摘要:

AbstractAs a first step toward understanding control of gene expression in early human development, an analysis of protein synthesis and amino‐acid transport in unfertilized mature oocytes was initiated. Qualitative patterns of protein synthesis were examined in individual oocytes cultured in medium containing radiolabeled methionine. No differences in synthetic pattern of proteins, resolved by one‐dimensional electrophoresis and fluorography, were observed in oocytes analyzed from times varying from 12 to 52 hr following collection by laparoscopy. Contamination by follicular or corona radiata cells was readily distinguished on the basis of increased relative synthesis of a polypeptide with Mr= 44,000, a dominant product of synthesis in follicular cells. Based on the specific activity of the methionine precursor, the absolute rate of synthesis was measured to be about 50 pg/oocytc/hr, a value higher than in mouse unfertilized eggs. No difference in protein synthetic rate was observed in oocytes analyzed at 12 hr postcollection versus later times up to 50 hr postcollection. Competition of methionine uptake by leucine, efflux of radiolabeled methionine from preloaded oocytes into medium containing methionine and uptake of methionine in medium with low sodium ion concentration was observed. These findings are consistent with the presence of an L (leucine‐preferring) system for neutral amino acid transport, similar to that in mouse and rabbit eggs. Total protein was measured to be about 150 ng/oocyte, a value five times that of the mouse. These studies provide basic data for further analysis of oocytes and perhaps preimplantation stage embryos in the f

ISSN:0148-7280    

DOI:10.1002/mrd.1120180202

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractWe have investigated the ability of the hamster oocyte to initiate DNA synthesis in nuclei differing in basic protein content. DNA synthesis was studied by autoradiography in oocytes that had been incubated in3H‐thymidine after being parthenogenetically activated by sham microinjection, or microinjected with hamster, mouse, rabbit, or fish sperm nuclei, or hamster hepatocyte nuclei. Within 6 hr of sham or nucleus microinjection, nuclei of each type underwent transformation into pronuclei and synthesized DNA. These results demonstrated that the hamster egg can access and utilize its own and each type of template provided, whether homologous or heterologous. However, pronuclei derived from hamster sperm nuclei were more likely to be synthesizing DNA at 6 hr than pronuclei derived from sperm nuclei of other species. We conclude that the mechanisms employed by the hamster oocyte to transform hamster sperm nuclei into pronuclei and to effect DNA synthesis in these nuclei are not specific for the hamster sperm nucleus. Nevertheless, these mechanisms apparently operate more efficiently when the hamster sperm nucleus, rather than a heterologous sperm nucleus, is presen

ISSN:0148-7280    

DOI:10.1002/mrd.1120180203

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractMammalian sperm hyperactivation (HA) is a change in motility that accompanies capacitation (CAP) and is dependent on calcium (Ca) (Yanagimachi and Usui, Exp Cell Res 89:161, 1974). HA may be important for transport through the female tract and/or for fertilization. To develop an objective and quantitative assay for HA in individual mouse sperm, a computer‐assisted motion‐analysis system was used to describe sperm trnaslational movements. To determine which movements were characteristic of HA, Ca‐dependent motility was identified. This was done by incubating sperm with or without calcium (Ca+ or Ca− sperm, respectively), and determining the range of values for each motility parameter that was present only among Ca+ sperm. To do this, we compared frequency distributions of motility parameter values at the time of maximal CAP (90 min). CAP was monitored by measuring the level of in vitro fertilization and by evaluating the pattern of chlortetracycline binding to individual sperm heads [Ward and Storey, Dev Biol 104:287, 1984]. Two Ca‐dependent motility subgroups were apparent: 1) a “slow‐speed” subgroup with a curvilinear velocity (Vc)169 μm/sec) and wider‐amplitude head movements as measured by curvilinear progressiveness ratio (PRc<0.56). The latter subgroup was selected as HA, since the frequencies and time course were similar to those for CAP in the same population. Two media components known to be important for CAP, bicarbonate and bovine serum albumin (BSA) were then tested to determine whether they were necessary for HA. Incubation of sperm without bicarbonate prevented HA, but omitting BSA did not affect HA during the first 3 hrs. These data suggest that HA is not tight

ISSN:0148-7280    

DOI:10.1002/mrd.1120180204

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractRat eggs treated with the calcium ionophore A23187 and subjected to long‐term observation by phase microscopy were found to undergo many developmental changes that are normally associated with fertilization. These included cortical granule exocytosis and the abstriction of the second polar body. In addition, time‐lapse video microscopy revealed that, unlike untreated eggs, whose surfaces remained relatively immotile, the ionophore‐treated eggs underwent a lengthy period of surface undulatory activity. Since all of these events were remarkably similar in timing and morphology to those seen in fertilized eggs, we conclude that A23187 is capable of activating rat eggs. Using NBD‐phallacidin, the distribution of F‐actin in ionophore‐activated eggs was determined. During most of the postactivation period the eggs possessed an uninterrupted, uniform band of polymerized actin encompassing the entire cortex of the egg. However, during a discrete 1.5‐h period after the formation of the second polar body, an area adjacent to the region of polar body abstriction exhibited more intense staining than the rest of the cortex. Cytochalasin B treatment caused a dramatic reduction and/or rearrangement in cortical NBD‐phallacidin staining in activated eggs as compared to activated controls not exposed to the drug. We observed that all the developmental changes described above could be produced in the absence of exogenous calcium, suggesting that the rat egg possesses internal stores of calcium sufficient to elicit an activational response. We conclude that the ionophore‐induced release of free calcium ions into the cytosol stimulates many of the developmental changes that are normally seen during fertilization. These results indicate that calcium influx and cytoskeletal activity are correlated during the activation o

ISSN:0148-7280    

DOI:10.1002/mrd.1120180205

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractSperm‐rich semen and washed porcine spermatozoa were incubated for up to 2 hr either in utero in the presence of oviduct fluid or in vitro at 37°C. Sperm lipids were extracted and separated into phospholid and neutral lipid fractions. Eleven phospholipid and five neutral lipid fatty acids were identified and quantified using GC and GC‐MS. The percentage of 22:5n6, the major phospholipid fatty acid, decreased slightly but significantly during 1.5 hr of in utero incubation (41.2–38.0%), but after 2.0 hr of in utero incubation no significant difference was observed (40.0%). None of the phospholipid fatty acids changed in concentration during in vitro incubation. The mole ratio of phospholipid to phospholipid fatty acid (1.00:1.27) did not change during incubation. The levels of neutral lipid‐bound 14:0 decreased (43.5% to 31.8%) and that of 18:0 increased (11.1% to 18.2%) during in utero incubation. Similar but less pronounced changes were observed during in vitro incubation. (43.5% to 36.0%; and 11.1% to 15.8%, respectively).Two major sterols, cholestrol (73%) and desmosterol (27%) were identified by gas chromatography–mass spectrometry. The mole ratio of phospholipid to sterol (2.47:1:00) did not change during

ISSN:0148-7280    

DOI:10.1002/mrd.1120180206

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractMotility patterns of caput epididymal chimpanzee sperm, caput epididymal chimpanzee sperm incubated in vitro with chimpanzee cauda epididymal fluid, and cauda epididymal chimpanzee sperm were assessed quantitatively. Sperm recovered from the caput epididymis showed no motility, whereas sperm recovered from cauda epididymis showed progressive forward motility. After incubation in cauda fluid, approximately 25% of caput epididymal sperm showed some motile activity. Electrophoretic analysis of125I‐labeled sperm plasma membrane preparations revealed that the surface of caput epididymal sperm, incubated in cauda fluid, was modified by the appearance of a major protein‐glycoprotein surface component with an apparent molecular weight of 27 kilodaltons (kD). THis 27‐kD component was not detected on caput epididymal sperm incubated in buffer or in caput fluid. However, it was present in cauda fluid and on cauda epididymal sperm. Binding to caput epididymal sperm was cell specific in that chimpanzee erythrocytes incubated in cauda fluid did not bind this 27‐kD cauda fluid component. Motility patterns of ejaculated chimpanzee sperm and of ejaculated chimpanzee sperm incubated in the uterus of adult female chimpanzees also were assessed quantitatively. Ejaculated sperm showed progressive forward motility, whereas in utero incubated ejaculated sperm showed hyperactivated motility typical of capacitated sperm. Electrophoretic analysis of125I‐labeled sperm plasma membrane preparations revealed the loss of a 27‐kD component from the surface of ejaculated sperm after in utero incubation. No significant change in the125I‐distribution pattern was detectable when ejaculated sperm were incubated in buffer. These results suggest that the lumenal fluid component, which becomes adsorbed to the surface of chimpanzee sperm during maturation in the epididymis and which is removed from the surface of mature chimpanzee sperm in the female reproductive tract, affects s

ISSN:0148-7280    

DOI:10.1002/mrd.1120180207

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractThe basic nuclear proteins of a fraction of elongating spermatids from human tests and of a fraction of motile spermatozoa from the ejaculate, separated by ion‐exchange chromatography, were compared. Analysis by acetic acid‐urea polyacrylamide gel electrophoresis (PAGE) showed that, in both fractions, four proteins of lower mobility were coeluted with protamine 1 by 23% guanidinium chloride (GuCI) while protamine 2 alone was eluted by 50% GuCI. Treatment with alkaline phosphatase identified those four proteins as phosphorylated protamines, and cyanogen bromide (CNBr) treatment of the dephosphorylated protamines distinguished them as variants of protamine 2 and not of protamine 1. Thus far, phosphorylated forms of protamine 1 have not been detected in either spermatids or spermatozoa. Those observations indicate that protamine 2 functions in the cycle of phosphorylation‐dephosphorylation, which is essential to the process of sperm chromatin condensation, while the role of protamine 1 in human spermiogenesis is not yet defined.The presence of phosphorylated protamine in motile, presumably mature spermatozoa appears to be characteristic of human sperm but not of the sperm of other mammals and is probably the basis for the heterogeneity of chromatin condensation frequently observed in human sperma

ISSN:0148-7280    

DOI:10.1002/mrd.1120180208

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractIn vivo fertilization of goat eggs has been studied by electron microscopy. Eggs were recovered from superovulated or natural cyclic goats, 32 to 52 hours after the onset of oestrus; only eggs recovered between 46 and 52 hours were fertilized.Spermatozoa penetrated the zona pellucida tangentially leaving vesiculated products of the acrosome reaction at the zona surface.As sperm penetrated into the ooplasm, the second meiotic division completed and cortical granule exocytosis occurred. However a few unreacted cortical granules usually remained in the cortex of the two fertilized eggs, adjacent to the plasma membrane.After swelling the two pronuclei presented similar ultrastructural morphology: they contained small, compact, agranular nucleoli and unevenly distributed chromatin. The cytoplasm in close vicinity to the apposed pronuclei contained large stacks of annulate lamellae, smooth endoplasmic reticulum, prominent Golgi complexes, as well as dense areas of unidentified material. The abundance of cytoplasmic organelles near the pronuclei might be the expression of intensive metabolic activity. Conversely, in the cortex of fertilized ova several large organelles‐free cytoplasmic areas were randomly distribute

ISSN:0148-7280    

DOI:10.1002/mrd.1120180209

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

ISSN:0148-7280    

DOI:10.1002/mrd.1120180201

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY