Expression of growth differentiation factor 9 (GDF-9) during in vitro maturation in canine oocytes

The aim of this study was to characterize in canine oocytes and cumulus cells the dynamic expression of growth differentiation factor 9 (GDF-9) in relation to meiotic development and cumulus expansion throughout in vitro maturation (IVM). Cumulus oocytes complexes (COCs) from ovaries of adult bitches were cultured intact for IVM during 0, 48, 72, and 96 hours. At 0 hours or after IVM, COCs were divided into two groups: one group remained with their cumulus cells and in the other group the cumulus cells were extracted. The expression levels of GDF-9 were determined in both groups using indirect immunofluorescence and Western blot analysis. For immunofluorescence assay, in vivo-matured oocytes collected from oviducts were also used as a positive control. The nuclear stage was analyzed in parallel with 4′-6-diamidino-2-phenylindole staining in denuded oocytes from all maturing groups. The intensity of fluorescence, indicative of GDF-9 expression level, decreased with time (P < 0.05). High expression was observed only in germinal vesicle nonmature oocytes; in contrast, second metaphase oocytes showed only low expression. Western blot analysis showed bands of approximately 56 kd and a split band of approximately 20 kd representing the proprotein and possibly two mature protein forms of GDF-9, respectively. The proprotein was detected in all samples, and it was highly expressed before IVM and in a lesser degree, during the first 48 hours, declining thereafter in coincidence with the expansion of the cumulus cell (P < 0.05). There was a negative correlation (r = −0.97; P < 0.05) between the expression level of GDF-9 and mucification. Mature forms were evident only in COCs, before culture and up to 48 hours of IVM. It was concluded that GDF-9 is expressed in canine oocytes and cumulus cells, mainly in the early developmental states, with low levels in mature oocytes in vitro and in vivo, representing the first approach of GDF-9 dynamic in dog oocyte maturation.     

Effect of cumulus cell coculture and oxygen tension on the in vitro developmental competence of bovine zygotes cultured singly

The customary practice in bovine in vitro embryo production (IVP) is to handle oocytes and embryos in groups; although there are several reasons for establishing an IVP system for individual embryos that allows for following a single oocyte from retrieval through development to the blastocyst stage. To date, reports of individual IVP are inconsistent, and in most cases, resulted in unsatisfactory blastocyst rates. The objective of this study was to develop an efficient system for routine in vitro culture of individual bovine embryos. Single culture of zygotes in 2 different culture volumes (20 and 500 μL) yielded less than 3% blastocysts in experiment 1. In an attempt to improve these results, cumulus cells were added to the culture medium in experiment 2, after which blastocyst rates increased from 2.9 to 21.8% (P < 0.05). The third experiment revealed that an atmospheric oxygen tension, which is commonly used with somatic cell coculture, was not beneficial during individual embryo-cumulus cell coculture, because it resulted in lower blastocyst rates (Odds ratio 0.57, P < 0.001) and in lower blastocyst cell numbers (P < 0.05), when compared to culture in 5% oxygen. Grouped vs. single culture and reduced oxygen tension did not have a significant effect on cleavage and hatching rates. In experiment 4, three different cumulus cell coculture conditions during individual culture were tested and compared with the cleavage, blastocyst and hatching rates, and cell number of group culture (73.2%, 36.4%, 66.7% and, 155.1 ± 7.26, respectively). The outcome variables after individual embryo culture on a 5-day-old cumulus cell monolayer (74.1%, 38.2%, 71.9% and 133.4 ± 9.16, respectively), and single culture in the presence of added cumulus cells (69.9%, 31.9%, 66.7% and 137.3 ± 8.01, respectively) were not significantly different from those obtained after group culture (P < 0.05). Though, individual culture in a cumulus cell conditioned medium significantly reduced both the cleavage (59.0%) and blastocyst rates (6.3%). These results demonstrate that single culture of bovine zygotes can be fully sustained by coculture with cumulus cells in a low oxygen environment; implementation of these findings in our IVP system produced blastocysts comparable in quantity and quality to those obtained by group culture. These results were consistently achieved after acquiring experience and expertise in the handling of single zygotes.     

Effects of co-culture and embryo number on the in vitro development of bovine embryos

It is generally accepted that culturing embryos in groups or with somatic cells improves both the yield and quality of the blastocysts obtained. The aims of this study were 1) to compare the yield and quality of the embryos obtained after culture in several number conditions and in several culture systems and 2) to assess the effect of co-culture started at various stages of embryo development. Under cell-free culture conditions (modified synthetic oviduct fluid [mSOF] supplemented with 10% fetal calf serum [FCS] 48 h post insemination, the rate of Day 10 blastocysts was lower when embryos were cultured in small groups (1 to 6 per drop) than in large groups (4 versus 23% ; P < 0.01). There was no group effect when embryos were co-cultured either with Buffalo rat liver (BRL) cells in TCM 199, or in a culture system allowing the progressive development of cumulus cells in mSOF, even if co-culture started at 66 or 114 h post insemination. However, embryos cultured singly had lower cell numbers than embryos cultured in large groups when co-culture started at 114 h post insemination. This suggests that 1) somatic cells improve the development of singly cultured bovine embryos up to the blastocyst stage after the 9-16 cell stage; 2) co-culture affects blastocyst cell number of singly cultured embryos by acting roughly between the 5-8 and the 9-16 cell stage; and 3) cooperation between embryos could replace the effect of co-culture either on the yield of blastocysts or on blastocyst cell number. Blastocysts appeared significantly earlier in co-culture with cumulus cells in mSOF than in co-culture with BRL cells in TCM 199 (detection of the blastocysts: 7.3 +/- 0.1 d post insemination with cumulus cells versus 8.1 +/- 0.1 d with BRL cells; P < 0.001) and had a significant higher number of cells (143 +/- 9 versus 85 +/- 11; P < 0.001). This system thus seems suitable for the culture of small numbers of embryos resulting from in vitro maturation and fertilization of oocytes from individual donor cows.     

Developmental stage of the oocyte during antral follicle growth and cumulus investment determines in vitro embryo development of sow oocytes

The aim of the study was to determine the contribution of cumulus cells on the developmental competence of porcine oocytes during follicle growth. Oocytes from large (5-8mm) and small (2-3mm) follicles were cultured with or without follicle stimulating hormone (FSH), subsequently examined for nuclear stage and spindle morphology, or fertilized and cultured for embryo development, or analyzed for glutathione content. Additionally, the significance of cumulus investment, corona radiata cells, cumulus cell number and origin of cumulus cells for oocyte maturation were investigated. Small follicle oocytes cultured without FSH exhibited the highest incidence of spindle aberrations. Oocytes cultured without FSH exhibited reduced sperm penetration and blastocyst rates, and a higher proportion monospermic oocytes developed to the blastocyst stage when derived from large follicles. The glutathione content in oocytes increased during follicle growth and oocyte maturation, but no direct correlation between oocyte glutathione content and oocyte developmental capacity was observed. Oocytes with a bigger cumulus investment exhibited better embryo development. Oocytes with a single corona radiata cell layer (CROs) exhibited similar progression through meiosis to oocytes with more cumulus cell layers, but showed reduced embryo development. More blastocysts were observed when CROs were cultured with disconnected cumulus cells during IVM, but no blastocyst increase was observed when CROs were cocultured with a higher number of cumulus cells or with cumulus cells from large follicles. We conclude that increased developmental capacity of oocytes during follicle growth is intrinsic and whether cumulus cells originate from large or small follicles, their contribution to oocyte maturation remains unchanged. Further, cumulus investment can be used as a variable to predict oocyte developmental capacity.     

Cumulus contributions during bovine fertilization in vitro

A mandatory step in performing micromanipulation techniques, studying sperm-oocyte interactions and evaluating morphological aspects of oocyte quality is the removal of cumulus cells from oocytes or zygotes at various stages. In cattle, cumulus removal shortly before fertilization in vitro strongly decreases sperm penetration rates. This study was conducted to evaluate the function of the cumulus oophorus during bovine fertilization in vitro. The importance of cumulus secretions during IVF was investigated by inseminating cumulus-denuded oocytes (CDOs) in fertilization medium supplemented with individual cumulus secretions, such as progesterone or hyaluronic acid. None of these substances increased the fertilization rate of CDOs. However, fertilizing CDOs in cumulus-conditioned medium or on a cumulus monolayer partially restored the reduction in fertilization rate (P<0.05). The fertilization rate of CDOs inseminated on a cumulus monolayer further increased when physical contact between the gametes and the monolayer was prevented by fertilizing them inside a culture plate insert placed on the monolayer (P<0.05). Finally, the importance of reactive oxygen species (ROS) generation and O(2) concentration during IVF was studied. Luminol-dependent chemiluminescence revealed a higher ROS load in conditioned medium of cumulus-enclosed oocytes (CEOs) than in that of CDOs after sperm-oocyte co-incubation (P<0.05). Furthermore, lowering the external O(2) concentration from 20 to 5% decreased the fertilization rate of both CEOs and CDOs, but had a higher impact on CEOs (P<0.05). In conclusion, this study provides evidence that the cumulus oophorus benefits the fertilizing ability of penetrating spermatozoa by creating a complex microenvironment of both cumulus secretions and metabolic products around the oocyte. Gap junctional communication between the oocyte and corona cells as well as sperm trapping by the cumulus oophorus seem to be essential factors in supporting fertilization.     

Effect of follicle cells on in vitro fertilization of pig follicular oocytes

We investigated the effect of cumulus and granulosa cells (follicle cells) on in vitro fertilization of pig follicular oocytes matured in vitro. Oocytes surrounded by cumulus and connected with a piece of parietal granulosa cells (complexes) were matured in vitro for 46hours and were then divided into 4 groups: Group I oocytes were surrounded by expanded cumulus and granulosa cells; Group II oocytes were surrounded by expanded cumulus cells; Group III were denuded oocytes; and Group IV were denuded oocytes with cumulus cells from other complexes. After incubation for 4 hours and 40 minutes with frozen, thawed and preincubated pig epididymal spermatozoa, the oocytes were cultured for 5 hours and 20 minutes. When oocytes were inseminated in the presence of cumulus cells, the penetration rates were higher (92.5% for Group II and 89.5% for Group IV) than when cumulus cells were not used for insemination (Group III, 66.8%) or when oocytes with follicle cells were inseminated (Group I, 72.3%). Denudation of follicle cells before insemination (Group III) decreased the percentage of male pronuclear formation (50.8%) compared with that of oocytes surrounded by follicle cells (66.7% for Group II and 80.2% for Group I). These results support the ability of a moderate number of follicle cells to facilitate sperm penetration of pig follicular oocytes and male pronuclear formation.     

Single in vitro bovine embryo production: coculture with autologous cumulus cells, developmental competence, embryo quality and gene expression profiles

Studies concerning oocyte quality markers, oocyte/embryo metabolism or commercial OPU settings treating donors with low oocyte yields, indicate a need for optimization of IVP protocols to culture single oocytes to the blastocyst stage. However, culture conditions for single oocyte usually impair development, although previous research showed that single oocyte culture on a monolayer of cumulus cells can lead to similar developmental competence than group oocyte culture. Aiming to develop a fully single IVP procedure, Experiment 1 and 2 revealed that individual maturation, fertilization and culture in 20 μL droplets, using a monolayer of heterologous (SSSm, Exp 1) or autologous cumulus cells in coculture (SSSa, Exp 2), resulted in 23.9% and 15.1% of blastocysts 8 days p.i., respectively, which is significantly less compared to regular group IVP (GGGc, 33.5% (Exp 1) and 26.2% (Exp 2), respectively). In a third Experiment, day 7 p.i. blastocyst quality was analyzed in four treatment groups: regular group IVP (GGGc), group IVP with coculture (GGGm), in group produced zygotes, singly cultured on a heterologous cumulus cell monolayer (GGSm) and individually matured and fertilized zygotes, singly cultured on a monolayer (SSSm). Mean cell number and apoptotic cell index, were similar for all treatment groups. Moreover, mRNA abundance relative to H2AFZ was equal for 9 qualitatively linked genes (TP53, BAX, SHC1 SHC, IGF2R, PTGS2, AKR1B1, PLAC8, SLC2A1, and MNSOD). Only GPX1, involved in detoxification and mtDNA protection to oxidative stress, was significantly downregulated (ANOVA, P < 0.05) in singly produced blastocysts (SSSm), compared to the other treatments. In conclusion, a valuable individual IVP system was established and autologous cumulus cells in coculture showed to partly neutralize hampered individual culture conditions. Additionally, to our knowledge this is the first report in which blastocyst quality, in terms of cell number, apoptosis and gene expression, of singly produced embryos was investigated and shown to be similar to in group produced embryos, implicating that the single IVP system can be applied as a tool in oocyte and embryo quality studies.     

Maturation of the rat cumulus-oocyte complex: structure and function

The cumulus cells that surround the mammalian oocyte become dispersed following the preovulatory surge of the pituitary gonadotropin, luteinizing hormone (LH). We have examined cumulus-oocyte complexes of PMSG-primed immature rats before and at 1, 2, 3, 4, 6, and 8 hr after injection of human chorionic gonadotropin (hCG), which acts on the rat ovary like the pituitary gonadotropin. Associations between projections of the cumulus cells and the oocyte were analyzed in thin sections. We observed that some cumulus projections were greatly enlarged where they associate with the oocyte. These enlarged regions were filled with numerous small vesicles. Gap junctions between cumulus cell projections and the oocytes were small. We quantitated the number and size of gap junctions between cumulus cells. The number of small gap junctions (less than 1 microM) between cumulus cells did not change significantly over the 8-hr period after hCG administration. Larger gap junctions, however, showed a general downward trend beginning after the third hour post hCG. Light microscopic observations of plastic sections revealed that dispersion of the cumulus oophorus is not observed until after 4 hr post-hCG, but between 4 and 8 hr after gonadotropin administration the cumulus becomes markedly dispersed. In the majority of the oocytes in these complexes the germinal vesicle (GV) displayed some irregularity in shape at 2 hr post-hCG, although absence of the GV was not observed until later. Our observations suggest a new means of communication in the cumulus-oocyte complex by the vesicle-filled enlargements of the cumulus cell projections at the oocyte surface. They further indicate that the decrease in metabolic coupling observed in rat cumulus-oocyte complexes soon after exposure to LH is not associated with a change in number and size of the gap junctions between the cumulus cells. We suggest that it is either the disruption of the gap junctions at the region of contact of the cumulus cell projections with the oocyte surface or the operation of a gating mechanism that blocks the junctional channels without affecting their morphological appearance that is responsible for uncoupling of the oocyte from the cumulus cells.     

Apoptotic index within cumulus cells is a questionable marker of meiotic competence of bovine oocytes matured in vitro

Information gained from most human studies indicate a negative correlation between the apoptotic index (AI) in cumulus cells (CC) and the quality of the corresponding oocytes. However, results obtained in other species are not so consistent. The rate of apoptosis-free COCs (cumulus oocytes complexes) subjected to IVM (in vitro maturation) also varies among studies. The aim of the present study was to investigate whether the AI in cumulus cells of post-IVM COCs is related to the morphology of pre-IVM COCs and to meiotic competence of bovine oocytes. COCs of known morphology (four grade scale) obtained from individual follicles were matured in a well-in-drop system. After IVM, the external layers of CC of each COC were analyzed by TUNEL. In order to determine the meiotic stage, oocytes were stained with DAPI. It was found that 25.6% of bovine COCs contained apoptosis-free cumulus cells. Moreover, the majority of COCs with apoptotic cells were characterized by apoptotic index lower than 15%. The level of apoptosis in CC was related neither to COC morphology nor to the oocyte meiotic stage. It is suggested that the extent of apoptosis in cumulus cells is not a reliable quality marker of the corresponding oocyte after IVM.     

Cumulus cells secrete a meiosi-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. The GVBD of unstimulated DO also increased significantly when cocultured with cumulus cells from primed CEO. The percentage of GVBD in unprimed DO and in DO isolated from primed CEO was the same. We suggest that within 1–2 hr, forskolin and cAMP stimulate cumulus cells to produce a diffusible meiosis-inducing substance which overcomes HX-inhibition and induces oocyte maturation, including both GVBD and PB formation. The CEO must be primed for more than 2 hr before the resumption of meiosis in DO isolated from such CEO is induced. Oocyte-cumulus connections are crucial as far as initiating the production of a meiosis-inducing substance is concerned. Oocyte-cumulus connections are not needed for transferring this substance to the oocyte. © 1994 Wiley-Liss, Inc.     

Beneficial influence of Vero cells on in vitro maturation and fertilization of bovine oocytes

The aim of this study was to examine the effects of Vero cells and other somatic cells on in vitro maturation of bovine oocytes. Both denuded oocytes and oocytes with intact cumuli (COCs) were cultured on monolayer of Vero cells, cumulus cells and granulosa cells. The effect of gonadotropins was investigated after the addition of gonadotropins to the culture medium. The evaluation using analysis of variance revealed that removal of cumulus cells generally reduced the percentage of oocytes completing their maturation in vitro and that this effect could not be overcome by the addition of gonadotropins to the culture medium. However, in individual experiments, when oocytes were co-cultured with different monolayers of somatic cells, Vero cells were able significantly support the maturation of denuded oocytes, and their beneficial effect was further enhanced by the addition of gonadotropins (76 vs 80.9%). We did not observe a similar effect after the co-culture of oocytes with a monolayer of cumulus cells (65.3 and 53%, respectively). Granulosa cell monolayer delayed maturation in the both COCs and denuded oocytes (10.5 and 16.5%, respectively). In vitro fertilization was successful in most of the experimental groups. However, when denuded oocytes were cultured without any somatic cell support, they did not decondense the penetrated sperm head after in vitro fertilization. This study demonstrates that 1) Vero cells beneficially affect the in vitro maturation of bovine oocytes; 2) cumulus cells in the form of monolayer lose their beneficial influence on in vitro maturation of bovine oocytes; and 3) granulosa cells and FSH and LH alone (without somatic cells) do not show positive effects on in vitro maturation of bovine oocytes.     

Pregnancies established from water buffalo (Bubalus bubalis) blastocysts derived from in vitro matured, in vitro fertilized oocytes and co-cultured with cumulus and oviductal cells

Buffalo ovaries were collected immediately after slaughter and were transported to laboratory in sterile saline at 37 degrees C. Follicular oocytes with the cumulus mass aspirated from 2 to 6 mm in diameter follicles were cultured in TCM-199 medium supplemented with 10% buffalo estrus serum (BES) in 5% CO(2) at 38.5 degrees C. After 20 to 24 h of incubation, the oocytes were inseminated with precapacitated frozen thawed spermatozoa for 6 h. The fertilization rate was 78.15% of the matured oocytes. Over an in vitro culture period of 3 to 9 d, 4.02% of the inseminated oocytes developed to the morula stage when cultured with cumulus cells alone and 17.83% when cumulus cells plus oviductal epithelial cells were used. The percentage of developed blastocysts was very low (0.57%) when the oocytes were co-cultured with cumulus cells from the original oocytes. However, 8% of the inseminated oocytes that were denuded 3 d after insemination developed to the blastocyst stage when they were co-cultured with cumulus and oviductal epithelial cells. Sixteen early/expanded blastocysts were transferred non-surgically to 16 recipients. Four of the 16 recipients became pregnant, of which 2 delivered normal buffalo male calves.     

An in vitro technique to study penetration of hamster oocyte-cumulus complexes by using physiological numbers of sperm

We have developed an inexpensive in vitro system for studying cumulus penetration and fertilization by using physiological numbers of sperm. This system simulates conditions believed to exist in vivo more closely than any in current usage. In this system, 1–100 hamster sperm are used to challenge fresh hamster oocyte-cumulus complexes (OCC). Only fresh (nonoviducal) OCC are used, as they present the most stringent challenge to sperm. Because sperm numbers are low, OCC do not disperse, and sperm can be studied microscopically during penetration of the cumulus oophorus and corona radiata. These conditions permit microscopic assessment of the sperm acrosome. Video tapes of experiments allow easy review and analysis of experiments. Results obtained employing this technique show that, in vitro, (1) capacitated, acrosome-intact hamster sperm can penetrate the extracellular matrix between cumulus cells and bind to the zona pellucida; (2) the “figure-eight motility” characteristic of hyperactivated hamster sperm swimming in culture medium is suppressed when sperm swim in the extracellular matrix between cumulus cells; and (3) fertilization occurs in capillary tubes when low numbers of sperm are used. The in vitro system that we have described will be useful in analyzing the mechanisms used by sperm to penetrate the cumulus and corona radiata and to clarify the role of the acrosomal enzymes in fertilization.     

Apoptosis in cumulus cells, but not in oocytes, may influence bovine embryonic developmental competence

Aim of our study was to clarify if the occurrence of apoptosis in oocytes and cumulus cells is correlated to bovine oocyte developmental competence. The cumulus-oocyte complexes (COCs) were selected according to cumulus status: G1 with more than five layers of compact cumulus cells, G2 with one to five layers of compact cumulus cells and G3 with expanded cumulus cells. The degree of apoptosis in cumulus cells and oocytes measured by caspase staining and TUNEL assay before and after maturation, and 24 h post-insemination was compared to the cleavage, blastocyst formation and hatching rates of each group. Highest cleavage, blastocyst and hatching rates were found in cumulus-oocyte complexes with more than five layers of compact cumulus cells, but no apoptosis was detected in immature or in vitro matured oocytes, regardless of the cumulus status. Many cumulus cells contained active caspases before maturation, but caspase activity declined dramatically after maturation. TUNEL positive cells were rarely observed in each cumulus-oocyte complex upon oocyte recovery, but a huge increase of them was seen after in vitro maturation. Significantly more TUNEL and caspase positive cells were found in G2 cumulus-oocyte complexes. Our results suggest that: (i) oocyte apoptosis does not account for the inferior oocyte quality of G2 and G3; (ii) apoptosis occurs in cumulus cells regardless of the number and compactness of cumulus cells; and (iii) the degree of apoptosis in the compact cumulus-oocyte complexes (G1 and G2) is negatively correlated to the developmental competence of oocyte.     

The collection of oocytes from bovine ovaries

Various needle sizes (17- and 20-g) and aspiration pressures (25, 50, 75 and 100 mmHg) were used to aspirate a total of 5,827 ovarian follicles from bovine ovaries from a slaughterhouse source to assess the impact on the quantity and quality of recovered immature oocytes. The cumulus oocyte complexes (COC's) were graded according to the presence and consistency of cumulus cells surrounding the oocyte and the data analyzed using general linear models. Overall recovery rates and the recovery of oocytes considered viable for IVM/IVF procedures (Classes A, B and C) were both significantly higher using a 17-g needle than a 20-g needle (P < 0.01). As the vacuum pressure increased so did the recovery rate of the total number of oocytes, although the number of viable oocytes reached a maximum at a calculated vacuum pressure of 55 mmHg for the 17-g needle and 77 mmHg for the 20-g needle, with an increased incidence of denuded oocytes at higher vacuum pressures. In a second experiment conducted on 1, 473 follicles, no significant difference was found between 17-g double (flushing) and 17-g single lumen needles in the recovery rate of either the total number or number of viable oocytes when using a vacuum pressure of 50 mmHg.     

Ca2+]i rise at in vitro maturation in bovine cumulus-oocyte complexes

An intracellular calcium ([Ca(2+)]i) rise has been described in cumulus-oocyte complexes (COCs) following luteinizing hormone (LH) exposure. Together with cAMP, Ca(2+) is a candidate signal for resumption of meiosis. Here, we analyzed if the most common hormones involved in oocyte maturation can induce the same Ca(2+) signal. In addition, we characterized the source of this signal. Immature, in vitro-matured, and roscovitine-meiotically arrested COCs were loaded with Fluo-4 AM, stimulated with hormones/growth factors, and tested for [Ca(2+)](i) variations in cumulus cells. Reagents known to inhibit or stimulate [Ca(2+)](i) rises were used to characterize these [Ca(2+)](i) dynamics. Finally, expression of LH receptors (LHRs) in COCs was analyzed by immunofluorescence. In immature COCs, follicle-stimulating hormone (FSH) elicited a single [Ca(2+)](i) rise that was higher than those induced by LH and growth hormone (GH), whereas epithelial growth factor failed to induce any changes in [Ca(2+)](i). The [Ca(2+)](i) rise induced by FSH was higher in immature COCs; was reduced in roscovitine-arrested, immature COCs; and was negligible in gonadotropin-induced, in vitro-matured COCs. In the case of spontaneous- and GH-matured COCs, however, FSH stimulation caused a lower [Ca(2+)](i) rise. The hormone-induced [Ca(2+)](i) rise was due to: (i) external Ca(2+) entry; (ii) intercellular communication; and (iii) intracellular Ca(2+) stores. Immunofluorescence revealed that LHRs were expressed throughout the cumulus cells. The above results show that: (i) gonadotropins and GH cause a [Ca(2+)](i) rise in cumulus cells; (ii) this [Ca(2+)](i) rise results from extra-, inter-, and intra-cellular cumulative Ca(2+) fluxes; and (iii) LHRs are distributed on either outer or inner cumulus cells.