Józefa Styrna

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.

The effect of a partial Y chromosome deletion in B10.BR-Ydel mice on testis morphology, sperm quality and efficiency of fertilization

Males of the mouse strain B10.BR/SgSn and its congenic mutant strain B10.BR-Ydel, with a partial deletion of the Y chromosome, were used to examine factors related to poor sperm quality and quantity in the mutant strain. The testes of males from the two strains did not differ in their immunohistochemical reaction to androgen receptors or in the number of Sertoli and germ cells in tubules with normal morphology. However, mutants showed a greater frequency of degenerated tubules, a higher level of X-Y chromosome dissociation at meiosis (18% v. 10% in control males), and a lower content of resistant sperm heads in testis homogenates. In the cauda epididymidis, there was a higher percentage of spermatozoa with abnormal heads (88% v. 31%) and of spermatozoa with a cytoplasmic droplet still attached (74% v. 51%). Many sperm heads with flat acrosomes, occurring only in mutants (30% of sperm population), were deficient in proteolytic enzymes, as evidenced by the reaction on gelatine membranes. Most copulations of mutant males (11/18) were sterile in spite of the presence of spermatozoa in the uterus, but in the remaining copulations the fertilization rate was reasonably good (79%). Low numbers of spermatozoa were recovered from the oviducts, and those with the most severely deformed heads were less frequent there than in the uterus. The results show that a partial deletion of the Y chromosome affects efficiency of spermatogenesis, morphology of spermatozoa, their epididymal maturation and capacity to reach the ampulla and fertilize eggs.

Gene mapping of sperm quality parameters in recombinant inbred strains of mice

The aim of this study was to map chromosomal regions containing hypothetical genes responsible for the following parameters of mouse semen quality: (1) the percentage of sperm with abnormal head morphology, (2) the level of dead spermatozoa, (3) the percentage of sperm tails with residual cytoplasmic droplets, and (4) the percentage of sperm with impaired sperm tail membrane integrity. We also analyzed any possible correlations between these parameters. The most appropriate animal model for mapping genes controlling quantitative traits (QTL, quantitative trait locus) is a set of recombinant inbred (RI) strains. The set of RI strains used in this study was derived from crosses between two inbred mouse strains, KE and CBA/Kw, which differ significantly in fertility parameters and gamete quality. We analyzed the four parameters of sperm quality in male mice from two parental strains and from 12 RI strains. The strain distribution pattern (SDP) of 187 polymorphic microsatellite markers was prepared for 20 chromosomes of the mouse genome in 12 RI strains. We correlated the SDP of these markers with the values of sperm quality parameters, using MapManager QTX software (ver. b18). The mapping procedure indicated that the percentage of sperm with abnormal head morphology is controlled by gene(s) located in chromosomal regions 11q24, 11q31 and 6q15.6. There was also a strong correlation between male body weight and the hypothetical gene(s) in chromosomal region 18q47. A detailed analysis of the genes located in these regions enabled us to prepare a list of candidate genes. We discuss the basis of the correlation between the measured parameters.

Effects of Copper Supplementation on the Structure and Content of Elements in Kidneys of Mosaic Mutant Mice

Menkes disease is an effect of ATP7A gene mutation in humans, coding the Cu-ATP-ase which is essential in intestinal copper absorption and its subsequent transfer to circulation. This mutation results in a deficiency of copper in all tissues except the epithelia of intestine and kidney tubules. Subcutaneous injection of copper ions is the main therapy for Menkes patients. Mosaic (Atp7amo-ms) mice closely simulate the situation in Menkes disease. The aim of this study was to evaluate the changes in structure and element content in kidneys of mosaic mice after copper supplementation. Hematoxylin–eosin staining was used to analyze tissue morphology and atomic absorption spectrometry to estimate Cu and Zn content. X-ray microanalysis was performed to measure Na, Mg, P, Cl, and K content in the cells of the proximal and distal tubules. Copper administration lengthened the lifespan of the mutants but led to its high accumulation and results in severe kidney damage. Karyomegalia, necrosis of tubular and Bowman’s capsule epithelium, lesions, and atrophy of glomeruli were observed in the treated mutants. Copper treatment afterwards led to sclerosis of glomeruli and tubules enhanced proliferation of epithelial cells and formation of both polycystic and papillary carcinoma patterns in kidney. We suggest that copper excess may impair the activity of Na+/K+ ATP-ase in renal tubules of ms/− males. The content of Mg, P, and Cl in kidneys in mutants was also changed after copper administration.

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

Summary: The mosaic (Atp7amo-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 strongsimilarities 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, alterationsin 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 renaltubules.

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.

Alterations in the expression of the Atp7a gene in the early postnatal development of the mosaic mutant mice (Atp7amo-ms) – An animal model for Menkes disease

Copper is a trace element that is essential for the normal growth and development of all living organisms. In mammals, the ATP7A Cu-transporting ATPase is a key protein that is required for the maintenance of copper homeostasis. In both humans and mice, the ATP7A protein is coded by the X-linked ATP7A/Atp7a gene. Disturbances in copper metabolism caused by mutations in the ATP7A/Atp7a gene lead to severe metabolic syndromes Menkes disease in humans and the lethal mottled phenotype in mice. Mosaic is one of numerous mottled mutations and may serve as a model for a severe Menkes disease variant. In Menkes patients, mutations in the ATP7A gene often result in a decreased level of the normal ATP7A protein. The aim of this study was to analyse the expression of the Atp7a gene in mosaic mutants in early postnatal development, a critical period for starting copper supplementation therapy in both Menkes patients and mutant mice. Using real-time quantitative RT-PCR, we analysed the expression of the Atp7a gene in the brain, kidney and liver of newborn (P0.5) and suckling (P14) mice. Our results indicate that in mosaic P0.5 mutants, the Atp7a mRNA level is decreased in all analysed organs in comparison with wild-type animals. In two week-old mutants, a significant decrease was observed only in the kidney. In contrast, their hepatic level of Atp7a tended to be higher than in wild-type mice. We speculate that disturbance in the expression of the Atp7a gene and, consequently, change in the copper concentration of the organs, may contribute to the early fatal outcome of mosaic males.

Sperm mitochondria diaphorase activity – a gene mapping study of recombinant inbred strains of mice

In order to study the genetic control of semen quality parameters, we derived a set of recombinant inbred (RI) mice from crosses between two inbred strains, KE and CBA/Kw, which differ significantly in gamete quality and fertility parameters. In this work, we used male mice from the two parental strains and from ten RI strains to map genes controlling quantitative traits such as sperm mitochondrial diaphorase activity, and assessed the correlation between this trait, sperm motility and in vivo fertilization efficiency. We analyzed sperm mitochondrial dehydrogenase (diaphorase) activity (NADH-dependent NBT assay) cytochemically by means of computerized image densitometry and obtained values for four parameters: 1) integrated optical density (IOD) for all pixels of the midpiece, 2) mean optical density (MOD) for the midpiece pixels, 3) length of sperm midpiece and 4) area of sperm midpiece. Polymorphic microsatellite marker profiles were prepared for 20 mouse chromosomes in the ten RI strains. We used Map Manager QTX software to correlate the strain distribution patterns (SDPs) of the four measured parameters with the SDPs of the analyzed markers. Hypothetical genes modifying diaphorase activity were mapped to chromosomal region 19q43-19q47, containing, for example, Poll, Sfxn2, Cyp17a1 and Usmg5 genes. Chromosomal regions 18q44 and 18q49-18q80 also showed correlation with the SDPs of diaphorase activity. Katnal2, Me2 and StARD6 candidate genes were proposed from this region. Diaphorase activity in the mouse sperm midpiece did not correlate with in vivo fertilization efficiency, but was negatively correlated with the linearity and straightness of sperm movement.

Impaired growth hormone-releasing hormone neurons ultrastructure and peptide accumulation in the arcuate nucleus of mosaic mice with altered copper metabolism.

A progressive decrease in body weight and retarded linear growth observed in mosaic male mice with the mutation linked to X-chromosome (Atp7a(mo-ms)) raised the question whether hypophysiotropic growth axis activity may be affected in these animals. A pathologically developed median eminence ultrastructure with very low somatostatin accumulation as well as an intensive phagocytosis of growth hormone cells observed in the anterior pituitary gland raised the question whether hypothalamic growth hormone-releasing hormone (GHRH) neuronal network is also affected in mosaic mice. In this study an arcuate nucleus GHRH neurons ultrastructure as well as GHRH peptide accumulation in normal and mutant mice were compared. An electron microscopic immunocytochemical method with colloidal-gold labeling was applied to compare the ultrastructural morphology of GHRH neuron and intracellular GHRH peptide distribution. Mosaic mice exhibited a pathologically developed ultrastructure of arcuate nucleus GHRH neurons, defective intracellular peptide localization as well as reduced peptide storage. Obtained results support the crucial role of unaltered copper metabolism in physiological development of hypophysiotropic growth axis activity. Consequently, a pathologically developed GHRH hypothalamic network may impact progressive decrease in body weight and retarded length growth observed in mosaic male mice.

Alternative splicing in the Atp7a gene in the Cu deficient mosaic mutation in mice.

The X-linked mosaic mutation in mice belongs to the mottled group of mutations. This group represents animal models of human copper deficiency disease, such as Menkes disease. It has been demonstrated that the disruption of copper metabolism is caused by a mutation in the Atp7a gene and leads to a lethal phenotype. Many similarities between mosaic and other mottled mutants give a strong indication that this mutation could occur in the cDNA of the Atp7a gene. In this paper, the cDNA of this gene was sequenced from 9 unrelated mutants and 7 unrelated control mice. It was found that a CAG insertion at the end of the 4th exon exists in the mutants but not in control cDNA. The same CAG insertion was previously described as a polymorphism in alternative splicing between BALB/c and C57BL/6 mice, therefore it is suggested that this changed sequence is a polymorphism strongly related to the phenotype rather than it is the cause of mutation. However, such a strong linkage between this polymorphism and the mosaic phenotype (lasting for 96 outbred generations), suggests that the mutation is in the Atp7a gene.