Sperm aging in the male after sexual rest: Contribution to chromosome anomalies

The chromosome complement was studied in first-cleavage metaphases of mouse zygotes resulting from sperm aged in the male physiologically, after sexual rest. Females were inseminated by control males mating at 3-day intervals while experimentals mated to males that had had a sexual rest of 14 or more days. A total of 1954 eggs were collected 33–35 h post-HCG from 101 superovulated females mated to 42 controls and 43 experimental males. The fertilization rate was similar in both groups, being 84% and 85%, respectively. G-banded or Q-banded chromosomes were analyzed in 301 (68.3%) controls and 392 (49%) experimental first-cleavage metaphases. The overall rate of chromosome anomalies in controls was 4.45% as compared to 10.94% in experimentals, a highly significant difference. In the experimental group compared to controls, the frequency of trisomy, triploidy, structural rearrangements, and tetraploidy increased from 3.9% to 6.9%, 0% to 1.6%, 0.8% to 2.8%, and 0% to 1.3%, respectively. The genomic source of origin of the abnormalities was determined on the basis of differential condensation of the genomes. In the experimentals, grossly unbalanced sperm (diploids, disomics, double disomics, and those with large fragments) fertilized significantly more oocytes compared to controls. Our results implicate an advantage either in numbers or fertilizing capability for chromosomally abnormal sperm in a physiologically aged population.     

Murine “housekeeping” enzyme (genetic locus:Idh-1) is regulated in an allele-specific manner

The murine “housekeeping” enzyme, cytosolic NADP-isocitrate dehydrogenase (E.C. 1.1.1.42) (genetic locus:Idh-1), exhibited a complex pattern of allele-specific expression. Protein electrophoresis on cellulose-acetate gels and determination of relative enzymatic activity by means of densitometry revealed that in heart tissue (but not liver tissue) of certain hybrid crosses the AA-homodimer was underrepresented relative to total enzymatic activity, and the degree of underrepresentation changed during development. In mixtures of homozygous tissue extracts of heart tissue (but not liver tissue) the AA-homodimer was underrepresented relative to the BB-homodimer. Relative activity of allelic isozymes varied as a function of tissue (heart versus liver), age, and the parental source of the Idh-1^a allele, but did not vary as a function of sex. Allele-specific expression was also exhibited in kidney tissue of the same animals. In adult male kidney tissue extracts from heterozygotes, the AA-hornodimer was underrepresented relative to total enzymatic activity; in adult female kidney tissue extracts from heterozygotes, a more codominant phenotype was observed. Tissue extracts from immature hybrid animals exhibited a phenotype midway between the adult male and adult female phenotypes. Tissue extracts from castrated males exhibited a phenotype equivalent to that seen in females. Relative activity of allelic isozymes in kidney varied as a function of age and sex, but did not vary as a function of the parental source of the Idh-1^a allele. While cytosolic NADP-IDH is a “housekeeping” enzyme, expressed in multiple tissues of the mouse, differences in the relative intensities of allelic isozyme bands provide evidence for tissue- and stage-specific regulatory variation.     

Toxicity and mutual interactions of cadmium and zinc ions in normal and carcinogen-transformed mouse cells

The toxicity of chloride salts of physiological (zinc, manganese, nickel) and non physiological (cadmium) bivalent metal ions was studied in normal or carcinogen-transformed mouse embryo fibroblast cells. The dose response curves for toxicity to both types of cells exhibited similar shapes. The transformed cells, however, were about twice as sensitive to zinc toxicity as normal cells. When normal and transformed cells were grown together and incubated for several hours with an appropriate concentration of zinc, the malignant cells were selectively killed. Cadmium was much more toxic than the three other metal ions in both types of cells. Its toxic effect was reversed by simultaneous addition of zinc at nontoxic concentrations.Abbrevications CFA colony forming ability - MCA 3-methylcholanthrene     

Cerebroside and Sulfatide Biosynthesis in the Brain of Snell Dwarf Mouse: Effects of Thyroxine and Growth Hormone in the Early Postnatal Period

Snell dwarf mice (dw/dw) and normal mice (+/?) were injected with thyroxine (T₄) (1 μg/animal, four injections) and growth hormone (GH) (20 μg/animal, four injections) from the 5th to the 15th day of life. In the untreated dw/dw mouse brain, the specific activities of UDP-galactose:ceramide galactosyltransferase (CGalT), PAPS:cerebroside sulfotransferase (CST), and 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) were decreased by 28, 25, and 37%, respectively, compared with the control untreated +/? mice. The major effect of T₄ was an increase of the brain CNP in the +/? mice (+40%) and dw/dw mice (+111%). The treatment with T₄ also brought to normal the level of CGalT in dw/dw brain; a somewhat less marked effect on CST was observed. The treatment with GH had a great stimulatory effect on CNP: the specific activity of this enzyme increased by 40 and 69% in +/? and dw/dw mouse brain, respectively. On the contrary, no effect of GH on the CGalT activity was observe in this study. Our results suggest that T₄ and GH may have both independent and complementary actions on the myelin-associated enzymes during the early postnatal period of brain development.     

Failure of spermatozoa from T/t mice to fertilize in vitro is overcome by zona drilling

Failure of epididymal spermatozoa from T/t mutant mice, but not from t/t individuals, to fertilize oocytes in vitro was partially overcome by opening a small aperture in the zona pellucida with acidified Tyrode's solution to permit direct access of the spermatozoon to the vitellus. This study provides a model system to evaluate requirements for successful zona drilling in the treatment of human infertility and further insights into the effects of the t complex on sperm fertility.     

Ultrastructural and histochemical changes in the murine zona pellucida during the final stages of oocyte maturation prior to ovulation

The ultrastructural morphology of the mouse zona pellucida was studied in preovulatory follicles from the ovaries of immature mice treated with exogenous gonadotrophins. The ovaries were fixed in the presence of cetylpyridinium chloride, which precipitates carbohydrates, so that their loss during fixation and processing is substantially reduced. The semi-thin araldite sections obtained from osmicated material were viewed by conventional light microscopy, while the ultra-thin sections were examined by transmission electron microscopy. A parallel series of semi-thin sections of non-osmicated ovaries was deresined and subsequently stained with periodic acid Schiff (PAS). The morphological appearance of the zona pellucida in preovulatory oocytes changed during the final stages of oocyte maturation. A close correlation was observed between the ultrastructural appearance of the zona pellucida and that observed following PAS staining during the period studied. Real differences were observed in the location, density, and distribution of glycoconjugates within the zona pellucida during the final stages of oocyte maturation prior to and immediately following germinal vesicle breakdown. Similar changes in the zona were observed in adult females autopsied during proestrus and oestrus. The changes in the density and distribution of glycoconjugates are likely to have important consequences for fertilization by affecting sperm-zona binding and sperm-egg interactions.     

Pregnant mares' serum gonadotropin source influences fertilization and fresh or thawed embryo development,but the effect is genotype specific

The influence of the source of pregnant mares' serum gonadotropin (PMSG) on the num ber, quality, and in vitro development of mouse embryos before and after freezing was evaluated among three genotypes: N:NIH(S), C57BL/6N, and C3H/HeN-MTV^?. Immature females were given PMSG from one of five commercial sources. Following col lection ( 116 hr later), embryos were evaluated for stage of development, and four-to eight-cell embryos were pooled within genotype and assigned to standardized fresh or freeze-thaw culture trials. Different PMSG sources stimulated the production of different num bers of total embryos (P < 0.05) but not necessarily more embryos suitable for freezing. Differences in embryo production among genotypes indicated that absolute embryo num bers using a single mouse genotype may not accurately reflect the potency of a specific gonadotropin source. The PMSG source also affected the ability of an embryo to survive in culture either immediately after collection or after frozen storage. The effect, however, was genotype specific, with some mouse strains being relatively insensitive to PMSG source, whereas gonadotropin source played a major role in determining in vitro viability in others. Development rates for freshly collected embryos differed, often inconsistently, from those of thawed embryos regardless of the PMSG source used, demonstrating that fresh embryo development cannot be used to estimate expected post-thaw survival. In vitro development of thawed embryos is influenced not only by genotype, but also the source of the gonadotropin used to promote follicular development and oocyte maturation. These findings may explain, in part, the wide variation in embryo viability and culture rates reported among laboratories and intraspecies animal populations.     

Radioactive labeling of proteins in cultured postimplantation mouse embryos II. Dose and time dependency

Summary The conditions for optimum incorporation of radioactive amino acids into proteins of cultured postimplantation mouse embryos were investigated under the aspect of using these proteins for two-dimensional electrophoretic separations and fluorography. The aim was to obtain highly radioactively labeled proteins under conditions as physiological as possible. Mouse embryos of Days 8, 10, and 11 of gestation were cultured in Tyrode’s solution. Incubation time and concentration of [³H (or¹⁴C)]amino acids in the culture medium were varied over a broad range. Embryos were prepared with placenta and yolk sac or without any embryonic envelopes. After culturing, the physiologic-morphologic state of the embryos was registered on the basis of several criteria. The radioactivity taken up by the total protein of each embryo was determined and calculated in disintegrations per minute per milligram protein per embryo. To approach our aim, embryos of different developmental stages had to be cultured under different conditions. A good compromise for Day-8, Day-10, and Day-11 embryos was: embryos prepared with yolk sac (opened) and placenta, 150 μCi radioactive amino acids added per milliliter medium, incubation for 4 to 5 h. For maximum labeling of proteins it is advisable to culture Day-10 embryos without embryonic envelopes under particular conditions. This work was supported by grants from the Deutsche Forschungsgemeinschaft awarded to the project K1 237/3-2 (Systematic analysis of cell proteins).     

Influence ofl-thyroxine,l-triiodothyronine,thyroid stimulating hormone,or estradiol on the cell kinetics of cultured mammary cancer cells

Summary We describe the in vitro influence of 3,5,3′-triiodo-l-thyronine (T₃),l-thyroxine (T₄), a thyroid-stimulating hormone (TSH), and/or estradiol (E₂: chosen as the control of the methodology) on the cell kinetics (cell distribution in the S+G₂+M phases) of mouse MXT and human MCF-7 mammary cancer cells. Experiments were performed by means of a cell image processor, analyzing MCF-7 or MXT cells that had been grown on glass cover slips and whose nuclei had been stained by the Feulgen reaction, which is selective and quantitative (stoichiometric) with respect to DNA. We show that T₃, T₄, and TSH at 0.01 μM dramatically stimulate the cell kinetics of the MXT mouse and the MCF-7 human mammary cancer cell lines. Indeed, the three hormones bring about a significant transient increase in the S+G₂+M fraction as does E₂. Furthermore, our data indicate that E₂ and TSH are antagonistic with regards to MXT or MCF-7 cell kinetics. This work is supported by grants awarded by the IRSIA and the Fonds de la Recherche Scientifique Médicale (FRSM, Belgium).