Identification of genes expressed in primate primordial oocytes

Jose A. Arraztoa1,2, Jian Zhou1, David Marcu1, Clara Cheng1, Robert Bonner3, Mei Chen4, Charlie Xiang4, Michael Brownstein4, Kevin Maisey5, Monica Imarai5 and Carolyn Bondy1,6

1 DEB, NICHD, 3 Laboratory of Pathology, NCI, 4 Laboratory of Genetics, NIMH, NIH, Bethesda, MD, USA, 2 Department Obstetric and Gynecology, University of Los Andes and 5 Laboratory of Biochemistry, University of Santiago, Chile

6 To whom correspondence should be addressed at: Building 10/10N262, 10 Center Dr, NIH, Bethesda, MD 20892, USA. Email: bondyc{at}mail.nih.gov


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The factors involved in oocyte survival and transition from quiescence to the growing phenotype remain unknown. Herein we report genes that are differentially expressed in the primordial oocyte revealed by DNA arrays. METHODS: Primordial oocytes were captured selectively in rhesus monkey ovary sections using laser capture microdissection. The RNA was extracted and amplified in two rounds by T7-based linear RNA amplification, fluorescence labelled and then hybridized to human cDNA arrays containing 7680 elements. RNA from human placenta served as a reference sample. RESULTS: Ninety-five genes were found to be consistently expressed at a higher level in primordial oocytes. Expression of several of these genes in the oocyte has been reported before, e.g. deleted in azoospermia (DAZ), prohibitin and transglutaminase 2. Oocyte expression of several novel transcripts revealed on array hybridization, such as gene 33, ubiquitin-conjugating enzyme E2A, G1 to S phase transition 1, growth arrest and DNA damage-inducible (GADD), and dendritic cell-derived ubiquitin-like protein (DC-UbP) was confirmed by in situ hybridization. Some array-identified gene products [integrin {beta}3, {alpha}-tubulin, regulatory telomere elongation protein (RAP1) and cellular repressor of EIA-stimulated genes (CREG protein)] were detected in human oocytes by immunofluorescence. Bioinformatic analysis of the oocyte-enriched transcripts reveals a functional profile summarized as follows: cell cycle (14%); transporter (13%); signal transduction (10%); cytoskeletal (7%); transcription factor (5%); immune response (5%); apoptosis-related (5%); RNA processing (5%); and the remainder of miscellaneous categories. CONCLUSIONS: These observations may contribute to the elucidation of molecular pathways involved in oocyte survival and maturation.

Key words: embryogenesis/fertility/folliculogenesis/microarray/oogenesis


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Oocyte-specific genes regulate follicle formation, fertility and early embryonic development (Tong et al., 2000Go; Dean, 2002Go; Eichenlaub-Ritter and Peschke, 2002Go). Primordial oocytes have been considered ‘quiescent’ germ cells of the ovary. These oocytes appear to be in a state of arrested development, suspended in the dictyate phase of prophase I of the first meiotic division. They are relatively small in diameter and are surrounded by a single layer of flattened, metabolically indolent granulosa cells, forming the ‘primordial follicle’ (Gougeon, 1993Go). There are 1–2 million of these primordial follicles in the human ovary at birth, but their numbers decrease dramatically through an unknown process of attrition, so that by the time of puberty, there are only ~300 000–400 000 primordial follicles forming the reproductive endowment (Gougeon, 1993Go). During the course of reproductive life, many more primordial oocytes undergo a mysterious demise, while relatively few begin to grow and mature into ovulatory follicles. The signal or signals that control the processes of oocyte demise or activation to growth and further development remain largely unknown.

To elucidate the pathways and signals involved in primate primordial oocyte survival and development, it is important to identify genes abundantly expressed in the oocyte. To obtain this information, we used laser capture microdissection (LCM) to selectively harvest primordial oocyte populations from rhesus monkey ovary tissue sections. We characterized the differentially expressed genes in these unique cells on high-density cDNA microarrays using human placental mRNA as a reference. We chose placenta as a reference because this tissue seemed more appropriate than typical somatic tissues (e.g. liver) and because it contains abundant RNA which provides good signals in most of the spots of microarray, facilitating reliable ratios between sample and reference signals. Furthermore, this strategy establishes a ready reference that could be used for comparisons in further studies against other ovary cells. Because normal, healthy human tissues are not available for this purpose, we used rhesus monkey ovary sections for this study. This species' reproductive cycle is very similar or identical to that of the human, and therefore we expect the differential gene expressed from the monkey oocyte to be informative for the human. This novel information is important to help comprehend physiological processes such as oocyte meiotic arrest and the initiation of folliculogenesis, and eventually to identify some molecular processes in ovarian diseases such as premature ovarian failure, polycystic ovarian syndrome and ovarian cancer.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Tissue samples
Female rhesus monkeys (Macacca mulatta) 6–13 years of age from the NIH Poolesville colony were used in accordance with a protocol approved by the NICHD Animal Care and Use Committee. All monkeys had regular menstrual cycles prior to participation in the study (average cycle length was 28±3 days) and they underwent ovariectomy under ketamine anaesthesia via a mid-ventral laparotomy. Ovaries from three monkeys were snap-frozen on dry ice, and stored at –70°C. Serial sections of 10 µm thickness were cut at –15°C and thaw-mounted onto poly-L-lysine-coated slides.

Laser capture microdissection and RNA preparation
Primordial oocytes, defined as an oocyte cell surrounded by a monolayer of flattened granulosa cells (Gougeon and Chainy, 1987Go), were stained with crystal violet and dissected under 20x microscopic visualization using the Arcturus Pixel II® LCM apparatus at the core facility at the National Institutes of Health. Using sections from the six ovaries, we were able to identify and capture ~10 000 oocyte sections. Oocytes with pycnotic or fragmented nuclei, shredded ooplasm or disintegrated follicular structures were regarded as possibly atretic and not collected. This material was pooled and RNA was extracted using the Absolutely RNATM Microprep Kit (Stratagene®, La Jolla, CA) in accordance with the manufacturer's instructions. Briefly, the cells were lysed for RNA extraction using 100 µl of RNA denaturing buffer (guanidine isothiocyanate) with 0.8 µl of {beta}-mercaptoethanol and stored at –70°C until use. The RNA isolation was performed according to the kit protocol and the RNA precipitated in 3 mol/l sodium acetate (pH 5.2) 1:10, 100% ethanol and glycogen. The RNA obtained was amplified in two rounds using oligo(dT) primers (Xiang and Brownstein, 2003Go). Briefly, for the first strand cDNA synthesis, 1 µl of a 100 pmol/µl solution of T7dT primer 5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTT-3' (Operon, Alameda, CA) was added to 23 µl of RNA. The RNA was denatured at 70°C for 10 min and chilled on ice for 10 min. A 1 µl aliquot of 10 mmol/l dNTPs (Amersham Pharmacia, Piscataway, NJ), 3 µl of 0.1 mmol/l dithiothreitol (DTT) and 2 µl of SuperScript II reverse transcriptase were added to the tubes and incubated at 42°C for 2 h. For the second strand cDNA synthesis, 81 µl of RNase-free water, 30 µl of 5x second strand buffer, 3 µl of 10 mmol/l dNTPs, 1 µl of DNA ligase, 4 µl of DNA polymerase I (Klenow fragment) and 1 µl of RNase H were added to the reaction, and the tubes were incubated at 16°C for 2 h. After the reaction, 2 µl of T4 DNA polymerase were added and the samples were incubated at 16°C for another 5 min. The resulting cDNA products were purified by Phase Lock Gel (Eppendorf, Westbury, NY), phenol–chloroform–isoamyl alcohol extraction and further purified as well as concentrated with MicroCon-30 columns (Millipore, Bedford, MA). In vitro RNA transcription and amplification were performed from these DNA template by using MEGA Script T7 reagents (Ambion, Austin, TX) according to the manufacturer's instructions, and purified with an RNeasy Mini kit (Qiagen, Valencia, CA). For the second and subsequent rounds of amplification, we used a T3N9 primer 5' -GCGCGAAATTAACCCTCACTAAAGGGAGAGGGNNNNNNNNN-3' (Invitrogen, Carlsbad, CA) to drive the first strand cDNA synthesis. The second strand cDNA synthesis and in vitro RNA transcription were done as described above. The majority of reagents and enzymes used in this section were obtained from Invitrogen Life Technologies (Carlsbad, CA), unless specified otherwise.

RNA labelling and array hybridization
The RNA labelling protocol was detailed in our previous publication (Xiang and Brownstein, 2003Go). Briefly 5 µg of total RNA (15.5 µl) were mixed with amine-modified random primer (2 µg/µl, 2 µl) and RNase inhibitor (5 U/µl, 1 µl), incubated at 70°C for 10 min, and then chilled on ice for 10 min. Primer/RNA solution was added to the RT mix [5x first strand buffer, 6 µl; 50x aa-dUTP/dNTPs (25 mmol/l dATP, dGTP and dCTP, 15 mmol/l dTTP and 10 mmol/l aminoallyl dUTP) 0.6 µl; 0.1 mol/l DTT, 3 µl; SSII RT, 2 µl] and incubated at 42°C for 2 h. EDTA (0.5 mol/l, 10 µl) was added to stop the reaction, and the RNA was hydrolysed with NaOH (1 mol/l, 10 µl) at 65°C for 30 min. The solution was neutralized with HCl (1 mol/l, 10 µl), and then purified by Qiagen MinElute PCR purification kits (Valenca, CA). A 3 µl aliquot of 1 mol/l sodium bicarbonate (pH 9.3) was added to the cDNA solution, followed by 1 µl of dye [NHS-ester Cy3 or Cy5, 62.5 µg/µl in dimethylsulphoxide (DMSO) (Amersham Pharmacia, Piscataway, NJ)]. The resulting solution was mixed by pipetting it up and down several times; the tubes were wrapped in aluminium foil, and incubated at room temperature for 1 h in an orbital shaker (USA Scientific, Ocala, FL). The labelling reaction was stopped with 4.5 µl of 4 mol/l hydroxylamine hydrochloride. Afterwards, the tubes were vortexed, briefly centrifuged, and incubated for 30 min at room temperature in the dark. The probes were cleaned with a Qia-quick PCR purification kit (Qiagen, Valenca, CA) and then hybridized in duplicate to the cDNA microarray in a hybridization chamber (Corning, Corning, NY) (Xiang and Brownstein, 2003Go).

The human 8K cDNA array (7361 human genes and 319 control spots, total of 7680 elements; list of target genes available on Human Reproduction website) was used in this study. We printed this human 8K cDNA microarrays on poly-L-lysine-coated glass slides using an OmniGrid arrayer (GeneMachines, San Carlos, CA). The human clones were originally purchased from Research Genetics (now Invitrogen, CA). A robotic arraying machine loaded ~1 µl of PCR-amplified fragments from corresponding wells of 96-well or 384-well plates and deposited ~5 nl of each sample onto each of 100 slides. The concentration of PCR products is ~500 ng/µl. Approximately 2.5 ng of DNA was printed on each slide for each sample. After printing, the slides are post-processed with denaturing, blocking and dehydration steps.

Please visit http://research.nhgri.nih.gov/microarray/fabrication.html for the details of making cDNA microarrays. Total RNA from human placenta (Clontech, originally BD Biosciences, San Jose, CA) was used as a reference. The arrays were incubated in a 65°C water bath for 16–24 h, and subsequently washed with 0.5x SSC, 0.01% SDS followed by 0.06x SSC at room temperature, 10 min each. The slides were next placed in 50 ml Falcon tubes and spun for 5 min at 800 r.p.m. at room temperature.

The arrays were read with a GenePix 4000A scanner (Axon, Foster City, CA) at 10 µm resolution and variable photomultiplier tube (PMT) voltage settings to obtain the maximal signal intensities with <1% probe saturation. The resulting images were analysed using IPLab (Fairfax, VA) and ArraySuite (NHGRI, Bethesda, MD) software. To determine the reliability of each ratio measurement, a set of quality indicators was used. To be considered reliable, intensity measurements had to satisfy the following criteria: (i) association of a sufficiently large number of pixels with the element; (ii) flat local background; (iii) uniform signal consistency within the target area; and (iv) unsaturation of the majority of the signal pixels. For each ratio measurement, R/G, one further condition was imposed—an average signal (R + G)/2 that is at least three times the noise level.

In situ hybridization
Details of the in situ hybridization protocol have been published (Arraztoa et al., 2002Go). To confirm the array results and establish oocyte localization, we labelled and hybridized six probes for targets identified in the array (cDNA clones were purchased from ATCC). These probes were hybridized to monkey ovary sections as previously described (Arraztoa et al., 2002Go).

Immunohistochemistry
Ovaries from a 28-week-old fetus that died spontaneously from a placental insufficiency were used to investigate array gene product expression in human primordial follicles. The ovaries were obtained during routine necropsy with informed consent from the parents and approval by the Biomedical Ethics Committee of University of Los Andes. The ovaries were fixed in 4% paraformaldehyde for 1 h at 4°C, before sequential transfer to 10% sucrose in phosphate-buffered saline (PBS) during 1 h and 30% sucrose in PBS overnight. The tissue was embedded in Cryo-M-Bed (Bright Instruments Co. Ltd, Huntingdon, UK) and frozen at –20°C. Slice of 4–10 µm thickness were obtained using a Bright Starlet Cryostat at –20°C, mounted on gelatin-coated slides and permeabilized using cold 70% ethanol (v/v) for 20 min at –20°C. After 2 h incubation in 1% (w/v) bovine serum albumin (Sigma) in PBS, sections were incubated overnight at 4°C with antibodies against cellular repressor of E1A-stimulated genes (CREG), integrin {beta}3, RAP1, laminin and {alpha}-tubulin (1:25, Santa Cruz Biotechnology). The sections were then washed several times in PBS and incubated with fluorescein isothiocyanate-conjugated anti-goat immunoglobulin G antibody (1:50) at room temperature for 1 h. Controls with non-immune serum or omitting primary antibody were routinely included in all experiments. Sections were counterstained using a solution of 1 µg /ml propidium iodide in PBS and mounted in a solution of PBS containing 10% (v/v) 1,4-diazobicyclo[2.2.2]octane (DABCO; Sigma) and 90% (v/v) glycerol (Gibco). The samples were examined using a Carl-Zeiss Confocal Laser Microscope, Model LSM 510.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Approximately 10 000 oocyte micro-sections from three monkeys were collected by LCM. The total pooled RNA was amplified, labelled with the fluorescent dye Cy5 and the control placenta RNA with dye Cy3, and then mix hybridized to a 7680 human cDNA array. To control the potential different labelling efficacy of the fluorescent dye, the reverse labelling was also conducted. A 2-fold difference consistently demonstrated on each microarray using the forward or reverse labelled probe set was used as a criterion to analyse the data. Ninety-five genes were found at >2-fold higher levels in primordial oocytes compared with placenta on both forward and reverse labelled arrays; the average ratio for oocyte versus placental expression was 9.3. A list of these genes grouped by function is presented in Table I. We did not investigate further genes that were expressed at similar or lower levels than our reference.


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Table I. Oocyte genes grouped by function

 
To evaluate the array results, we conducted a bioinformatic search using SOURCE, NLM and Ovarian Kaleidoscope databases. We found that 11 of the 95 array genes had been reported previously to be expressed within the ovary: caspase 6, apoptosis-related cysteine protease (Leo et al., 2000Go); cyclin D1 (Robker and Richards, 1998Go); growth arrest and DNA damage-inducible, {gamma} (Leo et al. 2000Go); prohibitin (Thompson et al., 1999Go); FK506-binding protein 12-rapamycin-associated protein 1 (Leo et al., 2000Go); peptidylprolyl isomerase C (cyclophilin C) (Friedman et al., 1994Go); FXYD domain-containing ion transport regulator 3 (Freiman et al. 2001Go); integrin {beta}3 (Burns et al., 2002Go); DAZ, deleted in azoospermia (Cooke et al., 1996Go; Pan et al., 2002Go); transglutaminase 2 (Lee et al., 2003Go); and hydroxysteroid (17-{beta}) dehydrogenase 4 (Leo et al., 2000Go). Table II shows the ovarian localization and the species where these genes have been described. Thus 84 genes identified by the array screen had not been reported previously to be expressed in the ovary or in oocytes.


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Table II. Ovarian localization and the species of reported genes

 
We investigated the cellular localization of six of the genes reported on the array using in situ hybridization with radiolabelled cRNA probes on rhesus monkey ovary sections. These experiments confirmed the expression of these six genes in oocytes and, in some cases, in surrounding granulosa cells (Table III and Figure 1). These findings validate the LCM and array methodology used in these experiments. To test our expectation that the non-human primate would be a good model for the human, we investigated some of the array-identified gene products in oocytes from human fetal ovaries. {alpha}-Tubulin showed a punctuate pattern in the ooplasm which differed from the classical fibrillar pattern in most cells (Figure 2A) and in our positive control (Figure 2a). Integrin {beta}3 (Figure 2B) showed a diffuse pattern in the ooplasm with discrete concentrations in a punctuate pattern which is in agreement with its cell membrane localization and its role in cell to cell communication and similar to our positive control using HeLa cells (Figure 2b). Regulatory telomere elongation protein (RAP1) immunoreactivity was localized in oocyte nuclei (Figure 2C) and CREG immunoreactivity in the ooplasm (Figure 2D). We did not detect laminin immunoreactivity in the fetal oocytes (Figure 2E).


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Table III. Cellular localization of six genes reported on the array

 


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Figure 1. Localization of ubiquitin-conjugating enzyme E2A (A and B) and G1 to S phase transition 1 (GSPT1, D and E) transcripts in primordial (A and D) and primary follicles (B and E) oocytes. In situ hybridization using radiolabelled riboprobes shows the hybrid signal as green grains in these micrographs taken from representative monkey ovary sections. Background signal from hybridization with a sense probe is seen in (C). Oo, oocyte; ST, stroma. Bar = 50 µm.

 


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Figure 2. Localization of array-identified gene products in human primordial oocytes. Upper panel and uppercase show protein immunoreactivities (green signal) in primordial oocytes. {alpha}-Tubulin (A) and integrin {beta}3 (B) are seen in a diffuse pattern through the ooplasm, with discrete concentrations in a punctuate pattern. RAP1 immunoreactivity (C) is focally concentrated in the oocyte nucleus, while CREG immunoreactivity (D) is found in focal deposits in the ooplasm. (E) A negative immunoreactivity for laminin. (F) Negative control where primary antibody was omitted. Lower panel and lowercase show positive control in HeLa cells for {alpha}-tubulin (a), integrin {beta}3 (b), RAP1 (c), CREG (d) and laminin (e), while the negative control illustrates absence of the primary antibody (f). In all pictures, red signal represents nucleus stained with propidium iodide.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Human oocytes are rare and precious cells, and there is great interest in improving our understanding of the survival and maturation processes critical for their role in human reproduction. The ability to study human oocytes is very limited, so that our knowledge of oocyte gene expression has been largely derived from mice, with a limited amount of information from human oocytes obtained after ovarian stimulation and follicular aspiration in the process of IVF (Neilson et al., 2000Go). The present study used normal healthy ovaries from cycling non-human primates and LCM to obtain primordial oocytes from a normal physiological environment as opposed to stimulated, superovulated material. This unique study has documented the expression of 95 genes in these normal, in situ oocytes. The expression of 84 of these genes in oocytes is novel. The LCM technique allows the study of RNA expression in specific cell types in a normal cellular microenvironment (Sirivatanauksorn et al., 1999Go). This is the first time, to the best of our knowledge, that such an approach has been used to study primordial oocytes.

The fact that several of the genes enriched on array analysis of LCM material previously had been found expressed in oocytes supports the reliability of our experimental approach. In addition, we confirmed a sampling of array data by in situ hybridization, demonstrating localization of the mRNAs in oocytes. We detected immunoreactivities in fetal human oocytes corresponding to the products of array-identified genes such as integrin {beta}3 and {alpha}-tubulin. Interestingly, this latter protein presents a pattern not typically seen in somatic cells. Since this is a descriptive study, this finding needs further investigation to be explained. We were not able to detect laminin immunoreactivity, however. This could be explained by a number of factors, including low expression levels in fetal human ovary compared with mature rhesus monkey.

Functional analysis of our array data showed that most highly expressed oocyte genes encode cell cycle-related proteins (14%), transporters (13%), signal transduction proteins (10%), structural proteins (7%), transcription factors (5%), immune response- (5%), apoptosis-related (5%) and RNA-processing proteins (5%). Neilson described the molecular phenotype of nine germinal vesicle (GV)-stage human oocytes obtained from two women who underwent ovarian stimulation and follicular aspiration (Neilson et al., 2000Go). The authors constructed a catalogue using the generation of expressed sequence tags (ESTs). Interestingly, many of the genes we detected using the microarray approach were similar to those described in the serial analysis of gene expression (SAGE)-PCR-generated GV-stage oocytes catalogue, including connexin 43, {alpha}-tubulin, cyclin A, cdk8 and RAS-related, among others.

These observations on genes expressed in primate primordial oocytes provide an infrastructure for further studies aimed at elucidation of the specific functional roles and interactions of these factors in primate oocyte biology. An important challenge is to compare expression levels for these factors at ensuing developmental stages. In addition, the abundance and functional profile of mRNAs found in these cells support the emerging view of primordial oocytes as dynamic cells, involved in active survival strategies and possibly as cycling cells, as suggested by recent investigations in the murine ovary (Johnson et al., 2004Go).


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This study was partially supported by the University of Los Andes grant #MED-003-03


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on February 10, 2004; resubmitted on June 15, 2004; accepted on August 5, 2004.





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