Follicle Size-Dependent Induction of Prostaglandin G/H Synthase-2 during Superovulation in Cattle'

Transcription

1 BIOLOGY OF REPRODUCTION 58, (1998) Follicle Size-Dependent Induction of Prostglndin G/H Synthse-2 during Superovultion in Cttle' Jinmin Liu nd Jen Sirois 2 Centre de recherche en reproduction nimle, Deprtement de biom6decine vterinire, Fcult6 de m6decine vet6rinire, Universite de Montr6l, C.P 5, Sint-Hycinthe, Qu6bec, Cnd J2S 7C6 ABSTRACT Under physiologicl conditions, prostglndin G/H synthse- 2 (PGHS-2) is induced in bovine preovultory follicles by the endogenous surge of gondotropins. To chrcterize the pttern of folliculr PGHS-2 expression during superovultion in cttle, heifers were treted with exogenous FSH nd ovultion ws induced with hcg. Animls were ovriectomized, 18, nd 24 h post-hcg, nd extrcts of follicles 6 mm were nlyzed by Western blotting. Folliculr fluid concentrtions of prostglndin (PG) E 2, PGF 2,,, progesterone, nd estrdiol-17p were determined by RIAs, nd the morphology of the cumulus oocyte complex ws exmined. Results showed tht PGHS-2 protein ws bsent in ll follicles isolted t h post-hcg (n = 119) nd in smll follicles (6 to < 8 mm) isolted between nd 24 h posthcg (n = 27 follicles). In contrst, 12.3% of medium (8 to < 1 mm) nd 43.7% of lrge ( 1 mm) follicles were PGHS-2- positive t 18 h post-hcg, nd these percentges rose t 24 h to 45.9% nd 91.% in medium nd lrge follicles, respectively (p <.5). Folliculr fluid concentrtions of PGE 2 nd PGF 2 were low in follicles isolted t h nd incresed only in PGHS- 2-positive follicles isolted 24 h post-hcg (p <.5). Concentrtions of progesterone nd estrdiol-1713 t h were nd ng/ml, respectively, nd shift from estrdiol-171 to progesterone dominnce (luteiniztion) occurred t 24 h post-hcg only in PGHS-2-positive follicles. Also, expnsion of the cumulus oocyte complex ws detected t 24 h posthcg only in PGHS-2-positive follicles. Lck of PGHS-2 induction in follicles of ovultory size (> 8 mm) ws ssocited with n pprent filure to respond to hcg (bsence of luteiniztion nd cumulus expnsion). Collectively, these results demonstrte the presence of time- nd follicle size-dependent induction of PGHS-2 in bovine follicles during superovultory tretment nd suggest tht PGHS-2 expression cn be used s mrker for folliculr commitment to ovultion during ovrin hyperstimultion protocols. INTRODUCTION Prostglndin G/H synthse (PGHS), lso known s cyclooxygense (COX), is key rte-limiting enzyme in the prostglndin biosynthetic pthwy, s it ctlyzes the conversion of rchidonic cid into prostglndin H 2 (PGH 2 ), common precursor for ll prostglndins, prostcyclins, nd thromboxnes [1-3]. Two genes encoding distinct PGHS isoforms, referred to s PGHS-1 nd PGHS-2, hve been chrcterized [4-6]. The PGHS-1 nd -2 proteins re bout 6% identicl t the mino cid level, but differences in their ptterns of expression nd regultion suggest tht the two isoforms could serve different biologicl functions [4, 5]. Accepted Februry 2, Received December 1, 'Supported by Medicl Reserch Council of Cnd Grnt MT-1319 nd FCAR of Quebec Grnt 97-NC-1456.S.). J.L. is supported by FCAR fellowship. 2 Correspondence. FAX: (514) ; e-mil: siroisje@medvet.umontrel.c 1527 Regultion of PGHS enzyme expression hs been demonstrted during vrious reproductive functions, including erly embryonic development nd implnttion [7-1], prturition [11-13], nd luteolysis [14, 15]. The process of ovultion is nother reproductive event during which the synthesis of prostglndins is required [16, 17]. The mrked increse in prostglndin synthesis in rt follicles before ovultion is relted to the induction of PGHS, nd more specificlly PGHS-2 [18-21]. The induction is rpid (2-4 h post-hcg), occurs only in grnulos cells, nd is cused by high (ovultory) levels of gondotropins. Comprtive studies in cttle nd horses showed tht the selective induction of PGHS-2 in grnulos cells is moleculr mechnism conserved in mono-ovultory species with long ovultory process [22-25]. The time course of PGHS-2 induction in bovine (18 h post-hcg) nd equine (3 h posthcg) preovultory follicles is delyed s compred to the rpid induction observed in rts (2-4 h post-hcg). However, the intervl from PGHS-2 induction to folliculr rupture is remrkbly conserved (-1 h) in ll species, suggesting tht PGHS-2 expression could serve s n lrm tht controls the mmmlin ovultory clock [26]. The dministrtion of exogenous gondotropins in cttle is used to override the intrinsic mechnisms of folliculr recruitment nd selection, nd to induce multiple ovultions [27-29]. Folliculr rupture during superovultory tretments ws shown to occur lmost exclusively in follicles tht hve reched dimeter of 8 mm [3]. However, despite incresed understnding of the control of bovine folliculr development nd the production of purified gondotropin preprtions, the outcome of current superovultion tretments remins unpredictble [28, 31]. One undesirble effect of these protocols is the development of proportion (up to 2%) of follicles greter thn 8 mm tht fil to ovulte nd tht eventully undergo tresi or become cystic [3, 32]. Understnding the development of such follicles during ovrin hyperstimultion hs been hmpered by the inbility to distinguish within popultion of follicles those committed to ovultion from those not committed. With the recent demonstrtion of gondotropin- nd time-dependent induction of PGHS-2 in bovine preovultory follicles during the estrous cycle [22, 23, 25], we hypothesized tht expression of PGHS-2 could be used during superovultory protocols s mrker of folliculr commitment to ovultion. Therefore, the objective of this study ws to chrcterize the pttern of expression of PGHS-2 in bovine superovultory follicles nd relte it to chnges in folliculr steroidogenesis, prostglndin production, nd cumulus-oocyte complex morphology. MATERIALS AND METHODS Mterils Diethyl ether, octyl -D-glucopyronoside (octyl glucoside), nd diethyldithiocrbmic cid were purchsed from

2 1528 LIU AND SIROIS Sigm (St. Louis, MO); Lutlyse from Upjohn (Klmzoo, MI); Folltropin-V from Vetrephrm Cnd Inc. (London, ONT, Cnd); APL (hcg) from Ayerst Lbortories (Montrel, PQ, Cnd); 25 I-Protein A nd Biotrns nylon membrnes (.2 plm) from ICN Phrmceuticls (Montrel, PQ, Cnd); prostglndin E 2 (PGE 2 ) nd prostglndin F 2 (PGF 2.) ntibodies from Advnced Mgnetics Inc. (Cmbridge, MA); 125 I-progesterone from Amershm (Okville, ON, Cnd); [ 3 H]PGE 2 nd [ 3 H]PGF 2. from DuPont NEN Reserch Products (Mississug, ON, Cnd); estrdiol-17 3 nd ntibody from Inter Medico (Willowdle, ON, Cnd); nitrocellulose membrnes (.45 pim) from Schleicher & Schuell (Keene, NH); rinbow moleculr weight mrkers from Amershm (Arlington Heights, IL); PBS from Gibco Bethesd Reserch Lbortories Life Technologies Inc. (Githersburg, MD); Bio-Rd protein ssy nd electrophoretic regents from Bio-Rd Lbortories (Richmond, CA); nd Kodk film X-OMAT AR from Estmn Kodk Compny (Rochester, NY). Animls, Superovultory Protocol, nd Ovriectomy Twelve Holstein heifers (2-3 yr old) were subjected to stndrd superovultory regimen initited between Dys 9 nd 1 of the estrous cycle. The tretment consisted of totl of 32 mg FSH (Folltropin-V) given in 8 decresing injections 12 h prt over period of 4 dys [33]. Lutel regression ws induced by injecting i.m. 25 mg PGF 2, (Lutlyse) t the fifth nd sixth FSH injection. To precisely control the time of ovultion, hcg (25 IU) ws dministered i.v. 36 h fter induction of luteolysis. Ovries were collected by ovriectomy vi colpotomy t (n = 4), 18 (n = 4), nd 24 h (n = 4) fter hcg. The ovries were trnsferred immeditely into ice-cold PBS supplemented with penicillin (5 U/ml)-streptomycin (5 jig/ml), nd trnsported to the lbortory. All niml procedures were pproved by the Comit6 de dontologie nimle of the University de Montrdl. Isoltion of Follicles All follicles - 6 mm were crefully dissected from the surrounding ovrin tissue with sclpel nd were mesured before being cut with fine scissors. The morphology of the cumulus oocyte complex (bsence or presence of expnsion) ws exmined under dissecting microscope, nd the folliculr fluid ws recovered nd stored t -7 C until ssyed for progesterone, estrdiol-1713, PGE 2, nd PGF 2,. The thec extern nd other surrounding tissues were dissected wy from the thec intern using fine forceps, s previously described [22, 25]. The resulting thec intern with ttched grnulos cells ws subsequently referred to s follicle wll preprtion. All smples were stored t -7 C until used for preprtion of cell extrcts. Cellulr Extrcts nd Western Blotting Solubilized cell extrcts were prepred from pieces of follicle wll s previously described [22, 25] nd were stored t -7 C. The protein concentrtion ws determined by the method of Brdford [34] using the Bio-Rd protein ssy kit. Protein extrcts (5,ug) were resolved by one-dimensionl SDS-PAGE nd electrophoreticlly trnsferred onto nitrocellulose membrnes s previously described [22, 25]. Membrnes were incubted 18 h t 4 C with ffinity-purified polyclonl ntibody 9181 rised in rbbits ginst ovine PGHS [2, 21]. The specificity of ntibody 9181 hs been chrcterized in rts [2, 21], sheep [11], cttle [22, 25], nd horses [24], with the ntibody recognizing both PGHS-1 nd PGHS-2 in ll species tested. 125 I-Lbeled protein A (1 X 16 cpm/ml Tris-buffered sline with 2% milk) ws used to visulize immunorective proteins. Filters were exposed to x-ry film t -7 C. Hormone Assys Nonextrcted liquots of folliculr fluid were ssyed for progesterone nd estrdiol-17 by specific RIAs, s previously described [25, 35, 36]. The sensitivities of the progesterone nd estrdiol-17 ssys were 3.12 nd.16 pg/ ssy tube, respectively. The intr- nd interssy coefficients of vritions for progesterone were 11.9% nd 8.%, respectively, nd were 9.6% nd 6.4%, respectively, for estrdiol-173. Smples of folliculr fluid were cidified to ph 3. with 1 N HC1, extrcted with diethyl ether, nd ssyed for PGE 2 nd PGF 2,, by specific RIAs, s previously described [22, 25]. The recovery rtes for PGE 2 nd PGF 2, were 82% nd 89%, respectively. The intr- nd interssy coefficients of vritions for PGE 2 were 1.7% nd 1.6%, respectively, nd were 1.1% nd 7.3% for PGF 2, respectively. Sttisticl Anlyses nd Follicle Size Ctegories One-wy ANOVA ws used to test the effect of time fter hcg on concentrtions of progesterone, estrdiol-173, PGE 2, nd PGF 2,. Scheffe's test ws used to compre individul mens when ANOVA indicted significnt differences (p <.5). Dt were trnsformed to logrithms before nlysis when heterogeneity of vrince ws observed with the Brtlett test. To test the effect of time fter hcg on folliculr expression of PGHS-2 nd cumulus oocyte complex expnsion, follicles were grouped into three size ctegories, including smll (6 to < 8 mm), medium (8 to < 1 mm) nd lrge follicles ( 1 mm). Results were nlyzed by one-wy ANOVA followed by the Tukey-Krmer HSD test for comprison of multiple mens. Dt were trnsformed rcsines before nlysis when heterogeneity of vrince ws observed with the Brtlett test. RESULTS Time- nd Follicle Size-Dependent Expression of PGHS-2 Figure 1 shows the expression of PGHS enzymes in nimls ovriectomized, 18, or 24 h post-hcg. No PGHS- 2 protein ws detected in group of 18 follicles of mm isolted t h post-hcg (Fig. 1A). However, fint but detectble PGHS-1 signl (7 kd) comigrting with the bovine PGHS-1 stndrd ws observed in severl follicles. Although the nture of the higher bnd detected with the nti-pghs ntibody in ll follicle extrcts remins unknown, its presence hs previously been reported in cttle [22] nd rts [37]. Immunorective PGHS-2 protein ws detected in 5 of 16 follicles isolted 18 h post-hcg, nd in 12 of 15 follicles isolted t 24 h (Fig. 1, B nd C). The enzyme ppered s 72-kD bnd (intct protein), nd smller proteolytic frgment of 62 kd [22, 25]. The reltive bundnce of the PGHS-2 signl t 24 h ppered higher thn t 18 h post-hcg. A cler time- nd follicle size-dependent expression of PGHS-2 ws observed when results from ll nimls were exmined (Fig. 2). PGHS-2 expression ws never observed

3 REGULATION OF PGHS-2 DURING SUPEROVULATION 1529 () ).o ' 2' n- smll -o ) co FIG. 1. Immunoblot nlysis of PGHS-2 protein in bovine follicles during superovultory tretment. Protein extrcts (5 [xg/lne) prepred from bovine follicles were nlyzed by one-dimensionl SDS-PAGE nd immunoblotting techniques using n ffinity-purified nti-pghs ntibody. Results from superovulted heifers ovriectomized t, 18, nd 24 h posthcg re shown in A-C, respectively. Mrkers on the right indicte migrtion of intct bovine PGHS-2 (72 -M, bnd) nd puttive proteolytic frgment (62 -M, bnd). An extrct of bovine pltelets ws used s PGHS-1 stndrd in ech blot (PGS, 2 ilg/lne) [22]. Filters were exposed to film t -7 C for 14 h. in smll follicles, whether they were isolted (n = 14 follicles), 18 (n = 7), or 24 h (n = 6) post-hcg (Fig. 2, upper pnel). Also, no PGHS-2 protein ws detected in medium (n = 41) or lrge (n = 64) follicles isolted t h post-hcg (Fig. 2). In contrst, n verge 12.3% nd 45.9% of medium follicles per niml were PGHS-2-positive t 18 nd 24 h post-hcg, respectively (p <.5; totl of n = 27 nd 19 follicles exmined t 18 nd 24 h, respectively). The proportion of lrge PGHS-2-positive follicles incresed with time, with n verge of 43.7% nd 91.% lrge follicles per niml being positive t 18 nd 24 h post-hcg, respectively (p <.5; totl of n = 39 follicles exmined t both 18 nd 24 h). Folliculr Fluid Concentrtions of PGs Folliculr fluid concentrtions of PGE 2 nd PGF 2 were nlyzed in reltion to PGHS-2 expression (Fig. 3). All follicles isolted h post-hcg were PGHS-2-negtive nd contined low concentrtions of PGE 2 (.47.7 ng/ml) nd PGF 2, ( ng/ml). No significnt chnges in prostglndin concentrtions were observed t 18 h posthcg, whether follicles were PGHS-2-negtive or -positive (Fig. 3). However, folliculr fluid concentrtions of PGE 2 nd PGF 2. incresed in PGHS-2-positive follicles isolted 24 h post-hcg, rising to ng/ml nd ng/ml, respectively (p <.5). Concentrtions of prostglndins in PGHS-2-negtive follicles isolted 24 h posthcg were not significntly different from those in follicles isolted t or 18 h (p >.5). (9, D lrge n M 18 Hours fter hcg FIG. 2. Follicle size- nd time-dependent expression of PGHS-2 in bovine follicles during superovultory tretment. Bovine follicles (- 6 mm) were isolted t, 18, nd 24 h post-hcg, nd were nlyzed by Western blotting. Follicles were defined s PGHS-2-negtive or PGHS-2-positive on the bsis of the bsence or presence of 72 -M, bnd on Western blots, nd were grouped into three size ctegories, including smll (upper pnel, n = 27 follicles), medium (middle pnel, n = 87 follicles), nd lrge follicles (lower pnel, n = 142 follicles). Results re shown s percentge of PGHS-2-positive follicles per niml per size ctegory nd time point post-hcg (men SEM). Brs with different superscripts re significntly different (p <.5). E S 3 L Hours fter hcg FIG. 3. Folliculr fluid concentrtions of PGE 2 nd PGF 2,, in bovine follicles during superovultory tretment. Bovine follicles ( 6 mm) were isolted t, 18, nd 24 h post-hcg, nd concentrtions of PGE 2 (top pnel) nd PGF 2 (bottom pnel) in folliculr fluid were nlyzed in reltion to folliculr PGHS-2 expression (negtive [-] or positive [+]). Note tht only PGHS-2-negtive follicles were present t h post-hcg. Results re shown s mens _ SEM, nd brs with different superscripts re significntly different (p <.5). C 24 24

4 153 LIU AND SIROIS C ) ) r wu Hours fter hcg FIG. 4. Folliculr fluid concentrtions of progesterone nd estrdiol-1 71 in bovine follicles during superovultory tretment. Bovine follicles (- 6 mm) were isolted t, 18, nd 24 h post-hcg, nd concentrtions of progesterone (top pnel) nd estrdiol-1 71 (bottom pnel) were nlyzed in reltion to folliculr PGHS-2 expression (- or +). Note tht only PGHS-2-negtive follicles were present t h post-hcg. Results re shown s mens SEM, nd brs with different superscripts re significntly different (p <.5). C x E 6- E O 4- ) _ 2- CL x - ) B 1. ) 8-6- _ Cumulus expnsion remined undetected in lmost ll smll follicles isolted between nd 24 h post-hcg (n = 27 follicles), except for one PGHS-2-negtive follicle isolted 18 h post-hcg. Cumulus expnsion ws rre or bsent in medium nd lrge PGHS-2-negtive follicles isolted nd 24 h post-hcg (Fig. 5, left pnels). However, n vmedium lrge I-:, b PGHS-2 (-) PGHS-2 (+) Hours fter hcg FIG. 5. Morphology of the cumulus oocyte complex in bovine follicles during superovultory tretment. The morphology of the cumulus-oocyte complex in smll (not shown), medium (upper pnel), nd lrge follicles (lower pnel) isolted t, 18, nd 24 h post-hcg ws nlyzed in reltion to folliculr PGHS-2 expression (negtive [-] or positive [+]). Results re shown s percentge of follicles with n expnded cumulus per niml per time point fter hcg in PGHS-2-negtive (left pnels) nd -positive follicles (right pnels). Brs with different superscripts re significntly different (p <.5). Note tht no follicles were PGHS-2-positive t h post-hcg nd, therefore, only results from PGHS-2-positive follicles t 18 nd 24 h post-hcg re presented. Folliculr Fluid Concentrtions of Progesterone nd Estrdiol- 1 7p Folliculr fluid concentrtions of progesterone nd estrdiol-1713 were nlyzed in reltion to folliculr PGHS-2 expression (Fig. 4). Results showed tht follicles isolted h post-hcg (ll PGHS-2-negtive) were estrogen-dominnt, with reltively low progesterone ( ng/ml) nd high estrdiol-17 ( ng/ml) levels (Fig. 4). After hcg tretment, PGHS-2-negtive nd -positive follicles hd distinct steroidogenic cpcities. At 24 h posthcg, PGHS-2-negtive follicles were still estrogen-dominnt, with reltively high estrdiol-171 ( ng/ ml) nd low progesterone ( ng/ml). In contrst, PGHS-2-positive follicles t 24 h post-hcg hd undergone luteiniztion s evidenced by mrked decrese in estrdiol-17p ( ng/ml, p <.1) nd increse in progesterone levels ( ng/ml, p <.1). At 18 h post-hcg, PGHS-2-positive follicles were lso luteinized, nd concentrtions of both steroids were not significntly different from those t 24 h post-hcg (Fig. 4). However, in contrst to results observed in PGHS-2-negtive follicles t 24 h, PGHS-2-negtive follicles t 18 h posthcg showed evidence of luteiniztion (Fig. 4). Morphology of the Cumulus-Oocyte Complex erge of 43.7% of medium nd 48.3% of lrge PGHS-2- negtive follicles isolted 18 h post-hcg hd n expnded cumulus (Fig. 5, left pnels). A distinct pttern of cumulus expnsion ws observed in medium nd lrge PGHS-2-positive follicles. No medium or lrge follicles isolted t h were PGHS-2-positive. However, cumulus expnsion ws observed in ll medium (n = 2 follicles) nd lrge (n = 44) PGHS-2-positive follicles isolted 18 nd 24 h post-hcg (Fig. 5). DISCUSSION The pttern of expression of PGHS-2 in bovine follicles during superovultory tretment clerly shows tht the enzyme is induced in time- nd follicle size-dependent mnner. The time course of PGHS-2 induction in bovine superovultory follicles ppers to be similr to tht observed in the preovultory follicle of norml estrous cycle (18 h post-hcg) [22, 251. A dimeter of 8 mm is n importnt developmentl stge for bovine follicles, becuse induction of PGHS-2 occurred only in follicles tht hd reched this size. The gondotropin-dependent expression of PGHS-2 t this developmentl stge is probbly relted to the coincident expression of LH receptors in grnulos cells [38, 39]. A similr developmentl- nd gondotropin-dependent induction of PGHS-2 hs been reported in rts, multiovultory species in which PGHS-2 expression is restricted to lrge preovultory follicles exposed to high levels of gondotropins [18, 2, 21]. Exogenous hcg ws dministered t the end of stndrd superovultory protocol to precisely control the induction of the ovultory process. The observed effect of hcg on PGHS-2 induction most likely mimics tht nor-

5 REGULATION OF PGHS-2 DURING SUPEROVULATION 1531 mlly cused by the endogenous LH surge. Previous studies hve shown tht both gondotropins (hcg nd LH) cuse similr pttern of PGHS-2 induction during the norml estrous cycle [22, 25]. The expression of PGHS-2 in superovultory follicles t 24 h post-hcg ws ssocited with n increse in prostglndin synthetic ctivities, but this ssocition ws not observed t 18 h post-hcg. A similr pttern is observed in equine preovultory follicles in which induction of PGHS-2 nd n increse in folliculr prostglndins re first detected t 3 nd 36 h, respectively [24]. Lower expression of PGHS-2 t erly time points nd the reltively lrge volume of folliculr fluid (dilution effect) could be responsible, t lest in prt, for the intervl observed in both species. Prostglndins hve been linked to ovultion for more thn 25 yr [16, 4-43]. Recent gene trgeting studies in mice hve provided convincing evidence for n obligtory role of prostglndin synthesis during the ovultory process [44, 45]. PGHS-2-deficient femle mice were shown to be infertile becuse of lck of ovultion. Absence of ovultion of follicles smller thn 8 mm reported during superovultion in cttle [3] my result, t lest in prt, from the inbility of gondotropins to induce PGHS-2 in these smll follicles. Similrly, the bsence of PGHS-2 expression in proportion of follicles lrger thn 8 mm t 24 h post-hcg could represent one of the mechnisms involved in the development of lrge novultory follicles during superovultion tretment. The incidence of PGHS-2-negtive follicles lrger thn 8 mm t 24 h post-hcg in this study (24%; n = 14 of 58 follicles) is very similr to the incidence of novultory follicles detected by ultrsonogrphy during superovultory tretment [3]. One importnt finding of this study is the identifiction. of PGHS-2 s mrker to differentite subpopultions of superovultory follicles with distinct steroidogenic cpcities. At 24 h post-hcg, PGHS-2-positive follicles were clerly luteinized, with high progesterone nd low estrdiol, wheres PGHS-2-negtive follicles were not luteinized nd hd low progesterone nd high estrdiol in folliculr fluid. Interestingly, the presence of high estrdiol in PGHS-2-negtive follicles indictes tht they were not tretic [46, 47]. Differences in LH receptor numbers nd/or ctivtion of intrcellulr signlling pthwys re potentil cuses for the lck of hcg responsiveness. In previous studies, the unintentionl grouping of these two subpopultions of follicles is presumbly responsible for the mrked heterogeneity reported in folliculr fluid concentrtions of steroids during superovultion in cttle [48, 49]. Similrly, the high vribility in concentrtions of progesterone nd estrdiol in PGHS-2-negtive follicles isolted t 18 h post-hcg in this study ws probbly cused by the indvertent pooling of different subpopultions of follicles (luteinized nd unluteinized). However, the problem t this erlier time point is tht induction of folliculr PGHS-2 is not completed. Since folliculr luteiniztion occurs before PGHS-2 induction in bovine follicles [22, 25, 5], we predict tht the popultion of PGHS-2-negtive follicles t 18 h post-hcg contined some follicles tht hd luteinized but hd not yet expressed PGHS-2. Therefore, PGHS-2 should be considered s good differentiting mrker just before ovultion. In ddition to its effects on ovultion nd luteiniztion, the preovultory gondotropin surge is the physiologicl trigger for resumption of oocyte mturtion. The structurl chnges of the bovine cumulus oocyte complex during its finl mturtion in vivo hve been well chrcterized in superovulted nd unstimulted nimls [51-54]. The present study shows tht PGHS-2 expression is closely relted to the morphology of the complex. All PGHS-2-positive follicles contined n expnded cumulus, wheres the vst mjority of smll, medium, nd lrge PGHS-2-negtive follicles isolted between nd 24 h post-hcg hd compct unexpnded complex. The only time t which cumulus expnsion ws observed in considerble number of PGHS- 2-negtive follicles ws t 18 h post-hcg. Considering tht cumulus expnsion precedes PGHS-2 induction [25, 52], we predict tht some PGHS-2-negtive follicles with expnded cumulus t 18 h would hve become positive shortly therefter. At 24 h post-hcg, ll medium nd lrge PGHS-2-negtive follicles contined n unexpnded cumulus, indicting tht the entire follicle unit remined unresponsive to hcg. In summry, induction of PGHS-2 in bovine follicles during superovultory tretment is time- nd size-dependent, nd results support our initil hypothesis tht PGHS- 2 is moleculr mrker for folliculr commitment to ovultion. The expression of PGHS-2 ws consistently ssocited with other signs of terminl folliculr differentition, including luteiniztion nd cumulus oocyte expnsion. In contrst, lck of PGHS-2 induction in follicles of ovultory size (> 8 mm) ws linked to n pprent filure to respond to the gondotropin preovultory signl (hcg), since no concomitnt chnges in folliculr steroidogenesis nd cumulus morphology were observed. Becuse these ltter follicles re likely to remin novultory, they should become the focus of future investigtions s they represent known drwbck of current superovultory protocols in cttle. ACKNOWLEDGMENT We thnk Dr. Aln K. Goff for providing the progesterone ntibody. REFERENCES 1. DeWitt DL. Prostglndin endoperoxide synthse: regultion of enzyme expression. Biochim Biophys Act 1991; 183: Smith WL. Prostnoid biosynthesis nd mechnisms of ction. Am J Physiol 1992; 263:F Funk CD. Moleculr biology in the eicosnoids field. Prog Nucleic Acid Res Mol Biol 1993; 45: Willims CS, DuBois RN. 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