Novel modulators of human sperm motility and migration

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Correia, Joao Natalino (2012). Novel modulators of human sperm motility and migration. University of Birmingham. Ph.D.

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Abstract

Overall, signaling events in human sperm, together with the identity of many of the molecules stimulating them, remain very poorly characterised. A two-pronged approach involving characterisation of sperm receptors in a model-cell system, followed by single cell [Ca2+]i imaging of large numbers of capacitated human spermatozoa, was employed to start to unravel some of these possibilities. Data were compared to responsiveness of the best-characterised physiological ligand of sperm – the steroid hormone progesterone.

Proteomic data strongly indicates the presence of OR2AE1, an olfactory receptor, in mature human sperm. OR2AE1 was cloned into the mammalian expression vector pcDNA3 and expressed in HEK293 cells. Functional expression of OR2AE1 (5.6±1.1% of cells showing correct localisation of FLAG-tagged recombinant receptor at the plasma membrane level) was demonstrated. Despite this relatively low level of functional expression, this system still allowed identification of a potent volatile agonist for OR2AE1 – omega () – via an elimination-panel system used alongside [Ca2+]i imaging. Around 3% of cells in the heterologous system generated a response to the agonist, indicating correct functional expression.

When sperm were exposed to 50 μM , [Ca2+]i imaging revealed a transient response which occurred in 30-40% of capacitated cells. 10 μM NNC 55-0396, an antagonist of the CatSper channels, inhibited around 80% of these responses. Similar inhibition levels were demonstrated by inhibitors of the cAMP pathway (100 μM SQ22536 and 25 μM cAMPS-Rp), but no substantial effect was observed by inhibition of PLC by 5 μM U73122. Chemotactic assays indicated that 50 μM  had an optimal chemoattractive effect, with 500 μM  significantly stimulating acrosome reaction.

An unrelated family of chemicals, the boronic acids, were also examined for their ability to affect sperm motility, migration and [Ca2+]i dynamics. A significant effect on the number of sperm which migrated from semen when exposed to these chemicals was observed, with pathway inhibition distinctly indicating a bifurcating signal propagating mainly via IP3 but also involving the cAMP pathway.

Manipulation of the signalling events described, with inherent modulation of sperm motility may underlie the ability to develop future novel therapy, including contraceptive and diagnostic treatment regimes for the human male

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

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