Vera D. Rinaldi

Reversal of Female Infertility by Chk2 Ablation Reveals the Oocyte DNA Damage Checkpoint Pathway

Eggs Well Done Germ cells can endure extensive DNA damage during their development. Programmed meiotic double-strand breaks (DSBs) are essential for proper segregation of chromosomes to oocytes and sperm. However, incomplete DSB repair by recombination activates a checkpoint that triggers cell death. Exogenous DNA damage is also lethal to oocytes via a highly sensitive checkpoint. Bolcun-Filas et al. (p. 533) show that the CHK2 kinase is a key component of both checkpoints in mouse oocytes. Deletion of Chk2 restored fertility to females that would otherwise be sterile because of a meiotic recombination mutation or radiation exposure. Elucidation of a system that monitors DNA damage in both meiotic and resting oocytes in mice is shown. Genetic errors in meiosis can lead to birth defects and spontaneous abortions. Checkpoint mechanisms of hitherto unknown nature eliminate oocytes with unrepaired DNA damage, causing recombination-defective mutant mice to be sterile. Here, we report that checkpoint kinase 2 (Chk2 or Chek2), is essential for culling mouse oocytes bearing unrepaired meiotic or induced DNA double-strand breaks (DSBs). Female infertility caused by a meiotic recombination mutation or irradiation was reversed by mutation of Chk2. Both meiotically programmed and induced DSBs trigger CHK2-dependent activation of TRP53 (p53) and TRP63 (p63), effecting oocyte elimination. These data establish CHK2 as essential for DNA damage surveillance in female meiosis and indicate that the oocyte DSB damage response primarily involves a pathway hierarchy in which ataxia telangiectasia and Rad3-related (ATR) signals to CHK2, which then activates p53 and p63.

Mutation in Spike Protein Cleavage Site and Pathogenesis of Feline Coronavirus

Feline coronaviruses (FCoV) exist as 2 biotypes: feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FECV causes subclinical infections; FIPV causes feline infectious peritonitis (FIP), a systemic and fatal disease. It is thought that mutations in FECV enable infection of macrophages, causing FIP. However, the molecular basis for this biotype switch is unknown. We examined a furin cleavage site in the region between receptor-binding (S1) and fusion (S2) domains of the spike of serotype 1 FCoV. FECV sequences were compared with FIPV sequences. All FECVs had a conserved furin cleavage motif. For FIPV, there was a correlation with the disease and >1 substitution in the S1/S2 motif. Fluorogenic peptide assays confirmed that the substitutions modulate furin cleavage. We document a functionally relevant S1/S2 mutation that arises when FIP develops in a cat. These insights into FIP pathogenesis may be useful in development of diagnostic, prevention, and treatment measures against coronaviruses.

Comparative Oncogenomics Implicates the Neurofibromin 1 Gene (NF1) as a Breast Cancer Driver

Identifying genomic alterations driving breast cancer is complicated by tumor diversity and genetic heterogeneity. Relevant mouse models are powerful for untangling this problem because such heterogeneity can be controlled. Inbred Chaos3 mice exhibit high levels of genomic instability leading to mammary tumors that have tumor gene expression profiles closely resembling mature human mammary luminal cell signatures. We genomically characterized mammary adenocarcinomas from these mice to identify cancer-causing genomic events that overlap common alterations in human breast cancer. Chaos3 tumors underwent recurrent copy number alterations (CNAs), particularly deletion of the RAS inhibitor Neurofibromin 1 (Nf1) in nearly all cases. These overlap with human CNAs including NF1, which is deleted or mutated in 27.7% of all breast carcinomas. Chaos3 mammary tumor cells exhibit RAS hyperactivation and increased sensitivity to RAS pathway inhibitors. These results indicate that spontaneous NF1 loss can drive breast cancer. This should be informative for treatment of the significant fraction of patients whose tumors bear NF1 mutations.

The DNA Damage Checkpoint Eliminates Mouse Oocytes with Chromosome Synapsis Failure.

Pairing and synapsis of homologous chromosomes during meiosis is crucial for producing genetically normal gametes and is dependent upon repair of SPO11-induced double-strand breaks (DSBs) by homologous recombination. To prevent transmission of genetic defects, diverse organisms have evolved mechanisms to eliminate meiocytes containing unrepaired DSBs or unsynapsed chromosomes. Here we show that the CHK2 (CHEK2)-dependent DNA damage checkpoint culls not only recombination-defective mouse oocytes but also SPO11-deficient oocytes that are severely defective in homolog synapsis. The checkpoint is triggered in oocytes that accumulate a threshold level of spontaneous DSBs (∼10) in late prophase I, the repair of which is inhibited by the presence of HORMAD1/2 on unsynapsed chromosome axes. Furthermore, Hormad2 deletion rescued the fertility of oocytes containing a synapsis-proficient, DSB repair-defective mutation in a gene (Trip13) required for removal of HORMADs from synapsed chromosomes, suggesting that many meiotic DSBs are normally repaired by intersister recombination in mice.

Pharmacological Inhibition of the DNA Damage Checkpoint Prevents Radiation-Induced Oocyte Death

Ovarian function is directly correlated with survival of the primordial follicle reserve. Women diagnosed with cancer have a primary imperative of treating the cancer, but since the resting oocytes are hypersensitive to the DNA-damaging modalities of certain chemo- and radiotherapeutic regimens, such patients face the collateral outcome of premature loss of fertility and ovarian endocrine function. Current options for fertility preservation primarily include the collection and cryopreservation of oocytes or in vitro-fertilized oocytes, but this necessitates a delay in cancer treatment and additional assisted reproductive technology procedures. Here, we evaluated the potential of pharmacological preservation of ovarian function by inhibiting a key element of the oocyte DNA damage checkpoint response, checkpoint kinase 2 (CHK2; CHEK2). Whereas nonlethal doses of ionizing radiation (IR) eradicate immature oocytes in wild-type mice, irradiated Chk2−/− mice retain their oocytes and, thus, fertility. Using an ovarian culture system, we show that transient administration of the CHK2 inhibitor 2-(4-(4-chlorophenoxy)phenyl)-1H-benzimidazole-5-carboxamide-hydrate (“CHK2iII”) blocked activation of the CHK2 targets TRP53 and TRP63 in response to sterilizing doses of IR, and preserved oocyte viability. After transfer into sterilized host females, these ovaries proved functional and readily yielded normal offspring. These results provide experimental evidence that chemical inhibition of CHK2 is a potentially effective treatment for preserving the fertility and ovarian endocrine function of women exposed to DNA-damaging cancer therapies such as IR.

Characterization of a recombinant canine coronavirus with a distinct receptor-binding (S1) domain.

Abstract Canine alphacoronaviruses (CCoV) exist in two serotypes, type I and II, both of which can cause severe gastroenteritis. Here, we characterize a canine alphacoronavirus, designated CCoV-A76, first isolated in 1976. Serological studies show that CCoV-A76 is distinct from other CCoVs, such as the prototype CCoV-1-71. Efficient replication of CCoV-A76 is restricted to canine cell lines, in contrast to the prototypical type II strain CCoV-1-71 that more efficiently replicates in feline cells. CCoV-A76 can use canine aminopeptidase N (cAPN) receptor for infection of cells, but was unable to use feline APN (fAPN). In contrast, CCoV-1-71 can utilize both. Genomic analysis shows that CCoV-A76 possesses a distinct spike, which is the result of a recombination between type I and type II CCoV, that occurred between the N- and C-terminal domains (NTD and C-domain) of the S1 subunit. These data suggest that CCoV-A76 represents a recombinant coronavirus form, with distinct host cell tropism.

Inhibition of influenza virus infection and hemagglutinin cleavage by the protease inhibitor HAI-2.

Influenza virus remains a significant concern to public health, with the continued potential for a high fatality pandemic. Vaccination and antiviral therapeutics are effective measures to circumvent influenza virus infection, however, multiple strains have emerged that are resistant to the antiviral therapeutics currently on the market. With this considered, investigation of alternative antiviral therapeutics is being conducted. One such approach is to inhibit cleavage activation of the influenza virus hemagglutinin (HA), which is an essential step in the viral replication cycle that permits viral-endosome fusion. Therefore, targeting trypsin-like, host proteases responsible for HA cleavage in vivo may prove to be an effective therapeutic. Hepatocyte growth factor activator inhibitor 2 (HAI-2) is naturally expressed in the respiratory tract and is a potent inhibitor of trypsin-like serine proteases, some of which have been determined to cleave HA. In this study, we demonstrate that HAI-2 is an effective inhibitor of cleavage of HA from the human-adapted H1 and H3 subtypes. HAI-2 inhibited influenza virus H1N1 infection in cell culture, and HAI-2 administration showed protection in a mouse model of influenza. HAI-2 has the potential to be an effective, alternative antiviral therapeutic for influenza.

Repair of Meiotic DNA Breaks and Homolog Pairing in Mouse Meiosis Requires a Minichromosome Maintenance (MCM) Paralog

The mammalian Mcm-domain containing 2 (Mcmdc2) gene encodes a protein of unknown function that is homologous to the minichromosome maintenance family of DNA replication licensing and helicase factors. Drosophila melanogaster contains two separate genes, the Mei-MCMs, which appear to have arisen from a single ancestral Mcmdc2 gene. The Mei-MCMs are involved in promoting meiotic crossovers by blocking the anticrossover activity of BLM helicase, a function presumably performed by MSH4 and MSH5 in metazoans. Here, we report that MCMDC2-deficient mice of both sexes are viable but sterile. Males fail to produce spermatozoa, and formation of primordial follicles is disrupted in females. Histology and immunocytological analyses of mutant testes revealed that meiosis is arrested in prophase I, and is characterized by persistent meiotic double-stranded DNA breaks (DSBs), failure of homologous chromosome synapsis and XY body formation, and an absence of crossing over. These phenotypes resembled those of MSH4/5-deficient meiocytes. The data indicate that MCMDC2 is essential for invasion of homologous sequences by RAD51- and DMC1-coated single-stranded DNA filaments, or stabilization of recombination intermediates following strand invasion, both of which are needed to drive stable homolog pairing and DSB repair via recombination in mice.

Evaluating a technology supported interactive response system during the laboratory section of a histology course

Monitoring of student learning through systematic formative assessment is important for adjusting pedagogical strategies. However, traditional formative assessments, such as quizzes and written assignments, may not be sufficiently timely for making adjustments to a learning process. Technology supported formative assessment tools assess student knowledge, allow for immediate feedback, facilitate classroom dialogues, and have the potential to modify student learning strategies. As an attempt to integrate technology supported formative assessment in the laboratory section of an upper‐level histology course, the interactive application Learning CatalyticsTM, a cloud‐based assessment system, was used. This study conducted during the 2015 Histology courses at Cornell University concluded that this application is helpful for identifying student misconceptions “on‐the‐go,” engaging otherwise marginalized students, and forming a new communication venue between students and instructors. There was no overall difference between grades from topics that used the application and grades from those that did not, and students reported that it only slightly helped improve their understanding of the topic (3.8 ± 0.99 on a five‐point Likert scale). However, they highly recommended using it (4.2 ± 0.71). The major limitation was regarding the image display and graphical resolution of this application. Even though students embrace the use of technology, 39% reported benefits of having the traditional light microscope available. This cohort of students led instructors to conclude that the newest tools are not always better, but rather can complement traditional instruction methods. Anat Sci Educ 10: 328–338.

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ovarian fitness is often assessed by visualizing and quantifying follicles and oocytes. Because the ovary is an opaque three-dimensional tissue, traditional approaches require laboriously slicing the tissue into numerous serial sections in order to visualize cells throughout the entire organ. Furthermore, because quantification by this method typically entails scoring only a subset of the sections separated by the approximate diameter of an oocyte, it is prone to inaccuracy. Here, a protocol is described that instead utilizes whole organ tissue clearing and immunofluorescence staining of mouse ovaries to visualize follicles and oocytes. Compared to more traditional approaches, this protocol is advantageous for visualizing cells within the ovary for numerous reasons: 1) the ovary remains intact throughout sample preparation and processing; 2) small ovaries, which are difficult to section, can be examined with ease; 3) cellular quantification is more readily and accurately achieved; and 4) the whole organ imaged.