Article Action of hypoxanthine and meiosis-activating sterol on oocyte maturation in the mouse is strain specific

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1 RBMOnline - Vol 8. No Reproductive BioMedicine Online; on web 27 April 2004 Article Action of hypoxanthine and meiosis-activating sterol on oocyte maturation in the mouse is strain specific Anne-Marie Griffin received her PhD in Molecular Biology from Paris X Orsay, France in Since 1997, she has worked in several Australian universities and research institutes and is presently a research associate in the Department of Reproductive Medicine, Westmead Hospital, Sydney, Australia. The research interests of the group she belongs to include oocyte maturation, folliculogenesis, endometrial differentiation and endometriosis. Dr Anne-Marie Griffin AM Griffin 1,3, C Grondahl 2, SD Fleming 1 1 Department of Obstetrics and Gynaecology, Westmead Hospital, University of Sydney, Australia; 2 Novo Nordisk A/S, Copenhagen, Denmark 3 Correspondence: Department of Reproductive Medicine, Westmead Hospital, New South Wales 2145, Australia. Tel: ; Fax: ; annemarg@westgate.wh.usyd.edu.au Abstract Follicular fluid meiosis-activating sterol (FF-MAS) is regarded as an important compound relevant to meiotic resumption in mammalian oocytes. The objective of this study was to investigate the influence of FF-MAS on germinal vesicle breakdown (GVBD) and first polar body (PBI) extrusion with regard to culture conditions, state of the oocyte and mouse strain. Denuded oocytes (DO) and cumulus-enclosed oocytes (CEO) were retrieved from PMSG-primed Quackenbush or C57BL/6J DBA/2 (C57) mice and cultured for 20 h in α-mem medium under the following conditions: (i) 250 μmol/l dibutyryl camp (dbcamp) ± EGF, 1 ng/ml or FF-MAS, 20 μmol/l; (ii) 4 mmol/l hypoxanthine (HX) ± EGF or FF-MAS; (iii) HX + EGF + FF-MAS; and (iv) HX + FF-MAS 5 h priming and subsequent culture with HX + EGF. Oocyte GVBD and PBI emission were recorded and stained with Hoechst Very limited meiotic inhibition was observed in Quackenbush mice in comparison with C57 mice. FF-MAS promoted maturation in C57 DO and CEO and Quackenbush DO. In Quackenbush DO and CEO and C57 DO a significant increase in atypical PBI extrusion occurred, but not in C57 CEO as well as in EGF-treated Quackenbush CEO primed or co-cultured with FF-MAS. These results support a meiosis resumption function for FF-MAS and suggest that in its presence, the quality of the MII oocytes retrieved appears to be influenced by the strain of the mice, the state of the oocyte and the presence or absence of growth factors in the culture medium. Keywords: FF-MAS, hypoxanthine, mouse, oocyte maturation Introduction Competence to undergo meiotic maturation by the mammalian egg is progressively acquired during the later stage of follicle growth. The meiotic hiatus is maintained until shortly before ovulation by the follicular environment, and several lines of evidence suggest that this inhibition derives principally through permanently elevated concentrations of cyclic adenosine monophosphate (camp). The physiological trigger for meiotic resumption (i.e. induced meiotic resumption) is the mid-cycle surge of gonadotrophins, FSH and LH, and is recognized morphologically by germinal vesicle breakdown (GVBD) followed by the extrusion of the first polar body (PBI). In vitro, GVBD can be induced by FSH or EGF in mouse cumulus-enclosed oocytes (CEO) maintained in meiotic arrest by purines such as hypoxanthine (HX), or the membrane permeable camp analogue dbcamp. Conversely, the removal of the oocyte from the follicular compartment, which contains meiosis-arresting factors such as HX, also induces GVBD (i.e. spontaneous meiotic resumption). It is important to note that from the antral stage onwards, the supply of FSH is essential for recruitment, initial growth up to the pre-ovulatory stages and development of fully mature oocytes. The chain of events leading to GVBD is still only partially understood and induced and spontaneous meiotic resumption in vitro appear to use, at least in mice, different signal transduction pathways (Coticchio and Fleming, 1998; Faerge et al., 2001; Su et al., 2001; Grondahl et al., 2003). 673

2 674 Furthermore, it has been shown that pre- and post-maturation conditions can have profound effects on oocyte developmental competence. Since its isolation from human follicular fluid in 1995, an intermediate of cholesterol biosynthesis (follicular fluid meiosis-activating sterol, FF-MAS) has been suggested to play a role in meiotic maturation in CEO as well as in denuded oocytes (DO) (Byskov et al., 1995; Hegele-Hartung et al., 2001). In fact, it has been proposed that FF-MAS is the positive stimulus by which granulosa cells, in response to acute gonadotrophin stimulation, instruct the oocyte to reinitiate meiosis. FF-MAS has also been suggested to improve certain cytoplasmic traits of in-vitro-matured mouse oocytes (Hegele-Hartung et al., 1999). However, specific data on the mechanism of action of FF-MAS, as well as its ability to decisively improve developmental potential, are still lacking. Furthermore, several publications have raised doubts regarding the universal meiosis-inducing function of this sterol (Tsafriri et al., 1998, 2002; Downs et al., 2001; Vaknin et al., 2001). The objective of this study was to investigate the influence of FF-MAS on GVBD and PBI extrusion with regard to culture conditions, state of the oocyte and mouse strain. Materials and methods Culture of mouse oocytes The in-vitro culture assay was followed for resumption of meiosis in mouse oocytes, as previously reported by Grondahl and co-workers (2003). Except when stated otherwise, all reagents were purchased from Sigma (Castle Hill, New South Wales). Briefly, oocytes were obtained from immature 3- week-old female mice (Quackenbush or C57BL/6J DBA/2, ARC, Western Australia), housed under controlled lighting and temperature and fed ad libitum. All animal procedures were carried out in accordance with the guidelines and regulations of Westmead Hospital Animal Ethics Committee. The mice were given an IP injection of 5 IU PMSG (Folligon ; Lyppard, Castle Hill, New South Wales) and were killed by cervical dislocation h later. The ovaries were dissected under oil in M2 medium supplemented with antibiotics and 4 mmol/l HX or 250 μmol/l dibutyryl camp (dbcamp). The oocytes were released by puncturing large antral follicles using a pair of Ultra-Fine needles (Becton Dickinson, North Ryde, New South Wales). CEO and DO displaying an intact germinal vesicle (GV) were transferred to α-mem (Gibco-BRL, Mt Waverley, Victoria) supplemented with 4 mmol/l HX or 250 μmol/l dbcamp, a final concentration of 8 mg/ml human serum albumin (HSA; SAGE BioPharma, Armadale North, Victoria), 0.23 mmol/l pyruvate, 2 mmol/l L-glutamine, 100 IU penicillin and 0.1 mg/ml streptomycin. These media were designated HX-medium and dbcamp-medium respectively. Following their release, the oocytes were washed repeatedly in fresh medium and transferred in groups of at least 30 to 0.5 ml medium in 4-well dishes for 20 h culture in a humidified incubator at 37 C under 5% CO 2. Control treatments with inhibitor-free media were also conducted to test spontaneous meiotic resumption. Oocyte treatments with the appropriate additional controls included: (i) 250 μmol/l dbcamp ± EGF 1 ng/ml or FF-MAS 20 μmol/l; (ii) 4 mmol/l HX ± EGF 1 ng/ml or FF-MAS 20 μmol/l; (iii) CEO primed for 5 h in α-mem medium containing 4 mmol/l HX and FF-MAS 20 μmol/l and subsequently cultured for 15 h in α-mem medium containing 4 mmol/l HX and EGF 1 ng/ml; and (iv) CEO cultured for 20 h in α-mem medium containing 4 mmol/l HX in the presence of FF-MAS 20 μmol/l and EGF 1 ng/ml. FF-MAS (4,4-dimethyl-5-α-cholest-8,14,24-trien-3β-ol) was generously provided by Novo Nordisk (Copenhagen, Denmark). Lyophilized FF-MAS aliquots containing recombinant human albumin (r-ha) were added to the preequilibrated medium at a final concentration of 20 μmol/l. Vials containing r-ha only were used as FF-MAS vehicle controls. Oocyte assessment On completion of the culture period, the CEO were mechanically denuded and the number of oocytes displaying an intact GV, GVBD, normal PBI, atypical PBI as well as the number of necrotic oocytes was recorded using a stereomicroscope (Olympus Optical Co. Ltd., Shinjuku-ku, Tokyo, Japan). Cumulus cell expansion was evaluated in CEO before denudation. The assignment of the oocytes to the different categories of meiotic maturation status was confirmed under fluorescence microscopy (Leica, DMLB; Leica Microsystems, Gladesville, New South Wales, Australia) after Hoechst staining. Statistical analysis The resumption of meiosis was defined as percentage GVBD = (number of GVBD + number of typical PBI + number of atypical PBI/total number of oocytes) 100. The percentage of oocytes having undergone a normal maturation (i.e. reaching metaphase II, MII) was defined as percentage typical MII = (number of typical MII/total number of oocytes) 100. The percentage of oocytes displaying atypical maturation was defined as percentage atypical MII = (number of atypical MII/number of atypical MII + number of typical MII) 100. All experiments were performed at least in triplicate. The statistical software package SPSS for Windows (version 11) was used to analyse the data. The results are presented as mean together with the 95% confidence interval (95% CI). The proportions were arcsin transformed and analysis of variance was used to test for differences between the treatments. Pairwise comparisons of treatments were adjusted for multiple comparisons using the Bonferroni method. Two-way analysis of variance of the transformed data was performed to investigate the effect of the mouse strain and treatment on GVBD. Statistical significance was set at <5%. Results No significant difference in percentage GVBD, percentage typical MII and percentage atypical MII were observed between the control HX-medium (containing HSA) and the control FF-MAS vehicle containing r-ha (data not shown). Spontaneous GVBD occurred in 91.2% of Quackenbush DO, 100% of Quackenbush CEO, 88.8% of C57 DO and 90.3% of C57 CEO.

3 Effect of HX in oocytes of two different mouse strains during meiotic maturation In HX-medium and regardless of the state of the oocyte (DO in Figure 1a and CEO in Figure 1b), meiosis maturation inhibition is significantly higher in C57 mouse oocytes than in Quackenbush mouse oocytes (P < 0.001). No significant interaction between the effect of strain and treatment was observed (P = 0.7 in DO, P = 0.15 in CEO). Treatments with EGF were used as controls. Effect of FF-MAS in HX-arrested oocytes in two different mouse strains In both strains, FF-MAS significantly promoted meiotic maturation in DO in comparison with HX-medium alone (76.4% GVBD versus 35.2% in Quackenbush and 81.2% GVBD versus 7.7% in C57 respectively, P < 0.001, Figure 2a, c). The percentage of atypical MII generated was also significantly greater in the presence of FF-MAS (81.3 versus 6.6% in Quackenbush P = 0.007, and 68.9 versus 0.0% in C57 respectively, P = 0.023). The proportion of oocytes reaching normal MII status was nevertheless not significantly affected by FF-MAS (7.6 versus 25.8% in Quackenbush and 7.6 versus 2.7% in C57 respectively). In accordance with previous data (Grondahl et al., 2003), FF- MAS significantly increased the proportion of C57 CEO resuming meiosis in comparison with HX-medium alone (42.1% GVBD versus 4.7% respectively, P = 0.04) and no atypical PBI extrusion was observed (Figure 2d). However, it was found that the proportion of oocytes reaching normal MII a was not affected by FF-MAS in comparison with the HXmedium control (11.6 versus 1.2% respectively), but was significantly lower than the proportion found in EGFsupplemented medium (40.8%, P = 0.05). Conversely, FF- MAS did not significantly increase the proportion of Quackenbush CEO resuming meiosis in comparison with HXmedium alone (59.9% GVBD versus 58.7% respectively, Figure 2b), but a significant increase in atypical PBI extrusion was observed (38.0 versus 6.0% respectively, P = 0.009). Finally, the proportion of oocytes reaching normal MII was not affected by FF-MAS in comparison with the HX-medium, but was significantly lower than the proportion found in EGFsupplemented medium (13.4 versus 42.8% respectively, P = 0.015). Atypical MII were recorded according to their phenotype. However, all of the oocytes were stained with Hoechst to check the chromatin status. The phenotype and chromatin status of atypical MII recovered from FF-MAS-treated Quackenbush oocytes are shown in Figure 3b f. For comparison, a control MII is shown in Figure 3a. Over 90% of the atypical MII recovered presented a giant polar body phenotype (Figure 3b). MII with fragmented PBI (Figure 3c) or more than one PBI (Figure 3d) was also recorded. With regards to chromatin status, a non-degenerate chromatin status was frequently observed in the giant polar body (Figure 3b) or apparent non-disjunction (Figure 3e). Interestingly, an atypical chromatin status was also observed in some phenotypically normal MII FF-MAS-treated oocytes (Figure 3f). Albeit an anecdotal observation in FF-MAS-treated oocytes, this type of atypical MII was not recorded in any other treatment. b Figure 1. (a) The effect of HX on meiotic arrest of mouse DO is strain specific. Two-way analysis of variance to investigate in DO the effect of strain with regard to meiotic resumption under hypoxanthine culture conditions. The box goes from lower quartile to upper quartile. The thick bar is the median and the whiskers extend to the usual range of the data. (b) The effect of HX on meiotic arrest of mouse CEO is strain specific. Two-way analysis of variance to investigate the effect of mouse strain in CEO with regard to meiotic resumption under hypoxanthine culture conditions. Data are shown as in (a). 675

4 676 Effect of FF-MAS in dbcamp-arrested Quackenbush oocytes Regardless of the oocyte state, FF-MAS failed to promote maturation in Quackenbush dbcamp-arrested oocytes when compared with the control (Figure 4). The combined percentage of atypical MII and necrotic oocytes never exceeded 3% in any experiment and was 2% in both FF-MAStreated DO and CEO. The percentage of FF-MAS-treated oocytes reaching normal MII was significantly lower than in the dbcamp-medium control in DO (0.5% GVBD versus 4.3% respectively, P = 0.01). In CEO, these proportions were similar (2.8 versus 2.3% normal MII). Effect of FF-MAS priming and FF-MAS co-culture in EGF-treated, HX-arrested oocytes in Quackenbush mice The proportion of oocytes resuming meiosis and reaching normal MII in EGF-treated oocytes was not affected by either 5 h of FF-MAS priming or co-culture with FF-MAS (Figure 5). Furthermore, no increase in atypical MII was observed. Cumulus cell expansion Under both meiotic-arresting factors no cumulus cell expansion was observed in FF-MAS-treated CEO (Figure 6a). In comparison, an extensive cumulus cell expansion was observed in all EGF treatments including those oocytes primed (Figure 6b) or co-cultured with FF-MAS (Figure 6c). Discussion So far as is known, this is the first time that the strain specificity of the meiotic resumption inhibitor HX has been shown. The Quackenbush strain was developed to produce a large litter size. However, the physiological/genetic basis of that trait has never been defined (Kirkpatrick et al., 1998). Since HX is a meiosis-arresting factor present naturally in the follicular compartment, further experiments are needed to test if the weak sensitivity to HX displayed by the Quackenbush mice may be a factor promoting their folliculogenesis and as a result, their litter size. FF-MAS promotes meiotic maturation in HX-arrested oocytes in both strains. The absence of significant promotion of meiotic maturation in Quackenbush CEO may be explained by the combination of a poor inhibition of maturation in the HX control, a poor sustainability of this inhibitory effect and the slow kinetics of FF-MAS usually observed in CEO. Percentages of GVBD observed in FF-MAS-treated CEO of both strains were not significantly different, so it is possible that any action of FF-MAS in Quackenbush CEO was masked by the high percentage of GVBD displayed by the HX control. Indeed, the extension of the culture period showed a lack of sustainability of the inhibition of maturation in the HX control. As a result, after 25 h, the increased percentage of GVBD observed in FF-MAS-treated CEO was not significantly different from the increased percentage of GVBD observed in the HX control (data not shown). Consequently, no conclusions can yet be drawn about the ability of FF-MAS to promote meiotic maturation in HX-arrested Quackenbush CEO. Because of this, the action of FF-MAS in Quackenbush CEO was further investigated using dbcamp, another meiotic maturation inhibitor. In contrast to HX, dbcamp proved to be very effective in that strain. However, the results show also that FF-MAS does not promote meiotic maturation in Quackenbush CEO and DO. These findings are consistent with previous data in CD1 (Coticchio et al., 2003) and C57 (Downs et al., 2001) mouse strains that confirm the fact that FF-MAS is not a universal stimulator of maturation. With regard to atypical PBI formation, a strain-specific and cumulus cell dependence of FF-MAS action was observed in HX-arrested oocytes. No atypical PBI extrusion was observed in C57 CEO, whereas a significant percentage of atypical PBI extrusion was observed in Quackenbush CEO. The incidence of atypical PBI extrusion in Quackenbush mice was noticeably greater in DO than in CEO. Atypical PBI extrusion in FF- MAS-treated Quackenbush mouse oocytes has been observed in different laboratories (G Coticchio, personal communication). However, this is the first time that it has been observed in FF-MAS-treated C57 mouse DO. In the present study, the rate of atypical MII observed in FF-MAS-treated Quackenbush mouse CEO and DO and C57 mouse DO support the existence of some strain-specific sensitivity to FF- MAS in this respect. FF-MAS has been shown to act through a MAPK-dependent pathway (Faerge et al. 2001). Interestingly, giant PBI can be observed in mos / mice, which lack MAPK activity (Choi et al., 1996; Verlhac et al., 2000; Su et al., 2002). Choi and coworkers (Choi et al., 1996) have shown that the Mos/MAPK pathway regulates the size and degradation of the PBI in maturing mouse oocytes. In vitro, oocytes from mos / mice are able to complete the first meiotic division. However, since the regulation of spindle formation is dependent on MAPK activation (Lu et al., 2002; Yu et al., 2002; Ye et al., 2003), these oocytes lose their ability to properly position the spindle apparatus. Consequently, they extrude a giant polar body. This specifically negatively affects the fertilization rate. In addition, the PBI usually persists instead of degrading (nondegenerative PBI) and sometimes undergoes an additional cleavage (Choi et al., 1996). These phenotypes can be categorized according to the classification of Hirao and Eppig (1997), and include oocytes with an extremely large PB or fragmented oocytes with up to two PB, and oocytes which have the appearance of 2-cell embryos. These phenotypes are similar to what was observed in experiments in HX-arrested, FF-MAS-treated oocytes. The exact state of the spindle, microfilament and microtubule organization in FF-MAStreated oocytes clearly needs to be further investigated (Hegele-Hartung et al., 1999; Cukurcam et al., 2003). Spindle alteration during meiotic maturation is highly likely to cause irreversible damage to the oocyte with regard to further developmental competence and quality (Eichenlaub-Ritter et al., 2002). In fact, an abnormal oocyte spindle is frequently associated with the infertility of aged women (Cheng et al., 2003). In that context, and despite the fact that the age of the oocyte remains critical, it might be interesting to look at MAS concentration pattern with regard to the age of the organism. The priming and co-culture experiments of Quackenbush CEO with FF-MAS in the presence of EGF examined a possible interaction between the apparent atypical effect of FF-MAS on

5 a b c d Figure 2. (a) FF-MAS promotes meiotic maturation in Quackenbush HX-arrested DO with a significant increase in atypical MII. Frequencies of GVBD, atypical MII and typical MII in Quackenbush DO cultured with FF-MAS or EGF in the presence of HX. Data are shown as the mean together with the 95% CI. Groups presenting different letters are significantly different. (b) FF-MAS promotes a significant increase in atypical MII in Quackenbush HX-arrested CEO. Frequencies of GVBD, atypical MII and typical MII in Quackenbush CEO cultured with FF-MAS or EGF in the presence of HX. Data are shown as in (a). (c) FF-MAS promotes meiotic maturation in (C57BL/6J DBA/2) HX-arrested DO with a significant increase in atypical MII. Frequencies of GVBD, atypical MII and typical MII in (C57BL/6J DBA/2) DO cultured with FF-MAS or EGF in the presence of HX. Data are shown as in (a). (d) FF- MAS promotes meiotic maturation in (C57BL/6J DBA/2) HX-arrested CEO without an increase in atypical MII. Frequencies of GVBD, atypical MII and typical MII in (C57BL/6J DBA/2) CEO cultured with FF-MAS or EGF in the presence of HX. Data are shown as in (a). 677

6 a b c d e f Figure 3. Chromatin status of atypical MII recovered from FF-MAS-treated mouse oocytes. (a) Typical MII (control); (b) nondegenerate giant PBI; (c) oocyte with two PBI; (d) fragmented PBI; (e) fragmented oocyte with 2-cell embryo appearance; (f) phenotypically normal looking MII with atypical chromatin appearance. 678

7 Figure 4. FF-MAS fails to promote meiotic maturation in Quackenbush dbcamp-arrested DO. Frequencies of GVBD and typical MII in Quackenbush DO cultured with FF-MAS or EGF in the presence of dbcamp. FF-MAS fails to promote meiotic maturation in Quackenbush dbcamp-arrested CEO. Frequencies of GVBD and typical MII in Quackenbush CEO cultured with FF-MAS or EGF in the presence of dbcamp. Data are shown as in Figure 2a. Figure 5. In the presence of EGF, FF-MAS does not promote an increase in atypical MII in Quackenbush CEO. Frequencies of GVBD, atypical MII and typical MII in Quackenbush CEO cultured with EGF with or after priming with FF-MAS in the presence of HX. Data are shown as in Figure 2a. a b c Figure 6. (a) In the presence of HX, FF-MAS does not promote cumulus cell expansion of Quackenbush CEO. Quackenbush CEO cultured for 25 h with FF-MAS in the presence of HX. (b) In the presence of HX, FF-MAS priming and subsequent culture with EGF does not impair cumulus cell expansion of Quackenbush CEO. Quackenbush CEO primed for 5 h with FF-MAS and subsequently cultured for 15 h with EGF in the presence of HX. (c) In the presence of HX and EGF, FF-MAS does not impair cumulus cell expansion of Quackenbush CEO. Quackenbush CEO co-cultured for 20 h with FF-MAS and EGF in the presence of HX. 679

8 680 PBI formation and EGF-mediated maturation. Since EGF and FSH have in common a mechanism of action that incorporates the IP 3 -Ca 2+ second messenger system, the present results suggest that in the presence of gonadotrophins, the apparent atypical effect of FF-MAS on PBI formation in CEO could be totally abolished. In the presence of EGF FF-MAS does not seem to further promote PBI extrusion, since the percentage of oocytes reaching MII is not significantly different between treatments. After 20 h of culture, no cumulus cell expansion was observed in HX-arrested, FF-MAS-treated CEO, while extensive cumulus cell expansion was observed when EGF was present in the medium, even after pretreatment with FF-MAS or when FF-MAS was present simultaneously in the culture medium. Cumulus expansion is an important process that must occur in pre-ovulatory follicles to enable ovulation (Eppig, 2001). It consists of cumulus cells secreting hyaluronic acid, which occurs in vivo after the pre-ovulatory gonadotrophin surge. It has been shown that MAPK activity in the cumulus cells, rather than the oocyte, is necessary for gonadotrophin-induced GVBD (Leonardsen et al., 2000; Su et al., 2001) and cumulus expansion of mos / or mos +/+ mouse CEO (Su et al., 2002). The results suggest that FF-MAS does not disrupt the MAPK pathway in the cumulus cells and thus does not impair the cumulus cell expansion process. In conclusion, this study demonstrates for the first time that the action of the meiotic resumption inhibitor HX is strain specific, at least at the routinely used concentration of 4 mmol/l. This source of variability should definitely be taken into account in future meiotic maturation studies and when comparing data using different strains of mouse. The present results support a meiosis resumption function for FF-MAS. It was found, however, that FF-MAS seems to be able, at least at the dosage used in this study, to disrupt the organization of the first meiotic spindle and consequently PBI extrusion. This phenomenon seems to be strain-dependent and dependent upon the state of the oocyte, but may not occur in the presence of gonadotrophins. Further experiments are currently being undertaken to determine the mode of action of FF-MAS on PBI extrusion mechanism. Acknowledgements This work was supported by NHMRC grant awarded to Steven Fleming. We thank Dr Christine Smyth from the Children s Medical Research Institute for her help with the fluorescent microscope and Dr Karen Byth from Westmead Millenium Institute for assistance with statistical analysis. Declaration of interest: Dr Christian Grondahl is Science Vice- President in the pharmaceutical company NOVO NORDISK A/S that has commercial interest in and has supplied FF-MAS to the study. 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