6 results
Participation of PLA2 and PLC in DhL-induced activation of Rhinella arenarum oocytes
- J. Zapata-Martínez, M.F. Medina, M.C. Gramajo-Bühler, G. Sánchez-Toranzo
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Rhinella arenarum oocytes can be artificially activated, a process known as parthenogenesis, by a sesquiterpenic lactone of the guaianolide group, dehydroleucodine (DhL). Transient increases in the concentration of cytosolic Ca2+ are essential to trigger egg activation events. In this sense, the 1-4-5 inositol triphosphate receptors (IP3R) seem to be involved in the Ca2+ transient release induced by DhL in this species. We analyzed the involvement of phosphoinositide metabolism, especially the participation of phospholipase A2 (PLA2) and phospholipase C (PLC) in DhL-induced activation. Different doses of quinacrine, aristolochic acid (ATA) (PLA2 inhibitors) or neomycin, an antibiotic that binds to PIP2, thus preventing its hydrolysis, were used in mature Rhinella arenarum oocytes. In order to assay the participation of PI-PLC and PC- PLC we used U73122, a competitive inhibitor of PI-PLC dependent events and D609, an inhibitor of PC-PLC. We found that PLA2 inhibits quinacrine more effectively than ATA. This difference could be explained by the fact that quinacrine is not a specific inhibitor for PLA2 while ATA is specific for this enzyme. With respect to the participation of PLC, a higher decrease in oocyte activation was detected when cells were exposed to neomycin. Inhibition of PC-PLC with D609 and IP-PLC with U73122 indicated that the last PLC has a significant participation in the effect of DhL-induced activation. Results would indicate that DhL induces activation of in vitro matured oocytes of Rhinella arenarum by activation of IP-PLC, which in turn may induce IP3 formation which produces Ca2+ release.
Histological and morphometric study of the epididymus of Chinchilla lanigera Grey under controlled conditions in captivity
- M.C. Gramajo-Bühler, F.J. Pucci, G. Sanchez-Toranzo
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Chinchilla lanigera, native to the Andean Mountains of Perú, Chile, Bolivia and Argentina, is a specimen of great economic importance because of its fur. In mammals, spermatozoa originate in testes and are transported to the epididymis, where they undergo morphological and biochemical modifications known as sperm maturation, a basic step in the acquisition of their fertilizing ability. The aim of this work is the macroscopic and microscopic analysis of the epididymis of Chinchilla lanigera Grey and its sectorization based on a histomorphological study. The epididymis presents a clear segmentation into four regions: initial segment, caput, corpus and cauda. The epithelium lining the seminiferous tubules is pseudostratified, with principal cells with stereocilia and basal, clear, apical, narrow and halo cells. The histological analysis showed that principal and basal cells are the prevailing populations in all regions, also revealing narrow cells and the absence of clear cells in the initial segment. Each segment presents its different histological and morphometric characteristics, which supports the idea of the specific behaviour of each region, giving a segment-specific character to the process of sperm maturation in this species. No significant differences were found in the morphometric measurements or in the histological evaluation of the epididymis of samples collected in April and October. The fact that no differences were found between the samples collected during the two periods when the reproductive ability in nature is different suggests the importance of external factors in the control of the reproductive cycle of Chinchilla lanigera.
Participation of IP3R, RyR and L-type Ca2+ channel in the nuclear maturation of Rhinella arenarum oocytes
- G. Sánchez Toranzo, M.C. Gramajo Bühler, M.I. Bühler
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During meiosis resumption, oocytes undergo a series of nuclear and cytosolic changes that prepare them for fertilization and that are referred to as oocyte maturation. These events are characterized by germinal vesicle breakdown (GVBD), chromatin condensation and spindle formation and, among cytosolic changes, organelle redistribution and maturation of Ca2+-release mechanisms. The progression of the meiotic cell cycle is regulated by M phase/maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK). Changes in the levels of intracellular free Ca2+ ion have also been implicated strongly in the triggering of the initiation of the M phase. Ca2+ signals can be generated by Ca2+ release from intracellular Ca2+ stores (endoplasmic reticulum; ER) or by Ca2+ influx from the extracellular space. In this sense, the L-type Ca2+ channel plays an important role in the incorporation of Ca2+ from the extracellular space. Two types of intracellular Ca2+ receptor/channels are known to mediate the intracellular Ca2+ release from the ER lumen. The most abundant, the inositol 1,4,5-trisphosphate receptor (IP3R), and the other Ca2+ channel, the ryanodine receptor (RyR), have also been reported to mediate Ca2+ release in several oocytes. In amphibians, MPF and MAPK play a central role during oocyte maturation, controlling several events. However, no definitive relationships have been identified between Ca2+ and MPF or MAPK. We investigated the participation of Ca2+ in the spontaneous and progesterone-induced nuclear maturation in Rhinella arenarum oocytes and the effect of different pharmacological agents known to produce modifications in the Ca2+ channels. We demonstrated that loading competent and incompetent oocytes with the intracellular calcium chelator BAPTA/AM produced suppression of spontaneous and progesterone-induced GVBD. In our results, the capacity of progesterone to trigger meiosis reinitiation in Rhinella in the presence of L-type Ca2+ channel blockers (nifedipine and lanthane) indicated that spontaneous and progesterone-induced maturation would be independent of extracellular calcium influx, but would be sensitive to intracellular Ca2+ deprivation. As demonstrated by the effect of thimerosal and heparin in Rhinella arenarum, the intracellular increase in Ca2+ during maturation is also mediated mainly by IP3R. In addition, our results using caffeine, an agonist of the RyR, could suggest that Ca2+ release from ryanodine-sensitive stores is not essential for oocyte maturation in Rhinella. The decrease in MPF activity with NaVO3 negatively affected the percentage of thimerosal-induced GVBD. This finding suggests that Ca2+ release through the IP3R could be involved in the signalling pathway that induces MPF activation. However, the inhibition of MAP/ERK kinase (MEK) by PD98128 or P90 by geldanamycin produced a significant decrease in the percentages of GVBD induced by thimerosal. This finding suggests that Ca2+ release per se cannot bypass the inhibition of the MAPK activity.
Effect on sperm quality of different cryoprotectants in sperm of Chinchilla lanigera
- M.C. Gramajo-Bühler, F. Pucci-Alcaide, G. Sánchez-Toranzo
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Chinchilla lanigera is an endangered species therefore the development of cryopreservation protocols for its gametes is a useful tool in the application of assisted reproduction techniques. A study of the functionality of the spermatozoa punctured from the cauda epididymis was performed on fresh or frozen–thawed samples with three cryoprotective media (test-yolk buffer, sucrose and glycerol). The effect that these media had on sperm physiology during the freezing, storage and later thawing process was analysed. A decrease in the percentages of viability, motility, membrane integrity and capacity to undergo the induced acrosome reaction was found with all the media assayed, an increase in the percentages of DNA fragmentation was also observed. The comparative analysis of the effect of the different cryoprotectants assayed showed that the best medium to use to cryopreserve epididymal sperm in this species is test-yolk buffer. This medium had the least effect on the abovementioned physiological parameters, especially at the level of genetic material.
Participation of MAPK, PKA and PP2A in the regulation of MPF activity in Bufo arenarum oocytes
- G. Sánchez Toranzo, F. Bonilla, M.C. Gramajo Bühler, M.I. Bühler
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The objectives of the present paper were to study the involvement and possible interactions of both cAMP-PKA and protein phosphatases in Bufo arenarum oocyte maturation and to determine if these pathways are independent or not of the MAP kinase (MAPK) cascade. Our results indicated that the inhibition of PKA by treatment with H-89, an inhibitor of the catalytic subunit of PKA, was capable of inducing GVBD in a dose-dependent manner by a pathway in which Cdc25 phosphatase but not the MAPK cascade is involved. The injection of 50 nl of H-89 10 μM produced GVBD percentages similar to those obtained with treatment with progesterone. In addition, the assays with okadaic acid (OA), a PP2A inhibitor, significantly enhanced the percentage of oocytes that resumed meiosis by a signal transducing pathway in which the activation of the MEK–MAPK pathway is necessary, but in which Cdc25 phosphatase was not involved. Treatment with H-89, was able to overcome the inhibitory effect of PKA on GVBD; however, the inhibition of Cdc25 activity with NaVO3 was able to overcome the induction of GVBD by H-89. Although the connections between PKA and other signalling molecules that regulate oocytes maturation are still unclear, our results suggest that phosphatase Cdc25 may be the direct substrate of PKA. In Xenopus oocytes it was proposed that PP2A, a major Ser/Thr phosphatase present, is a negative regulator of Cdc2 activation. However, in Bufo arenarum oocytes, inhibition of Cdc25 with NaVO3 did not inhibit OA-induced maturation, suggesting that the target of PP2A was not the Cdc25 phosphatase. MAPK activation has been reported to be essential in Xenopus oocytes GVBD. In B. arenarum oocytes we demonstrated that the inhibition of MAPK by PD 98059 prevented the activation of MPF induced by OA, suggesting that the activation of the MAPK cascade produced an inhibition of Myt1 and, in consequence, the activation of MPF without participation of the Cdc25 phosphatase. Our results suggest that in incompetent oocytes of B. arenarum two signal transduction pathways may be involved in the control of MPF activation: (1) the inhibition of phosphatase 2A that through the MEK–MAPK pathway regulates the activity of the Myt1; and (2) the inhibition of AMPc–PKA, which affects the activity of the Cdc25 phosphatase.
Involvement of the dehydroleucodine alpha-methylene-gamma-lactone function in GVBD inhibition in Bufo arenarum oocytes
- G. Sánchez Toranzo, L.A. López, J. Zapata Martínez, M.C. Gramajo Bühler, M.I. Bühler
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Dehydroleucodine (DhL), a sesquiterpenic lactone, was isolated and purified from aerial parts of Artemisia douglasiana Besser, a medicinal herb used in Argentina. DhL is an alpha-methylene butyro-gamma-lactone ring connected to a seven-membered ring fused to an exocyclic alpha,beta-unsaturated cyclopentenone ring
It has been previously shown that DhL selectively induces a dose-dependent transient arrest in G2 of both meristematic cells and vascular smooth muscle cells. Treatment with DhL induces an inhibition of spontaneous and progesterone-induced maturation in a dose-dependent manner in Bufo arenarum fully grown oocytes arrested at G2, at the beginning of meiosis I. However, the nature of the mechanisms involved in the process is still unknown.
The aim of this work was to analyse whether DhL's alpha-methylene-gamma-lactone function is responsible for the inhibition effect on meiosis reinitiation of Bufo arenarum oocytes as well as some of the transduction pathways that could be involved in this effect using a derivative of DhL inactivated for alpha-methylenelactone, the 11,13-dihydro-dehydroleucodine (2H-DhL).
The use of 2H-DhL in the maturation promoting factor (MPF) amplification experiments by injection of both cytoplasm with active MPF and of germinal vesicle content showed results similar to the ones obtained with DhL, suggesting that the hydrogenated derivative would act in a similar way to DhL.
Pretreatment with DhL or 2H-DhL did not affect the percentage of germinal vesicle breakdown (GVBD) induced by H89, a protein kinase A (PKA) inhibitor, which suggests that these lactones would act on another step of the signalling pathway that induces MPF activation. The fact that both DhL and 2H-Dhl inhibit GVBD induced by okadaic acid microinjection suggests that they could act on the activity of the Myt1 kinase. This idea is supported by the experiments of injection of GV contents in which an inhibitory effect of these lactones on GVBD was also observed.
Our results indicate that the inhibitory effect on meiosis progression of DhL does not depend only on the activity of the alpha-methylenelactone function, as its hydrogenated derivative, 2H-DhL, in which this function has been inactivated, causes similar effects on amphibian oocytes. However, 2H-DhL was less active than DhL as higher doses were required to obtain a significant inhibition. On the other hand, the analysis of the participation of certain mediators in some of the signalling pathways leading to MPF activation suggests that the Myt1 kinase could be a target of these lactones, while cdc25 phosphatase would not be affected. Besides, the PKA inhibition assays indicate that these lactones would act earlier in the signalling pathways.