Genes and proteins expressed specifically in testis

 
Principal Investigator :  Anil K Suri

Project Associates / Assistants
 Shalini Sen (till Jan 2003)
Shikha Yadav (till Sep 2002)
Vidhisha Tripathi (till Sep 2002)
Taran Jeet Saluja (till Sep 2002)
Bharkha Bhatnagar
Boohmika Rastongi

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 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, sperm functioning, and sperm-egg interaction.

Genomic structural organization and chromosomal mapping of A-kinase (PKRA) anchoring protein 4 (earlier called hi;AKAP 4, EMBL nomenclature) gene

Mammalian sperm motility is regulated by a cascade of cAMP-dependent protein phosphorylation events mediated by protein kinase A. A-kinase anchor proteins direct protein kinase A activity by tethering the enzyme near its physiologic substrates. The A-kinase anchor proteins (AKAPs) are a group of structurally diverse proteins, which have the common function of binding to the regulatory subunit of protein kinase A (PKA) and confining the holoenzyme to discrete locations within the cell. AKAP may be involved in the regulation of sperm motility.

In the course of identifying sperm proteins, we cloned the human hi mRNA from a testis cDNA expression library. This is referred as AKAP4 [A-kinase (PKRA) anchoring protein 4, EMBL nomenclature]. Northern blot analysis detected a 3.0-kb AKAP4 transcript exclusively expressed in the testis. The 860-amino acid polypeptide contains a secretory signal sequence, 5 glycosylation sites, 7 myristoylation sites, and numerous phosphorylation sites. The AKAP4 protein, has structural homology to other members of the AKAP family. In situ hybridization studies of human testis sections revealed that the AKAP4 protein is transcribed postmeiotically in round spermatids. Genomic sequence analysis of AKAP4 gene revealed that the gene has six exons (with introns-exons consensus sequence). Fluorescent in situ hybridization to normal human male metaphase chromosome was carried out. Assessment of well spread metaphase chromosomes indicated signals of AKAP4 gene on chromosome X, mapping on short arm of chromosome X at band p 11.2. It was further supported by the NCBI mapping view, localizing AKAP4 gene on chromosome X.

Transfection studies with AKAP4 gene

Transfection studies were carried out using AKAP4 constructs. Four constructs were prepared:

  1. Construct I having complete ORF, including Eukaryotic secretary signal (ESS), Target domain (T) and Regulatory domain (R).

  2. Construct II having target domain (T) and Regulatory domain (R), D ESS.

  3. Construct III having Regulatory domain only, D ESS, DTarget domain.

  4. Construct IV having Target domain only, D ESS and D Regulatory domain

Construct I was successfully expressed in mammalian cells. The protein assembly was localized in cytoplasm and anchored into the plasma membrane. Construct II showed cytoplasmic localization. Attempts are being made to transfect construct III and construct IV.

Characterization of SPAG9 (Sperm associated sperm antigen 9)

During this reporting year the recombinant protein was successfully expressed and purified. The recombinant protein was subjected to Mass spectrometery for confirming the sequence of SPAG9 protein. The predicted secondary structure of SPAG9 revealed that this protein is alpha helical in nature. Protein secondary structure is being studied using far UV-CD spectra.

Using Hampton Research crystal screen kits, recombinant SPAG9 was subjected to various condition to obtain crystals. The experiments were set up at room temperature and cold room. Various SPAG9 protein concentrations ranging from 7.5 mg/ml to 20 mg/ml were tried. Initial experiments revealed formation of crystals. The next step was to optimize the preliminary conditions, which will produce crystals and improve crystal size and quality, which will be used for diffraction. The experiments are being carried out.

Panel of infertile male semen samples were screened for the presence of SPAG9 protein on sperm. The study revealed that in 24-semen sample, SPAG9 protein was localized but intensity of localization varied among the semen samples. However it was also noted that 16 of the sperm sample did not show reactivity of SPAG9 indicating the absence of SPAG9 protein. The semen sample from fertile donor tested (110 samples) so far demonstrated the presence of SPAG9 protein.

Considering that the data indicated cytoplasmic and plasma membrane localization of SPAG9, further characterization of SPAG9 molecule involvement in human intact oocyte sperm interaction is under way.

Screening of mouse genomic library was carried out using Spag9 cDNA probe. The 29 Kb mouse genomic clone was isolated successfully. Attempts are being made to make constructs with deleting exons for studying the gene-deleted mice.