ARTICLE

Offspring From Intracytoplasmic Sperm Injection of AgedMouse Oocytes Treated With Caffeine or MG132

Tetsuo Ono,1,2* Eiji Mizutani,1 Chong Li,1,3 Kazuo Yamagata,1 and Teruhiko Wakayama1,2,3*

1Laboratory for Genomic Reprogramming, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan

2Department of Medical Science, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan

3Department of Bioscience, Graduate School of Science and Technology, Kwansei Gakuin University,Sanda 662-8501, Japan

Received 12 January 2011; Revised 1 April 2011; Accepted 10 April 2011

Summary: Postovulatory mammalian oocytes age sig-nificantly in culture. B6D2F1 or ICR strain mouseoocytes were collected 16 h after hCG injection andthen cultured for up to 40 h post hCG at 378C under 5%CO2 in air. After intracytoplasmic sperm injection (ICSI),B6D2F1 and ICR oocytes lost full-term developmentalpotential by 30 h and 26 h after hCG administration,respectively. However, using supplementation with 10mM caffeine or 1-5 lM of MG132, we could obtain liveoffspring from oocytes at 34 h (BDF1, 5%–21%) or 28 h(ICR, 5%–18%), whereas none were obtained fromuntreated aged oocytes. Caffeine maintained normalmeiotic spindle morphology, whereas MG132 main-tained maturation-promoting factor activity. Thesetreatments did not affect the potential of fresh oocytesfor fertilization and subsequent development. Thus, itshould be safe to use these chemicals in routine in vitrofertilization and offspring could be generated by ICSI ofaged fertilization failed oocytes. genesis 49:460–471,2011. VVC 2011 Wiley-Liss, Inc.

Key words: in vitro aging; oocyte; mouse; caffeine;MG132; ICSI; MPF

INTRODUCTION

It is well known that mammalian oocytes arrested atmetaphase II (MII) of meiosis have a short fertilizablelife span with a progressive decrease of their normal de-velopmental competence with time after ovulation. Forexample, mouse oocytes can be fertilized in vivo forabout 15 h after ovulation (Marston and Chang, 1964).If fertilization or spontaneous activation is delayed, the

mature oocytes undergo progressive aging (Whit-tingham and Siracusa, 1978; Yanagimachi and Chang,1961). Deterioration in the developmental potential ofthe mature ovulated oocyte during prolonged culture iscalled postovulatory aging and oocytes in which aginghas started but which have not yet degenerated fully arecalled aged oocytes (Kikuchi et al., 2002). Agingdecreases the potential of oocytes for fertilization andembryo development because of molecular changesoccurring in the aged ooplasm (Jones, 2008; Miao et al.,2009). One cause is the gradual decrease in maturation-promoting factor (MPF) activity with time. Regulationof MPF activity depends upon an association of the cata-lytic subunit of cdc2 kinase with the regulatory subunitof cyclin B. Inactivation of MPF is caused by proteolysisof cyclin B (Norbury and Nurse, 1992) and by phospho-rylation of cdc2 (Kikuchi et al., 1999). It has also beendemonstrated that aged eggs and their derivative

Current address for Eiji Mizutani: Research Team for Reproductive Biol-

ogy and Technology, National Institute of Livestock and Grassland Science,

Tsukuba 305-0901, Japan.

Current address for Kazuo Yamagata: Center for genetic Analysis of Bio-

logical Responses, Research Institute for Microbial Disease, Osaka 565-

0871, Japan.

* Correspondence to: T. Ono and T. Wakayama, Laboratory for Genomic

Reprogramming, Center for Developmental Biology, RIKEN, 2-2-3 Minato-

jima-minamimachi, Chuo-ku, Kobe 650-0047, Japan.

E-mail: onimarumaru@hotmail.com

Contract grant sponsor: Grants-in-Aid for Young Scientists B, Contract

grant number: 19780213, Contract grant sponsor: Scientific Research in

Priority Areas, Contract grant number: 15080211

Published online 18 April 2011 in

Wiley Online Library (wileyonlinelibrary.com).

DOI: 10.1002/dvg.20756

' 2011 Wiley-Liss, Inc. genesis 49:460–471 (2011)

embryos undergo molecular changes characteristic ofapoptosis or programmed cell death (Exley et al., 1999;Morita and Tilly, 1999; Perez et al., 1999). In addition,aged oocytes exhibit varying degrees of fragmentationor abnormal spindles and chromosomal alignment(Wakayama et al., 2004).

However, even if oocytes aged in vitro show variousabnormalities and a decrease in developmental capacity,these oocytes can still serve as valuable material for sev-eral purposes. For example, offspring can be producedfrom aged fertilization-failed (AFF) oocytes by intracyto-plasmic sperm injection (ICSI), termed rescue-ICSI(Nagy et al., 1993). This method could reduce the bur-den of human patients undergoing assisted reproduc-tive techniques who experience failed fertilization(Morton et al., 1997; Sjogren et al., 1995; Tsirigotiset al., 1995). On the other hand, we could establishmouse embryonic stem (ES) cell lines from somatic cellnuclei by nuclear transfer into AFF oocytes (Thuanet al., 2010; Wakayama et al., 2007). This method canavoid some of the ethical problems of human ES cellgeneration, in that it does not require oocyte donationfrom healthy young women. However, in both cases,the success rates of producing live offspring or estab-lishing ES cells were significantly lower for AFF oocytesthan for fresh oocytes (Cervera and Garcia-Ximenez,2003; Iwamoto et al., 2005; Wu et al., 2007; Zhouet al., 2003). Therefore, controlling or delaying oocyteaging in vitro might have many advantages for theseprocedures, as manipulation times could be extendedwith the sustained oocyte quality.

Several chemical treatments are reported to main-tain oocyte quality during aging in vitro. Caffeine, aphosphodiesterase inhibitor, has been reported to in-hibit Myt1/Wee1 activity (Smythe and Newport, 1992)and to delay oocyte aging in several species (Kikuchiet al., 2002; Miao et al., 2009; Mononmani et al.,2004; Ye et al., 2010). Similarly, MG132, an ubiquitin-mediated inhibitor of proteasome proteolysis, inhib-ited the degradation of cyclin B and maintained a highlevel of MPF activity in oocytes, but inhibited somenecessary degradation of proteins during in vitro fertil-ization (IVF) (Gao et al., 2005; Josefsberg et al., 2000;Zhou et al., 2003).

The aims of this study were as follows: first, to eval-uate when aging mouse oocytes lose their potentialfor development to full term; second, to evaluate howthe aging phenotypes of oocytes are related to full-term developmental potential by immunostaining andmeasuring MPF activity; third, to check whether wecould delay oocyte aging in vitro using caffeine orMG132 treatment. Finally, we had to confirm thatthese chemicals would show no toxicity for routineIVF and subsequent embryo development if they wereto be used in adjunct clinical or experimentaltreatments.

RESULTS

Pronuclear Formation and PreimplantationDevelopmental Capacity of In Vitro Aged Oocytesafter ICSI

Oocytes from B6D2F1 mice retained their capacityfor pronuclear (PN) formation after ICSI and showed asecond polar body extrusion capacity similar to non-aged (‘‘fresh") oocytes until 26 h after human chorionicgonadotropin (hCG) injection (93% at 22 h and 82% at26 h vs. 98% in 16 h controls; Fig. 1a, black column; Ta-ble 1). However, when oocytes were aged for morethan 30 h after hCG injection, the percentages of nor-mal PN stage oocytes decreased significantly (1–57%,P < 0.05; Fig. 1a, black column; Table 1). As postovula-tory aging proceeded, the proportions of abnormaloocytes (formation of three PN, abnormal cytokinesisand cytoplasmic fragmentation) increased at the PNstage (Fig. 1a, white column; Fig. 2a–d). At 40 h afterhCG injection, only one oocyte (0.6%) was activatednormally and 48% of oocytes were not activated by ICSI(Fig. 1a, gray column).

After culturing the ICSI embryos for 96 h (Fig. 1b; Ta-ble 1), 75% of the embryos using oocytes taken at 22 hafter hCG injection reached the morula/blastocyst stageand this rate did not differ significantly from that of thefresh controls (92%; Table 1). However, when oocyteswere aged for 26 h and 30 h, the development rate tothe morula/blastocyst stage decreased significantly com-pared with controls (56% at 26 h and 14% at 30 h, P <0.05; Fig. 1b; Table 1). None of the embryos usingoocytes aged for more than 34 h could reach the mor-ula/blastocyst stage (Fig. 1b; Table 1).

To examine the influence of mouse strain on posto-vulatory aging, we performed ICSI on ICR strainoocytes at 16, 22, 26, or 30 h after hCG. All controloocytes used at 16 h (fresh) fertilized by ICSI exhib-ited PN formation (Fig. 1d, black column; Table 1).There were no significant differences in the ability toform PN in oocytes aged for up to 26 h comparedwith the fresh group (22 h and 26 h: 87% and 83%,respectively; Fig. 1d, black column; Table 1). However,when oocytes were aged for more than 30 h, therewas a significant decrease in the rate of normal PNformation (64%, P < 0.05; Fig. 1d, black column; Ta-ble 1). Some oocytes aged for more than 22 h initiatedabnormal activation following ICSI; these oocytesshowed the formation of three PN, abnormal cytokine-sis and cytoplasmic fragmentation (Fig. 1d, white col-umn; Fig. 2). Thus, there was no significant differencebetween B6D2F1 and ICR strain oocytes in the abilityto form PN following aging.

After culturing the ICSI embryos for 96 h, the ratesof morula/blastocyst development were decreased sig-nificantly in aged ICR oocytes, even at 22 h after hCG

461OFFSPRING FROM AGED MOUSE OOCYTES

(22%, P < 0.05) compared with fresh oocytes (16 h,68%; Fig. 1e; Table 1). This rate was significantlyworse than with aged B6D2F1 oocytes (75% at 22 h;Fig. 1b,e; Table 1). At 26 h of aging, only 6% of theICR embryos reached the morula/blastocyst stage com-pared with 56% for the B6D2F1 embryos (Fig. 1b,e;Table 1). None of the embryos using oocytes aged for

more than 30 h could reach the morula/blastocyststage (Fig. 1e; Table 1).

Full-Term Development of In Vitro Aged Oocytesafter ICSI

As shown in Figure 1c and Table 2, � 50% of ICSIembryos using B6D2F1 mouse oocytes at 16 h (fresh),

FIG. 1. Pronucleus (PN) formation and developmental potential among in vitro aged oocytes. Percentages are based on the numbers ofoocytes surviving after intracytoplasmic sperm injection (ICSI). (a–c) B6D2F1 oocyte. (d–f) ICR oocyte. Fertilization rates (a,d), in vitro devel-opmental potential (b,e) and full-term development (c,f) of oocytes cultured for up to 40 h (B6D2F1) or 30 h (ICR) after hCG injection.B6D2F1 or ICR strain mouse oocytes maintained some developmental potential until 30 h or 26 h after hCG, respectively.

462 ONO ET AL.

Table 1Preimplantation Development of Zygotes after ICSI Using Fresh or In Vitro Aged Oocytes

StrainType of oocyte andtime post-hCG Injected Surviving (%)1 PN (%)2, 3

No. (%) of embryos, 96 h after activation2

Degradation 1-cell 2–8-cell M/B

B6D2F1 Control4 16 h 129 110 (85) 108 (98)a 8 (7) 0 1 (1) 101 (92)g

Aged in vitro 22 h 152 112 (74) 104 (93)a 16 (14) 5 (5) 7 (6) 84 (75)g, h

26 h 151 112 (74) 92 (82)a, b 44 (39) 2 (2) 3 (3) 63 (56)h

30 h 165 134 (81) 76 (57)b 104 (78) 1 (1) 10 (8) 19 (14)i

34 h 179 171 (96) 37 (22)c 146 (85) 20 (12) 5 (3) 040 h 179 161 (90) 1 (1)d 154 (96) 7 (4) 0 0

ICR Control4 16 h 105 98 (93) 98 (100)e 28 (29) 0 3 (3) 67 (68)j

Aged in vitro 22 h 94 77 (82) 67 (87)e, f 46 (60) 4 (5) 10 (13) 17 (22)k

26 h 91 82 (90) 68 (83)e, f 24 (29) 1 (1) 17 (21) 5 (6)l

30 h 99 87 (88) 56 (64)f 34 (39) 1 (1) 17 (20) 0

1Based on the number of injected oocytes.2Based on the number of surviving oocytes.3Normal pronuclear embryos.4Fresh oocytes.PN, pronuclear stage embryos; M/B, morula/blastocyst stage embryos.a to lValues in columns without a common superscript differ significantly (P < 0.05).

FIG. 2. Abnormal embryos after ICSI of oocytes cultured with caffeine or MG132. (a) Three PN. (b) Abnormal cytokinesis. (c) Fragmenta-tion. (d) Abnormal embryo. (e) Bright field image of metaphase (M) stage II oocytes. Arrows show the MII plate. (f) Spindles were visualizedby immunofluorescence staining for b-tubulin. Scale bar 5 20 lm. Key: 16 h, oocytes at 16 h after hCG injection; 34 h, oocytes at 34 h afterhCG injection; 34 h1Caff, oocytes at 34 h after hCG injection (with 10 mM caffeine); 34 h1MG, oocytes at 34 h after hCG injection (with 5lMMG132). (g) Illustration of metaphase (M)II spindle.

463OFFSPRING FROM AGED MOUSE OOCYTES

and at 22 h and 26 h after hCG supported full-term de-velopment; there were no significant differencesbetween these times. However, only 19% of zygotesusing oocytes subjected to ICSI at 30 h after hCG couldreach full term. None of the oocytes aged to 34 h coulddevelop to term when three 2-cell embryos were trans-ferred. It is known that when few embryos are trans-ferred into recipient females, the full-term developmentis less successful. Therefore, until we can perform moreembryo transfers, it is formally possible that we mightobtain full-term development from oocytes collected at34 h after hCG. However, as shown in Table 1, these 2-cell embryos failed even to develop to the blastocyststage in vitro. Probably, even if we transferred greaternumbers of 2-cell stage embryos, most would degener-ate before implantation and we would be very unlikelyto obtain offspring from those aged oocytes. On theother hand, no obvious abnormalities were found in theresulting progeny in terms of body weight, neonatal

weight gain, or sex ratio (Fig. 3; Table 2). These micewere fertile when mated with each other as adults.

To examine the developmental capacity to full termin the ICR mouse strain, 2-cell stage embryos weretransferred into pseudopregnant females. As shown inFigure 1f and Table 2, approximately 50% of embryosproduced by ICSI of oocytes at 16 h (fresh) or 22 h afterhCG supported full-term development and there wereno significant differences between treatments at thistime. However, only 18% of zygotes using oocytes agedfor 26 h could reach full term. These mice were fertilewhen mated with each other as adults.

Histone Acetylation in Aged Oocytes

The acetylation levels of various lysine residues in his-tones H3 or H4 were examined following in vitro agingof B6D2F1 mouse oocytes at 16 and 34 h after hCGadministration. Experiments were repeated three times,and more than ten oocytes were observed in eachexperiments. Immunocytochemistry with specific anti-bodies against H3K9, H3K14, H4K5, and H4K12showed no differences in fluorescence signal intensitiesfor any of the antibodies (see Fig. 4).

Effects of Caffeine or MG132

As demonstrated above, when oocytes were culturedin vitro to 34 h after hCG, all of them lost their potentialto develop to full term. Therefore, we performed experi-ments using oocytes aged for 34 h and compared treatedand untreated aged oocytes. When 10 mM caffeine wasadded to the culture medium, oocyte morphology wassimilar to fresh oocytes (Fig. 2e, ‘‘34h1Caff") and theproportions of normal PN stage zygotes and 2-cell stageembryos increased significantly compared with theuntreated group (27% vs. 15% for the PN stage, P < 0.05;17% vs. 6% for the 2-cell stage, P < 0.05; Table 3). Two-cell stage embryos were transferred into pseudopregnant

Table 2Full-Term Development of Zygotes after ICSI Using Fresh or In Vitro Aged Oocytes

Strain

Type of oocyteand time post-

hCG Injected Surviving (%)1 PN (%)2, 3 2-cell (%)2 ET (recipients) Offspring (%)2 Sex ratio (male/female)4

B6D2F1 Control5 16 h 108 98 (91) 97 (99) 93 (95) 93 (8) 47 (48)a 12/12Aged in vitro 22 h 205 137 (67) 127 (93) 116 (85) 116 (9) 70 (51)a 28/32

26 h 159 127 (80) 102 (80) 94 (74) 94 (7) 60 (47)a 33/2630 h 187 144 (77) 85 (59) 52 (36) 52 (4) 28 (19)b 5/634 h 122 107 (88) 21 (20) 3 (3) 3 (2) 0 –

ICR Control5 16 h 80 78 (98) 77 (99) 74 (95) 74 (5) 37 (47)c 17/13Aged in vitro 22 h 90 75 (83) 70 (93) 58 (77) 58 (5) 32 (43)c 16/8

26 h 97 87 (90) 63 (72) 56 (64) 56 (5) 16 (18)d 8/2

1Based on the number of injected oocytes.2Based on the number of surviving oocytes.3Normal pronuclear embryos.4Some pups died before being checked5Fresh oocytes.PN, pronuclear stage embryos; ET, embryo transfer.a to dValues in columns without a common superscript differ significantly (P < 0.05).

FIG. 3. Weight gain of offspring generated by ICSI using in vitroaged B6D2F1 oocytes. No obvious abnormalities were found in theresulting progeny in terms of body weight or weight gain.

464 ONO ET AL.

females and four offspring (13%) were obtained in thecaffeine-treated group (Table 3).

When MG132 was added to the culture medium, nor-mal morphology of these oocytes was maintained forlonger compared with untreated oocytes (Fig. 2e,

‘‘34h1MG") and we observed that PN formation wasdelayed when concentrations of MG132 greater than 5lM were used. The PN formation rates of oocytestreated with 1 or 5 lM MG132 were significantly higherthan those of the untreated control group (41% and

FIG. 4. Acetylation of lysines 9 and 14 on histone H3 and of lysines 5 and 12 on histone H4 during postovulatory aging of B6D2F1 mouseoocytes. Oocytes were immunostained with specific antibodies against acetylation for each site (H3K9, H3K14, H4K5, and H4K12). Key: 16h, oocytes at 16 h after hCG injection; 34 h, oocytes at 34 h after hCG injection (cultured in vitro for 18 h). DNA is stained red with propidiumiodide; H3K9, H3K14, H4K5, and H4K12 are stained green. Scale bar5 20 lm.

Table 3Full-term Development of Zygotes after ICSI Using B6D2F1 or ICR Mouse Strain Oocytes at 34 h or 28 h after hCG Cultured with

Caffeine or MG132

Strain

Treatment andtime post-

hCG Dose Injected Surviving (%)1 PN (%)2, 3 2 cell (%)2 ET (recipients) Implantation (%)4 Offspring (%)4

B6D2F1 Caffeine 34h 0 mM 203 173 (85) 26 (15)a 11 (6)h 11 (4) 0 01 mM 82 68 (83) 17 (25) 4 (6)h 4 (3) 0 010 mM 202 183 (91) 49 (27)b 31 (17)i 31 (5) 10 (32) 4 (13)20 mM 83 68 (82) 7 (10)a 5 (7) 5 (3) 0 0

MG132 34h 0 lM 105 92 (88) 18 (20)c 4 (4)j 4 (3) 0 01 lM 105 90 (86) 37 (41)d 19 (21)k 19 (3) 2 (11) 1 (5)5 lM 105 95 (91) 62 (65)d 28 (30)k 28 (3) 11 (39) 6 (21)25 lM 115 112 (97) 5 (5)e 3 (3)j 3 (2) 0 0

ICR MG132 28h 0 lM 108 84 (78) 60 (71)f 41 (49)l 41 (3) 0 01 lM 108 93 (86) 66 (71)f 40 (43)l, m 40 (3) 7 (18) 2 (5)5 lM 107 98 (92) 64 (65)f 38 (39)l, m 38 (3) 10 (26) 7 (18)25 lM 107 96 (90) 38 (40)g 25 (26)m 25 (3) 0 0

1Based on the number of injected oocytes.2Based on the number of surviving oocytes.3Normal pronuclear embryos.4Based on the number of transferred oocytes.PN, pronuclear stage embryos; ET, embryo transfer.a to mValues with different superscripts in the same column are significantly different (P < 0.05).

465OFFSPRING FROM AGED MOUSE OOCYTES

65%, respectively, vs. 20%, P < 0.05; Table 3), butdecreased significantly in the oocytes treated with 25lM MG132 (5% vs. 20%, P < 0.05; Table 3). After beingcultured, development to the 2-cell stage was alsodelayed in the MG132-treated groups, similar to the rateof PN formation. The rates of development to the 2-cellstage of oocytes cultured with 1 lM or 5 lM MG132were significantly greater than in the untreated group(21% and 30% vs. 4%, P < 0.05; Table 3). The 2-cellembryos were then transferred into pseudopregnantfemales to assess their developmental ability. Table 3shows that oocytes treated with 1 lM or 5 lM MG132supported full-term development: one and six pupswere born, respectively (5% and 21%; Table 3; Fig. 5a).

Unlike the B6D2F1 oocytes, when ICR oocytes werecultured with 1 lM or 5 lM MG132, no obvious differ-ences were found in the rate of forming PN or 2-cellstage embryos except for the group treated with 25 lMMG132 (Table 3). However, embryos from oocytes cul-tured with 1 or 5 lM MG132 could reach full term (5%and 18%, respectively; Table 3), even though none ofthe untreated embryos could do so.

Mice derived from caffeine- or MG132-treated oocyteswere fertile when mated as adults with each other orwith ICR strain mice (Fig. 5b).

The cdc2 Kinase Activity and Spindle Morphologyof In Vitro Aged MII Oocytes Treated WithCaffeine or MG132

To investigate whether caffeine or MG132 coulddelay in vitro aging, we examined the cdc2 kinase activ-ity (MPF level) and spindle morphology of oocytes at 16and 34 h after hCG injection with or without caffeine orMG132. The cdc2 kinase activity levels of aged oocyteswere decreased but not significantly different fromfresh oocytes (Table 4). However, the activity in caf-feine-treated oocytes was lower than controls, whereasoocytes treated with MG132 maintained activity similarto fresh controls (Table 4). It should be noted that MPFactivity in denuded oocytes was maintained at a levelsimilar to fresh oocytes, or decreased much moreslowly than reported for oocytes aged with cumuluscells (Liu et al., 2009; Miao et al., 2005). In this study,the surrounding cumulus cells were removed fromoocytes before ICSI so the MPF activity in untreatedaged oocytes was not reduced significantly.

Morphometric analysis of MII stage oocytes showedthat the pole-to-pole distance of spindles in agedoocytes was greater than in fresh controls (35.7 6 3.5lm vs. 29.3 6 2.4 lm, P < 0.001), but spindle diameterwas similar (Fig. 2f, ‘‘34h"; Table 4). When caffeine was

FIG. 5. Mice derived from aged B6D2F1 oocytes and their offspring. (a) Mice derived from B6D2F1 oocytes cultured with 5 lM MG132and subjected to ICSI at 34 h after hCG. (b) This mouse was fertile.

Table 4The cdc2 Kinase Activity (MPF Level) and Spindle Morphology

Type of oocyte Cdc2 kinase activity1 Width Height

Control2 100.06 6.9a 29.36 2.4 lme 16.06 1.3 lmg

Aged Untreated 92.16 6.8b 35.76 3.5 lmf 14.76 2.6 lmg

10 mM caffeine 80.76 9.6c 28.56 2.6 lme 12.36 1.7 lmh

5 lMMG132 108.56 9.1d 36.56 3.4 lmf 15.66 2.2 lmg

1Percentages based on fresh controls.2Fresh oocytes.a vs. c, b vs. c and d, c vs. d, e vs. f, g vs. h Values with different superscripts in the same column are significantly different (P < 0.001).MPF, maturation-promoting factor.

466 ONO ET AL.

used in the culture medium, the pole-to-pole distanceand spindle diameters were similar to or less than freshoocytes (12.3 6 1.7 lm vs. 16.0 6 1.3 lm, P < 0.001;Fig. 2f, ‘‘34h1Caff"; Table 4). There were no significantdifferences in spindle dimensions between MG132-treated and untreated oocytes (Fig. 2f, ‘‘34h1MG"; Ta-ble 4).

Production of Offspring From Fresh OocytesCultured in the Presence of Caffeine or MG132

The studies described above demonstrated that caf-feine and MG132 could delay oocyte aging. However, ifthese chemicals are toxic to oocytes and prevent nor-mal fertilization, they cannot be used for conventionalIVF. Therefore, we performed IVF in the presence or ab-sence of these chemicals. As shown in Table 5, the ratesof normal PN formation, blastocyst development, andfull-term development were not significantly differentbetween controls and chemically treated groups. Ran-domly selected offspring (10 pairs from each group)were fertile when they grew to adulthood. Thus, thesedata strongly suggest that caffeine and MG132 are nottoxic for carrying out IVF with fresh oocytes and do notimpair full-term development.

DISCUSSION

In this study, we successfully obtained healthy offspringfrom oocytes aged in vitro by ICSI. The maximumoocyte age that permitted fertility was 30 h after hCGinjection in B6D2F1 oocytes and 26 h in ICR oocytes.This could be extended to 34 h and 28 h, respectively,when caffeine or MG132 were added to the medium.Importantly, these chemicals had no toxic effects onfresh oocyte IVF and subsequent development.

The fertilizable life of ovulated mammalian eggs is lim-ited and many biochemical and cytoskeletal changesensue soon after ovulation (Kikuchi et al., 2000; Marstonand Chang, 1964). Postovulatory aged eggs also lose theirability to be fertilized normally and their capacity to giverise to fully viable embryos is reduced (Lanman, 1968;Tarin et al., 1998; Tesarik, 1993; Winston et al., 1993).

The present results showed that both B6D2F1 and ICRstrain mouse oocytes lost their developmental potentialprogressively and retained normal potential until 30 or26 h after hCG (18 and 14 h in vitro), respectively (Fig.1; Table 1). The rates of abnormalities were obviouslyincreased at the PN stage. These results are in agreementwith a previous report (Mononmani et al., 2004). Gordoet al. (2000, 2002) showed that initiation of internal cal-cium ion [Ca21]i oscillations by injection of sperm factor,by adenophostin A or by the sperm itself after ICSI, wereresponsible for inducing fragmentation and apoptosis ofoocytes aged in vitro.

In this study, we succeeded in obtaining live offspringfrom oocytes that had aged for several hours past thenormal limit using caffeine and MG132 (Table 3). It isknown that when the MII spindle becomes abnormal inoocytes aged in vitro—leading to misaligned chromo-somes or to dispersed, elongated or completely dis-rupted spindles—it will lead to poor rates of develop-ment (Wakayama et al., 2004). When MPF activitydecreases, it reduces the potential of oocytes for fertil-ization, increases cell cycle progression and decreasesthe potential for further development (Liu et al., 2009).Surprisingly, although treatment with caffeine main-tained spindle dimensions to be the same as seen infresh oocytes, MG132 did not influence this duringpostovulatory aging (Table 4). On the other hand,although treatment with MG132 maintained high MPFactivity as in fresh oocytes, caffeine reduced it com-pared with untreated aged oocytes. Therefore, it is notclear why these chemicals could delay oocyte aging andhigh levels of MPF activities and spindle dimensionsalone cannot indicate whether aged oocytes might beable to develop to full term after fertilization. Theseresults suggest that other unknown essential factors(apart from spindle morphology and MPF activity) areinvolved in maintaining developmental capacity in agedoocytes.

Qiao et al. (2008) have reported that cumulus cellsaccelerated the aging of mouse oocytes by secreting asoluble factor(s), whereas in our study, oocyte agingwas delayed even in the presence of many cumulus

Table 5Full-Term Development of Zygotes after IVF in the Presence of Caffeine or MG132

Treatment GroupNo. collected

embryos after IVFNo. with normalPN formation (%) 2-cell (%)1,2

Blastocyst(%) ET (recipients)

Implantation(%)

Offspring(%)

Control Culture 178 124 (70) 40 (100) 39 (98) – – –ET – – 84 (100) – 84 (4) 71 (85) 48 (57)

Caffeine Culture 489 329 (67) 161 (100) 142 (88) – – –ET – – 168 (100) – 168 (8) 125 (74) 91 (54)

MG132 Culture 444 370 (83) 159 (99) 155 (97) – – –ET – – 189 (100) – 189 (9) 154 (81) 97 (51)

1Based on the number of pronuclear zygotes.2All zygotes were divided into two groups for in vitro culture or embryo transfer (ET).IVF, in vitro fertilization; PN, pronuclear.

467OFFSPRING FROM AGED MOUSE OOCYTES

cells when caffeine was added to the culture medium.Therefore, it was not possible to discern whether caf-feine exerted its action on the oocytes directly or coun-teracted the release of the soluble factor(s) from cumu-lus cells. However, in a study on the pig, caffeineincreased MPF activity in aged oocytes lacking cumuluscells (Kikuchi et al., 2002), which suggests that caffeinecan affect the oocyte directly in preventing aging.

We examined the acetylation levels of H3K9, H3K14,H4K5, and H4K12 in B6D2F1 mouse oocytes (see Fig.4). A previous study examined the acetylation levels ofvarious lysine residues on histones H3 and H4 during invitro postovulatory aging of Kunming mouse oocytes(Huang et al., 2007). This showed that the acetylationlevels of H3K14 and H4K12 increased gradually withpostovulatory aging. However, in this study we couldnot observe any changes in fluorescence signal inten-sities for any of the antibodies examined (see Fig. 4).These differences were probably caused by differentmouse strains being used or by variations in cultureconditions. Although both H3 and H4 acetylation levelswere maintained in B6D2F1 oocytes until 34 h afterhCG injection, these could not develop to full term afterICSI. These results suggest that the loss of developmen-tal potential was not caused by changes in histone acet-ylation during postovulatory aging.

Although the rate of development to term wasdecreased with time in vitro (Fig. 1c), there were no dif-ferences in sex ratio and weight gain among offspringderived from fresh or various stages of aged oocytes(Fig. 3; Table 2). These offspring also showed normalreproductive potential, indicating that the aging did notaffect all oocytes equally and that a few oocytes still pos-sessed relatively normal potential for full-term develop-ment. The ICR strain oocytes lost developmentalcapacity faster than did the B6D2F1 oocytes. Probably,this was also caused by inherent strain differences, orperhaps the medium and culture conditions were notoptimal for ICR oocytes.

This is the first report documenting the time beforeICSI over which the developmental potential of B6D2F1and ICR mouse oocytes can be maintained in vitro. Wealso succeeded in producing live offspring by delayingthe in vitro aging of mouse oocytes using treatmentwith caffeine or MG132. The factors enabling full-termdevelopment in oocytes were clearly maintained tosome extent by these treatments during postovulatoryaging. Importantly, these two chemicals did not impairIVF or subsequent full-term development when freshoocytes were used. Therefore, we suggest that it wouldbe safe to use these chemicals in routine IVF culturemedium. Although complete failure of oocytes to be fer-tilized by IVF is rare, it can occur for unknown reasons.However, if the IVF medium were to contain caffeine orMG132, then live offspring could be generated by ICSIof these AFF oocytes.

METHODS

Animals

Adult female and male B6D2F1 strain mice (2-3months old) and adult female ICR mice (2–6 monthsold) were purchased from Shizuoka Laboratory AnimalCenter (Hamamatsu, Japan). All animal experimentsconformed to the Guide for the Care and Use of Labora-tory Animals and were approved by the InstitutionalCommittee of Laboratory Animal Experimentation ofthe RIKEN Kobe Institute.

Collection and In Vitro Aging of Mouse Oocytes

Mature B6D2F1 female mice were superovulated byintraperitoneal injections of 5 IU pregnant mare serumgonadotropin (PMSG; Teikokuzoki Co., Tokyo, Japan)and 5 IU human chorionic gonadotropin (hCG; Teikoku-zoki Co.) at 48-h intervals. COCs were collected fromthe oviducts about 16 h after the hCG injection. Forfresh control oocytes, cumulus cells were dispersedwith 0.1% bovine testicular hyaluronidase (type IV-S,embryo tested; Sigma-Aldrich, St. Louis, MO) in dropletsof HEPES-buffered CZB medium (HEPES-CZB) (Chatotet al., 1990). After several minutes, the oocytes weretransferred to fresh droplets of HEPES-CZB and weredenuded of almost all cumulus cells by gentle pipetting(Ono et al., 2008; Yamagata et al., 2009b). For in vitroaged oocytes, COCs were placed in droplets of TYH me-dium (Toyoda et al., 1971) with 0.1% bovine testicularhyaluronidase, which mimicked the presence of sper-matozoa, and covered with sterile mineral oil, until 22,26, 30, 34, or 40 h after hCG (i.e., 6, 10, 14, 18, or 24 hin vitro) at 378C under 5% CO2 in air. Note, the qualityof hyaluronidase is depend on the lot, therefore prelimi-nary experiments is essential before use. Oocytes werethen transferred to fresh droplets of TYH medium.Denuded oocytes with homogeneous ooplasm wereselected and resuspended in new droplets of CZB me-dium covered previously with mineral oil. The oocyteswere then cultured at 378C under 5% CO2 in air untilused.

Caffeine or MG132 Treatments

After collecting COCs, they were placed in dropletsof TYH with 0.1% bovine testicular hyaluronidase andcaffeine (1, 10, or 20 mM; Sigma-Aldrich) or MG132(1, 10, or 25 lM; Calbiochem, Darmstadt, Germany) for18 h (34 h after hCG, B6D2F1) or 12 h (28 h after hCG,ICR) at 378C under 5% CO2 in air. After treatment,oocytes were transferred to fresh droplets of TYH me-dium. Denuded oocytes with homogeneous ooplasmwere selected and resuspended in new droplets of CZBmedium, which had been covered with mineral oil. Theoocytes were then cultured at 378C under 5% CO2 in airuntil used.

468 ONO ET AL.

Sperm Collection, IVF, ICSI, Embryo Culture, andEmbryo Transfer

For ICSI, spermatozoa were collected from the caudaepididymidis of a mature B6D2F1 male for each experi-ment and incubated in CZB medium for 30 min at 378Cunder 5% CO2 in air. One-microliter aliquots of the sus-pension were placed in droplets of 10% polyvinyl pyrro-lidone (PVP)-HEPES-buffered HTF solution (Irvine Scien-tific, Santa Ana, CA) in a micromanipulation chamber.ICSI was performed as described (Kimura and Yanagi-machi, 1995; Ohta et al., 2008). Briefly, the sperm headwas separated from the tail by applying several piezopulses to the neck region and the head was theninjected into an oocyte. After 10 min of recovery atroom temperature, the oocytes were cultured in CZBmedium. In these experiments, aged oocytes were usedonly if they had normal surface features and were notactivated spontaneously. Usually, fewer than 10% ofoocytes were discarded as abnormal.

For IVF, B6D2F1 spermatozoa from the cauda epididy-midis were cultured in TYH medium for at least 1 h at378C under 5% CO2 in air. Cumulus-intact oocytes werecollected in 0.2 mL of TYH medium containing 10 mMcaffeine or 5 lM MG132 and inseminated with capaci-tated sperm (final concentration 100/lL).

After 5–6 h culture, PN formation and oocyte activa-tion were evaluated. The developmental competence ofoocytes was evaluated based on the percentage ofsperm-injected oocytes (ICSI) or pronuclear zygotes(IVF) that reached the morula or blastocyst stage after96 h in vitro.

Two-cell embryos (one day of culture) or 4- to 8-cellstage embryos (two days of culture) were transferredinto the oviducts of pseudopregnant ICR strain femalesat 0.5 days post coitus (dpc) and offspring were obtainedat 18.5 or 19.5 dpc through natural birth or Caesariansection. For B6D2F1 mouse oocytes, the weight gain ofoffspring was examined every two weeks.

Immunofluorescence Microscopy andMeasurement of Spindle Morphology inMetaphase (M) II Oocytes

B6D2F1 MII oocytes at 16 h or 34 h after hCG,untreated or treated with 10 mM caffeine or 5 lMMG132, were fixed in phosphate-buffered saline (PBS)containing 4% paraformaldehyde for 30 min. The fixedoocytes were washed twice in PBS containing 1% (w/v)bovine serum albumin (BSA; Nacalai Tesque, Kyoto, Ja-pan) (PBS-BSA) for 15 min each and then stored in PBS-BSA and 0.1% (v/v) Triton X-100 (Nacalai Tesque) over-night at 48C. All subsequent steps were carried out atroom temperature.

Primary antibody incubations were carried out inPBS-BSA for 2 h. Primary antibodies used here were rab-bit polyclonal anti-acetyl-histone H3K9 (1:250; Upstate,

Lake Placid, NY), H3K14 (1:250; Upstate), H4K5(1:100; Upstate), H4K12 (1:100; Upstate) and mousemonoclonal anti-b-tubulin (1:100; BD Biosciences, SanJose, CA). After the oocytes had been washed twice inPBS-BSA for 15 min each, they were incubated for 1 hwith conjugated secondary antibodies, Alexa-Fluor-488-labeled goat anti-rabbit IgG (Molecular Probes Inc.,Eugene, OR), Alexa-Fluor-488-labeled goat anti-mouseIgG (Molecular Probes) or Alexa-Fluor-568-labeled goatanti-mouse IgG (Molecular Probes). After the oocyteshad been washed twice in PBS-BSA for 15 min each, theDNA was stained with propidium iodide (PI; 1 lg/mL;Sigma-Aldrich). Serial images were taken using fluores-cence confocal microscopy. Three-dimensional imagesof oocytes were reconstructed and spindles were meas-ured using MetaMorph software (Universal Imaging,Downingtown, PA) (Yamagata et al., 2009a, 2009b). Foreach treatment, more than 30 samples collected onthree different experimental days were assayed.

Measurement of MPF in MII Oocytes

MII stage oocytes at 16 h and 34 h after hCG,untreated or treated with 10 mM caffeine or 5 lMMG132, were analyzed for the activity of the catalyticcdc2 kinase subunit of MPF using a MESACUP cdc2 Ki-nase Assay Kit (MBL, Nagoya, Japan). For each assay, 10oocytes were washed in sample buffer (50 mM Tris-HClpH 7.5, 0.5 M NaCl, 5 mM EDTA, 2 mM EGTA, 0.01%Briji35, 1 mM PMSF, 0.05 mg/mL leupeptin, 50 mM 2-mercaptoethanol, 25 mM b-glycerophosphate, and 1mM Na-orthovanadate) and stored in a 1.5 mL tube at2808C. The assay was performed according to the man-ufacturer’s instructions. The cdc2 kinase activity ineach well was detected at a wavelength of 490 nm usinga microplate reader (Wallac 1420 Multilabel Counter;PerkinElmer, Waltham, MA). Each treatment wasrepeated at least three times.

Statistical Analysis

Outcomes were evaluated using v2-tests or Student t-test, and P < 0.05 was regarded as statisticallysignificant.

ACKNOWLEDGMENTS

The authors thank H. Ohta, T. Oyanagi, S. Hirauchi, andY. Sakaide for preparing this manuscript. They are grate-ful to the Laboratory for Animal Resources and GeneticEngineering of RIKEN Kobe Center for housing themice.

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