OVERALL DIRECTIONS 

For the past two decades, our research studies have geared towards the understanding of the molecular mechanisms of sperm-egg interaction. Our ultimate goal is to have our basic science results translated into development of non-hormonal contraceptives and biomarkers of gamete fertilizing ability. Since the delivery of these contraceptives are likely most effective in the vagina, we are targeting molecules that would also contain anti-sexually transmitted disease (i.e., microbicidal and virucidal) activities. Our second line of research has therefore been initiated in more recent years to study: 1. the relationship between sperm and antimicrobial peptides (readily existing in the reproductive tract as part of the innate immunity system), and 2. the transmission mechanisms of HIV-1 and other microbes through vaginal and cervical epithelial cells. Our work has been continuously supported by Canadian Institutes of Health Research (CIHR) and Natural Science and Engineering Research Council. We have also received funding from  Bill and Melinda Gates Foundation, The Rockefeller Foundation, CONRAD, the Lalor Foundation, Health Canada, the University of Ottawa, Thailand Research Fund, and National Science and Technology Development Agency of Thailand.

1. SGG AND SGC AND THEIR BINDING PROTEINS IN CELL ADHESION 

Cell-cell/extracellular matrix interactions are the major processes that initiate the binding between gametes and between microbes/viruses to the host cell epithelia. Since glycolipids are generally known as adhesion molecules, we have been focusing our research study on SGG (sulfogalactosylglycerolipid) and its analog, SGC (sulfogalactosylceramide). In particular, we address the question of how surface sulfoglycolipids on sperm and vaginal/cervical epithelial cells contribute to these binding events.

1A. ROLES OF SGG IN MALE GERM CELLS IN REPRODUCTION

 

Sulfogalactosylglycerolipid (SGG, aka seminolipid, see Fig. 1 for the structure of its main molecular species with both hydrocarbon chains being C16:0) is a sulfoglycolipid that is present selectively in mammalian male germ cells; it is also the major glycolipid in these cells, constituting about 10 mole% of total lipids. Therefore, SGG can act as both an adhesion molecule and a structural lipid in mammalian male germ cells. In fact, we have shown that SGG is involved in sperm-zona pellucida interaction, and it is the structural lipid that significantly participates in the formation of sperm lipid rafts, which have ZP binding ability. We have further shown that SGG has a binding protein on the sperm surface, arylsulfatase A, which acts synergistically with SGG in sperm-ZP binding. We are currently profiling all SGG binding proteins that exist in sperm lipid rafts, using a proteomic approach. The identities of these proteins will provide us with a better understanding of the molecular mechanisms of sperm-ZP interaction, and in particular how SGG contributes to this interaction.Previous studies also demonstrate the significance of SGG in spermatogenesis. Male mice genetically null of two enzymes (Cgt and Cst) contain no SGG in their testes and spermatogenesis in these knockout mice are arrested at the primary spermatocyte stage. Currently, we are trying to discern the mechanisms through which SGG on the cell surface participates in the development of testicular germ cells. SGG binding proteins in both Sertoli cells and testicular male germ cells are being profiled using a proteomic approach.

1B. ROLES OF ARYLSULFATASE A (ARSA) ON SPERMATOGENESIS AND FERTILIZATION: RELATIONSHIP BETWEEN ASA AND SGG

 

We have recently confirmed the significance of ARSA in male reproduction in vivo in Arsa knockout mice. Although these knockout male mice can sire offspring, our mating study strongly suggests that Arsa knockout males are subfertile at an older age. In these mating experiments, wild type females were mated with Arsa null males or wild type males. During the age of 2 to 5 months, Arsa knockout males produce offspring with the same litter size and number of accumulated pups/mating pair as the wild type males. However, when Arsaknockout males become older than 5 months, the accumulated number of pups per mating pair is significantly lower than that from the wild type. The in vitro fertilizing ability of sperm from 8-month old Arsa knockout males was also nearly zero, whereas sperm from 5-month old knockout males fertilized eggs at rates similar to wild type sperm. Spermatogenesis also proceeds at a reduced rate in 8-month old Arsaknockout animals (8 months of age in mice ~40 years in humans). Since SGG has been demonstrated as a substrate of ARSA in vitro, we are characterizing the levels of SGG in male germ cells and Sertoli cells. Our recent results indicate aberrant SGG levels and appearance of new glycolipids in Arsa knockout mouse testis and sperm, and this may be the cause of subfertility in older Arsa knockout mice. Significantly, our findings suggest that ARSA present in male germ cells and Sertoli cells can regulate SGG levels and both ARSA and SGG play important roles in reproductive aging.

1C. ANTIMICROBIAL PEPTIDES (AMPs), LL-37 IN THE CATHELICIDIN FAMILY AND BIN1B AND HE2Beta1 IN THE Beta-DEFENSIN FAMILY, ARE ALSO SGG BINDING PROTEINS: THEIR POTENTIAL USE AS CONTRACEPTIVES WITH ANTI-STD ACTIVITIES

AMPs are effector molecules of the innate immunity system. They are cationic peptides usually of <100 amino acids, with a broad spectrum of antimicrobial/antiviral effects. A number of AMPs, including hCAP-18 (LL-37 precursor), Bin1b and HE2beta1 are expressed by the epididymal epithelial cells. Our microplate assays indicate affinity of LL-37, Bin1b, and HE2Beta1 for SGG, likely due to the electrostatic interaction between these cationic peptides and negatively charged SGG. This would explain the co-existence of hCAP18, Bin1b and HE2Beta1 with SGG on the sperm anterior head, and it is possible that these AMPs may act together SGG (in a similar manner to that observed with ARSA) in egg binding. However, a significant amount of hCAP18, Bin1b and HE2Beta1 is still present in the epididymal fluid/seminal plasma. hCAP18 is proteolytically processed into LL-37 several hours after intercourse. Mouse and human sperm, both non-capacitated and capacitated, can be deposited by exogenously added LL-37. This deposition leads to a drastic decrease in sperm motility. As expected, the in vitro fertilization rate of mouse sperm treated with LL-37 at the molar concentration equivalent to that of SGG is zero. Similar but less potent results were seen with purified recombinant Bin1b and HE2Beta1, exogenously added to mouse and human sperm suspensions, respectively. Our results suggest that cationic antimicrobial peptides, in particular LL-37, can potentially be developed into vaginal contraceptives with anti-sexually-transmitted-disease activity. 

2. DEVELOPMENT OF CATHELICIDIN ANTIMICROBIAL PEPTIDE (AMP) INTO A MULTIPURPOSE PREVENTION TECHNOLOGY AGENT

This research is the main direction in our lab now.  Based on our discovery described above that LL-37, the human cathelicidin AMP can bind to sperm SGG, we have further shown that LL-37 and its truncated peptides (GI-20 and GF-17) and its bioengineered peptide, 17BIPHE2 (resistant to protease degradation) have both spermicidal and anti-STI activities.  Their spermicidal activity is on both human and mouse sperm and we have shown in mice that all of these peptides, when administered into the female reproductive tract (FRT), can act as contraceptives and also anti-STI agents.  In particular, they have microbicidal activity against Neisseria gonorrhoeae, which is becoming resistant against conventional antibiotics.  We are currently developing formulation of 17BIPHE2 in a hydrogel form to be administered into the FRT.  We expect that formulated 17BIPHE2 will protect women on both unintended pregnancy and sexually transmitted infections.