Research article
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Lunatic fringe null female mice are infertile due to defects in meiotic maturation Katherine L. Hahn1,4, Joshua Johnson2,*, Brian J. Beres2,4, Sheena Howard3,4 and Jeanne Wilson-Rawls1,2,4,† 1
Molecular and Cellular Graduate Program, Arizona State University, Tempe, AZ 85284-4501, USA Biology Graduate Program, Arizona State University, Tempe, AZ 85284-4501, USA 3 Minority Access to Research Careers (MARC) Program at ASU, Arizona State University, Tempe, AZ 85284-4501, USA 4 School of Life Sciences, Box 4501, Arizona State University, Tempe, AZ 85284-4501, USA 2
*Present address: Vincent Center for Reproductive Biology, Department of Obstetrics and Gynecology, Massachusetts General Hospital, Harvard Medical School, Room 6607, Building 149, 149 13th Street, Charlestown, MA 02129, USA † Author for correspondence (e-mail:
[email protected])
Accepted 1 December 2004 Development 132, 817-828 Published by The Company of Biologists 2005 doi:10.1242/dev.01601
Development
Summary We have demonstrated that Notch genes are expressed in developing mammalian ovarian follicles. Lunatic fringe is an important regulator of Notch signaling. In this study, data are presented that demonstrate that radical fringe and lunatic fringe are expressed in the granulosa cells of developing follicles. Lunatic fringe null female mice were found to be infertile. Histological analysis of the lunatic fringe-deficient ovary demonstrated aberrant folliculogenesis. Furthermore, oocytes from these mutants
Introduction The Notch gene family encodes transmembrane receptors that are highly conserved evolutionarily (Kimble and Simpson, 1997; Lewis, 1998; Artavanis-Tsakonis et al., 1999). In mammals, there are four Notch receptors (Notch1-4) (Weinmaster et al., 1991; Weinmaster et al., 1992; Franco del Amo et al., 1992; Lardelli and Lendahl, 1993; Lardelli et al., 1994; Uyttendaele et al., 1996), and two families of ligands, Deltalike1 (Dll1) (Bettenhausen et al., 1996), Dll3 (Dunwoodie et al., 1997) and Dll4 (Shutter et al., 2000), and Jagged1 (Lindsell et al., 1995) and Jagged2 (Shawber et al., 1997). The receptors and ligands have overlapping expression patterns in many tissues (Lardelli and Lendahl, 1993; Williams et al., 1995; Lindsell et al., 1996; Johnson et al., 2001). Activation of Notch by ligand binding triggers cleavage of the receptor in a process known as regulated intramembrane proteolysis (RIP), generating the Notch intracellular domain (NotchIC) that then translocates to the nucleus (Schroeter et al., 1996; Kopan et al., 1996; Blaumueller et al., 1997; Logeat et al., 1998). In the nucleus, Notch forms transcriptional complexes with CSL transcription factors and activates the expression of downstream target genes (Tamura et al., 1995; Hsieh et al., 1996; Struhl and Adachi, 1998). These target genes are two families of basic helix-loop-helix (bHLH) proteins referred to as hairy enhancer of split (Hes), and a related family variously referred to as Hesr, HRT, Hey, CHF, and Gridlock (Jarriault et al., 1995; Jarriault et al., 1998; Ohtsuka et al., 1999; Nakagowa, 1999; Nakagowa, 2000; Kokubo et al., 1999; Chin et al., 2000; Maier and Gessler, 2000; Zhong et al., 2000).
did not complete meiotic maturation. This is a novel observation because this is the first report describing a meiotic defect that results from mutations in genes that are expressed in the somatic granulosa cells and not the oocytes. This represents a new role for the Notch signaling pathway and lunatic fringe in mammalian folliculogenesis. Key words: Lunatic fringe, Notch, Ovary, Follicle, Meiosis, Fertility
These proteins have been implicated in the repression of tissuespecific gene transcription (Jarriault et al., 1995; Jarriault et al., 1998; Ohtsuka et al., 1999; Hsieh et al., 1999; Nakagawa et al., 2000; Chin et al., 2000). The interaction between Notch and its ligands is modulated by O-linked fucose moieties that are added to the EGF repeats of the extracellular domain. Usually fucose is unaltered when it is added to proteins; however, on the Notch receptors fucose is modified with N-acetylglucosamine (GlcNac) added by the Fringe proteins (Moloney et al., 2000; Bruckner et al., 2000). The Fringe proteins are Golgi-localized and belong to a large family of β1,3-N-acetylglucosaminyl transferases (Moloney et al., 2000; Bruckner et al., 2000; Schwientek et al., 2002). Enzymes in this family have strong substrate and target specificity and diverse functions. The only known targets of the mammalian fringe proteins are the Notch receptors (Schwientek et al., 2000). In mammals, there are three fringe proteins, radical (Rfng), manic (Mfng) and lunatic fringe (Lfng) (Johnston et al., 1997). Modification of the extracellular domain of Notch by Lfng can potentiate or inhibit the interaction between a particular Notch receptor-ligand pair. For example, Lfng potentiates the interaction between Notch1 and Dll1, but inhibits Notch1-Jagged1 interactions. However, Lfng potentiates both Dll1 and Jagged1 mediated activation of Notch2 (Hicks et al., 2000). Lfng and Mfng reportedly modify different sites in the extracellular domain of Notch2 (Shimizu et al., 2001), indicating they may have different roles to play in regulating Notch signaling. In Drosophila, two Golgi localized β1,3-N-
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acetylglucosamine transferases, Fringe and Brainiac, play important roles in oogenesis and folliculogenesis (Goode et al., 1996a; Goode et al., 1996b; Hicks et al., 2000; Bruckner et al., 2000; Munro and Freeman, 2000; Schwientek, 2002). Brainiac activity is needed in the germ line for proper organization of the follicle (Goode, 1996). Fringe, the homolog of Lfng, is necessary for specification of the polar cells (Grammont and Irvine, 2001). Brainiac has been demonstrated to modify glycosphingolipids by adding GlcNac residues to mannose and galactose moieties on ceramide (Schwientek et al., 2002). In mice, a null mutation of the murine homolog of brainiac demonstrated that this protein is important for very early development, as braniac–/– embryos die prior to implantation (Vollrath et al., 2001). No role for this family of proteins in mammalian folliculogenesis has been described. It has been demonstrated that Lfng is an important regulator of Notch signaling. For example, Lfng null mutants have segmentation defects that are similar to those seen in null mutations of Notch1 and Dll1 (Evrard et al., 1997; Zhang and Gridley, 1997). In somites, where Lfng is the only family member expressed, Notch receptors and ligands are expressed normally in Lfng–/– mutants, but the Notch downstream target gene Hes5 was not detected, indicating a lack of Notch activation in the presence of ligand. However, Hes5 was expressed normally in the neural tube and developing brain of Lfng null mutants, probably due to expression of Mfng and Rfng in these tissues (Evrard et al., 1997). Interestingly, Rfngdeficient mice had no phenotype and Rfng/Lfng double null mutants had only defects associated with a lack of Lfng (Zhang et al., 2000). Folliculogenesis is the process by which oocytes develop in response to hormonal cues. This requires the coordination of the proliferation and differentiation of granulosa cells and the growth and maturation of the oocyte. Primordial follicles consist of a small oocyte surrounded by squamous somatic cells. When recruited to develop, the granulosa cells proliferate and become cuboidal. As these cells continue to proliferate, layers develop around the growing oocyte. Once a follicle has several layers of cells a fluid filled space, the antrum, will begin to form. The antrum spatially separates the two functionally distinct granulosa cell populations, cumulus and mural. During this time the oocyte has grown and at the time of antrum formation it becomes competent to resume meiosis in response to luteinizing hormone (LH). Resumption of meiosis is marked by the breakdown of the germinal vesicle (GVB). Meiosis continues to metaphase II (MII), and oocytes are blocked at this stage until fertilization. Studies done in mice have demonstrated that reciprocal signaling between the oocyte and the granulosa cells is necessary for the differentiation of the cumulus granulosa cells and meiotic maturation of the oocyte (Rodgers et al., 1999; Erickson and Shimasaki, 2000; Eppig, 2001; Matzuk et al., 2002). We have previously shown that Notch2, Notch3 and Jagged2 are expressed in granulosa cells, and Jagged1 is expressed in the oocytes of developing mammalian follicles (Johnson et al., 2001). Furthermore, transcripts of the Notch downstream target genes, Hes1, Hes5, Hesr1, Hesr2 and Hesr3 also were detected in the granulosa cells of follicle types 3b-8 (Johnson et al., 2001), indicating that Notch signaling was active. As all three mammalian fringe proteins can modify the Notch receptors when expressed in the same cell (Bruckner et al., 2000;
Research article Moloney et al., 2000; Hicks et al., 2000), we hypothesized that the fringe genes would also be expressed in the granulosa cells, and furthermore, that they would have a role in regulating folliculogenesis through Notch2 and Notch3. In this study, we demonstrate that Lfng is expressed in the granulosa cells and theca of developing follicles from primary to preovulatory in size. Rfng is expressed briefly in granulosa cells of early antral follicles. Mfng is only detected in the vasculature. Null mutations of the Notch receptors and ligands result in embryonic lethal phenotypes (Swiatek et al., 1994; Conlon et al., 1995; Hrabé de Angelis et al., 1997; Jiang et al., 1998; Hamada et al., 1999; Xue et al., 1999; McCright et al., 2001). Some Lfng–/– mice survive to adulthood, therefore we examined folliculogenesis in these mutants. Female Lfng null mutant mice have many aberrant follicles. When induced to ovulate they released oocytes into the oviduct, but only a small percentage could be fertilized in vitro. Examination of these oocytes demonstrated that cumulus expansion occurred in response to exogenous hormones, but the oocytes were not at metaphase of meiosis II, and had not completed meiotic maturation. Mutations that block the progression of meiosis have been described, but they are all germ-cell-specific genes. These are novel observations because the disregulation of meiosis is caused by a change in the somatic cells. This represents a new regulatory pathway in folliculogenesis and a new role for Notch signaling in mammals.
Materials and methods Mating study Eleven-week-old heterozygous male, and 8-week old null and heterozygous female, mice were paired. Each morning females were examined for the presence of a copulatory plug. If a plug was present, the female was removed and a new female introduced to the male cage. If after 6 days no copulatory plug was detected, the female was placed with a new male. Copulatory plugs and litter numbers were recorded and the genotype of the offspring was determined. Whole-mount thick section in situ hybridization (ISH) Whole-mount ISH was done on thick sections according to Johnson et al. (Johnson et al., 2001). Briefly, ovaries were fixed in 10% neutralbuffered formalin (NBF) (Richard-Allen Scientific, Kalamazoo, MI), and embedded in paraffin wax after stepwise dehydration in ethanol. Thirty micron (µm) sections were cut perpendicular to the axis of entry of ovarian blood vessels. Sections were dewaxed, rehydrated and antisense digoxigenin-labeled gene-specific RNA probes were hybridized. Transcripts were identified using anti-digoxigenin antibody (Roche, Indianapolis, IN) conjugated to alkaline phosphatase and the BM purple substrate (Roche, Indianapolis, IN). Replicates were performed on sections from at least 3 ovaries/genotype and probes were checked for specificity by ISH on embryos. Histology Tissues were fixed as above and sectioned to 10 µm. Sections were prepared by standard procedures and stained with Hematoxylin and Eosin. Bone and cartilage preparation The mice were skinned and eviscerated. The carcasses were placed in Alcian Blue (Sigma A3157) for 48 hours to stain the cartilage, followed by 2% KOH for 48 hours. Skeletons were then placed in Alizarin Red (Sigma A5533) to stain the bone for 72 hours.
Lfng null females are infertile
Development
Hormone treatment and isolation of OCC and oocytes Mice were injected intraperitoneally (ip) with 5 international units (IU) of pregnant mare’s serum gonadotropin (PMSG) (Calbiochem, Carlsbad, CA) and 48 hours later, were injected ip with 5 IU of human chorionic gonadotropin (hCG) (Calbiochem, Carlsbad, CA). Oocyte cumulus complexes (OCC) were harvested from the oviduct 16 hours later. OCC and ovaries were collected in KSOM (Specialty Media, La Jolla, CA) with 10% FBS. The OCC were incubated in KSOM containing hyaluronidase (300 µg/ml) for 30 seconds, then washed in KSOM, and fixed as described in LeMaire-Adkins et al. (LeMaireAdkins et al., 1997). Briefly, oocytes were fixed in 2% formaldehyde, 1% Triton X-100, 0.1 mM PIPES, 5 mM MgCl2, 1 mM DTT and 2.5 mM EGTA in D2O (Sigma, St Louis, MO) containing aprotinin (Sigma, St Louis, MO) and taxol (Sigma, St Louis, MO) at 37°C. Oocytes were washed in 0.1% normal goat serum (NGS) in PBS (GIBCO/BRL, Gaithersburg MD) and blocked at 37°C in PBS containing 10% NGS and 0.1% Triton X-100. Oocytes were stored in this at 4°C until staining was performed. For staining, oocytes were transferred to 1% Triton X-100 in PBS at room temperature then incubated with a monoclonal anti-α-tubulin (clone DM 1A, Sigma, St Louis, MO) conjugated to FITC at a 1:50 dilution. The oocytes were washed in PBS, incubated in PBS containing 1 µg/ml Hoechst 33258 (Molecular Probes, Eugene, OR). Oocytes were washed in PBS and mounted in glycerol with p-phenylenediamine and visualized by confocal microscopy. Confocal analysis was done using a Leica TCS NT, final magnification of 800. The FITC was visualized using an Ar laser, and Hoechst 33258 was visualized using an UV laser. In vitro fertilization (IVF) OCC were collected after hormone administration as above. Sperm were collected from the vas deferens and cauda epididymis in human tubal fluid (HTF) and capacitated for 2 hours at 37°C. Sperm (1106) were added to each OCC sample and fertilization allowed to proceed for 2 hours at 37°C. Eggs were washed three times in sperm free KSOM and incubated at 37°C. Eggs were scored as fertilized by the presence of two pronuclei and embryogenesis was scored daily. Immunohistochemistry (IHC) Sections were heated at 80°C for 30 minutes, cooled to room temperature, followed by xylenes, rehydrated through graded alcohols to 70% ethanol. Slides were incubated in water, then PBS, placed in 0.1 M sodium citrate (pH 6) and epitope retrieval done in the microwave. The sections were cooled to room temperature, rinsed in PBS, and incubated in 3% H2O2 in 60% methanol to destroy endogenous peroxidases. IHC was performed using the HistostainSP kit according to the manufacturer’s instructions (Zymed Labs, San Francisco, CA), and primary antibodies were diluted according to this protocol except for the following: polyclonal anti-c-Kit antibody (Ab1, Calbiochem, Carlsbad, CA) was diluted 1:25, and anti-connexin43 (Santa Cruz Biotechnology, Santa Cruz CA), 1:50. Proteins were detected with alkaline-phosphatase-conjugated anti-rabbit secondary antibody and exposed to colour reagent. No primary antibody controls were included in each experiment. Reverse transcription polymerase chain reaction (RT-PCR) Total ovary RNA was isolated using TRIzol (Life Technologies, Gaithersburg, MD), according to the manufacturer’s directions, from 3 different animals/genotype. For oocytes, 15 oocytes per sample were denuded using hyaluronidase and total RNA extracted. cDNA was synthesized using Superscript III (Invitrogen, Carlsbad CA), according to the manufacturer’s protocol. For each gene examined by semi-quantitative (sq) RT-PCR, 3 sets of samples comprising all three genotypes and no RT controls were amplified using α-[32P]dATP (Perkin-Elmer Life and Analytical Sciences, Boston, MA). For each gene-specific primer pair the minimum number of cycles to the linear range was determined and used for all subsequent experiments. All primer sets span at least one intron. Control experiments were done
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using total embryo RNA. All cDNA samples were normalized using the ribosomal gene L7 (Meyuhas et al., 1990), and quantified using a Storm 860 PhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). To detect the presence of transcripts in oocytes, a qualitative PCR protocol and amplification beyond the linear range after normalization was used (Münsterberg and Lassar, 1995). Kinase assays Kinase assays were carried out as described in Svoboda et al. (Svoboda et al., 2000). Single eggs were transferred in 1.5 µl of KSOM into 3.5 µl of double kinase lysis buffer (10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µM p-nitrophenyl phosphate, 20 mM βglycerophosphate, 0.1 mM sodium orthovanadate, 5 mM EGTA) and immediately frozen in liquid nitrogen, then stored at –80°C until the assay was performed. The kinase reaction was initiated by the addition of 5 µl of double kinase buffer (24 mM p-nitrophenyl phosphate, 90 mM β-glycerophosphate, 24 mM MgCl2, 24 mM EGTA, 0.2 mM EDTA, 4.6 mM sodium orthovanadate, 4 mM NaF, 1.6 mM dithiothreitol, 60 µg/ml aprotinin, 60 µg/ml leupeptin, 2 mg/ml polyvinyl alcohol, 2.2 mM protein kinase A inhibitor peptide (Sigma), 40 mM 3-(nmorpholino) propanesulfonic acid (MOPS), pH 7.2, 0.6 mM ATP, 2 mg/ml histone (type III-S, Sigma), 0.5 mg/ml MBP with 500 mCi/ml γ-[32P]ATP (3000 Ci/mmol) (Perkin-Elmer Life and Analytical Sciences). To determine the background level of phosphorylation, 5 µl of double kinase lysis buffer was added instead of egg lysate. Reactions were incubated for 30 minutes at 30°C, and terminated by the addition of 10 µl 2SDS-PAGE sample buffer and boiling for 3 minutes. Following 15% SDS-PAGE, the gel was dried and exposed to a phosphorimager screen and quantified. The mean value of the control samples was set to one and all others expressed as fold activity of control. Quantifying cumulus expansion OCC were collected from the oviducts of Lfng+/– and Lfng–/– mice (n=5/genotype) post-hormone administration. OCC were photographed at 70 and photomicrographs printed the same size. The widest diameter of each OCC was measured in mm and mean diameter±s.d. was determined. Statistical analysis Analysis was carried out using the SAS system and the FREQ procedure. Our data was found to be significant (P
