04_[Zhihua_Jiang,_Troy_L._Ott]_Reproductive_Genomics_479_2010

382 Genomics and Reproductive Biotechnology

in Smith 2007). Indeed two genes appear to gene of the platyfish Xiphophorus macula-
be strictly specific to the W-chromosome tus has been narrowed to a short male-
(FET1 and 2d-2D9) but different evidence, specific region on the Y chromosome.
such as their expression profiles, argue Interestingly, this region encompasses no
against the role of these genes as being DMRT1-related gene and is evolutionarily
ovarian-determining factors (Smith 2007, different from the sex-determining region of
and references therein). Presently, the the medaka, and from mammalian and avian
best female-determining candidate gene in sex chromosomes (reviewed in Schultheis
chicken remains HINTW (also called ASW et al. 2006 and in Volff et al. 2007).
or WPKCI). This gene is located on the W
chromosome and has a homolog HINTZ on Even in the closely related group of
the Z chromosome (Hori et al. 2000). HINT mammals, some species-specific differences
genes encode a family of nucleotide hydrox- exist, not in the sex-determining mecha-
ylase enzymes that need a histidine triad nism itself, but in some aspects of gonad
(HIT motif) to be functional. Interestingly, differentiation (e.g., estrogen production in
HINTZ has a HIT motif but HINTW does bovidae compared with that in pig or
not. Moreover, as these HINT genes act as rodents). Consequently, intending to control
dimers, it has been postulated that HINTW sex in a specific species requires a good
may act as a dominant negative in avian knowledge of the molecular mechanisms
sex determination by forming heterodimers involved in gonadal differentiation in the
with HINTZ (Hori et al. 2000; Moriyama species of interest.
et al. 2006).
Presently in domestic mammals, produc-
In conclusion on sex determination in tion of only male progeny will be possible
chicken, the actual view supposes that simply by adding an SRY transgene on the
DMRT1 acts as a testis-determining gene, X chromosome of an XY animal. This
having a dose-dependent effect because it is could be interesting in some breed-specific
located on the Z chromosome, being inhib- bovine lines for beef production. Such XY
ited in female by the presence of a single founder animals will, however, present some
locus together with a W-located female disadvantages: (1) 50% of the offspring will
determinant that could be HINTW or be sterile, being XX-SRY+ males; (2) such
another yet uncharacterized W-linked gene. an XY founder cannot reproduce naturally
(only via the animal cloning technology),
16.5 Future research directions and (3) this founder and 50% of his progeny
will fall into the highly controversial
Although sex is used as the mode of repro- Genetically Modified Organism (GMO)
duction in vertebrates, sex-determining category.
signals appear highly variable among the
different phyla. Actually, two genes have Consequently, today, the best way to
been equated as sex-determining genes, SRY control sex in a given species will be to create
in mammals and DMRT1bY in medaka. a heterogametic founder carrying an induc-
Fishes represent an inexhaustible resource ible killer transgene on one of his sexual
of mechanisms involved in sex determina- chromosomes. The idea is to specifically
tion. As an example, the sex-determining eliminate spermatozoa of a given sex consti-
tution (X or Y population depending on which
sex chromosome the transgene is located) by
activating the transgene in collected semen

Sex Determination in Domestic Animals 383

samples. Such a founder animal will cir- the gene for hornlessness. Science 99:
cumvent all three disadvantages linked to 124.
an X(SRY+)Y founder and will allow also Bagheri-Fam, S., Sim, H., Bernard, P.,
the production of only-male or only-female Jayakody, I., Taketo, M., Scherer, G., and
fertile and non-GMO progeny. Such strategy Harley, V. 2008. Loss of Fgfr2 leads to
could be an alternative to sperm separation partial XY sex reversal. Developmental
on a cell sorter that is used today in different Biology 314: 71–83.
species and that will be surely extended in Baroiller, J.-F., Guiguen, Y., and Fostier,
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17

Toxicogenomics of Reproductive Endocrine
Disruption

Ulf Magnusson and Lennart Dencker

17.1 Introduction picture more complex. Another challenging
aspect of the understanding of the endocrine
In the past few decades public awareness and disruption is that several of the chemicals of
scientific knowledge on how xenobiotics concern show non-monotic dose–response
exert hormone-like activity in humans and curves in experimental settings. Furthermore,
animals has expanded rapidly. The driving in real life humans or animals are rarely
force behind this expansion has been the exposed to one chemical at a time, but,
public and scientific concern that this phe- rather, to a mixture of chemicals that may
nomenon—called endocrine disruption—is act antagonistically, additively, or synergis-
a severe threat to human and wildlife tically. Moreover, the endocrine-disrupting
health. Attention has been paid to environ- chemicals generally act at relatively low
mental contaminants such as industrial and concentrations, which do not cause any
consumer chemicals as well as pesticides. overt effects on the individual at the time
However, this hormone-like activity may of exposure. Usually the disruption affects
also be exerted by natural compounds such the individual at certain vulnerable periods
as plant phytoestrogens. Overall, most sci- during development, and the effects become
entific reports within this field deal with the obvious first at adulthood. This also contrib-
disruption of the reproductive endocrine utes to making the study of endocrine dis-
system. ruption very complex.

The mechanism of action for these endo- Hence, there is a need for powerful
crine-disrupting chemicals was initially and precise research tools to dissect and
regarded to be restricted to activation via understand the world of endocrine disrup-
certain hormone receptors. However, it has tion. The disciplines of toxicogenomics and
now become increasingly clear that there are ecotoxicogenomics—that is, toxicology at
other mechanisms of action that make the the molecular level—may, by their precise

397

398 Genomics and Reproductive Biotechnology

readouts and generality across species, yield between cause and effects. Sometimes the
valuable contributions. causality has been confirmed by experi-
ments on wildlife in captivity under con-
17.2 Reproductive endocrine trolled conditions. In large part these
disruption observations are from species living in a
highly contaminated aquatic environment
17.2.1 The concept or from species high up in the food chain.
One classic example from the former cate-
Endocrine disruption, especially of the repro- gory is how the biocide tributylin causes
ductive endocrine system, is recognized as imposex in female prosobranch gastropods.
an important environmental concern. Public Imposex is an imposition of male reproduc-
research funding directed to the field has tive organs onto female snails that can
been substantial; the involvement of non- impair their reproductive ability. Imposex
governmental organizations and industries has been documented in some 150 species of
has been considerable; and the scientific these marine snails worldwide (Horiguchi
society has, in the last decade, generated 2006). The use of tributylin in anti-fouling
much new knowledge related to this paints of ships therefore became restricted
phenomenon. in many countries in the 1990s. This restric-
tion has been followed by the recovery of
The concept and concern of endocrine dis- several of the gastropod populations. Another
ruption emerged in the early 1990s (Colborn well-recognized association between endo-
and Clement 1992), and data from observa- crine-disrupting chemicals and reproductive
tions in wildlife, humans, laboratory, and disorders in wildlife is that between the
domestic animals have increased ever since. industrial chemical polychlorinated biphe-
There are some slightly different opinions on nyl (PCB) and uterine occlusion in seals in
how to define an endocrine-disrupting chem- the Baltic Sea (Helle et al. 1976). However,
ical. One of the most widely agreed upon is in this case the mechanism of action is more
that put forward by the International Program poorly understood than imposex in marine
on Chemical Safety in 2002: “An endocrine snails.
disruptor is an exogenous substance or
mixture that alters function(s) of the endo- 17.2.3 Indications in humans
crine system and consequently causes
adverse health effects in an intact organism, As for wildlife, the data on endocrine disrup-
or its progeny, or (sub)populations” (Damstra tion in humans result from studies where
et al 1992). Notably, in domestic animals the association between exposure to a certain
endocrine disruption of the reproductive chemical and effect is often rather weak.
system has long been recognized in the form However, the magnitude of the problem of
of phytoestrogens showing adverse effects on estrogenic effects was greatly spurred by an
grazing ruminants’ fertility. early disaster caused by administration of
the synthetic estrogen diethylstilbestrol
17.2.2 Observations in wildlife (previously an important drug in veterinary
medicine) during human pregnancy; this led
The data from wildlife are most often field to major malformations and dysfunction of
data with weak to moderate associations the reproductive organs in the offspring (for

Reproductive Endocrine Disruption 399

review, see Giusti et al. 1995). Lately, a par- to the male than to the female reproductive
ticularly interesting epidemiological study system. However, the concept of reproduc-
is one in which the exposure of the mother tive endocrine disruption has now been
to plastic softeners during pregnancy was expanded to include compounds with andro-
inversely related to the anogenital distance genic and anti-androgenic effects. The dis-
in newborn boys (Swan et al. 2005). A short- ruptive effects have been observed on the
ened anogenital distance in male laboratory morphology and function of the reproduc-
rodents is regarded as a sign of feminization tive systems as well as on the reproductive
achieved during development. The most behavior (for reviews, see Gray et al. 2004;
convincing data on endocrine disruption Stokes 2004). Interestingly, several of the
in humans, though, are from the field of experimental exposure–effect studies in lab-
occupational medicine or from cases of acci- oratory rodents do back up the associations
dental exposure such as the case of the between exposure and effects seen in the
diethylstilbestrol mentioned above. Other field and epidemiological studies. For
such examples are mothers who had PCB- instance, the so-called testicular dysgenesis
contaminated rice oil during pregnancy and syndrome in men, including cryptorchidism,
who gave birth to boys who in adulthood hypospadiasis, testicular cancer, and poor
displayed abnormal sperm morphology and semen quality, can be mimicked in rats by
motility (Guo et al. 2000) and male workers fetal exposure to plastic softeners (Hutchison
in a chemical industry that manufactured a et al. 2008).
stilbene derivate who had lower serum tes-
tosterone levels and suffered from decreased 17.2.5 Data from domestic animals
libido and impotence (Grajewski et al. 1996;
Whelan et al. 1996) The data on endocrine disruption in domes-
tic animals are sparse compared with those
17.2.4 Experimental evidence from from wildlife, humans, and laboratory
laboratory animals animals, and are mainly of two types: case
reports and controlled studies where farm
In laboratory animals there are solid experi- animals have been used as experimental
mental data showing endocrine-disrupting animals.
features in several groups of chemicals.
Mainly in these experimental settings the As mentioned above, reproductive endo-
exposure is at a relatively low dose and does crine disruption has a long history in farm
not cause any acute or general signs of intox- animals. The so-called sweet clover disease
ication. Typically the exposure has to occur is one of the earliest reported cases of endo-
during the pre- or early postnatal period in crine disruption affecting mammals. The
order to cause the most dramatic effects. disease is caused by the phytoestrogens
Effects have been observed in both female genestein and formononetin present in high
and male animals, although the majority concentrations in clover (Cox 1978). Typical
of studies concern the male reproductive effects observed in sheep grazing such clover
system. This is likely due to the fact that are prolapse of the uterus and reduced fertil-
several of the first reported endocrine- ity attributable to embryonic death. Another
disrupting chemicals were estrogenic and well-known case of reproductive endocrine
were therefore expected to cause more harm disruption in livestock is when pigs are
affected by the phytoestrogen zearalenon

400 Genomics and Reproductive Biotechnology

(reviewed by Diekman and Green 1992). risk assessment of chemicals for human and
This phytoestrogen is an acid lactone com- environmental health. A recent and remark-
pound produced by the fungi Fusarium. able finding that underpins the importance
Prepubertal gilts seem particularly sensitive of confirmatory studies is that transgenera-
and may show severe clinical signs of hyper- tional fertility problems caused by the anti-
estrogenecity such as vaginal or rectal androgenic compound vinclozolin after
prolapses. Sexually mature sows that have intraperitoneal injections to pregnant rats
eaten feedstuff contaminated with the (Anway et al. 2005) could not be repeated by
fungus have shown serious reproductive dis- the same substance given orally to pregnant
orders such as abortion, fetal mummifica- rats (although a different strain; Schneider
tion, stillbirths, and abnormal return to et al. 2008).
estrus intervals.
The second rationale is that farm animals
Besides these observations related to phy- can in fact be better models for human than
toestrogens there are very few reports on laboratory species when it comes to physi-
accidental exposure of farm animals to envi- ological aspects relevant for studies of
ronmental chemicals associated with dis- endocrine disruption. For instance, many
ruption of the reproductive endocrinology. endocrine-disrupting chemicals reach the
One possible example of such exposure and endocrine system through the oral route.
effect association is the case of dairy heifers Because the pig, for example, is an omnivo-
that were drinking surface water in direct rous and intermittent eater like humans, the
contact with sewerage overflow and that porcine digestive system shows many simi-
showed an increased age at first calving larities to the human digestive system
(Meijer et al. 1999). However, neither an (Moughan et al. 1994). It is therefore likely
analysis of chemicals nor any endocrinologi- that pigs have an advantage over laboratory
cal measurements were reported in that species as a model for human oral exposure
study. to endocrine-disrupting chemicals. The
kinetics of an endocrine-disrupting plastic
In addition to these observations from the softener following oral exposure in pigs is
field there is a set of controlled experimental indeed more similar to that in primates than
studies where endocrine disruption has been to that in rats (Ljungvall et al. 2004). In addi-
investigated in various species of farm tion, farm animals do have longer embryo-
animals. There are at least three rationales fetal and prepubertal periods compared with
for using farm animals as experimental laboratory species. Thus, they are, in this
animals (for review, see Magnusson 2005). sense, more similar to humans. Since expo-
sure to endocrine-disrupting chemicals is
The first rationale is that by using farm typically long-term and individuals are in
animals one tests or challenges the general- general particularly sensitive to exposure
ity of data generated in the classical labora- during these two periods, farm animals also
tory mammalians (mainly rabbits, mice, and have an advantage as a model for humans in
rats). The international testing strategy for this respect.
chemicals is much regulated by use of the
latter species. Confirmatory study in domes- The third rationale is that there might be
tic animals is, however, highly relevant, methodological advantages to use farm
given the diversity of the reproductive and animals as model species. Obviously, larger
metabolic systems throughout the animal samples can be collected from farm animals
kingdom, and may thus contribute to the

Reproductive Endocrine Disruption 401

Table 17.1 Examples of studies where farm animals have been used as models for studying reproductive
endocrine disruption.

Species Exposure Effect Reference

Goat Gestational and lactational; Reduced testosterone concentration and testis size Oskam et al. 2005
Goat PCB 153 and increased proportion damaged sperms Lyche et al. 2004
Pig Lowered prepubertal luteinizing hormone Ljungvall et al. 2005
Pig Gestational and lactational; concentration and delayed puberty in female goats Ljungvall et al. 2008
Sheep PCB 153 Increased testosterone concentration and Leydig cell Evans et al. 2004
Sheep number at adulthood Sweeney et al. 2000
Prepubertal; Precocious development of bulbourethral glands
Di(2-ethylhexyl)phthalate
Suppressed luteinizing hormone pulse frequency
Prepubertal; Decreased testis size and Sertoli cell number at birth
Di(2-ethylhexyl)phthalate

Prepubertal; Bisphenol A

Gestational; Octylphenol

than from smaller laboratory species, both tive endocrine disruption in farm animals,
in vivo and from euthanized animals. Also, for instance on bovine oocyte maturation
repeated sampling can more easily be per- and subsequent development (Pocar et al.
formed in farm animals. This holds true for 2001). Collectively the in vivo data in Table
instance for semen collection as well as 17.1 show that studies in farm animals may
blood sampling; the latter can, in cattle and contribute novel and sometimes opposing
pigs, be performed through venous catheters data compared with those generated in labo-
(Basu and Kindahl 1987; Rojkittikhun ratory species.
et al. 1991). Finally, since there are indica-
tions that sexually dimorphic behavior is 17.3 Reproductive endocrine
sensitive to endocrine-disrupting chemicals disruptors
(Palanza et al. 2002), the reproductive behav-
ior is of interest for studies in this context. 17.3.1 Chemicals of concern
The mating behavior of farm animals is well
described and is in some species relatively As indicated above there is a wide range of
long and complex compared with laboratory chemicals that may act as endocrine disrup-
species and is thus very suitable for study in tors. The majority of these are man-made,
the context of endocrine disruption, for but there are some disruptors that are natu-
instance, in the pig (Ljungvall et al. 2006 ) rally occurring in the ecosystem, such as the
or the quail (Brunström et al. 2003). previously discussed phytoestrogens. In the
so-called developed world, environmental
So which species of farm animals have pollution caused by chemicals hazardous to
been used in experimental studies? What humans and the environment has changed
kinds of compounds have been investigated over the last decades; thanks to cleaner
and which effects have been observed? In industrial procedures, pollution from indus-
Table 17.1 samples of studies on endocrine tries is very much reduced. However, there
disruption in vivo in farm animals is pre- is a more recently discovered type of
sented. It should be noted that there are also
some interesting in vitro data on reproduc-

402 Genomics and Reproductive Biotechnology

pollution, and that is the diffuse and con- the risk assessment in real life more complex.
stant exposure to consumer chemicals that To establish a solid causality between expo-
are abundant in our environment. This is sure to a chemical and effects on the envi-
called background exposure. Despite this ronment in the real-life situation is difficult.
change in the major source of pollution, Causality has instead been confirmed in
several of the hazardous industrial chemi- laboratory studies when associations
cals that have been banned for years are still between chemical exposure and effect on
an environmental concern due to their con- the reproductive system have been sus-
tinuous presence in the environment. In pected in the environment. In Table 17.2
addition, an emerging pollution concern some examples of associations between
that is particularly interesting in the context exposure and effect in the real-life situation
of endocrine disruption is pharmaceuticals. are presented. Very likely such a table may
Created to be biologically potent, when used be expanded over the coming years due to
in the wrong context they are potentially the intensive research within this field.
harmful to wildlife as well as to humans.
17.3.2 Vulnerable windows and
Another challenge when trying to esti- late effects
mate risks with individual, potential endo-
crine disruptors in the environment is that The effects of endocrine-disrupting com-
they are mostly present with other chemi- pounds differ in a general pattern depending
cals (Kolpin et al 2002). The vast majority of on when the organism or individual is
toxicological studies on endocrine disrup- exposed. Exposure during development
tion have been on single chemicals. However, generally causes irreversible organizational
it is well established that several of the effects on organs or organ systems, whereas
chemicals of concern may act as additives or exposure of the adult generally causes
even synergists on the endocrine system activational effects that are reversible
(Halldin et al. 2005). Obviously this makes

Table 17.2 Examples of associations between chemicals in the environment and effects on the reproduc-
tive system in humans or animals.

Chemicals Species affected Reproductive effect Reference

Industrial chemicals and pesticides

PCB, DDE White-tailed sea eagle Reduced reproductive success Helander et al. 1982
Reduced phallus size Guillette et al.1999
American alligator Swan et al. 2005
Decreased anogenital distance in
Consumer’s chemicals Human newborn boys Orlando et al. 2004.
Phthalates Vajda et al. 2008
Lower testicular testosterone
Pharmaceuticals Fathead minnow synthesis and smaller testis size Cox 1978
Trenbolone White sucker Female-biased sex ratio and
increased intersex
Ethinyl estradiol
Uterine prolapse, embryonic death
Phytoestrogens
Genestein and formononetin Sheep

Reproductive Endocrine Disruption 403

(McLachlan 2001). Most of the attention larger in exposed pigs compared with con-
to endocrine disruption has been given to trols. Interestingly, this effect was not seen
the irreversible effects during development, in littermates given the same exposure in
since a lower dose of exposure is usually parallel when examined directly after expo-
needed to produce an effect during this life sure (Ljungvall et al. 2005, 2008).
stage compared with adulthood. In other
words, the developing organism is in general While biologically intriguing, vulnerable
more sensitive to endocrine disruption than windows and late effects present trouble-
the adult. Notably, also during development some challenges both for regulatory toxicol-
there is a variability in sensitivity; the so- ogy when assessing the health risk for
called windows of vulnerability may vary by chemicals and for environmental monitor-
endocrine-disrupting compound, species, ing and tracking of effects of chemical expo-
and organ or physiological system affected. sure in the ecosystem.
In one elegant study showing the concept of
windows of vulnerability, male rabbits were 17.3.3 Different mechanisms of action
exposed to the plasticizer dibuthyl phthalate
in utero during adolescence or post puberty The chemicals that exert endocrine dis-
(Higuchi et al. 2003). The most dramatic ruption are structurally very diverse with
effects were seen in the group exposed in different physiochemical properties; one
utero with decreased number of sperm ejac- may therefore assume that they use various
ulated and reduced testis as adults. These mechanisms of action for their disrupting
windows may be very narrow, a matter of effects. Classically endocrine-disrupting
days, as shown by studies on the effect of chemicals have been regarded, or defined, as
estrogens on leopard frogs (Hogan et al. chemicals that modulate the endocrine
2008). system by binding to hormone receptors and
having an agonistic or antagonistic effect.
Another timing aspect of endocrine dis- Such a receptor-mediated mechanism has
ruption, particularly relevant to disruption been elegantly shown for the drug diethyl-
of the reproductive system, are the so-called stilbestrol by using estrogen receptor knock-
late effects. This means that there is a long out mice (Henley and Korach 2006). The
time gap between exposure, typically during downstream effect during the development
development, and overt effects on the repro- of the fetal mice by the diethylstilbestrol
ductive system, typically at adulthood when disruption seems then to be a decrease in
the individual is beginning to be sexually Hox and Wnt gene expression, critical for
functional. This is of course obvious for the development of the female genital tract.
several reproductive endpoints that are very
difficult to measure before puberty, such as Besides this orthodox receptor-mediated
number of sperm in an ejaculate or number endocrine disruption it has become increas-
of eggs at ovulation. However, in our own ingly clear that there are additional mecha-
split-litter designed studies in pigs that were nisms of action for endocrine-disrupting
exposed to a plastic softener for some weeks chemicals, such as hormone synthesis and
soon after birth, we showed that at adult- clearance. One such proposed mechanism of
hood, that is, 5–6 months after the end of particular interest for reproductive steroid
exposure, the plasma testosterone level was hormone metabolism is the increased aro-
elevated and bulbouthretral gland size was matase activity reported for atrazine in frogs
that increases the conversion of androgens

404 Genomics and Reproductive Biotechnology

to estrogens (Hayes et al. 2003). Another powerful research tools such as those of
example of modulating hormone metabo- toxicogenomics (and other “-omics”) in
lism is that exerted by methoxychlor, which molecular biology.
activates two nuclear receptors: the human
steroid and xenobiotic receptor/rodent pre- 17.4 Toxicogenomics
ganane X receptor and the constitutive
androstane receptor (reviewed by Tabb and 17.4.1 Complexity of
Blumberg 2006). These receptors are highly endocrine disruption
expressed in the liver and mediate the induc-
tion of cytochrome P450 and conjugation Endocrine-disrupting chemicals tradition-
enzymes, whereby they may severely affect ally consider those that mimic or block
steroid hormone metabolism. transcriptional activation by endogenous
hormones, and are not restricted to hor-
Yet another non-receptor-mediated mech- monal systems related to reproduction but
anism is modulation of the proteosome- include, for example, thyroid hormones.
mediated degradation of steroid receptors Working with endogenous substances or
(Wijayaratne and McDonnell. 2001). For their synthetic analogs acting on receptors
instance, it has been reported that Bisphenol directly involved in transcriptional activa-
A slows the degradation of the estrogen tion provides a “pure” study object when it
receptor alpha (Masuyama and Hiramatsu comes to various aspects on toxicogenom-
2004). In addition, it has been put forward ics. If then cells are also studied in vitro,
that endocrine-disrupting chemicals exert perhaps transfected by reporter genes directly
their effects by altering the levels of nuclear coupled to the receptor complex, one gets a
receptor coactivators (reviewed by Tabb and rather straightforward biological “answer.”
Blumberg 2006).
When investigating the effects of, for
Possibly the most intriguing, and alarm- example, environmental chemicals with sig-
ing, effects or mechanisms of action by nificant but much lower affinity for a recep-
endocrine-disrupting chemicals are the tor under study—as compared with the
transgenerational effects on the reproduc- endogenous ligand—one can demonstrate in
tive system. The mechanisms for these vitro or in vivo if that affinity also translates
effects are reported to be epigenetic, involv- into a biological effect. However, that par-
ing altered DNA methylation (reviewed by ticular chemical has to be administered at
Anway and Skinner 2006). This is an emerg- higher concentrations, or dose to an animal,
ing field for environmental research and making effects on other cellular systems
regulatory bodies. likely to occur. If we consider the fate and
biological activity of chemicals, be it recep-
Finally, regarding the diversity of mecha- tor ligands or not, in an organism (experi-
nisms of action for endocrine-disrupting mental or domestic animals, etc.) the picture
chemicals: the very same chemical can show rapidly becomes more complicated. It is
different effects in different species due to then reasonable to extend the definition of
downstream dissimilarities even though the endocrine disruptors from receptor active
initial interaction is the same (Tabb et al. substances only to include those exogenous,
2004). For the same reason a chemical may environmental molecules that affect, for
exert different effects in different tissues
within the same individual (Lonard et al.
2004). This complexity calls for precise and

Reproductive Endocrine Disruption 405

example, the synthesis, secretion, transport, information from transcriptomics experi-
metabolism, protein binding, and catabo- ments than from any of the other -omics
lism of natural hormones in the body. On a techniques in toxicology (for review, see,
cellular level, substances not only may act e.g., Gant 2007; Gomase and Tagore 2008).
through receptors but may also interfere in
various ways in the complex transcriptional Changes in gene expression as measured
regulatory machinery, including histone by mRNA levels using polymerase chain
deacetylase (HDAC) inhibition and protea- reaction (PCR) as well as globally by using
somal degradation of receptor complexes microarrays must, in order to give meaning-
(reviewed by Tabb and Blumberg 2006). ful information, somehow be related to mor-
Lately, the term “epigenetics” has come phological and/or physiological changes in
into fashion, being defined in this context as the cell/tissue. This is called “phenotypic
describing changes in gene expression that anchoring.” Typically, and of course based
are more or less stable even between genera- on temporal sequences, up- or downregula-
tions, without causing changes in the DNA tion of mRNA appears before observable
sequence (reviewed by Szyf 2007). Epigenetics cellular effects. This is important to con-
is discussed further below. sider in planning experiments. To get
information as close as possible to the
It has been shown that the potent environ- “source” of the change, that is, to more
mental compound 2,3,7,8-tetrachlorodibenso- reliably obtain mechanistic information,
p-dioxin (TCDD) is an endocrine disruptor, measurement of global mRNA should be
although the mechanism of action has been performed within hours after exposure.
obscure. Recently, it was found that the aryl Measuring later may of course give valuable
hydrocarbon receptor nuclear translocator information, but the information achieved
protein (ARNT), which is a necessary partner will be different, because one adds on sec-
for the TCDD or aryl hydrocarbon receptor ondary, tertiary, etc. inductions/repressions,
(AhR), acts as a coactivator for estrogen while initial responses may be attenuated.
receptors. Reducing the levels of available
ARNT by activating the AhR- or HIF Interesting results have come from studies
(hypoxia-inducible factor)-pathways, or by on temporal gene expression changes in the
targeted downregulation of ARNT by siRNA, endometrium after estrogen exposure in
decreased estrogen receptor (ER) transcrip- rodents and women (reviewed by Groothuis
tional activity, suggesting that competition et al. 2007). The endometrium may of course
for ARNT may be at least partly responsible be an interesting tissue to study in domestic
for the antiestrogenic effects of dioxins animals as well, considering its vital role
(Rüegg et al. 2007). in reproduction and perhaps as a sensitive
target organ to endocrine disruptors.
17.4.2 Global gene expression analysis
and phenotypic anchoring The following are some important general
considerations as derived from the review by
Toxicogenomics can be defined as an inte- Groothuis and collaborators.
gration of toxicology with genomics, which
in turn can be transcriptomics (gene expres- 1. There are dissimilarities in responses to
sion), proteomics, metabolomics, peptido- estrogens between the mouse and human
mics, etc. So far, there is more extensive endometrium. This brings into question
the likelihood of learning an easy lesson
from these species and transferring it to

406 Genomics and Reproductive Biotechnology

any domestic animal. Differences are During this built-up phase of the endo-
both temporal and qualitative. metrium, genes controlling proteins
2. Comparison of gene array studies in involved in anti-apoptosis were also
rodents, both between experiments in the upregulated, while pro-apoptotic genes
same lab and between labs, shows that were downregulated. One would expect a
rather few genes respond similarly, or in generality in these changes, but a com-
the same direction (up- or downregu- parison with other laboratories showed
lated), despite attempts to standardize that, surprisingly, only some 14 genes
experimental conditions. Differences in were affected similarly.
platforms used play a role, and it has even 4. As pointed out by Groothuis and collabo-
been proposed that EE and E2 may give rators in their review, it is well known
different responses. As far as comparison that when studying whole tissues from in
between mouse and humans—since they vivo studies, changes in gene expression
respond very differently physiologically may occur in a small population of cells,
anyway—very few genes are regulated in being of outmost physiological impor-
the same way. What is common is that tance, while the responsible mRNAs and
genes regulating the cell cycle are induced changes in their levels may not be detect-
by estrogens in both species. able due to dilution in the global mRNA.
3. In the mouse, which is the easiest to In such a case laser capture microdissec-
study, there are interesting consecutive, tion can be applied to pick mRNA from
temporal changes in gene expression, specific regions of a tissue slice. Of course,
which can be related to the physiological one must know which cells to look for.
state of the endometrium. In the first 4 h,
there was an increased influx of fluid into Yet another reason for phenotypic anchor-
the uterus, which may be due to the ing is that any perturbation such as exposure
increased expression of vascular endothe- to chemicals is likely to change gene expres-
lial growth factor (VEGF) and thus sion even in the absence of cellular changes;
increase in vascular permeability. Other this may be considered a stress response
vasoactive growth factors and vascular or “background noise” and not related to
endothelial receptors were also upregu- obvious toxicity. Thus it is not always trivial
lated. Following these changes, genes to relate changes in the expression of a single
involved in transcriptional regulation gene or gene family to a biological effect.
(e.g., mRNA and protein synthesis) and
signaling for growth and differentiation 17.4.3 Epigenetics
were seen to be upregulated. These gene
expression patterns were observed in the Alterations in the DNA-methylation (de-
absence of obvious histological changes. and remethylation) state of the embryo
After that, genes involved in controlling occur in a sophisticated way in waves,
chromosomal replication were upregu- and very specifically in germ line cells.
lated as a “worm up” in the cell cycle, Methylation patterns can be affected by
and at 24 h, a substantial increase in disease states, nutritional deficiencies, etc.,
mitotic index could be observed. These during pregnancy in a way that alters the
genes being initially upregulated then fell health of the offspring in adulthood. The
back, often to below control levels. same holds true for chemical exposure

Reproductive Endocrine Disruption 407

during pregnancy. As to endocrine disrup- 17.4.4 Advantage of domestic animals
tors, there is increasing evidence that the in toxicogenomics?
same mechanisms may be at play. For
example, for diethylstilbestrol, it is likely Artificial selection of domestic animals over
that vaginal tumors occurring at adolescence thousands of years has created a great
and later in women exposed transplacentally number of breeds (reviewed by Georges
as a result of maternal ingestion are caused 2007). Lately, the whole genome has been
by epigenetic changes. The other obvious mapped for the most common domestic
examples are that of the anti-androgenic animals. These late developments will
fungicide vinclozolin mentioned above and give animal breeding a new jump in refine-
also methoxychlor, which has been used as ment. There is no doubt that the new genetic
a replacement for DDT as an insecticide. If information and technologies as a spin-off
endocrine disruptors are administered to effect will give us tools to refine our under-
pregnant rats during the period of sex deter- standing of mechanisms of toxicology as
mination, increased spermatogenetic cell well, including endocrine disruption. For
apoptosis and decreased sperm number example, can positional identification of
and mobility are observed in the next genes underlying complex traits be used to
generation of males. Interestingly, this phe- study the influence of chemicals on charac-
notype is transmitted transgenerationally teristics such as behavior and sexual func-
through the male germ line. Not only were tion? To what extent does toxicological
signs of decreased fertility observed, but also influence depend on interaction with classi-
increased cancer rate, prostatic disease, and cal hormone receptors? To what extent
kidney and immune system problems. does it involve the epigenetic machinery?
Can information gathered by the different
At the molecular level it is likely that “-omics” techniques of transcriptomics,
methylation of critical genes during embry- proteomics, and peptidomics be integrated
onic gonadal sex determination can alter the into a more global understanding? It is dif-
male germ-line epigenetics, causing an epi- ficult to foresee which direction research
genetic reprogramming that appears to be in endocrine disruption will take, but con-
transmitted transgenerationally. At the tran- sidering the available tools and the under-
scriptome level, expression of 196 genes was standing of the biology of gene regulation,
found to be influenced (day 16 embryos), with the only limitations to development will
the majority of genes being silenced. Interest- be our imagination. To get a broader view
ingly, methyltransferases were affected in on all of these aspects of endocrine disrup-
the F1 and F2 vinclozolin generation (at tion, domestic animals have a place in
embryonic day 16) embryonic testis, being in research.
line with an effect on DNA methylation
(Anway et al. 2008). This complex area of 17.4.5 Experience on toxicogenomics
research, especially on vinclozin and diethyl- in avians
stilbestrol, has been reviewed (Anway and
Skinner 2006; Crews and McLachlan 2006). It has been shown, for example, by Brunström
Epigenetic effects of chemicals certainly give et al. (2003), that male quail embryos exposed
new, and to some extent frightening, aspects to estrogenic substance in the egg may have
on toxicology in general and on endocrine aberrant sexual behavior when adults. We
disruption in particular.

408 Genomics and Reproductive Biotechnology

thought that this would involve several 17.5 Future research directions
regions of the brain and that aberrant
morphology would be discovered close to The focus in research on endocrine disrup-
the time of exposure to ethinyl estradiol. tion so far has been on chemicals interacting
However, we found no change in gene with hormone receptors, mainly due to the
expression related to this exposure, either in fact that handy and high-throughput cellular
the quail or in the chicken. Interestingly, systems have been developed. We foresee
however, very stably expressed in the embry- that more in vivo studies will then be
onic brain even from day 4 of incubation (the required and that domestic animals will be
earliest time point studied) were a number valuable in these confirmatory studies, espe-
of sex-specific, sex-chromosome linked cially to support or reject the generality of
genes (Scholz et al. 2006). Yet another finding findings in the classical laboratory species.
in our work was the absence of evident The current mapping of the genome of
dosage compensation of sex-linked genes traditional domestic species will provide an
(Ellegren et al. 2007). excellent opportunity to combine research
on endocrine disruption with toxicogenom-
As indicated in Section 17.4.2, point 4, ics. In particular, phenotypic anchoring is an
it is possible that the effects of ethinyl area where research on domestic animals
estradiol are restricted to a few cells in a can contribute, since reproductive functions
restricted area, in this case, for example, in including behavior are very well character-
the preoptic medial nucleus (POM) in the ized in these species.
developing thalamus of the quail brain,
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18

Nutrigenomics for Improved Reproduction

John P. McNamara

18.1 Introduction describe the system as a whole. Using genetic
and genomic approaches recognizes that
Reproduction is a function of nutritional nutrient use traits and reproductive traits are
state, and also a director of nutrient flux, and heritable and specific gene sequences associ-
both have genetically inherited elements of ated with these traits can be identified.
control. These three systems function inte- Introducing nutrigenetic and nutrigenomic
grally in the animal, and as such there is no approaches recognizes that nutrient status
way that nutrition, reproduction, and genet- affects gene transcription in many organs,
ics can be separated in research. There are which in turn alters metabolic activity in
several systematic, dynamic controls of reproductive organs and thus fertility, and
nutrient flux involved in ovulation, gesta- gestational and lactational success. We now
tion, and lactation. Glucose can alter the have technical tools (primarily transcription
release of hormones from the hypothalamus arrays) to help define specific mechanisms
to direct ovulation, and it can also direct the that connect nutritional fluxes with repro-
secretion of other hormones such as insulin ductive success. The adipose tissue plays a
or IGFI that affect metabolic activity in central role in reproductive success, not just
reproductive organs. Once an animal ovu- as an energy storage and release organ, but
lates and fertilization occurs, additional perhaps also as a source of hormones and
interactive control systems are induced to control factors of reproduction. In order to
help direct nutrients to the developing move forward both in research and applica-
fetus(es) and then to the mammary gland. tion, we must use dynamic, integrated bio-
After thousands of years of human observa- mathematical modeling tools to help define
tions on the interactions of nutrition and those reproductive processes that respond to
fertility, and after two to three generations nutrient status and genetic selection and
(80 to 100 years) of reductionist research, we those changes in nutrient flux that respond
can now integrate our detailed knowledge to to genetic selection and reproductive state.

413

414 Genomics and Reproductive Biotechnology

Therefore, this chapter evaluates the effects while changes in transcriptome expression
of diet and nutrient management schemes present much more of a challenge to sort out
on gene expression and thus addresses some changes in known key controlling genes
of the nutrition limitations in reproductive from those of a more constitutive nature.
performance. For the purposes of this chapter, I am going
to concentrate primarily on nutrigenomics.

18.2 Nutritional physiology of 18.2.2 Body fat and reproduction
reproduction: A brief view
With that quick introduction to the present
18.2.1 Nutrigenomics and nutrigenetics and future, we need to visit the past to allow
us to understand the true role of nutrige-
Nutrigenomics is generally defined as the nomic work. The role of nutritional status
effect of dietary nutrients on gene transcrip- in reproductive fertility was recognized early
tion: “Nutrigenomics aims to determine the in human history. Ancient or historical
influence of common dietary ingredients on texts, drawings, and writings speak to tradi-
the genome, and attempts to relate different tions and perceptions of body fatness, shape,
phenotypes to differences in the cellular and size in human fertility. Likewise, domes-
and/or genetic response of the biological ticated animals were fattened to become
system” (Mutch et al. 2005). An example of fertile and sleek fat cattle were desired for
this is studying the effect of changes in diet their fertility. Initial fertility and postpar-
on gene transcription and metabolic flux tum anestrous varies among species or even
in the adipose tissue during pregnancy breeds, and can be attenuated or exacerbated
and lactation and relating those changes by nutritional status; while a certain amount
to differences in reproductive processes. of body fat might correlate with improved
“Nutrigenetics, on the other hand, aims to fertility, too much may be detrimental.
understand how the genetic makeup of an Today, we realize that in fact there is more
individual coordinates their response to diet, to fertility than just fatness—some animals
and thus considers genetic polymorphisms” alter fertility after increases or decreases in
(Mutch et al. 2005). The practical applica- body fatness (Wade and Schneider 1992). It
tion here is to identify the gene variants that is not simply the amount of body fat but flux
relate to differential response to nutrients. of glucose or other nutrients such as vita-
Obviously there is tremendous overlap here mins and minerals that can alter fertility or
and the two approaches can easily be related. gestational and lactational success (Wade
Nutrigenomic studies might find similari- and Schneider 1992; Wade and Jones 2004).
ties or differences in the nutrient effects on There have been several excellent summa-
the transcriptome of phenotypically similar ries and reviews on these complicated topics,
(or different!) animals, while nutrigenetics and the new reader to this field is strongly
might find that animals with specific gene encouraged to take the time to read them,
variants respond to a dietary change differ- as it is my experience that there is no real
ently, whether in the transcriptome or post- understanding or application of “genomics”
transcriptional processes. A variation in a outside of understanding the underlying
key controller like prolactin or IGF-I may be nutritional and reproductive physiology,
easy to explain (and perhaps manipulate), from the basic to applied in practice ( Staples

Nutrigenomics for Improved Reproduction 415

et al. 1990; Wade and Schneider 1992; Wade and reproductive success was clear (Wade
and Jones 2004; McNamara 2005; Roche and Schneider 1992). We will cover this in
2006; Vinsky et al. 2006; Chagas et al. 2007, more detail, but much of this work led to
and many of the older papers listed in the understanding the key role of glucose in
bibliography and the bibliographies of these reproduction. Yet other questions arose:
citations). “How can reproductive organs monitor
and respond to the amount of body fat, or
Early observations showed there was some vice versa?” A corollary and related line of
connection with being “well-fed,” if not fat, research asked the same question in relation
and fertility. Young females needed to to maintenance of body fat: “How does the
mature to a certain range of body shape and body monitor and maintain a fairly constant
fatness (species-dependent) before obvious body fat percentage, and what are the situa-
outward signs of potential fertility, such as tions in which this system can fail and
estrus behavior or receptivity, were observed. obesity (or extreme thinness) or reproduc-
Even if those signs were observed, and acted tive problems ensue?”
upon by a male, actual fertilization and suc-
cessful pregnancy, and lactation, also Then, research in many species tested
depended on some level of adequate nutri- theories on the presence of signaling mole-
tion. Human families and animal farmers cules or nervous activity to and from the
alike acted on these cues and attempted to adipose tissue. One major outcome of this
ensure adequate nutrient status prior to research effort (truly spanning from early
serious attempts at reproduction. post-World War II until today) was that the
amount of body fat alone did not account for
In the last 100 years or so, there have been even a small majority of the variation in
many studies of biological connections fertility. Large variations in the amount of
between nutritional status and fertility. It body fat, and rate of change of body fat, in
was noted that gross or even moderate stunt- reproductively successful females within
ing of growth delayed sexual maturity in and among species precluded body fat
most females. Even when outward signs of content as being the ultimate driving force
fertility were seen, a number of situations in (Butler and Smith 1989; Wade and Schneider
which actual fertility was delayed or reduced 1992). Although there is a perception that
were noted, including insufficient total food, body fat (or in dairy cows, body condition)
or the lack of certain food components. One directly relates to reproduction (Chagas et
eventual understanding was that some al. 2007), the preponderance of evidence is
amount of adipose tissue was necessary for that although body fat is a key part of the
a successful reproductive cycle of ovulation, system, it is the nutrient flux (energy balance
fertilization, implantation, pregnancy to or glucose supply) that is the mechanistic
term, and not to be overlooked, a successful cause of changes in reproductive status and
lactation. success (Wade and Jones 2004).

However, the story was not over. As noted The focus on body fat and potential signal-
above, there was a lot of variation in the ing pathways did in fact lead to important
amount of body fat, or body fat gain or loss discoveries, one is that adipose tissue is an
and fertility; it was not a direct and com- endocrine organ, secreting, among many
plete connection. Now we understand that other substances, insulin-like growth factor
there is in fact a connection between a posi- and the intake controlling hormone leptin.
tive energy balance (energy in > energy out)

416 Genomics and Reproductive Biotechnology

These two molecules are critically impor- (Girard et al. 1997). Several of these effects
tant in tissue growth, ovarian metabolism, relate directly or indirectly to reproductive
and food intake. In addition, glucose flux, processes.
even in ruminants, directly affects and is
affected by the amount and activity of Eventually the research led to the endo-
adipose tissue. The sum of all this work is, crine aspects of nutrient flux and reproduc-
in my opinion, a great success story in nutri- tion. Nutritional scientists started to realize
tional and reproductive biology. The results that “reproductive hormones” affected
of this research effort allow us to bring the nutrient use, while reproductive scientists
tools of nutrigenomics to bear in a focused started to explore the effects of nutrients on
fashion on the role of genetics and gene reproductive processes. We cannot fully
expression in nutrient use and reproduction. explain the connections between nutrient
The role of glucose interaction with the flux and reproduction without introducing
adipose tissue and reproduction may be the endocrine aspects. If one were to ask
expanded to changes in synthesis and secre- students of biology to list “reproductive hor-
tion of IGFI, leptin, or other control mole- mones,” estrogen, testosterone, and proges-
cules (e.g., cytokines; Zieba et al. 2008). terone would probably be the first answers,
also offered up would be luteinizing hormone
18.2.3 Metabolic flux and reproduction (LH), follicle stimulating hormone, prolac-
tin, placental lactogen, human chorionic
One of the subsequent lines of research gonadotropin, maybe oxytocin, and relaxin.
focused on the primary nutrient glucose. But not many students (other than those
Many studies were done to ask the question with really good nutritional or reproduction
“How does glucose status relate to reproduc- specialists as teachers!) would also list
tive success?” The majority of biological insulin, somatotropin, insulin-like growth
scientists now agree that a major driver of factor, thyroid hormone, and corticosteroids
reproductive success is sufficient glucose as involved in reproductive processes, yet,
flux in the body (Wade and Jones 2004; they are, both indirectly (regulating cell
Chagas et al. 2007). Glucose flux into many division and tissue growth) and directly
cell types, including brain, adipose, liver, (regulating glucose entry into the ovary, fol-
muscle, and ovary initiates many cascading licular growth and fetal and mammary
signals that direct metabolic flux, including gland differentiation, growth and metabo-
fat and protein synthesis. In addition to just lism). Recent studies also suggest roles
the use of glucose for energy generation for for cytokines and inflammatory molecules
anabolic reactions, included in these cas- (Trayhurn and Wood 2004; Loor et al. 2005,
cades in most instances are changes in gene 2006; Chagas et al. 2007). These findings
expression. The multivariate role of glucose have come out of the integration of many
in regulation of metabolism extends from different studies on many different aspects
the short term (seconds, minutes)—enzyme of nutrition and reproduction. Yet even
activation, increases in ATP and NADPH though all these hormones have a role in
concentrations—to longer term (days, weeks) various reproductive functions, the majority
changes in mRNA transcription and transla- of them respond to, or (or also) affect,
tion to make more or less of the enzymes the major driving force behind truly suc-
that catalyze many synthetic reactions cessful reproduction: the glucose flux in
the body.

Nutrigenomics for Improved Reproduction 417

18.2.4 Nutrigenomics for ties (Bergsma et al. 2007). Also, the interac-
improved reproduction tions of nutrition and reproduction have
been given strong attention, and the results
To prove the point of the reality of the genet- are impressive (Bergsma et al. 2007, and
ics if not the nutrigenomics of reproduction, many references therein) and the beginnings
recently, the Dairy Herd Improvement of a nutrigenomic awakening are present
Association has added daughters’ pregnancy (Dawson 2006; Bonnet et al. 2008). An inter-
rate, or days open, as a trait for bull selec- esting anecdote applies here. In the ancient
tion, and other countries have done similar past (late 1960s) when this author was
work (VanRaden et al. 2004; Harris 2005; slaving away helping to raise pigs (and milk
Weigel 2006). The mass of empirical genetic cows, you could do both at once then) in
data now show that in fact important traits Bureau County, Illinois, he was oft teased by
of reproduction are heritable, as we have rec- the wise old farmers that “you college types”
ognized for nutrient use for decades. We also could talk all you want about weaning nine
know that the genome must be “properly pigs per litter, getting 2.3 litters per year, or
fed” to fully express its potential. Much has you name it, but “it doesn’t work out here
been written in the last decade of the declin- in the real world.” Well, funny enough, now
ing fertility of Holstein dairy cattle, primar- the “real world” is showing us college types
ily in the United States, with a myriad of that in fact it does work and we better catch
suggested mechanisms, many of which actu- up with our research!
ally have little data to back them up (Royal
et al. 2002a, b; Chagas et al. 2007). The long overdue “admission” that repro-
ductive traits are heritable has already begun
It is oft repeated that “increasing milk to improve reproduction in dairy cattle and
production decreases fertility” and many pigs. If pregnancy rate or days open can be
statistics are cited to “prove” fertility is used as a selection trait, we should be able
lower in dairy cattle today. Yet on many to describe the biochemical mechanisms
herds and many hundreds of thousands of that make it so. The accepted generality that
cows, simultaneously fast rates of milk “well fed” cattle are more fertile, coupled
secretion, feed intake, and good fertility (any with the renewed focus on genetics of fertil-
way you measure it) occur all the time. Also, ity, and the simultaneously fast rates of
the recognition that the end result of suc- milk secretion and normal fertility support
cessful rebreeding during lactation can be a the concept that nutrigenomics and nutrige-
selectable trait in the bull proofs “proves the netics are the “Venn Diagram” of nutrient
point” that in fact, fertility is heritable, that use, genetic traits, and reproductive success.
it has many control factors involved, and
that there is no direct no overriding reason 18.3 Mechanistic connections
why all “high-producing dairy cattle” should between nutrient flux and
be sub-fertile. reproductive processes

The genetics of reproduction has not 18.3.1 Integration of reproductive
been ignored in the pork industry either. It processes and nutrient flux
has long been recognized and acted upon
that sow traits, including reproductive pro- The earlier observations and research
cesses such as ovulation, litter size, and findings introduced above have given us
return to estrus, have measurable heritabili-

418 Genomics and Reproductive Biotechnology

(Nutrients absorbed input from Molly Rumen Model, Baldwin et al. 1987a)

Nutritional inputs Glucose TAG NEFA FA isomers, Acetate Amino acids
linoleic, CLA

Endocrine Omega-3 fatty acids Omega-6 fatty acids
signals
in Molly Lactation hormone Adipose Liver Muscle Mammary gland
Anabolic hormone
Key organs
Catabolic hormone
Endocrine TRH IGF-I
controllers Pituitary
Hypothalamus Ant Post
GnRH
Leptin
(-) Prolactin FSH LH Oxytocin GH

Estrogen

Progesterone Temperature
pH ?
Reproductive Developing follicles Ovary Fertilized egg Developing embryo NH3?
organs & Follicle CL
processes Embryonic death
Ovulated egg

Outputs Uterus

Placenta CALF

Figure 18.1 Schematic flow diagram of a model of nutrient flux and reproductive functions.

sufficient knowledge to seriously study the nutrient use and reproductive function. In
integrated functions of nutrient use, genetic addition, care is given to use the major
expression, and reproductive processes. driving factors as states and signals (hor-
Figure 18.1 presents a simple flux diagram mones) as connections between states and
of a model of nutrient use and reproduction. fluxes. Lower levels of metabolic control
An old, simpler version was published previ- (specific enzymes or gene transcription
ously (McNamara 2005). Another more con- events) are in fact the mechanisms that
ceptual one can be found in the excellent define the “arrows” in the flux diagram.
review of Chagas et al. (2007). The flux
diagram is basically species-independent, In order to understand the context and
although there will be some variation among details of such a model, we must revisit in
species in the mechanisms of control. It is brief research that allowed this flux diagram
aggregated at the nutrient flux level, not at to be constructed. We can also point out
specific biochemical reactions or gene tran- components that have strong justification
scription events in order to describe the and validation, and others that are based on
basic processes in an animal that connect much less data. Then, we will revisit this
model with an eye to how to move forward

Nutrigenomics for Improved Reproduction 419

in an ordered nutrigenetic and nutrigenomic 18.3.2 The role of glucose
framework.
A key controller in the connection between
We can simplify the cycle of reproductive the brain and the ovary for follicular devel-
events to original sexual maturation, first opment and ovulation is glucose. Glucose is
ovulations and ability to conceive, to suc- known to have a direct effect on the hypo-
cessful gestation, to the first lactation, and thalamus that causes the release of GnRH,
then, often, to renewed ovarian cyclicity and which in turn causes LH release from the
a second (and subsequent) gestation and lac- pituitary (Wade and Jones 2004; Senger
tation. In most species, certainly cattle and 2004). In addition, glucose elicits increases
pigs, females must reach a certain physiolog- in circulating insulin and IGFI, which
ical maturity before the hypothalamus, pitu- have positive effects on follicular growth.
itary, and ovary can fully communicate and Although there appears to be a wide range of
function to develop an oocyte capable of “effective” glucose flux rates or circulating
becoming fertilized (Senger 2004). The spe- concentrations to allow these effects, they
cifics of development of these reproductive are still critical. This is one reason that in
organs are covered elsewhere in this book. most cases, fertility is not affected nega-
The nutritional development of fertility is tively until a serious deficit in glucose
both direct and indirect. There is likely not happens. In lactating sows and cattle, return
any one nutrient that directs the first fol- to estrus after parturition is also closely con-
licular waves, estrus behavior, and ovula- nected with adequate glucose flux returning
tion, but the end result of nutrient flux after the mammary gland starts to use large
allowing development of mature organs amounts of glucose and prior to sufficient
(such as adipose tissue), and adequate glucose increase in glucose. There are other aspects
availability. The growth rate of the animals, to the full return of fertility postpartum, but
a function of both genetics and nutrient glucose is an important factor. The role of
supply, dictates that animals will arrive at a glucose in stimulating insulin and IGFI is
body composition and glucose flux state that likely also important in the return to ovula-
supports the actions of the hypothalamus, tion of viable oocytes after parturition.
pituitary, and ovary. Early research discov-
ered that this initial fertility was much Thus it is not only nutrient flux effects on
less a function of age than of physiological ovulation that are important, but on devel-
maturity (roughly monitored by body com- opment of a viable oocyte and, perhaps,
position), and modern domestic breeds support of a uterine environment conducive
(cattle, pigs, poultry) certainly reach physi- to blastocyst development and implanta-
ological maturity, and become pregnant, at tion. Glucose likely plays a role via stimula-
much earlier ages than they did previously, tion of insulin and IGFI, which helps to
based on our selection pressure on growth support anabolic metabolism and oocyte
rate. Even when physiological maturity is development.
reached, in general, the animal must still
be “well-fed”—at maintenance or in posi- 18.3.3 The role of fatty acids
tive energy balance, with sufficient cir-
culating blood glucose to support follicular Certain classes of fatty acids, primarily the
development and eventual LH release and omega-3 series and omega-6 series and their
ovulation. metabolites, have also been identified as

420 Genomics and Reproductive Biotechnology

positive controllers (Ambrose et al. 2006; leading to changes in uterine ammonia and
Bilby et al. 2006a,b). Some intriguing results pH, but follow-up research failed to show a
have been reported in practical use of omega- strong connection. Lately, it has been sug-
3 and omega-6 fatty acids in improving fer- gested, based on larger empirical and mecha-
tility in lactating dairy cattle (Bilby et al. nistic studies, that even moderate heat
2006a,b) empirically, yet molecular mecha- stress, leading to an increase in body tem-
nisms here are not understood. Likely perature as little as 0.5 or 1°C can alter the
candidates include control of basic cell uterine environment (pH and ammonia con-
development and membrane function, and centration) that may hinder embryonic
in reduction of inflammation or inflamma- development. Students of physics and chem-
tory molecules that may hinder oocyte istry will recognize the potential of the
development (Trayhurn and Wood 2004; Arrhenius equation at work here: in general,
Webb et al. 2004; Ambrose et al. 2006) There for every 10°C decrease or increase in tem-
is ample evidence that specific fatty acids perature, all chemical reaction rates are
can alter gene expression in many tissues halved or doubled (McNaught and Wilkinson
(Al-Hasani and Joost 2005, and many refer- 1997). A 1°C difference could mean a 10 %
ences therein). Thus the ground for finding change in metabolic buffering reactions,
specific nutrigenomic mechanisms for fatty which could easily affect embryonic devel-
acids and reproduction is quite fertile, so to opment. This is an exciting ongoing area of
speak. An obvious problem is that the research, which promises to improve our
massive complexity and thus large number understanding of nutrient use, the environ-
of possible permutations will take the “will” ment, and fertility. Because gene transcrip-
for scientists to focus, together, on biomath- tion events are functions of metabolic
ematical solutions and complex models to reaction rates, there is (an admittedly
move forward. broad) potential involvement of nutrige-
nomic mechanisms.
18.3.4 Early embryonic losses and
nutritional status In addition to the glucose, fatty acid ,and
heat stress effects, there is potentially a role
Such challenges notwithstanding, as long as between protein nutrition, amino acid
the mother is in a reasonably good range of metabolism, genetics, and fertility. This is
nutrient supply, the embryo will develop likely not a major function of dietary protein,
normally. In dairy cattle, much work has but a subtle interaction between amino
been done to investigate early embryonic acids, gene transcription, and endocrine reg-
losses, usually categorized as animals first ulation. Genomic studies have suggested a
diagnosed pregnant (28 to 42 days post breed- connection between variants in the myo-
ing) then showing open. The other losses statin and calpastatin genes and fertility
from breeding to showing estrus again have in the cow (Garcia et al. 2006; Mitchell
myriad causes, including body temperature et al. 2006; Chagas et al. 2007; Szyda and
(Chagas et al. 2007) and uterine pH and Komisarek 2007). The protein myostatin
ammonia concentrations (in turn likely a may in fact regulate glucose uptake in
function of body temperature). Early research reproductive and other organs (Mitchell
connected, I think mistakenly, early embry- et al. 2006). These intriguing studies may
onic losses directly with excess protein provide initial evidence for a mechanistic
link between protein metabolism, gene

Nutrigenomics for Improved Reproduction 421

expression, and reproduction in a true nutri- sire would produce a smaller offspring in a
genetics and nutrigenomics way. smaller dam, and vice versa was known,
there was no understanding of why at the
18.4 History of integration of time. Why did not a large breed sire produce
physiological state, nutrient flux, a huge foal in all dams? Of course, there is
and reproduction a range—breeding large sires to small dams
will statistically result in larger foals, but
18.4.1 Hammond’s seminal work not as large as they would be in larger dams.
Dr. Hammond posited that there were some
We cannot write a chapter on nutrition, possible “special growth substances of
genetics, and reproduction without a nod to maternal origin” that directed the “parti-
what is likely the seminal work and first tioning of nutrients” to the organs most
true study in this area by Sir John Hammond: important in that physiological state, and
“Physiological Factors Affecting Birth also that “…some limiting internal secre-
Weight” (Hammond 1944). In this artilce the tion or metabolic substance produced by the
concept of the partitioning of nutrients was mother, as a controlling factor in foetal
first put forth. This concept captured the growth.” However, he recognized that “It is
idea that each organ has a priority for nutri- possible that the limitation of the size of the
ent use, with the brain having the highest crossbred foetus in the small mother is
priority, metabolic organs less, and muscle brought about by a higher rate of metabo-
and adipose even less. However, once preg- lism of the maternal tissues in the small
nancy or lactation occurred, these organs breed than in the large breed, that is, by the
moved up to or close to the priority for the greater competition of the maternal tissues.”
brain and in fact may alter the priority of
other tissues. Dr. Hammond did a tremen- The brain is always the most important
dous amount of research on genetics, nutri- (as the body will always sacrifice other
tion, and reproduction before our knowledge organs when faced with nutrient deficits).
allowed us to become much more specific, During reproduction, however, the fetal-
reductionist, and thus segregated as “geneti- placental unit “takes charge” and directs
cists,” “nutritionists,” and “reproductive nutrients to itself, even during periods of
physiologists.” He was the first to pose the nutrient deficit. When lactation begins, the
question (in print at least) of the connection mammary gland does the same thing. The
between the genetics of the sire and dam, work of Dr. Hammond, lacking in chemical
and thus the fetus, and the use of nutrients, and biochemical technology, laid the con-
for a specific reproductive outcome. ceptual framework for a large part of ensuing
work in the nutrition, genetic, and reproduc-
In an elegant experiment he bred large- tive biology of successful pregnancy and
breed horse sires to small-breed horse dams, lactation.
and small-breed sires to large-breed dams,
and proved the point (which any horse 18.4.2 Homeostasis
breeder knew but scientists had not figured
out yet) that the fetus can direct nutrients At that time the concept of homeostasis in
to itself to meet its “pre-programmed” physiological flux was well accepted. The
genetic pattern. Although that a large breed interactions of glucose and insulin were
known. But Dr. Hammond introduced the

422 Genomics and Reproductive Biotechnology

concept of a higher level, long-term regula- causative factor in this anabolism, which is
tion of metabolism, embodied in the term usually strongest in early to mid-pregnancy
“partitioning of nutrients.” Some factors is progesterone from the corpus luteum.
altered the normal homeostatic fluxes such Progesterone directs the ovary, uterus, and
that, while insulin still stimulated glucose hypothalamus to cease follicular develop-
uptake in insulin-sensitive cells, the set ment and ovulation (Senger 2004). However,
point of control was tipped toward ensuring progesterone also directly affects metabo-
that the fetal-placental unit, or the mammary lism in the liver, adipose tissue, and muscle,
gland, was the highest priority tissues for and promotes development of the mammary
glucose and other nutrient flux. Hormones gland! Liver, adipose tissue, and muscle
of pregnancy were later identified as the increase the synthesis of fat and protein to
factors controlling metabolism in maternal be used by the fetus, even at an unchanged
tissues, and mechanisms were later identi- feed or energy intake. To ensure support of
fied such as alterations in hormone receptor fetal development and growth, however,
content, activity, and/or cellular responses. progesterone also helps to increase food
intake. The nutrigenomic mechanism is
18.4.3 Homeorhesis such that progesterone alters the transcrip-
tion of anabolic enzymes to make fat or
Years later, based on a then large body of amino acids, and alters the gene expression
data on nutrition and endocrinology of preg- of insulin or beta-adrenergic receptors or
nancy and lactation (summarized below), downstream signaling molecules. Then,
the concept of homeorhesis, the long-term when sympathetic nervous system release of
alteration of metabolism in support of a norepinephrine or pancreatic release of
dominant physiological state was developed insulin occur to alter glucose or fatty acid
(Bauman and Currie 1980). This concept flux, the tissue responds, but at different
embodies the importance of changing the rates, such that glucose is used for body fat
partitioning of nutrients to support repro- synthesis or lipolysis is decreased more than
duction. Nutrigenetic and nutrigenomic normal to store body fat.
mechanisms certainly play a role in this
control. Then, in mid to later pregnancy, anabo-
lism switches to catabolism to use the stored
18.5 Nutritional physiology of energy. The fetal-placental unit secretes a
pregnancy and lactation protein hormone that directs the liver,
adipose tissue and muscle to supply an
18.5.1 Pregnancy increased amount of fat and protein to be
used by the fetus, even at an unchanged feed
Homeorhesis, nutrigenomics, and nutrige- or energy intake. The hormone is named
netics are exemplified by metabolic control differently among species, but is basically
in pregnancy. The anabolism of pregnancy the same protein: placental lactogen or cho-
has been recognized for a long time: even rionic gonadotropin. In some species (e.g.,
on somewhat restricted intakes, pregnant rabbits), the second cousin of this group, pro-
animals will accrue more adipose tissue lactin, serves the same role. These two hor-
than similar nonpregnant ones. A major mones are part of three called the “placental
lactogen gene family” and comprise a huge
role in the reproductive success of many

Nutrigenomics for Improved Reproduction 423

species (Harris et al. 2004). We will get to work such that permanent alterations in
the third member later. In addition to sup- fetal and later neonatal development ensue?
porting fetal development, these hormones I think the case for such a role in “extreme”
also help control mammary development. situations such as small or large birth weight
has been made. It is interesting to note that
The nutrigenomic mechanism involved this exciting new area of research, with tre-
is that pregnancy hormones bind to recep- mendous potential for improving human
tors in the adipose tissue, for example, and and livestock life and productivity, can be
alter the gene expression of insulin or beta- traced back directly to Dr. Hammond’s orig-
adrenergic receptors or downstream signal- inal work.
ing molecules. Then, when sympathetic
nervous system release of norepinephrine or 18.5.2 Lactation
pancreatic release of insulin occur to alter
glucose or fatty acid flux, the tissue responds, The same concepts apply to the final repro-
but at a different rates, such that glucose use ductive process in mammals—lactation. The
for body fat synthesis is decreased or lipoly- pathway to a successful independent next
sis is increased more than normal to supply generation must follow through the lacta-
nutrients to the fetus(es) or mammary gland tion for ultimate success. The hormones of
(McNamara 2005, 2006). pregnancy not only direct nutrients toward
the fetus, but also begin the process of
On the practical side, we use the knowl- mammary development. Progesterone, estro-
edge of pregnancy anabolism in many agri- gen, placental lactogen, prolactin, insulin,
cultural arenas, and often “limit feed” IGF1, and corticoids all play a role in
pregnant animals, especially swine, during mammary development. Significant amounts
gestation to avoid the animal becoming too of nutrients are not usually needed in preg-
fat, which can lead to problems in lactation. nancy for mammary development, but late
This useful practice embodies the concept of in pregnancy and at lactation, the mammary
partitioning of nutrients: we can limit feed gland goes “from 0 to 60” in a short period.
a pregnant animal knowing that, within a Modern sows can make 700 g of lactose, 600 g
range, the fetuses will “take care of them- of fat, and 450 g of protein a day. Work done
selves” and grow to a healthy weight, neither many years ago demonstrated that the use
too small nor too large. of nutrients was not just a function of “the
giant sucking sound” (also known as “meta-
There is a large body of knowledge in bolic pull” or more specifically “a demand
many species on nutrient requirements for function”) from the mammary glands, but a
a successful pregnancy (including optimal coordinated effort of the hormones of preg-
fetal development), and also on the nutri- nancy and lactation.
tional problems with pregnancy, including
small or low birth weight, pregnancy diabe- In late pregnancy, progesterone concentra-
tes, pregnancy toxemia, ketosis, and dysto- tion declines and allows the lactogenic hor-
cia (Senger 2004, and other). Although mones to initiate the expression of several
beyond the scope of this chapter, the study genes for catalysis of milk component syn-
of epigenetics, potential changes in the DNA thesis. In addition, these same hormones,
of developing fetuses, is one nutrigenetic primarily progesterone, prolactin (placental
mechanism that is most exciting: Can
specific nutrients or metabolites actually
change the genomic or transcriptomic frame-

424 Genomics and Reproductive Biotechnology

lactogen in some species) and somatotropin, lipolytic control. We demonstrated that
the final member of the placental lactogen there were clear differences in metabolic
gene family, coordinate metabolism in the control that were either functions of the
adipose tissue and muscle to help direct genetic merit of the animal, or functions of
glucose, fatty acids, and amino acids to the the diet, but that of course, were intercon-
mammary gland for milk synthesis. The nected. Although a direct connection to fer-
importance of the muscle cannot be over- tility had yet to be made, these studies
looked, as it must supply many of the essen- demonstrated that it was not just “body
tial and nonessential amino acids for milk condition score” that related to lactational
synthesis, and also glucogenic amino acids success.
to the liver for conversion to glucose for
lactose synthesis. We have tended to con- Just last year, we were able to finally
centrate on body fat, for good reason, but identify some of the genes involved in
a modern sow can lose 1 kg of muscle a day adipose tissue metabolic control in lacta-
for several days during lactation. Also, as tion (in dairy cattle) using RT-PCR and
noted above, amino acid or protein metabo- transcriptome arrays. We sampled several
lism may yet play a role in fertility at least animals with a range of genetic merit for
in dairy cattle (Mitchell et al. 2006; Chagas milk production and fed the same diets.
et al. 2007). We identified the beta-adrenergic receptors
(all three subtypes), hormone sensitive
We were able to conduct a series of lipase, and its cofactor perilipin as all
studies during the 1980s and the 1990s that increasing in transcription from pre-partum
detailed several of the enzymatic and flux to postpartum, in the adipose tissue of
changes that occurred during late pregnancy dairy cattle (Figure 18.2). In addition, we
and early lactation in dairy cattle and pigs extended and confirmed earlier findings
(McNamara et al. 1985; McNamara 1998; that there is a reduction in expression of
McNamara and Boyd 1998; McNamara 2005, enzymes controlling and supporting lipo-
2006, and several references therein), and genesis (Figure 18.3).
laid a framework for the nutrigenomic work
that was ongoing. In short, we investigated As expected, however, there was great
how animals of different genetic merit (in variation from animal to animal in both
dairy cattle), or litter size (in pigs), fed differ- lipogenic and lipolytic control genes (Figure
ent amounts of energy, expressed the recep- 18.4), suggesting that there is room for large
tors and enzymes of anabolism or catabolism. individual genetic responses to diet and
Expression and activity of enzymes and physiological state. The figure on the animal
beta-adrenergic receptors in adipose tissue variation is presented to make a strong point
were increased from late pregnancy to early here. Most nutritionists, physiologists, and
lactation (Parmley and McNamara 1996). reproduction specialists have been trained
Animals of greater genetic merit had a that to do an experiment, you need to reduce
greater activity of the receptors and enzymes as much among animal variation, to get as
that controlled lipolysis, and had a greater homogenous a genetic pool as possible to
fatty acid release, even at the same intakes. increase the likelihood that you can demon-
Animals limited in feed intake, however, strate “statistical significance” in whatever
reduced expression of lipogenic enzymes hypothesis you are testing. In this way,
dramatically and had a lower response in many would look at these figures and “wave
them away”: “you have nothing here.” This

Nutrigenomics for Improved Reproduction 425

Expression of beta adrenergic receptors subtypes in Expression of genes coding for lipid synthesis in
bovine adipose tissue during lactation bovine adipose tissue during lactation

1.8 Beta-1 450 Pre
1.6 Beta-2 400
1.4 Beta-3 350 Post
Fold change 1.2
90 270 300
1
0.8 250
0.6
0.4 200
0.2
150
0
30 100

50

0

Day relative to parturition SREBP TSH SP 14 Glut1 AcCoA ATP Citrate
Lyase

Expression of hormone-sensitive lipase in bovine Figure 18.3 Expression of sterol regulatory
adipose tissue during lactation element binding protein (SREBP), thyroid stimulat-
ing hormone spot 14, glucose transporter 1, acetyl
Fold change 4.0 CoA carboxylase, and ATP citrate lyase in adipose
3.5 tissue of dairy cattle at 30 days pre-partum (pre)
3.0 90 270 and 14 days postpartum (post). Data are in signal
2.5 strength from the Affymetrix Bovine Gene Array,
2.0 normalized to 125. Data show a consistent reduc-
1.5 tion in the family of control proteins that regulate
1.0 carbohydrate conversion to fatty acids. Reductions
0.5 in lipogenesis can affect overall reproductive effi-
0.0 ciency, from a successful lactation to rebreeding in
dairy cattle. Using gene array data and mathemati-
30 cal models, we can ask questions directly related to
the causative relationships underlying fertility and
Day relative to parturition lactation.

Fold change Expression of perilipin in bovine
adipose tissue during lactation
20 philosophy can be understood based on a
18 lack of understanding of complex systems.
16 In some cases you want to isolate the “nutri-
14 tion” from the “genetics” to identify spe-
12 cific mechanisms. However, it is this
10 author’s opinion (and shared by some others),
8 that in ignoring the genetic variation in
6 response to nutrition, tremendous knowl-
4 edge has been missed along the way, and it
2 took the “genomic” work to bring scientists
0 back together to understand the direct and
30 90 270 undeniable connections between nutrition,
reproduction, and genetics. The pace of
Day relative to parturition advancement in appreciating and under-
standing complex biological systems is
Figure 18.2 Expression of beta-adrenergic recep- already increasing because of this.
tor subtypes, hormone sensitive lipase, and its
cofactor perilipin in the adipose tissue of dairy cattle
in lactation. Data are the fold change measured
against the expression at 30 days pre-partum, mea-
sured by RT-PCR. Control of lipolysis in adipose
tissue is a major contributing factor to reproductive
success, including a successful lactation and fertil-
ity for rebreeding. Further mechanistic knowledge
on control of adipose tissue metabolic control
will help to define the specific roles in supporting
reproduction.

426 Genomics and Reproductive Biotechnology

AcCoA carboxlase transcript LPL transcript BG688620
NM_174224.2
7000
700

6000

600 Array signal strength

Array signal strength 500 5000

400 4000

300 3000

200 2000

100 1000

0 post 0
pre pre post

30 days before and 14 days after calving 30 days before and 14 days after calving

Beta 2 adrenergic receptor HSL mRNA transcript
transcript NM_174231.1 CK769629

350 800

300 700

Array signal strength 250 Array signal strength 600

200 500

150 400

100 300

50 200

0 100
pre post
0 post
30 days before and 14 days after calving pre

30 days before and 14 days after calving

Figure 18.4 Expression of mRNA sequences for two major anabolic control enzymes and two lipolysis
control proteins. Samples taken from 11 Holstein dairy cattle in same lactation, fed same diets. The ques-
tion arises—What are the causes and consequences of animal variation in gene expression in adipose
tissue to overall reproductive physiology?

We are presently conducting more specifi- It is through the continuous loop of nutri-
cally designed studies to determine the ent intake, hormonal response, gene expres-
range in nutrigenomic response to diet in sion, and nutrient partitioning to various
animals of varying genetic merit. Because organs of metabolism and reproduction that
the adipose tissue secretes several molecules the nutrigenetics and nutrigenomics of
that may affect ovarian function, there is reproduction occur. Now, with this brief
potential for identifying some important recap of 60 years of directed effort, we can
control factors through this approach. move forward.

Nutrigenomics for Improved Reproduction 427

18.6 Nutrigenetics and ever grounded in validated research data,
nutrigenomics approaches for will continue to improve our quantitative
improved fertility, pregnancy, understanding. It is this author’s experience
and lactation that information from genomic research can
only be understood with the means of
If one does a literature search (May 2008) on complex model systems, a philosophy shared
“nutrigenomics of reproduction” or various by others (Dawson 2006).
iterations, one does not find much. However,
“nutrition and reproduction (or fertility)” 18.6.1 The acceptance of integrative
yields a lifetime of reading material. The biology is critical
study of the role of gene expression in either
nutrition or reproduction has been fairly A major barrier to improvement of models
extensive. It is up to interested scientists to remains lack of an accurate description of
now “make the connections”, as we have the phenotype of the animal being modeled,
done in Figure 18.1. The success of efforts to expressed as, for example, gene transcription
unravel the nutrigenetics and nutrigenomics control, enzyme activity, hormone and
of reproduction will rely on the construc- receptor kinetics, and intracellular signaling.
tion, testing, and refinement of mechanistic, If we are going to integrate nutrient status
dynamic, biomathematical models of nutri- and reproductive physiology into research
ent use and reproductive processes. The and practical systems, we need an ordered
remainder of this chapter will present in approach centered on well-constructed bio-
brief two pertinent examples of such models, mathematical, dynamic models of nutrient
and some specific examples of transcrip- flux and reproduction.
tomic work focusing on nutrient effects on
gene transcription related to reproductive An additional barrier continues to be the
success, and how such knowledge can be thought processes of scientists who are not
integrated into mechanistic models. trained in more complex regulation and the-
ories and are uncomfortable with the ideas
The flood of information from the various or skeptical of the value of integrative
genome works and the ability to generate biology. The genome projects themselves
large volumes of transcriptome data from are starting to change those attitudes, espe-
animal studies have renewed calls for more cially in younger scientists, because the
integration of knowledge, including using central nature of gene transcription in meta-
biomathematical approaches. A model or a bolic regulation is better understood now
modeling approach to research may also be than before, and because the sheer mass of
defined as an ordered way of describing information generated in genomic and tran-
knowledge of some real complex system. scriptomic work dictates mathematical
Such models have been useful in practical methods and approaches to bring clarity
systems to describe, for example, drug from the data.
metabolism, biochemical pathways, and
nutrient requirements. A quantitative des- One underlying concept to such integra-
cription of metabolic transactions is critical tive work is that the amount and activity of
to enhance understanding and improvement all enzymes and hormones are genetically
of nutrient requirements, health, and lon- regulated, from immediate gene transcrip-
gevity. Models of increasing complexity, tion and translation, to heritability of varia-
tions in hormone and enzyme synthesis and

428 Genomics and Reproductive Biotechnology

secretion. Some examples may be found in 18.6.2 A present basis for a nutrient-
Girard et al. (1997) and Cornish-Bowden et reproduction model
al. (2007). However, some have small herita-
bilities, or are expressed constitutively, are For most research models, the objective is to
members of redundant control systems, and provide a framework to organize complex
are thus not relevant to metabolic control information to describe a system, set and
(Cornish-Bowden et al. 2007). To quote from test complex hypotheses, and evaluate use-
a recent review on nutrition and fertility: fulness of data for improving our quantita-
“…reduced fertility is not caused simply by tive understanding of complex systems. The
changes in management but also by changes objective of the model(s) described here are
in the genotype and underlying metabolic to develop dynamic, mechanistic models of
processes… . The demands impose by lacta- digestion and metabolism (in cows or pigs)
tion interact with the genetic makeup of suitable for evaluation of hypotheses regard-
the cow to have a major negative effect on ing underlying patterns of nutrient use and
the reproductive system…” (Chagas et al. reproductive processes.
2007). That summarizes the genetic connec-
tion to “everything”; now the devil is in the There already exist two solid and vali-
details. dated frameworks for models of nutrient
flux that can provide a basis for a nutritional
The statement “nutrigenomics for repro- reproduction model in cattle and swine. The
duction” explicitly recognizes (finally) that first is the 40 years of modeling work of
all three processes (genetics, nutrition, Baldwin and his many colleagues (Baldwin
reproduction) are integrated without possi- et al. 1987a,b; Baldwin 1995), which led to
bility of separation. That integration must tremendous improvement in understanding
be codified in a model of nutrient use and of the mechanistic connections between
reproduction. The objective of a model diet and animal performance. The model in
dictates (or should) the model components. question is titled “Molly” and the full
If an objective is to model metabolic flux in history and detail can be found in the previ-
any one species of animal, allowing for ous reference.
description of variation among animals,
then genetic control by definition must be In 1968, Dr. Baldwin published an article
included. There is not space to recapitulate titled “Estimation of Theoretical Calorific
all the proper and improper uses of models, Relationships as a Teaching Technique: A
or their importance to true understanding Review.” (Baldwin 1968). In it he described
of complex systems. The reader is directed the aggregate biochemical pathways that in
to some key references to pursue that fact were the components of the net energy
further, but regardless of one’s personal system of feeding cattle, a work that was
experience or opinion of the use of model just wrapping up after about 100 years of
systems in research, their importance effort across the world by many scientists
and utility and effectiveness cannot be (Lofgreen and Garrett 1968; NRC 1968).
denied (Carson et al. 1981; McNamara et al. This connection between the mechanisms
1991; Pettigrew et al. 1992a,b; Baldwin 1995; of nutrient flux and practical, empirical
NRC 2001; McNamara and Pettigrew, cattle feeding led to 40 years of work on
2002a,b; Baldwin 2005; McNamara 2005, developing biomathematical models of
2006; Cornish-Bowden et al. 2007). nutrient use, and “spun off” many other
related efforts.

Nutrigenomics for Improved Reproduction 429

Stemming from that work came the model connection to fertility and for its more
of nutrient use in the sow, “Susie,” devel- recent discovery as an endocrine organ
oped by Pettigrew and colleagues (1992a, b) (Mohamed-Ali et al. 1998). We are beginning
and since then developed and presently to understand that some of the aspects of
being extended to reproduction (McNamara initial fertility (puberty) and successful preg-
2005). This effort began more than 20 years nancy and lactation may in fact be the result
ago, and in 1992, Jim Pettigrew and col- of changes in endocrine activity of the
leagues gave a start to the first model of adipose tissue. In addition, it is also recog-
nutrition and reproduction in pigs, and a nized that many important reproductive
direct quote from that article is in order (as traits in sows are heritable and responsive to
I cannot say it any better!): nutrition (Quesnel et al. 2006; Schneider
et al. 2006; Bergsma et al. 2007) A transcrip-
The mechanisms connecting the diet to repro- tomic approach here can have great value
ductive performance are presently unknown in identifying the potential mechanisms
but may include variations in voluntary feed involved and ruling out those that are not.
intake, digestion, absorption, metabolism of
absorbed nutrients, and endocrine effects. The genetic elements of any metabolic
Clear understanding and manipulation of this reaction can be incorporated into flux control
connection to optimize long-term sow herd models. Maximal rates and substrate sensi-
performance requires ability to track, system- tivities are genetically inherited and in some
atically and quantitatively, dietary effects cases, have a measurable heritability. In an
through the various processes to reproductive aggregate pathway, changes in substrate sen-
performance. The project consists of the devel- sitivity can be measured (McNamara and
opment of a mathematical model of one com- Boyd 1998; McNamara 2005, 2006). We can
ponent of the connection, the metabolism of also envision the Vmax varying during the life
absorbed energy-containing nutrients, includ- cycle or by hormones related to environ-
ing amino acids, related to long-term feeding mental or physiological state.
strategies in the lactation phase of the
reproductive cycle of sows. (Pettigrew et al. Using lipogenesis as an example, kinetic
1992a) flux can be described through the two
Michaelis-Menten parameters of maximal
These models describe pathway biochem- velocity (Vmax) and substrate sensitivity (Km).
istry, as aggregated pathways in a simple and The equation below is used in Pettigrew’s
scientifically correct fashion. There is not an model of metabolism in lactating sows to
attempt to model every reaction, but to describe glucose conversion to body fat
model at the level of biological control most (Pettigrew et al. 1992a):
pertinent to the modeling objective. For a
thorough discussion of the purposes and UglTs = vGlTs (1 + (MGlTs cGl)) , and (1)
practices of metabolic models, see Baldwin (2)
(1995). We will focus on just two pathways ( )MGlTs = MAGlTs∗ (cGlr cGl)tAGlTs ;
in one tissue: lipogenesis and lipolysis in the
adipose tissue. These are two critical path- where MGlTs is the substrate sensitivity
ways in fertility, as they are an important constant for glucose, and is controlled by
mechanism by which animals utilize excess the concentration of glucose, and by an
glucose or respond to a deficit of glucose and “anabolic hormone,” representing primarily
direct fatty acids to reproductive tissues. insulin such that as glucose concentration
The adipose tissue is chosen for its historic rises (insulin increases); the sensitivity

430 Genomics and Reproductive Biotechnology

constant becomes smaller and reaction stores approach zero. The endocrine regula-
rate would increase. The representation tion of triacylglycerol lipolysis (UtsFa; Eq. 3)
of insulin (cGlr/cGl) is raised to a theta is recognized by introduction controlled by
value that can alter the sensitivity of the a maximal rate, as well as by “lactation
reaction. hormone” (Chl),

Let us explore the genetic elements in this The summary integration of metabolic
equation. The Vmax represents the total flux is given here, using glucose as an
amount of catalytic activity available, in example. If glucose flux (rate of entry, exit,
this case, in the sum of body adipose tissue or concentration) is important to reproduc-
(at other levels of aggregation, this may be tive organs (hypothalamus, ovary, uterus,
in a specific organ, cell or single enzyme or mammary gland) then we must have a math-
receptor). This is controlled genetically, ematical description of it to fully understand
inherited from the parents. The Vmax itself its mechanistic importance. It is glucose
may be variable, decreased by periods of around which the major regulatory processes
energy deficit that decreases the total mass of the body have evolved. The regulatory
of adipose tissue. The value of this parame- mechanisms invoked as glucose availability
ter for a population of animals may be deter- changes have major effects on metabolic
mined in studies combining direct measures rates in other organs. Altering glucose use
of enzyme activity and measures of total by food restriction, or by gene insertion
body adipose protein content. The K vari- for proteins such as the insulin-dependent
able, substrate sensitivity, is also inherited glucose transporter result in changes in tran-
(but may not have a high heritability, as the scription of thousands of genes in mice (Fu
catalytic sensitivity of this enzyme, as for et al. 2004).
most, is a function of the molecule, not con-
centration of the molecule). Changes in glucose concentration or pool
size (Gl) in the body are summed as:
The other important metabolic pathway
in adipose tissue is the breakdown of triac- DQGldt = PaaGl + PabGl + PpaGl + PgyTsFa
ylglycerol to fatty acids. This “fight or
flight” syndrome has in fact generated so − UglTs −UgyFaTm −UgyFaTs
much interest over the years; the history of
this quest is rich in itself, noted by many − UglCd − UGlGc −UgyGlTs
seminal research breakthroughs and leading
to several Nobel prizes given for discoveries − UglLm −UglTm (4)
at many levels of metabolic control.
We sum the uptake of glucose, gluconeo-
Triacylglycerol breakdown to free fatty genesis from amino acids (PAaGl), absorbed
acids is described as follows: glucose (PAbGl), glycerol from lipolysis
(PGyTsFa), and subtract the use of glucose
UtsFa = (vTsFa (1 + (MTsFa Chl)))* (3) for milk and body fat synthesis (UGlTm,
(Qts**0.67)* UGlTs), use for glycerol in TAG (UGyFaTm,
(1 − ((QsTs Qts)**thTsFa)) UGyFaTs), oxidation to carbon dioxide
(UGlCd), glycogen (UGlGc), lactose (Lm). In
Such that the maximal velocity of lipolysis this summative equation, all genetic effects
(vTsFa) is attenuated by a sensitivity con- are included in the equations describing
stant, a hormone or hormones (Chl; lacta- each pathway as exemplified above.
tion hormone) and is inhibited as body fat
The use of glucose has several dozens if
not hundreds of possible control points
throughout the body. Glucose use in the

Nutrigenomics for Improved Reproduction 431

muscle affects and is affected by every and NH3 at day 28 through day 45 after
single other use of glucose in the body. For conception.
example, the specific process in the muscle
may in fact have a major effect on glucose Ovulated Egg to Fertilized Egg
dynamics, or might in fact be so over-
whelmed or attenuated by other processes in Fertilized_Egg ∫ Σ [ovulated_egg, viable_
other organs that the true physiological sig- sperm]
nificance is minor. This becomes truly
obvious only when we start to construct Max_Fert_Egg = 0.75 ovulated_egg
models that must make the connection. An
example is that one animal or set of animals The equation here is based on data pre-
will have genetically controlled different sented by J. Santos at the Dairy Cattle
maxima for gluconeogenesis from others, Reproductive Council Meeting in Denver,
and this definitely will affect their glucose October 2006, in which he described that in
and amino acid use. In turn, this will affect fact, the rate of fertilization of an egg by the
nutrient and endocrine impacts on reproduc- sperm, in dairy cattle, is approximately 75%.
tive organs.
Follicular Development and Dominant
18.6.3 Integrating reproductive and Follicle to Ovulated Egg
nutritional functions
Ovulated_egg ∫ Σ [Dominant_follicle,
So now with a summary background, we can Luteinizing Hormone].
begin to construct equations that describe
the fluxes represented in Figure 18.1, and Dominant follicle, second wave follicle,
thus the mechanistic connections between first wave follicle ∫ Σ [follicle stimulating
nutrient use and reproductive processes. hormone, progesterone, 1/estrogen, IGFI,
Although the example given is for the cow, insulin, glucose, 1/NEFA, growth hormone].
the same concepts apply to the sow. All vari-
ables are in mass, concentration, or rate of The equations here capture the knowledge
flux. that a dominant follicle, which will ovulate,
is a function of three different waves in one
Fertilized Egg to Calf cycle of 21 days. The first wave (recruitment)
is a function, either directly or indirectly, of
Calf = Developing Embryo − Embryonic FSH, progesterone, insulin, glucose, and the
Death reciprocal function of NEFA, estrogen and
perhaps growth hormone. Here it is pertinent
Embryonic_Death_28 ∫ Σ [To, pH, NH3] to state that after the original construction of
Embryonic_Death_45 ∫ Σ [To, pH, NH3] potential equations, based on some knowl-
edge, the next step is to actually find (or
These equations capture the following pro- create through research) the data to set param-
cesses: a live calf is a function of a con- eters for those equations. If no data are avail-
ception, minus the rate of embryonic or able, or research shows no such relation, then
fetal death (here represented at 28 and 45 the equations are dropped from the model.
days post fertilization). Embryonic death
is a function of uterine temperature, pH The role of the hormones of reproduction,
in prose form as opposed to equations,
include: progesterone is a function of the
presence of corpus luteum, the placenta,