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Membrane dynamics during fertilization

This research line is embedded in the Research programs Biology of Reproductive Cells of the Faculty of Veterinary Medicine and in Growth & Differentiation of Utrecht University

Central topics in this research line are the dynamics in adhesive and fusion properties, molecular composition and architecture of the two gamete’s membranes leading to fertilization and embryo development.

 

Sperm membrane dynamics

The sperm cell has a characteristic polarized morphology and its surface is also highly differentiated into different membrane domains. Furthermore, lipid and membrane protein ordering is subjected to changes when sperm become capacitated. The forces that maintain the lateral polarity of membrane molecules over the sperm surface, as well as those that cause their dynamic redistribution, are only poorly understood.

Recent work allowed us to demonstrate specific SNARE-mediated membrane fusion events during the capacitation and acrosome reaction (see figure) (Tsai et al (2012) PloS One; Tsai et al (2010) PloS One).

Figure: Upon capacitation, SNARE proteins relocalized to the apical sperm head (marked with asterisk) and appeared as punctate aggregates indicating their association with other proteins (Tsai et al (2012) PloS One)

To study plasma membrane dynamics of sperm cells, we developed a procedure to isolate the apical sperm plasma membrane and solubilize the proteins from this membrane fraction (Flesh et al (1998) Biol Reprod). This method has been instrumental to show lipid-mediated signalling pathways during sperm activation (Gadella & Harrison (2000) Development; Flesh et al (2001) J. Cell Sci). Cholesterol extraction is an important step towards sperm cell activation. We now extent these studies by investigating the effect of cholesterol depletion on lipid raft dynamics and radical signalling in capacitating sperm. Preliminary evidence suggests that reactive oxygen species (ROS) effects are required for in vitro fertilization.

Figure: The bicarbonate/oxysterol pathway induces protein tyrosine phosphorylation and aggregation of lipid ordered domains.

 

Oocyte membrane dynamics

Membrane dynamics in the oocytes play an important role in oocyte maturation, fertilization, and oocyte quality. Secretory vesicles that are randomly dispersed throughout the cytoplasm of immature oocytes and migrate toward the cortical cytoplasm during maturation. Recently we could show that this surface-orientated redistribution behavior was also observed for the oocyte-specific SNARE proteins SNAP23 and VAMP1 that localized in the cortex area just under the oolemma (figure).

Figure: SNARE proteins migrate to the oocyte surface upon oocyte maturation. PNA-FITC was used to label secretory granules (green). DNA of the maturating oocytes was labeled with ToPro-3 (blue). Oocytes of two maturation stages (0 h: GV, and 44 h IVM: M-II) were used. An antibody against Vamp1 (V1, red) was used to indicate the localization at different maturation stages.

Recently a new research line was initiated in which metabolism, storage and mobilisation of lipids is investigated in relation to the quality of the oocyte. After fatty acids are taken up by somatic cells, they are esterified into triacylglycerols (TAG) and cholesterol-esters
and stored as neutral lipids in lipid droplets. In the oocyte it has been suggested that neutral lipids fulfil an important function in supplying energy and in biosynthesis of membranes during early embryonic development. The esterification of fatty acids and storage into lipid droplets may also protect the oocyte against fatty acid-induced lipotoxicity. In line with this, accumulation of neutral storage lipids in oocytes has been related to improved developmental competence.
Recently we could show that palmitic and stearic acid had a dose-dependent inhibitory effect on the amount of fat stored in lipid droplets and a concomitant detrimental effect on oocyte developmental competence (Aardema et al (2011) Biol. Reprod). Oleic acid, in contrast, had the opposite effect, causing an increase of lipid storage in lipid droplets and an improvement of oocyte developmental competence (Figure).

Figure: Confocal images of immature oocytes at the germinal vesicle (GV) stage (A) and mature oocytes at the metaphase II stage after maturation in control medium (B). Mature oocytes at metaphase II stage after exposure to 500 uM oleic acid (D) or 500 µM palmitic acid (E) during maturation. Merges show lipid droplets in green and DNA in blue. Bar=20 µm. PA, palmitic acid; OA, oleic acid; MII, metaphase- II oocytes.

These results suggest that the ratio and amount of saturated and unsaturated fatty acid is relevant for lipid storage in the maturing oocyte and that this relates to the developmental competence of maturing oocytes.