Various repair events following CRISPR/Cas9-based mutational correction of an infertility-related mutation in mouse embryos.

PURPOSE: Unpredictable genetic modifications and chromosomal aberrations following CRISPR/Cas9 administration hamper the efficacy of germline editing. Repair events triggered by double-strand DNA breaks (DSBs) besides non-homologous end joining and repair template-driven homology-directed repair have been insufficiently investigated in mouse. In this work, we are the first to investigate the precise repair mechanisms triggered by parental-specific DSB induction in mouse for paternal mutational correction in the context of an infertility-related mutation. METHODS: We aimed to correct a paternal 22-nucleotide deletion in Plcz1, associated with lack of fertilisation in vitro, by administrating CRISPR/Cas9 components during intracytoplasmic injection of Plcz1-null sperm in wild-type oocytes combined with assisted oocyte activation. Through targeted next-generation sequencing, 77 injected embryos and 26 blastomeres from seven injected embryos were investigated. In addition, low-pass whole genome sequencing was successfully performed on 17 injected embryo samples. RESULTS: Repair mechanisms induced by two different CRISPR/Cas9 guide RNA (gRNA) designs were investigated. In 13.73% (7/51; gRNA 1) and 19.05% (4/21; gRNA 2) of the targeted embryos, only the wild-type allele was observed, of which the majority (85.71%; 6/7) showed integrity of the targeted chromosome. Remarkably, for both designs, only in one of these embryos (1/7; gRNA 1 and 1/4; gRNA2) could repair template use be detected. This suggests that alternative repair events have occurred. Next, various genetic events within the same embryo were detected after single-cell analysis of four embryos. CONCLUSION: Our results suggest the occurrence of mosaicism and complex repair events after CRISPR/Cas9 DSB induction where chromosomal integrity is predominantly contained.

Comparative study of preimplantation development following distinct assisted oocyte activation protocols in a PLC-zeta knockout mouse model.

Mammalian fertilization encompasses a series of Ca2+ oscillations initiated by the sperm factor phospholipase C zeta (PLCζ). Some studies have shown that altering the Ca2+ oscillatory regime at fertilization affects preimplantation blastocyst development. However, assisted oocyte activation (AOA) protocols can induce oocyte activation in a manner that diverges profoundly from the physiological Ca2+ profiling. In our study, we used the newly developed PLCζ-null sperm to investigate the independent effect of AOA on mouse preimplantation embryogenesis. Based on previous findings, we hypothesized that AOA protocols with Ca2+ oscillatory responses might improve blastocyst formation rates and differing Ca2+ profiles might alter blastocyst transcriptomes. A total of 326 MII B6D2F1-oocytes were used to describe Ca2+ profiles and to compare embryonic development and individual blastocyst transcriptomes between four control conditions: C1 (in-vivo fertilization), C2 (ICSI control sperm), C3 (parthenogenesis) and C4 (ICSI-PLCζ-KO sperm) and four AOA groups: AOA1 (human recombinant PLCζ), AOA2 (Sr2+), AOA3 (ionomycin) and AOA4 (TPEN). All groups revealed remarkable variations in their Ca2+ profiles; however, oocyte activation rates were comparable between the controls (91.1% ± 13.8%) and AOA (86.9% ± 11.1%) groups. AOA methods which enable Ca2+ oscillatory responses (AOA1: 41% and AOA2: 75%) or single Ca2+ transients (AOA3: 50%) showed no significantly different blastocyst rates compared to ICSI control group (C2: 70%). In contrast, we observed a significant decrease in compaction (53% vs. 83%) and blastocyst rates (41% vs. 70%) in the absence of an initial Ca2+ trigger (AOA4) compared with the C2 group. Transcription profiles did not identify significant differences in gene expression levels between the ICSI control group (C2) and the four AOA groups.

Quantitative expression of phospholipase C zeta, as an index to assess fertilization potential of a semen sample.

BACKGROUND: Failed fertilization post-ICSI has been mainly attributed to the sperm's inability to induce oocyte activation. Phospholipase C zeta (PLCζ) is considered to be one of the factors for the induction of oocyte activation. The aim of this study was to quantitatively assess the expression of PLCζ in globozoospermic men or those with previously low or failed fertilization in comparison with fertile men or those with high fertilization potential. In addition, the relationship between expression of PLCζ and that of other sperm markers was evaluated. METHODS: Real-time PCR was carried out to evaluate relative expression of PLCζ mRNA. Chromatin maturity and acrosin activity were assessed by CMA3 staining and a colorimetric method. RESULTS: The expression of PLCζ was significantly lower in globozoospermic men (P< 0.01, n= 8) or individuals with previously low or failed fertilization (P< 0.01, n= 36) in comparison to fertile men (n= 24). In addition, a significant difference was observed between globozoospermic (P< 0.01) and individuals with previously low or failed fertilization (P= 0.003) in comparison to high fertilization individuals (n= 17). Expression of PLCζ was not correlated with either chromatin maturity or acrosin activity. However, a significant correlation was observed between the percentage of fertilization and relative expression of PLCζ (r= 0.4, P< 0.01). CONCLUSION: In this study, for the first time, we have shown that assessment of relative expression of PLCζ may provide a useful marker for the ability of sperm to induce oocyte activation after ICSI.

Reduced amounts and abnormal forms of phospholipase C zeta (PLCzeta) in spermatozoa from infertile men.

BACKGROUND: In mammals, oocyte activation at fertilization is thought to be induced by the sperm-specific phospholipase C zeta (PLCzeta). However, it still remains to be conclusively shown that PLCzeta is the endogenous agent of oocyte activation. Some types of human infertility appear to be caused by failure of the sperm to activate and this may be due to specific defects in PLCzeta. METHODS AND RESULTS: Immunofluorescence studies showed PLCzeta to be localized in the equatorial region of sperm from fertile men, but sperm deficient in oocyte activation exhibited no specific signal in this same region. Immunoblot analysis revealed reduced amounts of PLCzeta in sperm from infertile men, and in some cases, the presence of an abnormally low molecular weight form of PLCzeta. In one non-globozoospermic case, DNA analysis identified a point mutation in the PLCzeta gene that leads to a significant amino acid change in the catalytic region of the protein. Structural modelling suggested that this defect may have important effects upon the structure and function of the PLCzeta protein. cRNA corresponding to mutant PLCzeta failed to induce calcium oscillations when microinjected into mouse oocytes. Injection of infertile human sperm into mouse oocytes failed to activate the oocyte or trigger calcium oscillations. Injection of such infertile sperm followed by two calcium pulses, induced by assisted oocyte activation, activated the oocytes without inducing the typical pattern of calcium oscillations. CONCLUSIONS: Our findings illustrate the importance of PLCzeta during fertilization and suggest that mutant forms of PLCzeta may underlie certain types of human male infertility.

Iridium enrichment in airborne particles from kilauea volcano: january 1983.

Airborne particulate matter from the January 1983 eruption of Kilauea volcano was inadvertently collected on air filters at Mauna Loa Observatory at a sampling station used to observe particles in global circulation. Analyses of affected samples revealed unusually large concentrations of selenium, arsenic, indium, gold, and sulfur, as expected for volcanic emissions. Strikingly large concentrations of iridium were also observed, the ratio of iridium to aluminum being 17,000 times its value in Hawaiian basalt. Since iridium enrichments have not previously been observed in volcanic emissions, the results for Kilauea suggest that it is part of an unusual volcanic system which may be fed by magma from the mantle. The iridium enrichment appears to be linked with the high fluorine content of the volcanic gases, which suggests that the iridium is released as a volatile IrF(6).

Asian dust: seasonal transport to the hawaiian islands.

Analyses of atmospheric particles collected at Mauna Loa Observatory in Hawaii from February 1979 through September 1982 reveal strong influxes of Asian dust in the spring of each year. Concentrations of a typical crustal element, aluminum, are more than an order of magnitude greater between February and June than during the remainder of the year (71 +/- 51 versus 6.7 +/- 2.3 nanograms per cubic meter). The mass of crustal material transported during the relatively short dust episodes accounts for an average of 80 percent of the total yearly mass of atmospheric particles at 3400 meters on Mauna Loa.

Locus control region function and heterochromatin-induced position effect variegation.

Human CD2 locus control region (LCR) sequences are shown here to be essential for establishing an open chromatin configuration. Transgenic mice carrying an hCD2 mini-gene attached only to the 3' CD2 transcriptional enhancer exhibited variegated expression when the transgene integrated in the centromere. In contrast, mice carrying a transgene with additional 3' sequences showed no variegation even when the latter integrated in centromeric positions. This result suggests that LCRs operate by ensuring an open chromatin configuration and that a short region, with no enhancer activity, functions in the establishment, maintenance, or both of an open chromatin domain.

The pattern of localization of the putative oocyte activation factor, phospholipase Czeta, in uncapacitated, capacitated, and ionophore-treated human spermatozoa.

BACKGROUND: Recent studies suggest that in mammals, oocyte activation at fertilization is triggered by a sperm-specific phospholipase C, PLCzeta. We investigated PLCzeta localization in human spermatozoa. METHODS: A polyclonal antibody was generated against human PLCzeta and used in immunoblotting and immunofluorescence studies of ejaculated human sperm in uncapacitated and capacitated states. An ionophore was also used to induce the acrosome reaction in vitro. RESULTS: After verifying specificity of the anti-PLCzeta antibody by immunoblotting, immunofluorescence studies showed that the predominant localization of PLCzeta in uncapacitated sperm was in the equatorial region, a pattern maintained following capacitation and ionophore treatment. The analysis of pooled samples showed approximately 88% of uncapacitated sperm expressed PLCzeta in the equatorial region, whereas approximately 35% and approximately 21% of sperm expressed additional populations of PLCzeta in the acrosomal or post-acrosomal region, respectively. One population of PLCzeta was observed in the post-acrosomal region of approximately 12% of sperm. The proportion of cells with post-acrosomal PLCzeta increased following capacitation and ionophore treatment (P < 0.05). The same tendency was found in individual samples. There was a strong correlation (r = 0.716, P < 0.0001) between presence of an intact acrosome and proportion of sperm immunoreactive to PLCzeta in the acrosomal region. CONCLUSIONS: PLCzeta was variably detectable in three localities within the sperm head: the equatorial segment and acrosomal/post-acrosomal region. Variability in PLCzeta localization in sperm from fertile males may reflect differences in oocyte activation capabilities between individuals or within an ejaculate. This approach may help in investigating the possible links between PLCzeta and certain types of male infertility.

Transformation of growth factor-dependent myeloid stem cells with retroviral vectors carrying c-myc.

Myeloid progenitor cells and macrophages derived from bone marrow and spleen were efficiently transformed in vitro by infection with Moloney-based retroviral vectors carrying a human c-myc gene. Infected cells were plated in agar in the presence of combinations of the murine lymphokines CSF-1, IL-3, GM-CSF and IL-1. Between 20% and 100% of the colony-forming cells in the initial bone marrow or spleen population could be infected and gave rise to drug-resistant colonies. A large fraction of the infected cells showed continued proliferation after transfer to liquid media and we have derived over 200 growth factor-dependent cell lines. These include adherent and non-adherent CSF-1 or GM-CSF dependent macrophages and macrophage precursors and cell lines which require complex combinations of growth factors for optimal growth. Each of the cell lines displays a unique pattern of expression of surface markers specific for the myeloid lineage including the Mac-1, Mac-2, Mac-3, Ser-4 and F4/80 antigens. Surface markers not specifically associated with the myeloid lineage such as the MHC class II antigens and the Fc-receptor; and surface markers normally associated with the B-cell and T-cell lineages such as B220, L3T4 and Thy1.2 are also found on these cell lines.

Calcium oscillations, sperm factors and egg activation at fertilisation.

When an egg is fertilised by sperm, the first intracellular signalling event observed is a large transient increase in cytoplasmic free Ca2+ ions. Elevated Ca2+ is known to play a vital role as an intracellular messenger in all cells and the Ca2+ signal occurring in the egg at fertilisation triggers the subsequent events that mediate early embryo development. In mammalian eggs, the Ca2+ response is first observed as a Ca2+ wave that initiates near the point of sperm-egg fusion, spreads across the entire egg, and then continues as a series of intracellular Ca2+ oscillations. The way in which the fertilising sperm generates the Ca2+ response in the egg has been the subject of much debate over recent years. One proposal for which there is growing evidence suggests the mechanism of egg activation at fertilisation involves the introduction of a soluble sperm protein into the egg shortly after sperm-egg fusion.

The human glucosamine-6-phosphate deaminase gene: cDNA cloning and expression, genomic organization and chromosomal localization.

When mammalian eggs are fertilized by sperm, a distinct series of calcium oscillations are generated which serve as the essential trigger for egg activation and early embryo development. The identification of a soluble hamster sperm 33-kDa protein that co-migrated with calcium oscillation-inducing activity was recently described by Parrington et al. (Parrington, J., Swann, K., Shevchenko, V.I., Sesay, A.K. and Lai, F.A., 1996. Calcium oscillations in mammalian eggs triggered by a soluble sperm protein. Nature 379, 364-368). The hamster sperm 33 kDa protein was termed oscillin because it correlated with calcium oscillation-inducing activity in mammalian eggs. Sequence analysis of the hamster sperm 33 kDa protein indicated no similarity to any known cell signalling molecule, however, it displayed extensive homology with a bacterial glucosamine-6-phosphate deaminase. We have isolated the corresponding human testis homologue of the hamster sperm 33 kDa cDNA. Nucleotide sequence analysis reveals a high level of sequence identity between the hamster and human genes. The deduced protein sequence of the human gene also shares extensive amino acid identity with the bacterial glucosamine-6-phosphate deaminase enzyme. Heterologous expression of the human testis 33 kDa protein produced a glucosamine-6-phosphate deaminase activity. The genomic structure of the human glucosamine-6-phosphate deaminase has been mapped and the gene was localized by fluorescence in situ hybridization (FISH) to chromosome 5q31.

A mammalian sperm cytosolic phospholipase C activity generates inositol trisphosphate and causes Ca2+ release in sea urchin egg homogenates.

Injection of sperm extracts triggers Ca2+ oscillations in mammalian eggs similar to those seen at fertilisation. Here, we show that addition of sperm extracts to sea urchin egg homogenates causes Ca2+ release and inositol 1,4,5-trisphosphate (InsP3) production. Furthermore depleting homogenates of phosphatidylinositol lipids using a phosphatidylinositol-specific phospholipase C blocked the sperm extract from causing InsP3 production and a Ca2+ rise. A response could be recovered by the addition of phosphatidylinositol 4,5-bisphosphate to either sperm extracts or egg homogenates. These data indicate that sperm extracts contain an InsP3-generating phospholipase C which may play a role in Ca2+ release at fertilisation.

Presence and localization of oscillin in human spermatozoa in relation to the integrity of the sperm membrane.

We investigated the presence and localization of oscillin in human spermatozoa in relation to the integrity of the sperm membrane, which was assessed by the hypo-osmotic swelling (HOS) test. We found no gross differences in the presence of oscillin in semen samples from men who presented with 70%, 40%, 25% or 2% of membrane-intact spermatozoa. By immunofluorescence, membrane-intact (HOS-positive) spermatozoa showed staining of a single band at the equatorial region, whereas over 80% of HOS-negative spermatozoa consistently showed a diffuse distribution of oscillin over the sperm head. However, some individuals presented with up to 50% of HOS-positive spermatozoa showing an aberrant localization of oscillin. We found a significant correlation rate (r=0.70, P < 0.05) between the percentage of HOS-positive spermatozoa with an equatorial oscillin localization and the fertilization rates achieved after intracytoplasmic sperm injection. These data suggest that the localization of oscillin in human spermatozoa might have an impact on egg activation and fertilization rates.

Development and characterization of cisplatin-resistant human testicular and bladder tumour cell lines.

Cisplatin-resistant cells were derived in vitro from a human bladder carcinoma cell line (RT112) and a testicular tumour cell line (SuSa) by continuous exposure to increasing concentrations of cisplatin for 14 and 11 months, respectively. Both resistant cell lines had a four-fold level of resistance relative to their parental cell lines, comparing the cisplatin concentration to inhibit colony forming ability by 70%. These levels of resistance were retained in the absence of cisplatin for at least 3 months. In each case, four-fold fewer micronuclei were produced in the resistant lines by the same cisplatin concentrations. Cross-resistance to carboplatin and methotrexate was observed in both resistant cell lines, but neither line was resistant to doxorubicin. Isozyme and DNA analysis with hypervariable probes confirmed the origin of each resistant cell line from its parental line. Population doubling times and intermitotic times were similar in each of the pairs of cell lines. Karyotyping showed that the resistant cell lines had gained and lost marker chromosomes, but there were no changes common to both resistant cell lines.

The NAADP receptor: commentary on Billington et al.

NAADP is a recently described calcium-mobilizing messenger. First discovered as a potent calcium-releasing molecule in sea urchin eggs, its actions have now been reported in several mammalian cell types. In the sea urchin egg, NAADP-sensitive calcium release channels appear distinct from inositol trisphosphate or ryanodine receptors, and are mainly localized to acidic compartments. In this study, Billington et al. extend the pharmacology of the putative NAADP receptor utilizing molecules unrelated to NAADP itself. This work may provide an important step in developing selective NAADP receptor modulators that will help define the role of NAADP in cell signalling.

Spontaneous and induced chromosome damage in lymphocytes of medullary thyroid carcinoma patients and their family members.

Detailed chromosome analysis failed to reveal any evidence for spontaneous chromosome instability in lymphocytes from patients with multiple endocrine neoplasia, type 2 (MEN-2), whereas, with one exception, lymphocytes from MEN-2 patients were significantly more sensitive to chromosome damage by bleomycin and, to a lesser extent, MNNG than those from controls. The exceptional case suggests possible genetic heterogeneity in MEN-2.

The application of comparative genomic hybridization to previously karyotyped cervical cancer cell lines.

This investigation is concerned with the application of comparative genomic hybridization (CGH) to DNA from previously fully karyotyped cervical cancer cell lines using G-banding and fluorescence in situ hybridization (FISH) to compare the chromosome copy numbers observed in karyotypes with the profile shifts seen in CGH analysis. It has demonstrated that diploid DNA can be used as a reference to cohybridize with a test sample of any modal number because of the proportional representation of every chromosome arm and region in equal volumes of both test and reference DNAs. Profile shifts in the near-diploid line gave a clear indication of over and under-representation of either the whole or parts of chromosome arms. In near-tetraploid samples, profile shifts, either gain or loss due to copy number changes from four to five, five to six, or four to three were smaller and were not always seen; however, the points of profile shift would have allowed us to work out most of the breakpoints if karyotype information had not been available. The profiles, however, did not provide accurate information on the ploidy status; this would need to be measured by other means for the CGH data to be interpreted correctly. The 3q and 8q gain in all the squamous cell carcinoma cell lines appeared very clearly. Comparative genomic hybridization revealed a new breakpoint at 7q31 which was not detected originally on the karyotype in DE3. A breakpoint on 9q was reassigned on the basis of the profile shift from 9q13 to 9q22 in JE6. Clarification of the origin of a small fragment from chromosome 20 constantly present in JE6 showed it to be 20q22-qter.

Chromosome analysis of parallel short-term cultures from four testicular germ-cell tumors.

Cytogenetic analysis has been carried out on 17 parallel short-term cultures from four malignant testicular teratomas of intermediate differentiation (MTI), two of them combined with seminomas. Clonal development was seen in three tumors, with most of the variation involving different rearrangements of chromosome 1. Two copies of the i(12p), characteristic of testicular germ-cell tumors, were present in the two tumors with yolk sac elements, a single i(12p) and a 12q- were found in an MTI that had metastasized, and a rearrangement of chromosome 12 containing centromeric chromatin from chromosome 18 was found instead of the i(12p) in a mixed tumor. A 13p+ marker containing Q-negative material was seen in two of the tumors, with a der(7)t(Y;7) in one. Chromosomes 1, 3, 6, 7, 8, 12, 17, and X were invariably overrepresented either as complete or partial trisomies, and chromosomes 4, 5, 13, and 18 were underrepresented in all four tumors, strengthening the idea that tumor suppressor genes on the latter four chromosomes, all of which show some loss of heterozygosity with DNA markers, may be important in tumor progression.

Correlation of loss of heterozygosity with cytogenetic analysis using G-banding and fluorescence in situ hybridization in aneuploid cultures from two human testicular germ-cell tumors.

A detailed karyotype analysis using fluorescence in situ hybridization (FISH), with 24 chromosome-specific paint probes has been carried out on newly established cell lines from two testicular tumors, an i(12p)-positive teratoma, and an i(12p)-negative combined seminoma/teratoma. This has been correlated with loss of heterozygosity (LOH) and allelic imbalance, using DNA RFLP analysis to clarify the genetic changes and to identify any common regions of deletion or rearrangement. With G-banding alone, a total of 11 breakpoints were recognized. After FISH, the position of seven required revision, and 21 new ones were identified. The chromosomes involved most frequently in both tumors were numbers 1, 12, and 18. Breakpoints in 11q and 16q were also seen in both, and seven or more copies of 12p per cell were found in all clones. LOH was found for 18q in both tumors, and overall was much more frequent in underrepresented regions (one or two copies). On the whole, there was good agreement between the cytogenetic and DNA RFLP data; loci showing allelic imbalance generally had an odd number of copies of the chromosome region in which they were known to be located. Combined data on the chromosome 1 translocations in both tumors suggested that rearrangements were more complicated than cytogenetics alone had predicted.