Developmental Endocrinology Branch, NICHD, National Institutes of Health, Bethesda, MD 20892, USA
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Abstract |
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Key words: embryo/infertility/MATER/oocyte/premature ovarian failure
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Introduction |
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It is known that maternal factors govern early embryonic development before embryonic genome activation takes place (Latham, 1999; Latham and Schultz, 2001
) The maternal effect on early embryogenesis of Drosophila and Xenopus has been demonstrated by the identification of a number of maternal effect genes (Akam, 1987
; Droin, 1992
). However, the presence and importance of maternal effect genes in early mammalian development has been only inferred until the recent identification of maternal effect genes in mice, such as Mater (Tong et al., 2000a
) and Hsf1 (Christians et al., 2000
). In addition, a maternal effect in early mammalian embryogenesis has been demonstrated recently for some well-known proteins such as zona pellucida proteins (Rankin et al., 2000
, 2001
) and E-cadherin (Larue et al., 1994
), although their effects are not restricted to embryonic development. To date, little is known about the mechanisms by which maternal products direct early development in mammals. In mice, oocytes are transcriptionally active during oogenesis. As oocytes undergo growth and maturation, maternal proteins accumulate within the oocytes (Schultz, 1993
). After fertilization, activation of the embryonic genome is an essential event in early development (Flach et al., 1982
; Conover et al., 1991
; Bellier et al., 1997
). In mice the zygotic genome activation begins in the late stage of 1-cell zygotes and becomes dominant at the 2-cell stage of embryos (Latham et al., 1991a
,b
; Bouniol et al., 1995
; Nothias et al., 1995
; Aoki et al., 1997
). Metabolic inhibition of embryonic transcription does not block the first embryonic cleavage, but arrests development at the 2-cell stage in mice (Warner and Versteegh, 1974
; Flach et al., 1982
). This suggests that maternal products direct development before the embryo undergoes genome activation, while the products derived from embryonic genome are required for embryonic progression beyond the 2-cell stage. Although the molecular basis for this programmatic transition is poorly understood, clearly the maternal products are required to constitute appropriate conditions for activation of the embryonic genome.
In this study, we characterize human MATER gene and its protein. MATER serves as an autoantigen in mouse autoimmune oophoritis (Tong and Nelson, 1999), a model for human autoimmune premature ovarian failure (Kalantaridou and Nelson, 1998
). We are on a path to determine whether human MATER plays an antigenic role in the autoimmune pathogenesis of clinical premature ovarian failure. In addition, MATER as a maternal factor supports early embryonic development in mice, through which it determines female fertility (Tong et al., 2000a
). This raises the possibility that a MATER gene mutation or MATER protein deficiency might cause infertility in some women with normal ovulatory function. Thus, characterization of human MATER gene and its protein provides a new determinant with which to investigate ovarian autoimmunity and unexplained infertility in women.
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Materials and methods |
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Determination of human MATER locus and exon-intron map
Using the sequence of cloned human MATER cDNA, one human genomic clone (GenBank accession: AC011470) was determined to carry an entire gene locus of human MATER. Nearly full-length human MATER cDNA sequence was blasted to the DNA sequence of this human genomic clone to determine the size and numbers of exons and introns. The AGexonGT rule was applied to determine a splicing site between exon and intron (Breathnach and Chambon, 1981).
Northern and Southern hybridizations
Human ovarian total RNA from a 19 year old woman was obtained under an IRB-approved protocol (McGill University, Montreal, Canada) and our use of it was approved by the NIH Office of Human Subjects Research. Ovarian RNA from a 26 year old woman was purchased from BioChain Inc. (Hayward, CA, USA). Separation of human ovarian total RNA (10 µg) was performed by 1% agarose/formaldehyde gel electrophoresis. The RNA was transferred onto a nitrocellulose membrane to prepare a Northern blot. The PCR products were separated by 2% agarose gel electrophoresis and transferred onto a nylon membrane to prepare a Southern blot. Human MATER cDNA (~0.5 kbp) was labelled with -[32P]dCTP (3000 Ci/mmol; ICN, Costa Mesa, CA, USA) using Ready-To-Go DNA dCTP Beads (Pharmacia, Inc., Piscataway, NJ, USA). Both RNA and DNA blots were hybridized (68°C, 2 h) with 32P-labelled cDNA probe (~0.5 kbp) in QuikHyb solution (Stratagene, La Jolla, CA, USA). After washing the blots with a solution of 0.1xstandard saline citrate/0.1% sodium dodecyl sulphate (SDS) at 65°C, the hybridization signals were detected by autoradiography.
PCR
Human ovarian cDNA (Invitrogen; Origene), human ovarian cDNA library (Clontech) and the Human Rapid-Scan Gene Expression Panel (Origene) were used as templates for PCR reactions per manufacturers' instructions. Taq DNA polymerase (Life Technologies, Rockville, MD, USA) was used to amplify DNA products expected within 1.5 kbp at a condition of the PCR reaction (95°C, 5 min; 35 cycles of 95°C, 1 min; 60°C, 1 min; 72°C, 2 min; and 72°C, 10 min for further extention). Platinum PCR SuperMix (Life Technologies) was used for amplification of DNA products (25 kbp) with prolonging extension time at 72°C in every cycle, according to the product's manual. The PCR products were electrophoresed on agarose gel containing 0.1% ethidium bromide and subcloned into a TA cloning vector (Invitrogen) for DNA sequencing.
In-situ hybridizations
Both [35S]UTP-labelled sense and antisense probes were synthesized by in-vitro transcription using human MATER cDNA as templates and T3/T7 RNA polymerase (Ambion, Inc., Austin, TX, USA). Short probes (~300 nt) of alkaline hydrolysis were hybridized at 60°C for 24 h with frozen sections (8 µm) of human ovary obtained from the National Disease Research Institute (NDRI; Philadelphia, PA, USA). After dipping in Kodak NTB-2 emulsion, the slides were exposed for 1 week on an X-ray film for developing in Kodak D-19 and Kodak Fixer. The slides were finally stained with haematoxylin and eosin, and examined by microscopy under both dark and light fields.
Immunohistochemistry and immunoblotting
After blocking with 10% normal goat sera in phosphate-buffered saline (PBS), frozen sections (8 µm) of human ovary (NDRI) were incubated at 25°C for 2 h with rabbit antisera (1:200) specific to mouse MATER peptide. The slides were washed with 0.05% Tween-20 in PBS, and incubated with a goat anti-rabbit IgG antibody conjugated with fluorescein isothiocyanate (1:300; Sigma Chemical Co., St Louis, MO, USA). After washing and applying with glycerolglass cover, the slides were examined under fluorescent microscopy. Human immature oocytes were supplied for this study by a clinical IVF programme (Invitrogen; Origene) after obtaining consent and approval of the NIH Office of Human Subjects Research. For immunoblotting, human oocyte proteins were separated on 38% Trisacetate SDSpolyacrylamide gel electrophoresis. After transferring onto a nitrocellulose membrane, 5% dry milk in PBS was used to block non-specific binding in the blot. Subsequently, the blot was incubated with rabbit anti-MATER peptide antibody (1:1000) for 2 h at 25°C. After the blot was washed, the primary antibody-bound proteins were detected by a goat anti-rabbit IgG antibody (1:1000) labelled with horse-radish peroxidase using an enhanced chemiluminescence system according to the manufacturer's instructions (Amersham/Pharmacia, Inc., Piscataway, NJ, USA).
DNA sequencing and computational analysis
DNA was sequenced with a dRhodamine terminator cycle sequencing kit (PE Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. Sequencing reactions were conducted by PCR at 25 cycles of 95°C, 10 s; 50°C, 5 s; and 60°C, 4 min. Electrophoresis with 5% Long Ranger gels and sequence analysis were carried out on the ABI Prism 377 DNA Sequencer. Computational analyses of DNA sequences and deduced peptide sequences were conducted using software available at the National Center for Biotechnology Information at NIH (http://www.ncbi.nlm.nih.gov), the EMBL-EBI (http://www.ensembl.org), the Sanger Center (http://www.sanger.ac.uk) and the ExPASy Molecular Biology Server of the Swiss Institute for Bioinformatics (http://www.expasy.ch).
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Results |
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Expression of human MATER protein in oocytes was demonstrated by indirect immunofluorescence. Using a primary antibody of rabbit anti-mouse MATER peptide, the oocytes present in the human ovarian sections were visualized by FITC-conjugated second antibody (Figure 2A). No fluorescense was observed in the other somatic cells within ovarian sections. Normal rabbit sera did not stain the oocytes (data not shown). To determine the size of native MATER protein, human immature oocytes were used for immunoblotting. As shown in Figure 2C
, human MATER protein (~134 kDa) was detected in the preparation of the human oocyte proteins using anti-mouse MATER antibody. It was noted that there were two close signals of MATER proteins present in the immunoblotting. This might reflect a post-translational modification of human MATER protein or a possibility of an alternative initiation site in the synthesis of the polypeptide chain.
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The PCR products were generated to span ~98% of the coding region from nt 76 to nt 3596. The 3' non-coding regions were determined by sequencing cDNA clones isolated from the human ovarian cDNA library using the PCR-cloned cDNA as probe. The first 75 nt at the 5' end were derived from a predicted cDNA of the genomic clone AC011470 (http://www.ensembl.org). The non-coding region present at the 5' end of the MATER cDNA remains undetermined. Assuming the presence of a poly(A) tail (~150200 nt) and the 5' undetermined non-coding regions (~100150 nt), assembly of our determined human MATER cDNA sequence, its poly(A) tail and 5' undetermined non-coding region corresponded in size with the MATER transcripts detected in the Northern hybridization (4.2 kb). This suggests that the cloned cDNA represents a full length of the coding region. As shown in Figure 3, the open reading frame (nt 1 to nt 3600) of the cloned cDNA (3885 nt) encodes a protein composed of 1200 amino acids (Figure 3
). The entire polypeptide chain was predicted to have a pI 6.08 and a molecular mass of 134 235.76 Da, which corresponded to the size of the human oocyte protein recognized by the antisera specific to mouse MATER peptide. Expression of a recombinant protein further confirmed the cloned cDNA to encode the human MATER protein (data not shown).
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Analysis of human MATER polypeptide chain
A computational analysis was carried out to search for the structural features of human MATER protein. When the entire polypeptide chain of human MATER was compared with itself, the Dotplot analysis revealed the presence of repeat structural motifs at amino- and carboxyl-termini. These repeats were evidenced by a series of parallel but offset lines in these regions (Figure 4A), as also seen in the mouse MATER protein (Tong et al., 2000b
). A comparison between the polypeptide chains of human and mouse MATER indicated the similarities and homologies of their structural motifs and compositions (Figure 4B
).
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Discussion |
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The requirement of MATER for early embryonic development in mice is an example of how important the role of maternal products can be in the developmental programme of mammals (Tong et al., 2000a). The role of MATER appears to be specific to very early development. In mice, Mater null mutation does not affect oogenesis, folliculogenesis, oocyte maturation and ovulation, or fertilization. Phenotypic infertility in the null female mice lacking MATER is attributable to an arrested development at the 2-cell stage. The arrested embryos demonstrate a considerable decrease of embryonic gene transcription. The resultant 2-cell block appears similar to observations of mouse embryos treated with
-amanitin, in which the metabolic blockage of embryonic transcription results in an arrest of embryonic development specific to the 2-cell stage (Warner and Versteegh, 1974
; Flach et al., 1982
). MATER consistently remains within the embryonic cytoplasm during mouse development to the blastcyst stage. The exact function of MATER remains to be determined. The presence of an ATP-/GTP-binding domain in both human and mouse MATER proteins suggests their participation in an intracellular signal transduction (Saraste et al., 1990
). Our preliminary data indicate that MATER may be truly involved in cellular signaling (Z.-B.Tong et al., unpublished observations). In addition to the carboxyl-terminal leucine-rich repeat domain similar to the mouse MATER (Tong et al., 2000b
), human MATER has an amino-terminal PAAD_DAPIN domain (Pawlowski et al., 2001
; Staub et al., 2001
). Both domains are recognized as structural motifs for mediating proteinprotein interactions (Kobe and Deisenhofer 1995
; Pawlowski et al., 2001
), raising a possibility that MATER may interact with other protein(s) within the embryos. Different from the mouse MATER, human MATER protein has an amino-terminal aldo_ket_red domain, a motif of proteins with oxidoreductase activity (Bohren et al., 1989
), and a signal for nuclear localization. Further experiments are required to determine the significance of these structural motifs.
Although little is known about maternal embryonic effect in humans, increasing evidence indicates that maternal factors present in human oocytes perform a critical role in biological events following fertilization (Barritt et al., 2001). Among infertile women seeking reproductive assistance in clinical IVF programmes, not all women reach a desirable outcome since the in-vitro fertilized oocytes of some women fail to undergo normal early embryonic development (Alikani et al., 1999
; Kovacs et al., 2001
). It is a possibility that the maternal factor(s) necessary for supporting early development is defective in the fertilized ova of some infertile women. Indeed, recent reports suggest that injection of normal oocyte proteins into the oocytes of some infertile women might restore normal early development and successful pregnancy (Cohen et al., 1997
; Barritt et al., 2001
). However, specific factors rescuing early development have not been identified. Mater null female mice are infertile because the absence of MATER protein in the fertilized ova leads to failure in embryonic development. We propose that a homozygous human MATER mutation or its protein deficiency may cause infertility in some women. To test this hypothesis, we plan to examine infertile women with a history of successful fertilization but yet failure in embryonic development for gene mutations of human MATER. If such a deficiency is found, normal embryonic development might be induced by injection of recombinant human MATER protein into the oocytes before fertilization. This might provide IVF programmes with a novel therapy to treat infertile women who have a homozygous MATER mutation and the resulting oocyte protein deficiency.
Cloning and characterization of human MATER might also lead to insights regarding the pathogenesis of human autoimmune premature ovarian failure. This ovarian disease is a clinical syndrome characterized by amenorrhoea, infertility and menopausal symptoms attributable to hypoestrogenaemia and hypergonadotrophinaemia in women before age 40 (Kalantaridou and Nelson, 2000). However, specific ovarian antigenic targets involved in autoimmune premature ovarian failure have yet to be identified (Hoek et al., 1997
). There are striking similarities between human autoimmune premature ovarian failure and the autoimmune oophoritis in an animal model, which is generated by neonatal thymectomy in certain strains of mice (Taguchi et al., 1980
; Taguchi and Nishizuka, 1980
; Kalantaridou and Nelson, 2000
). MATER was first identified as an oocyte-specific antigen associated with autoimmune oophoritis in this thymectomy mouse model (Tong and Nelson, 1999
). A recent report has shown that the endogenous oocyte antigen may be essential for development of auotimmune ovarian disease in this animal model (Alard et al., 2001
). We are developing an assay using human recombinant MATER protein to determine whether patients with premature ovarian failure have circulating anti-MATER antibodies. If the outcome is positive, such an assay would help physicians classify patients with premature ovarian failure, and thus provide a basis for developing therapy for the disease.
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Acknowledgements |
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Notes |
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References |
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Submitted on September 26, 2001; accepted on November 27, 2001.