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Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Utah Health Sciences Center, Salt Lake City, Utah 84132
Address all correspondence and requests for reprints to: Dr. Eli Y. Adashi, Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Utah Health Sciences Center, 546 Chipeta Way, ARUP II, Box 20, Salt Lake City, Utah 84108. E-mail: eadashi{at}hsc.utah.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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-inhibin, and zona pellucida-3 genes proved sterile. A loss of
function mutation of the human FSH receptor gene established its
critical role in ovarian function. These data support the hypothesis
that genes expressed selectively or specifically in the ovary are
probably essential for the normal functioning of this organ system. We
have used the differential screening technique suppression subtractive
hybridization to systematically isolate and clone genes that are
expressed in an ovary-selective/specific manner. The resultant target
complementary DNA (cDNA) library has been exhaustively screened to a
point at which additional sequencing was increasingly unlikely (
4%)
to yield additional previously unencountered cDNAs. In
toto, 844 clones were sequenced and analyzed for homology to
known genes using the Basic Local Alignment Tool (BLAST). Of those, 342
were determined to be independent (nonredundant). One hundred and
fifty-nine independent clones proved identical to previously
characterized genes, whereas an additional 100 independent clones
proved significantly homologous (but not identical) to previously
characterized genes. Yet 83 other independent clones did not display
significant homology to previously characterized genes now listed in
the publicly accessible nonredundant databases. As such, these latter
genes were deemed novel. Of these 83 novel genes, a total of 36
displayed ovary-specific/selective expression, as determined by probing
mouse multitissue Northern blots with
32P-labeled/PCR-amplified cDNA inserts. Under these
circumstances, the false positive rate was minimal, as only one novel
clone was expressed at a higher level in nonovarian tissues relative to
ovary. Of the 36 ovary-specific/selective novel genes, 22 proved
subject to hormonal regulation during a simulated estrous cycle. In
this communication we focus on 2 such novel
ovary-specific/hormonally-dependent genes, the full-length sequences of
which were isolated using rapid amplification of 3'-cDNA ends
technology. Taken together, the present study accomplished systematic
identification of those genes that are restricted in their expression
to the ovary. These ovary-selective genes may have significant
implications for the understanding of ovarian function in molecular
terms and for the development of innovative strategies for the
promotion of fertility or its control. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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In the last few years several genes have been determined to be
expressed in an ovary-selective manner. Examples include members of the
transforming growth factor-ß superfamily, growth differentiation
factor-9 (GDF-9) and -inhibin (3, 4); c-mos, a 37-kDa
cytoplasmic serine/threonine kinase (5); as well as the oocyte-derived
zona pellucida (ZP) glycoproteins (ZP 1–3) (6). Each of the preceding
genes was isolated and studied on a case by case basis with regard to
ovarian function.
The critical role that ovary-selective genes may play in ovarian physiology was subsequently established through the generation of null mutant mice. For example, the ovaries of null mutants for GDF-9 possessed normal numbers of primordial and primary follicles but were devoid of all downstream follicular stages (7). Female null mutants for c-mos proved infertile in that meiotically active oocytes failed to arrest at metaphase II and, in fact, attempted a third round of meiosis (8, 9). Female null mutants for ZP-3 displayed markedly reduced (90%) ovulatory efficiency and consequent infertility, the complete absence of the ZP, and disorganization of the surrounding cumulus granulosa cells (10, 11). These studies, through the generation of null mutants, established that the above-mentioned ovary-selective genes are critical to murine ovarian function.
Taken together, these and other studies serve to demonstrate that several genes, whether restricted in their expression to the ovary or preferentially expressed in the ovary, constitute critical molecular determinants of ovarian function. It was, therefore, the purpose of this communication to systematically isolate genes that are expressed in an ovary-selective/specific manner.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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RNA isolation
Total RNA was isolated from the following nonovarian tissues of
immature 25-day-old female C57BL/6 mice: brain, heart, kidney, liver,
spleen, and lung. Total RNA was also isolated from the ovaries of
25-day-old female C57BL/6 mice undergoing superovulation as described
above. The isolation of total RNA was performed using the RNAeasy Kit
(QIAGEN, Santa Clara, CA) according to the manufacturer’s
directions. Polyadenylated [poly(A)+] RNA was
subsequently isolated by the use of an oligo(deoxythymidine) magnetic
sphere-based separation system (RNAatract, Promega Corp.,
Madison, WI).
Suppression subtractive hybridization (SSH)
SSH was performed with the PCR-Select Kit (CLONTECH Laboratories, Inc. San Diego, CA) according to the
manufacturer’s directions. Briefly, an equal amount of
poly(A)+ RNA isolated from each of the nonovarian
tissues was combined to generate a total of 1 µg
poly(A)+ RNA. This messenger RNA (mRNA) was used
to generate the driver complementary DNA (cDNA) through the use of the
SMART cDNA synthesis kit (CLONTECH Laboratories, Inc.)
according to the manufacturer’s instructions. Ovarian
poly(A)+ RNA (1 µg) isolated from mice
undergoing the above-described superovulation protocol was used to
construct the tester cDNA. Thirty primary and 12 secondary PCR cycles
were used in amplifying the target (subtracted) ovary-selective
cDNAs.
Cloning and sequencing of cDNAs
The PCR products generated by SSH were digested with
RsaI to generate blunt ends and to remove the adapters
previously ligated to both ends of the target cDNAs. These cDNAs were
subsequently purified using QIAGEN’s PCR purification
system, ligated into the vector pCR-SCRIPT (Stratagene,
San Diego, CA) and transformed into Escherichia coli strain
XL-1' Blue (Stratagene). The individual cDNA inserts were
isolated by PCR amplification using flanking T3 and T7 primer sites.
The plasmid template used in the PCR reaction was obtained by the
Wizard mini-prep kit (Promega Corp., Madison, WI) or by
directly using the bacterial cultures lysed in
ddH20 at a dilution of 1:50.
Purified/PCR-amplified cDNAs were sequenced using T7 primers at the DNA
sequencing core facility of the Huntsman Cancer Institute at the
University of Utah Health Sciences Center using Perkin-Elmer Corp. ABI 377 automated sequencers. The sequence data obtained
were analyzed for homology to previously characterized genes deposited
in the National Center for Biotechnology Informatics (NCBI) database,
which includes entries from GenBank, European Molecular Biology
Laboratory (EMBL), and DNA Database of Japan (DDBJ) databases using the
Basic Local Alignment Tool (BLAST) program.
Analysis of subtraction efficiency
An equal amount of cDNA from the (presubtraction) tester pool
and final SSH-subtracted product were used as a template for the
amplification of the housekeeping gene glyceraldehyde-3-phosphate
dehydrogenase (G3PDH). The forward (5'-TGAAGGTCGGTGTGAACGGATTTGGC-3')
and reverse (5'-CATGTAGGCCATGAGGTCCACCAC-3') G3PDH primers
(CLONTECH Laboratories, Inc.) were used to amplify a
983-bp product within the following PCR parameters: denaturation, 94 C
for 45 sec; annealing, 60 C for 45 sec; and extension, 72 C for 1 min
and 30 sec. Samples were removed following the completion of 14, 18,
22, 26, and 30 cycles. The resultant amplicon was resolved on a 2%
agarose gel stained with ethidium bromide.
Verification of ovary-selective expression (reverse Northern blot
analysis)
Poly(A)+ RNA from the previously described
nonovarian and ovarian tissues was used to generate double stranded
cDNA using the SMART cDNA synthesis system according to the
manufacturer’s directions (CLONTECH Laboratories, Inc.).
The nonovarian and ovarian cDNAs generated were purified, quantified,
separated on a 1% agarose gel, and blotted onto a Nylon membrane (MSI,
Westborough, MA). The membranes were then probed with
32P-labeled/PCR-amplified cDNA inserts according
to the method of Sambrook et al. (12). The blots were
imaged, and band density was determined by a phosphorimager
(Bio-Rad Laboratories, Inc., Hercules, CA). Equal cDNA
loading was confirmed by probing with a
32P-labeled/PCR-amplified 983-bp G3PDH
product.
Northern blot analysis
Total RNA (20 µg) isolated from ovaries at different stages of
the superovulation protocol was separated on denaturing 1%
agarose-formaldehyde gels and transferred to nylon membranes according
to the protocol of Sambrook et al. (12). Before transfer,
RNA quality and concentration were assessed by ethidium bromide
staining and visualization under UV light. Nonovarian tissue blots (20
µg total RNA) were purchased from Origene Technologies (Rockville,
MD). Nylon membranes were prehybridized for 2–6 h at 42 C in 5 x
SSPE (sodium chloride-sodium phosphate-EDTA), 50% formamide, 5 x
Denhardt’s solution, and 0.25% SDS. Probes were generated by
radiolabeling individual PCR-amplified cDNA inserts with
[32P]deoxy-CTP using the random priming method
(Amersham Pharmacia Biotech, Piscataway, NJ). The probes
were denatured in a boiling water bath for 5 min before quenching with
ice. Membranes were hybridized with the relevant probe overnight at 42
C in the same (above-mentioned) solution used for prehybridization. The
membranes were washed in 2 x SSC (standard saline citrate) and
0.1% SDS at room temperature three times for 5 min each time, followed
by two washes in 0.125 x SSC and 0.25% SDS for 15 min at 60 C.
The blots were rinsed in 4 x SSC and imaged through the use of a
phosphorimager (Bio-Rad Laboratories, Inc.). The intensity
of signals was quantified using Molecular Analyst software
(Bio-Rad Laboratories, Inc.). Equivalent RNA loading was
verified by probing the same (stripped) blots with radiolabeled PCR
products of the housekeeping genes ß-actin (for the ovarian blots)
and G3PDH (for the nonovarian tissue blots).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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). An additional 8–12 PCR
cycles were required for the subtracted (target) cDNA to achieve the
same level of G3PDH amplification as in the unsubtracted ovarian cDNA.
As PCR amplification is an exponential process, this differential in
cycle number translates into a 256- to 4096-fold depletion of G3PDH
cDNA in the subtracted ovarian material (Fig. 1B).
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To establish an appropriate conclusion for the screening process of the
subtracted ovary-selective library, the number of new sequences
obtained was plotted against the total number of clones sequenced (Fig. 2). Only 2 new clones were obtained upon
sequencing the last batch of 50 clones. These results suggested that
the screening of the subtracted ovarian cDNA library was all but
complete and that few new sequences were probably derived by further
screening.
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). Known genes were classified as
such if the homology was complete (>98% identity) between the
subtracted SSH-generated clone and the database match. The complete
list of known genes isolated by SSH was deposited in the Ovarian
Kaleidoscope Gene Database (http://ovary.stanford.edu). Clones were
classified as homologous if they did not completely match (<98%) but
exhibited an E probability value of <1-5
relative to entries in the nonredundant database of NCBI. Novel clones
were designated as such if no significant match (E probability value
>1-5) was obtained in the nonredundant
database. Clones not matching entries within the nonredundant database
were still designated as novel even if a complete sequence match was
uncovered within the NCBI EST database. Such EST entries represent
incomplete sequence information (
300–600 bp) of an uncharacterized
gene and thus do not detract from the novelty designation. cDNAs
possessing significant homology to genomic clones (i.e.
cosmid, BAC, and PAC) were also classified as novel, as these entries
are comprised of large uncharacterized DNA fragments from a variety of
different species. Of the 844 clones analyzed, 342 were determined to
be nonredundant (independent) sequences. It follows that different
clones containing the same cDNA insert were sequenced multiple times.
Of the 342 nonredundant genes sequenced, 159 corresponded to known
genes, whereas 100 proved partially homologous to entries in the
nonredundant NCBI database. Eighty-three clones were classified as
novel, of which 66 shared homology with entries within the NCBI EST
database.
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). Examples include
proteins encoded by genes involved in ovarian steroidogenesis [StAR,
3ß-hydroxysteroid dehydrogenase I (3ßHSD), 17ßHSD I, and
aromatase) and in cell to cell communication (connexin-43) (24, 25, 26, 27, 28).
Also noted were genes corresponding to secreted ovarian regulators
(-inhibin, ßB-inhibin, and GDF-9),and to the oocytic ZP-3
protein (3, 4, 6). Interestingly, among the clones sequenced, the Mti10
retrotransposon was highly represented in the ovary-selective cDNA
library (28 of 844 clones, or 3.3% of the total). The Mti10
retrotransposon, a mobile genetic element that relies on a RNA
intermediate to move throughout the genome, belongs to the MaLR
(mammalian apparent long terminal repeat-retrotransposons) family of
transposons (29). It has been estimated that most mammalian genomes
contain between 40,000–100,000 copies of family members of the MaLR
retrotransposon, thereby accounting for a significant proportion of the
genome (29).
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(31), germ cell nuclear factor (32), and c-mos
(5) were not found within the subtracted ovarian library. Likewise, the
granulosa cell-restricted FSH receptor cDNA (33) was not present within
the final subtracted target ovarian cDNAs. The absence of these genes
from the library may be due to an incomplete representation of the
relevant mRNA in the tester cDNA pool that was used in the subtraction
process. This hypothesis is supported by the observation that the genes
in question could not be detected by PCR in the unsubtracted ovarian
cDNAs used as the starting material for the SSH procedure (data not
shown). Both the tester and driver cDNA pools were generated by the use of the SMART (Switching Mechanism At 5'-end of RNA Transcript) cDNA synthesis kit (CLONTECH Laboratories, Inc.). This process relies on the addition of unique adapter oligonucleotides to the first strand cDNA. The unique adapters can then be used to prime the PCR amplification and the generation of double stranded cDNA. The advantage of this procedure is that it allows for the generation of large amounts of cDNA from limited quantities of RNA. Due to the utilization of PCR, however, some of the cDNAs may not be amplified as efficiently as others and may thus be lost from the SSH starting material. A similar inability to identify all of the expected known genes after a differential screening of retinal candidate genes was recently reported by others and ascribed to the incomplete representation of the total cDNA repertoire (34).
A significant number of subtracted/SSH-generated cDNAs
exhibited partial homology to entries in the nonredundant NCBI
database (Table 2
). Most of the cDNAs in this category probably
represent mouse homologues of genes identified in other species
(e.g. rat P450scc, rabbit 17-hydroxylase/17–20-lyase,
etc.). A large proportion (n = 104) of the clones
isolated from the subtracted/SSH-generated cDNA library exhibited
significant homology to rat P450scc, representing in all likelihood the
mouse counterpart. Only the 5'-region of the mouse P450scc gene has
previously been deposited in the NCBI database. The latter was mirrored
by 29 clones isolated from the subtracted/SSH-generated cDNA
library.
The degree of ovary-selective expression for 20 randomly chosen
(SSH-derived) cDNAs was verified by reverse Northern analysis. Of the
20 clones analyzed, only 1 exhibited a higher level of expression in
the nonovarian (driver) cDNA compared with the ovarian (tester) cDNA,
demonstrating the low false positive rate associated with SSH in this
experimental circumstance. Figure 3
depicts the ovary-selective expression of 3 cDNAs corresponding to
known genes. Genes previously reported to be ovary-selective (GDF-9 and
3ßHSD) (4, 26) were confirmed as such. A member of the MT transposon
family (Mti10) was also determined to be ovary selective by reverse
Northern blot analysis. Ovary-selective expression of Mti10 was further
supported by the observation that EST (expressed sequence tag)s
corresponding to this transposable element have been isolated
predominantly from unfertilized eggs and 2-cell embryo cDNA libraries.
Confirmation of equivalent cDNA loading was accomplished by probing for
the housekeeping gene G3PDH.
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). From this analysis, 17
clones were determined to be ovary specific and phase specific. Only 4
ovary-specific clones were constitutively expressed throughout a
simulated cycle. Fifteen of the 83 novel clones were classified as
ovary selective, in that their ovarian expression proved highest, with
limited expression in 1 or more of the nonovarian tissues. Of the 15
ovary-selective clones isolated, 5 were expressed in a phase-specific
manner, with 10 being expressed constitutively. The expression of 46 of
the novel cDNAs could not be detected by the Northern blot technique.
This negative outcome may reflect the low level of sensitivity of the
Northern blot methodology employed. Verification of ovary-selective or
-specific expression of these 46 negative clones will require the use
of a more sensitive methodology, such as RT-PCR, which is currently
underway. Only 1 of the novel clones was expressed at a higher level in
the nonovarian tissues relative to the ovarian RNA, demonstrating a low
false positive rate (2.1%) for the SSH technique as used under this
experimental circumstance.
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. The
expression of the two novel clones (M3-D2 and M7-A10) could not be
detected in any of the nonovarian RNA samples. The presence of RNA on
the membranes was verified by successfully reprobing for G3PDH
transcripts.
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. The expression of the
M3-D2 gene increased approximately 4.5-fold relative to that in
unstimulated ovaries 48 h after treatment with PMSG (Fig. 5, A and
C). Increases of 10- and 9.6-fold (relative to unstimulated ovaries)
were observed in the ovarian expression of the novel gene M3-D2 at 24
and 48 h after the injection of hCG to PMSG-primed mice
(P < 0.05), respectively. Another novel gene (M7-A10),
exhibited a 50% reduction in ovarian expression (relative to
unstimulated ovaries) 6 h after the administration of hCG to
PMSG-primed mice (P < 0.05; Fig. 5, B and D). However,
24 and 48 h post-hCG, the expression of M7-A10 was increased
significantly (P < 0.05) relative to the control value
(1.5- and 1.8-fold). The transcript sizes of the M3-D2 and M7-A10
genes, as calculated from the Northern blot analysis, were
approximately 1.3 and 1.5 kbp, respectively. Equivalent RNA loading was
verified by reprobing the same membrane for ß-actin transcripts.
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). The latter proved
highly acidic (theoretical pI of 3.9) due to the large number of
glutamic acid residues (49 of a total of 283). Based on its restricted
expression profile and highly acidic nature, we have termed this
protein ovary-specific acidic protein (OSAP). According to the PSORT
protein analysis tools (http://psort.nibb.ac.jp:8000), OSAP does not
possess a signal peptide, endoplasmic reticulum localization
motifs, peroxisomal targeting sequences, or transmembrane-spanning
domains. The PSORT cellular localization prediction algorithm suggested
that OSAP may be directed to reside in the mitochondria. By using
NCBI’s BLASTp program, low but significant homology (E value of
10-12) was observed between OSAP and the sea
lamprey neurofilament protein (NF-180). The acidic regions of
neurofilament proteins from other species were also found to possess
limited but significant homology to OSAP (data not shown).
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). PSORT analysis suggested that the
M7-A10 protein lacks a signal peptide, ER localization motifs,
peroxisomal targeting sequences, or transmembrane-spanning domains. The
PSORT cellular localization prediction algorithm suggested that M7-A10
localizes to the cytoplasm. By using NCBI’s BLASTp program, M7-A10 was
found to possess significant homology only at the amino acid level to 2
yeast proteins, termed MOB1 and MOB2 (Fig. 7). MOB1 (Mps One Binder) was originally
identified via a yeast two-hybrid system as a binding partner for the
yeast protein kinase MPS1 (35). MPS1, in turn, is necessary for proper
mitotic checkpoint control and spindle pole body duplication (36).
Conditional (temperature-sensitive) alleles for the yeast MOB1 gene
gave rise to a late nuclear division arrest at the restrictive
temperature (35). Another MPS1 binding protein was subsequently cloned
and named MOB2 (35). The MOB2 protein also exhibited significant
homology to the ovary-specific clone M7-A10. Based on the homology
among M7-A10, MOB1, and MOB2, we propose that this new putative protein
be named mammalian MOB homolog.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Obstetrics and Gynecology, St.
Marianna University School of Medicine, 2–16-1 Sugao, Miyamae-ku,
Kawasaki, Kanagawa, 216, Japan.
3 Current address: George Washington University School of Medicine,
2300 I Street, Washington, D.C. 20037.
Received March 13, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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, ßA, and ßB subunits in
various tissues predicts diverse functions. Proc Natl Acad Sci USA.
85:247–251
, a germ cell
specific transcription factor involved in the coordinate expression of
the zona pellucida genes. Development 124:4939–4947[Abstract]
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