QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 1 September 2004
doi: 10.1242/dev.01336

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ramos, S. B. V. Articles by Blackshear, P. J. Search for Related Content PubMed PubMed Citation Articles by Ramos, S. B. V. Articles by Blackshear, P. J. Social Bookmarking                         What's this?

The CCCH tandem zinc-finger protein Zfp36l2 is crucial for female fertility and early embryonic development

¹ Laboratory of Signal Transduction, National Institute of Environmental Health Science, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709, USA
² Duke Comprehensive Cancer Center, Duke University Medical Center, Durham, NC 27710, USA
³ The Office of Clinical Research, National Institute of Environmental Health Science, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709, USA
⁴ Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA
⁵ Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA

View larger version (46K): [in a new window]   Fig. 1. Generation of the N-Zfp36l2 mutant mouse. (A) The strategy used to disrupt Zfp36l2 is shown with a schematic representation of the endogenous (12 kb) and targeted (14 kb) alleles of Zfp36l2. Restriction sites within the wild-type (WT) and targeted gene (S, Sst I; X, Xba I; E, Eco RV; and N, Not I) are indicated. The various shadings represent, respectively, 5'-flanking region of the gene (white), protein coding sequences (black), 5'-UTR and 3'-UTR of the transcript (gray), intron (diagonal-hatch), and PGK-NEO and PGK-DTA cassettes (cross-hatch). A small double arrow indicates the exon 2 probe used for Southern blot analysis. Black bars indicate the location of the 5' and 3' probes used in Southern blot analysis to select heterozygous embryonic stem (ES) cells. Genotyping of DNA derived from the progeny of N-Zfp36l2 heterozygous crosses was performed by PCR and Southern blot analysis. (B) An example of the PCR results, in which the 0.6 kb and 2.4 kb fragments correspond to WT and Zfp36l2 mutant alleles, respectively. (C) Total DNA (10 µg) was digested with Sst I and probed with an exon 2 probe; the 10 kb and 2.5 kb bands correspond to the WT and Zfp36l2 mutant alleles, respectively. (D,E) Total RNA was isolated from the indicated tissues and bone marrow-derived macrophages (BMMac) of WT and mutant (Mut) mice and subjected to northern blot analysis. Each lane in D contains 10 µg of total RNA probed with Zfp36l2 exon 1 and Gapd probes, as indicated by the arrows. The positions of the 28S and 18S ribosomal RNAs are indicated. (E) RNA samples from the same tissues as described in D were subjected to northern blot analysis and hybridized with Zfp36l2 exon 2 (upper arrow) and Gapd (lower arrow) probes, showing the presence of a remaining Zfp36l2 transcript in the mutant mice. (F) Protein extracts from HEK 293 cells overexpressing Zfp36l2 (lane 1), and bone marrow-derived macrophages from WT and N-Zfp36l2 mutant mice (lane 2 and 3, respectively), were separated on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. Blotting with the Zfp36l2 amino-terminal peptide antibody revealed a broad band of 60 kDa corresponding to the predicted size of Zfp36l2 in the protein extracts from HEK 293 cells overexpressing Zfp36l2 and the cells from the WT mice, but not from the mutant cells (left panel). In the presence of competing peptide, the Zfp36l2 signal was blocked (right panel). (G) Western blot analysis using protein extracts from spleen (left) and BMMac (right) of WT and N-Zfp36l2 mutant (Mut) mice (lane 2 and 3, respectively) and HEK 293 cells overexpressing Zfp36l2-HA (left panel, lane 1) or not expressing Zfp36l2 (right panel, lane 4) were probed with a carboxyl-terminal peptide antiserum. This revealed that the band corresponding to Zfp36l2 exhibited a smaller size in tissues and cells from the mutant mice (arrow), and that the levels of expression of the protein were lower in the spleen, but approximately equal in BMMac. The dotted arrows point to the major non-specific (NS) bands recognized by this antiserum.

View larger version (52K): [in a new window]   Fig. 2. Presence of sequences from the single intron in the N-Zfp36l2 mutant transcript. (A) Total cellular RNA (10 µg per lane) was isolated from the indicated tissues and bone marrow-derived macrophages (BMMac) of wild-type (WT) and N-Zfp36l2 mutant mice, subjected to northern blot analysis and probed with Zfp36l2 intron and Gapd probes, as indicated by the arrows. (B) Cytosolic RNA was extracted from BMMac of WT and mutant mice, and subjected to northern blot analysis. Each lane contained 10 µg of cytosolic RNA probed, respectively, with Zfp36l2 exon 2, intron and Neo probes, as indicated at the top of each pair of lanes. The new transcript present in cytosolic samples from the mutant mice hybridized with both the exon 2 and intron probes, whereas the WT Zfp36l2 transcript hybridized only with the exon 2 probe. The new Zfp36l2 transcript was not fused with Neo, because it did not hybridize with a Neo probe; as shown in the last lane of this panel, the Neo probe hybridized only with the Neo transcript present in the sample from the N-Zfp36l2 mutant cells.

View larger version (45K): [in a new window]   Fig. 3. Expression of Zfp36l2 transcripts in tissues from mutant mice. (A) Total RNA was extracted from tissues of wild-type (WT) and N-Zfp36l2 mutant mice and subjected to northern blot analysis. Each lane contained 10 µg of total RNA, hybridized with Zfp36l2 exon 2 and Gapd 32P-labeled probes, as indicated by the arrows. Note the decreased size and expression levels of the mutant transcript. (B) The Zfp36l2 transcripts from the WT and N-Zfp36l2 mutant mice were quantified with a PhosphorImager, and normalized by Gapd mRNA levels. The normalized values were plotted for each tissue. (C) The normalized Zfp36l2 transcript levels in tissues from the N-Zfp36l2 mutant mice were expressed as percentages of WT transcript levels. The data for bone marrow-derived macrophages (BMMac) were taken from a separate experiment.

View larger version (63K): [in a new window]   Fig. 4. Anatomy of N-Zfp36l2 mutant female reproductive tract. Ovaries from adult (8-12 weeks old) female mice, synchronized with respect to the ovulatory cycle, were dissected from the fat pad, weighed and then fixed. The top and middle panels show the macroscopic anatomy of the female reproductive tract and the ovaries from wild-type (WT) (A,C) and N-Zfp36l2 mutant mice (B,D). The lower panel shows representative ovary sections stained with hematoxylin and eosin from females in post-estrus. No apparent abnormalities were noted in the N-Zfp36l2 mutant ovaries (F) in comparison with the WT (E); both exhibited follicles at various stages of development, as well as corpora lutea (CL). Scale bars: 1 mm. (G) Western blot analysis was performed using protein extracts from ovaries of WT (lanes 1) and N-Zfp36l2 mutant mice (lanes 2). These were probed with a carboxyl-terminal peptide antiserum (AS) or with pre-immune serum (PI). The band corresponding to the top band of Zfp36l2 was smaller in size in the ovaries from the mutant mice (arrow), and the level of expression of the protein appeared to be decreased in comparison with the WT. The smaller bands probably represent some combination of degraded fragments of Zfp36l2, phosphorylated isoforms, or possibly cross-reaction with the much smaller Zfp36l1 protein. In the presence of competing peptide (middle panel), the AS recognition of the Zfp36l2 band and the smaller bands was blocked.

View larger version (124K): [in a new window]   Fig. 5. Ovary and oocyte morphology in adult wild-type (WT) and N-Zfp36l2 mutant females. (A,B) Secondary follicles in which the small oocytes are surrounded by cuboidal cells. (C,D) Tertiary follicles are shown in which the cuboidal cells have divided actively, forming a stratified layer around the oocyte. Irregular spaces have appeared, forming the follicular antrum. (E,F) Graafian follicles, in which the large follicular cavity is distended with fluid, and the cumulus oophorus contains a mature oocyte surrounded by a thick zona pelucida and well-formed corona radiata. (G,H) The corpora lutea, which appear to form normally after ovulation. Scale bars: 40 µm in A-D; 100 µm in E-H.

View larger version (80K): [in a new window]   Fig. 6. Fertilization of ova in N-Zfp36l2 mice. Ova from wild-type (WT) (A-C, upper panels) and N-Zfp36l2 mutant females (D-F, lower panels) were collected from the oviduct 8-10 hours after in vivo fertilization of superovulated females, kept in culture for 4-5 hours, then fixed and stained with a chromosome dye (Hoechst 33258) and analyzed by confocal microscopy. These images are representative of three separate experiments. (A,D) Differential interference contrast images show apparently normal morphology, in which both ova are surrounded by zona pelucida. Chromosome staining of the same cells (C,F) demonstrated that ova from both the WT and N-Zfp36l2 mutant females showed typical markers of fertilization, such as the presence of two pronuclei and the formation of a second polar body (large filled arrow). A supernumerary sperm is indicated by the filled small arrow in E,F. In the middle panels (B,E), the left and right images were superimposed showing the localization of the second polar bodies (large filled arrows) and the two pronuclei (large open arrows) in relation to the surrounding zona pelucida, as well as the supernumerary sperm in E (small filled arrows).

View larger version (52K): [in a new window]   Fig. 7. Arrest of embryos from N-Zfp36l2 females at the two-cell stage. Wild-type (WT) and N-Zfp36l2 mutant females were superovulated and mated with WT stud males. Nineteen hours after the hCG injection, all potential in vivo fertilized ova were collected and incubated in M16 growth medium. After 5 hours in culture, the fertilized ova were sorted based on the morphological detection of two pronuclei and the presence of a second polar body, thus considered time zero for the fertilization. The unfixed embryos were photographed at 50x using Nomarski optics. The upper panel illustrates the normal development of embryos from WT females after 48 hours (A) and 72 hours (B) in culture; the embryos derived from N-Zfp36l2 mutant females at equivalent incubation periods are shown in C,D. The graph represents means ±s.d. results from three such experiments, in which the means of the numbers of actively dividing embryos derived from WT (black circles) and N-Zfp36l2 mutant females (white circles) were significantly different at 48 and 72 hours (P<0.01, using Student's t-test), but not at 24 hours.

View larger version (36K): [in a new window]   Fig. 8. Genetic models of mouse female infertility because of blockage of early embryonic development. The left column specifies the stage of development along the oviduct. The right column lists the genes associated with blockage at each stage. The zona pelucida protein Zp3 has been implicated in impaired fertilization (Liu et al., 1996; Rankin et al., 1996). Disruption of Npm2 (Burns et al., 2003), Zar1 (Wu et al., 2003) and Hsf1 (Christians et al., 2000) impedes the first cleavage step. Blockage at the two-cell stage is observed with disruption of Zfp36l2 (N-Zfp36l2) and MATER (Tong et al., 2000).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter