mhrep$0209
Molecular Human Reproduction vol.3 no.3 pp. 269–273, 1997
Sperm factor: what is it and what does it do?
Martin Wilding and Brian Dale1
Stazione Zoologica, Villa Comunale, 80121 Napoli, Italy
1To
whom correspondence should be addressed
There are two current hypotheses as to how the spermatozoon triggers the oocyte into activity; a
transmembrane receptor mechanism involving G-proteins and a soluble sperm-factor mechanism. In this
short review we show that the present data favours the idea of a soluble factor diffusing from the
spermatozoon into the oocyte following plasma membrane fusion of the two gametes that triggers calcium
release in the oocyte. Two categories of calcium release mechanisms, inositol-1,4,5-triphosphate-induced
calcium release (IICR) and calcium induced calcium release (CICR) are found in oocytes from a variety of
species and both appear to be activated at fertilization. Since these calcium release pathways are distinct it
is possible that sperm cytosol contains more that one activating factor. Finally, the fact that sperm extracts
‘activate’ oocytes from different phyla and trigger calcium oscillations in somatic cells infers calcium releasing
agents common to other cell types.
Key words: CICR/G-proteins/IICR/oocyte activation/soluble sperm factor
Introduction
A rise in intracellular calcium at fertilization is a fundamental
event in oocyte activation in invertebrates and vertebrates
(see Epel, 1990; Whitaker and Patel, 1990 for reviews).
However, despite the universal requirement for the calcium
signal, it is not known how spermatozoa trigger this rise in
calcium. In this review, we present current ideas of the
sperm-induced mechanism of intracellular calcium release
in oocytes. An idea of the components of spermatozoa that
might be capable of triggering calcium release depends on
a prior understanding of the calcium release machinery.
Therefore, we will discuss current knowledge on calcium
release, how these relate to oocyte activation and how these
may be harnessed into a suitable trigger for oocyte activation.
Intracellular calcium release pathways
Calcium release mechanisms may be divided into two
categories, depending on the type of receptor located on
the intracellular calcium store. These mechanisms are
commonly known as inositol 1,4,5-trisphosphate (IP3)induced calcium release (IICR) and calcium-induced calcium
release (CICR) (see Endo, 1977; Berridge, 1993). IICR is
triggered by the binding of IP3 to its receptor on the
endoplasmic reticulum (Terasaki and Sardet, 1991). IP3 is
produced by the action of phospholipase C on the plasma
membrane lipid phosphatidylinositol bisphosphate (PIP2) (see
Berridge, 1993). CICR is triggered through the opening of
the so-called ryanodine receptor on an intracellular store,
but can also be triggered in a mechanism involving the IP3
receptor (see Endo, 1977; Berridge, 1996). This can be
triggered by calcium itself, and appears to be modulated by
© European Society for Human Reproduction and Embryology
cyclic ADP ribose (see Galione and White, 1994). Cyclic
ADP ribose is in turn produced by metabolism of nicotinamide
adenine diphosphate (NAD1) by ADP ribosyl cyclase or
NAD1 glycohydrolase (see Galione and White, 1994;
Jacobson et al., 1995; Lee et al., 1995). More recently, other
calcium-releasing second messengers have been discovered
including cATP ribose and NADP1 (Lee and Aarhus, 1995;
Genazzani and Galione, 1996; Zhang et al., 1996). It is
possible that other calcium-releasing second messengers may
be discovered in the nicotinamide nucleotide family; evidence
for this comes from the observation that NAD1, NADH,
NADP1 and NADPH all can be metabolized to calciumreleasing second messengers in sea urchin microsomes
(Clapper et al., 1987). A pattern is emerging of two calciumreleasing second messenger families, one branch coming
from PIP2 and a second from nicotinamide nucleotides.
How are these second messengers formed? This subject
has been studied mainly in somatic cells and whether it
has any relevance to fertilization is as yet unclear. IP3
formation is well characterized in somatic cells. It appears
to occur by two mechanisms. The first involves a receptor
coupled G protein which stimulates the activity of phospholipase C on the plasma membrane, leading to PIP2 breakdown
(see Berridge, 1993). The second involves a receptor tyrosine
kinase, leading to the same result (see Berridge, 1993). The
metabolic pathway leading to the stimulation of cyclic ADP
ribose formation is not well established at present. In sea
urchins, cyclic ADP ribose is stimulated by cGMP (Galione
et al., 1993a). This suggests a role for the second messenger
nitric oxide, which stimulates guanylate cyclase activity (see
Bredt and Snyder, 1994).
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M.Wilding and B.Dale
Intracellular calcium release at fertilization
The mechanism of calcium release at fertilization varies
between species; however, there are common threads. Both
IICR and CICR have been demonstrated in many types of
oocyte (Galione et al., 1993b; see Whitaker and Swann, 1993).
However, this does not necessarily mean all oocytes contain
both IP3 and ryanodine receptors, because evidence suggests
that the IP3 receptor can support CICR (Galione et al., 1993b;
see Whitaker and Swann, 1993). In sea urchins, both IICR
and CICR are triggered at fertilization (see Whitaker and
Swann, 1993). This appears to be due to stimulation of two
calcium release pathways by the spermatozoa (Galione et al.,
1993b; Lee et al., 1993). In frog oocytes, IICR appears to be
uniquely activated at fertilization (Larabell and Nuccitelli,
1992, see Whitaker and Swann, 1993); however, what appears
to be IICR may in fact be CICR through the IP3 receptor
(Galione et al., 1993b, see Whitaker and Swann, 1993). The
urochordate Ciona intestinalis also releases calcium by both
IICR and CICR at fertilization (M.Wilding, unpublished data).
Ascidian oocytes also generate repetitive calcium transients
through meiosis I and II (Speksnijder et al., 1990; McDougall
and Sardet, 1995; Russo et al., 1996). These transients appear
to be triggered by an IP3-dependent mechanism because they
are blocked by heparin (McDougall and Sardet, 1995; Russo
et al., 1996). Mammalian oocytes are characterized by a large
increase in the sensitivity to CICR after fertilization together
with a series of repetitive calcium spikes (Igusa and Miyazaki,
1983; Cuthbertson and Cobbold, 1985; Miyazaki, 1988; Kline
and Kline, 1992; Taylor et al., 1993). These data again
suggest activation of both CICR and IICR at fertilization. The
mechanism of repetitive calcium spiking in mammalian oocytes
is not clear at present. Mammalian oocytes contain both
ryanodine and IP3 receptors (Miyazaki et al., 1992; Rickfords
and White, 1993; Ayabe et al., 1995; Yue et al., 1995; Sousa
et al., 1996a). However, it is not yet certain whether repetitive
calcium transients in mammalian oocytes are propagated by
IICR or CICR (Carrol and Swann, 1992; Swann, 1991, 1992;
Miyazaki et al., 1992; 1993; Kline and Kline, 1994; Fissore
et al., 1995; Tesarik et al., 1995; Berridge, 1996; Swann and
Lawrence, 1996; Tesarik and Sousa, 1996). Thimerosal, a
sulphydryl reagent, triggers repetitive calcium transients and
also sensitizes mammalian oocytes to CICR (Swann, 1991,
1992). The ability for thimerosal to mimic fertilization in
mammalian oocytes suggests that an element of sulphydration
may be involved in sperm-induced activation of mammalian
oocytes. This may involve protein kinase C (Sousa et al.,
1996b).
Sperm-induced calcium release at fertilization
How do spermatozoa from different species trigger the calcium
transient so vital at fertilization? The data above suggest that
we should look for agents that stimulate the production of IP3
and a second calcium releasing agent such as cADP ribose.
The hypotheses of sperm-induced oocyte activation fall into
two categories, both of which fit with the general hypothesis
summarized above.
270
Figure 1. Sperm/oocyte fusion revealed by electron microscopy. A
transmission electron micrograph showing cytoplasmic continuity
between the fertilising spermatozoon (marked S) and the oocyte
(marked O) in the sea urchin Paracentrotus lividus. Note that
sperm factor may flow through a pore of ~0.1 µm (marked FP), as
observed in the micrograph. The membrane-bound vesicles are an
intact cortical granule (l µm diameter, marked CG) and a pigment
granule (marked PG).
G proteins and oocyte activation
The formation of the G protein hypothesis of oocyte activation
comes from parallels with the calcium response to hormones
in somatic cells. Here, hormone-receptor binding on the outer
surface of the plasma membrane signals through a G protein
in the plasma membrane, and this signal triggers the activation
of phospholipase C, leading to the formation of IP3 and hence
calcium release (see Berridge, 1993). In the G protein model
of oocyte activation, the spermatozoon behaves as an ‘honorary
hormone’, which means that the attachment of spermatozoa
to the sperm receptor triggers IP3 formation through a G
protein linked to this receptor (Kline et al., 1988, see Foltz
and Schilling, 1993). There is some evidence supporting
this hypothesis. Firstly, antisera generated against the sperm
receptor on the oocyte plasma membrane or peptides from
spermatozoa were found to trigger oocyte activation when
added to the outside of oocyte membranes (Perlmann, 1954;
Gould and Stephano, 1987, 1991; Foltz and Lennarz, 1992;
Moore et al., 1993). Secondly, receptors introduced into oocytes
by mRNA injection can activate various oocytes on application
of their ligands to the plasma membrane, demonstrating that
the receptor-mediated oocyte activation pathways do exist in
oocytes (Kline et al., 1988, 1991; Shilling et al., 1991, 1993).
Furthermore, in-vitro G protein activators such as GTP-γ-S
and cholera toxin trigger intracellular calcium release in
oocytes with a short delay, which parallels the latent period
seen after sperm–oocyte fusion (Turner and Jaffe, 1989;
Crossley et al., 1991). In contrast to these data, inhibitors of
G proteins do not block the fertilization calcium wave in sea
Sperm factor
Figure 2. Mechanism of calcium release stimulated by soluble sperm extracts. The idea of a soluble extract, contained within sperm
cytoplasm, that is released into the oocyte cytosol triggering the release of intracellular calcium is shown. From what is known of
mechanisms of calcium release at fertilisation, both IICR and CICR are stimulated in most species of animal. We have suggested here that
CICR is triggered by cADPr, however this may not always be the case (outlined within the text). The fact that two independent pathways to
the release of intracellular calcium are stimulated suggests either the involvement of more than one factor within sperm cytosol, or a
common stimulatory mechanism. Spermatozoa and oocyte are marked for clarity.
urchins (Crossley et al., 1991). In summary, although the
above data show that the pathway for G protein-induced oocyte
activation does exist, it may not be the physiological pathway
to sperm-induced oocyte activation.
The sperm factor hypothesis
The sperm factor hypothesis is based on a diffusible messenger(s) in the cytoplasm of the spermatozoa that enters the oocyte
cytoplasm after sperm–oocyte fusion and triggers intracellular
calcium release. Evidence for this hypothesis stems from
different types of experiment. Spermatozoa are now known to
establish cytoplasmic continuity with oocytes in a definite
period before oocyte activation (Dale and Santella, 1985;
McCulloh and Chambers, 1987). This period is synonymous
with the latent period (Ginsberg, 1988). The latent period
between sperm–oocyte fusion and oocyte activation may allow
the diffusion of a messenger between the cytoplasm of the
spermatozoon and oocyte (Dale et al., 1985; Whitaker et al.,
1989; see Figure 1). The first direct evidence for a soluble
sperm factor was shown by Dale et al. who found that
microinjection of the soluble components from crushed spermatozoa into sea urchin and ascidian oocytes activated them
(Dale et al., 1985; Dale, 1988). Although this work has not been
repeated in sea urchins, similar findings were demonstrated in
mammals (Stice and Robl, 1990; Swann, 1990). In recent
years, human spermatozoa have been microinjected into human
oocytes, activating them after a delay which has been suggested
to be the time required for sperm plasma membrane breakdown
and the release of the cytoplasmic contents of the spermatozoa
(Tesarik et al., 1994). Soluble sperm extracts from human
spermatozoa trigger the activation current in human oocytes
(Dale et al., 1996). Sperm extracts also trigger repetitive
calcium transients in mammalian and ascidian oocytes which
closely mimic the pattern of calcium release seen at fertilization
(Swann et al., 1989; Swann, 1990, 1992; Miyazaki et al.,
1993; Homa and Swann, 1994; Wilding et al., 1997 for
ascidian). It therefore seems probable that a soluble component
of sperm cytoplasm is responsible for the calcium increase
that activates oocytes at fertilization.
So, what is the secret behind ‘sperm factor’? Spermatozoa
are known to contain many molecules capable of releasing
intracellular calcium, including cGMP, IP3, nicotinamide
nucleotide metabolites, calcium ions etc. (Iwasa et al., 1990;
Whitaker and Crossley, 1990; Tosti et al., 1993). However,
current data suggest that these molecules are not in themselves
responsible for triggering the fertilization calcium transient
(see Whitaker and Crossley, 1990; Whitaker and Swann, 1993).
Data from many laboratories point to a protein ‘sperm factor’,
at least in mammalian oocytes (Stice and Robl, 1990; Swann,
1990; Homa and Swann, 1994). In fact, a protein from
spermatozoa that triggers repetitive calcium release in hamster
oocytes has been sequenced (Parrington et al., 1996). However,
the protein found in hamster spermatozoa may not be the sole
oocyte activating substance contained within spermatozoa;
indeed recent data from our laboratory suggest that a small
molecule contained within human sperm extracts can activate
oocytes (M.Wilding and G.L.Russo, unpublished data),
although this is not in agreement with Swann (1990) who
found no activity in the low molecular weight fraction. The
supporters of the protein ‘sperm factor’ hypothesis suggest
that this protein is specific to spermatozoa (Swann, 1990;
Parrington et al., 1996). However, soluble extracts of human
spermatozoa can activate oocytes from different phyla as well
as different species (Homa and Swann, 1994; Wilding et al.,
1996a). Sperm extracts can also trigger calcium oscillations
in somatic cells (Currie et al., 1992; Berrie et al., 1996). These
data strongly suggest that sperm factors are common calciumreleasing agents and so indicate that they are not spermspecific molecules. Furthermore, recent data from several
sources point to the role of the spermatozoon as a stimulator
of at least two metabolic calcium-releasing systems within the
oocyte (M.Wilding, unpublished data; see also Osawa, 1994;
Ciapa and Epel, 1996; see Figure 2). Unless a common
activation pathway can be found for IICR and CICR, we could
assume that sperm factor includes at least two molecules.
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M.Wilding and B.Dale
Acknowledgements
The authors are supported by an EEC Human Capital and Mobility
grant CHRX-CT94–0646 and Medicult, Copenhagen, Denmark.
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Received on October 7, 1996; accepted on January 7, 1997
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