Cumulus Oocyte Research Articles

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 3 h 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 24 h 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.

278 GENE EXPRESSION PROFILE OF OVINE OOCYTES AND CUMULUS CELLS WITH REFERENCE TO PREMATING NUTRITION

Previous studies on sheep have demonstrated effects of maternal nutrition on fetal development with effects being observed on reproductive function (Rae et al. 2002 Anim. Reprod. Sci. 72, 63–71), blood pressure (Gopalakrishnan et al. 2004 Am. J. Physiol. Regul. Integr. Comp. Physiol. 287, R12–R20), and behavior (Erhard et al. 2004 Behav. Brain Res. 151, 25–35) in the adult offspring. It is known that gene expression in developing oocytes and very early embryos is influenced by their environment and some of the effects of nutrition on reproductive and other systems are likely to be expressed through such early changes (Hyttel 2000 Anim. Reprod. Sci. 60–61, 49–60). Effects of nutrition on oocyte function (i.e. before ovulation and fertilization) and resultant embryo quality have been observed (Gonzalez-Bulnes et al. 2004 Reprod. Fertil. Dev. 16, 421–435), but effects on oocyte gene expression have not been investigated. The aim of the present study was to investigate the effects of premating nutrition on the expression of selected genes in sheep cumulus cells and oocytes. For this purpose, 16 cast age ewes of proven fertility were used. At the time of manipulation and study, all ewes had a condition score of approximately 2.5. During the 2 weeks before slaughter, ewes of one group were fed a ration providing 0.5-live weight (n = 10) maintenance requirements and the other group 1.5-live weight (n = 6) maintenence requirements. This treatment ensured that all animals had similar numbers of potentially ovulatory follicles but different patterns of ovarian follicle maturation and, accordingly, different numbers of follicles and oocytes undergoing the final stages of maturation. The ewes's reproductive cycles were synchronized using intravaginal pessaries inserted 2 weeks before slaughter. At slaughter, ovaries were recovered and oocytes and associated cumulus cells were aspirated from all follicles >2 mm in diameter. For each ovary, oocytes derived from follicles with a diameter <4 mm and >4 mm were pooled separately, and stored for further analysis. Associated cumulus masses were collected and stored singly for subsequent investigations. As expected from previous observations, the number of visible follicles, the ratio of small-to-large antral follicles, and the total numbers of oocytes per ewe were similar for control and underfed ewes. Furthermore, the expression levels of the markers for oocyte growth, GDF-9, BMP-15, and c-kit, were investigated. Data were analyzed by ANOVA followed by Duncan test. Results showed no major differences between groups, independent of follicle diameter. These data suggest that the nutritional status of donor ewes did not significantly affect the expression profiles of these genes. However, further analyses on a greater panel of genes is required to rule out premating nutrition effects on oocyte gene expression. Furthermore, effects on the cumulus cell gene expression profile remain to be investigated. This work was supported by the University of Milan, FIRST 2006; Scottish Executive Environment and Rural Affairs Department.

Spermophagy by follicular cumulus cells in the common brushtail possum Trichosurus vulpecula following co-culture of sperm and immature ovarian oocytes.

This is a pilot study to investigate the effects of incubating cumulus oophorus-encased ovarian oocytes of the common brushtail possum (Trichosurus vulpecuila) with cauda epididymal sperm to determine if sperm could penetrate the cumulus oophous and thereby remove the need to mechanically denude in vitro matured ovarian oocyctes, and if sperm-zona binding would occur under these conditions. No fertilisation or sperm-zona pellucida binding took place. Some spermatozoa within the cumulus oophorus layer were phagocytosed.

The Impact of Nutrition of the Cumulus Oocyte Complex and Embryo on Subsequent Development in Ruminants

Cumulus-oocyte complexes (COCs) and early embryos rely on a histotrophic nutrition source for energy production and the synthesis of macromolecules. There is accumulating evidence suggesting that the balance of supply and demand for energy and other anabolic substrates during oocyte maturation and very early stages of development programmes subsequent developmental potential, and this may include subsequent fetal growth trajectory. One example is the role of glucose (Glc) during cumulus-oocyte complex maturation. Glucose is an essential nutrient for maturation, especially its role during cumulus expansion. Our laboratory has shown that during in vitro culture, too little glucose during cumulus-oocyte complex maturation affects meiotic competence. We have focussed on glucose (Glc) metabolism through the hexosamine biosynthesis pathway (HBP) during COC maturation in vitro. The HBP in somatic cells is regarded as a "fuel-sensing" pathway and its interaction with cell signalling systems and transcriptional regulation is increasingly apparent. Up-regulation of the HBP during oocyte maturation in vitro has negative consequences for subsequent development. Another example is the role of hypoxia (low O2) during peri-compaction development. My laboratory believes that ruminant embryos during compaction, blastulation and subsequent development in the uterine cavity lack a key hypoxia responsive element. Because of this, hypoxia is important for normal development in ruminants but perturbs further development in rodents. The implication of these examples to the fundamental concept of peri-conception nutritional programming of development are discussed.

159 LOCALIZATION OF CLAUDIN FAMILY PROTEINS IN PIG EMBRYOS DURING PRE-IMPLANTATION DEVELOPMENT

Tight junctions (TJ) are critical for blastocoel formation in mammalian pre-implantation embryos. Claudin family proteins, which are TJ proteins, are generally important for the barrier function of TJ. However, the expression and localization of claudin proteins are not clarified in mammalian pre-implantation embryos. The present study was designed to examine changes in the localization of claudin isoforms, claudin-1, -2, and -4, in pig parthenogenetic diploids through pre-implantation development. Oocyte–cumulus–granulosa cell complexes were collected from the follicles 4–6 mm in diameter, and then maturation-cultured. Only oocytes with a prominent first polar body were subjected to electro-stimulation after maturation, and they were treated with cytochalasin B to produce parthenogenetic diploids. Presumptive diploids were then cultured for 168 h and observed every 24 h. Embryos of each developmental stage from 2-cell to blastocyst were subjected to immunofluorescence staining of the anti-TJ proteins. Zona-free embryos were fixed and treated with rabbit anti-claudin-1 and -2 polyclonal antibodies or mouse anti-claudin-4 monoclonal antibody, followed by treatment with Alexa fluor 488-labeled goat anti-rabbit IgG antibody or goat anti-mouse IgG antibody. Some embryos were treated with rabbit anti-occludin antibody or anti-ZO-1 antibody for the detection of the TJ-net work. All embryos were counterstained with Hoechst 33342 and observed under an epifluorescence microscope after whole-mounting. The specific fluorescence for all TJ proteins examined was observed in embryos at all pre-implantation stages. Occludin and ZO-1 were detected in the cytoplasm before the morula stage (96 h), and localized toward the boundary region among cells from the morula and early blastocyst stages, reflecting the distribution of the tight junction in embryos. Claudin-1 and -2 localized in the cytoplasm at early-cleaving stages. They were detected in the nucleus after compaction, and the distribution of these proteins in the nucleus was dominant at the blastocyst stage. The distribution of the 2 claudin proteins in the nucleus was quite different; claudin-1 distributed rather homogeneously, but claudin-2 formed several bright spots in the nucleus. Claudin-4 also showed a unique distribution pattern in embryos; it was detected in the cytoplasm with strong fluorescence at the periphery of the nucleus of the 2- and 4-cell embryos. Claudin-4 changed its localization toward the boundary region of cells around the early blastocyst stage (120 h), and then the distribution of claudin-4 was restricted to the boundary region later in the blastocyst stage (144 h). These results indicate that claudin-4, but not claudin-1 and -2, is responsible for the formation of TJ in the pre-implantation embryos, although the function of these claudin proteins in the nucleus is unknown.

294 IN VITRO DEVELOPMENT OF RAT OOCYTES FOLLOWING MICROINJECTION OF A CUMULUS CELL INTO THE OOPLASM AND CHEMICAL ACTIVATION

If diploid zygotes constituted with a somatic and a maternal genome could successfully develop to term, a new reproductive method would be developed to produce animals. However, there appears to be little information on this subject. In the present study, in vitro early development of the constituted zygotes was examined. A cumulus cell was microinjected into a rat non-enucleated oocyte, the reconstructed oocyte was chemically activated, and the pronuclear formation and in vitro development of the embryo was observed. Prepubertal Wistar female rats (21–27 days old) were induced to superovulate with an IP injection of 15 IU of eCG, followed by 15 IU of hCG 48 h later. Cumulus cells were removed from oocytes by pipetting with 0.1% hyaluronidase. Experiment 1: The DNA content of cumulus cells for microinjection was evaluated by flow cytometry. Experiment 2: The optimal concentration of SrCl2 for activation of rat oocytes was examined. Experiment 3: Cumulus cells were injected into mature oocytes in BSA-free HEPES-buffered mKRB containing 0.1% polyvinyl alcohol (PVA) and cytochalasin B (5 �g mL-1), and were then chemically activated by treatment in Ca2+-free mKRB containing 5 mM SrCl2 for 20 min at 0 to 0.5 (A), 1 to 1.5 (B), or 3 to 3.5 h (C) after injection. Activated embryos were cultured in droplets of mKRB in an atmosphere of 5% CO2 in air at 37�C for 9 to 12 h. After being observed for pronuclear formation, the embryos were transferred into mR1ECM-PVA, and the cleavage and blastocyst formation rates were examined 24 and 120 h later, respectively. Results from 3 to 7 replicates were analyzed by ANOVA and Duncan's multiple range test. A total of 90.0 and 9.5% of cumulus cells derived from ovulated oocyte–cumulus complexes contained 2C and 4C DNA contents, respectively. Survival rates did not differ among oocytes stimulated with 0 to 5 mM SrCl2 (96.7–100%) but did differ between those stimulated with 1.25 and 10 mM SrCl2 (100 and 72.9%, respectively). Activation rates of oocytes increased at higher SrCl2 concentrations and were higher at 5 and 10 mM (92.6 and 98.5%, respectively) than at other concentrations. When cumulus-injected oocytes were activated after various periods after the injection, the incidences of pronuclear formation and cleavage did not differ among the periods (A: 95.0 and 81.3%; B: 85.6 and 85.0%; and C: 82.7 and 84.6%, respectively). Although a majority of the embryos developed to the 2- to 4-cell stages (78.7%; 152/208), the blastocyst formation rate was very low (0.8%; 2/208). In conclusion, rat non-enucleated oocytes injected with a cumulus cell can form pronuclei and cleave following chemical activation, but blastocyst formation of the embryos is very limited.

Die Cumulus-Oozyten-Regulationsschleife und deren Bedeutung für die Reproduktionsmedizin

Interaction between oocyte and follicle cells, cumulus oophorus and theca cells is the subject of research. Based both on fundamental research and first clinical experience, the role of cumulus cells for the oocyte within the framework of IVF/ICSI treatment is discussed. The connection between cumulus cells and oocytes via gap junctions makes the bidirectional exchange of chemical messengers possible. This exchange, in turn, contributes to the optimal development of the oocytes. FSH, LH and EGF as chemical messengers play an important role with regard to the development of oocytes. Furthermore, the cumulus-oocyte complex produces chemical messengers which attract sperm. The extent to which both the apoptosis of cumulus cells and the anti Mullerian hormone can be used as predictive factors for ART is discussed. The role that GnRH, FSH, LH and estradiol (E2) play in oocyte maturation within the framework of the cumulus-oocyte complex is described. FSH and LH are important factors for folliculogenesis and oogenesis. Threshold effects appear to exist for FSH and LH which affect oocyte quality and ultimately also pregnancy rate. The retrospective subanalysis of a comparative trial with hMG-HP vs. rFSH in GnRH agonist downregulated ovarian stimulation cycles showed that the hMG-HP stimulated cycles led to a significantly higher pregnancy rate among IVF patients than did those with rFSH. Further prospective randomized studies must now clarify whether, as a result of cumulus-oocyte communication, IVF patients profit more from hMG-HP stimulation than from rFSH with respect to pregnancy rate.

Intracellular cAMP and Calcium Signaling by Serotonin in Mouse Cumulus-Oocyte Complexes

cAMP and intracellular Ca2+ are important second messengers involved in mammalian follicular growth and oocyte meiotic maturation. We investigated the capacity of the neurohormone serotonin (5-hydroxytryptamine, 5-HT) to regulate intracellular cAMP and Ca2+ in mouse oocytes and surrounding cumulus cells. On the basis of a reverse transcription-polymerase chain reaction study, 5-HT7 receptor mRNA is expressed in cumulus cells, oocytes, and embryos up to the four-cell stage, and 5-HT2A and 5-HT2B receptor mRNAs are expressed in cumulus cells only, whereas 5-HT2C, 5-HT4, and 5-HT6 receptors are expressed in neither oocytes nor cumulus cells. The addition of 5-HT (10 nM to 10 microM) to isolated metaphase II oocytes had no effect on their internal cAMP or Ca2+ levels, whereas it caused dose-dependent cAMP and Ca2+ increases in cumulus cells. This cAMP increase in cumulus cells could be mimicked by 5-HT agonists with the following order of potency: 5-HT > 8-hydroxy-2-(di-n-propylamino) tetralin = alpha-methyl-5-HT = 5-carboxamidotryptamine maleate > 2-[1-(4-piperonyl)piperazinyl]benzo-triazole, thereby supporting a preferential involvement of 5-HT7 receptors. As measured with cumulus cells preloaded with fura-2/acetoxymethyl ester (AM), the addition of 5-HT also caused dose-dependent Ca2+ increases, which were probably linked to detected 5-HT2A and 5-HT2B receptors. Adding the Ca2+ ionophore ionomycin to cumulus cells resulted in both Ca2+ and cAMP elevations, whereas preincubation of cells with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM abolished the 5-HT-induced Ca2+ increase and reduced the cAMP increase, indicating cross-talk between the 5-HT-sensitive Ca2+ and cAMP pathways. Our results show that 5-HT may be a local regulator in mouse cumulus-oocyte complexes through its actions on cAMP and Ca2+ signaling, as mediated by 5-HT2A, 5-HT2B, and 5-HT7 receptors.

Highlight for Phenols, Quinolines, Indoles, Benzene and 2-Cyclopenten-1-ones Are Oviduct Toxicants in Cigarette Smoke, by Prue Talbot, Karen Riveles, and Ryan Rosa: List of Tobacco-Smoke Constituents That Are Harmful for Reproduction Grows—Passive Smokers May Be at Risk

Epidemiological studies provide compelling evidence that smoking has serious negative consequences on fertility and pregnancy outcome. Numerous epidemiological studies have linked smoking with higher frequency of ectopic pregnancies and problems related to the oviduct function (Castles et al., 1999). Smoking decreases the success rate of in vitro fertilization and, more specifically, reduces the rates of oocyte production (reviewed by Shiverick and Salafia, 1999). Clearly, we do not understand the exact mechanisms by which smoking decreases fertility and negatively affects pregnancy outcome. One of the reasons is the complex composition of the cigarette smoke, which contains over 4000 chemicals, and only few of them have been identified and tested toxicologically (nicotine, benzopyrenes, PAHs, carbon monoxide, heavy metals such as cadmium and lead.). The other reason is the lack of experimental models that provide specific, quantitative, and reproducible tests to assess the effect of defined tobacco constituents on specific biological processes of the reproductive cycle. The article by Riveles et al. in this issue of Toxicological Sciences is the most recent in the series of studies published by the same group of authors (DiCarlantonio and Talbot, 1999; Huang et al., 1997; Ji et al., 2002; Knoll and Talbot, 1998; Lam et al., 2000; Magers et al., 1995; Melkonian et al., 2003; Riveles et al., 2003, 2004; Talbot et al., 1999). This study confirms how important it is to have a well-characterized biological system in order to identify the effects and specific targets of different toxicants. The approach used by these authors to assay oviduct function is based on an in vitro culture of the hamster infundibulum (region of the oviduct closest to the ovary). The main functions of the oviduct are to pick up the oocyte cumulus complex after follicular rupture through the activity of the ciliary epithelium and to provide the entry into the oviduct lumen. Exposure to inappropriate conditions during this transition between peritoneal cavity and the tubal lumen can disrupt fertilization and early development. The oviduct assays used by these authors include measurements of ciliary beat frequency (CBC), oocyte cumulus complex pick-up rate (OPR) (Huang et al., 1997; Riveles et al., 2003), and smooth muscle contraction (SMC). Oocyte cumulus complex (OCC) pick-up rate depends on beating of cilia on the outer surface of the infundibulum and transient adhesion of the OCC to the tips of the cilia (Talbot et al., 1999). Movement of the zygote and preimplantation embryos through the oviduct depends on muscle contraction (Harper, 1994). To test the effect of different toxic compounds, explants of the infundibular portion of the oviduct were placed in culture medium in a holding pipette, and sequential measurements of CBC, OPR, and SMC were made before, during, and after exposure to test chemicals. These assays, thus, allow various cellular processes to be studied in a single explant and enable reversal of effects to be monitored. The results obtained using this system suggest that similar adverse effects can be potentially produced by the same chemicals in the other organs such as lungs (cilial beating) or smooth muscles (contractions). Prior work done by this group showed that a solution of mainstream cigarette smoke inhibits both CBF and OCC pick-up in the oviduct bioassay (Knoll and Talbot, 1998). To determine which chemicals of the over 4000 present in smoke solution could produce these effects, the authors passed mainstream cigarette smoke through various solid-phase extraction cartridges and screened the eluates for inhibitory activity using CBS, OPR, and SMC assays. Eluates that induced inhibition of oviduct activity by 80% or more were then analyzed using gas chromatography and mass spectrometry to identify the chemicals in the inhibitory eluates. Standards of the identified chemicals were purchased and used in the oviduct assays to 1 Towhomcorrespondence should be addressed. E-mail: genbacev@comcast. net.

Cigarette Smoke Inhibits Hamster Oocyte Pickup by Increasing Adhesion Between the Oocyte Cumulus Complex and Oviductal Cilia1

Previous research has shown that oocyte cumulus complex (OCC) pickup rate is inhibited in hamsters when oviducts and OCCs are simultaneously exposed to cigarette smoke solutions, independent of any effect on ciliary beat frequency. The purpose of this research was to determine whether smoke solutions caused a change in adhesion between the OCC and infundibulum of the oviduct and to determine whether a change in adhesion could account for decreased OCC pickup rate. OCC pickup rate and adhesion were measured before and after infundibula or OCCs recovered from acute in vitro exposures to mainstream and sidestream whole, gas, or particulate smoke solutions. Ciliary beat frequency was also measured on infundibula. Overall, smoke solutions decreased oocyte pickup rate 40% to 80% below control levels and increased adhesion 52% to 91% above control levels when infundibula were pretreated. A change in adhesion was observed in cases for which decreased OCC pickup rate could not be explained by a change in ciliary beat frequency. OCC pickup rate decreased 20% to 35% below control levels and adhesion increased 39% to 54% above control levels when OCCs were pretreated. These data show not only that cigarette smoke causes an increase in OCC adhesion to the oviduct, but also that there is a correlation between increased adhesion and decreased OCC pickup rate. The results also show that the both the OCC and oviduct are targets of cigarette smoke. The oviduct is more sensitive to the adverse effects of smoke; however, this may be caused by a combined impact on mechanisms involved in both adhesion and ciliary function.

Serotonin and Its Antidepressant-Sensitive Transport in Mouse Cumulus-Oocyte Complexes and Early Embryos1

Serotonin (5-hydroxytryptamine [5-HT]), is a neurohormone found in various nonneural tissues, including the gonads of many invertebrates, in which it regulates spawning and oocyte meiotic maturation. The possibility that a local serotonergic network might also exist in the female gonads of vertebrate species, including mammals, remains poorly documented. To clarify this possibility, we investigated mouse cumulus cells, oocytes, and embryos for three key serotonergic components, namely, 5-HT itself; the rate-limiting enzyme for its production, tryptophan hydroxylase 1 (TPH1); and the 5-HT-specific transporter (SLC6A4) required for modulating its cellular effects. Using a combination of reverse transcription-polymerase chain reaction analysis and immunofluorescence confocal microscopy, we showed that mouse cumulus cells, oocytes, and embryos contain 5-HT and SLC6A4, while only cumulus cells possess the 5-HT-producing enzyme TPH1 and may thus be the local source of 5-HT observed in their neighboring cells. With a semiquantitative assay in single cells, we demonstrated that 5-HT can actively be taken up by isolated oocytes when it is supplied exogenously in vitro. This 5-HT transport in isolated oocytes is driven by a classical serotonin transporter, expressed up to the blastocyst stage, that is sensitive to the antidepressants fluoxetine and fluvoxamine, which belong to the selective serotonin reuptake inhibitor family. All together, our results show that 5-HT may be produced locally by cumulus cells and that it can be actively taken up by mammalian oocytes and embryos as part of a likely larger serotonergic network possibly regulating various developmental processes much earlier than previously thought.

Nutrient concentrations in murine follicular fluid and the female reproductive tract

The culture of murine oocytes and preimplantation embryos in vitro has been used successfully for many years. However, this practice can result in cellular stress and reduced viability. Since this phenomenon is partly attributable to differences in nutrient composition between culture media and maternal tract fluids, we determined the concentrations of glucose, pyruvate, lactate and 19 amino acids in murine preovulatory follicles and oestrous oviductal and uterine fluids. Follicular fluids were aspirated from hyperstimulated ovaries, whereas oviductal fluids (with/without oocyte–cumulus complexes) and uterine fluids were collected from naturally cycling animals. Glucose, pyruvate and lactate concentrations were analysed using ultramicrofluorometric methods, whilst amino acid profiles were determined by reverse-phase high performance liquid chromatography. Mean glucose concentrations in follicular, oviduct (with/without cumulus cells) and uterine fluids were 0.46, 1.09/1.65 and 0.61 mmol l −1, respectively. Pyruvate concentrations were 0.38, 0.37/0.17 and 0.25 mmol l −1, respectively, and lactate concentrations were 17.34, 10.92/11.68 and 9.41 mmol l −1, respectively. Oviductal pyruvate concentration was significantly higher, and glucose significantly lower, in the presence of cumulus cells. Taurine, glycine, alanine, glutamine and glutamate were the major amino acids detected. Concentrations of amino acids differed among fluids, with highest levels being found in the oviduct. The follicular fluid and tract nutrient profiles differed from those of murine maturation, fertilisation and embryo culture media. These data extend our understanding of cellular metabolism and of nutritional environments of the oocyte and early embryo as they progress along the reproductive tract in vivo. These results may also contribute to the formulation of nutritionally more physiological media for mouse oocyte maturation and embryo culture.

261 IDENTIFICATION OF OOCYTE- AND CUMULUS-DERIVED CO-REGULATED AND DIFFERENTIALLY REGULATED TRANSCRIPTS ASSOCIATED WITH BOVINE MEIOTIC MATURATION

Understanding the process of oocyte maturation is critical for efficient application of biotechnologies such as in vitro embryo production and nuclear transfer/cloning. Intercellular communication between the oocyte and the encompassing somatic (cumulus) cells is pivotal for successful growth of ovarian follicles and oocyte maturation. Therefore, we utilized global gene expression profiling to determine changes in the transcriptome of oocytes and their adjacent cumulus cells during meiotic maturation in vitro to identify both co-regulated and differentially regulated transcripts within the two cell compartments of the cumulus oocyte complex (COC). Germinal vesicle (GV) and in vitro matured metaphase II (MII) COC (n = 5 pools of 5 COC per group) were denuded and separated into oocytes and cumulus cells. RNA was extracted from the oocytes and cumulus cells and subjected separately to microarray analysis using a bovine cDNA array containing expressed sequence tags (ESTs) representing 15 500 unique genes. A combined total of 1045 genes displaying greater mRNA abundance in GV oocytes and associated cumulus cells compared to MII samples were detected (P < 0.05; false discovery rate (FDR) = 5%). A combined total of 711 genes displaying greater mRNA abundance in MII oocytes and enclosing cumulus cells compared to GV samples were detected (P < 0.05; FDR = 5%). Fourteen transcripts were identified that were co-regulated and of greater abundance in GV or MII oocytes and in their matching cumulus cells (P < 0.05; FDR = 5%). The co-regulated transcripts identified are implicated in metabolism (e.g. heme oxygenase-2, leukotriene B4 12-hydroxydehydrogenase), signal transduction (e.g. caveolin 1, ring finger protein 31), and cell growth (e.g. BTG family member 2, myosin regulatory light chain 2). In contrast, thirteen transcripts differentially regulated in the GV oocyte versus MII cumulus cells were identified (P < 0.05; FDR = 5%). Similarly, nine transcripts differentially regulated in the MII oocyte versus GV cumulus cells were identified (P < 0.05; FDR = 5%). Some of the identified differentially regulated transcripts encode for genes associated with the cytoskeleton (e.g. tropomyosin 1), apoptotic activity (e.g. death effector domain containing protein 2) and DNA replication (e.g. epsilon polymerase). The results provide novel insights into the identity of transcripts whose abundance is co-regulated or differentially regulated between the oocyte and cumulus cells during the transition of a COC from the GV to the MII stage. Characterization of the signaling pathways driving changes in transcript abundance for co-regulated and differentially regulated genes in oocytes versus associated cumulus cells may lead to a better understanding of regulation of meiotic maturation and potential cross-talk between germ cells and somatic cells during the oocyte maturation cascade. This work was supported by the Rackham Foundation and the MI Agriculture Experiment Station.

283 EFFECTS OF CUMULUS CELL REMOVAL ON IN VITRO OOCYTE MATURATION, FERTILIZATION, AND EMBRYONIC DEVELOPMENT IN PIGS

It is generally accepted that cumulus cells (CCs) support the nuclear maturation of immature oocytes in mammals. However, the precise mechanism of interaction between cumulus cells and oocytes has not been clarified. Furthermore, the role of cumulus cells in embryonic development has not been reported. In the present study, the effect of denuding cumulus cells from porcine oocytes on oocyte maturation, ertilization, and their subsequent development to the blastocyst stage was examined in vitro. In vitro maturation, fertilization, and culture were carried out as previously reported (Kikuchi et al. 2002 Biol. Reprod. 66, 1033-1041). Porcine cumulus-oocyte complexes (COCs) were collected; some of them were completely denuded of cumulus cells immediately after the collection (DO-0 group). The remaining intact COCs and the DO-0 oocytes were cultured for 24 h in the presence of dbcAMP and hormones. After the initial culture, some of the intact COCs were denuded either completely (DO-24 group) or partially (H-DO-24 group). Additionally, some of DO-24 oocytes were co-cultured with the cumulus cells removed at 0 h and pre-cultured for 24 h (DO-24 + CCs group). The denuded oocytes in each experimental group and intact COCs (control) were further cultured for total 46 h. The remaining oocytes with a first polar body were either examined for the levels of intracellular glutathione (GSH) or fertilized in vitro with frozen-thawed boar spermatozoa. The inseminated oocytes were cultured and examined for their fertilization status after 10 h and for their developmental competence after 6 days. Data were analyzed by ANOVA, followed by the Duncan's multiple range tests. The maturation rates of all denuded groups were significantly lower (P < 0.05; 34.3 to 45.0%) than that of the control group (64.5%). Intracellular GSH concentrations of all denuded groups were also significantly lower (P < 0.05; 4.03 to 7.00 pmol/oocyte) than that of the control group (9.60 pmol/oocyte); however, the GSH level of H-DO-24 oocytes was significantly higher (P < 0.05) than the GSH levels in the other denuded groups. Male pronuclear formation rates of completely denuded oocytes (DO-0, DO-24, and DO-24 + CCs groups) were significantly lower (P < 0.05; 41.4 to 59.3%) than those of the control (89.4%) and the H-DO-24 (80.0%) groups. The blastocyst rate of the control group was significantly higher (P < 0.05; 19.9%) than that of H-DO-24 group (11.6%), and these rates were significantly higher (P < 0.05) than those of the completely denuded groups (3.0 to 4.5%). The results suggest that the presence of cumulus cells during maturation culture improves nuclear maturation of oocytes and plays an important role in embryonic development to the blastocyst stage in vitro.

281 MITOGEN-ACTIVATED PROTEIN KINASE IN PORCINE CUMULUS CELLS

Mitogen-activated protein kinase (MAPK) is involved in many signal processes within eukaryotic organisms. Its active form is phosphorylated. For meiotic resumption in oocytes the MAPK cascade plays a central role, because it participates in the transfer of the extracellular gonadotropin signal into the nucleus. In pigs it could be shown that for a gonadotropin-induced germinal vesicle breakdown (GVBD), an activation of MAPK in oocytes is not essential, but in the surrounding cumulus cells the MAPK has to be phosphorylated (Ohashi et al. 2003 Biol. Reprod. 68, 604–609). Because cumulus cells are very important for signal transfer, the present investigation dealt with the relevance of porcine cumulus cells and the phosphorylation of MAPK for the resumption of meiosis. Oocytes of slaughtered pigs were collected and cultured (medium: TCM 199, insulin, l-glutamine, gentamycin, 20% (v/v) FCS, and with or without 2.5 μg/mL FSH and 5.0 μg/mL LH). The proteins of isolated cumulus cells and oocytes were separated by gel electrophoresis (cumulus cells of 10 cumulus-oocyte complexes and 40 oocytes per lane, respectively) followed by an immunoblot with antibodies against MAPK and p90rsk (ERK 1 (sc-94) and Rsk-1 (sc-231), respectively; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Alternatively the nuclear maturation was determined by orceine staining. The following results were achieved: The phosphorylation of MAPK in cumulus cells began very early during the in vitro maturation period. This was demonstrated already after 0.5 h unlike in oocytes where phosphorylation of MAPK does not occur until 18 h. The phosphorylation in cumulus cells occurs both in the presence and in the absence of FSH/LH, but without FSH/LH almost no GVBD occurs (after 26 h IVM: 86.9% GV oocytes, n = 59). The phosphorylation in the absence of gonadotropins could be caused by components of FCS, but with an exchange against polyvinylpyrrolidone (0.3%), the phosphorylation without FSH/LH still existed. The specificity was examined with the MAPK kinase inhibitor U0126. A concentration of 10 μM U0126 prevented GVBD and phosphorylation of MAPK in oocytes. However, in cumulus cells the phosphorylation of MAPK was reduced only minimally. In the presence of 50 μM U0126, a distinct decrease was observed during the first hours of maturation. But after 26 h phosphorylated MAPK appeared in cumulus cells despite the high concentration of U0126. The p90rsk is an important substrate of MAPK, which is phosphorylated by activated MAPK in oocytes. In our investigations we could detect only unphosphorylated forms of p90rsk in the cumulus cells. It seems that there are different ways for phosphorylation of MAPK to occur in cumulus cells, but they do not have the same consequences. The phosphorylation of MAPK in cumulus cells is necessary for a gonadotropin induced meiotic resumption, but phosphorylation does not always lead to GVBD. Furthermore, the p90rsk appears not to have the same importance as a substrate of MAPK in cumulus cells as in oocytes.

026. The cumulus matrix in ovulation: inert packaging or active delivery vehicle for the oocyte?

Preovulatory follicles respond to the LH-surge with a cascade of molecular events. The ovulatory signal initially impinges on the mural granulosa layers triggering rapid tissue morphogenesis and ultimately terminal differentiation of these cells. Mural granulosa cells transiently produce a suite of transcriptional regulators, EGF-like ligands as well the extracellular matrix (ECM) proteoglycan, versican and the protease ADAMTS-1. These act in concert with permissive oocyte signals to induce and organise a complex hyaluronan (HA) rich ECM surrounding the cumulus cells and oocyte. This expanded cumulus matrix is analogous in composition to an extensive form of pericellular matrices actively associated with cell migration. During ovulation the cumulus matrix becomes anti-adhesive to the intra-follicular environment but is strongly pro-adhesive for the oviductal fimbria. When the follicle apex is perforated the COC is released binds to the fimbria and transports into the oviduct where fertilisation occurs. Success of ovulation and fertilisation is sensitive to the appropriate production and assembly of cumulus matrix components that are in turn dependent on an appropriate balance of oocyte and granulosa derived signals. Production of these cumulus matrix components is thus a potential checkpoint that assures ovulation of competent oocytes. The HA matrix is cross-linked by organiser molecules and also is enriched in proteases ADAMTS-1, 4, 5. Although these have potentially redundant functionality, ADAMTS-1 null female mice are profoundly sub-fertile and have reduced ovulation rate. Specific components of the cumulus matrix are disorganised in ADAMTS-1 null mice and cleavage of versican in these cumulus complexes is reduced. Thus ADAMTS-1 and versican have unique roles in normal cumulus matrix expansion that is important for ovulation. Altered interaction of the cumulus complex with neighbouring tissues alters transport through the oviduct, while abnormal persistence of COC matrix structure after ovulation is also likely to impair sperm interaction and penetration. Evidence thus indicates that the expanded cumulus matrix plays several active roles in oocyte release, transport and sperm interaction.

343 EFFECTS OF EPIDERMAL GROWTH FACTOR SUPPLEMENTATION ON IN VITRO MATURATION AND GENE EXPRESSION OF CANINE OOCYTES

Despite many efforts to improve canine in vitro maturation (IVM), the efficiency is still low compared to that of other mammalian species (Marie et al. 2004). Epidermal growth factor (EGF) has stimulatory effects on the resumption of oocyte maturation and cumulus expansion in vitro and on prei-mplantation embryonic development in mammals by either an autocrine or a paracrine pathway, or a combination of both systems (Paria et al. 2001 PNAS 98, 1047-1052). The present study investigated the effects of EGF supplementation on in vitro maturation and gene expression of canine oocytes. Oocytes were recovered by slicing ovaries recovered from 40 bitches after ovariohysterectomy at random stages of the estrous cycle. Cumulus-oocyte complexes (COCs) were matured in TCM-199 containing 10% FBS, 1 �g/mL FSH and LH, and EGF (0, 10, or 30 ng/mL) for 48 or 72 h at 39�C in a humidified atmosphere of 5% CO2 in air. In Experiment I (n = 2520 oocytes), the nuclear maturation status was assessed by fluorescence microscopy after bisbenzimide (Hoechst 33342) staining (10 �g/mL) at 0, 48, and 72 h of incubation. In Experiment II (n = 90 oocytes), expression of transcripts such as EGF receptor (EGFR), luteinizing hormone receptor (LHR), and gap junction protein (GJA5) were determined in 10 intact COCs each at 0, 48, and 72 h, respectively, by reverse transcription-polymerase chain reaction (RT-PCR). At 0 h 10-20% of the oocytes had undergone resumption of meiosis (GVBD<MII). After 48 h of IVM, rate of meiotic resumption for 0, 10, and 30 ng/mL EGF were 28, 35, and 30%, respectively. At 72 h of IVM, oocytes in the 10 ng/mL EGF group had resumed meiosis at a higher frequency (55%; P < 0.05) than in the 30 ng/mL EGF or the control group (39 and 42%, respectively). At 72 h of IVM, the frequency of maturation to the MII stage was significantly higher in the 10 ng/mL EGF group (9.6%) than in the 30 ng/mL EGF or the control group (4.2 and 3.3%, respectively). The expression of EGFR was significantly higher (P < 0.05) in 0 h oocytes than in the 48- or 72-h oocytes. Further EGFR expression levels were decreased in the presence of EGF in a dose dependent manner. Transcripts for LHR were detected at all maturation intervals and its expression patterns were not altered by supplementation with 10 ng/mL EGF. Expression of GJA5 was observed only after 48 h of IVM, and levels of expression were similar in oocytes supplemented with both 10 and 30 ng/mL EGF. In summary, our results indicate that supplementation of canine IVM medium with 10 ng/mL EGF had a positive influence on the progression of maturation to MII at 72 h. The effect may not be related to the alteration of mRNA expression of genes analyzed in the present study, due to the complex patterns regulating meiotic arrest in canine oocytes. This work was supported by Grant no. 204119-03-1-LG000 from ARPC, Republic of Korea.

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.

Effect of cumulus cell removal on the fertilization and the day 3 embryo quality in human IVF

The aim of this study was to examine the effect of partial cumulus removal on the fertilization and the day 3 embryo quality. Results indicated that fertilization and grade (G) 4/3 embryo (86.7% and 61.4%) rates in the large oocyte–cumulus complex (OCC, ≥2 mm) were significantly greater compared with the small OCC (<2 mm) (73.1% and 42.0%). Removal of cumulus cells decreased the fertilization rate and subsequent embryo quality. The cumulus cells of OCCs should be excised as little as possible at the time of blood removal.

Oocyte–somatic cell interactions during follicle development in mammals

Our current perspectives on the relationship between the oocyte and its surrounding somatic cells are changing as we gain a greater understanding of factors regulating folliculogenesis. It is now widely accepted that the oocyte plays a very active role in promoting follicle growth and directing granulosa cell differentiation. The oocyte achieves this, in part, by secreting soluble paracrine growth factors that act on its neighboring granulosa cells, which in turn regulate oocyte development. In preantral follicles, the oocyte directs granulosa cells to regulate oocyte growth, and oocytes may also directly drive follicle growth. In antral follicles, the oocyte governs the behaviour of cells in its immediate vicinity, thereby actively regulating its own microenvironment. As such, the oocyte establishes and maintains the distinct cumulus lineage of granulosa cells. This oocyte–cumulus cell interaction, in general, prevents luteinization of cumulus cells by promoting growth, regulating steroidogenesis and inhibin synthesis, and suppressing luteinizing hormone receptor expression. Conversely, mural granulosa cells in antral follicles, which have no direct physical contact with the oocyte and, presumably, experience a more diffuse concentration of oocyte-secreted factors, proceed to a different phenotype. In the ovulating follicle, oocyte-secreted factors also play vital roles in enabling cumulus cell expansion and regulating extracellular matrix stability, thus facilitating ovulation. The identities of these oocyte-secreted growth factors regulating such key ovarian functions remain unknown, although growth differentiation factor-9 (GDF-9), GDF-9B and/or bone morphogenetic protein-6 (BMP-6) are likely candidate molecules, probably forming complex local interactions with other related members of the transforming growth factor-β (TGF-β) superfamily. Elucidating the nature of oocyte–somatic cell interactions at the various stages of follicle development will have important implications for our understanding of factors regulating folliculogenesis, ovulation rate and fecundity.