The Testatin/Cres subgroup of family 2 cystatins in sexual development

The genetic sex in mammals is established at fertilization, where an XX chromosome complement will lead to female development and an XY genotype will result in male development. Thereafter, sex determination is initiated with expression of the Y-linked factor Sry in XY gonads, leading to testis formation, while in the absence of Sry expression an ovary is formed. A number of genes, both transcription factors and signaling molecules such as Sox9, Fgf9, Dhh, Dax1 and Wnt4 have been identified downstream of Sry. Disruption of these genes causes sex reversal and/or impaired gonad development in humans and/or mouse models. However, it is clear that key factors in gonad differentiation remain to be identified. Finding new genes would not only clarify basic mechanisms behind gonad development but could also improve diagnostics in patients with disorders of sex development.

Testatin was previously isolated by our group in a screen searching for novel genes expressed in early mouse sex differentiation. Testatin is specifically up regulated in the developing testis just after expression of Sry. Testatin belongs to the Testatin/Cres subgroup of cystatin family 2 protease inhibitors that show a reproductive tract restricted expression (testis, epididymis, ovary, pituitary) in contrast to the broad expression profiles of classical family 2 cystatins, implying specialized functions in reproduction.

To evaluate the role of Testatin in male sexual development, we generated a Testatin knockout mouse. Detailed phenotyping revealed normal testis development and fertility in male Testatin knockout animals (Paper I). An explanation for the lack of phenotype in the knockout mice could be functional redundancy between the subgroup members. Therefore, we evaluated the expression profiles of the Testatin/Cres subgroup genes in fetal testis using real-time PCR and in situ hybridization. We show that three of the subgroup members, namely Cres, cystatin SC, Cystatin TE-1, are expressed in mouse fetal testis together with Testatin (Paper II).

Ancestors of the Cystatins can be traced back to plants and to understand when and why the Testatin/Cres subgroup genes emerged, we performed an evolutionary study (Paper III). We have localized the evolutionary origin of the Testatin/Cres subgroup genes to the split between Marsupials and placental mammals and a model for the evolution of these genes illustrates that they constitute a dynamic group of genes, which has undergone several gene expansions. Furter, indications of a high degree of positive selection, in striking contrast to what is seen for the classical Cystatin C was found. We suggest a new nomenclature for the Testatin/Cres subgroup (TCS) genes, based on their syntenic gene location on mouse chromosome 2 and show with phylogenetic relations that the TCS genes are clustered into three original groups, a testatin (TCS7), a Cres (TCS5) and a CstL1 (TCS1) group.

We also evaluated the expression patterns of all human members of the subfamily (Paper III). Of a total of nine identified human genes, four express putative functional transcripts with a predominant expression in the male reproductive system which is in line with a suggested role in reproduction.

In conclusion, the expression profiles and the evolutionary history of the Testatin/Cres subgroup genes are compatible with a role of these genes in reproduction in placental mammals. Future studies, such as the generation of mice with targeted deletion encompassing additional TCS genes, could finally resolve this issue.

List of scientific papers

I. Töhönen V, Frygelius J, Mohammadieh M, Kvist U, Pelliniemi LJ, OBrien K, Nordqvist K, Wedell A (2005). Normal sexual development and fertility in testatin knockout mice. Mol Cell Biol. 25(12): 4892-902
https://pubmed.ncbi.nlm.nih.gov/15923608

II. Frygelius J, Oscarson M, Nordqvist K, Wedell A, Töhönen V (2007). The reproductive tissue specific cystatin subgroup of genes: expression during gonadal development in wildtype and testatin knockout animals. Sex Dev. 1(6): 363-72. Epub 2008 Jan 18
https://pubmed.ncbi.nlm.nih.gov/18391548

III. Frygelius J, Arvestad L, Wedell A, Töhönen V (2009). Evolution and human tissue expression of the TCS genes, a reproductive specific subgroup of the family 2 cystatins. [Manuscript]