Heavy metal ions exchange driven protein phosphorylation cascade functions in genomic instability in spermatocytes and male infertility.

DNA double-strand breaks (DSBs) are functionally linked to genomic instability in spermatocytes and to male infertility. The heavy metal cadmium (Cd) is known to induce DNA damage in spermatocytes by unknown mechanisms. Here, we showed that Cd ions impaired the canonical non-homologous end-joining (NHEJ) repair pathway, but not the homologous recombination (HR) repair pathway, through stimulation of Ser2056 and Thr2609 phosphorylation of DNA-PKcs at DSB sites. Hyper-phosphorylation of DNA-PKcs led to its premature dissociation from DNA ends and the Ku complex, preventing recruitment of processing enzymes and further ligation of DNA ends. Specifically, this cascade was initiated by the loss of PP5 phosphatase activity, which results from the dissociation of PP5 from its activating ions (Mn), that is antagonized by Cd ions through a competitive mechanism. In accordance, in a mouse model Cd-induced genomic instability and consequential male reproductive dysfunction were effectively reversed by a high dosage of Mn ions. Together, our findings corroborate a protein phosphorylation-mediated genomic instability pathway in spermatocytes that is triggered by exchange of heavy metal ions.

[Cypermethrin induced liver oxidative DNA damage via the JNK/c-Jun pathway].

OBJECTIVE: To clarify the adverse effect of cypermethrin(CYP) on the liver and explore the underlying role of the MAPK pathway. METHODS: Twenty-four Sprague-Dawley(SD) rats were exposed to 0, 5, 10 and 20 mg/(kg·d) ß-CYP by gavage for 31 days. Histomorphological and ultrastructural changes were evaluated by the hematoxylin & eosin(HE) staining and transmission electron microscope(TEM). Levels of MDA and 8-OHdG were detected by ELISA. Expressions of p-JNK and γ-H2A. X were assessed by IHC and IF respectively. RT-PCR was performed to examine mRNA levels of GPx1, GPx4, SOD1, and SOD2 in rat testes. Western blot was conducted to determine protein expressions of GPx1, SOD2, CAT, γ-H2A. X, and the MAPK pathway-associated proteins in rat testes. RESULTS: After ß-CYP exposure, the histomorphology and ultrastructures of rat livers were abnormally altered, as evidenced by hepatic sinusoidal dilation, hepatic plate space formation, mitochondrial crest fracture, etc. Moreover, ß-CYP induced mRNA levels of GPx1, GPx4, SOD1 and SOD2, as well as protein expressions of GPx1 and SOD2 in the liver. Compared to the control, GPx1 and SOD2 protein expressions were decreased by 57.9% and 50.0%(P<0.05), whereas the MDA level was increased by 56.2%(P<0.05) in the high-dose group. Additionally, the JNK/c-Jun pathway, one of MAPK pathways, in the liver was activated by ß-CYP, as shown by the increase of JNK and c-Jun phosphorylation, and protein expressions of p-JNK and p-c-Jun in the high-dose group were elevated by 47.7% and 46.5%(P<0.05) in comparison to the control, but the ERK and p38 pathways were not affected after ß-CYP exposure. Furthermore, ß-CYP promoted 8-OHdG and γ-H2A. X expressions in the liver. Compared to the control, γ-H2A. X protein expression in the mid-and high-dose group was upregulated by 16.9% and 33.9%(P<0.05), respectively. CONCLUSION: Cypermethrin had detrimental effects on the liver. CYP not only directly altered liver histomorphology and ultrastructures, but also caused oxidative stress, which activated the JNK/c-Jun pathway, finally inducing the DNA damage.

MicroRNA-3064-5p sponged by MALAT1 suppresses angiogenesis in human hepatocellular carcinoma by targeting the FOXA1/CD24/Src pathway.

Angiogenesis is critical for the development, progression, and metastasis of hepatocellular carcinoma (HCC), but the roles of miR-3064-5p in HCC angiogenesis are still unknown. In this study, the roles of miR-3064-5p in HCC angiogenesis were studied in 192 HCC patients, xenograft mouse models, and HCC cell lines. The results showed that miR-3064-5p expression was significantly decreased in HCC tissues and cells, and downregulated miR-3064-5p was associated with upregulated angiogenic potential of HCC. MiR-3064-5p inhibited proangiogenic VEGFA and angiogenin expressions but induced antiangiogenic endostatin and MMP12 expressions, finally leading to suppression of HCC angiogenesis, as shown by the decline in intratumoral microvessel density (MVD). Moreover, miR-3064-5p was inversely correlated with lncRNA MALAT1 and FOXA1. FOXA1 bound to and interacted with CD24 and then regulated Src phosphorylation. MiR-3064-5p played an antiangiogenic role by inhibiting the FOXA1/CD24/Src pathway, whereas oncogenic MALAT1 functioned as a competing endogenous RNA (ceRNA) by sponging miR-3064-5p to alleviate the suppressive effect on the FOXA1 pathway. HCC patients with high miR-3064-5p, low MALAT1, or low FOXA1 expression had a better prognosis with longer overall survival and recurrence-free survival. In univariate and multivariate analyses, miR-3064-5p was identified as the independent prognostic predicator for HCC progression and patient survival. Taken together, miR-3064-5p exerts an antiangiogenic role by targeting the FOXA1/CD24/Src pathway but oncogenic lncRNA MALAT1 acts as a ceRNA to sponge miR-3064-5p. MiR-3064-5p is of great clinical significance and is a novel prognostic indicator and an attractive therapeutic target for HCC.

Cypermethrin triggers YY1-mediated testosterone biosynthesis suppression.

Cypermethrin (CYP), an extensively-used broad-spectrum pyrethroid pesticide, is regarded as a potential environmental endocrine disruptor with the anti-androgenic characteristic. To explore underlying roles of non-coding RNAs and the Jak/Stat pathway in CYP-mediated testosterone biosynthesis suppression, SD rats and Leydig cells were employed in this work. Results displayed that ß-CYP decreased plasma testosterone levels and led to abnormal alterations of testicular histomorphology and ultrastructures. LncRNA XIST and miR-142-5p were co-localized in the cytoplasm of Leydig cells, but the expression of XIST was inhibited by ß-CYP while that of miR-142-5p was induced. Then overexpressed miR-142-5p dampened the Jak1/Stat1 pathway by directly targeting Jak1. Transcription factors NFκB and YY1 impeded by ß-CYP were positively regulated by the Jak1/Stat1 pathway. Bidirectional Co-IP and ChIP assays demonstrated that NFκB interacted with and modulated YY1 by directly binding to the promoter region of YY1. ChIP, qPCR, and YY1 knockdown/overexpression assays indicated that YY1 acted as a transcriptional activator to directly modulate steroidogenic StAR and 3ß-HSD in Leydig cells. Taken together, miR-142-5p sponged by lncRNA XIST directly targets the Jak1/Stat1 pathway, which regulates steroidogenic StAR and 3ß-HSD via NFκB and YY1, and ultimately dampens testosterone production in Leydig cells.

Triclosan Suppresses Testicular Steroidogenesis via the miR-6321/JNK/ Nur77 Cascade.

BACKGROUND/AIMS: Triclosan (TCS), a broad-spectrum antibacterial and antifungal compound and an endocrine disruptor, has anti-androgenic properties and could adversely affect male reproduction and fertility. METHODS: To elucidate the underlying roles of miRNAs and the MAPK pathway in TCS-mediated repression of testicular steroidogenesis, Sprague-Dawley male rats were dosed daily with TCS for 31 days, and TM3 cells were exposed to TCS for 24 h after the pretreatments with the activator of JNK, Nur77 siRNA, or recombinant lentivirus vector for Nur77. Tissues and/or cells were analyzed by several techniques including transmission electron microscopy, lentivirus production, overexpression, gene silencing, luciferase reporter assay, chromatin immunoprecipitation, western blot, and real-time PCR. RESULTS: TCS caused histopathologic alterations in the testis and reduced plasma LH and testicular testosterone. TCS induced miR-6321 expression, which in turn depressed its target gene, Map3k1. The inhibition of Map3k1 subsequently inactivated its downstream JNK/c-Jun pathway. ChIP and qPCR assays confirmed that c-Jun directly bound to the Nur77 DNA promoter regions to regulate Nur77 expression. The knockdown and overexpression of Nur77 demonstrated that the JNK/c-Jun-mediated decline in the transcription and translation of Nur77 resulted in the depression of steroidogenic proteins including SRB1, StAR, and 3ß-HSD. Intriguingly, the protein expressions of 5α-Reductases (SRD5A1 and SRD5A2) were also downregulated after TCS exposure. CONCLUSION: Taken together, the miR-6321/Map3k1-regulated JNK/c-Jun/ Nur77 cascade contributes to TCS-caused suppression of testicular steroidogenesis, and the decrease in 5α-Reductase expressions may be the compensatory mechanism.

The Protective Effect of Autophagy on DNA Damage in Mouse Spermatocyte-Derived Cells Exposed to 1800 MHz Radiofrequency Electromagnetic Fields.

BACKGROUND/AIMS: The effects of exposure to radiofrequency electromagnetic fields (RF-EMFs) on the male reproductive system have raised public concern and studies have shown that exposure to RF-EMFs can induce DNA damage and autophagy. However, there are no related reports on the role of autophagy in DNA damage in spermatocytes, especially after exposure to RF-EMFs. The aim of the present study was to determine the mechanism and role of autophagy induced by RF-EMFs in spermatozoa cells. METHODS: Mouse spermatocyte-derived cells (GC-2) were exposed to RF-EMFs 4 W/kg for 24 h. The level of reactive oxygen species (ROS) was determined by ROS assay kit. Comet assay was utilized to detect DNA damage. Autophagy was detected by three indicators: LC3II/LC3I, autophagic vacuoles, and GFP-LC3 dots, which were measured by western blot, transmission electron microscopy, and transfection with GFP-LC3, respectively. The expression of the molecular signaling pathway AMP-activated protein kinase (AMPK)/mTOR was determined by western blot. RESULTS: The results showed that RF-EMFs induced autophagy and DNA damage in GC-2 cells via ROS generation, and the autophagy signaling pathway AMPK/mTOR was activated by ROS generation. Furthermore, following inhibition of autophagy by knockdown of AMPKα, increased DNA damage was observed in GC-2 cells following RF-EMFs exposure, and overexpression of AMPKα promoted autophagy and attenuated DNA damage. CONCLUSIONS: These findings demonstrated that the autophagy which was induced by RF-EMFs via the AMPK/mTOR signaling pathway could prevent DNA damage in spermatozoa cells.

Exposures to Atmospheric PM10 and PM10-2.5 Affect Male Semen Quality: Results of MARHCS Study.

Studies have shown that the effects of ambient particulate matter (PM) may be related to particle's size. However, results on the relationships between different PM and reproductive health are controversial. To explore the impacts of various PM fractions on male reproductive health, a total of 796 eligible subjects recruited in 2013 baseline investigation. In addition, there were 656 (82.4%) and 568 (71.3%) subjects participated follow-up surveys in 2014 and 2015, respectively. We used multivariable regression analysis and mixed-effect model to investigate the associations between air pollutants PM10, PM10-2.5, and PM2.5 exposures and semen quality, sperm DNA fragmentation and serum reproductive hormones of subjects. In the preliminary regression analysis, PM10, PM10-2.5, and PM2.5 exposure all associated with sperm concentration, morphology, sperm high DNA stainability (HDS), serum estradiol and testosterone levels. However, in mixed models, we only found that PM10 exposure were negatively associated with sperm normal morphology (95% CI: -14.13, -24.47) but positively associated with sperm progressive motility (95% CI: 23.00, 8.49), and PM10-2.5 exposure was inversely associated with sperm concentration (95% CI: -9.06, -27.31) after multiplicity adjustment. Our results provide the evidence that air PM10 and PM10-2.5 exposures, not PM2.5, are risk factors of semen quality.

Exomic and Epigenomic Analyses in a Pair of Monozygotic Twins Discordant for Cryptorchidism.

Cryptorchidism represents one of the most common human congenital anomalies. In most cases, its etiology remains unclear and seems to be multifactorial. In the present study, a pair of monozygotic twins discordant for cryptorchidism was identified. Twin zygosity was confirmed by microsatellite genotyping. Whole exome sequencing and methylated DNA immunoprecipitation sequencing (MeDIP-Seq) of DNA extract from leucocytes were performed to, respectively, evaluate their exomes and epigenomes. No differences in exome sequencing data were found between the twins after validation. MeDIP-Seq analysis detected 5,410 differentially hypermethylated genes and 2,383 differentially hypomethylated genes. Bioinformatic analysis showed that these genes belonged to several biological processes and signaling pathways, including regulation of actin cytoskeleton, which has been previously implicated in the etiology of cryptorchidism. The findings of the present study suggest that non-genetic factors might contribute to the pathogenesis of cryptorchidism.

Research on the Vertical Transmission of Hepatitis C Gene from Father-to-child via Human Sperm.

BACKGROUND: The aim was to develop a better experimental model which could facilitate further studies assessing the vertical HCV gene transmission via human spermatozoa, and verify the possibility of father-to-child transmission of the HCV gene. METHODS: The recombinant plasmid pIRES2-EGFP-HCV C was constructed. Fluorescence in situ hybridization was performed to detect the integration of the HCV C gene in human sperm genome and in zygote's pronucleus. RESULTS: Successful construction of recombinant plasmid pIRES2-EGFP-HCV C was confirmed by restriction mapping, PCR, and sequencing. Positive HCV C DNA signals were observed in sperm heads, human sperm chromosomes and two-cell embryos in transfected samples. No positive signal was found in normal control and HCV infected groups. CONCLUSIONS: The recombinant plasmid pIRES2-EGFP-HCV C was successfully constructed. The HCV C gene was able to pass through the sperm membrane and integrate into the sperm genome. Human sperm carrying the HCV C gene was able to achieve normal fertilization. The replication of the sperm-mediated HCV C gene was synchronized with that of the host genome. Our results provide direct evidence for vertical transmission of the HCV C gene from father-to-child via human sperm.

Effects of Hepatitis C Virus Infection on Human Sperm Chromosomes.

BACKGROUND: To determine the degree of chromosomal aberrations in the sperm of men with hepatitis C. METHODS: 36 subjects (20 in the healthy control group and 16 in the HCV infection group [genotype 1b]) were recruited. The cause of viral transmission was unknown in all patients. Sperm samples from the subjects were used for interspecies in vitro fertilization of zona-free golden hamster ova. The frequencies of spermatozoan aberrations were compared between the healthy control group and the HCV infection group. RESULTS: A total of 280 sperm chromosome complements were studied, including 129 complements from the 16 donors in the HCV infection group and 151 from the healthy control group. Of the 129 analyzable sperm metaphase spreads in the HCV infection group, 14 (10.85%) complements contained chromosomal aberrations, which was significantly higher than the number (9/151, 5.96%) in the healthy control group (p < 0.01). Moreover, in the HCV infection group, chromosomes frequently showed anomalies such as stickiness, clumping, and failure to stain, which prevented their analysis. CONCLUSIONS: HCV infection has mutagenic effects on the chromosomes in sperm and may lead to extensive heredi-tary effects owing to genetic alterations and/or chromosomal aberrations. In addition, there is the possibility of vertical transmission of HCV via the germ line.

Association between polymorphisms in IL27 and risk for CHD in a Chinese population.

BACKGROUND: IL-27, a member of the IL-12 family, has been involved in maternal tolerance to the foetus and successful pregnancy. Growing evidences indicate that IL-27 plays a crucial role in pregnancy. Aim We carried out the present study in order to investigate whether polymorphisms in the IL27 are associated with the risk for CHDs, including atrial septal defect and ventricular septal defect. Patients and methods We conducted this case-control study among 247 atrial septal defect patients, 150 ventricular septal defect patients, and 368 healthy controls in a Chinese population using polymerase chain reaction-restriction fragment length polymorphism assay. RESULTS: Significantly increased risk for atrial septal defect (p=0.001, OR=1.490, 95% CI=1.178-1.887) and ventricular septal defect (p=0.004, OR=1.502, 95% CI=1.139-1.976) was observed to be associated with the allele G of rs153109. In a dominant model, we have also observed that increased susceptibilities for atrial septal defect (p<0.01, OR=1.89, 95% CI=1.35-2.63) and ventricular septal defect (p<0.01, OR=2.50, 95% CI=1.67-3.85) were statistically associated with rs153109; however, no association was found between CHD risk and rs17855750 in the IL27 gene. CONCLUSION: The 153109 of the IL27 gene may be associated with the susceptibility to CHD, including atrial septal defect and ventricular septal defect.

Racial/ethnic disparities in human DNA methylation.

The racial/ethnic disparities in DNA methylation patterns indicate that molecular markers may play a role in determining the individual susceptibility to diseases in different ethnic groups. Racial disparities in DNA methylation patterns have been identified in prostate cancer, breast cancer and colorectal cancer and are related to racial differences in cancer prognosis and survival.

Factors affecting sperm fertilizing capacity in men infected with HIV.

Studies on the sperm-fertilizing capacity of HIV-seropositive men show conflicting results for reasons that are not yet clear. The aim of this study was to investigate the effects and relationships of some factors such as patient age, CD4(+) cells count, fathering offspring, concomitant sexually transmitted diseases (STD), and receipt of highly active anti-retroviral therapy (HAART) on sperm fertilizing capacity. Semen samples were collected from 33 HIV-seropositive men. Data on the above factors were acquired from a self-designed questionnaire. Computer-assisted sperm analysis, a hypo-osmotic swelling, and zona-free hamster oocyte penetration tests were performed according to criteria of the World Health Organization. CD4(+) cells in peripheral blood were examined using a flow cytometric (FCM) analyzer. Sperm vitality, sperm motility (grades a + b), total sperm motility, and sperm penetration rates were significantly higher in patients whose CD4(+) counts were ≧350/µl than in those whose CD4(+) counts were <350/µl (P < 0.05), and the parameters mentioned above were also significantly correlated with CD4(+) cell number (all P < 0.05). Significant differences in total sperm count and sperm tail swelling rate between patients co-infected with STD and without STD were observed (P < 0.05). Sperm penetration rate in patients receiving HAART was significantly higher than in those not receiving HAART (P < 0.05). Blood CD4(+) cell counts are an important indicator for evaluating sperm fertilizing capacity of HIV-seropositive men. After receiving HAART, the sperm penetration rate of HIV-seropositive men can be improved.

Cadmium activates the innate immune system through the AIM2 inflammasome.

Cadmium (Cd) is a widely used heavy metal and has recently been recognized as a possible source of human toxicity due to its ability to accumulate in organs. Accumulation of heavy metals has several adverse effects, including inducing inflammation, in multiple organs, such as the testis. However, how Cd ions are sensed by host cells and how tissue inflammation eventually occurs remains unclear. Here, we show that Cd activates the AIM2 inflammasome by mediating genomic DNA release into the cytoplasm after DNA damage via oxidative stress, to trigger IL-1ß secretion and pyroptosis. Specifically, the toxicity effects induced by Cd in cells were prevented by melatonin, which served as an antagonist of oxidative stress. Accordingly, in a mouse model, Cd-induced inflammation in the testis and consequential male reproductive dysfunction were effectively reversed by melatonin. Thus, our results suggest a function of AIM2 in Cd-mediated testis inflammation and identify AIM2 as a major pattern recognition receptor in response to heavy metal Cd ions.

Identification of a Novel Frameshift Variant of ARR3 Related to X-Linked Female-Limited Early-Onset High Myopia and Study on the Effect of X Chromosome Inactivation on the Myopia Severity.

X-linked myopia 26 (Myopia 26, MIM #301010), which is caused by the variants of ARR3 (MIM *301770), is characterized by female-limited early-onset high myopia (eo-HM). Clinical characteristics include a tigroid appearance in the fundus and a temporal crescent of the optic nerve head. At present, the limited literature on eo-HM caused by ARR3 mutations shows that its inheritance mode is complex, which brings certain difficulties to pre-pregnancy genetic counseling, pre-implantation genetic diagnosis, and prenatal diagnosis. Here, we investigated the genetic underpinning of a Chinese family with eo-HM. Whole exome sequencing of the proband revealed a novel frameshift mutation in ARR3 (NM_004312, exon10, c.666delC, p. Asn222LysfsTer22). Although the mode of inheritance of the eo-HM family fits the X-linked pattern of ARR3, the phenotypes of three patients deviate from the typical early-onset high myopia. Through X-chromosome inactivation experiments, the patient's different phenotypes can be precisely explained. In addition, this study not only enhanced the correlation between ARR3 and early-onset high myopia but also provided explanations for different phenotypes, which may inspire follow-up studies. Our results enrich the knowledge of the variant spectrum in ARR3 and provide critical information for preimplantation and prenatal genetic testing, diagnosis, and counseling.

Environmental exposure to arsenic may reduce human semen quality: associations derived from a Chinese cross-sectional study.

BACKGROUND: Recent observations in in vitro and in vivo models suggest that arsenic (As) is an endocrine disruptor at environmentally-relevant levels. When exposed to As, male rats and mice show steroidogenic dysfunction that can lead to infertility. However, the possible effects of As on human male semen quality remain obscure. METHODS: We monitored the profile of As species in the urine of a reproductive-age human cohort and assessed its association with semen quality. Men (n = 96) were recruited in an infertility clinic from July 2009 to August 2010 in the Affiliated Hospital of Chongqing Institute for Population and Family Planning. Five urinary As species were analyzed by high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). Clinical information on the semen volume, sperm concentration and motility was employed to catalogue and evaluate semen quality according to WHO guidelines. As species concentrations in addition to other continuous variables were dichotomized by the medians and modelled as categorical variables in order to explore using the binary logistic regression possible associations between As exposure and semen quality. RESULTS: Urinary concentrations (geometric mean ± SD, µg g(-1) creatinine) of different As species were 7.49 (± 24.8) for AsB, 20.9 (± 13.7) for DMA, 2.77 (± 3.33) for MMA, and 4.03 (± 3.67) for Asi (Asi(III )and Asi(V)). DMA concentrations above the median were significantly associated with below-reference sperm concentrations (P = 0.02) after adjusting for age, body mass index (BMI), abstinence, smoking and drinking habits. In addition, smoking was positively associated with MMA. CONCLUSION: Reduced parameters in human semen quality are positively associated with As exposure in a reproductive-age Chinese cohort.

Down-regulation of KLF9 ameliorates LPS-caused acute lung injury and inflammation in mice via reducing GSDMD expression.

Acute lung injury (ALI) is considered as a severe respiratory disease with aggravated inflammatory responses. Krüppel-like factor 9 (KLF9), a member of KLF family, has been reported to be involved in inflammatory disorders. However, the effect of KLF9 in ALI has not been elucidated. Here the present study was to clarify the role of KLF9 and its mechanism in ALI. The ALI in vitro model was established with lipopolysaccharide (LPS)-treated RAW264.7 cells. Mice were injected with LPS to conduct an ALI in vivo model. The expression of KLF9 and gasdermin D (GSDMD) was examined using quantitative reverse transcription-PCR, haematoxylin-eosin/immunohistochemistry staining and western blot assays. Enzyme-linked immunosorbent assay was employed to detect the levels of inflammatory cytokines. JASPAR database was used to predict the recognition motif of KLF9, and the relationship between KLF9 and GSDMD was determined by luciferase reporter assay and chromatin immunoprecipitation analysis. The number of neutrophils in bronchoalveolar lavage fluid as well as the wet/dry weight ratio was caculated. The results showed that The expression of KLF9 in lung was significantly increased in LPS-stimulated mice. Moreover, KLF9 knockout relieved the lung injury in ALI mice. GSDMD is one of targets genes of the transcription factor KLF9. KLF9 knockout induced a decreased expression of GSDMD in LPS-treated mice. Furthermore, in RAW264.7 cells after LPS administration, KLF9 knockdown reduced the levels of inflammatory factors and suppressed the expression of GSDMD. In summarise, these findings exhibited that KLF9 knockout could mitigate the lung injury and inflammatory responses in ALI mice by directly regulating GSDMD.

BCKDK Promotes Ovarian Cancer Proliferation and Migration by Activating the MEK/ERK Signaling Pathway.

Background: Ovarian cancer (OC) is the most fatal gynecologic cancer. The branched-chain α-keto acid dehydrogenase kinase (BCKDK) plays an important role in many serious human diseases, including cancers. Its function in promoting cell proliferation and migration has been reported in various cancers. However, the biological role of BCKDK and its molecular mechanisms underlying OC initiation and progression are unclear. Methods: First, the expression level of BCKDK in OC cell lines or tissues was determined using tissue microarray- (TMA-) based immunohistochemistry or western blotting. Then, growth curve analysis, anchorage-independent cell transformation assays, wound healing assays, cell migration assays, and tumor xenografts were used to test whether BCKDK could promote cell transformation or metastasis. Finally, the signaling pathways involved in this process were investigated by western blotting or immunoprecipitation. Results: We found that the expression of BCKDK was upregulated in OC tissues and the high expression of BCKDK was correlated with an advanced pathological grade in patients. The ectopic overexpression of BCKDK promoted the proliferation and migration of OC cells, and the knockdown of BCKDK with shRNAs inhibited the proliferation and migration of OC ex vivo and in vivo. Moreover, BCKDK promoted OC proliferation and migration by activating MEK. Conclusions: Our results demonstrate that BCKDK promotes OC proliferation and migration by activating the MEK/ERK signaling pathway. Targeting the BCKDK-MEK axis may provide a new therapeutic strategy for treating patients with OC.

In vitro study on vertical transmission of the HIV-1 gag gene by human sperm.

HIV/AIDS is a major public health problem worldwide. To explore the feasibility of HIV vertical transmission by human sperm, plasmid construction and transfection, interspecific in vitro fertilization of zona-free hamster ova by human sperm, fluorescence in situ hybridization (FISH), RT-PCR, and immunofluorescence assay (IFA) were carried out. The FISH signals for HIV-1 gag DNA were observed in the nuclei and chromosomes of transfected human sperm, male pronuclei of zygotes, and nuclei of blastomeres of two-cell embryos, indicating that the HIV-1 gag gene could be transmitted via the sperm membrane and integrated into the sperm genome. In contrast, human sperm carrying the target gene achieved normal fertilization, and replication of the sperm-mediated target gene was synchronized with the host genome. Using RT-PCR, the positive bands for the target gene were observed in the transfected human sperm and two-cell embryos. These results further confirm that the target gene can be transcribed into mRNA in human sperm and embryonic cells. Positive signals for the HIV-1 p24 gag protein were shown by IFA in two-cell embryos containing the sperm-mediated target gene and not in the transfected human sperm, which indicated that the sperm-mediated target gene could be translated to make HIV-1 p24 gag protein in embryonic cells, but not in sperm cells. The results provide evidence for possible vertical transmission of the HIV-1 gag gene to the embryo by fertilizing sperm in vitro.