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).     

The Large Isoform of Myelin-Associated Glycoprotein Is Scarcely Expressed in the Quaking Mouse Brain

Two polypeptide isoforms of myelin-associated glycoprotein (MAG) with molecular masses of 72 and 67 kDa are produced by alternative splicing of the exon 12 portion. Our previous work has demonstrated that in the quaking mouse brain this alternative splicing is lacking and that the mRNA coding the large MAG isoform (L-MAG) is scarcely expressed, whereas that of small MAG isoform (S-MAG) is overexpressed. In the present study, we prepared antisera specific to the S-MAG and L-MAG amino acid residues, respectively. Immunoblots showed that the L-MAG band was scarcely detectable in the quaking mouse brain, whereas the S-MAG band had an apparently higher molecular mass than in the normal control. Our immunohistochemical study also showed that L-MAG was scarcely stained in the quaking mouse brain. These results seemed to reflect a reduction in content of L-MAG mRNA and abnormal glycosylation in the quaking mouse brain.     

Alteration of opioid receptors in seizure-susceptible El mouse brain

The distribution density of opioid receptors in the brain of El mice (seizure-susceptible strain) was examined to determine the relation between seizures and the opioid system. Saturation curves and Scatchard plots of [³H]2-d-alamine-5-d-leucine enkephalin binding revealed that the opioid delta receptor density in adult El mice during interictal periods was significantly increased in the cerebral cortex, hippocampus, and septal area. It was further shown that the concentration of such receptors in 25-day-old El mice that had no seizures was also significantly increased in the hippocampus and septal area, with no changes in apparent affinities, as compared with in the corresponding regions in ddY mice (seizure-nonsusceptible strain; the mother strain of El). Such up-regulation of opioid receptors in the El mouse brain could result from deficits in endogenous opioid peptides, which could be associated with the pathogenesis of seizure diathesis in the El mouse.     

Factors contributing to cerebral hypomyelination in the growth hormone-deficientlittle mouse

We attempted to delineate the events leading to hypomyelination in the brain of thelittle mouse, a promising murine model of isolated growth hormone deficiency. At 20 days of age, the mutant mouse brain weighed less than its normal counterpart, and this difference in brain weight persisted. Increase in CNPase activity was found to be suppressed in the cerebrum throughout the developmental stage, but not in the other parts of the brain. Differences in cerebral DNA content between thelittle and normal mice first became apparent on the 10th day of age. Thereafter, the rate of increase in thelittle brain consistently lagged behind the normal. [³H]Thymidine incorporation into the DNA fraction in vivo on the 7th day of age, when glial cell proliferation in the normal cerebrum is most active, was approximately half that of the controls in all parts of thelittle brain. These findings indicate that the hypomyelination of the mutant cerebrum might result from reduced oligodendroglial proliferation due to growth hormone deficiency.     

Metabolism of exogenous galactosylceramide in the twitcher mouse brain

The in vivo metabolism of galactosylceramide (gal-cer) in normal mice and in twitcher mice, a model of human GLD, was examined following intracerebral administration of gal-cer containing [1-¹⁴C]stearic acid. In normal mice, gal-cer was hydrolyzed to ceramide within 6 hours and ceramide was hydrolyzed to sphingosine and fatty acid. Most of the released fatty acid was immediately incorporated into other lipids. About 75% of injected gal-cer was hydrolyzed 80 hours after the injection, while in the twitcher mouse, only 17% of gal-cer was hydrolyzed. These results show that degradation of gal-cer is impaired in the twitcher mouse brain, but contradict to the fact that there was no evidence of any accumulation of gal-cer in the brain. This discrepancy may be due to the different sorting routes of biosynthesized and exogenously-administered gal-cer in the mouse brain. Most of the biosynthesized gal-cer is incorporated into myelin, while the injected gal-cer is incorporated into lysosomes.     

海南大田自然保护区海南坡鹿种群动态研究

笔者自1986年起对仅存于海南大田国家级保护区围栏内外两个坡鹿种群进行了连续4年追踪研究,就数量、年净增长率、年均增长率、年龄组成、性比、成幼比、母仔比、及补充率和死亡率进行了分析。围栏内外坡鹿年均增长率22.5％和10.6％,年净增长率逐年呈下降趋势。补充率下降是围栏内坡鹿年净增长率下降的主要原因,而这是由于密度过高、食物不足造成的;围栏外坡鹿年净增长率下降是死亡率增加的结果,偷猎是主要因素。对性比、成幼比、母仔比变化的分析也得出了同样的结论。本文最后提出了保护建议。     

Concentrations of essential elements after repeated administrations of tin and selenium

To study effects of simultaneous administration of tin (Sn) and selenium (Se) on concentrations of several essential elements, mice were injected with either SnCl2 (ip) or Na2SeO3 (sc), alone or both compounds at a daily dose of 5 mumol/kg each for 12 consecutive days. Mice were sacrificed at 20 h after the last injection and concentrations of Sn, Se, Na, Ca, Zn, P, Fe, K, and Mg in the liver, kidney, spleen, pancreas, testis, seminal vesicle, lung, femoral muscle, and femoral bone were determined. In the control mice, Sn and Se concentrations were the highest in bone (0.69 micrograms Sn and 6.93 micrograms Se/g dry wt). Administered Sn was found to accumulate in all organs except the testis. Among the essential elements determined, Na was the most affected in terms of concentration in the organs and Mg was the least affected element in these organs. Among the organs tested, each elemental concentration in the pancreas was most affected. Simultaneous injections of Sn and Se appeared to keep the correlation coefficients between elements similar to those found in the control mice.