8
Functional Genomics Studies of Ovarian Function
in Livestock: Physiological Insight Gained and
Perspective for the Future
Beau Schilling and George W. Smith
8.1 Introduction The dawning of the genomics and genome
sequencing era in livestock has provided
Reproductive efficiency is a major limiting tremendous potential for advancement in
factor in the success of livestock operations. understanding of the molecular mecha-
The ovarian cycle is central to the reproduc- nisms involved in the regulation of the
tive process because only mature ovarian above aspects of ovarian function. The
follicles release eggs competent to be fertil- recent increase in availability and econo-
ized. Once a follicle develops to the preovu- mic feasibility of platforms for expressed
latory stage, it undergoes one of two fates: sequence tag (EST) sequencing, microarrays,
ovulation or atresia. The corpus luteum and proteomics appropriate for large-scale
(CL), formed from remnants of the ovulated studies of RNA and protein expression in
follicle, secretes the steroid hormone proges- livestock species has provided reproductive
terone, which sustains embryo growth and biologists opportunities to characterize
survival critical to pregnancy success. In the changes in RNA transcript (transcriptome)
absence of appropriate embryonic signals, and protein (proteome) profiles of ovarian
the CL will regress and a new reproductive tissues/cell types at key developmental
cycle is initiated. An increased understand- time points, in response to hormonal treat-
ing of the intrinsic and extrinsic factors ments and in animals of different repro-
regulating ovarian function (follicular devel- ductive potential. Such approaches hold
opment and atresia, oocyte maturation and considerable potential for advancing under-
function, and CL growth and regression) is standing of regulation of ovarian function
critical to improvements in reproductive and factors contributing to reproductive
efficiency of livestock species. efficiency.
183
184 Physiological Genomics of Reproduction
Expression profiling strategies have been such libraries are essential to the character-
successfully applied to the study of follicular ization of transcriptome composition of
growth and development, CL function, and tissues and species of interest and can hence
oocyte biology in cattle and swine. While facilitate the discovery of new genes of func-
amounts of raw information obtained from tional significance. Such efforts have also
the limited number of studies applying such accelerated the development of microarray
technologies in livestock have been exten- resources in livestock species required for
sive, new insight of verified biologic/physi- comparative transcriptome profiling. To
ological relevance obtained has been more date, EST sequencing efforts in livestock
limited, and overall rate of utilization of pro- have been most extensive in cattle and pigs,
filing data in subsequent physiological or with over 1.5 million bovine and 1.4 million
functional studies has been low. Some would porcine EST sequences deposited in the
argue that the limited physiological insight Genbank in contrast to the approximately
obtained from the application of such emerg- 39,000 equine ESTs present. Despite the fact
ing technologies is a consequence of a move that ovarian tissues are represented in many
from hypothesis-driven research to descrip- of the cDNA libraries from which ESTs
tive discovery research. While technological were generated, drawing inferences about
and logistical impediments to elucidation of ovarian gene expression, or ovary-specific
physiologically relevant information from gene expression in particular via data mining
expression profiling studies in livestock of these large electronic resources, is com-
are still evident, rigid criteria for data analy- promised because in many instances, mixed
sis, utilization of the wealth of available bio- tissue libraries were utilized (Smith et al.
informatics tools for mining of expression 2001).
profiling data, and a commitment to subse-
quent downstream functional studies neces- 8.2.1 EST sequence analysis of the
sary to prove biologic significance can greatly follicular and luteal transcriptomes
enhance the utility of data obtained.
Significant effort has been placed on the
The objective of this review is to highlight characterization of ESTs from cDNA librar-
the results of published expression profiling ies generated from porcine ovarian (follicu-
studies of ovarian tissues/cell types (follicu- lar and luteal) tissues. Caetano et al. (2003)
lar tissue and cells, luteal tissue, and oocytes) reported the construction of a normalized
in livestock species. Emphasis will be on cDNA library from swine ovarian follicles
studies utilizing high-throughput approaches ranging in size from 2 to 10 mm and genera-
for expression profiling (EST sequencing, tion of 3479 unique EST sequence clusters
microarrays, and proteomics). Emphasis in from this library. Physiologically relevant
the discussion of such results will be on information reported was limited because
documented or potential physiological sig- the depth of EST sequencing was limited
nificance of findings. and because emphasis was on details of
library construction and limited gene ontol-
8.2 Transcriptomics of ovarian ogy analysis for sequences obtained.
tissues: EST sequencing
The most extensive EST sequencing efforts
Generation of cDNA libraries from specific published to date, focused specifically on
tissues and characterization of ESTs from ovarian tissues in livestock, were conducted
by the investigators at the University of
Ovarian Function in Livestock 185
Missouri-Columbia using pigs (Jiang et al. acteristic of the extensive extracellular
2004). Eleven porcine cDNA libraries were matrix remodeling characteristic of the
constructed from whole ovary and (or) developmental transition from follicular to
various ovarian structures harvested at spe- luteal tissue in sheep (Smith et al. 1994a),
cific stages of differentiation including fetal, swine (Smith et al. 1994b), and cattle (Smith
neonatal, and prepubertal ovaries; specific et al. 1996).
ovarian structures collected on day 0 (folli-
cle), day 5 (follicle and CL), and day 12 of While a greater number of total EST
the estrous cycle; and follicles of different sequences have been reported for the cow
size classifications harvested from weaned versus the pig, ovarian tissue-specific EST
sows. Approximately 15,613 EST sequences, projects are more limited in scope in cattle.
representing 8507 gene clusters were gener- Casey and colleagues (2004) generated a
ated from these libraries. The majority of cDNA library from bovine CL collected on
clusters (68%) had consensus sequences days 6, 8, and 14 after estrus. Three hundred
homologous to tentative consensus sequences fifty-one unique sequences were obtained
for mature transcripts represented in The from 960 ESTs characterized. Genes encod-
Institute for Genomic Research Porcine ing for proteins with documented or poten-
Gene Index. tial importance to luteal function were
represented in ESTs generated, including
Gene ontology analysis revealed most of regulators of steroidogenesis (e.g., STAR),
the cDNA-encoded proteins that function in transcription (e.g., CTCF), antioxidants (e.g.,
binding, catalysis, and transport, and (or) are CAT), and tissue remodeling factors (e.g.,
involved in cell growth and maintenance TIMP2). Temporal expression of select genes
and (or) metabolism. Since cDNAs were of interest (identified from EST sequencing)
not subtracted or libraries normalized, fre- in CL on days 6, 8, 14, and 17 of the estrous
quency of the detection of clones of interest cycle was examined using Northern blot
across libraries was quantified using “elec- analysis. Messenger RNA (mRNA) for SQLE
tronic Northern analysis” as a means to (squalene epoxidase), which catalyzes a rate-
approximate expression levels across tissues limiting step in cholesterol synthesis was
from which libraries were constructed. For decreased sevenfold in regressed CL col-
example, cDNA encoding for FTH1, INHA, lected on day 17, coinciding with the char-
and AKR1C3 were represented at the highest acteristic decrease in systemic progesterone.
frequency in the library generated from Results suggest that reduced cholesterol
highly estrogenic 6-mm follicles versus 2-, synthesis may accompany the decrease in
4-, or 8-mm follicles. Authors proposed progesterone production characteristic of
a key role for FTH1 in storage of iron for luteal regression.
cytochrome-containing enzymes (Jiang et al.
2004) involved in steroidogenesis. 8.2.2 Oocyte EST sequencing projects
Comparison of frequency of individual The oocyte is a key regulator of ovarian fol-
clones sequenced from the day 12 follicle licular development and early embryogene-
versus the day 12 CL library by virtual sis. A developmental program intrinsic to
Northern analysis revealed 12 differentially the oocyte controls the rate of follicular
expressed genes. For example, frequency development (Eppig et al. 2002), and bidirec-
of clones for TIMP1 was greater in the tional communication between the oocyte
day 12 CL library than the day 12 follicle and nearby cumulus and granulosa cells is
library. Increased TIMP1 expression is char-
186 Physiological Genomics of Reproduction
critical for the normal growth of ovarian of all cell types (e.g., RPL15) and some
follicles (Eppig 2001; Matzuk et al. 2002). represent genes previously known to be
Furthermore, during the initial cleavage expressed both in oocytes and other tissues
divisions after fertilization, embryonic (e.g., CKS1B), the majority of the ESTs
development is supported by the products of encoded either for genes whose expression
maternal effect genes (mRNAs and proteins) in mammalian oocytes to our knowledge
synthesized and stored in oocytes during had not previously been reported (e.g.,
oogenesis (Telford et al. 1990; De Sousa CART) or for genes of unknown function
et al. 1998). Such products of maternal effect (Yao et al. 2004). Results of this small-scale
genes are critical for the interval between EST sequencing project stimulated a series
fertilization and the maternal-to-embryonic of functional studies (described below) that
transition when transcriptional activity of enhanced understanding of oocyte regula-
the embryonic genome becomes robust, and tion of follicular development and early
regulation of embryogenesis is shifted from embryogenesis in cattle (Bettegowda et al.
control by maternal (oocyte-derived) mRNA 2007).
and proteins to control by products of the
embryonic genome (Bettegowda et al. 2008a). 8.2.3 Oocyte regulation of follicular
Despite the important regulatory role of the development and early embryogenesis:
oocyte in the control of follicular develop- The story of JY-1
ment and early embryogenesis, composition
of the oocyte transcriptome and identities Among the oocyte EST described above (Yao
and functions of key oocyte-specific genes et al. 2004) encoding for genes of unknown
involved in the above processes are rela- function, one EST sequence (represented by
tively unknown in livestock species, and 14 fully sequenced clones with two different
the products of important oocyte-expressed sizes: 455 and 355 bp) was selected for further
genes were likely underrepresented in the analysis because it was completely novel
results of ovarian EST projects and hence and did not show significant homology to
unidentified. sequences of any known genes or ESTs
deposited in the Genbank. The name JY-1
To gain a better understanding of the was assigned to the putative novel gene
composition of the transcriptome of bovine encoding for this transcript. Identification
oocytes, we constructed a cDNA library of this novel EST was significant because
from a pool of 200 immature, germinal at the time of its discovery, there were
vesicle (GV) and mature, metaphase II (MII) 4.9 million human, 3.7 million mice, and
stage oocytes and sequenced a limited approximately 228,000 bovine EST sequences
number of ESTs (Yao et al. 2004). Our initial deposited in the Genbank. Since this time,
analysis of 230 EST sequences from the additional JY-1 EST obtained from two-cell
bovine oocyte cDNA library (unnormalized) embryos as part of the MU bovine genome
revealed novel information about oocyte- project (genome.rnet.missouri.edu/bovine)
expressed genes. The 230 ESTs represented have been deposited in the Genbank. Based
102 unique sequences of which 46 displayed on these results, we hypothesized that
significant similarities to sequences encod- the expression of JY-1 is oocyte-specific
ing for known genes present in the Genbank and of potential functional significance to
database. While some ESTs correspond to female fertility, and conducted a series of
housekeeping genes critical to the function
Ovarian Function in Livestock 187
experiments to further characterize JY-1 Results demonstrate pronounced effects of
expression and actions relevant to follicular rJY-1 on granulosa cell function and mimic
development and early embryogenesis. those initiated in preovulatory granulosa
cells during the luteinization process.
Our published results (Bettegowda et al.
2007) established that the JY-1 gene encodes Our results also support an important
for a species-specific secreted protein belong- regulatory role for JY-1 in bovine early
ing to a novel protein family. JY-1 mRNA embryonic development. Abundance of JY-1
and protein are expressed in an ovary-specific mRNA is temporally regulated during early
fashion, present throughout follicular devel- embryonic development in a manner char-
opment in primordial through antral follicles acteristic of maternal effect genes, and the
and restricted exclusively to the oocyte. The JY-1 gene is not transcribed in early embryos.
oocyte-specific expression of JY-1 supports a JY-1 small interfering RNA (siRNA) injec-
potential specific role for JY-1 in the regula- tion into zygotes revealed that oocyte-
tion of follicular development. derived JY-1 is required for embryos to reach
the blastocyst stage, and most JY-1 siRNA-
Recombinant JY-1 protein (rJY-1) is bio- injected embryos do not progress past the
logically active, and treatment with rJY-1 8- to 16-cell stage (Figure 8.2). The mecha-
in combination with follicle-stimulating nisms involved in JY-1 regulation of early
hormone (FSH) reduces granulosa cell estra- embryonic development are currently under
diol production but stimulates progesterone investigation.
production (Figure 8.1). Biologic actions of
JY-1 on bovine granulosa cells are novel and Given the fact that originally identified
do not mimic the reported effects of other JY-1 sequences in bovine oocyte cDNA
well-known oocyte-specific growth factors library were totally novel, we investigated
(GDF9 and BMP15) on bovine granulosa the presence of JY-1 orthologs in other
cells (McNatty et al. 2005; Spicer et al. 2006). species using available genome sequence
Figure 8.1 Effect of recombinant JY-1 protein (rJY-1) on granulosa cell estradiol and progesterone produc-
tion. (A) Effect of rJY-1 on FSH-stimulated estradiol production by bovine granulosa cells. (B) Effect of rJY-1
on progesterone production by FSH-treated bovine granulosa cells. Concentrations of estradiol and pro-
gesterone were normalized to 30,000 cells. Data are depicted as mean ± SEM (a,b; P < 0.05). Copyright
2007 National Academy of Sciences, U.S.A.
188 Physiological Genomics of Reproduction
(A) %8- to 16-cell embryos (72h) a a
80 64.0 66.7
60
40 b b
36.3 35.5
20
0 Sham water JY-1 siRNA JY-1 siRNA
Uninjected Species 1 Species 2
(B) a a
27.7 30.0
35
30 %blastocysts (d7) Uninjected bb
25 9.0 8.3
20
15 Sham water JY-1 siRNA JY-1 siRNA
10 Species 1 Species 2
5
0
Figure 8.2 Effect of microinjection of individual JY-1 siRNA species on the development of IVF embryos.
Presumptive one-cell IVF embryos were subjected to one of the following microinjection treatments:
(1) uninjected control, (2) sham water injection, (3) JY-1 siRNA 1 (25 μM), or (4) JY-1 siRNA 2 (25 μM).
Microinjected embryos were cultured in vitro for 7 days with rate of development to 8- to 16-cell stage
denoted on day 3 and rate of development to blastocyst stage denoted on day 7. (A) Effect of microinjection
of individual JY-1 siRNA species on percentage of embryos reaching the 8- to 16-cell stages. (B) Effect of
microinjection of individual JY-1 siRNA species on percentage of embryos reaching the blastocyst stage.
Data are shown as mean ± SEM (a,b; P < 0.0001). Copyright 2007 National Academy of Sciences, U.S.A.
resources. JY-1-like sequences are present at genesis EST sequencing project (Whitworth
chromosomal locations of other vertebrate et al. 2004). Approximately 1668 oocyte EST
species (e.g., mice, rats, humans) syntenic to sequences were reported, including those
the JY-1 locus on bovine chromosome 29, encoding known oocyte-specific genes such
but lack exons 1 and 2 and do not encode for as ZP1 and ZP3 and many unique sequences.
a functional protein (Figure 8.3). Results Virtual Northern blot analysis revealed 37
support species specificity in evolution and EST clusters whose representation in the
function of this novel oocyte-specific gene GV oocyte versus four cell embryo cDNA
initially identified through oocyte EST libraries differed significantly. Many of such
sequencing efforts. clusters correspond to new products of the
embryonic genome in swine or maternal
8.2.4 Oocyte EST sequencing in swine transcripts that were depleted or degraded
during the maternal-to-embryonic transi-
Sequencing of GV oocyte ESTs has also been tion whose functional significance has not
performed in swine as part of a pig embryo- been investigated.
Ovarian Function in Livestock 189
Figure 8.3 JY-1 gene structure and potential gous platforms for gene expression profiling
orthologs in other species. Genomic DNA data- in livestock species was a significant impedi-
bases at NCBI for bovine, human, chimpanzee, ment to the application of microarray tech-
dog, mouse, and rat were also searched with the nology to the study of ovarian gene expression.
nucleotide sequence of the 1.5-kb bovine JY-1 The above-described EST sequencing efforts
cDNA. Structure of JY-1 gene on bovine chromo- have facilitated advances in the application
some 29 is denoted. The JY-1 gene has three of microarray technology to the study of
exons (E1, E2, and E3) separated by two introns. gene expression in ovarian tissues, either
The start (ATG) and stop (TAG) codons of the open through the use of custom cDNA microar-
reading frame are indicated within the exons. JY-1- rays generated by individual investigators or
like sequences corresponding to exon 3 (length via the use of high-density arrays commer-
and sequence identity noted) were identified on cially available for cattle and swine. The
syntenic chromosomes in human (chromosome following section summarizes prominent
11), chimpanzee (chromosome 11), dog (chromo- microarray studies of gene expression in
some 21), mouse (chromosome 7), and rat (chro- ovarian tissues (follicle, CL, oocyte) in live-
mosome 1), but lack exons 1 and 2 and do not stock species with emphasis on studies
encode for a functional protein. Copyright 2007 where physiological insight was gained.
National Academy of Sciences, U.S.A.
8.3.1 Follicular growth and development
8.3 Transcriptomics of ovarian
tissues: Microarray studies Antral follicle growth in cattle is gonadotro-
pin (FSH and luteinizing hormone [LH])
The recent development of tools for expres- dependent and occurs in a characteristic
sion profiling in livestock has provided wave-like pattern (Ireland et al. 2000;
reproductive biologists new opportunities to Fortune et al. 2001). Two or three waves of
examine global changes in gene expression follicular growth are typical of the bovine
in ovarian tissues during key developmental estrous cycle. Follicular waves are generally
time points, in response to hormonal treat- characterized by growth of a cohort of mul-
ments, and as a tool for phenotyping or pre- tiple small antral follicles and subsequent
dicting reproductive potential. Several years atresia of all follicles (termed subordinate
ago, the availability of appropriate homolo- follicles) but one that continues to grow
(termed the dominant follicle). During each
wave, a dominant follicle grows to ovulatory
size and either undergoes atresia or ovulates
if present during the follicular phase of the
estrous cycle.
Evans et al. (2004) published the first
microarray study of mechanisms regulating
follicular development in cattle. Granulosa
and theca cells were collected from individ-
ual dominant and the largest subordinate
follicles on day 3 after the initiation of
a follicular wave. A bovine cDNA micro-
array (BOTL-4) representing over 1300 genes,
190 Physiological Genomics of Reproduction
including receptors, signaling proteins, tran- duction proteins were found to be differen-
scription factors, and apoptosis regulators, tially expressed in granulosa (45 genes) and
was utilized. Approximately 261 differen- theca cells (38 genes) of dominant versus
tially expressed genes in granulosa and (or) subordinate follicles (Forde et al. 2008).
theca cells of dominant versus subordinate Differential expression of a subset of such
follicles were detected by microarray analy- genes was examined using Q-RT-PCR.
sis. Authors hypothesized that granulosa Expression of mRNA for BCAR1 was greater
and theca cells from growing dominant fol- in the granulosa cells of dominant follicles,
licles would have greater expression of genes whereas mRNA for TGFBR3, FIBP, SIPA1,
linked to inhibition of apoptosis and lower PPID, and RANGAP1 was higher in the
expression of proapoptotic genes than granulosa cells of subordinate follicles. In
cells from subordinate follicles. Differential contrast, mRNA for FRAP1, GNAI3,
expression of 11 genes linked to the regula- CAMK1, FIBP, STX5, WNT2B, DGCR2, and
tion of apoptosis in granulosa and theca cells FMNL3 was higher in the theca of dominant
of dominant versus subordinate follicles was versus subordinate follicles, and EPHA4
detected by microarray analysis and con- expression was increased in the theca layer
firmed by quantitative real time PCR. In the of subordinate versus dominant follicles.
granulosa layer, mRNA for DICE1 and MCL1 Expression of mRNA for the genes above
was greater in granulosa cells of dominant was further examined in granulosa and (or)
follicles, whereas mRNA for PTGS1, TNF, theca cells of follicles collected at specific
CAD, and DRAK2 was greater in the granu- stages of a follicular wave representing
losa cells of subordinate follicles. Within the wave emergence, selection, and dominance.
theca layer, mRNA abundance for CASP13, Expression profiles supported important
P58IPK, APAF1, BTG3, and TSBCLL was roles for CAMK1 and the receptor tyrosine
greater in subordinate versus dominant fol- kinase EphA4 in theca cells and BCAR1 in
licles. Authors also noted increased expres- granulosa cells for the development of domi-
sion of the inhibin co-receptor betaglycan nant follicles. The authors also utilized
(TGFBR3) in granulosa and theca cells of siRNA-based RNA knockdown procedures
subordinate versus dominant follicles. Given to investigate the contribution of TGFBR3
the marked enhanced production of estradiol and FIBP to granulosa cell estradiol produc-
and the presence of estradiol receptors in tion. Transfection of FIBP siRNA into granu-
dominant versus subordinate follicles early losa cells, and accompanying knockdown
in development, it was suggested that the of FIBP mRNA led to a significant increase
genes above may in fact represent estradiol in estradiol production and the ratio of
target genes functionally associated with the estradiol to progesterone produced by cul-
selection of a dominant follicle during fol- tured bovine granulosa cells. Knockdown
licular waves in cattle (Evans et al. 2004). of TGFBR3 RNA using similar procedures
However, the regulation and functional sig- also significantly increased the estradiol to
nificance of most of the genes above to fol- progesterone ratio. Although accompanying
licular development in cattle, with the demonstration of knockdown of TGFBR3
exception of TGFBR3 (described below) has and FIBP protein following transfection of
not yet been determined. their respective siRNAs would further
increase confidence in the results presented,
From the same microarray study described described studies (Forde et al. 2008) support
above, 83 genes encoding for signal trans-
Ovarian Function in Livestock 191
a functional role for TGFBR3 and FIBP3 in ing molecular evidence supporting a shift in
negative regulation of estradiol production control of dominant follicle development
and illustrate how results of microarray from FSH to LH dependence in cattle (Mihm
studies can be utilized as a platform to for- et al. 2006).
mulate novel, specific hypotheses and gener-
ate accompanying functional data that can Gene expression profiling was also used
enhance understanding of regulation of fol- to characterize differences in ovary and
licular growth and development. ovarian follicle transcriptome composition
between a randomly selected control line
To investigate the molecular mechanisms and a selected index line of pigs that ovu-
controlling FSH and LH signaling in growing lates approximately 6.7 more oocytes per
dominant follicles, Mihm et al. (2006) con- cycle than the control line (Caetano et al.
ducted microarray studies of dominant fol- 2004). A porcine follicle microarray spotted
licles collected from cows at 2, 2.5, 3, 3.5, 4, with cDNAs representing 3636 unique
5, and 5.5 days after the emergence of the clones was interrogated with labeled cDNA
first follicular wave. An expanded version derived from RNA isolated from ovarian
of the above-described cDNA microarray, tissue and isolated follicles (2–7 mm) from
referred to as BOTL-5, was used to screen for the control and index lines. Differential
genes that were differentially regulated in expression of 71 genes in ovarian tissue and
dominant follicles across different days of 59 additional genes in follicular samples
development. Alterations in mRNA abun- across days of the follicular phase was noted.
dance for 60 genes in the granulosa layer The design of this study precludes definitive
were observed from day 2 to day 5.5 of the conclusions about the physiological signifi-
follicular wave. From the microarray results, cance of differential gene expression observed
Q-RT-PCR analysis was performed for genes relative to the ovulation rate phenotype of
from above linked to apoptosis regulation, interest. However, 12 of the differentially
cell proliferation, steroidogenesis, growth expressed genes above were subsequently
factor signaling, and for known FSH-, LH-, mapped to quantitative trait loci associated
and estradiol-regulated genes. with the ovulation rate in swine (Caetano et
al. 2005), which illustrates the utility expres-
A decrease in the follicular fluid estradiol sion profiling data holds for molecular genet-
to progesterone ratio, characteristic of a ics applications relevant to reproductive
decrease in estradiol-producing capacity, traits of interest.
was coincident with a specific gene expres-
sion profile in granulosa cells during domi- 8.3.2 Luteinization of the
nant follicle growth. Concentrations of dominant follicle
mRNA encoding for FSHR, ESR2, INHA,
ACVR1, CCND2, and the proapoptotic gene Using the porcine ovarian ESTs (Jiang et al.
SIVA decreased in granulosa cells, while 2004) described above, Agca et al. (2006) gen-
mRNA for LHCGR increased from day 2 to erated a porcine ovary cDNA array repre-
day 5.5 of the first follicular wave. The senting 8009 unique genes and used such
observed reduction in mRNA for FSHR and array to investigate changes in transcrip-
known genes positively regulated by FSH tome composition associated with luteiniza-
(ESR2, ACVR1, INHA, CCND2), coupled tion of preovulatory follicles in the pig. RNA
with an observed increase in granulosa cell samples isolated from whole preovulatory
LHCGR mRNA, provided new and convinc-
192 Physiological Genomics of Reproduction
porcine follicles >6 mm in diameter obtained in luteinized follicles included MAOB and
before estrus versus similar sized luteinized LAP3.
follicles collected approximately 1 day after
the preovulatory LH surge (day 2 post estrus) The porcine ovarian microarray study
were utilized to interrogate the array. There described above (Agca et al. 2006) identified
were 107 and 43 genes with mRNA identi- distinct differences in the transcriptome of
fied as decreased or increased in luteinized preovulatory estrogenic versus luteinized
follicles, respectively (Agca et al. 2006). porcine follicles. Previously undocumented
Pronounced changes in mRNA abundance changes in gene expression associated with
for specific genes functionally associated the luteinization process were identified. It
with the characteristic shift in follicular is worth noting that there were commonali-
steroidogenic and proliferative capacity asso- ties in changes in gene expression (CYP19A1,
ciated with the luteinization process were LRP8, CJA1, LHCGR, JAK3) accompanying
detected. Steroidogenic factors with greater luteinization in the study above and in a
mRNA concentrations in the estrogenic fol- previously published study in the bovine
licles collected prior to the LH surge included using suppressive subtractive hybridization
STAR, CYP17A1, POR (donates electrons (SSH) (Ndiaye et al. 2005), providing further
to P450 complexes), and HSD3B1. INHBB, confidence in microarray results and mecha-
PTGES, and AKR1C3 mRNA concentrations nistic changes in gene expression accom-
were also greater in estrogenic versus lutein- panying the luteinization process across
ized follicles. Furthermore, dimeric dihy- species.
drodiol dehydrogenase (SUS2DD), which
converts progesterone to its inactive form, 8.3.3 CL regression
was more highly expressed in estrogenic fol-
licles collected prior to the LH surge, which The principle endocrine factors governing
indicates that progesterone may be metabo- luteal development and regression have been
lized by estrogenic preovulatory follicles. well characterized, but the intracellular
Greater mRNA abundance for CCND2 and mechanisms by which these processes occur
members of the Wnt/β-catenin pathways are not thoroughly understood. Casey et al.
were also observed in preovulatory estro- (2005) used the above-described 351 CL ESTs
genic follicles relative to luteinized follicles (Casey et al. 2004) combined with 83 ESTs
collected after the LH surge, reflective of from an ovarian cortex library to generate a
differences in proliferative status. 434 gene cDNA array. Corpora lutea were
obtained from heifers on days 16 through 19
Following the LH surge, luteinized folli- following estrus, then classified as regressing
cles had greater expression of cell adhesion- or non-regressing based on systemic proges-
and migration-related mRNAs and mRNA terone concentrations and evidence of apop-
for factors regulating blood flow. These tosis (oligonucleosome formation) in luteal
included mRNA for cell surface antigens tissue. Results of array analysis indicated
CD9, CD24, and LGALS3. Six mRNAs that mRNA for steroidogenic factors includ-
encoding for proteins with growth inhibi- ing HSD3B1, STAR, SCARB1, and CYP11A1
tory functions, including PPP2CB and were downregulated in regressing as com-
PDCD4, were upregulated in luteinized fol- pared with non-regressing CL, as was GSTA1,
licles collected after the LH surge. Messenger which functions to protect lipids from oxida-
RNAs for vasodilatory proteins upregulated tive stress. In contrast, mRNA for CLU, a
Ovarian Function in Livestock 193
factor linked to mammary tissue involution In contrast, in a recent microarray study of
and tissue structural/remodeling compo- changes in the oocyte transcriptome associ-
nents (COLA1 and MGP) was increased in ated with in vitro oocyte maturation, Fair
regressing corpora lutea. Such results (Casey et al. (2007) reported that approximately
et al. 2005) illustrate the utility of array 54% of the 23,000 transcripts represented on
approaches for the characterization of tem- the bovine Affymetrix array were detected.
poral changes in mRNA abundance during Abundance of 209 transcripts was increased
luteal regression, but alone, provide limited and 612 transcripts decreased in MII oocytes
new biologic insight into mechanisms following in vitro maturation relative to
involved in luteal regression due to lack of their immature GV stage counterparts. Gene
precisely timed samples relative to initiation ontology classification revealed prominent
of luteolysis, limited number of genes repre- changes in genes involved in the regulation
sented on the array, and absence of comple- of mitogen-activated protein (MAP) kinase
mentary hormonal regulation or functional activity, translation initiation, and tran-
studies. scription accompanying meiotic maturation.
While descriptive, this data set will provide
8.3.4 Oocyte maturation a foundation for future studies focused on
the identification of components of the
One of the first array-based attempts to maternal mRNA pool and associated path-
characterize the oocyte transcriptome and ways aberrantly regulated during meiotic
changes in RNA transcript profiles associ- maturation in vitro versus in vivo. It is well
ated with bovine oocyte maturation was established that the maternal pool of mRNA
reported by Dalbies-Tran and Mermillod and proteins is critical to early embryonic
(2003). In this study, mRNA was collected development until control is transferred
from bovine oocytes before and after in to products of the embryonic genome
vitro maturation. 32P-labeled probes gener- (Bettegowda et al. 2008a). Given that rates of
ated from cDNA amplified from the two embryonic development to blastocyst stage
oocyte populations were hybridized to Atlas in vitro are 2.5- to 3-fold greater when in vivo
human arrays consisting of cDNA fragments matured oocytes are fertilized and cultured
representing 1176 known genes spotted on versus in vitro matured counterparts (Rizos
nylon membranes. Positive signals for 300 et al. 2002), it appears likely that alterations
genes were detected, and mRNA abundance in the maternal RNA pool associated with in
for 37 and 33 genes was decreased and vitro oocyte maturation are functionally
increased, respectively, within in vitro associated with poor oocyte developmental
matured MII versus immature GV oocytes competence and reduced rates of embryonic
harvested prior to maturation. At the time development.
of this study, platforms for gene expression
profiling in livestock species were limited. 8.3.5 Oocyte competence
Hence, conclusions about lack of expression
for specific genes could be attributed to Oocyte competence, defined as the ability of
insufficient sequence homology between an oocyte to be fertilized and develop to the
human cDNAs spotted on the array and blastocyst stage is progressively acquired
mRNA sequences for corresponding bovine during the period of oocyte growth accom-
genes. panying follicular development (Eppig et al.
194 Physiological Genomics of Reproduction
2002; Matzuk et al. 2002). Pituitary gonado- mRNA abundance for FST in oocytes with
tropins and bidirectional local communica- oocyte competence and a potential func-
tion between the oocyte and adjacent tional role for FST in bovine early embryonic
cumulus cells are critical for both nuclear development (Patel et al. 2007). Real-time
and cytoplasmic maturation (acquisition of reverse transcription–polymerase chain reac-
the ability to complete meiosis, to ensure tion (RT-PCR) analysis using a set of samples
monospermic fertilization, and to undergo distinct from those used in microarray exper-
preimplantation development) (Eppig et al. iments confirmed lower amounts of mRNA
2002; Gosden 2002). Competencies acquired for FST in oocytes collected from prepubertal
by the nuclear and cytoplasmic compart- (low quality oocytes) versus adult animals
ments during the final stages of maturation and in late cleaving versus early cleaving
support the notion that oocyte quality two-cell stage bovine embryos (Figure 8.4).
depends on a multitude of factors, many Early cleaving two-cell stage embryos
of which can be assessed only at the mole- develop to the blastocyst stage at an approxi-
cular level. mately fourfold greater rate than their late
cleaving counterparts. Given that activation
We have utilized functional genomics of the embryonic genome and transition
approaches to identify the differences in from oocyte to embryonic control of devel-
RNA transcript profiles of both the oocyte opment occurs later (eight-cell stage), differ-
and adjacent cumulus cells associated with ences in transcript abundance between early
poor developmental competence of bovine and late cleaving bovine embryos are reflec-
oocytes and established a series of markers tive of differences in competence of oocytes
predictive of oocyte competence (Patel et al. from which they were derived. Furthermore,
2007; Bettegowda et al. 2008b). The prepu- preliminary results indicate that follistatin
bertal calf model of poor oocyte competence supplementation during the initial stages
has been foundational to such studies (Revel of in vitro embryo culture (prior to embry-
et al. 1995; Damiani et al. 1996). Oocyte onic genome activation) can enhance rates
RNA transcript profiling experiments using of blastocyst development (Lee et al. 2007).
a bovine cDNA array with approximately Results support a positive association of
15,000 genes represented (Suchyta et al. FST mRNA abundance with oocyte quality
2003) revealed a total of 193 genes coding and suggest the potential for a functional
for transcripts displaying greater mRNA role for FST in bovine early embryonic
abundance in adult oocytes and 223 genes development.
coding for transcripts displaying greater
mRNA abundance in compromised pre- Available evidence (Hagemann 1999) indi-
pubertal oocytes (Patel et al. 2007). Such cates that competence of oocytes for in vitro
results formed the foundation for subsequent embryo production in cattle is influenced by
studies focused on elucidation of the func- stage of the follicular wave during which
tional significance of the markers identified. oocytes were collected, with developmental
Of particular interest from the oocyte micro- competence of oocytes from small follicles
array studies were genes in the regulation of greater when recovered during the growth
hormone secretion ontology category (FST, phase (early in a wave) than the dominance
INHBA, INHBB), which were overrepresented phase (when a dominant follicle is present).
in the adult oocyte samples. Subsequent Using a bovine cDNA array containing 2304
studies revealed a positive association of bovine oocyte/embryo-enriched ESTS and
Ovarian Function in Livestock 195
(A) (B) a
8 4
Relative expressionb
6 Relative expression
4 2
2a b
0
0 Late
Adult Prepubertal cleaving
Early
Follistatin mRNA cleaving
Follistatin mRNA
Figure 8.4 Positive relationship between follistatin mRNA abundance and oocyte competence in two
distinct models of poor oocyte competence. (A) Quantitative real-time RT-PCR analysis of follistatin mRNA
abundance in oocytes collected from adult (black bars) and prepubertal (white bars) animals (model of poor
oocyte competence). (B) Quantitative real-time RT-PCR analysis of follistatin mRNA abundance in two-cell
stage bovine embryos (collected before embryonic genome activation) that cleaved early (≤30 h post fertil-
ization) versus those that cleaved late (30–36 h post fertilization; model of poor oocyte competence).
Approximately fourfold greater development to blastocyst stage was observed for embryos cleaving early
versus late. Data were normalized relative to abundance of RPS18 (endogenous control) and are shown
as mean ± SEM (a,b; P < 0.05).
various control genes (Sirard et al. 2005), tence, a similar relationship between mRNA
Ghanem et al. (2007) identified 51 tran- abundance and oocyte competence was
scripts differentially expressed between observed for PTTG1, MSX1, PP, and RPL24
oocytes collected from small follicles in the as was observed when stage of the follicular
growth (good quality oocytes) versus domi- wave at collection was used as criteria for
nance phase (poor quality oocytes). Greater the classification of oocyte competence, pro-
mRNA abundance for ANXA2, S100A10, viding evidence of a relationship between
PP, and RPL24 in the oocytes of small fol- oocyte mRNA abundance for the genes
licles collected during the growth phase and above and developmental competence in a
for MSX1 and BMP15 in oocytes of small second model predictive of oocyte quality.
follicles collected during the dominance The functional contribution of observed dif-
phase was confirmed by Q-RT-PCR. The ferences in mRNA abundance of PTTG1,
authors also investigated the relationship MSX1, PP, and RPL24 to oocyte competence
between G6PDH activity in oocytes col- remains to be elucidated.
lected from an abattoir (determined by bril-
liant cresyl blue [BCB] staining) and mRNA We (Bettegowda et al. 2008b) have utilized
abundance for five genes associated with a similar approach to identify cumulus
oocyte competence in the above model. cell markers associated with poor quality
Several reports indicate that the activity of oocytes in the prepubertal model system.
G6PDH is negatively associated with bovine Approximately 110 genes encoding for tran-
oocyte competence (Pujol et al. 2004; Alm scripts displaying greater mRNA abundance
et al. 2005). Using BCB staining as the crite- in cumulus cells surrounding GV oocytes
ria for the classification of oocyte compe- collected from adult animals and 45 genes
encoding for transcripts displaying greater
196 Physiological Genomics of Reproduction
mRNA abundance in cumulus cells sur- petence were obtained, were then subjected
rounding compromised prepubertal oocytes. to RNA isolation. Using ddRT-PCR and SSH,
Genes in the cysteine-type endopeptidase 5 and 18 potential transcripts, respectively,
(cathepsin) activity category (CTSB, CTSS, were identified as potentially differentially
CTSZ) were overrepresented in the cumulus expressed in the granulosa cells of follicles
cell samples harvested from oocytes of pre- bearing oocytes that did or did not develop
pubertal animals. Q-RT-PCR analysis con- into blastocysts following IVF. While more
firmed that higher amounts of mRNA for research is needed, results of this study and
the cathepsins above are present in cumulus the cumulus cell transcript profiling study
cells surrounding poor quality oocytes har- described above (Bettegowda et al. 2008b)
vested from the ovaries of prepubertal versus illustrate the potential diagnostic applicabil-
adult animals. We also established a nega- ity of ovarian cumulus and granulosa cells as
tive relationship between cumulus cell indicators of oocyte competence.
cathepsin B, S, and Z expression and embry-
onic phenotype (blastocyst development) 8.4 Proteomics of ovarian tissues
for oocytes collected from adult animals.
Furthermore, addition of a cathepsin inhibi- While a vast amount of novel information
tor during in vitro oocyte maturation can about the biology of the oocyte has been
enhance subsequent rates of blastocyst obtained using RNA transcript profiling
development following parthenogenetic approaches, oocytes display pronounced
activation or in vitro fertilization (IVF) and posttranscriptional regulatory mechanisms
is associated with reduced rates of cumulus that control RNA translation and stability
cell apoptosis. Collectively, results support (Bettegowda and Smith 2007). Hence, it is
a potential functional relationship between the protein products that modulate such
cumulus cell cathepsin expression and processes as nuclear and cytoplasmic
oocyte competence in cattle and suggest maturation and development through the
that cumulus cell cathepsin expression is maternal-to-embryonic transition following
predictive of an oocyte’s embryo develop- fertilization. Ellederova et al. (2004) used
ment potential. two-dimensional gel electrophoresis and
matrix-assisted laser desorption/ionization
The relationship between granulosa cell (MALDI) mass spectrometry to characterize
gene expression and oocyte competence has composition of the porcine oocyte proteome.
also been investigated in the bovine model From 350 spots on the gel, proteins in 35
using SSH and differential display RT-PCR spots were identified by mass spectrometry
(ddRT-PCR) procedures (Robert et al. 2001). with 18 spots representing individual pro-
Cumulus oocyte complexes were aspirated teins. Proteins in greatest abundance (equal
from small (<4 mm) or large (>5 mm) follicles to or greater than β-actin) in porcine oocytes
of heifers treated with FSH and pooled in included peroxiredoxins, spermine synthase,
groups of five. Granulosa cells from indi- and ubiquitin carboxyl-terminal hydrolase
vidual pools of follicles were harvested and isozyme L1. When examining changes in the
stored for subsequent analysis and pools oocyte proteome at different stages of porcine
of oocytes subjected to in vitro maturation, in vitro maturation, intensity of all but six
IVF, and embryo culture to assess develop- spots remained stable, most of which did not
mental competence. Granulosa cells from
follicle pools, where 100% versus 0% com-
Ovarian Function in Livestock 197
contain a single protein. The exception was indicate that overall number of phosphory-
antiquitin, a member of the aldehyde dehy- lated proteins increases in response to matu-
drogenase family whose abundance increased ration signals. Analysis of two-dimensional
during meiotic maturation. gels revealed that proteins in seven spots
were shown to be differentially phosphory-
In a subsequent study from the same lated at the above stages of meiotic matura-
group, Susor et al. (2007) characterized tion, including cyclin E2 and a truncated
changes in the proteome of porcine oocytes form of cyclin E2, protein disulfide isomer-
during in vitro maturation, but used 35S ase 3 ER 60 precursor, peroxiredoxin 2,
incorporation to identify changes in synthe- β-actin, aldose reductase, and uridine mono-
sis of specific proteins during in vitro matu- phosphate (UMP) synthase. However, the
ration. Intensity of 16 protein spots changed importance of changes in abundance and (or)
from GV to MII stages, with intensity of four phosphorylation of the above proteins to pro-
spots increased. The identity of proteins in gression of meiotic maturation (nuclear and
one such spot that increased in intensity cytoplasmic) remains to be elucidated.
was determined to be ubiquitin C-terminal
hyhydrolase-L1 (UCHL1) by mass spectrom- The most comprehensive study on charac-
etry. Addition of a specific inhibitor of teristics of the bovine oocyte and cumulus
UCHL1 during in vitro maturation signifi- cell proteome was reported by Memili et al.
cantly blocked the progression of porcine (2007). In this study, 5253 and 1950 proteins
oocytes to MII, with the majority of oocytes were identified in cumulus cells and oocytes,
arrested at metaphase I (MI) in response to respectively, using differential detergent frac-
treatment with the highest concentration tionation two-dimensional liquid chroma-
of inhibitor. These biologically relevant tography followed by electrospray ionization
results obtained using proteomics approaches tandem mass spectrometry. Approximately
suggest an important role for UCHL1 in 12% of proteins were common to the oocyte
completion of the first meiosis and transi- and cumulus cells. The importance of this
tion to anaphase in porcine oocytes. study as a resource for future investigations
relevant to oocyte and cumulus cell function
Bhojwani et al. (2006) used two- is highly significant. For example, proteins
dimensional gel electrophoresis, MALDI- representing 338 transcription factors and
time of flight (TOF) mass spectrometry, 241 receptor-ligand pathways present in the
and Pro-Q diamond phosphoprotein staining cumulus cells and oocyte were detected,
to assess changes in the bovine oocyte including 18 pathways for growth factors.
phosphoproteome (phosphorylated proteins) Such information provides a tremendous
during in vitro maturation. Oocytes were foundation for formulation of specific hypo-
cultured for 0, 10, and 24 h to represent GV, theses and future studies of differential
MI, and MII stages of meiotic maturation. expression and potential function.
Approximately 550 spots were detected for
total proteins at each stage of maturation 8.5 Future research directions
and identity of proteins in 40 spots obtained,
four of which differed in abundance at differ- The above-described results illustrate the
ent stages of in vitro maturation. Approxi- contribution of EST sequencing at a species
mately 190, 270, and 250 spots representing level to gene discovery and the value of EST
phosphorylated proteins were detected at
GV, MI, and MII stages, respectively, which
198 Physiological Genomics of Reproduction
projects for specific cell types (e.g., oocyte) may soon become routine. Development of
likely underrepresented in libraries used to technology for high-density gene expression
generate the majority of currently reported profiling in other farm species is certainly
EST sequences. While gene predictions from not far behind. While the wet lab component
available genome sequence hold value, con- of microarray experiments is not technically
firmation of expression is still important. demanding, data analysis and interpretation
Results also illustrate how gene discovery can present significant obstacles due to the
from EST sequencing projects can be linked sheer volume of data generated and accom-
to functional studies of biologic significance. panying statistical challenges (e.g., multiple
While virtual Northern analysis procedures testing). Hence, ovarian biologists applying
described above do hold potential for char- such technologies face an oncoming explo-
acterization of differences in transcriptome sion of information and potential data over-
composition, such approaches are biased load. To emphasize results of functional and
toward the detection of differences in fre- biologic significance, a logical and system-
quency for abundant transcripts. Such efforts atic approach grounded in sound experimen-
can only be undertaken using clone fre- tal design with sufficient biologic replication,
quency data obtained from libraries that appropriate statistical analysis incorporating
were not subjected to normalization and are control of false discovery rate, and utiliza-
limited in depth by the number of clones tion of available tools to facilitate interpreta-
sequenced from individual libraries. Results tion of biologic themes can relieve the
of biologic interest for future study should enormity of such experiments and facilitate
be confirmed using appropriate quantitative the generation of new biologically signifi-
procedures, such as real-time RT-PCR cant data relevant to an individual’s model
(Q-RT-PCR). In silico analysis of EST fre- system of interest (Smith and Rosa 2007).
quency cannot replace the scope and power Although challenging due to less extensive
afforded by microarray approaches for global annotation/ontology classification for genes
investigation of tissue-specific gene expres- in livestock species, functional categories of
sion, but such questions remain relatively co-regulated genes and gene pathways can be
unexplored in livestock species. Further mined, and hypotheses about common regu-
improvements in parallel platforms for EST latory elements can be formulated and inves-
sequencing at greatly reduced costs may tigated, given the availability of genome
ultimately result in greater application of sequence information. The application of
EST sequencing as a means for transcrip- such data mining approaches will move end
tome characterization between specific cell points of experiments beyond the simplicity
types of interest, stages of development, and of solely reporting lists of upregulated and
so on, but cannot currently replace the downregulated transcripts and also form a
power and practicality of microarray tech- foundation for delineation of results of great-
nology for addressing such questions. est potential biologic relevance and subse-
quent testing of significance in functional
Widespread availability of platforms for studies. Advances in technologies for testing
performing RNA transcript profiling experi- gene function (e.g., siRNA, morpholinos)
ments in important livestock species (cattle and appropriate methods of delivery into
and swine) has advanced incorporation of primary cultures of ovarian cells now make
such technology into studies of ovarian func- such studies not only possible but also
tion of farm animals, and such approaches
Ovarian Function in Livestock 199
necessary to confirm physiological signifi- Prather, R.S., and Lucy, M.C. 2006.
cance of microarray data. Luteinization of porcine preovulatory
follicles leads to systematic changes in
Documented use of proteomics approaches follicular gene expression. Reproduction
to investigate the regulation of ovarian 132: 133–145.
function in farm animals is much more Alm, H., Torner, H., Lohrke, B., Viergutz, T.,
limited than other approaches described. Ghoneim, I.M., and Kanitz, W. 2005.
Nevertheless, the challenges associated with Bovine blastocyst development rate in
the application of such technologies and the vitro is influenced by selection of oocytes
potential rewards are illustrated in the above by brilliant cresyl blue staining before
studies. The most up-to-date and highly IVM as indicator for glucose-6-phosphate
sensitive procedures for protein identifica- dehydrogenase activity. Theriogenology
tion must be utilized to stretch sensitivity 63: 2194–2205.
from mere detection of the most highly Bettegowda, A., Lee, K.B., and Smith, G.W.
abundant proteins to that necessary to 2008a. Cytoplasmic and nuclear deter-
obtain a “snapshot” reflective of the pro- minants of the maternal-to-embryonic
teome composition of cell/tissue types of transition. Reproduction, Fertility, and
interest. Sophisticated separation proce- Development 20: 45–53.
dures are also required to facilitate the iden- Bettegowda, A., Patel, O.V., Lee, K.B., Park,
tification of individual proteins at a high K., Ireland, J.J., and Smith, G.W. 2008b.
frequency. While cost and logistics of Identification of novel cumulus cell
technologies are not conducive to high- molecular markers predictive of oocyte
throughput analysis of multiple samples in competence: Functional and diagnostic
most settings, the value of information on implications. Biology of Reproduction 79:
proteome composition for ovarian tissues/ 301–309.
cell types of interest cannot be underesti- Bettegowda, A. and Smith, G.W. 2007.
mated, and the potential for subsequent Mechanisms of maternal mRNA regula-
functional studies derived from results of tion: Implications for mammalian early
proteomics investigations is evident. Major embryonic development. Frontiers in
investment in categorizing the proteome Bioscience 12: 3713–3726.
composition of ovarian tissues/cell types Bettegowda, A., Yao, J., Sen, A., Li, Q., Lee,
at key stages of development and archiving K.B., Kobayashi, Y., Patel, O.V., Coussens,
of such results in a searchable, categorized P.M., Ireland, J.J., and Smith, G.W. 2007.
publicly accessible database would greatly JY-1, an oocyte-specific gene, regulates
accelerate rate of advancement for reproduc- granulosa cell function and early embry-
tive biologists in unlocking the secrets of onic development in cattle. Proceedings
ovarian function beyond that possible using of the National Academy of Sciences of
traditional functional genomics approaches. the United States of America 104: 17602–
17607.
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9
Physiological Genomics of Preimplantation Embryo
Development in Production Animals
Luc J. Peelman
9.1 Introduction unique genome resulting from the fusion of
sperm and oocyte, each contributing unique
A mammalian genome contains on average genetic material and molecular toolbox, and
between 20,000 and 25,000 protein coding nutrients supplied by the mother and the
genes. With this relatively small amount, an environment in general. Each of these factors
organism needs to produce all the different puts constraints on the possibilities of the
metabolites necessary for its development developing organism. In order to cope with
and function. To be able to do this, several this enormous variation, the organism and
layers of regulation and modification exist. its individual cells have developed an admi-
It has become clear that much more of the rable flexibility. However, there are limits
genome is transcribed than was previously to this flexibility, and many developing
anticipated. A new box of RNAs has been embryos do not make it to adulthood. A
opened, the regulation and function of which newly fertilized oocyte soon encounters the
remains, for the most part, an enigma. It has first formidable hurdle (Figure 9.1).
been estimated that in mouse preimplanta-
tion development around 15,500 genes are To survive to the blastocyst stage, a fertil-
expressed at one time or another and that ized oocyte must switch on its own genome
this number is probably similar to that for after several rounds of cell division and
other mammals (Stanton et al. 2003). stop using the material that was stored
in the oocyte. Not much later, during the
In the long run toward adulthood, many early morula stage, cell differentiation com-
molecular hurdles have to be overcome by mences. The morula develops into a blasto-
an organism, which is equipped with a cyst with an inner cell mass (ICM) containing
205
206 Physiological Genomics of Reproduction
Two-cell Four-cell Eight-cell Morula Blastocyst
Maternal RNA Embryonic RNA
Bovine–Ovine
Maternal RNA Embryonic RNA
Porcine
Figure 9.1 Schematic representation of preimplantation embryo development from the 2-cell stage with
indication of maternal RNA degradation and embryo genome activation.
the embryonic stem cells and an outer tro- 9.2 Preimplantation developmental
phoblast layer that will form the extra stages and transcriptomics
embryonic tissue. The blastocyst must then
hatch from the zona pellucida before being 9.2.1 Timing of the first cleavage
able to implant. All of these transitions need division—Developmental competence
well-orchestrated sweeps of gene expression.
The study of gene expression during preim- Developmental competence, which is the
plantation embryo development is a real ability of an oocyte to proceed through mat-
challenge mainly due to the innate complex- uration, fertilization, and embryo develop-
ity of expression profiling, the individual ment, is largely determined by the quality
embryo variation in expression patterns, the of the oocyte. Oocytes competent to com-
difficulty of obtaining in vivo embryos, and plete nuclear maturation, meaning to be
the small amount of starting material with able to progress through meiosis, do not all
which to work. Many of the studies are per- show the same capacity to reach the blasto-
formed using in vitro–produced embryos, cyst stage. This difference has been linked
introducing an extra level of variation due to variations in cytoplasmic maturation,
to the differences in media and culture con- including differences in oocyte activation,
ditions used. The small amount of starting pronucleus formation, and preimplantation
material necessitates an extra amplification development. Molecular mechanisms gov-
step or pooling of embryos for several appli- erning these processes are only barely under-
cations like the use of microarrays for tran- stood. Oocytes of lower quality have a
scription profiling. delayed first cell cycle, slower cleavage rate,
Preimplantation Embryo Development 207
and lower blastocyst yield (Lonergan et al. development. Ped homologs have been
1999). identified in humans as the HLA-G gene
(Juriscova et al. 1996), and in bovine as the
Leoni et al. (2007) used ovine oocytes from MHC I4221.1 gene (Fair et al. 2004a). In this
prepubertal animals (30–40 days old) as a study, the fast allele of the bovine Ped
model of low-quality oocytes and compared gene had a threefold higher transcription in
their developmental capacity and gene tran- fast-cleaving two-cell embryos than in slow-
scription profile with oocytes from adult cleaving ones. Fair et al. also found lower
animals. They observed similar maturation transcription in in vitro-produced embryos
and fertilization rates but a delayed first cell from different stages up to the blastocyst
cycle, slower cleavage rate, and lower blas- compared to in vivo embryos, with the
tocyst yield in the prepubertal oocyte group. largest difference starting at the 16-cell
Of the 11 genes studied, seven (activin, stage, which coincides with the onset of a
p34cdc2, glucose-transporter 1, Na+K+ATPase, major activation stage of the embryonic
E-cadherin, zona occludens protein 2, and expression.
poly(A)polymerase) showed a significant
reduction in relative mRNA abundance, Fair et al. (2004b) performed a limited
indicating that the lower developmental suppression subtractive hybridization (SSH)
competence in prepubertal oocytes is associ- experiment between early and late-cleaving
ated with deficiencies in the mRNA storage ttwo-cell bovine embryos to identify genes
of germinal vesicle oocytes. associated with developmental competence.
Between 30 and 40 clones from each library
Several genes associated with the timing were sequenced. Of these clones, three
of the first cell division and developmental (H3A, cyclin B1, and BMP15) were chosen
competence have been identified. Compari- for further analysis using real-time poly-
son of mRNA contents of slow and fast merase chain reaction (PCR). H3A was found
cleaving bovine embryos revealed differ- to be more abundant in early-cleaving
ences in histone 3 (H3A), preimplantation embryos, whereas the transcription of the
embryo development (Ped; Fair et al. 2004a,b), two other genes was variable. In another
HPRT, G6PD, IGF-I and IGF-IR (Lonergan study, the transcription levels of H2A were
et al. 2000), GLUT-5, sarcosine (SOX), Mn- also higher in fast-cleaving bovine embryos
superoxide dismutase (MnSOD), Cx43, IFN- than in slow-cleaving embryos (Dode et al.
τ, IGF-II, BAX (Gutierrez-Adan et al. 2004), 2006), and histone methyltransferase G9a,
histone 2 (H2A), isocitrate dehydrogenase which regulates lysine 9-acetylated histone
(IDH), and YY1- and E4TF1-associated factor H3 methylation, was found to be essential
1 (YEAF1) genes in cattle (Dode et al. 2006). for early embryo development (Tachibana et
al. 2002). Taken together, these results indi-
The Ped gene was identified in the mouse cate that early embryos must have enough
as the Qa-2 antigen gene located in the Q histone mRNAs in store for normal develop-
region of the mouse major histocompatibil- ment, and the abundance can eventually be
ity complex (MHC; Warner et al. 1987). The used as a marker for competence. Histone
mouse Ped gene has two alleles indicated as genes are often used as reference genes
fast and slow in reference to their effect on in real-time reverse transcriptase (RT)-PCR
the preimplantation developmental growth experiments. However, as these and other
rate. The fast allele gives a higher expression studies have shown, expression of histones
of the Qa-2 antigen in early embryos and is
correlated with a faster cleavage rate and
208 Physiological Genomics of Reproduction
is variable during preimplantation develop- and hypoxanthine phosphoribosyl trans-
ment and more than one reference gene ferase (HPRT), two genes that also play
should be used for accurate normalization an important role in energy metabolism.
(Goossens et al. 2005). Differential expression of both genes was
found between male and female blastocysts
In the study of Dode et al. (2006), two (Gutierrez-Adan et al. 2000), and a correla-
other genes were found to be upregulated in tion with cleavage rate was found in another
early-cleaving embryos. YEAF1 is involved study (Lonergan et al. 2000). No difference
in transcription. Its role in early embryo in expression was found in two-cell embryos,
development is not yet known. IDH cata- but blastocysts derived from fast-cleaving
lyzes a key regulatory step in the tricarbox- embryos had a higher expression of G6PD
ylic acid (TCA) cycle, which potentially and HPRT than blastocysts from slow-
plays an important role in bovine oocyte cleaving embryos. In the mouse, adenylation
maturation (Cetica et al. 2003), and IDH also of the poly(A)-tail of HPRT occurred during
has an important role in protection against oocyte maturation and was associated with
oxidative stress (Lee et al. 2002). an increase in its translation (Paynton and
Bachvarova 1994). Low oocyte competence,
Abundance of mRNA for insulin like indeed, seems to be associated with altered
growth factor I (IGF-I) may be an indicator poly-adenylation patterns and differences in
of embryonic developmental competence, maternal RNA degradation (Brevini et al.
at least in cattle. It was found in all early- 2002). It has been shown that a shorter
cleaving bovine two-cell embryos, but in poly(A)-tail is correlated with low develop-
none of the late-cleaving embryos. IGF-I mental competence (Brevini-Gandolfi et al.
receptor (IGF-IR) mRNA was found in all 1999, 2000). In conjunction with this, the
two-cell embryos. IGF-I and IGF-IR mRNA relative abundance of the poly(A) polymerase
were found in all blastocysts, regardless of (PAP) mRNA was found to be lower in ovine
cleavage status (Lonergan et al. 2003). prepubertal oocytes than in oocytes derived
from adult ewes (Leoni et al. 2007). It has
It has been observed that male bovine in been shown that maternal mRNAs stored
vitro–produced embryos have higher devel- in the cytoplasm of the oocyte have short
opmental rates than female (Hasler et al. poly(A)-tails and become translationally
1995; Massip et al. 1996). Male embryos active only after lengthening of the poly(A)-
have a higher competency, develop faster to tail by PAP and associated factors. Regulation
the blastocyst stage, and reach the expanded of this process is an important step in
blastocyst stage faster. It was postulated that early embryonic development (Gandolfi and
this difference is due to a gene located on Gandolfi, 2001).
the Y chromosome or a consequence of dif-
ferences in expression between male and Genes that may be stress induced, such as
female X chromosome-linked genes, with SOX, MnSOD, BAX, interferon tau (IFN-τ),
females having a higher expression because and G6PD, were found to be expressed in
they possess two gene copies before X inac- greater amounts in slow-cleaving embryos
tivation (Gutierrez-Adan et al. 1996). Oxygen and in in vitro-produced embryos than in
radicals are necessary for normal embryonic fast-cleaving embryos and in in vivo embryos,
development. Regulation of the amount respectively, whereas genes functioning
of oxygen radicals is influenced by, among in metabolism, growth, and differentiation
others, two X chromosome-linked genes,
glucose-6-phosphate dehydrogenase (G6PD)
Preimplantation Embryo Development 209
such as GLUT-5, Cx43, IGF-II, and IGF-IIR starting around the eight- to 16-cell stage (48
had higher mRNA concentrations in fast- to 64 h after fertilization). Due to this late
cleaving embryos and in in vivo embryos onset, EGA in cattle is somewhat easier to
(Gutierrez-Adan et al. 2004). The two study than in other species where EGA
patterns may be a reflection of the health starts earlier (see also Figure 9.1).
status of the embryo. However, some con-
tradicting results exist for the influence of Generally, two approaches have been used:
IFN-τ. In some studies, higher transcription (1) comparing transcript profiles of oocytes
of IFN-τ was related to reduced competence and two- to four-cell stage embryos with blas-
(Kubisch et al. 1998; Wrenzycki et al. 2001b), tocyst profiles and (2) blocking transcription
whereas in other studies higher transcription by addition of α-amanitin to the culture
of IFN-τ was linked to higher quality medium. Alpha-amanitin is a specific inhibi-
(Hernandez-Ledezma et al. 1993; Russell tor of RNA polymerase II and blocks de novo
et al. 2006). mRNA synthesis. The last approach was
used by Misirlioglu and coworkers (2006) in
9.2.2. Major onset of combination with microarray analysis using
embryonic expression the Affymetrix GeneChip Bovine Genome
Array (Affymetric Inc., Santa Clara, CA).
Probably the most crucial period of preim- They found 258 genes that were at least
plantation development is the activation of twofold increased in in vitro eight-cell
the embryonic genome. The embryo starts embryos compared with in vitro matured
development under the control of maternal oocytes (MII). Gene ontology analysis was
RNAs and proteins and has to switch on its carried out using NetAffx Analysis Center
own genome for further development. Failing and Cowbase (http://www.agbase.msstate.
to do this adequately will lead to the death edu). Ontology analysis identified regulators
of the embryo. Minor genome activation of transcription (NFYA, USF2), cell adhesion
starts in most mammalian species around (DSC2, COL12A1), signal transduction
the first cell division. Timing of the major (PTGER4, ADRBK1), transporters (CRABP1),
embryonic genome activation (EGA) is metabolism-related genes, and immune
somewhat different from species to species. response-related genes. On the other hand,
In mice, EGA occurs rapidly (in late one-cell 124 genes were found to be increased in MII
embryos), whereas in bovine and ovine EGA compared with eight-cell stage embryos.
is more delayed (eight- to 16-cell stage). Transcriptome comparison of eight-cell stage
In pigs and humans, EGA occurs around embryos with α-amanitin-treated eight-cell
the two-cell stage (Telford et al. 1990). An stage embryos revealed 233 genes with a
important aspect of the maternal to zygotic twofold or more increase. Among these genes
transition (MZT) next to EGA is the degra- were DSC2 and CRABP1, which were also
dation of maternal, oocyte-specific tran- found in the comparison between eight-cell
scripts. Given the importance of EGA, it has embryos and MII. Another gene, purine
been studied extensively. Several research nucleoside phorphorylase (NP), was 149-fold
groups compared the transcription profile of higher in eight-cell embryos and not detect-
the embryo, before and after EGA, with gene able in the α-amanitin-treated eight-cell
expression profiles. In cattle, the major onset embryos. Maternal degraded transcripts were
of embryonic expression is relatively late, identified by comparing MII with α-amaniti-
treated eight-cell embryos. A total of 147
210 Physiological Genomics of Reproduction
transcripts were at least twofold increased. starts at the 32- to 64-cell stage (Koyama
Among these were several genes involved et al. 1994). The cell differentiation is
in DNA methylation and metabolism accompanied by clear morphological changes
(Misirlioglu et al. 2006). in the embryo. The spherical blastomeres
flatten onto each other, forming a morula.
The expression of some important nucleo- Cell-to-cell adhesion occurs by formation
lar proteins was studied using the α-amanitin of epithelial zonula adherens (ZA) of which
block in pig preimplantation embryos. It the transmembrane E-cadherin protein is
was shown that RNA polymerase I and RNA an important constituent. E-cadherin binds
Pol I-associated factor PAF53 are transcribed homotypically extracellularly and with
de novo from the embryonic genome and catenin cytoplasmically (Figure 9.2).
that activation of these genes is delayed in
in vitro embryos compared with in vivo The catenins are linked to the actin cyto-
embryos (Bjerregaard et al. 2004). skeleton (Aberle et al. 1996). Following for-
mation of ZA, tight junctions (TJs) are
Using a microarray with 15,529 human formed. TJs are ring-like structures around
cDNAs, Adjaye et al. (2007) found 164 tran- a cell which are responsible for sealing
scripts that were bovine oocyte specific com- cells together and allowing the establish-
pared with blastocyst, and 1324 that were ment of apical and basal polarity. They
blastocyst specific compared with oocyte. consist of integral membrane proteins
Oocyte and blastocyst had 419 transcripts in (occludin [OCLN], claudins, and junction
common. Pathway analysis revealed differ- adhesion molecule [JAM]) that are linked
ential expression of genes involved in 107 to the actin cytoskeleton by a number of
distinct signaling and metabolic pathways. cytoplasmic plaque proteins (including dif-
A comparable but small-scale study in which ferent isoforms of zona occludens 1-3 [ZO-
the transcription profile of oocytes was com- 1-3]; (Stevenson and Keon 1998). Miller and
pared with that of blastocysts was performed coworkers (2003) studied six bovine TJ genes
by Mamo et al. (2006). They used a combina- (JAM, OCLN, Pan ZO-1, Pan ZO-2, ZO-1α+,
tion of cDNA array analysis and real-time and ZO-2β+) by semiquantitative RT-PCR.
RT-PCR to study the transcription of 82 They found a dramatic increase in total TJ
selected genes. Of these genes, 35 were found transcripts during transition from morula to
to be differently regulated. blastocyst stage just before cavitation, which
indicates stage-dependent rather than time-
9.2.3 Compaction dependent control.
The first cell differentiation processes start Further aspects of cell polarization include
as a consequence of EGA and involve com- distribution of microvilli, restriction of
paction and cell allocation. The timing of the plasma membrane components to the apical
first cell differentiation is, just as for EGA, surface, and cytoplasmic polarization. The
different from species to species. It is earliest developmental fate of blastomeres is depen-
(eight-cell) in mouse embryos (Reeve 1981) dent on their location in the embryo, with
and latest in pig embryos, where the first cell inner cells becoming part of the ICM,
differentiation starts only shortly before for- containing the embryonic stem cells, and
mation of the blastocyst (Reima et al. 1993). the outer cells forming the trophectoderm
In bovine, the first cell differentiation occurs that will develop into extra embryonic
at the 16- to 32-cell stage, and in rabbit it membranes.
Preimplantation Embryo Development 211
Apical surface Zona pellucida
Actin TJ
Occludins/claudins/
JAM
ZO-1, 2, 3 Cadherins
Catenins
ZA
Plakoglobin/plakophilin
Desmosome Desmocollins/desmogleins
Basolateral surface Desmoplakin
Intermediate filaments (keratin 8/18) Blastocoel
Figure 9.2 Schematic representation of the cell–cell junctions in trophectoderm cells of the morula and
blastocyst.
9.2.4 Blastocyst formation genes involved in cell–cell contact and TJ
formation.
After formation of the morula, the outer
cells start pumping sodium into the inter- Transcription in the blastocyst is com-
stitial spaces and, due to osmosis, water pared with that of previous stages in dif-
follows, expanding the spaces to a fluid- ferent studies using techniques such as
filled cavity, the so-called blastocoel. After differential display RT-PCR, SSH, and, more
differentiation of the trophectoderm cells recently, microarray analysis, mostly in
and cavitation, the embryo is called a blas- combination with real-time RT-PCR for
tocyst and contains around 32 to 64 cells. verification of results. Subtraction between
bovine morula and blastocyst was used
Important gene families controlling the to establish a cDNA library for studying
different facets of blastocyst formation have expression of the genes during blastocyst
been previously mentioned: E-cadherin– formation. Seventy-one clones representing
catenin cell adhesion family, the TJ gene 33 different expressed sequence tags (ESTs)
family, the Na/K-ATPase gene family, and were generated (Ponsuksili et al. 2002). Of
the aquaporin gene family (Watson and these, 19 were verified by real-time RT-PCR
Barcroft 2001). Also important are other and 84% (16/19) followed the SSH pattern
212 Physiological Genomics of Reproduction
(El-Halawany et al. 2004). Goossens et al. bovine preimplantation, from zygote to blas-
(2007a) used SSH between two- to eight-cell tocyst, as detected by conventional RT-PCR.
embryos and blastocysts. Sixty-five clones In the same study, isoform β1 was detected
representing 36 known genes, five sequences only in the morula and blastocyst stages,
homologous to genomic sequences, and two and isoform β2 was detected from the eight-
sequences with no match in the database cell stage on (Betts et al. 1997). Isoforms α1
were sequenced. Twelve genes were verified and α3 were localized by immunofluores-
by real-time RT-PCR and 75% (9/12) were cence to encircle the entire margin of each
found to be in agreement with results from blastomere in bovine embryos from zygote
SSH. Of the non-ribosomal and mitochon- up to morula. In blastocysts, isoform α1 was
drial genes commonly found in these types confined to the basolateral membranes of
of experiments, the following genes were trophectoderm cells and to the periphery of
detected in both studies: keratin 18 (KRT18), ICM cells. Isoform α3 was confined to apical
fibronectin (FN1), adenine nucleotide trans- cell surfaces of the trophectoderm and was
locator 2, and elongation factor 1 alpha. not detected in ICM, indicating an involve-
One of these genes, KRT18, is not expressed ment in blastocyst formation (Betts et al.
in two- to eight-cell embryos, in vitro 1998). The presence of isoform α1 in troph-
and in vivo, and becomes relatively abun- ectoderm and ICM of bovine blastocysts was
dant in blastocysts (El-Halawany et al. 2004; later confirmed (Wrenzycki et al. 2003). The
Goossens et al. 2007a). Immunofluorescent mRNA amount of isoform β3 (ATP1B3) was
staining showed the KRT18 protein is local- found to be significantly lower in in vitro
ized at cell–cell contact sites of the trophec- two- to eight-cell embryos compared with in
toderm, but not at those of the ICM. Hence, vitro blastocysts, but no significant differ-
KRT18 is a potential marker for trophoblast ence was found between in vivo two- to
differentiation (Goossens et al. 2007a). eight-cell embryos and blastocysts (Goossens
Knockout of KRT18 in the mouse leads to et al. 2007a). It was shown that in vivo-
trophoblast fragility and early embryonic produced morulas had a more firm and more
lethality (Hesse et al. 2000). A similar mRNA prolonged compaction and that they started
pattern was observed for keratin 8 (KRT8; blastulation at a later embryonic age and cell
El-Halawany et al. 2004). KRT8, together number (Van Soom et al. 1997), which might
with KRT18, comprises the intermediate be related to a slower increase in ATP1B3
filaments and influences the three-dimen- expression.
sional formation of cell–cell contacts in
embryonic visceral endoderm (Jackson et al. E-cadherin and beta-catenin transcripts
1980). are present in all stages throughout (in vitro)
bovine preimplantation development. The
Na/K-ATPase is confined to the basolat- proteins were detected by immunocyto-
eral membrane domain of the trophecto- chemistry to encircle the cell margins of all
derm, and enzyme activity increases just blastomeres up to the eight-cell stage. From
prior to blastocyst formation in the mouse the morula on, protein distribution became
(Watson and Barcroft 2001). Na/K-ATPase similar to that of Na/K-ATPase α1 (Barcroft
consists of a catalytic α subunit and a non- et al. 1998). In the same study, the expres-
catalytic, glycosylated β subunit, which are sion of zonula occludens protein 1 (ZO-1)
both encoded by different genes. Isoforms was not detected before the morula stage,
α1, α2, and α3 were found in all stages of where it appeared as punctae between the
Preimplantation Embryo Development 213
outer cells. In the blastocyst, the protein and in vivo-produced embryos (Ponsuksili
was confined to continuous rings at the et al. 2002; El-Halawany et al. 2004; Mohan
apical points of the trophectoderm cell et al. 2004; Goossens et al. 2007a). The FN1
contact. The protein was not found in the protein was predominantly expressed in the
ICM; however, ZO-1 mRNA was found ICM and formed filamentous structures
in ICM cells (Wrenzycki et al. 2003). Expres- between the ICM and the trophectoderm
sion of E-cadherin was suppressed in one (Goossens et al. 2007a). FN1 interacts with
of the first RNA interference studies per- several types of ligands, such as heparin,
formed in bovine preimplantation embryos fibrin, immunoglobulins, and DNA, and
(Nganvongpanit et al. 2006). The E-cadherin acts as a bridge between the collagen matrix
mRNA in morula stage embryos was reduced and integrins at the cytotrophoblast surface
by 80% compared with the controls, and the (Aplin et al. 1999). FN1 knockout mice die
number of embryos reaching the blastocyst shortly after gastrulation (George et al. 1993).
stage was reduced from around 40% to 22%. A new FN1 splice variant specific for bovine
blastocysts was detected, bringing the total
Another type of junction, called desmo- of FN1 splice variants in bovine to nine. This
somes, is formed in the trophectoderm from splice variant was different from the one
the time of cavitation on (see Figure 9.2). present in cumulus cells surrounding the
Desmosomes are spot-like junctions that oocyte (Goossens et al. 2007b).
maintain the integrity of the epithelium
during blastocyst expansion. The extracel- Another gene involved in cytoskeletal
lular domain is formed by several proteins organization that is significantly upregu-
such as desmogleins and desmocollins. The lated in blastocysts is MYL6, which is the
intracellular domain comprises plakoglobin smooth muscle isoform of myosin light
and plakophilin, which link desmoplakin chain. The protein was found mainly around
(DSP) to E-cadherin. DSP also attaches to the the blastocoel cavity, and no difference in
intermediate filaments (see keratin 8 and 18) expression was found between in vivo and
of the cell. No transcripts of desmoglein 1 in vitro embryos (Goossens et al. 2007a).
(DG1) and desmocollin I (DSC1) were found
in bovine preimplantation embryos. DSC2 Bovine embryos become dependent on
and DSC3 transcripts were found in two- to aerobic metabolism from the blastocyst
four-cell embryos up to hatched blastocysts. stage on. One aspect of this change is that
DSC2 was predominantly found in trophec- the embryos start utilizing glucose instead
toderm cells as is the mRNA of plakophilin of pyruvate and lactate, as can be seen in the
(Plako;Wrenzycki et al. 1998; Wrenzycki expression profile of several glucose trans-
et al. 2003). No difference in transcription porters (Wrenzycki et al. 2003). Transcription
was found between in vitro and in vivo profiles of GLUT-1, GLUT-2, GLUT-3,
embryos. GLUT-4, and GLUT-8 have been studied
in bovine preimplantation development.
Another structural gene involved in blasto- GLUT-2 is not transcribed in preimplanta-
cyst formation, cell proliferation, cell adhe- tion embryos up to the blastocyst stage
sion, and cell mobility is FN1. Expression of (Augustin et al. 2001; Lazzari et al. 2002).
FN1 mRNA and protein was found to be GLUT-1 and GLUT-4 have higher mRNA
significantly higher in blastocysts than in content in trophectoderm cells compared
earlier stages, and it was also differently with ICM cells, whereas mRNA content is
expressed between in vitro-produced embryos similar for GLUT-3 (Wrenzycki et al. 2003).
214 Physiological Genomics of Reproduction
Expression of GLUT genes is influenced opposite the ICM, and some specialized
by environmental conditions (Lazzari et al. trophectoderm cells seem to be involved
2002), except for GLUT-1, and is species- (Sathananthan et al. 2003). However, rela-
specific (Wrenzycki et al. 2003). GLUT-3, tively little is known about the genes
GLUT-4, and GLUT-8 mRNA abundance is involved in the process. A lower caspase
higher in in vitro-produced embryos com- activity was found in expanded blastocysts
pared with in vivo blastocysts, and these and also in the non-expanded blastocysts
genes are possibly involved in the develop- with a higher hatching rate (Jousan et al.
ment of Large Offspring Syndrome (Lazzari 2008), but this is more an indication of the
et al. 2002; Knijn et al. 2005). developmental capacity of the embryo and
not the hatching capabilities per se.
OCT-4, a member of the POU transcrip-
tion factor family, is involved in transcrip- 9.3 Preimplantation developmental
tional regulation during early development systems and transcriptomics
and cell differentiation and is often used as
a marker for totipotency as it acts as a tran- 9.3.1 In vivo preimplantation
scription factor for many genes specifically development
expressed in pluripotent cells in the mouse
(Yeom et al. 1996). However, OCT-4 expres- Systematic functional genomic analyses of
sion was observed in bovine and porcine in vivo preimplantation embryo develop-
ICM and trophectoderm cells from in vivo– ment are very scarce in production animals.
and in vitro–produced embryos, which is in Several studies have been performed com-
contrast with the mouse, indicating that in paring transcription profiles of in vivo with
pig and cattle, OCT-4 is also expressed in in vitro and/or somatic cell nuclear transfer
non-pluripotent cells (Kirchhof et al. 2000; (SCNT) embryos of certain stages, but a
Kurosaka et al. 2004). OCT-4 transcription transcription profile of all important devel-
is also influenced by the culture conditions. opmental stages from oocyte to implanta-
Transcription was lower in blastocysts tion has not yet been made. The main reason
derived from oocytes matured in TCM-199 for this is that it is difficult and expensive
supplemented with bovine serum albumin to obtain enough in vivo embryos for making
(BSA) or 10% serum. This could indicate large, representative, stage-specific cDNA
that these blastocysts would reach the dif- libraries and ESTs or large-scale microarray
ferentiated stage earlier (Russell et al. 2006). studies. The situation is improving with
new technologies for linear amplification of
9.2.5 Hatching the RNA before hybridization with the
arrays. A large-scale study was published by
During the development of the embryo up Adjaye et al. (2007) comparing the transcrip-
to the blastocyst stage, it is protected by a tomes of bovine oocytes and blastocyst
glycoprotein membrane, called the zona pel- against 15,529 human cDNAs.
lucida (ZP), which surrounds the plasma
membrane. Before the blastocyst can expand One of the more extensive studies of tran-
and implant, it needs to hatch from the ZP. scription in in vivo pig embryos was per-
Hatching involves the embryonic produc- formed by Whitworth and coworkers (2004).
tion of proteases that will digest the ZP. The They made cDNA libraries from germinal
blastocyst normally hatches out at the pole
Preimplantation Embryo Development 215
vesicle-stage oocytes and in vivo- and in ferences in the adaptability of embryos to
vitro-produced two-cell and blastocyst-stage culture conditions.
embryos and sequenced the 3′ ends. In this
way, the expression of 2489 gene clusters 9.3.2 The effect of in vitro
was scored and virtual Northern blotting production (IVP)
was used to compare the expression of in
vivo and in vitro porcine embryos. Thirty- It is generally acknowledged that in vivo-
eight clusters were found to be different derived embryos are of superior quality in
between in vitro and in vivo two-cell comparison with in vitro-produced embryos.
stage embryos and thirty-seven between Many differences between in vitro and in
in vitro and in vivo blastocysts. In a follow- vivo embryos have been observed: cytoplasm
up study, Whitworth et al. (2005) used a color and density (Pollard and Leibo 1994),
15-K microarray and found 1409 and 1696 general morphology (Van Soom et al. 1997),
differentially detected cDNAs between in metabolism (Khurana and Niemann 2000),
vitro and in vivo two-cell and blastocysts, tolerance of lower temperature (Leibo and
respectively. Loskutoff 1993), developmental capacity
(van Wagtendonk-de Leeuw et al. 2000), inci-
The ewe oviduct is sometimes used as dence of chromosomal abnormalities (Viuff
a surrogate in vivo system for the produc- et al. 1999), pregnancy rate, and frequency of
tion of bovine preimplantation embryos. heavier fetuses after transfer (Hasler 2000).
Approximately 100 zygotes can be trans-
ferred and cultured in the ligated ewe Several research groups compared the
oviduct. The quality of the blastocysts transcriptome of in vivo with in vitro-
obtained is similar to that of in vivo- produced embryos starting from oocytes up
produced blastocysts (Galli and Lazzari to hatching blastocysts. The first group of
1996; Enright et al. 2000), and it has been those studies focused on candidate genes
reported that the transcription pattern of chosen because of their known functions in
some developmentally important genes is early development from different species
similar to that of in vivo-derived bovine (Wrenzycki et al. 1998; Eckert and Niemann
embryos (Lazzari et al. 2002). However, devi- 1998; Lequarré et al. 2001; Knijn et al. 2002;
ations for some genes may be observed. It is Lazzari et al. 2002; El-Halawany et al. 2004)
also of note that there exist considerable or identified by means of SSH (Tesfaye et al.
transcription differences and thus gene regu- 2004; Goossens et al. 2007a). It was shown
lation between sheep and cow. Rizos et al. that the relative abundance of the transcripts
(2004) compared the transcription of eight studied varied through the preimplantation
genes between ovine and bovine embryos period and that changes in transcript abun-
cultured under the same in vitro conditions, dance at the blastocyst stage was, in many
and found significantly higher mRNA abun- cases, a consequence of perturbation in an
dance for MnSOD, survivin, and GLUT-5 in earlier stage (Lonergan et al. 2003).
ovine blastocysts, whereas that of connexin
31 (Cx31), IFN-τ, and sarcosine (SOX) was Most studies comparing in vivo with in
higher in bovine blastocysts. The two other vitro-produced embryos focus on the blasto-
genes investigated (E-cad and Na/K ATPase) cyst stage. Corcoran and coworkers (2006)
showed no difference. The differences were used the bovine BOLT5 microarray with
thought to be related to species-specific dif- 3888 spots representing 932 bovine EST
clones from a bovine total leukocyte cDNA
216 Physiological Genomics of Reproduction
library and complemented this with 459 effects on gene expression in preimplanta-
additional amplicons to compare in vitro tion embryos through epigenetic modifica-
and in vivo (ewe oviduct) transcriptomes. In tions. Epigenetic modifications in mammals
total, 384 genes were found to be differently primarily result from changes in the meth-
transcribed, with 85% downregulated in ylation/demethylation of DNA, mostly Cs
vitro compared with in vivo. Many of these in CpG dinucleotide motifs, and alterations
genes are involved in the regulation of tran- of histones (ref). DNA methylation is,
scription and translation and point to a in general, an expression-repressive mecha-
deregulation of these processes in in vitro- nism that possibly developed to protect
produced embryos. Miles and coworkers the genome against the uncontrolled action
(2006) used small amplified RNA (SAR)- of transposons (Yoder et al. 1997). Two main
SAGE to compare the transcription profile waves of DNA methylation reprogramming
of porcine in in vitro- and in vivo-produced take place, one during germ cell develop-
embryos. A total of 20,029 and 23,453 unique ment and one during preimplantation devel-
putative transcripts were detected in in vitro opment (Reik and Walter 2001). Highly
and in vivo porcine blastocysts, respectively. methylated primordial germ cells undergo
Of these, around 900 were differentially rapid genome-wide demethylation and par-
expressed. The in vitro blastocysts showed ent-of-origin methylation of certain genes.
reduced transcription in biological processes The second wave of DNA methylation
such as cellular metabolism, organization, reprogramming, between fertilization and
and response to stress. blastocyst formation, starts with a rapid
active paternal-specific demethylation inde-
Mohan et al. (2004) used SSH to compare pendent of replication, which is then fol-
transcription in in vitro and in vivo bovine lowed by a stepwise maternal methylation
blastocysts. Both categories of embryos decline up to the morula stage. This passive
yielded 32 ESTs. These corresponded to 32 decline is linked to the absence of DNMT1,
and 22 known genes for in vivo- and in vitro- the primary DNA methyltransferase. Active
produced embryos, respectively. Only three paternal-specific demethylation has been
of the genes (galectin-1, FN1, and filamin A) found in pig, cattle, and, to a lesser degree,
were tested using real-time RT-PCR. No dif- sheep (Dean et al. 2003). Methylation starts
ference was found for filamin A. Galectin-1 again during the first cell differentiation
mRNA was about three times more abundant steps in the blastocyst, leading to hyper-
in in vivo blastocysts than in in vitro blasto- methylation of ICM cells and undermethyl-
cysts. Galectin-1 is involved in cell–cell and ation of trophectoderm cells (Dean et al.
cell–matrix interactions and is a regulator 2001).
of cell transformation (Liu et al. 2002).
Transcription of another galectin, galectin-3, Transcription of DNA methyltransferases
was found to be three times higher in blasto- has been studied in bovine preimplantation
cysts than in morulas (Ponsuksili et al. 2002). embryos. Transcription of DNMT1, DNMT2,
DNMT3a, and DNMT3b was found by
9.3.3 Epigenetic modifications Golding and Westhusin (2003) in all stages,
and SCNT from two-cell up to blastocyst. The same
authors found a new DNMT2 splice variant
In vitro culture systems and somatic in the embryos, and the embryo-specific
cell (SCNT) procedures can have profound variant, Dnmt1o, found in mice, was not
Preimplantation Embryo Development 217
detected, indicating differences in regulation clones (Santos et al. 2003). It was shown by
between species. Different transcription using differential methylation hybridization
between male and female blastocysts (DMH) and bisulfite sequencing that meth-
was reported for DNMT3a and DNMT3b, ylation remodeling in in vitro-produced
along with hnRNP methyltransferase-like 1 porcine blastocysts produced porcine blasto-
(HMT1) and interleukin enhancer binding cysts that deviated considerably from in
factor 3 (ILF3), pointing to epigenetic differ- vivo blastocysts, whereas remodeling in par-
ences between in vitro male and female thenogenetic and SCNT-derived blastocysts
bovine blastocysts (Bermejo-Alvarez et al. was more similar to in vivo blastocysts
2008). Significantly higher mRNA amounts (Bonk et al. 2008).
of the histone methyltransferases SUV39H1
and G9A and of the heterochromatin protein The effect of SCNT on gene expression in
1 (HP1) were found in blastocysts derived preimplantation embryos was studied by
from male donor cells when compared RT-PCR for individual bovine genes (Daniels
with in vivo blastocysts (Nowak-Imialek et al. 2000, 2001; Wrenzycki et al. 2001a,
et al. 2007). A difference was also observed 2004; Park et al. 2003; Camargo et al. 2005;
between blastocysts derived from male Jang et al. 2005; Sawai et al. 2005) and
and female fibroblasts, indicating that the porcine genes (McElroy et al. 2008). A whole
epigenetic modifications are influenced by genome approach has been applied using
donor cell line. Differences in the transcrip- microarray platforms (Pfister-Genskow et al.
tion patterns of other genes involved in 2005; Smith et al. 2005; Somers et al. 2006).
chromatin remodeling and histone acetyla- All of these studies reported significant dif-
tion/deacetylation during bovine preimplan- ferences in transcription between SCNT and
tation development have been described in vivo or in vitro-produced embryos. Beyhan
(McGraw et al. 2007). et al. (2007) also took into account the source
of the donor nuclei, implying that the source
Cloning of livestock animals through of the donor nuclei can have important con-
nuclear transfer (NT) has been hindered by sequences for embryonic development. A
low efficiency. Early gestational losses of striking result from comparing the four
NT embryos are often associated with microarray studies is that the genes found
aberrant placental development linked to differently expressed between SCNT and in
deregulation of gene expression, mainly by vitro embryos are mostly different in all of
epigenetic modifications (Wells et al. 2004). the studies. The reasons for this observation
Aberrant transcription of some genes (acro- can be many, ranging from differences in
granin, estrogen receptor-like 2 [ERR2], and culture conditions to donor cells and micro-
caudal-related homeobox gene 2 [CDX2]) array platforms used. Another important
involved in preimplantation and early pla- aspect was that in all four studies less than
cental development has been reported in 1% of the genes were differently transcribed
cloned bovine blastocysts (Hall et al. 2005). between SCNT and in vitro fertilized (IVF)
blastocysts, indicating that reprogramming
The epigenetic status of the donor cell has fails only for a limited number of genes.
to be efficiently erased and reprogrammed However, as mentioned earlier, the tran-
after transfer for SCNT to be successful. scription profile of IVF blastocysts can also
Chromatin remodeling is often incomplete, be called into question. Also, there are indi-
as is reflected in aberrant DNA methylation cations that the failure to reprogram is not
and histone modification in bovine embryo
218 Physiological Genomics of Reproduction
gene specific but rather is a random process. tion of BAX and BCL-2 under different
This may also account for (some) differences culture systems. They, however, found dif-
found between the studies. ferences for XIAP, an X chromosome-linked
inhibitor of apoptosis. Also, no difference in
9.3.4 Effect of culture medium on early transcription of BAX and BCL-2 could be
embryo development found in porcine blastocysts after addition of
melatonin (Rodriguez-Osorio et al. 2007). On
Several studies have been performed to gauge the other hand, a higher BAX mRNA amount
the effect of the environment on gene expres- was found in blastocysts produced in syn-
sion on preimplantation development. Until thetic oviductal fluid (SOF) medium com-
now, most of these studies looked at the pared with in vivo blastocysts (Rizos et al.
transcription of individual genes, often 2002), which may be due to the presence of
focusing on stress, imprinting, and apopto- calf serum. It has been shown that calf serum
sis-related genes. In one such study, McElroy has an influence on the expression of several
et al. (2008) studied the effect of culture con- genes (Rizos et al. 2003). Addition of leptin
ditions and SCNT on the expression of during bovine oocyte maturation had no
HSP70.2, integrin beta 1 (ITGB1), phospho- effect on cleavage rate, but increased number
glycerate kinase 1 (PGK1), BAX, and IGF2R developed to blastocysts and the proportion
in porcine preimplantation embryos. The of apoptotic cells was reduced. Transcription
amount of BAX mRNA was higher in in of the leptin receptor (LEPR), signal trans-
vitro-produced blastocysts and SCNT blas- ducer and activator of transcription 3
tocysts cultured in a medium with addition (STAT3), and baculoviral inhibitor of apop-
of 10% fetal bovine serum (FBS) on day 4 tosis protein repeat-containing 4 (BIRC4)
compared with in vivo blastocysts, whereas was increased, whereas that of BAX was
the mRNA content was lower for HSP70.2, reduced, indicating a positive effect of leptin
IGF2R, and ITGB1 in in vitro than in in vivo on preimplantion embryo development
blastocysts. BAX is a pro-apoptotic mole- (Boelhauve et al. 2005).
cule. Lower developmental capacity of in
vitro-produced embryos has been linked Given that HSP70.2 is a molecular
to aberrant apoptosis. Therefore, it can be chaperone that is generally upregulated in
expected that the expression of pro-apoptotic response to stress, it is surprising that the
genes is higher in in vitro and SCNT pro- transcription of HSP70.2 was lower in in
duced embryos than in in vivo embryos. vitro-produced embryos than in in vivo
However, it has been shown that the mRNA porcine blastocysts (McElroy et al. 2008). It
content of a certain gene can change quickly is also contrary to the transcription pattern
over time and that for several genes involved found for the HSC70 gene (Bernardini et al.
in apoptosis, such as BAX, BCL-2, caspase 3, 2003). However, regulation of expression of
and caspase 7, the mRNA content bears no heat shock proteins is complex and sensitive
relation to active protein content; hence, the to minor changes in environment and
mRNA content of these genes cannot be manipulation. Some reports mention no dif-
used as a reliable marker for apoptosis ference in HSP70.1 transcription between
in preimplantation development (Vandaele in vitro-produced embryos and in vivo
et al. 2008). In agreement with this, Knijn bovine blastocysts (Wrenzycki et al. 2001b;
et al. (2005) found no difference in transcrip- Lazzari et al. 2002), whereas others do (Knijn
et al. 2005). Also, addition of serum, BSA, or
Preimplantation Embryo Development 219
polyvinyl alcohol (PVA) influences tran- tal capacity in bovine preimplantation
scription of HSP70.1 (Wrenzycki et al. 1999; development (May-Panloup et al. 2005).
Lazzari et al. 2002). A higher relative mRNA
abundance was observed in preimplantation An interesting experiment to study the
embryos cultured in a medium with addi- relationship between transcription profile
tion of serum compared with addition of and in vitro bovine embryo production
BSA or PVA. On the other hand, no differ- success was performed by El-Sayed et al.
ence in transcription for HSP70.1 was found (2006). These authors took biopsies of
under different culture conditions by de bovine blastocysts (day 7), and the remain-
Oliveira et al. (2006). ing embryo (60%–70%) was transferred to
recipients after re-expansion. Based on the
Addition of BSA or PVA was found to have success of the pregnancy, the embryos were
a beneficial effect on the incidence of Large divided in three groups and the biopsies
Offspring Syndrome (Thompson et al. 1995; were used in microarray analysis. A home-
van Wagtendonk-de Leeuw et al. 2000). It made array containing 219 clones and the
was also shown that the transcription pro- BlueChip bovine cDNA microarray contain-
files of some developmentally important ing around 2000 clones (Sirard et al. 2005)
genes were more similar to that in in vivo were used to profile the transcription differ-
bovine embryos in PVA embryos compared ences between embryos giving no pregnancy
with embryos grown in medium supple- (G1) versus embryos leading to calf delivery
mented with BSA or serum (Wrenzycki (G3), or embryos that were resorbed (G2)
et al. 1999; Wrenzycki et al. 2001b). Several versus embryos leading to calf delivery (G3).
indications exist that in vitro culture Fifty-two and fifty-eight genes were differ-
systems put a considerable amount of oxida- ently transcribed between G1 and G3, and
tive stress on embryos. This can be seen in between G2 and G3, respectively. Biopsies
a significant upregulation of antioxidative from G3 embryos had higher transcription
enzymes such as copper–zinc containing of genes involved in implantation (COX2,
superoxide dismutase (Cu/Zn-SOD; Lazzari CDX2), carbohydrate metabolism (ALOX15),
et al. 2002). growth (BMP15), and signal transduction
(PLAU), whereas those from embryos result-
Salazar et al. (2007) used differential ing in no pregnancy were enriched for tran-
hybridization to identify genes differently scripts from genes involved in inflammation
expressed in porcine morula after in vitro (TNF-α), transcription (MSX1, PTTG1),
culture with malathion, a widely used glucose metabolism (PGK1, AKR1B1), and
organophosphate insecticide, added to the implantation inhibition (CD9).
medium. Nine genes, of which three were
unknown, were found to be downregulated 9.4 Future research directions
by malathion. These include cytochrome c
oxidase I and III and MHC I. Malathion may Functional genomics of preimplantation
interfere with mitochondrial electron trans- development is a thriving discipline that has
port. This is in agreement with the finding a promising future in bridging basic science
that genes involved in mitochondrial bio- and practical applications. As in many
genesis such as cytochrome oxidase I (COXI) related disciplines, data are gathered at an
and nuclear respiratory factor I (NRFI) astounding rate using the new technologies
and mitochondrial transcription factor A
(mtTFA) have an influence on developmen-
220 Physiological Genomics of Reproduction
exploring whole genomes. However, the embryos. Molecular Reproduction and
data flow too often stops at presenting lists Development 60: 370–376.
of differently transcribed genes. What is defi- Barcroft, L.C., Hay-Schmidt, A., Caveney,
nitely needed is a follow-up of these studies A., Golfoyle, E., Overstrom, E.W., Hyttel,
with research on protein expression, and P., and Watson, A.J. 1998. Trophectoderm
functional analysis. Integration of transcrip- differentiation in the bovine embryo:
tomics and proteomics in biological systems Characterization of a polarized epithe-
is necessary to make meaning of all the data. lium. Journal of Reproduction and Ferti-
lity 114: 327–329.
Acknowledgments Bernardini, C., Fantinati, P., Castellani, G.,
Forni, M., Zannoni, A., Seren, E., and
The author wishes to thank Karen Goossens Bacci, M.L. 2003. Alteration of constitu-
for critically reading the manuscript, correc- tive heat shock protein 70 (HSC70)
tions, and suggestions. production by in vitro culture of porcine
preimplantation embryos. Veterinary
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10
Physiological Genomics of Conceptus–Endometrial
Interactions Mediating Corpus Luteum Rescue
Troy L. Ott and Thomas E. Spencer
10.1 Introduction unravel the key signaling events designed to
inhibit regression of the corpus luteum (CL).
Placental mammals require luteal progester-
one for part or all of gestation. The domestic Common domestic animals belong to the
animals covered in this chapter utilize pros- Perissodactyla (equidae) and Artiodactyla
taglandin F2α (PGF) of uterine origin for (bovidae, ovidae, caprinae, suidae) orders,
mediating luteal regression. In this regard, which are both members of the superorder
pregnancy establishment has necessitated Laurasiatheria. Horses, swine, and domestic
evolution of conceptus (embryo/fetus and ruminants exhibit uterine-dependent ovarian
associated extraembryonic membranes) stra- cycles. Placental mammals have evolved
tegies to alter uterine PGF production so reproductive strategies that rely on extended
that it no longer induces luteal regression. periods of intrauterine development fol-
Early studies examining the effects of lowed by birth of live offspring that exhibit
the conceptus on uterine PGF production a broad range of development and mobility
revealed complexity both on the part of reg- at birth, including some that are substan-
ulation of endometrial PGF production and tially mobile within minutes to hours of
release and on the part of conceptus signal- birth. Without exception, Eutherian domes-
ing designed to abrogate the luteolytic pro- ticated farm animals require conceptus-
duction of PGF. Although we are learning mediated rescue of CL function and luteal
more and more about the complex biochem- progesterone production for part or all of
ical dialogue that is initiated at maternal gestation (Bazer et al. 2008). In contrast,
recognition of pregnancy signaling, this the dog and cat exhibit an extended luteal
review will focus only on those studies phase in the absence of conceptus signaling.
using genomic and proteomic approaches to It is thought that a reproductive strategy
that requires CL rescue ensures repeated
231
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