Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing.

Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2), plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI). Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops), and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx⁻/⁻ revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.

Expression of ovarian tumour suppressor OPCML in the female CD-1 mouse reproductive tract.

Opioid binding protein/cell adhesion molecule-like gene (OPCML) is frequently inactivated in epithelial ovarian cancer, but the role of this membrane protein in normal reproductive function is unclear. The ovarian surface epithelium (OSE) is thought to be the cell of origin of most epithelial ovarian cancers, some of which arise after transformation of OSE cells lining ovarian inclusion cysts, formed during ovulation. We used immunohistochemistry, immunoblotting and quantitative RT-PCR (qRT-PCR) to investigate OPCML expression in the uteri and ovaries of cycling 3-month CD-1 mice, as well as in ovaries from older mice containing inclusion cysts derived from rete ovarii tubules. Immunoblotting showed OPCML bands in uterine, but not whole ovarian or muscle extracts. Strong OPCML immunoreactivity was observed in oviduct, rete ovarii and uterus, whereas in ovary more immunoreactivity was seen in granulosa cells than OSE. No staining was observed in OSE around ovulation sites, where OSE cells divide to cover the site. OPCML immunoreactivity was also weaker in more dysplastic cells lining large ovarian inclusion cysts, compared with normal rete ovarii. No significant changes in Opcml mRNA expression were observed in whole ovarian and uterine extracts at different stages of the cycle. We conclude that murine OPCML is more consistently expressed in cells lining the uterus, oviduct and rete ovarii than in ovary and is not expressed in OSE associated with ovulation sites. This observation supports the hypothesis that a proportion of epithelial ovarian cancers arise from ductal cells and other epithelia of the secondary Mullerian system, rather than the OSE.

Targeted disruption of Brca1 in restricted compartments of the mouse mammary epithelia.

Tumours arising in BRCA1 mutation carriers have a characteristic phenotype, the molecular and cellular basis of which is unknown. To address the hypothesis that this phenotype reflects a role for BRCA1 in either in the basal or the stem cell compartments of the mammary epithelia, we have targeted its disruption to K14 and K6a expressing cells of the mouse. Unlike MMTV and WAP driven conditional knockout models of Brca1, these two models did not result in any observable changes in the mammary gland. Our results suggest that BRCA1-associated tumours arise either in K14 and K6a negative basal cells of the mammary gland, or possibly from transdifferentiation of luminal epithelia.

A high-resolution anatomical ontology of the developing murine genitourinary tract.

Cataloguing gene expression during development of the genitourinary tract will increase our understanding not only of this process but also of congenital defects and disease affecting this organ system. We have developed a high-resolution ontology with which to describe the subcompartments of the developing murine genitourinary tract. This ontology incorporates what can be defined histologically and begins to encompass other structures and cell types already identified at the molecular level. The ontology is being used to annotate in situ hybridisation data generated as part of the Genitourinary Development Molecular Anatomy Project (GUDMAP), a publicly available data resource on gene and protein expression during genitourinary development. The GUDMAP ontology encompasses Theiler stage (TS) 17-27 of development as well as the sexually mature adult. It has been written as a partonomic, text-based, hierarchical ontology that, for the embryological stages, has been developed as a high-resolution expansion of the existing Edinburgh Mouse Atlas Project (EMAP) ontology. It also includes group terms for well-characterised structural and/or functional units comprising several sub-structures, such as the nephron and juxtaglomerular complex. Each term has been assigned a unique identification number. Synonyms have been used to improve the success of query searching and maintain wherever possible existing EMAP terms relating to this organ system. We describe here the principles and structure of the ontology and provide representative diagrammatic, histological, and whole mount and section RNA in situ hybridisation images to clarify the terms used within the ontology. Visual examples of how terms appear in different specimen types are also provided.

E-cadherin expression and bromodeoxyuridine incorporation during development of ovarian inclusion cysts in age-matched breeder and incessantly ovulated CD-1 mice.

BACKGROUND: Female CD-1/Swiss Webster mice subjected to incessant ovulation for 8 months and 12-month breeder mice both developed ovarian inclusion cysts similar to serous cystadenomas. The majority of cysts appeared to be dilated rete ovarii tubules, but high ovulation number resulted in more cortical inclusion cysts. We hypothesized that comparison of inclusion cyst pathology in animals of the same age, but with differences in total lifetime ovulation number, might allow us to determine distinguishing characteristics of the two types of cyst. METHODS: Ovaries from breeder mice (BR) or females subjected to incessant ovulation (IO) were compared at 6-, 9- and 12-months of age. Ovaries were serially sectioned and cysts characterized with regard to location and histology, E-cadherin immunoreactivity and rates of BrdU incorporation. RESULTS: Inclusion cysts developed with age in BR and IO ovaries. The majority of cysts were connected to the ovarian hilus. Two cortical inclusion cysts were observed in ten IO ovaries and one in ten BR ovaries. Low or no E-cadherin immuno-staining was seen in the OSE of all mice studied. Conversely, strong membrane immuno-staining was observed in rete ovarii epithelial cells. Variable E-cadherin immunoreactivity was seen in cells of hilar inclusion cysts, with strong staining observed in cuboidal ciliated cells and little or no staining in flat epithelial cells. Two of the three cortical cysts contained papillae, which showed E-cadherin immuno-staining at the edge of cells. However hilar and cortical cysts were not distinguishable by morphology, cell type or E-cadherin immunoreactivity. BrdU incorporation in cyst cells (1.4% [95% CI: 1.0 to 2.1]) was greater than in OSE (0.7% [95% CI: 0.4 to 1.2]) and very few BrdU-labeled cells were observed in rete ovarii at any age. Incessant ovulation significantly increased BrdU incorporation in OSE of older animals. CONCLUSION: These experiments confirm ovarian inclusion cysts develop with age in the CD-1 mouse strain, irrespective of total ovulation burden. We conclude longer periods of incessant ovulation do not lead to significant changes in inclusion cyst formation or steroidogenesis in CD-1 mice and inclusion cyst type can not be distinguished by morphology, cell proliferation rate or E-cadherin immunoreactivity.

Incessant ovulation, inflammation and epithelial ovarian carcinogenesis: revisiting old hypotheses.

Epithelial ovarian cancer (EOC) is often a lethal disease because in many cases early symptoms go undetected. Although research proceeds apace, as yet there are few reliable and specific biomarkers for the early stages of the disease. EOC is an umbrella label for a highly heterogeneous collection of cancers, which includes tumours of low malignant potential, serous cystadenomas, mucinous and clear cell carcinomas, all of which are likely to arise from a number of epithelial cell types and a variety of progenitor lesions. Many, but not all types of EOC are thought to arise from the cells lining ovarian inclusion cysts. In this review, we discuss the hypotheses that have driven our ideas on epithelial ovarian carcinogenesis and examine the morphological and genetic evidence for pathways to EOC. The emergence of laser-capture microdissection and expression profiling by microarray technologies offers the promise of defining these pathways more accurately, as well as providing us with the tools for earlier diagnosis.

Rat activin-betaE mRNA expression during development and in acute and chronic liver injury.

Activin-betaE mRNA expression was investigated in male and female rats using gel-based and quantitative RT-PCR, in fetal and post-natal liver during development and in a variety of tissues from 200 gm adult animals. Activin-betaE expression was also assessed in rat liver after partial hepatectomy, and after repeated toxic insult. Male Sprague Dawley rats were subjected to partial hepatectomy or sham operations. Samples were collected from the caudate liver lobe during regeneration, from 12 to 240 hr after surgery. Three groups of 5 male rats were treated with CCl(4) for 0 (control), 5 or 10 weeks, to induce liver fibrosis and cirrhosis. Activin-betaE mRNA was predominantly expressed in liver, with much lower amounts of mRNA observed in pituitary, adrenal gland and spleen, in both males and females. Low activin-betaE expression was observed in liver at fetal day 16, with higher levels seen between post-natal days 3 and 35 and a further increase noted by day 47, in both males and females. Liver activin-betaE mRNA concentrations did not change from control values 12-72 hr after PHx, but significantly increased over six fold, 168 hr post-hepatectomy, when liver mass was restored. Activin-betaE mRNA was up-regulated after 5 weeks of CCl(4) treatment, but not after 10 weeks. The changes in activin-betaE mRNA concentrations after liver insult did not always parallel those reported for the activin-betaC subunit, suggesting activin-betaE may have an independent role in liver under certain conditions.

Location of inclusion cysts in mouse ovaries in relation to age, pregnancy, and total ovulation number: implications for ovarian cancer?

Benign ovarian cysts are thought to be precursor lesions that differentiate and transform into carcinoma. With the aim of testing the hypothesis that increased ovulation number increases the frequency or number of ovarian cysts, the development and appearance of ovarian cysts was investigated in mice of differing ages and total lifetime ovulation number. High total ovulation number was induced by keeping mice in cages divided by a screen, with a male on one side and two females on the other side. Significantly more cysts were observed in animals subjected to incessant ovulation for 8 months and in 12 month breeding mice than in 3-month virgin mice or 1-month prepubertal animals. These cysts had the appearance of benign serous inclusion cysts. When cystic ovaries were serial sectioned, 47% of cysts had a connection to the ovarian hilus and potentially to the tubules of the rete ovarii, 31% were adjacent to the hilus, and 22% had an intra-ovarian location. A significant increase in intra-ovarian cysts was observed in the 8-month incessant ovulation group, implying that high ovulation number leads to ovarian surface invagination and inclusion cyst formation. In conclusion, ovarian inclusion cysts may be derived from more than one epithelial source, but incessant ovulation may increase the proportion derived from the ovarian surface epithelium. Because the cysts observed resembled human serous inclusion cysts these results have possible implications for epithelial ovarian carcinoma.

Cell-specific expression of betaC-activin in the rat reproductive tract, adrenal and liver.

betaC-activin expression was assessed in rat tissues, using reverse transcription and real-time polymerase chain reaction, Western blotting and immunohistochemistry with a specific monoclonal antibody. betaC-activin mRNA was predominantly expressed in liver, but significant amounts were found in rat whole pituitary extracts (n = 5), and in three of five extracts of ovary, testis, and adrenal gland. Specific betaC-activin immunoreactivity was demonstrated in the cytoplasm of hepatocytes, neurosecretory cell terminals in posterior pituitary, ovarian primordial follicles, theca interna, large luteal cells and rete ovarii, spermatogonia, pachytene spermatocytes and Leydig cells of the testis, uterine endometrium, oviduct epithelium and zona glomerulosa of the adrenal. The observation of stage-specific expression in gonadal cells suggests this activin subunit has specific roles, different from those of other activin/inhibin subunits. Small amounts of mRNA in the presence of significant betaC-activin protein highlights the importance of examining betaC-activin expression at both the mRNA and protein level.

Effects of scrotal heating on sperm surface protein PH-20 expression in sheep.

Sperm surface protein PH-20 expression was studied during spermatogenesis in pubertal and adult sheep, using molecular and histological methods. The effects of 24 hr of insulation raising scrotal temperatures to 39 degrees C on PH-20 expression in ejaculated sheep sperm were also determined. A 282 nt cDNA fragment of ovine PH-20 was identified in total RNA extracts of sheep testes, which exhibited 76% identity at the nucleotide level with the equivalent region of the human sequence. Ovine PH-20 mRNA and immunoreactivity were identified only in adult ram testis and not in peri-pubertal ram testis tubules lacking round spermatids, nor in adult sheep brain, pituitary, heart, spleen, lung, liver, kidney, epididymis, or ovary. Ovine PH-20 protein was distributed predominantly on the postacrosomal membrane and was also present on the anterior membrane of the sperm head in fresh, unheated sheep semen. Scrotal heating caused a significant, transient decrease in the percentage of PH-20 immunoreactive sperm, but did not change the pattern of PH-20 staining on the sperm head. The results strongly suggest that ovine PH-20 is postmeiotically expressed in haploid germ cells in sheep testis and is arrayed on the membrane of the mature ovine spermatozoon. Scrotal heating appears to have few effects on PH-20 expression and distribution on ejaculated sperm.

Ethical, cultural and spiritual objections to genetically modified organisms: a review of the New Zealand process and perspective.

The New Zealand Royal Commission on Genetic Modification was directed to investigate the strategic options available to address the use of genetically modified organisms and products. The Commission spent 14 months hearing submissions in public meetings and formal hearings. Over 10,000 written public submissions were received. Most were against any use of the technology in food, and many were angry at the lack of product labelling and therefore choice. Few were supportive, although there was little objection to the use of genetic technology or modified organisms in containment, especially for medical research. Many New Zealanders had strong spiritual objections to the creation of transgenic animals containing human DNA, which they described as "playing God" or "interfering with Nature". Many expressed lack of trust in scientists and biotechnology companies. Despite these views, the Commission concluded that New Zealand should keep its options open and proceed carefully, minimising and managing risks. The Commission recommended that Government establish a Bioethics Council to act as a transparent advisory body and prepare guidelines on biotechnology, enabling public education and participation in decision-making.

Changes in activin and activin receptor subunit expression in rat liver during the development of CCl4-induced cirrhosis.

Amounts of betaA-activin, betaC-activin, activin receptor subunits ActRIIA and ActRIIB mRNA, and betaA- and betaC-activin subunit protein immunoreactivity were investigated in male Lewis rats, either untreated or after 5 or 10 weeks of CCl(4) treatment to induce cirrhosis. Apoptosis was assessed histologically and with an in situ cell death detection kit (TUNEL). Reverse transcription and polymerase chain reaction were used to evaluate mRNA levels. Activin betaA- and betaC-subunit immunoreactivity was studied by immunohistochemistry using specific monoclonal antibodies. Hepatocellular apoptosis (P<0.001), increased betaA- and betaC-activin mRNAs (three- to fourfold; P<0.01) and increased betaA- and betaC-activin tissue immunoreactivity were evident, whereas ActRIIA mRNA concentrations fell (30%; P<0.01) after 5 weeks of CCl(4) treatment. The mRNA concentrations at 10 weeks were not significantly different from controls, despite extensive hepatic nodule formation. We conclude that the increased activin subunit expression is associated with apoptosis, rather than hepatic fibrosis and nodule formation.

Current treatments for hepatitis.

Common viral agents known to cause inflammation of the liver (hepatitis) are hepatitis A virus (HAV), hepatitis B virus (HBV), and hepatitis C virus (HCV). Some other viral agents that can cause hepatitis are Epstein Barr virus, herpes simplex virus, and cytomegalovirus. Some patients infected with these viral agents progress to develop chronic viral hepatitis. Approximately 45% of chronic hepatitis cases are associated with hepatitis C and approximately 15% are associated with hepatitis B. In addition to being a leading cause of chronic hepatitis, HCV is most frequently associated with liver cirrhosis and hepatocellular carcinoma. Although much has been published about HAV and HBV, health professionals have learned about HCV only in recent years. For this reason, this article will emphasize the epidemiologic challenges and current treatments for hepatitis C; hepatitis A and B will be discussed in brief.