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PubMed1999

Oocytectomy does not influence synthesis of hyaluronic acid by pig cumulus cells: retention of hyaluronic acid after insulin-like growth factor-I treatment in serum-free medium.

Mouse oocytes secrete a factor that enables cumulus cells to undergo expansion in response to FSH (1 microg/ml), whereas expansion of the porcine cumulus oophorus has been shown to be independent of the oocyte. The aim of this study was to assess FSH-induced synthesis of hyaluronic acid (HA) by porcine cumulus cells before and after oocytectomy. In addition, we studied the effect of insulin-like growth factor-I (IGF-I) on the ability of cumulus cells to synthesize and retain HA in response to FSH in serum-free medium. Porcine oocyte-cumulus complexes and complexes from which the oocytes had been removed by oocytectomy were cultured for 24 h in the presence of 2.5 microCi of D-[6-(3)H]glucosamine hydrochloride, fetal calf serum (FCS, 5%), and FSH. After 24 h, incorporation of [(3)H]glucosamine into HA was measured either in complexes alone (retained HA) or in medium plus complexes (total HA). Specificity of incorporation of radioactivity into HA was confirmed by the sensitivity to highly specific Streptomyces hyaluronidase. Our results suggest that 1) the synthesis of HA by pig cumulus cells in vitro is stimulated by FSH and that oocytectomy does not change this synthesis; 2) oocytes do not influence retention of HA within the complex; 3) FSH-induced synthesis of HA by cumulus cells is decreased in medium with polyvinylpyrrolidone (PVP)-supplemented (total and retained HA) compared to FCS-supplemented medium; 4) IGF-I enabled cumulus cells to synthesize HA in response to FSH in PVP-supplemented medium in a manner similar to that observed when serum is present in the medium.

Animals↗
PubMed2007

Luteinizing hormone receptors expression in cumulus cells closely related to mouse oocyte meiotic maturation.

In the process of oocyte maturation, gonadotrophins are believed as main stimulators for oocyte meiosis resumption. However, which gonadotrophin (i.e. FSH or LH) is the key hormone in this process is still unknown. This study indicated a close relationship between LH and FSH on activating meiotic maturation of oocyte in vitro. FSH efficiently induced oocyte meiosis at concentration of 50 IU/L, while LH alone had no effect on oocyte meiotic initiation. Using RT-PCR and in situ hybridization, a tight corelationship between FSH-stimulated oocyte meiotic resumption and LHR mRNA expression in cumulus cells was found. LHR mRNA was present in cumulus cells of oocyte-cumulus cell complexes. Except the expression of LHRs was present in cumulus cells surrounding all maturing oocytes, low level expression was also detected in cells associated with oocytes that were still at GV stage. Its expression was enhanced by FSH stimulation before oocyte maturation. However, LHRs did not express in cumulus cells associated with oocytes that were completely arrested at GV stage by IBMX. Furthermore, increased progesterone concentration was found in the medium in which CEOs were cultured with FSH and LH, but not in those with FSH or LH alone. LHR expression in cumulus cells increases with time in culture, and the levels of expression are enhanced in the presence of FSH. Oocyte meiotic resumption may create conditions favorable for LHR expression. LHR expression may be considered as a potential marker for oocytes maturation initiation.

Animals↗
PubMed2005

Strong expression of steroid sulfatase in human cumulus cells in patients with endometriosis.

OBJECTIVE: To investigate the localization and expression of steroid sulfatase (STS) in cumulus cells obtained from subjects with and without endometriosis. DESIGN: Case-control study. SETTING: In vitro fertilization program at the Showa University School of Medicine. PATIENT(S): Cumulus cells (142 samples) were obtained from 49 patients for whom IVF was indicated. Some of these samples were taken from cases complicated by endometriosis (35 samples), polycystic ovary syndrome (PCOS; 16 samples), or latent hyperprolactinemia (16 samples). INTERVENTION(S): Immunohistochemical staining for STS. Measurement of STS mRNA expression by real-time polymerase chain reaction (PCR). MAIN OUTCOME MEASURE(S): Expression of STS mRNA and localization of STS. RESULT(S): Steroid sulfatase was localized in the cytoplasm of the cumulus cells, and expression of STS mRNA was observed. The expression level of STS mRNA from patients with endometriosis was significantly higher (11.8-fold) than that of patients without endometriosis. CONCLUSION(S): These results suggest a local steroidal regulation mechanism in cumulus cells. Although the physiological role of STS in cumulus cells remains unclear, STS may be involved in the quality of eggs in patients with endometriosis.

Adult↗
PubMed1994

Cumulus cells secrete a meiosis-inducing substance by stimulation with forskolin and dibutyric cyclic adenosine monophosphate.

The role of the cumulus cells in initiating the resumption of meiosis after exposure to forskolin and dbcAMP was studied in the mouse. The resumption of meiosis was monitored by the percentage of germinal vesicle breakdown (GVBD) and polar body formation (PB). The cumulus-enclosed oocytes (CEO) and denuded oocytes (DO) were cultured with and without hypoxanthine (HX) in the culture medium. Three types of experiments were performed: (1) Effect of forskolin on spontaneous resumption of meiosis, i.e. cultures without HX, and two experiments in which HX is present throughout the culture: (2) Effect of transient exposure to forskolin or dibutyric-cyclic adenosinemonophosphate (dbcAMP) on GVBD prior to continued culture without forskolin or dbcAMP (oocyte priming). (3) Priming of CEO with forskolin for 2 hr, separation of cumulus cells and oocytes, followed by coculture of rejoined cumulus cells and oocytes, or coculture of the cumulus cells and new, unprimed DO. (1) Forskolin inhibited a spontaneous resumption of meiosis in a dose-dependent manner during the first 5 hr of culturing. After 22 hr all controls and CEO resumed meiosis, whereas only half of the DO did. (2) At least 1 hr of priming the CEO with forskolin is needed to induce GVBD and PB formation, but forskolin inhibited the resumption of meiosis when present for 24 hr. Similar results were obtained with a high concentration of dbcAMP. (3) A separation and rejoining of oocytes and cumulus cells after priming induced the resumption of meiosis in a significantly greater number of oocytes than in the control oocytes which were not primed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Effects of cumulus cells and follicle-stimulating hormone during in vitro maturation on parthenogenetic activation of bovine oocytes.

The response to parthenogenetic activation (calcium ionophore A23187) of bovine oocytes after 30 hr of culture in different maturation conditions was evaluated. The activation rates of oocytes in response to 10 mu M A23187 were similar (86% +/- 4 vs. 89% +/- 8) when the oocytes matured with or without cumulus cells were cultured in TCM-199 + 10% fetal calf serum (maturation medium [MM]) alone. However, the activation rates were significantly lower in oocytes matured with than without cumulus cells (48 +/- 10 vs. 79% +/- 16; P < 0.05) when MM was supplemented with 0.5 microgram/ml follicle-stimulating hormone (FSH), 50 micrograms/ml luteinizing hormone (LH), and 1.0 microgram/ml estradiol-1 beta (E2). When oocytes with cumulus cells were cultured in different media, MM along, MM + 0.5 microgram/ml FSH, MM + 50 micrograms/ml LH, MM + 1.0 microgram/ml E2, and MM + all three hormones, the percentages of activated oocytes were 91% +/- 3.52% +/- 14.83% +/- 7, 83% +/- 13, and 54% +/- 7, respectively. Oocytes with or without cumulus cells during culture began to form a pronucleus 5 hr after activation (5% +/- 1 and 7% +/- 1, respectively). Finally, the proteins synthesized by oocytes matured with or without cumulus cells before (3 hr) and after (10 hr) activation was labelled with [35S]methionine for analysis. Before activation, a 47 kD protein complex was produced by cumulus cell-intact oocytes. An active protein synthesis of 60 kD was characteristic for the oocytes with cumulus cells cultured in MM containing 0.5 micrograms/ml FSH. After activation, there was an appearance of proteins at 75 kD and 47 kD in the oocytes with cumulus cells but not in the oocytes without cumulus cells during the maturation period. These results indicate that bovine oocytes matured in vitro have different capacities for parthenogenetic activation depending on the presence or absence of cumulus cells and the presence of FSH in MM during culture.

Animals↗
PubMed2006

Lower apoptosis rate in human cumulus cells after administration of recombinant luteinizing hormone to women undergoing ovarian stimulation for in vitro fertilization procedures.

OBJECTIVE: To investigate the effects of recombinant (r-) LH supplementation in "low responder" patients undergoing ovarian stimulation with r-FSH for an IVF program. The apoptosis rate in cumulus cells was used as an indicator of oocyte quality. DESIGN: Comparison of the rate of DNA fragmentation and caspase-3 activity in cumulus cells in women stimulated with r-LH and r-FSH, versus patients treated with r-FSH alone (control). SETTING: In vitro fertilization (IVF) laboratory. PATIENT(S): Forty patients undergoing assisted fertilization programs treated with a GnRH agonist, or r-FSH treatment begun on day 3 of the cycle (control). In the r-LH group, from day 8 of gonadotropin stimulation, 150 IU per day of r-LH were administered. INTERVENTION(S): Terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine-triphosphate (dUTP) nick-end labeling (TUNEL) assay, and anti-caspase-3 cleaved immunoassay, to detect apoptosis in human cumulus cells. MAIN OUTCOME MEASURE(S): Difference in DNA fragmentation rate between cumulus cells derived from r-LH treatment and cumulus cells derived from control patients. RESULT(S): No differences were observed between the two groups in the total amount of r-FSH administered and in the number of retrieved oocytes per patient. A statistically significant increase in the number of immature oocytes and in the E(2) serum peak was observed in the control group. The number of transferred embryos was significantly higher in the r-LH group. Pregnancy and implantation rates were higher in the r-LH group, but without statistical significance. The apoptosis rate in cumulus cells was higher in the control group than in the r-LH group. CONCLUSION(S): This study suggests that supplementation with r-LH improves the chromatin quality of cumulus cells involved in the control of oocyte maturation.

Adult↗
PubMed2005

Predictive value of cumulus cell apoptosis with regard to blastocyst development of corresponding gametes.

OBJECTIVE: To test if a high degree of apoptosis in cumulus cells could indicate diminished oocyte quality and developmental competence. DESIGN: Prospective analysis. SETTING: Public hospital and university. PATIENT(S): Thirty seven women who gave written consent to participate in this study. INTERVENTION(S): Cumulus-oocyte complexes were denuded separately and the resulting cumulus cell suspensions were analyzed for presence of apoptosis using a terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay detection kit. MAIN OUTCOME MEASURE(S): Percentage of apoptotic cumulus cells and blastocyst formation rate. RESULT(S): Younger patients (< or = 35 years) showed significantly fewer apoptotic cumulus cells than older ones. In all patients gamete maturity was shown to be highly correlated to the rate of apoptosis in cumulus cells. At zygote and cleavage stages (days 1 to 4) no morphologic features were related to the degree of programmed cell death. However, blastocyst development was predictable taking into account the percentage of apoptosis in associated somatic cells. In addition, there was a trend toward better quality blastocysts from follicles with a lower rate of apoptotic cells. No influence on pregnancy and implantation rate was observed. CONCLUSION(S): Apoptotic processes within follicles seem to impair oocyte maturation. Though not manifested in the morphologic appearance, gametes and embryos derived from cumulus complexes with no or minor apoptosis have an increased chance of giving rise to optimal blastocysts.

Adult↗
PubMed2005

Follicular fluid and cumulus cells synergistically improve mouse early embryo development in vitro.

The development of preimplantation mammalian embryos in vitro is less than optimal. Follicular fluid and cumulus cells have both been used independently, to improve preimplantation embryo quality in culture. This study was undertaken to evaluate the influence of a cumulus cell monolayer in human follicular fluid on mouse early embryo development in vitro. One-cell embryos were obtained from NMRI mice after superovulation with eCG and hCG. Cumulus cells were prepared from mouse egg-cumulus mass. These cells were separated from red blood cells using a Percoll gradient. Follicular fluid was collected from patients undergoing an IVF program during oocyte pick-up. The cumulus cell monolayer was prepared in follicular fluid (FC) and Ham's F10 (HC). Mouse one-cell embryos were cultured in FC, HC, Ham's F10 (HF) and follicular fluid (FF) for 120 h. Only 10.5% of embryos passed the two-cell block in HF. However, the proportions of embryos passing the two-cell block were 23.1%, 21.4% and 68.5% in FF, HC, and FC treatments, respectively; which were significantly different from HF (p<0.05). The differences between FC and the two other treatments were also significant (p<0.001). In FC, 33.7% of one-cell embryos continued to grow to the blastocyst stage whereas only 2.1% and 1.9% of one-cell embryos in FF and HC reached this stage and no embryos developed to blastocyst in HF. The proportion of blastocysts in FC was significantly higher than all other treatments (p<0.001). It can be concluded that follicular fluid and cumulus cells in monolayer form synergistically improve the early embryo culture condition.

Animals↗
PubMed1995

Human follicular fluid and mouse cumulus cells act synergistically to enhance preimplantation mouse Balb/cJ embryo development.

PROBLEM: The development of preimplantation mammalian embryos in vitro is less than optimal. Follicular fluid and cumulus cells have both been used, independently, to improve preimplantation embryo quality in culture. METHOD: To determine the ability of mouse cumulus cell coculture in the presence of human follicular fluids to support preimplantation mouse Balb/cJ embryo development in vitro. RESULTS: Culture of preimplantation mouse Balb/cJ embryo's independently in human follicular fluid or on mouse cumulus cells had no significant affect on blastocyst. The coculture of mouse Balb/cJ preimplantation-stage embryos on mouse cumulus cells in the presence of human follicular fluid significantly (P < 0.01) improved blastocyst development and the total number of cells per blastocyst. CONCLUSION: Cumulus cells and follicular fluid have a positive synergistic affect on preimplantation mouse Balb/cJ embryo development and formation in vitro.

Animals↗
PubMed2005

Role of cumulus cells during maturation of porcine oocytes in the rise in intracellular Ca2+ induced by inositol 1,4,5-trisphosphate.

At the time of fertilization, release of inositol 1,4,5-trisphosphate (IP3) into the cytoplasm of oocytes is said to be induced by hydrolysis of phosphatidylinositol bis phosphate (PI2) via activation of phospholipase C and is responsible for the Ca2+ oscillation in oocytes immediately after sperm penetration. On the other hand, cumulus cells have been reported to play an important role in cytoplasmic maturation of mammalian oocytes and to affect embryonic development after fertilization. To obtain more information on the role of cumulus cells in cytoplasmic maturation of oocytes, the effects of cumulus cells on the rise in [Ca2+]i and the rates of activation and development of porcine mature oocytes induced by IP3 injection were investigated. Mature porcine oocytes that had been denuded of their cumulus cells in the early stage of the maturation period had a depressed rise in [Ca2+]i (4.0-6.0) and reduced rates of activation (31.4-36.8%) and development (10.0-24.4%) induced by IP3 injection compared with those of their cumulus-enclosed counterparts (7.3, 69.1% and 43.8%; P < 0.05). The [Ca2+]i rise and the rates of activation and development depressed by the removal of cumulus cells were restored by adding pyruvate to the maturation medium. Furthermore, the IP3 injection-induced depression of [Ca2+]i rise in mature oocytes derived from cumulus-denuded oocytes (DOs) was restored when they were cultured in a medium with pyruvate (3.9-6.3, P < 0.05). Also, mature oocytes from cumulus-oocyte complexes (COCs) cultured in a medium without glucose had a lower rise in [Ca2+]i than that in mature oocytes from COCs cultured with glucose (7.4-6.0, P < 0.05). Cumulus cells supported porcine oocytes during maturation in the rise in [Ca2+]i induced by IP3 and the following activation and development of porcine oocytes after injection of IP3. Moreover, we inferred that a function of cumulus cells is to produce pyruvate by metabolizing glucose and to provide oocytes with pyruvate during maturation, thereby promoting oocyte sensitivity to IP3.

Animals↗
PubMed2002

FSH and LH induce progesterone production and progesterone receptor synthesis in cumulus cells: a requirement for meiotic resumption in porcine oocytes.

The aim of this study was to investigate the role of progesterone in the meiotic resumption of porcine oocytes. Progesterone production and progesterone receptor (PR) immunoreactivity in cumulus cells were not detected in porcine cumulus-oocyte complexes (COC) when observations were made either just after collection from the follicles or after 28 h cultivation without LH and FSH. However, the addition of LH and FSH induced PR expression in cumulus cells, concomitant with increased progesterone production. To assess the role of progesterone in the COC, an inhibitor of progesterone production, aminoglutethimide (AGT), was administered. The addition of AGT to the medium with LH and FSH significantly suppressed progesterone production in a dose-dependent fashion. When COC were cultured with LH, FSH and 0.5 x 10(-3) mol/l AGT, almost complete inhibition of progesterone production and of germinal vesicle breakdown (GVBD) was seen. However, this inhibitory effect on GVBD was overcome by additional progesterone. Moreover, 0.5 x 10(-3) mol/l AGT also suppressed the reduction in connexin43, a gap junctional protein, in cumulus cells after 28 h cultivation, and increased the level of cyclic AMP in oocytes. These results support the hypothesis that the binding of progesterone, which was secreted by LH- and FSH-stimulated cumulus cells, to its newly synthesized receptor induces GVBD in porcine oocytes, possibly through a reduction of connexin43 in cumulus cells.

Animals↗
PubMed2001

Phosphatidylinositol 3-kinase in cumulus cells and oocytes is responsible for activation of oocyte mitogen-activated protein kinase during meiotic progression beyond the meiosis I stage in pigs.

The roles of phosphatidylinositol 3-kinase (PI 3-kinase) during meiotic progression beyond the meiosis I (MI) stage in porcine oocytes were investigated. PI 3-kinase exists in cumulus cells and oocytes, and the PI 3-kinase inhibitor, LY294002, suppressed the activation of mitogen-activated protein (MAP) kinase in denuded oocytes during the beginning of the treatment. However, in denuded oocytes cultured with LY294002, the MAP kinase activity steadily increased, and at 48 h of cultivation MAP kinase activity, p34(cdc2) kinase activity, and proportion of oocytes that had reached the meiosis II (MII) stage were at a similar level to those of oocytes cultured without LY294002. In contrast, LY294002 almost completely inhibited the activation of MAP kinase, p34(cdc2) kinase activity, and meiotic progression to the MII stage in oocytes surrounded with cumulus cells throughout the treatment. Treating cumulus oocyte complexes (COCs) with LY294002 produced a significant decrease in the phosphorylation of connexin-43, a gap junctional protein, in cumulus cells compared with that in COCs cultured without LY294002. These results indicate that PI 3-kinase activity in cumulus cells contributes to the activation of MAP kinase and p34(cdc2) kinase, and to meiotic progression beyond the MI stage. Moreover, gap junctional communications between cumulus cells and oocytes may be closed by phosphorylation of connexin-43 through PI 3-kinase activation in cumulus cells, leading to the activation of MAP kinase in porcine oocytes.

Animals↗
PubMed1989

Periovulatory expression of cholesterol side-chain cleavage cytochrome P-450 in cumulus cells.

Immunocytochemical staining methods were used to examine the appearance of cholesterol side-chain cleavage cytochrome P-450 (P-450scc) in mitochondria of cumulus cells during follicular development. The cumulus-oocyte complexes were isolated from pregnant mare serum gonadotropin (PMSG)/human CG (hCG)-treated 25 day rats and examined in culture. It is shown that P-450scc is not expressed in the cumulus cell earlier than 2-3 h before ovulation. After ovulation, the expression of P-450scc rapidly increased, so that postovulatory cumuli contained ample amounts of the cytochrome. RIA of progestins secreted by the cumulus-oocyte complexes in culture corroborated the immunocytochemical observations. A single administration of LH or PMSG treatment of hypophysectomized rat did not result in P-450scc accumulation. However, this failure of hormonal responses in vivo was not due to lack of available receptors, since both FSH and LH could induce cAMP accumulation and P-450scc when added to isolated cumuli in culture. Therefore, these findings suggest the presence of a putative intraovarian suppressive factor(s) which disappears before ovulation and thus render(s) the cumulus cells permissive for P-450scc responsiveness. An additional intriguing aspect of P-450scc responsiveness to gonadotropins was revealed in experiments showing that 60% of the cultured cumulus complexes failed to accumulate P-450scc in response to hormones, if collected from 21 day animals. Interestingly, those P-450scc negative cumuli were always associated with an oocyte which did not resume meiotic maturation. We may therefore suggest that meiotic incompetence of the oocyte is also accompanied by functional incompetence of its embracing cumulus cells which cannot, for yet unclear reasons, acquire their steroidogenic capacity.

Animals↗
PubMed2002

The effects of oocyte storage and cumulus cell presence on canine zona penetration by domestic dog spermatozoa.

Zona penetration assays (ZPAs) have been developed in numerous species to evaluate sperm fertilizing potential. Preservation methods to stockpile oocytes would be beneficial because of the difficulty in obtaining sufficient numbers of fresh oocytes. Using a canine ZPA, the objectives of this study were to evaluate: (1) two methods of storing canine oocytes (salt storage and intrafollicular cooling) and (2) the effects of cumulus cells on oocyte penetration. In experiment 1. oocytes from fresh ovaries were assigned at random to 3 categories: fresh control (FRE), salt storage in solution 1 (1.5 M MgCl2.6H2O; SS1) and salt storage in solution 2 (0.5 M (NH4)2SO4, 0.75 M MgCl2.6H2O, 0.2 mM ZnCl2; SS2). Each category was subdivided into two treatments: cumulus cells intact (intact) and cumulus cells removed (denuded), resulting in a total of six treatments with n > 15 oocytes per treatment for each ejaculate. Fresh (FRE) intact oocytes demonstrated greater sperm-oocyte interaction than other treatments, including FRE denuded oocytes (11.7 +/- 0.6 versus <4.1 +/- 0.5 sperm-oocyte and 94.9 versus <55.6% penetration; P < 0.01). Poor sperm-oocyte interaction was demonstrated with all salt-stored oocytes (< or = 1.6 +/- 0.2 sperm-oocyte and < 51% penetration), but was further attenuated in the absence of cumulus cells. In experiment 2, oocytes obtained from fresh (FRESH) or cooled (24 h COOL, 48 h COOL) ovaries were used with cumulus cells intact for a total of three treatments with n > 15 oocytes per treatment for each ejaculate. No significant difference was observed in sperm interaction between oocytes from fresh, 24 and 48 h COOL ovaries ( 12.3 +/- 0.5 to 13.1 +/- 0.4 sperm-oocyte and 92.2-97.7% penetration; P > 0.01). These results indicate that salt storage may cause damage to canine oocytes, subsequently impairing sperm penetration, whereas short-term intrafollicular cooling does not affect the oocyte's penetrability. Furthermore, greater sperm interaction in oocytes with an intact cumulus suggests a possible role for cumulus cells in canine gamete interaction.

Acrosome↗
PubMed2005

Easy and rapid method for the determination of gene expression in cumulus cells incubated for oocyte maturation.

BACKGROUND: The objectives of this study were to develop an easy and rapid method for measuring gene expression in a small number of cells by real-time PCR without RNA extraction and purification, and to use this method to determine more precisely IGF-I gene expression in the cumulus cells surrounding oocytes. METHODS: First, after small numbers of cumulus cells were lysed in cell lysis buffer, they were digested with various concentrations of DNase I for different periods at 37 degrees C to determine the optimal conditions for digestion of genomic DNA in the lysate. Since nonspecific amplification was liable to occur when the non-purified RT product of the cell lysate was used for real-time PCR with the given primers, the optimal conditions for Mg2+ and annealing temperature were well investigated. Further, to create the same conditions as in the actual sample reaction for measurement by real-time PCR, RT-minus product was added to the reaction mixture of the standard curve, and then the amplification efficiency was assessed. Next, IGF-I gene expression in cumulus cells collected from cumulus oocyte complexes (COCs) every 4 h during maturation was determined using the developed method. RESULTS: The optimal conditions for measuring gene expression using the cell lysate from a small number of cells were as follows: incubation of the cell lysate with 0.16 U/microL DNase I with 10 U/microL for 30 min, an Mg concentration of 1.5 mM for amplification of target gene by real-time PCR using RT-product of the cell lysate. When the RT-minus products added to the reaction mixture for the standard curve, which was prepared for purified 18SrRNA plasmid, the PCR efficiency was similar between the sample and the standard. The IGF-I gene expression in the cumulus cells was elevated up through the first 8 h of the culture and then declined gradually by the end of maturation, with the maximal gene expression (778-fold) seen at 8 h. CONCLUSION: It can be concluded that the method developed here, in which equivalent to cumulus cells collected from 0.03-0.075 COCs were employed per reaction, permits rapid and easy determination of target gene expression in a limited number of cells using real-time PCR without RNA extraction.

Animals↗
PubMed2000

Protection of porcine oocytes against apoptotic cell death caused by oxidative stress during In vitro maturation: role of cumulus cells.

The present study was conducted to examine the protective effect of cumulus cells on oocyte damage caused by reactive oxygen species (ROS), generated by the hypoxanthine-xanthine oxidase (XOD) system, during in vitro maturation of porcine oocytes. Cumulus-oocyte complexes (COCs) and cumulus-denuded oocytes (DOs) were cultured for 44 h in NCSU37 supplemented with cysteine, gonadotropins, 10% porcine follicular fluid, and hypoxanthine in the presence or absence of XOD. DNA cleavage and damage were analyzed using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and single cell microgel electrophoresis (comet) assay, respectively, and caspase-3 activity and glutathione (GSH) content were measured in each experimental group. Exposure of DOs to ROS resulted in meiotic arrest and the increase of degenerated oocytes. These degenerated DOs underwent apoptosis, as shown by the TUNEL-positive reaction within their germinal vesicles and the activation of caspase-3. The length of DNA migration in DOs treated with XOD was significantly longer than that of untreated DOs (P: < 0.05). However, irreparable cell damage caused by ROS was not observed in COCs, and no difference was observed in the caspase-3 activity of both COCs treated with and without XOD. A significantly (P: < 0.05) high level of GSH was found in COCs after 44 h of culture, compared with that of oocytes freshly isolated from their follicles, whereas GSH content in DOs markedly decreased after treatment with or without XOD. These findings suggest that cumulus cells have a critical role in protecting oocytes against oxidative stress-induced apoptosis through the enhancement of GSH content in oocytes.

Animals↗
PubMed1989

Quantitative inhibitory influence of porcine cumulus cells upon the maturation of pig and cattle oocytes in vitro.

Porcine cumulus oocyte complexes (COCs) were cultured together in 10-microliters droplets of culture medium. When 10 COCs were cultured for 24 h, germinal vesicle breakdown (GVBD) occurred in 81% of them. When more COCs (20 or 40) were put into the same volume of medium the frequency of GVBD gradually decreased. This inhibition was not observed in denuded oocytes. The process of GVBD was adversely influenced when 10 COCs were cultured in cumulus-preconditioned medium. It is concluded that porcine cumulus cells produced a factor inhibiting GVBD. After removing the inhibitory block and extensive washing, GVBD of arrested oocytes was significantly accelerated. The addition of LH or heparin only partially overcame the inhibitory action. This factor produced by porcine cumulus cells negatively influenced maturation of bovine oocytes; however, a similar effect was not demonstrated in the mouse. Our results suggest that a high concentration of porcine cumulus cells exerts a quantitative inhibitory effect upon GVBD of porcine and cattle oocytes cultured in vitro.

Animals↗
PubMed2005

Reduction of progesterone receptor expression in human cumulus cells at the time of oocyte collection during IVF is associated with good embryo quality.

BACKGROUND: It has been reported that the progesterone receptor (PR) level is transiently increased within the follicle by LH stimulation and controls cumulus cells in follicles and oocyte maturation. The purpose of this study was to predict developmental competence of human oocytes during IVF via analysis of PR in cumulus cells surrounding mature oocytes. METHODS: Prior to oocyte retrieval, the follicular diameter was measured and follicular fluid was collected from each mature follicle. Cumulus cells were manually separated from the oocyte-cumulus complex under a microscope. PR and PR mRNA were assessed by immunohistochemistry and real-time quantitative polymerase chain reaction (RT-PCR) measurement in human cumulus cells. RESULTS: Immunoreactive PR-A was mainly localized in the cytoplasm and PR-B was localized in the nuclei. There was no significant relationship between PR expression and follicular diameter, follicular fluid concentration of steroids, or LH. There was no significant relationship between expression of PRs and fertilization or cleavage rate. However, PR expression was lower in the good morphology group (blastomeres > or =7 cells with fragmentation > or =5% on day 3) when compared to the other groups (P<0.05). CONCLUSIONS: These results suggest that follicular LH or steroids do not affect PR expression, and full reduction of total PR expression on cumulus cells at the time of oocyte collection is associated with good morphology in human oocytes.

Adult↗