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Biology of Reproduction 67, 953-960 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Identification of Genes Encoding Mouse Oocyte Secretory and Transmembrane Proteins by a Signal Sequence Trap1

Robert A. Tafta, James M. Denegrea, Frank L. Pendolaa, and John J. Eppig2,a

a Jackson Laboratory, Bar Harbor, Maine 04609


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The oocyte plays a key role in follicular development. At all stages of follicular development, oocytes interact with surrounding granulosa cells and promote their differentiation into the types of cells that support further oocyte growth and developmental competence. These interactions suggest the existence of an oocyte-granulosa cell regulatory loop that includes both secreted proteins and cell surface receptors on both cell types. Factors involved in the regulatory loop will therefore contain a signal sequence, which can be used to identify them through a signal sequence trap (SST). A screen of an oocyte SST library identified three classes of oocyte-expressed sequences: known mouse genes, sequences homologous to known mammalian genes, and novel sequences of unknown function. Many of the recovered genes may have roles in the oocyte-granulosa cell regulatory loop. For several of the known mouse genes, new roles in follicular development are implied by identification of their expression, for the first time, in the oocyte. The future characterization of novel sequences may lead to the identification of novel proteins participating in the regulatory loop.

developmental biology, gamete biology, gametogenesis, oocyte, ovum


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The oocyte is an active participant in ovarian follicular development; it orchestrates and coordinates the development of follicles. The rate of follicular development is based on a developmental program intrinsic to the oocyte [1]. Oocytes affect granulosa cell differentiation, steroidogenesis, and proliferation (see [2] for review). Nevertheless, the function of the oocyte's companion granulosa cells is important for oocyte growth and development. Thus, an oocyte-granulosa cell regulatory loop is essential for the development of both the oocyte and somatic cell follicular components. A major challenge for the future is to identify the key molecules and pathways involved in this loop.

Communication between oocytes and companion granulosa cells occurs through both gap junctions (membrane specializations that allow exchange of low-molecular-weight molecules) and secreted paracrine signals. Gap junctions provide the pathway for metabolic cooperation and signaling by low molecular weight compounds, and their function is key for the development of both oocytes and their companion somatic cells [3–6]. Paracrine signals, which are too large for transmission through gap junctions, are secreted into the extracellular space and bind transmembrane receptors on target cells. Proteins also may be integrated into the plasma membrane of the originating cell and signal by direct contact with receptors on the target cell. Included among the known paracrine factors secreted by oocytes are growth differentiation factor (GDF)-9 [7–10], bone morphogenetic protein (BMP)-15 [11–14], and BMP-6 [15]. In addition, Oosp1, encoding an oocyte-secreted protein (OOSP1), is expressed by oocytes in a developmental pattern similar to that of Gdf9 and Bmp15, but its function is unknown [16]. Granulosa cells express kit ligand (KL) [17–20], whose cognate receptor KIT is expressed by the oocyte [17, 21, 22]. Thus, GDF-9, BMP-15, KL, KIT, and possibly BMP-6 and OOSP1 are among the very few known components of the oocyte-granulosa cell regulatory loop despite the strong probability that many more components exist [23].

Elements of the regulatory loop could be identified as a class of secretory (paracrine factors) and membrane-bound and/or transmembrane proteins (receptors) that have in common a short peptide sequence known as a signal sequence. Signal sequences are usually found at the amino terminus of the protein and are heterogeneous in their amino acid/nucleotide composition [24], making it difficult to identify secreted/transmembrane proteins based solely on sequence analysis.

Signal sequences exhibit specific characteristics enabling them to target a protein to the endoplasmic reticulum, leading to the protein being secreted or integrated into the plasma membrane. Furthermore, they are interchangeable between proteins and organisms. This property makes it possible to identify signal sequences using a protocol based on their function rather than their sequence. This protocol is called a signal sequence trap (SST) and is used to identify secreted and transmembrane proteins based on the ability of a cloned cDNA to drive secretion of a selection gene in a genetically modified host cell.

Here, we describe the application of a yeast-based SST, as described by Jacobs et al. [25, 26], to identify genes encoding oocyte-secreted and transmembrane proteins with potential roles in the oocyte-granulosa cell regulatory loop. Use of this protocol greatly enhanced the identification of these genes expressed in the ovary only by the oocyte. The SST revealed expression in mouse oocytes of previously unknown genes and genes that were previously described but not known to be expressed in oocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
SST Vector

The SST vector, pSUC2T7M13ORI (pSUC2), is a disabled yeast plasmid designed to allow for selection of cDNAs with a 5' functional domain containing a signal sequence [25, 26]. Upon cloning of cDNAs containing a signal sequence, the vector, which carries a defective invertase gene, is repaired, thus allowing for the synthesis and secretion of invertase. pSUC2- strains of yeast, such as YTK12, that have plasmids with cDNAs lacking a signal sequence are not capable of secreting recombinant invertase. Consequently, they are selected against by growth inhibition because of their inability to metabolize sucrose or raffinose. Yeast that are capable of secreting recombinant invertase as a result of being transformed with the functional SST plasmid will grow, allowing for the identification of secreted and transmembrane proteins. A flow chart for the SST strategy is presented in Figure 1.



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  		FIG. 1. Flow chart of the essential steps in the SST strategy

Library Construction

Approximately 18 000 oocytes were collected from C57BL/6J x SJL/J F1 mice. Oocytes were collected on Day 12 to obtain growing oocytes from preantral follicles and on Day 22 to obtain fully grown germinal vesicle (GV) stage oocytes. Approximately twice as many Day 12 as Day 22 oocytes were collected to compensate for the difference in oocyte volume between these two stages of development. Oocytes from both stages were then pooled. The Fast Track kit (Invitrogen, San Diego, CA), modified as described previously [27], was used to isolate mRNA, after which the protocol of Jacobs et al. [25, 26] was followed to generate and screen an oocyte library for secreted proteins. First- and second-strand syntheses were carried out using the Superscript Choice System (Invitrogen) for cDNA. Diversity of selected cDNA species was generated using random nonamers with a flanking XhoI site. These random nonamers reduced the 3' bias and the likelihood of capturing full-length sequences while enriching the selection of the 5' functional domain of the transcripts, which would include putative signal sequences. Following cDNA synthesis, custom adapters containing an EcoRI site were ligated to the cDNA, phosphorylated, and digested with XhoI, yielding cDNA with XhoI and EcoRI cohesive ends. The cDNAs were size-fractionated by column chromatography. Appropriate fractions (200–800 base pairs) were pooled and directionally cloned into the SST vector. The library was amplified by transformation into Escherichia coli (ElectroMAX DH10B; Invitrogen), grown to 1 L, aliquoted, and stored at -80°C.

Transformation and Signal Sequence Trapping

Ten micrograms of plasmid library was used to transform yeast strain YTK12 by the lithium acetate method [28]. Transformed yeast were plated onto complete minimal medium lacking tryptophan and containing sucrose (2%) and dextrose (0.1%, CMS/loD-W). Cultures were allowed to grow for 3 days and then replica plated to raffinose and antimycin A (YPRAA) medium, simulating anaerobic conditions. Replicas were grown for 7 days, then single colonies were streaked onto YPRAA plates and grown for 7 days. Individual colonies were transferred to wells of a deep-well microtiter plate containing complete minimal medium with dextrose but lacking tryptophan (CMD-W) and grown overnight. Plasmids were prepared by lysis of 3 µl of overnight cultures in 10 µl zymolase (ICN T20; ICN Pharmaceuticals, Costa Mesa, CA) in 1.2 M sorbitol, 0.1 M sodium phosphate at 37°C for 1 h followed by 95°C for 5 min. Remaining overnight cultures were archived at -80°C. Plasmid inserts were amplified by polymerase chain reaction (PCR) using 3 µl of zymolase reaction in 30-µl reactions, using primers as previously described [26]. Sizes of PCR products were determined on agarose gels to confirm the presence of an insert.

Sequence Analysis

Purified PCR products (Qiaquick; Qiagen, Valencia, CA) were sequenced using the primer from the 3' side, and sequences were compared with those in GenBank utilizing National Center for Biotechnology Information (NCBI) BLAST search algorithms (BLASTN and BLASTX). Clones with a high degree of homology to known sequences were classified according to the expected location of the protein they encoded (i.e., secretory, membrane, nuclear, or mitochondrial).

Differential Screening

PCR products were spotted in replicate 96-well patterns on nitrocellulose membranes, denatured, and hybridized against random-primed oocyte or granulosa cell cDNAs. Complementary DNAs were made by the SMART cDNA method (Clontech, Palo Alto, CA). Those PCR products that hybridized exclusively to oocyte cDNA were selected for further analysis.

Effectiveness of Trapping

To determine whether the SST was an effective approach for identifying secreted and transmembrane proteins, the number of genes and the proportion of secreted/transmembrane proteins were compared before and after trapping. One hundred sequences were obtained from bacteria transformed with plasmid from the oocyte library described above and were considered to represent the unselected pool of sequences. The number of unique sequences obtained and the proportion of sequences representing secreted/transmembrane proteins were then compared with those values obtained from yeast following trapping (100 sequences).

In Situ Hybridization

SST clones chosen for further characterization were digested with EcoRI and XhoI to remove primer sequences and then subcloned into the transcription vector pBS SK (Stratagene, La Jolla, CA). Riboprobes were made from the appropriate promoter using 33P CTP. In situ hybridization was carried out as described previously [29] using 12- and 22-day-old mouse ovaries.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Final selection on antimycin A medium yielded approximately 1400 colonies, 543 of which had inserts long enough (>50–60 bases) to yield useful information following sequencing. A total of 129 different sequences were identified. Homologies to known sequences were determined by using the NCBI BLAST search algorithms. Criteria for homology were a BLAST score of >=200, using a minimum of 150 bases of sequence.

Range of Trapped Sequences

Of the unique sequences, 76 clones had a high degree of homology to previously characterized mammalian genes, and 28 sequences were determined to be novel, i.e., demonstrating little or no homology with sequences of known function. Of the previously characterized genes, approximately 75% encoded proteins known to be secreted or expected to have signal sequences. The other 25% represented false positives, i.e., genes not expected to be secreted. This percentage of false positives is similar to the rate reported previously for this method [25]. Genes for all classes of proteins with an expected signal sequence were recovered: secreted proteins, transmembrane proteins, membrane-bound proteins, and endoplasmic reticulum proteins. All of the unique sequences recovered by SST were organized by organism, homology, and subcellular location (Tables 1–3).


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  		TABLE 1. Mouse genes with known function or subcellular localization (false positives not listed)

Those sequences homologous to known mouse genes are listed in Table 1 and are categorized according to their subcellular location. Of the 11 genes identified encoding secreted proteins, 4 have already been demonstrated to be expressed by the oocyte: Plat, Zp2, Gdf9, and Bmp6 [7, 14, 30, 31]. Absent from the recovered genes are Zp1 and Zp3, zona pellucida genes that are coordinately expressed with Zp2 [31]. Also absent is another known oocyte paracrine factor, BMP-15. However the SST did identify the crumbs homology 1 gene (Crb1), a predicted paracrine factor in other cell types [32, 33]. This is the first report of Crb1 expression in oocytes.

Sequences encoding transmembrane and membrane proteins, which are likely involved in cell-cell signaling and signal transduction, were also trapped. For example, the receptor gene Plxnb1 (plexin B1) was identified; however the oocyte-expressed receptor gene Kit has yet to be recovered. The gene encoding the cell-signaling and extracellular matrix assembly protein syndecan 2 (Sdc2) was identified, as was Pkd2l2 (polycystic kidney disease 2L2), an ion channel member. Ncstn (nicastrin), Cd63 (tetraspanin), Inpp4a (inositol polyphosphate 4 phosphatase), and Gpiap1 (GPI-anchored membrane protein 1), all genes encoding transmembrane proteins participating in signal transduction [34–37], were also trapped.

Sequences with matches to only human or rat genes are listed in Table 2, also categorized according to subcellular location. These sequences presumably encode mouse homologues that have not been previously identified. Of particular interest are the genes encoding the Cdc42 receptor IQGAP [38] and chimaerin, a phorbol ester receptor with racGAP activity [39].


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  		TABLE 2. Mouse oocyte SST sequences with homologies to human and rat genes with known subcellular localization

Novel genes, meaning sequences with functions yet to be assigned, were also recovered (Table 3) and are listed based on homology to mouse or human sequences and/or expressed sequence tags (ESTs) in the mammalian databases. Sequences are indicated as homologous when the BLAST score was 200 or greater. Scores <200 were not considered a match to reported ESTs are were listed numerically. Because these sequences do not match any known ESTs, they are of particular interest as potential regulatory loop factors. Novel sequences account for 40% of all the genes recovered by the SST screen, an indication of the value of SST for identifying previously unknown genes.


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  		TABLE 3. Novel sequences (no known function)

Effectiveness of Trapping

To determine the effectiveness of trapping, the proportion of known transcripts encoding secreted and transmembrane proteins was compared before and after trapping. Similar numbers of unique sequences were identified from bacteria transformed with plasmids from the SST library (34 of 100 random colonies) and from sequences obtained after signal sequence trapping (32 of 100). In the unselected group, only 13% of the known sequences encoded secreted/transmembrane proteins, whereas 66% were classified as such after trapping, a 5-fold improvement. These results demonstrate that the SST dramatically enriches searches for sequences encoding oocyte secreted and transmembrane proteins.

Oocyte-Specific Expression

The localization of expression of trapped sequences was determined by differential hybridization against oocyte and granulosa cell cDNAs (data not shown) and in situ hybridization. Expression in the ovary was exclusive to the oocyte for approximately 50% of the sequences characterized. Some genes encoding signal sequence-containing proteins were expressed by both granulosa cells and oocytes. As detected by in situ hybridization (data not shown), both synaptogamin-like 4 (Sytl4) and chimaerin (CHN1) are expressed by oocytes of preantral and antral follicles and by their companion granulosa cells. This pattern of expression was expected because some proteins are secreted by multiple cell types.

Examples of oocyte-specific expression, as detected by in situ hybridization, are shown in Figure 2 for genes encoding three classes of signal sequence-containing proteins: secreted, transmembrane, and membrane-anchored proteins. These genes include Zp2, Crb1, Pkd2l2, and Gpiap1; this is the first report of expression of the latter three genes in oocytes.



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  		FIG. 2. Gene expression of SST-recovered clones by in situ hybridization against Day 22 mouse ovaries. Note oocytes of preantral follicles (arrowheads) and antral follicles (arrows). Zp2, Secreted extracellular matrix protein; Crb1, predicted paracrine factor CRUMBS; Pkd2l2, transmembrane ion channel member; Gpiap1, GPI-anchored protein. Bar = 100 µm

ZP2 is an oocyte-secreted protein of the zona pellucida, the extracellular matrix surrounding the oocyte. ZP2 is highly expressed by and specific to the oocyte [31]. As expected, the Zp2 sequence recovered by SST demonstrates strong localization specific to the oocyte, as seen for antral and preantral follicles in Figure 2.

Mouse Crb1 is an orthologue of human CRB1 and Drosophila crb, which codes for the crumbs protein [33]. Human CRUMBS protein is a predicted paracrine factor, establishing and maintaining cellular polarities. Crb1 expression in the ovary is restricted to the oocytes in both preantral and antral follicles (Fig. 2). PKD2L2 is a member of the polycystin family, integral membrane proteins forming cation channels [40]. Pkd2l2 transcripts are synthesized in the ovary by only the oocyte (Fig. 2). Glycosylphosphatidylinositol (GPI)-anchored proteins, as a class, are mainly involved in signal transduction via kinases, G-proteins, and immunoreceptors [41]. GPI-anchored protein p137 cycles between the cell surface and the cytoplasm. Expression of Gpiap1 is exclusive to the oocyte in both preantral and antral follicles (Fig. 2).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The complexity of the oocyte-granulosa cell regulatory loop is now being realized, with many elements still to be identified. Many components of the regulatory loop will contain signal sequences, allowing them to be identified by an SST approach. Versions of SSTs have been developed and tested using mammalian or yeast vectors [25, 42–44]. Most traps utilizing mammalian systems are not useful for targeting oocyte-specific sequences because of the requirement for expression of trapped genes in established cell lines. The SST of Jacobs et al. [25], a yeast-based method to screen inserted cDNA clones for putative signal sequences, was therefore used as a functional test of genes expressed in the oocyte. This method was used successfully for the recovery of all classes of secretory and transmembrane proteins, many of which are oocyte specific within the ovary.

Three categories of sequences were recovered: sequences representing known mouse genes (Table 1), sequences homologous only to known human or rat genes (Table 2), and novel mouse or human sequences of unknown function (Table 3). Among the known genes, many with potential relevance to the oocyte-granulosa cell regulatory loop were identified based on their function in cell signaling. These include Crb1, encoding a predicted paracrine factor, and the receptor genes Plxnb1 and Sdc2 and the CHN1 orthologue. Two others encode proteins that process receptor signaling at the membrane: Ncstn (nicastrin), which regulates proteolytic cleavage of NOTCH [34], and Ranbp9 [36, 45]. The product of one gene, Cd63 (tetraspanin) transduces signals through membrane structures and the cytoskeleton [35, 46]. Analysis of gene expression for several types of sequences has more directly implied roles in the regulatory loop. We report here for the first time that Crb1, Gpiap1, and Pkd2l2 are, within the ovary, all expressed specifically in the oocyte. Equally important are the novel sequences that have been recovered, because of the high potential for uncovering novel proteins with roles in the regulatory loop. The novel sequences are expected to consist of a similar variety of genes for secretory and transmembrane proteins.

Effectiveness of the SST

Use of this yeast-based SST resulted in a 5-fold increase in the selection of cDNAs encoding authentic secreted/transmembrane proteins. However, not all proteins with a signal sequence are of interest for this screen. Proteins destined for the endoplasmic reticulum will also carry a signal sequence and are not expected to play roles in oocyte-granulosa cell communication. A 5-fold enrichment in selection, in conjunction with rapid sequencing and database searching, quickly limits the data to a productive subset of clones.

Not all the clones recovered by signal sequence trapping have signal sequences. For example, mitochondrial and nuclear proteins may have transmembrane sequences that behave as a signal sequence in yeast. For this SST screen, the false-positive rate was 25%, which is the same rate as that originally described for this vector using human activated peripheral blood mononuclear cells as a source of cDNA [25]. Other yeast vectors have reported rates of 17% and 20% [44, 47]. These rates are lower than those described for mammalian systems. False positives are recovered at rates of up to 50% for the ES cell lines or the COS cell lines, using vectors with different reporter genes [42, 43]. All SSTs reported, whether by gene trapping or cDNA screening, demonstrate enrichment, but not a clear-cut selection, for signal sequences. From these data and from genomic analysis of signal sequences [24], it is clear that the minimum requirements for a signal sequence can, in general, be promiscuous. Achieving a lower rate of false positives may not be practical or may require a combination of techniques.

It is not possible to assess accurately the rate of false negatives. Not all of the known genes encoding oocyte-secreted or transmembrane proteins were detected by this technique; notably missing were Zp1, Zp3, Bmp15, and Kit. All of these genes are oocyte specific and have relatively high levels of expression. Genes encoding other secreted proteins also may have been missed. False negatives could arise for a number of reasons: 1) the sequence is not present in the library because of primer or extension failure, 2) the protein signal sequence is not recognized by yeast, 3) biological reasons, i.e., the yeast expressing these proteins did not grow well or expression of the proteins caused yeast lethality, or 4) analysis of the trapped library has not been saturated. Continued screening of the library might reveal these genes as well as new, unexpected sequences.

Novel Expression Patterns for Known Genes

Many of the known genes uncovered by the SST have not been previously identified as expressed in the oocyte and were not expected as targets of the trap. Reported functions for some of these genes have no immediate analogy in oocyte biology. The discovery that their transcripts are highly localized to the oocyte within the ovary suggests that the genes may play a role in follicular development and implicates new classes of cell signaling molecules in the oocyte-granulosa cell regulatory loop.

Of particular interest is Crb1, a predicted paracrine factor and orthologue of Drosophila crb and human CRB1 [33]. Drosophila crumbs protein is an important factor in establishing and maintaining cellular polarity through interactions with the cytoskeleton [48, 49]. Human CRB1 is highly conserved relative to Drosophila crb yet has splice variants that lead to fundamentally different molecules. One transcript splice variant encoded by CRB1 is reduced in the transmembrane domain, and the protein is probably secreted. The other splice variant includes the cytoplasmic domain and the transmembrane region and is functionally identical to Drosophila crumbs. Human CRB1 is expressed in the retina and brain, whereas mouse Crb1 is present in the eye and central nervous system [32, 33]. Mutations of CRB1 lead to retinal dystrophies [50]. Mouse and human CRUMBS proteins presumably establish or maintain the highly polar epithelium of the retina, and Drosophila crumbs protein plays a similar role in photoreceptor cells [51, 52]

Although crumbs protein is critical to follicular epithelial formation in the Drosophila ovary, it is also present in the membrane of the Drosophila oocyte [53]. However, crumbs is not required for oocyte development nor is it known whether oocyte crumbs is a nurse cell derivative or an oocyte product [54]. We demonstrated the presence of Crb1 transcripts in the mouse ovary, and these transcripts were restricted to the oocyte. This finding was unexpected because there is no strong evidence for functional polarity in the immature mouse oocyte [55, 56]. A possible role for Crb1 in the mouse ovary is the organization of the granulosa cells in the developing follicle. Granulosa cells can be seen as analogous to the follicular epithelium in the Drosophila ovary but lack the highly defined apical/basal polarity. However, the granulosa cells do have distinct morphologies that are developmentally regulated and may be under the control of the oocyte. CRUMBS protein is a likely candidate for an element of the oocyte-granulosa cell regulatory loop.

Proteins of the polycystin (PKD) family interact to form calcium-permeable channels and are presumed ion channel regulators [40, 57]. These genes are important to proper functioning of the kidney; mutations result in polycystic kidney disease. Because no other functions for polycystin family members has appeared, a first assumption would be that PKD2L2 protein is also involved in calcium channel formation [58]. Such a function is anticipated because intracellular calcium plays important roles in oocyte and egg biology. Intracellular calcium is released during germinal vesicle breakdown as meiosis resumes [59] and at fertilization as the sperm and egg membranes fuse [60]. The regulation of intracellular free calcium is important for maintaining cell viability in general. Physiological studies indicate that calcium channels and exchangers operate in the oocyte [61], but specific channels, or even channel components, have not been identified. The finding of a calcium channel protein in the oocyte is important because it suggests that the polycystin family of ion channels may play a role in calcium events during oocyte development.

Based on phospholipase biochemistry, the presence of GPI-anchored proteins on the oocyte surface has been inferred [62, 63]. Here, we report for the first time identification of the protein moiety for a GPI-anchored protein in the oocyte. GPI-anchored proteins are organized in microdomains of the plasma membrane (rafts) and are usually involved in cell signaling [41]. The human homologue to GPIAP1 is present in the polarized intestinal cell line CaCo-2. It transcytoses bidirectionally, and the protein function has not yet been determined. Its presence in the oocyte is completely unexpected, and its function is not at all apparent. The identification of a GPI-anchored protein in the oocyte leads to the expectation that other members of this class of proteins may also be present.

Conclusions

The SST approach appears to be more efficient than other genomic approaches for identifying genes encoding oocyte-secreted proteins. For example, a catalog of human oocyte-specific genes created by serial amplification of gene expression (SAGE) included only an overlapping subset of secreted/transmembrane sequences homologous to the mouse sequences reported here [64–66]. Although SAGE analysis revealed the presence of GDF-9 and ZP2 in human oocytes, it failed to identify ZP2, CRB-1, BMP-6, PKD2L2, granuphilin, tPA, and hyaluronidase. More receptor-encoding genes, presumably containing signal sequences, were identified by SAGE than by the SST. The larger size of transcripts for receptors, compared with secreted proteins, may bias cDNA synthesis in favor of the secreted protein sequences. Thus, the assembly of a complete catalog of oocyte-expressed genes encoding secreted or transmembrane proteins will require application of multiple gene discovery modalities. Nevertheless, this yeast-based SST screen was used successfully to identify several known and novel oocyte-expressed genes that may be essential components of the oocyte-granulosa cell regulatory loop.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Kenneth A. Jacobs and the Women's Health Research Institute of Wyeth Research for providing reagents needed for the SST. We also thank Drs. Janan Eppig and Martin Matzuk for their helpful suggestions in the preparation of this report and Dr. Verity Letts for giving us the benefit of her knowledge of yeast genetics and for the care and nurturing of yeast.


   FOOTNOTES
 
First decision: 5 April 2002.

1 This research was supported by grant HD23839 (J.J.E.) from the NICHD. R.A.T. was supported by NIH NRSA grant F32 HD08730. Scientific Resources at the Jackson Laboratory are supported in part by a Cancer Center Core grant (CA34194) from the National Cancer Institute. Back

2 Correspondence. FAX: 207 288 6073; jje{at}jax.org Back

3 These authors contributed equally Back

Accepted: April 17, 2002.

Received: March 13, 2002.


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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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