Angiotensin II, progesterone, and prostaglandins are sequential steps in the pathway to bovine oocyte nuclear maturation

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1 Availale online at Theriogenology 77 (2012) Angiotensin II, progesterone, and prostaglandins are sequential steps in the pathway to ovine ooyte nulear maturation Luas Carvalho Siqueira a, Maros Henrique Barreta a, Bernardo Gasperin a, Rodrigo Bohrer a, Joael Tonellotto Santos a, José Buratini Junior, João Franiso Oliveira a, Paulo Bayard Gonçalves a, * a Laoratory of Biotehnology and Animal Reprodution - BioRep, Federal University of Santa Maria, Santa Maria, RS, Brazil Departament of Physiology, State University of São Paulo (UNESP), Botuatu, SP, Brazil Reeived 28 April 2011; reeived in revised form 12 Deemer 2011; aepted 15 Deemer 2011 Astrat Ooyte meioti resumption is triggered y the ovulatory gonadotropin surge; in attle, angiotensin II (AngII) and prostaglandins (PG) are key mediators of this gonadotropin-indued event. Here, we tested the hypothesis that progesterone (P 4 ) is also involved in ooyte meioti resumption indued y the gonadotropin surge. In Experiment I, P 4 indued nulear maturation in a dose-dependent manner using a oulture of folliular hemisetions and umulus-ooyte omplexes. In the seond experiment, using an in vivo model, an injetion of mifepristone (MIFE; P 4 reeptor antagonist) at the antrum of preovulatory folliles prevented GnRH-indued ooyte meioti resumption in vivo. In Experiment III (oulture system similar to that of Experiment I), MIFE prevented stimulatory effets of AngII on resumption of meiosis, ut saralasin (AngII reeptor antagonist) did not inhiit P 4 ations. In Experiments IV and V, firolast growth Fator 10 (; known to suppress steroidogenesis in granulosa ells), loked AngII-ut not P 4 -indued ooyte meioti resumption. Therefore, we inferred that AngII is upstream to P 4 in a asade to indue meioti resumption. Previously, we had reported that AngII ated throughout the PGs pathway to modulate nulear progression. In Experiment V, indomethain inhiited resumption of meiosis indued y P 4, providing further support to the AngII-P 4 sequential effet on meioti resumption. In onlusion, we inferred that AngII, P 4 and PGs are sequential steps in the same pathway that ulminates with ovine ooyte maturation Elsevier In. All rights reserved. Keywords: Ovulation; Meioti resumption; Angiotensin; Steroid; Eiosanoid 1. Introdution The preovulatory gonadotropin surge triggers a asade of events that ulminates with ovulation and nulear ooyte maturation. Reently, angiotensin II (AngII) has een reognized as one of the earliest mediators of gonadotropin-indued ovulation and ooyte maturation * Corresponding author. Tel: ; fax: address: ayard@ufsm.r (P.B. Gonçalves). [1 3]. The positive effet of AngII in these proesses is mediated through a Type 2 reeptor [1]. Furthermore, the onentration of AngII and expression of its reeptors (AT2) within the follile inreased during the interval etween the gonadotropin surge and ovulation (Siqueira, et al, unpulished data). Other studies provided additional evidene that AngII regulated seretion of progesterone (P 4 ) and prostaglandins (Pg), hormones involved in ovulation [4,5]. In granulosa ell ulture, AngII upregulated expression of ylooxygenase 2 (COX-2), the rate-limiting enzyme for PG prodution [3] X/$ see front matter 2012 Elsevier In. All rights reserved. doi: /j.theriogenology

2 1780 L.C. Siqueira et al. / Theriogenology 77 (2012) During follile development, ovine ooytes remain arrested at prophase of the first meioti division, and resume meiosis after the preovulatory LH surge [6], or after removal from the folliular environment [7]. The presene of folliular wall fragments in a oulture system with umulus-ooyte omplexes (COCs) prevents meioti resumption [8]. This oulture system is a good model to study the role of fators that at through folliular ells on ooyte nulear maturation [9,10]. Using this oulture system, we reported that AngII ated through a PG pathway to mediate gonadotropin-indued ooyte meioti resumption [2]. The ylooxygenase pathway is a lassial mediator of LH-indued ovulation and nulear ooyte maturation in attle [11 15]. Progesterone is another key element in the ovulatory asade and ooyte maturation [13,14,16]. Indeed, there are indiations that PGs are downstream fators to this steroid; in that regard, a gonadotropin surge stimulates an inrease in intrafolliular P 4, whih ats y inding to its nulear reeptor and inreasing aundane of mrna for COX2 [14]. The role of P 4 on ooyte nulear maturation in attle remains ontroversial. Nulear and memrane progesterone reeptors are present in ovine COCs, and regulated during in vitro maturation in the presene of FSH and LH [16]. Although Sirotkin [17] reported a stimulatory effet on ooyte meioti resumption, more reent studies onluded that P 4 was not neessary to promote nulear maturation, umulus expansion, and early emryo development [18,19]. Folliular ells serete fators that prevent ooyte meioti resumption efore the LH surge. The family of firolast growth fators (FGFs) is omposed of more than 20 fators, largely studied for their roles in emryogenesis and oogenesis. Buratini, et al [20] reported that the ovine thea ells and ooytes expressed. Expression of reeptor (FGFR2III) was identified in thea [21], granulosa [20], and umulus ells [22]. Furthermore, in the granulosa ell ulture inhiited steroidogenesis [20] and AT2 expression [23]. Ativation of FGF reeptors (FGFRs) appeared to e involved in inhiition of germinal vesile reakdown (GVBD) in mie [24]. Conversely, Zhang, et al [25] reported that improved ovine ooyte maturation, umulus expansion and susequently emryo development in medium ontaining estradiol and in the asene of folliular ells. The information summarized aove provided an impetus to investigate interations etween and fators involved in triggering ovine ooyte meioti resumption. In the present study, a omination of in vivo and in vitro experiments were onduted to test the hypothesis that P 4 plays a role in regulation of ooyte meioti progression indued y gonadotropin surge in onert with AngII and PGs. In an in vitro experiment, interations of P 4 and AngII with (an antisteroidogeni fator reently desried as an important regulator of folliular development) were studied, with regards to their roles in resumption of meiosis. 2. Materials and methods All experimental proedures were reviewed and approved y the Federal University of Santa Maria Animal Care and Use Committee ( / CCR/UFSM). All hemials used were purhased from Sigma Chemial Company (St. Louis, MO, USA), unless otherwise indiated in the text Preparation of folliular hemisetions, ooyte reovery and nulear maturation Bovine ovaries at various stages of the estrous yle were otained from an aattoir and transported to the laoratory in saline solution (0.9% NaCl) at 30 C ontaining 100 IU ml 1 peniillin and 50 g ml 1 streptomyin sulfate. Proedures for follile dissetion and ulture proedures were previously validated in our laoratory [2,9,10]. Briefly, transparent folliles, 2 to 5 mm in diameter, were seleted and disseted from ovarian stromal tissue, and setioned into halves. Folliular hemi-setions were washed in TCM 199 ontaining 0.4% ovine serum alumin (BSA) and randomly distriuted into four-well ulture dishes (Nun, Roskilde, Denmark) ontaining ulture medium with the desired treatment. There were eight folliular halves per 200 l of medium. Dishes were inuated for 2 h efore adding COCs. The COCs were aspirated from folliles 3 to 8 mm in diameter, reovered under a stereomirosope, and seleted aording to Leifred and First [26]. Grades 1 and 2 COCs (n 10 to 30) were randomly distriuted into treatments and ultured in an inuator at 39 C in a saturated humidity atmosphere ontaining 5% CO 2 in air and 95% air, for either 7, 15, or 24 h, depending on the experiment. The ulture medium used was TCM 199 ontaining Earle s salts and L-glutamine (Gio BRL, Grand Island, NY, USA) supplemented with 25 mm HEPES, 0.2 mm pyruvi aid, 2.2 mg ml 1 sodium iaronate, 5.0 g/ml LH (lutropin-v, Bionihe, ON, Canada), 0.5 g/ml FSH (Folltropin-V, Bionihe), 0.4% fatty aid-free BSA, 100 IU ml 1 peniillin, and 50 g ml 1 streptomyin sulfate. At the end of the

3 L.C. Siqueira et al. / Theriogenology 77 (2012) ulture period, umulus ells were removed y vortexing for 5 min and ooytes were fixed with 4% paraformldehyde for 15 min, followed y permeailization of the nulear memranes with 0.5% Triton X-100. After 2 h, ooytes were fixed, stained with Hoehst (33,342) and mounted under a overslip with Vetashield (Vetor Laoratories, Burlington, Ontario, L7N 3J5, Canada) for nulear evaluation. Ooytes were lassified aording to their nulear hromatin onfiguration using a fluoresent mirosope as germinal vesile (GV), GV reakdown (GVBD), metaphase I (MI), anaphase I (AI), telophase I (TI), and metaphase II (MII). In all experiments, all treatments were repeated three times Cattle, superovulation protool, and ultrasoundguided intrafolliular injetion The superovulation protool and intrafolliular injetion proedures were previously desried [2]. Five yling ows (Bos taurus taurus), multiparous, with ody ondition sores of 3 and 4 (1 thin to 5 oese) were sumitted to the 9-d progesterone/fshased superovulation protool. On day 9 of the progesterone treatment, the numer of folliles in eah ovary was evaluated y transretal ultrasonography, and all folliles 5 to 11 mm in diameter were aspirated using a vauum pump, leaving no more than the three largest folliles in eah ovary. On the afternoon of Day 10, after the intravaginal devie had een removed, eah ovary was examined with transretal ultrasonography, a map of the folliles was prepared, and all folliles 12 mm in diameter were sujeted to intrafolliular injetions. Intrafolliular injetions were done with a 7.5 MHz transduer attahed to a iopsy guide and a sanner (AquilaVet Sanner; Pie Medial Equipment BV, Maastriht, the Netherlands). A system with two sterile needles was used, as previously desried [1]. Briefly, the ovary was manipulated to introdue the needle into the follile via the ovarian stroma at the ase of the follile. When the ovary and follile were in position, the outer needle was advaned until the image of its tip eame visile on the sreen, 3 to 5 mm from the follile. At this moment, a seond operator pushed the inner needle forward until the image of the needle tip was visile within the follile. Treatments were then injeted into the follile. To otain the desired final onentration inside the follile, the dose of eah treatment was alulated ased on the volume of folliular fluid, estimated y the linear regression equation V D, where V orresponded to the estimated folliular volume and D to the diameter of the follile to e injeted [1]. The injetion volume per follile ranged from 80 to 110 l, approximately 10% of folliular fluid volume. Cows were exluded from the experiment if the injeted follile had a redution in diameter 1 mm within 2 h after injetion (evidene of leakage) Experiment I: progesterone indued ooyte nulear maturation in vitro The first experiment was designed to assess the P 4 effet on nulear maturation. Ooytes (n 565) ultured with folliular hemisetions treated were with 0, 10, 100, 1,000 or 10,000 ng/ml of P 4. After 22 h of ulture, ooytes were onsidered mature when lassified as AI, TI, or MII Experiment II: effet of progesterone antagonist on lh-indued meioti resumption in vivo Five ows were primed for superovulation and manipulated to have no more than three folliles 12 mm in eah ovary at the time of injetion. For eah ow, folliles in the right ovary were treated to otain a final onentration in folliular fluid of 1 M of mifepristone (MIFE group; n 10), whereas those from the left ovary were treated with 0.9% saline (ontrol group; n 10). Immediately after the intrafolliular injetions, the ows were given 100 g of gonadorelin aetate im (Profertil, Tortuga, Brazil), a GnRH agonist. Fifteen h after GnRH treatment, ows were ovarietomized y olpotomy. The COCs were reovered and proessed as desried aove. Ooytes at GVBD or MI stages were onsidered as having resumed meiosis Experiment III: progesterone mediates AngIIindued meioti resumption in vitro The COCs (n 540) were seleted and distriuted among the following seven groups for 15 h of ulture: positive and negative ontrols; AngII (10 11 M); AngII plus MIFE (1 M; P 4 antagonist); P 4 (100 ng/ml), P 4 plus saralasin (10 5 M; AngII antagonist); and AngII plus saralasin. In all groups, exept the positive ontrol, folliular hemisetions and COCs were oultured. Ooytes in MI or latter stages were onsidered to have normal resumption of meiosis. To determine if there was a toxi effet of the P 4 antagonist, COCs were ultured for 22 h, without folliular hemisetions, in the asene or presene of MIFE (1 M). Ooytes were onsidered mature when lassified as AI, TI or MII.

4 1782 L.C. Siqueira et al. / Theriogenology 77 (2012) Experiment IV: effet of on AngIIindued meioti resumption in vitro Control COCs were ultured in medium in the asene (positive ontrol; n 84) or presene (negative ontrol; n 88) of folliular hemisetions for 7 h. Four treatment groups were estalished; the COCs were ultured in the presene of: a) AngII (10 11 M;n 83) with folliular hemisetions; ) AngII and (100 ng/ml) with folliular hemisetions (AngII group; n 82); ) with folliular hemisetions ( ells group; n 80); and d) without folliular hemisetions ( group; n 88). Ooyte nulear hromatin onfiguration was lassified as GV or germinal vesile reakdown (GVBD) Experiment V: effet of or indomethain on progesterone-indued meioti resumption in vitro Control COCs were ultured for 7 h in the asene (positive ontrol; n 85) or presene (negative ontrol; n 82) of folliular hemisetions. Three treatment groups were estalished. The COCs were oultured with folliular ells in the presene of: a) progesterone (100 ng/ml; P 4 group; n 84); ) P 4 plus (100 ng/ml; P 4 group; n 80) and ) P 4 plus indomethain (a COX nonseletive inhiitor; 10 M, P 4 indo group; n 85). Ooyte nulear hromatin onfiguration was lassified as GV or germinal vesile reakdown (GVBD) Statistial analysis Data from Experiments I, III, IV, and V were analyzed using the ANOVA test in a statistial model for ategorial data, using the PROC CATMOD (Categorial Data Analysis Proedures). All in vitro experiments were performed in tripliate. When there were signifiant differenes, independent variales were ompared using the ontrast test. All data were analyzed using statistial analysis software (SAS; SAS Institute, In., Cary, NC, USA). In Experiment II, meioti resumption was ompared using the generalized linear models from JMP software (SAS Institute, In.). various onentrations of P 4. Progesterone indued nulear maturation in ovine ooytes ultured with folliular ells in a dose-dependent manner (Fig. 1). The MII rate was greatest for ooytes ultured with folliular ells treated with 100 ng/ml of P 4 (P 0.01) Experiment II: effet of progesterone antagonist on LH-indued meioti resumption in vivo One P 4 stimulated nulear maturation in vitro, whether the LH-indued resumption of meiosis was mediated y progesterone was tested using an in vivo model. The mean initial diameter of folliles treated with progesterone antagonist (MIFE; mm) did not differ from those injeted with saline ( mm; P 0.05). From the injeted folliles, 20 ooytes were reovered and evaluated (10 per group). The aility of the LH surge (indued y the GnRH agonist) to indue resumption of meiosis was impaired when folliles were treated with the progesterone reeptor antagonist (MIFE; 70, 10 and 20% were GV, GVBD, and MI, respetively; P 0.01; Fig. 2B). As expeted, the GnRH agonist indued 90% of meioti resumption in ooytes from saline-treated folliles (10, 10, and 80% were GV, GVBD, and MI) Experiment III: progesterone mediated AngIIindued meioti resumption in vitro Sine the role of AngII in resumption of meiosis and ovulation is well estalished, we tested the hypothesis that AngII is an upstream fator to P 4 in the asade of meioti resumption. Meioti resumption Metaphase II (%) y = x x x P < Results 3.1. Experiment I: progesterone indued ooyte nulear maturation in vitro The hypothesis tested in this experiment was that P 4 indues nulear maturation in ovine ooytes. Bovine COCs, reovered from aattoir-derived ovaries, were oultured with folliular hemisetions for 22 h with Progesterone (ng/ml) Fig. 1. Effet of progesterone on nulear maturation of ovine ooytes (metaphase II, Anaphase I and Telophase I; Experiment I). Rates of ooyte maturation (solid ars) and predited regression line after oulture of ooytes (n 565) and folliular hemisetions treated for 22 h with various onentrations of P 4. The experiment was performed in tripliate.

5 L.C. Siqueira et al. / Theriogenology 77 (2012) A a B 80 a GVBD (%) Positive ontrol Negative ontrol AngII AngII + MIF P4 P4 + saralasin AngII + saralasin Saline MIF Folliular ells Fig. 2. Effet of angiotensin II (AngII), progesterone (P 4 ) or progesterone antagonist on the asade of ooyte meioti resumption in vitro (Panel A) or in vivo (Panel B). in vitro, ovine umulus ooyte omplexes (n 540) were oultured for 15 h with folliular ells and AngII, AngII plus mifepristone (MIFE), P 4,P 4 plus saralasin, and AngII plus saralasin (Experiment III). in vivo, folliles ( 12 mm) were hallenged with GnRH and intrafolliular injeted with saline (n 10) or MIFE (n 10). After 15 h, ooytes were otained y folliular aspiration to aess the nulear maturation stage (Experiment II). a- Within a panel, olumns without a ommon supersript differed (P 0.05). was inhiited when the COCs were oultured with folliular hemisetions (Fig. 2A; positive vs. negative ontrols). With this model, AngII or P 4 indued meioti resumption (61 and 66%, respetively, ompared with 32% of the negative ontrol; P 0.01). However, AngII did not indue resumption of meiosis when saralasin or MIFE was present in the maturation medium. Independent of the presene of saralasin, most ooytes reahed MI in the presene of P 4. A further experiment was done, ulturing COCs without folliular hemisetions for 22 h, with or without MIFE, to exlude a detrimental effet on ooyte maturation. Ooytes treated with MIFE reahed a similar rate of nulear maturation (88%) to that of ooytes ultured in the ontrol medium (85%) Experiment IV: effet of on AngIIindued meioti resumption in vitro Our hypothesis was that has a negative role in the resumption of meiosis indued y AngII. In the asene of folliular ells, the rate of meioti resumption rate was not different etween the positive ontrol and -treated COCs after 7 h of ulture (Fig. 3A). Also, did not affet the aility of folliular ells to prevent ooytes from resuming meiosis. However, inhiited the AngII effet in folliular ells. When ooytes were ultured simultaneously with AngII and, 32% ahieved GVBD, whereas 62% of those ultured only with AngII ahieved GVBD (P 0.01; Fig. 3A) Experiment V: effet of or indomethain on P 4 -indued meioti resumption in vitro The role of in the ooyte meioti resumption indued y P 4 was examined. Using a oulture system of ooytes and folliular hemisetions, the P4 effet on the meiosis resumption was not affeted y (P 0.05). However, when indomethain (a nonseletive PG antagonist) was inluded in the oulture system of ooytes and folliular hemisetions, the P 4 effet was inhiited, impliating prostaglandins in P4-indued meiosis resumption (Fig. 3B). 4. Disussion In the present study, we tested the hypothesis that P 4 is an intermediate fator etween AngII and PGs in the meioti resumption stimulatory asade. The main findings were 1) progesterone indued meiosis resumption, in a onentration-dependent manner, of ovine umulus-enlosed ooytes ultured with follile walls; 2) MIFE inhiited GnRH-indued ooyte meioti resumption in vivo; 3) MIFE inhiited ooyte meioti resumption indued y AngII in vitro, whereas an AngII reeptor antagonist did not interfere with the P 4 stimulatory effet; 4) P 4 -indued ooyte meioti resumption was loked y indomethain (ox non-seletive inhiitor) in vitro; and 5) inhiited AngII-ut not P 4 -indued ooyte meioti resumption in vitro. Previously, using the same in vitro oulture system of ovine umulus-enlosed ooytes and folliular hemisetions, we reported that AngII ated through

6 1784 L.C. Siqueira et al. / Theriogenology 77 (2012) GVBD (%) GVBD (%) A B a Positive ontrol a Positive ontrol Negative ontrol Negative ontrol AngII AngII + Folliular ells P4 P4 Folliular ells P4 + INDO PGs to mediate LH-indued ooyte meioti resumption [2] and that AngII, in synergism with LH, indued P 4 and PG synthesis in the ovine dominant follile (Siqueira, et al unpulished). Based on all of these data, we inferred that AngII is upstream to P 4 in a pathway to indue ooyte nulear maturation that is initiated y a gonadotropin surge and stimulates prodution of PGs. In this study, we used two experimental models already estalished. In the first approah, spontaneous meioti progression was inhiited in a oulture system with ooyte and folliular hemisetions [8,9]. With this model, P 4 stimulated ooyte nulear maturation in a dose-dependent manner. In the seond model, ows were primed for superovulation and, after a GnRH hallenge, intrafolliular injetions guided y ultrasongraphy were performed in the right (treatment) and a Fig. 3. Effet of firolast growth Fator 10 () or indomethain (INDO-) on AngII- or P4-indued meioti resumption. Bovine umulus-ooyte omplexes were ultured for 7 h, with or without folliular hemisetions. Both experiments were performed in tripliate. a- Within a panel, olumns without a ommon supersript differed (P 0.05). left (ontrol) ovaries [1,2]. In this experiment, MIFE inhiited ooyte meioti resumption. Progesterone also partiipates in the ooyte nulear maturation in primates and swine [27,28]. In monkeys, inhiition of folliular progesterone prodution y trilostane (steroid synthesis inhiitor) did not redue gonadotropin-indued ooyte maturation, ut inreased the perentage of degenerated ooytes [27]. In pigs, treatment of COCs with MIFE modified the pattern of expression of P 4 reeptors in umulus and redued progesterone synthesis [28]. The reason of the lower positive effet at higher doses of progesterone was unlear. A similar progesterone dose response effet was oserved in oxytoin seretion in ovine granulosa ells ultured in vitro [29]. Nevertheless, 1,000 and 10,000 ng/ml are supraphysiologi doses; therefore, redued support for ooyte maturation with these doses may not e physiologially relevant. Previous studies demonstrated that progesterone onentrations in folliular fluid in vivo inreased etween Time 0 and 3.5 h after GnRH, dereased etween 6 and 18 h, with a seond inrease in progesterone evident at 24 h [14]. These inreases were onomitant to the upregulation of progesterone reeptor mrna expression in folliular wall [4]. Nevertheless, the maximum onentration of progesterone in folliular fluid etween the LH surge and ovulation in attle is 250 ng/ml [14]. Previously, others and we reported that P 4 (mediated y AngII) [2] and PGs [30,31] are in the pathway of ooyte meioti resumption. Herein, we onfirmed the hypothesis that AngII is upstream to P 4 in the asade of resumption of meiosis. In Experiment III, a P 4 reeptor antagonist prevented AngII stimulatory effets on resumption of meiosis, ut saralasin did not inhiit P 4 ations. There are indiations that the stimulatory effets of AngII on ooyte nulear maturation are mediated y PGs [2]. In Experiment V, indomethain inhiited resumption of meiosis indued y P 4, suggesting that PGs also mediate this steroid ations. Progesterone is essential to indue PG seretion during the ovulatory proess [5] and we reently demonstrated that AngII has a synergisti ation with LH to indue prodution of P 4 and PGs y granulosa ells from large dominant folliles (Siqueira, et al, unpulished). Based on these data, we inferred that AngII, P 4 and PGs are sequential steps from the same pathway. We previously demonstrated that Ang II has no effet on meioti resumption in vitro in the asene of folliular ells [9]. Nevertheless, we also demonstrated that Ang II is indispensale for ovine ooyte meioti

7 L.C. Siqueira et al. / Theriogenology 77 (2012) resumption in vivo [2]. Cumulus-ooyte omplexes matured in vitro in media supplemented with BSA and gonadotrophins, (in similar onentrations to those used in the present experiments) an synthesize progesterone, reahing onentrations of 40 ng/ml after 16 h of ulture [32]. Perhaps these onentrations are not enough to overome the negative effet of folliular ells. Unfortunately, progesterone seretion y COCs oultured with folliular hemisetions was not measured. Ooytes remain arrested in germinal vesile during follile development and are ale to reinitiate meiosis after the LH surge. The oulture of ooytes and folliular hemisetions effiiently inhiits ooyte meioti resumption, proaly eause during the ulture period, ells from 3 to 8 mm folliles produe inhiitory fators. Using the oulture system, we an reprodue the inhiitory effet of the follile environment and test if LH-indued fators, e.g. Ang II, progesterone and prostaglandins, are ale to overome the negative effet of folliular ells-sereted fator on meioti resumption. There were no indiations that toxiity was responsile for the inhiitory effets of the antagonists used. Saralasin, MIFE, and indomethain are safe for ell viaility and funtion [2,5]. Indeed, in the present study, saralasin did not affeted P4-indued meioti resumption. Also, MIFE (1 M) in the asene of folliular hemisetions did not impair ovine ooyte nulear maturation (Experminent III), nor did it affet susequent emryo development [16]. Reently, it was demonstrated that progesterone signaling is not essential for ovine ooyte meioti resumption in vitro using trilostane [16]. Based on these data, we inferred that the progesterone positive effet on ooyte meioti resumption is mediated through folliular ells in vivo. In the present study, inhiited the positive effet of AngII, ut not of P4 on ooyte meioti resumption. Although umulus ells also express reeptors [22], did not affet meioti resumption rate in the asene of folliular ells. Therefore, we inferred that inhiited meioti progression y ating on the folliular wall. Indeed, may e ating on AngII-indued meioti resumption y modulating steroid prodution in folliular ells. Type II reeptors for AngII (AT2) are responsile for transduing AngII positive signal for resumption of meiosis in ooytes and ovulation [1]. Furthermore, downregulates the expression of AT2 reeptors in folliular ells [33] and inhiits steroidogenesis [20]. Ativation of FGFR2III ( reeptor) inhiits gonadotropin indued progesterone seretion in granulosa ells [34]. Therefore, ould e exerting its negative effet through downregulation of AT2 expression, and onsequently, dereasing AngII-stimulated progesterone synthesis or diretly inhiiting folliular ell steroidogenesis. The disrepany etween our results and those reently reported y Zhang, et al [25] ould e due to differenes in ulture onditions, suh as the presene of estradiol and the asene of folliular ells in the system. Nevertheless, further studies are neessary to eluidate the role of on ovine ooyte nulear maturation. Taken together with other studies from our group, it is possile to propose a model (Fig. 4) in whih the gonadotropin surge stimulates a single asade of events to indue ovulation and nulear ooyte maturation. In this model, gonadotropin surge stimulates AngII seretion and upregulates AT2 expression in folliular ells, whereas AngII inreases folliular ells seretion of, P4 that stimulates PGs. Ultimately, this sequene of events ulminates with ovulation of a fertile ooyte. LH AT2 P4 ACE AngII AT2 P4 AngI AngII Cox-2 AA PG? Fig. 4. Proposed model for a single asade of events to indue ovulation and nulear ooyte maturation in attle. Preovulatory gonadotropin surge indues an up regulation of Type II angiotensin reeptors (AT2) and folliular Angiotensin onverting enzyme expression (ACE). The inhiitory effet to AT2 expression is overome y the gonadotropin surge. Upegulation of ACE indues an inrease in folliular Angiotensin II (AngII) synthesis, whih will ind to AT2 to stimulate synthesis of progesterone (P4) and PG.

8 1786 L.C. Siqueira et al. / Theriogenology 77 (2012) In summary, ased on the present work, we onluded that P 4 in attle, similar to AngII and PGs, mediated the resumption of meioti progression indued y gonadotropin surge. Indeed, ased on our study, we speulated that AngII, P 4 and PGs are sequential steps in the same pathway that ulminates with ooyte maturation. Aknowledgments The authors thank Adalerto Siqueira for providing animals and Silva Aattoir that kindly provided ovine ovaries. This study was supported y the Brazilian Counil of Sientifi and Tehnologial Development (CNPq) and CAPES. Referenes [1] Ferreira R, Oliveira JF, Fernandes R, Moraes JF, Gonçalves PB. The role of angiotensin II in the early stages of ovine ovulation. Reprodution 2007;134: [2] Barreta MH, Oliveira JF, Ferreira R, Antoniazzi AQ, Gasperin BG, Sandri LR, et al. Evidene that the effet of angiotensin II on ovine ooyte nulear maturation is mediated y prostaglandins E2 and F2alpha. 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