Testicular cell lines

Molecular and Cellular Endocrinology 228 (2004) 53–65

Testicular cell lines Nafis A. Rahman a , Ilpo T. Huhtaniemi a,b,∗ b

a Department of Physiology, University of Turku, 20520 Turku, Finland Faculty of Medicine, Institute of Reproductive and Developmental Biology (IRDB), Imperial College London, Du Cane Road, London W12 0NN, UK

Received 30 December 2002; accepted 7 May 2003

Abstract The range of in vivo or in vitro immortalized cell lines currently available provides a variety of model systems for studies of normal and pathological cell functions. The cell lines have been derived from spontaneous or experimentally induced tumors, or through in vitro immortalization. The transgenic (TG) techniques provide a powerful approach, allowing the production of in vivo animal models for a variety of diseases, including malignant tumors, through tissue-specific expression of oncogenes or other tumor-promoting genes. The TG techniques also enable the production of cell lines with specific characteristics, through insertion of desired genes into specific cell types, which can then be immortalized upon cell culture. The use of temperature-sensitive immortalizing genes offers an additional advantage of controlling gene expression, including the proliferation and differentiation of the cells to be immortalized. As regards the male reproductive system, a number of cell lines of testicular somatic cells are currently available. This review covers mainly the immortalized cell lines of testicular Leydig and Sertoli cells, with special reference to murine cell lines for the study of testicular endocrine function and tumorigenesis. These cell lines also provide useful tools to investigate the molecular basis of hormone actions and testicular cell interactions. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Gonadal tumorigenesis; Gene targeting; Transgenic mice; Leydig and Sertoli cell lines; Spermatogenic cells

1. Aspects of cell culture in endocrine research Cell and tissue cultures are today a routine laboratory method, with versatile application in almost all types of basic biomedical research. A major challenge in the development of cell lines is to maintain as closely as possible their normal functional properties upon prolonged culture. The origin of cell lines can be: (1) normal tissue, when occasional cells survive in culture and continue to grow; (2) cells becoming spontaneously transformed; (3) cells originating from spontaneous tumors or those induced by X-ray irradiation, hormonal stimulation or chemical carcinogens; (4) cells arising from fusion of normal cells with transformed cells, followed by selection of a cell line with desired properties; and finally, (5) introduction of a foreign DNA into cells either in vivo (transgenesis) or in vitro (transfection). Cell cultures are indispensable in many types of endocrine research, such as signal transduction of cell membrane receptors and regulation of gene activity by nuclear receptors ∗ Corresponding author. Tel.: +44 20 7594 2104; fax: +44 20 7594 2184. E-mail address: [email protected] (I.T. Huhtaniemi).

(Bosland, 1996). The use of cultured cell lines (normal or malignant) of endocrine origin is playing an important role in furthering our understanding on almost all aspects of hormone biosynthesis, action and target cell responses (Ascoli, 1981a,b; Dilworth, 1990). The highly complex process of carcinogenesis has been difficult to approach in experimental conditions in vivo. TG mouse models have proven extremely useful in this process for instance by facilitating the study of malignant transformation from hyperplasia to malignant growth. Tumor formation can be selectively targeted in TG mice to the tissues of interest using tissue or cell-specific promoters and genes encoding cellular or viral oncogenes. This process of “targeted oncogenesis” involves transgenic expression of an immortalizing oncogene, such as the commonly used Simian virus 40 T-antigen (Tag) (Livingston and Bradley, 1987; Dilworth, 1990). TG tumor mice are a valuable tool in studies of the onset, progression and regulation of tumor growth (Thomas and Balkwill, 1994). In addition, we can in this way immortalize in vivo even specific rare cell types and establish permanent cell lines for the study of their functions in vitro (Kananen et al., 1995, 1996b; Lahti et al., 2001). Such cells enable for example the study of the sequential

0303-7207/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2003.05.001

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stages of differentiation in a given cell lineage (Alarid et al., 1996).

2. The strategies of targeted tumorigenesis 2.1. Genetically targeted tumorigenesis through oncogene expression The use of TG mice has over the last decade led to major breakthroughs in research areas such as genetics, physiology, embryology and oncology. TG mice are produced by three main methods, i.e. transfection with viral vectors, pronucleus injection, and by homologous recombination in embryonic stem cells. The commonest oncogene in TG animal studies is the Tag (see further). Cellular proto-oncogenes encoding growth factors (e.g. wnt-1, int-2), signal transduction proteins (e.g. c-ras), tyrosine and serine/threonine kinases (e.g. c-src, c-mos), and transcription factors (e.g. c-myc, c-fos) have also been utilized with success in TG constructs, especially in studies on mammary gland carcinogenesis (Kwan et al., 1992; Thomas and Balkwill, 1994; Elson and Leder, 1995). The large and middle T-antigen coding sequences of polyoma virus, E1 region gene of adenovirus type 12, v-Harvey-ras coding sequence, mutated p53 and human papilloma virus type 18 E6 and E7 have also been used to target tumorigenesis to various TG mouse tissues (Paquis-Flucklinger et al., 1993; Kondoh et al., 1994; Bourdon et al., 1998). 2.2. Tag — the viral oncogene Tag is a multifunctional protein of 90 kDa size, controlling the replication and transcription of the viral genome, possessing both immortalizing and transforming capacity in cells transfected by the virus. In this way, Tag is a unique oncogene, since most cellular and viral oncogenes are only capable of either immortalization (class I oncogenes) or transformation (class II oncogenes). Transformation implies a change in phenotype dependent on the uptake of new genetic material. Although it is now possible in mammalian cells, it has been called transfection to distinguish it from transformation, which in tissue culture implies spontaneous or induced permanent phenotype change that does not necessarily involve the uptake of new genetic material. Transformation can arise from infection with a transforming virus such as polyoma and from incorporation of new genomic DNA, it can also arise spontaneously or following exposure to a chemical carcinogen. Immortalization is the acquisition of an infinite lifespan, presumed to be due to the deletion or mutation of one or more senescence genes or overexpression or mutation of one or more oncogenes that override the action of the senescence genes. The multiple functions of Tag in vivo and in vitro include binding to double-stranded DNA and activation of cellular promoters, autokinase, helicase and non-DNA-dependent ATPase activity, stimulation of trans-

fer of G1 cells into the S phase, and stimulation of polymerase I transcription for rRNA overproduction (Livingston and Bradley, 1987). The immortalization and transformation of rodent and human cells is usually dependent on persistent Tag expression. The transformation capacity of Tag resides in its ability to bind to the cellular anti-oncogene, p53 (Livingston and Bradley, 1987; Dilworth, 1990). The first study using Tag to induce tumors in TG mice was reported in 1984 (Brinster et al., 1984). They showed that the SV40 early region, i.e. enhancer element and the large and small T-antigen coding sequences, under the metallothioneine (MT) promoter, resulted in TG mice in choroid plexus tumors and pathological changes in the thymus and kidney. The upstream region of the rat insulin II gene was the first tissue-specific promoter fragment used to direct Tag expression (Hanahan, 1989). These mice developed pancreatic ␤-cell tumors, proving that cellular promoters are able to induce high enough expression of Tag to induce tumorigenesis in target tissues (Hanahan, 1989).

3. Testicular tumorigenesis The etiology of human testicular tumors remains poorly defined (Fowler et al., 2000). Although some association between testicular cancer risk and such factors as prenatal estrogen exposure, low birth weight, premature birth, intrauterine growth retardation, familial aggregation of risk or genetic predisposition, mumps-orchitis, testicular trauma, inguinal hernia, cryptorchidism and vasectomy has been shown, the evidence of these factors as potential risk factor are still inconclusive (Bosland, 1996). Specific chemical carcinogens implicated with increased testicular cancer risk have yet to be identified, although environmentally or genetically determined ethnic factors could be candidates for testicular tumorigenesis (Bosland, 1996). Thus, there is increased demand for experimental in vivo models for testicular tumors and for in vitro models to study the mechanisms of testicular tumorigenesis.

4. Testicular somatic cell tumors and immortalized cell lines TG tumor mouse models have enabled the immortalization of numerous cell lines from isolated tumor cells (see further). The cells are usually immortalized at an intermediate step of their cytodifferentiation, hence often differing from normal, terminally differentiated cells. The SV40 genome or other oncogenic sequences have also been utilized in immortalizing gonadal somatic cells in vitro, but their stability is questionable, since the oncogenic plasmids stay mostly episomal and can be eliminated during cell division (Windle et al., 1990). Cell lines derived by in vivo immortalization in TG mice carry chromosomally integrated oncogenic sequences, and therefore are considered more stable. How-

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ever, it has to be kept in mind that immortalized cells seldom represent exact counterparts of the original cell type. The immortalized cells might undergo further transformation during prolonged cultures, which sometimes is accompanied by secondary changes through chromosomal mutations (Amsterdam and Rotmensch, 1987; Rao et al., 1993). 4.1. Development of immortalized testicular Sertoli cell lines The use of primary Sertoli cell cultures has been vital for establishing the role of these cells in protein secretion (Kissinger et al., 1982; Skinner and Griswold, 1982) and responsiveness to FSH stimulation (Dorrington and Armstrong, 1979). Between days 6 and 10 in culture, primary Sertoli cells lose the ability to respond to hormonal stimulation. However, these cultures are often contaminated by germ cells and/or peritubular cells (McGuinness et al., 1994). For this reason there is a demand of a Sertoli cell line to facilitate the understanding of their specific functions (McGuinness et al., 1994). The establishment of a well-characterized Sertoli cell line was a prerequisite for subsequent in vitro analysis of the physiological role of this cell type in germ cell proliferation and differentiation. Sertoli cell lines have been established either from spontaneous immortalization (like TM4 cell line) (Mather, 1980), from transfected somatic cell lines (Lopez et al., 1999), or from targeted testicular tumors (Peschon et al., 1992; Paquis-Flucklinger et al., 1993; Dutertre et al., 1997; Bourdon et al., 1998). Some studies have also been made on rat Sertoli cells from primary cultures with the temperature-sensitive mutant of Tag (Roberts et al., 1995; Jiang et al., 1997), or normal Tag (Pognan et al., 1997), but not with as much success as with the murine Sertoli cells. Table 1 presents the most successful spontaneous or clonal cell lines immortalized from spontaneously occurring testicular tumors. Table 2 presents the most successful spontaneous or clonal cell lines immortalized from spontaneously occurred testicular tumors, as well as the targeted tumorigenesis in TG and knock-out (KO) mice with testicular tumors and eventual in vivo immortalization of cell lines. However, these testicular cell lines were derived mainly from testis tumors.

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4.1.1. Immortalization of murine testicular Sertoli cell lines with the large T-antigen of polyomavirus (PyLT) The large T-antigen of polyomavirus efficiently immortalizes rodent fibroblasts, but, unlike the Tag, it is not sufficient to achieve complete oncogenic transformation. Sertoli cell tumors were produced in TG mice using the polyoma virus large Tag coding sequence under control of the viral enhancer-promoter region (Paquis-Flucklinger et al., 1993). It has been reported that PyLT provides an efficient source of untransformed testicular cell lines from the tumors and from the apparently normal looking testes before the onset of tumor growth that occurs beyond 12 months of life (Paquis-Flucklinger et al., 1993). The testicular cell line 15P-1 from mature PyLT expressing animals exhibited some characteristics of Sertoli cells, but it also contained a significant fraction of germ cells as suggested by the expression of the germ-specific genes LDH-X, Hox 1.4 and c-kit (Paquis-Flucklinger et al., 1993). For this reason, the isolation and characterization of a clonal Sertoli cell line (42GPA9) has been carried out (Bourdon et al., 1998). This 42GPA9 cell line demonstrated biochemical features of normal Sertoli cells. Messages and proteins of transferrin, sulfated glycoprotein-2, and c-kit ligand were detected by RT-PCR and Western blot analyses (Bourdon et al., 1998), and zymographic analysis indicated that the 42GPA9 cell line secreted tissue-type plasminogen activator. These cells also retained their FSHR expression, as suggested by their responsiveness to this gonadotropin (morphological and phagocytic changes, stimulation of cAMP production) and the presence of FSHR mRNA by RT-PCR (Bourdon et al., 1998). Another interesting aspect of the 42GPA9 cells is their ability to form tight junctions at confluency, as demonstrated by electron microscopy and immunolocalization of the tight junction-associated protein zonula occludens-1 (Bourdon et al., 1998). The 42GPA9 cell line, due to retention of several important hallmarks of normal Sertoli cells, might prove useful for studies on Sertoli cell function and on Sertoli-germ cell interactions (Bourdon et al., 1998). 4.1.2. Immortalization of murine testicular Sertoli cells with Tag A successfully immortalized Sertoli cell line, (MSC-1) (Fig. 1) was established from TG mice expressing Tag under the anti-Müllerian hormone promoter (Peschon et al.,

Table 1 Reports on some existing murine cell lines immortalized from spontaneous testicular tumors or from non-tumorigenic primary cultures, which underwent spontaneous immortalization in vitro (TM3 and TM4) Tumor type

Mouse strain

Reference

Immortalized cell line/name of cell line

Testicular epithelial (Leydig) cells Leydig cell tumor Leydig cell tumor Leydig cell tumor Sertoli cell tumor Testicular epithelial (Sertoli) cells

BALB/c C57Bl/M5480 C57Bl/M5480P BALB/cJ and BALB/cByJ C3H BALB/c

Mather (1980) Ascoli and Puett (1978) Rebois (1982) Mahler and Sundberg (1997) Franks (1968) Mather (1980)

+/TM3 +/MA-10 and MA-12 +/mLTC-1 −/− −/− +/TM4

“−”, No cell line have been immortalized; “+”, cell line have been immortalized.

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Table 2 Studies on targeted tumorigenesis in TG and KO mice with testicular tumors and immortalized in vivo cell lines Expressed/disrupted (KO) gene

Tumor type

Reference

Immortalized cell line/ name of cell line

Promoter

Structural gene

MTa

hAMH

Leydig cell hyperplasia

Behringer et al. (1990)

−/−

MT

PV large T-antigen

Leydig cell hyperplasia and tumors

Chalifour et al. (1992)

−/−

Lebel and Mes-Masson (1994a)

+/MT-PVLT-10

SV40 T-antigen/pSVtsA58

Leydig cells

Ohta et al. (2002b)

+/TTE1

AMH

SV40 T-antigen

Sertoli cell tumors

P53 (KO)

Teratocarcinomas, gonadoblastomas, seminomas, Leydig cell tumors

Peschon et al. (1992) Dutertre et al. (1997) Donehower et al. (1992)

+/MSC-1 +/− −/−

SV40 T-antigen/pSVtsA58

Sertoli cells

Tabuchi et al. (2002)

+/TTE3

Viral

Sertoli cell tumors

Paquis-Flucklinger et al. (1993)

+/15P-1

Bourdon et al. (1998)

+/42GPA9

Leydig cell tumors

Kondoh et al. (1994)

−/−

Leydig cell tumors Leydig cell tumors Leydig cell hyperplasia/adenoma

Kananen et al. (1996a) Fowler et al. (2000) Li et al. (2001)

+/BLT-1 −/− −/−

Viral Inhibin-␣ MMTV UbC

PV large T-antigen enhancer/promoter Human PV(HPV 15 E6E7) enhancer/promoter SV40 T-antigen int-5/aromatase P450 aromatase

a AMH, anti-Müllerian hormone; MT, metallothioneine; PV, polyoma virus; SV, simian virus; MMTV, mouse mammary tumor virus; UbC, ubiquitin; SV40 T-antigen/pSVtsA58, temperature-sensitive SV40 large T-antigen gene pSVtsA58; “−“, no cell line have been immortalized; “+”, cell line have been immortalized.

1992). The MSC-1 cells retained certain features of normal Sertoli cells, such as expression of transferrin, sulfated glycoprotein-2 and inhibin-␤B, but no longer expressed the FSHR. The authors speculated that the lack of inhibin-␣ expression could be connected to the loss of the FSHR expression (Peschon et al., 1992). Comparing various characteristics of MSC-1 cells to primary cultures of Sertoli cells from immature rats or adult mice indicated that the MSC-1 cells were ultrastructurally similar to mouse Sertoli cells in culture (Peschon et al., 1992; McGuinness et al., 1994). It has also been proven that the MSC-1 cell line is composed of a single cell type displaying numerous characteristics of Sertoli cells, which may provide a unique source of cells for studying various aspects of Sertoli cell function (Peschon et al., 1992; McGuinness et al., 1994). Recently, a conditionally immortalized testicular Sertoli cell line from 8-week-old male transgenic mice bearing the temperature-sensitive Tag gene pSVtsA58 was developed and partially characterized (Tabuchi et al., 2002). When the cells were cultured at a permissive (33 ◦ C) or non-permissive (39 ◦ C) temperature on a collagen type I pre-coated culture vessel, they grew at 33 ◦ C but not at 39 ◦ C, and large T-antigen was expressed only in the nuclei at 33 ◦ C, indicating that the temperature-sensitive growth phenotype of the cells arose as a result of the function of temperature-sensitive simian virus 40 large T-antigen. The cells did not show any colony-forming activity in soft agar or form tumors in subcutaneous tissue in nude mice, showing that TTE3 cells were not transformed

(Tabuchi et al., 2002). The cells expressed mRNAs encoding steel factor, inhibin-␣, transferrin, follicle-stimulating hormone receptor and sulfated glycoprotein-2. Moreover, expression of vimentin and zonula occludens-1 was observed in the cytoplasm and on the boundaries of the cells, respectively, but the expression levels of transferrin and zonula occludens-1 were significantly elevated at 39 ◦ C (Tabuchi et al., 2002). No data about the steroidogenic function of this cell line, showing closer physiological resemblance to normal Sertoli cells, has yet been shown. As this cell line expresses the endogenous FSHR, it could be used for studying Sertoli cell function in vitro along with the above-mentioned Sertoli cell lines. 4.1.3. Tag immortalized Sertoli cell lines with characteristics of prepubertal Sertoli cells Two murine Sertoli cell lines were generated from 6.5-day-old pre-tumoral testes of TG mice expressing the AMH promoter/Tag fusion gene (Dutertre et al., 1997). One line (SMAT1), displayed expression pattern that was characteristic of prepubertal Sertoli cells, by high levels of SF-1, AMH, but no transferrin mRNAs. SMAT1 cells also secreted the AMH protein into the culture medium and expressed the AMH receptor (Dutertre et al., 1997). This study showed that the timing of cell line derivation might play a role even when using a developmentally regulated promoter (Dutertre et al., 1997). Further studies are needed to characterize these cell lines.

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Fig. 1. A microscopic view of the mLTC-1 cells (A and C), MSC-1 cells (B) and BLT-1 cells (D) showing homogenous, fibroblast-like appearance (except the mLTC-1 cells, which are bigger cells) (the bar in panel (A) is 40 ␮m, the same magnification in (B–D)).

4.2. Murine Sertoli cell lines and the FSHR expression In the testis, the FSHR gene is expressed only in Sertoli cells. To date, the mechanism(s) responsible for Sertoli cell-specific FSHR expression are unknown. Among the Sertoli cell lines reported in the literature, only few are responsive to FSH stimulation (Bourdon et al., 1998). The FSH stimulated cAMP response reported in this study was 2.5-fold compared to basal cAMP production (Bourdon et al., 1998), and further functional studies are needed to show whether the FSH response persists upon prolonged culture in this cell line. Additional cell lines expressing endogenous FSHR were established from the immature mouse testis (Mather, 1980; Walther et al., 1996; Hofmann and Millan, 1998). Since the physiological responses to FSH change continuously during sexual maturation, these cell lines are not representative of mature Sertoli cells (Bourdon et al., 1998). The MSC-1 Sertoli cells (Peschon et al., 1992) were stably transfected with a plasmid expressing the rat FSHR cDNA under the Tag promoter, resulting in high level of FSHR expression in the transfected cells (Eskola et al., 1998). The FSHR signaling pathway was intact in the FSHR express-

ing MSC-1 cells and they also expressed the inhibin-␣ gene. Since adult Sertoli cells do not proliferate in vitro, the proliferating rFSHR/MSC-1 cells stably expressing the FSHR provide a useful in vitro model for studying some facets of Sertoli cell function, though keeping in mind that these transformed cells are not identical to adult Sertoli cells in vivo. The constitutive nature of FSHR expression in these cells mainly allows the study of post-transcriptional events in the FSHR related functions (Eskola et al., 1998). 4.2.1. Site-specific methylation of the FSHR promoter Some mammalian genes exhibit inverse correlation between the extent of DNA methylation and gene activity (Eden and Cedar, 1994; Siegfried and Cedar, 1997). As the murine MSC-1 Sertoli cells have an inactive FSHR promoter (McGuinness et al., 1994), in a recent study, changes in DNA methylation at specific sites in the FSHR promoter were shown to lead to changes in the DNA–protein interactions at those sites and, subsequently, to transcriptional repression of the FSHR gene (Griswold and Kim, 2001). The extent of methylation of cytosine residues within the core promoter region of genomic DNA isolated from cells/tissues that expressed, or did not express, the FSHR

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gene was analyzed by the sodium bisulfite conversion technique (Griswold and Kim, 2001). All seven cytosine residues in CpG dinucleotides within the core promoter region were found to be unmethylated in primary cultures of rat Sertoli cells that expressed actively the FSHR mRNA. In contrast, in tissues not expressing FSHR the same region of the gene was methylated at each of the CpG dinucleotides examined (Griswold and Kim, 2001). The inactive state of transcription in MSC-1 cells correlates with cytosine methylation in the core promoter of the FSHR gene (Griswold and Kim, 2001). The demethylation of these four sites by treatment of the MSC-1 cells with 5-aza-2 -deoxycytidine (5-azaCdR), known to block DNA methylation in newly replicated DNA molecules, activated the transcription of the FSHR gene (Griswold and Kim, 2001). This study opens up new possibilities for discovering the factors involved in repressing FSHR expression. 4.3. Development of testicular Leydig cell lines 4.3.1. Fetal-type Leydig cells The ontogenesis of Leydig cells in mammals involves at least two generations of cells. The fetal generation of Leydig cells is responsible for masculinization of the primary sex characteristics in fetal and neonatal life. These cells regress thereafter in all the species studied to date. This is followed by the emergence of the adult population of Leydig cells, which is responsible for pubertal masculinization and Leydig cell functions in adult age (for a review, see Saez, 1994). One of the most striking functional differences between fetal and adult rat Leydig cells is the resistance of fetal cells to LH/hCG-induced LHR downregulation and blockage of steroidogenesis (desensitization) (Huhtaniemi et al., 1981, 1984; Warren et al., 1982; Huhtaniemi and Pelliniemi, 1992). In fetal rat Leydig cells, LH/hCG treatment upregulates LH/hCG receptor mRNA and binding sites and increases steroidogenic activity, in contrast to the desensitization, seen in adult rat Leydig cells. Human, rhesus monkey, and rabbit fetal Leydig cells are also resistant to LH/hCG-induced desensitization (Leinonen and Jaffe, 1985). This resistance explains why, in the human, sustained high hCG concentrations are associated with testicular steroidogenic activity in the male fetus (Saez, 1994). The biochemical mechanisms of this resistance are not yet understood. It has been proposed that, at least in the rat, it might be linked to the low aromatase activity and the low level of estradiol receptors in fetal-type Leydig cells, since hCG-stimulated desensitization involves an increase in intracellular estradiol in the adult rat (Dufau, 1988). Although some factors responsible for the proliferation and differentiation of adult Leydig cells have been identified, the molecules triggering the proliferation and differentiation of fetal Leydig cells remain unknown (Habert et al., 2001). Until now, to the best of our knowledge, a fetal-type immortalized Leydig cell line does not exist. It would be of great interest to immortalize such a cell line for

studying the special features of fetal-type Leydig cells in vitro. 4.3.2. Adult-type Leydig cells The adult-type Leydig cells appear in the mouse testis around postnatal day 10 and increase considerably in number after day 15, to reach 25 × 105 cells per testis by the end of puberty (day 56) (Saez, 1994). They are mainly formed through differentiation of mesenchymal cells but also assumed to be recruited by division of pre-existing Leydig cells (Fouquet and Kann, 1987; Hardy et al., 1989; Saez, 1994). The increase in the number of Leydig cells is controlled by both LH and FSH (Odell and Swerdloff, 1976; Kerr and Sharpe, 1985). Leydig cell tumors are the commonest tumors of gonadal stroma (Hawkins and Miaskowski, 1996). Some rare spontaneously developing Leydig cell tumors have been reported in non-inbred mouse and rat strains (Mahler and Sundberg, 1997). As the normal adult Leydig cells do not proliferate and cease to grow after the first trypsinisation for cell culturing (Nagpal et al., 1994), immortalized Leydig cell lines are desired for studying the physiological characteristics of these cells and the molecular pathogenesis of their tumors in vitro. Leydig cell lines have been established either from spontaneous immortalization (like the TM3 cell line) (Mather, 1980), from primary cells transfected with viral oncogenes (Nagpal et al., 1994), by hybridizing freshly isolated murine Leydig cells with existing immortalized Leydig tumor cells (Finaz et al., 1987), or from genetically targeted TG mouse testicular Leydig cell tumors (for a review, see (Rahman et al., 1998). Some studies also have been carried out on rat Leydig cell primary cultures transfected in vitro with Tag (Nagpal et al., 1994), but with less success than with the murine Leydig cell lines. Table 2 presents the cell lines developed by targeted tumorigenesis in TG and KO mice with testicular Leydig cell tumors and eventual immortalization of in vivo cell lines. 4.4. Immortalization of murine testicular Leydig cell lines 4.4.1. Leydig cell tumors with human papillomavirus type 16 (HPV16) TG mice expressing the human papillomavirus type 16 E6 and E7 oncogenes develop testicular tumors in vivo (Kondoh et al., 1994). The HPV16 virus has also been used successfully for in vitro immortalization of Leydig cells from estrogen receptor KO mice (Mueller and Korach, 2001). On cross-matings with other inbred strains, the HPV transgene seems to be dominant in genetic background (Mueller and Korach, 2001). The expression of gonad-specific 3␤-hydroxysteroid dehydrogenase (3␤-HSD) and other steroidogenic enzymes for androgen metabolism suggested Leydig cell origin for this tumor (Mueller and Korach, 2001), although its further functional characterization is needed. No cell lines were yet developed from these immortalized cells.

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4.4.2. Leydig cell tumors overexpressing aromatase Overexpression of aromatase under the MMTV-int-5 promoter resulted in increased estrogen production and altered hormone milieu, leading to the induction of testicular Leydig cell tumors (Fowler et al., 2000). TG mice bearing the human ubiquitin C promoter/human P450 aromatase fusion gene have been shown to have cryptorchidism associated with Leydig cell hyperplasia and Leydig cell adenomas (Li et al., 2001). 4.4.3. Immortalization of testicular Leydig cell lines with PyLT MT-PVLT-10 TG mice expressing the large T-antigen of polyomavirus under control of the mouse metallothionein-1 promoter have been produced (Lebel and Mes-Masson 1994a,b; Lebel et al., 1996). The males of this TG line developed testicular tumors and seminal vesicle engorgement in advanced age (Lebel and Mes-Masson, 1994a). Histological analysis of the adenomatous testes indicated a predominance of Leydig cells, with few normal Sertoli cells or seminiferous tubules remaining, and permanent cell lines were derived from the pre- and post-adenomatous testes (Lebel and Mes-Masson, 1994a). All primary cultures and cell lines expressed the large T-antigen. A primary culture (D-37) derived from an MT-PVLT-10 male with normal testes but enlarged seminal vesicles has been maintained for over 2 years, but injected D-37 cells were unable to form tumors in nude mice. In contrast, an injection of the primary immortal culture (D-4) derived from adenomatous testes of a MT-PVLT-10 mouse consistently resulted in tumor formation in nude mice (Lebel and Mes-Masson, 1994a). Cloning of the D-4 culture resulted in pure Sertoli or Leydig cell clones. However, neither of them alone could form tumors upon injection into nude mice. Injection of a mixture of both cell types (Sertoli and Leydig) resulted in tumor formation, suggesting dynamic interaction between these cell types in MT-PVLT-10-induced tumorigenesis (Lebel and Mes-Masson, 1994a). Differential display technique was employed to compare mRNA expression from immortalized cell lines derived from normal or adenomatous testis from MT-PVLT-10 transgenic males (Lebel et al., 1996), and a complementary DNA fragment corresponding to the mouse Fas antigen receptor was recovered from normal testicular cells but not from tumor cells (Lebel et al., 1996). 4.4.4. Immortalization of “clonal” Leydig cell lines Cloned cell lines have proven to be useful models for exploring the biosynthetic pathways and regulation of endocrine cells (Kilgore and Stocco, 1989). One Leydig cell tumor originating from a mouse of the C57Bl/6 strain, designated M5480, appeared to be hormone responsive, and was adapted for serial transplantation (Moyle and Ramachandran, 1973). Leydig cells derived from the M5480 tumor were adapted to multiply in culture by growing them alternately in culture and in host animals. Mass cultures obtained from culture-derived tumors were cloned, and

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clonal lines were isolated and characterized (Ascoli and Puett, 1978; Ascoli, 1981a). MA-10 and MA-12 (and also mLTC-1, described later on) clonal Leydig cell populations from the M5480 tumor were obtained and shown to have retained several functional characteristics of normal Leydig cells, such as LH responsiveness, gonadotropin binding and steroidogenic response (Ascoli and Puett, 1978; Lacroix et al., 1979; Ascoli, 1981a). These Leydig cells possess the LHR and respond to LH/hCG by increased progesterone production, and only one tumor produced about equal amounts of progesterone and testosterone (Ascoli and Puett, 1978; Lacroix et al., 1979). However, the steroidogenesis of MA-10 and mLTC-1 (see further) is partly impaired, since it did not proceed beyond the progesterone step. An attempt of restoration of testosterone production by MA-10 cells succeeded by hybridizing them with freshly isolated murine Leydig cells (Finaz et al., 1987). This resulted in a subclone, K9, expressing all the steroidogenic enzymes needed for testosterone production (Lefevre et al., 1994). The MA-10 cells also possess the receptors for mouse epidermal growth factor (mEGF), and exposure of the MA-10 cells to mEGF results in a substantial (80–90%) reduction in the number of hCG receptors per cell accompanied by a corresponding reduction in hCG-stimulated steroidogenesis (Ascoli, 1981b). The steroidogenic responses to cholera toxin and 8-Br-cAMP, however, are not affected. The number of LH/hCG receptors per cell varied from 1100 to 2200, in different tumor clones (Ascoli and Puett, 1978). Another clonal Leydig tumor cell line, mLTC-1 (Fig. 1), was established by adapting the transplantable Leydig tumor, M548OP (same origin as for the MA-10 and MA-12 cells), and it was characterized with regard to the gonadotropin-responsive adenylate cyclase system (Rebois, 1982). As MA-10 cells, also these cells express the LHR and display LH/hCG-stimulated cAMP and steroid production, with progesterone being the main steroid produced (Rebois, 1982). mLTC-1 cells were shown to possess a gonadotropin-responsive adenylate cyclase system consisting of a specific hormone receptor, a regulatory component, and a catalytic subunit (Rebois, 1982). The MA-10 and mLTC-1 cells seems to be very similar with regard to their functional characteristics (Rebois, 1982). There are interesting reports on a rat R2C Leydig tumor cell line (Shin et al., 1968), which, unlike MA-10 or mLTC-1 cells, secretes large amounts of progesterone in the absence of hormonal stimulation like most tumors of steroidogenic cell origin (Freeman, 1987) and constitutively express high levels of the steroidogenic acute regulatory (StAR) protein (Stocco and Chen, 1991). Studies to explain the constitutive nature of R2C cell steroidogenesis revealed that steroid synthesis in R2C cells is independent of cAMP production (Stocco and Chen, 1991). In an attempt to further investigate the mechanisms that render R2C cells constitutively steroidogenic, key components involved in the uptake and mobilization of cholesterol were compared and contrasted with both MA-10 and R2C cells to determine whether their

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steroidogenic response could be attributed to the capacity to internalize and efficiently utilize exogenous cholesterol (Rao et al., 2002; Rao et al., 2003). It has been shown that, constitutive StAR expression in R2C cells alone does not appear to be sufficient to elicit the extremely high steroidogenic response, although the rate-limiting step in steroid hormone biosynthesis is the transfer of cholesterol from the outer to the inner mitochondrial membrane (Rao et al., 2003). Indeed, transfection of both MA-10 and COS-1 cells (rendered steroidogenic by cotransfection with the P450scc enzyme system) with StAR cDNA showed only a modest increase in steroid production (3–10-fold, respectively) (King et al., 1995; Lin et al., 1995), while trophic hormone (or cAMP analog) stimulation of MA-10 cells resulted in a robust (500-fold or greater) induction of steroid hormone synthesis. These observations provide further credence to the idea that additional factors may be required to maintain a constant supply of cholesterol precursor to support high rates of steroid synthesis in immortalized cells. 4.4.5. Immortalization of Leydig cell lines with Tag in vitro Recently, a Leydig cell line, TTE1, was established from transgenic mice harboring a temperature-sensitive simian virus 40 (tsSV40) large T-antigen gene (Ohta et al., 2002b). The cells showed temperature-sensitive growth characteristics and a differentiated phenotype at non-permissive temperature. They grew at a permissive temperature (33 ◦ C), but growth was markedly prevented at a non-permissive temperature (39 ◦ C). T-antigen was expressed in the nuclei at 33 ◦ C but disappeared at 39 ◦ C, indicating that the cells show a temperature-sensitive growth phenotype reflected by the tsSV40 large T-antigen. These cells did not show any colony-forming activity in soft agar or form tumors in subcutaneous tissue in nude mice, indicating that the cells were not transformed. TTE 1 cells expressed mRNAs encoding 17␤-HSD types 1 and 3, and inhibin-␣. To identify differentially expressed genes in the process of Leydig cell differentiation, the authors also carried out microarray analysis of TTE1 cells cultured at permissive and non-permissive temperatures (Ohta et al., 2002a). Of the 1081 genes analyzed, the levels of 31 genes were changed, with 24 genes showing increased levels of expression and the remaining 7 genes showing decreased levels. One of the tyrosine kinases Tie2, displayed the greatest change, with a 13.5-fold upregulation under the differentiated condition (Ohta et al., 2002a). These cells could therefore be useful to study the function of Leydig cells. 4.4.6. Immortalization of Leydig cell lines with Tag in vivo We will concentrate further on a gonadal tumor mouse model that was produced in our laboratory (Kananen et al., 1995, 1996a), and on an in vivo immortalized Leydig tumor cell line from these mice. These TG mice (inh␣/Tag) express the Tag under control of a 6-kb fragment of the mouse inhibin ␣-subunit promoter (inh␣) (Kananen et al., 1995). We have utilized the mouse line for establishing permanent cell

lines from ovarian, testicular and adrenal tumors, in studies on inhibin-␣ expression, in studies on gonadotropin dependence of the gonadal tumorigenesis, and for endocrine and gene therapy of the tumors (Kananen et al., 1995, 1996a; Mikola et al., 2001). Histopathological analysis of the tumor sections indicated that the testicular tumorigenesis originated from interstitial cells. At earlier stages of tumor development, the tumorigenesis begun as local hyperplasia of interstitial cells, leaving most of the testis intact, thereby allowing normal spermatogenesis and fertility (Kananen et al., 1996a). We have established a steroidogenic Leydig cell line, BLT-1, from a tumor of a founder inh␣/Tag mouse (Kananen et al., 1996a). Partial characterization of the BLT-1 cells showed that they closely resemble the previously established MA-10 and mLTC-1 cell lines (see earlier). The BLT-1 cells are fairly stable in culture, since LH response is maintained even after 40 passages and these cells are easy to transfect (Fig. 1). As the former cells, BLT-1 cells respond to hCG stimulation by elevated cAMP and progesterone production (Fig. 2a), but display only low testosterone production. The BLT-1 cells possess high-affinity LH receptors up to 36000 sites per cell (Fig. 2b), which is close to the LH receptor number found in primary Leydig cells isolated from mouse testis (Stalvey and Payne, 1983) and showed specific [125 I]iodo-recombinant human LH (rhLH) binding (Fig. 2c). These cells also express all six splice variants of the LH receptor mRNA, the same as mLTC-1 cells (Fig. 2d) (Kananen et al., 1996a), as earlier described (Wang et al., 1991). The BLT-1 cells displayed good stability upon prolonged culture, even at passage number 45, maintaining their functional characteristics (Kananen et al., 1996a). Therefore, despite their lack of testosterone production, BLT-1 cell can be used in studies on the endocrine, paracrine and autocrine regulation of Leydig cells. 4.5. Testicular peritubular cell tumors (GCT) and immortalized peritubular cell lines Immortalized peritubular cells were identified by their spindle-like appearance and their high expression of alkaline phosphatase and the intermediary filament desmin, as well as production of high amounts of collagen. It has been shown that peritubular myoid cells derived from immature rat testes produce factors that modulate Sertoli cell function (P-Mod-S) and that this secretion is controlled in part by androgens (Hoeben et al., 1994). Establishment of a peritubular myoid-like cell line (TR-M) and their interactions with other testicular cell lines have been attempted (Mather and Phillips, 1984), although later on cultured prostatic stromal cells with strong resemblance of peritubular myoid cells and production of mediators with similar activity have been more successful (Hoeben et al., 1994). Heparin and other heparin-like glycosamingoglycans synthesized by Sertoli cells are thought to modulate the growth of peritubular myoid cells during co-cultures (Tung and Fritz, 1991).

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Fig. 2. Example of functional characterization of a testicular Leydig cell line, BLT-1. (a) hCG-stimulated cAMP and progesterone responses of the BLT-1 cells during 1 or 8 h of incubation times, respectively. ∗∗ P < 0.001 vs. control (0 h). Modified data from (Kananen et al., 1996a); reproduced with permission. (b) Top panel (A): Scatchard analysis of LH binding to BLT-1 cells (䊊) and mouse testes homogenate (䊐). The BLT-1 cells possess high affinity LHR with Ka = 1.5 × 1010 L/mol (vs. Ka = 1.1 × 1010 L/mol in mouse testes homogenate). Figure reproduced under the kind permission of the authors (Kananen et al., 1996a). Bottom panel (B): Regulation of LH binding to BLT-1 cells showing increase with 8Br-cAMP, dramatic decrease with 5 mg of hCG/L and loss of binding with 50 mg of hCG/L (48 h incubation). Figure reproduced under the kind permission of the authors (Kananen et al., 1996a). (c) Northern hybridization analysis of LHR mRNA expression in murine mLTC-1, BLT-1 cells and in testes as a control. Each lane contains 20 ␮g of total RNA from the testes, BLT-1 and mLTC-1 cells. The migration of the 18S and 28S rRNAs is depicted on the left of the panels, and the sizes of the different LHR mRNA splice variants (in kb) on the right. One lane for each type of sample are depicted. The top panels show Northern hybridization for LHR mRNA. The bottom panel shows EtBr staining of the 28S ribosomal RNA for control of RNA loading.

It is hypothesized that heparin may play a similar role in maintaining the quiescent peritubular myoid cell phenotype in vivo (Tung and Fritz, 1991). It has also been shown that activin-A is secreted by the peritubular cells in vitro and that this peptide is similar in many ways to the elusive P-mod-S, stimulating basal and FSH-induced inhibin and transferrin production by Sertoli cells (de Winter et al., 1994). Still, there remains the task of further in vivo immortalization attempts and establishment of new peritubal myoid cell lines.

5. Testicular germ cell tumors (GCT) and immortalized germ cell lines Testicular germ cell tumors are subdivided based largely on pathological aspects, into two major groups: seminomas and non-seminomatous germ cell tumors (NSGCT). The relationship of seminomas to NSGCT remains controversial

(Damjanov, 1993a,b). The pathogenesis of NSGCT has been studied experimentally in mice, but there are not yet adequate experimental models of seminomas in laboratory rodents (Damjanov, 1993a,b). Experiments with tumor cell lines derived from animal tumors such as embryonal carcinoma and yolk sac tumor have generated useful data for better understanding of the biology of stem cells (for a review, see Damjanov, 1993b). Until recently, the molecular mechanisms of germ cell tumor transformation, differentiation, or sensitivity and resistance to chemotherapy were poorly understood (Chaganti and Houldsworth, 2000). The final answer to questions pertaining to the cytohistogenesis and classification of human germ cell tumors will however be obtained only after appropriate tumor cell lines of human origin are cloned (Damjanov, 1993b). The germ cell-specific alkaline phosphatase isoenzyme, which is developmentally expressed in primordial germ cells is reported to be a clinically useful marker of germ cell differentiation in the management of germ cell tumors (Hofmann and Millan, 1993;

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Narisawa et al., 1993). Immortalization using the Tag of all cell types contributing to developing seminiferous tubules of the mouse testis was reported (Hofmann et al., 1992), but further improvements of in vitro conditions are necessary for the maintenance of germ cell development (Hofmann et al., 1992). Until now, four established germ cell lines have been described in the literature (Hofmann et al., 1992, 1994; Tascou et al., 2000), all expressing the Tag. Based on its characteristics in phase contrast and electron microscopy, the first established germ cell line (GC-1spg) corresponds to a stage between spermatogonia type B and primary spermatocytes. These cells exhibited testicular cytochrome ct and lactate dehydrogenase-C4 isozyme (Hofmann et al., 1992). The other germ cell lines have characteristics of more advanced germ cell types. GC-2spd(ts) has spermatocyte characteristics and undergoes a conditional meiosis in vitro (Hofmann et al., 1994), GC-3spc(ts) has characteristics of spermatocytes (Hofmann et al., 1994) and GC-4spc has characteristics between preleptotene and early pachytene spermatocytes (Tascou et al., 2000). The latter recently established murine germ cell line used a novel directed promoter-based selection strategy, namely a vector that contains the Tag and the neomycin phosphotransferase II genes under control of the SV40 early promoter and a spermatocyte-specific promoter for human phosphoglycerate kinase 2, respectively (Tascou et al., 2000). Very recently, a rat spermatogonial germ cell line (GC-5spg) was established with characteristics of A spermatogonia, by transfecting a mixed population of purified rat As (stem cells) and Apr spermatogonia with SV40 T-antigen (van Pelt et al., 2002). These cells expressed characteristics of rat spermatogonial stem cells and thus may facilitate investigations into the mechanisms that regulate stem cell proliferation and differentiation. There still remains the need to establish germ cell lines immortalized in vivo in order to enhance our ability to study stage-specific cellular interactions and paracrine regulation of spermatogenesis. There also remains the need for immortalized seminoma cell lines that would be essential for the study of the biology of seminomas. In conventional culture conditions, seminoma cells usually die within the first 3 days after plating (Berends et al., 1991). Tissue culture conditions for establishing such cell lines and significant improvement (viable until 11th day of culture) of survival of the seminoma cells in vitro has been reported by the use of a rat Sertoli cell feeder layer and serum-free medium (Berends et al., 1991). Recent studies have shown that targeted overexpression of glial cell line derived neurotrophic factor (GDNF) in undifferentiated spermatogonia promotes malignant testicular tumors expressing germ cell markers (Meng et al., 2001). The tumors are invasive and contain aneuploid cells, but no distant metastases have been found. The GDNF-induced tumors mimic classic seminomas in men, representing a useful experimental model for testicular germ cell tumors (Meng et al., 2001).

6. Concluding remarks This review provides evidence for usefulness of numerous testicular cell lines in the exploration of the cellular and molecular mechanisms of testicular biology. We reviewed many of these murine cellular models as tools to study the hormonal regulation of testicular germinal and somatic cell functions. These models should further our understanding of testicular physiology and pathophysiology, and probably our ability to develop important new therapies to prevent and treat testicular endocrine and neoplastic diseases and also to assess chemicals for their endocrine-disrupting activities on the testes. TG mice with overexpression or targeted disruption of a regulating factor or its receptor will provide further evidence defining their role in vivo adding to the in vitro data obtained with testicular cell lines. A promising new approach is to test the cytotoxicity of new anticancer drugs in vitro using cell lines. Taken together, the immortalized cell lines provide invaluable tools for in-depth studies of normal and pathological function of testicular somatic and germ cells.

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