Mechanisms of displacement of sperm basic nuclear proteins in mammals. An in vitro simulation of post-fertilization results

1982 ◽  
Vol 53 (1) ◽  
pp. 227-244
Author(s):  
T.C. Rodman ◽  
F.H. Pruslin ◽  
V.G. Allfrey
Keyword(s):  
Sperm Nucleus ◽  
Nuclear Proteins ◽  
Mouse Sperm ◽  
Basic Proteins ◽  
Molecular Events ◽  
In Vitro Simulation ◽  
Sperm Nuclei ◽  
Two Parameters

A standardized cytological preparation of mature mouse sperm has been devised to serve as an in vitro system for probing the intra-ooplasmic molecular events of transformation of the fertilizing sperm. Two parameters of the early phase of transformation in vivo are defined at the resolution of the light microscope: deletion of sperm-unique nuclear proteins, detectable by immunofluorescence, and retention of homogeneity of the residual DNA complex, with intact chromatin boundaries detectable by ethidium bromide staining. These studies show that both parameters are conserved when in vitro sperm preparations are treated with NaCl under reducing conditions. The deletion of 2 different classes of the unique basic proteins of mouse sperm nuclei is specified by the NaCl concentration: 0.7 M-NaCl displaces the non-protamine class but not the protamines, while 1 M-NaCl displaces both. On the other hand the effects of treatment with trypsin at various concentrations and intervals are less consistent with the in vivo parameters, indicating fragmentation and displacement, not only of the sperm-unique basic proteins, but also of structural proteins believed to maintain the fundamental cohesive organization of the DNA matrix. These observations suggest that mechanisms other than proteolysis, e.g. localized changes in ionic concentrations, may participate in the post-fertilization displacement of the sperm-unique nuclear proteins in vivo. This study also supports the validity of the in vitro simulation as a model with which to probe the progression of transformation of the sperm nucleus to the zygote pronucleus.

scholarly journals Turnover of basic chromosomal proteins in fertilized eggs: a cytoimmunochemical study of events in vivo.

1981 ◽  
Vol 90 (2) ◽  
pp. 351-361 ◽  
Author(s):  
T C Rodman ◽  
F H Pruslin ◽  
H P Hoffmann ◽  
V G Allfrey
Keyword(s):  
Ethidium Bromide ◽  
Basic Protein ◽  
Protein S ◽  
Sperm Nucleus ◽  
Cross Reactivity ◽  
Chromosomal Proteins ◽  
Basic Proteins ◽  
Embryonic Genome ◽  
Sperm Nuclei

The chromosomal complements of mouse oocytes, ova, and fertilizing sperm have been studied by immunofluorescence with specific antisera to the basic protein fraction of sperm nuclei and to histones H2b and H4, and by staining with ethidium bromide. These studies support the hypothesis, previously proposed (Rodman and Barth, 1979, Dev. Biol. 68:82-95), that the chromosomes of the oocyte in maturation incorporate unique basic protein(s) similar to those incorporated during spermiogenesis. That similarity is characterized, here, by immunologic cross-reactivity. The basic proteins of the fertilizing sperm nucleus and the cross-reactive moiety of the two haploid complements of the ovum are displaced simultaneously, shortly after sperm entry. However, because the unique basic proteins incorporated into the oocyte chromosomes do not, as in the spermatogenic sequence, entirely replace the histones, the maternal chromosomes display histones H2b and H4 at all postfertilization stages examined, whereas the decondensing paternal complement, for an interval before maturation of the pronuclei, contains neither sperm basic chromosomal proteins nor histones. Sequential staining of the same specimens with ethidium bromide revealed well-organized nuclear morphology of the residual DNA complex. Those observations suggest that, for an as yet undefined period in the transformation from spermatozoal to embryonic genome, the chromatin is devoid of a complement of basic proteins.

scholarly journals Maternal Dppa2 and Dppa4 are dispensable for zygotic genome activation but important for offspring survival

Development ◽  
2021 ◽  
Vol 148 (24) ◽  
Author(s):  
Oana Kubinyecz ◽  
Fatima Santos ◽  
Deborah Drage ◽  
Wolf Reik ◽  
Melanie A. Eckersley-Maslin
Keyword(s):  
Null Allele ◽  
Embryonic Stem ◽  
Offspring Survival ◽  
Zygotic Genome Activation ◽  
Molecular Events ◽  
Genome Activation ◽  
Mitotic Chromatin ◽  
Zygotic Genome

ABSTRACT Zygotic genome activation (ZGA) represents the initiation of transcription following fertilisation. Despite its importance, we know little of the molecular events that initiate mammalian ZGA in vivo. Recent in vitro studies in mouse embryonic stem cells have revealed developmental pluripotency associated 2 and 4 (Dppa2/4) as key regulators of ZGA-associated transcription. However, their roles in initiating ZGA in vivo remain unexplored. We reveal that Dppa2/4 proteins are present in the nucleus at all stages of preimplantation development and associate with mitotic chromatin. We generated conditional single and double maternal knockout mouse models to deplete maternal stores of Dppa2/4. Importantly, Dppa2/4 maternal knockout mice were fertile when mated with wild-type males. Immunofluorescence and transcriptome analyses of two-cell embryos revealed that, although ZGA took place, there were subtle defects in embryos that lacked maternal Dppa2/4. Strikingly, heterozygous offspring that inherited the null allele maternally had higher preweaning lethality than those that inherited the null allele paternally. Together, our results show that although Dppa2/4 are dispensable for ZGA transcription, maternal stores have an important role in offspring survival, potentially via epigenetic priming of developmental genes.

scholarly journals Association of CD44−/CD24− Breast Cancer Cells with Late Stage Tumor Recurrence

2020 ◽  
Author(s):  
Xinbo Qiao ◽  
Yixiao Zhang ◽  
Lisha Sun ◽  
Qingtian Ma ◽  
Jie Yang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Tumor Cells ◽  
Tumor Metastasis ◽  
Cancer Prognosis ◽  
Molecular Events ◽  
Cell Conversion ◽  
Tissue Specimens ◽  
Distant Tumor

AbstractTumor metastasis remains the main cause of breast cancer-related deaths, especially the later breast cancer distant metastasis. This study assessed CD44−/CD24− tumor cells in 576 tissue specimens for associations with clinicopathological features and metastasis and then investigated the underlying molecular events. The data showed that level of CD44−/CD24− cells was associated with later postoperative distant tumor metastasis. Furthermore, CD44−/CD24− triple negative cells could spontaneously convert into CD44+/CD24− cancer stem cells (CSCs) with properties similar to CD44+/CD24− CSCs from parental MDA-MB-231 cells in terms of gene expression, tumor cell xenograft formation, and lung metastasis in vitro and in vivo. Single-cell RNA sequencing identified RHBDL2 as a regulator that enhanced spontaneous CD44+/CD24− CSC conversion, whereas knockdown of RHBDL2 expression inhibited YAP/NF-κB signaling and blocked spontaneous CD44−/CD24− cell conversion to CSCs. These data suggested that the level of CD44−/CD24− tumor cells could predict breast cancer prognosis, metastasis, and response to adjuvant therapy.

scholarly journals Interspecies Differences in the Stability of Mammalian Sperm Nuclei Assessed in Vivo by Sperm Microinjection and in Vitro by Flow Cytometry1

1988 ◽  
Vol 39 (1) ◽  
pp. 157-167 ◽  
Author(s):  
Sally D. Perreault ◽  
Randy R. Barbee ◽  
Kenneth H. Elstein ◽  
Robert M. Zucker ◽  
Carol L. Keefer
Keyword(s):  
Interspecies Differences ◽  
Mammalian Sperm ◽  
Sperm Nuclei ◽  
The Stability

scholarly journals Histone carbonylation in vivo and in vitro

2000 ◽  
Vol 351 (3) ◽  
pp. 769-777 ◽  
Author(s):  
Georg T. WONDRAK ◽  
Daniel CERVANTES-LAUREAN ◽  
Elaine L. JACOBSON ◽  
Myron K. JACOBSON
Keyword(s):  
Histone H1 ◽  
Nuclear Proteins ◽  
Carbonyl Groups ◽  
Cell Nuclei ◽  
Strand Breaks ◽  
Core Histones ◽  
Dna Strand ◽  
And Function

Non-enzymic damage to nuclear proteins has potentially severe consequences for the maintenance of genomic integrity. Introduction of carbonyl groups into histones in vivo and in vitro was assessed by Western blot immunoassay and reductive incorporation of tritium from radiolabelled NaBH4 (sodium borohydride). Histone H1 extracted from bovine thymus, liver and spleen was found to contain significantly elevated amounts of protein-bound carbonyl groups as compared with core histones. The carbonyl content of nuclear proteins of rat pheochromocytoma cells (PC12 cells) was not greatly increased following oxidative stress induced by H2O2, but was significantly increased following alkylating stress induced by N-methyl-N´-nitro-N-nitrosoguanidine or by combined oxidative and alkylating stress. Free ADP-ribose, a reducing sugar generated in the nucleus in proportion to DNA strand breaks, was shown to be a potent histone H1 carbonylating agent in isolated PC12 cell nuclei. Studies of the mechanism of histone H1 modification by ADP-ribose indicate that carbonylation involves formation of a stable acyclic ketoamine. Our results demonstrate preferential histone H1 carbonylation in vivo, with potentially important consequences for chromatin structure and function.

Oestrogens in breast tumours and fat

1989 ◽  
Vol 95 ◽  
pp. 161-168
Author(s):  
J. H. H. Thijssen ◽  
M. A. Blankenstein
Keyword(s):  
Biological Effects ◽  
Plasma Concentrations ◽  
Normal Breast ◽  
Malignant Tumours ◽  
Breast Tumours ◽  
Two Parameters ◽  
Breast Tissues

SynopsisThe levels of endogenous steroids in the target tissues are thought to be more closely related to the biological effects than their concentrations in plasma. Therefore studies on oestrogen levels in malignant and non-malignant breast tissues (expressed per g wet weight) were conducted and the following conclusions were drawn:(1) malignant tumours contained higher oestradiol levels than normal or benign breast tissues, whereas oestrone levels were more comparable;(2) in contrast to the large decrease in plasma concentrations after menopause, the levels of oestradiol in tumours and in normal breast tissue did not change with advancing age;(3) the oestradiol levels in breast tissues were lower than in uterine tissues, particularly in women before menopause; oestrone levels were very similar in all tissues studied;(4) the mean oestradiol level was higher in oestrogen-receptor-positive tumours, but no correlation between the two parameters was found;(5) preliminary results indicated lower oestradiol levels in tumours obtained from countries with a lower incidence of breast cancer;(6) as far as available, oestrone levels were comparable and those of oestradiol were lower in fat tissues than in breast tumours;(7) neither in vitro studies with breast tumours, nor in vivo results using myometrial tissues support a prominent role of the metabolism of oestrogens at the 16α-position in the development of tumours;(8) the role of local factors in the production, retention and metabolism of oestradiol in the breast remains to be elucidated.

Survival of Bacillus cereus Vegetative Cells and Spores during In Vitro Simulation of Gastric Passage

2012 ◽  
Vol 75 (4) ◽  
pp. 690-694 ◽  
Author(s):  
SIELE CEUPPENS ◽  
MIEKE UYTTENDAELE ◽  
KATRIEN DRIESKENS ◽  
ANDREJA RAJKOVIC ◽  
NICO BOON ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Acid Resistance ◽  
In Vitro Experiments ◽  
Vegetative Cells ◽  
Ph Values ◽  
Constant Ph ◽  
In Vitro Simulation ◽  
Ph Range

The enteric pathogen Bacillus cereus must survive gastric passage in order to cause diarrhea by enterotoxin production in the small intestine. The acid resistance and the survival after gastric passage were assessed by in vitro experiments with acidified growth medium and gastric simulation medium with B. cereus NVH 1230-88 vegetative cells and spores. First, batch incubations at constant pH values for 4 h, which represented different physiological states of the stomach, showed that spores were resistant to any gastric condition in the pH range of 2.0 to 5.0, while vegetative cells were rapidly inactivated at pH values of ≤4.0. Second, a dynamic in vitro gastric experiment was conducted that simulated the continuously changing in vivo conditions due to digestion dynamics by gradually decreasing the pH from 5.0 to 2.0 and fractional emptying of the stomach 30 to 180 min from the start of the experiment. All of the B. cereus spores and 14% (±9%) of the vegetative cells survived the dynamic simulation of gastric passage.

scholarly journals Oxidative stress and ageing of the post-ovulatory oocyte

Reproduction ◽  
2013 ◽  
Vol 146 (6) ◽  
pp. R217-R227 ◽  
Author(s):  
Tessa Lord ◽  
R John Aitken
Keyword(s):  
Oxidative Stress ◽  
Molecular Mechanisms ◽  
Embryo Quality ◽  
Fertilization Rates ◽  
Molecular Events ◽  
Post Implantation ◽  
Potential Use

With extended periods of time following ovulation, the metaphase II stage oocyte experiences deterioration in quality referred to as post-ovulatory oocyte ageing. Post-ovulatory ageing occurs both in vivo and in vitro and has been associated with reduced fertilization rates, poor embryo quality, post-implantation errors and abnormalities in the offspring. Although the physiological consequences of post-ovulatory oocyte ageing have largely been established, the molecular mechanisms controlling this process are not well defined. This review analyses the relationships between biochemical changes exhibited by the ageing oocyte and the symptoms associated with the ageing phenotype. We also discuss molecular events that are potentially involved in orchestrating post-ovulatory ageing with a particular focus on the role of oxidative stress. We propose that oxidative stress may act as the initiator for a cascade of events that create the aged oocyte phenotype. Specifically, oxidative stress has the capacity to cause a decline in levels of critical cell cycle factors such as maturation-promoting factor, impair calcium homoeostasis, induce mitochondrial dysfunction and directly damage multiple intracellular components of the oocyte such as lipids, proteins and DNA. Finally, this review addresses current strategies for delaying post-ovulatory oocyte ageing with a particular focus on the potential use of compounds such as caffeine or selected antioxidants in the development of more refined media for the preservation of oocyte integrity during IVF procedures.

scholarly journals Transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of maturing oocytes of the mouse.

1986 ◽  
Vol 102 (3) ◽  
pp. 1039-1046 ◽  
Author(s):  
H J Clarke ◽  
Y Masui
Keyword(s):  
Morphological Changes ◽  
Small Mass ◽  
Sperm Nucleus ◽  
Metaphase Chromosomes ◽  
Oocyte Meiosis ◽  
Maturation In Vitro ◽  
Sperm Penetration ◽  
Sperm Nuclei ◽  
Metaphase I

Zona-free oocytes of the mouse were inseminated at prometaphase I or metaphase I of meiotic maturation in vitro, and the behavior of the sperm nuclei within the oocyte cytoplasm was examined. If the oocytes were penetrated by up to three sperm, maturation continued during subsequent incubation and became arrested at metaphase II. Meanwhile, each sperm nucleus underwent the following changes. First, the chromatin became slightly dispersed. By 6 h after insemination, this dispersed chromatin had become coalesced into a small mass, from which short chromosomal arms later became projected. Between 12 and 18 h after insemination, each mass of chromatin became resolved into 20 discrete metaphase chromosomes. In contrast, if oocytes were penetrated by four to six sperm, oocyte meiosis was arrested at metaphase I, and each sperm nucleus was transformed into a small mass of chromatin rather than into metaphase chromosomes. If oocytes were penetrated by more than six sperm, the maternal chromosomes became either decondensed or pycnotic, and the sperm nuclei were transformed into larger masses of chromatin. As control experiments, immature and fully mature metaphase II oocytes were inseminated. In the immature oocytes, which were kept immature by exposure to dibutyryl cyclic AMP, no morphological changes in the sperm nucleus were observed. On the other hand, in the fully mature oocytes, which were activated by sperm penetration, the sperm nucleus was transformed into the male pronucleus. Therefore, the cytoplasm of the maturing oocyte develops an activity that can transform the highly condensed chromatin of the sperm into metaphase chromosomes. However, the capacity of an oocyte is limited, such that it can transform a maximum of three sperm nuclei into metaphase chromosomes. Furthermore, the presence of more than six sperm causes a loss of the ability of the oocyte to maintain the maternal chromosomes in a metaphase state.

scholarly journals Granulocyte macrophage colony stimulating factor produced in lesioned peripheral nerves induces the up-regulation of cell surface expression of MAC-2 by macrophages and Schwann cells.

1996 ◽  
Vol 133 (1) ◽  
pp. 159-167 ◽  
Author(s):  
A Saada ◽  
F Reichert ◽  
S Rotshenker
Keyword(s):  
Cell Surface ◽  
Schwann Cells ◽  
Interleukin 1 ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Gm Csf ◽  
Molecular Events ◽  
Macrophage Colony

Peripheral nerve injury is followed by Wallerian degeneration which is characterized by cellular and molecular events that turn the degenerating nerve into a tissue that supports nerve regeneration. One of these is the removal, by phagocytosis, of myelin that contains molecules which inhibit regeneration. We have recently documented that the scavenger macrophage and Schwann cells express the galactose-specific lectin MAC-2 which is significant to myelin phagocytosis. In the present study we provide evidence for a mechanism leading to the augmented expression of cell surface MAC-2. Nerve lesion causes noneuronal cells, primarily fibroblasts, to produce the cytokine granulocyte macrophage-colony stimulating factor (GM-CSF). In turn, GM-CSF induces Schwann cells and macrophages to up-regulate surface expression of MAC-2. The proposed mechanism is based on the following novel observations. GM-CSF mRNA was detected by PCR in in vitro and in vivo degenerating nerves, but not in intact nerves. The GM-CSF molecule was detected by ELISA in medium conditioned by in vitro and in vivo degenerating peripheral nerves as of the 4th h after injury. GM-CSF activity was demonstrated by two independent bioassays, and repressed by activity blocking antibodies. Significant levels of GM-CSF were produced by nerve derived fibroblasts, but neither by Schwann cells nor by nerve derived macrophages. Mouse rGM-CSF enhanced MAC-2 production in nerve explants, and up-regulated cell surface expression of MAC-2 by Schwann cells and macrophages. Interleukin-1 beta up-regulated GM-CSF production thus suggesting that injury induced GM-CSF production may be mediated by interleukin-1 beta. Our findings highlight the fact that fibroblasts, by producing GM-CSF and thereby affecting macrophage and Schwann function, play a significant role in the cascade of molecular events and cellular interactions of Wallerian degeneration.

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