Adult Germ Cells Research Articles

Infantile and adult testicular germ cell tumors: a different pathogenesis?

Most adult testicular germ cell tumors have a characteristic chromosomal abnormality that is an isochromosome 12p [i(12p)]. Furthermore, these tumors are characterized by a chromosome number in the triploid range and gains and losses of (parts of) specific chromosomes. Cytogenetic investigation of three cases of infantile testicular germ cell tumors, all diagnosed as yolk sac tumors, revealed highly abnormal karyotypes. We found one case to be diploid; the other two cases were in the hypertriploid/hypotetraploid range. Structural abnormalities of chromosomes 1, 3, and 6 were recurrent and no i(12p) was found. Our results, together with data from the literature, suggest that infantile and adult testicular germ cell tumors have a different origin and pathogenetic pathway. Aberrations of chromosomes 1, 3, and 6 may play an important role in the pathogenesis of infantile testicular yolk sac tumors.

Obox, a Family of Homeobox Genes Preferentially Expressed in Germ Cells

We used in silico (electronic database) subtraction to identify ESTs that are preferentially expressed in the adult mouse germ cells. During our analysis, we identified Obox1 and Obox2 transcripts as preferentially expressed in the mouse unfertilized egg libraries. Obox1 and Obox2 transcripts encode homeodomain proteins of 204 amino acids that share 97% identity with each other. Further characterization of mouse BACs encoding Obox1 and Obox2, as well as available BAC and EST sequences in GenBank, identified four closely related genes: Obox3, Obox4, Obox5, and Obox6. Northern blot analyses and RT-PCR from 10 different adult mouse tissues showed that the six Obox family transcripts are preferentially expressed in the gonads. In situ hybridization detected Obox1 and Obox6 transcripts exclusively in oocytes as early as one-layer follicles and throughout folliculogenesis. Obox1, Obox2, Obox3, Obox4, Obox5, and Obox6 map to proximal chromosome 7 in the mouse. The Obox1 and Obox2 genomic structures revealed the presence of six exons each. The Obox genes represent a new family of tissue-specific homeobox genes preferentially expressed in gonads.

Low Expression of DNA Polymerase β in Human Testicular Germ Cell Tumours: Impact on Foetal Gonocytic Origin Theory

Different models of pathogenesis of adult testicular germ cell tumours (TGCTs) are presented. Analysis of telomeric length and DNA polymerase g expression suggests that seminoma and nonseminoma, two main histological types of TGCTs, derive independently from transformed foetal primordial cells.

Chromosomal imbalances associated with carcinoma in situ and associated testicular germ cell tumours of adolescents and adults

Carcinoma in situ (CIS) or intratubular germ cell neoplasia is generally considered the precursor lesion of adult testicular germ cell tumours (TGCT). The chromosomal imbalances associated with CIS and the corresponding seminoma (SE) or nonseminoma (NS) have been determined by comparative genomic hybridization (CGH) analysis of microdissected material from seven cases. Significantly, the CIS showed no gain of 12p material whereas in the invasive components of all cases gain of 12p was found, in 2 cases associated with amplification of the 12p11.2–12.1 region. Interphase fluorescence in situ analysis was consistent with this and provided evidence for the i(12p) or 12p11.2–12.1 amplification in the SE and NS but not in the corresponding CIS. This suggests a role for these changes in progression of CIS to invasive testicular cancer or progression of the invasive disease. Other imbalances such as gain of material from chromosomes 1, 5, 7, 8, 12q and X and loss of material from chromosome 18 were frequently identified (> 40% of cases) in the CIS associated with both SE and NS as well as in the invasive components. Loss of material from chromosome 4 and 13 and gain of 2p were more frequently found in the invasive components. The results shed light on the genetic relationship between the non-invasive and invasive components of testicular cancer and the stage at which particular chromosomal changes may be important. © 2001 Cancer Research Campaign http://www.bjcancer.com

Definition of chromosome aberrations in testicular germ cell tumor cell lines by 24-color karyotyping and complementary molecular cytogenetic analyses

Many of the reported karyotypes for adult testicular germ cell tumors (GCTs) are complex and incomplete, although the presence of an isochromosome 12p, i(12p), and gain of 12p material have consistently been found. Here, an accurate definition of the chromosome aberrations associated with four cell lines derived from GCTs (GCT27, H12.1, Tera1, and Tera2) has been produced using 24-color karyotyping by mulifluor in situ hybridization, comparative genomic hybridization analysis, and further fluorescence in situ hybridization analysis to confirm some chromosomal assignments and refine involvement of specific regions of 12p. There was karyotypic heterogeneity. Isochromosomes in addition to i(12p) were found, as were other rearrangements with breakpoints at or near centromeric regions. The most frequent non-centromeric breakpoints were at 1p31∼p32, 1p21∼p22, 11q13, and Xq22, although consistent partner chromosomes were not involved. One cell line (Tera1) showed a subtle dosage increase in the copy number of a 12p probe known to be within the smallest overlapping region of amplification that has been defined in a number of testicular GCTs with amplicons at 12p11∼p12. The chromosome rearrangements and associated imbalances may be significant in GCT progression and the characterized cell lines can be used to investigate these further.

The role of the histopathologist in the management of testicular germ cell tumour in adults.

In the last 20--30 years the availability of effective chemotherapy and more accurate clinical staging has greatly improved the prognosis for patients with testicular germ cell tumours. Initially, such treatment appeared to diminish the role of histopathology to the distinction between seminoma and nonseminomatous germ cell tumour (NSGCT) in the primary specimen. However, histopathology has evolved as a prognostic tool indicating the risk of relapse in various defined clinical contexts thereby facilitating therapeutic decisions. The clinical emphasis has been on quality of life and reduction of therapy both in terms of the number of patients treated and the number of chemotherapy courses given to each patient. The treatment of adult testicular germ cell tumours may differ between countries but protocols are established. Therefore it is appropriate to discuss the role of histopathology during this era of relative therapeutic stability.

Germ cell nuclear factor is a transcriptional repressor essential for embryonic development.

GCNF is an orphan member of the nuclear receptor superfamily. The nuclear receptor superfamily is a large superfamily of transcription factors, the majority of which are designated as orphan receptors because their ligands and functions are currently unknown. GCNF (Germ Cell Nuclear Factor) is so named because of its restricted expression pattern in the adult. In the testis, GCNF is expressed only in the post meiotic round spermatids. Likewise in the ovary, GCNF's expression is restricted to the growing oocyte. To date nothing is known of GCNF's putative ligand; however, much is known about its physiological function through the use of gene targeting. Inactivation of the GCNF gene showed that it was essential for normal embryonic development. In addition to being expressed in the germ cells of the adult, it is expressed widely throughout the embryo after gastrulation. Significant strides have also been made in understanding GCNF's mechanism of action using molecular biology. The DNA binding properties of GCNF have been investigated and its response element identified. GCNF binds as a homodimer to a direct repeat element with zero nucleotides between the reiterated sequence AGGTCA. GCNF target genes have been identified that contain this DR0 element in their promoters. Such genes as Protamines 1 and 2 and Oct4 are regulated by GCNF through this element. GCNF has been shown to be a repressor of the protamine and Oct4 genes. GCNF's repression function has been shown to be mediated by interaction with the co-repressors N-CoR and SMRT in the absence of ligand. Our current efforts are to explore GCNF function in the adult germ cells using tissue specific gene targeting to specifically knock out the GCNF gene in oocytes and spermatogenic cells. In addition, efforts are being made to identify the endogenous ligand that regulates GCNF's transcriptional properties.

Microsatellite Instability in Germ Cell Tumors of the Testis and Ovary

Objectives. Widespread replication-type error (RER) is a genetic alteration that has been observed in many different neoplasms and has been associated with defective DNA repair activity. There are conflicting data regarding the role that this type of genetic instability plays in the development and progression of adult germ cell tumors.Methods. Universal amplification was performed on 104 paired specimens of tumor and constitutional DNA isolated from adult male and female germ cell tumors, in addition to subpopulations of carcinoma in situ (CIS), the precursor of testicular germ cell tumors (TGCTs). Preamplified DNA samples of TGCTs and ovarian germ cell tumors (OGCTs) were assayed for the presence of RER at 78 and 64 microsatellite loci, respectively.Results. RER was observed at a single microsatellite locus in 7 of 24 individual testicular germ cell tumors, including subpopulations of CIS isolated from one of these patients. There was some evidence of RER clustering for microsatellite loci mapping to the short arm of chromosome 12. Genetic instability was more frequent in OGCTs, with widespread RER observed at 38 of 64 microsatellite loci. These alterations were noted in 12 of 36 malignant OGCTs showing RER at 1 or more loci, including 3 OGCTs demonstrating RER at more than 6 separate microsatellite loci.Conclusions. The pathogenetic significance of genetic instability in germ cell tumors remains uncertain, although the results of this study suggest a lesser role in TGCTs compared to that in OGCTs.

The human VASA gene is specifically expressed in the germ cell lineage

To understand the origins and function of the human germ cell lineage and to identify germ cell-specific markers we have isolated a human ortholog of the Drosophila gene vasa. The gene was mapped to human chromosome 5q (near the centromere) by radiation hybrid mapping. We show by Northern analysis of fetal and adult tissues that expression of the human VASA gene is restricted to the ovary and testis and is undetectable in somatic tissues. We generated polyclonal antibodies that bind to the VASA protein in formalin-fixed, paraffin-embedded tissue and characterized VASA protein expression in human germ cells at various stages of development. The VASA protein is cytoplasmic and expressed in migratory primordial germ cells in the region of the gonadal ridge. VASA protein is present in fetal and adult gonadal germ cells in both males and females and is most abundant in spermatocytes and mature oocytes. The gene we have isolated is thus a highly specific marker of germ cells and should be useful for studies of human germ cell determination and function.

Mitochondrial morphology in human fetal and adult female germ cells

The aim of this study has been to observe, by electron microscopy, the morphological changes affecting mitochondria and associated organelles in the human female germ cell during oogenesis, maturation and fertilization. In the primordial germ cell (PGC), rounded mitochondria with a pale matrix and small vesicular cristae are disposed near the nucleus and significantly increase in number during PGC migration and settlement in the gonadal ridge, where they differentiate into oogonia. In these early stages of mammalian oogenesis, aggregates of mitochondria are typically clustered around or in close relationship with the nuage. In oocytes at early prophase stage, mitochondria proliferate while aligned along the outer surface of the nuclear membrane, contain a more dense matrix than before, and have lamellar cristae. Oocytes of primordial and primary follicles mostly contain round or irregular mitochondria whose matrix has become very light. These mitochondria show typical parallel, arched cristae, and are clustered near the nucleus with other organelles forming the Balbiani's vitelline body. When follicles grow, the mitochondria of the oocytes become even more numerous and are dispersed in the ooplasm. Both paranuclear accumulation and subsequent dispersion of mitochondria in the cytoplasm are likely to be regulated by microtubules. By ovulation, mitochondria are the most prominent organelles in the ooplasm. They form voluminous aggregates with smooth endoplasmic reticulum (SER) tubules and vesicles. These mitochondrial-SER aggregates (M-SER) and the mitochondrial-vesicle complexes (MV) could be involved in the production of a reservoir of substances or membranes anticipating subsequent fertilization and early embryogenesis. Just after fertilization, the mitochondria of the oocyte undergo a further substantial change in size, shape, and microtopography. In the pronuclear zygote, mitochondria concentrate around the pronuclei. During the first embryonic cleavage divisions, round or oval mitochondria with a dense matrix and few arched cristae are gradually replaced by elongated ones with a less dense matrix and numerous transverse cristae. A progressive reduction in size and number of M-SER aggregates and MV complexes also occurs. In summary, oocyte mitochondria show dynamic morphological changes as they increase in number and populate different cell domains within the oocyte. They form complex relationships with other cell organelles, according to the different energetic -metabolic needs of the cell during differentiation, maturation, and fertilization, and are ultimately inherited by the developing embryo, where they eventually assume a more typical somatic cell form.

Regulation of the aromatase gene expression in adult male rat germ cells: effects of TGFß and TNFα

Les fonctions de reproduction chez le mâle sont sous le controle des hormones hypophysaires. Outre cette regulation endocrine, il existe une regulation locale intragonadique dont depend le bon deroulement de la spermatogenese. Cette regulation locale fait notamment intervenir les steroides, les facteurs de croissance et les cytokines. L’aromatase est l’enzyme responsable de la conversion irreversible des androgenes en estrogenes. Dans la gonade mâle de differentes especes de mammiferes, l’aromatase a ete immunolocalisee non seulement dans les cellules de Leydig, mais aussi dans les cellules germinales. L’objectif de notre travail est d’etudier les facteurs susceptibles de controler la production des estrogenes dans les cellules germinales du rat mâle adulte en precisant notamment, les effets du TGFs et du TNFα sur l’expression du gene de l’aromatase. Les spermatocytes pachytenes et les spermatides rondes ont la capacite d’aromatiser les androgenes en estrogenes; cette activite augmente en fonction de stade de developpement des cellules germinales: elle est 5 fois plus elevee dans les spermatides que dans les spermatocytes pachytenes. A l’aide d’amorces specifiques, nous avons pu mettre en evidence par RT-PCR, l’ARNm de l’aromatase dans les spermatocytes pachytenes et les spermatides rondes. Nos resultats preliminaires obtenus par RT-PCR quantitative competitive ont montre que la testosterone (200ng/ml) stimule la synthese de l’ARNm de l’aromatase dans les deux types de cellules germinales etudies. Cette stimulation est fortement inhibee dans les spermatides rondes par l’ajout du TGFs (1ng/ml); l’effet de ce facteur sur les spermatocytes reste a determiner. L’ajout du TNFα (10ng/ml) aux cellules traitees par la testosterone induit une legere stimulation de la synthese des messagers de l’aromatase dans les spermatocytes pachytenes; cependant, cette cytokine exerce un effet inhibiteur dans les spermatides rondes. En somme, dans le testicule des rongeurs, les cellules germinales meiotiques et post-meiotiques representent une nouvelle source d’estrogene; ces cellules possedent en outre des recepteurs (ERs) pour ces hormones. Ces observations permettent de suggerer un controle estrogenique de la spermiogenese chez certains mammiferes.

Identification of a mouse germ cell-less homologue with conserved activity in Drosophila

Drosophila Germ cell-less (Gcl) has previously been shown to be important in early events during the formation of pole cells, which are the germ cell precursors in the fly. We have isolated a 524 amino acid mouse gene with 32% identity and 49% similarity to Drosophila gcl, termed mgcl-1. Like Drosophila Gcl, mGcl-1 localizes to the nuclear envelope. Ectopic expression of mgcl-1 in Drosophila rescues the gcl-null phenotype, indicating that mGcl-1 is a functional homologue of Gcl. mgcl-1 maps to chromosome 6 at 47.3 cM, and is expressed at low levels at all embryonic stages examined from 8.5 to 18.5 d.p.c. as well as in many adult tissues. Different from Drosophila gcl, mgcl-1 is not highly expressed at the time the primordial germ cells appear in the mouse, but high mgcl-1 expression is found in selected mouse adult male germ cells. The differences in these expression patterns in light of conserved activity between the two genes is discussed.

P Granules in the Germ Cells of Caenorhabditis elegans Adults Are Associated with Clusters of Nuclear Pores and Contain RNA

The germ cells, and germ cell precursors, in the nematode Caenorhabditis elegans contain distinctive granules called P granules. During early embryogenesis, P granules are segregated asymmetrically into those blastomeres that eventually produce the germ line. Because of the correlation between P granule distribution and the development of the germ line, P granules are widely thought to function in some aspect of germ line specification or differentiation. Most of the analysis of P granule structure and localization has focused on the early embryo, when P granules are located in the cytoplasm. However, during most of development P granules are associated with germ cell nuclei. We report here an ultrastructural analysis of the nuclear-associated P granules in the germ cells of the adult hermaphrodite gonad. We show that P granules are tightly associated with nuclear pores and that the positions of certain structures within the P granules correspond to the positions of pores on the nuclear envelope. We present immunocytochemical and ultrastructural data suggesting that P granules can associate, or remain associated, with pore-like structures even after they detach from the nuclear envelope during oogenesis. Finally, we show that nuclear-associated P granules in the gonad contain RNA, complementing previous studies showing that cytoplasmic P granules in embryos contain RNA.

Expression of the RNA component of human telomerase in adult testicular germ cell neoplasia

During human development, telomerase is repressed in most somatic cells, whereas it is maintained in male germline cells. Reactivation of telomerase has been associated with somatic cancers. To the authors' knowledge, the role of telomerase in germ cell derived malignancies has not previously been evaluated. A radioactive in situ hybridization method was used to study the expression of the RNA component of human telomerase (hTR) in 22 cases of adult testicular germ cell neoplasia encompassing all major histomorphologic types. For precise cell identification, hTR in situ hybridization was combined with immunohistochemistry in select cases. Testicular germ cell tumors showed differential expression of hTR. The highest level of expression was seen in embryonal carcinoma. Seminoma and unclassified intratubular germ cell neoplasia exhibited moderate levels of expression. Yolk sac tumor was characterized by a range of expression, which mirrored its morphologic variation. Immature teratoma recapitulated the down-regulation of telomerase manifested during human embryogenesis. Mature teratoma represented the adult pattern of somatic repression. Notably, choriocarcinoma showed modest expression. The expression of spermatocytic seminoma was intermediate between that of classic seminoma and embryonal carcinoma. No difference in expression was evident between matching intratubular and invasive components. In nonneoplastic testis, hTR expression was down-regulated during spermatogenesis and was absent in spermatozoa. Expression was negligible in rete testis and interstitial Leydig cells, and low in epididymis. Unexpectedly, Sertoli cells, which are testicular accessory somatic cells, displayed the most intense expression observed in this study. In testicular germ cell tumors of young adults (and during spermatogenesis), hTR expression is down-regulated with differentiation, irrespective of the aggressiveness of the tumors. Spermatocytic seminoma, regarded as a low grade malignancy, shows moderately intense expression.

Expression of the RNA component of human telomerase in adult testicular germ cell neoplasia

Expression of the RNA component of human telomerase in adult testicular germ cell neoplasia

Expression of the homophilic adhesion molecule, Ep-CAM, in the mammalian germ line.

During normal embryonic development, mammalian germ cells use both cell migration and aggregation to form the primitive sex cords. Germ cells must be able to interact with their environment and each other to accomplish this; however, the molecular basis of early germ cell adhesion is not well characterized. Differential adhesion is also thought to occur in the adult seminiferous tubules, since germ cells move from the periphery to the lumen as they differentiate. In a screen for additional adhesion molecules expressed by the germ line, expression of the homophilic adhesion molecule, Ep-CAM, was identified in embryonic, neonatal and adult germ cells using immunocytochemistry and flow cytometry with an Ep-CAM-specific monoclonal antibody. At embryonic stages, germ cells were found to express Ep-CAM during migration at embryonic day 10.5 and early gonad assembly at embryonic day 12.5. Expression of Ep-CAM was also found on neonatal male and female germ cells. In the adult testis, Ep-CAM was detected only on spermatogonia, and was absent from more differentiated cells. Finally, embryonic stem cells were shown to express this receptor. It is proposed that Ep-CAM plays a role in the development of the germ line and the behaviour of totipotent cells.

Loss of the Y-Chromosome in the Primary Metastasis of a Male Sex Cord Stromal Tumor: Pathogenetic Implications

The first published chromosomal pattern of the retroperitoneal lymph node metastasis of a malignant gonadal stroma cell tumor of the adult testis is presented. Karyotyping showed structural chromosomal abnormalities and loss of the Y-chromosome. This loss was confirmed in primary tumor and metastasis using fluorescence in situ hybridization (FISH). The characteristic chromosomal abnormality of adult testicular germ cell tumors, an i(12p), was not present. The results are compared with other data of testicular and ovarian sex cord stromal tumors. From the comparison of the male tumors, it is concluded that loss of the Y-chromosome might have a pathogenetic significance.

Genomic imprinting of H19 and insulin‐like growth factor‐2 in pediatric germ cell tumors

BACKGROUND Insulin-like growth factor-2 (IGF2) and H19 are reciprocally imprinted genes on chromosome 11; IGF2 is expressed paternally and H19 is expressed maternally. Loss of imprinting (LOI) at both H19 and IGF2 has been reported in seven fully informative adult testicular germ cell tumors (GCTs) and may contribute to germ cell carcinogenesis. METHODS Genomic DNA from 61 pediatric GCTs was amplified by polymerase chain reaction (PCR) and screened for heterozygosity at both IGF2 and H19 using either ApaI or RsaI, respectively. If heterozygous, polyadenylated RNA was isolated and reversed-transcribed into cDNA. cDNA then was amplified by PCR and the products were digested with restriction enzymes to evaluate GCT expression of IGF2 and H19. RESULTS Eleven pediatric GCTs were fully informative for H19 and IGF2, including 5 ovarian GCTs, 2 testicular GCTs, and 4 extragonadal GCTs. Consistent with prior studies, both testicular GCTs showed LOI at both H19 and IGF2. In contrast, three of the five ovarian GCTs had LOI at both IGF2 and H19; one had LOI at IGF2 only, and one retained imprinting at both loci. Only one of the four extragonadal GCTs had LOI at IGF2 whereas three of the four had LOI at H19. CONCLUSIONS These data suggest that LOI at H19 and IGF2 also may be common in pediatric testicular GCTs. However, ovarian and extragonadal pediatric GCTs showed variable patterns of LOI that may indicate differences in the timing of carcinogenesis in germ cells at these sites. Cancer 1999;85:1389–94. © 1999 American Cancer Society.

Genomic imprinting of H19 and insulin-like growth factor-2 in pediatric germ cell tumors

Insulin-like growth factor-2 (IGF2) and H19 are reciprocally imprinted genes on chromosome 11; IGF2 is expressed paternally and H19 is expressed maternally. Loss of imprinting (LOI) at both H19 and IGF2 has been reported in seven fully informative adult testicular germ cell tumors (GCTs) and may contribute to germ cell carcinogenesis. Genomic DNA from 61 pediatric GCTs was amplified by polymerase chain reaction (PCR) and screened for heterozygosity at both IGF2 and H19 using either ApaI or RsaI, respectively. If heterozygous, polyadenylated RNA was isolated and reversed-transcribed into cDNA. cDNA then was amplified by PCR and the products were digested with restriction enzymes to evaluate GCT expression of IGF2 and H19. Eleven pediatric GCTs were fully informative for H19 and IGF2, including 5 ovarian GCTs, 2 testicular GCTs, and 4 extragonadal GCTs. Consistent with prior studies, both testicular GCTs showed LOI at both H19 and IGF2. In contrast, three of the five ovarian GCTs had LOI at both IGF2 and H19; one had LOI at IGF2 only, and one retained imprinting at both loci. Only one of the four extragonadal GCTs had LOI at IGF2 whereas three of the four had LOI at H19. These data suggest that LOI at H19 and IGF2 also may be common in pediatric testicular GCTs. However, ovarian and extragonadal pediatric GCTs showed variable patterns of LOI that may indicate differences in the timing of carcinogenesis in germ cells at these sites.

The human SRY protein is present in fetal and adult Sertoli cells and germ cells.

Sex determination in mammals is controlled by the Y chromosome located SRY gene. Despite recent advances towards understanding the mechanisms that regulate sex determination in mammals, the expression profile of the SRY protein in human tissues is unknown. To localize the SRY protein and determine its cellular distribution, we prepared monoclonal antibodies (mAb) against the recombinant SRY protein. One antibody, LSRY1.1, recognizes a SRY-specific epitope and was used to localize the protein in different cells and tissues. The mAb recognizes a protein of 27 kDa in total lysates of HeLa SRYB3 cells. Immunocytochemical staining showed a nuclear localization of the protein. Immunohistochemical studies performed on gonadal tissue of a fetus, a one month-old boy and an adult man, demonstrated the presence of SRY protein in the nucleus of Sertoli and germ cells. In addition two 46,XX SRY(+) males had the SRY protein in their gonadal tissues. All other samples were negative, including all female tissue studied and the testis of a 46,XX SRY(-) male. The presence of SRY protein in fetal and adult gonadal tissues including germ cells suggests that SRY may have other male-specific functions in addition to sex determinism.