Changes in membrane potential during the cell cycle

The membrane potential of isolated synchronized Chinese hamster lung cells (V79) has been determined as a function of their position in the cell cycle. During G 1 the cells exhibit a low but increasing membrane potential which rises sharply at the onset of the S phase. The elevated membrane potential is maintained throughout S and G 2 and declines again when the cells enter mitosis. Membrane potentials in an unsynchronized culture, which was recorded from both mitotic and interphase cells physically associated in groups and clusters, were similar to the plateau level obtained during S and G 2 in isolated synchronized cells, and exhibited little variation. It is concluded that although the membrane potential of isolated cells fluctuates during the cell cycle, it plays no causal role as a regulator of mitotic activity.     

Analysis of human cytomegalovirus nucleoprotein complexes

When chromatin was isolated from cells infected with human cytomegalovirus, the virus DNA remained with the chromatin fraction. If deproteinized virus DNA was added to either isolated nuclei or chromatin, the DNA was lost during the chromatin isolation. When isolated chromatin from cytomegalovirus-infected cells was banded in isopycnic metrizamide gradients, a single peak with a density of 1.18 g/cm3 was present. Analysis of this peak in isopycnic neutral CsCl gradients indicated that it contained both human cytomegalovirus and human embryonic lung cell DNAs. When infected nuclei were treated with micrococcal nuclease, 11S subunit particles which cosedimented with cell nucleosomes and contained virus DNA were isolated.     

Acquisition of a beta-adrenergic response by adult rat hepatocytes during primary culture

In freshly isolated parenchymal hepatocytes of adult rats, the beta-adrenergic agonist isoproterenol (Ip) did not stimulate cAMP formation, protein kinase activity, or glycogenolysis, although glucagon markedly stimulated all these activities. However, the beta-adrenergic response appeared when rat hepatocytes were cultured as monolayers. This response had already appeared after 2-h culture and increased during further culture. The appearance of the beta-adrenergic response during culture was blocked by cycloheximide, actinomycin D, or alpha-amanitin. Thus adult rat hepatocytes acquired marked ability to respond to Ip during culture through the syntheses of mRNA and protein. Freshly isolated hepatocytes from postnatal rats showed a high beta-adrenergic response that did not increase further during culture. This response gradually decreased during development and had almost disappeared about 60 days after birth. In plasma membranes prepared from freshly isolated cells of adult rats the basal and NaF-stimulated activities of adenylate cyclase (EC 4.6.1.1) were similar to those of cultured cells and the enzyme activity was also stimulated by guanyl-5'-yl imidodiphosphate. However, in plasma membranes of freshly isolated cells Ip scarcely stimulated adenylate cyclase, but glucagon did. The intact cells, whether they were freshly isolated or cultured, accumulated cAMP when exposed to cholera toxin. Moreover, the two subunits of GTP-binding regulatory protein (also named G/F or Ns site) were detected by [32P]ADP ribosylation with cholera toxin and [32P]NAD+ in freshly isolated cells as well as in cultured cells. These results indicate that freshly isolated and cultured hepatocytes of adult rats contain sufficient levels of all the components of the postreceptor-adenylate cyclase system for activity. However, the number of beta-adrenergic receptors measured by binding of [125I]iodocyanopindolol, a potent beta-adrenergic antagonist, was very low in purified plasma membranes of freshly isolated cells (20 fmol/mg of protein), and the number increased about 6-fold without change in the dissociation constant (Kd = 132 pM) when the cells were cultured for 7 h. This increase in beta-adrenergic receptor sites was completely abolished by cycloheximide and alpha-amanitin. Thus it is concluded that the unresponsiveness of adult rat hepatocytes to Ip was due to a very low amount of beta-adrenergic receptor and that the appearance of a beta-adrenergic response during primary culture was due to new synthesis of beta-adrenergic receptor through synthesis of mRNA.     

An alternative methylation pathway in lignin biosynthesis in Zinnia.

S-Adenosyl-L-methionine:trans-caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT) is implicated in disease resistant response, but whether it is involved in lignin biosynthesis is not known. We isolated a cDNA clone for CCoAOMT in differentiating tracheary elements (TEs) induced from Zinnia-isolated mesophyll cells. RNA gel blot analysis showed that the expression of the CCoAOMT gene was markedly induced during TE differentiation from the isolated mesophyll cells. Tissue print hybridization showed that the expression of the CCoAOMT gene is temporally and spatially regulated and that it is associated with lignification in xylem and in phloem fibers in Zinnia organs. Both CCoAOMT and caffeic acid O-methyltransferase (COMT) activities increased when the isolated Zinnia mesophyll cells were cultured, whereas only CCoAOMT activity was markedly enhanced during lignification in the in vitro-differentiating TEs. The induction pattern of the OMT activity using 5-hydroxyferuloyl CoA as substrate during lignification was the same as that using caffeoyl CoA. Taken together, the results indicate that CCoAOMT is associated with lignification during xylogenesis both in vitro and in the plant, whereas COMT is only involved in a stress response in vitro. We propose that CCoAOMT is involved in an alternative methylation pathway in lignin biosynthesis. In Zinnia in vitro-differentiating TEs, the CCoAOMT mediated methylation pathway is dominant.     

Effect of Lipopolysaccharide on Progesterone Production during Luteinization of Granulosa and Theca cells In Vitro

The aim of this study is to examine the effect of lipopolysaccharide (LPS) on progesterone production during luteinization of granulosa and theca cells isolated from bovine large follicles. Granulosa and theca cells isolated from large follicles of bovine ovaries were exposed to LPS under appropriate hormone conditions in vitro. Progesterone (P4) production in theca cells, but not granulosa cells, was decreased by long‐term exposure of LPS. Long‐term exposure of LPS suppressed the gene expression of luteinizing hormone receptor in theca cells. Although long‐term exposure of LPS did not affect the expression of steroidogenic acute regulatory protein (StAR) and 3β‐hydroxy‐steroid dehydrogenase (3β‐HSD) genes, it did inhibit the protein expression of StAR and 3β‐HSD in theca cells. These findings suggest that theca cells, rather than granulosa cells, are susceptible to LPS during luteinization and that LPS inhibits P4 production by decreasing protein levels of StAR during luteinization of theca cells.     

Characteristics of insulin and glucagon release from the perfused pancreas, intact isolated islets, and dispersed islet cells

There are a variety of different tissue preparations which have been used to study secretion from the endocrine pancreas and there are considerable differences in the results obtained from these. The purpose of this study was to compare several preparations in one laboratory using the same rats, buffers, and radioimmunoassays. The preparations included the isolated perfused rat pancreas, fresh isolated intact islets and dispersed cells, and cultured islets and cells. Insulin release from the perfused rat pancreas at 2.8 mM glucose was so low that it could not be measured, such that over a 90-min time period the amount of insulin released was less than 0.004% of pancreatic insulin content. In contrast, islets in static incubation appear to release 2.0% of their stored content and dispersed cells appear to release 2.6% of their content. Samples were taken at early time points during incubations of fresh islets and dispersed cells, and it was found that almost all of the insulin found at the end of a 90-min incubation period was present during the first 5 min. It is therefore suspected that the true secretory rate of insulin at a low glucose concentration is far lower than had been generally appreciated. Glucagon release patterns showed similarities in that with isolated islets and dispersed cells a disproportionate amount of glucagon release was found during a 0- to 30-min incubation period when compared with the 30- to 90-min period. In summary, artifacts have been identified in some of the in vitro systems used for the study of endocrine pancreatic secretion and these deserve greater recognition.     

The increase of microRNA-21 during lung fibrosis and its contribution to epithelial-mesenchymal transition in pulmonary epithelial cells

Background

The excess and persistent accumulation of fibroblasts due to aberrant tissue repair results in fibrotic diseases such as idiopathic pulmonary fibrosis. Recent reports have revealed significant changes in microRNAs during idiopathic pulmonary fibrosis and evidence in support of a role for microRNAs in myofibroblast differentiation and the epithelial-mesenchymal transition in the context of fibrosis. It has been reported that microRNA-21 is up-regulated in myofibroblasts during fibrosis and promotes transforming growth factor-beta signaling by inhibiting Smad7. However, expression changes in microRNA-21 and the role of microRNA-21 in epithelial-mesenchymal transition during lung fibrosis have not yet been defined.

Methods

Lungs from saline- or bleomycin-treated C57BL/6 J mice and lung specimens from patients with idiopathic pulmonary fibrosis were analyzed. Enzymatic digestions were performed to isolate single lung cells. Lung epithelial cells were isolated by flow cytometric cell sorting. The expression of microRNA-21 was analyzed using both quantitative PCR and in situ hybridization. To induce epithelial-mesenchymal transition in culture, isolated mouse lung alveolar type II cells were cultured on fibronectin-coated chamber slides in the presence of transforming growth factor-β, thus generating conditions that enhance epithelial-mesenchymal transition. To investigate the role of microRNA-21 in epithelial-mesenchymal transition, we transfected cells with a microRNA-21 inhibitor. Total RNA was isolated from the freshly isolated and cultured cells. MicroRNA-21, as well as mRNAs of genes that are markers of alveolar epithelial or mesenchymal cell differentiation, were quantified using quantitative PCR.

Results

The lung epithelial cells isolated from the bleomycin-induced lung fibrosis model system had decreased expression of epithelial marker genes, whereas the expression of mesenchymal marker genes was increased. MicroRNA-21 was significantly upregulated in isolated lung epithelial cells during bleomycin-induced lung fibrosis and human idiopathic pulmonary fibrosis. MicroRNA-21 was also upregulated in the cultured alveolar epithelial cells under the conditions that enhance epithelial-mesenchymal transition. Exogenous administration of a microRNA-21 inhibitor prevented the increased expression of vimentin and alpha-smooth muscle actin in cultured primary mouse alveolar type II cells under culture conditions that induce epithelial-mesenchymal transition.

Conclusions

Our experiments demonstrate that microRNA-21 is increased in lung epithelial cells during lung fibrosis and that it promotes epithelial-mesenchymal transition.     

Protein synthesis by isolated pachytene spermatocytes in the absence of Sertoli cells

Isolated rat pachytene spermatocytes were incubated in chemically defined medium supplemented with pyruvate and lactate, which are known to be essential energy substrates for these germ cells. Protein synthesis by the isolated cells was investigated by means of two-dimensional polyacrylamide gel electrophoresis. The electrophoretic patterns of (35S)-labeled proteins, synthesized by the pachytene spermatocytes during incubation in the presence of (35S)methionine either from 0-2 h or from 24-26 h after isolation, were almost completely identical. The patterns of newly synthesized proteins of freshly isolated spermatocytes and spermatids, however, showed several stage-specific proteins in addition to many proteins common to both spermatogenic cell types. Hence, it was concluded that a stage-specific pattern of protein synthesis can be maintained by pachytene spermatocytes during incubation for a period of 24 h in the absence of Sertoli cells but in the presence of a proper energy source.     

Purification of 14C-labelled deoxyribonuclease II from HeLa S3 lysosomes and its use as a marker for the study of nuclear deoxyribonuclease II

Deoxyribonuclease II (DNAase II) in mammalian cells has generally been considered to be located in the lysosomes. Several recent studies have indicated that some DNAase II activity is present in purified nuclei; this, however, could have been due to some contamination of the nuclear fraction by lysosomes, or alternatively, it could have been caused by specific binding of lysosomal DNAase II to the nuclear fraction during isolation. Our previous studies have eliminated the possibility that lysosomal contamination was the cause of the presence of DNAase II in isolated nuclei. In this study I have purified (14)C-labelled lysosomal DNAase II and added it to cells during isolation of their nuclei. This study demonstrates that there is no specific binding of lysosomal DNAase II to the nuclear fraction and concludes that DNAase II activity observed in isolated nuclei represents an intrinsic activity that might be involved in nuclear DNA metabolism.     

Kinetic aspects of hemoglobin.haptoglobin-receptor interaction in rat liver plasma membranes, isolated liver cells, and liver cells in primary culture

125I-Hemoglobin.haptoglobin injected intravenously into rats was incorporated into liver parenchymal cells as evidenced by a cell separation technique. A mixture of freshly isolated liver parenchymal and nonparenchymal cells failed to internalize and degrade the 125I-hemoglobin.haptoglobin added, although it retained the ability to bind the molecule. The liver parenchymal cells in primary culture also lacked the ability to degrade 125I-hemoglobin.haptoglobin, although they bound the molecule more extensively as compared with the freshly isolated liver cells. It was confirmed that the 125I-hemoglobin.haptoglobin which was bound to the freshly isolated liver parenchymal cells localized on the outer surface of liver plasma membranes. Scatchard plots revealed the existence of two binding sites for 125I-hemoglobin-haptoglobin on the isolated liver plasma membrane: an apparent high affinity binding site (Kd = 1.3 X 10(-7) M) and an apparent low affinity binding site (Kd = 4.0 X 10(-6) M) at 37 degrees C. In contrast, freshly isolated liver parenchymal cells had only an apparent low affinity binding site (Kd = 1.4 X 10(-6) M) at 37 degrees C. Impairment of the apparent high affinity binding site during the isolation procedure with collagenase seemed to be related to loss of the ability to internalize and degrade the 125I-hemoglobin.haptoglobin molecules into the freshly isolated liver parenchymal cells or liver parenchymal cells in primary culture.     

Synthesis of adenosine triphosphate in isolated nuclei and intact cells

1. It has previously been demonstrated that nuclei isolated from normal and neoplastic lymphoid cells are capable of oxygen-dependent ATP synthesis. In this paper it is shown that also the corresponding intact cells can synthesize ATP under those conditions in which nuclei can synthesize ATP. 2. In nuclei isolated from liver, kidney, rhabdomyosarcoma and osteosarcoma, oxygen-dependent ATP synthesis could not be demonstrated. The cells isolated from these tissues or tumours could not synthesize ATP either. The alternatives that such nuclei lost their ability for oxidative phosphorylation during the isolation procedure or that the process does not occur in these nuclei were explored. 3. Janus Green B, a vital stain for mitochondria, was used as a differential inhibitor of mitochondrial and nuclear ATP synthesis in intact cells. 4. Oxidative phosphorylation in mitochondria isolated from cells that had been incubated with various concentrations of Janus Green B (1–10μm) was seriously uncoupled, whereas at these concentrations oxygen-dependent ATP synthesis in isolated nuclei and in isolated cells were only inhibited to a small extent. 5. The results suggest that oxygen-dependent ATP synthesis in isolated cells measured under `nuclear' conditions and in the presence of Janus Green B and Ca²⁺ is mainly due to nuclear oxygen-dependent ATP synthesis. The stimulation of cellular ATP synthesis by glucose was completely inhibited by Janus Green B. 6. It is tentatively concluded that the stimulation of ATP synthesis in isolated cells by glucose, which is not found in isolated nuclei, represents mitochondrial ATP synthesis, and nuclear and mitochondrial ATP synthesis can then be studied differentially in the intact cell. The possibility is considered that oxygen-dependent nuclear ATP synthesis is not a general property of cell nuclei.     

Development of a bioassay from isolated digestive gland cells of the cuttlefish Sepia officinalis L. (Mollusca Cephalopoda): effect of Cu, Zn and Ag on enzyme activities and cell viability

The purpose of this study was to establish a bioassay from isolated digestive gland cells of the cuttlefish Sepia officinalis in order to observe the effect of heavy metals on digestive enzyme activities. Digestive cells were isolated using a pronase enzyme that was removed by several washings of the cell suspension. Cell viability was tested by the MTT assay (3-4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium) and microscopic analysis. The results showed that isolated digestive cells could be maintained 24 h with preservation of whole digestive functionality, measured in terms of MTT test. In fact, the viability was maintained at a high level during 24 h and the intra- and extracellular digestive enzyme activities became stabilised rapidly. Furthermore, suspension cells responded to calcium ionophore and 8-Bromo-cAMP by an unspecific secretion of extracellular digestive enzyme, trypsin, which demonstrated that isolated digestive cells were functional. Using the bioassay, ecotoxicological studies showed that heavy metals could have effects on digestive enzyme activities after 24 h of an incubation time of the metal with the cells. In fact, zinc and silver affected trypsin and/or cathepsins specific activity of the cells. On the contrary, copper had no effect on digestive enzyme activities. Zinc, which is a trace element in all living animals, generated two different responses of cathepsins and cell viability. At a low concentration (0.02 μM), it increased viability and cathepsins specific activity, whereas at a high concentration (0.02 mM), zinc inhibited the cathepsins specific activity with an inhibition of cathepsins. For silver, whatever the tested concentration (0.02 mM or 0.02 μM), it has no impact on digestive gland isolated cell viability. Nevertheless, heavy metal induced high disturbance of enzymatic systems.     

小鼠骨髓间充质干细胞的分离、培养及鉴定

目的建立一种从小鼠骨髓中分离培养间充质干细胞（MSCs）的高效方法。方法采取贴壁细胞分离法分离和纯化小鼠骨髓间充质干细胞（mMSCs）,检测mMSCs在不同诱导条件下向成骨细胞及脂肪细胞分化能力,用流式细胞术及显微镜分别检测mMSCs纯度和形态特征。结果mMSCs贴壁生长后形态较均一,细胞形态呈成纤维细胞样,流式细胞术检测：CD45、CD11b、CD44及CD29分别为（3.34）%、（2.41）%、（98.46）%及（99.36）%。第4代mMSCs经诱导后可向成骨细胞和脂肪细胞分化。结论通过贴壁培养可以从小鼠骨髓中分离培养出高纯度mMSCs,该方法效率高,稳定性好。     

Cellular Commitment for Post-Gastrular Increase in Alkaline Phosphatase Activity in Xenopus laevis Development

Xenopus embryos were dissociated into cells and cultured in Ca²⁺-free medium to study the relationship between the cell-to-cell interaction and macromolecular synthesis. Under the conditions, cells did not aggregate at all, and remained isolated even while they were dividing actively. Synthesis of DNA and protein as studied by the incorporation of (³H)thymidine and (³H)leucine proceeded as in the aggregating cells. Also, the activity to synthesize rRNA, 5S RNA, and heterogeneous RNA as determined by the incorporation of (³H)uridine was not impaired. Such an increase in the activity of alkaline phosphatase, as occurs in embryos after the gastrula stage, was found to be inhibited greatly when early-blastula cells were cultured in the non-aggregating conditions. However, we found here that the inhibition was not observed with cells isolated from late-blastulae. Therefore, it appears that the increase in the activity of alkaline phosphatase during post-gastrular stages is dependent on some cellular commitment which may be established by cell-to-cell contact during the blastula stage.     

The bases for and timing of regional specification during larval development in Phoronis

A fate map has been constructed for Phoronis vancouverensis. The animal pole of the egg gives rise to the apical plate in the hood of the actinotroch larva. The vegetal pole of the egg marks the site of gastrulation. During the initiation of gastrulation the cells of the animal pole of the embryo are directly opposite those at the vegetal pole of the embryo. The plane of the first cleavage always goes through the animal-vegetal pole of the egg. In about 70% of the cases the plane of the first cleavage is perpendicular to the future anterior-posterior axis of the actinotroch larva; in the remaining cases the plane of the first cleavage is either oblique with reference to, or occurs along, the future anterior-posterior axis of the larva. Following gastrulation catecholamine-containing cells first make their appearance in the apical plate and gut cells first produce esterase. The timing of regional specification in these embryos has been examined by isolating animal or vegetal, anterior or posterior, or lateral regions at different time periods between the initiation of cleavage and gastrulation and examining their ability to differentiate. Animal halves isolated from early cleavage through late blastula stages do not gastrulate and do not form catecholamine-containing cells. When animal halves are isolated with endoderm during gastrulation, they differentiate catecholamine-containing cells. Vegetal halves isolated at the 8- to 16-cell stage gastrulate and form normal actinotroch larvae with esterase-positive gut and catecholamine-containing apical plate cells. When this same region is isolated at blastula stages it does not gastrulate and does not differentiate these cell types. Vegetal halves isolated during gastrulation subsequently form esterase-positive gut cells, but they do not form catecholamine-containing apical plate cells. When presumptive anterior, posterior, or lateral halves are isolated from early cleavage through blastula stages, each half forms a normal actinotroch larva. Lateral halves isolated during gastrulation also form normal larvae. Anterior halves isolated during late gastrulation differentiate only the anterior end of the actinotroch larva. These isolates have a hood with catecholamine-containing apical plate cells and the first part of an esterase-positive gut but lack the anlagen of the intestine and protonephridia. Posterior halves isolated during late gastrulation differentiate only the posterior end of the actinotroch which lacks a hood with catecholamine-containing cells but has an esterase-positive gut, protonephridia, and the anlagen of the intestine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Use of a selectively permeabilized isolated rat hepatocyte preparation to study changes in the properties of overt carnitine palmitoyltransferase activity in situ.

1. A permeabilized isolated rat liver cell preparation was developed to achieve selective permeabilization of the cell membrane to metabolites and to allow the assay of mitochondrial overt carnitine palmitoyltransferase (CPT I) activity in situ. By performing the digitonin-induced permeabilization in the presence of fluoride and bivalent-metal-cation sequestrants, it was possible to demonstrate that the activity of other enzymes, which are regulated by reversible phosphorylation, was preserved during the procedure and subsequent washing of cells before assay. 2. CPT activity at a sub-optimal palmitoyl-CoA concentration was almost totally (approximately 90%) inhibited by malonyl-CoA, indicating that mitochondrial CPT I was largely measured in this preparation. 3. The palmitoyl-CoA-saturation and malonyl-CoA-inhibition curves for CPT activity in permeabilized cells were very similar to those obtained previously for the enzyme in isolated liver mitochondria. Moreover, starvation and diabetes had the same effects on enzyme activity, affinity for palmitoyl-CoA and malonyl-CoA sensitivity of CPT I in isolated cells as found in isolated mitochondria. These physiologically induced changes persisted through the cell preparation and incubation period. 4. Neither incubation of cells with glucagon or insulin nor incubation with pyruvate and lactate before permeabilization resulted in alterations of these parameters of CPT I in isolated cells. 5. The results are discussed in relation to the temporal relationships of changes in the activity and properties of CPT I in vivo in relation to the effects of insulin and glucagon on fatty acid metabolism in vivo.     

Morphological observations on the formation and stability of the crystalline arrays in the plasma membrane of Saccharomyces cerevisiae

Two-dimensional crystalline arrays of freeze-fracture particles are known to occur in abundant quantities in the plasma membrane of stationary state yeast cells. Although these crystalline arrays are seen only infrequently in cells during mid-exponential growth, we now observe that formation of crystalline arrays can be induced in such cells by a “metabolic starvation” protocol. Surprisingly, starvation-induced formation of crystalline patches can be prevented by inhibition of new protein synthesis during the starvation period. The size and quantity of crystalline arrays can be increased by removal of the cell wall prior to starvation. Induction of crystalline arrays in protoplasts has made it possible to investigate the surface morphology of the crystalline particles in isolated membranes as well as at the extracellular surface of intact protoplasts. The stability of isolated crystalline arrays to several detergents has been investigated and conditions have been found that result in improved morphological purity of the isolated crystalline patches.     

The presence of professional phagocytes dictates the number of host cells targeted for Yop translocation during infection

Type III secretion systems deliver effector proteins from Gram‐negative bacterial pathogens into host cells, where they disarm host defences, allowing the pathogens to establish infection. Although Yersinia pseudotuberculosis delivers its effector proteins, called Yops, into numerous cell types grown in culture, we show that during infection Y. pseudotuberculosis selectively targets Yops to professional phagocytes in Peyer's patches, mesenteric lymph nodes and spleen, although it colocalizes with B and T cells as well as professional phagocytes. Strikingly, in the absence of neutrophils, the number of cells with translocated Yops was significantly reduced although the bacterial loads were similar, indicating that Y. pseudotuberculosis did not arbitrarily deliver Yops to the available cells. Using isolated splenocytes, selective binding and selective targeting to professional phagocytes when bacteria were limiting was also observed, indicating that tissue architecture was not required for the tropism for professional phagocytes. In isolated splenocytes, YadA and Invasin increased the number of all cells types with translocated Yops, but professional phagocytes were still preferentially translocated with Yops in the absence of these adhesins. Together these results indicate that Y. pseudotuberculosis discriminates among cells it encounters during infection and selectively delivers Yops to phagocytes while refraining from translocation to other cell types.     

Isolation of two populations of sperm cells and microelectrophoresis of pairs of sperm cells from pollen tubes of tobacco (Nicotiana tabacum)

Prior research has indicated that the two sperm cells of Nicotiana tabacum are dimorphic, suggesting that they may participate in preferential fertilization during in vivo fusion with the egg and central cells. To probe the mechanism of potential preferential fertilization in this plant, it will be necessary to use modern sensitive molecular techniques. For this purpose, two individual populations of two sperm cells, constituting the Svn (associated with the vegetative nucleus) and Sua (unassociated with the vegetative nucleus), were isolated in the thousands from tobacco pollen tubes with a micromanipulator as a preliminary step toward research on gametic recognition using molecular techniques. Microelectrophoresis of paired sperm cells from a single pollen tube was conducted at different developmental stages. Sperm cells isolated from 1-, 2-, 3- and 4-cm stylar lengths migrated to the negative pole, with the Sua displaying significantly greater electrophoretic mobility than the Svn, reflecting a more positively charged cell surface on the Sua. The sperm cells isolated from 1-cm style are very sensitive to electron potential in an electrophoretic field, presumably reflecting that they are still in a young state. Differences in cell surface charge between the Sua and Svn may be related with cell fate during fertilization. Supported by National Natural Science Foundation of CHINA (30170060)     

Expression of inhibin α-subunit gene during mouse gametogenesis

Mammalian gametogenesis is regulated through complex interactions between germ and somatic cells. To investigate the mechanism underlying the differentiation of functional gametes, some genes specifically expressed during gametogenesis have been isolated and characterized. In a search for further examples of such genes, we have isolated from a newborn mouse testis cDNA library, a clone corresponding to mouse inhibin alpha-subunit. Although it is known that the inhibin alpha-subunit molecule is abundantly produced in ovarian follicle and in testicular Sertoli cells, the spatial and temporal patterns of expression of this gene remain to be elucidated. In this study, the patterns of expression of inhibin alpha-subunit mRNA during mouse gametogenesis were examined by RNA blot, cytoplasmic dot and in situ hybridization techniques. In the testis, the concentration of inhibin alpha-subunit mRNA increased from about 16 dpc (days post coitum), peaked at birth and then gradually decreased, paralleling testicular development. Inhibin alpha-subunit mRNA was localized in Sertoli cells of wild type as well as W/Wv testes. In adult testis, mRNA was restricted to the perinuclear cytoplasm of Sertoli cells. Inhibin alpha-subunit mRNA was expressed in follicle cells of adult ovary more abundantly than in adult testis. Analysis of expression during folliculogenesis showed that the accumulation of this mRNA began in preantrum follicles and the level of expression reached a maximum in Graafian follicles.