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Theriogenology 68S (2007) S22–S29

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, UKb 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: bob.webb@nottingham.ac.uk (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

R. Webb et al. / Theriogenology 68S (2007) S22–S29 S23

Fig. 1. Mean (�S.E.D.) log10 (a) progesterone and (b) estradiol pro-

duction by porcinegranulosa 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].

both species, the dominant follicle(s) transfers depen-

dency 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 non-

carriers, 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 homo-

zygous 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 synth-

esis 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].

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, therewere a

number of differences in the secreted proteome of GVand

MII oocytes, which is consistent with the co-culture

R. Webb et al. / Theriogenology 68S (2007) S22–S29S24

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 dehydrogen-

ase 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 sup-

pressed significantly in both granulosa and theca cells.

Furthermore the active phosphorylated downstream

BMPR-regulated signaling molecule Smad-1 (p-Smad-

1) was upregulated in cells treated with BMP [17].

These findings provide evidence for the presence of a

complex signaling mechanism in poly-ovulatory spe-

cies 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 (BMPR-

IA, -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 compo-

nents 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 invitro [31]. It is not until

the early antral stage of follicular development that IGF-

II 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

R. Webb et al. / Theriogenology 68S (2007) S22–S29 S25

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

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]. Interest-

ingly, 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 follista-

tin. 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 poly-

ovulatory 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

R. Webb et al. / Theriogenology 68S (2007) S22–S29S26

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 develop-

ment 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 supplementa-

tion 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

R. Webb et al. / Theriogenology 68S (2007) S22–S29 S27

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 mono-

and 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 opportu-

nities 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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