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Ovary |
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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conditional knockout, Cre, folliculogenesis, gamete biology, gametogenesis, oocyte development, ovary, ovum, oogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Folliculogenesis is the development of the follicle, an essential functional unit of the ovary containing an oocyte and granulosa and thecal cells, from the primordial stage through the primary, preantral, antral, and preovulatory follicle stages [9]. Along with follicular development, oocytes also undergo growth in the postnatal ovary, giving rise to mature eggs [10, 11]. Oocyte-expressing genes are involved not only in folliculogenesis and oogenesis but also in preimplantation embryonic development [12–15]. There are many genes expressed in the oocytes that are not oocyte specific [4, 16, 17]. Deletion of these genes using the conventional ES-targeting strategy may cause embryonic lethality, which prevents analysis of their functions in oogenesis and folliculogenesis [5]. Even if germline deletion of oocyte-expressing genes could bypass embryonic lethality, their female reproductive phenotypes (if there are any) may not be interpreted as a result of the loss of their gene products in the oocytes, but rather could be due to the loss of their expression in other tissues, such as hypothalamus and pituitary (if expressed). Also, if genes of interest are expressed in oocytes at all stages of follicular development, their roles in later follicular stages, such as secondary and preovulatory follicles, will be unlikely to be characterized using the conventional knockout mouse models. In addition, it is impossible to determine the role of maternal gene products during preimplantation embryonic development using the conventional ES cell knockout strategy. In an effort to understand the physiological roles of genes, which are expressed in oocytes, during folliculogenesis, oogenesis, and preimplantation embryonic development, we have sought to adapt the Cre/loxP system to delete floxed genes specifically in oocytes, at different stages of follicular development. Several Cre transgenic mouse lines, such as Zp3Cre and Msx2Cre, in which the Cre gene is driven by zona pellucida protein 3 (Zp3) promoter and a homeobox gene Msx2 promoter, respectively, have been generated [18, 19]. Zp3Cre was shown to express the Cre recombinase in the oocytes at primary and/or later follicular stages [12, 18]. Recently, these transgenic mice have been used successfully to delete an oocyte-expressing gene, germ cell nuclear factor (GCNF), in the female germline [12, 20]. Both Zp3Cre and Msx2Cre transgenic mouse lines are valuable for generating conditional knockouts in the oocytes at primary and/or later follicular stages. However, both transgenic mouse lines are unlikely to be useful for achieving complete deletion of oocyte genes at early follicular stages, such as at primordial and even early primary stages. To date, there is no Cre transgenic mouse line expressing Cre recombinase in the oocytes in the primordial follicles during postnatal ovarian development. In this report, we describe the generation and characterization of an iCre-expressing transgenic mouse line under the promoter of an oocyte-expressing gene, growth differentiation factor-9 (GDF-9) [21, 22] and its comparison with Zp3Cre and Msx2Cre.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The floxed ROSA, R26R [23], mice were generously provided by Dr. Phillip Soriano (Fred Hutchinson Research Cancer Center, Seattle, WA). The floxed GCNF mice were generated as described previously [12, 20]. Transgenic mice, Zp3Cre [18] and Msx2Cre [19], were generously provided by Drs. Barbara Knowles (The Jackson Laboratory) and Gail Martin (University of California at San Francisco), respectively. Healthy adult C57B6/SJL mice were purchased from the Jackson Laboratory (Bar Harbor, ME). All mice were maintained on a 14L:10D cycle, with free access to food and water, in the vivarium of the Baylor College of Medicine. All experiments in this study were approved by the Animal Welfare Committee of Baylor College of Medicine.
Generation of Transgenic GDF-9-iCre Mice
A 1.1-kilobase (kb) improved Cre (iCre) fragment, which contains a consensus Kozak sequence [24], the nuclear localization signal peptide of the a simian virus 40 (SV40) large T antigen, and coding sequences of the iCre gene [8], was generated by PCR using primers iCre-F (5'-gatatcaagcttgtccaccatggtg-3') and iCre-R (5'-caactctagactcagtttcagtccccatc-3'), and pBlue.iCre plasmid (generously provided by Dr. R. Sprengel) [8] as a template. This 1.1-kb iCre fragment was then digested with HindIII and XbaI, and the digested iCre fragment was subcloned into the HindIII/XbaI sites of the pGL3-Basic-Luc vector (Promega, Madison, WI) to replace the original luciferase coding sequences to generate a promoterless mini-iCre expression cassette, pGL3B-iCre. GDF-9 promoter fragment (-3277-+48) was amplified from the pGDF-9-LUC plasmid (generously provided by Dr. Martin Matzuk) [12] and cloned into the KpnI/HindIII sites of the pGL3B-iCre plasmid to generate GDF-9-iCre transgene expression vector, pGL3B-GDF-9-iCre. The nucleotide sequences of this transgene vector, pGL3B-GDF-9-iCre, were confirmed by sequence analysis.
A 4.68-kb KpnI/SalI DNA fragment from the iCre expression vector, pGL3B-GDF-9-iCre, which contains 3.3-kb GDF-9 promoter, 1.1-kb iCre coding sequence, and a SV40 late poly(A) signal (Fig. 1), was purified from an agarose gel and then microinjected into the pronuclei of fertilized eggs of C57-B6/SJL mice. The microinjected eggs were then transferred to pseudopregnant mothers to generate GDF-9-iCre founder mice. The resulting transgenic mouse lines were named GDF-9-iCre.
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Mouse Genotyping
Mouse tail genomic DNA was extracted [25] and genotyped by PCR. GDF-9-iCre mice were genotyped by PCR analysis using primers iCre-PCR-F (5'-tctgatgaagtcaggaagaacc-3') and iCre-PCR-R (5'-gagatgtccttcactctgattc-3'). PCR reactions for the iCre transgene were carried out for 30 cycles (94°C, 1 min; 58°C, 2 min; 72°C, 2 min) in a buffer containing 2.5 mM MgCl2, while the PCR conditions for genotyping the Cre transgene in Zp3Cre and Msx2Cre mice were described previously [12]. PCR conditions for genotyping the GCNF+, GCNFfl, GCNFlox, and R26R allele were reported previously [12, 25].
ß-Galactosidase Staining
GDF-9-iCre, Zp3Cre, and Msx2Cre male mice were crossed with R26R females to generate GDF-9-iCre;R26R, Zp3Cre;R26R, and Msx2Cre;R26R mice, respectively. Ovaries at Days 1, 3, 5, 12, and 24 after birth were collected and then subjected to ß-galactosidase staining according to the manufacturer's protocol (Specialty Media, NJ). After staining, tissues were postfixed, dehydrated, embedded, and then sectioned. Tissue sections were counterstained with 0.1% nuclear fast red. These tissues and their sections were examined under a Zeiss Axioskop 2 microscopy (Carl Zeiss, Inc., Thornwood, NY).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Microinjection of the GDF-9-iCre transgene (Fig. 1), which contains 3.3-kb GDF-9 promoter (-3277-+48) [12], 1.1-kb iCre coding sequence [8], and a SV40 late poly(A) signal, into the pronuclei of fertilized eggs of C57-B6/SJL mice followed by transfer of the microinjected eggs into pseudopregnant mothers produced eight founder pups. Genomic PCR analysis showed that four of these eight pups carried the GDF-9-iCre transgene (data not shown). Among these four founders, three were females (5090, 5091, and 5092) and the other one was male (5085). All founders except the male founder successfully transmitted the transgene to their offspring after breeding with wild-type mice.
Cre Recombinase Activity in the Ovary of GDF-9-iCre Mice
R26R reporter mice [23] were used to determine the tissue and cell specificity of the Cre recombinase in three germline-transmitted GDF-9-iCre mouse lines. Breeding of the founder mice carrying the GDF-9-iCre transgene with R26R mice produced R26R and GDF-9-iCre;R26R mice. A variety of tissues, including heart, kidney, liver, lung, spleen, brain, testis, and ovary, of 24-day-old R26R and GDF-9-iCre;R26R mice were collected postmortem and subjected to ß-galactosidase staining. Positive blue staining in tissues indicates that Cre recombinase is active in turning on the LacZ gene expression in R26R mice. As expected, no ß-galactosidase staining was observed in all tested tissues of R26R mice (data not shown). All tested tissues of GDF-9-iCre;R26R mice generated from the GDF-9-iCre mouse line 5091 had a various degree of blue staining (data not shown), indicating ectopic expression of Cre recombinase in this transgenic mouse line. In contrast with the ectopic Cre expression in the GDF-9-iCre transgenic mouse line, 5091, the other GDF-9-iCre transgenic mouse lines, 5090 and 5092, displayed tissue-specific Cre expression (Fig. 2). ß-Galactosidase staining of tissues derived from the GDF-9-iCre transgenic mouse line, 5092, is shown in Figure 2. No ß-galactosidase activity was observed in the heart, kidney, liver, lung, spleen, brain, testis, uterus, or oviduct of 24-day-old GDF-9-iCre;R26R mice (Fig. 2, a– g). However, positive ß-galactosidase staining was observed in the ovary of 24-day-old GDF-9-iCre;R26R mice (Fig. 2h). Ovarian sections showed that positive blue staining was observed only in the oocytes, not the somatic cells (Fig. 2i). These results suggested that Cre recombinase from this transgenic mouse line is active only in the oocytes turning on the LacZ expression in GDF-9-iCre;R26R mice. Similar results were obtained using the GDF-9-iCre transgenic mouse line, 5090 (data not shown). Therefore, we have successfully generated a GDF-9-iCre transgenic mouse line that will be useful for achieving oocyte-specific deletion of the floxed genes of interest in the postnatal ovary.
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Stage-Specific Expression of Cre Recombinase in GDF-9-iCre, Zp3Cre, and Msx2Cre Transgenic Mice
To determine the ontogeny of the expression of Cre recombinase in the ovary during postnatal development, ovaries of GDF-9-iCre;R26R derived from the 5092 GDF-9-iCre transgenic line were collected at Days 1, 3, 5, 12, and 24 after birth and then subjected to ß-galactosidase staining. Similarly, the Cre recombinase activities of Msx2Cre transgenic mice [19] and Zp3Cre transgenic mice [18] in the ovaries at Days 1, 3, 5, 12, and 24 after birth were also analyzed. Ovaries from R26R littermates were used as negative controls for ß-galactosidase staining. As expected, there was no blue staining in the ovarian sections from R26R mice at the age of 1–24 days after birth (Fig. 3, a– e). In GDF-9-iCre;R26R mice, positive blue staining was observed in the oocytes of 3- to 24-day-old ovaries, but not in 1-day-old ovary (Fig. 3, f–j). At Day 3, positive blue staining was observed in the oocytes of some primordial follicles in GDF-9-iCre;R26R mice (Fig. 3g). At Days 5, 12, and 24, positive blue staining was detected in the oocytes of a number of primordial follicles in GDF-9-iCre; R26R ovaries (Figs. 3, h–j, and 4). Positive blue staining was also observed in the oocytes, not the somatic cells of all follicles at the primary, secondary, and later follicular stages (Figs. 3 and 4). In the Zp3Cre;R26R females, positive blue staining was observed in the ovaries at the ages of 5 days or older (Fig. 3, k–o). There was no positive blue staining in the primordial follicles in any Zp3Cre;R26R females tested. Similar to previous reports [12, 18], positive blue staining was observed only in the oocytes at the primary, secondary, and later follicular stages, but not in the adjacent somatic cells in Zp3Cre;R26R ovaries (Figs. 3 and 4). In Msx2Cre;R26R mice, positive blue staining was observed in the ovaries of 12-day- and 24-day-old animals, but not 1-, 3-, and 5-day-old animals (Fig. 3, p–t). Positive blue staining was observed in the oocytes of follicles at the early secondary and later follicular stages, but not in the oocytes of primordial follicles nor in the primary follicles of Msx2Cre;R26R ovaries (Figs. 3 and 4). Therefore, the Cre recombinase in the GDF-9-iCre mice was active earlier than that of Zp3Cre and Msx2Cre in the ovary not only at the postnatal age but also at the follicular stage, indicating that GDF-9-iCre, Zp3Cre, and Msx2Cre can be used to delete oocyte-specific genes of interest at specific stages of follicular development in the postnatal ovary.
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Deletion of the Floxed GCNF Allele in Female Germline by GDF-9-iCre Mice
The floxed GCNF mice [20] were used to test the efficiency of the Cre recombinase in GDF-9-iCre transgenic mice in female germline. Breeding of GDF-9-iCre males with the floxed GCNF females produced GDF-9-iCreGCNFfl/+ mice (Fig. 5a). GDF-9-iCreGCNFfl/+ females were then crossed with wild-type male mice, and the progeny of this breeding was genotyped to determine whether the floxed GCNF allele was lost in the progeny and a recombined GCNFlox allele was detected in the progeny (Fig. 5a). If Cre recombinase is active in the oocyte, the floxed GCNF allele (GCNFfl) will be converted to the recombined GCNFlox allele regardless of the presence or absence of GDF-9-iCre transgene in the progeny. We found that no floxed GCNF allele was detected in any of the 47 pups generated from three GDF-9-iCreGCNFfl/+ females, and a recombined GCNFlox allele was observed in 22 of these 47 progeny. A representative genotyping result is shown in Figure 5b. The recombined GCNFlox allele but not the floxed GCNF allele was detected in pups 1, 4, and 7, even though there was no GDF-9-iCre transgene in pups 4 and 7 (Fig. 5b). These results show that the Cre recombinase expressed in adult GDF-9-iCre females is sufficient to delete the floxed GCNF allele in the female germline.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Having determined that Cre recombinase in the GDF-9-iCre transgenic line can efficiently induce excision of the artificial DNA fragment in the ROSA locus, we asked the question whether the Cre recombinase in this GDF-9-iCre transgenic line could delete a floxed endogenous gene fragment. Using the floxed GCNF mice recently generated [20], we found that the floxed GCNF allele was completely deleted in the female germline by GDF-9-iCre (Fig. 5), similar to Zp3Cre and Msx2Cre reported previously [12, 20]. These results indicate that GDF-9-iCre transgenic mice can be used for conditional deletion of oocyte genes in the postnatal ovary, thus allowing dissection of the physiological functions of genes expressed in the oocytes during folliculogenesis, oogenesis, and preimplantation embryonic development.
Using the conventional ES cell-targeting strategies, it is impossible for us to determine the roles of oocyte genes at different stages of follicular development if these genes are expressed continuously in the oocytes during follicular development. The Cre/loxP system provides an excellent strategy to understand gene function during folliculogenesis and oogenesis. The sequential oocyte-specific Cre expression in primordial, primary, or secondary follicles of GDF-9-iCre, Zp3Cre, and Msx2Cre mouse lines characterized in this report (Figs. 3 and 4) would allow conditional ablation of oocyte genes at a specific follicular stage, thus facilitating understanding of their roles at each follicular developmental stage. In addition, these three transgenic mouse lines can be used for conditional expression of transgenes specifically in various follicular developmental stages in the ovary.
In conclusion, we generated and characterized a GDF-9-iCre transgenic mouse line that can express Cre recombinase in oocytes as early as at the primordial follicular stage. We also reported the sequential oocyte-specific Cre expression in primordial, primary, or secondary follicles of GDF-9-iCre, Zp3Cre, and Msx2Cre mouse lines. We believe that GDF-9-iCre transgenic mice along with Zp3Cre and Msx2Cre transgenic mice will allow us to dissect the physiological functions of genes expressed in the oocytes during folliculogenesis, oogenesis, and preimplantation embryonic development.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Austin J. Cooney, Department of Molecular and Cellular Biology, Baylor College of Medicine, BCMM-M626, One Baylor Plaza, Houston, TX 77030. FAX: 713 790 1275; acooney{at}bcm.tmc.edu
Received: 6 May 2004.
First decision: 19 May 2004.
Accepted: 16 June 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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