Loss of Bmal1 decreases oocyte fertilization, early embryo development and implantation potential in female mice.

PubMed

Xu, Jian; Li, Yan; Wang, Yizi; Xu, Yanwen; Zhou, Canquan

2016-10-01

Biological clock genes expressed in reproductive tissues play important roles in maintaining the normal functions of reproductive system. However, disruption of female circadian rhythm on oocyte fertilization, preimplantation embryo development and blastocyst implantation potential is still unclear. In this study, ovulation, in vivo and in vitro oocyte fertilization, embryo development, implantation and intracellular reactive oxygen species (ROS) levels in ovary and oviduct were studied in female Bmal1+/+ and Bmal1-/- mice. The number of naturally ovulated oocyte in Bmal1-/- mice decreased (5.2 ± 0.8 vs 7.8 ± 0.8, P < 0.001), with an increasing abnormal oocyte ratio (20.4 ± 3.5 vs 11.7 ± 2.0%, P = 0.001) after superovulation. Significantly lower fertilization rate and obtained blastocyst number were observed in Bmal1-/- female mice either mated with wild-type in vivo or fertilized by sperm from wild-type male mice in vitro (all P < 0.05). Interestingly, in vitro fertilization rate of oocytes derived from Bmal1-/- increased significantly compared with in vivo study (P < 0.01). After transferring blastocysts derived from Bmal1+/+ and Bmal1-/- female mice to pseudopregnant mice, the implantation sites of the latter decreased 5 days later (8.0 ± 0.8 vs 5.3 ± 1.0, P = 0.005). The intracellular ROS levels in the ovary on proestrus day and in the oviduct on metestrus day increased significantly in Bmal1-/- mice compared with that of Bmal1+/+ mice. Deletion of the core biological clock gene Bmal1 significantly decreases oocyte fertilization rate, early embryo development and implantation potential in female mice, and these may be possibly caused by excess ROS levels generated in ovary and oviduct.

Embryo apoptosis identification: Oocyte grade or cleavage stage?

PubMed Central

Bakri, Noraina Mohd; Ibrahim, Siti Fatimah; Osman, Nurul Atikah; Hasan, Nurhaslina; Jaffar, Farah Hanan Fathihah; Rahman, Zulaiha Abdul; Osman, Khairul

2015-01-01

Apoptosis is a programed cell death that is vital for tissue homeostasis. However, embryo apoptosis had been known to be related to embryo fragmentation which should be avoided in in vitro fertilization (IVF). The purpose of this study was to evaluate the relationship of embryo apoptosis with the grade of immature oocytes and cleavage stage of in vitro produced (IVP) cattle embryos. This study consisted of 345 oocytes collected through ovary slicing. Immature oocytes were graded as A, B and C. This grading was based on cumulus cell thickness and compactness. All oocytes then underwent an in vitro maturation (IVM) procedure. An IVF was done 24 h after IVM culture. Prior to staining, stage of cleaved embryos was determined and classified as either 2, 4, 8 or >8-cell embryo stage. Apoptosis status of cleaved IVP embryos was determined by using annexin V-FITC staining technique at 48 and 72 h post insemination (hpi). Apoptosis status for each embryo was classified as either early or late. The result showed that there was no significant difference (p > 0.05) of apoptosis status among grade A, B and C embryos. All grades of oocytes showed embryo apoptosis where 1.5% late apoptosis for grade A, 4.5% and 10.4% of early and late apoptosis for grade B and grade C. Early apoptosis was not seen in grade A embryo. We also noted no significant difference (p > 0.05) of apoptosis status between 2, 4, 8 and >8-cell embryo stage. Early apoptosis was also not seen in >8-cell stage. Even though there were no differences in apoptosis expression between the three classes, the cleavage rate of grade A oocytes was significantly higher (p < 0.01) than grade B and grade C. In conclusion, the apoptosis expression in the embryo can occur regardless of the oocyte quality and the cleavage stage of the embryo produced. PMID:26858565

Effect of recombinant-LH and hCG in the absence of FSH on in vitro maturation (IVM) fertilization and early embryonic development of mouse germinal vesicle (GV)-stage oocytes.

PubMed

Dinopoulou, Vasiliki; Drakakis, Peter; Kefala, Stella; Kiapekou, Erasmia; Bletsa, Ritsa; Anagnostou, Elli; Kallianidis, Konstantinos; Loutradis, Dimitrios

2016-06-01

During in vitro maturation (IVM), intrinsic and extrinsic factors must co-operate properly in order to ensure cytoplasmic and nuclear maturation. We examined the possible effect of LH/hCG in the process of oocyte maturation in mice with the addition of recombinant LH (r-LH) and hCG in our IVM cultures of mouse germinal vesicle (GV)-stage oocytes. Moreover, the effects of these hormones on fertilization, early embryonic development and the expression of LH/hCG receptor were examined. Nuclear maturation of GV-stage oocytes was evaluated after culture in the presence of r-LH or hCG. Fertilization rates and embryonic development were assessed after 24h. Total RNA was isolated from oocytes of different stages of maturation and from zygotes and embryos of different stages of development in order to examine the expression of LH/hCG receptor, using RT-PCR. The in vitro nuclear maturation rate of GV-stage oocytes that received hCG was significantly higher compared to the control group. Early embryonic development was increased in the hCG and LH cultures of GV oocytes when LH was further added. The LH/hCG receptor was expressed in all stages of in vitro matured mouse oocytes and in every stage of early embryonic development. Addition of hCG in IVM cultures of mouse GV oocytes increased maturation rates significantly. LH, however, was more beneficial to early embryonic development than hCG. This suggests a promising new technique in basic science research or in clinical reproductive medicine. Copyright © 2016 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

The beneficial effects of cumulus cells and oocyte-cumulus cell gap junctions depends on oocyte maturation and fertilization methods in mice.

PubMed

Zhou, Cheng-Jie; Wu, Sha-Na; Shen, Jiang-Peng; Wang, Dong-Hui; Kong, Xiang-Wei; Lu, Angeleem; Li, Yan-Jiao; Zhou, Hong-Xia; Zhao, Yue-Fang; Liang, Cheng-Guang

2016-01-01

Cumulus cells are a group of closely associated granulosa cells that surround and nourish oocytes. Previous studies have shown that cumulus cells contribute to oocyte maturation and fertilization through gap junction communication. However, it is not known how this gap junction signaling affects in vivo versus in vitro maturation of oocytes, and their subsequent fertilization and embryonic development following insemination. Therefore, in our study, we performed mouse oocyte maturation and insemination using in vivo- or in vitro-matured oocyte-cumulus complexes (OCCs, which retain gap junctions between the cumulus cells and the oocytes), in vitro-matured, denuded oocytes co-cultured with cumulus cells (DCs, which lack gap junctions between the cumulus cells and the oocytes), and in vitro-matured, denuded oocytes without cumulus cells (DOs). Using these models, we were able to analyze the effects of gap junction signaling on oocyte maturation, fertilization, and early embryo development. We found that gap junctions were necessary for both in vivo and in vitro oocyte maturation. In addition, for oocytes matured in vivo, the presence of cumulus cells during insemination improved fertilization and blastocyst formation, and this improvement was strengthened by gap junctions. Moreover, for oocytes matured in vitro, the presence of cumulus cells during insemination improved fertilization, but not blastocyst formation, and this improvement was independent of gap junctions. Our results demonstrate, for the first time, that the beneficial effect of gap junction signaling from cumulus cells depends on oocyte maturation and fertilization methods.

Metaphase II oocytes from human unilaminar follicles grown in a multi-step culture system.

PubMed

McLaughlin, M; Albertini, D F; Wallace, W H B; Anderson, R A; Telfer, E E

2018-03-01

fixed for analysis. Pieces of human ovarian cortex cultured in serum free medium for 8 days (Step 1) supported early follicle growth and 87 secondary follicles of diameter 120 ± 6 μm (mean ± SEM) could be dissected for further culture. After a further 8 days, 54 of the 87 follicles had reached the antral stage of development. COCs were retrieved by gentle pressure from the cultured follicles and those with adherent mural granulosa cells (n = 48) were selected and cultured for a further 4 days (Step 3). At the end of Step 3, 32 complexes contained oocytes >100 μm diameter were selected for IVM (Step 4). Nine of these complexes contained polar bodies within 24 h and all polar bodies were abnormally large. Confocal immuno-histochemical analysis showed the presence of a Metaphase II spindle confirming that these IVG oocytes had resumed meiosis but their developmental potential is unknown. This is a small number of samples but provides proof of concept that complete development of human oocytes can occur in vitro. Further optimization with morphological evaluation and fertilization potential of IVG oocytes is required to determine whether they are normal. The ability to develop human oocytes from the earliest follicular stages in vitro through to maturation and fertilization would benefit fertility preservation practice. Funded by MRC Grants (G0901839 and MR/L00299X/1). No competing interests.

The Role of Microfilaments in Early Meiotic Maturation of Mouse Oocytes

NASA Astrophysics Data System (ADS)

Calarco, Patricia G.

2005-04-01

Mouse oocyte microfilaments (MF) were perturbed by depolymerization (cytochalasin B) or stabilization (jasplakinolide) and correlated meiotic defects examined by confocal microscopy. MF, microtubules, and mitochondria were vitally stained; centrosomes ([gamma]-tubulin), after fixation. MF depolymerization by cytochalasin in culture medium did not affect central migration of centrosomes, mitochondria, or nuclear breakdown (GVBD); some MF signal was localized around the germinal vesicle (GV). In maturation-blocking medium (containing IBMX), central movement was curtailed and cortical MF aggregations made the plasma membrane wavy. Occasional long MF suggested that not all MF were depolymerized. MF stabilization by jasplakinolide led to MF aggregations throughout the cytoplasm. GVBD occurred (unless IBMX was present) but no spindle formed. Over time, most oocytes constricted creating a dumbbell shape with MF concentrated under one-half of the oocyte cortex and on either side of the constriction. In IBMX medium, the MF-containing half of the dumbbell over time sequestered the GV, MF, mitochondria, and one to two large cortical centrosomes; the non-MF half appeared empty. Cumulus processes contacted the oocyte surface (detected by microtubule content) and mirrored MF distribution. Results demonstrated that MF play an essential role in meiosis, primarily through cortically mediated events, including centrosome localization, spindle (or GV) movement to the periphery, activation of (polar body) constriction, and establishment of oocyte polarity. The presence of a cortical “organizing pole” is hypothesized.

From fresh heterologous oocyte donation to autologous oocyte banking.

PubMed

Stoop, D

2012-01-01

Today, oocyte donation has become well established, giving rise to thousands of children born worldwide annually. The introduction of oocyte cryopreservation through vitrification allows the introduction of egg banking, improving the efficiency and comfort of oocyte donation. Moreover, the vitrification technique can now enable autologous donation of oocytes to prevent future infertility. We evaluated fresh heterologous oocyte donation in terms of obstetrical and perinatal outcome as well as of the reproductive outcome of past donors. We then evaluated the efficiency of a closed vitrification device and its clinical applications within ART. Thirdly, we evaluated the opinion of women with regard to preventive egg freezing and the efficiency of a human oocyte in relation to age. Oocyte donation is associated with an increased risk of first trimester bleeding and pregnancy induced hypertension. Donating oocytes does not seem to increase the likelihood for a later need of fertility treatment. The chance of an oocyte to result in live birth (utilization rate) in women <37 years old remains constant with a mean of 4.47%. A significant proportion of young women would consider safeguarding their reproductive potential through egg freezing or are at least open to the idea. The introduction of efficient oocyte cryopreservation has revolutionized oocyte donation through the establishment of eggbank donation. The technique also enables women to perform autologous donation after preventive oocyte storage in order to circumvent their biological clock.

From fresh heterologous oocyte donation to autologous oocyte banking

PubMed Central

Stoop, D.

2012-01-01

Introduction: Today, oocyte donation has become well established, giving rise to thousands of children born worldwide annually. The introduction of oocyte cryopreservation through vitrification allows the introduction of egg banking, improving the efficiency and comfort of oocyte donation. Moreover, the vitrification technique can now enable autologous donation of oocytes to prevent future infertility. Methods: We evaluated fresh heterologous oocyte donation in terms of obstetrical and perinatal outcome as well as of the reproductive outcome of past donors. We then evaluated the efficiency of a closed vitrification device and its clinical applications within ART. Thirdly, we evaluated the opinion of women with regard to preventive egg freezing and the efficiency of a human oocyte in relation to age. Results: Oocyte donation is associated with an increased risk of first trimester bleeding and pregnancy induced hypertension. Donating oocytes does not seem to increase the likelihood for a later need of fertility treatment. The chance of an oocyte to result in live birth (utilization rate) in women <37 years old remains constant with a mean of 4.47%. A significant proportion of young women would consider safeguarding their reproductive potential through egg freezing or are at least open to the idea. Discussion and Conclusion: The introduction of efficient oocyte cryopreservation has revolutionized oocyte donation through the establishment of eggbank donation. The technique also enables women to perform autologous donation after preventive oocyte storage in order to circumvent their biological clock. PMID:24753920

The effect of immature oocytes quantity on the rates of oocytes maturity and morphology, fertilization, and embryo development in ICSI cycles.

PubMed

Halvaei, Iman; Ali Khalili, Mohammad; Razi, Mohammad Hossein; Nottola, Stefania A

2012-08-01

The goal was to evaluate the role of the number of retrieved immature oocytes on mature oocyte counts and morphology, and also the rates of fertilization and embryo development in ICSI cycles. 101 ICSI cycles were included in this prospective evaluation. Patients were divided into 2 groups of A (≤ 2 immature oocytes) and B (> 2 immature oocytes). In sub-analysis, the impacts of the number of GV and MI oocytes were assessed on the rates of fertilization and embryo development. Also, correlations between the numbers of immature and mature oocytes, as well as maternal age between two groups were analyzed. Assessments of oocyte morphology, fertilization, embryo quality and development were done accordingly. There was no correlation between the immature oocytes quantity with the number of mature ones. There were insignificant differences for embryo development between two groups, but fertilization rate was higher in group A (P = 0.03). In sub-analysis, insignificant differences were observed between two groups of ≤ and >2 GV and MI oocytes for rates of fertilization and embryo development. Also, the rates of clinical pregnancy and delivery were insignificant between groups. The rate of morphologically abnormal oocytes had no significant difference between two groups, except for wide perivitelline space (PVS) which was higher in group A (P = 0.03). There was no significant difference for maternal age between two groups. In cases with few retrieved immature oocytes, rates of fertilization and incidence of wide PVS may increase, although immature oocytes may not have any negative impacts on early embryo development, or the rates on number of mature oocytes.

Mechanisms of oocyte development in European sea bass (Dicentrarchus labrax L.): investigations via application of unilateral ovariectomy.

PubMed

García-López, Ángel; Sánchez-Amaya, María I; Tyler, Charles R; Prat, Francisco

2011-08-01

Unilateral ovariectomy (ULO) was performed in European sea bass (Dicentrarchus labrax L.) during late pre-vitellogenesis/early vitellogenesis. Plasma steroid levels and the expression of a suite of potential oogenesis-relevant genes in the ovary, brain, and pituitary were evaluated with the aim of understanding their involvement in the compensatory oocyte development occurring within the remaining ovarian lobe. After 69 days of surgery the remaining ovarian lobe in ULO fish was gravimetrically equivalent to an intact-paired ovary of sham operated, control fish. This compensatory ovarian growth was based on an increased number of early perinucleolar oocytes and mid-late stage vitellogenic follicles without an apparent recruitment of primary oocytes into the secondary growth phase. Plasma steroid levels were similar in ULO and control females at all time points analyzed, suggesting an increased steroid production of the remaining ovarian lobe in hemi-castrated females. Results of the gene expression survey conducted indicate that the signaling pathways mediated by Fsh and Gnrh1 constitute the central axes orchestrating the observed ovarian compensatory growth. In addition, steroid receptors, Star protein, Igfs, and members of the transforming growth factor beta superfamily including anti-Mullerian hormone and bone morphogenetic protein 4 were identified as potentially relevant players within this process, although their specific actions and interactions remain to be established. Our results demonstrate that ULO provides an excellent in vivo model for elucidating the interconnected endocrine and molecular mechanisms controlling oocyte development in European sea bass.

[Wide support for oocyte donation and banking in the Netherlands].

PubMed

Bos, Annelies M E; Klapwijk, Petra; Fauser, Bart C J M

2012-01-01

To assess the general consensus on the cryopreservation of oocytes and the introduction of oocyte banking facilities in the Netherlands. Poll investigation A poll with the use of an online questionnaire was conducted among nearly 19,000 participants of the Dutch EenVandaag opinion panel in May 2011. The poll results were adjusted to the Dutch population based on data from the Dutch Central Office for Statistics for age, gender, education, marital status, geographical area and political preference (measured according to the lower house elections of 2010). The primary endpoints were the percentages of supporters of oocyte freezing for own future use and of the concept of introducing oocyte banking facilities in The Netherlands. The secondary endpoints were the demographic differences between supporters and opponents. Approximately half of 18.911 participants supported oocyte freezing (47%). Fifty-percent of all participants supported oocyte banking in the Netherlands. Supporters of oocyte freezing were mainly women ≤ 45 years of age, who are highly educated and have no children. Four percent of the participating women aged ≤ 45 years would seriously consider obtaining donor oocytes from an available oocyte banking facility. Twelve percent of the participating women ≤ 45 years of age said they would definitely donate their oocytes or would seriously consider donating. Thirty-seven percent of all participants were against the introduction of oocyte banking facilities. The most important arguments against oocyte freezing were that women should reproduce during normal reproductive years and that it was not medically necessary. Poll results showed much support for oocyte freezing and for the introduction of oocyte banking facilities in the Netherlands. In addition, the poll shows that oocyte banking facilities would fulfil a need in the population.

Maternal hCG concentrations in early IVF pregnancies: associations with number of cells in the Day 2 embryo and oocytes retrieved.

PubMed

Tanbo, T G; Eskild, A

2015-12-01

Do number of cells in the transferred cleavage stage embryo and number of oocytes retrieved for IVF influence maternal hCG concentrations in early pregnancies? Compared with transfer of a 2-cell embryo, transfer of a 4-cell embryo results in higher hCG concentrations on Day 12 after transfer, and more than 20 oocytes retrieved were associated with low hCG concentrations. Maternal hCG concentration in very early pregnancy varies considerably among women, but is likely to be an indicator of time since implantation of the embryo into the endometrium, in addition to number and function of trophoblast cells. We followed 1047 pregnancies after IVF/ICSI from oocyte retrieval until Day 12 after embryo transfer. Women were recruited in Norway during the years 2005-2013. Successful pregnancies after transfer of one single embryo that had been cultured for 2 days were included. Maternal hCG was quantified on Day 12 after embryo transfer by chemiluminescence immunoassay, which measures intact hCG and the free β-hCG chain. Information on a successful pregnancy, defined as birth after >16 weeks, was obtained by linkage to the Medical Birth Registry of Norway. Transfer of a 4-cell embryo resulted in higher maternal hCG concentrations compared with transfer of a 2-cell embryo (134.8 versus 87.8 IU/l, P < 0.05). A high number of oocytes retrieved (>20) was associated with low hCG concentrations (P < 0.05). The factors studied explain a limited part of the total variation of hCG concentrations in early pregnancy. Although embryo transfer was performed at the same time after fertilization, we do not know the exact time of implantation. A further limitation to our study is that the number of pregnancies after transfer of a 2-cell embryo was small (27 cases). Number of cells in the transferred embryo and number of oocytes retrieved may influence the conditions and timing for embryo implantation in different ways and thereby influence maternal hCG concentrations. Such knowledge may be

Discovery of a novel oocyte-specific Krüppel-associated box domain-containing zinc finger protein required for early embryogenesis in cattle.

PubMed

Hand, Jacqelyn M; Zhang, Kun; Wang, Lei; Koganti, Prasanthi P; Mastrantoni, Kristen; Rajput, Sandeep K; Ashry, Mohamed; Smith, George W; Yao, Jianbo

2017-04-01

Zinc finger (ZNF) transcription factors interact with DNA through zinc finger motifs and play important roles in a variety of cellular functions including cell growth, proliferation, development, apoptosis, and intracellular signal transduction. One-third of ZNF proteins in metazoans contain a highly conserved N-terminal motif known as the Krüppel-associated box (KRAB) domain, which acts as a potent, DNA-binding dependent transcriptional repression module. Analysis of RNA-Seq data generated from a bovine oocyte cDNA library identified a novel transcript, which encodes a KRAB-containing ZNF transcription factor (named ZNFO). Characterization of ZNFO mRNA expression revealed that it is exclusively expressed in bovine oocytes and early embryos. A GFP reporter assay demonstrated that ZNFO protein localizes specifically to the nucleus, supporting its role in transcriptional regulation. To test the role of ZNFO in early embryonic development, zygotes were generated by in vitro maturation and fertilization of oocytes, and injected with small interfering RNA (siRNA) designed to knockdown ZNFO. Cleavage rates were not affected by ZNFO siRNA injection. However, embryonic development to 8- to 16-cell stage and blastocyst stage was significantly reduced relative to the uninjected and negative control siRNA-injected embryos. Further, interaction of ZNFO with the highly conserved co-factor, KRAB-associated protein-1 (KAP1), was demonstrated, and evidence supporting transcriptional repression by ZNFO was demonstrated using a GAL4-luciferase reporter system. Results of described studies demonstrate that ZNFO is a maternally-derived oocyte-specific nuclear factor required for early embryonic development in cattle, presumably functioning by repressing transcription. Copyright © 2017 Elsevier B.V. All rights reserved.

Nucleoli from two-cell embryos support the development of enucleolated germinal vesicle oocytes in the pig.

PubMed

Kyogoku, Hirohisa; Ogushi, Sugako; Miyano, Takashi

2012-11-01

Recent research has shown that nucleoli of oocytes at the germinal vesicle (GV) stage (GV nucleoli) are not necessary for oocyte maturation but are essential for early embryonic development. Nucleoli of 2-cell embryos (2-cell nucleoli) have morphology similar to that of nucleoli in oocytes at the GV stage. In this study, we examined the ability of 2-cell nucleoli to substitute for GV nucleoli in terms of supporting early embryonic development by nucleolus aspiration (enucleolation) and transfer into metaphase II (MII) oocytes or 2-cell embryos that were derived from enucleolated oocytes at the GV stage in the pig. When 2-cell embryos were centrifuged to move the lipid droplets to one side of the blastomere, multiple nucleoli in the nucleus fused into a single nucleolus. The nucleoli were then aspirated from the 2-cell embryos by micromanipulation. The injection of 2-cell nucleoli to GV enucleolated oocytes at the MII stage rescued the embryos from the early embryonic arrest, and the resulting oocytes developed to blastocysts. However, the injection of 2-cell and GV nucleoli to 2-cell embryos derived from GV enucleolated oocytes rarely restored the development to blastocysts. These results indicate that 2-cell nucleoli support early embryonic development as GV nucleoli and that the presence of nucleoli is essential for pig embryos before the 2-cell stage.

Oocyte-specific gene Oog1 suppresses the expression of spermatogenesis-specific genes in oocytes.

PubMed

Honda, Shinnosuke; Miki, Yuka; Miyamoto, Yuya; Kawahara, Yu; Tsukamoto, Satoshi; Imai, Hiroshi; Minami, Naojiro

2018-05-03

Oog1, an oocyte-specific gene that encodes a protein of 425 amino acids, is present in five copies on mouse chromosomes 4 and 12. In mouse oocytes, Oog1 mRNA expression begins at embryonic day 15.5 and almost disappears by the late two-cell stage. Meanwhile, OOG1 protein is detectable in oocytes in ovarian cysts and disappears by the four-cell stage; the protein is transported to the nucleus in late one-cell to early two-cell stage embryos. In this study, we examined the role of Oog1 during oogenesis in mice. Oog1 RNAi-transgenic mice were generated by expressing double-stranded hairpin Oog1 RNA, which is processed into siRNAs targeting Oog1 mRNA. Quantitative RT-PCR revealed that the amount of Oog1 mRNA was dramatically reduced in oocytes obtained from Oog1-knockdown mice, whereas the abundance of spermatogenesis-associated transcripts (Klhl10, Tekt2, Tdrd6, and Tnp2) was increased in Oog1 knockdown ovaries. Tdrd6 is involved in the formation of the chromatoid body, Tnp2 contributes to the formation of sperm heads, Tekt2 is required for the formation of ciliary and flagellar microtubules, and Klhl10 plays a key role in the elongated sperm differentiation. These results indicate that Oog1 down-regulates the expression of spermatogenesis-associated genes in female germ cells, allowing them to develop normally into oocytes.

Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy

PubMed Central

Bradley, Josephine; Pope, Iestyn; Masia, Francesco; Sanusi, Randa; Langbein, Wolfgang; Borri, Paola

2016-01-01

Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos. PMID:27151947

Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy.

PubMed

Bradley, Josephine; Pope, Iestyn; Masia, Francesco; Sanusi, Randa; Langbein, Wolfgang; Swann, Karl; Borri, Paola

2016-06-15

Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos. © 2016. Published by The Company of Biologists Ltd.

Genetic influences on ovulation of primary oocytes in LT/Sv strain mice.

PubMed

Everett, Clare A; Auchincloss, Catherine A; Kaufman, Matthew H; Abbott, Catherine M; West, John D

2004-11-01

A high proportion of LT/Sv strain oocytes arrest in meiotic metaphase I (MI) and are ovulated as diploid primary oocytes rather than haploid secondary oocytes. (Mus musculus castaneus x LT/SvKau)F1 x LT/SvKau backcross females were analysed for the proportion of oocytes that arrested in MI and typed by PCR for a panel of microsatellite DNA sequences (simple sequence repeat polymorphisms) that differed between strain LT/SvKau and M. m. castaneus. This provided a whole genome scan of 86 genetic markers distributed over all 19 autosomes and the X chromosome, and revealed genetic linkage of the MI arrest phenotype to markers on chromosomes 1 and 9. Identification of these two chromosomal regions should facilitate the identification of genes involved in mammalian oocyte maturation and the control of meiosis.

Oocyte Activation and Fertilisation: Crucial Contributors from the Sperm and Oocyte.

PubMed

Yeste, Marc; Jones, Celine; Amdani, Siti Nornadhirah; Coward, Kevin

2017-01-01

This chapter intends to summarise the importance of sperm- and oocyte-derived factors in the processes of sperm-oocyte binding and oocyte activation. First, we describe the initial interaction between sperm and the zona pellucida, with particular regard to acrosome exocytosis. We then describe how sperm and oocyte membranes fuse, with special reference to the discovery of the sperm protein IZUMO1 and its interaction with the oocyte membrane receptor JUNO. We then focus specifically upon oocyte activation, the fundamental process by which the oocyte is alleviated from metaphase II arrest by a sperm-soluble factor. The identity of this sperm factor has been the source of much debate recently, although mounting evidence, from several different laboratories, provides strong support for phospholipase C ζ (PLCζ), a sperm-specific phospholipase. Herein, we discuss the evidence in support of PLCζ and evaluate the potential role of other candidate proteins, such as post-acrosomal WW-binding domain protein (PAWP/WBP2NL). Since the cascade of downstream events triggered by the sperm-borne oocyte activation factor heavily relies upon specialised cellular machinery within the oocyte, we also discuss the critical role of oocyte-borne factors, such as the inositol trisphosphate receptor (IP 3 R), protein kinase C (PKC), store-operated calcium entry (SOCE) and calcium/calmodulin-dependent protein kinase II (CaMKII), during the process of oocyte activation. In order to place the implications of these various factors and processes into a clinical context, we proceed to describe their potential association with oocyte activation failure and discuss how clinical techniques such as the in vitro maturation of oocytes may affect oocyte activation ability. Finally, we contemplate the role of artificial oocyte activating agents in the clinical rescue of oocyte activation deficiency and discuss options for more endogenous alternatives.

Developmental consequences of cryopreservation of mammalian oocytes and embryos.

PubMed

Smith, Gary D; Silva E Silva, Cristine Ane

2004-08-01

During the last three decades, significant advances have been made in successful cryopreservation of mammalian preimplantation embryos, and more recently oocytes. The ability to cryopreserve, thaw, and establish pregnancies with supernumerary preimplantation embryos has become an important tool in fertility treatment. Human oocyte cryopreservation has practical application in preserving fertility for individuals at risk of compromised egg quality due to cancer treatments or advanced maternal age. While oocyte/embryo cryopreservation success has increased over time, there is still room for improvement. Oocytes and embryos are susceptible to cryo-damage, which collectively entails cellular damage caused by mechanical, chemical, or thermal forces during the freeze-thaw process. Basic studies focused on understanding cellular structures, their composition, and more importantly their functions, in normal cell developments will continue to be critical in assessing, understanding, and correcting oocyte/embryo cryo-damage. This review will delineate many of the oocyte/embryo intracellular and extracellular structures that are or may be compromised during cryopreservation. A global theme presented throughout this review is that many structural components of the oocyte/embryo also have essential functional roles in development. Compromising these cellular structures, and thus their cellular homeostatic functions, can deleteriously influence initial cryo-survival or compromise subsequent normal development through effects on the oocyte and/or early embryo.

Raman-microscopy investigation of vitrification-induced structural damages in mature bovine oocytes

PubMed Central

De Canditiis, Carolina; Zito, Gianluigi; Rubessa, Marcello; Roca, Maria Serena; Carotenuto, Rosa; Sasso, Antonio; Gasparrini, Bianca

2017-01-01

Although oocyte cryopreservation has great potentials in the field of reproductive technologies, it still is an open challenge in the majority of domestic animals and little is known on the biochemical transformation induced by this process in the different cellular compartments. Raman micro-spectroscopy allows the non-invasive evaluation of the molecular composition of cells, based on the inelastic scattering of laser photons by vibrating molecules. The aim of this work was to assess the biochemical modifications of both the zona pellucida and cytoplasm of vitrified/warmed in vitro matured bovine oocytes at different post-warming times. By taking advantage of Principal Component Analysis, we were able to shed light on the biochemical transformation induced by the cryogenic treatment, also pointing out the specific role of cryoprotective agents (CPs). Our results suggest that vitrification induces a transformation of the protein secondary structure from the α-helices to the β-sheet form, while lipids tend to assume a more packed configuration in the zona pellucida. Both modifications result in a mechanical hardening of this cellular compartment, which could account for the reduced fertility rates of vitrified oocytes. Furthermore, biochemical modifications were observed at the cytoplasmic level in the protein secondary structure, with α-helices loss, suggesting cold protein denaturation. In addition, a decrease of lipid unsaturation was found in vitrified oocytes, suggesting oxidative damages. Interestingly, most modifications were not observed in oocytes exposed to CPs, suggesting that they do not severely affect the biochemical architecture of the oocyte. Nevertheless, in oocytes exposed to CPs decreased developmental competence and increased reactive oxygen species production were observed compared to the control. A more severe reduction of cleavage and blastocyst rates after in vitro fertilization was obtained from vitrified oocytes. Our experimental

Distribution and viability of spermatozoa in the canine female genital tract during post-ovulatory oocyte maturation

PubMed Central

2012-01-01

Background Unlike other domestic mammals, in which metaphase-II oocytes are ovulated, canine ovulation is characterized by the release of primary oocytes, which may take 12 to up to 36 hours. Further 60 hours are needed for maturation to secondary oocytes which then remain fertile for about 48 hours. Oestrus takes 7 to 10 days on average and may start as early as a week before ovulation. This together with the prolonged process of post-ovulatory oocyte maturation requires an according longevity of spermatozoa in the female genital tract in order to provide a population of fertile sperm when oocytes have matured to fertilizability. Therefore the distribution and viability of spermatozoa in the bitch genital tract was examined during post-ovulatory oocyte maturation. Methods Thirteen beagle bitches were inseminated on the day of sonographically verified ovulation with pooled semen of two beagle dogs containing one billion progressively motile spermatozoa. Ovariohysterectomy was performed two days later (group 1, n = 6) and four days later (group 2, n = 7). The oviduct and uterine horn of one side were flushed separately and the flushing’s were checked for the presence of gametes. The oviducts including the utero-tubal junction and the uterine horns, both the flushed and unflushed, were histologically examined for sperm distribution. Results The total number of spermatozoa recovered by flushing was low and evaluation of viability was limited. Prophase-I oocytes were collected from oviduct flushing in group 1, whereas unfertilized metaphase-II oocytes were detected in group 2. From day 2 to day 4 after ovulation a significant decrease in the percentage of glands containing sperm (P<0.05) and a marked reduction of the mean sperm number in uterine horn glands were observed. A concomitant diminution of spermatozoa was indicated in the utero-tubal junction accompanied by a slight increase in sperm numbers in the mid oviduct. Conclusions Oocyte maturation to

Nucleoli from growing oocytes inhibit the maturation of enucleolated, full-grown oocytes in the pig.

PubMed

Kyogoku, Hirohisa; Ogushi, Sugako; Miyano, Takashi; Fulka, Josef

2011-06-01

In mammals, the nucleolus of full-grown oocyte is essential for embryonic development but not for oocyte maturation. In our study, the role of the growing oocyte nucleolus in oocyte maturation was examined by nucleolus removal and/or transfer into previously enucleolated, growing (around 100 µm in diameter) or full-grown (120 µm) pig oocytes. In the first experiment, the nucleoli were aspirated from growing oocytes whose nucleoli had been compacted by actinomycin D treatment, and the enucleolated oocytes were matured in vitro. Most of non-treated or actinomycin D-treated oocytes did not undergo germinal vesicle breakdown (GVBD; 13% and 12%, respectively). However, the GVBD rate of enucleolated, growing oocytes significantly increased to 46%. The low GVBD rate of enucleolated, growing oocytes was restored again by the re-injection of nucleoli from growing oocytes (23%), but not when nucleoli from full-grown oocytes were re-injected into enucleolated, growing oocytes (49%). When enucleolated, full-grown oocytes were injected with nucleoli from growing or full-grown oocytes, the nucleolus in the germinal vesicle was reassembled (73% and 60%, respectively). After maturation, the enucleolated, full-grown oocytes injected with nucleoli from full-grown oocytes matured to metaphase II (56%), whereas injection with growing-oocyte nucleoli reduced this maturation to 21%. These results suggest that the growing-oocyte nucleolus is involved in the oocyte's meiotic arrest, and that the full-grown oocyte nucleolus has lost the ability. Copyright © 2011 Wiley-Liss, Inc.

Cytoskeleton-associated protein 5 and clathrin heavy chain binding regulates spindle assembly in mouse oocytes

PubMed Central

Wang, Dong-Hui; Han, Zhe; Kong, Xiang-Wei; Ma, Yu-Zhen; Yun, Zhi-Zhong; Liang, Cheng-Guang

2017-01-01

Mammalian oocyte meiotic maturation is the precondition of early embryo development. Lots of microtubules (MT)-associated proteins participate in oocyte maturation process. Cytoskeleton-associated protein 5 (CKAP5) is a member of the XMAP215 family that regulates microtubule dynamics during mitosis. However, its role in meiosis has not been fully studied. Here, we investigated the function of CKAP5 in mouse oocyte meiotic maturation and early embryo development. Western blot showed that CKAP5 expression increased from GVBD, maintaining at high level at metaphase, and decreased after late 1-cell stage. Confocal microscopy showed there is no specific accumulation of CKAP5 at interphase (GV, PN or 2-cell stage). However, once cells enter into meiotic or mitotic division, CKAP5 was localized at the whole spindle apparatus. Treatment of oocytes with the tubulin-disturbing reagents nocodazole (induces MTs depolymerization) or taxol (prevents MTs depolymerization) did not affect CKAP5 expression but led to a rearrangement of CKAP5. Further, knock-down of CKAP5 resulted in a failure of first polar body extrusion, serious defects in spindle assembly, and failure of chromosome alignment. Loss of CKAP5 also decreased early embryo development potential. Furthermore, co-immunoprecipitation showed that CKAP5 bound to clathrin heavy chain 1 (CLTC). Taken together, our results demonstrate that CKAP5 is important in oocyte maturation and early embryo development, and CKAP5 might work together with CLTC in mouse oocyte maturation. PMID:28177917

Hydroxypropyl cellulose supplementation in vitrification solutions: a prospective study with donor oocytes.

PubMed

Gallardo, Miguel; Hebles, María; Migueles, Beatriz; Dorado, Mónica; Aguilera, Laura; González, Mercedes; Piqueras, Paloma; Lucas, Alejandro; Montero, Lorena; Sánchez-Martín, Pascual; Sánchez-Martín, Fernando; Risco, Ramón

2017-03-01

Hydroxypropyl cellulose (HPC), a polysaccharide that forms a viscous gel under low temperatures, is a promising substitute of the blood-derived macromolecules traditionally used in cryopreservation solutions. The performance of a protein-free, fully synthetic set of vitrification and warming solutions was assessed in a matched pair analysis with donor oocytes. A prospective study including 219 donor MII oocytes was carried out, comparing the laboratory outcomes of oocytes vitrified with HPC-based solutions and their fresh counterparts. The primary performance endpoint was the fertilization rate. Secondary parameters assessed were embryo quality on days 2 and 3. 70/73 (95.9%) vitrified MII oocytes exhibited morphologic survival 2 h post-warming, with 49 (70.0%) presented normal fertilization, compared to 105 of 146 (71.9%) MII fresh oocytes. Similar embryo quality was observed in both groups. A total of 18 embryos implanted, out of 38 embryos transferred (47.3%), resulting in 13 newborns.

Uptake and selective partitioning of dietary lipids to ovarian and muscle tissue of maturing female coho salmon, Oncorhynchus kisutch, during secondary oocyte growth.

PubMed

Johnson, Ronald B; Kroeger, Eric L; Reichert, William L; Carter, Cameron S; Rust, Michael B

2017-06-01

Female coho salmon, Oncorhynchus kisutch, were fed one of two experimental feeds containing lipids with markedly different stable 13 C isotope signatures during the late cortical alveolus, lipid droplet, and vitellogenesis stages of secondary oocyte growth. Ovarian and muscle lipids fatty acid concentrations were significantly affected by treatment during all three stages of development. Stable 13 C isotope analyses confirmed that dietary lipids were incorporated into both ovarian and muscle lipids during all three stages and revealed that ovarian lipids were more affected than muscle lipids during vitellogenesis. Arachidonic acid (ARA) was incorporated into ovarian lipids at the highest rate of all fatty acids examined with the greatest uptake observed during the cortical alveolus and lipid droplet stages of development. Docosahexaenoic acid (DHA) was incorporated into ovarian lipids at the next highest rate with the greatest uptake observed during the lipid droplet stage of development. The presence of an ovary specific, fatty acid transfer mechanism is proposed. Results from this study demonstrate the ability to greatly alter the fatty acid composition of ovarian lipids through a dietary change during secondary oocyte growth and may be of great interest to producers of farmed salmon and salmon broodstock programs. Published by Elsevier Inc.

The fertilization ability and developmental competence of bovine oocytes grown in vitro

PubMed Central

MAKITA, Miho; UEDA, Mayuko; MIYANO, Takashi

2016-01-01

In vitro growth culture systems for oocytes are being developed in several mammalian species. In these growth culture systems, in vitro grown oocytes usually have lower blastocyst formation than in vivo grown oocytes after in vitro fertilization. Furthermore, there have been a few reports that investigated the fertilization ability of in vitro grown oocytes in large animals. The purpose of this study was to investigate the fertilization process and developmental competence of bovine oocytes grown in vitro. Oocyte-granulosa cell complexes collected from bovine early antral follicles (0.4−0.7 mm in diameter) were cultured for growth with 17β-estradiol and androstenedione for 14 days and matured in vitro. These oocytes were then inseminated for 6 or 12 h, and further cultured for development up to 8 days in vitro. After growth culture, oocytes grew from 95 µm to around 120 µm and acquired maturation competence (79%). Although fertilization rates of in vitro grown oocytes were low after 6 h of insemination, 34% of in vitro grown oocytes fertilized normally after 12 h of insemination, having two polar bodies and two pronuclei with a sperm tail, and 22% of these oocytes developed into blastocysts after 8 days of culture. The fertilization and blastocyst formation rates were similar to those of in vivo grown oocytes. In addition, blastocyst cell numbers were also similar between in vitro and in vivo grown oocytes. In conclusion, in vitro grown bovine oocytes are similar to in vivo grown oocytes in fertilization ability and can develop into blastocysts. PMID:27151093

The fertilization ability and developmental competence of bovine oocytes grown in vitro.

PubMed

Makita, Miho; Ueda, Mayuko; Miyano, Takashi

2016-08-25

In vitro growth culture systems for oocytes are being developed in several mammalian species. In these growth culture systems, in vitro grown oocytes usually have lower blastocyst formation than in vivo grown oocytes after in vitro fertilization. Furthermore, there have been a few reports that investigated the fertilization ability of in vitro grown oocytes in large animals. The purpose of this study was to investigate the fertilization process and developmental competence of bovine oocytes grown in vitro. Oocyte-granulosa cell complexes collected from bovine early antral follicles (0.4-0.7 mm in diameter) were cultured for growth with 17β-estradiol and androstenedione for 14 days and matured in vitro. These oocytes were then inseminated for 6 or 12 h, and further cultured for development up to 8 days in vitro. After growth culture, oocytes grew from 95 µm to around 120 µm and acquired maturation competence (79%). Although fertilization rates of in vitro grown oocytes were low after 6 h of insemination, 34% of in vitro grown oocytes fertilized normally after 12 h of insemination, having two polar bodies and two pronuclei with a sperm tail, and 22% of these oocytes developed into blastocysts after 8 days of culture. The fertilization and blastocyst formation rates were similar to those of in vivo grown oocytes. In addition, blastocyst cell numbers were also similar between in vitro and in vivo grown oocytes. In conclusion, in vitro grown bovine oocytes are similar to in vivo grown oocytes in fertilization ability and can develop into blastocysts.

Criteria to assess human oocyte quality after cryopreservation.

PubMed

Coticchio, G; Bonu, M A; Bianchi, V; Flamigni, C; Borini, A

2005-10-01

Oocyte cryopreservation certainly represents one of the most attractive developments in the field of assisted reproduction, with the aim of preserving female fertility and circumventing the ethical and legal drawbacks associated with embryo freezing. Despite the achievement of the first pregnancy from frozen oocytes dating back as early as 1987, since then fewer than 150 pregnancies have been reported. Over a long period of time, application of oocyte storage on a large scale has been prevented by various factors, namely poor post-thaw survival. Fertilization rates remained low even after the introduction of intracytoplasmic sperm injection. Modifications of slow-freezing protocols, mainly based on the increase of the concentration of sucrose used as non-penetrating cryoprotectant (CPA) and the replacement of sodium with choline, appear to have decisively improved survival rates to over 80%. Investigations at the cellular level on thawed oocytes are largely lacking. Fertilization rates have also benefited from protocol modifications, reaching values indistinguishable from those normally obtained with fresh material. Vitrification protocols have also been tested, giving rise to improvements whose reproducibility is still uncertain. Data on the dynamics of fertilization and preimplantation development of embryos derived from frozen oocytes are extremely scarce. At the moment, clinical efficiency of oocyte cryopreservation cannot be precisely assessed because of the lack of controlled studies, although it appears to be considerably lower than that achieved with embryo freezing. In summary, encouraging advances have been made in the field of oocyte cryopreservation, but presently no protocol can ensure standards of success and safety comparable to those guaranteed by embryo storage.

Increased incidence of preeclampsia in mothers of advanced age conceiving by oocyte donation.

PubMed

Dior, Uri P; Laufer, Neri; Chill, Henry H; Granovsky-Grisaru, Sorina; Yagel, Simcha; Yaffe, Haim; Gielchinsky, Yuval

2018-05-01

The aim of this study was to evaluate the risk of preeclampsia in women of advanced age who conceived through donated oocytes as compared with natural conceptions. A historical prospective study of singleton live births of parturients ≥ 45 years of age at four university hospitals was conducted. For the purpose of the study, the population was divided by the mode of conception into two groups: oocyte donation and natural conception. The main outcome variable in this study was preeclampsia. Secondary outcomes included pregnancy-induced hypertension and Small for Gestational Age. Two hundred and seventy pregnancies were achieved naturally and 135 women conceived by oocyte donation. Mean age at delivery for the natural conception and oocyte donation groups was 45.7 and 47.8, respectively. Preeclampsia complicated 3 out of 270 (1.1%) natural conception pregnancies and 17 out of 135 (12.6%) oocyte donation conceptions. After adjusting for confounders, oocyte donation pregnancies were found to be associated with a 12-fold increased risk for preeclampsia (P = 0.001). Among oocyte donation pregnancies, the risk of preeclampsia was not affected by parity or age. A substantially increased risk for preeclampsia was found in oocyte donation pregnancies, suggesting that the foreign oocyte may play a specific biologic role in the development of preeclampsia after the age of 45.

Toxic effects and possible mechanisms of hydrogen sulfide and/or ammonia on porcine oocyte maturation in vitro.

PubMed

Yang, Lei-Lei; Zhao, Yong; Luo, Shi-Ming; Ma, Jun-Yu; Ge, Zhao-Jia; Shen, Wei; Yin, Shen

2018-03-15

Previous studies suggest that hydrogen sulfide (H 2 S) and ammonia (NH 3 ) are two major air pollutants which can cause damage to porcine health. However, the mechanisms underlying toxic effects of these compounds on porcine oocyte maturation are not clear. To clarify the mechanism, we evaluated the oocyte quality by detecting some events during oocytes maturation. In our study, porcine oocytes were cultured with different concentrations of Na 2 S and/or NH 4 Cl in vitro and the rate of the first polar body extrusion decreased significantly. Also, actin filament was seriously disrupted to damage the cytoskeleton which resulted in reduced rate of oocyte maturation. We explored the reactive oxygen species (ROS) generation and found that the ROS level was increased significantly after Na 2 S treatment but not after NH 4 Cl treatment. Moreover, early stage apoptosis rate was significantly increased and autophagy protein LC3 B expression level was higher in oocytes treated with Na 2 S and/or NH 4 Cl, which might be caused by ROS elevation. Additionally, exposure to Na 2 S and/or NH 4 Cl also caused ROS generation and early apoptosis in cumulus cells, which might further affect oocyte maturation in vitro. In summary, our data suggested that exposure to H 2 S and/or NH 3 decreased porcine oocyte maturation in vitro, which might be caused by actin disruption, ROS generation, early apoptosis and autophagy. Copyright © 2017 Elsevier B.V. All rights reserved.

Caffeine delays oocyte aging and maintains the quality of aged oocytes safely in mouse.

PubMed

Zhang, Xia; Liu, Xiaoyan; Chen, Li; Wu, Dan-Ya; Nie, Zheng-Wen; Gao, Ying-Ying; Miao, Yi-Liang

2017-03-28

Caffeine, as an oocyte aging inhibitor, was used in many different species to control or delay oocyte aging. However, the safety of caffeine and developmental competence of aged oocytes inhibited by caffeine has not been studied systematically. So we detected the spindle morphology, distribution of cortical granules, zona pellucida hardening and pronucleus formation to assess oocyte quality of caffeine treated oocytes. We found that aged oocytes treated by caffeine maintained weak susceptibility to activating stimuli and regained normal competent after aged further 6 hr. Caffeine maintained the spindle morphology, changed cortical granules distribution of aged oocytes and could not prevent zona pellucida hardening. Furthermore, caffeine increased pronucleus formation of aged oocytes and decreased fragmentation after fertilization. These results suggested that caffeine could maintain the quality of aged oocytes safely in mouse.

Optimum culture duration for growing oocytes to attain meiotic and fertilization competence.

PubMed

Yamochi, Takayuki; Hashimoto, Shu; Yamanaka, Masaya; Nakaoka, Yoshiharu; Morimoto, Yoshiharu

2017-12-15

To determine the optimum culture duration for porcine growing oocytes (GOs) to attain maturation competence, we examined the meiotic competence, chromatin configuration, and fertilization ability of porcine oocytes obtained from early antral follicles and cultured for 10-16 days. The survival rate of oocytes after 10 days of culture (62.8%) was similar to that of oocytes after 12 days of culture (55%) and significantly higher than that of oocytes cultured for 14 and 16 days (52.9 and 24.3%, respectively). No significant difference was observed in the diameter of ooplasm from oocytes cultured for different durations (117.4-118.3 μm). The maturation rates of surviving oocytes after 10 and 16 days of culture (38.3 and 22.7%, respectively) were significantly lower than those of oocytes cultured for 12 and 14 days, and their in vivo counterparts (52.8-62.4%). The number of oocytes with surrounded-nucleolus chromatin was significantly lower in the 10-day culture group (78.4%) as compared with 14-day culture and in vivo counterpart groups (93.6 and 95.1%, respectively). After in vitro maturation and intracytoplasmic sperm injection, no significant difference was observed in the rate of fertilization among oocytes cultured for 12 and 14 days, and their in vivo counterparts (40.5-47.2%). Thus, porcine GOs required at least 12 days to acquire meiotic and fertilization competence, and the culture duration to maximize the number of mature oocytes ranged from 12 to 14 days.

Mutations in TUBB8 and Human Oocyte Meiotic Arrest

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feng, Ruizhi; Sang, Qing; Kuang, Yanping

BACKGROUND: We present that human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to the arrest of human oocyte maturation are unknown. METHODS: We sequenced the exomes of five members of a four-generation family, three of whom had infertility due to oocyte meiosis I arrest. We performed Sanger sequencing of a candidate gene, TUBB8, in DNA samples from these members, additional family members, and members of 23 other affected families. The expression of TUBB8 and all other β-tubulin isotypes was assessed in human oocytes, earlymore » embryos, sperm cells, and several somatic tissues by means of a quantitative reverse- transcriptase-polymerase-chain-reaction assay. We evaluated the effect of the TUBB8 mutations on the assembly of the heterodimer consisting of one α-tubulin polypeptide and one β-tubulin polypeptide (α/β-tubulin heterodimer) in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes. RESULTSL: We identified seven mutations in the primate-specific gene TUBB8 that were responsible for oocyte meiosis I arrest in 7 of the 24 families. TUBB8 expression is unique to oocytes and the early embryo, in which this gene accounts for almost all the expressed β-tubulin. The mutations affect chaperone-dependent folding and assembly of the α/β-tubulin heterodimer, disrupt microtubule behavior on expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle-assembly defects and maturation arrest on expression in mouse and human oocytes. CONCLUSIONS: Lastly, TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility.« less

Mutations in TUBB8 and Human Oocyte Meiotic Arrest

DOE PAGES

Feng, Ruizhi; Sang, Qing; Kuang, Yanping; ...

2016-01-21

BACKGROUND: We present that human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to the arrest of human oocyte maturation are unknown. METHODS: We sequenced the exomes of five members of a four-generation family, three of whom had infertility due to oocyte meiosis I arrest. We performed Sanger sequencing of a candidate gene, TUBB8, in DNA samples from these members, additional family members, and members of 23 other affected families. The expression of TUBB8 and all other β-tubulin isotypes was assessed in human oocytes, earlymore » embryos, sperm cells, and several somatic tissues by means of a quantitative reverse- transcriptase-polymerase-chain-reaction assay. We evaluated the effect of the TUBB8 mutations on the assembly of the heterodimer consisting of one α-tubulin polypeptide and one β-tubulin polypeptide (α/β-tubulin heterodimer) in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes. RESULTSL: We identified seven mutations in the primate-specific gene TUBB8 that were responsible for oocyte meiosis I arrest in 7 of the 24 families. TUBB8 expression is unique to oocytes and the early embryo, in which this gene accounts for almost all the expressed β-tubulin. The mutations affect chaperone-dependent folding and assembly of the α/β-tubulin heterodimer, disrupt microtubule behavior on expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle-assembly defects and maturation arrest on expression in mouse and human oocytes. CONCLUSIONS: Lastly, TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility.« less

Recent advances in understanding oogenesis: interactions with the cytoskeleton, microtubule organization, and meiotic spindle assembly in oocytes

PubMed Central

Marlow, Florence L.

2018-01-01

Maternal control of development begins with production of the oocyte during oogenesis. All of the factors necessary to complete oocyte maturation, meiosis, fertilization, and early development are produced in the transcriptionally active early oocyte. Active transcription of the maternal genome is a mechanism to ensure that the oocyte and development of the early embryo begin with all of the factors needed for successful embryonic development. To achieve the maximum maternal store, only one functional cell is produced from the meiotic divisions that produce the oocyte. The oocyte receives the bulk of the maternal cytoplasm and thus is significantly larger than its sister cells, the tiny polar bodies, which receive a copy of the maternal genome but essentially none of the maternal cytoplasm. This asymmetric division is accomplished by an enormous cell that is depleted of centrosomes in early oogenesis; thus, meiotic divisions in oocytes are distinct from those of mitotic cells. Therefore, these cells must partition the chromosomes faithfully to ensure euploidy by using mechanisms that do not rely on a conventional centrosome-based mitotic spindle. Several mechanisms that contribute to assembly and maintenance of the meiotic spindle in oocytes have been identified; however, none is fully understood. In recent years, there have been many exciting and significant advances in oogenesis, contributed by studies using a myriad of systems. Regrettably, I cannot adequately cover all of the important advances here and so I apologize to those whose beautiful work has not been included. This review focuses on a few of the most recent studies, conducted by several groups, using invertebrate and vertebrate systems, that have provided mechanistic insight into how microtubule assembly and meiotic spindle morphogenesis are controlled in the absence of centrosomes.

Nanoliter droplet vitrification for oocyte cryopreservation.

PubMed

Zhang, Xiaohui; Khimji, Imran; Shao, Lei; Safaee, Hooman; Desai, Khanjan; Keles, Hasan Onur; Gurkan, Umut Atakan; Kayaalp, Emre; Nureddin, Aida; Anchan, Raymond M; Maas, Richard L; Demirci, Utkan

2012-04-01

Oocyte cryopreservation remains largely experimental, with live birth rates of only 2-4% per thawed oocyte. In this study, we present a nanoliter droplet technology for oocyte vitrification. An ejector-based droplet vitrification system was designed to continuously cryopreserve oocytes in nanoliter droplets. Oocyte survival rates, morphologies and parthenogenetic development after each vitrification step were assessed in comparison with fresh oocytes. Oocytes were retrieved after cryoprotectant agent loading/unloading, and nanoliter droplet encapsulation showed comparable survival rates to fresh oocytes after 24 h in culture. Also, oocytes recovered after vitrification/thawing showed similar morphologies to those of fresh oocytes. Additionally, the rate of oocyte parthenogenetic activation after nanoliter droplet encapsulation was comparable with that observed for fresh oocytes. This nanoliter droplet technology enables the vitrification of oocytes at higher cooling and warming rates using lower cryoprotectant agent levels (i.e., 1.4 M ethylene glycol, 1.1 M dimethyl sulfoxide and 1 M sucrose), thus making it a potential technology to improve oocyte cryopreservation outcomes.

Identification of candidate miRNAs and expression profile of yak oocytes before and after in vitro maturation by high-throughput sequencing.

PubMed

Xiong, X R; Lan, D L; Li, J; Zi, X D; Li, M Y

2016-12-01

Small RNA represents several unique non-coding RNA classes that have important function in a wide range of biological processes including development of germ cells and early embryonic, cell differentiation, cell proliferation and apoptosis in diverse organisms. However, little is known about their expression profiles and effects in yak oocytes maturation and early development. To investigate the function of small RNAs in the maturation process of yak oocyte and early development, two small RNA libraries of oocytes were constructed from germinal vesicle stage (GV) and maturation in vitro to metaphase II-arrested stage (M II) and then sequenced using small RNA high-throughput sequencing technology. A total of 9,742,592 and 12,168,523 clean reads were obtained from GV and M II oocytes, respectively. In total, 801 and 1,018 known miRNAs were acquired from GV and M II oocytes, and 75 miRNAs were found to be significantly differentially expressed: 47 miRNAs were upregulated and 28 miRNAs were downregulated in the M II oocytes compared to the GV stage. Among the upregulated miRNAs, miR-342 has the largest fold change (9.25-fold). Six highly expressed miRNAs (let-7i, miR-10b, miR-10c, miR-143, miR-146b and miR-148) were validated by real-time quantitative PCR (RT-qPCR) and consistent with the sequencing results. Furthermore, the expression patterns of two miRNAs and their potential targets were analysed in different developmental stages of oocytes and early embryos. This study provides the first miRNA profile in the mature process of yak oocyte. Seventy-five miRNAs are expressed differentially in GV and M II oocytes as well as among different development stages of early embryos, suggesting miRNAs involved in regulating oocyte maturation and early development of yak. These results showed specific miRNAs in yak oocytes had dynamic changes during meiosis. Further functional and mechanistic studies on the miRNAs during meiosis may beneficial to understanding the role of mi

Nanoliter droplet vitrification for oocyte cryopreservation

PubMed Central

Zhang, Xiaohui; Khimji, Imran; Shao, Lei; Safaee, Hooman; Desai, Khanjan; Keles, Hasan Onur; Gurkan, Umut Atakan; Kayaalp, Emre; Nureddin, Aida; Anchan, Raymond M; Maas, Richard L; Demirci, Utkan

2011-01-01

Aim Oocyte cryopreservation remains largely experimental, with live birth rates of only 2–4% per thawed oocyte. In this study, we present a nanoliter droplet technology for oocyte vitrification. Materials & methods An ejector-based droplet vitrification system was designed to continuously cryopreserve oocytes in nanoliter droplets. Oocyte survival rates, morphologies and parthenogenetic development after each vitrification step were assessed in comparison with fresh oocytes. Results Oocytes were retrieved after cryoprotectant agent loading/unloading, and nanoliter droplet encapsulation showed comparable survival rates to fresh oocytes after 24 h in culture. Also, oocytes recovered after vitrification/thawing showed similar morphologies to those of fresh oocytes. Additionally, the rate of oocyte parthenogenetic activation after nanoliter droplet encapsulation was comparable with that observed for fresh oocytes. This nanoliter droplet technology enables the vitrification of oocytes at higher cooling and warming rates using lower cryoprotectant agent levels (i.e., 1.4 M ethylene glycol, 1.1 M dimethyl sulfoxide and 1 M sucrose), thus making it a potential technology to improve oocyte cryopreservation outcomes. PMID:22188180

Skewed segregation of the mtDNA nt 8993 (T-->G) mutation in human oocytes.

PubMed Central

Blok, R B; Gook, D A; Thorburn, D R; Dahl, H H

1997-01-01

Rapid changes in mtDNA variants between generations have led to the bottleneck theory, which proposes a dramatic reduction in mtDNA numbers during early oogenesis. We studied oocytes from a woman with heteroplasmic expression of the mtDNA nt 8993 (T-->G) mutation. Of seven oocytes analyzed, one showed no evidence of the mutation, and the remaining six had a mutant load > 95%. This skewed expression of the mutation in oocytes is not compatible with the conventional bottleneck theory. A possible explanation is that, during amplification of mtDNA in the developing oocyte, mtDNA from one mitochondrion is preferentially amplified. Thus, subsequent mature oocytes may contain predominantly wild-type or mutant mitochondrial genomes. Images Figure 2 Figure 3 PMID:9199572

Identification of Maturation-Specific Proteins by Single-Cell Proteomics of Human Oocytes

PubMed Central

Virant-Klun, Irma; Leicht, Stefan; Hughes, Christopher; Krijgsveld, Jeroen

2016-01-01

Oocytes undergo a range of complex processes via oogenesis, maturation, fertilization, and early embryonic development, eventually giving rise to a fully functioning organism. To understand proteome composition and diversity during maturation of human oocytes, here we have addressed crucial aspects of oocyte collection and proteome analysis, resulting in the first proteome and secretome maps of human oocytes. Starting from 100 oocytes collected via a novel serum-free hanging drop culture system, we identified 2,154 proteins, whose function indicate that oocytes are largely resting cells with a proteome that is tailored for homeostasis, cellular attachment, and interaction with its environment via secretory factors. In addition, we have identified 158 oocyte-enriched proteins (such as ECAT1, PIWIL3, NLRP7)1 not observed in high-coverage proteomics studies of other human cell lines or tissues. Exploiting SP3, a novel technology for proteomic sample preparation using magnetic beads, we scaled down proteome analysis to single cells. Despite the low protein content of only ∼100 ng per cell, we consistently identified ∼450 proteins from individual oocytes. When comparing individual oocytes at the germinal vesicle (GV) and metaphase II (MII) stage, we found that the Tudor and KH domain-containing protein (TDRKH) is preferentially expressed in immature oocytes, while Wee2, PCNA, and DNMT1 were enriched in mature cells, collectively indicating that maintenance of genome integrity is crucial during oocyte maturation. This study demonstrates that an innovative proteomics workflow facilitates analysis of single human oocytes to investigate human oocyte biology and preimplantation development. The approach presented here paves the way for quantitative proteomics in other quantity-limited tissues and cell types. Data associated with this study are available via ProteomeXchange with identifier PXD004142. PMID:27215607

Identification of Maturation-Specific Proteins by Single-Cell Proteomics of Human Oocytes.

PubMed

Virant-Klun, Irma; Leicht, Stefan; Hughes, Christopher; Krijgsveld, Jeroen

2016-08-01

Oocytes undergo a range of complex processes via oogenesis, maturation, fertilization, and early embryonic development, eventually giving rise to a fully functioning organism. To understand proteome composition and diversity during maturation of human oocytes, here we have addressed crucial aspects of oocyte collection and proteome analysis, resulting in the first proteome and secretome maps of human oocytes. Starting from 100 oocytes collected via a novel serum-free hanging drop culture system, we identified 2,154 proteins, whose function indicate that oocytes are largely resting cells with a proteome that is tailored for homeostasis, cellular attachment, and interaction with its environment via secretory factors. In addition, we have identified 158 oocyte-enriched proteins (such as ECAT1, PIWIL3, NLRP7)(1) not observed in high-coverage proteomics studies of other human cell lines or tissues. Exploiting SP3, a novel technology for proteomic sample preparation using magnetic beads, we scaled down proteome analysis to single cells. Despite the low protein content of only ∼100 ng per cell, we consistently identified ∼450 proteins from individual oocytes. When comparing individual oocytes at the germinal vesicle (GV) and metaphase II (MII) stage, we found that the Tudor and KH domain-containing protein (TDRKH) is preferentially expressed in immature oocytes, while Wee2, PCNA, and DNMT1 were enriched in mature cells, collectively indicating that maintenance of genome integrity is crucial during oocyte maturation. This study demonstrates that an innovative proteomics workflow facilitates analysis of single human oocytes to investigate human oocyte biology and preimplantation development. The approach presented here paves the way for quantitative proteomics in other quantity-limited tissues and cell types. Data associated with this study are available via ProteomeXchange with identifier PXD004142. © 2016 by The American Society for Biochemistry and Molecular Biology

Clinical benefit of metaphase I oocytes

PubMed Central

Vanhoutte, Leen; De Sutter, Petra; Van der Elst, Josiane; Dhont, Marc

2005-01-01

Background We studied the benefit of using in vitro matured metaphase I (MI) oocytes for ICSI in patients with a maximum of 6 mature metaphase II (MII) oocytes at retrieval. Methods In 2004, 187 ICSI cycles were selected in which maximum 6 MII oocytes and at least one MI oocyte were retrieved. MI oocytes were put in culture to mature until the moment of ICSI, which was performed between 2 to 11 hours after oocyte retrieval (day 0). In exceptional cases, when the patient did not have any mature oocyte at the scheduled time of ICSI, MI oocytes were left to mature overnight and were injected between 19 to 26 hours after retrieval (day 1). Embryos from MI oocytes were chosen for transfer only when no other good quality embryos from MII oocytes were available. Outcome parameters were time period of in vitro maturation (IVM), IVM and fertilization rates, embryo development, clinical pregnancy rates, implantation rates and total MI oocyte utilization rate. Results The overall IVM rate was 43%. IVM oocytes had lower fertilization rates compared to in vivo matured sibling oocytes (52% versus 68%, P < 0.05). The proportion of poor quality embryos was significantly higher in IVM derived oocytes. One pregnancy and live birth was obtained out of 13 transfers of embryos exclusively derived from IVM oocytes. This baby originated from an oocyte that was injected after 22 hrs of IVM. Conclusion Fertilization of in vitro matured MI oocytes can result in normal embryos and pregnancy, making IVM worthwhile, particularly when few MII oocytes are obtained at retrieval. PMID:16356175

Cloning and characterization of a novel oocyte-specific gene encoding an F-Box protein in rainbow trout (Oncorhynchus mykiss)

USDA-ARS?s Scientific Manuscript database

Oocyte-specific genes play critical roles in oogenesis, folliculogenesis and early embryonic development. Through analysis of expressed sequence tags (ESTs) from a rainbow trout oocyte cDNA library, we identified a novel transcript which is represented by multiple ESTs derived only from the oocyte c...

Cloning and characterization of a novel oocyte-specific gene encoding an F-Box protein in rainbow trout (Oncorhynchus mykiss)

USDA-ARS?s Scientific Manuscript database

Oocyte-specific genes play critical roles in oogenesis, folliculogenesis and early embryonic development. Through analysis of expressed sequence tags (ESTs) from a rainbow trout oocyte cDNA library, we identified a novel transcript which is represented by ESTs only from the oocyte library. The novel...

Correlating microscopy techniques and ToF-SIMS analysis of fully grown mammalian oocytes.

PubMed

Gulin, Alexander; Nadtochenko, Victor; Astafiev, Artyom; Pogorelova, Valentina; Rtimi, Sami; Pogorelov, Alexander

2016-06-20

The 2D-molecular thin film analysis protocol for fully grown mice oocytes is described using an innovative approach. Time-of-flight secondary ion mass spectrometry (ToF-SIMS), scanning electron microscopy (SEM), atomic force microscopy (AFM) and optical microscopy imaging were applied to the same mice oocyte section on the same sample holder. A freeze-dried mice oocyte was infiltrated into embedding media, e.g. Epon, and then was cut with a microtome and 2 μm thick sections were transferred onto an ITO coated conductive glass. Mammalian oocytes can contain "nucleolus-like body" (NLB) units and ToF-SIMS analysis was used to investigate the NLB composition. The ion-spatial distribution in the cell components was identified and compared with the images acquired by SEM, AFM and optical microscopy. This study presents a significant advancement in cell embryology, cell physiology and cancer-cell biochemistry.

Direct real-time measurement of intra-oocyte nitric oxide concentration in vivo.

PubMed

Goud, Pravin T; Goud, Anuradha P; Najafi, Tohid; Gonik, Bernard; Diamond, Michael P; Saed, Ghassan M; Zhang, Xueji; Abu-Soud, Husam M

2014-01-01

Nitric oxide (NO) is reported to play significant a role in oocyte activation and maturation, implantation, and early embryonic development. Previously we have shown that NO forms an important component of the oocyte microenvironment, and functions effectively to delay oocyte aging. Thus, precise information about intra-oocyte NO concentrations [NO] will result in designing more accurate treatment plans in assisted reproduction. In this work, the direct, real-time and quantitative intra-oocyte [NO] was measured utilizing an L-shaped amperometric integrated NO-selective electrode. This method not only provides an elegant and convenient approach to real-time the measurement of NO in physiological environments, but also mimics the loss of NO caused by rapid NO diffusion combined with its reactivity in the biological milieu. This experiment suggests that the NO levels of oocytes obtained from young animals are significantly higher than those of oocytes obtained from old animals. Additionally the NO levels stay constant during the measurements; however, the intra-oocyte [NO] is reduced significantly (70-75% reduction) in response to L-NAME incubation, suggesting that NO measurements are truly NOS based rather than caused by an unknown interfering substance in our system. We believe this first demonstration of the direct quantitative measurement of [NO] in situ in an intact cellular complex should be useful in tracking real-time and rapid changes at nanomolar levels. Moreover, this finding confirms and extends our previous work showing that supplementation with NO delays the oocyte aging process.

Direct Real-Time Measurement of Intra-Oocyte Nitric Oxide Concentration In Vivo

PubMed Central

Goud, Pravin T.; Goud, Anuradha P.; Najafi, Tohid; Gonik, Bernard; Diamond, Michael P.; Saed, Ghassan M.; Zhang, Xueji; Abu-Soud, Husam M.

2014-01-01

Nitric oxide (NO) is reported to play significant a role in oocyte activation and maturation, implantation, and early embryonic development. Previously we have shown that NO forms an important component of the oocyte microenvironment, and functions effectively to delay oocyte aging. Thus, precise information about intra-oocyte NO concentrations [NO] will result in designing more accurate treatment plans in assisted reproduction. In this work, the direct, real-time and quantitative intra-oocyte [NO] was measured utilizing an L-shaped amperometric integrated NO-selective electrode. This method not only provides an elegant and convenient approach to real-time the measurement of NO in physiological environments, but also mimics the loss of NO caused by rapid NO diffusion combined with its reactivity in the biological milieu. This experiment suggests that the NO levels of oocytes obtained from young animals are significantly higher than those of oocytes obtained from old animals. Additionally the NO levels stay constant during the measurements; however, the intra-oocyte [NO] is reduced significantly (70–75% reduction) in response to L-NAME incubation, suggesting that NO measurements are truly NOS based rather than caused by an unknown interfering substance in our system. We believe this first demonstration of the direct quantitative measurement of [NO] in situ in an intact cellular complex should be useful in tracking real-time and rapid changes at nanomolar levels. Moreover, this finding confirms and extends our previous work showing that supplementation with NO delays the oocyte aging process. PMID:24887331

Ultrastructural Studies on Oocyte Development and Vitellogenesis associated with Follicle Cells in Female Scapharca subcrenata (Pelecypoda: Arcidae) in Western Korea

PubMed Central

Kim, Sung Han

2016-01-01

Ultrastructural studies on oocyte development and vitellogenesis in oocytes, and the functions of follicle cells during oogenesis and oocyte degeneration were investigated to clarifyb the reproductive mechanism on vitellogenesis of Scapharca subcrenata using electron microscope observations. In this study, vitellogenesis during oogenesis in the oocytes occured by way of autosynthesis and heterosynthesis. Of two processes of vitellogenesis during oogenesis, the process of endogenous autosynthesis involved the combined activity of the Golgi complex, mitochondria and rough endoplasmic reticulum. However, the process of exogenous heterosynthesis involved endocytotic incorporation of extraovarian precursors at the basal region of the oolema of the early vitellogenic oocytes before the formation of the vitelline coat. In this study, follicle cells, which attached to the previtellogenic and vitellogenic oocytes, were easily found. In particular, the follicle cells were involved in the development of previtellogenic oocytes by the supply of nutrients, and vitellogenesis in the early and late vitellogenic oocytes by endocytosis of yolk precursors. Based on observations of follicle cells attached to degenerating oocytes after spawning, follicles of this species are involved in lysosomal induction of oocyte degeneration for the resorption phagosomes (phagolysosomes) in the cytoplasm for nutrient storage, as seen in other bivalves. In this study, the functions of follicle cells can accumulate reserves of lipid granules and glycogen particles for vitellogenesis from degenerating oocytes after spawning. PMID:27796004

Cryopreserved oocyte versus fresh oocyte assisted reproductive technology cycles, United States, 2013.

PubMed

Crawford, Sara; Boulet, Sheree L; Kawwass, Jennifer F; Jamieson, Denise J; Kissin, Dmitry M

2017-01-01

To compare characteristics, explore predictors, and compare assisted reproductive technology (ART) cycle, transfer, and pregnancy outcomes of autologous and donor cryopreserved oocyte cycles with fresh oocyte cycles. Retrospective cohort study from the National ART Surveillance System. Fertility treatment centers. Fresh embryo cycles initiated in 2013 utilizing embryos created with fresh and cryopreserved, autologous and donor oocytes. Cryopreservation of oocytes versus fresh. Cancellation, implantation, pregnancy, miscarriage, and live birth rates per cycle, transfer, and/or pregnancy. There was no evidence of differences in cancellation, implantation, pregnancy, miscarriage, or live birth rates between autologous fresh and cryopreserved oocyte cycles. Donor cryopreserved oocyte cycles had a decreased risk of cancellation before transfer (adjusted risk ratio [aRR] 0.74, 95% confidence interval [CI] 0.57-0.96) as well as decreased likelihood of pregnancy (aRR 0.88, 95% CI 0.81-0.95) and live birth (aRR 0.87, 95% CI 0.80-0.95); however, there was no evidence of differences in implantation, pregnancy, or live birth rates when cycles were restricted to those proceeding to transfer. Donor cryopreserved oocyte cycles proceeding to pregnancy had a decreased risk of miscarriage (aRR 0.75, 95% CI 0.58-0.97) and higher live birth rate (aRR 1.05, 95% CI 1.01-1.09) with the transfer of one embryo, but higher miscarriage rate (aRR 1.28, 95% CI 1.07-1.54) and lower live birth rate (aRR 0.95, 95% CI 0.92-0.99) with the transfer of two or more. There was no evidence of differences in ART outcomes between autologous fresh and cryopreserved oocyte cycles. There was evidence of differences in per-cycle and per-pregnancy outcomes between donor cryopreserved and fresh oocyte cycles, but not in per-transfer outcomes. Published by Elsevier Inc.

Ovarian ageing: the role of mitochondria in oocytes and follicles.

PubMed

May-Panloup, Pascale; Boucret, Lisa; Chao de la Barca, Juan-Manuel; Desquiret-Dumas, Valérie; Ferré-L'Hotellier, Véronique; Morinière, Catherine; Descamps, Philippe; Procaccio, Vincent; Reynier, Pascal

2016-11-01

There is a great inter-individual variability of ovarian ageing, and almost 20% of patients consulting for infertility show signs of premature ovarian ageing. This feature, taken together with delayed childbearing in modern society, leads to the emergence of age-related ovarian dysfunction concomitantly with the desire for pregnancy. Assisted reproductive technology is frequently inefficacious in cases of ovarian ageing, thus raising the economic, medical and societal costs of the procedures. Ovarian ageing is characterized by quantitative and qualitative alteration of the ovarian oocyte reserve. Mitochondria play a central role in follicular atresia and could be the main target of the ooplasmic factors determining oocyte quality adversely affected by ageing. Indeed, the oocyte is the richest cell of the body in mitochondria and depends largely on these organelles to acquire competence for fertilization and early embryonic development. Moreover, the oocyte ensures the uniparental transmission and stability of the mitochondrial genome across the generations. This review focuses on the role played by mitochondria in ovarian ageing and on the possible consequences over the generations. PubMed was used to search the MEDLINE database for peer-reviewed original articles and reviews concerning mitochondria and ovarian ageing, in animal and human species. Searches were performed using keywords belonging to three groups: 'mitochondria' or 'mitochondrial DNA'; 'ovarian reserve', 'oocyte', 'ovary' or 'cumulus cells'; and 'ageing' or 'ovarian ageing'. These keywords were combined with other search phrases relevant to the topic. References from these articles were used to obtain additional articles. There is a close relationship, in mammalian models and humans, between mitochondria and the decline of oocyte quality with ageing. Qualitatively, ageing-related mitochondrial (mt) DNA instability, which leads to the accumulation of mtDNA mutations in the oocyte, plays a key role in

Microinjection of Follicle-Enclosed Mouse Oocytes

NASA Astrophysics Data System (ADS)

Jaffe, Laurinda A.; Norris, Rachael P.; Freudzon, Marina; Ratzan, William J.; Mehlmann, Lisa M.

The mammalian oocyte develops within a complex of somatic cells known as a follicle, within which signals from the somatic cells regulate the oocyte, and signals from the oocyte regulate the somatic cells. Because isolation of the oocyte from the follicle disrupts these communication pathways, oocyte physiology is best studied within an intact follicle. Here we describe methods for quantitative microinjection of follicle-enclosed mouse oocytes, thus allowing the introduction of signaling molecules as well as optical probes into the oocyte within its physiological environment.

Oocyte transport: Developmental competence of bovine oocytes arrested at germinal vesicle stage by cycloheximide under air.

PubMed

Hashimoto, Shu; Kimura, Kouji; Iwata, Hisataka; Takakura, Ryo

2003-02-01

The effects of the medium (TCM 199 or SOFaa) and temperature (20 or 39 C) during meiotic arrest by cycloheximide (CHX) under air on the developmental competence of bovine oocytes after in vitro maturation (IVM) and fertilization (IVF) were investigated. Oocytes were maintained in meiotic arrest by 10 microg/ml CHX in a 50-microl droplet of 25-mM HEPES-buffered TCM 199 (H199) at 39 C or synthetic oviduct fluid (HSOFaa) at 20 or 39 C in air for 24 h. After release from the arrest, the oocytes was matured and fertilized in vitro and their developmental competence was examined. The developmental rate of oocytes arrested in HSOFaa at 20 C to the blastocyst stage was similar to that of non-arrested oocytes but was significantly higher (P<0.05) than that of oocytes arrested at 39 C in H199 or in HSOFaa. In consideration of oocyte transport conditions, we also investigated the meiotic arrest of oocytes maintained in a 0.25-ml straw by CHX individually with 10 microl HSOFaa or as a group (40-50 oocytes) with 170-200 microl HSOFaa at 20 C in air for 24 h. After release from meiotic arrest, the developmental competence of these oocytes was assessed similarly. The developmental rate of oocytes treated with CHX individually was similar to that of those treated with CHX in 50-microl droplet of HSOFaa at 20 C. However, the developmental rate of oocytes treated with CHX as a group was lower than that of oocytes treated with CHX in a 50-microl droplet. Five blastocysts developed from oocytes maintained in meiotic arrest in a plastic straw were transferred to five recipient heifers. Consequently, three recipients became pregnant and 2 calves were delivered. The results of the present study indicate that bovine oocytes treated with CHX in HSOFaa at 20 C under air retain the same developmental competence as non-arrested oocytes.

Oocyte cryopreservation: where are we now?

PubMed

Argyle, Catrin E; Harper, Joyce C; Davies, Melanie C

2016-06-01

Since the first live birth from oocyte cryopreservation three decades ago, oocyte cryopreservation has become an important component of ART. Cryopreservation techniques have evolved, leading to higher success rates and the introduction of oocyte cryopreservation into IVF clinics worldwide. Concurrently, there has been an increase in patient demand, especially for so-called 'social egg freezing' that allows women to preserve their fertility in anticipation of age-related fertility decline. This review addresses a need to evaluate the current status of oocyte cryopreservation. It explores current techniques and success rates, clinical applications, the rise of elective oocyte cryopreservation, and future implications. A search was performed using Web of Science and PubMed databases for publications between January 1980 and December 2015. Keywords used included 'egg freezing', 'oocyte freezing', 'oocyte cryopreservation', 'oocyte vitrification', and 'fertility preservation'. The success rate of oocyte cryopreservation has risen, and the increasing use of vitrification offers has improved outcomes, with IVF pregnancy rates now similar to those achieved with fresh oocytes. There are conflicting opinions about the comparative success rates of open and closed vitrification. Patients are accessing and receiving oocyte cryopreservation for a wide range of indications, and there has been a marked increase in patient numbers and oocyte cryopreservation cycles. Oocyte cryopreservation for circumventing age-related infertility is becoming more widely accepted. Oocyte cryopreservation is an established component of ART, with vitrification now being the cryopreservation technique of choice. Increasing numbers of women undergo oocyte cryopreservation for both medical and social reasons. It is important to continue auditing outcomes and reporting long-term follow-up of children born from frozen-thawed oocytes. © The Author 2016. Published by Oxford University Press on behalf of

Human oocyte cryopreservation.

PubMed

Tao, Tao; Zhang, Wenling; Del Valle, Alfonso

2009-06-01

This review summarized the clinical breakthroughs in the human oocyte cryopreservation field in the past 2 years and gave special emphasis on the role of vitrification method. Human oocyte cryopreservation is an attractive strategy to preserve female fertility, as it offers more opportunities to the future destination of the female gametes and also raises fewer legal and ethical questions compared with embryo cryopreservation. It became promising in recent years because of dramatic improvement in cryopreservation technologies. Human oocyte cryopreservation would not become a clinical routine until the availability of reliable cryopreservation methods and long-term follow-up results of the babies born by this technique. Oocyte cryopreservation produced very exciting results with pregnancy and implantation rates comparable to embryo cryopreservation and in some cases comparable to fresh in-vitro fertilization cycles with both modified slow-freezing and vitrification methods. A cancer patient conceived and delivered her own babies by this technology after recovery from the disease. Oocyte cryopreservation became a new focus in assisted reproductive technology. We witnessed the advanced development of human oocyte cryopreservation in the past years because of increasing demand, medically, legally and ethically, and also because of the dramatic improvement of the freezing technique. There is still a long way to go to integrate it into a routine clinical procedure to benefit more patients and encourage clinicians to follow the standard protocols.

Cyclin B in mouse oocytes and embryos: importance for human reproduction and aneuploidy.

PubMed

Polański, Zbigniew; Homer, Hayden; Kubiak, Jacek Z

2012-01-01

Oocyte maturation and early embryo development require precise coordination between cell cycle progression and the developmental programme. Cyclin B plays a major role in this process: its accumulation and degradation is critical for driving the cell cycle through activation and inactivation of the major cell cycle kinase, CDK1. CDK1 activation is required for M-phase entry whereas its inactivation leads to exit from M-phase. The tempo of oocyte meiotic and embryonic mitotic divisions is set by the rate of cyclin B accumulation and the timing of its destruction. By controlling when cyclin B destruction is triggered and by co-ordinating this with the completion of chromosome alignment, the spindle assembly checkpoint (SAC) is a critical quality control system important for averting aneuploidy and for building in the flexibility required to better integrate cell cycle progression with development. In this review we focus on cyclin B metabolism in mouse oocytes and embryos and illustrate how the cell cycle-powered clock (in fact cyclin B-powered clock) controls oocyte maturation and early embryo development, thereby providing important insight into human reproduction and potential causes of Down syndrome.

Cryopreserved oocyte versus fresh oocyte assisted reproductive technology cycles, United States, 2013

PubMed Central

Crawford, Sara; Boulet, Sheree L.; Kawwass, Jennifer F.; Jamieson, Denise J.; Kissin, Dmitry M.

2017-01-01

Objective To compare characteristics, explore predictors, and compare assisted reproductive technology (ART) cycle, transfer, and pregnancy outcomes of autologous and donor cryopreserved oocyte cycles with fresh oocyte cycles. Design Retrospective cohort study from the National ART Surveillance System. Setting Fertility treatment centers. Patient(s) Fresh embryo cycles initiated in 2013 utilizing embryos created with fresh and cryopreserved, autologous and donor oocytes. Intervention(s) Cryopreservation of oocytes versus fresh. Main Outcomes Measure(s) Cancellation, implantation, pregnancy, miscarriage, and live birth rates per cycle, transfer, and/or pregnancy. Result(s) There was no evidence of differences in cancellation, implantation, pregnancy, miscarriage, or live birth rates between autologous fresh and cryopreserved oocyte cycles. Donor cryopreserved oocyte cycles had a decreased risk of cancellation before transfer (adjusted risk ratio [aRR] 0.74, 95% confidence interval [CI] 0.57–0.96) as well as decreased likelihood of pregnancy (aRR 0.88, 95% CI 0.81–0.95) and live birth (aRR 0.87, 95% CI 0.80–0.95); however, there was no evidence of differences in implantation, pregnancy, or live birth rates when cycles were restricted to those proceeding to transfer. Donor cryopreserved oocyte cycles proceeding to pregnancy had a decreased risk of miscarriage (aRR 0.75, 95% CI 0.58–0.97) and higher live birth rate (aRR 1.05, 95% CI 1.01–1.09) with the transfer of one embryo, but higher miscarriage rate (aRR 1.28, 95% CI 1.07–1.54) and lower live birth rate (aRR 0.95, 95% CI 0.92–0.99) with the transfer of two or more. Conclusion(s) There was no evidence of differences in ART outcomes between autologous fresh and cryopreserved oocyte cycles. There was evidence of differences in per-cycle and per-pregnancy outcomes between donor cryopreserved and fresh oocyte cycles, but not in per-transfer outcomes. PMID:27842997

Transcriptome analyses of rhesus monkey preimplantation embryos reveal a reduced capacity for DNA double-strand break repair in primate oocytes and early embryos

PubMed Central

Wang, Xinyi; Liu, Denghui; He, Dajian; Suo, Shengbao; Xia, Xian; He, Xiechao; Han, Jing-Dong J.; Zheng, Ping

2017-01-01

Preimplantation embryogenesis encompasses several critical events including genome reprogramming, zygotic genome activation (ZGA), and cell-fate commitment. The molecular basis of these processes remains obscure in primates in which there is a high rate of embryo wastage. Thus, understanding the factors involved in genome reprogramming and ZGA might help reproductive success during this susceptible period of early development and generate induced pluripotent stem cells with greater efficiency. Moreover, explaining the molecular basis responsible for embryo wastage in primates will greatly expand our knowledge of species evolution. By using RNA-seq in single and pooled oocytes and embryos, we defined the transcriptome throughout preimplantation development in rhesus monkey. In comparison to archival human and mouse data, we found that the transcriptome dynamics of monkey oocytes and embryos were very similar to those of human but very different from those of mouse. We identified several classes of maternal and zygotic genes, whose expression peaks were highly correlated with the time frames of genome reprogramming, ZGA, and cell-fate commitment, respectively. Importantly, comparison of the ZGA-related network modules among the three species revealed less robust surveillance of genomic instability in primate oocytes and embryos than in rodents, particularly in the pathways of DNA damage signaling and homology-directed DNA double-strand break repair. This study highlights the utility of monkey models to better understand the molecular basis for genome reprogramming, ZGA, and genomic stability surveillance in human early embryogenesis and may provide insights for improved homologous recombination-mediated gene editing in monkey. PMID:28223401

What does the cryopreserved oocyte look like? A fresh look at the characteristic oocyte features following cryopreservation.

PubMed

Hosseini, Sayyed Morteza; Nasr-Esfahani, Mohammad Hossein

2016-04-01

In October 2012, the American Society for Reproductive Medicine (ASRM) and, in March 2012, the European Society of Human Reproduction and Embryology (ESHRE), lifted the categorization of oocyte cryopreservation as being "experimental" and endorsed its entrance into the mainstream of assisted reproductive techniques. This change in policy, with the considerable advantages that oocytes offer over embryos for cryopreservation, has increased applications of oocyte cryopreservation in assisted reproduction techniques. A deep understanding of oocyte cryobiology, however, is lagging behind the forces propelling the clinical application of oocyte cryopreservation. We have drawn attention to this shortcoming by initiating a debate on whether a vitrified-warmed oocyte has the same characteristics as its fresh sibling. The answer to this question may explain why the oocyte cryopreservation success rate is as yet far from satisfactory and why cryopreserved oocytes should be treated differently from their fresh siblings. A fresh look at the characteristic features of oocytes after cryopreservation is the main scope of this review as a stimulus to further improvement of oocyte cryopreservation. Copyright © 2016. Published by Elsevier Ltd.

Polypeptide profiles of human oocytes and preimplantation embryos.

PubMed

Capmany, G; Bolton, V N

1993-11-01

The polypeptides that direct fertilization and early development until activation of the embryonic genome occurs, at the 4-8 cell stage in the human, are exclusively maternal in origin, and are either synthesized during oogenesis or translated later from maternal mRNA. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and silver stain, we have visualized and compared the polypeptides present in different populations of human oocytes and cleavage stage embryos obtained after superovulation and insemination in vitro. Two polypeptide patterns were resolved, differing in the region of mol. wt 69 kDa. The distribution of these patterns showed no correlation with the ability of individual oocytes to achieve fertilization and develop normally to the 8-cell stage.

Oocyte glutathione and fertilisation outcome of Macaca nemestrina and Macaca fascicularis in in vivo- and in vitro-matured oocytes.

PubMed

Curnow, E C; Ryan, J P; Saunders, D M; Hayes, E S

2010-01-01

Fertilisation and development of IVM non-human primate oocytes is limited compared with that of in vivo-matured (IVO) oocytes. The present study describes the IVM of macaque oocytes with reference to oocyte glutathione (GSH). Timing of maturation, comparison of IVM media and cysteamine (CYS) supplementation as a modulator of GSH were investigated. A significantly greater proportion of oocytes reached MII after 30 h compared with 24 h of IVM. Following insemination, IVM oocytes had a significantly lower incidence of normal fertilisation (i.e. 2PN = two pronuclei and at least one polar body) and a higher rate of abnormal fertilisation (1PN = one pronucleus and at least one polar body) compared with IVO oocytes. Immunofluorescence of 1PN zygotes identified incomplete sperm head decondensation and failure of male pronucleus formation as the principal cause of abnormal fertilisation in IVM oocytes. The IVO oocytes had significantly higher GSH content than IVM oocytes. Cumulus-denuded oocytes had significantly lower GSH following IVM compared with immature oocytes at collection. Cysteamine supplementation of the IVM medium significantly increased the GSH level of cumulus-intact oocytes and reduced the incidence of 1PN formation, but did not improve GSH levels of the denuded oocyte. Suboptimal GSH levels in macaque IVM oocytes may be related to reduced fertilisation outcomes.

An oocyte-specific ELAVL2 isoform is a translational repressor ablated from meiotically competent antral oocytes

PubMed Central

Chalupnikova, Katerina; Solc, Petr; Sulimenko, Vadym; Sedlacek, Radislav; Svoboda, Petr

2014-01-01

At the end of the growth phase, mouse antral follicle oocytes acquire full developmental competence. In the mouse, this event is marked by the transition from the so-called non-surrounded nucleolus (NSN) chromatin configuration into the transcriptionally quiescent surrounded nucleolus (SN) configuration, which is named after a prominent perinucleolar condensed chromatin ring. However, the SN chromatin configuration alone is not sufficient for determining the developmental competence of the SN oocyte. There are additional nuclear and cytoplamic factors involved, while a little is known about the changes occurring in the cytoplasm during the NSN/SN transition. Here, we report functional analysis of maternal ELAVL2 an AU-rich element binding protein. Elavl2 gene encodes an oocyte-specific protein isoform (denoted ELAVL2°), which acts as a translational repressor. ELAVL2° is abundant in fully grown NSN oocytes, is ablated during the NSN/SN transition and remains low during the oocyte-to-embryo transition (OET). ELAVL2° overexpression during meiotic maturation causes errors in chromosome segregation, indicating the significance of naturally reduced ELAVL2° levels in SN oocytes. On the other hand, during oocyte growth, prematurely reduced Elavl2 expression results in lower yields of fully grown and meiotically matured oocytes, suggesting that Elavl2 is necessary for proper oocyte maturation. Moreover, Elavl2 knockdown showed stimulating effects on translation in fully grown oocytes. We propose that ELAVL2 has an ambivalent role in oocytes: it functions as a pleiotropic translational repressor in efficient production of fully grown oocytes, while its disposal during the NSN/SN transition contributes to the acquisition of full developmental competence. PMID:24553115

Serum human chorionic gonadotropin levels on the day before oocyte retrieval do not correlate with oocyte maturity.

PubMed

Levy, Gary; Hill, Micah J; Ramirez, Christina; Plowden, Torrie; Pilgrim, Justin; Howard, Robin S; Segars, James H; Csokmay, John

2013-05-01

To evaluate the correlation of preretrieval quantitative serum hCG level with oocyte maturity. Retrospective cohort study. Military assisted reproductive technology (ART) program. Fresh autologous ART cycles. Serum hCG level the day before oocyte retrieval. Linear regression was used to correlate serum hCG levels and oocyte maturity rates. Normal oocyte maturity was defined as ≥75% and the Wilcoxon rank sum test was used to compare serum hCG levels in patients with normal and low oocyte maturity. Threshold analysis was performed to determine hCG levels that could predict oocyte maturity. A total of 468 ART cycles were analyzed. Serum hCG level was not correlated with hCG dose; however, it was negatively correlated with body mass index (BMI). Serum hCG levels did not differ between patients with oocyte maturity of <75% and ≥75%. Serum hCG levels did not correlate with oocyte maturity rates. Receiver operator characteristic and less than efficiency curves failed to demonstrate thresholds at which hCG could predict oocyte maturity. Serum hCG levels were not correlated with oocyte maturity. Although a positive hCG was reassuring that mature oocytes would be retrieved for most patients, the specific value was not helpful. Copyright © 2013. Published by Elsevier Inc.

Ovarian and oocyte cryopreservation.

PubMed

Lornage, Jacqueline; Salle, Bruno

2007-08-01

The present article is an update on progress in the two available techniques of oocyte and ovarian cryopreservation: slow cooling/rapid thawing and vitrification. A new line of research has opened in recent years: freezing the whole ovary with its vascular pedicle, so as to enable vascular grafts limiting ischemia-related follicle reserve loss. The technique of mature oocyte vitrification has advanced significantly, with improved oocyte physiology, increased safety, and higher clinical pregnancy rates. The number of studies on whole ovary freezing has grown, and there has been a large-mammal (sheep) live birth by orthotopic graft with vascular anastomosis of a cryopreserved ovary. Ovarian and oocyte cryopreservation is essential to conserving the fertility of young women. Results of mature oocyte freezing techniques have improved significantly over the past few years, but remain poorer than those with embryo freezing. Mature oocyte vitrification is progressing well, but requires safety validation in view of the high cryoprotectant concentrations used. Ovarian cortex fragment freezing is widely used in patients, with two live births after orthotopic graft, worldwide. The problem of rapid graft exhaustion has led to a focus on whole ovary cryopreservation which has resulted in one live birth in a ewe.

Oocyte glutathione and fertilisation outcome of Macaca nemestrina and Macaca fascicularis in in vivo- and in vitro-matured oocytes

PubMed Central

Curnow, E. C.; Ryan, J. P.; Saunders, D. M.; Hayes, E. S.

2010-01-01

Fertilisation and development of IVM non-human primate oocytes is limited compared with that of in vivo-matured (IVO) oocytes. The present study describes the IVM of macaque oocytes with reference to oocyte glutathione (GSH). Timing of maturation, comparison of IVM media and cysteamine (CYS) supplementation as a modulator of GSH were investigated. A significantly greater proportion of oocytes reached MII after 30 h compared with 24 h of IVM. Following insemination, IVM oocytes had a significantly lower incidence of normal fertilisation (i.e. 2PN = two pronuclei and at least one polar body) and a higher rate of abnormal fertilisation (1PN = one pronucleus and at least one polar body) compared with IVO oocytes. Immunofluorescence of 1PN zygotes identified incomplete sperm head decondensation and failure of male pronucleus formation as the principal cause of abnormal fertilisation in IVM oocytes. The IVO oocytes had significantly higher GSH content than IVM oocytes. Cumulus-denuded oocytes had significantly lower GSH following IVM compared with immature oocytes at collection. Cysteamine supplementation of the IVM medium significantly increased the GSH level of cumulus-intact oocytes and reduced the incidence of 1PN formation, but did not improve GSH levels of the denuded oocyte. Suboptimal GSH levels in macaque IVM oocytes may be related to reduced fertilisation outcomes. PMID:20591337

Evidence for an absence of deleterious effects of ultrasound on human oocytes.

PubMed

Mahadevan, M; Chalder, K; Wiseman, D; Leader, A; Taylor, P J

1987-10-01

Animal and human data would suggest that ultrasound causes deleterious effects to oocytes during meiosis. We directly compared the fertilization rate and embryonic development following in vitro fertilization and embryo transfer of those oocytes exposed to ultrasound and those not exposed in the same patient. In 39 unscreened patients a combination of laparoscopy and ultrasound was used for oocyte recovery. Laparoscopy was performed first on the most accessible ovary (usually the right) and at least one oocyte was obtained. Ultrasound-guided oocyte recovery was successful in the other inaccessible ovary. To assess how oocytes obtained by ultrasound or laparoscopy related to the pregnancy rate, two groups of patients were evaluated in whom the embryos transferred either had been exposed to ultrasound or had not been. The fertilization and the embryo cleavage rates were not significantly different between the ultrasound-exposed and the unexposed groups. The pregnancy rate was also not significantly different [9 of 49 (18.4%) for ultrasound exposed versus 14 of 74 (18.9%) for unexposed]. There was one early spontaneous abortion in each group. Further analysis of a group of 40 patients, in whom the oocytes were exposed to ultrasound in situ, after the endogenous luteinizing hormone (LH) surge had begun 1-27 hr earlier, revealed that 6 became pregnant (15%). This preliminary study suggests that exposure of human oocytes to ultrasonic waves, either during the different phases of meiosis or after the completion of meiosis, did not significantly influence the developmental potential of the in vitro fertilized embryos.

Serum human chorionic gonadotropin levels on the day before oocyte retrieval do not correlate with oocyte maturity

PubMed Central

Levy, Gary; Hill, Micah J.; Ramirez, Christina; Plowden, Torrie; Pilgrim, Justin; Howard, Robin S.; Segars, James H.; Csokmay, John

2014-01-01

Objective To evaluate the correlation of preretrieval quantitative serum hCG level with oocyte maturity. Design Retrospective cohort study. Setting Military assisted reproductive technology (ART) program. Patient(s) Fresh autologous ART cycles. Intervention(s) Serum hCG level the day before oocyte retrieval. Main Outcome Measure(s) Linear regression was used to correlate serum hCG levels and oocyte maturity rates. Normal oocyte maturity was defined as ≥ 75% and the Wilcoxon rank sum test was used to compare serum hCG levels in patients with normal and low oocyte maturity. Threshold analysis was performed to determine hCG levels that could predict oocyte maturity. Result(s) A total of 468 ART cycles were analyzed. Serum hCG level was not correlated with hCG dose; however, it was negatively correlated with body mass index (BMI). Serum hCG levels did not differ between patients with oocyte maturity of <75% and ≥ 75%. Serum hCG levels did not correlate with oocyte maturity rates. Receiver operator characteristic and less than efficiency curves failed to demonstrate thresholds at which hCG could predict oocyte maturity. Conclusion(s) Serum hCG levels were not correlated with oocyte maturity. Although a positive hCG was reassuring that mature oocytes would be retrieved for most patients, the specific value was not helpful. PMID:23375205

Zona-free oocyte fertilized with intracytoplasmic sperm injection and underwent further division: case report and literature review.

PubMed

Hsieh, Y Y; Chang, C C; Tsai, H D

2001-09-01

The zona pellucida (ZP) plays a protective role during fertilization and early embryonic development. It is related to sperm binding, the acrosome reaction, prevention of polyspermic fertilization, and holding blastomeres together before the morular stage. Zona-free oocytes are accidentally encountered. If these oocytes are healthy, they can be fertilized normally by intracytoplasmic sperm injection (ICSI). We reported on a couple with male infertility undergoing oocyte retrieval after ovarian hyperstimulation. Before the ICSI procedure, cumulus cells surrounding the oocytes were removed, which resulted in one oocyte escaping from its ZP. The zona-free oocyte was fertilized normally with ICSI and developed to the 8-cell stage. We observed that the zona-free zygote had the ability to further divide, despite its loose contact. The zona-free embryo was transferred with other zona-intact embryos, but the implantation failed. We conclude that zona-free oocytes can be rescued, fertilized with ICSI, and cultured for further transfer or cryopreservation.

Ultrastructural characteristics of the follicle cell-oocyte interface in the oogenesis of Ceratophrys cranwelli.

PubMed

Villecco, Evelina I; Genta, Susana B; Sánchez Riera, Alicia N; Sánchez, Sara S

2002-05-01

In this work we carried out an ultrastructural analysis of the cell interface between oocyte and follicle cells during the oogenesis of the amphibian Ceratophrys cranwelli, which revealed a complex cell-cell interaction. In the early previtellogenic follicles, the plasma membrane of the follicle cells lies in close contact with the plasma membrane of the oocyte, with no interface between them. In the mid-previtellogenic follicles the follicle cells became more active and their cytoplasm has vesicles containing granular material. Their apical surface projects cytoplasmic processes (macrovilli) that contact the oocyte, forming gap junctions. The oocyte surface begins to develop microvilli. At the interface both processes delimit lacunae containing granular material. The oocyte surface has endocytic vesicles that incorporate this material, forming cortical vesicles that are peripherally arranged. In the late previtellogenic follicle the interface contains fibrillar material from which the vitelline envelope will originate. During the vitellogenic period, there is an increase in the number and length of the micro- and macrovilli, which become regularly arranged inside fibrillar tunnels. At this time the oocyte surface exhibits deep crypts where the macrovilli enter, thus increasing the follicle cell-oocyte junctions. In addition, the oocyte displays coated pits and vesicles evidencing an intense endocytic activity. At the interface of the fully grown oocyte the fibrillar network of the vitelline envelope can be seen. The compact zone contains a fibrillar electron-dense material that fills the spaces previously occupied by the now-retracted microvilli. The macrovilli are still in contact with the surface of the oocyte, forming gap junctions.

Ovarian Grafts 10 Days after Xenotransplantation: Folliculogenesis and Recovery of Viable Oocytes

PubMed Central

Campos-Junior, Paulo Henrique Almeida; Alves, Thalys Jair Melo; Dias, Marco Tulio; Assunçao, Carolina Marinho; Munk, Michele; Mattos, Matheus Silvério; Kraemer, Lucas Rocha; Almeida, Brígida Gomes; Russo, Remo Castro; Barcelos, Lucíola; Camargo, Luiz Sérgio Almeida; Viana, Joao Henrique Moreira

2016-01-01

Ovarian xenotransplantation is a promising alternative to preserve fertility of oncologic patients. However, several functional aspects of this procedure remained to be addressed. The aim of this study was evaluate the feasibility of xenotransplantation as a strategy to maintain bovine ovarian grafts and produce oocytes. Adult ovarian cortical pieces were xenotransplanted to the dorsal subcutaneous of female NOD-SCID mice (n = 62). Grafts were recovered ten days after xenotransplantation. Host and graft weights; folliculogenesis progression; blood perfusion, relative gene expression and number of macrophage and neutrophil of xenografts; in vitro developmental competence of graft-derived oocytes were evaluated. Folliculogenesis was supported in the grafts, as indicated by the presence of primordial, primary, secondary, antral, and atretic follicles. The xenografts showed a greater volumetric density of atretic follicles and higher hyperemia and number of host-derived macrophage and neutrophil (P<0.05), when compared to non-grafted fragments. There was a higher blood perfusion under the back skin in the transplantation sites of host animals than in control and non-grafted (P<0.01). BAX and PRDX1 genes were up-regulated, while BCL2, FSHR, IGF1R and IGF2R were down-regulated, when compared to the control (P<0.01). Twenty seven oocytes were successfully harvested from grafts, and some of these oocytes were able to give rise to blastocysts after in vitro fertilization. However, cleavage and blastocyst rates of xenograft derived oocytes were lower than in control (P<0.01). Despite showing some functional modifications, the ovarian xenografts were able to support folliculogenesis and produce functional oocytes. PMID:27362486

Cryo-survival, fertilization and early embryonic development of vitrified oocytes derived from mice of different reproductive age

PubMed Central

Yan, Jie; Suzuki, Joao; Yu, Xiaomin; Kan, Frederick W. K.

2010-01-01

Purpose To evaluate the effect of female reproductive age on oocyte cryo-survival, fertilization and the subsequent embryonic development following vitrification using the mouse model in order to address the question of how maternal reproductive age is related to fertility preservation. Methods Oocytes were collected from mice of different reproductive age: (1) 8–10 weeks, (2) 16–20 weeks, (3) 32–36 weeks, and (4) 44–48 weeks. Following vitrification and warming, the oocytes in each group were assessed for cryo-survival, fertilization and embryonic development as well as for the quality of blastocysts. Fresh oocytes without undergoing vitrification were used in each age group as controls. Results The mean number of oocytes retrieved following superovulation was found to reduce significantly (P < 0.05) in mice from 32–36 weeks of age (18.1 ± 8.5) compared with 8–10 weeks of age (26.8 ± 9.8) and 16–20 weeks of age (23.9 ± 4.2) respectively. The cryo-survival rate of oocytes was reduced significantly (P < 0.05) in mice of 44–48 weeks of age (90.4% ± 7.9) compared with the other 3 groups (98.8% ± 2.1, 98.0% ± 3.3 and 98.5% ± 2.2, respectively). The cleavage rate of vitrified oocytes declined significantly following the increase in maternal age in mice of 32–36 weeks of age (69.7% ± 20.8) forward (63.6% ± 9.2). However, no significant difference in the cleavage rate was found among the control groups of different maternal ages. The rate of embryo development to the blastocyst stage in the vitrified oocytes also significantly declined following the increase in maternal age (71.8% ± 8.8, 66.4% ± 10.7, 64.2% ± 17.4 and 4.1% ± 8.3 respectively). There were no such differences in the rates of embryo development to the blastocyst stage among the control groups following the increase in maternal age (75.9% ± 12.2, 79.5% ± 28.9, 70.2% ± 17.4 and 69.3% ± 19

Human oocyte developmental potential is predicted by mechanical properties within hours after fertilization

PubMed Central

Yanez, Livia Z.; Han, Jinnuo; Behr, Barry B.; Pera, Renee A. Reijo; Camarillo, David B.

2016-01-01

The causes of embryonic arrest during pre-implantation development are poorly understood. Attempts to correlate patterns of oocyte gene expression with successful embryo development have been hampered by the lack of reliable and nondestructive predictors of viability at such an early stage. Here we report that zygote viscoelastic properties can predict blastocyst formation in humans and mice within hours after fertilization, with >90% precision, 95% specificity and 75% sensitivity. We demonstrate that there are significant differences between the transcriptomes of viable and non-viable zygotes, especially in expression of genes important for oocyte maturation. In addition, we show that low-quality oocytes may undergo insufficient cortical granule release and zona-hardening, causing altered mechanics after fertilization. Our results suggest that embryo potential is largely determined by the quality and maturation of the oocyte before fertilization, and can be predicted through a minimally invasive mechanical measurement at the zygote stage. PMID:26904963

Deoxynivalenol exposure induces autophagy/apoptosis and epigenetic modification changes during porcine oocyte maturation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Jun; Wang, Qiao-Chu; Zhu, Cheng-Cheng

Deoxynivalenol (DON) is a widespread trichothecene mycotoxin which contaminates agricultural staples and elicits a complex spectrum of toxic effects on humans and animals. It has been shown that DON impairs oocyte maturation, reproductive function and causes abnormal fetal development in mammals; however, the mechanisms remain unclear. In the present study, we investigate the possible reasons of the toxic effects of DON on porcine oocytes. Our results showed that DON significantly inhibited porcine oocyte maturation and disrupted meiotic spindle by reducing p-MAPK protein level, which caused retardation of cell cycle progression. In addition, up-regulated LC3 protein expression and aberrant Lamp2, LC3more » and mTOR mRNA levels were observed with DON exposure, together with Annexin V-FITC staining assay analysis, these results indicated that DON treatment induced autophagy/apoptosis in porcine oocytes. We also showed that DON exposure increased DNA methylation level in porcine oocytes through altering DNMT3A mRNA levels. Histone methylation levels were also changed showing with increased H3K27me3 and H3K4me2 protein levels, and mRNA levels of their relative methyltransferase genes, indicating that epigenetic modifications were affected. Taken together, our results suggested that DON exposure reduced porcine oocytes maturation capability through affecting cytoskeletal dynamics, cell cycle, autophagy/apoptosis and epigenetic modifications. - Highlights: • DON exposure disrupted meiotic spindle by reducing p-MAPK expression. • DON exposure caused retardation of cell cycle progression in porcine oocytes. • DON triggered autophagy and early-apoptosis in porcine oocytes. • DON exposure led to aberrant epigenetic modifications in porcine oocytes.« less

Detailed lipid analysis of yolk platelets of amphibian (Bufo arenarum) oocytes.

PubMed

Buschiazzo, Jorgelina; Bruzzone, Ariana; Alonso, Telma Susana

2003-06-01

Yolk platelets, the principal components of amphibian oocytes, have been generally considered as material reservoirs. Their biochemical composition and function during oogenesis and early development have not been fully elucidated. The aim of this study was to carry out a lipidic characterization of yolk platelets from full-grown Bufo arenarum oocytes. Ovarian oocytes were manually obtained and the subcellular fraction was isolated by centrifugation at low velocity. Lipids were separated by thin-layer chromatography. For compositional analysis, they were derived by methanolysis, being identified and quantified in a gas-liquid chromatograph. Phospholipid content indicates that phosphatidylcholine and phosphatidylethanolamine are the main phospholipids followed by phosphatidylinositol, sphingomyelin, phosphatidylserine, and phosphatidic acid. Phospholipidic profile is similar to that in whole oocytes except for the absence of diphosphatidylglycerol in yolk platelets. Oleic, palmitic, and linoleic acids are the main fatty acids in phosphatidylcholine, and oleic acid is the principal one in phosphatidylethanolamine. In phosphatidic acid, palmitic, estearic, palmitoleic, and oleic acids represent 68 mol% of the total acyl groups. Phosphatidylinositol, enriched in arachidonic acid, is the most unsaturated phospholipid while sphingomyelin shows the lowest unsaturation index. The acyl group distribution in triacylglycerols is similar when yolk platelets and whole oocytes are compared. Polar and neutral lipids of yolk platelets determine the lipidic profile of the whole oocyte. The presence of unusual fatty acids as 14:0, 15:0, 15:1, 17:0, and 17:1 in phospholipids and triacylglycerols may indicate an oxidation mechanism different from beta-oxidation in yolk platelets and/or a structural and functional relation with mitochondria. Given that yolk platelets in amphibian oocytes may act in a dynamic fashion in development, their role should be reconsidered.

Obesity adversely impacts the number and maturity of oocytes in conventional IVF not in minimal stimulation IVF.

PubMed

Zhang, John J; Feret, Maciej; Chang, Lyndon; Yang, Mingxue; Merhi, Zaher

2015-05-01

The objective of this study was to assess the relationship between BMI and oocyte number and maturity in participants who underwent minimal stimulation (mini-) or conventional IVF. Participants who underwent their first autologous cycle of either conventional (n = 219) or mini-IVF (n = 220) were divided according to their BMI to analyze IVF outcome parameters. The main outcome measure was the number of oocytes in metaphase II (MII). Secondary outcomes included the number of total oocytes retrieved, fertilized (2PN) oocytes, cleavage and blastocyst stage embryos, clinical pregnancy (CP), and live birth (LB) rates. In conventional IVF, but not in mini-IVF, the number of total oocytes retrieved (14.5  ±  0.8 versus 8.8  ±  1.3) and MII oocytes (11.2 ± 0.7 versus 7.1 ± 1.1) were significantly lower in obese compared with normal BMI women. Multivariable linear regression adjusting for age, day 3 FSH, days of stimulation, and total gonadotropin dose demonstrated that BMI was an independent predictor of the number of MII oocytes in conventional IVF (p = 0.0004). Additionally, only in conventional IVF, BMI was negatively correlated with the total number of 2PN oocytes, as well as the number of cleavage stage embryos. Female adiposity might impair oocyte number and maturity in conventional IVF but not in mini-IVF. These data suggest that mild ovarian stimulation might yield healthier oocytes in obese women.

Growing Mouse Oocytes Transiently Activate Folate Transport via Folate Receptors As They Approach Full Size.

PubMed

Meredith, Megan; MacNeil, Allison H; Trasler, Jacquetta M; Baltz, Jay M

2016-06-01

The folate cycle is central to cellular one-carbon metabolism, where folates are carriers of one-carbon units that are critical for synthesis of purines, thymidylate, and S-adenosylmethionine, the universal methyl donor that forms the cellular methyl pool. Although folates are well-known to be important for early embryo and fetal development, their role in oogenesis has not been clearly established. Here, folate transport proteins were detected in developing neonatal ovaries and growing oocytes by immunohistochemistry, Western blot, and immunofluorescence. The folate receptors FOLR1 and FOLR2 as well as reduced folate carrier 1 (RFC1, SLC19A1 protein) each appeared to be present in follicular cells including granulosa cells. In growing oocytes, however, only FOLR2 immunoreactivity appeared abundant. Localization of apparent FOLR2 immunofluorescence near the plasma membrane increased with oocyte growth and peaked in oocytes as they neared full size. We assessed folate transport using the model folate leucovorin (folinic acid). Unexpectedly, there was a transient burst of folate transport activity for a brief period during oocyte growth as they neared full size, while folate transport was otherwise undetectable for the rest of oogenesis and in fully grown germinal vesicle stage oocytes. This folate transport was inhibited by dynasore, an inhibitor of endocytosis, but insensitive to the anion transport inhibitor stilbene 4-acetamido-40-isothiocyanato-stilbene-2,20-disulfonic acid, consistent with folate receptor-mediated transport but not with RFC1-mediated transport. Thus, near the end of their growth, growing oocytes may take up folates that could support the final stage of oogenesis or be stored to provide the endogenous folates needed in early embryogenesis. © 2016 by the Society for the Study of Reproduction, Inc.

Both nuclear and cytoplasmic components are defective in oocytes of the B6.Y(TIR) sex-reversed female mouse.

PubMed

Amleh, A; Smith, L; Chen, H; Taketo, T

2000-03-15

In the mammalian gonadal primordium, activation of the Sry gene on the Y chromosome initiates a cascade of genetic events leading to testicular organization whereas its absence results in ovarian differentiation. An exception occurs when the Y chromosome of Mus musculus domesticus from Tirano, Italy (Y(TIR)), is placed on the C57BL/6J (B6) genetic background. The B6.Y(TIR) progeny develop only ovaries or ovotestes despite Sry transcription in fetal life. Consequently, the XY offspring with bilateral ovaries develop into apparently normal females, but their eggs fail to develop after fertilization. Our previous studies have shown that the primary cause of infertility can be attributed to oocytes rather than their surrounding somatic cells in the XY ovary. This study attempted to identify the defects in oocytes from the B6.Y(TIR) female mouse. We examined the developmental potential of embryos from XY and XX females after exchanging their nuclear components by microsurgery following in vitro maturation and fertilization. The results suggest that both nuclear and cytoplasmic components are defective in oocytes from XY females. In the XY fetal ovary, most germ cells entered meiosis and their autosomes appeared to synapse normally while the X and Y chromosomes remained unpaired during meiotic prophase. This lack of X-Y pairing probably caused aneuploidy in some secondary oocytes following in vitro maturation. However, normal numbers of chromosomes in the rest of the secondary oocytes indicate that aneuploidy alone can not explain the nuclear defect in oocytes. Copyright 2000 Academic Press.

Developmental competence of Dromedary camel (Camelus dromedarius) oocytes selected using brilliant cresyl blue staining.

PubMed

Fathi, Mohamed; Ashry, Mohamed; Salama, Ali; Badr, Magdy R

2017-08-01

The objectives of the present studies were to investigate the developmental capacity of dromedary camel oocytes selected by brilliant cresyl blue (BCB) staining and to investigate the expression of select transcripts in germinal vesicle (GV) stage oocytes. These transcripts included BMP15 and GDF9 as important transcripts for folliculogenesis and oocyte development, Zar1 and Mater as maternal transcripts required for embryonic development, Cyclin B1 and CDK1 as cell cycle regulators and Oct4 and STAT3 as transcription factors. Dromedary camel oocytes were retrieved from ovaries collected at a local slaughterhouse. After exposure to BCB staining, cumulus-oocyte complexes (COCs) from BCB+, BCB- and control (selected based on morphological criteria) groups were subjected to in vitro maturation, in vitro fertilization and in vitro culture. For gene expression studies, after BCB staining cumulus cells were stripped off and the completely denuded GV stage oocytes were used for RT-PCR analysis of selected transcripts. BCB+ oocytes showed higher maturation, and fertilization rates compared with BCB- and control groups. Indices of early embryonic development, namely, cleavage at 48 hours post insemination (hpi), and development to morula at day 5 and day 7 blastocyst rates were also significantly higher in the BCB+ group. RT-PCR revealed a higher expression of BMP15, GDF9, Zar1, Mater, Cyclin B1, CDK1, OCT4 and STAT3 in good quality oocytes that stained positively for BCB (BCB+). Collectively, results provide novel information about the use of BCB screening for selecting good quality oocytes to improve in vitro embryo production in the dromedary camel.

RNA SYNTHESIS IN THE MOUSE OOCYTE

PubMed Central

Moore, G. P. M.; Lintern-Moore, Sue; Peters, Hannah; Faber, M.

1974-01-01

RNA synthesis in the oocyte and granulosa cell nuclei of growing follicles has been studied in the mouse ovary. The RNA precursor [3H]uridine was administered intraperitoneally to adult mice and the amount of label incorporated into ovarian RNA was quantitated autoradiographically using grain-counting procedures. Uridine incorporation into the nucleus is low in oocytes of small, resting follicles but increases during follicle growth and reaches a peak prior to the beginning of antrum formation. Thereafter uptake rapidly declines and is very low in the oocytes of maturing follicles. Uridine incorporation into granulosa cell nuclei, in contrast to that found in the oocyte, increases gradually during most of the period of follicle growth. Qualitative studies of the activity of endogenous, DNA-dependent RNA polymerases have also been made in fixed oocytes isolated from follicles at different stages of growth. Polymerase activity is demonstrable in the nucleolus and nucleoplasm of oocytes from growing follicles, but is absent from maturing oocytes of large follicles. PMID:4813213

Partial Recovery of Mitochondrial Function of Vitrified Porcine MII Stage Oocytes During Post-Thaw Incubation.

PubMed

Dai, J J; Yang, J H; Zhang, S S; Niu, Y F; Chen, Y N; Wu, C F; Zhang, D F

　　The survival of porcine oocytes is still very low after cryopreservation. To investigate whether and when the mitochondrial function of vitrified porcine oocytes could be recovered post-thaw. Mitochondrial potential, ROS level, ATP content, apoptotic rate, caspase activity, and parthenogenetics developmental ability of thawed porcine oocytes were measured after culture in vitro for 0, 1, 2 or 4 h. Mitochondrial potential after 2 h and 4 h post-thaw culture were 1.19 and 1.26, significantly lower than that of fresh oocytes but much higher than the groups cultured for 0 h and 1 h (P<0.05). Cryopreservation increased the ROS level in oocytes considerably, which decreased only after 2 to 4 h incubation following thaw. ATP content increased gradually over time and recovered to the level comparable to that of fresh oocytes after 4 h. Pan caspase levels increased after cryopreservation and reached the highest level at 1 h incubation. Thereafter it decreased to a low value, but still higher than fresh oocytes. Oocytes showing an early apoptotic event decreased upon 2 to 4 h incubation. The parthenogenetic cleavage and blastocyst rates were the highest (19.8% and 5.6%) after 2 h incubation. The recovery of mitochondrial function could complete after 2 to 4 h post-thaw incubation. Post-thaw incubation for 2 to 4 h reduced apoptotic events and improved parthenogenetic developmental ability of vitrified porcine MII stage oocytes.

Relationship between time post-ovulation and progesterone on oocyte maturation and pregnancy in canine cloning.

PubMed

Kim, Joung Joo; Park, Kang Bae; Choi, Eun Ji; Hyun, Sang Hwan; Kim, Nam-Hyung; Jeong, Yeon Woo; Hwang, Woo Suk

2017-10-01

Canine oocytes ovulated at prophase complete meiosis and continue to develop in presence of a high progesterone concentration in the oviduct. Considering that meiotic competence of canine oocyte is accomplished in the oviductal environment, we postulate that hormonal milieu resulting from the circulating progesterone concentration may affect oocyte maturation and early development of embryos. From 237 oocyte donors, 2620 oocytes were collected and their meiotic status and morphology were determined. To determine optimal characteristics of the mature oocytes subjected to nuclear transfer, a proportion of the meiotic status of the oocytes were classified in reference to time post-ovulation as well as progesterone (P4) level. A high proportion of matured oocytes were collected from >126h (55.5%) post-ovulation or 40-50ngmL -1 (46.4%) group compared to the other groups. Of the oocyte donors that provided mature oocytes in vivo, there was no correlation between serum progesterone of donors and time post ovulation, however, time post-ovulation were significantly shorter for <30ng/mL group (P<0.05). Using mature oocytes, 1161 cloned embryos were reconstructed and transferred into 77 surrogates. In order to determine the relationship between pregnancy performance and serum progesterone level, embryos were transferred into surrogates showing various P4 serum levels. The highest pregnancy (31.8%) and live birth cloning efficacy (2.2%) rates were observed when the embryos were transferred into surrogates with circulating P4 levels were from 40 to 50ngmL -1 . In conclusion, measurement of circulating progesterone of female dog could be a suitable an indicator of the optimal time to collect quality oocyte and to select surrogates for cloning. Copyright © 2017 Elsevier B.V. All rights reserved.

Equine sperm-oocyte interaction: results after intraoviductal and intrauterine inseminations of recipients for oocyte transfer.

PubMed

Carnevale, E M; Maclellan, L J; Coutinho da Silva, M A; Checura, C M; Scoggin, C F; Squires, E L

2001-12-03

Insemination of recipients for oocyte transfer and gamete intrafallopian transfer (GIFT) in five experiments were reviewed, and factors that affected pregnancy rates were ascertained. Oocytes were transferred into recipients that were (1) cyclic and ovulated at the approximate time of oocyte transfer, (2) cyclic with aspiration of the preovulatory follicle, and (3) noncyclic and treated with hormones. Recipients were inseminated before, after, or before and after transfer. Intrauterine and intraoviductal inseminations were done. Pregnancy rates were not different between cyclic and noncyclic recipients (8/15, 53% and 37/93, 39%). The highest numerical pregnancy rates resulted when recipients were inseminated with fresh semen from fertile stallions before oocyte transfer or inseminated with cooled transported semen before and after oocyte transfer. Oxytocin was administered to recipients before oocyte transfer when fluid was imaged within the uterus. Administration of oxytocin to recipients at the time of oocyte transfer resulted in significantly higher pregnancy rates than when oxytocin was not administered (17/26, 65% and 28/86, 33%). Intraoviductal and intrauterine inseminations of recipients during oocyte transfer resulted in similar embryo development rates when fresh semen was used (12/22, 55% and 14/26, 55%). However, embryo development rates significantly reduced when frozen (1/21, 5%) versus fresh sperm were inseminated into the oviduct. Results suggest that insemination of a recipient before and after transfer could be beneficial when semen quality is not optimal; however, a single insemination before transfer was adequate when fresh semen from fertile stallions was used. Absence of a preovulatory follicle did not appear to affect pregnancy rates in the present experiments. The transfer of sperm and oocytes (GIFT) into the oviduct was successful and repeatable as an assisted reproductive technique in the equine.

Effects of trehalose vitrification and artificial oocyte activation on the development competence of human immature oocytes.

PubMed

Zhang, Zhiguo; Wang, Tianjuan; Hao, Yan; Panhwar, Fazil; Chen, Zhongrong; Zou, Weiwei; Ji, Dongmei; Chen, Beili; Zhou, Ping; Zhao, Gang; Cao, Yunxia

2017-02-01

Sucrose and trehalose are conventional cryoprotectant additives for oocytes and embryos. Ethanol can artificially enhance activation of inseminated mature oocytes. This study aims to investigate whether artificial oocyte activation (AOA) with ethanol can promote the development competence of in vitro matured oocytes. A total of 810 human immature oocytes, obtained from 325 patients undergoing normal stimulated oocyte retrieval cycles, were in vitro maturated (IVM) either immediately after collection (Fresh group n = 291)) or after being vitrified as immature oocytes (Vitrified group n = 519). These groups were arbitrarily assigned. All fresh and vitrified oocytes which matured after a period of IVM then underwent intra-cytoplasmic sperm injection (ICSI). Half an hour following ICSI, they were either activated by 7% ethanol (AOA group) or left untreated (Non-AOA group). Fertilization, cleavage rate, blastocyst quality and aneuploidy rate were then evaluated. High-quality blastocysts were only obtained in both the fresh and vitrified groups which had undergone AOA after ICSI. Trehalose vitrification slightly, but not significantly, increased the formation rates of high-quality embryos (21.7% VS 15.4%, P > 0.05) and blastocysts (15.7% VS 7.69%, P > 0.05)) when compared with sucrose vitrification. Aneuploidy was observed in 12 of 24 (50%) of the AOA derived high quality blastocysts. High-quality blastocysts only developed from fresh or vitrified immature oocytes if the ICSI was followed by AOA. This information may be important for human immature oocytes commonly retrieved in normal stimulation cycles and may be particularly important for certain patient groups, such as cancer patients. AOA with an appropriate concentration of ethanol can enhance the developmental competence of embryos. Copyright © 2016 Elsevier Inc. All rights reserved.

Liposome-encapsulated diacyl glycerol and inositol triphosphate-induced delayed oocyte activation and poor development of parthenotes.

PubMed

Nair, Ramya; Manikkath, Jyothsna; Hegde, Aswathi R; Mutalik, Srinivas; Kalthur, Guruprasad; Adiga, Satish Kumar

2017-09-01

To explore the ability of diacyl glycerol (DAG) and inositol triphosphate (IP3), two major secondary messengers in the calcium signaling pathway, in activating oocytes. Oocyte cumulus complex obtained from superovulated Swiss albino mice were incubated in M16 medium with liposome-encapsulated 1,2-Dipalmitoyl-sn-glycerol (LEDAG) and/or IP3 for 3 h. Strontium chloride was used as positive control. The activation potential, ploidy status, and blastocyst rate was calculated. Both DAG and IP3, individually, induced activation in ~98% of oocytes, which was significantly higher (p<0.01) than activation induced by strontium chloride (60%). Delayed pronucleus formation and a higher percentage of diploid parthenotes was observed in oocytes activated with LEDAG and/or IP3. However, these embryos failed to progress beyond the 6-8-cell stage. Only when the medium was supplemented with LEDAG (5 μg/mL) and IP3 (10 μg/mL) could activated oocytes progress till the blastocyst stage (5.26%), which was lower than the blastocyst rate in the positive controls (13.91%). The results of the present study indicate that DAG and IP3 can induce delayed oocyte activation and poor development of parthenotes in vitro.

Elective oocyte cryopreservation for deferred childbearing.

PubMed

Goldman, Kara N; Grifo, Jamie A

2016-12-01

Elective oocyte cryopreservation for deferred childbearing has gained popularity worldwide, commensurate with increased knowledge regarding age-related fertility decline. The purpose of this review is to summarize recent data regarding trends in delayed childbearing, review recent findings surrounding age-related fertility decline, acknowledge significant gaps in knowledge among patients and providers regarding fertility decline and review outcomes following elective oocyte cryopreservation. Despite an inevitable decline in fertility and increase in miscarriage with increasing female age, there is a growing worldwide trend to delay childbearing. Patients and providers alike demonstrate large gaps in knowledge surrounding age-related fertility decline. Oocyte cryopreservation is clinically approved for medically indicated fertility preservation, but a growing number of women are using oocyte cryopreservation to defer childbearing and maintain reproductive autonomy. Mounting data support the efficacy and safety of oocyte cryopreservation when used to electively defer childbearing, with recent studies demonstrating rates of euploidy, implantation and live birth rates equivalent to in-vitro fertilization (IVF) with fresh oocytes. Oocyte cryopreservation provides women with an option to defer childbearing and maintain reproductive autonomy, with IVF success rates on par with fresh IVF. However, it is critical that patients understand the limitations of oocyte cryopreservation. Greater education regarding age-related fertility decline should be geared toward patients and providers to prevent unintended childlessness.

Outcomes of pregnancies achieved by double gamete donation: A comparison with pregnancies obtained by oocyte donation alone.

PubMed

Preaubert, Lise; Vincent-Rohfritsch, Aurélie; Santulli, Pietro; Gayet, Vanessa; Goffinet, François; Le Ray, Camille

2018-03-01

Women increasingly resort to oocyte donation to become pregnant. The high risk of preeclampsia found in oocyte donation pregnancies and the separate risk of preeclampsia associated with sperm donation may be cumulative in double donation pregnancies. We aimed to study the obstetrical and perinatal outcomes of pregnancies obtained by double donation (both oocyte and sperm) in comparison with those obtained by oocyte donation alone (oocyte donation and partner's sperm). This cohort study included all women aged 43 and older who became pregnant after oocyte donation and gave birth between 2010 and 2016 in a tertiary maternity center. Primary outcomes were preeclampsia and hypertensive gestational disorders. Secondary outcomes were gestational diabetes, placental abnormalities, postpartum hemorrhage, perinatal death, and preterm delivery. We used univariate and multivariate analysis to compare IVF with double donation and IVF with oocyte donation alone for obstetric and perinatal outcomes. 247 women, 53 with double donations and 194 with oocyte donations alone, gave birth to 339 children. We observed no significant differences between groups for any obstetric or perinatal complications, except for the risk of gestational diabetes, which was more frequent in women with double donations compared with oocyte donation alone (26.4% vs. 12.9%, P = 0.02) and remained significant after adjustment (aOR = 2.80 95%CI[1.26-6.17]). Rates of gestational hypertension and preeclampsia were high, but similar between groups (20.7% vs. 26.3%, P = 0.41, and 18.9% vs. 17.5%, P = 0.82). Women undergoing oocyte donation should be fully informed of its high rates of obstetric and perinatal risks. However, except for a higher observed risk of gestational diabetes, double donation does not appear to be associated with a higher risk of complications than oocyte donation alone. Copyright © 2017 Elsevier B.V. All rights reserved.

High Fat Diet Induced Developmental Defects in the Mouse: Oocyte Meiotic Aneuploidy and Fetal Growth Retardation/Brain Defects

PubMed Central

Purcell, Scott H.; Chi, Maggie; Jimenez, Patricia T.; Grindler, Natalia; Schedl, Tim; Moley, Kelle H.

2012-01-01

Background Maternal obesity is associated with poor outcomes across the reproductive spectrum including infertility, increased time to pregnancy, early pregnancy loss, fetal loss, congenital abnormalities and neonatal conditions. Furthermore, the proportion of reproductive-aged woman that are obese in the population is increasing sharply. From current studies it is not clear if the origin of the reproductive complications is attributable to problems that arise in the oocyte or the uterine environment. Methodology/Principal Findings We examined the developmental basis of the reproductive phenotypes in obese animals by employing a high fat diet mouse model of obesity. We analyzed very early embryonic and fetal phenotypes, which can be parsed into three abnormal developmental processes that occur in obese mothers. The first is oocyte meiotic aneuploidy that then leads to early embryonic loss. The second is an abnormal process distinct from meiotic aneuploidy that also leads to early embryonic loss. The third is fetal growth retardation and brain developmental abnormalities, which based on embryo transfer experiments are not due to the obese uterine environment but instead must be from a defect that arises prior to the blastocyst stage. Conclusions/Significance Our results suggest that reproductive complications in obese females are, at least in part, from oocyte maternal effects. This conclusion is consistent with IVF studies where the increased pregnancy failure rate in obese women returns to the normal rate if donor oocytes are used instead of autologous oocytes. We postulate that preconceptional weight gain adversely affects pregnancy outcomes and fetal development. In light of our findings, preconceptional counseling may be indicated as the preferable, earlier target for intervention in obese women desiring pregnancy and healthy outcomes. PMID:23152876

Oocyte-granulosa-theca cell interactions during preantral follicular development

PubMed Central

Orisaka, Makoto; Tajima, Kimihisa; Tsang, Benjamin K; Kotsuji, Fumikazu

2009-01-01

The preantral-early antral follicle transition is the penultimate stage of follicular development in terms of gonadotropin dependence and follicle destiny (growth versus atresia). Follicular growth during this period is tightly regulated by oocyte-granulosa-theca cell interactions. Formation of the theca cell layer is a key event that occurs during this transitional stage. Granulosal factor(s) stimulates the recruitment of theca cells from cortical stromal cells, while oocyte-derived growth differentiation factor-9 (GDF-9) is involved in the differentiation of theca cells during this early stage of follicular development. The preantral to early antral transition is most susceptible to follicular atresia. GDF-9 promotes follicular survival and growth during transition from preantral stage to early antral stage by suppressing granulosa cell apoptosis and follicular atresia. GDF-9 also enhances preantral follicle growth by up-regulating theca cell androgen production. Thecal factor(s) promotes granulosa cell proliferation and suppress granulosa cell apoptosis. Understanding the intraovarian mechanisms in the regulation of follicular growth and atresia during this stage may be of clinical significance in the selection of the best quality germ cells for assisted reproduction. In addition, since certain ovarian dysfunctions, such as polycystic ovarian syndrome and gonadotropin poor-responsiveness, are consequences of dysregulated follicle growth at this transitional stage, understanding the molecular and cellular mechanisms in the control of follicular development during the preantral-early antral transition may provide important insight into the pathophysiology and rational treatment of these conditions. PMID:19589134

Bisphenol A alters oocyte maturation by prematurely closing gap junctions in the cumulus cell-oocyte complex.

PubMed

Acuña-Hernández, Deyanira Guadalupe; Arreola-Mendoza, Laura; Santacruz-Márquez, Ramsés; García-Zepeda, Sihomara Patricia; Parra-Forero, Lyda Yuliana; Olivares-Reyes, Jesús Alberto; Hernández-Ochoa, Isabel

2018-04-01

In ovarian follicles, cumulus cells communicate with the oocyte through gap junction intercellular communication (GJIC), to nurture the oocyte and control its meiosis arrest and division. Bisphenol A (BPA) is a monomer found in polycarbonate-made containers that can induce functional alterations, including impaired oocyte meiotic division and reduced molecule transfer in GJIC. However, how BPA alters oocyte meiotic division is unclear. We investigated whether BPA effects on oocyte meiotic division were correlated with reduced transfer in GJIC. Cumulus cell-oocyte complexes (COCs) isolated from mouse preovulatory follicles were cultured with 0, 0.22, 2.2, 22, 220, and 2200 nM BPA for 2 h. An additional 16-h incubation with epidermal growth factor (EGF) was performed to promote the occurrence of meiotic resumption and progression to metaphase II. Without EGF stimulus, BPA treatment increased the percentage of oocytes undergoing meiotic resumption, decreased GJIC in the COCs, and did not modify GJIC gene (Cx43 and Cx37) and protein (CX43) expression. Following EGF stimulus, BPA increased the percentage of oocytes that remained at the anaphase and telophase stages, and decreased the percentage of oocytes reaching the metaphase II stage. Concomitantly, BPA reduced the expansion of cumulus cells. Carbenoxolone (a GJIC inhibitor) and 6-diazo-5-oxo-l-norleucine (a cumulus cell-expansion inhibitor) exerted effects on meiotic division similar to those exerted by BPA. These data suggest that BPA accelerates meiotic progression, leading to impaired prophase I-to-metaphase II transition, and that this adverse effect is correlated with reduced bidirectional communication in the COC. Copyright © 2018 Elsevier Inc. All rights reserved.

Mouse oocytes nucleoli rescue embryonic development of porcine enucleolated oocytes.

PubMed

Morovic, Martin; Strejcek, Frantisek; Nakagawa, Shoma; Deshmukh, Rahul S; Murin, Matej; Benc, Michal; Fulka, Helena; Kyogoku, Hirohisa; Pendovski, Lazo; Fulka, Josef; Laurincik, Jozef

2017-12-01

It is well known that nucleoli of fully grown mammalian oocytes are indispensable for embryonic development. Therefore, the embryos originated from previously enucleolated (ENL) oocytes undergo only one or two cleavages and then their development ceases. In our study the interspecies (mouse/pig) nucleolus transferred embryos (NuTE) were produced and their embryonic development was analyzed by autoradiography, transmission electron microscopy (TEM) and immunofluorescence (C23 and upstream binding factor (UBF)). Our results show that the re-injection of isolated oocyte nucleoli, either from the pig (P + P) or mouse (P + M), into previously enucleolated and subsequently matured porcine oocytes rescues their development after parthenogenetic activation and some of these develop up to the blastocyst stage (P + P, 11.8%; P + M, 13.5%). In nucleolus re-injected 8-cell and blastocyst stage embryos the number of nucleoli labeled with C23 in P + P and P + M groups was lower than in control (non-manipulated) group. UBF was localized in small foci within the nucleoli of blastocysts in control and P + P embryos, however, in P + M embryos the labeling was evenly distributed in the nucleoplasm. The TEM and autoradiographic evaluations showed the formation of functional nucleoli and de novo rRNA synthesis at the 8-cell stage in both, control and P + P group. In the P + M group the formation of comparable nucleoli was delayed. In conclusion, our results indicate that the mouse nucleolus can rescue embryonic development of enucleolated porcine oocytes, but the localization of selected nucleolar proteins, the timing of transcription activation and the formation of the functional nucleoli in NuTE compared with control group show evident aberrations.

[New possibilities resulting from oocyte banking].

PubMed

Revel, Ariel; Revel, Michel; Laufer, Neri; Kasher, Asa

2011-06-01

Oocyte cryopreservation solves the legal and ethical problems associated with the cryopreservation of embryos in patients undergoing in vitro fertilization procedures. Furthermore, it may also offer the possibility of extending the reproductive capability of young women with malignant diseases in cases where the treatment may compromise the ovarian reserve. Moreover, it may also offer alternatives for infertile patients who are subject to ovarian hyper-stimulation syndrome or premature ovarian faiLure or who require oocyte donation. The creation of banks for cryopreserved oocytes avoids the need for cycle synchronization or the formation of an over-supply of embryos destined for cryopreservation. If a Large number of oocytes is obtained it could possibly enable women and couples the opportunity to postpone childbirth according to their wishes. This paper reviews the revolution obtained by oocyte vitrification, reports on ethical issues and discusses the pros and cons of oocyte banking and its potential effects on society.

Zona pellucida gene mRNA expression in human oocytes is related to oocyte maturity, zona inner layer retardance and fertilization competence.

PubMed

Canosa, S; Adriaenssens, T; Coucke, W; Dalmasso, P; Revelli, A; Benedetto, C; Smitz, J

2017-05-01

Do the mRNA expression levels of zona pellucida (ZP) genes, ZP1, 2, 3 and 4 in oocyte and cumulus cells (CC) reveal relevant information on the oocyte? The ZP mRNA expression in human oocytes is related to oocyte maturity, zona inner layer (IL) retardance and fertilization capacity. ZP structure and birefringence provide useful information on oocyte cytoplasmic maturation, developmental competence for embryonic growth, blastocyst formation and pregnancy. In order to understand the molecular basis of morphological changes in the ZP, in the current study, the polarized light microscopy (PLM) approach was combined with analysis of the expression of the genes encoding ZP1, 2, 3 and 4, both in the oocytes and in the surrounding CC. This is a retrospective study comprising 98 supernumerary human cumulus oocyte complexes (COC) [80 Metaphase II (MII), 10 Metaphase I (MI) and 8 germinal vesicle (GV)] obtained from 39 patients (median age 33.4 years, range 22-42) after controlled ovarian stimulation. Single oocytes and their corresponding CC were analysed. Oocytes were examined using PLM, and quantitative RT-PCR was performed for ZP1, 2, 3 and 4 in these individual oocytes and their CC. Ephrin-B2 (EFNB2) mRNA was measured in CC as a control. Presence of ZP3 protein in CC and oocytes was investigated using immunocytochemistry. Data were analysed using one-parametric and multivariate analysis and were corrected for the potential impact of patient and cycle characteristics. Oocytes contained ZP1/2/3 and 4 mRNA while in CC only ZP3 was quantifiable. Also ZP3 protein was detected in human CC. When comparing mature (MII) and immature oocytes (MI/GV) or their corresponding CC, ZP1/2 and 4 expression was lower in mature oocytes compared to the expression in immature oocytes (all P < 0.05) and ZP3 expression was lower in the CC of mature oocytes compared to the expression in CC of immature oocytes (P < 0.05). This coincided with a significantly smaller IL-ZP area and thickness in

Blood clots in the cumulus-oocyte complex predict poor oocyte quality and post-fertilization development.

PubMed

Ebner, T; Moser, M; Shebl, O; Sommergruber, M; Yaman, C; Tews, G

2008-06-01

Assessment of oocyte maturity and quality (morphological appearance) at the time of retrieval is difficult as the egg is obscured by a large cumulus mass that hinders adequate scoring. Since no data are available on the possible relationship between the cumulus-oocyte complex (COC) and oocyte morphology, this prospective intracytoplasmic sperm injection study was set up in 87 consecutive patients. COC were grouped according to expansion of both corona radiata and cumulus matrix. Special emphasis was placed on recording morphological anomalies of COC (inclusion of blood clots and amorphous clumps). For all mature ovae, quality was assessed and preimplantation development followed up to blastocyst stage if fertilized. The risk of not harvesting an oocyte was higher in COC with blood clots compared with normal cumulus matrices (P = 0.004). COC expansion did not allow for prediction of either nuclear status or quality of the egg. The presence of blood clots within the cumulus matrix was associated with reduced oocyte quality (dense central granulation), fertilization rate and blastocyst formation, compared with unaffected COC (P < 0.05). It may be postulated that COC showing blood inclusions derive from poor quality follicles, which has a detrimental effect on oocyte quality and further cleavage to blastocyst stage. Consequently, mechanical removal of blood clots cannot rescue the corresponding embryo.

Apoptosis in mammalian oocytes: a review.

PubMed

Tiwari, Meenakshi; Prasad, Shilpa; Tripathi, Anima; Pandey, Ashutosh N; Ali, Irfan; Singh, Arvind K; Shrivastav, Tulsidas G; Chaube, Shail K

2015-08-01

Apoptosis causes elimination of more than 99% of germ cells from cohort of ovary through follicular atresia. Less than 1% of germ cells, which are culminated in oocytes further undergo apoptosis during last phases of oogenesis and depletes ovarian reserve in most of the mammalian species including human. There are several players that induce apoptosis directly or indirectly in oocytes at various stages of meiotic cell cycle. Premature removal of encircling granulosa cells from immature oocytes, reduced levels of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate, increased levels of calcium (Ca(2+)) and oxidants, sustained reduced level of maturation promoting factor, depletion of survival factors, nutrients and cell cycle proteins, reduced meiotic competency, increased levels of proapoptotic as well as apoptotic factors lead to oocyte apoptosis. The BH3-only proteins also act as key regulators of apoptosis in oocyte within the ovary. Both intrinsic (mitochondria-mediated) as well as extrinsic (cell surface death receptor-mediated) pathways are involved in oocyte apoptosis. BID, a BH3-only protein act as a bridge between both apoptotic pathways and its cleavage activates cell death machinery of both the pathways inside the follicular microenvironment. Oocyte apoptosis leads to the depletion of ovarian reserve that directly affects reproductive outcome of various mammals including human. In this review article, we highlight some of the important players and describe the pathways involved during oocyte apoptosis in mammals.

Total number of oocytes and zygotes are predictive of live birth pregnancy in fresh donor oocyte in vitro fertilization cycles.

PubMed

Hariton, Eduardo; Kim, Keewan; Mumford, Sunni L; Palmor, Marissa; Bortoletto, Pietro; Cardozo, Eden R; Karmon, Anatte E; Sabatini, Mary E; Styer, Aaron K

2017-08-01

To evaluate the association of oocyte donor-recipient characteristics, oocyte donor response, and live birth pregnancy rate following fresh donor oocyte IVF-ET. Retrospective cohort study. Academic reproductive medicine practice. Two hundred thirty-seven consecutive fresh donor oocyte IVF-ET cycles from January 1, 2007 to December 31, 2013 at the Massachusetts General Hospital Fertility Center. None. Live birth rate per cycle initiated. The mean (±SD) age of oocyte donors and recipients was 27.0 ± 3.7 and 41.4 ± 4.6 years, respectively. Oocyte donor demographic/reproductive characteristics, ovarian reserve testing, and peak serum E 2 during ovarian stimulation were similar among cycles which did and did not result in live birth, respectively. Overall implantation, clinical pregnancy, and live birth pregnancy rates per cycle initiated were 40.5%, 60.8%, and 54.9%, respectively. The greatest probability of live birth was observed in cycles with >10 oocytes retrieved, mature oocytes, oocytes with normal fertilization (zygote-two pronuclear stage), and cleaved embryos. The number of oocytes (total and mature), zygotes, and cleaved embryos are associated with live birth following donor oocyte IVF cycles. These findings suggest that specific peri-fertilization factors may be predictive of pregnancy outcomes following donor oocyte IVF cycles. Copyright © 2017 American Society for Reproductive Medicine. All rights reserved.

Storage time does not modify the gene expression profile of cryopreserved human metaphase II oocytes.

PubMed

Stigliani, Sara; Moretti, Stefano; Anserini, Paola; Casciano, Ida; Venturini, Pier Luigi; Scaruffi, Paola

2015-11-01

Does storage time have any impact on the transcriptome of slowly frozen cryopreserved human metaphase II (MII) oocytes? The length of cryostorage has no effect on the gene expression profile of human MII oocytes. Oocyte cryopreservation is a widely used technique in IVF for storage of surplus oocytes, as well as for fertility preservation (i.e. women undergoing gonadotoxic therapies) and oocyte donation programs. Although cryopreservation has negative impacts on oocyte physiology and it is associated with decrease of transcripts, no experimental data about the effect of storage time on the oocyte molecular profile are available to date. This study included 27 women, ≤38 years aged, without any ovarian pathology, undergoing IVF treatment. Surplus MII oocytes were donated after written informed consent. A total of 31 non-cryopreserved oocytes and 68 surviving slow-frozen/rapid-thawed oocytes (32 oocytes cryostored for 3 years and 36 cryostored for 6 years) were analyzed. Pools of ≈10 oocytes for each group were prepared. Total RNA was extracted from each pool, amplified, labeled and hybridized on oligonucleotide microarrays. Analyses were performed by R software using the limma package. Comparison of gene expression profiles between surviving thawed oocytes after 3 and 6 years of storage in liquid nitrogen found no differently expressed genes. The expression profiles of cryopreserved MII oocytes significantly differed from those of non-cryopreserved oocytes in 107 probe sets corresponding to 73 down-regulated and 29 up-regulated unique transcripts. Gene Ontology analysis by DAVID bioinformatics resource disclosed that cryopreservation deregulates genes involved in oocyte function and early embryo development, such as chromosome organization, RNA splicing and processing, cell cycle, cellular response to DNA damage and to stress, DNA repair, calcium ion binding, malate dehydrogenase activity and mitochondrial activity. Among the probes significantly up-regulated in

Regulation of the formin Cappuccino is critical for polarity of Drosophila oocytes

PubMed Central

Bor, Batbileg; Bois, Justin S.; Quinlan, Margot E.

2014-01-01

The Drosophila formin Cappuccino (Capu) creates an actin mesh-like structure that traverses the oocyte during mid-oogenesis. This mesh is thought to prevent premature onset of fast cytoplasmic streaming which normally happens during late-oogenesis. Proper cytoskeletal organization and cytoplasmic streaming are crucial for localization of polarity determinants such as osk, grk, bcd and nanos mRNAs. Capu mutants disrupt these events, leading to female sterility. Capu is regulated by another nucleator, Spire, as well as by autoinhibition in vitro. Studies in vivo confirm that Spire modulates Capu’s function in oocytes; however, how autoinhibition contributes is still unclear. To study the role of autoinhibition in flies, we expressed a Capu construct that is missing the Capu Inhibitory Domain, CapuΔN. Consistent with a gain of activity due to loss of autoinhibition, the actin mesh was denser in CapuΔN oocytes. Further, cytoplasmic streaming was delayed and fertility levels decreased. Localization of osk mRNA in early stages, and bcd and nanos in late stages, were disrupted in CapuΔN-expressing oocytes. Finally, evidence that these phenotypes were due to a loss of autoinhibition comes from co-expression of the N-terminal half of Capu with CapuΔN, which suppressed the defects in actin, cytoplasmic streaming and fertility. From these results, we conclude that Capu can be autoinhibited during Drosophila oocyte development. PMID:25557988

Telomere lengthening early in development.

PubMed

Liu, Lin; Bailey, Susan M; Okuka, Maja; Muñoz, Purificación; Li, Chao; Zhou, Lingjun; Wu, Chao; Czerwiec, Eva; Sandler, Laurel; Seyfang, Andreas; Blasco, Maria A; Keefe, David L

2007-12-01

Stem cells and cancer cells maintain telomere length mostly through telomerase. Telomerase activity is high in male germ line and stem cells, but is low or absent in mature oocytes and cleavage stage embryos, and then high again in blastocysts. How early embryos reset telomere length remains poorly understood. Here, we show that oocytes actually have shorter telomeres than somatic cells, but their telomeres lengthen remarkably during early cleavage development. Moreover, parthenogenetically activated oocytes also lengthen their telomeres, thus the capacity to elongate telomeres must reside within oocytes themselves. Notably, telomeres also elongate in the early cleavage embryos of telomerase-null mice, demonstrating that telomerase is unlikely to be responsible for the abrupt lengthening of telomeres in these cells. Coincident with telomere lengthening, extensive telomere sister-chromatid exchange (T-SCE) and colocalization of the DNA recombination proteins Rad50 and TRF1 were observed in early cleavage embryos. Both T-SCE and DNA recombination proteins decrease in blastocyst stage embryos, whereas telomerase activity increases and telomeres elongate only slowly. We suggest that telomeres lengthen during the early cleavage cycles following fertilization through a recombination-based mechanism, and that from the blastocyst stage onwards, telomerase only maintains the telomere length established by this alternative mechanism.

Cyclic AMP Affects Oocyte Maturation and Embryo Development in Prepubertal and Adult Cattle

PubMed Central

Bernal-Ulloa, Sandra Milena; Heinzmann, Julia; Herrmann, Doris; Hadeler, Klaus-Gerd; Aldag, Patrick; Winkler, Sylke; Pache, Dorit; Baulain, Ulrich; Lucas-Hahn, Andrea; Niemann, Heiner

2016-01-01

High cAMP levels during in vitro maturation (IVM) have been related to improved blastocyst yields. Here, we employed the cAMP/cGMP modulators, forskolin, IBMX, and cilostamide, during IVM to unravel the role of high cAMP in early embryonic development produced from prepubertal and adult bovine oocytes. Oocytes were collected via transvaginal aspiration and randomly assigned to three experimental groups: TCM24 (24h IVM/control), cAMP30 (2h pre-IVM (forskolin-IBMX), 30h IVM-cilostamide), and DMSO30 (Dimethyl Sulfoxide/vehicle control). After IVM, oocytes were fertilized in vitro and zygotes were cultured in vitro to blastocysts. Meiotic progression, cAMP levels, mRNA abundance of selected genes and DNA methylation were evaluated in oocytes. Blastocysts were used for gene expression or DNA methylation analyses. Blastocysts from the cAMP30 groups were transferred to recipients. The cAMP elevation delayed meiotic progression, but developmental rates were not increased. In immature oocytes, mRNA abundance of PRKACA was higher for cAMP30 protocol and no differences were found for PDE3A, SMAD2, ZAR1, PRDX1 and SLC2A8. EGR1 gene was up-regulated in prepubertal cAMP30 immature oocytes and down-regulated in blastocysts from all in vitro treatments. A similar gene expression profile was observed for DNMT3b, BCL2L1, PRDX1 and SLC2A8 in blastocysts. Satellite DNA methylation profiles were different between prepubertal and adult oocytes and blastocysts derived from the TCM24 and DMSO30 groups. Blastocysts obtained from prepubertal and adult oocytes in the cAMP30 treatment displayed normal methylation profiles and produced offspring. These data indicate that cAMP regulates IVM in prepubertal and adult oocytes in a similar manner, with impact on the establishment of epigenetic marks and acquisition of full developmental competency. PMID:26926596

Apoptosis in human unfertilized oocytes after intracytoplasmic sperm injection.

PubMed

Bosco, Liana; Ruvolo, Giovanni; Morici, Giovanni; Manno, Maurizio; Cittadini, Ettore; Roccheri, Maria C

2005-11-01

To investigate the presence of programmed cell death in unfertilized oocytes after intracytoplasmic sperm injection (ICSI), assuming that previous apoptotic events could be correlated with the fertilization failure. Comparison of the rate of DNA fragmentation in human oocytes at different stages of maturation soon after pick-up (control) and in unfertilized oocytes after ICSI treatment. In vitro fertilization (IVF) laboratory with extensive ICSI experience. Sixty-three patients undergoing assisted fertilization by ICSI. Terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay and anticaspase-3 cleaved immunoassay to detect apoptosis in control and ICSI-treated oocytes. Differences in the percentage of oocytes demonstrating DNA fragmentation between control oocytes and unfertilized ICSI treated oocytes at different stages of maturation. The DNA fragmentation, by TUNEL assay, appeared in all the immature control oocytes, but only 37% of mature oocytes showed DNA fragmentation. This DNA fragmentation was observed in 88.8% of the oocytes unfertilized after ICSI; furthermore, DNA fragmentation appeared as well in the sperm injected into the cytoplasm. The study has shown DNA fragmentation in human oocytes unfertilized after ICSI. The evidence is confirmed as well in control oocytes, free from in vitro culture or manipulation stress. Caspase-3 immunoassay suggests the presence of apoptosis. The high percentage of oocytes demonstrating DNA fragmentation in the unfertilized oocytes could be correlated with fertilization failure.

[A prospective study to compare the efficiency of oocyte vitrification using closed or open devices].

PubMed

Sarandi, S; Herbemont, C; Sermondade, N; Benoit, A; Sonigo, C; Poncelet, C; Benard, J; Gronier, H; Boujenah, J; Grynberg, M; Sifer, C

2016-05-01

Oocyte vitrification using an open device is thought to be a source of microbiological and chemical contaminations that can be avoided using a closed device. The principal purpose of this study was to compare the two vitrification protocols: closed and open system. The secondary aim was to study the effects of the storage in the vapor phase of nitrogen (VPN) on oocytes vitrified using an open system and to compare it to those of a storage in liquid nitrogen (LN). Forty-four patients have been included in our study between November 2014 and May 2015. Two hundred and fourteen oocytes have been vitrified at germinal vesicle (GV), metaphase I (0PB) and metaphase II (1PB) stages. We vitrified 96 oocytes (59 GV/37 0PB) using a closed vitrification device and 118 oocytes (57 GV/31 0PB/30 1PB) using an open device. The vitrified oocytes were then stored either in LN or in VPN. The main outcome measures were the survival rate after warming (SR), meiosis resumption rate (MRR) and maturation rate (MR). The global post-thaw SR was significantly higher for oocytes vitrified using an open system (93.2%) compared to those vitrified using a closed one (64.5%; P<0.001). On the contrary, there was no significant difference in terms of global MRR and MR (82.1% vs. 87.5% and 60.7% vs. 61.2% using closed and open system respectively). The SR, MRR and the MR were not significantly different when vitrified oocytes were stored in VPN or LN (91.6, 83.8, 64.5% vs. 93.9, 89.8, 59.1% respectively). Taking into account the limits of our protocol, the open vitrification system remains the more efficient system. The use of sterile liquid nitrogen for oocyte vitrification and the subsequent storage in vapor phase of nitrogen could minimize the hypothetical risks of biological and chemical contaminations. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

The RNA m6A Reader YTHDF2 Is Essential for the Post-transcriptional Regulation of the Maternal Transcriptome and Oocyte Competence.

PubMed

Ivanova, Ivayla; Much, Christian; Di Giacomo, Monica; Azzi, Chiara; Morgan, Marcos; Moreira, Pedro N; Monahan, Jack; Carrieri, Claudia; Enright, Anton J; O'Carroll, Dónal

2017-09-21

YTHDF2 binds and destabilizes N 6 -methyladenosine (m 6 A)-modified mRNA. The extent to which this branch of m 6 A RNA-regulatory pathway functions in vivo and contributes to mammalian development remains unknown. Here we find that YTHDF2 deficiency is partially permissive in mice and results in female-specific infertility. Using conditional mutagenesis, we demonstrate that YTHDF2 is autonomously required within the germline to produce MII oocytes that are competent to sustain early zygotic development. Oocyte maturation is associated with a wave of maternal RNA degradation, and the resulting relative changes to the MII transcriptome are integral to oocyte quality. The loss of YTHDF2 results in the failure to regulate transcript dosage of a cohort of genes during oocyte maturation, with enrichment observed for the YTHDF2-binding consensus and evidence of m 6 A in these upregulated genes. In summary, the m 6 A-reader YTHDF2 is an intrinsic determinant of mammalian oocyte competence and early zygotic development. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Treatment with acetyl-l-carnitine during in vitro maturation of buffalo oocytes improves oocyte quality and subsequent embryonic development.

PubMed

Xu, Hui-Yan; Yang, Xiao-Gan; Lu, Sheng-Sheng; Liang, Xing-Wei; Lu, Yang-Qing; Zhang, Ming; Lu, Ke-Huan

2018-06-01

Oocyte quality is one of the important factors in female fertility, in vitro maturation (IVM), and subsequent embryonic development. In the present study, we assessed whether acetyl-l-carnitine (ALC) supplementation during in vitro maturation of buffalo oocytes could improve oocyte quality and subsequent embryonic development. To determine the optimal level of ALC supplementation, we matured cumulus-oocyte complexes in maturation medium supplemented with 0, 2.5, and 5 mM ALC. The oocytes with a polar body were selected for parthenogenetic activation (PA) and in vitro fertilization (IVF). We found that oocytes matured in 2.5 mM ALC had significantly higher PA blastocyst rate (P < 0.05) and blastocyst cell number than those of unsupplemented oocytes (P < 0.05) and a significantly higher IVF blastocyst rate than that of oocytes matured in 5 mM ALC (P < 0.05). In all further experiments, we supplemented the maturation medium with 2.5 mM ALC. We then tested whether ALC supplementation could improve various markers of oocytes and cumulus cells. We compared cell proliferation; concentrations of reactive oxygen species (ROS), intracellular ATP, estradiol, and progesterone; mitochondrial distribution; mitochondrial DNA copy number (mtDNA); and expression levels of four genes encoding oocyte-derived factors (GDF9, BMP15) and steroid hormones (StAR, P450scc) between the supplemented and unsupplemented oocytes and cumulus cells. Cumulus cells matured with ALC supplementation were more prolific than those matured without ALC supplementation (P < 0.05). Oocytes treated with ALC had lower concentrations of intracellular ROS (P < 0.05) and a higher rate of diffuse mitochondrial distributions (P < 0.05) than those of untreated oocytes. Additionally, the mtDNA was higher in the ALC-treated oocytes (P < 0.05) and cumulus cells (P < 0.05) than that in the untreated cells. The ALC-treated maturation medium had a higher postmaturation

Ethical issues in transnational "mail order" oocyte donation.

PubMed

Heng, B C

2006-12-01

The rising demand for donor oocytes in developed countries has led to what is referred to as transnational or international oocyte donation, or the outsourcing of oocyte donation to poorer countries. In a further twist, frozen sperm from a recipient's partner can also be mailed to a foreign clinic to fertilize donor oocytes, and the resulting embryos are mailed back, cryopreserved, for transfer to the recipient. Among the numerous ethical concerns raised by this practice of mail order oocyte donation, the most obvious are that underprivileged women from poorer countries are often exploited; fertility physicians from richer counties abdicate responsibility for the welfare of donors; and responsibility could become an issue of contention if transmission of disease to the oocyte recipient or congenital defects in offspring born from such oocyte donation were to occur. Moreover, savings from utilizing donors from poorer countries ought to be shared with oocyte recipients.

Comparative proteomic analysis of Populus trichocarpa early stem from primary to secondary growth.

PubMed

Liu, Jinwen; Hai, Guanghui; Wang, Chong; Cao, Shenquan; Xu, Wenjing; Jia, Zhigang; Yang, Chuanping; Wang, Jack P; Dai, Shaojun; Cheng, Yuxiang

2015-08-03

Wood is derived from the secondary growth of tree stems. In this study, we investigated the global changes of protein abundance in Populus early stems using a proteomic approach. Morphological and histochemical analyses revealed three typical stages during Populus early stems, which were the primary growth stage, the transition stage from primary to secondary growth and the secondary growth stage. A total of 231 spots were differentially abundant during various growth stages of Populus early stems. During Populus early stem lignifications, 87 differential spots continuously increased, while 49 spots continuously decreased. These two categories encompass 58.9% of all differential spots, which suggests significant molecular changes from primary to secondary growth. Among 231 spots, 165 unique proteins were identified using LC-ESI-Q-TOF-MS, which were classified into 14 biological function groups. The proteomic characteristics indicated that carbohydrate metabolism, oxido-reduction, protein degradation and secondary cell wall metabolism were the dominantly occurring biochemical processes during Populus early stem development. This study helps in elucidating biochemical processes and identifies potential wood formation-related proteins during tree early stem development. It is a comprehensive proteomic investigation on tree early stem development that, for the first time, reveals the overall molecular networks that occur during Populus early stem lignifications. Copyright © 2015. Published by Elsevier B.V.

The current challenges to efficient immature oocyte cryopreservation.

PubMed

Brambillasca, Fausta; Guglielmo, Maria Cristina; Coticchio, Giovanni; Mignini Renzini, Mario; Dal Canto, Mariabeatrice; Fadini, Rubens

2013-12-01

Oocyte cryopreservation represents an important tool for assisted reproductive technology. It offers the opportunity to preserve fertility in women at risk of loss of the ovarian function for various pathologies. It also represents a treatment alternative for couples that cannot benefit from embryo cryopreservation because of moral, religious, or legal constrains. On the other hand, in vitro oocyte maturation has a range of applications. It can be applied in patients with a contraindication to ovarian stimulation to prevent ovarian hyperstimulation syndrome or to eliminate the risk of stimulation of hormone-sensitive tumours in cancer patients. However, while mature oocyte cryopreservation has found wide-spread application and oocyte in vitro maturation has a place for the treatment of specific clinical conditions, data on the efficiency of freezing of immature or in vitro matured oocytes are poorer. In this review we will focus on the combination of oocyte in vitro maturation with oocyte cryopreservation with particular emphasis on the biological implications of the cryopreservation of immature or in vitro matured oocytes. The two cryopreservation approaches, slow freezing and vitrification, will be discussed in relation to possible cryodamage occurring to subcellular structures of the oocyte and the functional interaction between oocyte and cumulus cells.

Successful ongoing pregnancies after vitrification of oocytes.

PubMed

Lucena, Elkin; Bernal, Diana Patricia; Lucena, Carolina; Rojas, Alejandro; Moran, Abby; Lucena, Andrés

2006-01-01

To demonstrate the efficiency of vitrifying mature human oocytes for different clinical indications. Descriptive case series. Cryobiology laboratory, Centro Colombiano de Fertilidad y Esterilidad-CECOLFES LTDA. (Bogotá, Colombia). Oocyte vitrification was offered as an alternative management for patients undergoing infertility treatment because of ovarian hyperstimulation syndrome, premature ovarian failure, natural ovarian failure, male factor, poor response, or oocyte donation. Mature oocytes were obtained from 33 donor women and 40 patients undergoing infertility treatment. Oocytes were retrieved by ultrasound-guided transvaginal aspiration and vitrified with the Cryotops method, with 30% ethylene glycol, 30% dimethyl sulfoxide, and 0.5 mol/L sucrose. Viability was assessed 3 hours after thawing. The surviving oocytes were inseminated by intracytoplasmic sperm injection. Fertilization was evaluated after 24 hours. The zygotes were further cultured in vitro for up to 72 hours until time of embryo transfer. Recovery, viability, fertilization, and pregnancy rates. Oocyte vitrification with the Cryotop method resulted in high rates of recovery, viability, fertilization, cleavage, and ongoing pregnancy. Vitrification with the Cryotop method is an efficient, fast, and economical method for oocyte cryopreservation that offers high rates of survival, fertilization, embryo development, and ongoing normal pregnancies, providing a new alternative for the management of female infertility.

Differential nuclear remodeling of mammalian somatic cells by Xenopus laevis oocyte and egg cytoplasm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alberio, Ramiro; Johnson, Andrew D.; Stick, Reimer

2005-07-01

The mechanisms governing nuclear reprogramming have not been fully elucidated yet; however, recent studies show a universally conserved ability of both oocyte and egg components to reprogram gene expression in somatic cells. The activation of genes associated with pluripotency by oocyte/egg components may require the remodeling of nuclear structures, such that they can acquire the features of early embryos and pluripotent cells. Here, we report on the remodeling of the nuclear lamina of mammalian cells by Xenopus oocyte and egg extracts. Lamin A/C is removed from somatic cells incubated in oocyte and egg extracts in an active process that requiresmore » permeable nuclear pores. Removal of lamin A/C is specific, since B-type lamins are not changed, and it is not dependent on the incorporation Xenopus egg specific lamin III. Moreover, transcriptional activity is differentially regulated in somatic cells incubated in the extracts. Pol I and II transcriptions are maintained in cells in oocyte extracts; however, both activities are abolished in egg extracts. Our study shows that components of oocyte and egg extracts can modify the nuclear lamina of somatic cells and that this nuclear remodeling induces a structural change in the nucleus which may have implications for transcriptional activity. These experiments suggest that modifications in the nuclear lamina structure by the removal of somatic proteins and the incorporation of oocyte/egg components may contribute to the reprogramming of somatic cell nuclei and may define a characteristic configuration of pluripotent cells.« less

Novel mutations in PATL2 cause female infertility with oocyte germinal vesicle arrest.

PubMed

Huang, Lingli; Tong, Xianhong; Wang, Fengsong; Luo, Lihua; Jin, Rentao; Fu, Yingyun; Zhou, Guixiang; Li, Daojing; Song, Gaojie; Liu, Yusheng; Zhu, Fuxi

2018-06-01

Do PATL2 mutations account for female infertility with oocyte germinal vesicle (GV) arrest? Four of nine independent families with oocyte GV arrest were identified with biallelic PATL2 mutations, suggesting that these mutations may be responsible for oocyte maturation arrest in primary infertile women. Recently, two independent studies have demonstrated that infertility in some women with oocyte maturation arrest at the GV stage was caused by biallelic mutations in PATL2. PATL2 encodes protein PAT1 homolog 2, an RNA-binding protein that may act as a translational repressor. In this study, nine unrelated primary infertile females presenting with oocyte GV arrest were recruited during the treatment of early rescue ICSI or ICSI from January 2013 to December 2016. Genomic DNA was isolated from blood samples obtained from all nine affected individuals and all of their available family members. All the coding regions of PATL2 were sequenced by Sanger sequencing. The pathogenicity of the identified variants and their possible effects on the protein were evaluated in silico. Five novel point mutations and one recurrent splicing mutation in PATL2 were identified in four of nine (44.4%) unrelated patients. We found a consanguineous family with a homozygous missense mutation in two affected sisters, and their fertile brother. There were no clear phenotypic differences in oocytes between the patient with the homozygous missense mutation, patients with nonsense mutations and undiagnosed patients. n/a. The function of PATL2 remains largely unknown. Both the exact pathogenic mechanism(s) of mutated PATL2 causing human oocyte maturation arrest and the strategies to overcome this condition should be further investigated in the future. According to our data, mutations in PATL2 account for 44.4% of the individuals with oocyte GV arrest. Our study further confirms that PATL2 is required for human oocyte maturation and female fertility, which indicates a potential prognostic value of

Birth after 12 hours of oocyte refrigeration.

PubMed

Coban, Onder; Hacifazlioglu, Oguzhan; Ciray, H Nadir; Ulug, Ulun; Tekin, H Ibrahim; Bahceci, Mustafa

2010-12-01

To assess cycle outcome after oocyte refrigeration. Case report. Private IVF center. One couple in a donor oocyte program. Intracytoplasmic sperm injection and blastocyst culture after refrigeration of oocytes for 12 hours. Birth. Fourteen two-pronuclei zygotes from 17 metaphase II refrigerated oocytes resulted in transfer of two blastocysts at day 5 and cryopreservation of six excess embryos at day 6. The patient delivered one healthy male baby after 38 weeks' gestation. The successful outcome of oocyte refrigeration indicates that this protocol could be useful in circumstances in which a delay in obtaining spermatozoa arises. Copyright © 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Early Development of Cloned Bovine Embryos Produced from Oocytes Enucleated by Fluorescence Metaphase II Imaging Using a Conventional Halogen-Lamp Microscope

PubMed Central

Iwamoto, Daisaku; Yamagata, Kazuo; Kishi, Masao; Hayashi-Takanaka, Yoko; Kimura, Hiroshi; Wakayama, Teruhiko

2015-01-01

Abstract Enucleation of a recipient oocyte is one of the key processes in the procedure of somatic cell nuclear transfer (SCNT). However, especially in bovine species, lipid droplets spreading in the ooplasm hamper identification and enucleation of metaphase II (MII) chromosomes, and thereby the success rate of the cloning remains low. In this study we used a new experimental system that enables fluorescent observation of chromosomes in living oocytes without any damage. We succeeded in visualizing and removing the MII chromosome in matured bovine oocytes. This experimental system consists of injecting fluorescence-labeled antibody conjugates that bind to chromosomes and fluorescent observation using a conventional halogen-lamp microscope. The cleavage rates and blastocyst rates of bovine embryos following in vitro fertilization (IVF) decreased as the concentration of the antibody increased (p<0.05). The enucleation rate of the conventional method (blind enucleation) was 86%, whereas all oocytes injected with the antibody conjugates were enucleated successfully. Fusion rates and developmental rates of SCNT embryos produced with the enucleated oocytes were the same as those of the blind enucleation group (p>0.05). For the production of SCNT embryos, the new system can be used as a reliable predictor of the location of metaphase plates in opaque oocytes, such as those in ruminant animals. PMID:25826723

Current trends and progress in clinical applications of oocyte cryopreservation

PubMed Central

Cil, Aylin P.; Seli, Emre

2013-01-01

Purpose of review To delineate the current trends in the clinical application of oocyte cryopreservation. Recent findings Although the first live birth from oocyte cryopreservation was reported approximately three decades ago, significant improvement in the clinical application of oocyte cryopreservation took place only over the past decade. On the basis of the available evidence suggesting that success rates with donor oocyte vitrification are similar to that of IVF with fresh donor oocytes, the American Society of Reproductive Medicine has recently stated that oocyte cryopreservation should no longer be considered experimental for medical indications, outlying elective oocyte cryopreservation. Meanwhile, a few surveys on the attitudes toward oocyte cryopreservation revealed that elective use for the postponement of fertility is currently the most common indication for oocyte cryopreservation. Most recently, a randomized controlled trial revealed important evidence on the safety of nondonor oocyte cryopreservation, and confirmed that the clinical success of vitrification is comparable to that of IVF with fresh oocytes. Summary The evidence suggesting similar IVF success rates with both donor and nondonor cryopreserved oocytes compared with fresh oocytes will increase the utilization of elective oocyte cryopreservation. Appropriate counseling of women for oocyte cryopreservation requires the establishment of age-based clinical success rates with cryopreserved oocytes for various indications. PMID:23562954

Current trends and progress in clinical applications of oocyte cryopreservation.

PubMed

Cil, Aylin P; Seli, Emre

2013-06-01

To delineate the current trends in the clinical application of oocyte cryopreservation. Although the first live birth from oocyte cryopreservation was reported approximately three decades ago, significant improvement in the clinical application of oocyte cryopreservation took place only over the past decade. On the basis of the available evidence suggesting that success rates with donor oocyte vitrification are similar to that of IVF with fresh donor oocytes, the American Society of Reproductive Medicine has recently stated that oocyte cryopreservation should no longer be considered experimental for medical indications, outlying elective oocyte cryopreservation. Meanwhile, a few surveys on the attitudes toward oocyte cryopreservation revealed that elective use for the postponement of fertility is currently the most common indication for oocyte cryopreservation. Most recently, a randomized controlled trial revealed important evidence on the safety of nondonor oocyte cryopreservation, and confirmed that the clinical success of vitrification is comparable to that of IVF with fresh oocytes. The evidence suggesting similar IVF success rates with both donor and nondonor cryopreserved oocytes compared with fresh oocytes will increase the utilization of elective oocyte cryopreservation. Appropriate counseling of women for oocyte cryopreservation requires the establishment of age-based clinical success rates with cryopreserved oocytes for various indications.

Gonadotropin-releasing hormone agonist trigger in oocyte donors co-treated with a gonadotropin-releasing hormone antagonist: a dose-finding study.

PubMed

Vuong, Thi Ngoc Lan; Ho, Manh Tuong; Ha, Tan Duc; Phung, Huy Tuan; Huynh, Gia Bao; Humaidan, Peter

2016-02-01

To determine the optimal GnRH agonist dose for triggering of oocyte maturation in oocyte donors. Single-center, randomized, parallel, investigator-blinded trial. IVFMD, My Duc Hospital, Ho Chi Minh City, Vietnam. One hundred sixty-five oocyte donors (aged 18-35 years, body mass index [BMI] <28 kg/m(2), antimüllerian hormone level >1.25 ng/mL, and antral follicle count ≥6). Ovulation trigger with 0.2, 0.3, or 0.4 mg triptorelin in a GnRH antagonist cycle. The primary end point was number of metaphase II oocytes. Secondary end points were fertilization and cleavage rates, number of embryos and top-quality embryos, steroid levels, ovarian volume, and ongoing pregnancy rate (PR) in recipients. There were no significant differences between the triptorelin 0.2, 0.3, and 0.4 mg trigger groups with respect to number of metaphase II oocytes (16.0 ± 8.5, 15.9 ± 7.8, and 14.7 ± 8.4, respectively), embryos (13.2 ± 7.8, 11.7 ± 6.9, 11.8 ± 7.0), and number of top-quality embryos (3.8 ± 2.9, 3.6 ± 3.0, 4.1 ± 3.0). Luteinizing hormone levels at 24 hours and 36 hours after trigger was significantly higher with triptorelin 0.4 mg versus 0.2 mg and 0.3 mg (9.8 ± 7.1 IU/L vs. 7.3 ± 4.1 IU/L and 7.2 ± 3.7 IU/L, respectively; 4.6 ± 3.2 IU/L vs. 3.2 ± 2.3 IU/L and 3.3 ± 2.1 IU/L, respectively. Progesterone level at oocyte pick-up +6 days was significantly higher in the 0.4-mg group (2.2 ± 3.7 ng/ml) versus 0.2 mg (1.1 ± 1.0 ng/ml) and 0.3 mg (1.2 ± 1.6 ng/ml). One patient developed early-onset severe ovarian hyperstimulation syndrome (OHSS). No significant differences between triptorelin doses of 0.2, 0.3, and 0.4 mg used for ovulation trigger in oocyte donors were seen with regard to the number of mature oocytes and top-quality embryos. NCT02208986. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Expression of XNOA 36 in the mitochondrial cloud of Xenopus laevis oocytes.

PubMed

Vaccaro, M C; Wilding, M; Dale, B; Campanella, C; Carotenuto, R

2012-08-01

In Xenopus laevis oocytes a mitochondrial cloud (MC) is found between the nucleus and the plasma membrane at stages I-II of oogenesis. The MC contains RNAs that are transported to the future vegetal pole at stage II of oogenesis. In particular, germinal plasm mRNAs are found in the Message Transport Organiser (METRO) region, the MC region opposite to the nucleus. At stages II-III, a second pathway transports Vg1 and VegT mRNAs to the area where the MC content merges with the vegetal cortex. Microtubules become polarized at the sites of migration of Vg1 and VegT mRNAs through an unknown signalling mechanism. In early meiotic stages, the centrioles are almost completely lost with their remnants being dispersed into the cytoplasm and the MC, which may contain a MTOC to be used in the later localization pathway of the mRNAs. In mammals, XNOA 36 encodes a member of a highly conserved protein family and localises to the nucleolus or in the centromeres. In the Xenopus late stage I oocyte, XNOA 36 mRNA is transiently segregated in one half of the oocyte, anchored by a cytoskeletal network that contains spectrin. Here we found that XNOA 36 transcript also localises to the nucleoli and in the METRO region. XNOA 36 protein immunolocalization, using an antibody employed for the library immunoscreening that depicted XNOA 36 expression colonies, labels the migrating MC, the cytoplasm of stage I oocytes and in particular the vegetal cortex facing the MC. The possible role of XNOA 36 in mRNA anchoring to the vegetal cortex or in participating in early microtubule reorganization is discussed.

[Can one authorize oocyte donation in the Grand Duchy of Luxembourg?].

PubMed

Arendt, J

2012-01-01

In the case of early ovary extinction, the only way to have a child is either adoption or egg/embryo reception by donation. To day, egg donation is prohibited in Luxembourg by ministerial decision in 2003. Germ cell donation is part of artificial reproductive therapy. Oocyte donation, in particular, needs to be done by IVF treatment, which makes it more complicated then sperm donation What makes it more difficult is the fact that there are no oocyte bank yet. Today, prohibition encourages procreative tourism what only wealthy people can afford. Although donation programs are well established many questions arise about egg donation such as refunds, divulging information, women's age limit, health insurance participation.

Impact of prolonged oocyte incubation time before vitrification on oocyte survival, embryo formation, and embryo quality in mice.

PubMed

Karami, Azade; Bakhtiari, Mitra; Azadbakht, Mehri; Ghorbani, Rostam; Khazaei, Mozafar; Rezaei, Mansour

2017-06-01

Oocyte incubation time before freezing is one of the factors affecting oocyte vitrification. In the assisted reproductive technology (ART) clinics, it is sometimes decided to perform oocyte vitrification after a long period of incubation time due to various conditions, such as inability to collect semen samples, unsuccessful urological interventions (PESA, TESE, etc.), or unexpected conditions. A time factor of up to 6 h has been studied in the available reports. Therefore, this study was designed to evaluate oocyte incubation time before freezing at 0, 6, 12, 18, and 24 h after retrieval. Metaphase II (MII) oocytes were obtained from NMRI female mice after being randomly divided into the five groups of 0, 6, 12, 18, and 24 h of freezing via hormonal stimulation following retrieval and entered into the vitrification-warming process. The thawed oocytes were evaluated according to the survival criteria and then inseminated with the sperms of male mice for in vitro fertilization. The next day, the embryo formation rate and embryo quality were assessed. Our results demonstrated that even after 24 h of incubation, the survival rate of oocytes was 51.35% with the embryo formation rate of 73.21%. However, the survival and embryo formation rates significantly decreased within 12, 18, and 24 h after retrieval compared to the groups vitrified at 0 h. The embryo quality was significantly reduced by vitrification at 0 to 24 h after retrieval. According to our data, although a prolonged incubation time before freezing reduced the survival rate, there was still a chance for oocytes to stay alive with acceptable embryo formation and quality rates after vitrification warming of oocytes.

The human cumulus--oocyte complex gene-expression profile

PubMed Central

Assou, Said; Anahory, Tal; Pantesco, Véronique; Le Carrour, Tanguy; Pellestor, Franck; Klein, Bernard; Reyftmann, Lionel; Dechaud, Hervé; De Vos, John; Hamamah, Samir

2006-01-01

BACKGROUND The understanding of the mechanisms regulating human oocyte maturation is still rudimentary. We have identified transcripts differentially expressed between immature and mature oocytes, and cumulus cells. METHODS Using oligonucleotides microarrays, genome wide gene expression was studied in pooled immature and mature oocytes or cumulus cells from patients who underwent IVF. RESULTS In addition to known genes such as DAZL, BMP15 or GDF9, oocytes upregulated 1514 genes. We show that PTTG3 and AURKC are respectively the securin and the Aurora kinase preferentially expressed during oocyte meiosis. Strikingly, oocytes overexpressed previously unreported growth factors such as TNFSF13/APRIL, FGF9, FGF14, and IL4, and transcription factors including OTX2, SOX15 and SOX30. Conversely, cumulus cells, in addition to known genes such as LHCGR or BMPR2, overexpressed cell-tocell signaling genes including TNFSF11/RANKL, numerous complement components, semaphorins (SEMA3A, SEMA6A, SEMA6D) and CD genes such as CD200. We also identified 52 genes progressively increasing during oocyte maturation, comprising CDC25A and SOCS7. CONCLUSION The identification of genes up and down regulated during oocyte maturation greatly improves our understanding of oocyte biology and will provide new markers that signal viable and competent oocytes. Furthermore, genes found expressed in cumulus cells are potential markers of granulosa cell tumors. PMID:16571642

Human oocyte cryopreservation and the fate of cortical granules.

PubMed

Ghetler, Yehudith; Skutelsky, Ehud; Ben Nun, Isaac; Ben Dor, Liah; Amihai, Dina; Shalgi, Ruth

2006-07-01

To examine the effect of the commonly used oocyte cryopreservation protocol on the cortical granules (CGs) of human immature germinal vesicle (GV) and mature metaphase II (MII) oocytes. Laboratory study. IVF unit. Unfertilized, intracytoplasmic sperm injected (ICSI) oocytes, and immature oocytes were cryopreserved using a slow freezing-rapid thawing program with 1,2-propanediol (PROH) as a cryoprotectant. Cortical granule exocytosis (CGE) was assessed by either confocal microscopy or transmission electron microscopy (TEM). The survival rates of frozen-thawed oocytes (mature and immature) were significantly lower compared with zygotes. Both mature and immature oocytes exhibited increased fluorescence after cryopreservation, indicating the occurrence of CGE. Mere exposure of oocytes to cryoprotectants induced CGE of 70% the value of control zygotes. The TEM revealed a drastic reduction in the amount of CGs at the cortex of frozen-thawed GV and MII oocytes, as well as appearance of vesicles in the ooplasm. The commonly used PROH freezing protocol for human oocytes resulted in extensive CGE. This finding explains why ICSI is needed to achieve fertilization of frozen-thawed human oocytes.

DNase I and II present in avian oocytes: a possible involvement in sperm degradation at polyspermic fertilisation.

PubMed

Stepińska, Urszula; Olszańska, Bozenna

2003-02-01

During polyspermic fertilisation in birds numerous spermatozoa enter the eggs, in contrast to the situation in mammals where fertilisation is monospermic. However, in birds only one of the spermatozoa which have entered an egg participates in zygote nucleus formation, while the supernumerary spermatozoa degenerate at early embryogenesis. Our previous work has demonstrated the presence in preovulatory quail oocytes of DNase I and II activities able to digest naked lambdaDNA/HindIII substrate in vitro. In the present studies, the activities of both DNases in quail oocytes at different stages of oogenesis and in ovulated mouse oocytes were assayed in vitro using the same substrate. Degradation of quail spermatozoa by quail oocyte extracts was also checked. Digestion of the DNA substrate was evaluated by electrophoresis on agarose gels. The activities of DNase I and II in quail oocytes increased during oogenesis and were the highest in mature oocytes. The activities were present not only in germinal discs but also in a thin layer of cytoplasm adhering to the perivitelline layer surrounding the yolk. At all stages of oogenesis the activity of DNase II was much higher than that of DNase I. DNA contained in spermatozoa was also degraded by the quail oocyte extracts under conditions optimal for both DNases. In contrast to what is observed in quail oocytes, no DNase activities were detected in ovulated mouse eggs; this is logical as they would be useless or even harmful in monospermic fertilisation. The possible role of DNase activities in avian oocytes, in degradation of accessory spermatozoa during polyspermic fertilisation, is discussed.

Prediction of Developmentally Competent Chromatin Conformation in Mouse Antral Oocytes.

PubMed

Daszkiewicz, Regina; Szymoniak, Magdalena; Gąsior, Łukasz; Polański, Zbigniew

Mouse prophase oocytes isolated from antral follicles may possess two alternative types of chromatin configuration: NSN configuration represents more dispersed chromatin and is characteristic mainly for growing oocytes whereas SN configuration, attained upon oocyte growth, comprises more condensed chromatin with a significant fraction concentrated around the nucleolus. Importantly, fully grown oocytes isolated from antral follicles represent a non-homogenous population in which some oocytes posses NSN-type and others SN-type of chromatin conformation. From these two, only oocytes with SN configuration are able to complete full development upon fertilization. We show that among mouse oocytes isolated from antral follicles, those surrounded by cumulus cells were larger and more frequently possessed SN chromatin than oocytes lacking the complete cumulus cell layer. Females primed with PMSG gave a higher number of oocytes with a complete layer of cumulus cells and the frequency of oocytes with SN chromatin was also elevated. Within the whole population of isolated antral oocytes, we observed subtle variation in size which allowed fractionation of oocytes under a stereomicroscope into groups representing oocytes of slightly different sizes. The occurrence of SN chromatin configuration was highly dependent on the oocyte size and its frequency increased gradually in subsequent size groups reaching 95-100% in the group representing the largest oocytes. These findings demonstrate that the subtle differences in the size of antral oocytes allow prediction of the status of their chromatin, thus providing a simple, fast, non-invasive and non-expensive way to select good quality oocytes for ART purposes in mammals.

Characterization of urea transport in Bufo arenarum oocytes.

PubMed

Silberstein, Claudia; Zotta, Elsa; Ripoche, Pierre; Ibarra, Cristina

2003-07-01

Xenopus laevis oocytes have been extensively used for expression cloning, structure/function relationships, and regulation analysis of transporter proteins. Urea transporters have been expressed in Xenopus oocytes and their properties have been described. In order to establish an alternative system in which urea transporters could be efficiently expressed and studied, we determined the urea transport properties of ovarian oocytes from Bufo arenarum, a toad species common in Argentina. Bufo oocytes presented a high urea permeability of 22.3 x 10(-6) cm/s, which was significantly inhibited by the incubation with phloretin. The urea uptake in these oocytes was also inhibited by mercurial reagents, and high-affinity urea analogues. The urea uptake was not sodium dependent. The activation energy was 3.2 Kcal/mol, suggesting that urea movement across membrane oocytes may be through a facilitated urea transporter. In contrast, Bufo oocytes showed a low permeability for mannitol and glycerol. From these results, we propose that one or several specific urea transporters are present in ovarian oocytes from Bufo arenarum. Therefore, these oocytes cannot be used in expression studies of foreign urea transporters. The importance of Bufo urea transporter is not known but could be implicated in osmotic regulation during the laying of eggs in water. Copyright 2003 Wiley-Liss, Inc.

Ethics of medical and nonmedical oocyte cryopreservation.

PubMed

Patrizio, Pasquale; Molinari, Emanuela; Caplan, Arthur

2016-12-01

To assess the effectiveness and ethical dimensions of oocyte cryopreservation for both medical and social indications. As more women are postponing motherhood for a variety of reasons, including lack of partner, for completing career plans and reaching financial stability, they are resorting to oocyte cryopreservation. To make informed choices, women rely on their primary care physicians (PCPs) for initial advice, but PCPs are not always fully prepared to discuss oocyte cryopreservation. Interestingly, there are mixed feelings among obstetricians/gynecologists on whether oocyte cryopreservation should be used for elective reasons, whereas it is fully supported for medical indications. Oocyte vitrification has become an established procedure for safeguarding future reproductive chances for medical reasons, and its use is progressively expanding. There is an urgent need in preparing future PCPs and obstetricians/gynecologists as to how to initiate discussions with their patients about elective oocyte banking consistent with fully respecting patient autonomy so as to facilitate informed decisions.

Extending prematuration with cAMP modulators enhances the cumulus contribution to oocyte antioxidant defence and oocyte quality via gap junctions.

PubMed

Li, H J; Sutton-McDowall, M L; Wang, X; Sugimura, S; Thompson, J G; Gilchrist, R B

2016-04-01

Can bovine oocyte antioxidant defence and oocyte quality be improved by extending the duration of pre-in vitro maturation (IVM) with cyclic adenosine mono-phosphate (cAMP) modulators? Lengthening the duration of cAMP-modulated pre-IVM elevates intra-oocyte reduced glutathione (GSH) content and reduces hydrogen peroxide (H2O2) via increased cumulus cell-oocyte gap-junctional communication (GJC), associated with an improvement in subsequent embryo development and quality. Oocytes are susceptible to oxidative stress and the oocyte's most important antioxidant glutathione is supplied, at least in part, by cumulus cells. A temporary inhibition of spontaneous meiotic resumption in oocytes can be achieved by preventing a fall in cAMP, and cyclic AMP-modulated pre-IVM maintains cumulus-oocyte GJC and improves subsequent embryo development. This study consisted of a series of 10 experiments using bovine oocytes in vitro, each with multiple replicates. A range of pre-IVM durations were examined as the key study treatments which were compared with a control. The study was designed to examine if one of the oocyte's major antioxidant defences can be enhanced by pre-IVM with cAMP modulators, and to examine the contribution of cumulus-oocyte GJC on these processes. Immature bovine cumulus-oocyte complexes were treated in vitro without (control) or with the cAMP modulators; 100 µM forskolin (FSK) and 500 µM 3-isobutyl-1-methyxanthine (IBMX), for 0, 2, 4 or 6 h (pre-IVM phase) prior to IVM. Oocyte developmental competence was assessed by embryo development and quality post-IVM/IVF. Cumulus-oocyte GJC, intra-oocyte GSH and H2O2 were quantified at various time points during pre-IVM and IVM, in the presence and the absence of functional inhibitors: carbenoxolone (CBX) to block GJC and buthionine sulfoximide (BSO) to inhibit glutathione synthesis. Pre-IVM with FSK + IBMX increased subsequent blastocyst formation rate and quality compared with standard IVM (P < 0.05), regardless of

Motility contrast imaging of live porcine cumulus-oocyte complexes

NASA Astrophysics Data System (ADS)

An, Ran; Turek, John; Machaty, Zoltan; Nolte, David

2013-02-01

Freshly-harvested porcine oocytes are invested with cumulus granulosa cells in cumulus-oocyte complexes (COCs). The cumulus cell layer is usually too thick to image the living oocyte under a conventional microscope. Therefore, it is difficult to assess the oocyte viability. The low success rate of implantation is the main problem for in vitro fertilization. In this paper, we demonstrate our dynamic imaging technique called motility contrast imaging (MCI) that provides a non-invasive way to monitor the COCs before and after maturation. MCI shows a change of intracellular activity during oocyte maturation, and a measures dynamic contrast between the cumulus granulosa shell and the oocytes. MCI also shows difference in the spectral response between oocytes that were graded into quality classes. MCI is based on shortcoherence digital holography. It uses intracellular motility as the endogenous imaging contrast of living tissue. MCI presents a new approach for cumulus-oocyte complex assessment.

Chronic unpredictable stress decreases expression of brain-derived neurotrophic factor (BDNF) in mouse ovaries: relationship to oocytes developmental potential.

PubMed

Wu, Li-Min; Hu, Mei-Hong; Tong, Xian-Hong; Han, Hui; Shen, Ni; Jin, Ren-Tao; Wang, Wei; Zhou, Gui-Xiang; He, Guo-Ping; Liu, Yu-Sheng

2012-01-01

Brain-derived neurotropic factor (BDNF) was originally described in the nervous system but has been shown to be expressed in ovary tissues recently, acting as a paracrine/autocrine regulator required for developments of follicles and oocytes. Although it is generally accepted that chronic stress impairs female reproduction and decreases the expression of BDNF in limbic structures of central nervous system, which contributes to mood disorder. However, it is not known whether chronic stress affects oocytes developments, nor whether it affects expression of BDNF in ovary. Mice were randomly assigned into control group, stressed group, BDNF-treated group and BDNF-treated stressed group. The chronic unpredictable mild stress model was used to produce psychosocial stress in mice, and the model was verified by open field test and hypothalamic-pituitary-adrenal (HPA) axis activity. The methods of immunohistochemistry and western blotting were used to detect BDNF protein level and distribution. The number of retrieved oocytes, oocyte maturation, embryo cleavage and the rates of blastocyst formation after parthenogenetic activation were evaluated. Chronic unpredictable stress decreased the BDNF expression in antral follicles, but didn't affect the BDNF expression in primordial, primary and secondary follicles. Chronic unpredictable stress also decreased the number of retrieved oocytes and the rate of blastocyst formation, which was rescued by exogenous BDNF treatment. BDNF in mouse ovaries may be related to the decreased number of retrieved oocytes and impaired oocytes developmental potential induced by chronic unpredictable stress.

Chronic Unpredictable Stress Decreases Expression of Brain-Derived Neurotrophic Factor (BDNF) in Mouse Ovaries: Relationship to Oocytes Developmental Potential

PubMed Central

Tong, Xian-Hong; Han, Hui; Shen, Ni; Jin, Ren-Tao; Wang, Wei; Zhou, Gui-Xiang; He, Guo-Ping; Liu, Yu-Sheng

2012-01-01

Background Brain-derived neurotropic factor (BDNF) was originally described in the nervous system but has been shown to be expressed in ovary tissues recently, acting as a paracrine/autocrine regulator required for developments of follicles and oocytes. Although it is generally accepted that chronic stress impairs female reproduction and decreases the expression of BDNF in limbic structures of central nervous system, which contributes to mood disorder. However, it is not known whether chronic stress affects oocytes developments, nor whether it affects expression of BDNF in ovary. Methods Mice were randomly assigned into control group, stressed group, BDNF-treated group and BDNF-treated stressed group. The chronic unpredictable mild stress model was used to produce psychosocial stress in mice, and the model was verified by open field test and hypothalamic-pituitary-adrenal (HPA) axis activity. The methods of immunohistochemistry and western blotting were used to detect BDNF protein level and distribution. The number of retrieved oocytes, oocyte maturation, embryo cleavage and the rates of blastocyst formation after parthenogenetic activation were evaluated. Results Chronic unpredictable stress decreased the BDNF expression in antral follicles, but didn’t affect the BDNF expression in primordial, primary and secondary follicles. Chronic unpredictable stress also decreased the number of retrieved oocytes and the rate of blastocyst formation, which was rescued by exogenous BDNF treatment. Conclusion BDNF in mouse ovaries may be related to the decreased number of retrieved oocytes and impaired oocytes developmental potential induced by chronic unpredictable stress. PMID:23284991

Fourier analysis of mitochondrial distribution in oocytes

NASA Astrophysics Data System (ADS)

Hollmann, Joseph L.; Brooks, Dana H.; Newmark, Judith A.; Warner, Carol M.; DiMarzio, Charles A.

2011-03-01

This paper describes a novel approach to quantifying mitochondrial patterns which are typically described using the qualitative terms "diffuse" "aggregated" and are potentially key indicators for an oocyte's health and survival potential post-implantation. An oocyte was isolated in a confocal image and a coarse grid was superimposed upon it. The spatial spectrum was calculated and an aggregation factor was generated. A classifier for healthy cells was developed and verified. The aggregation factor showed a clear distinction between the healthy and unhealthy oocytes. The ultimate goal is to screen oocytes for viability preimplantation, thus improving the outcome of in vitro fertilization (IVF) treatments.

Injurious effects of curcumin on maturation of mouse oocytes, fertilization and fetal development via apoptosis.

PubMed

Chen, Chia-Chi; Chan, Wen-Hsiung

2012-01-01

Curcumin, a common dietary pigment and spice, is a hydrophobic polyphenol derived from the rhizome of the herb Curcuma longa. Previously, we reported a cytotoxic effect of curcumin on mouse embryonic stem cells and blastocysts and its association with defects in subsequent development. In the present study, we further investigated the effects of curcumin on oocyte maturation and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, curcumin induced a significant reduction in the rate of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with curcumin during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased fetal weight. Experiments with an in vivo mouse model disclosed that consumption of drinking water containing 40 μM curcumin led to decreased oocyte maturation and in vitro fertilization as well as early embryonic developmental injury. Finally, pretreatment with a caspase-3-specific inhibitor effectively prevented curcumin-triggered injury effects, suggesting that embryo impairment by curcumin occurs mainly via a caspase-dependent apoptotic process.

Ovine secondary follicles vitrified out the ovarian tissue grow and develop in vitro better than those vitrified into the ovarian fragments.

PubMed

Lunardi, Franciele Osmarini; de Aguiar, Francisco Leo Nascimento; Duarte, Ana Beatriz Graça; Araújo, Valdevane Rocha; de Lima, Laritza Ferreira; Ribeiro de Sá, Naiza Arcângela; Vieira Correia, Hudson Henrique; Domingues, Sheyla Farhayldes Souza; Campello, Cláudio Cabral; Smitz, Johan; de Figueiredo, José Ricardo; Ribeiro Rodrigues, Ana Paula

2016-04-15

Cryopreservation of preantral follicles is a promising technique to preserve female fertility. The aim of this study was to evaluate the effect of vitrification on the development of secondary follicles included in ovarian tissue or isolated after microdissection. An important end point included is the capacity of grown oocytes to resume meiosis. Sheep ovarian cortexes were cut into fragments and split into three different groups: (1) fresh (control): secondary follicles isolated without any previous vitrification; (2) follicle-vitrification (follicle-vit): secondary follicles vitrified in isolated form; and (3) tissue-vitrification (tissue-vit): secondary follicles vitrified within fragments of ovarian tissue (in situ former) and subsequently subjected to isolation. From the three groups, isolated secondary follicles were submitted to IVC for 18 days. After IVC, cumulus-oocyte complexes (COCs) were harvested from follicles. As an additional control group, in vivo grown, in vivo-grown COCs were collected from antral ovarian follicles. All, recovered COCs were matured and the chromatin configuration was evaluated. Data were analyzed by ANOVA, and the means were compared by Student-Newman-Keuls test, and by chi-square. Differences were considered to be significant when P < 0.05. Isolated preantral follicles from all treatments had normal morphology, antrum formation, and low follicle degeneration after IVC. The growth rate between control and follicle-vit did not differ (P > 0.05), and was higher (P < 0.05) than for tissue-vit. The percentage of follicles that decreased diameter during IVC was significantly higher in tissue-vit than the in follicle-vit. Recovery rate of oocytes from normal follicles was higher in follicle-vit than in tissue-vit. Furthermore, oocyte viability was lower in tissue-vit than other treatments, and follicle-vit did not differ from control and in vivo grown. The percentage of oocytes meiosis resuming was not different between

The evolution of oocyte donation in China.

PubMed

Wang, Fang; Sun, Yingpu; Kong, Huijuan; Li, Jing; Su, Yingchun; Guo, Yihong

2010-07-01

To review the experience with and clinical outcomes for recipients of embryos from oocytes donated under different regulatory standards in China. Initially, the oocytes were provided by one of the patient's consanguineous sisters. Then, the oocytes were obtained from another patient treated with assisted reproduction techniques (ART). Presently, oocytes thus produced are cryopreserved for at least 6months before transfer. The records from all women treated with ART at First Affiliated Hospital of Zhengzhou University since 2001 were reviewed and the pregnancy rates and clinical outcomes were determined for each of the 3 periods. In the second period, the mean implantation and clinical pregnancy rates were significantly higher for the 22 oocyte recipients than for their donors. In the third period, the rates for the 56 recipients were compared with the 78 other regular ART patients fertilized with their own oocytes. There were 40 live births for 32 of the recipients over 28 cycles, and the rates of implantation and clinical pregnancy were much higher for the recipients than for the other ART patients (P<0.001). Using freshly donated eggs yields a higher pregnancy rate but there is a risk of infectious disease. Using frozen oocytes can significantly decrease this risk but implantation rates are lower. Copyright (c) 2010 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.

Oocyte maturation in the sloth's giant tick Amblyomma varium (Acari: Ixodidae) in an ecological context.

PubMed

Sanches, Gustavo S; André, Marcos R; do Prado, Angelo P; Allegretti, Silmara M; Remedio, Rafael N; Nunes, Pablo H; Machado, Rosangela Z; Bechara, Gervásio H; Camargo-Mathias, Maria I

2014-12-01

The sloth's giant tick Amblyomma varium Koch, which is a neotropical species that inhabits tropical rainforests, is the largest tick reported to date. The adult stage of this tick parasitizes mammals from the families Bradypodidae and Magalonychidae (Xenarthra) nearly exclusively. This study aimed to describe morphological and histological features of the reproductive system and the oocyte maturation process of this tick species. The ovary of A. varium is a long single tubular organ that is horseshoe-shaped, winding and arranged in the posterior part of the body. Two oviducts are connected to the ovary on each side; these thicken at certain region forming the uterus (common oviduct), followed by a muscular connecting tube, vagina and genital aperture. A large number of oocytes at different stages of development are attached to the ovary wall by the pedicel, as they reach maturity they are released into the ovary lumen and from there to the genital aperture. These oocytes develop simultaneously and asynchronically along the ovary. Amblyomma varium oocytes were classified into five development stages (i.e., I-V), and specific characteristics were observed; the processes of yolk and chorion deposition begin early in oocytes stage II, and oocytes V exhibit a very thick chorion and eggs of a large size. These characteristics are likely adaptations that enhance the survival and the reproductive success of this extremely host-specific tick, which is limited to a particular environment.

High-throughput optofluidic system for the laser microsurgery of oocytes

NASA Astrophysics Data System (ADS)

Chandsawangbhuwana, Charlie; Shi, Linda Z.; Zhu, Qingyuan; Alliegro, Mark C.; Berns, Michael W.

2012-01-01

This study combines microfluidics with optical microablation in a microscopy system that allows for high-throughput manipulation of oocytes, automated media exchange, and long-term oocyte observation. The microfluidic component of the system transports oocytes from an inlet port into multiple flow channels. Within each channel, oocytes are confined against a microfluidic barrier using a steady fluid flow provided by an external computer-controlled syringe pump. This allows for easy media replacement without disturbing the oocyte location. The microfluidic and optical-laser microbeam ablation capabilities of the system were validated using surf clam (Spisula solidissima) oocytes that were immobilized in order to permit ablation of the 5 μm diameter nucleolinus within the oocyte nucleolus. Oocytes were the followed and assayed for polar body ejection.

Myo-inositol may improve oocyte quality in intracytoplasmic sperm injection cycles. A prospective, controlled, randomized trial.

PubMed

Papaleo, Enrico; Unfer, Vittorio; Baillargeon, Jean-Patrice; Fusi, Francesco; Occhi, Francesca; De Santis, Lucia

2009-05-01

To determine the effects of myo-inositol on oocyte quality in polycystic ovary syndrome (PCOS) patients undergoing intracytoplasmic sperm injection (ICSI) cycles. A prospective, controlled, randomized trial. Assisted reproduction centers. Sixty infertile PCO patients undergoing ovulation induction for ICSI. All participants underwent standard long protocol. Starting on the day of GnRH administration, 30 participants received myo-inositol combined with folic acid (Inofolic) 2 g twice a day and 30 control women received folic acid alone, administrated continuously. Primary end points were number of morphologically mature oocytes retrieved, embryo quality, and pregnancy and implantation rates. Secondary end points were total number of days of FSH stimulation, total dose of gonadotropin administered, E(2) level on the day of hCG administration, fertilization rate per number of retrieved oocytes, embryo cleavage rate, live birth and miscarriage rates, cancellation rate, and incidence of moderate or severe ovarian hyperstimulation syndrome. Total r-FSH units (1,958 +/- 695 vs. 2,383 +/- 578) and number of days of stimulation (11.4 +/- 0.9 vs. 12.4 +/- 1.4) were significantly reduced in the myo-inositol group. Furthermore, peak E(2) levels (2,232 +/- 510 vs. 2,713 +/- 595 pg/mL) at hCG administration were significantly lower in patients receiving myo-inositol. The mean number of oocytes retrieved did not differ in the two groups, whereas in the group cotreated with myo-inositol the mean number of germinal vesicles and degenerated oocytes was significantly reduced (1.0 +/- 0.9 vs. 1.6 +/- 1.0), with a trend for increased percentage of oocytes in metaphase II (0.82 +/- 0.11% vs. 0.75 +/- 0.15%). These data show that in patients with PCOS, treatment with myo-inositol and folic acid, but not folic acid alone, reduces germinal vesicles and degenerated oocytes at ovum pick-up without compromising total number of retrieved oocytes. This approach, reducing E(2) levels at h

Oocyte holding in the Iberian red deer (Cervus elaphus hispanicus): Effect of initial oocyte quality and epidermal growth factor addition on in vitro maturation.

PubMed

Macías-García, B; González-Fernández, L; Matilla, E; Hernández, N; Mijares, J; Sánchez-Margallo, F M

2018-02-01

Current in vitro embryo production protocols in the Iberian red deer (Cervus elaphus hispanicus) need to be optimized; oocyte harvesting in situ followed by overnight holding could reduce the human effort and shipping costs. In our work, post-mortem ovaries were retrieved, and the oocytes were harvested and allocated to G1 group (good quality) or G2 + G3 group (low quality). The oocytes were separately subjected to immediate in vitro maturation (IVM) or held overnight in a holding medium composed of 40% of TCM 199 with Earle's salts, 40% TCM 199 with Hanks' salts and 20% fetal bovine serum (FBS), at room temperature (16 hr). In vitro maturation was carried out in a basal medium supplemented or not with 50 ng/ml of epidermal growth factor (EGF). Our data showed that addition of EGF to the maturation medium increases the percentage of G1 oocytes reaching metaphase II (3.9% vs. 50%, basal vs. EGF; p < .001) and decreased their degeneration rate (69.9% vs. 22.2%, basal vs. EGF; p < .01) when oocytes were immediately matured. Overnight holding increased the meiotic competence of G1 oocytes (37.5% matured in basal medium) and EGF increased prophase arrest in G2 + G3 oocytes (16.1% vs. 38.8% in germinal vesicle [GV] stage in basal medium vs. EGF added medium; p < .05). Our data demonstrate that oocyte holding can be used in Iberian red deer oocytes. Interestingly, EGF addition increases the oocytes' meiotic competence in immediately matured oocytes but not after oocyte holding depending upon initial oocyte quality. © 2017 Blackwell Verlag GmbH.

Phospholipid transfer activities in toad oocytes and developing embryos. [Bufo arenarum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rusinol, A.; Salomon, R.A.; Bloj, B.

1987-01-01

The role of lipid transfer proteins during plasma membrane biogenesis was explored. Developing amphibia embryos were used because during their growth an active plasma membrane biosynthesis occurs together with negligible mitochondrial and endoplasmic reticulum proliferation. Sonicated vesicles, containing /sup 14/C-labeled phospholipids and /sup 3/H-labeled triolein, as donor particles and cross-linked erythrocyte ghosts as acceptor particles were used to measure phospholipid transfer activities in unfertilized oocytes and in developing embryos of the toad Bufo arenarum. Phosphatidylcholine transfer activity in pH 5.1 supernatant of unfertilized oocytes was 8-fold higher than the activity found in female toad liver supernatant, but dropped steadily aftermore » fertilization. After 20 hr of development, at the stage of late blastula, the phosphatidylcholine transfer activity had dropped 4-fold. Unfertilized oocyte supernatant exhibited phosphatidylinositol and phosphatidylethanolamine transfer activity also, but at the late blastula stage the former had dropped 18-fold and the latter was no longer detectable under our assay conditions. Our results show that fertilization does not trigger a phospholipid transport process catalyzed by lipid transfer proteins. Moreover, they imply that 75% of the phosphatidylcholine transfer activity and more than 95% of the phosphatidylinositol and phosphatidylethanolamine transfer activities present in pH 5.1 supernatants of unfertilized oocytes may not be essential for toad embryo development. Our findings do not rule out, however, that a phosphatidylcholine-specific lipid transfer protein could be required for embryo early growth.« less

Dynamics of intracellular phospholipid membrane organization during oocyte maturation and successful vitrification of immature oocytes retrieved by ovum pick-up in cattle.

PubMed

Aono, Akira; Nagatomo, Hiroaki; Takuma, Tetsuya; Nonaka, Rika; Ono, Yoshitaka; Wada, Yasuhiko; Abe, Yasuyuki; Takahashi, Masashi; Watanabe, Tomomasa; Kawahara, Manabu

2013-05-01

The objective was to determine if immature bovine oocytes with cumulus cells at the germinal vesicle (GV) stage could be vitrified by aluminum sheets (AS; pieces of sheet-like aluminum foil). Cleavage rates in fertilized oocytes previously vitrified by the AS procedure were higher than those vitrified by a nylon-mesh holder (NM) procedure (89.3 ± 2.1% vs. 65.0 ± 3.7%). Cleaved embryos derived from the AS but not from the NM procedures developed to blastocysts. Furthermore, to investigate the effects of vitrifying GV oocytes on cytoplasmic structure and on the ability to undergo cytoplasmic changes, the intracellular phospholipid membrane (IM) was stained with the lipophilic fluorescent dye, 3,3'-dioctadecyloxa-carbocyanine perchlorate. After vitrification by AS, the IM remained intact relative to that of oocytes vitrified by NM. During in vitro maturation, reorganization of the IM was also undamaged in oocytes vitrified by AS before oocyte maturation, and the IM within oocytes vitrified by the NM procedure was evidently impaired. Finally, vitrification (AS) was used for GV oocytes collected using the ovum pick-up method. A bull calf was born after in vitro production and subsequent embryo transfer. The vitrification techniques described herein should facilitate generation of viable in vitro production bovine blastocysts using oocytes recovered using the ovum pick-up method. Copyright © 2013 Elsevier Inc. All rights reserved.

Role of UHRF1 in de novo DNA methylation in oocytes and maintenance methylation in preimplantation embryos

PubMed Central

Toh, Hidehiro; Ohishi, Hiroaki; Sharif, Jafar; Koseki, Haruhiko; Sasaki, Hiroyuki

2017-01-01

oocytes and early embryos. PMID:28976982

Human oocyte calcium analysis predicts the response to assisted oocyte activation in patients experiencing fertilization failure after ICSI.

PubMed

Ferrer-Buitrago, M; Dhaenens, L; Lu, Y; Bonte, D; Vanden Meerschaut, F; De Sutter, P; Leybaert, L; Heindryckx, B

2018-01-10

Can human oocyte calcium analysis predict fertilization success after assisted oocyte activation (AOA) in patients experiencing fertilization failure after ICSI? ICSI-AOA restores the fertilization rate only in patients displaying abnormal Ca2+ oscillations during human oocyte activation. Patients capable of activating mouse oocytes and who showed abnormal Ca2+ profiles after mouse oocyte Ca2+ analysis (M-OCA), have variable responses to ICSI-AOA. It remains unsettled whether human oocyte Ca2+ analysis (H-OCA) would yield an improved accuracy to predict fertilization success after ICSI-AOA. Sperm activation potential was first evaluated by MOAT. Subsequently, Ca2+ oscillatory patterns were determined with sperm from patients showing moderate to normal activation potential based on the capacity of human sperm to generate Ca2+ responses upon microinjection in mouse and human oocytes. Altogether, this study includes a total of 255 mouse and 122 human oocytes. M-OCA was performed with 16 different sperm samples before undergoing ICSI-AOA treatment. H-OCA was performed for 11 patients who finally underwent ICSI-AOA treatment. The diagnostic accuracy to predict fertilization success was calculated based on the response to ICSI-AOA. Patients experiencing low or total failed fertilization after conventional ICSI were included in the study. All participants showed moderate to high rates of activation after MOAT. Metaphase II (MII) oocytes from B6D2F1 mice were used for M-OCA. Control fertile sperm samples were used to obtain a reference Ca2+ oscillation profile elicited in human oocytes. Donated human oocytes, non-suitable for IVF treatments, were collected and vitrified at MII stage for further analysis by H-OCA. M-OCA and H-OCA predicted the response to ICSI-AOA in 8 out of 11 (73%) patients. Compared to M-OCA, H-OCA detected the presence of sperm activation deficiencies with greater sensitivity (75 vs 100%, respectively). ICSI-AOA never showed benefit to overcome

Effects of Simulated Weightlessness on Mammalian Development. Part 2: Meiotic Maturation of Mouse Oocytes During Clinostat Rotation

NASA Technical Reports Server (NTRS)

Wolgemuth, D. J.; Grills, G. S.

1985-01-01

In order to understand the role of gravity in basic cellular processes that are important during development, the effects of a simulated microgravity environment on mammalian gametes and early embryos cultured in vitro are examined. A microgravity environment is simulated by use of a clinostat, which essentially reorients cells relative to the gravity vector. Initial studies have focused on assessing the effects of clinostat rotation on the meiotic progression of mouse oocytes. Modifications centered on providing the unique in vitro culture of the clinostat requirements of mammalian oocytes and embryos: 37 C temperature, constant humidity, and a 5% CO2 in air environment. The oocytes are observed under the dissecting microscope for polar body formation and gross morphological appearance. They are then processed for cytogenetic analysis.

Bovine non-competent oocytes (BCB-) negatively impact the capacity of competent (BCB+) oocytes to undergo in vitro maturation, fertilisation and embryonic development.

PubMed

Salviano, M B; Collares, F J F; Becker, B S; Rodrigues, B A; Rodrigues, J L

2016-04-01

Competent oocyte selection remains a bottleneck in the in vitro production (IVP) of mammalian embryos. Among the vital assays described for selecting competent oocytes for IVP, the brilliant cresyl blue (BCB) test has shown consistent results. The aim of the first experiment was to observe if oocytes directly submitted to IVM show similar cleavage and blastocyst rates as those obtained with oocytes maintained under the same in vitro conditions as the oocytes that undergo the BCB test. Bovine cumulus-oocyte complexes (COCs) were recovered from slaughterhouse-derived ovaries and, after morphological evaluation, were randomised grouped into three groups: (1) directly submitted to IVM; (2) oocytes submitted to the BCB test without the addition of BCB stain (BCB control group); and (3) submitted to the BCB test. The results showed that oocytes directly submitted to IVM reached similar cleavage (48/80 - 60%) and embryonic development rates to the blastocyst stage (10/48 - 21%) as the results obtained with the BCB control group oocytes (45/77 - 58% and 08/45 - 18%, respectively). The aim of the second experiment was to determine the cleavage and blastocyst rates obtained from BCB+ oocytes undergoing IVM in the presence of BCB- oocytes at a ratio of 10:1. COCs were recovered from slaughterhouse-derived ovaries and, after morphological evaluation, were randomised into two groups that were submitted to IVM either directly (1: control group) or submitted to the BCB test prior to IVM. After the BCB test, the COCs were classified as either BCB+ (blue cytoplasm) or BCB- (colourless cytoplasm) and then divided into four experimental groups: (2) BCB+; (3) BCB-; and (4) BCB+ matured in same IVM medium drop as (5) BCB- at a ratio of 10:1. After IVM (24 h), oocytes from the different experimental groups were submitted to in vitro fertilisation (IVF) and in vitro culture (IVC) under the same culture conditions until they reached the blastocyst stage (D7). With regards to the cleavage

Construction of conditional acid ceramidase knockout mice and in vivo effects on oocyte development and fertility.

PubMed

Eliyahu, Efrat; Shtraizent, Nataly; Shalgi, Ruth; Schuchman, Edward H

2012-01-01

The number of resting follicles in the ovary and their successful maturation during development define the fertile female lifespan. Oocytes, enclosed within follicles, are subject to natural selection, and the majority will undergo apoptosis during prenatal life through adulthood. Our previous studies revealed high levels of the lipid hydrolase, acid ceramidase (AC), in human and mouse oocytes, follicular fluid and cumulus cells. In addition, supplementation of in vitro fertilization media with recombinant AC enhanced the survival of oocytes and preimplantation embryos. Herein we constructed and used a conditional knockout mouse model of AC deficiency (cACKO) to further investigate the role of this enzyme in oocyte survival in vivo. Immunohistochemical staining, activity assays, and western blot analysis revealed that AC expression was high in the ovaries of normal mice, particularly in the theca cells. After induction of the AC gene knockout with tamoxifen (TM), AC levels decreased in ovaries, and ceramide was correspondingly elevated. A novel immunostaining method was developed to visualize follicles at various stages, and together with light microscopic examination, the transition of the follicle from the secondary to antral stage was found to be defective in the absence of AC. Western blot analysis showed elevated BAX and PARP expression in TM-treated cACKO mouse ovaries compared to control animals. In parallel, the levels of BCL-2 and anti-Mullerian hormone, a marker of ovarian reserve, were decreased. In addition to the above, there was a significant decrease in fertility observed in the TM-treated cACKO mice. Together, these data suggest that AC plays an important role in the preservation of fertility by maintaining low ceramide levels and preventing apoptosis of theca cells, thereby promoting survival of the follicle during the transition from the secondary to antral stage. Copyright © 2012 S. Karger AG, Basel.

Oocyte-specific deletion of N-WASP does not affect oocyte polarity, but causes failure of meiosis II completion.

PubMed

Wang, Zhen-Bo; Ma, Xue-Shan; Hu, Meng-Wen; Jiang, Zong-Zhe; Meng, Tie-Gang; Dong, Ming-Zhe; Fan, Li-Hua; Ouyang, Ying-Chun; Snapper, Scott B; Schatten, Heide; Sun, Qing-Yuan

2016-09-01

There is an unexplored physiological role of N-WASP (neural Wiskott-Aldrich syndrome protein) in oocyte maturation that prevents completion of second meiosis. In mice, N-WASP deletion did not affect oocyte polarity and asymmetric meiotic division in first meiosis, but did impair midbody formation and second meiosis completion. N-WASP regulates actin dynamics and participates in various cell activities through the RHO-GTPase-Arp2/3 (actin-related protein 2/3 complex) pathway, and specifically the Cdc42 (cell division cycle 42)-N-WASP-Arp2/3 pathway. Differences in the functions of Cdc42 have been obtained from in vitro compared to in vivo studies. By conditional knockout of N-WASP in mouse oocytes, we analyzed its in vivo functions by employing a variety of different methods including oocyte culture, immunofluorescent staining and live oocyte imaging. Each experiment was repeated at least three times, and data were analyzed by paired-samples t-test. Oocyte-specific deletion of N-WASP did not affect the process of oocyte maturation including spindle formation, spindle migration, polarity establishment and maintenance, and homologous chromosome or sister chromatid segregation, but caused failure of cytokinesis completion during second meiosis (P < 0.001 compared to control). Further analysis showed that a defective midbody may be responsible for the failure of cytokinesis completion. The present study did not include a detailed analysis of the mechanisms underlying the results, which will require more extensive further investigations. N-WASP may play an important role in mediating and co-ordinating the activity of the spindle (midbody) and actin (contractile ring constriction) when cell division occurs. The findings are important for understanding the regulation of oocyte meiosis completion and failures in this process that affect oocyte quality. None. This work was supported by the National Basic Research Program of China (No. 2012CB944404) and the National Natural

The Role of Maternal Nutrition on Oocyte Size and Quality, with Respect to Early Larval Development in The Coral-Eating Starfish, Acanthaster planci.

PubMed

Caballes, Ciemon Frank; Pratchett, Morgan S; Kerr, Alexander M; Rivera-Posada, Jairo A

2016-01-01

Variation in local environmental conditions can have pronounced effects on the population structure and dynamics of marine organisms. Previous studies on crown-of-thorns starfish, Acanthaster planci, have primarily focused on effects of water quality and nutrient availability on larval growth and survival, while the role of maternal nutrition on reproduction and larval development has been overlooked. To examine the effects of maternal nutrition on oocyte size and early larval development in A. planci, we pre-conditioned females for 60 days on alternative diets of preferred coral prey (Acropora abrotanoides) versus non-preferred coral prey (Porites rus) and compared resulting gametes and progeny to those produced by females that were starved over the same period. Females fed ad libitum with Acropora increased in weight, produced heavier gonads and produced larger oocytes compared to Porites-fed and starved females. Fed starfish (regardless of whether it was Acropora or Porites) produced bigger larvae with larger stomachs and had a higher frequency of normal larvae that reached the late bipinnaria / early brachiolaria stage compared to starved starfish. Females on Acropora diet also produced a higher proportion of larvae that progressed to more advanced stages faster compared to Porites-fed starfish, which progressed faster than starved starfish. These results suggest that maternal provisioning can have important consequences for the quality and quantity of progeny. Because food quality (coral community structure) and quantity (coral abundance) varies widely among reef locations and habitats, local variation in maternal nutrition of A. planci is likely to moderate reproductive success and may explain temporal and spatial fluctuations in abundance of this species.

Signal recognition particle assembly in relation to the function of amplified nucleoli of Xenopus oocytes.

PubMed

Sommerville, John; Brumwell, Craig L; Politz, Joan C Ritland; Pederson, Thoru

2005-03-15

The signal recognition particle (SRP) is a ribonucleoprotein machine that controls the translation and intracellular sorting of membrane and secreted proteins. The SRP contains a core RNA subunit with which six proteins are assembled. Recent work in both yeast and mammalian cells has identified the nucleolus as a possible initial site of SRP assembly. In the present study, SRP RNA and protein components were identified in the extrachromosomal, amplified nucleoli of Xenopus laevis oocytes. Fluorescent SRP RNA microinjected into the oocyte nucleus became specifically localized in the nucleoli, and endogenous SRP RNA was also detected in oocyte nucleoli by RNA in situ hybridization. An initial step in the assembly of SRP involves the binding of the SRP19 protein to SRP RNA. When green fluorescent protein (GFP)-tagged SRP19 protein was injected into the oocyte cytoplasm it was imported into the nucleus and became concentrated in the amplified nucleoli. After visiting the amplified nucleoli, GFP-tagged SRP19 protein was detected in the cytoplasm in a ribonucleoprotein complex, having a sedimentation coefficient characteristic of the SRP. These results suggest that the amplified nucleoli of Xenopus oocytes produce maternal stores not only of ribosomes, the classical product of nucleoli, but also of SRP, presumably as a global developmental strategy for stockpiling translational machinery for early embryogenesis.

Arachidonic and linoleic acid derivatives impact oocyte ICSI fertilization--a prospective analysis of follicular fluid and a matched oocyte in a 'one follicle--one retrieved oocyte--one resulting embryo' investigational setting.

PubMed

Ciepiela, Przemysław; Bączkowski, Tomasz; Drozd, Arleta; Kazienko, Anna; Stachowska, Ewa; Kurzawa, Rafał

2015-01-01

To evaluate human oocyte ability to undergo fertilization and subsequent preimplantation embryonic development in relation to a wide panel of follicular fluid (FF) arachidonic acid derivatives (AAD) and linoleic acid derivatives (LAD) of prospectively selected patients undergoing intracytoplasmic sperm injection (ICSI). Study was designed as a two center (a university clinic and a private clinic) prospective study. 54 women of 181 consecutive couples undergoing ICSI were prospectively found to be eligible for analysis. 'One follicle - one retrieved oocyte - one resulting embryo' approach was used. Each individual follicle was aspirated independently and matched to an oocyte growing in this particular follicular milieu. FF samples were assessed for AAD and LAD by high-performance liquid chromatography; additionally, activity of secretory phospholipase A (sPLA2) was determined by enzyme-linked immunosorbent assay. Increased activity of sPLA2 and significantly higher AAD and LAD levels were found in FF of oocytes that did not show two pronuclei or underwent degeneration after ICSI in comparison to oocytes with the appearance of two pronuclei. Receiver operating characteristics curve analysis identified acids with the highest sensitivity and specificity: 5oxo-hydroxyeicosatetraenoic, 16-hydroxyeicosatetraenoic, 9-hydroxyoctadecadieneoic and 12-hydroxyeicosatetraenoic. No significant differences between AAD and LAD related to embryo quality were found. Our study demonstrates for the first time that elevated concentrations of AAD and LAD in FF at the time of oocyte retrieval significantly decrease the ability of oocytes to form pronuclei after ICSI. This may serve as a new tool for non-invasive assessment of oocyte developmental capacity. However, levels of AAD and LAD are not associated with subsequent embryo quality or pregnancy rate, and therefore more studies are needed to determine their usefulness in human IVF procedure.

Elevated intracellular pH appears in aged oocytes and causes oocyte aneuploidy associated with the loss of cohesion in mice

PubMed Central

Cheng, Jin-Mei; Li, Jian; Tang, Ji-Xin; Chen, Su-Ren; Deng, Shou-Long; Jin, Cheng; Zhang, Yan; Wang, Xiu-Xia; Zhou, Chen-Xi; Liu, Yi-Xun

2016-01-01

ABSTRACT Increases in the aneuploidy rate caused by the deterioration of cohesion with increasing maternal age have been well documented. However, the molecular mechanism for the loss of cohesion in aged oocytes remains unknown. In this study, we found that intracellular pH (pHi) was elevated in aged oocytes, which might disturb the structure of the cohesin ring to induce aneuploidy. We observed for the first time that full-grown germinal vesicle (GV) oocytes displayed an increase in pHi with advancing age in CD1 mice. Furthermore, during the in vitro oocyte maturation process, the pHi was maintained at a high level, up to ∼7.6, in 12-month-old mice. Normal pHi is necessary to maintain protein localization and function. Thus, we put forward a hypothesis that the elevated oocyte pHi might be related to the loss of cohesion and the increased aneuploidy in aged mice. Through the in vitro alkalinization treatment of young oocytes, we observed that the increased pHi caused an increase in the aneuploidy rate and the sister inter-kinetochore (iKT) distance associated with the strength of cohesion and caused a decline in the cohesin subunit SMC3 protein level. Young oocytes with elevated pHi exhibited substantially the increase in chromosome misalignment. PMID:27472084

Sex aneuploidy of unfertilized human oocytes after intracytoplasmic sperm injection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, G.; Ward, D.C.; Jones, E.E.

1994-09-01

Intracytoplasmic sperm injection (ICSI) has recently achieved successful fertilization and pregnancy in human in vitro fertilization, particularly in cases of severe male factor infertility. One criticism of this novel clinical technique is that it bypasses the natural selection process of fertilization. We use fluorescence in situ hybridization (FISH) to analyze oocytes which fail to fertilize after ICSI in the Yale IVF Program. The purpose of this study is to determine whether failed fertilization after ICSI can be attributed to sex chromosome aneuploidy in the oocyte. Fertilization of oocytes is determined by the presence of two pronuclei on light microscopic examinationmore » (400X). Multi-probe FISH with DAPI (4,6,-diamino-2-phenyl-indole) counterstain is then performed to determine oocyte ploidy and the presence of decondensed sperm. Centromeric probes for X, Y and 17 are used simultaneously in each oocyte for in situ hybridization to oocyte chromatin. In all oocytes examined after ICSI to date, unfertilized oocytes have decondensed sperm DNA present confirming appropriate intracytoplasmic placement of the sperm. Preliminary results obtained from 31 oocytes have not identified any sex chromosome aneuploidies. The FISH technique used in post-ICSI oocytes is a model system for delineating genetic causes of failed fertilization in the human.« less

How do oocytes disappear?

PubMed

Bonilla-Musoles, F; Renau, J; Hernandez-Yago, J; Torres, J

1975-07-29

It has been study using transmission and scanner electron microscopy the mean procedures of dessaparence of the oocytes. On described three methods: 1. The necrosis of the oocytes. 2. The autolysis and fagocitosis by granulosa cells. 3. The migration of those to the superphicie and fall into the peritoneal cavity. Using the scanner electron microscopy in ovaries of fetus and newborn it seems the latest method to bee the most important during the intrauterine life. After the birth, this last phenomenon seems to disappear.

Role of arachidonic acid cascade in Rhinella arenarum oocyte maturation.

PubMed

Ortiz, Maria Eugenia; Arias-Torres, Ana Josefina; Zelarayán, Liliana Isabel

2015-08-01

There are no studies that document the production of prostaglandins (PGs) or their role in Rhinella arenarum oocyte maturation. In this study, we analysed the effect of arachidonic acid (AA) and prostaglandins (PGs) on maturation, activation and pronuclear formation in R. arenarum oocytes. Our results demonstrated that AA was capable of inducing maturation in time-dependent and dose-dependent manner. Arachidonic acid-induced maturation was inhibited by indomethacin. PGs from AA hydrolysis, such as prostaglandin F2α (PGF2α) and, to a lesser extent, PGE2, induced meiosis resumption. Oocyte maturation in response to PGF2α was similar to that produced by progesterone (P4). Oocyte response to PGE1 was scarce. Rhinella arenarum oocyte PGF2α-induced maturation showed seasonal variation. From February to June, oocytes presented low sensitivity to PGF2α. In following periods, this response increased until a maximum was reached during October to January, a close temporal correlation with oocyte response to P4 being observed. The effect of PGF2α on maturation was verified by analysing the capacity of oocytes to activate and form pronuclei after being injected with homologous sperm. The cytological analysis of activated oocytes demonstrated the absence of cortical granules in oocytes, suggesting that PGF2α induces germinal vesicle breakdown (GVBD) and meiosis resumption up to metaphase II. In turn, oocytes matured by the action of PGF2α were able to form pronuclei after fertilization in a similar way to oocyte maturated by P4. In microinjection of mature cytoplasm experiments, the transformation of pre-maturation promoting factor (pre-MPF) to MPF was observed when oocytes were treated with PGF2α. In summary, our results illustrated the participation of the AA cascade and its metabolites in maturation, activation and pronuclei formation in R. arenarum.

Vitrification, in vitro fertilization, and development of Atg7 deficient mouse oocytes.

PubMed

Bang, Soyoung; Lee, Geun-Kyung; Shin, Hyejin; Suh, Chang Suk; Lim, Hyunjung Jade

2016-03-01

Autophagy contributes to the clearance and recycling of macromolecules and organelles in response to stress. We previously reported that vitrified mouse oocytes show acute increases in autophagy during warming. Herein, we investigate the potential role of Atg7 in oocyte vitrification by using an oocyte-specific deletion model of the Atg7 gene, a crucial upstream gene in the autophagic pathway. Oocyte-specific Atg7 deficient mice were generated by crossing Atg7 floxed mice and Zp3-Cre transgenic mice. The oocytes were vitrified-warmed and then subjected to in vitro fertilization and development. The rates of survival, fertilization, and development were assessed in the Atg7 deficient oocytes in comparison with the wildtype oocytes. Light chain 3 (LC3) immunofluorescence staining was performed to determine whether this method effectively evaluates the autophagy status of oocytes. The survival rate of vitrified-warmed Atg7(f/f) ;Zp3-Cre (Atg7(d/d) ) metaphase II (MII) oocytes was not significantly different from that of the wildtype (Atg7(f/f) ) oocytes. Fertilization and development in the Atg7(d/d) oocytes were significantly lower than the Atg7(f/f) oocytes, comparable to the Atg5(d/d) oocytes previously described. Notably, the developmental rate improved slightly in vitrified-warmed Atg7(d/d) MII oocytes when compared to fresh Atg7(d/d) oocytes. LC3 immunofluorescence staining showed that this method can be reliably used to assess autophagic activation in oocytes. We confirmed that the LC3-positive signal is nearly absent in Atg7(d/d) oocytes. While autophagy is induced during the warming process after vitrification of MII oocytes, the Atg7 gene is not essential for survival of vitrified-warmed oocytes. Thus, induction of autophagy during warming of vitrified MII oocytes seems to be a natural response to manage cold or other cellular stresses.

Injurious effects of emodin on maturation of mouse oocytes, fertilization and fetal development via apoptosis.

PubMed

Chang, Mei-Hui; Chang, Shao-Chung; Chan, Wen-Hsiung

2012-10-29

Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a major constituent of rhubarb, has a wide range of therapeutic applications. Previous studies have established that emodin induces apoptosis in the inner cell mass and trophectoderm of mouse blastocysts and leads to decreased embryonic development and viability, indicating a role as an injury risk factor for normal embryonic development. However, the mechanisms underlying its hazardous effects have yet to be characterized. In the current study, we further investigated the effects of emodin on oocyte maturation and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, emodin induced a significant reduction in the rates of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with emodin during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased fetal weight. Experiments using an in vivo mouse model disclosed that consumption of drinking water containing 20-40 μM emodin led to decreased oocyte maturation and in vitro fertilization, as well as early embryonic developmental injury. Notably, pretreatment with a caspase-3-specific inhibitor effectively prevented emodin-triggered injury effects, suggesting that impairment of embryo development occurs via a caspase-dependent apoptotic process.

[Successful pregnancies after oocyte and embryo vitrification].

PubMed

Salazar, Francisco Hernández; Loza, Erik Omar Okhuysen; Lucas, Maria Teresa Huerta J; Gutiérrez, Gustavo Romero

2008-02-01

Cryopreservation of human oocytes represents a solution for ethic conflict about frozen embryo storage for patients with risk to develop ovarian hyperstimulation syndrome; also is an available technique to preserve fertility in women with cancer under treatment, in poor response patients, in case of premature ovarian failure or aging and for other medical or social conditions that require to delay pregnancies, as well as to make easier oocyte donation programs. This paper reports two cases of successful pregnancies after embryo and oocyte vitrification, as well as their results. The technique of vitrification with the cryotop method is an excellent alternative, efficient, fast and cheap for oocyte and embryo cryopreservation with high ranges of fertilization, cleavage and pregnancies with a normal evolution.

Oocyte Degeneration Associated with Follicle Cells in Female Mactra chinensis (Bivalvia: Mactridae)

PubMed Central

Kim, Sung Han; Chung, Ee-Yung; Lee, Ki-Young

2014-01-01

Ultrastructural studies of oocyte degeneration in the oocyte, and the functions of follicle cells during oocyte degeneration are described to clarify the reproductive mechanism on oocyte degeneration of Mactra chinensis using cytological methods. Commonly, the follicle cells are attached to the oocyte. Follicle cells play an important role in oocyte degeneration. In particular, the functions of follicle cells during oocyte degeneration are associated with phagocytosis and the intracellular digestion of products. In this study, morphologically similar degenerated phagosomes (various lysosomes), which were observed in the degenerated oocytes, appeared in the follicle cells. After the spawning of the oocytes, the follicle cells were involved in oocyte degeneration through phagocytosis by phagolysosomes. Therefore, it can be assumed that follicle cells reabsorb phagosomes from degenerated oocytes. In this study, the presence of lipid granules, which occurred from degenerating yolk granules, gradually increased in degenerating oocytes. The function of follicle cells can accumulate reserves of lipid granules and glycogen in the cytoplasm, which can be employed by the vitellogenic oocyte. Based on observations of follicle cells attached to degenerating oocytes after spawning, the follicle cells of this species are involved in the lysosomal induction of oocyte degeneration for the reabsorption of phagosomes (phagolysosomes) in the cytoplasm for nutrient storage, as seen in other bivalves. PMID:25949203

Expression of mammalian beta-adrenergic receptors in Xenopus laevis oocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bahouth, S.W.; Malbon, C.C.

1987-05-01

Xenopus laevis oocytes are a useful transcription and expression system for DNA and RNA, respectively. Total cellular RNA was extracted from mouse lymphoma S49 cells and poly(A)/sup +/mRNA prepared by affinity chromatography of RNA on oligo(dT) cellulose. The membranes of S49 cells contain beta-adrenergic receptors that display pharmacological characteristics of beta/sub 2/-subtype. Xenopus laevis oocytes were injected with 50 ng of mRNA/oocyte. Expression of beta-adrenergic receptors in oocytes incubated for 30 hr after microinjection was assessed in membranes by radioligand binding using (/sup 3/H) dihydroalprenolol. The injected oocytes displayed 0.34 fmol receptor/oocyte as compared to 0.02 fmol receptor/oocyte in themore » control oocytes. The affinity of beta-adrenergic receptors in injected oocytes for this radioligand was 2 nM, a value similar to the affinity of beta-adrenergic receptors for DHA in S49 cell membranes. The potency of beta-adrenergic agonists in competing for DHA binding to oocytes membranes was isoproterenol > epinephrine > norepineprine, indicating that the expressed beta-adrenergic receptors were of the beta/sub 2/-subtype. The K/sub I/ of these agonists for the beta-adrenergic receptor in oocyte membranes was 0.03, 0.15 and 1.2 ..mu..M, respectively. The role of post-translational modification in dictating receptor subtype is analyzed using mRNA of beta/sub 1/- as well as beta/sub 2/-adrenergic receptors.« less

Cortical granule exocytosis in Bufo arenarum oocytes matured in vitro.

PubMed

Oterino, J; Sanchez Toranzo, G; Zelarayán, L; Valz-Gianinet, J N; Bühler, M I

2001-08-01

Denuded Bufo arenarum oocytes matured in vitro by progesterone treatment exhibited abnormal segmentation due to the penetration of more than one sperm. These oocytes were able to respond to activation stimuli and exhibited the external signs characteristic of activation. However, the prevention of polyspermy was not effective in these oocytes, which exhibited numerous sperm in their cytoplasm. The aim of this work was to analyse the cortical reaction in polyspermic Bufo arenarum oocytes matured in vitro. The result indicate that the cortical reaction of these oocytes seems to occur with a chronological sequence similar to that described for ovoposited oocytes of this species. In addition, when, 1 min after pricking, cortical granule exocytosis occurred, the oocytes became refractory to sperm entry, suggesting that they are able to establish a slow block to polyspermy.

Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA.

PubMed

Shishova, Kseniya V; Lavrentyeva, Elena A; Dobrucki, Jurek W; Zatsepina, Olga V

2015-01-15

It is well known that fully-grown mammalian oocytes, rather than typical nucleoli, contain prominent but structurally homogenous bodies called "nucleolus-like bodies" (NLBs). NLBs accumulate a vast amount of material, but their biochemical composition and functions remain uncertain. To clarify the composition of the NLB material in mouse GV oocytes, we devised an assay to detect internal oocyte proteins with fluorescein-5-isothiocyanate (FITC) and applied the fluorescent RNA-binding dye acridine orange to examine whether NLBs contain RNA. Our results unequivocally show that, similarly to typical nucleoli, proteins and RNA are major constituents of transcriptionally active (or non-surrounded) NLBs as well as of transcriptionally silent (or surrounded) NLBs. We also show, by exposing fixed oocytes to a mild proteinase K treatment, that the NLB mass in oocytes of both types contains nucleolar proteins that are involved in all major steps of ribosome biogenesis, including rDNA transcription (UBF), early rRNA processing (fibrillarin), and late rRNA processing (NPM1/nucleophosmin/B23, nucleolin/C23), but none of the nuclear proteins tested, including SC35, NOBOX, topoisomerase II beta, HP1α, and H3. The ribosomal RPL26 protein was detected within the NLBs of NSN-type oocytes but is virtually absent from NLBs of SN-type oocytes. Taking into account that the major class of nucleolar RNA is ribosomal RNA (rRNA), we applied fluorescence in situ hybridization with oligonucleotide probes targeting 18S and 28S rRNAs. The results show that, in contrast to active nucleoli, NLBs of fully-grown oocytes are impoverished for the rRNAs, which is consistent with the absence of transcribed ribosomal genes in the NLB mass. Overall, the results of this study suggest that NLBs of fully-grown mammalian oocytes serve for storing major nucleolar proteins but not rRNA. Copyright © 2014 Elsevier Inc. All rights reserved.

On-chip enucleation of an oocyte by untethered microrobots

NASA Astrophysics Data System (ADS)

Ichikawa, Akihiko; Sakuma, Shinya; Sugita, Masakuni; Shoda, Tatsuro; Tamakoshi, Takahiro; Akagi, Satoshi; Arai, Fumihito

2014-09-01

We propose a novel on-chip enucleation of an oocyte with zona pellucida by using a combination of untethered microrobots. To achieve enucleation within the closed space of a microfluidic chip, two microrobots, a microknife and a microgripper were integrated into the microfluidic chip. These microrobots were actuated by an external magnetic force produced by permanent magnets placed on the robotic stage. The tip of the microknife was designed by considering the biological geometric feature of an oocyte, i.e. the oocyte has a polar body in maturation stage II. Moreover, the microknife was fabricated by using grayscale lithography, which allows fabrication of three-dimensional microstructures. The microgripper has a gripping function that is independent of the driving mechanism. On-chip enucleation was demonstrated, and the enucleated oocytes are spherical, indicating that the cell membrane of the oocytes remained intact. To confirm successful enucleation using this method, we investigated the viability of oocytes after enucleation. The results show that the production rate, i.e. the ratio between the number of oocytes that reach the blastocyst stage and the number of bovine oocytes after nucleus transfer, is 100%. The technique will contribute to complex cell manipulation such as cell surgery in lab-on-a-chip devices.

Tetraspanins and Mouse Oocyte Microvilli Related to Fertilizing Ability.

PubMed

Benammar, Achraf; Ziyyat, Ahmed; Lefèvre, Brigitte; Wolf, Jean-Philippe

2017-07-01

Our electron microscopy observations demonstrate for the first time that the number of microvilli on the mice oocyte membrane decreases when meiosis progresses from prophase I to metaphase II (MII) stage, and the morphology of the microvilli also changes. Microvilli are significantly shorter and larger on the ovulated oocyte membrane than at the previous stages. Although clathrin vesicles clearly disappear during oocyte maturation, exosome-like vesicles begin to be secreted at the metaphase I stage, more strongly at the MII stage. Multivesicular bodies are visible only at the MII stage. Since several oocyte tetraspanins are involved in the gamete interaction, Cd9 being congregated on the MII oocyte microvilli, we analyzed the effect of tetraspanin deletion on oocyte membrane morphology. The Cd9 -/- and Cd9 -/- Cd81 -/- deletions are associated with a decreased microvilli density on the MII oocyte surface. Microvilli thickness is significantly increased whatever the deleted tetraspanin gene be. Only Cd9 deletion clearly disturbs the vesicular traffic, increasing the number of clathrin and exosome vesicles. Additional investigations are necessary to elucidate how tetraspanins modulate the microvilli morphology, likely in relation with cytoskeleton. The role of oocyte exosomes in gamete adhesion/fusion remains to be further studied.

Evolution of human oocyte cryopreservation: slow freezing versus vitrification.

PubMed

Levi-Setti, Paolo Emanuele; Patrizio, Pasquale; Scaravelli, Giulia

2016-12-01

The purpose is to determine the efficiency and efficacy of oocyte cryopreservation by slow freezing versus vitrification, recent data collected from the Italian National Assisted Reproductive Technology Register during the period 2009-2014 will be presented and reviewed. The data on oocyte cryopreservation were also compared with the results obtained with embryo cryopreservation and relative IVF with fresh oocytes. During the period 2009-2014 preservation of oocytes by vitrification had a significantly higher survival rate, implantation, and pregnancy rate than slow freezing; however, there are still large variations in success rates among centers in relation to the number of procedures performed. Vitrification has now become the method of choice for oocyte cryopreservation because of better results than slow freezing, but still requires a more standardized utilization. The transfer of fresh or cryopreserved embryo still shows a statistically significant better performance than transfers with embryos obtained with cryopreserved oocytes. Only in a few centers with much experience in cryopreservation are the results between transfers of frozen embryos or embryos obtained from oocyte cryopreservation comparable.

Onset and progress of meiotic prophase in the oocytes in the B6.YTIR sex-reversed mouse ovary.

PubMed

Park, E-H; Taketo, T

2003-12-01

When the Y chromosome of a Mus musculus domesticus male mouse (caught in Tirano, Italy) is placed on a C57BL/6J genetic background, approximately half of the XY (B6.YTIR) progeny develop into normal-appearing but infertile females. We have previously reported that the primary cause of infertility can be attributed to their oocytes. To identify the primary defect in the XY oocyte, we examined the onset and progress of meiotic prophase in the B6.YTIR fetal ovary. Using bromo-deoxyuridine incorporation and culture, we determined that the germ cells began to enter meiosis at the developmental ages and in numbers comparable to those in the control XX ovary. Furthermore, the meiotic prophase appeared to progress normally until the late zygotene stage. However, the oocytes that entered meiosis early in the XY ovary failed to complete the meiotic prophase. On the other hand, a considerable number of oocytes entered meiosis at late developmental stages and completed the meiotic prophase in the XY ovary. We propose that the timing of entry into meiosis and the XY chromosomal composition influence the survival of oocytes during meiotic prophase in the fetal ovary.

Identification of oocyte progenitor cells in the zebrafish ovary.

PubMed

Draper, Bruce W

2012-01-01

Zebrafish breed year round and females are capable of producing thousands of eggs during their lifetime. This amazing fecundity is due to the fact that the adult ovary, contains premeiotic oocyte progenitor cells, called oogonia, which produce a continuous supply of new oocytes throughout adult life. Oocyte progenitor cells can be easily identified based on their expression of Vasa, and their characteristic nuclear morphology. Thus, the zebrafish ovary provides a unique and powerful system to study the genetic regulation of oocyte production in a vertebrate animal. A method is presented here for identifying oocyte progenitor cells in the zebrafish ovary using whole-mount confocal immunofluorescence that is simple and accurate.

Oocyte Polarization Is Coupled to the Chromosomal Bouquet, a Conserved Polarized Nuclear Configuration in Meiosis

PubMed Central

Elkouby, Yaniv M.; Jamieson-Lucy, Allison; Mullins, Mary C.

2016-01-01

The source of symmetry breaking in vertebrate oocytes is unknown. Animal—vegetal oocyte polarity is established by the Balbiani body (Bb), a conserved structure found in all animals examined that contains an aggregate of specific mRNAs, proteins, and organelles. The Bb specifies the oocyte vegetal pole, which is key to forming the embryonic body axes as well as the germline in most vertebrates. How Bb formation is regulated and how its asymmetric position is established are unknown. Using quantitative image analysis, we trace oocyte symmetry breaking in zebrafish to a nuclear asymmetry at the onset of meiosis called the chromosomal bouquet. The bouquet is a universal feature of meiosis where all telomeres cluster to one pole on the nuclear envelope, facilitating chromosomal pairing and meiotic recombination. We show that Bb precursor components first localize with the centrosome to the cytoplasm adjacent to the telomere cluster of the bouquet. They then aggregate around the centrosome in a specialized nuclear cleft that we identified, assembling the early Bb. We show that the bouquet nuclear events and the cytoplasmic Bb precursor localization are mechanistically coordinated by microtubules. Thus the animal—vegetal axis of the oocyte is aligned to the nuclear axis of the bouquet. We further show that the symmetry breaking events lay upstream to the only known regulator of Bb formation, the Bucky ball protein. Our findings link two universal features of oogenesis, the Bb and the chromosomal bouquet, to oocyte polarization. We propose that a meiotic—vegetal center couples meiosis and oocyte patterning. Our findings reveal a novel mode of cellular polarization in meiotic cells whereby cellular and nuclear polarity are aligned. We further reveal that in zygotene nests, intercellular cytoplasmic bridges remain between oocytes and that the position of the cytoplasmic bridge coincides with the location of the centrosome meiotic—vegetal organizing center. These

Ethical Dilemmas for Oocyte Donations: Slippery Slope for Conflicts of Interest.

PubMed

Tulay, Pinar

2016-01-01

Oocyte donations have increased with improvements in oocyte cryopreservation procedures in recent years. Women with medical conditions that require chemotherapy or radiotherapy have begun to opt for oocyte cryo¬preservation prior to their treatment or to enroll in an oocyte donation program. Alternatively, some women apply for "third-party" oocyte donation programs for nonmedical reasons such as delayed childbearing. Although society seems to accept oocyte donations for medical reasons, it appears that there are still some moral issues surrounding nonmedical oocyte donations. In this review, the ethical aspects of oocyte donations and donors' perspectives are discussed. With developing technologies, the genetic screening of donors has expanded to include diseases. This review explores the ethical issues involved in genetic screening of gamete donors.

Vitrification of oocytes from endangered Mexican gray wolves (Canis lupus baileyi).

PubMed

Boutelle, S; Lenahan, K; Krisher, R; Bauman, K L; Asa, C S; Silber, S

2011-03-01

Careful genetic management, including cryopreservation of genetic material, is central to conservation of the endangered Mexican gray wolf. We tested a technique, previously used to vitrify human and domestic animal oocytes, on oocytes from domestic dogs as a model and from the endangered Mexican wolf. This method provided a way to conserve oocytes from genetically valuable older female Mexican wolves as an alternative to embryos for preserving female genes. Oocytes were aspirated from ovaries of 36 female dogs in December and March (0 to 65 oocytes per female) and from six female wolves (4 to 73 per female) during their physiologic breeding season, or following stimulation with the GnRH agonist deslorelin. Oocytes from dogs were pooled; half were immediately tested for viability and the remainder vitrified, then warmed and tested for viability. All oocytes were vitrified by being moved through media of increasing cryoprotectant concentration, placed on Cryotops, and plunged into liquid nitrogen. There was no difference in viability (propidium iodide staining) between fresh and vitrified, warmed dog oocytes (65.7 and 61.0%, respectively, P = 0.27). Oocyte viability after warming was similarly assessed in a subset of wolves (4 to 15 oocytes from each of three females; total 29 oocytes). Of these, 57.1% of the post-thaw intact oocytes were viable, which was 41.4% of all oocytes warmed. These were the first oocytes from a canid or an endangered species demonstrated to have maintained viability after vitrification and warming. Furthermore, our results demonstrated that vitrification of oocytes with the Cryotop technique was an option for preserving female gametes from Mexican wolves for future use in captive breeding programs, although in vitro embryo production techniques must first be developed in canids for this technique to be used. Copyright © 2011 Elsevier Inc. All rights reserved.

Association of Intrauterine and Early-Life Exposures With Age at Menopause in the Sister Study

PubMed Central

Steiner, Anne Z.; D'Aloisio, Aimee A.; DeRoo, Lisa A.; Sandler, Dale P.; Baird, Donna D.

2010-01-01

Oocytes are formed in utero; menopause occurs when the oocyte pool is depleted. The authors hypothesized that early-life events could affect the number of a woman's oocytes and determine age at menopause. To test their hypothesis, the authors conducted a secondary analysis of baseline data from 22,165 participants in the Sister Study (2003–2007) who were aged 35–59 years at enrollment. To estimate the association between early-life events and age at natural menopause, the authors used Cox proportional hazards models to estimate hazard ratios with 95% confidence intervals, adjusting for current age, race/ethnicity, education, childhood family income, and smoking history. Earlier menopause was associated with in-utero diethylstilbestrol exposure (hazard ratio (HR) = 1.45, 95% confidence interval (CI): 1.27, 1.65). Suggestive associations included maternal prepregnancy diabetes (HR = 1.33, 95% CI: 0.89, 1.98) and low birth weight (HR = 1.09, 95% CI: 0.99, 1.20). Having a mother aged 35 years or older at birth appeared to be associated with a later age at menopause (HR = 0.95, 95% CI: 0.89, 1.01). Birth order, in-utero smoke exposure, and having been breastfed were not related to age at menopause. In-utero and perinatal events may subsequently influence age at menopause. PMID:20534821

Influence of co-culture with denuded oocytes during in vitro maturation on fertilization and developmental competence of cumulus-enclosed porcine oocytes in a defined system.

PubMed

Appeltant, Ruth; Somfai, Tamás; Kikuchi, Kazuhiro; Maes, Dominiek; Van Soom, Ann

2016-04-01

Co-culture of cumulus-oocyte complexes (COCs) with denuded oocytes (DOs) during in vitro maturation (IVM) was reported to improve the developmental competence of oocytes via oocyte-secreted factors in cattle. The aim of the present study was to investigate if addition of DOs during IVM can improve in vitro fertilization (IVF) and in vitro culture (IVC) results for oocytes in a defined in vitro production system in pigs. The maturation medium was porcine oocyte medium supplemented with gonadotropins, dbcAMP and β-mercaptoethanol. Cumulus-oocyte complexes were matured without DOs or with DOs in different ratios (9 COC, 9 COC+16 DO and 9 COC+36 DO). Consequently; oocytes were subjected to IVF as intact COCs or after denudation to examine if DO addition during IVM would affect cumulus or oocyte properties. After fertilization, penetration and normal fertilization rates of zygotes were not different between all tested groups irrespective of denudation before IVF. When zygotes were cultured for 6 days, no difference could be observed between all treatment groups in cleavage rate, blastocyst rate and cell number per blastocyst. In conclusion, irrespective of the ratio, co-culture with DOs during IVM did not improve fertilization parameters and embryo development of cumulus-enclosed porcine oocytes in a defined system. © 2015 Japanese Society of Animal Science.

How FSH and AMH reflect probabilities of oocyte numbers in poor prognosis patients with small oocyte yields.

PubMed

Gleicher, Norbert; Darmon, Sarah K; Kushnir, Vitaly A; Weghofer, Andrea; Wang, Qi; Zhang, Lin; Albertini, David F; Barad, David H

2016-11-01

In poor prognosis patients undergoing in vitro fertilization, advance determinations of likely oocyte yields are especially important since oocyte numbers to large degree determine in vitro fertilization cycle outcomes. Based on baseline follicle stimulating hormone and anti-müllerian hormone levels at time of initial presentation, we here, therefore, determined at all ages the probabilities of obtaining 1-≥5 oocytes in a retrospective analysis of 1554 consecutive patients undergoing in vitro fertilization cycles at an academically affiliated private fertility center. At lowest levels (≤2.5 mIU/mL), Follicle stimulating hormone at all ages was highly predictable for ≥1 oocyte (88-96 %). Probabilities declined and diverged between ages with increasing follicle stimulating hormone, though narrowed again at high follicle stimulating hormone. Anti-Müllerian hormone demonstrated at higher levels (2.5-≥5 ng/ml) at all ages almost perfect probabilities (99-100 %). With declining anti-Müllerian hormone, age categories, however, increasingly diverged, though to lesser degree than follicle stimulating hormone. In poor prognosis patients, follicle stimulating hormone and anti-Müllerian hormone, thus, offer at different ages very specific probabilities for retrieval of 1-≥5 oocytes. Since oocyte numbers are associated with embryo numbers, and numbers of transferable embryos with live birth rates, here presented probability tables should facilitate improved prognostication of poor prognosis patients. Discrepancies in here reported probabilities between follicle stimulating hormone and anti-müllerian hormone also further define follicle stimulating hormone and anti-müllerian hormone in their respective abilities to represent functional ovarian reserve at different ages.

Journey of oocyte from metaphase-I to metaphase-II stage in mammals.

PubMed

Sharma, Alka; Tiwari, Meenakshi; Gupta, Anumegha; Pandey, Ashutosh N; Yadav, Pramod K; Chaube, Shail K

2018-08-01

In mammals, journey from metaphase-I (M-I) to metaphase-II (M-II) is important since oocyte extrude first polar body (PB-I) and gets converted into haploid gamete. The molecular and cellular changes associated with meiotic cell cycle progression from M-I to M-II stage and extrusion of PB-I remain ill understood. Several factors drive oocyte meiosis from M-I to M-II stage. The mitogen-activated protein kinase3/1 (MAPK3/1), signal molecules and Rho family GTPases act through various pathways to drive cell cycle progression from M-I to M-II stage. The down regulation of MOS/MEK/MAPK3/1 pathway results in the activation of anaphase-promoting complex/cyclosome (APC/C). The active APC/C destabilizes maturation promoting factor (MPF) and induces meiotic resumption. Several signal molecules such as, c-Jun N-terminal kinase (JNK2), SENP3, mitotic kinesin-like protein 2 (MKlp2), regulator of G-protein signaling (RGS2), Epsin2, polo-like kinase 1 (Plk1) are directly or indirectly involved in chromosomal segregation. Rho family GTPase is another enzyme that along with cell division cycle (Cdc42) to form actomyosin contractile ring required for chromosomal segregation. In the presence of origin recognition complex (ORC4), eccentrically localized haploid set of chromosomes trigger cortex differentiation and determine the division site for polar body formation. The actomyosin contractile activity at the site of division plane helps to form cytokinetic furrow that results in the formation and extrusion of PB-I. Indeed, oocyte journey from M-I to M-II stage is coordinated by several factors and pathways that enable oocyte to extrude PB-I. Quality of oocyte directly impact fertilization rate, early embryonic development, and reproductive outcome in mammals. © 2018 Wiley Periodicals, Inc.

N-octanoylated ghrelin peptide inhibits bovine oocyte meiotic resumption.

PubMed

Xu, X L; Bai, J H; Feng, T; Xiao, L L; Song, Y Q; Xiao, Y X; Liu, Y

2018-07-01

Studies have shown that ghrelin plays an important role in the mammalian reproductive system, including the central, gonadal levels, and also during in vitro maturation of oocytes; however, the functions of ghrelin in bovine oocyte meiosis require further investigation. We aimed to evaluate the effects of an n-octanoylated ghrelin peptide on oocyte meiotic resumption and the developmental competence of mature oocytes in vitro. design: The expression of GHRL (encoding ghrelin) mRNA and its receptor (the growth hormone secretagogue receptor, GHSR) in the cumulus-oocyte complex (COCs), denuded oocytes (DOs), and cumulus cells (CCs) was assessed using quantitative real-time reverse transcription PCR (qRT-PCR), and the effects of the n-octanoylated ghrelin peptide on meiotic resumption were studied at four different doses (0, 10, 50, and 100 ng/mL) in a 6 h culture system. qRT-PCR analysis showed that GHRL and GHSR mRNAs were expressed in all tested samples; however, GHRL was predominantly expressed in DOs, and GHSR was predominantly expressed in CCs. Germinal vesicle breakdown was inhibited significantly by 50 ng/mL ghrelin compared with that in the negative control (P < 0.05). Further studies showed that n-octanoylated ghrelin increased the levels of cAMP and cGMP in the CCs and DOs, which inhibited the meiotic resumption of bovine oocytes. And the inhibitory role in the developmental competence of mature oocytes were also included, ghrelin could significantly improve the cleavage rate (P < 0.05) and blastocyst rate (P < 0.05). N-octanoylated ghrelin maintained bovine oocytes meiotic arrest and further improved their developmental competence; therefore, n-octanoylated ghrelin could be considered as a potential pharmaceutical inhibitor of meiosis for the in vitro maturation of bovine oocytes. Copyright © 2018 Elsevier Inc. All rights reserved.

Quality of common marmoset (Callithrix jacchus) oocytes collected after ovarian stimulation.

PubMed

Kanda, Akifumi; Nobukiyo, Asako; Yoshioka, Miyuki; Hatakeyama, Teruhiko; Sotomaru, Yusuke

2018-01-15

The common marmoset (Callithrix jacchus) is an experimental animal that is considered suitable for the creation of next-generation human disease models. It has recently been used in the reproductive technology field. Oocytes can be effectively collected from female marmosets via ovarian stimulation with injections of follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG). The oocytes, collected about 28 h after the hCG injection, include both premature oocytes and postmature (in vivo matured; IVO) oocytes, and the premature oocytes can be matured by in vitro culture (in vitro matured; IVM). Although IVM and IVO oocytes are equivalent in appearance at the MII stage, it remains unclear whether there are differences in their properties. Therefore, we investigated their in vitro fertilization and developmental capacities and cytoskeletal statuses. Our findings revealed that the IVM and IVO oocytes had similar fertilization rates but that no IVO oocytes could develop to the blastocyst stage. Additionally, IVO oocytes showed abnormal cytoskeletal formation. It is concluded that IVM oocytes maintain normal function, whereas IVO oocytes would be affected by aging and other factors when they remain for a long time in the ovary. Copyright © 2017 Elsevier Inc. All rights reserved.

Early Career Challenges in Secondary School Music Teaching

ERIC Educational Resources Information Center

Welch, G.; Purves, R.; Hargreaves, D.; Marshall, N.

2011-01-01

The article reports an Economic and Social Research Council-funded study of the early career experiences of secondary school music teachers in England, set within a wider national picture of decreasing age-related pupil engagement with school music, career perceptions of music teaching, variable patterns of teacher recruitment and possible…

Oocyte cryopreservation for donor egg banking.

PubMed

Cobo, Ana; Remohí, José; Chang, Ching-Chien; Nagy, Zsolt Peter

2011-09-01

Oocyte donation is an efficient alternative to using own oocytes in IVF treatment for different indications. Unfortunately, 'traditional' (fresh) egg donations are challenged with inefficiency, difficulties of synchronization, very long waiting periods and lack of quarantine measures. Given the recent improvements in the efficiency of oocyte cryopreservation, it is reasonable to examine if egg donation through oocyte cryopreservation has merits. The objective of the current manuscript is to review existing literature on this topic and to report on the most recent outcomes from two established donor cryobank centres. Reports on egg donation using slow freezing are scarce and though results are encouraging, outcomes are not yet comparable to a fresh egg donation treatment. Vitrification on the other hand appears to provide high survival rates (90%) of donor oocytes and comparable fertilization, embryo development, implantation and pregnancy rates to traditional (fresh) egg donation. Besides the excellent outcomes, the ease of use for both donors and recipients, higher efficiency, lower cost and avoiding the problem of synchronization are all features associated with the benefit of a donor egg cryobank and makes it likely that this approach becomes the future standard of care. Oocyte donation is one of the last resorts in IVF treatment for couples challenged with infertility problems. However, traditional (fresh) egg donation, as it is performed today, is not very efficient, as typically all eggs from one donor are given to only one recipient, it is arduous as it requires an excellent synchronization between the donor and recipient and there are months or years of waiting time. Because of the development of an efficient oocyte cryopreservation technique, it is now possible to cryo-store donor (as well as non-donor) eggs, maintaining their viability and allowing their use whenever there is demand. Therefore, creating a donor oocyte cryobank would carry many advantages

Amino acid turnover by human oocytes is influenced by gamete developmental competence, patient characteristics and gonadotrophin treatment

PubMed Central

Hemmings, K.E.; Maruthini, D.; Vyjayanthi, S.; Hogg, J.E.; Balen, A.H.; Campbell, B.K.; Leese, H.J.; Picton, H.M.

2013-01-01

STUDY QUESTION Can amino acid profiling differentiate between human oocytes with differing competence to mature to metaphase II (MII) in vitro? SUMMARY ANSWER Oocytes which remained arrested at the germinal vesicle (GV) stage after 24 h of in vitro maturation (IVM) displayed differences in the depletion/appearance of amino acids compared with oocytes which progressed to MII and patient age, infertile diagnosis and ovarian stimulation regime significantly affected oocyte amino acid turnover during IVM. WHAT IS KNOWN ALREADY Amino acid profiling has been proposed as a technique which can distinguish between human pronucleate zygotes and cleavage stage embryos with the potential to develop to the blastocyst stage and implant to produce a pregnancy and those that arrest. Most recently, the amino acid turnover by individual bovine oocytes has been shown to be predictive of oocyte developmental competence as indicated by the gamete's capacity to undergo fertilization and early cleavage divisions in vitro. STUDY DESIGN, SIZE, DURATION The study was conducted between March 2005 and March 2010. A total of 216 oocytes which were at the GV or metaphase I (MI) stages at the time of ICSI were donated by 67 patients. PARTICIPANTS/MATERIALS, SETTINGS, METHODS The research was conducted in university research laboratories affiliated to a hospital-based infertility clinic. Oocytes were cultured for 24 h and the depletion/appearance of amino acids was measured during the final 6 h of IVM. Amino acid turnover was analysed in relation to oocyte meiotic progression, patient age, disease aetiology and controlled ovarian stimulation regime. MAIN RESULTS AND THE ROLE OF CHANCE The depletion/appearance of key amino acids was linked to the maturation potential of human oocytes in vitro. Oocytes which arrested at the GV stage (n = 9) depleted significantly more valine and isoleucine than those which progressed to MI (n = 32) or MII (n = 107) (P < 0.05). Glutamate, glutamine, arginine and

Xenopus laevis oocyte maturation is affected by metal chlorides.

PubMed

Marin, Matthieu; Slaby, Sylvain; Marchand, Guillaume; Demuynck, Sylvain; Friscourt, Noémie; Gelaude, Armance; Lemière, Sébastien; Bodart, Jean-François

2015-08-01

Few studies have been conducted using Xenopus laevis germ cells as oocytes, though these cells offer many advantages allowing both electrophysiological studies and morphological examination. Our aim was to investigate the effects of metal (cadmium, lead, cobalt and zinc) exposures using cell biology approaches. First, cell survival was evaluated with both phenotypical and electrophysiological approaches. Secondly, the effect of metals on oocyte maturation was assessed with morphological observations and electrophysiological recordings. From survival experiments, our results showed that metal chlorides did not affect cell morphology but strongly depolarized X. laevis oocyte resting potential. In addition, cadmium chloride was able to inhibit progesterone-induced oocyte maturation. By contrast, zinc, but also to a lesser extent cadmium, cobalt and lead, were able to enhance spontaneous oocyte maturation in the absence of progesterone stimulation. Finally, electrophysiological recordings revealed that some metal chlorides (lead, cadmium) exposures could disturb calcium signaling in X. laevis oocyte by modifying calcium-activated chloride currents. Our results demonstrated the high sensitivity of X. laevis oocytes toward exogenous metals such as lead and cadmium. In addition, the cellular events recorded might have a predictive value of effects occurring later on the ability of oocytes to be fertilized. Together, these results suggest a potential use of this cellular lab model as a tool for ecotoxicological assessment of contaminated fresh waters. Copyright © 2015 Elsevier Ltd. All rights reserved.

The Role of Maternal Nutrition on Oocyte Size and Quality, with Respect to Early Larval Development in The Coral-Eating Starfish, Acanthaster planci

PubMed Central

Pratchett, Morgan S.; Kerr, Alexander M.; Rivera-Posada, Jairo A.

2016-01-01

Variation in local environmental conditions can have pronounced effects on the population structure and dynamics of marine organisms. Previous studies on crown-of-thorns starfish, Acanthaster planci, have primarily focused on effects of water quality and nutrient availability on larval growth and survival, while the role of maternal nutrition on reproduction and larval development has been overlooked. To examine the effects of maternal nutrition on oocyte size and early larval development in A. planci, we pre-conditioned females for 60 days on alternative diets of preferred coral prey (Acropora abrotanoides) versus non-preferred coral prey (Porites rus) and compared resulting gametes and progeny to those produced by females that were starved over the same period. Females fed ad libitum with Acropora increased in weight, produced heavier gonads and produced larger oocytes compared to Porites-fed and starved females. Fed starfish (regardless of whether it was Acropora or Porites) produced bigger larvae with larger stomachs and had a higher frequency of normal larvae that reached the late bipinnaria / early brachiolaria stage compared to starved starfish. Females on Acropora diet also produced a higher proportion of larvae that progressed to more advanced stages faster compared to Porites-fed starfish, which progressed faster than starved starfish. These results suggest that maternal provisioning can have important consequences for the quality and quantity of progeny. Because food quality (coral community structure) and quantity (coral abundance) varies widely among reef locations and habitats, local variation in maternal nutrition of A. planci is likely to moderate reproductive success and may explain temporal and spatial fluctuations in abundance of this species. PMID:27327627

[Analysis of the Localization of Fibrillarin and Sites of Pre-rRNA Synthesis in the Nucleolus-Like Bodies of Mouse GV Oocytes after Mild Treatment with Proteinase K].

PubMed

Shishova, K V; Khodarovich, Yu M; Lavrentyeva, E A; Zatsepina, O V

2015-01-01

Postnatal development of mammalian oocytes is accompanied by functional and structural remodeling of the nucleolar apparatus: the final stage of this process is the formation of large objects (up to 10 μm in diameter) termed nucleolus-like bodies (NLBs) in preovulatory GV oocytes. N LB material was shown to be essential for early embryonic development, but its composition is still uncharacterized. In the present study, the protein-binding dye fluorescein-5-isothiocyanate (FITC) was used to show that proteins characterized by a high local concentration are essential NLB components in mouse GV oocytes. One of these proteins was able to be identified for the first time using a mild treatment of oocytes with proteinase K; the protein identified was fibrillarin, a factor of early pre-rRNA processing. Fibrillarin is present in the inner NLB mass of all oocytes capable of synthesizing rRNA; however, it is not colocalized with BrUTP microinjected into oocytes in order to identify transcribed ribosomal genes, in contrast to the "surface" fibrillarin. These observations imply the accumulation of nucleolar proteins not involved in ribosome biogenesis inside the NLB. All NLBs present in an individual nucleus of an NSN-type GV oocyte contain fibrillarin and are associated with active ribosomal genes. The results obtained in the present work demonstrate that proteinase K treatment of GV mouse oocytes allows for: (1) identification of "cryptic" proteins inside the densely packed NLB material and (2) the enhancement of oocyte image quality during BrUTP-based identification of rRNA synthesis sites but (3) not for the detection of active ribosomal genes in the inner mass of the NLB. The fluorescent dye FITC can be recommended for assessment of intracellular protein localization in the oocytes of all mammalian species.

No difference in mitochondrial distribution is observed in human oocytes after cryopreservation.

PubMed

Stimpfel, Martin; Vrtacnik-Bokal, Eda; Virant-Klun, Irma

2017-08-01

The primary aim of this study was to determine if any difference in mitochondrial distribution can be observed between fresh and cryopreserved (slow-frozen/thawed and vitrified/warmed) oocytes when oocytes are stained with Mitotracker Red CMXRos and observed under a conventional fluorescent microscope. Additionally, the influence of cryopreservation procedure on the viable rates of oocytes at different maturation stages was evaluated. The germinal vesicle (GV) and MII oocytes were cryopreserved with slow-freezing and vitrification. After thawing/warming, oocytes were stained using Mitotracker Red CMXRos and observed under a conventional fluorescent microscope. Mitotracker staining revealed that in GV oocytes the pattern of mitochondrial distribution appeared as aggregated clusters around the whole oocyte. In mature MII oocytes, three different patterns of mitochondrial distribution were observed; a smooth pattern around the polar body with aggregated clusters at the opposite side of the polar body, a smooth pattern throughout the whole cell, and aggregated clusters as can be seen in GV oocytes. There were no significant differences in the observed patterns between fresh, vitrified/warmed and frozen/thawed oocytes. When comparing the viable rates of oocytes after two different cryopreservation procedures, the results showed no significant differences, although the trend of viable MII oocytes tends to be higher after vitrification/warming and for viable GV oocytes it tends to be higher after slow-freezing/thawing. Mitotracker Red CMXRos staining of mitochondria in oocytes did not reveal differences in mitochondrial distribution between fresh and cryopreserved oocytes at different maturity stages. Additionally, no difference was observed in the viable rates of GV and MII oocytes after slow-freezing/thawing and vitrification/warming.

Factors affecting the electrofusion of mouse and ferret oocytes with ferret somatic cells.

PubMed

Li, Ziyi; Sun, Xingshen; Chen, Juan; Leno, Gregory H; Engelhardt, John F

2005-09-01

The domestic ferret, Mustela putorius furos, holds great promise as a genetic model for human lung disease, provided that key technologies for somatic cell nuclear transfer (SCNT) are developed. In this report, we extend our understanding of SCNT in this species by defining conditions for efficient cell fusion by electrical pulse. Two experimental systems were employed in this study. First, in vivo-matured mouse oocytes and ferret somatic cells were used to establish general parameters for fusion. One fibroblast, or cumulus cell, was agglutinated to nucleate, zona pellucida-free, mouse oocytes, and subjected to an electrical pulse. Similar electrical pulse conditions were also tested with 1 or 2 somatic cells inserted into the perivitelline space (PVS) of intact mouse oocytes. The fusion rate for a single fibroblast with a zona-free oocyte was 80.2%, significantly higher (P < 0.05) than that observed for 1, or 2, fibroblasts placed in the PVS (52.0% and 63.8%, respectively). The fusion rate (44.1%) following insertion of two cumulus cells was significantly higher (P < 0.05) than that following insertion of one cumulus cell (25.1%). Second, in vitro-matured ferret oocytes were enucleated, and one to three fibroblasts or cumulus cells were inserted into the PVS. Zona pellucida-free ferret oocytes were fragile and excluded from the study. The fusion rates with two or three fibroblasts were 71.4% and 76.8%, respectively; significantly higher (P < 0.05) than that for one fibroblast (48.6%). This cell number-dependent difference in fusion efficiency was also observed with cumulus cells. Fusion-derived (ferret-ferret) NT embryos cleaved, formed blastocysts in vitro, and underwent early-stage fetal development following embryo transfer. The rate of development was cell type-independent, in contrast to the cell type-dependent differences observed in fusion efficiency. In conclusion, fibroblasts fused more efficiently than cumulus cells and the efficiency of single cell

Social oocyte cryopreservation: a portrayal of Brazilian women.

PubMed

Santo, Elisangela V Espirito; Dieamant, Felipe; Petersen, Claudia G; Mauri, Ana L; Vagnini, Laura D; Renzi, Adriana; Zamara, Camila; Oliveira, João Batista A; Baruffi, Ricardo L R; Franco, José G

2017-06-01

This study aimed to determine what Brazilian childless women of reproductive age think about oocyte cryopreservation to postpone pregnancy and their reasons for performing or not performing this procedure. Women of reproductive age were randomly selected from the general population using different e-mail lists and were invited to participate in the study by completing an online web survey regarding social oocyte cryopreservation. The survey was also distributed through social media to women of reproductive age. Although most of the responders had a partner (86.9%) and had already planned the pregnancy of their first child (69.6%), 85.4% (379) considered the potential of social oocyte freezing to improve their chances of giving birth later in life. Those that had already planned pregnancy were two times more likely to intend to freeze their oocytes (p=0.03). The most important barrier for not undergoing oocyte cryopreservation was cost. The women who indicated that they could not currently undergo the procedure now because of cost were two times (p=0.03) more likely to intend to cryopreserve their oocytes than women who thought that they would not need to delay pregnancy. Brazilian women who think that they are not ready to have a family are discovering the option of oocyte cryopreservation. Most participants considered safeguarding their reproductive potential. Making the procedure more accessible could give women the opportunity to make proactive decisions about the future of their fertility.

Dynamic distribution of spindlin in nucleoli, nucleoplasm and spindle from primary oocytes to mature eggs and its critical function for oocyte-to-embryo transition in gibel carp.

PubMed

Sun, Min; Li, Zhi; Gui, Jian-Fang

2010-10-01

Spindlin (Spin) was thought as a maternal-effect factor associated with meiotic spindle. Its role for the oocyte-to-embryo transition was suggested in mouse, but its direct evidence for the function had been not obtained in other vertebrates. In this study, we used the CagSpin-specific antibody to investigate CagSpin expression pattern and distribution during oogenesis of gibel carp (Carassius auratus gibelio). First, the oocyte-specific expression pattern and dynamic distribution was revealed in nucleoli, nucleoplasm, and spindle from primary oocytes to mature eggs by immunofluorescence localization. In primary oocytes and growth stage oocytes, CagSpin accumulates in nucleoli in increasing numbers along with the oocyte growth, and its disassembly occurs in vitellogenic oocytes, which implicates that CagSpin may be a major component of a large number of nucleoli in fish growth oocytes. Then, co-localization of CagSpin and β-tubulin was revealed in meiotic spindle of mature egg, indicating that CagSpin is one spindle-associated factor. Moreover, microinjection of CagSpin-specific antibody into the fertilized eggs blocked the first cleavage, and found that the CagSpin depletion resulted in spindle assembly disturbance. Thereby, our study provided the first direct evidence for the critical oocyte-to-embryo transition function of Spin in vertebrates, and confirmed that Spin is one important maternal-effect factor that participates in oocyte growth, oocyte maturation, and oocyte-to-embryo transition.

Ultrastructure of the human preovulatory oocyte.

PubMed

Szöllösi, D; Mandelbaum, J; Plachot, M; Salat-Baroux, J; Cohen, J

1986-08-01

The ultrastructure of preovulatory human oocyte-cumulus complexes was described after inducing maturation by clomiphene, human menopausal gonadotropin (hMG), human chorionic gonadotropin (hCG) treatment. The majority of the oocytes was at metaphase II of meiosis, with a radially orientated spindle. The oocyte surface was covered by a multitude of microvilli. Cortical granules were nonuniformly distributed along the cortex. A cytoplasmic polarization was observed. The cytoplasmic organelles were in general uniformly dispersed, with the exception of a narrow segment within which cytoplasmic membranes and mitochondria formed clusters. The spindle was usually found at the borderline between the two regions of the cytoplasm. The functional significance of this polarization is not yet known.

SIRT1, 2, 3 protect mouse oocytes from postovulatory aging.

PubMed

Zhang, Teng; Zhou, Yang; Li, Li; Wang, Hong-Hui; Ma, Xue-Shan; Qian, Wei-Ping; Shen, Wei; Schatten, Heide; Sun, Qing-Yuan

2016-04-01

The quality of metaphase II oocytes will undergo a time-dependent deterioration following ovulation as the result of the oocyte aging process. In this study, we determined that the expression of sirtuin family members (SIRT1, 2, 3) was dramatically reduced in mouse oocytes aged in vivo or in vitro. Increased intracellular ROS was observed when SIRT1, 2, 3 activity was inhibited. Increased frequency of spindle defects and disturbed distribution of mitochondria were also observed in MII oocytes aged in vitro after treatment with Nicotinamide (NAM), indicating that inhibition of SIRT1, 2, 3 may accelerate postovulatory oocyte aging. Interestingly, when MII oocytes were exposed to caffeine, the decline of SIRT1, 2, 3 mRNA levels was delayed and the aging-associated defective phenotypes could be improved. The results suggest that the SIRT1, 2, 3 pathway may play a potential protective role against postovulatory oocyte aging by controlling ROS generation.

Current status of human oocyte and embryo cryopreservation.

PubMed

Herrero, Leyre; Martínez, Mónica; Garcia-Velasco, Juan A

2011-08-01

To summarize recent advances in oocyte and embryo cryopreservation techniques and outcomes. Vitrification is gradually replacing slow freezing due to a better survival rate after thawing. Most units use vitrification for both oocyte and blastocyst cryopreservation, as these two biological structures did not perform very well with slow freezing technique. Basic experiments show that cellular damage seems lower after vitrification. Taken all together, this is helping vitirification to be expanding rapidly, and new clinical indications are being incorporated as well (i.e., fertility preservation). Cryopreservation has been used as a complement to IVF, and recent publications indicate that pregnancy rates achieved with frozen oocytes and embryos are comparable with those achieved in fresh cycles. Multiple publications studying oocyte and embryo physiology during cryopreservation have been published recently; however, larger studies are needed to verify the efficacy of new cryopreservation techniques. Vitrification is a simple and robust technique that is being incorporated into the majority of IVF units, mainly for oocyte and blastocyst cryopreservation.

Developmental Arrest and Mouse Antral Not-Surrounded Nucleolus Oocytes1

PubMed Central

Monti, Manuela; Zanoni, Mario; Calligaro, Alberto; Ko, Minoru S.H.; Mauri, Pierluigi; Redi, Carlo Alberto

2013-01-01

ABSTRACT The antral compartment in the ovary consists of two populations of oocytes that differ by their ability to resume meiosis and to develop to the blastocyst stage. For reasons still not entirely clear, antral oocytes termed surrounded nucleolus (SN; 70% of the population of antral oocytes) develop to the blastocyst stage, whereas those called not-surrounded nucleolus (NSN) arrest at two cells. We profiled transcriptomic, proteomic, and morphological characteristics of antral oocytes and observed that NSN oocyte arrest is associated with lack of cytoplasmic lattices coincident with reduced expression of MATER and ribosomal proteins. Cytoplasmic lattices have been shown to store maternally derived mRNA and ribosomes in mammalian oocytes and embryos, and MATER has been shown to be required for cytoplasmic lattice formation. Thus, we isolated antral oocytes from a Matertm/tm mouse and we observed that 84% of oocytes are of the NSN type. Our results provide the first molecular evidence to account for inability of NSN-derived embryos to progress beyond the two-cell stage; these results may be relevant to naturally occurring preimplantation embryo demise in mammals. PMID:23136301

Melatonin protects against maternal obesity-associated oxidative stress and meiotic defects in oocytes via the SIRT3-SOD2-dependent pathway.

PubMed

Han, Longsen; Wang, Haichao; Li, Ling; Li, Xiaoyan; Ge, Juan; Reiter, Russel J; Wang, Qiang

2017-10-01

Maternal obesity in humans is associated with poor outcomes across the reproductive spectrum. Emerging evidence indicates that these defects are likely attributed to factors within the oocyte. Although various molecules and pathways may contribute to impaired oocyte quality, prevention of fertility issues associated with maternal obesity is a challenge. Using mice fed a high-fat diet (HFD) as an obesity model, we document spindle disorganization, chromosome misalignment, and elevated reactive oxygen species (ROS) levels in oocytes from obese mice. Oral administration of melatonin to HFD mice not only reduces ROS generation, but also prevents spindle/chromosome anomalies in oocytes, consequently promoting the developmental potential of early embryos. Consistent with this finding, we find that melatonin supplement during in vitro maturation also markedly attenuates oxidative stress and meiotic defects in HFD oocytes. Finally, by performing morpholino knockdown and acetylation-mimetic mutant overexpression assays, we reveal that melatonin ameliorates maternal obesity-induced defective phenotypes in oocytes through the SIRT3-SOD2-dependent mechanism. In sum, our data uncover the marked beneficial effects of melatonin on oocyte quality from obese females; this opens a new area for optimizing culture system as well as fertility management. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography.

PubMed

Payne, D; Flaherty, S P; Barry, M F; Matthews, C D

1997-03-01

arose from oocytes that had more uniform timing from injection to pronuclear abuttal and tended to have a longer cytoplasmic wave. In conclusion, we have shown that time-lapse video cinematography is an excellent tool for studying fertilization and early embryo development, and have demonstrated that human fertilization comprises numerous complex dynamic events.

Early College High Schools: A Proposed Solution to Secondary Transition Services

ERIC Educational Resources Information Center

Bridges, Jeanne M.; Maxwell, Gerri M.

2015-01-01

This qualitative case study examines the challenges facing rural secondary schools in transitioning youth from high school to post-secondary education and careers, and whether the interventions, strategies and support built into the Early College High School could offer a solution to this long-standing challenge to better meet the needs of special…

In vitro embryos production after oocytes treatment with forskolin.

PubMed

Paschoal, Daniela Martins; Maziero, Rosiára Rosária Dias; Sudano, Mateus José; Guastali, Midyan Daroz; Vergara, Luis Eduardo; Crocomo, Letícia Ferrari; Lima-Neto, João Ferreira de; Magalhães, Luis Carlos Oña; Monteiro, Bianca Andriolo; Rascado, Tatiana da Silva; Martins, Alício; Leal, Claudia Lima Verde; Landim-Alvarenga, Fernanda da Cruz

2016-04-01

The inhibition of nuclear maturation allows time for the oocyte to accumulate molecules that are important for embryonic development. Thus, the objective of this work was to evaluate the effect of blocking oocyte meiosis with the addition of forskolin, an efficient inhibitor of nuclear maturation, in in vitro maturation (IVM) medium. Forskolin was added to the IVM medium for 6 h at concentrations of 0.1 mM, 0.05 mM or 0.025 mM, then the oocytes were allowed to mature in drug-free medium for 18 h. The oocytes were assessed for the stage of nuclear maturation, the activity and distribution of mitochondria, oocyte ultrastructure, the number of viable cells and the apoptosis rate. After forskolin treatment, the oocytes were fertilized in vitro and cultured for 7 days. On day 7, the blastocyst rate, the ultrastructure, the number of intact cells and the apoptosis rate of the blastocysts were measured. No differences were observed for the stage of nuclear maturation of the oocyte, the mitochondrial activity and distribution, the blastocyst rate or total number of intact cells. However, a higher rate of apoptosis was observed in the blastocysts produced from oocytes blocked for 6 h with the higher concentration of forskolin (P < 0.05). We conclude that all the experimental groups reached the MII stage after the addition of forskolin and that the highest concentration of forskolin caused cellular degeneration without harming embryo production on the 7th day.

Chromatin remodeling in somatic cells injected into mature pig oocytes.

PubMed

Bui, Hong-Thuy; Van Thuan, Nguyen; Wakayama, Teruhiko; Miyano, Takashi

2006-06-01

We examined the involvement of histone H3 modifications in the chromosome condensation and decondensation of somatic cell nuclei injected into mature pig oocytes. Nuclei of pig granulosa cells were transferred into in vitro matured intact pig oocytes, and histone H3 phosphorylation, acetylation, and methylation were examined by immunostaining with specific antibodies in relation to changes in chromosome morphology. In the condensed chromosomes of pig oocytes at metaphase II, histone H3 was phosphorylated at serine 10 (H3-S10) and serine 28 (H3-S28), and methylated at lysine 9 (H3-K9), but was not acetylated at lysine 9, 14 and 18 (H3-K9, H3-K14 and H3-K18). During the first 2 h after nuclear transfer, a series of events were observed in the somatic nuclei: nuclear membrane disassembly; chromosome condensation to form a metaphase-like configuration; an increase in histone H3 phosphorylation levels (H3-S10 and H3-S28). Next, pig oocytes injected with nuclei of somatic cells were electroactivated and the chromosome morphology of oocytes and somatic cells was examined along with histone modifications. Generally, chromosomes of the somatic cells showed a similar progression of cell cycle stage to that of oocytes, through anaphase II- and telophase II-like stages then formed pronucleus-like structures, although the morphology of the spindles differed from that of oocyte spindles. The chromosomes of somatic cells also showed changes in histone H3 dephosphorylation and reacetylation, similar to oocytes. In contrast, histone H3 methylation (H3-K9) of somatic cell nuclei did not show any significant change after injection and electroactivation of the oocytes. These results suggest that nuclear remodeling including histone H3 phosphorylation and acetylation of injected somatic nuclei took place in the oocytes under regulation by the oocyte cytoplasm.

Oocyte stem cells: fact or fantasy?

PubMed

Horan, Corrina J; Williams, Suzannah A

2017-07-01

For many decades, the dogma prevailed that female mammals had a finite pool of oocytes at birth and this was gradually exhausted during a lifetime of reproductive function. However, in 2004, a new era began in the field of female oogenesis. A study was published that appeared to detect oocyte-stem cells capable of generating new eggs within mouse ovaries. This study was highly controversial and the years since this initial finding have produced extensive research and even more extensive debate into their possibility. Unequivocal evidence testifying to the existence of oocyte-stem cells (OSCs) has yet to be produced, meanwhile the spectrum of views from both sides of the debate are wide-ranging and surprisingly passionate. Although recent studies have presented some convincing results that germ cells exist and are capable of creating new oocytes, many questions remain. Are these cells present in humans? Do they exist in physiological conditions in a dormant state? This comprehensive review first examines where and how the dogma of a finite pool was established, how this has been challenged over the years and addresses the most pertinent questions as to the current status of their existence, their role in female fertility, and perhaps most importantly, if they do exist, how can we harness these cells to improve a woman's oocyte reserve and treat conditions such as premature ovarian insufficiency (POI: also known as premature ovarian failure, POF). © 2017 Society for Reproduction and Fertility.

Metabolic control of oocyte development: linking maternal nutrition and reproductive outcomes

PubMed Central

Liu, Honglin; Gu, Xi; Boots, Christina; Moley, Kelle H.

2015-01-01

Obesity, diabetes, and related metabolic disorders are major health issues worldwide. As the epidemic of metabolic disorders continues, the associated medical comorbidities, including the detrimental impact on reproduction, increase as well. Emerging evidence suggests that the effects of maternal nutrition on reproductive outcomes are likely to be mediated, at least in part, by oocyte metabolism. Well-balanced and timed energy metabolism is critical for optimal development of oocytes. To date, much of our understanding of oocyte metabolism comes from the effects of extrinsic nutrients on oocyte maturation. In contrast, intrinsic regulation of oocyte development by metabolic enzymes, intracellular mediators, and transport systems is less characterized. Specifically, decreased acid transport proteins levels, increased glucose/lipid content and elevated reactive oxygen species in oocytes have been implicated in meiotic defects, organelle dysfunction and epigenetic alteration. Therefore, metabolic disturbances in oocytes may contribute to the diminished reproductive potential experienced by women with metabolic disorders. In-depth research is needed to further explore the underlying mechanisms. This review also discusses several approaches for metabolic analysis. Metabolomic profiling of oocytes, the surrounding granulosa cells, and follicular fluid will uncover the metabolic networks regulating oocyte development, potentially leading to the identification of oocyte quality markers and prevention of reproductive disease and poor outcomes in offspring. PMID:25280482

Injurious Effects of Emodin on Maturation of Mouse Oocytes, Fertilization and Fetal Development via Apoptosis

PubMed Central

Chang, Mei-Hui; Chang, Shao-Chung; Chan, Wen-Hsiung

2012-01-01

Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a major constituent of rhubarb, has a wide range of therapeutic applications. Previous studies have established that emodin induces apoptosis in the inner cell mass and trophectoderm of mouse blastocysts and leads to decreased embryonic development and viability, indicating a role as an injury risk factor for normal embryonic development. However, the mechanisms underlying its hazardous effects have yet to be characterized. In the current study, we further investigated the effects of emodin on oocyte maturation and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, emodin induced a significant reduction in the rates of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with emodin during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased fetal weight. Experiments using an in vivo mouse model disclosed that consumption of drinking water containing 20–40 μM emodin led to decreased oocyte maturation and in vitro fertilization, as well as early embryonic developmental injury. Notably, pretreatment with a caspase-3-specific inhibitor effectively prevented emodin-triggered injury effects, suggesting that impairment of embryo development occurs via a caspase-dependent apoptotic process. PMID:23203041

Oocyte-activating capacity of fresh and frozen-thawed spermatids in the common marmoset (Callithrix jacchus).

PubMed

Ogonuki, Narumi; Inoue, Hiroki; Matoba, Shogo; Kurotaki, Yoko K; Kassai, Hidetoshi; Abe, Yukiko; Sasaki, Erika; Aiba, Atsu; Ogura, Atsuo

2018-02-19

The common marmoset (Callithrix jacchus) represents a promising nonhuman primate model for the study of human diseases because of its small size, ease of handling, and availability of gene-modified animals. Here, we aimed to devise reproductive technology for marmoset spermatid injection using immature males for a possible rapid generational turnover. Spermatids at each step could be identified easily by their morphology under differential interference microscopy: thus, early round spermatids had a round nucleus with a few nucleolus-like structures and abundant cytoplasm, as in other mammals. The spermatids acquired oocyte-activating capacity at the late round spermatid stage, as confirmed by the resumption of meiosis and Ca 2+ oscillations upon injection into mouse oocytes. The spermatids could be cryopreserved efficiently with a simple medium containing glycerol and CELL BANKER®. Late round or elongated spermatids first appeared at 10-12 months of age, 6-8 months before sexual maturation. Marmoset oocytes microinjected with frozen-thawed late round or elongated spermatids retrieved from a 12-month-old male marmoset developed to the 8-cell stage without the need for artificial oocyte activation stimulation. Thus, it might be possible to shorten the intergeneration time by spermatid injection, from 2 years (by natural mating) to 13-15 months including gestation. © 2018 Wiley Periodicals, Inc.

Effect of dehydroleucodine on meiosis reinitiation in Bufo arenarum denuded oocytes.

PubMed

Sánchez Toranzo, G; Giordano, O S; López, L A; Bühler, M I

2007-05-01

In amphibian oocytes meiosis, the transition from G2 to M phase is regulated by the maturation promoting factor (MPF), a complex of the cyclin-dependent kinase p34/cdc2 and cyclin B. In immature oocytes there is an inactive complex (pre-MPF), in which cdc2 is phosphorylated on both Thr-161 and Thr-14/Tyr-15 residues. The dephosphorylation of Thr-14/Tyr-15 is necessary for the start of MPF activation and it is induced by the activation of cdc25 phosphatase. Late, to complete the activation, a small amount of active MPF induces an auto-amplification loop of MPF stimulation (MPF amplification). Dehydroleucodine (DhL) is a sesquiterpenic lactone that inhibits mammalian cell proliferation in G2. We asked whether DhL interferes with MPF activation. For this question, the effect of DhL (up to 30 microM) on the resumption of meiosis was evaluated, and visualized by germinal vesicle break down (GVBD), of Bufo arenarum oocytes induced in vitro by either: (i) removing follicle cells; (ii) progesterone stimulation; (iii) VG-content injection; or (iv) injection of mature cytoplasm. The results show that DhL induced GVBD inhibition, in a dose-dependent manner, in spontaneous and progesterone-induced oocyte maturation. Nevertheless, DhL at the doses assayed had no effect on GVBD induced by mature cytoplasm injection, but exerted an inhibitory effect on GVBD induced by GV content. On the basis of these results, we interpreted that DhL does not inhibit MPF amplification and that the target of DhL is any event in the early stages of the cdc25 activation cascade.

High Peak Estradiol/Mature Oocyte Ratio Predicts Lower Clinical Pregnancy, Ongoing Pregnancy, and Live Birth Rates in GnRH Antagonist Intracytoplasmic Sperm Injection Cycles.

PubMed

Sandoval, Juan S; Steward, Ryan G; Chen, Chen; Li, Yi-Ju; Price, Thomas M; Muasher, Suheil J

2016-01-01

To define the relationship between peak estradiol (E2)/mature oocyte ratio and pregnancy outcomes in gonadotropin-releasing hormone (GnRH) antagonist intracytoplasmic sperm injection (ICSI) cycles. Retrospective cohort study in the setting of an academic reproductive medicine practice. Records from 162 fresh, autologous, GnRH antagonist ICSI cycles performed between 2009 and 2012 .were analyzed. The main outcome measures were rates of clinical pregnancy (CPR), ongoing pregnancy (OPR), and live birth (LBR). For the primary analysis, 4 groups were created based on peak E2/mature oocyte ratio (group 1: <200, group 2: 200-300, group 3: 300-400, and group 4: >400 pg/mL/oocyte). After adjusting for age, basal FSH, and the number of mature oocytes, a significantly lower OPR was seen in group 4 as compared to group I (OR 0.15, 95% CI 0.03-0.86; p=0.032) and group 3 (OR 0.17, 95% CI 0.03-0.98; p=0.048), respectively. The adjusted LBR was also significantly lower in group 4 as compared to group 1 (OR 0.15, 95% CI 0.03-0.83; p=0.030). In a secondary analysis, 3 ranges of peak E2/ mature oocyte ratio (<200, 200-400, and >400 pg/ mL/oocyte) were compared between low, normal, and high responders (<6, 6-15, and >15 mature oocytes, respectively). Clinical pregnancy rate, OPR, and LBR were all lower in normal responders when the E2/oocyte ratio exceeded 400 pg/mL/oocyte as compared to <200 pg/mL/oocyte and 200-300 pg/mL/oocyte (CPR 1% vs. 16% and 32%, respectively, p=0.017; OPR 0 vs. 15% and 27%, respectively, p=0.011; and LBR 0 vs. 13% and 26%, respectively, p=0.018). Very elevated peak E2/mature oocyte ratio is associated with a lower CPR, OPR, and LBR in fresh, autologous, GnRH antagonist ICSI cycles.

Parthenogenic Blastocysts Derived from Cumulus-Free In Vitro Matured Human Oocytes

PubMed Central

McElroy, Sohyun L.; Byrne, James A.; Chavez, Shawn L.; Behr, Barry; Hsueh, Aaron J.; Westphal, Lynn M.; Reijo Pera, Renee A.

2010-01-01

Background Approximately 20% of oocytes are classified as immature and discarded following intracytoplasmic sperm injection (ICSI) procedures. These oocytes are obtained from gonadotropin-stimulated patients, and are routinely removed from the cumulus cells which normally would mature the oocytes. Given the ready access to these human oocytes, they represent a potential resource for both clinical and basic science application. However culture conditions for the maturation of cumulus-free oocytes have not been optimized. We aimed to improve maturation conditions for cumulus-free oocytes via culture with ovarian paracrine/autocrine factors identified by single cell analysis. Methodology/Principal Finding Immature human oocytes were matured in vitro via supplementation with ovarian paracrine/autocrine factors that were selected based on expression of ligands in the cumulus cells and their corresponding receptors in oocytes. Matured oocytes were artificially activated to assess developmental competence. Gene expression profiles of parthenotes were compared to IVF/ICSI embryos at morula and blastocyst stages. Following incubation in medium supplemented with ovarian factors (BDNF, IGF-I, estradiol, GDNF, FGF2 and leptin), a greater percentage of oocytes demonstrated nuclear maturation and subsequently, underwent parthenogenesis relative to control. Similarly, cytoplasmic maturation was also improved as indicated by development to blastocyst stage. Parthenogenic blastocysts exhibited mRNA expression profiles similar to those of blastocysts obtained after IVF/ICSI with the exception for MKLP2 and PEG1. Conclusions/Significance Human cumulus-free oocytes from hormone-stimulated cycles are capable of developing to blastocysts when cultured with ovarian factor supplementation. Our improved IVM culture conditions may be used for obtaining mature oocytes for clinical purposes and/or for derivation of embryonic stem cells following parthenogenesis or nuclear transfer. PMID

Elective oocyte cryopreservation: who should pay?

PubMed

Mertes, Heidi; Pennings, Guido

2012-01-01

Despite the initial reactions of disapproval, more and more fertility clinics are now offering oocyte cryopreservation to healthy women in order to extend their reproductive options. However, so-called social freezing is not placed on an equal footing with 'regular' IVF treatments where public funding is concerned. In those countries or states where IVF patients receive a number of free cycles, we argue that fertilization and transfer cycles of women who proactively cryopreserved their oocytes should be covered. Moreover, when the argument of justice is consistently applied, coverage should also include the expenses of ovarian stimulation, oocyte retrieval and storage. Different modalities are possible: full coverage from the onset, reimbursement in cash or reimbursement in kind, by offering more free transfer cycles.

A Voltage Dependent Non-Inactivating Na+ Channel Activated during Apoptosis in Xenopus Oocytes

PubMed Central

Englund, Ulrika H.; Gertow, Jens; Kågedal, Katarina; Elinder, Fredrik

2014-01-01

Ion channels in the plasma membrane are important for the apoptotic process. Different types of voltage-gated ion channels are up-regulated early in the apoptotic process and block of these channels prevents or delays apoptosis. In the present investigation we examined whether ion channels are up-regulated in oocytes from the frog Xenopus laevis during apoptosis. The two-electrode voltage-clamp technique was used to record endogenous ion currents in the oocytes. During staurosporine-induced apoptosis a voltage-dependent Na+ current increased three-fold. This current was activated at voltages more positive than 0 mV (midpoint of the open-probability curve was +55 mV) and showed almost no sign of inactivation during a 1-s pulse. The current was resistant to the Na+-channel blockers tetrodotoxin (1 µM) and amiloride (10 µM), while the Ca2+-channel blocker verapamil (50 µM) in the bath solution completely blocked the current. The intracellular Na+ concentration increased in staurosporine-treated oocytes, but could be prevented by replacing extracellular Na+ whith either K+ or Choline+. Prevention of this influx of Na+ also prevented the STS-induced up-regulation of the caspase-3 activity, suggesting that the intracellular Na+ increase is required to induce apoptosis. Taken together, we have found that a voltage dependent Na+ channel is up-regulated during apoptosis and that influx of Na+ is a crucial step in the apoptotic process in Xenopus oocytes. PMID:24586320

Anti-Müllerian hormone is produced heterogeneously in primate preantral follicles and is a potential biomarker for follicle growth and oocyte maturation in vitro.

PubMed

Xu, Jing; Xu, Fuhua; Letaw, John H; Park, Byung S; Searles, Robert P; Ferguson, Betsy M

2016-12-01

The main goals of this study were to investigate the expression of anti-Müllerian hormone (AMH) and its receptor (AMHR2) during follicular development in primates, and to evaluate the potential of AMH as a biomarker for follicle growth and oocyte maturation in vitro. The mRNA and protein expression of AMH and AMHR2 were determined using isolated follicles and ovarian sections from rhesus macaques (n = 4) by real-time PCR and immunohistochemistry, respectively. Isolated secondary follicles were cultured individually. Follicle growth and media AMH concentrations were assessed by ELISA. The mRNA expression profiles, obtained from RNA sequencing, of in vitro- and in vivo-developed antral follicles were compared. Secondary follicles from additional animals (n = 35) were cultured. Follicle growth, oocyte maturation, and media AMH concentrations were evaluated for forecasting follicular development in vitro by AMH levels. AMH immunostaining was heterogeneous in the population of preantral follicles that were also stained for AMHR2. The mRNA expression profiles were comparable between in vivo- and in vitro-developed follicles. AMH levels produced by growing follicles were higher than those of nongrowing follicles in culture. With a cutoff value of 1.40 ng/ml, 85 % of nongrowing follicles could be identified while eliminating only 5 % of growing follicles. Growing follicles that generated metaphase II-stage oocytes secreted greater amounts of AMH than did those yielding immature germinal vesicle-stage oocytes. AMH, co-expressed with AMHR2, was produced heterogeneously by preantral follicles in macaques with levels correlated positively with follicle growth and oocyte maturation. AMH may serve as a biomarker for primate follicular development in vitro.

[Recovery and light microscopic evaluation of follicular oocytes of swine and relationship between the degeneration rate of oocytes and the estrus phase].

PubMed

Schnurrbusch, U; Schmette, C; Elze, K

1990-10-01

Cumulus-oocyte complexes were recovered from 25 gilts by aspiration of follicular fluid or cutting of follicles from all Graafian follicles of greater than or equal to 3 mm in diameter during diestrus, proestrus or estrus. In 5 gilts the oocytes were collected post ovulation by flushing of oviducts. The recovery rate of follicular oocytes differed between 75.5% during the late diestrus (days 13-17) and 43.5% during the proestrus (days 18-21). During the proestrus and on day 1 of the estrus the recovery of oocytes was more difficult as a result of the higher viscosity of follicular fluid and the mucification of cumulus-oocyte complexes. The degeneration rate of oocytes was high during the diestrus with a peak at the time of regression of corpora lutea. From diestrus to the estrus the degeneration rate decreased. Following degeneration rates were found in the oocytes during the cycle: days 7-12: 38.8%, days 13-17: 50.0%, days 18-21: 29.6%, day 1 of the estrus: 10.8%, day 2 of the estrus ante ovulation: 11.8%, day 2 of the estrus post ovulation: 6.2%. Signs of degeneration were: Loss of cumulus cells (during diestrus and proestrus), damaged zona pellucida, enlargement of perivitelline space, deformation of oocyte, alteration of structure of the ooplasm, diameter of vitellus less than 100 microns. It was concluded that the selection of dominant follicles takes place in pigs during a long time of the cycle, especially during the diestrus. There were not any indications of a 2-wave hypothesis of follicular growth during the cycle in pig.

Progressive obesity alters the steroidogenic response to ovulatory stimulation and increases the abundance of mRNAs stored in the ovulated oocyte.

PubMed

Pohlmeier, William E; Xie, Fang; Kurz, Scott G; Lu, Ningxia; Wood, Jennifer R

2014-08-01

Obese women who are able to attain pregnancy are at increased risk for early-pregnancy loss due, in part, to reduced oocyte quality. We and others have demonstrated that female Lethal Yellow (LY) mice and female C57BL/6 mice fed a high fat diet (B6-HFD) exhibit phenotypes consistent with human obesity. These studies also showed that zygotes collected from LY and B6-HFD females have reduced developmental competence. The current hypothesis is that LY and B6-HFD females exhibit an abnormal response to gonadotropin stimulation compared to C57BL/6 controls fed normal rodent chow (B6-ND), resulting in the ovulation of oocytes with an altered molecular phenotype which may contribute to its reduced developmental competence. To test this hypothesis, age-matched B6-ND, B6-HFD, and LY females were stimulated with exogenous gonadotropins, then circulating hormone levels and the phenotypes of ovulated oocytes were analyzed. There was no difference in ovulation rate or in the percentage of morphologically abnormal oocytes collected from the oviduct of any females. Progesterone and progesterone/estradiol ratios, however, were increased in B6-HFD and LY compared to B6-ND females 16 hr post-human chorionic gonadotropin treatment. The transcript abundance of several candidate oocyte genes was also increased in B6-HFD- and LY-derived oocytes compared to B6-ND-derived oocytes. These data suggest that increased insulin and leptin levels of obese females elevated circulating progesterone concentrations, altered transcriptional activity during oocyte growth, and/or impaired mechanisms of RNA translation and degradation during oocyte maturation. These changes in mRNA abundance likely contribute to reduced oocyte quality and the subsequent poor embryogenesis associated with obesity. © 2014 Wiley Periodicals, Inc.

Transcription and chromatin determinants of de novo DNA methylation timing in oocytes.

PubMed

Gahurova, Lenka; Tomizawa, Shin-Ichi; Smallwood, Sébastien A; Stewart-Morgan, Kathleen R; Saadeh, Heba; Kim, Jeesun; Andrews, Simon R; Chen, Taiping; Kelsey, Gavin

2017-01-01

Gametogenesis in mammals entails profound re-patterning of the epigenome. In the female germline, DNA methylation is acquired late in oogenesis from an essentially unmethylated baseline and is established largely as a consequence of transcription events. Molecular and functional studies have shown that imprinted genes become methylated at different times during oocyte growth; however, little is known about the kinetics of methylation gain genome wide and the reasons for asynchrony in methylation at imprinted loci. Given the predominant role of transcription, we sought to investigate whether transcription timing is rate limiting for de novo methylation and determines the asynchrony of methylation events. Therefore, we generated genome-wide methylation and transcriptome maps of size-selected, growing oocytes to capture the onset and progression of methylation. We find that most sequence elements, including most classes of transposable elements, acquire methylation at similar rates overall. However, methylation of CpG islands (CGIs) is delayed compared with the genome average and there are reproducible differences amongst CGIs in onset of methylation. Although more highly transcribed genes acquire methylation earlier, the major transitions in the oocyte transcriptome occur well before the de novo methylation phase, indicating that transcription is generally not rate limiting in conferring permissiveness to DNA methylation. Instead, CGI methylation timing negatively correlates with enrichment for histone 3 lysine 4 (H3K4) methylation and dependence on the H3K4 demethylases KDM1A and KDM1B, implicating chromatin remodelling as a major determinant of methylation timing. We also identified differential enrichment of transcription factor binding motifs in CGIs acquiring methylation early or late in oocyte growth. By combining these parameters into multiple regression models, we were able to account for about a fifth of the variation in methylation timing of CGIs. Finally

Belongingness in Early Secondary School: Key Factors that Primary and Secondary Schools Need to Consider

PubMed Central

Vaz, Sharmila; Falkmer, Marita; Ciccarelli, Marina; Passmore, Anne; Parsons, Richard; Black, Melissa; Cuomo, Belinda; Tan, Tele; Falkmer, Torbjörn

2015-01-01

It is unknown if, and how, students redefine their sense of school belongingness after negotiating the transition to secondary school. The current study used longitudinal data from 266 students with, and without, disabilities who negotiated the transition from 52 primary schools to 152 secondary schools. The study presents the 13 most significant personal student and contextual factors associated with belongingness in the first year of secondary school. Student perception of school belongingness was found to be stable across the transition. No variability in school belongingness due to gender, disability or household-socio-economic status (SES) was noted. Primary school belongingness accounted for 22% of the variability in secondary school belongingness. Several personal student factors (competence, coping skills) and school factors (low-level classroom task-goal orientation), which influenced belongingness in primary school, continued to influence belongingness in secondary school. In secondary school, effort-goal orientation of the student and perception of their school’s tolerance to disability were each associated with perception of school belongingness. Family factors did not influence belongingness in secondary school. Findings of the current study highlight the need for primary schools to foster belongingness among their students at an early age, and transfer students’ belongingness profiles as part of the hand-over documentation. Most of the factors that influenced school belongingness before and after the transition to secondary are amenable to change. PMID:26372554

Belongingness in Early Secondary School: Key Factors that Primary and Secondary Schools Need to Consider.

PubMed

Vaz, Sharmila; Falkmer, Marita; Ciccarelli, Marina; Passmore, Anne; Parsons, Richard; Black, Melissa; Cuomo, Belinda; Tan, Tele; Falkmer, Torbjörn

2015-01-01

It is unknown if, and how, students redefine their sense of school belongingness after negotiating the transition to secondary school. The current study used longitudinal data from 266 students with, and without, disabilities who negotiated the transition from 52 primary schools to 152 secondary schools. The study presents the 13 most significant personal student and contextual factors associated with belongingness in the first year of secondary school. Student perception of school belongingness was found to be stable across the transition. No variability in school belongingness due to gender, disability or household-socio-economic status (SES) was noted. Primary school belongingness accounted for 22% of the variability in secondary school belongingness. Several personal student factors (competence, coping skills) and school factors (low-level classroom task-goal orientation), which influenced belongingness in primary school, continued to influence belongingness in secondary school. In secondary school, effort-goal orientation of the student and perception of their school's tolerance to disability were each associated with perception of school belongingness. Family factors did not influence belongingness in secondary school. Findings of the current study highlight the need for primary schools to foster belongingness among their students at an early age, and transfer students' belongingness profiles as part of the hand-over documentation. Most of the factors that influenced school belongingness before and after the transition to secondary are amenable to change.

Develop to Term Rat Oocytes Injected with Heat-Dried Sperm Heads

PubMed Central

Lee, Kyung-Bon; Park, Ki-Eun; Kwon, In-Kiu; Tripurani, Swamy K.; Kim, Keun Jung; Lee, Ji Hye; Niwa, Koji; Kim, Min Kyu

2013-01-01

This study investigated the development of rat oocytes in vitro and in vivo following intracytoplasmic injection of heads from spermatozoa heat-dried at 50°C for 8 h and stored at 4°C in different gas phases. Sperm membrane and chromosome are damaged by the process of heat-drying. Oocyte activation and cleavage of oocytes were worse in oocytes injected with spermatozoa heat-dried and stored for 1 week than unheated, fresh spermatozoa, but in heat-dried spermatozoa, there were no differences in these abilities of oocytes between the samples stored in nitrogen gas and in air. The oocytes injected with heat-dried spermatozoa stored for 1 week could develop to the morula and blastocyst stages without difference between the samples stored in nitrogen gas and in air after artificial stimulation. Cleavage of oocytes and development of cleaved embryos were higher when heat-dried spermatozoa were stored for 3 and 6 months in nitrogen gas than in air. However, the ability of injected oocytes to develop to the morula and blastocyst stages was not inhibited even when heat-dried spermatozoa stored in both atmosphere conditions for as long as 6 months were used. When 2-cell embryos derived from oocytes injected with heads from spermatozoa heat-dried and stored for 1 week and 1 month were transferred, each 1 of 4 recipients was conceived, and the conceived recipients delivered 1 live young each. These results demonstrate that rat oocytes can be fertilized with heat-dried spermatozoa and that the fertilized oocytes can develop to term. PMID:24223784

Activation of amphibian oocytes by sperm extracts.

PubMed

Bonilla, F; Ajmat, M T; Sánchez Toranzo, G; Zelarayán, L; Oterino, J; Bühler, M I

2008-11-01

In the fertilization of most animals, egg activation is accompanied by an increase in cytoplasmatic Ca2+; however, the mechanism through which the fertilizing sperm induce this phenomenon is still controversial. An increase in intracellular free Ca2+ is required to trigger egg activation events, a process that includes cortical granule exocytosis, resumption and completion of meiosis and DNA replication, and culminates in the first mitotic cleavage. In this work, we investigated the effect of microinjection and incubation of different fractions of homologous sperm extract on the activation of Bufo arenarum oocytes matured in vitro. Two heat treatment-sensitive fractions obtained by chromatography were able to induce oocyte activation. The sperm fraction, which contained a 24 kDa protein, induced 90% activation when it was microinjected into the oocytes. Whilst the sperm fraction, which contained a 36 kDa protein, was able to induce about 70% activation only when it was applied on the oocyte surface.

Single-cell analysis of differences in transcriptomic profiles of oocytes and cumulus cells at GV, MI, MII stages from PCOS patients

PubMed Central

Liu, Qiwei; Li, Yumei; Feng, Yun; Liu, Chaojie; Ma, Jieliang; Li, Yifei; Xiang, Huifen; Ji, Yazhong; Cao, Yunxia; Tong, Xiaowen; Xue, Zhigang

2016-01-01

Polycystic ovary syndrome (PCOS) is a common frequent endocrine disorder among women of reproductive age. Although assisted reproductive techniques (ARTs) are used to address subfertility in PCOS women, their effectiveness is not clear. Our aim was to compare transcriptomic profiles of oocytes and cumulus cells (CCs) between women with and without PCOS, and assess the effectiveness of ARTs in treating PCOS patients. We collected oocytes and CCs from 16 patients with and without PCOS patients to categorize them into 6 groups according to oocyte nuclear maturation. Transcriptional gene expression of oocyte and CCs was determined via single-cell RNA sequencing. The ratio of fertilization and cleavage was higher in PCOS patients than in non-PCOS patients undergoing ARTs, and there was no difference in the number of high-quality embryos between the groups. Differentially expressed genes including PPP2R1A, PDGFRA, EGFR, GJA1, PTGS2, TNFAIP6, TGF-β1, CAV1, INHBB et al. were investigated as potential causes of PCOS oocytes and CCs disorder at early stages, but their expression returned to the normal level at the metaphase II (MII) stage via ARTs. In conclusion, ARTs can improve the quality of cumulus-oocyte complex (COC) and increase the ratio of fertilization and cleavage in PCOS women. PMID:28004769

Single-cell analysis of differences in transcriptomic profiles of oocytes and cumulus cells at GV, MI, MII stages from PCOS patients.

PubMed

Liu, Qiwei; Li, Yumei; Feng, Yun; Liu, Chaojie; Ma, Jieliang; Li, Yifei; Xiang, Huifen; Ji, Yazhong; Cao, Yunxia; Tong, Xiaowen; Xue, Zhigang

2016-12-22

Polycystic ovary syndrome (PCOS) is a common frequent endocrine disorder among women of reproductive age. Although assisted reproductive techniques (ARTs) are used to address subfertility in PCOS women, their effectiveness is not clear. Our aim was to compare transcriptomic profiles of oocytes and cumulus cells (CCs) between women with and without PCOS, and assess the effectiveness of ARTs in treating PCOS patients. We collected oocytes and CCs from 16 patients with and without PCOS patients to categorize them into 6 groups according to oocyte nuclear maturation. Transcriptional gene expression of oocyte and CCs was determined via single-cell RNA sequencing. The ratio of fertilization and cleavage was higher in PCOS patients than in non-PCOS patients undergoing ARTs, and there was no difference in the number of high-quality embryos between the groups. Differentially expressed genes including PPP2R1A, PDGFRA, EGFR, GJA1, PTGS2, TNFAIP6, TGF-β1, CAV1, INHBB et al. were investigated as potential causes of PCOS oocytes and CCs disorder at early stages, but their expression returned to the normal level at the metaphase II (MII) stage via ARTs. In conclusion, ARTs can improve the quality of cumulus-oocyte complex (COC) and increase the ratio of fertilization and cleavage in PCOS women.

Arachidonic and Linoleic Acid Derivatives Impact Oocyte ICSI Fertilization – A Prospective Analysis of Follicular Fluid and a Matched Oocyte in a ‘One Follicle – One Retrieved Oocyte – One Resulting Embryo’ Investigational Setting

PubMed Central

Bączkowski, Tomasz; Drozd, Arleta; Kazienko, Anna

2015-01-01

Objective To evaluate human oocyte ability to undergo fertilization and subsequent preimplantation embryonic development in relation to a wide panel of follicular fluid (FF) arachidonic acid derivatives (AAD) and linoleic acid derivatives (LAD) of prospectively selected patients undergoing intracytoplasmic sperm injection (ICSI). Methodology Study was designed as a two center (a university clinic and a private clinic) prospective study. 54 women of 181 consecutive couples undergoing ICSI were prospectively found to be eligible for analysis. 'One follicle – one retrieved oocyte – one resulting embryo' approach was used. Each individual follicle was aspirated independently and matched to an oocyte growing in this particular follicular milieu. FF samples were assessed for AAD and LAD by high-performance liquid chromatography; additionally, activity of secretory phospholipase A (sPLA2) was determined by enzyme-linked immunosorbent assay. Principal Findings Increased activity of sPLA2 and significantly higher AAD and LAD levels were found in FF of oocytes that did not show two pronuclei or underwent degeneration after ICSI in comparison to oocytes with the appearance of two pronuclei. Receiver operating characteristics curve analysis identified acids with the highest sensitivity and specificity: 5oxo-hydroxyeicosatetraenoic, 16-hydroxyeicosatetraenoic, 9-hydroxyoctadecadieneoic and 12-hydroxyeicosatetraenoic. No significant differences between AAD and LAD related to embryo quality were found. Conclusions/Significance Our study demonstrates for the first time that elevated concentrations of AAD and LAD in FF at the time of oocyte retrieval significantly decrease the ability of oocytes to form pronuclei after ICSI. This may serve as a new tool for non-invasive assessment of oocyte developmental capacity. However, levels of AAD and LAD are not associated with subsequent embryo quality or pregnancy rate, and therefore more studies are needed to determine their

Time-lapse evaluation of human embryo development in single versus sequential culture media--a sibling oocyte study.

PubMed

Ciray, Haydar Nadir; Aksoy, Turan; Goktas, Cihan; Ozturk, Bilgen; Bahceci, Mustafa

2012-09-01

To compare the dynamics of early development between embryos cultured in single and sequential media. Randomized, comparative study. Private IVF centre. A total of 446 metaphase II oocytes from 51 couples who underwent oocyte retrieval procedure for intracytoplasmic sperm injection. Forty-nine resulted in embryo transfer. Oocytes were split between single and sequential media produced by the same manufacturer and cultured in a time-lapse incubator. Morphokinetic parameters until the embryos reached the 5-cell stage (t5), utilization, clinical pregnancy and implantation rates. Embryos cultured in single media were advanced from the first mitosis cycle and reached 2- to 5-cell stages earlier. There was not any difference between the durations for cell cycle two (cc2 = t3-t2) and s2 (t4-t3). The utilization, clinical pregnancy and implantation rates did not differ between groups. The proportion of cryopreserved day 6 embryos to two pronuclei oocytes was significantly higher in sequential than in single media. Morphokinetics of embryo development vary between single and sequential culture media at least until the 5-cell stage. The overall clinical and embryological parameters remain similar regardless of the culture system.

Expanding reproductive lifespan: a cost-effectiveness study on oocyte freezing.

PubMed

van Loendersloot, L L; Moolenaar, L M; Mol, B W J; Repping, S; van der Veen, F; Goddijn, M

2011-11-01

The average age of women bearing their first child has increased strongly. This is an important reproductive health problem as fertility declines with increasing female age. Unfortunately, IVF using fresh oocytes cannot compensate for this age-related fertility decline. Oocyte freezing could be a solution. We used the Markov model to estimate the cost-effectiveness of three strategies for 35-year-old women who want to postpone pregnancy till the age of 40: Strategy 1: women undergo three cycles of ovarian hyperstimulation at age 35 for oocyte freezing, then at age 40, use these frozen oocytes for IVF; Strategy 2: women at age 40 attempt to conceive without treatment; and the reference strategy: women at age 40 attempt to conceive and, if not pregnant after 1 year, undergo IVF. Sensitivity analyses were carried out to investigate assumptions of the model and to identify which model inputs had most impact on the results. Oocyte freezing (Strategy 1) resulted in a live birth rate of 84.5% at an average cost of €10,419. Natural conception (Strategy 2) resulted in a live birth rate of 52.3% at an average cost of €310 per birth. IVF (the reference strategy) resulted in a cumulative live birth rate of 64.6% at an average cost of €7798. The cost per additional live birth for the oocyte freezing strategy was €13,156 compared to the IVF strategy. If at least 61% of the women return to collect their oocytes, and if there is a willingness to pay €19,560 extra per additional live birth, the oocyte freezing strategy is the most cost-effective strategy. Oocyte freezing is more cost effective compared to IVF, if at least 61% of the women return to collect their oocytes and if one is willing to pay €19,560 extra per additional live birth. Our Markov model shows that, considering all the used assumptions, oocyte freezing provides more value for money than IVF.

In vitro penetration of swine oocytes by homologous spermatozoa: Distinct systems for gamete's co-incubation and oocyte's cryopreservation.

PubMed

Macedo, M C; Lucia, T; Rambo, G; Ferreira Filho, E B; Rosa, A P; Fabiane, C; Cabral, M; Deschamps, J C

2010-02-01

In vitro penetration (IVP) of swine oocytes by homologous spermatozoa was evaluated in two experiments using four boars as semen donors. In experiment 1, the IVP rate and the number of penetrating spermatozoa (PSP) were compared using three co-incubation systems for vitrified oocytes and fresh sperm: (1) 35mL petri dishes in a CO(2) incubator, (2) 35mL petri dishes in bags (submarine system) and (3) glass flasks partially submerged in water bath with the same gas mixture used for the bag system. Mean PSP was 8.2+/-10.1 and the IVP rate was 90.5%. The PSP differed across all systems (P=0.0006): 15.5+/-0.5 for flasks, 6.3+/-0.4 for CO(2), and 3.9+/-0.4 for bags. The IVP rate for flasks (95.0%) was greater (P=0.01) than for CO(2) and bags (90.8% and 85.0%, respectively), but it did not differ between flasks and CO(2) for three boars (P>0.05). In experiment 2, co-incubation was done as described for glass flasks in experiment 1. The IVP rate and PSP were compared for cryopreserved oocytes: either vitrified in open pulled straws (OPS), or frozen in cryotubes. Mean PSP was 5.4+/-6.5 and IVP rate was 89.6%. Both PSP and IVP rate were greater (P<0.0001) for oocytes frozen in cryotubes (7.0+/-0.3% and 95.8%, respectively) than those frozen in OPS (3.7+/-0.3% and 83.4%, respectively), with no differences found for three boars (P>0.05). In summary, successful IVP of swine oocytes by homologous spermatozoa can be achieved using gametes incubated in glass flasks and oocytes frozen in cryotubes.

Effect of heat stress on the survival and development of in vitro cultured bovine preantral follicles and on in vitro maturation of cumulus-oocyte complex.

PubMed

Paes, V M; Vieira, L A; Correia, H H V; Sa, N A R; Moura, A A A; Sales, A D; Rodrigues, A P R; Magalhães-Padilha, D M; Santos, F W; Apgar, G A; Campello, C C; Camargo, L S A; Figueiredo, J R

2016-09-01

The deleterious effect of heat stress (HS) on competence of oocytes from antral follicles is well recognized, but there is a lack of data regarding its impact on the viability and growth of preantral follicles. In this study, we used in vitro preantral follicle cultures to investigate the effects of HS on the following parameters: survival and development of primordial follicles after in vitro culture of ovarian fragments (experiment I); growth and antrum formation of isolated advanced secondary follicles (experiment II); and maturation rates after in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) from antral follicles (>2-6 mm) grown in vivo (experiment III). Furthermore, the following end points were evaluated in all experiments: follicle/oocyte survival, reactive oxygen species (ROS), estradiol (E2) and progesterone (P4) production, as well as mRNA expression for select genes related to stress (HSP70) and apoptosis (MCL1 and BAX). In all experiments, HS consisted of exposing the structures (ovarian fragments, isolated preantral follicles and COCs) to 41 °C for 12 hours and then to 38.5 °C until the end of the culture (7 days for experiments I and II and 24 hours for experiment III). The temperature for the control group was held at 38.5 °C for the entire culture period. Heat stress increased (P < 0.05) the percentage of developing follicles (intermediate, primary, and secondary follicles) at 12 hours and increased levels of ROS at all evaluated time points (12, 24 hours, and D7), when compared to the control (experiment I). Heat stress did not affect (P > 0.05) any identified end points when preantral follicles were cultured in their isolated form (experiment II). However, in experiment III, HS decreased (P < 0.05) both the rates of metaphase II after 24 hours and E2 production at 12 hours of IVM. Moreover, HS increased (P < 0.0001) levels of P4 after IVM and ROS production at every evaluated time point, compared with the

Expression of G protein estrogen receptor (GPER) on membrane of mouse oocytes during maturation.

PubMed

Li, Yi-Ran; Ren, Chun-E; Zhang, Quan; Li, Ji-Chun; Chian, Ri-Cheng

2013-02-01

To determine expression of G-protein estrogen receptor (GPER) in mouse oocyte membrane during maturation. The expression of GPER from different maturation stages of oocytes, in vivo and in vitro matured oocytes as well as aging oocytes was examined by immune-fluorescence GPR30 antibody and the images were analyzed by laser scanning confocal microscope. Further confirmation was performed by Western blots for cell fractionation. Significant fluorescent signal was observed on the surface of mouse oocytes. The image expression was lower in germinal vesicle (GV) stage than mature metaphase-II (M-II) stage oocytes. There was high expression in in-vivo matured oocytes compared to in vitro matured oocytes. The highest expression was observed in aging oocytes compared with other oocytes. The changes of expression of GPER on mouse oocytes plasma membrane confirm oocyte membrane maturation, suggesting that those changes of GPER may be related to the functional role of oocyte maturation.

PTK2b function during fertilization of the mouse oocyte

DOE Office of Scientific and Technical Information (OSTI.GOV)

Luo, Jinping; McGinnis, Lynda K.; Carlton, Carol

Highlights: • PTK2b is expressed in oocytes and is activated following fertilization. • PTK2b suppression in oocytes prevents fertilization, but not parthenogenetic activation. • PTK2b suppression prevents the oocyte from fusing with or incorporating bound sperm. • PTK2b suppressed oocytes that fail to fertilize do not exhibit calcium oscillations. - Abstract: Fertilization triggers rapid changes in intracellular free calcium that serve to activate multiple signaling events critical to the initiation of successful development. Among the pathways downstream of the fertilization-induced calcium transient is the calcium-calmodulin dependent protein tyrosine kinase PTK2b or PYK2 kinase. PTK2b plays an important role in fertilizationmore » of the zebrafish oocyte and the objective of the present study was to establish whether PTK2b also functions in mammalian fertilization. PTK2b was activated during the first few hours after fertilization of the mouse oocyte during the period when anaphase resumption was underway and prior to the pronuclear stage. Suppression of PTK2b kinase activity in oocytes blocked sperm incorporation and egg activation although sperm-oocyte binding was not affected. Oocytes that failed to incorporate sperm after inhibitor treatment showed no evidence of a calcium transient and no evidence of anaphase resumption suggesting that egg activation did not occur. The results indicate that PTK2b functions during the sperm-egg fusion process or during the physical incorporation of sperm into the egg cytoplasm and is therefore critical for successful development.« less

Establishment of an oocyte donor program. Donor screening and selection.

PubMed

Quigley, M M; Collins, R L; Schover, L R

1991-01-01

IVF with donated oocytes, followed by embryo placement in the uterus of a recipient who has been primed with exogenous steroids, is a successful treatment for special cases of infertility. Preliminary results indicate that the success rate in this situation is even greater than that usually seen with normal IVF (with placement of the embryos back into the uteri of the women from whom the oocytes were recovered). Although different sources for donated oocytes have been identified, the use of "excess" oocytes from IVF cycles and the attempted collection of oocytes at the time of otherwise indicated pelvic surgery have ethical and practical problems associated with their use. We have herein described the establishment of a successful program relying on anonymous volunteers who go through ovarian stimulation, monitoring, and oocyte recovery procedures solely to donate oocytes. The potential donors go through an exhaustive screening and education process before they are accepted in the program. Psychological evaluation of our potential donors indicated a great degree of turmoil in their backgrounds and a wide variety of motivations for actually participating. Despite the extensive educational and screening process, a substantial percentage of the donors did not complete a donation cycle, having either voluntarily withdrawn or been dropped because of lack of compliance. Further investigation of the psychological aspects of participating in such a program is certainly warranted. The use of donated oocytes to alleviate specific types of infertility is quite successful, but the application of this treatment is likely to be limited by the relative unavailability of suitable oocyte donors.

Oocyte vitrification for elective fertility preservation: the past, present, and future.

PubMed

Gunnala, Vinay; Schattman, Glenn

2017-02-01

Oocyte cryopreservation is no longer experimental and one of its rapidly growing indications is elective fertility preservation. Currently there is no sufficient evidence to support its practice and therefore its place in IVF remains uncertain. Vitrification has superior post-thaw survival and fertilization outcomes compared with oocytes that were frozen with the slow-freeze technique. Oocyte vitrification produces similar IVF outcomes compared with fresh oocytes and is not associated with further obstetrical or perinatal morbidity. Undergoing elective oocyte cryopreservation between ages 35 and 37 will optimize live birth rates as well as cost effectiveness from mathematical models. In women who delay child bearing, elective oocyte cryopreservation in the mid 30s may be beneficial in terms of live birth rates and cost effectiveness. Prospective studies of women who have undergone oocyte cryopreservation and are now attempting conception are needed before official recommendations can be made regarding elective egg freezing.

Full-grown oocytes from Xenopus laevis resume growth when placed in culture

PubMed Central

Wallace, Robin A.; Misulovin, Ziva; Etkin, Laurence D.

1981-01-01

When most full-grown, follicle cell-invested oocytes from Xenopus laevis are placed in an appropriate culture medium, they resume growth and remain physiologically healthy for at least 2-3 weeks. Rates of growth by full-grown oocytes in vitro generally approximate and can even exceed the most rapid growth rate achieved by vitellogenic oocytes in vivo. Resumption of oocyte growth can be correlated with the loss of investing follicle cells, which under normal conditions appear to interfere with vitellogenin and nutrient access to the oocyte. The final size reached by the oocyte within the ovary is thus not an intrinsic property of the oocyte but is extrinsically imposed by the somatic environment. Images PMID:16593019

Oocytes express an endogenous red fluorescent protein in a stony coral, Euphyllia ancora: a potential involvement in coral oogenesis

PubMed Central

Shikina, Shinya; Chiu, Yi-Ling; Chung, Yi-Jou; Chen, Chieh-Jhen; Lee, Yan-Horn; Chang, Ching-Fong

2016-01-01

To date,the molecular and cellular mechanisms underlying coral sexual reproduction remain largely unknown. We then performed a differential screen to identify genes related to oogenesis in the stony coral Euphyllia ancora. We identified a clone encoding a novel red fluorescent protein cDNA of E. ancora (named EaRFP). Microscopic observation and quantitative RT-PCR revealed that EaRFP is almost exclusively expressed in the ovary of the adult coral. The combination of the ovarian-cell separation method and the RT-PCR analysis revealed that the oocytes, but not the ovarian somatic cells, are the cells expressing EaRFP. Immunohistochemical analysis revealed that the expression of EaRFP starts in the early stage of the oocyte and continues until the maturation period. Furthermore, recombinant EaRFP was shown to possess an H2O2 degradation activity. These results raise the possibility that EaRFP plays a role in protecting the oocytes from oxidative stress from the early to late stages of oogenesis. The present study provides not only the first evidence for the potential involvement of FPs in coral oogenesis but also an insight into a cellular strategy underlying coral sexual reproduction. PMID:27167722

Oocytes express an endogenous red fluorescent protein in a stony coral, Euphyllia ancora: a potential involvement in coral oogenesis.

PubMed

Shikina, Shinya; Chiu, Yi-Ling; Chung, Yi-Jou; Chen, Chieh-Jhen; Lee, Yan-Horn; Chang, Ching-Fong

2016-05-11

To date,the molecular and cellular mechanisms underlying coral sexual reproduction remain largely unknown. We then performed a differential screen to identify genes related to oogenesis in the stony coral Euphyllia ancora. We identified a clone encoding a novel red fluorescent protein cDNA of E. ancora (named EaRFP). Microscopic observation and quantitative RT-PCR revealed that EaRFP is almost exclusively expressed in the ovary of the adult coral. The combination of the ovarian-cell separation method and the RT-PCR analysis revealed that the oocytes, but not the ovarian somatic cells, are the cells expressing EaRFP. Immunohistochemical analysis revealed that the expression of EaRFP starts in the early stage of the oocyte and continues until the maturation period. Furthermore, recombinant EaRFP was shown to possess an H2O2 degradation activity. These results raise the possibility that EaRFP plays a role in protecting the oocytes from oxidative stress from the early to late stages of oogenesis. The present study provides not only the first evidence for the potential involvement of FPs in coral oogenesis but also an insight into a cellular strategy underlying coral sexual reproduction.

Short-term preservation of porcine oocytes in ambient temperature: novel approaches.

PubMed

Yang, Cai-Rong; Miao, De-Qiang; Zhang, Qing-Hua; Guo, Lei; Tong, Jing-Shan; Wei, Yanchang; Huang, Xin; Hou, Yi; Schatten, Heide; Liu, ZhongHua; Sun, Qing-Yuan

2010-12-07

The objective of this study was to evaluate the feasibility of preserving porcine oocytes without freezing. To optimize preservation conditions, porcine cumulus-oocyte complexes (COCs) were preserved in TCM-199, porcine follicular fluid (pFF) and FCS at different temperatures (4°C, 20°C, 25°C, 27.5°C, 30°C and 38.5°C) for 1 day, 2 days or 3 days. After preservation, oocyte morphology, germinal vesicle (GV) rate, actin cytoskeleton organization, cortical granule distribution, mitochondrial translocation and intracellular glutathione level were evaluated. Oocyte maturation was indicated by first polar body emission and spindle morphology after in vitro culture. Strikingly, when COCs were stored at 27.5°C for 3 days in pFF or FCS, more than 60% oocytes were still arrested at the GV stage and more than 50% oocytes matured into MII stages after culture. Almost 80% oocytes showed normal actin organization and cortical granule relocation to the cortex, and approximately 50% oocytes showed diffused mitochondria distribution patterns and normal spindle configurations. While stored in TCM-199, all these criteria decreased significantly. Glutathione (GSH) level in the pFF or FCS group was higher than in the TCM-199 group, but lower than in the non-preserved control group. The preserved oocytes could be fertilized and developed to blastocysts (about 10%) with normal cell number, which is clear evidence for their retaining the developmental potentiality after 3d preservation. Thus, we have developed a simple method for preserving immature pig oocytes at an ambient temperature for several days without evident damage of cytoplasm and keeping oocyte developmental competence.

SMC5/6 is required for the formation of segregation-competent bivalent chromosomes during meiosis I in mouse oocytes

PubMed Central

Hwang, Grace; Sun, Fengyun; Eppig, John J.; Handel, Mary Ann

2017-01-01

SMC complexes include three major classes: cohesin, condensin and SMC5/6. However, the localization pattern and genetic requirements for the SMC5/6 complex during mammalian oogenesis have not previously been examined. In mouse oocytes, the SMC5/6 complex is enriched at the pericentromeric heterochromatin, and also localizes along chromosome arms during meiosis. The infertility phenotypes of females with a Zp3-Cre-driven conditional knockout (cKO) of Smc5 demonstrated that maternally expressed SMC5 protein is essential for early embryogenesis. Interestingly, protein levels of SMC5/6 complex components in oocytes decline as wild-type females age. When SMC5/6 complexes were completely absent in oocytes during meiotic resumption, homologous chromosomes failed to segregate accurately during meiosis I. Despite what appears to be an inability to resolve concatenation between chromosomes during meiosis, localization of topoisomerase IIα to bivalents was not affected; however, localization of condensin along the chromosome axes was perturbed. Taken together, these data demonstrate that the SMC5/6 complex is essential for the formation of segregation-competent bivalents during meiosis I, and findings suggest that age-dependent depletion of the SMC5/6 complex in oocytes could contribute to increased incidence of oocyte aneuploidy and spontaneous abortion in aging females. PMID:28302748

Intraovarian markers of follicular and oocyte maturation.

PubMed

Pellicer, A; Diamond, M P; DeCherney, A H; Naftolin, F

1987-08-01

The use of ovulation induction for multiple follicular growth in in vitro fertilization (IVF) has introduced the problem of follicular asynchrony. As a consequence of the asynchrony, the parameters most commonly used by IVF groups to assess follicular and oocyte quality within those follicles are not sufficiently sensitive or specific. Thus, each follicle must be considered separately, and specific markers of follicular and/or oocyte maturation must be sought from within the follicle. In this review we analyze previous reports of potential markers of follicular and oocyte maturation. In regards to the follicular fluid constituents, the level of estradiol in follicular fluid correlates with fertilization and pregnancy in stimulated cycles. Other steroids are only helpful when specific stimulation protocols are used. The level of some follicular proteins such as alpha-1-antitrypsin and fibrinogen also correlates with fertilization and pregnancy outcome. Cyclic AMP levels in follicular fluid are significantly reduced in follicles leading to conception. Regulators of oocyte maturation, such as the Oocyte Maturation Inhibitor (OMI) or the Meiosis Inducing Substance (MIS) have also been correlated with IVF outcome, but their exact structure remains still unknown. In addition, other sophisticated parameters, such as chemotactic activity of human leukocytes, or simple methods, such as the presence of intrafollicular echoes, have also been used as successful markers in predicting IVF outcome.

Membrane currents in the oocyte of the toad Bufo arenarum.

PubMed

Kotsias, Basilio A; Damiano, Alicia E; Godoy, Sebastian; Assef, Yanina; Ibarra, Cristina; Cantiello, Horacio F

2002-03-01

The amphibian oocyte cell model is widely used for heterologous expression of ionic channels and receptors. Little is known, however, about the physiology of oocyte cell models other than Xenopus laevis. In this study, the two-electrode voltage clamp technique was used to assess the most common electrical patterns of oocytes of the South American toad Bufo arenarum. Basal membrane resistance, resting potential, and ionic currents were determined in this cell model. The oocyte transmembrane resistance was 0.35 M(Omega), and the resting potential in normal saline was about -33 mV with a range between -20 mV and -50 mV. This is, to our knowledge, the first attempt to begin an understanding of the ion transport mechanisms of Bufo arenarum oocytes. This cell model may provide a viable alternative to the expression of ion channels, in particular those endogenously observed in Xenopus laevis oocytes. Copyright 2002 Wiley-Liss, Inc.

Chapter 10 Human Oocyte Vitrification.

PubMed

Rienzi, Laura; Cobo, Ana; Ubaldi, Filippo Maria

2017-01-01

Discovery and widespread application of successful cryopreservation methods for MII-phase oocytes was one of the greatest successes in human reproduction during the past decade. Although considerable improvements in traditional slow-rate freezing were also achieved, the real breakthrough was the result of introduction of vitrification. Here we describe the method that is most commonly applied for this purpose, provides consistent survival and in vitro developmental rates, results in pregnancy and birth rates comparable to those achievable with fresh oocytes, and does not result in higher incidence of gynecological or postnatal complications.

Live Birth from Slow-Frozen Rabbit Oocytes after In Vivo Fertilisation

PubMed Central

Jiménez-Trigos, Estrella; Vicente, José S.; Marco-Jiménez, Francisco

2013-01-01

In vivo fertilisation techniques such as intraoviductal oocyte transfer have been considered as alternatives to bypass the inadequacy of conventional in vitro fertilisation in rabbit. There is only one study in the literature, published in 1989, that reports live offspring from cryopreserved rabbit oocytes. The aim of the present study was to establish the in vivo fertilisation procedure to generate live offspring with frozen oocytes. First, the effect of two recipient models (i) ovariectomised or (ii) oviduct ligated immediately after transfer on the ability of fresh oocytes to fertilise were compared. Second, generation of live offspring from slow-frozen oocytes was carried out using the ligated oviduct recipient model. Throughout the experiment, recipients were artificially inseminated 9 hours prior to oocyte transfer. In the first experiment, two days after unilateral transfer of fresh oocytes, oviducts and uterine horns were flushed to assess embryo recovery rates. The embryo recovery rates were low compared to control in both ovariectomised and ligated oviduct groups. However, ligated oviduct recipient showed significantly (P<0.05) higher embryo recovery rates compared to ovariectomised and control-transferred. In the second experiment, using bilateral oviduct ligation model, all females that received slow-frozen oocytes became pregnant and delivered a total of 4 live young naturally. Thus, in vivo fertilisation is an effective technique to generate live offspring using slow-frozen oocytes in rabbits. PMID:24358281

IVF versus ICSI for the fertilization of in-vitro matured human oocytes.

PubMed

Walls, M; Junk, S; Ryan, J P; Hart, R

2012-12-01

Traditional dogma suggests that intracytoplasmic sperm injection (ICSI) should be performed to ensure successful oocyte fertilization in an in-vitro maturation (IVM) cycle. This study postulated that there would be no difference in the fertilization rate when ICSI was compared with IVF. This hypothesis was tested in a randomized trial of IVF versus ICSI in IVM. A total of 150 immature oocytes were collected in eight cycles of IVM for patients diagnosed with polycystic ovarian syndrome (PCOS). Patients were primed with minimal FSH before transvaginal oocyte aspiration. Sibling oocytes were inseminated by 50% IVF and 50% ICSI. There was no significant difference in fertilization, useable or total blastocyst development between the two insemination technique groups. Clinical pregnancy results for combined fresh and cryopreserved transfers were identical between the two insemination techniques with a total of two fresh and five cryopreserved IVF-inseminated embryos resulting in three clinical pregnancies (42.9%) and five fresh and two cryopreserved ICSI-derived embryos resulting in three clinical pregnancies (42.9%). This research has shown IVF to be a legitimate fertilization technique for IVM oocytes in PCOS patients and provides a greater awareness of the use of a fertilization method previously not utilized with IVM. In-vitro maturation (IVM) is an alternative treatment method to traditional IVF. Due to the minimal use of stimulating hormones in this treatment, IVM has a lower risk of ovarian hyperstimulation syndrome, it can be used for fertility preservation in cancer patients and it is more cost conservative. Early research into the effects of IVM showed a hardening effect on the membrane surrounding the egg (the zona pellucida). It was initially believed that, to overcome this hardening in order to allow the egg to be fertilized, spermatozoa would need to be injected into the egg using intracytoplasmic sperm injection. Due to recent advances in hormonal

Effect of different types of sesquiterpene lactones on the maturation of Rhinella arenarum oocytes.

PubMed

Sánchez-Toranzo, G; Zapata-Martínez, J; Catalán, C; Bühler, M I

2015-06-01

The sesquiterpene lactones (STLs) are a large class of plant secondary metabolites that are generally found in the Asteraceae family and that have high diversity with respect to chemical structure as well as biological activity. STLs have been classified into different groups, such as guaianolides, germacranolides, and melampolides etc., based on their carboxylic skeleton. In amphibians, fully grown ovarian oocytes are arrested at the beginning of meiosis I. Under the stimulus of progesterone, this meiotic arrest is released and meiosis progresses to metaphase II, a process known as oocyte maturation. The purpose of this work was to determine whether sesquiterpene lactones from the germacranolide and melampolide groups act as inhibitor agents on the meiosis of amphibian oocytes in vitro. Results for germacranolides indicated that the addition of deoxyelephantopins caused a high degree of inhibition and that minimolide showed a moderate inhibitory effect, whereas glaucolide A was inactive. Furthermore, the addition of melampolides (uvedalin, enhydrin, polymatin A and polymatin B) showed inhibitory effects. For enhydrin and uvedalin, inhibitory effects were observed at the higher concentrations assayed. The results of this study suggest that the inhibitory activity of the tested sesquiterpene lactones on the meiosis of Rhinella arenarum oocytes is not dependent on the group to which they belong, i.e. not on the carboxylic skeleton, but probably due to the arrangement and type of function groups present in the molecules. All assayed lactones in the germacranolide group showed low toxicity. In contrast, important differences in toxicity were observed for lactones from the melampolide group: enhydrin and uvedalin showed low toxicity, but polymatin A and B were highly toxic.

The presence of acylated ghrelin during in vitro maturation of bovine oocytes induces cumulus cell DNA damage and apoptosis, and impairs early embryo development.

PubMed

Sirini, Matias A; Anchordoquy, Juan Mateo; Anchordoquy, Juan Patricio; Pascua, Ana M; Nikoloff, Noelia; Carranza, Ana; Relling, Alejandro E; Furnus, Cecilia C

2017-10-01

The aim of this study was to investigate the effects of acylated ghrelin supplementation during in vitro maturation (IVM) of bovine oocytes. IVM medium was supplemented with 20, 40 or 60 pM acylated ghrelin concentrations. Cumulus expansion area and oocyte nuclear maturation were studied as maturation parameters. Cumulus-oocyte complexes (COC) were assessed with the comet, apoptosis and viability assays. The in vitro effects of acylated ghrelin on embryo developmental capacity and embryo quality were also evaluated. Results demonstrated that acylated ghrelin did not affect oocyte nuclear maturation and cumulus expansion area. However, it induced cumulus cell (CC) death, apoptosis and DNA damage. The damage increased as a function of the concentration employed. Additionally, the percentages of blastocyst yield, hatching and embryo quality decreased with all acylated ghrelin concentrations tested. Our study highlights the importance of acylated ghrelin in bovine reproduction, suggesting that this metabolic hormone could function as a signal that prevents the progress to reproductive processes.

In vitro maturation of human oocytes for assisted reproduction.

PubMed

Jurema, Marcus W; Nogueira, Daniela

2006-11-01

To describe and evaluate the current practice of in vitro maturation of oocytes for assisted reproduction. Review of the available and relevant literature regarding in vitro maturation of oocytes. In vitro maturation of human oocytes retrieved from antral ovarian follicles is an emerging procedure quickly being incorporated into the realm of assisted reproductive technologies. This new technology has several potential advantages over traditional controlled ovarian hyperstimulation for IVF, such as reduction of costs by minimizing gonadotropin and GnRH analogue use, elimination of ovarian hyperstimulation syndrome, and simplicity of protocol. In vitro maturation of oocytes for assisted reproduction in human beings still is undergoing refinement but currently is providing efficacy and safety outcome comparable to that of traditional IVF in recent selected studies. Implementing in vitro maturation into an established IVF practice is feasible and requires only a few simple adjustments. Crucial to the advancement and optimization of the technology is a better understanding of how to maximize immature oocyte developmental competence and endometrial receptivity.

Oocyte spindle proteomics analysis leading to rescue of chromosome congression defects in cloned embryos

PubMed Central

Duan, Xunbao; Zhong, Zhisheng; Potireddy, Santhi; Moncada, Camilo; Merali, Salim; Latham, Keith E.

2015-01-01

Embryos produced by somatic cell nuclear transfer (SCNT) display low term developmental potential. This is associated with deficiencies in spindle composition prior to activation and at early mitotic divisions, including failure to assemble certain proteins on the spindle. The protein-deficient spindles are accompanied by chromosome congression defects prior to activation and during the first mitotic divisions of the embryo. The molecular basis for these deficiencies and how they might be avoided are unknown. Proteomic analyses of spindles isolated from normal metaphase II (MII) stage oocytes and SCNT constructs, along with a systematic immunofluorescent survey of known spindle-associated proteins were undertaken. This was the first proteomics study of mammalian oocyte spindles. The study revealed four proteins as being deficient in spindles of SCNT embryos in addition to those previously identified; these were clathrin heavy chain (CLTC), aurora B kinase, dynactin 4, and casein kinase 1 alpha. Due to substantial reduction in CLTC abundance after spindle removal, we undertook functional studies to explore the importance of CLTC in oocyte spindle function and in chromosome congression defects of cloned embryos. Using siRNA knockdown we demonstrated an essential role for CLTC in chromosome congression during oocyte maturation. We also demonstrated rescue of chromosome congression defects in SCNT embryos at the first mitosis using CLTC mRNA injection. These studies are the first to employ proteomics analyses coupled to functional interventions to rescue a specific molecular defect in cloned embryos. PMID:20883044

Efficiency of metaphase II oocytes following minimal/mild ovarian stimulation in vitro fertilization.

PubMed

Zhang, John J; Yang, Mingxue; Merhi, Zaher

2016-01-01

An inverse relationship between oocyte efficiency and ovarian response was reported in conventional IVF. The purpose of this study was to report metaphase II (MII) oocyte efficiency according to oocyte yield in minimal/mild stimulation IVF (mIVF) and to assess whether oocyte yield affects live birth rate (LBR). Infertile women ( n  = 264) aged < 39 years old with normal ovarian reserve who had mIVF were recruited. All participants received the same protocol for ovarian stimulation. All the embryos were cultured to the blastocyst stage and vitrified using a freeze-all approach. This was followed by a single blastocyst transferred to each participant in subsequent cycles over a 6-month period. Ovarian response was categorized according to the number of MII oocyte yield (low: 1-2, intermediate: 3-6 and high ≥ 7 MII oocytes). MII oocyte utilization rate was calculated as the number of live births divided by the number of MII oocytes produced after only one oocyte retrieval and subsequent transfers of vitrified/warmed blastocysts. The main outcome measure was cumulative LBR over a 6-month period. Among all the participants, 1173 total retrieved oocytes (4.4 ± 0.2 per patient) resulted in 1019 (3.9 ± 0.2 per patient) total MII oocytes, a clinical pregnancy rate of 48.1 % and a LBR of 41.2 %. Oocyte utilization rate was inversely related to ovarian response where it was 30.3 % in the "low" vs. 9.3 % in the "intermediate" vs. 4.3 % in the "high" oocyte yield groups ( p  < 0.05). Implantation rate significantly dropped as the number of MII oocytes increased and was highest in the "low" oocyte yield group ( p  < 0.0001). Cumulative LBR was similar in "low," "intermediate," and "high" oocyte yield groups ( p  > 0.05). The number of MII oocytes had poor sensitivity and specificity for predicting a live birth. These data extend the hypothesis of oocyte efficiency reported in conventional IVF protocols to mIVF protocols. Registration

Coordination of cellular differentiation, polarity, mitosis and meiosis - New findings from early vertebrate oogenesis.

PubMed

Elkouby, Yaniv M; Mullins, Mary C

2017-10-15

A mechanistic dissection of early oocyte differentiation in vertebrates is key to advancing our knowledge of germline development, reproductive biology, the regulation of meiosis, and all of their associated disorders. Recent advances in the field include breakthroughs in the identification of germline stem cells in Medaka, in the cellular architecture of the germline cyst in mice, in a mechanistic dissection of chromosomal pairing and bouquet formation in meiosis in mice, in tracing oocyte symmetry breaking to the chromosomal bouquet of meiosis in zebrafish, and in the biology of the Balbiani body, a universal oocyte granule. Many of the major events in early oogenesis are universally conserved, and some are co-opted for species-specific needs. The chromosomal events of meiosis are of tremendous consequence to gamete formation and have been extensively studied. New light is now being shed on other aspects of early oocyte differentiation, which were traditionally considered outside the scope of meiosis, and their coordination with meiotic events. The emerging theme is of meiosis as a common groundwork for coordinating multifaceted processes of oocyte differentiation. In an accompanying manuscript we describe methods that allowed for investigations in the zebrafish ovary to contribute to these breakthroughs. Here, we review these advances mostly from the zebrafish and mouse. We discuss oogenesis concepts across established model organisms, and construct an inclusive paradigm for early oocyte differentiation in vertebrates. Copyright © 2017 Elsevier Inc. All rights reserved.

The Chromosomal Constitution of Embryos Arising from Monopronuclear Oocytes in Programmes of Assisted Reproduction

PubMed Central

2014-01-01

The assessment of oocytes showing only one pronucleus during assisted reproduction is associated with uncertainty. A compilation of data on the genetic constitution of different developmental stages shows that affected oocytes are able to develop into haploid, diploid, and mosaic embryos with more or less complex chromosomal compositions. In the majority of cases (~80%), haploidy appears to be caused by gynogenesis, whereas parthenogenesis or androgenesis is less common. Most of the diploid embryos result from a fertilization event involving asynchronous formation of the two pronuclei or pronuclear fusion at a very early stage. Uniparental diploidy may sometimes occur if one pronucleus fails to develop and the other pronucleus already contains a diploid genome or alternatively a haploid genome undergoes endoreduplication. In general, the chance of obtaining a biparental diploid embryo appears higher after conventional in vitro fertilization than after intracytoplasmic sperm injection. If a transfer of embryos obtained from monopronuclear oocytes is envisaged, it should be tried to culture them up to the blastocyst since most haploid embryos are not able to reach this stage. Comprehensive counselling of patients on potential risks is advisable before transfer and a preimplantation genetic diagnosis could be offered if available. PMID:25763399

Influence of Meiotic Stages on Developmental Competence of Goat’ Oocyte After Vitrification

NASA Astrophysics Data System (ADS)

Wahyuningsih, S.; Ihsan, M. N.

2018-02-01

This objective of this research was to investigate effect of goat oocyte meiotic stages on developmental competence after cryopreservation. Ovaries were collected from slaugterhouse and oocytes was aspirated from2-6 mm of follicles. Oocyte with compacted cumulus cells and evenly granulated ooplasm were selected for this experiment. The lenght of in vitro maturation before vitrification was 8 or 22 h in IVM media TCM 199 + FCS 10 % + PMSG 10 IU + hCG 10 IU at 38.5 °C in a humidified atmosphere of 5 % CO2 in air and were vitrified. After vitrification process, GVBD and MII oocyte were matured for 18 or 4 h to fullfill 26 h maturation requirement and then oocytes were subjected to IVF and culture. Cleavage and blastocyst formation rate were to asses their developmental competence. Cleavage rates were obtained for both GVBD ( 56.78 %) and MII (69.64 % ) oocytes (P<0.05). Proportion of cleaved embryos from vitrified MII oocytes develop into blastocysts higher (P<0.05) than those from vitrified GVBD oocytes (10.25% vs 3.54%) repectively. Goat oocytes in different maturation stages response to vitrification differently and MII stages have better developmental competence than GVBD.

Characterization of oocyte retrieval cycles with empty zona pellucida.

PubMed

Oride, Aki; Kanasaki, Haruhiko; Hara, Tomomi; Ohta, Hiroko; Kyo, Satoru

2018-01-01

To identify the factors that characterize cycles with empty zona pellucida (EZP). Thirty-six oocyte retrieval cycles from which EZP were collected and another 36 cycles from which no EZP was collected were compared. The patients were divided into three groups: those with no EZP collected during any cycle, those with EZP collected during all cycles, and those experiencing cycles both with and without EZP. The mean number of oocytes collected per cycle was higher in the cycles with EZP than without EZP. The fertilization rate of the collected oocytes and the rate of good embryo formation were significantly lower in the cycles with EZP. No significant difference was observed between the three groups in terms of age, number of oocytes collected, or hormone levels before and after the oocyte retrieval. The fertilization and pregnancy rates were highest in the patients with no EZP being collected during any cycle, followed by those experiencing cycles both with and without EZP, and then by those with EZP collected during all cycles. The observation of lower fertilization, poor embryo formation, and a low pregnancy rate in the patients with EZP suggests the poor quality of oocytes that were collected with EZP in the same cycle.

The signaling pathways by which the Fas/FasL system accelerates oocyte aging.

PubMed

Zhu, Jiang; Lin, Fei-Hu; Zhang, Jie; Lin, Juan; Li, Hong; Li, You-Wei; Tan, Xiu-Wen; Tan, Jing-He

2016-02-01

In spite of great efforts, the mechanisms for postovulatory oocyte aging are not fully understood. Although our previous work showed that the FasL/Fas signaling facilitated oocyte aging, the intra-oocyte signaling pathways are unknown. Furthermore, the mechanisms by which oxidative stress facilitates oocyte aging and the causal relationship between Ca2+ rises and caspase-3 activation and between the cell cycle and apoptosis during oocyte aging need detailed investigations. Our aim was to address these issues by studying the intra-oocyte signaling pathways for Fas/FasL to accelerate oocyte aging. The results indicated that sFasL released by cumulus cells activated Fas on the oocyte by increasing reactive oxygen species via activating NADPH oxidase. The activated Fas triggered Ca2+ release from the endoplasmic reticulum by activating phospholipase C-γ pathway and cytochrome c pathway. The cytoplasmic Ca2+ rises activated calcium/calmodulin-dependent protein kinase II (CaMKII) and caspase-3. While activated CaMKII increased oocyte susceptibility to activation by inactivating maturation-promoting factor (MPF) through cyclin B degradation, the activated caspase-3 facilitated further Ca2+releasing that activates more caspase-3 leading to oocyte fragmentation. Furthermore, caspase-3 activation and fragmentation were prevented in oocytes with a high MPF activity, suggesting that an oocyte must be in interphase to undergo apoptosis.

Prospective randomized comparison of human oocyte cryopreservation with slow-rate freezing or vitrification.

PubMed

Smith, Gary D; Serafini, Paulo C; Fioravanti, Joyce; Yadid, Isaac; Coslovsky, Marcio; Hassun, Pericles; Alegretti, José Roberto; Motta, Eduardo L

2010-11-01

To compare cryopreservation of mature human oocytes with slow-rate freezing and vitrification and determine which is most efficient at establishing a pregnancy. Prospective randomized. Academically affiliated, private fertility center. Consenting patients with concerns about embryo cryopreservation and more than nine mature oocytes at retrieval were randomized to slow-rate freezing or vitrification of supernumerary (more than nine) oocytes. Oocytes were frozen or vitrified, and upon request oocytes were thawed or warmed, respectively. Oocyte survival, fertilization, embryo development, and clinical pregnancy. Patient use has resulted in 30 thaws and 48 warmings. Women's age at time of cryopreservation was similar. Oocyte survival was significantly higher following vitrification/warming (81%) compared with freezing/thawing (67%). Fertilization was more successful in oocytes vitrified/warmed compared with frozen/thawed. Fertilized oocytes from vitrification/warming had significantly better cleavage rates (84%) compared with freezing/thawing (71%) and resulted in embryos with significantly better morphology. Although similar numbers of embryos were transferred, embryos resulting from vitrified oocytes had significantly enhanced clinical (38%) pregnancy rates compared with embryos resulting from frozen oocyte (13%). Miscarriage and/or spontaneous abortion rates were similar. Our results suggest that vitrification/warming is currently the most efficient means of oocyte cryopreservation in relation to subsequent success in establishing pregnancy. Copyright © 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Effects of mineral supplements on ovulation and maturation of dog oocytes.

PubMed

Kim, Min Jung; Oh, Hyun Ju; Park, Jung Eun; Kim, Geon A; Park, Eun Jung; Jang, Goo; Lee, Byeong Chun

2012-07-01

The aim of this study was to assess the effects of trace mineral supplements near the time of ovulation on the number of ovulated oocytes, in vivo oocyte maturation and pregnancy for dog cloning. Sixteen oocyte donor dogs were used in each control and mineral supplement group, and 136 and 166 corpora lutea were counted from each group. No significant difference was observed between oocyte recovery rates in the control (91.2 ± 2.7%) and mineral (89.9 ± 2.7) groups. Proportions of mature (86.2 ± 7.2 and 88.4 ± 6.8%) and aged (13.8 ± 7.2 and 11.6 ± 6.8%) oocytes were not different in the control and mineral groups, respectively. Oocytes with fair (91.5 ± 3.6 and 93.6 ± 2.1%) and poor (8.5 ± 3.6 and 6.4 ± 2.1%) quality also showed no difference between the control and mineral groups. The concentrations of manganese and ferrous iron were higher and lower on the day of ovulation, respectively, in both groups, but trace element concentrations in peripheral blood were not affected by mineral treatment. Oocytes were used to make cloned embryos; after embryo transfer, four and two pups were delivered from the control and mineral group, respectively, but there was no difference in the delivery rate (4.6 and 2.7%). In conclusion, intravenous mineral supplements administered once close to the LH surge in oocyte donor dogs and recipients had no effect on the number of ovulated oocytes, in vivo oocyte maturation or pregnancy in dog cloning in this study. Copyright © 2012 Elsevier Inc. All rights reserved.

Effects of tributyltin chloride on developing mouse oocytes and preimplantation embryos.

PubMed

Huang, Xian-Ju; Shen, Ming; Wang, Lizhong; Yu, Fengxiang; Wu, Wangjun; Liu, Hong-Lin

2015-04-01

Tributyltin, an organotin, is ubiquitous in estuaries and freshwater systems. Previous reports suggest that tributyltin is an endocrine disruptor in many wildlife species and it inhibits aromatase in mammalian placental and granulosa-like tumor cell lines. However, no evidence showing the effects of tributyltin on oocytes or preimplantation embryonic developmental competence exists. Therefore, we investigated the role of tributyltin chloride (TBTCl) in the development of female oocytes and preimplantation embryos. Briefly, female ICR mice were gavaged with 0 (vehicle), 4, and 8 mg/kg of TBTCl each day for 18 days. The fluorescence intensity analysis showed that the 5-methylcytosine level decreased after TBTCl treatment, indicating that the general DNA methylation level decreased in the treated oocytes. Our results demonstrate that TBTCl treatment results in decreased mRNA levels of imprinted genes H19, Igf2r, and Peg3 during oocyte growth. The TBTCl-treated oocytes showed a significant increase in reactive oxygen species levels in germinal vesicle oocytes. In TBTCl-treated oocytes, there was no difference in GPx and Sod1 expression, but a decreased mRNA level of Cat occurred when compared with control. Moreover, the blastocysts with TBTCl exposure displayed higher apoptotic signals. These results suggest that TBTCl induces developmental defects in oocytes and preimplantation embryos.

Propofol versus thiopental sodium as anaesthetic agents for oocyte retrieval: a randomized controlled trial.

PubMed

Goutziomitrou, Evangelia; Venetis, Christos A; Kolibianakis, Efstratios M; Bosdou, Julia K; Parlapani, Aggeliki; Grimbizis, Gregoris; Tarlatzis, Basil C

2015-12-01

Clinical outcomes of IVF cycles using propofol or thiopental sodium as anaesthetic agents for oocyte retrieval were compared. The primary outcome measure was fertilization rate per patient. One hundred and eighty patients undergoing ovarian stimulation with gonadotrophins and gonadotrophin-releasing hormone antagonists for IVF were randomized to receive either propofol (n = 90) or thiopental sodium (n = 90). No significant differences in baseline characteristics were present between the two groups. Overall fertilization rates were similar between propofol and thiopental sodium groups, respectively: median (IQR): 54.8 (29.2) versus 54.6 (29.7); fertilization rates for intracytoplasmic sperm injection only: median (IQR): 70 (50) versus 75 (50), respectively. For secondary outcome measures, time under anaesthesia was significantly increased in the thiopental sodium group: median (IQR): 12(5) versus 10 (4.5) min, P = 0.019 compared with the propofol group. Number of cumulus oocyte complexes retrieved [median (IQR): 7.1 (6.3) versus 6.5 (5.6)] did not differ significantly between the two groups. A non-significant difference in live birth rates per randomized patient of +4.4% (95% CI: -5.7 to +14.6) in favour of propofol was observed. Use of propofol compared with thiopental sodium for general anaesthesia during oocyte retrieval results in similar fertilization rates and IVF outcomes. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Trichloroethylene Metabolism in the Rat Ovary Reduces Oocyte Fertilizability

PubMed Central

Wu, Katherine Lily; Berger, Trish

2007-01-01

Exposure to trichloroethylene (TCE, an environmental toxicant) reduced oocyte fertilizability in the rat. In vivo, TCE may be metabolized by cytochrome P450 dependent oxidation or glutathione conjugation in the liver or kidneys, respectively. Cytochrome P450 dependent oxidation is the higher affinity pathway. The primary isoform of cytochrome P450 to metabolize TCE in the liver, cytochrome P450 2E1, is present in the rodent ovary. Ovarian metabolism of TCE by the oxidative pathway and the production of reactive oxygen species may occur given the presence of the metabolizing enzyme. The objectives of this study were to define the sensitive interval of oocyte growth to TCE exposure, and to determine if TCE exposure resulted in the formation of ovarian protein carbonyls, an indicator of oxidative damage. Rats were exposed to TCE in drinking water (0.45% TCE (v/v) in 3% Tween) or 3% Tween (vehicle-control) during three 4–5 day intervals of oocyte development preceding ovulation. Oocytes from TCE-exposed females were less fertilizable compared with vehicle-control oocytes. Immunohistochemical labeling of ovaries and Western blotting of ovarian proteins demonstrated TCE treatment induced a greater incidence of protein carbonyls compared with vehicle controls. Protein carbonyl formation in the ovary is consistent with TCE metabolism by the cytochrome P450 pathway. Oxidative damage following ovarian TCE metabolism or the presence of TCE metabolites may contribute to reduced oocyte fertilizability. In summary, these results indicate maturing oocytes are susceptible to very short in vivo exposures to TCE. PMID:17673192

Cytoskeleton and Cytoskeleton-Bound RNA Visualization in Frog and Insect Oocytes.

PubMed

Kloc, Malgorzata; Bilinski, Szczepan; Kubiak, Jacek Z

2016-01-01

The majority of oocyte functions involves and depends on the cytoskeletal elements, which include microtubules and actin and cytokeratin filaments. Various structures and molecules are temporarily or permanently bound to the cytoskeletal elements and their functions rely on cytoskeleton integrity and its timely assembly. Thus the accurate visualization of cytoskeleton is often crucial for studies and analyses of oocyte structure and functions. Here we describe several reliable methods for microtubule and/or microfilaments preservation and visualization in Xenopus oocyte extracts, and in situ in live and fixed insect and frog (Xenopus) oocytes. In addition, we describe visualization of cytoskeleton-bound RNAs using molecular beacons in live Xenopus oocytes.

Liver condition of Holstein cows affects mitochondrial function and fertilization ability of oocytes

PubMed Central

TANAKA, Hiroshi; TAKEO, Shun; ABE, Takahito; KIN, Airi; SHIRASUNA, Koumei; KUWAYAMA, Takehito; IWATA, Hisataka

2016-01-01

The aim of the present study was to examine the fertilization ability and mitochondrial function of oocytes derived from cows with or without liver damage. Oocytes were collected from the ovaries of cows with damaged livers (DL) and those of cows with healthy livers (HL), subjected to in vitro maturation, and fertilized in vitro. A significantly high abnormal fertilization rate was observed for oocytes from DL cows compared to oocytes from HL cows. The time to dissolve the zona pellucida by protease before fertilization was similar between the two liver conditions, whereas after fertilization treatment this time was shorter for DL cows than for HL cows. The percentage of oocytes with equivalent cortical granule distributions underneath the membrane was greater for in vitro matured oocytes from HL cows, whereas an immature distribution pattern was observed for oocytes from DL cows. In addition, a greater percentage of oocytes derived from HL cows released cortical granules following fertilization compared with oocytes from DL cows. Mitochondrial function determined by ATP content and membrane potential were similar at the germinal vesicle stage, but post-in vitro maturation, the oocytes derived from HL cows showed higher values than DL cows. The mitochondrial DNA copy number in oocytes was similar between the two liver conditions for both the germinal vesicle and post-in vitro maturation oocytes. In conclusion, liver damage induces low fertilization, likely because of incomplete cortical granule distribution and release, and the maturation of oocytes from DL cows contain low-functioning mitochondria compared to their HL counterparts. PMID:26832309

Liver condition of Holstein cows affects mitochondrial function and fertilization ability of oocytes.

PubMed

Tanaka, Hiroshi; Takeo, Shun; Abe, Takahito; Kin, Airi; Shirasuna, Koumei; Kuwayama, Takehito; Iwata, Hisataka

2016-06-17

The aim of the present study was to examine the fertilization ability and mitochondrial function of oocytes derived from cows with or without liver damage. Oocytes were collected from the ovaries of cows with damaged livers (DL) and those of cows with healthy livers (HL), subjected to in vitro maturation, and fertilized in vitro. A significantly high abnormal fertilization rate was observed for oocytes from DL cows compared to oocytes from HL cows. The time to dissolve the zona pellucida by protease before fertilization was similar between the two liver conditions, whereas after fertilization treatment this time was shorter for DL cows than for HL cows. The percentage of oocytes with equivalent cortical granule distributions underneath the membrane was greater for in vitro matured oocytes from HL cows, whereas an immature distribution pattern was observed for oocytes from DL cows. In addition, a greater percentage of oocytes derived from HL cows released cortical granules following fertilization compared with oocytes from DL cows. Mitochondrial function determined by ATP content and membrane potential were similar at the germinal vesicle stage, but post-in vitro maturation, the oocytes derived from HL cows showed higher values than DL cows. The mitochondrial DNA copy number in oocytes was similar between the two liver conditions for both the germinal vesicle and post-in vitro maturation oocytes. In conclusion, liver damage induces low fertilization, likely because of incomplete cortical granule distribution and release, and the maturation of oocytes from DL cows contain low-functioning mitochondria compared to their HL counterparts.

Consequences of metaphase II oocyte cryopreservation on mRNA content.

PubMed

Chamayou, S; Bonaventura, G; Alecci, C; Tibullo, D; Di Raimondo, F; Guglielmino, A; Barcellona, M L

2011-04-01

We studied the consequences of freezing/thawing processes on mRNA contents in MII oocytes after slow-freezing/rapid thawing (SF/RT) and vitrification/warming (V/W) protocols, and compared the results to fresh MII oocytes. We quantified the nuclear transcript mRNA responsible for the translation of proteins belonging either to trans-regulatory protein family or to functional structural proteins such as proteins involved in DNA structural organization (NAP1L1, TOP1, H1F0H1), chromosomal structure maintenance (SMC, SCC3, RAD21, SMC1A, SMC1B, STAG3, REC8), mitochondrial energetic pathways (ATP5GJ, SDHC), cell cycle regulation and processes (CLTA, MAPK6, CKS2) and staminal cell potency-development competence stage (DPPA3, OCT4, FOXJ2). Surplus MII oocytes were donated from patients in IVF cycles and divided in three groups of 15 oocytes. Group 1 was comprised of non-cryopreserved oocytes and Groups 2 and 3 underwent SF/RT and V/W procedures, respectively. There was an overall decrease of mRNA extracted from cryopreserved oocytes compared to control group. Only 39.4% of mRNA content were preserved after SF/RT while 63.3% of mRNA content were maintained after V/W. Oocyte cryopreservation is associated with molecular injury associated with the decrease of stored mRNA. However the V/W protocol is more conservative than SF/RT resulting in a level of mRNA sufficient to maintain biologic functions in the subsequent fertilized oocyte. Copyright © 2011 Elsevier Inc. All rights reserved.

Mature Oocyte Cryopreservation for Fertility Preservation.

PubMed

Liang, Tina; Motan, Tarek

2016-01-01

In recent decades, advances in cancer treatment have led to a dramatic improvement in long term survival. This has led to an increasing focus on quality of life after surviving cancer treatment, with fertility being an important aspect. Given the known reproductive risks of cancer therapies, there has been a growing interest in the field of fertility preservation (also referred to as oncofertility). Mature oocyte cryopreservation is no longer considered experimental and has become a realistic option for reproductive aged women prior to undergoing cancer treatment. Additionally, as cryopreservation techniques improve, mature oocyte cryopreservation is increasing being marketed to healthy women without cancer wishing to delay child bearing, also termed "social egg freezing". This chapter provides a review of the current technology, use, and outcomes of mature oocyte cryopreservation. It also outlines the ethical debate surrounding social egg freezing and directions for future research in female fertility preservation.

The Human Oocyte Preservation Experience (HOPE) a phase IV, prospective, multicenter, observational oocyte cryopreservation registry.

PubMed

Ezcurra, Diego; Rangnow, Jennifer; Craig, Maryellen; Schertz, Joan

2009-05-27

It has been recommended by the American Society of Clinical Oncology and the American Society of Reproductive Medicine that options to preserve fertility be presented at the outset of treatment for cancer. This recommendation may have arisen, in part, to the increasing survival of patients with cancer and the realization that certain forms of cancer treatment can lead to infertility. One option for these patients, particularly those with ethical or religious objections to freezing embryos is oocyte cryopreservation. However universal acceptance of these procedures has yet to be established, most likely due to a poor history of success and concerns that there has yet to be a comprehensive approach to evaluating these techniques. In light of this, a registry of patients undergoing oocyte cryopreservation, called the HOPE registry, is being implemented. The intent of the HOPE Registry is to enroll approximately 400 women of reproductive age who will undergo thawing/warming of oocytes and subsequent transfer. Data from the patients enrolled will be collected via a uniform, standardized form and will document important parameters such as demographics, laboratory procedures and outcomes, including following the outcomes of babies born for one year after birth. The results of the registry will be published on a yearly basis. A patient registry has been established in order to systematically document the techniques and outcomes of oocyte cryopreservation procedures. The results will be published in order to provide a widely accessible resource that will allow patients who are considering these procedures validated information in order to make informed decisions as to how their treatment will proceed.

Differential sensitivity of mouse oocytes to colchicine-induced aneuploidy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mailhes, J.B.; Yuan, Z.P.

1987-01-01

Unpublished results from our laboratory showed that colchicine increased the incidence of hyperploid mouse metaphase II (MII) oocytes when injected at the same time as human chorionic gonadotrophin (HCG). The objective of the present study was to determine whether the time of administering colchicine influenced the incidence of aneuploidy in MII oocytes. CD-1 mice were given pregnant mare's serum (PMS) and, 48 hr later, HCG. An intraperitoneal injection of 0.2 mg/kg colchicine was given at +4, +2, 0, -2, or -4 hr relative to HCG. Oocytes were collected 17 hr post-HCG and processed, and chromosomes were subsequently C-banded. The percentagemore » of hyperploid oocytes was 0.77, 2.56, 5.71, 7.79, 3.54, and 2.70 for control, +4, +2, 0, -2, or -4 hr pre/post-HCG, respectively. Chi-square analyses of these data demonstrated that colchicine significantly increases the proportion of aneuploid oocytes, and that the relative sensitivity of colchicine-induced aneuploidy depends upon the time that this drug is administered relative to HCG.« less

Fibroblast growth factor 10 enhances bovine oocyte maturation and developmental competence in vitro.

PubMed

Zhang, Kun; Hansen, Peter J; Ealy, Alan D

2010-12-01

The ability of oocytes to resume meiosis, become fertilized, and generate viable pregnancies is controlled during folliculogenesis by several endocrine and paracrine factors. The aim of this work is to determine whether fibroblast growth factor 10 (FGF10) is an oocyte competent factor. Transcripts for each of the four FGF receptor types (FGFR) were present in cumulus and oocytes after their extraction from the follicles. FGFR1 transcripts predominated in cumulus cells whereas FGFR2 was most abundant in oocytes. Exposing the cumulus-oocyte complexes to FGF10 during in vitro maturation did not affect cleavage rates, but increases (P<0.05) in the percentage of embryos at the 8-16-cell stage on day 3 and at the blastocyst stage on day 7, which were evident in FGF10-supplemented oocytes. The progression of oocytes through meiosis and cumulus expansion was increased (P<0.05) by FGF10. The importance of the endogenous sources of FGFs was examined by adding anti-FGF10 IgG during oocyte maturation. Blocking endogenous FGF10 activity decreased (P<0.05) the percentage of oocytes developing into blastocysts and limited (P<0.05) cumulus expansion. Expression profiles of putative cumulus and oocyte competency markers were examined for their involvement in FGF10-mediated responses. FGF10 influenced the expression of CTSB and SPRY2 in cumulus cells and BMP15 in oocytes. In summary, this work provides new insight into the importance of FGFRs and locally derived FGF10 during oocyte maturation in cattle. Its subsequent impact on in vitro embryo development implicates it as a noteworthy oocyte competent factor.

SMC5/6 is required for the formation of segregation-competent bivalent chromosomes during meiosis I in mouse oocytes.

PubMed

Hwang, Grace; Sun, Fengyun; O'Brien, Marilyn; Eppig, John J; Handel, Mary Ann; Jordan, Philip W

2017-05-01

SMC complexes include three major classes: cohesin, condensin and SMC5/6. However, the localization pattern and genetic requirements for the SMC5/6 complex during mammalian oogenesis have not previously been examined. In mouse oocytes, the SMC5/6 complex is enriched at the pericentromeric heterochromatin, and also localizes along chromosome arms during meiosis. The infertility phenotypes of females with a Zp3-Cre -driven conditional knockout (cKO) of Smc5 demonstrated that maternally expressed SMC5 protein is essential for early embryogenesis. Interestingly, protein levels of SMC5/6 complex components in oocytes decline as wild-type females age. When SMC5/6 complexes were completely absent in oocytes during meiotic resumption, homologous chromosomes failed to segregate accurately during meiosis I. Despite what appears to be an inability to resolve concatenation between chromosomes during meiosis, localization of topoisomerase IIα to bivalents was not affected; however, localization of condensin along the chromosome axes was perturbed. Taken together, these data demonstrate that the SMC5/6 complex is essential for the formation of segregation-competent bivalents during meiosis I, and findings suggest that age-dependent depletion of the SMC5/6 complex in oocytes could contribute to increased incidence of oocyte aneuploidy and spontaneous abortion in aging females. © 2017. Published by The Company of Biologists Ltd.

Human oocyte cryopreservation in infertility and oncology.

PubMed

Porcu, Eleonora; Bazzocchi, Antonia; Notarangelo, Leonardo; Paradisi, Roberto; Landolfo, Chiara; Venturoli, Stefano

2008-12-01

To evaluate the present state of research and clinical application of human oocyte cryopreservation in infertility and oncology. Recent literature documents have an increasing interest in cryopreserving human eggs. A number of studies report on different freezing protocols and various types of clinical application. Increasing attention is paid to vitrification as an alternative to slow cooling for oocyte cryopreservation. Several studies cover the modification of meiotic spindle during cryopreservation in order to assess the less damaging cryopreservation system. The first births with cryopreserved oocytes in cancer patients are reported. Egg freezing may circumvent the ethical and legal concerns regarding embryo cryopreservation, increase assisted reproduction flexibility and be a concrete option to save fertility in women with cancer. Recently, egg survival and pregnancy rates improved, with the birth of more than 500 children. The birth rate per thawed oocyte is around 5-6%. As regards safety, data on birth defects seems to be reassuring so far but must be monitored by an international registry. Comparative studies between slow freezing and vitrification in the same patient population are needed to elucidate pros and cons of each technique.

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

PubMed

Nikiforaki, D; Vanden Meerschaut, F; Qian, C; De Croo, I; Lu, Y; Deroo, T; Van den Abbeel, E; Heindryckx, B; De Sutter, P

2014-01-01

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

Physiology and Endocrinology Symposium: influence of cattle genotype (Bos indicus vs. Bos taurus) on oocyte and preimplantation embryo resistance to increased temperature.

PubMed

Paula-Lopes, F F; Lima, R S; Satrapa, R A; Barros, C M

2013-03-01

High environmental temperatures during the hot months of the year reduce reproductive performance in cattle. Summer heat stress depression in fertility is a multifactorial problem; however, there is evidence that the bovine germinal vesicle and maturing oocyte, as well as the early embryo, are major targets of the deleterious effects of heat stress. Such adverse effects are less pronounced in heat-tolerant breeds (Bos indicus) than heat-sensitive breeds (Bos taurus). This genetic variation results from the greater thermoregulatory ability and cellular thermoresistance of heat-tolerant breeds. Heat-induced oocyte cellular damage occurs in both cytoplasmic and nuclear compartments. Heat shock has been shown to reduce oocyte nuclear maturation, induce apoptosis, compromise oocyte cytoskeleton, and impair oocyte mitochondrial function and developmental competence. However, the oocyte cytoplasm is more susceptible to heat shock than the nucleus. This effect is greater for Bos taurus than Bos indicus oocytes. The detrimental effects of heat shock are also critical during the first cleavage divisions when most of the embryonic genome is inactive; however, the bovine embryo becomes more resistant to increased temperature as it proceeds through development. Several studies demonstrated that Bos indicus embryos are more thermotolerant than Bos taurus embryos. Adaptive changes involved in acquisition of thermotolerance are likely derived from changes in gene expression and (or) activity of biochemical molecules that control cellular functions against stress. Recently, molecules such as IGF-I and caspase inhibitor z-DEVD-fmk have been shown to exert a thermoprotective role, rescuing heat-induced oocyte and embryo cellular damage and developmental competence. Therefore, cattle genotype and thermoprotective molecules can be considered as an alternative to modulate the effects of increased temperature in reproductive function.

Femtosecond laser based enucleation of porcine oocytes for somatic cell nuclear transfer

NASA Astrophysics Data System (ADS)

Kütemeyer, K.; Lucas-Hahn, A.; Petersen, B.; Hassel, P.; Lemme, E.; Niemann, H.; Heisterkamp, A.

2009-07-01

Cloning of several mammalian species has been achieved by somatic cell nuclear transfer (SCNT) in recent years. However, this method still results in very low efficiencies around 1% which originate from suboptimal culture conditions and highly invasive techniques for oocyte enucleation and injection of the donor cell using micromanipulators. In this paper, we present a new minimal invasive method for oocyte imaging and enucleation based on the application of femtosecond (fs) laser pulses. After imaging of the oocyte with multiphoton microscopy, ultrashort pulses are focused onto the metaphase plate of MII-oocytes in order to ablate the DNA molecules. We show that fs laser based enucleation of porcine oocytes completely inhibits the first mitotic cleavage after parthenogenetic activation while maintaining intact oocyte morphology in most cases. In contrast, control groups without previous irradiation of the metaphase plate are able to develop to the blastocyst stage. Further experiments have to clarify the suitability of fs laser based enucleated oocytes for SCNT.

A comparison of the Cook single lumen immature ovum IVM needle to the Steiner-Tan pseudo double lumen flushing needle for oocyte retrieval for IVM.

PubMed

Rose, B I; Laky, D

2013-06-01

This study compared the impact of using the Steiner-Tan pseudo double lumen needle for antral follicle oocyte retrieval to using a conventional non-flushing needle. The Steiner-Tan needle has a much smaller dead space than the needles commonly used for IVM oocyte retrievals. This was a retrospective cohort study. The patient population was determined by the time period in which a patient underwent IVM in a single physician's IVF practice. The following data was abstracted from clinical and embryology records: oocytes retrieved, oocytes matured, early maturing oocytes, oocytes fertilized, embryo quality measures, retrieval time, needle punctures, clot formation, and clinical pregnancy rate. The Steiner-Tan needle did not increase the number of oocytes retrieved. It also did not increase the time required for retrieval. However, flushing of antral follicles significantly decreased clot formation in fluid aspirates. Use of the Steiner-Tan needle also significantly decreased the number of vaginal needle punctures during each case. There was a trend toward improved embryo quality, but statistical power was inadequate to show a difference. The primary benefit of the Steiner-Tan needle was on the embryological aspects of IVM. Decreased blood and blood clots in the aspirates made an IVM retrieval more like conventional IVF for the embryologist. The patient also experienced less tissue trauma without increasing anesthesia or surgical time. There was no improvement in the number of oocytes retrieved, but based on the results, we hypothesized that oocytes were more commonly retrieved from slightly large follicles than when using a routine needle.

Supporting and Inhibiting the Well-Being of Early Career Secondary School Teachers: Extending Self-Determination Theory

ERIC Educational Resources Information Center

Hobson, Andrew J.; Maxwell, Bronwen

2017-01-01

This paper reports an original examination of the well-being of early career secondary school teachers in England, which extends the evidence bases relating to early career teachers' working lives, teacher well-being, self-determination theory and performativity, respectively. Drawing on a secondary analysis of qualitative data generated for four…

Effects of dimethyl sulfoxide on asymmetric division and cytokinesis in mouse oocytes.

PubMed

Zhou, Dongjie; Shen, Xinghui; Gu, Yanli; Zhang, Na; Li, Tong; Wu, Xi; Lei, Lei

2014-06-21

Dimethyl sulfoxide (DMSO) is used extensively as a permeable cryoprotectant and is a common solvent utilized for several water-insoluble substances. DMSO has various biological and pharmacological activities; however, the effect of DMSO on mouse oocyte meiotic maturation remains unknown. In DMSO-treated oocytes, we observed abnormal MII oocytes that contained large polar bodies, including 2-cell-like MII oocytes, during in vitro maturation. Oocyte polarization did not occur, due to the absence of actin cap formation and spindle migration. These features are among the primary causes of abnormal symmetric division; however, analysis of the mRNA expression levels of genes related to asymmetric division revealed no significant difference in the expression of these factors between the 3% DMSO-treated group and the control group. After each "blastomere" of the 2-cell-like MII stage oocytes was injected by one sperm head respectively, the oocytes still possessed the ability to extrude the second polar body from each "blastomere" and to begin cleavage. However, MII oocytes with large polar bodies developed to the blastocyst stage after intracytoplasmic sperm injection (ICSI). Furthermore, other permeable cryoprotectants, such as ethylene glycol and glycerol, also caused asymmetric division failure. Permeable cryoprotectants, such as DMSO, ethylene glycol, and glycerol, affect asymmetric division. DMSO disrupts cytokinesis completion by inhibiting cortical reorganization and polarization. Oocytes that undergo symmetric division maintain the ability to begin cleavage after ICSI.

Melatonin accelerates maturation inducing hormone (MIH): induced oocyte maturation in carps.

PubMed

Chattoraj, Asamanja; Bhattacharyya, Sharmistha; Basu, Dipanjan; Bhattacharya, Shelley; Bhattacharya, Samir; Maitra, Saumen Kumar

2005-02-01

The present communication is an attempt to demonstrate the influence of melatonin on the action of maturation inducing hormone (MIH) on the maturation of oocytes in carps. The oocytes from gravid female major carp Labeo rohita were isolated and incubated separately in Medium 199 containing (a) only MIH (1 microg/ml), (b) only melatonin (at concentrations of 50, 100 or 500 pg/ml), and (c) both melatonin and MIH, but at different time intervals. In the latter group, melatonin was added to the incubating medium either (i) 4 h before addition of MIH, (ii) 2 h before addition of MIH, (iii) co-administered with MIH (0 h interval) or (iv) 2 h after addition of MIH. In each case, oocytes were further incubated for 4, 8, 12 or 16 h post- administration of MIH, and the effects of treatment on oocyte maturation were evaluated by considering the rate (%) of germinal vesicle breakdown (GVBD). Incubation of oocytes in a medium containing only melatonin did not result in GVBD of any oocyte. Nearly all the oocytes underwent GVBD when incubated with MIH for 16 h. Administration of melatonin along with MIH (at 0 h interval) or 2 h after addition of MIH did not result in any significant change in the rate of GVBD compared to that in a medium containing only MIH. However, it was quite interesting to observe that incubation of oocytes with melatonin especially 4 h prior to addition of MIH in the medium, led to an accelerated rate of GVBD in the oocytes. Experiments with the oocytes of another major carp Cyprinus carpio following an identical schedule depicted similar results except a difference in the optimum melatonin dose. In L. rohita, 50 pg/ml melatonin had maximum acceleratory effect on MIH-induced GVBD of oocytes, while it was 100 pg/ml in C. carpio. Further study revealed that pre-incubation with melatonin accelerates the action of MIH on the formation of a complex of two proteins (MPF), a regulatory component called cyclin B and the catalytic component protein kinase known as

Transcription factors SOHLH1 and SOHLH2 coordinate oocyte differentiation without affecting meiosis I.

PubMed

Shin, Yong-Hyun; Ren, Yu; Suzuki, Hitomi; Golnoski, Kayla J; Ahn, Hyo Won; Mico, Vasil; Rajkovic, Aleksandar

2017-06-01

Following migration of primordial germ cells to the genital ridge, oogonia undergo several rounds of mitotic division and enter meiosis at approximately E13.5. Most oocytes arrest in the dictyate (diplotene) stage of meiosis circa E18.5. The genes necessary to drive oocyte differentiation in parallel with meiosis are unknown. Here, we have investigated whether expression of spermatogenesis and oogenesis bHLH transcription factor 1 (Sohlh1) and Sohlh2 coordinates oocyte differentiation within the embryonic ovary. We found that SOHLH2 protein was expressed in the mouse germline as early as E12.5 and preceded SOHLH1 protein expression, which occurred circa E15.5. SOHLH1 protein appearance at E15.5 correlated with SOHLH2 translocation from the cytoplasm into the nucleus and was dependent on SOHLH1 expression. NOBOX oogenesis homeobox (NOBOX) and LIM homeobox protein 8 (LHX8), two important regulators of postnatal oogenesis, were coexpressed with SOHLH1. Single deficiency of Sohlh1 or Sohlh2 disrupted the expression of LHX8 and NOBOX in the embryonic gonad without affecting meiosis. Sohlh1-KO infertility was rescued by conditional expression of the Sohlh1 transgene after the onset of meiosis. However, Sohlh1 or Sohlh2 transgene expression could not rescue Sohlh2-KO infertility due to a lack of Sohlh1 or Sohlh2 expression in rescued mice. Our results indicate that Sohlh1 and Sohlh2 are essential regulators of oocyte differentiation but do not affect meiosis I.

Oocyte vitrification as an efficient option for elective fertility preservation.

PubMed

Cobo, Ana; García-Velasco, Juan A; Coello, Aila; Domingo, Javier; Pellicer, Antonio; Remohí, José

2016-03-01

To provide a detailed description of the current oocyte vitrification status as a means of elective fertility preservation (EFP). Retrospective observational multicenter study. Private university-affiliated center. A total of 1,468 women who underwent EFP because of age or having associated a medical condition other than cancer (January 2007 to April 2015). None. Survival and cumulative live birth rate (CLBR) per consumed oocyte. Mean age was higher with EFP due to age versus having an associated medical reason (37.7 y [95% confidence interval (CI) 36.5-37.9] vs. 35.7 y [95% CI 34.9-36.3]). In total, 137 patients (9.3%) returned to use their oocytes. Overall survival rate was 85.2% (95% CI 83.2-87.2). Live birth rate per patient was higher in women ≤35 years old than ≥36 years old (50% [95% CI 32.7-67.3] vs. 22.9% [95% CI 14.9-30.9]). CLBR was higher and increased faster in younger women. The gain in CLBR was sharp from 5 (15.4%, 95% CI -4.2 to 35.0) to 8 oocytes (40.8%, 95% CI 13.2-68.4), with an 8.4% gain per additional oocyte, in the ≤35-year-old group. The increase was slower with 10-15 oocytes, reaching a plateau CLBR of 85.2%. A milder increase (4.9% gain) was observed in the ≥36-year-old group (from 5.1% [95% CI -0.6 to 10.7] to 19.9% [95% CI 8.7-31.1] when 5-8 oocytes were consumed), reaching the plateau with 11 oocytes (CLBR 35.6%). Forty babies were born. At least 8-10 metaphase II oocytes are necessary to achieve reasonable success. Numbers should be individualized in women >36 years old. We suggest encouraging women who are motivated exclusively by a desire to postpone childbearing because of age, to come at younger ages to increase success possibilities. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Artificial intelligence techniques for embryo and oocyte classification.

PubMed

Manna, Claudio; Nanni, Loris; Lumini, Alessandra; Pappalardo, Sebastiana

2013-01-01

One of the most relevant aspects in assisted reproduction technology is the possibility of characterizing and identifying the most viable oocytes or embryos. In most cases, embryologists select them by visual examination and their evaluation is totally subjective. Recently, due to the rapid growth in the capacity to extract texture descriptors from a given image, a growing interest has been shown in the use of artificial intelligence methods for embryo or oocyte scoring/selection in IVF programmes. This work concentrates the efforts on the possible prediction of the quality of embryos and oocytes in order to improve the performance of assisted reproduction technology, starting from their images. The artificial intelligence system proposed in this work is based on a set of Levenberg-Marquardt neural networks trained using textural descriptors (the local binary patterns). The proposed system was tested on two data sets of 269 oocytes and 269 corresponding embryos from 104 women and compared with other machine learning methods already proposed in the past for similar classification problems. Although the results are only preliminary, they show an interesting classification performance. This technique may be of particular interest in those countries where legislation restricts embryo selection. One of the most relevant aspects in assisted reproduction technology is the possibility of characterizing and identifying the most viable oocytes or embryos. In most cases, embryologists select them by visual examination and their evaluation is totally subjective. Recently, due to the rapid growth in our capacity to extract texture descriptors from a given image, a growing interest has been shown in the use of artificial intelligence methods for embryo or oocyte scoring/selection in IVF programmes. In this work, we concentrate our efforts on the possible prediction of the quality of embryos and oocytes in order to improve the performance of assisted reproduction technology

Resveratrol Improves the Mitochondrial Function and Fertilization Outcome of Bovine Oocytes

PubMed Central

TAKEO, Shun; SATO, Daichi; KIMURA, Koji; MONJI, Yasunori; KUWAYAMA, Takehito; KAWAHARA-MIKI, Ryoka; IWATA, Hisataka

2013-01-01

The aim of the present study was to address the effect of resveratrol-mediated upregulation of sirtuin 1 (SIRT1) during oocyte maturation on mitochondrial function, the developmental ability of oocytes and on mechanisms responsible for blockage of polyspermic fertilization. Oocytes collected from slaughterhouse-derived ovaries were cultured in TCM-199 medium supplemented with 10% FCS and 0 or 20 µM resveratrol (Res). We examined the effect of Res on SIRT1 expression in in vitro-matured oocytes (Exp 1); fertilization and developmental ability (Exp 2); mitochondrial DNA copy number (Mt number), ATP content and mitochondrial membrane potential in matured oocytes (Exp 3); and the time required for proteinase to dissolve the zona pellucida following in vitro fertilization (as a marker of zona pellucida hardening), as well as on the distribution of cortical granules before and after fertilization (Exp 4). In Exp 1, the 20 µM Res treatment upregulated protein expression of SIRT1 in oocytes. In Exp 2, Res treatment improved the ratio of normal fertilization and the total cell number of blastocysts. In Exp 3, Res treatment significantly increased the ATP content in matured oocytes. Additionally, Res increased the overall Mt number and mitochondrial membrane potential, but the effect was donor-dependent. In Exp 4, Res-induced zona hardening improved the distribution and exocytosis of cortical granules after in vitro fertilization. In conclusion, Res improved the quality of oocytes by improving mitochondrial quantity and quality. In addition, Res added to the maturation medium enhanced SIRT1 protein expression in oocytes and improved fertilization via reinforcement of the mechanisms responsible for blockage of polyspermic fertilization. PMID:24390595

Abnormal chromosome behavior in human oocytes which remained unfertilized during human in vitro fertilization.

PubMed

Spielmann, H; Krüger, C; Stauber, M; Vogel, R

1985-09-01

Chromosomal abnormalities and abnormal embryonic development have previously been observed after human in vitro fertilization (IVF). Chromosomal abnormalities may arise not only after fertilization but even earlier during meiotic maturation of human oocytes in culture. Since chromosomal analysis is simple in oocytes during meiotic maturation, the chromosomal status was analyzed in oocytes which remained unfertilized in a human in vitro fertilization program. In 50 fertilization attempts the chromosomes of 62 unfertilized oocytes could be analyzed; 45 of them were in the process of meiotic maturation. In three oocytes two small polar bodies were observed 16-18 hr after insemination in the absence of fertilization. In one oocyte abnormal chromosome behavior was found during the first meiotic division, and in four oocytes during metaphase of the second meiotic division. These data suggest that chromosomal analysis of unfertilized oocytes in human IVF may improve the understanding human oocyte maturation and fertilization.

N-hexane alters the maturation of oocytes and induces apoptosis in mice.

PubMed

Liu, Jin; Huang, Lei; Sun, Yan; Li, Yu Chen; Zhu, Jian Lin; Wang, Wen Xiang; Zhang, Wen Chang

2013-09-01

This study was aimed to determine the effects of n-hexane on the maturation of mouse oocytes. Cell culture was used to observe the maturation of mouse oocytes and CLSM was employed to determine their apoptosis. Germinal vesicle breakdown (GVBD) and extrusion of the first polar body in mouse oocytes were significantly inhibited by n-hexane. After fertilization, the number of eggs in the mouse was significantly reduced by n-hexane. Mitochondrial membrane potentials (ΔΨm) were altered in mouse oocytes that were leading to apoptosis of the oocytes. N-hexane might have affected the maturation of oocytes, causing alteration of ΔΨm and leading to apoptosis which maybe one of the most important mechanisms. Copyright © 2013 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Oocyte cryopreservation beyond cancer: tools for ethical reflection.

PubMed

Linkeviciute, Alma; Peccatori, Fedro A; Sanchini, Virginia; Boniolo, Giovanni

2015-08-01

This article offers physicians a tool for structured ethical reflection on challenging situations surrounding oocyte cryopreservation in young healthy women. A systematic literature review offers a comprehensive overview of the ethical debate surrounding the practice. Ethical Counseling Methodology (ECM) offers a practical approach for addressing ethical uncertainties. ECM consists of seven steps: (i) case presentation; (ii) analysis of possible implications; (iii) presentation of ethical question(s); (iv) explanation of ethical terms; (v) presentation of the ethical arguments in favor of and against the procedure; (vi) examination of the individual patient's beliefs and wishes; and (vii) conclusive summary. The most problematic aspects in the ethical debate include the distinction between medical and non-medical use of oocyte cryopreservation, safety and efficiency of the procedure, and marketing practices aimed at healthy women. Female empowerment and enhanced reproductive choices (granted oocyte cryopreservation is a safe and efficient technique) are presented as ethical arguments supporting the practice, while ethical reservations towards oocyte cryopreservation are based on concerns about maternal and fetal safety and wider societal implications. Oocyte cryopreservation is gaining popularity among healthy reproductive age women. However, despite promised benefits it also involves risks that are not always properly communicated in commercialized settings. ECM offers clinicians a tool for structured ethical analysis taking into consideration a wide range of implications, various ethical standpoints, and patients' perceptions and beliefs.

A feasibility study of prepubertal and over mature aged local goat in relation to results of In Vitro growth culture to obtain additional M-II oocyte resources

NASA Astrophysics Data System (ADS)

Ciptadi, Gatot; Ihsan, M. Nur; Rahayu, Sri; Widjaja, D. H. K.; Mudawamah, Mudawamah

2017-11-01

The aims of this research are to study the potential source of mature (M-II) oocytes of domestic animals using follicles isolated from prepubertal and over mature aged Indonesian local goats, resulting from an in vitro growth (IVG) method. This method of IVG could provide a new source of M-II oocytes for embryo production. In Indonesia, a very limited number of a good quality oocytes are available for research purposes, as there is a limited number of reproductive females slaughtered, which is dominated by prepubertal and old mature aged animals. IVG culture systems could be improved as an alternative method to provide a new source of a good quality oocytes for in vitro maturation of M-II oocytes. From a number of prepubertal and mature aged goats slaughtered in a local abattoir, the small oocytes in the preantral follicles were cultured in vitro to normal oocyte growth. The methods used in this research are experimental. Follicles were isolated, cultured in vitro for 14 days individually using a sticky medium containing 4% (w/v) polyvinylpyrrolidone in TCM 199 10% Fetal Bovine Serum supplemented with Follicle Stimulating Hormone, which was then evaluated for their follicle development and oocyte quality. The research results showed that a minimum follicle size and oocyte diameter is needed (>100 um) for early evaluation of maturation to be achieved, meanwhile oocytes recovered from IVG after being cultured in vitro for maturation resulted in a very low rate of maturation. However, in the future, IVG of the preantral follicles of Indonesian local goat could be considered as an alternative source of oocytes for both research purposes and embryo production in vitro.

Naturally occurring mastitis disrupts developmental competence of bovine oocytes.

PubMed

Roth, Z; Dvir, A; Kalo, D; Lavon, Y; Krifucks, O; Wolfenson, D; Leitner, G

2013-10-01

We examined the effects of naturally occurring mastitis on bovine oocyte developmental competence in vitro. Specifically, we investigated the effects of intramammary infection on the ovarian pool of oocytes (i.e., follicle-enclosed oocytes) and their ability to undergo in vitro maturation, fertilization, and further development to the blastocyst stage. Culled Holstein cows (n=50) from 9 commercial dairy farms in Israel were allotted to 3 groups according to somatic cell count (SCC) records of the last 3 monthly milk tests as well as of quarter samples collected before slaughter: (1) low SCC (n=7), (2) medium SCC (n=16), or (3) high SCC (n=27). Means of SCC values differed among low-, medium-, and high-SCC groups: 148,000, 311,000 and 1,813,000 cell/mL milk, respectively. Milk yield and days in milk did not differ among the 3 groups. Bacterial isolates included coagulase-negative staphylococci, Escherichia coli, Streptococcus dysgalactiae, or no bacteria found. Ovaries were collected at the abattoir and brought to the laboratory. Cumulus oocyte complexes were recovered separately from each cow and subjected individually to in vitro maturation and fertilization, followed by 8d in culture. The number of aspirated oocytes did not differ among groups, with a range of 17 to 21 oocytes per cow. The proportion of oocytes that cleaved into 2- to 4-cell-stage embryos (86.1 ± 3.4%) did not differ among groups. In contrast, mean percentages of embryos developed to the blastocyst stage on d 7 and 8 after fertilization were less in both medium- and-high SCC groups than in the low-SCC group (5.6 ± 2.3 and 4.1 ± 1.8 vs. 18.1 ± 4.6%, respectively). Additional analysis indicated that cleavage and blastocyst-formation rates did not differ among the bacterial types in the low-, medium-, and high-SCC groups. These are the first results to demonstrate that naturally occurring mastitis disrupts the developmental competence of the ovarian pool of oocytes, (i.e., oocytes at the

Low-Dose Urinary Human Chorionic Gonadotropin Is Effective for Oocyte Maturation in In Vitro Fertilization/ Intracytoplasmic Sperm Injection Cycles Independent of Body Mass Index

PubMed Central

R. Hoyos, Luis; Khan, Sana; Dai, Jing; Singh, Manvinder; P. Diamond, Michael; E. Puscheck, Elizabeth; O. Awonuga, Awoniyi

2017-01-01

Background: Currently, there is no agreement on the optimal urinary derived human chorionic gonadotropin (u-hCG) dose requirement for initiating final oocyte maturation prior to oocyte collection in in vitro fertilization (IVF), but doses that range from 2500- 15000 IU have been used. We intended to determine whether low dose u-hCG was effective for oocyte maturation in IVF/intracytoplasmic sperm injection (ICSI) cycles independent of body mass index (BMI). Materials and Methods: We retrospectively evaluated a cohort of 295 women who underwent their first IVF/ICSI cycles between January 2003 and December 2010 at the Division of Reproductive Endocrinology and Infertility, Wayne State University, Detroit, MI, USA. Treatment cycles were divided into 3 groups based on BMI (kg/ m2): <25 (n=136), 25- <30 (n=84), and ≥30 (n=75) women. Patients received 5000, 10000 or 15000 IU u-hCG for final maturation prior to oocyte collection. The primary outcome was clinical pregnancy rates (CPRs) and secondary outcome was live birth rates (LBRs). Results: Only maternal age negatively impacted (P<0.001) CPR [odds ratio (OR=0.85, confidence interval (CI: 0.79-0.91)] and LBR (OR=0.84, CI: 0.78-0.90). Conclusion: Administration of lower dose u-hCG was effective for oocyte maturation in IVF and did not affect the CPRs and LBRs irrespective of BMI. Women’s BMI need not be taken into consideration in choosing the appropriate dose of u-hCG for final oocyte maturation prior to oocyte collection in IVF. Only maternal age at the time of IVF negatively influenced CPRs and LBRs in this study. PMID:28367299

Improved low-CPA vitrification of mouse oocytes using quartz microcapillary.

PubMed

Choi, Jung Kyu; Huang, Haishui; He, Xiaoming

2015-06-01

Cryopreservation by low-cryoprotectant (CPA) vitrification has the potential to combine all the advantages of the conventional high-CPA vitrification and slow-freezing approaches while avoiding their drawbacks. However, current low-CPA vitrification protocol for cryopreservation of oocytes requires a lengthy and multi-step procedure for unloading CPAs. In this study, we report a much-simplified procedure of using quartz microcapillary (QMC) for low-CPA vitrification of mouse oocytes with only one step for unloading CPAs. The immediate viability of oocytes after the improved low-CPA vitrification was determined to be more than 90%. Moreover, no significant difference was observed in terms of embryonic development from the two-cell to blastocyst stages between the fresh and vitrified oocytes after in vitro fertilization (IVF). This improved low-CPA vitrification technology has the potential for efficient cryopreservation of oocytes to preserve the fertility of mammals including humans for assisted reproductive medicine, maintenance of animal resource and endangered species, and livestock management. Copyright © 2015 Elsevier Inc. All rights reserved.

Betaine is accumulated via transient choline dehydrogenase activation during mouse oocyte meiotic maturation.

PubMed

McClatchie, Taylor; Meredith, Megan; Ouédraogo, Mariame O; Slow, Sandy; Lever, Michael; Mann, Mellissa R W; Zeisel, Steven H; Trasler, Jacquetta M; Baltz, Jay M

2017-08-18

Betaine ( N,N,N -trimethylglycine) plays key roles in mouse eggs and preimplantation embryos first in a novel mechanism of cell volume regulation and second as a major methyl donor in blastocysts, but its origin is unknown. Here, we determined that endogenous betaine was present at low levels in germinal vesicle (GV) stage mouse oocytes before ovulation and reached high levels in the mature, ovulated egg. However, no betaine transport into oocytes was detected during meiotic maturation. Because betaine can be synthesized in mammalian cells via choline dehydrogenase (CHDH; EC 1.1.99.1), we assessed whether this enzyme was expressed and active. Chdh transcripts and CHDH protein were expressed in oocytes. No CHDH enzyme activity was detected in GV oocyte lysate, but CHDH became highly active during oocyte meiotic maturation. It was again inactive after fertilization. We then determined whether oocytes synthesized betaine and whether CHDH was required. Isolated maturing oocytes autonomously synthesized betaine in vitro in the presence of choline, whereas this failed to occur in Chdh -/- oocytes, directly demonstrating a requirement for CHDH for betaine accumulation in oocytes. Overall, betaine accumulation is a previously unsuspected physiological process during mouse oocyte meiotic maturation whose underlying mechanism is the transient activation of CHDH. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

New technique for mouse oocyte injection via a modified holding pipette.

PubMed

Lyu, Q F; Deng, L; Xue, S G; Cao, S F; Liu, X Y; Jin, W; Wu, L Q; Kuang, Y P

2010-11-01

To improve mouse oocyte survival from intracytoplasmic sperm injection, the sharp tip of the injection pipette has been modified to have a flat end. Here, for the same goal but for a more convenient manipulation, a sharp injection pipette was kept whereas the holding pipette was modified to have a trumpet-shaped opening, which allows deeper injection into the oocyte as it is held. Mouse oocyte injection with mouse and human spermatozoa was performed at 37°C. For the injection of mouse oocyte with mouse sperm head, a significantly higher survival rate (83%) was achieved by utilizing the modified holding pipette than the conventional one (21%; P<0.001) and the fertilization rates were normal and comparable for both methods (82% versus 81%). A superior survival rate (82%) and acceptable normal fertilization rate (71%) were also achieved by utilizing the modified holding pipette for interspecies ICSI (injecting mouse oocyte with human spermatozoon). Taken together, by utilizing a holding pipette with a trumpet-shaped opening, acceptable rates of mouse oocyte survival and fertilization can be achieved using a sharp injection pipette under conditions usual for human oocyte injection. Copyright © 2010 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Reproductive experiences of women who cryopreserved oocytes for non-medical reasons.

PubMed

Hammarberg, Karin; Kirkman, Maggie; Pritchard, Natasha; Hickey, Martha; Peate, Michelle; McBain, John; Agresta, Franca; Bayly, Chris; Fisher, Jane

2017-03-01

What are the reproductive experiences of women who cryopreserve oocytes for non-medical reasons? One in three women had been pregnant at some stage in their lives and while most still wanted to have a child or another child, very few had used their stored oocytes, predominantly because they did not want to be single parents. The number of healthy women who freeze oocytes to avoid age-related infertility is increasing. Evidence about reproductive outcomes after oocyte cryopreservation for non-medical reasons is needed to help women make informed decisions. A cross-sectional survey was carried out. Study packs which included a self-administered questionnaire were mailed by clinic staff to 193 eligible women. Women who had stored oocytes for non-medical reasons at Melbourne IVF, a private ART clinic, between 1999 and 2014 were identified from medical records and invited to complete an anonymous questionnaire about their reproductive histories and experience of oocyte cryopreservation. A total of 10 survey packs were returned to the clinic marked 'address unknown'. Of the 183 potential respondents, 96 (53%) returned the questionnaire. One respondent provided only free-text comments, thus data from 95 respondents were compiled. The mean age at the time of freezing oocytes was 37.1 years (SD ± 2.6, range: 27-42) and the average number of oocytes stored was 14.2 (SD ± 7.9, range: 0-42); 2% had attempted to store oocytes but had none suitable for freezing, 24% had stored <8 oocytes, 35% had 8-15, 25% had 16-23 and 14% had stored >23 oocytes. About one-third of respondents (34%) had been pregnant at some point in their lives. Six women (6%) had used their stored oocytes and three of them had given birth as a result. The main reason for not using stored oocytes was not wanting to be a single parent. Of the 87 (91%) women who still had oocytes stored, 21% intended to use them while 69% indicated that their circumstances would determine usage. The mean number of children

Effects of dimethyl sulfoxide on asymmetric division and cytokinesis in mouse oocytes

PubMed Central

2014-01-01

Background Dimethyl sulfoxide (DMSO) is used extensively as a permeable cryoprotectant and is a common solvent utilized for several water-insoluble substances. DMSO has various biological and pharmacological activities; however, the effect of DMSO on mouse oocyte meiotic maturation remains unknown. Results In DMSO-treated oocytes, we observed abnormal MII oocytes that contained large polar bodies, including 2-cell–like MII oocytes, during in vitro maturation. Oocyte polarization did not occur, due to the absence of actin cap formation and spindle migration. These features are among the primary causes of abnormal symmetric division; however, analysis of the mRNA expression levels of genes related to asymmetric division revealed no significant difference in the expression of these factors between the 3% DMSO-treated group and the control group. After each “blastomere” of the 2-cell–like MII stage oocytes was injected by one sperm head respectively, the oocytes still possessed the ability to extrude the second polar body from each “blastomere” and to begin cleavage. However, MII oocytes with large polar bodies developed to the blastocyst stage after intracytoplasmic sperm injection (ICSI). Furthermore, other permeable cryoprotectants, such as ethylene glycol and glycerol, also caused asymmetric division failure. Conclusion Permeable cryoprotectants, such as DMSO, ethylene glycol, and glycerol, affect asymmetric division. DMSO disrupts cytokinesis completion by inhibiting cortical reorganization and polarization. Oocytes that undergo symmetric division maintain the ability to begin cleavage after ICSI. PMID:24953160

Effects of endocrine-disrupting contaminants on amphibian oogenesis: methoxychlor inhibits progesterone-induced maturation of Xenopus laevis oocytes in vitro.

PubMed Central

Pickford, D B; Morris, I D

1999-01-01

There is currently little evidence of pollution-induced endocrine dysfunction in amphibia, in spite of widespread concern over global declines in this ecologically diverse group. Data regarding the potential effects of endocrine-disrupting contaminants (EDCs) on reproductive function in amphibia are particularly lacking. We hypothesized that estrogenic EDCs may disrupt progesterone-induced oocyte maturation in the adult amphibian ovary, and tested this with an in vitro germinal vesicle breakdown assay using defolliculated oocytes from the African clawed frog, Xenopus laevis. While a variety of natural and synthetic estrogens and xenoestrogens were inactive in this system, the proestrogenic pesticide methoxychlor was a surprisingly potent inhibitor of progesterone-induced oocyte maturation (median inhibitive concentration, 72 nM). This inhibitory activity was specific to methoxychlor, rather than to its estrogenic contaminants or metabolites, and was not antagonized by the estrogen receptor antagonist ICI 182,780, suggesting that this activity is not estrogenic per se. The inhibitory activity of methoxychlor was dose dependent, reversible, and early acting. However, washout was unable to reverse the effect of short methoxychlor exposure, and methoxychlor did not competitively displace [3H]progesterone from a specific binding site in the oocyte plasma membrane. Therefore, methoxychlor may exert its action not directly at the site of progesterone action, but downstream on early events in maturational signaling, although the precise mechanism of action is unclear. The activity of methoxychlor in this system indicates that xenobiotics may exert endocrine-disrupting effects through interference with progestin-regulated processes and through mechanisms other than receptor antagonism. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:10090707

Truths and myths of oocyte sensitivity to controlled rate freezing.

PubMed

Coticchio, G; Bonu, M A; Sciajno, R; Sereni, E; Bianchi, V; Borini, A

2007-07-01

The mammalian oocyte is especially sensitive to cryopreservation. Because of its size and physiology, it can easily undergo cell death or sub-lethal damage as a consequence of intracellular ice formation, increase in the concentration of solutes and other undesired effects during the conversion of extracellular water into ice. This has generated the belief that oocyte storage cannot be achieved with the necessary efficiency and safety. However, many concerns raised by oocyte freezing are the result of unproven hypotheses or observations conducted under sometimes inappropriate conditions. For instance, spindle organization can undergo damage under certain freezing conditions but not with other protocols. The controversial suggestion that cryopreservation induces cortical granule discharge and zona pellucida hardening somehow questions the routine use of sperm microinjection. Damage to mouse oocytes caused by solute concentration is well documented but, in the human, there is no solid evidence that modifications of freezing mixtures, to prevent this problem, provide an actual advantage. The hope of developing oocyte cryopreservation as a major IVF option is becoming increasingly realistic, but major efforts are still required to clarify the authentic implications of oocyte cryopreservation at the cellular level and identify freezing conditions compatible with the preservation of viability and developmental ability.

REACTIVE OXYGEN SPECIES AND OOCYTE AGING: ROLE OF SUPEROXIDE, HYDROGEN PEROXIDE AND HYPOCHLOROUS ACID

PubMed Central

GOUD, ANURADHA P.; GOUD, PRAVIN T.; DIAMOND, MICHAEL P.; GONIK, BERNARD; ABU-SOUD, HUSAM M.

2009-01-01

Aging of the unfertilized oocyte inevitably occurs following ovulation, limiting its fertilizable life-span. However, the mechanisms that regulate oocyte aging are still unclear. We hypothesize that reactive oxygen species such as superoxide (O2•−), hydrogen peroxide (H2O2), and hypochlorous acid (HOCl) are likely candidates that may initiate these changes in the oocyte. In order to test this hypothesis, we investigated direct effects of O2•− [hypoxanthine/xanthine oxidase system generating 0.12 (n=42) and 0.25 μM O2•−/min (n=45)], H2O2 (20 or 100 μM, n=60) and HOCl, (1, 10 and 100 μM, n=50) on freshly ovulated or relatively old mouse oocytes, while their sibling oocytes were fixed immediately or cultured under physiological conditions (n=96). The aging process was assessed by the zona pellucida dissolution time (ZPDT), ooplasm microtubule dynamics (OMD), and cortical granule (CG) status. The ZPDT increased 2-fold in relatively old, compared to young, untreated oocytes (P<0.0001). Exposure to O2•− increased it even further (P<0.0001). Similarly, more O2•− exposed oocytes exhibited increased OMD and major CG loss, with fewer having normal OMD and intact CG compared to untreated controls. Interestingly, young oocytes resisted “aging”, when exposed to 20 μM H2O2, while the same enhanced the aging phenomena in relatively old oocytes (P<0.05). Exposure to even very low levels of HOCl induced aging phenomena in young and relatively old oocytes, and higher concentrations of HOCl compromised oocyte viability. Overall, O2•−, H2O2 and HOCl each augment oocyte “aging”, more so in relatively old oocytes, suggesting compromised antioxidant capacity in aging oocytes. PMID:18177745

Src-family Tyrosine Kinases in Oogenesis, Oocyte Maturation, and Fertilization: An Evolutionary Perspective

PubMed Central

Kinsey, William H.

2015-01-01

The oocyte is a highly specialized cell poised to respond to fertilization with a unique set of actions needed to recognize and incorporate a single sperm, complete meiosis, reprogram maternal and paternal genomes and assemble them into a unique zygotic genome, and finally initiate the mitotic cell cycle. Oocytes accomplish this diverse series of events through an array of signal transduction pathway components that include a characteristic collection of protein tyrosine kinases. The src-family protein kinases figure importantly in this signaling array and oocytes characteristically express certain SFKs at high levels to provide for the unique actions that the oocyte must perform. The SFKs typically exhibit a distinct pattern of subcellular localization in oocytes and perform critical functions in different subcellular compartments at different steps during oocyte maturation and fertilization. While many aspects of SFK signaling are conserved among oocytes from different species, significant differences exist in the extent to which src-family -mediated pathways are used by oocytes from species that fertilize externally vs those which are fertilized internally. The observation that several oocyte functions which require SFK signaling appear to represent common points of failure during assisted reproductive techniques in humans, highlights the importance of these signaling pathways for human reproductive health. PMID:25030759

The developmental potential of parthenogenetic and somatic cell nuclear-transferred rat oocytes in vitro.

PubMed

Mizumoto, Shigetoshi; Kato, Yoko; Tsunoda, Yukio

2008-12-01

We examined the optimal conditions for somatic cell nuclear transfer (SCNT) in rat oocytes. First, we compared the effects of two types of inhibitors of spontaneous activation, MG132 and demecolcine, on the developmental potential of parthenogenetic oocytes. The potential of activated oocytes to develop into blastocysts significantly decreased 2 h after oocyte recovery (77% vs. 7%). The developmental potential of oocytes preserved in MG132-supplemented medium for 1 to 4 h was high (62% to 77%), but the potential of those preserved in demecolcine-supplemented medium for 3 and 4 h was low (77% vs. 41% and 37%, respectively). Second, the effect of the duration of parthenogenetic activation on the developmental potential was examined. When oocytes preserved in MG132 for 4 h were treated with 10 mM strontium for 5 or 6 h, the potential of activated oocytes to develop into blastocysts was high (78% and 70%, respectively). Using the optimal conditions for parthenogenetic activation, we examined the potential of rat enucleated oocytes receiving cumulus cells to develop into blastocysts. In contrast to parthenogenotes, the potential of SCNT rat oocytes to develop into blastocysts was low (2%) even if then oocytes were treated with the histone deacetylation inhibitor trichostatin A. The reason for the low developmental potential of rat SCNT oocytes is discussed.

Cytoplasmic regionalization in starfish oocyte occurrence and localization of cytoplasmic determinants responsible for the formation of archenteron and primary mesenchyme in starfish ( asterias amurensis) oocytes

NASA Astrophysics Data System (ADS)

Zhang, Shicui; Wu, Xianhan; Zhou, Jing; Wang, Renxue; Wu, Shangqin

1990-09-01

Starfish oocytes with intact germinal vesicles (GVs) were cut along desired planes with glass needles or ligated using silk thread loops into two parts and allowed to mature in vitro, and inseminated. The experimental results showed that (1) only the parts with GVs or partial GV contents (PGVCs) cleaved, those without any GV materials did not; but nucleated and non-nucleated fragments cut from mature eggs were able to divide; (2) the development of animal parts of oocytes containing GVs or PGVCs was like that of animal fragments of matured oocytes with female pronuclei; most of them gave rise to permanent blastulae, and just a few formed ectodermal vesicles with a little primary mesenchyme; (3) a large part of vegetal fragments with GVs or PGVCs, and the vegetal parts of mature eggs without female pronuclei developed into small but normal embryos; (4) the fragments containing GVs or PGVCs obtained from the oocytes along a plane parallel to the animal-vegetal (A-V) axis developed as normally as the halves (with or without female pronuclei) severed from mature eggs along the same axis. Based on the data above, it was concluded that (1) the non-chromatin materials in the oocyte GVs are indispensable for successful fertilization and cleavage of starfish eggs; (2) some factor (s) located asymmetrically in the vegetal hemispheres of starfish oocytes is (are) responsible for formation of the archenteron and primary mesenchyme. It is evident from the above findings that the oocyte cytoplasm of the starfish had already regionalized before the GV break-down.

Current results with slow freezing and vitrification of the human oocyte.

PubMed

Boldt, Jeffrey

2011-09-01

The past decade has witnessed renewed interest in human oocyte cryopreservation (OCP). This article reviews the two general methods used for OCP, slow freezing and vitrification, compares the outcomes associated with each technique and discusses the factors that might influence success with OCP (such as oocyte selection or day of transfer). Based on available data, OCP offers a reliable, reproducible method for preservation of the female gamete and will find increasing application in assisted reproductive technology. Oocyte cryopreservation can provide a number of advantages to couples undergoing assisted reproduction or to women interested in fertility preservation. Two methods, slow freezing and vitrification, have been used successfully for oocyte cryopreservation. This article reviews and compares these methods, and discusses various factors that can impact upon success of oocyte cryopreservation. Copyright © 2011 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Kif4 Is Essential for Mouse Oocyte Meiosis.

PubMed

Camlin, Nicole J; McLaughlin, Eileen A; Holt, Janet E

2017-01-01

Progression through the meiotic cell cycle must be strictly regulated in oocytes to generate viable embryos and offspring. During mitosis, the kinesin motor protein Kif4 is indispensable for chromosome condensation and separation, midzone formation and cytokinesis. Additionally, the bioactivity of Kif4 is dependent on phosphorylation via Aurora Kinase B and Cdk1, which regulate Kif4 function throughout mitosis. Here, we examine the role of Kif4 in mammalian oocyte meiosis. Kif4 localized in the cytoplasm throughout meiosis I and II, but was also observed to have a dynamic subcellular distribution, associating with both microtubules and kinetochores at different stages of development. Co-localization and proximity ligation assays revealed that the kinetochore proteins, CENP-C and Ndc80, are potential Kif4 interacting proteins. Functional analysis of Kif4 in oocytes via antisense knock-down demonstrated that this protein was not essential for meiosis I completion. However, Kif4 depleted oocytes displayed enlarged polar bodies and abnormal metaphase II spindles, indicating an essential role for this protein for correct asymmetric cell division in meiosis I. Further investigation of the phosphoregulation of meiotic Kif4 revealed that Aurora Kinase and Cdk activity is critical for Kif4 kinetochore localization and interaction with Ndc80 and CENP-C. Finally, Kif4 protein but not gene expression was found to be upregulated with age, suggesting a role for this protein in the decline of oocyte quality with age.

Spontaneous and LH-induced maturation in Bufo arenarum oocytes: importance of gap junctions.

PubMed

Toranzo, G Sánchez; Oterino, J; Zelarayán, L; Bonilla, F; Bühler, M I

2007-02-01

It has been demonstrated in Bufo arenarum that fully grown oocytes are capable of meiotic resumption in the absence of a hormonal stimulus if they are deprived of their follicular envelopes. This event, called spontaneous maturation, only takes place in oocytes collected during the reproductive period, which have a metabolically mature cytoplasm. In Bufo arenarum, progesterone acts on the oocyte surface and causes modifications in the activities of important enzymes, such as a decrease in the activity of adenylate cyclase (AC) and the activation of phospholipase C (PLC). PLC activation leads to the formation of diacylglycerol (DAG) and inositol triphosphate (IP(3)), second messengers that activate protein kinase C (PKC) and cause an increase in intracellular Ca(2+). Recent data obtained from Bufo arenarum show that progesterone-induced maturation causes significant modifications in the level and composition of neutral lipids and phospholipids of whole fully grown ovarian oocytes and of enriched fractions in the plasma membrane. In amphibians, the luteinizing hormone (LH) is responsible for meiosis resumption through the induction of progesterone production by follicular cells. The aim of this work was to study the importance of gap junctions in the spontaneous and LH-induced maturation in Bufo arenarum oocytes. During the reproductive period, Bufo arenarum oocytes are capable of undergoing spontaneous maturation in a similar way to mammalian oocytes while, during the non-reproductive period, they exhibit the behaviour that is characteristic of amphibian oocytes, requiring progesterone stimulation for meiotic resumption (incapable oocytes). This different ability to mature spontaneously is coincident with differences in the amount and composition of the phospholipids in the oocyte membranes. Capable oocytes exhibit in their membranes higher quantities of phospholipids than incapable oocytes, especially of PC and PI, which are precursors of second messengers such as

Predictive value of bovine follicular components as markers of oocyte developmental potential.

PubMed

Matoba, Satoko; Bender, Katrin; Fahey, Alan G; Mamo, Solomon; Brennan, Lorraine; Lonergan, Patrick; Fair, Trudee

2014-01-01

The follicle is a unique micro-environment within which the oocyte can develop and mature to a fertilisable gamete. The aim of this study was to investigate the ability of a panel of follicular parameters, including intrafollicular steroid and metabolomic profiles and theca, granulosa and cumulus cell candidate gene mRNA abundance, to predict the potential of bovine oocytes to develop to the blastocyst stage in vitro. Individual follicles were dissected from abattoir ovaries, carefully ruptured under a stereomicroscope and the oocyte was recovered and individually processed through in vitro maturation, fertilisation and culture. The mean (±s.e.m.) follicular concentrations of testosterone (62.8±4.8 ngmL(-1)), progesterone (616.8±31.9 ngmL(-1)) and oestradiol (14.4±2.4 ngmL(-1)) were not different (P>0.05) between oocytes that formed (competent) or failed to form (incompetent) blastocysts. Principal-component analysis of the quantified aqueous metabolites in follicular fluid showed differences between oocytes that formed blastocysts and oocytes that degenerated; l-alanine, glycine and l-glutamate were positively correlated and urea was negatively correlated with blastocyst formation. Follicular fluid associated with competent oocytes was significantly lower in palmitic acid (P=0.023) and total fatty acids (P=0.031) and significantly higher in linolenic acid (P=0.036) than follicular fluid from incompetent oocytes. Significantly higher (P<0.05) transcript abundance of LHCGR in granulosa cells, ESR1 and VCAN in thecal cells and TNFAIP6 in cumulus cells was associated with competent compared with incompetent oocytes.

Oocyte quality in mice is affected by a mycotoxin-contaminated diet.

PubMed

Hou, Yan-Jun; Xiong, Bo; Zheng, Wei-Jiang; Duan, Xing; Cui, Xiang-Shun; Kim, Nam-Hyung; Wang, Qiang; Xu, Yin-Xue; Sun, Shao-Chen

2014-05-01

Mycotoxins, such as deoxynivalenol (DON), zearalenone (ZEN), and aflatoxin (AF), are commonly found in many food commodities and may impair the growth and reproductive efficiency of animals and humans. We investigated the effects of a mycotoxin-contaminated diet on mouse oocyte quality. Maize contaminated with DON (3.875 mg/kg), ZEN (1,897 μg/kg), and AF (806 μg/kg) was incorporated into a mouse diet at three different levels (0, 15, and 30% w/w). After 4 weeks, ovarian and germinal vesicle oocyte indices decreased in mycotoxin-fed mice. Oocytes from these mice exhibited low developmental competence with reduced germinal vesicle breakdown and polar body extrusion rates. Embryo developmental competence also showed a similar pattern, and the majority of embryos could not develop to the morula stage. Actin expression was also reduced in both the oocyte cortex and cytoplasm, which was accompanied by decreased expression of the actin nucleation factors profilin-1 and mDia1. Moreover, a large percentage of oocytes derived from mice that were fed a mycotoxin-contaminated diet exhibited aberrant spindle morphology, a loss of the cortical granule-free domain, and abnormal mitochondrial distributions, which further supported the decreased oocyte quality. Thus, our results demonstrate that mycotoxins are toxic to the mouse reproductive system by affecting oocyte quality. Copyright © 2013 Wiley Periodicals, Inc.

Investigations of oocyte in vitro maturation within a mouse model.

PubMed

Chin, Alexis Heng Boon; Chye, Ng Soon

2004-02-01

This study attempted to develop a 'less meiotically competent' murine model for oocyte in vitro maturation (IVM), which could more readily be extrapolated to human clinical assisted reproduction. Oocyte meiotic competence was drastically reduced upon shortening the standard duration of in vivo gonadotrophin stimulation from 48 h to 24 h, and by selecting only naked or partially naked germinal vesicle oocytes, instead of fully cumulus enclosed oocyte complexes. With such a less meiotically competent model, only porcine granulosa coculture significantly enhanced the oocyte maturation rate in vitro, whereas no significant enhancement was observed with macaque and murine granulosa coculture. Increased serum concentrations and the supplementation of gonadotrophins, follicular fluid and extracellular matrix gel within the culture medium did not enhance IVM under either cell-free or coculture conditions. Culture medium conditioned by porcine granulosa also enhanced the maturation rate, and this beneficial effect was not diminished upon freeze-thawing. Enhanced IVM in the presence of porcine granulosa coculture did not, however, translate into improved developmental competence, as assessed by in vitro fertilization and embryo culture to the blastocyst stage.

PP2A regulates kinetochore-microtubule attachment during meiosis I in oocyte.

PubMed

Tang, An; Shi, Peiliang; Song, Anying; Zou, Dayuan; Zhou, Yue; Gu, Pengyu; Huang, Zan; Wang, Qinghua; Lin, Zhaoyu; Gao, Xiang

2016-06-02

Studies using in vitro cultured oocytes have indicated that the protein phosphatase 2A (PP2A), a major serine/threonine protein phosphatase, participates in multiple steps of meiosis. Details of oocyte maturation regulation by PP2A remain unclear and an in vivo model can provide more convincing information. Here, we inactivated PP2A by mutating genes encoding for its catalytic subunits (PP2Acs) in mouse oocytes. We found that eliminating both PP2Acs caused female infertility. Oocytes lacking PP2Acs failed to complete 1(st) meiotic division due to chromosome misalignment and abnormal spindle assembly. In mitosis, PP2A counteracts Aurora kinase B/C (AurkB/C) to facilitate correct kinetochore-microtubule (KT-MT) attachment. In meiosis I in oocyte, we found that PP2Ac deficiency destabilized KT-MT attachments. Chemical inhibition of AurkB/C in PP2Ac-null oocytes partly restored the formation of lateral/merotelic KT-MT attachments but not correct KT-MT attachments. Taken together, our findings demonstrate that PP2Acs are essential for chromosome alignments and regulate the formation of correct KT-MT attachments in meiosis I in oocytes.

Assessment of aneuploidy in human oocytes and preimplantation embryos by chromosome painting

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rougier, N.; Viegas-Pequignot, E.; Plachot, M.

1994-09-01

The poor quality of chromosome preparations often observed after fixation of oocytes and embryos did not usually allow accurate identification of chromosomes involved in non-disjunctions. We, therefore, used chromosome painting to determine the incidence of abnormalities for chromosomes 1 and 7. A total of 50 oocytes inseminated for IVF and showing no signs of fertilization as well as 37 diploid embryos donated for research were fixed according to the Dyban`s technique. Fluorescence in situ hybridization was carried out using whole chromosome painting DNA probes specific for human chromosome 1 and 7. The incidence of aneuploidy was 28%, 10% and 60%more » for metaphase II, polar body and sperm chromosomes, respectively. The high incidence of aneuploidy observed in sperm prematurely condensed sperm chromosomes is due to the fact that usually far less than 23 sperm chromatids are observed, maybe as a consequence of incomplete chromosome condensation. Thirty seven embryos were analyzed with the same probes. 48% of early embryos were either monosomic 1 or 7 or mosaics comprising blastomeres with 1, 2 or 3 signals. Thus, 8 among the 11 abnormal embryos had hypodiploid cells (25 to 37 chromosomes) indicating either an artefactual loss of chromosomes or a complex anomaly of nuclear division (maltinucleated blastomeres, abnormal migration of chromosomes at anaphase). We therefore calculated a {open_quotes}corrected{close_quotes} incidence of aneuploidy for chromosomes 1 or 7 in early embryos: 18%. 86% of the blastocysts showed mosaicism 2n/3 or 4n as a consequence of the formation of the syncitiotrophoblast. To conclude, chromosome painting is an efficient method to accurately identify chromosomes involved in aneuploidy. This technique should allow us to evaluate the incidence of non-disjunction for all chromosome pairs. Our results confirm the high incidence of chromosome abnormalities occurring as a consequence of meiotic or mitotic non-disjunctions in human oocytes and

In vitro growth and maturation of isolated caprine preantral follicles: Influence of insulin and FSH concentration, culture dish, coculture, and oocyte size on meiotic resumption.

PubMed

Silva, G M; Brito, I R; Sales, A D; Aguiar, F L N; Duarte, A B G; Araújo, V R; Vieira, L A; Magalhães-Padilha, D M; Lima, L F; Alves, B G; Silveira, L B R; Lo Turco, E G; Rodrigues, A P; Campello, C C; Wheeler, M B; Figueiredo, J R

2017-03-01

The aims of this study were: (1) to evaluate the effect of different insulin concentrations, alone or in combination with either a fixed FSH concentration or increasing FSH concentrations on the in vitro culture of isolated caprine preantral follicles and (2) to analyze the efficiency of two IVM media and maturation culture systems (with or without coculture with in vivo grown oocytes) on the meiosis resumption. Secondary follicles were cultured for 18 days in a basic medium supplemented with low- or high-insulin concentration alone or with a fixed FSH concentration or with increasing FSH concentrations. Oocytes grown in vivo or in vitro were matured alone or cocultured. The high-insulin concentration associated with fixed FSH treatment had higher meiotic resumption rate (P < 0.05) and was the only treatment capable of producing oocytes in metaphase II. The rates of germinal vesicle, germinal vesicle breakdown, metaphase I, metaphase II (MII), meiotic resumption, and oocyte diameter were similar between the maturation media. In conclusion, a basic medium supplemented with 10-μg/mL insulin and 100-μg/mL FSH throughout the culture period improved meiotic resumption rate and produced MII oocytes from caprine preantral follicles cultured in vitro. The MII rate was similar between in vivo and in vitro grown oocytes ≥110 μm. Copyright © 2016 Elsevier Inc. All rights reserved.

Prophase I Mouse Oocytes Are Deficient in the Ability to Respond to Fertilization by Decreasing Membrane Receptivity to Sperm and Establishing a Membrane Block to Polyspermy1

PubMed Central

Kryzak, Cassie A.; Moraine, Maia M.; Kyle, Diane D.; Lee, Hyo J.; Cubeñas-Potts, Caelin; Robinson, Douglas N.; Evans, Janice P.

2013-01-01

ABSTRACT Changes occurring as the prophase I oocyte matures to metaphase II are critical for the acquisition of competence for normal egg activation and early embryogenesis. A prophase I oocyte cannot respond to a fertilizing sperm as a metaphase II egg does, including the ability to prevent polyspermic fertilization. Studies here demonstrate that the competence for the membrane block to polyspermy is deficient in prophase I mouse oocytes. In vitro fertilization experiments using identical insemination conditions result in monospermy in 87% of zona pellucida (ZP)-free metaphase II eggs, while 92% of ZP-free prophase I oocytes have four or more fused sperm. The membrane block is associated with a postfertilization reduction in the capacity to support sperm binding, but this reduction in sperm-binding capacity is both less robust and slower to develop in fertilized prophase I oocytes. Fertilization of oocytes is dependent on the tetraspanin CD9, but little to no release of CD9 from the oocyte membrane is detected, suggesting that release of CD9-containing vesicles is not essential for fertilization. The deficiency in membrane block establishment in prophase I oocytes correlates with abnormalities in two postfertilization cytoskeletal changes: sperm-induced cortical remodeling that results in fertilization cone formation and a postfertilization increase in effective cortical tension. These data indicate that cortical maturation is a component of cytoplasmic maturation during the oocyte-to-egg transition and that the egg cortex has to be appropriately primed and tuned to be responsive to a fertilizing sperm. PMID:23863404

A Flexure-Guided Piezo Drill for Penetrating the Zona Pellucida of Mammalian Oocytes.

PubMed

Johnson, Wesley; Dai, Changsheng; Liu, Jun; Wang, Xian; Luu, Devin K; Zhang, Zhuoran; Ru, Changhai; Zhou, Chao; Tan, Min; Pu, Huayan; Xie, Shaorong; Peng, Yan; Luo, Jun; Sun, Yu

2018-03-01

Mammalian oocytes such as mouse oocytes have a highly elastic outer membrane, zona pellucida (ZP) that cannot be penetrated without significantly deforming the oocyte, even with a sharp micropipette. Piezo drill devices leverage lateral and axial vibration of the micropipette to accomplish ZP penetration with greatly reduced oocyte deformation. However, existing piezo drills all rely on a large lateral micropipette vibration amplitude ( 20 ) and a small axial vibration amplitude (0.1 ). The very large lateral vibration amplitude has been deemed to be necessary for ZP penetration although it also induces larger oocyte deformation and more oocyte damage. This paper reports on a new piezo drill device that uses a flexure guidance mechanism and a systematically designed pulse train with an appropriate base frequency. Both simulation and experimental results demonstrate that a small lateral vibration amplitude (e.g., 2 ) and an axial vibration amplitude as large as 1.2 were achieved. Besides achieving 100% effectiveness in the penetration of mouse oocytes (n = 45), the new piezo device during ZP penetration induced a small oocyte deformation of 3.4 versus larger than 10 using existing piezo drill devices.

Biotin-deficient diet induces chromosome misalignment and spindle defects in mouse oocytes.

PubMed

Tsuji, Ai; Nakamura, Toshinobu; Shibata, Katsumi

2015-01-01

Increased abnormal oocytes due to meiotic chromosome misalignment and spindle defects lead to elevated rates of infertility, miscarriage, and trisomic conceptions. Here, we investigated the effect of biotin deficiency on oocyte quality. Three-week-old female ICR mice were fed a biotin-deficient or control diet (0, 0.004 g biotin/kg diet) for 21 days. On day 22, these mouse oocytes were analyzed by immunofluorescence. Due to biotin, undernutrition increased the frequency of abnormal oocytes (the biotin deficient vs. control: 40 vs. 16%). Next, the remaining mice in the biotin-deficient group were fed a control or biotin-deficient diet from day 22 to 42. Although biotin nutritional status in the recovery group was restored, the frequency of abnormal oocytes in the recovery group was still higher than that in the control group (48 vs. 18%). Our results indicate that steady, sufficient biotin intake is required for the production of high-quality oocytes in mice.

[Effect of kuntai capsule on the number of retrieved oocytes, high-quality oocytes and embryos in in vitro fertilization of poor ovarian response patients].

PubMed

Lian, Fang; Jiang, Xiao-Yuan

2014-08-01

To observe the effect of Kuntai Capsule (KC) on the number of retrieved oocytes, the quality of high-quality oocytes and embryos in in vitro fertilization of poor ovarian response (POR) patients. Totally 70 POR patients preparing for in vitro fertilization-embryo transfer (IVF-ET) were randomly assigned to the observation group and the control group, 35 cases in each group. KC was administered to patients in the observation group in the preparation cycle (i.e., three menstrual cycles before IVF-ET) and during the superovulation process. Those in the control group took placebo during this period. Before and after medication the improvement of Shen yin deficiency syndrome (SYDS) was observed in the two groups. The basal follicle-stimulating hormone (bFSH), luteinizing hormone (LH), estradiol (E2), anti-Mullerian hormone (AMH), the ratio of FSH to LH, and antral follicle count (AFC) were observed. Besides, the E2 level of a single ovum on the day of HCG injection, the number of retrieved oocytes, the high-quality oocyte rate, and the high-quality embryos were observed. Compared with the control group, the SYDS, decreased bFSH and LH levels, increased ACF numbers, the E2 level of a single ovum on the day of HCG injection, the number of retrieved oocytes, high-quality oocytes, and high-quality embryos were superior in the observation group (P < 0.05). There was no statistical difference in the decreased FSH/LH level (P > 0.05). E2 and AMH increased after medication of KC in the observation group, while they decreased after administration of placebos in the control group. There was statistical difference in the post-pre treatment difference of E2 and AMH between the two groups (P < 0.05). KC could increase the number of retrieved oocytes, and elevate the quality of occytes and embryos in the IVF-ET.

Oocyte developmental failure in response to elevated nonesterified fatty acid concentrations: mechanistic insights.

PubMed

Van Hoeck, V; Leroy, J L M R; Arias Alvarez, M; Rizos, D; Gutierrez-Adan, A; Schnorbusch, K; Bols, P E J; Leese, H J; Sturmey, R G

2013-01-01

Elevated plasma nonesterified fatty acid (NEFA) concentrations are associated with negative energy balance and metabolic disorders such as obesity and type II diabetes. Such increased plasma NEFA concentrations induce changes in the microenvironment of the ovarian follicle, which can compromise oocyte competence. Exposing oocytes to elevated NEFA concentrations during maturation affects the gene expression and phenotype of the subsequent embryo, notably prompting a disrupted oxidative metabolism. We hypothesized that these changes in the embryo are a consequence of modified energy metabolism in the oocyte. To investigate this, bovine cumulus oocyte complexes were matured under elevated NEFA conditions, and energy metabolism-related gene expression, mitochondrial function, and ultrastructure evaluated. It was found that expression of genes related to REDOX maintenance was modified in NEFA-exposed oocytes, cumulus cells, and resultant blastocysts. Moreover, the expression of genes related to fatty acid synthesis in embryos that developed from NEFA-exposed oocytes was upregulated. From a functional perspective, inhibition of fatty acid β-oxidation in maturing oocytes exposed to elevated NEFA concentrations restored developmental competence. There were no clear differences in mitochondrial morphology or oxygen consumption between treatments, although there was a trend for a higher mitochondrial membrane potential in zygotes derived from NEFA-exposed oocytes. These data show that the degree of mitochondrial fatty acid β-oxidation has a decisive impact on the development of NEFA-exposed oocytes. Furthermore, the gene expression data suggest that the resulting embryos adapt through altered metabolic strategies, which might explain the aberrant energy metabolism previously observed in these embryos originating from NEFA-exposed maturing oocytes.

Fatty Acid Synthesis and Oxidation in Cumulus Cells Support Oocyte Maturation in Bovine

PubMed Central

Sanchez-Lazo, Laura; Brisard, Daphné; Elis, Sébastien; Maillard, Virginie; Uzbekov, Rustem; Labas, Valérie; Desmarchais, Alice; Papillier, Pascal; Monget, Philippe

2014-01-01

Oocyte meiotic maturation requires energy from various substrates including glucose, amino acids, and lipids. Mitochondrial fatty acid (FA) β-oxidation (FAO) in the oocyte is required for meiotic maturation, which is accompanied by differential expression of numerous genes involved in FAs metabolism in surrounding cumulus cells (CCs) in vivo. The objective was to elucidate components involved in FAs metabolism in CCs during oocyte maturation. Twenty-seven genes related to lipogenesis, lipolysis, FA transport, and FAO were chosen from comparative transcriptome analysis of bovine CCs before and after maturation in vivo. Using real-time PCR, 22 were significantly upregulated at different times of in vitro maturation (IVM) in relation to oocyte meiosis progression from germinal vesicle breakdown to metaphase-II. Proteins FA synthase, acetyl-coenzyme-A carboxylase, carnitine palmitoyltransferase, perilipin 2, and FA binding protein 3 were detected by Western blot and immunolocalized to CCs and oocyte cytoplasm, with FA binding protein 3 concentrated around oocyte chromatin. By mass spectrometry, CCs lipid profiling was shown to be different before and after IVM. FAO inhibitors etomoxir and mildronate dose-dependently decreased the oocyte maturation rate in vitro. In terms of viability, cumulus enclosed oocytes were more sensitive to etomoxir than denuded oocytes. In CCs, etomoxir (150μM) led to downregulation of lipogenesis genes and upregulated lipolysis and FAO genes. Moreover, the number of lipid droplets decreased, whereas several lipid species were more abundant compared with nontreated CCs after IVM. In conclusion, FAs metabolism in CCs is important to maintain metabolic homeostasis and may influence meiosis progression and survival of enclosed oocytes. PMID:25058602

The development of oocytes in the ovary of a parthenogenetic tick, Haemaphysalis longicornis.

PubMed

Mihara, Ryo; Umemiya-Shirafuji, Rika; Abe, Yasuyuki; Matsuo, Tomohide; Horiuchi, Noriyuki; Kawano, Suguru; Fujisaki, Kozo; Suzuki, Hiroshi

2018-04-17

Haemaphysalis longicornis is an important vector of various pathogens in domestic animals and humans. The tick is a unique species with bisexual and parthenogenetic races. Although mating induces oocyte development, it is possible in the parthenogenetic race to complete oogenesis without copulation. Here we examined the developmental process of oocytes from unfed to the oviposition period in parthenogenetic H. longicornis. We classified the developmental stages of oocytes into five stages: stage I, germinal vesicle occupies more than half of the cytoplasm; stage II, germinal vesicle occupies less than half of the cytoplasm; stage III, germinal vesicle migrates from the center in the oocyte to the vicinity of the pedicel cells; stage IV, the cytoplasm is filled with yolk granules of various sizes; stage V, the cytoplasm is occupied by large yolk granules. Oocytes at the unfed period were undeveloped and classified as stage I. Stage I and II oocytes were observed at the rapid feeding period, indicating that oocyte development began after the initiation of blood feeding. All developmental stages of oocytes were observed at the pre-oviposition period. At 10 days after the beginning of the oviposition period, the ratios of stage I and II oocytes were higher than those of the previous period, suggesting that the ovarian development and activity may be continuing. Based on these findings, we propose classification criteria for the oocyte development in the parthenogenetic H. longicornis. The criteria will be useful for understanding the mechanisms of tick reproduction and transovarial transmission of pathogens. Copyright © 2018 Elsevier B.V. All rights reserved.

Repair and recombination of X-irradiated plasmids in Xenopus laevis oocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sweigert, S.E.; Carroll, D.

1990-11-01

Plasmid DNA substrates were X-irradiated and injected into the nuclei of Xenopus laevis oocytes. After incubation for 20 h, DNA was recovered from the oocytes and analyzed simultaneously for repair and for intermolecular homologous recombination by electrophoresis and bacterial transformation. Oocyte-mediated repair of DNA strand breaks was observed with both methods. Using a repair-deficient mutant Escherichia coli strain and its repair-proficient parent as hosts for the transformation assay, we also demonstrated that oocytes repaired oxidative-type DNA base damage induced by X-rays. X-irradiation of a circular DNA stimulated its potential to recombine with a homologous linear partner. Recombination products were detectedmore » directly by Southern blot hybridization and as bacterial transformant clones expressing two antibiotic resistance markers originally carried separately on the two substrates. The increase in recombination was dependent on X-ray dose. There is some suggestion that lesions other than double-strand breaks contribute to the stimulation of oocyte-mediated homologous recombination. In summary, oocytes have considerable capacity to repair X-ray-induced damage, and some X-ray lesions stimulate homologous recombination in these cells.« less

Oocyte exposure to ZnO nanoparticles inhibits early embryonic development through the γ-H2AX and NF-κB signaling pathways.

PubMed

Liu, Jing; Zhao, Yong; Ge, Wei; Zhang, Pengfei; Liu, Xinqi; Zhang, Weidong; Hao, Yanan; Yu, Shuai; Li, Lan; Chu, Meiqiang; Min, Lingjiang; Zhang, Hongfu; Shen, Wei

2017-06-27

The impacts of zinc oxide nanoparticles on embryonic development following oocyte stage exposure are unknown and the underlying mechanisms are sparsely understood. In the current investigation, intact nanoparticles were detected in ovarian tissue in vivo and cultured cells in vitro under zinc oxide nanoparticles treatment. Zinc oxide nanoparticles exposure during the oocyte stage inhibited embryonic development. Notably, in vitro culture data closely matched in vivo embryonic data, in that the impairments caused by Zinc oxide nanoparticles treatment passed through cell generations; and both gamma-H2AX and NF-kappaB pathways were involved in zinc oxide nanoparticles caused embryo-toxicity. Copper oxide and silicon dioxide nanoparticles have been used to confirm that particles are important for the toxicity of zinc oxide nanoparticles. The toxic effects of zinc oxide nanoparticles emanate from both intact nanoparticles and Zn2+. Our investigation along with others suggests that zinc oxide nanoparticles are toxic to the female reproductive system [ovaries (oocytes)] and subsequently embryo-toxic and that precaution should be taken regarding human exposure to their everyday use.

Oocyte exposure to ZnO nanoparticles inhibits early embryonic development through the γ-H2AX and NF-κB signaling pathways

PubMed Central

Liu, Jing; Zhao, Yong; Ge, Wei; Zhang, Pengfei; Liu, Xinqi; Zhang, Weidong; Hao, Yanan; Yu, Shuai; Li, Lan; Chu, Meiqiang; Min, Lingjiang; Zhang, Hongfu; Shen, Wei

2017-01-01

The impacts of zinc oxide nanoparticles on embryonic development following oocyte stage exposure are unknown and the underlying mechanisms are sparsely understood. In the current investigation, intact nanoparticles were detected in ovarian tissue in vivo and cultured cells in vitro under zinc oxide nanoparticles treatment. Zinc oxide nanoparticles exposure during the oocyte stage inhibited embryonic development. Notably, in vitro culture data closely matched in vivo embryonic data, in that the impairments caused by Zinc oxide nanoparticles treatment passed through cell generations; and both gamma-H2AX and NF-kappaB pathways were involved in zinc oxide nanoparticles caused embryo-toxicity. Copper oxide and silicon dioxide nanoparticles have been used to confirm that particles are important for the toxicity of zinc oxide nanoparticles. The toxic effects of zinc oxide nanoparticles emanate from both intact nanoparticles and Zn2+. Our investigation along with others suggests that zinc oxide nanoparticles are toxic to the female reproductive system [ovaries (oocytes)] and subsequently embryo-toxic and that precaution should be taken regarding human exposure to their everyday use. PMID:28487501

Triplet pregnancy after intracytoplasmic sperm injection of cryopreserved oocytes: case report.

PubMed

Young, E; Kenny, A; Puigdomenech, E; Van Thillo, G; Tiverón, M; Piazza, A

1998-08-01

To report a triplet pregnancy that occurred after intracytoplasmic injection of sperm into cryopreserved oocytes. Case report. Instituto de Ginecología y Fertilidad (IFER), Buenos Aires, Argentina. A 36-year-old infertile patient with premature ovarian failure and a previous term pregnancy with fresh donated oocytes. We administered leuprolide acetate for pituitary down-regulation followed by E2 valerianate in incremental doses until an endometrial lining of >8 mm was observed by ultrasound. Thawing of frozen donated oocytes, intracytoplasmic sperm injection (ICSI), and translaparoscopic fallopian tube ET also were performed. Natural micronized progesterone was administered intravaginally (600 mg/d) before ET. Ultrasound at the 8th week of gestation revealed a triplet pregnancy with active fetal heartbeats. A triple intrauterine gestation was achieved with the use of microinjection into cryopreserved oocytes. This case illustrates the feasibility of oocyte cryopreservation for clinical use in the era of ICSI.

A minimally invasive method for extraction of sturgeon oocytes

USGS Publications Warehouse

Candrl, James S.; Papoulias, Diana M.; Tillitt, Donald E.

2010-01-01

Fishery biologists, hatchery personnel, and caviar fishers routinely extract oocytes from sturgeon (Acipenseridae) to determine the stage of maturation by checking egg quality. Typically, oocytes are removed either by inserting a catheter into the oviduct or by making an incision in the body cavity. Both methods can be time-consuming and stressful to the fish. We describe a device to collect mature oocytes from sturgeons quickly and effectively with minimal stress on the fish. The device is made by creating a needle from stainless steel tubing and connecting it to a syringe with polyvinyl chloride tubing. The device is filled with saline solution or water, the needle is inserted into the abdominal wall, and eggs are extracted from the fish. Using this device, an oocyte sample can be collected in less than 30 s. Such sampling leaves a minute wound that heals quickly and does not require suturing. The extractor device can easily be used in the field or hatchery, reduces fish handling time, and minimizes stress.

Translational control by cytoplasmic polyadenylation during Xenopus oocyte maturation: characterization of cis and trans elements and regulation by cyclin/MPF.

PubMed

McGrew, L L; Richter, J D

1990-11-01

The expression of certain maternal mRNAs during oocyte maturation is regulated by cytoplasmic polyadenylation. To understand this process, we have focused on a maternal mRNA from Xenopus termed G10. This mRNA is stored in the cytoplasm of stage 6 oocytes until maturation when the process of poly(A) elongation stimulates its translation. Deletion analysis of the 3' untranslated region of G10 RNA has revealed that two sequence elements, UUUUUUAU and AAUAAA were both necessary and sufficient for polyadenylation and polysomal recruitment. In this communication, we have defined the U-rich region that is optimal for polyadenylation as UUUUUUAUAAAG, henceforth referred to as the cytoplasmic polyadenylation element (CPE). We have also identified unique sequence requirements in the 3' terminus of the RNA that can modulate polyadenylation even in the presence of wild-type cis elements. A time course of cytoplasmic polyadenylation in vivo shows that it is an early event of maturation and that it requires protein synthesis within the first 15 min of exposure to progesterone. MPF and cyclin can both induce polyadenylation but, at least with respect to MPF, cannot obviate the requirement for protein synthesis. To identify factors that may be responsible for maturation-specific polyadenylation, we employed extracts from oocytes and unfertilized eggs, the latter of which correctly polyadenylates exogenously added RNA. UV crosslinking demonstrated that an 82 kd protein binds to the U-rich CPE in egg, but not oocyte, extracts. The data suggest that progesterone, either in addition to or through MPF/cyclin, induces the synthesis of a factor during very early maturation that stimulates polyadenylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of triiodothyronine on developmental competence of bovine oocytes.

PubMed

Costa, N N; Cordeiro, M S; Silva, T V G; Sastre, D; Santana, P P B; Sá, A L A; Sampaio, R V; Santos, S S D; Adona, P R; Miranda, M S; Ohashi, O M

2013-09-01

Developmental competence of in vitro-matured bovine oocytes is a limiting factor in production of embryos in vitro. Several studies have suggested a potential positive effect of thyroid hormones on cultured oocytes and/or their supporting cells. Therefore, the aim of the present study was to ascertain whether medium supplementation with triiodothyronine (T3) improved subsequent developmental competence of in vitro-matured bovine oocytes. For this purpose, we first documented (using reverse transcription PCR) that whereas bovine cumulus cells expressed both thyroid hormone receptor (TR)-α and TRβ, immature bovine oocytes expressed TRα only. Thereafter, to test the effects of TH on developmental competence, abattoir-derived oocytes were matured in vitro in a medium containing 0, 25, 50, or 100 nM T3 and subjected to in vitro fertilization. Embryo quality was evaluated by assessing cleavage and blastocyst rates, morphological quality, development kinetics, and total cell number on Day 8 of culture. Notably, addition of 50 or 100 nM T3 to the in vitro maturation medium increased (P < 0.05) the rate of hatched blastocysts on the eighth day of culture, as compared with other groups (62.4 ± 11.7, 53.1 ± 16.3, and 32.4 ± 5.3, respectively). Next, the relative expression levels of genes related to embryo quality POU-domain transcription factor (POU5F1) and glucose transporter-1 (GLUT 1) were compared between in vivo- and in vitro-produced blastocysts. On the basis of the previous experiments, IVP embryos originating from oocytes that were matured in vitro in the presence or absence of 50 nM T3 were evaluated. The treatment had no effect (P > 0.05) on gene expression. We concluded that supplementation of bovine oocyte in vitro maturation medium with T3 may have a beneficial effect on the kinetics of embryo development. Copyright © 2013 Elsevier Inc. All rights reserved.

Noninvasive assays of in vitro matured human oocytes showed insignificant correlation with fertilization and embryo development.

PubMed

Ashourzadeh, Sareh; Khalili, Mohammad Ali; Omidi, Marjan; Mahani, Seyed Nooraldin Nematollahi; Kalantar, Seyed Mehdi; Aflatoonian, Abbas; Habibzadeh, Victoria

2015-08-01

Recently, the upgrading of in vitro maturation (IVM) of human oocytes as a promising strategy has emerged in assisted reproductive technology (ART). The goal was to evaluate the correlation of the in vitro matured oocytes selected on the basis of the zona pellucida (ZP) birefringence and meiotic spindles (MS) detection with fertilization and subsequent embryo development in ICSI program. A total of 168 immature oocytes [germinal vesicle (n = 140) and metaphase I (n = 28)] obtained from patients undergoing oocytes retrieval for ICSI. After in vitro culture for 24-40 h, 112 (67 %) oocytes reached to MII stage. Using a polarized microscopy, the presence of MS and ZP birefringence were assessed in matured oocytes, followed by ICSI performance. The rates of fertilization in oocytes with spindles (51.3 %) were similar to that of the oocytes without spindles (50.7 %; P = 1.00). Moreover, the fertilization rates in high birefringence (HB) oocytes was not statistically different than oocytes with low birefringence (LB) (P = 0.44). The findings also showed that 64.9 % of the fertilized oocytes developed to embryos, in which 33.3 % were derived from spindle-detected oocytes. Regarding the ZP birefringence, 35.5 % of the embryos were derived from HB oocytes. There were insignificant relationships between the MS detection and ZP birefringence score with the rates of fertilization and embryo development in IVM oocytes.

Exposure to bisphenol A in young adult mice does not alter ovulation but does alter the fertilization ability of oocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moore-Ambriz, Teresita Rocio; Acuña-Hernández, Deyanira Guadalupe; Ramos-Robles, Brenda

Follicle growth culminates in ovulation, which allows for the expulsion of fertilizable oocytes and the formation of corpora lutea. Bisphenol A (BPA) is present in many consumer products, and it has been suggested that BPA impairs ovulation; however, the underlying mechanisms are unknown. Therefore, this study first evaluated whether BPA alters ovulation by affecting folliculogenesis, the number of corpora lutea or eggs shed to the oviduct, ovarian gonadotropin responsiveness, hormone levels, and estrous cyclicity. Because it has been suggested (but not directly confirmed) that BPA exerts toxic effects on the fertilization ability of oocytes, a second aim was to evaluatemore » whether BPA impacts the oocyte fertilization rate using an in vitro fertilization assay and mating. The possible effects on early zygote development were also examined. Young adult female C57BL/6J mice (39 days old) were orally dosed with corn oil (vehicle) or 50 μg/kg bw/day BPA for a period encompassing the first three reproductive cycles (12–15 days). BPA exposure did not alter any parameters related to ovulation. Moreover, BPA exposure reduced the percentage of fertilized oocytes after either in vitro fertilization or mating, but it did not alter the zygotic stages. The data indicate that exposure to the reference dose of BPA does not impact ovulation but that it does influence the oocyte quality in terms of its fertilization ability. - Highlights: • Bisphenol A targets the fertilization ability of oocytes. • Bisphenol A does not alter ovulation. • Young adult females may be susceptible to the effects of bisphenol A on fertilization.« less

Oxygen consumption is a quality marker for human oocyte competence conditioned by ovarian stimulation regimens.

PubMed

Tejera, Aberto; Herrero, Javier; de Los Santos, M J; Garrido, Nicolás; Ramsing, Niels; Meseguer, Marcos

2011-09-01

To evaluate the effect of different ovarian stimulation protocols on oocyte respiration and to investigate the relationship between oocyte oxygen consumption and reproductive outcome. Prospective observational cohort study. Infertility clinic in a university hospital. A total of 349 oocytes from 56 IVF treatment cycles in our oocyte donation program. None. Average oocyte oxygen consumption rate in fmol/s. We correlated oxygen consumption values with ovarian stimulation features, fertilization, embryo quality on days 2 and 3, and implantation. Differences in the measured oxygen consumption rates were found depending on which type of gonadotropins were used in the stimulation protocol. Higher consumption rates were found for oocytes that underwent normal fertilization compared with rates from nonfertilized or abnormal oocytes (odds ratio = 1.340; 95% confidence intervals = 1.037-1.732). Furthermore, higher oxygen consumption was observed for those oocytes which generated embryos that implanted compared with those that did not implant (6.21 ± 0.849 fmol/s vs. 5.23 ± 0.345 fmol/s. Measurement of oxygen consumption rates for individual oocytes before fertilization provides a noninvasive marker of oocyte quality and hence a quantitative assessment of the reproductive potential for the oocyte. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Lectin from embryos and oocytes of Xenopus laevis. Purification and properties.

PubMed

Roberson, M M; Barondes, S H

1982-07-10

Soluble extracts of Xenopus laevis blastula stage embryos, oocytes, and adult liver contain lectin activities detected by agglutination of trypsinized, glutaraldehyde-fixed rabbit erythrocytes. Lectin from the embryos and oocytes was purified by affinity chromatography on a column derivatized with melibiose. Trace contaminants were removed either by preparative isoelectric focusing or by gel filtration. Based on its behavior on Sepharose 6B the purified oocyte lectin has an apparent molecular weight of approximately 480,000. On sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions there were two major bands with molecular weight ranges of about 43,000 and 45,000, with diffuse trails. Since the purified lectin contains about 20% saccharides by weight and since both bands are glycosylated, diffuseness might be due to variable glycosylation. Heterogeneity was indicated by isoelectric focusing in polyacrylamide gels, which showed four protein bands with isoelectric points ranging from 4.4 to 4.9. Lectins from both embryos and oocytes comprised about 1 to 2% of the total soluble protein and could not be distinguished by sodium dodecyl sulfate polyacrylamide gel electrophoresis. However, the specific hemagglutination activity of the purified oocyte lectin was, on the average, 7-fold higher. Levels in crude extracts of liver were 3 orders of magnitude lower than those from oocytes. The hemagglutination activities of the lectins from embryos, oocytes, and adult liver required Ca2+ and were blocked by similar concentrations of both alpha- and beta-galactosides.

Cholesterol Depletion Disorganizes Oocyte Membrane Rafts Altering Mouse Fertilization

PubMed Central

Buschiazzo, Jorgelina; Ialy-Radio, Come; Auer, Jana; Wolf, Jean-Philippe; Serres, Catherine

2013-01-01

Drastic membrane reorganization occurs when mammalian sperm binds to and fuses with the oocyte membrane. Two oocyte protein families are essential for fertilization, tetraspanins and glycosylphosphatidylinositol-anchored proteins. The firsts are associated to tetraspanin-enriched microdomains and the seconds to lipid rafts. Here we report membrane raft involvement in mouse fertilization assessed by cholesterol modulation using methyl-β-cyclodextrin. Cholesterol removal induced: (1) a decrease of the fertilization rate and index; and (2) a delay in the extrusion of the second polar body. Cholesterol repletion recovered the fertilization ability of cholesterol-depleted oocytes, indicating reversibility of these effects. In vivo time-lapse analyses using fluorescent cholesterol permitted to identify the time-point at which the probe is mainly located at the plasma membrane enabling the estimation of the extent of the cholesterol depletion. We confirmed that the mouse oocyte is rich in rafts according to the presence of the raft marker lipid, ganglioside GM1 on the membrane of living oocytes and we identified the coexistence of two types of microdomains, planar rafts and caveolae-like structures, by terms of two differential rafts markers, flotillin-2 and caveolin-1, respectively. Moreover, this is the first report that shows characteristic caveolae-like invaginations in the mouse oocyte identified by electron microscopy. Raft disruption by cholesterol depletion disturbed the subcellular localization of the signal molecule c-Src and the inhibition of Src kinase proteins prevented second polar body extrusion, consistent with a role of Src-related kinases in fertilization via signaling complexes. Our data highlight the functional importance of intact membrane rafts for mouse fertilization and its dependence on cholesterol. PMID:23638166

Reproductive choices and outcomes after freezing oocytes for medical reasons: a follow-up study.

PubMed

Dahhan, T; Dancet, E A F; Miedema, D V; van der Veen, F; Goddijn, M

2014-09-01

What reproductive choices do women make after they have cryopreserved oocytes for medical reasons? Women who had cryopreserved oocytes for medical reasons and tried to become pregnant, either attempted natural conception or resorted to assisted reproduction with fresh oocytes. Women confronted with a risk of premature ovarian insufficiency, due to gonadotoxic therapy, ovarian surgery or genetic predisposition, have an indication to cryopreserve oocytes. Many of these women will retain ovarian function, thus will retain the possibility of natural conception. The added value of cryopreserved oocytes to reproductive outcomes is unknown as there is a lack of follow-up of women who have cryopreserved oocytes for medical reasons. This follow-up study included a cohort of 85 women who cryopreserved their oocytes for medical reasons between 2009 and 2012. Medical data from women who cryopreserved their oocytes at the Centre for Reproductive Medicine in the Academic Medical Centre in Amsterdam were extracted and self-report questionnaires were disseminated. The collected data considered demographics, outcomes of ovarian stimulation, fertility-threatening treatments, menstrual cycle changes, pregnancy attempts and outcomes and intended plans for the cryopreserved oocytes. A total of 68 women, followed up for an average 25.3 months, returned the questionnaire (response rate: 80%). None of the women had used her cryopreserved oocytes although 16 women had tried to conceive. Of these women, eight were trying to conceive naturally, five had conceived naturally within 2 months and three had conceived with assisted reproduction not requiring cryopreserved oocytes (two women with conventional IVF because of tubal pathology and endometriosis and one woman with IUI because of polycystic ovary syndrome). Three out of the eight pregnancies had resulted in live births, two resulted in miscarriages and three were ongoing. Most women (71%) intended to conceive with their cryopreserved

Ultrastructural Interactions and Genotoxicity Assay of Cerium Dioxide Nanoparticles on Mouse Oocytes

PubMed Central

Courbiere, Blandine; Auffan, Mélanie; Rollais, Raphaël; Tassistro, Virginie; Bonnefoy, Aurélie; Botta, Alain; Rose, Jérôme; Orsière, Thierry; Perrin, Jeanne

2013-01-01

Cerium dioxide nanoparticles (CeO2 ENPs) are on the priority list of nanomaterials requiring evaluation. We performed in vitro assays on mature mouse oocytes incubated with CeO2 ENPs to study (1) physicochemical biotransformation of ENPs in culture medium; (2) ultrastructural interactions with follicular cells and oocytes using Transmission Electron Microscopy (TEM); (3) genotoxicity of CeO2 ENPs on follicular cells and oocytes using a comet assay. DNA damage was quantified as Olive Tail Moment. We show that ENPs aggregated, but their crystal structure remained stable in culture medium. TEM showed endocytosis of CeO2 ENP aggregates in follicular cells. In oocytes, CeO2 ENP aggregates were only observed around the zona pellucida (ZP). The comet assay revealed significant DNA damage in follicular cells. In oocytes, the comet assay showed a dose-related increase in DNA damage and a significant increase only at the highest concentrations. DNA damage decreased significantly both in follicular cells and in oocytes when an anti-oxidant agent was added in the culture medium. We hypothesise that at low concentrations of CeO2 ENPs oocytes could be protected against indirect oxidative stress due to a double defence system composed of follicular cells and ZP. PMID:24185910

Dimethyl Sulfoxide Perturbs Cell Cycle Progression and Spindle Organization in Porcine Meiotic Oocytes

PubMed Central

Li, Xuan; Wang, Yan-Kui; Song, Zhi-Qiang; Du, Zhi-Qiang; Yang, Cai-Xia

2016-01-01

Meiotic maturation of mammalian oocytes is a precisely orchestrated and complex process. Dimethyl sulfoxide (DMSO), a widely used solvent, drug, and cryoprotectant, is capable of disturbing asymmetric cytokinesis of oocyte meiosis in mice. However, in pigs, DMSO’s effect on oocyte meiosis still remains unknown. We aimed to evaluate if DMSO treatment will affect porcine oocyte meiosis and the underlying molecular changes as well. Interestingly, we did not observe the formation of the large first polar body and symmetric division for porcine oocytes treated with DMSO, contrary to findings reported in mice. 3% DMSO treatment could inhibit cumulus expansion, increase nuclear abnormality, disturb spindle organization, decrease reactive oxygen species level, and elevate mitochondrial membrane potential of porcine oocytes. There was no effect on germinal vesicle breakdown rate regardless of DMSO concentration. 3% DMSO treatment did not affect expression of genes involved in spindle organization (Bub1 and Mad2) and apoptosis (NF-κB, Pten, Bcl2, Caspase3 and Caspase9), however, it significantly decreased expression levels of pluripotency genes (Oct4, Sox2 and Lin28) in mature oocytes. Therefore, we demonstrated that disturbed cumulus expansion, chromosome alignment, spindle organization and pluripotency gene expression could be responsible for DMSO-induced porcine oocyte meiotic arrest and the lower capacity of subsequent embryo development. Our results provide new insights on DMSO’s effect on porcine oocyte meiosis and raise safety concerns over DMSO’s usage on female reproduction in both farm animals and humans. PMID:27348312

Dimethyl Sulfoxide Perturbs Cell Cycle Progression and Spindle Organization in Porcine Meiotic Oocytes.

PubMed

Li, Xuan; Wang, Yan-Kui; Song, Zhi-Qiang; Du, Zhi-Qiang; Yang, Cai-Xia

2016-01-01

Meiotic maturation of mammalian oocytes is a precisely orchestrated and complex process. Dimethyl sulfoxide (DMSO), a widely used solvent, drug, and cryoprotectant, is capable of disturbing asymmetric cytokinesis of oocyte meiosis in mice. However, in pigs, DMSO's effect on oocyte meiosis still remains unknown. We aimed to evaluate if DMSO treatment will affect porcine oocyte meiosis and the underlying molecular changes as well. Interestingly, we did not observe the formation of the large first polar body and symmetric division for porcine oocytes treated with DMSO, contrary to findings reported in mice. 3% DMSO treatment could inhibit cumulus expansion, increase nuclear abnormality, disturb spindle organization, decrease reactive oxygen species level, and elevate mitochondrial membrane potential of porcine oocytes. There was no effect on germinal vesicle breakdown rate regardless of DMSO concentration. 3% DMSO treatment did not affect expression of genes involved in spindle organization (Bub1 and Mad2) and apoptosis (NF-κB, Pten, Bcl2, Caspase3 and Caspase9), however, it significantly decreased expression levels of pluripotency genes (Oct4, Sox2 and Lin28) in mature oocytes. Therefore, we demonstrated that disturbed cumulus expansion, chromosome alignment, spindle organization and pluripotency gene expression could be responsible for DMSO-induced porcine oocyte meiotic arrest and the lower capacity of subsequent embryo development. Our results provide new insights on DMSO's effect on porcine oocyte meiosis and raise safety concerns over DMSO's usage on female reproduction in both farm animals and humans.

Effect of Nanoparticles on the Survival and Development of Vitrified Porcine GV Oocytes.

PubMed

Li, W J; Zhou, X L; Liu, B L; Dai, J J; Song, P; Teng, Y

BACKGROUND: Some mammalian oocytes have been successfully cryopreserved by vitrification. However, the survival and developmental rate of vitrified oocytes is still low. The incorporation of nanoparticles into cryoprotectant (CPA) may improve the efficiency of vitrification by changing the properties of solutions. The toxicity of different concentrations of hydroxy apatite (HA), silica dioxide (SO 2 ), aluminum oxide (Al 2 O 3 ) and titanium dioxide (TiO 2 ) nanoparticles (20 nm in diameter) to oocytes was tested and the toxicity threshold value of each nanoparticle was determined. Porcine GV oocytes were vitrified in optimized nano-CPA, and effects of diameter and concentration of nanoparticles on the survival rate and developmental rate of porcine GV oocytes were compared. HA nanoparticles have demonstrated the least toxicity among four nanoparticles and the developmental rate of GV-stage porcine oocytes was 100% when its concentration was lower than 0.5%. By adding 0.1% HA into VS, the developmental rate of GV-stage porcine oocytes (22%) was significantly higher than other groups. The effect of vitrification in nano-CPA on oocytes was related to the concentration of HA nanoparticles rather than their size. By adding 0.05% HA nanoparticles (60nm in diameter), the developmental rate increased dramatically from 14.7% to 30.4%. Nano-cryopreservation offers a new way to improve the effect of survival and development of oocytes, but the limitation of this technology shall not be ignored.

Lithobates catesbeianus (American Bullfrog) oocytes: a novel heterologous expression system for aquaporins

PubMed Central

2018-01-01

ABSTRACT Xenopus laevis oocytes are a valuable tool for investigating the function of membrane proteins. However, regulations around the world, specifically in Brazil, render the import of Xenopus laevis frogs impractical, and, in some cases, impossible. Here, as an alternative, we evaluate the usefulness of the North American aquatic bullfrog Lithobates catesebeianus, which is commercially available in Brazil, for the heterologous expression of aquaporin (AQP) proteins. We have developed a method that combines a brief collagenase treatment and mechanical defolliculation for isolating individual oocytes from Lithobates ovaries. We find that they have a similar size, shape, and appearance to Xenopus oocytes and can tolerate and survive following injections with cRNA or water. Furthermore, surface biotinylation, western blot analysis, and measurements of osmotic water permeability (Pf) show that Lithobates oocytes can express AQPs to the plasma membrane and significantly increase the Pf of the oocytes. In fact, the Pf values are similar to historical values gathered from Xenopus oocytes. Due to the presence of a mercury sensitive cysteine (Cys or C) in the throat of the water channel, the Pf of oocytes expressing human (h) AQP1, hAQP1FLAG [FLAG, short protein tag (DYKDDDDK) added to the N-terminus of AQP1], hAQP8, and rat (r) AQP9 was inhibited with the mercurial compound p-chloromercuribenzene sulfonate (pCMBS), whereas AQPs lacking this Cys – hAQP1C189S mutant [residue Cys 189 was replaced by a serine (Ser or S)] and hAQP7 – were mercury insensitive. Contrary to previous studies with Xenopus oocytes, rAQP3 was also found to be insensitive to mercury, which is consistent with the mercury-sensitive Cys (Cys 11) being located intracellularly. Thus, we consider Lithobates oocytes to be a readily accessible system for the functional expression and study of membrane proteins for international researchers who do not currently have access to Xenopus oocytes. PMID

Functional signaling and gene regulatory networks between the oocyte and the surrounding cumulus cells.

PubMed

Biase, Fernando H; Kimble, Katelyn M

2018-05-10

The maturation and successful acquisition of developmental competence by an oocyte, the female gamete, during folliculogenesis is highly dependent on molecular interactions with somatic cells. Most of the cellular interactions identified, thus far, are modulated by growth factors, ions or metabolites. We hypothesized that this interaction is also modulated at the transcriptional level, which leads to the formation of gene regulatory networks between the oocyte and cumulus cells. We tested this hypothesis by analyzing transcriptome data from single oocytes and the surrounding cumulus cells collected from antral follicles employing an analytical framework to determine interdependencies at the transcript level. We overlapped our transcriptome data with putative protein-protein interactions and identified hundreds of ligand-receptor pairs that can transduce paracrine signaling between an oocyte and cumulus cells. We determined that 499 ligand-encoding genes expressed in oocytes and cumulus cells are functionally associated with transcription regulation (FDR < 0.05). Ligand-encoding genes with specific expression in oocytes or cumulus cells were enriched for biological functions that are likely associated with the coordinated formation of transzonal projections from cumulus cells that reach the oocyte's membrane. Thousands of gene pairs exhibit significant linear co-expression (absolute correlation > 0.85, FDR < 1.8 × 10 - 5 ) patterns between oocytes and cumulus cells. Hundreds of co-expressing genes showed clustering patterns associated with biological functions (FDR < 0.5) necessary for a coordinated function between the oocyte and cumulus cells during folliculogenesis (i.e. regulation of transcription, translation, apoptosis, cell differentiation and transport). Our analyses revealed a complex and functional gene regulatory circuit between the oocyte and surrounding cumulus cells. The regulatory profile of each cumulus-oocyte complex is likely

HIGH INCIDENCE OF POLYSPERMIC FERTILIZATION IN BOVINE OOCYTES MATURED IN VITRO AFTER CRYOTOP VITRIFICATION.

PubMed

Hwang, In-Sul; Kwon, Dae-Jin; Im, Gi-Sun; Tashima, Kazuya; Hochi, Shinichi; Hwang, Seongsoo

2016-01-01

Vitrification with the Cryotop device is the most promising technique for oocyte cryopreservation, but the high post-warming morphological survival of bovine oocytes does not guarantee high developmental competence after in vitro fertilization (IVF). This study was designed to examine achievement of normal fertilization in bovine oocytes vitrified-warmed with the Cryotop device. Oocytes were matured in vitro and vitrified-warmed after complete removal of the cumulus layers. Distribution of cortical granules (CGs) was assessed by Lens culinaris agglutinin (LCA) lectin staining. Ten hours after IVF, presumptive zygotes were analyzed for pronuclear formation. Day-8 blastocysts were harvested and stained with Hoechst-33342 for total cell counting. Both yield and mean cell number of the blastocysts were impaired by Cryotop vitrification. Incidence of polyspermic fertilization was three-times higher in vitrified oocytes compared to fresh oocytes. No difference in CG distribution was found between vitrified and fresh oocytes. Polyspermic fertilization induced in vitrified-warmed bovine oocytes may be one of the possible causes responsible for their low developmental potential.

In vitro Maturation of Oocytes in High Altitude Women with Polycystic Ovaries.

PubMed

Tominaga, Luis A Vargas; Cáceres, Ricardo E Pella; Lechuga, Jose A Vargas; Durán, Livia S Bartolo; Vargas, Mariela Serrano

2015-05-01

Determine the effectiveness of in vitro maturation of oocytes in the infertility treatment in high altitude women with polycystic ovaries. descriptive and retrospective study. Women with polycystic ovaries and infertility. there were 11 women from locations above 7,546 feet above sea level with polycystic ovaries and infertility in which were performed in vitro maturation of oocytes, followed by intracytoplasmic sperm injection, culture and embryo vitrification. After that, the endometrium was prepared and the embryos were thawed and transferred. Main results mesurements: oocytes maturation, fertilization, clinical pregnancy and implantation rates. Oocytes maturation rate was 86.1%; fertilization rate 90.3%; clinical pregnancy rate 36.4% and implantation rate 17.4%. In vitro maturation of oocytes is an effective technique in the infertility treatment of high altitude women with polycystic ovaries.

Reconstruction of mammalian oocytes by germinal vesicle transfer: A systematic review

PubMed Central

Darbandi, Sara; Darbandi, Mahsa; Khorram Khorshid, Hamid Reza; Shirazi, Abolfazl; Sadeghi, Mohammad Reza; Agarwal, Ashok; Al-Hasani, Safaa; Naderi, Mohammad Mehdi; Ayaz, Ahmet; Akhondi, Mohammad Mehdi

2017-01-01

Nuclear transfer procedures have been recently applied for clinical and research targets as a novel assisted reproductive technique and were used for increasing the oocyte activity during its growth and maturation. In this review, we summarized the nuclear transfer technique for germinal vesicle stage oocytes to reconstruct the maturation of them. Our study covered publications between 1966 and August 2017. In result utilized germinal vesicle transfer techniques, fusion, and fertilization survival rate on five different mammalian species are discussed, regarding their potential clinical application. It seems that with a study on this method, there is real hope for effective treatments of old oocytes or oocytes containing mitochondrial problems in the near future. PMID:29387825

Role of human oocyte-enriched factors in somatic cell reprograming.

PubMed

El-Gammal, Zaynab; AlOkda, Abdelrahman; El-Badri, Nagwa

2018-06-08

Cellular reprograming paves the way for creating functional patient-specific tissues to eliminate immune rejection responses by applying the same genetic profile. However, the epigenetic memory of a cell remains a challenge facing the current reprograming methods and does not allow transcription factors to bind properly. Because somatic cells can be reprogramed by transferring their nuclear contents into oocytes, introducing specific oocyte factors into differentiated cells is considered a promising approach for mimicking the reprograming process that occurs during fertilization. Mammalian metaphase II oocyte possesses a superior capacity to epigenetically reprogram somatic cell nuclei towards an embryonic stem cell-like state than the current factor-based reprograming approaches. This may be due to the presence of specific factors that are lacking in the current factor-based reprograming approaches. In this review, we focus on studies identifying human oocyte-enriched factors aiming to understand the molecular mechanisms mediating cellular reprograming. We describe the role of oocyte-enriched factors in metabolic switch, chromatin remodelling, and global epigenetic transformation. This is critical for improving the quality of resulting reprogramed cells, which is crucial for therapeutic applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Enhanced polarizing microscopy as a new tool in aneuploidy research in oocytes.

PubMed

Shen, Ying; Betzendahl, Ilse; Tinneberg, Hans-Rudolf; Eichenlaub-Ritter, Ursula

2008-03-12

Chromosomal non-disjunction in female meiosis gives rise to reduced fertility and trisomy in humans. Human oocytes, especially from aged women, appear especially susceptible to non-disjunction. The oocyte spindle is crucial for high fidelity of chromosome segregation at meiotic divisions, and alterations in spindle morphology are therefore indicators of adverse conditions during oocyte development that may result in meiotic aneuploidy. In the past, oocytes had to be fixed for spindle analysis, precluding direct non-invasive identification of aneugens and adverse maturation conditions that affect spindle integrity and chromosome behaviour. Aneuploidy research for detection of spindle aberrations was therefore mainly focused on in vivo or in vitro exposed, fixed animal oocytes or cytogenetic analysis of spread oocytes. Orientation independent enhanced polarizing microscopy with nearly circularly polarized light and electronically controlled liquid crystal compensator optics is a new tool to study spindle morphology non-invasively in vivo for qualitative as well as quantitative analysis. Image generation by polarization microscopy depends on the intrinsic optical properties of the spindle with its paracrystalline microtubule lattice. When polarized light passes through such a lattice it induces a splitting of the beam and shift in the plane of vibration and retardation of light (termed birefringence and retardance). Studies of animal oocytes and follicle-cell denuded human oocytes fertilized by intracytoplasmic sperm injection for assisted conception have demonstrated the safety and efficacy of enhanced polarization microscopy. The method can be employed in aneuploidy research for non-invasive dose-response studies to detect spindle aberrations, for instance, in combination with cytogenetic analysis. Due to the non-invasive nature of the technique it may be employed in routine analysis of human oocytes to assess risks by lifestyle factors, and occupational and adverse

Expression of functional neurotransmitter receptors in Xenopus oocytes after injection of human brain membranes

NASA Astrophysics Data System (ADS)

Miledi, Ricardo; Eusebi, Fabrizio; Martínez-Torres, Ataúlfo; Palma, Eleonora; Trettel, Flavia

2002-10-01

The Xenopus oocyte is a very powerful tool for studies of the structure and function of membrane proteins, e.g., messenger RNA extracted from the brain and injected into oocytes leads to the synthesis and membrane incorporation of many types of functional receptors and ion channels, and membrane vesicles from Torpedo electroplaques injected into oocytes fuse with the oocyte membrane and cause the appearance of functional Torpedo acetylcholine receptors and Cl channels. This approach was developed further to transplant already assembled neurotransmitter receptors from human brain cells to the plasma membrane of Xenopus oocytes. Membranes isolated from the temporal neocortex of a patient, operated for intractable epilepsy, were injected into oocytes and, within a few hours, the oocyte membrane acquired functional neurotransmitter receptors to -aminobutyric acid, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, and glycine. These receptors were also expressed in the plasma membrane of oocytes injected with mRNA extracted from the temporal neocortex of the same patient. All of this makes the Xenopus oocyte a more useful model than it already is for studies of the structure and function of many human membrane proteins and opens the way to novel pathophysiological investigations of some human brain disorders.

Oocyte cryopreservation: a feasible fertility preservation option for reproductive age cancer survivors

PubMed Central

Labella, Patty Ann; Grifo, James; Knopman, Jaime M.

2010-01-01

Purpose To compare oocyte cryopreservation cycles performed in cancer patients to those of infertile women. Methods Cancer patients referred for fertility preservation underwent counseling in compliance with the ASRM; those electing oocyte cryopreservation were included. Ovarian stimulation was achieved with injectable gonadotropins and freezing was performed using slow-cooling and vitrification methods. Results Fifty cancer patients (mean age 31 y) underwent oocyte cryopreservation; adequate ovarian stimulation was achieved in 10 ± 0.3 days. The outcome from these cycles included a mean peak estradiol of 2,376 pg/ml and an average of 19 oocytes retrieved (15 mature oocytes were cryopreserved/cycle). All patients tolerated ovarian hyperstimulation. There were no significant differences noted between cryopreservation cycles performed in cancer patients and in women without malignancy. Conclusions Oocyte cryopreservation appears to be a feasible fertility preservation method for reproductive-age women diagnosed with cancer. This modality is not only effective but also, providing a multidiscipline effort, can be completed in timely fashion. PMID:20480389

Intact cell MALDI-TOF mass spectrometry on single bovine oocyte and follicular cells combined with top-down proteomics: A novel approach to characterise markers of oocyte maturation.

PubMed

Labas, Valérie; Teixeira-Gomes, Ana-Paula; Bouguereau, Laura; Gargaros, Audrey; Spina, Lucie; Marestaing, Aurélie; Uzbekova, Svetlana

2018-03-20

Intact cell MALDI-TOF mass spectrometry (ICM-MS) was adapted to bovine follicular cells from individual ovarian follicles to obtain the protein/peptide signatures (<17kDa) of single oocytes, cumulus cells (CC) and granulosa cells (GC), which shared a total of 439 peaks. By comparing the ICM-MS profiles of single oocytes and CC before and after in vitro maturation (IVM), 71 different peaks were characterised, and their relative abundance was found to vary depending on the stage of oocyte meiotic maturation. To identify these endogenous biomolecules, top-down workflow using high resolution MS/MS (TD HR-MS) was performed on the protein extracts from oocytes, CC and GC. The TD HR-MS proteomic approach allowed for: (1) identification of 386 peptide/proteoforms encoded by 194 genes; and (2) characterisation of proteolysis products likely resulting from the action of kallikreins and caspases. In total, 136 peaks observed by ICM-MS were annotated by TD HR-MS (ProteomeXchange PXD004892). Among these, 16 markers of maturation were identified, including IGF2 binding protein 3 and hemoglobin B in the oocyte, thymosins beta-4 and beta-10, histone H2B and ubiquitin in CC. The combination of ICM-MS and TD HR-MS proved to be a suitable strategy to identify non-invasive markers of oocyte quality using limited biological samples. Intact cell MALDI-TOF mass spectrometry on single oocytes and their surrounding cumulus cells, coupled to an optimised top-down HR-MS proteomic approach on ovarian follicular cells, was used to identify specific markers of oocyte meiotic maturation represented by whole low molecular weight proteins or products of degradation by specific proteases. Copyright © 2017 Elsevier B.V. All rights reserved.

Morphological records of oocyte maturation in the parthenogenetic tick Amblyomma rotundatum Koch, 1844 (Acari: Ixodidae).

PubMed

Sanches, Gustavo S; Araujo, Andrea M; Martins, Thiago F; Bechara, Gervásio H; Labruna, Marcelo B; Camargo-Mathias, Maria I

2012-02-01

Oocyte maturation in the thelytokous parthenogenetic tick Amblyomma rotundatum was examined for the first time using light and scanning electron microscopy. The panoistic ovary lacks nurse and follicular cells and is a single continuous tubular structure forming a lumen delimited by the ovarian wall. Oocytes of tick species are usually classified according to cytoplasm appearance, the presence of germinal vesicle, the presence of yolk granules, and the chorion. However, for this species, we also use oocyte size as an auxiliary tool since most oocytes were in stages I-III and were histologically very similar. Oocytes were classified into five development stages, and specific characteristics were observed: mature oocytes with thin chorion, pedicel cells arranged forming an epithelium with two or more oocytes attached by the same structure, and a large number of oocytes in the process of reabsorption. Copyright © 2011 Elsevier GmbH. All rights reserved.

Transcriptome dynamics and molecular cross-talk between bovine oocyte and its companion cumulus cells

PubMed Central

2011-01-01

Background The bi-directional communication between the oocyte and its companion cumulus cells (CCs) is crucial for development and functions of both cell types. Transcripts that are exclusively expressed either in oocytes or CCs and molecular mechanisms affected due to removal of the communication axis between the two cell types is not investigated at a larger scale. The main objectives of this study were: 1. To identify transcripts exclusively expressed either in oocyte or CCs and 2. To identify those which are differentially expressed when the oocyte is cultured with or without its companion CCs and vice versa. Results We analyzed transcriptome profile of different oocyte and CC samples using Affymetrix GeneChip Bovine Genome array containing 23000 transcripts. Out of 13162 genes detected in germinal vesicle (GV) oocytes and their companion CCs, 1516 and 2727 are exclusively expressed in oocytes and CCs, respectively, while 8919 are expressed in both. Similarly, of 13602 genes detected in metaphase II (MII) oocytes and CCs, 1423 and 3100 are exclusively expressed in oocytes and CCs, respectively, while 9079 are expressed in both. A total of 265 transcripts are differentially expressed between oocytes cultured with (OO + CCs) and without (OO - CCs) CCs, of which 217 and 48 are over expressed in the former and the later groups, respectively. Similarly, 566 transcripts are differentially expressed when CCs mature with (CCs + OO) or without (CCs - OO) their enclosed oocytes. Of these, 320 and 246 are over expressed in CCs + OO and CCs - OO, respectively. While oocyte specific transcripts include those involved in transcription (IRF6, POU5F1, MYF5, MED18), translation (EIF2AK1, EIF4ENIF1) and CCs specific ones include those involved in carbohydrate metabolism (HYAL1, PFKL, PYGL, MPI), protein metabolic processes (IHH, APOA1, PLOD1), steroid biosynthetic process (APOA1, CYP11A1, HSD3B1, HSD3B7). Similarly, while transcripts over expressed in OO + CCs are involved in

Role of animal pole protuberance and microtubules during meiosis in sea cucumber Apostichopus japonicus oocytes

NASA Astrophysics Data System (ADS)

Pang, Zhenguo; Chang, Yaqing; Sun, Huiling; Yu, Jiaping

2010-05-01

Fully grown oocytes of Apostichopus japonicus have a cytoplasmic protuberance where the oocyte attaches to the follicle. The protuberance and the oolamina located on the opposite side of the oocyte indicate the animal-vegetal axis. Two pre-meiotic centrosomes are anchored to the protuberance by microtubules between centrosomes and protuberance. After meiosis reinitiation induced by DTT solution, the germinal vesicle (GV) migrates towards the protuberance. The GV breaks down after it migrates to the oocyte membrane on the protuberance side. The protuberance then contracts back into the oocyte and the first polar body extrudes from the site of the former protuberance. The second polar body forms beneath the first. Thus the oocyte protuberance indicates the presumptive animal pole well before maturation of the oocyte.

Obox4 critically regulates cAMP-dependent meiotic arrest and MI-MII transition in oocytes.

PubMed

Lee, Hyun-Seo; Kim, Eun-Young; Kim, Kyeoung-Hwa; Moon, Jisook; Park, Kyung-Soon; Kim, Kwang-Soo; Lee, Kyung-Ah

2010-07-01

Extra follicular oocytes spontaneously resume meiosis in vitro, but the intact germinal vesicle (GV) is retained if the oocytes are cultured in medium containing phosphodiesterase (PDE) inhibitors or cAMP analogues. On the basis of our finding that Obox4 is prominently expressed in oocytes, the present study was conducted to determine the functional role of the homeodomain-containing factor Obox4 during in vitro oocyte maturation. After microinjection of Obox4 dsRNA into the cytoplasm of GV oocytes cultured in M16 medium, oocytes were arrested at metaphase I (MI, 77.7%) and metaphase II (MII, 22.3%). Surprisingly, however, 89% of Obox4 RNAi-treated oocytes resumed meiosis and developed to MI and MII when cultured in medium containing 0.2 mM 3-isobutyl-1-methyl-xanthine (IBMX), in which untreated oocytes maintain intact GVs. Spindles were aberrant, and chromosomes were severely aggregated with decreased MPF and MAP kinase activities in arrested MI oocytes after exposure to Obox4 RNAi. Oocytes overexpressing Obox4 retained intact GVs when cultured in M16 medium. Taken together, for the first time to our knowledge, these findings indicate that Obox4 plays a key role in the cAMP-dependent signaling cascades that maintain GV arrest. Oocytes not expressing Obox4 failed to maintain intact GVs in IBMX-supplemented medium, while GVs remained intact when oocytes were kept in plain medium and overexpressing Obox4, suggesting that Obox4 plays a critical role in cAMP-dependent cascade for maintaining intact GVs.

Optimization of cryoprotectant loading into murine and human oocytes.

PubMed

Karlsson, Jens O M; Szurek, Edyta A; Higgins, Adam Z; Lee, Sang R; Eroglu, Ali

2014-02-01

Loading of cryoprotectants into oocytes is an important step of the cryopreservation process, in which the cells are exposed to potentially damaging osmotic stresses and chemical toxicity. Thus, we investigated the use of physics-based mathematical optimization to guide design of cryoprotectant loading methods for mouse and human oocytes. We first examined loading of 1.5 M dimethyl sulfoxide (Me(2)SO) into mouse oocytes at 23°C. Conventional one-step loading resulted in rates of fertilization (34%) and embryonic development (60%) that were significantly lower than those of untreated controls (95% and 94%, respectively). In contrast, the mathematically optimized two-step method yielded much higher rates of fertilization (85%) and development (87%). To examine the causes for oocyte damage, we performed experiments to separate the effects of cell shrinkage and Me(2)SO exposure time, revealing that neither shrinkage nor Me(2)SO exposure single-handedly impairs the fertilization and development rates. Thus, damage during one-step Me(2)SO addition appears to result from interactions between the effects of Me(2)SO toxicity and osmotic stress. We also investigated Me(2)SO loading into mouse oocytes at 30°C. At this temperature, fertilization rates were again lower after one-step loading (8%) in comparison to mathematically optimized two-step loading (86%) and untreated controls (96%). Furthermore, our computer algorithm generated an effective strategy for reducing Me(2)SO exposure time, using hypotonic diluents for cryoprotectant solutions. With this technique, 1.5 M Me(2)SO was successfully loaded in only 2.5 min, with 92% fertilizability. Based on these promising results, we propose new methods to load cryoprotectants into human oocytes, designed using our mathematical optimization approach. Copyright © 2013 Elsevier Inc. All rights reserved.

Optimization of Cryoprotectant Loading into Murine and Human Oocytes

PubMed Central

Karlsson, Jens O.M.; Szurek, Edyta A.; Higgins, Adam Z.; Lee, Sang R.; Eroglu, Ali

2014-01-01

Loading of cryoprotectants into oocytes is an important step of the cryopreservation process, in which the cells are exposed to potentially damaging osmotic stresses and chemical toxicity. Thus, we investigated the use of physics-based mathematical optimization to guide design of cryoprotectant loading methods for mouse and human oocytes. We first examined loading of 1.5 M dimethylsulfoxide (Me2SO) into mouse oocytes at 23°C. Conventional one-step loading resulted in rates of fertilization (34%) and embryonic development (60%) that were significantly lower than those of untreated controls (95% and 94%, respectively). In contrast, the mathematically optimized two-step method yielded much higher rates of fertilization (85%) and development (87%). To examine the causes for oocyte damage, we performed experiments to separate the effects of cell shrinkage and Me2SO exposure time, revealing that neither shrinkage nor Me2SO exposure single-handedly impairs the fertilization and development rates. Thus, damage during one-step Me2SO addition appears to result from interactions between the effects of Me2SO toxicity and osmotic stress. We also investigated Me2SO loading into mouse oocytes at 30°C. At this temperature, fertilization rates were again lower after one-step loading (8%) in comparison to mathematically optimized two-step loading (86%) and untreated controls (96%). Furthermore, our computer algorithm generated an effective strategy for reducing Me2SO exposure time, using hypotonic diluents for cryoprotectant solutions. With this technique, 1.5 M Me2SO was successfully loaded in only 2.5 min, with 92% fertilizability. Based on these promising results, we propose new methods to load cryoprotectants into human oocytes, designed using our mathematical optimization approach. PMID:24246951

Mammalian nuclear transplantation to Germinal Vesicle stage Xenopus oocytes – A method for quantitative transcriptional reprogramming

PubMed Central

Halley-Stott, R.P.; Pasque, V.; Astrand, C.; Miyamoto, K.; Simeoni, I.; Jullien, J.; Gurdon, J.B.

2010-01-01

Full-grown Xenopus oocytes in first meiotic prophase contain an immensely enlarged nucleus, the Germinal Vesicle (GV), that can be injected with several hundred somatic cell nuclei. When the nuclei of mammalian somatic cells or cultured cell lines are injected into a GV, a wide range of genes that are not transcribed in the donor cells, including pluripotency genes, start to be transcriptionally activated, and synthesize primary transcripts continuously for several days. Because of the large size and abundance of Xenopus laevis oocytes, this experimental system offers an opportunity to understand the mechanisms by which somatic cell nuclei can be reprogrammed to transcribe genes characteristic of oocytes and early embryos. The use of mammalian nuclei ensures that there is no background of endogenous maternal transcripts of the kind that are induced. The induced gene transcription takes place in the absence of cell division or DNA synthesis and does not require protein synthesis. Here we summarize new as well as established results that characterize this experimental system. In particular, we describe optimal conditions for transplanting somatic nuclei to oocytes and for the efficient activation of transcription by transplanted nuclei. We make a quantitative determination of transcript numbers for pluripotency and housekeeping genes, comparing cultured somatic cell nuclei with those of embryonic stem cells. Surprisingly we find that the transcriptional activation of somatic nuclei differs substantially from one donor cell-type to another and in respect of different pluripotency genes. We also determine the efficiency of an injected mRNA translation into protein. PMID:20123126

Successful pregnancy and live birth from a hypogonadotropic hypogonadism woman with low serum estradiol concentrations despite numerous oocyte maturations: a case report.

PubMed

Matsumoto, Kaori; Imakawa, Kazuhiko; Hayashi, Chuyu

2017-09-20

The increase in serum estradiol (E 2 ) concentrations during the follicular phase becomes the index of oocyte maturation in vivo. When ovarian stimulation is performed to hypogonadotropic hypogonadism (HH) patients with only follicle stimulating hormone (FSH), proper increase in serum E 2 concentrations is not observed. Even if oocytes are obtained, which usually have low fertilization rate. In this report, we would like to present an unique case, in which under low E 2 concentrations and without luteinizing hormone (LH) administration, numerous mature oocytes could be obtained and a healthy baby delivered. During controlled ovarian stimulation (COS) with only recombinant follicular stimulating hormone (rFSH) administrations, a 26-year-old Japanese woman with hypothalamic amenorrhea (i.e., hypogonadotropic hypogonadism) developed numerous follicles despite low serum E 2 , 701 pg/ml, and high progesterone (P 4 ) concentrations, 2.11 ng/ml, on the day of induced ovulation. However, 33 cumulus-oocyte complexes (COCs) were successfully obtained; following the embryo culture, four early embryos and six blastocysts were cryopreserved. This patient received hormone replacement therapy (HRT), during which one of six cryopreserved blastocysts was thawed and transferred into the uterine lumen. The patient became pregnant from the first transfer, went through her pregnancy without any complications, and delivered a healthy male baby in the 39th week. Low E 2 concentrations in follicular fluids (FFs) are suggestive that aromatase and/or 17β-hydroxysteroid dehydrogenase (17β-HSD) could be low. Serum E 2 concentrations may not be the most important index for oocyte maturation during COS, and suggested that oocyte maturation was in progress even under low serum E 2 and high P 4 conditions. Even if serum E 2 concentrations did not properly increase, numerous mature oocytes could be obtained, resulting in the birth of a healthy baby.

Effect of milrinone on the developmental competence of growing lamb oocytes identified with brilliant cresyl blue.

PubMed

Wang, Liqin; Jiang, Xiangjiu; Wu, Yangsheng; Lin, Jiapeng; Zhang, Li; Yang, Nan; Huang, Juncheng

2016-11-01

Juvenile in vitro embryo transfer is a novel technique that can be used to increase the rate of genetic gain in a population and presents an alternative to embryo technologies on the basis of adult animals. However, oocytes from prepubertal animals have a lower viability than those obtained from adult ewe oocyte donors. In this research, we aimed to determine the optimum concentration and time of treatment of oocytes from prepubertal lambs with brilliant cresyl blue (BCB) stain and milrinone during IVM. This would improve the developmental rate of lamb oocytes and embryos after IVF. First, lamb cumulus-oocyte complexes were cultured under different concentrations (13 or 26 μM) of BCB staining. Treated lamb oocytes were then divided into BCB- (colorless cytoplasm) and BCB+ (colored cytoplasm) groups on the basis of their glucose-6-phosphate dehydrogenase activity. The blastocyst efficiency rate of BCB+ oocytes treated with 13 μM BCB (37.03%) was significantly higher than that of BCB+ oocytes treated with 26 μM BCB (23.25%) and that of nontreated BCB control oocytes (15.37%), as well as that of BCB- oocytes (6.28%). Both control oocytes and BCB+ oocytes exhibited significantly higher cleavage rates (60.15% and 73.44%, respectively) than that of BCB- oocytes (36.19%). Moreover, the diameter and glutathione content of BCB+ oocytes were found to be significantly greater than those of BCB- oocytes (163.37 vs. 159.25 μm and 6.39 vs. 0.26 pM, respectively). After culturing BCB- oocytes in different concentrations of milrinone (0, 50, 75, and 100 μM) for 3, 6, or 9 hours, results reported that supplementation of IVM medium with 75 μM milrinone for 6 hours yielded a significantly higher proportion of blastocysts than the other treatments. These results show that the staining of lamb cumulus-oocyte complexes with 13 μM BCB before IVM may be used to select developmentally competent lamb oocytes. Furthermore, they suggest that milrinone can be used to promote

Influence of follicular fluid and cumulus cells on oocyte quality: clinical implications.

PubMed

Da Broi, M G; Giorgi, V S I; Wang, F; Keefe, D L; Albertini, D; Navarro, P A

2018-03-02

An equilibrium needs to be established by the cellular and acellular components of the ovarian follicle if developmental competence is to be acquired by the oocyte. Both cumulus cells (CCs) and follicular fluid (FF) are critical determinants for oocyte quality. Understanding how CCs and FF influence oocyte quality in the presence of deleterious systemic or pelvic conditions may impact clinical decisions in the course of managing infertility. Given that the functional integrities of FF and CCs are susceptible to concurrent pathological conditions, it is important to understand how pathophysiological factors influence natural fertility and the outcomes of pregnancy arising from the use of assisted reproduction technologies (ARTs). Accordingly, this review discusses the roles of CCs and FF in ensuring oocyte competence and present new insights on pathological conditions that may interfere with oocyte quality by altering the intrafollicular environment.

Potassium salt microinjection into Xenopus oocytes mimics gonadotropin treatment.

PubMed

Lau, Y T; Yassin, R R; Horowitz, S B

1988-06-03

Gonadotropin stimulates protein synthesis and growth in ovarian oocytes. The hormone is also known to modify transfollicular K+ fluxes and is now shown to cause increased intraoocytic K+ activity (aK). The hormone's effect on aK was duplicated by microinjecting K+ salts into oocytes which were incubated in paraffin oil. This treatment mimicked the influence of gonadotropin on both the rate of protein synthesis and the synthesis of specific polypeptides. These findings suggest that gonadotropin-stimulated oocyte growth is attributable largely to the hormone's influence on transfollicular K+ fluxes. They support the hypothesis that the K+ flux and aK changes observed during cell activation are critical in causing subsequent increases in protein synthesis and growth.

Effect of potential oocyte transport protocols on blastocyst rates after intracytoplasmic sperm injection in the horse.

PubMed

Foss, R; Ortis, H; Hinrichs, K

2013-12-01

Intracytoplasmic sperm injection (ICSI) is used to produce foals from otherwise infertile mares and from stallions with limited sperm stores, but requires expensive equipment and is technically demanding. Methods to transport oocytes to ICSI laboratories would allow collection of oocytes by the referring veterinarian and enable greater application of this technique. This study was conducted to evaluate protocols that could be used to transport immature and maturing oocytes for ICSI. In vitro experiment. Oocytes were recovered by transvaginal ultrasound-guided follicular aspiration either from dominant follicles 24 h after deslorelin administration (dominant stimulated follicle [DSF]), or from subordinate (immature) follicles at the same time. To mimic transport, DSF oocytes were incubated overnight under differing conditions before ICSI; immature oocytes were placed in varying conditions overnight before in vitro maturation, followed by ICSI. The rate of blastocyst production was compared among treatments. Blastocysts were produced in all groups. Dominant stimulated follicle oocytes held in sealed tubes in pre-equilibrated control maturation medium maintained at 37°C yielded blastocyst development equal to that obtained for control incubated oocytes (70%). Dominant stimulated follicle oocytes held similarly in a warm passive device yielded poor blastocyst development (10%). Immature oocytes held for one or 2 nights in modified M199 medium, or for one night in commercial embryo holding solution, in air at room temperature, yielded 35-37% blastocyst development per injected oocyte. A commercially available medium can be used for shipping immature oocytes at room temperature with good resulting blastocyst rates. Better blastocyst rates per oocyte are obtained from DSF oocytes; however, these require maintenance at 37°C and as they are already maturing at the time of collection, are more sensitive to delays. This new, practical information supporting transport of

Ethical issues in paying for long-distance travel and accommodation expenses of oocyte donors.

PubMed

Heng, Boon Chin

2005-11-01

In many countries where the sale and purchase of donor oocytes is banned, a legal loophole often exploited is the use of free air tickets and hotel stay to entice prospective oocyte donors, in lieu of monetary payment. Such a means of procuring much-needed donor oocytes is ethically unsound. There is a lack of transparency and the personal motivation of the oocyte donor may be clouded by the desire for a 'free' holiday. Moreover, such a system is open to abuse by medical professionals. Private fertility clinics may source for oocyte donors to attract patients. The oocyte donor is paid nothing (except free travel and hotel stay), while the medical professional makes a handsome profit from treating infertile patients, which is not equitable. Medical professionals can also easily make a profit by marking up the price of air tickets and hotel stay to the patient (oocyte recipient). This would be thoroughly unprofessional, since the money earned is not directly related to the medical skills and expertise of the fertility specialist. Hence, it is imperative that various regulatory authorities should critically re-examine the giving of free travel and accommodation to oocyte donors, instead of monetary compensation.

Loss of TIGAR induces oxidative stress and meiotic defects in oocytes from obese mice.

PubMed

Wang, Haichao; Cheng, Qing; Li, Xiaoyan; Hu, Feifei; Han, Longsen; Zhang, Hao; Li, Ling; Ge, Juan; Ying, Xiaoyan; Guo, Xuejiang; Wang, Qiang

2018-05-18

Maternal obesity has been reported to impair oocyte quality in mice, however, the underlying mechanisms remain unclear. In the present study, by conducting a comparative proteomic analysis, we identified a reduced expression of TIGAR protein in ovulated oocytes from high-fat diet (HFD)-fed mice. Specific depletion of TIGAR in mouse oocytes results in the marked elevation of reactive oxygen species (ROS) levels and the failure of meiotic apparatus assembly. Importantly, forced expression of TIGAR in HFD oocytes not only attenuates ROS production, but also partly prevents spindle disorganization and chromosome misalignment during meiosis. Meantime, we noted that TIGAR knockdown in oocytes induces a strong activation of autophagy, while overexpression of TIGAR significantly reduces the LC3 accumulation in HFD oocytes. By anti-oxidant treatment, we further demonstrated that such an autophagic response is dependent on the TIGAR-controlled ROS production. In summary, our data indicate a role for TIGAR in modulating redox homeostasis during oocyte maturation, and uncover that loss of TIGAR is a critical pathway mediating the effects of maternal obesity on oocyte quality. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Evaluating methods of mouse euthanasia on the oocyte quality: cervical dislocation versus isoflurane inhalation.

PubMed

Roustan, Audrey; Perrin, Jeanne; Berthelot-Ricou, Anaïs; Lopez, Erica; Botta, Alain; Courbiere, Blandine

2012-04-01

Cervical dislocation is a commonly used method of mouse euthanasia. Euthanasia by isoflurane inhalation is an alternative method which allows the sacrifice of several mice at the same time with an anaesthesia, in the aim to decrease pain and animal distress. The objective of our study was to assess the impact of these two methods of euthanasia on the quality of mouse oocytes. By administering gonadotropins, we induced a superovulation in CD1 female mice. Mice were randomly assigned to euthanasia with cervical dislocation and isoflurane inhalation. Oviducts were collected and excised to retrieve metaphase II oocytes. After microscopic examination, oocytes were classified into three groups: intact, fragmented/cleaved and atretic. Intact metaphase II oocytes were employed for biomedical research. A total of 1442 oocytes in the cervical dislocation group were compared with 1230 oocytes in the isoflurane group. In the cervical dislocation group, 93.1% of the oocytes were intact, versus 65.8% in the isoflurane group (P ≤ 0.001). In light of these results, we conclude that cervical dislocation is the best method of mouse euthanasia for obtaining intact oocytes for biomedical research.

RNA-Seq transcriptome profiling of mouse oocytes after in vitro maturation and/or vitrification.

PubMed

Gao, Lei; Jia, Gongxue; Li, Ai; Ma, Haojia; Huang, Zhengyuan; Zhu, Shien; Hou, Yunpeng; Fu, Xiangwei

2017-10-16

In vitro maturation (IVM) and vitrification have been widely used to prepare oocytes before fertilization; however, potential effects of these procedures, such as expression profile changes, are poorly understood. In this study, mouse oocytes were divided into four groups and subjected to combinations of in vitro maturation and/or vitrification treatments. RNA-seq and in silico pathway analysis were used to identify differentially expressed genes (DEGs) that may be involved in oocyte viability after in vitro maturation and/or vitrification. Our results showed that 1) 69 genes were differentially expressed after IVM, 66 of which were up-regulated. Atp5e and Atp5o were enriched in the most significant gene ontology term "mitochondrial membrane part"; thus, these genes may be promising candidate biomarkers for oocyte viability after IVM. 2) The influence of vitrification on the transcriptome of oocytes was negligible, as no DEGs were found between vitrified and fresh oocytes. 3) The MII stage is more suitable for oocyte vitrification with respect to the transcriptome. This study provides a valuable new theoretical basis to further improve the efficiency of in vitro maturation and/or oocyte vitrification.

Efficiency and kinetics of the in vitro fertilization process in bovine oocytes with different meiotic competence.

PubMed

Horakova, J; Hanzalova, K; Reckova, Z; Hulinska, P; Machatkova, M

2007-08-01

The aim of the study was to investigate the efficiency and kinetics of fertilization in oocytes with different meiotic competence, as defined by the phase of the follicular wave and follicle size. Oocytes were recovered from cows with synchronized estrus cycles, slaughtered in either the growth (day 3) or the dominant (day 7) phase, separately from large, medium and small follicles. The oocytes were matured and fertilized by a standard protocol. Twenty-four hours after fertilization, the oocytes were denuded from cumulus cells, fixed and stained with bisbensimid Hoechst-PBS. Fertilization was more efficient and the first cleavage was accelerated in growth phase-derived oocytes, as shown by significantly higher (p < or = 0.01) proportions of both normally fertilized and cleaved oocytes (68.8 and 25.1%), in comparison with dominant phase-derived oocytes (44.2 and 10.3%). In the growth-phase derived oocytes, proportions of normally fertilized and cleaved oocytes were significantly higher (p < or = 0.01) in oocytes from large (100.0 and 36.4%) and medium (83.3 and 36.5%) follicles than in those from small (54.8 and 14.6%) follicles. The dominant phase-derived oocytes showed higher proportions of normally fertilized and cleaved oocytes in the populations recovered from small (51.5 and 10.0%) and medium (43.1 and 12.0%) follicles than in those from large (25.0 and 0%) follicles; however, the differences were not significant. It can be concluded that: (i) efficiency and kinetics of fertilization differ in relation to oocyte's meiotic competence; (ii) improved development of embryos from oocytes with greater meiotic competence is associated with a more effective fertilization process.

Genetics and imaging to assess oocyte and preimplantation embryo health.

PubMed

Warner, C M; Newmark, J A; Comiskey, M; De Fazio, S R; O'Malley, D M; Rajadhyaksha, M; Townsend, D J; McKnight, S; Roysam, B; Dwyer, P J; DiMarzio, C A

2004-01-01

Two major criteria are currently used in human assisted reproductive technologies (ART) to evaluate oocyte and preimplantation embryo health: (1) rate of preimplantation embryonic development; and (2) overall morphology. A major gene that regulates the rate of preimplantation development is the preimplantation embryo development (Ped) gene, discovered in our laboratory. In mice, presence of the Ped gene product, Qa-2 protein, results in a fast rate of preimplantation embryonic development, compared with a slow rate of preimplantation embryonic development for embryos that are lacking Qa-2 protein. Moreover, mice that express Qa-2 protein have an overall reproductive advantage that extends beyond the preimplantation period, including higher survival to birth, higher birthweight, and higher survival to weaning. Data are presented that suggest that Qa-2 increases the rate of development of early embryos by acting as a cell-signalling molecule and that phosphatidylinositol-32 kinase is involved in the cell-signalling pathway. The most likely human homologue of Qa-2 has recently been identified as human leukocyte antigen (HLA)-G. Data are presented which show that HLA-G, like Qa-2, is located in lipid rafts, implying that HLA-G also acts as a signalling molecule. In order to better evaluate the second criterion used in ART (i.e. overall morphology), a unique and innovative imaging microscope has been constructed, the Keck 3-D fusion microscope (Keck 3DFM). The Keck 3DFM combines five different microscopic modes into a single platform, allowing multi-modal imaging of the specimen. One of the modes, the quadrature tomographic microscope (QTM), creates digital images of non-stained transparent cells by measuring changes in the index of refraction. Quadrature tomographic microscope images of oocytes and preimplantation mouse embryos are presented for the first time. The digital information from the QTM images should allow the number of cells in a preimplantation embryo to

Patterns of oocyte development in natural habitat and captive Salminus hilarii Valenciennes, 1850 (Teleostei: Characidae).

PubMed

Honji, R M; Narcizo, A M; Borella, M I; Romagosa, E; Moreira, R G

2009-03-01

Fecundity and oocyte development in Salminus hilarii female brood stock were analyzed with the aim of investigating the impact of migration impediment on oogenesis. Histological analyses of the ovaries were performed in adult females caught in two different environments--the Tietê River (natural) and captivity--and the gonadossomatic index, oocyte diameter and fecundity determined. Five germ cell development stages (oogonium, perinucleolar, cortical alveoli, vitellogenic, ripe) and two other structures (postovulatory follicles and atretic oocytes) were observed in females caught in the river. Captive animals lacked the ripe oocytes and postovulatory follicles and had a relatively higher number of atretic oocytes. Females in captivity are known to produce larger oocytes, and they release fewer eggs in each spawn (absolute fecundity) when compared with animals that are able to migrate. Our results suggest that the Tietê River is undergoing alterations which are being reflected in the reproductive performance of S. hilarii, mainly due to the presence of atretic oocytes in females caught in the river. The lack of postovulatory follicles and ripe oocytes in captive animals reveals that migratory impediment negatively impacts final oocyte maturation. However, the stage of maturation reached is adequate for ovulation induction with hormone manipulation.

Merotelic kinetochore attachment in oocyte meiosis II causes sister chromatids segregation errors in aged mice.

PubMed

Cheng, Jin-Mei; Li, Jian; Tang, Ji-Xin; Hao, Xiao-Xia; Wang, Zhi-Peng; Sun, Tie-Cheng; Wang, Xiu-Xia; Zhang, Yan; Chen, Su-Ren; Liu, Yi-Xun

2017-08-03

Mammalian oocyte chromosomes undergo 2 meiotic divisions to generate haploid gametes. The frequency of chromosome segregation errors during meiosis I increase with age. However, little attention has been paid to the question of how aging affects sister chromatid segregation during oocyte meiosis II. More importantly, how aneuploid metaphase II (MII) oocytes from aged mice evade the spindle assembly checkpoint (SAC) mechanism to complete later meiosis II to form aneuploid embryos remains unknown. Here, we report that MII oocytes from naturally aged mice exhibited substantial errors in chromosome arrangement and configuration compared with young MII oocytes. Interestingly, these errors in aged oocytes had no impact on anaphase II onset and completion as well as 2-cell formation after parthenogenetic activation. Further study found that merotelic kinetochore attachment occurred more frequently and could stabilize the kinetochore-microtubule interaction to ensure SAC inactivation and anaphase II onset in aged MII oocytes. This orientation could persist largely during anaphase II in aged oocytes, leading to severe chromosome lagging and trailing as well as delay of anaphase II completion. Therefore, merotelic kinetochore attachment in oocyte meiosis II exacerbates age-related genetic instability and is a key source of age-dependent embryo aneuploidy and dysplasia.

Ultrastructural analysis of oocytes of Rhipicephalus (Boophilus) annulatus during postengorgement period as a tool to evaluate the cytotoxic effects of amitraz and deltamethrin on the germinative cells.

PubMed

Sreelekha, Kanapadinchareveetil; Chandrasekhar, Leena; Kartha, Harikumar S; Ravindran, Reghu; Juliet, Sanis; Ajithkumar, Karapparambu G; Nair, Suresh N; Ghosh, Srikanta

2017-11-30

The present study utilizes the ultrastructural analysis of the fully engorged female Rhipicephalus (Boophilus) annulatus ticks, as a tool to evaluate the cytotoxic potential of deltamethrin and amitraz on the germinative cells. The ultrastructural analysis of the ovary of the normal (untreated) R (B.) annulatus revealed, oocytes in different stages of development, attached to the ovary wall by pedicel cells. The attachment site of oocyte to the pedicel cell was characterized by indentations of the plasma membrane. The oocyte was bound by three cell membranes viz., plasma membrane, chorion and basal lamina. The stages of oocytes were differentiated ultrastructurally based on the features of their outer membrane and the number and size of lipid and yolk droplets. Detailed day wise analysis of ultrastructural changes in the ovary during the post-engorgement period revealed the occurrence of the degenerative changes from day five onwards. These appeared first in the oocytes followed by the germinal epithelium. The ovary of ticks treated with methanol (control), revealed similar topographies as that of a normal ovary except for the presence of very few oocytes with ring shaped nucleoli. Ultrastructurally, treatment with deltamethrin produced more prominent and extensive morphological alterations when compared to amitraz. In the case of ticks treated with amitraz, the oocytes of stage IV and V showed wavy and disrupted outer boundaries along with the loss of integrity of the yolk droplets. Uneven nuclear membranes of stage II oocytes and cristolysis of mitochondria of mature oocytes were the other changes noticed. Ticks treated with deltamethrin revealed prominent modifications such as, detachment of the basal lamina, wrinkled boundary, inconsistent nuclear membrane, ring shaped nucleoli and chromatin clumping in the case of the early stage oocytes (I and II), whereas swelling and cristolysis of mitochondria were seen in mature oocytes. The study further indicated that

Pronuclear formation by ICSI using chemically activated ovine oocytes and zona pellucida bound sperm.

PubMed

Hernández-Pichardo, J E; Ducolomb, Y; Romo, S; Kjelland, M E; Fierro, R; Casillas, F; Betancourt, M

2016-01-01

In order to improve ICSI, appropiate sperm selection and oocyte activation is necessary. The objective of the present study was to determine the efficiency of fertilization using ICSI with chemically activated ovine oocytes and sperm selected by swim up (SU) or swim up + zona pellucida (SU + ZP) binding. Experiment 1, 4-20 replicates with total 821 in vitro matured oocytes were chemically activated with ethanol, calcium ionophore or ionomycin, to determine oocyte activation (precense of one PN). Treatments showed similar results (54, 47, 42 %, respectively) but statistically differents ( P  < 0.05) than mechanical activated oocytes in sham, ICSI and sham injection (13, 25, 32 %, respectively) (10-17 replicates; n  = 429). Experiment 2: Twelve ejaculates and 28 straws of semen were used (11-19 replicates). Sperm were selected by SU in BSA-TCM 199-H medium. A total of 2,294 fresh sperm and 2,760 from frozen-thawed semen were analyzed after SU or SU + ZP binding. Fresh sperm selected by SU showed acrosome reaction (AR) of 59 %, the sperm selected by SU + ZP binding increased AR to 91 %. In comparison, the AR of frozen-thawed sperm using SU or SU + ZP binding was 77 and 86 %, respectively ( P  < 0.05). Experiment 3: fertilization in 200 mechanical activativated oocytes (17 replicates) was 4 %, but fertilization increased in ethanol activated oocytes after ICSI (12-28 %) (5-6 replicates). When fresh sperm only selected by SU were injected to 123 oocytes, a fertilization rate (28 %) was achieved; in sperm selected by SU + ZP was 25 % (73 oocytes). In comparison, in frozen-thawed sperm selected by SU, fertilization was 13 % (70 oocytes), whereas sperm from SU + ZP binding displayed 12 % (51 oocytes) ( P  > 0.05). Chemical activation induces higher ovine oocyte activation than mechanical activation. Ethanol slightly displays higher oocyte activation than calcium ionophore and ionomicine. Sperm selection with SU�

Application of auxin-inducible degron technology to mouse oocyte activation with PLCζ.

PubMed

Miura, Kento; Matoba, Shogo; Ogonuki, Narumi; Namiki, Takafumi; Ito, Junya; Kashiwazaki, Naomi; Ogura, Atsuo

2018-05-05

In mammals, spermatozoa activate oocytes by triggering a series of intracellular Ca 2+ oscillations with phospholipase C zeta (PLCζ), a sperm-borne oocyte-activating factor. Because the introduction of PLCζ alone can induce oocyte activation, it might be a promising reagent for assisted reproductive technologies. To test this possibility, we injected human PLCζ (hPLCζ) mRNA into mouse oocytes at different concentrations. We observed the oocyte activation and subsequent embryonic development. Efficient oocyte activation and embryonic development to the blastocyst stage was achieved only with a limited range of mRNA concentrations (0.1 ng/μl). Higher concentrations of mRNA caused developmental arrest of most embryos, suggesting that excessive PLCζ protein might be harmful at this stage. In a second series of experiments, we aimed to regulate the PLCζ protein concentration in oocytes by applying auxin-inducible degron (AID) technology that allows rapid degradation of the target protein tagged with AID induced by auxin. Injection of the hPLCζ protein tagged with AID and enhanced green fluorescent protein (hPLCζ-AID-EGFP) demonstrated that high EGFP expression levels at the late 1-cell stage were efficiently reduced by auxin treatment, suggesting efficient hPLCζ degradation by this system. Furthermore, the defective development observed with higher concentrations of hPLCζ-AID-EGFP mRNA was rescued following auxin treatment. Full-term offspring were obtained by round spermatid injection with optimized hPLCζ-AID activation. Our results indicate that this AID technology can be applied to regulate the protein levels in mouse oocytes and that our optimized PLCζ system could be used for assisted fertilization in mammals.

Donor motivations, associated risks and ethical considerations of oocyte donation.

PubMed

Boutelle, Amy L

2014-01-01

Three decades after the first reported successful cases, oocyte donation continues to grow in popularity and regard as an established method to aid women in achieving their reproductive goals. As a result of the increased demand for donated oocytes, many young women in the U.S. volunteer to undergo complex medical procedures to donate their oocytes in return for financial compensation. To best care for these women before, during and after donation, it is important to explore donor characteristics and motivations, discuss the safety of the donation procedure and examine the ethical issues related to this process. © 2014 AWHONN.

Two Dimensional Finite Element Model to Study Calcium Distribution in Oocytes

NASA Astrophysics Data System (ADS)

Naik, Parvaiz Ahmad; Pardasani, Kamal Raj

2015-06-01

Cytosolic free calcium concentration is a key regulatory factor and perhaps the most widely used means of controlling cellular function. Calcium can enter cells through different pathways which are activated by specific stimuli including membrane depolarization, chemical signals and calcium depletion of intracellular stores. One of the important components of oocyte maturation is differentiation of the Ca2+ signaling machinery which is essential for egg activation after fertilization. Eggs acquire the ability to produce the fertilization-specific calcium signal during oocyte maturation. The calcium concentration patterns required during different stages of oocyte maturation are still not completely known. Also the mechanisms involved in calcium dynamics in oocyte cell are still not well understood. In view of above a two dimensional FEM model has been proposed to study calcium distribution in an oocyte cell. The parameters such as buffers, ryanodine receptor, SERCA pump and voltage gated calcium channel are incorporated in the model. Based on the biophysical conditions the initial and boundary conditions have been framed. The model is transformed into variational form and Ritz finite element method has been employed to obtain the solution. A program has been developed in MATLAB 7.10 for the entire problem and executed to obtain numerical results. The numerical results have been used to study the effect of buffers, RyR, SERCA pump and VGCC on calcium distribution in an oocyte cell.

Production of giant mouse oocyte nucleoli and assessment of their protein content.

PubMed

Fulka, Helena; Martinkova, Stanislava; Kyogoku, Hirohisa; Langerova, Alena; Fulka, Josef

2012-01-01

Compared with advanced developmental stage embryos and somatic cells, fully grown mammalian oocytes contain specific nucleolus-like structures (NPB - nucleolus precursor bodies). It is commonly accepted that they serve as a store of material(s) from which typical nucleoli are gradually formed. Whilst nucleoli from somatic cells can be collected relatively easily for further biochemical analyses, a sufficient number of oocyte nucleoli is very difficult to obtain. We have found that isolated oocytes nucleoli fuse very efficiently when contact is established between them. Thus, well visible giant nucleoli can be obtained, relatively easily handled and then used for further biochemical analyses. With the use of colloidal gold staining, we estimated that a single fully grown mouse oocyte nucleolus contains approximately 1.6 ng of protein. We do believe that this approach will accelerate further research aiming at analyzing the composition of oocyte nucleoli in more detail.

Oocyte mitochondrial deletions and heteroplasmy in a bovine model of ageing and ovarian stimulation.

PubMed

Hammond, Elizabeth R; Green, Mark P; Shelling, Andrew N; Berg, Martin C; Peek, John C; Cree, Lynsey M

2016-04-01

Maternal ageing and ovarian stimulation result in the accumulation of mitochondrial DNA (mtDNA) deletions and heteroplasmy in individual oocytes from a novel bovine model for human assisted reproductive technology (ART). The levels of mtDNA deletions detected in oocytes increased with ovarian ageing. Low levels of mtDNA heteroplasmy were apparent across oocytes and no relationship was identified with respect to ovarian ageing or ovarian stimulation. Oocyte quality decreases with ovarian ageing and it is postulated that the mtDNA may have a role in this decline. The impact of ovarian stimulation on oocyte quality is poorly understood. Human studies investigating these effects are often limited by the use of low quality oocytes and embryos, variation in age and ovarian stimulation regimens within the patients studied, as well as genetic and environmental variability. Further, no study has investigated mtDNA heteroplasmy in individual oocytes using next-generation sequencing (NGS), and little is known about whether the oocyte accumulates heteroplasmic mtDNA mutations following ageing or ovarian stimulation. A novel bovine model for the effect of stimulation and age in human ART was undertaken using cows generated by somatic cell nuclear transfer (SCNT) from one founder, to produce a homogeneous population with reduced genetic and environmental variability. Oocytes and somatic tissues were collected from young (3 years of age; n = 4 females) and old (10 years of age; n = 5 females) cow clones following multiple natural ovarian cycles, as well as oocytes following multiple mild (FSH only) and standard (based on human a long GnRH agonist protocol) ovarian stimulation cycles. In addition, oocytes were recovered in a natural cycle from naturally conceived cows aged 4-13.5 years (n = 10) to provide a heterogeneous cohort for mtDNA deletion studies. The presence or absence of mtDNA deletions were investigated using long-range PCR in individual oocytes (n = 62). To determine

Activin Decoy Receptor ActRIIB:Fc Lowers FSH and Therapeutically Restores Oocyte Yield, Prevents Oocyte Chromosome Misalignments and Spindle Aberrations, and Increases Fertility in Midlife Female SAMP8 Mice.

PubMed

Bernstein, Lori R; Mackenzie, Amelia C L; Lee, Se-Jin; Chaffin, Charles L; Merchenthaler, István

2016-03-01

Women of advanced maternal age (AMA) (age ≥ 35) have increased rates of infertility, miscarriages, and trisomic pregnancies. Collectively these conditions are called "egg infertility." A root cause of egg infertility is increased rates of oocyte aneuploidy with age. AMA women often have elevated endogenous FSH. Female senescence-accelerated mouse-prone-8 (SAMP8) has increased rates of oocyte spindle aberrations, diminished fertility, and rising endogenous FSH with age. We hypothesize that elevated FSH during the oocyte's FSH-responsive growth period is a cause of abnormalities in the meiotic spindle. We report that eggs from SAMP8 mice treated with equine chorionic gonadotropin (eCG) for the period of oocyte growth have increased chromosome and spindle misalignments. Activin is a molecule that raises FSH, and ActRIIB:Fc is an activin decoy receptor that binds and sequesters activin. We report that ActRIIB:Fc treatment of midlife SAMP8 mice for the duration of oocyte growth lowers FSH, prevents egg chromosome and spindle misalignments, and increases litter sizes. AMA patients can also have poor responsiveness to FSH stimulation. We report that although eCG lowers yields of viable oocytes, ActRIIB:Fc increases yields of viable oocytes. ActRIIB:Fc and eCG cotreatment markedly reduces yields of viable oocytes. These data are consistent with the hypothesis that elevated FSH contributes to egg aneuploidy, declining fertility, and poor ovarian response and that ActRIIB:Fc can prevent egg aneuploidy, increase fertility, and improve ovarian response. Future studies will continue to examine whether ActRIIB:Fc works via FSH and/or other pathways and whether ActRIIB:Fc can prevent aneuploidy, increase fertility, and improve stimulation responsiveness in AMA women.

Contributions of UBE2C and UBE2S to meiotic progression of porcine oocytes.

PubMed

Fujioka, Yoshie A; Onuma, Asuka; Fujii, Wataru; Sugiura, Koji; Naito, Kunihiko

2018-06-22

Vertebrate oocytes arrested at the first meiotic prophase must proceed to the second meiotic metaphase (MII) before fertilization. This meiotic process requires the precise control of protein degradation. Part of the protein degradation in oocytes is controlled by members of the ubiquitin-conjugating enzyme family, UBE2C and UBE2S, which are known to participate in mono-ubiquitination and poly-ubiquitination, respectively. Although UBE2 enzymes have been well studied in mitosis, their contribution to mammalian oocyte meiosis is relatively unknown and has been studied only in mice. Here, we investigated the contribution of UBE2C and UBE2S to porcine oocyte maturation using an RNA injection method. Overexpression of UBE2S prevented MII arrest of oocytes and led to the formation of a pronucleus (PN) at 48 h of culture. This effect was also observed for prolonged cultures of UBE2C-overexpressing oocytes, suggesting the effectiveness of poly-ubiquitination in the rapid escape from M-phase in porcine oocytes. Although the inhibition of either UBE2C or UBE2S by antisense RNA (asRNA) injection had no effect on oocyte maturation, asRNA-injected oocytes showed inhibited PN formation after parthenogenetic activation. These results indicated that ubiquitination of certain factors by UBE2S and UBE2C plays a role in the escape from MII arrest in porcine oocytes. Further investigations to identify the factors and how mono- and/or poly-ubiquitination contributes to protein degradation could provide a better understanding of UBE2 roles in oocyte maturation.

The polarized distribution of poly(A+)-mRNA-induced functional ion channels in the Xenopus oocyte plasma membrane is prevented by anticytoskeletal drugs.

PubMed

Peter, A B; Schittny, J C; Niggli, V; Reuter, H; Sigel, E

1991-08-01

Foreign mRNA was expressed in Xenopus laevis oocytes. Newly expressed ion currents localized in defined plasma membrane areas were measured using the two-electrode voltage clamp technique in combination with a specially designed chamber, that exposed only part of the surface on the oocytes to channel agonists or inhibitors. Newly expressed currents were found to be unequally distributed in the surface membrane of the oocyte. This asymmetry was most pronounced during the early phase of expression, when channels could almost exclusively be detected in the animal hemisphere of the oocyte. 4 d after injection of the mRNA, or later, channels could be found at a threefold higher density at the animal than at the vegetal pole area. The pattern of distribution was observed to be similar with various ion channels expressed from crude tissue mRNA and from cRNAs coding for rat GABAA receptor channel subunits. Electron microscopical analysis revealed very similar microvilli patterns at both oocyte pole areas. Thus, the asymmetric current distribution is not due to asymmetric surface structure. Upon incubation during the expression period in either colchicine or cytochalasin D, the current density was found to be equal in both pole areas. The inactive control substance beta-lumicolchicine had no effect on the asymmetry of distribution. Colchicine was without effect on the amplitude of the expressed whole cell current. Our measurements reveal a pathway for plasma membrane protein expression endogenous to the Xenopus oocyte, that may contribute to the formation and maintenance of polarity of this highly organized cell.

Molecular and cellular mechanisms of sperm-oocyte interactions opinions relative to in vitro fertilization (IVF).

PubMed

Anifandis, George; Messini, Christina; Dafopoulos, Konstantinos; Sotiriou, Sotiris; Messinis, Ioannis

2014-07-22

One of the biggest prerequisites for pregnancy is the fertilization step, where a human haploid spermatozoon interacts and penetrates one haploid oocyte in order to produce the diploid zygote. Although fertilization is defined by the presence of two pronuclei and the extraction of the second polar body the process itself requires preparation of both gametes for fertilization to take place at a specific time. These preparations include a number of consecutive biochemical and molecular events with the help of specific molecules and with the consequential interaction between the two gametes. These events take place at three different levels and in a precise order, where the moving spermatozoon penetrates (a) the outer vestments of the oocyte, known as the cumulus cell layer; (b) the zona pellucida (ZP); where exocytosis of the acrosome contents take place and (c) direct interaction of the spermatozoon with the plasma membrane of the oocyte, which involves a firm adhesion of the head of the spermatozoon with the oocyte plasma membrane that culminates with the fusion of both sperm and oocyte membranes (Part I). After the above interactions, a cascade of molecular signal transductions is initiated which results in oocyte activation. Soon after the entry of the first spermatozoon into the oocyte and oocyte activation, the oocyte's coat (the ZP) and the oocyte's plasma membrane seem to change quickly in order to initiate a fast block to a second spermatozoon (Part II). Sometimes, two spermatozoa fuse with one oocyte, an incidence of 1%-2%, resulting in polyploid fetuses that account for up to 10%-20% of spontaneously aborted human conceptuses. The present review aims to focus on the first part of the human sperm and oocyte interactions, emphasizing the latest molecular and cellular mechanisms controlling this process.

Vitrification: an effective new approach to oocyte banking and preserving fertility in cancer patients.

PubMed

Cobo, A; Domingo, J; Pérez, S; Crespo, J; Remohí, J; Pellicer, A

2008-05-01

Oocyte cryopreservation is a useful tool for preserving the fertility of cancer patients at risk of losing ovarian function due to undergoing potentially sterilising therapies. Results obtained with different cryopreservation protocols have been disappointing, particularly those obtained with slow cooling procedures. The efficacy of vitrification as an application in clinical practice has recently been demonstrated. The aim of this study is to report results obtained with the Cryotop method of oocyte vitrification in a population of healthy women and to point out its potential usefulness for fertility preservation in oncological patients. The study population consisting of non-oncological patients included 47 oocyte donors and 57 recipients undergoing an oocyte donation cycle of assisted reproductive technology (ART). A total of 693 mature metaphase II oocytes were collected following ovarian stimulation using long protocol down-regulation plus gonadotropin administration. Vitrification was carried out by means of the Cryotop method. Oocytes were donated to a compatible recipient after endometrial preparation. Of the 693 oocytes, 666 (96.1%) survived. A total of 487 (73.1%) were fertilised successfully. One hundred and seventeen embryos were transferred to 57 recipients. Pregnancy rate per transfer and implantation rates were 63.2% and 38.5% respectively. Twenty-eight healthy babies were later born. Oocyte cryo-banking by means of the Cryotop vitrification method represents a viable option for healthy women, producing excellent survival rates and a clinical outcome similar to that obtained with fresh oocytes. This approach could potentially be used in cancer patients who want to safeguard their fertility. Cancer patients could potentially benefit from this approach by storing their oocytes before the onset of the oncological therapy.

Participation of inositol trisphosphate and ryanodine receptors in Bufo arenarum oocyte activation.

PubMed

Ajmat, M T; Bonilla, F; Zelarayán, L; Bühler, M I

2011-05-01

Calcium is considered the most important second messenger at fertilization. Transient release from intracellular stores is modulated through both agonist-gated channels, IP₃Rs and RyRs, which can be found individually or together depending on the oocyte species. Using the four commonly used compounds (thimerosal, caffeine, heparin and ruthenium red), we investigated the existence and interdependence of both IP₃Rs and RyRs in mature Bufo arenarum oocytes. We found that caffeine, a well known specific RyRs agonist, was able to trigger oocyte activation in a dose-dependent manner. Microinjection of 10 mM caffeine showed 100% of oocytes exhibiting characteristic morphological criteria of egg activation. Ruthenium red, the specific RyR blocker, was able to inhibit oocyte activation induced either by sperm or caffeine. Our present findings provide the first reported evidence of the existence of RyR in frogs. We further explored the relationship between IP₃Rs and RyRs in B. arenarum oocytes by exposing them to the agonists of one class after injecting a blocker of the other class of receptor. We found that thimerosal overcame the inhibitory effect of RyR on oocyte activation, indicating that IP₃Rs function as independent receptors. In contrast, previous injection of heparin delayed caffeine-induced calcium release, revealing a relative dependence of RyRs on functional IP₃Rs, probably through a CICR mechanism. Both receptors play a role in Ca²+ release mechanisms although their relative contribution to the activation process is unclear.

A Simplified Method for Three-Dimensional (3-D) Ovarian Tissue Culture Yielding Oocytes Competent to Produce Full-Term Offspring in Mice

PubMed Central

Higuchi, Carolyn M.; Maeda, Yuuki; Horiuchi, Toshitaka; Yamazaki, Yukiko

2015-01-01

In vitro growth of follicles is a promising technology to generate large quantities of competent oocytes from immature follicles and could expand the potential of assisted reproductive technologies (ART). Isolated follicle culture is currently the primary method used to develop and mature follicles in vitro. However, this procedure typically requires complicated, time-consuming procedures, as well as destruction of the normal ovarian microenvironment. Here we describe a simplified 3-D ovarian culture system that can be used to mature multilayered secondary follicles into antral follicles, generating developmentally competent oocytes in vitro. Ovaries recovered from mice at 14 days of age were cut into 8 pieces and placed onto a thick Matrigel drop (3-D culture) for 10 days of culture. As a control, ovarian pieces were cultured on a membrane filter without any Matrigel drop (Membrane culture). We also evaluated the effect of activin A treatment on follicle growth within the ovarian pieces with or without Matrigel support. Thus we tested four different culture conditions: C (Membrane/activin-), A (Membrane/activin+), M (Matrigel/activin-), and M+A (Matrigel/activin+). We found that the cultured follicles and oocytes steadily increased in size regardless of the culture condition used. However, antral cavity formation occurred only in the follicles grown in the 3-D culture system (M, M+A). Following ovarian tissue culture, full-grown GV oocytes were isolated from the larger follicles to evaluate their developmental competence by subjecting them to in vitro maturation (IVM) and in vitro fertilization (IVF). Maturation and fertilization rates were higher using oocytes grown in 3-D culture (M, M+A) than with those grown in membrane culture (C, A). In particular, activin A treatment further improved 3-D culture (M+A) success. Following IVF, two-cell embryos were transferred to recipients to generate full-term offspring. In summary, this simple and easy 3-D ovarian culture

A morphologic study of unfertilized oocytes and abnormal embryos in human in vitro fertilization.

PubMed

Bałakier, H; Casper, R F

1991-04-01

The morphology of human, unfertilized oocytes and abnormal embryos cultured in vitro for 48-72 hr was examined in an attempt to learn more about oocyte maturation and reproductive failure in in vitro fertilization (IVF). About 21% of the unfertilized oocytes were totally degenerated. The majority (56%) of the remaining oocytes was arrested at the metaphase II stage. They contained coherent chromosomal plates and had extruded the first polar body with nuclear material. About 13% of oocytes underwent spontaneous activation. In most of these cases the second polar body was retained and many subnuclei or one big nucleus was formed. Five percent of metaphase II oocytes penetrated by sperm were not activated, likely as a result of oocyte immaturity. The developmental ability of abnormal embryos was poor. Several one-cell-stage zygotes were arrested at the pronuclear stage or at mitosis of the first mitotic division. Polyspermic embryos, especially those which contained four or more pronuclei, did not divide or formed uneven, multinucleated blastomeres. However, some triploid and tetraploid embryos often appeared normal morphologically despite their lethal chromosomal abnormalities.

Effect of sericin supplementation in maturation medium on cumulus cell expansion, oocyte nuclear maturation, and subsequent embryo development in Sanjabi ewes during the breeding season.

PubMed

Aghaz, F; Hajarian, H; Shabankareh, H Karami; Abdolmohammadi, A

2015-12-01

The purpose of this study was to evaluate the effect of sericin with different concentrations (0% [control], 0.1%, 0.5%, 1.0%, and 2.5%) added to the IVM medium on cumulus cell expansion, oocyte nuclear maturation, and subsequent embryo development in Sanjabi ewes during the breeding season. The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown and the first polar body extrusion. After IVF with fresh ram semen, presumptive zygotes were cultured 8 days in potassium simplex optimization medium supplemented by amino acids, and the percentages developing to the two-cell and blastocyst stages were measured as the indicators of early embryonic developmental competence. More cumulus-oocyte complexes matured with 0.5% sericin underwent germinal vesicle breakdown and reached metaphase II stage compared with the control cumulus-oocyte complexes matured without sericin (P ≤ 0.05). The present findings indicated that supplementation with 0.5% sericin during the maturation culture may improve the nuclear maturation and the cumulus cell expansion. Furthermore, the percentage of blastocysts obtained from 0.5% and 0.1% sericin (37.8 ± 1.76% and 34.8 ± 1.09%, respectively) was higher (P ≤ 0.05) than that of the control medium (29.60 ± 1.67%). However, addition of 1% and 2.5% of sericin to the IVM medium oocytes had a negative effect on nuclear maturation and cumulus cell expansion. Furthermore, the percentage of cleavage and blastocyst rate was significantly lower in the 1% and 2.5% sericin groups than in the control group. These findings showed that supplementation of IVM medium with 0.5% sericin may improve the meiotic competence of oocytes and early embryonic development in Sanjabi ewes during the breeding season. Copyright © 2015 Elsevier Inc. All rights reserved.

Promoting pedagogical content knowledge development for early career secondary teachers in science and technology using content representations

NASA Astrophysics Data System (ADS)

Williams, John; Eames, Chris; Hume, Anne; Lockley, John

2012-11-01

Background: This research addressed the key area of early career teacher education and aimed to explore the use of a 'content representation' (CoRe) as a mediational tool to develop early career secondary teacher pedagogical content knowledge (PCK). This study was situated in the subject areas of science and technology, where sound teacher knowledge is particularly important to student engagement. Purpose: The study was designed to examine whether such a tool (a CoRe), co-designed by an early career secondary teacher with expert content and pedagogy specialists, can enhance the PCK of early career teachers. The research questions were: How can experts in content and pedagogy work together with early career teachers to develop one science topic CoRe and one technology topic CoRe to support the development of PCK for early career secondary teachers? How does the use of a collaboratively designed CoRe affect the planning of an early career secondary teacher in science or technology? How has engagement in the development and use of an expert-informed CoRe developed an early career teacher's PCK? Sample: The research design incorporated a unique partnership between two expert classroom teachers, two content experts, four early career teachers, and four researchers experienced in science and technology education. Design: This study employed an interpretivist-based methodology and an action research approach within a four-case study design. Data were gathered using qualitative research methods focused on semi-structured interviews, observations and document analysis. Results: The study indicated that CoRes, developed through this collaborative process, helped the early career teachers focus on the big picture of the topic, emphasize particularly relevant areas of content and consider alternative ways of planning for their teaching. Conclusions: This paper presents an analysis of the process of CoRe development by the teacher-expert partnerships and the effect that had on

Effects of simulated weightlessness on mammalian development. Part 1: Development of clinostat for mammalian tissue culture and use in studies on meiotic maturation of mouse oocytes

NASA Technical Reports Server (NTRS)

Wolegemuth, D. J.; Grills, G. S.

1984-01-01

The effects of weightlessness on three aspects of mammalian reproduction: oocyte development, fertilization, and early embryogenesis was studied. Zero-gravity conditions within the laboratory by construction of a clinostat designed to support in vitro tissue culture were simulated and the effects of simulated weightlessness on meiotic maturation in mammalian oocytes using mouse as the model system were studied. The timing and frequency of germinal vesicule breakdown and polar body extrusion, and the structural and numerical properties of meiotic chromosomes at Metaphase and Metaphase of meiosis are assessed.

Collaborative Leadership Practices among Ohio's Early College High School Principals and Their Post-Secondary Partners

ERIC Educational Resources Information Center

Carter, Allia L.

2012-01-01

This constructivist multiple-case study examined the collaborative leadership practices of seven secondary and seven post-secondary leaders who participate in Ohio's Early College High School Initiative (ECHSI). The 14 educational leaders in this study partnered in an effort to respond to the access and success of traditionally underrepresented…

Maturation capacity, morphology and morphometric assessment of human immature oocytes after vitrification and in-vitro maturation

PubMed Central

Nazari, Saeedeh; Khalili, Mohammad Ali; Esmaielzadeh, Forouzan; Mohsenzadeh, Mehdi

2011-01-01

Background: In general, 15% of oocytes collected in ART cycles are immature. These oocytes may be cryopreserved further for use in in-vitro maturation (IVM) program. Objective: The aim of this study was to determine maturation capacity, morphometric parameters and morphology of human immature oocytes in both fresh IVM (fIVM) and vitrified-IVM (vIVM) oocytes. Materials and Methods: 93 women who underwent controlled ovarian stimulation for ART were included. The immature oocytes (n=203) were divided into two groups: the first group (n=101) directly matured in vitro; and the second group (n=102) first vitrified, then matured in vitro. All oocytes underwent IVM in Ham’s F10 supplemented with LH+FSH and human follicular fluid. After 48h of incubation, the oocyte maturation rates, as well as morphometric and morphologic characteristics were assessed using cornus imaging and were compared. Results: Oocyte maturation rates were reduced in vIVM, (40.4%), in comparison with fIVM (59.4%, p<0.001). Following morphometric assessment, there was no difference in the mean oocyte diameters (µm) between fIVM and vIVM, 156.3±6.8 and 154.07±9.9, respectively. Other parameters of perimeters, egg areas, as well as oocyte and ooplasm volumes were similar in two groups. In addition, more morphologic abnormalities, such as, vacuole, and dark oocyte were observed in vIVM oocytes. Conclusion: fIVM was more successful than vIVM groups. No statistical differences were noticed in morphometry assessment in two groups. This suggests that morphometric parameters can not be applied as prognosis factor in oocyte maturation outcome in IVM program. PMID:26396566

G-protein coupled estrogen receptor (GPER) inhibits final oocyte maturation in common carp, Cyprinus carpio.

PubMed

Majumder, Suravi; Das, Sumana; Moulik, Sujata Roy; Mallick, Buddhadev; Pal, Puja; Mukherjee, Dilip

2015-01-15

GPR-30, now named as GPER (G protein-coupled estrogen receptor) was first identified as an orphan receptor and subsequently shown to be required for estrogen-mediated signaling in certain cancer cells. Later studies demonstrated that GPER has the characteristics of a high affinity estrogen membrane receptor on Atlantic croaker and zebra fish oocytes and mediates estrogen inhibition of oocyte maturation in these two distantly related teleost. To determine the broad application of these findings to other teleost, expression of GPER mRNA and its involvement in 17β-estradiol mediated inhibition of oocyte maturation in other cyprinid, Cyprinus carpio was investigated. Carp oocytes at pre-vitellogenic, late-vitellogenic and post-vitellogenic stages of development contained GPER mRNA and its transcribed protein with a maximum at late-vitellogenic oocytes. Ovarian follicular cells did not express GPER mRNA. Carp oocytes GPER mRNA was essentially identical to that found in other perciformes and cyprinid fish oocytes. Both spontaneous and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P)-induced oocyte maturation in carp was significantly decreased when they were incubated with either E2, or GPER agonist G-1. On the other hand spontaneous oocyte maturation was significantly increased when carp ovarian follicles were incubated with an aromatase inhibitor, fadrozole, GPER antagonist, G-15 and enzymatic removal of the ovarian follicle cell layers. This increase in oocyte maturation was partially reversed by co-treatment with E2. Consistent with previous findings with human and fish GPR30, E2 treatment in carp oocytes caused increase in cAMP production and simultaneously decrease in oocyte maturation, which was inhibited by the addition of 17,20β-P. The results suggest that E2 and GPER play a critical role in regulating re-entry in to meiotic cell cycle in carp oocytes. Copyright © 2014 Elsevier Inc. All rights reserved.