Biomedical subjects

E Ko

Publications and source records attributed to E Ko.

At least 19 recordsLinked to original sources

PubMed2006

Analysis of non-crossover bivalents in pachytene cells from 10 normal men.

BACKGROUND: Bivalents with no recombination foci (possible achiasmates) are unable to orient properly on the metaphase plate or to segregate chromosomes to daughter cells. Non-crossover bivalents are known to cause meiotic arrest in various organisms. METHODS: Individual non-crossover bivalents were identified in 886 pachytene cells (19 492 bivalents) from testicular biopsies of 10 normal men. Fluorescence staining combined with centromere-specific multicolour fluorescence in situ hybridization (cenM-FISH) was used to identify mismatch repair gene mutation of human mutL homologue 1 (MLH1) recombination foci along each bivalent synaptonemal complex (SC). RESULTS: A total of 60 autosomal non-crossovers (SCs without an MLH1 focus) were found, and of these, chromosomes 21 (2.1%) and 22 (1.7%) had a significantly higher proportion than chromosomes 11, 12, 19 (each 0.1%), 13 (0.2%), 14 (0.6%), 16 (0.5%) and 15, 17, 18, 20 (each 0.3%) (P < 0.05). Sex chromosome univalents had a frequency of 27%, higher than that observed in any autosomal bivalent (P < 0.0001). CONCLUSIONS: These results suggest that G-group chromosomes and sex chromosomes are most susceptible to having no recombination foci and thus would be more susceptible to non-disjunction during spermatogenesis. This is consistent with previous observations from sperm karyotyping and FISH analysis, which demonstrate that chromosomes 21 and 22 and the sex chromosomes have a significantly increased frequency of aneuploidy compared with other autosomes.

Aged↗

PubMed2005

Meiotic studies in an azoospermic human translocation (Y;1) carrier.

A reciprocal translocation between the long arm of the Y chromosome and the long arm of chromosome 1 was observed in an infertile man with non-obstructive azoospermia. The study was performed using a combination of techniques: immunocytogenetic analysis, which allows the detection of synaptonemal complexes (SCs) and recombination sites (MLH1) simultaneously, and fluorescence in-situ hybridization analysis. Meiotic pairing analysis on 100 pachytene spreads showed the presence of a quadrivalent containing chromosomes 1 and Y. There were many abnormalities in chromosome pairing and recombination. These abnormalities included a great reduction of recombination events (as many as one fifth of the SCs had no MLH1 foci), and high proportions of unpaired regions and discontinuities in the SCs. We discuss the possibility that infertility in this patient may be due to transcriptional repression of part of chromosome 1 involved in the translocation, silencing some genes necessary for the progression of meiosis and causing defective meiotic pairing and recombination.

Adult↗

PubMed2005

Meiotic studies in two human reciprocal translocations and their association with spermatogenic failure.

BACKGROUND: Reciprocal translocations are often associated with infertility in male carriers. However, some carriers present normal semen profiles and are identified because of repetitive pregnancy failures. METHODS: Here, we report two different cases of reciprocal translocations. The first patient carried a t(10;14) and was normozoospermic. The second patient carried a t(13;20) and was azoospermic. Synaptonemal complexes from both carriers were analysed using immunocytogenetic techniques and multi-centromere fluorescent in situ hybridization (cenM-FISH). RESULTS: Associations between the quadrivalent and the sex body or other autosomes were seen only in the t(13;20) carrier. Heterosynapsis was observed only in the t(10;14) carrier. Synaptic pairing abnormalities were seen in 71% of the spreads in the t(13;20) carrier and 30% of the spreads in the t(10;14) carrier. Recombination frequency was decreased in the t(13;20) carrier, but not in the t(10;14) carrier. CONCLUSIONS: By comparing these two different translocation carriers with different fertility outcomes, we discuss the possible mechanisms by which translocations might cause the spermatogenesis process to fail.

Adult↗

PubMed2005

Temporal progression of recombination in human males.

To date, immunocytology has been used in humans to detect a limited number of meiotic proteins: components of the synaptonemal complex (SCP1 and SCP3) and some proteins known to participate in recombination events, such as MLH1 or RAD51. However, the colocalization or coexistence of proteins known to participate during the different stages of human meiosis remains largely unstudied, and these studies could provide important clues about the mechanics of recombination. This work reports the relative timing and localization of five different meiotic proteins that have previously been implicated in human homologous recombination [RAD51, replication protein A (RPA), MSH4, MLH1 and MLH3]. MSH4 foci appear concurrently with synapsis initiation at zygotene, shortly after the first RAD51 foci are detected. The presence of RPA in MSH4 foci was noted, suggesting that these two proteins may act co-operatively. Both RPA and MSH4 foci reach maximal numbers at the end of zygotene, when synapsis is concluding. From this point, RPA foci all but disappear by the end of pachytene, whereas MSH4 foci decline to a stable number at mid-pachytene, where they localize with MLH1/MLH3 recombination sites. We discuss a possible role for MSH4 in synapsis initiation and/or maintenance.

Carrier Proteins↗

PubMed2005

Male infertility in reciprocal translocation carriers: the sex body affair.

Previous reports have linked chromosomal reorganization and spermatogenic failure. In this context, it has long been known that reciprocal translocation carriers are more likely to have anomalies in the meiotic process, including fertility failures. It has also been proposed that this fertility failure may be a consequence of an association between the translocated chromosomes and the sex body. In this work, we review different hypotheses explaining meiotic failure in these carriers, and propose a model that relates meiotic abnormalities with both sex body-translocation association and different checkpoints that are known to operate during meiosis.

Animals↗

PubMed2005

Immunofluorescent synaptonemal complex analysis in azoospermic men.

The molecular cause of germ cell meiotic defects in azoospermic men is rarely known. During meiotic prophase I, a proteinaceous structure called the synaptonemal complex (SC) appears along the pairing axis of homologous chromosomes and meiotic recombination takes place. Newly-developed immunofluorescence techniques for SC proteins (SCP1 and SCP3) and for a DNA mismatch repair protein (MLH1) present in late recombination nodules allow simultaneous analysis of synapsis, and of meiotic recombination, during the first meiotic prophase in spermatocytes. This immunofluorescent SC analysis enables accurate meiotic prophase substaging and the identification of asynaptic pachytene spermatocytes. Spermatogenic defects were examined in azoospermic men using immunofluorescent SC and MLH1 analysis. Five males with obstructive azoospermia, 18 males with nonobstructive azoospermia and 11 control males with normal spermatogenesis were recruited for the study. In males with obstructive azoospermia, the fidelity of chromosome pairing (determined by the percentage of cells with gaps [discontinuities]/splits [unpaired chromosome regions] in the SCs, and nonexchange SCs [bivalents with 0 MLH1 foci]) was similar to those in normal males. The recombination frequencies (determined by the mean number of MLH1 foci per cell at the pachytene stage) were significantly reduced in obstructive azoospermia compared to that in controls. In men with nonobstructive azoospermia, a marked heterogeneity in spermatogenesis was found: 45% had a complete absence of meiotic cells; 5% had germ cells arrested at the zygotene stage of meiotic prophase; the rest had impaired fidelity of chromosome synapsis and significantly reduced recombination in pachytene. In addition, significantly more cells were in the leptotene and zygotene meiotic prophase stages in nonobstructive azoospermic patients, compared to controls. Defects in chromosome pairing and decreased recombination during meiotic prophase may have led to spermatogenesis arrest and contributed in part to this unexplained infertility.

Humans↗

PubMed2004

Meiotic defects in a man with non-obstructive azoospermia: case report.

Infertile men have an increased frequency of aneuploid sperm. We have determined that decreased recombination is associated with the production of aneuploid sperm in humans. The aim of this study was to determine whether some cases of infertility are associated with decreased meiotic recombination. Analysis of the early stages of meiosis was performed in a 33-year-old man with non-obstructive azoospermia. Newly developed immunocytogenetic techniques were used to identify the synaptonemal complex (SC) in various stages of prophase. Antibodies to meiotic proteins identified the SC (SYN1/SCP3), the centromere (CREST) and recombination sites (MLH1). Only 36 meiotic spreads were recovered from the infertile man, compared with hundreds available from controls. One-third of the cells were in zygotene compared with 4% in controls, demonstrating an inability of bivalents to synapse and progress to pachytene. The infertile man had a greatly reduced frequency of recombination, with a mean of only 32.7 MLH1 foci/cell (range 1-60) compared with 46.0 (range 21-62) in control donors. A high proportion of cells (73%) contained at least one autosomal bivalent with zero MLH1 foci, compared with only 4.5% in control donors. Discontinuities in the SC were also more prevalent (68% of cells versus 26% in controls). This is the first demonstration of dramatic pachytene-stage abnormalities in an infertile man using these powerful new immunocytogenetic techniques.

Adult↗

PubMed2004

The effect of cold storage on recombination frequencies in human male testicular cells.

Meiotic recombination is essential for the segregation of homologous chromosomes and formation of normal haploid gametes. Decreased recombination is associated with the production of aneuploid sperm in humans. MLH1, a DNA mismatch repair protein, was recently found to mark the sites of recombination in humans. Newly developed immunofluorescence techniques to identify MLH1 foci on synaptonemal complexes (SCs) in pachytene cells from testicular tissue have opened up a new avenue of research on meiotic recombination. Future studies on normal and abnormal recombination in early meiosis will further research in human reproduction and genetics. However, the availability of testicular material will always be a major limiting factor in this kind of study. In order to obtain an adequate number of samples and samples of particular research interest, it is often of benefit to obtain samples from distant regions. Therefore, it is necessary to determine whether the quality of samples and accuracy of MLH1 frequencies change after transporting testicular samples from a distance. In the present study, we examined the recombination frequencies (numbers of MLH1 foci using immunofluorescence techniques) in 6 normal testicular samples. Each sample was split and analyzed in the fresh state and after storage on ice for two days, mimicking overnight courier air transport. The results showed no significant difference in the quality of the SC preparations or in the number of MLH1 foci between these two groups. These results demonstrate that testicular specimens may be shipped on ice without compromising data on chromosome pairing and recombination in early meiosis.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Regulation of the expression of the prostate-specific antigen by claudin-7.

Claudins are a family of proteins involved in forming tight junctions between cells. Here we describe two forms of claudin-7 (CLDN-7), a full-length form of CLDN-7 with 211 amino-acid residues and a C-terminal truncated form with 158 amino-acid residues. These two forms of CLDN-7 are able to regulate the expression of a tissue-specific protein, the prostate-specific antigen (PSA), in the LNCaP prostate cancer cell line. We also found that the expression of CLDN-7 is responsive to androgen stimulation in the LNCaP cell line, suggesting that this protein is involved in the regulatory mechanism of androgen. Both forms of claudin-7 are expressed in human prostate, kidney and lung samples, and in most samples, the full-length form of claudin-7 was predominant. However, in some prostate samples from healthy individuals, the truncated form of claudin-7 is predominantly expressed. Our results demonstrated that unlike other claudins, CLDN-7 has both structural and regulatory functions, and the two forms of CLDN-7 may be related to cell differentiation in organ development.

Adenocarcinoma↗

PubMed2001

Ca2+-dependent membrane currents in vascular smooth muscle cells of the rabbit.

The membrane potential in vascular smooth muscle cells contributes to the regulation of cytosolic [Ca2+], which in turn regulates membrane potential by means of Ca2+i-dependent ionic currents. We investigated the characteristics of Ca2+i-dependent currents in rabbit coronary and pulmonary arterial smooth muscle cells. Ca2+i-dependent currents were recorded using the whole-cell patch-clamp technique while cytosolic [Ca2+] was increased by caffeine. The reversal potentials of caffeine-induced currents were between -80 and -10 mV under normal ionic conditions, whereas they were about 0 mV when K+-free NaCl solutions were used both in pipette and bath. The total substitution of extracellular Na+ with membrane-impermeable cation N-Methyl-D-glucamine did not affect caffeine-induced currents, implying no significant contribution of Na+ as a permeant ion to the currents. The substitution of extracellular NaCl with sucrose reduced outward component of the currents and shifted the reversal potentials according to the change in Cl- equilibrium potential. Upon application of the niflumic acid under K+-free conditions, most of the current induced by caffeine was inhibited. Taken together, the results of the present study indicate that K+ and Cl- currents are major components of Ca2+i-dependent currents in vascular smooth muscles isolated from coronary and pulmonary arteries of the rabbit, and the relative contribution of each type of current to total currents are not different between the two arteries.

Animals↗

PubMed2001

Fos expression in orexin neurons varies with behavioral state.

The neuropeptide orexin (also known as hypocretin) is hypothesized to play a critical role in the regulation of sleep-wake behavior. Lack of orexin produces narcolepsy, which is characterized by poor maintenance of wakefulness and intrusions of rapid eye movement (REM) sleep or REM sleep-like phenomena into wakefulness. Orexin neurons heavily innervate many aminergic nuclei that promote wakefulness and inhibit REM sleep. We hypothesized that orexin neurons should be relatively active during wakefulness and inactive during sleep. To determine the pattern of activity of orexin neurons, we recorded sleep-wake behavior, body temperature, and locomotor activity under various conditions and used double-label immunohistochemistry to measure the expression of Fos in orexin neurons of the perifornical region. In rats maintained on a 12 hr light/dark cycle, more orexin neurons had Fos immunoreactive nuclei during the night period; in animals housed in constant darkness, this activation still occurred during the subjective night. Sleep deprivation or treatment with methamphetamine also increased Fos expression in orexin neurons. In each of these experiments, Fos expression in orexin neurons correlated positively with the amount of wakefulness and correlated negatively with the amounts of non-REM and REM sleep during the preceding 2 hr. In combination with previous work, these results suggest that activation of orexin neurons may contribute to the promotion or maintenance of wakefulness. Conversely, relative inactivity of orexin neurons may allow the expression of sleep.

Animals↗

PubMed2001

Single sperm typing demonstrates that reduced recombination is associated with the production of aneuploid 24,XY human sperm.

To account for the increased proportion of paternal nondisjunction in 47,XXY males as compared to other trisomies, it has been suggested that the XY bivalent, with its reduced region of homology, is particularly susceptible to nondisjunction. Molecular studies of liveborn Klinefelter syndrome (47,XXY) individuals have reported an association between the absence of recombination in the pseudoautosomal region and nondisjunction of the XY bivalent. In this study we examined single sperm from a normal 46,XY male to determine if there is any alteration in the recombination frequency of aneuploid disomic 24,XY sperm compared to unisomic sperm (23,X or Y). Two DNA markers STS/STS pseudogene and DXYS15 were typed in sperm from a heterozygous man to determine if recombination had occurred in the pseudoautosomal region. Individual unisomic sperm (23,X or Y) were isolated using a FACStar(Plus) flow cytometer into PCR tubes. To identify disomic 24,XY sperm, 3-colour FISH analysis was performed with probes for chromosomes X,Y and 1. The 24,XY cells were identified using fluorescence microscopy, each disomic sperm was scraped off the slide using a glass needle attached to a micromanipulator and then put into a PCR tube. Hemi-nested PCR analysis of the two markers was performed to determine the frequency of recombination. A total of 329 unisomic sperm and 150 disomic sperm have been typed. The frequency of recombination between the two DNA markers was 38.3% for the unisomic sperm, similar to frequencies previously reported. The 24,XY disomic sperm had an estimated recombination frequency of 25.3%, however, a highly significant decrease compared to the unisomic 23,X or 23,Y sperm (chi(2) = 10.7, P = 0.001). This direct analysis of human sperm indicates that lack of recombination in the pseudoautosomal region is a significant cause of XY nondisjunction and thus Klinefelter syndrome.

Aneuploidy↗

PubMed2001

Cigarette smoking and aneuploidy in human sperm.

Cigarette smoke contains chemicals which are capable of inducing aneuploidy in experimental systems. These chemicals have been shown to reach the male reproductive system, increasing oxidative DNA damage in human sperm and lowering semen quality. We have examined the association between smoking and aneuploid sperm by studying 31 Chinese men with similar demographic characteristics and lifestyle factors except for cigarette smoking. None of the men drank alcohol. These men were divided into three groups: nonsmokers (10 men), light smokers (< 20 cigarettes/day, 11 men), and heavy smokers (> or = 20 cigarettes/day, 10 men). There were no significant differences in semen parameters or in age across groups. Two multi-color fluorescence in situ hybridizations (FISH) were performed: two-color FISH for chromosomes 13 and 21, and three-color FISH for the sex chromosomes using chromosome 1 as an internal autosomal control for diploidy and lack of hybridization. The mean hybridization efficiency was 99.78%. The frequency of disomy 13 was significantly higher in light and heavy smokers than in non-smokers, while no significant differences in the frequency of disomy 21, X or Y were observed across groups. Significant inter-donor heterogeneity in every category of disomic sperm examined was found in both light and heavy smokers, while in nonsmokers only XY disomy showed significant inter-donor differences. Thus, we conclude that cigarette smoking may increase the risk of aneuploidy only for certain chromosomes and that men may have different susceptibilities to aneuploidy in germ cells induced by cigarette smoking. Mol. Reprod. Dev. 59: 417-421, 2001.

Adult↗

PubMed2001

Microwave decondensation and codenaturation: a new methodology to maximize FISH data from donors with very low concentrations of sperm.

To increase analyzable sperm numbers when dealing with semen samples from donors with extremely low quantities of sperm, a microwave decondensation/codenaturation technique for fluorescence in situ hybridization (FISH) was developed and results were compared to those obtained from conventional FISH techniques. There was no significant difference between the frequencies of disomy and diploidy obtained using the two techniques.

Humans↗

PubMed2000

Segregation of a supernumerary del(15) marker chromosome in sperm.

Supernumerary marker chromosomes (SMC) can be associated with both normal and abnormal phenotypes. In addition, SMC are found at higher frequency in males with infertility. We identified a SMC, characterized as a del(15)(q11.2) chromosome, in a phenotypically normal male. Using fluorescence in situ hybridization (FISH), we examined the segregation of the del(15) chromosome in sperm from this patient. Only 6.23% of sperm nuclei showed disomy using a chromosome 15 alpha-satellite FISH probe, instead of the expected 50%. In addition, FISH analysis showed no increase for non-disjunction of chromosome 18, excluding an interchromosomal effect for this chromosome. The significant decrease in sperm bearing the del(15) may be due to tissue-specific mosaicism or a result of some form of selection against the del(15) during spermatogenesis. This finding provides a basis for the observation that SMC(15) are less likely to be inherited from a paternal carrier.

Adult↗

PubMed2000

Analysis of aneuploidy frequencies in sperm from patients with hereditary nonpolyposis colon cancer and an hMSH2 mutation.

Hereditary nonpolyposis colon cancer (HNPCC) has been shown to be caused by mutations in the mismatch repair genes hMSH2, hMLH1, hPMS1, and hPMS2. Recent evidence has demonstrated that mutations in mismatch repair genes disrupt meiosis in mice. A large HNPCC kindred in Newfoundland, Canada, has an hMSH2 mutation-an A-->T transversion at the +3 position of the splice-donor site of exon 5. We have studied sperm from men with this hMSH2 mutation, since it is possible that mismatch repair mutations in humans might also have an effect on meiosis and normal segregation of chromosomes. The frequencies of aneuploid and diploid sperm were determined in 10 men with the hMSH2 mutation, by use of multicolor FISH analysis for chromosomes 13, 21, X, and Y. A minimum of 10,000 sperm per man was studied per chromosome probe. Control individuals consisted of men in the same kindred with HNPCC who did not carry the mutation and of other normal men from Newfoundland. A total of 321,663 sperm were analyzed: 200,905 sperm were from men carrying the hMSH2 mutation and 120,758 sperm were from control men. There was a significantly increased frequency of disomy 13, disomy 21, XX, and diploidy in mutation carriers compared with control men. These results suggest that the hMSH2 mutation may affect meiosis in humans.

Adult↗

PubMed2000

Chromosome analysis of spermatozoa extracted from testes of men with non-obstructive azoospermia.

Infertile men with azoospermia now have the possibility of fathering children by testicular sperm extraction combined with intracytoplasmic sperm injection. However, there are concerns about the risk of chromosomal abnormalities in their spermatozoa. We have studied aneuploidy frequencies for chromosomes 13, 21, X and Y by multicolour fluorescence in-situ hybridization (FISH) in testicular spermatozoa extracted from three men with non-obstructive azoospermia. The men were 34-37 years of age and had normal follicle-stimulating hormone (FSH) concentrations and normal 46,XY somatic karyotypes. A total of 3324 spermatozoa was analysed. The infertile patients had an elevated frequency of disomy for chromosomes 13, 21, XY disomy compared to controls but none of these reached statistical significance. Also there was no significant difference in the sex ratio or the frequency of diploidy in azoospermic patients compared to normal control donors. This first report on chromosomal aneuploidy in spermatozoa extracted from testes of patients with non-obstructive azoospermia suggests that some azoospermic men do not have a substantially increased risk of chromosomally abnormal spermatozoa.

Adult↗

PubMed2000

Direct measurement of islet amyloid polypeptide fibrillogenesis by mass spectrometry.

A novel method for monitoring fibrillogenesis is developed and applied to the amyloidogenic peptide, islet amyloid polypeptide (IAPP). The approach, based on electrospray ionization mass spectrometry, is complementary to existing assays of fibril formation as it monitors directly the population of precursor rather than product molecules. We are able to monitor fiber formation in two modes: a quenched mode in which fibril formation is halted by dilution into denaturant and a real time mode in which fibril formation is conducted within the capillary of the electrospray source. Central to the method is the observation that fibrillar IAPP does not compromise the ionization of monomeric IAPP. Furthermore, under mild ionization conditions, fibrillar IAPP does not dissociate and contribute to the monomeric signal. Critically, we introduce an internal standard, rat IAPP, for analysis on the mass spectrometer. This standard is sufficiently similar in sequence in that it ionizes identically to human IAPP. Furthermore, the sequence is sufficiently different in that it does not form fibrils and is distinguishable on the basis of mass. Applied to IAPP fibrillogenesis, our technique reveals that precursor consumption in seeded reactions obeys first-order kinetics. Furthermore, a consistent level of monomer persists in both seeded and unseeded experiments after the fibril formation is complete. Given the inherent stability of fibrils, we expect this approach to be applicable to other amyloid systems.

Amyloid↗