Human TSPY was originally identified as the first Y-chromosomal gene, which expression was elusively restricted to the testis (Arnemann et al., 1987). Nowadays it is known that TSPY is predominantly expressed in the testes and to much lesser extent expressed in the native prostate and the human brain (Kido and Lau, 2005; Lau et al., 2003).
The organization of human TSPY on the human Y chromosome as a heterogeneous repetitive gene family with varying copy number ranging from 9-101 among individuals is unusual in the human genome (Nickkholgh et al., 2010; Giachini et al., 2009; Shen et al., 2013). Besides a single separate functional TSPY gene (TSPY3) on Yp are most TSPY copies (TSPY1) embedded in 20.4 kb repeat units of the DYZ5 tandem array (Skaletsky et al., 2003). Especially this ampliconic TSPY1 gene array on Yp11.2 is a hotspot for intrachromosomal recombination likely by unequal sister chromatid exchange (Skaletsky et al., 2003) that leads to differences in copy number among men. It has to be mentioned that the TSPY nomenclature is far from being established right now. Besides the TSPY1 and TSPY3 genes were also TSPY2, TSPY4, TSPY8 and TSPY10 genes and multiple TSPY pseudogenes (TSPYPn) annotated on Yp11.2 on the human Y chromosome (reference NC_00024.9). Controversially, TSPY1 was earlier designated as one out of the multiple TSPY genes within the TSPY tandem array (Bhowmick et al., 2007).

The prototypic human TSPY gene is composed of six exons and five introns and a promoter region of yet unknown length. Functional TSPY genes can differ in sequence within coding and promoter regions of up to 1% (Vogel and Schmidtke, 1998).

Besides the TSPY1 main transcript TSPY-L (NM_003308.3), which was originally named as TSPY^major (Schnieders et al., 1996) or type 1 TSPY transcript (Lau et al., 2003), eleven different transcript isoforms that differ in sequence and in length were detected in the testis, and in prostatic and testicular tumors (Zhang et al., 1992; Dechend et al., 2000; Schnieders et al., 1996; Lau et al., 2003; Li et al., 2007). One of these TSPY1 minor transcripts is the TSPY-S variant (NM_001197242.1) which uses an alternative splice acceptor site 11 bp upstream of the native intron 5/exon 5 border of the main TSPY-L transcript (Zhang et al., 1992). TSPY-S is translated in a shortened distinct peptide (NP_001184171.1) of 294 amino acids with frameshifted ORF at amino acid position 275 in comparison to TSPY-L. The TSPY-L transcript has a length of 1160 bp and encodes for a TSPY-L peptide of 308 amino acids (NP_003299.2).

Multiple annotated TSPY pseudogenes (TSPYPn) are available on the human Y chromosome. These TSPY pseudogenes can show a nucleotide divergence in coding and promoter regions of up to 10% (Vogel and Schmidtke, 1998).

Human TSPY1 encodes the TSPY1 protein. The main protein is named TSPY-L and is composed of 308 amino acids (size 33 kDa). TSPY-L occurs mainly as a phosphoprotein with a predicted size of 38 kDa (Schnieders et al., 1996). TSPY is sharing with its X-encoded homolog TSPYL2 (designated also as TSPX, CDA1, DENTT or NP79), autosomal TSPY-like paralogs and with SET and NAP-1 a highly-conserved type B cyclin binding SET/NAP domain (amino acids residues 121-265 according to Li and Lau, 2008).
For human TSPY the translation elongation factor eEF1A1 has also been identified as binding partner of the SET/NAP domain in a yeast-two-hybrid screen (Kido and Lau, 2008). An interaction of TSPY with eEF1A1 and eEF1A2 was also shown by in vitro GST-pull down assays by Kido and Lau (2008).
An in vitro binding of the SET/NAP domain of TSPY and the calcium/calmodulin-dependent serine protein kinase (CASK), which is among other things essential for synapsis formation and memory, was also demonstrated in transfected COS7 cells (Kido et al., 2011).
Besides the cyclin B, eEF1A1 and CASK interactive SET/NAP domain is TSPY-L harbouring an N-terminus of 120 and a C-terminus of 43 amino acids.
Two putative homodimer forming α-helices (α1, aa 52-95 and α2, aa 105-141) were predicted within the N-terminal region by in silico analysis (Kido and Lau, 2008). It was further shown by co-immunprecipitation of TSPY from transfected COS7 cells that TSPY can form homodimers, which is presumably mediated through the α-helices of its N-terminal region (Kido and Lau, 2008).
The N-terminal region of TSPY is also capable to bind to the androgen receptor (AR) AF-2 region (Akimoto et al., 2010). For rat TSPY it was also shown that the N-terminal region of rat TSPY is preferentially binding to core histones, although a much lower binding affinity for core histones was also observed for the acidic part of the SET/NAP domain of rat TSPY (Kido and Lau, 2006).

Human TSPY is mainly expressed in testicular germ cells and weakly expressed in epithelial cells of the prostate. However, some TSPY ESTs originated from the medulla of human brains are also available in databases (Kido and Lau, 2005). TSPY is expressed in gonocytes and prespermatogonia of the fetal testis and in prespermatogonia of the neonatal testis (Honecker et al., 2004). Within the adult testis are highest TSPY expression levels observed in spermatogonia and in primary spermatocytes of the preleptotene to zygotene stage of the meiotic prophase I, while expression level decreases gradually during meiosis form primary spermatocytes of the pachytene stage to round spermatids (Lau et al., 2011).

TSPY is predominantly localized in the cytoplasm but also to lesser extent present in the nucleus of male germ cells and epithelial cells of the prostate. It is supposed that the phosphorylation status of the CK2 phosphorylation site (T300) at the C-terminus of human TSPY is important for its nucleo-cytoplasmatic shuttling (Krick et al., 2006).

Currently the biological role of TSPY within the testis and in the prostate are unknown but TSPY proposed functions are diverse. Especially the conservation of functional TSPY genes in different mammalian lineages indicates an important biological role of TSPY in spermatogenesis. Expression studies of human TSPY within the human testis suggest that TSPY could act as proliferation factor of testicular germ cells, such as gonocytes, prespermatogonia and spermatogonia in the fetal, neonatal, pubertal and adult testis and could act as a catalyst in meiotic differentiation and division of spermatocytes within the pubertal and adult testis (Schnieders et al., 1996; Honecker et al., 2004; Lau et al., 2011). These proposed functions are founded on the following observations:
- TSPY shares a highly conserved SET/NAP domain with other members of a protein family that was designated as TSPY/TSPY-like/SET/NAP (TTSN)-family (Schnieders et al., 1996; Vogel and Schmidtke, 1998). Besides TSPY itself, are its X-encoded homologue TSPYL2, the oncogene SET (suppressor of variegation, enhancer of zeste and Trithorax), the neucleosome assembly protein-1 (NAP1) and the autosomal encoded paralogs TSPYL1, TSPYL3, TSPYL4, TSPYL5, TSPYL6 members of this protein family. Members of the SET/NAP-protein-family play function in cell cycle regulation, transcription, translation, signal transduction, DNA replication and chromatin condensation (Lau et al., 2009; Lau et al., 2011 and included references).
- Ectopic expression of TSPY in transfected human HeLa and murine NIH3T3 cells stimulates cell growth by promoting the G2/M-phase transition of the cell cycle (Oram et al., 2006). It was later shown in transiently transfected HeLa and HEK293 cells that this effect is mediated by a direct interaction of the SET/NAP domain of TSPY with the activated cyclin B1/CDK complex. TSPY accelerates the G2/M-phase transition of host cells by stimulating the kinase activity of the activated cyclin B1/CDK complex (Li et al., 2008). The co-localization of TSPY and cyclin B1 in spermatogonia and primary spermatocytes suggests a putative interaction of both proteins during spermatogonial renewal and during the prophase I of the first meiotic division (Lau et al., 2011). However, the relevance of TSPYs ability to stimulate the kinase activity of an activated cyclin B1/cdk complex in transiently transfected human HEK293 cells to its in vivo function within the testis is still unclear.
- Kido and Lau (2008) have identified the eucaryotic elongation factor 1 alpha (eEF1A) in a yeast two-hybrid screen as interaction partner for human and rat TSPY. It was shown by in vitro GST pull down assays that eEF1A1 and eEF1A2, which both are essential for the elongation phase of protein translation, can bind to the SET/NAP domain of human TSPY. Kido and Lau (2008) have also demonstrated that ectopic expression of TSPY in HEK293 cells enhances expression of a cotransfected luciferase reporter transcript. Whether this effect is mediated by a synergistically interaction of TSPY and eEF1A in reporter gene transcription and translation is currently unknown. It is also still unclear whether TSPY could affect the expression of specific genes in spermatogonia and spermatocytes by interaction with eEF1A. The co-expression and co-localization of TSPY and eEF1A in spermatogonia and spermatocytes makes an interaction of both proteins in the human testis not unlikely. The findings of the latter study suggest that TSPY and eEF1A could synergistic exert progrowth functions on germ cells by promoting gene expression.
Other TSPY proposed functions are:
- It is also supposed that rat TSPY could affect spermatogenesis by acting as histone chaperone in elongated spermatids during the maturation process when histones are replaced by basic protamines (Kido and Lau, 2006). It was shown by in vitro GST pull down assays that human TSPY and rTSPY can both bind to the core histones H2A, H2B, H3 and H4, respectively (Kido and Lau, 2006). The non overlapping expression pattern of human TSPY and rTSPY in spermatids makes a function of human TSPY as histone chaperone in spermatids rather unlikely.
- TSPY is coexpressed with the androgen receptor in testicular germ cell tumors (seminomas and nonseminomas) and in prostatic tumors (Akimoto et al., 2010). TSPY can trap the androgen-bound androgen receptor in the cytoplasm of TSPY stably expressing NEC8-TSPY cells (a model nonseminoma cell line) and in TSPY endogenously expressing LNCaP cells, thereby repressing AR mediated gene transcription (Akimoto et al., 2010). It is supposed that TSPY represses androgen dependent cell proliferation in TGCTs, presumably by preventing AR nuclear translocation. However TSPY is not coexpressed with the human AR in testicular cells, which exclude such a function in the native testis.
- TSPY and CASK are coexpressed and co-localized in the brains of TSPY transgenic founder mice, carrying a human TSPY 12,5 kb genomic fragment, which includes 7,1 kb TSPY promoter region, a 2,8 kb structural TSPY gene and 2,6 kb 3-region. Whether an in vivo co-localization and interaction of both proteins exist in humans is currently unknown (Kido et al., 2011).

Especially the SET/NAP domain of TSPY shares high homology to other members of the TTSN-protein family.

Due to the multi-copy status of TSPY genes and the presence of multiple TSPY pseudogenes on the Y chromosome no disease associated mutations have been identified so far. Recently, multiple TSPY-variants within the 5-UTR, exon 1 and 3-UTR have been identified in a screening approach of 72 infertile men and 31 fertile men as controls (Svacinova et al., 2011). Out of these were 39 variants in exon 1 elusively restricted to the infertile cohort (Svacinova et al., 2011).