Germ Cell Gene Research Articles

Where Do Babies Come From?

T his is a question parents inevitably hear from their children. It's hard enough to find an answer that will satisfy a 4-year-old, much less a reproductive biologist. Understanding the basic biology of reproduction, researchers hope, will guide treatments for infertility, pregnancy-related disorders, and diseases of the reproductive tract, as well as aid in the development of contraceptives, hormone replacements, and methods for assisted reproductive technology (ART). This reproductive biology special issue assembles articles from several active areas of research. Brinster (p. [2174][1]) describes male germ-cell transplantation, a method that can be used in the study of stem cell biology and infertility and in transgenesis. Sassone-Corsi (p. [2176][2]) illustrates the special case of male germ-cell gene expression and how this mechanism differs from that seen in all the other cells of the body. Matzuk et al. (p. [2178][3]) shift attention to the female germ cell and describe the communication between the oocyte and surrounding somatic cells. In order to contribute the appropriate chromosomal complement, germ cells undergo meiosis. Hunt and Hassold (p. [2181][4]) explain sexual dimorphism in meiotic disruption and its consequences. ![Figure][5] CREDIT: RUBBERBALL PHOTOGRAPHY Once germ cells are fully differentiated, fertilization can take place. Primakoff and Myles (p. [2183][6]) outline the fertilization process. After egg and sperm fusion, a blastocyst develops; Paria et al. (p. [2185][7]) describe the molecular events of its implantation. When normal fertilization is impeded, couples often turn to ART. Schultz and Williams (p. [2188][8]) outline recent advances but also warn that basic science must be applied to make protocols safer and more effective. In a related Editorial, Braude (p. [2101][9]) cautions that the success of ART should not be measured only in terms of live births; the health of the children must be considered. Reproduction is a perilous process. In the News section, Jon Cohen (p. [2164][10]) examines the most common cause of miscarriage—aneuploidy—and describes some of the latest techniques aimed at addressing the problem. Even successful pregnancies can leave babies at risk of certain diseases; Jennifer Couzin (p. [2167][11]) reports that disruptions in a fetus's environment can reverberate 50 years later in a propensity for heart disease and other adult ailments. Likewise, some of the physical costs of bearing a child aren't apparent for decades. Marcia Barinaga (p. [2169][12]) explores microchimerism: Cells exchanged during pregnancy can live on indefinitely in the mother and child, spurring autoimmune and other diseases. Those who choose not to reproduce are mostly stuck with contraceptive methods that have been around for a generation; Constance Holden (p. [2172][13]) finds that few new strategies are being researched, despite a growing need for appropriate contraceptives, particularly in the developing world. It is unlikely that we will come up with a satisfactory answer to the 4-year-old's question of where babies come from, for any response is sure to be followed by, “But why?” Similarly, as each hypothesis about reproduction is tested, new questions spring to mind. [1]: /lookup/doi/10.1126/science.1071607 [2]: /lookup/doi/10.1126/science.1070963 [3]: /lookup/doi/10.1126/science.1071965 [4]: /lookup/doi/10.1126/science.1071907 [5]: pending:yes [6]: /lookup/doi/10.1126/science.1072029 [7]: /lookup/doi/10.1126/science.1071601 [8]: /lookup/doi/10.1126/science.1071741 [9]: /lookup/doi/10.1126/science.296.5576.2101 [10]: /lookup/doi/10.1126/science.296.5576.2164 [11]: /lookup/doi/10.1126/science.296.5576.2167 [12]: /lookup/doi/10.1126/science.296.5576.2169 [13]: /lookup/doi/10.1126/science.296.5576.2172

Generation and in vitro differentiation of a spermatogonial cell line.

Spermatogenesis is the process by which spermatogonial stem cells divide and differentiate to produce sperm. In vitro sperm production has been difficult to achieve because of the lack of a culture system to maintain viable spermatogonia for long periods of time. Here we report the in vitro generation of spermatocytes and spermatids from telomerase-immortalized mouse type A spermatogonial cells in the presence of stem cell factor. This differentiation can occur in the absence of supportive cells. The immortalized spermatogonial cell line may serve as a powerful tool in elucidating the molecular mechanisms of spermatogenesis. Furthermore, through genomic modification and transplantation techniques, this male germ cell line may be used to generate transgenic mice and to develop germ cell gene therapy.

Expression of a linker histone-like gene in the primordial germ cells in zebrafish

Similar to many other organisms, specification of primordial germ cells (PGCs) in zebrafish occurs during early development and depends on inheritance of ‘germ plasm’ determinants. Following their specification, the PGCs exhibit characteristic transcriptional profile and cell behaviour. Here we describe the cloning, expression pattern and sub-cellular localization of the zebrafish H1 type linker histone, H1M, which is specifically expressed in the PGCs in the second phase of their development. H1M transcripts are uniformly distributed during blastula stages, but become restricted to the PGCs during gastrulation and remain visible in PGCs as they migrate towards the region of the gonad.

Allele-specific expression of imprinted genes in mouse migratory primordial germ cells

In somatic cells, imprinted genes are expressed monoallelically according to parent-of-origin. In contrast, in 11.5 days post-coitum primordial germ cells (PGCs), and later stage germ cells, these same genes are expressed biallelically, suggesting that imprints inherited from the gametes are largely erased by this stage. To determine when in germ cell development this biallelic expression phenomenon commences, we isolated migrating PGCs by flow cytometry and determined the allele-specific expression of four imprinted genes – Snrpn, Igf2, H19 and Igf2r. The first three genes were expressed monoallelically, while the latter gene was expressed biallelically. These results show that inherited imprints regulating monoallelic expression are largely intact in migrating PGCs.

Methylation Dynamics of Imprinted Genes in Mouse Germ Cells

DNA methylation differences between maternal and paternal alleles of many imprinted genes are inherited from the male and female gametes and subsequently maintained during development. However, the stages of gametogenesis during which methylation imprints are established have not been well defined. In this study, we used bisulfite sequencing to determine the methylation dynamics of the imprinted genes small nuclear ribonucleoprotein N (Snrpn), insulin-like growth factor 2 receptor (Igf2r), mesoderm-specific transcript (Mest; formerly Peg1), paternally expressed gene 3 (Peg3), and H19 fetal liver mRNA (H19). We identified regions in the maternally imprinted genes (Snrpn, Mest, and Peg3) that were unmethylated in sperm but 100% methylated in mature oocytes. Igf2r, which is expressed from the maternal allele, was completely methylated within intronic differentially methylated region 2 in oocytes and unmethylated in sperm. The 5′ region of H19, a paternally imprinted gene, was completely methylated in sperm and unmethylated in oocytes. We examined the methylation status of Snrpn during oocyte growth and maturation. Whereas the DNA of non-growing oocytes was mostly unmethylated, mid-size growing oocytes had a mosaic pattern of allelic methylation, and full acquisition of the methylation imprint was complete by metaphase II. We have identified regions within imprinted genes that show gamete-specific methylation patterns in mature germ cells and demonstrated that maternal methylation imprints on at least one imprinted gene, Snrpn, are established during the postnatal growth phase of oogenesis. Thus, whereas paternal imprints seem to be established early (in diploid gonocytes well before the onset of meiosis), maternal imprints are established late (in growing oocytes that are arrested in the diplotene stage of meiosis). These findings raise the possibility that assisted reproductive technologies that involve in vitro maturation of oocytes may result in developmental abnormalities due to incomplete methylation imprints in immature oocytes.

Chronic cyclophosphamide treatment alters the expression of stress response genes in rat male germ cells.

Increases in the survival rate of men treated with chemotherapeutic drugs and their desire to have children precipitate concerns about the effects of these drugs on germ cells. Azoospermia, oligospermia, and infertility are common outcomes resulting from treatment with cyclophosphamide, an alkylating agent. Exposure of male rats to cyclophosphamide results in dose-dependent and time-specific adverse effects on progeny outcome. Elucidation of the effects of chronic low-dose cyclophosphamide treatment on the expression of stress response genes in male germ cells may provide insight into the mechanisms underlying such adverse effects. Male rats were gavaged with saline or cyclophosphamide (6 mg/kg) for 4-5 wk; pachytene spermatocytes, round spermatids, and elongating spermatids were isolated; RNA was extracted and probed on cDNA arrays containing 216 cDNAs. After saline treatment, 125 stress response genes were expressed in pachytene spermatocytes (57% of genes studied), 122 in round spermatids (56%), and 83 in elongating spermatids (38%). Cyclophosphamide treatment reduced the number of genes detected in all germ cell types. The predominant effect of chronic cyclophosphamide exposure was to decrease the expression level of genes in pachytene spermatocytes (34% of genes studied), round spermatids (29%), and elongating spermatids (4%). In elongating spermatids only, drug treatment increased the expression of 8% of the genes studied. The expression profiles of genes involved in DNA repair, posttranslational modification, and antioxidant defense in male germ cells were altered by chronic cyclophosphamide treatment. We hypothesize that the effects of cyclophosphamide exposure on germ cell gene expression during spermatogenesis may have adverse consequences on male fertility and progeny outcome.

Genes associated with the development of the male germ line

The development of the mammalian germ line has been well studied, from the designation of primordial germ cells and their migration in the embryo to their progression through gametogenesis. The pattern of germ cell development, as established through classical studies, is now being overlaid with molecular, genetic and epigenetic data. Eventually, proteonomics will lead to a deeper understanding of the function of these genes. Through knowledge of germ cell gene expression patterns, it is now possible to develop transgenic molecular tools for the isolation of germ cells at different stages of development. By linking stage-specific germ cell promoter regions to the green fluorescent protein (GFP) reporter gene it is possible to tag these cells genetically for histological identification and cell sorting. Our long-term goal is to develop male germ cells as stem cells for therapeutic purposes. It is hoped that this goal will be achieved by purifying germ cells at different stages in development and gaining a deeper understanding of them by studying their gene expression patterns, potency and plasticity, both in vivo and in vitro.

Male germ cell gene expression.

Formation of the male gamete occurs in sequential mitotic, meiotic, and postmeiotic phases. Many germ cell-specific transcripts are produced during this process. Their expression is developmentally regulated and stage specific. Some of these transcripts are product of genes that are male germ cell-specific homologs of genes expressed in somatic cells, while some are expressed from unique genes unlike any others in the genome. Others are alternate transcripts derived from the same gene as transcripts in somatic cells but differing from them in size and/or overall sequence. They are generated during gene expression by using promoters and transcription factors that activate transcription at different start sites upstream or downstream of the usual site, by incorporation of alternate exons, by germ cell-specific splicing events, and by using alternate initiation sites for polyadenylation. Male germ cell development consists of an assortment of unique processes, including meiosis, genetic recombination, haploid gene expression, formation of the acrosome and flagellum, and remodeling and condensation of the chromatin. These processes are intricate, highly ordered, and require novel gene products and a precise and well-coordinated program of gene expression to occur. The regulation of gene expression in male germ cells occurs at three levels: intrinsic, interactive, and extrinsic. A highly conserved genetic program "intrinsic" to germ cells determines the sequence of events that underlies germ cell development. This has been underscored by recent studies showing that meiosis involves many genes that have been conserved during evolution from yeast to man. During meiosis and other processes unique to germ cells, the intrinsic program determines which genes are utilized and when they are expressed. In the postmeiotic phase, it coordinates the expression of genes whose products are responsible for constructing the sperm. The process of spermatogenesis occurs in overlapping waves, with cohorts of germ cells developing in synchrony. The intrinsic program operating within a particular germ cell requires information from and provides information to neighboring cells to achieve this coordination. Sertoli cells are crucial for this "interactive" process as well as for providing essential support for germ cell proliferation and progression through the phases of development. The interactive level of regulation is dependent on "extrinsic" influences, primarily testosterone and follicle-stimulating hormone (FSH). Studies during the last 4 years have established that FSH is not essential for germ cell development but instead serves an important supportive role for this process. While testosterone is essential for maintenance of spermatogenesis, it acts on Sertoli cells and peritubular cells and has indirect effects on germ cells. The extrinsic and interactive processes are extremely important for establishing and maintaining an optimum environment within which gametogenesis occurs. Nevertheless, an intrinsic evolutionarily conserved genetic program regulates male germ cell gene expression and development.

Absence of germline infection in male mice following intraventricular injection of adenovirus.

The possibility of inadvertent exposure of gonadal tissue to gene therapy vectors has raised safety concerns about germline infection. We show here that the receptor for coxsackie B viruses and adenoviruses 2 and 5 (CXADR) is expressed in mouse germ cells, suggesting the possibility that these viruses could infect germ cells. To directly assess the risk of germline infection in vivo, we injected an adenovirus carrying the germ-cell-specific protamine promoter fused to the bacterial lacZ reporter gene into the left ventricular cavity of mice and then monitored expression of the reporter gene in germ cells. To differentiate between infection of stem cells and differentiating spermatogenic cells, we analyzed expression of the reporter cassette at different times after viral delivery. Under all conditions tested, mice did not express the Escherichia coli beta-galactosidase protein in developing spermatids or in mature epididymal spermatozoa. Primary germ cells cultured in vitro were also refractory to adenoviral infection. Our data suggest that the chance of vertical germline transmission and insertional mutagenesis is highly unlikely following intracoronary adenoviral delivery.

Expression of stress response genes in germ cells during spermatogenesis.

During germ cell development different spermatogenic cell types show remarkable variation in their susceptibility to stressful stimuli. Various cellular mechanisms are triggered in germ cells after exposure to stress, but the expression of only a few of the genes involved in such pathways has been studied during spermatogenesis. In the present study we determined the expression profiles of 216 stress response genes in isolated rat germ cells (pachytene spermatocytes, and round and elongating spermatids) using cDNA atlas arrays. Of the 216 genes studied, 86 were detected in pachytene spermatocytes, 82 in round spermatids, and 52 in elongating spermatids. Fifty percent (48) of the total number of genes detected during spermatogenesis were detected in all three cell types while nearly 25% (25) were expressed exclusively in pachytene spermatocytes and round spermatids; some cell specific transcripts were observed also. The use of the K means clustering method allowed us to group genes by their pattern of expression during spermatogenesis; five specific expression profiles were obtained and analyzed. To determine how stress response genes are regulated throughout spermatogenesis, we examined the expression of genes involved in stress response mechanisms such as heat shock proteins-chaperones, DNA repair, and oxidative stress. Genes belonging to these families were differentially expressed during germ cell development. We suggest that the differential expression of stress response genes during spermatogenesis contributes to the selectivity of the susceptibility of germ cells to stress.

Aromatase messenger RNA is derived from the proximal promoter of the aromatase gene in Leydig, Sertoli, and germ cells of the rat testis.

It has long been recognized that individual cell types within the testes possess the capacity to synthesize estrogen. A number of studies on different species have demonstrated that the levels of aromatase expression and the patterns of regulation are distinct between the different cell types of the testes. Whereas a variety of promoters have been shown to contribute to the patterns of aromatase expression in different cell lineages, studies using ovarian RNA, testis RNA, and Leydig cell tumor lines have demonstrated that the same promoter (promoter II) was used in each. Recent experiments using potent aromatase inhibitors or analysis of animals in which the genes encoding the estrogen receptor-alpha (ER-alpha) or the aromatase, P450, are defective, have confirmed the importance of local estrogen formation in normal testicular function. In order to permit experiments to identify the elements controlling aromatase expression in the individual cell compartments of the testes, we prepared RNA from purified preparations of Leydig, Sertoli, and germ cells. Using specific oligonucleotide primers, the sites of initiation of the aromatase mRNA were determined using rapid amplification of cDNA ends (RACE) and nucleotide sequence analysis of the resulting cDNA fragments. Our results indicate that aromatase mRNA is derived from the proximal promoter (PII) of the aromatase gene in each of the major cell types of the rat testes.

Isolation and characterization of differentially expressed genes in invasive and non-invasive immortalized murine male germ cells in vitro

In an attempt to elucidate the potential of premeiotic male germ cells to malignant transformation both the invasiveness and the differential gene expression of several putative tumor markers of the spermatogonia-derived cell line GC-1spg and the spermatocyte-derived cell line GC-4spc were analyzed. Studies, using RT-PCR analysis, of the expression pattern of the alkaline phosphatase isoenzymes which serve as markers for testicular germ cell tumors demonstrated that the expression of the endogenous mouse embryonic alkaline phosphatase (EAP) is upregulated in the GC-1spg cell line. Additionally, after transfection of GC-1spg cells and GC-4spc cells with a GCAP-CAT construct, an increased promoter activity of the human germ cell alkaline phosphatase (GCAP), the equivalent human isoenzyme of EAP, was shown in GC-1spg. Furthermore, an in vitro Matrigel invasion assay revealed a significant higher invasive potential of GC-1spg cells as compared to GC-4spc cells. Finally, a suppression subtractive hybridization on RNA of invasive GC-1spg cells and non-invasive GC-4spc cells was performed. In total, 31 cDNA sequences were isolated and further analyzed. Among these, 18 known sequences and 13 unknown sequences were determined. Northern blot analysis revealed that one unknown gene and eight known genes, namely integrin alpha 6, L6 antigen, annexin VIII, BVL-1 retrotransposon, protective protein, replacement variant histone 3.3, alpha-catenin and LPS-binding protein, are over-expressed in invasive GC-1spg cells. Taken together, both the enhanced invasive activity of GC-1spg cells and the upregulated expression of genes involved in the process of tumor progression suggest that the immortalized spermatogonia-derived cell line GC-1spg does have a higher potential to malignant transformation than the immortalized spermatocyte-derived cell line GC-4spc.

Ethical issues of human germ-cell therapy: a preparation for public discussion.

Public debate over the use of techniques that result in heritable changes to human germ cells (i.e., sperm and ova), called germ-cell gene interventions, lags far behind the development of such therapies. Such a debate is particularly needed now because the first steps in somatic-cell, or non-heritable, gene therapy have taken place and may accelerate the beginning of germ-cell gene therapy trials. Because germ-cell therapy affects future generations, its moral status differs considerably from that of somatic-cell therapy. To stimulate and inform public discussion, the authors review the major ethical arguments for and against germ-cell therapy that are found in the literature. (These arguments include the dangers of "playing God," of moving on the "slippery slope" to germ-cell manipulations for enhancement rather than therapy, and of causing irreversible changes to the genomes of future generations.) They demonstrate that these arguments do not apply uniquely to such therapy, since most of the properties of germ-cell therapy are present in other medical interventions or elsewhere in social interactions. For example, there are many examples of human activities that are irreversible and have effects on future generations. However, this lack of uniqueness does not necessarily imply that the moral status of germ-cell gene therapy is unique, or that it should be banned forever. The public, and especially health policymakers, researchers, and clinicians, must reflect on and discuss the issues outlined in this article before efforts are made to introduce changes into the germ cells of human beings.

Identification of meiotic and postmeiotic gene expression in testicular tissue of patients histologically classified as Sertoli cell only

Objective: To determine whether meiotic and postmeiotic germ cell gene products could be detected in biopsy specimen from patients with Sertoli cell only (SCO) and maturation arrest. Design: Prospective clinical study. Setting: University-based departments and laboratories. Patient(s): Nine patients, seven with nonobstructive azoospermia (12 biopsies) and two with obstructive azoospermia (controls) (2 biopsies). Intervention(s): Specimens were divided into three parts: IVF laboratory, histology, and molecular analysis. Germ cell-specific messenger RNAs (mRNAs) were detected by extracting total RNA for Northern blotting or reverse transcription-polymerase chain reaction. Main Outcome Measure(s): Detection of meiotic (lactate dehydrogenase C4) and postmeiotic (transition protein 1 and protamine 1 and 2) gene expression and correlation with histologic and IVF laboratory findings. Result(s): The IVF laboratory identified spermatozoa in 3 of 14 biopsies (controls and severe hypospermatogenesis). Histologically, 6 of 14 biopsies (43%) were diagnosed as SCO, 4 (29%) maturation arrest, 2 (14%) severe hypospermatogenesis, and 2 normal. Molecular analysis showed mRNA for meiotic and postmeiotic genes in 12 of 14 biopsies (86%) ( P = .006), of which 4 (67%) in SCO and 3 (75%) in maturation arrest. Conclusion(s): Differentiated germ cells are present in biopsies of men histologically diagnosed as SCO. Absence of these molecular markers strengthens the histologic diagnosis and helps the physician in counseling the infertile couple.

Rearrangement of immunoglobulin genes in shark germ cells.

The variable (V), (diversity [D]), and joining (J) region recombinases (recombination activating genes [RAGs]) can perform like transposases and are thought to have initiated development of the adaptive immune system in early vertebrates by splitting archaic V genes with transposable elements. In cartilaginous fishes, the immunoglobulin (Ig) light chain genes are organized as multiple VJ-constant (C) clusters; some loci are capable of rearrangement while others contain fused VJ. The latter may be key to understanding the evolutionary role of RAG. Are they relics of the archaic genes, or are they results of rearrangement in germ cells? Our data suggest that some fused VJ genes are not only recently rearranged, but also resulted from RAG-like activity involving hairpin intermediates. Expression studies show that these, like some other germline-joined Ig sequences, are expressed at significant levels only early in ontogeny. We suggest that a rejoined Ig gene may not merely be a sequence restricting antibody diversity, but is potentially a novel receptor no longer tied to somatic RAG expression and rearrangement. From the combined data, we arrived at the unexpected conclusion that, in some vertebrates, RAG is still an active force in changing the genome.

The Drosophila gene stand still encodes a germline chromatin-associated protein that controls the transcription of the ovarian tumor gene.

The Drosophila gene stand still (stil) encodes a novel protein required for survival, sexual identity and differentiation of female germ cells. Using specific antibodies, we show that the Stil protein accumulates in the nucleus of all female germ cells throughout development, and is transiently expressed during early stages of male germline differentiation. Changes of Stil subnuclear localization during oogenesis suggest an association with chromatin. Several mutant alleles, which are point mutations in the Stil N-terminal domain, encode proteins that no longer co-localized with chromatin. We find that Stil binds to many sites on polytene chromosomes with strong preference for decondensed chromatin. This localization is very similar to that of RNA polymerase II. We show that Stil is required for high levels of transcription of the ovarian tumor gene in germ cells. Expression of ovarian tumor in somatic cells can be induced by ectopic expression of Stil. Finally, we find that transient ubiquitous somatic expression of Stil results in lethality of the fly at all stages of development.

Germ Cell Development in the XXY Mouse: Evidence That X Chromosome Reactivation Is Independent of Sexual Differentiation

Prior to entry into meiosis, XX germ cells in the fetal ovary undergo X chromosome reactivation. The signal for reactivation is thought to emanate from the genital ridge, but it is unclear whether it is specific to the developing ovary. To determine whether the signals are present in the developing testis as well as the ovary, we examined the expression of X-linked genes in germ cells from XXY male mice. To facilitate this analysis, we generated XXY and XX fetuses carrying X chromosomes that were differentially marked and subject to nonrandom inactivation. This pattern of nonrandom inactivation was maintained in somatic cells but, in XX as well as XXY fetuses, both parental alleles were expressed in germ cell-enriched cell populations. Because testis differentiation is temporally and morphologically normal in the XXY testis and because all germ cells embark upon a male pathway of development, these results provide compelling evidence that X chromosome reactivation in fetal germ cells is independent of the somatic events of sexual differentiation. Proper X chromosome dosage is essential for the normal fertility of male mammals, and abnormalities in germ cell development are apparent in the XXY testis within several days of X reactivation. Studies of exceptional germ cells that survive in the postnatal XXY testis demonstrated that surviving germ cells are exclusively XY and result from rare nondisjunctional events that give rise to clones of XY cells.

Methylation Levels at Selected CpG Sites in the Factor VIII and FGFR3 Genes, in Mature Female and Male Germ Cells: Implications for Male-Driven Evolution

Transitional mutations at CpG dinucleotides account for approximately a third of all point mutations. These mutations probably arise through spontaneous deamination of 5-methylcytosine. Studies of CpG mutation rates in disease-linked genes, such as factor VIII and FGFR3, have indicated that they more frequently originate in male than in female germ cells. It has been speculated that these sex-biased mutation rates might be a consequence of sex-specific methylation differences between the female and the male germ lines. Using the bisulfite-based genomic-sequencing method, we investigated the methylation status of the human factor VIII and FGFR3 genes in mature male and female germ cells. With the exception of a single CpG, both genes were found to be equally and highly methylated in oocytes and spermatocytes. Whereas these observations strongly support the notion that DNA methylation is the major determining factor for recurrent CpG germ-line mutations in patients with hemophilia and achondroplasia, the higher mutation rate in the male germ line is apparently not a simple reflection of sex-specific methylation differences.

K-FGF Protoncogene Expression is Associated With Murine Testicular Teratogenesis, But is Not Involved During Mouse Testicular Development

The k-FGF gene, which belongs to the family of the fibroblast growth factor genes, is implicated in tumoral and developmental processes. It is expressed in embryonal carcinoma cells, in embryonic stem cells, during limb and tooth formation and in some germ cell tumors. However, the expression of this protooncogene during testicular development as well its relationship to spontaneous teratogenesis have not been determined. Here we investigate k-FGF expression during testicular development in mice, as well as in a spontaneous testicular teratoma (STT) and in the OTT6050 teratocarcinoma (TC) by Northern blotting, RT-PCR and it situ hybridization. Several data indicate that k-FGF gene contains downstream regulatory sequences which bind octamer factors. One of these transcription factors which binds to k-FGF enhancer is Oct-4. Although the k-FGF gene is activated by Oct-4 in embryonal carcinoma and embryonic stem cells and Oct-4 is expressed in the germ cells of the embryo, our results indicate that there is no detectable k-FGF expression in mouse testicular germ cells at any stage of development. This indicates that Oct-4 does not activate transcription of the k-FGF gene in mouse germ cells, and that k-FGF is not implicated during testicular development. We also show that there is a high k-FGF expression in the experimental OTT6050 TC, but only very low levels in a murine differentiated STT, suggesting that k-FGF activation may be responsible for the genesis and development of STT, behaving as a marker of malignancy in these neoplasms.

HAPLOID MALE GERM CELLS SHOW NO SUSCEPTIBILITY TO TRANSFORMATION BY SIMIAN VIRUS 40 LARGE TUMOUR ANTIGEN IN TRANSGENIC MICE

Cell-type specific tumorigenesis can be induced in transgenic mice by the directed expression of simian virus 40 (SV 40) large tumour antigen (TAg). In an attempt to determine the susceptibility of haploid male germ cells to neoplastic transformation by this oncogene, transgenic mice were generated that harboured a chimeric gene composed of the SV40 T antigen genes fused to the 2.3-kb 5′ flanking sequences of the rat proacrosin gene. It was previously shown that this regulatory sequence is able specifically to direct the expression of CAT reporter gene in male germ cells with the onset of translation in early haploid male germ cells. The transgene showed regulated expression in male germ cells. Although T antigen immunostaining was detected specifically in spermatids, no testicular pathology was observed. This indicates that spermatids show no susceptibility to transformation by oncogene TAg. However, in about 10% of animals of two independent transgenic lines, we could find non-testicular tumours in abdomen with a sarcoma-like structure in advanced age which showed SV40 TAg expression.