Expression of miRNAs in Bull Spermatozoa Correlates with Fertility Rates.

Bull spermatozoa are rich in active miRNAs, and it has been shown that specific spermborne miRNAs can be linked to fertility. Thus, expression profiling of spermatozoa could be helpful for understanding male fertility and the ability of spermatozoa to initiate and sustain zygotic, embryonic and foetal development. Herein we hypothesized that bulls with moderate to high fertility can be identified by differences in amounts of certain miRNAs between their ejaculates. RNA samples from spermatozoa of eight brother pairs (one bull with high and one with moderate NRR in each pair) of the Holstein breed were prepared. miRNA was isolated, and the expression of 178 miRNAs was determined by RT-qPCR. Important findings were that highly expressed miRNAs, not linked to NRR status, were identified in the bull sperm samples, which indicate that these miRNAs have an important role in early embryogenesis. A large fraction of the targets genes were phosphoproteins and genes involved in the regulation of transcription. Seven miRNAs (mir-502-5p, mir-1249, mir-320a, mir-34c-3p, mir-19b-3p, mir-27a-5p and mir-148b-3p) were differentially expressed between bulls with moderate and high NRR with a strong tendency towards a higher expression of miRNAs in bulls with moderate fertility. Thus, bulls with a moderate NRR negatively regulate the expression of protein-coding genes, which leads to problems during the pregnancy.

High-resolution genomic profiling of carboplatin resistance in early-stage epithelial ovarian carcinoma.

Chemotherapy resistance remains a major obstacle to successful treatment of ovarian cancer patients. Therefore, increased knowledge of underlying mechanisms and identification of predictive factors are of great importance. Standard treatment for ovarian carcinoma is surgery followed by platinum-based chemotherapy. In this study, we aimed to search for genes or genomic regions involved in platinum resistance in ovarian carcinoma. Array-based comparative genomic hybridization (CGH) was used to identify genetic alterations in 32 early-stage epithelial ovarian carcinomas homogeneously treated with single-agent carboplatin. The arrays contain 33,370 bacterial artificial chromosome (BAC) clones and form a contiguous and tiling coverage of the human genome with an average resolution of approximately 100 kb. We found certain genetic changes associated with carboplatin response. Gains in 1q25.1-q41 were significantly more frequent in carboplatin-resistant tumours. In this region, we further detected two smallest regions of overlap (SRO) at 1q25.2 and 1q32.2 (approximately 690 and approximately 830 kb in size, respectively). Interestingly, we found some regions that were lost exclusively in the sensitive tumours 17q24.1, Xq21.33-q22.1, and 6 regions in 15q. We also detected genetic differences with regard to histologic subtype. Gain in 8q was found highly associated with serous and clear cell subtypes, and an SRO was identified at 8q24.22-q24.23. The genomic regions found altered in this study confirm some of our previous metaphase CGH results. The alteration found in chromosome arm 1q was verified and specified, and is therefore of great interest as a candidate for predictive markers. Identifying predictive markers of chemosensitive and chemoresistant disease would greatly help in the choice of chemotherapy in the clinic, and thus improve treatment of women with ovarian cancer.

Testing multiple hypotheses for the maintenance of male homosexual copulatory behaviour in flour beetles.

Diverse animal groups exhibit homosexual interactions, yet the evolutionary maintenance of such behaviours remains enigmatic as they do not directly increase reproductive success by generating progeny. Here, we use Tribolium castaneum flour beetles, which exhibit frequent male homosexual copulations, to empirically test several hypotheses for the maintenance of such behaviours: (1) establishing social dominance; (2) practice for future heterosexual encounters; and (3) indirect sperm translocation. We found no evidence that Tribolium males use homosexual copulations either to establish dominance or to practice behaviours that increase their subsequent heterosexual reproductive performance. Our results provide limited support for the hypothesis of indirect sperm translocation: when males from two genetic strains mated with females immediately following a homosexual copulation, females produced progeny sired not only by the directly mating male, but also by that male's homosexual partner. However, this phenomenon was detected in only 7% of homosexual pairs, and in each case such indirectly sired progeny accounted for < 0.5% of females' total progeny. Direct observations indicated that mounting males often released spermatophores during homosexual copulations. These observations suggest that homosexual copulations may be a behavioural mechanism that allows males to expel older, potentially low-quality sperm. Additional work is needed to test this new hypothesis, and to determine whether sperm release during homosexual copulations occurs in other groups.

Chromosomal alterations in 98 endometrioid adenocarcinomas analyzed with comparative genomic hybridization.

The aim of the present study was to investigate chromosomal alterations in a large set of homogeneous tumors, 98 endometrioid adenocarcinomas. We also wanted to evaluate differences in chromosomal alterations in the different groups of tumors in relation to stage, survival and invasive or metastatic properties of the tumors. Comparative genomic hybridization (CGH) was used to detect chromosomal alterations in tissue samples from 98 endometrioid adenocarcinomas. All chromosomes were involved in DNA copy number variations at least once in the tumor material, but certain changes were recurrent and rather specific. Among the specific changes, it was possible to identify 39 chromosomal regions displaying frequent DNA copy number alterations. The most frequent alteration was detected at 1q25-->q42, in which gains were found in 30 cases (30%). Gains at 19pter-->p13.1 were detected in 26 tumors (26%) and at 19q13.1-->q13.3 in 19 tumors (19%). Increased copy numbers were also detected at 8q (8q21-->q22 and 8q22-->qter), at a relatively high rate, in 17 cases (17%). Furthermore, gains at 10q21-->q23 and 10p were found in 14 (14%) and 13 cases (13%), respectively. The most common losses were found in the three regions 4q22-->qter, 16q21-->qter and 18q21-->qter, all of which were detected in eight of the 98 tumors (8%). We also detected differences between the tumors from deceased patients and from survivors. Gain at 1q25-->q42 was more commonly detected in the tumors from patients who died of cancer. We noted that the regions most affected differed in the different surgical stages (I-IV). The results of the CGH analysis identify specific chromosomal regions affected by copy number changes, appropriate objects for further genetic studies.

Genetic analysis of susceptibility to endometrial adenocarcinoma in the BDII rat model.

Most cancers are genetically complex and heterogeneous, a serious obstacle to identifying specific genes underlying the disease. If inbred animal models are used, then both the genetic constitution and environmental influences can be carefully controlled. Females of the BDII inbred rat strain are genetically predisposed to endometrial cancer; more than 90% of virgin BDII females will develop endometrial adenocarcinoma (EAC) during their life span. BDII females were crossed to males from inbred strains with low EAC incidence (SPRD or BN). When F(1) males were backcrossed to BDII females to generate N(1) populations of offspring, about one fourth of the female progeny developed EAC. With transmission disequilibrium test analysis, significant association was detected in three chromosomal regions (on RNO1, RNO11, and RNO17) in the SPRD crosses and in the short arm of RNO20 in the BN crosses. It appears that several susceptibility genes with minor but cooperating effects are responsible for the susceptibility. Furthermore, it seems clear from the interstrain crosses not only that the onset of tumors depends on the presence of susceptibility alleles from the EAC-prone BDII strain, but also that tumor development is affected by the contribution of a genetic component derived from the nonsusceptible strains.

A low-toxicity method for the separation of lanosterol and dihydrolanosterol from commercial mixtures.

We describe an inexpensive, low-toxicity and high-yielding method for the production of pure lanosterol and dihydrolanosterol from the commercially available mixture. Optimum conditions are presented for the one-pot production of the intermediate 24,25 vicinal diol of lanosterol acetate (via either epoxidation or hydroxyhalogenation) which is readily separated from the unreacted dihydrolanosterol acetate. The lanosterol diol can then be converted to pure (>97%) lanosterol. Hypophosphorous acid was used for both the conversion of the epoxide to the diol, and as a catalyst for the hydroxyhalogenation by N-halosuccinimides of the olefinic bond.

Efficient routes to epimerically-pure side-chain derivatives of lanosterol.

A technically simple route is described to individual epimers of side-chain derivatives of lanosterol (3beta-hydroxy-5alpha-lanosta-8,24-diene). Epimerically pure 24,25-epoxy-, 24,25-dihydroxy- and 24-bromo-25-hydroxy-lanosterol have been prepared in good yield from commercial (50-60%) lanosterol. Hypophosphorous acid was used as a catalyst for the cohalogenation of the Delta24(25) bond and also for the efficient conversion of 24,25-epoxy- and 24-bromo-25-hydroxylanosterol to epimerically pure 24(R) or 24(S)-24,25-dihydroxylanosterols.

Evolutionary aspects of the genomic organization of rat chromosome 10.

Using FISH and RH mapping a chromosomal map of rat chromosome 10 (RNO10) was constructed. Our mapping data were complemented by other published data and the final map was compared to maps of mouse and human chromosomes. RNO10 contained segments homologous to mouse chromosomes (MMU) 11, 16 and 17, with evolutionary breakpoints between the three segments situated in the proximal part of RNO10. Near one of these breakpoints (between MMU17 and 11) we found evidence for an inversion ancestral to the mouse that was not ancestral to the condition in the rat. Within each of the chromosome segments identified, the gene order appeared to be largely conserved. This conservation was particularly clear in the long MMU11-homologous segment. RNO10 also contained segments homologous to three human chromosomes (HSA5, 16, 17). However, within each segment of conserved synteny were signs of more extensive rearrangements. At least 13 different evolutionary breakpoints were indicated in the rat-human comparison. In contrast to what was found between rat and mouse, the rat-human evolutionary breaks were distributed along the entire length of RNO10.

Genetic identification of multiple susceptibility genes involved in the development of endometrial carcinoma in a rat model.

There are clear indications that inheritance plays an essential role in certain cases of human endometrial cancer, and there are at least 2 forms of early-onset heritable endometrial adenocarcinomas (EACs). Females of the BDII inbred rat strain are known to be genetically predisposed to endometrial carcinoma, and we have performed a genetic analysis of susceptibility to endometrial cancer in this strain. F(2) populations were generated by crossing BDII females with males from 2 different strains with a low incidence of EAC, and the occurrence of endometrial cancer was studied. Three chromosome regions associated to EAC susceptibility were identified, and the susceptibility genes in these regions were designated Ecs1, Ecs2 and Ecs3. Our results indicate that the genes affecting susceptibility to EAC are different in the 2 crosses, suggesting that the genes behind the susceptibility in BDII animals may interact with different genes in different genetic backgrounds.

High-density marker loss of heterozygosity analysis of rat chromosome 10 in endometrial adenocarcinoma.

Endometrial cancer is a disease with serious impact on the human population, but not much is known about genetic factors involved in this complex disease. Female BDII rats are genetically predisposed to spontaneous endometrial carcinoma, and the BDII inbred strain provides an experimental animal model for endometrial carcinoma development. In the present study, BDII females were crossed with males from two nonsusceptible inbred rat strains. Endometrial adenocarcinomas (EACs) developed in a proportion of the F1 and F2 progeny. We screened 18 EAC solid tumors and 9 EAC cell cultures for loss of heterozygosity (LOH) using fluorescent-PCR-based marker allelotyping methodology with 47 microsatellite markers covering the proximal part of rat chromosome 10 (RNO10). Conclusive evidence was obtained for LOH/deletion involving about 56 cM in the proximal part of RNO10 in DNA from six out of seven informative tumor cell cultures. Analysis of the solid tumors confirmed the presence of LOH in this part of RNO10 in 14 of 17 informative tumors. However, from the studies in the solid tumors it appeared that in fact three separate segments in the proximal part of RNO10 were affected. These three LOH/deletion regions were located approximately in cytogenetic bands 10q11-12, 10q22, and 10q24.

Analysis of genetic changes in rat endometrial carcinomas by means of comparative genomic hybridization.

Animals of the BDII inbred rat strain are known to be genetically predisposed to endometrial adenocarcinoma (EAC). Using them as models of human EACs, we studied tumors arising in F1 and F2 progeny from BDII animals crossed to animals from two other inbred strains, in which EACs were quite rare. In order to identify chromosomal regions exhibiting DNA copy number changes, comparative genomic hybridization (CGH) was applied in a series corresponding to 27 different solid tumors, most of which were classified as EACs, from these animals. The main findings from the study were that, although many different chromosomes were involved in copy number variation, some of the changes detected were recurrent and quite specific. Among specific changes found were gains in rat chromosome (RNO) regions 4q12 approximately q22, 6q14 approximately q16, and whole chromosome arms in some of the small metacentric chromosomes (e.g., RNO14, 16, and 18). RNO10 was involved in gain in the terminal and proximal regions. Each of these regions contains previously identified cancer-related genes representing possible candidates to be involved in the development of EAC. Furthermore, it was observed that there were clear differences in the pattern of copy number changes between tumors occurring in the two different crosses, and also between solid tumors and cell cultures. Endometrial cancer is the most common human gynecological cancer, but not much is known about specific genetic changes influencing this disease. Two genetic alterations that have been reported from human endometrial cancer are amplification of the ERBB2 gene and mutations in the 12 codon of the KRAS gene. One case of Erbb2 amplification was found but there were no Kras mutations in the rat material studied. We conclude that molecular genetic analysis of the rat BDII model will provide important new information about EAC in mammals.

Herpes simplex virus 1 ICP27 is required for transcription of two viral late (gamma 2) genes in infected cells.

The herpes simplex virus infected cell protein 27 (ICP27) is required for the expression of certain early viral proteins and for many late proteins during productive infection. Expression of at least one late (gamma 2) gene, that encoding glycoprotein C, is severely restricted in the absence of functional ICP27. The exact mode of action by which ICP27 induces late gene expression is not known, but the effect is apparent at the mRNA level as demonstrated by Northern blot analysis. To determine whether ICP27 activates late genes via transcriptional or posttranscriptional mechanisms, we initially used nuclear run-on assays to measure transcription of viral genes in Vero cells infected with wild-type (WT) virus or an ICP27 nonsense mutant virus, n504. We observed a 4-fold reduction in the nuclear run-on signal from the coding strand of the gC gene for n504-infected cells compared to that of WT-infected cells. However, interpretation of the results was complicated by the observation of a significant signal from the noncoding strand in these experiments. To obviate the problem of symmetrical transcription, we utilized in vivo RNA pulse-labeling to measure the amount of transcription of viral genes in cells infected with either WT virus or n504 virus. We found a 5- to 10-fold reduction in the transcription of the gC and U(L)47 genes, two late genes, in cells infected with n504 compared to that in cells infected with WT virus. In contrast, transcription of the ICP8 gene, an early gene, was similar in WT and n504 virus-infected cells. We also examined the stability of the gC and U(L)47 gene transcripts in n504-infected cells, and we found it to be comparable to that in WT virus-infected cells, further supporting an effect on transcription. Transcription of the gC and U(L)47 genes by n504 was normal in a cell line that expresses WT ICP27. From these results we conclude that ICP27 is required for transcription of the late gC and U(L)47 genes during productive infection.

Genomewide assessment of genetic alterations in DMBA-induced rat sarcomas: cytogenetic, CGH, and allelotype analyses reveal recurrent DNA copy number changes in rat chromosomes 1, 2, 4, and 7.

Rat sarcomas, induced by subcutaneous injections of 7,12-dimethylbenz[a]anthracene (DMBA), were studied with the objective of identifying critical chromosome regions associated with tumorigenesis. We employed three genomewide screening techniques-cytogenetics, CGH, and allelotyping-in 19 DMBA-induced sarcomas in F1 (BN/Han x LE/Mol) rats. The most conspicuous finding in the cytogenetic analysis was a high incidence of trisomy for rat chromosome 2 (RNO2). Signs of gene amplification (hsr) were also seen in several tumors. The CGH analysis revealed that gains in copy number were much more common than losses. The gains mostly affected RNO2 (10/19), RNO12q (7/19), and RNO19q (5/19), as well as the proximal part of RNO4 (8/19) and the distal part of RNO7 (7/19). Reduction in copy number was seen in RNO17 (2/19). For the allelotyping, we used 318 polymorphic microsatellite marker loci covering the entire genome. We identified regions of allelic imbalance affecting most of the rat chromosomes. The highest incidences of recurrent allelic imbalance were observed at loci in certain regions in RNO1, 2, 4, and 7 and at lower incidences in parts of RNO12, 16, 18, and 19. The combined results suggested that genetic alterations detected in RNO2 and RNO12 usually corresponded to complete or partial trisomy, whereas those in RNO1 and RNO7 seemed to involve regional deletions and/or gains. Furthermore, we could confirm that copy number gains occur proximally in RNO4, where a previous study showed amplification of the Met oncogene in a subset of these tumors.

A dual-color FISH framework map for the characterization of the Sai1 tumor suppression region on rat chromosome 5.

The analysis of cell hybrids between malignant mouse hepatoma cells and normal rat fibroblasts has previously demonstrated the critical role of a deletion in rat chromosome 5 (RNO5) that was related to an anchorage independent phenotype. Those hybrids that were anchorage independent displayed loss of the entire RNO5 or an interstitial deletion in RNO5. These findings suggested that a putative tumor suppressor gene, Sai1 (suppression of anchorage independence 1), was located within the deleted region. To explore the molecular basis of the tumor suppressor activity of the Sai1 region, we analyzed the RNO5q23-q36 region with several genes and microsatellite markers that could be assigned to the region, as well as with new markers derived by representational difference analysis (RDA) or by microdissection. Dual-color FISH was used to construct a detailed physical map of the entire RNO5. These new data can be used to connect the physical and linkage maps in the rat, as well as to identify the details of the comparative map with other mammalian species including humans and mice. Using as FISH reagents genomic YAC, P1, or phage lambda clones corresponding to RNO5 markers, the order and unique positions of 18 markers could be established. The map provided a framework for the detailed characterization of the deletion found in anchorage independent hybrids. All markers within the bands RNO5q31.3-q35 were shown to be lost, including known cancer-related genes such as Ifna (5q32), Cdkn2a, -b (5q32), Jun (5q34), and Cdkn2c (5q35). However, the aberration in the deletion chromosome turned out to be more complex than originally thought in that we detected the presence of a paracentric inversion in addition to a deletion. The inversion led to the juxtaposition of the gene markers Tal2 (5q24.1) and Cd30lg (5q24.3). The framework map will provide the basis for the detailed physical YAC clone contig mapping of this region, and facilitate the identification and characterization of the Sai1 locus.

Amplification and overexpression of the hepatocyte growth factor receptor (HGFR/MET) in rat DMBA sarcomas.

In the present study subcutaneous fibrosarcomas were induced by the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) in rats from F1 generation cross breedings of two different inbred strains. Comparative genomic hybridization (CGH) analysis, which allows detection of DNA sequence copy changes, was applied to one of the tumors and it was found that there were increased copy numbers of sequences at chromosome 4q12-q21 in this tumor. We have previously determined that the loci for the hepatocyte growth factor (Hgf) and hepatocyte growth factor receptor (Hgfr/Met), a protooncogene, are situated in this particular chromosome region. Using probes for the two genes in FISH (fluorescence in situ hybridization) and in Southern blots we found that the Hgfr/Met gene was amplified in five of the 19 sarcomas studied, and that the Hgf gene was coamplified in two of them. Northern and Western blots and tyrosine phosphorylation analysis showed that the HGF receptor was overexpressed and functional in all five tumors, as well as in two additional tumors. In summary, both amplification and overexpression of the Hgfr/Met gene was found in about 25% of DMBA-induced experimental rat sarcomas, and HGF receptor overexpression alone was seen in two additional tumors. Possibly this reflects an involvement in paracrine or autocrine stimulation of growth and invasiveness by HGF. Our finding could provide a rodent model system to increased knowledge about causality and therapy, which may be applicable to the sizeable fraction of human musculoskeletal tumors displaying MET overexpression.

Rat Chromosome 2: assignment of the genes encoding cyclin B1, interleukin 6 signal transducer, and proprotein convertase 1 to the Mcs1-containing region and identification of new microsatellite markers.

The rat Chromosome (Chr) 2 harbors several genes controlling tumor growth or development, blood pressure, and non-insulin-dependent diabetes mellitus. We report that the region (2q1) containing the mammary susceptibility cancer gene Mcs1 also harbors the genes encoding cyclin B1, interleukin 6 signal transducer (gp130), and proprotein convertase 1. We also generated 13 new anonymous microsatellite markers from Chr 2-sorted DNA. These markers, as well as a microsatellite marker in the cyclin B1 gene, were genetically mapped in combination with known markers. A cyclin B1-related gene was also cytogenetically assigned to rat Chr 11q22-q23.

Gene-based anchoring of the rat genetic linkage and cytogenetic maps: new regional localizations, orientation of the linkage groups, and insights into mammalian chromosome evolution.

In order to generate anchor points connecting the rat cytogenetic and genetic maps, the cytogenetic position of 62 rat markers (including 55 genes) already localized genetically was determined by fluorescence in situ hybridization. Whenever possible, markers located near one end of the linkage groups were included. These new localizations allowed us to unambiguously orient the 20 autosomal and the X chromosome linkage groups. The position of the centromere in the linkage map could also be determined in the case of several metacentric chromosomes. In addition, the regional localization of 15 other rat genes was determined. These new data bring useful information with respect to comparative mapping with the mouse and the human and to mammalian evolution. They illustrate, for instance, that groups of genes can remain syntenic during mammalian evolution while being subjected to intrachromosomal rearrangements in some lineages (synteny is conserved while gene order is not). This analysis also disclosed cases of synteny conservation in one the two rodent species and the human, while the synteny is split in the other rodent species: such configurations are likely examples of lineage-specific interchromosomal rearrangements associated with speciation.