Production of offspring after sperm chromosome screening: an experiment using the mouse model.

STUDY QUESTION: Is it possible to produce offspring after sperm chromosome screening? SUMMARY ANSWER: It is possible to produce zygotes after examining the genome of individual spermatozoa prior to embryo production. WHAT IS KNOWN ALREADY: Chromosomal aberrations in gametes are a major cause of pregnancy loss in women treated with assisted reproductive technology. However, to our knowledge, there are no reports on the successful genomic screening of spermatozoa, although some attempts have been made using the mouse as a model. STUDY DESIGN: To prevent the transmission of chromosomal aberrations from fathers to offspring, we performed sperm chromosome screening (SCS) prior to fertilization using the mouse as a model. The production of offspring after SCS consists of (i) replication of the sperm chromosomes, (ii) analysis of one copy of the replicated sperm chromosomes, (iii) construction of a zygote using another set of chromosomes and (iv) production of a transferable embryo. MATERIALS, SETTING, METHODS: A single spermatozoon of a male mouse, with or without a Robertsonian translocation, was injected into an enucleated oocyte to allow the replication of sperm chromosomes. One of the sister blastomeres of a haploid androgenic 2-cell embryo was used for chromosome analysis. The other blastomere was fused with an unfertilized oocyte, activated and allowed to develop to a blastocyst before transfer to a surrogate mother. MAIN RESULTS AND ROLE OF CHANCE: With high efficiency, we were able to analyze sperm chromosomes in a blastomere from the androgenic 2-cell embryos and culture zygotes, with and without aberrant chromosomes, to the blastocyst stage before embryo transfer. The karyotypes of the offspring faithfully reflected those of the blastomeres used for SCS. LIMITATIONS, REASONS FOR CAUTION: This study was conducted using a mouse model; whether or not the method is applicable to humans is not known. WIDER IMPLICATIONS OF THE FINDINGS: This study has shown that it is possible to produce zygotes without any paternally inherited aberrations by examining the genome of individual spermatozoa prior to embryo production.

Annexin V decreases PS-mediated macrophage efferocytosis and deteriorates elastase-induced pulmonary emphysema in mice.

Efferocytosis is believed to be a key regulator for lung inflammation in chronic obstructive pulmonary disease. In this study we pharmacologically inhibited efferocytosis with annexin V and attempted to determine its impact on the progression of pulmonary emphysema in mouse. We first demonstrated in vitro and in vivo efferocytosis experiments using annexin V, an inhibitor for phosphatidylserine-mediated efferocytosis. We then inhibited efferocytosis in porcine pancreatic elastase (PPE)-treated mice. PPE-treated mice were instilled annexin V intranasally starting from day 8 until day 20. Mean linear intercept (Lm) was measured, and cell apoptosis was assessed in lung specimen obtained on day 21. Cell profile, apoptosis, and mRNA expression of matrix metalloproteinases (MMPs) and growth factors were evaluated in bronchoalveolar lavage (BAL) cells on day 15. Annexin V attenuated macrophage efferocytosis both in vitro and in vivo. PPE-treated mice had a significant higher Lm, and annexin V further increased that by 32%. More number of macrophages was found in BAL fluid in this group. Interestingly, cell apoptosis was not increased by annexin V treatment both in lung specimens and BAL fluid, but macrophages from mice treated with both PPE and annexin V expressed higher MMP-2 mRNA levels and had a trend for higher MMP-12 mRNA expression. mRNA expression of keratinocyte growth factor tended to be downregulated. We showed that inhibited efferocytosis with annexin V worsened elastase-induced pulmonary emphysema in mice, which was, at least partly, attributed to a lack of phenotypic change in macrophages toward anti-inflammatory one.

Photoelectrochemical dimethoxylation of furan via a bromide redox mediator using a BiVO4/WO3 photoanode.

Photoelectrochemical dimethoxylation of furan with methanol using a BiVO4/WO3 photoanode and Br+/Br- as a mediator was demonstrated with low applied potential. The faradaic efficiency for the dimethoxylation with a Et4NBF4 co-supporting electrolyte at +0.1 V vs. SHE was almost quantitative up to 99%.

Survey of thorotrast-associated liver cancers in Japan.

Data on 93 autopsy cases (group A) of thorotrast-associated liver cancers were obtained from the "Annual of Pathological Autopsy Cases in Japan" from 1958 to 1979, and data on 78 autopsy cases (group B) of thorotrast-associated liver cancers were obtained from the Japanese literature from 1953 to 1980. Cholangiocarcinoma (CLC) constituted 58% of group A and 55% of group B. The curve of the cumulative numbers of patients with CLC versus year in group A was almost linear, showing an increasing risk per surviving patients with advancing time. Angiosarcoma (AGS) occurred in 25% of group A and 24% of group B. The number of patients with AGS increased significantly after 1969 in both groups (P less than 0.05). In group B, age and years after thorotrast injection of patients with AGS were statistically higher than those of patients with CLC (age: P less than 0.05; years after thorotrast injection: P less than 0.0001). Hepatocellular carcinoma (HPC) accounted for 17 and 21% of groups A and B, respectively. When yearly distribution, age, and time after thorotrast injection of patients with HPC were correlated with those of patients with other liver cancers, the only statistically significant difference between patients with HPC and patients with CLC (P less than 0.02) was in the years after thorotrast administration. Since 1977 multiple primary liver cancers including AGS developed in thorotrast-administered patients in both groups.

Immunohistochemical localization of rhamnose-binding lectins in the steelhead trout (Oncorhynchus mykiss).

The localization of three -rhamnose-binding lectins named STL1, STL2, and STL3 from eggs of steelhead trout (Oncorhynchus mykiss) was analyzed by indirect immunohistochemical staining with specific antisera against individual lectins. In early previtellogenic oocyte, STL1 was localized not only in the cortical vesicles, but also in the plasma membrane and germinal vesicle. On the other hand, STL2 and STL3 were localized only in the cortical vesicles. In pre-fertilization mature egg, STLs were localized in a thin layer of cortical granules at the cytoplasmic side of the plasma membrane. STLs were accumulated on the surface of cytoplasm and inner membrane 30 min after fertilization. The strong staining with anti-STL1 antiserum was observed in several tissues and cells of the steelhead trout, such as spleen, thrombocytes, and blood leukocytes, but not erythrocytes. STL1 was also identified in exocrine cells, such as goblet cells of intestine and mucous cells of gill. These results indicate that the multiple lectins found in eggs of the steelhead trout play physiological roles not only in eggs, but also in various cells related to the innate immunity.

Primary adenoid cystic carcinoma of the trachea: a case report of a twelve year survivor.

Malignant tumors arising from trachea are not common. This paper presents an example of primary tracheal adenoid cystic carcinoma treated by surgical resection with good prognosis. A 46-year-old woman presented with a short history of dyspnea. Five months before the onset of dyspnea, the patient had mild wheezing. She had no history of smoking. Physical examination suggested primary tracheal tumor. The patient underwent a V-shaped resection of 3.5 cm of trachea followed by reconstruction with the trough method. Histological examination revealed adenoid cystic carcinoma. Adjuvant chemotherapy was indicated with 50 mg of adriamycin postoperatively. The patient has done well for 12 years with no further treatment. The tumor was an adenoid cystic carcinoma that was slow-growing, infiltration of mucus membrane was few and growth fraction (mitotic index was less than 1%) was low. Those were considered the reason for good prognosis.

Chromosomal characteristics of tumorigenic cells derived from a spontaneous uterine leiomyosarcoma of the Chinese hamster.

A tumorigenic cell line (CHAUT) derived from a spontaneous uterine leiomyosarcoma of the Chinese hamster was established, and two clones (CHAUT-C and CHAUT-G) were characterized cytogenetically by both G- and C-band techniques. In both clones, all cells analyzed (53 in the C clone and 65 in the G clone) were distributed within a diploid range (24-26) with a modal number of 25. Their karyotypes were also characterized by four common changes; translocation of heterochromatic segment onto chromosome 2, tetrasomy of chromosome 10, monosomy of X chromosome, and one to three additional marker chromosomes of unknown origin.

Presence of Streptococcus infection in extra-oropharyngeal head and neck squamous cell carcinoma and its implication in carcinogenesis.

To investigate the involvement of S. anginosus infection in head and neck cancer in the extra-oropharyngeal cavity, we analyzed 3 DNA samples prepared from squamous cell carcinoma of the external auditory canal and one from squamous cell carcinoma of the skin using polymerase chain reaction (PCR) analysis and Southern blot analysis to detect the DNA sequence of S. anginosus. We also examined these four specimens by Gram's stain to detect the streptococcal bacterial bodies. By PCR analysis, the DNA sequence of S. anginosus was found in 4 out of 4 (100%) DNA samples obtained from these tumors. By Southern blot analysis, positive bands were detected in one out of the 3 (33%) samples from the tumor taken from the external auditory canal. We detected streptococcal bacterial bodies in one of the three specimens from the tumor obtained from cancer of the external auditory canal and in the one specimen from the skin cancer by the method of Gram's stain. Contrary to our expectations, these bacterial bodies were located in the middle of the tumor. Since S. anginosus is thought to exist in the mouth as a normal flora and to be located mainly in the gingiva and dental plaque, these data strongly indicate that S. anginosus infection is implicated in the carcinogenesis of head and neck squamous cell carcinoma.

Expression of 11 beta-hydroxysteroid dehydrogenase type 2 and mineralocorticoid receptor in primary lung carcinomas.

We examined the immunohistochemical distribution of 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta HSD2) and mineralocorticoid receptor (MR) in 63 primary lung carcinomas. Immunoreactivity of 11 beta HSD2 and MR was detected in 37 cases and in 32 cases of 42 adenocarcinomas, respectively. There was a significant correlation between 11 beta HSD2 and MR immunoreactivity. In three adenosquamous carcinomas, both 11 beta HSD2 and MR were detected only in adenocarcinomatous components. Neither 15 squamous cell carcinomas, 2 small cell carcinomas nor 1 large cell carcinoma expressed 11 beta HSD2 or MR. In papillary and acinar adenocarcinomas, both 11 beta HSD2 and MR immunoreactivity was significantly correlated with the grade of histological differentiation. The patterns of 11 beta HSD2 and MR expression in 10 lymph-node metastases were similar to those determined in the primary lesions. These data suggest that the patterns of 11 beta HSD2 and MR expression may reflect cellular origin and differentiation status of primary lung adenocarcinomas and serve as a new useful marker of differentiation.

Immunohistochemistry of Caspase3/CPP32 in human stomach and its correlation with cell proliferation and apoptosis.

Caspase3/CPP32 is a member of the interleukin-1 beta-converting enzyme (ICE) or cell death effector (CED)-3 family, which is involved in the induction of apoptosis and has been considered to be correlated with apoptosis because of the most downstream enzyme in their apoptosis inducing pathway. We immunolocalized Caspase3/CPP32 in both normal and neoplastic human gastric mucosa. We then correlated the findings with cell proliferation studied by Ki67 immunostaining and apoptosis, which was tested for by DNA fragmentation in situ using TdT-mediated dUTP biotin nick end labeling (TUNEL) method in order to examine possible biological significance in cell turnover of normal and pathological human gastric tissues. Caspase3/CPP32 immunoreactivity was detected in both the cytoplasm and nuclei of glandular epithelial cells, predominantly in the Ki67 positive proliferative zone and TUNEL positive foveolar epithelium of normal non-neoplastic gastric mucosa (n = 10) and tumor cells of both adenoma (n = 17) and carcinoma (n = 33). We determined the labeling index (LI) of Ki67, Caspase3/CPP32 and TUNEL positive cells by evaluating the number of positive cells in the same areas of serial tissue sections using computer-assisted image analysis. Ki67 LI in adenocarcinoma (78.6 +/- 12.6%) was significantly [p < 0.0001] higher than that of adenoma (43.8 +/- 8.9%) and non-neoplastic gastric mucosa (24.2 +/- 9.0%). Caspase3/CPP32 LI in adenocarcinoma (17.1 +/- 10.3%) was significantly lower [p < 0.0001] than that of gastric adenoma (33.1 +/- 19.8%) and non-neoplastic gastric mucosa (42.4 +/- 15.8%). TUNEL LI in adenocarcinoma (1.9 +/- 2.1%) was significantly [p < 0.0001] lower than that of non-neoplastic gastric mucosa (6.0 +/- 3.5%), but not significantly different from that of adenoma (3.0 +/- 2.9%). These results indicate that gastric adenocarcinoma is associated with an inhibition of apoptosis and the augmentation of proliferative activity of tumor cells compared to non-neoplastic gastric mucosa. There was a tendency to a positive correlation between the Caspase3/CPP32 and TUNEL LI and an inverse correlation between the Caspase3/CPP32 and Ki67 LI, when evaluating all the specimens, although the correlation did not reach statistical significance. These results also suggest that Caspase3/CPP32 is involved in the development or regulation of apoptotic cell death in cell turnover of normal and neoplastic mucosa of the human stomach.

Relative biological effectiveness (RBE) of 252Cf fission neutrons for the induction of chromosome damage in human spermatozoa.

Effects of 60Co gamma-rays and 252Cf neutrons on human sperm chromosomes were studied using our interspecific in vitro fertilization system to estimate relative biological effectiveness (RBE) of neutrons. Semen samples were exposed to 0.5, 1.0 and 2.0 Gy of 60Co gamma-rays at 1.7 cGy/ min and 0.25, 0.5 and 1.0 Gy of 252Cf radiation at 1.3-1.7 cGy/ min. In the 60Co experiment, 509 spermatozoa from controls and 902 spermatozoa from the irradiated groups were karyotyped, while in the 252Cf experiment 460 control and 804 irradiated spermatozoa were analysed. In both 60Co and 252Cf experiments, incidences of spermatozoa with radiation-induced structural chromosome aberrations increased linearly with increase of dosage. The RBE of 252Cf neutrons for the induction of chromosomally abnormal spermatozoa was estimated to be 1.6. The number of induced structural chromosome aberrations per spermatozoon also increased linearly. The RBE of neutrons for this index was 2.0. Among structural chromosome aberrations observed, chromosome-type breaks were predominant in both 60Co and 252Cf experiments, and they showed a significant linear dose-dependent increase. Other types of aberrations such as chromosome-type exchanges and chromatid-type breaks also increased linearly with increase in dose. The RBEs of 252Cf neutrons for the induction of these three types of aberrations were 1.6, 3.2 and 3.9, respectively. Thus, the RBEs of neutrons for the induction of chromosome aberrations were smaller in human spermatozoa than in human lymphocytes, and mouse spermatogonia and embryos. This result is discussed from the point of view of DNA-repairing capacity of oocytes.

Plasma eotaxin level and severity of asthma treated with corticosteroid.

Our understanding of asthma severity was advanced by the identification of biomarkers which account for differences in lung function impairment. We tried to examine the effects of corticosteroid treatment on known correlates of asthma severity including peripheral eosinophil counts, total IgE, IL-5, and eotaxin Levels in plasma. We compared these biomarkers among groups of stable asthmatics categorized by the dose of corticosteroid (N: steroid-free, n = 25; L: low-dose inhaled, n = 27; MH: medium or high-dose inhaled, n= 19; O: inhaled plus oral, n= 8). Next we compared these markers and peak expiratory flow rate (PEFR) in unstable asthmatics before and after treatment with steroids (n = 22). Eotaxin levels in the O group were higher than those in the N and MH groups (P < 0.05). Logistic regression analysis demonstrated that plasma eotaxin level was correlated with the severity of asthma defined by treatment intensity (P = 0.01) and % FEV1 (P = 0.04) while the other markers were not. Eotaxin levels did not change after steroid treatment in unstable patients, whereas eosinophil counts decreased in parallel with PEFR. Among biomarkers of asthma severity studied, plasma eotaxin level was not significantly affected by corticosteroid treatment, and was associated with the severity even in the presence of steroid therapy.

Meiotic stage-dependent induction of chromosome aberrations in Chinese hamster primary oocytes exposed to topoisomerase II inhibitor etoposide.

To investigate the chromosomal effects of topoisomerase II (topo-II)-interactive drugs on mammalian primary oocytes, female Chinese hamsters were treated with etoposide (VP-16) at various intervals pre- and post-human chorionic gonadotropin (hCG) injections. Chromosome analysis of oocytes at metaphase II (M II) showed that treatment with VP-16 at 50h pre-hCG had no effect, but the treatments between 24h pre-hCG and 2h post-hCG often caused structural chromosome aberrations. Although treatment at 4h post-hCG had no effect, subsequent treatments at 6 and 8h post-hCG produced a significant increase in structural chromosome aberrations. No effect was found following treatment at 10h post-hCG. The incidence of aneuploidy following exposure to VP-16 was also dependent on the time of hCG injection. Taking the time course of meiotic progression in primary oocytes following hCG injection and pharmacokinetics of VP-16 into consideration, it is likely that meiotic stages from late dictyate to diakinesis are highly sensitive to VP-16, while stages at dictyate and from metaphase I (M I) to telophase I (telo I) are relatively insensitive to the drug. Moreover, the effect of VP-16 on structural chromosome aberrations and aneuploidy was dose-dependent. Chromosome analysis at M I detected a frequent occurrence of structural chromosome aberrations in treated oocytes. This suggests that structural aberrations may be caused by disruption of cleavable complexes during chromosome condensation. Detection of chromosome bridges during anaphase I/telophase I (ana I/telo I) may support the hypothesis that induction of aneuploidy by VP-16 is due to failure in decatenation of recombinant homologous chromosomes.

Micronucleus test in 2-cell embryos as a simple assay system for human sperm chromosome aberrations.

A micronucleus test method to assess radiation-induced chromosomal damage in human spermatozoa is described, and its efficiency examined by comparison with that of sperm chromosome analysis. Human spermatozoa were exposed in vitro to 1.11 and 2.13 Gy of 137Cs gamma-rays at a dose rate of 1.36 Gy/min. After interspecific in vitro fertilization of irradiated spermatozoa with zona-free hamster oocytes, a total of 193 monospermic eggs were examined with the micronucleus test at the 2-cell stage, and a total of 304 male pronuclear chromosome plates were analyzed according to our established method. The incidence of 2-cell embryos with micronuclei coincided well with the incidence of spermatozoa with chromosomal breaks and fragments (51.6% vs. 50.3% in the 1.11-Gy group and 82.7% vs. 79.3% in the 2.13-Gy group). A similar correlation was also found between the number of micronuclei per embryo and the number of breaks and fragments per spermatozoon (0.85 vs. 0.88 and 1.50 vs. 1.45 in the 2 dose groups, respectively). These results indicate that our micronucleus test is useful as a simple and rapid method for assessing the clastogenic effects of various environmental mutagens on human sperm chromosomes.

Dose-response relationship for the induction of structural chromosome aberrations in human spermatozoa after in vitro exposure to tritium beta-rays.

The effects of tritium (HTO) beta-rays on human sperm chromosomes were studied using our interspecific in vitro fertilization system between human spermatozoa and zona-free hamster oocytes. Semen samples were treated with media containing 1.53-24.3 mCi/ml HTO for about 80 min. 1290 spermatozoa from the controls and 1842 spermatozoa from the irradiated groups were karyotyped. The incidence of spermatozoa with structural chromosome aberrations increased linearly with increasing dosage. Breakage-type aberrations occurred far more frequently than exchange-type. Chromosome-type aberrations appeared far more frequently than chromatid-type. All of these types of aberrations showed linear dose-dependent increases. The RBE values of HTO beta-rays relative to X-rays were calculated for the above-mentioned 5 indices, respectively. Their RBE values ranged from 1.89 to 3.00 when the absorbed dose was estimated to be the minimum, whereas the values ranged between 1.04 and 1.65 when the absorbed dose was estimated to be the maximum.

Types of structural chromosome aberrations and their incidences in human spermatozoa X-irradiated in vitro.

We studied the effects of in vitro X-irradiation on human sperm chromosomes, using our interspecific in vitro fertilization system between human spermatozoa and zona-free hamster oocytes. 28 semen samples from 5 healthy men were exposed to 0.23, 0.45, 0.91 and 1.82 Gy of X-rays. Totals of 2098 and 2862 spermatozoa were karyotyped in the control and the irradiated groups, respectively. The incidence of spermatozoa with X-ray-induced structural chromosome aberrations (Y) increased linearly with increasing dosage (D), being best expressed by the equation, Y = 0.08 + 34.52 D. The incidence of breakage-type aberrations was more than 9 times higher than that of exchange-type aberrations. Both of them showed linear dose-dependent increases, which were expressed by the regression lines, Y = -0.014 + 0.478 D and Y = -0.010 + 0.057 D, respectively. The incidence of chromosome-type aberrations was about 6 times higher than that of chromatid-type aberrations. Their dose-dependent increases were expressed by the regression lines, Y = -0.015 + 0.462 D and Y = -0.006 + 0.079 D, respectively. These results are discussed in relation to the previous data obtained with gamma-rays. The repair mechanism of X-ray-induced sperm DNA lesions is also discussed.

Chromosome analysis of human spermatozoa exposed to antineoplastic agents in vitro.

We studied in vitro the cytogenetic effects of six antineoplastic agents, bleomycin (BM), cyclophosphamide (CP), daunomycin (DM), methyl methanesulfonate (MMS), mitomycin C (MMC) and triethylenemelamine (TEM) on spermatozoa, using an interspecific in vitro fertilization system between zona-free hamster oocytes and human or bull spermatozoa. In preliminary experiments with bull spermatozoa, clastogenic effects were clearly shown with BM, DM, MMS and TEM, but not with CP and MMC. In main experiments, the effects of the first four chemicals were studied in detail with human spermatozoa. Total numbers of 585 and 512 spermatozoa were karyotyped in the control and the chemical-treated groups respectively. The incidence of spermatozoa with structural chromosome aberrations was 34.5%, 53.0%, 59.3%, and 55.6% in the BM (50 micrograms/ml, 90 min), DM (0.1 microgram/ml, 90 min), MMS (100 micrograms/ml, 120 min) and TEM (0.1 micrograms/ml, 120 min) groups respectively, each showing a significantly higher incidence than the matched controls (10.1-13.5%). Breakage-type aberrations were more frequent than exchange-type aberrations in the BM, MMS and TEM groups, while the exchange-type aberrations were more frequent in the DM group. Exchanges were mainly of the chromatid type in the DM, MMS and TEM groups, while chromosome-type exchanges occurred more frequently in the BM group. These results are discussed in relation to previous data on chemical-induced chromosome aberrations in mammalian somatic cells and in mouse spermatozoa.

Difference in types of radiation-induced structural chromosome aberrations and their incidences between Chinese and Syrian hamster spermatozoa.

The effects of ionizing radiations on sperm chromosomes were studied in the Chinese hamster (Crisetulus griseus) and the Syrian (golden) hamster (Mesocrisetus auratus). Testes of mature male Chinese hamsters (CH) were irradiated with X-rays (0.91, 1.82 and 3.63 Gy) and gamma-rays (1.10, 2.15, 2.95 and 4.01 Gy) at a single acute dosage, whereas the irradiation was done with lower doses of X-rays (0.45, 0.91 and 1.82 Gy) and gamma-rays (0.49, 0.99 and 1.98 Gy) in mature male Syrian hamsters (SH), taking the higher radiosensitivity of this species into consideration. They were mated with normal females within 6 days of exposure. Sperm-derived chromosomes were analyzed in 1125 and 1966 fertilized ova of the CH and the SH, respectively. In both species, there was no great difference in the induction of structural chromosome aberrations between X-irradiated and gamma-irradiated spermatozoa. Chromosome-type aberrations were predominantly induced. The incidence of breakage-type aberrations increased linearly, and that of exchange-type aberrations linear-quadratically with increase of dosage. A species-specific difference in chromosomal radiosensitivity of spermatozoa was clear. In spite of the same radiation dosage, the incidence of chromosomally abnormal spermatozoa in the SH was about twice as high as that in the CH (e.g. 27.0% vs. 14.7% at 0.91 Gy of X-rays). The incidences of breakage-type aberrations (69-89%) were far higher than those of exchange-type aberrations (11-31%) in the SH, while the disparity of the two incidences was much smaller in the CH (46-65% vs. 35-54%). Exchange-type aberrations consisted of both chromosome-type and chromatid-type in the SH, while almost all of them were of the chromosome-type in the CH. These results suggest that the DNA-repairing capacity of oocytes is much higher in the CH than in the SH. Moreover, it seems likely that radiation-induced sperm DNA damage is repaired with both pre-replication repair (excision repair) and post-replication repair systems in SH oocytes, whereas the excision repair system operate most exclusively in CH oocytes.