A co-culture system with preantral follicular granulosa cells in vitro induces meiotic maturation of immature oocytes

2011 ◽  
Vol 135 (5) ◽  
pp. 513-522 ◽  
Author(s):  
Zhanbiao Li ◽  
Pan Zhang ◽  
Zhipeng Zhang ◽  
Bo Pan ◽  
Huhe Chao ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Culture System ◽  
Meiotic Maturation ◽  
Immature Oocytes

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.

Spatiotemporal dynamics of intracellular [Ca2+]i oscillations during the growth and meiotic maturation of mouse oocytes

Development ◽  
1994 ◽  
Vol 120 (12) ◽  
pp. 3507-3517 ◽  
Author(s):  
J. Carroll ◽  
K. Swann ◽  
D. Whittingham ◽  
M. Whitaker
Keyword(s):  
Spatial Organization ◽  
Calcium Release ◽  
Calcium Oscillations ◽  
Meiotic Maturation ◽  
Similar Proportion ◽  
Calcium Waves ◽  
Apparent Difference ◽  
Immature Oocytes ◽  
Mouse Oocytes

Calcium oscillations occur during meiotic maturation of mouse oocytes. They also trigger activation at fertilization. We have monitored [Ca2+]i in oocytes at different stages of growth and maturation to examine how the calcium release mechanisms alter during oogenesis. Spontaneous calcium oscillations occur every 2–3 minutes in the majority of fully grown (but immature) mouse oocytes released from antral follicles and resuming meiosis. The oscillations last for 2–4 hours after release from the follicle and take the form of global synchronous [Ca2+]i increases throughout the cell. Rapid image acquisition or cooling the bath temperature from 28 degrees C to 16 degrees C did not reveal any wave-like spatial heterogeneity in the [Ca2+]i signal. Calcium appears to reach highest levels in the germinal vesicle but this apparent difference of [Ca2+] in nucleus and cytoplasm is an artifact of dye loading. Smaller, growing immature oocytes are less competent: about 40% are able to resume meiosis and a similar proportion of these oocytes show spontaneous calcium oscillations. [Ca2+]i transients are not seen in oocytes that do not resume meiosis spontaneously in vitro. Nonetheless, these oocytes are capable of [Ca2+]i oscillations since they show them in response to the addition of carbachol or thimerosal. To examine how the properties of calcium release change during meiotic maturation, a calcium-releasing factor from sperm was microinjected into fully grown immature and mature oocytes. The sperm-factor-induced oscillations were about two-fold larger and longer in mature oocytes compared to immature oocytes. Calcium waves travelling at 40–60 microns/second were generated in mature oocytes, but not in immature oocytes. In some mature oocytes, successive calcium waves had different sites of origin. The modifications in the size and spatial organization of calcium transients during oocyte maturation may be a necessary prerequisite for normal fertilization.

scholarly journals C-kit signaling promotes human pre-implantation 3PN embryonic development and blastocyst formation

2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Jun Tan ◽  
Yang Zou ◽  
Zhi-Hui Huang ◽  
Zhi-Qin Zhang ◽  
Li-Ping Wu ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Embryo Culture ◽  
Culture System ◽  
Target Genes ◽  
Regulatory Mechanism ◽  
Control Group ◽  
Blastocyst Formation ◽  
High Quality ◽  
Immature Oocytes

Abstract Background Although in vitro culture system has been optimized in the past few decades, the problem of few or no high quality embryos has been still not completely solved. Accordingly, fully understanding the regulatory mechanism of pre-implantation embryonic development would be beneficial to further optimize the in vitro embryo culture system. Recent studies have found the expression of c-kit in mouse embryo and its promotion effects on mouse embryonic development. However, it is unclear the expression, the role and the related molecular regulatory mechanism of c-kit in human pre-implantation embryo development. Therefore, the present study is to determine whether c-kit is expressed in human pre-implantation embryos, and to investigate the possible regulatory mechanism of c-kit signaling in the process of embryonic development. Methods The present study includes human immature oocytes and three pronucleus (3PN) embryos collected from 768 women (28–32 ages) undergoing IVF, and normal 2PN embryos collected from ICR mice. Samples were distributed randomly into three different experimental groups: SCF group: G-1™ (medium for culture of embryos from the pro-nucleate stage to day 3) or G-2™ (medium for culture of embryos from day3 to blastocyst stage) + HSA (Human serum album) solution + rhSCF; SCF + imanitib (c-kit inhibitor) group: G-1™ or G-2™ + HSA solution + rhSCF + imanitib; SCF + U0126 (MEK/ERK inhibitor) group: G-1™ or G-2™ + HSA solution + rhSCF + U0126; Control group: G-1™ or G-2™ + HSA solution + PBS; The rate of good quality embryos at day 3, blastulation at day 6 and good quality blastulation at day 6 were analysis. RT-PCR, western blot and immunofluorescence staining were applied to detect the target genes and proteins in samples collected from human or mice, respectively. Results c-kit was expressed ubiquitously in all human immature oocytes, 3PN embryos and 3PN blastocysts. In the experiment of human 3PN embryos, compared with other groups, SCF group showed obviously higher rate of good quality at day 3, better rate of blastocyst formation at day 6 and higher rate of good quality blastocyst formation at day 6. Furthermore, we observed a higher ETV5 expression in SCF group than that in other groups. Similar results were also found in animal experiment. Interestingly, we also found a higher phosphorylation level of MEK/ERK signal molecule in mice embryos from SCF group than those from other groups. Moreover, inhibition of MEK/ERK signaling would remarkably impeded the mice embryonic development, which might be due to the reduced ETV5 expression. Conclusions The present study firstly revealed that c-kit signaling might promote the human pre-implantation embryonic development and blastocyst formation by up-regulating the expression of ETV5 via MEK/ERK pathway. Our findings provide a new idea for optimizing the in vitro embryo culture condition during ART program, which is beneficial to obtain high quality embryos for infertile patients.

Effects of gonadotropins and insulin-like growth factors-I and -II on in vitro steroid production by bovine granulosa cells

1998 ◽  
Vol 78 (4) ◽  
pp. 587-597 ◽  
Author(s):  
M. Y. Yang ◽  
R. Rajamahendran
Keyword(s):  
Growth Factors ◽  
Granulosa Cells ◽  
Culture System ◽  
Physiological Role ◽  
Serum Free ◽  
Igf I ◽  
The Media ◽  
Estradiol 17Β ◽  
Insulin Like Growth Factors

The objectives of this study were: 1) to develop a bovine granulosa cell (GC) culture system; and 2) to use this system to evaluate the effects of gonadotropins (FSH and LH) and insulin-like growth factors-I and -II (IGF-I and IGF-II) on steroidogenesis of bovine GC derived from small, medium, and large antral follicles (diameters ≤4, 5–8 and >8 mm, respectively). Granulosa cells were cultured (concentration, 5 × 105 cells per well) in serum-free medium for 48 h with variable doses of hormones and growth factors. Concentrations of progesterone (P4) and estradiol-17β (E2) in the media were determined by radioimmunoassay. Basal E2 production by GC from follicles of all sizes decreased with time of culture (P < 0.01) while basal P4 production increased (P < 0.01). Basal E2 and P4 production increased with increasing size of follicles (P < 0.01). Only very low concentrations of FSH stimulated E2 production from medium and large follicles. Follicle-stimulating hormone stimulated P4 production by GC of follicles of all sizes (P < 0.05). Luteinizing hormone inhibited E2 production by GC in medium and large follicles (P < 0.05), suggesting that LH is responsible for the rise in plasma E2 through effects on both theca cells and GC. A dose of 100 ng mL−1 of IGF-I increased E2 production by GC from medium and large follicles (P < 0.05). Progesterone production by GC from all categories of follicles was also stimulated by IGF-I (P < 0.05). Estradiol-17β production by GC from large follicles decreased in response to IGF-II (P < 0.05). The physiological role of IGF-II on steroidogenesis in the bovine ovary remains to be elucidated. In summary, these results demonstrate the development of a serum-free culture system for bovine GC, and that FSH, LH, IGF-I and IGF-II have different effects on steroidogenesis by bovine GC from different size follicles. Key words: Granulosa cells, gonadotropins, Insulin-like growth factors, progesterone, estradiol-17β, cows

Ultrastructural localisation of calcium deposits in pig oocytes maturing in vitro: effects of verapamil

Zygote ◽  
2003 ◽  
Vol 11 (3) ◽  
pp. 253-260 ◽  
Author(s):  
J. Rozinek ◽  
Z. Vaňourková ◽  
M. Sedmíkova ◽  
V. Lánská ◽  
J. Petr ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Channel Blocker ◽  
Meiotic Maturation ◽  
Oocyte Nucleus ◽  
Calcium Dependent ◽  
Calcium Deposits ◽  
In Vitro Effects ◽  
Oocyte Meiotic Maturation ◽  
Metaphase I

The culture of pig oocytes in the presence of the calcium channel blocker verapamil (0.02 mM) resulted in the blocking of meiosis at the metaphase I stage, and only a small fraction (about 28%) of the oocytes were able to continue their maturation to the stage of metaphase II. Hence, meiotic maturation in pig oocytes is a calcium-dependent process. After isolation of the pig oocytes from their follicles, the intracellular calcium deposits in the oocyte and granulosa cells, detectable using the combined oxalate-pyroantimonate method, are depleted. The amount of calcium deposits in the oocyte and granulosa cells increased during oocyte meiotic maturation in vitro, especially in the nucleus, mitochondria, vacuoles and cytoplasm. The replenishment of calcium deposits is significantly changed under the effect of verapamil. The increase in calcium deposits in the oocyte nucleus was delayed, a much larger amount of deposits was formed in the mitochondria, and the amount of deposits in the vacuoles was demonstrably smaller. A significant peak in the accumulation of calcium deposits was observed in the cytoplasm of verapamil-treated oocytes after 16 h of in vitro culture. We propose that an altered pattern in the replenishment of calcium deposits can disturb intracellular signalling and prevent the exit of oocytes from the metaphase I stage.

scholarly journals Regulation of mitochondrial DNA accumulation during oocyte growth and meiotic maturation in the mouse

Reproduction ◽  
2012 ◽  
Vol 144 (2) ◽  
pp. 177-185 ◽  
Author(s):  
Enas Mahrous ◽  
Qin Yang ◽  
Hugh J Clarke
Keyword(s):  
Granulosa Cells ◽  
Oocyte Quality ◽  
Meiotic Maturation ◽  
Mt Dna ◽  
Oocyte Growth ◽  
Α Subunit ◽  
Insufficient Amount ◽  
Mtdna Replication ◽  
Replication Factors

Oocytes accumulate an enormous quantity of mitochondrial (mt) DNA, and an insufficient amount of mtDNA may underlie some cases of poor oocyte quality leading to infertility. Little is known, however, about the mechanisms that govern the timing and regulation of mtDNA accumulation during oogenesis. We report, through analysis of the mtDNA content of individual oocytes of the mouse, that mtDNA accumulates steadily during oocyte growth to reach a value of ∼175 000 copies per cell. MtDNA content ceases to increase once oocytes reach full size and remains unchanged during meiotic maturation. To test whether mtDNA accumulation depends on oocyte growth, we inhibited growth in vitro in two ways – by exposing complexes comprising partially grown oocytes enclosed by granulosa cells to a chemical inhibitor of the phosphatidylinositol-3-kinase signaling pathway and by removing the surrounding granulosa cells from partially grown oocytes. Under both conditions, the oocytes fail to grow, but mtDNA accumulation is unaffected, indicating that the two processes can be mechanistically uncoupled. Quantitative analysis of the mRNAs encoding proteins required for mtDNA replication revealed that Polg (Polga) (polymerase-γ, α-subunit), Polg2 (Polgb), and Tfam (transcription factor A, mitochondrial) increase during oocyte growth but then decrease after fully grown oocytes become transcriptionally silent as indicated by the non-surrounded nucleolus-to-surrounded nucleolus transition. Thus, there is a correlation between the decline in the quantity of mRNAs encoding mtDNA replication factors in fully grown oocytes and the arrest of mtDNA accumulation in these cells, suggesting that the two events may be causally linked.

82 VITRIFICATION OF IMMATURE OVINE OOCYTES WITH CRYOLOOP™: EFFECT ON MEIOTIC MATURATION

2008 ◽  
Vol 20 (1) ◽  
pp. 122
Author(s):  
A. R. Moawad ◽  
K. H. S. Campbell
Keyword(s):  
Genetic Material ◽  
Basal Medium ◽  
Germinal Vesicle ◽  
Meiotic Maturation ◽  
Control Group ◽  
High Survival ◽  
Chi Square ◽  
Immature Oocytes ◽  
The Difference

Oocyte cryopreservation will become an important tool for the creation of genetic resources bank in domestic animals. Many problems have been described following cryopreservation of MII oocytes, including spindle disorganization (Ledda et al. 2007 Reprod. Fertil. Dev. 19, 13–23) and loss or clumping of microtubules resulting in some scattered chromosomes (Sathananthan et al. 1988 Hum. Reprod. 3, 968–977). Freezing of immature oocytes at the geminal vesicle stage might circumvent these problems because at this stage the genetic material is contained within the contours of a nuclear envelope (Siqeira-Pyles and Landim-Alvarenga 2005 Anim. Reprod. Sci. 193, 176–182). However, the number of reports relating to cryopreservation of immature oocytes remains low and it has been suggested that immature oocytes are more susceptible to cryoinjuries (Ledda et al. 2007). The aim of the present work was to study the effect of CryoLoop™ (Hampton Research, Aliso Viejo, CA, USA) vitrification on survival and subsequent meiotic maturation of immature ovine oocytes. Cumulus–oocyte complexes (COCs) were isolated from ovaries of slaughtered sheep. COCs were washed three times in basal medium [BM (HEPES–TCM-199 supplemented with 10% fetal bovine serum)], and then exposed to equilibration solution (10% ethylene glycol (EG) and 0.25 m trehalose (T) in BM for 3 min. After that, COCs were exposed to vitrification solution (20% EG and 20% DMSO in BM), loaded into the CryoLoop within 1 min, and immersed in liquid nitrogen. Following cryopreservation, COCs were warmed by exposure of oocytes to (1) 10% EG and 1 mT in BM, (2) 0.5 mT in BM, and (3) BM for 3 min in each solution at 39�C. Then oocytes were matured in vitro for 24 h in maturation medium, as previously described (Lee and Campbell 2006 Biol. Reprod. 74, 691–698). The nuclear maturation of oocytes was determined using aceto-orcein staining. Oocytes were classified into four stages: germinal vesicle (GV), metaphase-I (MI), anaphase-I + telophase-I (AI/TI), and metaphase-II (MII). Oocytes at MII stage were considered matured. Data were analyzed using chi-square test. The survival rate of oocytes after vitrification and warming based on oocyte morphology was 72.6% (61/84). Numbers of oocytes remaining at the GV stage were significantly higher in the vitrified group than in the control group (43.4 v. 12.6%, respectively; P < 0.01). Maturation to MII stage was higher (63.2%, 55/87) in the control group than in the vitrified group (43.4%, 23/53); however, the difference was not significant. Also, there were no significant differences between vitrified and control groups in terms of MI and AI/TI (7.5 and 5.7 v. 10.3 and 13.8%, respectively). In conclusion, immature ovine oocytes can be vitrified using CryoLoop with high survival and maturation rates.

Bovine granulosa cell culture for assessment of potency and specificity of antibodies to pregnant mares' serum gonadotrophin

1996 ◽  
Vol 134 (4) ◽  
pp. 497-500 ◽  
Author(s):  
Mehmet Kuran ◽  
Peter J Broadbent ◽  
JS Morley Hutchinson
Keyword(s):  
Cell Culture ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Culture System ◽  
Dependent Manner ◽  
Progesterone Production ◽  
Neutralizing Capacity ◽  
Dose Dependent

Kuran M, Broadbent PJ, Hutchinson JSM. Bovine granulosa cell culture for assessment of potency and specificity of antibodies to pregnant mares' serum gonadotrophin. Eur J Endocrinol 1996;134:497–500. ISSN 0804–4643 Antibodies to pregnant mares' serum gonadotrophin (PMSG) neutralize the effect of PMSG in vivo and increase the number of transferable embryos when administered at the optimum time relative to the preovulatory luteinizing hormone (LH) surge in PMSG-stimulated cows. The objective of the present study was to investigate the possible use of bovine granulosa cells in a serum-free culture system as a bioassay for antibodies to PMSG. Granulosa cells (2–3 × 105 viable cells) were cultured with varying doses of PMSG and/or an anti-PMSG for 4 days. Whilst progesterone production (ng/μg DNA) of granulosa cells was stimulated by PMSG (p < 0.01) in a dose-dependent manner, increasing amounts of anti-PMSG neutralized (p < 0.01) this stimulatory effect of either follicle-stimulating hormone or LH on progesterone production of bovine granulosa cells in vitro. The bovine granulosa cell culture system is a potential in vitro bioassay method for testing the specificity and the neutralizing capacity of different anti-PMSG preparations. Mehmet Kuran, Ondokuz Mayis Universitesi, Ziraat Fakultesi, Zootekni Bolumu, 55149 Samsun, Turkey

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