Principal Investigator :    Anil K Suri

Project Associates/Assistants
Shalini Sen
P John Leema Rose
Shikha Yadav
Taran Jeet Saluja

Collaborators
John C Herr, Univ Virginia, USA

Mammalian spermatogenesis involves differential expression of many testis specific genes. The precise regulation of germ cells differentiation requires a strict program of stage and cell specific gene expression in germ cells. During this process, many germ cell specific genes excluding housekeeping genes are differentially expressed with respect to space and time. This project’s goal is to investigate testis specific genes expressed in a restricted, temporal and spatial manner during spermatogenesis.

Morphological and biochemical observations along the molecular biological approaches suggest that germ cell differentiation is accompanied by selective gene expression. However, the molecular mechanisms regulating the cell type specifically gene expression during spermatogenesis is yet to be determined. Identification of testis specific genes and their characterization would thus enhance our understanding about the functioning of sperm at the molecular level. This would possibly highlight the mechanism of sperm development, maturation and sperm-egg interaction.

Cloning, expression and purification of HSS recombinant protein: Immunogenicity studies with recombinant HSS protein

The recombinant HSS protein was successfully expressed and purified as reported earlier. The recombinant protein was subjected to mass spectrometry for confirming the sequence of protein. It was confirmed that HSS deduced amino acid was infact expressed. Immunogenicity studies were carried out in rats, rabbits and monkeys. In rats and rabbits, subcutaneous immunizations were performed with 10 micrograms and 50 micrograms doses respectively. Three injection schedule at 0, 30 and 60 days were conducted. Zero day immunization was done using Freund’s complete adjuvant and subsequent immunizations were done using incomplete Freund’s adjuvant. In bonnet monkeys recombinant protein was adsorbed on alum and was used for immunizations. Intramuscularly immunizations were carried out with 500 microgram dose on three injection schedule at 0, 30, and 60 days. The first immunization was performed using recombinant protein adsorbed on alum along with 1 mg of adjuvant sodium pathalyl lippopolysaccharide (SPLPS) in order to enhance the humoral response. Subsequent booster immunizations were done using alum alone. The antibodies thus generated against recombinant protein were subjected to various functional assays and immunolocalization by flow cytometery analysis, indirect immunofluorescence and electron microscopy. The functional assays revealed that HSS protein was present on the surface of the sperm. Flow cytometery and indirect immunofluorescence revealed the localization on the sperm head in the acrosomal compartment. Electron microscopy studies revealed clearly the presence of HSS protein on the sperm surface on the acrosomal compartment.

Transfection studies with hss gene

Transfection studies were carried out using hss gene. Three constructs were prepared:

Construct A will be having complete ORF: LZ, A1-A3, T, and C

Construct C will be having LZ, A1-A3, DT and DC

Construct D will be having only A1-A3, DLZ, DT and DC

All these constructs were cloned in pcDNA 3.1/Myc-His A eukaryotic expression vector for transfection assay. Transfection assays showed that construct A protein was localized on to the surface of the cell. This study supported the role of leucine zipper motif in directing the protein to the surface of the cell.

Genomic structural organization of hss gene

It was earlier reported that we have cloned a human sperm surface gene designated as hss gene, which encodes for a protein having Leuzine zipper (LZ), antigenic determinant sites (A1-A3), transmembrane domain (T) and cytosolic domain (C). Tissue distribution of hss gene was studied using different tissue mRNA which revealed that hss gene was exclusively expressed in testis only. Expression of hss transcript was observed only in round spermatids indicating post meiotic haploid gene expression. Human genomic leucocytic library was screened using hss cDNA. Genomic clone was successfully isolated and was sequenced. Sequence analysis revealed the hss gene has 17 exons (with introns-exons consensus sequence). The sequence analysis also revealed that hss gene did not harbor CAATT or TATA box (TATAAT box) in 5’ upstream sequences. Fluorescent in situ hybridization to normal human male metephase chromosome was carried out. Assessment of well spread metaphase chromosomes indicated signals of hss gene on both homologous chromosome 17, mapping long arm of chromosome 17 at band 17q 21.3-q 22.

Studies on post-transcriptional regulation of human hss gene during spermatogenesis

Cloning and characterization of RNA binding protein involved in binding to palindromic motifs of hss gene involved in post-transcriptional control were carried out. The cDNA testis library was screened to fish out the clone of our interest. The clones are being characterized. Protein interacting with our unique motifs was identified and was cored from SDS-PAGE gel for amino acid sequencing by mass spectrometry for the further studies.