Testatin has been implicated in fetal testis development due to its restricted expression in pre-Sertoli cells immediately after the onset of Sry gene expression. However, testatin knockout mice showed normal testis development and fertility. We investigated the spatial and temporal expression pattern of the Cres/testatin subgroup of genes, including the novel gene Cstl1/Cres4, in fetal mouse gonads and in adult testis, epididymis and ovary. The genes are related to the family 2 cystatins of protease inhibitors. Using real-time PCR and in situ hybridization we could show that 4 subgroup genes, testatin, CstSC, CstTE-1/Cres3 and Cres are expressed in fetal testis. We also confirmed the expression of testatin, CstE2, CstSC, CstTE-1/Cr es3, Cres, CstT and Cstl1/Cres4 in adult testis and CstE2, CstTE-1/Cres3, Cres and CstE1/Cres2 in adult epididymis. In testatin knockout animals, the expression of CstE2 was heavily downregulated in adult testis, but not in adult epididymis, compared to wildtype controls. In conclusion, an explanation for the lack of phenotype in testatin knockout mice could be functional redundancy with another member of the Cres/testatin subgroup. The most likely candidate/s would be CstSC, CstTE-1/Cres3 or Cres as they are expressed in the fetal testicular tubules in early testis differentiation together with testatin.

Keywords:
Cystatin type 2 family, Gonad development, Testatin knockout mouse
1.
Abrahamson M, Barrett AJ, Salvesen G, Grubb A: Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids. J Biol Chem 261:11282–11289 (1986).
2.
Barrett AJ, Fritz H, Grubb A, Isemura S, Jarvinen M, et al: Nomenclature and classification of the proteins homologous with the cysteine-proteinase inhibitor chicken cystatin. Biochem J 236:312 (1986).
3.
Brennan J, Capel B: One tissue, two fates: molecular genetic events that underlie testis versus ovary development. Nat Rev Genet 5:509–521 (2004).
4.
Canaff L, Bennett HP, Hou Y, Seidah NG, Hendy GN: Proparathyroid hormone processing by the proprotein convertase-7: comparison with furin and assessment of modulation of parathyroid convertase messenger ribonucleic acid levels by calcium and 1,25-dihydroxyvitamin D3. Endocrinology 140:3633–3642 (1999).
5.
Cornwall GA, Hann SR: Transient appearance of CRES protein during spermatogenesis and caput epididymal sperm maturation. Mol Reprod Dev 41:37–46 (1995).
6.
Cornwall GA, Hsia N: A new subgroup of the family 2 cystatins. Mol Cell Endocrinol 200:1–8 (2003).
7.
Cornwall GA, Orgebin-Crist MC, Hann SR: The CRES gene: a unique testis-regulated gene related to the cystatin family is highly restricted in its expression to the proximal region of the mouse epididymis. Mol Endocrinol 6:1653–1664 (1992).
8.
Cornwall GA, Cameron A, Lindberg I, Hardy DM, Cormier N, Hsia N: The cystatin-related epididymal spermatogenic protein inhibits the serine protease prohormone convertase 2. Endocrinology 144:901–908 (2003).
9.
Hacker A, Capel B, Goodfellow B, Lovell-Badge R: Expression of Sry, the mouse sex determining gene. Development 121:1603–1614 (1995).
10.
Hamil KG, Liu Q, Sivashanmugam P, Yenugu S, Soundararajan R, et al: Cystatin 11: a new member of the cystatin type 2 family. Endocrinology 143:2787–2796 (2002).
11.
Hasegawa K, Chuma S, Tada T, Sakurai T, Tamura M, et al: Testatin transgenic and knockout mice exhibit normal sex-differentiation. Biochem Biophys Res Commun 341:369–375 (2006).
12.
Hsia N, Cornwall GA: Cres2 and Cres3: new members of the cystatin-related epididymal spermatogenic subgroup of family 2 cystatins. Endocrinology 144:909–915 (2003).
13.
Huh C, Nagle JW, Kozak CA, Abrahamson M, Karlsson S: Structural organization, expression and chromosomal mapping of the mouse cystatin-C-encoding gene (Cst3). Gene 152:221–226 (1995).
14.
Kanno Y, Tamura M, Chuma S, Sakura T, Machida T, Nakatsuji N: A cystatin-related gene, testatin/cresp, shows male-specific expression in germ and somatic cells from the initial stage of murine gonadal sex-differentiation. Int J Dev Biol 43:777–784 (1999).
15.
Li Y, Friel PJ, Robinson MO, McLean DJ, Griswold MD: Identification and characterization of testis- and epididymis-specific genes: cystatin SC and cystatin TE-1. Biol Reprod 67:1872–1880 (2002).
16.
Li Y, Friel PJ, McLean DJ, Griswold MD: Cystatin E1 and E2, new members of male reproductive tract subgroup within cystatin type 2 family. Biol Reprod 69:489–500 (2003).
17.
Mruk D, Zhu LJ, Silvestrini B, Lee WM, Cheng CY: Interactions of proteases and protease inhibitors in Sertoli-germ cell cocultures preceding the formation of specialized Sertoli-germ cell junctions in vitro. J Androl 18:612–622 (1997).
18.
Sharpe RM, McKinnell C, Kivlin C, Fisher JS: Proliferation and functional maturation of Sertoli cells, and their relevance to disorders of testis function in adulthood. Reproduction 125:769–784 (2003).
19.
Shoemaker K, Holloway JL, Whitmore TE, Maurer M, Feldhaus AL: Molecular cloning, chromosome mapping and characterization of a testis-specific cystatin-like cDNA, cystatin T. Gene 245:103–108 (2000).
20.
Sutton HG, Fusco A, Cornwall GA: Cystatin- related epididymal spermatogenic protein colocalizes with luteinizing hormone-beta protein in mouse anterior pituitary gonadotropes. Endocrinology 140:2721–2732 (1999).
21.
Syntin P, Cornwall GA: Immunolocalization of CRES (Cystatin-related epididymal spermatogenic) protein in the acrosomes of mouse spermatozoa. Biol Reprod 60:1542–1552 (1999).
22.
Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680 (1994).
23.
Tohonen V, Osterlund C, Nordqvist K: Testatin: a cystatin-related gene expressed during early testis development. Proc Natl Acad Sci USA 95:14208–14213 (1998).
24.
Tohonen V, Frygelius J, Mohammadieh M, Kvist U, Pelliniemi LJ, et al: Normal sexual development and fertility in testatin knockout mice. Mol Cell Biol 25:4892–4902 (2005).
25.
Turk V, Bode W: The cystatins: protein inhibitors of cysteine proteinases. FEBS Lett 285:213–219 (1991).
26.
Wassler M, Syntin P, Sutton-Walsh HG, Hsia N, Hardy DM, Cornwall GA: Identification and characterization of cystatin-related epididymal spermatogenic protein in human spermatozoa: localization in the equatorial segment. Biol Reprod 67:795–803 (2002).
27.
Yanagimachi R: Fertility of mammalian spermatozoa: its development and relativity. Zygote 2:371–372 (1994).
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2008
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