scholarly journals A Method for Chronological Intravital Imaging of Bovine Oocytes during In Vitro Maturation

2008 ◽  
Vol 14 (6) ◽  
pp. 549-560 ◽  
Author(s):  
Morten R. Petersen ◽  
Michael Hansen ◽  
Birthe Avery ◽  
Ingrid B. Bøgh
Keyword(s):  
Embryonic Development ◽  
Oocyte Maturation ◽  
Laser Scanning ◽  
Subsequent Development ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Laser Scanning Microscopy ◽  
Intravital Imaging ◽  
Bovine Oocytes

AbstractOocyte maturation is known to affect the chances for successful fertilization, embryonic development, establishment of pregnancy and delivery of a live, healthy, and viable offspring. Two-photon laser scanning microscopy (TPLSM) has previously been used to evaluate early embryonic development without a detectable impairment of subsequent development, but has never been applied to assess mammalian oocytes throughout in vitro maturation (IVM). Visualization of structures within live oocytes during IVM, followed by fertilization and embryo culture, may improve the understanding of oocyte maturation. To visualize structures within bovine oocytes using TPLSM, it is necessary to remove the cumulus cells that normally surround the oocyte during maturation. Repeated visualization of structures within the same oocyte is possible, if movement of the oocyte can be avoided. In this article, we describe the development of a method for repeated intravital imaging of denuded bovine oocytes using an upright TPLSM equipped with a specially constructed incubator. Oocytes were stained with Hoechst 33258, and the nuclear structures were evaluated. Oocyte fertilization rate was not affected by TPLSM exposure, but the developmental capacity of the denuded oocytes was significantly reduced. This is, to our knowledge, the first article describing repeated intravital imaging during mammalian oocyte maturation using TPLSM.

scholarly journals 295 COMPARISON OF TWO METHODS TO AVOID MOVEMENT OF BOVINE OOCYTES DURING IN VITRO MATURATION

2005 ◽  
Vol 17 (2) ◽  
pp. 298 ◽  
Author(s):  
M.M. Petersen ◽  
B. Avery ◽  
T. Greve ◽  
I.B. Bøgh
Keyword(s):  
Oocyte Maturation ◽  
Laser Scanning ◽  
Early Stage ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Laser Scanning Microscopy ◽  
Control Group ◽  
Exact Test ◽  
Standard Procedures

Development of two-photon laser scanning microscopy (TPLSM) has made it possible to conduct several recordings over time of early stage embryos without compromising viability. To use TPLSM to study structures within the oocyte it is necessary to remove at least part of the cumulus cells to prevent emitted light from being blocked. Aspiration of cumulus oocyte complexes (COC) through a denudation pipette creates a “window” through which the emitted light can escape and be recorded. To allow repeated recordings of the same location within an object it is important to avoid movement of the object. Gelatine (Gel) and poly-l-lysine (PLL) have previously been used to promote adhesion of cells in culture. The aim of our study was to develop a method to avoid movement during IVM of partially denuded COCs without compromising oocyte viability. Previous experiments in our lab showed that partial denudation of COC had no effect on embryo development (unpublished). Bovine COCs were obtained from abattoir ovaries. In the control group COCs were placed in non-treated dishes. In the experimental groups, they were placed in Gel- or PLL-coated dishes, either intact or partially denuded, where the length of cumulus cell “tails” was shortened to around 200 μm on each side of the oocyte. The coated dishes were prepared 24 h prior to IVM with 200 μL of 0.1% Gel (Sigma, Copenhagen, Denmark, G2500) or 200 μL 0.01% PLL (Sigma, P-4832). Partial denudation of COCs was performed with a 127–129 μm diameter denudation pipette. Standard procedures were used for IVM (23 h in DMEM with 5% serum and eCG/hCG), IVF (23 h in TALP), and IVC (SOF with 10% serum); IVM and IVF were incubated at 38.5°C in 5% CO2 in air, and IVC at 5% CO2 in 5% O2. The study was based on a total of 1151 oocytes and 3 replicates. Day 8 blastocyst (BL) rates, BL kinetics, and morphology were used as endpoints to assess oocyte maturation. Kinetics/morphology were graded by a scoring system: hatched/excellent 3, expanded/good 2, non-expanded/poor 1. COCs placed in Gel- or PLL-coated dishes did not move during handling of the dishes. The BL rates in the Gel group were 37%, 25%, and 17%, and in the PLL group 24%, 21%, and 12%, for the control, intact, and partially denuded COCs, respectively. In the Gel group the BL rates showed a decreasing trend (P < 0.0036), whereas in only the PLL group the BL rates from the partially denuded COC differed from the control and the intact COCs (P < 0.008). No significant differences were seen between blastocyst kinetics (Gel/PLL 1.9/1.9, 1.8/1.9, 1.6/1.7) or morphology (Gel/PLL 2.2/2.4, 2.0/2.5, 2.2/2.1) in the control, intact or partially denuded groups. Fisher's exact test used. We conclude that it is possible to avoid movement of COCs during IVM without compromising oocyte maturation in dishes coated with Gel or PLL, if the cumulus layer is intact. The BL rates are compromised if COCs are partially denuded and the “cumulus tails” shortened before IVM in Gel or PLL coated dishes, whereas kinetics and morphology are unaffected. This research was funded by the Danish Research Agency, no. 23-023-0133.

scholarly journals Proline-rich tyrosine kinase2 is involved in F-actin organization during in vitro maturation of rat oocyte

Reproduction ◽  
2006 ◽  
Vol 132 (6) ◽  
pp. 859-867 ◽  
Author(s):  
Xiao-Qian Meng ◽  
Ke-Gang Zheng ◽  
Yong Yang ◽  
Man-Xi Jiang ◽  
Yan-Ling Zhang ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Actin Filaments ◽  
Laser Scanning ◽  
Polar Body ◽  
Laser Scanning Microscopy ◽  
Meiotic Spindle ◽  
Confocal Laser ◽  
Cell Cortex ◽  
First Polar Body

Microfilaments (actin filaments) regulate various dynamic events during meiotic maturation. Relatively, little is known about the regulation of microfilament organization in mammalian oocytes. Proline-rich tyrosine kinase2 (Pyk2), a protein tyrosine kinase related to focal adhesion kinase (FAK) is essential in actin filaments organization. The present study was to examine the expression and localization of Pyk2, and in particular, its function during rat oocyte maturation. For the first time, by using Western blot and confocal laser scanning microscopy, we detected the expression of Pyk2 in rat oocytes and found that Pyk2 and Try402 phospho-Pyk2 were localized uniformly at the cell cortex and surrounded the germinal vesicle (GV) or the condensed chromosomes at the GV stage or after GV breakdown. At the metaphase and the beginning of anaphase, Pyk2 distributed asymmetrically both in the ooplasm and the cortex with a marked staining associated with the chromosomes and the region overlying the meiotic spindle. At telophase, Pyk2 was observed in the cleavage furrows in addition to its cortex and cytoplasm localization. The dynamics of Pyk2 were similar to that of F-actin, and this kinase was found to co-localize with microfilaments in several developmental stages during rat oocyte maturation. Microinjection of Pyk2 antibody demolished the microfilaments assembly and also inhibited the first polar body (PB1) emission. These findings suggest an important role of Pyk2 for rat oocyte maturation by regulating the organization of actin filaments.

scholarly journals Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen-thawed spermatozoa

Reproduction ◽  
2002 ◽  
pp. 455-465 ◽  
Author(s):  
YH Choi ◽  
CC Love ◽  
LB Love ◽  
DD Varner ◽  
S Brinsko ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
First Polar Body ◽  
Bovine Oocytes

This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment after swim-up for 20 min in sperm-Tyrode's albumen lactate pyruvate. Frozen-thawed spermatozoa from the same stallion were treated in a similar way. Spermatozoa were immobilized and injected into the oocytes using a Piezo drill. Presumptive zygotes were cultured in G1.2 medium for 20 or 96 h after the injection was administered, or were transferred to the oviducts of recipient mares and recovered 96 h later. In addition, bovine oocytes with first polar bodies were injected with the two types of stallion spermatozoa and fixed 20 h after injection to examine pronuclear formation. Fertilization rate (pronucleus formation and cleavage) at 20 h after injection of spermatozoa was not significantly different between fresh and frozen-thawed sperm groups in either equine or bovine oocytes. Pronucleus formation after injection of spermatozoa into bovine oocytes was significantly higher than that for equine oocytes (P < 0.05). There were no significant differences in cleavage rate or average number of nuclei at 96 h between equine oocytes injected with fresh or frozen-thawed spermatozoa. However, embryos developed in vivo for 96 h had a significantly higher number of nuclei in both sperm treatments compared with those cultured in vitro. These results indicate that good activation rates may be obtained after injection of either fresh or frozen-thawed equine spermatozoa without additional activation treatment. Injection of frozen-thawed equine spermatozoa results in similar embryo development to that obtained with fresh equine spermatozoa. In vitro culture of equine zygotes in G1.2 medium results in a similar cleavage rate but reduced number of cells compared with in vivo culture within the oviduct. Bovine oocytes may be useful as models for assessing sperm function in horses.

scholarly journals Penambahan Glutathione pada Medium Fertilisasi Efektif Mendukung Pembentukan Pronukleus dan Perkembangan Awal Embrio Sapi (SUPPLEMENTATION OF GLUTATHIONE IN FERTILIZATION MEDIUM EFFECTIVELY SUPPORT NORMAL PRONUCLEUS FORMATION AND EARLY BOVINE EMBRYONIC DE

2017 ◽  
Vol 18 (3) ◽  
pp. 327
Author(s):  
Aras Prasetiyo Nugroho ◽  
Iman Supriatna ◽  
Mohamad Agus Setiadi
Keyword(s):  
Tissue Culture ◽  
Embryonic Development ◽  
Culture Medium ◽  
Fertilization Rate ◽  
Tissue Culture Medium ◽  
Randomized Design ◽  
Oviduct Fluid ◽  
And Control ◽  
Bovine Oocytes

The objective of this study was to determine fertilization rate effectiveness and early embryonic development competency with glutathione (GSH) supplementation in fertilization medium and culture This study consisted of two experiments comprising each of the four treatment and six repetitions with completely randomized design (CRD) using 651 oocytes. In the first experiment, a total of 317 bovine oocytes were matured in tissue culture medium (TCM) 199 at incubator 5% CO2 with temperature 39 ºC for24 h, then fertilized with sperm separated by swim up technique. Oocyte and sperms were incubated in fertilization medium supplemented with 0.25 mM, 0.50 mM, 1.00 mM GSH. In the second experiment, bovine oocytes were matured in maturation medium and fertilized with same procedure as mentioned before, then cultured in modified synthetic oviduct fluid (mSOF) with the following treatment: supplementation GSH only in fertilization medium (T1), supplementation GSH only in culture medium (T2), and supplementation GSH in both fertilization and culture medium (T3), while control not supplementation GSH (T0). Result of the first experiment showed that supplementation 1.00 mM GSH in fertilization medium can increase higher normal pronucleus (2PN) formation (86,9%) compared to other treatments, 0.50 mM (80.3%), 0.25 mM (73.8%), and control (58.9%) (P<0.05). In the second experiment showed that early bovine embryonic development on 2nd day cultured which reached 5-8 cell on treatment T1 (56.0%) and T3 (53.6%) were higher (P<0.05) compared to treatment T2 (26.2%) and T0 (control) (31.3%). Result of the other were showed that early bovine embryonic development on 4th day cultured which reached 9-16 cell on treatment T1 (26.2%) and T3 (27.4%) were higher (P<0.05) compared to that T2 (11.9%) and T0 (control) (10.8%). In conclusion, 1.00 mM GSH supplementation in medium was more effective in supporting normal pronucleus formation and early fertilization bovine embryonic development compared to in culture medium. ABSTRAK Penelitian ini bertujuan untuk mengetahui tingkat fertilisasi dan kompetensi perkembangan awal embrio sapi dengan penambahan glutathione (GSH) pada medium fertilisasi in vitro (IVF) dan kultur in vitro (IVC). Penelitian ini terdiri atas dua penelitian yang terdiri dari masing-masing empat perlakukan dan enam kali ulangan dengan rancangan acak lengkap (RAL) menggunakan 651 oosit. Penelitian I, sebanyak 317 oosit sapi dalam tissue culture medium (TCM) 199 dimatangkan pada inkubator 5% CO2 dan suhu 39°C selama 24 jam, kemudian difertilisasi dengan spermatozoa yang telah diseleksi menggunakan teknik swim up. Oosit dan spermatozoa diinkubasi pada medium fertilisasi dengan penambahan 0,25 mM, 0,50 mM, dan 1,00 mM GSH. Penelitian II, sebanyak 334 oosit sapi dimatangkan pada medium pematangan dan difertilisasi, kemudian dikultur pada medium modified synthetic oviduct fluid (mSOF), dengan perlakuan: penambahan GSH hanya pada medium fertilisasi (T1), penambahan GSH hanya pada medium kultur (T2), dan kombinasi penambahan GSH pada medium fertilisasi dan kultur (T3). Hasil penelitian I, menunjukkan bahwa penambahan 1,00 mM GSH pada medium fertilisasi dapat meningkatkan pembentukan pronukleus normal (2PN) yang lebih tinggi (86,9%) dibandingkan dengan perlakuan yang lain yaitu 0,50 mM (80,3%), 0,25 mM (73,8%), dan kontrol (58,9%) (P<0,05). Penelitian II menujukkan bahwa perkembangan awal embrio sapi pada hari ke-2 kultur yang mencapai pembelahan 5-8 sel pada perlakukan T1 (56,0%) dan T3 (53,6%) lebih tinggi (P<0,05) dibandingkan dengan perlakuan T2 (26,2%) dan T0 (kontrol) (31,3%). Hasil penelitian lain menunjukkan bahwa perkembangan awal embrio sapi pada hari ke-4 kultur yang mencapai pembelahan 9-16 sel pada perlakuan T1 (26,2%) dan T3 (27,4%) lebih tinggi dibandingkan dengan perlakukan T2 (11,9%) dan T0 (kontrol) (10,8%) (P<0,05). Dapat disimpulkan bahwa penambahan 1,00 mM GSH pada medium fertilisasi lebih efektif dalam mendukung pembentukan pronukleus normal dan perkembangan awal embrio sapi dibandingkan pada medium kultur.

230 ROLE OF PITUITARY HORMONES AND CUMULUS CELLS IN MODULATING THE DEVELOPMENTAL CAPACITY OF AGING BOVINE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 204
Author(s):  
G. Singina ◽  
I. Lebedeva ◽  
T. Taradajnic ◽  
N. Zinovieva
Keyword(s):  
Embryonic Development ◽  
Tyrosine Kinases ◽  
Cumulus Cells ◽  
Pituitary Hormones ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Prolonged Culture ◽  
Bovine Oocytes

The competence for embryonic development acquired during the oocyte maturation attenuates during the subsequent oocyte aging both in vivo and in vitro. Thus, the successful control of the female fertility requires information regarding factors responsible for the oocyte protection from early aging. The aim of the present research was to study the pattern and pathways of actions of two closely related pituitary hormones, prolactin (PRL), and growth hormone (GH), on the developmental potential of bovine oocytes during their aging in vitro. Therefore, we analysed (1) effects of PRL and GH during the prolonged culture of bovine oocytes on their subsequent development up to the blastocyst stage and (2) the role of cumulus cells (CC) and tyrosine kinases, the well-known mediators of PRL and GH signalling, in these effects. Bovine cumulus-enclosed oocytes (CEO) were cultured for 22 h in the following maturation medium: TCM 199 containing 10% fetal calf serum (FCS), 10 μg mL–1 of porcine FSH, and 10 μg mL–1 of ovine LH. After IVM, CEO or denuded oocytes (DO) were transferred to the aging medium consisting of TCM 199 supplemented with 10% FCS and cultured for 10 h in the absence (Control) or presence of 50 ng mL–1 bovine PRL or 10 ng mL–1 recombinant bovine GH and/or 10 μg mL–1 genistein (a non-selective inhibitor of tyrosine kinases). Genistein was not applied in the case of aging DO, since their developmental potential was not affected by both hormones. Following the prolonged culture, oocytes underwent IVF and IVC. Embryos were cultured in CR1aa medium until Day 5 post-insemination and then transferred to the same medium supplemented with 5% FCS and cultured up to Day 8. The embryo development was evaluated at Days 2 and 8 for cleavage and blastocyst formation. The data from 5 to 6 replicates using 135–184 oocytes per treatment were analysed by ANOVA. Aging of oocytes in the control medium had no effect on the cleavage rate, but caused the blastocyst yield to decline (P < 0.001) from 31.1 ± 2.3% (CEO fertilized immediately after maturation) to 10.5 ± 2.4% (aged CEO) and 7.9 ± 1.9% (aged DO). Cleavage rates of aging CEO and DO were unaffected by both PRL and GH. In the case of CEO, the addition of PRL (but not GH) to the aging medium raised the blastocyst yield from 8.2 ± 0.9% to 15.2 ± 2.1% (P < 0.05), whereas the removal of CC abolished this effect, reducing the yield up to 9.1 ± 2.7% (P < 0.05). At the same time, genistein did not influence the blastocyst yield in the PRL-treated group. The findings demonstrate that PRL can inhibit the attenuation of the developmental competence of bovine oocytes aging in vitro, with this effect being achieved via cumulus cells. Tyrosine kinases are unlikely to mediate the beneficial action of PRL on the CEO capacity for embryonic development. Meanwhile, closely related GH does not affect the developmental competence of aging bovine oocytes.This research was supported by RFBR (project No. 13-04-01888).

Effects of ghrelin on activation of Akt1 and ERK1/2 pathways during in vitro maturation of bovine oocytes

Zygote ◽  
2017 ◽  
Vol 25 (2) ◽  
pp. 183-189 ◽  
Author(s):  
Thomas-Markos Chouzouris ◽  
Eleni Dovolou ◽  
Fotini Krania ◽  
Ioannis S. Pappas ◽  
Konstantinos Dafopoulos ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Cumulus Cells ◽  
Treated Group ◽  
Control Group ◽  
Dependent Manner ◽  
Nuclear Maturation ◽  
Bovine Oocytes ◽  
Phosphorylation Rate ◽  
Matured Oocyte

SummaryThe purpose of this study was to investigate the possible molecular pathways through which ghrelin accelerates in vitro oocyte maturation. Bovine cumulus–oocyte complexes (COCs), after 18 or 24 h maturation in the absence or the presence of 800 pg ml–1 of acylated ghrelin were either assessed for nuclear maturation or underwent in vitro fertilization in standard media and putative zygotes were cultured in vitro for 8 days. In a subset of COCs the levels of phosphorylated Akt1 and ERK1/2 (MAPK1/3) were assessed at the 0th, 6th, 10th, 18th and 24th hours of in vitro maturation (IVM). At 18 and 24 h no difference existed in the proportion of matured oocytes in the ghrelin-treated group, while in the control group more (P < 0.05) matured oocyte were found at 24 h. Oocyte maturation for 24 h in the presence of ghrelin resulted in substantially reduced (P < 0.05) blastocyst yield(16.3%) in comparison with that obtained after 18 h (30.0%) or to both control groups (29.3% and 26.9%, for 18 and 24 h in maturation, respectively). Ghrelin-treated oocytes expressed lower Akt1 phosphorylation rate at the 10th hour of IVM, and higher ERK1/2 at the 6th and 10th hours of IVM compared with controls. In cumulus cells, at the 18th and 24th hours of IVM Akt1 phosphorylation rate was higher in ghrelin-treated oocytes. Our results imply that ghrelin acts in a different time-dependent manner on bovine oocytes and cumulus cells modulating Akt1 and ERK1/2 phosphorylation, which brings about acceleration of the oocyte maturation process.

scholarly journals Live offspring from vitrified blastocysts derived from fresh and cryopreserved ovarian tissue grafts of adult mice

Reproduction ◽  
2009 ◽  
Vol 138 (3) ◽  
pp. 527-535 ◽  
Author(s):  
Xiaoqian Wang ◽  
Sally Catt ◽  
Mulyoto Pangestu ◽  
Peter Temple-Smith
Keyword(s):  
Cancer Patients ◽  
Oocyte Maturation ◽  
Ovarian Tissue ◽  
Subsequent Development ◽  
Cumulus Cells ◽  
Ovarian Tissue Cryopreservation ◽  
Blastocyst Formation ◽  
Adult Mice ◽  
Tissue Grafts

Ovarian tissue cryopreservation and transplantation can be used to preserve fertility for cancer patients. In this study, we assessed the viability and function of ovarian tissue from adult mice that was cryopreserved by solid surface vitrification or traditional slow-cooling using variousin vitroandin vivotechniques, including allotransplantation,in vitrooocyte maturation, embryo culturein vitro, blastocyst cryopreservation, embryo transfer, and development. The importance of cumulus cells for oocyte maturation, fertilization, and embryo development was investigated. Graft recovery, follicle survival, and oocyte retrieval was similar in control, vitrified, and slow-cooled groups. High rates of oocyte maturation, cleavage, and blastocyst formation were achieved, with no significant differences between the control, vitrified or slow-cooled ovarian tissue grafts. The presence of cumulus cells was important for oocyte maturation, fertilization, and subsequent development. Cumulus–oocyte complexes with no surrounding cumulus cells (N-COCs) or with an incomplete layer (P-COCs) had significantly lower rates of oocyte maturation and blastocyst formation than cumulus–oocyte complexes with at least one complete layer of cumulus cells (F-COCs; maturation rate: 63, 78 vs 94%; blastocyst rate: 29, 49 vs 80%). Live births were achieved using vitrified blastocysts derived from oocytes taken from vitrified and slow-cooled ovarian tissue heterotypic allografts. Successful production of healthy offspring from these vitrified blastocysts suggests that this technique should be considered as a useful stage to pause in the assisted reproduction pathway. This provides an alternative protocol for restoring fertility and offering cancer patients a better indication of their chances of pregnancy and live birth.

scholarly journals Early embryonic development of bovine oocytes challenged with LPS in vitro or in vivo

Reproduction ◽  
2019 ◽  
Vol 158 (5) ◽  
pp. 453-463
Author(s):  
Joao Alveiro Alvarado Rincón ◽  
Patricia Carvalho Gindri ◽  
Bruna Mion ◽  
Ferronato Giuliana de Ávila ◽  
Antônio Amaral Barbosa ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Embryo Development ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Cleavage Rate ◽  
Early Embryo Development ◽  
Lps Challenge ◽  
Bovine Oocytes

The aim of this study was to evaluate the effect of exposing bovine oocytes to lipopolysaccharides (LPS) in vivo and in vitro on early embryo development. In experiment 1, cumulus oocyte complexes (COCs, n = 700/group) were challenged with 0, 0.1, 1.0 or 5.0 μg/mL of LPS during in vitro maturation (IVM). Later, in vitro fertilization (IVF) and in vitro culture (IVC) were performed. In experiment 2, COCs (n = 200/group) matured and in vitro fertilized without LPS were subjected to IVC with the same doses of LPS from experiment 1. In experiment 3, heifers received two injections of saline solution (n = 8) or 0.5 μg/kg of LPS (n = 8) 24 h apart, and 3 days later, COCs were recovered and submitted to IVM, IVF, and IVC. In experiments 1 and 3, the expression of TLR4, TNF, AREG and EREG genes in cumulus cells was evaluated. Exposure to 1 and 5 μg/mL of LPS during IVM decreased nuclear maturation (39.4 and 39.6%, respectively) compared with control (63.6%, P < 0.05). Despite that, no effect on cleavage and blastocyst rates were observed. Exposure to LPS during IVC did not affect embryonic development. In vivo exposure to LPS decreased the in vitro cleavage rate (54.3 vs 70.2%, P = 0.032), but cleaved embryos developed normally. Number of cells per embryo and gene expression were not affected by the LPS challenge in any experiment. In conclusion, although in vitro exposure to LPS did not affect early embryo development, in vivo LPS exposure reduced cleavage rate.

Follicular cells affect the fertilizability and developmental competency of bovine oocytes in vitro

1997 ◽  
Vol 9 (8) ◽  
pp. 763 ◽  
Author(s):  
K. S. Kim ◽  
N. Minami ◽  
M. Yamada ◽  
K. Utsumi
Keyword(s):  
Subsequent Development ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Follicular Cells ◽  
Maturation Period ◽  
Nuclear Maturation ◽  
Maturation In Vitro ◽  
Further Development ◽  
Bovine Oocytes

The present study examined the time-dependent effects of follicular cells on the fertilizability of oocytes and their subsequent development to blastocysts. The percentages of oocytes reaching the metaphase-II stage of maturation rose from 51·3% after 16 h of culture to 86·2% at 28 h (cumulus-intact oocytes; CIO) and, for the same time points, from 65·4% to 83·3% (corona-enclosed oocytes; CO) and 54·3% to 88·9% (denuded oocytes; DO), respectively. When DO were cultured for more than 24 h before insemination, fertilization rates were significantly lower compared with CIO and CO. The maximum rates of development to blastocysts were observed when the oocytes were cultured for 24 h in the CIO group (22·1%), 20 h in the CO group (19· 7%) and 18 h in the DO group (9·2%), respectively. These results suggest that (i) the presence of cumulus cells or corona cells during maturation is not necessary for nuclear maturation of oocytes; (ii) the attachment of corona cells to the oocytes during maturation is important for the further development to the blastocyst stage, and (iii) the presence of attached cumulus and/or corona cells during maturation in vitro extends the maturation period required for further development to the blastocyst stage.

3 ANALYSIS OF HOX EXPRESSION IN BOVINE OOCYTES AND EARLY EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 159
Author(s):  
D. Paul ◽  
W. Sonnet ◽  
R. Rezsohazy ◽  
I. Donnay
Keyword(s):  
Embryonic Development ◽  
Rna Polymerase Ii ◽  
Oocyte Maturation ◽  
Hox Genes ◽  
Early Stage ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Main Body ◽  
Bovine Oocytes

HOX genes encode transcription factors known to play a major role in patterning the main body axis of vertebrate embryos from the gastrulation stage onward. A few studies have provided evidence that some HOX genes might be expressed before implantation in mammalian embryos. Translation of maternally inherited transcripts is regulated by modifications of the poly(A) tail length until embryonic genome activation (EGA), occurring during the 4th cell cycle in the bovine. The objective of this work was to establish the expression pattern of various HOX genes and to study the polyadenylation of their transcripts during oocyte maturation and early embryonic development. Pools of 20 bovine oocytes before and after in vitro maturation and 20 in vitro-produced embryos at different stages of development up to the blastocyst stage were collected. Three to 12 pools were used for each stage. RNA was extracted and reverse transcribed (RT) using random hexamers. Quantitative real-time PCR (qPCR) was performed to establish expression profiles of 4 HOX genes: HOXD1, HOXA3, HOXB9, and HOXC9. Two distinct patterns of expression were observed. First, relative amounts of HOXD1, HOXA3, and HOXC9 were lower in morulae and blastocysts than in oocytes. On the other hand, relative expression of HOXB9 increased between the 5 to 8 cell stage and the morula stage (Mann-Whitney, P < 0.05). Those expression patterns were not modified when embryos were cultured in presence of α-amanitin, a RNA polymerase II inhibitor, indicating the maternal origin of the transcripts until EGA. Total amount of mRNAs, estimated by RT-qPCR with random hexamers, was stable for all studied genes during oocyte maturation. The relative amount of polyadenylated GAPDH mRNAs, estimated by RT-qPCR with poly(dT), decreased greatly in mature oocytes compared with immature oocytes indicating massive deadenylation of those transcripts. The relative amount of polyadenylated HOXC9 transcripts decreased slightly but significantly during oocyte maturation (Mann-Whitney, P < 0.05).The relative amount of polyadenylatedm RNAs corresponding to HOXD1, HOXA3, and HOXB9 was stable during oocyte maturation. This indicates that those transcripts escape the default deadenylation pathways followed by housekeeping genes. This experiment has been repeated 3 to 4 times. In conclusion, we confirmed the presence of HOXD1, HOXA3, HOXB9, and HOXC9 transcripts in bovine oocytes and early-stage embryos. Their role during oocyte maturation and the first stages of embryonic development will be investigated through loss of function studies. This work is funded by the Fonds National de la Recherche Scientifique (Belgium).

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