JY-1, an oocyte-specific gene, regulates granulosa cell
function and early embryonic development in cattle
Anilkumar Bettegowda*†, Jianbo Yao‡, Aritro Sen*†, Qinglei Li*†, Kyung-Bon Lee*†, Yasuhiro Kobayashi*†,
Osman V. Patel*†, Paul M. Coussens†, James J. Ireland†§, and George W. Smith*†§¶
*Laboratory of Mammalian Reproductive Biology and Genomics, and Departments of†Animal Science and§Physiology, Michigan State University,
East Lansing, MI 48824; and‡Division of Animal and Nutritional Sciences, West Virginia University, Morgantown, WV 26506
Edited by R. Michael Roberts, University of Missouri, Columbia, MO, and approved September 21, 2007 (received for review July 7, 2007)
Oocyte-specific gene products play a key role in regulation of
fertility in mammals. Here, we describe the discovery, molecular
characterization, and function of JY-1, a bovine oocyte-expressed
gene shown to regulate both function of ovarian granulosa cells
and early embryogenesis in cattle and characteristics of JY-1 loci in
other species. The JY-1 gene encodes for a secreted protein with
multiple mRNA transcripts containing an identical ORF but differ-
ing lengths of 3? UTR. JY-1 mRNA and protein are oocyte-specific
tein regulates function of follicle-stimulating hormone-treated
ovarian granulosa cells, resulting in enhanced progesterone syn-
thesis accompanied by reduced cell numbers and estradiol produc-
tion. JY-1 mRNA of maternal origin is also present in early bovine
embryos, temporally regulated during the window from meiotic
maturation through embryonic genome activation, and is required
for blastocyst development. The JY-1 gene has three exons and is
located on bovine chromosome 29. JY-1-like sequences are present
on syntenic chromosomes of other vertebrate species, but lack
exons 1 and 2, including the protein-coding region, suggestive of
species specificity in evolution and function of this oocyte-specific
embryogenesis (1). The advent of oocyte genomics and EST
sequencing projects have led to a dramatic increase in our
understanding about the identities and functions of oocyte-
specific genes in female reproduction (2, 3). However, inherent
species-specific differences exist in the ovulation quota, follic-
ular waves, duration of the ovarian cycle, and number of
embryonic cell cycles required for embryonic genome activation
(4) between the traditional animal model (polyovulatory mouse)
versus monoovulatory species such as cattle and primates, in-
cluding humans. Numerous examples suggest that oocyte-
specific genes identified in the mouse may not have identical
functions in other species. For instance, Belclare and Cambridge
ewes with naturally occurring heterozygous mutations in the
GDF9 gene have an increased ovulation rate and litter size (5),
but mice heterozygous for the GDF9 gene disruption exhibit no
obvious phenotype (6). Similarly, Inverdale and Hanna strains of
sheep with homozygous mutations in BMP15 are infertile (7, 8),
whereas homozygous BMP15 mutant mice are subfertile with
defects in ovulation and fertilization (9). Thus, comparative
genomics approaches coupled to functional studies in nontradi-
tional model systems are needed to address dissimilarities in
transcriptome composition between model organisms and pro-
vide information on existence of genes or gene families that may
play important regulatory roles in fertility in nonmurine models,
including the human. With this goal in mind, we previously
constructed a bovine oocyte cDNA library and sequenced a
number of ESTs (2). A highly abundant transcript (designated as
JY-1) was identified and selected for further analysis, because it
is entirely novel despite 7.95 million human, 4.74 million mouse,
and ?1.31 million bovine EST sequences in GenBank and
because its expression is ovary specific. We thus hypothesized
he oocyte is a key regulator of multiple aspects of female
fertility, including ovarian follicular development and early
that JY-1 encodes for an oocyte-specific gene with important
functions during folliculogenesis and early embryonic develop-
ment. Here, we report the characterization and intraovarian
localization of JY-1 mRNA and protein during folliculogenesis,
evidence for a regulatory role for JY-1 in regulation of granulosa
cell function and early embryonic development, and pronounced
differences in characteristics of JY-1 loci in the genome of cattle
versus other species examined (human, mouse, rat, chimpanzee,
Tissue Distribution and Characterization of JY-1 mRNA Transcripts.
Screening of RNA from various tissues by RT-PCR detected
JY-1 mRNA only in fetal ovaries collected at days 180 and 210
of gestation but not in any other tissues examined [supporting
JY-1 mRNA. Northern analysis revealed three predominant
JY-1 transcripts in RNA isolated from fetal ovaries (Fig. 1A).
Further analysis of adult germinal vesicle (GV) oocytes (GVOs)
transcripts of different lengths (?1.8 kb, 1.2 kb, and 700 bp) (Fig.
1B). Because all 14 JY-1 inserts sequenced from the oocyte
library were small (the longest is ?455 bp in length) and could
be partial cDNAs or represent the smaller predominant tran-
script detected by Northern analysis, a fetal ovary cDNA library
was screened and two additional clones containing larger inserts
other clone had an insert of ?1.0 kb in length (GenBank
accession nos. EF642496 and EF642497). Sequence analysis of
these two larger JY-1 cDNAs and two original smaller cDNAs
(455 and 355 bp) from the oocyte library revealed that the four
cDNA represent four different transcripts of the JY-1 gene (SI
Fig. 6B). Experiments using 5?RACE did not reveal any addi-
tional 5? sequence confirming that the sequence observed at the
5? end of all JY-1 transcripts is indeed complete (data not
shown). Thus, the minor differences in the length of JY-1
transcripts observed in fetal ovary versus adult GVOs is most
likely attributed to polyadenylation status of the mRNA tran-
scripts. An identical ORF of 255 bp encoding for a predicted
protein of 84 aa was identified in all four transcripts derived from
the oocyte and fetal ovary libraries (SI Fig. 6B). Sequences
K.-B.L. performed research; Y.K., O.V.P., and P.M.C. contributed new reagents/analytic
tools; A.B., J.Y., and G.W.S. analyzed data; and A.B. and G.W.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Abbreviations: FSH, follicle-stimulating hormone; GV, germinal vesicle; GVO, GV oocyte;
rJY-1, recombinant JY-1; MII, metaphase II; IVF, in vitro fertilized; Chr, chromosome.
database (accession nos. EF642496 and EF642497).
¶To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
November 6, 2007 ?
vol. 104 ?
upstream of the defined start AUG in all four JY-1 transcripts
did not contain any additional in-frame start codons. All four
transcripts differ in length of the 3? UTR but have an identical
5?UTR. The AU-rich putative cytoplasmic polyadenylation el-
ements (AUUUUAAAA and UAUUUUAAUA) were also
noted in the 3? UTR of the two longest transcripts (SI Fig. 6B).
Characterization of JY-1 Protein. The Signal IP3 program (10)
predicted a signal peptide of 21 aa, indicating that JY-1 protein
is likely to be secreted from the oocyte. The predicted molecular
weight of JY-1 is ?9,000 Mr, but the NetOGlyc-3.1 program (11)
predicted two O-linked glycosylation sites in the deduced JY-1
amino acid sequence, suggesting probable glycosylation of the
JY-1 protein. Polyclonal antiserum raised against recombinant
JY-1 (rJY-1) protein (mature form without the signal peptide)
was used in Western blot analysis to detect JY-1 protein.
Immunoreactive JY-1 protein of ?11,000 Mr and additional
higher Mrbands were detected in extracts of adult GVOs (Fig.
1C). The polyclonal antiserum also detected the rJY-1 protein
(6,700 Mr, mature form lacking the signal peptide) that was used
to generate the antiserum (Fig. 1C). Preincubation of JY-1
antiserum with excess antigen (rJY-1) blocked binding of the
antibody specifically to the 11,000 Mrprotein and rJY-1 protein,
but not to the higher Mr bands (Fig. 1D), which represent
nonspecific cross-reactivity. Immunoreactive JY-1 protein was
detected in adult GVOs but not in any other cell/tissue samples
examined (Fig. 1E). The 11,000 MrJY-1 protein also was not
detected in GVOs when blots were incubated with preimmune
rabbit serum (Fig. 1F). Publicly available databases were
searched with the predicted amino acid sequence of JY-1 to
identify functional domains and predict the structure of the JY-1
protein, and no significant orthologs of the JY-1 protein were
found. A putative secondary structure for JY-1 protein was
predicted by using the PSIPRED program (12), but we have not
identified any motifs that are indicative of functional domains by
using the conserved domain database (CDD) (13). Similarly,
pFam A and B (14) and a PSI?BLAST search of the Protein Data
Bank at the National Center for Biotechnology Information (15)
designed to designate sequences to protein families based on
homology and identify 3D structures for homology modeling
were unsuccessful. Thus, we conclude JY-1 is a member of a
novel protein family.
Oocyte-Specific Localization of JY-1 mRNA and Protein within Ovarian
Follicles. Intraovarian expression of JY-1 mRNA and protein was
restricted exclusively to oocytes. In situ hybridization localized
JY-1 mRNA specifically to oocytes of preantral and antral
follicles (SI Fig. 7). No significant hybridization to somatic
ovarian cell types (granulosa, theca, and stroma) was noted. JY-1
protein was localized to oocytes of growing follicles at the
primordial (single layer, with ?10 flattened granulosa cells),
primary (single layer with cuboidal granulosa cells) through
antral follicle stages (Fig. 2) in fetal ovaries collected at day 230
of gestation. Immunoreactivity was not detected when tissue
sections were incubated with preimmune rabbit IgG or the JY-1
antibody was preabsorbed with immunogen peptide (SI Fig. 8).
analysis of JY-1 mRNA in multiple bovine tissues (A) and adult GVOs (B). (C–E)
Western blot detection of JY-1 protein in bovine oocytes by using antisera
generated against rJY-1 protein (mature form lacking signal peptide). (C)
Representative Western blot demonstrating detection of immunoreactive
JY-1 protein of ?11,000 Mrin lysates of 150 GVOs and immunoreactivity of
rJY-1 (6,700 Mr). (D) Duplicate blot of C incubated with JY-1 antiserum
preabsorbed with excess rJY-1 protein, which blocked binding of antibody to
JY-1 protein in GVO and to rJY-1. (E) Representative Western blots demon-
strating tissue specificity of JY-1 immunoreactivity. Note the absence of
immunoreactive JY-1 in the samples of bull serum, granulosa cells, liver, and
adrenal gland. (F) Duplicate blot of E demonstrating the absence of JY-1
immunoreactivity when the blot was incubated with preimmune serum.
Characterization of JY-1 mRNA and protein. (A and B) Northern
calization of JY-1 protein to the oocytes of growing follicles (A), primordial
follicles (B), primary follicles (C), and antral follicle (D). Arrows in B and C
indicate a primordial and a primary follicle. (Magnification: A, ?200; B and C,
?1,000; D, ?100.)
Intraovarian localization of JY-1 protein. Immunohistochemical lo-
Bettegowda et al.
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Effect of rJY-1 Protein on Cell Number and Production of Estradiol and
Progesterone by Cultured Granulosa Cells. The rJY-1 protein was
used to test the ability of JY-1 to regulate bovine granulosa cell
proliferation and steroidogenesis. Addition of rJY-1 to cultured
granulosa cells inhibited the follicle-stimulating hormone
(FSH)-induced increase in granulosa cell numbers at the 0.5
ng/ml dose (P ? 0.05), and the response was maximal at 1 and
10 ng/ml doses (P ? 0.05; Fig. 3A). Addition of rJY-1 at 0.1 ng/ml
had no effect on granulosa cell numbers (Fig. 3A). However, in
vitro production of estradiol was inhibited 2-fold in FSH-
supplemented granulosa cells (P ? 0.05) treated with the 0.1
ng/ml rJY-1 dose where significant effects on granulosa cell
numbers were not observed (Fig. 3B). Further, the inhibitory
effect on estradiol production was maximal at 0.5 ng/ml rJY-1,
and supplementation with 1 and 10 ng/ml rJY-1 did not inhibit
estradiol production. In contrast, addition of rJY-1 increased
production of progesterone in a dose-dependent manner (P ?
3C). Even though total cell numbers decreased by ?50% in
response to treatment with 1 and 10 ng/ml rJY-1, progesterone
production was doubled compared with cells cultured without
rJY-1 (Fig. 3 A and C). No effects of rJY-1 on granulosa cell
numbers or estradiol and progesterone production were ob-
served for granulosa cells cultured in the absence of FSH (data
Quantification of JY-1 mRNA During the Oocyte-to-Embryo Transition
and Effect of JY-1 Knockdown on Early Embryonic Development.
Given the observed oocyte-specific localization of JY-1 mRNA
and protein, we hypothesized that JY-1 mRNA is regulated
during meiotic maturation and early embryonic development.
Temporal changes in abundance of polyadenylated versus total
JY-1 transcripts during early development were characterized by
quantitative real-time PCR. Abundance of polyadenylated JY-1
transcripts [cDNAs synthesized from oligo(dT) primers] de-
creased during meiotic maturation (P ? 0.0001) increased (P ?
0.05) at the pronuclear and four-cell stages relative to the
metaphase II (MII) stage, and then decreased to nearly unde-
tectable levels after the 16-cell stage of embryo development
(Fig. 4A and SI Fig. 9A). In contrast, the amount of total JY-1
transcripts (cDNAs synthesized from random hexamers) grad-
ually decreased from GV through 16-cell stages to nearly
in abundance of polyadenylated versus total transcripts is prob-
ably the result of JY-1 mRNA deadenylation. Further, results of
embryo culture experiments in the presence of the transcription
inhibitor ?-amanitin suggest that the JY-1 gene is not transcribed
during the first and second embryonic cell cycles (SI Fig. 10),
thus the JY-1 mRNA detected in early bovine embryos is
To test the requirement of JY-1 during early embryonic
development, we validated procedures for siRNA-mediated
gene silencing in bovine embryos (SI Text). Multiple siRNA
species were tested for efficacy and specificity of JY-1 mRNA
knockdown via microinjection into MII oocytes followed by
to test the efficacy of JY-1 knockdown in embryos because it is
easier to manipulate and allows for cumulus cell removal and
siRNA injection earlier in development. The two most effective
JY-1 mRNA abundance by ?90% in four-cell stage embryos (SI
Fig. 11), and a mixture of both siRNAs reduced JY-1 mRNA
abundance by ?95% in two-cell embryos (Fig. 4B). JY-1 siRNA
mixture specifically reduced JY-1 mRNA in four-cell embryos
(SI Fig. 12). Specificity of the JY-1 siRNA was further confirmed
via measurement of JY-1 protein abundance in 8- to 16-cell
embryos. JY-1 siRNA specifically reduced JY-1 protein to
terone production. (A) Effect of rJY-1 on total granulosa cell numbers for
FSH-treated cells. Note the decrease in cell numbers with increasing concen-
tration of rJY-1 (P ? 0.05). (B) Effect of rJY-1 on FSH-stimulated estradiol
production by bovine granulosa cells. Concentrations of estradiol were de-
creased in response to 0.1 ng/ml rJY-1, and the response was maximal at 0.5
ng/ml rJY-1 (P ? 0.05). (C) Effect of rJY-1 on progesterone production by
FSH-treated bovine granulosa cells. Note dose-dependent increase in proges-
terone in response to increasing concentrations of rJY-1 (P ? 0.01). Concen-
trations of estradiol and progesterone were normalized to 30,000 cells. Data
are depicted as mean ? SEM. Letters a and b indicate significant differences.
Effect of rJY-1 on granulosa cell numbers and estradiol and proges-
and early embryogenesis and the effect of JY-1 knockdown on blastocyst
development. (A) Relative abundance of polyadenylated JY-1 mRNA tran-
scripts during meiotic maturation through embryonic genome activation
[GVOs and MII-stage oocytes, pronucleus (PN), two-cell (2C), four-cell (4C),
Effect of JY-1 siRNA microinjection on abundance of polyadenylated JY-1
mRNA in samples of two-cell embryos. Denuded MII oocytes were either
microinjected with sham water or JY-1 siRNA mixture followed by partheno-
genetic activation. Data were normalized relative to abundance of endoge-
nous control 18S rRNA and shown as mean ? SEM. (C and D) Effect of JY-1
blastocyst stage. Denuded MII oocytes or presumptive zygotes were microin-
jected with sham water, JY-1 siRNA mixture, negative (?) control (Ctrl) siRNA
or served as uninjected controls. Average rates of blastocyst development
were calculated and data are shown as mean ? SEM. Time points without a
common letter (a, b, c, and e) are significantly different. P ? 0.05.
Quantification of JY-1 mRNA abundance during oocyte maturation
www.pnas.org?cgi?doi?10.1073?pnas.0706383104Bettegowda et al.
undetectable levels compared with uninjected control embryos
(SI Fig. 13).
JY-1 siRNA mixture injection strikingly decreased the pro-
portion of parthenogenetic embryos developing to the blastocyst
stage (7.4%) relative to uninjected (31.7%), sham-injected
(31.5%), and negative control siRNA-injected (33.7%) embryos
(P ? 0.05; Fig. 4C). Cleavage rates of embryos were not different
between the groups. Similarly, JY-1 siRNA mixture injection
into in vitro-fertilized (IVF) embryos did not affect the cleavage
rates but dramatically reduced the proportion of IVF embryos
developing to the blastocyst stage (4.2%) relative to uninjected
(23.5%), sham-injected (24.1%), and negative control siRNA-
injected (23.6%) embryos (P ? 0.01; Fig. 4D). To further ensure
the specificity of JY-1 siRNA in inhibiting embryonic develop-
ment, experiments were repeated with the individual siRNAs
injected separately. A reduction in the proportion of IVF
embryos developing to the 8- to 16-cell stage (P ? 0.0001; SI Fig.
14A) and proportion of embryos developing to the blastocyst
stage (P ? 0.0001; SI Fig. 14B) relative to uninjected and
sham-injected controls was noted after injection of each siRNA
Identification of JY-1-Like Sequences in Other Species. Southern blot
analysis was used to investigate the presence of the JY-1 gene in
the genome of cattle and other species. The 450-bp JY-1 cDNA
strongly hybridized to an EcoRI genomic fragment in bovine
genomic DNA, and weaker hybridization to sheep, pig, and
human genomic DNA was also noted (Fig. 5A and SI Fig. 15).
No significant hybridization to mouse, chicken, rainbow trout,
and zebrafish genomic DNA was detected. The bovine JY-1 gene
has three exons (25, 92, and 1,400 bp in length) separated by two
introns (12.8 and 1.5 kb in length) (Fig. 5B and SI Fig. 16A) and
is 16 kb in length and located on chromosome (Chr) 29 in the
bovine genome. To identify putative cis elements that may
confer tissue/cell-specific expression of JY-1, the 5? flanking
sequence of the JY-1 gene was visually inspected. Five putative
E-boxes [canonical sequence CANNTG; known to mediate
oocyte-specific expression (16) in other species] were identified
within 500 bp of the 5? flanking sequence of the bovine JY-1 gene
(SI Fig. 16B).
JY-1-like sequences corresponding to exon 3 of the gene
were also found in the human genomic and EST databases.
JY-1-like sequence was identified on human Chr 11 (syntenic
with bovine Chr 29) with the region of similarity corresponding
to a portion (187 bp) of the protein-coding region and 850 bp
in the 3? UTR of the 1.5-kb JY-1 cDNA (Fig. 5C). In the human
EST database, a single EST derived from a human erythroid
precursor cell (adult stem cell) cDNA library and lacking an
ORF was identified (GenBank accession no. BU656412). The
region of sequence similarity in the human EST is 187 bp and
maps to human Chr 11 (11q14) with 100% identity and to the
exact location where the sequence similar to bovine JY-1 is
present (SI Fig. 17). JY-1-like sequences corresponding to exon
3 of the gene were also identified in the genome of additional
vertebrate species. JY-1-like sequences were identified on
chimpanzee Chr 11, dog Chr 21, mouse Chr 7, and rat Chr 1
(syntenic Chrs to human Chr 11 and bovine Chr 29; Fig. 5C).
Results of the present studies demonstrate that the bovine JY-1
gene encodes for an oocyte-specific protein with important
regulatory roles in granulosa cell function and early embryonic
development and suggest that evolution of a functional JY-1 gene
may be species-specific. Multiple oocyte-specific genes have
been described in mice that directly regulate either folliculogen-
esis or early embryonic development (1). However, to our
knowledge, establishment of a functional role for a single
oocyte-specific gene (JY-1) in regulation of function of ovarian
granulosa cells and early embryogenesis is unprecedented.
Identification of JY-1-like sequences corresponding to a small
3? portion of the ORF and/or the 3? UTR portion of the bovine
JY-1 cDNA on syntenic Chrs to bovine Chr 29 in human,
chimpanzee, mouse, rat, and dog (17–20) raises the possibility
that the JY-1 locus is conserved in multiple vertebrate species.
The sequence identity of the human EST (from erythroid
precursor cells) with JY-1-like sequence on human Chr 11
(syntenic to bovine Chr 29) suggests that a mRNA transcript may
be transcribed from the above locus. However, the syntenic loci
do not encode for the complete JY-1 gene and lack sequences
corresponding to exons 1 and 2 and thus a significant portion of
the protein coding region. It appears unlikely based on extensive
sequence analysis that the above loci in other species, including
humans, encode for a protein of similar identity to bovine JY-1.
Therefore, evolution of the oocyte-specific JY-1 protein is most
likely species-specific. However, the presence of a functional
ortholog performing similar roles as JY-1 in other mammalian
species cannot be ruled out. Further, the significance of the
conserved JY-1 3? UTR in multiple species is not known, but
based on accumulating evidence for important regulatory roles
of noncoding RNAs (21), a functional role for the observed
JY-1-like sequence in the genome of other species cannot be
To suit the diverse reproductive functions in mammals, certain
genes or gene families may have evolved by selection pressure
during the course of evolution. For example, the trophoblast cell
derived pregnancy recognition factor IFN-? is produced in
mammals within the Ruminantia suborder (e.g., cattle, sheep,
goats), but not in unrelated species (22). Recent identification of
the trophoblast kunitz domain protein (TKDP) gene family
specifically expressed in ruminants (23) further supports the
concept that certain genes in the reproductive system may have
and structure of JY-1 gene. (A) Genomic Southern blot hybridized with
of the 3?UTR. Note strong hybridization to a bovine genomic DNA fragment
and weaker hybridization to sheep, pig, and human genomic DNA. (B) Gene
structure of bovine JY-1. The JY-1 gene has three exons (E1, E2, E3) separated
additional species. Genomic DNA databases at the National Center for Bio-
technology Information for human, chimpanzee, dog, mouse, rat, chicken,
zebrafish, and Drosophila were searched with the nucleotide sequence of the
1.5-kb bovine JY-1 cDNA. JY-1-like sequences corresponding to exon 3 were
rat Chr 1 (syntenic Chrs to human Chr 11). JY-1-like sequences were not
identified in genomic DNA databases for chicken, zebrafish, and Drosophila.
Detection of JY-1-like sequences in the genome of multiple species
Bettegowda et al.
November 6, 2007 ?
vol. 104 ?
no. 45 ?
evolved in a species-specific fashion and been selected for
specialized functions. The evolution of the above genes in
ruminants may be attributed to clear species-specific differences
in the trophoblast and the type of placentation mediating
maternal-fetal communication (24). Species-specific attributes
of oocyte function in general are not well understood, but bovine
versus mouse oocytes do differ in their requirement for cumulus
cell expansion and ability to promote glucose uptake by such
cells (25–29). Studies in closely related species (e.g., sheep and
goats) will be necessary to further determine the specificity in
structure and function of the JY-1 gene.
Results support an important role for JY-1 in early embry-
onic development in cattle. Our evidence indicates that JY-1
mRNA is dynamically regulated during the window from
meiotic maturation through embryonic genome activation.
Results of siRNA-mediated gene knockdown experiments in
two different in vitro models of early embryogenesis support a
requirement of JY-1 for development to the blastocyst stage in
cattle. Results also suggest that the maternal JY-1 mRNA is
translated during bovine early embryogenesis because siRNA-
mediated mRNA knockdown prevented the accumulation of
JY-1 protein in early embryos. Our results further suggest that
JY-1 is required during the early embryonic stages before
embryonic genome activation, because JY-1 siRNA injection
reduced development of embryos to the 8- to 16-cell stages by
?40%, and only 25% of injected embryos reaching the 8-to
16-cell stage developed into blastocysts. Gene targeting ap-
proaches have demonstrated the role of oocyte-specific
MATER, ZAR1, and NPM2 genes for early embryo develop-
ment in mice (30). Embryonic genome activation occurs much
later in domestic ruminants (e.g., 8- to 16-cell stages in cattle
and sheep) compared with the mouse (at the two-cell stage),
thus additional maternal effect genes may be required to
promote early embryogenesis in such species. While MATER
and ZAR1 expression in bovine oocytes/embryos has been
reported (31, 32), experimental evidence is lacking to support
the requirement of the above genes for bovine early embry-
ogenesis. To our knowledge, JY-1 is the only known oocyte-
specific maternal factor demonstrated to govern early embry-
onic development in nonmurine species.
Oocyte regulation of folliculogenesis and phenotype/function
of ovarian somatic (cumulus and granulosa) cells has been well
established (1, 33). Results of the present studies demonstrate
pronounced effects of rJY-1 protein on the granulosa cell
phenotype in a manner mimicking preovulatory events charac-
teristic of the luteinization process. Biological actions of JY-1 on
bovine granulosa cells do not mimic the reported effects of the
well known oocyte-specific growth factors GDF9 and BMP15 on
bovine granulosa cell function (34, 35). In vivo, the preovulatory
gonadotropin (luteinizing hormone) surge results in decreased
estradiol and increased progesterone levels in the follicular fluid
of bovine preovulatory follicles (36), and preovulatory granulosa
cells exit from the cell cycle and are transformed into nondi-
viding terminally differentiated luteal cells capable of producing
high levels of progesterone (37, 38). In our culture studies, the
increase in progesterone was accompanied by a suppression of
the FSH-stimulated increase in granulosa cell numbers and
estradiol production, mimicking the in vivo preovulatory follic-
ular environment. Further studies will be required to determine
whether JY-1 can enhance granulosa cell luteinization induced
by luteinizing hormone.
A direct physiological role for JY-1 in modulating granulosa
cell function depends on secretion of JY-1 from the oocyte in
physiologically significant quantities. Our prediction analysis
using the signal IP and NetOGlyc-3.1 programs suggests that
the JY-1 protein is most likely secreted and glycosylated.
However, the Mrof immunoreactive JY-1 detected in lysates
of GVOs is larger than the predicted Mr of the mature/
processed form of JY-1. Future experiments will be required
to resolve whether the 11,000 Mrform of immunoreactive JY-1
protein detected represents the proform of JY-1 (before signal
peptide cleavage), directly detect secretion of the mature/
processed form of the JY-1 protein, and determine whether the
JY-1 protein is subjected to additional posttranslational mod-
ifications that would account for the difference in observed
versus predicted Mr. It will also be of interest to see whether
JY-1 has similar effects on proliferation and steroidogenesis of
bovine cumulus cells, the somatic cells in closest proximity to
the oocyte and plausibly exposed to the highest concentrations
of JY-1 in vivo.
In summary, results of the present studies establish JY-1 as an
oocyte-specific protein in cattle and demonstrate multiple dis-
tinct roles for an oocyte-specific gene related to both folliculo-
genesis and early embryonic development. Structure function
studies and future investigation of the signaling pathways or
mechanisms mediating JY-1 action will provide further insight
into functional domains that mediate JY-1 activity and into
fundamental mechanisms regulating folliculogenesis and early
Materials and Methods
Northern Blot Analysis. Northern blotting was performed as de-
scribed (39). For details see SI Text.
Bovine Fetal Ovary cDNA Library Construction. See SI Text.
Western Blotting. Recombinant JY-1 protein (rJY-1, predicted
mature protein without signal peptide) was expressed in BL21
Escherichia coli and purified commercially by C & P Biotech
(Thornhill, ON, Canada) using the pET15b vector. Polyclonal
antiserum was generated in rabbits against rJY-1 protein by
Affinity Bioreagents (Golden, CO). Western blotting was per-
formed with our established protocols (39). See SI Text.
In Situ Hybridization. In situ hybridization was performed accord-
ing to our established procedure (40). See SI Text.
Immunohistochemistry. Polyclonal antiserum was generated
against a 20-aa synthetic peptide corresponding to a portion of
the carboxyl terminus of the predicted amino acid sequence of
bovine JY-1 (C55-A74). Peptide synthesis, conjugation to key-
hole limpet hemocyanin, immunization, and immunoaffinity
purification was conducted commercially by Bethyl Laboratories
(Montgomery, TX). Immunocytochemical localization of JY-1
protein was performed according to our published procedures
(n ? 3 samples) (39). See SI Text.
Effect of rJY-1 on Granulosa Cell Function. Serum-free long-term
granulosa cell culture was performed as described (41, 42). See
Quantification of JY1 mRNA in Oocytes and Early Embryos. Oocyte
recovery, in vitro maturation, in vitro fertilization, embryo cul-
ture, and quantification of mRNA by real-time PCR were
performed as described (43). See SI Text.
Synthesis and Validation of siRNA Species by Microinjection. To
determine the effects of JY-1 knockdown on blastocyst devel-
opment, microinjection experiments were performed in two
different in vitro models of embryo development: parthenogen-
esis and IVF (n ? 4–5 replicates per treatment) (see SI Text).
Genomic Southern Blot Analysis, Genomic Library Screening, and
Bioinformatics Analysis. See SI Text.
www.pnas.org?cgi?doi?10.1073?pnas.0706383104 Bettegowda et al.
Statistical Analysis. For real-time PCR experiments, differences Download full-text
in mRNA abundance were determined by one-way ANOVA
using the GLM procedure of SAS. For microinjection exper-
iments, rates of embryo development to 8- to 16-cell and
blastocyst stages were analyzed after arcsin transformation by
using the Mixed Linear Models procedure of SAS. Similarly,
differences in progesterone, estradiol, and cell numbers were
determined by the Mixed Linear Models procedure of SAS.
Mean comparisons were performed with Tukey’s test. The
dose–response relationship between rJY-1 and progesterone
was determined by regression analysis. Differences of P ? 0.05
were considered significant.
We thank F. Jimenez-Krassel, J. L. H. Ireland, and L. Lv for help with
follicle cutting; L. Chapin for help with statistical analysis; R. Huang for
help with in situ hybridization experiments; and P. Ross and Z. Beyhan
for help with microinjection procedures. This work was supported by the
Rackham Foundation, the Michigan State University Office of the Vice
President for Research and Graduate Studies, and the Michigan Agri-
cultural Experiment Station.
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