scholarly journals Superovulation, fertilization and in vitro embryo development in mice after administration of an inhibin-neutralizing antiserum

Reproduction ◽  
2001 ◽  
pp. 809-816 ◽  
Author(s):  
H Wang ◽  
CB Herath ◽  
G Xia ◽  
G Watanabe ◽  
K Taya
Keyword(s):  
Chorionic Gonadotrophin ◽  
Developmental Competence ◽  
Alpha Subunit ◽  
Control Group ◽  
Control Groups ◽  
Blastocyst Development ◽  
Embryo Production ◽  
Adult Mice

The present study was conducted to investigate the effect of immunoneutralization against endogenous inhibin on oocyte and embryo production in adult and immature mice. At 12:00 h on day 2 of oestrus (day 1 of dioestrus), a single i.p. injection of inhibin antiserum (50, 100, 200 or 400 microl per animal) or equine chorionic gonadotrophin (eCG; 10 or 20 iu per animal) or control goat serum (100 microl per animal) was administered to adult female mice. After 48 h, the mice in each of the three groups were given a single i.p. injection of hCG (10 iu per animal). At 42 h after hCG injection, ova were collected from oviducts and cultured in KSOM solution. Treatments with both inhibin antiserum-hCG and eCG-hCG induced superovulation in all the animals tested. The number of oocytes in animals treated with inhibin antiserum was significantly higher (P < 0.05) compared with the control group, and the number of oocytes ovulated in animals treated with 200 or 400 ml inhibin antiserum was significantly (P < 0.05) higher than that in animals treated with 10 or 20 iu eCG. The superovulated oocytes that were fertilized normally in vivo were able to form blastocysts in vitro. The rate of blastocyst development for animals treated with 50-200 ml inhibin antiserum was significantly (P < 0.05) higher than that of the eCG-treated animals. Irrespective of the day of the oestrous cycle, 200 microl inhibin antiserum administered at 12:00 h on each of 4 days induced superovulation in all the animals tested. The rates of oocyte and embryo production by these animals were significantly (P < 0.05) higher than in the control groups. Furthermore, administration of inhibin antiserum at doses of 50, 100, 200 or 400 ml produced similar results in 26-day-old immature mice. These results indicate that passive immunoneutralization of endogenous inhibin alpha-subunit induces superovulation in immature and adult mice. The superovulated oocytes obtained by administration of inhibin antiserum have normal embryonic developmental competence. Thus, it is concluded that this inhibin antiserum method is a new practical alternative for induction of superovulation in mice instead of the more commonly used eCG-hCG protocol.

Supplementation of c-type natriuretic peptide during in vitro growth period benefits the development of murine preantral follicles

Zygote ◽  
2020 ◽  
pp. 1-5
Author(s):  
Li Ang ◽  
Cao Haixia ◽  
Li Hongxia ◽  
Li Ruijiao ◽  
Guo Xingping ◽  
...  
Keyword(s):  
Natriuretic Peptide ◽  
Granulosa Cells ◽  
Culture System ◽  
Early Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Follicle Development ◽  
Control Groups ◽  
Preantral Follicles

Summary The present study investigated the effects of c-type natriuretic peptide (CNP) on the development of murine preantral follicles during in vitro growth (IVG). Preantral follicles isolated from ovaries of Kunming mice were cultured in vitro. In the culture system, CNP was supplemented in the experimental groups and omitted in the control groups. In Experiment 1, CNP was only supplemented at the early stage and follicle development was evaluated. In Experiments 2 and 3, CNP was supplemented during the whole period of in vitro culture. In Experiment 2, follicle development and oocyte maturity were evaluated. In Experiment 3, follicle development and embryo cleavage after in vitro fertilization (IVF) were assessed. The results showed that in the control groups in all three experiments, granulosa cells migrated from within the follicle and the follicles could not reach the antral stage. In the experimental groups in all three experiments, no migration of granulosa cells was observed and follicle development was assessed as attaining the antral stage, which was significantly superior to that of the control group (P < 0.0001). Oocyte meiotic arrest was effectively maintained, hence giving good developmental competence. In conclusion, CNP supplementation in the culture system during IVG benefited the development of murine preantral follicles.

75 EFFECT OF PHENAZINE ETHOSULFATE ON PORCINE BLASTOCYST DEVELOPMENT, APOPTOSIS, AND CRYOTOLERANCE AFTER OPEN PULLED STRAW VITRIFICATION

2008 ◽  
Vol 20 (1) ◽  
pp. 118
Author(s):  
B. Gajda ◽  
Z. Smorag ◽  
M. Bryla
Keyword(s):  
Cell Number ◽  
Developmental Competence ◽  
Control Group ◽  
Tunel Staining ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Morula Stage ◽  
Phenazine Ethosulfate ◽  
And Control

It is possible to improve the success of cryopreservation of in vitro-produced bovine embryos by modifying the embryos with the metabolic regulator phenazine ethosulfate (PES) (Seidel 2006 Theriogenology 65, 228–235). The PES treatment increased glucose matabolism, tended to increase the pentose phosphate pathway flux of glucose, and clearly reduced accumulation of lipids in cultured bovine embryos (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 597–607). It is known that porcine embryos have a considerably high content of lipids, and the success rates of their cryopreservation appear to be highly correlated with cytoplasmic lipid content. In our preliminary study, we observed that supplementation of NCSU-23 medium with PES has a positive effect on efficiency of pig blastocysts of good quality (Gajda et al.. 2007 Acta Biochim. Pol. 54(Suppl 1), 52 abst). In the present study, the effects of PES on pig blastocyst development, apoptosis, and survival after vitrification were investigated. In Exp. 1, porcine zygotes obtained from superovulated gilts were cultured in NCSU-23 medium supplemented with 0 (control), 0.025, 0.05, or 0.075 µm PES. The culture was performed at 39�C, with 5% CO2 in air, for 96–120 h. Embryo quality criteria were developmental competence (cleavage, morula stage, and blastocyst stage), cell number per blastocyst, and the degree of apoptosis as assessed by TUNEL staining. In Exp. 2, expanded blastocysts cultured with 0.025 µm PES were vitrified in a ethylene glycol and dimethyl sulfoxide mixture using open pulled straw (OPS) technology (Vajta et al. 1997 Acta Vet. Scand. 38, 349–352). After thawing, the blastocysts were cultured in vitro for re-expansion or transferred to synchronized recipients. Data were analyzed by chi-square test. There was a difference between the 0.025 µm PES-treated and the control group in percentage of cleaved embryos (99.0 and 91.4%, respectively; P < 0.05), between all experimental groups and control in percentage of morula stage (90.7, 87.8, 83.8, and 80.0%, respectively), and between 0.025 and 0.05 µm PES-treated and control in percentage of blastocyst rates (70.0, 75.5, and 65.7%, respectively). The number of cells and percentage of TUNEL-positive nuclei per blastocyst were lower in the PES-treated than in the control group. The survival rate of blastocysts after vitrification and thawing was enhanced in the presence of PES compared to that in the PES-free group (45.2 and 38.9%, respectively; P < 0.05). After transfer of 56 expanded blastocysts cultured with PES and vitrified into 3 recipients, two gilts were confirmed pregnant at 35 days of gestation. In conclusion, a higher blastocyst percentage with a low incidence of apoptosis was obtained in the presence of PES compared to control. These blastocysts also had an increased ability to survive cryopreservation.

207 IN VITRO MATURATION TREATMENT AFFECTS DEVELOPMENTAL COMPETENCE OF LAPAROSCOPIC OVUM PICKUP-DERIVED OOCYTES IN FOLLICLESTIMULATING HORMONE-STIMULATED GOATS

2008 ◽  
Vol 20 (1) ◽  
pp. 182 ◽  
Author(s):  
Y. Locatelli ◽  
N. Poulin ◽  
G. Baril ◽  
J.-L. Touzé ◽  
A. Fatet ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Follicular Fluid ◽  
Developmental Competence ◽  
Postmortem Changes ◽  
Blastocyst Development ◽  
Embryo Production ◽  
Development Rates ◽  
Epidermal Growth

The aim of the present study was to assess the effect of IVM treatment on the developmental competence of oocytes recovered from repeated laparoscopic ovum pickukp (LOPU) in goats. A total of 94 LOPU sessions were performed on 33 adult goats of the Saanen and Alpine breeds. Females were synchronized (Day 0) during the nonbreeding season by inserting vaginal sponges (45 mg of fluorogestone acetate, Intervet, Boxmeer, The Netherlands). At Day 8, an i.m. injection of 50 μg of cloprostenol (Estrumate; Schering-Plough Animal Health, Pointe-Claire, Quebec, Canada) was administered. Porcine FSH (Stimufol, Merial, Brussels, Belgium, 160 mg/goat) was administered in 5 injections at 12-h intervals, starting on Day 8. The LOPU took place under general anesthesia on Day 11, and follicles ≥2 mm were aspirated with an 18-gauge needle connected to a controlled vacuum system. Vaginal sponges were removed at the time of LOPU. Treatments were repeated 2 times in a 2-week interval scheme (2 goats and 1 goat were excluded from the experiment during the second and third LOPU sessions, respectively). Cumulus–oocyte complexes were washed and evaluated for quality (graded from 1 to 3). Oocytes recovered from unstimulated slaughterhouse-derived ovaries served as a control. Cumulus–oocytes complexes from Grades 1 and 2 were submitted to IVM in TCM-199, supplemented with 100 μm of cysteamine and either 10 ng mL–1 of epidermal growth factor (EGF) or 10% follicular fluid and 100 ng mL–1 of ovine FSH (FF-FSH). Matured oocytes were then submitted to IVF and in vitro development as described by Cognié et al. (2004 Reprod. Fertil. Dev. 16, 437–445). Over the 94 LOPU sessions, 20.4 ± 0.9 follicles were aspirated (mean ± SEM), allowing the recovery of 12.3 ± 0.7 COC per goat and per session, of which 80.1% were suitable for IVM (Grades 1 and 2). Results of in vitro production are detailed in the table. The IVM treatment did not significantly affect cleavage or blastocyst development rates in oocytes derived from slaughterhouse ovaries. Cleavage rates were significantly decreased in LOPU-derived oocytes when compared with control oocytes. For LOPU-derived oocytes, cleavage and final blastocyst development rates were increased significantly and kinetics of embryo development were accelerated when FF-FSH was used during IVM as compared with EGF. The IVM with FF-FSH allowed us to produce 4.1 blatocysts per goat per LOPU session. These results demonstrate the interest in LOPU for goat embryo production once appropriate IVM treatment is used. The difference observed between LOPU and slaughterhouse oocytes in terms of response to IVM treatments may be related to FSH stimulation prior to the LOPU session or to postmortem changes in oocyte responsiveness in the slaughterhouse group. Table 1. Effects of oocyte origin [laparoscopic ovum pickukp (LOPU) or slaughterhouse derived] and maturation treatment [epidermal growth factor (EGF) or follicular fluid (FF)-FSH] on in vitro embryo production (6 replicates)

scholarly journals Use of oocytes selected by brilliant cresyl blue staining enhances rabbit cloned embryo development in vitro

Zygote ◽  
2019 ◽  
Vol 27 (3) ◽  
pp. 166-172 ◽  
Author(s):  
Linying Jia ◽  
Bo Ding ◽  
Chong Shen ◽  
Shiwei Luo ◽  
Yanru Zhang ◽  
...  
Keyword(s):  
Cell Mass ◽  
Developmental Competence ◽  
Blue Staining ◽  
Control Group ◽  
Control Groups ◽  
Brilliant Cresyl Blue ◽  
Positive Group ◽  
Cresyl Blue ◽  
And Control

SummaryRabbits play an important role in people’s lives due to their high nutritional value and high-quality hair that can be used as raw material for textiles. Furthermore, rabbits are an important animal model for human disease, as genome-edited animals are particularly valuable for studying gene functions and pathogenesis. Somatic cell nuclear transfer (SCNT) is an important technique for producing genome-edited animals and it has great value in saving endangered species and in clone stem cell therapy. However, the low efficiency of SCNT limits its application, with the selection of suitable rabbit oocytes being crucial to its success. In the present study, we collected oocytes from ovarian follicles and stained them with 26 μM brilliant cresyl blue (BCB). We then matured the oocytes in vitro and used them for SCNT. Comparison of the BCB-positive oocytes with BCB-negative oocytes and the control group showed that the BCB-positive group had a significantly higher maturation rate (81.4% vs. 48.9% and 65.3% for the negative and control groups, respectively), cleavage rate (86.6% vs. 67.9% and 77.9%), blastocyst rate (30.5% vs. 12.8% and 19.6%), total number of blastocysts (90±7.5 vs. 65.3±6.3 and 67.5±5.7), and inner cell mass (ICM)/ trophectoderm (TE) index (42.3±4.2 vs. 30.2±2.1 and 33.9±5.1) (P<0.05). The BCB-positive group had a significantly lower apoptosis index (2.1±0.6 vs. 8.2±0.9 and 6.7±1.1 for the negative and control groups, respectively) (P<0.05). These findings demonstrate that BCB-positive oocytes have a higher maturation ability and developmental competence in vitro, indicating that BCB staining is a reliable method for selecting oocytes to enhance the efficiency of SCNT.

329 SUPPRESSION OF BOVINE OOCYTE IVM BY SERUM AND FSH DEPRIVATION OR BY α-AMANITIN: EFFECT ON FERTILIZATION AND EMBRYO DEVELOPMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 272
Author(s):  
K. Kananen-Anttila ◽  
M. Eronen ◽  
J. Matilainen ◽  
M. Kallio ◽  
J. Peippo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Blastocyst Development ◽  
Nuclear Maturation ◽  
Embryo Cleavage ◽  
Bovine Oocytes ◽  
Sperm Aster

We have studied the effect of suppressed IVM on the developmental competence of bovine oocytes, aiming at elucidating the importance of cytoplasmic maturation in fertilization and embryo development. Six replicates of abattoir-derived oocytes were randomly divided into three IVM groups. Control (n = 950): TCM-199 with glutamax-I (Gibco, Grand Island, NY, USA), 0.25 mM Na-pyruvate, 100 IU mL−1 penicillin and 100 μg mL−1 streptomycin, 50 ng mL−1 FSH, and 10% fetal bovine serum (FBS) (Gibco); Serum+FSH-free (n = 944): same as control but without FSH and FBS; α-amanitin (n = 977): same as control but with 10 μg mL−1 α-amanitin. Nuclear maturation of oocytes was studied 24 h after the onset of IVM, the formation of sperm aster structure 10 hours post-insemination (hpi) and the formation of pronuclei 20 hpi. Sperm aster was visualized with β-tubulin antibody (modified from Navara et al. 1999 Dev. Biol. 162, 29–40). Presumptive zygotes were cultured until Day 7 in modified SOFaaci + 4 mg mL−1 fatty acid-free BSA in 5% O2. Cumulus cell expansion was seen only in the control group. The results of nuclear maturation, fertilization, and embryo development are summarized in Table 1. Serum and FSH deprivation did not have a statistically significant effect on the parameters studied (vs. control). α-amanitin exposure during IVM reduced nuclear maturation, fertilization, and Day 3 embryo cleavage vs. control, and resulted in total blockage of Day 7 blastocyst development. The treatment groups had significantly smaller mean diameters of male pronuclei (control: 14 ± 0.6 μ­m; serum+FSH-free: 12 ± 0.5 μ­m, P < 0.05; α-amanitin: 10 ± 0.6 μ­m, P < 0.001) and sperm asters (control: 86 ± 4 μ­m; serum+FSH-free: 82 ± 4 μ­m, P < 0.01; α-amanitin: 49 ± 7 μm, P < 0.001) (nonparametric Kruskall Wallis and Mann-Whitney U tests) vs. control group. Despite reduction in pronucleus and sperm aster diameter, serum and FSH deprivation during IVM did not affect in vitro developmental competence of bovine oocytes, suggesting a need for re-evaluation of the components of IVM. α-Amanitin exposure in IVM disturbed nuclear maturation, fertilization, and embryo development, indicating the essence of early transcription. Table 1. Average percentages ± (n) for nuclear maturation, fertilization (min two pronuclei), embryo cleavage, and blastocyst development

359 ICSI AND ACTIVATION OF OOCYTES RECOVERED FROM TRANSITIONAL AND CYCLING MARES

2006 ◽  
Vol 18 (2) ◽  
pp. 286 ◽  
Author(s):  
T. Suh ◽  
S. Purcell ◽  
G. Seidel Jr
Keyword(s):  
Growth Factor ◽  
Polar Body ◽  
Follicular Development ◽  
Transitional Period ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
First Polar Body

Ovarian follicular development in mares during the transitional period before the breeding season leads to an accumulation of antral follicles of various sizes. The quality of oocytes at this stage may be compromized until the first seasonal ovulation. In this study, we evaluated the developmental competence of oocytes recovered from transitional and cyclic mares, and the effect of zygote activation after intracytoplasmic sperm injection (ICSI). A 2 × 2 × 2 factorial experiment consisting of oocytes from transitional and cyclic mares, two follicle sizes (10 to 20 and 20+ mm), and two treatments (control and activated) was conducted. Follicular oocytes of 14 mares were aspirated in March and April (transitional) and May to July (cyclic) five times per each period at 10-day intervals, without use of hCG. Oocytes aspirated from mares were matured in vitro in a defined medium similar to SOF plus FSH, LH, epidermal growth factor (EGF), insulin-like growth factor (IGF), estradiol (E2), prostaglandin (P4) and 10% FCS, for 30 ± 1 h under 5% CO2 in air at 38.5°C; oocytes with a first polar body were used for ICSI. Motile sperm from frozen-thawed semen were used for sperm injection with a piezo-driven pipet. For activation after ICSI, presumptive zygotes were cultured in G1.3 containing 0.02 µM phorbol 12-myristate 13-acetate (PMA) for 2 h, and then in 2 mM 6-dimethylaminopurine (6-DMAP) for 3 h under 6% CO2 in air at 38.5°C. Zygotes were cultured in 50 µL drops of DMEM/F12 containing 10% FCS for 9 days at 38.5°C in 5% CO2/5% O2/90% N2. Medium was replaced every 3 days. Cleavage and blastocyst rates were calculated based on non-degenerating injected oocytes. Data were analyzed by Fisher's exact test. A total of 115 and 78 oocytes were recovered from cyclic and transitional mares. Average maturation rates to MII in the respective groups were 76.5 and 65.4%, respectively (P < 0.07), and those of 10 to 20 and 20+ mm follicle groups were 70.6 and 80.0%, respectively (P > 0.05). The average cleavage rate in cyclic mares was higher than in transitional mares, and that of the activated group averaged over follicle sizes was higher than that of controls (P < 0.05; Table 1); those of 10 to 20 and 20+ mm follicle groups were not different (P < 0.05; Table 1). Blastocyst rates per oocyte within main effects were not different (P < 0.05; Table 1). Oocytes from transitional mares had lower cleavage rates than those of cyclic mares, but blastocyst development was similar. Activation of zygotes clearly improved cleavage rates of in vivo-derived immature equine oocytes after ICSI. Table 1. Main effect means of responses after ICSI

58 ISOLATION OF BOVINE TROPHOBLAST AND ITS REPROGRAMMING BY NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 134
Author(s):  
I. M. Saadeldin ◽  
B. H. Kim ◽  
B. Roibas da Torre ◽  
O. J. Koo ◽  
G. Jang ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Quantitative Polymerase Chain Reaction ◽  
Cell Mass ◽  
Donor Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Embryonic Cells ◽  
Blastocyst Development

Nuclear transfer (NT) has been used to produce many cloned offspring using several types of cells, including embryonic cells. Even though inner cell mass cells have been used as donor karyoplast for producing cloned animals, there are few studies using trophoblast. In mice, clones were born by nuclear transfer of trophoblasts from the expanded blastocyst into enucleated oocytes as a trial to show the totipotency of both inner cell mass and trophectoderm cells isolated from blastocysts (Tsunoda and Kato 1998 J. Reprod. Fertil. 113, 181–184). However, bovine trophoblast cell (TC) lines have not been used in NT to date. The purpose of this study was to elucidate whether TC as donor cell can be reprogrammed in bovine enucleated oocyte and determine the relative abundance of interferon tau (IFNτ) expression in the resulting cloned preimplantational embryos. Hatched blastocysts produced by IVF were used to isolate TCs on mouse embryonic fibroblasts treated with mitomycin C as feeder cells. TCs and adult fibroblasts (AF, control group for NT) were microinjected to perivitelline space of in vitro mature enucleated oocytes and electrically fused. Reconstructed embryos were chemically activated and cultured in a 2-step chemically defined medium. Levels of IFNτ expression in IVF-, TC-, and AF-derived blastocysts were analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). IVF produced embryos were used as reference to analyze the linear progressive expression of IFNτ through mid-, expanded, and hatching blastocysts. As a result, TCs expressing IFNτ were successfully isolated and cultured on feeder layers. It grew as cell sheets of cuboidal epithelium with high proliferation capacity as a single colony originated from a small clump of cells measured 0.5 cm within 7 days of culture. TCs were reprogrammed in the enucleated oocytes to blastocyst with similar efficiency to AF (14.5% and 15.6%, respectively; P ≤ 0.05). RT-qPCR studies showed that IFNτ expression was higher in TC-derived blastocysts than IVF- and AF-derived blastocysts. Both IVF- and TC-derived blastocysts, showed progressive increase of IFNτ expression through the advancement of blastocyst development when it was compared to AF-derived blastocysts. In conclusion, using TCs expressing IFNτ as donor cell for bovine NT could increase the developmental competence of cloned embryos as indicated by progressive linear increase in IFNτ expression. This study was supported by grants from IPET (#109023-05-1-CG000), NRF (#M10625030005-10N250300510), MKE (#2009-67-10033839, #2009-67-10033805), and BK21 program. Saadeldin I. M. is supported by Islamic Development Bank (IDB) merit scholarship, Jeddah, Saudi Arabia.

scholarly journals Developmental Competence of Domestic Cat Vitrified Oocytes in 3D Enriched Culture Conditions

Animals ◽  
2019 ◽  
Vol 9 (6) ◽  
pp. 329 ◽  
Author(s):  
Martina Colombo ◽  
Maria Giorgia Morselli ◽  
Mariana Riboli Tavares ◽  
Maricy Apparicio ◽  
Gaia Cecilia Luvoni
Keyword(s):  
Developmental Stages ◽  
Culture Conditions ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Control Group ◽  
Vitro Fertilization ◽  
In Vitro Embryo Production ◽  
Physical And Chemical

Cryoinjuries severely affect the competence of vitrified oocytes (VOs) to develop into embryos after warming. The use of culture conditions that provide physical and chemical support and resemble the in vivo microenvironment in which oocytes develop, such as 3D scaffolds and coculture systems, might be useful to improve VOs outcomes. In this study, an enriched culture system of 3D barium alginate microcapsules was employed for the in vitro embryo production of domestic cat VOs. Cryotop vitrified-warmed oocytes were in vitro matured for 24 h in the 3D system with or without fresh cumulus-oocyte complexes (COCs) in coculture, whereas a control group of VOs was cultured in traditional 2D microdrops of medium. After in vitro fertilization, presumptive embryos were cultured in 3D or 2D systems according to the maturation conditions. Vitrified oocytes were able to mature and develop into embryos in 3D microcapsules (17.42 ± 11.83%) as well as in 2D microdrops (14.96 ± 8.80%), but the coculture with companion COCs in 3D resulted in similar proportions of VOs embryo development (18.39 ± 16.67%; p = 1.00), although COCs presence allowed for blastocyst formation (0.95 ± 2.52%). In conclusion, embryos until late developmental stages were obtained from cat VOs, and 3D microcapsules were comparable to 2D microdrops, but improvements in post-warming conditions are still needed.

scholarly journals 237 INCREASE OF BLASTOCYST DEVELOPMENTAL RATE IN VITRO BY SELECTION OF DEVELOPMENTALLY COMPETENT COW OOCYTES BEFORE IVM USING A STAINING TEST

2005 ◽  
Vol 17 (2) ◽  
pp. 269
Author(s):  
H. Alm ◽  
H. Torner ◽  
B. Loehrke ◽  
T. Viergutz ◽  
I. Ghoneim ◽  
...  
Keyword(s):  
Developmental Rate ◽  
Cell Number ◽  
Developmental Competence ◽  
Control Group ◽  
G6pdh Activity ◽  
Blastocyst Development ◽  
Brilliant Cresyl Blue ◽  
Immature Oocytes ◽  
Cresyl Blue

A large proportion of bovine oocytes fail to develop to blastocyst stage following maturation, fertilization, and culture in vitro. While suboptimal culture conditions undoubtedly contribute to this poor development, it is recognized that immature oocytes, especially from cows with reduced reproductive performance or which are slaughtered on the end of their use, are heterogeneous in quality and developmental competence (Gordon 2003). The aim of the present study was to increase the efficiency of blastocyst production from cows after IVM/IVF by oocyte selection before maturation. Immature oocytes are known to synthesize a variety of proteins (Wassarman PM 1988, Annu. Rev. Biochem. 57, 415–442), among them, glucose-6-phosphate dehydrogenase (G6PDH). This enzyme is active in the growing oocyte, but has decreased activity in oocytes that have finished their growth phase. Brilliant cresyl blue (BCB) has been used to measure G6PDH activity. The BCB test is based on the capability of the G6PDH to convert the BCB stain from blue to colorless (Erisson et al. 1993 Theriogenology 39, 214). The ovaries were obtained from a slaughterhouse and transported to the laboratory; cumulus-oocyte complexes (COCs) were recovered by slicing the surface of the ovary. Only oocytes with a compact cumulus investment were used. Oocytes were placed into three groups: (1) control – placed immediately into culture; (2) holding control – COCs kept in PBS containing 0.4% BSA for 90 min at 38.5°C before placement into culture; and (3) treatment – incubation with brilliant cresyl blue for 90 min at 38.5°C before culture. Treated oocytes were then divided into BCB− (colorless cytoplasm, increased G6PDH) and BCB+ (colored cytoplasm, low G6PDH) on their ability to metabolize the stain. Activity of G6PDH was determined via measurement of NADP reduction in control, BCB−, and BCB+ groups; activity was significantly increased in BCB− COCs in comparison to the control and BCB+ COCs. After IVM, oocytes were fertilized in vitro. Embryos were cultured to Day 8. The rate of maturation to metaphase II was significantly higher for control and BCB+ oocytes (77.1 and 72.5%, respectively) than for BCB− oocytes (58.1%). The BCB+ oocytes yielded a significantly higher proportion of blastocysts (34.1%) than either control group (18.3 and 19.2%); and both controls and BCB+ oocytes had significantly higher blastocyst development than did BCB− oocytes (3.9%). The number of nuclei in the blastocysts was comparable in BCB+ and both control groups (105.5 ± 5.8 and 117.5 ± 8.5, 101.8 ± 6.2, respectively). Blastocysts in the BCB− group had a significantly lower cell number (61.0 ± 2.6) than did controls. The results show that the staining of COCs from cows before IVM may be useful in increasing the efficiency of blastocyst production during standard IVF procedures. In addition, classification of G6PDH activity on the basis of BCB staining may be used to effectively select cow oocytes with further developmental competence. To our knowledge, this is the first study to evaluate the association between G6PDH activity in oocytes and further blastocyst development in cows.

202 KNOCK-DOWN AND DEVELOPMENTAL EFFICIENCY OF Arhgap15 GENE KNOCK-DOWN EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 200
Author(s):  
B.-C. Yang ◽  
H.-C. Lee ◽  
S. Hwang ◽  
I.-S. Jeon ◽  
D.-K. Lee ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Rho Gtpase ◽  
Mrna Level ◽  
Developmental Competence ◽  
Control Group ◽  
Dependent Manner ◽  
Blastocyst Development ◽  
Knock Down ◽  
Donor Cells

The technique of RNA interference (RNAi) knock-down is a powerful tool for the analysis of gene function in mammalian cells and eggs. Rho GTPase-activating protein 15 (Arhgap15) is closely related to anaemia and immunity, especially as caused by trypanosome. This study was performed to investigate the effect of Arhgap15 gene knock-down on the developmental competence of bovine embryos in vitro. Bovine fibroblast cells were treated with 50 and 100 nM concentrations of Arhgap15 RNAi, respectively. After 24 h of transfection, the control group showed no change in Arhgap15 mRNA level, whereas mRNA expression in the RNAi-treated donor cells was obviously decreased in a dose-dependent manner. These RNAi-treated cells were then transferred into enucleated bovine oocytes to analyse the consequence of RNAi-mediated Arhgap15 gene knock-down. In the control group, cleavage and blastocyst development rates were 75% (102/136) and 14.7% (20/136), whereas those in the knock-down embryos were 81.6% (120/147) and 24.5% (36/147), respectively. The occurrence of cell death by apoptosis was examined in Day 7 blastocysts. Apoptotic cells numbered 12 ± 3.2 in control embryos and 8.9 ± 4.8 in knock-down embryos. Therefore, it can be concluded that RNAi-mediated Arhgap15 gene knock-down in somatic cells did not affect the developmental competence of bovine cloned embryos. This work was supported by grant 120080401034062 from the BioGreen 21 Program, Rural Development Administration, Republic of Korea.

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