Role of Sirt3 in mitochondrial biogenesis and developmental competence of human in vitro matured oocytes.

Does Sirt3 dysfunction result in poor developmental outcomes for human oocytes after in vitro maturation (IVM)? Inefficient Sirt3 expression induced decreased mitochondrial DNA copy number and biogenesis, and therefore impaired the developmental competence of human IVM oocytes. Cytoplasmic immaturity in IVM oocytes may lead to reduced developmental competence. Mitochondrial dysfunction results in the accumulation of free radicals and leads to DNA mutations, protein damage, telomere shortening and apoptosis. SIRT3 (in the Sirtuin protein family) has emerged as a mitochondrial fidelity protein that directs energy generation and regulates reactive oxygen species scavenging proteins. In vivo matured metaphase II (IVO-MII) oocytes and IVM-MII oocytes were donated by 324 infertile patients undergoing assisted reproductive technology cycles (12 patients for 60 IVO oocytes, and 312 patients for 403 IVM oocytes). Five oocytes each in the germinal vesicle (GV), IVM and IVO groups were compared with respect to mRNA levels for Sirt1-7 mRNA, and five samples at each developmental stage were analysed for Sirt3 mRNA. IVM-MII oocytes were injected with in vitro transcribed mRNA (n = 59) or small interfering RNA (siRNA) (n = 78). In human and mouse, IVM, mRNA-injection IVM, and siRNA-injection IVM groups (n = 5 each) were analysed for mitochondrial DNA copy number and abundance of Sirt3 and Pgc1α (an inducer of mitochondrial biogenesis) mRNAs. Human blastocysts in the IVO (n = 12), IVM (n = 9), mRNA-injection IVM (n = 13) and siRNA-injection IVM (n = 6) groups were used to generate embryonic stem cells (ESCs). In addition, 587 IVO-MII and 1737 IVM-MII oocytes from 83 mice were collected to compare the preliminary human oocyte data with another species. mRNA abundance was analysed by single-cell real-time PCR. Karyotyping of human embryos was performed with an array comparative genomic hybridization method, and that of ESCs by cytogenetic analysis. The function of the Sirt3 gene was investigated using siRNA and in vitro transcribed mRNA injection. Markers of ESCs were identified using immunofluorescence. A retrospective analysis revealed a higher spontaneous abortion rate (P < 0.01) and decrease in high quality embryo rate (P < 0.01) in patients with IVM versus controlled ovarian stimulation (COS) cycles. A decrease in abundance of Sirt3 mRNA (P < 0.01) and mitochondrial biogenesis (P < 0.05) were identified in human IVM compared with IVO oocytes. The developmental potential of human IVM-MII oocytes to the blastocyst stage was significantly reduced when Sirt3 mRNA was inhibited by siRNA (P < 0.05 versus IVM-MII group) but could be up-regulated by injection of Sirt3 mRNAs. Compared with IVO-MII group, comparable generation efficiency of human ESCs can be obtained using blastocysts from IVM-MII oocytes with Sirt3 mRNA injection. Sirt3 mRNA was significantly increased in mouse zygotes after IVF (P < 0.001 versus MII oocytes) but gradually declined until the blastocyst stage. In mice, lower Sirt3 mRNA levels were observed IVM-MII oocytes and preimplantation embryos compared with in vivo controls, and mitochondrial biogenesis and the developmental efficiency from oocytes to blastocyst were affected by the abundance of Sirt3 mRNA in accordance with human. Therefore a similar role for Sirt3 mRNA in IVM-MII oocytes was observed in mouse and human. The couples in the study had a variety of different simple and complex factors causing infertility. Additional studies with a larger number of oocytes are required to confirm the present results owing to the limited number of human oocytes in the present study. To our knowledge, this is the first study investigating a role of the Sirt3 gene in mitochondrial biogenesis and the developmental competence of human IVM-MII oocytes. The observation may help to improve clinical application of the IVM procedure. This work was supported in part by the National Natural Science Foundation of Key Program (31230047), Ministry of Science and Technology of China Grants (973 program; 2014CB943203), the National Natural Science Foundation of General Program (31371521 and 81571400), Beijing Nova Program (xxjh2015011), and Specialized Research Fund for the Doctoral Program of Higher Education (20120001130008) and the National Natural Science Foundation of Young Scholar (31501201). The authors have declared that no conflict of interest exists.

Zhao HC ，Ding T ，Ren Y ，Li TJ ，Li R ，Fan Y ，Yan J ，Zhao Y ，Li M ，Yu Y ，Qiao J ... -  《-》

Improved cryotolerance and developmental potential of in vitro and in vivo matured mouse oocytes by supplementing with a glutathione donor prior to vitrification.

Can supplementation of media with a glutathione (GSH) donor, glutathione ethyl ester (GEE), prior to vitrification protect the mouse oocyte from oxidative damage and critical changes in redox homeostasis, and thereby improve cryotolerance? GEE supplementation supported redox regulation, rapid recovery of spindle and chromosome alignment after vitrification/warming and improved preimplantation development of mouse metaphase II (MII) oocytes. Cryopreservation may affect mitochondrial functionality, induce oxidative stress, and thereby affect spindle integrity, chromosome segregation and the quality of mammalian oocytes. GEE is a membrane permeable GSH donor that promoted fertilization and early embryonic development of macaque and bovine oocytes after IVM. Two experimental groups consisted of (i) denuded mouse germinal vesicle (GV) oocytes that were matured in vitro in the presence or absence of 1 mM GEE (IVM group 1) and (ii) in vivo ovulated (IVO) MII oocytes that were isolated from the ampullae and exposed to 1 mM GEE for 1 h prior to vitrification (IVO group 2). Recovery of oocytes from both groups was followed after CryoTop vitrification/warming for up to 2 h and parthenogenetic activation. Reactive oxygen species (ROS), spindle morphology and chromosome alignment were analyzed by confocal laser scanning microscopy (CLSM) and polarization microscopy in control and GEE-supplemented MII oocytes. The relative overall intra-oocyte GSH content was assessed by analysis of monochlorobimane (MBC)-GSH adduct fluorescence in IVM MII oocytes. The GSH-dependent intra-mitochondrial redox potential (EmGSH) of IVM MII oocytes was determined after microinjection with specific mRNA at the GV stage to express a redox-sensitive probe within mitochondria (mito-Grx1-roGFP2). The absolute negative redox capacity (in millivolts) was determined by analysis of fluorescence of the oxidized versus the reduced form of sensor by CLSM and quantification according to Nernst equation. Proteome analysis was performed by quantitative 2D saturation gel electrophoresis (2D DIGE). Since microinjection and expression of redox sensor mRNA required removal of cumulus cells, and IVM of denuded mouse oocytes in group 1 induces zona hardening, the development to blastocysts was not assessed after IVF but instead after parthenogenetic activation of vitrified/warmed MII oocytes from both experimental groups. IVM of denuded mouse oocytes in the presence of 1 mM GEE significantly increased intra-oocyte GSH content. ROS was not increased by CryoTop vitrification but was significantly lower in the IVM GEE group compared to IVM without GEE before vitrification and after recovery from vitrification/warming (P < 0.001). Vitrification alone significantly increased the GSH-dependent intra-mitochondrial redox capacity after warming (EmGSH, P < 0.001) in IVM oocytes, presumably by diffusion/uptake of cytoplasmic GSH into mitochondria. The presence of 1 mM GEE during IVM increased the redox capacity before vitrification and there was no further increase after vitrification/warming. None of the reproducibly detected 1492 spots of 2D DIGE separated proteins were significantly altered by vitrification or GEE supplementation. However, IVM of denuded oocytes significantly affected spindle integrity and chromosome alignment right after warming from vitrification (0 h) in group 1 and spindle integrity in group 2 (P < 0.05). GEE improved recovery in IVM group as numbers of oocytes with unaligned chromosomes and aberrant spindles was not significantly increased compared to unvitrified controls. The supplementation with GEE for 1 h before vitrification also supported more rapid recovery of spindle birefringence. GEE improved significantly development to the 2-cell stage for MII oocytes that were activated directly after vitrification/warming in both experimental groups, and also the blastocyst rate in the IVO GEE-supplemented group compared to the controls (P < 0.05). None LIMITATIONS, REASONS FOR CAUTION: The studies were carried out in a mouse model, in IVM denuded rather than cumulus-enclosed oocytes, and in activated rather than IVF MII oocytes. Whether the increased GSH-dependent intra-mitochondrial redox capacity also improves male pronuclear formation needs to be studied further experimentally. The influence of GEE supplementation requires also further examination and optimization in human oocytes before it can be considered for clinical ART. Although GEE supplementation did not alter the proteome at MII, the GSH donor may support cellular homeostasis and redox regulation and, thus, increase developmental competence. While human MII oocyte vitrification is an established procedure, GEE might be particularly beneficial for oocytes that suffer from oxidative stress and reduced redox capacity (e.g. aged oocytes) or possess low GSH due to a reduced supply of GSH from cumulus. It might also be of relevance for immature human oocytes that develop without cumulus to MII in vitro (e.g. in ICSI cycles) for ART. The study has been supported by the German Research Foundation (DFG FOR 1041; EI 199/3-2). There are no conflict of interests.

Trapphoff T ，Heiligentag M ，Simon J ，Staubach N ，Seidel T ，Otte K ，Fröhlich T ，Arnold GJ ，Eichenlaub-Ritter U ... -  《-》

A pre-in vitro maturation medium containing cumulus oocyte complex ligand-receptor signaling molecules maintains meiotic arrest, supports the cumulus oocyte complex and improves oocyte developmental competence.

Can a pre-in vitro maturation (pre-IVM) medium containing signaling molecules rather than chemical/pharmaceutical agents, sustain meiotic arrest and improve developmental competence of in vitro matured oocytes in CF1 outbred mice? A short 2 h period of pre-IVM prevents spontaneous meiotic resumption, improves mitochondria activity in subsequently matured oocytes, and increases developmental competence, pregnancy rate and implantation of resulting embryos. Spontaneous resumption of meiosis in vitro is detrimental for oocyte developmental competence. Pre-IVM systems that prevent spontaneous meiotic resumption with chemical/pharmaceutical agents are a promising approach to improving IVM oocyte competence; however, the success of these methods has proven to be inconsistent. This study consisted of a series of experiments using cumulus oocyte complexes (COC) derived from outbred mice following ovarian stimulation. The study was designed to examine if a novel, ligand/receptor-based pre-IVM treatment could sustain meiotic arrest in vitro and improve oocyte developmental competence, compared to control IVM. Two pre-IVM durations (2 h and 24 h) were evaluated, and the effect of the mitochondrial stimulator PQQ during 24 h pre-IVM was studied. Murine (outbred CF1) immature COC were cultured in vitro in the presence of C-type natriuretic peptide (CNP) (30 nM), estradiol (100 nM), FSH (1 × 10-4 IU/ml) and bone morphogenic protein 15 (BMP15) (100 ng/ml) for 2 h or 24 h prior to IVM. Meiotic status during pre-IVM and IVM was analyzed using orcein staining, and functionality of gap junction communication was confirmed using the functional gap junction inhibitor carbenoxolone (CBX). Oocytes exposed to pre-IVM treatment were compared to control oocytes collected on the same day from the same females and undergoing standard IVM. Developmental competence and embryo viability was assessed by oocyte mitochondrial activity and ATP concentration, in vitro embryo development following IVF and in vitro culture, blastocyst cell number and allocation, embryo morphokinetics, and embryo transfer. Differences were determined to be significant when P < 0.05. Both a short (2 h) and long (24 h) pre-IVM period successfully prevented spontaneous resumption of meiosis. Moreover, gap junctions remained open during the pre-IVM period, as shown by the resumption of meiosis (95.9 ± 2.1%) in the presence of CBX during pre-IVM. A 2 h pre-IVM treatment improved blastocyst development after 96 h of culture per cleaved embryo compared to control (71.9 ± 7.4% versus 53.3 ± 6.2%, respectively), whereas a longer 24 h pre-IVM had no effect on development. A short 2 h period of pre-IVM increased mitochondrial activity in mature oocytes. On the contrary, mitochondrial activity was reduced in mature oocytes following 24 h of arrest and IVM. Treatment of arrested COC with pyrroloquinoline quinone (PQQ) during the 24 h pre-IVM period successfully maintained mitochondrial activity equal to control. However, PQQ was not able to improve blastocyst development compared to pre-IVM 24 h without PQQ. Moreover, ATP concentration in mature oocytes following pre-IVM and/or IVM, did not differ between treatments. A 2 h pre-IVM period prior to IVM improved pregnancy rate following transfer to recipient females. Implantation was also improved after transfer of embryos derived from oocytes arrested for either 2 h or 24 h prior to IVM, compared to control IVM derived embryos (41.9 ± 9%, 37.2 ± 9.5% and 17.2 ± 8.3%, respectively), although fetal development did not differ. Slower meiotic resumption and enhanced mitochondrial activity likely contribute to improved developmental competence of oocytes exposed to pre-IVM for 2 h, but further experiments are required to identify specific mechanisms. Maintaining oocytes in meiotic arrest for 24 h with this approach could be a potential window to improve oocyte quality. However, an initial attempt to utilize this period of arrest to manipulate quality with PQQ, a mitochondrial stimulator, did not improve oocyte competence. IVM could be an attractive clinical alternative to conventional IVF, with reduced time, cost and reliance on high doses of exogenous hormones to stimulate follicle growth, thus eliminating ovarian hyperstimulation syndrome (OHSS). Currently IVM is not widely used as it results in reduced embryo development and lower pregnancy outcomes compared to embryos produced from in vivo matured oocytes. Our approach to IVM, incorporating a ligand/receptor pre-IVM period, could improve human oocyte quality following IVM leading to routine adoption of this patient friendly technology. In addition, our methodology of pre-IVM containing signaling molecules rather than chemical/pharmaceutical agents may prove to be more consistent at improving oocyte quality than those focusing only on cAMP modulation with pharmacological agents. Finally, a reliable method of maintaining oocytes in meiotic arrest in vitro provides a novel window of opportunity in which the oocyte may be manipulated to address specific physiological deficiencies prior to meiotic resumption. N/A. This study was supported by the Colorado Center for Reproductive Medicine (CCRM, Lone Tree, Colorado USA). We declare no conflict of interest.

Santiquet NW ，Greene AF ，Becker J ，Barfield JP ，Schoolcraft WB ，Krisher RL ... -  《-》

l-carnitine supplementation during vitrification of mouse germinal vesicle stage-oocytes and their subsequent in vitro maturation improves meiotic spindle configuration and mitochondrial distribution in metaphase II oocytes.

How does l-carnitine (LC) supplementation during vitrification and in vitro maturation (IVM) of germinal vesicle stage (GV)-oocytes improve the developmental competence of the resultant metaphase II (MII) oocytes? LC supplementation during both vitrification of GV-oocytes and their subsequent IVM improved nuclear maturation as well as meiotic spindle assembly and mitochondrial distribution in MII oocytes. Vitrification of GV-oocytes results in a lower success rate of blastocyst development compared with non-vitrified oocytes. LC supplementation during both vitrification and IVM of mouse GV-oocytes significantly improves embryonic development after IVF. GV-oocytes were collected from (B6.DBA)F1 and B6 mouse strains and subjected to vitrification and warming with or without 3.72 mM LC supplementation. After IVM with or without LC supplementation, the rate of nuclear maturation and the quality of MII oocytes were evaluated. At least 20 oocytes/group were examined, and each experiment was repeated at least three times. All experiments were conducted during 2013-2014. Extrusion of the first polar body in IVM oocytes was observed as an indication of nuclear maturation. Spindle assembly and chromosomal alignment were examined by immunostaining of α-tubulin and nuclear staining with 4,6-diamidino-2-phenylindole (DAPI). Mitochondrial distribution and oxidative activity were measured by staining with Mitotracker Green Fluorescence Mitochondria (Mitotracker Green FM) and chloromethyltetramethylrosamine (Mitotracker Orange CMTMRos), respectively. ATP levels were determined by using the Bioluminescent Somatic Cell Assay Kit. LC supplementation during both vitrification and IVM of GV-oocytes significantly increased the proportions of oocytes with normal MII spindles to the levels comparable with those of non-vitrified oocytes in both mouse strains. While vitrification of GV-oocytes lowered the proportions of MII oocytes with peripherally concentrated mitochondrial distribution compared with non-vitrified oocytes, LC supplementation significantly increased the proportion of such oocytes in the (B6.DBA)F1 strain. LC supplementation decreased the proportion of oocytes with mitochondrial aggregates in both vitrified and non-vitrified oocytes in the B6 strain. The oxidative activity of mitochondria was mildly decreased by vitrification and drastically increased by LC supplementation irrespective of vitrification in both mouse strains. No change was found in ATP levels irrespective of vitrification or LC supplementation. Results were considered to be statistically significant at P < 0.05 by either χ(2)- or t-test. It remains to be tested whether beneficial effect of LC supplementation during vitrification and IVM of GV-oocytes leads to fetal development and birth of healthy offspring after embryo transfer to surrogate females. This protocol has the potential to improve the quality of vitrified human oocytes and embryos during assisted reproduction treatment. Partially supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant and Mitacs Elevate Postdoctoral Fellowship, Canada.

Moawad AR ，Xu B ，Tan SL ，Taketo T ... -  《-》

Human cumulus-enclosed germinal vesicle oocytes from early antral follicles reveal heterogeneous cellular and molecular features associated with in vitro maturation capacity.

Are oocyte size, chromatin remodelling, transcriptional activity and mitochondrial distribution in human immature oocytes from early antral follicles retrieved for in vitro maturation (IVM) associated with the acquisition of meiotic competence? Oocyte size, chromatin compaction, cessation of RNA synthesis and mitochondria rearrangement around the nucleus are associated with the oocyte's potential to resume meiosis in vitro. Information on oocyte features that confer meiotic competence in human mainly derives from germinal vesicle (GV) oocytes that failed to resume meiosis following an hCG trigger after ovulation induction cycles. Characterization of cumulus-enclosed GV oocytes from small antral follicles prior to IVM provides knowledge on the initial oocyte status and suggests culture requirements in order to promote oocyte competence in vitro. Prospective collection of 107 oocytes immediately after retrieval (before IVM) and of 293 GV oocytes that had failed to resume meiosis (after IVM). Human oocytes were collected from women with polycystic ovary syndrome (PCOS), receiving in total 450 IU of highly purified-hMG for IVM treatment (patients) or who donated oocytes for IVM research (donors). Oocytes at GV-stage were retrieved from follicles <10 mm (range 2-10 mm) diameter, before IVM (oocytes at retrieval) or those that failed to mature after IVM (meiotically incompetent). Oocytes were allocated for either mitochondrial staining, by incubating in mitotracker red and then fixed; or for nascent RNA staining, which was assessed by fluorescent labelling (Click-iT(®) RNA Assay). In every case, oocyte diameter was recorded and chromatin was stained after oocyte fixation. GV-stage oocytes were analysed by confocal laser-scanning microscopy and their characteristics were compared and related to their meiotic competence. Analysis of oocytes at the immature GV-stage revealed that oocytes at retrieval were significantly larger than those that failed to resume meiosis after IVM (112.7 versus 109.6 µm, P < 0.0001). Oocytes assessed at retrieval showed that 50.6% had a condensed chromatin configuration (perinucleolar chromatin rim) and were consistently transcriptionally silent. This rate matched maturation rates in our current in vitro culture system (49%). However, oocytes that had not reinitiated meiosis after 30 h IVM demonstrated, apart of being smaller in diameter, significantly higher rates of dispersed or intermediate chromatin (P = 0.005). Analysis of mitochondrial distribution revealed that many oocytes at retrieval displayed mitochondrial internalization towards the nucleus (12/30) or a perinuclear mitochondrial distribution (6/30). These mitochondrial patterns were observed more rarely in GV incompetent oocytes following 30 h IVM (16/98 and 1/98, respectively). Most of the analyses involved the use of invasive techniques. Hence, despite the fact that these data deliver essential information on the intrinsic oocyte maturational and developmental status, a direct match with embryological outcomes could not be established. The evidence described here can aid in tailoring IVM systems in order to promote completion of nuclear and cytoplasmic maturation of unexpanded cumulus-oocyte complexes. This study was supported by research grants by the Institute for the Promotion of Innovation by Science and Technology in Flanders, project numbers IWT 130327 and 110680; the Fund for Research Flanders, project number FWO G.0343.13, the Belgian Foundation Against Cancer (HOPE project) and COOK Medical. None of the authors has any competing interest to declare.

Sánchez F ，Romero S ，De Vos M ，Verheyen G ，Smitz J ... -  《-》