Biochemical Society Transactions (1997) 25 S635
99
Avian acrosin: the relationship between
biological role and specificity
FIONA C. M. MILNE, GRAHAM J. WISHART, LAURA
ROBERTSON and A. JANET HORROCKS.
School of Molecular and Life Sciences, University of
Abertay Dundee, Bell Street, Dundee, DD1 1HG
Acrosin is a proteolytic enzyme released by spermatozoa
to facilitate penetration of the proteinaceous layer
surrounding the mature ovum and allow fusion of the
plasma membranes surrounding the gametes. The avian
ovum is surrounded at the time of fertilisation by a
proteinaceous layer known as the inner perivitelline layer
(IPVL) which is in many ways analogous to the
mammalian zona pellucida. It must however contain a
much larger ovum (a typical chicken ovum has a volume
millions of times greater than a mammalian ovum) and
unlike the mammalian ovum where normally only one
spermatozoa can penetrate the zona pellucida, the inner
perivitelline layer can be penetrated by thousands of
spermatozoa, although only one pronucleus fuses with
the female pronucleus [l]. Such multiple punctures of the
IPVL, particularly at the animal pole, could potentially
compromise the mechanical stability of the large ovum
[2]. A further problem exists in that sperm penetration of
1
.
2
-
3-
4
5-
6-
7
(Figure 1)however several prominent protease resistant
fragments principally of 36kDa, 30.5 kDa and 21 kDa
remained after prolonged (48 hour) incubation. Trypsin
rapidly hydrolysed the major IPVL proteins with only
traces of the 36kDa, 30.5kDa and 2lkDa fragments
remaining after prolonged incubation. When pieces of
isolated and washed IPVL were treated with
acrosin-containing extracts disintegration was observed to
occur in a series of stages, the final stage consisting of a
fibre mesh. We have some evidence that these fibres
consist principally of the protease-resistant fragments.
These experiments suggest that acrosin has a more
defined specificity than trypsin enabling sufficent
hydrolysis of IPVL protein to allow penetration of the
spermatozoa without compromising the mechanical
integrity of the ovum.
8
66 0 kDa
45 0 kDa
36 0 kDa
A
29OkDa
24 0 kDa
20 1 kDa
14 2 kDa
1 2 3 4
5 6 7 8
66 0 kDa
B
the IPVL is both an enzymatic and a mechanical evelit.
Penetration is accompanied by "burrowing" of attached
sperm through IPVL [3]. Thus whilst digesting the IPVL
spermatozoa must remain attached to (and therefore
preserve) binding sites within the zone of hydrolysis.
We have investigated the specificity of avian
acrosin from a biological perspective. Acrosin (from all
species) is said to have trypsin-like specificity,
preferentially hydrolysing peptide bonds on the carboxyl
side of arginine and lysine [2]. An extract containing avian
acrosin was prepared from washed sperm by freeze thaw
disruption and centrifugation. This extract could
hydro1yse benzoyl-DL-arginininep-nitroanilide (BAPNA),
a the trypsin substrate. When the acrosin-containing
extract was incubated with a protein substrate (bovine
serum albumin) no significant hydrolysis was observed
even after prolonged (48hour) incubation (Figure 1).If a
similar number of units of trypsin activity (with respect to
BAPNA hydrolysis) was incubated with bovine serum
albumin (BSA) after one hour of incubation fragmentation
was observed and after a prolonged (48hour) incubation
the protein was extensively fragmented (Figure 1).
Essentially acrosin was unable to hydrolyse bovine serum
albumin.
The physiological acrosin substrate (homogenised
IPVL) was fragmented by acrosin-containing extracts
46 0 kDa
36 0 kDa
29 0 kDa
24 0 kDa
20 1 kDa
14 2 kDa
Figure 1 SDS PAGE of the fragmentation by acrosin containinE extract
and trypsin of BSA and IPVL. Panel A Fragmentation of BSA by acrosin containing extract (2,3 and 4)
or trypsin (lanes 5,6 and 7) after 1 hour (3 and 6), 48 hours (4 and 7) and
0 hours (2 and 5). Panel B: Fragmentation of IPVL by acrosin containing
extract (lanes 2 3 and 4) or trypsin (lanes 5 , 6 and 7) after 1 hour (3 and
6), 48 hours (4 and 7) and 0 hours (2 and 5).
All digests were stopped by the addition of Soybean trypsin inhibitor
(resulting in a characteristic band at 20kDa) and fragmentation patterns
detected using a silver stain.
Y
1.Perry, M. M. (1987) J. Anatomy 150,99-109.
2. Howarth, B. and Digby, S. T. (1973) J. Reprod. Fert. 33,
123-125.
3. Bakst, M. R. and Howarth, 8. (1977) Biol. Reprod. 17,
370-379.