The effects of MEK and PKA inhibition on Spire proteins during oocyte maturation.

Asymmetric division of oocytes driven by chromosome migration is a crucial step of oocyte maturation. Actin filaments take key roles in chromosome migration in oocytesThe aim of this study was to determine the effects of MEK and PKA inhibition on the levels of Spire-1 and Spire-2 proteins that are known to be related to actin nucleation.MEK inhibitor PD98059 and PKA inhibitor H89 were applied during IVM to the oocytes retrieved from preovulatory ovarian follicles of PMSG induced 3-5 weeks old female BalbC mice. GVBD and PBE rates were determined. Spire-1 and Spire-2 proteins were detected by immunofluorescence and western blot in oocytes at different maturation stages.Though GVBD rates were similar in different groups, PBE rates were lower in the MEK inhibition group. Through immunofluorescence, cortical localizations of Spire-1 and Spire-2 were determined. MEK inhibition resulted in a decrease in cortical Spire-1 and Spire-2 levels in PBE oocytes. PKA inhibition led to an increase in cortical Spire-1 levels in spindle migration stage oocytes, and an increase in cortical and total Spire-2 levels in PBE oocytes. Application of both MEK and PKA inhibition resulted in compensation of the decrease in Spire-1, while Spire-2 levels remained low with no compensation of PKA inhibition.According to the results of this study, chemical inhibition of MEK and PKA during oocyte maturation alters Spire-1 and Spire-2 protein levels.

Interactions of Ingested Polystyrene Microplastics with Heavy Metals (Cadmium or Silver) as Environmental Pollutants: A Comprehensive In Vivo Study Using Drosophila melanogaster.

Living organisms are now constantly exposed to microplastics and nanoplastics (MNPLs), and besides their toxic potential, they can also act as carriers of various hazardous elements such as heavy metals. Therefore, this study explored possible interactions between polystyrene microplastics (PSMPLs) and two metal pollutants: cadmium chloride (CdCl2) and silver nitrate (AgNO3). To better understand the extent of biological effects caused by different sizes of PSMPLs, we conducted in vivo experiments with five doses (from 0.01 to 10 mM) that contained polystyrene particles measuring 4, 10, and 20 µm in size on Drosophila larvae. Additional experiments were performed by exposing larvae to two individual metals, CdCl2 (0.5 mM) and AgNO3 (0.5 mM), as well as combined exposure to PSMPLs (0.01 and 10 mM) and these metals, in an attempt to gain new insight into health risks of such co-exposure. Using transmission electron microscopy imaging, we managed to visualize the biodistribution of ingested PSMPLs throughout the fly's body, observing the interactions of such plastics with Drosophila intestinal lumen, cellular uptake by gut enterocytes, the passage of plastic particles through the intestinal barrier to leak into the hemolymph, and cellular uptake by hemocytes. Observations detected size and shape changes in the ingested PSMPLs. Egg-to-adult viability screening revealed no significant toxicity upon exposure to individual doses of tested materials; however, the combined exposure to plastic and metal particles induced aggravated genotoxic effects, including intestinal damage, genetic damage, and intracellular oxidative stress (ROS generation), with smaller sized plastic particles + metals (cadmium and silver) causing greater damage.

Hazard Assessment of the Effects of Acute and Chronic Exposure to Permethrin, Copper Hydroxide, Acephate, and Validamycin Nanopesticides on the Physiology of Drosophila: Novel Insights into the Cellular Internalization and Biological Effects.

New insights into the interactions between nanopesticides and edible plants are required in order to elucidate their impacts on human health and agriculture. Nanopesticides include formulations consisting of organic/inorganic nanoparticles. Drosophila melanogaster has become a powerful model in genetic research thanks to its genetic similarity to mammals. This project mainly aimed to generate new evidence for the toxic/genotoxic properties of different nanopesticides (a nanoemulsion (permethrin nanopesticides, 20 ± 5 nm), an inorganic nanoparticle as an active ingredient (copper(II) hydroxide [Cu(OH)2] nanopesticides, 15 ± 6 nm), a polymer-based nanopesticide (acephate nanopesticides, 55 ± 25 nm), and an inorganic nanoparticle associated with an organic active ingredient (validamycin nanopesticides, 1177 ± 220 nm)) and their microparticulate forms (i.e., permethrin, copper(II) sulfate pentahydrate (CuSO4·5H2O), acephate, and validamycin) widely used against agricultural pests, while also showing the merits of using Drosophila-a non-target in vivo eukaryotic model organism-in nanogenotoxicology studies. Significant biological effects were noted at the highest doses of permethrin (0.06 and 0.1 mM), permethrin nanopesticides (1 and 2.5 mM), CuSO4·5H2O (1 and 5 mM), acephate and acephate nanopesticides (1 and 5 mM, respectively), and validamycin and validamycin nanopesticides (1 and 2.5 mM, respectively). The results demonstrating the toxic/genotoxic potential of these nanopesticides through their impact on cellular internalization and gene expression represent significant contributions to future nanogenotoxicology studies.

The interaction of Wnt signaling members with growth factors in cultured granulosa cells.

Wnt family members have recently been distinguished in the adult ovary with potential roles in ovarian function. Though particular growth factors interact with Wnt signaling members in extraovarian cell types, it is unclear whether this interaction is applicable in the granulosa cells. Therefore, the current study aimed to determine the effect of insulin-like growth factor-1 (IGF-I), epidermal growth factor (EGF) and basic fibroblast growth factor (FGF-ß) on Wnt ligands WNT2 and WNT4 and Wnt receptor Frizzled-4 (FZD4) protein levels in cultured mouse granulosa cells. Granulosa cells were isolated from antral follicles of adult Balb/C mice and cultured for 24 hours in the presence of 100 ng/mL of IGF-I, or EGF or FGF-ß. WNT2, WNT4 and FZD4 protein levels were evaluated through western blotting after the culture process. IGF-I treated granulosa cells had significantly the highest level of WNT2 and WNT4 as well as FZD4 when compared to FGF-ß and EGF groups. FGF-ß group had a significantly higher level of WNT2, WNT4 and FZD4 expression when compared to EGF group. FZD4 expression was at the highest level in the IGF-I group and this difference was statistically significant for all groups including uncultured cells and vehicle group. In addition, FGF-ß was shown to positively affect the adhesion of granulosa cells. This study demonstrates that IGF-I, FGF-ß and EGF have differential effects on the expressions of WNT2, WNT4, and FZD4 in cultured mouse granulosa cells, suggesting that particular growth factors related to ovarian function might conduct their roles in the ovary through Wnt signaling.

Enteric apelin enhances the stress-induced stimulation of colonic motor functions.

In response to stress, apelin and corticotropin-releasing factor (CRF) are upregulated in the gastrointestinal (GI) tract. This study was designed to investigate the effect of stress on endogenous apelin in colon and its regulatory role on colonic motor functions. Colon transit (CT) was measured in rats exposed to acute restraint stress (ARS). APJ and CRF receptor antagonists F13A and astressin were administered intraperitoneally 30 min before ARS loading. Colonic muscle contractions were evaluated by in-vivo motility recording and in-vitro organ bath studies. Detection of apelin or CRF was performed using immunohistochemistry in proximal and distal colon of non-stressed (NS) and ARS-loaded rats. Immunoreactivity of CRF1 with apelin or APJ receptor was detected with double-labeled immunofluorescence in colonic myenteric neurons. Compared with NS rats, ARS accelerated the CT which was attenuated significantly by F13A or astressin. Following ARS, the expression of CRF was increased remarkably in distal colon, while the stress-induced change was not prominent in proximal colon. Apelin-positive cells were detected in myenteric ganglia of distal colon, while no apelin immunoreactivity observed in myenteric neurons of proximal colon. Both apelin and APJ receptor are colocalized with CRF1 in myenteric neurons of distal colon. In the in-vivo colonic motility experiments, apelin-13 exhibited a rapid stimulatory effect. CRF administration increased the motility which was abolished by F13A. Apelin-induced contractions in muscle strips were no longer observed with preadministration of F13A. These results suggest that enteric apelin contributes to the action of CRF on colonic motor functions under stressed conditions.LAY SUMMARYIt has been suggested in rodents that acute stress increases the expression of apelin in gastrointestinal tissues. We have found that under stressed conditions, enteric apelin contributes to the CRF-induced alterations in colonic motor functions through APJ receptor.

The effect of GnRH antagonist cetrorelix on Wnt signaling members in pubertal and adult mouse ovaries.

Wide application of gonadotropin-releasing hormone (GnRH) agonists and antagonists for clinical purposes determines their effects on ovarian signaling pathways. Our study aimed to determine the localization, expression levels of Wnt signaling members in the pubertal and adult mouse ovary and the impact of GnRH antagonist cetrorelix on these signaling members. 0.5 mg/kg of cetrorelix was injected to 3-and 6-week-old mice for 2 weeks. At the end of injection, ovaries from 5 (5Ce)- to 8-week (8Ce)-old mice were embedded in paraffin for immunohistochemistry and homogenized for western blot to compare with control (5C-8C) and sham groups (5S-8S). WNT2 and WNT4 showed higher expression in thecal and stromal cells in adult mouse ovaries and only WNT4 expression was affected by cetrorelix. FZD1 was localized mainly in oocytes of pubertal ovaries and granulosa cells and oocytes of adult ovaries. FZD1 was reduced by cetrorelix in pubertal ovaries. FZD4 was abundantly localized in thecal and stromal cells of all groups and protein level was not affected by cetrorelix. LRP-6 was expressed mainly in oocytes and stromal cells of pubertal, oocytes of adult ovaries and its expression was reduced by cetrorelix in adult ovaries. CTNNB1 intensity in granulosa cells was the lowest in pubertal and the highest in adult ovaries and its expression was decreased by cetrorelix in adult ovaries. Cetrorelix affected the expression of specific members of the Wnt signaling depending on the developmental stage of mice, pointing out its possible interaction with gonadotropins during pubertal and adult stages.

Decreased expression of DNA methyltransferases in the testes of patients with non-obstructive azoospermia leads to changes in global DNA methylation levels.

DNA methylation plays key roles in epigenetic regulation during mammalian spermatogenesis. DNA methyltransferases (DNMTs) function in de novo and maintenance methylation processes by adding a methyl group to the fifth carbon atom of the cytosine residues within cytosine-phosphate-guanine (CpG) and non-CpG dinucleotide sites. Azoospermia is one of the main causes of male infertility, and is classified as obstructive (OA) or non-obstructive (NOA) azoospermia based on histopathological characteristics. The molecular background of NOA is still largely unknown. DNA methylation performed by DNMTs is implicated in the transcriptional regulation of spermatogenesis-related genes. The aim of the present study was to evaluate the cellular localisation and expression levels of the DNMT1, DNMT3A and DNMT3B proteins, as well as global DNA methylation profiles in testicular biopsy samples obtained from men with various types of NOA, including hypospermatogenesis (hyposperm), round spermatid (RS) arrest, spermatocyte (SC) arrest and Sertoli cell-only (SCO) syndrome. In the testicular biopsy samples, DNMT1 expression and global DNA methylation levels decreased gradually from the hyposperm to SCO groups (P P P <0.05). Although both DNMT1 and DNMT3A were localised in the cytoplasm and nucleus of the spermatogenic cells, staining for DNMT3B was more intensive in the nucleus of spermatogenic cells. In conclusion, the findings suggest that significant changes in DNMT expression and global DNA methylation levels in spermatogenic cells may contribute to development of male infertility in the NOA groups. Further studies are needed to determine the molecular biological effects of the altered DNMT expression and DNA methylation levels on development of male infertility.

Acute restraint stress induces cholecystokinin release via enteric apelin.

Stress increases the apelin content in gut, while exogenous peripheral apelin has been shown to induce cholecystokinin (CCK) release. The present study was designed to elucidate (i) the effect of acute stress on enteric production of apelin and CCK, (ii) the role of APJ receptors in apelin-induced CCK release depending on the nutritional status. CCK levels were assayed in portal vein blood samples obtained from stressed (ARS) and non-stressed (NS) rats previously injected with APJ receptor antagonist F13A or vehicle. Duodenal expressions of apelin, CCK and APJ receptor were detected by immunohistochemistry. ARS increased the CCK release which was abolished by selective APJ receptor antagonist F13A. The stimulatory effect of ARS on CCK production was only observed in rats fed ad-libitum. Apelin and CCK expressions were upregulated by ARS. In addition to the duodenal I cells, APJ receptor was also detected in CCK-producing myenteric neurons. Enteric apelin appears to regulate the stress-induced changes in GI functions through CCK. Therefore, apelin/APJ receptor systems seem to be a therapeutic target for the treatment of stress-related gastrointestinal disorders.

Peripheral apelin mediates stress-induced alterations in gastrointestinal motor functions depending on the nutritional status.

Exposure to stress induces gastrointestinal (GI) dysmotility. In rodents, acute restraint stress (ARS) inhibits gastric emptying (GE) and intestinal transit (IT) via central and peripheral corticotropin-releasing factor (CRF)-mediated pathways. Peripherally administered apelin-13 was shown to inhibit GI motor functions; moreover, stress-induced upregulation of gastric apelin content was demonstrated in rats suggesting that peripheral apelin may mediate stress-induced alterations in GI motility. We investigated the role of endogenous peripheral apelin in stress-induced GI dysfunction. GE, IT and gastro-duodenal fasting motility were measured in non-stressed (NS), CRF-injected and ARS-loaded rats. CRF and apelin receptor antagonists astressin or F13A was administered before ARS or peripheral CRF injection. Apelin and APJ receptor expressions were determined using immunohistochemistry and quantified by qRT-PCR. Double immunofluorescence was performed for enteric neuronal apelin. GE and IT were delayed by CRF and ARS. ARS-induced changes were attenuated by F13A, whereas astressin was ineffective. CRF-induced alterations in GE and IT were restored completely by astressin, while they were diminished by F13A. Antral phase III-like contractions were disturbed following ARS which were preserved by preadministration of astressin, but not F13A. CRF impaired gastric and duodenal fasting contractions, while these changes were not altered by F13A. ARS increased apelin expression in stomach and duodenum. Apelin immunoreactivity was detected in mucosa, smooth muscles and myenteric plexi, whereas dense APJ receptor expression was observed within tunica muscularis. APJ receptor was downregulated in rats fasted overnight. These results suggest that enteric apelin acts as an inhibitor stress mediator in the postprandial state.

Corrigendum to: Decreased expression of DNA methyltransferases in the testes of patients with non-obstructive azoospermia leads to changes in global DNA methylation levels.

DNA methylation plays key roles in epigenetic regulation during mammalian spermatogenesis. DNA methyltransferases (DNMTs) function in de novo and maintenance methylation processes by adding a methyl group to the fifth carbon atom of the cytosine residues within cytosine-phosphate-guanine (CpG) and non-CpG dinucleotide sites. Azoospermia is one of the main causes of male infertility, and is classified as obstructive (OA) or non-obstructive (NOA) azoospermia based on histopathological characteristics. The molecular background of NOA is still largely unknown. DNA methylation performed by DNMTs is implicated in the transcriptional regulation of spermatogenesis-related genes. The aim of the present study was to evaluate the cellular localisation and expression levels of the DNMT1, DNMT3A and DNMT3B proteins, as well as global DNA methylation profiles in testicular biopsy samples obtained from men with various types of NOA, including hypospermatogenesis (hyposperm), round spermatid (RS) arrest, spermatocyte (SC) arrest and Sertoli cell-only (SCO) syndrome. In the testicular biopsy samples, DNMT1 expression and global DNA methylation levels decreased gradually from the hyposperm to SCO groups (P P P <0.05). Although both DNMT1 and DNMT3A were localised in the cytoplasm and nucleus of the spermatogenic cells, staining for DNMT3B was more intensive in the nucleus of spermatogenic cells. In conclusion, the findings suggest that significant changes in DNMT expression and global DNA methylation levels in spermatogenic cells may contribute to development of male infertility in the NOA groups. Further studies are needed to determine the molecular biological effects of the altered DNMT expression and DNA methylation levels on development of male infertility.

Central apelin administration and restraint stress induce hypothalamic cholecystokinin release via the APJ receptor.

Exposure to an acute stressor induces up-regulation of apelin and cholecystokinin (CCK) in the hypothalamic paraventricular nucleus (PVN), which is the key brain centre integrating the stress-induced alterations in neuroendocrine, autonomic and behavioural functions. We tested the hypothesis that the release of CCK from the PVN is increased by centrally administered or stress-induced up-regulated endogenous apelin via the APJ receptor. Additionally, the effect of hypothalamic CCK on autonomic outflow was investigated under basal and stressed conditions. In vivo brain microdialysis was performed in rats that received (i) intra-PVN administration of apelin-13 or (ii) acute restraint stress (ARS). For chemical stimulation of the neurones in the PVN, a high concentration of KCl was applied by reverse microdialysis. CCK-8 levels in microdialysates were quantified by an enzyme immunoassay. The immunoreactivity of the APJ receptor and CCK was detected by immunofluorescence in hypothalamic sections. Heart rate variability was assessed in rats that received PVN stimulation or ARS following pre-administration of vehicle or CCK1 receptor antagonist lorglumide. Both intra-PVN exogenous apelin-13 and ARS increased the CCK-8 levels in dialysates significantly. The ARS-induced elevations in CCK levels were reversed by intra-PVN pre-administration of the APJ receptor antagonist F13A. Within the PVN, robust APJ receptor expression was detected on the CCK-producing mediocellular cells, in addition to the parvocellular neurones in the periventricular region. Dual immunoreactivity of APJ/CCK was observed in magnocellular cells to a lesser degree. Both exogenous apelin and ARS increased the CCK immunoreactivity markedly within the PVN, which was diminished significantly by F13A. Sympathetic tonus was increased markedly both by PVN stimulation and ARS, which was attenuated by lorglumide. These results revealed the interaction between apelin and CCK in the brain, suggesting that hypothalamic CCK may contribute to the apelin-induced alterations in autonomic outflow under stressed conditions.

Apelin-APJ system is responsible for stress-induced increase in atrial natriuretic peptide expression in rat heart.

BACKGROUND: The cardiovascular system is a primary target of stress and stress is the most important etiologic factor in cardiovascular diseases. Stressors increase expressions of atrial natriuretic peptide (ANP) and apelin in cardiac tissue. AIM: The aim of the present study was to investigate whether stress-induced apelin has an effect on the expression of ANP in the right atrium of rat heart. METHODS: The rats were divided into the control, stress and F13A+stress groups. In the stress and F13A+stress groups, the rats were subjected to water immersion and restraint stress (WIRS) for 6h. In the F13A+stress group, apelin receptor antagonist F13A, was injected intravenously immediately before application of WIRS. The plasma samples were obtained for the measurement of corticosterone and atrial natriuretic peptide. The atrial samples were used for immunohistochemistry and western blot analysis. RESULTS: F13A administration prevented the rise of plasma corticosterone and ANP levels induced by WIRS. While WIRS application increased the expressions of apelin, HIF-1α and ANP in atrial tissue, while F13A prevented the stress-induced increase in the expression of HIF-1α and ANP. CONCLUSION: Stress-induced apelin induces ANP expression in atrial tissue and may play a role in cardiovascular homeostasis by increasing ANP expression under WIRS conditions.

Superovulation alters DNA methyltransferase protein expression in mouse oocytes and early embryos.

PURPOSE: DNA methylation is an epigenetic mechanism that plays critical roles during mammalian oocyte and preimplantation embryo development. It is achieved by adding a methyl group to the fifth carbon atom of cytosine residues within cytosine-phosphate-guanine (CpG) and non-CpG dinucleotide sites using DNA methyltransferase (DNMT) enzymes for de novo and maintenance methylation processes. DNMT1, DNMT3A, and DNMT3B play important roles in establishing methylation of developmentally related genes in oocytes and early embryos. The purpose of this study is to identify the effect of superovulation on the expression and subcellular localizations of these three DNMT enzymes in the mouse oocytes and early embryos. METHODS: Three groups composed of control, normal dose [5 IU pregnant mare serum gonadotropin (PMSG) and 5 IU human chorionic gonadotropin (hCG)], and high dose [7.5 IU PMSG and 7.5 IU hCG] were created from 4-5-week-old female BALB/c mice. The relative expression and subcellular localizations of the DNMT proteins in the control and experiment groups have been characterized by using immunofluorescence staining subsequently analyzed in detailed. RESULTS: DNMT1, DNMT3A, and DNMT3B protein expression in the germinal vesicle and metaphase II oocytes and in one-cell and two-cell embryos differed significantly when some of the normal- and high-dose groups were compared with the control counterparts. CONCLUSION: This study has demonstrated for the first time that superovulation alters expression levels of the DNMT proteins, a finding that indicates that certain developmental defects in superovulated oocytes and early embryos may result from impaired DNA methylation processes.

DNMT1, DNMT3A and DNMT3B proteins are differently expressed in mouse oocytes and early embryos.

DNA methylation is one of the epigenetic mechanisms and plays important roles during oogenesis and early embryo development in mammals. DNA methylation is basically known as adding a methyl group to the fifth carbon atom of cytosine residues within cytosine-phosphate-guanine (CpG) and non-CpG dinucleotide sites. This mechanism is composed of two main processes: de novo methylation and maintenance methylation, both of which are catalyzed by specific DNA methyltransferase (DNMT) enzymes. To date, six different DNMTs have been characterized in mammals defined as DNMT1, DNMT2, DNMT3A, DNMT3B, DNMT3C, and DNMT3L. While DNMT1 primarily functions in maintenance methylation, both DNMT3A and DNMT3B are essentially responsible for de novo methylation. As is known, either maintenance or de novo methylation processes appears during oocyte and early embryo development terms. The aim of the present study is to investigate spatial and temporal expression levels and subcellular localizations of the DNMT1, DNMT3A, and DNMT3B proteins in the mouse germinal vesicle (GV) and metaphase II (MII) oocytes, and early embryos from 1-cell to blastocyst stages. We found that there are remarkable differences in the expressional levels and subcellular localizations of the DNMT1, DNMT3A and DNMT3B proteins in the GV and MII oocytes, and 1-cell, 2-cell, 4-cell, 8-cell, morula, and blastocyst stage embryos. The fluctuations in the expression of DNMT proteins in the analyzed oocytes and early embryos are largely compatible with DNA methylation changes and genomic imprintestablishment appearing during oogenesis and early embryo development. To understand precisemolecular biological meaning of differently expressing DNMTs in the early developmental periods, further studies are required.

DNA methyltransferases exhibit dynamic expression during spermatogenesis.

DNA methylation is one of the epigenetic marks and plays critically important functions during spermatogenesis in mammals. DNA methylation is catalysed by DNA methyltransferase (DNMT) enzymes, which are responsible for the addition of a methyl group to the fifth carbon atom of the cytosine residues within cytosine-phosphate-guanine (CpG) and non-CpG dinucleotide sites. Structurally and functionally five different DNMT enzymes have been identified in mammals, including DNMT1, DNMT2, DNMT3A, DNMT3B and DNMT3L. These enzymes mainly play roles in two DNA methylation processes: maintenance and de novo. While DNMT1 is primarily responsible for maintenance methylation via transferring methyl groups to the hemi-methylated DNA strands following DNA replication, both DNMT3A and DNMT3B are capable of methylating unmodified cytosine residues, known as de novo methylation. However, DNMT3L indirectly participates in de novo methylation, and DNMT2 carries out methylation of the cytosine 38 in the anticodon loop of aspartic acid transfer RNA. To date, many studies have been performed to determine spatial and temporal expression levels and functional features of the DNMT in the male germ cells. This review article comprehensively discusses dynamic expression of the DNMT during spermatogenesis and their relationship with male infertility development in the light of existing investigations.

Immunohistochemistry of Paraffin Sections from Mouse Ovaries.

Immunohistochemistry (IHC) is an efficient technique to detect cellular localizations of the proteins in paraffin-embedded tissues. It allows specific proteins to be visualized by the interaction of antibodies with an enzyme-substrate-chromogen system. Here, we describe indirect immunohistochemistry method for paraffin-embedded mouse ovaries fixed with Bouin's Fixative.

The effect of FSH and activin A on Akt and MAPK1/3 phosphorylation in cultured bovine ovarian cortical strips.

BACKGROUND: rhFSH and rhActA have been used in mammalian ovarian follicle culture systems for activation of follicular growth in vitro and suggested to be responsible for primordial follicle survival through MAPK and Akt pathways. The aim of our study was to determine the effects of rhFSH and rhActA on Akt, pAkt, MAPK1/3 and pMAPK1/3 protein levels in bovine ovarian cortical strips cultured in vitro. METHODS: Ovarian cortical strips from heifers were cultured in the presence of rhFSH (50 ng/mL), rhActA (100 ng/mL) or combination of these factors for 6 days. The strips were embedded in paraffin for histological observations and homogenized for western blot to determine Akt, pAkt, MAPK1/3 and pMAPK1/3 protein levels after the culture. Determination of primordial, primary and secondary follicle proportions at the end of culture as well as comparison of healthy follicle for each developmental stage after the culture was performed to quantify follicle survival and activation. RESULTS: pAkt protein levels were significantly lower in rhFSH + rhActA group among the other groups, whereas pMAPK1/3 levels were not significantly changed. Follicular activation and survival was measured to be significantly lower in rhFSH + rhActA group. Percentage of healthy primordial follicles was higher in control group whereas healthy secondary follicle proportion was higher in both rhActA and rhFSH groups. rhActA alone had a better impact on follicular activation, since the percentage of the secondary follicles was significantly higher than other treatment groups. CONCLUSIONS: The use of rhActA and rhFSH alone or in the combined form results in differential levels of Akt and MAPK proteins. Both rhActA and rhFSH alone has a remarkable contribution in survival and activation of the follicles in accordance with higher levels of these proteins. Thus, the manipulation of Akt and MAPK pathways with appropriate activators might contribute to proper activation and development of ovarian follicles in vitro.

The poly(A)-binding protein genes, EPAB, PABPC1, and PABPC3 are differentially expressed in infertile men with non-obstructive azoospermia.

PURPOSE: Azoospermia is one of the major causes of male infertility and is basically classified into obstructive (OA) and non-obstructive azoospermia (NOA). The molecular background of NOA still largely remains elusive. It has been shown that the poly(A)-binding proteins (PABPs) essentially play critical roles in stabilization and translational control of the mRNAs during spermatogenesis. METHODS: In the present study, we aim to evaluate expression levels of the PABP genes, EPAB, PABPC1, and PABPC3, in the testicular biopsy samples and in the isolated spermatocyte (SC) and round spermatid (RS) fractions obtained from men with various types of NOA including hypospermatogenesis (hyposperm), RS arrest, SC arrest, and Sertoli cell-only syndrome (SCO). RESULTS: In the testicular biopsy samples, both PABPC1 and PABPC3 mRNA expressions were gradually decreased from hyposperm to SCO groups (P < 0.05), whereas there was no remarkable difference for the EPAB expression among groups. The expression levels of cytoplasmically localized PABPC1 and PABPC3 proteins dramatically reduced from hyposperm to SCO groups (P < 0.05). In the isolated SC and RS fractions, the EPAB, PABPC1, and PABPC3 mRNA expressions were gradually decreased from hyposperm to SC arrest groups (P < 0.05). Similarly, both PABPC1 and PABPC3 proteins were expressed at higher levels in the SC and RS fractions from hyposperm group when compared to the SC and RS fractions from either RS arrest or SC arrest group (P < 0.05). CONCLUSION: Our findings suggest that observed significant alterations in the PABPs expression may have an implication for development of different NOA forms.

2100-MHz electromagnetic fields have different effects on visual evoked potentials and oxidant/antioxidant status depending on exposure duration.

The purpose of the present study was to investigate the duration effects of 2100-MHz electromagnetic field (EMF) on visual evoked potentials (VEPs) and to assess lipid peroxidation (LPO), nitric oxide (NO) production and antioxidant status of EMF exposed rats. Rats were randomized to following groups: Sham rats (S1 and S10) and rats exposed to 2100-MHz EMF (E1 and E10) for 2h/day for 1 or 10 weeks, respectively. At the end of experimental periods, VEPs were recorded under anesthesia. Brain thiobarbituric acid reactive substances (TBARS) and 4-hydroxy-2-nonenal (4-HNE) levels were significantly decreased in the E1 whereas increased in the E10 compared with their control groups. While brain catalase (CAT), glutathione peroxidase (GSH-Px) activities and NO and glutathione (GSH) levels were significantly increased in the E1, reduction of superoxide dismutase (SOD) activity was detected in the same group compared with the S1. Conversely, decreased CAT, GSH-Px activities and NO levels were observed in the E10 compared with the S10. Latencies of all VEP components were shortened in the E1 compared with the S1, whereas latencies of all VEP components, except P1, were prolonged in the E10 compared with the S10. There was a positive correlation between all VEP latencies and brain TBARS and 4-HNE values. Consequently, it could be concluded that different effects of EMFs on VEPs depend on exposure duration. In addition, our results indicated that short-term EMF could provide protective effects, while long-term EMF could have an adverse effect on VEPs and oxidant/antioxidant status.