A new dimension of the forensic DNA expertise - the need for training experts and expertise recipients.

Forensic genetics is a rapidly developing discipline. Nowadays, human genetic identification relies on the application of complex solutions ensuring high sensitivity and resistance to the inhibition and degradation of biological traces, and revealing maximum information which has relevance for the justice system. However, recent improvements in forensic DNA identification testing are associated with problems including secondary transfer, DNA mixtures and incompleteness of DNA profiles, which were formerly less significant. It also seems that the potential of the national DNA database in Poland has not been fully developed, and it is necessary to implement an appropriate information policy in order to improve it. Novel methods that can be applied at the level of investigation include analysis of biogeographic ancestry, prediction of visible traits, and estimation of human chronological age. Moreover, next-generation sequencing has a potential to entirely replace capillary electrophoresis in forensic genetics. Further works are necessary to ensure a proper implementation of uniform standards of data interpretation and evaluation of DNA evidence in forensic genetics. In order to maintain proper standards of forensic DNA assessment, continuous training of DNA experts and appropriate information policy for recipients of DNA assessments are required.

Curcumin disrupts meiotic and mitotic divisions via spindle impairment and inhibition of CDK1 activity.

OBJECTIVES: Curcumin, a natural compound, is a potent anti-cancer agent, which inhibits cell division and/or induces cell death. It is believed that normal cells are less sensitive to curcumin than malignant cells; however, the mechanism(s) responsible for curcumin's effect on normal cells are poorly understood. The aim of this study was to verify the hypothesis that curcumin affects normal cell division by influencing microtubule stability, using mouse oocyte and early embryo model systems. MATERIALS AND METHODS: Maturating mouse oocytes and two-cell embryos were treated with different concentrations of curcumin (10-50 microm), and meiotic resumption and mitotic cleavage were analysed. Spindle and chromatin structure were visualized using confocal microscopy. In addition, acetylation and in vitro polymerization of tubulin, in the presence of curcumin, were investigated and the damage to double-stranded DNA was studied using gammaH2A.X. CDK1 activity was measured. RESULTS AND CONCLUSIONS: We have shown for the first time, that curcumin, in a dose-dependent manner, delays and partially inhibits meiotic resumption of oocytes and inhibits meiotic and mitotic divisions by causing disruption of spindle structure and does not induce DNA damage. Our analysis indicated that curcumin affects CDK1 kinase activity but does not directly affect microtubule polymerization and tubulin acetylation. As our study showed that curcumin impairs generative and somatic cell division, its future clinical use or of its derivatives with improved bioavailability after oral administration, should take into consideration the possibility of extensive side-effects on normal cells.

Influence of the CBA genetic background on sperm morphology and fertilization efficiency in mice with a partial Y chromosome deletion.

Males of the B10.BR-Ydel mouse strain, with a deletion in the long arm of the Y chromosome, were backcrossed to CBA females to introduce the Ydel chromosome to the genetic background of the CBA mice. The CBA-Ydel males (sixth backcross generation) had similar symptoms to those previously described for B10.BR-Ydel males (deterioration of sperm quality and of efficiency of fertilization), but these effects were much less pronounced, showing a favourable influence of the CBA genetic background. The CBA-Ydel males produced only 12% severely misshapen spermatozoa, and mating with B10.BR females gave 100% successful fertilization. Although nearly all sperm heads were abnormal (92% versus 6% in control males), most of the spermatozoa (76%) had deformation only in the acrosomal part, that is, flat heads, which were not found in the control males. These abnormalities were analysed in detail. As shown by differential staining, the acrosomes of the spermatozoa with flat heads were deformed; 18% of these acrosomes looked damaged, and often contained a vesicle, which stained in a similar way to the acrosome but lacked the reaction for acrosomal proteinase. Electron microscopy of testis sections revealed that deformations appeared already in round spermatids as distortion of the acrosomal vesicle and asymmetrical position of the acrosomal granule; in many elongating spermatids the proximal end had a flat or concave shape, and the acrosomes contained a translucent vesicle. It is possible that the genes that are missing in the Yq deletion have some important regulatory function in the course of spermiogenesis, which may explain the various sperm defects observed in Y-del males.

Immunohistochemical localization of androgen receptors in testicular cells of mice with mosaic mutation.

Mice with mosaic mutation could be one of the models of human Menkes disease, which is associated with abnormal cooper metabolism. The aim of the present study was to localize androgen receptors (ARs) in the testes by means of immunohistochemistry. AR expression was observed in the nuclei of all somatic cells such as Leydig cells, Sertoli cells, and peritubular cells in sections from testes of control and mosaic mutant males. In the latter, very strong immunoreactivity for AR as well as higher levels of steroid hormones in homogenates were noticed in comparison to control mice. No positive immunoreaction for ARs was seen in control sections incubated without the primary antibody.

Pathological structure of the kidney from adult mice with mosaic mutation.

The mosaic (Atp7a(mo-ms)) is an X-linked, lethal mutation in mice. In mosaic mutant males, many clinical features characteristic of defective copper metabolism have been observed and they die at the age of 15 days, exhibiting strong similarities to the brindled and macular mutants. About 4% of the mutant males live to sexual maturity and some of them are fertile. In this paper, alterations in the structure of the kidney from adult mutants are described. Owing to an inherited defect of efflux, copper is accumulated in the kidney of the mutants up to a toxic level and this leads to severe damage of the renal cortex. Pathological changes in the kidney mostly affected the structure of the renal corpuscle and renal tubules.

Sperm select penetration test reveals differences in sperm quality in strains with different Y chromosome genotype in mice.

Mouse sperm penetration into a hyaluronic acid polymer (Sperm Select), hypoosmotic swelling in distilled water (water test), and eosin-Y staining were studied in sperm samples from males belonging to five inbred strains of mice (CBA, KE, KE-YCBA, B10.BR, and B10.BR-Ydel), which differ in the proportion of abnormal sperm heads and in efficiency of fertilization. Correlation coefficients, calculated from mean values for each strain, showed that Sperm Select penetration (to the depth of 1-2 cm) was significantly correlated with both sperm motility and fertilization efficiency, while correlation with the proportion of normal spermatozoa did not reach the level of significance. The indices of the water test and erosin Y staining were correlated with each other but not with Sperm Select penetration. In comparison with the B10.BR strain, its mutant strain B10.BR-Ydel, with a large deletion in the long arm of the Y chromosome and characterized by highly elevated proportion of abnormal sperm and low fertilization efficiency, showed lower values of the indices of all tests, suggesting that the partial deletion of the Y chromosome has affected various aspects of sperm morphology and function. On the other hand, for two consomic strains (KE and KE-YCBA with the Y chromosome introduced from the CBA strain), the difference in sperm morphology and fertilization rate was correlated with Sperm Select penetration but not with hypoosmotic swelling or eosin-Y staining; this suggests that only some aspects of sperm physiology are due to genetic differences between YKE and YCBA chromosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of sperm quality in recombinant inbred mouse strains: correlation of sperm head shape with sperm abnormalities and with the incidence of supplementary spermatozoa in the perivitelline space.

Recombinant inbred strains were developed from reciprocal crosses between two inbred strains of mice (CBA and KE) differing in sperm head shape, proportion of normal sperm heads (CBA, 95%; KE, 78%) and fertilization efficiency (CBA, 100% of fertilized ova; KE, 72%), to determine whether the indices of sperm morphology and function were correlated. The following parameters were analysed in recombinant inbred and progenitor strains: index of sperm head shape (head width in the middle of its length/head length), percentage of abnormal sperm heads, percentage of spermatozoa with progressive movements, efficiency of penetration of hyaluronic acid polymer (Sperm Select) and percentage of fertilized ova after mating males from the tested strains with females from an outbred stock. For each investigated character, recombinant inbred strains, recombinant inbred EXCB and CBXE, could be divided into at least three categories: KE-like, CBA-like and intermediate, suggesting that in each case a minimum of two genes was involved. Recombinant strains derived from the reciprocal crosses of progenitor strains differed only with respect to the proportion of abnormal sperm heads, showing the involvement of the Y chromosome in determining this character. Penetration into Sperm Select was significantly correlated both with fertilization efficiency and sperm motility, while correlation with the proportion of normal spermatozoa did not reach the level of significance. However, there was a significant negative correlation of both sperm abnormalities and the incidence of supplementary spermatozoa in the perivitelline space with the index of sperm head shape.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial deletion of the Y chromosome removes the effect of paternal genome imprinting on periovum sensitivity to hyaluronidase in mice.

Cumulus-oocyte complexes from the two consomic strains of mice, B10.BR and B10.BR-Ydel (with a partial deletion of the Y chromosome) and the BALB/c strain, as well as reciprocal F1 and F2 hybrids, were tested for their susceptibility to attack by hyaluronidase. The cumulus cell dispersal was significantly more rapid in the eggs of B10.BR females and of F1 and F2 hybrid females sired by the B10.BR males than in those of BALB/c females and hybrids sired by BALB/c males. These results confirm the earlier data of Bander et al. (1989) which were interpreted as evidence of paternal imprinting of the C57BL genome. In contrast, cumulus cell dispersal in the eggs of females sired by B10.BR-Ydel males was significantly slower than in females sired by B10.BR males, and did not differ from that in the BALB/c strain. The results suggest that the partial deletion of the Y chromosome abolished the effect of paternal genome imprinting which is observed in the B10.BR strain.

Analysis of Hya locus in B10.BR males with partial deletion of the Y chromosome.

Skin transplantation technique was used to check the presence of the Hya gene in two consomic strains, B10.BR and B10.BR-Ydel, which differ in the length of the Y chromosome. In both consomic strains the skin grafts from males to females were rejected in approximately the same time (B10.BR- mean = 45.65 +/- 3.12; B10.BR-Ydel- mean = 46.56 +/- 4.00). These results show that the deletion in the B10.BR-Ydel males does not include the Hya gene.

Analysis of genetic markers Svp-1 and Svp-3 in recombinant mouse strains CBXE and EXCB.

New genetic markers; seminal vesicle proteins (Svp-1, Svp-3) and agouti (A) locus were tested in five RI EXCB and 15 RI CBXE strains developed from CBA/Kw and KE inbred strains of mice. For agouti and Svp-1 loci, on the chromosome 2, there are five recombinant strains out of 20 strains which gives r = 0.1. It is close to the known map distance (10cM) between these two loci.

Influence of partial deletion of the Y chromosome on mouse sperm phenotype.

Two congenic strains of mice (control, B10.BR/SgSn; mutant, B10.BR-Ydel/Ms with partial deletion of the Y chromosome) were examined. In control males, 22.6% of spermatozoa had abnormal heads; in mutant males, there were 64.2%, the most common being heads with flat acrosomes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of mature sperm proteins, followed by acrosin assay and acrosome silver staining, revealed a reduced concentration of acrosin in acrosomal caps in 35.8% of the spermatozoa in mutant males. Electron microscope analysis showed that some of the round, early spermatids in the mutants had normally formed acrosomal caps but lacked the proacrosomal granule and had no, or only scarce, acrosomal material. These observations indicate that formation of the acrosomal cap is controlled separately from the synthesis of the acrosomal material and suggest that some factors linked on the Y chromosome are involved in the control of acrosome development.

Linkage between sperm abnormality level and major urinary protein phenotype in mice.

Segregation of sperm abnormality level and the pattern of major urinary proteins (MUPs) were investigated in F2 and B1 hybrid males obtained from crosses involving two contrasting inbred strains of mice: CBA/Kw (Mup-1a1a, 3.3% abnormal sperm) and C57BL/Kw (Mup-1b1b, 21.9% abnormal sperm). In the progeny of both crosses mean levels of abnormal spermatozoa were significantly higher for males typed as Mup-1b1b than for heterozygous Mup-1a1b males. Moreover, all F2 hybrid males showing very high percentages of abnormal sperm were Mup-1b1b homozygotes. Similarly, among B1 males with a high level of deformed spermatozoa, a statistically significant majority were Mup-1b1b genotypes. Our results suggest that at least two genes which influence sperm abnormality level are segregating in these crosses. Both appear to be recessive for high sperm abnormality level, and one shows weak linkage to Mup-1 on chromosome 4.

An increased level of sperm abnormalities in mice with a partial deletion of the Y chromosome.

Two congenic lines of mice, one with a partial deletion of the Y chromosome, differ in the percentage of spermatozoa with abnormal heads: B10.BR/SgSn males give 22.6% and B10.BR-Ydel/Ms males give 64.2% abnormal sperm. The F1s resulting from crosses of B10.BR/SgSn males with females of five common inbred strains exhibited significantly lower levels of abnormal sperm than the parental strains, as opposed to F1 hybrids sired by B10.BR-Ydel/Ms mutant males, where very high levels of abnormal spermatozoa were found. About 30% of abnormal spermatozoa, produced by males with deletion on the Y chromosome, were characterized by a flat acrosomal cap. This class of abnormality was never observed in non-mutant males, suggesting a mutant-specific defect. These results demonstrate the important role of the Y chromosome in spermatogenesis.

Effect of Y chromosome and H-2 complex derived from Japanese wild mouse on sperm morphology.

Segregation of sperm abnormality level and H-2 haplotypes was investigated in F2 hybrid males obtained from reciprocal crosses involving two B10.congenic strains carrying H-2 and the Y chromosome of Japanese wild mice: B10.MOL-OHM (H-2wm4, 23.1% of sperm abnormalities) and B10.MOL-OKB (H-2wm8, 11.1% of sperm abnormalities). In both types of crosses mean levels of abnormal spermatozoa were significantly higher for males typed as H-2wm4/H-2wm4 than for heterozygous H-2wm4/H-2wm8 or homozygous H-2wm8/H-2wm8. These results suggest that the gene for high sperm abnormality is linked to H-2 complex of the B10.MOL-OHM strain.