Scaramuzzi 2011 PDF

Review CSIRO PUBLISHING
Reproduction, Fertility and Development, 2011, 23, 444–467 www.publish.csiro.au/journals/rfd
Regulation of folliculogenesis and the determination

of ovulation rate in ruminants
R. J. ScaramuzziA,B,M, D. T. BairdC, B. K. CampbellD, M.-A. DriancourtE,

J. DupontA, J. E. FortuneF, R. B. GilchristG, G. B. MartinH, K. P. McNatty I,
A. S. McNeillyJ, P. MongetA, D. Monniaux A, C. ViñolesH,K and R. WebbL
A
INRA, UMR85 Physiologie de la Reproduction et des Comportements, Centre INRA de Tours,
37380 Nouzilly, France.
B
Department of Veterinary Basic Sciences, The Royal Veterinary College, Hawkshead Lane,
North Mimms, Hertfordshire AL9 7TA, UK.
C
University of Edinburgh, Centre for Reproductive Biology, The Queen’s Medical
Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, UK.
D
University of Nottingham, Division of Obstetrics and Gynaecology, The Queen’s
Medical Centre, Nottingham NG7 2UH, UK.
E
Intervet Schering Plough Animal Health, Intervet Pharma R & D, BP 67131,
49071 Beaucouzé, France.
F
College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.
G
Robinson Institute, School of Paediatrics and Reproductive Health, Medical School South,
University of Adelaide, SA 5005, Australia.
H
Animal Production Systems, UWA Institute of Agriculture, The University of Western Australia,
Crawley, WA 6009, Australia.
I
School of Biological Sciences, Victoria University of Wellington, Wellington 6140, New Zealand.
J
Medical Research Council Human Reproductive Sciences Unit, The University of Edinburgh
Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, UK.
K
INIA Tacuarembó, Ruta 5, km 386, Tacuarembó, Uruguay.
L
University of Nottingham, Division of Animal Science, School of Biosciences,
Loughborough, Leicestershire LE12 5RD, UK.
M
Corresponding author. Email: rex.scaramuzzi@orange.fr
Abstract. The paper presents an update of our 1993 model of ovarian follicular development in ruminants, based on
knowledge gained from the past 15 years of research. The model addresses the sequence of events from follicular
formation in fetal life, through the successive waves of follicular growth and atresia, culminating with the emergence
of ovulatory follicles during reproductive cycles. The original concept of five developmental classes of follicles,
defined primarily by their responses to gonadotrophins, is retained: primordial, committed, gonadotrophin-responsive,
gonadotrophin-dependent and ovulatory follicles. The updated model has more extensive integration of the morpholo-
gical, molecular and cellular events during folliculogenesis with systemic events in the whole animal. It also incorporates
knowledge on factors that influence oocyte quality and the critical roles of the oocyte in regulating follicular develop-
ment and ovulation rate. The original hypothetical mechanisms determining ovulation rate are retained but with some
refinements; the enhanced viability of gonadotrophin-dependent follicles and increases in the number of gonadotrophin-
responsive follicles by increases in the throughput of follicles to this stage of growth. Finally, we reexamine how these two
mechanisms, which are thought not to be mutually exclusive, appear to account for most of the known genetic and
environmental effects on ovulation rate.
Additional keywords: ewe, follicle, nutrition, oocyte, ovary.
Ó CSIRO 2011 10.1071/RD09161 1031-3613/11/030444

Regulation of folliculogenesis in ruminants Reproduction, Fertility and Development 445
Dedication stress. Cattle have also been useful because the processes
We dedicate this review to the memory of a colleague and friend, leading to ovulation in the cow are more tightly controlled, so
Dr Hannah Peters (b 20/03/1911: d 04/03/2009), who made many multiple ovulations are rarer than in the ewe.
significant and insightful contributions to our understanding of Progress in this field has been aided by a steady escalation
ovarian function. Our review addresses several basic events of of our knowledge of reproductive physiology leading to con-
ovarian function in ruminants including follicular formation and siderable depth in our understanding of the processes that
growth and the roles of intra-ovarian factors and gonadotrophins control folliculogenesis. To summarise our knowledge of these
in these processes. These were topics investigated by Hannah processes and to stimulate international collaborative research,
Peters in the 1960s and 1970s. In 1975, at an ovarian workshop in a workshop was held in 1991 when eleven researchers from
Glasgow, she presented a memorable and insightful summary laboratories around the world met at Terrigal, Australia. Parti-
of her studies of ovarian biology in rodents and humans (Peters cular attention was directed towards the mechanisms in sheep
et al. 1975). The views expressed in this summary have stood the that produce different ovulation rates among breeds and among
test of time and represent a remarkably accurate understanding of environmental or experimental conditions. The outcome was a
our current concepts of follicular physiology. In brief, Peters and review paper representing a consensus of the group (Scaramuzzi
her colleagues concluded that: (1) the initiation of follicular et al. 1993). It outlined models of follicular development and
growth is continuous during infancy and adult life, irrespective selection and of the determination of ovulation rate based on
of reproductive status and whether mammals ovulate or not; knowledge of the physiological, rather the than anatomical,
(2) follicles grow sequentially and continue to grow without rest changes during these processes. These models were therefore
until they die or ovulate; (3) the number of follicles that start to functional rather than descriptive and since then, they have
grow is dependent upon the size of the pool of small follicles (e.g. been used to develop and test hypotheses on the regulation of
the larger the pool, the more follicles leave the pool per unit time) folliculogenesis and ovulation rate.
and inferred that intraovarian mechanisms via factors from The present paper is the result of a follow-up workshop
growing follicles were likely to be involved; (4) the initiation of held in July 2008 at Tours, France, that aimed for an updated
follicular growth is independent of gonadotrophins and involves consensus of the state of our knowledge. In the intervening
intraovarian factors; (5) the continued growth of medium and 17 years, five of the original participants had left the field, but
large follicles is dependent upon gonadotrophins and the larger the remaining six agreed to hold an update-workshop. The scope
that follicles become, the more important gonadotrophins was extended by inviting additional researchers to cover new
become; and (6) exogenous gonadotrophins reduce the incidence developments, for example, the role of the oocyte in folliculo-
of atresia in large follicles. In addition, Hannah Peters provided genesis. Some of the scientific changes have been profound,
the first classification system for the sequential stages of folli- either because of major contributions from techniques in mole-
cular growth and these have been utilised for our classifications cular biology or from dedicated effort in previously neglected
in ruminants. Her published works include studies on duration of areas. In particular, we have new perspectives on the develop-
amenorrhea and age of menarche in Indian women, ovarian ment of ovarian follicles in the fetus, the role of the oocyte in the
development during fetal life, radiation sensitivity and DNA process of follicular development leading to ovulation and on
synthesis in oocytes, the kinetics of follicular growth and the the ways in which metabolic factors can directly affect follicular
effects of breast cancer, Down’s syndrome and leukaemia on function.
ovarian function. In her retirement, she continued to mentor her The scope of the workshop, and thus this review, was limited
colleagues and remained interested in whether we had learnt to the events and processes in ovarian folliculogenesis from the
anything new about how follicles start to grow or how the formation of the follicle in the fetus to the final selection of the
environment (e.g. life-style, nutrition) affects reproductive ovulatory follicle(s). We have omitted the processes of ovula-
function. She often expressed concern about the ethics of modern tion and luteinisation, not because we believe they are unim-
reproductive technologies and was puzzled as to why many of us portant, but because of the necessity to limit the scope of the
have avoided researching the mechanisms as to how the ovary workshop. Again, there has been a strong focus on mechanisms
constantly remodels itself while disposing of degenerating fol- at various levels of analysis from molecular and cellular to the
licles and luteal tissue. She remained fascinated by the dynamics whole organism. This paper reviews major conceptual advances
of ovarian function and perhaps this can be summarised by a since 1991 and re-addresses the functional model of folliculo-
favourite quotation of hers from Plato ‘nothing ever is, but is genesis incorporating new information and exploring how this
always becoming’. affects our interpretation of follicular responses to nutrition. The
model for the control of ovulation rate is updated and, finally,
primary foci for future research are considered.
Background
Most mammals precisely regulate their ovulation rate. In
Update on the mechanisms that control folliculogenesis
ruminants, this number seldom exceeds three and is an important
determinant of litter size. In studies of the mechanisms Primordial and committed follicles
responsible for follicular development and differentiation, Our original view (Scaramuzzi et al. 1993) was that primordial
sheep have proven to be an excellent experimental model follicles were quiescent but we now know this to be incorrect
because their ovulation rate is affected by genetic differences because of significant new information from studies in sheep
and by environmental factors such as photoperiod, diet and and, to a lesser extent, in cattle, concerning the ontogeny of
446 Reproduction, Fertility and Development R. J. Scaramuzzi et al.
follicular formation, including the origins of the granulosa cells Table 1. The temporal patterns of expression and location of selected
in primordial follicles (Sawyer et al. 2002). Moreover, as we genes expressed during the primordial, primary and preantral stages of
shall see below, many of the genes expressed in follicles during folliculogenesis
their formation and the early stages of growth have now been All genes were localised by in situ hybridisation (Juengel et al. 2007;
identified. The process leading to the formation of follicles takes McNatty et al. 2007). For definitions of follicular Types 1–4 see Lundy et al.
(1999). O, oocyte; G, granulosa cells; T, theca cells
place during fetal life. In sheep, it begins shortly after sexual
differentiation, ,35 days after conception. Initially, in the cor-
Primordial Primary Preantral Preantral
tical region of the ovary, mitotically active oogonia containing c-
(Type 1) (Type 2) (Type 3) (Type 4)
kit interact with adjacent mesonephric cells that contain the kit
ligand to form the ovigerous cords that subsequently isolate the ALK3O,G ActRIIBG ActRIIBT ARG,T
oogonia from the interstitium, but not the cells of the surface ALK5O ALK6G ALK3T 3bHSDT
epithelium, which also contain kit ligand. Once the ovigerous ALK6O AMHG ALK5G,T ERaG
cords are formed, mitotically active oogonia can only recruit BetaglycanO AMHRIIG ALK6T? ERbT
somatic pre-granulosa cells from the surface epithelium. In 3bHSDG bB activinG BetaglycanG,T IGF2T
BMP6O BMP15O BMPRIIT a InhibinG
both sheep and cattle, the oogonia form tight junctions with
BMPRIIO,G Connexin 43G FollistatinG LHRT
the mesonephric or surface epithelial cells that subsequently c-kitO FIGaO FSRPG,T PRT
become pre-granulosa cells (Sawyer et al. 2002). The oogonia Connexin 37O FSH-RG IGFR1T SF1T
then attach to pre-granulosa cells and enter meiosis, during ERbO,G IGFRIG TGF-b1, 2T StART
which at least 80% of the germ cells, but not their pre-granulosa GDF9O TGF-bR11T 17aOH lyaseT
cells, undergo apoptosis. In sheep, the evidence suggests that the Kit ligandG
pre-granulosa cells then associate with nearby surviving oocytes StARG
and thus each oocyte recruits different numbers of pre-granulosa WT1G
cells from different sources. After Day 75 of fetal life in sheep,
and Day 90 in cattle, the first follicles separate themselves by
producing a basement membrane at the base of the ovigerous of the proteins in primordial follicles are associated with cell
cords (Sawyer et al. 2002; Garverick et al. 2010). In sheep, it maintenance and preparation for growth (Table 1). In primordial
can be calculated that 90% or more of the granulosa cells are follicles of rodents, the kit-ligand–c-kit system and components
recruited from the surface epithelium (Sawyer et al. 2002) and of the phosphoinositol (PI3) signalling system are present as well
that primordial follicles contain a mean of 16 (range 3–52) as phosphatase and tensin homologue (PTEN), which acts as a
granulosa cells (Lundy et al. 1999). In sheep and cattle, follicular negative regulator of PI3 signalling (Reddy et al. 2008). The kit-
growth is initiated before the last primordial follicles are formed ligand–c-kit system is present in sheep primordial follicles
and then continues throughout fetal, neonatal and adult life. (Tisdall et al. 1999); however, unlike in rodents it seems that
The delay between the appearance of the first primordial and PTEN is not present (Froment et al. 2005). Nevertheless, given
the first primary follicles is relatively long at 25 days in sheep that primordial and committed follicles have numerous recep-
and 50 days in cattle (McNatty et al. 2007). In cattle, this delay tors and ligands for growth factors of the transforming growth
is associated with progression through to meiotic prophase I factor (TGF)-b super family and oestradiol (Table 1), it is likely
and arrest at diplotene. In vitro studies suggest that the capacity that several growth-promoting factors are involved, both nega-
of the first-formed bovine follicles to initiate growth is inhibited tively and positively, in the initiation of follicular growth.
reversibly by oestradiol and is associated with inhibition of the In vitro studies with cortical fractions of bovine and human
progression to meiotic prophase (Yang and Fortune 2008). It ovaries show that the majority of primordial follicles are
remains to be established if this is also the case in sheep. activated within a few days of isolation from the ovary,
Ovine primordial follicles have been used to generate ,2500 suggesting that inhibitory factors may be important for con-
expressed sequence tags and these have been matched to 500 trolled exit from the pool of primordial follicles and there is
mRNAs linked to cytoskeletal events, DNA repair, mRNA evidence that anti-Müllerian hormone (AMH) inhibits follicle
processing, ribosomal function, protein synthesis and ubiquiti- activation and early follicular growth in bovine ovaries (Fortune
nation, and signalling pathways (McNatty et al. 2007). Exam- 2003). Thus, although we have learnt much about the character-
ples of genes expressed in oocytes, granulosa cells and theca istics of primordial follicles, the fact remains that, as reported
cells of primordial, primary and preantral follicles are listed in by Scaramuzzi et al. (1993), the processes that control the
Table 1. Collectively, this evidence indicates that the primordial departure of primordial follicles from their pool remain to be
follicle is expressing hundreds of genes that fulfil housekeeping determined.
and signalling functions. Moreover, given that the oocyte has Once follicles have left the pool of primordial follicles they
receptors for oestradiol and several growth factors, it is either are ‘committed’ to gonadotrophin-independent growth. As a
receptive or developing receptivity, to its environment. follicle grows to the primary stage, the granulosa cells increase
In ruminants, all current evidence is consistent with follicular in number and change shape, becoming uniformly cuboidal. In
formation being completed during fetal life (Garverick et al. the ewe, the number of granulosa cells increases to a mean of 128
2010) and with a decline in the number of remaining follicles (range 30–520; Fig. 1). The oocyte also enlarges, with 3- to 10-
throughout life. In sheep, counts of primordial follicles suggest fold increases in the volume of smooth endoplasmic reticulum,
that 25–30 begin growing each day (McNatty et al. 2007). Many mitochondria, ribosomes and lipid droplets, and the zona
pellucida, absent in primordial follicles, is deposited (Lundy granulosa cells, a zona pellucida and a theca interna that
et al. 1999). Analysis of expressed sequence tags from primary contains luteinising hormone receptor (LH-R) (Table 1). The
follicles shows that several hundred genes not found in primor- acquisition of the enzymes required for thecal androgen produc-
dial follicles are activated during this stage of growth, including tion is essentially complete before antrum formation (McNatty
some involved in mitochondrial function, cell signalling and et al. 2007). The collective evidence suggests that the rate of
communication, and the synthesis of the zona pellucida follicular atresia (i.e. apoptosis) is very low during the preantral
(Table 1). stages of growth. In sheep, a large preantral follicle contains
1000–3500 granulosa cells and the oocyte is 70–120 mm in
Formation of large preantral follicles diameter (Fig. 1). Thus, the granulosa cells undergo about seven
As ovine and bovine follicles develop to form large preantral to eight doublings in population before the formation of an
follicles (Lundy et al. 1999), they gain multiple layers of antrum and the emergence of the distinct phenotypes of the
Growth
150
Oocyte diameter (µm)
100
50
0
100
Granulosa cells (⫻104)
10
1
0.1
0.01
0
10
Follicular diameter (mm)
1.0
0.1 Emergence of follicular waves

and granulosa cell LH receptors
0
Increasing steroidogenic competence
Antrum formation and differentiation of cumulus cell phenotype
Differentiation of theca interna with LH receptors

Morphology and functions
Zona pellucida present
Granulosa cell FSH receptors and oocyte-granulosa cell gap-junctions present
Primordial Primary Preantral Small-medium antral Medium-large antral

Type 1 1a 2 3 4 5 5⫹
Preovulatory
growth to
ovulation
Non-growing Committed Gonadotrophin- Gonadotrophin-

pool responsive dependent
Indefinite ⱕ130 days ⱕ12 days ⱕ6 days
Fig. 1. A summary of folliculogenesis in the ewe, developed with data from several sources (Lundy et al. 1999; K. P. McNatty, unpubl. data). The upper panel
shows the mean (central line) and range (shaded band) time lines of growth of the follicle and oocyte and the number of granulosa cells, from primordial to
ovulatory stages. The lower panel shows the progressive emergence of several critical functional and morphological characteristics of follicles as they develop.
The stages of development have been defined by two different systems – one based on morphology and the other on functional characteristics of follicles.
cumulus and mural cells (McNatty et al. 2007). Very similar phase of oocyte development has been termed ‘oocyte
changes in the number of granulosa cells and in the diameter of capacitation’.
the oocyte take place in cattle. In both species, the transition to At about the time of antrum formation, granulosa cells
an antral follicle is accompanied by a significant increase in the differentiate into two anatomically and functionally distinct
rate of atresia, suggesting that it is physiologically challenging lineages; the mural granulosa cells that line the wall of the
to remodel the avascular granulosa to form an antrum and to follicle and that have principally, a steroidogenic role and the
maintain the granulosa–oocyte syncytium. cumulus cells, that form an intimate life-support association
with the oocyte. As we shall see below, this process of differ-
Follicle–oocyte interactions entiation of granulosa cells is driven by the oocyte and the
It has long been known that oogenesis and folliculogenesis are outcome is a follicular microenvironment that is critical to the
inextricably linked with the oocyte growing and developing final acquisition of developmental competence by the oocyte.
in an intimate and mutually dependent relationship with the However, the oocyte–cumulus cell communication system is
somatic cells of the follicle (Thibault 1977). During the course both complex and dynamic and we have a relatively poor
of folliculogenesis, oocytes first acquire meiotic competence understanding of the nature and the diversity of molecules that
and then gradually acquire developmental competence – a transfer from the cumulus cells to the oocyte during follicular
biochemical and molecular state that allows a mature oocyte to growth or of how they contribute to developmental competence
undergo normal fertilisation, support normal preimplantation (Gilchrist and Thompson 2007). Unravelling the intricate rela-
embryo development and subsequent healthy growth of the tionships between the follicle and its oocyte will likely generate
implanted embryo to term (Hyttel et al. 1997). Support for the new insights into fundamental mechanisms regulating follicu-
acquisition of meiotic competence is the most important func- logenesis and oocyte development.
tion of the follicle, but we are still striving to understanding the
molecular processes involved (Sirard et al. 2003). The oocyte as a regulator of follicular development
The traditional perspective on the relationship between One of the most exciting new concepts to emerge since our
oocyte and follicle has been that the oocyte is passive and that 1993 review is the concept that the oocyte has an essential, very
its growth and development are dictated by the endocrine system active role in folliculogenesis. The concept was confirmed when
and follicular somatic cells. However, we now know that the Matzuk and colleagues identified candidate factors using a
oocyte is not simply a passenger in the process of folliculogen- knockout mouse model (Dong et al. 1996). It is now clear that
esis. In sheep, for example, it produces at least two growth the oocyte secretes growth factors that play a central role in the
factors that regulate follicular development from its earliest regulation of folliculogenesis, granulosa and cumulus cell
stages (McNatty et al. 2006a). Their role in determining ovula- differentiation, ovulation rate and litter size (Gilchrist et al.
tion rate was unexpected because an equivalent effect 2004; McNatty et al. 2004). The functional consequences of the
was not evident from studies in mice, perhaps because rodents processes driven by oocyte-secreted factors have been revealed
already have a high ovulation rate, in contrast to the typically through two lines of experimentation: (1) bioassays of granulosa
low ovulation rate of sheep. In this section, we review or cumulus cells treated with oocyte-secreted factors and
both aspects of the active bi-directional relationship between recombination-derived candidate molecules generated from
oocyte and follicle, and develop perspectives on how these transfected cell-lines; and (2) animals deficient in candidate
interactions can affect the outcomes of folliculogenesis, includ- molecules through natural genetic mutation or immunisation.
ing ovulation rate. The outcome is a powerful new perspective on the differentia-
tion and functional control of granulosa cells in committed
The follicle as a regulator of oocyte development follicles during their transition to the gonadotrophin-responsive
and gonadotrophin-dependent stages.
In primordial and committed follicles (i.e. gonadotrophin-
independent), the oocyte is meiotically and developmentally
incompetent. During the gonadotrophin-responsive phase, Oocyte regulation of granulosa cell differentiation
oocytes actively synthesise RNA as evidenced by dispersed Oocyte-secreted factors regulate several important functions
chromatin configurations and transcriptionally active nucleoli of granulosa and cumulus cells; including regulation of cellular
(de Smedt et al. 1994). The initiation of these processes in the growth, enhancement of cell survival, modulation of steroido-
oocyte probably involves local paracrine mechanisms. Towards genesis, regulation of the expansion of cumulus cells and the
the end of the gonadotrophin-independent period, and after metabolism of cumulus cells (Eppig 2001; Gilchrist et al. 2004).
formation of the follicular antrum, oocytes from livestock Interestingly, the oocyte has the greatest capacity to regulate
species complete their growth phase and acquire the capacity many of these functions during the gonadotrophin-dependent
to resume, but not complete, meiosis. During the early stages phase of folliculogenesis, after the oocyte has completed its
of antral follicle growth, the oocyte acquires the capacity to growth phase (Gilchrist et al. 2008). During pre-ovulatory
complete meiosis. Oocytes from mid-sized antral follicles maturation, FSH and LH drive the differentiation of mural
possess low developmental competence but as follicles progress granulosa cells towards a luteinised phenotype. However, in
towards pre-ovulatory size, the oocyte acquires the full comple- cumulus cells, this process is actively inhibited by the oocyte
ment of cytoplasmic machinery necessary to support complete (Eppig et al. 1997), which secretes paracrine factors that
embryo and fetal development (Sirard et al. 2006). This latter establish concentration gradients that effectively become
morphogenic gradients (Hussein et al. 2005). Hence, through infertile women. In 2001, three laboratories independently
the actions of oocyte-secreted factors, the oocyte actively directs identified a point mutation (FecBB) in the ALK6 gene in ewes
the lineage of its neighbouring granulosa cells towards the carrying the Booroola mutation (Mulsant et al. 2001; Souza
cumulus cell phenotype (Eppig et al. 1997; Li et al. 2000). et al. 2001; Wilson et al. 2001). This point mutation has now
We suggest that the oocyte, with its unique nutritional and been identified in many breeds including the Merino, Javanese
hormonal requirements for its development, must regulate its Thin-Tail, Garole, Hu and Han breeds. Animals heterozygous or
microenvironment to keep it distinct from the rest of the follicle homozygous for this mutation have ovulation rates that are 1–4
(Eppig et al. 1997; Hussein et al. 2005). How does the oocyte and 4–9 times higher, respectively, than wild-type type ewes of
do this? We believe that the oocyte maintains a population of the same breed. Since 1991, studies of inherited patterns of
cumulus cells that it then directs to regulate the microenviron- ovulation rate in sheep have revealed at least six other point
ment of the oocyte, a function for which it has a poor capacity mutations in either the pro- or mature regions of the BMP15
(Sutton et al. 2003a, 2003b; Sugiura et al. 2005, 2007). The gene and at least two point mutations in the mature region of the
ability of the oocyte to control cumulus cell development and to GDF9 gene. Animals homozygous for mutations to BMP15
maintain a regulatory loop with them is likely to be a critical have streak ovaries and do not ovulate whereas those that are
developmental function for oocytes in general (Gilchrist et al. heterozygous, surprisingly, have higher ovulation rates than
2008). This is supported by the observation that adding exo- wild-type ewes (McNatty et al. 2006a; Bodin et al. 2007;
genous oocyte-secreted factors to oocytes maturing in vitro Martinez-Royo et al. 2008; Monteagudo et al. 2009). For one
improves the pre- and post-implantation development of their of the GDF9 point mutations, (FecGH), the homozygous and
subsequent embryos (Hussein et al. 2006; Yeo et al. 2008). heterozygous phenotypes are similar to those reported for the
Details of the molecular nature of the paracrine communica- BMP15 mutations (Hanrahan et al. 2004). Of particular interest
tion among oocytes, granulosa cells and cumulus cells are still is the finding that animals heterozygous for FecGH and FecXB
emerging as specific oocyte-secreted factors are identified and (another mutation of BMP15) have additive effects on ovulation
characterised and their signalling pathways in granulosa cells rate, suggesting that GDF9 and BMP15 cooperate with respect
and cumulus cells are elucidated. At this time it is already clear to regulating ovulation rate (Hanrahan et al. 2004); Research is
that two growth factors expressed primarily in oocytes have continuing on several other breeds of sheep that have genetic
important functions: growth differentiation factor 9 (GDF9) and mutations affecting ovulation rate (e.g. Lacaune, FecL, Thoka,
bone morphogenetic protein 15 (BMP15) otherwise known as and Woodlands FecX2W). The FecL, Thoka and Woodlands
GDF9b (Dong et al. 1996; Galloway et al. 2000). They are both FecX2W mutations have been mapped to sheep Chromosomes
closely related members of the TGF-b superfamily and they are 11, 5 and X respectively and work is in progress to identify these
produced as pre-pro-proteins consisting of a signal peptide, a genes. The ovarian phenotype in Thoka ewes is similar to that of
large pro-region and a mature region (Shimasaki et al. 2004). GDF9 mutants so it has been suggested that the Thoka mutation
Most unusually for this superfamily, both factors lack the is in the GDF9 gene (Nicol et al. 2009). Most, if not all, of these
otherwise conserved fourth cysteine residue that is required phenotypes can be reproduced by immunisation of ewes with
for the formation of disulfide bridges between the subunits, BMP15 or GDF9 peptide constructs. Animals with high anti-
so their dimers are non-covalent. They signal to granulosa body titres leading to total immuno-neutralisation of BMP15
and cumulus cells through the TGF-b superfamily receptors or GDF9 activity have inhibited follicular growth from the
(Juengel and McNatty 2005). Granulosa and cumulus cells primary and primordial stages. In contrast, partial immuno-
express a large complement of cell-specific receptors, co- neutralisation against either GDF9 or BMP15, or both, results in
receptors and SMADs of the TGF-b superfamily. Bone mor- higher than normal ovulation rates and increased numbers of
phogenetic protein receptor II (BMP-RII) is a critical receptor lambs (McNatty et al. 2006a). Immunisation of cattle against
for GDF9 and BMP15. When GDF9 binds to it, activin-like GDF9 or BMP15, or both, has also been shown to affect
kinase 5 (ALK5) is activated and phosphorylates SMAD2/3. ovulation rate (Hudson et al. 2007).
Thereafter, SMAD2/3 associates with the common SMAD4 and In the ovary of the ewe and cow, GDF9 is produced
this complex translocates to the nucleus where it interacts with exclusively by the oocyte (Bodensteiner et al. 1999). Moreover,
specific DNA motifs and transcriptional regulators leading GDF9 and its mRNA have been identified in oocytes at the time
to the transcription of target genes. When BMP15 binds to of follicular formation and throughout all stages of follicular
BMP-RII, it causes recruitment and activation of ALK6, leading development (McNatty et al. 2006a). In contrast, BMP15 and its
to signalling through the alternative BMP pathway mediated by mRNA were first identified in sheep, at the primary stage of
SMADs 1, 5 and 8 (Juengel and McNatty 2005). follicular growth and at all stages thereafter. Both BMP15 and
GDF9 are thought to be important during pre-ovulatory matura-
Oocyte-derived factors that affect folliculogenesis tion because passive immunisation of ewes during the follicular
Sheep are proving to be an excellent model for investigating phase of the oestrous cycle inhibited either ovulation or the
the roles of the TGF-b superfamily in the control of ovula- normal function of the corpus luteum (Juengel et al. 2002).
tion rate and infertility in mono-ovular species. In sheep, Evidence from in vitro studies suggests that BMP15 and GDF9
over 12 different, naturally-occurring, point mutations in the cooperate as a complex to influence proliferation and steroido-
BMP15, GDF9 and ALK6 genes that affect ovulation rate have genesis in granulosa cells (McNatty et al. 2005). Given that both
been discovered. In humans, several point mutations have been BMP15 and GDF9 are present in follicular fluid and that
identified in the pro- or mature regions of the BMP15 gene of immunisation interferes with their actions, the current view is
that these oocyte-secreted factors act in a concentration- discussed above, the follicle contains several other paracrine
dependent paracrine manner on adjacent cumulus and granulosa and autocrine systems and, of these, the IGF system appears
cells. Moreover, reduced concentrations of BMP15 and GDF9 to be particularly significant for folliculogenesis. It has been
alter only slightly the responsiveness of granulosa cells to either defined in detail and its role in folliculogenesis has been firmly
FSH or LH while the absence of BMP15 or GDF9 prevents normal established in several species, including some ruminant species
follicular growth beyond the primordial or primary stages. (Silva et al. 2009).
Within the ovary, ALK6 and its mRNA have been localised
to oocytes from the primordial stage and in granulosa cells from The role of IGF in folliculogenesis
the primary stage of growth and thereafter in both cell types
Studies using several in vivo animal models (e.g. overexpres-
throughout follicular development. It is evident from morpho-
sion of IGF-I in transgenic mice, targeted deletion of IGF-I and
logical studies that follicles in homozygous ALK6 (FecBB)
IGF-deficient animals) have shown that IGF-I is not required for
mutants mature and ovulate at significantly smaller diameters
ovarian organogenesis, the recruitment of primordial follicles
and with fewer granulosa cells than in the wild-type. Despite the
or growth of gonadotrophin-independent follicles. Rather, they
homozygous mutant ovulating 5–9 follicles compared with 1–2
show that IGF-I increases the sensitivity of small follicles
in the wild-type, the overall secretion rates of steroids and
(diameter 200 mm in the mouse, 2 mm in the sheep, 5 mm in
inhibin are similar among these genotypes. Another important
cattle and humans) to gonadotrophin stimulation and simulates
and relatively subtle difference is that oocytes in homozygous
their transition from the gonadotrophin-responsive to the
FecBB ewes reach their mature diameter of 130 mm during the
gonadotrophin-dependent stages (Mazerbourg et al. 2003).
preantral stage of development whereas the oocytes in the wild-
In ovarian tissue in vitro, IGF-I stimulated steroidogenesis by
type do not reach this diameter until after antrum formation.
thecal cells and both proliferation and differentiation of granu-
Thus, the effects of the ALK6 mutation can be detected both
losa cells (Mazerbourg et al. 2003). In sheep, IGF-I stimulated
morphologically and functionally during preantral development
proliferation of granulosa cells in small follicles (1–3 mm
in committed follicles.
diameter), and the secretion of progesterone by granulosa cells
In ewes carrying the FecBB mutation, the oocyte reaches full
of large (45 mm diameter) but not small follicles (Monniaux
size earlier with fewer granulosa cells which, in turn, undergo
and Pisselet 1992). The secretion of oestradiol by granulosa cells
fewer doublings to reach their ovulatory size compared with
in culture was also stimulated by IGF-I (Campbell et al. 1996)
wild-type ewes. In vitro evidence suggests that granulosa cells
and in the ewe, in vivo data have shown that follicular oestradiol
from FecBB mutants develop earlier responsiveness to FSH
and inhibin are stimulated by IGF-I during the follicular phase
(with respect to cyclic AMP synthesis) and to LH than granulosa
of the oestrous cycle (Campbell 1988). Therefore, it seems that
cells from wild-type ewes. This earlier responsiveness to gona-
IGF-I can stimulate either the proliferation or the differentiation
dotrophins is further illustrated by superovulation treatments
and differentiated functions of granulosa cells, depending on the
where the ovulation rates in ewes carrying the FecBB mutation
stage of development of the follicle.
are significantly higher than in wild-type ewes (McNatty et al.
2006b). This is in contrast to ewes carrying the BMP15 or GDF9
mutations. It is not known how the FecBB mutation has such The IGF binding proteins (IGFBPs) as modulators
profound effects on folliculogenesis and ovulation rate. How- of IGF activity in the follicle
ever, in vitro studies have shown that the mutation in the ALK6 It is clear IGF-I can exert profound effects on the later stages
receptor causes a loss of function with respect to the action of of folliculogenesis and steroidogenesis, and both high and low
BMP ligands (Fabre et al. 2003) and perhaps this explains the levels of IGF-I activity are probably deleterious to the follicle
higher responsiveness to FSH observed in related studies (Fabre and its oocyte. Consequently, IGF activity within the follicle
et al. 2006). Potential molecular mechanisms and signalling needs to be regulated within narrow limits. The problem is that,
pathways including the Type I and Type II receptors and their in follicular fluid, there is no correlation between the concentra-
second-messenger pathways are reviewed elsewhere (Kaivo-oja tion of total IGF-I and follicle size (Monget and Monniaux
et al. 2006). 1995) because most IGF-I in follicular fluid is derived from
blood (Mazerbourg et al. 2003). Critically, follicular fluid
Regulation of folliculogenesis by the insulin-like concentrations of total IGF-I are always within the range that
growth factor (IGF) system will stimulate proliferation and steroidogenesis of granulosa
The concept of local modulation of gonadotrophin-stimulated cells in culture.
folliculogenesis was new in 1991, although it was becoming The supply of IGF-I to the follicle is outside the control of the
clear to us and others that ‘FSH action is modulated at the ovary reproductive axis, therefore intra-follicular IGF activity is
by the actions of metabolic hormones, growth factors, other regulated locally by intra-ovarian factors, primarily the IGF
peptides and steroids’ (Scaramuzzi and Campbell 1990). This binding proteins (IGFBPs). The low-molecular weight IGFBPs
concept is now widely accepted, although precisely how endo- (BP-2, -4 and -5) are inhibitory to IGF actions because they bind
crine and metabolic signals to the follicle interact with paracrine IGF, preventing it from binding to its receptor. Thus, it is the
and autocrine modulators is not fully established. Recent years bioavailability of IGF-I, rather than its total concentration, that
have seen very substantial increases in our knowledge of the changes during folliculogenesis. In the ewe, sow, cow and mare
intra-ovarian regulation of both gonadotrophin-responsive and the intrafollicular concentrations of IGFBP-2 and IGFBP-4
gonadotrophin-dependent follicles. In addition to those already decrease as follicles grow from 1–2 mm to pre-ovulatory size
(Mazerbourg et al. 2003), and in ruminants the intrafollicular relationships among reproduction, nutrition and metabolism,
concentrations of these IGFBPs and of IGFBP-5 increase in probably depend to a great degree on IGF-I and on other
follicles as they become atretic. For IGFBP-3, intrafollicular metabolic factors that directly affect the follicle. This topic is
concentrations do not change during folliculogenesis, except the focus of the next section of this review.
in the ewe, where they decrease in small follicles that become
atretic. The removal of the smaller (o40 kDa) IGFBP-2, -4 and
-5 from pre-ovulatory follicles and their accumulation in atretic Nutrition and folliculogenesis
follicles suggests that there are major changes in the local Nutritional influences, either directly through dietary nutrients
bioavailability of IGF-I (Monget et al. 1993). or through metabolic intermediates, affect folliculogenesis at
multiple levels in the hypothalamo–pituitary–ovarian axis
The role of follicular pregnancy-associated plasma (Scaramuzzi et al. 2006). At a hypothalamic level, conditions
protein-A (PAPP-A) in the regulation of the IGF system resulting in hypoglycaemia and negative energy balance inhibit
The role of pregnancy-associated plasma protein-A (PAPP- LH pulsatility in sheep, cattle and humans (Jorritsma et al.
A) in ovulatory follicles is highly conserved across mammalian 2005), most probably by inhibiting GnRH secretion resulting in
species. The low concentrations of IGFBPs in healthy growing a failure of the LH surge mechanism and anovulation. Severe
follicles are caused by increased rates of proteolytic degradation negative energy balance can also have deleterious influences on
of IGFBP-2, -4 and -5 by PAPP-A and by low rates of gene folliculogenesis and the oocyte; these have been most clearly
transcription for IGFBP-2 (Mazerbourg et al. 2003). In bovine shown using induced ovulation in postpartum dairy cows.
granulosa cells the expression of the mRNA for PAPP-A is Increasing the supply of energy or improving body condition to
greatest in ovulatory follicles and is positively correlated with produce a state of positive energy balance appears to have little
aromatase and LH receptor (Mazerbourg et al. 2001). In rodents, effect on LH pulsatility (Rhind et al. 1989a, 1989b), perhaps
pregnant mare serum gonadotrophin (PMSG) stimulates PAPP- because negative feedback rapidly counters any stimulatory
A mRNA in granulosa (Hourvitz et al. 2002) and FSH induces effect on the follicle unless there are concomitant direct ovarian
the degradation of both IGFBP-4 and -5 (Resnick et al. 1998). or peripheral effects that modify negative feedback efficiency.
These observations suggest that gonadotrophin-stimulated Thus, there appears to be an energy threshold for GnRH and
PAPP-A gene expression is associated with selection of the increasing the energy supply above the threshold has no
dominant follicle. However, the regulation of PAPP-A expres- apparent effect on ovulation (Loucks and Thuma 2003).
sion by FSH and intra-ovarian factors is not verified for At an ovarian level, the situation is reversed and increased
ruminants, although in the rodent BMP15 appears to be involved energy supply stimulates folliculogenesis in sheep and cattle
in the induction of PAPP-A (Matsui et al. 2004). (Webb et al. 2004; Viñoles et al. 2005; Letelier et al. 2008).
In cattle PAPP-A activity was higher in dominant than in There is now good evidence showing that the stimulatory effects
subordinate follicles on Days 2 and 3 of the first follicular wave of nutrition on folliculogenesis are mediated directly at an
of the oestrous cycle and the levels of activity correlated ovarian level (Scaramuzzi et al. 2006) and glucose, fatty acids
positively with oestradiol and negatively with low-molecular and several metabolic hormones have all been shown to have
weight IGFBPs in follicular fluid (Rivera et al. 2001). When direct actions on the follicle. Since 1991, research into the
cattle were treated with low doses of recombinant bovine FSH nutritional regulation of folliculogenesis has explored in con-
for 2 days shortly after wave emergence, two co-dominant siderable detail and two general themes have emerged. They are
follicles were selected, both with higher PAPP-A activities the identification of the nutrients and metabolic signals that
and oestradiol concentrations and lower amounts of IGFBP-4 mediate nutritional effects in the follicle and the elaboration of
in their follicular fluid than subordinate follicles (Rivera and the local intra-follicular nutrient-sensing and integrative path-
Fortune 2001). These results suggest that the action of FSH ways and mechanisms that modulate gonadotrophin-stimulated
on dominant follicles is to increase PAPP-A activity. Careful folliculogenesis.
analyses of various characteristics of follicles just before and When discussing nutritional influences on folliculogenesis,
after follicle emergence showed that an increase in PAPP-A there are some important generalities that should be borne in
activity in one follicle of the wave was detected before any mind: (1) the energy requirement for folliculogenesis when
detectable difference in diameter or in the concentrations of considered in relation to whole body energy turnover is insig-
oestradiol or IGFBP-4 or -5 in follicular fluid (Rivera and nificant. Thus, the effect of energy on folliculogenesis is most
Fortune 2003). These results point to changes in the IGF system likely regulatory rather than providing metabolic fuel; (2) the
as an integral component of selection for dominance. stimulatory effect of nutritional energy on folliculogenesis is
In conclusion, it is clear that the IGF system is essential for relatively small and its increase induces minor alterations in
follicular growth and development. However, a successful out- folliculogenesis. More importantly, these effects can be over-
come requires fine-scale local modulation of the bioactivity of ridden by other physiological mechanisms and by many com-
externally derived IGF-I because of the profound effects it can mon experimental models. Thus, in sheep where short-term
exert on the differentiation and function of follicular cells. This energy supplements stimulate folliculogenesis, only 30–50%
modulation is controlled by the IGF binding proteins and PAPP- of treated ewes will convert increases in follicle number to
A. Their importance is illustrated by situations in which there is increased ovulation rate. In cattle, energy supplements also
a breakdown in the regulation of IGF-I production by the liver, stimulate folliculogenesis but this is rarely converted into
such as the high-producing dairy cow (Lucy 2008). Indeed, the increased ovulation rate. Presumably in the cow a more
powerful mechanism of follicle selection completely overrides detrimental irrespective of their effect on folliculogenesis. In
the stimulatory effect of nutrition; and (3) nutritional effects on cattle there is a link between nutrient intake and the develop-
folliculogenesis have been linked to absolute bodyweight (i.e. mental competence of oocytes and it was shown that high-fat
adiposity), the rate of gain in bodyweight and short-term nutrient diets enhanced the developmental competence of oocytes
fluxes that have no effect on bodyweight (Scaramuzzi et al. (Fouladi-Nashta et al. 2007; Garnsworthy et al. 2009). Cows
2006). In each of these three situations the nutrient and meta- were fed a diet formulated to stimulate insulin in order to restore
bolic profiles will be different as will metabolic signals that ovarian cyclicity and were then switched to a diet that lowered
reach the follicle. Thus, it is unlikely that there is a single insulin and increased fatty acids during the mating period
mechanism or a single metabolic mediator of nutritional influ- (Garnsworthy et al. 2009). The results showed that this two-
ences on folliculogenesis, although it may be possible that diet strategy increased the pregnancy rate from 27% to 60%.
mechanisms converge in the follicle. Thus, it appears that optimal dietary strategies can enhance
oocyte quality. However, it is clear that further research is
Identification of metabolic and nutritional mediators required to fully elucidate the mechanisms and to determine if
of folliculogenesis they are mediated by follicular somatic cells or if they involve
direct actions on the oocyte.
The list of metabolites and nutrients that have direct effects
on follicular function is extensive. The most studied of the intra- Metabolic-sensing systems in the follicle
follicular mediators of nutritional influences on folliculogenesis
It is now widely accepted that there are direct nutritional
are leptin, glucose–insulin, growth hormone (GH) and IGF.
effects on folliculogenesis, suggesting that there are specific
The blood concentrations of leptin, glucose and insulin are all
nutrient-sensing mechanisms in the follicle. Also emerging is
elevated when animals are in positive energy balance as well as
an understanding of how these sensing mechanisms affect
immediately following feeding and can convey metabolic
folliculogenesis.
information to the follicle. In contrast, GH is elevated during
negative energy balance (Downing et al. 1995) when its princi- The leptin system in follicles
pal function is to mobilise the body’s energy reserves to counter
All of the components of a functional leptin system are found
negative energy balance. Thus, during negative energy balance
in follicles. In ruminants, leptin protein and its mRNA have been
GH can convey negative metabolic information to the follicle
detected in theca cells and the oocyte (Ryan et al. 2002; Muñoz-
via follicular GH receptors (Eckery et al. 1997). The signalling
Gutiérrez et al. 2005; Pisani et al. 2008). In granulosa cells,
role of GH is complicated because GH stimulates the production
however, leptin is only weakly expressed and attempts to
of hepatic IGF-I and prolonged exposure to high concentrations
identify its mRNA have been unsuccessful (Muñoz-Gutiérrez
of circulating GH can induce insulin resistance.
et al. 2005; Pisani et al. 2008). Both mRNA and the protein of
The IGF system is also usually listed among the potential
the functional long form of the leptin receptor have also been
mediators of nutritional effects on folliculogenesis. The blood
detected in granulosa cells, theca cells and the oocyte (Muñoz-
concentrations of IGF-I were reduced during severe negative
Gutiérrez et al. 2005; Pisani et al. 2008). These findings suggest
energy balance (Lucy 2008) and thus the IGF system can convey
that leptin may also have paracrine and autocrine functions in
metabolic information to the follicle. The action of IGF-I in the
the follicle in addition to its endocrine functions.
follicle is modulated by complex interactions with its receptor,
Much of the in vivo research dealing with the reproductive
the various IGF binding proteins and IGF-II, as discussed earlier
effects of leptin have used the mutant obese (ob/ob) mouse or
in this paper. IGF-I, a potent stimulator of follicular growth and
have been descriptive in nature; that is, measuring leptin in
follicular oestradiol secretion (Scaramuzzi et al. 1999), is almost
blood and follicular fluid or describing the presence and dis-
certainly essential for normal folliculogenesis and follicle selec-
tribution of leptin and the various forms of its receptor in
tion (Mihm and Evans et al. 2008). When used exogenously
reproductive tissues. The few physiological studies suggest that
in the sheep auto-transplant model it induced severe hyper-
exogenous leptin stimulates folliculogenesis in sheep (Kendall
stimulation of both folliculogenesis and follicular oestradiol
et al. 2004; Muñoz-Gutiérrez et al. 2005). Some in vitro and
and inhibin secretion (Campbell 1988; Scaramuzzi et al. 1999).
in vivo studies have also provided data on the effects of leptin on
Thus, under normal physiological conditions the activity of IGF-
follicular steroidogenesis. For example, passive immunisation
I in the follicle is maintained within precise limits by systems in
against leptin increases ovarian oestradiol secretion in ewes and,
the follicle whose critical function is to prevent over-activity of
conversely, infusion of leptin directly into the ovarian artery
intra-follicular IGF-I. During negative energy balance low IGF-
reduces ovarian oestradiol secretion (Kendall et al. 2004). The
I inhibits folliculogenesis, but during positive energy balance
intrafollicular actions of leptin have been studied in vitro using
elevated IGF-I is likely to have only small effects on the follicle
cultured granulosa cells and the published data suggest that
because of its intra-follicular inhibition by the IGFBPs and
leptin inhibits hormonally stimulated oestradiol production by
sequestration of the IGF-I receptor by IGF-II.
granulosa cells in vitro (Zachow and Magoffin 1997; Spicer
2001) and by the follicle in vivo (Kendall et al. 2004). The
Nutritional influences on the oocyte inhibitory influence of leptin on oestradiol secretion appears to
There is now good evidence that diet and body condition can involve the IGF system; immuno-neutralisation of IGF-I
affect the quality of oocytes. Thus, depending on composition, reverses the inhibitory effect of leptin on oestradiol secretion
some diets are beneficial to oocyte quality whereas others are (Sirotkin et al. 2005) and leptin inhibits the effect of IGF-I on
FSH-stimulated oestradiol production by granulosa cells in vitro important for the production of hyaluronic acid during cumulus
(Zachow and Magoffin 1997). In cultured bovine granulosa expansion (Thompson et al. 2007). Perturbations in glucose flux
cells, leptin attenuates the effect of IGF-I on FSH-stimulated (hyperglycaemia) through this pathway may have substantial
oestradiol production (Spicer et al. 2000). Similarly, it inhibits negative effects on oocyte competence (Thompson et al. 2007).
the effect of IGF-I on LH-stimulated androstenedione produc- It has been suggested that this nutrient-sensing pathway may
tion in theca cells (Spicer et al. 2000). also mediate nutritionally stimulated folliculogenesis (Muñoz-
Gutiérrez et al. 2004).
The glucose–insulin system in follicles
The mammalian follicle also has a fully functional glucose– Local integrative pathways and crosstalk between
insulin system. The follicle has insulin receptors (Poretsky et al. insulin and gonadotrophin signalling
1999) and insulin has been shown to act primarily through its A concept not widely appreciated in 1991, but now generally
own receptor and not the closely related IGF-I receptor (Willis accepted, is that a cascade of divergent intracellular signalling
and Franks 1995). The effects of insulin on cellular function pathways are activated when gonadotrophins stimulate follicu-
are mediated by a complex array of intracellular pathways lar cells (Richards et al. 2002; Hunzicker-Dunn and Maizels
(Taniguchi et al. 2006) with a multitude of phosphatases, inter- 2006). By replacing the linear models of hormone signalling
mediary and terminal kinases as well as numerous scaffold and with functional interactions among signalling pathways (often
docking proteins that impart hormone- and tissue-specificity on referred to as cross-talk) and networks in the follicle (Taniguchi
the cellular responses to insulin. The insulin receptor substrate et al. 2006), we can begin to understand how short-term
proteins and the downstream kinases Akt, PI3K and ERK and the nutritional stimulation might modify gonadotrophin-stimulated
phosphatase PTEN have all been identified in the follicle. In a follicular function. In a recent experiment a short-term infusion
rodent study, it was shown that selective deletion of the gene for of glucose at 10 mM per hour increased the total number of
IRS-2 was associated with impaired folliculogenesis (Neganova follicles 41 mm in diameter but decreased the amount of
et al. 2007). Insulin-dependent glucose transport proteins have phosphorylated Akt and AMPK as well as aromatase in granu-
also been reported in granulosa and theca cells in ovarian follicles losa cell lysates (Gallet et al. 2009). The molecular details of
from sheep (Williams et al. 2001; Nishimoto et al. 2006). The how and when these pathways interact (cross-talk) in granulosa
presence of GLUT4 in the follicle suggests a role for insulin- cells and their implications for folliculogenesis remain to be
mediated uptake of glucose in the follicle. explored.
Overall, there is no doubt that the follicle contains a func-
tional glucose–insulin system, but we are left with a critical Systemic endocrinology
unanswered question – what is its role in folliculogenesis and Endocrinology of the oestrous cycle, follicle dynamics
oocyte development? There are at least two possibilities. The and follicle waves
first, and simplest, is that the glucose–insulin system, or insulin
The temporal patterns of secretion for the major reproductive
itself, has only non-specific functions in the maintenance of
hormones during the oestrous cycle had been well described by
cellular health and integrity, as it does for all cells and tissues.
1991 (fig. 1 in Scaramuzzi et al. 1993). The secretion of GnRH
Alternatively, the glucose–insulin system may also have spe-
is inhibited by ovarian feedback exerted through a synergistic
cific functions that affect granulosa and theca cells. This second
interaction between oestradiol and progesterone acting on
possibility is supported by an overwhelming body of evidence,
centres in the hypothalamus. This process explains the induction
derived principally from in vitro studies.
of the follicular phase and the sequence of events leading to
the LH surge and ovulation. For the hormonal control of the
Adenosine monophosphate-activated kinase (AMPK) terminal stages of folliculogenesis and the determination of
Adenosine monophosphate-activated kinase (AMPK) is a ovulation rate, the secretion of FSH is important and FSH
serine/threonine kinase that is activated by an increase in the secretion is controlled primarily by negative feedback at the
ratio of AMP to ATP that is associated with a depletion of pituitary level through a synergistic interaction between inhibin
ATP in response to nutritional and environmental stress and oestradiol. In the 17 years since our initial workshop, we
(Hardie 2004). Adenosine phosphate-activated kinase has have seen the emergence of more precise descriptions of the
been identified in theca cells, granulosa cells and oocytes patterns of secretion of inhibin A and B and the close association
(Bilodeau-Goeseels et al. 2007; Tosca et al. 2007). Adenosine between the wave-like patterns of terminal follicle growth and
phosphate-activated kinase is often viewed as a metabolic regression (described in the next section) and the patterns of
master molecule and in the ovary it may regulate metabolic secretion of reproductive hormones, particularly FSH, oestra-
influences on folliculogenesis and oocyte maturation. Thus, the diol and inhibin.
AMPK signalling pathway is a potential modulator of interac-
tions between energy balance and folliculogenesis. Inhibins and activins
It was clear in 1991 that inhibin suppressed FSH secretion
The hexosamine pathway both in vivo and in vitro, acting with oestradiol to suppress
The hexosamine nutrient sensing system has been reported in secretion of FSH by a direct inhibitory action on the expression
muscle and adipose tissue (Marshall et al. 1991). Hexosamine of the mRNA for FSHb. However, in 1991 the only available
biosynthesis is active in bovine follicles and is particularly assays for inhibin detected its a sub-unit and thus were unable to
discriminate between the biologically inactive free a sub-unit on folliculogenesis cannot be resolved until we can measure
and the intact and biologically active inhibin a–bA and a–bB peripheral inhibin concentrations. There is now good evidence
dimers. An increase in inhibin concentrations was seen in the showing that nutrition suppresses follicular oestradiol secretion
follicular phase and inhibin secretion was stimulated by FSH, while increasing the number of small and medium follicles. So
but the adrenal cortex also secreted inhibin a sub-unit (McNeilly why does FSH not increase? Perhaps it is because nutrition
et al. 1994), so interpretation of these data was problematic. stimulates inhibin secretion! This would be consistent with the
All of the inhibin subunits, as well as activin receptors and stimulation of folliculogenesis in small and medium-sized
follistatin, have been detected in ovarian follicles from sheep follicles. The combined effect of lower oestradiol and increased
(Engelhardt et al. 1993). In addition, betaglycan, the putative inhibin could, in effect, counter each other so that there is no
inhibin receptor (Gray et al. 2001) has been identified in sheep alteration in FSH secretion. One report showed that ewes in
ovarian follicles (McNatty et al. 2007). Multiple isoforms of the moderate body condition had higher concentrations of circulat-
inhibin dimers have been identified in bovine follicular fluid ing inhibin-a than ewes in low body condition (Rhind and
(Glister et al. 2006) but nothing is known of the dimers’ Schanbacher 1991). Clearly this is a critical area where know-
presence in sheep follicles or of their relative patterns of ledge is deficient.
secretion and physiological functions in either species. A further complexity is that the plasma concentrations of
The successful development of two-site enzyme linked FSH measured using current homologous radioimmunoassays
immunosorbent assays (ELISA) that were able to differentiate show that the mean concentrations of FSH are similar within
the two biologically active forms of dimeric inhibin namely, ewes across several oestrous cycles and during anoestrus (A.S.
inhibin A and inhibin B (Groome et al. 1994, 1996), was a major McNeilly, unpubl. data). This suggests that the FSH threshold
technical advance in the field of inhibin physiology. These concentration for the emergence of gonadotrophin-dependent
assays showed that sheep produced only inhibin A (McNeilly follicles varies among individuals according to age, season and
et al. 2002). In follicles from sheep, much less of the bB subunit other as-yet unidentified variables as was shown in one study
of inhibin B was expressed compared with the bA subunit of (Picton and McNeilly 1991). The large variation in ovulation
inhibin A. In the ewe, the plasma concentrations of inhibin A rates after PMSG or FSH suggests that differing FSH thresholds
increased during the follicular phase up to the point of ovulation are a significant factor in maintaining breed-specific ovulation
(Souza et al. 1997). rates. However, the nature of this threshold and unravelling
While early studies concentrated on investigating inhibin as how it affects selection of the ovulatory follicle remains to be
an endocrine regulator of FSH secretion, it is now clear in sheep determined.
(Campbell and Baird 2001) as well as other species (Knight and
Glister 2006) that inhibin also acts locally within the follicle to
augment LH-stimulated production of androgen by theca cells Conceptual issues – the feedback–ovulation rate paradox
and FSH-induced aromatase activity in granulosa cells. The The hypothalamo–hypophyseal–ovarian axis, with its feed-
activins appear to inhibit these effects. The only identified forward and feedback endocrine links, is essentially a homeo-
inhibin receptor, betaglycan, does not appear to mediate a static system that will tend to neutralise the effects of external
signalling response to inhibin (Gray et al. 2001), so inhibin factors (e.g. a nutritional supplement) on folliculogenesis. Any
probably acts to block the action of activin and possibly some stimulus that leads to the emergence of more ovulatory follicles
BMPs. This remains to be confirmed. Activin A enhanced the would induce higher rates of secretion of follicular hormones,
growth of follicles in rodents and humans (Knight and Glister leading to greater inhibitory feedback, and thus a reduction in
2006) and improved the competence of bovine oocytes (Silva gonadotrophin secretion, thereby reducing follicular growth and
et al. 2003) as well as the in vitro development of ovine oocytes re-establishing the status quo. Thus, based on first principles,
and preantral follicles (Thomas et al. 2003). However, activin A we have two mutually incompatible a priori hypotheses: (1) an
had no effect on the development of bovine primordial and increase in the secretion of FSH is needed to stimulate follicu-
primary follicles in vitro (Fortune et al. 2000) and inhibin A logenesis; and (2) increased folliculogenesis leads to lower
reduced the competence of bovine oocytes in vitro (Silva et al. concentrations of FSH. This conflict probably explains the
2003). How these effects are modulated in vivo is unclear variety of experimental observations in the literature, with
because little is known about the control of activin secretion studies supporting both hypotheses and others showing no
due to the lack of assays sufficiently sensitive to detect activin A, significant change in FSH concentrations. Arguably, lack of
activin AB, and particularly activin B, in sheep. Furthermore, change is the correct and normal outcome. This is only feasible if
both inhibin and follistatin can modulate the action of activin, there are changes in the balance of the feedback loop such as the
suggesting that the physiological effects of activin are tightly ratio of oestradiol to inhibin, or there is another variable, other
regulated within individual follicles. than concentration, involved in the response to the hormones
comprising the feed-forward and feedback loops (e.g. variable
time lags).
Technical issues – the measurement of inhibin and FSH With respect to the balance of the feedback equilibrium, two
The sensitivity and specificity of hormone assays, particu- options can be considered: (1) there is variation in the respon-
larly for peripheral inhibin and FSH remain a problem. There is siveness of follicles to FSH, for both proliferation and
an almost total lack of data on the effects of nutrition on inhibin hormone production; and (2) there is a change in clearance of
and the role of ovarian feedback in mediating nutritional effects the feedback hormones, so that concentrations reaching the
hypothalamo–hypophyseal axis do not directly reflect secretion Gonadotrophin-dependent follicles grow and regress in a regular
rates. These are not mutually exclusive and there is evidence sequential pattern of waves, a phenomenon first described in cattle
for both. For example, when ewes are fed a supplement, more in 1960 and now detailed by ultrasound-based observations. A
ovarian follicles develop but the amount of oestradiol produced wave begins under the influence of a small rise in FSH concentra-
per follicle is reduced (Viñoles et al. 2005; Somchit 2008). In tions that is caused by the momentary disappearance of inhibitory
addition, diet affects the rate of metabolic clearance of oestra- feedback (oestradiol and inhibin) when a dominant follicle ovu-
diol (Adams et al. 1994) so the overall effect is less inhibition of lates or when a potentially ovulatory follicle undergoes atresia.
FSH secretion than might be expected for the number of follicles The rise in FSH induces a cohort of gonadotrophin-dependent
that are developing. follicles to emerge from a pool of gonadotrophin-responsive
follicles (Souza et al. 1997, 1998; Viñoles et al. 1999). About
Dynamic patterns in the growth of gonadotrophin- three days after emergence, one (or a few) of these follicles each
responsive, gonadotrophin-dependent achieve potentially ovulatory status (diameter 5–8 mm in the
and ovulatory follicles ewe, 15–18 mm in the cow) by developing LH receptors on their
granulosa cells and becoming independent of FSH; they, in fact,
The last 17 years have seen a rapid expansion in the application
switch their absolute dependence on gonadotrophins from FSH
to farm animals of ultrasound imaging for monitoring follicle
to LH. They also secrete large amounts of oestradiol, androste-
development, a technique that had been widely used in clinical
nedione and inhibin A, reducing FSH concentrations to
medicine for monitoring of ovarian structures in women (Kerin
below the threshold needed to sustain the other gonadotrophin-
et al. 1981). It was initially adapted for use in cattle (Pierson and
dependent follicles (Fig. 2). Thus, dominance by the potentially
Ginther 1984) and then in small ruminants. Repeated, precise
ovulatory follicle(s) causes atresia in any remaining members of
measurement of individually identified ovarian follicles
the cohort that have failed to switch their absolute dependence on
allowed a clear description of the wave-like patterns of terminal
gonadotrophins from FSH to LH and thus cannot continue to
follicular growth and has led to considerable refinement of
function in the presence of low concentrations of FSH (Campbell
hypotheses and experiments.
et al. 1999). If the potentially ovulatory follicle(s) achieve their
In small ruminants, ultrasound imaging of ovarian follicles is
status soon after luteolysis, they will continue to develop and
technically challenging because of the small size of the animals,
ovulate; otherwise, they will stop producing oestradiol, even
the position of the reproductive tract, and the size of the ovaries
if provided with normally adequate gonadotrophic support
and its structures. For follicles larger than 2 mm in diameter, the
(Dobson et al. 1997), although they will persist and be identifi-
technique is accurate but, below this limit, the resolution is
able by ultrasonography for several days thereafter (Souza et al.
insufficient to provide quality data (Viñoles et al. 2004). Some
1997; Viñoles et al. 1999). Ultimately, they also undergo
of these limitations can be overcome by coupling ultrasound with
structural and functional atresia, thus initiating the next follicular
ovarian autotransplantation, in which the left ovary and its vascular
wave in the sequence.
pedicle are relocated to a subcutaneous site in the neck (Souza et al.
During the oestrous cycle, the ewe has two to four follicle
1997, 1998). This allows transdermal imaging in two planes and,
waves each of 4–8 days. The reason for this variation is
when it is allied with contemporaneous sampling of ovarian
unknown, but body condition has been suggested (Murphy
venous blood, the physiological and morphological changes
et al. 1991). The variation in waves per cycle might explain
within the follicle population can be followed over considerable
much of the variability that is encountered in experiments
periods of time. The transplant model has highlighted a primary
designed to study the effects of short-term nutritional supple-
limitation of conventional ultrasound by showing that large antral
ments on follicular development. In a randomly-selected group
follicles can remain morphologically normal, as assessed by
of ewes, during any random period of the oestrous cycle, there
ultrasound, for several days after losing hormonal function (Souza
will be a mix of animals going through any of the final stages
et al. 1997). The value of the technology is that it allows us to study
of follicular development, from gonadotrophin-responsive to
the processes of transformation that follicles undergo as they
gonadotrophin-dependent to atretic, despite treatments used to
progress through these functional stages of development. Ultra-
synchronise oestrous cycles. Unless we can control this varia-
sound-based studies have been particularly useful for studying
tion, it will be difficult to obtain robust data in detailed
follicle selection and follicle dominance, critical aspects of the
physiological studies of ovulation rate. In concluding this
very final stages of folliculogenesis that determine the outcome for
section, we need to point out that there is debate over whether
ovulation and the ovulation rate.
follicular dominance actually exists in the ewe (Duggavathi
et al. 2005). In the ewe, there is ample evidence suggesting that
Dominance and waves of development in each follicle wave, a potentially ovulatory follicle is present
in gonadotrophin-dependent follicles and that in the mono-ovulatory ewe, one follicle becomes larger
The development of the pool of gonadotrophin-responsive and more highly oestrogenic (or deviates). However, the oestro-
follicles can be considered hierarchical with most, if not all, genic follicle is not always the largest follicle and the ewe can
being at a different stage of growth. Subsequently, the most rapidly promote replacement follicles into the ovulatory role so
advanced of these gonadotrophin-responsive follicles (i.e. that further recruitment and selection can occur during the
during the antral growth stage) are those that emerge concomi- follicular phase (Souza et al. 1997). This debate thus concerns
tantly with the increases in FSH to form what is commonly the mechanisms responsible for the wave pattern and not the
referred to as the cohort of gonadotrophin-dependent follicles. existence of the wave pattern itself.
Non-ovulatory wave Ovulatory wave

Ovulation
Potentially
ovulatory
Atresia
Gonadotrophin-
dependent
Atresia Atresia
Gonadotrophin-
responsive
FSH
FSH
Time
Fig. 2. The wave-like pattern in the terminal stages of follicle growth in ruminants. Growth to the gonadotrophin-responsive stage is approximately linear
ensuring a continuous supply of gonadotrophin-responsive follicles. Under the influence of FSH, gonadotrophin-responsive follicles become gonadotrophin-
dependent in a process that reflects the dynamics of the negative feedback loop between oestradiol and inhibin and pituitary FSH. A peak of FSH (indicated by
the arrows) leads to the emergence of a group of gonadotrophin-dependent follicles, visible by ultrasound, with a higher threshold for FSH than gonadotrophin-
responsive follicles. A potentially ovulatory follicle or follicles emerge from this group and, while present and fully functional, it is dominant, secreting
sufficient oestradiol and inhibin to suppress FSH, promote atresia in the remaining gonadotrophin-dependent follicles and preventing the emergence of a new
cohort of gonadotrophin-responsive follicles. In the presence of a corpus luteum (a non-ovulatory wave), the dominant follicle(s) becomes atretic after 4–5
days, secreting less oestradiol and inhibin so that FSH can increase and start a new wave. If the corpus luteum has regressed, an ovulatory wave results and the
dominant follicle(s) ovulate(s).
AMH and the transition from gonadotrophin-responsive transformation of a follicle from one stage of development to the
to gonadotrophin-dependent follicles next. However, there is information that is not easily incorpo-
In rodents, the granulosa cells of committed and gonadotro- rated into a general model. In particular, in vitro culture systems
phin-responsive follicles produce AMH (Themmen 2005). First using oocytes and follicular somatic cells have generated detail
detected in granulosa cells of committed follicles, AMH reaches on the molecular and cellular mechanisms responsible for local
its highest level in granulosa cells of gonadotrophin-responsive control systems. The problem is that, at this time, it is difficult to
follicles and then gradually diminishes until no longer detect- decide the relative physiological importance of many of these
able in gonadotrophin-dependent follicles. Similarly, in the cow newly described intrafollicular systems in comparison with, say,
the concentrations of AMH are elevated in follicular fluid from the gonadotrophins or IGF-I. Therefore, to provide a durable
healthy gonadotrophin-responsive follicles and decrease during model, we have retained a general model (Fig. 3) based on that
terminal follicular development (Rico et al. 2009) and AMH is put forward in 1991 and included a molecular-based model that
not present in atretic follicles (Rico et al. 2009). Furthermore, describes events between the oocyte, cumulus and granulosa
in rat and pig granulosa cells, AMH was able to reduce cells (Fig. 4). Here we have sifted information, omitting
FSH-stimulated aromatase and the expression of LH receptors some possibilities from the model. Clearly, this is a relati-
(di Clemente et al. 1994). These data suggest that AMH may vely subjective process. The most important additions to the
regulate the responsiveness of follicles to FSH. Overall, the models are:
evidence suggests that AMH modulates both early and terminal (1) The interactions between the oocyte and the granulosa cells
stages of follicle development in rodents. However, in rumi- including the differentiation of the granulosa cells into
nants more research is required to clarify the patterns and levels mural and cumulus phenotypes.
of expression as well as the bioactivity of AMH isoforms during (2) The initiation of growth and development in the primordial
follicle development and in particular the role of gonadotrophins follicle, a process that was essentially a mystery in 1991, has
in the control of AMH. been updated along with an updated understanding of the
development of committed follicles.
(3) Advances in our understanding of the physiology of antral
Update on models of folliculogenesis, ovulation rate
follicle growth and differentiation of gonadotrophin-
and nutritional influences on folliculogenesis
responsive follicles into gonadotrophin-dependent and ovu-
Models of folliculogenesis latory follicles. Here progress has been relatively modest
With respect to the general mechanistic model proposed in 1991 compared with the above two areas.
(fig. 3 in Scaramuzzi et al. 1993), the basic model still holds, (4) Confirmation of the concept that metabolic factors can
although significant new information has been added and directly influence follicular development and thus ovulation
additional mechanisms proposed to explain the progressive rate.
Primordial follicles (Type 1 or 1a): Largest

population of follicles present, oocyte and granulosa
cells have receptors for some growth factors, but not
LH or FSH
Committed follicles (Types 2 and 3; primary and small

preantral): FSH and LH not essential; continuous
growth controlled by the oocyte; low rate of atresia,
development of zona pellucida
Gonadotrophin-responsive follicles (Types 4

and 5; large preantral; small-medium antral):
Antrum Development of LH-responsive theca interna and
FSH-responsive granulosa cells; oocyte-mediated
forms differentiation of a cumulus phenotype; increasing
atresia and progressive increase in requirement
for gonadotrophins
Gonadotrophin-dependent follicles (Type 5+;

medium-large antral): Become atretic if FSH
concentrations are low before granulosa cells
develop LH receptor; high rate of atresia
associated with emergence of ovulatory follicles
Ovulatory follicle(s): have LH

receptors on granulosa cells; can
survive low FSH concentrations
Ovulation
Induced by the LH
surge about 72 h after
emergence of the
Atresia ovulatory follicle(s)
Fig. 3. A model for folliculogenesis in the ewe based on that developed by Scaramuzzi et al. (1993). Folliculogenesis is illustrated as a cascade of
development during which follicles emerge from a pool of primordial follicles to enter a process of growth and development that is continuous and ends in either
atresia or ovulation. In most mammals this process is approximately linear to the gonadotrophin-responsive stage and, especially in ruminants, becomes wave-
like in the gonadotrophin-dependent stage, as shown in Fig. 2.
The models portray the developmental path of a single The continued orderly development of these follicles (Lundy et al.
follicle from the primordial stage to ovulation. Ultimately, we 1999) appears to be a linear process controlled primarily by the
anticipate a model encompassing the entire follicle population oocyte. As follicle development progresses follicles gradually
functioning in a physiological context to produce a cooperative become more and more reliant on gonadotrophins, first as
outcome: the ovulatory quota. gonadotrophin-responsive follicles and then as gonadotrophin-
dependent follicles. The antrum forms under the influence of FSH
An updated model for folliculogenesis and thus gonadotrophin-responsive follicles have a more variable
An updated general model for folliculogenesis is presented in morphology; they can be preantral, early antral (with antral
Fig. 3. This model retains the functional classification system gaps yet to coalesce into a complete antrum) or fully antral.
of the 1991 model. This classification system is based on the In gonadotrophin-responsive follicles and then as gonadotrophin-
interrelationship of the follicle with the gonadotrophins. Primor- dependent follicles inadequate support from gonadotrophins leads
dial and committed follicles are largely independent of gonado- to atresia. It is the developing dependence on gonadotrophins that
trophins and even though they are affected by gonadotrophins transforms folliculogenesis from a linear process in the preantral
their essential feature is that they do not require gonadotrophins and early antral stages of development into a wave-like process
for their survival and continued development. They are sometimes during the terminal stages of folliculogenesis. The final class of
called gonadotrophin-independent follicles; however, we have follicle is the potentially ovulatory follicle. We have chosen
preferred the term committed follicle to describe this class of to use the term ‘potentially ovulatory’ to describe this class of
follicle as they are preantral and have a consistent morphology. follicle rather than alternatives such as ‘selected’, ‘dominant’ or
Granulosa GDF9?
Kit ligand?
Primordial
cells
GDF9/BMP15 Committed
Kit ligand (primary)
Gonadotrophin-
GDF9/BMP15 responsive
(preantral, secondary)
Luteinisation
H
FS
GDF
9/BM
P15
cAM Gonadotrophin-
P
responsive (antral)
Luteinisation
FSH
GDF
9/BM
P15
cAM Gonadotrophin-
P dependent
FSH
Luteinisation
LH
GDF
9/BM
P15
cAM Preovulatory
P (until LH surge)
Mural Cumulus Oocyte

granulosa granulosa
Fig. 4. A scheme illustrating the complex and highly integrated nature of folliculogenesis involving interactions between the oocyte and granulosa cells of
developing follicles and indicating pathways of granulosa cell differentiation (into cumulus and mural granulosa cells) and function, oocyte growth and the
acquisition of meiotic and developmental competence. Up to antrum formation, oocytes and granulosa cells are intimately associated and signal to each other
via gap-junctions and with paracrine growth factors such as GDF9, BMP15 and kit-ligand. From antrum formation onwards, a critical role of oocyte-secreted
factors (principally GDF9 and BMP15) is to prevent luteinisation of the oocyte’s neighbouring granulosa cells, thereby establishing and maintaining the
distinctive cumulus cell phenotype. During the antral phase, oocyte-secreted factors also continue to act on mural granulosa cells to promote mitogenic activity
and simultaneously inhibit luteinisation. In turn, both granulosa cell types, but in particular the cumulus cells, provide the oocyte with paracrine factors and
multiple metabolites and regulatory molecules via gap-junctions (e.g. cyclic AMP), required for the oocyte to acquire developmental competence in the
follicle. Broken arrows indicate pathways for which the evidence is limited. Increasingly heavy arrows indicate stronger effects. The figure provides a model
for future research.
‘oestrogenic’. Ovulatory follicles are those that are capable of Molecular model of folliculogenesis and interactions
ovulation given the correct endocrine milieu of high oestradiol, between the oocyte and somatic cells
low progesterone and high LH pulse frequency. Selected and This is an area of conceptual revolution since 1991. The
oestrogenic follicles are indeed the physiological equivalent of oocyte has been confirmed as a major regulator of preantral
ovulatory follicles and they can ovulate if challenged exogenously and early antral follicular growth, affecting ovulation rate and
with an LH surge (Webb et al. 1992). The dominant follicle is a the differentiation and metabolism of the cumulus oophorus,
morphological description and refers to the largest follicle. How- including its capacity to expand and the oocyte’s acquisition of
ever, the concept of the dominant follicle has doubtful physiolo- developmental competence. The oocyte secretes two important
gical relevance in small ruminants because the largest follicle growth factors, GDF9 and BMP15, and other potentially impor-
is not necessarily the ovulatory follicle and may even be atretic tant oocyte secreted factors including BMP6, the fibroblast
(Souza et al. 1997; Viñoles et al. 1999). growth factor (FGF), TGF-a and a2-macroglobulin (ovostatin).
(a)
Known and suspected interactions between the oocyte and
Plasma FSH
Threshold
follicular somatic cells during the different stages of folliculo- of FSH
genesis are shown in Fig. 4.
Models for the regulation of ovulation rate

The model for the regulation of ovulation rate proposes a Duration of ‘window’
metaphorical wall with a gate that regulates the number of
Ovulatory Single ovulatory
gonadotrophin-dependent follicles that pass through it and
follicle follicle
avoid atresia to become ovulatory follicles (Baird 1987). The
gate represents the period of time during a follicle wave when
the concentration of FSH remains above the minimum or
threshold value required by gonadotrophin-dependent follicles Atresia Atresia Atresia
to avoid atresia. A narrow gate only allows a single follicle to Follicle
avoid atresia and if the timing is right, to go on and ovulate ‘cohort’
(Fig. 5a). A wider gate allows for multiple ovulations
(Fig. 5b). Alternatively, for a gate of fixed width, there may be (b)
Plasma FSH
a larger pool of gonadotrophin-dependent follicles undergoing Threshold
development and statistically more will be able to pass of FSH
through to become ovulatory (Fig. 5c). The number of gona-
dotrophin-dependent follicles at the gate depends on two
factors: (1) a genetically determined constant that does not
change; and (2) non-genetic environmental influences that are Duration of ‘window’
not constant and may change even from follicle wave to fol- Two
Ovulatory
licle wave. ovulatory
follicle
There are two ways of increasing the number of ovulatory follicles
follicles (Baird and Campbell 1998):
(1) Widening the gate by decreasing the sensitivity of the
Atresia Atresia
hypothalamo–pituitary–ovarian axis to negative feedback Follicle
to maintain the concentration of FSH above the threshold ‘cohort’
level for longer, thus allowing more time for follicles to
pass through the widened gate (Fig. 5b). This is the case in
(c)
the Romanov and Finnish Landrace breeds of sheep. The
Plasma FSH
Threshold
administration of oestradiol antiserum to sheep or anti- of FSH
oestrogens to women has the same effect.
(2) Increasing the number of potentially ovulatory follicles
available to pass through the gate before it is closed by
suppression of FSH below its threshold level (Fig. 5c). In Duration of ‘window’
this case the levels of FSH and the feedback mechanisms
Ovulatory Two ovulatory
are the same in ewes with single and multiple ovulations
follicle follicles
but follicles are more sensitive to FSH and hence develop
precociously. This is the case for ewes carrying the Booroola
mutation and in ewes treated with low doses of FSH.
(3) In reality both mechanisms may operate simultaneously in
Atresia Atresia
some highly fecund breeds, for example, the Finnish Land- Follicle
race. These hypothetical mechanisms need to be tested ‘cohort’
against evidence. Two points need to be made: (1) the
hypotheses are not mutually exclusive; and (2) the evidence Time
is not strong for either. In reality, the correct experiments
have never been done. Having said this, what does the Fig. 5. A model of the interactions that control ovulation rate. To become
evidence, such as it is, suggest? ovulatory, members of a cohort of gonadotrophin-dependent follicles need
their stage of development to coincide with a selection window, the width of
which is determined by the period of time that FSH concentrations exceed
(1) Widening the gate through elevated FSH the critical threshold needed to prevent atresia (a). Ovulation rate can be
The fact that exogenous FSH or PMSG can increase the increased by increasing the width of the window (b) or by increasing the
number of ovulatory follicles tells us that this mechanism is number of gonadotrophin-dependent follicles that are ready to pass through
feasible, but its feasibility does not conclusively demonstrate the window (c).
that it is a physiologically normal, regulatory process. Much of (3) Increasing the number of gonadotrophin-
the evidence demonstrating that the length of time during which dependent follicles
the FSH remains above its threshold before being suppressed This can be achieved by reducing the size at which follicles
by rising oestradiol and inhibin is based on experiments using become gonadotrophin responsive (Fig. 5c), as happens, for
exogenous FSH. In the ewe, when the concentration of FSH was example, in ewes carrying the Booroola mutation. These ewes
maintained above its threshold for different lengths of time by are comparable with wild-type controls with respect to the
the infusion of exogenous FSH there was a time-related stimula- concentrations of FSH and timing of events around ovulation,
tion of folliculogenesis (Baird and McNeilly 1987) and in a from luteal regression to the LH surge, from the onset of oestrus
similar experiment in women (Schipper et al. 1998) the con- to the LH surge and from the LH surge to ovulation. Concentra-
tinuous infusion of FSH over several days produced a much tions of oestradiol are also similar between the wild-type and
greater increase in the number of ovulatory follicles than the FecBB mutant genotypes because the total secretion of oestra-
same dose given as a single injection. These data provide diol from several smaller preovulatory follicles of ewes with the
evidence supporting the widening of the window model through FecBB mutation is approximately the same as from a single large
elevated FSH. pre-ovulatory follicle in wild-type ewes. Thus, in ewes with the
The measurement of FSH concentrations in jugular venous FecBB mutation follicles become gonadotrophin-responsive and
blood throughout the cycle presents a confusing picture that is not form an antrum at a smaller diameter, thus effectively increasing
always consistent with the widening of the window model. The the size of the pool of gonadotrophin-responsive follicles avail-
confusion in the literature is not surprising given what we now able to pass through the selection gate without any change in the
know about follicle waves, and studies in which follicle waves are overall numbers of follicles, and unaltered negative feedback
synchronised may help to resolve the confusion. A more relevant mechanisms.
approach may be to study the ovulatory wave during the follicular Alternatively the number of gonadotrophin-dependent
phase of the oestrous cycle. In the ewe, there is a consistent fall in follicles may be increased by increasing the population of
FSH during the follicular phase of the cycle ,24–36 h before gonadotrophin-responsive and gonadotrophin-dependent folli-
ovulation and the number of ovulatory follicles and the ovulation cles, without changes in follicle size as happens, for example, in
rate can be easily determined. In the luteal phase of the oestrous the Romanov breed (Fig. 5c). Ewes of this breed have ovaries
cycle the major regulator of oestradiol secretion and hence the that contain three times more growing follicles (including
level of inhibition of FSH is the frequency of LH pulses, which, in gonadotrophin-responsive and gonadotrophin-dependent folli-
turn, is dependent on the level of progesterone. cles) than those of non-prolific breeds. Thus, more gonadotro-
phin-responsive follicles pass through the selection gate. In
(2) Widening the gate by lowering the threshold for FSH
addition, at ovulation, each of their potentially ovulatory folli-
The gate will also be widened if the FSH threshold in the cles has a granulosa cell population of a similar size to that of a
follicle is lowered. The evidence for this is almost all indirect single potentially ovulatory follicle in a non-prolific breed. As a
and circumstantial and there is no direct evidence that the consequence of these follicular features, oestradiol concentra-
threshold concentration of FSH necessary to activate medium- tions before ovulation are 3-fold higher than in non-prolific
size antral follicles is different in ewes with a single or a multiple breeds. Recently, a reduced sensitivity to oestradiol feedback in
ovulation. The experimental demonstration of a change in the this breed has been shown by the observation of a longer latency
threshold concentration of FSH in vivo is a difficult challenge between the insertion of an oestradiol implant and the LH surge
and we do not know the threshold concentration of FSH. At induced by oestradiol in Romanov ewes compared with Île de
the Terrigal meeting (Scaramuzzi et al. 1993) its value for the France ewes (Ben Saı̈d et al. 2007). Roger Land had also shown
mono-ovulatory ewe was estimated as 1.0 ng mL1 of National in 1976 that the ovulation rate in Finish Landrace ewes was less
Institute of Health, Reference preparation 1 (NIH RP-1) and sensitive to the suppressive effect of oestradiol than in Scottish
although this estimate has never been confirmed or refuted it Blackface ewes (Land 1976). The concentrations of FSH in the
seems probable that a single FSH threshold value is unlikely. A Romanov and the Île-de-France are similar but the decline of
suitable model to examine the FSH threshold is the antagonist- FSH after luteal regression is less profound and delayed in the
suppressed ewe given replacement FSH. Only one limited study Romanov. From these data, one concludes that in Finish Land-
has suggested that each ewe has its own FSH threshold (Picton race and Romanov ewes the hypothalamo–pituitary axis is more
and McNeilly 1991). Thus, it seems likely that the FSH threshold resistant to oestradiol. Thus, the available evidence presented
can vary widely among animals depending on age, genotype and above suggests that multiple mechanisms are involved in the
other unidentified factors. The indirect evidence, on the other determination of ovulation rate in sheep and these mechanisms
hand, is considerable. Both insulin and IGF-I can, in a dose- appear to be additive, at least in some models of extreme
dependent manner, alter the sensitivity of granulosa cells to FSH- prolificacy.
stimulated oestradiol production as can several other paracrine
and autocrine growth factors. Some of these factors (insulin,
IGF-I and TGF-b) reduce the dose of FSH required in vitro for (4) The link with nutrition
the maximum stimulation of oestradiol production. Others (EGF, What is the relationship between nutrition and the mechan-
FGF and TFGa) inhibit aromatase activity (Scaramuzzi and isms that determine ovulation rate? It is undisputed that nutrition
Campbell 1990) and thus increase the minimum concentration stimulates folliculogenesis in ruminants. Furthermore, it would
of FSH required to stimulate oestradiol production. be reasonable to assume that the mechanism of this effect will be
one of those proposed for the control of ovulation rate in general. A model for nutritional and metabolic influences
Available evidence suggests that nutrition (1) increases the on folliculogenesis
number of small and medium-sized gonadotrophin-responsive A mechanistic model for the influence of nutrition on folliculo-
follicles; and (2) reduces atresia among large gonadotrophin- genesis is shown in Fig. 6. This model attempts to integrate
dependent follicles without a change in FSH, suggesting that the known influences of nutrition and body condition with the
nutrition increases the number of ovulatory follicles by increas- metabolic effects on folliculogenesis that we consider to be the
ing the cohort size of gonadotrophin-responsive follicles more significant. The model is a conceptual framework that
(Fig. 5c). Reduced atresia amongst gonadotrophin-dependent attempts to make physiological sense of known facts and high-
follicles can be regarded as a direct consequence of a widened light critical areas where knowledge is deficient or contradictory,
gate (Fig. 5b) associated with a low FSH threshold rather than thus providing direction and focus for future research. It is
increased concentrations of circulating FSH. clear that body condition and nutrition can act throughout the
⫺ Central nervous system Short-term

Body weight GnRH, GHRH, somatostatin ... nutrition
Adiposity
⫹
⫺ Pituitary gland
GH FSH, LH Small intestine
Glucose
⫹ ⫹ ⫹
Negative
feedback ⫹
Liver
IGF-I
Adipose tissue Pancreas
Leptin ⫹
Insulin
LH, FSH
Function Proliferation Survival

⫹
IGF-I ⫹
⫹
⫹
– Insulin
⫹
⫹
Glucose
Leptin ⫹
⫺
Aromatase Cell cycle Anti-apoptosis
Negative Reduced Increased Increased Reduced

feedback oestradiol inhibin? folliculogenesis atresia
Fig. 6. A model for nutritional and metabolic inputs into folliculogenesis. Short-term nutritional influences are mediated primarily by glucose–insulin while
the effect of bodyweight or adiposity is mediated by leptin and IGF. Short-term nutritional treatment that leads to the increased post-ruminal absorption of
glucose stimulates folliculogenesis by direct intrafollicular effects of insulin or insulin-mediated glucose uptake; these inhibit oestradiol and probably stimulate
inhibin. The feedback consequences of altered secretion of follicular hormones are unclear but they appear to have little effect on FSH secretion. Thus,
increased folliculogenesis resulting from short-term nutrition may be the result of subtle changes in negative feedback or the result of direct follicular action of
insulin or glucose. The effect of insulin may also be mediated via leptin. The effect of short-term nutrition on the IGF system is uncertain (dashed line). High
bodyweight and adiposity lead to increased circulating IGF-I and leptin. The leptin inhibits oestradiol secretion and stimulates folliculogenesis but its effect on
the follicular inhibin system is not known. The effect of GH folliculogenesis is probably mediated by IGF-I. Known intra-ovarian interactions are shown as
solid lines while dashed lines indicate speculative interactions that have not been confirmed and may not exist. Some of the intracellular mediators for these
interactions have been elucidated and are discussed in the text.
hypothalamus–pituitary–ovarian axis to affect the follicle and genome for other ruminants) and proteomics specific to ruminants.
that the later stages of folliculogenesis have a primary require- Third, there is a need for improved in vitro systems for single
ment for LH and FSH (Webb and Campbell 2007). It is probable follicles and ovarian cells. Technical breakthroughs in these areas
that nutritional influences on folliculogenesis involve both direct will help us to overcome several conceptual barriers. Most
intra-follicular and negative feedback mechanisms (Scaramuzzi attractive is the ultimate goal of being able to track the dynamic,
et al. 2006), but with a primary effect at the level of the follicle functional history of a follicle in real time, either in vitro or in vivo.
that evokes alterations in negative feedback. Implicit in this is the ability to study the processes that determine
Three effects of nutrition on folliculogenesis have been the rate of recruitment of primordial follicles.
described. Increased bodyweight and higher body condition
scores are both associated with increased folliculogenesis. The Conclusions
rate of increase of bodyweight is also associated with increased We have only recently begun to appreciate how folliculogenesis
ovulation rate but this is a difficult experimental model to work is affected by the functional interactions between the oocyte and
with. At this point we assume without supporting data that granulosa and cumulus cells. We need to further develop this
its effect is also mediated by increased folliculogenesis; it is field of study so we can address questions such as how the
not included in the model. Short-term nutritional stimulation developing endocrine functions of the follicle are coordinated
that does not affect bodyweight or adiposity also stimulates among the oocyte, cumulus and granulosa cells as well as
folliculogenesis in sheep and cattle without significant changes between the granulosa and theca cells, why granulosa cells
in peripheral gonadotrophin concentrations. This plethora of undergo apoptosis preferentially and why oocytes are protected
experimental models has led to many superficially comparable during early atresia? Additionally we need to understand the
nutritional studies that are, in fact, difficult to compare because internal workings of the follicle to explain, for example, the high
the experimental manipulation can involve short-term supple- degree of redundancy, the local diffusion of regulatory factors,
ments, prolonged changes in body mass, static differences in the role of the extracellular matrix and the basal lamina in reg-
body mass and fasting. ulating diffusion (Irving-Rodgers and Rodgers 2006), the origin
There are some important omissions in the model; these are and role of the theca and its androgens and the role of TGF
the effect of nutrition on the oocyte and the effects of dietary signalling (BMP, AMH).
polyunsaturated fatty acids (PUFAs) on folliculogenesis. These
have been deliberately excluded either because of limited data or Acknowledgements
because of a need to impose limits on the extent of the review.
We wish to acknowledge the financial support of the UMR Physiologie de la
Importantly, the follicle is the vehicle for the oocyte. There is
Reproduction et des Comportements, L’Institut National de la Reproduc-
increasing but limited evidence that nutrition affects the devel- tion, Intervet Schering Plough Animal Health, Intervet Pharma R and D,
opmental potential of the oocyte and the subsequent survival of Beaucouzé, France and the European Union Marie Curie Program (MEXC-
the embryo and may be a significant factor affecting the success CT-2006–042499). R.J.S. is the recipient of an EU Marie Curie Chair of
of assisted reproduction. Thus, interesting questions arise: is the Excellence (MEXC-CT-2006–042499).
oocyte a direct target for nutrition and is the nutritional influence
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doi:10.1530/REP.1.00536 Manuscript received 11 July 2009, accepted 5 October 2010
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Review CSIRO PUBLISHING

Reproduction, Fertility and Development, 2011, 23, 444–467 www.publish.csiro.au/journals/rfd

Regulation of folliculogenesis and the determination

R. J. ScaramuzziA,B,M, D. T. BairdC, B. K. CampbellD, M.-A. DriancourtE,

Additional keywords: ewe, follicle, nutrition, oocyte, ovary.

Ó CSIRO 2011 10.1071/RD09161 1031-3613/11/030444

0.1 Emergence of follicular waves

Antrum formation and differentiation of cumulus cell phenotype

Differentiation of theca interna with LH receptors

Zona pellucida present

Granulosa cell FSH receptors and oocyte-granulosa cell gap-junctions present

Primordial Primary Preantral Small-medium antral Medium-large antral

Non-growing Committed Gonadotrophin- Gonadotrophin-

Non-ovulatory wave Ovulatory wave

Primordial follicles (Type 1 or 1a): Largest

Committed follicles (Types 2 and 3; primary and small

Gonadotrophin-responsive follicles (Types 4

Gonadotrophin-dependent follicles (Type 5+;

Ovulatory follicle(s): have LH

Mural Cumulus Oocyte

Models for the regulation of ovulation rate

⫺ Central nervous system Short-term

Function Proliferation Survival

Aromatase Cell cycle Anti-apoptosis

Negative Reduced Increased Increased Reduced

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