d u b t p s © Available online at www.sciencedirect.com Theriogenology 77 (2012) 1779–1787 0 d Angiotensin II, progesterone, and prostaglandins are sequential steps in the pathway to bovine oocyte nuclear maturation Lucas Carvalho Siqueiraa, Marcos Henrique Barretaa, Bernardo Gasperina, Rodrigo Bohrera, Joabel Tonellotto Santosa, José Buratini Juniorb, João Francisco Oliveiraa, Paulo Bayard Gonçalvesa,* a Laboratory of Biotechnology and Animal Reproduction - BioRep, Federal University of Santa Maria, Santa Maria, RS, Brazil b Departament of Physiology, State University of São Paulo (UNESP), Botucatu, SP, Brazil Received 28 April 2011; received in revised form 12 December 2011; accepted 15 December 2011 Abstract Oocyte meiotic resumption is triggered by the ovulatory gonadotropin surge; in cattle, angiotensin II (AngII) and prostaglan- dins (PG) are key mediators of this gonadotropin-induced event. Here, we tested the hypothesis that progesterone (P4) is also involved in oocyte meiotic resumption induced by the gonadotropin surge. In Experiment I, P4 induced nuclear maturation in a ose-dependent manner using a coculture of follicular hemisections and cumulus-oocyte complexes. In the second experiment, sing an in vivo model, an injection of mifepristone (MIFE; P4 receptor antagonist) at the antrum of preovulatory follicles prevented GnRH-induced oocyte meiotic resumption in vivo. In Experiment III (coculture system similar to that of Experiment I), MIFE prevented stimulatory effects of AngII on resumption of meiosis, but saralasin (AngII receptor antagonist) did not inhibit P4 actions. In Experiments IV and V, fibroblast growth Factor 10 (FGF10; known to suppress steroidogenesis in granulosa cells), locked AngII-but not P4-induced oocyte meiotic resumption. Therefore, we inferred that AngII is upstream to P4 in a cascade o induce meiotic resumption. Previously, we had reported that AngII acted throughout the PGs pathway to modulate nuclear rogression. In Experiment V, indomethacin inhibited resumption of meiosis induced by P4, providing further support to the AngII-P4 sequential effect on meiotic resumption. In conclusion, we inferred that AngII, P4 and PGs are sequential steps in the ame pathway that culminates with bovine oocyte maturation. 2012 Elsevier Inc. All rights reserved. Keywords: Ovulation; Meiotic resumption; Angiotensin; Steroid; Eicosanoid www.theriojournal.com 1. Introduction The preovulatory gonadotropin surge triggers a cas- cade of events that culminates with ovulation and nuclear oocyte maturation. Recently, angiotensin II (AngII) has been recognized as one of the earliest mediators of gonadotropin-induced ovulation and oocyte maturation * Corresponding author. Tel: �55 55 3220 8752; fax: �55 55 3220 8484. E-mail address: bayard@ufsm.br (P.B. Gonçalves). 093-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. oi:10.1016/j.theriogenology.2011.12.022 [1–3]. The positive effect of AngII in these processes is mediated through a Type 2 receptor [1]. Furthermore, the concentration of AngII and expression of its receptors (AT2) within the follicle increased during the interval between the gonadotropin surge and ovulation (Siqueira, et al, unpublished data). Other studies provided additional evidence that AngII regulated secretion of progesterone (P4) and prostaglandins (Pg), hormones involved in ovu- lation [4,5]. In granulosa cell culture, AngII upregulated expression of cyclooxygenase 2 (COX-2), the rate-limit- ing enzyme for PG production [3]. mailto:bayard@ufsm.br www.theriojournal.com a t [ A o o i i [ d g l r t u a l s m c i B m 1780 L.C. Siqueira et al. / Theriogenology 77 (2012) 1779–1787 During follicle development, bovine oocytes remain arrested at prophase of the first meiotic division, and resume meiosis after the preovulatory LH surge [6], or after removal from the follicular environment [7]. The presence of follicular wall fragments in a coculture system with cumulus-oocyte complexes (COCs) pre- vents meiotic resumption [8]. This coculture system is good model to study the role of factors that act hrough follicular cells on oocyte nuclear maturation 9,10]. Using this coculture system, we reported that ngII acted through a PG pathway to mediate gonad- tropin-induced oocyte meiotic resumption [2]. The cyclooxygenase pathway is a classical mediator f LH-induced ovulation and nuclear oocyte maturation n cattle [11–15]. Progesterone is another key element n the ovulatory cascade and oocyte maturation 13,14,16]. Indeed, there are indications that PGs are ownstream factors to this steroid; in that regard, a onadotropin surge stimulates an increase in intrafol- icular P4, which acts by binding to its nuclear receptor and increasing abundance of mRNA for COX2 [14]. The role of P4 on oocyte nuclear maturation in cattle emains controversial. Nuclear and membrane proges- erone receptors are present in bovine COCs, and reg- lated during in vitro maturation in the presence of FSH nd LH [16]. Although Sirotkin [17] reported a stimu- atory effect on oocyte meiotic resumption, more recent tudies concluded that P4 was not necessary to promote nuclear maturation, cumulus expansion, and early em- bryo development [18,19]. Follicular cells secrete factors that prevent oocyte meiotic resumption before the LH surge. The family of fibroblast growth factors (FGFs) is composed of more than 20 factors, largely studied for their roles in em- bryogenesis and oogenesis. Buratini, et al [20] reported that the bovine theca cells and oocytes expressed FGF10. Expression of FGF10 receptor (FGFR2IIIb) was identified in theca [21], granulosa [20], and cumu- lus cells [22]. Furthermore, FGF10 in the granulosa cell culture inhibited steroidogenesis [20] and AT2 expres- sion [23]. Activation of FGF receptors (FGFRs) ap- peared to be involved in inhibition of germinal vesicle breakdown (GVBD) in mice [24]. Conversely, Zhang, et al [25] reported that FGF10 improved bovine oocyte maturation, cumulus expansion and subsequently em- bryo development in medium containing estradiol and in the absence of follicular cells. The information summarized above provided an im- petus to investigate interactions between FGF10 and factors involved in triggering bovine oocyte meiotic resumption. In the present study, a combination of in 5 vivo and in vitro experiments were conducted to test the hypothesis that P4 plays a role in regulation of oocyte eiotic progression induced by gonadotropin surge in oncert with AngII and PGs. In an in vitro experiment, nteractions of P4 and AngII with FGF10 (an antis- teroidogenic factor recently described as an important regulator of follicular development) were studied, with regards to their roles in resumption of meiosis. 2. Materials and methods All experimental procedures were reviewed and ap- proved by the Federal University of Santa Maria Ani- mal Care and Use Committee (23081.004717/2010–53 CCR/UFSM). All chemicals used were purchased from Sigma Chemical Company (St. Louis, MO, USA), un- less otherwise indicated in the text. 2.1. Preparation of follicular hemisections, oocyte recovery and nuclear maturation Bovine ovaries at various stages of the estrous cycle were obtained from an abattoir and transported to the laboratory in saline solution (0.9% NaCl) at 30 °C containing 100 IU ml�1 penicillin and 50 �g ml�1 streptomycin sulfate. Procedures for follicle dissection and culture procedures were previously validated in our laboratory [2,9,10]. Briefly, transparent follicles, 2 to 5 mm in diameter, were selected and dissected from ovar- ian stromal tissue, and sectioned into halves. Follicular hemi-sections were washed in TCM 199 containing 0.4% bovine serum albumin (BSA) and randomly dis- tributed into four-well culture dishes (Nunc, Roskilde, Denmark) containing culture medium with the desired treatment. There were eight follicular halves per 200 �l of medium. Dishes were incubated for 2 h before add- ing COCs. The COCs were aspirated from follicles 3 to 8 mm in diameter, recovered under a stereomicroscope, and selected according to Leibfred and First [26]. Grades 1 and 2 COCs (n � 10 to 30) were randomly distributed into treatments and cultured in an incubator at 39 °C in a saturated humidity atmosphere containing 5% CO2 in air and 95% air, for either 7, 15, or 24 h, depending on the experiment. The culture medium used was TCM 199 containing Earle’s salts and L-glutamine (Gibco RL, Grand Island, NY, USA) supplemented with 25 M HEPES, 0.2 mM pyruvic acid, 2.2 mg ml�1 sodium bicarbonate, 5.0 �g/ml LH (lutropin-V, Bioniche, ON, Canada), 0.5 �g/ml FSH (Folltropin-V, Bioniche), 0.4% fatty acid-free BSA, 100 IU ml�1 penicillin, and 0 �g ml�1 streptomycin sulfate. At the end of the o t d l 2 n c fi 2 a n i f c ( o n t ( a c d c 2 i a p s l u i r 1781L.C. Siqueira et al. / Theriogenology 77 (2012) 1779–1787 culture period, cumulus cells were removed by vortex- ing for 5 min and oocytes were fixed with 4% para- formldehyde for 15 min, followed by permeabilization of the nuclear membranes with 0.5% Triton X-100. After 2 h, oocytes were fixed, stained with Hoechst (33,342) and mounted under a coverslip with Vectashield (Vector Laboratories, Burlington, Ontario, L7N 3J5, Canada) for nuclear evaluation. Oocytes were classified according to their nuclear chromatin config- uration using a fluorescent microscope as germinal ves- icle (GV), GV breakdown (GVBD), metaphase I (MI), anaphase I (AI), telophase I (TI), and metaphase II (MII). In all experiments, all treatments were repeated three times. 2.2. Cattle, superovulation protocol, and ultrasound- guided intrafollicular injection The superovulation protocol and intrafollicular in- jection procedures were previously described [2]. Five cycling cows (Bos taurus taurus), multiparous, with body condition scores of 3 and 4 (1 � thin to 5 � obese) were submitted to the 9-d “progesterone/FSH- based” superovulation protocol. On day 9 of the pro- gesterone treatment, the number of follicles in each ovary was evaluated by transrectal ultrasonography, and all follicles 5 to 11 mm in diameter were aspirated using a vacuum pump, leaving no more than the three largest follicles in each ovary. On the afternoon of Day 10, after the intravaginal device had been removed, each ovary was examined with transrectal ultrasonog- raphy, a map of the follicles was prepared, and all follicles �12 mm in diameter were subjected to intra- follicular injections. Intrafollicular injections were done with a 7.5 MHz transducer attached to a biopsy guide and a scanner (AquilaVet Scanner; Pie Medical Equipment BV, Maastricht, the Netherlands). A system with two sterile needles was used, as previously described [1]. Briefly, the ovary was manipulated to introduce the needle into the follicle via the ovarian stroma at the base of the follicle. When the ovary and follicle were in position, the outer needle was advanced until the image of its tip became visible on the screen, 3 to 5 mm from the follicle. At this moment, a second operator pushed the inner needle forward until the image of the needle tip was visible within the follicle. Treatments were then injected into the follicle. To obtain the desired final concentration inside the follicle, the dose of each treat- ment was calculated based on the volume of follicular fluid, estimated by the linear regression equation V � �685.1�120.7 D, where V corresponded to the esti- mated follicular volume and D to the diameter of the follicle to be injected [1]. The injection volume per follicle ranged from 80 to 110 �l, approximately 10% f follicular fluid volume. Cows were excluded from he experiment if the injected follicle had a reduction in iameter �1 mm within 2 h after injection (evidence of eakage). .2.1. Experiment I: progesterone induced oocyte uclear maturation in vitro The first experiment was designed to assess the P4 effect on nuclear maturation. Oocytes (n � 565) cul- tured with follicular hemisections treated were with 0, 10, 100, 1,000 or 10,000 ng/ml of P4. After 22 h of ulture, oocytes were considered mature when classi- ed as AI, TI, or MII. .2.2. Experiment II: effect of progesterone ntagonist on lh-induced meiotic resumption in vivo Five cows were primed for superovulation and ma- ipulated to have no more than three follicles �12 mm n each ovary at the time of injection. For each cow, ollicles in the right ovary were treated to obtain a final oncentration in follicular fluid of 1 �M of mifepristone MIFE group; n � 10), whereas those from the left vary were treated with 0.9% saline (control group; � 10). Immediately after the intrafollicular injections, he cows were given 100 �g of gonadorelin acetate im Profertil, Tortuga, Brazil), a GnRH agonist. Fifteen h fter GnRH treatment, cows were ovariectomized by olpotomy. The COCs were recovered and processed as escribed above. Oocytes at GVBD or MI stages were onsidered as having resumed meiosis. .2.3. Experiment III: progesterone mediates AngII- nduced meiotic resumption in vitro The COCs (n � 540) were selected and distributed mong the following seven groups for 15 h of culture: ositive and negative controls; AngII (10�11 M); AngII plus MIFE (1 �M; P4 antagonist); P4 (100 ng/ml), P4 plus aralasin (10�5 M; AngII antagonist); and AngII plus sara- asin. In all groups, except the positive control, follic- lar hemisections and COCs were cocultured. Oocytes n MI or latter stages were considered to have normal esumption of meiosis. To determine if there was a toxic effect of the P4 antagonist, COCs were cultured for 22 h, without fol- licular hemisections, in the absence or presence of MIFE (1 �M). Oocytes were considered mature when classified as AI, TI or MII. n w m m w d 0 o T a w c G e r 1 3 i a p c 1782 L.C. Siqueira et al. / Theriogenology 77 (2012) 1779–1787 2.2.4. Experiment IV: effect of FGF10 on AngII- induced meiotic resumption in vitro Control COCs were cultured in medium in the ab- sence (positive control; n � 84) or presence (negative control; n � 88) of follicular hemisections for 7 h. Four treatment groups were established; the COCs were cul- tured in the presence of: a) AngII (10�11 M; n � 83) with follicular hemisections; b) AngII and FGF10 (100 ng/ml) with follicular hemisections (AngII�FGF10 group; n � 82); c) FGF10 with follicular hemisections (FGF10�cells group; n � 80); and d) FGF10 without follicular hemisections (FGF10 group; n � 88). Oocyte nuclear chromatin configuration was classified as GV or germinal vesicle breakdown (GVBD). 2.2.5. Experiment V: effect of FGF10 or indomethacin on progesterone-induced meiotic resumption in vitro Control COCs were cultured for 7 h in the absence (positive control; n � 85) or presence (negative control; n � 82) of follicular hemisections. Three treatment groups were established. The COCs were cocultured with follicular cells in the presence of: a) progesterone (100 ng/ml; P4 group; n � 84); b) P4 plus FGF10 (100 g/ml; P4 � FGF10 group; n � 80) and c) P4 plus indomethacin (a COX nonselective inhibitor; 10 �M, P4�indo group; n � 85). Oocyte nuclear chromatin configuration was classified as GV or germinal vesicle breakdown (GVBD). 2.3. Statistical analysis Data from Experiments I, III, IV, and V were ana- lyzed using the ANOVA test in a statistical model for categorical data, using the PROC CATMOD (Categor- ical Data Analysis Procedures). All in vitro experi- ments were performed in triplicate. When there were significant differences, independent variables were compared using the contrast test. All data were ana- lyzed using statistical analysis software (SAS; SAS Institute, Inc., Cary, NC, USA). In Experiment II, mei- otic resumption was compared using the generalized linear models from JMP software (SAS Institute, Inc.). 3. Results 3.1. Experiment I: progesterone induced oocyte nuclear maturation in vitro The hypothesis tested in this experiment was that P4 induces nuclear maturation in bovine oocytes. Bovine COCs, recovered from abattoir-derived ovaries, were cocultured with follicular hemisections for 22 h with various concentrations of P4. Progesterone induced nu- clear maturation in bovine oocytes cultured with follic- ular cells in a dose-dependent manner (Fig. 1). The MII rate was greatest for oocytes cultured with follicular cells treated with 100 ng/ml of P4 (P � 0.01). 3.2. Experiment II: effect of progesterone antagonist on LH-induced meiotic resumption in vivo Once P4 stimulated nuclear maturation in vitro, hether the LH-induced resumption of meiosis was ediated by progesterone was tested using an in vivo odel. The mean initial diameter of follicles treated ith progesterone antagonist (MIFE; 12.8 � 0.4 mm) id not differ from those injected with saline (13.1 � .5 mm; P � 0.05). From the injected follicles, 20 ocytes were recovered and evaluated (10 per group). he ability of the LH surge (induced by the GnRH gonist) to induce resumption of meiosis was impaired hen follicles were treated with the progesterone re- eptor antagonist (MIFE; 70, 10 and 20% were GV, VBD, and MI, respectively; P � 0.01; Fig. 2B). As xpected, the GnRH agonist induced 90% of meiotic esumption in oocytes from saline-treated follicles (10, 0, and 80% were GV, GVBD, and MI). .3. Experiment III: progesterone mediated AngII- nduced meiotic resumption in vitro Since the role of AngII in resumption of meiosis nd ovulation is well established, we tested the hy- othesis that AngII is an upstream factor to P4 in the ascade of meiotic resumption. Meiotic resumption Progesterone (ng/mL) 0 10 100 1000 10000 M et ap ha se II (% ) 0 20 40 60 80 3824 101.1102.111.03.7 xxxy −− ×+×−+= 01.0