Role of the Y-located putative gonadoblastoma gene in human spermatogenesis

Abstract

The gonadoblastoma locus on the human Y chromosome (GBY) is postulated to serve normal functions in spermatogenesis, but could exert oncogenic properties in predisposing susceptible germ cells to tumorigenesis in incompatible niches such as streaked gonads in XY sex reversed patients or dysfunctional testis in males. The testis-specific protein Y-linked (TSPY) repeat gene has recently been demonstrated to be the putative gene for GBY, based on its location on the GBY critical region, expression patterns in early and late stages of gonadoblastoma and ability to induce gonadoblastoma-like structures in the ovaries of transgenic female mice. Over-expression of TSPY accelerates G₂/M progression in the cell cycle by enhancing the mitotic cyclin B-CDK1 kinase activities. Currently the normal functions of TSPY in spermatogenesis are uncertain. Expression studies of TSPY, and its X-homologue, TSPX, in normal human testis suggest that TSPY is co-expressed with cyclin B1 in spermatogonia and various stages of spermatocytes while TSPX is principally expressed in Sertoli cells in the human testis. The co-expression pattern of TSPY and cyclin B1 in spermatogonia and spermatocytes suggest respectively that 1) TSPY is important for male spermatogonial cell replication and renewal in the testis; and 2) TSPY could be a catalyst/meiotic factor essential for augmenting the activities of cyclin B-cyclin dependent kinases, important for the differentiation of the spermatocytes in prophase I and in preparation for consecutive rounds of meiotic divisions without an intermediate interphase during spermatogenesis.

Keywords:

• cyclin B
• gonadoblastoma
• TSPX
• TSPY
• Y chromosome

Introduction

Gonadoblastoma is a benign germ cell tumor that arises with high frequency in dysgenetic gonads of XY sex-reversed or intersex individuals and in dysfunctional testes of some phenotypic males [Verp and Simpson 1987; Lau 1999; Cools et al. 2006; Looijenga et al. 2007]. It possesses similar properties as carcinoma-in-situ (CIS), the premalignant precursor of testicular germ cell tumors [Jorgensen et al. 1997; Rajpert-De Meyts 2006]. If untreated, gonadoblastoma could develop into aggressive germ cell tumors, such as dysgerminoma in females and testicular seminoma/nonseminoma in males [Oosterhuis and Looijenga 2005]. In 1987, David Page hypothesized the existence of a gonadoblastoma locus on the Y chromosome (GBY) to explain the high incidence of gonadoblastoma development in patients with gonadal dysgenesis and Y chromosome materials in their genomes [Page 1987]. He predicted that the GBY serves a normal function(s) in spermatogenesis of the testis and only predisposes susceptible germ cells to tumorigenesis in dysgenetic gonadal environment. Deletion mapping of XY sex-reversed and intersex patients has localized the GBY locus to a small region on the short arm, proximal to the centromere, on the Y chromosome [Salo et al. 1995; Tsuchiya et al. 1995]. Since the testis-specific protein Y-linked (TSPY) gene [Arnemann et al. 1991; Zhang et al. 1992; Schnieders et al. 1996] is located in the GBY critical region, it has been a subject of intensive studies on its candidacy as the gene for GBY. Although additional regions, e.g., the AZFa on the long arm, proximal to the centromere, and genes, such as USPY, DDX3Y, and UTY, located herein, could also contribute to the overall gonadoblastoma development [Vogt et al. 2007], results from various studies, as described below, support the role of the TSPY gene in this special type of germ cell tumors.

TSPY as the gene for gonadoblastoma locus on the Y chromosome

Numerous studies demonstrated that TSPY is present in XY sex-reversed patients and is expressed abundantly at early stages of tumorigenesis in gonadoblastoma, as well as testicular CIS, seminomas and selected nonseminomas, such as embryonal carcinoma and yolk sac tumors [Lau et al. 2009]. TSPY is co-expressed with numerous germ cell tumor markers, such as placental alkaline phosphatase, OCT4 and c-KIT, and hence is postulated to be involved in testicular and extragonadal germ cell tumorigenesis [Hoei-Hansen et al. 2006; Li et al. 2007a; 2007b]. Over-expression of TSPY in cultured cells accelerates cell proliferation and tumorigenicity in athymic mice [Oram et al. 2006]. TSPY specifically expedites the G₂/M progression in the cell cycle by interacting with the mitotic cyclin B1 and stimulating the phosphorylation activities of an activated cyclin B1-cyclin dependent kinase 1 (CDK1) complex (Fig. 1B and 1C) [Li and Lau 2008]. Most significantly, female transgenic mice harboring a TSPY transgene could develop gonadoblastoma-like structures within their ovaries, which also expressed the TSPY transgene at high levels [Lau et al. 2009]. Collectively, these studies demonstrated that TSPY is the putative proto-oncogene for GBY.

Figure 1.  Diagrammatic illustration of TSPY and TSPX domains and effects on cyclin B1-CDK1 kinase activities. (A) TSPY and TSPX proteins share significant similarity at their SET/NAP (NAP) domain, but diverge at flanking sequences, particularly at their carboxyl termini. TSPX harbors an acidic tail, which is absent in TSPY. (B) Cyclin B-CDK1 complex phosphorylates target proteins in an energy dependent manner involving ATP. (C) TSPY interacts with cyclin B and enhances while (D) TSPX also interacts with cyclin B but inhibits the cyclin B-CDK1 kinase activities. (E) Conjugating the acidic domain of TSPX to the carboxyl terminus of TSPY renders the recombinant protein to be inhibitory in the same cyclin B-CDK1 kinase assay, suggesting that the TSPX acidic domain is responsible for its repression on cyclin B-CDK1 kinase activities [Li and Lau 2008].

TSPY and its X-homologue, TSPX, possess contrasting properties in cell cycle regulation

TSPY is a tandemly repeated gene, whose 2.8-kb transcriptional unit harbors 6 exons and is embedded in a homologous 20.4-kb repeat unit [Skaletsky et al. 2003; Repping et al. 2006]. The copy number of TSPY-containing repeat units varies from the low 20's to over 70 among normal individuals [Repping et al. 2006; Giachini et al. 2009], which encompasses tandem arrays of 0.4 to >1.4 MB and constitutes the largest block of functional non-ribosomal repeat sequences in the human genome. The variation in copy number suggests that the TSPY arrays are hotspots for genetic instability and/or epigenetic dysregulation [Lau et al. 2009]. Preliminary reports suggest that TSPY copy number variations could be associated with human fertility and cancer [Vijayakumar et al. 2006; Giachini et al. 2009]. TSPY and its X-located single-copy homologue, designated as TSPX, share a similar genomic organization [Chai et al. 2001; Ozbun et al. 2001; Delbridge et al. 2004; Wang et al. 2004; Lau et al. 2007]. The 6.3-kb TSPX gene contains 7 exons and 6 introns. They encode proteins of 308 and 693 amino acids respectively, which share significant homology at a protein-interacting domain, termed SET/NAP domain, initially identified in the SET oncoprotein and the nucleosome assembly protein-1 (NAP-1) [von Lindern et al. 1992; Adachi et al. 1994; Kellogg et al. 1995; Kawase et al. 1996]. The SET/NAP domain is encoded by respective exons 2-5 in both genes, which show highly conserved exon-intron junction structures. TSPY and TSPX are postulated to have evolved from an ancestral gene on the proto-X and proto-Y chromosome, which gave rise to the present day X and Y chromosome [Lau et al. 2007]. Since they are located on the non-pairing regions of the sex chromosomes, they have diverged significantly through time, yielding various sequence differences outside the conserved SET/NAP domain. One of the key differences is the absence and presence of an acidic domain in the carboxyl termini of TSPY and TSPX, respectively (Fig. 1A). Various studies showed that TSPY and TSPX possess contrasting properties in cell cycle regulation [Chai et al. 2001; Oram et al. 2006; Kandalaft et al. 2008]. Over-expression of TSPX retards cell cycle progression, particularly at the G₂/M phase, and inhibits tumorigenicity in athymic mice. It interacts with cyclin B and represses the kinase activities of an active cyclin B1-CDK1 complex (Fig. 1D) [Li and Lau 2008]. Domain swapping experiments demonstrate that the carboxyl acidic domain of TSPX, which is absent in TSPY, is responsible for such inhibitory actions on cyclin B1-CDK1 activities. Transposition of this acidic domain to the carboxyl terminus of TSPY renders the recombinant TSPY-TSPX protein to be inhibitory in the same kinase activity assay (Fig. 1E). Hence, TSPX is postulated to be a tumor suppressor with a normal function(s) in modulating G₂/M progression [Kandalaft et al. 2008; Lau et al. 2009].

Immunofluorescence analysis of transfected cells demonstrated that TSPY is localized in both the cytoplasm and nucleus in the interphase while cyclin B1 is distributed in the cytoplasm in interphase and migrates to the nucleus prior to the onset of mitosis (Fig. 2). TSPY is preferentially localized on the microtubules in the mitotic spindles at metaphase and continues through anaphase while cyclin B1 is localized similarly at the mitotic spindles at metaphase but is degraded at anaphase. The co-localization of TSPY and cyclin B1 at the mitotic spindles and the TSPY enhancement of the cyclin B1-CDK1 kinase activities suggest that TSPY might potentially exert some degrees of oncogenic function(s) by expediting a rapid transit through this mitotic checkpoint. TSPX is also preferentially localized at the mitotic spindles of metaphase cells [Lau et al. 2009] and could have a modulating effect(s) on cyclin B1-CDK1 kinase activities at this checkpoint, thereby ensuring an orderly passage through mitosis in the cell cycle.

Figure 2.  Co-localization of TSPY and cyclin B1 in COS7 cells. COS7 cells were transiently transfected with a human TSPY expression vector (pCS2-hTSPY) and were analyzed by immunofluorescence using a mouse monoclonal antibody against human TSPY (red in left panels) and a rabbit polyclonal antibody against human cyclin B1 (green in left panels; red in right panels) [Kido and Lau 2005; Li and Lau 2008]. On the right panel, a mouse monoclonal antibody against α-tubulin was used to detect the microtubule in the mitotic spindle (green). Binding of the primary antibodies were visualized with respective fluorescence dye labelled secondary antibodies. DNA was visualized by DAPI staining (blue). Note that, at M-phase, both transfected TSPY and endogenous cyclin B1 were localized on mitotic spindle. Scale bar = 20 μm. Color figure shown in electronic copy.

TSPY is co-expressed with cyclin B1 in spermatogonia and spermatocytes in meiotic prophase I

The contrasting properties of TSPY and TSPX could have a significant effect on spermatogenesis in normal testis. To shed light on their probable functions, immunofluorescence studies were conducted with specific antibodies against these two SET/NAP proteins and cyclin B1 on normal human testis. Our results showed that TSPY is primarily expressed in spermatogonia and spermatocytes at meiotic prophase I while TSPX is expressed mostly in the somatic cells, principally in Sertoli cells and to a less extent the spermatogonia within the seminiferous tubules (Fig. 3A). Interestingly, TSPX is primarily localized on the nuclei of somatic cells, but could be localized in both nucleus and cytoplasm of spermatogonial cells. TSPY is co-localized with cyclin B1 in the cytoplasm and nucleus of the spermatogonia in both pre-mitotic and mitotic phases (Fig. 3B, arrows).

Figure 3.  Immunofluorescence analysis of TSPY, TSPX and cyclin B1 in normal adult human testis. (A) Five-micron tissue sections were incubated with anti-TSPY monoclonal antibody (red) [Kido and Lau 2005] or rabbit polyclonal anti-TSPX antibody (green) [Lau et al. 2007]. DNA was visualized by DAPI staining (blue). Bottom panels show the magnified images of boxed area indicated in top panel. TSPY was specifically expressed in spermatogonia and spermatocytes (Spg and Spc, respectively), while TSPX was preferentially expressed in the nuclei of Sertoli cells (Ser) and only at low levels in spermatogonia (Spg). (B) Immunofluorescence of TSPY (red) and cyclin B1 (green) in normal human testis. Pre-mitotic spermatogonia (arrowheads in left panel) and mitotic spermatogonia (arrowheads in right panel) are presented at high magnification. TSPY and cyclin B1 were co-localized in both pre-mitotic and mitotic spermatogonia. Experiments were conducted under an approved protocol by the Institutional Committee on Human Research, VA Medical Center, San Francisco. Scale bar = 20 μm. Color figure shown in electronic copy.

To further delineate the pattern of co-expression of TSPY and cyclin B1, immunofluorescence analysis was conducted in detail with respect to various stages of spermatogenesis (Fig. 4A). Our results showed that TSPY is primarily located in the spermatogonia and prophase I spermatocyes at the preleptotene, leptotene, and zygotene stages. Its expression gradually reduces in early pachytene following the presumably inactivation of the sex chromosomes and is at residual levels in diplotene, and is barely detectable during meiotic divisions and in the haploid spermatids (Fig. 4A and 4B, top panel). TSPY is capable of generating alternatively spliced transcripts encoding isoforms harboring the same SET/NAP domain [Lau et al. 2003]. In some preparations, residual levels of TSPY protein could still be detected in stages as late as the round spermatids [Kido and Lau 2005]. Cyclin B1 is expressed similarly in the spermatogonia and spermatocytes at various stages of the meiotic prophase I, but its expression seems to persist further beyond the pachytene and possibly diplotene stages. Similar to TSPY, its expression is at residual levels during meiotic divisions and stages beyond. The expression patterns of both TSPY and cyclin B1 suggest that they are essential for spermatogonial cell renewal and meiotic prophase I spermatocyte differentiation.

Figure 4.  Relative expression levels of TSPY and cyclin-B1 during different spermatogenic stages in human testis. (A) Immunofluorescence of TSPY (red) and cyclin B1 (green) in spermatogonia, spermatocytes, and spermatids at various spermatogenic stages of a normal human testis [Kido and Lau 2005]. The specific spermatogenic cell types are indicated by brackets with adjacent numbers that correspond to respective stages of spermatogenesis, as illustrated in B. Scale bar = 20 μm. (B) Relative immunofluorescence signals of TSPY and cyclin B1 were diagrammatically illustrated along with schematic representation of spermatogonia, spermatocytes at prophase I and meiosis divisions. Color figure shown in electronic copy.

Probable functions of TSPY in spermatogenesis

Proteins with SET/NAP domain serve diverse functions in transcription, translation, DNA replication, signal transduction, neuronal differentiation, and cell cycle regulation [Estanyol et al. 1999; Chai et al. 2001; Qu et al. 2002; Canela et al. 2003; Kandilci et al. 2004; Gamble et al. 2005; Karetsou et al. 2005; Carujo et al. 2006; Kido and Lau 2006; Oram et al. 2006; Muto et al. 2007]. Several studies conducted in our laboratory and those of others have identified additional TSPY-like genes in the human and other mammalian genomes [Vogel et al. 1998; Dechend et al. 2000; Lau et al. 2007]. These TSPY-like genes could be derived from retrotransposition events involving TSPY transcripts. They contain a single exon and encode proteins harboring a conserved SET/NAP domain, similar to that of TSPY. Although the exact functions of these TSPY-like genes are uncertain, mutations in TSPYL1, have been associated with the sudden infant death with dysgenesis of the testis syndrome [Hering et al. 2006]. Hence, it is conceivable that the TSPY family of proteins could serve various functions, similar to those prescribed for the SET/NAP proteins.

TSPY has been demonstrated to interact with the translation elongation factors, eEF1A1 and eEF1A2 [Kido and Lau 2008], which are involved in protein synthetic machinery in the cells and are highly expressed in various types of human cancers, including testicular germ cell tumors, ovarian, breast, and prostate cancers [Sonenberg 1993; Anand et al. 2002; Tomlinson et al. 2005; Kulkarni et al. 2006; Yu et al. 2006]. TSPY interactions with eEF1A enhance protein synthesis and transcriptional activities of a reporter gene in the host cells [Kido and Lau 2008]. Hence, TSPY could potentially be involved in stimulation of protein synthesis and other cellular functions, such as nuclear export and transcriptional regulation ascribed to eEF1A [Khacho et al. 2008], in spermatogonia and spermatocytes. TSPY is also capable of interacting with the core histones and is postulated to serve a function(s) in chromatin modification and/or organization during spermatogenesis [Kido and Lau 2006]. Despite these probable functions, its interactions with type B cyclins and its stimulating effects on cyclin B-CDK complexes could be the most important properties affecting the biological processes in spermatogonial cell renewal and in prophase I spermatocyte differentiation, critical in consecutive meiotic divisions and progression of spermatogenesis in the testis. Spermatogonia are pluripotent germ cells, i.e., type A spermatogonia, essential for the maintenance of continuous sources of stem germ cells as well as committed germ cells, i.e., type B spermatogonia, for spermatogenesis and sperm production [Morgan 2007; Sun and Handel 2008]. We surmise that TSPY functions in the renewal of spermatogonial pluripotent cells could be similar to those involved in mitotic cycle regulation. Interestingly, the preferentially expression of TSPX in Sertoli cells and its cell cycle modulation/repression function(s) suggest that it is associated with cells not actively involved in mitotic division or meiotic differentiation.

Although similar genes are involved in the processes, meioses in males and females are different in mammals [Morgan 2007]. In females, oogenesis is arrested at meiotic prophase I during embryonic life. A subset of oocytes undergoes maturation at regular intervals starting at puberty, occurring years after birth in humans, and progresses through metaphase I and differentiates into unfertilized eggs at metaphase meiosis II, whose division will only be completed upon fertilization. Male meiosis is a continuous process, once the spermatogonial cells are committed to differentiation. As in the case of man, spermatogenesis usually takes approximately 64 days from spermatogonia to spermatozoa [Heller and Clermont 1963]. Similar to late G₂/antephase in mitosis [Morgan 2007; Chin and Yeong 2010], the preleptotene spermatocytes possess 4N of DNA content in preparation for meiotic divisions. These spermatocyes enter the meiotic prophase I, in which chromosome condensation, chromosome pairing, synaptonemal complex formation, synapsis, and desynapsis/synaptonemal complex degradation take place in leptotene, zygotene, pachytene, and diplotene stage, respectively. The spermatocytes (4N) go through two consecutive rounds of meiotic divisions without an intermediate interphase, resulting in haploid round spermatids (1N). As mitotic/meiotic cyclins, type B cyclins could form active complexes with cyclin dependent kinases, important for phosphorylation activities at meiotic prophase I in preparation for such consecutive meiostic divisions [Refik-Rogers et al. 2006; Wolgemuth 2008]. The co-expression of TSPY with cyclin B1 and its catalytic properties on cyclin B1-CDK1 kinase activities suggest that the TSPY actions could ensure the proper progression of the spermatocytes through different stages of the meiotic prophase. Currently, the exact targets of TSPY-associated cyclin B-cyclin dependent kinases are still uncertain. We postulate that these phosphorylation events are essential for chromosome alignments, bivalent pairing, synaptonemal complex and chiasmata formations, and the consecutive meiosis divisions [Inselman and Handel 2004; Refik-Rogers et al. 2006; Sun and Handel 2008; Wolgemuth 2008; Yu and Wu 2008; Brar et al. 2009; Kang and Yu 2009]. The present observations suggest that TSPY serves as a specialized meiotic catalyst on cyclin B1-CDKs activities, important in the meiotic prophase, and perhaps the consecutive meiotic divisions.

TSPY is evolutionarily conserved as moderately repetitive sequences on the Y chromosome and is preferentially expressed in the testis of many mammals, including bovine and primates [Zhang et al. 1992; Vogel et al. 1997; Rottger et al. 2002], suggesting that it might serve a conserved function(s), as hypothesized above, in spermatogenesis. Significantly, Tspy genes in the laboratory mouse and Mongolian gerbil are incapable of encoding any Tspy proteins [Mazeyrat and Mitchell 1998; Schubert et al. 2000a; 2000b; Karwacki et al. 2006]. We surmise that other autosomal Tspy-like gene(s) could have assumed the function(s) of the original Y-located Tspy gene before/during such Y chromosome decays in these mammalian species.

Several studies demonstrated that other type B cyclins, i.e., cyclin B2 and cyclin B3, as well as type A and D cyclins are also expressed at various spermatogenic stages [Yu and Wu 2008]. In particular, cyclin B2 is preferentially expressed in diplotene and metaphase I and meiosis II and cyclin B3 is preferentially expressed in zygotene stage, overlapping that of cyclin B1. Since cyclin B1 shares significant similarity with cyclin B2, the immunofluorescence observed with the polyclonal antibody against the human cyclin B1 could potentially detect the cyclin B2 protein as well. Since TSPY has been demonstrated to interact with cyclin B2 and cyclin B3 [Li and Lau 2008], TSPY could also potentially stimulate cyclin B2/B3-cyclin dependent kinase activities in stages, in which both TSPY and these cyclin Bs are co-expressed. Hence, in addition to its role in the mitotic cycle of spermatogonial renewal, TSPY could play significant roles in the progression of prophase I and meiotic divisions during spermatogenesis in humans. The present hypothesis is supported by a recent study by Schubert and colleagues [Schoner et al. 2010], who introduced a human TSPY transgene into the homozygous Kit^W-v genetic background, which is normally associated with total germ cell loss in the fetal testis and infertility in adult animals. These investigators showed that human TSPY transgene was capable of restoring spermatogenesis and fertility in some of these mutant mice, suggesting that TSPY could play key roles in fetal and adult germ cell proliferation and meiotic division, as postulated here.

Declaration of Interest: The authors report no conflicts of interests. The authors alone are responsible for the content and writing of this paper.

Abbreviations

GBY:	=	gonadoblastoma locus on the human Y chromosome
TSPY:	=	testis-specific protein Y-linked (X-homologue, TSPX)
CIS:	=	carcinoma-in-situ
CDK1:	=	cyclin B1-cyclin dependent kinase 1
SET:	=	Patient SE Translocation
NAP-1:	=	nucleosome assembly protein-1
eEF:	=	translation elongation factors.

Acknowledgment

This work was partially supported by a VA Merit grant to Y-FC Lau, who is also a Research Career Scientist of the Department of Veterans Affairs.