|
Molecular characterization of zona pellucida glycoproteins: Role in fertilization and regulation of fertility |
Principal Investigator : Satish K Gupta Project Associates/Assistants Ph D
Students
Collaborators The
major emphases of this project are molecular and biochemical characterization
of zona pellucida (ZP) glycoproteins and delineation of their role during
fertilization, and evaluation of the immunocontraceptive potential of ZP based
immunogens. In addition, the studies pertaining to the relevance of Y
chromosome microdeletion in male infertility have also been initiated. The
specific objectives are (i) expression of human, bonnet monkey and canine ZP
glycoproteins in various expression systems, (ii) delineation of structural
and functional attributes of purified recombinant ZP proteins/glycoproteins,
(iii) immunization strategies aiming to inhibit fertility and provide
protection against rabies infection and (iv) analysis of human Y-chromosome
microdeletion in non-obstructive oligospermia or azoospermia. Characterization
of the binding domain of bmZP3 to capacitated spermatozoa In
order to delineate the segment of bmZP3 that is responsible for its binding to
the spermatozoa, we reported last year that E.coli expressed r-bmZP3-C
(223-348 aa) was able to bind to the head region of capacitated spermatozoa.
Using competitive inhibition assays, r-bmZP3-C inhibited the binding of both E.coli
as well as baculovirus expressed r-bmZP3 (23-348 aa) to the capacitated
spermatozoa. However, E.coli expressed r-bmZP3-C failed to induce
acrosomal exocytosis when incubated with capacitated spermatozoa. To ascertain
if glycosylated r-bmZP3-C could induce acrosome reaction in capacitated bonnet
monkey spermatozoa, bmZP3-C was cloned in baculovirus expression system. The
purified baculovirus expressed r-bmZP3-C resolved as a closely spaced doublet
of ~27 kDa in SDS-PAGE. Binding of recombinant protein with Concanvalin A (ConA),
Peanut agglutinin (PNA), Lens culinaris (LCA) and Jacalin lectins
revealed the presence of both N- as well as O-linked sugars on its polypeptide
backbone. Baculovirus expressed r-bmZP3-C bound to capacitated spermatozoa but
could not induce acrosome reaction. Interestingly, it could inhibit induction
of acrosome reaction mediated by baculovirus expressed r-bmZP3. Identification
of ZP proteins binding ligand(s) on bonnet monkey spermatozoa An
attempt was made to characterize the spermatozoa associated proteins
responsible for binding to ZP glycoproteins, using two different strategies.
In the first one, a phage display peptide library (12 mer) was screened for
binding to baculovirus expressed r-bmZP3. After three rounds of affinity
selection, 7 phage clones exhibited strong reactivity with r-bmZP3. However,
these clones also showed varied binding with baculovirus expressed r-bmZP1 and
r-bmZP2. The DNA from 7 phage clones was eluted and sequenced. However, no
conserved sequence could be determined among 7 phage clones. Alternatively,
a “Pull Down Assay” was performed wherein the individual biotinylated
recombinant bonnet monkey ZP glycoproteins immobilized on to streptavidin
beads were used as a “bait” to capture the spermatozoa surface protein
(“prey”). As a negative control, a non-specific biotinylated glycoprotein
i.e., ovalbumin was used as a “bait”. Analysis by SDS-PAGE of the eluate
fractions from all the “Pull Down Assays” involving the three bonnet
monkey ZP glycoproteins seem to have a more or less same profile with some
minor differences. Six bands of approximate molecular weights of 122, 96, 52,
35, 29 and 21 kDa were observed in the eluates from all three zona proteins.
These bands are absent in the eluates obtained by using biotinylated ovalbumin
as the “bait” or with uncoated and blocked streptavidin beads, suggesting
the specificity of the bands eluted in the pull downs with ZP glycoproteins.
The ~122 kDa band observed in the r-bmZP2 pull down migrated slightly higher
than that observed in r-bmZP1 and r-bmZP3 pull downs. In the r-bmZP3 “Pull
Down Assay”, a doublet band was observed around 35 and 29 kDa instead of the
broad bands observed in r-bmZP1 and r-bmZP2 “Pull Down Assays”. Amino acid
sequencing of the ~37 kDa band observed in the r-bmZP3-pull down, revealed it
to be b-form
of ram spermatozoa acrosin. Currently, efforts are on to characterize other
protein bands. Delineation
of biological activity of recombinant human ZP glycoproteins We
have reported last year the cloning and expression of human zona pellucida
glycoproteins-1 (hZP1), -2 (hZP2) and -3 (hZP3) in the E.coli as well
as the baculovirus expression system. As in the monkey system, the recombinant
human zona proteins were also tested for their ability to induce acrosome
reaction in capacitated human spermatozoa. Baculovirus expressed r-hZP3
induced acrosomal exocytosis in the capacitated human spermatozoa, whereas the
E.coli expressed protein failed to do so. The induction of acrosomal
exocytosis, in this case was observed as early as 30 min after incubation with
r-hZP3. A significant increase in acrosomal exocytosis, as compared to
control, was observed at as low as 100 ng/ml of r-hZP3. Pertussis toxin, an
inhibitor of Gi protein mediated signal transduction pathway was shown to
inhibit the r-hZP3 induced acrosome reaction in capacitated human spermatozoa,
suggesting that r-hZP3 induced acrosomal exocytosis operates through a Gi
protein mediated pathway. Strategies
to obtain immunogens with ability to inhibit fertility and provide protection
against rabies infection in canines 1. Recombinant canine zona pellucida
glycoproteins and rabies glycoprotein–G Previously,
we have reported the expression in baculovirus of recombinant dog zona
pellucida glycoprotein-2 (r-dZP2), dog zona pellucida glycoprotein-3 (r-dZP3)
and rabies glycoprotein-G (r-rG) as polyhistidine tagged fusion proteins. In
addition, we have also reported earlier the cloning of a chimeric cDNA
construct encoding fusion recombinant protein encompassing dZP3 and rG
(r-dZP3-rG) in the baculovirus expression system. The above glycoproteins were
purified using Ni-NTA resin under denaturing conditions. 2. Active immunization studies (a)
Rodents (i)
Immunogenicity: To
check the immunogenicity of the recombinant proteins, groups of female mice (Balb/cJ;
5 animals/group) were immunized, either with renatured or denatured form (4 M
urea) of r-rG and r-dZP3-rG. In addition, another group of mice was immunized
with r-dZP3 conjugated to diphtheria toxoid (DT) [r-dZP3-DT]. The mice were
immunized subcutaneously with these recombinant proteins in CFA/IFA
(equivalent to 50 µg protein/mouse/injection). To determine the efficacy of
the adjuvant to generate antibody response, a group of mice was also immunized
with denatured r-dZP3-rG fusion protein (equivalent to 50 µg
protein/mouse/injection) in various adjuvants such as CFA/IFA, TiterMax or
Squalene: Arlacel-A (4:1 ratio with 100 µg SPLPS/mouse only in the first
injection). Two booster injections of 50 µg protein/mouse/injection with
appropriate adjuvants were administered intraperitoneally at 30 days interval.
Mice were bled at day 15 after the second and third injection. ELISA
experiments showed that the immunization of mice with both the forms of r-rG
(refolded and denatured), resulted in the generation of anti-r-rG antibodies.
The antibody response generated in mice as a result of immunization with
renatured r-dZP3-rG showed weaker anti-r-rG antibody response as compared to
anti-r-dZP3 antibody response. A comparison of the antibody response among the
different groups of mice immunized with r-dZP3-rG (denatured, in 4 M urea),
revealed that the highest antibody response was generated with the Squalene:
Arlacel-A group and lowest antibody response was observed with TiterMax group.
The mice immunized with r-dZP3-DT conjugate generated antibodies against
r-dZP3 as well as DT. However, the antibody titers generated against DT were
higher than the antibody titers generated against r-dZP3. (ii)
Rabies virus neutralizing antibody (RVNA) titers:
The ability of the mouse anti-r-rG antibodies thus generated to neutralize the
rabies virus in vitro was tested by Rapid Fluorescence Focus Inhibition
Test (RFFIT). The protective RVNA responses were observed in the mice sera
immunized with denatured rG at day 45 as well as day 75. None of the mice
sera, immunized with renatured r-rG or r-dZP3-rG, could generate a protective
RVNA response. A comparative study for the RVNA titers among different groups
of mice immunized with denatured r-dZP3-rG showed that the highest geometric
mean RVNA titers were observed with the mice sera immunized with denatured
r-dZP3-rG in Squalene:Arlacel-A group whereas, the mice sera immunized with
denatured r-dZP3-rG in TiterMax generated the lowest mean RVNA titers. (iii)
Reactivity of immune sera with native zona pellucida:
The ability of mouse polyclonal antibodies, generated subsequent to
immunization with r-dZP3-rG and r-dZP3-DT, to recognize native ZP was
performed by an indirect immunofluorescence assay on dog or bonnet monkey
ovarian cryosections. Anti-r-dZP3 antibodies showed positive fluorescence with
dog or bonnet monkey native zona pellucida. No fluorescence was observed when
pre-immune serum from the same immunized animal was used. The specificity of
the antibody reactivity with dog zona was further confirmed by inhibition of
the fluorescence in the presence of E.coli expressed r-dZP3. The immune
serum from mouse immunized with baculovirus expressed r-dZP3-rG fusion protein
also exhibited distinct fluorescence with native dog ZP, which was inhibited
in the presence of E.coli expressed r-dZP3. (b)
Female dogs In
light of the results obtained from mice experiments, active immunization
studies in female non-descript dogs were initiated to evaluate the feasibility
of developing immunization strategy for blocking fertility and to
simultaneously generate rabies virus neutralization antibodies. The female
dogs (n = 22) used for immunization were reared at the Central Military
Laboratory, Meerut Cantt. The animals were divided randomly into five groups.
The female dogs were immunized intramuscularly at two sites with r-dZP3-rG
(n=6), physical mixture of r-dZP3-DT and r-rG (n=5), physical mixture of
r-dZP2-DT and r-rG (n=5), r-rG (n=3) and DT (n=3) emulsified in Squalene and
Arlacel-A in 4:1 ratio. The dosage of respective recombinant protein was
equivalent to 250 µg protein/animal. SPLPS was also included in the first
injection as an additional adjuvant. Primary immunization of dogs comprised of
three injections of the respective protein at monthly intervals. Blood samples
were collected from the ante-cubital vein for determination of antibody titers
and progesterone concentrations. Immunization
of female dogs with the respective recombinant protein resulted in the
generation of anti-r-rG, anti-r-dZP3, anti-r-dZP2 and anti-DT antibodies as
determined by ELISA. An increase in the antibody response was observed in all
the animals after the booster injections. Preliminary data of the mating
experiments of the immunized female dogs suggests that immunization with
r-DZP3-DT block fertility. Immunization of female dogs with r-dZP2-DT failed
to block fertility. Y-chromosome
microdeletions in infertile male A
genetic factor located on the long arm of Y-chromosome has been postulated to
be an important factor for male germ cell development and is referred to as
“Azoospermia Factor” (AZF). In the AZF region, deletions in AZFa, AZFb,
and AZFc loci are linked with infertility. Our aim is to study microdeletion
in these regions in infertile males with non-obstructive oligospermia or
azoospermia. For this purpose, 13 sets of primers were made, each set
belonging to different Sequence Tagged Sites (STSs) of AZFa, AZFb, and AZFc
regions. For analysis of the AZF deletions, so far genomic DNA has been
isolated from 37 infertile and 4 fertile males attending the Out Patients
Department, AIIMS, New Delhi. PCR amplification with the respective set of
primers followed by analysis of amplicons by agarose gel electrophoresis,
revealed Y chromosome microdeletions in 4 out of 37 infertile subjects. Two
subjects revealed deletion in sY86 STS region and one both in sY86 and sY127
STS regions. In one particular case (IF23), a very large deletion in the AZFc
region including sY153, sY157, sY158, sY254 and sY255 STSs was found. Six new
primers are in the process of standardization in addition to Multiplex PCRs.
Publications Original peer-reviewed articles 1.
Gahlay GK and Gupta SK (2003) Glycosylation of zona pellucida glycoprotein-3
is required for inducing acrosomal exocytosis in the bonnet monkey. Cell Mol
Biol 49:389-398. 2.
*Rath A, Batra D, Kaur R, Vrati S and Gupta SK (2003) Characterization of
immune response in mice to plasmid DNA encoding dog zona pellucida
glycoprotein-3. Vaccine 21:1913-1923 (*in press last year, since published). 3. *Sivapurapu N, Upadhyay A, Hasegawa A, Koyama K and Gupta SK (2003) Efficacy of antibodies against Escherichia coli expressed chimeric recombinant protein encompassing multiple epitopes of zona pellucida glycoproteins to inhibit in vitro human sperm-egg binding. Mol Reprod Dev 65:309-317 (*in press last year, since published).
Reviews/Proceedings 1.
Gupta SK, Srivastava N, Choudhury S, Rath A, Sivapurapu N, Gahlay GK and Batra
D (2004) Update on zona pellucida glycoproteins based contraceptive vaccine. J
Reprod Immunol (in press). 2.
Suraj K, Chakravarty S and Gupta SK Genital mucosal immunity against sexually
transmitted infections. In: Sexually transmitted infections (Eds: Kumar B and
Gupta S), Elsevier Science, New Delhi (in press). 3.
*Gupta SK (2003) Status of immunodiagnostic and immunocontraceptive vaccines
in India. Adv Biochem Engineer Biotech 85:181-214. (*in press last year, since
published). 4.
Gupta SK (2003) Monoclonal antibodies: Basics to therapeutics. In: Therapeutic
antibodies (Eds: Talwar GP and Sood OP), Ranbaxy Science Foundation, Ninth
Annual Symposium, New Delhi, 3-9. 5.
Gupta SK, Choudhury S, Srivastava N and Ravi C (2003) Zona pellucida
glycoproteins based immunocontraceptive vaccines: strategies for development
and their applications. Ind J Exp Biol 41:682-693. |