Cystatin-related epididymal spermatogenic protein colocalizes with luteinizing hormone-beta protein in mouse anterior pituitary gonadotropes

The CRES (cystatin-related epididymal spermatogenic) protein, a member of t he cystatin superfamily of cysteine protease inhibitors, exhibits highly re stricted expression in the mouse testis and epididymis, suggesting roles in reproduction. Considering the well-established relationship that exists be tween the gonads and the neuroendocrine system, the present studies were un dertaken to determine whether the CRES messenger RNA and protein are expres sed in the anterior pituitary gland and, if so, whether the expression is r egulated by hormones. RT-PCR analysis of whole pituitary gland RNA preparat ions, and Northern blot analyses of pituitary gland cell lines, demonstrate d that the CRES gene is expressed in the male and female anterior pituitary gland gonadotropes. Furthermore, Western blot analysis demonstrated that C RES protein was present in whole mouse pituitary glands and was synthesized and secreted by the L beta T2 gonadotrope cell Line. Interestingly, wherea s the predominant CRES proteins present in epididymal lysates, L beta T2 se cretory granules, and whole pituitary gland lysates were 19 and 14 kDa, the predominant CRES proteins present in the cell culture conditioned media we re 17 and 12 kDa. Deglycosylation studies revealed that the higher-molecula r-mass CRES proteins (19 and 17 kDa) were the result of N-linked glycosylat ion, caused by the presence of high mannose residues. Double-label immunofl uorescence and confocal microscopic analysis of male and female mouse pitui tary gland tissue confirmed the RNA studies and showed that CRES protein co localized with LH beta protein in the gonadotropes. Finally, gonadectomy an d hormone replacement studies suggest that CRES protein in the gonadotropes is hormonally regulated. These studies suggest that CRES protein may perfo rm a role in the gonadotrope-mediated control of reproduction.