Examination of the ovotoxicity of 5-fluorouracil in mice.

PURPOSE: Undesirable side effects of cancer treatments are common and include damage to the ovary, and depletion of the follicle reserve, which if severe enough, can lead to infertility and early menopause. Antimetabolite drugs, such as 5-fluorouracil (5-FU), are not considered to be detrimental to the ovary, but the ovotoxicity of 5-FU has not been evaluated in any detail. The purpose of this study was to evaluate the effects of 5-FU on follicle number. METHODS: In this study, adult female C57Bl6 mice (n = 4-6 animals/group) received a single dose of saline or 5-FU (150 mg/kg) and markers of ovarian damage and follicle depletion were assessed 12 h and 7 days later. RESULTS: Exposure to 5-FU did not alter primordial and primary follicle numbers. Atresia of secondary and antral follicles was increased significantly 12 h after 5-FU treatment, but atresia rates returned to levels similar to that of saline treated controls at 7 days. The number of corpora lutea were reduced 7 days after exposure to 5-FU, possibly as a consequence of earlier follicular atresia. CONCLUSIONS: These findings suggest that a single dose of 5-FU is mildly ovotoxic, but any effects on ovarian function are likely transient because the primordial follicle population is not depleted. Collectively, these data support the notion that 5-FU is unlikely to impact on the long-term fertility of women.

NOD1 mediates interleukin-18 processing in epithelial cells responding to Helicobacter pylori infection in mice.

The interleukin-1 family members, IL-1ß and IL-18, are processed into their biologically active forms by multi-protein complexes, known as inflammasomes. Although the inflammasome pathways that mediate IL-1ß processing in myeloid cells have been defined, those involved in IL-18 processing, particularly in non-myeloid cells, are still not well understood. Here we report that the host defence molecule NOD1 regulates IL-18 processing in mouse epithelial cells in response to the mucosal pathogen, Helicobacter pylori. Specifically, NOD1 in epithelial cells mediates IL-18 processing and maturation via interactions with caspase-1, instead of the canonical inflammasome pathway involving RIPK2, NF-κB, NLRP3 and ASC. NOD1 activation and IL-18 then help maintain epithelial homoeostasis to mediate protection against pre-neoplastic changes induced by gastric H. pylori infection in vivo. Our findings thus demonstrate a function for NOD1 in epithelial cell production of bioactive IL-18 and protection against H. pylori-induced pathology.

Differential expression profiles of conserved Snail transcription factors in the mouse testis.

Snail transcription factors are key regulators of cellular transitions during embryonic development and tumorigenesis. The closely related SNAI1 and SNAI2 proteins induce epithelial-mesenchymal transitions (EMTs), acting predominantly as transcriptional repressors, while the functions of SNAI3 are unknown. An initial examination of Snai2-deficient mice provided evidence of deficient spermatogenesis. To address the hypothesis that Snail proteins are important for male fertility, this study provides the first comprehensive cellular expression profiles of all three mammalian Snail genes in the post-natal mouse testis. To evaluate Snail transcript expression profiles, droplet digital (dd) PCR and in situ hybridization were employed. Snai1, 2 and 3 transcripts are readily detected at 7, 14, 28 days post-partum (dpp) and 7 weeks (adult). Unique cellular expression was demonstrated for each by in situ hybridization and immunohistochemistry using Western blot-validated antibodies. SNAI1 and SNAI2 are in the nucleus of the most mature germ cell types at post-natal ages 10, 15 and 26. SNAI3 is only detected from 15 dpp onwards and is localized in the Sertoli cell cytoplasm. In the adult testis, Snai1 and Snai2 transcripts are detected in spermatogonia and spermatocytes, while Snai3 is in both germ and Sertoli cells. SNAI1 protein is evident in nuclei of spermatogonia, spermatocytes, round spermatids and elongated spermatids (Stages IX-XII). SNAI2 is present in the nuclei of spermatogonia and spermatocytes, with a faint signal detected in round spermatids. SNAI3 was detected only in Sertoli cell cytoplasm, as in juvenile testes. Additionally, colocalization of SNAI1 and SNAI2 with previously identified key binding partners, LSD1 and PRC2 complex components, provides strong evidence that these important functional interactions are conserved during spermatogenesis to control gene activity. These distinct expression profiles suggest that each Snail family member has unique functions during spermatogenesis.

The murine A33 antigen is expressed at two distinct sites during development, the ICM of the blastocyst and the intestinal epithelium.

The expression pattern of the murine A33 antigen has been defined during development using wholemount immunohistochemistry. Two temporally and spatially distinct sites of expression were identified: the inner cell mass of the blastocyst and the endoderm cell layer of the intestinal tract where expression is initiated at E14.5 in the hindgut and subsequently extends throughout the length of the intestine. The onset of mA33 antigen expression in the gut occurs at the beginning of an extensive phase of cell movement involved in the conversion of the endoderm cell layer to a single cell layer of polarized epithelium. Expression of mA33 antigen is then maintained into adulthood, where it is a definitive marker of intestinal epithelium.

Dynamic expression of alternate splice forms of D-cbl during embryogenesis.

The Cbl family of proteins act as E3 ubiquitin-protein ligases and have been associated with the down regulation of a variety of receptor tyrosine kinases. Cbl proteins associate with many different cell signalling molecules suggesting that they may have functions outside of the RING finger-mediated ubiquitin ligase activity. The Drosophila melanogaster cbl gene (D-cbl) encodes two splice forms (Oncogene 19 (2000) 3299). Here we report on the differential expression of these isoforms during Drosophila embryogenesis. Both isoforms are maternally expressed but the long isoform of D-cbl is also transiently expressed in the invaginating mesoderm and later is specifically expressed in neurons of the central nervous system (CNS). Cbl protein is shown to be localised to axons of the longitudinal connectives and commissures in the central nervous system.

Multiple functions of fibroblast growth factors in vertebrate development.

The biological consequences of ectopic FGF-4 expression were examined in chimaeric mouse embryos prepared with ES cells constitutively expressing FGF-4. The embryos exhibit abnormalities of the limbs and the anterior central nervous system. The limb phenotype consisted of the induction of early phases of limb development along the lateral ridge between the definitive fore and hind limb buds, where normally no limb outgrowth takes place. This phenotype was examined further by the implantation of beads soaked in FGF into the flank of developing chick embryos. This resulted in the induction of complete supernumerary limbs containing skeletal structures. These results indicated that FGFs have the ability to induce the outgrowth of limbs in an ectopic site and implicate FGFs in the initiation of normal limb development.

Drosophila spermatogenesis: insights into testicular cancer.

Geneticists have a long history of studying reproduction in the fruitfly, Drosophila melanogaster, and in recent years it has become apparent that many of the genes that regulate invertebrate reproduction have been conserved through vertebrate evolution. As with other higher eukaryotes, spermatogenesis in Drosophila is characterized by a regenerative germline stem cell population that divides asymmetrically to produce mitotic spermatogonia which will eventually differentiate into spermatocytes. Germline tumours consisting of undifferentiated germ cells have been associated with both loss-of-function mutations and ectopic gene expression. While the genesis of these tumours may not be identical to human germ cell tumours many of the genes that regulate stem cell proliferation and aberrant over-proliferation in the Drosophila testis provide candidate molecules that may underlie the genetic programmes that contribute to human testicular oncogenesis.

Mutation in Sos1 dominantly enhances a weak allele of the EGFR, demonstrating a requirement for Sos1 in EGFR signaling and development.

We have investigated the role of the mammalian Son of sevenless 1 (Sos1) protein in growth factor signaling in vivo by generating mice and cell lines that lacked the Sos1 protein. Homozygous null embryos were smaller than normal, died mid-gestation with cardiovascular and yolk sac defects, and their fibroblasts showed reduced mitogen-activated protein kinase activation in response to epidermal growth factor (EGF). An intercross of mice mutant for Sos1 and the EGF receptor (EGFR) demonstrated that a heterozygous mutation in Sos1 dominantly enhanced the phenotype of a weak allele of the EGFR allele (wa-2). These animals had distinctive eye defects that closely resembled those seen in mice that were null for the EGFR or its ligand, TGF alpha. Our findings provide the first demonstration of a functional requirement for Sos1 in growth factor signaling in vivo. They also show that the genetic test of enhancement of weak receptor allele by heterozygous mutation in one component represents a powerful tool for analyzing the ras pathway in mammals.

Ectopic expression of Fgf-4 in chimeric mouse embryos induces the expression of early markers of limb development in the lateral ridge.

The biological consequences of constitutive fibroblast growth factor-4 (fgf-4) expression were investigated in chimeric embryos prepared between wild-type host embryos and murine ES cells transfected with a construct in which expression of the murine fgf-4 gene was directed by the phosphoglycerate kinase (PGK-1) promoter. The embryos exhibit abnormalities of the limbs and the anterior central nervous system (CNS). The limb phenotype comprised the induction of outgrowth along the lateral ridge between the definitive fore and hind limbs resembling the early phases of limb development. The CNS defects comprised a complete absence, or marked reduction in forebrain and midbrain structures and rudimentary or absent eye development. Constitutive expression of fgf-4 was also accompanied by ectopic expression of the sonic hedgehog (shh) and msx-1 genes in the lateral ridge. These findings indicate that FGF exhibits multiple activities in early development which include the ability to induce the expression of early markers of limb development in the lateral ridge.

Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types.

Telomerase is a multicomponent reverse transcriptase enzyme that adds DNA repeats to the ends of chromosomes using its RNA component as a template for synthesis. Telomerase activity is detected in the germline as well as the majority of tumors and immortal cell lines, and at low levels in several types of normal cells. We have cloned a human gene homologous to a protein from Saccharomyces cerevisiae and Euplotes aediculatus that has reverse transcriptase motifs and is thought to be the catalytic subunit of telomerase in those species. This gene is present in the human genome as a single copy sequence with a dominant transcript of approximately 4 kb in a human colon cancer cell line, LIM1215. The cDNA sequence was determined using clones from a LIM1215 cDNA library and by RT-PCR, cRACE and 3'RACE on mRNA from the same source. We show that the gene is expressed in several normal tissues, telomerase-positive post-crisis (immortal) cell lines and various tumors but is not expressed in the majority of normal tissues analyzed, pre-crisis (non-immortal) cells and telomerase-negative immortal (ALT) cell lines. Multiple products were identified by RT-PCR using primers within the reverse transcriptase domain. Sequencing of these products suggests that they arise by alternative splicing. Strikingly, various tumors, cell lines and even normal tissues (colonic crypt and testis) showed considerable differences in the splicing patterns. Alternative splicing of the telomerase catalytic subunit transcript may be important for the regulation of telomerase activity and may give rise to proteins with different biochemical functions.

Characterization of mouse A33 antigen, a definitive marker for basolateral surfaces of intestinal epithelial cells.

The murine A33 antigen is emerging as a definitive marker of intestinal epithelial cells. Cloning and sequence determination of cDNAs encoding mA33 antigen predict a novel type 1 transmembrane protein of 298 amino acids, comprising an extracellular domain with two immunoglobulin-like domains, a single-span transmembrane domain, and a highly acidic cytoplasmic domain. On the basis of conservation of amino acid sequence and genomic structure, the mA33 antigen is a member of a growing subfamily within the immunoglobulin superfamily, which includes transmembrane proteins CTX/ChT1, CTM/CTH, and CAR. During embryonic development, mA33 antigen expression is first observed in the inner cell mass of blastocysts before implantation. Intestinal expression of mA33 antigen is initiated in the hindgut at E14.5 and increases steadily throughout late embryonic and postnatal life into adulthood. The protein is specifically expressed on the basolateral surfaces of intestinal epithelial cells of all lineages, independent of their position along the rostrocaudal and crypt-villus axes. Thus the mA33 antigen appears to be a novel marker for both proliferating and differentiating intestinal epithelial cells.