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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: embryogenesis/fertility/folliculogenesis/microarray/oogenesis
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Laser capture microdissection and RNA preparation
Primordial oocytes, defined as an oocyte cell surrounded by a monolayer of flattened granulosa cells (Gougeon and Chainy, 1987), 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
-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, 2003
). 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), phenolchloroformisoamyl 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, 2003). 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, 2003
).
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 1624 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 imposedan 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., 2002). 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., 2002
).
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 410 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 3, RAP1, laminin and
-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 3 and
-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., 2000). 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,
-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., 2004).
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Brown KA, Boerboom D, Bouchard N, Dore M, Lussier JG and Sirois J (2004) Human chorionic gonadotropin-dependent regulation of 17beta-hydroxysteroid dehydrogenase type 4 in preovulatory follicles and its potential role in follicular luteinization. Endocrinology 145, 19061915.
Burns KH, Owens GE, Fernandez JM, Nilson JH and Matzuk MM (2002) Characterization of integrin expression in the mouse ovary. Biol Reprod 67, 743751.
Cooke HJ, Lee M, Kerr S and Ruggiu M (1996) A murine homologue of the human DAZ gene is autosomal and expressed only in male and female gonads. Hum Mol Genet 5, 513516.
Crackower MA, Kolas NK, Noguchi J, Sarao R, Kikuchi K, Kaneko H, Kobayashi E, Kawai Y, Kozieradzki I, Landers R, Mo R, Hui CC, Nieves E, Cohen PE, Osborne LR, Wada T, Kunieda T, Moens PB and Penninger JM (2003) Essential role of Fkbp6 in male fertility and homologous chromosome pairing in meiosis. Science 300, 12911295.
Dean J (2002) Oocyte-specific genes regulate follicle formation, fertility and early mouse development. J Reprod Immunol 53, 171180.[CrossRef][ISI][Medline]
Eichenlaub-Ritter U and Peschke M (2002) Expression in in-vivo and in-vitro growing and maturing oocytes: focus on regulation of expression at the translational level. Hum Reprod Update 8, 2141.
Freiman RN, Albright SR, Zheng S, Sha WC, Hammer RE and Tjian R (2001) Requirement of tissue-selective TBP-associated factor TAFII105 in ovarian development. Science 293, 20842087.
Friedman J, Weissman I and Alpert S (1994) An analysis of the expression of cyclophilin C reveals tissue restriction and an intriguing pattern in the mouse kidney. Am J Pathol 144, 12471256.[Abstract]
Gougeon A (1993) Dynamics of human follicular growth: a morphologic perspective. In Adashi EY and Leung PCK (eds) The Ovary. Raven Press, New York, pp. 2140.
Gougeon A and Chainy GB (1987) Morphometric studies of small follicles in ovaries of women at different ages. J Reprod Fertil 81, 433442.[ISI][Medline]
Johnson J, Canning J, Kaneko T, Pru JK and Tilly JL (2004) Germline stem cells and follicular renewal in the postnatal mammalian ovary. Nature 428, 145150.[ISI]
Kim SW, Lee ZW, Lee C, Im KS and Ha KS (2001) The role of tissue transglutaminase in the germinal vesicle breakdown of mouse oocytes. Biochem Biophys Res Commun 286, 229234.[CrossRef][ISI][Medline]
Lee CJ, Do BR, Lee JM, Song KW, Kang JS and Park MH (2003) Differential expression of tissue transglutaminase protein in mouse ovarian follicle. In Vivo 17, 235238.[ISI][Medline]
Leo CP, Vitt UA and Hsueh AJ (2000) The Ovarian Kaleidoscope database: an online resource for the ovarian research community. Endocrinology 141, 30523054.
Neilson L, Andalibi A, Kang D, Coutifaris C, Strauss JF, 3rd, Stanton JA and Green DP (2000) Molecular phenotype of the human oocyte by PCR-SAGE. Genomics 63, 1324.[CrossRef][ISI][Medline]
Nnene IO, Nieto JJ, Crow JC, Sundaresan M, MacLean AB, Perrett CW and Hardiman P (2004) Cell cycle and apoptotic proteins in relation to ovarian epithelial morphology. Gynecol Oncol 92, 247251.[CrossRef][ISI][Medline]
Pan HA, Tsai SJ, Chen CW, Lee YC, Lin YM and Kuo PL (2002) Expression of DAZL protein in the human corpus luteum. Mol Hum Reprod 8, 540545.
Robker RL and Richards JS (1998) Hormone-induced proliferation and differentiation of granulosa cells: a coordinated balance of the cell cycle regulators cyclin D2 and p27Kip1. Mol Endocrinol 12, 924940.
Sirivatanauksorn Y, Drury R, Crnogorac-Jurcevic T, Sirivatanauksorn V and Lemoine NR (1999) Laser-assisted microdissection: applications in molecular pathology. J Pathol 189, 150154.[CrossRef][ISI][Medline]
Thompson WE, Powell JM, Whittaker JA, Sridaran R and Thomas KH (1999) Immunolocalization and expression of prohibitin, a mitochondrial associated protein within the rat ovaries. Anat Rec 256, 4048.[CrossRef][ISI][Medline]
Thompson WE, Asselin E, Branch A, Stiles JK, Sutovsky P, Lai L, Im GS, Prather RS, Isom SC, Rucker IE and Tsang BK (2004) Regulation of prohibitin expression during follicular development and atresia in the mammalian ovary. Biol Reprod.
Tong ZB, Gold L, Pfeifer KE, Dorward H, Lee E, Bondy CA, Dean J and Nelson LM (2000) Mater, a maternal effect gene required for early embryonic development in mice. Nat Genet 26, 267268.[CrossRef][ISI][Medline]
Xiang C and Brownstein MJ (2003) Fabrication of cDNA microarrays. In Khodursky MJBaA (ed.) Methods in Molecular Biology, Vol 224. Humana Press Inc, Totowa, NJ, pp. 17.
Yuan YQ, Peelman LJ, Williams JL, Van Zeveren A, Kruif A, Law A and Van Soom A (2004) Mapping and transcription profiling of CASP1, 3, 6, 7 and 8 in relation to caspase activity in the bovine cumulusoocyte complex. Anim Genet 35, 234237.[CrossRef][ISI][Medline]
Submitted on February 10, 2004; resubmitted on June 15, 2004; accepted on August 5, 2004.
|