Periodic Reporting for period 4 - Sperm-Egg Phusion (Unexpected connections between a phagocytic machinery and mammalian fertilization)
Reporting period: 2024-04-01 to 2024-09-30
Fertilization is essential for a species to survive. Mammalian sexual reproduction requires the fusion between the haploid gametes sperm and egg to create a new diploid organism. Although fertilization has been studied for decades, and despite the remarkable recent discoveries of the critical ligand-receptor pair Izumo (on sperm) and Juno (on oocytes), it is clear that other players on both the sperm and the oocytes must be involved. While the focus of our laboratory over the years has been in understanding apoptotic cell clearance by phagocytes, we accidentally discovered that viable, motile, and fertilization-competent sperm exposes the phospholipid phosphatidylserine (PtdSer). PtdSer is normally exposed during apoptosis and functions as an ‘eat-me’ signal for phagocytosis. In further experiments, we found that masking this PtdSer on sperm inhibits its ability to fertilize oocytes in vitro. In this ERC proposal, we have been testing the hypothesis that PtdSer on viable sperm and the complementary PtdSer receptors on oocytes are key players in mammalian fertilization. We use a combination of molecular, biochemical, cellular, functional, and genetic approaches. We addressing: (1) the mechanisms regulate the exposure of PtdSer on viable sperm; (2) the genetic relevance of different PtdSer receptors on oocytes in fertilization; and (3) understand what novel signals are induced by PtdSer within oocytes. By combining the tools and knowledge from the field of phagocytosis with tools from spermatogenesis/fertilization, this proposal integrates fields that normally do not intersect. From the perspective of the societal impact, currently 1 in 4 couples seek some form of therapy for having children and there is a remarkable drop in overall birthrate of which only some factors are understood. We expect that our approaches will open up new research routes of high relevance to both male and female reproductive health and fertility.
This project has progressed well in several ways :
1. The preliminary data that were presented as part of the original ERCAdG application in 2018 were solidified and this was published as a paper (Rival CM, Xu W, Shankman LS, Morioka S, Arandjelovic S, Lee CS, Wheeler KM, Smith RP, Haney LB, Isakson BE, Purcell S, Lysiak JJ, KS Ravichandran. 2019. Phosphatidylserine on viable sperm and phagocytic machinery in oocytes regulate mammalian fertilization. Nature Communications. 10:4456. doi: 10.1038/s41467-019-12406-z). Importantly, this work has now established that phosphatidylserine exposed on viable and motile sperm is a key component of sperm:egg fusion, and the existence of specific phosphatidylserine receptors on oocytes that mediate the interaction between sperm and egg during mammalian fertilization. Further, as proposed in the original application, we have now identified multiple receptors on oocytes that engage phosphatidylserine on sperm.
2. Using new approaches, we have now identified multiple proteins that are in the neighbourhood of phosphatidylserine. This is a true breakthrough for this whole field, and we are now addressing the function of the specific molecules that appear to be in close proximity to phosphatidylserine on sperm and how these may regulate sperm:egg fusion. Further, as a comparison, we have ‘forced’ other live cells (such as lymphocytes) to expose phosphatidylserine and we now discover both overlapping and unique players, and this is being further pursued through a combination of CRISPR/Cas-mediated genetic approaches, biochemistry, and functional studies in cell lines and mice.
3. We have designed several new technical approaches to detect PS proximal proteins on sperm as well as other cell types that will be broadly inter-disciplinary. Further, we have generated new mice that can conditionally delete both the major enzymes that synthesize PtdSer in cells including sperm. Further, we have designed new mice to specifically address how the PtdSer on sperm induces an anti-inflammatory tone in the female reproductive tract.
4. These have led to multiple high-impact publications in journals such as Nature and we are currently preparing one more manuscript for publication in Nature. Further, multiple personnel associated with this project have progressed positively in their careers (including three who acquired faculty positions as independent investigators).
1. The preliminary data that were presented as part of the original ERCAdG application in 2018 were solidified and this was published as a paper (Rival CM, Xu W, Shankman LS, Morioka S, Arandjelovic S, Lee CS, Wheeler KM, Smith RP, Haney LB, Isakson BE, Purcell S, Lysiak JJ, KS Ravichandran. 2019. Phosphatidylserine on viable sperm and phagocytic machinery in oocytes regulate mammalian fertilization. Nature Communications. 10:4456. doi: 10.1038/s41467-019-12406-z). Importantly, this work has now established that phosphatidylserine exposed on viable and motile sperm is a key component of sperm:egg fusion, and the existence of specific phosphatidylserine receptors on oocytes that mediate the interaction between sperm and egg during mammalian fertilization. Further, as proposed in the original application, we have now identified multiple receptors on oocytes that engage phosphatidylserine on sperm.
2. Using new approaches, we have now identified multiple proteins that are in the neighbourhood of phosphatidylserine. This is a true breakthrough for this whole field, and we are now addressing the function of the specific molecules that appear to be in close proximity to phosphatidylserine on sperm and how these may regulate sperm:egg fusion. Further, as a comparison, we have ‘forced’ other live cells (such as lymphocytes) to expose phosphatidylserine and we now discover both overlapping and unique players, and this is being further pursued through a combination of CRISPR/Cas-mediated genetic approaches, biochemistry, and functional studies in cell lines and mice.
3. We have designed several new technical approaches to detect PS proximal proteins on sperm as well as other cell types that will be broadly inter-disciplinary. Further, we have generated new mice that can conditionally delete both the major enzymes that synthesize PtdSer in cells including sperm. Further, we have designed new mice to specifically address how the PtdSer on sperm induces an anti-inflammatory tone in the female reproductive tract.
4. These have led to multiple high-impact publications in journals such as Nature and we are currently preparing one more manuscript for publication in Nature. Further, multiple personnel associated with this project have progressed positively in their careers (including three who acquired faculty positions as independent investigators).
The progress beyond the state of the art include:
1. Exploration of new molecules on the sperm surface that contribute to sperm:egg fusion, especially in the context of phosphatidylserine that is important to the fusion process per se.
2. Discovery/exploration of new sperm-associated molecules involved in fertilization and the possible design of a new test for PtdSer on sperm for further testing of male fertility
3. We have recently begun addressing while one sperm is successful in fertilizing the oocytes, what happens to the millions of other sperm that 'do not make it'. In particular, we are addressing whether the phosphatidylserine on the other non-fertilizing sperm might contribute to the 'tolerance' of the 'foreign' sperm in a female host. We are using a combination of sophisticated genetic and biochemical approaches to address this question.
4. We are also generating nano bodies to specific sperm proteins - this could be used in human diagnostics as roughly 1 in 4 couples go through some form of fertility treatments. On the opposite end, some of these nano bodies may also prove useful in contraception.
1. Exploration of new molecules on the sperm surface that contribute to sperm:egg fusion, especially in the context of phosphatidylserine that is important to the fusion process per se.
2. Discovery/exploration of new sperm-associated molecules involved in fertilization and the possible design of a new test for PtdSer on sperm for further testing of male fertility
3. We have recently begun addressing while one sperm is successful in fertilizing the oocytes, what happens to the millions of other sperm that 'do not make it'. In particular, we are addressing whether the phosphatidylserine on the other non-fertilizing sperm might contribute to the 'tolerance' of the 'foreign' sperm in a female host. We are using a combination of sophisticated genetic and biochemical approaches to address this question.
4. We are also generating nano bodies to specific sperm proteins - this could be used in human diagnostics as roughly 1 in 4 couples go through some form of fertility treatments. On the opposite end, some of these nano bodies may also prove useful in contraception.