scholarly journals Role of ascorbic acid in promoting follicle integrity and survival in intact mouse ovarian follicles in vitro

Reproduction ◽  
2001 ◽  
pp. 89-96 ◽  
Author(s):  
AA Murray ◽  
MD Molinek ◽  
SJ Baker ◽  
FN Kojima ◽  
MF Smith ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Basement Membrane ◽  
Growth And Development ◽  
Culture Media ◽  
Membrane Integrity ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Follicular Growth ◽  
Intact Mouse

Ascorbic acid has three known functions: it is necessary for collagen synthesis, promotes steroidogenesis and acts as an antioxidant. Within the ovary, most studies have concentrated on the role of ascorbic acid in luteal formation and regression and little is known about the function of this vitamin in follicular growth and development. Follicular growth and development were investigated in this study using an individual follicle culture system that allows the growth of follicles from the late preantral stage to Graafian morphology. Follicles were isolated from prepubertal mice and cultured for 6 days. Control media contained serum and human recombinant FSH. Further groups of follicles were cultured in the same media but with the addition of ascorbic acid at concentrations of either 28 or 280 micromol l(-1). Addition of ascorbic acid at the higher concentration significantly increased the percentage of follicles that maintained basement membrane integrity throughout culture (P < 0.001). Ascorbic acid had no effect on the growth of the follicles or on oestradiol production. Metalloproteinase 2 activity tended to increase at the higher concentration of ascorbic acid and there was a significant concomitant increase in the activity of tissue inhibitor of metalloproteinase 1 (P < 0.01). Follicles cultured without the addition of serum but with FSH and selenium in the culture media underwent apoptosis. Addition of ascorbic acid to follicles cultured under serum-free conditions significantly reduced apoptosis (P < 0.05). From these data it is concluded that ascorbic acid is necessary for remodelling the basement membrane during follicular growth and that the ability of follicles to uptake ascorbic acid confers an advantage in terms of granulosa cell survival.

IMPACT OF SORBITOL- INDUCED OSMOTIC STRESS ON SOME BIOCHEMICAL TRAITS OF POTATO IN VITRO

2020 ◽  
Vol 51 (4) ◽  
pp. 1038-1047
Author(s):  
Mawia & et al.
Keyword(s):  
Ascorbic Acid ◽  
Osmotic Stress ◽  
Cytoplasmic Membrane ◽  
Genotypic Variation ◽  
Membrane Damage ◽  
Membrane Integrity ◽  
Culture Collection ◽  
Nutritive Medium ◽  
Starting Point

This study had as principal objective identification of osmotic-tolerant potato genotypes by using "in vitro" tissue culture and sorbitol as a stimulating agent, to induce water stress, which was added to the  culture nutritive medium in different concentration (0,50, 110, 220, 330 and 440 mM).  The starting point was represented by plantlets culture collection, belonging to eleven potato genotypes: Barcelona, Nectar, Alison, Jelly, Malice, Nazca, Toronto, Farida, Fabulla, Colomba and Spunta. Plantlets were multiplied between two internodes to obtain microcuttings (in sterile condition), which were inoculated on medium. Sorbitol-induced osmotic stress caused a significant reduction in the ascorbic acid, while the concentration of proline, H2O2 and solutes leakage increased compared with the control. Increased the proline content prevented lipid peroxidation, which played a pivotal role in the maintenance of membrane integrity under osmotic stress conditions. The extent of the cytoplasmic membrane damage depends on osmotic stress severity and the genotypic variation in the maintenance of membranes stability was highly associated with the ability of producing more amounts of osmoprotectants (proline) and the non-enzymic antioxidant ascorbic acid in response to osmotic stress level. The results showed that the genotypes Jelly, Nectar, Allison, Toronto, and Colomba are classified as highly osmotic stress tolerant genotypes, while the genotypes Nazca and Farida are classified as osmotic stress susceptible ones.

scholarly journals The role of IGFs in the regulation of ovarian follicular growth in the brushtail possum (Trichosurus vulpecula)

Reproduction ◽  
2010 ◽  
Vol 140 (2) ◽  
pp. 295-303 ◽  
Author(s):  
Jennifer L Juengel ◽  
Lisa J Haydon ◽  
Brigitta Mester ◽  
Brian P Thomson ◽  
Michael Beaumont ◽  
...  
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Cell Function ◽  
Antral Follicle ◽  
Brushtail Possum ◽  
Follicular Growth ◽  
Theca Cells ◽  
Ovarian Follicular Growth

IGFs are known to be key regulators of ovarian follicular growth in eutherian mammals, but little is known regarding their role in marsupials. To better understand the potential role of IGFs in the regulation of follicular growth in marsupials, expression of mRNAs encoding IGF1, IGF2, IGF1R, IGF-binding protein 2 (IGFBP2), IGFBP4 and IGFBP5 was localized by in situ hybridization in developing ovarian follicles of the brushtail possum. In addition, the effects of IGF1 and IGF2 on granulosa cell function were tested in vitro. Both granulosa and theca cells synthesize IGF mRNAs, with the theca expressing IGF1 mRNA and granulosa cell expressing IGF2 mRNA. Oocytes and granulosa cells express IGF1R. Granulosa and theca cells expressed IGFBP mRNAs, although the pattern of expression differed between the BPs. IGFBP5 mRNA was differentially expressed as the follicles developed with granulosa cells of antral follicles no longer expressing IGFBP5 mRNA, suggesting an increased IGF bioavailability in the antral follicle. The IGFBP protease, PAPPA mRNA, was also expressed in granulosa cells of growing follicles. Both IGF1 and IGF2 stimulated thymidine incorporation but had no effect on progesterone production. Thus, IGF may be an important regulator of ovarian follicular development in marsupials as has been shown in eutherian mammals.

scholarly journals Cathepsin B Localizes in the Caveolae and Participates in the Proteolytic Cascade in Trabecular Meshwork Cells. Potential New Drug Target for the Treatment of Glaucoma

2020 ◽  
Vol 10 (1) ◽  
pp. 78
Author(s):  
April Nettesheim ◽  
Myoung Sup Shim ◽  
Angela Dixon ◽  
Urmimala Raychaudhuri ◽  
Haiyan Gong ◽  
...  
Keyword(s):  
Trabecular Meshwork ◽  
Cathepsin B ◽  
Molecular Mechanisms ◽  
Culture Media ◽  
Ecm Remodeling ◽  
Trabecular Meshwork Cells ◽  
Proteolytic Cascade

Extracellular matrix (ECM) deposition in the trabecular meshwork (TM) is one of the hallmarks of glaucoma, a group of human diseases and leading cause of permanent blindness. The molecular mechanisms underlying ECM deposition in the glaucomatous TM are not known, but it is presumed to be a consequence of excessive synthesis of ECM components, decreased proteolytic degradation, or both. Targeting ECM deposition might represent a therapeutic approach to restore outflow facility in glaucoma. Previous work conducted in our laboratory identified the lysosomal enzyme cathepsin B (CTSB) to be expressed on the cellular surface and to be secreted into the culture media in trabecular meshwork (TM) cells. Here, we further investigated the role of CTSB on ECM remodeling and outflow physiology in vitro and in CSTBko mice. Our results indicate that CTSB localizes in the caveolae and participates in the pericellular degradation of ECM in TM cells. We also report here a novel role of CTSB in regulating the expression of PAI-1 and TGFβ/Smad signaling in TM cells vitro and in vivo in CTSBko mice. We propose enhancing CTSB activity as a novel therapeutic target to attenuate fibrosis and ECM deposition in the glaucomatous outflow pathway.

scholarly journals The Periplasmic α-Carbonic Anhydrase Activity of Helicobacter pylori Is Essential for Acid Acclimation

2005 ◽  
Vol 187 (2) ◽  
pp. 729-738 ◽  
Author(s):  
Elizabeth A. Marcus ◽  
Amiel P. Moshfegh ◽  
George Sachs ◽  
David R. Scott
Keyword(s):  
Helicobacter Pylori ◽  
Membrane Potential ◽  
Carbonic Anhydrase ◽  
Membrane Integrity ◽  
Carbonic Anhydrase Activity ◽  
Knockout Mutant ◽  
Inner Membrane ◽  
Acid Acclimation

ABSTRACT The role of the periplasmic α-carbonic anhydrase (α-CA) (HP1186) in acid acclimation of Helicobacter pylori was investigated. Urease and urea influx through UreI have been shown to be essential for gastric colonization and for acid survival in vitro. Intrabacterial urease generation of NH3 has a major role in regulation of periplasmic pH and inner membrane potential under acidic conditions, allowing adequate bioenergetics for survival and growth. Since α-CA catalyzes the conversion of CO2 to HCO3 −, the role of CO2 in periplasmic buffering was studied using an α-CA deletion mutant and the CA inhibitor acetazolamide. Western analysis confirmed that α-CA was bound to the inner membrane. Immunoblots and PCR confirmed the absence of the enzyme and the gene in the α-CA knockout. In the mutant or in the presence of acetazolamide, there was an ∼3 log10 decrease in acid survival. In acid, absence of α-CA activity decreased membrane integrity, as observed using membrane-permeant and -impermeant fluorescent DNA dyes. The increase in membrane potential and cytoplasmic buffering following urea addition to wild-type organisms in acid was absent in the α-CA knockout mutant and in the presence of acetazolamide, although UreI and urease remained fully functional. At low pH, the elevation of cytoplasmic and periplasmic pH with urea was abolished in the absence of α-CA activity. Hence, buffering of the periplasm to a pH consistent with viability depends not only on NH3 efflux from the cytoplasm but also on the conversion of CO2, produced by urease, to HCO3 − by the periplasmic α-CA.

scholarly journals The Role of Androgens in Mammals Folliculogenesis

2018 ◽  
Vol 44 (1) ◽  
pp. 15
Author(s):  
Livia Brunetti Apolloni ◽  
Jamily Bezerra Bruno ◽  
Benner Geraldo Alves ◽  
José Ricardo de Figueiredo
Keyword(s):  
Androgen Receptor ◽  
In Vitro Culture ◽  
Steroid Hormones ◽  
Oocyte Maturation ◽  
Follicular Development ◽  
Follicular Growth ◽  
Preantral Follicles ◽  
Ovarian Cells

Introduction: Steroid hormones production is a physiological process termed steroidogenesis. An important stage of this process is the conversion of androgens into estrogens through aromatase enzyme. Furthermore, androgens are important in the process of folliculogenesis, promoting follicular growth in different species. Thus, the aim of this review was to present the process of synthesis, mechanism of action, and importance of androgens in folliculogenesis. Additionally, the main results of in vitro culture of ovarian cells in the presence of these hormones were emphasized.Review: Folliculogenesis begins in prenatal life in most of species and can be defined as the process of formation, follicular growth, and oocyte maturation. Preantral follicles represent 95% of the follicular population and assisted reproductive technologies have been developed (e.g., Manipulation of Oocytes Enclosed in Preantral Follicles - MOEPF) in order to avoid the great follicle loss that occurs naturally in vivo by atresia. The MOEPF aim to obtain a large number of competent oocytes from preantral follicles and then subject to in vitro maturation, fertilization, and culture for embryo production. However, the development of an efficient medium to ensure the follicular survival and oocyte maturation is the major challenge of this biotechnology. To achieve the success on in vitro culture, the effects of substances as androgens on follicular development have been evaluated. Androgens are steroid hormones produced in theca cells (TC) that are fundamental for follicular growth. These cells provide all the androgens required by the developing follicles for conversion into estrogens by the granulosa cells (GC). Androgens receptors (AR) are localized in cell cytoplasm of all follicular categories, being more expressed in preantral follicles. The androgen pathway initiates through its connection to its receptor, making a complex androgen-AR, that in the nucleus helps on the process of gene transcription related with follicular survival. This mechanism is androgen receptor genomic activity. In addition to genomic action, there is an androgen receptor non-genomic activity. This occurs through activation of AR and its interaction with different signaling molecules located on the cell membrane, triggering events that aid in the follicular development. Regardless of the androgens actions, ovarian cells of several species subjected to in vitro culture have shown the importance of these hormones on the follicle development. Recent studies demonstrated that androgens addition on the culture medium stimulated the activation of preantral follicles (bovine and caprine), antrum formation (swine), survival (non-primate), and oocyte maturation (antral follicles; bovine). Also, some studies suggest that the addition of these hormones on in vitro culture is dose-dependent and species-specific.Conclusion: This review shows the role of androgens in different stages of follicular development and its action as a substrate for steroidogenesis and transcription of genes related to follicular survival and oocyte maturation. However, when these hormones should be added during in vitro follicular culture and which concentration is required remains unclear, being necessary more studies to elucidate these aspects.

scholarly journals Membrane lysis during biological membrane fusion: collateral damage by misregulated fusion machines

2008 ◽  
Vol 183 (2) ◽  
pp. 181-186 ◽  
Author(s):  
Alex Engel ◽  
Peter Walter
Keyword(s):  
Membrane Fusion ◽  
Biological Membrane ◽  
Membrane Integrity ◽  
Fusion Pore ◽  
Mechanistic Models ◽  
Collateral Damage ◽  
Vacuole Fusion ◽  
Membrane Lysis

In the canonical model of membrane fusion, the integrity of the fusing membranes is never compromised, preserving the identity of fusing compartments. However, recent molecular simulations provided evidence for a pathway to fusion in which holes in the membrane evolve into a fusion pore. Additionally, two biological membrane fusion models—yeast cell mating and in vitro vacuole fusion—have shown that modifying the composition or altering the relative expression levels of membrane fusion complexes can result in membrane lysis. The convergence of these findings showing membrane integrity loss during biological membrane fusion suggests new mechanistic models for membrane fusion and the role of membrane fusion complexes.

Bovine granulosa cells express extracellular matrix proteins and their regulators during luteinization in culture

1996 ◽  
Vol 8 (2) ◽  
pp. 259 ◽  
Author(s):  
Y Zhao ◽  
MR Luck
Keyword(s):  
Extracellular Matrix ◽  
Granulosa Cells ◽  
Culture Media ◽  
Gelatin Zymography ◽  
Fold Increase ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Subunit Gene ◽  
Gelatinase Activity ◽  
Cell Extracts

This study investigated the ability of bovine granulosa cells to express and secrete collagen, metalloproteinase (MMP) activity and a tissue inhibitor of metalloproteinase (TIMP-1) during luteinization in vitro. Cells from mature (1-2 mL fluid volume) bovine follicles were cultured over 4 days in serum-free medium. Their luteinization during culture was confirmed by a 10-fold increase in progesterone secretion. Samples of cell extracts, culture media and follicular fluid were subjected to Western blotting to identify secreted proteins and to gelatin zymography to detect enzyme activity. Poly A+ RNA, isolated from cells before and after culture, was probed to detect expression of collagen alpha 1(I), collagen alpha 3(IV) and TIMP-1. The results revealed that: (1) the collagen alpha 1(I) subunit gene was expressed in cells before culture but with greater intensity by Day 4 culture; collagen I protein, on the other hand, was not detectable in culture medium; (2) the collagen alpha 3(IV) subunit gene was expressed at a low level in uncultured cells and could be detected on Day 4 of culture; low amounts of the protein were detected in medium; (3) a 92-kDa band of gelatinase activity (presumed MMP-9) was present in all medium samples, together with bands of unidentified activity; and (5) the TIMP-1 gene was expressed in uncultured cells but its expression increased markedly up to Day 4 of culture. These results show that granulosa luteinization is associated with an increase in the expression of collagen, collagen-degrading enzymes and TIMP-1. Collagen protein, however, may be only poorly synthesized in this culture model. The results suggest that granulosa-derived cells are a likely source of components of the extracellular matrix during post-ovulatory remodelling of early luteal tissue.

The Role of Dystroglycan and Its Ligands in Physiology and Disease

Physiology ◽  
2000 ◽  
Vol 15 (5) ◽  
pp. 255-259 ◽  
Author(s):  
Thomas Meier ◽  
Markus A. Ruegg
Keyword(s):  
Extracellular Matrix ◽  
Cell Membrane ◽  
Basement Membrane ◽  
Embryonic Development ◽  
Bacterial Pathogens ◽  
Membrane Integrity ◽  
Muscle Disease ◽  
Entry Point ◽  
Cell Membrane Integrity

Dystroglycan contributes to the formation of basement membrane during embryonic development and enforces cell membrane integrity by bridging cytoskeleton and components of the extracellular matrix. In several forms of muscle disease, dystroglycan is reduced in abundance. Moreover, human viral and bacterial pathogens use dystroglycan as their cellular entry point.

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