Discovery in Silico and Characterization in Vitro of Novel Genes Exclusively Expressed in the Mouse Epididymis

Abstract

Epididymal proteins interact with sperm during their passage through the epididymis and thus contribute to the maturation and fertilizing capacity of the spermatozoa. In the present study we have discovered five novel epididymis-specific genes through in silico analysis of expressed sequence tags (ESTs) at the UniGene library collection. The strategy used is a powerful way to discover novel epididymis-specific genes. The full-length cDNA sequences were determined, and computational tools were used to characterize the genomic structures and to predict putative functions for the encoded proteins. In vitro analyses revealed that all five genes characterized were highly expressed in the defined areas of the epididymis, and they were not expressed at significant levels in any other tissue. Three of the genes were named on the basis of their putative functions: Spint4 (serine protease inhibitor, Kunitz type 4), and Rnase9 and Rnase10 (ribonuclease, Rnase A family 9 and 10), while for the ESTs AV381130 and AV381126 no putative functions could be predicted. The expression of Spint4, Rnase9, and AV381130 was found to be under a direct or indirect regulation by androgens, while the expression of Rnase10 is regulated by a testicular factor(s) other than androgen. None of the genes were expressed in the immature epididymis, while mRNAs were detected from d 17 onward, at the time of maturation of epididymal epithelium. However, the expression of AV381130 was not detected until d 30 after birth, indicating a close connection between gene expression and puberty.

Human ribonuclease 9, a member of ribonuclease A superfamily, specifically expressed in epididymis, is a novel sperm-binding protein

“…We show here that human RNase 9 was expressed uniquely in the epididymis while remaining undetectable in twelve other tissues examined. It is consistent with the expression of rhesus monkey Esc461 [22] (homolog of human RNase 9) and mouse RNase 9 [13]. This indicates that human RNase 9 may have functions related to male reproduction system.…”

“…Asian Journal of Andrology | http://www.asiaandro.com; aja@sibs.ac.cn npg members (human RNases 9-13) [11][12][13] of the RNase A superfamily, forming a cluster of ~550 kb. Figure 6 shows the amino acid sequence alignment of human RNases 1-13.…”

“…They also exhibit a divergent physiological functions, including degradation of dietary RNA in the digestive gut (RNase 1) [6], angiogenesis (RNase 5) [7], and innate immunity (RNases 2, 3, 7) [8,9,10]. With the discovery of RNases-9, -10, -11, -12 and -13 [11][12][13], these requirements have been somewhat relaxed because these genes are clearly the derivatives of the RNase A lineage based on sequence similarity and the presence of the characteristic disulfide bridges, but they lack the active site signature motif and, as such, are unlikely to have ribonucleolytic activity.…”

“…Penttinen et al [13] reported the in silico discovery of mouse RNase 9, a new member of the RNase A superfamily, exclusively expressed in epididymis; nevertheless, the function of mouse RNase 9 remains unknown. With the completion of the human genome sequence, human RNase 9 was identified in the human genome sequence by computational search.…”

Characterisation of Lyzls in mice and antibacterial properties of human LYZL6

“…[17][18][19][20] There are probably four types of regulation: (i) positive androgen control: the mRNAs decrease dramatically following bilateral castration and then recover to pre-orchidectomy levels following the administration of testosterone; 21 (ii) negative androgen control: the mRNA levels increase following castration with a subsequent decrease in mRNA following testosterone replacement; 22 (iii) testicular factor regulation: the mRNAs decrease dramatically following castration, but remain low to undetectable following testosterone supplementation; 23 (iv) both androgen and testicular factor regulation: the mRNA levels decrease to undetectable following castration and then recover incompletely after androgen supplementation. 24 On the basis of the castration experiment, in the present study, we presume that Lyzl1, Lyzl3 and Lyzl6 mRNA expression belongs to testicular factor regulation, since there is a postcastration decline and no response to testosterone.…”