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.
1D. INVOLVEMENT OF SGG, SGC AND GC ON THE VAGINAL/ CERVICAL EPITHELIAL CELL SURFACE IN HIV-1 TRANSMISSION
SGG, and its analog, sulfogalactosylceramide (SGC) and SGC's desulfated form, GC, have all been shown for their affinity for gp120 and have been implicated in HIV-1 interaction. Our recent results indicate the existence of SGG, SGC and GC in human vaginal and ecto-and endocervical epithelial cells. As expected, these glycolipids exist in isolated lipid raft membranes, which are also known as host cell surface microdomains that first interact with HIV-1 in the infection/transmission process. These results strongly suggest that SGG, SGC and GC may act as the portals for initial binding of HIV-1 to these epithelia, which do not have CD4. The current concept is that HIV-1 is transcytosed through these epithelial cells to infect CD4+ T cells underneath the epithelial cell layer, a phenomenon already demonstrated in the intestinal mucosa. Our ongoing work will demonstrate whether SGG, SGC and GC on the epithelial cell surface are involved in HIV-1 transmission through the vaginal and cervical mucosa. Finally, we will investigate whether aqueous soluble glycolipid analogs of SGG, SGC and GC have competitively inhibitory effects on HIV-1 transmission through the vaginal/cervical epithelia. We expect that our results will be beneficial for future translation work in curbing HIV-1 transmission rates.
2. ROLES OF PROPROTEIN CONVERTASE IN MALE REPRODUCTION
Proprotein convertase 4 (PCSK4) is selectively expressed in male germ cells, and male mice genetically null of Pcsk4 are infertile despite the production of caudal epididymal sperm. We have shown that sperm from Pcsk4 knockout mice have a precocious capacitation rate leading to the much higher sensitivity of ZP-induced acrosome reaction. In other words, Pcsk4 null sperm would have undergone the acrosome reaction right after the initial adhesion to the ZP, thus losing the ability to remain bound to the ZP, and this would lead to minimal in vitro fertilization rates and fertility in vivo. However, whether proprotein convertase enzymatic activity is significant in fertilization cannot be concluded from the Pcsk4 knockout mouse study. We have recently demonstrated that PC activity is indeed present on live mouse sperm and it is localized mainly to the sperm plasma membrane. Inhibition of this proprotein convertase activity using a specific peptide inhibitor of PCSK4 results in a concentration dependent inhibition of mouse in vitro fertilization. To further understand the mechanisms of how sperm PCSK4 contributes to the fertilization process, we have searched for its natural substrates that are pertinent to this process, and have found that ADAM2 is one of PCSK4 substrates We hope to develop proprotein convertase inhibitors as vaginal contraceptives.
3. REPRODUCTIVE TOXICOLOGY: ADVERSE EFFECTS OF TRICHLOROETHYLENE (TCE) IN MALE REPRODUCTION
Increasing evidence indicates that various environmental toxins are the cause of human infertility. We have described higher levels of TCE in seminal plasma of mechanics, who attended the Ottawa Fertility Centre for infertility treatment. Our experiments in mice further confirm that male mice with prolonged inhalation of TCE produced sperm with minimal fertilizing ability. These results were communicated to the public through the Toxic Substance Research Initiative Program. We have ongoing interests in reproductive toxicology research and will be interested to collaborate with toxicologists to discern molecular mechanisms of adverse effects of other environmental toxins on male reproduction.
