Identification and functional charecterization of Prostate and Testis expressed (Pate) genes in the rat

Abstract

Sperm maturation is a complex process that occurs in the epididymis and thought to be facilitated by a wide variety of proteins. Among them, the cysteine rich proteins that belong to different families, such as defensins, cystatins, cysteine rich secretory proteins, whey acidic proteins, etc. Recently a new class of cysteine rich proteins that belong to prostate and testis expressed (PATE) family were identified. The PATE proteins identified till date are thought to resemble the three fingered protein/urokinase-type plasminogen activator receptor proteins and are not well characterized in many species including rats. In this study, we report the identification and characterization of Pate genes in the rat. The rat Pate genes (Pate-P, Pate-Q, Pate- F, Pate-A, Pate-C, Pate-E, Pate-N, Pate, Pate-Dj, Pate-B) are clustered on chromosome 8 and their corresponding predicted proteins retained the ten cysteine signature characteristic to TFP/Ly-6 protein family. Though Pate gene expression is thought to be prostate and testis specific, we observed that the rat Pate genes are also expressed in seminal vesicle and epididymis and in tissues beyond the male reproductive tract. In the developing rats (20 – 60 day old), expression of Pate genes seem to be androgen dependent in the epididymis and testis. In the adult rats, androgen ablation resulted in down regulation of the Pate genes in the epididymides. PATE and PATE-F proteins were found to be expressed abundantly in the epididymis and on the sperm. In this study, for the first time, we report that rearrangement of PATE and PATE-F proteins occur on the rat sperm head during capacitation. In vitro neutralization of PATE and PATE-F on the rat sperm using specific antiserum resulted in significant reduction of capacitation and acrosome reaction. To further understand the role of PATE and PATE-F proteins in sperm function, rats were immunized with Abstract iii these proteins to achieve neutralization of these proteins endogenously. In the immunized rats, a very high antibody titer was observed in the serum, epididymal and testicular fluids. Spermatozoa obtained from the PATE-F immunized rats failed to undergo capacitation and acrosome reaction, where as their counterparts obtained from PATE immunized animals displayed these properties. Concomitant with the loss of sperm function, PATE-F animals when mated with females produced reduced litter size. Basing on the results obtained using immunization strategies, the role of these proteins was further examined by knocking down the mRNA expression of Pate in the cauda. Spermatozoa obtained from these cauda failed to undergo capacitation and acrosome reaction. Further, infertility was observed in rats when Pate was knock down in cauda. These results suggest that PATE proteins may have role in sperm function and thus fertility. Besides role in sperm function, their contribution to immune function was analysed. PATE exhibited potent antibacterial activity and was inducible during LPS challenge both in vitro and in vivo. PATE-F on the other hand was not antibacterial. In conclusion, we report that PATE and PATE-F proteins in the rat contribute to sperm function innate immunity as well.