Light modulates the melanophore response to alpha-MSH in Xenopus laevis: An analysis of the signal transduction crosstalk mechanisms involved

Melanin granule (melanosome) dispersion within Xenopus laevis melanophores is evoked either by light or alpha-MSH. We have previously demonstrated that the initial biochemical steps of light and alpha-MSH signaling are distinct, since the increase in cAMP observed in response to alpha-MSH was not seen after light exposure. cAMP concentrations in response to alpha-MSH were significantly lower in cells pre-exposed to light as compared to the levels in dark-adapted melanophores. Here we demonstrate the presence of an adenylyl cyclase (AC) in the Xenopus melanophore, similar to the mammalian type IX which is inhibited by Ca(2+)-calmodulin-activated phosphatase. This finding supports the hypothesis that the cyclase could be negatively modulated by a light-promoted Ca(2+) increase. In fact, the activity of calcineurin PP2B phosphatase was increased by light, which could result in AC IX inhibition, thus decreasing the response to alpha-MSH. St-Ht31, a disrupting agent of protein kinase A (PKA)-anchoring kinase A protein (AKAP) complex totally blocked the melanosome dispersing response to alpha-MSH, but did not impair the photo-response in Xenopus melanophores. Sequence comparison of a melanophore AKAP partial clone with GenBank sequences showed that the anchoring protein was a gravin-like adaptor previously sequenced from Xenopus non-pigmentary tissues. Co-immunoprecipitation of Xenopus AKAP and the catalytic subunit of PKA demonstrated that PKA is associated with AKAP and it is released in the presence of alpha-MSH. We conclude that in X laevis melanophores, AKAP12 (gravin-like) contains a site for binding the inactive PKA thus compartmentalizing PKA signaling and also possesses binding sites for PKC. Light diminishes alpha-MSH-induced increase of cAMP by increasing calcineurin (PP2B) activity, which in turn inhibits adenylyl cyclase type IX, and/or by activating PKC, which phosphorylates the gravin-like molecule, thus destabilizing its binding to the cell membrane. (C) 2009 Elsevier Inc. All rights reserved.

Melatonin and IP(3)-induced Ca(2+) Release from Intracellular Stores in the Malaria Parasite Plasmodium falciparum within Infected Red Blood Cells

IP(3)-dependent Ca(2+) signaling controls a myriad of cellular processes in higher eukaryotes and similar signaling pathways are evolutionarily conserved in Plasmodium, the intracellular parasite that causes malaria. We have reported that isolated, permeabilized Plasmodium chabaudi, releases Ca(2+) upon addition of exogenous IP(3). In the present study, we investigated whether the IP(3) signaling pathway operates in intact Plasmodium falciparum, the major disease-causing human malaria parasite. P. falciparum-infected red blood cells (RBCs) in the trophozoite stage were simultaneously loaded with the Ca(2+) indicator Fluo-4/AM and caged-IP(3). Photolytic release of IP(3) elicited a transient Ca(2+) increase in the cytosol of the intact parasite within the RBC. The intracellular Ca(2+) pools of the parasite were selectively discharged, using thapsigargin to deplete endoplasmic reticulum (ER) Ca(2+) and the antimalarial chloroquine to deplete Ca(2+) from acidocalcisomes. These data show that the ER is the major IP(3)-sensitive Ca(2+) store. Previous work has shown that the human host hormone melatonin regulates P. falciparum cell cycle via a Ca(2+)-dependent pathway. In the present study, we demonstrate that melatonin increases inositol-polyphosphate production in intact intraerythrocytic parasite. Moreover, the Ca(2+) responses to melatonin and uncaging of IP(3) were mutually exclusive in infected RBCs. Taken together these data provide evidence that melatonin activates PLC to generate IP(3) and open ER-localized IP(3)-sensitive Ca(2+) channels in P. falciparum. This receptor signaling pathway is likely to be involved in the regulation and synchronization of parasite cell cycle progression.

Vitellin-binding proteins in the nematode Oscheius tipulae (Nematoda, Rhabditida)

We describe the first application of a non-radioactive ligand-blotting technique to the characterization of proteins interacting with nematode vitellins. Chromatographically purified vitellins from the free-living nematode Oscheius tipulae were labeled with fluorescein in vitro. Ligand-blotting assays with horseradish peroxidase-conjugated anti-fluorescein antibodies showed that labeled vitellins reacted specifically with a polypeptide of approximately 100 kDa, which we named P100. This polypeptide is a specific worm`s vitellin-binding protein that is present only in adult worms. Blots containing purified O. tipulae vitellin preparations showed no detectable signal in the 100 kDa region, ruling out any possibility of yolk polypeptides self-assembling under the conditions used in our assay. Experiments done in the presence of alpha-methyl mannoside ruled out the possibility of vitellins binding to P100 through mannose residues. Triton X-114 fractionation of whole worm extracts showed that P100 is either a membrane protein or has highly hydrophobic regions. (C) 2008 Elsevier Inc. All rights reserved.

Oscheius tipulae as an example of eEF1A gene diversity in nematodes

We characterized four eEF1A genes in the alternative rhabditid nematode model organism Oscheius tipulae. This is twice the copy number of eEF1A genes in C. elegans, C. briggsae, and, probably, many other free-living and parasitic nematodes. The introns show features remarkably different from those of other metazoan eEF1A genes. Most of the introns in the eEF1A genes are specific to O. tipulae and are not shared with any of the other genes described in metazoans. Most of the introns are phase 0 (inserted between two codons), and few are inserted in protosplice sites (introns inserted between the nucleotide sequence A/CAG and G/A). Two of these phase 0 introns are conserved in sequence in two or more of the four eEF1A gene copies, and are inserted in the same position in the genes. Neither of these characteristics has been detected in any of the nematode eEF1A genes characterized to date. The coding sequences were also compared with other eEF1A cDNAs from 11 different nematodes to determine the variability of these genes within the phylum Nematoda. Parsimony and distance trees yielded similar topologies, which were similar to those created using other molecular markers. The presence of more than one copy of the eEF1A gene with nearly identical coding regions makes it difficult to define the orthologous cDNAs. As shown by our data on O. tipulae, careful and extensive examination of intron positions in the eEF1A gene across the phylum is necessary to define their potential for use as valid phylogenetic markers.

Regulation of Radial Glial Motility by Visual Experience

Radial glia in the developing optic tectum express the key guidance molecules responsible for topographic targeting of retinal axons. However, the extent to which the radial glia are themselves influenced by retinal inputs and visual experience remains unknown. Using multiphoton live imaging of radial glia in the optic tectum of intact Xenopus laevis tadpoles in conjunction with manipulations of neural activity and sensory stimuli, radial glia were observed to exhibit spontaneous calcium transients that were modulated by visual stimulation. Structurally, radial glia extended and retracted many filopodial processes within the tectal neuropil over minutes. These processes interacted with retinotectal synapses and their motility was modulated by nitric oxide (NO) signaling downstream of neuronal NMDA receptor (NMDAR) activation and visual stimulation. These findings provide the first in vivo demonstration that radial glia actively respond both structurally and functionally to neural activity, via NMDAR-dependent NO release during the period of retinal axon ingrowth.

Samba, a Xenopus hnRNP Expressed in Neural and Neural Crest Tissues

RNA binding proteins regulate gene expression at the posttranscriptional level and play important roles in embryonic development. Here, we report the cloning and expression of Samba, a Xenopus hnRNP that is maternally expressed and persists at least until tail bud stages. During gastrula stages, Samba is enriched in the dorsal regions. Subsequently, its expression is elevated only in neural and neural crest tissues. In the latter, Samba expression overlaps with that of Slug in migratory neural crest cells. Thereafter, Samba is maintained in the neural crest derivatives, as well as other neural tissues, including the anterior and posterior neural tube and the eyes. Overexpression of Samba in the animal pole leads to defects in neural crest migration and cranial cartilage development. Thus, Samba encodes a Xenopus hnRNP that is expressed early in neural and neural crest derivatives and may regulate crest cells migratory behavior. Developmental Dynamics 238:204-209, 2009. (C) 2008 Wiley-Liss, Inc.

Effect of Hormonal Supplementation Periods and In Vitro Maturation Media on Developmental Competence of Pig Oocytes

Background: The oocyte ability to undergo successful fertilization, cleavage and embryonic development depends on meiotic maturation and developmental competence acquisition. In vitro maturation (IVM) protocols currently use eCG, hCG or a combination of both, the effect of these gonadotrophins during IVM and subsequent embryonic development is still controversial. Several media have been used for IVM of porcine oocytes: TCM199, Whitten's and NCSU23 have also been shown to support pig oocyte IVM. This study was designed to determine the effect of hormonal supplementation period and maturation media during in vitro maturation of pig oocytes (1) and subsequent embryonic development (2). Materials, Methods & Results: Oocytes with intact cumulus oophurus layers and homogeneous cytoplasm were collected from prebubertal gilts. IVM was subjected in NCSU23, TCM199 or Whitten's media supplemented with 10 IU/mL eCG and 10 IU/mL hCG for the first 24 or 48 h of IVM. In each replicate the oocytes were fixed every 4 h from 32 to 48 h IVM or the past 48 h after IVM, oocytes were fertilized in vitro in mTBM medium for six hours and cultured in NCSU23 medium for nine days. Cleavage, blastocyst and hatching rates were evaluated at 48 h (day 2), 168 h (day 7) and 216 h (day 9), respectively. The addition of eCG and hCG during the first 24 h IVM increased the proportion of oocytes that reached MII stage at 44 h of maturation in NCSU23 medium. This effect was also observed in Whitten medium at 44 and 48 h (P < 0.05). However, it was not observed in the TCM199 medium. No effect of maturation medium on oocyte nuclear maturation (P > 0.05) was observed in oocytes matured in the presence of eCG and hCG during the first 24 h IVM or during 48 h IVM. A progressive increase of maturation indexes was observed on oocytes matured with hormonal supplementation in Whitten media for 24 h. Higher indexes were obtained at 44 and 48 h. When NCSU23 media was used, no difference after 36 h of maturation was observed. The same result was observed in TCM199. A progressive increase of maturation indexes was observed on oocytes matured with hormonal supplementation for 48 h in Whitten media. Higher indexes were obtained in 36 and 40 h. When NCSU23 or TCM199 were used, no difference was observed. No effect of IVM media on the percentage of fertilized oocytes and polyspermic oocytes or number of spermatozoa per fertilized oocytes was observed. Also, no effect of IVM media on cleavage and blastocyst rates was seen. However, the proportion of hatched blastocysts was lower in NCSU23 compared to Whitten or TCM199. Discussion: Similar results were reported by Marques et al. [13], that it no differences between hormonal supplementation for 22 or 44 h were observed. Therefore, more studies are needed to elucidate the role of these hormones in nuclear in vitro maturation in pig oocytes. In conclusion, no effect of maturation media on meiotic progression was observed. However, the proportion of oocytes that reached metaphase II (MII) stage was higher when eCG + hCG were added for 24 h than 48 h mainly at the 44 h of maturation. In addition, no differences were observed in cleavage and blastocyst rates of the cultured embryos. However, embryos cultured in NCSU23 showed lower rates of hatching compared to other media. These results indicated no effect of maturation media on the fertilization and embryonic development even in the presence of cysteine, PFF and EGF, except for hatched embryos that these rates were lower in NCSU23.

Sequential analysis of global gene expression profiles in immature and in vitro matured bovine oocytes: potential molecular markers of oocyte maturation

Background: Without intensive selection, the majority of bovine oocytes submitted to in vitro embryo production (IVP) fail to develop to the blastocyst stage. This is attributed partly to their maturation status and competences. Using the Affymetrix GeneChip Bovine Genome Array, global mRNA expression analysis of immature (GV) and in vitro matured (IVM) bovine oocytes was carried out to characterize the transcriptome of bovine oocytes and then use a variety of approaches to determine whether the observed transcriptional changes during IVM was real or an artifact of the techniques used during analysis. Results: 8489 transcripts were detected across the two oocyte groups, of which similar to 25.0% (2117 transcripts) were differentially expressed (p < 0.001); corresponding to 589 over-expressed and 1528 under-expressed transcripts in the IVM oocytes compared to their immature counterparts. Over expression of transcripts by IVM oocytes is particularly interesting, therefore, a variety of approaches were employed to determine whether the observed transcriptional changes during IVM were real or an artifact of the techniques used during analysis, including the analysis of transcript abundance in oocytes in vitro matured in the presence of a-amanitin. Subsets of the differentially expressed genes were also validated by quantitative real-time PCR (qPCR) and the gene expression data was classified according to gene ontology and pathway enrichment. Numerous cell cycle linked (CDC2, CDK5, CDK8, HSPA2, MAPK14, TXNL4B), molecular transport (STX5, STX17, SEC22A, SEC22B), and differentiation (NACA) related genes were found to be among the several over-expressed transcripts in GV oocytes compared to the matured counterparts, while ANXA1, PLAU, STC1and LUM were among the over-expressed genes after oocyte maturation. Conclusion: Using sequential experiments, we have shown and confirmed transcriptional changes during oocyte maturation. This dataset provides a unique reference resource for studies concerned with the molecular mechanisms controlling oocyte meiotic maturation in cattle, addresses the existing conflicting issue of transcription during meiotic maturation and contributes to the global goal of improving assisted reproductive technology.

P2X4 receptors interact with both P2X2 and P2X7 receptors in the form of homotrimers

BACKGROUND AND PURPOSE The P2X receptor family consists of seven subunit types - P2X1-P2X7. All but P2X6 are able to assemble as homotrimers. In addition, various subunit permutations have been reported to form heterotrimers. Evidence for heterotrimer formation includes co-localization, co-immunoprecipitation and the generation of receptors with novel functional properties; however, direct structural evidence for heteromer formation, such as chemical cross-linking and single-molecule imaging, is available in only a few cases. Here we examined the nature of the interaction between two pairs of subunits - P2X2 and P2X4, and P2X4 and P2X7. EXPERIMENTAL APPROACH We used several experimental approaches, including in situ proximity ligation, co-immunoprecipitation, co-isolation on affinity beads, chemical cross-linking and atomic force microscopy (AFM) imaging. KEY RESULTS Both pairs of subunits co-localize upon co-transfection, interact intimately within cells, and can be co-immunoprecipitated and co-isolated from cell extracts. Despite this, chemical cross-linking failed to show evidence for heteromer formation. AFM imaging of isolated receptors showed that all three subunits had the propensity to form receptor dimers. This self-association is likely to account for the observed close interaction between the subunit pairs, in the absence of true heteromer formation. CONCLUSIONS AND IMPLICATIONS We conclude that both pairs of receptors interact in the form of distinct homomers. We urge caution in the interpretation of biochemical evidence indicating heteromer formation in other cases.

Global poly(A) mRNA expression profile measured in individual bovine oocytes and cleavage embryos

The objective of this article was to estimate quantitative differences for GAPDH transcripts and poly(A) mRNA: (i) between oocytes collected from cumulus-oocyte complexes (COCs) qualified morphologically as grades A and B; (ii) between grade A oocytes before and after in vitro maturation (IVM); and (iii) among in vitro-produced embryos at different developmental stages. To achieve this objective a new approach was developed to estimate differences between poly(A) mRNA when using small samples. The approach consisted of full-length cDNA amplification (acDNA) monitored by real-time PCR, in which the cDNA from half of an oocyte or embryo was used as a template. The GAPDH gene was amplified as a reverse transcription control and samples that were not positive for GAPDH transcripts were discarded. The fold differences between two samples were estimated using delta Ct and statistical analysis and were obtained using the pairwise fixed reallocation randomization test. It was found that the oocytes recovered from grade B COCs had quantitatively less poly(A) mRNA (p < 0.01) transcripts compared with grade A COCs (1 arbitrary unit expression rate). In the comparison with immature oocytes (I arbitrary unit expression rate), the quantity of poly(A) mRNA did not change during IVM, but declined following IVF and varied with embryo culture (p < 0.05). Amplification of cDNA by real-time PCR was an efficient method to estimate differences in the amount of poly(A) mRNA between oocytes and embryos. The results obtained from individual oocytes suggested an association between poly(A) mRNA abundance and different morphological qualities of oocytes from COCs. In addition, a poly(A) mRNA profile was characterized from oocytes undergoing IVM, fertilization and blastocyst heating.

Spindle and chromosome configurations of in vitro-matured oocytes from polycystic ovary syndrome and ovulatory infertile women: a pilot study

To evaluate the meiotic spindle and chromosomal distribution of in vitro-matured oocytes from infertile nonobese women with PCOS and male or tubal causes of infertility (controls), and to compare in vitro maturation (IVM) rates between groups. Seventy four patients (26 with PCOS and 48 controls) undergoing stimulated cycles of oocyte retrieval for ICSI were selected prospectively. Thirteen PCOS patients and 27 controls had immature oocytes retrieved submitted to IVM. After IVM, oocytes showing extrusion of the first polar body were fixed and processed for evaluation of the meiotic spindle and chromosome distribution by immunofluorescence microscopy. There were no differences between PCOS and control groups with respect to IVM rates (50.0% and 42.9%, respectively) nor the percentage of meiotic abnormalities in metaphase II oocytes (35.3% and 25%, respectively). In vitro-matured oocytes obtained from stimulated cycles of nonobese PCOS did not have an increased ratio of meiotic abnormalities.

Preimplantation development and expression of Hsp-70 and Bax genes in bovine blastocysts derived from oocytes matured in alpha-MEM supplemented with growth factors and synthetic macromolecules

In vitro culture conditions affect both the maternal and embryonic expression of genes and is likely to alter both oocyte and embryo developmental competence. The search for better and less variable culture conditions simulating those in vivo has led to the development of defined culture media, with lower impact on the molecular reprogramming of oocytes and embryos. We evaluated embryo development and relative abundance (RA) of Hsp-70 and Bax transcripts in bovine blastocysts produced from oocytes matured in a chemically defined IVM system with synthetic polymers. Immature cumulus oocyte complexes (COCs) were matured for 22-24 h in alpha-MEM supplemented with IGF-1, insulin, 0.1% polyvinyl alcohol (PVA), or 0.1% polyvinylpyrrolidone (PVP), but without FSH or LH. The control group consisted of COCs matured it, TCM plus FSH and 10% estrous cow serum. After fertilization. presumptive zygotes were co-cultured with cumulus cells until 224 h post-insemination. Total RNA was isolated from embryo pools, reverse transcribed into cDNA, and subjected to transcript analysis by real-time PCR. Cleavage rate was higher (P < 0.05) for the control group (68.3%) than for the PVA (54.4%) and PVP-40 (58.3%) groups. Nevertheless. there was no difference among the PVA, PVP-40 and control groups in blastocyst or hatching rates. similarly, no difference in relative abundance of Hsp-70 and Bax transcripts was detected in comparison to the control group. We inferred that bovine oocytes can be matured in serum- and gonadotrophin-free medium supplemented with PVA or PVP, enriched with IGF-I and insulin, without altering post-cleavage development and relative abundance of some genes associated with stress and apoptosis. (C) 2009 Elsevier Inc. All rights reserved.

Supplemented tissue culture medium 199 is a better medium for in vitro maturation of oocytes from women with polycystic ovary syndrome women than human tubal fluid

Objective: To compare oocyte maturation, fertilization and cleavage rates, and embryonic developmental quality after culture of human immature oocytes from polycystic ovary syndrome (PCOS) patients in human tubal fluid (HTF) or tissue culture medium (TCM) 199. Design: Prospective, randomized, controlled trial. Setting: University hospital. Patient(s): Thirteen women undergoing 23 in vitro maturation cycles, from whom 119 oocytes were retrieved. Intervention(s): Cumulus-enclosed germinal vesicle-stage oocytes matured in TCM-199-supplemented or HTF-supplemented media. Main Outcome Measure(s): Oocyte maturation and fertilization rates, embryonic developmental quality. Result(s): Significant differences were observed between TCM 199 and HTF regarding maturation rate (82% vs. 56.9%), fertilization rate (70% vs. 39.4%), and embryo quality (81.3% vs. 41.7%). Conclusion(S): Human tubal fluid medium, although widely used for embryo fertilization and maintenance in IVF techniques, is not,in appropriate medium for the maturation of oocytes obtained from PCOS patients in nonstimulated cycles. (Fertil Steril(R) 2009;91:509-13. (C)2009 by American Society for Reproductive Medicine.)

Synergistic Effect of Porcine Follicular Fluid and Dibutyryl Cyclic Adenosine Monophosphate on Development of Parthenogenetically Activated Oocytes from Pre-Pubertal Gilts

This study investigated the effect of porcine follicular fluid (PFF) and dibutyryl cyclic adenosine monophosphate (dbcAMP) during in vitro maturation (IVM) of porcine oocytes on meiotic maturation, fertilization and embryo development, and compared the effect of supplementing the embryo culture media with PFF or foetal bovine serum (FBS) on embryo development. Oocytes from pre-pubertal gilts were IVM for 44 h, and parthenogenetically activated or in vitro-fertilized. Embryos were cultured in porcine zygote medium (PZM3) for 7 days. Cleavage and blastocyst rates were evaluated at 48 h and 7 days of culture. The supplementation of the IVM medium with 25% PFF and 1 mm dbcAMP for the first 22 h resulted in more (p < 0.05) embryos developing to the blastocyst stage as compared with the inclusion of dbcAMP alone. The dbcAMP + PFF combination increased (p < 0.05) the average number of nuclei per blastocyst as compared with either of these components alone or in its absence. A synergistic effect of dbcAMP + PFF during IVM was also reflected in the capacity of oocytes to regulate sperm penetration and prevent polyspermy, as twice as many oocytes from the control group were penetrated by more than one sperm as compared with those matured in the presence of both dbcAMP and PFF. The supplementation of PZM3 with 10% FBS from days 5 to 7 of culture significantly improved the total cell quantity in embryos derived either from control or dbcAMP + PFF matured oocytes. There was no effect on the total cell quantity when FBS was replaced by the same concentration of PFF. These studies showed that dbcAMP, PFF and FBS can improve both the quantity (57.3% vs 41.5%) and quality (74.8 vs 33.3 nuclei) of porcine blastocysts derived from oocytes recovered of pre-pubertal gilts.

Oestrogen and Progesterone Receptor Gene Expression in Canine Oocytes and Cumulus Cells Throughout the Oestrous Cycle

The aim of this research was to analyze oestrogen receptor-alpha (ER alpha), ER beta and progesterone receptor (PR) gene expression in the canine oocyte and cumulus cells throughout the oestrous cycle. Ovaries from 38 bitches were recovered after ovariohysterectomy and sliced. The phase of the oestrous cycle was determined by vaginal cytology, vaginoscopy and serum hormonal measurements. Oocytes were mechanically denuded by repeated pipetting. For each phase of the cycle, a sample was composed by a pool of 50 oocytes (sample number: prooestrus = 3, oestrus = 8, dioestrus = 5 and anoestrus = 5) or a pool of cumulus cells (prooestrus = 4, oestrus = 7, dioestrus = 4 and anoestrus = 6). Oocyte and cumulus cells` total RNA was isolated and reverse transcription was conducted to perform real-time PCR. Oestrogen receptor-alpha was expressed throughout the cycle in the oocyte (33.33%, 25.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively) and cumulus cells (50.0%, 47.14%, 25.0% and 66.67% for prooestrus, oestrus, dioestrus and anoestrus, respectively). In the oocyte, the ER beta was also expressed in all phases of the cycle (33.33%, 50.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively), whereas in cumulus cells, ER beta was only expressed during prooestrus (50%) and oestrus (14.29%). Interestingly, while the oocyte PR was not detected in any phase of the cycle, this receptor was expressed during prooestrus (50%), oestrus (42.86%) and anoestrus (16.67%) in cumulus cells. In conclusion, canine oocytes express ER alpha and ER beta throughout the oestrous cycle, however, there is a lack of PR expression in all these phases. Moreover, in cumulus cells, only ER alpha was expressed throughout the oestrous cycle.