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Intra-ovarian regulation of follicular development and oocyte competence in farm animals
Theriogenology 68S (2007) S22–S29 www.theriojournal.com
Intra-ovarian regulation of follicular development and oocyte competence in farm animals R. Webb a,*, P.C. Garnsworthy a, B.K. Campbell b, M.G. Hunter a a
School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK b Department of Obstetrics and Gynaecology, School of Human Development, Queens Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK
Abstract In both mono-ovulatory species, such as cattle, and poly-ovulatory species, such as pigs, the interactions among extra-ovarian gonadotropins, metabolic hormones and intra-ovarian growth factors determine the continued development of follicles, the number of follicles that ovulate and the developmental competence of the ovulated oocyte. FSH and then subsequently LH are the main hormones regulating antral follicle growth in both mono- and poly-ovular species. However, a range of intra-ovarian growth factors, such as insulin-like growth factors (IGFs) and bone morphogenetic proteins (BMPs), are expressed throughout follicle and oocyte development and interact with gonadotropins to control follicle maturation. In addition, environmental factors such as nutrition, including both the amount and composition of the diet consumed prior to ovulation, can influence follicle development and the quality of the oocyte. Recent progress in our understanding has resulted in the development of diets that enhance oocyte quality and improve pregnancy rate in both pigs and cattle. In conclusion, despite some species-specific differences, similar interacting mechanisms control follicular development and influence oocyte quality. # 2007 Elsevier Inc. All rights reserved. Keywords: Ovary; Cattle; Pigs; Follicle; Growth factors; Gonadotropins
1. Introduction In both mono-ovulatory and poly-ovulatory species follicular growth is a continuum, controlled by the interaction between extra-ovarian factors, including gonadotropins and metabolic factors, and locally produced growth factors [1–3]. In addition, ovulation rate is a key determinant of reproductive efficiency and is tightly controlled in all species through mechanisms involving both extra-follicular factors and locally produced growth factors. This review will discuss the influence of intra-follicular factors, and how they interact
* Corresponding author. Tel.: +44 1159516056; fax: +44 1159516069. E-mail address: [email protected] (R. Webb). 0093-691X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2007.04.036
with extra-ovarian factors, on follicular growth, oocyte quality and embryo survival. Comparison between mono-ovulatory and poly-ovular species will be made to assist in identifying the key mechanisms involved. 2. Role of gonadotropins In both cattle and pigs, although gonadotropins do not appear to be involved in the initiation of follicular growth they do influence the early stages of follicular development [1,2,4]. Gonadotropins are definitely required for the final stages of follicular growth. In both species the emergence of follicular waves is preceded by a transient increase in FSH [1,5]. FSH then declines, due to ovarian negative feedback, below the threshold for further follicular selection and then, in
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both species, the dominant follicle(s) transfers dependency to LH [1,5]. Using experimental GnRH agonist models it was demonstrated in cattle that follicles grow to 8 mm in diameter when pulsatile LH release is inhibited and to only 4 mm when peripheral concentration of FSH is also reduced, as well as LH pulses being inhibited [6]. Likewise in pigs, when gonadotropins are reduced by GnRH agonist treatment, follicles do not grow beyond 4 mm in diameter [4]. Such results demonstrate the definitive requirement for gonadotropins in the final stages of follicular development in species with large differences in ovulation rate. Genetic differences in ovulation rate have been observed in different breeds of sheep, which demonstrate the importance of intra-ovarian mechanisms. For example in Booroola (FecB) sheep, carrying a single point mutation in the intracellular kinase domain of the BMPR-1B gene, follicles mature at a smaller diameter [7] and undergo precocious differentiation of granulosa cells with expression of LH receptors and increased expression of aromatase and inhibin bA genes [8,9]. Indeed Campbell et al. [10] demonstrated that the FecB mutation acts primarily within the ovaries of sheep resulting in increased ovulation rate compared with noncarriers, even when receiving similar patterns of gonadotropins. In addition to the FecB gene in sheep, genetic studies in Inverdale (FecXI) sheep have identified a point mutation in the BMP-15 gene [11], which affects follicle development and ovulation quota. Heterozygous ewes have an increased ovulation rate whilst homozygous ewes have small non-functional ovaries and are infertile [12]. More recently, BMP-15 point mutations have been identified in the Belclare (FecXB) and in the Cambridge (FecXC) and again, although the point at which the mutation occurs is breed dependent, they all exhibit the same X-linked phenotype [13]. 3. Bone morphogenetic proteins (BMP) and other oocyte secreted factors Recent research on oocyte secreted factors has focussed on murine systems; however we have extended these findings to the pig and shown that the porcine oocyte can modulate both granulosa and theca cell growth and function [14]. Oocyte secreted factors suppressed progesterone, but stimulated estradiol synthesis by granulosa cells throughout a 6-day culture period (Fig. 1). Furthermore, oocyte-derived suppression of progesterone was also observed in cultured theca cells and interestingly, both androstenedione and estradiol synthesis were influenced by oocyte derived factors [14].
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Fig. 1. Mean (S.E.D.) log10 (a) progesterone and (b) estradiol production by porcine granulosa cells after 144 h in serum free culture in the presence (&) and absence (&) of oocyte conditioned medium (OCM), 1 ng/ml pFSH and with either an optimal dose of Long R3 IGF-1 (agonist) (100 ng/ml) or no (0 ng/ml) Long R3 IGF-1 (agonist). (c) Number of viable granulosa cells after 144 h in serum free culture in the presence (&) and absence (&) of oocyte conditioned medium (OCM), 1 ng/ml pFSH and either an optimal dose of Long R3 IGF-1 (agonist) (100 ng/ml) or no (0 ng/ml) IGF-1. Values are from three independent cultures, each treatment having four replicates [modified from 14].
In addition, we have recently shown that the secretion of these factors is developmentally regulated, as oocytes at the germinal vesicle stage suppressed progesterone production, whereas oocytes that had matured to the metaphase II stage did not [15]. Furthermore, there were a number of differences in the secreted proteome of GVand MII oocytes, which is consistent with the co-culture
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findings [15]. Further work is required to identify more of these secreted proteins and their biological activity, although likely candidates include the BMPs and GDF-9. In summary, these findings support the proposal that oocytes secrete a factor(s) that modulates both cell proliferation and steroidogenesis and confirm that these factors are active inhibitors of luteinization. More specifically, we have demonstrated that BMPs decrease expression of 3b-hydroxysteroid dehydrogenase protein (3b-HSD, BMP-2;-6) and steroidogenic acute regulatory protein (StAR; BMP-6) [16] in cultured porcine granulosa cells. Cyclic adenosine monophosphate (cAMP) production was also suppressed significantly in both granulosa and theca cells. Furthermore the active phosphorylated downstream BMPR-regulated signaling molecule Smad-1 (p-Smad1) was upregulated in cells treated with BMP [17]. These findings provide evidence for the presence of a complex signaling mechanism in poly-ovulatory species as in mono-ovulatory species [18], and support the hypothesis that BMP-2 and BMP-6 act in a paracrine manner to control granulosa cell function, one aspect of which is to inhibit luteinization. BMPs exert their effects via BMP receptors (BMPRIA, -IB and -II) and in pigs immunohistochemistry for these receptors showed the presence of all three receptors in the fetal egg nests, oocytes and in the granulosa cell layer of follicles ranging from primordial to late antral stages [19]. Some immunostaining was also observed in the theca layer, corpus luteum and ovarian surface epithelium [19]. Actual protein expression of BMP-2 in pigs was identified by Western blotting in the oocyte, follicular fluid and to a lesser extent granulosa cells [16]. Similarly, GDF-9 has been shown to be present in the oocyte similar to BMP-15 where mRNAwas localized by in situ hybridization to the oocyte exclusively [20]. Similar to observations in the pig, BMP-6 and BMP receptors (BMPR) have been shown to be present in cattle fetal ovaries [21,22], with a similar pattern of mRNA expression in sheep [23]. These findings agree with those of Souza et al. [24] who demonstrated strong expression of BMPR in the oocyte and granulosa cells of ovine follicles from the primordial to preovulatory stages. As for pigs, the presence of both the ligand (BMP-6) and the receptors in cattle follicles, even at this early stage of development, illustrates the presence of all the components of a fully functional BMP system. Similarly in the adult ovaries of sheep [24] and cattle [25] there appears to be a fully functional BMP system and BMP2, 4, 6 and 7 have all been shown to exert effects on somatic cell function in vitro [24–27]. As discussed, recent studies by Hanrahan et al. [13] have also demonstrated a role for
BMP-15 in sheep. Also ovine granulosa cell progesterone production was inhibited while immunoreactive a inhibin levels increased when BMP15 and GDF9 were given together in culture [28]. However, the precise role of BMP-15 in bovine and porcine ovarian follicular development has not been elucidated; BMP-15 mRNA has recently been localized to small bovine and porcine preantral follicles [20], although temporal patterns of expression during follicle growth have still to be determined. In conclusion, both mono-and poly-ovular species appear to possess a fully functional BMP system, which is already present during fetal development. Members of the BMP system have been shown to be intricately linked with significant changes in ovulation rate [18,29]. Hence possible differences in BMP action and/or expression may explain the differences in ovulation rate between species, although this needs to be investigated further. 4. Insulin-like growth factors Another well-characterized local growth factor system is the insulin-like growth factors. In cattle, even by the preantral stage of development, follicles possess both IGFBP-2 and type 1 IGF receptors [30]. It appears that IGFs control preantral follicle growth primarily via endocrine mechanisms, with IGFBPs regulating the bioavailability of extra-ovarian IGF-I [18]. In support of this suggestion, IGF-I has also been shown to stimulate bovine preantral follicle growth in vitro [31]. It is not until the early antral stage of follicular development that IGFII expression in the thecal cells is first detected [32,33], when there appears to be a fully function IGF system. IGF-II has been shown to stimulate steroidogenesis of bovine thecal cells, acting via IGF type 1 receptors [34]. Thus IGF-II, like IGF-1, may have a significant role in thecal cell steroidogenesis during follicular development in mono-ovulatory species like cattle and sheep. As indicated, the actions of IGF-I and -II are regulated by locally produced IGF binding proteins [2,9]. In healthy bovine antral follicles up to 9 mm in diameter, the approximate size when granulosa cell LH receptors are first expressed, IGFBP-2 and -4 mRNA expression was restricted to granulosa and theca cells respectively [35]. IGFBP-2, and possibly IGFBP-4 and 5 concentrations, are higher in the follicular fluid of small and medium-sized bovine antral follicles, but are significantly reduced in follicular fluid of large and/or dominant bovine follicles [36]. Hence the conversion of a subordinate follicle to a future dominant follicle in cattle has been associated with a decrease in IGFBP-2 [18,35,37]. This reduction in follicular fluid IGFBP-2
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and -4 concentrations, coupled with an increase in estradiol concentrations, have been associated with the selection of the dominant follicle in cattle [35,38]. IGFs are also expressed within the porcine follicle; however, unlike cattle, IGF-I is expressed predominantly in the granulosa cells of follicles from 2 to 8 mm in diameter [39]. However, similar to cattle, IGF-II expression is higher in theca of 6 mm follicles and remains high until after the LH surge, indicating a role for IGF-II in ovulation and/or luteinization in the pig. In addition, IGF-I and the type I IGF receptor are required for all phases of preovulatory growth [40]. Similar to cattle, IGFBP-2 expression in porcine follicles is also inversely correlated with the diameter of follicles [40], suggesting that the potential for IGF action in large follicles is increased as the follicle grows. Similarly in the pig, increased follicular growth has been associated with greater circulating IGF-I [41]. Likewise, lower IGF-I concentrations are associated with reduced ovulation rates [41,42]. Hence changes in bio-active IGF-1 appear to be associated with changes in ovulation rate. These developmentally regulated changes in the patterns of expression of IGFs are associated with the action of gonadotropins. Utilizing a physiologically relevant culture system in both cattle and pigs [43,44], it has been demonstrated that FSH can induce estradiol production by granulosa cells and this induction is related to an increase in P450arom mRNA expression [43,45,46]. In both species, IGF-1, as well as insulin, interacts with FSH to stimulate granulosa cell estradiol production. In conclusion, despite species differences, in pigs, cattle and sheep, the development of follicles through to ovulation is controlled in part by the interaction between gonadotropins and IGFs. However in addition to IGFs a panoply of other locally produced factors, in addition to BMPs, appear to interact together to impact on follicle development and are involved in the control of ovulation rate. 5. Interaction between intra-follicular factors The effects of BMPs on both porcine [16,47] and ovine [27] granulosa cell function and their potential interactions with FSH and IGF-I have been investigated. In pigs BMPs can significantly suppress progesterone production in vitro. For example, there are significant interactions between both BMP-2 and -6 and IGF-I on progesterone production [16] and BMP-2, -6 and -15 also modified estradiol synthesis. Furthermore BMP-2 and -6 interacts with both IGF-I and FSH, whereas BMP-15 appears to interact with FSH only. In porcine
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theca cells, which express BMP receptors [19], all BMPs investigated (2, 6 and 15) stimulated cell proliferation in vitro [48], but in contrast both progesterone and estradiol synthesis were suppressed by BMP-2 and -6, but stimulated by BMP-15. Hence a range of BMPs can alter the pattern of steroid production and interactions have been observed between BMPs and both IGF-I and LH [48]. Interestingly, a significant modification of the response of both granulosa and theca cells to the BMPs occurs when cells are co-cultured with five oocytes/well compared to BMPs or oocytes alone, indicating a complex feedback loop involving BMPs, oocytes and somatic cells [48]. Collectively therefore, there is good evidence for a functional BMP system in the porcine ovary which interacts with other locally produced growth factors and gonadotropins and show that BMPs modulate somatic cell function and hence follicular development. Similarly for mono-ovulatory species, BMP-2 and -4 enhance FSH-stimulated estradiol production in sheep [24,27]. Furthermore, as for pigs, BMP-6 acts on bovine granulosa cells to enhance estradiol secretion whilst suppressing progesterone secretion [25]. There is also an interaction between BMPs and other local factors, showing that BMPs can enhance basal and IGF-induced secretion of estradiol, inhibin-A, activin-A and follistatin. More recently, Campbell et al. [27] confirmed BMP-6 protein expression in sheep and demonstrated a significant interaction between BMPs and IGFs [27] in stimulating granulosa cell differentiation. Further, it was demonstrated that the FecB mutation, which as discussed increases ovulation rate and litter size, results in an increased differentiative response of both granulosa and thecal cells to BMPs, IGFs and gonadotropins. These results demonstrate a major role for local growth factors such as BMPs and IGFs in both mono- and polyovulatory species in modulating proliferative and differentiative responses of theca and granulosa to gonadotropins (see Fig. 2). They may also be involved in controlling the number of follicles that are available for ovulation. 6. Impact of extra-ovarian factors on oocyte quality In addition to the paracrine interactions within the follicle of both mono- and poly-ovulatory species, oocyte quality is influenced by extra-follicular factors such as nutrition. In high yielding dairy cows the decline in fertility has been associated with negative energy balance postpartum and associated changes in metabolic hormones including reduced IGF-I and
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Fig. 2. Diagram showing the influence of nutrition, the role of gonadotropins and the interaction with the intra-follicular IGF and BMP systems on antral follicle development in both mono- and poly-ovulatory farm animal species. The top section describes a follicular wave and when the dominant follicle(s) transfers its dependence from FSH to LH. The bottom two sections illustrate some of the key members of two local growth factor systems (IGFs and BMPs) shown to be important in follicular development. It also highlights the additive effect of the IGF and BMP systems on FSH and LH stimulated follicular development [Adapted from 1,18,27].
insulin concentrations [2,49,50]. Feeding diets that increase insulin concentrations can advance the first ovulation postpartum [51] and stimulate follicle development in heifers [52]. Also in larger follicles in ruminants, IGF-I and insulin have been found to stimulate granulosa cell proliferation and mitogenesis and enhance FSH induced steroidogenesis by granulosa cells [44,53]. However, nutritionally-induced changes in circulating and local concentrations of IGF-I that are optimal for follicular growth may not be necessarily optimal for bovine oocyte maturation [54] and may even have a negative effect on oocyte growth [55]. Endocrine and metabolic signals that regulate follicular growth may also influence oocyte development either through changes in hormone/growth factor concentrations in follicular fluid or via granulosa– oocyte interaction. For example, short-term changes in dietary energy intake influence both oocyte morphology and developmental potential [56–58] and supplementation of rations with fats, can result in an increase in
energy intake and energy status of the cow [59,60]. We and others have also demonstrated that fatty acids may influence oocyte developmental potential in high yielding diary cows [60,61]. More recently we have utilized this information to demonstrate that pregnancy rate in high yielding dairy cows can be significantly improved by feeding diets that can influence follicle development and oocyte quality (Garnsworthy and Webb, unpublished observations). Similarly in pigs we have demonstrated that feeding a high plane of nutrition to gilts can improve oocyte quality [47,62]. Increased feed intake was not only associated with an increase in the proportion of oocytes at metaphase II, but also with increased IGF-I, leptin and LH concentrations. Furthermore the composition of the diet was shown to alter oocyte maturation and prenatal survival. Studies involving alterations in the protein, starch and/or fiber content of the pre-mating diet showed that dietary fiber can improve embryo survival [63,64]. There was 18% higher embryo survival
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on Days 27–29 of pregnancy in gilts [63] and nearly one extra piglet per litter when tested in multiparous sows in a commercial environment [65]. In addition, higher blastocyst yields and blastocyst cell numbers on Days 6–7 following in vitro fertilization have been achieved when gilts are fed a high fiber diet [66]. These results provide some of the first evidence of a direct link between oocyte maturity and embryo survival in the pig. They also demonstrate that diets that increase embryo survival are also associated with improved oocyte maturity and quality. Collectively, these findings indicate in both monoand poly-ovulatory species that nutritional regimens that increase embryo survival are also associated with beneficial effects on oocyte maturity and quality, supporting the idea that embryo viability originates during oocyte development. In summary, in both cattle and pigs changes in extra-ovarian factors such as metabolic hormones are associated with changes in follicular growth patterns, oocyte quality and embryo survival. Hence producing a good quality oocyte is essential for embryo survival, the maintenance of litter size in pigs and pregnancy in cattle and sheep. 7. Conclusions In both cattle and pigs many of the mechanisms involved with the development of the follicle involve the interaction of a panoply of intra-follicular growth factors (see Fig. 2). Indeed many of these mechanisms are similar in cattle and pigs, although with some species-specific differences. In addition, extra-ovarian follicular factors interact with these local factors to determine whether follicles continue to develop and the quality of the ovulated oocyte. Recent metabolomic studies, where a range of peripheral metabolites and metabolic hormones were measured, have demonstrated that diet can also influence oocyte maturity and quality, supporting the concept that embryo viability originates during oocyte development. These interactions are of key importance since they influence the developmental potential of the embryo and subsequent maintenance of pregnancy. Recent progress in understanding this multifactorial process has highlighted new opportunities for improving pregnancy rate in both mono- and multi-ovulatory farm animal species using nutritional approaches. Acknowledgements Much of the authors, own cited work was kindly supported by the BBSRC, Defra and SEERAD.
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R. Webb et al. / Theriogenology 68S (2007) S22–S29 [47] Hunter MG, Brankin V, Quinn RL, Ferguson EF, Edwards SA, Ashworth CJ. Oocyte-somatic cell–endocrine interactions in pigs. Dom Anim Endocrinol 2005;29:371–84. [48] Brankin V, Quinn RL, Webb R, Hunter MG. BMP-2 and -6 modulate porcine theca cell function alone and co-cultured with granulosa cells. Dom Anim Endocrinol 2005;29:593–604. [49] Beam SW, Butler WR. Effects of energy balance on follicular development and first ovulation in postpartum dairy cows. J Reprod Fertil 1999;Suppl. 4:411–24. [50] Butler WR. Nutritional interactions with reproductive performance in dairy cattle. Anim Reprod Sci 2000;60–61:449–57. [51] Gong JG, Lee WJ, Garnsworthy PC, Webb R. Effect of dietaryinduced increases in circulating insulin concentrations during the early postpartum period on reproductive function in dairy cows. Reproduction 2002;123:419–27. [52] Gutierrez CG, Oldham J, Bramley TA, Gong JG, Campbell BK, Webb R. The recruitment of ovarian follicles is enhanced by increased dietary intake in heifers. J Anim Sci 1997;75:1876–84. [53] Campbell BK, Scaramuzzi RJ, Webb R. Induction and maintenance of oestradiol and immunoreactive inhibin production with FSH by ovine granulosa cells cultured in serum free media. J Reprod Fertil 1996;106:7–16. [54] Armstrong DG, Gong JG, Webb R. Interactions between nutrition and ovarian activity in cattle: physiological, cellular and molecular mechanisms. Reprod Suppl 2003;61:403–14. [55] McCaffery FH, Leask R, Riley SC, Telfer EE. Culture of bovine pre-antral follicles in a serum-free system: markers for assessment of growth and development. Biol Reprod 2000;63:267–73. [56] McEvoy TG, Robinson JJ, Aitken RP, Findlay PA, Palmer RM, Robinson IS. Dietary-induced suppression of preovulatory progesterone concentrations in superovulated ewes impairs subsequent in vivo an in vitro development of ova. Anim Reprod Sci 1995;39:89–107. [57] O’Callaghan D, Boland MP. Nutritional effects on ovulation, embryo development and the establishment of pregnancy in ruminants. Anim Sci 1999;68:299–314.
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[58] O’Callaghan D, Yaakub H, Hyttel P, Spicer LJ, Boland MP. Effect of nutrition and superovulation on oocyte morphology, follicular fluid composition and systemic hormone concentrations in ewes. J Reprod Fertil 2000;118:303–13. [59] Staples CR, Thatcher WW. Effects of fatty acids on reproduction of dairy cows. In: Garnsworthy PC, Wiseman J, editors. Recent advances in animal nutrition. Nottingham University Press; 2005 . p. 229–56. [60] Thatcher WW, Bilby TR, Bartolome JA, Silvestre F, Staples CR, Santos JE. Strategies for improving fertility in the modern dairy cow. Theriogenology 2006;65:30–44. [61] Fouladi-Nashta AA, Gutierrez CG, Gong JG, Garnsworthy PC, Webb R. Impact of dietary fatty acids on oocyte quality and development in lactating dairy cows. Biol Reprod 2007; in press. [62] Ferguson EM, Ashworth CJ, Edwards SA, Hawkins N, Hepburn N, Hunter MG. Effect of feeding different nutritional regimes prior to ovulation on plasma concentrations of metabolic and reproductive hormones, ovulation rate and oocyte maturation in gilts. Reproduction 2003;126:61–71. [63] Ferguson EM, Slevin J, Edwards SA, Hunter MG, Ashworth CJ. Effect of alterations in the quantity and composition of the premating diet on embryo survival and fetal growth in the pig. Anim Reprod Sci 2006;96:89–103. [64] Ferguson EM, Slevin J, Hunter MG, Edwards SA, Ashworth CJ. Beneficial effects of a high fibre diet on oocyte maturity and embryo survival in gilts. Reproduction 2007;133:433–9. [65] Ferguson EM, Ashworth CJ, Hunter MG, Penny P, Slevin J, Edwards SA. The effect of feeding a high fibre diet from mid lactation until breeding on subsequent litter size of sows. In: Thompson JE, Gill BP, Varley MA, editors. The appliance of pig science. Nottingham University Press; 2004. p. 175–9. [66] Ashworth CJ, Ferguson EM, Edwards SA, Hunter MG. Nutritional Insights into the origins of embryonic loss in the pig. Proceedings of Havemeyer Foundation Workshop on Embryonic and Fetal Nutrition;. Havemeyer Foundation Series 2007; in Press.
Intra-ovarian regulation of follicular development and oocyte competence in farm animals R. Webb a,*, P.C. Garnsworthy a, B.K. Campbell b, M.G. Hunter a a
School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK b Department of Obstetrics and Gynaecology, School of Human Development, Queens Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK
Abstract In both mono-ovulatory species, such as cattle, and poly-ovulatory species, such as pigs, the interactions among extra-ovarian gonadotropins, metabolic hormones and intra-ovarian growth factors determine the continued development of follicles, the number of follicles that ovulate and the developmental competence of the ovulated oocyte. FSH and then subsequently LH are the main hormones regulating antral follicle growth in both mono- and poly-ovular species. However, a range of intra-ovarian growth factors, such as insulin-like growth factors (IGFs) and bone morphogenetic proteins (BMPs), are expressed throughout follicle and oocyte development and interact with gonadotropins to control follicle maturation. In addition, environmental factors such as nutrition, including both the amount and composition of the diet consumed prior to ovulation, can influence follicle development and the quality of the oocyte. Recent progress in our understanding has resulted in the development of diets that enhance oocyte quality and improve pregnancy rate in both pigs and cattle. In conclusion, despite some species-specific differences, similar interacting mechanisms control follicular development and influence oocyte quality. # 2007 Elsevier Inc. All rights reserved. Keywords: Ovary; Cattle; Pigs; Follicle; Growth factors; Gonadotropins
1. Introduction In both mono-ovulatory and poly-ovulatory species follicular growth is a continuum, controlled by the interaction between extra-ovarian factors, including gonadotropins and metabolic factors, and locally produced growth factors [1–3]. In addition, ovulation rate is a key determinant of reproductive efficiency and is tightly controlled in all species through mechanisms involving both extra-follicular factors and locally produced growth factors. This review will discuss the influence of intra-follicular factors, and how they interact
* Corresponding author. Tel.: +44 1159516056; fax: +44 1159516069. E-mail address: [email protected] (R. Webb). 0093-691X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2007.04.036
with extra-ovarian factors, on follicular growth, oocyte quality and embryo survival. Comparison between mono-ovulatory and poly-ovular species will be made to assist in identifying the key mechanisms involved. 2. Role of gonadotropins In both cattle and pigs, although gonadotropins do not appear to be involved in the initiation of follicular growth they do influence the early stages of follicular development [1,2,4]. Gonadotropins are definitely required for the final stages of follicular growth. In both species the emergence of follicular waves is preceded by a transient increase in FSH [1,5]. FSH then declines, due to ovarian negative feedback, below the threshold for further follicular selection and then, in
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both species, the dominant follicle(s) transfers dependency to LH [1,5]. Using experimental GnRH agonist models it was demonstrated in cattle that follicles grow to 8 mm in diameter when pulsatile LH release is inhibited and to only 4 mm when peripheral concentration of FSH is also reduced, as well as LH pulses being inhibited [6]. Likewise in pigs, when gonadotropins are reduced by GnRH agonist treatment, follicles do not grow beyond 4 mm in diameter [4]. Such results demonstrate the definitive requirement for gonadotropins in the final stages of follicular development in species with large differences in ovulation rate. Genetic differences in ovulation rate have been observed in different breeds of sheep, which demonstrate the importance of intra-ovarian mechanisms. For example in Booroola (FecB) sheep, carrying a single point mutation in the intracellular kinase domain of the BMPR-1B gene, follicles mature at a smaller diameter [7] and undergo precocious differentiation of granulosa cells with expression of LH receptors and increased expression of aromatase and inhibin bA genes [8,9]. Indeed Campbell et al. [10] demonstrated that the FecB mutation acts primarily within the ovaries of sheep resulting in increased ovulation rate compared with noncarriers, even when receiving similar patterns of gonadotropins. In addition to the FecB gene in sheep, genetic studies in Inverdale (FecXI) sheep have identified a point mutation in the BMP-15 gene [11], which affects follicle development and ovulation quota. Heterozygous ewes have an increased ovulation rate whilst homozygous ewes have small non-functional ovaries and are infertile [12]. More recently, BMP-15 point mutations have been identified in the Belclare (FecXB) and in the Cambridge (FecXC) and again, although the point at which the mutation occurs is breed dependent, they all exhibit the same X-linked phenotype [13]. 3. Bone morphogenetic proteins (BMP) and other oocyte secreted factors Recent research on oocyte secreted factors has focussed on murine systems; however we have extended these findings to the pig and shown that the porcine oocyte can modulate both granulosa and theca cell growth and function [14]. Oocyte secreted factors suppressed progesterone, but stimulated estradiol synthesis by granulosa cells throughout a 6-day culture period (Fig. 1). Furthermore, oocyte-derived suppression of progesterone was also observed in cultured theca cells and interestingly, both androstenedione and estradiol synthesis were influenced by oocyte derived factors [14].
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Fig. 1. Mean (S.E.D.) log10 (a) progesterone and (b) estradiol production by porcine granulosa cells after 144 h in serum free culture in the presence (&) and absence (&) of oocyte conditioned medium (OCM), 1 ng/ml pFSH and with either an optimal dose of Long R3 IGF-1 (agonist) (100 ng/ml) or no (0 ng/ml) Long R3 IGF-1 (agonist). (c) Number of viable granulosa cells after 144 h in serum free culture in the presence (&) and absence (&) of oocyte conditioned medium (OCM), 1 ng/ml pFSH and either an optimal dose of Long R3 IGF-1 (agonist) (100 ng/ml) or no (0 ng/ml) IGF-1. Values are from three independent cultures, each treatment having four replicates [modified from 14].
In addition, we have recently shown that the secretion of these factors is developmentally regulated, as oocytes at the germinal vesicle stage suppressed progesterone production, whereas oocytes that had matured to the metaphase II stage did not [15]. Furthermore, there were a number of differences in the secreted proteome of GVand MII oocytes, which is consistent with the co-culture
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findings [15]. Further work is required to identify more of these secreted proteins and their biological activity, although likely candidates include the BMPs and GDF-9. In summary, these findings support the proposal that oocytes secrete a factor(s) that modulates both cell proliferation and steroidogenesis and confirm that these factors are active inhibitors of luteinization. More specifically, we have demonstrated that BMPs decrease expression of 3b-hydroxysteroid dehydrogenase protein (3b-HSD, BMP-2;-6) and steroidogenic acute regulatory protein (StAR; BMP-6) [16] in cultured porcine granulosa cells. Cyclic adenosine monophosphate (cAMP) production was also suppressed significantly in both granulosa and theca cells. Furthermore the active phosphorylated downstream BMPR-regulated signaling molecule Smad-1 (p-Smad1) was upregulated in cells treated with BMP [17]. These findings provide evidence for the presence of a complex signaling mechanism in poly-ovulatory species as in mono-ovulatory species [18], and support the hypothesis that BMP-2 and BMP-6 act in a paracrine manner to control granulosa cell function, one aspect of which is to inhibit luteinization. BMPs exert their effects via BMP receptors (BMPRIA, -IB and -II) and in pigs immunohistochemistry for these receptors showed the presence of all three receptors in the fetal egg nests, oocytes and in the granulosa cell layer of follicles ranging from primordial to late antral stages [19]. Some immunostaining was also observed in the theca layer, corpus luteum and ovarian surface epithelium [19]. Actual protein expression of BMP-2 in pigs was identified by Western blotting in the oocyte, follicular fluid and to a lesser extent granulosa cells [16]. Similarly, GDF-9 has been shown to be present in the oocyte similar to BMP-15 where mRNAwas localized by in situ hybridization to the oocyte exclusively [20]. Similar to observations in the pig, BMP-6 and BMP receptors (BMPR) have been shown to be present in cattle fetal ovaries [21,22], with a similar pattern of mRNA expression in sheep [23]. These findings agree with those of Souza et al. [24] who demonstrated strong expression of BMPR in the oocyte and granulosa cells of ovine follicles from the primordial to preovulatory stages. As for pigs, the presence of both the ligand (BMP-6) and the receptors in cattle follicles, even at this early stage of development, illustrates the presence of all the components of a fully functional BMP system. Similarly in the adult ovaries of sheep [24] and cattle [25] there appears to be a fully functional BMP system and BMP2, 4, 6 and 7 have all been shown to exert effects on somatic cell function in vitro [24–27]. As discussed, recent studies by Hanrahan et al. [13] have also demonstrated a role for
BMP-15 in sheep. Also ovine granulosa cell progesterone production was inhibited while immunoreactive a inhibin levels increased when BMP15 and GDF9 were given together in culture [28]. However, the precise role of BMP-15 in bovine and porcine ovarian follicular development has not been elucidated; BMP-15 mRNA has recently been localized to small bovine and porcine preantral follicles [20], although temporal patterns of expression during follicle growth have still to be determined. In conclusion, both mono-and poly-ovular species appear to possess a fully functional BMP system, which is already present during fetal development. Members of the BMP system have been shown to be intricately linked with significant changes in ovulation rate [18,29]. Hence possible differences in BMP action and/or expression may explain the differences in ovulation rate between species, although this needs to be investigated further. 4. Insulin-like growth factors Another well-characterized local growth factor system is the insulin-like growth factors. In cattle, even by the preantral stage of development, follicles possess both IGFBP-2 and type 1 IGF receptors [30]. It appears that IGFs control preantral follicle growth primarily via endocrine mechanisms, with IGFBPs regulating the bioavailability of extra-ovarian IGF-I [18]. In support of this suggestion, IGF-I has also been shown to stimulate bovine preantral follicle growth in vitro [31]. It is not until the early antral stage of follicular development that IGFII expression in the thecal cells is first detected [32,33], when there appears to be a fully function IGF system. IGF-II has been shown to stimulate steroidogenesis of bovine thecal cells, acting via IGF type 1 receptors [34]. Thus IGF-II, like IGF-1, may have a significant role in thecal cell steroidogenesis during follicular development in mono-ovulatory species like cattle and sheep. As indicated, the actions of IGF-I and -II are regulated by locally produced IGF binding proteins [2,9]. In healthy bovine antral follicles up to 9 mm in diameter, the approximate size when granulosa cell LH receptors are first expressed, IGFBP-2 and -4 mRNA expression was restricted to granulosa and theca cells respectively [35]. IGFBP-2, and possibly IGFBP-4 and 5 concentrations, are higher in the follicular fluid of small and medium-sized bovine antral follicles, but are significantly reduced in follicular fluid of large and/or dominant bovine follicles [36]. Hence the conversion of a subordinate follicle to a future dominant follicle in cattle has been associated with a decrease in IGFBP-2 [18,35,37]. This reduction in follicular fluid IGFBP-2
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and -4 concentrations, coupled with an increase in estradiol concentrations, have been associated with the selection of the dominant follicle in cattle [35,38]. IGFs are also expressed within the porcine follicle; however, unlike cattle, IGF-I is expressed predominantly in the granulosa cells of follicles from 2 to 8 mm in diameter [39]. However, similar to cattle, IGF-II expression is higher in theca of 6 mm follicles and remains high until after the LH surge, indicating a role for IGF-II in ovulation and/or luteinization in the pig. In addition, IGF-I and the type I IGF receptor are required for all phases of preovulatory growth [40]. Similar to cattle, IGFBP-2 expression in porcine follicles is also inversely correlated with the diameter of follicles [40], suggesting that the potential for IGF action in large follicles is increased as the follicle grows. Similarly in the pig, increased follicular growth has been associated with greater circulating IGF-I [41]. Likewise, lower IGF-I concentrations are associated with reduced ovulation rates [41,42]. Hence changes in bio-active IGF-1 appear to be associated with changes in ovulation rate. These developmentally regulated changes in the patterns of expression of IGFs are associated with the action of gonadotropins. Utilizing a physiologically relevant culture system in both cattle and pigs [43,44], it has been demonstrated that FSH can induce estradiol production by granulosa cells and this induction is related to an increase in P450arom mRNA expression [43,45,46]. In both species, IGF-1, as well as insulin, interacts with FSH to stimulate granulosa cell estradiol production. In conclusion, despite species differences, in pigs, cattle and sheep, the development of follicles through to ovulation is controlled in part by the interaction between gonadotropins and IGFs. However in addition to IGFs a panoply of other locally produced factors, in addition to BMPs, appear to interact together to impact on follicle development and are involved in the control of ovulation rate. 5. Interaction between intra-follicular factors The effects of BMPs on both porcine [16,47] and ovine [27] granulosa cell function and their potential interactions with FSH and IGF-I have been investigated. In pigs BMPs can significantly suppress progesterone production in vitro. For example, there are significant interactions between both BMP-2 and -6 and IGF-I on progesterone production [16] and BMP-2, -6 and -15 also modified estradiol synthesis. Furthermore BMP-2 and -6 interacts with both IGF-I and FSH, whereas BMP-15 appears to interact with FSH only. In porcine
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theca cells, which express BMP receptors [19], all BMPs investigated (2, 6 and 15) stimulated cell proliferation in vitro [48], but in contrast both progesterone and estradiol synthesis were suppressed by BMP-2 and -6, but stimulated by BMP-15. Hence a range of BMPs can alter the pattern of steroid production and interactions have been observed between BMPs and both IGF-I and LH [48]. Interestingly, a significant modification of the response of both granulosa and theca cells to the BMPs occurs when cells are co-cultured with five oocytes/well compared to BMPs or oocytes alone, indicating a complex feedback loop involving BMPs, oocytes and somatic cells [48]. Collectively therefore, there is good evidence for a functional BMP system in the porcine ovary which interacts with other locally produced growth factors and gonadotropins and show that BMPs modulate somatic cell function and hence follicular development. Similarly for mono-ovulatory species, BMP-2 and -4 enhance FSH-stimulated estradiol production in sheep [24,27]. Furthermore, as for pigs, BMP-6 acts on bovine granulosa cells to enhance estradiol secretion whilst suppressing progesterone secretion [25]. There is also an interaction between BMPs and other local factors, showing that BMPs can enhance basal and IGF-induced secretion of estradiol, inhibin-A, activin-A and follistatin. More recently, Campbell et al. [27] confirmed BMP-6 protein expression in sheep and demonstrated a significant interaction between BMPs and IGFs [27] in stimulating granulosa cell differentiation. Further, it was demonstrated that the FecB mutation, which as discussed increases ovulation rate and litter size, results in an increased differentiative response of both granulosa and thecal cells to BMPs, IGFs and gonadotropins. These results demonstrate a major role for local growth factors such as BMPs and IGFs in both mono- and polyovulatory species in modulating proliferative and differentiative responses of theca and granulosa to gonadotropins (see Fig. 2). They may also be involved in controlling the number of follicles that are available for ovulation. 6. Impact of extra-ovarian factors on oocyte quality In addition to the paracrine interactions within the follicle of both mono- and poly-ovulatory species, oocyte quality is influenced by extra-follicular factors such as nutrition. In high yielding dairy cows the decline in fertility has been associated with negative energy balance postpartum and associated changes in metabolic hormones including reduced IGF-I and
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Fig. 2. Diagram showing the influence of nutrition, the role of gonadotropins and the interaction with the intra-follicular IGF and BMP systems on antral follicle development in both mono- and poly-ovulatory farm animal species. The top section describes a follicular wave and when the dominant follicle(s) transfers its dependence from FSH to LH. The bottom two sections illustrate some of the key members of two local growth factor systems (IGFs and BMPs) shown to be important in follicular development. It also highlights the additive effect of the IGF and BMP systems on FSH and LH stimulated follicular development [Adapted from 1,18,27].
insulin concentrations [2,49,50]. Feeding diets that increase insulin concentrations can advance the first ovulation postpartum [51] and stimulate follicle development in heifers [52]. Also in larger follicles in ruminants, IGF-I and insulin have been found to stimulate granulosa cell proliferation and mitogenesis and enhance FSH induced steroidogenesis by granulosa cells [44,53]. However, nutritionally-induced changes in circulating and local concentrations of IGF-I that are optimal for follicular growth may not be necessarily optimal for bovine oocyte maturation [54] and may even have a negative effect on oocyte growth [55]. Endocrine and metabolic signals that regulate follicular growth may also influence oocyte development either through changes in hormone/growth factor concentrations in follicular fluid or via granulosa– oocyte interaction. For example, short-term changes in dietary energy intake influence both oocyte morphology and developmental potential [56–58] and supplementation of rations with fats, can result in an increase in
energy intake and energy status of the cow [59,60]. We and others have also demonstrated that fatty acids may influence oocyte developmental potential in high yielding diary cows [60,61]. More recently we have utilized this information to demonstrate that pregnancy rate in high yielding dairy cows can be significantly improved by feeding diets that can influence follicle development and oocyte quality (Garnsworthy and Webb, unpublished observations). Similarly in pigs we have demonstrated that feeding a high plane of nutrition to gilts can improve oocyte quality [47,62]. Increased feed intake was not only associated with an increase in the proportion of oocytes at metaphase II, but also with increased IGF-I, leptin and LH concentrations. Furthermore the composition of the diet was shown to alter oocyte maturation and prenatal survival. Studies involving alterations in the protein, starch and/or fiber content of the pre-mating diet showed that dietary fiber can improve embryo survival [63,64]. There was 18% higher embryo survival
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on Days 27–29 of pregnancy in gilts [63] and nearly one extra piglet per litter when tested in multiparous sows in a commercial environment [65]. In addition, higher blastocyst yields and blastocyst cell numbers on Days 6–7 following in vitro fertilization have been achieved when gilts are fed a high fiber diet [66]. These results provide some of the first evidence of a direct link between oocyte maturity and embryo survival in the pig. They also demonstrate that diets that increase embryo survival are also associated with improved oocyte maturity and quality. Collectively, these findings indicate in both monoand poly-ovulatory species that nutritional regimens that increase embryo survival are also associated with beneficial effects on oocyte maturity and quality, supporting the idea that embryo viability originates during oocyte development. In summary, in both cattle and pigs changes in extra-ovarian factors such as metabolic hormones are associated with changes in follicular growth patterns, oocyte quality and embryo survival. Hence producing a good quality oocyte is essential for embryo survival, the maintenance of litter size in pigs and pregnancy in cattle and sheep. 7. Conclusions In both cattle and pigs many of the mechanisms involved with the development of the follicle involve the interaction of a panoply of intra-follicular growth factors (see Fig. 2). Indeed many of these mechanisms are similar in cattle and pigs, although with some species-specific differences. In addition, extra-ovarian follicular factors interact with these local factors to determine whether follicles continue to develop and the quality of the ovulated oocyte. Recent metabolomic studies, where a range of peripheral metabolites and metabolic hormones were measured, have demonstrated that diet can also influence oocyte maturity and quality, supporting the concept that embryo viability originates during oocyte development. These interactions are of key importance since they influence the developmental potential of the embryo and subsequent maintenance of pregnancy. Recent progress in understanding this multifactorial process has highlighted new opportunities for improving pregnancy rate in both mono- and multi-ovulatory farm animal species using nutritional approaches. Acknowledgements Much of the authors, own cited work was kindly supported by the BBSRC, Defra and SEERAD.
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