Lgr5‐expressing stem cells are not the cells of origin of pyloric neuroendocrine carcinomas in mice

In intestinal and pyloric epithelia, leucine‐rich repeat‐containing G protein‐coupled receptor 5 (Lgr5)‐expressing cells represent long‐lived adult stem cells that give rise to all epithelial cell types, including endocrine cells. Ablation of the Apc gene in Lgr5‐expressing cells leads to intestinal and pyloric adenomas. To assess whether all epithelial tumours of the gastrointestinal tract are derived from LGR5‐positive stem cells, we crossed Lgr5–EGFP–IRES–creER^T2 mice, which express EGFP and Cre recombinase driven by the Lgr5 promoter, with CEA424–SV40–TAg mice, which develop pyloric neuroendocrine carcinomas of epithelial origin. In 19 day‐old mice, single SV40 T antigen (TAg)‐positive cells were identified preferentially at the the bases of pyloric glands, close to the stem cell compartment. However, contrary to previous publications describing subpopulations of LGR5‐positive cells in gastrointestinal neoplasia, we could not detect Lgr5–EGFP‐positive tumour cells in malignant lesions. The lack of expression of the Wnt target gene Lgr5 is probably not caused by suppression of Wnt signalling by TAg, since β‐catenin‐mediated Wnt signalling, as measured by the TOPflash assay, was not inhibited. To determine the cellular origin of CEA424–SV40–TAg tumours, we performed tracing experiments using Lgr5–EGFP–IRES–creERT2:CEA424–SV40–TAg:ROSA26–tdRFP mice. Following tamoxifen induction, it was possible to efficiently trace the progeny of Lgr5‐expressing cells in gastrointestinal tissue via red fluorescent protein (RFP) expression. No RFP‐positive tumour cells were detected, even when RFP gene activation occurred in 7 day‐old mice well before the appearance of TAg‐positive tumour cells. Hence, we conclude that Lgr5‐expressing stem cells probably do not constitute the cells of origin in CEA424–SV40–TAg mice. Consequently, not all epithelial tumours in the pyloric region are initiated by transformation of LGR5‐positive stem cells. Thus, additional long‐lived LGR5‐negative stem cells or progenitor cells with a low turnover rate might exist in the pyloric region, which could give rise to tumours. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Investigation of simian virus 40 large T antigen in 18 autopsied malignant mesothelioma patients in Japan

It has been reported that Simian virus 40 (SV40) is linked to human beings by inoculation of contaminated poliovaccines and may have a role in the etiology of malignant mesothelioma. However, there have been no reports describing the relationship between SV40 and malignant mesothelioma in Japan. A study was undertaken to investigate whether SV40 was related to patients of malignant mesothelioma in Japan by the polymerase chain reaction (PCR) assay, DNA sequence analysis, and immunohistochemical methods. Paraffin-embedded samples of the 18 autopsied patients with pleural malignant mesothelioma were collected from five hospitals in Japan. After isolation of DNA from paraffin blocks, PCR analyses followed by sequencing were performed using three different sets of primers for detection of SV40 large T antigen (TAg) gene. All 18 malignant mesothelioma samples were also immunohistochemically evaluated for expression of SV40 TAg protein with two different anti-SV40 TAg antibodies. SV40 TAg genome was detected in eight malignant mesothelioma cases. Only one of three primer pairs successfully amplified SV40 genome in the samples, whereas all pairs yielded a PCR product in the controls, suggesting a low content of virus DNA. No immunopositive staining for SV40 TAg was found in any of the samples. This study shows that SV40 genome was present in a subset of Japanese malignant mesothelioma patients who were unlikely to have received a contaminated polio vaccine based on their age.     

NF-kappa B is developmentally regulated during spermatogenesis in mice.

To analyze NF-kappa B activity in the testis, we used murine transgenic lines carrying a LacZ reporter gene under the control of a NF-kappa B-responsive promoter (Schmidt-Ullrich et al. [1996] Dev 122:2117-2128). We constructed three independent lines containing the promoter of the gene encoding p105, the precursor of the p50 subunit. This promoter contains three NF-kappa B-binding sites in its proximal part. Our results show that in adult mice, the beta-galactosidase activity which reflects nuclear NF-kappa B activity, is first detected in spermatocytes at the pachytene stage and remains activated in the following steps of germ cell differentiation and maturation. Using transgenic mice carrying a p105nlslacZ construct with the 3 NF-kappa B sites mutated in the p105 promoter, we found a significant reduction in the transgene activity, confirming the important role of NF-kappa B in the activation of the transgene. To confirm the stage of induction during spermatogenesis, we analysed the beta-galactosidase activity in the testes from prepuberal mice in which cells synchrouneously enter meiosis. We detected the transgene activity at 18 days after birth, corresponding to the pachytene stage in spermatocytes. In nuclear extracts prepared from prepuberal mice, we found a peak of NF-kappa B DNA-binding activity made of p50 and p65 subunits at day 18 after birth, which remains high in the later stages. Further analysis showed that I kappa B alpha and beta, but not epsilon are expressed in the testes. Altogether, these data suggest that NF-kappa B factors are stage specifically controlled and may play a role during the development of sperm cells.     

A genetically engineered ovarian cancer mouse model based on fallopian tube transformation mimics human high‐grade serous carcinoma development

Recent evidence suggests that ovarian high‐grade serous carcinoma (HGSC) originates from the epithelium of the fallopian tube. However, most mouse models are based on the previous prevailing view that ovarian cancer develops from the transformation of the ovarian surface epithelium. Here, we report the extensive histological and molecular characterization of the mogp–TAg transgenic mouse, which expresses the SV40 large T‐antigen (TAg) under the control of the mouse müllerian‐specific Ovgp‐1 promoter. Histological analysis of the fallopian tubes of mogp–TAg mice identified a variety of neoplastic lesions analogous to those described as precursors to ovarian HGSC. We identified areas of normal‐appearing p53‐positive epithelium that are similar to ‘p53 signatures’ in the human fallopian tube. More advanced proliferative lesions with nuclear atypia and epithelial stratification were also identified that were morphologically and immunohistochemically reminiscent of human serous tubal intraepithelial carcinoma (STIC), a potential precursor of ovarian HGSC. Beside these non‐invasive precursor lesions, we also identified invasive adenocarcinoma in the ovaries of 56% of the mice. Microarray analysis revealed several genes differentially expressed between the fallopian tube of mogp–TAg and wild‐type (WT) C57BL/6. One of these genes, Top2a, which encodes topoisomerase IIα, was shown by immunohistochemistry to be concurrently expressed with elevated p53 and was specifically elevated in mouse STICs but not in the surrounding tissues. TOP2A protein was also found elevated in human STICs, low‐grade and high‐grade serous carcinoma. The mouse model reported here displays a progression from normal tubal epithelium to invasive HGSC in the ovary, and therefore closely simulates the current emerging model of human ovarian HGSC pathogenesis. This mouse therefore has the potential to be a very useful new model for elucidating the mechanisms of serous ovarian tumourigenesis, as well as for developing novel approaches for the prevention, diagnosis and therapy of this disease. Published 2014. This article has been contributed to by US Government employees and their work is in the public domain in the USA.     

Identification of stem cells during prepubertal spermatogenesis via monitoring of nucleostemin promoter activity

The nucleostemin (NS) gene encodes a nucleolar protein found at high levels in several types of stem cells and tumor cell lines. The function of NS is unclear but it may play a critical role in S-phase entry by stem/progenitor cells. Here we characterize NS expression in murine male germ cells. Although NS protein was highly expressed in the nucleoli of all primordial germ cells, only a limited number of gonocytes showed NS expression in neonatal testes. In adult testes, NS protein was expressed at high levels in the nucleoli of spermatogonia and primary spermatocytes but at only low levels in round spermatids. To evaluate the properties of cells expressing high levels of NS, we generated transgenic reporter mice expressing green fluorescent protein (GFP) under the control of the NS promoter (NS-GFP Tg mice). In adult NS-GFP Tg testes, GFP and endogenous NS protein expression were correlated in spermatogonia and spermatocytes but GFP was also ectopically expressed in elongated spermatids and sperm. In testes of NS-GFP Tg embryos, neonates, and 10-day-old pups, however, GFP expression closely coincided with endogenous NS expression in developing germ cells. In contrast to a previous report, our results support the existence in neonatal testes of spermatogonial stem cells with long-term repopulating capacity. Furthermore, our data show that NS expression does not correlate with cell-cycle status during prepuberty, and that strong NS expression is essential for the maintenance of germline stem cell proliferation capacity. We conclude that NS is a marker of undifferentiated status in the germ cell lineage during prepubertal spermatogenesis.     

促甲状腺激素释放激素受体-2在大鼠生后睾丸不同发育阶段的表达和定位

目的研究促甲状腺激素释放激素受体-2（TRH-R2）在大鼠睾丸出生后不同发育阶段的表达,探讨其在生殖发育调节中的作用。方法应用蛋白质免疫印迹杂交技术以及免疫组织化学ABC法检测8d、15d、20d、35d、60d和90d大鼠睾丸中TRH-R2的表达和定位,并结合图像分析技术对免疫组织化学结果进行统计学分析,观察其在发育过程中的变化。结果免疫印迹杂交发现TRH-R2蛋白表达于出生后15d以后各阶段的大鼠睾丸;而免疫组织化学在第8d以后的各个发育阶段均检测到TRH-R2的表达,TRH-R2定位于大鼠睾丸的间质细胞,TRH-R2免疫反应阳性物位于细胞膜和细胞质内,细胞核为阴性;图像分析结果显示随着大鼠睾丸的发育,TRH-R2表达量逐渐增多,35d达到最大值,此后维持稳定水平,表达量在35d同其他阶段之间具有统计学差异（P〈O．01）。结论TRH-R2在大鼠睾丸发育过程中均有表达,定位于睾丸的间质细胞,其表达量随着增龄变化而变化,即同发育过程相关。     

恶性间皮瘤可能与致瘤性猿猴病毒SV40无关

目的探讨致瘤性猿猴病毒SV40（simian virus 40）是否与中国人恶性间皮瘤的发生相关。方法从蜡块中提取17例恶性间皮瘤组织中的DNA后,用三组引物对SV40大T抗原（TAg）的基因片段分别进行聚合酶链反应（PCR）扩增,另外,用两种SV40相关抗体（Pab101和Ab-2）分别进行免疫组织化学染色,检测肿瘤组织中是否存在SV40 TAg。结果（1）一组引物的PCR反应仅有3例扩增出了SV40TAg的基因片段,其余两组引物的PCR反应均为阴性。（2）两种抗体的免疫组织化学染色均未检测出SV40TAg。结论中国人恶性间皮瘤与SV40感染的关系可能不密切。     

Cytogenetic analysis of hepatic cell lines derived from SV40-T antigen gene-harboring transgenic mice.

A total of five hepatic cell lines were established from SV40-T transgenic mice at different stages of development. Karyotype analysis performed on these cell lines revealed 1) chromosomal instability manifested by numerous karyotypic alterations, 2) non-random numerical changes of chromosome number 18 and 3) frequent occurrence of marker chromosomes in four of the five lines. These findings indicate that genetic abnormalities occur very frequently in hepatocytes of the transgenic mice, most probably caused by the mutagenic action of T-antigen, raising the possibility that they play essential roles in initiation and progression in this model of murine hepatocarcinogenesis.     

Low frequency of SV40, JC and BK polyomavirus sequences in human medulloblastomas, meningiomas and ependymomas

Several reports have suggested a role for polyomaviruses in the pathogenesis of human brain tumors. This potential involvement is not conclusively resolved. For the present study, a highly sensitive PCR-assay with fluorescence-labelled primers was developed to search for polyomavirus sequences in human brain tumor and control DNA samples. The assay was shown to detect approximately one viral large T-antigen (TAg) gene per 250 cells. We identified simian virus 40 (SV40)-like sequences in 2/116 medulloblastomas, in 1/131 meningiomas, in 1/25 ependymomas and in 1/2 subependymomas. A single case of ependymoma contained SV40 VP-1 late gene sequences. Moreover, one of the meningioma samples showed JC virus sequences. In contrast, 60 hepatoblastoma samples and 31 brain samples from schizophrenic patients were consistently negative. BK virus sequences were not detectable in any of our samples. Immunohistochemical analysis of two SV40 positive tumor biopsies failed to detect large TAg in the tumor cells. In the JC positive meningioma, immunoreactivity for the viral late gene product (VP-1) was not observed. Our data do not entirely rule out SV40 and JC virus as an initiative agent with a hit-and-run mechanism. However the low frequency of virus sequences and the absence of TAg protein expression argue against a major role of these viruses in the pathogenesis of human medulloblastomas, meningiomas and ependymomas.     

Differential expression of estrogen receptors (ERalpha/ERbeta) in testis of mature and immature pigs

High affinity estrogen receptors (ERs) mediate estrogen action in male reproductive tissues. The objective of the present study was the immunolocalization of estrogen receptor alpha and estrogen receptor beta in immature and mature testes of pig, a species in which the role of estrogens on gonadal function is scarcely known. Testes from 3 and 18 month-old pigs were investigated. Immunohistochemistry was performed on paraffin embedded-tissues using both mouse anti-human monoclonal IgG ERalpha and IgG ERbeta 1 isoform. Western blot analysis demonstrated antibody specificity. ERalpha staining was not observed in immature testes, but it was detected in spermatogonia, spermatocytes and in the most Leydig cells of mature testes. ERbeta immunoreactivity was observed in spermatogonia and Leydig cells of immature gonads, while it was clearly detected in spermatogonia and in spermatocytes of adult pig testes. The differential ERalpha/ERbeta expression in germ and somatic cells of the gonads suggest a role of estrogens in function and in development of pig testis.     

类固醇生成因子-1对青春期小鼠睾丸中睾酮生成及精子发生的调节

目的 探讨类固醇生成因子-1(SF-1)对青春期小鼠睾丸内分泌功能及精子发生过程的调节作用，并推测其可能机制。方法 用免疫组织化学方法定位SF-1在不同年龄小鼠睾丸中的细胞分布，进一步分离有SF-1阳性表达信号的青春期小鼠Leydig细胞在体外进行培养，用反义转染方法抑制细胞内SF-1蛋白质的表达，检测细胞的睾酮分泌量及睾酮生成酶P450scc的mRNA水平变化。结果 1．SF-1在青春期Leydig细胞核有表达；反义抑制细胞内SF-1蛋白质的表达，则细胞的睾酮分泌量及P450scc mRNA水平均显著下降；2．SF-1在青春期小鼠睾丸B型精原细胞及细线期、偶线期、粗线期的初级精母细胞核中也有表达。结论 1．SF-1参与调节青春期睾丸Leydig细胞中P450scc基因的转录，影响睾酮分泌；2．SF-1作为一种核受体，可能也是生精过程中重要的转录调控因子，调节B型精原细胞向初级精母细胞分化及初级精母细胞第1次减数分裂过程中特异表达的基因转录过程，从而影响青春期小鼠精子发生过程。     

Male infertility and thiamine-dependent erythroid hypoplasia in mice lacking thiamine transporter Slc19a2

Thiamine-responsive megaloblastic anemia with diabetes and deafness (TRMA) is an autosomal recessive disease caused by mutations in the high-affinity thiamine transporter gene SLC19A2. To study the role of thiamine transport in the pathophysiology of TRMA syndrome and of each of the component disorders, we created a targeted disruption of the Slc19a2 gene in mice. Slc19a2 -/- mice are viable and females are fertile. Male -/- mice on a pure 129/Sv background are infertile with small testes (testis/body weight=0.13 +/- 0.04 knockout vs. 0.35 +/- 0.05 wild type, P<0.000005). The lack of developing germ cells beyond primary spermatocytes suggests an arrest in spermatogenesis prior to meiosis II. Nuclear chromatin changes indicative of apoptosis are present. No mature sperm are found in the tubules or epididymis. This phenotype suggests a previously unknown role for thiamine transport in spermatogenesis and male fertility. Slc19a2 -/- mice on a pure 129/Sv background develop reticulocytopenia after two weeks on thiamine-depleted chow with a virtual absence of reticulocytes in the peripheral blood (0.12% knockout vs. 2.58% wild type, P=0.0079). Few erythroid precursors are found in the bone marrow. Contrary to human TRMA syndrome, we see no evidence of megaloblastosis or ringed sideroblasts in the bone marrow of Slc19a2 -/- mice in thiamine-replete or thiamine-deficient dietary states. Phenotypic differences between TRMA patients and Slc19a2 -/- mice might be explained by dissimilar tissue expression patterns of the transporter, as well as by differing metabolic needs and possible different species-specific contributions of the related thiamine transporter Slc19a3.     

Expression of rck/p54, a DEAD-box RNA helicase, in gametogenesis and early embryogenesis of mice.

rck/p54 is a DEAD-box RNA helicase protein with ATP-dependent RNA-unwinding activity. Its ortholog is required for sexual reproduction in yeast and for oocyte survival and sperm fertility in Caenorhabditis elegans. In the current study, we investigated the expression of rck/p54 in mouse gametogenesis and early embryogenesis. Western blot analysis revealed that rck/p54 was highly expressed in both the ovary and testis. In the ovary, maturing oocytes strongly expressed rck/p54 in their cytoplasm. In contrast, in the testis, spermatogonia and primary spermatocytes highly expressed rck/p54 in their cytoplasm, but its expression decreased in the spermatids. Interestingly, rck/p54 was concentrated in the heads of spermatozoa; and then its expression gradually decreased as these cells matured along the epididymal duct. After fertilization, rck/p54 protein and its mRNA remained present in the pronucleus phase; and then their expression levels slightly but definitely decreased in morulae and blastocytes. The injection of a CMV-based rck/p54 expression vector into the pronuclei of fertilized eggs caused a delay in early embryogenesis. In generating RCK transgenic mice, the birth rate of the mice was significantly lower than those of other gene transgenic mice. These findings indicate that rck/p54 may play an important role in gametogenesis and early embryogenesis in mammals.