Date of Award

Summer 1995

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program/Concentration

Biomedical Sciences

Committee Director

Sergio Oehninger

Committee Member

Frank J. Castora

Committee Member

James H. Yuan

Committee Member

Ke-wen Dong

Committee Member

Mark S. Elliot

Abstract

Human zona pellucida protein 3 (hZP3) is the putative receptor on the zona pellucida of the mature oocyte that recognizes and binds sperm, and therefore plays a critical role in fertilization.

A full length cDNA of hZP3 (1278 bp) was amplified from the human ovary mRNA by reverse transcription-polymerase chain reaction (RT-PCR). The hZP3 cDNA was subcloned into PSK and pREP4 expression vectors. The cDNA of hZP3 was further characterized by restriction mapping, PCR, auto-sequencing and Southern blot analysis by using an internal oligonucleotide probe, and found to be identical to the one reported by J. Dean. Using autosequencing, 289 bases were identified and more than 98% homology was found at the 3'-end with the reported sequence, whereas, 224 bases were identified and more than 91% homology was found at the $5\sp\prime$-end. In vitro translation was performed to ensure the capacity of the cloned cDNA for producing a full length, non-glycosylated hZP3 protein (47 kDa). Stable transfection of a human ovarian tumor cell line, PA-1, was produced by introduction of the hZP3/pREP4 expression vector construct using the calcium phosphate precipitation method. Approximately 200 single colonies of the stable-transfected cells were isolated by the cloning-ring method. Direct PCR, RT-PCR, and ELISA were used to identify and select clones; ten of the isolated clones were found to be positive.

Two of the ten selected clones (SKC-P3-12 and SKC-P3-19) were used for further studies. Culture medium was collected from transfected and non-transfected cells (negative control), and the samples were purified by wheat germ agglutinin (WGA)-lectin chromatography and anti-WGA immunoaffinity chromatography; the final eluted fraction was concentrated by ultrafiltration with a limiting molecular weight cut off at 30 kDa. The secreted glycoproteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining, Western blot analysis using a specific anti-ZP3 antibody, and by a metabolic labelling study using 3H-galactose. Several bands including a band at 65 kDa, were observed in both non-transfected and transfected medium by silver staining. Distinct bands were identified by Western blot analysis at 65 kDa and 100 kDa for the recombinant product; solubilized human zonae pellucidae demonstrated a molecular mass of native hZP3 at approximately 65 kDa, and was at the same position identified for the recombinant product. In the metabolic study, several distinct bands including the band at 65 kDa, were identified as biosynthesized glycosylated recombinant proteins.

In the hemizona assay (a test for sperm-zona pellucida binding), a significant inhibition (>$40%) of binding was observed in competition studies using a 1:4 dilution of the WGA-purified recombinant glycoprotein solution. This material did not affect sperm viability or motility, and induced a more than 50% increase in the acrosome reaction of sperm after overnight incubation.

Comments

Dissertation submitted to the Faculty of Eastern Virginia Medical School and Old Dominion University in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biomedical Sciences.

DOI

10.25777/x546-tp65

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