Effect of temporary nuclear arrest by phosphodiesterase 3-inhibitor on morphological and functional aspects of in vitro matured mouse oocytes

The present study aimed to analyze detailed morphological and functional characteristics of mouse in vitro matured oocytes after a pre-maturation culture (PMC) by temporary nuclear arrest with the specific phosphodiesterase 3-inhibitor (PDE3-I) Cilostamide. In a first experiment the lowest effective dose of Cilostamide was determined. Cumulus-oocyte complexes (COCs), isolated from small antral follicles, were exposed to different concentrations of Cilostamide (ranging from 0 (control) to 10 microM) for 24 hr. Afterwards, oocytes were removed from PDE3-I-containing medium and underwent in vitro maturation (IVM) for 16-18 hr. A concentration of 1 microM Cilostamide was the lowest effective dose for maximum level of inhibition and reversibility of meiosis inhibition. This concentration was used in further experiments to evaluate oocyte quality following IVM in relation to different parameters: kinetics of meiotic progression, metaphase II (MII) spindle morphology, aneuploidy rate, fertilization, and embryonic developmental rates. The results were compared to nonarrested (in vitro control) and in vivo matured oocytes (in vivo control). Following withdrawal of the inhibitor, the progression of meiosis was more synchronous and accelerated in arrested when compared to nonarrested oocytes. A PMC resulted in a significant increase in the number of oocytes constituting a MII spindle of normal morphology. None of the oocytes exposed to PDE3-I showed numerical chromosome alterations. In addition, fertilization and embryonic developmental rates were higher in the PMC group compared to in vitro controls, but lower than in vivo controls. These results provide evidence that induced nuclear arrest by PDE3-I is a safe and reliable method to improve oocyte quality after IVM.     

雷公藤多甙对小鼠卵母细胞成熟和体外受精的影响

采用超排卵技术研究雷公藤多甙（GTW）对小鼠卵母细胞的成熟和体外受精以及脏器等的影响,GTW对小鼠卵母细胞生发泡破裂没有影响,但可以抑制卵母细胞第一极体的释放,影响卵母细胞的存活率并可降低体外受精率和超排卵的卵母细胞数量。GTW可以破坏卵母细胞成熟,降低卵母细胞的体外受精能力,影响小鼠的正常生殖功能。     

甲醛对雌性小鼠生殖功能及脏器的影响

采用超排卵技术研究甲醛对小鼠卵母细胞的成熟和体外受精以及脏器等的影响.甲醛对小鼠卵母细胞生发泡破裂没有影响,但可以抑制卵母细胞第一极体的释放,影响卵母细胞的存活率和破坏卵母细胞成熟,并可降低体外受精率和超排卵的卵母细胞数量,影响小鼠的正常生殖功能.而且,高剂量的甲醛还对小鼠的肝脏以及卵巢有明显的毒副作用.     

Cows are not mice: the role of cyclic AMP, phosphodiesterases, and adenosine monophosphate-activated protein kinase in the maintenance of meiotic arrest in bovine oocytes

Meiotic maturation in mammalian oocytes is initiated during fetal development, and is then arrested at the dictyate stage - possibly for several years. Oocyte meiosis resumes in preovulatory follicles in response to the lutenizing hormone (LH) surge or spontaneously when competent oocytes are removed from follicles and cultured. The mechanisms involved in meiotic arrest and resumption in bovine oocytes are not fully understood, and several studies point to important differences between oocytes from rodent and livestock species. This paper reviews earlier and contemporary studies on the effects of cAMP-elevating agents and phosphodiesterase (PDE) enzyme inhibitors on the maintenance of meiotic arrest in bovine oocytes in vitro. Contrary to results obtained with mouse oocytes, bovine oocyte meiosis is inhibited by activators of the energy sensor adenosine monophosphate-activated protein kinase (AMPK, mammalian gene PRKA), which is activated by AMP, the degradation product of cAMP. It is not clear whether or not the effects were due to AMPK activation, and they may depend on culture conditions. Evidence suggests that other signaling pathways (for example, the cGMP/nitric oxide pathway) are involved in bovine oocyte meiotic arrest, but further studies are needed to understand the interactions between the signaling pathways that lead to maturation promoting factor (MPF) being inactive or active. An improved understanding of the mechanisms involved in the control of bovine oocyte meiosis will facilitate better control of the process in vitro, resulting in increased developmental competence and increased efficiency of in vitro embryo production procedures.     

Development of mouse and rat oocytes in chimeric reaggregated ovaries after interspecific exchange of somatic and germ cell components

The germ cell and somatic cell compartments of newborn rat and mouse ovaries, which contain only primordial stage follicles, were completely exchanged and reaggregated to produce xenogeneic chimeric ovaries. The reaggregated ovaries were grafted beneath the renal capsules of ovariectomized SCID mice to develop for periods up to 21 days. Xenogeneic follicles developed with essentially normal morphological characteristics. Both rat and mouse oocytes with species-specific characteristics grew within follicles that were composed of somatic cells exclusively of the alternative species. Rat oocytes grown in mouse follicles became competent to resume meiosis, and progressed to metaphase II when they were removed from follicles and cultured. In addition, mouse oocytes grown in rat follicles underwent fertilization and preimplantation development in vitro, and developed to term after embryos were transferred to pseudopregnant mouse foster mothers. Therefore, despite an estimated 11 million years of divergent evolution, oocytes and somatic cells of rat and mouse ovaries can be exchanged and can produce functional oocytes. It is concluded that factors involved in oocyte-somatic cell interactions necessary to support oocyte development and appropriate differentiation of the oocyte-associated granulosa cells are conserved between rats and mice. Moreover, although granulosa cells play important roles in oocyte development, the development of species-specific characteristics of oocytes occurs without apparent modification by a xenogeneic follicular environment.     

Improvement in in?vitro fertilization outcome following in?vivo synchronization of oocyte maturation in mice

Synchronization of oocyte maturation in vitro has been shown to produce higher in vitro fertilization (IVF) rates than those observed in oocytes matured in vitro without synchronization. However, the increased IVF rates never exceeded those observed in oocytes matured in vivo without synchronization. This study was therefore designed to define the effect of in vivo synchronization of oocyte maturation on IVF rates. Mice were superovulated and orally treated with 7.5 mg cilostazol (CLZ), a phosphodiesterase 3A (PDE3A) inhibitor, to induce ovulation of immature oocytes at different stages depending on frequency and time of administration of CLZ. Mice treated with CLZ ovulated germinal vesicle (GV) or metaphase I (MI) oocytes that underwent maturation in vitro or in vivo (i.e. in the oviduct) followed by IVF. Superovulated control mice ovulated mature oocytes that underwent IVF directly upon collection. Ovulated MI oocytes matured in vitro or in vivo had similar maturation rates but significantly higher IVF rates, 2–4 cell embryos, than those observed in control oocytes. Ovulated GV oocytes matured in vitro showed similar maturation rates but significantly higher IVF rates than those observed in control oocytes. However, ovulated GV oocytes matured in vivo had significantly lower IVF rates than those noted in control oocytes. It is concluded that CLZ is able to synchronize oocyte maturation and improve IVF rates in superovulated mice. CLZ may be capable of showing similar effects in humans, especially since temporal arrest of human oocyte maturation with other PDE3A inhibitors in vitro was found to improve oocyte competence level. The capability of a clinically approved PDE3A inhibitor to improve oocyte fertilization rates in mice at doses extrapolated from human therapeutic doses suggests the potential scenario of the inclusion of CLZ in superovulation programs. This may improve IVF outcomes in infertile patients.     

Live offspring produced by mouse oocytes derived from premeiotic fetal germ cells

Mature mouse oocytes currently can be generated in vitro from the primary oocytes of primordial follicles but not from premeiotic fetal germ cells. In this study we established a simple, efficient method that can be used to obtain mature oocytes from the premeiotic germ cells of a fetal mouse 12.5 days postcoitum (dpc). Mouse 12.5-dpc fetal ovaries were transplanted under the kidney capsule of recipient mice to initiate oocyte growth from the premeiotic germ cell stage, and they were recovered after 14 days. Subsequently, the primary and early secondary follicles generated in the ovarian grafts were isolated and cultured for 16 days in vitro. The mature oocytes ovulated from these follicles were able to fertilize in vitro to produce live offspring. We further show that the in vitro fertilization offspring were normal and able to successfully mate with both females and males, and the patterns of the methylated sites of the in vitro mature oocytes were similar to those of normal mice. This is the first report describing premeiotic fetal germ cells able to enter a second meiosis and support embryonic development to term by a combination of in vivo transplantation and in vitro culture. In addition, we have shown that the whole process of oogenesis, from premeiotic germ cells to germinal vesicle (GV)-stage oocytes, can be carried out under the kidney capsule.     

Role of phosphodiesterase type 3A in rat oocyte maturation.

It is generally accepted that cyclic nucleotides are key signaling molecules in the control of oocyte meiotic resumption. Given the role of phosphodiesterases (PDEs) in cyclic nucleotide degradation, this study was undertaken to investigate the properties and regulation of PDEs expressed in rat oocytes. Cilostamide-sensitive PDE3 was the major activity detected in denuded oocytes, whereas no PDE3 activity could be detected in cumulus cells. Moreover, comparable levels of PDE3 activity were measured in cumulus-oocyte complexes (COCs) and in denuded oocytes. The oocyte PDE was recovered in the soluble fraction of the homogenate and immunoprecipitated with a specific PDE3A antibody. A significant and transient increase (P < 0.05) in PDE3 activity was measured in the oocytes after 30 min of culture (70 min after isolation) compared with immediately after collection (10 min after isolation). Conversely, no changes in activity were observed when denuded oocytes or cumulus cells were incubated for up to 130 min. Evaluation of oocyte maturation indicated that only 10% of oocytes had resumed meiosis at the peak of the PDE3 activity. A significant increase (P < 0.05) in PDE3 activity was measured in COCs when follicle-enclosed oocytes were cultured in the presence of hCG. Again, this increase preceded oocyte maturation. In conclusion, these data demonstrate that PDE3A is the major PDE form expressed in mammalian oocytes. PDE3A activity increases prior to resumption of meiosis in both spontaneous and gonadotropin-stimulated maturation. These findings strongly support the hypothesis that an increase in oocyte PDE3A activity is one of the intraoocyte mechanisms controlling resumption of meiosis in rat oocytes, at least in vitro.     

Chronic low-dose antiprogestin impairs preimplantation embryogenesis, but not oocyte nuclear maturation or fertilization in rhesus monkeys

Continual administration of low doses of the antiprogestin ZK137316 permits ovarian/menstrual cyclicity, but prevents pregnancy in female rhesus monkeys. The sites of contraceptive action remain unknown. This study determined whether chronic, low-dose antiprogestin exposure during follicular development impairs oocyte maturation in vivo, as well as fertilization and preimplantation embryogenesis in vitro. Adult, female rhesus monkeys exhibiting normal menstrual cycles received vehicle (n=9) or 0.03 mg ZK137316 (n=8)/kg body weight i.m. daily for 3 months. Controlled ovarian stimulation with recombinant gonadotropins was initiated in the 3rd month. Oocytes collected from preovulatory follicles were evaluated for nuclear maturity and inseminated in vitro. Preimplantation embryonic development was monitored in vitro. The total number of oocytes and percentage collected at each nuclear stage were similar in both groups. More (P<0.05) atretic oocytes were recovered following antiprogestin relative to vehicle treatment. Fertilization rates and percentages of embryos that progressed to the morula stage were similar between groups, but antiprogestin-treated females exhibited less (P<0.05) normal cleavage. Embryonic development was accelerated by 1 day (P<0.05) from the 16-cell to the morula stage in the antiprogestin group relative to vehicle. Despite this, the majority of embryos became blastocysts within 6 days in vitro in the antiprogestin group, but fewer expanded (P=0.09) and hatched (P<0.05) compared to vehicle. During in vivo treatment with chronic, low-dose antiprogestin, oocytes retained their ability to resume and complete meiosis as well as fertilize following insemination in vitro. However, preimplantation embryogenesis in vitro was impaired, particularly during the later stages of blastocyst development. Thus, antiprogestin exposure during follicular development altered oocyte functions that are critical for normal preimplantation embryogenesis; this may contribute to pregnancy prevention.     

Human oocytes reversibly arrested in prophase I by phosphodiesterase type 3 inhibitor in vitro

This study addresses the role of cAMP hydrolytic isoenzyme phosphodiesterase type 3 (PDE 3) modulation on human oocyte maturation in vitro. Presence of phosphodiesterase type 3 A (PDE 3A) mRNA was confirmed in human germinal vesicle-stage (GV) oocytes. Making use of a selective PDE 3 inhibitor, Org 9935 (10 microM), oocytes retrieved from immature follicles were arrested in prophase I with a high efficiency for up to 72 h. Cumulus oocyte complexes (COCs) were retrieved in the follicular phase of the cycle before or after exposure to endogenous LH or hCG administration in vivo and randomly distributed into maturation medium with or without the PDE 3 inhibitor. Previous exposure of small follicles to LH activity in vivo had no influence on the arresting capacity of the PDE 3 inhibitor. Reversal from pharmacological arrest leads to a progression through meiosis in a normal time frame with formation of a well-aligned metaphase plate. Ultrastructure analysis of COC derived from follicles between 8 and 12 mm showed that the induced extension of prophase I arrest in vitro resulted in cytoplasm changes but not in apparent nuclear changes during culture.     

Psychological Stress on Female Mice Diminishes the Developmental Potential of Oocytes: A Study Using the Predatory Stress Model

Although the predatory stress experimental protocol is considered more psychological than the restraint protocol, it has rarely been used to study the effect of psychological stress on reproduction. Few studies exist on the direct effect of psychological stress to a female on developmental competence of her oocytes, and the direct effect of predatory maternal stress on oocytes has not been reported. In this study, a predatory stress system was first established for mice with cats as predators. Beginning 24 h after injection of equine chorionic gonadotropin, female mice were subjected to predatory stress for 24 h. Evaluation of mouse responses showed that the predatory stress system that we established increased anxiety-like behaviors and plasma cortisol concentrations significantly and continuously while not affecting food and water intake of the mice. In vitro experiments showed that whereas oocyte maturation and Sr²⁺ activation or fertilization were unaffected by maternal predatory stress, rate of blastocyst formation and number of cells per blastocyst decreased significantly in stressed mice compared to non-stressed controls. In vivo embryo development indicated that both the number of blastocysts recovered per donor mouse and the average number of young per recipient after embryo transfer of blastocysts with similar cell counts were significantly lower in stressed than in unstressed donor mice. It is concluded that the predatory stress system we established was both effective and durative to induce mouse stress responses. Furthermore, predatory stress applied during the oocyte pre-maturation stage significantly impaired oocyte developmental potential while exerting no measurable impact on nuclear maturation, suggesting that cytoplasmic maturation of mouse oocytes was more vulnerable to maternal stress than nuclear maturation.     

Cumulus cells accelerate aging of mouse oocytes

The role of cumulus cells (CCs) that surround oocytes in maturation, ovulation, and fertilization has been extensively studied, yet little is known about their role in oocyte aging. Although early studies have shown that when ovulated oocytes are aged in vitro displayed similar morphological alterations as those aged in vivo, a recent study found that vitro culture of mouse oocytes retarded oocyte aging. The objective of this study was to test the hypothesis that CCs would accelerate oocyte aging. During in vitro aging with CCs of both in vivo-matured and in vitro-matured mouse oocytes, activation rates increased, whereas the maturation-promoting factor (MPF) activity decreased significantly as during in vivo aging of the ovulated oocytes. During aging after denudation of CCs, however, activation rates of both in vivo-matured and in vitro-matured oocytes remained low and the MPF activity decreased much more slowly compared to that of oocytes aged with CCs. Although many oocytes aged in vivo and in vitro with CCs showed a partial cortical granule (CG) release, very few cumulus-free oocytes released their CGs during in vitro aging. When denuded oocytes were cultured with cumulus-oocyte-complexes at a 1:2 ratio or on a CC monolayer, activation rates increased, while MPF activity decreased significantly. The results strongly suggested that CCs accelerated the aging progression of both in vivo-matured and in vitro-matured mouse oocytes.     

Reduced expression and function of aquaporin-3 in mouse metaphase-II oocytes induced by controlled ovarian hyperstimulation were associated with subsequent low fertilization rate.

BACKGROUND/AIMS: Aquaporin-3 (AQP3), one isoform of water channel family, has been found to be expressed in mouse oocytes. The present study aimed to investigate whether functional AQP3 was expressed in oocytes induced by controlled ovarian hyperstimulation (COH), and whether altered oocyte AQP3 expression was associated with changes in fertilization rate. METHODS: Sixty ICR female mice were divided into two groups: COH and control. AQP3 mRNA expression of mouse metaphase II (MII) oocytes was quantified by real-time RT-PCR. The water permeability of oocytes was assessed with cell swelling test. The fertilization profiles of oocytes were generated via in vitro fertilization. RESULTS: AQP3 mRNA was expressed in both natural and COH-induced mouse oocytes. COH significantly reduced AQP3 mRNA expression. The volume of oocytes was significantly increased after exposure to hypotonic medium and pretreatment with HgCl(2) attenuated hypotonic medium-induced increase in oocyte volume and water permeability coefficient (Pf). Furthermore, the expression of AQP3, Pf and the fertilization rate were significantly lower in COH oocytes than those in control. CONCLUSION: AQP3 might play an important role in controlling oocyte quality and a low in vitro fertilization rate of COH mice might, in part, result from reduced AQP3 expression and water permeability in mouse oocytes.     

Synergistic roles of BMP15 and GDF9 in the development and function of the oocyte-cumulus cell complex in mice: genetic evidence for an oocyte-granulosa cell regulatory loop

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-specific growth factors that appear to play key roles in granulosa cell development and fertility in most mammalian species. We have evaluated the role(s) of these paracrine factors in the development and function of both the cumulus cells and oocytes by assessing cumulus expansion, oocyte maturation, fertilization, and preimplantation embryogenesis in Gdf9+/-Bmp15-/- [hereafter, double mutant (DM)] mice. We found that cumulus expansion, as well as the expression of hyaluronon synthase 2 (Has2) mRNA was impaired in DM oocyte-cumulus cell complexes. This aberrant cumulus expansion was not remedied by coculture with normal wild-type (WT) oocytes, indicating that the development and/or differentiation of cumulus cells in the DM, up to the stage of the preovulatory luteinizing hormone (LH) surge, is impaired. In addition, DM oocytes failed to enable FSH to induce cumulus expansion in WT oocytectomized (OOX) cumulus. Moreover, LH-induced oocyte meiotic resumption was significantly delayed in vivo, and this delayed resumption of meiosis was correlated with the reduced activation of mitogen-activated protein kinase (MAPK) in the cumulus cells, thus suggesting that GDF9 and BMP15 also regulate the function of cumulus cells after the preovulatory LH surge. Although spontaneous in vitro oocyte maturation occurred normally, oocyte fertilization and preimplantation embryogenesis were significantly altered in the DM, suggesting that the full complement of both GDF9 and BMP15 are essential for the development and function of oocytes. Because receptors for GDF9 and BMP15 have not yet been identified in mouse oocytes, the effects of the mutations in the Bmp15 and Gdf9 genes on oocyte development and functions must be produced indirectly by first affecting the granulosa cells and then the oocyte. Therefore, this study provides further evidence for the existence and functioning of an oocyte-granulosa cell regulatory loop.     

The effect of sera, bovine serum albumin and follicular cells on in vitro maturation and fertilization of porcine oocytes

The effects of fetal calf serum (FCS), estrus gilt serum (EGS) BSA, dispersed granulosa cells, hemi-sections of follicular wall, and replacement of medium after 24 hours on in vitro maturation and fertilization of porcine oocytes were studied. The results indicate that the use of BSA for 24 or 48 hours inhibited the expansion of cumulus cells and the maturation of oocytes. An incubation of 24 hours culture in FCS followed by a second 24 hours in BSA containing medium did not decrease the rate of maturation but significantly decreased the polyspermy and mean number of spermatozoa penetrated/oocyte. Renewing the medium with or without removal of cumulus cells during the second incubation increased the maturation rate. Removal of cumulus cells decreased the penetrability, the polyspermy rates of the oocyte and the mean number of spermatozoa/oocyte penetrated. The EGS-supplemented medium, dispersed granulosa cells or hemi-sections of follicular wall did not affect the maturation or fertilization rates. In conclusion, BSA, a protein supplement in maturation medium, inhibited cumulus cell expansion and maturation of porcine oocytes. After resumption of meiosis triggered by FCS, BSA did not influence maturation. The FCS-BSA treatment reduced the incidence of polyspermy and the mean number of spermatozoa penetrated/oocyte without decreasing the rate of maturation and fertilization.     

Localization of microfilaments during oocyte maturation of golden hamster

The localization and changes in microfilaments (MF) during golden hamster oocyte maturation were examined by an immunofluorescein method and confocal laser scanning microscopy (CLSM). We also studied the relationship between the changes in MF and oocyte nuclear and cytoplasmic maturation. During in vivo maturation, generalized submembranous MF were found initially which gradually became more prominent at the site of the first polar body extrusion. However, 43.7% of the in vitro matured metaphase 2 stage oocytes lacked the submembranous MF structure. This fact may partly account for the low fertilization rate of in vitro matured oocytes. MF were not found in the folicular oocytes cultured in cytochalasin-D-containing medium, and metaphase-like chromosomes were located at the center of the oocyte and first polar body extrusion did not occur. Twenty-five percent of the oocytes, which were arrested at meiosis by hypoxanthine, synthesized submembranous MF structure although the nuclear stage of these oocytes was germinal vesicle. These facts suggest that MF plays a role in nuclear behavior but there are some differences in the changes taking place within the nucleus and MF. MF may play a role in oocyte cytoplasmic maturation although the details of this have yet to be established. © 1995 Wiley-Liss, Inc.     

Regulation of mouse oocyte meiotic maturation: implication of a decrease in oocyte cAMP and protein dephosphorylation in commitment to resume meiosis

Mouse oocytes are reversibly inhibited from resuming meiotic maturation in vitro by cAMP phosphodiesterase inhibitors such as 3-isobutyl-1-methyl xanthine (IBMX) and cAMP analogs such as dibutyryl cAMP (dbcAMP). Oocytes cultured in IBMX-containing medium were transferred to and cultured in IBMX-free medium for various periods of time prior to their return to either IBMX- or dbcAMP-containing medium. Results from these experiments defined a period of time in which oocytes became committed to resuming meiosis. Forskolin, which elevated the intracellular oocyte cAMP concentration, transiently inhibited oocytes from resuming meiosis. Levels of cAMP were determined in oocytes incubated in medium that allows resumption of meiosis. The level of oocyte cAMP decreased significantly during the time in which oocytes become committed to resuming meiosis. This decrease in oocyte cAMP was not observed in oocytes inhibited from resuming meiosis by IBMX. In addition, cAMP levels were determined in preovulatory antral follicles, cumulus cell-oocyte complexes, and oocytes during gonadotropin-induced resumption of meiosis in vivo. A decrease in oocyte cAMP preceded resumption of meiosis as manifested by germinal vesicle breakdown (GVBD). This decrease apparently occurred before or during a period of time in which follicle and cumulus cell cAMP were increasing. Associated with commitment to resume meiosis was a characteristic set of changes in oocyte phosphoprotein metabolism that preceded GVBD. These changes are, to date, some of the first reported biochemical changes that precede GVBD. Results from these experiments are discussed in terms of a possible role cAMP may play in regulation of resumption of meiosis in mammals.     

Ion currents involved in oocyte maturation, fertilization and early developmental stages of the ascidian Ciona intestinalis

Electrophysiological techniques were used to study the role of ion currents in the ascidian Ciona intestinalis oocyte plasma membrane during different stages of growth, meiosis, fertilization and early development. Three stages of immature oocytes were discriminated in the ovary, with the germinal vesicle showing specific different features of growth and maturation. Stage-A (pre-vitellogenic) oocytes exhibited the highest L-type calcium current activity and were incompetent for meiosis resumption. Stage-B (vitellogenic) oocytes showed a progressive disappearance of calcium currents and the first appearance of sodium currents that remained high during the maturation process, up to the post-vitellogenic stage-C oocytes. The latter had acquired meiotic competence, undergoing spontaneous in vitro maturation and interacting with the spermatozoon. However, fertilized oocytes did not produce normal larvae, suggesting that cytoplasmic maturation may affect embryo development. In mature oocytes at the metaphase I stage, sodium currents were present and remained high up to the zygote stage. Oocytes fertilized in the absence of sodium showed significant reduction of the fertilization current amplitude and high development of anomalous "rosette" embryos. Current amplitudes became negligible in embryos at the 2- and 4-cell stage, whereas resumption of all the current activities occurred at the 8-cell embryo. Taken together, these results suggest: (i) an involvement of L-type calcium currents in initial oocyte meiotic progression and growth; (ii) a role of sodium currents at fertilization; (iii) a role of the fertilization current in ensuring normal embryo development.     

Bovine cumulus cell-oocyte gap junctional communication during in vitro maturation in response to manipulation of cell-specific cyclic adenosine 3',5'-monophosophate levels

In the growing follicle, communication between the oocyte and its surrounding follicular cells is essential for normal oocyte and follicular development. Maturation of the fully grown oocyte in vivo is associated with the loss of cumulus cell-oocyte gap junctional communication, preventing entry of meiotic-modulating factors such as cAMP into the oocyte. We have previously shown that oocyte and cumulus cell cAMP levels can be independently regulated using inhibitors of cell-specific phosphodiesterase (PDE) isoenzymes. The objectives of this study were to examine the effects of cell type-specific PDE inhibitors on the maintenance of cumulus cell-oocyte gap junction communication (GJC) and oocyte meiotic progression. Cumulus-oocyte complexes (COCs) were aspirated from antral follicles of abattoir-derived ovaries. Cumulus cell-oocyte GJC during oocyte maturation was quantified using the fluorescent dye, calcein-AM. COCs were cultured in the presence of specific PDE inhibitors, milrinone (an oocyte PDE3 inhibitor) or rolipram (a cumulus cell PDE4 inhibitor), and were pulsed with calcein-AM to allow dye transfer between the two cell types. Following cumulus cell removal, fluorescence in denuded oocytes was measured by microphotometry, and meiotic progression was assessed. In control COCs, dye transfer from cumulus cells to the oocyte fell progressively from 0 to 9 h, after which oocyte-cumulus cell GJC was completely lost. Loss of GJC was significantly attenuated (P < 0.05) during this time in response to treatment with milrinone and rolipram. Forskolin maintained GJC at the initial 0 h level until 3-4 h of culture, whereas treatment with milrinone and forskolin together actually increased the level of dye transfer above that in COCs treated with forskolin alone. Importantly, all treatments that prolonged GJC also delayed meiotic resumption, with meiosis generally resuming when fluorescence had fallen to approximately 40% of initial levels. These results, together with our previous studies, demonstrate that treatments that maintain or elevate cAMP levels in cumulus cells, oocytes, or both result in prolonged oocyte-cumulus cell communication and delayed meiotic resumption.