Temporal effects of exogenous oocyte-secreted factorson bovine oocyte developmental competence during IVM

Tamer S. HusseinA, Melanie L. Sutton-McDowallA, Robert B. GilchristA,B

and Jeremy G. ThompsonA,B,C

ARobinson Institute, Research Centre for Reproductive Health, School of Paediatrics

and Reproductive Health, The University of Adelaide, Adelaide, SA 5005, Australia.BJoint last authors.CCorresponding author. Email: jeremy.thompson@adelaide.edu.au

Abstract. We investigated whether paracrine signalling between the bovine oocyte and cumulus cells is altered during

the course of in vitro maturation (IVM). Bovine COCs were cocultured with denuded oocytes or treated with specificoocyte-secreted factors, namely recombinant bone morphogenetic protein (BMP)-15 or growth differentiation factor(GDF)-9, beginning from 0 or 9 h IVM. To generate a 9-h denuded oocyte (DO) group, COCs were cultured intact for thefirst 9 h of IVM and then denuded. Coculturing intact COCs with DOs denuded immediately after collection or following

9 h of maturation did not affect cleavage rate, but improved blastocyst yield (P, 0.05) on Day 8 (51 and 61%,respectively; P, 0.05) and cell number compared with COCs cultured alone (41%). Significantly, we observed higherlevels of endogenous GDF-9 and BMP-15 protein in oocytes of COCs matured for 9 h compared with no incubation.

The addition of 175 ngmL�1 GDF-9 or 10%v/v BMP-15 from partially purified transfected 293H cell supernatant for24 h IVM significantly enhanced development to the blastocyst stage from 40% (control) to 51 and 47%, respectively(P, 0.05). However, treatment of COCs with GDF-9 or BMP-15 between 9 and 24 h of IVM did not increase blastocyst

yield. These results provide evidence of quantitative and possibly qualitative temporal changes in oocyte paracrine factorproduction during IVM.

Additional keywords: bone morphogenetic proteins, cumulus–oocyte complex, growth differentiation factors.

Introduction

From the earliest stages of follicular growth, intercellular

communication between all cell types within the ovarian follicleis important for the acquisition of oocyte developmental com-petence, which entails the capacity of the oocyte to produce a

normal and viable embryo after fertilisation and, followingembryo transfer, will support development to term (Gilchristand Thompson 2007). During the antral phase of folliculo-

genesis, oocyte-secreted factors (OSFs) play a crucial role indifferentiating granulosa cells (GCs) into two anatomically andfunctionally distinct subtypes: (1) the mural granulosa cells(MGC), the cells lining the follicle wall; and (2) the cumulus

cells (CCs), the cells surrounding the oocyte (Eppig et al. 1997;Li et al. 2000; Gilchrist et al. 2004a). The oocyte maintainsintimate contact with CCs via an extensive network of gap

junctions, forming the cumulus–oocyte complex (COC;Albertini et al. 2001). The close association between the oocyteand CCs is fundamental to CC development and the mainte-

nance of oocyte health (Buccione et al. 1990; Gosden et al.1997; Gilchrist et al. 2004a).

The CCs play a major role in the maintenance of oocyte

meiotic arrest by the direct transfer, via gap junctions, of smallmolecules, in particular cyclic nucleotides (Anderson and

Albertini 1976; Moor et al. 1980; Eppig 1982; Sirard andBilodeau 1990). Moreover, CCs play a crucial role in promoting

oocyte maturation and the acquisition of full embryonic devel-opmental competence (Ka et al. 1997; Fulka et al. 1998; Tangheet al. 2002). The CCs influence both transcription and post-

translational modification of oocyte proteins (Cecconi et al.1991) and their removal from bovine oocytes before in vitro

maturation (IVM) is detrimental to oocyte maturation, fertilisa-

tion and embryo development (Fukui and Sakuma 1980; Zhanget al. 1995; Fatehi et al. 2002).

In recent years it has become clear that oocytes potentlyregulate many of the distinctive functions of CCs, including

proliferation, steroidogenesis, differentiation, metabolism,apoptosis and expansion (Eppig 2001; Gilchrist et al.2004a). These paracrine actions of the oocyte are largely

attributed to members of the transforming growth factor(TGF)-b superfamily, in particular growth differentiationfactor (GDF)-9 and bone morphogenetic protein (BMP)-15.

For example, GDF-9 and BMP-15 are known to stimulateMGC and CC proliferation (Gilchrist et al. 2004b, 2006;McNatty et al. 2005), CC expansion (Gilchrist et al. 2004a;

Dragovic et al. 2005) and metabolism (Eppig et al. 2005;Sugiura and Eppig 2005) and BMP-15 also prevents CC

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Reproduction, Fertility and Development, 2011, 23, 576–584 www.publish.csiro.au/journals/rfd

� CSIRO 2011 10.1071/RD10323 1031-3613/11/040576

apoptosis by maintaining a localised gradient of anti-apoptoticfactors within the COC (Hussein et al. 2005).

Despite considerable efforts over the past four decadesto improve efficiency there has been little real success andthe IVM of oocytes remains inefficient at producing viable

embryos and offspring compared with in vivo-matured andharvested oocytes (Thompson et al. 1995; Eppig et al. 2009).We have shown previously that the addition of native OSFs to

cattle COCs (by the coculture of denuded oocytes with intactCOCs) or the addition of either recombinant GDF-9 or BMP-15to both cattle and mouse COCs significantly enhances thedevelopmental competence of IVM oocytes, as assessed by

enhanced blastocyst yield and quality (Hussein et al. 2006; Yeoet al. 2008). Furthermore, the addition of GDF-9 during mouseIVM improves fetal survival after transfer (Yeo et al. 2008).

These studies suggest a deleterious consequence of IVM ineither the dilution, or reduced signalling, of OSFs that facilitatethe communication from the oocyte to the CCs. In other studies,

we have reported that by 9 h after the removal of the bovineCOC from the follicular environment, gap junction communi-cation is lost between the oocyte and CCs and that the rate ofloss of gap junctions is dependent on IVM culture conditions

(Thomas et al. 2004a, 2004b). In the present study, we investi-gated the effect of CC on bovine oocyte developmental compe-tence during the course of IVM. Specifically, we were

interested in what proportion of the oocyte’s developmentalcompetence was bestowed during the first 9 h ofmaturation (i.e.during the time of gap junctional communication) compared

with the following 15 h and whether exogenous native orrecombinant OSFs influenced oocyte developmental compe-tence during or after the period of gap junction communication.

As such, we hypothesised that the benefit of OSFs (either nativeor recombinant) in improving the developmental competenceof bovine oocytes during IVM is mediated in the first 9 h ofmaturation. We found that this was the case for the addition

of recombinant GDF-9 and BMP-15, but that supplementingnative OSFs continued to positively affect oocyte competencebeyond the first 9 h of maturation.

Materials and methods

Collection of oocytes and culture conditions

Unless specified otherwise, all chemicals and reagents werepurchased fromSigma (St Louis,MO,USA). Oocytematuration

and embryo production techniques have been described previ-ously (Hussein et al. 2006). Briefly, bovine ovaries were col-lected from abattoirs and transported to the laboratory in warm

saline (30–358C). TheCOCswere aspirated from 3–8-mmantralfollicles. Maturation was performed in bovine maturationmedium (IVF Vet Solutions, Adelaide, SA, Australia), a medi-

um based on the ionic composition of bovine follicular fluid(Sutton-McDowall et al. 2005). All IVM treatments were sup-plemented with 0.1 IUmL�1 FSH (Puregon; Organon, Oss,Netherlands). Complexes (1 per 10 mL) were cultured in pre-

equilibrated 200-mL drops overlaid with mineral oil and incu-bated at 398C with 5% CO2 in humidified air for 24 h. Denudedoocytes (DOs) were derived by removing CCs from COCs by

vortexing as described by Hussein et al. (2006).

IVF and embryo culture

In vitro production of embryos was undertaken using defined,serum-free media (IVF Vet Solutions) and methodology

described previously (Hussein et al. 2006). Briefly, motilespermatozoa were separated from thawed semen using a dis-continuous (45% : 90%) Percoll gradient (Amersham Biosci-

ence, Uppsala, Sweden). Spermatozoa were resuspended in IVFmedium (Bovine VitroFert; IVF Vet Solutions), then added tothe fertilisation media drops at a final concentration of sper-

matozoa 1� 106mL�1. COCs or DOs were inseminated at adensity of 10 mL IVF medium per COC or DO for 24 h, at 398Cin 6% CO2 in humidified air.

In treatment groups in which the COC remained intact at the

time of fertilisation, the CCs were removed by gentle pipetting23–24 h after insemination. Five presumptive zygotes weretransferred into each 20-mL drop of pre-equilibrated cleavage

medium (IVF Vet Solutions) and cultured under mineral oil at38.58C in 7%O2, 6%CO2 and balance N2 for 5 days (Days 1–5).On Day 5, embryos were transferred in groups of five to six

20-mL drops of pre-equilibrated blastocyst medium (IVF VetSolutions) at 38.58C, overlaid with mineral oil, cultured untilDay 8 and assessed for quality on Day 8, as described previously

(Hussein et al. 2006). Assessments were performed indepen-dently and blinded by an experienced bovine embryologist(independent evaluator).

Differential staining

Cell countswere performed as described byHussein et al. (2006)

using a modified version of the technique described by Fouladi-Nashta et al. (2005). Briefly, blastocysts were placed intoprotein-free acidTyrode’s solution to remove the zona pellucida.

Zona-free embryos were washed and then incubated at 48Cfor 10min in 10mM trinitrobenzene sulfonic acid (TNBS) inphosphate-buffered saline (PBS)–polyvinyl alcohol (PVA), thensubsequently incubated at 378C for 10min with 0.1mgmL�1

anti-dinitrophenol–BSA antibody (Molecular Probes, Eugene,OR,USA). Following complement-mediated lysis using guinea-pig complement, embryos were washed in PBS/PVA and incu-

bated in 10mgmL�1 propidium iodide for 20min at 378C (tostain the trophectoderm, TE), followed by 4mgmL�1 bisbenzi-mide (Hoechst 33342; Sigma-Aldrich, St Louis, MO, USA)

in 100% ethanol at 48C overnight (to stain both the inner cellmass (ICM) and trophectoderm (TE)). Embryos were examinedat 400� under a fluorescence microscope (Olympus, Tokyo,

Japan) equipped with an ultraviolet filter and a digital camera todetermine total and compartment cell counts, where ICM nucleiappeared blue and TE nuclei stained pink.

Recombinant OSFs

Recombinant mouse GDF-9 and recombinant ovine BMP-15were produced and partially purified in-house as describedpreviously (Gilchrist et al. 2004b; Hickey et al. 2005; Hussein

et al. 2005) using transfected 293 human embryonic kidneycell lines (293H) donated by O. Ritvos (University of Helsinki,Helsinki, Finland). Control conditioned medium (hereafter

designated ‘293H’) was also produced from untransfected 293Hcells and partially purified (Hickey et al. 2005).

Temporal actions of oocyte-secreted factors Reproduction, Fertility and Development 577

Experiment 1: assessment of oocyte developmentalcompetence following coculture of intact COCswith DOs during IVM

This experiment was designed to assess embryo developmentalcompetence after coculture of COCs with DOs during IVM, inwhich exposure was either from 0 or 9 h until 24 h of maturation.

An outline of the experimental design is shown in Fig. 1. TheCOCs were randomly allocated into one of six treatment groupsas follows (see Fig. 1).

1. Treatment 1: 20 COCs underwent standard IVM for 24 h.2. Treatment 2: 20 DOs oocytes underwent standard IVM for

24 h.

3. Treatment 3: 20 COCs were cocultured from 0 to 24 h with100 DOs, after which the COCs and DOs were separated toform Treatments 3 and 4, respectively (see Fig. 1).

4. Treatments 5 and 6: 20COCswere initially cultured alone for9 h but, from 9 to 24 h of IVM, were cocultured with 100DOs, which had been cultured from 0 to 9 h as intact COCs.

At the end of 24 h, these COCs and DOs were separated toform Treatments 5 and 6, respectively (Fig. 1).

All IVMwas performed in 200-mL drops and COCs and DOswere fertilised after 24 h of IVM. The number and quality of

blastocysts formed was assessed on Day 8 of culture. Sevenreplicate experiments were performed.

Experiment 2: localisation of BMP-15 and GDF-9within COCs at 0 or 9 h of IVM

Because results from Experiment 1 suggested either qualitative

and/or quantitative differences in OSF production withinoocytes after 0 and 9 h of IVM,we examined whether there weremeasurable differences in intraoocyte GDF-9 and BMP-15

content after these periods of maturation. The COCs werewashed once in PBS and fixed in 4% paraformaldehyde for30min before being transferred onto polylysine-coated micro-

scope slides. Further washing with Ca2þ- and Mg2þ-free PBSwas followed by permeabilisation for 90minwith 0.25%Triton-X plus 0.1% sodium citrate in PBS at room temperature in

humidified chambers. The COCs were further washed with PBSand incubated with 10% normal donkey serum in PBS for120min. Primary antibodies against BMP-15 (A-20; Santa CruzBiotechnology, Santa Cruz, CA, USA) and GDF-9 (SC-C20;

Santa Cruz Biotechnologywere diluted 1 : 250with 10%normaldonkey serum in PBS and cocultured with COCs overnight at48C. Following three washes with PBS, COCs were cultured

with a secondary antibody (fluorescein isothiocyanate (FITC)-labelled donkey anti-goat IgG, 1 : 200 dilution in PBS; SantaCruz Biotechnology) for 120 min, washed in PBS, then coun-

terstained with 40,60-diamidino-2-phenylindole (DAPI) for30min before being mounted in PBS. Dual fluorescenceintensity was visualised using a Nikon C1 confocal scanninghead attached to a Nikon TE2000E microscope (Nikon, Tokyo,

Japan). BMP-15/GDF-9 staining (FITC) was visualised at laserexcitation 488 nm and emission 500–530 nm, whereas DAPIcounterstaining (nuclear) was visualised at laser excitation

405 nm and emission 425–455 nm. Negative controls werecreated by replacing the primary antibody with 10% donkeyserum in PBS and coincubating 2mL primary antibody with

10 mL blocking peptide (BMP-15, SC-18337P and GDF-9,SC-7407P; Santa Cruz) overnight at 48C before use, accordingto the manufacturer’s instructions.

Fluorescence intensity within cross-sections of oocytes wasdetermined using Photoshop CS3 (Adobe Systems, San Jose,CA, USA) and Scanalytics IPlab software version 3.6(Scanalytics, Fairfax, VA, USA). The mid-section of an oocyte

was divided into four equally sized regions (Fig. 2a). Allmeasure-ments were corrected for background fluorescence and differ-ences in intensity within regions (CCs and intraoocyte) were

compared. Three experimental replicates were performed with15 oocytes used per replicate and time point.

Experiment 3: assessment of oocyte developmentalcompetence following temporal treatment of COCswith GDF-9 or BMP-15 during IVM

Previously, we showed that the addition of recombinant GDF-9

or BMP-15 to COCs during IVM improved oocyte develop-mental competence (Hussein et al. 2006). In the present study,we conducted Experiment 3 to determine whether there is a

temporal effect of the addition of these OSFs during IVM. Weexamined whether GDF-9 or BMP-15 from 0 or 9 h of IVM

24 h0 h

24 h0 h

Treatment 5

Treatment 4

Treatment 3

24 h0 h

Treatment 2

Treatment 1

24 h9 h0 h

9 h0 h

Treatment 6

Fig. 1. Schematic illustration of the coculture system of cumulus–oocyte

complexes (COCs) in the presence or absence of denuded oocytes (DOs)

during IVM.TheCOCs andDOswere divided into six treatment groups after

IVM. Treatment 1¼COC; Treatment 2¼DO; Treatment 3¼COCs cocul-

tured with DOs from 0 to 24 h; Treatment 4¼DOs cocultured with COCs

from 0 to 24 h; Treatment 5¼COCs cultured for 9 h alone, then cocultured

with DOs for the last 15 h of IVM; Treatment 6¼ oocytes cultured for 9 h

before denudation, then cocultured with COCs for the final 15 h of IVM.

578 Reproduction, Fertility and Development T. S. Hussein et al.

(i.e. present for 0–24 or 9–24 h) improves subsequent oocytedevelopmental competence.TheCOCswerematured as describedabove, with the following treatment groups established: (1) con-

trol; (2) 175 ngmL�1 GDF-9 from 0h of IVM (0–24 h); (3)175 ngmL�1 GDF-9 from 9h of IVM (9–24 h); (4) 10%v/v

BMP-15 from 0h of IVM (0–24h); (5) 10%v/v BMP-15 from 9hof IVM (9–24 h); (6) 10%v/v 293H from 0h of IVM; and

(7)10%v/v293Hfrom9hof IVM.After IVM,allcomplexeswerefertilised and blastocyst formation was assessed on Day 8. Fourreplicatesof these experimentswere performedusing30COCsper

treatment group per replicate experiment. A further cohort ofCOCs (n¼ 80) was subjected to either no treatment or treatmentwithGDF-9, BMP-15 or 293H from0 to 24 h using concentrations

as described above and was assessed for meiotic competencefollowing 24 h of maturation by orcein staining.

Experiment 4: temporal addition of native OSFsor exogenous BMP-15 during IVM

This experiment was conducted to determine whether adding

native OSFs or exogenous recombinant BMP-15 has similartemporal effects on enhancing oocyte developmental compe-tence. The COCs were either cultured alone, cocultured with

DOs from 0 or 9 h of IVM (0–24 or 9–24 h, respectively) orcocultured with DOs for just the first 9 h of IVM (0–9 h; at 9 h,the COCs were removed and then matured in the absence of DO

from 9 to 24 h of IVM). These treatments were replicated using10%v/v BMP-15 from 0 or 9 h or for just the first 9 h of IVM(0–9 h). After IVM, all complexes were fertilised and blastocyst

formation was assessed on Day 8. Three replicates were per-formed using 30 COCs per treatment group per replicateexperiment.

Statistical analyses

All replicated proportional developmental data were arcsine

transformed before analysis. Statistical analyses were per-formed by ANOVA using SigmaStat software (SPSS Inc.,Chicago, IL, USA), and significant differences between means

were determined using the Tukey–Kramer post hoc test forcomparison of multiple means. Differences were consideredsignificant at P, 0.05. Analysis of cell numbers and arbitrary

fluorescence units was conducted using one-way ANOVA.

Results

Experiments 1 and 2: assessment of oocyte developmentalcompetence following coculture of intact COCswith DOs during IVM

A diagram of the experimental design for Experiment 1 is pro-vided in Fig. 1 and the embryo development data are given inTables 1 and 2. Exposure of intact COCs to native OSFs from

DOs significantly increased the number of oocytes that reachedthe blastocyst stage at Day 8 after insemination compared withCOCs cultured alone. Although blastocyst rates were signifi-cantly increased above control (Treatment 1¼ 41%) by cocul-

turing with DOs from 0 h of IVM (Treatment 3¼ 51%;P, 0.05; Table 1), interestingly blastocysts rates were signifi-cantly (P, 0.05) higher than either of these treatments when

DOs were added from 9 h of IVM (Treatment 5¼ 61%). Fur-thermore, coculturing COCs with DOs (Treatments 3 and 5)significantly increased total cell numbers of the resulting

blastocysts, with DOs added at 9 h (Treatment 5) producingblastocysts with the highest number of trophectoderm cells

10

20

30

40

50

60

70

BMP-15

GDF-9

0 h 9 h Blocking

0 h 9 h Blocking

Flu

ores

cenc

e in

tens

ity

*

**

*

4321

(a)

(b)

1 2 3 4

Flu

ores

cenc

e in

tens

ity

20

30

40

50

60

70

80

90

Intraoocyte regions

1 2 3 4

Intraoocyte regions

*

*

*

*(c)

Fig. 2. Immunohistochemical quantification of bone morphogenetic pro-

tein (BMP)-15 and growth differentiation factor (GDF)-9 proteins within

cumulus–oocyte complexes (COCs) after 0 and 9 h of maturation in vitro.

(a) A cross-section of the COC was divided into four evenly spaced regions

and fluorescence intensity was measured within each region. (b, c) Fluores-

cence intensity of BMP-15 (b) and GDF-9 (c) staining. Data are the

means� s.e.m. *P, 0.001 compared with 0 h.

Temporal actions of oocyte-secreted factors Reproduction, Fertility and Development 579

(P, 0.05; Table 2). Because these data suggest a temporal

effect during IVM on the production of OSFs, we quantifiedintraoocyte GDF-9 and BMP-15 levels at 0 and 9 h of IVM. Insupport of the data in Tables 1 and 2, after 9 h ofmaturation as an

intact COC, there were significantly higher levels of BMP-15and GDF-9 protein within the oocyte compared with levels infreshly isolated COCs (Fig. 2b, c; P, 0.001), suggesting an

accumulation and/or production of these proteins during IVM.As expected, removal of CCs before IVM significantly

decreased the number of oocytes that reached the blastocyst

stage at Day 8 after insemination compared with intactCOCs (Treatment 1: COC¼ 41% v. Treatment 2: DO¼ 13%;P, 0.05; Table 1). Furthermore, the presence of CCs (fromneighbouring COCs) did not improve the developmental capa-

bility of DOs (Treatment 4: DO), because blastocyst rates weresimilar to DOs cultured alone (Treatment 2: DO; Table 1).However, maturing oocytes with an intact cumulus for the first

9 h before denuding significantly improved the blastocyst ratecompared with DOs cultured alone (Treatment 2: DO¼ 13% v.

Treatment 6: DO¼ 25%; P, 0.05; Table 1). Furthermore, the

presence of CCs (from neighbouring COCs) improved subse-quent embryo quality from the denuded oocyte (Treatments 4and 6), regardless of whether they were denuded immediately orafter 9 h ofmaturation (as evidenced by increased total, ICMand

TE cell numbers; P, 0.05; Table 2). The incidence of poly-spermy (as assessed by separate cohorts of oocytes stained withbisbenzimide, Hoescht 33342) did not differ between DOs and

COCs (data not shown).

Experiment 3: assessment of oocyte developmentalcompetence following treatment of COCswith GDF-9 or BMP-15 during IVM

The addition of GDF-9 or BMP-15 to maturing COCs had nosignificant effect onmeiotic competence, as evaluated by orcein

staining after 24 h maturation (between 80 and 95% MII). Bothtreatments significantly enhanced embryo development to theblastocyst stage compared with control COCs (5 and 47% v.

40%, respectively; P, 0.05; Table 3). However, the addition ofGDF-9 or BMP-15 tomaturingCOCs at 9 h after the initiation ofmaturation did not increase the blastocyst yield after insemi-

nation compared with control COCs (Table 3). Conditionedmedium from the parent 293H cell line, when added from 0 h,adversely affected oocyte developmental potential, loweringblastocyst rates by 12% compared with control COCs

(P, 0.05). Conversely, there was no adverse effect on oocytedevelopmental potential when 293H conditioned medium wasadded from 9 h. Embryo cleavage was not significantly affected

by any of the treatments.

Experiment 4: effects of temporal addition of native OSFsor exogenous BMP-15 during IVM on oocytedevelopmental competence

In this experiment, the effects of the addition of DOs or BMP-15to COC for only the first 9 h of IVM were compared withtreatment from 0 h (0–24 h) or from 9 h (9–24 h) of IVM. Blas-

tocyst development rates were increased by the addition of

Table 1. Development of in vitro-produced embryos following coculture of intact cumulus]oocyte complexes with denuded

oocytes during IVM

See Fig. 1 for details of the different treatment groups (Treatments 1–6). Data are the mean� s.e.m. Values with different superscript

letters within the same column differ significantly (P, 0.05). COCs, cumulus–oocyte complexes; DOs, denuded oocytes

Treatments Period of DO–COC

coculture (h)

No. oocytes Cleavage rate (%) % Blastocysts from cleaved

Treatment 1: COC – 163 82.6� 2.7ab 40.7� 1.4a

Treatment 2: DO – 176 58.7� 4.6c 12.7� 0.8b

Treatment 3: COC 0–24 166 89.1� 2.6a 50.6� 1.9c

Treatment 4: DO 0–24 191 69.7� 2.0bc 15.4� 1.6b

Treatment 5: COC 9–24 158 91.8� 1.8a 61.3� 1.9d

Treatment 6: DO 9–24 178 76.1� 3.5b 25.0� 1.5e

Table 2. Number of total, inner cell mass and trophectoderm cells and proportional ratios of Day 8 expanded and hatched

blastocysts following coculture of intact cumulus]oocyte complexes with denuded oocytes during IVM

See Fig. 1 for details of the different treatment groups (Treatments 1–6). Data are the mean� s.e.m. Values with different superscript

letters within the same column differ significantly (P, 0.05). COCs, cumulus–oocyte complexes; DOs, denuded oocytes

Treatments Period of DO–COC

coculture (h)

n Total no. cells Inner cell mass Trophectoderm

Mean� s.e.m. Proportion Mean� s.e.m. Proportion

Treatment 1: COC – 40 148.3� 1.2a 50.0� 1.3a 33.6� 0.6a 98.2� 0.8a 66.4� 0.6a

Treatment 2: DO – 12 118.5� 2.4b 32.2� 1.2b 27.3� 1.1b 86.3� 2.6b 72.7� 1.1b

Treatment 3: COC 0–24 51 156.1� 1.3c 49.4� 1.1a 31.5� 0.5c 105.7� 1.3c 68.5� 0.5c

Treatment 4: DO 0–24 19 137.2� 1.3d 39.7� 1.2c 28.9� 0.7bd 97.5� 0.9a 71.1� 0.7b

Treatment 5: COC 9–24 61 159.9� 0.9c 50.2� 0.9a 31.2� 0.4cd 109.8� 0.4d 68.8� 0.4c

Treatment 6: DO 9–24 31 138.0� 1.1d 36.7� 0.9c 26.5� 0.5b 101.3� 0.7a 73.5� 0.5b

580 Reproduction, Fertility and Development T. S. Hussein et al.

either DOs or BMP-15 to COCs for the full duration of IVM, asobserved in the Experiments 1 and 3 (Table 4). Culturing COCs

with either DOs or BMP-15 for just the first 9 h of IVM, fol-lowed bywashing andmaturation alone for the last 15 h of IVM,did not alter blastocyst rates compared with rates in COCs

cultured with DOs or BMP-15 from the beginning of IVM(Table 4). Consistent with Experiments 1 and 3, treatment ofCOCs with BMP-15 from 9 h did not increase blastocyst yields

compared with control (Table 4). In contrast, treatment of COCswith DOs from 9 h significantly increased blastocyst develop-ment, compared with control (Table 4). Embryo cleavage was

not affected by any oocyte treatment.

Discussion

The results of the present study further substantiate the data ofHussein et al. (2006), and more recently Romaguera et al.(2010), demonstrating that exogenous OSFs directly enhance

oocyte developmental competence during IVM of intact COCs.However, the results here go further in showing that OSFquantity, and possibly quality or characteristics, is also tempo-

rally regulated during maturation.The evidence for temporal regulation is threefold. First,

denuding oocytes at 9 h of maturation and then using these

oocytes in coculture with intact COCs resulted in a higherdevelopmental competence of those COCs compared withCOCs cocultured with oocytes denuded immediately at the time

of collection. Second, using quantitative immunohistochemistrywe investigated whether endogenousGDF-9 and BMP-15 levels

changed during COC maturation. We found that by 9 h ofmaturation these specific OSFs were present in significantlyhigher levels in the oocyte compared with levels in freshly

collected COCs. Third, the addition of native OSFs by coculturewith denuded oocytes influenced oocyte quality within cocul-tured COCs throughout the oocyte maturation period, yet the

beneficial effects of exogenous BMP-15 and GDF-9 on oocytedevelopmental competence were restricted to the first 9 h ofIVM alone.

There are other reports of temporally regulated proteinand/or mRNA production of OSFs, particularly BMP-15(Gueripel et al. 2006; Yoshino et al. 2006; Li et al. 2008; Zhuet al. 2008), or at least temporal effects of native OSFs during

oocytematuration (Gilchrist et al. 2001). However, only Li et al.(2008) have described temporally regulated protein levels ofBMP-15 during IVM in the pig. It is likely that such changes are

regulated by CC–oocyte cross-talk (Gittens et al. 2005) and thatIVM alters this communication network compared with post-LH oocyte maturation in vivo (Hussein et al. 2006; Gilchrist

2011). Furthermore, it is also likely that denuding oocytes eitherbefore or during maturation creates a different OSF secretionprofile for the remainder ofmaturation compared with an oocyte

within an intact COC. This may explain why there are differ-ences in the ability of oocytes denuded at 0 or 9 h to influencedevelopmental competence.

Table 4. Effect of temporal exposure of bovine cumulus]oocyte complexes to denuded oocytes or bone morphogenetic

protein-15 on subsequent bovine embryo development

Data are the mean� s.e.m. Values with different superscript letters within the same column differ significantly (P, 0.05). OSF, oocyte

secreted factor; BMP-15, bone morphogenetic protein-15; COCs, cumulus–oocyte complexes; DOs, denuded oocytes

Treatment Period of OSF exposure (h) No. oocytes Cleavage rate (%) % Blastocysts

COC – 93 86.3� 1.3 38.8� 0.3a

COCþDO 0–9 104 85.5� 1.8 49.8� 1.2b

COCþBMP-15 0–9 104 83.8� 1.9 45.8� 1.0ab

COCþDO 0–24 104 87.0� 1.9 49.8� 1.7b

COCþBMP-15 0–24 106 84.8� 1.1 49.0� 0.6b

COCþDO 9–24 103 88.5� 0.6 58.5� 0.9c

COCþBMP-15 9–24 100 87.3� 2.7 41.5� 0.6a

Table 3. Effects of temporal exposure to recombinant oocyte-secreted factors (growth differentiation factor-9, bone

morphogenetic protein-15 or cell supernatant (293H)) during IVM on the production of bovine embryos

Data are the mean� s.e.m. Values with different superscript letters within the same column differ significantly (P, 0.05). GDF-9,

growth differentiating factor-9; BMP-15, bonemorphogenetic protein-15; 293H, vehicle control (partially purified conditionedmedium

from untransfected 293H cells; Hickey et al. 2005)

Treatment Period of OSF exposure (h) No. oocytes Cleavage rate (%) % Blastocysts

Control – 121 82.8� 1.3a 39.5� 1.3a

GDF-9 0–24 119 80.8� 1.7a 50.8� 1.9b

GDF-9 9–24 127 84.3� 1.1a 41.3� 1.0ac

BMP-15 0–24 118 83.0� 2.4a 47.0� 1.0bc

BMP-15 9–24 129 82.3� 0.9a 39.8� 1.4a

293H 0–24 119 73.3� 2.4b 28.0� 0.9d

293H 9–24 123 80.0� 3.5ab 36.0� 1.6a

Temporal actions of oocyte-secreted factors Reproduction, Fertility and Development 581

The data also show an improvement in denuded oocytecompetence if matured initially as an intact COC for the first

9 h of maturation and then subsequently denuded and coculturedwith COCs compared with other DO treatments. Similar effectshave been reported previously (Luciano et al. 2005) and can be

attributed to gap junction communication rather than mereproximity to the cumulus, because oocytes denuded from freshlycollected COCs have a lower developmental competence.

Consistent with this suggestion, several studies have shownthat gap junction communication between the oocyte andCCs during early maturation is important for the promotion ofoocyte growth and development in vitro (Buccione et al. 1990;

Carabatsos et al. 2000; Kidder and Mhawi 2002).We have reported previously that gap junction communi-

cation between CCs and the oocyte is no longer measurable

after 9 h of IVM (Thomas et al. 2004a, 2004b). Nevertheless,our data suggest that a proportion of the acquisition ofdevelopmental competence is derived from signalling between

the oocyte and surrounding CCs after this 9 h period. Under ourexperimental conditions, this effect appears restricted to theoocyte within the COC, as opposed to the neighbouring DOs.The most plausible explanation is that there remains an

element of junctional communication after this period that isnot measured by themethod described in Thomas et al. (2004a,2004b). Indeed, as argued by Tanghe et al. (2002), the different

techniques for measuring gap junction communication yielddiffering results. An alternative possibility is that CC paracrinesignalling is strongly unidirectional within the intact COC.

This is in contrast with results from a recent paper by McElroyet al. (2010), who found that human denuded immatureoocytes matured in medium containing a cocktail of CC

growth factors and oestradiol produced more embryos follow-ing parthenogenesis. This suggests that when DOs are coin-cubated with COCs, they have poor access to such CC factorsunder our incubation conditions. This could be due to the

critical dilution of such factors or that they are bound to theextracellular matrix and only directed towards the oocyte lyingwithin the COC complex.

It is well established that BMP-15 and GDF-9 regulatedifferentiation of the CC phenotype and, subsequently, oocytecompetence (Gilchrist et al. 2004a, 2008; Juengel and McNatty

2005). Our data here and elsewhere (Hussein et al. 2006; Yeoet al. 2008) demonstrate that the addition ofGDF-9 andBMP-15during IVM improves oocyte competence. However, rathersurprisingly, in the present study we found that these two OSFs

had little influence if added after 9 h from the initiation ofmaturation. This suggests that the exogenous recombinantBMP-15 and GDF-9 influence oocyte developmental compe-

tence primarily within the first 9 h of IVM (perhaps viadecreased sensitivity to GDF-9 and/or BMP-15 signallingwithin CCs after this time). However, this is in contrast with

the results described for native OSFs, where their influenceextended beyond 9 h. Perhaps otherOSFs are responsible for thiseffect, but they remain to be identified. Candidate molecules

may include other members of the TGF-b superfamily, such asTGF-bs, activins and BMP-6 (Juengel and McNatty 2005;Trombly et al. 2009), or fibroblast growth factors (Sugiuraet al. 2007; Portela et al. 2010).

It is not yet clear which of the CC processes affected byOSFsduring maturation are involved in the enhancement of oocyte

competence and increased developmental outcomes. One clearcandidate from the work of Eppig et al. is the influence of OSFson CC metabolism (Eppig et al. 2005; Sugiura and Eppig 2005;

Sugiura et al. 2007). Recently studies have also demonstrated acomplex level of interaction between OSFs and epidermalgrowth factor receptor-mediated extracellular signal-regulated

kinase 1/2 signalling in CCs that may be perturbed during IVM(Gilchrist 2011; Sasseville et al. 2010; Su et al. 2010).

Collectively, the evidence presented in the present studydemonstrates that there are temporally regulated levels of OSF

signalling that influence the acquisition of developmental com-petence of bovine oocytes during IVM.

Acknowledgements

T. S. Hussein was supported, in part, by the Faculty of Health Sciences,

University of Adelaide. This project was supported by a National Health and

Medical Research Council of Australia (NHMRC) program grant (250306)

and the Research Centre for Reproductive Health, The University of Ade-

laide. The authors thank Samantha Schulz, Fred Amato, Alexandra Harvey,

Lesley Ritter, David Mottershead and Karen Kind for helpful technical and

editorial suggestions.

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Manuscript received 29 November 2010, accepted 16 December 2010

http://www.publish.csiro.au/journals/rfd

584 Reproduction, Fertility and Development T. S. Hussein et al.