Niels Graem

Stem cell pluripotency factor NANOG is expressed in human fetal gonocytes, testicular carcinoma in situ and germ cell tumours.

Aims :u2002NANOG is a key regulator of embryonic stem cell (ESC) self‐renewal and pluripotency. Our recent genome‐wide gene expression profiling study of the precursor of testicular germ cell tumours, carcinoma in situ testis (CIS), showed close similarity between ESC and CIS, including high NANOG expression. In the present study we analysed the protein expression of NANOG during normal development of human testis and in a large series of neoplastic/dysgenetic specimens.

Transcription Factor AP-2γ Is a Developmentally Regulated Marker of Testicular Carcinoma In situ and Germ Cell Tumors

Purpose: Transcription factor activator protein-2γ (TFAP2C, AP-2γ) was reported previously in extraembryonic ectoderm and breast carcinomas but not in the testis. In our recent gene expression study we detected AP-2γ in carcinoma in situ testis (CIS, or intratubular germ cell neoplasia), precursor of testicular germ cell tumors. In this study we aimed to investigate the expression pattern of AP-2γ and to shed light on this factor in germ cell differentiation and the pathogenesis of germ cell neoplasia. Experimental Design: We analyzed expression pattern of AP-2γ at the RNA and protein level in normal human tissues and a panel of tumors and tumor-derived cell lines. In the gonads, we established the ontogeny of expression of AP-2γ in normal and dysgenetic samples. We also investigated the regulation of AP-2γ by steroids and retinoic acid. Results: We detected abundant AP-2γ in testicular CIS and in testicular germ cell tumors of young adults and confirmed differential expression of AP-2γ in somatic tumors. We found that AP-2γ expression was regulated by retinoic acid in an embryonal carcinoma cell line (NT2). The investigation of ontogeny of AP-2γ protein expression in fetal gonads revealed that it was confined to oogonia/gonocytes and was down-regulated with germ cell differentiation. In some prepubertal intersex cases, AP-2γ was detected outside of the normal window of expression, probably marking neoplastic transformation of germ cells. Conclusions: AP-2γ is developmentally regulated and associated with the undifferentiated phenotype in germ cells. This transcription factor may be involved in self-renewal and survival of immature germ cells and tissue-specific stem cells. AP-2γ is a novel marker of testicular CIS and CIS-derived tumors.

Cryptorchidism and testis cancer. Atypical infantile germ cells followed by carcinoma in situ and invasive carcinoma in adulthood

Atypical germ cells closely resembling carcinoma in situ (CIS) cells of the adult testis were found in a biopsy specimen from an undescended testis of a 10‐year old prepubertal boy who was admitted for orchiopexy. The atypical cells were distributed as normal infantile germ cells in the seminiferous tubules, but in successive postpubertal biopsies an adult CIS pattern developed. Microspectrophotometric DNA measurements showed aneuploidy of both the prepubertal and postpubertal atypical germ cells, and the malignant potential of these cells was proven as small foci of invasive germ cell carcinoma in addition to widespread CIS were found when an orchiectomy was performed at the age of 20 years. Thus, premalignant changes in the testis may be found before puberty, and may precede a clinically detectable tumor by more than 10 years.

Presumed pluripotency markers UTF-1 and REX-1 are expressed in human adult testes and germ cell neoplasms

BACKGROUNDnUTF-1 and REX-1/ZFP42 are transcription factors involved in pluripotency. Because of phenotypic similarities between pluripotent embryonic stem cells and testicular germ cell tumours (TGCT) and the derivation of pluripotent cells from testes, we investigated the expression of UTF-1 and REX-1 during human gonadal development and in TGCT.nnnMETHODSnExpression of UTF-1 and REX-1 was studied in 52 specimens from human gonadal development and in 86 samples from TGCT.nnnRESULTSnUTF-1 and REX-1 were expressed throughout male gonadal development. In the mature testis, UTF-1 was expressed in spermatogonia, whereas REX-1 was expressed in meiotic cells and, together with OCT-3/4, in primary oocytes. Both UTF-1 and REX-1 were expressed in testicular carcinoma in situ and in TGCT. Contrarily to REX-1, UTF-1 was expressed in all spermatocytic seminomas.nnnCONCLUSIONSnUnlike other pluripotency markers NANOG and OCT-3/4, UTF-1 and REX-1 are expressed throughout human testes development. The expression pattern indicated that UTF-1 plays a possible role in spermatogonial self-renewal, whereas expression of REX-1 in meiotic cells from both testes and ovary indicate a role in meiosis. UFT-1 and REX-1 are expressed in TGCT and the high abundance of UTF-1 in spermatocytic seminomas is consistent with the hypothesis that this tumour type originates from spermatogonia.

Autopsy Standards of Body Parameters and Fresh Organ Weights in Nonmacerated and Macerated Human Fetuses

Standards for body parameters and organ weights are important tools in fetal and perinatal pathology. Previously there has been only a weak emphasis on the effect of maceration on dimensions and weights. This study provides autopsy standards for body weight, body dimensions, and fresh organ weights for nonmacerated fetuses and for mildly, moderately, and markedly macerated fetuses at 12 to 43 weeks of gestation. Cases were selected from a consecutive series of 1800 fetal and perinatal autopsies. Cases with malformations, hydrops, infection, or chromosomal abnormality, fetuses from multiple births, and infants who lived longer than 24xa0h were excluded. In each case the maceration was graded and body weight and 4 body dimensions were recorded before organ examination. Organs were weighed immediately and before fixation. Growth curves were fitted according to appropriate mathematical methods and the effects of maceration on each value were tested statistically. We found that weights of the liver, thymus, and spleen markedly decrease with increasing maceration. The weights of the lungs, kidneys, and adrenals decreased modestly, whereas weights of the heart and brain changed only slightly. Body length increased slightly with maceration, whereas body weight and head circumference were unaffected. User-friendly charts and tables of mean values and standard deviations for nonmacerated and macerated fetuses are provided.

Analysis of meiosis regulators in human gonads: a sexually dimorphic spatio-temporal expression pattern suggests involvement of DMRT1 in meiotic entry

The mitosis-meiosis switch is a key event in the differentiation of germ cells. In humans, meiosis is initiated in fetal ovaries, whereas in testes meiotic entry is inhibited until puberty. The purpose of this study was to examine the expression pattern of meiosis regulators in human gonads and to investigate a possible role of DMRT1 in the regulation of meiotic entry. The expression pattern of DMRT1, STRA8, SCP3, DMC1, NANOS3, CYP26B1 and NANOS2 was investigated by RT-PCR and immunohistochemistry in a series of human testis samples from fetal life to adulthood, and in fetal ovaries. DMRT1 was expressed in testes throughout development but with marked spatio-temporal changes. At the early fetal period of 8-20 gestational weeks (GW) and at infantile mini-puberty, DMRT1 was predominantly expressed in Sertoli cells, whereas at later stages of gestation (22-40 GW), during childhood and in post-pubertal testes, DMRT1 was most abundant in spermatogonia, except in the A-dark type. In fetal ovaries, DMRT1 was detected in oogonia and oocytes until 20 GW, but was completely down-regulated following meiotic entry. STRA8, SCP3 and DMC1 were expressed mainly in oocytes and spermatogonia in accordance with their role in initiation and progression of meiosis. The putative meiosis inhibitors, CYP26B1 and NANOS2, were primarily expressed in Leydig cells and spermatocytes, respectively. In conclusion, the expression pattern of the investigated meiotic regulators is largely conserved in the human gonads compared with rodents, but with some minor differences, such as a stable expression of CYP26B1 in human fetal ovaries. The sexually dimorphic expression pattern of DMRT1 indicates a similar role in the mitosis-meiosis switch in human gonads as previously demonstrated in mice. The biological importance of the changes in expression of DMRT1 in Sertoli cells remains to be established, but it is consistent with DMRT1 reinforcing the inhibition of meiosis in the testis.

OCT2, SSX and SAGE1 reveal the phenotypic heterogeneity of spermatocytic seminoma reflecting distinct subpopulations of spermatogonia

Spermatocytic seminoma (SS) is a rare testicular neoplasm that occurs predominantly in older men. In this study, we aimed to shed light on the histogenesis of SS by investigating the developmental expression of protein markers that identify distinct subpopulations of human spermatogonia in the normal adult testis. We analysed the expression pattern of OCT2, SSX2‐4, and SAGE1 in 36 SS cases and four intratubular SS (ISS) as well as a series of normal testis samples throughout development. We describe for the first time two different types of SS characterized by OCT2 or SSX2‐4 immunoexpression. These findings are consistent with the mutually exclusive antigenic profile of these markers during different stages of testicular development and in the normal adult testis. OCT2 was expressed predominantly in Adark spermatogonia, SSX2‐4 was present in Apale and B spermatogonia and leptotene spermatocytes, whilst SAGE1 was exclusively present in a subset of post‐pubertal germ cells, most likely B spermatogonia. The presence of OCT2 and SSX2‐4 in distinct subsets of germ cells implies that these markers represent germ cells at different maturation stages. Analysis of SAGE1 and SSX2‐4 in ISS showed spatial differences suggesting ongoing maturation of germ cells during progression of SS tumourigenesis. We conclude that the expression pattern of OCT2, SSX2‐4, and SAGE1 supports the origin of SS from spermatogonia and provides new evidence for heterogeneity of this tumour, potentially linked either to the cellular origin of SS or to partial differentiation during tumour progression, including a hitherto unknown OCT2‐positive variant of the tumour likely derived from Adark spermatogonia. Copyright

Neural tube defects and associated anomalies in a fetal and perinatal autopsy series

Neural tube defects (NTDs) are congenital malformations of the central nervous system (CNS) secondary to abnormal closure of the neural tube during embryonic development. This study provides information on NTD rate, distribution, associated morphologic anomalies and organ weights in a Danish fetal and perinatal autopsy series during a 16 year period. The data were extracted from the autopsy reports of a consecutive series of 1984 fetal and perinatal autopsies from the Copenhagen area performed in the period 1989–2004. Registered parameters included: The location and morphology of the NTD, associated morphological anomalies, and organ weights. Organ weights were evaluated according to new fetal autopsy standards and grouped as low, normal or high. Ninety‐seven NTD cases were found (4.9%): Spina bifida (38 cases), cephalocele (17 cases) and anencephaly (42 cases). 63% of NTD cases had associated morphologic anomalies. Among the most common were hydrocephalus, NTD in another region, and anomalies in the urogenital system. 58% of the NTD cases had abnormal weight of one or more organs. Most notable was low adrenal weight not only in anencephalic fetuses but also in cases with cephalocele, suggesting a possible association.

AZFa protein DDX3Y is differentially expressed in human male germ cells during development and in testicular tumours: new evidence for phenotypic plasticity of germ cells

BACKGROUNDnDDX3Y (DBY), located within AZoospermia Factor a (AZFa) region of the human Y chromosome (Yq11), encodes a conserved DEAD-box RNA helicase expressed only in germ cells and with a putative function at G1-S phase of the cell cycle. Deletion of AZFa results most often in germ cell aplasia, i.e. Sertoli-cell-only syndrome. To investigate the function of DDX3Y during human spermatogenesis, we examined its expression during development and maturation of the testis and in several types of testicular germ cell tumours (TGCTs), including the pre-invasive carcinoma in situ (CIS) precursor cells which are believed to originate from fetal gonocytes.nnnMETHODSnDDX3Y protein expression was analysed during development in different tissues by western blotting. The localization of DDX3Y in normal fetal and prepubertal testis tissue of different ages as well as in a series of distinct TGCT tissue samples (CIS, classical seminoma, spermatocytic seminoma, teratoma and embryonal carcinoma) was performed by immunohistochemistry.nnnRESULTSnGerm cell-specific expression of DDX3Y protein was revealed in fetal prospermatogonia but not in gonocytes and not before the 17th gestational week. After birth, DDX3Y was expressed at first only in the nuclei of Ap spermatogonia, then also in the cytoplasm similarly to that seen after puberty. In CIS cells, DDX3Y was highly expressed and located predominantly in the nuclei. In invasive TGCT, significant DDX3Y expression was found in seminomas of the classical and spermatocytic type, but not in somatically differentiated non-seminomas, consistent with its germ-cell specific function.nnnCONCLUSIONSnThe fetal germ cell DDX3Y expression suggests a role in early spermatogonial proliferation and implies that, in men with AZFa deletion, germ cell depletion may begin prenatally. The strong expression of DDX3Y in CIS cells, but not in gonocytes, indicates phenotypic plasticity of CIS cells and suggests partial maturation to spermatogonia, likely due to their postpubertal microenvironment.

Acardiac Twin with Preserved Brain

The fatal acardiac syndrome is a rare complication of monochorionic twinning and is reported in 1 of 35,000 births. It is caused by arterioarterial and venovenous placental anastomoses leading to circulatory predominance of 1 twin. The donor ‘pump’ twin provides circulation for itself and for the recipient acardiac twin. The acardiac twin is usually grossly abnormal with severe reduction anomalies of the upper part of the body. We report a twin pregnancy, where a recipient twin initially by ultrasound was misdiagnosed as dead. In the third trimester the supposedly dead twin presented as an edematous acardiac twin without peripheral reduction defects and a nearly normally developed brain. An acardiac twin with a nearly normal external appearance and an almost normally developed brain, nourished by a surviving twin brother, has not previously been described in the literature.