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PubMed1995

DNA measurements and ploidy determination of developmental stages in the life cycles of Theileria annulata and T. parva.

The relative DNA levels of different developmental stages of Theileria annulata and T. parva in the cow and the tick were measured by the cytophotometric DNA technique using the fluorochrome Hoechst 33258 as a staining dye. The results revealed that sporozoites, merozoites, gamonts, and gametes were haploid, whereas multinucleated intralymphocytic schizonts were polyploid. No difference was observed between T. parva and T. annulata in these stages. For both Theileria species, the DNA measurements revealed that fusion of gametes occurred in the gut of the final host, thus providing evidence of sexual reproduction. However, differences were observed between the two parasites in the tick. Whereas T. parva zygotes underwent a two-step meiotic division, a comparable reduction division could not be unequivocally detected in T. annulata. Differences could also be detected in the further development of kinetes, indicating that Theileria species are not characterized by only one life cycle, which is specific for this genus.

Animals↗

PubMed1976

Originalarbeiten.

In 5 cases of polycythaemia vera and 2 cases with other myeloproliferative disorders accompanied by thrombocythaemia (megakaryocytic myelosis), the megakaryocytes were differentiated and studied by use of the combined application of cytophotometric determination of the DNA content and autoradiography with tritiated thymidine (3H-TdR) in vitro. A shift to the right of the megakaryocyte series, occurence of high polyploidy cells at 64c and a decrease of the 3H-TdR-labeling indices were observed. The data suggest a disturbance of the rhythmical polyploidization of the megakaryocytes, consisting of an elevated proportion of rest cells at the different ploidy stages. The maturation capacity of megakaryocytes may be related more to the resting than to the DNA synthesizing cells.

Adult↗

PubMed1979

A quantitative cytochemical study of acid phosphatases in rat liver parenchymal cells of different ploidy values.

The Vickers M86 integrating microdensitometer has been used to quantify the cytochemical reaction for acid naphthol AS-BI phosphatase activity in isolated, rat liver parenchymal cells. Data are presented which validate the method. The use of this method together with that of the Feulgen reaction to estimate nuclear ploidy value in the same cell, has shown that there is an increase in acid phosphatase activity of up to 100% when the euploidy value of the liver cell doubles. It has been further shown that 70--80% of this enzyme activity is ouabain-sensitive, regardless of the euploidy value. The data may be interpreted to indicate that the extra gene copies of the polyploid cells are operative.

Acid Phosphatase↗

PubMed1989

Super high estradiol response to gonadotropin stimulation in patients undergoing in vitro fertilization.

The likelihood of establishment of a term pregnancy from in vitro fertilization (IVF) is related to the estradiol (E2) pattern and peak level. To examine the influence of super high E2 peak levels (greater than or equal to 2000 pg/ml), we reviewed the clinical outcomes of all IVF cycles with follicular phase E2 levels greater than 2000 pg/ml from May 1982 through June 1987. Among 1651 IVF cycles initiated during this time, 102 cycles (6.2%) had super high E2 levels. Twenty-seven of these cycles occurred in 34 IVF attempts in 12 women. Stimulation was performed with human menopausal gonadotropin (hMG) in 96 cycles and follicle-stimulating hormone (FSH) in 6 cycles. A mean of 9.3 +/- 0.7 oocytes per cycle was recovered, of which 5.5 +/- 0.5 fertilized and underwent cleavage. In 11 cycles, with a mean of 6.8 oocytes recovered, none fertilized. Polyploid fertilization occurred in 23 of 90 cycles (25.6%), and 40 of 558 fertilized oocytes (7.2%). From these cycles, 10 clinical pregnancies (9.8%) have resulted: 6 pregnancies in 59 cycles with luteal-phase progesterone support (10.2%) and 4 pregnancies in 31 cycles without luteal-phase progesterone support (12.9%). Among the 1549 cycles with peak E2 levels less than or equal to 2000 pg/ml, 143 (9.2%) resulted in clinical pregnancies. We conclude that there is a small subset of patients who will have super high E2 responses to gonadotropin stimulation and that there is a tendency to stimulate repetitively in this fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

Chorionic Gonadotropin↗

PubMed1984

In vitro fertilization and embryo replacement at the Department of Obstetrics and Gynecology, University of Kiel, F.R.G.

From July 1982 until September 1983, 194 pelviscopies were performed in Kiel (phase I). The patients were stimulated either with Clomid/human menopausal gonadotropin hMG)/human chorionic gonadotropin (hCG) or with Clomid/hCG or hMG/hCG alone. Follicular maturation was monitored by ultrasonography and the daily measured E2 and luteinizing hormone (LH) response. Surgic therapeutical pelviscopy with follicular puncture followed 36 hr after hCG application. Oocytes were incubated in either Ham's F-10 or Menezo B2 medium in an automatically gas-controlled exsiccator. Forty-eight hours after insemination normal-looking four- to eight-cell embryos were replaced into the uterine cavity. Oocytes were successfully collected in 87.4% of the patients, with an average of 2.2 oocytes per patient. Eighteen pregnancies resulted of 101 embryo replacements. The overall pregnancy rate was 16.2% per replacement and 9.3% per pelviscopy. Undivided oocytes and polyploid embryos were analyzed cytogenetically. From October 1983 to October 1984 the overall pregnancy rate after 144 pelviscopies and 88 embryo replacements (phase II) improved to 23.9% per replacement and 14.6% per pelviscopy.

Blastocyst↗

PubMed1991

Treatment of severe male-factor infertility with high concentrations of motile sperm by microinsemination in embryo cryopreservation straws.

A microinsemination technique was evaluated for treating our program's most severe cases of male-factor infertility. Oocytes were inseminated with high concentrations of motile sperm (1 to 9 x 10(6)/ml) in 10 to 150 microliters within embryo cryopreservation straws. Fertilization was obtained in 20 of 29 (69%) couples treated by this technique. In the 15 patients in which only embryos generated from the straw technique were transferred, 7 clinical pregnancies resulted (46.7% per transfer). The implantation rate for couples receiving embryos from the straw technique only (12/58; 20.7%) compared favorably to that observed for other cases treated during this same time period with regular insemination techniques (111/766; 14.5%). Clinical pregnancy rates per transfer for IVF-ET, TET, and PROST were 33.0% (1/3), 0% (0/2), and 60.0% (6/10), respectively. The percentage of polyploidic embryos was significantly lower (P less than 0.0001) for male-factor patients treated by the straw technique with high sperm concentrations than for non-male-factor patients treated during this same time period with standard sperm concentrations. Normal births have resulted from straw inseminations with 3.4 x 10(6) and ongoing pregnancies with 5.0 x 10(6) motile sperm/ml. The results of this study suggest that some cases of male-factor infertility can be successfully treated by insemination with high concentrations of motile sperm in embryo cryopreservation straws. A technique of centrifuging sperm in straws was also developed to concentrate the entire fraction of washed sperm into 10 microliters. Further development of this technique may allow treatment of more severe cases of oligo/asthenospermia by microinsemination with high concentrations of motile sperm than is presently possible with standard washing techniques.

Adult↗

PubMed1986

The use of synthetic culture medium and patient serum for human in vitro fertilization and embryo replacement.

The use of heat-inactivated patient serum as both fertilization and embryo replacement medium was compared in a prospective randomized study with a fully synthetic culture medium containing human serum albumin without serum addition (B3 INRA Menezo). Another series of the author's IVF program was analyzed retrospectively when a commercially available synthetic medium with bovine serum albumin (B2 INRA Menezo) or B3, as mentioned above, was used without serum addition for fertilization but with 50-100% patient serum as embryo replacement medium. Fertilization rates were significantly higher in the synthetic culture media (70%) than in serum (57%). The rate of polyploid fertilization was significantly lowest in B3 medium. There was a clear trend toward better pregnancy rates when high-percentage or 100% patient serum was used for embryo replacement, no matter if one, two, three, four or more embryos were replaced. We conclude that there should be no need for any kind of serum addition to fertilization media. The present study proves our previous observation that the use of serum seems to be beneficial as embryo replacement medium. This might well be explained by a "protein stick effect" due to the high macromolecular contents of serum rather than by viscosity measurements, since no significant increase in viscosity was observed when a high percentage of serum was added to culture media.

Blood↗

PubMed1979

Chromomeres and puffing in experimentally induced polytene chromosomes of Calliphora erythrocephala.

In the most advanced types of meroistic ovaries the synthesis of RNA for the growing oocyte in each follicle is taken over by nurse cells, i.e., sister cells of the definite egg cell. In calliphora, the highly polyploid nurse cells (NC) develop a polytene karyotype under conditions of strict brother-sister inbreeding and using a controlled selection technique. A comparison of the polytene NC-chromosomes with those from the pupal bristle forming cells reveals an unexpected discrepancy: while both chromosome complements exhibit a constant banding pattern it is not possible to homologize the two tissue specific patterns by identifying homologous band-sequences. Puffing in NC likewise turns out to be unusual in its extent as well as in that is remains constant during long periods of oogenesis. In a more detailed discussion an interpretation and evaluation of these findings will be attempted.

Animals↗

PubMed1975

Simian virus 40-chinese hamster kidney cell interaction. I. Relationship of chromosome changes to transformation.

After approximately 20 in vitro passages, Chinese hamster kidney (CHK) cell cultures transformed upon exposure to different strains of SV 40 can show a diploid modal chromosome number of 22 with chromosome counts exclusively or essentially in the diploid range (20-25). In primary culture and at the 5th-7th subculture, tumors produced in nude mice by such cells can display a diploid modal value of 22 chromosomes. Numerical chromosome variations and karyotype abnormalities observed in CHK cells transformed upon infection with SV 40, are apparently indistinguishable from those that can be observed in uninfected CHK cells undergoing "spontaneous"-transformation. These results indicate that polyploidization is not a necessary step either in the process of transformation or in that of tumorigenic conversion with SV 40.

Animals↗

PubMed1976

Microcalorimetric investigation of the metabolism of yeasts. V. Influence of ploidy on growth and metabolism.

Microcalorimetric experiments on growth and maintenance metabolism of Saccharomyces strains ranging from haploid to hexaploid are described. During growth, the mean dry weight, the mean volume and the maximum heat flux of the cells are nearly linear functions of ploidy. These parameters are correlated with the cell concentration in such a manner that the weight-specific heat production and the grown biomass are independent of ploidy. For the metabolism of maintenance, two levels of the specific heat flux are found, the lower of which is occupied by the haploids, diploids and triploids. The higher polyploids exhibit the higher level.

Energy Metabolism↗

PubMed1976

New approach to brain tumour chemoradiotherapy with cellular synchronization by colcemid.

Cellular synchronization using Colcemid as pretreatment for combined chemoradiotherapy was investigated. C6 rat brain tumour was cultured in RPMI medium containing 10(-5)-10(-7) Mol. of Colcemid for 24 hours. The basic cell kinetics were analysed with a Pulse Cytophotometer, which facilitated the analysis of tumour cell cycle phase distribution according to the DNA content. The effect of Colcemid depended on the concentration, and the minimal concentration showing continuous blocking during 48 hours after removal of the drug was 10(-6) Mol. G1 fraction of 2 C DNA content was reduced from 74% to 36%. G2-M phase of 4 C DNA content increased from 9% to 28%. S phase cells increased from 17% to 31%. Polyploid cells in the Tetraploid cell cycle could be recognized. The remaining 36% of cells within the GO + G1 peak of 2 C DNA content were considered to be diploid GO cells.

Brain Neoplasms↗

PubMed1978

Identification of young megakaryocytes by immunofluorescence and cytophotometry.

The DNA-content of fluoresceine-labeled platelet antigen containing cells of mouse bone marrow was measured. For immunofluorescence highly specific anti-mouse-platelet-serum and fluoresceine-conjugated antigammaglobuline was used, applying the "sandwich" technique. Three hundred panoptically identifable megakaryocytes served as control group. The DNA-polyploidization pattern of megakaryocytes and immunofluorescence positive cells was almost identical. However, among the immunofluorescence positive cells a considerable amount of cells showed DNA-values lower than 4c, whereas the megakaryocytes of the Pappenheim stained smears revealed no DNA-values lower than 4c. The percentages of diploid and tetraploid cells, respectively, was 6 and 7% compared with 0 and 1% of panoptically identifiable megakaryoctyes. The results suggest that young megakaryocytic cells with diploid and tetraploid DNA-values can be detected by immunofluorescence technique, indicating that the flow from the uncommited to the committed megakaryocytic precursor cell appears at this early stage of megakaryocyte production.

Age Factors↗

PubMed1976

A CHO-cell mutant with a defect in cytokinesis.

In a selection procedure designed to enrich for temperature-sensitive mutant cells blocked in mitosis a CHO-cell mutant was isolated which has a defect in cytokinesis as the basis of its temperature-sensitive phenotype. Cultures of the mutant had an abnormally high percentage (ie. 34%) of polyploid cells at the permissive temperature of 34 degress C and showed further increased frequencies of polyploidy as well as many multinucleated cells at 38.5 degrees 39.5 degrees. When the mutant cells were synchronized in metaphase by Colcemid arrest and then placed into fresh medium at nonpermissive temperature, they did not divide although the completion of mitosis appeared cytologically normal. Ultrastructural examination by electron microscopy of such synchronized cells at telophase revealed no specific defects in cellular components other than failure of development of a normal midbody. The sensitivity of the mutant to cytochalasin B and to Colcemid was the same as for wild-type cells. This mutation behaved as recessive in tetraploid cell hybrids constructed by fusing the mutant with a CHO strain which was wild-type with respect to temperature sensitivty.

Animals↗

PubMed1994

Genetic modification of an echinocandin B-producing strain of Aspergillus nidulans to produce mutants blocked in sterigmatocystin biosynthesis.

The production of echinocandin B (ECB), a lipopolypeptide used for chemical manufacture of the anti-Candida agent Cilofungin, was accomplished by fermentation using a strain of Aspergillus nidulans. In addition to ECB, this fermentation also produces a significant amount of sterigmatocystin (ST), a potent carcinogen structurally related to the aflatoxins. Mutants blocked in the ST biosynthetic pathway were created by genetic modification of the polyploid production strain C747. The following steps were involved: (i) reduction of the genotype to haploid by treatment with the spindle fiber poison methyl 1-(butylcarbamoyl)-2-benzimidazole carbamate (MBC), using colony morphology, conidia size, and the ability to obtain 5-fluoro-orotic acid (5-FOA)-resistant mutants as criteria for ploidy; (ii) mutagenesis of a haploid isolate using UV irradiation; and (iii) screening of mutants for inability to produce ST by thin layer chromatography. Six mutants blocked in ST production were isolated. All six remained capable of producing ECB equivalent in quantity to the haploid strain C747-GR14. One of the mutants was shown to be the result of a chromosomal translocation.

Anti-Bacterial Agents↗

PubMed1978

[Transsphincteric approach to the rectum in cases of low early cancer (author's transl)].

Based on 4 years of experience with the transsphincteric approach to the anorectum in 24 cases of low-situated cancers of the rectum, an attempt is made to demonstrate which tumors are treatable by this method and which are not. Exophytic or polyploid tumors with a diameter of not more than 3-4 cm, that are easily palpated and appear freely mobile, and have histologically a differentiated pattern may be approached by transsphincteric resection, under the condition that the tumor has not spread across the wall of the rectum or into adjacent lymph nodes. The method is not recommended in any other instances becauses of the high risk of recurrencies.

Anal Canal↗

PubMed1987

Dry mass, DNA and non-histone protein determinations in lung cancer cells.

Cell nuclei from two biopsies of bronchial mucosa, seven squamous-cell carcinomas and six small-cell carcinomas of the lung were investigated. DNA and non-histone protein (NHP) content were simultaneously determined in Feulgen-Naphthol Yellow S-stained smears by means of multiple plug cytophotometry. In addition, the nuclear dry mass of cells originating from the same populations was measured by interference microscopy. DNA histograms of carcinomas were characterized by DNA stemlines being situated in the diploid range in four out of six small-cell carcinomas and in the hyperdiploid to hypertetraploid range in six out of seven squamous-cell carcinomas. Polyploid values (up to 8 c) were seldom found in small-cell carcinomas whereas squamous-cell carcinomas showed a broader dispersion approaching the 16 c level. The similarity of the NHP distributions with the DNA histograms was more marked in small-cell carcinomas than in the squamous-cell carcinomas. In comparison with the NHP distributions of normal bronchial epithelial cells, those of carcinomas were shifted to higher values. The increased NHP content was found to be a more prominent sign of malignancy in small-cell carcinomas than the DNA mass. The increase in nuclear dry mass in carcinomas was mainly caused by the accumulation of NHP in tumour cell nuclei.

Bronchi↗

PubMed1988

Flow cytometric analysis of cellular DNA content in human astrocytomas and oligodendrogliomas.

This report presents a flow-cytometric analysis of cellular DNA in biopsies and primary cell cultures of 21 human astrocytomas and 19 oligodendrogliomas. A distinct correlation between histological dedifferentiation and pathological DNA distribution was found. Classification was made according to increasing histological anaplasia, corresponding to a four-grade scale and proliferation index (PI). Four types of gliomas were defined according to characteristic DNA patterns and proliferative activities in comparison to their histological grading: 1. purely diploid DNA patterns with low 4C (premitotic) peaks and PI values up to 10 in well-differentiated gliomas; 2. increase of tetraploid cells and PI of 10-16 in tumors with histological grades II or II-III; 3. diploid-tetraploid DNA distribution with PI values up to 30-31 and malignancy grade III; 4. polyploid and aneuploid karyograms with excessive 4C increase, emerging in grade III and especially grade III-IV of these gliomas. Varying DNA distribution during tumor development could be observed in a malignant transformation of an oligodendroglioma I to a glioblastoma after a course of 3 1/2 years. A more detailed subdivision of these tumors according to their DNA content and proliferative activity was achieved. With the exception of occasional variation in karyograms, DNA distribution usually remained stable in primary tissue cultures (PTC).

Adolescent↗

PubMed1976

Chloral hydrate induced haploidization in Aspergillus nidulans.

This is the first report of induction of haploidization in Aspergillus nidulans by chloral hydrate, which is an efficient polyploidizing agent for higher plants and a psychotropic drug for man. A new procedure has been described to isolate haploids from diploids with a very high frequency, as compared to p-fluorophenylalanine, which is generally used for this purpose.

Aspergillus nidulans↗