Integrative metabolome and transcriptome analyses reveal the coloration mechanism in Camellia oleifera petals with different color.

BACKGROUND: Camellia olelfera petals are colorful, and have high ornamental value. However, the color formation mechanism of C. olelfera petals with different color is still unclear. In our study, WGCNA method was applied to integrate metabolites and transcriptomes to investigate the coloration mechanism of four C. olelfera cultivars with different petal colors. RESULTS: Here, a total of 372 flavonoids were identified (including 27 anthocyanins), and 13 anthocyanins were significantly differentially accumulated in C. olelfera petals. Among them, cyanidin-3-O-(6''-O-p-Coumaroyl) glucoside was the main color constituent in pink petals, cyanidin-3-O-glucoside, cyanidin-3-O-galactoside, cyanidin-3-O-rutinoside, and cyanidin-3-O-(6''-O-malonyl) glucoside were the main contributors to candy pink petals, and peonidin-3-O-glucoside was the important color substance responsible for the red petals of C. oleifera. Furthermore, six structural genes (Co4CL1, CoF3H1, CoF3'H, CoANS, CoUGT75C1-4, and CoUGT75C1-5), three MYBs (CoMYB1, CoMYB4, and CoMYB44-3), three bHLHs (CobHLH30, CobHLH 77, and CobHLH 79-1), and two WRKYs (CoWRKY7 and CoWRKY22) could be identified candidate genes related to anthocyanins biosynthesis and accumulation, and lead to the pink and red phenotypes. The regulatory network of differentially accumulated anthocyanins and the anthocyanins related genes in C. olelfera petals were established. CONCLUSIONS: These findings elucidate the molecular basis of the coloration mechanisms of pink and red color in C. olelfera petals, and provided valuable target genes for future improvement of petals color in C. olelfera.

Multi-environment evaluations across ecological regions reveal climate and soil effects on amides contents in Chinese prickly ash peels (Zanthoxylum bungeanum Maxim.).

BACKGROUND: Environmental factors difference is the key factor for the difference in the production, transformation and accumulation of effective components in plants. UPLC-MS/MS and multivariate statistical methods were applied to describe the region difference of amides compounds in Chinese prickly ash peels from different regions and their correlation with climatic factors and soil factors. RESULTS: Amides compounds contents were significantly higher in high altitude areas, with obvious altitude change trend. Two ecotypes were classified based on the amides compounds contents, one was the high altitude-cool type from Qinghai, Gansu, Sichuan and western Shaanxi province, and the other one was low altitude-warm type from eastern Shaanxi, Shanxi, Henan, Hebei and Shandong province. Amides compounds content were negatively correlated with annual mean temperature, max temperature of warmest month, mean temperature of wettest quarter and mean temperature of warmest quarter (P < 0.01). Except for hydroxy-γ-sanshool and ZP-amide A, the residual amides contents were significantly positively correlated with organic carbon, available nitrogen, phosphorus and potassium in soil and negatively correlated with soil bulk density. Low temperature, low precipitation and high organic carbon in soil were conducive to amides accumulation. CONCLUSIONS: This study aided in site specific exploration of high amides contents yielding samples, enriched the environment factors effects on amides compounds, and provided scientific foundation for the improvement of Chinese prickly ash peels quality and the location of high-quality production areas.

[Micro-dissection testicular sperm extraction for non-obstructive azoospermia patients with the history of secondary testicular injury].

Objective: To investigate the value of micro- dissection testicular sperm extraction (micro-TESE) in the treatment of non-obstructive azoospermia (NOA) in patients with the history of secondary testicular injury. METHODS: Totally, 121 NOA patients with the history of secondary testicular injury underwent micro-TESE in our hospital from September 2014 to December 2017. We analyzed the correlation of the sperm retrieval rate with the causes of testicular injury and compared the outcomes of the ICSI cycles with the sperm retrieved from the NOA males by micro-TESE (the micro-TESE group) and those with the sperm ejaculated from severe oligospermia patients (sperm concentration <1×106/ml, the ejaculate group). Comparisons were also made between the two groups in the female age, two-pronucleus (2PN) fertilization rate, transferrable embryos on day 3 (D3), D3 high- quality embryos, D14 blood HCG positive rate, embryo implantation rate, and clinical pregnancy rate. RESULTS: Testicular sperm were successfully retrieved by micro-TESE in 86.0% of the patients (104/121), of whom 98.4% had the history of orchitis, 75.5% had been treated surgically for cryptorchidism, and 63.6% had received chemo- or radiotherapy. No statistically significant differences were observed between the micro-TESE and ejaculate groups in the 2PN fertilization rate (59.4% vs 69.3%, P > 0.05), D14 blood HCG positive rate (44.6% vs 57.9%, P > 0.05), embryo implantation rate (31.8 %% vs 32.6%, P > 0.05) and clinical pregnancy rate (41.5% vs 48.7%, P > 0.05). However, the rate D3 transferrable embryos was significantly lower in the micro-TESE than in the ejaculate group (40.5% vs 52.2%,P < 0.05), and so was that of D3 high-quality embryos (32.5% vs 42.1%, P < 0.05). CONCLUSIONS: Micro-TESE can be applied as the first choice for NOA patients with the history of secondary testicular injury, but more effective strategies are to be explored for the improvement of ICSI outcomes with the sperm retrieved by micro- TESE.

Prematuration with cyclic adenosine monophosphate modulators alters cumulus cell and oocyte metabolism and enhances developmental competence of in vitro-matured mouse oocytes.

Oocyte in vitro maturation (IVM) is an important assisted reproductive technology and research tool. The adoption of IVM into routine clinical practice has been hindered by its significantly lower success rates compared to conventional in vitro fertilization. Cyclic AMP (cAMP) modulation and follicle-stimulating hormone (FSH), independently, have long been known to improve IVM oocyte developmental competence. This study comprehensively examined the effects of FSH and cAMP/cGMP modulation, alone and in combination, on IVM oocyte metabolism and developmental outcomes. Mouse cumulus-oocyte complexes (COCs) were subjected to a 1 h prematuration phase ± the cAMP modulator forskolin and cAMP/cGMP modulator 3-isobutyl-1-methylxanthine followed by IVM ± FSH. Prematuration with these cyclic nucleotide modulators or IVM with FSH significantly improved oocyte developmental competence and reduced spindle abnormalities compared to spontaneous IVM (no treatment); however, these two treatments in combination endowed even greater developmental competence (improved subsequent blastocyst rates and quality; P < 0.05), albeit blastocyst yield and quality remained significantly lower than that of oocytes matured in vivo. A significant additive effect of combined IVM treatments was evident as increased COC lactate production and oxygen consumption and enhanced oocyte oxidative metabolism, ATP production, ATP:ADP ratio, and glutathione levels (P < 0.05). Nevertheless, IVM increased reactive oxygen species production, particularly as a consequence of FSH addition, relative to in vivo matured oocytes. In conclusion, improvements in the embryo yield following IVM is associated with increased COC oxygen consumption and oocyte oxidative metabolism, but these remain metabolically and developmentally less competent relative to in vivo derived oocytes.

Heparin and cAMP modulators interact during pre-in vitro maturation to affect mouse and human oocyte meiosis and developmental competence.

STUDY QUESTION: Does heparin ablate the advantageous effects of cyclic adenosine mono-phosphate (cAMP) modulators during pre-in vitro maturation (IVM) and have a deleterious effect in standard oocyte IVM? SUMMARY ANSWER: Heparin interrupts energy metabolism and meiotic progression and adversely affects subsequent development of oocytes under conditions of elevated cAMP levels in cumulus-oocyte complexes (COCs) after pre-IVM treatment with forskolin. WHAT IS KNOWN ALREADY: In animal IVM studies, artificial regulation of meiotic resumption by cAMP-elevating agents improves subsequent oocyte developmental competence. Heparin has no effect on spontaneous, FSH- or epidermal growth factor (EGF)-stimulated meiotic maturation. STUDY DESIGN, SIZE, DURATION: An in vitro cross-sectional study was conducted using immature mouse and human COCs. Depending on individual experimental design, COCs were treated during pre-IVM with or without heparin, in the presence or absence of forskolin and/or 3-isobutyl-1-methylxanthine (IBMX), and then COC function was assessed by various means. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Forty-two women with polycystic ovaries (PCOs) or polycystic ovarian syndrome (PCOS) donated COCs after oocyte retrieval in a non-hCG-triggered IVM cycle. COCs were collected in pre-IVM treatments and then cultured for 40 h and meiotic progression was assessed. COCs from 21- to 24-day-old female CBA F1 mice were collected 46 h after stimulation with equine chorionic gonadotrophin. Following treatments, COCs were checked for meiotic progression. Effects on mouse oocyte metabolism were measured by assessing oocyte mitochondrial membrane potential using JC-1 staining and oocyte ATP content. Post-IVM mouse oocyte developmental competence was assessed by in vitro fertilization and embryo production. Blastocyst quality was evaluated by differential staining of inner cell mass (ICM) and trophectoderm (TE) layers. MAIN RESULTS AND THE ROLE OF CHANCE: In the absence of heparin in pre-IVM culture, the addition of cAMP modulators did not affect human oocyte MII competence after 40 h. In standard IVM, heparin supplementation in pre-IVM did not affect MII competence; however, when heparin was combined with cAMP modulators, MII competence was significantly reduced from 65 to 15% (P < 0.05). In mouse experiments, heparin alone in pre-IVM significantly delayed germinal vesicle breakdown (GVBD) so that fewer GVBDs were observed at 0 and 1 h of IVM (P < 0.05), but not by 2 or 3 h of IVM. Combined treatment with IBMX and forskolin in the pre-IVM medium produced a large delay in GVBD such that no COCs exhibited GVBD in the first 1 h of IVM, and the addition of heparin in pre-IVM further significantly delayed the progression of GVBD (P < 0.05), in a dose-dependent manner (P < 0.01). Combined IBMX and forskolin treatment of mouse COCs during pre-IVM significantly increased mitochondrial membrane potential and ATP production in the oocyte at the end of pre-IVM (P < 0.05), and significantly improved fertilization, embryo development and quality (P < 0.05). However, heparin abolished the IBMX + forskolin-stimulated increase in mitochondrial membrane potential and ATP production (P < 0.05), and adversely affected embryonic cleavage, development rates and embryo quality (P < 0.05). This latter adverse combinational effect was negated when mouse COCs were collected in heparin and IBMX for 15 min, washed and then cultured for 45 min in IBMX and forskolin without heparin. LIMITATION, REASONS FOR CAUTION: Experiments in mice found that heparin ablation of the advantageous effects of cAMP modulators during pre-IVM was associated with altered oocyte metabolism, but the mechanism by which heparin affects metabolism remains unclear. WIDER IMPLICATIONS OF THE FINDINGS: This study has revealed a novel and unexpected interaction between heparin and cAMP modulators in pre-IVM in immature mouse and human oocytes, and established a means to collect oocytes using heparin while modulating oocyte cAMP to improve developmental potential.

MiR-708-5p/Pit-1 axis mediates high phosphate-induced calcification in vascular smooth muscle cells via Wnt8b/ß-catenin pathway.

Recently, the underlying mechanism of vascular calcification (VC) has been partially elucidated. However, it is still high incidence, and no effective treatment has been found. This study aims at figuring out the underlying mechanisms of microRNA-708-5p (miR-708-5p)/sodium-phosphate transporter 1 (Pit-1) axis in high phosphate (HP)-induced VC of T/G HA-VSMCs. Alizarin Red S staining was used to evaluate calcium salt deposition, and the activity of alkaline phosphatase (ALP) was determined by measuring the absorbance at 405 nm. RT-qPCR and Western blot were performed to assess the levels of miR-708-5p and Pit-1, the levels of ALP, Pit-1, ß-catenin, glycogen synthesis kinase 3 ß (GSK3ß), and p-GSK3ß proteins, respectively. The interaction between miR-708-5p and Pit-1 was validated by luciferase reporter assay. Our findings illustrated that miR-708-5p was downregulated and Pit-1was upregulated in HP-induced VC. MiR-708-5p mimics inhibited HP-induced VC. Further experiments demonstrated that miR-708-5p targets Pit-1. In addition, miR-708-5p inactivates the Wnt8b/ß-catenin pathway via targeting Pit-1 to reduce HP-induced VC. MiR-708-5p has a crucial effect on VC via targeting Pit-1 and inhibiting Wnt8b/ß-catenin pathway, it may serve as a new target for VC treatment.

MISSIM: An Incremental Learning-Based Model With Applications to the Prediction of miRNA-Disease Association.

In the past few years, the prediction models have shown remarkable performance in most biological correlation prediction tasks. These tasks traditionally use a fixed dataset, and the model, once trained, is deployed as is. These models often encounter training issues such as sensitivity to hyperparameter tuning and "catastrophic forgetting" when adding new data. However, with the development of biomedicine and the accumulation of biological data, new predictive models are required to face the challenge of adapting to change. To this end, we propose a computational approach based on Broad learning system (BLS) to predict potential disease-associated miRNAs that retain the ability to distinguish prior training associations when new data need to be adapted. In particular, we are introducing incremental learning to the field of biological association prediction for the first time and proposed a new method for quantifying sequence similarity. In the performance evaluation, the AUC in the 5-fold cross-validation was 0.9400 +/- 0.0041. To better assess the effectiveness of MISSIM, we compared it with various classifiers and former prediction models. Its performance is superior to the previous method. Besides, the case study on identifying miRNAs associated with breast neoplasms, lung neoplasms and esophageal neoplasms show that 34, 36 and 35 out of the top 40 associations predicted by MISSIM are confirmed by recent biomedical resources. These results provide ample convincing evidence of this approach have potential value and prospect in promoting biomedical research productivity.

Shear Stress Triggers Angiogenesis of Late Endothelial Progenitor Cells via the PTEN/Akt/GTPCH/BH4 Pathway.

BACKGROUND: Shear stress is an effective modulator of endothelial progenitor cells (EPCs) and has been suggested to play an important role in angiogenesis. The phosphatase and tensin homolog (PTEN)/Akt and guanosine triphosphate cyclohydrolase (GTPCH)/tetrahydrobiopterin (BH4) pathways regulate the function of early EPCs. However, the role of these pathways in the shear stress-induced angiogenesis of late EPCs remains poorly understood. Therefore, we aim to investigate whether shear stress could upregulate the angiogenesis capacity of late EPCs and to further explore the possible underlying mechanisms. METHODS: Late EPCs were subjected to laminar shear stress (LSS), and their in vitro migration, proliferation, and tube formation capacity were determined. In addition, the in vivo angiogenesis capacity was explored, along with the expression of molecules involved in the PTEN/Akt and GTPCH/BH4 pathways. RESULTS: LSS elevated the in vitro activities of late EPCs, which were accompanied by downregulated PTEN expression, accelerated Akt phosphorylation, and GTPCH/BH4 pathway activation (all P < 0.05). Following Akt inhibition, LSS-induced upregulated GTPCH expression, BH4, and NO level of EPCs were suppressed. LSS significantly improved the migration, proliferation, and tube formation ability (15 dyn/cm2 LSS vs. stationary: 72.2 ± 5.5 vs. 47.3 ± 7.3, 0.517 ± 0.05 vs. 0.367 ± 0.038, and 1.664 ± 0.315 vs. 1 ± 0, respectively; all P < 0.05) along with the in vivo angiogenesis capacity of late EPCs, contributing to the recovery of limb ischemia. These effects were also blocked by Akt inhibition or GTPCH knockdown (P < 0.05, respectively). CONCLUSIONS: This study provides the first evidence that shear stress triggers angiogenesis in late EPCs via the PTEN/Akt/GTPCH/BH4 pathway, providing a potential nonpharmacologic therapeutic strategy for promoting angiogenesis in ischemia-related diseases.

Effects of cilostamide and forskolin on the meiotic resumption and embryonic development of immature human oocytes.

BACKGROUND: In an attempt to allow for acquisition of oocyte cytoplasmic maturation, PDE3 specific inhibitor, cilostamide and adenylate cyclase activator, forskolin were used to extend pre-maturation culture of immature human oocytes. METHODS: Cumulus-oocyte complexes retrieved from unstimulated ovaries were continuously cultured under 20 microM cilostamide or 50 microM forskolin, alone or in combination for 6, 12, 24 or 48 h, respectively. Levels of intercellular gap junction communication (GJC) and maturational status were examined at these designated time points. Metaphase II oocytes obtained following 54 h biphasic culture (with meiotic inhibitors from 0 to 24 h, no meiotic inhibitors from 24 to 54 h) were subject to intracytoplasmic sperm injection and embryos were cultured for five more days. RESULTS: Both cilostamide and forskolin delayed spontaneous meiotic progression after continuous culture with immature human oocytes. Combined treatment of cilostamide and forskolin significantly lowered the rates of germinal vesicle breakdown (GVBD) at 6, 12, 24 or 48 h after meiotic inhibitory culture, when compared with the control (all P < 0.05). A delay of 6 h for the loss of GJC was also observed under the combined treatment of cilostamide and forskolin. The fertilization rate was significantly higher under the combined treatment of cilostamide and forskolin than that of the control. Although the rates of oocyte maturation and embryo cleavage were similar among groups, there was a slight but non-significant increase in blastocyst formation rate with the treatment of cilostamide and forskolin. CONCLUSIONS: Combined treatment of cilostamide and forskolin positively influences oocyte developmental competence by exhibiting a synergistic effect on the prevention of GJC loss and resumption of meiosis.

SIRT1 prevents atherosclerosis via liver­X­receptor and NF­κB signaling in a U937 cell model.

Atherosclerosis is a chronic immunoinflammatory disease associated with blood lipid disorders. Previous studies in mice have demonstrated that liver X receptor (LXR)­ATP­binding cassette (ABC) A1/ABCG1/C­C chemokine receptor type 7 (CCR7) and nuclear factor κB (NF­κB) signaling pathways are important for atherosclerotic plaque formation. In addition, Sirtuin 1 (SIRT1) has been reported as a key regulator in the protection from risk of atherosclerosis. However, the exact mechanism by which SIRT1 prevents atherosclerosis remains largely unknown. To explore the possible mechanisms, the expression of SIRT1 and the association between SIRT1, LXR and NF­κB in the process of foam cell formation was investigated in an in vitro human mononuclear U937 cell line. Monocyte­derived foam cells were induced by palmitate and Ox­LDL treatment. Oil Red O staining revealed an accumulation of a large number of lipid droplets in foam cells. Results of reverse transcription polymerase chain reaction (RT-PCR) revealed that SIRT1 expression was downregulated during foam cell formation. In addition, the expression of LXRα and its targets, ABCA1, ABCG1 and CCR7, were downregulated. However, NF­κB and its targets, tumor necrosis factor α (TNFα) and interleukin (IL)­1ß, were upregulated in foam cells. Following activation of SIRT1 by SRT1720, the expression of LXRα and its targets increased, whereas expression of NF­κB and its targets decreased. Furthermore, the formation of foam cells was blocked. The SIRT1 inhibitor, nicotinamide, was found to eliminate the effects of SRT1720. Results of the present study indicate that SIRT1 may prevent the formation and progression of atherosclerosis by enhancing the LXR­ABCA1/ABCG1/CCR7 and inhibiting the NF­κB pathways.

Preimplantation genetic diagnosis for Duchenne muscular dystrophy by multiple displacement amplification.

OBJECTIVE: To evaluate the use of multiple displacement amplification (MDA) in preimplantation genetic diagnosis (PGD) for female carriers with Duchenne muscular dystrophy (DMD). DESIGN: MDA was used to amplify a whole genome of single cells. Following the setup on single cells, the test was applied in two clinical cases of PGD. One mutant exon, six short tandem repeats (STR) markers within the dystrophin gene, and amelogenin were incorporated into singleplex polymerase chain reaction (PCR) assays on MDA products of single blastomeres. SETTING: Center for reproductive medicine in First Affiliated Hospital, Sun Yat-sen University, China. PATIENT(S): Two female carriers with a duplication of exons 3-11 and a deletion of exons 47-50, respectively. INTERVENTION(S): The MDA of single cells and fluorescent PCR assays for PGD. MAIN OUTCOME MEASURE(S): The ability to analyze single blastomeres for DMD using MDA. RESULT(S): The protocol setup previously allowed for the accurate diagnosis of each embryo. Two clinical cases resulted in a healthy girl, which was the first successful clinical application of MDA in PGD for DMD. CONCLUSION(S): We suggest that this protocol is reliable to increase the accuracy of the PGD for DMD.

In vitro-matured rat oocytes have low mitochondrial deoxyribonucleic acid and adenosine triphosphate contents and have abnormal mitochondrial redistribution.

OBJECTIVE: To study the development and function of mitochondria in in vitro-matured rat oocytes derived from follicles of different sizes. DESIGN: Experimental animal study. SETTING: Department of Anatomy at the University of Hong Kong. ANIMAL(S): Immature female Sprague-Dawley rats that were 25 days of age. INTERVENTION(S): Immature oocytes were collected from rat ovarian follicles of different sizes and were induced to mature in vitro. MAIN OUTCOME MEASURE(S): The number of copies of mitochondrial DNA, mitochondrial activity, adenosine triphosphate content of matured oocytes, and rates of fertilization and blastulation were determined. RESULT(S): The mitochondrial DNA copy number of oocytes increased linearly with the diameter of antral follicles. The mitochondrial DNA copy number, adenosine triphosphate content, and proportion of oocytes with peripheral distribution of mitochondria in in vitro-matured oocytes from small antral follicles were significantly lower than those from preovulatory follicles and in vivo-matured oocytes. Compared with in vitro-matured oocytes from small antral follicles, those from preovulatory follicles and in vivo-matured oocytes also had significantly better fertilization potential and higher blastulation rate. CONCLUSION(S): The inferior developmental potential of in vitro-matured oocytes may be attributed partly to a reduced number of mitochondria, resulting in insufficient production of adenosine triphosphate for required developmental events.

Biology of sperm capacitation: evidence for multiple signalling pathways.

The endpoint of in vitro/in vivo capacitation is the ability of sperm surface receptors to bind to their complementary ligands on zona pellucida, the extracellular glycocalyx that surrounds the egg, and undergo the Ca2+-dependent signal transduction. The net result is the fenestration and fusion of the sperm plasma membrane and the underlying outer acrosomal membrane at multiple sites and exocytosis of acrosomal contents. The hydrolytic action of glycohydrolases and proteinases, released at the site of sperm-zona binding, along with the enhanced thrust generated by the hyperactivated flagellar motility of the bound spermatozoon, are important factors that regulate the fertilization process. This report discusses the physiological significance of calmodulin, a 17 kDa Ca2+ sensor protein, in sperm function. The in vitro experimental approaches described in this article provide evidence strongly suggesting that calmodulin plays an important role in the priming (that is, capacitation) of mouse spermatozoa as well as in the agonist-induced acrosome reaction. In addition, we have used several calmodulin antagonists in an attempt to characterize further the morphological and biochemical changes associated with sperm capacitation. Data presented in this report suggest that calmodulin antagonists prevent capacitation by interfering with multiple regulatory pathways and do so either with or without adverse effects on sperm motility and protein tyrosine phosphorylation of sperm components.

Low mitochondrial DNA and ATP contents contribute to the absence of birefringent spindle imaged with PolScope in in vitro matured human oocytes.

BACKGROUND: Birefrigent meiotic spindle in live human oocytes can be visualized by the PolScope. This study investigated the relationship between birefrigent meiotic spindle and cytoplasmic mitochondrial DNA (mtDNA) and ATP contents in in vitro matured human oocytes. METHODS: Oocytes at germinal vesicle stage were collected and cultured for 24-48 h with or without the metabolic inhibitor, carbonyl cyanide p-(tri-fluromethoxy) phenyl-hydrazone (FCCP). All in vitro matured oocytes were examined by PolScope for the presence of meiotic spindle, then the oocytes were used for either intracytoplasmic sperm injection or the measurement of mitochondrial quantity and ATP content. RESULTS: Meiotic spindles were observed in 51.3% (60/117) of the in vitro matured oocytes. Oocytes with detectable meiotic spindle contained significantly higher mtDNA copies (637 250 +/- 237 606 versus 491 454 +/- 153 406, P = 0.027) and ATP content (1.97 +/- 0.38 versus 1.65 +/- 0.32 pmol, P = 0.028) when compared with those without detectable meiotic spindle. However, in vitro matured oocytes showed a significantly reduced rate of positive meiotic spindle and a lower ATP content when cultured with FCCP. A lower incidence of normal fertilization and good quality embryos were observed if meiotic spindles were not detected. CONCLUSIONS: Low mtDNA and ATP content might contribute to the absence of birefringent spindle imaged with the PolScope in human in vitro matured oocytes.

Oridonin induces apoptosis via PI3K/Akt pathway in cervical carcinoma HeLa cell line.

AIM: To investigate the apoptosis-inducing effect of oridonin, a diterpenoid isolated from Rabdosia rubescens, in the human cervical carcinoma HeLa cell line. METHODS: A morphological analysis, nuclear condensation, and fragmentation of chromatin were monitored using Hoechst 33342 staining. Cell viability was assessed using the 3-(4, 5-dimethylthiazol-(2)-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Cell apoptosis and the apoptosis-related activation in the HeLa cell line were evaluated by flow cytometry and Western blotting. RESULTS: Oridonin suppressed the proliferation of the HeLa cell line in a dose- and time-dependent fashion. Oridonin treatment downregulated the activation of protein kinase B (Akt), the expression of forkhead box class O (FOXO) transcription factor, and glycogen synthase kinase 3 (GSK3). Oridonin also induced the release of cytochrome c accompanied by the activation of caspase-3 and poly-adenosine diphosphate- ribose polymerase cleavage. In addition, Z-D(OMe)-E(OMe)-V-D(OMe)- FMK (z-DEVD-fmk), an inhibitor of caspases, prevented caspase-3 activation and abrogated oridonin-induced cell death. Finally, oridonin treatment of the HeLa cell line downregulated the expression of the inhibitor of the apoptosis protein. CONCLUSION: Our results showed that oridonin-induced apoptosis involved several molecular pathways. Oridonin may suppress constitutively activated targets of phosphatidylinositol 3-kinase (Akt, FOXO, and GSK3) in the HeLa cell line, inhibiting the proliferation and induction of caspase-dependent apoptosis.

Calmodulin antagonists differentially affect capacitation-associated protein tyrosine phosphorylation of mouse sperm components.

Sperm capacitation in vitro is thought to be correlated with the increased protein tyrosine phosphorylation of a subset of sperm components. Our group recently used a pharmacological approach to demonstrate that calmodulin (CaM), a 17 kDa calcium sensor protein, has a role in sperm capacitation. In the present study, we have used several CaM antagonists in an attempt to characterize further the role of CaM in capacitation-associated protein tyrosine phosphorylation of sperm components. Our data demonstrate, first, that mouse spermatozoa incubated in a medium that favors capacitation undergo increased protein tyrosine phosphorylation in a time-dependent manner. Second, inclusion of six CaM antagonists individually in an in vitro incubation medium prevented sperm capacitation, as demonstrated by their diminished ability to undergo agonist-induced acrosome reaction. Third, half of the CaM antagonists (compound 48/80, W13 and CaM-binding domain) had no effect on protein tyrosine phosphorylation or sperm motility. Fourth, by contrast, three CaM antagonists (W7, ophiobolin A and calmidazolium) significantly inhibited protein tyrosine phosphorylation of sperm components (42, 56, 66, 82 and 95 kDa) and adversely affected their motility without altering viability as assessed by propidium iodine staining. Finally, inclusion of purified CaM in the capacitation medium significantly increased tyrosine phosphorylation of 82 kDa and 95 kDa components. Combined, these data suggest that CaM antagonists prevent capacitation by interfering with multiple regulatory pathways, and do so either with or without adverse effects on sperm motility and protein tyrosine phosphorylation.

Assessment of acrosomal status in rat spermatozoa: studies on carbohydrate and non-carbohydrate agonists.

In the mouse and several other species, including man, capacitated acrosome-intact spermatozoa interact with natural [soluble zona pellucida (ZP) and progesterone (P4)] and synthetic [neoglycoproteins (ngps) and calcium (Ca(2+)) ionophore] agonists, prior to the initiation of a Ca(2+)-dependent signal transduction cascade. The net result is the fusion of the sperm plasma membrane overlying the outer acrosomal membrane at multiple sites and exocytosis of acrosomal contents [i.e., induction of the acrosome reaction (AR)]. This step is believed to be a prerequisite that enables the acrosome-reacted spermatozoon to penetrate the ZP and fertilize the egg. Although the rat is one of the most commonly used laboratory animals, very little is known about the chemical nature of agonists that induce the AR in this species. The lack of this information is primarily due to the fact that the rat sperm acrosome is a relatively thin structure. Thus, it is difficult to assess the status of the sperm acrosome in this species. In this report, we describe the use of a Coomassie brilliant blue dye staining procedure to assess the status of the rat sperm acrosome by light microscopy. The procedure is highly reproducible and has allowed us to determine the effects of carbohydrate (ngps and mouse ZP) and noncarbohydrate (P4 and Ca(2+) ionophore) agonists on capacitated spermatozoa. In addition, we have used a pharmacological approach to examine the functional significance of calmodulin (CaM), a Ca(2+)-binding protein, in induction of the AR in spermatozoa. Data presented in this report demonstrate that several ngps, solubilized mZP, P4, and Ca(2+) ionophores induce the AR in rat spermatozoa. Furthermore, we demonstrate that, whereas CaM antagonists blocked P4-induced AR, most of the inhibitors used had no significant effect on the Ca(2+) ionophore-induced (nonphysiological) AR.