aims in this R03 application are: 1) To investigate the expression pattern and secretion of PMCA4 in all three regions of the murine epididymis by analyzing the mRNA and protein isoforms in tissues as well as in ELF and sperm, and 2) To determine if, and how, PMCA4/a is acquired in vitro on the surface of WT and Pmca4 null caudal sperm, and if acquisition increases PMCA4 enzymatic activity, Ca2+ clearance, and motility. We will focus on the role of membranous vesicles or epididymosomes, in delivering PMCA4/a to the sperm surface, assisted by the presence of CD9 tetraspanin which mediates cell-to-cell protein transfer via membrane fusion. Sperm PM - vesicle interaction will be visualized in ultrastructural images using immunogold labeling to begin to understand the process of delivery of PMCA4 to the PM. Together, these experiments will provide new information on the mechanism of uptake of TM proteins and the role of epididymal PMCA4a in sperm maturation and function. Pilot data from this exploratory project will allow us to apply (through the R01 mechanism) for funding to fully mechanistically elucidate the role of vesicles in the delivery of PMCA4a and other TM proteins, and to determine the possibility of using artificial vesicles in ART (assisted reproductive technology) to deliver the proteins to deficient sperm to improve their fertilizing capabilities, via IVF.
Funding Period: 2013-07-01 - 2015-06-30
more information: NIH RePORT

Detail Information

Research Grants31

  1. Phosphorylation Events During Sperm Capacitation
    PABLO EDUARDO VISCONTI; Fiscal Year: 2013
    ..Accomplishment of these goals will lead to novel insights in clinical diagnosis of male infertility and provide tools for evaluating pharmacological contraceptive targets. ..
  2. Strategies for Improved Shock Wave Lithotripsy
    JAMES ALEXANDER MCATEER; Fiscal Year: 2013
    ..and the session can be ended * Determine the mechanism by which cavitation within a vessel causes hemorrhage * Develop numerical models to understand the role of cavitation and non-cavitational mechanisms in causing tissue damage ..
  3. Immune Responses To AAV-Mediated FIX Gene Transfer
    Hildegund C J Ertl; Fiscal Year: 2013
    ..HC ErtI): T Cells to AAV and AAV-Encoded Transgene Products Project 3 (RW Herzog, C Terliorst): Pathways Towards Immune Tolerance to Coagulation Factors Core A (HC ErtI): Administrative Core Core B (S Zliou): Vector Core ..
  4. Baylor Intellectual and Developmental Disabilities Research Center
    Huda Y Zoghbi; Fiscal Year: 2013
    ..abstract_text> ..
  5. Serotonin as a Regulator of Bone Mass Accrual: Basic and Clinical
    Gerard Karsenty; Fiscal Year: 2013
    ..Together our studies should provide important and novel insights in the genetic and molecular control of bone remodeling as well as in the pathogenesis and treatment of osteoporosis, a major disease of aging. ..
  6. Transplant Tolerance in Non-Human Primates
    STUART JOHNSTON KNECHTLE; Fiscal Year: 2013
    ..This goal will be accomplished via four interrelated projects and two supporting cores. ..
  7. Cell Adhesion Mechanisms in Vascular Disease &Thrombosis
    MARK HOWARD GINSBERG; Fiscal Year: 2013
    ..abstract_text> ..
  8. Role of epididymal dendritic cells in male reproductive function
    Nicolas Da Silva; Fiscal Year: 2013
    ....
  9. Rocky Mountain Regional Center of Excellence or Biodefense and Emerging Infectiou
    John T Belisle; Fiscal Year: 2013
    ..abstract_text> ..
  10. Protein Amyloidogenesis in the Epididymis: Mechanisms and Biological Significance
    Gail Cornwall; Fiscal Year: 2013
    ..abstract_text> ..
  11. MRNA PROCESSING IN REPRODUCTION AND FERTILIZATION
    Clinton C MacDonald; Fiscal Year: 2013
    ..Using a variety of techniques, we want to learn the molecular causes of why sperm production fails in males missing the CSTF2T gene, to better understand how to assist these infertile couples. ..
  12. Molecular Mechanisms linking Aging, Abeta Proteotoxicity and Neurodegeneration
    Jeffery W Kelly; Fiscal Year: 2013
    ..abstract_text> ..
  13. Molecular Mechanisms that Control Ca2+ Signaling in Human Spermatozoa
    Yuriy Kirichok; Fiscal Year: 2013
    ..The results will help in the development of new treatments for male infertility as well as new methods of male contraception. ..
  14. Expanding Excellence in Developmental Biology in Oklahoma
    Linda F Thompson; Fiscal Year: 2013
    ..abstract_text> ..
  15. "Physical and Chemical Cues that Guide Sperm Migration"
    Mingming Wu; Fiscal Year: 2013
    ..Binding is reduced by capacitation, so we propose to test its effects on BSPs and on release of sperm from oviductal epithelium, as well as the role that the interactions of BSPs with ANXAs could play in guiding sperm to the egg. ..
  16. The MRAD9 Radioresistance Gene
    Howard B Lieberman; Fiscal Year: 2013
    ..abstract_text> ..
  17. Intercellular networks and luminal acidification in the male reproductive tract
    Sylvie Breton; Fiscal Year: 2013
    ..Data generated here will provide novel insights into the male reproductive tract in particular, as well as the physiology of acid/base transporting epithelia in general. ..
  18. Chemistry and Biology of Coagulation
    Sriram Krishnaswamy; Fiscal Year: 2013
    ..This program seeks to develop new information by which key reactions of blood clotting are regulated. This information will lead to new concepts and strategies for the treatment of blood clotting-related human disease. ..
  19. Soluble adenylyl cyclase isoforms essential for male fertility
    Lonny R Levin; Fiscal Year: 2013
    ..The studies proposed here may reveal unique aspects to the regulation of sperm cAMP signal transduction which could define a new target for contraceptive intervention. ..
  20. Genetic Basis of Oligozoospermia in Infertile Males
    Alexander N Yatsenko; Fiscal Year: 2013
    ..The long-term career goals of the applicant is to become an independent physician-scientist focused on studying genetic disorders that affect male reproductive system. ..