1 Brigham and Women's Hospital, Harvard Medical School, Department of Obstetrics and Gynecology, Boston, MA 02115, 2 University of Massachusetts, Department of Veterinary and Animal Sciences, Amherst, MA 01003 and 3 Tufts University School of Medicine, Department of Anatomy and Cellular Biology, Boston, MA 02111, USA 4 Current address: The University of Kansas Medical Center, Department of Molecular and Integrative Physiology, Kansas City, KS 66160, USA
5 To whom correspondence should be addressed at: Biology Department, McCardell Bicentennial Hall 350, Middlebury College, Middlebury, VT 05753, USA. Email: ccombell{at}middlebury.edu
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Abstract |
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Key words: co-culture/cumulus cells/in vitro maturation/oocyte/three-dimensional collagen gel
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Introduction |
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Although a variety of culture media have been utilized for human IVM, no single system has been shown to be clearly superior for the production of developmentally competent oocytes. Culture conditions, including the formulation of the base medium, supplementations, and the in vitro physical environment (such as the oxygen tension and presence of cumulus cells) all influence multiple events that are paramount to oocyte maturation and subsequent embryonic development (Van de Sandt et al., 1990; Sutton et al., 2003
). The culture medium has been shown to modulate not only the metabolism of human oocytes (Roberts et al., 2002
), but also the maturation to MII, the kinetics of cell cycle progression, and spindle/chromatin organization (Cekleniak et al., 2001
; Trounson et al., 2001
).
The majority of human IVM studies have utilized immature oocytes denuded of surrounding cumulus cells that were obtained after ovarian stimulation with gonadotrophins (Cha and Chian, 1998; Trounson et al., 2001
). We have shown previously that these denuded human oocytes exhibit accelerated meiotic resumption in vitro, a deficiency in the ability of the cytoplasm to maintain M-phase characteristics while meiosis is progressing, a propensity to activate spontaneously after M-phase arrest, and a lack of coordination between nuclear and cytoplasmic maturation (Combelles et al., 2002
). What contributes to these deficiencies remains to be determined, but given the interdependence of oocytes and cumulus cells for their normal development and function (Eppig, 1991
), an immediate shortcoming in the type of culture system used to date has been the absence of somatic cell support.
Co-cultures of oocytes and cumulus cells have been employed in vitro to restore support from the surrounding cumulus cells to the oocyte and/or to probe interactions between the two cell compartments. Oocytes were cultured either over monolayers of cumulus or granulosa cells or along with (in suspension or not) pieces of mural granulosa cells or follicular shells in several species, including the mouse (Eppig, 1979; Herlands and Schultz, 1984
; Cecconi et al., 1991
; Downs and Mastropolo, 1994
), cow (Sirard and Bilodeau, 1990
; Osaki et al., 1997
) and pig (Moor et al., 1990
; Motlik et al., 1996
). While preliminary reports exist for the co-culture of human oocytes with different cell types (Dandekar et al., 1991
; Janssenswillen et al., 1995
; Coskun et al., 1998
; Haberle et al., 1999
), to our knowledge, there has been no previous description of an efficacious co-culture system of human denuded oocytes and cumulus cells for IVM.
The objective of the current work was to develop a co-culture system that will favour the optimal development and function of not only the oocyte but also the cumulus cells. Of relevance in designing such a system is the well-established importance of the extracellular matrix (ECM) and a three-dimensional (3D) environment when studying cell behaviour and function in vitro (Abbott, 2003). Indeed, the ECM influences a multitude of cell functions, including morphogenesis, survival, migration, proliferation, communication, metabolism, and response to external stimuli (Weaver et al., 1997
; Bissell, 1998
). In addition, it is equally important to culture cells in a dimension that mirrors, as closely as possible, the 3D environment in vivo. Indeed, in comparison with a two-dimensional (2D) environment, a 3D environment results in cell behaviour, signalling and gene expression profiles most resembling those observed in living tissues (Cukierman et al., 2001
, 2002
). Granulosa cells also behave and function in distinct ways when cultured in the presence or absence of ECM (Aharoni et al., 1996
; Hwang et al., 2000
; Richardson et al., 2000
; Huet et al., 2001
). In addition, follicles and intact cumulusoocyte complexes from several species have been cultured embedded in a 3D collagen gel (mouse: Torrance et al., 1989
; Gomes et al., 1999
; pig: Hirao et al., 1994
; cow: Osaki et al., 1997
; human: Abir et al., 1999
). However, a 3D system making use of an ECM has not been applied to the co-culture of oocytes and cumulus cells in any species to date.
The present study was designed to develop and characterize a novel co-culture 3D system for IVM of immature denuded human oocytes. We compared oocytes matured in microdrop or in co-culture with respect to their ability to progress to metaphase II (MII) and with regard to the organization of their cytoskeleton and chromatin. Lastly, given the importance of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) in the regulation of oocyte maturation (Heikinheimo and Gibbons, 1998; Trounson et al., 2001
) and the paucity of reports on MPF and MAP kinase activities in human oocytes (Pal et al., 1994
; Sun et al., 1999
; Anderiesz et al., 2000
), we investigated the dynamics of cell cycle kinases during human IVM.
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Materials and methods |
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Source of oocytes and cumulus cells
Immature human oocytes were aspirated from ovaries of women undergoing ovarian stimulation for ICSI. These immature oocytes were donated to research given their otherwise clinically-useless nature. Complete institutional review board approval (protocol #02-001986) and written consents were obtained prior to placing oocytes in culture. After enzymatic and mechanical removal of the cumulus and corona cells, the meiotic status of oocytes was determined, and immature oocytes [germinal vesicle (GV) or metaphase I (MI)] were used for IVM experiments. GV oocytes had an intact nucleus, and MI oocytes were defined by the absence of a nucleus and polar body. A total of 231 oocytes retrieved from 44 patients (35.2±4.5 years; mean±SD) were included in this study. In vivo-matured MII oocytes (n=13) were donated from three patients (37.0 ± 3.8 years) who elected to have only a limited number of their oocytes inseminated. Oocytes were photographed prior to culture and the vitelline diameters (not including the zona pellucida) of fresh oocytes were determined from the maximum and minimum diameters of each oocyte.
Autologous cumulus cells were collected for use in co-cultures after their removal from cumulusoocyte complexes in preparation for ICSI. All cumulus cells were pooled together within each patient regardless of the meiotic state of the associated oocytes.
Culture systems
Immature oocytes were matured in vitro in either a microdrop (C) or co-culture (CC) system. For the C group, denuded oocytes were placed in 25 µl microdrops of culture medium (see composition below) overlaid with embryo-tested light mineral oil (Irvine Scientific, USA) in a humidified atmosphere of 5% CO2 at 37 °C. After coronacumulus cell removal, the medium (Ham's F-10 with 5% human serum albumin (HSA); InVitroCare Inc., USA) containing the removed cells was collected into a conical tube with an equal volume of warm (37 °C) HEPES-buffered Hanks' balanced salt solution (HBSS) without magnesium and calcium supplemented with 5% HSA. Following centrifugation at 200 g for 5 min, the cell pellet was resuspended in 1.5 ml of warm HBSS with 5% HSA and laid gently over a density gradient consisting of 1.5 ml of 90% Isolate (Irvine Scientific) and 1.5 ml of HBSS with 5% HSA. After centrifugation for 10 min to pellet red blood cells, purified cumulus cells were aspirated from the interface and washed with HBSS with 5% HSA prior to use. Cells were counted using a haemacytometer, and only cell preparations with a viability 90% as assessed by Trypan Blue exclusion were used. For the co-culture experiments, the cell pellet was resuspended in neutralized 1% collagen (4.12 mg/ml rat tail collagen, type I; BD Biosciences, USA) kept at 4 °C. Aliquots of 4 µl each were seeded in 4-well Nunc tissue culture dishes (Nunclon, Denmark) at a cell density of 1 x 106 cells/ml, creating gels that contained
4000 cumulus cells each. A single immature oocyte was added carefully to each gel, which was then allowed to polymerize at 37 °C for 10 min before adding 400 µl of culture medium to each well. The culture medium used for all IVM studies consisted of M-199 with Earle's salts (Invitrogen Life Technologies, USA) supplemented with 5% HSA; 0.075 IU/ml recombinant FSH (Gonal-F; Serono Laboratories, USA); 0.075 IU/ml hCG (Profasi; Serono Laboratories); 1 µg/ml estradiol; 0.30 mmol/l sodium pyruvate; 1 mmol/l glutamine; 0.032 mg/ml penicillin; and 0.050 mg/ml streptomycin. This supplemented tissue culture medium (M199-S) supports the maturation of denuded human oocytes to MII (Cekleniak et al., 2001
; Chian and Tan, 2002
), and it was chosen because of the need to support two distinct cell compartments, the somatic and germ cells.
Comparison of oocytes matured either in microdrops or co-cultures was performed within a single patient to account for potential inter-patient variability. Only patients with at least four immature oocytes and 10 cumulusoocyte complexes on the day of retrieval were included. Also, only oocytes with a diameter of 110 µm (116.1 ± 4.7 µm, mean ± SD; n=135) were placed in culture given that these oocytes are known to represent meiotically competent oocytes (McNatty et al., 1979
; Durinzi et al., 1995
; Combelles et al., 2002
). Oocytes were randomly allocated within each patient to the two culture groups.
In an initial study aimed at evaluating the influence of the environment, cumulus cells from each of seven patients were cultured either on a 2D surface (with or without collagen) or in a 3D collagen gel. Cell handling for 3D culture was done as described previously. For cells cultured on a 2D surface, purified cell preparations were resuspended in M199-S and plated at a concentration of 1 x 106 cells/ml. After allowing cell attachment to the culture surface (12 mm sterile glass coverslips), culture wells were flooded with M199-S. For 2D cultures in the presence of collagen, coverslips were coated with 50 µg/ml of collagen type I for 2 h at room temperature, followed by three washes with phosphate-buffered saline and air-drying before use.
Processing of samples for immunofluorescence analysis
Cumulus cell cultures on glass coverslips and collagen gels containing cells with or without oocytes were fixed and extracted for 15 min at 37 °C in a microtubule stabilizing buffer (0.1 mol/l PIPES, pH 6.9, 5 mmol/l MgCl2·6H2O, 2.5 mmol/l EGTA) containing 2% formaldehyde, 0.1% Triton X-100, 1 µmol/l taxol, 10 IU/ml aprotinin, and 50% deuterium oxide. Samples were washed and stored at 4 °C in a blocking solution of phosphate-buffered saline containing 2% bovine serum albumin, 2% powdered milk, 2% normal goat serum, 0.1 mol/l glycine and 0.01% Triton X-100 containing 0.2% sodium azide. Prior to processing, collagen gels were extracted with 0.5% Triton X-100 for 20 min at 37 °C with shaking. Microtubules were detected using a 1:1 mixture of monoclonal anti--tubulin and anti-
-tubulin antibodies (1:250) for 2 h at 37 °C with shaking, followed by three 15 min washes and Alexa-fluor 488 goat anti-mouse IgG for 2 h (1:600; Molecular Probes, USA). All samples were subsequently washed and stained with 1 µg/ml Hoechst 33258 (Polysciences Inc., USA) for 30 min to label chromatin prior to mounting in 50% glycerol and phosphate-buffered saline solution containing 25 mg/ml sodium azide. To preserve the three-dimensional integrity, each collagen gel was mounted uncompressed using wax recess. Samples were analysed using an Axiovert 200 inverted microscope (Zeiss, USA) or a TCS SP2 laser scanning confocal microscope (Leica Microsystems Inc., USA). Digital images were collected with an ORCA ER digital camera (Hamamatsu Corp., USA) and the Metamorph image analysis software (Universal Imaging Corp., USA). The pyknotic index, as defined by the percentage of cells with condensed or fragmented chromatin, was calculated from random fields (three per culture condition for each patient) that were imaged with a x40 objective under both phase and fluorescent modes.
In vitro protein kinase assays
MPF and MAP kinase activities were measured simultaneously using histone 1 (H1) and myelin basic protein (MBP) as their respective substrates. Lysates of single human oocytes were prepared by six cycles of freezing and thawing in 4.5 µl of kinase lysis buffer consisting of 80 mmol/l glycerophosphate (pH 7.3), 20 mmol/l EGTA, 15 mmol/l MgCl2, 1 mmol/l dithiothreitol, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 500 nmol/l cAMP-dependent protein kinase inhibitor. Lysates were stored at 80 °C before use. Kinase reactions were started by adding 5 µl of substrate buffer containing 1.98 mg/ml histone H1 (type III-S), 1.0 mg/ml MBP, 0.9 mmol/l ATP, and 25 µCi of [-32P]ATP (Amersham Biosciences, USA). The kinase reaction was conducted for 30 min at room temperature and stopped by the addition of 5 µl of double-strength electrophoresis sample buffer (Laemmli, 1970
). After boiling for 3 min, samples were separated by standard polyacrylamide gel electrophoresis (15% sodium dodecyl sulphatepolyacrylamide gels). Detection of phosphorylation levels was done by autoradiography at 80 °C with several film exposures of the same gel performed until quantification of activities was obtained in the linear range and without saturation of the counting system for each kinase. The mean pixel intensity of a preselected, set area was quantified using Adobe Photoshop (Adobe Systems, USA). Kinase activities were expressed relative to phosphorylation levels, which were set arbitrarily at a value of 1 in control samples consisting of lysis buffer alone without oocyte.
Statistical analysis
Proportions and relative kinase activity levels were analysed by 2 and one-way ANOVA respectively (SPSS 10.0; Statistics Package for Social Sciences, USA). P<0.05 was considered significant.
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Results |
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Discussion |
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As demonstrated using other cell types, cumulus cells appear to behave differently and display distinct morphologies whether cultured with or without collagen, and whether in a 2D or a 3D environment. Cell viability, based on the presence of homogeneous chromatin in nuclei, is highest in a 3D collagen gel and lowest in cultures on a flat surface with an intermediate pyknotic index if cultured in a 2D environment with matrix. While cell survival and apoptosis remain to be specifically assayed, of relevance here are previous reports that apoptosis is more elevated when cells are cultured on plastic without matrix and in serum-free media (Frisch and Francis, 1994; Aharoni et al., 1996
). The use of an ECM during IVM is all the more significant when considering the impetus to design a chemically defined culture medium for human IVM and notably serum-free conditions as were used here. Cumulus cells allowed to interact with a matrix displayed multiple neuronal-like processes (Figure 1), which may reflect cells exploring their surrounding environment and thereby becoming interconnected to each other in a 3D space, as demonstrated for fibroblasts grown in a 3D collagen gel (Grinnell et al., 2003
). Further studies need to assess metabolic coupling between cumulus cells under different culture conditions. In addition, given the close relationship between shape and function of both granulosa and cumulus cells (Aharoni et al., 1996
; Bar-Ami et al., 1997
; Huet et al., 2001
), future analysis of cumulus cell function under 2D or 3D cultures in the presence or absence of ECM should include not only the release and use of metabolites, but also the production of steroid hormones, growth factors, and ECM components.
Influences between the two cell populations used here may be exerted by junctional and/or paracrine actions. Whether gap junctional communication exists between cumulus cells and oocytes in the present co-culture system remains to be determined. Notably, dynamics of gap junctional communication between oocyte and cumulus cells are unknown in the human, and it is conceivable that these are absent or are highly dynamic at such an advanced stage of oocyte maturation. Also, the current supplementation of the culture medium may not favour, or may even inhibit, gap junction communication. A number of paracrine factors with regulatory local roles within COC are produced during oogenesis, including kit ligand, GDF-9, BMP-15, and other members of the transforming growth factor superfamily (Eppig, 1991; Elvin and Matzuk, 1998
), and paracrine signalling is likely to take place when co-culturing oocytes with cumulus cells. Furthermore, culturing cumulus cells in a 3D ECM environment may permit the mobilization and modulate the activity of growth factors as demonstrated for other cell types (Rosso et al., 2004
). Future investigation into this possibility must include a comparison of oocytecumulus cells co-cultured in the presence (2D versus 3D) or absence of collagen. It is noteworthy that microtubule-rich processes resembling transzonal projections previously described in human cumulusoocyte complexes (Motta et al., 1994
) were abundant at the oocytecumulus cell interface (Figure 2). The possibility that these transzonal projections serve as conduits for the transport of paracrine factors (Albertini et al., 2001
) remains to be determined in the co-culture system used here.
MPF and MAPK regulate cell cycle progression and a number of events during oocyte maturation (Heikinheimo and Gibbons, 1998). While the dynamics of these kinase activities has been extensively studied in oocytes from other species, very limited attention has been given to their analysis in human oocytes (Pal et al., 1994
; Sun et al., 1999
; Anderiesz et al., 2000
; Trounson et al., 2001
)this is despite the fact that the kinetics of cell cycle progression are known to differ significantly among studies of human IVM (Cha and Chian, 1998
). In our study, MAPK levels continued to rise from the GV stage through meiosis I until arrest at metaphase of meiosis II (Figure 3); this pattern is similar to previous findings in other species (Verlhac et al., 1994
; Heikinheimo and Gibbons, 1988
; Wehrend and Meinecke, 2001
). Also, MPF activity increased from the GV stage to MI as described previously in oocytes of several species (Hashimoto and Kishimoto, 1988
; Wu et al., 1997
; Wehrend and Meinecke, 2001
); no further significant increase in MPF level was observed between MI and MII human oocytes (Figure 3).
While no differences were observed in MPF levels at MII between oocytes matured in vitro (in microdrop or collagen co-culture) or in vivo, enhanced MAPK activity was detected in MII oocytes matured in collagen co-culture versus microdrops and a further increase was detectable in in vivo-matured oocytes (Figure 4). Here again, there is an immediate need to perform an internal comparison of oocytes and cumulus cells co-cultured with or without collagen in a 2D or 3D environment. Culture media and maturation conditions can influence cell cycle kinase activities as documented in pig (Naito et al., 1992; Motlik et al., 1996
), horse (Goudet et al., 1998
), and cow (Sakaguchi et al., 2002
) oocytes. A hypothesis to explain why MAPK levels are reduced in denuded oocytes cultured in microdrop may be that by abrogating support from the surrounding somatic cells, phosphorylation events and more generally the activation of signalling pathways known to regulate kinase activity may be deficient. Indeed, cumulus cells modulate protein synthesis and phosphorylation levels in the oocyte (Colonna et al., 1989
; Cecconi et al., 1991
; Trounson et al., 2001
). In addition, Su et al. (2002)
demonstrated further the influence of somatic cells on oocyte cell cycle kinases, with meiotic resumption requiring the participation of MAPK in the cumulus cells. It is interesting to note that denuded human oocytes matured in vitro exhibit a recurrent incidence of microtubule arrays typical of interphase in the oocyte cytoplasm with clear M-phase progression (Combelles et al., 2002
). Therefore, given the involvement of MAPK in regulating microtubule dynamics in mouse oocytes (Verlhac et al., 1994
, 1996
), one may speculate that a causal relationship exists between compromised MAPK activity and interphase microtubules in denuded human oocytes matured in vitro in microdrops. Also, oocytes cultured in microdrops exhibited a greater incidence of spontaneous activation after MII arrest (Combelles et al., 2002
; data presented herein); in contrast, oocytes co-cultured with cumulus cells in 3D collagen gels rarely exited MII arrest. Therefore, a certain threshold of MAPK activity may be required to maintain M-phase arrest after polar body extrusion in human oocytes; this is not surprising given the interplay between the MosMAPK pathway and the cytostatic factor (CSF)-induced arrest in MII (Kosako et al., 1994
; Fan and Sun, 2004
). Interplays are known to exist between MAPK and MPF activity in mammalian oocytes (Motlik et al., 1998
; Gordo et al., 2001
), a possibility that warrants further investigation in human oocytes. It is worth noting that diminished MAPK activity may be associated with an increased predisposition of oocytes to apoptosis; indeed, apoptosis may be regulated by MPF and MAPK levels (Fissore et al., 2002
) and furthermore, the role of the MosMAPK pathway in inhibiting apoptotic events was demonstrated in Xenopus egg extracts (Tashker et al., 2002
).
The kinetics of maturation also remains to be assessed in relation to cell cycle kinase activities using the novel co-culture system defined here for human IVM. It may be speculated that the pace of maturation is slowed down in the co-culture when compared to the microdrop system, although the exact influence of cumulus cells on the progression of oocyte maturation is unknown. Interestingly, previous studies in bovine oocytes indicate that the kinetics of maturation is faster when kinase levels are elevated (Fissore et al., 1996; Sakaguchi et al., 2002
).
One cannot disregard the inherent limitations of utilizing immature oocytes that failed to resume meiosis in vivo following gonadotrophin stimulation and retrieved from patients undergoing fertility treatment. Therefore, similar analyses must be undertaken with not only human oocytes from alternate sources but also oocytes from other animals, which may also provide the opportunity to compare mechanisms in vivo. In addition, while efforts were made in the design of the present co-culture system to mirror the physiological situation, the fact that MAPK levels were lower in oocytes matured in this system as compared to in vivo-matured oocytes indicates that deficiencies still remain. Given that diminished MAPK activity may be associated with normal microtubule organization and spindle function (Verlhac et al., 1996), spontaneous activation (Kosako et al., 1994
; Fan and Sun, 2004
; data presented herein), and/or apoptosis (Fissore et al., 2002
; Tashker et al., 2002
), all of which result in compromised oocyte developmental competence, the utilization of in vitro-matured human oocytes should not be encouraged until conditions are thoroughly tested.
Although additional studies first need to confirm a direct beneficial influence of ECM and a 3D environment on oocyte quality when using a co-culture approach, there is undoubtedly a need to continue optimizing culture conditions for human IVM. Additional improvements for the co-culture of cumulus cells and oocytes may include the use of extracellular matrices other than simple collagen gels. Indeed, the exact composition of the matrix influences cell function (Abbott, 2003), and while commercial ECM preparations, such as Matrigel, are readily available, the concept of using matrix components normally found in the cell system of interest in the body is gaining increased interest in other fields (Rosso et al., 2004
). A diversity of ECM molecules is present in ovarian follicles (Rodgers et al., 2003
), and proteoglycans, laminin, collagen IV and fibronectin are all present in the hyaluronan-rich matrix characteristic of cumulusoocyte complexes (Zhuo and Kimata, 2001
). With the technical challenges associated with designing and/or extracting the natural ECM of cumulusoocyte complexes, synthetic 3D products may represent another attractive alternative. Artificial 3D scaffolds are receiving a great deal of attention in tissue engineering applications, thereby permitting a superior level of control over the use of natural ECM components (Bouhadir and Mooney, 1998
). Alginate-based hydrogels have been applied to the culture of intact mouse granulosa celloocyte complexes (Pangas et al., 2003
), granulosa cells (Kreeger et al., 2003a
) and follicles (Kreeger et al., 2003b
). Indeed, one may envision constructing a 3D-engineered follicle in vitro with its multiple compartments by taking advantage of powerful and novel approaches now available.
Clinical IVM still awaits a breakthrough, and while the developmental competence of in vitro-matured oocytes using our 3D collagen co-culture system ultimately remains to be tested, we believe that this work opens exciting new avenues of research. This system may also prove valuable for use in the arrest of oocyte maturation with type-specific phosphodiesterase inhibitors (Nogueira et al., 2003); as a result, immature oocytes may be prepared and undergo prematuration events (Hendriksen et al., 2000
) in a controlled microenvironment that simulates physiological conditions, so that their subsequent developmental competence is improved.
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Acknowledgements |
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Submitted on August 17, 2004; accepted on December 21, 2004.