Cell surface changes in preimplantation mouse embryos during compaction investigated using FITC conjugated lectins after proteolytic enzyme treatment

Summary Fluorescein-conjugated lectins were used to examine the reappearance of glycoproteins on the surface of 8-cell mouse embryos after treatment with proteolytic enzymes. Embryos were decompacted in calcium free medium, treated with various proteases and the process of recompaction monitored. The most effective enzymes in delaying recompaction were subtilopeptidase A and proteinase K at 1 mg/ml; the initiation of recompaction was delayed by about 5 h and 90% recompaction by 14–18 h. Papain and -chymotrypsin were only effective in the absence of calcium. The reappearance of receptors for fluorescein-conjugated Con-A, MPA, RCA-I, FBP, BSL-II and DBA was examined photometrically at 0,8–10 and 17–18 h after proteinase K treatment. There was an increase in binding of MPA, RCA-I, FBP and BSL-II in control embryos during the period of the experiment, between approx. 61 and 80 h post coitum in which embryos passed from the 8-cell stage to the 16–32 cell stage. Con-A binding remained the same and that of DBA decreased. By the time that 50% of enzyme treated embryos had recompacted (8–10 h) binding of Con-A was similar to control embryos. Binding of FBP had almost reached control levels while that of BSL-II, DBA, RCA-I and MPA had reached 60–85% of control levels. When embryos were fully compact (17–18 h) Con-A, FBP and DBA were bound in equal or slightly greater amounts to enzyme treated as to control embryos, and receptors for BSL-II, MPA and RCA-I had recovered almost to control levels. The results clearly show that the recovery of glycoproteins on the surface of 8–16 cell embryos parallels recompaction, providing further evidence for the role of these molecules in compaction.     

胎肝细胞输注与全胚注射液治疗再生障碍性贫血的机理探讨

本文对胎肝细胞输注或全胚注射液治疗再生障碍性贫血的可能机理作了一些实验性探讨。研究结果表明: 1.胎肝细胞在培养或解体过程中释放某些刺激红系造血的因子,有利于已经损伤的造血功能的恢复。 2.对正常小鼠注射无细胞胎肝制剂或全胚注射液后,骨髓红系细胞的分裂指数明显升高,骨髓中粒/红比值趋于降低,反映了骨髓中红系细胞增生活跃的状态。 3.对正常小鼠注射无细胞胎肝制剂或全胚注射液后,外周血网织红细胞和腹腔巨噬细胞的吞噬指数趋于平行增高,其增高程度和持续时间随注射次数的增加而加强。 小鼠注射无细胞胎肝制剂或全胚注射液后,巨噬细胞吞噬指数的增加,反映了巨噬细胞激活,这种作用除了提高机体的非特异性免疫功能,增强机体的抵抗力外,还可能通过巨噬细胞的活化,直接或间接地调控机体红系细胞的增殖,因而,对巨噬细胞在造血调控中的作用以及它在再生障碍性贫血发病机理研究中的意义提出了讨论。     

Effects of fructose on human fibroblast metabolism: the application of DNA measurements as a basis for interpretation

Summary A fluorometric procedure for measuring DNA was used to study growth and metabolic responses of eight cell strains of human foreskin fibroblasts. In preliminary studies this procedure gave more precise specific activity changes inN-acetyl-β-d-glucosaminidase (NAG) than did a protein activity basis, when changes in this enzyme's specific activity were investigated as a function of experimental cell manipulation. When fibroblast growth in eight cell strains was compared in 134 mM d-fructose vs. 13.4 mM glucose-supplemented minimum essential media, a significant increase in cellular DNA (50%) and protein (45%) occurred over an 11-d period. No significant differences in media pH change, lactate production, or carbohydrate uptake occurred on a DNA basis when cell metabolism was compared over the last 24 h of culture in the two media. Cells grown in fructose-containing media tended to show a reduction in NAG specific activity when compared with those grown in glucose-containing media.     

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.     

The human pronuclear ovum: Fine strcture of monospermic and polyspermic fertilization in vitro

The fine structure of pronuclear ova (monospermy and polyspermy) and one-cell embryos has been investigated in our IVF programme. Sixteen oocytes were collected at laparoscopy after appropriate hormonal stimulation and were matured and fertilized in vitro by methods that have given rise to normal pregnancies. Pronuclear ova showing monospermic fertilization had two vesicular pronuclei surrounded by aggregations of cellular organelles. The male pronucleus was closely associated with a sperm axoneme, while the female pronucleus was dismantling its envelope and condensing its chromatin ahead of its counterpart in late pronuclear ova. Each pronucleus had dispersed chromatin, dense compact nucleoli, and intranuclear annulate lamellae. Smooth endoplasmic reticulum, annulate lamellae, Golgi complexes, and mitochondria formed a conspicuous part of the perinuclear ooplasm. The one-cell embryos were either in syngamy or in the process of undergoing first cleavage. Positive evidence of cortical granule release and second polar bodies were detected in the perivitelline space. A block to polyspermy seemed to operate at the level of the inner zona. Dispermic and polyspermic ova had 3–16 pronuclei resembling those of monospermic ova and had sperm tails in the ooplasm. Sperm were also seen penetrating the inner zona and were occasionally found in the perivitelline space. Incomplete cortical granule release and early signs of cytoplasmic fragmentation were noted in polyspermic ova. Both normal and abnormal features of these ova are reported and compared with pronuclear structure in vivo and in vitro.     

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.     

Abscisic acid induction of cloned cotton late embryogenesis-abundant (Lea) mRNAs

Summary Earlier studies found that cotton (Gossypium hirsutum L.) cotyledons contain several mRNAs which are more abundant during late embryogenesis than in mid-embryogenesis or early germination. They are here termed Late embryogenesis-abundant mRNAs, encoded by Lea loci. Complementary DNA clones for 18 such mRNA sequences, defined at a hybridization criterion of T_m-15°C, were identified in a mature embryo cDNA library by differential cDNA hybridization. At a lower hybridization criterion, some sequence homology was found within several of these cloned Lea mRNA sequences. Each Lea mRNA sequence comprises 0.04–1.3% of mature embryo poly(A)⁺ mRNA, a level ten-fold to several hundred-fold higher than in young embryo or 24 h seedling poly(A)⁺ mRNA. Of 18 Lea mRNA sequences examined in cultured young embryos, the level of at least 13 are specifically increased by exogenous abscisic acid (ABA), several to a level near that in normal mature embryos. However, the abundance of several of the sequences does not appear to be significantly modulated by ABA. The LEA polypeptides encoded by 10 Lea mRNA sequences were identified by hybrid-arrested translation. They include most of the late embryogenesis-abundant, ABA-inducible, polypeptides previously identified. Preliminary results suggest that many of the individual Lea mRNA sequences are transcribed from 1–3 genes in each of cotton's two subgenomes.     

The use of embryo culture for the recovery of plants from cassava (Manihot esculenta Crantz) seeds

Cassava fertility and seed viability are frequently low, which can be a disadvantage in a breeding programme. An embryo culture method is described whereby embryonic axes are excised from mature seeds and placed on a culture medium containing 1.23 M indolebutyric acid (IBA) at 30°C under continuous light. The number of plants recovered by embryo culture was much greater than the number recovered from conventional seed germination procedures.     

Ontogenetic development of corticotropin-releasing factor (CRF)-containing neural elements in the brain of the chicken during incubation and after hatching

Summary In chicken embryos of different ages and in young chickens after hatching, neural elements reacting with antibodies generated against synthetic ovine corticotropin-releasing factor (CRF) were studied by means of the peroxidase-anti-peroxidase (PAP) technique at the lightmicroscopic level. CRF-immunoreactivity was first observed in perikarya located in the periventricular part of the hypothalamus on the 14th day of the incubation period. CRF-containing neural elements were detected on the same day of incubation in the external zone of the median eminence, but not in all investigated animals. In extrahypothalamic sites, immunoreactive perikarya were demonstrable in the central gray of the mesencephalon on the 15th day of incubation. Furthermore, immunoreactive cells appeared in other brain regions such as nucleus accumbens and dorsomedial nucleus of the thalamus after hatching. The present observations provide information regarding the functional development of the hypothalamo-hypophyseal-adrenal axis in the chick embryo.     

Embryonic cholinesterase activity during morphogenesis of the mouse genital tract

Summary In the genital tract of male and female mouse embryos cholinesterase activity is described that is independent from innervation. The enzyme activity is localized in the mesenchyme at the junction of Wolffian and Müllerian ducts with the urogenital sinus. During male development prostate buds and vesicular glands grow out into the cholinesterase-active mesenchyme. During female development the active mesenchyme participates in the downgrowth of the vaginal anlage. Ultrastructurally the cholinesterase activity is localized in the perinuclear cisterna and in smooth endoplasmic reticulum of the mesenchymal cells. The enzyme activity disappears with definitive differentiation of the tissue. The embryonic cholinesterase is a component of a primitive muscarinic system. Its relation to the morphogenetic action of testosterone and its possible general functions are discussed.