Nongnuj Tanphaichitr, PhD
ntanphaichitr@ohri.ca
Senior Scientist
,
Chronic Disease
, Ottawa Hospital Research Institute
Professor, Departments of Obstetrics & Gynecology and Biochemistry, Microbiology & Immunology, University of Ottawa RESEARCH INTERESTS
Fertilization, sperm-egg interaction, sperm capacitation, sperm membranes, epididymis, vaginal and cervical epithelial cells, sulfoglycolipids, lipidomics, lipid rafts, proteomics, glycolipid-protein interaction, antimicrobial peptides, HIV-1, proprotein convertase, contraceptive development, biomarkers for sperm functions, reproductive toxicology, reproductive aging
RESEARCH PROJECTS (for more details on research click here)
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 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 (ASA) ON SPERMATOGENESIS AND FERTILIZATION: RELATIONSHIP BETWEEN ASA AND SGG
We have recently confirmed the significance of ASA in male reproduction in vivo in ASA knockout mice. Although ASA knockout male mice can sire offspring, our mating study strongly suggests that ASA knockout males are subfertile at an older age. In these mating experiments, wild type females were mated with ASA null males or wild type males. During the age of 2 to 5 months, ASA knockout males produce offspring with the same litter size and number of accumulated pups/mating pair as the wild type males. However, when ASA knockout 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 ASA 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 ASA knockout animals (8 months of age in mice ~40 years in humans). Since SGG has been demonstrated as a substrate of ASA 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 ASA knockout mouse testis and sperm, and this may be the cause of subfertility in older ASA knockout mice. Significantly, our findings suggest that ASA present in male germ cells and Sertoli cells can regulate SGG levels and both ASA 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 ASA) 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-STD 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 (PC4) is selectively expressed in male germ cells, and male mice genetically null of Pc4 are infertile despite the production of caudal epididymal sperm. We have shown that sperm from Pc4 knockout mice have a precocious capacitation rate leading to the much higher sensitivity of ZP-induced acrosome reaction. In other words, Pc4 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 PC enzymatic activity is significant in fertilization cannot be concluded from the Pc4 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 PC activity using a specific peptide inhibitor of PC results in a concentration dependent inhibition of mouse in vitro fertilization. To further understand the mechanisms of how sperm PC contributes to the fertilization process, attempts are being made to search for its substrates that are pertinent to this process. We hope to develop PC inhibitors as vaginal contraceptives, and thus we are investigating whether PC inhibitors, when administered into the female reproductive tract, can effectively inhibit fertilization.
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.
PUBLICATIONS (past 10 years)
1. Quantification of seminolpid by LC-ESI-MS/MS-multiple reaction monitoring: compensatory levels in Cgt+/- mice. Kongmanas K, Xu H, Yaghoubian A, Franchini L, Panza L, Ronchetti F, Faull KF, Tanphaichitr N. J Lipid Res. 2010 Sep 3. [Epub ahead of print]
2. Phillips K and Tanphaichitr N. Mechanisms of obesity-induced male infertility. Expert Rev Endocrin Metabol, 2010; 5: 229-251.
3. Schenk M, Costa Santos D, Koppisetty C, Carmona E, Bhatia S, Brisson J-R, Nyholm P-G*, Tanphaichitr N*. Interaction of arylsulfatase-A (ASA) with its natural sulfoglycolipid substrates: a computational and site-directed mutagenesis study. Glycoconjugate J 2009 Nov;26(8):1029-45.*shared senior authorship
4. Nixon B, Harman A, McLaughlin EA, Tanphaichitr N, Aitken RJ. Composition and significance of lipid rafts in mouse spermatozoa. J Cell Physiol 2009; 218:122-134.
5. Orimoto AM, Dumaresq-Dorion K, Jiang J-J, Tanphaichitr N, Tsang BK, Carmona E. Mammalian hyaluronidase induces ovarian granulose cell apoptosis and is involved in follicular atresia. Endocrinology 2008; 149:5835-5847.
6. Anupriwan A, Schenk M, Kongmanas K, Vanichviriyakit V, Costa Santos D, Wu A, Yaghoubian A, Berger T, Nyholm P-G, Faull K, Sretarugsa P, Tanphaichitr N. Presence of arylsulfatase A and sulfogalactosylglycerolipid in ovaries: Localization to the corpus luteum. Endocrinology 2008; 149:3942-3951.
7. Kruevaisayawan H, Vanichviriyakit R, Weerachatyanukul W, Taweepreda P, Basak A, Withyachumnarnkul B, Tanphaichitr N*, Sobhon P*. Sperm acrosome reaction in Penaeus monodon: Roles of the binding components in egg water and sperm acrosomal trypsin. Biol. Reprod. 2008;79:134-141.
*shared senior authorship
8. Franchini L, Panza L, Kongmanas K, Tanphaichitr N, Faull K, Ronchetti F. An efficient and convenient synthesis of deuterium-labelled seminolipid isotopomers and their ESI-MS characterization. Chem Phys Lipids. 2008; 152:78-85.
9. Phillips K, Tanphaichitr N. Human exposure to endocrine disrupters and semen quality. J. Toxicol. Environ. Health, Part B, 2008; 11:188-220
10. Wu A, Anupriwan A, Iamsaard S, Chakrabandhu K, Costa Santos D, Rupar T, Tsang BK, Carmona E, Tanphaichitr N. Sperm surface arylsulfatase A can disperse the cumulus matrix of cumulus oocyte complexes. J. Cell. Physiol. 2007; 213(1): 203-211.
11. Tanphaichitr N, Faull KF, Yaghoubian A, and Xu H. Roles of sperm lipid rafts and
sulfogalactosyglycerolipid (SGG) in sperm functions: Consensus and controversy. Trends Glycosci. Glycotech. 2007; 19:67-83.
12. Tanphaichitr N, Carmona E, Bou Khalil M, Xu H, Berger T, Gerton GL. New insights into sperm-zona pellucida interaction:involvement of sperm lipid rafts. Front. Biosci. 2007; 12:1748-1766.
13. 10. Weerachatyanukul W, Probodh I, Kongmanas K, Tanphaichitr N*, Johnston LJ*. Visualizing the localization of sulfoglycolipids in lipid raft domains in model membranes and sperm membrane extracts. Biochim. Biophys. Acta, Biomembranes, 2007; 1768: 299-310.
*shared senior authorship
14. Bou Khalil M, Chakrabandhu K, Weerachatyanukul W, Xu H, Vuong N, Buhr M, Berger T, Kumarathasan P, Carmona E, Wong PTT, Carrier D and Tanphaichitr N. Pig sperm rafts have zona pellucida binding ability and contain the mammalian male germ cell specific sulfogalactosylglycerolipid. Dev. Biol. 2006; 290:220-235.
15. Gyamera-Acheampong C, Tantibhedhyangkul J, Tadros H, Sirois F, Weerachatyanukul W, van de Loo J, Pelletier R-M, Tanphaichiatr N*, MbiKay M*. Proprotein convertase 4 (PC4) is located on the acrosomal plasma membrane and its deficiency renders spermatozoa less efficient at egg-binding. Biol. Reprod. 2006; 74: 666 - 673.
*shared senior authorship
16. Furimsky A, Vuong N, Xu H, Kumarathasan P, Weerachatyanukul W, Bou Khalil M, Kates M, and Tanphaichitr N. Percoll-gradient centrifuged capacitated mouse sperm have increased fertilizing ability and higher contents of sulfogalactosylglycerolipid and docosahexaenoic acid- containing phosphatidylcholine than washed capacitated mouse sperm. Biol. Reprod. 2005; 72:574-583.
17. Xu H, Tanphaichitr N, Forkert G, Anupriwan A, Weerachatyanukul W, Vincent R, Leader A and Wade MG. Exposure to trichloroethylene and its metabolites causes impairment of sperm fertilizing ability in mice. Toxicol. Sci. 2004 Dec; 82(2):590-7.
18. Forkert PG, Lash L, Tardif R, Tanphaichitr N, Vandevoort C, and Moussa M. Identification of trichloroethylene and its metabolites in human seminal fluid of workers exposed to trichloroethylene. Drug Metab Dispos. 2003; 31:306-311.
19. Weerachatyanukul W, Xu H, Anupriwan A, Wade M, Carmona E, Hermo L., Rippstein P, Sobhon P and Tanphaichitr N. Acquisition of arylsulfatase A (AS-A) onto the mouse sperm surface during epididymal transit. Biol. Reprod. 2003, 69:1183-92.
20. Tanphaichitr N, Haebe J, Leader A, Carmona E, Harris JD, Silva SM, Antunes TT, Chakrabandhu K, Léveillé MC. Towards a more precise assay of sperm function in egg binding which ART is best suited for treating male infertility. J. Obst. Gynec. Canada 2003; 25:461-470
21. Carmona E, Weerachatyanukul W, Xu H, Fluharty AL, Anupriwan A, Shoushtarian A, Chakrabandhu K and Tanphaichitr N. Binding of arylsulfatase A to mouse sperm inhibits gamete interaction and induces the arosome reaction. Biol. Reprod. 2002; 66: 1820-1827.
22. Tantibhedhyangkul J, Weerachatyanukul W, Carmona E, Xu H, Anupriwan A, Michaud D, and Tanphaichitr N. Role of sperm surface arylsulfatase A in mouse sperm-zona pellucida binding. Biol. Reprod. 2002; 67:212-219.
23. Carmona E, Weerachatyanukul W, Soboloff T, Fluharty AL, Shite D, Promdee L, Ekker M, Berger T, Buhr M, and Tanphaichitr N. Arylsulfatase A is present on the pig sperm surface and is involved in sperm-zona pellucida binding. Devel. Biol. 2002; 247:182-196.
24. Mai A, Weerachatyanukul W, Tomietto M, Wayner DDM, Wells G, Balhorn R, Leader A, Cyr J-L and Tanphaichitr N. Use of atomic force microscopy for morphological and morphometric analyses of acrosome intact and acrosome reacted human sperm. Molec. Reprod. Dev. 2002; 63:471-479.
25. Bou Khalil M, Carrier D, Wong PTT and Tanphaichitr N. Polymorphic phases of galactocerebrosides: spectroscopic evidence of lamellar crystalline structures. Biochim. Biophys. Acta. 2001; 1512:158-170.
26. Rattanachaiyanont M, Leveille MC, Taylor T, D'Amours D, Weerachatyanukul W, Rivers D, Leader A, and Tanphaichitr N. Presence of sulfolipidimmobilizing protein in human sperm and its role in human zona binding. Molec. Human Reprod. 2001; 7:633-640.
27. Weerachatyanukul W, Rattanachaiyanont M, Carmona E, Furimsky A, Mai A, Shoushtarian A, Sirichotiyakul S, Ballakier H, Leader A and Tanphaichitr N. Sulfogalactosylglycerolipid is involved in human gamete interaction. Molec. Reprod. Dev. 2001; 60: 569-578.
28. White D, Weerachatyanukul W, Gadella B, Kamolvarin N, Suwajanakorn S, Attar M and Tanphaichitr N. Role of sperm sulfogalactosylglycerolipid (SGG) on murine sperm-zona pellucida binding. Biol. Reprod. 2000; 63:147-155.
29. Attar M, Kates M, Carrier D, Bou Khalil M and Tanphaichitr N. A Fourier-transform infrared spectroscopic study of the interaction between germ-cell specific sulfogalactosylglycerolipid and dimyristoylphosphatidylcholine. Chem. Phys. Lipids. 2000; 106:101-114.
30. Jarrell JF, Leader A, Leveille M-C, Daya S, Ward S, Pierson R, Yuzpe A, Tanphaichitr N, and Orr-Mongeau S. Accreditation of reproductive medicine in Canada. J. Soc. Obstet. Gynaecol. Can. 2000; 22:800-803.
31. Ahnonkitpanit V, White D, Suwajanakorn S, Kan FW, Namking M, Wells G and Tanphaichitr N. Role of egg sulfolipidimmobilizing protein 1 on sperm-egg plasma membrane binding. Biol. Reprod. 1999; 61:749-756.
Book Chapter
1. Tanphaichitr N, Bou Khalil M, Weerachatyanukul W, Kates M, Xu H, Carmona E, Attar M and Carrier D. Physiological and biophysical properties of male germ cell sulfogalactosylglycerolipid. In: Lipid Metabolism and Male Fertility.(Ed. SR DeVriese and AB Christophe) AOCS Press, 2003. pp. 125-148.
OTHER INFORMATION
1. I have been training students/postdoctoral fellows from various countries. These include graduate students and postdoctoral fellows at the University of Ottawa, as well as exchange graduate students, postdoctoral fellows and visiting scholars from various countries: Thailand (Mahidol University, Chulalongkorn University, Khon Kaen University), China (Shanghai Institute of Biological Science), Japan (Nagoya University), Sweden (Goteborg University), Italy (University of Milan) and Brazil (Institute of Butantan). The opportunity to have trainees from other countries is through our active international collaborations.
2. I have been participating as an active member of the Society for the Study of Reproduction. I was Chair of Minority Affairs Committee (MAC) from 2005-2008. Currently, I am an active member of MAC and Publication Committee, and also serve on the Board of Reviewing Editors of the SSR journal, Biology of Reproduction.
3. I set up the Human In Vitro Fertilization (IVF) Laboratory of the Ottawa Fertility Centre in 1987 and was the Lab Director and then Scientific Director of the Human IVF Program until 2006.
4. I was a faculty member in the Department of Obstetrics/Gynecology and Department of Anatomy and Cell Biology at Harvard Medical School from 1984-1987. I set up and ran the Andrology Lab in the Human IVF Program at Beth Israel during this time period.
