Hai-tao Zeng

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.

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

AbstractAim: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.

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.

Prematuration with Cyclic Adenosine Monophosphate Modulators Alters Cumulus Cell and Oocyte Metabolism and Enhances Developmental Competence of In Vitro-Matured Mouse Oocytes

ABSTRACT 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.

Oocyte maturation and quality: role of cyclic nucleotides

The cyclic nucleotides, cAMP and cGMP, are the key molecules controlling mammalian oocyte meiosis. Their roles in oocyte biology have been at the forefront of oocyte research for decades, and many of the long-standing controversies in relation to the regulation of oocyte meiotic maturation are now resolved. It is now clear that the follicle prevents meiotic resumption through the actions of natriuretic peptides and cGMP - inhibiting the hydrolysis of intra-oocyte cAMP - and that the pre-ovulatory gonadotrophin surge reverses these processes. The gonadotrophin surge also leads to a transient spike in cAMP in the somatic compartment of the follicle. Research over the past two decades has conclusively demonstrated that this surge in cAMP is important for the subsequent developmental capacity of the oocyte. This is important, as oocyte in vitro maturation (IVM) systems practised clinically do not recapitulate this cAMP surge in vitro, possibly accounting for the lower efficiency of IVM compared with clinical IVF. This review particularly focuses on this latter aspect - the role of cAMP/cGMP in the regulation of oocyte quality. We conclude that clinical practice of IVM should reflect this new understanding of the role of cyclic nucleotides, thereby creating a new generation of ART and fertility treatment options.

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.

Increased endothelial progenitor cells and nitric oxide in young prehypertensive women

This study investigated the effect of sex differences on circulating endothelial progenitor cells (EPCs) in prehypertension and its underlying mechanism. The authors found that premenopausal women show increased number and activity of circulating EPCs when compared with men, which was similar to enhanced nitric oxide (NO) level in plasma or culture medium. There was no difference in the number and activity of circulating EPCs and NO level between normotensive and prehypertensive premenopausal women. There was also no difference seen in levels of vascular endothelial growth factor and granulocyte macrophage colony‐stimulating factor. Both number and activity of circulating EPCs were correlated with the level of NO. The present study firstly demonstrated that the number and activity of circulating EPCs were preserved in prehypertensive premenopausal women, which was related to the restoration of NO production. The sex differences in EPCs in prehypertension may be involved in the mechanism underlying vascular protection in premenopausal women.

The expression of mammalian target of rapamycin in Ishikawa and HEC-1A cells

The activation of mammalian target of rapamycin (mTOR) signaling pathway in endometrial carcinoma cells Ishikawa and HEC-1A was investigated. The expression of mTOR was detected by confocal fluorescence microscopy in Ishikawa and HEC-1A cells. The mRNA levels of PTEN and mTOR, the downstream substrate S6K1 and 4E-BP1 protein were assayed by RT-PCR and Western blot, respectively. The expression of PTEN in Ishikawa cells was deficient, but intact in HEC-1A cells respectively (P<0.01). There was mTOR expression in both Ishikawa and HEC-1A cells and the phosporylated substrate levels in Ishikawa cells were higher than those in HEC-1A cells (P<0.05). mTOR signaling pathway is activated in two endometrial carcinoma cell strains and the status of activation is related with PTEN expression of the cells. The activation level of mTOR is higher in PTEN-deficient endometrial carcinoma cells than that in PTEN-intact endometrial carcinoma cells.SummaryThe activation of mammalian target of rapamycin (mTOR) signaling pathway in endometrial carcinoma cells Ishikawa and HEC-1A was investigated. The expression of mTOR was detected by confocal fluorescence microscopy in Ishikawa and HEC-1A cells. The mRNA levels of PTEN and mTOR, the downstream substrate S6K1 and 4E-BP1 protein were assayed by RT-PCR and Western blot, respectively. The expression of PTEN in Ishikawa cells was deficient, but intact in HEC-1A cells respectively (P<0.01). There was mTOR expression in both Ishikawa and HEC-1A cells and the phosporylated substrate levels in Ishikawa cells were higher than those in HEC-1A cells (P<0.05). mTOR signaling pathway is activated in two endometrial carcinoma cell strains and the status of activation is related with PTEN expression of the cells. The activation level of mTOR is higher in PTEN-deficient endometrial carcinoma cells than that in PTEN-intact endometrial carcinoma cells.

Effect of small interfering RNAs of cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17) on androgen biosynthesis in theca cells

Polycystic ovary syndrome (PCOS) is associated with a variety of endocrinologic and metabolic abnormalities, with clinical features of hyperandrogenism and hyperandrogenemia. Cytochrome P450 17α‐hydroxylase/17,20‐lyase (CYP17) is critical in androgen biosynthesis, and CYP17 mRNA expression was proven augmented in PCOS theca cells. To demonstrate whether RNA interference (RNAi) could lower the androgen concentration in theca cells, small interfering RNA (siRNA) targeting the CYP17 gene was co‐cultured with exogenous CYP17 in HeLa cells and endogenous CYP17 of theca cells. CYP17 gene expression was measured by fluorescent microscopy, flow cytometry and real‐time reverse transcription‐polymerase chain reaction analysis. Androstenedione and progesterone concentrations were measured by ELISA. RNAi effectively reduced the expression of exogenous CYP17 in HeLa cells by up to 50%. The CYP17 mRNA and androstenedione production of theca cells were slightly, but not significantly, reduced when compared with non‐specific siRNA.