Cycloheximide speeds the porcine oocyte nuclear kinetics and retains embryonic developmental potential in the presence of gonadotrophins

2010 ◽  
Vol 90 (2) ◽  
pp. 189-196
Author(s):  
X -L. Sun ◽  
W -Z. Ma ◽  
Y -B. Zhu ◽  
Z -H. Wu ◽  
L. An ◽  
...  
Keyword(s):  
Embryo Development ◽  
Germinal Vesicle ◽  
Chorionic Gonadotrophin ◽  
Developmental Competence ◽  
Electrical Activation ◽  
Control Group ◽  
Germinal Vesicle Breakdown ◽  
Developmental Potential ◽  
Oocyte Meiosis

Animal embryo engineering requires large amounts of synchronized mature oocytes in vitro. However, porcine cumulus-oocyte complexes aspirated from 3-8 mm follicles are at different germinal vesicle stages. They reach metaphase II stages asynchronously when cultured in vitro. In this study, we examined the effects of pretreatment with or without cycloheximide (CHX), equine chorionic gonadotrophin (eCG), human chorionic gonadotrophin (hCG), and their combinations on meiotic synchronization and the developmental competence of porcine oocytes in vitro following electrical activation. The COCs were pretreated for 12 h with either control medium (TCM 199), CHX (TCM 199 + CHX), eCG/hCG (TCM 199 + eCG/hCG) or eCG/hCG + CHX (TCM 199 + CHX + eCG/hCG), and then cultured for up to 32 h with TCM199 + eCG/hCG. After 12 h pretreatment, the rates of germinal vesicle breakdown (GVBD) were lower (P < 0.05) in the CHX (8.4%) and eCG/hCG + CHX (1.5%) groups compared with control (55.4%) and eCG/hCG (27.2%) groups. After removal of CHX and culture for an additional 12 h in vitro, the majority of the oocytes were synchronized at the GVBD stage in CHX (75.6%) and eCG/hCG + CHX (65.0%) groups. At additional 32 h of culture, the rate of oocytes in metaphase II in eCG/hCG + CHX group (68.3%) was significantly (P < 0.05) higher than the eCG/hCG group (54.8%), but did not differ from other groups (control: 61.3%, CHX: 58.8%). After electrical activation, the cleavage and blastocyst formation rates in the CHX group (80.3%; 19.5%) were significantly (P < 0.05) lower than those in the control group (95.5%; 45.3%), while no difference was found between eCG/hCG + CHX (82.2%; 34.4%) and control groups. Our data, hence, demonstrate pretreatment with CHX hastened nuclear kinetics of porcine oocytes cultured in vitro; however, embryo development potential was retained only when gonadotrophins is present in the in vitro maturation (IVM) medium. Thus, CHX should be used in the two-step culture systems in combination with gonadotrophins. Key words: Oocyte meiosis, synchronization, cycloheximide, embryo development, pig

Histone deacetylase inhibitor trichostatin A affects porcine oocyte maturation in vitro

2014 ◽  
Vol 26 (6) ◽  
pp. 806 ◽  
Author(s):  
Yong-Xun Jin ◽  
Ming-Hui Zhao ◽  
Zhong Zheng ◽  
Jung-Suk Kwon ◽  
Seul-Ki Lee ◽  
...  
Keyword(s):  
Trichostatin A ◽  
Germinal Vesicle ◽  
Oocyte Quality ◽  
Histone Deacetylation ◽  
Developmental Competence ◽  
Mitogen Activated Protein Kinase ◽  
Control Group ◽  
Germinal Vesicle Breakdown ◽  
Porcine Oocyte

Previous studies show that porcine oocyte aging resulting from asynchronised IVM impairs embryo developmental competence. In the present study we investigated whether trichostatin A (TSA; an inhibitor of histone deacetylation) prolongs the maturation time and prevents the aging of oocytes. Porcine oocytes were cultured in medium containing increasing concentrations of TSA (300 nM) for 24, 44 or 64 h. The percentage of oocytes that underwent germinal vesicle breakdown was significantly lower in the TSA-treated group (300 nM) than in the control group. TSA did not affect oocyte quality at MII based on levels of maturation-promoting factor, the phosphorylation status of mitogen-activated protein kinase or histone H3K9 acetylation analysis. We also compared the preimplantation developmental competence and the viability of pathenogenetic embryos treated with 100 nM TSA for 24 h and then continuously cultured for another 24 h in TSA free condition. No significant differences were observed for either parameter between the TSA-treated and control groups. These results indicate that TSA prolongs the IVM of porcine oocytes but that oocyte quality and aging are not affected. These findings provide a feasible option by which to adjust the initiation time of downstream experiments based on porcine matured oocytes.

scholarly journals 241 EFFECT OF MEIOTIC ARREST BY ROSCOVITINE AND SUBSEQUENT IVM TIME ON DEVELOPMENTAL COMPETENCE OF IMMATURE BOVINE OOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 271
Author(s):  
L. Campos-Chillon ◽  
T. Suh ◽  
E. Carnevale ◽  
G. Seidel
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Sperm Concentration ◽  
Germinal Vesicle ◽  
Developmental Competence ◽  
Control Group ◽  
Meiotic Arrest ◽  
Cell Stage ◽  
Bovine Oocytes

Maintaining immature bovine oocytes at the germinal vesicle stage by inhibiting M-phase promoting factor (MPF) activity is a reversible process when using roscovitine, and this can improve cytoplasmic maturation in vitro. However, optimum meiotic arrest times and subsequent IVM times have not been determined, so we evaluated the developmental competence of oocytes in relation to these times. Two by two factorial treatments consisting of 2 arrest times (8 h, 16 h) and 2 subsequent IVM times (16 h, 22 h) plus a control were replicated 6 times in this study. Semen from two bulls was used three times. Chemically defined media (CDM) were used throughout (Olson and Seidel 2000 J. Anim. Sci. 78, 152–157). Slaughterhouse-derived oocytes were arrested in meiosis in 1 mL of CDM-M without any hormones, but containing 50 μM roscovitine and 0.5% fatty acid-free (FAF)-BSA under 5% CO2 in air at 38.5°C. After 8 or 16 h of meiotic arrest, oocytes were washed and matured in 1 mL of CDM-M containing 0.5% FAF-BSA, 2 mM glucose, 50 ng/mL EGF, 15 ng/mL NIDDK-oFSH-20, 1 μg/mL USDA-LH-B-5, 1 μg/mL E2, and 0.1 mM cysteamine for 16 or 22 h under 5% CO2 in air at 38.5°C. Oocytes for the control group were matured in 1 mL of the CDM-M with hormones for 22 h. Ten oocytes from each group were fixed after IVM, stained with orcein, and evaluated for maturation to MII. For fertilization, motile sperm recovered from frozen-thawed semen were co-incubated for 18–20 h with ∼20 oocytes/group at a final sperm concentration of 0.5 × 106 sperm/mL in F-CDM. Presumptive zygotes were cultured in 0.5 mL of CDM-1 for 2.5 days and then in CDM-2 for 5.5 days in 5% CO2, 5% O2, 90% N2 in a humidified incubator at 39°C. Cleavage rates were evaluated after culture in CDM-1. Blastocyst rate, blastocyst stage (5 = early, 6 = full, 6.5 = expanding, 7 = expanded, 7.5 = hatching, 8 = hatched), and embryo quality (1 = excellent, 2 = good, 3 = fair, 4 = poor) were evaluated after CDM-2. Data were subjected to ANOVA; the arc sin transformation was used for percentage data, and least-squares means are presented. There were no significant differences in % cleavage (Cle), cell stage, or blastocyst quality among treatments (P > 0.1). However, meiotic arrest of oocytes for 16 h and subsequent IVM for 16 h improved embryo development to blastocysts compared to other roscovitine treatments (Table 1, P < 0.05). A bull effect for % blastocysts was observed, 19.9% and 25.2% for bulls 1 and 2, respectively (P < 0.08). Blastocyst production was improved by shortening oocyte maturation time from 22 to 16 h, when meiotic progression was previously inhibited for 16 h with roscovitine. Table 1. Effect of meiotic arrest and IVM times on oocyte maturation and embryo development

scholarly journals 289 EFFECTS OF THE STORAGE OF BOVINE OVARIES ON THE NUCLEAR MATURATION AND DEVELOPMENT OF IN VITRO PRODUCED EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 295
Author(s):  
M. Narita ◽  
S. Goda ◽  
Y. Inaba ◽  
K. Imai ◽  
S. Matoba ◽  
...  
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Saline Group ◽  
The Other ◽  
Control Group ◽  
Maturation Time ◽  
Germinal Vesicle Breakdown ◽  
Nuclear Stage

The objectives of this study were to investigate effects of storage of bovine ovaries on the maturation of oocytes and to determine the optimal maturation time for oocytes obtained from the stored ovaries. Ovaries were obtained at a local abattoir and transported in physiological saline to the laboratory (18°C, 3 h; storage group). As a control, oocytes were collected from ovaries without storage. Other ovaries were kept in a plastic bag without solution (Bag-group) or with saline (Saline-group). These ovaries were preserved at 20°C for 18 h. Then cumulus-oocyte complexes were collected and maturated in TCM-199 + 5% CS. In Experiment 1, to investigate effects of the storage methods of bovine ovaries on the timing of germinal vesicle breakdown (GVBD) and the progression to MII in oocytes obtained from ovaries, oocytes were fixed every 2 h (0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 h) from the start of in vitro maturation, and then stained for examination of their nuclear stage. In Experiment 2, to investigate effects of length of in vitro maturation (18, 20, 22, 24 h) of oocytes (18-h, 20-h, 22-h and 24-h group, respectively) obtained from the ovaries stored in a saline for 18 h at 20°C on the subsequent in vitro development after IVF and IVC. Following insemination, the presumptive zygotes were cultured in CR1aa + 5% CS for 6 days to assess the development of embryos on Day 2 (Day 0 = the day of IVF) for rates of cleavage and on Day 6 for rates of embryo development to morulae (M), compacted morulae (CM), and blastocyst (BL) stages. The data of nuclear stage were analyzed by ANOVA after transformation to arcsine, and the rates of embryo development were analyzed by chi-square. There were two peaks of GVBD in the storage group, one occurred at 2 h of maturation culture, the other at 4–8 h of culture as control. There were between-treatment differences in the timing of increase in the rates of oocytes to reach MII. After 12 h of culture 21.2 ± 1.1% of oocytes in the Saline-group and 11.6 ± 4.6% of oocytes in the Bag-group reached MII, but no oocytes in the control group reached MII (P < 0.05). Furthermore, the rate of oocytes in the Saline-group matured to MII at 20 h of culture was lower than that of the control group (Bag-group: 67.9 ± 7.3%; Saline-group: 61.2 ± 14.5%; control: 82.9 ± 5.3%) (P < 0.05). The rates of embryos that cleaved after IVF of IVM oocytes in the 18-h group (90.2 ± 7.0%) was higher than those of the other groups (20-h group: 81.3 ± 8.2%, 22-h group: 80.5 ± 13.2%, 24-h group: 75.8 ± 6.0%) (P < 0.05). The rate of embryos developed to M, CM, and BL stages in the 18-h group (48.4 ± 6.7%) was the highest among the treatments, and significantly higher than that of the 24-h group (36.2 ± 6.7%) (P < 0.05). These results indicated that the timing of undergoing GVBD and reaching MII of oocytes obtained from the stored ovaries was earlier than that of oocytes obtained from the non-preserved ovaries, and the optimal maturation time for oocytes obtained from stored ovaries was 18 h. This work was supported by The Ito Foundation, Tokyo, Japan.

Modification of ovary stock solution with magnesium and raffinose improves the developmental competence of oocytes after long preservation

Zygote ◽  
2005 ◽  
Vol 13 (4) ◽  
pp. 303-308 ◽  
Author(s):  
H. Iwata ◽  
T. Hayashi ◽  
H. Sato ◽  
K. Kimura ◽  
T. Kuwayama ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Germinal Vesicle ◽  
Storage Period ◽  
Cell Number ◽  
Developmental Competence ◽  
Germinal Vesicle Breakdown ◽  
Storage Solutions ◽  
Stock Solutions ◽  
Phosphate Buffered Saline

During ovary storage oocytes lose some of their developmental competence. In the present study, we maintained storage solutions of phosphate-buffered saline (PBS) at various temperatures (20 or 35 °C) or supplemented them with magnesium (Mg), raffinose and sucrose. Subsequently, we examined the kinetics of electrolytes in the follicular fluid (FF) during the ovary storage period (9h), the survival rate of granulosa cells in the follicles, and the developmental competence of oocytes after the storage. Lowering the temperature from 35 to 20 °C increased the total cell number of blastocysts that developed at 7 days after in vitro maturation and in vitro fertilization of oocytes. In stock solution with supplements of 15 mM Mg or a combination of 5 mM Mg and 10 mM raffinose or sucrose, a significantly higher number of oocytes developed into blastocysts with a large number of cells in each blastocyst, and a significantly higher number of living granulosa cells were obtained as compared with stock solutions without any supplements. During ovary storage, the concentrations of potassium and chloride in the FF were increased, and the addition of Mg to the stock solution increased the concentration of Mg in the FF. Germinal vesicle breakdown in oocytes that were collected from ovaries stored in the solution supplemented with 15 mM Mg or a combination of 5 mM Mg and 10 mM of raffinose occurred at a slower rate than that in oocytes collected from ovaries stored in PBS alone. On the other hand, the oocytes collected from ovaries stored in the solution supplemented with 15 mM Mg or a combination of 5 mM Mg and 10 mM raffinose reached the metaphase II (MII) stage more rapidly than the oocytes collected from ovaries stored in the PBS alone. In conclusion, the modification of stock solution by the addition of Mg and raffinose improved the developmental competence of oocytes obtained from ovaries preserved for a long period.

Improvement of the developmental competence of canine oocyte using caffeine supplementation during IVM at different maturation time

Zygote ◽  
2018 ◽  
Vol 26 (2) ◽  
pp. 162-167 ◽  
Author(s):  
Mohamed Fathi ◽  
A. Salama ◽  
Magdy R. Badr
Keyword(s):  
Developmental Competence ◽  
Control Group ◽  
Free Medium ◽  
Maturation Time ◽  
Fluid Medium ◽  
Developmental Potential ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Oviductal Fluid

SummaryThe aim of the current study was to investigate the effect of caffeine supplementation during in vitro maturation (IVM) for different maturation times on the developmental potential of canine oocytes recovered from ovariohysterectomized bitches. The recovered cumulus–oocytes complexes were in vitro matured for 72 h. Here, 10 mM caffeine was added to the maturation medium for different incubation times (caffeine from 0–72 h maturation, caffeine for the first 24 h of maturation only, caffeine addition from 24 to 48 h maturation time, caffeine addition from 48 to 72 h maturation or in caffeine-free medium, control group). The matured oocytes were in vitro fertilized using frozen–thawed spermatozoa. The presumptive zygotes were in vitro cultured in synthetic oviductal fluid medium for 5 days. The results showed that both maturation and fertilization rates were significantly higher (P ˂ 0.05) using caffeine-treated medium for the first 24 h of maturation compared with the control and other two groups of caffeine treatment (from 24 to 48 h and from 48 to 72 h), whereas use of caffeine-treated medium for a 0–72 h incubation time did not affect these rates (P > 0.05). Interestingly, the matured oocytes in caffeine-supplemented medium for the first 24 h or from 0–72 h showed a significant (P ˂ 0.05) increase in the total number of cleaved embryos compared with the control group. In conclusion, supplementation of the maturation medium with 10 mM caffeine for the first 24 h of maturation or during the whole maturation time (0–72 h) improved nuclear maturation and subsequent embryo development preimplantation following in vitro fertilization.

161 EFFECT OF OVIDUCTAL FLUID ON BOVINE OOCYTE ZONA PELLUCIDA HARDENING AND SPERM BINDING, AND ON EARLY EMBRYO DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 210
Author(s):  
P. Hugon ◽  
J. Lamy ◽  
E. Corbin ◽  
P. Mermillod ◽  
M. Saint-Dizier
Keyword(s):  
Corpus Luteum ◽  
Embryo Development ◽  
Zona Pellucida ◽  
Developmental Stages ◽  
Early Embryo ◽  
Developmental Competence ◽  
Control Group ◽  
Vitro Fertilization ◽  
Oviductal Fluid

This study was designed to evaluate the effects of oviductal fluid at different periovulatory times on oocyte maturation, modification of the zona pellucida (ZP), fertilization and embryo development. Bovine oviducts were collected at a slaughterhouse and classified as preovulatory (pre-ov: 1 pre-ov follicle and a regressing corpus luteum) or post-ovulatory (post-ov: a corpus haemorrhagicum or recent corpus luteum; n = 10 cows/stage). Both oviducts were flushed with 1 mL of sterile TCM-199, and oviductal flushes (OF) were aliquoted and stored at –80°C. Abattoir-derived bovine ovaries were aspirated and cumulus‐oocyte complexes (COC) with at least 3 cumulus layers and homogeneous oocyte cytoplasm were in vitro matured for 22 h in standard maturation medium (control group, n = 319) or in standard medium with 2× concentrated additives supplemented (50% v/v) with pre-ov OF (n = 255) or post-ov OF (n = 248). After in vitro maturation (IVM), subgroups of COC were denuded, and the time of digestion of the ZP by pronase 0.1% (v/v in TCM-199) was determined to evaluate ZP hardening. After IVM, COC were fertilised in vitro for 18–20 h at a final concentration of 1.106 million spermatozoa (spz)/mL. After in vitro fertilization (IVF), COC were denuded, washed twice and cultured for 8 days more under standard conditions. After IVM, IVF, and embryo culture, oocytes/embryos were fixed with ethanol, stained with Hoescht, and examined under fluorescence microscopy for determination of (1) maturation and developmental stages, (2) numbers of fertilised and polyspermic oocytes, and (3) spz bound to the ZP. Percentages were compared between groups by chi-square. Times of ZP digestion were compared by Kruskal‐Wallis test. Numbers of spz bound to the ZP were compared by ANOVA on normalised data followed by Newman-Keuls tests. Data are presented as mean ± SEM. A P < 0.05 was considered significant. Addition of OF during IVM had no effect on maturation rates compared with the control. However, the digestion time of the ZP by pronase was reduced after IVM with pre-ov OF (313 ± 21 s; n = 26) compared with post-ov OF (459 ± 23 s; n = 23) but not with the control (416 ± 30 s; n = 25). After IVF, the number of spermatozoa bound to the ZP was increased after IVM with pre-ov OF (57 ± 5 spz/oocyte; n = 67) and decreased after IVM with post-ov OF (34 ± 3 spz/oocyte; n = 76) compared with the control (42 ± 5 spz/oocyte; n = 60). Addition of OF during IVM had no effect on rates of IVF and polyspermia. However, the rate of development to the blastocyst stage was less after IVM with post-ov OF (10%, n = 97 cleaved oocytes) compared with control (24%, n = 130) and pre-ov OF (29%, n = 101). In conclusion, the OF collected before ovulation decreased the resistance of the ZP to protease digestion and increased its ability to bind spz, whereas it was the opposite for the post-ov OF. Furthermore, the post-ov OF decreased the developmental competence of fertilised oocytes.

scholarly journals Delay of nuclear maturation and reduction in developmental competence of pig oocytes after mineral oil overlay of in vitro maturation media

Reproduction ◽  
2002 ◽  
pp. 557-564 ◽  
Author(s):  
M Shimada ◽  
N Kawano ◽  
T Terada
Keyword(s):  
Steroid Hormones ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Mineral Oil ◽  
Developmental Competence ◽  
Cdc2 Kinase ◽  
Germinal Vesicle Breakdown ◽  
High Concentrations ◽  
P34 Cdc2

Steroid hormones, such as progesterone, oestrogen, androgen and meiosis activating sterols, are secreted from cumulus cells that are stimulated by gonadotrophins during maturation of oocytes in vitro. These steroid hormones may be absorbed by mineral oil or paraffin oil; however, in vitro maturation of pig oocytes is commonly performed using medium covered by oil. In this study, high concentrations of progesterone, oestradiol and testosterone were detected in the culture medium after pig cumulus-oocyte complexes (COCs) were cultured with FSH and LH for 44 h in medium without an oil overlay. However, high concentrations of these steroid hormones were not detected in medium when COCs were cultured with the mineral oil overlay. When high concentrations of these steroid hormones were secreted by COCs, germinal vesicle breakdown (GVBD) and the activation of p34(cdc2) kinase and mitogen-activated protein (MAP) kinase in oocytes occurred earlier in comparison with oocytes cultured in medium covered with mineral oil. Moreover, a decrease in p34(cdc2) kinase activity during meiotic progression beyond metaphase I was observed in oocytes cultured in conditions under which high concentrations of steroid hormones were secreted by COCs. In addition, the rate of development to the blastocyst stage after IVF was higher in oocytes matured in medium without an oil overlay. These adverse effects of oil may be explained by absorption by the oil of cumulus-secreted steroids or by the release of toxic compounds into the medium.

Effect of trichostatin A on fertilization and embryo development during extended culture of mouse oocyte

Zygote ◽  
2011 ◽  
Vol 20 (1) ◽  
pp. 27-32 ◽  
Author(s):  
Byung Chul Jee ◽  
Jun Woo Jo ◽  
Jung Ryeol Lee ◽  
Chang Suk Suh ◽  
Seok Hyun Kim ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Embryo Development ◽  
Trichostatin A ◽  
Formation Rate ◽  
Control Group ◽  
Blastocyst Formation ◽  
Developmental Potential ◽  
Extended Culture ◽  
Mouse Oocytes

SummaryWe performed this study to investigate the effect of histone deacetylase inhibition during extended culture of in vitro matured mouse oocytes. In vitro matured mouse (BDF1) oocytes were cultured in vitro for 6, 12, and 24 h, respectively, and then inseminated. During in vitro culture for 6 and 12 h, two doses of trichostatin A (TSA), a histone deacetylase inhibitor, were added (100 nM and 500 nM) to the culture medium and the oocytes were then inseminated. During the 24-h in vitro culture, two doses of TSA were added (100 nM and 500 nM) to the medium and the oocytes were activated with 10 mM SrCl2. After the 6-h culture, the fertilization rate was similar to that of the control group, but the blastocyst formation rate was significantly decreased. After the 12-h culture, both the fertilization and blastocyst formation rates were significantly decreased. After the 24-h culture, total fertilization failure occurred. In the oocytes cultured for 6 and 12 h, the fertilization and blastocyst formation rates did not differ between the TSA-supplemented and control groups. Although extended culture of the mouse oocytes significantly affected their fertilization and embryo development, TSA supplementation did not overcome their decreased developmental potential.

scholarly journals Graft site and gonadotrophin stimulation influences the number and quality of oocytes from murine ovarian tissue grafts

Reproduction ◽  
2006 ◽  
Vol 131 (5) ◽  
pp. 851-859 ◽  
Author(s):  
Hsiao Yun Yang ◽  
Shae-Lee Cox ◽  
Graham Jenkin ◽  
Jock Findlay ◽  
Alan Trounson ◽  
...  
Keyword(s):  
Ovarian Tissue ◽  
Germinal Vesicle ◽  
Fertilization Rate ◽  
Ovarian Tissue Cryopreservation ◽  
Control Group ◽  
Developmental Potential ◽  
Oocyte Yield ◽  
Tissue Grafts

Ovarian tissue cryopreservation and subsequent transplantation can restore fertility in cancer patients. This study used a mouse ovarian grafting model to investigate whether the graft site (bursal cavity, the kidney capsule or subcutaneous) influences the number, fertilization rate and developmental potential of oocytes recovered from grafts and whether using a standard gonadotrophin stimulation protocol would increase oocyte yield from the grafts. Mouse ovarian tissue was grafted into four week old mice and collected three weeks later. Graft recipients were treated either with or without exogenous gonadotrophin stimulation prior to graft collection. Grafted ovaries yielded oocytes that were either at the germinal vesicle (GV) stage or mature metaphase II (MII) stage at collection. These GV oocytes were matured beforein vitrofertilization (IVF), while the MII oocytes underwent IVF immediately. Oocytes collected from the oviducts of non-grafted superovulated mice of the same age served as controls. Two-cell embryos were transferred to pseudopregnant recipients and recovered at day 15 of gestation or left to go to term. Graft retrieval and the number of oocytes from each graft were lowest from the subcutaneous graft site. The number of two-cell embryos produced was significantly higher for oocytes from the grafts to the bursa as compared with the other sites. All graft sites gave rise to embryos with comparable implantation rates and developmental potential to fetuses and offspring following transfer. However, the oocytes from grafted ovaries had a significantly lower developmental potential when compared with the control group. Stimulation with exogenous gonadotrophins did not significantly increase oocyte yield from grafted ovaries but did enhance oocyte maturation and development. In conclusion, graft site affects the number and quality of oocytes produced from ovarian grafts.

scholarly journals Supplementation of c-type natriuretic peptide during the whole in vitro growth period benefits the development of murine secondary follicles

2020 ◽  
Author(s):  
Ang Li ◽  
Haixia Cao ◽  
Hongxia Li ◽  
Ruijiao Li ◽  
Huaixiu Wang ◽  
...  
Keyword(s):  
Natriuretic Peptide ◽  
Ovulation Induction ◽  
Germinal Vesicle ◽  
Antral Follicle ◽  
Control Group ◽  
Follicle Development ◽  
In Vitro Growth ◽  
Germinal Vesicle Breakdown ◽  
Preovulatory Follicles

Abstract Background Supplementation of c-type natriuretic peptide (CNP) in the culture medium shortly before in vitro maturation (IVM) has been reported to be effective in delaying meiotic resumption of murine oocyte. The present study investigated the effect of CNP supplementation during the whole period of in vitro growth (IVG) on the development of murine secondary ovarian follicles.Methods Late secondary ovarian follicles isolated from ovaries of Kunming mice were cultured in vitro with and without supplementation of CNP. In experiment 1, CNP was supplemented at the early stage and the follicle development was evaluated. In experiment 2 and 3, CNP was supplemented during the whole period of IVG. In experiment 2, follicle development and oocyte maturity were evaluated. In group 3, follicle development and rate of cleaved embryos after in vitro fertilization (IVF) was assessed.Results In control group in all 3 experiments, granulosa cells migrated from within follicle and adhered to the plate at different degrees. The follicles flattened and could not reach antral stage. About 39.8% (39/98) of the oocytes ovulated nakedly. As no antral follicle was obtained, IVF was not performed in control group in experiment 3. In experiment group in all 3 experiments, no migration of guanulosa cells was observed and the follicles grew three-dimensionally. Ovulation of naked oocyte decreased substantially. The rate of antral stage follicle were 45% (18/40) in experiment 1. This parameter was 75.9% (44/58) in experiment 2 and 3 combined. In experiment 2, in preovulatory follicles without ovulation induction, oocytes at germinal vesicle (GV) stage and germinal vesicle breakdown (GVBD) stage were 87.5% (14/16) and 12.5% (2/16), respectively. In preovulatory follicles with ovulation induction, no GV stage oocyte was retrieved, oocytes at GVBD and metaphase II (MII) stage were 50% (8/16), respectively. In experiment 3, among 18 follicles cultured, 12 cumulus-oocyte complexes (COC) ovulated automatically after ovulation induction. Eleven oocytes were fertilized and cleaved. Compared with control groups, the follicle development assessed by naked oocyte ovulation and follicle stage (preantral follicle and antral follicle) in experiment groups were significantly superior (p<0.0001). CNP effectively maintained oocytes’ meiotic arrest and enhanced fertilization competency.Conclusions The supplementation of CNP in culture system of murine late secondary follicle during the whole period of IVG could sustain the 3-dimensional structure of follicle, increase the antral formation rate. As a result, the oocyte’s competency to be fertilized was greatly improved.

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