04_[Zhihua_Jiang,_Troy_L._Ott]_Reproductive_Genomics_479_2010

232 Physiological Genomics of Reproduction

opportunities for mating and pregnancy growth factor 2 receptor (IGF2R) and an
establishment at shortened intervals. This increase in expression of the apoptosis-
evolutionary strategy is taken to the extreme related gene, clusterin (CLU), in regressing
in rodents, which exhibit 4- to 5-day estrous CL. Together these changes in gene expres-
cycles and do not form a functional CL unless sion paint a fairly intuitive picture of CL
mated. regression involving decreased steroid bio-
synthesis and metabolic activity associated
10.2 Physiological genomics of with functional regression of the CL, along
luteal regression with increases in ECM remodeling and
apoptosis associated with structural regres-
All domestic farm species covered in this sion of the CL.
chapter have uteri that produce PGF, which
acts on the CL to initiate its functional and Recently, Goravanahally and coworkers
structural regression. Recently, genomics (2009) compared gene expression profiles in
techniques have been utilized to examine bovine CL before (day 4) and after (day 10)
the transcriptomes of CL at various stages responsiveness to PGF develops. Because
of development and regression. Casey and CL of both statuses contained receptors for
coworkers (2005) conducted one of the PGF, the differences in patterns of gene
earliest studies of differentially expressed expression might reveal critical signaling
genes in the functional and regressing CL of pathways mediating PGF-induced CL regres-
cattle using a custom ovarian cDNA micro- sion. Of the 167 differentially expressed
array. This analysis yielded 15 differentially genes detected, the majority were divided
expressed genes, of which seven increased equally (∼18% each) between genes involved
and eight decreased in regressed CL com- in protein synthesis and modification and
pared with non-regressed CL. These genes genes involved in transcriptional regulation
fell broadly into the following categories: and DNA synthesis. Slightly lower per-
extracellular matrix (ECM), cell structure, centages were involved in cell signaling
oxygen metabolism, apoptosis, steroid bio- (∼12%) and steroidogenesis and metabolism
synthesis, and metabolism. In general, genes (∼10%). In response to PGF, expression of
in the ECM category increased during lute- both calcium/calmodulin-dependent protein
olysis, with decorin (DCN) showing the kinase kinase 2, beta (CAMKK2) and guanine
greatest increase (Casey et al. 2005). DCN is nucleotide binding protein (G protein), beta
a small proteoglycan associated with endo- polypeptide 1 (GNB1) were increased in the
thelial cell angiogenesis, particularly that CL of cattle. The authors suggested that the
associated with inflammation (Nelimarkka combined increase in expression of CAMKK2
et al. 2001), and collagen fibril assembly due to the developmental transition (day 4
(Casey et al. 2005). There was also upregula- to day 10) and PGF treatment may have a
tion of collagen genes associated with for- critical role in increased luteolytic sensitiv-
mation of type I collagen. As expected, all ity to PGF in cattle. This increase in
the genes in the steroid biosynthesis cate- CAMKK2 occurred at a developmental stage
gory were reduced in regressing CL com- (day 10), when PGF had an increased ability
pared with functional CL. In addition, there to elicit a rise in intracellular calcium
was a large decrease in the insulin-like concentrations compared with those in day
4 CL. CAMKK2 is thought to increase
intracellular calcium via phosphorylation

Conceptus–Endometrial Interactions 233

of calcium/calmodulin-dependent protein the PGF receptor (PTGFR) and phospholi-
kinases such as CAMK1 and CAMK4. Once pase C, gamma 1 (PLCG1), which mediates
phosphorylated, kinase activity increased PFG signaling. In addition, they showed that
10- to 20-fold. Furthermore, CAMKs can PRL inhibited expression of transforming
activate mitogen-activated protein kinase 1 growth factor beta 1 (TGFB1), a pro-apop-
(MAPK1) and MAPK3 in several ligand-stim- totic cytokine (Stocco et al. 2001). Consistent
ulated pathways. with this hypothesis, PRL induced expres-
sion of a number of genes involved in steroid
Although there are a large number of can- biosynthesis, whereas PGF inhibited these
didate genes identified and characterized same genes and reduced expression of the
in CL of farm species, there are few studies luteinizing hormone (LH) receptor.
that have attempted to characterize the
transcriptomes of the CL at various stages Foyouzi and coworkers (2005) found that
of function or regression. In this regard, genes involved in steroidogenesis and in
the rodent provides little help as a compara- maintaining the antioxidant status of the CL
tive model. Rodents exhibit 4- to 5-day were regulated by PGF-induced luteolysis in
estrous cycles but lack a true luteal phase. the day 19 mouse CL. Using microarray
In the absence of mating, the CL does not analysis, the same authors found that
become fully functional and produces AKR1C1 expression increased in response to
scant progesterone for about 2 days and exogenous PGF, which would result in the
increased activity of aldo-keto reductase conversion of progesterone to its inactive
family 1, member C1 (dihydrodiol dehydro- metabolite 20α-hydroxyprogesterone. They
genase 1; 20-alpha [3-alpha]-hydroxysteroid further showed that CL undergoing induced
dehydrogenase [AKR1C1]) producing 20α- regression expressed higher concentrations
hydroxyprogesterone, which does not support of cytochrome P450, family 17, subfamily A,
pregnancy or the decidual reaction (see polypeptide 1 (CYP17A1), which would
Bachelot and Binart 2005). Mating-induced result in increased production of androstene-
surges in prolactin (PRL) are the signal, which dione. Androgens have previously been
is initially responsible for establishing a fully shown to induce abortion in mice (Sridaran
functional CL that will support gestation in et al. 1991). Interestingly, Sidran et al. (1991)
rodents. also showed that CYP19A1 and HSD17B7
(hydroxysteroid (17-beta) dehydrogenase 7)
Stocco and coworkers (2001) conducted expression decreased in response to PGF.
cDNA expression array experiments to CYP19A1 converts androstenedione to
examine the effects of PRL and PGF on the estrone, and HSD17B7 converts estrone to
transcriptome of the rat CL. In response to the more biologically active 17β-estradiol.
mating, the rodents produce diurnal surges These results suggest that PGF action on the
of PRL from the anterior pituitary which rodent CL involves increased metabolism of
support CL function for the first half of ges- progesterone to an inactive metabolite and
tation (Soares et al. 2007). As in the farm a reduction in the ability of the CL to produce
species, PGF is responsible for luteal regres- estrogens, perhaps resulting in further
sion in rodents, but its origin is the ovary increases in androstenedione.
and not the uterus. Stocco and coworkers
(2001) hypothesized that PRL effects were Another family of genes involved in
mediated in part by antagonizing the effects luteal function is the superoxide dismutase
of PGF, including decreasing expression of (SOD) family. These genes are involved in

234 Physiological Genomics of Reproduction

converting the superoxide radical to hydro- receptor (LHCGR). Bogan and coworkers
gen peroxide. Foyouzi and coworkers (2005) (2008) used genomic approaches to identify
found a decrease in SOD2, SOD3, and copper differentially expressed genes at different
chaperone of SOD1 (CCS) expression at the stages of CL development in the rhesus
end of pregnancy in mice, which may reduce macaque CL. They identified changes in
the capacity of luteal cells to cope with gene families associated with immune func-
superoxide accumulation. In that study, tion; hormone and growth factor signaling;
other members of the free radical scavenging steroidogenesis; and prostaglandin biosyn-
family were high in functioning CL and thesis, metabolism, and signaling. They
reduced in response to PGF. Oxidative stress divided CL developmental stages in early
enzymes in the glutathione S-transferase (3–5), mid (7–8), mid-late (10–12), late (14–
(GST) family were also shown to be increased 16), and very late (18–19) after the mid-cycle
by PRL and decreased by PGF in the rat CL LH surge. Using an Affymetrix® (Affymetrix,
(Stocco et al. 2001). Thus, it is likely that Santa Clara, CA) rhesus macaque genome
responses to PGF include an increase in free DNA microarray, they identified 3234 dif-
radicals that coincides with luteal regres- ferentially expressed genes (1418 up and
sion. Whether this is actually inducing 1816 down; Bogan et al. 2008). Early in
regression or merely a result of CL regres- CL development, most transcripts were
sion remains to be determined. increased, whereas later in development the
converse was true. One of the strengths of
In a recently published study using the this study was their extensive validation
bonnet monkey (Macaca radiata), PGF and of microarray results using both quantita-
chorionic gonadotropin (CG), the luteotro- tive polymerase chain reaction (qPCR) and
pin in humans and subhuman primates, protein quantification. Not surprisingly,
were found to have opposite effects on a genes involved in immune function were
number of genes that are thought to be criti- expressed at highest concentrations in the
cal for CL function (Priyanka et al. 2009). late and very late stage CL, supporting a role
Not surprisingly, genes associated with for the immune system in CL regression.
steroid biosynthesis, including steroidogenic Bishop and coworkers (2009) used the
acute regulatory protein (STAR), CYP11A1, macaque array to examine the effects of LH
hydroxy-delta-5-steroid dehydrogenase, 3 and progesterone on CL gene expression.
beta- and steroid delta-isomerase 1 (HSD3B1), They identified nearly 1500 LH-regulated
and CYP19A1 were increased by CG and genes in the macaque CL, with less than
decreased by PGF. This analysis also showed one-third of these being affected by steroid
that the monkey CL contained all the neces- ablation and progestin replacement. Never-
sary enzymes for de novo cholesterol bio- theless, several genes in the canonical
synthesis and that these enzymes were steroid biosynthetic pathway were affected
increased by CG (Priyankda et al. 2009). The similarly by LH or steroid withdrawal
authors suggested that the effect of PGF was and replacement including STAR, sterol-
to interrupt LH signaling downstream of C4-methyl oxidase-like (SC4MOL), and
receptor binding. This is supported by the CYP19A1 (Bishop et al. 2009). Results from
fact that CL regression in monkeys occurs these genomic studies fit with the concept
without noticeable changes in LH secretion. that the primate conceptus rescues the CL
In addition, PGF treatment also reduced using a different mechanism from that of the
expression of the LH/choriogonadotropin

Conceptus–Endometrial Interactions 235

farm species. Namely, CG mimics LH func- function, was termed the period of maternal
tion and abrogates the luteolytic effects of recognition of pregnancy by R.V. Short
PGF produced in the ovary. In addition, (1967), a designation that persists today.
these studies provide further support for the Work conducted during the late 1960s and
concept that intraluteal prostaglandin E early 1970s by a number of laboratories iden-
(PGE) and PGF are important regulators of tified the luteolytic signal as PGF. A number
CL function in primates. of studies, most notably those from the
McCracken laboratory, showed that the
10.3 Physiological genomics of uterus of sheep and cattle produced pulses
blocking luteal regression of PGF around the time of luteal regression
and that these pulses were absent in early
10.3.1 Conceptus signals pregnant animals (McCracken et al. 1999).

In the early part of the 20th century, Loeb Once the luteolytic signal and the critical
was the first to suggest and later demon- window for the conceptus to block this
strate that the uterus could influence CL signal were defined, work began to deter-
function in guinea pigs (Short 1967). Later mine the nature of the conceptus factor(s)
research by Moor and Rowson in the 1960s responsible. Early work on effects of the con-
showed that products of the ovine conceptus ceptus on endometrial function utilized con-
were responsible for blocking luteal regres- ceptus and endometrial tissue cultured in
sion (Short 1967). This set off a flurry of the presence of radiolabeled amino acids
work to identify factors produced by the to identify proteins that were produced
conceptus that could extend the lifespan of during the period of maternal recognition of
the CL (see McCracken et al. 1999). This pregnancy (see Bazer and Spencer 2006).
early work ruled out a systemic effect of the This approach allowed identification of de
conceptus on CL lifespan and found that the novo synthesized proteins following two-
conceptus must be present in the uterus by dimensional gel electrophoresis and Western
day 12 to extend CL function in sheep (Bazer blotting, which are referred to today as pro-
and Roberts 1983; Spencer and Bazer 2004). teomic techniques. These studies revealed a
This was followed by studies in cattle that group of low-molecular-weight proteins pro-
showed that the critical period was 2 to 3 duced by the conceptus that, when intro-
days later. Similar work in pigs established duced into the uterine lumen in their purified
that conceptus signaling commenced around form, extended CL lifespan in cattle, sheep,
days 11 to 12. These studies were important and goats (Bazer 1992). These proteins were
for two reasons. First, they narrowed the later cloned and determined to be members
search for the conceptus signal responsible of the interferon (IFN) family of genes that
for blocking luteal regression to a defined were most closely related to the Type I IFN
window of early pregnancy. Second, they omega family (see Roberts et al. 2008).
demonstrated that there were no conceptus Conceptus IFNT was shown to alter the
signals prior to this period that were abso- pattern of PGF release and abolish the large
lutely required for pregnancy establishment. pulses of PGF that were, by then, known to
This time of early pregnancy, when the con- mediate CL regression. Other studies also
ceptus must signal the dam to extend CL suggested that there was an increase in the
PGE : PGF ratio that might also contribute to
the maintenance of CL function (Arosh et al.

236 Physiological Genomics of Reproduction

2009), although the significance of PGE in extend CL function for a period close to the
luteal maintenance is not clear because length of gestation in swine (114 days;
hysterectomy (and removal of any uterine Geisert et al. 1982a).
PGE) results in extension of CL lifespan in
sheep (Kiracofe and Spies 1966). In either Our understanding of the effects of the
case, there is now evidence for significant conceptus on luteal maintenance in horses
increases in endometrial and luteal PGE lags well behind that for ruminants and
production during early pregnancy in rumi- swine. Attempts to establish roles for equine
nants (Asselin and Fortier 2000) and swine conceptus steroid or protein hormones have
(Waclawik and Ziecik 2007). Part of this not been met with much success (Sharp
increase in PGE is due to the activity of et al. 1989). Although the horse conceptus
9 keto reductase (20-beta hydroxysteroid does produce steroid and protein hormones,
dehydrognease or carbonyl reductase [CBR]), these have not been shown to be responsible
which can convert PGF to PGE (Waclawik for alterations in uterine prostaglandin pro-
and Ziecik 2007). duction in horses (Bettridge 2007). Interest-
ingly, recent evidence suggests that the
The conceptus signals mediating luteal horse conceptus produces a unique Type I
maintenance in pigs differ from those in IFN, IFN delta (IFND), similar to that pro-
ruminants in two important ways. The first duced by the pig conceptus (Cochet et al.
was the effect of the conceptus on uterine 2009). Whether this IFN induces changes in
PGF production. Bazer and Thatcher (1977) the endometrium associated with maintain-
first proposed the endocrine–exocrine theory ing CL function remains to be determined.
for CL rescue in swine, which established The most compelling evidence for a signal
that in cyclic pigs PGF was released toward mediating CL rescue in horses comes from
the uterine vasculature (endocrine secretion) studies showing that rapid and sustained
and that the conceptus changed the direc- conceptus migration between uterine horns,
tion of secretion toward the uterine lumen which occurs between days 12 and 17 after
(exocrine secretion). This hypothesis was mating, was critical for CL rescue. If the
built upon Bazer and Thatcher’s observa- conceptus was confined to the tip of one
tions that a purple iron transport protein, uterine horn, it could not rescue CL func-
uteroferrin, was secreted into the uterine tion (Sharp et al. 1989). Furthermore, intro-
lumen during early pregnancy but toward duction of a glass ball (∼30 mm) into the
the uterine vasculature in cyclic pigs. uterine lumen of cyclic mares inhibited
Discussion of this phenomenon with a col- estrous behavior and extended progesterone
league and eminent fetal physiologist, Dr. production in about 40% of mares (Nie et al.
Donald Barron, formed the initial concept of 2001), suggesting that physical contact of
the endocrine–exocrine theory (F.W. Bazer, the endometrium had the ability to alter the
personal communication). The second major luteolytic mechanism.
difference was that the conceptus signal
mediating this effect was the steroid estro- One can summarize from the studies just
gen and not an IFN as in ruminants. Work mentioned that ruminants utilize IFNT and
from the Bazer lab was the first to show that swine estrogen as the initial conceptus
there were two periods of conceptus estro- signals responsible for blocking luteal regres-
gen production, around day 12 and again at sion. Both of these conceptus signals alter
day 15 after mating, which were required to PGF release by the endometrium to main-
tain CL function. In addition, introduction

Conceptus–Endometrial Interactions 237

of these hormones into the uterus at the approximately 5 days after pregnancy signal-
appropriate times will extend CL function. ing commences. In addition, there are a large
Horse conceptus signaling involves embryo number of additional studies that have been
migration between uterine horns, and, conducted using candidate gene approaches
undoubtedly, some conceptus produced that grew out of discoveries using proteomic
factors that have yet to be determined. analysis of endometrial- and conceptus-con-
ditioned culture media (Wolf et al. 2003;
10.3.2 Uterine responses to Spencer and Bazer 2004). Of course, early
conceptus signals studies in sheep and cattle focused on known
IFN-stimulated genes (ISGs), and later work
Ruminants focused on the interaction between proges-
A number of genomic studies of the physi- terone and IFNT in regulating uterine gene
ological responses to conceptus signaling expression. This work was recently reviewed
have been conducted in recent years by Spencer and coworkers (2007, 2008) and
(reviewed by Spencer et al. 2008). These Bazer and coworkers (2008) and summarized
include studies examining the effects of in Figure 10.1.
pregnancy and progesterone on endometrial
gene expression in sheep (Spencer et al. 1999; The difficulty with global gene expression
Gray et al. 2006; Satterfield et al. 2010); preg- profiling studies is teasing out endometrial
nancy on endometrial gene expression in responses to the conceptus that are involved
cattle (Klein et al. 2006; Bauersachs et al. in CL rescue from the responses mediating
2006, 2007, 2008, 2009; Forde et al. 2009); conceptus growth, attachment, elongation,
pregnancy on gene expression profiles in the and placentation, which all occur in response
caruncular and intercaruncular endome- to continued conceptus signaling and main-
trium of cattle (Mansouri-Attia et al. 2009); tenance of progesterone production. Clearly,
and pregnancy on gene expression in porcine there are dramatic changes in gene expres-
endometrium (Ka et al. 2009). In addition, sion associated with induction of uterine
Chen and coworkers (2007) examined the secretion, remodeling, and immune accom-
effects of IFNT on gene expression in an modation at the fetal–maternal interface.
immortalized ovine luminal epithelial cell Relevant to the present discussion, however,
line, and Kim and coworkers (2003), in oxytocin receptor (OXTR) and estrogen
human cell lines. Of these, the work by Gray receptor alpha (ESR1) expression are both
and coworkers (2006), Klein and coworkers reduced in pregnant or IFNT-treated endo-
(2006), Forde and coworkers (2009), and Ka metrium compared with cyclic endome-
and coworkers (2009) are most relevant to trium. In the absence of OXTR and ESR1,
the present discussion on physiological the endometrium does not respond to oxy-
genomics of CL rescue because endometrial tocin (OT) from the CL and posterior pitu-
tissues analyzed in these studies were from itary and cannot produce the pulsatile
the early period of the window of pregnancy pattern of PGF that induces luteal regression
recognition signaling (e.g., day 14 for sheep, (Spencer and Bazer 2004). Spencer and Bazer
day 18 for cattle, days 13 and 16 for cattle, (2004) developed a model for conceptus-
and day 12 for swine, respectively). The work mediated CL rescue in ruminants. In this
by Mansouri-Attia and coworkers (2009) model IFNT, binds the Type I IFN receptor
focused on day 20 of pregnancy in cattle, on the endometrial luminal epithelium and
activates a signaling pathway resulting in

10/11 Day of pregnancy 18/20
12/13 14/15 16/17 IFNT

Conceptus CSH1
Uterine
Lumen PGs

Uterine n.d. GLYCAM1
Luminal LGALS15
epithelium
(LE) SPP1

Uterine Glucose
Glandular
epithelium Amino acids
(GE)
CTSL
Uterine
CST3

CXCL10

PGR

MUC1

GLYCAM1

LGALS15

CTSL

CST3

WNT7A

PTGS2

IGFBP1

HSD11B1

SLC2A1

SLC5A1

SLC7A2

VEGFA

ISG15

RSAD2
PGR

PRLR

SPP1

CTSL

CST3

GRP

UTMP

STC1

SLC5A11

SLC7A2

STAT1

STAT2

IRF9

IRF1

GBP2

IFIT1

IFIH1

RSAD2

IFI27
ISG15
MIC
B2M

RSAD2

ISG15
PGR

Figure 10.1 Temporal and spatial roadmap of progesterone and IFNT stimulated genes in the uterus
during establishment of pregnancy in sheep. The relative abundance of mRNA or protein in the uterus
across days of pregnancy is indicated as well as regulation of genes by progesterone and/or IFNT.

= downregulated by progesterone; = induced or stimulated by progesterone; =induced or stimulated
by IFNT; = induced by progesterone and stimulated by IFNT; n.d. = not determined due to inability to
flush intact conceptuses from the uterine lumen on days 18–20 of pregnancy.

Conceptus–Endometrial Interactions 239

inhibition of ESR1 gene transcription, which ably due to the potent IRF2 repressor that is
abrogates OXTR expression and thus pro- specifically expressed in the endometrial
duction of luteolytic pulses of PGF. Available lumenal and superficial glandular epithe-
data support the idea that IFN regulatory lium and increased during early pregnancy.
factor two (IRF2), a potent transcriptional Thus, the canonical JAK-STAT pathway
repressor, is involved in IFNT inhibition of mediating the effects of IFNT in the stroma
ESR1 gene transcription. The luminal epi- and glandular epithelium is not active in the
thelia of the endometrium is responsible for endometrial lumenal epithelium of preg-
production of the bulk of PGF during luteal nancy. Consequently, most classical ISGs
regression. Interestingly, PTGS2 (prosta- (interferon-stimulated gene 15 [ISG15], beta-
glandin-endoperoxide synthase 2 [prosta- 2-microglobulin [B2M], radical S-adenosyl-
glandin G/H synthase and cyclooxygenase]), containing domain two [RSAD2], etc.) are
the enzyme responsible for producing the not expressed or induced by IFNT in the
precursor to PGF, PGG2, is not suppressed endometrial lumenal epithelium of early
by the conceptus or IFNT. PTGS2 is pregnancy, which may have implications in
expressed during early pregnancy in the maternal tolerance of the conceptus allograft
ovine endometrium as well as conceptus (Johnson et al. 2002; Choi et al. 2003; Song
trophectoderm and is postulated to mediate et al. 2007). There is at least one classical
production of prostaglandins that are critical ISG, MX1, that does not comply with this
for uterine receptivity and conceptus sur- model and is induced the endometrial
vival in rodents, including PGE2 and PGI2 lumenal epithelium of pregnant sheep (Ott
(Wang and Dey 2005). et al. 1998; Johnson et al. 2002; Hicks et al.
2003) and cattle (Mansouri-Attia et al. 2009).
In the sheep endometrium, the tran- The mechanism of induction of MX1 (Assiri
scriptional repressor IRF2 is specifically et al. 2007), as well as many nonclassical
and constitutively expressed in the endome- IFNT-stimulated genes in the endometrial
trial luminal epithelia and increases during luminal epithelium, remains to be deter-
early pregnancy. IRF2 is a potent repressor mined but is postulated to involve a unique,
of IFN-stimulated gene transcription (see STAT1-independent signaling pathway
Spencer et al. 2007; Bazer et al. 2009). (Spencer and Bazer 2004; Bazer et al. 2009).
Therefore, the signaling components that
mediate induction of classical ISGs are Available data support the idea that
apparently suppressed in the endometrial ovarian progesterone and conceptus IFNT
luminal epithelium but remain functional act synergistically on the endometrial epi-
in the glandular epithelium and stroma. thelia of the ruminant uterus to regulate
Although the receptors for IFNT (interferon genes important for conceptus development
alpha subunit receptors one and two [IFNAR1 and production of IFNT as well as uterine
and IFNAR2]) are most abundant on the receptivity conceptus implantation. Expres-
endometrial epithelia, the transcription sion of many endometrial lumenal epithe-
factors that govern classical Type I IFN lium genes is initiated between days 10
responses in many different cell types (signal and 12 post-estrus/mating in both cyclic
transducer and activator of transcription one and pregnant ewes (Figure 10.1). Hormone
and two [STAT1, STAT2] and IFN regula- replacement studies in sheep found that P4
tory factor nine [IRF9], which forms the induces the expression of many genes in the
ISGF3 complex) are not present, most prob- endometrial lumenal epithelium that encode

240 Physiological Genomics of Reproduction

adhesion proteins (galectin-15 [LGALS15], hundred fold increase in IFNG mRNA as the
insulin-like growth factor binding protein conceptus develops from its spherical to its
one [IGFBP1]), enzymes (PTGS2), a protease filamentous forms between days 10 and 14
(cathepsin L [CTSL1]), a protease inhibitor (Ross et al. 2009). It is clear that pig concep-
(cystatin C [CST3]), cell proliferation factors tus IFN is affecting endometrial function via
(gastrin-releasing peptide [GRP]), glucose induction of STAT1 (Joyce et al. 2007);
transporters (SLC2A1, SLC5A1), and cat- however, it does not appear that this IFN
ionic amino acid (arginine, lysine, and orni- plays a role in blocking luteal regression.
thine) transporter (SLC7A2). In the glandular
epithelium, P4 induces genes that encode a Green and coworkers (2006) used a custom
cell proliferation factor (GRP), a glucose cDNA microarray to identify 4827 genes
transporter (SLC5A11), adhesion protein that were differentially expressed in the
(secreted phosphoprotein one [SPP1]), regu- porcine endometrium across the estrous
lator of calcium/phosphate homeostasis cycle. This report also presents an excellent
(stanniocalcin one [STC1]), and an immuno- overview of the technologies, databases, and
modulatory factor (uterine milk protein challenges associated with transcriptional
[UTMP]). Of particular note, IFNT stimu- profiling experiments. Although the experi-
lates a number of those P4-induced genes ments examined changes in gene expression
that encode secreted proteins (including at days 0, 3, 6, 10, 12, 14, and 18, days 10
CST3, CTSL1, GRP, LGALS15) as well as and 12 are most relevant to the present dis-
transporters for glucose (SLC2A1, SLC5A11) cussion because they represent the tran-
and amino acids (SLC7A1, SLC7A2). IFNT scriptome of the endometrium at the time
stimulation of most of these genes requires when conceptus signals are first received
P4 action. The combinatorial effects of P4 (http://genome.rnet.missouri.edu/swine).
and IFNT on the endometrium are hypoth- Interestingly, day 12 endometrium exhib-
esized to result in specific changes in the ited the highest number of differentially
intrauterine milieu necessary for conceptus expressed genes (542) compared with the
elongation and development (see Spencer other days. The largest proportion of differ-
et al. 2008). entially expressed genes from days 10–14
were associated with signal transduction,
Swine particularly those associated with receptor
Swine conceptuses produce estrogen tyrosine kinase activity (Green et al. 2006).
between days 11 and 15 after mating which This is in contrast to differentially expressed
is responsible for changing the direction of genes from days 0 and 18, which clustered
endometrial PGF secretion towards the predominantly in the immune function
uterine lumen and away from the uterine theme. This experiment is an excellent
vasculature (Bazer and Thatcher 1977; Bazer example of how global gene expression pro-
et al. 1998). In addition, the conceptus also filing can be used to survey the physiological
produces significant amounts of IFN gamma “landscape” at the time when the conceptus
(IFNG; LeFevre et al. 1990; Murphy et al. is first signaling the endometrium to rescue
2009) and delta (Lefevre and Boulay 1993), as CL function.
well as interleukin 1, beta (IL1B; Ross et al.
2003), during the initial period of conceptus Pregnancy recognition signaling in swine
elongation. For example, there is a several coincides with conceptus and endometrial
estrogen production that is responsible for
the endocrine–exocrine switch in endome-

Conceptus–Endometrial Interactions 241

trial PGF secretion (Perry et al. 1973; Bazer endometrium collected from cyclic or preg-
and Thatcher 1977; Bazer et al. 1998; Tayade nant pigs at day 12 after estrus. Day 12 rep-
et al. 2007; Franczak and Bogacki 2009). resents the earliest period that conceptus
It is now clear that although PGF and estrogen-mediated responses could be
other prostaglandins are detrimental to CL detected and changes in gene expression are
function when secreted into the uterine vas- likely involved in mediating the endocrine–
culature, they are critical to successful con- exocrine switch in pigs. It was interesting
ceptus growth, attachment, and placentation that three of the conceptus-induced endo-
(Ashworth et al. 2006; Waclawik et al. 2006). metrial genes identified using this approach,
Although the functioning of the endocrine– S100A7A (S100 calcium binding protein
exocrine switch has been known for some A7A), GSN (gelsolin [amyloidosis, Finnish
time (Gross et al. 1988), the actual genes and type]), and TRPV6 (transient receptor poten-
physiological pathways mediating this effect tial cation channel, subfamily V, member 6),
have not been established. Moreover, few are involved in calcium regulation (Eckert
studies have attempted to define the tran- et al. 2004; Sun et al. 1999; Hoenderop et al.
scriptome of the pregnant endometrium in 2005). Prior work has shown that calcium
swine in response to conceptus estrogen increases in uterine secretions at the time of
(Jiang et al. 2003; Ka et al. 2009). conceptus elongation (Geisert et al. 1982a,b)
and that the calcium ionophore A23187 is
Candidate gene approaches have defined able to activate the endocrine–exocrine
a critical role for OT in inducing uterine switch (Gross et al. 1990). Whether any of
PGF production during luteolysis in swine these genes are involved in the endocrine–
(Carnahan et al. 1996). Regulation appears to exocrine switch remains to be determined.
occur both at the level of OXTR number
(Ludwig et al. 1998; Franczak et al. 2005; Horse
Oponowicz et al. 2006) and OXTR coupling It has been over two years since the first
to its second messenger system in the endo- assembly of the horse genome was published
metrium (Ludwig et al. 1998). These changes (see Ramery et al. 2009). The horse genome
occur on a backdrop of elevated PGFS and has now been sequenced at close to seven
endometrial capacity to produce PGF. The times coverage, which is similar to that
effects of conceptus estrogen on pulsatile available for mice, rats, and dogs. However,
release of endometrial PGF likely involves the horse genome lags behind these species
changes in OXTR numbers that are medi- in terms of annotation. To date, there have
ated via the nuclear ESR1 (Franczak and been no genomic or proteomic studies
Bogacki 2009). Recently, however, there has focused on the conceptus signals mediating
been evidence that estrogen may also act via CL rescue in the mare. What is available
a membrane-bound ESR1 through activation stems from early “proteomic” studies that
of Akt to alter translation initiation in examined the array of horse conceptus secre-
porcine endometrial cells (Wollenhaupt tory products and their effects on CL rescue
et al. 2007). in mares (see Sharp et al. 1989; Allen 2001).

Recently, Ka and coworkers (2009) uti- The physiological genomics of CL rescue
lized a technique called annealing control in the mare remains enigmatic. Neither
primer-based reverse transcription PCR conceptus estrogen nor proteins have been
(ACP RT-PCR; Hwang et al. 2003) to iden- determined to rescue CL function when
tify differentially expressed genes between

242 Physiological Genomics of Reproduction

introduced into the uterine lumen of the must take place to confirm genes identified
mare. Clearly, however, the equine uterus using global screening methods. In addition,
exhibits reduced responsiveness to OT and proteomic technologies have matured and
lowered PGF production during early preg- been automated to allow cataloging of pro-
nancy (Sharp et al. 1989; Starbuck et al. teins that regulate critical cellular processes.
1998). Most intriguing is the fact that the As the robustness and sensitivity of these
horse conceptus is propelled rapidly between techniques improves, they will provide new
uterine horns between days 10 and 14 after opportunities for evaluating the transcrip-
mating and that this movement is critical tional and translational control of gene
for blocking luteolytic production of PGF by expression in the endometrium, conceptus,
the endometrium (Allen 2001). This move- and CL. With publication of human and
ment is clearly affected by conceptus pro- animal genomes and their more complete
duction of prostaglandins (Stout et al. 2001; annotation, newer bioinformatic and statis-
2002); however, plastic spheres introduced tical tools are allowing these genes and their
into the uterine lumen are also propelled protein products to be organized into fami-
between uterine horns (although to a lesser lies for evaluation of pathways activated
extent) and extended CL function in approx- in the endometrium and CL by conceptus
imately 75% of mares (Rivera del Alamo signals. However, we are still well away
et al. 2008). The current evidence suggests from a complete understanding of the
that either some factor on the surface of the process. What these technologies have pro-
equine capsule or present in low concentra- vided in essence is the cast of characters for
tions adjacent to the conceptus mediates a story that is not near fully written. Future
this response, or the physical contact of the advances will rely on the painstaking deter-
equine capsule (or glass bead) is responsible mination of how these characters interact
for altering uterine PGF production (Rivera during the process of establishment of preg-
del Alamo et al. 2008). Both in the area of nancy. This will allow determination of
conceptus signals and in the area of endome- the etiology of pregnancy failure, and guide
trial responses to those signals, the physio- attempts to regulate reproductive processes
logical genomics of CL rescue is certainly to improve efficiency of animal agriculture.
ripe for investigation in equids. This holistic or systems approach to repro-
ductive biology represents a great advance
10.4 Future research directions over the incremental approaches of the past
(Hiendleder et al. 2005). Only by taking this
Although global transcriptional profiling has approach can the physiological genomics
been widely used for less than a decade in of conceptus–endometrial interactions be
livestock species, it has greatly expanded the understood and, even more importantly, be
known universe of genes participating in manipulated to improve animal agriculture.
conceptus–endometrial interactions that
meditate rescue of CL function in domestic Acknowledgments
farm animals. In many cases these studies
have confirmed previous work utilizing can- The research reported here was supported
didate gene approaches. However, there still in part by National Research Initiative
remains quite a bit of validation work that Competitive Grant No. 2005-35203-16252

Conceptus–Endometrial Interactions 243

from the USDA Cooperative State Research, genetic models in mice. Current Topics in
Education, and Extension Service to TES, Developmental Biology 68: 49–85.
and National Research Initiative Competitive Bauersachs, S., Mitko, K., Blum, H., and
Grant No. 2000-02398 from the USDA Wolf, E. 2007. Technical note: Bovine
Cooperative State Research, Education, and oviduct and endometrium array version 1:
Extension Service to TLO. Thanks to Ms. A tailored tool for studying bovine endo-
Shannon Boone for help preparing this metrium biology and pathophysiology.
chapter. Journal of Dairy Science 90: 4420–4423.
Bauersachs, S., Mitko, K., Ulbrich, S.E.,
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11

Physiological Genomics of Placental Growth
and Development

Sukanta Mondal

11.1 Introduction which contribute to successful pregnancy
and establishment of placenta.
The placenta (Greek, plakuos: flat cake) is a
functional connection between the embryo The recent developments in molecular
and the uterus which meets the significant biology and biotechnology have resulted in
challenge of accommodating the nutritional unlimited access to the genome and have
and growth regulatory needs of the develop- enhanced the pace and precision of creating
ing fetus. This feto-maternal organ begins at gene sequences and functional genomics to
implantation of the blastocyst and is deliv- meet the challenges of food, agriculture,
ered with the fetus at birth. The placenta and animal improvement. The development
plays a critical role in (1) mediating implan- of new innovative technologies for sequenc-
tation, (2) establishing the interface for ing of whole genomes and for the mass
nutrient and gas exchange between maternal screening of transcriptomes has revolution-
and fetal circulation, (3) regulating maternal ized genetic profiling, mapping as well
recognition of pregnancy, (4) altering the as our understanding of underlying phy-
local immune environment, and (5) stimu- siological mechanisms. These molecular
lating maternal cardiovascular and meta- technologies have provided new ways of
bolic functions through production of evaluating reproductive potential and the
paracrine and endocrine hormones. It pro- basic physiological mechanisms that limit
duces a plethora of functional molecules, reproductive performance. These technolo-
viz. prolactin (PRL), growth hormone (GH), gies will also provide new tools for manag-
placental lactogen (PL), insulin-like growth ing and monitoring livestock fertility.
factors (IGFs), prolactin-related proteins Therefore, understanding of genetic mecha-
(PRPs), and prolactin-like proteins (PLPs), nisms and pathways involved in plancetal
growth and function has opened new vistas

251

252 Physiological Genomics of Reproduction

for improving livestock. Furthermore, these the maternal decidua. The trophoblast cells
genomic and transcriptomic approaches produce vascular endothelial growth factor
are helpful in functional genomics and (VEGF), placenta-derived growth factor
have led to newer ways of evaluating the (PDGF), and fibroblast growth factor (FGF),
genetic components of phenotypes in live- which augment angiogenesis and placental
stock species. Advance biotechnological development.
approaches such as microarray, siRNA,
and bioinformatics tools have tremendous 11.2.2 Primary cell types of placenta
importance in understanding the complex
mechanisms of poor reproductive efficiency, In general, placentas of various species
which could lead to safe and effective gene- consist of two primary cell populations:
based strategies for enhancement of repro- an outer epithelial layer derived from the
duction and production. trophoectoderm (trophoblast), and inner
vascular and stromal layers derived from
11.2 Placental development: Basics the allantois (extra embryonic mesoderm).
Trophoblast cells are one of the earliest dif-
11.2.1 The origin of placenta ferentiating cells and show species to species
variation in their development and organiza-
After fertilization, the next major event is tion into placental structure. The tropho-
trophoblast differentiation, which is required blast layer generates the extensive area for
for implantation. For the first 4 to 6 days, nutrient exchange as well as interacting
preimplantation development takes place closely with the uterus to produce a plethora
within the oviduct. During this period the of macromolecules that promote maternal
zygote undergoes cleavage division and dif- blood flow to the implantation site. The tro-
ferentiation of innermost cells into the inner phoblast layer is covered by extensive micro-
cell mass and the surrounding cells into tro- villi, which spread diffusely across the
phoectoderm, which occurs around the 16- placenta in pigs and are clustered into micro-
cell stage. The inner cell mass subsequently cotyledons in horses or macro-cotyledons in
develops into the fetus and the trophoecto- ruminants. Rodents and primates possess a
derm gives rise to the placenta. This process hemochorial placenta in which maternal
differs somewhat between species but blood directly bathes fetal chorionic villi
also shares substantial similarities across a cells. In ruminants, the separation between
broad range of species. For example, in maternal and fetal blood is more extensive.
humans, after the trophoblast attaches to However, ruminants do form transient syn-
the endometrium, the embryo invades the epitheliochorial placentas. The maternal
endometrium through differentiation of the endometrial epithelium eventually regrows,
trophoectoderm into cytotrophoblast (inner and there is minimal invasion and maximum
layer) and syncytiotrophoblast (outer layer). cellular separation between maternal and
The syncytiotrophoblast cells migrate and fetal compartments.
ultimately line the maternal spiral arteries
opening them and causing maternal blood to 11.2.3 Placenta—An endocrine organ
flow across the cytotrophoblast. The cyto-
trophoblast forms columns of cells (villi) The placenta is directly responsible for
that invade the endometrium and anchor to mediating and/or modulating the maternal

Placental Growth and Development 253

environment necessary for fetal growth tion caused arrested development at mid-
and development. As an active endocrine gestation and depressed mouse placental
organ, the placenta is capable of secreting a lactogen-I (Prl3d1) gene expression (Ma et al.
plethora of hormones, growth factors, cell 1997). In primates, the CHS genes (PL homo-
adhesion molecules, extracellular matrix logs) are derivatives of the ancestral growth
metalloproteinase, and cytokines, which hormone gene, whereas in rodents and rumi-
play crucial roles in implantation and pla- nants, the PL genes are derived from the
centation. Although several transcription ancestral prolactin (PRL) gene. In rodents,
factors are involved in placental develop- Prl3d1 gene is specifically expressed in tro-
ment, the exact role of specific transcription phoblast giant cells and Prl3d1 mRNA is
factors is unclear. Considerable evidence reduced in Hand1 mutants (Firulli et al.
indicates that basic helix-loop-helix (bHLH) 1998). Co-transfection of Hand1 with a
transcription factors are involved in placen- Prl3d1 promoter reporter gene construct
tal trophoblast cell development. Hand1, a results in dose-dependent transactivation
bHLH transcription factor, is essential for (Cross et al. 1995). Deletion of an 86-bp
the differentiation of trophoblast giant cells. region of the promoter (between -274 and -88
The expression of Hand1 mRNA is not relative to the transcription start site) pre-
detectable at early postimplantation stages vents transactivation by Hand1, suggesting
and highly expressed in the trophoblast giant that Hand1 regulates Prl3d1 gene promoter
cell layer surrounding the implanted con- activity.
ceptus (Cross et al. 1995; Scott et al. 2000).
Firulli et al. (1998) observed that mouse 11.3 Placental hormones
embryos that are homozygous for a Hand1 and peptides
null mutation did not survive beyond day 8.
The outer layer of trophoblast cells in Hand1 11.3.1. PRL
knockout mice failed to undergo the charac-
teristic morphological giant cell appearance Prolactin plays a crucial role in placental
(Riley et al. 1998). Mash 2, another bHLH growth and development, mammary gland
gene, is required for the maintenance of development, and immune responses. The
giant cell precursors (Guillemot et al. 1994), members of the PRL family of genes include
and its overexpression in Rcho-1 cells pre- PLs, PLPs, proliferins (PLF), and PLF-related
vented giant cell differentiation. In contrast, proteins (PLF-RP). The PRL and GH genes
formation of syncytiotrophoblast cells in are closely related and evolved from a
mice is controlled by a distinct genetic common ancestral gene. The PRL family
pathway that is governed by glial cell genes in human, rat, mouse, and cow are
missing-1 (GCM-1). In humans, GCM-1 was located in chromosomes 6, 17, 13, and 22,
shown to regulate the activity of the syncy- respectively. Two exon–intron organizations
tiotrophoblast aromatase gene (CYP19). have been described; (1) five exon–four intron
Anson-Cartwright et al. (2000) reported that structure for both PRL and other members
the labyrinth layer of the placenta does not of PRL families, and (2) a six exon–five intron
form in GCM-1 null mutants and embryonic structure for members of the rodent PLP-C
death occurs at day 10. The zinc finger pro- subfamily. The members of the PRL family
teins GATA-2 and GATA-3 are expressed possess four conserved cysteine residues
in trophoblast giant cells, and their disrup-

254 Physiological Genomics of Reproduction

(Nicoll et al. 1986). However, placental (Ain et al. 2003; Wiemers et al. 2003). In
lactogen-I (PL-I), PL-I variants, (PL-Iv), and mice and rats, at least 19 different genes
prolactin-like protein-A (PLP-A) possess a with some similarity to PRL, such as PL,
fifth cysteine (Cohick et al. 1996), and PRL, PLPs, PRPs, PLF, and PLF-RP, have been
PLF, and members of the PLP-C family identified. In the ruminant placenta, unlike
has six cysteine residues (Roby et al. 1993). GH, no PRL activity has been reported.
The PRL family members possess post- However, PRP genes are expressed in binu-
translational addition of carbohydrates, cleate cells of cow and sheep placentae
except PL-II and PLP-Cv. Glycosylation is (Anthony et al. 1995). In cattle and sheep,
an important post-translational modifica- binucleate trophoblast cells and endometrial
tion in eukaryotic cells which influences heterokaryons (fusion of binucleate tro-
the structure and biological function of phoblast cells with endometrial epithelial
proteins, including effects on protein stabil- cells) produce PLs. In cattle, PRP-1 is
ity, protein secretion, protein half-life, recep- expressed in the placenta during the early
tor interaction, and subsequent downstream peri-implantation period and before bPL can
biological activities. PRL family members be detected (Yamada et al. 2002).
expressed by spongiotrophoblast cell possess
distinct glycosylation patterns that involve The members of the PRL family exhibit
Asn-linked oligosaccharides containing both two types of biological functions: classical
N-acetyl galactosamine (GalNAC) and sialic and nonclassical. Classical actions involve
acid. There are differences in glycosylation biological effects mediated through PRL
patterns between mouse and rat PRL. There and/or GH receptors, whereas nonclassical
are two putative N-linked glycosylation actions represent the mechanisms of ligand-
sites that correspond to two glycoproteins mediated biological activities. The cellular
of 29 and 33 kDa in rat, whereas mice possess targets for nonclassical members of the PRL
a single putative N-linked glycosylation family include endothelial cells (angiogene-
site that corresponds to a 29 kDa protein sis), erythrocyte and megakaryocyte precur-
species. sors (erythropoiesis), natural killer cells
(immune response), eosinophils, and hepato-
The members of the PRL family are cytes. The PRL receptor gene, a member
expressed in cell type-, location- and tempo- of the cytokine receptor superfamily, is
ral-specific patterns in the uteroplacental comprised of 10 exons and 9 introns. The
compartment and the anterior pituitary. receptor has two 5′ promoters that direct
Rodent trophoblast giant cells, spongiotro- transcription of a 598 amino acid protein,
phoblast cells, and invasive trophoblast cells which is composed of an extracellular
each produce a unique subset of PRL family domain (ECD), a hydrophobic transmem-
members. Transcriptional control of tropho- brane domain, and a cytoplasmic region
blast giant cell-specific gene expression homologous to GH receptors.
has been studied using the Rcho-I tropho-
blast cell line (Lu et al. 1994; Peters et al. Species differences exist in ligand–
2000). During the last week of gestation, a receptor interaction. In some species, ligands
population of trophoblast cells exits the that are produced at the feto-maternal inter-
chorioallantoic placenta and invades the face bind with the PRL receptor, whereas in
uterine mesometrial compartment. Here other species hormones/cytokines are pro-
they express a subset of PRL family members duced to activate both PRL and GH recep-
tors. PRL binding to the receptor causes

Placental Growth and Development 255

dimerization, which induces protein tyro- and goats are evolutionarily related, they
sine phosphorylation and activation of JAK2 differ from each other in the ways their pla-
kinase and STATS 1 to 5 (Prigent-Tessier cental GH and PRL-like hormones evolved.
et al. 2001). The auto-paracrine effects of Humans possess a cluster of five highly
PRL in decidua are mediated by the activa- related genes. One of these is expressed in
tion of PRL signal transduction, which the pituitary (GH-N) while four are expressed
involves stimulation of Jak2-STAT5 and in the placenta: placental GH variant (GHv)
activation of phosphatidyl inositol 3 kinase/ and the chorionic somatomammotropins,
Akt signaling (Prigent-Tessier et al. 2001). CS-A, CS-B, and CS-L (Lacroix et al. 2002).

11.3.2 GH Placental GH is the product of the GHv
gene expressed in the syncytiotrophoblast of
The GH gene is a member of a multigene human placenta. During pregnancy, GHv
family that includes chorionic somatomam- expression gradually replaces pituitary GH
motropin and prolactin as well as several expression, which becomes undetectable by
other genes, which evolved through a series the end of the gestation (Lacroix et al. 2002).
of gene duplications (Gootwine 2004). GH In primates, at least five genes code for GH-
stimulates cell growth and proliferation like proteins. One is expressed in pituitary
either directly or indirectly through insulin- and four in the placenta. This cluster of GH-
like growth factor I (IGF-I). GH receptors like genes has evolved from duplications of
(GHRs) are expressed in the bovine (Scott the single GH gene.
et al. 1992; Kolle et al. 1997) and sheep pla-
centa (Lacroix et al. 1999). Although the Duplication at the GH locus occurred
activity of GH is first detected in the fetal both in sheep and goat (Yamamo et al.
pituitary and fetal circulation around days 1991), but not in cattle (Woychick et al.
50 to 60 of pregnancy in ruminants, fetal GH 1982). Two types of GH transcripts encoding
is the main source of GH activity in the feto- two GH-like proteins have been detected in
placental unit. However, GH concentrations sheep (Lacroix et al. 1996). The sequence of
in the maternal circulation are less than that one of these is identical to that for the pitu-
in the fetal umbilical cord during early preg- itary oGH gene (Orian et al. 1988). However,
nancy, suggesting that the placenta may be the other differs from pituitary oGH by the
an additional source (Lacroix et al. 1999). substitution of three amino acids: one in the
Lacroix et al. (1996, 1999) reported expres- signal peptide, the second at the border of
sion of GH by the trophoectoderm and syn- helix 1 of the GH molecule, and the third in
cytial cells of placenta between days 27 and the loop structure at the binding site (de Vos
75 of pregnancy in sheep. et al. 1992). In sheep, there are two alleles:
the GH1 allele contains a single gene copy
The GH and PRL genes are structurally (GH1), whereas in the GH2 allele, the gene
similar and evolved from a common ances- is duplicated (GH2-N and GH2-Z). The
tral precursor. The GH genes are located in sequence of the GH1 allele is identical to
chromosomes 17, 10, 11, and 22 in human, that of the pituitary oGH gene (Ofir and
rat, mouse, and cow, respectively. Gene Gootwine 1997). The sequence of the GH2-N
duplication is one of the mechanisms that gene is similar to that of the pituitary GH
allowed the evolution of placental-specific gene, but the duplicated GH2-Z gene copy
endocrine activity. Although cattle, sheep, of the GH2 allele has a three amino acid
substitution similar to GH placental cDNA

256 Physiological Genomics of Reproduction

variant (Lacroix et al. 1996). The GH activity subsequently Forsyth (1974) and Kelly et al.
in sheep placenta during early pregnancy (1974) identified PL in sheep placental tissue.
evolved to include both extrapituitary PLs are produced by binucleate cells of the
expression of the original pituitary GH gene, conceptus trophoectoderm and are secreted
and through creation of a new GH gene copy into both the maternal and fetal circulation
by gene duplication that codes for slightly in ruminants. PL is detectable in trophoblas-
modified protein (Gootwine et al. 1996). tic tissue by days 16 and 36 in ewe and cow,
Yamano et al. (1991) investigated GH genes respectively, and continues to be synthe-
in a goat genomic library and found two sized throughout pregnancy (Gootwine
types of fragments, one containing a single 2004). It is thought that fully granulated
GH gene (gGH1) and the other containing binucleate cells migrate across the microvil-
two genes arranged in tandem (gGH2 and lar junction in the placenta and fuse with
gGH3). The tandem arrangement of the maternal uterine epithelial cells to form
gGH2 and gGH3 genes is similar to that seen either transiently surviving trinucleate cells
for ovine GH2-N and GH2-Z. The ovine GH in cattle or a persistent feto-maternal syncy-
gene is expressed in uninucleate and binu- tia in sheep and goat (Soares 2004). In ewes,
cleate trophoblasts and heterokaryons of oPL can be detected in maternal circulation
placenta during days 35 to 70 of pregnancy. by day 50 with maximum levels between
Transcripts for members of GH family (GHv, days 120 and 140 and declining thereafter
CS-A, CS-B, and CS-L) were detected in the until parturition. The concentration of this
human placenta. Transcription of GH family hormone is lower in the fetus but shows a
genes is regulated by a locus control region similar decline with the advancement of
located 23 kb upstream of the cluster. In pri- pregnancy (Kappes et al. 1992). The concen-
mates, four members of the GH family, trations of PL in bovine maternal and fetal
CS-1, CS-2, CS-3, and GHv, possess exten- circulation are similar to those in sheep,
sive homology with human GH family and except that PL levels are much lower than
are expressed in chorio-allantoic placenta that in sheep (Gootwine 2004). In goat, the
(Golos et al. 1993). concentration starts to increase at approxi-
mately days 45 to 60 of the gestation and
11.3.3 PL either peaks or reaches a plateau during the
last third of pregnancy. The lower levels in
Placental lactogen, a member of the GH/ sheep and goat compared with cow suggest
PRL gene family, is secreted from the pla- that there has been some divergence in the
centa of primate, rodent, and ruminant. function of PL in these species.
Although its function and secretory control
are not completely understood, it has myriad PL arose during mammalian evolution
effects during pregnancy, such as placental through three independent events. One was
angiogenesis, maternal, and fetal intermedi- a duplication of the GH gene in primate
ary metabolism, mammary gland growth (Chen et al. 1986) and two separate duplica-
and development, ovarian and placental ste- tions of the PRL gene to give PLs and other
roidogenesis, and luteal function (Corbacho prolactin-like placental proteins in rodent
et al. 2002; Gertler and Djiane 2002; (Lin et al. 2000) and ruminant (Anthony
Gootwine 2004). The first ruminant PL was et al. 1995). Bovine and ovine PL are structur-
detected in goat (Buttle et al. 1972), and ally more similar to PRL than to GH. Schuler
et al. (1988) cloned and characterized the

Placental Growth and Development 257

ruminant PL, which is 67% identical with tion of the receptor ECD and subsequent
oPL, 51% with bPRL, 30% with bovine pro- trans-phosphorylation of receptor-associated
lactin-related cDNAI, 30% with rodent pla- JAK2 or other related kinases (Kelly et al.
cental hormone, and 20% with human PL 1974). Ruminant PLs can bind to both PRL
and bGH. Both oPL and bPL possess an and GH receptors (Anthony et al. 1995),
N-terminal disulfide loop that is character- whereas oPL can mimic the action of oPRL
istic of mammalian prolactin but is not (Sakal et al. 1997). In ruminants, PLs signal
present in somatotropins (Nicoll et al. 1986). through PRL-R homodimers and PRL-R/
Ovine PL is a non-glycosylated, single-chain, GH-R heterodimers and, in the absence
23-kDa polypeptide consisting of 198 amino of PRL-R, may act as GH-R antagonist.
acids. However, bovine PL is structurally Some of the GH-like effects of PLs may
different from oPL, having the apparent be mediated through the interaction with
molecular weight of 32 to 34 kDa, and PRL-R/GH-R heterodimers. Ruminant PLs
is secreted as multiple isoforms due to dif- have somatogenic activity in a heterologous
ferential splicing of bPL transcripts and system, but in a homologous system rumi-
allelic variants of the gene (Kessler and nant PLs antagonize GH activity (Herman
Schuler 1991; Yamakawa et al. 1990). Bovine et al. 1999; Warren et al. 1999; Gertler and
PL is heavily glycosylated and contains Djiane 2002). Studies on the interaction of
N-linked triantennary oligosaccharides and oPLs with the ECDs of oGHR and bovine
one or more O-linked carbohydrate chains. and ovine PRL receptor (PRL-Rs) revealed
However, other members of this gene family that oPL can heterodimerize GH-Rs and
such as ovine, porcine, and human PRL are PRL-Rs (Gertler and Djiane 2002). In rumi-
glycosylated, and N-linked carbohydrates nants, PLs activate Jak/STAT and mitogen-
are attached at a different portion of the mol- activated protein kinase signaling pathways
ecule (asparagines 31 in PRL compared with (Anthony et al. 1998; Gertler and Djiane
asparagine 53 in bPL). Moreover, the pla- 2002). The nature of PL signal transduction
centa does not secrete non-glycosylated bPL, differs depending on whether PRL-R forms
in contrast to PRL, of which only a portion homodimers or PRL-R/GH-R heterodimers
of the secreted protein is glycosylated (Byatt are formed. Heterodimer interaction results
et al. 1992). Depending on the protein, gly- in prolonged STAT3 activation leading
cosylation can dramatically affect biological to distinct cellular responses (Gertler and
activity. Enzymatic removal of N-linked oli- Djiane 2002).
gosaccharides increases the affinity of bPL
for the bovine somatotropin receptor by 11.3.4 PRPs
twofold (Byatt et al. 1992). However, degly-
cosylation did not affect activity using a Prolactin-related proteins are nonclassical
somatotropin bioassay or in a lactogenic bio- members of the PRL/GH family that have
assay. Therefore, although glycosylation been found in the cow, sheep, goat, mouse,
may affect receptor binding affinity, the bio- and rat placentas. They play important roles
logical activity of bPL is not dependent on in the regulation of implantation and pla-
the presence of oligosaccharides. cental formation in mammals. In cattle, at
least 13 placental PRPs have been identified
The members of the PL family exhibit a and are thought to play vital roles in implan-
similar mechanism of action and receptor tation and formation of placentomes in
activation via homo- and hetero-dimeriza-

258 Physiological Genomics of Reproduction

cattle (Kesssler et al. 1991; Yamada et al. two consensus sequences for N-glycosylation
2002). Prolactin-related protein-I belongs to and Asn X Ser/Thr at positions 60–62 and
the PRL/GH family and shares a 63% simi- 233–235, whereas bPRP-IX had four consen-
larity to bovine PRL and 45% to bovine GH sus sequences for N-glycosylation at posi-
(Schuler and Hurley 1987). The N-terminal tions 70–72, 92–94, 146–148, and 160–162.
regions of the bPRP-I and bPRP-VI proteins Ushizawa et al. (2007b) cloned and charac-
are rich in hydrophobic amino acids charac- terized PRP-I and PRP-VI cDNA in goat.
teristic of a signal peptide (Schuler and The full length cPRP-I and cPRP-VI cDNA
Hurley 1987). Bovine PRP-I and PRP-VI contained 717- and 720-bp open reading
mature proteins have three disulfide bonds frames corresponding to proteins of 238 and
with six cysteine residues at positions 39, 239 amino acids, respectively. The inferred
42, 97, 215, 232, and 238. bPRP-I has three amino acid sequence of cPRP-VI is 74%
N-glycosylation sites at positions 70-72, identical to bPRP-VI. The cPRP-I showed
92–94, and 159–161. Recently, bPRP-VII, 72% homology to bPRP-I, 61% to bRPR-II,
bPRP-VIII, and bPRP-IX were cloned and 72% to bPRP-IV, 76% to bPRP-IX, and 71%
characterized from the bovine placenta to bPRP-XII (Ushizawa et al. 2007b). Like
(Ushizawa et al. 2005a, b). Bovine PRP-VII bPRP-I, cPRP-I contains three disulfide
contains a 929 nucleotide ORF, which bonds with six cysteine residues. In contrast
encodes a protein of 238 amino acids. The to bPRP-VI, cPRP-VI has eight cysteine resi-
predicted amino acid sequence is 63% dues with six residues at positions 39,
homologous to bPRP-I and 70% to bPRP-VI 42, 43, 97,174, and 215, and an extra two
(Ushizawa et al. 2005a). Bovine PRP-VII has cysteines at positions 232 and 239. Caprine
eight cysteine residues with four disulfide PRP-I possesses two consensus sequences
bonds, which is more than other bPRPs. The for N-glycosylation at positions 70–72 and
bPRP-VIII and bPRP-IX cDNAs consist of 92–94 and an atypical N-glycosylation site,
909 and 910 bp ORFs, which correspond to Asn X Cys, at position 95–97 (Ushizawa
proteins of 236 and 238 amino acids, respec- et al. 2007b). Unlike bovine PRP-VI,
tively. The inferred amino acid sequence of which has only one consensus sequence,
bPRP-VIII was 69% identical to bPRP-VI, cPRP-VI has three consensus sequences for
66% to bPRP-VII, 61% to bPRP-I and bPRP- N-glycosylation at positions 48–50, 60–62,
III, 58% to bPRP-IV and bPRP-V, 57% to and 70–72, with the atypical glycosylation
bPRP-IX, and 42% to bPRP-II (Ushizawa site (Asn X Cys) at positions 95–97.
et al. 2005b). The deduced amino acid
sequence of bPRP-IX protein showed 81% Recently, Ushizawa et al. (2007a) cloned
homology to bPRP-IV, 76% to bPRP-I, 70% two novel ovine PRPs: oPRP-I and oPRP-II.
to bPRP-II, 60% to bPRP-VII, 57% to bPRP- Ovine PRP-II had a typical PRP sequence
VI and bPRP-VIII, and 53% to bPRP-III and similar to bovine PRP-I. Ovine PRP-I had a
bPRP-V. Phylogenetic analysis revealed that shorter sequence lacking 52 bp from the
bPRP-VIII, bPRP-III, bPRP-VI, and bPRP-VII coding region of other PRP sequences
comprise one clade, whereas bPRP-IX, (positions 529–580). Phylogenetic analysis
bPRP-II, and bPRP-IV comprise another revealed that oPRP-I and bPRP-I, bPRP-II,
clade (Ushizawa et al. 2005b). The N-terminal bPRP-IV, bPRP-IX, bPRP-XII, bPRP-XIV,
regions of bPRP-VIII and bPRP-IX possess and cPRP-I are closely related. In contrast,
oPRP-II was more distant from bPRP-I,

Placental Growth and Development 259

bPRP-II, bPRP-IV, bPRP-IX, bPRP-XII, bPRP- possesses two putative N-glycosylation sites
IV, and cPRP-I. Both oPRP-I and oPRP-II are and eight cysteine residues, of which six are
expressed in trophoblast binucleate cells in highly conserved in the placental PRL family
cattle and goats. Ovine PRP-I expression (Iwatsuki et al. 1998). Similar to PLP-C and
declined from early to mid-gestation, PLP-D, PLP-H mRNA first appears on day
whereas oPRP-II expression remained con- 14 of pregnancy and expression increases
stant throughout the gestation period. until term. Iwatsuki et al. (1996) cloned a rat
PLP-D cDNA which encodes a protein of
11.3.5 PLPs 240 amino acids including a signal peptide
of 29 amino acids. It contains a putative
Prolactin-like proteins belong to the GH/ N-glycosylation site and six cysteine resi-
PRL family and have structural similarity to dues that are highly conserved in the placen-
PRL and PL. Lin et al. (1997) cloned, charac- tal PRL family. The deduced amino acid
terized, and expressed three members of sequence of PLP-D is 80% homologous to
mouse PLP, including PRL-like protein A PLP-E and 73% to decidual PRL-related
(PLP-A), PLP-B, and decidual/trophoblast protein. Like PLP-H, PLP-D mRNA is also
PRL-related protein (d/t PRP). Mouse PLP-A expressed in spongiotrophoblast and tropho-
is synthesized as a 227 amino acid precursor blast giant cells and is first detected at day
and is secreted as a glycoprotein of 196 14 of pregnancy and increases until term
amino acid, which is 78% homologous to rat (Iwatsuki et al. 1996). In the bovine placenta,
PLP-A. PLP-B encodes a protein of 230 amino two prolactin-like proteins (bPLP-I and
acids consisting of a mature glycoprotein of bPLP-II) were identified which resemble
201 amino acids which shares 66% identity bovine prolactin but are different from
with rat PLP-B. Decidual/trophoblast PRP bovine PL or PRPs (Yamakawa et al. 1990).
encodes a precursor protein of 240 residues The inferred amino acid sequences of bPLP-I
and a secreted glycoprotein of 211 amino and bPLP-II share 45–51% identity with
acids with 64% homology with rat d/t PRP. bPRL and 23–24% with bGH (Yamakawa
PLP-A, PLP-B, and d/t PRP are expressed in et al. 1990). At the nucleotide and amino
placenta or decidua. Expression of PLP-A acid level, bPLP-I and bPLP-II share 62% and
mRNA is maximum on day 12 in rodent 39% homology, respectively. Bovine PLP-I,
trophoblast giant cells, whereas PLP-B bPLP-II, PLs, PRLs, and other prolactin-like
mRNA is high on day 10 in decidual cells proteins from cow, mouse, and rat possess
and on day 12 in spongiotrophoblast (Lin et seven common amino acid residues: five are
al. 1997). Decidual/trophoblast PRP mRNA conserved among other members of the
is abundant in the decidual layer on day 8 of family, and the other two residues are con-
gestation (Lin et al. 1997). In rats, nine PLP served in bovine, mouse, and rat PLs, PRLs,
genes have been identified that are structur- and PRL-like proteins.
ally similar to PRL and GH. Iwatsuki et al.
(1998) characterized PLP-H which encodes a 11.3.6 IGFs
mature protein of 239 amino acids including
a 31 amino acid signal sequence. At the Insulin-like growth factors IGF-I and IGF-II
amino acid level, PLP-H shares 78% homol- play key roles in the regulation of embryonic
ogy with PLP-C and 67% with PLP-D. PLP-H and fetal growth and development. IGF-I is

260 Physiological Genomics of Reproduction

a single-chain basic protein of 70 amino Westwood 2008). The role of IGFBP-1 in the
acids, and IGF-II is a slightly acidic, single- placenta is controversial. IGFBP-1 has been
chain, peptide of 67 amino acids (Sara and found to enhance both basal and IGF-II-
Hall 1990; Forbes and Westwood 2008 for induced extravillous trophoblast migration
reviews). IGFs exert their biological effects (Irving and Lala 1995). IGFBP-3 is predomi-
by binding to cell surface receptors. Two nantly expressed in trophoblast, fibroblasts
distinct subtypes of receptors for IGFs have of the villous stroma, amnion, and chorion
been identified. Type I IGF receptor binds of fetal membranes (Han et al. 1996; Rogers
IGF-I with equal or greater affinity than et al. 1996). IGFBP-3 has been found to
IGF-II and also binds insulin with low affin- inhibit IGF-stimulated mitogenesis of pla-
ity. In contrast, Type II IGF receptor typi- cental fibroblasts (Rogers et al. 1996).
cally binds IGF-II with greater affinity than
IGF-I and does not bind insulin. Human As mentioned previously, IGFs induce
IGF-I and IGF-II are present in the placenta their effects on cellular proliferation, differ-
as early as 6 weeks of gestation (Han et al. entiation, and survival by binding to and
1996) and augment the proliferation and sur- activating specific receptors. Two forms of
vival of placental fibroblast. IGF-I has been IGF receptors have been identified: a hetero-
found to regulate both the differentiation of tetrameric type-1 receptor, which resembles
cytotrophoblasts into syncytiotrophoblasts the insulin receptor, and a monomeric type-II
(Bhaumick et al. 1992) and into extravillous receptor, which is structurally different from
cells (Lacey et al. 2002). In mice, knockdown the insulin or type-1 receptor. IGF-IR is a
of IGF-II in the placenta reduced diffusional heterotetrameric glycoprotein consisting of
exchange surface area and reduced permea- two alpha subunits of 706 amino acids
bility for nutrients. Conversely, in guinea and two transmembrane β subunits of 627
pigs Sferruzzi-Perri et al. (2006) observed residues (Sara and Hall 1990). The IGF-IIR
that maternal IGF-II increases the total is a single-chain, membrane-spanning, gly-
surface area of the placenta for nutrient coprotein that is also known as cation-
exchange, whereas IGF-I did not affect the independent mannose-6-phosphate receptor.
surface area of the placenta but diverted The IGF-IR is found in trophoblast, villous
nutrients from mother to fetus. endothelium, and the mesenchymal core of
the placenta. Studies on transgenic mice
IGFs circulate in the blood bound to IGF- lacking the IGF-IR revealed that a reduction
binding proteins (IGF-BPs). IGF-BPs abolish in the number of placental IGF-IR might be
the acute insulin-like actions, restricts their a contributory factor in pregnancies compli-
permeability through capillaries, and inhib- cated by intrauterine growth restriction
its their access to membrane receptors. In (IUGR). Binding of IGF-I to its receptor results
humans, six separate IGF-BPs have been in the activation of two signaling cascades:
described, viz. IGF-BP1, IGFBP-2, IGFBP-3, the PI3K pathway or the mitogen-activated
IGFBP-4, IGFBP-5, and IGFBP-6 (Han et al. protein kinase (MAPK/ERK1/2) pathway.
1996). IGF-BP3 is primarily responsible for Activation of IGF-IR results in autophos-
maintaining IGF levels in the blood. Other phorylation of tyrosine residues in the intra-
IGF-BPs found in the blood stream, includ- cellular β subunits and subsequent activation
ing IGFBP-1 and IGFBP-2 can cross endothe- of PI3K and MAPK pathways, resulting in the
lial barriers and transport IGFs from the transcription of target genes involved in cel-
circulation to peripheral tissues (Forbes and lular proliferation and differentiation.

Placental Growth and Development 261

11.4 Transcriptomics of cells. Recently, Ushizawa et al. (2007c) eval-
placental development uated gestational stage-specific gene expres-
sion profiles in bovine placentomes using
11.4.1 Assessment of transcriptional microarray and in silico analysis. They sug-
regulation of placental genes gested that the genes TFAP2A, TFAP2B, and
through microarray TFAP2C may have different roles in the
differentiation and proliferation of tropho-
Evaluation of gene expression is an effective blasts. Ishida et al. (2007) analyzed cDNA
way of identifying genes important in the from mid- to late-stage mouse placenta to
regulation of traits that are of economic understand the molecular basis of placental
importance in livestock production. Precise development and function, using micro-
knowledge of gene expression profiles is nec- array. They reported that the expression
essary to improve the reproductive efficiency patterns of apolipoprotein A-II (Apoa 2), apo-
of mammals. Using microarrays as tools for lipoprotein C-II (Apoc 2), CEA-related cell
screening for expression of thousands or tens adhesion molecule 14 (Ceacam 14), cell
of thousands of genes has been a revolution- repressor of E1A-stimulated genes (Creg 1),
ary breakthrough in identifying candidate flavin-containing monooxygenase 1 (Fmo1),
genes that are critical during early preg- insulin-like growth factor-II (IGF-II), serine
nancy. The detailed gene expression profiles protease inhibitor, Kazal type 3 (Spink 3),
in the preimplantation embryo and placenta serine protease inhibitor 1-1(Spi 1-1), and
provide insights into the molecular mecha- trophoblast-specific protein alpha (Tpbpa)
nisms that are vital to furthering our knowl- were similar to mouse PL.
edge of embryogenesis, implantation, and
placental development. Ushizawa et al. 11.4.2 Genomic imprinting
(2007c) evaluated global gene expression in
the placenta and classified them into 10 The underlying genetic mechanisms that
clusters. Increased expression was found control interactions between different cell
for PL, pregnancy-associated glycoprotein-1 types within the feto-maternal interface and
(PAG-1), and the sulfotransferase family the relative combinations of the maternal
member estrogen preferring member I and zygotic genes are poorly understood.
(SULTIEI) gene. Expression of transcription Genomic imprinting is an epigenetic phe-
factor AP-2 alpha (TFAP2A) was high, nomenon that results in the differentiated
whereas that of transcription factor AP-2 expression of a gene or chromosomal region
beta (TFAP2B) was low and was inter- according to the parental origin of inheri-
mediate to that of transcription factor tance (Joyce and Ferguson-Smith 1999).
AP-2 gamma (TFAP2C). In situ hybri- Imprinted genes play a crucial role in feto-
dization revealed that TFAP2A, TFAP2B, placental development by affecting the
and TFAP2C mRNA were localized in dif- growth and nutrient transfer capacity of the
ferent sets of trophoblast cells (Ushizawa placenta in mammals. Georgiades et al.
et al. 2007c). In cow placenta, TFAP2A was (2000) investigated the in vivo function
expressed in cotyledonary epithelial cells of mouse chromosome 12 imprinting by gen-
including binucleate cells; TFAP2B was spe- erating conceptuses that inherited both
cifically expressed in binucleate cells; and copies of this chromosome from either
TFAP2C was expressed in mononucleate the father (paternal uniparental disomy for

262 Physiological Genomics of Reproduction

chromosome 12 [pUPD-12]) or the mother members of large datasets; the analysis and
(mUPD-12). Maternal UPD-12 animals died interpretation of various types of data includ-
perinatally and exhibited embryonic and pla- ing nucleotide and amino acid sequences,
cental growth retardation. In contrast, pUPD- protein domains, and protein structures; and
12 conceptuses died late in gestation and had the development and implementation of
a variety of defects including placentomeg- tools that enable efficient access and man-
aly. Georgiades et al. (2001) identified a agement of different types of information.
variety of defects in cell function at the feto- It involves the creation of extensive elec-
maternal interface, such as compromised tronic databases on genomes and protein
invasion of the maternal decidualized endo- sequences, and techniques such as the three-
metrium and the central maternal artery, dimensional modeling of biomolecules and
abnormalities in the wall of the central biological systems. These revolutionary
maternal artery, and defects within the technologies have provided a new under-
zygote-derived cellular layer of the labyrinth. standing of biology, with widespread appli-
Recently, Zhou et al. (2007) investigated cations to medicine, agriculture, and ecology.
the imprinting status of L-arginine : glycine Large databases of cDNA sequences of
amidinotransferase (GATM) and paternally tens of thousands of genes in thousands of
expressed gene (PEG10) on days 75 and 90 of tissue samples provide the source data for
pregnancy in pig placentas. Biallelic expres- identifying candidate genes that are associ-
sion of the GATM gene was observed in the ated with placental and fetal growth and
placenta of pigs. In contrast, the PEG10 gene development.
was monoallelically expressed in the porcine
placenta on days 75 and 90 of gestation. It was Ming Wong and Walker (2001) studied the
observed that deletion of IGF-II gene in the expression patterns of IGF and placental
labyrinthine trophoblast of the placenta steroid synthesis (PSS) genes in human
restricted placental growth by interfering cDNA libraries. They observed that either
with the permeability of the placenta to IGF/PSS genes, including placental lacto-
nutrients (Hemberger et al. 2002). gen-4 (PL-4), human growth hormone (hGH),
pregnancy-associated plasma protein-A
11.4.3 Tracking gene expression (PAPP-A), eosinophil major basic protein
signatures using bioinformatics tools (EMBP), placental alkaline phosphatases
(PLAP), placental aromatose P450; choles-
Bioinformatics allows the conceptualization terol side chain cleavage enzyme (P450scc),
of biology in terms of molecules and then and 3 beta hydroxy steroid dehydrogenase (3
applying informatics techniques to under- beta-HSD) share a similar expression profile
standing and organizing the information across these libraries. They chose these eight
associated with these molecules and their genes as their bait to look for other genes
expression patterns. It involves developing that showed very similar expression. They
and applying computational methods for observed 10 genes that were not previously
managing and analyzing information about linked to IGF/PSS that had expression
the sequence, structure, and function of bio- patterns similar to the eight genes. Out of
logical molecules and systems. It involves 10 genes, six genes including malignant
the development of new algorithms and sta- melanoma metastasis suppressor, placenta
tistics to assess the relationships among specific-1 (PLAC-1), pregnancy specific gly-
coprotein 10 (PSG-10), pregnancy specific

Placental Growth and Development 263

beta 1 glycoprotein (PSG-beta1), serine pal- molecular biology and biotechnology, par-
mitoyl transferase (SPT), and TONDU are ticularly functional genomics (DNAarrays),
associated with cell growth in fetal and /or have allowed the identification of embry-
cancer tissues. Four are EST sequences, onic and maternal genes potentially involved
namely, PLAC2, PLAC3, PLAC4, and in embryo survival and placental develop-
PLAC5, which occur predominantly in pla- ment. Validation of the functional involve-
cental/fetal tissue or tumors suggesting the ment of genes that have been identified
involvement of PLAC genes in tissue growth. requires extensive in vitro studies before in
Other known IGF/PSS genes such as metal- vivo therapy can be applied. Recently, oligo-
loprotease ADAM 12, early placenta insulin based and cDNA microarray technologies
like peptide (EPIL), IGF binding proteins, made it possible to understand many of the
and placental growth factor are also found to factors controlling the regulation of gene
be co-expressed less consistently with transcription and to globally evaluate gene
PLAC2, PLAC3, PLAC4 and PLAC5 genes. expression profiles. Thus, a genome-wide
The genes identified by coexpression analy- screening approach coupled with functional
sis are useful candidates for exploring their assays will help elucidate the complex
roles in placental and fetal development. embryo–uterine crosstalk. The application
Recently, Jiang et al. (2004) analyzed ESTs of molecular genetic technologies to animal
and identified 5024 genes in bovine placenta agriculture will definitely bring about excit-
with human orthologs. A total of 24 pre- ing changes in livestock production and the
ferentially expressed genes (PEG) and 39 tailoring of animals to produce products
highly expressed genes (HEG) were found in needed by humans.
the placenta. Transcriptional profiles were
similar in the placenta, ovary, and mammary Acknowledgments
gland.

11.5 Future research directions The author wishes to express his deep sense
of gratitude to Director, NIANP, Bangalore,
Exploring how genomes affect reproductive for granting permission to write the chapter.
efficiency will undoubtedly lead to the Sincere help rendered by Mrs. Rekha is
development of tools to optimize reproduc- duly acknowledged. I should not forget to
tive management. acknowledge Shelby Hayes, Editorial Assis-
tant, Wiley-Blackwell, for prompt and helpful
This has been greatly aided by the advent service when required. Finally, thanks are
of genomic and bioinformatics technologies. due to my wife, Shrabanti, and daughter,
Understanding the mechanisms of preim- Anoushka, who cheerfully tolerated and sup-
plantation embryo development and placen- ported the many hours of absence involved
tation has been a challenge to reproductive in writing the chapter.
and developmental biologists. Recent tech-
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Kizaki, K., Abe, Y., Sasada, H., Sato, E., hormone gene family in bovine placenta:
and Hashizume, K. 2007b. Gene expres- Isolation and characterization of two pro-
sion profiles of novel caprine placental lactin-like cDNA clones. Journal of
prolactin related proteins similar to Biological Chemistry 265: 8915–8920.
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12

Cellular, Molecular, and Genomic Mechanisms
Regulating Testis Function in Livestock

Kyle Caires, Jon Oatley, and Derek McLean

12.1 Introduction germ cells that differentiate through a
unique cellular process, meiosis, which
The production of sperm occurs in the occurs continually throughout the life of the
testis and is essential for male fertility male. In addition, the germ cells interact
and ultimately the production of offspring. with, and are regulated by, somatic cells
Structurally, the testis is organized with through intimate contact within the semi-
seminiferous tubules that produce sperm niferous tubule. The cells present in the
and the cells in the interstitial space between interstitial space also influence germ cell
the tubules. The seminiferous tubules lack differentiation by providing factors that may
blood vessels and include differentiating directly regulate the germ cells or regulate
germ cells and Sertoli cells that are con- the somatic cells of the seminiferous tubule.
tained within a single cell layer of peritubu-
lar myoid cells that form the final cell barrier Testis development and sperm production
or outside ring of the tubule. The interstitial are controlled during development and
space comprises Leydig cells, fibroblasts, in mature animals by the hypothalamus
some immune cells, and the cells that and pituitary gland. Gonadotropin-releasing
make up blood vessels. This complex orga- hormone (GnRH) from the hypothalamus
nization presents some challenges when stimulates the pituitary gland to produce the
investigating how individual cell types gonadotropin follicle-stimulating hormone
contribute to the overall process of sperm (FSH) and luteinizing hormone (LH), which
production. For example, spermatogenesis regulate the Sertoli and interstitial Leydig
includes mitosis of undifferentiated germ cells, respectively. LH stimulates Leydig
cells, followed by meiosis for chromosomal cells to produce testosterone. FSH and tes-
reduction to produce haploid gametes. tosterone act on Sertoli cells to stimulate
Therefore, the testis has a complex set of these cells to proliferate during development
and support germ cell differentiation in

269

270 Physiological Genomics of Reproduction

mature animals. Testosterone and inhibin, a from laboratory and livestock species and
protein hormone produced by Sertoli cells the ability to isolate the complete set of
under the regulation of FSH, feed back to the transcripts from a cell or tissue (transcrip-
hypothalamus and pituitary gland to sup- tome) has greatly aided the characterization
press the production of GnRH and the of genes and proteins regulating testicular
gonadotropins. This feedback is critical to function. The impact of these research
controlling the pulsatile release of GnRH to approaches has improved our understanding
maintain serum hormone concentrations of testis biology. However, most research in
within normal physiological ranges. Disrup- testis biology using genomics-focused tech-
tion of the positive or negative signaling niques has been conducted using mice or
associated with the hypothalamic–pituitary– rats as model organisms.
testis loop results in complete or partial loss
of sperm production. The aim of this chapter is to provide infor-
mation about techniques that are used to
The complex environment of the testis to investigate spermatogenesis in livestock
support germ cell differentiation and sperm and the information that has been learned
production creates challenges for identifying from experimentation. In addition, we will
and characterizing the mechanisms that provide background on spermatogenesis and
regulate spermatogenesis. The ability to discuss several projects that used genomics-
simulate the seminiferous tubule environ- based approaches to determine basic mecha-
ment to support germ cell differentiation nisms that regulate somatic or germ cell
from immature diploid spermatogonia into development in the testis. Examples of how
haploid sperm in vitro has not been consis- genomics-based approaches have generated
tently achieved in mammals. Therefore, the large databases of information regarding
specific signals and genes responsible for gene expression profiles of the testis in
each development step must be investigated rodents will be referenced to provide infor-
in combination with all other signals from mation and resources for the reader to under-
germ cells and somatic cells at each devel- stand how these datasets can be developed
opmental stage. Primary culture of Sertoli and interpreted to gain insight into testis
cells and germ cells for short periods of biology.
time has been used to investigate aspects of
hormone action and signal transduction in 12.2 Spermatogenesis
these cell types. However, removal of the
cells from the physiological environment of 12.2.1 Germ cell differentiation: Basics
the testis eliminates cell interactions and
any potential paracrine signaling. Although The entire process of spermatogenesis is
the results from these projects provide valu- dependent on the formation of the testis
able information, they must be interpreted during embryonic and postnatal develop-
with caution. Fortunately, the development ment (Cupp and Skinner 2005). During
of multiple experimental approaches includ- embryonic development the SRY gene is
ing transgenic animals, cloning, germ cell expressed in primitive Sertoli cells, stimu-
transplantation, and ectopic testis grafting lating a cascade of events leading to the for-
enables scientists to investigate physiologi- mation of sex cords in the embryonic gonad.
cal mechanisms within the testis. The elu- These cords result from the aggregation of
cidation of the complete genome sequence

Testis Function in Livestock 271

primordial germ cells (PGCs), primitive nia, and give rise to differentiating A-type
Sertoli cells, and pre-peritubular cells that spermatogonia that undergo another series
have migrated from the mesonephros. These of amplifying mitotic divisions. These dif-
cells go through periods of proliferation or ferentiating A-type spermatogonia mature
mitotic arrest in the case of germ cells during into intermediate and B-type spermatogonia,
the remaining time of embryonic develop- which enter meiosis, becoming primary and
ment and at birth have formed seminiferous secondary spermatocytes, and eventually
tubules (Cupp and Skinner 2005). PGCs haploid spermatids are produced, which
migrate from the yolk sac to the embryonic undergo a transformation into spermatozoa.
gonad and proliferate for a period of time. In Collectively, the SSCs (also termed As), Apr,
the embryonic testis, the PGCs differentiate and Aal germ cells, are referred to as prolif-
into gonocytes, the most primitive male erating spermatogonia and all share very
germ cell, and then stop proliferating. similar phenotypic and likely molecular
characteristics (Oatley and Brinster 2008).
Germ cells resume mitosis after birth and SSCs are rare, estimated to be present in
migrate from the center of the seminiferous approximately 1 in 3000 cells of the adult
tubule to the base of the tubule. The time of mouse testis (Tegelenbosch and de Rooij
this migration and cell division varies 1993).
between species, starting around postnatal
day 2 in mice (Nagano et al. 2000) to several Following the initial steps of germ cell
weeks after birth in bull calves (Curtis and differentiation, germ cells in the testis
Amann 1981). The paracrine or autocrine undergo mitosis as spermatogonia then dif-
signals that stimulate this process are not ferentiate into spermatocytes that undergo
known, but cells that do not migrate to meiosis. After meiosis, the haploid germ
the base of the tubule undergo apoptosis. cells are called spermatids, and upon com-
Migration of germ cells to the basal portion pletion of nuclear repackaging and the for-
of the tubule and resumption of mitosis mation of the axoneme, these cells spermiate
during this time is important for the estab- into the lumen for transport to the epididy-
lishment of the spermatogonial stem cell mis. In the bull, spermatogonia first appear
(SSC) population in mice (McLean et al. at around 12–16 weeks of age, and these cells
2003). differentiate into spermatocytes at around
24 weeks of age (Curtis and Amann 1981).
The initial steps of germ cell differentia-
tion have been determined by analysis of 12.2.2 Germ cell
histological sections from the testes of many differentiation: Regulation
species. Morphological differences in differ-
entiating germ cells were used to distinguish The factors that influence germ cell differ-
between different germ cell developmental entiation are likely regulated by the gonado-
stages. Initiation of sperm production occurs tropins and testosterone. For example,
when SSC differentiation results in produc- plasma FSH levels are fairly stable from 4
tion of daughter cells, termed Apaired (Apr) weeks of age through puberty in bull calves
spermatogonia, which are committed to dif- (Amann 1983), indicating that the action of
ferentiation rather than self-renewal (de this hormone is regulated by FSH receptor
Rooij and Russell 2000). The Apr spermato- expression by Sertoli cells. In contrast,
gonia then undergo a series of mitotic cell plasma LH levels increase from 8 to 12
divisions, becoming Aaligned (Aal) spermatogo-

272 Physiological Genomics of Reproduction

weeks of age and then begin to fluctuate a variety of mammalian genome projects
during sexual development (Amann 1983). (Lewin 2003; Rothschild 2003; Womack
Testosterone, produced by Leydig cells 2005), microarray platforms are available for
under the influence of LH, has a slight several domestic livestock species, includ-
increase at around 4 weeks of age, then ing bovine. Information from protein–
declines, and gradually increases from 12 protein interaction and metabolism and cell
weeks of age to reach maximal levels at signaling research has facilitated the devel-
24–28 weeks of age. The peak of serum tes- opment of pathway analysis software on a
tosterone concentrations coincide with the genome-wide level and extend the useful-
appearance of meiotic cells (Amann 1983). ness of microarray data to provide additional
Although there is a general understanding information regarding bioactive molecules
of hormone regulation and morphological in gene expression studies.
changes that occur during the establishment
of the testis leading to sperm production, a Thus, Schmidt et al. (2007) sought to
detailed understanding of the factors required investigate the factors critical for bovine
to initiate this process is lacking. One testis development in vivo and determine
approach for identifying the proteins asso- the mechanisms that may be responsible for
ciated with testis development, the estab- donor-age-related differences in the ability
lishment of spermatogenesis, and germ of bovine testis tissue grafts to produce
cell differentiation is to profile the genes elongated spermatids (Schmidt et al. 2006a).
expressed in the testis during development. To accomplish this objective, testis tissue
Several platforms are available for this type obtained from 2-, 4-, and 8-week-old bull
of characterization including the production calves were grafted on immunodeficient
of cDNA libraries from isolated testicular mice and removed at several tissue age time
germ and somatic cells. Gene expression points. To determine factors potentially
profiling with gene microarrays is the most responsible for the age-related differences in
prevalent approach for characterizing the sperm production in bovine testis grafts, and
genes involved in development or function therefore testis development, the tran-
of specific cells or tissues. scriptomes of donor tissues were assayed
using Affymetrix Bovine GeneChips (Santa
12.3 Transcriptomics of testis Clara, CA) and deposited in the NCBI
in bulls Gene Expression Omnibus (GEO) database
(http://www.ncbi.nlm.nih.gov/geo; series
12.3.1 Microarray analysis on testis GEO series # GSE5970). On average, 56% of
tissue grafts transcripts present on the GeneChips were
expressed in 2-, 4-, and 8-week-old bull testis
The use of microarray technology in male (Schmidt et al. 2007).
reproductive biology has allowed the charac-
terization of large numbers of genes that are Data processing identified approximately
important for sperm production and testis 200 transcripts for further analysis, and
development in humans (He et al. 2006) and ontological clustering of transcripts (twofold
rodents (McLean et al. 2002; Shima et al. difference in expression) indicated signifi-
2004; Small et al. 2005; Johnston et al. 2008). cant differences in expression of genes
With sequence information obtained from involved in cell communication, mainte-
nance, and signal transduction between 2
and 8 weeks of bovine testis development

Testis Function in Livestock 273

in situ (Schmidt et al. 2007). Thus, age- expression of GDNF and FGF2, two proteins
related gene expression (and subsequent important for SSC self-renewal and Sertoli
protein expression) in the donor testis tissue cell function, also decreased with donor age.
before grafting likely affected the ability of Abundance of transcripts for both these genes
grafted tissues to be accepted by the host in 8-week-old donor tissues were signifi-
environment and/or support germ cell dif- cantly lower than other donor ages evaluated
ferentiation. Microarray analysis identified (Schmidt et al. 2007). Together these findings
angiogenin, early growth response 1, insu- provide insights into factors important for
lin-like growth factor 2, insulin-like growth bovine testis development and thus may
factor-binding protein 3, transgelin 2, and provide novel targets for improving fertility
thrombomodulin as candidate genes respon- in bulls and testis tissue grafts.
sible for donor-tissue age variation in the
production of sperm by testis grafts (Schmidt 12.3.2 Ectopic testis xenografting
et al. 2007). The authors confirmed the
expression patterns of these genes by quan- Scientists have been experimenting with
titative polymerase chain reaction (qPCR) testis transplantation since the late 1800s
(Schmidt et al. 2007), and these findings for a variety of purposes, including hormone
underscore the importance of genes involved therapy (Turner 1938). Ectopic subcutane-
in cell growth and vascular biology for estab- ous testicular grafting has been utilized as
lishment of spermatogenesis in the bull. another technique to investigate spermato-
genesis of lab and livestock species ex situ
Schmidt et al. (2007) also evaluated the (Johnson et al. 1996; Honaramooz et al.
expression of genes previously known to be 2002; Oatley et al. 2004b, 2005a). Ectopic
important for germ and Sertoli cell biology in grafting of testicular tissue is a method in
rodents by qPCR in bovine tissue after graft- which a portion of testicular parenchyma
ing. The interaction between KIT ligand from a donor animal is placed into a recipi-
(KITL), also known as stem cell factor, pro- ent animal, usually under the skin on the
duced by the Sertoli cells and its receptor KIT back of the animal. This recipient animal is
on germ cells is important for germ cell dif- usually an immunodeficient nude male
ferentiation (Mauduit et al. 1999). KIT expres- mouse (nu+/nu+). The nu/nu mouse strain
sion was significantly lower in bovine testis lacks a thymus and hence does not have the
tissue grafts when compared with bovine characteristic immune system T cells that
testis in situ, but transcript abundance of KIT could mount an immune response and ulti-
and KITLG increased as grafts developed in mately reject donor-derived testis tissue.
the recipient mouse (Schmidt et al. 2007). Remarkably, spermatogenesis will initiate
Other Sertoli cell-expressed genes, including in the grafted tissue and elongated sperm are
clusterin and GATA4, were also found to produced (ref). Offspring have been gener-
increase as grafted tissues developed, and ated by intracytoplasmic sperm injection
these transcripts were significantly lower (ICSI) into oocytes using elongated sperma-
in grafted tissues from 8-week-old donors tids recovered from mice testis tissue grafted
when compared with grafts originating from onto mice (Schlatt et al. 2003). To date, no
donors of other ages (Schmidt et al. 2007). offspring generated using sperm from cross-
Immunohistochemical analysis confirmed species testis grafts (e.g., pig grafted on
expression of clusterin and GATA4 protein mouse) have been reported. Some of the
in these tissues (Schmidt et al. 2007). The

274 Physiological Genomics of Reproduction

potential applications of testis tissue graft- production suppresses FSH and LH produc-
ing include male germline preservation tion. Therefore, grafting tissue from neona-
including the conservation of endangered tal animals onto intact, adult animals does
species, investigation of the effects of toxi- not provide a similar endocrine environment
cants on spermatogenesis, investigation of that is present in the donor animal. It appears
endocrine regulation of spermatogenesis, likely that removing the gonad of the recipi-
and the production of transgenic spermato- ent mouse prior to testis transplantation
zoa following the genetic manipulation of results in an ideal environment for cell dif-
testis tissue before grafting. Although testis ferentiation, the initiation of spermatogen-
tissue grafting does produce sperm, it is by esis, and hormone production in the grafted
no means normal spermatogenesis when testis tissue, resulting in sperm production.
considering sperm production and sperm However, the high gonadotropin concentra-
transport. However, this technique provides tions present in castrated mice may influ-
a novel tool for investigating the fundamen- ence cell differentiation. For example, pig
tal aspects of testis function for large, agri- testis tissue that was grafted on nude mice
culturally significant species. showed complete spermatogenesis earlier
than in a normal pig testis (Honaramooz
Ectopic tissue xenografting provides a et al. 2002). This means that germ cell dif-
useful means for researchers to investigate ferentiation was accelerated in grafted pig
factors and mechanisms important to germ testis tissue compared with the normal time
cell differentiation, Sertoli cell populations, required for germ cell differentiation in an
and production of sperm in various livestock intact pig. Similarly, many seminiferous
species without maintaining those bulls and tubules in grafted tissue have larger diame-
boars, respectively. For example, in bovine ters than tubules in testes that are still
testis tissue grafts, only about 10% of the attached to the animal (Oatley et al. 2004b;
seminiferous tubules are capable of undergo- Schmidt et al. 2006a; Caires et al. 2008). This
ing complete spermatogenesis after a 24- may be a result of high FSH concentrations
week grafting period (Oatley et al. 2004a,b). stimulating Sertoli cell proliferation without
As a result of this relatively low efficiency, the normal feedback mechanisms regulating
factors that upregulate or downregulate the arrest of Sertoli cell mitosis just before
Sertoli cell proliferation and germ cell dif- puberty. Although a doubling of the Sertoli
ferentiation in the testis tissue grafts can be cell number increases sperm output in rats,
more easily observed. hyperproliferation of Sertoli cells in ectopic
grafted testis tissue may result in seminifer-
The differentiation of testis tissue follow- ous tubules that are too large to support germ
ing grafting varies depending on the age cell differentiation.
of the donor animal (Oatley et al. 2004a;
Schmidt et al. 2007; Caires et al. 2008). Variation in spermatid production in testis
During development, testicular cells are grafts following the grafting period repre-
exposed to increasing concentrations of FSH sents a unique method for evaluating the
and LH. FSH is critical for the establishment timing necessary for germ cells to differenti-
of the Sertoli cell population, and LH sti- ate in different species. For example, bovine
mulates developing Leydig cells to produce testis tissue from 2-week-old calves grafted
testosterone. Both of these processes are onto mice and removed 24 weeks later has
essential for germ cell differentiation. In few seminiferous tubules with elongating
adults, negative feedback from testosterone

Testis Function in Livestock 275

spermatids. However, if the grafting period et al. 1998). VEGF receptors are differentially
is extended to 36 weeks, the percent of semi- expressed on developing germ cells. VEGFR-2
niferous tubules supporting germ cell dif- is present on spermatogonia, whereas VEGFR-
ferentiation is significantly higher (Schmidt 1 is present on spermatids (Nalbandian et al.
et al. 2006a). These results suggest that, to 2003). These results indicate that VEGF may
some extent, the intrinsic mechanisms regu- have non-endothelial cell targets in the testis.
lating germ cell differentiation do not change This is interesting because VEGF appears to
when the tissue is grafted onto mice or have a positive effect on Sertoli and germ cell
exposed to a different endocrine environ- differentiation in the testis.
ment. Similarly, manipulation of the endo-
crine environment of the host mouse may The action of VEGF on endothelial cells is
represent a novel approach for investigating an active area of research, and much is
how systemic factors regulate testis devel- known about its mechanisms of action in
opment and germ cell differentiation. this cell type. VEGF receptors are receptor
tyrosine kinases (RTK), which are enzymes
12.3.3 Manipulation of testis tissue that can transfer a phosphate group (via
before xenografting autophosphorylation) to a tyrosine residue
in a protein (on the c-terminus end of a recep-
The success of bovine testis grafts depends on tor) following ligand binding and dimeriza-
many factors including: donor age, endocrine tion. Receptor protein tyrosine kinases
environment of the recipient, and endoge- (PTKs) possess an extracellular ligand-
nous treatments of testis tissue prior to (or binding domain, a transmembrane domain,
during) the grafting period with factors that and an intracellular catalytic domain. The
may potentially regulate testis function. transmembrane domain anchors the recep-
Schmidt et al. (2006b) treated testis tissue at tor in the plasma membrane, while the
the time of grafting with 1 μg/ml of vascular extracellular domains bind growth factors.
endothelial growth factor (VEGF), a potent Characteristically, the extracellular domains
angiogenic factor. The hypothesis was that of VEGF comprise immunoglobulin-like
testis tissue treated with VEGF before graft- domain structural motifs (Shibuya and
ing would significantly increase angiogenesis Claesson-Welsh 2006). Phosphorylation is
in the grafts, leading to improved graft sur- an important function in signal transduction
vival. Results showed that VEGF treatment to regulate enzyme activity.
increased graft weight and spermatogenesis
in grafted tissue but did not increase blood VEGFR-1, which is present in transmem-
vessel numbers in grafted tissue. VEGF is brane and soluble forms, inhibits angiogen-
produced in the testis and gene expression is esis during early embryogenesis, but it also
induced by human chorionic gonadotropin stimulates angiogenesis and inflammatory
(hCG) treatment (Haggstrom Rudolfsson responses in postnatal life, playing a role in
et al. 2003). In the human testis, VEGF and its several human diseases such as rheumatoid
receptors, VEGFR-1 and VEGFR-2, are local- arthritis and cancer. The soluble VEGFR-1
ized to both the Sertoli and Leydig cells. Addi- is overexpressed in placental trophoblast
tionally, VEGFR-1 and VEGFR-2 are found cells (Shibuya and Claesson-Welsh 2006).
on the testicular capillary endothelial cells VEGFR-2 has critical functions in physiolog-
(Ergun et al. 1997) and germ cells (Korpelainen ical and pathological angiogenesis through
distinct signal transduction pathways regu-
lating the proliferation and migration of

276 Physiological Genomics of Reproduction

endothelial cells (Shibuya and Claesson- genetically manipulated by electroporation
Welsh 2006). The downstream targets of and that the germ cells survive this treat-
both receptors include activation of many ment. Thus, nontargeted introduction of
signaling pathways (PI3K/Akt, Ras/Raf- genes is possible with ectopic testis grafting.
MEK/Erk, eNOS/NO, and IP3/Ca2+, PKC, However, targeted gene deletion has not
PKA) that lead to changes in gene tran- been attempted, and more precise methods
scription responsible for their functions as for accomplishing this goal need to be
endothelial cell mitogens and vascular per- developed.
meability factors (Namiecinska et al. 2005).
We demonstrated that VEGF treatment sup- Manipulation of testis tissue before graft-
ports spermatogonial survival in the bovine ing can also improve our understanding of
testis by blocking apoptosis pathways (Caires the factors that regulate spermatogenesis.
et al. 2009). Intracellular signaling cross-talk Testis tissue maintained in culture for 5–7
between VEGF and glial cell line-derived days before grafting is capable of producing
neurotrophic factor (GDNF) occurs in neu- elongating spermatids after the grafting
ronal cells. As will be described in the SSC period (Schmidt et al. 2006b). Similarly,
section of this chapter, GDNF regulates SSC testis tissue cryopreserved before grafting
proliferation and self-renewal. Therefore, can be grafted following thaw, and produce
interaction between GDNF and VEGF in sperm (Caires et al. 2008). As a result, several
cattle and possibly other mammalian testis powerful applications of the technique can
may be an important mechanism for estab- be employed, mainly pertaining to investi-
lishing the spermatogonia population during gating the molecular mechanism regulating
testis development. Sertoli and germ cell proliferation and
differentiation in the testis. For example,
Ectopic testis tissue grafting may provide culturing tissue provides a useful means of
an effective method for genetically manipu- directly assessing the effects of growth
lating male germ cells before differentiation. factors on germ cell and Sertoli cell survival
This approach would generate a large number in ectopic testis tissue grafts. Also, because
of genetically modified sperm for the pro- testis tissue can be cryopreserved before
duction of transgenic animals. Several tech- grafting and still achieve successful sper-
niques can be used to genetically modify the matogenesis, male germ-line preservation is
undifferentiated germ cells in the testis another potential use of this technique.
tissue, including lipofection, electropora-
tion, or virus-mediated methods. Bovine 12.3.4 SSC transplantation
testis tissue was electroporated with a β-
galactosidase expression vector prior to Spermatogenesis is the process by which
grafting the tissue on mice (Oatley et al. millions of sperm are produced daily within
2004b). The grafts were removed 24 weeks the testis. Spermatogenesis commences at
later, stained for β-galactosidase activity, puberty and continues throughout the life of
and evaluated for germ cell differentiation the male. At the foundation of this process
and transgene expression. Histological anal- are the SSCs, which undergo both self-
ysis showed that transgene expression was renewal and differentiation. SSC transplan-
present in both Sertoli and differentiated tation experiments pioneered by Brinster
germ cells but not in interstitial cells, sug- and Zimmermann (1994) provided the first
gesting that SSCs or spermatogonia can be and only functional assay for SSCs. This

Testis Function in Livestock 277

procedure involves injecting a suspension of SSCs be utilized. Because there are currently
testicular cells into the seminiferous tubules no known specific morphological or pheno-
of an infertile recipient. The SSCs translo- typic markers for SSCs, they cannot be iso-
cate to the basement membrane of the semi- lated as a pure cell population from the testis.
niferous tubule and colonize the recipient Selection strategies rely on collection of cell
testis. SSC transplantation has enabled sci- fractions enriched for SSCs, which are effec-
entists to characterize the biological activity tive because nearly all somatic cells (e.g.,
of SSCs, generate transgenic mice, and assess testicular fibroblasts, myoid, Leydig, and
factors important in the culture of SSCs Sertoli cells) and more mature germ cells
(Jeong et al. 2003; Oatley et al. 2007; McLean (e.g., differentiating spermatogonia, sper-
2008). Transplantation of testicular cells matocytes, and spermatids) are removed.
from many species into the seminiferous Currently, techniques for isolating SSC-
tubules of immunodeficient mice has been enriched cell fractions from total testis cell
used to determine if SSCs are present in a populations are available for mice (Kubota et
cell population (Dobrinski et al. 1999; al. 2004a), rats (Hamra et al. 2004; Ryu et al.
Dobrinski et al. 2000; Oatley et al. 2002). 2004), and nonhuman primates (Müller et al.
Transplantation of SSCs from species that 2008). In rodents and primates, isolation of
are closely related to mice (i.e., rats and cells expressing specific surface molecules
hamsters) will result in complete germ cell has provided the most efficient means for
differentiation and sperm formation from collecting SSC-enriched fractions. In the
the donor SSCs (Clouthier et al. 1996). adult mouse, isolation of testis cells that
However, transplantation of SSCs from express the surface marker Thy1 results in
species that are more divergent from mice 300-fold enrichment for SSCs compared with
does not result in germ cell differentiation. total testis cell populations (Kubota et al.
Interestingly, the SSCs in these experiments 2004a). In the rat, selection of epithelia cell
survived but did not differentiate beyond the adhesion molecule (Ep-CAM)-positive testis
undifferentiated spermatogonia cell type. cells results in 120-fold enrichment of SSCs
However, there are examples of transplanta- (Ryu et al. 2004). Similarly, isolation of testis
tion of donor germ cells from livestock cell populations that preferentially bind to
species into the testes of the same species, laminin also results in enrichment of SSCs
resulting in donor-derived spermatogenesis from both mouse and rat testes (Shinohara
(Honaramooz et al. 2003). et al. 2003; Hamra et al. 2004). Laminin is a
major component of the seminiferous tubular
Research focused on SSCs in mice and live- basement membrane of most mammals
stock have provided valuable information including livestock. Thus, it is reasonable to
about the factors that regulate the initiation hypothesize that laminin-binding cells from
of germ cell differentiation leading to the testes of livestock species will also be
production of sperm. Enrichment and culture enriched for SSCs; however, this possibility
of primary SSCs is the most direct way of has not been tested. In addition, isolation of
determining the mechanisms regulating SSC SSC-enriched fractions from testes of any
self-renewal and proliferation. Rarity of SSCs livestock species based on expression of
in the testis poses a challenge for establishing the surface molecules Thy1 or Ep-CAM has
long-term cultures of SSCs when total testis not been reported. Studies by Aponte et al.
cell populations are utilized. Thus, it is (2006) and Izadyar et al. (2002, 2003) used
essential that a cell fraction enriched for

278 Physiological Genomics of Reproduction

gravity sedimentation through bovine serum embryonic stem cells, require the addition
albumin (BSA) gradients to isolate spermato- of fetal bovine serum (FBS) in basal media to
gonia-enriched fractions from bull testes; support growth. The richness of nutrients in
however, the SSC content of these cell popu- FBS preferentially supports proliferation of
lations was not determined. rapidly dividing cells. Because SSCs divide
relatively slowly (Kubota et al. 2004b), other
Maintenance of SSCs in vitro for extended rapidly dividing cell types such as testicular
periods, in conditions that support their fibroblasts outgrow SSCs when cultured in
self-renewal, allows for expansion of SSC serum-containing media, resulting in loss of
numbers. Currently, techniques for long- SSCs over time. Also, FBS appears to have
term culture of SSCs are only available for toxic effects on mouse and rat SSCs in
mice (Kubota et al. 2004b; Oatley and Brinster culture (Kubota et al. 2004a; Ryu et al. 2005).
2008), rats (Ryu et al. 2005), and hamsters Previous attempts at culturing bovine SSCs
(Kanatsu-Shinohara et al. 2008). Previous have included FBS in basal media (Dobrinski
studies have resulted in short-term prolifera- et al. 2000; Oatley et al. 2002; Oatley et al.
tion of bovine SSCs, but long-term mainte- 2004a,c; Aponte et al. 2006). In those studies,
nance has not been achieved (Oatley et al. short-term expansion of bovine SSCs was
2004a,c; Aponte et al. 2008). Additionally, observed, followed by a rapid decline of SSC
culture of SSCs from other livestock species numbers at which time fibroblast takeover
has not been reported. In rodents, long-term was observed, which likely impaired SSC
maintenance of SSCs requires culture on proliferation and survival (Dobrinski et al.
mitotically inactive feeder cell monolayers 2000; Oatley et al. 2002; Oatley et al. 2004c).
in optimized serum-free media with specific To date, attempts to maintain SSCs of any
nutrient and growth factor supplementa- livestock species in serum-free media condi-
tions. These types of conditions have not tions have not been reported.
been evaluated for SSCs of any livestock
animal. With rodents, feeder cells derived SSC self-renewing proliferation in serum-
from mouse embryos are effective at free conditions is limited without the addi-
supporting long-term self-renewing SSC tion of specific growth factors. Inclusion of
expansion. Immortalized STO feeder cell the growth factor GDNF is essential for
monolayers were shown to support both expansion of mouse, rat, and hamster SSCs
mouse (Kubota et al. 2004b) and rat (Ryu when cultured in defined conditions (Kubota
et al. 2005) SSC expansion for greater than et al. 2004b; Ryu et al. 2005; Kanatsu-
5 months in culture. With bulls, primary Shinohara et al. 2008). Additionally, prelimi-
bovine embryonic fibroblasts (BEF) have nary bovine studies showed that addition of
been used as feeders to support short- GDNF into cultures of bovine germ cells in
term expansion of bovine SSCs (Oatley nonoptimized medium enhanced short-term
et al. 2004a). In those studies, SSC numbers expansion over a 14-day period (Oatley et al.
increased over a 7-day period but rapidly 2004c). These results suggest that there is
declined after 14 days, suggesting that long- conservation among mammalian species
term self-renewal cannot be supported. for specific growth factors that influence
SSC self-renewal. Additional mouse studies
Effective expansion of rodent SSCs in have shown that insulin-like growth factor
vitro has relied on the use of serum-free con- 1 (IGF-1), epidermal growth factor (EGF),
ditions (Kubota et al. 2004b; Ryu et al. 2005). basic fibroblast growth factor (bFGF), and
In contrast, several cell types, including

Testis Function in Livestock 279

leukemia inhibitory factor (LIF) also for sperm production. The timeline for
influence SSC proliferation in serum-free Sertoli cell proliferation and differentiation
conditions (Kubota et al. 2004a,b; Kanatsu- events are highly species dependent, occur
Shinohara et al. 2008). To date, influences of before puberty, and are essential for fertility.
these factors on the self-renewal of SSCs in In rodents, it is established that during
vitro from any livestock species has not prenatal and postnatal testis development,
been evaluated. The entire milieu of growth FSH stimulates Sertoli cells to proliferate
factors that control SSC self-renewal has yet (Griswold 1993). In contrast, Sertoli cells
to be discovered, and SSCs of each species halt proliferation and initiate terminal
may require specific combinations to differentiation in response to thyroid
promote self-renewing proliferation. hormones, testosterone, and retinoic acid
signaling (Orth 1982; Buzzard et al. 2003;
12.4 Reproductive genomics Holsberger and Cooke 2005).
in boars
The majority of research in porcine testis
12.4.1 Boar testis development development has focused on biological
events occurring between 1 month after
Efficient sperm production in boars is depen- birth and pubertal age (approximately 5
dent on germ and somatic cell maturation months of age in European breeds), and it is
during neonatal and prepubertal develop- known that Sertoli cell expansion in the
ment. As with all mammals, during this boar testis occurs between birth and approx-
time, somatic cells undergo critical prolif- imately 20 weeks of age (Erickson 1964;
eration and differentiation events that ulti- Putra and Blackshaw 1985). In mice, ablat-
mately determine the baseline for future ing the biological activity of thyroid hor-
sperm production in the adult. However, the mones during postnatal life results in an
mechanisms responsible for these biological extended period of Sertoli cell proliferation,
processes in the developing boar testis larger testes, and an increase in spermato-
remain unclear. The majority of published genic capacity (Joyce et al. 1993). A similar
work regarding male reproductive biology study evaluating the effect of postnatal
and testis development has been conducted hypothyroidism was conducted in 3-week-
in rodent models, and thus a better under- old boars, and the authors concluded that
standing of factors regulating the onset and no increase in Sertoli cell proliferation
maintenance of spermatogenesis in boars is occurred following treatment (Klobucar
lacking. et al. 2003), in striking contrast to findings
in rodents.
Somatic cells, including Sertoli cells and
Leydig cells, account for the majority of 12.4.2 The Meishan model
estrogen and testosterone synthesis in the
male but also produce nutrients and growth The Meishan breed presents a unique model
factors essential for regulating germ cell dif- for investigating the endocrine regulation of
ferentiation. Although in theory the boar spermatogenesis in swine. The Meishan is
can generate millions of sperm daily, the a slow-growing breed of swine originating
number of Sertoli cells contained within the from the Taihu Lake region outside of
testes ultimately determines the capacity Shanghai, China. Evidence suggests that
divergence between European breeds and the

280 Physiological Genomics of Reproduction

Meishan occurred roughly between 2000 the contralateral testis in place) results
and 500,000 years ago (Paszek et al. 1998; in compensatory testicular hypertrophy in
Giuffra et al. 2000) while domestication of mammals as a result of increased numbers
wild boars took place approximately 9000 of germ and Sertoli cells (Brown and
years ago (Bokonyi 1974). Chakraborty 1991; Orth 1993). Thus, prepu-
bertal hemicastration is a useful model
When compared with boars originating for studying factors governing germ and
from conventional breeds of swine, Meishan somatic cell proliferation in the mammalian
boars experience hastened puberty and sig- testis. A study evaluated the proliferative
nificantly elevated (2- to 10-fold) levels of response following hemicastration in cross-
gonadotropins and testosterone during estab- bred Meishan × White Composite boars by
lishment of spermatogenesis (Lunstra et al. dividing animals into large (Lg) and small
1997). These endocrine differences are main- (Sm) testis groups. Hemicastration stimu-
tained in mature animals due to a larger lated Leydig and Sertoli cell proliferation in
population of pituitary gonadotrophs and Lg testis boars (Lunstra et al. 2003). In con-
increased expression of genes encoding the trast, populations of Leydig and Sertoli cells
subunits of FSH and LH (Li et al. 1997). in Sm testis boars expanded due to increases
Furthermore, Meishan boars have a unique in size, but not number (Lunstra et al. 2003).
testicular composition that is characterized The number of Sertoli cells per testis was
by a twofold increase in the proportion of maximal 56 days after birth in Sm testis
interstitial tissue compared with total tes- boars. However, a longer duration of Sertoli
ticular volume. In addition, mean Leydig cell proliferation was observed in Lg testis
cell size is threefold larger when compared boars as Sertoli cell number per testis reached
with boars of European descent (Okwun a maximum of 112 days after birth (Lunstra
et al. 1996a,b; McCoard et al. 2003a). In et al. 2003). The mechanisms responsible for
contrast, both testis size and the number differences in somatic cell growth and dif-
of Sertoli cells are significantly reduced in ferentiation are unknown but could provide
Meishan boars when compared with boars of insight into methods for increasing sperm
European descent (McCoard et al. 2003a,b). production in males.
Interestingly, sperm production is not adver-
sely affected by this phenomenon; in fact, 12.4.3 Candidate genes and
the efficiency of spermatogenesis (daily quantitative trait loci (QTLs)
sperm production per Sertoli cell) is twofold for boar phenotypes
greater in Meishan boars (Okwun et al.
1996a,b). Moreover, Sertoli cell volume is In order to provide an effective means
significantly larger in the Meishan boar for understanding the genetic mechanisms
testes (McCoard et al. 2003a). Thus, the responsible for the reproductive phenotypes
unique physiology of the Meishan boar pro- in the Meishan boars, a resource population
vides an excellent model for the investiga- was developed by scientists from the US
tion of factors regulating testis development Meat Animal Research Center’s (MARC)
and affords the opportunity to better under- Swine Resource Population. Reciprocal
stand mechanisms governing germ and matings of purebred Meishan (Ms) and White
somatic cell biology in the male. Composite (WC; Chester White, Landrace,
Large White, and Yorkshire) were conducted
Hemicastration of males before puberty
(i.e., removal of one testis while leaving

Testis Function in Livestock 281

to produce F1 and F3 generations, respec- size. The A allele was associated with
tively. A genomic scan identified a centro- increased affinity of mature TBG for thyroid
metric region (∼80 cM) of the Sus scrofa X hormones, a reduction in circulating free T4
chromosome that was associated with puber- and T3, and a significant increase in testis
tal gonadotropin concentrations and testis size in WC boars (Nonneman et al. 2005).
size in boars (Ford et al. 2001; Rohrer et al. Similar biochemical activities were observed
2001). The gene encoding thyroxine-binding in a variant form of human TBG in vitro
globulin (TBG), the primary transporter and (Bertenshaw et al. 1992). The frequency of
regulator of thyroid hormone availability in the A and C alleles in the boar population
serum (Bartalena and Robbins 1993), resides was 0.71 and 0.29, respectively (Nonneman
within QTL for testis size and plasma FSH et al. 2005). The His226Asn SNP also resides
as demonstrated by using a comparative within the QTL for increased circulating
mapping approach of the porcine X chromo- FSH in boars. Thus, variation in testis size
some (McCoard et al. 2002). of boars is due, in part, to the effects of
thyroid hormones despite conflicting obser-
As discussed previously, thyroid hor- vations in swine (Nonneman et al. 2005).
mones regulate Sertoli cell proliferation in
rodents (Cooke et al. 1994) and bulls (Majdic Another unique breed of swine with atypi-
et al. 1998), and thus TBG represents a can- cal testicular characteristics is the Piau,
didate gene influencing testis development which originated in Brazil (França et al.
in swine. In a follow-up study, Nonneman 2000). Piau swine are similar to the Meishan
et al. (2005) developed the hypothesis that in growth rate and testis size, but the adult
TBG is responsible for causing decreased cir- Piau boars contain a significantly greater
culating levels of triiodothyronine (T3) and proportion of seminiferous tubules relative
thyroxine (T4), resulting in an extended to total testicular volume when compared
period of Sertoli cell proliferation responsi- with Meishan boars (França et al. 2000).
ble for the increased testis size characteristic Thus, longitudinal studies with Piau,
of European breeds (WC) when compared Meishan, and European breeds provide valu-
with Asiatic breeds like the Meishan (Ms). able insight into testis biology and how
To test this hypothesis, germplasm (F8 and selection pressure for growth and carcass
F10) from the original research population traits affect reproductive performance in
consisting of 3/4 WC X 1/4 Ms was used domestic swine. Two distinct phases of
to identify positional single nucleotide Sertoli cell proliferation occur during testis
polymorphisms (SNPs) in TBG that affect development in Piau boars (França et al.
endocrine parameters and testis growth in 2000). The first phase of Sertoli cell prolif-
developing boars (Nonneman et al. 2005). eration is responsible for a sixfold increase
The porcine TBG gene was sequenced and a in cell numbers between birth and 30 days
nonconservative adenine to cytosine poly- post partum (dpp), similar to observations
morphism (codon 226) in exon 2 was identi- and proposed mechanism in mice (Vergouwen
fied, which resulted in a consensus change et al. 1991; Joyce et al. 1993) and rats (Orth
of a histidine (Ms) to an asparagine (WC) in 1982). However, a second period of Sertoli
the ligand-binding domain of the mature cell proliferation occurred in Piau boars
TBG protein (Nonneman et al. 2005). The between 90- and 120-dpp, as evidenced by a
consensus C allele was found to be Meishan- twofold increase in Sertoli cell number per
specific and is associated with reduced testis testis during this time (França et al. 2000),