Molecular Human Reproduction vol.3 no.8 pp. 646–650, 1997
2. Sperm–zona interaction and recombinant DNA technology
Neil R.Chapman1,2 and Christopher L.R.Barratt2,3
1Departments of Molecular Biology and Biotechnology, Firth Court, University of Sheffield, S10 2UH, UK and
2Department of Obstetrics and Gynaecology, Jessop Hospital for Women, Leavygreave Road, Sheffield S3 7RE,
UK
3To
whom correspondence should be addressed at: University Department of Obstetrics and Gynaecology,
University of Birmingham, Birmingham Womens Hospital, Edgbaston, Birmingham B15 2TG, UK;
email: [email protected]
Recombinant DNA technology has revolutionized our understanding of many biological systems. However,
such techniques and their application have not been fully exploited in the study of sperm zona interaction.
Using examples from other biological systems, we ourline several experimental approaches that are likely to
significantly enhance our understanding of the gamete recognition process.
Key words: plasmid/recombinant ZP3/recombinant ZP2/spermatozoa
Introduction
Recombinant DNA technologies represent extremely powerful
tools to study and dissect both the gross and the fine details of
biological systems. However, despite the significant advantages
that these technologies provide, and their widespread use,
reproductive biologists working in the field of sperm–zona
interaction have yet to fully exploit their potential. In this
article we will highlight some examples of using recombinant
expression systems for studying sperm–zona interaction and
provide insights for future experiments.
Background
To date, expression of recombinant zona pellucida proteins
(rZP) from a range of mammals has facilitated the study of
gamete interaction. Kinlock et al. (1991) first demonstrated
that it was possible to express biologically active (defined as
the ability of the recombinant material to induce acrosomal
exocytosis) recombinant mouse ZP3 (rmZP3). Furthermore,
exon swapping and mutagenesis experiments with the mouse
ZP3 gene demonstrated that a region within the C-terminus
was responsible for initiating the acrosome reaction, and that
O-linked carbohydrates within this region were important for
biological activity (Kinloch et al., 1995).
Expression of rZP is not restricted to those of the mouse,
since recombinant zona proteins from other species, namely
rabbits and humans have been reported. For example, Prasad
et al. (1996) demonstrated that recombinant rabbit 55 kDa
protein (which is thought to be the rabbit homologue of mouse
ZP1) purified from a baculovirus expression system was used
to generate a polyclonal antiserum which was then employed
to study the localization of the native 55 kDa protein in rabbit
zonae. Cloning the human ZP3 gene (Chamberlin and Dean,
1990) has facilitated the study of this glycoprotein in human
gamete interaction. Recombinant human ZP3 has been
expressed using several different approaches, e.g. Escherichia
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coli (Chapman and Barratt, 1996), in-vitro transcription and
translation (Whitmarsh et al., 1996) and in Chinese hamster
ovary (CHO) cells (Van Duin et al., 1994; Barratt and Hornby,
1995; Brewis et al., 1996). A brief outline detailing the
expression of rHuZP3 in E.coli is given in Figure 1A,B. A
full length cDNA encoding the human ZP2 protein has also
been produced, although to the best of our knowledge no
reports of the expression and purification of recombinant
human ZP2 have been documented.
Furthermore, the use of transgenic mice that were homozygous for mutations that abolished the expression of ZP3 has
demonstrated the importance of this protein in zona biosynthesis and mouse fertility (Liu et al., 1995 and Rankin et al.,
1996). Although these mutants were phenotypically normal,
they were sterile and their oocytes lacked zonae pellucidae.
Transgenic mice have also been used to investigate the role
of certain oligosaccharides in mouse sperm–ZP3 interaction
(Thall et al., 1995). Interestingly, Thall et al. showed that
when expression of the enzyme involved in the synthesis of
the terminal galactose-α1,3-galactose epitope on glycoproteins
was abolished, female mice homozygous for this mutation
were phenotypically normal and still fully fertile.
There are few examples in the literature investigating the
activity of recombinant spermatozoa-bound receptors for the
zona from any species of mammal. This is because the exact
nature of such a molecule(s) remains elusive. However, there
are two notable examples. The first concerns the putative
zona receptor galactosyltransferase (GalTase). Using transgenic
mice, Youakim et al. (1994) demonstrated that when GalTase
was over-expressed in the mouse testis, giving rise to a greater
number of functional receptors on the mature spermatozoon,
there was actually a decrease in the number of spermatozoa
bound to intact mouse zonae pellucidae. This anomaly was
thought to be because the mutant spermatozoa underwent
increased rates of acrosomal exocytosis upon binding to the
zona and hence did not remain bound to the zona for a
© European Society for Human Reproduction and Embryology
Sperm–zona interaction and recombinant DNA technology
Figure 1. Expression of recombinant GST-HuZP3 in Escherichia coli. (A) The lac repressor protein is synthesized from the lacIq gene. In
the absence of a natural inducer of protein expression (such as lactose) or more commonly a synthetic inducer [e.g. isopropyl-β-Dthiogalactoside (IPTG)] this protein binds to the tac promoter. E.coli RNA polymerase is prevented from binding to the tac promoter by the
lac repressor protein. Transcription of recombinant GST-HuZP3 is thus prevented. The dashed line signifies that the gst cDNA is fused to
the N-terminus of the huzp-3 cDNA. (B) IPTG binds to the lac repressor protein and prevents it from binding to the tac promoter. E.coli
RNA polymerase is now able to bind to the tac promoter and initiate transcription of recombinant GST-HuZP3. The GST protein moiety
(encoded by the gst cDNA) facilitates purification of rHuZP3 by glutathione-agarose affinity chromatography.
GST 5 glutathione S-transferase.
sufficient length of time to permit secondary binding. The
second example concerns the sperm serine protease acrosin.
Until recently the nature of the residues within acrosin that
are responsible for binding to the zona pellucida (ZP) were
unknown. Deletion mutagenesis of recombinant boar proacrosin expressed in E.coli identified a central region, Gly 93-Ala
275 (with important contributions from His 47 and Arg 50)
which was primarily responsible for binding to polysulphate
groups on native ZP glycoproteins (Jansen et al 1995). Complementary analysis of synthetic polypeptide fragments suggested
that protein folding, possibly to confer spatial configuration of
positively charged residues, was important (see also Fini et al.,
1996). Richardson and O’Rand (1996), using site directed
mutagenesis, demonstrated that when residues Arg 50 and Arg
51 of rabbit proacrosin were mutated to Ala, the mutant
recombinant protein had little zona-binding ability. Complementary deletion mutagenesis experiments using human recombinant acrosin have yet to be reported.
Future experimentation using recombinant DNA
technology
Production of biologically active recombinant human
ZP is a primary objective
With regard to the human system, production of purified
glycosylated zona proteins in a biologically active form is
fraught with technical difficulties (see van Duin et al., 1994,
Barratt and Hornby, 1995). Reliable and repeatable systems
need to be established so that these proteins can be made
available, in large quantities, to the scientific community.
Rapid production of small quantities of rZP can be made using
in-vitro transcription and translation (see Whitmarsh et al.,
1996). Such methods are ideally suited for site-directed mutagenesis experiments, for example, to determine the active site
of ZP3 and ZP2 binding to spermatozoa. However, to date,
only core glycosylation of the recombinant protein is possible.
Expression of recombinant human ZP3, using an Escherichia
coli system, leads to the production of mainly insoluble protein,
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N.R.Chapman and C.L.R.Barratt
although small amounts of purified solubilized protein are
available for a limited number of experiments. Expression
in E.coli clearly allows the contribution of protein versus
carbohydrate recognition to be determined with confidence but
to date, refolding of insoluble protein has not resulted in the
maintenance of biological activity (Chapman and Barratt,
1996). To make significant and meaningful progress, the
production of human rZP in various forms (glycosylated and
non-glycosylated) is a priority.
Insolubility is a common problem associated with the
expression of rHuZP3 in other host cell lines. For example, in
our laboratory, when expressed in sf9 insect cells, the rHuZP3
is still seen to partition into the insoluble fraction thus
complicating the purification of biologically active material
(N.R.Chapman, C.L.R.Barratt and D.P.Hornby, unpublished
observations). More detailed analyses aimed at optimizing
expression conditions to yield milligram quantities of soluble,
biologically active rHuZP3 are thus required.
To our knowledge there are no reports in the literature
detailing expression of rHuZP3 in the methylotrophic yeast
Pichia pastoris. This expression system has been used to
obtain substantial amounts (mg recombinant proteins/l culture
medium) of a number of commercially interesting proteins
including tumour necrosis factor and human epidermal growth
factor (reviewed in Cregg et al., 1993). However, one should
appreciate the fact that the structure of the oligosaccharide
chains attached to secreted proteins expressed in P.pastoris
does not reflect the pattern seen for glycans attached to proteins
expressed in mammalian cells (Cregg et al., 1993). Whether
such differences would alter the biological activity of rHuZP3
is equivocal.
Contraception research
Interestingly, a primary goal in the production of rZPs is to
explore their use for contraceptive research. rZPs have been
used as antigens to stimulate an immune response, however,
the initial enthusiasm for this approach has somewhat subsided
due to the subsequent ovarian dysfunction following immunization in several species, e.g. marmosets (Paterson et al., 1996).
Ovarian pathology may be due to induction of a cell mediated
immune response caused by cytotoxic T cell epitopes residing
within the ZP3 sequence (Miller et al., 1989; Rhim et al.,
1992). Interestingly, careful selection of mouse ZP3 epitopes
has allowed induction of limited suppression of fertility without
accompanying ovarian pathology (Lou et al., 1995). However,
the possible presence of ZP3 on primordial follicles may
contribute to ovarian pathology and thus complicate the choice
of peptide antigens (Grootenhuis et al., 1996). Production of
defined recombinant zona peptides can be used for epitope
mapping studies. For example, Gupta et al. (1996) used such
an approach to identify amino acid residues 133–144 and 205–
216 of porcine ZP3α (homologous to human ZP1) which are
important in the sperm–zona recognition process and thus may
be important candidate epitopes for contraceptive design. Such
an experimental approach, for example with rHuZPs, is now
warranted.
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Complementary zona receptor(s) on spermatozoa
The difficulties in developing zona proteins as targets for
contraceptive research has focused attention on interrupting
fertilization by targeting the sperm membrane. Following
the production of various forms of biological rHuZPs, the
identification of complementary receptors on spermatozoa
becomes a possibility. Studies using native zona pellucida
have identified several candidate molecules, e.g. zona receptor
kinase (ZRK; Burks et al., 1995). As the cDNA for ZRK
is available it is theoretically possible to express recombinant
ZRK (rZRK) and examine the kinetics of interaction with
rHuZPs. An equivalent approach using rabbit recombinant
Sp17 and native rabbit zona pellucida has been applied
(Yamasaki et al., 1995). If rZRK interacts with rHuZPs in
a similar manner to native zona proteins then the next step
is to study the primary cell signalling mechanisms associated
with ZRK in the spermatozoa. This can be performed by
adding rHuZPs to non-spermatozoan cells transfected with
rZRK and detecting activation, e.g. calcium influx. This
approach is currently used to dissect many other signalling
pathways (see White and Khan, 1994). Subsequent mutation
of ZRK, and formation of chimeric receptors, are a logical
extension. An alternative but complementary strategy to
identify other possible receptors on the spermatozoa is to
use combinatorial expression libraries (see Georgiou et al.,
1997, for review)
Yeast two-hybrid system
There are numerous other examples of recombinant DNA
technology which can be utilized to study sperm–zona
interaction. A particularly interesting technique is the yeast
two-hybrid system (illustrated in Figure 2A–C). This system
is extensively used to study protein–protein interactions, e.g.
the interaction between FAS and FADD in the regulation
of apoptosis (Chinnaiyan et al., 1995). It was first developed
to detect the interaction between two yeast proteins, SNF1,
a protein kinase and SNF4, a protein associated with this
kinase (Fields and Song, 1989). The major advantage of
the system over other available techniques, e.g. cross-linking
(see Bleil and Wassarman, 1990) is that since the assay is
performed in vivo, the interacting proteins are more likely
to be in their native conformation. The two-hybrid system
exploits the ability of a pair of interacting proteins to bring
together a transcription activating domain and a DNA
binding domain that regulates the expression of an adjacent
reporter gene (Fields and Song, 1989). It could be used to
determine the nature of the interaction between human ZP2
and ZP3. In the mouse, where all three genes encoding
mouse zona proteins have been cloned (Epifano et al.,
1995), one could investigate the nature of the interactions
between ZP1/ZP2, ZP1/ZP3 and ZP2/ZP3. Furthermore, the
availability of the mouse sp56 cDNA (a putative sperm
receptor for mouse ZP3) (Bookbinder et al., 1995) would
allow investigations into the binding (if any) between sp56
and the mouse zona proteins. A similar approach in the
human could use ZRK (see above). A potential drawback
of the yeast two-hybrid system to detect interactions between
Sperm–zona interaction and recombinant DNA technology
Figure 2. Model of transcriptional activation by reconstitution of GAL4 activity. GAL4 controls expression of galactose utilization genes in
Sacchromyces cerevisiae. (Figure reproduced from Fields and Song, 1989 with kind permission). (A) The native GAL4 protein contains both
DNA-binding and activating regions and induces GAL1-lacZ transcription; (B) hybrids containing either the DNA-binding domain (upper) or
activating domain (lower) are incapable of inducing transcription; (C) protein–protein interaction between proteins X and Y brings the
GAL4 domains into close proximity and results in transcriptional activity.
zona proteins is that the system has been used, in the main,
for intra-cellular interactions. Whether the nature of the
interaction between zona proteins (extra-cellular proteins)
would be influenced by conditions within the cell is at
present not clear.
The ultimate goal
Ideally, the crystal structure of rZPs in either human and/or
animal systems should be solved. Developing a crystal structure
can be an arduous task. For example, it is difficult to predict if
suitable crystals can be formed, and mg of recombinant material
are necessary before crystallization trials can be initiated. Yet,
successfully obtaining a structure is a worthwhile goal as it
allows significant advances in the understanding of how molecules interact (see Stuart and Jones, 1995; Wells, 1996). Such
information can be used to provide the baseline for more critical
studies, e.g. using site-directed mutagenesis (see Wieczorek
et al., 1996 for example of p53). In many cases, the structure
provides unexpected results, e.g. a novel serine protease fold in
human cytomegalovirus protease (Chen et al., 1996) and, the
construction of the active site of the tyrosine kinase c-Src (Xu
et al., 1997). The structure also highlights the way for the rational
design of drugs/molecules which can inhibit/activate the protein.
This information is likely to be pivotal in the design of a new
generation of contraceptive agents to interrupt the fertilization
process. To date, the structure of only one fertilization-associated
protein has been obtained, i.e. lysin (Shaw et al., 1993). The leap
in our understanding of the action of lysin based on this model
(see Shaw et al., 1995) must encourage the search for the first
zona protein structure.
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N.R.Chapman and C.L.R.Barratt
Conclusion
Limited progress in the study of sperm–zona interaction has
been made. Significant advances in our knowledge of the
fertilization process will be achieved when we can successfully
extrapolate information and techniques from other disciplines
where recombinant DNA technology has revolutionized the
understanding of biological systems.
Acknowledgements
The authors would like to thank D.P.Hornby for critical reading of
this manuscript. We are grateful for the financial support provided
by the Infertility Research Trust (Sheffield), Wellbeing and the
University of Sheffield to undertake this work.
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Received on April 4, 1997; accepted on May 9, 1997