The Y-encoded TSPY protein: a significant marker potentially plays a role in the pathogenesis of testicular germ cell tumors

Abstract

The testis-specific protein Y-encoded (TSPY) gene is the putative gene for the gonadoblastoma locus on the Y chromosome (GBY) that predisposes dysgenetic gonads of intersex patients to gonadoblastoma development. TSPY is expressed at high levels in gonadoblastoma tissues, supporting its possible oncogenic function in this type of germ cell tumors. To explore the possibility that this Y chromosome gene is also involved in pathogenesis of the more common testicular germ cell tumors (TGCTs), we have conducted various expression studies using immunohistochemistry, Western blotting, and reverse transcription-polymerase chain reaction analysis on 171 cases of TGCTs and selected normal testis controls. Our results demonstrated that TSPY protein is abundantly expressed in the precursor, carcinoma in situ or intratubular germ cell neoplasia unclassified, and seminoma, but only minimally or not expressed in various types of nonseminomas. TSPY coexpresses with established germ cell tumor markers (such as placental-like alkaline phosphatase, c-KIT, OCT4) and proliferative markers (such as Ki-67 and cyclin B1) in the same tumor cells at both RNA and protein levels. Ectopic TSPY expression in cultured cells up-regulates progrowth genes, including those at chromosome 12p13, frequently gained/amplified in TGCTs. Our results suggest that TSPY, in combination with other markers, could be an important marker for diagnosis and subclassification of TGCTs and support its role in the pathogenesis of both gonadoblastoma and TGCTs.

Figure 1

Preferential TSPY expression in testicular seminoma and its precursor, CIS/ITGCNU. A) An example of intense TSPY immunostaining of seminoma (right) harboring adjacent CIS/ITGCNU cells (left) in a 34-year old patient. B–E) Enlarged views of boxed areas in A showing CIS/ITGCNU (B, C) and seminoma (D, E) components. Immunostaining of TSPY on F) a case of metastatic seminoma in lymph node, and G–I) selected nonseminomas harboring adjacent CIS/ITGCNU cells. TSPY was intensely positive for metastatic seminoma cells (J), and CIS/ITGCNU cells but not the respective nonseminomas (K–M). N) Double immunostaining of TSPY and OCT4 on an embryonal carcinoma (left) with adjacent CIS/ITGCNU (right) of a 35-year old patient. O–P) Enlarged views of corresponding boxed areas in N. LN = lymph node; YS = yolk sac tumor; EC = embryonal carcinoma. Bar in F represents 400 μm in F–I; bar in N represents 100 μm in A, J–N; and bar in P represents 20 μm in B–E, O–P respectively.

Figure 2

Co-expression of TSPY and various tumor markers in CIS/ITGCNU (left 3 columns) and seminoma (right 3 columns) tumor germ cells. Double immunofluorescence of TSPY (green in A, D, G, J, M, P) and PLAP (red in B, E), OCT4 (red in H, K), and c-KIT (red in N, Q) showed that these tumor markers were expressed in the same tumor germ cells, as indicated in merged images (yellow-orange in C, F, I, L, O and R). TSPY (green in S and V) was co-expressed with cyclin B1 (a potential interactive partner, red in T) and the Ki-67 proliferative marker (red in W) in the same tumor germ cells, as revealed in respective merged images (yellow-orange in U and X). These specimens were derived from patients with ages between 27 to 56 years old. Bar in X represents 20 μm in all images.

Figure 3

Protein and RNA analyses of gene expression in normal testes, seminoma and nonseminoma specimens. A) Western blot analyses of various samples with a TSPY antibody showed multiple reactive protein bands, representing different isoforms of TSPY in protein lysates of normal testes (lanes 1–3, 11), seminomas (lanes 4–9) and nonseminomas (lane 10, 12, 13). Some of these TSPY bands corresponded to those from HEK293 cells transfected with DNA plasmids expressing various TSPY isoforms (left 3 lanes, type 1 and 2 = full length cDNA, Exon1A = cDNA of alternatively spliced transcript). The amounts of TSPY proteins were the highest among the seminoma specimens, low but detectable levels in normal testes and minimal amounts or none among the nonseminomas. Re-probing of the same or parallel filters showed similar expression patterns for PLAP, c-KIT and, to a certain extent, OCT4 and cyclin B1. The latter markers also expressed at high levels in selected nonseminoma specimens (e.g. mixed germ cell tumor in lane 10 and embryonal carcinomas in lanes 12 and 13). The relative amounts of tubulin seemed to be quite even among all samples. B) RT-PCR analyses of transcripts for TSPY isoforms (TSPY=total transcripts; Exon1A, Exon1B, Exon1C and Int4 = alternatively spliced transcripts), and germ cell tumor markers (PLAP, c-KIT, OCT4, and cyclin B1) in the same samples used in Western blotting in A. Successful amplification of RT-PCR products with primer sets specific for the transcripts of the respective isoforms suggested that these variant TSPYs were present in the corresponding samples, particularly the seminomas (lanes 4–9). Although semi-quantitative in nature, the amounts of the RT-PCR products paralleled the intensities of Western blot signals (detected in A) while the same analysis of a reference gene (HPRT) showed relatively even amounts of RT-PCR products among all samples. All RT-PCR images were obtained from negative printing of the respective ethidium bromide staining of the agarose gels. C) Diagrammatic illustration of major variant transcripts originated from the expression of the TSPY tandem arrays on the human Y chromosome. Arrows indicate the positions of respective primers in the structural gene used to detect these variant transcripts by RT-PCR analysis (B). Solid and open boxes represent coding and non-coding sequences of the variant transcripts.

Figure 4

Relative TSPY immunostaining of tumor cells in various subtypes of TGCTs, based on a crude grading system (as described in Table 3). The CIS/ITGCNU, precursor for all TGCTs, showed the most intense and extensive staining while seminomas at clinical stages I, II and III were positive but less intense than the CIS/ITGCNU. Nonseminomas at all clinical stages showed minimal staining with the TSPY antibody under the same conditions, suggesting that they did not express TSPY to any significant levels. TSPY seemed to express at higher levels in early germ cell tumors, but declined towards more advanced clinical stages among the CIS/ITGCNU and seminoma samples. Such differential TSPY expression suggests that TSPY could be an efficient marker for identification of pre-malignant precursors (CIS/ITGCNU) and classification of various subtypes of TGCTs. Student’s t-test showed that the p value of these samples were <0.05 when compared with nonseminomas (NS), suggesting that they were significantly different from the NS samples in TSPY immunostaining.

Figure 5

TSPY expression is associated with up-regulation of selected chromosome 12p genes, involved in TGCTs. A–B) Hierarchical clustering analysis of microarray data on TSPY and selected 12p13 genes from GEO datasets (A, see Methods) and published data (B, Looijenga, 2006). Gene candidates among the list may occur more than once due to duplicate probes, indicating the consistent trend of gene expression in the group. The expression patterns of CIS/ITGCNU and seminoma samples (vertical boxed regions in A and B) resemble closely that for HeLa cells ectopically expressing TSPY transgene (C). C–D) Ectopic TSPY expression up-regulates 12p genes. C) Hierarchical clustering analysis of selected 12p genes in HeLa cells over-expressing TSPY (HeLa) and control cells (control). D) Semi-quantitative RT-PCR analysis of RNAs derived from HeLa cells expressing (+) and lacking (−) TSPY, using specific primers for 12p genes. GAPDH was used as a control. The relative ratios of TSPY expression were calculated as signals from HeLa+TSPY/HeLa+control, as from microarray and Q-RT-PCR analyses (lower panel).