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 ◽  
...  

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.

2020 ◽  
Author(s):  
Ang Li ◽  
Haixia Cao ◽  
Hongxia Li ◽  
Ruijiao Li ◽  
Huaixiu Wang ◽  
...  

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.


Zygote ◽  
2020 ◽  
pp. 1-5
Author(s):  
Li Ang ◽  
Cao Haixia ◽  
Li Hongxia ◽  
Li Ruijiao ◽  
Guo Xingping ◽  
...  

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.


2014 ◽  
Vol 26 (6) ◽  
pp. 806 ◽  
Author(s):  
Yong-Xun Jin ◽  
Ming-Hui Zhao ◽  
Zhong Zheng ◽  
Jung-Suk Kwon ◽  
Seul-Ki Lee ◽  
...  

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.


2018 ◽  
Vol 30 (1) ◽  
pp. 190
Author(s):  
G. L. Vasconcelos ◽  
R. Maculan ◽  
N. Alves ◽  
A. L. A. P. L. Ribeiro ◽  
A. W. B. Silva ◽  
...  

Embryo production may be enhanced when associated with cows selected on the basis of fertility markers, which should be easy to measure, such as antral follicle count (AFC) and genital tract morphometrics. The objective was to evaluate the effects of AFC class on oocyte 24-h outcome and in vitro embryo production in Bos indicus beef cows. Brahman (n = 151) cows (2-13 years old, 344-803 kg of BW, and 7-9 BCS). Low (LAFC), intermediate (IAFC), and high (HAFC) antral follicle classes were defined as follows: LAFC ≤ 30; IAFC 30-49; and HAFC ≥50 AFC. All follicles ≥3 mm in diameter were aspirated by conventional ovum pick-up technique. Only cumulus–oocyte complexes with at least 2 layers of granulosa cells and homogeneous cytoplasm were used for in vitro culture. They were matured in TCM-199 plus supplements for 24 h at 38.7°C in a 5% CO2 humidified atmosphere. After 24 h of maturation, a subset of oocytes (n = 319) was fixed and analysed under fluorescent microscopy and oocyte outcome was evaluated by classification, as follows: germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I/anaphase I/telophase I (MIAITI), and metaphase II (MII). The second subset of oocytes (n = 797) was fertilized in Ferti-TALP (10-15 oocytes per 60-µL drop) with frozen–thawed semen (18-22 h at 38.7°C in 5% CO2 after Percoll) from a single bull previously tested for good in vitro fertility. Presumptive zygotes were cultivated in CR2 medium for 48 h at 37.8°C in 5% CO2. For the remaining 96 h, embryos were transferred to 10% FCS-supplemented TCM-199 drops until the final evaluation. Data were analysed by the GENMOD, GLM, and CORR procedures of SAS (SAS Institute Inc., Cary, NC, USA). Viable oocytes, total embryos, and embryo production efficiency (viable oocyte/total embryos produced; P < 0.05) to AFC in various degrees (r2 = 0.87, 0.86, 0.30, respectively). The proportion of oocytes in GV, GVBD, MIAITI, and MII were different (P < 0.05) between LAFC, IAFC, and HAFC classes [GV: 12.3% (13/106)a, 3.1% (3/96)a and 4.3% (5/117)b, respectively]; [GVBD: 32.1% (34/106)a, 8.3% (8/96)a and 6.0 (7/117)b]; [MIAITI: 14.2% (15/106)a, 26.0% (25/96)b and 8.5% (10/117)c, respectively] and [MII: 41.5% (44/106)b, 62.5% (60/96)a and 81.2% (95/117)c, respectively). In conclusion, high AFC is positively related to better in vitro embryo fertility and to 24-h oocyte outcome after in vitro maturation.


2010 ◽  
Vol 90 (2) ◽  
pp. 189-196
Author(s):  
X -L. Sun ◽  
W -Z. Ma ◽  
Y -B. Zhu ◽  
Z -H. Wu ◽  
L. An ◽  
...  

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


2005 ◽  
Vol 17 (2) ◽  
pp. 295
Author(s):  
M. Narita ◽  
S. Goda ◽  
Y. Inaba ◽  
K. Imai ◽  
S. Matoba ◽  
...  

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.


2012 ◽  
Vol 26 (7) ◽  
pp. 1158-1166 ◽  
Author(s):  
Yorino Sato ◽  
Yuan Cheng ◽  
Kazuhiro Kawamura ◽  
Seido Takae ◽  
Aaron J.W. Hsueh

Abstract C-type natriuretic peptide (CNP) encoded by the NPPC (Natriuretic Peptide Precursor C) gene expressed in ovarian granulosa cells inhibits oocyte maturation by activating the natriuretic peptide receptor (NPR)B (NPRB) in cumulus cells. RT-PCR analyses indicated increased NPPC and NPRB expression during ovarian development and follicle growth, associated with increases in ovarian CNP peptides in mice. In cultured somatic cells from infantile ovaries and granulosa cells from prepubertal animals, treatment with CNP stimulated cGMP production. Also, treatment of cultured preantral follicles with CNP stimulated follicle growth whereas treatment of cultured ovarian explants from infantile mice with CNP, similar to FSH, increased ovarian weight gain that was associated with the development of primary and early secondary follicles to the late secondary stage. Of interest, treatment with FSH increased levels of NPPC, but not NPRB, transcripts in ovarian explants. In vivo studies further indicated that daily injections of infantile mice with CNP for 4 d promoted ovarian growth, allowing successful ovulation induction by gonadotropins. In prepubertal mice, CNP treatment alone also promoted early antral follicle growth to the preovulatory stage, leading to efficient ovulation induction by LH/human chorionic gonadotropin. Mature oocytes retrieved after CNP treatment could be fertilized in vitro and developed into blastocysts, allowing the delivery of viable offspring. Thus, CNP secreted by growing follicles is capable of stimulating preantral and antral follicle growth. In place of FSH, CNP treatment could provide an alternative therapy for female infertility.


Zygote ◽  
2020 ◽  
pp. 1-3
Author(s):  
Burcu Ozbakir ◽  
Pinar Tulay

Summary Alcohol consumption has long been shown to affect both fetal health and pregnancy. In this study, antral follicle count, maturation level of oocytes including morphological assessment and number of metaphase I (MI), metaphase II (MII) and germinal vesicle (GV) stage oocytes obtained from young women (age < 30 years old) with or without alcohol consumption were investigated. In total, 20 healthy women who were social drinkers and 36 healthy women who do not consume alcohol were involved in this study. Women in both study and control groups were undergoing controlled ovarian stimulation. The antral follicle count and the number and quality of the oocytes retrieved were evaluated and recorded. In total, 635 antral follicles, 1098 follicles and 1014 oocytes with 820 MII, 72 MI and 78 GV stage oocytes were collected from the social drinkers. In the control group, 628 antral follicles, 1136 follicles and 1085 oocytes with 838 MII, 93 MI and 102 GV stage oocytes were evaluated. The results of this study showed that the antral follicle count was very similar in both groups. The number of oocytes and MII stage oocytes was slightly higher in the control group, although it was not a significant difference. This study showed that although the consumption of alcohol may have adverse effects post-implantation, it may not have a solid effect during oogenesis in young women. The results of this study are especially important in clinical settings as some women who are social drinkers undergo in vitro fertilization treatments.


1992 ◽  
Vol 12 (7) ◽  
pp. 3192-3203 ◽  
Author(s):  
K M Pickham ◽  
A N Meyer ◽  
J Li ◽  
D J Donoghue

The p34cdc2 protein kinase is a component of maturation-promoting factor, the master regulator of the cell cycle in all eukaryotes. The activity of p34cdc2 is itself tightly regulated by phosphorylation and dephosphorylation. Predicted regulatory phosphorylation sites of Xenopus p34cdc2 were mutated in vitro, and in vitro-transcribed RNAs were injected into Xenopus oocytes. The cdc2 single mutants Thr-14----Ala and Tyr-15----Phe did not induce germinal vesicle breakdown (BVBD) upon microinjection into oocytes. In contrast, the cdc2 double mutant Ala-14/Phe-15 did induce GVBD. Both the Ala-14 and Ala-14/Phe-15p34cdc2 mutants were shown to coimmunoprecipitate cyclin B1 and to phosphorylate histone H1 in immune complex kinase assays. Microinjection of antisense oligonucleotides to c-mosXe was used to demonstrate the role of mos protein synthesis in the induction of GVBD by the Ala-14/Phe-15 cdc2 mutant. Thr-161 was also mutated. p34cdc2 single mutants Ala-161 and Glu-161 and triple mutants Ala-14/Phe-15/Ala-161 and Ala-14/Phe-15/Glu-161 failed to induce GVBD in oocytes and showed a decreased binding to cyclin B1 in coimmunoprecipitations. Each of the cdc2 mutants was also assayed by coinjection with cyclin B1 or c-mosXe RNA into oocytes. Several of the cdc2 mutants were found to affect the kinetics of cyclin B1 and/or mos-induced GVBD upon coinjection, although none affected the rate of progesterone-induced maturation. We demonstrate here the significance of Thr-14, Tyr-15, and Thr-161 of p34cdc2 in Xenopus oocyte maturation. In addition, these results suggest a regulatory role for mosXe in induction of oocyte maturation by the cdc2 mutant Ala-14/Phe-15.


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