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HOXBES2: A Novel Epididymal HOXB2 Homeoprotein and Its Domain-Specific Association with Spermatozoa¹

Departments of Biochemistry³ and Structural Biology,⁴ National Institute for Research in Reproductive Health (NIRRH), Indian Council of Medical Research, Parel 400 012, Mumbai, India

ABSTRACT

The sperm from the testis acquires complete fertilizing ability and forward progressive motility following its transit through the epididymis. Acquisition of these characteristics results from the modification of the sperm proteome following interactions with epididymal secretions. In our attempts to identify epididymis-specific sperm plasma membrane proteins, a partial 2.83-kb clone was identified by immunoscreening a monkey epididymal cDNA library with an agglutinating monoclonal antibody raised against washed human spermatozoa. The sequence of the 2.83-kb clone exhibited homology to the region between 1 and 1097 bp of the homeobox gene, Hoxb2. This sequence was found to be species conserved, as revealed by RT-PCR analysis. To obtain a full-length clone of the sequence, 5' RACE-PCR (rapid amplification of cDNA ends PCR) was carried out using rat epididymal RNA as the template. It resulted in a full-length 1.657-kb cDNA encoding a 32.9-kDa putative protein. The protein designated HOXBES2 exhibited homology to the conserved 61-amino acid homeodomain region of the HOXB2 homeoprotein. However, characteristic differences were noted in its amino and carboxyl termini compared with HOXB2. A putative 30-kDa protein was detected in the tissue extracts from adult rat epididymis and caudal spermatozoa, and a 37-kDa protein was detected in the rat embryo when probed with a polyclonal antibody against HOXB2 protein. Multiple tissue Western blot and immunohistochemical analysis further indicated its expression in the cytoplasm of the principal and basal epithelial cells, with maximal expression in the distal epididymal segments. Northern blot analysis detected a single ~2.5-kb transcript from the adult epididymis. Indirect immunofluorescence localized the protein to the acrosome, midpiece, and equatorial segments of rat caudal and ejaculated human and monkey spermatozoa, respectively. In conclusion, we have identified and characterized a novel epididymal homeoprotein different from HOXB2 protein and hereafter referred to as HOXBES2, (HOXB2 homeodomain containing epididymis-specific sperm protein) with a probable role in fertilization.

epididymis, gene regulation, sperm, sperm maturation, testosterone

¹This work (NIRRH/MS/7/2006) was supported by grants from the Indian Council of Medical Research (ICMR). E.P. is a recipient of the ICMR Senior Research Fellowship.

Correspondence: ²Vijaya P. Raghavan, Department of Biochemistry, National Institute for Research in Reproductive Health (ICMR), J.M. Street, Parel 400 012, Mumbai, India. FAX: 91 22 2413 9412; e-mail: vijuraghavan{at}hotmail.com

This article has been cited by other articles:

				E. Prabagaran, U. C. Hegde, S. B. Moodbidri, A. H. Bandivdekar, and V. P. Raghavan Postnatal Expression and Androgen Regulation of HOXBES2 Homeoprotein in Rat Epididymis J Androl, September 1, 2007; 28(5): 755 - 771. [Abstract] [Full Text] [PDF]