Comparison between effects of 3-isobutyl-1-methylxanthine and FSH on gap junctional communication, LH-receptor expression, and meiotic maturation of cumulus-oocyte complexes in pigs

We investigated cAMP content, gap junctional communications (GJCs) status, and LH-receptor (LH-R) expression in porcine cumulus-oocyte complexes (COCs) during in vitro maturation treated with the phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) or with FSH. COCs were cultured for 20 hr (1st culture) in M199 containing 10% FBS (basic medium, BM group) or BM supplemented with FSH (FSH group) or IBMX (IBMX group). Each COC was then transferred into BM containing both FSH and LH and cultured for an additional 24 hr (2nd culture). The proportions of metaphase-II (M-II) oocytes at the end of the 2nd culture did not differ between the FSH (75.7%) and IBMX (68.2%) groups, whereas only 10.1% of oocytes in the BM group reached the M-II stage. During the 1st culture, the cAMP content of COCs and oocytes became significantly higher in the FSH and IBMX groups than in the BM group; the FSH group had a far greater increment than did the IBMX group. GJCs in the FSH and BM groups gradually closed with increasing duration of the 1st culture, whereas a significantly higher proportion of COCs in the IBMX group still had open GJCs than in the other two groups. Furthermore, LH-R mRNA expression significantly increased in both the FSH and IBMX groups compared with the BM group. These results suggest that inhibition of PDEs in porcine COCs make the oocyte ready for release from meiotic arrest, and that maintenance of a moderate cAMP content may prolong GJCs and stimulate LH-R expression.     

Cytoplasmic glutathione regulated by cumulus cells during porcine oocyte maturation affects fertilization and embryonic development in vitro

It is generally accepted that cumulus cells support the nuclear maturation of mammalian oocytes. In the present study, we examined relationships between the cytoplasmic glutathione (GSH) content of porcine oocytes, and oocyte nuclear maturation, fertilization or subsequent embryonic development. Cumulus-oocyte complexes (COCs; control group) and oocytes denuded of cumulus cells after collection (DO 0h group) were cultured for 24h with dibutyryl cAMP, eCG and hCG (first culture step) and then for a further 20h without supplements (second culture step; 44h total culture). After the first culture step, some of the COCs were denuded, either completely (DO 24h group) or partly (H-DO 24h group), and then matured by the second culture step. Also, in the second culture step, some DOs were co-cultured with cumulus cells that had been pre-cultured for 24h (DO 24h+CC group). The maturation rates of all the cumulus-removed groups (DO 0h, DO 24h, H-DO 24h and DO 24h+CC groups) were lower (34.3-45.0%) than that of the control group (64.5%; P<0.05). The GSH contents of matured oocytes in the completely denuded groups (DO 0h, DO 24h and DO 24h+CC groups) were lower (4.03-5.26pmol/oocyte) than that of the control group (9.60pmol/oocyte; P<0.05); however, the H-DO 24h group had an intermediate value (7.0pmol/oocyte). The male pronuclear formation rates of completely denuded oocytes were lower (41.4-59.3%) than that of the control group (89.4%; P<0.05), whereas the H-DO 24h group had an intermediate rate (80.0%). The blastocyst formation rates of the completely denuded oocytes were lower (3.0-4.5%) than that of the control group (19.9%; P<0.05), and the H-DO 24h group again had an intermediate rate (11.6%). The GSH content was correlated with the rates of male pronuclear formation (P<0.01) and blastocyst formation (P<0.01), and also with the number of cells per blastocyst (P<0.01). In conclusion, we inferred that GSH synthesized by intact cumulus cells during maturation culture improved oocyte maturation and played an important role in fertilization and embryonic development.     

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.     

Roles of gap junctional communication of cumulus cells in cytoplasmic maturation of porcine oocytes cultured in vitro

Cumulus cells of the oocyte play important roles in in vitro maturation and subsequent development. One of the routes by which the factors are transmitted from cumulus cells to the oocyte is gap junctional communication (GJC). The function of cumulus cells in in vitro maturation of porcine oocytes was investigated by using a gap junction inhibitor, heptanol. Cumulus-oocyte complexes (COCs) were collected from the ovaries of slaughtered gilts by aspiration. After selection of COCs with intact cumulus cell layers and uniform cytoplasm, they were cultured in a medium with 0, 1, 5, or 10 mM of heptanol for 48 h. After culture in vitro, one group of oocytes was assessed for nuclear maturation and glutathione (GSH) content, and another group was assigned to in vitro fertilization and assessed for the penetrability of oocytes and the degree of progression to male pronuclei (MPN) of penetrated spermatozoa. At the end of in vitro maturation, the oocytes reached metaphase II at a high rate (about 80%) regardless of the presence of heptanol at various concentrations. Cumulus cell expansion and the morphology of oocytes cultured in the medium with heptanol were similar to those of control COCs matured without heptanol. The amount of GSH in cultured oocytes tended to decrease as the concentration of heptanol in the medium was increased. Although there was no difference in the rates of penetrated oocytes cultured in media with different concentrations of heptanol, the proportion of oocytes forming MPN after insemination decreased significantly (P < 0.01) at all concentrations tested. A higher rate of sperm (P < 0.01) failed to degrade their nuclear envelopes after penetration into the oocytes that were treated with heptanol. GJC between the oocyte and cumulus cells might play an important role in regulating the cytoplasmic factor(s) responsible for the removal of sperm nuclear envelopes as well as GSH inflow from cumulus cells.     

Cumulus cells suppress meiotic progression in pig oocytes cultured in vitro

The objective of this study was to examine the effect of cumulus cell removal from cumulusoocyte complexes (COCs) on meiotic progression. In Experiments 1, 2 and 3, pig COCs were cultured for 16, 20 and 24 h, respectively. The cumulus cells were then removed, and the denuded oocytes were incubated in fresh medium for another 32 h in Experiment 1, for 28 h in Experiment 2 and for 24 h in Experiment 3. In Experiment 4, the denuded oocytes and COCs were co-cultured in a drop of fresh medium from 24 h of cultivation to the end of the culture period (48 h). Removal of the cumulus cells after 16 h of cultivation had no effect on the proportions of oocytes both undergoing germinal vesicle breakdown (GVBD) and reaching MII. When the denuded oocytes were further cultured for 24 h, following the removal of their cumulus cells after 24 h of cultivation, the proportion of oocytes undergoing GVBD was significantly higher (90%, P < 0.05) than that of oocytes that were continuously cultured for 48 h without removing the cumulus cells (80%). Removal of the cumulus cells after 20 and 24 h of incubation produced a significant increase in the proportion of oocytes reaching the MII stage (84%, P < 0.05 and 76%, P < 0.01, respectively) as compared with COCs cultured continuously for 48 h without removing cumulus cells (71% and 55%, respectively). The maturation rate of denuded oocytes co-cultured with COCs for the second 24 h of cultivation was comparable to that of denuded oocytes cultured without COCs (77 and 74%, respectively). From these results, it was concluded that cumulus cells surrounding oocytes suppressed meiosis of both the GVBD process and progression from GVBD to MII in pig oocytes cultured in vitro, and that the suppressive factor in meiotic progression produced by the cumulus cells might be transferred to the oocytes through gap junctions rather than through the medium.     

Luteinizing hormone receptor formation in cumulus cells surrounding porcine oocytes and its role during meiotic maturation of porcine oocytes

We investigated the formation of LH receptor (LHR) in cumulus cells surrounding porcine oocytes and the role of LHR in meiotic maturation of oocytes. At least three splice variants of LHR mRNA were detected in cumulus cells, in addition to the full-length form. Low levels of three types of products were seen in cumulus cells from cumulus oocytes complexes (COCs), whereas the full-length form was significantly increased by 12-h cultivation with FSH. The addition of FSH also significantly increased the binding level of biotinylated hCG to COCs. The formation of LHR in FSH-stimulated cumulus cells was not affected by additional 0.5 mM phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), and the oocytes were synchronized to the germinal vesicle (GV) II stage by exposure to 0.5 mM IBMX and FSH for 20 h. The binding of LH to its receptor induced a further increase in cAMP level and progesterone production and acceleration of meiotic progression to the metaphase I stage. The oocytes cultured with LH for 24 h following cultivation with FSH and IBMX were used for in vitro fertilization. At 6 days after in vitro fertilization, blastocyst rate in oocytes matured under these conditions was significantly higher than that of oocytes cultured in the absence of LH. Treatment of oocytes with FSH and 0.5 mM IBMX to express LH receptor in cumulus cells while holding oocytes at the GV II stage is a very beneficial way to produce in vitro-matured oocytes, which have high developmental competence.     

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.     

Effect of growth hormone releasing hormone (GHRH) and vasoactive intestinal peptide (VIP) on in vitro bovine oocyte maturation

The aim of this study was to investigate the effects of growth hormone releasing hormone (GHRH) and the structural-related peptide vasoactive intestinal peptide (VIP) on nuclear maturation, cortical granule distribution and cumulus expansion of bovine oocytes. Bovine cumulus oocyte complexes (COCs) were cultured in M199 without FCS and gonadotropins and in the presence of either 100 ng/mL bovine GHRH or 100 ng/mL porcine VIP. The COCs were incubated at 39 degrees C in a humidified atmosphere with 5% CO2 in air, and the nuclear stage was assessed after 16 or 24 h of incubation using DAPI staining. Cortical granule distribution was assessed after 24 h of incubation using FITC-PNA staining. To assess the effects of GHRH and VIP on cumulus expansion, COCs were incubated for 24 h under the conditions described above. In addition, 0.05 IU/mL recombinant human FSH was added to GHRH and VIP groups. Cultures without GHRH/VIP/FSH or with only FSH served as negative and positive controls, respectively. At 16 h neither GHRH (42.9%) nor VIP (38.5%) influenced the percentage of MII stage oocytes compared with their respective controls (44.2 and 40.8%). At 24 h there also was no difference in the percentage of MII oocytes between GHRH (77.0%), VIP (75.3%) and their respective controls (76.0 and 72%). There was no significant cumulus expansion in the GHRH or VIP group, while FSH induced significant cumulus expansion compared with the control groups, which were not inhibited by GHRH or VIP. Distribution of cortical granules was negatively affected by GHRH and VIP. The percentage of oocytes showing more or less evenly dispersed cortical granules in the cortical cytoplasm aligning the oolemma (Type 3) was lower in the GHRH (2.7%) and VIP (7.8%) groups than in the control group (15.9%). In conclusion, GHRH and VIP have no effect on nuclear maturation or cumulus expansion of bovine COCs but retard cytoplasmic maturation, as reflected by delayed cortical granule migration.     

Effect of gonadotropins during in vitro maturation of feline oocytes on oocyte-cumulus cells functional coupling and intracellular concentration of glutathione

Information about the mechanisms of meiotic arrest and resumption of meiosis in feline oocytes is still limited. The aim of this study was to investigate the effect of the presence of gonadotropins during IVM, on meiotic progression in relation to the status of gap junction mediated communications between oocyte and cumulus cells, to the cAMP intracellular content, and to the intra-oocyte concentration of glutathione (GSH) in feline oocytes. Our results indicated that about 50% of cumulus-oocyte complexes (COCs) showed functionally open communications at the time of collection, while the remainder were partially or totally closed. After 3h of culture, the percentage of COCs with functional gap junctions was significantly greater in the group matured in the presence of gonadotropins than in those matured without them, where an interruption of communications was observed. Moreover, this precocious uncoupling was associated with a moderate increase of cAMP concentration in the oocyte, lower than in the group exposed to gonadotropins. Intra-oocyte glutathione levels decreased significantly after 24h of IVM, whether gonadotropins were present or absent during the culturing process. The presence of thiol compounds in the IVM medium induced an intra-oocyte GSH concentration significantly higher than that found in oocytes cultured without these compounds, and similar to the GSH content of immature oocytes. Moreover, the intracellular GSH concentration increased as meiosis progressed. The present study suggests that in feline oocytes, gonadotropins affect the dynamic changes in communications between oocyte and cumulus cells during IVM. However, the intracellular concentration of GSH is not influenced by the gonadotropin stimulation. Moreover, the presence of gonadotropins and thiol compounds results in an increase of GSH levels along with meiotic progression of the oocytes.     

Effect of human chorionic gonadotrophin supplementation during different culture periods on in vitro maturation of canine oocytes

The IVM of canine oocytes is characterized by low rates of metaphase II. The objective of this study was to evaluate the effects of hCG on meiotic development of canine oocytes for culture periods up to 96 h. Oocytes were collected after ovariohysterectomy. Only oocytes >110 microm in diameter, with a homogeneous dark cytoplasm and three or more layers of compact cumulus cells were used. For IVM, the COCs were cultured in TCM-199+10% fetal calf serum, without (medium A control) or supplement with 10 IU/mL of hCG (medium B), or with a combination of both media (treatment B/A). The COCs were randomly allocated into three groups. The first and second groups were cultured in either medium A or B, respectively for 24, 48, 72, and 96 h. Oocytes of the third group (treatment B/A) were incubated in medium with hCG (medium B) the first 48 h and then transferred to medium without hCG (medium A) for an additional 24 or 48 h. The proportion of COCs with cumulus cell expansion was also evaluated before fixation. Oocytes were stained with propidium iodide prior to nuclear assessment (with epifluorescence microscopy). COCs with cumulus expansion were evident after 48 h of culture. The proportion of COCs with cumulus expansion was higher (P<0.05) for media containing hCG (B or B/A) than for meda lacking hCG (A); this difference was maintained for 72 and 96 h in culture. In media A, B and B/A, 23.3, 31.7 and 29.5%, respectively, of oocytes were at metaphase II after 72 h, with 20.7, 33.1 and 43.4% at this stage after 96 h. The advancement of meiosis was directly proportional to the time of incubation; the highest percentage (P<0.05) of oocytes at metaphase II was observed after 96 h of culture when 10 IU/mL hCG was present for only the first 48 h of culture.     

Positive feedback loop between prostaglandin E2 and EGF-like factors is essential for sustainable activation of MAPK3/1 in cumulus cells during in vitro maturation of porcine cumulus oocyte complexes

During in vitro maturation of porcine cumulus-oocyte complexes (COCs), follicle-stimulating hormone (FSH) increases both prostaglandin E2 (PGE2) production and the expression levels of EGF-like factors. The ligands act on cumulus cells by the autocrine system due to their specific receptors, EP2, EP4, or EGF receptor. When each pathway is suppressed by inhibitors, complete cumulus expansion and oocyte maturation do not occur. In this study, we examined the relationship between both of these pathways in cumulus cells of porcine COCs. When COCs were cultured with FSH, Fshr mRNA expression was immediately decreased within 5 h, whereas Ptger2, Ptger4, and Ptgs2 expression levels were significantly increased in cumulus cells in the culture containing FSH for 5 or 10 h. The PTGS2 inhibitor NS398 significantly suppressed not only PGE2 secretion at any culture time point but also Areg, Ereg, and Tace/Adam17 expression in cumulus cells at 10 and 20 h but not at 1 or 5 h. During the early culture period, phosphorylation of MAPK3 and MAPK1 (MAPK3/1) was not affected by NS398; however, at 10 and 20 h, phosphorylation was suppressed by the drug. Furthermore, down-regulations of MAPK3/1 phosphorylation and expression of the target genes by NS398 was overcome by the addition of either PGE2 or EGF. FSH-induced cumulus expansion and meiotic progression to the MII stage were also suppressed by NS398, whereas these effects were also overcome by addition of either PGE2 or EGF. These results indicated that PGE2 is involved in the sustainable activation of MAPK3/1 in cumulus cells via the induction of EGF-like factor, which is required for cumulus expansion and meiotic maturation of porcine COCs.     

Changes in cumulus-oocyte complexes of pregnant and non-pregnant camels (Camelus dromedarius) during maturation in vitro

The aim of the present study was to examine the cumulus morphology and the oocyte chromatin quality of camel cumulus-oocyte complexes (COCs) at the time of recovery, and to monitor changes in oocyte chromatin configuration and apoptosis in cumulus cells from camel COCs during in vitro maturation (IVM) (0, 12, 24, 32, 36, 42, and 48 p.IVM) depending on pregnancy of donors. A total of 1023 COCs were isolated from sliced ovaries after slaughtering of 47 pregnant and 43 non-pregnant camels in an abattoir. The mean number of COCs per donor was 10.3 in pregnant and 12.5 in non-pregnant donors. The cumulus morphology of COCs was independent of the type of donor and was divided in COCs with compact (26.9 and 28%), dispersed (39.3 and 46%), corona radiata cumulus investment (27.9 and 21.7%) and without cumulus (6 and 4.2%), respectively for pregnant and non-pregnant donors. The highest proportion of COCs exhibited dispersed cumulus (P<0.05). Oocytes with meiotic stages of diplotene >50% were found only in compact (55 and 56.5%) and in dispersed COCs (58.4 and 60%), respectively for pregnant and non-pregnant donors. During IVM (0-48h) the first significant onset of specific meiotic stages were different in oocytes from pregnant donors: metaphase 1 (24-32h), metaphase 2 (36-42h), versus oocytes from non-pregnant donors: metaphase 1 (24h), metaphase 2 (32-48h) (P<0.05). The level of apoptotic cells in cumuli of matured COCs increased during IVM and was higher in matured COCs from non-pregnant donors for each time point during IVM (P<0.01). Camel oocytes meiosis during IVM is accompanied by a drastic increase of apoptosis in the surrounding cumulus cells 0-32 and 0-24h during IVM, respectively for pregnant and non-pregnant donors. The oocytes of pregnant camels require 36h of maturation to reach levels of >50% metaphase 2 stage in comparison to oocytes from non-pregnant donors where 32h are sufficient. The earlier onset of apoptosis in the COCs derived from non-pregnant donors possibly determines the faster progression of the oocytes through the final stages of meiosis.     

Relationship between the morphological changes of somatic compartment and the kinetics of nuclear and cytoplasmic maturation of oocytes during in vitro maturation of porcine follicular oocytes

Based on the morphology and expansion of the cumulus cells, several different classes of porcine cumulus-oocyte complexes (COCs) can be distinguished, during their maturation in vitro. The goal of the present study was to find out the rate of each morphologic category in case of COCs and granulosa-cumulus-oocyte complexes (GCOCs), the characteristics of their nuclear progression, cytoplasmic maturation, and the frequency of monospermy after IVF. It was found that the frequency of cumulus expansion is higher in case of GCOCs than that of COCs. Nuclear progression of COCs was more accelerated than that of GCOCs. Oocytes attached to the bottom of culture dish with dark, compact cumulus underwent nuclear and acquired their ability to be activated earlier than that of oocytes showing normal cumulus expansion. The rate of monospermic fertilization after IVF of normal COCs showing normal cumulus expansion was higher than that of COCs attached to the dish. These results suggest that diverse behavior of cumulus cells during in vitro culture affects nuclear and cytoplasmic maturation of porcine oocytes, which also affects IVF results. It can be concluded that granulosa cells promote normal cumulus expansion thus decrease heterogeneity in nuclear and cytoplasmic maturation amongst oocytes.     

Production of progesterone from de novo-synthesized cholesterol in cumulus cells and its physiological role during meiotic resumption of porcine oocytes

To investigate the role of factors secreted by cumulus cells during meiotic resumption of porcine oocytes, 1, 5, 10, or 20 cumulus-oocyte complexes (COCs) were cultured in each well of a culture dish containing 300 microl of maturation medium for 20 h. There was a significant positive correlation between the rate of germinal vesicle breakdown (GVBD) and the number of COCs cultured in each well for 20 h. The level of progesterone in the medium in which COCs had been cultured for 20 h also rose significantly with an increase in the number of COCs cultured in each well. A significantly small proportion of GVBD in oocytes when one COC was cultured in each well for 20 h was improved by the addition of progesterone. This proportion of GVBD was fully comparable to that of COCs cultured in the absence of additional progesterone with 20 COCs. Thus, progesterone secreted by COCs plays a positive role in GVBD induction in porcine oocytes. Furthermore, we also examined the role of sterol biosynthesis on progesterone production by cumulus cells and in oocyte GVBD. The results showed that the addition of ketoconazole, which suppressed the sterol biosynthetic pathway produced by demethylation of lanosterol, decreased the rate of GVBD, as well as progesterone production in COCs cultured for 20 h. However, the suppression of GVBD by ketoconazole was overtaken by the addition of progesterone. These results demonstrate that a high level of progesterone produced by cumulus cells was responsible for an acceleration of GVBD in porcine oocytes.     

Chronological and ultrastructural changes in camel (Camelus dromedarius) oocytes during in vitro maturation

Cumulus-oocyte complexes (COCs) were collected from non-pregnant camels at a local slaughterhouse by aspiration from antral follicles (2-6 mm). In Experiment I, camel COCs (n=304) were matured in vitro in Hams-F10, fixed at different time intervals (6, 12, 18, 24, 30, 36, 42, or 48 h) and stained with 1% aceto-orcein to assess nuclear changes in culture. A majority of the oocytes (81.5%) underwent germinal vesicle break down (GVBD) between 6 and 12h. Forty-eight percent of the oocytes were observed at the metaphase I (M I) stage by 18 h culture. The percentage of matured oocytes (M II stage) at 30 and 42 h were 66.5 and 71% respectively, which were significantly (p<0.05) different to that observed at 24 h (42.5%). In Experiment II, after different periods of culture (12, 24, 36, or 48 h), the COCs (n=26) were processed for transmission electron microscopy. Expansion of both the cumulus and corona radiate cells occurred between 12 and 24 h in the majority of oocytes concomitant with enlargement of the cumulus cell process endings (CCPEs) in the developed perivitelline space. After 12 h of culture disruption of the junctions between CCPEs and the oolemma was observed together with and breakdown of the GV. For 24-36 h of culture cortical granules had spread and aligned along the oolemma. Signs of degeneration in the cytoplasmic organelles of the oocytes were also observed from less than 36 h. After 48 h of culture, larger vesicles and lipid droplets had appeared in the central part of the oocytes and showed uneven distribution throughout the ooplasm. Predominantly non-penetrating CCPEs were also observed in four oocytes by 48 h. In conclusion, based on both light and electron microscopic evaluations, the optimal culture time for the development of competent Camelus dromedarius oocytes in vitro appears to be 30 h using Hams-F10 medium.     

Production of plasminogen activators (PAs) in bovine cumulus-oocyte complexes during maturation in vitro: effects of epidermal growth factor on production of PAs in oocytes and cumulus cells.

We examined whether plasminogen activators (PAs) are produced by bovine cumulus-oocyte complexes (COCs) during maturation in vitro. The effects of epidermal growth factor (EGF) on production of PAs in oocytes and cumulus cells were also examined. When COCs were cultured for 24 h with 30 ng/ml EGF, three plasminogen-dependent lytic zones (58.5 +/- 3.5 kDa, 79.0 +/- 3.0 kDa, and 113.5 +/- 6.5 kDa) were observed. Addition of amiloride, a competitive inhibitor of urokinase-type PA (uPA), to the zymogram eliminated the activity of the 58.5 +/- 3.5-kDa zone, suggesting that this band is a uPA. However, since the activity of the remaining two bands was not eliminated, it was suggested that the 79.0 +/- 3.0-kDa band is a tissue-type PA (tPA) and the 113.5 +/- 6.5-kDa band is possibly a tPA-PA inhibitor (tPA-PAI) complex. In COCs before culture, however, no activity of PAs was detected. At 6 h of culture, the same level of uPA activity was detected in COCs cultured both in the absence and in the presence of EGF. The uPA activity was increased at 12 h of culture but without further increase at 24 h of culture, with higher activity in the presence than in the absence of EGF. The activity of tPA and tPA-PAI was first detected at 24 h of culture in the absence of EGF. In the presence of EGF, however, some activity of tPA-PAI was detected at 12 h of culture. At 24 h of culture, the activity of all PAs was detected in cumulus cells, but only uPA activity was detected in oocytes, with higher activity in the presence than in the absence of EGF. The uPA activity in oocytes was not detected when they were cultured without cumulus cells in either the presence or absence of EGF, although cumulus expansion was stimulated by EGF, exhibiting a time-course similar to that observed in PA production. These results suggest that uPA, tPA, and tPA-PAI are all produced by bovine COCs, but only uPA by oocytes, during maturation in vitro. However, cumulus cells play an essential role or roles in the production of uPA by oocytes, and EGF enhances the roles of cumulus cells.