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Reproduction (2006) 132 549-557
DOI: 10.1530/rep.1.01066
Copyright © 2006 Society for Reproduction and Fertility
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RESEARCH

Abilities of cumulus and granulosa cells to enhance the developmental competence of bovine oocytes during in vitro maturation period are promoted by midkine; a possible implication of its apoptosis suppressing effects

S Ikeda, K Saeki1, H Imai and M Yamada

Laboratory of Reproductive Biology, Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan and 1 Department of Genetic Engineering, Kinki University, Kinokawa, Wakayama 649-6493, Japan

Correspondence should be addressed to M Yamada; Email: masayasu{at}kais.kyoto-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
We previously reported that when midkine (MK), a heparin-binding growth differentiation factor was used in in vitro maturation (IVM) culture of bovine cumulus-enclosed oocytes (CEOs), their developmental competence to the blastocyst stage after in vitro fertilization (IVF) was enhanced and the effect of MK might be mediated by its action upon mural granulosa cells and cumulus cells that closely surround the oocyte. In the present study, when denuded oocytes (DOs) were matured in IVM medium with or without MK (200 ng/ml) in the presence or absence of isolated cumulus cell masses and subjected to IVF, the enhancing effects of MK on the developmental competence of DOs to the blastocyst stage after IVF were exerted only in the presence of cumulus cells. In addition, we prepared the conditioned media of granulosa cells cultured with or without 200 ng MK/ml (CMMK+ or CMMK– respectively) and examined their effects on the IVM of DOs in terms of their developmental competence to the blastocyst stage after IVF. The supplementation of CMMK+ into IVM medium at 40% (v/v) significantly enhanced the blastocyst development compared with the no additive control and the CMMK– supplemented groups. Furthermore, the effects of MK during IVM of bovine CEOs on the cumulus cell apoptosis were investigated. CEOs were cultured up to 24 h in IVM medium without (control) or with 200 ng MK/ml. The genomic DNA was extracted from CEOs at 0, 6, 12, 18 and 24 h of IVM and subjected to ligation-mediated PCR (LM-PCR) to detect the apoptotic internucleosomal DNA fragmentation. DNA fragmentation was scarcely detected at the start of IVM, whereas it increased time-dependently as the IVM culture progressed. The degree of the fragmentation was significantly lower in the MK-treatment group compared with the control group at 18 and 24 h of IVM. The apoptosis-suppressing effect of MK on cumulus cells was further confirmed in situ by using TUNEL on CEOs. In conclusion, data from the present study further confirmed that MK enhances the developmental competence of bovine oocytes via cumulus and granulosa cells. It was also demonstrated that MK suppresses the apoptosis that occurs in cumulus cells during the period of IVM of bovine CEOs. The putative soluble factor(s) from cumulus cells was suggested from the experiment using CMMK+ . MK may promote the production of such factors in part by its anti-apoptotic effects on cumulus cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
In the course of growth and maturation of oocytes in vivo, follicular cells surrounding oocytes secrete various bioactive substances, some of which might affect the characteristics of the oocyte (Driancourt & Thuel 1998). Mammalian oocytes removed from the follicles can spontaneously undergo meiotic maturation in vitro, resulting in the formation of morphologically normal secondary oocytes arrested at the metaphase stage (MII) of the second meiotic division (Edwards 1965). However, post-fertilization development to the blastocyst stage of oocytes that have reached the MII stage in in vitro maturation (IVM) culture is generally less successful than that of oocytes matured in vivo (Leibfried-Rutledge et al. 1987, van de Leemput et al. 1999, Rizos et al. 2002). This is often attributed to the lack of physiological factors in IVM that regulate the acquisition of the developmental competence of oocytes during the maturation period. Several growth factors contained in the follicular fluid have been listed as developmental competence-enhancing factors, and include epidermal growth factor (Harper & Brackett 1993, Kobayashi et al. 1994, Lonergan et al. 1996), transforming growth factor-{alpha} (Kobayashi et al. 1994), insulin-like growth factor-I (Harper & Brackett 1992), activin A (Stock et al. 1997, Silva & Knight 1998), inhibin A (Stock et al. 1997), vascular endothelial growth factor (Einspanier et al. 2002, Luo et al. 2002), brain-derived neurotrophic factor (Kawamura et al. 2005, Martins da Silva et al. 2005) and midkine (MK) (Ikeda et al. 2000a, 2000b).

MK is one of the heparin-binding growth differentiation factors that is known to be quite rich (125 ng/ml) in bovine follicular fluid. We previously found that the addition of recombinant MK into IVM medium of bovine cumulus-enclosed oocytes (CEOs), enhanced the developmental competence of oocytes to the blastocyst stage after in vitro fertilization (IVF) and suggested that the MK effects are not exerted by its direct action upon the oocytes, but mediated through the cumulus cells in CEOs. However, the concrete action of MK upon cumulus cells remains unknown. Recently, we reported that cumulus cells undergo apoptosis during the IVM period and suggested the negative correlation between the developmental competence of CEOs and the degree of apoptosis of the cumulus cells during IVM (Ikeda et al. 2003). On the other hand, MK has been reported to have anti-apoptotic effects on certain types of cells, including neuronal (Owada et al. 1999a and 1999b) and tumour (Qi et al. 2000, Ohuchida et al. 2004) cells.

In the present study, the requirement of cumulus and granulosa cells for the enhancing effects of MK on the developmental competence of bovine oocytes was examined using the following IVM culture systems: (1) IVM of denuded oocytes (DOs) co-cultured with isolated cumulus cells and (2) IVM of DOs with granulosa cell derived conditioned medium prepared in the presence or absence of MK. Furthermore, the effects of MK on apoptosis of cumulus cells in CEOs during the IVM period were also investigated on the basis of our hypothesis that MK enhances the viability of the cumulus cells through its anti-apoptotic property.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
All chemicals, except where specified otherwise were purchased from Sigma.

Culture media
The washing medium for the collection and sampling of CEOs was modified PBS (mPBS) that contained 0.9 mmol CaCl2/l, 0.49 mmol MgCl2/l, 1.19 mmol NaHCO3/l, 0.33 mmol sodium pyruvate/l, 1.5 mmol glucose/l and 0.5 mg polyvinylalcohol/ml (PVA). The basal medium for IVM (IVM medium) was a modified synthetic oviduct fluid (mSOF) described by Ikeda et al. (2000a). This medium consisted of SOF (Tervit et al. 1972), 2% (v/v) basal medium Eagle amino acids solution, 1% (v/v) minimum essential medium non-essential amino acids solution, 0.5 mg PVA/ml, 1–2 µg oestradiol-17ß/ml and 100 IU human chorionic gonadotrophin/ml (Sankyo Co., Tokyo, Japan).

The medium for IVF was modified Tyrode’s balanced salt solution (BO) (Brackett & Oliphant 1975). The washing medium for frozen-thawed bull spermatozoa was BO supplemented with 10 mmol caffeine sodium benzoate/l and 20 µg heparin/ml (BO-1). The medium used for the co-incubation of spermatozoa and oocytes was a 50:50 (v/v) mixture of BO-1 and BO supplemented with 20 mg BSA/ml (BO-2). The medium for in vitro culture (IVC) of embryos after IVF (IVC medium) was the mSOF described above, supplemented with 1 or 5% (v/v) heat-inactivated foetal bovine serum (FBS; JRH Biosciences, Lenexa, KS) without PVA and hormones.

Preparation of MK
Recombinant bovine MK was produced by means of a baculovirus expression system as described previously (Ikeda et al. 2000a).

Preparation of denuded oocytes (DOs)
Ovaries collected from Japanese beef cattle at a local abattoir were transported to the laboratory within 3 h in saline (0.9% (w/v) NaCl) at 35–38 °C. Follicular contents were aspirated from follicles with a diameter of 2–5 mm and diluted with mPBS. The CEOs consisting of oocytes with homogeneous and evenly granulated cytoplasm and an intact cumulus cell mass were selected from the follicular contents. To prepare DOs, groups of ~100 CEOs were transferred into microcentrifuge tubes with 100 µl mPBS and cumulus cells were removed from the CEOs by vortexing for about 5 min.

Experiment 1: Effects of MK in the presence or absence of isolated cumulus cells during IVM of DOs on their post-fertilization development
IVM medium drops containing a cumulus cell mass were prepared as follows: ten CEOs were introduced into 50 µl drops of IVM medium with or without 200 ng MK/ml under mineral oil. Oocytes were mechanically removed from the cumulus mass by pipetting and only the cumulus mass was left in the drop. Ten to twelve DOs prepared as described above were introduced into the drop and matured with or without MK in combination with the presence or absence of cumulus mass (see Fig. 1Go) at 39 °C under 5% CO2 in air for 24 h. To estimate the number of cells per drop, cumulus cells in ten CEOs were counted as follows. Ten CEOs were vortexed in 100 µl mPBS as described earlier, so that completely denuded oocytes and suspension of dispersed cumulus cells were prepared. The numbers of cumulus cells in suspension were calculated using haemocytometer. The cell number from ten CEOs was calculated as 8 x 104. The DOs after IVM culture were subjected to the IVF and IVC as described elsewhere (Ikeda et al. 2000a, 2000b). Briefly, after thawing frozen bull semen, spermatozoa were washed with BO-1, resuspended at a concentration of 4–5 x 106/ml in BO-2 and prepared as 100 µl suspension. Groups of 10–12 DOs after IVM were transferred into the sperm suspension and incubated for 6 h. Thereafter, oocytes were transferred into 50 µl drops of IVC medium containing 1% (v/v) FBS and cultured at 39 °C under 5% CO2, 5% O2 and 90% N2. At 48 h post-insemination, the morphologically normal cleaved embryos were transferred to IVC medium containing 5% (v/v) FBS and cultured until day 8 (fertilization = day 0). The cleavage rates were assessed at 48 h post-insemination. The percentages of embryos reaching the blastocyst stage were recorded on day 8.


Figure 1
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  		Figure 1 Bovine denuded oocytes (DOs, A) and DOs with isolated cumulus cell mass (B) at the beginning of IVM culture. Bar represents 150 µm.

 
Experiment 2: Effects of conditioned media of granulosa cells cultured with or without MK
The granulosa cells obtained from small follicles at CEO collection were washed in mPBS twice by centrifugation at 200 g for 5 min. After the second centrifugation, the granulosa cells were resuspended in 10 ml IVM medium and then washed using centrifugation. After the supernatant was discarded, the granulosa cell pellet was resuspended in 5 ml IVM medium with or without 200 ng MK/ml and cultured for 24 h at 39 °C under 5% CO2 in air in a 15 ml plastic tube. To estimate the number of cells per tube, GC pellet was dispersed by vortexing and cell number was calculated by haemocytometer. Cell density was adjusted to 4 x 106 cells/ml. After culturing, the granulosa cells were sedimented by centrifugation at 1500 g for 20 min. The supernatant was filtered and stored at – 80 °C until use. The medium conditioned by granulosa cells in the presence or absence of MK was designated as CMMK+ or CMMK– respectively. Ten to twelve DOs were introduced into the 50 µl drop of IVM medium containing 10, 20, 40 and 50% (v/v) of CMMK+ or CMMK– and 40% (v/v) CMMK– containing IVM medium supplemented with 200 ng MK/ml and cultured for 24 h at 39 °C under 5% CO2 in air. No additive group served as control. Sixty to hundred and twenty-two oocytes in each group were examined in three replicates. The DOs after IVM culture were subjected to IVF and IVC and the developmental rates were recorded as in experiment 1.

Experiment 3: Effects of MK on apoptosis of cumulus cells in CEOs during IVM culture
Ten CEOs collected as described earlier were introduced into a 50 µl drop of IVM medium with or without 200 ng MK/ml under mineral oil. CEOs were cultured for 0, 6, 12, 18 and 24 h at 39 °C under 5% CO2 in air. The detection of the nucleosomal DNA ladder of apoptotic cumulus cells was performed using an ApoAlert ligation-mediated PCR (LM-PCR) ladder assay kit (Clontech) as reported previously (Ikeda et al. 2003). The LM-PCR assay has been reported to be semiquantitative, allowing the comparison of the relative extent of apoptosis in different samples (Staley et al. 1997). The individual groups of ten CEOs were washed with mPBS and transferred into microcentrifugal tubes with 50 µl of mPBS. The tubes, each containing ten CEOs, were immersed in liquid nitrogen and stored at – 80 °C until DNA extraction. After thawing, the CEOs were lysed by adding 50 µl of two times strengthened lysis buffer (20 mmol Tris–HCl/l, 200 mmol NaCl/l, 50 mmol EDTA/l, 1% (w/v) SDS, 0.2 mg proteinase K/ml, pH 8.0) and incubating at 50 °C for 18 h by gentle shaking. The DNA was extracted by the phenol/chloroform/isoamyl alcohol (25:24:1, v:v:v, PCI) method and ethanol precipitated. The DNA pellets were dried and resuspended in 20 µl TE buffer (10 mmol Tris–HCl/l, 1 mmol EDTA/l, pH 8.0). This solution (20 µl) contained DNA corresponding to eight CEOs. To verify the equality of the amounts of the DNA extracted, PCRs upon the ß-actin gene as an internal control were performed using the DNA solution. Two microlitres of DNA solution (corresponding to 0.8 CEO) were mixed in a 50 µl reaction mixture containing 200 µmol dNTPs/l, 2.5 U Ex-Taq DNA polymerase (Takara, Japan) and a pair of primers (0.25 µmol/l) specific for the ß-actin gene and amplified for 30 cycles of 30 s at 94 °C, 30 s at 60 °C and 1 min at 72 °C. DNA corresponding to three CEOs was mixed with 1 nmol each of 24 bp (5'-AGCACTCTCGAGCCTCTCACCGCA-3') and 12 bp (5'-TGCGGTGAGAGG-3') unphosphorylated oligonucleotides in a 49 µl reaction volume with 1 x Ligation mix (Clontech). The oligonucleotides were annealed by heating to 55 °C for 10 min and cooling gradually to 10 °C over 1 h followed by incubation at 10 °C for 10 min. Four hundred units (1 µl) of T4 DNA ligase (Clontech) were added and ligation was performed at 16 °C for 16 h. This procedure allowed the unphosphorylated adaptors to ligate to the 5'-phosphorylated blunt ends of the DNA fragments generated during the IVM culture. Adaptor-ligated DNA solution (50 µl) was stored at – 20 °C until PCR was performed.

Adaptor-ligated DNA solution (8 µl) corresponding to half of a CEO was mixed with 5 µl 10 x LM-PCR mix (Clontech) containing the 24 bp oligonucleotides (linker primer) and 36.5 µl water in a PCR tube. The tube was heated to 72 °C for 3 min followed by the addition of 2.5 U (0.5 µl) of Ex-Taq DNA polymerase and additional incubation at 72 °C for 5 min to fill in the 5' protruding ends of the ligated DNA. The 24 bp oligonucleotide could now serve as a primer and the DNA fragments with adaptors on both ends could be exponentially amplified. PCRs were performed for 24–26 cycles of 1 min at 94 °C and 3 min at 72 °C. All the PCR products (50 µl) were subjected to electrophoresis through 2% (w/v) agarose gels containing 0.8 µg ethidium bromide/ml. After electrophoresis for 60 min at 8 V/cm in Tris–borate/ EDTA buffer, the gels were photographed on a UV transilluminator.

The band intensities of the PCR products were measured by densitometry using a model 4.0 Atto densitograph (Atto, Tokyo, Japan). The intensity of ladder-like bands derived from the apoptotic DNA fragments (< 1 kb) was expressed relative to the intensity of the band for ß-actin. The relative intensity for the onset of IVM (0 h) was subtracted from that for each time point and the difference was designated as the apoptotic index. Experiments were repeated at least twice for each time point in each group.

Effects of MK on cumulus cell apoptosis were further examined using TUNEL assay. After the 24 h IVM without (control) or with 200 ng MK/ml, CEOs were fixed in 4% (w/v) paraformaldehyde in PBS and permeabilised with PBS containing 0.5% (v/v) Triton-X 100. After washing with PBS containing 0.01% (w/v) PVA (PBS–PVA), the samples were incubated for 1 h at 37 °C in a TUNEL reaction mixture (In situ Cell Death Detection Kit, Roche). The negative control did not contain TdT, while the positive control was subjected to TUNEL after treatment with 100 U DNase I/ml for 1 h at 37 °C. After the TUNEL reaction, all the nuclei were stained with 0.001% (w/v) Hoechst 33342 diluted in PBS–PVA for 5 min. The CEOs were mounted onto a slide with a droplet of VECTASHIELD Mounting Medium (Vector Laboratories, Burlingame, CA) and flattened with a coverslip. Slides were examined under a Carl Zeiss Axiophot2 microscope (Oberkochen, Germany) equipped with No. 2 filter (excitation 365 nm, emission 420 nm for Hoechst stain) and No. 10 filter (excitation 450–490 nm, emission 515–565 nm for TUNEL assay). The fluorescence images were then analysed with AQUACOSMOS system (Version 2.6, Hamamatsu Photonics, Hamamatsu, Japan). The TUNEL index was calculated as total intensity of TUNEL staining divided by that of Hoechst staining per CEO. Thirty CEOs in each group from three replicates were examined.

Statistical analysis
The statistical analyses of data were performed using the StatView4.02 (Abacus Concepts, Berkeley, CA, USA). Developmental percentages were arcsine transformed to stabilize the variances and subjected to one-factor ANOVA followed by post hoc Fisher’s PLSD (experiment 1) or Tukey’s test (experiment 2) to detect significant differences among the treatments. In experiment 1, data were further subjected to 2-factor ANOVA to detect the overall effect of each factor (presence or absence of MK and cumulus cells) and interaction between them. In experiment 3, apoptotic and TUNEL indexes were subjected to t-test. Significance was accepted at P< 0.05. All data were expressed as mean ± S.E.M.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
In experiment 1, DOs were matured in IVM medium supplemented with or without 200 ng MK/ml in the presence or absence of cumulus cell mass and subjected to IVF and IVC. The percentages of embryos that developed to the cleavage and blastocyst stages in each treatment group are shown in Table 1Go. More than 60% of the oocytes were cleaved and the rates were not significantly different between the treatments. In the absence of cumulus cells, the blastocyst rates were low regardless of MK addition, but the rates increased by MK addition in the presence of cumulus cells. The overall effects of MK and cumulus cells during IVM on each developmental rate and the interaction between the two factors (MK and cumulus cells) were not significant with regard to cleavage rates, but significant regarding blatocyst rates.


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  		Table 1 Effects of MK in combination with the presence or absence of isolated cumulus cells during IVM of bovine DOs on post-fertilization development.
 
In experiment 2, DOs were matured in IVM medium containing various dosages of CMMK+ or CMMK– and in 40% (v/v) of CMMK– containing IVM medium supplemented with 200 ng MK/ml, followed by IVF and IVC. The developmental rates are shown in Fig. 2Go. When the DOs were matured in basic IVM medium, their developmental rates to blastocysts after IVF were very low (1.5% for IVM/IVF oocytes and 3.0% for cleaved oocytes). Forty percent (v/v) of CMMK+ significantly enhanced the blastocyst development compared with the same dose of CMMK– . The most effective dose of CMMK+ was 40% (12.3% for IVM/IVF oocytes and 23.4% for cleaved oocytes). The 40% (v/v) CMMK– supplementation showed no significant effect on the blastocyst yield, even if MK was added. From the results of experiment 1 and 2, it was suggested that the enhancing effects of MK on developmental competence are mediated through its action on cumulus and granulosa cells. We therefore propose that MK may enhance the viability of cumulus cells in CEOs by its anti-apoptotic property.


Figure 2
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  		Figure 2 Effects of conditioned media of granulosa cells cultured with or without MK (CMMK+ or CMMK– respectively) on the IVM of denuded oocytes in terms of (A) development to the cleavage stage, (B) blastocyst yield from tested oocytes and (C) blastocyst yield from cleaved embryos after in vitro fertilization. Data are presented as mean with the S.E.M. from three replicates. Numbers of oocytes examined per group are indicated in the column. Values without common superscripts differ significantly (P< 0.05). NONE and MK mean no additive control and midkine respectively.

 
In experiment 3, the MK action on the apoptosis of cumulus cells was investigated. Figure 3Go shows the results of LM-PCR using genomic DNA from bovine CEOs matured in serum-free IVM medium with or without MK addition. The ladder-like PCR products in CEOs were hardly detectable at 0 h of IVM, while they increased after 12 h of culture and toward the end of IVM (24 h), irrespective of MK addition. The sizes of the ladders observed were, as expected, 185 bp multiples with additional 24 bp oligonucleotides at each DNA end corresponding to the apoptotic internucleosomal fragments (Ikeda et al. 2003). When compared at each time point, the apoptotic index was significantly lower in the MK-treatment group at 18 and 24 h of IVM than in the control. No ladder could be obtained from the oocytes that were freed from CEOs after 24 h of culturing (data not shown).


Figure 3
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  		Figure 3 (A) LM-PCR analysis of internucleosomal DNA fragmentation in CEOs during IVM culture with or without MK (200 ng/ml) addition. PCR products derived from adaptor-ligated DNA corresponding to half of a CEO were subjected to electrophoresis and then photographed. The lower panel shows a control PCR assay for the ß-actin gene to confirm that equivalent amounts of DNA were analysed. 1–3 nuc: the PCR ladder derived from 1–3 nucleosomal units of DNA fragments. Ctrl. and MK mean no additive control and MK-treated group respectively. (B) Apoptotic index calculated from the results of the LM-PCR during IVM culture. Numbers in parentheses indicate the number of replications for each value. Different letters (a and b) depict significant differences between the treatments at the same time point (P< 0.05).

 
The effects of MK on apoptosis of cumulus cells were further examined in situ by means of TUNEL (Fig. 4Go). TUNEL index was calculated as intensity of TUNEL staining divided by that of all DNA staining (Hoechst) in CEOs, which had been IVM cultured in the presence or absence (control) of MK for 24 h. MK added into IVM medium significantly reduced apoptotic index compared with no additive control (0.226 ± 0.00236 vs 0.181 ± 0.00143, P = 0.0035).


Figure 4
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  		Figure 4 (A) A representative image of bovine cumulus enclosed oocytes (CEOs) after IVM (24 h) subjected to TUNEL (green label) to detect DNA fragmentation. All cell nuclei are also stained by Hoechst (blue). Scale bar represents 50 µm. (B) TUNEL index calculated as intensity of TUNEL staining divided by that of Hoechst staining in the control and MK-treatment group. Numbers in parentheses indicate the number of CEOs subjected to the analysis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
MK was first identified as a product of a retinoic acid-inducible gene in a teratocarcinoma cell line (Kadomatsu et al. 1988), which forms a unique family with pleiotrophin, also known as HB-GAM (Rauvala 1989, Li et al. 1990). MK was reported to be rich (125 ng/ml) in bovine follicular fluid (Ohyama et al. 1994). Furthermore, it was reported that follicular granulosa cells produced MK under the control of gonadotrophin (Karino et al. 1995, Minegishi et al. 1996), suggesting its role in follicular development, including oocyte growth and maturation.

We previously reported that the recombinant MK added into the IVM medium of bovine oocytes enhanced their developmental competence to the blastocyst stage after IVF (Ikeda et al. 2000a, 2000b). In the previous study, since the enhancing effects of MK on the developmental competence of DOs were exerted only in the presence of isolated granulosa cells, we suggested that the MK effects are mediated by granulosa cells rather than by a direct action on oocytes (Ikeda et al. 2000b). In experiment 1, in the present study, we investigated the effects of MK on the IVM of DOs in the presence or absence of isolated cumulus cells that are a sub-population of granulosa cells and demonstrated that MK exerted its developmental competence-enhancing effects only under the presence of cumulus cells (Table 1Go). This finding confirmed the involvement of follicular cumulus and granulosa cells in MK action on oocytes during the maturation period.

Furthermore, in experiment 2, the granulosa cell-conditioned medium prepared in the presence of MK (CMMK+ ) also enhanced the developmental competence of DOs (Fig. 2Go). The inability of MK added into CMMK– containing IVM medium to promote the developmental competence indicates that the enhancing effect of CMMK+ during IVM is not due to MK included in CMMK+ in the preparation of the conditioned medium. In experiment 1, the addition of cumulus cells into IVM medium did not affect the developmental competence of DOs to the cleavage stage (Table 1Go). On the contrary, even with no statistical significance, the granulosa cell-conditioned medium at 40% (v/v) increased the cleavage rate compared to the no addition group, consistent with the previous study, in which, addition of granulosa cells into IVM culture of DOs had a significant effect on the cleavage rate (Ikeda et al. 2000b). The difference in the cleavage rates of DOs between the effects of cumulus cells and of granulosa cells may be due to the mutually unique function of these cells (Gilchrist et al. 2004). Whatever the uniqueness, the results of experiment 2 further confirmed the MK action via cumulus and granulosa cells.

These findings prompted us to explore the action of MK on the cumulus and granulosa cells. We previously showed that apoptosis occurs in cumulus cells during IVM of CEOs (Ikeda et al. 2003). We tentatively refer to this phenomenon as IVM-induced apoptosis because apoptosis is unlikely to occur in cumulus cells during natural (i.e. in vivo and not artificially stimulated) oocyte maturation (see the discussion in the reference, Ikeda et al. 2003). It was also found that apoptosis during IVM was induced irrespective of serum addition, although serum has been shown to protect cultured granulosa cells from apoptosis (Hu et al. 2001, Johnson et al. 2001). On the other hand, MK has been reported to have anti-apoptotic effects on certain types of cells, including neuronal (Owada et al. 1999a, 1999b) and tumour (Qi et al. 2000, Ohuchida et al. 2004) cells. In the present study, we demonstrated for the first time that MK could suppress the apoptosis that occurred in cumulus cells during the IVM of bovine CEOs (Figs 3Go and 4Go). It was revealed from the results that the enhancing effects of MK on the developmental competence of bovine CEOs could be exerted in part by its effect of improving the viability of cumulus cells. After we reported that cumulus cells undergo apoptosis during the IVM period and proposed the negative correlation between the degree of cumulus cell apoptosis during IVM and the developmental competence of CEOs (Ikeda et al. 2003), supportive results were successively published (Alisch et al. 2003, Corn et al. 2005, Leroy et al. 2005, Yuan et al. 2005).

The results from experiment 2 also suggest that MK exerts its enhancing effects via soluble factor(s) from cumulus and granulosa cells. Recently, Luciano et al.(2005) found that the addition of intact CEOs during IVM of DOs could restore their developmental competence to the blastocyst stage and reached the presumption of diffusible factor(s) from cumulus cells, which enhances the developmental competence.

So far, many reports have suggested the importance of the presence of cumulus and granulosa cells during IVM culture in terms of the developmental competence of oocytes (Zhang et al. 1995, Kim et al. 1997, Hashimoto et al. 1998, Luciano et al. 2005), and the importance may at least be attributed to soluble factor(s), as mentioned earlier. It is therefore suggested that MK promotes the production of such soluble factor(s) in cumulus and granulosa cells. MK has been reported to activate a cell-surface receptor, resulting in the activation of a transcription factor, STAT1{alpha} (Ratovitski et al. 1998). The activation of such transcription factor(s) by MK signal transduction may be directly involved in the expression of the developmental competence enhancing factor(s) at transcriptional levels. Alternatively, MK may promote the production of the enhancing factor(s) as a consequence of inhibition of the cumulus cell death by its anti-apoptotic effects elucidated in this study.

In conclusion, data from previous and the present study indicate that MK enhances the developmental competence of oocytes via surrounding cumulus and granulosa cells and suppresses the cumulus cell apoptosis that occurs spontaneously in bovine CEOs during the IVM period. The putative soluble factor(s) from cumulus and granulosa cells was suggested and MK may promote the production of such factors in part by its anti-apoptotic effects on cumulus cells.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
The authors thank the staff at the Second Wholesale Markets of Kyoto City, especially K Naka and T Ohnisi, for allowing access to bovine ovaries. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan and by a grant from Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the Japan Science and Technology Corporation. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.


   Footnotes
 
Received 12 December 2005
First decision 8 February 2006
Revised manuscript received 9 June 2006
Accepted 20 June 2006

S Ikeda is now at Department of Genetic Engineering, Kinki University, Kinokawa, Wakayama 649-6493, Japan


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

Alisch A, Ruping K, Koster F, Schopper B, Baum M, Finas D, Felberbaum R, Dor S, Al-Hasani S & Diedrich K 2003 Cumulus cell apoptosis as a predictor for oocyte quality in artificial reproduction technique. Zentralblatt fur Gynakologie 125 452–457.[CrossRef][Medline]

Brackett BG & Oliphant G 1975 Capacitation of rabbit spermatozoa in vitro. Biology of Reproduction 12 260–274.[Abstract]

Corn CM, Hauser-Kronberger C, Moser M, Tews G & Ebner T 2005 Predictive value of cumulus cell apoptosis with regard to blastocyst development of corresponding gametes. Fertility and Sterility 84 627–633.[CrossRef][Web of Science][Medline]

Driancourt MA & Thuel B 1998 Control of oocyte growth and maturation by follicular cells and molecules present in follicular fluid. Reproduction, Nutrition, Development 38 345–362.[Web of Science]

Edwards RG 1965 Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature 208 349–351.[CrossRef][Medline]

Einspanier R, Schonfelder M, Muller K, Stojkovic M, Kosmann M, Wolf E & Schams D 2002 Expression of the vascular endothelial growth factor and its receptors and effects of VEGF during in vitro maturation of bovine cumulus–oocyte complexes (COC). Molecular Reproduction and Development 62 29–36.[CrossRef][Web of Science][Medline]

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