Hum. Reprod. Advance Access originally published online on September 13, 2007
Human Reproduction 2007 22(11):2842-2850; doi:10.1093/humrep/dem277
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Altered composition of the cumulus-oocyte complex matrix during in vitro maturation of oocytes
Discipline of Obstetrics and Gynaecology, Research Centre for Reproductive Health, The University of Adelaide, Adelaide, South Australia 5005, Australia
1 Correspondence address. Tel: +61-8-8303-4096; Fax: +61-8-8303-4099; E-mail: darryl.russell{at}adelaide.edu.au
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Abstract |
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BACKGROUND: In vitro maturation (IVM) of mammalian oocytes has potential health benefits for patients undergoing assisted reproduction as an alternative to gonadotrophin treatment. This procedure is also useful for studying the process of oocyte and early embryo development. However, oocytes undergoing IVM have much lower competence than in vivo matured oocytes. Efforts to optimize IVM success have focused on replicating in vivo timing, hormonal milieu and cumulus cell responses associated with maturing oocytes. We have previously identified two extracellular matrix proteins, the protease Adamts1 and hyaluronan-binding proteoglycan Versican, produced by mural granulosa cells that selectively incorporate into the periovulatory cumulus-oocyte complex (COC).
METHODS: Murine COC were cultured in the presence of epidermal growth factor and/or FSH. mRNA and protein were measured by real time PCR and Western blot and compared to in vivo derived COC.
RESULTS: COCs from mice that underwent IVM for 6 or 20 h in the presence of epidermal growth factor, FSH or in combination had a > 10-fold reduction in mRNA (P < 0.05) for Adamts1 and Vcan when compared with in vivo matured COCs. Hyaluronan synthase 2 expression was up-regulated up to 8-fold (P < 0.05) over the unstimulated control, demonstrating successful induction of cumulus gene expression by the IVM conditions. While in vivo matured COCs showed abundant levels of these proteins, COCs that underwent IVM had neither detectable Adamts1, nor intact or Adamts1-cleaved Vcan. Human cumulus and granulosa cells matured in vivo contained abundant mRNA for Adamts1 and Vcan, demonstrating the potential relevance to human IVM.
CONCLUSION: These results indicate that extensively altered COC matrix composition is present during IVM and may contribute to the observed poorer competence of the derived oocytes.
Key words: oocyte maturation/ovarian function/cumulus cells/mural granulosa cells/gene regulation
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Introduction |
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In all mammals, the LH surge initiates a sequence of events required to coordinate ovulation and maturation of the oocyte (Richards et al., 2002
). One of these is cumulus expansion, whereby synthesis of hyaluronan (HA) and several interacting extracellular matrix (ECM) proteins produces a unique visco-elastic matrix enveloping the cumulus-oocyte complex (COC) (Russell and Salustri, 2006). The cumulus cell matrix is essential for successful ovulation and fertilization (Russell and Robker, 2007) as exemplified by disruptions to these processes in response to null mutation of genes that either express or regulate expression of COC matrix components in mice (Lim et al., 1997; Robker et al., 2000; Zhuo et al., 2001; Varani et al., 2002; Ochsner et al., 2003). The degree of cumulus matrix expansion has also been correlated with oocyte developmental competence (Ball et al., 1983; Allworth and Albertini, 1993; Chen et al., 1993; McKenzie et al., 2004).
Formation of the COC matrix requires synthesis of HA by cumulus cells. The cumulus cell expression of a number of matrix genes is dependent on oocyte secreted growth factors in addition to induction by LH. Hyaluronan synthase 2 (Has2) is regulated in this way (Tirone et al., 1997; Su et al., 2004; Dragovic et al., 2005) and its expression has been correlated with developmental competence of the oocyte. Versican (Vcan) is a widely expressed HA-binding proteoglycan that has distinctive expression and regulation in the ovary. Vcan mRNA has been localized to mouse mural granulosa cells (mGCs) after LH stimulation, while the secreted protein localizes to the ECM of expanding COC (Russell et al., 2003b). Vcan may function in the COC by binding HA via its N-terminal link protein-domain, while the C-terminal domains interact with cell surface proteins including sulphated glycolipids (Miura et al., 1999), heparan sulphate prostaglandins (Ujita et al., 1994), growth factor receptors (Xiang et al., 2006) and integrins (Wu et al., 2002).
Vcan is a substrate for the protease Adamts1 (a disintegrin and metalloproteinase with thrombospondin motifs 1) (Sandy et al., 2001), and Adamts1 mediated Vcan cleavage occurs in ovulating mouse COC (Russell et al., 2003a). Adamts1 cleavage of Vcan in the mouse COC accounts for > 60% of all proteolytic activity, while Adamts4 may be responsible for the remaining cleavage (Russell et al., 2003a). The expression of Adamts1 mRNA is induced in mGCs in response to the LH surge in mice (Robker et al., 2000), rat (Espey et al., 2000), cow (Madan et al., 2003) and mare (Boerboom et al., 2003), and is strongly expressed in normal human ovaries (Jansen et al., 2004). Adamts1 deficient COCs also show significantly reduced rates of fertilization (H.M. Brown et al., manuscript in preparation). Thus Vcan in its intact and/or cleaved forms may alter the functional properties of the COC matrix during oocyte maturation, ovulation and/or fertilization.
In human assisted reproduction therapies, in vitro maturation (IVM) of oocytes is an appealing alternative to exogenous gonadotrophin stimulation as it reduces the expense and risks associated with infertility therapies (Wang and Gill, 2004). However, the use of IVM has limitations as oocytes matured in vitro are of poorer quality (Wang and Gill, 2004). When compared with in vivo matured oocytes, embryos derived from IVM oocytes have decreased cleavage rates, increased levels of embryo growth retardation and poor implantation and pregnancy rates (Greve et al., 1987; Barnes et al., 1996; Goud et al., 1998; Kim et al., 2000; Trounson et al., 2001; Wang and Gill, 2004). The cumulus complex surrounding oocytes is important to oocyte maturation and embryogenesis (Goud et al., 1998; Atef et al., 2005). Embryo quality from IVF of in vivo matured oocytes is correlated with cumulus expression of several ECM genes (McKenzie et al., 2004; Zhang et al., 2005). Cumulus cells also control critical energy metabolites in the oocyte environment (Eppig, 2005; Sugiura et al., 2005), and limit exposure to excessive glucose which is detrimental to oocyte quality (Hashimoto et al., 2000; Colton et al., 2002; Sutton et al., 2003). Thus, the microenvironment maintained by cumulus cells and their ECM influence oocyte maturation during IVM.
We examined the hypothesis that the COC matured in vitro is deficient in components secreted from mGCs that normally form a part of the microenvironment of maturing oocytes. To determine whether Vcan and Adamts1 are deficient in COCs undergoing IVM, we analysed their mRNA expression and protein levels both during in vivo maturation and IVM of mouse COCs. Additionally, we determined whether these matrix components are normally present in human cumulus and mGCs following in vivo maturation.
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Materials
Equine chorionic gonadotropin (eCG/Gestyl) was purchased from Professional Compounding Centre of Australia, (Sydney, NSW). HCG (Pregnyl) was purchased from Organon, Australia (Sydney, NSW). Chondroitinase ABC was from Seikagaku (East Falmouth, MA). Secondary antibody (horse-radish peroxidase labelled goat-anti-rabbit) was purchased from Millipore Corporation (Billerica, MA). Plastic culture dishes were purchased from SARSTEDT Australia Pty. Ltd. (Adelaide, South Australia). Culture media was purchased from GIBCO, Invitrogen Australia Pty. Ltd. (Victoria, Australia).
Isolation and culture of mouse COCS
All mice (F1 C57Bl/6 X CBA) were maintained on a 12 :12 h day/night cycle with rodent chow and water provided ad libitum. All murine experiments were approved by the University of Adelaide’s ethics committee and were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. For IVM, COCs were isolated from pre-pubertal 23-day-old mice 44–48 h after i.p. injection of 4 IU eCG. COCs were washed in complete mimimum essential medium (MEM) [alpha MEM media supplemented with 5% (v/v) fetal calf serum, 0.25 mM sodium pyruvate (GIBCO, Invitrogen Australia Pty. Ltd), penicillin G (100 U/ml) and streptomycin sulphate (100 mg/ml)] and cultured in groups of 30 at 37°C in 5%CO2, 95% air for either 6 or 20 h in drops of 100 µl of complete MEM media with suitable treatments and overlaid with sterile mineral oil (Sigma-Aldrich Pty. Ltd., Castle Hill, NSW, Australia). Culture treatments involved media supplemented with either 50 mIU/ml recombinant human FSH (Sigma-Aldrich Pty. Ltd) or 3 ng/ml epidermal growth factor (EGF) (Sigma-Aldrich Pty. Ltd) or both EGF and FSH at these doses.
Expression analysis was performed following 6 or 20 h of IVM culture. After 6 h, the expression of Adamts1, Vcan and Has2 has previously been shown to be strongly up-regulated in the ovary (Fulop et al., 1997; Robker et al., 2000; Ochsner et al., 2003; Russell et al., 2003b), while 20-h IVM culture has previously been shown to be the time for completion of oocyte maturation in vitro (Eppig, 1979; Downs, 1989). Groups of 30 COCs were harvested for RNA isolation after 6 or 20 h of culture. Gene expression at these times was compared with in vivo matured COCs obtained following 6, 12 (preovulatory) or 16 h (postovulatory) hCG treatment. In vivo matured COCs were obtained from pre-pubertal mice treated with i.p. administration of eCG (4 IU) followed after 44 h by hCG (5 IU) and collected after 6 or 12 (preovulatory) or 16 h (postovulatory). Preovulatory mGC and COCs were collected by puncture of large antral follicles with a 26-gauge needle to release cells. COCs were cleaned of large adherent masses of mGCs before collection for RNA or protein isolation. Ovulated COCs (16 h after hCG) were isolated by puncture of the ampulla of the oviduct. Corresponding mGCs were also collected for RNA or protein isolation by puncture of the large follicles. Cell pellets were snap frozen and stored at –80°C for later mRNA or protein extraction and PCR or western blot analysis, as described below.
Assessment of cumulus expansion
The degree of cumulus expansion of in vitro matured oocytes was assessed in 20-h IVM cultures by two independent blinded assessors using the scale previously described (Vanderhyden et al., 1990; Dragovic et al., 2005). Briefly, a score of 0 indicates no expansion of cumulus cells; +1 the most outer layers of cumulus cells expand; +2 expansion of the outer half of cumulus cells; +3 all layers expanded except the corona radiata; +4 expansion of all layers of cumulus cells. For each treatment a mean cumulus expansion index (CEI) (0.0–4.0) was calculated.
Isolation of human cumulus and granulosa cells
Ethical approval for the use of human samples was obtained from the Women’s and Children’s Hospital Human Research Ethics Committee, Adelaide, Australia. Written consent for the use of cumulus and mGCs was obtained from couples undergoing infertility treatment at Repromed, Dulwich, South Australia. Patients with clinical indications of polycystic ovary syndrome were not included in this study. Of the 14 patients included in the study (average age 34.5 ± 3.7 years), infertility was attributed to male factor (n = 5), tubal defects (n = 4), or unexplained (n = 5). When patients began their menstrual cycle, they were administered s.c. daily FSH (150–300 IU, Gonal-F, Serono or Puregon, Organon) and following 8–14 days, patients with a minimum of three follicles of > 18 mm diameter observed by transvaginal ultrasound scans were scheduled for oocyte retrieval. At this point, women were administered 10 000 IU hCG (Pregnyl, Organon) s.c. and after 36 h follicles were aspirated transvaginally. Using this procedure all patients yielded 8–23 COCs. All patients had successful fertilization, with an average 60 ± 30% of oocytes fertilized (76% for ICSI n = 5, or 52% for IVF n = 9). Fifty percent of patients achieved pregnancy from a single-embryo transferred in this round of treatment, indicated by fetal heartbeat after 8-weeks. We obtained matched cumulus and mGC pairs from follicular aspirates at the time of oocyte retrieval. Cumulus cells were isolated from COCs by either manual trimming of the outer cumulus cell mass before IVF (n = 9), or hyaluronidase treatment of COCs before ICSI for the five patients with male factor infertility These different cell collection regimens showed no significant differences in the expression of Vcan, Adamts1 or Has2. Mural and cumulus cells were stored as cell pellets at –80°C until RNA isolation was performed.
Real time RT–PCR
Total RNA was isolated using Trizol (Invitrogen Australia Pty. Ltd), as per manufacturer’s instructions, with the inclusion of 7.5 µg Blue Glycogen (Ambion Inc., Austin, TX, USA) during precipitation. Total RNA was then treated with 1U of DNase as per manufacturer’s instructions (Ambion Inc). First-strand complementary DNA (cDNA) was synthesized from total RNA (350 or 400 ng) using random hexamer primers (Geneworks, Hindmarsh SA, Australia) and Superscript III reverse transcriptase (Invitrogen Australia Pty. Ltd).
Specific gene primers for real time RT–PCR were designed against published mRNA sequences (NCBI Pubmed database) using Primer Express software (PE Applied Biosystems, Foster City, CA) and synthesized by Sigma Genosys (Sigma-Aldrich Pty. Ltd). Primer pairs and sequences for murine Adamts1, Vcan, Has2 and RpL19, and human Adamts1, Vcan, Has2 and RPL19 are listed in Table 1. Primer sequences that detect human V1 Vcan splice variant and human Adamts1 were described previously (Corps et al., 2004; Cross et al., 2005). Real time RT–PCR was performed in triplicate for each sample on an ABI GeneAmp 5700 sequence detection system (PE Applied Biosystems). In each reaction, 2 µl of cDNA (equivalent to 10 ng of total RNA), 0.2 µl of forward and reverse primers and 10 µl of SYBR Green master mix were added, and H2O was added to a final volume of 25 µl. All primers were used at a concentration of 50 µM with the exception of human V1 Vcan primers, which were used at 25 µM, and human Adamts1, which were used at 6.25 µM. PCR cycling conditions were 50°C for 2 min, 95°C for 10 min, followed by 40 amplification cycles of 95°C for 15 s and 60°C for 1 min. Controls included omission of the cDNA template or RT enzyme in otherwise complete reaction mixtures; each showed no evidence of product amplification or primer dimers. For mouse and human samples, gene expression was normalized to the RpL19-internal control. All values were then expressed relative to calibrator samples using the 2–(
CT) method (K. Livak PE-ABI, Sequence Detector User Bulletin 2) (Livak and Schmittgen, 2001). Results for each PCR were normalized to the calibrator, which was given the arbitrary value of 1. For murine Vcan and Adamts1 real time RT–PCR, the calibrator was RNA from mGCs after eCG + hCG 12-h in vivo treatment. For Has2, the calibrator was RNA from COCs after eCG + hCG 6-h in vivo treatment. For human V1 Vcan, Adamts1 and Has2 real time RT–PCR, the calibrator was RNA from the human embryonic kidney cell line 293T. Following real time RT–PCR, analysis of the dissociation curves confirmed that a single product was amplified in all reactions.
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Western blot
In two replicate experiments protein extracts of COCs and mGCs were prepared by homogenization in 0.05 M sodium acetate, 6 M urea and 0.1% Triton buffer and protease inhibitors [0.1 M EDTA, 5 mM benzamidine, 0.005 M phenylmethylsufonyl fluoride, 10 IU/ml aprotinin and 10 µl/ml Sigma protease inhibitor cocktail (Sigma-Aldrich Pty. Ltd)]. Protein concentrations were determined by Bradford assay (Bio-Rad, Regents Park, NSW, Australia). Samples were chondroitinase treated by diluting 1:5 in 0.4 M sodium acetate, 0.1% bovine serum albumin and 0.4 M Tris–HCl, pH 8.0 containing 0.02 U/ml chondroitinase ABC and incubated for 1 h at 3
C. The equivalent of 21 COCs was loaded on the gel for each treatment. For granulosa cell extracts, collected at the same time as in vivo matured COCs, the protein quantity loaded was equalized to the respective COC sample to allow comparison of the relative abundance of Adamts1 and Vcan in each tissue compartment. Proteins were separated on 4–15% Tris–HCl acrylamide gels (Bio-Rad) and transferred to polyvinylidene difuoride membrane (Immobilon-P, Millipore Corporation). All gels for western blotting included pre-stained protein molecular weight markers (Bio-Rad). Membranes were blocked in TBST [10 mM Tris (pH 7.5), 150 mM NaCl and 0.05% Tween 20] containing 3% (w/v) non-fat milk for 1 h at room temperature. Membranes were then incubated with primary antibodies for 1 h at room temperature in 3% milk/TBST. Primary antibodies were diluted at 1:5000 for anti-Adamts1 (Russell et al., 2003a) or 1:200 for anti-JSCDPE (previously termed anti-DPEAAE) which detects the cryptic ectodomain of Vcan revealed after Adamts1 cleavage (Sandy et al., 2001). Blots were then washed in TBST and incubated with horse-radish peroxidase-linked anti-rabbit immunoglobulin G at 1:10 000 (Millipore Corporation). Enhanced chemiluminescence detection was used as per manufacturer’s instructions (Amersham, GE Healthcare Life Sciences, Rydalmere NSW, Australia). The bands detected by western blot were quantitatively analysed using QuantityOne (Bio-Rad) software and expressed arbitrarily as values of relative density.
Statistical analysis
Murine real time RT–PCR expression analysis and cumulus expansion experiments were performed in triplicate and analysed by performing a one-way analysis of variance and a Tukey post hoc test using the Statistical Package for the Social Sciences version 13 (SPSS, Chicago, IL, USA). A P-value < 0.05 was considered statistically significant. For the human real time RT–PCR experiments, the expression of each gene was compared between cumulus and mGCs using a t-test. Differences were considered significant at a P-value P < 0.05.
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Induction of Adamts1 and Vcan mRNA in IVM versus in vivo matured cumulus complexes. Adamts1 and Vcan to be induced in IVM cumulus complexes
As expected, COCs cultured without EGF or FSH stimuli failed to undergo COC matrix expansion (mean CEI = 0.82, data not shown) while consistently high CEIs were observed for COCs treated with EGF (3 ng/ml), FSH (50 mIU/ml) or EGF + FSH (3 ng/ml +50 mIU/ml), with no significant differences in CEI between the stimulation regimens. These experiments demonstrate that our IVM conditions were optimal and able to replicate previous experiments (Downs, 1989
; Vanderhyden et al., 1990; Park et al., 2004).
Expression of Adamts1 and Vcan was induced by 6 and 24-fold, respectively, in COCs after 6-h hCG in vivo compared with COCs from eCG only treated mice (Fig. 1A and B). However, after IVM treatment for 6 h there was no significant induction of either Adamts1 or Vcan by FSH, EGF or their combination. The level of Adamts1 or Vcan expression in IVM COCs was 10 and 15-fold lower, respectively, after 6 h of culture than in COCs after 6-h in vivo hCG treatment. In vivo stimulated COCs had 
40% lower expression of Adamts1 and Vcan compared with mGCs collected at the same time, but this difference was not significant.
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We confirmed that the response to stimulation was normal in IVM COCs, by quantifying Has2 expression (Fig. 1C). As expected, culture of COCs for 6 h without stimuli caused a small, but non significant induction of Has2 expression when compared with COCs before culture, however, maturation of COCs in vitro through treatment with either EGF, FSH or EGF + FSH significantly increased Has2 expression 8, 4 and 6-fold, respectively, over the untreated control and 240, 120 and 200-fold over COCs without culture. The in vivo induction of Has2 mRNA after 6 h of hCG treatment was 70-fold increased over the non-cultured control COC, and was significantly lower than the induction in EGF-treated IVM COC (Fig. 1C).
To determine whether Adamts1 and Vcan expression is delayed under IVM conditions we examined mRNA levels in 20-h IVM cultures which is the accepted time when expansion and maturation are complete (Eppig, 1979; Downs, 1989; De La Fuente et al., 1999). (Fig. 2). Expression levels of both Adamts1 and Vcan in IVM COCs were negligible and similar to immature control COCs after eCG treatment alone. In contrast expression for both Adamts1 and Vcan were markedly induced in COCs matured in vivo for 12 h (21 and 16-fold, respectively) or 16 h (3 and 5-fold, respectively) compared with immature control COCs. The level of Adamts1 and Vcan mRNA in mGCs was significantly higher than in COCs from the same follicles after 12 h hCG (2 and 1.3-fold, respectively).
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In vitro matured COCs are deficient in adamts1 protein, as well as intact and cleaved Vcan
To verify our observations of mRNA expression, we compared the protein levels of Adamts1, Vcan and the 70 kDa cleavage product of Vcan generated by Adamts1 action (Russell et al., 2003a
). Western blot analysis was performed following 20 h of IVM as this has previously been shown to be sufficient time for complete matrix synthesis in vitro (Eppig, 1979; Downs, 1989). We compared this to COCs and mGC obtained immediately pre- or post-ovulation (12 or 16 h hCG, respectively) as it has previously been shown that expanded COCs shortly following ovulation in vivo have the equivalent amount of HA per cell as in vitro expanded COC (Salustri et al., 1992). Abundant intact 250 kDa Vcan was present in COC and mGC extracts after hCG 12-h treatment (Fig. 3A, Lanes 4 and 6) but not detectablein COCs from eCG treated mice (Fig. 3A, Lane 1). There was no detectable full length Vcan in COCs cultured without stimulus (Lane 2) or treated for 20 h with EGF (Lane 3). The 70 kDa cleavage product of Vcan has been verified as predominantly an Adamts1-generated cleavage product in COCs (Russell et al., 2003a). We detected this fragment specifically in COCs after 12 and 16-h hCG treatment (Fig. 3A, Lanes 4 and 5), however this Vcan fragment was undetectablein mGCs (Fig. 3B Lanes 6–7, Fig. 3E), in unstimulated IVM COCs or IVM COCs (Fig. 3B, Lanes 2 and 3, respectively). In accordance with this, mature and active Adamts1 of 85 kDa was most abundant in in vivo matured COCs. The active protease was 3.4-fold predominant over the pro-protein (110 kDa) in 12-h hCG in vivo matured COCs (Fig. 3C, Lane 4), and in COCs collected from oviducts 16 h after hCG only active Adamts1 was detectable (Fig. 3C, Lane 5, Fig. 3E). In mGCs from the same follicles, pro- and active Adamts1 were present in equal abundance (Fig. 3C, Lanes 6 and 7, Fig. 3E). Both the mature and precursor forms of Adamts1 were absent in non-cultured or IVM COCs (Fig. 3C, Lanes 1, 2 and 3).
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Adamts1 and Vcan mRNA in human cumulus and mGCs
We next confirmed the expression of Adamts1 and Vcan mRNA in human cumulus and granulosa cells of women during collection of matured oocytes for assisted reproduction. In matched cumulus and mGC samples from 14 patients, Adamts1, Vcan and Has2 mRNA were abundantly present (Fig. 4). The expression of Has2 mRNA was significantly 4.3-fold higher in cumulus cells than their matched mGCs, confirming the purity of each isolated cell type. Interestingly, Vcan mRNA was also significantly 8.2-fold higher in cumulus cells than in the mural compartment (Fig. 4). Expression of Adamts1 at this time showed similar abundance of mRNA in both cell compartments.
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Discussion |
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Maturation of oocytes in vitro deprives them of the normal follicular environment, including potentially important secretions from the mGCs and results in reduced developmental potential when compared with in vivo maturation (Combelles et al., 2002
; Wang and Gill, 2004; Combelles et al., 2005). In the present study, we have shown that murine COCs matured by IVM are deficient in the matrix components, Adamts1 and Vcan, normally present in vivo.
We found that the expression of Adamts1 and Vcan was up-regulated within 6 h following hCG administration both in COCs and mGCs reaching maximal levels at 12 and 6–12 h, respectively, as previously reported for mGCs (Robker et al., 2000; Russell et al., 2003b). However, mouse COCs undergoing IVM for 6 or 20 h show no significant induction of Adamts1 or Vcan mRNA after FSH and/or EGF treatment. These COCs had 10–500-fold reduced expression when compared with COCs from eCG/12 h hCG treated mice. This result demonstrates that in vivo there is a considerable cumulus cell induction of these genes, while in IVM with FSH and/or EGF stimuli this normal gene induction is deficient. The same IVM COCs all showed a significant induction of Has2 mRNA, as previously shown (Dragovic et al., 2005), which was even greater than that seen after 6-h stimulation in vivo confirming that this critical cumulus gene induction pathway was activated in all IVM conditions tested. Previously it has been suggested that Adamts1 and Vcan are predominantly expressed in the mGC layer and not the cumulus cells in vivo (Robker et al., 2000; Russell et al., 2003b). However, the in situ hybridization technique used previously has a less sensitive threshold than the quantitative PCR of the current study in which both genes were strongly detected in COCs. We did however, confirm that at the peak of induction (eCG/12 h hCG) Adamts1 and Vcan expression were significantly greater in mGCs than COCs. This is consistent with reports that Adamts1 gene promoter is responsive to LH as well as progesterone mediated transactivation ( Robker et al., 2000; Doyle et al., 2004), receptors for which are also selectively mGC expressed genes in mouse preovulatory follicles (Eppig et al., 1997; Robker et al., 2000). Furthermore, a spatial pattern of pro-Adamts1 protein predominantly in mGCs and the active protease predominant in COCs was found in this and a previous study (Russell et al., 2003a). This spatial pattern indicates that mGCs are the primary site of Adamts1 synthesis in mouse follicles in vivo. We conclude that both Adamts1 and Vcan are LH-responsive genes predominantly expressed in mGCs, but also induced in mouse COCs in vivo.
The regulation of human Vcan expression appears to vary slightly from that in mouse follicles. We found that after 36-h hCG stimulation, human Vcan and Has2 were predominantly cumulus expressed genes. In contrast, Adamts1 mRNA was equivalent in both cumulus cells and mGCs following stimulation, similar to the expression in mouse follicles. Our evidence is the first to suggest that Adamts1 and Vcan are involved in human COC expansion and maturation, but whether cumulus Vcan or Adamts1 expression are induced normally during human IVM needs further investigation.
That these gene products of the normal in vivo COC environment are absent in IVM indicates that full responsiveness of the COC in IVM is inadequate and the environment of the maturing oocyte may be correspondingly inadequate. Whether Adamts1 and Vcan play essential roles in the maturation of oocytes, either direct or indirect, is uncertain. It is known that Vcan can modulate growth factor receptor interactions (Wu et al., 2004), and that Vcan and its cleavage products have EGF-like signalling activity (Xiang et al., 2006). Adamts1 modulates activity of growth factors, including vascular endothelial growth factor, fibroblast growth factor and the EGF-like factors (Luque et al., 2003; Liu et al., 2006; Suga et al., 2006). In maturing COCs, Adamts1 cleaves Vcan (Russell et al., 2003a) but in several cultured cell lines this protease also activates heparin sulphate-bound EGF and amphiregulin (Liu et al., 2006). The action of EGF-like growth factors is emerging as critical to acquisition of full oocyte competence (see review by Russell and Robker, 2007). Alteration in the normal oocyte–somatic interactions mediated by growth factors in the absence of Adamts1 and/or Vcan may be detrimental to IVM oocyte quality.
Previous studies have aimed at improving nutrient balance in IVM culture media, supplying additional antioxidants to oocytes undergoing IVM. Culture conditions lead to an increase in reactive oxygen species due to higher oxygen and glucose concentrations and in bovine IVM this has a negative impact on embryo development (Luvoni et al., 1996; Hashimoto et al., 2000). Antioxidant properties of pericellular Vcan, as described in other culture systems (Wu et al., 2005), may protect cumulus cells and oocytes exposed to free radicals. Vcan may play a role in maintaining the concentrations of reactive oxygen species associated with inflammatory properties during ovulation in vivo.
Our findings show that mouse COCs matured in vitro are deficient in both Adamts1 and Vcan mRNA and protein when compared with in vivo matured COCs. Human in vivo matured COCs normally express these factors also, although whether this expression is deficient in human IVM conditions is unknown. The dramatically altered matrix environment of oocytes which undergo IVM potentially contributes to the poor health of embryos derived from IVM. The relationship between expression of these genes as a marker of oocyte quality, as well as the effects of supplementation of Adamts1 and Vcan protein on IVM success, should be further investigated.
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The authors would like to gratefully acknowledge the expert assistance of Cadence Minge and Lindsay Chura in the recruitment of patients and acquisition of human granulosa cell samples. This project was supported by the Australian NHMRC project grant 299063.
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References |
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Submitted on January 4, 2007; resubmitted on July 1, 2007; accepted on July 11, 2007.
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