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:
WEIGEL MUÑOZ M; BATTISTONE A; ERNESTO JI; COHEN D.J; CUASNICU PS
Lugar:
Sao Paulo
Reunión:
Congreso; Fifth International Conference on Epididymis; 2010
Resumen:
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, synthesizedby 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.