Unveiling the impact of DNA methylation machinery: Dnmt1 and Dnmt3a in orchestrating oocyte development and cellular homeostasis.

DNA methylation can be considered the most prominent in controlling the gene expression responsible for the balance between cell proliferation and cell death. In this study, we aimed to analyze the distinct contributions of Dnmt1 and Dnmt3a enzymes in oocyte maturation, survival, autophagy, reactive oxygen species (ROS) production, and compensation capacity of Dnmt3b and Dnmt3l enzymes in mouse oocytes. Following confirming the suppression of Dnmt1or Dnmt3a through siRNA application, the assessment involved immunofluorescence staining for Dnmts, 5mC, p62, and ROS levels. Cell death rates showed a noticeable increase while oocyte maturation rates exhibited significant reduction. Global DNA methylation showed a decline, concomitant with elevated p62 and ROS levels upon Dnmt1 or Dnmt3a knockdown. Remarkably, silencing of Dnmt1 led to an upsurge in Dnmt3a expression, whereas Dnmt3a knockdown triggered an increase in Dnmt1 levels. Furthermore, Dnmt3l expression exhibited a notable decrease after silencing of either Dnmt1 or Dnmt3a, while Dnmt3b levels remained comparable between control and siRNA-treated groups. Collectively, this study underscores the pivotal roles of Dnmt1 and Dnmt3a in orchestrating various facets of oocyte development, encompassing maturation, survival, autophagy, and ROS production. These findings offer valuable insights into the intricate regulatory network governed by DNA methylation machinery within the context of oocyte physiology.

Antioxidant supplementation may effect DNA methylation patterns, apoptosis, and ROS levels in developing mouse embryos.

This study was designed to address the question: does antioxidant-containing embryo culture media affect DNA methyltransferases, global DNA methylation, inner cell mass/trophoblast differentiation, intracellular reactive oxygen species (ROS) levels, and apoptosis? Mouse zygotes were cultured in embryo culture media containing MitoQ, N-acetyl-L-cysteine (NAC), acetyl-L-carnitine (ALC), α-lipoic acid (ALA), or the mixture of NAC + ALC + ALA (AO) until the blastocyst stage, whereas in vivo-developed blastocysts were used as control. Protein expression levels of Dnmt1, 3a, 3b, and 3l enzymes were analyzed by immunofluorescence and western blot, while global DNA methylation, apoptosis, and ROS levels were evaluated by immunofluorescence. NAC, ALC, and MitoQ significantly increased the levels of all Dnmts and global methylation. ALA significantly induced all Dnmts, whereas global methylation did not show any difference. NAC and mixture AO applications significantly induced Nanog levels, ALA and MitoQ increased Cdx2 levels, while the other groups were similar. ALA and MitoQ decreased while ALC increased the levels of intracellular ROS. This study illustrates that antioxidants, operating through distinct pathways, have varying impacts on DNA methylation levels and cell differentiation in mouse embryos. Further investigations are warranted to assess the implications of these alterations on the subsequent offspring.

Histone acetyltransferases and histone deacetyl transferases play crucial role during oogenesis and early embryo development.

Dynamic epigenetic regulation is critical for proper oogenesis and early embryo development. During oogenesis, fully grown germinal vesicle oocytes develop to mature Metaphase II oocytes which are ready for fertilization. Fertilized oocyte proliferates mitotically until blastocyst formation and the process is called early embryo development. Throughout oogenesis and early embryo development, spatio-temporal gene expression takes place, and this dynamic gene expression is controlled with the aid of epigenetics. Epigenetic means that gene expression can be altered without changing DNA itself. Epigenome is regulated through DNA methylation and histone modifications. While DNA methylation generally ends up with repression of gene expression, histone modifications can result in expression or repression depending on type of modification, type of histone protein and its specific residue. One of the modifications is histone acetylation which generally ends up with gene expression. Histone acetylation occurs through the addition of acetyl group onto amino terminal of the core histone proteins by histone acetyltransferases (HATs). Contrarily, histone deacetylation is associated with repression of gene expression, and it is catalyzed by histone deacetylases (HDACs). This review article focuses on what is known about alterations in the expression of HATs and HDACs and emphasizes importance of HATs and HDACs during oogenesis and early embryo development.

High-fat diet induced obesity alters Dnmt1 and Dnmt3a levels and global DNA methylation in mouse ovary and testis.

Obesity impairs reproductive capacity, and the link between imprinting disorders and obesity has been discussed in many studies. Recent studies indicate that a high-fat diet may cause epigenetic changes in maternal and paternal genes, which may be transmitted to offspring and negatively affect their development. On this basis, our study aims to reveal the changes in DNA methylation and DNA methyltransferase enzymes in the ovaries and testes of C57BL/6 mice fed a high-fat diet and created a model of obesity, by comparing them with the control group. For this purpose, we demonstrated the presence and quantitative differences of DNA methyltransferase 1 and DNA methyltransferase 3a enzymes as well as global DNA methylation in ovaries and testis of C57BL/6 mice fed a high-fat diet by using immunohistochemistry and western blot methods. We found that a high-fat diet induces the levels of Dnmt1 and Dnmt3a proteins (p < 0.05). We observed increased global DNA methylation in testes but, interestingly, decreased global DNA methylation in ovaries. We think that our outcomes have significant value to demonstrate the effects of obesity on ovarian follicle development and testicular spermatogenesis and may bring a new perspective to obesity-induced infertility treatments. Additionally, to the best of our knowledge, this is the first study to document dynamic alteration of Dnmt1 and Dnmt3a as well as global DNA methylation patterns during follicle development in healthy mouse ovaries.

DNMT enzymes differentially alter global DNA methylation in a stage-dependent manner during spermatogenesis.

DNA methylation plays important roles during spermatogenesis. This mechanism includes maintenance and de novo methylation which are catalysed by DNA methyltransferase enzymes. DNMT1 plays role in maintenance methylation, while DNMT3A, DNMT3B and DNMT3L are primarily responsible for de novo methylation. Both maintenance and de novo methylation processes appears during primordial germ cell development and spermatogenesis. However, the function(s) of the methylation and DNMTs during spermatogenesis still remain elusive. The aim of the study was to evaluate the relationship between DNMTs levels and global DNA methylation in total testis and during spermatogenesis. For this purpose, DNMTs were analysed using Western blot and immunohistochemistry techniques. We also analysed global DNA methylation level by 5mC staining. We found that DNMTs expression and global DNA methylation levels were significantly differed in total testes and spermatogenetic cells in a stage-dependent manner. DNMT3B and DNMT3L were more abundant in testes, while DNMT1 and DNMT3A were comparatively low. Interestingly, no DNMTs signal was seen in elongated spermatid whereas global DNA methylation was at the highest level. To understand the meaning of differential expressions of DNMTs in the testes, further molecular biological studies are required.

Hydroxychloroquine induces endothelium-dependent and endothelium-independent relaxation of rat aorta.

BACKGROUND: Hydroxychloroquine (HCQ) is an antimalarial that is widely used in the management of rheumatoid arthritis and other autoimmune diseases. In this study, we aimed to examine the vascular effects of HCQ on rat aorta (RA). METHODS: The RA rings were suspended in isolated organ baths and tension was recorded isometrically. HCQ-induced relaxations were tested in the presence of the nitric oxide synthase inhibitor, nitro-L-arginine methyl ester (L-NAME, 100 mM); the cyclooxygenase enzyme inhibitor, indomethacin (10 mM); the calcium (Ca2+) ion channel blocker, nilvadipine (10 µM); and the K+ ion channel inhibitors, tetraethylammonium (1 mM), glibenclamide (10 mM), 4-aminopyridine (1 mM), and barium chloride (30 mM). The effect of HCQ on Ca2+ channels was examined using Ca2+-free Krebs solution, and adding calcium chloride (CaCl2 , 10-5- 10-2 M) cumulatively to baths incubated with HCQ. RESULTS: Removing the endothelium resulted in less relaxation of RA rings compared to endothelium-intact rings (p < 0.05). The effect of endothelium was supported by using L-NAME where HCQ produced-vasorelaxation was decreased (p < 0.05). The contraction of vascular rings was inhibited to a significant degree following the addition of CaCl2 , PE, or KCl on HCQ-incubated RA rings (p < 0.05). The incubation of the RA rings with the Ca2+ channel blocker, the K+ channel blockers, and the COX inhibitor, indomethacin did not significantly affect vascular relaxation induced by HCQ. DISCUSSION: HCQ produced relaxation of RA rings. The relaxation mechanism differs according to the concentration of HCQ. At con-centrations of 10-6 and 10-5 M, the relaxation is endothelium-dependent and mediated by NO. We strongly suggest that Ca2+ channel inhibition is involved at concentrations of 10-5 and 10-4 M, as well as NO.

Knockdown of Dnmt1 and Dnmt3a gene expression disrupts preimplantation embryo development through global DNA methylation.

PURPOSE: DNA methylation is one of the epigenetic mechanisms that plays critical roles in preimplantation embryo development executed by DNA methyltransferase (Dnmt) enzymes. Dnmt1, responsible for the maintenance of methylation, and Dnmt3a, for de novo methylation, are gradually erased from the zygote in succeeding stages and then reestablished in the blastocyst. This study was designed to address the vital role of Dnmt1 and Dnmt3a enzymes by silencing their gene expressions in embryonic development in mice. METHODS: Groups were (i) control, (ii) Dnmt1-siRNA, (iii) Dnmt3a-siRNA, and (iv) non-targeted (NT) siRNA. Knockdown of Dnmt genes using siRNAs was confirmed by measuring the targeted proteins using Western blot and immunofluorescence. Following knockdown of Dnmt1 and Dnmt3a in zygotes, the developmental competence and global DNA methylation levels were analyzed after 96 h in embryo cultures. RESULTS: A significant number of embryos arrested at the 2-cell stage or had undergone degeneration in the Dnmt1 and Dnmt3a knocked-down groups. By 3D observations in super-resolution microscopy, we noted that Dnmt1 was exclusively found in juxtanuclear cytoplasm, while the Dnmt3a signal was preferentially localized in the nucleus, both in trophoblasts (TBs) and embryoblasts (EBs). Interestingly, the global DNA methylation level decreased in the Dnmt1 knockdown group, while it increased in the Dnmt3a knockdown group. CONCLUSION: Precisely aligned expression of Dnmt genes is highly essential for the fate of an embryo in the early developmental period. Our data indicates that further analysis is mandatory to designate the specific targets of these methylation/demethylation processes in mouse and human preimplantation embryos.

Decreased expression of TERT and telomeric proteins as human ovaries age may cause telomere shortening.

OBJECTIVE: Telomeres are repetitive sequences localized at the ends of eukaryotic chromosomes comprising noncoding DNA and telomere-binding proteins. TRF1 and TRF2 both bind to the double-stranded telomeric DNA to regulate its length throughout the lifespan of eukaryotic cells. POT1 interacts with single-stranded telomeric DNA and contributes to protecting genomic integrity. Previous studies have shown that telomeres gradually shorten as ovaries age, coinciding with fertility loss. However, the molecular background of telomere shortening with ovarian aging is not fully understood. METHODS: The present study aimed to determine the spatial and temporal expression levels of the TERT, TRF1, TRF2, and POT1 proteins in different groups of human ovaries: fetal (n = 11), early postnatal (n = 10), premenopausal (n = 12), and postmenopausal (n = 14). Also, the relative telomere signal intensity of each group was measured using the Q-FISH method. RESULTS: We found that the telomere signal intensities decreased evenly and significantly from fetal to postmenopausal groups (P < 0.05). The TERT, TRF1, TRF2, and POT1 proteins were localized in the cytoplasmic and nuclear regions of the oocytes, granulosa and stromal cells. Furthermore, the expression levels of these proteins reduced significantly from fetal to postmenopausal groups (P < 0.05). CONCLUSION: These findings suggest that decreased TERT and telomere-binding protein expression may underlie the telomere shortening of ovaries with age, which may be associated with female fertility loss. Further investigations are required to elicit the molecular mechanisms regulating the gradual decrease in the expression of TERT and telomere-binding proteins in human oocytes and granulosa cells during ovarian aging.

The loss of global DNA methylation due to decreased DNMT expression in the postnatal mouse ovaries may associate with infertility emerging during ovarian aging.

Ovarian aging is one of the main causes of female infertility, and its molecular background is still largely unknown. As DNA methylation regulates many oogenesis/folliculogenesis-related genes, the expression levels and cellular localizations of DNA methyltransferases (DNMTs) playing key roles in this process is important in the ovaries from early to aged terms. In the present study, we aimed to evaluate the spatial and temporal expression of the Dnmt1, Dnmt3a, Dnmt3b, and Dnmt3l genes as well as global DNA methylation levels in the mouse ovaries during aging. For this purpose, the following groups were created: young (1- and 2-week old; n = 3 from each week), prepubertal (3- and 4-week-old; n = 3 from each week), pubertal (5- and 6-week-old; n = 3 from each week), postpubertal (16- and 18-week-old; n = 3 from each week), and aged (52-, 60- and 72-week-old; n = 3 from each week). We found here that Dnmt1, Dnmt3a, and Dnmt3l genes' expression at mRNA and protein levels as well as global DNA methylation profiles were gradually and significantly decreased in the postnatal ovaries from young to aged groups (P < 0.05). In contrast, there was a remarkable increase of Dnmt3b expression in the pubertal, postpubertal and aged groups (P < 0.05). Our findings suggest that the significantly altered DNMT expression and global DNA methylation levels during ovarian aging may contribute to female infertility development at the later terms of lifespan. Also, new researches are required to determine the molecular biological mechanism(s) that how altered DNMT expression and decreased DNA methylation lead to ovarian aging.

Glabridin attenuates airway inflammation and hyperresponsiveness in a mice model of ovalbumin-induced asthma.

Asthma is an inflammatory disease of the airways of the lungs, which is characterized by airflow obstruction and bronchospasms. Glabridin is a major flavonoid, especially found in root of Glycyrrhiza glabra, and has several pharmacological activities, including antioxidant and anti-inflammatory effects. The anti-asthmatic effect and possible mechanism of glabridin, however, have not been revealed so far. The aim of this study is to investigate the effects and possible mechanisms of glabridin against ovalbumin (OVA)-induced airway hyperresponsiveness (AHR) and inflammation in mice. In male BALB/c mice, asthma was induced by intraperitoneal (i.p) injection of OVA mixed with 2 mg aluminium hydroxide on days 0, 14 and boosted with OVA aerosol challenge on days 21, 22, and 23. Mice were either treated with dexamethasone (i.p, 1 mg/kg) or glabridin (10, 20, and 30 mg/kg) from days 18-23. Pulmonary function parameters such as peak inspiratory flow, peak expiratory flow, tidal volume, expiratory volume, the frequency of breathing, enhanced pause values were evaluated by using whole-body plethysmography. Measurements were performed at baseline and following methacholine (50 mg/mL) challenges. In addition, white blood cells (WBC) count, total protein, and IgE levels were measured in bronchial alveolar lavage fluid (BALF), lung, and serum, respectively. Glabridin (20 or 30 mg/kg) significantly attenuated (p < 0.05) OVA-induced alteration in respiratory parameters. Elevated counts of total WBC, differential WBC (neutrophils, lymphocytes, monocytes, and eosinophils) in BALF and the total protein in lungs and BALF were significantly decreased (p < 0.05) by glabridin (20 or 30 mg/kg). It also significantly attenuated the increased serum IgE levels (p < 0.05). As glabridin reduces the level of serum IgE, the total protein and the count of WBC and improves respiratory function, it may be a novel therapeutic agent in asthma.

PFA is superior to glyoxal in preserving oocyte, embryo, and stem cell proteins evidenced by super-resolution microscopical surveys of epitopes.

PURPOSE: Chemical fixation is a critical step to retaining cellular targets as naturally as possible. Recent developments in microscopy allow sophisticated detection and measuring techniques with which spatio-temporal molecular alterations are conceivable. In this study, we compare two members of aldehyde fixatives [i.e., glyoxal (Gly) and paraformaldehyde (PFA)] to determine whether Gly, a less toxic dialdehyde fixative that is considered to retain immunoreactivity could provide a successful and consistent cell fixation in favor of PFA in various cell preparations and types. METHODS: We document the fixation competence of Gly and PFA side-by-side (with or without Triton X-100 permeabilization) in live- and fixed-cell preparations in mouse oocytes, embryos, and human somatic cells (human umbilical cord-derived mesenchymal stromal cells) using protein quantification by Western blot assay and super-resolution microscopy. RESULTS: Although Gly seemed to act faster than PFA, catastrophic consequences were found not acceptable, especially in oocytes and embryos. Due to cell lysate and immunocytochemistry surveys, it was obvious that PFA is superior to Gly in retaining cellular proteins in situ with little/no background staining. In many samples, PFA revealed more reliable and consistent results regarding the protein quantity and cellular localization corresponding to previously defined patterns in the literature. CONCLUSION: Although the use of Gly is beneficial as indicated by previous reports, we concluded that it does not meet the requirement for proper fixation, at least for the tested cell types and proteins. However, PFA alone with no addition of TX displayed a significant cytoplasmic loss by generating membrane blebs during fixation.

Embryonic poly(A)-binding protein is differently expressed and interacts with the messenger RNAs in the mouse oocytes and early embryos.

The embryonic poly(A)-binding protein (EPAB) functions in the translational regulation of the maternal messenger RNAs (mRNAs) required during oocyte maturation, fertilization, and early embryo development. Since there is no antibody specific to mammalian EPAB protein, all studies related to the Epab gene could be performed at the mRNA levels except for the investigations in the Xenopus. In this study, we have produced an EPAB-specific antibody. When we examined its expressional distribution in the mouse gonadal and somatic tissues, the EPAB protein was found to be expressed only in the mouse ovary and testis tissues, but it is undetectable level in the somatic tissues including stomach, liver, heart, small intestine, and kidney. Additionally, the spatial and temporal expression patterns of the EPAB and poly(A)-binding protein cytoplasmic 1 (PABPC1) proteins were analyzed in the mouse germinal vesicle (GV) and metaphase II (MII) oocytes, one-cell, and two-cell embryos. While EPAB expression gradually decreased from GV oocytes to two-cell embryos, the PABPC1 protein level progressively increased from GV oocytes to one-cell embryos and remarkably declined in the two-cell embryos ( P < 0.05). We have also described herein that the EPAB protein interacted with Epab, Pabpc1, Ccnb1, Gdf9, and Bmp15 mRNAs dependent upon the developmental stages of the mouse oocytes and early embryos. As a result, we have first produced an EPAB-specific antibody and characterized its expression patterns and interacting mRNAs in the mouse oocytes and early embryos. The findings suggest that EPAB in cooperation with PABPC1 implicate in the translational control of maternal mRNAs during oogenesis and early embryo development.

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.

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.

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.

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.

Potential roles of the poly(A)-binding proteins in translational regulation during spermatogenesis.

Spermatogenesis is briefly defined as the production of mature spermatozoa from spermatogonial stem cells at the end of a strictly regulated process. It is well known that, to a large extent, transcriptional activity ceases at mid-spermiogenesis. Several mRNAs transcribed during early stages of spermatogenesis are stored as ribonucleoproteins (RNPs). During the later stages, translational control of these mRNAs is mainly carried out in a time dependent-manner by poly(A)-binding proteins (PABPs) in cooperation with other RNA-binding proteins and translation-related factors. Conserved PABPs specifically bind to poly(A) tails at the 3' ends of mRNAs to regulate their translational activity in spermatogenic cells. Studies in this field have revealed that PABPs, particularly poly(A)-binding protein cytoplasmic 1 (Pabpc1), Pabpc2, and the embryonic poly(A)-binding protein (Epab), play roles in the translational regulation of mRNAs required at later stages of spermatogenesis. In this review article, we evaluated the spatial and temporal expression patterns and potential functions of these PABPs in spermatogenic cells during spermatogenesis. The probable relationship between alterations in PABP expression and the development of male infertility is also reviewed.

Telomere length and telomerase activity during folliculogenesis in mammals.

Telomeres are repetitive non-coding DNA sequences located at the ends of chromosomes in eukaryotic cells. Their most important function is to protect chromosome ends from being recognized as DNA damage. They are also implicated in meiosis and synapse formation. The length of telomeres inevitably shortens at the end of each round of DNA replication and, also, as a consequence of the exposure to oxidative stress and/or genotoxic agents. The enzyme telomerase contributes to telomere lengthening. It has been reported that telomerase is exclusively expressed in germ cells, granulosa cells, early embryos, stem cells, and various types of cancerous cells. Granulosa cells undergo many mitotic divisions and either granulosa cells or oocytes are exposed to a variety of genotoxic agents throughout folliculogenesis; thus, telomerase plays an important role in the maintenance of telomere length. In this review article, we have comprehensively evaluated the studies focusing on the regulation of telomerase expression and activity, as well as telomere length, during folliculogenesis from primordial to antral follicles, in several mammalian species including mice, bovines, and humans. Also, the possible relationships between female infertility caused by follicular development defects and alterations in the telomeres and/or telomerase activity are discussed.

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.

Poly(A)-binding proteins are required for translational regulation in vertebrate oocytes and early embryos.

Poly(A)-binding proteins (PABPs) function in the timely regulation of gene expression during oocyte maturation, fertilisation and early embryo development in vertebrates. To this end, PABPs bind to poly(A) tails or specific sequences of maternally stored mRNAs to protect them from degradation and to promote their translational activities. To date, two structurally different PABP groups have been identified: (1) cytoplasmic PABPs, including poly(A)-binding protein, cytoplasmic 1 (PABPC1), embryonic poly(A)-binding protein (EPAB), induced PABP and poly(A)-binding protein, cytoplasmic 3; and (2) nuclear PABPs, namely embryonic poly(A)-binding protein 2 and nuclear poly(A)-binding protein 1. Many studies have been undertaken to characterise the spatial and temporal expression patterns and subcellular localisations of PABPC1 and EPAB in vertebrate oocytes and early embryos. In the present review, we comprehensively evaluate and discuss the expression patterns and particular functions of the EPAB and PABPC1 genes, especially in mouse and human oocytes and early embryos.