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First published online 31 January 2007
doi: 10.1242/dev.02795
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1 Division of Neuroscience, Oregon National Primate Research Center/Oregon
Health and Science University, 505 N.W. 185th Avenue, Beaverton, OR,
USA.
2 Division of Animal Resources, Oregon National Primate Research Center/Oregon
Health and Science University, 505 N.W. 185th Avenue, Beaverton, OR,
USA.
3 University of Sassari Medical School/INBB, Italy.
4 The Salk Institute, San Diego, CA, USA.
5 The Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10
1SA, UK.
Author for correspondence (e-mail:
ojedas{at}ohsu.edu)
Accepted 18 December 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Ovarian development, Peptidases, Follicular assembly, Follicular growth, siRNAs
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), stem
cell factor (SCF) necessary for germ cell survival during migration towards
the genital ridge (Godin et al.,
1991), and wingless-related MMTV integration site 4 (Wnt4), a
member of the Wnt family of locally acting cell signals, which is required for
intragonadal survival of newly formed oocytes
(Vainio et al., 1999). The
formation of primordial follicles requires a transcription factor termed
factor in the germline alpha (FIG
)
(Liang et al., 1997).
Subsequent differentiation and growth of primordial follicles is regulated by
several factors produced locally by either granulosa cells or the oocyte
itself. Granulosa cell factors that facilitate follicle growth include the kit
ligand KL (Kitl) (Huang et al.,
1993; Parrott and Skinner,
1999), basic fibroblast growth factor (bFGF; also known as Fgf2)
(Nilsson et al., 2001),
leukemia inhibitory factor (Lif) (Nilsson
et al., 2002), the neurotrophins nerve growth factor (NGF),
neurotrophin-4/5 (NT-4/5; also known as Ntf5), and brain-derived neurotrophic
factor (Bdnf) (Dissen et al.,
2001; Paredes et al.,
2004; Spears et al.,
2003), and others (Skinner,
2005). Oocyte factors implicated in the control of follicular
growth include: growth differentiation factor 9 (Gdf9)
(Dong et al., 1996), the
homeobox gene Nobox (newborn ovary homeobox-encoding gene)
(Rajkovic et al., 2004;
Suzumori et al., 2002) and the
TrkB receptors, which are high-affinity tyrosine kinase receptors for NT-4/5
and Bdnf (Paredes et al.,
2004; Spears et al.,
2003).
Identification of these key molecules has made it evident that follicular
assembly, and the subsequent initiation of gonadotropin-independent follicular
growth, require not only genes necessary for the specific development of germ
and somatic cells, but also genes that promote and maintain the structural
organization of the gland. We have now identified a gene that appears to be
required for the structural organization of the rodent ovary. This gene, which
we have termed Felix-ina (Fxna), encodes a novel member of the M28
family of metallopeptidases (Rawlings and
Barrett, 1995). Our results suggest that Fxna plays a crucial role
in processing proteins required for the organization of somatic cells and
oocytes into follicular structures. A partial report of these findings has
appeared in an abstract form (Garcia-Rudaz
et al., 2004).
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)
for use in the gene differential display and RNase-protection assays, or using
TRI Reagent (Molecular Research Center, Cincinnati, OH)
(Mungenast and Ojeda, 2005)
for use in all other procedures.
Gene differential display
For identification of genes differentially expressed at the time of
follicular assembly we used a Differential Display Kit (GeneHunter, Nashville,
TN), as described (Paredes et al.,
2005). The sequences obtained were compared with gene sequences
available in GenBank using the Basic Local Alignment Search Tool (BLAST)
algorithm applied to searches of the non-redundant (nr) and Expressed Sequence
Tag (EST) NCBI databases.
RNase-protection assay and cRNA probes
RNase-protection assays (Gilman,
1993) were carried out as described previously
(Dissen et al., 1995;
Ma et al., 1996) using 10
µg total RNA per tube. Cyclophilin mRNA, which is constitutively expressed
in the ovary, was used to normalize Fxna mRNA values. The antisense
RNA probes used to detect Fxna transcripts were transcribed from two
DNA templates derived from the coding region of Fxna mRNA (probe A,
292 nt, complementary to nt 4846-5138 in NM_184050; probe B, 488 nt,
complementary to nt 1591-2079). The cyclophilin probe (158 nt) is
complementary to nt 265-422 of rat cyclophilin mRNA (M19533).
Northern blots
Polyadenylated RNA was isolated from total RNA extracted from various
tissues of 2-day-old female rats using the MicroPolyA-Purist Kit (Ambion,
Austin, TX). Northern blotting was performed as described
(Lara et al., 1990;
Trzeciak et al., 1987), using
5 µg of mRNA per lane.
In situ hybridization
The in situ hybridization procedure employed
(Simmons et al., 1989) was
carried out as previously reported (Dissen
et al., 1991; Dissen et al.,
1995) using 14 µm cryostat sections and Fxna cRNA
probe A labeled with 35S-UTP. Control sections were hybridized to a
sense Fxna probe transcribed from the same cDNA template but in the opposite
direction.
PCR cloning
To sequence Fxna mRNA, we PCR-amplified overlapping portions of
the mRNA from ovarian RNA beginning from the 3' end using, as a starting
point, the sequence of the C5-530a2 cDNA identified by gene differential
display. The primers used are listed in
Table 1.
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A tagged Fxna construct was generated by PCR-amplifying the Fxna coding region from ovarian RNA with a sense primer (5'-GGATCCGCTGCCGCCATGGAGTGG-3') and an antisense primer (5'-GATATCATATTACTTGTCGTCATCGTCTTTGTAGTCAAACACAAAGAGACTATA-3') that contains a FLAG epitope-coding sequence (in italics); BamH1 and EcoRV sequences added to the sense and antisense primers, respectively, are underlined. The resulting construct was ligated into pcDNA-Zeo (Invitrogen).
siRNA synthesis and transfection
Five siRNAs were synthesized by in vitro transcription with T7 RNA
polymerase using the Silencer siRNA Construction Kit (Ambion). The siRNAs were
named according to their position in the coding region of Fxna mRNA
(i.e. starting at 436, 571, 724, 975 and 1239 nt, where ATG=+1).
siRNA 436: sense, 5'-AACAGCCTCCACAGAATCTCA-3'; antisense, 5'-AATGAGATTCTGTGGAGGCTG-3';
siRNA 571: sense, 5'-AAGTACGCTGTCCTGGCTAAC-3'; antisense, 5'-AAGTTAGCCAGGACAGCGTAC-3';
siRNA 724: sense 5'-AATGGTGCAGAGGAAAATGTC-3'; antisense, 5'-AAGACATTTTCCTCTGCACCA-3';
siRNA 975: sense 5'-AATCTACAGGGATTTTGGGAA-3'; antisense, 5'-AATTCCCAAAATCCCTGTAGA-3'; and
siRNA 1239: sense, 5'-AATAAACTACATGGTGGTAAT-3'; antisense, 5'-AAATTACCACCATGTAGTTTA-3'.
The effectiveness of these siRNAs was tested using rat kidney embryonic cells (RK3E) and human 293T cells. The cells were maintained in DMEM (Sigma, St Louis, MO) supplemented with 10% heat-inactivated FBS (HiClone, Logan, UT), and containing penicillin G (100 U/ml; Sigma) and streptomycin sulfate (100 µg/ml; Sigma). The medium for 293T cells also contained 50 µg/ml gentamycin. Cells were cultured at 37°C under a 5%-CO2 95%-air atmosphere. Cells were seeded into 6-well plates (3x105 cells per well) 24 hours prior to transfection, in antibiotic-free DMEM medium. Each siRNA was transfected (at a 10 nM concentration) using the Gene Eraser Reagent (Stratagene). Cells were harvested 48 hours posttransfection, and total RNA extracted for PCR amplification. The sense primer used (5'-CCATCGGCCCCAGGACTA-3') corresponds to nt 361-378 of rat Fxna mRNA (NM_184050), and the antisense primer (5'-AACACGAGAAGGGTACGCAATGAC-3') is complementary to nt 1224-1247. As an internal control, we amplified a fragment from rat cyclophilin mRNA using a forward primer (5'-ACGCCGCTGTCTCTTTTC-3') corresponding to nt 344-364 of rat cyclophilin mRNA (NM_19533), and a reverse primer (5'-CTTGCCACCAGTGCCATTAT-3') complementary to nt 545-565. PCR amplification was performed using HotStart Taq polymerase (Qiagen, Valencia, CA). Equal volumes were electrophoresed on 2% agarose gels stained with ethidium bromide. Gel images were quantitated using Quantity One software (Bio-Rad Laboratories, Hercules, CA). The optical densities of Fxna mRNA were normalized to cyclophilin mRNA.
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Design and cloning of shRNA-producing cassettes
Delivery of siRNAs via lentiviruses was carried out as described
(Tiscornia et al., 2003). A
cassette derived from the vector pSilencer 1.0-U6 (Ambion) was cloned into the
3' long terminal repeat (3'-LTR; BbsI site) of a
lentiviral vector modified from that described by others
(Follenzi et al., 2000). In
this cassette, transcription of siRNAs is directed by the mouse RNA Polymerase
III U6 promoter. In our plasmid, termed LV-EGFP, the original PGK promoter
driving expression of an enhanced green fluorescent protein (EGFP)
(Follenzi et al., 2000) was
replaced by a cytomegalovirus (CMV) promoter. The DNA sequence encoding the
most potent siRNA (siRNA 436) was cloned into the ApaI-EcoRI
sites of the multiple cloning site of the U6 cassette (see Fig. S1 in the
supplementary material). We refer to this construct as LV-sh436. A DNA
fragment encoding siRNA 436 with nucleotide mismatches (LV-sh436 mism,
5'-CAGGCTCGACAGTATCTGATTCAAGAGATCAGATACTGTCGAGCCTGTTTTTT-3';
the hairpin loop and the polyT sequences are in italics; mismatches are
underlined) was similarly cloned.
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;
Follenzi et al., 2000),
consists of two packaging plasmids (pMDLgpRRE, pRSV-Rev), an envelope plasmid
(pMD.G) and a lentiviral vector plasmid (pLV, the `delivery vector') that
encodes an EGFP marker. The 3'-LTR is self-inactivating. Recombinant
lentiviruses were produced by cotransfecting the four plasmids into 293T cells
using the calcium phosphate method (Dull
et al., 1998). The medium containing infectious viral particles
was collected 24 and 48 hours after transfection and filtered through a 0.22
µm membrane (Steriflip; Millipore, Bedford, MA). Lentiviruses were
concentrated by ultracentrifugation (2 hours, 53,000xg)
and purified through 20% sucrose (2 hours, 45,000xg)
(Tiscornia et al., 2003).
Lentivirus titer was determined by flow cytometry (FACScalibur; BD
Biosciences, San Jose, CA) using serial dilutions of the virus, and EGFP as
the marker. The titer is expressed as transducing units (TU) per ml.
Interferon-like responses
To rule out non-specific mRNA degradation resulting from activation of the
interferon system, semi-quantitative PCR was used to measure the mRNA content
of 2'-5' oligoadenylate synthase (Oas1), a major
interferon target gene (Bridge et al.,
2003), in untreated cultured ovaries and ovaries treated with
LV-sh436, LV-sh436 mism or LV-EGFP. The primers used to detect Oas1
were: sense, 5'-TACAATCCTGATCCCAAGA-3' corresponding to nt 538-559
of rat Oas1 mRNA (NM_138913); and antisense,
5'-GAGCTCCGTGAAGCAGGTAGA-3' complementary to nt 613-633.
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). One
ovary from each animal was treated for 4 days with LV-EGFP and the
contralateral ovary with LV-sh436, each at 4x106 TU per well.
Some ovaries (two pools containing three glands per pool) were treated with
LV-sh436 mism.
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), embedded in paraffin and sectioned at 6 µm. BrdU
immunohistochemistry was performed using a monoclonal antibody (Mab-2531,
1:1000; Sigma) and the reaction was developed with DAB-nickel chloride
(Dees et al., 1995). Ovary
sections were imaged on a Marianas Imaging Workstation using Slidebook
(Intelligent Imaging, Denver, CO), and a 10x NA 0.5 objective.
BrdU-positive cells were counted using Metamorph (Universal Imaging, West
Chester, PA) in 30 identical rectangles (70x52 pixels) corresponding to
an area of 1456 µm2, each uniformly distributed in a random
fashion over four sections from each group. Apoptotic ovarian cells were detected in 14 µm cryostat sections using the TUNEL assay according to the manufacturer's specifications (Roche Diagnostics).
Follicular development
Cultured ovaries were fixed in Kahle's fixative, embedded in paraffin,
serially sectioned at 6 µm, and stained with Weigert's iron Hematoxylin and
counterstained with picric acid-Methyl Blue
(Dissen et al., 2001). Every
third section was imaged on a Zeiss Axioplan (Carl Zeiss, Jenna, Germany),
using a CoolSnap camera (Roper Scientific, Stillwater, MN). Follicles were
counted using the manual count feature of MetaMorph (Universal Imaging, West
Chester, PA) as reported (Dissen et al.,
2001; Paredes et al.,
2004).
Immunohistofluorescence-confocal microscopy
Ovarian cells infected by the lentivirus were visualized by
immunofluorescence in 14 µm frozen sections using an anti-EGFP polyclonal
antibody (Molecular Probes, Eugene, OR; 1:800, overnight incubation at
4°C). Immunoreactivity was developed using an Alexa 488-conjugated donkey
anti-rabbit gamma globulin antibody (Molecular Probes; 1:200). Control
sections were incubated without primary antibody. Images were acquired as
previously reported (Dissen et al.,
2001). Cell nuclei were stained with Hoescht 33258 (Invitrogen;
0.1 µg/ml KPBS for 1 minute after completion of the immunohistochemical
reactions).
To determine the intracellular localization of Fxna, COS-7 cells (1.5x105 cells per well in 6 well plates, i.e. 15,000 cells/cm2) were transfected with either 650 ng per well of Fxna-Flag-pcDNA or the empty vector (pcDNA3.1 Zeo) using Lipofectamine 2000. Twenty-four hours later the cells were fixed in acetone (1 minute at -20°C). Fxna-Flag was detected with an anti-Flag monoclonal antibody (M2, Sigma) at 1:1000 dilution, and the reaction was developed using an Alexa 488-conjugated donkey anti-mouse gamma globulin antibody (1:500). To visualize the endoplasmic reticulum, we used a rabbit polyclonal antibody (sc-20132, Santa Cruz; 1:250) against oxidoreductase-protein disulfide isomerase (PDI), an endoplasmic reticulum-specific protein. The PDI reaction was developed using Alexa 594-conjugated donkey anti-rabbit gamma globulin antibody (1:500). Cell nuclei were stained with Hoescht.
Statistical analysis
Comparisons between two groups were performed using either the unpaired
(cell transfection studies) or paired (organ culture, morphometric studies)
Student's t-test. Sequence comparisons were performed using BlastP
(Altschul et al., 1990) against
the non-redundant protein sequence database at NCBI
(Benson et al., 2006), and
using FastA (Lipman and Pearson,
1985) against the peptidase sequence collection compiled by the
MEROPS database team (Rawlings et al.,
2006).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Rajah et al., 1992), we
compared the expression profiles of RNA extracted from pools of F21 ovaries
with those derived from ovaries dissected at PN 48 hours, using the technique
of gene differential display (Martin and
Pardee, 1999). An -33P-ATP-labeled band
(C5-530a2) of approximately 300 bp (arrow in
Fig. 1A) showing increased
intensity at PN 48 hours was selected for further analysis. The PCR product
showed strong similarity to a mouse hypothetical gene (BC031519) located on
chromosome 19, predicted to encode a 3.3 kb mRNA species and a protein of 317
amino acids.
C5-530a2 mRNA is larger than the predicted mRNA and is expressed in several tissues of 2-day-old rats
The size of the predicted C5-530a2 mRNA was estimated by northern blot
hybridization using a cRNA probe transcribed from C5-530a2 cDNA. The mRNA is
widely expressed in rat tissues (Fig.
1B), with the greatest abundance seen in ovary, kidney and two
areas of the brain (hypothalamus and hippocampus). Contrary to the predicted
mRNA size of 3.3 kb, the northern blot showed the existence of a major mRNA
species of 5.4 kb. The heavier, but minor, hybridizing species observed in
ovary, kidney and adrenal gland may correspond to a transcript(s) resulting
from the use of alternative transcription start sites or alternative
polyadenylation signals. Because no hybridizing species smaller than 5.4 kb
was detected, we conclude that the C5-530a2 mRNA is larger than the size
predicted by computational analysis.
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RNA from ovaries at different developmental phases was analyzed by RNase-protection assay using two cRNA probes. Probe A (289 nt) corresponds to the C5-530a2 sequence obtained by gene differential display; probe B (406 nt) recognizes a sequence spanning the originally predicted ATG site in BC031519 (Fig. 3A). Consistent with the gene differential display results, C5-530a2 mRNA abundance detected with probe A increased significantly (P<0.02) at PN 48 hours, decreasing thereafter (P<0.02) to lower levels in adult ovaries (Fig. 3B). A similar profile was observed when probe B was hybridized to a pool of RNA samples from selected ages (Fig. 3C). Probe B recognized a single mRNA species, suggesting the absence of a C5-530a2 mRNA variant conforming to the mRNA predicted in BC031519. Based on these observations, we named the new gene (NM_184050) Fxna (for Felix-ina).
Fxna is a transmembrane peptidase of the M28 family localized to the endoplasmic reticulum
The predicted Fxna protein sequence is similar to members of peptidase
family M28 (Rawlings and Barrett,
1995) (Fig. 4).
Fig. 5 depicts the nucleotide
sequence of Fxna and its predicted amino acid composition, showing the
peptidase unit (underlined), the catalytic residues Asp201 and Glu245
(circled), and the metal ligand residues His199, Asp211, Glu246, Glu272 and
His 348 (in squares).
Further analysis of the protein (http://www.cbs.dtu.dk/services/SignalP) predicted the absence of a canonical signal peptide. Thus, it is unlikely that Fxna is a secreted protein, a conclusion supported by a second analysis using a different software package (http://www.cbs.dtu.dk/services/SecretomeP). Instead, Fxna appears to be embedded into cellular membranes, via eight transmembrane domains located towards its C-terminus (Fig. 6A). The peptidase domain of Fxna is located toward the N-terminus (amino acid residues 163-393), a portion of the protein predicted to be localized on the external surface of cellular membranes (http://www.cbs.dtu.dk).
To identify the cellular membrane where Fxna is localized, we transfected COS-7 cells with Fxna-Flag and examined the cells by immunohistofluorescence-confocal microscopy using antibodies against the Flag epitope and against PDI, an endoplasmic reticulum (ER)-specific marker. Fxna immunoreactivity colocalized with PDI (Fig. 6B-D), indicating that under basal conditions (i.e. in the absence of experimentally induced ligand-dependent activation of cell membrane receptors), Fxna is associated with ER membranes.
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Lentiviral-mediated delivery of siRNAs to the ovary decreases Fxna mRNA levels without triggering an interferon response
To determine the role of Fxna in the development of the ovary we employed
an siRNA-mediated gene knock-down approach. Of five siRNAs tested, one (siRNA
436) silenced Fxna expression by more than 80% in the rat kidney cell
line RK3E that expresses Fxna mRNA
(Fig. 8A). None of the siRNAs
tested showed off-target effects, as indicated by the unchanging levels of
cyclophilin mRNA in the treated cells in comparison with untreated controls
(Fig. 8A). After determining
the biological potency and specificity of siRNA 436, the DNA sequence encoding
siRNA 436 was cloned into pLV to generate viral particles producing shRNA.
Using this construct, a `rescue' experiment was performed using an Fxna mRNA in which the region targeted by siRNA 436 carries silent nucleotide substitutions. 293T cells were transfected with expression vectors encoding the wild-type or the mutated Fxna mRNA form, plus the virus producing siRNA 436 (LV-sh436), and were cultured for 4 days. LV-sh436 suppressed wild-type Fxna mRNA expression, but failed to decrease mutant Fxna mRNA levels (Fig. 8B), demonstrating the specificity of the siRNA effect.
To evaluate the ability of LV-sh436 to decrease Fxna gene expression in the ovary, 1-day-old rat ovaries were placed in organ culture and treated for 4 days with LV-sh436 (4x106 TU per well, in 750 µl of medium). Fxna mRNA abundance was reduced by more than 90% in the LV-sh436-treated group as compared with LV-EGFP-treated controls (Fig. 9A). Ovaries treated with LV-siRNA 436 mism had Fxna mRNA levels indistinguishable from those seen in LV-EGFP-treated controls (LV-siRNA mism levels were 98.5% of those of the LV-EGFP group, data not shown). Despite this ineffectiveness we did not use this mutated siRNA further to avoid potential off-site effects resulting from the hypothetical ability of mismatched siRNAs to inhibit the translation of an unrelated gene(s) via a microRNA-like action (see the symposium on Understanding the RNAissance at www.nature.com/horizon).
A major difficulty encountered when using siRNAs
(Sledz et al., 2003) and/or
their delivery vectors (Bridge et al.,
2003) is activation of the interferon system
(Bridge et al., 2003;
Sledz et al., 2003). To
address this issue we adopted a recommended strategy
(Bridge et al., 2003) and
measured the mRNA encoded by the major interferon target gene
2'-5' oligoadenylate synthase (Oas1)
(Pebernard and Iggo, 2004;
Samuel, 2001) in the same
samples used to validate the effect of LV-sh436 on Fxna mRNA levels.
Oas1 mRNA levels were similar in ovaries treated with LV-EGFP,
LV-sh436 and LV-sh436 mism (Fig.
9B), indicating the absence of an interferon response in the
Fxna-knock-down ovaries.
Fxna deficiency disrupts follicular development and alters the structural organization of the ovary
To determine the cellular sites of LV expression, newborn rat ovaries were
cultured for 4 days in the presence of LV-EGFP (4x106 TU per
well in 750 µl of medium), before immunohistochemical detection of EGFP. As
shown in Fig. 9C,D, infection
was widespread, with EGFP-immunoreactive material observed in both somatic
cells and oocytes. Infection of newborn ovaries with LV-sh436 for 4 days
severely compromised the follicular structure as compared with that observed
in control, LV-EGFP infected glands (Fig.
9E,F). The LV-sh436-infected ovaries exhibited aggregates of
somatic cells excluding oocytes, and oocytes not encapsulated by granulosa
cells (Fig. 9G,H).
|
), i.e. with many primordial follicles, but also showing an
abundance of primary follicles (Fig.
10A,C). By contrast, the ovaries treated with LV-sh436 exhibited
areas of striking immaturity (Fig.
10B) and overall structural disorganization characterized by
solitary follicles containing up to four oocytes, nests of oocytes not
associated with somatic cells, and aggregates of somatic cells not associated
with oocytes (Fig. 10D). The
number of `naked' oocytes per ovary was similar in Fxna-knock-down
ovaries and LV-EGFP-treated controls (Fig.
10E, left panel), but Fxna-deficient ovaries exhibited a marked
(P<0.01) decrease in the total number of follicles per ovary
(Fig. 10E, right panel). A
more detailed analysis of the follicle populations affected revealed that all
categories of follicles (primordial, primary and secondary) were significantly
reduced in Fxna-deficient ovaries (Fig.
10F). These results indicate that the deficiency in follicular
formation seen in the absence of Fxna is not secondary to a primary loss of
oocytes.
The effects of Fxna deficiency on ovarian histogenesis are not related to alterations in either apoptotic or proliferative rates of ovarian cells
To determine if the alterations in ovarian histogenesis observed in
Fxna-deficient ovaries were due to abnormal proliferation of somatic cells or
somatic cell/oocyte death, we performed apoptosis and proliferation assays in
LV-EGFP and LV-sh436-treated ovaries. No differences in the number of
apoptotic cells per ovary as detected by TUNEL, or in the incorporation of
BrdU into cell nuclei as detected by immunohistochemistry, were apparent
between the two groups (data not shown).
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), deserves
further consideration.
|
).
The structural characteristics of Fxna predict its existence as a
membrane-bound protein, and our results demonstrate that in contrast to most
peptidases expressed in reproductive organs
(Fujiwara et al., 1999), Fxna
is localized to the ER instead of the cell membrane. Previously described ER
peptidases include endoplasmic reticulum aminopeptidase 1 (ERAP1)
(Fruci et al., 2006), the
related aminopeptidase leukocyte-derived arginine aminopeptidase (L-RAP, also
called ERAP2) (Saveanu et al.,
2005; Tanioka et al.,
2003), and vitellogenic carboxypeptidase-like protein
(Harris et al., 2006). The
presence in Fxna of up to eight putative transmembrane domains resembles the
hydropathy profile of site-2 protease (S2P), a zinc metallo-aminopeptidase
required for the intramembranous proteolysis of
sterol-regulatory-element-binding proteins (SREBPs)
(Rawson et al., 1997). S2P is
embedded in the ER via 5 transmembrane domains and releases the N-terminal
domain of SREBPs within the ER membrane
(Zelenski et al., 1999).
Peptidases bound to intracellular membranes may have fascinating functional
properties as shown by the insulin-regulated aminopeptidase (IRAP), which is
bound to intracellular membranes under basal conditions, but redistributes to
the cell membrane in response to insulin stimulation
(Keller, 2003). In type 2
diabetes, insulin action is impaired and the translocation of IRAP to the cell
membrane fails to occur, suggesting that the absence of IRAP-mediated peptide
processing on the cell membrane is an intrinsic component of this pathology.
Although our results do not identify the mode of action of Fxna, they do
demonstrate that Fxna is essential for the normalcy of definitive ovarian
histogenesis. Silencing the Fxna gene strikingly disrupts ovarian
structural organization. In Fxna-deficient ovaries, both the rate of follicle
assembly and initiation of follicle growth are compromised, without detectable
prior loss of oocytes. An even more remarkable feature is a widespread loss of
somatic germ cell organization. Pregranulosa cells appear to encircle more
than one oocyte, and many oocytes remain clustered in isolation without having
been infiltrated by somatic cells, as would normally occur during follicular
formation (Hirshfield,
1991).
Very few substrates for metallopeptidases have been identified. We do not
know at present which proteins are hydrolyzed by Fxna. Because of its
intracellular localization, Fxna might be necessary for the correct processing
of proteins involved in cell signaling
(Wolfe and Kopan, 2004) and/or
transcriptional control (Rawson et al.,
1997). Alternatively, it may act as a general degrading protease
of transmembrane protein precursors. Nevertheless, the alterations in
structural organization resulting from the loss of Fxna function in the ovary
argue against the latter possibility, and suggest that systems affected by the
deficiency may be those cell-to-cell communication pathways required for
definitive ovarian histogenesis. The two major morphogenic signaling complexes
required for the organization of the very processes affected by Fxna
deficiency are the Jagged-Notch
(Artavanis-Tsakonas et al.,
1999; Lai, 2004)
and the Wnt-Frizzled (Cadigan and Nusse,
1997; Miller et al.,
1999) signaling systems. Of note, both Notch2 and Frizzled1
receptors are expressed in granulosa cells throughout preantral development
(Johnson et al., 2001;
Richards et al., 2002), and in
both cases loss of function results in abnormalities similar to those seen in
Fxna-deficient ovaries (Hahn et al.,
2005; Vainio et al.,
1999). Should Fxna be involved in processing any of the basic
and/or associated components of these systems, one would have to assume that
it acts early in the processing pathway, as a proteolytic event that precedes
the transport of such target proteins to the Golgi apparatus for covalent
modifications and intracellular targeting.
In summary, our results identify a new regulatory system that operates in somatic granulosa cells to control ovarian development, and which appears to be required for proper structural organization of the ovary. The cellular systems targeted by the action of Fxna remain to be identified.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/5/945/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Facultad de Ciencias Quimicas, Benemerita Universidad
Autonoma de Puebla, Puebla, Mexico
Present address: Laboratorio de Bioquimica, Departmento de Ob/Gyn, Hospital
Clinico, Universidad de Chile, Santiago, Chile
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