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Gamete Biology |
Physiologie de la Reproduction et des Comportements, UMR 6175 Institut National de la Recherche Agronomique/ Centre National de la Recherche Scientifique/Université François Rabelais de Tours/Haras Nationaux, F-37380 Nouzilly, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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gamete biology, gene regulation, oocyte development, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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It remained to be elucidated whether these maternal-effect genes were conserved in cattle. Given their suggested role in embryo transcription activation, whether expression is required for the second division as in mouse or rather until the maternal/embryonic transition (8/16-cell) appeared an intriguing question. In this study, we have cloned bovine homologue of MATER. We have analyzed the expression in somatic and gonadal tissues of five bovine genes known as markers of germ cells or oocytes in other species: VASA, MATER, ZAR1, GDF9, and BMP15. We have followed the fate of these last four transcripts during oocyte maturation, fertilization, and preimplantation embryo development.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult bovine ovaries were collected at a slaughterhouse and the oocyte-cumulus complexes were aspirated from 3- to 6-mm follicles, selected based on morphological criteria, and washed in saline solution (for details, see [23]). Denuded immature and in vitro-matured oocytes were obtained as previously described [24]. Briefly, oocyte-cumulus complexes were denuded by mechanical treatment either before or after in vitro maturation in TCM199 (Sigma, France) supplemented with 10 ng/ml epidermal growth factor for 24 h at 39°C in water-saturated air with 5% carbon dioxide. For in vitro fertilization, groups of 50 intact in vitro-matured oocyte-cumulus complexes were transferred into 500 µl fertilization medium containing 5 x 105 motile spermatozoa. Twenty-four hours later, presumptive zygotes were denuded. Twenty of them were set aside, while groups of 25 zygotes were transferred into 25-µl droplets (under paraffin oil) of modified synthetic oviduct fluid [25] supplemented with 5% fetal calf serum. Embryos were cultured at 39°C in a water-saturated atmosphere with 5% CO2:5% O2:90% N2. Groups of 20 embryos were collected over preimplantation development period: two-cell and four-cell embryos (Day 1), five- to eight-cell embryos (Day 2), morulae (Days 4 and 5), and expanded blastocysts (Days 6 and 7). All oocyte and embryo samples were stored frozen at –80°C in RNAlater (Ambion, UK) until RNA extraction. Two independent sets of oocytes and embryos were collected.
Collection of Organ Biopsies
Biopsies of bovine ovary and somatic tissues (uterus, heart, spleen, intestine, liver, lung, mammary gland, muscle) were sampled from an adult cow killed in an experimental setting. The animal was taken care of and killed in INRA farm/research center. All procedures were approved by the Agricultural and Scientific Research Government Committees in accordance with the guidelines for Care and Use of Agricultural Animals in Agricultural Research and Teaching (approval A37801). Calf ovary and adult testis were obtained from animals at a commercial abattoir. Tissues were frozen in liquid nitrogen and stored at –80°C until RNA extraction.
RNA Isolation, Reverse Transcription, PCR, and Southern Hybridization
Total RNA was extracted from groups of 20 oocytes or embryos, after adding 10 pg of rabbit globin mRNA (Gibco, France) using Tripure Isolation Reagent (Roche Diagnostics, Mannheim, Germany), and from 0.3– 0.5 g biopsies of gonadic and somatic tissues using TriZol reagent (Invitrogen, France) following the manufacturer's instructions. Aliquots of RNA from the tissue biopsies were further incubated with 10 U of RQ1 DNase (Promega, France) for 20 min at 37°C, followed by organic extraction, precipitation, and resuspension into water; RNA concentration was deduced from optical density and RNA integrity was checked by electrophoresis on denaturing gel. Reverse transcription was performed on RNA amounts equivalent to five oocytes or embryos and on 2.5 µg of RNA from tissue biopsies. The cDNA was extended from oligo(dT)15 primers (Promega, France) during 50 min at 37°C by mouse Moloney leukemia virus reverse transcriptase (Invitrogen, France) in a total volume of 20 µl. Parallel negative control reactions were set up without enzyme.
For PCR, we used as template cDNA amounts equivalent to 0.05 oocyte/embryo or, for other tissues, 1 µl of the reverse transcribed products or of a 1:10 dilution (only for ß-actin). Depending on the target gene, PCR was run for 30–38 thermal cycles, with the corresponding primers designed as summarized in Table 1. Primer sequences for rabbit globin were from [26]. Most primer pairs were designed in regions spanning at least one intron in the bovine gene (ß-actin [27]) or, when unknown, in the human gene (VASA, MATER, ZAR1, GDF9 primers sense1 and antisense1); in addition, we checked by PCR that no product of a similar size as the expected cDNA fragments was amplified from bovine genomic DNA (included in Advantage 2 PCR kit, Ozyme, France) with these primer pairs. PCR products were separated by agarose gel electrophoresis and observed under ultraviolet illumination in the presence of ethidium bromide. The products were then transferred onto Hybond N+ membrane (Amersham Biosciences, Orsay, France) and hybridized with the corresponding cDNA fragment labeled with 
[32P]-dCTP (Amersham, France) using Ready-To-Go DNA Labelling Beads (Amersham, France) following the manufacturer's instructions. We had previously sequenced these fragments, the lengths of which was as expected and which were 90% (VASA), 73% (MATER), and 92% (ZAR1) homologous to the human sequences or identical (GDF9, BMP15) to the known bovine sequences.
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Virtual Northern Blot
Total RNA was isolated from 50 bovine immature denuded oocytes as described above. The cDNAs were synthesized and amplified using Smart cDNA synthesis kit (Ozyme, France) following the manufacturer's instruction. The cDNAs were separated onto agarose gel, transferred to Hybond N+ membrane, and hybridized with the radio-labeled MATER probe, in Church solution (EDTA [1 mM], Na2HPO4 [0.5 M, pH = 7.5], SDS 7%), overnight at 65°C, followed by several washes. The membrane was finally subjected to autoradiography.
5'- and 3'-Rapid Amplification of cDNA Ends and Sequence Analysis
Total RNA was isolated from 100 bovine immature denuded oocytes as described above. 5'-and 3' rapid-amplification of cDNA ends (RACE) were performed using Smart Race cDNA Amplification Kit (Ozyme, France) according to the manufacturer's instructions. MATER-sense2 and antisense2 primers (Table 1) were used for 3' and 5' RACE, respectively. PCR products were subcloned using the TA cloning kit (Invitrogen, France) and selected inserts were sequenced. Alignments were performed using BLASTn (http://www.ncbi.nlm.nih.gov/BLAST/) and Dialign (http://www.infobiogen.fr/services/analyseq/cgi-bin/clustalw_in.pl). Protein sequence was analyzed using the Interpro database (http://www.ebi.ac.uk/interpro).
In Situ Hybridization
Ovaries from 6-mo-old calves were embedded in Tissue-Tek medium (Sekura, Bayer Diagnostics, France), frozen in liquid nitrogen, and then serially sectioned (10 µm) with a cryostat. The sections were fixed in 4% paraformaldehyde and washed in PBS. Mater and Zar1 antisense and sense probes were synthesized from linearized Dual Promoter pCRII plasmids (Invitrogen, France) containing the 391-base pair (bp) MATER (amplified with sense2 and antisense1 primers) and 331 bp ZAR1 cDNA inserts, respectively, using Riboprobe combined system SP6/T7 (Promega, France) and labeled [35S]-UTP. In situ hybridization was performed as described elsewhere [28].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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We found in GenBank a 439-bp bovine oocyte-specific expressed sequence tag (Z86039, gi 2398536) about 80% homologous with the 3' region of human MATER cDNA. We designed MATER-sense2 and antisense1 primers (see Table 1) to amplify by RT-PCR from oocyte total RNA and clone a 391-bp fragment (nt 2819–3209 in bovine coding sequence). Then we designed MATER-sense1 primer (see Table 1) in a region highly conserved with mouse in human exon 7. MATER-sense1 and antisense1 primers were used to amplify and clone a 1391-bp-long fragment (nucleotides 1819–3209), which displayed 73% sequence identity with human MATER cDNA. Used as hybridization probe in virtual Northern blot, this fragment revealed in the oocyte a unique, approximately 4-kbp-long cDNA (Fig. 1A). This length does exclude the polyA tail because it is not reverse transcribed in our protocol. To obtain the full-length sequence, we set 5' and 3' rapid-amplification of cDNA ends using primers MATER-antisense2 and sense1 (see Table 1), respectively. The corresponding overlapping 2.5-kb and 2-kb fragments were subcloned and sequenced. Within the 3.8-kbp-long deduced sequence, the longest open reading frame (ORF) is 3297 nucleotides long and 70% homologous to the human MATER coding sequence (Fig. 1B). The 5' and 3' noncoding regions were about 270 bp and 250 bp long, respectively. A polyadenylation signal (AATAAA) was localized 22 nucleotides upstream of the polyA tail. This ORF encodes a putative protein of 1098 aa with a predicted molecular mass of 121 kDa (Fig. 1C). A search within the Interpro database of proteins and domains was performed. Three major regions were identified: a DAPIN or Pyrin domain at the amino terminus (aa 1–97), followed by a NACHT domain (aa 180–349), and a region containing 12 leucine-rich repeats of the ribonuclease inhibitor subtype (LRR-RI) toward the carboxy terminus (aa 679– 706, 735–762, 764–791, 792–819, 821–848, 849–876, 878–905, 906–933, 935–962, 963–990, 991–1018, 1019– 1046) (Fig. 1D). Sequence is also well conserved in the aa 372–678 region, including the NAD (NACHT-associated domain) in the human protein. This global protein organization is typical of the recently discovered NALP (Nacht, leucine-rich repeat and pyrin domain containing) family that includes human MATER, also named NALP5 [29].
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Expression ofMATER, ZAR1, GDF9, BMP15,andVASAin Somatic and Gonadic Tissues
RT-PCR (with sense1 and antisense1 primers) was used to analyze mRNA expression pattern of MATER, ZAR1, GDF9, BMP15, and VASA in somatic (uterus, heart, spleen, intestine, liver, lung, mammary gland, muscle) and gonadic (testis, ovary) tissues and in immature oocytes. An estimated 1000 times less oocyte cDNA than other gonadic/ somatic tissues cDNA was subjected to the PCR reaction. PCR products were analyzed by gel electrophoresis, followed by Southern hybridization with the specific cDNA fragment (Fig. 2). MATER, ZAR1, GDF9, and BMP15 were strongly expressed in the oocyte as compared with other tissues. ZAR1, BMP15, GDF9, and VASA could be detected both in the testis and ovary. MATER was not detected in the ovary, but was detected in the testis. Actually, in addition to a band of similar length as in the oocyte, additional, slightly shorter fragments could be amplified from testis material, that are under current investigation. To check that signal was not due to contaminating DNA, we did analyze, in parallel, bovine genomic DNA and RNA samples subjected to mock reverse-transcription (not shown). As a positive control, ß-actin cDNA was amplified from all samples using a 100-fold excess of substrate cDNA for all tissues over the oocyte (Fig. 2).
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To further characterize the localization of MATER and ZAR1 transcripts within the ovary, we performed in situ hybridization on ovarian sections. Specific staining of the oocyte was observed with both MATER and ZAR1 antisense probes. ZAR1 was expressed as early as the primary follicle up to the antral stage (Fig. 3, A–I). MATER transcript was detected in oocytes from secondary and antral follicles (Fig. 3, J–P); it was unclear whether it was present at the primary stage (not shown). Only nonlocalized background staining was visible with the corresponding sense probes.
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Expression in Oocytes and Preimplantation Embryos
Expression of MATER, ZAR1, GDF9, and BMP15 was analyzed by RT-PCR during oocyte in vitro maturation and preimplantation embryo development (Fig. 4). Primers were as above except for GDF9 (GDF9-sense2 and antisense2). Transcripts were detected in oocytes before as well as after in vitro maturation. MATER, ZAR1, BMP15, and GDF9 transcripts were detected until the five- to eight-cell stage or at trace levels in morulae. No product was amplified from blastocysts. By contrast, our ß-actin control was detected in all samples and, in fact, its transcription was seen to resume between the eight-cell and morula stages, coincident with the maternal-to-embryo transition (MET). Exogenous rabbit globin was amplified from all samples.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Analysis of BovineMATERSequence
The bovine 4-kb cDNA detected by virtual Northern blot has a similar size as the 4.2-kb human transcript and the 3.75-kb mouse transcript observed by Northern blot [20, 21]. In rat, the coding sequence itself is over 6.3 kb, as predicted by automated computational analysis (accession XM_218237). In bovine, the longest ORF is 3297 nucleotides long and encodes a putative protein of 1098 aa with a predicted molecular mass of 121 kDa (Fig. 1). Thus, the bovine coding sequence and protein are similar to their mouse counterparts (3336 nucleotides and 1111 aa long), but shorter than in human (3603 nucleotides and 1200 aa long). Sequence alignment reveals that this discrepancy is mostly attributable to deletion at the 5' end. Compared with the human, the bovine cDNA lacks exon 1, beginning of exon 2, most of exon 4, and exons 5 and 6 (Fig. 1B); the latter two exons encode identical peptide sequences also missing in mouse. In this region, the human end of exon 2 and exon 3 are remarkable exceptions because they are well conserved in bovine. Conservation is strong in the rest of the cDNA, starting at human exon 7, bovine nucleotides 377–3297. Global alignment scores in the coding region are 70% and 57% with human and mouse, respectively. At the protein level, respective levels of identity with human and mouse are 52% and 41% (similarity 66% and 60%). Divergence at the N-terminus, resulting from the above-mentioned deletions in the cDNA 5' region, is also observed between human and mouse. Despite these deletions, all three domain characteristics of the NALP family [29] are indeed retained in the protein: an N-terminal DAPIN domain, followed by a NACHT domain and 12 C-terminal LRR-RI repeats (Fig. 1D). These characteristics support the identification of this cDNA as MATER rather than a homologue of mouse Mater2 cDNA discovered during the course of this study: Mater2 transcript is shorter (1878 bp) and the ORF (269 amino acids) does not encode a leucine-rich region [30]. Within the bovine 3'UTR, the AAUAAA consensus polyadenylation signal is present, 22 bp upstream of the polyA tail. In relation to the essential role of Mater in the early embryo, we also looked for a potential cytoplasmic polyadenylation element. No canonical signal could be distinguished within the 3'UTR, neither for polyadenylation during oocyte maturation ((A)U3(U/A)(U)A(A)(U/A)) nor after fertilization (Un>12). Therefore, maturational or embryonic posttranscriptional regulation of Mater messenger RNA cannot be excluded but is unlikely.
Expression Pattern in Adult Tissues
We analyzed expression of five selected genes in bovine adult gonadic and somatic tissues. Altogether, our results indicate preferential expression in male and female gonads, but mostly spectacular upregulation of MATER, ZAR1, GDF9, and BMP15 in the oocyte, as evidenced by the intense band observed even with an estimated 3 log ratio of substrate for the PCR.
VASA
VASA was strongly expressed in the testis and the ovary and was also detected in oocytes isolated from antral follicles. At first sight, our results contrast with previous studies in mice and human, where the transcript was only detected in adult testis but not ovary by Northern blot [7, 8]. However, it is possible that VASA is expressed in the ovary below the detection level by Northern blotting. In the human study, the authors themselves argue that too few oocytes may be represented in their RNA sample to permit detection. In fact, immunostaining detected the protein in mouse oocytes from primordial until early antral follicle, and in human oocytes up to the antral follicle stage [7]. VASA has been characterized as a marker of primordial germ cells and as such is usually studied in the early stages of germ cell differentiation. Detection of the transcript in oocytes from antral follicles is the first evidence of persistence of this transcript at such a late stage in mammals. Whether it is merely residual and awaiting degradation or to support later translation during maturation or pre-MET development deserves future experimental investigation.
MATER, ZAR1
By RT-PCR, in addition to the oocyte itself, ZAR1 transcript was detected in the ovary and in the testis. In human, expression is higher in the testis whereas the opposite is true in mouse [19, 22]. Within the ovary, oocyte restricted expression was confirmed by in situ hybridization. As expected from the RT-PCR data, ZAR1 transcript was present in oocytes from antral follicles (Fig. 3, G–I). In addition, in situ hybridization revealed that ZAR1 was indeed expressed at earlier stages, in primary and secondary follicles (Fig. 3, A–F). This pattern parallels that in mouse, where the transcript is first observed by RNase protection in type 3a follicles, is present at high level in growing oocytes, and persists in type 8 follicles [19]. By RT-PCR, MATER could not be detected in any tissue except for the testis and the oocyte itself. Additional shorter fragments detected in the testis might be derived from transcription of a distinct but related gene arisen by duplication, as was recently found in mouse [30]. MATER transcript had previously been detected in ovaries of mouse and young women by Northern blot or RT-PCR [20, 21]. Below detection level in the bovine ovary might reflect a lower copy number and/or a later stage of transcription as compared with ZAR1, with too few oocytes of the corresponding stage represented in our ovary sample. Although in situ hybridization is not a quantitative method, our results tend to support this hypothesis: oocyte staining in the antral follicle was more intense than in the secondary follicle (Fig. 3, K–P). On the other hand, in human ovary, the transcript was detected in what appears to be growing oocytes, based on the oocyte diameter (however, no indication of follicle stage is given by the authors) [21]. In mouse, the transcript is first observed in type 2 primary follicles, accumulates during oocyte growth up until type 5 follicles, and then decreases [31]. Thus MATER might be transcribed later in bovine than in mouse or human, a timing still compatible with its role in embryo development rather than oogenesis or folliculogenesis, as evidenced using knockout mouse [18].
GDF9, BMP15
Gdf9 was first reported as expressed solely in the oocyte [10, 32], but was later detected not only in both female and male gonads but also in nongonadic tissues including mouse hypothalamus, human pituitary, uterus, and bone marrow, and ewe pituitary [33, 34]. In agreement with such preferential gonadic expression, we detected GDF9 specifically in the testis and the ovary, and in none of the somatic tissues tested, including the uterus. In bovine, it was previously shown to be expressed in oocytes from primordial follicles onward and persisted after in vitro maturation [17, 35, 36]. We detected BMP15 in testis and ovary, similar to a previous report in human based on virtual Northern blot [14]. In mouse, it is expressed in the ovary and at a low level in the pituitary [11, 37].
While oocyte preferential expression of both bone morphogenetic proteins family members is common to all mammals studied so far, their relative functional importance varies between species. Gdf9 knockout female mice are infertile due to a block in folliculogenesis at the primary follicle stage, while Bmp15 null females exhibit only subfertility with decreased ovulation and fertilization rates [12, 38]. By contrast, in sheep and potentially in other mammals with a low ovulation rate phenotype, both genes are essential for normal early and late ovarian follicular development [39]. In addition to this functional difference, there are differences between species in the timing of appearance of the transcripts in the course of folliculogenesis. In mice, Gdf9 and Bmp15 transcripts were first detected beginning at the primary follicle stage, whereas in ovine and bovine ovaries, GDF9 was expressed even earlier in primordial follicles [10, 11, 35]. Our detection in the ovary supports an earlier expression compared with Mater, as discussed above.
Expression in Oocytes and Preimplantation Embryos
MATER, ZAR1, BMP15, and GDF9 transcripts were present in immature oocytes from antral follicles, after in vitro maturation, and after in vitro fertilization in embryos from one-cell up to five-/eight-cell or morula stage (at trace level). In the blastocyst, it was below detection level in our experimental conditions. GDF9 data are in agreement with a previous report [17]. Recently, BMP15 was identified in two-cell bovine embryos and potentially overrepresented in fast-cleaving versus slow-cleaving embryos [40]. Our data are particularly interesting in view of a recent study of global expression evolution during mouse preimplantation development [3]. In this article, genes were classified into clusters based on evolution of the transcript abundance in the embryo: Zar1 and Gdf9 belonged to the same cluster exhibiting abrupt decrease from zygote to eight-cell, while Mater displayed a slower decrease from unfertilized egg to blastocyst. Bmp15 appeared in a distinct category and showed temporary and weak reactivation between the four- and eight-cell stages. In line with this classification, Zar1 could be amplified by RT-PCR from mouse one- and two-cell but not later stage embryos [19]. BMP15 was deduced from subtractive suppressive hybridization to decrease between oocytes and eight-cell embryos [4]. A failure to detect Mater transcript in mouse preimplantation embryos by RNase protection may be attributable to a lower sensitivity of this technique; the protein was present at all stages through blastocyst [31]. Quantitative data by real-time PCR will be necessary to analyze whether these maternal transcripts follow parallel relative evolutions in the bovine as in the mouse. Finally, our temporal expression profile shows that transcription was clearly not reactivated at the time of bovine maternal to embryonic transition, i.e., 8/16 cell, in contrast with our ß-actin control. Because embryonic genome activation is usually described as a rather promiscuous phenomenon, these four oocyte-specific transcripts indeed follow a singular pattern.
We have demonstrated preferential expression in bovine oocytes of four genes, GDF9, BMP15, MATER, and ZAR1, the messengers of which persist in early embryos but the transcription of which is not reactivated at maternal-to-embryonic transition. Two of them, MATER and ZAR1, were previously unreported in this species. Their tissue distribution and temporal pattern of expression support a similar role in early embryonic development as in mouse. MATER and ZAR1 are indeed the first germ cell-specific maternal-effect genes identified in bovine.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Rozenn Dalbiès-Tran, INRA-PRC F-37380 Nouzilly, France. FAX: 33 247 42 77 43; dalbies{at}tours.inra.fr
Received: 29 March 2004.
First decision: 20 April 2004.
Accepted: 2 June 2004.
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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