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Molecular characterization of zona
pellucida glycoproteins: role in fertilization and regulation of fertility |
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Principal Investigator : Satish K Gupta Project Associates/Assistants Ph D Students Collaborators Major
themes of this group are i) molecular and biochemical characterization of zona
pellucida (ZP) glycoproteins and delineation of their role during
fertilization and ii) evaluation of the immunocontraceptive potential of ZP
based immunogens. Binding
characteristics of recombinant bonnet monkey (bm) ZP proteins to spermatozoa During the year of report, the binding of the purified
r-bmZP1, r-bmZP2, and r-bmZP3, expressed in E.coli, to the capacitated and
acrosome reacted spermatozoa was assessed by indirect immunofluorescence and
also by the direct binding of the respective biotinylated recombinant protein.
Simultaneously, the acrosomal status of the spermatozoa was assessed by the
binding pattern of tetramethylrhodamine isothyiocynate (TRITC)-conjugated
Pisum sativum in double labeling experiments. The binding of the r-bmZP1 was
localized to the principal segment of the acrosomal cap of the capacitated
spermatozoa whereas a shift in the binding to the equatorial segment,
postacrosomal domain and midpiece region was observed in acrosome-reacted
spermatozoa. The binding of the biotinylated r-bmZP1 to the capacitated sperm
was inhibited by the cold r-bmZP1 as well as by the monoclonal and polyclonal
antibodies generated against r-bmZP1. The r-bmZP2 binds to the principal
segment of capacitated spermatozoa. However, in the acrosome reacted
spermatozoa the binding of r-bmZP2 was observed to the equatorial segment, tip
of the inner acrosomal membrane, and midpiece. The r-bmZP3 binds to the head
region of capacitated sperm, which was lost subsequent to acrosome reaction.
These results suggest, for the first time, that polypeptide backbone may be
sufficient for binding of ZP proteins to spermatozoa and ZP1, in contrast to
mice, may have a functional role during fertilization in non-human primates. Immunogenicity of ZP glycoproteins based synthetic
peptides In our quest to develop ZP glycoproteins based
synthetic peptide immunogens capable of eliciting antibodies reactive with
native ZP and inhibiting specifically gamete interaction, 9 peptides were made
during this year. Peptide sequences were selected on the basis of
computational analysis with respect to hydrophilicity, conservation of amino
acid (aa) sequence between bonnet monkey and human zona proteins, antigenicity
and putative glycosylation sites. Four peptides corresponding to bmZP1
(bmZP1A58-79, bmZP1B136-153, bmZP1C212-228 and bmZP1D248-273) and 4
corresponding to bmZP2 (bmZP2A85-109, bmZP2B103-125, bmZP2C394-406 and
bmZP2D532-549) were synthesized using standard F-moc chemistry. The synthetic
peptides were cleaved from resin and purified on C18 reverse phase HPLC column
and their authenticity confirmed by ESI-MS. All the 4 peptides corresponding
to bmZP1 were conjugated individually to DT at 10:1 molar ratio using
glutaraldehyde as coupling reagent. Four groups of female BALB/cJ mice were
immunized with the respective ZP1-peptide-DT conjugates. The antibodies
generated against bmZP1A58-79 and bmZP1D248-273 peptides recognized r-bmZP1 in
ELISA and bonnet monkey ZP in an indirect immunofluorescence assay. Antibodies
against bmZP1C212-228 peptide though reacted with the peptide but failed to
react with r-bmZP1 in an ELISA as well as native zona. Anti-bmZP1B136-153
peptide antibodies though reacted with r-bmZP1 in an ELISA but failed to react
with native zona. All the 4 peptides corresponding to bmZP2 have been purified
and conjugated with DT. Another synthetic peptide corresponding to bmZP3 (aa
residues 334-341) was synthesized co-linearly with the “promiscuous” T
cell epitope of circumsporozoite protein of Plasmodium falciparum. Female BALB/cJ
mice immunized with this synthetic peptide generated good anti-peptide
antibodies. Anti-peptide antibodies recognized both r-bmZP3 as well as native
ZP. Evaluation of in vivo contraceptive efficacy of
recombinant dog zona proteins in female dogs Last year, in collaboration with Central Military
Veterinary Laboratory, Meerut Cantt, India, we initiated the active
immunization studies in female stray dogs to evaluate the immunocontraceptive
efficacy of recombinant dog ZP3 (r-dZP3) and ZP2 (r-dZP2) expressed in E.coli.
Twelve female dogs in three groups (4 animals/group) were immunized with
r-dZP3-DT (equivalent to 250 mg r-dZP3 per animal), r-dZP2-DT (equivalent to
250 mg r-dZP2 per animal) and DT (equivalent to 250 mg DT per animal)
respectively, emulsified in squalene and arlacel in 4:1 ratio. Primary
immunization comprising of three injections at 4 weekly intervals led to the
generation of adequate antibody response against the respective recombinant
zona protein and carrier as analyzed by an ELISA. Animals were boosted at
varying intervals to maintain the antibody titers until the dogs came in heat.
Mating experiments were initiated when the animals came in estrous cycle
(heat), which was confirmed by increase in serum progesterone concentrations
as well as by vaginal cytology (presence of cornified epithelial cells).
During mating studies, two animals immunized with r-dZP3-DT (Dog 1 and Dog 5)
failed to conceive in presence of high anti-r-dZP3 antibody titers (>5.7 x
103 AU). Dog 3 and Dog 4 conceived when anti-r-dZP3 antibody titer at the time
of mating was 0.8 X103 AU and 1.0 X103 AU respectively. All the 4 female dogs,
immunized with r-dZP2-DT became pregnant in spite of high anti-r-dZP2 antibody
titers (ranged from 2.0 -10 X 103 AU) at the time of mating, suggesting
thereby that antibodies generated against r-dZP2 failed to block fertility. In
control group (dogs immunized with DT), 3 female dogs conceived subsequent to
mating during heat. The fourth female (Dog 8) failed to conceive, in spite of
successful mating, which may be due to the low serum progesterone levels (<
1.0 ng/ml) and absence of cornified epithelial cell in vaginal cytology. These
results, though preliminary, suggest the possibility of fertility regulation
in female dogs by immunization with r-dZP3. However, further active
immunization studies incorporating more potent adjuvants with glycosylated or
non-glycosylated r-dZP3 using large number of animals will be desirable for
final inference. Characterization of
the immune response to plasmid DNA encoding ZP glycoproteins and rabies
glycoprotein-G The success of DNA vaccine is influenced by many
factors, including expression levels of the cloned gene product subsequent to
DNA delivery. Following constructs were made to optimize expression of bmZP1: i) Cloning of the full
length bmZP1 cDNA, retaining the native signal sequence and C-terminal
transmembrane-like domain, in pCI-neo vector under CMV promoter, ii)
Cloning of bmZP1 cDNA without C-terminal transmembrane-like domain in
pCI-neo vector, and iii)
Cloning of bmZP1 cDNA without the native signal sequence and
transmembrane-like domain in VR1020 vector under TPA signal sequence. Lipofectamine-mediated in vitro transfection of
mammalian cell lines with the purified plasmid DNA followed by localization of
bmZP1 by indirect immunofluorescence assay showed better expression of bmZP1
cloned in VR1020 vector. Host cells used for transfection also influenced the
expression levels of bmZP1 as better expression was observed in COS-I cell
line as compared to HEK-293, CHO, UM449 or COS-7 cell lines. The cDNA
corresponding to rabies glycoprotein-G gene was also cloned in VR1020.
Localization studies further revealed that bmZP1 and rabies glycoprotein-G are
not present on the membrane of transfected cells but are localized in the
cytoplasm. Immunoprecipitation studies are in progress to check if these
proteins remain inside the cell or are secreted in to the medium. Publications Original peer-reviewed articles 1. *Govind CK and Gupta SK (2000) Failure of female baboons (Papio anubis) to conceive following immunization with recombinant non-human primate zona pellucida glycoprotein-B expressed in Escherichia coli. Vaccine 18:2970-2978 (*in press last year, since published). 2. Patra AK, Gahlay GK, Reddy BVV, Gupta SK and Panda AK (2000) Refolding, structural transition and spermatozoa-binding of recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-C expressed in Escherichia coli. Eur J Biochem 267:7075-7081. 3. Govind CK, Gahley GK, Choudhury S and Gupta SK (2001) Purified and refolded recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-B expressed in Escherichia coli binds to spermatozoa. Biol Reprod 64:1147-1152. 4. Talwar GP, Gupta R, Gupta SK, Malhotra R, Khanna R, Mitra DK, Sehgal S, Minz R and Kumar A (2001) A monoclonal antibody cytolytic to androgen independent DU145 and PC3 human prostatic carcinoma cells. Prostate 46:207-213. 5. Kaul R, Sivapurapu N, Afzalpurkar A, Srikant V, Govind CK and Gupta SK (2001) Immunocontraceptive potential of recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-C expressed in Escherichia coli and its corresponding synthetic peptide. Reproductive BioMedicine Online (in press). 6. Gahlay GK, Srivastava N, Govind, CK and Gupta SK (2001) Primate recombinant zona pellucida proteins expressed in Escherichia coli binds to spermatozoa. J Exp Clin Reprod Immunobiol (in press). Reviews/Proceedings 1. *Gupta SK, Srivastava N, Govind CK, Sivapurpu N and Gahlay GK (2000) Comparative molecular biology and immunobiology of zona pellucida glycoproteins: Fundamental and applied aspects for contraception. Proc Indian Natl Sci Acad -B 66:33-48 (*in press last year, since published). 2. N, Upadhyay A, Rath A, Chaudhury S and Gupta SK (2001) Prospects of contraceptive vaccines for females. In: Current status in fertility regulation: Indigenous and modern approaches (Eds: Chowdhury SR, Gupta CM and Kamboj VP), Central Drug Research Institute, Lucknow, 239-253. |