scholarly journals Synergism between albumin, bicarbonate and cAMP upregulation for cholesterol efflux from ram sperm

Reproduction ◽  
2020 ◽  
Vol 160 (2) ◽  
pp. 269-280
Author(s):  
Naomi C Bernecic ◽  
Bart M Gadella ◽  
Simon P de Graaf ◽  
Tamara Leahy
Keyword(s):  
Cholesterol Efflux ◽  
Functional Relationship ◽  
Mammalian Species ◽  
Dibutyryl Camp ◽  
Pde Inhibitors ◽  
Sperm Plasma Membrane ◽  
Ram Sperm ◽  
First Time ◽  
Dependent Processes

Compared to other mammalian species, ram spermatozoa are difficult to capacitate in vitro. Dibutyryl cAMP (db-cAMP) and the phosphodiesterase (PDE) inhibitors, caffeine and theophylline (cAMP up-regulators), must be added to traditional capacitation media (containing bicarbonate, calcium and BSA) to elicit a capacitation response. In this exploratory study, we assessed whether bicarbonate was still required for ram spermatozoa if cAMP is up-regulated by the addition of db-cAMP and PDE inhibitors and what role BSA plays in cholesterol efflux under these conditions. In this study, the validated BODIPY-cholesterol assay was used for the first time in ram spermatozoa to quantify cholesterol efflux by tracking the loss of BODIPY-cholesterol from the sperm plasma membrane using flow cytometry. The results show that under cAMP up-regulated conditions, an increase in membrane fluidity and tyrosine phosphorylation of sperm proteins remain as bicarbonate-dependent processes. In fact, the supplementation of bicarbonate under these conditions was necessary to further enhance cAMP production in ram spermatozoa, which correlated with the presence of these capacitation-related processes. When BSA was supplemented with cAMP up-regulators (as well as bicarbonate), there was a loss of approximately 20–23% of BODIPY-cholesterol (79.5 ± 30.5% to 76.9 ± 12.3% remaining from 10 min), indicating that BSA is essential for mediating cholesterol efflux in ram spermatozoa as measured by the BODIPY-cholesterol assay. The current study identifies the functional relationship between bicarbonate, BSA and cAMP up-regulators that is required to support capacitation-related processes in ram spermatozoa, specifically cholesterol efflux.

scholarly journals Penicillamine prevents ram sperm agglutination in media that support capacitation

Reproduction ◽  
2016 ◽  
Vol 151 (2) ◽  
pp. 167-177 ◽  
Author(s):  
T Leahy ◽  
J P Rickard ◽  
R J Aitken ◽  
S P de Graaf
Keyword(s):  
Sperm Capacitation ◽  
Dibutyryl Camp ◽  
Phosphorylated Proteins ◽  
Bicarbonate Ions ◽  
Merocyanine 540 ◽  
Highly Effective ◽  
Lactate And Pyruvate ◽  
Ram Sperm ◽  
Sperm Population

Ram spermatozoa are difficult to capacitate in vitro. Here we describe a further complication, the unreported phenomenon of head-to-head agglutination of ram spermatozoa following dilution in the capacitation medium Tyrodes plus albumin, lactate and pyruvate (TALP). Sperm agglutination is immediate, specific and persistent and is not associated with a loss of motility. Agglutination impedes in vitro sperm handling and analysis. So the objectives of this study were to investigate the cause of sperm agglutination and potential agents which may reduce agglutination. The percentage of non-agglutinated, motile spermatozoa increased when bicarbonate was omitted from complete TALP suggesting that bicarbonate ions stimulate the agglutination process. d-penicillamine (PEN), a nucleophilic thiol, was highly effective at reducing agglutination. The inclusion of 250 μM PEN in TALP reduced the incidence of motile, agglutinated spermatozoa from 76.7±2.7% to 2.8±1.4%. It was then assessed if PEN (1 mM) could be included in existing ram sperm capacitation protocols (TALP +1 mM dibutyryl cAMP, caffeine and theophylline) to produce spermatozoa that were simultaneously capacitated and non-agglutinated. This protocol resulted in a sperm population which displayed high levels of tyrosine phosphorylated proteins and lipid disordered membranes (merocyanine-540) while remaining motile, viable, acrosome-intact and non-agglutinated. In summary, PEN (1 mM) can be included in ram sperm capacitation protocols to reduce sperm agglutination and allow for the in vitro assessment of ram sperm capacitation.

scholarly journals Update on mammalian sperm capacitation: how much does the horse differ from other species?

Reproduction ◽  
2019 ◽  
Vol 157 (5) ◽  
pp. R181-R197 ◽  
Author(s):  
Bart Leemans ◽  
Tom A E Stout ◽  
Catharina De Schauwer ◽  
Sonia Heras ◽  
Hilde Nelis ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Zona Pellucida ◽  
Mammalian Species ◽  
Cumulus Cells ◽  
Sperm Cells ◽  
Cholesterol Depletion ◽  
Sperm Capacitation ◽  
Sperm Plasma Membrane ◽  
Species Specific

In contrast to various other mammalian species, conventional in vitro fertilization (IVF) with horse gametes is not reliably successful. In particular, stallion spermatozoa fails to penetrate the zona pellucida, most likely due to incomplete activation of stallion spermatozoa (capacitation) under in vitro conditions. In other mammalian species, specific capacitation triggers have been described; unfortunately, none of these is able to induce full capacitation in stallion spermatozoa. Nevertheless, knowledge of capacitation pathways and their molecular triggers might improve our understanding of capacitation-related events observed in stallion sperm. When sperm cells are exposed to appropriate capacitation triggers, several molecular and biochemical changes should be induced in the sperm plasma membrane and cytoplasm. At the level of the sperm plasma membrane, (1) an increase in membrane fluidity, (2) cholesterol depletion and (3) lipid raft aggregation should occur consecutively; the cytoplasmic changes consist of protein tyrosine phosphorylation and elevated pH, cAMP and Ca2+ concentrations. These capacitation-related events enable the switch from progressive to hyperactivated motility of the sperm cells, and the induction of the acrosome reaction. These final capacitation triggers are indispensable for sperm cells to migrate through the viscous oviductal environment, penetrate the cumulus cells and zona pellucida and, finally, fuse with the oolemma. This review will focus on molecular aspects of sperm capacitation and known triggers in various mammalian species. Similarities and differences with the horse will be highlighted to improve our understanding of equine sperm capacitation/fertilizing events.

Cyclic-AMP initiates protein tyrosine phosphorylation independent of cholesterol efflux during ram sperm capacitation

2008 ◽  
Vol 20 (6) ◽  
pp. 649 ◽  
Author(s):  
Carmen Colas ◽  
Peter James ◽  
Liz Howes ◽  
Roy Jones ◽  
José A. Cebrian-Perez ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Maximal Response ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Phosphorylated Proteins ◽  
Pde Inhibitors ◽  
Flagellar Proteins ◽  
Ram Sperm ◽  
Camp Levels

Unlike most other species, ram spermatozoa are difficult to capacitate in vitro. Bicarbonate and Ca2+ are necessary, whereas bovine serum albumin does not appear to be obligatory. In the present investigation we have assessed (1) the ability of the cholesterol-sequestering agent, methyl-β-cyclodextrin (M-β-CD), to initiate protein tyrosine phosphorylation, and (2) the importance of phosphodiesterases (PDEs) in controlling the levels of cAMP. Results show that despite removing significant amounts of membrane cholesterol, as assessed by filipin staining, M-β-CD treatment did not stimulate major increases in protein tyrosine phosphorylation. Addition of a cocktail of PDE inhibitors (theophylline and caffeine), a phosphatase inhibitor (okadaic acid) and dibutyryl-cAMP (db-cAMP), however, stimulated specific tyrosine phosphorylation of several proteins between 30 and 120 kDa. On their own, none of the above reagents were effective but a combination of db-cAMP + PDE inhibitors was sufficient to achieve a maximal response. H-89, a protein kinase-A inhibitor, suppressed tyrosine phosphorylation significantly. Immunofluorescence revealed that the newly-phosphorylated proteins localised mainly in the sperm tail. These findings suggest that in ram spermatozoa cAMP levels are too low to initiate tyrosine phosphorylation of flagellar proteins that are indicative of the capacitation state and that this is caused by unusually high levels of intracellular PDEs.

scholarly journals Effect of caffeine on functions of cooling-stored ram sperm in vitro

2014 ◽  
Vol 83 (1) ◽  
pp. 19-25 ◽  
Author(s):  
Eliška Špaleková ◽  
Alexander V. Makarevich ◽  
Elena Kubovičová ◽  
Alexander Ostró ◽  
Peter Chrenek
Keyword(s):  
Sperm Motility ◽  
Membrane Integrity ◽  
Annexin V ◽  
Plasma Membrane Integrity ◽  
Progressive Movement ◽  
Viability Assay ◽  
Ram Sperm ◽  
First Time ◽  
Membrane Destabilization

Caffeine is a well-known sperm motility stimulator, however, its effects on cooling-stored ram semen are unknown. The aim of the study was to examine the effect of caffeine on selected motility and viability indices of cooling-stored ram spermatozoa. Sperm ejaculates from 4 rams were diluted (1:3) in a Triladyl extender. Samples were stored for 96 h at 4-5 °C in two sets. In the first set used for motility analysis, caffeine at concentrations of 1, 2 or 4 mmol·l-1 was added to sperm aliquots on the day of analysis. In the second set used for viability assay, caffeine at the same concentrations (1, 2 or 4 mmol·l-1) was added at the beginning of storage. Control was left without caffeine addition. Sperm motility was analyzed at 0, 24, 48 and 72 h of cooling-storage. Viability assays were done after 72–96 h of cooling-storage. Caffeine significantly (P < 0.05) increased sperm motility and progressive movement and maintained this value for 72 h. Caffeine at the dose of 2 mmol·l-1 and 4 mmol·l-1 significantly (P < 0.05) reduced the proportion of dead/necrotic sperm detected by propidium iodide and proportion of apoptotic sperm detected by Yo-Pro-1, respectively. No effect of caffeine on plasma membrane integrity was noted. Proportion of sperm with membrane destabilization (annexin V-Fluos) was reduced by caffeine given at 1 and 4 mmol·l-1 compared to control. Our study for the first time demonstrates that caffeine maintains motility and viability of cooling-stored ram sperm for longer time compared to control.

scholarly journals Ultrastructural characteristics and immune profile of equine MSCs from fetal adnexa

Reproduction ◽  
2017 ◽  
Vol 154 (4) ◽  
pp. 509-519 ◽  
Author(s):  
Eleonora Iacono ◽  
Luisa Pascucci ◽  
Barbara Rossi ◽  
Cinzia Bazzucchi ◽  
Aliai Lanci ◽  
...  
Keyword(s):  
Mammalian Species ◽  
Cell Bank ◽  
Transmission Electron ◽  
Ultrastructural Characteristics ◽  
Hanging Drop Culture ◽  
Means Of Transmission ◽  
Different Characteristics ◽  
First Time ◽  
Active Genes

Both in human and equine species, mesenchymal stem cells (MSCs) from amniotic membrane (AM) and Wharton’s jelly (WJ), may be particularly useful for immediate use or in later stages of life, after cryopreservation in cell bank. The aim of this study was to compare equine AM- and WJ-MSCs in vitro features that may be relevant for their clinical employment. MSCs were more easily isolated from WJ, even if MSCs derived from AM exhibited more rapid proliferation (P < 0.05). Osteogenic and chondrogenic differentiation were more prominent in MSCs derived from WJ. This is also suggested by the lower adhesion of AM cells, demonstrated by the greater volume of spheroids after hanging drop culture (P < 0.05). Data obtained by PCR confirmed the immunosuppressive function of AM and WJ-MSCs and the presence of active genes specific for anti-inflammatory and angiogenic factors (IL-6, IL 8, IL-β1). For the first time, by means of transmission electron microscopy (TEM), we ascertained that equine WJ-MSCs constitutively contain a very impressive number of large vesicular structures, scattered throughout the cytoplasm. Moreover, an abundant extracellular fibrillar matrix was located in the intercellular spaces among WJ-MSCs. Data recorded in this study reveal that MSCs from different fetal tissues have different characteristics that may drive their therapeutic use. These finding could be noteworthy for horses as well as for other mammalian species, including humans.

scholarly journals Mechanisms of Kinesin-1 activation by Ensconsin/MAP7 in vivo

2018 ◽  
Author(s):  
Mathieu Métivier ◽  
Brigette Y. Monroy ◽  
Emmanuel Gallaud ◽  
Renaud Caous ◽  
Aude Pascal ◽  
...  
Keyword(s):  
Large Cell ◽  
Binding Domain ◽  
Microtubule Network ◽  
Centrosome Separation ◽  
Microtubule Binding Domain ◽  
Dual Activation ◽  
First Time ◽  
Dependent Processes

AbstractCentrosome separation in Drosophila larval neuroblasts and asymmetric transport of embryonic determinants in oocytes are both microtubule-dependent processes that require Kinesin-1 activation by Ensconsin/microtubule-associated protein 7 (MAP7). However, the molecular mechanism used by Ensconsin to activate Kinesin-1 remains elusive. Ensconsin/ MAP7 contains an N-terminal microtubule-binding domain (MBD) and a C-terminal Kinesin-binding domain (KBD). Using rescue experiments in live flies, we show that KBD expression alone is sufficient to fully rescue Ensconsin-dependent centrosome separation defects, but not the fast oocyte streaming and the localization patterns of Staufen and Gurken proteins. Interestingly, we show here for the first time that KBD binds and stimulates Kinesin-1 binding to Mts in vivo and in vitro. We propose that the KBD/Kinesin-1 motor represents a minimal activation module that stimulates Kinesin-1 binding to Mts. Addition of the MBD, present in the full length Ensconsin allows this activation to occur directly on the Mt. Our data also suggest that in a very large cell with a complex microtubule network, but not in smaller cells, this dual activation by Ensconsin is essential for optimal Kinesin-1 targeting to the microtubule cytoskeleton.

scholarly journals NLRP7 is expressed in the ovine ovary and associated with in vitro pre-implantation embryo development

Reproduction ◽  
2019 ◽  
Vol 158 (5) ◽  
pp. 415-427 ◽  
Author(s):  
Guangdong Li ◽  
Xiuzhi Tian ◽  
Dongying Lv ◽  
Lu Zhang ◽  
Zhenzhen Zhang ◽  
...  
Keyword(s):  
Embryo Development ◽  
Homo Sapiens ◽  
Mammalian Species ◽  
Ovarian Follicles ◽  
The Future ◽  
First Time ◽  
Hydatidiform Moles ◽  
Parthenogenetic Embryos

NLRP (NACHT, LRR and PYD domain-containing proteins) family plays pivotal roles in mammalian reproduction. Mutation of NLRP7 is often associated with human recurrent hydatidiform moles. Few studies regarding the functions of NLRP7 have been performed in other mammalian species rather than humans. In the current study, for the first time, the function of NLRP7 has been explored in ovine ovary. NLRP7 protein was mainly located in ovarian follicles and in in vitro pre-implantation embryos. To identify its origin, 763 bp partial CDS of NLRP7 deriving from sheep cumulus oocyte complexes (COCs) was cloned, it showed a great homology with Homo sapiens. The high levels of mRNA and protein of NLRP7 were steadily expressed in oocytes, parthenogenetic embryos or IVF embryos. NLRP7 knockdown by the combination of siRNA and shRNA jeopardized both the parthenogenetic and IVF embryo development. These results strongly suggest that NLRP7 plays an important role in ovine reproduction. The potential mechanisms of NLRP7 will be fully investigated in the future.

Somatic Embryogenesis and In vitro Plant Regeneration in Vigna trilobata (L.) Verd. - A Potential Pasture Legume

1970 ◽  
Vol 19 (1) ◽  
pp. 89-99
Author(s):  
K. Choudhary ◽  
M. Singh ◽  
M. S. Rathore ◽  
N. S. Shekhawat
Keyword(s):  
Somatic Embryogenesis ◽  
Growth Regulators ◽  
Somatic Embryos ◽  
Basal Medium ◽  
Long Term Study ◽  
Continuous Application ◽  
First Time ◽  
Pasture Legume

This long term study demonstrates for the first time that it is possible to propagate embryogenic Vigna trilobata and to subsequently initiate the differentiation of embryos into complete plantlets. Initiation of callus was possible on 2,4-D. Somatic embryos differentiated on modified MS basal nutrient medium with 1.0 mg/l  of 2,4-D and 0.5 mg/l  of Kn. Sustained cell division resulted in globular and heart shape stages of somatic embryos. Transfer of embryos on to a fresh modified MS basal medium with 0.5 mg/l of Kn and 0.5 mg/l of GA3 helped them to attain maturation and germination. However, the propagation of cells, as well as the differentiation of embryos, were inhibited by a continuous application of these growth regulators. For this reason, a long period on medium lacking these growth regulators was necessary before the differentiation of embryos occurred again. The consequences for improving the propagation of embryogenic cultures in Vigna species are discussed. Key words: Pasture  legume, Vigna trilobata, Globular, Heart shape, somatic embryogenesis D.O.I. 10.3329/ptcb.v19i1.4990 Plant Tissue Cult. & Biotech. 19(1): 89-99, 2009 (June)

Follicle-stimulating hormone-dependent estrogen secretion by rat Sertoli cells in vitro: Modulation by calcium

1991 ◽  
Vol 125 (3) ◽  
pp. 280-285 ◽  
Author(s):  
J. Alan Talbot ◽  
Ann Lambert ◽  
Robert Mitchell ◽  
Marek Grabinski ◽  
David C. Anderson ◽  
...  
Keyword(s):  
Sertoli Cells ◽  
Culture Medium ◽  
Follicle Stimulating Hormone ◽  
Dibutyryl Camp ◽  
Immature Rat ◽  
Estradiol Secretion ◽  
Old Rats ◽  
Stimulating Hormone

Abstract We have investigated the role of Ca2+ in the control of FSH-induced estradiol secretion by Sertoli cells isolated from 8-10 days old rats. Exogenous Ca2+ (4-8 mmol/1) inhibited FSH-stimulated E2 secretion such that, with 8 mmol/l Ca2+ and FSH (8 IU/l) E2 secretion decreased from 2091±322 to 1480±84 pmol/l (p<0.002), whilst chelation of Ca2+ in the culture medium with EGTA (3 mmol/l) increased E2 secretion from 360±45 to 1242±133 pmol/l) in the absence of FSH. Further, EGTA (3 mmol/l) markedly potentiated FSH (8 IU/l), forskolin (1 μmol/l) and dibutyryl cAMP (1 mmol/l)-stimulated E2 secretion. Addition of the Ca2+ ionophores, ionomycin (2-5 μmol/l) and A23187 (2 μmol/l), inhibited FSH (8 IU/l)-stimulated E2 secretion by >80%. The effect of ionomycin was totally reversible, whereas that of A23187 was irreversible. Ionomycin (5 μmol/l) had no effect on EGTA-induced E2 secretion in the absence of FSH, but reduced EGTA-provoked E2 secretion by 59% in the presence of FSH (8 IU/l). Similarly, forskolin- and dibutyryl cAMP-provoked E2 production was inhibited 46-50% by ionomycin (5 μmol/l). We conclude that FSH-induced E2 secretion from immature rat Sertoli cells is modulated by intra- and extracellular Ca2+.

Stimulation of Ca(2+)-dependent exocytosis of the sperm acrosome by cAMP acting downstream of phospholipase A2

Reproduction ◽  
2000 ◽  
pp. 57-68 ◽  
Author(s):  
J Garde ◽  
ER Roldan
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Phosphodiesterase Inhibitors ◽  
Dibutyryl Camp ◽  
Camp Phosphodiesterase ◽  
Ram Sperm ◽  
Camp Formation ◽  
Stimulation Of

Spermatozoa undergo exocytosis in response to agonists that induce Ca2+ influx and, in turn, activation of phosphoinositidase C, phospholipase C, phospholipase A2, and cAMP formation. Since the role of cAMP downstream of Ca2+ influx is unknown, this study investigated whether cAMP modulates phospholipase C or phospholipase A2 using a ram sperm model stimulated with A23187 and Ca2+. Exposure to dibutyryl-cAMP, phosphodiesterase inhibitors or forskolin resulted in enhancement of exocytosis. However, the effect was not due to stimulation of phospholipase C or phospholipase A2: in spermatozoa prelabelled with [3H]palmitic acid or [14C]arachidonic acid, these reagents did not enhance [3H]diacylglycerol formation or [14C]arachidonic acid release. Spermatozoa were treated with the phospholipase A2 inhibitor aristolochic acid, and dibutyryl-cAMP to test whether cAMP acts downstream of phospholipase A2. Under these conditions, exocytosis did not occur in response to A23187 and Ca2+. However, inclusion of dibutyryl-cAMP and the phospholipase A2 metabolite lysophosphatidylcholine did result in exocytosis (at an extent similar to that seen when cells were treated with A23187/Ca2+ and without the inhibitor). Inclusion of lysophosphatidylcholine alone, without dibutyryl-cAMP, enhanced exocytosis to a lesser extent, demonstrating that cAMP requires a phospholipase A2 metabolite to stimulate the final stages of exocytosis. These results indicate that cAMP may act downstream of phospholipase A2, exerting a regulatory role in the exocytosis triggered by physiological agonists.

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