Our research aims at understanding the roles of seminolipid (sulfogalactosylglycerolipid or SGG) and its associated membrane domains in male reproduction. SGG is a sulfoglycolipid present selectively and abundantly in mammalian male germ cells. Therefore, information on its properties would be relevant towards the development of male fertility biomarkers and spermicide-based contraceptives. We have shown that SGG has direct affinity for zona pellucida (ZP, egg extracellular matrix) and plays a role in the formation of sperm lipid rafts, the ZP-binding platforms on the sperm anterior head plasma membrane (APM), the initial ZP binding site. For a better understanding of mechanisms underlying sperm-ZP interaction, I performed proteomic characterization of APM vesicles (SGG-associated membrane domains with ZP affinity) isolated from sperm before and after capacitation, a process through which sperm gain maximal ZP affinity. Proteomic results revealed that capacitated APM vesicles contained high-molecular-weight protein complexes, with higher ZP affinity and levels of ZP-binding proteins as compared with those of the non-capacitated samples. ZP-binding proteins known to exist in the acrosome (i.e., zonadhesin, proacrosin/acrosin) were found in these APM protein complexes. Immunofluorescence suggested that a fraction of these proteins trafficked from the acrosome to APM during capacitation. These findings provided a new mechanism on how sperm gain full ZP-binding ability during capacitation. Since SGG is a major component of APM, proper SGG levels at this site would be important for male fertility. Levels of sperm SGG are regulated through the synthesis and degradation. In fact, lack of SGG-synthesis enzymes causes a spermatogenesis disruption, resulting in male infertility. However, significance of SGG degradation remains unknown. SGG can be desulfated in vitro by arylsulfatase A (ARSA), an enzyme existing in the acrosomes of sperm/spermatids and lysosomes of Sertoli cells, testicular somatic cells that nurture developing germ cells. Sertoli cells also phagocytose ~50% of germ cells that become apoptotic during spermatogenesis. To understand physiological importance of SGG degradation, the fertility status and SGG levels of Arsa-/- male mice were determined. We found that Arsa-/- males became subfertile when they were older than 5 months, and when they were 8-month-old (~40-year-old men) they produced sperm at 50% wild type rate. Arsa-/- sperm had minimal in vitro fertilizing ability and a number of them showed abnormal morphology. Quantitative mass spectrometry revealed that SGG levels in Sertoli cells of 8-month-old Arsa-/- mice were increased to ~250% of the wild type level; this SGG accumulation may lead to a decrease in Sertoli cell ability to support spermatogenesis. However, SGG levels in sperm of 8-month-old Arsa-/- mice were ~50% of the wild type value, a result that partly explained the decreased fertilizing ability of these sperm. The reduced SGG level of Arsa-/- sperm was likely due to a lack of SGG’s building-block lipid (palmitylpalmitoylglycerol) putatively generated in Arsa-/- Sertoli cells and recycled to the next generation of primary spermatocytes for SGG synthesis. Hence, levels of sperm SGG are a promising bioindex for male fertility. Since Sertoli cells also regulate SGG homeostasis, their functionality should be now included in male fertility/subfertility diagnosis.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/32509 |
| Date | January 2015 |
| Creators | Kongmanas, Kessiri |
| Contributors | Tanphaichitr, Nongnuj |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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