The relation of radiation sensitivity to pronuclear chromosome structure

After treatment of spermatozoa of Phryne cincta from a Berlin population (bs) with low crossing-over and from an Alpine population (as) with high crossing-over frequency, egg mortality was ascertained and chromosomal mutation rate determined by examination of the polytene chromosomes of the F₁ progeny. — The as stock exhibits a 1.33 times greater radiation sensitivity than the bs stock, both in chromosomal mutation and in egg mortality rates. There is no evidence that factors other than structural ones account for the differences in radiation sensitivity. From inversion length measurements, it was proposed that a reduction of the spiral diameter of the paternal pronucleus chromosomes is responsible for a higher site number and thereby for a greater radiation sensitivity.Dedicated to Prof. Dr. B.E. Wolf, initiator of this work, in honour of his 70th birthday on September 27, 1978     

X-ray induced visible alterations in the giant chromosomes of Phryne cincta (Nematocera,Diptera)

In order to induce chromosomal rearrangements, males were exposed to x-rays and then mated to non-irradiated females. The number of each type of structural alteration was determined by examination of the polytene chromosomes of the F1 progeny. -- A comparison of the results with similar studies made on Drosophila revealed a significantly greater sensitivity in Phryne. Parallel to that an extremely high frequency of small inversions was ascertained in Phryne, and the observed ratio of inversions to translocations was the inverse of that which would be expected from purely mathematical considerations based on the lengths of the different chromosomes. These facts allow the conclusion that the paternal pronuclear chromosomes in Phryne are highly spiralized. Besides, the kinetochore-to-translocation-breakpoint distance was measured in both of the chromosomes involved in each reciprocal translocation and the differences (kinetochore-break distance differences) were registered and from them the arrangement of the chromosomes in the pronucleus of Phryne deduced. The data obtained support the assumption of an ordered, polar-field type of orientation. In Drosophila, in contrast, the comparable data showed that the pronuclear chromosomes are not spiralized and are randomly arranged (Bauer, 1939). -- These results seem to indicate that a close correlation exists between the different radiation sensitivities of Drosophila and Phryne and the different states of spiralisation and arrangements of their chromosomes in the pronucleus stage. It is hypothesized that the influence of the maternal genome on the degree of spiralization of the paternal chromosomes could account for differences in the pronuclear chromosome structure of both species.     

Analysis of structural and numerical chromosome abnormalities in sperm of normal men and carriers of constitutional chromosome aberrations. A review

Sperm chromosome analysis offers the opportunity to gather information about the origin of chromosome aberrations in human germ cells. Over the last 20 years more than 20 000 sperm chromosome complements from normal donors and almost 6000 spermatozoa from men with constitutional chromosome aberrations (inversions, translocations) have been analyzed for structural and numerical chromosome abnormalities, as well as for segregation of the constitutional chromosome aberrations after the sperm had penetrated hamster oocytes. On the other hand, it took only 6 years to screen more than 3 million mature spermatozoa from healthy probands for disomy rates of 20 autosomes (chromosomes 19 and 22 not evaluated) and the sex chromosomes, and for diploidy rates by in situ hybridization techniques. In the present paper the results arising from both methods are compiled and compared. Received: 29 January 1997 / Accepted: 5 March 1997     

Frequency and distribution of aneuploidy in human gametes: differences as a function of sex]

The frequency and the distribution of aneuploidies were analysed in both spermatozoa and mature oocyte. The present study has pooled 13,975 human sperm chromosome complements and 1,897 oocyte chromosome complements examined to date. The overall frequency of aneuploidy is 10% in spermatozoa and 22.4% in oocytes. Human sperm is characterized by a significant excess of hypo-haploidies and an equitable distribution of aneuploidies among all chromosome groups, whereas mature oocytes display an equal ratio of hypo-haploidies: hyper-haploidies and a high variability in the distribution of non-disjunctions; in the A, B, C and especially in D and G groups, there is a significant difference between the observed and estimated rates of non-disjunction and the frequencies expected from an equal partitioning of non-disjunctions among all chromosomes. This indicates that non-disjunction is not a random event in female meiosis, and consequently that there are differences in the meiotic process between the sexes.     

Increased incidence of hyperhaploid 24,XY spermatozoa detected by three-colour FISH in a 46,XY/47,XXY male

Meiotic segregation of gonosomes from a 46,XY/47,XXY male was analysed by a three-colour fluorescence in situ hybridisation (FISH) procedure. This method allows the identification of hyperhaploid spermatozoa (with 24 chromosomes), diploid spermatozoa (with 46 chromosomes) and their meiotic origin (meiosis I or 11). Alpha satellite DNA probes specific for chromosomes X, Y and 1 were observed on 27,097 sperm nuclei. The proportions of X-and Y -bearing sperm were estimated to 52.78% and 43.88%, respectively. Disomy (24,XX, 24,YY, 24,X or Y,+1) and diploidy (46,XX, 46,YY, 46,XY) frequencies were close to those obtained from control sperm, whereas the frequency of hyperhaploid 24,XY spermatozoa (2.09%) was significantly increased compared with controls (0.36%). These results support the hypothesis that a few 47,XXY germ cells would be able to complete meiosis and to produce mature spermatozoa.     

Mixotrophy in the newly described phototrophic dinoflagellate Woloszynskia cincta from western Korean waters: feeding mechanism, prey species and effect of prey concentration

Woloszynskia species are dinoflagellates in the order Suessiales inhabiting marine or freshwater environments; their ecophysiology has not been well investigated, in particular, their trophic modes have yet to be elucidated. Previous studies have reported that all Woloszynskia species are photosynthetic, although their mixotrophic abilities have not been explored. We isolated a dinoflagellate from coastal waters in western Korea and established clonal cultures of this dinoflagellate. On the basis of morphology and analyses of the small/large subunit rRNA gene (GenBank accession number=FR690459), we identified this dinoflagellate as Woloszynskia cincta. We further established that this dinoflagellate is a mixotrophic species. We found that W. cincta fed on algal prey using a peduncle. Among the diverse prey provided, W. cincta ingested those algal species that had equivalent spherical diameters (ESDs) ≤12.6 μm, exceptions being the diatom Skeletonema costatum and the dinoflagellate Prorocentrum minimum. However, W. cincta did not feed on larger algal species that had ESDs≥15 μm. The specific growth rates for W. cincta increased continuously with increasing mean prey concentration before saturating at a concentration of ca. 134 ng C/ml (1,340 cells/ml) when Heterosigma akashiwo was used as food. The maximum specific growth rate (i.e. mixotrophic growth) of W. cincta feeding on H. akashiwo was 0.499 d(-1) at 20 °C under illumination of 20 μE/m(2) /s on a 14:10 h light-dark cycle, whereas its growth rate (i.e. phototrophic growth) under the same light conditions without added prey was 0.040 d(-1). The maximum ingestion and clearance rates of W. cincta feeding on H. akashiwo were 0.49 ng C/grazer/d (4.9 cells/grazer/d) and 1.9 μl/grazer/h, respectively. The calculated grazing coefficients for W. cincta on co-occurring H. akashiwo were up to 1.1 d(-1). The results of the present study suggest that grazing by W. cincta can have a potentially considerable impact on prey algal populations.     

Prae- and postreplication repair in pronucleus chromosomes of Phryne after sperm irradiation

On the basis of differences in crossing-over frequency and in radiation sensitivity, two stocks of Phryne cincta can be distinguished. The higher sensitivity of the Alpine stock (as), compared with the Berlin stock (bs), seemed to be due to its higher site number in tightly spiralized pronucleus chromosomes (Israelewski, 1979). A mathematical model was tested to arrive at an estimate of the maximal site number in Phryne. Deducing from this model, repair in a 2-break site would result with the probability of 1/6 in restitution, 1/6 in balanced chromosome aberration and 4/6 in unbalanced chromosome aberration. The maximal site number available is 13 in bs and 20 in as. It is suggested that a high proportion of 2-break sites are repaired after replication of the paternal pronucleus chromosomes. The proportion of prae- and postreplication repair is estimated to be 50% each, possibly in agreement with the distribution of nucleosomal and internucleosomal DNA in eukaryotic chromatin.     

Sperm nuclei analysis of a Robertsonian t(14q21q) carrier,by FISH,using three plasmids and two YAC probes

The meiotic segregation of chromosomes 14 and 21 was analysed in 1116 spermatozoa from an oligoasthenospermic carrier of a Robsertsonian translocation t(14q21q), and in 16 392 spermatozoa from a control donor, using two-colour fluorescence in situ hybridisation (FISH). Two YAC probes (cloned in yeast artificial chromosomes) specific for regions on the long arms of these chromosomes were co-hybridised. Of the spermatozoa, 12% were unbalanced, resulting from adjacent segregations. Chromosomes X, Y and 1 were also simultaneously detected in 1335 spermatozoa from the same carrier. Whereas gonosomal disomy rates were not significantly different from those of the control donors, disomy 1 were slightly but significantly increased to 0.7%. The diploidy rate was also slightly increased to approximately 1% in the translocation carrier.     

Chromosome aberrations in mouse embryos and fetuses produced by assisted reproductive technology

The occurrence of structural chromosome aberrations in mouse one-cell embryos produced by intracytoplasmic sperm injection (ICSI) with mature spermatozoa was dependent on the type of sperm incubation medium and sperm incubation time. When cauda epididymal spermatozoa were used following incubation in bicarbonate-buffered TYH medium for 0h (no incubation) and 0.5h, the chromosome aberration rates (6.9% and 7.4%, respectively) in the resultant embryos were significantly higher than that (2.3%) in the IVF embryos. However, when the spermatozoa were incubated for 2-2.5h and 6h in the same medium, the chromosome aberration rates were reduced to the IVF embryo level (3.8% and 4.3%, respectively). When spermatozoa incubated in Hepes-buffered H-mCZB and phosphate-buffered PB1 media were used for ICSI, chromosome aberration rates in embryos were significantly high (8.6-28.1%) and increased in a time-dependent manner. On the other hand, when immature testicular spermatozoa were incubated in those three media for 0.5h and 6h, the incidences of resultant embryos with structural chromosome aberrations ranged between 7.4% and 11.7%, and there was no medium- and time-dependent change in these aberration rates. To evaluate transmissible risk of chromosome aberrations to offspring, two- or four-cell embryos derived from cauda epididymal spermatozoa were transferred into the oviducts of pseudopregnant females and chromosomes of live fetuses were examined on gestational day 16. One (2.0%) mosaic fetus was found when spermatozoa were incubated in TYH for 2-2.5h, and there were four (6.7%) fetuses displaying a structurally abnormal karyotype when spermatozoa were incubated in H-mCZB for 2-2.5h, indicating that structural chromosome aberrations generated in ICSI one-cell embryos are transmissible to offspring. The causal mechanism of structural chromosome aberrations in ICSI one-cell embryos is discussed in relation to the acrosomal plasma membrane cholesterol and the acrosome.     

Rate of homologous chromosome bivalents in spermatocytes may predict completion of spermatogenesis in azoospermic men

Abstract.The rate of bivalent formation during meiosis was correlated with the presence and amount of spermatozoa in the testes of azoospermic men. Four pairs of chromosomes, X-Y, 9, 15, and 18, were evaluated. In addition, left and right testes were compared. Three biopsies from each testis were undertaken to extract spermatozoa for intracytoplasmic sperm injection. In addition, one sample from each testis was used for histological definition, spermatozoa count and detection of chromosome bivalents in spermatocytes. A significantly higher rate of bivalents of all homologous chromosomes was found whenever spermatozoa were detected. The rate of bivalent X-Y was found to be the most sensitive predictor for detection of spermatozoa, with a cut-off value of 47%. The R(2) was 27% ( P=0.003) for the percent of spermatozoa in the minced sample as well as the number of mature spermatids per tubule in the histological section. All pairs of testes were in concord in regard to the likelihood of finding spermatozoa. In the testes where no spermatozoa were found on biopsy, the rate of X-Y bivalent indicated the presence of spermatozoa in the opposite side. Thus, it may be concluded that the rate of X-Y bivalent formation in spermatocytes may predict the presence and amount of spermatozoa in the testicular tissue of azoospermic men. It is suggested that when no spermatozoa are located by testicular fine-needle aspiration, X-Y bivalent evaluation may be conducted if spermatocytes are evinced. A high rate of X-Y bivalents may impel one to continue with testicular open biopsies.     

Cytogenetic Analysis of Spermatozoa in a Patient with Chromosome Constitution 45,X/46,X,r(Y) by Intracytoplasmic Injection into Mouse Oocytes

The chromosome set of human spermatozoa was studied by intracytoplasmic injection into mouse oocytes. A total of 85 metaphase plates of male pronuclei of a patient with chromosome constitution 45,X/46,X,r(Y) and 108 metaphase plates of patients with normal sperm parameters (control group) were examined. The ratio between X- and Y-bearing chromosomes in the 45,X/46,X,r(Y) patient and in the control group did not differ from 1 : 1. A significant increase in the rates of diploidy, hypoploidy, hyperploidy of sex chromosomes, and chromosome structure rearrangements in spermatozoa of the patient in comparison with spermatozoa in the control group was recorded.     

Genetic and reproductive potential of spermatozoa of diploid and triploid males obtained from interspecific hybridization of Misgurnus anguillicaudatus female with M. mizolepis male

Diploid and triploid interspecific hybrid male progeny obtained from mating Misgurnus anguillicaudatus with M. mizoleis were reported to have histologically fertile and sterile testes, respectively. However, their reproductive capacity is still unclear because mating tests have not been examined using mature hybrids. Here, we examined physiological and genetic characteristics of spermatozoa of diploid and triploid hybrids. In diploid hybrid males, 1n, 2n and 4n spermatozoa showing low motility were detected. However, spermatozoa of three diploid hybrid males could generate 2n larvae. Therefore, only 1n spermatozoa of diploid hybrid males was fertile to produce larva. The chromosomes of diploid hybrid males were transmitted to spermatozoa by random segregation between the homologous chromosomes because most larvae had one allele derived from both M. anguillicaudatus and M. mizolepis at all loci examined. In triploid hybrid males, spermatozoa could be categorized to three different types based on their ploidy status. Type 1: In the first and second males, sperm samples mainly comprised 6n spermatozoa. Motility and fertility were not recorded. Type 2: The third male gave a large proportion of 6n spermatozoa as well as a small proportion of 1n spermatozoa. Although no motility was observed, larvae arose from eggs inseminated with such spermatozoa. Type 3: In the fourth male, only 1n spermatozoa were detected and their motility was vigorous. When eggs were fertilized with such 1n spermatozoa, normal larvae hatched. 1n spermatozoa of the triploid hybrid male only included the M. anguillicaudatus genome. In Misgurnus fishes, diploid hybrid males exhibited semi-sterility or slight fertility. On the contrary, triploid hybrid males were sometime fertile due to the production of 1n spermatozoa by a kind of transformation of meiosis like meiotic hybridogenesis.     

Assessment of aneuploidy for chromosomes 8, 9, 13, 16, and 21 in human sperm by using primed in situ labeling technique.

The incidence of aneuploidy was estimated for chromosomes 8, 9, 13, 16, and 21 in mature human spermatozoa by primed in situ (PRINS) labeling technique. This method allows us to perform a chromosome-specific detection by in situ annealing of a centromeric specific primer. A dual color PRINS protocol was adapted to human sperm. The decondensation and the denaturation of sperm nuclei were simultaneously performed by 3-M NaOH treatment. Double labeling of spermatozoa was obtained in <2 h. A total of 96,292 sperm nuclei were analyzed by two independent observers. The estimates of disomy were 0.31% for chromosome 8, 0.28% for chromosome 9, 0.28% for chromosome 13, 0.26% for chromosome 16, and 0.32% for chromosome 21. These homogeneous findings suggest an equal distribution of aneuploidies among autosomal chromosomes in males.     

Cytogenetic analysis of spermatozoa in a patient with chromosome constitution 45,X/46,X,r(Y) by intracytoplasmic injection into mouse oocytes

The chromosome set of human spermatozoa was studied by intracytoplasmic injection into mouse oocytes. A total of 85 metaphase plates of male pronuclei of a patient with chromosome constitution 46,X,r(Y)/45,X and 108 metaphase plates of patients with normal sperm parameters (control group) were examined. The ratio between X- and Y-bearing chromosomes in the 46,X,r(Y)/45,X patient and in the control group did not differ from 1:1. A significant increase in the rates of diploidy, hypoploidy, hyperploidy of sex chromosomes, and chromosome structure rearrangements in spermatozoa of the patient in comparison with spermatozoa in the control group was recorded.     

Meiotic behaviour of sex chromosomes investigated by three-colour FISH on 35 142 sperm nuclei from two 47,XYY males

Meiotic segregation of sex chromosomes from two fertile 47,XYY men was analysed by a three-colour fluorescence in situ hybridisation procedure. This method allows the identification of hyperhaploidies (spermatozoa with 24 chromosomes) and diploidies (spermatozoa with 46 chromosomes), and their meiotic origin (meiosis I or II). Alpha-satellite probes specific for chromosomes X, Y and 1 were observed simultaneously in 35 142 sperm nuclei. For both 47,XYY men (24 315 sperm nuclei analysed from one male and 10 827 from the other one) the sex ratio differs from the expected 1:1 ratio (P < 0.001). The rates of disomic Y, diploid YY and diploid XY spermatozoa were increased for both 47,XYY men compared with control sperm (142 050 sperm nuclei analysed from five control men), whereas the rates of hyperhaploidy XY, disomy X and disomy 1 were not significantly different from those of control sperm. These results support the hypothesis that the extra Y chromosome is lost before meiosis with a proliferative advantage of the resulting 46,XY germ cells. Our observations also suggest that a few primary spermatocytes with two Y chromosomes are able to progress through meiosis and to produce Y-bearing sperm cells. A theoretical pairing of the three gonosomes in primary spermatocytes with an extra sex chromosome, compatible with active spermatogenesis, is proposed. Received: 12 April 1996 / Revised: 26 August 1996     

X-ray induction of dominant lethals in late spermatids and mature spermatozoa of Drosophila melanogaster: the role of oxygenation

The role of oxygenation in determining the sensitivity to the induction of dominant lethals was examined in late spermatids and mature spermatozoa of Drosophila melanogaster. 0–2-h-old or 7-day-old Oregon-K males were irradiated with different X-ray exposures in nitrogen, air or in oxygen and the frequencies of dominant lethals induced in these stages were studied. The results obtained confirm and extend Sobels' earlier observations and the interpretation derived therefrom namely, that under normal conditions in air, mature spermatozoa are characterised by a higher degree of oxygenation than late spermatids and this difference is sufficient to explain the differential response of these stages. Similar Oxygen-Enhancement-Ratios(OERs) (of about 2) were obtained for both the cell stages. The present data also revealed that late spermatids are significantly less sensitive than mature spermatozoa to the X-ray-induction of dominant lethals in a nitrogen atmosphere. A plausible mechanism is suggested to explain this observation.     

Effects of osmolality, bicarbonate and buffer on the metabolism and motility of testicular, epididymal and ejaculated spermatozoa of boars.

Spermatozoa were collected from the rete testis of conscious boars, from the cauda epididymidis by retro-flushing, and by ejaculation. Testicular spermatozoa showed no progressive motility, and that of ejaculated was greater than that of epididymal spermatozoa. Glycolysis and respiration of testicular spermatozoa, while lower than that of the more mature cells, were only slightly affected by the incubation conditions. Epididymal spermatozoa converted 83% of the glucose they utilized to CO2 or lactate, but testicular cells converted only 35% to these metabolites. Synthesis of lipid was greatest by testicular spermatozoa. With the more mature cells hyperosmolar conditions depressed CO2 production, but increased lactate production, and these changes were greater for ejaculated than for epididymal spermatozoa. Glycolysis plus respiration of these cells was related to their motility. These results were interpreted as showing increasing motility, glycolysis and respiration with maturation, but also decreased synthetic capacity and increased sensitivity to the environment.     

A method for chromosome analysis of ram spermatozoa using zona-free hamster oocytes

A method for displaying ram spermatozoan chromosomes using the interspecific zona-free hamster oocyte penetration was described to distinguish X- and Y-bearing spermatozoa. Semen samples from four rams were frozen and stored in liquid nitrogen. After thawing the samples, motile spermatozoa were collected by the swim-up method and treated with ionophore A23187 for the purpose of facilitating their capacitation. Slides were prepared by the gradual fixation-air dry method. The rates of oocyte penetration, first cleavage metaphase, and the number of ova that were karyotyped successfully were 67.9, 60.8 and 40.6%, respectively. The overall success rate (number of spermatozoa karyotyped/number of oocytes used for insemination) was 47.9%. A total of 1009 spermatozoa were analyzed, and the ratio of X- and Y-bearing spermatozoa was 508:501.     

Scrotal heat stress effects on sperm viability, sperm DNA integrity, and the offspring sex ratio in mice

Evidence exists to suggest detrimental effects of heat stress on male fertility. This study was designed to assess the effects of scrotal heat stress on mature and developing sperm in a mouse model. After receiving shock heat treatment (42 degrees C for 30 min), mature spermatozoa were recovered from the epididymis hours (6) or Days (7, 14, 21, 28, 60) later, to determine the variables: number of spermatozoa, sperm viability, motility and progressive motility, sperm DNA integrity as established by the TUNEL method, embryo implantation rate, and sex ratio of the fetuses conceived using the heat-exposed spermatozoa. Our results indicate that transient mild heat treatment does not affect in the same way the different types of male germ cells. Spermatocytes present within the testis at the time of heat stress resulted into a lower concentration of spermatozoa with reduced viability and low motility. Even though, DNA integrity of spermatozoa resulting from spermatocytes was also compromised by heat stress, the higher degree of DNA damage was found among spermatozoa resulting from spermatids present within the testis at the time of heat stress. At last, heat shock effect on spermatozoa present in the epididymis at the time of thermal stress resulted into a sex ratio distortion. These findings point to a higher sensitivity of spermatocytes to heat exposure and also suggest a different response of X and Y chromosome-bearing spermatozoa to heat stress that warrants further investigation.     

Evidence of an essential difference between point mutations and chromosome breaks induced by triethylene melamine inDrosophila spermatozoa

Summary Storing of triethylene melamine-treated mature spermatozoa in untreated females was found to result in increased frequencies of both sex-linked recessive lethals and translocations involving the Y, II and III chromosomes. Frequencies of these mutations in effectively unstored spermatozoa were determined from progenies produced using sperm 2–4 days after treatment. The increase in translocation frequencies was on the order of 12-fold in progenies from sperm utilized 11–13 days after treatment when the sperm were stored at 25°C, and 3- to 6-fold when comparable sperm were stored at 12.5°C. Consistent but much smaller increases in frequencies of sex-linked lethals were found, with the increase in lethals tending to be correlated with relative increase in translocation frequency in a given experiment. On the assumption that sex-linked lethals related to chromosome breakage would be expected to increase in frequency in the same proportion as do translocations, approximate agreement was obtained when the proportions of breakage-related lethals among unstored lethals were estimated from the data in the four experimental series. The data are thus consistent with the hypothesis that chromosome breaks but not point mutations are realized during storage of treated spermatozoa. Possible interpretations of a differential effect of storage on treated chromosomes are discussed.Studies carried out while the author was a guest investigator at the Institute of Animal Genetics on sabbatical leave from the University of Minnesota.