Mature cystic teratomas arise from meiotic oocytes, but not from pre-meiotic oogonia.

Mature cystic teratomas (MCTs) in the ovaries have been thought to originate from germ cells from all developmental stages, i.e., from pre-meiotic oogonia through meiotic oocytes to mature post-meiotic ova. This view was based on research on MCTs by classical methods, including those involving centromeric heteromorphisms in karyotypes, enzyme polymorphisms, and DNA polymorphisms. However, insufficient genomic information was obtained in those studies. The current study aimed to confirm the cytogenetic origin of ovarian MCTs by using short tandem repeat (STR) polymorphism analysis to obtain sufficient genomic information, especially in connection with centromeric loci. Tissue samples of MCTs (57 ovaries from 51 patients, 91 MCTs, 156 specimens in total) obtained from cystectomies or oophorectomies were used. We categorized the specimens into two groups: i) solid components of MCTs and ii) cyst walls. The numbers of solid components of MCTs from pre-meiotic oogonia, primary oocytes, secondary oocytes, and ova were 0, 33, 16, and 15, respectively. There were no pre-meiotic oogonia in this series of solid-component specimens. We propose a hypothesis for the tumorigenesis of ovarian MCTs: the precursors of ovarian MCTs are not functional oocytes or ova, but are primary oocytes that have escaped from meiotic arrest. This hypothesis could satisfactorily explain the lack of pre-meiotic teratomas observed in this study and the nearly equal distribution of teratomas originating from primary oocytes, secondary oocytes, and ova in previous studies. Furthermore, this hypothesis could provide a starting point for determining the mechanism underlying tumorigenesis of ovarian MCTs.

Ex vivo culture of human fetal gonads: manipulation of meiosis signalling by retinoic acid treatment disrupts testis development.

STUDY QUESTION: What are the effects of experimentally manipulating meiosis signalling by addition of retinoic acid (RA) in cultured human fetal gonads? SUMMARY ANSWER: RA-treatment accelerated meiotic entry in cultured fetal ovary samples, while addition of RA resulted in a dysgenetic gonadal phenotype in fetal testis cultures. WHAT IS KNOWN ALREADY: One of the first manifestations of sex differentiation is the initiation of meiosis in fetal ovaries. In contrast, meiotic entry is actively prevented in the fetal testis at this developmental time-point. It has previously been shown that RA-treatment mediates initiation of meiosis in human fetal ovary ex vivo. STUDY DESIGN, SIZE, DURATION: This was a controlled ex vivo study of human fetal gonads treated with RA in 'hanging-drop' tissue cultures. The applied experimental set-up preserves germ cell-somatic niche interactions and the investigated outcomes included tissue integrity and morphology, cell proliferation and survival and the expression of markers of meiosis and sex differentiation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Tissue from 24 first trimester human fetuses was included in this study, all from elective terminations at gestational week (GW) 7-12. Gonads were cultured for 2 weeks with and without addition of 1 µM RA. Samples were subsequently formalin-fixed and investigated by immunohistochemistry and cell counting. Proteins investigated and quantified included; octamer-binding transcription factor 4 (OCT4), transcription factor AP-2 gamma (AP2γ) (embryonic germ cell markers), SRY (sex determining region Y)-box 9 (SOX9), anti-Müllerian hormone (AMH) (immature Sertoli cell markers), COUP transcription factor 2 (COUP-TFII) (marker of interstitial cells), forkhead box L2 (FOXL2) (granulosa cell marker), H2A histone family, member X (γH2AX) (meiosis marker), doublesex and mab-3 related transcription factor 1 (DMRT1) (meiosis regulator), cleaved poly ADP ribose polymerase (PARP), cleaved Caspase 3 (apoptosis markers) and Ki-67 antigen (Ki-67) (proliferation marker). Also, proliferation was determined using a 5'-bromo-2'-deoxyuridine (BrdU) incorporation assay. MAIN RESULTS AND THE ROLE OF CHANCE: A novel ex vivo 'hanging-drop' culture model for human fetal gonads was successfully established. Continued proliferation of cells without signs of increased apoptosis was observed after 2 weeks of culture. In cultured fetal ovaries treated with RA, an increased number of meiotic germ cells (P < 0.05) and DMRT1-positive oogonia initiating meiosis (P < 0.05) was observed, which is in agreement with a previous study. In fetal testes, RA-treatment resulted in a decreased number of gonocytes (P < 0.05), a reduced percentage of proliferating gonocytes (P < 0.05), altered expression pattern of the somatic cell markers AMH and COUP-TFII, as well as disrupted seminiferous cord structure and testis morphology. LIMITATIONS, REASONS FOR CAUTION: The number of samples included in this study was relatively small due to the limited availability of human fetal tissue. WIDER IMPLICATIONS OF THE FINDINGS: The hanging-drop culture, similarly to other organ culture approaches, allows studies of germ cell-somatic niche interactions and determination of effects after manipulating specific signalling pathways. Our novel finding of disrupted fetal testis development after treatment with RA indicates that abnormal meiosis regulation can potentially cause gonadal dysgenesis. Further studies will elucidate the exact mechanisms and timing of observed effects. STUDY FUNDING/COMPETING INTERESTS: This work was supported in part by an ESPE Research Fellowship, sponsored by Novo Nordisk A/S to A.Jø. Additional funding for this project was obtained from The Research Council of the Capital Region of Denmark (E.R.-D.M.), The Research Fund at Rigshospitalet (A.Ju. and J.E.N.), Familien Erichssens Fund (A.Jø.), Dagmar Marshalls Fund (A.Jø.) and Aase & Ejnar Danielsens Fund (A.Jø.). The authors have no conflicts of interest.

Ovarian dysgerminomas are characterised by frequent KIT mutations and abundant expression of pluripotency markers.

BACKGROUND: Ovarian germ cell tumours (OGCTs) typically arise in young females and their pathogenesis remains poorly understood. We investigated the origin of malignant OGCTs and underlying molecular events in the development of the various histological subtypes of this neoplasia. RESULTS: We examined in situ expression of stem cell-related (NANOG, OCT-3/4, KIT, AP-2gamma) and germ cell-specific proteins (MAGE-A4, NY-ESO-1, TSPY) using a tissue microarray consisting of 60 OGCT tissue samples and eight ovarian small cell carcinoma samples. Developmental pattern of expression of NANOG, TSPY, NY-ESO-1 and MAGE-A4 was determined in foetal ovaries (gestational weeks 13-40). The molecular genetic part of our study included search for the presence of Y-chromosome material by fluorescence in situ hybridisation (FISH), and mutational analysis of the KIT oncogene (exon 17, codon 816), which is often mutated in testicular GCTs, in a subset of tumour DNA samples. We detected a high expression of transcription factors related to the embryonic stem cell-like pluripotency and undifferentiated state in OGCTs, but not in small cell carcinomas, supporting the view that the latter do not arise from a germ cell progenitor. Bilateral OGCTs expressed more stem cell markers than unilateral cases. However, KIT was mutated in 5/13 unilateral dysgerminomas, whereas all bilateral dysgerminomas (n = 4) and all other histological types (n = 22) showed a wild type sequence. Furthermore, tissue from five phenotypic female patients harbouring combined dysgerminoma/gonadoblastoma expressed TSPY and contained Y-chromosome material as confirmed by FISH. CONCLUSION: This study provides new data supporting two distinct but overlapping pathways in OGCT development; one involving spontaneous KIT mutation(s) leading to increased survival and proliferation of undifferentiated oogonia, the other related to presence of Y chromosome material and ensuing gonadal dysgenesis in phenotypic females.

Genetic analysis results of mature cystic teratomas of the ovary in Taiwan disagree with the previous origin theory of this tumor.

The most accepted theory regarding mature cystic teratomas of the ovary is that they are of parthenogenetic origin from oocyte after the completion of first division. Our previous study demonstrated that the origin of mature cystic teratoma of the uterus is not related to the parthenogenetic process, but is most likely pluripotential stem cell or primordial germ cell before meiosis I. Further studies are needed to clarify the origin of benign mature cystic teratomas of the ovary in Taiwan. In the present study, we investigated the DNA profiles of 9 mature cystic teratomas of the ovary using short tandem repeat analysis with AmpFLSTR SGM Plus, Profiler PCR amplification kits. The methylation statuses of the HhaI sites in the SNRPN, H19DMR, and KvDMR regions were determined on methylation-sensitive multiplex ligation-dependent probe amplification analysis. DNA profiling data from the 9 mature cystic teratomas of the ovary excluded parthenogenetic origin, as most of the 15 short tandem repeat loci were heterozygous on genotyping. There were varying degrees of hypermethylation of SNRPN gene and KvDMR locus in the presence of maternal uniparental disomy in all 9 mature cystic teratomas of the ovary. In light of these results, we further postulated that the origin of these mature cystic teratomas of the ovary is oogonia or primary oocyte before germinal vesicle stage failure of meiosis I.

Description of primordial germ cells, oogonia, oocytes and embryo-like growth in squash preparations of tissues from hematological malignancies.

This study evidences the presence of primordial germ cells, in tissue squash preparations and sections from hematological malignancies. Primordial germ cells were identified by their morphology, the intense PAS, PAS-D reaction and presence of calcium-activated neutral proteinase. Primordial germ cells gave rise to nuclear vlimata. Immature oogonia exhibited a nuclear envelope and a star-shaped nuclear core, arising from acellular globose bodies impregnated by a nuclear vlima of primordial germ cell. Bone marrow tissue oogonia were PAS and PAS-D positive, identical to fungal ones. Calcium-activated neutral proteinase was demonstrated in the plasma of the acellular globose bodies, the nuclear envelope and the conglomerated primordial germ cells. Immature bone marrow oogonia progressed into mature ones, leptotene, diplotene, dictyotene and mature oocytes. Nuclear vlimata fertilized primordial germ cells, oogonia and oocytes, giving rise to round embryos at the morula and hatching morula-like stages. Embryos consisted of a zonapellucida-like cortex, composed of glycosaminoglycans, glycoproteins, protease and diffuse nuclear material, enclosing developing cells. Primordial germ cells, oogonia and embryos were also demonstrated in squash preparations of adult rat testis and sections of normal rat bone marrow tissues. The observations document that primordial germ cells are the primary stem cells which give rise to nuclear vlimata and oogonia, which constitute the secondary stem germ cells. The results are discussed in terms of stem cell renewal according to the events: primordial germ cells-gametes-fertilization-embryos-primordial germ cells.

Number of ovarian follicles in human fetuses with the 45,X karyotype.

OBJECTIVE: To evaluate the numbers of ovarian follicles during fetal life in the gonads of human female fetuses with the 45,X karyotype (Turner syndrome, TS) and to compare them with those from age-matched 46,XX fetuses. DESIGN: Retrospective study. SETTING: An academic hospital. PATIENT(S): Ovarian samples from TS fetuses (aged 17-37 weeks), mainly obtained after induced abortion, and ovaries from eight control fetuses (matching gestational ages). INTERVENTION(S): Embedded blocks of ovaries were collected from anatomy-pathology departments of three university hospitals and were sectioned and stained with hematoxylin-eosin. MAIN OUTCOME MEASURE(S): Observation of primordial and growing follicles. RESULT(S): In the fetal ovaries of controls, numerous oogonia were observed at 18 weeks. Primordial follicles were present in all ovaries from 20 weeks' gestation onward, whereas preantral and antral follicles were observed from 26 weeks onwards. In ovaries from 45,X TS fetuses, oogonia were observed in some ovaries, but no primordial, preantral, or antral follicles were found, even in ovaries from the third trimester of gestation. CONCLUSION(S): Follicle formation and growth are severely reduced in ovaries from aborted 45,X TS fetuses.

Recurrent Down's syndrome due to maternal ovarian trisomy 21 mosaicism.

A family with three children who had Down's syndrome and one healthy child is reported. Cytogenetic studies of the peripheral blood revealed trisomy 21 in the affected children, and normal karyotypes in both the parents and the healthy child. However, a biopsy of the mother's right ovary showed a mosaic trisomy 21 cell line (8/20 cells). By DNA polymorphism analysis, segregation of trisomy oogonia appeared to be the cause of recurrent trisomy 21.