IBYME   02675
INSTITUTO DE BIOLOGIA Y MEDICINA EXPERIMENTAL
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
ASSOCIATION OF HUMAN CRISP3 WITH SPERM AND ITS BEHAVIOUR DURING CAPACITATION
Autor/es:
MARIANA WEIGEL MUÑOZ, ; AGUSTINA BATTISTONE; JUAN I ERNESTO; , DEBORA J COHEN; PATRICIA S CUASNICU
Lugar:
SAN PABLO
Reunión:
Congreso; Fifth International Conference on The Epididymis.; 2010
Resumen:
ASSOCIATION OF HUMAN CRISP3 WITH SPERM AND ITS BEHAVIOUR DURING
CAPACITATION
Mariana Weigel Muñoz*,
Agustina Battistone, Juan I Ernesto, Debora J Cohen and Patricia S Cuasnicu
Instituto de Biología y Medicina Experimental
(IBYME-CONICET) Buenos Aires, Argentina
Keywords: CRISP3, sperm, epididymis, capacitation
*Correspondence: Mariana Weigel Muñoz,
Instituto de Biología y Medicina Experimental (IBYME), Vuelta de Obligado 2490,
(1428) Buenos Aires, Argentina (FAX: 54-114-786-2564; E-mail: weigel@dna.uba.ar
The cysteine-rich secretory
protein (CRISP) family is a large group of secreted proteins characterized by
the presence of 16 conserved cysteine residues.In mammals, four main groups have been described: CRISP1, synthesized by the epididymis,
CRISP2, of testicular origin, CRISP3, with a wider
tissue distribution, including reproductive and non-reproductive tissue, and
CRISP4, also expressed in the epididymis. In our
laboratory, we have studied the association of epididymal CRISP1 and testicular
CRISP2 with human, rat and mouse sperm observing
that both proteins remain associated with sperm after capacitation and acrosome
reaction and, in agreement with this behaviour, both molecules have a role in
the fertilization process. Considering that CRISP3 is secreted by the
epididymal epithelium and could also be involved in the acquisition of sperm
fertilizing ability during maturation, in the present work we studied the
association of human CRISP3 with sperm as well as its behaviour during
capacitation as a first approach to investigate its potential role in
fertilization. For this purpose, human sperm were subjected to different extraction
treatments and the permanence of CRISP3 in sperm was evaluated by Western blots
and indirect immunofluorescence (IIF) using a polyclonal antibody against human
CRISP3. Results revealed the presence of
two bands of 31kDa and 29kDa corresponding to the two already described forms
of CRISP3 in protein extracts from fresh, untreated sperm. While the protein
corresponding to the 31kDa band was easily removed by washing the cells with PBS,
the one corresponding to the 29kDa band remained on sperm after their exposure
to high ionic strength (0.6M NaCl), and was completely removed from the gamete only
by treatment with detergent (1% Triton X-100) indicating the tight association
of this protein to human sperm. Interestingly, the 29kDa band was also detected
in protein extracts from sperm that have already undergone both capacitation
and calcium ionophore-induced acrosome reaction. Subsequent analysis of these
sperm by IIF revealed the presence of fluorescent labelling in the acrosome and
tail of capacitated sperm and in the equatorial segment of the acrosome reacted
cells. The existence of a strongly bound population of CRISP3 in human sperm
and its permanence after capacitation and the acrosome reaction supports the
potential participation of this protein in the fertilization process.
Financial Support: National Research Council of Argentina grant, National
Agency of Scientific and Technological Promotion grant and World Health
Organization (WHO) RMG grant