Mammalian oocytes within Graafian follicles are arrested at prophase I of meiosis. C-type natriuretic peptide (NPPC), secreted by mural granulosa cells (MGCs), maintains oocyte meiotic arrest via binding to its cognate receptor natriuretic peptide receptor 2 (NPR2) and producing cyclic guanosine monophosphate (cGMP). NPR2 is most concentrated in the cumulus cells. In addition, cAMP, gap junction, inosine monophosphate dehydrogenase (IMPDH) and other important regulatory factors are also involved in meiotic arrest. Luteinizing hormone (LH) then rapidly decreases cGMP and induces oocyte meiotic resumption. In this paper, advances in the molecular mechanisms of meiotic arrest and LH-induced meiotic resumption were reviewed. This paper may provide new ideas for the prevention, diagnosis and treatment of related reproductive diseases.
Animals ; Cumulus Cells ; Female ; Luteinizing Hormone ; Meiosis ; Natriuretic Peptide, C-Type ; genetics ; Oocytes

OBJECTIVE: In vitro maturation (IVM) of immature oocytes can be useful for some infertile patients. In IVM programs, the rates of embryo formation and pregnancy are low. Therefore, it is essential to recognize the main factors involved in regulating oocyte maturation in vitro. The purpose of this study was to investigate the effects of growth differentiation factor 9 (GDF9) and cumulus cell (CC) supplementation in IVM medium on the rates of embryo formation and viability of human blastocysts.METHODS: A total of 80 germinal vesicle oocytes from stimulated cycles underwent an IVM program. The oocytes were divided into four groups, where group I consisted of IVM media only and served as the control, group II consisted of IVM+CCs, group III consisted of IVM+GDF9 (200 ng/mL), and group IV consisted of IVM+CCs+GDF9 (200 ng/mL). Intracytoplasmic sperm injection was performed on the IVM oocytes, and the cleavage embryos that were generated were vitrified. Following thawing, the embryos were cultured for 3 additional days, and the viability rates of the developed blastocysts were determined.RESULTS: The maturation rate of the oocytes did not differ significantly across the four groups. The fertilization rate in group II was significantly higher than that in the control group (76.5% vs. 46.2%). Embryo formation was significantly more frequent in all experimental groups than in the control group, while blastocyst formation did not show significant differences in the three experimental groups compared to the control. The mean viability rates in groups II, III, and IV were 58.16%, 55.91%, and 55.95%, respectively, versus 37.78% in the control group (p<0.05).CONCLUSION: Supplementation of IVM culture media with GDF9 and CCs enhanced the fertilization, embryo formation, and viability rates of blastocysts generated from vitrified cleavage embryos.
Blastocyst ; Culture Media ; Cumulus Cells ; Embryonic Structures ; Fertilization ; Growth Differentiation Factor 9 ; Humans ; In Vitro Techniques ; Oocytes ; Pregnancy ; Sperm Injections, Intracytoplasmic

OBJECTIVE: This study was performed to explore the possibility that each oocyte and its surrounding cumulus cells might have different genetic expression patterns that could affect human reproduction. METHODS: Differential gene expression analysis was performed for 10 clusters of cumulus cells obtained from 10 cumulus-oocyte complexes from 10 patients. Same procedures related to oocyte maturation, microinjection, and microarray analyses were performed for each group of cumulus cells. Two differential gene expression analyses were performed: one for the outcome of clinical pregnancy and one for the outcome of live birth. RESULTS: Significant genes resulting from these analyses were selected and the top 20 affected pathways in each group were analyzed. Circadian entrainment is determined to be the most affected pathway for clinical pregnancy, and proteoglycans in cancer pathway is the most affected pathway for live birth. Circadian entrainment is also amongst the 12 pathways that are found to be in top 20 affected pathways for both outcomes, and has both lowest p-value and highest number of times found count. CONCLUSION: Although further confirmatory studies are necessary, findings of this study suggest that these pathways, especially circadian entrainment in cumulus cells, may be essential for embryo development and pregnancy.
Circadian Clocks ; Cumulus Cells ; Embryonic Development ; Female ; Gene Expression ; Granulosa Cells ; Humans ; Infertility ; Live Birth ; Microarray Analysis ; Microinjections ; Oocytes ; Ovarian Follicle ; Pregnancy ; Proteoglycans ; Reproduction ; Reproductive Techniques, Assisted

This study was conducted to determine the effects of biophoton treatment during in vitro maturation (IVM) and/or in vitro culture (IVC) on oocyte maturation and embryonic development in pigs. An apparatus capable of generating homogeneous biophoton energy emissions was placed in an incubator. Initially, immature pig oocytes were matured in the biophoton-equipped incubator in medium 199 supplemented with cysteine, epidermal growth factor, insulin, and gonadotrophic hormones for 22 h, after which they were matured in hormone-free medium for an additional 22 hr. Next, IVM oocytes were induced for parthenogenesis (PA) or provided as cytoplasts for somatic cell nuclear transfer (SCNT). Treatment of oocytes with biophoton energy during IVM did not improve cumulus cell expansion, nuclear maturation, intraoocyte glutathione content, or mitochondrial distribution of oocytes. However, biophoton-treated oocytes showed higher (p < 0.05) blastocyst formation after PA than that in untreated oocytes (50.7% vs. 42.7%). In an additional experiment, SCNT embryos produced from biophoton-treated oocytes showed a greater (p < 0.05) number of cells in blastocysts (52.6 vs. 43.9) than that in untreated oocytes. Taken together, our results demonstrate that biophoton treatment during IVM improves developmental competence of PA- and SCNT-derived embryos.
Blastocyst ; Cumulus Cells ; Cysteine ; Embryonic Development ; Embryonic Structures* ; Epidermal Growth Factor ; Female ; Glutathione ; Gonadotrophs ; In Vitro Techniques ; Incubators ; Insulin ; Mental Competency ; Oocytes* ; Parthenogenesis* ; Pregnancy ; Swine

OBJECTIVE: This study was conducted to compare the effects of static culture, dynamic culture, and the combination of dynamic culture with specialized surfaces involving co-culture on human embryonic development. Embryos cultured using conventional static culture (SC) techniques served as a control group. We compared dynamic culture using micro-vibration culture (MVC) and micro-vibration with co-culture (MCoC), in which autologous cumulus cells were used as a specialized surface. METHODS: We conducted a chart review of patients who were treated between January 2011 and November 2014 in order to compare embryonic development rates and pregnancy rates among the groups. Zygotes were cultured in micro-droplets, and embryos were subsequently selected for transfer. Some surplus embryos were cryopreserved, and the others were cultured for blastocyst development. A micro-vibrator was set at the frequency of 42 Hz for duration of 5 seconds per 60 minutes to facilitate embryo development. RESULTS: No significant differences among the groups were present in patient's characteristics. However, the clinical pregnancy rates were significantly higher in the MVC group and the MCoC group than in the SC group. No significant differences were found in the blastocyst development rate between the SC group and the MVC group, but the blastocyst development rate in the MCoC group was significantly higher than in the SC and MVC groups. CONCLUSION: The clinical pregnancy rate was significantly increased by the application of micro-vibration to the embryonic cultures of poor responders. The blastocyst development rate was significantly increased by the application of MCoC to surplus embryos.
Blastocyst ; Coculture Techniques* ; Cumulus Cells ; Embryo Culture Techniques ; Embryonic Development ; Embryonic Structures ; Female ; Humans ; Pregnancy ; Pregnancy Rate ; Zygote

OBJECTIVE: The long interspersed elements (LINE-1, L1s) are a group of genetic elements found in large numbers in the human genome that can translate into phenotype by controlling genes. Growing evidence supports the role of epigenetic in polycystic ovary syndrome (PCOS). The purpose of this study is to evaluate the DNA methylation levels in LINE-1 in a tissue-specific manner using cumulus cells from patients with PCOS compared with normal controls. METHODS: The study included 19 patients with PCOS and 22 control patients who were undergoing controlled ovarian hyperstimulation. After oocyte retrieval, cumulus cells were extracted. LINE-1 DNA methylation levels were analysed by bisulfite treatment, polymerase chain reaction, and restriction enzyme digestion. The Connection Up- and Down-Regulation Expression Analysis of Microarrays software package was used to compare the gene regulatory functions of intragenic LINE-1. RESULTS: The results showed higher LINE-1 DNA methylation levels in the cumulus cells of mature oocytes in PCOS patients, 79.14 (±2.66) vs. 75.40 (±4.92); p=0.004, but no difference in the methylation of cumulus cells in immature oocytes between PCOS and control patients, 70.33 (±4.79) vs. 67.79 (±5.17); p=0.155. However, LINE-1 DNA methylation levels were found to be higher in the cumulus cells of mature oocytes than in those of immature oocytes in both PCOS and control patients. CONCLUSION: These findings suggest that the epigenetic modification of LINE-1 DNA may play a role in regulating multiple gene expression that affects the pathophysiology and development of mature oocytes in PCOS.
Cumulus Cells* ; Digestion ; DNA ; DNA Methylation ; Down-Regulation ; Epigenomics* ; Fertilization in Vitro ; Gene Expression ; Genome, Human ; Humans ; Infertility ; Long Interspersed Nucleotide Elements ; Methylation ; Oocyte Retrieval ; Oocytes* ; Phenotype ; Polycystic Ovary Syndrome* ; Polymerase Chain Reaction

OBJECTIVE: The goal of this study was to evaluate the pregnancy potential of immature (metaphase I or germinal vesicle stage) oocytes retrieved in intracytoplasmic sperm injection (ICSI) cycles. METHODS: A total of 1,871 couples with infertility underwent 2,984 ICSI cycles. Cycles in which three or fewer oocytes were retrieved were included in this study in order to evaluate the pregnancy potential of immature oocytes. Cycles were divided into five groups (group I-V), according to the maturation status of the oocytes at the time of cumulus cell removal and ICSI. The fertilization and pregnancy rates after ICSI were analyzed and compared among the study groups based on the maturation status of the retrieved oocytes. RESULTS: The retrieval of only immature oocytes was associated with a significant decrease in the fertilization rate (76.1%+/-37.3% vs. 49.0%+/-49.1%, 66.7%+/-48.7%; group I vs. group II, group III, respectively) and the average number of transferred embryos (1.5+/-0.7 vs. 1.1+/-0.4, 1.1+/-0.6). The cycle cancellation rate was significantly higher when only immature oocytes were retrieved. The clinical pregnancy rate decreased significantly when the transferred embryos had originated from immature oocytes (16.9% vs. 10.3%, 1.2%). CONCLUSION: In ICSI cycles, the fertilization potential and pregnancy potential of the immature oocytes retrieved in ICSI cycles were inferior to those of mature oocytes. Therefore, increasing the number of injectable oocytes and transferrable embryos by using immature oocytes after their spontaneous in vitro maturation does not necessarily improve pregnancy outcomes.
Cumulus Cells ; Embryonic Structures ; Family Characteristics ; Female ; Fertilization* ; Infertility ; Oocytes* ; Pregnancy Outcome ; Pregnancy Rate ; Pregnancy* ; Sperm Injections, Intracytoplasmic*

OBJECTIVE: We found previously that interferon regulatory factor (Irf)-1 is a germinal vesicle (GV)-selective gene that highly expressed in GV as compared to metaphase II oocytes. To our knowledge, the function of Irf-1 in oocytes has yet to be examined. The present study was conducted to determine the relationship between retinoic acid (RA) and RA-mediated expression of Irf-1 and the mouse oocyte maturation. METHODS: Immature cumulus-oocyte-complexes (COCs) were collected from 17-day-old female mice and cultured in vitro for 16 hours in the presence of varying concentrations of RA (0-10 microM). Rate of oocyte maturation and activation was measured. Gene expression was measured by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and cytokine secretion in the medium was measured by Bio-Plex analysis. Apoptosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. RESULTS: The rates of oocyte maturation to metaphase II and oocyte activation increased significantly with RA treatment (10 nM-1 microM). With 100 nM RA treatment, lowest level of Irf-1 mRNA and cumulus cell's apoptosis was found. Among 23 cytokines measured by Bio-Plex system, the substantial changes in secretion of tumor necrosis factor-alpha, macrophage inflammatory protein-1beta, eotaxin and interleukin-12 (p40) from COCs in response to RA were detected. CONCLUSION: We concluded that the maturation of oocytes and Irf-1 expression are negatively correlated, and RA enhances the developmental competence of mouse immature oocytes in vitro by suppressing apoptosis of cumulus cells. Using a mouse model, results of the present study provide insights into improved culture conditions for in vitro oocyte maturation and relevant cytokine production and secretion in assisted reproductive technology.
Animals ; Apoptosis ; Cumulus Cells ; Cytokines ; DNA Nucleotidylexotransferase ; Female ; Gene Expression ; Humans ; In Vitro Oocyte Maturation Techniques ; Interferon Regulatory Factor-1 ; Interferons ; Interleukin-12 ; Macrophages ; Mental Competency ; Metaphase ; Mice ; Oocytes ; Reproductive Techniques, Assisted ; RNA, Messenger ; Tretinoin ; Tumor Necrosis Factor-alpha

Fertilization is a process involving multiple steps that lead to the final fusion of one sperm and the oocyte to form the zygote. One of the steps, acrosome reaction (AR), is an exocytosis process, during which the outer acrosome membrane fuses with the inner sperm membrane, leading to the release of acrosome enzymes that facilitate sperm penetration of the egg investments. Though AR has been investigated for decades, the initial steps of AR in vivo, however, remain largely unknown. A well elucidated model holds the view that AR occurs on the surface of the zona pellucida (ZP), which is triggered by binding of sperm with one of the ZP glycosylated protein, ZP3. However, this model fails to explain the large number of 'falsely' acrosome-reacted sperms found within the cumulus layer in many species examined. With the emerging evidence of cross-talk between sperm and cumulus cells, the potential significance of AR in the cumulus oophorus, the outer layer of the egg, has been gradually revealed. Here we review the acrosome status within the cumulus layer, the cross-talk between sperm and cumulus cells with the involvement of a novel sperm-released factor, NYD-SP8, and re-evaluate the importance and physiological significance of the AR in the cumulus in fertilization.
Acrosome Reaction ; physiology ; Cell Communication ; Cumulus Cells ; metabolism ; Female ; Fertilization ; physiology ; Humans ; Male ; Membrane Proteins ; metabolism ; Oocytes ; metabolism ; Progesterone ; physiology ; Spermatozoa ; metabolism

The objective of the present study was to examine the feasibility of the production of autologous porcine somatic cell nuclear transfer (SCNT) blastocysts using oocytes and donor cells from slaughtered ovaries. Therefore, we attempted to optimize autologous SCNT by examining the effects of electrical fusion conditions and donor cell type on cell fusion and the development of SCNT embryos. Four types of donor cells were used: 1) denuded cumulus cells (DCCs) collected from in vitro-matured (IVM) oocytes; 2) cumulus cells collected from oocytes after 22 h of IVM and cultured for 18 h (CCCs); 3) follicular cells obtained from follicular contents and cultured for 40 h (CFCs); and 4) adult skin fibroblasts. The DCCs showed a significantly (p > 0.01) lower rate of fusion than the CCCs when two pulses of 170 V/mm DC were applied for 50 microsec (19 +/- 2% vs. 77 +/- 3%). The rate of DCC fusion with oocytes was increased by the application of two DC pulses of 190 V/mm for 30 microsec, although this was still lower than the rate of fusion in the CCCs (33 +/- 1% vs. 80 +/- 2%). The rates of cleavage (57 +/- 5%) and blastocyst formation (1 +/- 1%) in the DCC-derived embryos did not differ from those (55 +/- 6% and 3 +/- 1%, respectively) in the CCC-derived SCNT embryos. Autologous SCNT embryos derived from CFCs (5 +/- 2%) showed higher levels of blastocyst formation (p > 0.01) than CCC-derived autologous SCNT embryos (1 +/- 0%). In conclusion, the results of the present study show that culturing cumulus and follicular cells before SCNT enhances cell fusion with oocytes and that CFCs are superior to CCCs in the production of higher numbers of autologous SCNT blastocysts.
Animals ; *Animals, Genetically Modified ; Cloning, Organism ; Cumulus Cells/metabolism ; Electric Stimulation ; Embryo Culture Techniques/veterinary ; Embryonic Development ; Female ; Fibroblasts/metabolism ; Nuclear Transfer Techniques/*veterinary ; Oocytes/*metabolism ; Ovarian Follicle/metabolism ; Swine/embryology/*physiology