Effects of N6-methyladenosine modification on the function of the female reproductive system.

In recent years, the rate of female infertility in China has been increasing, posing an urgent challenge to improve fertility. A healthy reproductive system is essential for successful reproduction, and N6-methyladenosine (m6A) is the most abundant chemical modification in eukaryotes and plays a critical role in cellular processes. Recent studies have shown that m6A modifications also have a keying effect in various physiological and pathological processes in the female reproductive system, although their regulatory mechanisms and biological functions remain unclear. In this review, we first introduce the reversible regulatory mechanisms of m6A and its functions, discuss the role of m6A in female reproductive function and disorders of the reproductive system, and present recent advances in m6A detection technologies and methods. Our review provides new insights into the biological role of m6A and its potential application in the treatment of female reproductive disorders.

Identification of a WRKY transcriptional activator from Camellia sinensis that regulates methylated EGCG biosynthesis.

Naturally occurring methylated catechins, especially methylated EGCG in tea leaves are known to have many health benefits. Although the genes involved in methylated EGCG biosynthesis have been studied extensively, the transcriptional factors controlling methylated EGCG biosynthesis are still poorly understood. In the present study, a WRKY domain-containing protein termed CsWRKY57like was identified, which belongs to group IIc of the WRKY family, and contains one conserved WRKY motif. CsWRKY57like was found to localize in the nucleus, function as a transcriptional activator, and its expression positively correlated with methylated EGCG level. In addition, CsWRKY57like activated the transcription of three genes related to methylated EGCG biosynthesis, including CCoAOMT, CsLAR, and CsDFR by specifically interacting with their promoters via binding to the cis-acting element (C/T)TGAC(T/C). Further assays revealed that CsWRKY57like physically interacts with CsVQ4, and participates in the metabolic regulation of O-methylated catechin biosynthesis. Collectively, we conclude that CsWRKY57like may positively impact the biosynthesis of methylated EGCG in the tea plant, which comprehensively enriches the regulatory network of WRKY TFs associated with methylated EGCG and provide a potential strategy for the breeding of specific tea plant cultivars with high methylated EGCG .

[Molecular mechanisms of recursive splicing events in long introns of eukaryotes].

Recursive splicing refers to the biological process that long introns are removed in multiple steps during pre-mRNA splicing. In comparison to large introns (>10 kb), most introns in higher eukaryotic genomes are removed in one step during transcription. Previous studies have revealed that recursive splicing events play important roles in many biological processes, including the pathogenesis and development of diseases. In recent years, more researchers have focused on recursive splicing events and found that recursive splicing occurs in Drosophila and many other vertebrates. Multiple recursive splicing sites have been predicted by different bioinformatics methods and verified by experiments. Current researches focus on the process of recursive splicing, recursive splicing site recognition and its influence on biological processes. In this review, we summarize the molecular mechanism of recursive splicing events in eukaryotic genomes and the present development in this field, aiming to lay the basis for further understanding of the mechanisms of RNA splicing.

Expression of Cyt-c-Mediated Mitochondrial Apoptosis-Related Proteins in Rat Renal Proximal Tubules during Development.

BACKGROUND: Apoptosis regulates embryogenesis, organ metamorphosis and tissue homeostasis. Mitochondrial signaling is an apoptotic pathway, in which Cyt-c and Apaf-1 are transformed into an apoptosome, which activates procaspase-9 and triggers apoptosis. This study evaluated Cyt-c, Apaf-1 and caspase-9 expression during renal development. METHODS: Kidneys from embryonic (E) 16-, 18-, and 20-day-old fetuses and postnatal (P) 1-, 3-, 5-, 7-, 14-, and 21-day-old pups were obtained. Immunohistochemical analysis, dual-labeled immunofluorescence, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique assay and Western blot were performed in addition to histological analysis. RESULTS: Immunohistochemistry showed that Cyt-c was strongly expressed in proximal and distal tubules (DTs) at all time points. Caspase-9 and Apaf-1 were strongly expressed in proximal tubules (PTs) but only weakly expressed in DTs. Dual-labeled immunofluorescence showed that most tubules expressed both Cyt-c and Apaf-1, except for some tubules that only expressed Cyt-c. The TUNEL assay showed a greater percentage of apoptotic cells in PTs compared to DTs. Apaf-1 and cleaved caspase-9 protein expression gradually increased during the embryonic period and peaked during the early postnatal period but apparently declined from P7. Cyt-c protein expression was weak during the embryonic period but obviously increased after P1. CONCLUSION: This study showed that PTs are more sensitive to apoptosis than DTs during rat renal development, even though both tubule segments contain a large number of mitochondria. Furthermore, Cyt-c-mediated mitochondrial apoptosis-related proteins play an important role in PTs during the early postnatal kidney development.

Differential activation of CD95-mediated apoptosis related proteins in proximal and distal tubules during rat renal development.

The CD95-mediated apoptotic pathway is the best characterized of the death receptor-mediated apoptotic pathways. The present study characterized localization and expression of proteins involved in CD95-mediated apoptosis during rat renal development. Kidneys were obtained from embryonic (E) 18 and 20-day-old fetuses and postnatal (P) 1-, 3-, 5-, 7-, 14-, and 21-day-old pups. Immunohistochemical characterization revealed that CD95, FasL and cleaved caspase-3 were strongly expressed in proximal tubules and weakly expressed in distal tubules, but that expression of caspase-8 in distal tubules was stronger than that in proximal tubules. Results from terminal deoxynucleotidyl transferase dUTP nick end labeling assays showed that levels of apoptosis in proximal tubules slowly increased after E18, while those of distal tubules slowly decreased after P5. Western blotting demonstrated that expression of CD95, FasL and FADD was very weak during embryonic development, but rapidly increased at P14. Expression of cleaved caspase-3 was maintained at high levels after P1, while caspase-8 expression gradually reached a peak at P7. Results from this study reveal that the CD95-mediated apoptotic pathway is a key driver of apoptosis in proximal tubules during late postnatal kidney development in rats and suggest that apoptosis in distal tubules is mediated by a different apoptotic pathway.

[Expression analysis of green fluorescent protein in tissues and organs in α-1,3 galactosyltransferase knockout pigs].

The pig is an ideal source to provide organs because its organ size and physiology are similar to humans. However, an acute rejection will ensue after pig-to-human xenotransplantation. The α-1,3 galactosyltransferase gene knockout (GTKO) pigs were generated in recent years, and could solve the problem of hyperacute rejection. But due to lack of reporting genes, the rejection status of cells and organs post pig-to-human xenotransplantation cannot be visualized. In this study, we introduced the enhanced green fluorescent protein (EGFP) gene driven by the CAG promoter into GTKO porcine ear fibroblasts. Then we produced transgenic pigs expressing the EGFP gene by nuclear transfer technology. Expression levels of EGFP in different tissues and organs of the cloned pig were investigated by Nightsea DFP-1 Fluorescent Protein Flashlight, fluorescence microscope and quantitative PCR assays. The results showed that the protein and transcript of EGFP were expressed in all tissues and organs of the GTKO pig, but the expression was weak in the liver and central nervous system. In conclusion, we have successfully produced the transgenic GTKO pigs expressing EGFP in all tested tissues and organs, which builds up a good basis to track transplanted cells or tissues.

[Spectroscopic analysis and photocatalytical activity of carbon nitride materials].

Graphited carbon nitride materials (g-C3N4) with high visble-light response were synthesized by thermal condensation of melamine at varied temperature. The microstructure and optical property of as achieved catalysts were investigated by XRD, SEM and UV-Vis techniques, respectively. Moreover, rhodamine B solution was applied to measure the catalytical performance under the irradiation of different sources of light. The results showed that the major structures of g-C3N4 were kept, though lots of blocks were scattered on the surface because of the damage of lamellar structure caused by the high temperature. As the thermal temperature was increased, the adsorptions of light were greatly enhanced in both UV and Vis region, which might be due to the decrease in reflection and the increase in refraction at the lumpy surface. In the degradation of rhodamine B solution, all the samples showed high photocatalytic activities under the irradiation of both Visible-light and sunlight, and 94. 8% (60min, under Vis-light) and 91. 1% (90min, under sunlight) of RhB were degraded when the thermal temperature was 580°C. This research would greatly enlighten the studies of environmental purification using clean green energy.

MicroRNA-1 promotes apoptosis of hepatocarcinoma cells by targeting apoptosis inhibitor-5 (API-5).

Although microRNA-1 (miR-1) is a known liver cancer suppressor, the role of miR-1 in apoptosis of hepatoma cells has remained largely unknown. Our study shows that ectopic miR-1 overexpression induced apoptosis of liver hepatocellular carcinoma (HepG2) cells. Apoptosis inhibitor 5 (API-5) was found to be a potential regulator of miR-1 induced apoptosis, using a bioinformatics approach. Furthermore, an inverse relationship between miR-1 and API-5 expression was observed in human liver cancer tissues and adjacent normal liver tissues. Negative regulation of API-5 expression by miR-1 was demonstrated to promote apoptosis of HepG2 cells. Our study provides a novel regulatory mechanism of miR-1 in the apoptosis of hepatoma cells.

[Expression and synergistic function of ENHANCIN-like gene of Agrotis Segetum granulovirus].

ENHANCIN is an enhancing protein chiefly found in insect baculoviruses. One ENHANCIN homologue was identified, by blast method, in Agrotis Segetum granulovirus (AgseGV) genome, named enhancin-like. Sequence analysis indicated that this gene includes the conserved domains, conserved in other ENHANCIN, and it has no signal peptide or a-transmembrane helix. A proline-rich domain, which is similar to those of mammals, is present at its C-terminal. To analyze the synergistic function of AgseGV enhancin-like gene, prokaryotic expression vectors of its whole gene and the 5'-truncated fragment (1, 017bp) were constructed. Expression product of truncated fragment was purified by chromatography, and then it was used to prepare antibody. The expression product of whole gene was identified by Western blot with specific antibody and anti-His-Tag antibody. Bioassay proved that the expression product of whole gene can increase the mortality with 16.25% to 3th instar larvae of Helicoverpa armigera (HaNPV: 1.17 x 10(2) PIBS/mL), while the truncated fragment has no obvious synergistic function.

Expression of Bcl-2 and Bax in mouse renal tubules during kidney development.

Bcl-2 and Bax play an important role in apoptosis regulation, as well as in cell adhesion and migration during kidney morphogenesis, which is structurally and functionally related to mitochondria. In order to elucidate the role of Bcl-2 and Bax during kidney development, it is essential to establish the exact location of their expression in the kidney. The present study localized their expression during kidney development. Kidneys from embryonic (E) 16-, 17-, 18-day-old mouse fetuses, and postnatal (P) 1-, 3-, 5-, 7-, 14-, 21-day-old pups were embedded in Epon. Semi-thin serial sections from two E17 kidneys underwent computer assisted 3D tubule tracing. The tracing was combined with a newly developed immunohistochemical technique, which enables immunohistochemistry on glutaraldehyde fixated plastic embedded sections. Thereby, the microstructure could be described in detail, and the immunochemistry can be performed using exactly the same sections. The study showed that Bcl-2 and Bax were strongly expressed in mature proximal convoluted tubules at all time points, less strongly expressed in proximal straight tubules, and only weakly in immature proximal tubules and distal tubules. No expression was detected in ureteric bud and other earlier developing structures, such as comma bodies, S shaped bodies, glomeruli, etc. Tubules expressing Bcl-2 only were occasionally observed. The present study showed that, during kidney development, Bcl-2 and Bax are expressed differently in the proximal and distal tubules, although these two tubule segments are almost equally equipped with mitochondria. The functional significance of the different expression of Bcl-2 and Bax in proximal and distal tubules is unknown. However, the findings of the present study suggest that the mitochondrial function differs between mature proximal tubules and in the rest of the tubules. The function of Bcl-2 and Bax during tubulogenesis still needs to be investigated.

Genetic variation of the hemagglutinin of avian influenza virus H9N2.

Avian influenza virus H9N2 has become the dominant subtype of influenza which is endemic in poultry. The hemagglutinin, one of eight protein-coding genes, plays an important role during the early stage of infection. The adaptive evolution and the positively selected sites of the HA (the glycoprotein molecule) of H9N2 subtype viruses were investigated. Investigating 68 hemagglutinin H9N2 avian influenza virus isolates in China and phylogenetic analysis, it was necessary that these isolates were distributed geographically from 1994, and were all derived from the Eurasian lineage. H9N2 avian influenza virus isolates from domestic poultry in China were distinct phylogenetically from those isolated in Hong Kong, including viruses which had infected humans. Seven amino acid substitutions (2T, 3T, 14T, 165D, 197A, 233Q, 380R) were identified in the HA possibly due to positive selection pressure. Apart from the 380R site, the other positively selected sites detected were all located near the receptor-binding site of the HA1 strain. Based on epidemiological and phylogenetics analysis, the H9N2 epidemic in China was divided into three groups: the 1994-1997 group, the 1998-1999 group, and the 2000-2007 group. By investigating these three groups using the maximum likelihood estimation method, there were more positive selective sites in the 1994-1997 and 1998-1999 epidemic group than the 2000-2007 groups. This indicates that those detected selected sites are changed during different epidemic periods and the evolution of H9N2 is currently slow. The antigenic determinant or other key functional amino acid sites should be of concern because their adjacent sites have been under positive selection pressure. The results provide further evidence that the pathogenic changes in the H9N2 subtype are due mainly to re-assortment with other highly pathogenic avian influenza viruses.

A novel specific application of pyruvate protects the mouse retina against white light damage: differential stabilization of HIF-1α and HIF-2α.

PURPOSE: To mimic hypoxia preconditioning by a novel specific pyruvate treatment and to study its retinal protection against white light damage. METHODS: Six-to-eight-week-old BALB/c mice were exposed to strong white light calculated to produce photoreceptor degeneration. Some were given injections of pyruvate in a preordained protocol because evidence exists that proves pyruvate can affect the concentration of hypoxia inducible factor (HIF). Western blotting and real-time PCR were used to determine the concentration of proteins and mRNAs in retinas. Morphology was analyzed with toluidine blue staining and was plotted using a spidergraph. A free nucleosome cell death assay was used to examine apoptosis. Retina explant cultures were used to investigate the background mechanism. RESULTS: Pyruvate administration stabilized hypoxia inducible factor (HIF)-1α but not HIF-2α. Expression of the downstream genes hemoxygenase-1 and erythropoietin mirrored the changes of the two HIFs, respectively. Importantly, pyruvate given not only before but also after exposure to light protected photoreceptors against apoptosis. In the retinal explant system, addition or depletion of pyruvate caused only changes of HIF-1α and prolyl hydroxylase (PHD)-2, while HIF-2α and PHD1 were not affected. However, under hypoxic conditions, HIF-2α was stabilized by pyruvate but not HIF-1α. CONCLUSIONS: Pyruvate evoked a hypoxia-like response under normoxic conditions and was retina-protective against strong white light. This response included stabilization of HIF-1α but not HIF-2α. This differential stabilization might be related to the distinct preference of their degrading enzyme of PHD2 and PHD1 in response to pyruvate treatment.

Adaptive evolution of rotavirus VP7 and NSP4 genes in different species.

Rotavirus is recognized as the major enteric pathogen associated with high burden of worldwide epidemic diarrhea disease in human and animals. Its outer capsid structural protein VP7 elicits the production of distinct neutralizing antibodies in the host and also determines the serotype of the virus strain. As the diarrhea-related protein of rotavirus, NSP4 is becoming an attractive candidate for vaccine development. It is not clear whether rotavirus VP7 or NSP4 evolved in the same way or not, and how the rotavirus VP7 and NSP4 evolved in specific species. Using the different models, we analyzed Datasets A composed of 12 coding sequences representing 12 species and Dataset B composed of nine coding sequences representing nine species. Computational results indicate that rotavirus experienced strong purifying selection in VP7 and NSP4 across species, and there exist some positive selective sites in specific species by Branch-site model A (119S in Bovine lineage and 199T in Canine lineage for Datasets A, 69Y and 70H in Murine lineage for Datasets B). Since these sites are located in different functional sequence segments, it may be concluded that these sites are crucial to related virus function. Therefore, the results of this study would provide potential values to vaccine research and development.

[Effects of Di (2-ethylhexyl) phthalate on the testis and testicular gubernaculum of fetal KM mice].

OBJECTIVE: To explore the effects of Di (2-ethylhexyl) phthalate (DEHP) on the testis and testicular gubernaculum of fetal KM mice in vivo and to investigate the mechanism of DEHP-induced cryptorchidism. METHODS: Thirty healthy pregnant KM mice were randomly and equally divided into a blank control group, a corn oil control group and a DEHP group. The pregnant mice in the latter group were exposed to DEHP by gavage at the dose of 500 mg/kg body weight per day from gestation day 12 (GD12) through gestation day 19 (GD19). The effects of DEHP were observed on the number of fetuses per pregnancy, the ratio of male to female pups, the weight of the testis, the morphology and location of the testis and gubernaculum, the relative testis-bladder neck distance (TBD) and cranial suspensory ligament (CSL) residual. The expressions of the androgen receptor (AR), estrogen receptor (ER) and actin and proliferating cell nuclear antigen (PCNA) in the gubernaculum were detected by immunohistochemistry. RESULTS: DEHP reduced the testis weight and TBD, induced different degrees of testis maldescent, but produced no obvious effect on the body weight, the number of fetuses per pregnancy, the sex ratio and the testis gubernacular morphology. Under the light microscope, hypotrophy was seen in all the testis seminiferous tubules, spermatogenic cells and Sertoli cells, marked Leydig cell hyperplasia was noted, and the positive expression of AR in the gubernaculum was decreased in the DEHP group (P < 0.01). CONCLUSION: DEHP could cause dysfunction of the testis gubernaculum via its anti-androgen effect, induce cryptorchidism, and cause dysplasia and dysfunction of Sertoli cells, Leydig cells and spermatogenic cells in fetal mice.

Long-term coculture of spermatogonial stem cells on sertoli cells feeder layer in vitro.

OBJECTIVE: To construct a cell model of spermatogonial stem cells (SSCs) cocultured on Sertoli cells feeder layer in vitro, and study the proliferation characteristics of SSCs. METHODS: Sertoli cells and SSCs were separated from testes of 14-15 days and 6 days KM mice respectively by two-step enzyme digestion. SSCs were seeded on the Sertoli cells layer at 5 days in culture. The clones of SSCs on the sertoli cells layer were detected, and cast-off cells in culture medium were counted. RESULTS: SSCs began to proliferate and differentiate 24 hours after being cultured on the Sertoli cells layer, and there were a few paired (Ap) cell clones. With more time of culture, the number of Ap cell clones decreased gradually, meanwhile the number of aligned (Aal) cell clones increased, then Aal cell colonies retained stable quantity after 120 hours in culture, it could retain (51.2 +/- 5.8) days under the condition of the culture medium being changed every 4 or 5 days. CONCLUSION: SSCs can proliferate colonially on the feeder layer of Sertoli cells, and retain stable morphous and quantity. SSCs cultured on Sertoli cell feeder layer provide a cell model for studying SSCs biological behavior and interruptions of drugs or toxins on spermatogenesis in vitro. Coculture

[Di(2-ethylhexyl) phthalate affects the testes and leydig cells of neonatal KM mice].

OBJECTIVE: To explore the effects of di(2-ethylhexyl)phthalate (DEHP) on neonatal mice's testes and Leydig cells in vivo. METHODS: Pregnant mice were exposed to DEHP at the dose of 100 mg/kg, 200 mg/kg or 500 mg/kg (body weight) per day by gavage from gestation day 12 (GD 12) through postnatal day 3 (PND 3), respectively. The testis and body weights, testicular histopathology and the activity of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) of the neonatal mice were investigated. RESULTS: The body and testis weights of the male mice's offspring were significantly reduced following DEHP exposure. Leydig cell morphology was affected significantly by DEHP as compared with the controls. Leydig cells obviously increased in the neonatal mice's testes on PND 15 and PND 30 when exposed to DEHP (500 mg/[kg x d]). Activities and positive area of the steroidogenic enzymes 3beta-HSD immunoexpression decreased markedly when exposed to DEHP (100 mg/[kg x d] or 200 mg/[kg x d]). Image analysis showed a decrease in the activities of 3beta-HSD in the animals exposed to DEHP (500 mg/[kg x d]), but an increase in the positive area of 3beta-HSD immunoexpression as compared with the control animals on PND 15 (P < 0.01). CONCLUSION: DEHP affects the Leydig cell morphology, the activity of 3beta-HSD, the testis and body weights and the testicular histopathology of neonatal mice, and it may function as an antiandrogenic agent.

[Isolation, cultivation, identification and functional study of fetal mice Leydig cells in vitro].

OBJECTIVE: To explore the methods of isolation, cultivation, purification, identification of the fetal mice testis Leydig cell and to observe its biological characteristics in vitro. METHODS: Leydig cells were isolated by 0.03% collagenase (type I) from fetal mice testis and cultured in DMEM/F12 medium. The identity and purity of Leydig cell were assessed by 3beta-hydroxysteroid dehydrogenase delta4-delta5 isomerase (3beta-HSD). Cell viability was measured by trypan blue. Testosterone level in the medium of cultured Leydig cells was measured in various culture phases and cell density by radioimmunoassay. RESULTS: The purity of Leydig cell was (45.10 +/- 1.66)% before culture, and (81.17 +/- 2. 32)% 72 h after culture. The level of testosterone secreted by Leydig cells could be detected in the medium and its level was associated with the density and time of cultured Leydig cells. The secretion capacity of testosterone by single Leydig cell decreased gradually during the culturing period. CONCLUSION: The fetal Leydig cells isolated from fetal mice testis have high purity. It can be cultured and kept the secretion ability of testosterone for a few days in vitro. This system can provide a valuable model for further study on the cellular function of the Leydig cells of fetal mice.