Effect of superstimulatory treatments on the ... - CSIRO Publishing

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Reproduction, Fertility and Development, 2013, 25, 17–25 http://dx.doi.org/10.1071/RD12271

Effect of superstimulatory treatments on the expression of genes related to ovulatory capacity, oocyte competence and embryo development in cattle Ciro M. Barros A,C, Rafael A. Satrapa A, Anthony C. S. Castilho A, Patrıćia K. Fontes A, Eduardo M. Razza A, Ronaldo L. Ereno A and Marcelo F. G. Nogueira B A

Department of Pharmacology, Institute of Bioscience, University of Sao Paulo State (UNESP), Rubiao Jr S/N, Botucatu, 18618-970, SP, Brazil. B Department of Biological Science, University of Sao Paulo State (UNESP), Assis, 19806-900, SP, Brazil. C Corresponding author. Email: [email protected]

Abstract. Multiple ovulation (superovulation) and embryo transfer has been used extensively in cattle. In the past decade, superstimulatory treatment protocols that synchronise follicle growth and ovulation, allowing for improved donor management and fixed-time AI (FTAI), have been developed for zebu (Bos indicus) and European (Bos taurus) breeds of cattle. There is evidence that additional stimulus with LH (through the administration of exogenous LH or equine chorionic gonadotrophin (eCG)) on the last day of the superstimulatory treatment protocol, called the ‘P-36 protocol’ for FTAI, can increase embryo yield compared with conventional protocols that are based on the detection of oestrus. However, inconsistent results with the use of hormones that stimulate LH receptors (LHR) have prompted further studies on the roles of LH and its receptors in ovulatory capacity (acquisition of LHR in granulosa cells), oocyte competence and embryo quality in superstimulated cattle. Recent experiments have shown that superstimulation with FSH increases mRNA expression of LHR and angiotensin AT2 receptors in granulosa cells of follicles .8 mm in diameter. In addition, FSH decreases mRNA expression of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in oocytes, but increases the expression of both in cumulus cells, without diminishing the capacity of cumulus–oocyte complexes to generate blastocysts. Although these results indicate that superstimulation with FSH is not detrimental to oocyte competence, supplementary studies are warranted to investigate the effects of superstimulation on embryo quality and viability. In addition, experiments comparing the cellular and/or molecular effects of adding eCG to the P-36 treatment protocol are being conducted to elucidate the effects of superstimulatory protocols on the yield of viable embryos. Additional keywords: bovine, embryo transfer, FSH, LH, LH receptor.

Introduction Reproductive biotechnologies, such as AI and embryo transfer, are important for the genetic improvement of cattle (Pinheiro et al. 1998; Barros et al. 2000; Barros and Nogueira 2001; Fernandes et al. 2001; Nogueira et al. 2004; Mapletoft and Hasler 2005; Baruselli et al. 2006; Bo´ et al. 2006, 2008). The detection of oestrus is a major factor affecting the success of AI programmes, requiring both time and properly trained personnel. However, the short duration of behavioural oestrus in zebu cows (,11 h; Pinheiro et al. 1998; Sartori and Barros 2011) makes detection difficult and limits the use of conventional AI programmes. Greater understanding of follicular dynamics (for reviews, see Fortune et al. 2001; Ginther et al. 2001) has increased the possibility of controlling follicular development through hormonal treatment protocols, which allow for fixed-timed AI Journal compilation Ó IETS 2013

(FTAI) in both Bos taurus (Pursley et al. 1995; Martinez et al. 2002; Perry et al. 2002; Bucher et al. 2009; Larson et al. 2009) and Bos indicus (Barros et al. 2000; Fernandes et al. 2001; Ayres et al. 2008; Carvalho et al. 2008; Meneghetti et al. 2009; Pinheiro et al. 2009; Sa Filho et al. 2009) cattle. Similarly, follicular development and ovulation have been manipulated to improve the management of superovulation and embryo transfer (Barros and Nogueira 2001; Mapletoft et al. 2002; Mapletoft and Hasler 2005; Baruselli et al. 2006; Nogueira et al. 2007b; Bo´ et al. 2008). Numerous treatment protocols to induce multiple ovulations in cattle, using different gonadotropins, doses, routes of administration and various hormone combinations and schedules, have been proposed in an attempt to improve embryo yield (Barros and Nogueira 2001; Baruselli et al. 2006; Bo´ et al. 2006; Barros et al. 2010). So far, these superstimulatory treatment protocols www.publish.csiro.au/journals/rfd

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have improved donor management, but have not significantly improved embryo yield (for reviews, see Barros et al. 2001, 2010; Baruselli et al. 2006). Treatments to induce multiple ovulations Synchronisation of follicle wave emergence for superstimulation Considering that the presence of a dominant follicle at the beginning of the superstimulatory treatment protocol decreases embryo yield (Guilbault et al. 1991), strategies such as starting FSH administration on the first day of the oestrous cycle (Goulding et al. 1990; Nasser et al. 1993, 2011; Roberts et al. 1994) or synchronising follicular wave emergence by aspirating follicles (Berfelt et al. 1994; Bodensteiner et al. 1996) or with oestradiol and progestins (Bo´ et al. 2006), have been used. Regarding the last strategy, Bo´ et al. (2003) showed that the combined use of an intravaginal device containing a progestin and intramuscular oestradiol administration induced follicular atresia and the onset of a new follicular wave approximately 4 days after treatment. To avoid the presence of a dominant follicle, FSH treatment is initiated at the beginning of a new follicular wave, 4 days after the combined use of progesterone and oestradiol. Two days after the first FSH injection, a luteolytic dose of prostaglandin (PG) F2a is administered and, 12 h later, the progesterone device is removed. Finally, the donors are inseminated 12 and 24 h after oestrus detection and, 7 days later, embryos are collected, classified and transferred or cryopreserved. Synchronising ovulation for FTAI The use of protocols to synchronise follicle wave emergence has partially facilitated the management of donor cows; however, this has not improved the number of viable embryos obtained or the variability of the donors’ responses to treatment. The inconsistent number of viable embryos obtained after superovulation can be explained, in part, by the asynchrony between nuclear and cytoplasmic maturation in the oocytes of superstimulated cows (intrafollicular asynchrony), as well as by the anomalous steroidogenic profiles of preovulatory follicles, which can result in deficient sperm transportation and oocyte maturation (Hyttel et al. 1986). Furthermore, preparations containing high levels of LH may cause premature activation of the oocyte (Mapletoft et al. 2002). In addition, inconsistent results have been associated with premature ovulation during FSH-induced superstimulation, followed by early formation of a corpus luteum secreting sub-luteal levels of progesterone at the corresponding time of oestrus, which may inhibit the preovulatory LH surge, ovulation and sperm transport (Callesen et al. 1987; Stock et al. 1996). Effects of exogenous LH on ovulation It has been suggested that follicles that do not ovulate after FSH superstimulation are abnormally developed or may not have sufficient LH receptors (LHR) to respond to the preovulatory LH surge (Xu et al. 1995; D’Occhio et al. 1997; Liu and Sirois 1998). Therefore, it has been proposed that postponing the preovulatory LH surge would allow for FTAI (Barros and Nogueira 2001) and increase embryo production

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after superovulation (D’Occhio et al. 1997; Liu and Sirois 1998; van de Leemput et al. 2001). Based on this information, Barros and Nogueira (2005) designed a protocol called the P-36 protocol in which the progesterone-releasing device is left in place for up to 36 h after administration of PGF2a and ovulation is induced by the administration of exogenous LH 12 h after the withdrawal of the progesterone device (i.e. 48 h after PGF2a administration). Because exogenous LH induces ovulation in 24 and 36 h after administration (Nogueira and Barros 2003), FTAI is scheduled for 12 and 24 h later, eliminating the need for the detection of oestrus. The P-36/LH48 protocol has proven effective in Nelore cattle (Barros and Nogueira 2001; Baruselli et al. 2006; Nogueira et al. 2007b). Conversely, the number of viable embryos obtained from European breeds was reduced after application of the P-36 protocol compared with conventional protocols using oestrus detection. For this reason, the P-36 protocol has been adjusted for European breeds. In Holstein (Baruselli et al. 2006) and Angus (Chesta et al. 2007) cows, the P-36 protocol was more effective (i.e. increased viable embryo production) when the ovulation-inducing treatment (porcine (p) LH or gonadotrophin-releasing hormone (GnRH)) was delayed for 12 h; that is, when pLH or GnRH were administered 60 h (P-36/LH60), rather than 48 h (P-36/ LH48), after PGF2a administration. Effects of LH activity on terminal follicle growth Improved success of the P-36 protocol is observed when the last two doses of FSH were replaced with administration of equine chorionic gonadotrophin (eCG). It was hypothesised that, on the last day of the superstimulatory treatment, most of the follicles would have acquired LHR and would grow in response to eCG due to its capacity to stimulate both LH and FSH receptors (Murphy and Martinuk 1991). Indeed, administration of 200 IU eCG twice rather than FSH on the last day of the P-36 superstimulation protocol has improved embryo yield in Bonsmara (Barcelos et al. 2006), Nelore (Barcelos et al. 2007), Brangus (Reano et al. 2009) and Sindhi (Mattos et al. 2011) cattle. These results indicate that eCG stimulates final follicle growth, possibly due to the presence of LHR in follicles .7 mm in diameter from B. indicus cows (Nogueira et al. 2007a; Simo˜es et al. 2012). To verify the relevance of LH activity, Rosa et al. (2010) replaced eCG with pLH on the last day of the superstimulatory treatment protocol. In this experiment, Angus cows (n ¼ 22) were submitted to four treatments: P-36/LH60 (control group); P-36/eCG/LH60, in which the last two doses of FSH were replaced with two doses of 200 IU eCG; P-36/LH/LH60, in which the last two doses of FSH were replaced with two doses of 1.0 mg LH; and P-36/FSHþLH/LH60, in which the last two doses of FSH were administered simultaneously with 1.0 mg LH. The results of that study indicated that replacement of eCG with LH alone (P-36/LH/LH60 group) decreased the number of viable embryos compared with that seen in the P-36/FSHþLH/ LH60, P-36/eCG/LH60 and control groups (0.8 0.2 vs 5.4 1.0, 3.9 0.8 and 2.5 0.7, respectively; P , 0.05). Hence, in Angus cattle, 400 IU eCG cannot be replaced with 2.0 mg LH; however, the combination of FSH plus LH may be

Gene expression after superstimulatory treatments

used as a substitute for eCG on the last day of superstimulatory treatment. This interesting observation must be confirmed. In a complementary study, Oliveira et al. (2010) tested four treatments in Nelore cows (n ¼ 25): P-36/LH48 (control); P-36/ eCG/LH48; P-36/LH2.0/LH48, in which the last two doses of FSH were replaced with two doses of 1.0 mg pLH; and P-36/ LH4.0/LH48, in which the last two doses of FSH were replaced with two doses of 2.0 mg pLH. Oliveira et al. (2010) showed that, in Nelore cows, the substitution of eCG with either 2 or 4.0 mg pLH did not significantly reduce viable embryo yield (3.7 0.8 and 4.2 1.0, respectively) compared with the control (3.3 0.7), P-36/eCG/LH48 (4.5 0.5) and P-36/LH2.0 (3.7 0.8) groups. These interesting results have led to our designing further studies to investigate the role of LH and its receptors in ovulatory capacity (acquisition of LHR in granulosa cells), oocyte competence and embryo quality in cows submitted to these various superstimulatory protocols. Ovulatory capacity and regulation of signalling mechanisms for the LHR It is known that LH plays a key role in controlling physiological processes in the ovary, such as in the development of antral follicles, ovulation and luteal development and maintenance. (Xu et al. 1995). These events highlight the importance of LHR expression in bovine granulosa cells for the transition from FSH to LH dependency. For many years, the expression of LHR was considered to occur exclusively in gonadal cells (i.e. in the Leydig cells of the testes and in the theca, granulosa and luteal cells of the ovaries). However, there is evidence of the presence of LHR in extragonadal tissues, such as the uterus, the mucosa of the oviduct (Zhang et al. 2001), human spermatozoa, the prostate, the seminal vesicles and the lactating mammary gland of rodents (Lei et al. 1992; Zhang et al. 2001). This wide tissue and physiological distribution reflects the importance of LHR, which belong to the large family of G-protein-coupled receptors (GPCR). Like all GPCR, the LHR is characterised as having three distinct domains: a large N-terminal domain, predicted to be an extracellular domain; seven transmembrane segments connected by three extracellular loops; and three intracellular loops and a C-terminal tail (predicted to be intracellular; Ji et al. 1998). This protein conformation of LHR is very similar among species and is encoded by a single gene (for a review, see Ascoli et al. 2002). The LHR gene consists of 11 exons and 10 introns (Ji and Ji 1991; Koo et al. 1991; Tsai-Morris et al. 1991; Atger et al. 1995). The entire transmembrane region and C-terminal domain are encoded by exon 11, whereas the N-terminal region is encoded by the alternative splicing (gene rearrangement) of exons 1–10. In granulosa cells, the transcriptional regulation of the LHR gene is FSH dependent and is important for the differentiation of this cell type (Shi and Segaloff 1995). During the growth and differentiation of granulosa cells, there is an increase in the abundance of LHR transcript (dependent on oestradiol and FSH), and these actions can be mimicked by factors that result in an increase in cAMP concentrations. During in vitro culture of granulosa cells, FSH can induce increases in the four major transcripts of bovine LHR (Nogueira et al. 2007a).


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In general, most of the available data in the literature agree that the effects of LHR on the regulation of granulosa cell differentiation are mediated mainly via the Gs-protein/adenylate cyclase/cAMP/protein kinase A (PKA) system, although other pathways can be used for the activation of LHR (for a review, see Ascoli et al. 2002). In general, GPCR activation induces two types of G-protein intracellular signalling: adenylate cyclase via Gs or phospholipase C (PLC)/inositol 1,4,5-trisphosphate (IP3) via Gai or Gaq/11 (Shemesh 2001). Although the use of these two pathways has been reported during the activation of LHR in granulosa cell rats (Herrlich et al. 1996), PLC/IP3 signalling remains contentious. Donadeu et al. (2011) verified the expression of G-proteins and PLC subtypes in bovine antral follicles of different sizes. These authors reported increases in mRNA and protein levels of heterotrimeric G-protein subunits (i.e. as (GNAS), aq (GNAQ) and a11 (GNA11)), concomittant with an increase in LHR expression in ovulatory-sized follicles compared with small follicles. Of the four known PLCb isoforms, PLCb3 showed higher expression in cells from ovulatory-sized follicles, in association with the predominantly cytoplasmic location of PLCB3 in these cells and with a significant IP3 response to LH stimulation. Furthermore, RNA interferencemediated PLCB3 downregulation reduced the ability of LH to induce both aromatase expression and oestradiol production. In summary, Donadeu et al. (2011) provided evidence of the physiological involvement of PLCb3 signalling in ovulatorysized follicles. To gain an insight into the effects of superstimulation with FSH (P-36 protocol) on LHR mRNA expression and the proteins involved in LHR signalling, ovaries from Nelore cows submitted to the P-36 protocol or untreated controls were dissected and the mRNA of the target genes (LHR, GNAS, GNAQ, GNA11, PLCB3) were assessed to determine the effects of FSH treatment. No differences in GNAS, GNAQ, GNA11 or PLCB3 mRNA levels were observed in granulosa cells regardless of whether cows received the superstimulation treatment or not (A. C. S. Castilho, R. A. Satrapa, C. M. Barros, unpubl. data). Although the P-36 protocol did not regulate the mRNA expression of the proteins involved in the signalling mechanisms of the cAMP/IP3 system, the constant presence of GNAS, GNAQ, GNA11 and PLCB3 mRNA indicated the participation of both LHR pathways during granulosa cell differentiation, corroborating the results of Donadeu et al. (2011). In addition, there was significantly higher expression of LHR mRNA in the granulosa cells of cows submitted to the P-36 protocol than in non-superstimulated control animals (Fig. 1). These in vivo results confirmed the results of previous studies in which LHR mRNA transcripts were increased by FSH treatment of cultured bovine granulosa cells (Nogueira et al. 2007a). Another important factor that seems to be involved in ovulation is angiotensin (Ang) II, which is the major bioactive peptide of the renin–angiotensin system (Paul et al. 2006). Infusion of AngII induced ovulation in rabbits and the use of AngII antagonists inhibited ovulation in rabbits, rats and cattle (Pellicer et al. 1988; Yoshimura et al. 1993, 1996; Kuji et al. 1996; Ferreira et al. 2007). AngII acts through two distinct transmembrane receptors, namely AT1 (encoded by the AGTR1

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LHR mRNA

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Treatments Fig. 1. Mean ( s.e.m.) relative abundance of LHR and AT2 receptor mRNA in granulosa cells from superstimulated Nelore cows (P-36 group; n ¼ 24–27) or untreated controls (control group; n ¼ 14). Levels of both LHR and AGTR2 mRNA were higher in the P-36 group (P , 0.05).

Total LHR mRNA

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ax b b

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Days after ovulation Fig. 2. Mean ( s.e.m.) relative abundance of total LH receptor (LHR; no isoform distinction) and the LRBP isoform mRNA in granulosa cells from the largest follicle (black bars) and second largest follicle (grey bars) around the time of follicle deviation in Nelore heifers (Day 2, n ¼ 3; Day 2.5, n ¼ 4; Day 3, n ¼ 3). Within days, different letters (a, b) indicate significant differences (P , 0.05) between the largest and second largest follicles; between days, different letters (x, y) indicate significant differences (P , 0.05) in the largest follicle.

gene) and AT2 (encoded by the AGTR2 gene; Paul et al. 2006). In rabbits, the receptors are mostly AT2 receptors and they are expressed in the granulosa cells of preovulatory follicles, consistent with the role of AngII in ovulation. A similar role has been suggested in cattle by Portela et al. (2008), who observed that AGTR2 mRNA in bovine granulosa cells was more abundant in healthy compared with atretic follicles. Recently, we observed higher levels of AT2 receptor mRNA in cows from the P-36 compared with the control group, as with LHR (Fig. 1). These findings substantiate those of Portela et al. (2008), who observed increases in AT2 receptor mRNA and protein levels after adding FSH to granulosa cell culture. Role of LHR in follicular deviation in cattle There are differences of opinion regarding the time at which follicles acquire LHR in granulosa cells. Some authors have reported that the future dominant follicle acquires LHR before follicular deviation (Beg et al. 2001; Ginther et al. 2001, 2003), whereas others have reported that the expression of LHR occurs

after follicular deviation (Xu et al. 1995; Bao et al. 1997; Fortune 2001; Nogueira et al. 2007a). In a previous study in which semiquantitative polymerase chain reaction (PCR) was used to determine LHR mRNA isoforms (LHRB) in Nelore cattle, we detected LHR expression in only a few samples during the expected time of follicular deviation (Day 2.5) and much more LHR expression after deviation (Day 3; Barros et al. 2010). However, in a recent study using real-time PCR to assess LHR mRNA expression (no isoform distinction) in the granulosa cells of the two largest follicles from Nelore heifers on Days 2, 2.5 and 3 after ovulation, we detected LHR mRNA at the predicted time of follicle deviation (Day 2.5) and after deviation (Fig. 2; unpubl. obs.). The second largest follicles (subordinate follicles) had lower levels of LHR mRNA compared with the largest follicles (future dominant follicles) on Days 2.5 and 3. Because LHR mRNA binding protein (LRBP) is responsible for the downregulation of LHR mRNA, thereby controlling steady state levels, we investigated the presence of LRBP mRNA in granulosa cells. We observed that, in contrast with


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Treatments Fig. 3. Mean ( s.e.m.) relative abundance of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) mRNA in cumulus cells and oocytes in cumulus–oocyte complexes from superstimulated Nelore cows (P-36 group; n ¼ 4) or untreated controls (control group; n ¼ 6). All comparisons between treatments were significant (P , 0.05).

LHR mRNA expression, LRBP is expressed more in the subordinate follicles than in the largest follicles 2.5 and 3.0 days after ovulation (Fig. 2), indicating that it may be necessary to decrease LRBP expression in future dominant follicles to increase expression of the LHR. This inverse relationship between LHR and LRBP mRNA has also been demonstrated in luteal cells from rats (Nair et al. 2002). Regarding the participation of LH/LHR in ovulation, it has been suggested that ovulatory capacity is linked to the acquisition of sufficient LHR (Xu et al. 1995; D’Occhio et al. 1997). Gimenes et al. (2008) observed that the administration of 25 mg pLH induced ovulation in 33.3%, 80.0% and 90.0% of B. indicus heifers when follicles were 7.0–8.0, 8.5–10 and .10 mm in diameter, respectively. The relationship between ovulation and LHR isoform gene expression was recently investigated by Simo˜es et al. (2012) in Nelore cattle. Increases in follicular diameter from 7 to 8 mm, from 8.1 to 9 mm and from 9.1 to 10 mm corresponded to increases in the incidence of ovulation of 9%, 36% and 90% (6.25 mg, i.m., LH), respectively, and increases in the expression of LHR isoforms in granulosa cells of 16.5%, 21% and 37.6%, respectively. Sartori et al. (2001) also evaluated the ovulatory capacity of Holstein cows (B. taurus) and found that follicles 7.0 and 8.5 mm in diameter did not ovulate, even after the administration of as much as 40 mg pLH. However, 80% of follicles $10 mm in diameter ovulated in cows after pLH treatment. Taking into account that follicle deviation occurs when the follicles reach

6.0 mm in diameter in B. indicus (Sartorelli et al. 2005; Gimenes et al. 2008) and 8.5 mm in diameter in B. taurus (for reviews, see Ginther et al. 1996; Fortune 2001; Fortune et al. 2001), these collective results indicate that the dominant follicle acquires the capacity to ovulate only after follicle deviation. Bovine cumulus–oocyte complex competence and embryonic development Superovulation with exogenous gonadotropins has been used extensively to produce in vivo-derived embryos for embryo transfer in cattle. However, the quality of the embryos varies and may be affected by oocyte competence (Sirard et al. 2006) or during embryo development in the oviduct (Killian 2004; Looney and Pryor 2010). Oocyte competence seems to be related to the abundance of specific mRNA that is transcribed and accumulated during oocyte growth and the final phases of folliculogenesis. Several genes have been identified as potential markers of oocyte competence in generating embryos (Donnison and Pfeffer 2004; Fair et al. 2004; Dode et al. 2006; Mourot et al. 2006; Caixeta et al. 2009). Various factors can improve the capacity of the cumulus to support oocyte maturation during antral follicle growth, including members of the transforming growth factor (TGF)-b superfamily, such as growth differentiation factor (GDF) 9 and bone morphogenetic protein (BMP) 15 (Lucidi et al. 2003; Pangas and Matzuk 2005), and the gonadotropins

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FSH and LH (Adriaens et al. 2004; Ali and Sirard 2005; Sirard 2007). It has been reported that GDF9 and BMP15 mRNA is expressed in bovine oocytes from primary and primordial follicles and expression is maintained until the 8-cell stage after fertilisation (Hussein et al. 2006). Moreover, the addition of exogenous GDF9 and BMP15 to cumulus– oocyte complexes (COCs) during IVM increases blastocyst yield (Hussein et al. 2006). Previously, mRNA expression was detected only in oocytes, but recently Hosoe et al. (2011) detected BMP15 and GFD9 mRNA (at low levels compared with oocytes and their proteins in bovine cumulus cells). In that study, higher levels of BMP15 and GDF9 mRNA were observed in cumulus cells from cows than from calves, suggesting that the low levels in calves could explain the inferior competence of calf oocytes (Hosoe et al. 2011). Another important aspect of COC development is the role of LH. Although it has been established that LH controls important functions in both the oocyte and cumulus cell layer, there is no agreement about the presence of LHR proteins in cumulus cells, suggesting an indirect pathway for COCs (Peng et al. 1991) by the epidermal growth factor (EGF)/EGF receptor (EGFR) network (Panigone et al. 2008; Reizel et al. 2010) and by EGF-related growth factors, such as amphiregulin, epiregulin and betacellulin (for reviews, see Conti et al. 2006; Gilchrist 2011). To better understand the effects of ovarian superstimulation with FSH on oocyte competence and blastocyst development, we recently determined the profiles of BMP15, GDF9, histone H2A, FSH receptor (FSHR), EGFR and pentraxin 3 (PTX3) mRNA in immature cumulus–oocyte cells in Nelore cattle treated (P-36) or not (control) with FSH (A. C. S. Castilho, R. A. Satrapa, C. M. Barros, unpubl. data). In addition, we investigated the effect of this superovulatory treatment (P-36) on oocyte competence to develop to the blastocyst stage. We found that immature bovine oocytes (i.e. oocytes obtained before the LH surge) from cows submitted to the P-36 protocol had lower levels of BMP15 and GDF9 mRNA compared with non-stimulated control cows (Fig. 3), suggesting a negative effect of FSH treatment on oocyte competence or a decline in mRNA levels, possibly due to post-transcriptional events inducing protein expression. In contrast with members of the TGF-b family, histone H2A mRNA levels did not change with the superstimulatory protocol; similarly, the expression of EGFR, FSHR and PTX3 mRNA was similar in superstimulated and non-superstimulated cattle. In contrast, the expression of GDF9 and BMP15 in cumulus cells from the superstimulated group was significantly higher than in the control group (Fig. 3), indicating that FSH may act by different mechanisms in COCs. Indeed, the COCs obtained from the P-36 and control groups produced similar in vitro blastocyst rates (40% and 37%, respectively; A. C. S. Castilho, R. A. Satrapa, C. M. Barros, unpubl. data). In summary, although important genes were suppressed in oocytes from superstimulated cows, the maintenance of histone H2A in the oocytes, as well as PTX3, EGFR and FSHR mRNA, and the increases in GDF9 and BMP15 expression in the cumulus cells seem to ensure sufficient oocyte competence to produce embryos.

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Final remarks The response to superstimulatory treatments varies depending on different factors, such as breed, animal, superstimulatory protocol and type of gonadotropin used. In addition, ovarian superstimulation can affect embryo quality; however, reports have varied from improvement (Mattos et al. 2011) to decreases (Lerner et al. 1986) in embryo quality in superovulated cattle. Our recent data show that FSH superstimulatory treatment increases LHR expression in granulosa cells and decreases GDF9 and BMP15 expression in oocytes, but increases GDF9 an BMP15 expression in cumulus cells and does not decrease the capacity of COCs to generate blastocysts. These preliminary results indicate that superstimulation with FSH is not detrimental to oocytes. Nevertheless, additional experiments are necessary to investigate the effects of superstimulatory treatments on the mRNA levels of marker genes involved in embryo quality, to compare the effects of the P-36/eCG protocol with those observed after P-36 treatment and to analyse how these superstimulatory protocols may affect bovine oviduct function. Acknowledgements The authors are grateful to FAPESP (Saõ Paulo, Brazil) for funding and to CAPES, CNPq (Brasilia, Brazil) and FAPESP for fellowships.

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