Factors influencing resumption of meiotic maturation and cumulus expansion of porcine oocyte-cumulus cell complexes in vitro

The present study was undertaken to examine effects of various combinations of epidermal growth factor (EGF), transforming growth factor-b?₁ (TGF-b?₁), follicle-stimulating hormone (FSH), luteinizing hormone (LH), androstenedione (A₄), and estradiol-17b? (E₂) on meiotic maturation and cumulus expansion in the pig using an in vitro model system. Oocyte-cumulus cell complexes (OCC) were cultured in the media containing the abovementioned agents for 24 hr and were observed for germinal vesicle breakdown (GVBD), indicative of initiation of meiotic maturation, and for expansion of their cumulus cells. Treatment with EGF significantly increased (P < 0.05) incidence of GVBD, with maximal stimulation occurring at 1 ng/ml (55% vs. 12% in the control). Concentrations of EGF as low as 100 pg/ml significantly stimulated GVBD over control (37% vs. 12%). Addition of EGF (1 ng/ml) and FSH (1.5 μg/ml) together and LH (2 μg/ml) and FSH (1.5 μg/ml) together resulted in significantly higher (P < 0.01) GVBD levels than were observed in response to EGF, FSH, or LH alone. Addition of E₂ (1 μg/ml) had no effect by itself but significantly decreased the incidence of GVBD in the presence of FSH and of LH + FSH. Addition of A₄ (1 μg/ml) significantly reduced the percentage of oocytes undergoing GVBD when added alone or with FSH. Although both EGF and LH stimulated cumulus expansion, FSH was more effective in stimulating cumulus expansion than EGF or LH. TGF-b?₁ had no effect on GVBD or cumulus expansion. These studies indicate that these hormones may have differing roles in oocyte maturation and that their interactions may be part of an intricate system regulating the maturation of oocytes during follicular development in vivo. © 1993 Wiley-Liss, Inc.     

Antagonistic actions of estradiol and tamoxifen upon forskolin-dependent meiotic arrest, intercellular coupling, and the cyclic AMP content of hamster oocyte-cumulus complexes

The effects of estradiol (E2) and the anti-E2 tamoxifen (tam) on forskolin (F)-dependent meiotic arrest in hamster oocytes were investigated. The hypotheses tested were that 1) the arresting action of F is enhanced by E2 and suppressed by tam and 2) the extent of heterologous metabolic coupling and the concomitant transfer of cumulus cell cAMP into the oocyte is increased and decreased by E2 and tam, respectively. E2 was tested with the ID25 F (where ID25 is the dose of F previously shown to arrest the meiosis of 25% cultured oocytes; intact, 3 microM; denuded, 10 microM) and tam was tested with the ID75 F (intact, 10 microM; denuded, 100 microM). E2 induced reversible dose-dependent increases in the percent germinal vesicle (%GV; determined cytogenetically) of both intact and denuded oocytes in the presence of ID25 F (intact: ID50 E2 = 18.0 microM; denuded: ID50 E2 = 17.2 microM) but, in contrast to intact oocytes, E2 also exerted a dose-dependent action on denuded oocytes in the absence of F (ID50 = 26.1 microM). E2 induced dose-dependent increases in the cAMP content (determined by RIA) of intact oocytes (cAMP-oo) and of cumulus masses (cAMP-cm) and in the ratio of cAMPooo:cAMP-cm but failed to elevate F-stimulated cAMP in denuded oocytes. Heterologous metabolic coupling, as assessed by determination of the fraction of radiolabeled uridine marker that was transferred from the cumulus cells to the oocyte, was not significantly enhanced by E2. In contrast to denuded oocytes, tam induced dose-dependent decreases in the %GV and cAMP content of intact oocytes in the presence of ID75 F and significantly depressed heterologous metabolic coupling. While tam failed to antagonize the E2 action on denuded oocytes in the presence of ID25 F, in intact oocytes cultured with E2 and the ID25 F, the anti-E2 significantly decreased the %GV, the cAMP-oo and cAMP-cm, and the extent of heterologous metabolic coupling. These data show that while E2 can directly arrest the maturation of denuded hamster oocytes with no associated elevation of cAMP-oo, E2-enhancement of arrest in intact oocytes is correlated with both elevation of cAMP within the oocyte-cumulus complex and maintenance of heterologous metabolic coupling and is accompanied by an increase in cAMP-oo.     

Metabolic, fluorescent dye and electrical coupling between hamster oocytes and cumulus cells during meiotic maturation in vivo and in vitro

Heterologous intercellular communication was determined qualitatively by lucifer yellow dye transfer and quantitatively by transfer of radiolabeled uridine metabolites and electrical current in hamster oocyte-cumulus complexes during meiotic maturation in vitro and in vivo. In addition, changes in cell resting potentials during maturation were recorded. Significantly less time was required for germinal vesicle breakdown (GVBD) in oocytes matured in vitro than in oocytes stimulated in vivo (1.81 +/- 0.06 hr, N = 13 vs 2.46 +/- 0.07 hr, N = 18, respectively, P less than 0.001). Resting potentials of the oocyte (RP-o) and cumulus cells (RP-c) significantly increased contemporaneously with GVBD in vitro (RP-o: from -18.9 +/- 3.2 mV to -33.2 +/- 2.9 mV, P less than 0.001; RP-c: from -16.3 +/- 1.9 mV to -27.5 +/- 2.6 mV, P less than 0.001) and in vivo after hCG injection (RP-o: from -16.8 +/- 5.9 mV to -30.1 +/- 3.9 mV, P less than 0.001; RP-c: from -15.5 +/- 3.8 mV to -26.3 +/- 3.2 mV, P less than 0.001). RP-o and RP-c progressively increased with time of culture up to 7 hr (maximum time examined) while the values reached maxima in in vivo matured oocytes 4.5 hr post-hCG and subsequently declined concomitant with the onset of cumulus expansion. Cumulus to oocyte coupling decreased progressively with time after release from meiotic arrest both in vitro and in vivo, as assessed by a progressive reduction in transfer of either uridine marker or lucifer yellow from the cumulus cell to the oocyte. By 4.5 hr after hCG injection, cumulus expansion had begun in 100% of complexes examined. Expansion was extensive by 7 hr post-hCG and spread of lucifer yellow from a cumulus cell was limited to very few adjacent cumulus cells. Oocyte to cumulus cell metabolic coupling also decreased progressively with time in both treatment groups. Examination of the extent of heterologous ionic coupling revealed that ionic coupling exhibited biphasic and, bidirectionally parallel, increases during meiotic maturation. While these temporal changes were observed in both groups, the coupling ratios were much greater in those complexes matured in vitro than in vivo. These results show that dye, metabolic, and electrical coupling exist between the immature hamster oocyte and its surrounding cumulus cells but that during the early stages of meiosis, metabolic and dye coupling decrease, while electrical coupling increases biphasically.     

Metabolic coupling and ligand-stimulated meiotic maturation in the mouse oocyte-cumulus cell complex.

Cumulus cells are metabolically coupled to the mammalian oocyte via heterologous gap junctions. One function attributed to the gap junctional communications is the transfer of regulatory signals that direct the meiotic state of the oocyte. However, the precise role of these junctions in meiotic maturation is still unclear. The aim of this study was to test the hypothesis that meiotic resumption is induced by the transfer of a stimulatory signal(s) from the cumulus cells to the oocyte through the gap junctional coupling pathway. We have previously shown that the mitogenic lectin concanavalin A (Con A) induces oocyte maturation in isolated cumulus cell-enclosed oocytes (CEO) when meiotic arrest is maintained with a number of different inhibitory agents [Biol Reprod 1990; 42:413-423]. In the present study, Con A stimulated maturation in dibutyryl cAMP (dbcAMP)-arrested CEO but not in denuded oocytes cocultured with cumulus cells. Heptanol, a known gap junction uncoupler, effectively prevented Con A- and FSH-induced maturation of intact CEO and dramatically reduced metabolic coupling between cumulus cells and the oocyte. However, this alcohol had no effect on denuded oocytes (DO) or on dbcAMP-arrested CEO in the absence of stimulating ligand. Con A and FSH produced only a minimal loss of coupling. When the effects of heptanol were compared with those of the n-alkanols hexanol and decanol, the efficacies of these agents as suppressors of Con A-stimulated oocyte maturation was directly related to their relative abilities to suppress metabolic coupling.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of MEK-mitogen-activated protein kinase pathway in follicle-stimulating hormone-induced but not spontaneous meiotic resumption of mouse oocytes

Mitogen-activated protein (MAP) kinase has been reported to be activated during oocyte meiotic maturation in a variety of mammalian species. However, the mechanism(s) responsible for MAP kinase activation and the consequence of its premature activation during gonadotropin-induced oocyte meiotic resumption have not been examined. The present experiments were conducted to investigate the possible role of MAP kinase in FSH-induced and spontaneous oocyte meiotic resumption in the mouse. MAP kinase kinase (MAPKK, MEK) inhibitor, PD98059 or U0126, produced a dose-dependent inhibitory effect on both FSH-induced oocyte meiotic resumption and MAP kinase activation in the oocytes. However, the same inhibitor did not block spontaneous meiotic resumption of either denuded or cumulus cell-enclosed mouse oocytes, despite the activity of MAP kinase being totally inhibited. Immunoblotting the oocytes and the cumulus cells with the anti-active MAP kinase antibody showed that MAP kinase activity in the oocytes was detected at 8 h of FSH treatment, prior to germinal vesicle breakdown and increased as maturation progressed in the following culture period. In the cumulus cells, MAP kinase was activated even faster, its activity was detected at 1 h of FSH stimulation and increased gradually until 8 h of FSH treatment, then decreased and diminished after 12 h of FSH action. These data demonstrated that the MEK-MAP kinase pathway is implicated in FSH-induced but not spontaneous oocyte meiotic resumption.     

A G(s)-linked receptor maintains meiotic arrest in mouse oocytes, but luteinizing hormone does not cause meiotic resumption by terminating receptor-G(s) signaling

The maintenance of meiotic prophase arrest in fully grown vertebrate oocytes depends on the activity of a G(s) G-protein that activates adenylyl cyclase and elevates cAMP, and in the mouse oocyte, G(s) is activated by a constitutively active orphan receptor, GPR3. To determine whether the action of luteinizing hormone (LH) on the mouse ovarian follicle causes meiotic resumption by inhibiting GPR3-G(s) signaling, we examined the effect of LH on the localization of Galpha(s). G(s) activation in response to stimulation of an exogenously expressed beta(2)-adrenergic receptor causes Galpha(s) to move from the oocyte plasma membrane into the cytoplasm, whereas G(s) inactivation in response to inhibition of the beta(2)-adrenergic receptor causes Galpha(s) to move back to the plasma membrane. However, LH does not cause a change in Galpha(s) localization, indicating that LH does not act by terminating receptor-G(s) signaling.     

Chromatin, microtubules, and kinases activities during meiotic resumption in bitch oocytes

In contrast to the majority of mammals, canine oocytes are ovulated at immature germinal vesicle (GV) stage and complete meiotic maturation to metaphase II during 48-72 hr within the oviducts. This study aims to characterize meiotic maturation process in bitch oocytes, with both morphological and biochemical approaches. The follow-up of chromatin and microtubules during maturation was described, and MPF and MAP kinase activities were quantified at different stages of maturation. Since bitch oocyte cytoplasm is darkly pigmented, the first step was to setup an appropriate staining method for DNA. We thus compared the efficiency of two visualization techniques and demonstrated that propidium iodide coupled to confocal microscopy was a better method than Hoechst/fluorescence microscopy for nuclear stage observation (determination rates: 98.6 vs. 69.5%, respectively; P < 0.01, n = 1622 oocytes). Microtubule organization, evaluated by tubulin immunodetection, revealed subcortical and perinuclear alpha-tubulin and asters in GV oocytes and a clear network of microtubules in GVBD oocytes. In MI and MII oocytes, a symmetrical, barrel-shaped, and radially located spindle was observed. MPF and MAP kinase activities were assayed concomitantly using histone H1 and MBP as substrates. Kinase activities were detected at low levels in oocytes at GV and GVBD stages and were significantly higher at MI and MII stages. In conclusion, despite the particular pattern of meiotic resumption in canine oocytes (ovulated at GV stage), cytoskeleton/chromatin organization and kinase activities follow a similar pattern to those observed in other mammalian species.     

Hydralazine, but not Captopril, Decreases Free Radical Production and Apoptosis in Neurons and Thymocytes

The effects of captopril and hydralazine, two commonly used antihypertensive drugs, on free radical generation and the onset of apoptosis in neuron and thymocyte preparations from 10-12 day old rats have been studied. Apoptosis was induced in neurons by kainate or N-methyl-D-aspartate and in thymocytes by heat shock. Intracellular free radical production was measured by 2',7'-dichlorofluorescein fluorescence, and apoptotic cells were detected by cell staining with fluorescein-labelled annexin V. Captopril was found to have no effect on intracellular free radical generation and also had no significant effect on the early stages of apoptosis in neurons and thymocytes. In contrast, hydralazine was found to decrease free radical generation in both neurons and thymocytes, and it also significantly decreased the numbers of apoptotic cells when neurons and thymocytes were stimulated for apoptosis. Hydralazine had a greater effect on decreasing free radical generation in neurons than in thymocytes, but it had a more pronounced effect on decreasing apoptosis in thymocytes compared to neurons, suggesting that apoptosis, under our experimental conditions, may not solely be triggered by free radical generation. These results contrast with earlier reports that captopril is a free radical scavenger and can decrease apoptosis in T-lymphocytes and cardiomyocytes, and the results obtained with hydralazine are in apparent disagreement with earlier reports that this drug is a free radical generator and can cause intracellular damage suggestive of enhanced free radical formation.     

Effect of forskolin on maintenance of meiotic arrest and stimulation of cumulus expansion, progesterone and cyclic AMP production by pig oocyte-cumulus complexes

Forskolin induced biphasic responses of cumulus progesterone secretion (determined by RIA) and cumulus mass expansion, with maximal increases occurring at 6.25 microns, and subsequent dose-dependent declines observed up to 10 microns-forskolin. The diterpene induced dose-dependent responses in the % germinal vesicle (GV) of cumulus-enclosed and denuded oocytes (0.23 and 4.84 microns maintained 50% GV, respectively), it increased the cAMP content of cumulus masses, cumulus-enclosed oocytes and denuded oocytes, and increased heterologous metabolic coupling (determined by measuring transfer of radiolabelled uridine marker from the cumulus mass to the oocyte). A significant correlation was established between the amount of cAMP within the cumulus mass and that in the corresponding oocyte (r = 0.58). Above 10 microns-forskolin, the cAMP content of cumulus-enclosed oocytes was significantly greater than that of denuded oocytes (100 microns-forskolin: 0.118 +/- 0.082 and 0.006 +/- 0.001 pmol/oocyte respectively; P less than 0.001, paired t test), and the enhanced arresting action of forskolin upon cumulus-enclosed oocytes was correlated with an increase in intra-oocyte cAMP. Maintenance of meiotic arrest and stimulation of oocyte-cumulus cAMP were reversible. During 48 h of culture, the arresting action of forskolin (50 microns) was maintained on denuded and cumulus-enclosed oocytes but heterologous metabolic coupling significantly declined. The cAMP content of the cumulus mass and corresponding oocyte significantly declined, while that of the denuded oocyte remained unchanged. The cAMP content of arrested cumulus-enclosed oocytes cultured for 48 h in 50 microns-forskolin was significantly greater than that of maturing oocytes cultured for 24 h in 50 microns-forskolin and then for 24 h in control medium. These results show that (1) forskolin stimulates progesterone secretion and expansion of pig cumuli, but at high doses the drug inhibits these functions while cumulus cAMP remains elevated; (2) when heterologous metabolic coupling is maintained, cumulus cAMP may be transferred to the oocyte; (3) the pig oocyte can synthesize cAMP; and (4) forskolin-maintenance of meiotic arrest of pig oocytes is correlated with elevated intra-oocyte cAMP but a 'factor' other than cAMP is also involved in maintenance of meiotic arrest.     

Inositol 1,4,5-triphosphate microinjection triggers activation, but not meiotic maturation in amphibian and starfish oocytes

Inositol 3,4,5-triphosphate (InsP3) brought about cortical granule exocytosis and elevation of a fertilization membrane, due to a rapid increase of free calcium in cytoplasm, when injected into oocytes of the amphibian Xenopus laevis arrested at second meiotic metaphase. The same result was observed when injection was performed into oocytes of the starfish Marthasterias glacialis arrested either at the first meiotic prophase or after completion of meiosis. Although meiotic maturation was induced in both animals by specific hormones which have been previously shown to release Ca2+ within cytoplasm, InsP3 microinjection into prophase-arrested oocytes did not release them from prophase block.     

Heterologous cell contacts and metabolic coupling in bovine cumulus oocyte complexes

The integrity of the cumulus cell processes were studied in four categories of bovine cumulus oocyte complexes (COCs) selected on their morphological characteristics. Three different types of cumulus cell process endings (CCPEs) were identified, one penetrating the cortex, another not penetrating the cortex, and a third form was intermediate and more rare in appearance. The process endings that penetrated the cortex frequently made gap junctions with the oolemma. The division of the three types of CCPEs over the four different COC categories was specific for three of the four categories. The first-category COC predominantly possessed the penetrating CCPE, the fourth-category COC possessed predominantly the nonpenetrating CCPE, and the second and third categories had both types of CCPEs. The metabolic coupling of the cumulus-oocyte contacts was assessed by means of incorporation of 3H-choline into the oocyte. The majority of category 4 COCs transferred low levels of choline into the oocyte while the majority of the oocytes of the other three categories transferred high levels of choline into the oocyte. Category 4 includes a smaller proportion of oocytes capable of cleaving after fertilization than the other three categories. This reduced developmental capacity is probably due to the loss of metabolic coupling before the onset of culture.     

Metabolic coupling, cumulus expansion and meiotic resumption in mouse cumuli oophori cultured in vitro in the presence of FSH or dcAMP, or stimulated in vivo by hCG

The temporal changes of metabolic coupling between the mouse oocyte and the cumulus cells which follow hCG injection in vivo and FSH treatment in vitro were studied by measuring what fraction of [3H]uridine taken up by cumulus cells was transferred to the oocyte. Meiotic resumption and a partial coupling loss (to 35% of the initial value) spontaneously occurred in cumuli cultured in control medium. The addition of 1 microgram FSH/ml in vitro, or the injection of hCG in vivo caused a delay of about 3 h in both phenomena and a near total uncoupling, together with cumulus expansion. FSH caused uncoupling even if cumulus expansion was prevented by the addition of heparin. The presence of 2 mM-dcAMP prevented meiotic resumption in cumulus-enclosed oocytes and maintained a high level of co-operation for at least 6 h. The slow uncoupling observed at later times was due to cumulus expansion, because it was totally prevented by heparin. We suggest that metabolic co-operation with the cumulus oophorus and meiotic resumption are both regulated by FSH through variations of intracellular levels of cAMP.     

Association between muscular strength and metabolic risk in Japanese women, but not in men

We examined whether cardiorespiratory fitness (maximal oxygen uptake, VO(2)max) and muscular strength (grip strength) are associated with individual and clustered metabolic risk factors independently of abdominal adiposity in Japanese men (n=110) and women (n=110) aged 20-69 years. Blood pressure, triglycerides (TG), HDL cholesterol, and fasting plasma glucose (FPG) were assessed and metabolic risk score was calculated, which is the sum of the z scores for each individual risk factor. Waist circumference was measured and the area of visceral fat was assessed by MRI. Multiple linear regression analysis revealed that VO(2)max was inversely associated with TG in men (p<0.05) and grip strength was negatively associated with FPG and metabolic risk score in women (p<0.001 and p<0.05, respectively), independently of waist circumference. Adjusting for visceral fat instead of waist circumference, similar results were obtained in women (p<0.01 and p<0.05, respectively), but the association between VO(2)max and TG in men was attenuated to nonsignificant. This cross-sectional study demonstrates that muscular strength is inversely associated with plasma glucose levels and clustered metabolic risk factors independently of abdominal adiposity in Japanese women, but not in men.     

Proteolytic activity of the 26S proteasome is required for the meiotic resumption, germinal vesicle breakdown, and cumulus expansion of porcine cumulus-oocyte complexes matured in vitro

The resumption of oocyte meiosis in mammals encompasses the landmark event of oocyte germinal vesicle (GV) breakdown (GVBD), accompanied by the modification of cell-to-cell communication and adhesion between the oocyte and surrounding cumulus cells. The concomitant cumulus expansion relies on microfilament-cytoskeletal remodeling and extracellular matrix (ECM) deposition. We hypothesized that this multifaceted remodeling event requires substrate-specific proteolysis by the ubiquitin-proteasome pathway (UPP). We evaluated meiotic progression, cytoskeletal dynamics, and the production of cumulus ECM in porcine cumulus-oocyte complexes (COCs) cultured with or without 10-200 microM MG132, a specific proteasomal inhibitor, for the first 22 h of in vitro maturation, followed by 22 h of culture with or without MG132. Treatment with 10 microM MG132 arrested 28.4% of oocytes in GV stage (vs. 1.3% in control), 43.1% in prometaphase I, and 16.2% in metaphase I, whereas 83.7% of control ova reached metaphase II (0% of MG132 reached metaphase II). The proportion of GV-stage ova increased progressively to >90% with increased concentration of MG132 (20-200 microM). Furthermore, MG132 blocked the extrusion of the first polar body and degradation of F-actin-rich transzonal projections (TZP) interconnecting cumulus cells with the oocyte. The microfilament disruptor cytochalasin E (CE) prevented cumulus expansion but accelerated the breakdown of TZPs. Ova treated with a combination of 10 microM MG132 and 10 microM CE underwent GVBD, despite the inhibition of proteasomal activity. However, 90.0% of cumulus-free ova treated with 10 microM MG132 remained in GV stage, compared with 16.7% GV ova in control. Cumulus expansion, retention of hyaluronic acid, and the deposition of cumulus ECM relying on the covalent transfer of heavy chains of inter-alpha trypsin inhibitor (IalphaI) were also inhibited by MG132. Cumulus expansion in control COCs was accompanied by the degradation of ubiquitin-C-terminal hydrolase L3, an important regulator of UPP. RAC1, a UPP-controlled regulator of actin polymerization was maintained at steady levels throughout cumulus expansion. We conclude that proteasomal proteolysis has multiple functions in the progression of oocyte meiosis beyond GV and metaphase I stage, polar body extrusion, and cumulus expansion.     

FANCM-associated proteins MHF1 and MHF2, but not the other Fanconi anemia factors,limit meiotic crossovers

Genetic recombination is important for generating diversity and to ensure faithful segregation of chromosomes at meiosis. However, few crossovers (COs) are formed per meiosis despite an excess of DNA double-strand break precursors. This reflects the existence of active mechanisms that limit CO formation. We previously showed that AtFANCM is a meiotic anti-CO factor. The same genetic screen now identified AtMHF2 as another player of the same anti-CO pathway. FANCM and MHF2 are both Fanconi Anemia (FA) associated proteins, prompting us to test the other FA genes conserved in Arabidopsis for a role in CO control at meiosis. This revealed that among the FA proteins tested, only FANCM and its two DNA-binding co-factors MHF1 and MHF2 limit CO formation at meiosis.     

mus309 mutation, defective in DNA double-strand break repair, affects intergenic but not intragenic meiotic recombination in Drosophila melanogaster

The effect was investigated of the hypomorphic DNA double-strand break repair, notably synthesis-dependent strand annealing, deficient mutation mus309 on the third chromosome of Drosophila melanogaster on intergenic and intragenic meiotic recombination in the X chromosome. The results showed that the mutation significantly increases the frequency of intergenic crossing over in two of three gene intervals of the X chromosome studied. Interestingly the increase was most prevalent in the tip of the X chromosome where crossovers normally are least frequent per physical map unit length. In particular crossing over interference was also affected, indicating that the effect of the mus309 mutation involves preconditions of crossing over but not the event of crossing over itself. On the other hand, the results also show that most probably the mutation does not have any effect on intragenic recombination, i.e. gene conversion. These results are fully consistent with the present molecular models of meiotic crossing over initiated by double-strand breaks of DNA followed by formation of a single-end-invasion intermediate, or D-loop, which is subsequently processed to generate either crossover or non-crossover products involving formation of a double Holliday junction. In particular the results suggest that the mus309 gene is involved in resolution of the D-loop, thereby affecting the choice between double-strand-break repair (DSBR) and synthesis-dependent strand annealing (SDSA) pathways of meiotic recombination.     

Heterogeneous function of ryanodine receptors, but not IP3 receptors, in hamster cremaster muscle feed arteries and arterioles

The roles played by ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP?Rs) in vascular smooth muscle in the microcirculation remain unclear. Therefore, the function of both RyRs and IP?Rs in Ca(2+) signals and myogenic tone in hamster cremaster muscle feed arteries and downstream arterioles were assessed using confocal imaging and pressure myography. Feed artery vascular smooth muscle displayed Ca(2+) sparks and Ca(2+) waves, which were inhibited by the RyR antagonists ryanodine (10 μM) or tetracaine (100 μM). Despite the inhibition of sparks and waves, ryanodine or tetracaine increased global intracellular Ca(2+) and constricted the arteries. The blockade of IP?Rs with xestospongin D (5 μM) or 2-aminoethoxydiphenyl borate (100 μM) or the inhibition of phospholipase C using U-73122 (10 μM) also attenuated Ca(2+) waves without affecting Ca(2+) sparks. Importantly, the IP?Rs and phospholipase C antagonists decreased global intracellular Ca(2+) and dilated the arteries. In contrast, cremaster arterioles displayed only Ca(2+) waves: Ca(2+) sparks were not observed, and neither ryanodine (10-50 μM) nor tetracaine (100 μM) affected either Ca(2+) signals or arteriolar tone despite the presence of functional RyRs as assessed by responses to the RyR agonist caffeine (10 mM). As in feed arteries, arteriolar Ca(2+) waves were attenuated by xestospongin D (5 μM), 2-aminoethoxydiphenyl borate (100 μM), and U-73122 (10 μM), accompanied by decreased global intracellular Ca(2+) and vasodilation. These findings highlight the contrasting roles played by RyRs and IP?Rs in Ca(2+) signals and myogenic tone in feed arteries and demonstrate important differences in the function of RyRs between feed arteries and downstream arterioles.