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Dr. Michelle Rhoads

Dr. Michelle Rhoads is an Assistant Professor in the Animal Sciences Department at the
University of Arizona. She was born and raised on a dairy farm in a rural community near
Jefferson City, Missouri. She completed her Ph.D. and B.S. at the University of Missouri
Columbia and earned her M.S. from Cornell University. Dr. Rhoads research encompasses
factors affecting reproductive physiology in dairy cattle with a particular interest in metabolic
and nutritional influences on fertility.

Reproduction and Hormones that Regulate Energy Balance

M.L. Rhoads, M.E. Field, S.E. Cossel, X. Li, T.R. Bilby, R.J. Collier, L.H. Baumgard and
R.P. Rhoads
Department of Animal Sciences
The University of Arizona
Corresponding Author: rhoadsm@email.arizona.edu

SUMMARY

Dairy cattle typically experience a period of negative energy balance during the early
postpartum period.
The circulating concentrations of several hormones and metabolites change during
periods of negative energy balance, including growth hormone, insulin-like growth
factor I and ghrelin (a growth hormone secretagogue).
Altered concentrations of these three hormones may serve as metabolic signals to the
reproductive tract of early lactation dairy cows, thereby decreasing fertility.

INTRODUCTION

It is typical for lactating dairy cows to experience a period of negative energy balance during the
early postpartum period. Depending on the severity and duration of this energy deficit, the
physiological events that precede a successful pregnancy are often disrupted. Therefore,
understanding the metabolic- and nutrient-mediated control of the reproductive system of dairy
cattle is a constant pursuit. Several hormones and metabolites have been investigated for
interactions with the reproductive system, including growth hormone (GH), insulin-like growth
factor I (IGF-I) and ghrelin (a potent GH secretagogue). And in spite of all that weve learned
about these hormones, it seems that our current knowledge of these metabolic signals to the
reproductive system is just the tip of a physiological iceberg.

NUTRITION AND METABOLISM DURING THE TRANSITION PERIOD

The transition period in dairy cattle is defined as three weeks before parturition to three weeks
after parturition (Ingvartsen and Andersen, 2000), and involves the adaptation of several
physiological systems. Some of the most profound changes observed during the transition
period occur in the digestive system. It is no surprise that dairy cattle typically reduce their dry
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matter intake (DMI) before parturition. The decline in DMI has traditionally been studied
during the last few weeks before calving, but DMI is actually depressed throughout the last
trimester of pregnancy in cows and heifers (Ingvartsen and Andersen, 2000). One of the most
popular explanations for the observed decrease in DMI is that the rumen is physically
compressed by the expanding uterus and the accumulation of abdominal fat, thereby limiting the
amount of feed the cow can consume. Research has shown that this relationship does exist in
both cattle and sheep (Ingvartsen and Andersen, 2000), but it is unlikely that it is the only factor
contributing to the decline in DMI during late pregnancy. If physical compression of the rumen
were the only factor limiting DMI at this time, relief of that compression (parturition) would
result in a rapid increase in feed intake. Instead, however, the increase in DMI following
parturition is relatively slow with maximum DMI generally achieved between eight and twenty-
two weeks postpartum (Ingvartsen and Andersen, 2000; Jorritsma et al., 2003). This increase in
DMI lags behind that of milk production (Lucy et al., 2001) suggesting that the control of DMI
involves a combination of factors, including (but not limited to) the metabolic and endocrine
changes that occur during late pregnancy, diet composition, parity, body condition score and
disease (Grummer, 1995; Ingvartsen and Andersen, 2000).

Periparturient changes in digestion and metabolism further complicate matters during this period
by altering nutrient availability. The onset of lactation is associated with hypertrophy of the
digestive tract and increased capacity for nutrient absorption, thereby increasing the utilization
of the nutrients contained in the feed (Bauman and Currie, 1980). Despite this increase in
absorption efficiency, the nutrient demands of early lactation exceed consumption, resulting in
the mobilization of body reserves of fat and protein in order to sustain milk production.

Lipid Metabolism

One of the key aspects of the metabolic shift that occurs during the transition period is a change
in lipid metabolism. Blood concentrations of nonesterified fatty acids (NEFA) are an indication
of lipid mobilization and are elevated during late pregnancy. The NEFA concentrations peak at
calving and decrease thereafter (Bell, 1995; Drackley, 1999; Grummer, 1995; Ingvartsen and
Andersen, 2000). Much of the circulating NEFA are absorbed by the liver, esterified and stored
as triglycerides. The release of triglycerides from the liver lags behind triglyceride synthesis
and accumulation. Therefore, excessive rates of lipid mobilization from adipose tissue results in
increased hepatic triglyceride accumulation, which can develop into hepatic lipidosis (fatty
liver). Excessive hepatic triglyceride accumulation is associated with an increased incidence of
ketosis, which may be a consequence of decreased hepatic glucose synthesis following
triglyceride accumulation (Drackley, 1999; Grummer, 1995).

Protein Metabolism

Inadequate protein intake associated with severely depressed DMI during the periparturient
period may predispose dairy cows to retained placenta and ketosis (Grummer, 1995). If protein
consumption during the postpartum period is insufficient to meet the demands of early lactation,
the dairy cow will mobilize protein from body reserves. Intrinsically, there is a transient
decrease in hepatic protein synthesis at calving. Fortunately, thereafter dairy cows have
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increased levels of hepatic protein synthesis (Grummer, 1995), thereby increasing the amount of
protein available for milk production.

GROWTH HORMONE

Growth hormone, which is synthesized in and released from the somatotroph cells of the
anterior pituitary gland, is involved in a variety of biological processes. These processes include
longitudinal bone growth, skeletal muscle growth, lipolysis, the inhibition of lipogenesis,
lactation and reproduction. Perhaps most significantly, GH stimulates liver IGF-I synthesis.

The synthesis and secretion of GH are under the control of GH releasing hormone (stimulatory)
and somatostatin (inhibitory) from the hypothalamus. Growth hormone is stored in secretory
granules within the somatotroph cells of the anterior pituitary gland and released into the
circulation upon stimulation. In many species, including cattle, some of the GH in the
circulatory system is bound to a GH binding protein (Davis et al., 1994; Devolder et al., 1993;
Massart et al.; 1996). This soluble binding protein consists of the extracellular domain of the
GH receptor (GHR), and is the result of limited proteolysis of the membrane-bound molecule.
The biological significance of the GH binding protein is not completely understood (Kopchick
and Andry, 2000). Despite being capable of competing with the GHR for GH binding, it is most
likely responsible for enhancing GH action by increasing its half-life (Ketelslegers et al., 1996).

Circulating concentrations of GH are elevated during the periparturient period (Bell, 1995) and
may play an important role in the metabolic transition that occurs with the initiation of lactation
(Bauman and Currie, 1980). Growth hormone increases the sensitivity and responsiveness of
adipose tissue to catecholamines (Bauman and Vernon, 1993) resulting in increased lypolysis
and subsequent increases in circulating NEFA concentrations. The additional NEFA can be
directly incorporated into milk fat or oxidized in the liver or extra-hepatic tissues (Lucy et al.,
2001). Growth hormone also diminishes the ability of insulin to stimulate lipogenesis (Bauman
and Vernon, 1993) and enhances lactose synthesis by stimulating hepatic gluconeogenesis (Lucy
et al., 2001). Thus, GH is critical to the physiological processes involved in the initiation of
lactation.

Growth Hormone Receptor

Membrane-bound GHR are located in tissues throughout the body, enabling GH to have a wide
array of physiological functions. In cattle, the gene encoding the GHR is located on
chromosome 20 (Moody et al., 1995). The mRNA consists of a 5 untranslated region, an open
reading frame transcribed from exons 2 to 10 and a 3 untranslated region. The second exon
encodes the signal peptide, exons 3 to 7 encode the majority of the extracellular domain, exon 8
encodes the transmembrane domain, and exons 9 and 10 encode the intracellular domain (Edens
and Talamantes, 1998).

In cattle, there are three promoters that drive the expression of the GHR. The resulting mRNA
variants have unique exon 1 sequences which are spliced onto exons 2-10 (Edens and
Talamantes, 1998; Lucy et al., 2001). Promoter P1 drives transcription of GHR 1A, which is
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found exclusively in the liver. Promoters P2 and P3 transcribe GHR 1B and 1C, respectively,
which are found in many tissues, including the liver (Jiang et al., 1999).

The GHR is a member of the class I cytokine receptor superfamily. The physiological actions
of GH are initiated as GH sequentially binds to two GHR molecules. The GH binds the first
receptor at a high affinity binding site and a second receptor at a lower affinity binding site.
Interactions between the receptors stabilize the dimer complex (Kopchick and Andry, 2000).
The GHR lacks intrinsic tyrosine kinase activity. Upon ligand binding, the cytoplasmic tyrosine
kinase, janus kinase 2 (JAK2), is activated by tyrosine phosphorylation. The activated JAK2
phosphorylates the intracellular domain of the GHR. The phosphorylated JAK2 and GHR
interact with various signaling molecules to activate three signaling pathways: the janus kinase -
signal transducers and activators of transcription (JAK-STAT), mitogen-activated protein kinase
(MAPK) and protein kinase C (PKC) signaling pathways (Kopchick and Andry, 2000; Le Roith
et al., 2001; Zhu et al., 2001).

Roles of Growth Hormone in Reproduction

The mRNA for the GHR is found throughout the female reproductive tract, indicating potential
roles for GH in the establishment and/or maintenance of pregnancy. Indeed, GH has been
implicated in a number of reproductive processes including the onset of puberty, follicular
development, oocyte maturation and ovulation (Chandrashekar et al., 2004).

Follicular development. The administration of exogenous GH increases the number of both
small (2 to 5 mm in diameter;(Gong, 2002) and medium-sized (6 to 9 mm in diameter; (Kirby et
al., 1997) ovarian follicles in cattle. However, it is difficult to distinguish the direct effects of
GH from other metabolic hormones since administration of GH also increases circulating
concentrations of IGF-I and insulin. As the follicle matures from the primary through the
tertiary stages, the greatest abundance of GHR mRNA and protein shifts from the oocyte to the
cells of the cumulus oophorus (Kolle et al., 1998). Evidence suggests that GH plays little more
than a supportive role during late follicular development.

Development of the corpus luteum. The large luteal cells of the corpus luteum (CL) contain the
greatest abundance of GHR mRNA relative to other female reproductive tissues (Kolle et al.,
1998; Lucy et al., 1993; Yuan and Lucy, 1996). Growth hormone receptor mRNA increases
during the early luteal period and reaches maximal concentration during the functional luteal
phase (Schams et al., 1999). During luteolysis, the expression of GHR mRNA steadily declines
(Neuvians et al., 2003).

The effects of GH on CL development and function are unclear. Again, it is difficult to separate
the direct effects of GH from those of IGF-I. The weight of the CL in hypophysectomized ewes
is restored to nearly normal levels when exogenous LH and GH are administered (Juengel et al.,
1997). However, cattle with high circulating concentrations of GH and low IGF-I (due to a
mutation in GH or undernutrition) have smaller CL than control animals (Chase et al., 1998;
McCormack et al., 2004; Vandehaar et al., 1995). Studies examining the effects of exogenous
GH on CL development and function in cattle have contradictory results, with some reporting
increased CL weight in response to GH administration (Lucy et al., 1995), and others finding no
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change in the size of the CL (Kirby et al., 1997). Effects of exogenous GH on plasma
progesterone concentrations are equally variable: at times increased (Schemm et al., 1990),
decreased (Kirby et al., 1997) or unchanged (Lucy et al., 1995).

The oviduct. There is little information available on the role(s) of GH within the oviduct. The
mRNA for the GHR is found in the bovine oviduct, albeit at low levels compared to other
female reproductive tissues (Kirby et al., 1996). Cultured bovine oviductal epithelial cells
respond to GH treatment by increasing IGF-I synthesis and secretion. In addition, PGF
2

production by the oviduct epithelium is inhibited by GH treatment (Makarevich and Sirotkin,
1997). The significance of these effects in terms of embryo development and survival are not
completely understood.

The uterus. The GHR mRNA is found in both the endometrium and myometrium of the bovine
uterus (Kirby et al., 1996). There is limited information on the direct effects of GH on the
bovine uterus. Administration of GH to pregnant ewes increased the weight of the uterus
(Jenkinson et al., 1999) and stimulated uterine gland proliferation (Spencer et al., 1999).
Similar effects in the bovine uterus may enhance embryo survival by increasing the amount of
tissue available to produce the uterine secretions necessary for pre-implantation embryo
development.

INSULIN-LIKE GROWTH FACTOR I

The IGF family contains two ligands: IGF-I and IGF-II. Despite their structural similarity, IGF-
I production and secretion is GH dependant whereas IGF-II is not. Insulin-like growth factor I
is a relatively small peptide, made up of 70 amino acids with a molecular weight of 7649 Da
(Laron, 2001). The IGF-I molecule consists of A and B chains as well as C and D domains, the
latter two of which are lacking in the mature insulin molecule (McCusker, 1998). The gene
encoding IGF-I contains six exons and is over 30 kb in length. The third and fourth exons
encode the mature protein (Ohlsen et al., 1993; Ohlsen et al., 1994; Rotwein et al., 1986;
Shimatsu and Rotwein, 1987). There are two promoters (promoter 1 and 2), which produce
transcripts containing either exon 1 or exon 2 (Phillips et al., 1998). Promoter 2 is
predominantly active in the liver, whereas promoter 1 is active in extrahepatic tissues
(O'Sullivan et al., 2002). In addition, multiple transcription start sites, alternative splicing of the
primary IGF-I gene transcript and use of numerous polyadenylation sites produce various
mRNA species (Jones and Clemmons, 1995).

Insulin-like growth factor I plays an important role in basic physiological processes, including
growth, development, immune function, reproduction and metabolism (Jones and Clemmons,
1995; Stewart and Rotwein, 1996). Most tissues and cell types are capable of producing IGF-I,
although the liver is the primary site of IGF-I production. Under normal physiological
conditions, GH stimulates hepatic IGF-I production. Physiological states that decrease GH
activity (such as undernutrition) result in depressed IGF-I concentrations. The IGF-I secretory
response is slow, taking several days to fully respond to elevated GH concentrations (McCusker,
1998).

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Plasma IGF-I concentrations reach a nadir during the first week of lactation (Block et al., 2001;
Radcliff et al., 2003; Reist et al., 2003), concurrent with a period of severe negative energy
balance (Block et al., 2001; Rhoads et al., 2004). At this time, plasma GH concentrations are
elevated and the ability of GH to increase plasma IGF-I is attenuated (Vicini et al., 1991) as a
result of decreased GHR concentrations in the liver. Specifically, the hepatic expression of the
GHR1A mRNA is severely depressed at parturition. Hepatic GHR1A expression does not begin
to rebound until the second week of lactation, and continues to increase into late lactation. The
depression in GHR1A expression around the time of calving limits the ability of circulating GH
to affect IGF-I synthesis and secretion (Kobayashi et al., 1999; Radcliff et al., 2003).

IGF Binding Proteins

Most IGF-I molecules within the circulation or in the extracellular space are bound to IGF
binding proteins (IGFBP). Six IGFBPs have been characterized (IGFBP-1 through IGFBP-6).
Under normal circumstances, more than 95% of circulating IGF is bound in a ternary complex
with IGFBP-3 and the acid labile subunit. Insulin-like growth factor can also form a ternary
complex with IGFBP-5 and the acid labile subunit (Firth and Baxter, 2002). Incorporation into
the ternary complex increases the half-lives of the IGFs from about 10 minutes (in their free
form) to approximately 12 hours. This is considerably longer than the half-lives of IGFs in
binary complexes with IGFBP-1 (30 minute half-life) or IGFBP-2 (2 hour half-life; (Jones and
Clemmons, 1995; McCusker, 1998).

In addition to prolonging the half-life of the IGFs in the circulation, IGFBPs also act as transport
proteins in plasma that control the efflux of IGFs from the vascular compartment, mediate the
interaction of the IGFs with their receptors, provide a means of storage within the extracellular
matrices and exert IGF-independent effects on target cells (Jones and Clemmons, 1995; Rajaram
et al., 1997; Ranke and Elmlinger, 1997).

The ternary complex containing IGFBP-3 and the acid labile subunit is approximately 150 kDa
in size. Because of their large size, IGFs bound in the ternary complex are unable to escape the
vascular compartment. However, research has shown that the lower molecular weight IGFBPs
are capable of crossing endothelial barriers. In fact, IGFBP-1 is thought to act as a shuttle
protein, assisting in the transport of the IGFs from the circulation to target tissues (Jones and
Clemmons, 1995; Ranke and Elmlinger, 1997).

Initially, it appeared that the IGFBPs inhibited the interactions of the IGFs with their receptors.
However, this is not always the case. In some instances IGFBPs actually enhance the action of
the IGFs. Insulin-like growth factor binding proteins inhibit IGF action by occupying the
location for receptor binding. Since there is no evidence that the IGFs are capable of binding to
two sites simultaneously, IGFs bound to IGFBPs are incapable of activating their receptor
(Jones and Clemmons, 1995; McCusker, 1998; Rajaram et al., 1997; Ranke and Elmlinger,
1997). Insulin-like growth factor binding proteins enhance the activity of the IGFs by
delivering them to the surface of target cells. This is accomplished when the IGFBP complex
binds to the target cell surface. As this occurs, the affinity of the IGFBP for the IGF drops. The
IGF is released from the IGFBP and becomes available to bind to its receptor, which is likely in
close proximity (Jones and Clemmons, 1995; McCusker, 1998).
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Insulin-like Growth Factor I Receptor

There are two receptors for the IGFs: the IGF-I receptor (type 1 IGF receptor), and the IGF-II
receptor (type 2 IGF receptor or mannose-6-phosphate receptor). The IGF-I receptor is
structurally similar to the insulin receptor, and is responsible for mediating the cellular effects of
both IGF-I and IGF-II. The IGF-II receptor is not associated with intracellular IGF signaling
mechanisms, and is mainly responsible for the clearance of IGF-II. Receptors on the cell
surface bind IGF-II with high affinity, and facilitate the internalization and degradation of the
ligand (Jones and Clemmons, 1995; Le Roith et al., 2001).

The IGF-I receptor is a heterotetramer, consisting of two extracellular subunits and two
transmembrane subunits. The and subunits are linked to each other with disulfide bonds
to form half of the receptor structure. Two half-receptors are then linked to each other with
disulfide bonds between the subunits to form the mature receptor (Jones and Clemmons,
1995; Laron, 2001). Upon activation of the IGF-I receptor, an IRS protein (IRS-1, -2, -3 or -4)
is phosphorylated on multiple tyrosines (Le Roith et al., 2001). At least two intracellular
signaling cascades are then activated: the phosphatidylinositol-3 (PI-3) kinase and MAPK
pathways.

Roles of Insulin-like Growth Factor I in Reproduction

Follicular development. Members of the IGF family are found in both the granulosa and theca
interna cells of the ovarian follicle. Studies agree that the expression of IGF-I mRNA in the
granulosa cells increases from early to mid-dominance (Schams et al., 2002; Yuan et al., 1998).
The IGF-I receptor is expressed at low levels in both the granulosa and theca interna cells of
small follicles (Armstrong et al., 2000; Schams et al., 2002) and increases in both cell types as
follicles mature (Schams et al., 2002).

The IGF system plays a number of important roles in the growth and development of ovarian
follicles. Perhaps the most significant of these is the synergistic interaction between IGF and
the gonadotropins. Together, the gonadotropins and the follicular IGF system amplify the
steroidogenic capacity of the ovarian follicle and hasten the maturation process. Ovarian IGF-I
is synthesized and secreted by the granulosa cells. The IGF-I binds to its local receptor and
increases the expression of the gonadotropin receptors as well as the activity of the second
messenger systems activated by the gonadotropins. In turn, the gonadotropins increase ovarian
IGF-I synthesis, as well as IGF-I receptor expression (Lucy, 2000).

Selection of the dominant follicle. The follicular IGF system also appears to play an important
role in dominant follicle selection. The concentration of IGF-I mRNA is higher in the dominant
follicle than in subordinate follicles. In contrast, subordinate follicles have higher
concentrations of IGFBP-2 than the dominant follicle, suggesting that much of the IGF-I present
in the subordinate follicles may not be available to activate the IGF-I receptor (Yuan et al.,
1998). Dominant follicles also have higher levels of proteolytic activity against IGFBP-4 and
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IGFBP-5 than subordinate follicles, resulting in increased concentrations of free intrafollicular

IGF-I and estradiol (Rivera and Fortune, 2003; Spicer, 2004).

The corpus luteum. The mRNAs for IGF-I and the IGF-I receptor are found in the bovine CL
(Perks et al., 1999). However, patterns of IGF-I and IGF-I receptor expression are not clearly
defined. Some have reported an increase in luteal IGF-I mRNA from day 5 to day 10 of the
estrous cycle and an even greater IGF-I mRNA concentration in the regressing CL (Woad et al.,
2000). Similar patterns of IGF-I mRNA expression have been reported in the ovine CL (Juengel
et al., 1997). Information regarding IGF-I receptor mRNA concentrations during the luteal
phase has been limited and contradictory (Neuvians et al., 2003). Despite conflicting results
regarding IGF-I and IGF-I receptor expression, there is general agreement that the luteal IGF
system is important for the maintenance, function and perhaps the regression of the CL.

The oviduct. All three regions of the bovine oviduct (infundibulum, ampulla and isthmus),
contain IGF-I and IGF-I receptor mRNA. Expression of IGF-I mRNA in the oviduct is higher
within the first few days following estrus than at later stages of the estrous cycle (Pushpakumara
et al., 2002). Interestingly, the time period during which oviductal IGF-I concentrations are
elevated corresponds to the point at which an embryo would be present in the oviduct. These
observations suggest that the IGF system within the oviduct has the potential to affect
embryonic development. In support of this hypothesis, treatment of bovine oviduct epithelial
cells with IGF-I attenuates the production of PGF
2
, which is known to be detrimental to
embryonic development (Makarevich and Sirotkin, 1997).

The uterus. The expression of IGF-I mRNA in the bovine uterus is at least partially localized to
a band of dense caruncular stroma underling the luminal epithelium (Wathes et al., 1998a), but
is also expressed in the intercaruncular endometrial tissue (Pershing et al., 2002). Secretion of
IGF-I into the uterine lumen peaks at estrus in response to high estradiol concentrations in both
the bovine and ovine species. Therefore, uterine IGF-I concentrations are maximal several days
before the embryo enters the uterus (Wathes et al., 1998a).

The receptor for IGF-I is mainly expressed in the epithelium of the endometrial glands, but has
also been identified in the caruncles and myometrium. Insulin-like growth factor I receptor
mRNA concentrations are highest during the mid-luteal phase in the bovine and do not appear to
be influenced by estradiol or progesterone concentrations (Wathes et al., 1998a).

THE NUTRITIONALLY OR METABOLICALLY ALTERED IGF-I SYSTEM:
EFFECTS ON REPRODUCTION

Reproductive Effects of the Insulin-like Growth Factor System in Cattle

Cattle with low circulating concentrations of IGF-I that occur during a period of negative energy
balance typically experience disrupted reproductive processes, including extended postpartum
intervals to first ovulation. This phenomenon is likely due to decreased follicular
responsiveness to LH, decreased GnRH secretion and decreased LH secretion. As a result,
estradiol production is suppressed to levels that are unable to stimulate the preovulatory GnRH
surge. Indeed, in dairy cattle, ovulation of the first postpartum dominant follicle is associated
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with higher IGF-I concentrations the first two weeks after calving. In fact, positive linear
relationships exist between plasma IGF-I concentrations and the initiation or resumption of
ovulation. (Beam and Butler, 1997; Diskin et al., 2003; Roberts et al., 1997; Zulu et al., 2002a;
Zulu et al., 2002b). Thus, negative energy balance inhibits the LH secretion necessary to
support follicle development in addition to decreasing the ability of the follicle to respond to
LH. Together, these mechanisms result in sustained and extended anestrus periods.

The Insulin-like Growth Factor System in the Early Embryo

The oocyte expresses mRNA for GH and the GHR, and the addition of GH to culture media
enhances oocyte competency, resulting in more oocytes developing to cleaved embryos and
blastocysts following fertilization (Izadyar et al., 1996). The blastocyst, itself, does not express
GH, however it does contain the GHR (Izadyar et al., 2000; Kolle et al., 2001). In vitro, GH
treatment increased the number of 8-cell or greater embryos, enhanced blastocyst formation and
increased blastocyst hatching (Izadyar et al., 2000). In cattle, the IGF-I receptor is expressed at
all developmental stages of the preimplantation embryo (Lonergan et al., 2000; Lonergan et al.,
2003; Wathes et al., 1998b; Yaseen et al., 2001), suggesting that the embryo is capable of
responding to IGF-I concentrations throughout the reproductive tract. Furthermore, the
embryonic expression of the IGF-I ligand is positively associated with the developmental
competence of mouse and bovine embryos (Kowalik et al., 1999; Lonergan et al., 2000).
Therefore, low IGF-I concentrations may limit embryonic development, ultimately resulting in
early embryonic loss.

GHRELIN

In 1999, Kojima and co-workers identified an endogenous ligand for the growth hormone
secretagogue receptor which they called ghrelin (the Proto-Indo-European root ghre meaning
grow'; Cruz et al., 2008). In cattle, ghrelin is 27 amino acids in length and the active form
contains an octanoyl group at the serine 3 residue (Dickin et al., 2004). In addition to being a
growth hormone secretagogue, ghrelin is known for being a potent regulator of metabolism and
appetite.

The Ghrelin Receptor

The G protein-coupled receptor known as the growth hormone secretagogue receptor (GHSR)
was initially cloned from porcine anterior pituitary tissue (Howard et al., 1996). The GHSR is a
seven-transmembrane domain G-protein-coupled (GPCR) and is fairly homologous to other
GPCRs (Kojima et al., 2004). The GHSR gene has two exons coding for the seven-
transmembrane domain protein, which is a 366 amino acid polypeptide with a molecular mass
of 41 kDa. The primary target cells for ghrelin are somatotrophs in the anterior pituitary
(Kojima et al., 2005). The mRNA for GHSR1a (the active form of the receptor) has also been
detected in other locations throughout the body including gastrointestinal, cardiac and
reproductive tissues (Giampiero et al., 2004).

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Ghrelin

Ghrelin is secreted in two forms: the active (acyl) and inactive (desacyl) forms, with activity
conferred by an n-octanoylation, at Ser3 (Dickin et al., 2004). The gene encoding ghrelin has
five exons including (1) a short non-coding segment, (2) two exons which code for ghrelin, and
(3) two exons with a proghrelin sequence. Mature ghrelin is the result of a splice at Arg28-
Ala29 (Pemberton et al., 2008). The vast majority of circulating ghrelin is the inactive form
(lacks the posttranslational modification) which does not have the GH releasing properties of its
acylated counterpart (Kojima et al., 2008).

Because of ghrelins similarity to a gut peptide (motilin), research to identify predominant sites
of synthesis began in gastrointestinal tissues. Early research reported that the novel protein was
produced by oxyntic glands and secreted into the circulation (not into gastrointestinal fluids;
Tomasetto et al., 2000). Ghrelin is primarily produced by X/A-like cells within the oxyntic
gland, which remained ill defined in purpose until the recent identification of ghrelin. These
cells are minimal in fetal oxyntic tissue and progressively increase in number with growth.
Ghrelin is also produced in cells found in the duodenum, jejunum, ileum, colon, kidney, and
pancreas (although the pancreatic cell type is not well defined; Kojima et al., 2005).

In addition to GH, ghrelin influences other regulators of metabolism and appetite. The
GHSR1a-containing neurons in the hypothalamic arcuate nucleus extend to communicate with
neuropeptide Y (NPY) and agouti-related protein (AgRP) neurons as a primary route of action
for ghrelin (Kojima et al., 2005; Korbonits et al., 2004). The brain may also be a source of
small amounts of ghrelin which act locally on receptors enclosed by the blood-brain barrier (van
der Lely et al., 2004). Furthermore, GHSR1a has been found on vagal afferent nerves (Sakata et
al., 2003), and disruption of the vagus nerve system inhibits ghrelin-mediated stimulation of
growth hormone release and feed intake (Date et al., 2002).

Ghrelin Secretion. Ghrelin secretion has been investigated on both chronic and acute bases. It
is collectively agreed upon that ghrelin is secreted in response to periods of fasting and low
body fat content. In turn, ghrelin secretion is inhibited by elevated body fat content and obesity
(Nonogaki et al., 2008). Studies in humans have reported that circulating ghrelin concentrations
are lower in obese patients and higher in anorexic or bulimic patients, relative to normal patients
(Shiiya et al., 2002; Tschp et al., 2001). Likewise, body fat mass is correlated with circulating
ghrelin concentrations, though it has proven difficult to separate the influence of energy status
versus that of ghrelin on intake (Tolle et al., 2004).

In cattle, transient increases in plasma ghrelin concentrations occur prior to feeding (Bradford
and Allen, 2008), and then decrease thereafter (Miura et al., 2004; Wertz-Lutz et al., 2006).
However, it appears that these acute changes in ghrelin concentrations are limited to specific
physiological states, as calves (as opposed to mature cows) and late lactation (as opposed to
early lactation) cows did not exhibit this secretion pattern (Miura et al., 2004; Wertz-Lutz et al.,
2006; Bradford and Allen, 2008).

Effects on growth hormone secretion. Receptors for ghrelin are located on somatotroph cells of
the anterior pituitary, allowing ghrelin to directly affect GH synthesis and secretion (Howard et
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al., 1996). In terms of synthesis, ghrelin stimulates Pit-1: a transcription factor that regulates the
expression of the GH gene (Diguez et al., 2004). Growth hormone secretion is achieved
following activation of the GHSR1a. Upon activation, the G
q/11
subunit dissociates from the
receptor resulting in the activation of phospholipase C (PLC) and subsequent hydrolyzation of
membrane bound PIP
2
into DAG and IP
3
. Inositol 1,4,5-trisphosphate is then responsible for
initiating calcium release from the endoplasmic reticulum. Diacylglycerol works via PKC to
allow an influx of calcium (Cooper et al., 2004; Cruz et al., 2008). Finally, GH is released from
somatotrophs into the blood via endocytosis in response to elevated intracellular calcium levels
(Anderson et al., 2004).

In cattle, ghrelin potentiates its trademark effect of stimulating GH secretion in cultured
adenohypophysial cells as well as when injected into the medial basal hypothalamus in vivo
(Hashizume et al., 2003; 2005). However, the contribution of ghrelin to the stimulation of GH
secretion varies in relation to physiological state (Itoh et al., 2005).

Effects of Ghrelin on Reproduction

The plasma ghrelin concentrations of early lactation, high producing dairy cows are elevated on
both a chronic and acute basis (relative to feeding; Roche et al., 2006; Bradford and Allen,
2008). In other words, the dairy cows that are exhibiting the poorest fertility typically have
higher circulating concentrations of ghrelin than their late-lactation counterparts. Yet little is
known about the effects that ghrelin might be exerting on bovine reproductive processes.
Considerable evidence exists for an influential role of ghrelin in sexual development,
maintenance of reproductive functionality and fetal development in other species. Thus, it is
likely that ghrelin is involved in conveying nutritional and metabolic signals to the reproductive
tract of dairy cattle as well.

Ghrelin and steroid hormones. In recent years, research has indicated that interactions may
exist between steroid hormone production and ghrelin. Although much of this research has been
conducted in humans, similar interactions in cattle could be extremely detrimental to fertility.
For example, ghrelin treatment suppresses luteinizing hormone pulses (which stimulate steroid
hormone production) in men (Kluge et al., 2007). Similar suppression of luteinizing hormone in
cows would potentially result in decreased estradiol secretion from the dominant follicle and
decreased progesterone secretion from the corpus luteum. In agreement with this hypothesis,
progesterone, testosterone and estradiol secretion from chicken ovarian follicular fragments are
inhibited by ghrelin (Sirotkin et al., 2008). Likewise, ghrelin inhibited progesterone secretion
from luteal cells cultured with human chorionic gonadotropin and ghrelin (Tropea et al., 2007).
The same studies also found that ghrelin decreases PGE
2
and increases PGF
2
release from luteal
cells, suggesting that ghrelin is capable of inhibiting luteal function (Tropea et al., 2007).

Ghrelin and embryonic development. Ghrelin and the GHSR have been detected in a number of
reproductive tissues in humans, rats, mice and sheep. Our laboratory has detected both ghrelin
and GHSR-1a mRNA and protein in the ovaries, oviducts, uterus, oocytes and early embryos of
Holstein heifers (Rhoads et al., 2007). The presence of ghrelin and GHSR-1a in the bovine
reproductive tract is significant in light of the observed increase in plasma ghrelin
concentrations during early lactation. Rodent studies have shown that when ghrelin increased in
134

the circulation, it was also elevated in the secretions of the reproductive tract (perhaps to an
even greater extent than in plasma; (Kawamura et al., 2003). During culture of murine embryos,
ghrelin significantly decreased embryonic development and viability. Furthermore, rodents with
elevated circulating concentrations of ghrelin (either by exogenous ghrelin injection or
underfeeding) experienced decreased embryonic development and increased embryonic loss
(Barreiro and Tena-Sempere, 2004). Taken together, this evidence suggests that as circulating
ghrelin concentrations increase during early lactation in dairy cattle, ghrelin concentrations in
the reproductive tract are also likely increasing and directly compromising embryonic
development and survival. Thus, increased ghrelin concentrations within the reproductive tract
during periods of negative energy balance may be a metabolic mediator of reproductive
performance in dairy cattle.

CONCLUSIONS

Clearly, periparturient dairy cattle experience significant metabolic stress as they transition into
lactation. The hormones involved in this transition (and the associated state of negative energy
balance) are the very hormones that may be conveying metabolic signals to the reproductive
system. Characteristic alterations in GH, IGF-I and ghrelin may be responsible for the observed
decrease in fertility in lactating dairy cattle. Gaining a more complete understanding of their
actions and interactions within the reproductive tract is critical to our efforts to increase the
reproductive efficiency of dairy cattle.

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