Research in our lab

Molecular mechanisms of mammalian fertilization

A series of carefully orchestrated molecular events ensures monospermic fertilization, which is essential for successful embryonic development and a healthy pregnancy outcome. First, a taxon-specific gamete recognition at the zona pellucida, the envelope surrounding mammalian oocytes, mediates sperm binding and penetration through the zona. After penetration, the sperm reach the perivitelline space, a space enclosed between the inner aspect of the zona and the egg plasma membrane. Here, a second taxon-specific gamete recognition process occurs prior to gamete fusion. Following fertilization, molecular changes in the zona pellucida and in the egg plasma membrane establish effective blocks to polyspermy, preventing polyploidy, which is embryonic lethal in mammals.

Sperm binding to the zona pellucida

 

Sperm binding to the zona pellucida (ZP) is a necessary step for successful fertilization and which ZP protein mediates sperm binding has been a compelling and debated biological question over the past few decades. Using mouse genetics we demonstrated that a single domain on ZP2 is necessary and sufficient to mediate sperm binding. In particular using galK recombineering, we established a number of transgenic mouse lines that express chimeric (mouse/human) or mutant ZP2 which lack the ZP2 N terminal region.  We used mouse or human sperm (unable to fuse with mouse eggs) to interrogate transgenic mouse eggs: mouse sperm bind to zonae pellucidae expressing mouse ZP2, but do not bind to zonae lacking ZP2 or expressing truncated mouse ZP2 lacking the N terminal region (top panels). Human sperm bind to transgenic mouse zonae expressing human ZP2

or chimeric mouse ZP2 expressing the human ZP2 N terminal region (bottom panels), but do not bind to eggs expressing a chimeric human ZP2 in which the N terminus of ZP2 has been replaced with the mouse N terminus (bottom panels). 

Competitive sperm binding to mouse eggs

 

We compare  sperm binding avidity of Acr/EGFP sperm freshly released from the epididymis (green, left) with Acr/mCherry sperm after 1 hour of incubation in culture media, alone (left and middle panels, respectively) or mixed (1:1) with the freshly released sperm (right panel). Confocal Z projections images. Sperm nuclei are stained with Hoechst.

Sperm transit in the female genital tract

 

To track the sperm transit in the female genital tract, we use transgenic mouse lines with sperm that accumulate EGFP in the nucleus and cytoplasm as well as mCherry in the acrosome (Top panel). Vottom panels show ejaculated sperm as they migrate from the uterus into the oviduct and within the ampulla of the oviduct where they approach the eggs prior to fertilization.

Uterus-Oviduct

Oviduct-Ampulla

References

Avella, M. A., Baibakov, B. A., Jimenez-Movilla, M., Sadusky, A. B. & Dean, J. ZP2 peptide beads select human sperm in vitro, decoy mouse sperm in vivo, and provide reversible contraception. Science Translational Medicine 8, 336ra60 (2016).

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Avella, M. A., Baibakov, B. & Dean, J. A single domain of the ZP2 zona pellucida protein mediates gamete recognition in mice and humans. Journal of Cell Biology 205, 801–809 (2014).