Biology Reference
In-Depth Information
can better represent cells and subcellular districts and that can analyze functional
relationships between various signaling pathways. This method is based on the
“biological networks theory” (Weng et al.
1999
), which uses mathematical models
and special software to represent cells as networks of molecules (the network
nodes) that interact with each other, resulting in different interaction types (the
links between nodes). Through subsequent statistical and topological analysis, it is
possible to define the main parameters of each network and identify the key
molecules of metabolic processes.
Recently, our research group developed a network to model the biochemical
events occurring during the acquisition of fertilizing ability in boar spermatozoa
(Bernab` et al.
2009
). Such a cell type is particularly suitable for this analytical
approach, since it is transcriptionally silent and, therefore, its protein content is
almost constant. In addition, some biological events involving spermatozoa can be
recreated and modulated in vitro, making possible the acquisition of molecular and
biological information. In fact, it is possible to reproduce in vitro capacitation, the
set of morpho-functional changes that allow the sperm to acquire full fertilizing
power and induce acrosome reaction (AR); that is, the exocytosis of acrosome
content in response to species-specific interactions with zona pellucida proteins
(Abou-haila and Tulsiani
2009
).
In this work, we use a computational model to aggregate and interpret the effects
induced by inhibition of actin polymerization during capacitation and the AR
promoted in vitro in boar spermatozoa.
6.2 Materials and Methods
Semen of three boars of proven fertility was used. Samples were divided into two
different groups: control group spermatozoa (CTR) were cultured using validated
conditions that promote capacitation (Maccarrone et al.
2005
), while inhibition of
actin polymerization was obtained by treating the sperm with a specific inhibitor,
cytochalasin D (20
m
M), from the moment of ejaculation and during the whole
culture period, conducted as in the CTR group (CD). Both samples were incubated
for 4 h at a final concentration of 1
10
8
spermatozoa/-ml. After incubation, the
acquisition of fertilizing ability was verified, evaluating the incidence of AR in
sperm exposed to solubilized zonae pellucidae (sZP) and staining spermatozoa
with FITC-PSA (Barboni et al.
1995
). In addition, the main signaling pathways
were analyzed (a) acquisition of a pattern indicative of capacitated state by staining
with chlortetracycline (CTC) (Mattioli et al.
1996
); (b) pattern of tyrosine phos-
phorylation by western blotting; (c) localization of PLC
1 in cytosolic and mem-
brane fractions by western blotting and transmission microscopy (Spungin et al.
1995
); and (d) concentration of intracellular calcium ([Ca
2+
]
i
), analyzed kinetically
via confocal microscopy, using the fluorescent probe fluo-3-AM both before and
after exposure to sZP. The biological results were included in the computational
model recently validated by our group on mammalian sperm. This model was
g
