Hum. Reprod. Advance Access published online on March 8, 2007
Human Reproduction, doi:10.1093/humrep/dem023
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Effect of fibronectin on proteasome activity, acrosome reaction, tyrosine phosphorylation and intracellular calcium concentrations of human sperm
Department of Biomedicine, Faculty of Health Sciences, University of Antofagasta, Antofagasta, Chile
1 To whom correspondence should be addressed at: Department of Biomedicine, Faculty of Health Sciences, University of Antofagasta, PO Box 170, Antofagasta, Chile. E-mail: pmorales{at}uantof.cl
BACKGROUND: Previously we showed that the human sperm proteasome plays significant roles during mammalian fertilization. Here we studied the effect of fibronectin (Fn), an extracellular matrix protein present in the cumulus oophorus of the oocyte, on proteasome activity, acrosome reaction, intracellular calcium concentration ([Ca2+]i) and protein tyrosine phosphorylation of human sperm.
METHODS: Aliquots of motile sperm were incubated for 15 min (T0), 5 h (T5) and 18 h (T18), at 37°C, 5% CO2 and 95% air with Fn (0–100 µg/ml). The chymotrypsin- and trypsin-like activity of the proteasome was measured using the fluorogenic substrates, Suc-Leu-Leu-Val-Tyr-AMC and Boc-Gln-Ala-Arg-AMC, respectively. At T18, sperm aliquots were incubated for 15 min with Fn and/or progesterone in the presence or absence of epoxomicin (a proteasome inhibitor). The percentage of viable acrosome reacted sperm was evaluated using the Fluorescein isothiocyanate (FITC)-labeled Pisum sativum agglutinin. Tyrosine phosphorylation was evaluated by western blot and [Ca2+]i using fura 2.
RESULTS: Fn stimulated both enzymatic activities of the proteasome and the acrosome reaction of human sperm. Progesterone enhanced and epoxomicin drastically inhibited the effect of Fn. Fn treatment also increased the [Ca2+]i. Western blot analysis revealed that Fn increased tyrosine protein phosphorylation and that some proteasome subunits became tyrosine phosphorylated upon Fn treatment.
CONCLUSIONS: These results suggest that Fn activates the proteasome and induces the acrosome reaction in human sperm. This effect may involve binding with specific receptors (integrins) on the sperm surface and the activation of tyrosine kinases.
Key words: acrosome reaction/extracellular matrix/human sperm/proteasome/tyrosine phosphorylation
Submitted on November 3, 2006; resubmitted on December 29, 2006; resubmitted on January 9, 2007; accepted on January 16, 2007.
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