GV Stage Research Articles

Addition of lysophosphatidic acid to mouse oocyte maturation media can enhance fertilization and developmental competence

Does exposure to lysophosphatidic acid (LPA) during in vitro maturation (IVM) enhance the maturation and developmental competence of mouse oocytes? Supplementation of IVM medium with 30 μM LPA enhanced the developmental competence of in vitro matured oocytes and so made them more comparable to in vivo matured control oocytes. LPA is a small phospholipid that acts as an extracellular signaling molecule by binding to and activating at least five G protein-coupled receptors. LPA has various biological actions, with both developmental and physiological effects. During IVM, LPA at six different doses (0, 1, 10, 30, 50 or 100 μM) was added into the TCM-199 medium. After maturation, the developmental competence and other parameters of the oocytes were assessed. Immature GV stage oocytes from 5- to 6-week-old female BDF-1 mice were incubated for 17-18 h in IVM medium containing 0, 1, 10 or 30 μM LPA and then either fertilized in vitro with epididymal sperm, or assessed for spindle morphology, mitochondrial membrane potential (ΔΨm) or the mRNA expression of a meiotic checkpoint gene (Mad2), a microtubule structure gene (Hook1), two maternally derived genes (Mater and Hsf1) and an apoptosis-related gene (Caspase6). The fertilized embryos were grown in vitro to assess blastocyst-formation rates, differential cell counts and apoptosis. Rates of maturation, fertilization and blastocyst formation and hatching were significantly higher in the 30 μM LPA-supplemented group (94.3, 96.3, 79.1 and 51.3%, respectively) than in the unsupplemented control (0 μM) group (80.5, 87.5, 61.3 and 37.8%, respectively) and more comparable to that of the in vivo matured oocytes (100, 96.5, 95.3 and 92.9%, respectively). LPA did not adversely affect mitochondrial activity, spindle integrity, blastocyst cell number, caspase positivity or Mad2 expression. Oocytes matured in 30 μM LPA had reduced Caspase6 expression, but Hook1, Mater and Hsf1 were up-regulated in all of the LPA-supplemented groups. Chromosomal aneuploidy in the resultant blastocysts and the production of normal pups were not assessed. Only mouse oocytes were assessed. Supplementation of IVM medium with 30 μM LPA may enhance the developmental competence of mouse oocytes without affecting apoptosis, spindle normalcy or mitochondrial integrity. This study was supported by a research grant (02-2012-021) from the Seoul National University Bundang Hospital. The authors declare that they have no competing interests.

Oocyte cryopreservation and in vitro culture affect calcium signalling during human fertilization

What are the precise patterns of calcium oscillations during the fertilization of human oocytes matured either in vivo or in vitro or aged in vitro and what is the effect of cryopreservation? Human oocytes matured in vivo exhibit a specific pattern of calcium oscillations, which is affected by in vitro maturation, in vitro ageing and cryopreservation. Oscillations in cytoplasmic calcium concentration are crucial for oocyte activation and further embryonic development. While several studies have described in detail the calcium oscillation pattern during fertilization in animal models, studies with human oocytes are scarce. This was a laboratory-based study using human MII oocytes matured in vivo or in vitro either fresh or after cryopreservation with slow freezing or vitrification. Altogether, 205 human oocytes were included in the analysis. In vivo and in vitro matured human oocytes were used for this research either fresh or following vitrification/warming (V/W) and slow freezing/thawing (F/T). Human oocytes were obtained following written informed consent from patients undergoing ovarian hyperstimulation. For the calcium pattern analysis, oocytes were loaded with the ratiometric calcium indicator fluorescent dye Fura-2. Following ICSI using sperm from a single donor, intracellular calcium was measured for 16 h at 37°C under 6% CO(2). The calcium oscillation parameters were calculated for all intact oocytes that showed calcium oscillations and were analyzed using the Mann-Whitney U-test. Human in vivo MII oocytes display a specific pattern of calcium oscillations following ICSI. This pattern is significantly affected by in vitro ageing, with the calcium oscillations occurring over a longer period of time and with a lower frequency, shorter duration and higher amplitude (P < 0.05). In vitro matured oocytes from the GV and MI stage exhibit a different pattern of calcium oscillations with calcium transients being of lower frequency and shorter duration compared with in vivo matured MII. In MI oocytes that reached the MII stage within 3 h the calcium oscillations additionally appear over a longer period of time (P < 0.05). In vivo MII oocytes show a different calcium oscillation pattern following V/W with calcium oscillations occurring over a longer period of time, with a higher amplitude and a lower frequency (P < 0.05). In vitro matured oocytes, either from the GV or the MI stage, also display an altered pattern of calcium oscillations after V/W and the parameters that were similarly affected in all these oocyte groups are the frequency and the amplitude of the calcium transients. Slow freezing/thawing differentially affects the calcium oscillation pattern of in vitro matured and in vitro aged oocytes. The relationship between a specific pattern of calcium oscillations and subsequent human embryonic development could not be evaluated since the calcium indicator used and the high-intensity excitation light impair development. Furthermore, all oocytes were derived from stimulated cycles and immature oocytes were denuded prior to in vitro maturation. Our data show for the first time how calcium signalling during human fertilization is affected by oocyte in vitro maturation, in vitro ageing as well as V/W and slow freezing/thawing. The analysis of calcium oscillations could be used as an oocyte quality indicator to evaluate in vitro culture and cryopreservation techniques of human oocytes. This work was supported by a clinical research mandate from the Flemish Foundation of Scientific Research (FWO-Vlaanderen, FWO09/ASP/063) to F.V.M, a fundamental clinical research mandate from the FWO-Vlaanderen (FWO05/FKM/001) to P.D.S and a Ghent University grant (KAN-BOF E/01321/01) to B.H. The authors have no conflict of interest to declare.

L-Carnitine Supplementation During Vitrification of Mouse Oocytes at the Germinal Vesicle Stage Improves Preimplantation Development Following Maturation and Fertilization In Vitro

Oocyte cryopreservation is important for assisted reproductive technologies (ART). Although cryopreservation of metaphase II (MII) oocytes has been successfully used, MII oocytes are vulnerable to the damage inflicted by the freezing procedure. Cryopreservation of germinal vesicle stage oocytes (GV-oocytes) is an alternative choice; however, blastocyst development from GV-oocytes is limited largely due to the need for in vitro maturation (IVM). Herein, we evaluated the effects of l-carnitine (LC) supplementation during vitrification and thawing of mouse GV-oocytes, IVM, and embryo culture on preimplantation development after in vitro fertilization (IVF). We first compared the rate of embryonic development from the oocytes that had been collected at the GV stage from three mouse strains, (B6.DBA)F1, (B6.C3H)F1, and B6, and processed for IVM and IVF, as well as that from the oocytes matured in vivo, i.e. ovulated (IVO). Our results demonstrated that the rate of blastocyst development was the highest in the (B6.DBA)F1 strain and the lowest in the B6 strain. We then supplemented the IVM medium with 0.6 mg/ml LC. The rate of blastocyst development improved in the B6 but not in the (B6.DBA)F1 strain. Vitrification of GV-oocytes in the basic medium alone reduced the rate of blastocyst development in both of those mouse strains. LC supplementation to the IVM medium alone did not change the percentage of blastocyst development. However, LC supplementation to both vitrification and IVM media significantly improved blastocyst development to the levels comparable with those obtained from vitrified/thawed IVO oocytes in both of the (B6.DBA)F1 and B6 strains. We conclude that LC supplementation during vitrification is particularly efficient in improving the preimplantation development from the GV-oocytes that otherwise have lower developmental competence in culture.

MBTD1 is associated with Pr-Set7 to stabilize H4K20me1 in mouse oocyte meiotic maturation

H4K20me1 is a critical histone lysine methyl modification in eukaryotes. It is recognized and “read” by various histone lysine methyl modification binding proteins. In this study, the function of MBTD1, a member of the Polycomb protein family containing four MBT domains, was comprehensively studied in mouse oocyte meiotic maturation. The results showed that depletion of MBTD1 caused reduced expression of histone lysine methyl transferase Pr-Set7 and H4K20me1 as well as increased oocyte arrest at the GV stage. Increased γH2AX foci were formed, and DNA damage repair checkpoint protein 53BP1 was downregulated. Furthermore, depletion of MBTD1 activated the cell cycle checkpoint protein Chk1 and downregulated the expression of cyclin B1 and cdc2. MBTD1 knockdown also affected chromosome configuration in GV stage oocytes and chromosome alignment at the MII stage. All these phenotypes were reproduced when the H4K20 methyl transferase Pr-Set7 was depleted. Co-IP demonstrated that MBTD1 was correlated with Pr-Set7 in mouse oocytes. Our results demonstrate that MBTD1 is associated with Pr-Set7 to stabilize H4K20me1 in mouse oocyte meiotic maturation.

Hec1-Dependent Cyclin B2 Stabilization Regulates the G2-M Transition and Early Prometaphase in Mouse Oocytes

SummaryThe functions of the Ndc80/Hec1 subunit of the highly conserved Ndc80 kinetochore complex are normally restricted to M phase when it exerts a pivotal kinetochore-based role. Here, we find that in mouse oocytes, depletion of Hec1 severely compromises the G2-M transition because of impaired activation of cyclin-dependent kinase 1 (Cdk1). Unexpectedly, impaired M phase entry is due to instability of the Cdk1-activating subunit, cyclin B2, which cannot be covered by cyclin B1. Hec1 protects cyclin B2 from destruction by the Cdh1-activated anaphase-promoting complex (APCCdh1) and remains important for cyclin B2 stabilization during early M phase, required for the initial stages of acentrosomal spindle assembly. By late M phase, however, Hec1 and cyclin B2 become uncoupled, and although Hec1 remains stable, APCCdc20 triggers cyclin B2 destruction. These data identify another dimension to Hec1 function centered on M phase entry and early prometaphase progression and challenge the view that cyclin B2 is completely dispensable in mammals.

TBP Dynamics during Mouse Oocyte Meiotic Maturation and Early Embryo Development

To maintain cell lineage, cells develop a mechanism which can transmit the gene activity information to the daughter cells. In mitosis, TBP (TATA-binding protein), a transcription factor which belongs to TFIID was associated with M phase chromosomes and was proved to be a bookmark for cellular memory. Although previous work showed that TBP was dispensable for mouse oocyte maturation and early embryo development, exogenous TBP protein was detected in the nuclear of oocytes and early embryos. It is still unknown whether exogenous TBP can associate with condensed chromosomes during meiosis and mouse early embryo development. In present study by the injection of GFP-tagged TBP mRNA we for the first time investigated TBP dynamics in mouse early embryos and confirmed its localization pattern in oocytes. The exogenous TBP enriched at germinal vesicle at GV stage but disappeared from the chromosomes after GVBD. Moreover, exogenous TBP was still dispersed from the chromosomes of somatic donor nuclear in oocytes by nuclear transfer (NT), further proving that oocyte has some mechanism to remove TBP. During mouse embryo development, the exogenous TBP was removed from the chromosomes of M phase zygotes, but was found to express weakly at the M phase of 2-cell. Moreover, in the blastocyst TBP was also detected at the M phase chromosomes. Overexpression of TBP caused the failure of oocyte maturation and embryo development. Our results supported the idea that TBP might be a marker for transmitting cellular memory to daughter cells.

The Role of 14-3-3ε Interaction with Phosphorylated Cdc25B at Its Ser321 in the Release of the Mouse Oocyte from Prophase I Arrest

The protein kinase A (PKA)/Cdc25B pathway plays a critical role in maintaining meiotic arrest in mouse oocytes. However, the molecular mechanism underlying this interchange is not known. In this study, we assessed the role of 14-3-3ε interaction with phosphorylated Cdc25B at its Ser321 as the mouse oocyte is released from prophase I arrest. The 14-3-3ε isoform is a highly conserved protein with various regulatory roles, including maintenance of meiotic arrest. Cdc25B phosphatase is also a key cell cycle regulator. 14-3-3ε binds to Cdc25B-WT, which was abrogated when Ser321 of Cdc25B was mutated to Ala. In addition, we found that 14-3-3ε and Cdc25B were co-localized. Cdc25B was translocated from the cytoplasm to the nucleus shortly before germinal vesicle breakdown (GVBD) during the primary oocyte stage of oogenesis. However, mutation of Ser321 to Ala completely abolished the cytoplasmic localization of Cdc25B. Furthermore, oocytes co-expressing of Cdc25B-WT or Cdc25B-Ser321D and 14-3-3ε were unable to undergo GVBD. In contrast, co-expression of 14-3-3ε and Cdc25B-Ser321A induced GVBD and allowed the process to continue. Down-regulation of 14-3-3ε caused partial meiotic resumption. Taken together, these data indicate that Ser321 of Cdc25B is the specific binding site for 14-3-3ε binding, and that 14-3-3ε is the significant factor in Cdc25B regulation during meiotic resumption of GV stage.

Overexpression of SETβ, a protein localizing to centromeres, causes precocious separation of chromatids during the first meiosis of mouse oocyte

Chromosome segregation in mammalian oocyte meiosis is an error-prone process, and any mistake in this process may result in aneuploidy, which is the main cause of infertility, abortion and many genetic diseases. It is now well known that shugoshin and protein phosphatase 2A (PP2A) play important roles in the protection of centromeric cohesion during the first meiosis. PP2A can antagonize the phosphorylation of rec8, a member of the cohesin complex, at the centromeres and thus prevent cleavage of rec8 and so maintain the cohesion of chromatids. SETβ is a protein that physically interacts with shugoshin and inhibits PP2A activity. We thus hypothesized that SETβ might regulate cohesion protection and chromosome segregation during oocyte meiotic maturation. Here we report for the first time the expression, subcellular localization and functions of SETβ during mouse oocyte meiosis. Immunoblotting analysis showed that the expression level of SETβ was stable from the germinal vesicle stage to the MII stage of oocyte meiosis. Immunofluorescence analysis showed SETβ accumulation in the nucleus at the germinal vesicle stage, whereas it was targeted mainly to the inner centromere area and faintly localized to the interchromatid axes from germinal vesicle breakdown to MI stages. At the MII stage, SETβ still localized to the inner centromere area, but could relocalize to kinetochores in a process perhaps dependent on the tension on the centromeres. SETβ partly colocalized with PP2A at the inner centromere area. Overexpression of SETβ in mouse oocytes caused precocious separation of sister chromatids, but depletion of SETβ by RNAi showed little effects on the meiotic maturation process. Taken together, our results suggest that SETβ, even though it localizes to centromeres, might not be essential for chromosome separation during mouse oocyte meiotic maturation, although its forced overexpression causes premature chromatid separation.

Folliculogenesis, Ovulation and Endocrine Control of Oocytes and Embryos in the Dog

Reproductive physiology in dogs is quite unusual compared with that in other mammalian species. The peculiarities include the presence of numerous polyoocyte follicles, the ovulation of an immature oocyte (GV stage, non-fertilizable) and a peri-ovulatory period during which concentrations of circulating progesterone are particularly high. The aim of this review is to examine the unusual aspects of the reproductive physiology of dogs and how this relates to the clinical biology of this species.

Vitrification of immature bovine cumulus-oocyte complexes: effects of cryoprotectants, the vitrification procedure and warming time on cleavage and embryo development

BackgroundThe present studies evaluated the effects of cryoprotectants, the vitrification procedure and time in the warming solution containing sucrose on cleavage and embryo development of immature (GV stage) bovine cumulus-oocyte complexes (COCs).MethodsTwo experiments were conducted. In Experiment 1, COCs (n = 420) were randomly assigned to four groups: 1) Control group: no treatment; 2) VS1 group: COCs were exposed to vitrification solution 1 (VS1) containing 7.5% ethylene glycol [EG] + 7.5% dimethyl sulfoxide [DMSO] + 20% calf serum [CS] in TCM-199 at 37 C for 5 min; 3) VS1 + VS2 group: COCs were exposed to VS1 for 5 min followed by VS2 (15% EG + 15% DMSO + 17.1% sucrose + 20% CS) at 37 C for 45–60 sec; and 4) Vitrified group: COCs were exposed to VS1 and VS2, loaded on cryotops, vitrified in liquid nitrogen and then warmed in TCM-199 + 17.1% sucrose + 20% CS at 37 C for 1 min. In Experiment 2, COCs (n = 581) were assigned to the same groups, but those in VS1, VS1 + VS2 and Vitrified groups were sub-divided and exposed to the warming solution for either 1 or 5 min. After treatment and/or warming, all COCs in both experiments underwent in vitro maturation, in vitro fertilization and in vitro culture.ResultsCleavage and blastocyst rates did not differ among Control, VS1 and VS1 + VS2 groups in either experiment. In Experiment 2, there was no effect of time in the warming solution.However, both cleavage and blastocyst rates were lower (P < 0.001) in the Vitrified group than in the Control, VS1 and VS1 + VS2 groups (40.9 and 1.6% vs 92.2 and 34.4%, 79.4 and 25.2%, and 80.2 and 20.8%, respectively in Experiment 1, and 25.0 and 1.7% vs 75.3 and 27.2%, 67.9 and 19.5%, and 62.7 and 22.5%, respectively in Experiment 2).ConclusionsThe permeating cryoprotectants (EG and DMSO) present in VS1 and VS2 solutions and the time in the warming solution containing sucrose had no adverse effects on cleavage and blastocyst rates of immature bovine COCs. However, cleavage rate and early embryo development were reduced following the vitrification and warming.

Septin 7 is required for orderly meiosis in mouse oocytes

Septin 7 is a conserved GTP-binding protein. In this study, we examined the localization and functions of Septin 7 during mouse oocyte meiotic maturation. Immunofluorescent analysis showed that intrinsic Septin 7 localized to the spindles from the pro-MI stage to the MII stage. Knockdown of Septin 7 by siRNA microinjection caused abnormal spindles and affected extrusion of the first polar body. Septin 7 mRNA tagged with myc was injected into GV stage oocytes to overexpress Septin 7. Overexpressed Myc-Septin 7 localized to the spindle and beneath the plasma membrane displaying long filaments. Fluorescence intensity of spindle α-tubulin in myc-Septin 7-injected oocytes was weaker than that of the control group, demonstrating that Septin 7 may influence recruitment of α-tubulin to spindles. MII oocytes injected with myc-Septin 7 exhibited abnormal chromosome alignment, and parthenogenetic activation failed to allow extrusion of the second polar body, suggesting that overexpression of Septin 7 may affect extrusion of the polar body by disturbing the alignment of chromosomes and regulating α-tubulin recruitment to spindles. In summary, Septin 7 may regulate meiotic cell cycle progression by affecting microtubule cytoskeletal dynamics in mouse oocytes.

The dynamics of in vitro maturation of germinal vesicle oocytes

To evaluate the dynamics of the nuclear maturation (NM) of in vitro-matured (IVM) oocytes and to determine the most favorable duration of meiosis II (MII) arrest in relation to the normal activation response. Experimental. University-affiliated infertility clinic. Donated immature germinal vesicle oocytes (GV). The GV underwent spontaneous IVM and the dynamics of NM studied by real-time monitoring. The IVM oocytes were parthenogenetically activated at different MII arrest points and their response assessed. Moment of GV breakdown; extrusion of the first polar body; duration of MI and MII arrest; activation rate (AR) and type. Two GV populations-early (E-IVM, 18.4 ± 2.7 hours) and late (L-IVM, 26.3 ± 3.8 hours) maturing-were defined according to the time required for extrusion of the first polar body. Significantly more E-IVM than L-IVM exhibited a normal activation response (61.3% vs. 34.6%), but AR were similar (average, 88.6%) in both groups. Duration of the GV stage differed between the two groups, but MI arrest (14.0 ± 0.3 hours) was constant. The E-IVM arrested at MII for at least 4.3 hours displayed significantly lower AR and similar normal activation rates (61.3%) to E-IVM arrested for a shorter time (83.9% vs. 100%). The L-IVM displayed a similar AR (80.8%), but lower normal activation rates than E-IVM (34.6%), regardless of when activation took place. The success of IVM depends on the NM timing rather than on the length of MII arrest.

The Beneficial Effects of Antifreeze Proteins in the Vitrification of Immature Mouse Oocytes

Antifreeze proteins (AFPs) are a class of polypeptides that permit organismal survival in sub-freezing environments. The purpose of this study was to investigate the effect of AFP supplementation on immature mouse oocyte vitrification. Germinal vesicle-stage oocytes were vitrified using a two-step exposure to equilibrium and vitrification solution in the presence or absence of 500 ng/mL of AFP III. After warming, oocyte survival, in vitro maturation, fertilization, and embryonic development up to the blastocyst stage were assessed. Spindle and chromosome morphology, membrane integrity, and the expression levels of several genes were assessed in in vitro matured oocytes. The rate of blastocyst formation was significantly higher and the number of caspase-positive blastomeres was significantly lower in the AFP-treated group compared with the untreated group. The proportion of oocytes with intact spindles/chromosomes and stable membranes was also significantly higher in the AFP group. The AFP group showed increased Mad2, Hook-1, Zar1, Zp1, and Bcl2 expression and lower Eg5, Zp2, Caspase6, and Rbm3 expression compared with the untreated group. Supplementation of the vitrification medium with AFP has a protective effect on immature mouse oocytes, promoting their resistance to chilling injury. AFPs may preserve spindle forming ability and membrane integrity at GV stage. The fertilization and subsequent developmental competence of oocytes may be associated with the modulation of Zar1, Zp1/Zp2, Bcl2, Caspase6, and Rbm3.

Differences in transcriptomic profiles of human cumulus cells isolated from oocytes at GV, MI and MII stages after in vivo and in vitro oocyte maturation

Oocyte maturation and competence to development depends on its close relationship with cumulus cells (CCs). However, the maturation conditions of human cumulus-oocyte complexes (COCs) might affect gene expression in both oocyte and CCs. We thus compared the transcriptome profiles of CCs isolated from in vivo and in vitro matured COCs at different nuclear maturation stages. Three groups of CCs from patients who underwent ICSI were included: CCs of patients with polycystic ovary syndrome (PCOS) referred for in vitro maturation (IVM), CCs from patients with PCOS for in vivo maturation (used as controls) and CCs from normal responders referred for in vivo maturation. CCs were isolated from COCs at the germinal vesicle, metaphase I and metaphase II stages. Microarray technology was used to analyse the global gene expression and significance analysis of microarray to compare the expression profiles of CCs from COCs at different nuclear maturation stages following IVM or in vivo maturation. Selected genes were validated by RT-qPCR. In CCs isolated after IVM, genes related to cumulus expansion and oocyte maturation, such as EREG, AREG and PTX3, were down-regulated, while cell cycle-related genes were up-regulated in comparison with CCs from in vivo matured COCs from PCOS and normal responder patients. Moreover, irrespective of the stage of oocyte maturation, genes involved in DNA replication, recombination and repair were up-regulated in CCs after IVM. The CC transcriptomic signature varies according to both the oocyte maturation stage and the maturation conditions. Our findings suggest a delay in the acquisition of the mature CC phenotype following IVM, opening a new perspective for the improvement in IVM conditions.

Successful pregnancy following a novel endometrial preparation in a PCOS patient undergoing IVM: a case report

In-vitro fertilization (IVF) relies heavily on controlled ovarian stimulation to increase the number of available oocytes and embryos, and therefore the pregnancy rate. However, the side effects, such as nausea, mood swings, menopausal symptoms, ovarian hyperstimulation syndrome (OHSS), and potential cancer risk of ovarian stimulation may not be avoided. Therefore, the recovery of immature oocytes followed by in-vitro maturation (IVM) and fertilization is an attractive alternative to avoid the side effects of using gonadotrophin stimulation. In IVM, immature oocytes are retrieved from unstimulated or minimally stimulated cycles, then cultured using IVM culture media for maturation in vitro [1]. In comparison with conventional IVF, IVM technology is generally associated with the absence of OHSS, lower cost, and fewer complications. However, the efficiency of existing IVM technique is suboptimal because embryos generated from IVM exhibit frequent cleavage blocks and low implantation rate [2]. Women with polycystic ovarian syndrome (PCOS) are considered as the target population for this approach. This group of patients is extremely sensitive to ovarian stimulation and have a high potential for developing of OHSS [3]. In recent years, many improvements have been made in clinical and laboratory aspects of IVM treatment which have resulted in better pregnancy outcomes [4]. It was shown that pretreatment with FSH in the early days of folliculogenesis phase may increase the number of retrieved immature oocytes and/or maturation potential [5, 6]. In addition, hCG priming promotes some GV oocytes to reach the MI stage and increases the maturation rate of immature oocytes in vitro setting. Despite increased pregnancy rates following IVM in recent years, implantation rates are still lower than conventional IVF cycles. One possible explanation may be related to insufficient endometrial preparation during IVM cycles before ET [4, 7]. Here, we aim to report a novel endometrial preparation method in a PCOS case undergoing IVM. Case report A 33 years old patient with history of primary infertility for 3 years was referred to our program for infertility treatment. She was diagnosed as PCOS according to Rotterdam criteria [8] with history of acne, hirsutism and oligomenorrhea. The clinical profile showed that she had BMI = 28, LH = 4, FSH = 1.5, LH/FSH > 2. The patient had history of both clomiphen failure and metformin administration for more than 6 months. She also underwent laparoscopy and ovarian cauterization approximately 1 year before. Infertility work up for the husband showed abnormal sperm analysis including a count of 28 millions/ml, <20% progressive motility and <14% normal morphology according to WHO criteria. A recent varicoceletomy did not improve semen quality for her husband. In her first treatment cycle and due to a high response to the antagonist protocol and to avoid the risk of severe OHSS, it was decided to cancel the cycle, and to continue the treatment using IVM techniques. After progesterone withdrawal bleeding, FSH priming was delayed until 13th day of the cycle when endometrial thickness reached 7 mm. At that time, FSH priming started, 75 IU/day of FSH (Serono, Aubnne, Switzerland) was injected for 3 days, followed by an injection of 10,000 IU hCG (Pregnyl, Organon, Netherlands). Follicular diameters were less than 10 mm at the time of hCG injection. On the day of hCG injection, both endometrial thickness and level of estradiol were measured. Oocytes were retrieved 36 h after hCG injection using a 19-gauge aspiration needle under transvaginal ultrasound guidance. A total of 10 immature oocytes were retrieved, including 3 at GV stage, 5 at MI stage, and 2 were degenerated. Immature oocytes were transferred to IVM culture media (SAGE Co., CT) for 28 h, when 5 oocytes reached MII stage. The rest of oocytes were arrested. The mature oocytes were microinjected with husbands’ sperm and normal fertilization was confirmed when two polar bodies with two pronuclei were observed 18–20 h after insemination. A total of 3 oocytes were fertilized and 3 four cells embryos (grade B) were transferred 2 days after microinjection using ET catheter (Labotect, Gottingen, Germany). Lutheal phase support was done with 100 mg progesterone in oil (Aburaihan Co, Tehran, Iran) injected daily. Estradiol valerate (Aburaihan C,Tehran, Iran) was taken orally at the dose of 6 mg/day from day of oocytes retrieval, and continued until confirmation of fetal heart by sonography. βhCG was measured 14 days after ET, which was reported positive (>180 IU/L). A healthy baby girl was born at 39 weeks of pregnancy by cesarean section. The baby weigh at the time of birth was 3,500 g with the apgar of 9/10.

Effects of inhibiting nitric oxide synthase on cumulus expansion and nuclear maturation of sheep oocytes

Nitric oxide (NO) is a biological signaling molecule that plays a crucial role in oocyte maturation of mammalians. It is generated by the nitric oxide synthase (NOS) enzyme from l-arginine. Although the effect of NO has been shown in oocyte maturation of some species, there is no report about its effect on the in vitro maturation of sheep oocyte. So, this study aimed to investigate the importance of NO/NOS system in the in vitro maturation of ovine oocytes. Different concentrations of L-NAME (a NOS inhibitor) (0.1, 1 and 10mM) were added to maturation medium to evaluate the effect of inhibiting NOS on cumulus expansion and meiotic resumption of sheep oocytes. After 26 h culture, low and medium concentrations of L-NAME (0.1 and 1mM) had no significant effect on cumulus expansion, however, its higher concentration (10mM) decreased percentage of oocytes with total cumulus expansion as compared to control (P &amp;lt; 0.05). The extrusion of the first polar body was also suppressed in a dose-dependent manner, so that the addition of 10mM L-NAME to maturation medium significantly stopped oocytes in GV stage (P &amp;lt; 0.05). Moreover, to confirm the results and to evaluate if this effect is reversible, 0.1mM sodium nitroprusside (SNP, a NO donor) was added only to the maturation medium which had the highest concentration of L-NAME (10mM). The concomitant addition of NOS inhibitor with NO donor reversed the inhibitory effect of L-NAME on cumulus expansion and meiotic maturation. These results indicated that NO/NOS system is involved in the maturation of sheep oocytes.

Molecular cloning and expression of bovine nucleoplasmin 2 (NPM2): a maternal effect gene regulated by miR-181a

BackgroundNucleoplasmin 2 (NPM2) is an oocyte-specific nuclear protein essential for nuclear and nucleolar organization and early embryonic development. The aims of this study were to clone the bovine NPM2 gene, determine its temporal expression during oocyte development and early embryogenesis, and evaluate the potential role of miRNA-181a in regulation of its expression.MethodsA 329 bp cDNA fragment was amplified from bovine fetal ovary using primers designed based on the conserved regions of the human and mouse NPM2 cDNA sequences. RACE experiments were performed to obtain the 5' and 3' ends of the bovine NPM2 cDNA. Real time PCR and Western blot analysis were used to examine the expression of bovine NPM2 in oocytes and early embryos. Co-expression of bovine NPM2 and miRNA-181a in Hela cells was performed to determine if expression of bovine NPM2 is regulated by miRNA-181a.ResultsThe bovine NPM2 cDNA is 851 bp in length encoding a protein of 200 amino acids. The protein contains the conserved bipartite nuclear localization sequence and shows 53% and 62% identity with mouse and human NPM2, respectively. Expression of bovine NPM2 mRNA is restricted to ovaries. NPM2 mRNA is abundant in GV and MII stage oocytes, decreases in early cleavage stage embryos, and barely detectable in morula and blastocyst stage embryos. Similarly, expression of NPM2 protein is high in oocytes and early embryos but extremely low in blastocysts. The abundance of NPM2 mRNA is significantly lower in oocytes isolated from persistent versus growing dominant follicles (P < 0.05). A miR-181a binding site in the 3'UTR of the NPM2 transcript was identified. Transfection experiments showed that bovine NPM2 protein expression is reduced in Hela cells expressing miR-181a compared to control cells without miR-181a, indicating that translation of NPM2 is repressed by miR-181a.ConclusionsOur data suggest that expression of bovine NPM2 is temporally regulated during early embryogenesis and miR-181a may play a role in its regulation.

A combination of FSH and dibutyryl cyclic AMP promote growth and acquisition of meiotic competence of oocytes from early porcine antral follicles

Growing porcine oocytes from early antral follicles (1.2–1.5 mm in diameter) do not mature to metaphase II (MII, 4%) under culture conditions which supported maturation (MII, 95%) of fully grown oocytes from large (4–6 mm) antral follicles. We hypothesized that FSH and dbcAMP supported growth and acquisition of meiotic competence. Growing oocytes (113.0 ± 0.4 μm, mean ± SEM) were cultured for 5 d in medium supplemented with 1 mM dbcAMP, 0.01 IU/mL FSH or both; in these media, oocytes reached, 120.5 ± 0.4, 123.5 ± 0.4 and 125.7 ± 0.2 μm, respectively, after 5 d, and then were matured in vitro for 48 h. Oocytes remained enclosed by cumulus cells when cultured with FSH (82%) or both FSH and dbcAMP (80%), but not with dbcAMP alone (0%). Furthermore, oocytes cultured with FSH maintained trans-zonal projections of cumulus cells. Oocytes remained at the GV stage at higher rates when cultured with dbcAMP and FSH (99%), or dbcAMP (97%), than with FSH (64%), or without either (75%). Following in vitro maturation, oocytes reached MII after in vitro growth with dbcAMP (19%), FSH (11%), or both (68%). When oocytes were cultured with both FSH and dbcAMP, activation of Cdc2 and MAP kinases in growing oocytes was similar to fully grown oocytes. In conclusion, growing porcine oocytes grew and acquired meiotic competence in medium supplemented with dbcAMP and FSH; the former maintained oocytes in meiotic arrest, whereas the latter maintained trans-zonal projections of cumulus cells to oocytes during in vitro growth culture.

Ultrastructural changes of sheep cumulus–oocyte complexes following different methods of vitrification

To determine the ultrastructural changes of sheep cumulus-oocyte complexes (COCs) following different methods of vitrification, good quality isolated COCs (GV stage) were randomly divided into the non-vitrified control, conventional straw, cryotop and solid surface vitrification groups. In both conventional and cryotop methods, vitrified COCs were respectively loaded by conventional straws and cryotops, and then plunged directly into liquid nitrogen (LN2); whereas in the solid surface group, vitrified COCs were first loaded by cryotops and then cooled before plunging into LN2. Post-warming survivability and ultrastructural changes of healthy COCs in the cryotop group especially in comparison with the conventional group revealed better viability rate and good preservation of the ooplasm organization. However in all vitrification groups except the cryotop group, mitochondria were clumped. Solely in the conventional straw group, the mitochondria showed different densities and were extremely distended. Moreover in the latter group, plenty of large irregular connected vesicles in the ooplasm were observed and in some parts their membrane ruptured. Also, in the conventional and solid surface vitrification groups, cumulus cells projections became retracted from the zona pellucida in some parts. In conclusion, the cryotop vitrification method as compared with other methods seems to have a good post-warming survivability and shows less deleterious effects on the ultrastructure of healthy vitrified-warmed sheep COCs.

Effect of H89 on the meiotic resumption of pig oocytes.

We studied the effect of H89, an inhibitor of protein kinase A (PKA), on the meiotic resumption of pig oocytes. Pig cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) were cultured for 27 h to induce meiotic resumption. COCs and DOs were exposed to H89 for different periods. Oocyte PKA activity was assessed by in vitro kinase assay and immunocytochemistry using an antibody against fully active PKA catalytic subunits. Oocyte serine/threonine (Ser/Thr)-phosphorylated proteins were detected by Western blotting and immunocytochemistry using an anti-pSer/pThr PKA substrate antibody. H89 suppressed germinal vesicle break down (GVBD) in COCs and DOs. To determine whether the suppression was due to inhibition of oocyte PKA, we analyzed oocyte PKA. Kinase assay showed that both types of oocytes possessed PKA activity throughout the culture period. Immunocytochemistry showed that fully active PKA catalytic subunits and Ser/Thr phosphorylated proteins were present in the oocytes at the GV stage and after GVBD. Western blotting indicated that both types of oocytes contained Ser/Thr phosphorylated proteins at the GV stage, and that several proteins became phosphorylated after GVBD. Pig oocytes contain active PKA during the occurrence of GVBD, and H89 suppresses the GVBD.